From the Department of Women’s and Children’s Health

Karolinska Institutet, Stockholm, Sweden

MOLECULAR AND CLINICAL STUDIES OF INTESTINAL MALROTATION

Karin Salehi Karlslätt

Stockholm 2022

Cover by Karin Salehi Karlslätt Pictures in the thesis are generated from open access sources with general permissions or illustrated by Karin Salehi Karlslätt, if nothing else is stated. All previously published papers were reproduced with permission from the publisher. Published by Karolinska Institutet. Printed by Universitetsservice US-AB, 2022 © Karin Salehi Karlslätt, 2022 ISBN 978-91-8016-526-6

Molecular and clinical studies of intestinal malrotation THESIS FOR DOCTORAL DEGREE (PhD)

By

Karin Salehi Karlslätt

Principal Supervisor:

Agneta Nordenskjöld, professor

Karolinska Institutet

Department of Women’s and Children’s Health

Division of Pediatric Oncology and Pediatric

Surgery

Co-supervisors:

Britt Husberg, med dr

Karolinska Institutet

Division of clinical sciences

Department of Surgery at Ersta Hospital

Ersta Hospital

Anna Lindstrand, professor

Karolinska Institutet

Department of Molecular Medicine and Surgery

Division of Rare Diseases

Tomas Wester, professor

Karolinska Institutet

Department of Women’s and Children’s Health

Division of Pediatric Oncology and Pediatric

Surgery

Opponent:

Martin Salö, docent

Lund University

Department of Clinical Sciences

Division of Pediatric Surgery

Examination Board:

Greger Lindberg, professor

Karolinska Institutet

Department of Medicine

Division of Medical Gastroenterology and

Hepatology

Catharina Lavebratt, docent

Karolinska Institutet

Department of Molecular Medicine and Surgery

Division of Neurogenetics

Anders Elfvin, docent

Sahlgrenska Academy, University of Gothenburg

Division of Clinical Sciences

Department of Pediatrics

Division of Neonatology

To David, Arvid and Jennifer

Human beings are members of a whole,

in creation of one essence and soul.

If one member is inflicted with pain,

other members uneasy will remain.

If you have no sympathy for human pain,

the name of human you cannot retain.

Saadi Shirazi (1210-1292) from Gulistan, 1258

Translation from Persian by M. Aryanpoor

POPULAR SCIENCE SUMMARY OF THE THESIS

Intestinal malrotation (IM) is a congenital malformation of the intestines characterized by

deficient attachment of the intestines to the posterior abdominal wall and increases the risk

for midgut volvulus. It may be as common as in 1/500 individuals. Volvulus requires

immediate surgery to derotate the intestines. The symptoms can also be of a more chronic

character due to a mechanical obstruction of the duodenum that may cause abdominal

discomfort, sometimes during several years. This has mainly been described in children, but

during recent years also among adults. The treatment is Ladd’s procedure, when the

intestines are placed in the abdomen in an organized way so that an intestinal strangulation

cannot happen and also embryonal adhesive bands, called Ladd’s bands, that can obstruct

the intestines are removed.

The knowledge that also adults can have symptoms from intestinal malrotation is relatively

new and not well-studied so we wanted to study all age groups regarding symptoms,

diagnostics and outcomes and compare them to better understand the variants of the

disease. In addition, the molecular knowledge on how IM develops is limited. There are

familial cases as well as patients with malrotation and genetic syndromes or different

chromosomal aberrations. These data together speak for genetic causes in at least some

cases of malrotation.

We performed two clinical studies (study I and II) on 138 children and 39 adults with

intestinal malrotation and we compared initial symptoms, other associated malformations,

treatment and the outcome after treatment. Other malformations were common and

symptoms were age-dependent to some extent. We found that in children up to five years of

age the main symptom was vomiting, and that is was bile-stained up to the same age. From

five years of age abdominal pain is the main symptom at diagnosis. Children born

extremely preterm and patients with severely affected circulation of the intestines had a

dismal outcome in the pediatric study. In addition, it is more common with a need for

emergency surgery in children but recurrence of volvulus was more prevalent in the adult

group. Longer periods of diffuse or intermittent acute abdominal discomfort before

diagnosis was often described among adult patients and it was not uncommon with residual

abdominal discomfort also after surgery.

We also performed two molecular projects (study III and IV) to investigate molecular

causes for malrotation. In study III we did ¨array comparative genomic hybridization¨

(array CGH), in order to find submicroscopic chromosome changes. We found six such

variants in five of the 47 DNA samples that we examined, whereof four were disease

causing. Among these patients, all had one or several other malformations or a

developmental disability, which lead us to recommend that a genetic investigation should

be done in intestinal malrotation with an additional malformation or developmental delay.

In study IV we performed DNA sequencing of all genes in ten patients with only intestinal

malrotation, to look for possibly pathological variants among a few hundred candidate

genes. One patient had two gene variants in the same gene, the TTC7A-gene. Further

studies showed that the mother also had the same variants as well as a milder form of

malrotation. Interestingly, TTC7A-deficiency is a rare disorder that is autosomal

recessively inherited, meaning that you can inherit the disorder if you get one pathogenic

variant from each of your parents. Generally, these patients have several stops in the

intestines (atresias) in combination with an immunodeficiency, and also some patients have

been born with intestinal malrotation. We hypothesize that intestinal malrotation is a mild

manifestation of TTC7A-deficiency and these findings call for further studies in a larger

material.

In summary this thesis describes symptoms and treatment outcome in patients with

intestinal malrotation of all ages and also molecular studies are performed using DNA from

patients in order to elucidate the background for the malformation.

NORMAL INTESTINES

INTESTINAL MALROTATION MIDGUT VOLVULUS

POPULÄRVETENSKAPLIG SAMMANFATTNING

Malrotation av tarmen är en missbildning av tarmens vidhäftning mot bakre bukväggen

som medför en ökad risk att utveckla tarmvred. Det finns olika uppgifter om hur vanligt

malrotation är, men det förekommer hos ungefär 1/500 och ungefär 1/6000 spädbarn

uppvisar symptom. Av tarmvred, får man generellt akut magsmärta, men även kräkningar,

vilket är speciellt påtagligt hos spädbarn och man behöver opereras akut. Symptombilden

kan även variera i svårighetsgrad med kroniska besvär som tidig mättnadskänsla eller

återkommande buksmärta. Behandlingen är Ladds operation, då tarmarna placeras på ett

organiserat sätt i buken så att inte tarmvred ska kunna utvecklas, samt att embryonala

bindvävsstråk som kan obstruera tarmen tas bort.

Vi ville kartlägga hur symptomen skiljer sig genom olika skeden i livet, som generellt mest

varit kända under neonatalperioden och vi ville även se över diagnostik, behandling och

postoperativa komplikationer i de olika åldersgrupperna. Kunskapen om malrotation är

begränsad och det finns familjära fall som tyder på att det kan vara ärftligt, vilket vi också

ville utforska.

I avhandlingen presenteras två kliniska studier (I och II), där 138 barn respektive 39 vuxna,

som fått diagnosen malrotation, studerades med avseende på symptom, andra

missbildningar, behandling samt kliniska resultat efter operationen. Andra samtidigt

förekommande missbildningar är vanliga och uppvisade symptom är till viss del

ålderstypiska. Vi fann att hos barn upp till fem års ålder var kräkningar det främsta

symptomet. Från fem år till vuxen ålder dominerade symptomet buksmärta.

Extremprematurer och patienter med kraftig cirkulationspåverkan på tarmen hade störst risk

för komplikationer i barnstudien, där var det även dubbelt så många pojkar som drabbades.

Det var även vanligare med ett akut behov av operation i barnstudien och de uppvisade mer

sällan recidiv efter operation jämfört med den vuxna gruppen. Hos äldre barn och vuxna var

det vanligt att man haft långa perioder av oklara magbesvär innan man fick sin diagnos och

för vuxna var det inte ovanligt med kvarvarande lite mer diffusa magbesvär även efter

operation.

Två molekylära studier (III och IV) har också gjorts, för att leta efter en möjlig orsak till

malrotation. I studie III gjorde vi ¨array comparative genomic hybridization¨ (array CGH),

som är en metod att leta efter submikroskopiska kromosomförändringar, där vi fann sex

förändringar hos fem av 47 patienter, varav 4 av dem var direkt sjukdomsorsakande. De

som hade sjukdomsorsakande fynd hade alla någon ytterligare missbildning eller

utvecklingsförsening, vilket ledde till slutsatsen att patienter med malrotation i kombination

med någon annan missbildning eller utvecklingsavvikelse borde genomgå en genetisk

utredning. Vi gjorde även helgenomsekvensering på tio patienter som bara hade malrotation

utan annan missbildning, för att leta efter avvikelser i kandidatgener hos dessa patienter. Av

dessa fann vi en patient som hade två genavvikelser i samma gen, TTC7A-genen. Utredning

visade att även mamman hade samma genavvikelser och en liknande mildare avvikelse av

tarmrotationen. TTC7A-brist är ovanligt och recessivt nedärvt, dvs man får normalt sett

sjukdomen om ens båda arvsanlag är skadade dvs har varsin mutation. Denna sjukdom

innebär att man föds med stopp (atresier) på flera ställen i tunntarmen och immunbrist, men

även fall med malrotation är rapporterade. I detta fall var det alltså bara en av de två

genkopiorna som bar på avvikelserna och i association med rotationsavvikelser i tarmen.

Detta behöver studeras ytterligare i större omfång.

Sammanfattningsvis beskriver vi symptom och behandlingsutfall för patienter med

malrotation i alla åldrar och vi beskriver våra molekylära studier där vi med DNA från

patienterna letat efter submikroskopiska kromosomförändringar och mer specifika

gensekvensförändringar.

ABSTRACT

Intestinal malrotation (IM) can present as a potentially life-threatening condition with

midgut volvulus and need of immediate surgery, but also as a chronic condition with long-

lasting symptoms. It is a well-known neonatal condition but it is not as well “explored” up

to adulthood. Familial cases and chromosomal aberrations have been described, as well as

genetic syndromes associated with intestinal malrotation, but the molecular cause is not

known.

The aim of study I and II was to describe symptoms, management and outcomes of IM

throughout all age groups by scrutinizing medical records and through interviews. In study

III and IV the aim was to study the molecular background of IM.

In study I, 39 adult patients, 15-67 years old, were included. 31 (79%) underwent a Ladd’s

procedure and among them 12 (31%) as an emergency procedure. A triple contrast CT scan

confirmed the diagnosis and many patients, who often had symptoms for several years were

relived of symptoms after Ladd’s procedure, even though six needed re-do surgery because

of recurrence.

In study II, 138 children were included based on strict inclusion criteria, 1-15 years old.

125 (91%) had Ladd’s procedure and only one had recurrence needing re-do surgery.

The major findings were that the most common diagnostic symptom up to 5 years of age

was vomiting, while abdominal pain was a more relevant symptom in older children and

adults. Complications were mainly detected among extremely premature children, with an

overall mortality of 50%, and also among children who had severely affected circulation.

Four children had a chronic intestinal failure, two because of midgut volvulus and 14

patients (11%) had adhesive bowel obstruction.

In study III, by using array CGH we detected six different copy number variants (CNVs)

in five patients of the 47 DNA samples that were analyzed. Five were found in patients with

other malformations or developmental delay and one in a patient with isolated IM had a

variant of unknown significance in the GALNT14 gene.

In study IV, we performed whole genome sequencing (WGS) in ten children with severe

isolated IM, and found two maternally inherited allelic mutations in the TTC7A gene in a

boy with isolated IM. Further studies showed that the mother also had a rotational

abnormality. The rare autosomal recessively inherited TTC7A deficiency disorder causes

multiple intestinal atresia and immunodeficiency, but also IM in some case reports.

In conclusion, the results of this thesis show that IM should be regarded as a malformation

affecting all age groups, and midgut volvulus can occur in all ages. The symptoms vary

with age and calls for an active strategy for diagnosis and treatment. Extreme prematurity

as well as midgut volvulus with severely affected circulation, increase the risk for short-

term complications after surgery. We recommend genetic screening for CNVs in cases with

IM and other associated malformations or a neurodevelopmental disorder and the TTC7A

gene needs to be further studied in larger groups of cases with IM.

LIST OF SCIENTIFIC PAPERS

I. Britt Husberg, Karin Salehi, Trevor Peters, Ulf Gunnarsson, Margareta

Michanek, Agneta Nordenskjöld, Karin Strigård. Congenital intestinal

malrotation in adolescent and adult patients: a 12-year clinical and

radiological survey. Springerplus. 2016; 5: 245

II. Karin Salehi Karlslätt, Britt Husberg, Ulla Ullberg, Agneta Nordenskjöld,

Tomas Wester. Intestinal malrotation in children: Clinical presentation and

outcomes. Manuscript.

III. Karin Salehi Karlslätt*, Maria Pettersson*, Nina Jäntti, Przemyslaw

Szafranski, Tomas Wester, Britt Husberg, Ulla Ullberg, Pawel Stankiewicz,

Ann Nordgren, Johanna Lundin, Anna Lindstrand, Agneta Nordenskjöld.

Rare copy number variants contribute pathogenic alleles in patients with

intestinal malrotation. Molecular Genetics and Genomic Medicine. 2019;

7(3): e549

IV. Karin Salehi Karlslätt, Maria Pettersson, Kristina Lagerstedt-Robinson,

Ulla Ullberg, Anna Lindstrand, Agneta Nordenskjöld. Gene panel-based

screening of patients with isolated intestinal malrotation: An extended

phenotype for mutations in the TTC7A gene. Manuscript.

* Equal contribution

CONTENTS

1 INTRODUCTION ......................................................................................................... 5 1.1 Historical Background ...................................................................................... 5 1.2 Embryological development of the midgut ....................................................... 7

Definitions of intestinal rotation abnormalities ................................... 10 Secondary malrotation ......................................................................... 12

1.3 Clinical perspectives on IM ............................................................................ 12 Associated anomalies .......................................................................... 13 Radiological diagnostics ..................................................................... 13 Management and surgical treatment ................................................... 16 Outcomes ............................................................................................. 18 Adult patients and IM .......................................................................... 18

1.4 Molecular perspectives on IM ......................................................................... 19 Human genetics ................................................................................... 19 Experimental data ................................................................................ 21 Familial cases ...................................................................................... 22 Chromosomal aberrations associated with IM .................................... 22 Monogenic IM ..................................................................................... 24 Ciliopathies .......................................................................................... 24

2 RESEARCH AIMS ..................................................................................................... 27 3 CLINICAL STUDIES I & II ....................................................................................... 29

3.1 Methods ........................................................................................................... 29 Study design, Setting and Participants ................................................ 29 Variables .............................................................................................. 30 Statistical analysis ............................................................................... 31

3.2 Main results and discussion ............................................................................ 32 Study I inclusion .................................................................................. 32 Study II inclusion and variants of IM .................................................. 32 Associated malformations ................................................................... 33 Gender and age at diagnosis ................................................................ 34 Clinical presentation ............................................................................ 35 Diagnostic procedures ......................................................................... 37 Management ........................................................................................ 39 Outcome .............................................................................................. 40 Adult patients and IM .......................................................................... 42

4 MOLECULAR STUDIES III & IV ............................................................................ 43 4.1 Methods ........................................................................................................... 43

Study design, Setting and Participants ................................................ 43 Molecular analyses .............................................................................. 44

4.2 Main results and discussion Study III ............................................................. 48 Inclusion .............................................................................................. 48 Reported CNVs ................................................................................... 48 Clinical relevance ................................................................................ 50

4.3 Main results and discussion Study IV: ............................................................ 51 Inclusion .............................................................................................. 51 Reported variants ................................................................................. 51 The TTC7A-gene and IM ..................................................................... 52 Clinical relevance ................................................................................ 53

5 STRENGTHS AND LIMITATIONS ......................................................................... 55 6 ETHICAL CONSIDERATIONS ................................................................................ 56

7 CONCLUSIONS ......................................................................................................... 57 8 POINTS OF PERSPECTIVE ...................................................................................... 59 9 ACKNOWLEDGEMENTS ........................................................................................ 60 10 REFERENCES ............................................................................................................ 65

LIST OF ABBREVIATIONS

A

ALL Array CGH

C CAD

CADD CI

CID

Adenine

Acute lymphoblastic leukemia Array comparative genomic hybridization

Cytosine Congenital alveolar dysplasia

Combined Annotation Dependent Depletion Confidence Interval

Combined immunodeficiency CDH

CLDS CLMP

CNV CNS

CT DNA

Congenital diaphragmatic hernia

Cornelia de Lange syndrome CXADR Like Membrane Protein

Copy number variants Central nervous system

Computed tomography Deoxyribonucleic acid

EOIBD FLNA

FOXF1 FTR

G GA

GALNT14 HPO

IM IMA

Early onset inflammatory bowel disease Filamin A

Forkhead Box F1 Failure to rescue

Guanine Gestational Age

Polypeptide N-Acetylgalactosaminyltransferase 14 Human Phenotype Ontology

Intestinal malrotation Inferior mesenteric artery

IRA IUGR

kb MIA

MRI mRNA

MZ OMIM

OR

Intestinal rotation abnormalities Intrauterine growth retardation

kilobase multiple intestinal atresias

Magnetic Resonance Imaging Messenger ribonucleic acid

Monozygotic Online Mendelian Inheritance in Man

Odds Ratio

PI4IIIα-complex

PN SBS

SMA SMV

SNV SV

T TPR

TTC7A/B UGI study

VOUS VUR

VSD WGS

3D

Phosphatidylinositol 4-kinase IIIα-complex

Parenteral nutrition Short bowel syndrome

Superior mesenteric artery Superior mesenteric vein

Single nucleotide variant structural variants

Thymine Tetratricopeptide Repeat

Tetratricopeptide Repeat Domain 7A/7B Upper gastrointestinal contrast study

Variant of uncertain significance Vesicoureteral reflux

Ventricular septal defect Whole genome sequencing

Three-dimensional

5

1 INTRODUCTION Intestinal malrotation means that the intestines are positioned abnormally in the abdomen

constituting a predisposition for midgut volvulus, which causes obstruction and ischemia of

the intestines. This is a potentially life-threatening condition and needs immediate surgery.

1.1 HISTORICAL BACKGROUND

The historical background of intestinal malrotation displays the beginning of a thrilling

story on how a malformation is discovered, diagnosed, treated and later studied concerning

molecular background. This thesis pursues to be a small chapter of this adventure, and a

summary on how far this expedition has come, which still has a long way to go until it is

finished.

To define a malformation, the normal anatomy must first be understood. Václav Treitz

(1819-1872), worked as a professor in anatomy in Prague and published "Ueber einen

neuen Muskel am Duodenum des Menschens" (On a New Muscle in the Duodenum of Man)

in 1853 where he described the ligament of Treitz. This suspensory muscle of duodenum is

the fixating unit of the duodeno-jejunal junction in the left upper quadrant, and its absence

has become a hallmark for intestinal malrotation (IM) (Lampl 2009).

In 1898, Mall was the first to describe the normal rotation and fixation of the intestine

during embryogenesis (Mall 1898), a theory which has remained unchanged until recently.

IM was described before 1900 from autopsy- and surgical findings, and in 1923, Dott

pointed out the relationship between incomplete embryological intestinal rotation and its

need for surgical treatment because of its predisposition to midgut volvulus (Dott 1923).

The Halifax Disaster (1917) during the First World War was an accidental collision of two

ships in the Halifax harbor in Canada. The explosion killed 2000 people and over 9000

people were harmed, including children. William E. Ladd (1880-1967), as well as other

surgeons, were sent there to help. He was strongly affected by the disaster, especially by the

wounded children, so after returning to his hospital in Boston, USA, he devoted his career

to treat children (Lampl 2009). He has since contributed to pediatric surgery in many ways

but his name is immortal due to the Ladd´s procedure that he described in 1932 on how to

treat intestinal malrotation and volvulus, which is still the gold standard treatment for

intestinal malrotation (Ladd 1932, Ladd 1936).

The cause of the deviating embryonal development is not known, but it is thought to be

partly environmental and partly genetic in cause. The understanding of genetic causes of

6

human disease started first through the possibility to study chromosomes by karyotyping

from 1959 and then later the possibility to look for single nucleotide variants (SNVs) in the

genome.

In the 1990s, Sanger sequencing was clinically used. In 2001, the entire human genome was

sequenced from the important insight of a need to have a common, unbiased reference for

scientists throughout the world in order to accelerate biomedical research (Lander 2001).

Mass sequencing and softwares to compare and interpret changes in the genome has turned

the knowledge of about 300 rare monogenic diseases in 1990 to 7000 in 2022 and complex

diseases from zero to almost 70 000.

In order to study the genetic backgrounds different methods are used that can discover

different genetic mechanisms. Karyotyping when chromosomes are sorted and studied in a

light microscope has a resolution of down to 50-100 genes (≈5-10 Mb) (Figure 1). Array

CGH that is a method we used in this thesis can detect submicroscopic changes, sometimes

down to part of genes. In order to examine smaller gene variants different methods for

DNA sequencing are used, like here whole genome sequencing (WGS) although we only

examined a subset of all genes, regarded as candidates to cause a certain disease. Thus

different molecular methods can be used for different research questions and have different

advantages and disadvantages. Methods can also be used together. For example, if DNA

from a patient with a rare disease shows a chromosomal aberration, this can indicate that

disease genes are located in that specific chromosome region and those genes can further be

studied with other genetic methods like sequencing.

Figure 1: Normal male 46, XY human karyotype, 44 autosomes and 2 sex chromosomes

(here X and Y). The entire genome is packed into 23 pairs of chromosomes with one copy

from each parent. (Wessex Reg. Genetics Centre. Attribution 4.0 International (CC BY

4.0))

7

1.2 EMBRYOLOGICAL DEVELOPMENT OF THE MIDGUT The different stages of embryologic development are important when studying the etiology

of intestinal malrotation. Current knowledge is based on experimental observations and

hitherto not completely revealed.

Rotation of the intestines occurs between the fourth and twelfth weeks of gestation. The

mechanism leading to the “intestinal rotation” and the exact order of events has been

discussed since the 19th century based on observations of the normal development and of

malformations such as IM and abdominal wall defects (Mall 1889, Frazer 1915). These

concepts are recently reviewed in human and rat fetuses and are now considered to occur

somewhat differently (Soffers 2015, Kluth 2003, Metzger 2011, Ginzel 2021).

The embryology of gut rotation has been described to start during the fourth gestational

week when the primitive gut develops into three sub-divisions: Foregut, midgut and

hindgut (Figure 2). The midgut will develop into the future duodenum, jejunum, ileum,

ascending colon and right two thirds of the transverse colon. At four to five weeks of

gestation the midgut is a linear tube lying straight in the midline. The midgut elongates

Figure 2. Schematic drawing of an embryo at week 5. The gut-tube is marked in yellow and

the midgut is highlighted in light yellow. The arteries branching from the aorta from

cranial to caudal are 1) the celiac trunk, 2) the superior mesenteric artery (SMA) and 3) the

inferior mesenteric artery (IMA).

3

2

1

8

rapidly compared to the embryo’s growth. As the midgut grows, it turns into a loop with a

sagittal orientation, the primary loop, with the superior mesenteric artery as its axis, which

is connected to the yolk sac by the vitelline duct (Figure 2, figure 3D). The loop herniates

into the base of the umbilical cord during the sixth week of gestation. It is hypothesized that

the herniation takes place due to the small size of the abdominal cavity of the embryo. As

the midgut loop herniates into the umbilical cord, in the sixth week, it appears to turn 90°

counterclockwise (viewed from the front) with the superior mesenteric artery as its axis

(Figure 3E1). It is probably the adjacent surrounding structures that move that cause the

“rotation”, rather than an actual primary rotation of the SMA and the dorsal mesentery.

Figure 3. A) The primary loop herniating into the umbilical ring. B) The duodenum

forming its secondary loop under SMA, progressing towards the final position of the

duodenojejunal junction. C) The embryo is not in a straight line, rather in a chiral

formation. D-E) demonstrating the formation of the primary, secondary and tertiary loops,

as well as the return of the intestines into the abdominal cavity. F) Final position of the

colon with the cecum in the right lower quadrant. (Soffers 2015) 3: SMA, 7: Right vitelline

vein, 8: Vitelline duct, 9: Duodenum, Dp: Dorsal pancreas, Vp: Ventral pancreas, Gb:

Gallbladder, Li: Liver, St: Stomach.

Experiments using 3D visualization techniques on serial sections from human embryos

have shown that the embryo actually has a chiral formation (Figure 3C), with the caudal

C

A

B

9

part being located on the right side of the cranial part (Ramasubramanian 2013). When the

midline structures (upper abdominal and thoracic) in the embryo descend, the cranial part

of the intestinal loop will descend rightwards and be located to the right side of the SMA

and the caudal part of the loop goes to the left where after the loop then attains a transverse

position (Soffers 2015) referred to as “rotating” 90° (Figure 3E1).

As the intestines are herniated, the secondary loops form. The first secondary loop is

located in the abdominal cavity, comprising the duodenum and first part of jejunum. It

expands rightward and caudally on the right side of the SMA. During the eighth week of

gestation, prior to the return of the intestines, the first secondary loop, (the duodenal part)

grows substantially and continues to expand below the SMA towards the left approaching

its final position of the duodenojejunal junction (Metzger 2011, Soffers 2015) (Figure 3B).

The second to fourth secondary loop forms in the herniated part (Figure 3 E2). Of the

colon, only the cecum and most proximal part is herniated. The intestines grow further and

tertiary loops develop within the areas of the secondary loops, but not in the first secondary

loop (the duodenal part) (Figure 3 E3). The primary and secondary loops seem

predetermined, and the second secondary loop will always for unknown reasons, position

itself to the left in the herniation when developing the tertiary loops (Soffers 2015, Ginzel

2021).

The return of the intestines occurs in a cranial-to-caudal manner into the abdominal cavity

as it expands, at 8.5 – 9.5 weeks of gestation, previously explained as rotating 180°

counterclockwise, en bloc. First the second secondary loop to the left, then the third in the

midline, and the fourth in a ventral position in the abdomen. Previously, it was understood

that the cecum is the last portion to return into the abdominal cavity, however the cecum

returns before the distal part of the ileum, indicating that the colon is co-migrating (Ginzel

2021, Soffers 2015). In week 10, the cecum has taken its definitive position in the right

fossa (Soffers 2015). The cause of the return has many theories, among them that there is a

traction of the vessels, and that the umbilical ring opens up to allow for an intestinal return

(Ginzel 2021).

Altogether, a 270° counterclockwise rotation is said to occur, even though this is not true

for all parts of the midgut. The ascending colon and most of the descending colon fuse with

the posterior abdominal wall and becomes retroperitoneal during the fourth and fifth month

of gestation (Langer 2017). Thereby the duodenojejunal junction, marked by the ligament

of Treitz, and the cecum in the right lower quadrant has maximum distance in the abdomen

in a diagonal manner, leading to a wide dorsal mesentery (dotted line), which carries the

superior mesenteric artery and vein to the intestines (Figure 4).

10

Figure 4. Normal intestines. The dotted line demonstrates a wide dorsal mesenteric base.

Kluth et al showed that it is rather the elongation of the intestines that seem to push them to

rotate than an actual rotational process. Also, the duodenojejunal junction is pushed under

the mesentery through lengthening and ends up in its correct position. The development of

the duodenum is crucial in IM, and interestingly the duodenum is not being a part of the

herniating and rotating process. The driving force is not an actual rotation, but rather

elongation in selective parts of the intestines at different time periods. The elongation of the

intestines with anisotropic growth is shown to be self-consistent by smooth muscle

contractions; peristalsis (Khalipina 2019) and the stereotypical looping pattern seem to be

driven by growth as well (Savin 2011).

1.2.1 Definitions of intestinal rotation abnormalities A disturbance in this embryological process is thought to cause intestinal malrotation and

other intestinal rotation abnormalities (IRA) (Langer 2017). There are some concepts that

are used in pediatric surgery in order to describe these intestinal rotational abnormalities.

When there is “no rotation” around the superior mesenteric artery, this is called

“nonrotation”. The cranial part of the midgut, including duodenojejunal junction and most

Duodenojejunal junction

Cecum

11

part of the small intestine, will lay to the right in the abdomen, while the caudal part of the

midgut, including the colon and terminal ileum, lays to the left in the abdomen (Nehra

2011). “Incomplete rotation” or intestinal malrotation (IM) occurs when there is only a

“partial rotation”. As a result, the cecum will stay located in the right upper quadrant, often

with adhesive peritoneal bands overriding the duodenum, fixed to the right sided abdominal

wall and sometimes also to the gallbladder and duodenum. These embryonic fibrous bands

constitute a failed attempt of cecal fixation, also referred to as Ladd’s bands. In both

incomplete rotation and non-rotation, duodenum goes straight caudally without overriding

to the left of the vertebral spine towards the position of the ligament of Treitz. IM

predisposes to midgut volvulus due to a narrow mesenteric base and non-fixated intestines.

There is also a risk of duodenal obstruction due to Ladd’s bands causing more chronic

symptoms.

Figure 5. Intestinal malrotation (A) and Midgut volvulus (B)

In non-rotation, the risk of midgut volvulus might be smaller due to a wider mesenteric

base (Langer 2017) though there is still the element of non-fixated intestines. Other

rotational abnormalities, not necessarily included in the topic but worth mentioning, are

“reversed rotation”, “para-duodenal hernias” and atypical malrotation. Reversed rotation

occurs when there is a 90° clockwise rotation from the original sagittal plane, leading to

colon located behind the superior mesenteric vessels and the duodenum in front. Para-

A B

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duodenal hernias occur when the right or left mesocolon fails to fuse to the posterior body

wall. There will be an opening into which the small intestine can herniate, causing potential

obstruction. Atypical malrotation is defined as the duodenojejunal junction being positioned

to the left of the midline, but below the level of the pylorus (Graziano 2015). All grades

may exist, from atypical malrotation to nonrotation.

1.2.2 Secondary malrotation IM can also arise as an intrinsic component of congenital diaphragmatic hernia,

omphalocele or gastroschisis. This is also called secondary malrotation, since it is

secondary to another major abdominal malformation interrupting the normal developmental

process of the midgut. This is generally in the form of non-rotation.

1.3 CLINICAL PERSPECTIVES ON IM

The true incidence of intestinal malrotation is not known, from radiological studies, it is

reported to be up to 0.2% (Kantor 1934), while autopsy studies estimate an incidence of

some form of intestinal rotation abnormalities (IRA) up to 1% (Kapfer 2004). The

incidence of symptomatic IM (in neonates) is reported to be 1/2500-6000 infants (Adams

2014, Kapfer 2004) Traditionally it has been reported that up to 90% of patients present

within the first year of life, usually within the first month (Ford 1992, Stewart 1976).

However a recent study suggests that 60% present below one year of age (Aboagye 2014).

The risk for midgut volvulus in patients with intestinal malrotation is present regardless of

age. Midgut volvulus is a clockwise rotation of the base of the dorsal mesentery,

obstructing the blood- and lymphatic flow to and from the intestines through the superior

mesenteric vessels. If not treated, it will lead to intestinal gangrene and death. The

symptoms are usually acute bilious vomiting, abdominal discomfort and distension. The

abdominal symptoms, followed by metabolic acidosis and hypovolemic shock, will get

worse within hours if not treated. Since the blood supply to and from the damaged

intestines is strangulated, sometimes lab-tests, such as leukocytes, lactate and electrolytes

can be normal, even if the damage is severe.

Other less acute symptoms of intestinal malrotation can arise from intermittent duodenal

obstruction due to Ladd’s bands, intermittent volvulus or chronic volvulus. In addition,

there are dysmotility disorders that have been shown to sometimes coexist with intestinal

malrotation (Coombs 1991, Erez 2001, Devane 1992). Duodenal obstruction causes

13

symptoms like gastroesophageal reflux, failure to thrive, feeling of fullness after small

meals and mild abdominal discomfort. Chronic volvulus can show similar symptoms, due

to malfunctioning nutrient absorption resulting from insufficient blood supply and chronic

irritation.

1.3.1 Associated anomalies Associated anomalies are relatively common; intestinal atresia, 5-26%; imperforate anus, 0-

9%; cardiac anomalies, 7-13%; duodenal web, 1-2%; Meckel’s diverticulum, 1-4%;

inguinal hernia, 0-7%; trisomy 21, 3-10% (Little 2010). Hirschsprung’s disease and CNS-

anomalies are also associated with intestinal malrotation (Stratulat-Chiriac 2016). Intestinal

rotation abnormalities are known to coexist with heterotaxy syndrome. Volvulus in utero

may also cause multiple intestinal atresias (Budd 1988).

1.3.2 Radiological diagnostics The clinical symptoms of IM are very variable, and IM may present as an incidental finding

during a radiographic evaluation of another diagnosis or with peritonitis and shock from a

midgut volvulus.

A plain abdominal radiograph is usually the first step to investigate abdominal symptoms.

This can be inconclusive, but especially in new-borns there can be a “double bubble”,

which corresponds to a distended ventricle and a dilated proximal part of the duodenum.

This can appear in duodenal atresia, presence of Ladd’s bands or midgut volvulus. In

volvulus, there might be an edema and thickening of the bowel wall. Free gas in the

abdomen is a sign of intestinal perforation and requires emergency surgery.

The next step in children to establish the course of the duodenum is an upper

gastrointestinal contrast study, which was introduced in the 1960s and is the gold standard

to diagnose IM (Binu 2021). Barium enema or iodine-based water-soluble contrast is given

orally or through a nasogastric tube. Before that, when suspecting midgut volvulus, barium

enemas were used, looking for the position of the cecum (Lampl 2009). Normally, the

duodenojejunal junction is in the anatomical position of the ligament of Treitz to the left of

the spine at the level of the duodenal bulb (McVay 2007). In IM, the duodenojejunal

junction will be located on the right side of the spine (Figure 5A). In patients with duodenal

obstruction, the suspicion of midgut volvulus should be high. Other pathological findings

are “corkscrew” appearance of the duodenum (Figure 5B) and “bird’s beak” appearance of

the duodenum, both being signs of midgut volvulus. There can also be the “coil spring”

14

sign, which is a sign of intramural hematoma or edema and blood infiltration (Freeman

2008). In patients with signs of duodenal obstruction and deteriorating general condition,

midgut volvulus cannot be excluded which calls for an acute surgical intervention. Saline-

aided doppler ultrasound can also be used to show a dilated duodenum and an inversion of

the superior mesenteric artery and vein in midgut volvulus (Chen 2022).

Figure 5. UGI studies, demonstrating A) Intestinal malrotation B) Corkscrew sign

In older children and adults, a computed tomography (CT) with double contrast,

intravenous and oral, is recommended. The course of the duodenum can be followed, as

well as the colon if “triple” contrast investigation is done. In addition, the abnormal

positioning of the superior mesenteric artery and vein can be seen (Strouse 2004) (Figure

6). In midgut volvulus the “whirlpool sign”, a clockwise rotation of the superior mesenteric

vein, bowel, and the mesentery around the superior mesenteric artery, can be visualized

(Strouse 2004, Brinkley 2016) (Figure 7A). If strangulation of the SMA is complete, the

“whirlpool sign” might not be seen, so one also has to look beyond the signs. In children

the “whirlpool sign” can be demonstrated by Doppler ultrasound with a 97% positive

predictive value (Brinkley 2016, Orzech 2006), also prenatal diagnosis of IM with midgut

volvulus was performed from 25 weeks GA with Doppler ultrasonography by the

“whirlpool sign” (Yang 2022).

A B

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Figure 6. A) A CT-scan, coronal section, demonstrating the normal position of SMA (red)

to the left of SMV (blue). SMV and the splenic vein join to form the portal vein. B) IM

demonstrating the SMA on the right side of the SMV.

Figure 7. A) Whirlpool sign in an adult patient, coronal secion B) Normal finding of SMA

in the same section as aorta C) SMA drifting towards the right, not found in the same

section as the aorta. Sometimes seen in intestinal malrotation and more common in midgut

volvulus.

A B

A

B C

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1.3.3 Management and surgical treatment In the typical case with acute symptoms and radiological signs of intestinal malrotation an

operative treatment is indicated, often as an emergency procedure. The volvulus is reduced

by de-rotation and Ladd’s procedure prevents volvulus from recurring. The Ladd’s bands

are divided, the small intestine mesentery is widened to avoid relapse, the small intestines

are placed to the right and the large intestines to the left in the abdominal cavity (Swenson

1945). Usually an appendectomy is performed, since the atypical position may delay a later

diagnosis of appendicitis.

When the radiographic examination is inconclusive and the symptoms are mild, generally

follow-up or repeated contrast study is recommended. In asymptomatic patients with

incidental findings, there is a lack of evidence on whether to operate. Prophylactic surgery

on asymptomatic patients could be considered in younger patients with low risk of

postoperative complications, while older asymptomatic patients could benefit from follow-

up and information (Graziano 2015).

The surgical procedure can also be performed laparoscopically, which was first reported in

1995 in a 7-days old neonate with midgut volvulus (van der Zee 1995). Laparoscopic

approach may be preferred in elective cases when radiological examinations are

inconclusive and the diagnosis needs to be confirmed. A four-port technique is usually

applied. In acute volvulus, the accessibility can be limited due to bowel edema and chylous

ascites. Comparative retrospective studies have shown that the laparoscopic Ladd’s

procedure takes a longer time, but shortens the time to full enteral feeding and hospital stay.

Open surgery was related to more post-operative complications, however, midgut volvulus

after Ladd’s surgical procedure was encountered in 3.5% of patients in the laparoscopic

group and 1.4% in the open group (Catania 2016).

17

Figure 9. Ladd’s procedure with photographs taken during surgical procedures on adults (Brungardt 2021 with permission, photos by Britt Husberg and from study I)

Midgut volvulus

Ladd’s bands Widening of the mesentery and placement of the small intestines to the right, and the large intestines to the lef.

18

1.3.4 Outcomes After Ladd’s procedure, adhesive small bowel obstruction is the most common

complication, with a reported incidence of 5-10% (Fischer 2011). Recurrence of midgut

volvulus may occur and is encountered in 0.5-7% (Dilley 2000, El-Gohary 2010, Feitz

1997). The mortality of midgut volvulus depends on the damage on the small bowel and if

there is systemic shock. The overall mortality rate after surgical correction of intestinal

malrotation is 3-9%. The risk increases if there is intestinal necrosis, other abnormalities, or

prematurity (Fischer 2011). In one study, the mortality rate in patients undergoing surgery

for small bowel volvulus were 10-35%. In cases of gangrenous bowel, the mortality

increased to 20-60% (Iwuagwu 1999).

IM with volvulus is one of the most common etiologies of intestinal failure in children.

This condition is a malabsorptive state often following an extensive resection of the small

intestine (Vanderhoof 1997). The ability to absorb nutrients and balance fluid excretion is

disrupted and adaption and recovery depend on the amount and function of the remaining

small intestine. Generally, the ileum has a greater adaptive ability than the jejunum and the

importance of preserving the ileocecal valve has been put forward. Supportive therapy is a

combination of enteral feeding, parenteral nutrition, medical therapies and occasionally

intestinal transplantation.

In adults, the most common cause of intestinal failure is mesenteric ischemia due to

thromboembolic events whereas in children, it is necrotizing enterocolitis (Fischer 2011).

Intestinal failure among neonates is caused by volvulus in 10% (Wales 2010).

Approximately 2% of children and adults with intestinal volvulus develop intestinal failure,

in adults most commonly caused by intra-abdominal adhesions, and in children due to IM

(Koffeman 2003).

The overall incidence of intestinal failure is 24.5 per 100 000 live births (Wales 2004) and

the risk for intestinal failure is higher in low birth-weight infants, as well as in live births

before 37 weeks of gestation (Wales 2010). The mortality rate in pediatric intestinal failure

was previously reported to be between 20-40% (Wales 2010) but more recent advances

have increased the pediatric intestinal failure survival rate to >90% (Duggan 2017). The

mortality depends primarily on the need of parenteral nutrition, which can lead to central

venous catheter infections and intestinal failure-associated liver disease.

1.3.5 Adult patients and IM Intestinal malrotation has until recently been considered a strict pediatric diagnosis and was

rarely diagnosed in adult patients. Nowadays, it has become more accepted that it is an

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important diagnosis also in the adult population. Symptoms like abdominal pain, early

satiety and dyspepsia can be caused by malrotation. Surgery may also prevent development

of an acute volvulus, even though the risk generally seems to decrease with age (Nehra

2011). The incidence of midgut volvulus in adults is reported to be between 0.00001% to

0.19% (Leow 2016).

1.4 MOLECULAR PERSPECTIVES ON IM

1.4.1 Human genetics

Figure 10. DNA transcription

and translation into a protein (a

folded polypeptide consisting of

amino acids). Only about 1% of

the human genome consists of

coding genes and they are

approximately 20 000 in number.

The genome is composed of

deoxyribonucleic acid (DNA),

which consists of different

nucleotides, adenine (A), thymine

(T) cytosine (C) and guanine (G).

They are referred to as bases and

pair as A-T and C-G, called base pairs. (Picture used with license from shutterstock.com)

The DNA is to a very high degree identical between individuals especially concerning the

parts of the DNA that codes for proteins. Overall there are still non-disease-causing base

variants that can be used for “finger printing”. When you study a disorder and find a genetic

variant you need to evaluate how damaging the variant is, for example how deleterious

(harming to the protein product) it is, if it is conserved over different species and how

common it is in the general population as well as its original function compared to the

phenotype of the carrier of the gene. Some specific sequences are rather constant, and also

between species (conserved), and they are generally more important for normal

development and function and a sequence variant in these regions are generally more

damaging.

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We have two sets of each gene, one of each of the two autosomal chromosome pairs. If a

genetic disease is caused by a mutation in one of the genes, it is an autosomal dominant

disease that can be inherited in 50% of the offspring. If both genes need to be mutated for

the patient to be affected by a disease, it is an autosomal recessive inheritance, meaning that

each parent is a carrier of one mutation and then 25% of the offspring will get the disorder.

Additionally, 50% will be carriers.

Figure 11. Autosomal dominant, autosomal recessive and X-linked recessive inheritance

patterns. (Picture used with license from shutterstock.com)

1.4.1.1 Genetic variants

Genes that code for proteins consist of intervals of DNA called exons and introns, where

only the exons are translated to proteins. In this thesis we focused on the protein coding

genome. Smaller changes in the DNA can have different consequences on proteins. There

are synonymous variants that do not change an amino acid and the final protein will not be

altered. Missense variants will code for a different amino acid, possibly altering the

function of the protein and nonsense variants changes the code into a stop codon, resulting

in premature termination of translation. Frameshift variants are deletions, duplications,

insertions, or a combination, that alter the number of bases usually causing lots of changes

in the protein or more commonly a premature stop codon. All these variants will generally

be found with sequencing methods such as whole genome sequencing (WGS).

Larger deletions or duplications or insertions, referred to as copy number variants

(CNVs) are unbalanced structural variants (SVs), meaning the total gene dosage will

change. For example it may either increase (if a duplication) or decrease (if a deletion) the

expression of a gene which can affect regulatory mechanisms. If a SV is balanced it means

21

that there is an inversion or translocation, not changing the gene dosage. Only unbalanced

SVs can be detected when performing array CGH.

1.4.2 Experimental data A disturbance in left-right axis development in embryogenesis might be a possible cause of

intestinal malrotation, even though gut tube elongation is also considered to be involved. It

is known that Nodal is the start of laterality, probably through Pitx2 that activates Wnt5a.

Examples of genes that are important for left-right asymmetry in humans are for example

LEFTYA, CFC1, ZIC3, NKX2.5, AVCR2B (Maclean 2004). Sequence variants in those

genes have been found in patients with heterotaxy, sometimes in combination with IM

(Bamford 2000). Furthermore, the motile cilia are important in establishing normal LR

asymmetry of the body. Hundreds of genes are involved in cilia formation and function and

many of those have been linked to human disease, collectively called ciliopathies (Table 2).

The molecular landscape during embryologic development of the gut has been investigated

extensively in chicken embryos and in mice. In those model systems, rotation of the midgut

is initiated by differences in the cellular architecture of the right and left side in the dorsal

mesentery (Davis 2008). Different cascades of transcription factors cause the epithelial

(columnar shape) and mesenchymal cells on the left side of the dorsal mesentery to become

more condensed whilst the right-side epithelium is cuboidal and the mesenchyme remains

in an uncondensed state. A few key components are Nodal, regulating factors downstream

and is expressed only on the left side, also Pitx2, Fzd4, Fzt8, Gpc3, Daam2 and Isl1, are

only expressed on the left side and Tbx18 is expressed on the right side. It is suggested that

Wnt5 activates expression of the left-sided genes including Daam2, that regulates cell-cell

contacts to create a condensed state of the mesenchyme. Right-sided expression inhibits the

Wnt5a-pathway by for instance Sfrp1 and Sfrp2. Therefore, Daam2 is not activated and the

cells on the right side are not condensed. The result will be a leftward tilt of the dorsal

mesentery, suggesting the beginning of rotation of the midgut. (Davis 2008) (Figure 12).

Wnt5a is not expressed in the dorsal mesentery, but in the gut tube, and is important for

elongation of the tube (Sadler 2004, Welsh 2013). Also, Foxf1 is important in the formation

of the dorsal mesentery and is also a target for sonic hedgehog signaling, that is essential in

embryonic development (Mahlapuu & Enerbäck 2001, Mahlapuu & Ormestad 2001).

22

Figure 12. Transverse section of an embryo, left from 30 days of gestation and in the right

picture the dorsal mesentery shows a leftward tilt. The dorsal mesentery in red and the gut-

tube in yellow. (Martin 2010)

1.4.3 Familial cases Even though most IM cases are isolated and sporadic there are several reports on familial

cases (Erez 2001, Smith 1972, Beaudoin 2005, Budd 1988), mainly siblings, and thus

malrotation is regarded as a complex disorder depending on genetic as well as

environmental factors. Familial cases with IM together with other malformations have been

reported. One known familial disorder is congenital short bowel associated with

malrotation, intestinal dysmotility and short bowel (Erez 2001) and hereditary MIA and

malrotation is also previously reported (Gibson 1987).

1.4.4 Chromosomal aberrations associated with IM In the literature autosomal dominant, autosomal recessive, X-linked and chromosomal

forms of intestinal malrotation have been reported, mostly as syndromes with unknown

genetic etiology (Martin 2010) (Table 1).

23

Involved chromosome region

Associated malformations apart from IM Reference

Del 13q32 and dup 4q25

IUGR, cleft lip-palate, absent thumbs, bicuspid pulmonary valve, pulmonary hypoplasia. Father and paternal grandmother and great-grandmother with balanced translocations

Najafzadeh 1983

Ring chromosome 15 IUGR, microcephaly, a large VSD, dysmorphic ears, fingers and feet

Otto 1984

Partial distal 6p trisomy Abnormal lung lobulation, renal hypoplasia, crossed renal ectopia. Fetus 19 week GA, Maternal balanced translocation.

Fryns 1986

Del 13q21.2q32.3 Hirschsprung disease, developmental delay Lamont 1989

Mosaic tetrasomy in 8p Developmental disabilities, skeletal anomalies, rectal atresia, horse-shoe kidney, VUR. I(8p) in 68% of lymphocytes and 44% in skin fibroblasts.

Fisher 1993

Del 13q14.3q22.1 Hirschsprung disease, developmental delay Shanske 2001

1p36 del syndrome Growth delay, seizures, craniofacial dysmorphism and annular pancreas

Minama 2005

Mosaic trisomy 22, de novo

Total colon aganglionosis, congenital hearing loss, IUGR

Hall 2009

Mosaic trisomy 18 VACTERL including renal agenesis, cardiac laterality defect

Harewood 2010

t(2:6)(p23:q14), de novo Renal agenesis, cardiac and respiratory laterality defect

Harewood 2010

Partial trisomy 2p and monosomy 16p.

Dysmorphic features and developmental delay. Developed a neuroblastoma

Morgenstern 2013

3p14 interstitial del, 6.88 Mb, de novo

MZ twins, intellectual disability, autism, facial dysmorphism

Okumura 2014

Distal 15q25 dup and 12p13 del

Dysmorphic, craniosynostosis, horse-shoe kidney, pyloric stenosis

McLaughlin 2015

22q11.1q11.22 dup 6.6 Mb de novo

Dysmorphic face, CHD and later developed AML

Vaisvilas 2018

13q21.31q33.1 interstitial del (38.8 Mb)

Craniofacial dysmorphism, Mb Hirschsprung and hearing loss

Chatmetakul 2019

12p del syndrome Not specified Oliveira 2020

Mosaic trisomy 12 Dysmorphic face, bicornuate uterus, broad thumbs, partial anomalous pulmonary venous return. TOP week 22.

Bonasoni 2020

Table 1. Examples of chromosomal aberrations reported in the literature. Del deletion, dup

duplication, GA gestational age, IUGR intrauterine growth retardation, VSD ventricular

septal defect, VUR vesicoureteral reflux, MZ monozygotic twins, CHD congenital heart

defects, ALL acute lymphoblastic leukemia, TOP termination of pregnancy.

24

1.4.5 Monogenic IM One syndrome associated with IM and with known genetic etiology is congenital alveolar

dysplasia (CAD) due to mutations in the FOXF1 gene located in chromosome 16q24 that

usually leads to death within the first months after birth. This syndrome also presents with

intestinal malrotation, congenital short bowel, duodenal stenosis, annular pancreas,

pulmonary isomerism, cranio-facial features, cardiovascular and genitourinary

malformations (Stankiewicz 2009). Another syndrome is pediatric idiopathic intestinal

pseudo-obstruction, associated with intestinal malrotation, pyloric stenosis, hydronephrosis,

undescended testis, patent ductus arteriosus and thrombocytopenia. It is caused by an X-

linked recessive inheritance pattern due to mutations in the FLNA gene at Xq28 (Martin

2010, Gargiulo 2007). So far there are, to our knowledge, no such reports on patients with

isolated IM.

1.4.6 Ciliopathies Cilia are found on almost every human cell. There are motile and primary (non-motile)

sensory cilia (Wheway 2018, Fliegauf 2007). Examples of motile cilia are those found in

the airways to mobilize mucous and particles that need to be excreted. Also, the Node is an

important transient embryological structure that initiates the process of laterality through

motile cilia (Sharma 2008, Dasgupta 2016). Primary cilia (non-motile, sensory cilia) are

found on almost every other human cell that does not carry motile cilia and mediates

signals through sensation or signaling pathways (Reiter 2017). Primary cilia are important

during embryonic development and are involved in the Wnt-signaling pathway and in

Hedgehog signaling, both being associated with laterality defects in animal models

(Hildebrandt 2011).

A defect in the cilia, or in proteins important for the function of the cilia, can result in a

ciliopathy (Badano 2006, Fliegauf 2007, Reiter 2017), many of which include situs defects

(Table 2). Intestinal rotational abnormalities and heterotaxy are known to coexist, the latter

being associated to ciliopathies. Interestingly, there seem to be a male predominance in IM,

and the male to female ratio of heterotaxy is 2:1 (Ware 2011). There are cases with

ciliopathies presenting with IM, for example in a case with Jeune asphyxiating thoracic

dystrophy, hypothesizing that intestinal malrotation may be a ciliopathy (Hall 2009).

25

Table 2. Primary ciliopathies where LR axis defects have been described (Pollara 2022).

Disease Main clinical phenotypes Main gene(s) Inheritance

Nephronophthisis tubulointerstitial fibrosis, liver fibrosis, situs inversus, cardiac defects

NPHP1-4, IQCB1, CEP290, GLIS2, RPGRIP1L, NEK8,

SDCCAG8, TMEM67, TTC21B, WDR19, ZNF423, CEP164,

ANKS6, CEP83, INVS, DCDC2, MAPKBP1

Autosomal recessive

Bardet-Biedl syndrome

Obesity, polydactyly, retinitis pigmentosa, anosmia, congenital heart defects, situs inversus

BBS1, BBS2, ARL6, BBS4, BBS5, MKKS, BBS7, TTC8, BBS9, BBS10, TRIM32, BBS12, MKS1, CEP290, C2ORF86, IFT74, CFAP418, LZTFL1, IFT172, IFT27, SDCCAG8, PTHB1, BBIP1, TRIM32, WDPCP

Autosomal recessive

Meckel syndrome Cystic kidneys, encephalocele, polydactyly, liver fibrosis, congenital heart defects, situs defects

MKS1, TMEM216, TMEM67, CEP290, RPGRIP1L, CC2D2A,

NPHP3, B9D2, TMEM231, TCTN2, TMEM107, KIF14

Autosomal recessive

Joubert syndrome Cerebellar-brainstem malformation (molar tooth sign), developmental delay, ataxia, ocular-motor apraxia, retinal dystrophy, polydactyly, nephronophthisis

INPP5E, TMEM216, AHI1, NPHP1, CEP290, TMEM67, RPGRIP1L, ARL13B, CC2D2A, CPLANE1, OFD1, ZNF423, B9D2, TMEM107, CEP41, TMEM237, CSPP1, PDE6D, PIBF1, TCTN2, KIAA0586, KIAA0556, KIAA0753, CEP104, CEP120, TMEM231, TCTN1, ARMC9, ARL3, B9D1, MKS1, FAM149B1, SUFU

Autosomal recessive, X linked recessive (OFD1)

Alström syndrome Dilated cardiomyopathy, obesity, sensorineural hearing loss, retinitis pigmentosa, endocrine, renal and hepatic defects

ALMS1 Autosomal recessive

Jeune asphyxiating thoracic dystrophy/Short rib polydactylies

Skeletal dysplasia, polydactyly, cystic kidneys, retinal degeneration, cardiac, liver and brain anomalies

NEK1, DYNC2H1, DYNC2LI1, KIAA0586, WDR35, WDR60, IFT52, IFT43, IFT81, IFT80, WDR34, IFT140, TCTEX1D2, TTC21B, IFT172, WDR19, CEP120, CSPP1, DYNC2I1-2

Autosomal recessive

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2 RESEARCH AIMS

The overall aim of this thesis was to better understand the molecular background and the

clinical spectrum of IM especially according to age and to increase the knowledge about the

outcome after treatment.

Specific aims:

- To understand the symptoms, diagnostics, treatments and outcomes of IM

throughout life. (Study I and Study II)

- To examine the prevalence of structural genomic variants in IM. (Study III)

- To examine underlying genetic causes of isolated IM. (Study IV)

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3 CLINICAL STUDIES I & II

3.1 METHODS

3.1.1 Study design, Setting and Participants

To understand and describe the symptoms of IM throughout life we have described IM in

both adult and pediatric patients.

3.1.1.1 Study I – Adult patients

This was a retrospective case series at the Department of Surgery at Karolinska University

Hospital, where patients diagnosed and treated for IM between 2002 and 2013 from 15

years of age were included. Patients were continuously (prospectively) recruited into the

study during the time-period when diagnosed with IM. Inclusion criteria was IM, including

secondary IM.

3.1.1.2 Study II – Pediatric patients

This was a retrospective case series at the Department of Pediatric Surgery at Karolinska

University Hospital with children 0-15 years old who were diagnosed and treated for IM

from January 1983 to December 2016 were included. The hospital’s administrative

database was used, applying the ICD-codes for IM (ICD-10 Q433, ICD-9 751.4/751E) and

volvulus (ICD-10 K562, ICD-9 560.2/560C) and additional searches in the operation

planning system were made. We paired the temporary social security numbers and removed

double registrations. The medical records were reviewed and unclear cases were discussed

further with an experienced pediatric radiologist and two experienced pediatric surgeons.

When going through this large material we realized that many different phenotypes were

included in this group of patients. Every medical record needed extended scrutiny together

with evaluation of the radiologic findings to understand the specific malformation of each

patient for a valid inclusion.

Inclusion criteria was IM. Exclusion criteria were incorrect diagnose code, insufficient

information, incomplete variants of IM and cases with volvulus and no IM. Also patients

older than 15 years at onset of symptoms and patients with secondary IM were excluded.

30

3.1.2 Variables Data were retrieved from the medical records concerning age, sex, associated

malformations, symptoms, diagnostic and surgical procedures, previous disorders and

outcomes.

The patients were further divided into age groups.

Study II: <1 month, 1 month-1 year, 1-5 years, 6-15 years

Study I: 15-20 years, 21-50 years, 51-67 years

3.1.2.1 Study I, outcomes and follow-up

The patients were assessed 6 weeks, 6 months and 12 months postoperatively with a

possibility to resume contact if they had further complaints. Semi-structured telephone

interviews were performed 2012-2013 by an experienced research nurse regarding the

patients’ current symptoms as well as onset of symptoms, such as pain, nausea, vomiting,

constipation, and if they regarded their general physical condition as improved to a high

degree, improved with some reservation, or without any notable improvement.

3.1.2.2 Study II, outcomes and follow-up

Clavien-Dindo classification (Clavien 2009) was used to evaluate short-term complications

(<30 days) where grade IIIb or higher were taken into consideration (Table 3).

The patients were further divided into two groups based on severity in order to assess if

there was a greater risk for complications. The severely affected group still had

compromised bowel after de-rotation of midgut volvulus, defined by needing intestinal

resection or rescue strategies such as “second look” or “open abdomen” procedures.

Long term outcomes were collected from medical records: surgical procedures, intestinal

failure and mortality. In addition, a postoperative follow-up telephone interview was

conducted by an experienced research nurse with patients or parents according to a semi-

structured questionnaire. The questions were focused on the onset of symptoms, as well as

later gastrointestinal symptoms such as abdominal pain, nausea, vomiting, bloating,

constipation or diarrhea. If no symptoms were reported, the patients were considered “free

from symptoms”. Questions were also asked about perceived health after surgery and

current disorders.

31

Grade Definition

Grade I Any deviation from the normal postoperative course without the need for pharmacological treatment or surgical, endoscopic and radiological interventions.

Grade II Requiring pharmacological treatment with drugs other than such allowed for grade I complication.

Grade IIIa Requiring surgical, endoscopic or radiological intervention, not under general anesthesia.

Grade IIIb Requiring surgical, endoscopic or radiological intervention, under general anesthesia.

Grade Iva Life-threatening complication (including CNS complications) requiring IC/ICU management with single organ dysfunction.

Grade Ivb Life-threatening complication (including CNS complications) requiring IC/ICU management with multiorgan dysfunction.

Grade V Death of a patient

Table 3. Clavien-Dindo classification of surgical complications. (Clavien 2009)

3.1.2.3 Radiological diagnostics

Two experienced radiologists analyzed the radiological examinations, one in all adult

patients (Study I) and one in the difficult pediatric cases (Study II) respectively. In the adult

cases most patients had a CT scan with triple-contrast; per oral, intravenous, and intrarectal

contrast. This allowed us to visualize and evaluate the position of the duodenum, but also

the orientation of the superior mesenteric artery and vein as well as the position of the

cecum.

3.1.3 Statistical analysis Categorical variables are presented as frequencies and percentages in all studies. In Study

II, Chi-square test or Fisher’s exact test was used to analyze the association between age-

groups and symptoms, diagnosis and treatments. Univariable, as well as stepwise logistic

regression was used to analyze whether Clavien-Dindo complications could be caused by

variables that we assessed as risk factors. Results are presented as odds ratios (OR) and

95% confidence intervals (CI). P-values <0.05 were considered statistically significant.

32

3.2 MAIN RESULTS AND DISCUSSION

3.2.1 Study I inclusion

Between 2002 to 2013, 39 patients aged between 15 and 67 years were diagnosed with IM

and were all included in the study.

3.2.2 Study II inclusion and variants of IM

There were 319 patients taken into consideration for the study through ICD-codes after

doubles were removed and temporary social security numbers were matched, 138 patients

were included in the study. One hundred eighty-one patients were excluded.

Cause of exclusion N =

Secondary IM 18

Incomplete variants of IM (IRA) 47

Volvulus without IM 49

Insufficient information 25

No IM, IRA or volvulus 34

Age >15 years at onset 8

Total: 181

Table 4. Patients excluded in Study II.

Many excluded patients had volvulus without IM because the ICD-code for volvulus was

used in the search and the most common type was partial small intestinal volvulus (n=37).

A large group of the patients with the ICD-code Q43.3, that were excluded had intestinal

rotation abnormalities (IRA), but not IM. Thirty-nine had mobile cecum, six had an

abnormal variant of duodenums position, but did not fulfill the criteria for IM and two had

both. An interesting aspect, especially from a molecular perspective, is that patients with

IRA harbored almost identical rates of associated malformations, except for the CNS-

malformations (p=0.018) (Table 5). Within the IRA group one patient had a midgut

volvulus, another patient had a partial small intestinal volvulus and a third patient had a

cecal volvulus. All three had a normally positioned duodenum. Nine patients had

33

intussusception, indicating that this group of patients should not be completely dismissed

after IM by definition is excluded. Among the IM-patients, six had Waugh syndrome

(intussusception and IM).

3.2.3 Associated malformations

Pediatric patients IM

(N=138)

IRA

(N=47)

Gastrointestinal malformations 28% (38) 28% (13)

Cardiovascular malformations 16% (22) 15% (7)

Urogenital malformations 11% (15) 13% (6)

Malformation in the central nervous

system or developmental delay

14% (malformations

n=11, developmental

delay n=9)

2% (1 had

developmental delay)

Genetics syndromes/chromosomal

aberrations

12% (17) 11% (5)

Table 5. Malformations Study II. IRA: Intestinal rotation abnormality

Altogether 50% (69 patients) of the pediatric cases had an associated malformation, and

just over one third (n=25) of them had more than one additional malformation. In the adult

group, patients with secondary IM (n=3) were included and there were 15 patients (38%)

who had other malformations.

In the pediatric group, the most common additional malformation was duodenal stenosis or

atresia and it is interesting whether these cases would be undetected IM if there was no

duodenal atresia causing intestinal obstruction. Twenty percent of the patients with

duodenal atresia have been reported to have IM (Mirza 2012). This shows the importance to

look for intraluminal intestinal obstruction when operating on neonates with IM. Our

findings correlate well with previous studies, even if associated malformations were more

common in this study than in some of the previous ones (30%) (Stewart 1976, Murphy

2006).

34

Among the adult patients, six had gastrointestinal motility disturbances and among the

pediatric patients, two cases of gastrointestinal dysmotility were reported. Both groups

included one case of Hirschsprung.

Many patients in the pediatric group had a known syndrome or chromosomal aberration,

which also was present in the adult group. IM with multiple malformations was also noted

in previously reported cohorts, indicating that syndromes among these patients is probably

not that uncommon (Ford 1992, Martin 2010).

In the pediatric group hepatobiliary malformations were present, like biliary tract

malformations in five and pancreas annulare in three children. Maybe variants in the

hepatobiliary system could partly explain why eight of the adult patients had a history of

disease in the hepatobiliary and pancreatic system, including four cases of pancreatitis.

3.2.4 Gender and age at diagnosis

In the pediatric group (up to 15 years) the male:female ratio was 2.1:1, with no significant

difference between age groups. However, in the adult group the sex ratio was 0.8:1. At the

same time previous reports have shown a male to female ratio 1.29:1 – 2:1 (Spigland 1990,

Feitz 1997, El-Gohary 2010, Nagdeve 2012, Kulayalat 2015, Butterworth 2018, Black

2020) indicating a male predominance.

In the pediatric group 91/138 (66%) presented before one year of age, which is in

accordance with another recent study suggesting that 60% present under one year of age

(Aboagye 2014) and differs from previous reports on 90%. Interestingly, recently acquired

figures from the National Board of Health and Welfare show that 645 patients received the

diagnosis Q4.33 in Sweden between 2008 and 2020 and of them, only 37% were diagnosed

under one year of age and 31% being over 15 years when diagnosed (data received through

personal communication). In patients up to 15 years, 53% presented under one year of age,

which does not deviate as much compared to our findings in the pediatric study. The sex

ratio was equal, as well as in the IRA-group. It must be taken into consideration that the

diagnose code Q4.33 includes IM, and milder variants. The numbers may therefore differ

due to other diagnosis than strict IM.

Among the adult patients, presenting symptoms did not correlate with age. Even though

some patients had experienced symptoms for a long period of time, the symptoms seemed

to be aggravated at some point and needed assessment in all age groups.

35

3.2.4.1 Preterm birth and IM

In the pediatric group 33/138 (24%) were born preterm, the median gestational age among

preterm infants was 32 weeks (range 23-36 weeks, one missing data). One third of the

infants diagnosed with IM within the first month, as well as in the first year of life, were

born preterm. The reason for the high number of preterm patients could be the general

increased incidence of malformations in preterm infants (Rasmussen 2001).

Eight were born extremely preterm with a median gestational age 24.5 weeks; range 23-27

weeks and five of them presented within the first year of life. Half of the extremely preterm

infants died related to surgery, and two of them had necrotic bowel. There are many

comorbidities in extremely preterm patients and there is a higher risk to attain infections. It

can be a challenge to differentiate midgut volvulus from other conditions such as

necrotizing enterocolitis and sepsis. At the same time, in a recent study symptomatic IM

was diagnosed in 1.4% of 514 extremely premature infants, while only in 0.2% born after

28 weeks of gestation. They were all admitted to a neonatal intensive care unit, thus not

representing all newborns, but indicating an increased representation among the extremely

preterm patients (Mishra 2021).

3.2.5 Clinical presentation

A previous study divided 170 IM-patients into three groups: under one year of age, one to

18 years of age, and adults (Nehra 2011). We further divided the pediatric and adult groups

in order to more specifically describe the symptoms at presentation according to age. The

presented data shows that symptoms of IM will vary with age (Figure 13). The most

significant finding was that vomiting is the predominant symptom until five years of age,

whereas abdominal pain is the most common presenting symptom from 6 years of age and

through adulthood.

Bile-stained vomiting was seen up to five years of age and was the cardinal sign in the

neonatal group, but also common in the age group from one month to one year together

with abdominal pain and distended abdomen. This confirmed Nehra’s findings showing

that 93% of patients presented with vomiting if they were less than 1 year of age. He also

described that abdominal pain was the most common symptom in the age group one to18

years. In the present study, it was abdominal pain and vomiting that predominated from one

to five years of age, but also faltering growth was reported among one third. From 6 years

and through adulthood abdominal pain was the predominant symptom, even if vomiting, or

in the adult group as signs of intestinal obstruction, was common until 20 years of age.

36

Another recent publication on children with IM over one year of age (Dekonenko 2019)

reported abdominal pain, bilious vomiting and feeding intolerance as the most common

symptoms which confirmed our findings even though they reported bilious vomiting as

common up to 18 years of age, which differed from the present study. Blood-stained stool

was present in about 10% until 5 years of age and was then an alarming sign, all having

strangulating midgut volvulus except for one patient with chronic constipation.

Figure 13. Symptoms in children and adults related to age (%). *Referred to as obstruction

in adults.

Figure 14. Duration of symptoms. Pediatric patients up to 15 years diagnosed 1983-2016,

and adult patients from 15 years diagnosed 2002-2013.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

<1 month 1 month-1year

1-5 years 6-15 years 15-20 years 21-50 years >50 years

Hours/days Weeks Months Years

0

10

20

30

40

50

60

70

80

90

100

<1 month 1 month-1 year 1-5 years 6-15 years 15-20 years 21-50 years >50 years

Vomiting* Bilious vomiting Abdominal pain Faltering growth

37

Infants had an early diagnosis, older children and adults, however, often had long-lasting

symptoms, in many cases for several years, which correlates with previous reports (Nehra

2011) (Figure 14). Almost half of the adult patients had symptoms during childhood, many

of them ongoing since. Other adult patients experienced a relatively sudden onset of

abdominal pain, interestingly sometimes associated with stressful events or life changes,

developing into chronic symptoms and intermittent abdominal pain.

The reason why the diagnosis is more rapidly established among neonates could be that IM

is a more recognized diagnosis in in that age-group who generally present emergency

symptoms. In the older age-groups, often with less alarming and more chronic symptoms,

probably other diagnoses were primarily taken into consideration. In addition,

gastroenterologists and surgeons treating adult patients with abdominal symptoms,

probably do not have a congenital malformation as a primary differential diagnosis.

In both studies, incidental findings were rare, but they were more common in the pediatric

group with 16%, mostly because of prenatal diagnosis of intestinal obstruction by duodenal

atresia in the neonatal period, as compared to other studies with incidental findings up to

36% (Dekonenko 2019, Nehra 2011).

3.2.6 Diagnostic procedures

The gold standard UGI-study were most often used in the pediatric cases, and often led to

diagnosis, given that the examination was done properly. In adults a multi-detector

computed tomography with triple contrast was used and was preferred as other diseases

generally are suspected as a cause for intestinal obstruction.

3.2.6.1 Upper gastrointestinal series

In the pediatric study, UGI-series were done in 83 patients and in 56 of them IM was

confirmed by the exam, nine were inconclusive, two were not available and one was

assessed as having normal anatomy. This demonstrates the importance of not excluding IM

based on a normal UGI-study. Fourteen patients had a non-acute duodenal obstruction, 15

patients had signs of midgut volvulus, such as a bird’s beak (n=5) or corkscrew appearance

(n=5) of the intestines, and in all but one of them malrotation was also described. One

patient had a follow-up CT-scan with oral and IV contrast after an inconclusive UGI study.

Another option to obtain a diagnose after an inconclusive UGI study, was a diagnostic

laparoscopic procedure.

38

3.2.6.2 Computed Tomography

The pediatric patients who had a CT-scan were given intravenous contrast, sometimes oral

contrast, and revealed cases of midgut volvulus but not always IM. Among the 19 patients

who had a CT-scan, 14 were 6 years or older. According to the medical records, the

abnormal position of the duodenum was confirmed only in three, inversion of SMA and

SMV in five, abnormal positioning of colon in eight, and signs of midgut volvulus in five

(with whirlpool sign in three of them and strangulation of the SMA in the other two).

In the 33 adult patients who had a CT-scan (one had an MRI) the duodenum was located on

the right side of the abdomen, with the duodenojejunal junction not traversing the vertical

spine, except for in one case, where the duodenal course initially crossed the midline to the

left but as a loop turned back again to the right side. The cecum had an abnormal position in

22 cases and SMA and SMV were mispositioned in 26 cases, whereas all three signs were

confirmed in 19 cases (Figure 15). This confirms that the positioning of the duodenum is

the most reliable diagnostic feature to look for in a CT-scan as in coherence with previous

studies (Zissin 1999).

Figure 15. Radiological findings of the 33 adult cases who had a CT-scan with triple-

contrast (one had MRI). AC- Ascending colon. SMA- Superior mesenteric artery. SMV-

Superior mesenteric vein.

Interestingly, “whirlpool sign” was noted in seven of the adult patients, not just in

emergency cases, but also in chronic ones, and none of them needed intestinal resection.

The three children with “whirlpool sign” did not have an affected circulation, two were

noted as incidental findings in a trauma and in a case of appendicitis and one had chronic

symptoms from midgut volvulus. The two children who showed midgut volvulus by

strangulation of the SMA, had severely affected circulation at exploration as expected.

SMA and SMV n= 26

Duodenum n= 33

Cecum n= 22

n= 19

100%

AC mobile at surgery

39

3.2.7 Management

The treatment of IM was either surgery, using Ladd’s procedure or clinical follow-up

depending on comorbidities and symptoms. Diagnostic laparoscopy was sometimes

performed in the pediatric group (n=10), and when diagnose was established, converted to

open surgery in six cases. Laparoscopic Ladd’s procedure was performed in the remaining

four cases. The same open surgical procedure was performed in pediatric and adult cases.

Ninety-one % (125/138) of the pediatric group underwent Ladd’s procedure and 79%

(31/39) in the adult group. In the pediatric group 75 patients only had Ladd’s procedure,

additional procedures were done in the remaining 50 because of duodenal atresia or

stenosis, small bowel atresia, resection of Meckel’s diverticulum, other malformations,

planned gastrostomy and reduction of intussusception. In the adult group Ladd’s surgery

was the only procedure.

One third of the adult cases underwent emergency surgery, compared to half of the

pediatric cases. Of the pediatric cases there were 24 who had a midgut volvulus with

impaired bowel circulation. Midgut volvulus with severely compromised bowel was found

in eight patients, they needed intestinal resection, second look or open abdomen rescue to

reevaluate bowel vitality and to prevent compartment syndrome. Among the adult patients

there were eight who had midgut volvulus with compromised circulation, with one needing

resection of the entire necrotic small bowel and three who needed a minor resection. Two

of these patients were over 50 years old.

3.2.7.1 Appendectomy

An incidental appendectomy was performed in all the adult patients and in 58% of the

pediatric patients. One patient had acute appendicitis eight years after Ladd’s procedure. It

has been tradition to perform an appendectomy as part of Ladd’s procedure because it

decreases the risk of misdiagnosing appendicitis. The different recent approach to save the

appendix is based on reasonings such as diagnosing appendicitis radiologically has become

more accessible and an appendectomy is not free from complications. Furthermore, some

children with pseudo-obstruction (that is related to IM) may need a future appendicostomy

for antegrade enemas (Healy 2016).

40

3.2.8 Outcome

3.2.8.1 Recurrent midgut volvulus

Recurrence was more common in the adult group with six patients, compared to one patient

in the pediatric group. There are studies supporting that it is more common among adult

patients (Ferreira 2020) and one could speculate that a cause could be that the intestines

have grown and settled into a certain way, and sometimes into a chronic rotated state that it

returns to. If that is the case, this is also a reason to diagnose and treat IM early.

3.2.8.2 Intestinal failure

In the pediatric group four patients developed chronic intestinal failure, defined as having

the need for parenteral nutrition for at least 90 days (Duggan 2017). Two because of short

bowel syndrome from a midgut volvulus, one because of necrotizing enterocolitis and one

because of intestinal dysmotility. Two patients were weaned off parenteral nutrition after

nine and 24 months, one patient needed intestinal transplantation and the one patient with

necrotizing enterocolitis died at 11 months of age. Among the adult patients, one patient

died due to short bowel after midgut volvulus. Another 17 of 124 patients (one missing

data) needed parenteral nutrition for more than two weeks postoperatively in the pediatric

group and two of 31 in the adult group.

3.2.8.3 Short-term complications (Study I)

The Clavien-Dindo classification scale was used to look for short-term complications

within 30 days. Postoperative data on one of the 125 patients who had surgery were missing

and only 124 cases could be assessed. Complications from type IIIb-V were considered

because of the difficulty to ensure that the correct data would be collected on grade I-IIIa

from the retrospective cohort.

The risk to develop short term complications grade IIIb-V were 20% (25/124), which was

similar compared to a previous study where 16% developed complications graded III-V

(Salö 2019). Predictive factors to develop complications were midgut volvulus with

severely affected circulation, OR 8 (95% CI 1.77-36.22). Of the eight patients who had a

severely affected circulation, defined by intestinal resection of necrotic intestines, “second

look” and “open abdomen” procedure, 63% (n=5) had a complication with 25% (n=2)

mortality, while in the other group with Ladd’s procedure with or without midgut volvulus,

17% (n=20) had a complication, with 2% (n=2) mortality.

Another predictive factor was extreme prematurity (gestational age < week 28), OR 30.95

(95% CI 3.52-271.93), which is in accordance with a recent large study showing that short

41

term postoperative complications and failure to rescue (FTR) increases with decreasing

gestational age (GA) (Mehl 2021), even though the OR to develop complications among

patients born week 28 – 37 was not significant in the present study.

3.2.8.4 Long-term complications

In the pediatric study 11% developed small bowel obstruction which correlates with

previous studies (Fredriksson 2016, Salö 2019). In the adult group it was only 8%, maybe

partly because no other surgical procedures were done simultaneously.

The pediatric and adult patients who had Ladd’s surgical procedure were followed-up by a

telephone interview. In the adult group 26 patients were available for an interview 1-12

years after surgery, and in the pediatric group 61 patients 1-36 years after surgery (median

8 years).

Adult group

The symptom of acute intermittent pain that was present before surgery in almost all of the

interviewed patients was mainly gone postoperatively, leaving only one patient who did not

experience a relieve. However, abdominal pain of a more chronic character remained in 12

patients (46%). Among 13 patients with constipation, it remained in eight postoperatively.

When asked about their general life quality, it was improved in all but one patient

postoperatively.

Pediatric group

Nineteen patients reported constipation prior to surgery and 12 patients postoperatively

where 10 claimed need of pharmacological treatment. Constipation in one fifth of the

patients who had surgery for IM, is in coherence with previous reports on constipation

associated with IM (Salö 2019) and may be expected as motility disturbances are associated

with IM. Seventeen patients (28%) still had abdominal pain after surgery and of them it was

four who experienced the same pain as before the surgery.

The telephone interview created an opportunity for the parents to express their experiences

and in 45 of the cases they described it as a traumatic experience with words as “horrific”,

“a hopeless situation” and “shocking”.

3.2.8.5 Mortality

In the adult group one patient died because of intestinal failure after midgut volvulus with

total bowel necrosis, four other patients died due to other comorbidities. In the pediatric

group, four died within five days postoperatively, they were all born extremely preterm,

were aged between two to six months and had a short history of abdominal symptoms. Two

of them had midgut volvulus with necrotic intestines at exploration. Five patients,

42

previously operated for IM died due to other comorbidities. The mortality rate was

relatively high, but within previous reported data, 2-9%, and the cause of death as in our

case, usually were not related to IM (El-Gohary 2010, Murphy 2006). Also, there was an

increased mortality <30 days postoperatively among the patients with severely affected

bowel (P=0.021).

3.2.9 Adult patients and IM

With increased knowledge about IM, the diagnosis needs to be considered as a possible

cause of acute and chronic symptoms also in adults, which has also been pointed out in

previous studies (Coe 2015, Nehra 2011). In this group, concurrent developmental

disorders and lower age, were red flags and should raise suspicion. Due to age-related

comorbidities and a seemingly decreased risk for midgut volvulus in the older age group, a

more conservative approach regarding surgery could be considered, even though other

studies recommend surgery on incidental adult cases (Moldrem 2008). It is impossible to

predict who will develop a strangulating midgut volvulus in the future, especially

considering the oldest patient being 62 years old in this material.

43

4 MOLECULAR STUDIES III & IV

4.1 METHODS

4.1.1 Study design, Setting and Participants

To study the molecular background of IM we both screened for submicroscopic structural

chromosome variants and performed whole genome sequencing (WGS). In the clinical

studies, patients were asked to donate blood for molecular studies on DNA and were in the

semi-structured interviews asked about if there was any knowledge about affected relatives.

Among the children, also the parents were asked to donate blood.

4.1.1.1 Study III

A custom designed array CGH, targeting 1989 genes involved in ciliopathies and other

genes important for early development was used, to screen for small intragenic and large

deletions and duplications in a single experiment. The study included data from two

cohorts.

Cohort 1: All adult and pediatric patients with IM, who had donated blood for molecular

studies n=42.

Cohort 2: Five patients with an IM diagnosis that had undergone clinical genetic

investigation at the Department of Clinical Genetics (Karolinska University Hospital).

Using the terms “intestinal malrotation” or “midgut volvulus” they were identified in the

internal database of >5000 clinical array CGH analyses.

4.1.1.2 Molecular Study IV

We also wanted to investigate if there was a genetic predisposition in patients with isolated

IM without any other comorbidities. We used WGS to look for sequence variants in

candidate genes, but also used the data to look for larger structural variants as in Study III.

This study included pediatric patients with IM, who had donated blood for further

molecular studies. Inclusion criteria was isolated IM with early onset midgut volvulus

where surgery had also verified the diagnosis and whole blood for DNA extraction was

collected from both parents.

44

4.1.2 Molecular analyses

4.1.2.1 Array comparative genomic hybridization (array CGH)

array CGH is a cytogenetic technique to detect submicroscopic chromosomal copy number

variants (CNVs) with a high resolution and covering the whole genome. In clinical

diagnostics, the resolution is usually about 25-50 kilo-bases, but with custom made targeted

array designs the resolution can be a few hundred base pairs in specific regions (Lindstrand

2016).

Figure 16. array CGH. DNA from patient and control is labelled with different fluorescent

dyes. When the labelled and denatured single stranded DNA hybridizes to the array it will

bind to the matching oligonucleotide probes in a dose dependent way. The patient DNA is

labelled in green (cy3) and the control DNA in red (cy5) and when they bind in an equal

amount the signal will be yellow. When there is a deletion, there will be more cy5-labeled

control-DNA causing emission of red fluorescence and when there is a duplication there

will be green fluorescence as there is more cy3-labeled DNA. The ratio of the two

fluorescent signals is quantified by a scanner and visualized as a log2 plot in an array

analysis software. Two examples are shown, a duplication and a deletion.

45

Databases with CNVs that are often present in healthy controls, are used to filter out

common CNVs, regarded as normal variants, found in the patient.

In Study III, cohort 1, array CGH analysis was performed in accordance with a previously

published method (Pettersson 2017). A custom designed microarray, consisting of 400 000

oligonucleotide probes per patient was used. The probes target 1989 genes, including all

genes in the cilia proteome and genes involved in malformation syndromes. Within the

targeted genes, intragenic variants can be detected, with the resolution 1 probe per 100 base

pairs (bp) in the coding regions and 1 probe per 500 pb in the noncoding regions. The

remaining probes, cover the genome with an approximate resolution of 25-50 kilobases

(kb).

Genomic DNA from the patients and reference DNA were labeled and hybridization were

performed according to protocols provided by Agilent Technologies. The slides were

scanned, using a DNA Microarray Scanner. The scanned file was processed and then

analyzed using Cytosure Interpret Software.

In cohort 2, array CGH analyses were done due to clinical reasons at the Department of

Clinical Genetics at Karolinska University Hospital, where a 180K microarray was used

with whole genome coverage.

Classification Description

Benign Not in individuals with certain phenotypes, reported in multiple peer-reviewed publications and repeatedly found in the normal population.

VOUS Contains genes of interest, but little is known about them.

Likely pathogenic

Described previously in a patient with a similar phenotype, or a gene has an associated function to the patient’s phenotype.

Pathogenic Well documented as clinically significant in multiple peer-reviewed publications.

Table 6. Classification of CNVs at a glance. VOUS, Variant of uncertain significance

46

4.1.2.2 Whole genome sequencing (WGS)

Whole genome sequencing (WGS) methods sequence the whole genome and the main

difference from array CGH is that SNVs can be identified, as well as SVs not targeted by

the array CGH, such as smaller duplications and deletions and insertions. In addition, some

chromosomal abnormalities are not seen in array CGH, such as balanced translocations.

WGS provides an opportunity to look for specific candidate genes, and since the data is

stored additional candidate genes can be analyzed later on. Detection of structural variants

is based on read depth. Lower read depth would indicate a deletion and vice versa.

Sequence variants are detected using filtering programs that first compare the reads to the

human reference genome, and then filter against the common variants seen in the general

population.

Figure 17: Whole genome sequencing. Briefly a DNA-library is prepared with DNA

fragmented to around 300bp and use short signature tags of approximately 20-30

nucleotides on both the 5’ and 3’ end of the DNA-fragment for paired-end sequencing. The

DNA-fragments are introduced to a flow cell, a glass slide full of oligonucleotides, that the

DNA can bind to and will be used for further sequencing. For the fluorescent signal to be

strong, cluster amplification is the next step, generating multiple clonal DNA-strands in a

cluster on the flow cell by a polymerase. In the beginning of the amplification-step the

paired-end reads will bind on both ends as an upside-down U and strands from both sides

will be copied and further amplified. The sequencing takes place as fluorescently tagged

nucleotides attach to the DNA-strand and emit a characteristic fluorescent signal that is

read by a laser scanner. This happens in multiple clusters of DNA at the same time and is

scanned in a parallel manner. For each cycle a fluorescently tagged nucleotide adapts to

the chain and the number of cycles will determine the length of the read. All scanned reads

will be further processed for data analysis.

Librarypreparation

Cluster amplification

Sequencing

Data analysis

47

In Study IV, DNA from whole blood from each patient was sequenced using the national

genomics infrastructure (NGI) with an average read depth of 30x with Illumina TruSeq

PCR-free paired end protocol on a Illumina NovaSeq 6000. In brief, Scout v63.0 software

was used for calling variants among the 429 candidate genes through a built-in ranking

pipeline, prioritizing the variants from most to least interesting (Stranneheim 2021). Then

vcf2cytosure (Lindstrand 2019) was used to visualise SVs in a similar way as in the

previous array CGH in the four patients who did not previously have an array CGH done.

4.1.2.3 Candidate genes

HPO-panels for IM (HP:0002566) and heterotaxy (HP:0030853) was used (143 genes), as

well as gene panel for cilipathies (204 genes). Through literature review in PubMed and

searches in OMIM, 103 additional genes involved in intestinal development and IM were

identified, as well as 22 genes that are paralogues to or interact with the TTC7A and TTC7B

genes.

4.1.2.4 Sanger sequencing

Sequence variants were confirmed, and carrier testing of parental DNA was performed in

some cases.

48

4.2 MAIN RESULTS AND DISCUSSION STUDY III

4.2.1 Inclusion

Cohort 1

Forty-two patients were included, treated between 2012 and 2015, 20 male and 22 female.

Median age at inclusion were 20.5 years and forty patients had abdominal surgery. 52%

(n=22) had isolated IM. Associated malformations were: Situs inversus n=1, cardiac

malformations n=4 (including one heterotaxy syndrome), stenosis or atresia of the intestine

n=6, urogenital malformations n=2, CNS-malformations n=5, cleft lip palate n=1. At least

five patients were born preterm, four patients had intellectual disability or developmental

delay and three had cerebral palsy. Secondary malrotation in three cases was caused by

omphalocele, gastroschisis and congenital diaphragmatic hernia (CDH). All patients in the

study had symptomatic IM.

Cohort 2

Five patients were included, none had isolated IM. Two patients had cardiac malformations,

one patient had omphalocele and Meckel’s diverticulum, and one had duodenal atresia.

Two patients had developmental delay or intellectual disability.

4.2.2 Reported CNVs

We found six rare CNVs in five patients.

Cohort Array result

Coordinates Affected gene/ loci Classification IM previously reported

1 Deletion 2p23.1 GALNT14 VOUS No

1 Duplication 7q11.23 7q11.23 microduplication syndrome

Likely pathogenic

Yes

Duplication 16p13.11 16p13.11 microduplication syndrome

VOUS

1 Deletion 18q22.1q23 18q deletion syndrome

Pathogenic No

1 Deletion Xq13.1q13.2 HDAC8 (Cornelia de Lange typ 5)

Pathogenic No

2 Duplication 16q24 FOXF1 Pathogenic Yes

Table 7. Genotype of patients with rare CNVs and IM. VOUS, Variant of uncertain

Significance.

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Patient one had isolated IM and had an intragenic heterozygous deletion in the GALNT 14-

gene, inherited from the healthy father and not previously implicated in human disease, and

thus was considered as of uncertain significance. The GALNT14 gene was of interest

because it is important during embryonal development and overexpression increases

mRNA expression of N-cadherin (Huanna 2015). It is a cell adhesion protein important in

asymmetric cell-formations in the dorsal mesentery (Davis 2008, Plageman 2011, Welsh

2013). Knockdown of the gene inhibits cellular migration and affects cell morphology

(Wang 2013). This patient was the only one of 22 patients with isolated IM that harboured a

rare CMV.

Patient two, had two heterozygous rare de novo separate microduplications, both described

as having varying penetrance (Kirov 2014). The 7q11.23 microduplication syndrome

(MIM#609757) is known and causes language and developmental delay, also intellectual

disability with an estimated 44% penetrance (Kirov 2014). In addition, congenital heart

defects, neonatal hypotonia and cryptorchidism are common. One case of congenital

diaphragmatic hernia and one case of umbilical hernia was reported (van der Aa 2009) and

one previous report with IM (Morris 2015). This patient had IM, expressional language

disorder and cryptorchidism which fits the above features. The other duplication, located in

the 16p13.11 region is associated with congenital anomalies and neurodevelopmental

disorders, however only exhibiting a 8.4% penetrance (Kirov 2014, Sahoo 2011). The

patient’s phenotype was likely caused by a combinatory effect of both detected

rearrangements.

Patient three had a heterozygous terminal deletion 18q22.1-q23, previously associated with

many congenital anomalies (Strathdee 1995). The patient had congenital nystagmus,

delayed myelination, global developmental delay, left-sided clubfoot, and umbilical hernia

which was in concordance with the 18q deletion syndrome (MIM#601808). The patient

also had IM and an accessory spleen which was not previously reported in patients with 18q

terminal deletions (Cody 1999).

Patient four had a de novo heterozygous deletion, Xq13.1-q13.2, involving HDAC8

(MIM#300269), associated with X-linked dominant Cornelia de Lange syndrome (CDLS)

type 5 (MIM#300882). The patient had IM, delayed psychomotor development, speech

delay, strabismus, dysmorphic facial features, nevus flammeus, brachydactyly and

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hirsutism, all in concordance with CDLS. CDLS is associated with gastrointestinal

malformations, but only 2.3% has IM (Kapoor 2014) and to our knowledge, this is the first

published case of HDAC8-related CDLS with IM.

Patient five had a large de novo duplication on 16q24.1, including the FOXF1 gene. The

patient had IM, mild intellectual disability, short stature, mild pulmonary artery stenosis,

mild dysmorphic facial features. Duplications involving the FOXF1 gene, from 15kb to

1,7Mb in size have previously been described in four families with intellectual disability,

speech delay, and gastrointestinal abnormalities including IM, thus IM in a syndromic

context (Dharmadhikari 2014).

4.2.3 Clinical relevance

We could present, to our knowledge, the first cases of 18q terminal deletion syndrome and

CDLS type 5 with IM which could be rare phenotypes. Since IM can be asymptomatic, it

can have been missed in previous cases. In addition, these findings could be incidental,

since the cause of IM is probably often multifactorial.

We identified likely pathogenic or pathogenic CNVs in 8,5% (4/47) patients with IM. All

with those findings had associated malformations and neurological symptoms. Therefore,

we suggest that genetic investigation with CNV screening is indicated in cases with IM and

additional extra-gastrointestinal malformations or developmental delay.

We did not find any SVs in ciliopathy genes thus we found no support for the theory that

IM is a ciliopathy.

To fully understand the impact on rare CNVs on IM, further genetic studies on larger

cohorts are needed. For this, array CGH is an effective and well-established tool to identify

disease causing genes in both isolated and syndromic gastrointestinal malformations

(Dworschak 2015, Genesio 2018, Tsai 2015). Further advances have led to more accessible

whole genome sequencing (WGS). With tools, to find structural variants (SVs), it is an

appealing approach to also be able to screen for SNVs in gene panels and to be able to

reanalyze data when discovering future candidate genes.

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4.3 MAIN RESULTS AND DISCUSSION STUDY IV:

4.3.1 Inclusion

This was a pilot study of WGS in ten patients with isolated IM and no other malformations

or comorbidities, except for one who had intrauterine growth retardation and was born in

week 35+0. Of the ten, nine were male. Early symptoms were a prerequisite to attain a

severe phenotype and five had symptoms since birth. Seven were diagnosed within the first

month, two were one year old and one patient was four years old when diagnosed and

treated. All had midgut volvulus at the time of diagnosis and surgery, except for one who

had recurrent midgut volvulus later on and needed redo-surgery.

4.3.2 Reported variants

After filtering and ranking the WGS data on all ten cases, we identified two rare allelic

variants in the Tetratricopeptide Repeat Domain 7A, TTC7A gene: c.433G>A p.Ala145Thr

and c.1057G>A p.Glu353Lys in one patient (Table 8).

Variant c.433G>A p.(Ala145Thr) c.1057G>A p.(Glu353Lys)

Exon 3 8

Allele frequency (GnomAD) 0.0004 0.000008

Conservation Conserved Conserved

CADD score 22.8 25.9

TPR-domain Yes No

Table 8: Description of the discovered variants in TTC7A.

The male patient had surgery when he was 16 days old, and was followed up to 14 years of

age. No other malformations or comorbidities were reported except for gluten intolerance

as well as some abdominal complaints with fecal urgency.

Carrier testing showed that both variants were inherited from the mother, who has an

autoimmune disease and a mild intestinal rotation variant with the cecum pathologically

located in the right upper quadrant discovered when an experienced radiologist analyzed

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previous radiologic investigations due to other causes. She also suffers from fecal urgency,

as does her sister. The maternal grandmother also has an autoimmune disease (Figure 19).

Figure 19. Pedigree of the family with two variants in cis in the TTC7A-gene (from study IV).

Familial cases with IM can easily be missed so several affected could be more common

than reported. Abdominal complaints are not uncommon but with a family history of IM,

further investigation could be indicated.

4.3.3 The TTC7A-gene and IM

The TTC7A gene is expressed in the intestines and involved in intestinal development. It

has 20 exons, is located on chromosome 2p21 and contains nine tetratricopeptide repeat-

domains (TPR-domains) that are important in protein-protein interactions.

TTC7A-deficiency is an autosomal recessive condition, causing multiple intestinal atresias

(MIA) and immune defects; combined immunodeficiency (CID) (Lemoine 2014, Woutsas

2015, Yang 2015). Another expression of the disease is early onset inflammatory bowel

disease (EOIBD). So far 55 cases are reported, with homozygous or compound

heterozygous mutations in the TTC7A gene (Jardine 2019).

IM has been reported in at least five cases with MIA, in two of the cases, who also had

CID, genetic testing was done and showing smaller homozygous deletions in the TTC7A

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gene; c.53344_53347del, and c.313_216delTATC respectively (Bilodeau 2004, Mandiá

2018, Guanà 2014).

4.3.4 Clinical relevance

In this first confirmed familial case in our cohort, the mother who has an IRA, also has an

autoimmune disease which could cohere with a mild phenotype of TTC7A-deficiency.

Our findings could be incidental, but there are examples of autosomal recessive disorders

where carriers express a milder phenotype. (Wigley 1994, Nordenström 2019). Even

though carriers of TTC7A variants generally do not have symptoms, it is interesting to

speculate that IM could be a mild manifestation of a milder form of TTC7A-deficiency,

when in this family both carriers have a rotational anomaly and the mother also manifests

an autoimmune disease.

Possible mechanisms connecting TTC7A to IM are the Phosphatidylinositol 4-kinase IIIα-

complex (PI4IIIα-complex) where TTC7A is one of four proteins. PI4IIIα is another protein

in the complex, and translates from the PI4KA gene. A homozygous missense mutation in

the PI4KA gene was reported in a consanguineous family, with 13 patients with MIA and

inflammatory changes in the intestines. Two of them also had IM and the variant had a

negative impact on PI4KIIIα and TTC7A-interaction as identified by biochemical studies

(Salter 2021). A TTC7A protein defect also leads to worsened interaction, and causes the

same disease, including some cases with IM. Also, the TTC7A protein regulates the

RhoA/ROCK-signaling pathway (Ras homolog family member A/Rho-associated protein

kinase signaling pathway). Studies on Xenopus have shown that the gut-tube formation and

elongation is dependent on Rho/ROCK/MyosinII activity where mild cases had IM and

more severe cases had elongation deformities (Reed 2009). This would support the theory

that embryological development and “rotation” of the midgut is driven by elongation. The

full role of the PIKIIIα-complex and of TTC7A in the RhoA/ROCK pathway is however

still under investigation. The involvement in elongation of the gut tube is interesting in the

light of the embryological development.

Genes in fetal development not only associated to laterality and dorsal mesentery, but also

genes associated with intestinal elongation, function and development have been high-

lighted. For example, CLMP and FLNA genes are associated with short bowel and

malrotation (Negri 2020, Wang 2021), also pseudo-obstruction and IM is associated with the

FLNA-gene (Clayton-Smith 2009). These genes were included in our gene panels and without

any findings. Pathogenic CNVs in patients with IM are found in association with other

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malformations and not in isolated cases. We do not believe that IM is a monogenic disease

possibly due to many compensating systems in fetal development. Clinically, there are

isolated cases (in the pediatric study 50%) and the rest have additional malformations

showing a failed embryonal mechanism, affecting several organs. The isolated cases could

derive from either variants that have well established compensatory mechanisms in other

organs but fail in the intestinal development. Alternatively, they act locally on the intestines,

or due to local insults such as ischemia or environmental factors acting at a critical stage of

intestinal development.

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5 STRENGTHS AND LIMITATIONS The clinical studies (I and II) consist of well-defined large cohorts considering that IM is an

uncommon disease, so they are a valuable addition to previous studies in the field. The

purpose of the statistical analysis is to simplify interpretation of the data, and its limitations

should to be taken into consideration such as an increased risk to miss or to overestimate an

association. Previous studies are also valuable in order to validate our findings. One could

consider a selection bias considering so many excluded patients in the clinical study in

children. However, we used a wide search in an effort not to miss anyone and defined the

diagnosis through strict inclusion criteria from surgical records and radiological findings. It

was also important to have a defined phenotype for further molecular studies.

Like in other retrospective cohorts, limitations such as recall- and misclassification bias, as

well as missing data, should be considered and an attempt to counterbalance this somehow

was by having a strict attitude when assessing the data.

In study III the median age at inclusion is high compared to the typical IM-patient and at

the time of the study the response rates among older patients were higher, explaining the

skewed median age. This can be a strength since malformations and comorbidities, not

discovered at birth, have been revealed by time. For the molecular studies we have well

characterized phenotypes, and it is a relatively large group with a diagnosis that has not

previously been studied this way. We have the possibility to follow up the families and

gather blood samples for further studies on relatives. During the studies we came to realize

that identifying familial cases is extremely challenging since the spectrum of intestinal

rotation varies and some are asymptomatic. Some families that we thought we had

identified had to be excluded for different reasons. Further comorbidities can develop over

time, that we have not yet detected so the estimation of associated comorbidities may be

understated. Array CGH is a well-established method to look for unbalanced SVs, even

though balanced SVs, such as balanced translocations and inversions, are missed. In study

IV there are challenges that arise when analyzing WGS, we screened for candidate genes

and for larger duplications or deletions in the genome. Bad reads could have caused us to

exclude potentially interesting findings, also balanced SVs might be missed as well as

findings in the genes we did not yet screen for. Study IV was a small pilot study and will

need further confirmation in larger materials.

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6 ETHICAL CONSIDERATIONS

The studies were approved by the Regional Ethical Review Board, Dnr; 2008/670,

2012/957-31/3, 2013/944-31, and they were designed in accordance with the Declaration of

Helsinki/Good Clinical Practice. Written informed consent was collected from all

participants or parents/legal guardians where blood was collected or interviews were

performed.

Parents to patients with malformations want to know the cause, possible treatments and

how the future for the patient will be affected. The aim of these studies was to learn how to

better diagnose and treat IM, as well as to understand the genetic background. Such

increased knowledge can sometimes ease the worry that parents to patients with a

malformation have, like a feeling of guilt when they do not know the cause of the disease.

Earlier experiences are that patients, and their parents, generally are positive to these

studies and see it as an opportunity to increase knowledge about their disease.

This is a rather rare disease and patients who have been treated might feel that they can

recognize themselves in the studies. This is avoided by grouping the results and be as

general as possible when describing a phenotype.

Blood was donated from patients and relatives, they received written and oral information

and it was voluntary with clear information on that they could change their decision about a

consent at any time. If possible, blood was collected in association with other blood tests.

The advancement in mass sequencing has opened up new possibilities in many ways,

among rare disease and cancer treatment strategies. One possible dilemma may be

incidental findings of a hereditary diagnosis. The analyzed data is always coded or double-

coded and is only used in relation to the specific research question/ diagnosis.

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7 CONCLUSIONS

The studies have contributed to an expanded knowledge or confirmed previous finding on

the clinical picture and outcome from birth to adulthood, as well as increased knowledge on

the molecular etiology of IM.

In study I, we concluded that IM should be regarded as a malformation affecting all age-

groups, with acute obstruction by midgut volvulus occurring in all ages. Surgery generally

relieves symptoms, even long-lasting pain and a contrast enhanced computed tomography

reveals the diagnosis. The most common presenting symptom is abdominal pain.

In study II, we concluded that vomiting is the most common presenting symptom until 5

years of age, and abdominal pain from six years of age. About 60% of pediatric cases

present before one year of age, and older children often have delayed diagnosis, as in the

adult cases. Risk factors to develop complications after surgery are extreme prematurity and

midgut volvulus with severely affected circulation of the intestines.

In study III, based on our findings, we suggest that genetic investigation with CNV

screening should be performed in cases with IM and additional malformations or

intellectual disability. We could also present, to our knowledge, the first cases of 18q

terminal deletion syndrome and CDLS type 5 with IM.

In study IV, we suggest that IM can be a mild manifestation of TTC7A-deficiency. The

TTC7A gene needs to be studied further in DNA from a larger cohort of patients born with

IM.

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8 POINTS OF PERSPECTIVE

It was recently put to light that also adult patients can develop acute and chronic symptoms

from IM and not much was known about this group of patients. They are rare and the

elective treatment is centralized to surgeons who have also had training in pediatric surgery.

The emergency cases are not centralized and knowledge on how many patients who are

diagnosed in these settings is unknown. If the diagnosis is not detected, the outcome may be

fatal, emphasizing the importance to ensure that general surgeons are aware about the

condition. Minimally invasive surgical methods are becoming more established in the

clinical practice and further evaluation on their efficiency is needed.

Among pediatric patients we found that extremely preterm patients have a dismal outcome

and the only way to avoid complications is to have a low threshold for suspicion and trying

to avoid a severely affected circulation, which would increase the risk further. Knowledge,

clinical skills and experience seem to be the most important factors in diagnosing and

treating IM, so it is important to keep discussing and providing information about the

malformation. When investigating abdominal complaints, it is especially important to have

the diagnosis in mind when encountering a patient with other malformations,

developmental disorders or who might for other reasons lack the ability to express clinical

symptoms. A workup can relieve years of future discomfort or even be lifesaving. Further if

the diagnosis is confirmed genetic investigation is suggested.

Recently a better knowledge on the embryological development has contributed to a better

understanding of the pathomechanism and new techniques for molecular studies such as

WGS provide favorable conditions to further investigate the molecular cause of IM. Larger

studies are needed to confirm our recent findings, in addition, using WGS in trios we could

more easily look for autosomal recessive disorders as well as de novo mutations.

I look forward to use my obtained knowledge from this project in my clinical work, to

recognize the pediatric patients who would benefit from genetic counseling, and to

contribute to the important collaboration between the clinical work setting and the research

lab. Here, the exciting story about intestinal malrotation has come to an end, but it is only a

chapter of this endless adventure.

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9 ACKNOWLEDGEMENTS

First of all I would like to thank the patients and their families for participating in the

studies.

I would like to express my sincere gratitude to all researchers, clinicians, co-authors and

friends who helped and supported me during these years. We made it through the

pandemic. Many people have made this possible and especially I would like to thank

Agneta Nordenskjöld, my main supervisor for seeing potential in me a long time ago and

helping me grow in the field of research but also on a personal level with kindness,

enthusiasm and experience as leading lights. Words cannot express my gratitude. You are a

fantastic person as well as researcher and clinician. You always found a solution where

there was none and believed in me when I did not. Thank you for taking me in as your

PhD-student.

Anna Lindstrand my co-supervisor and a visionary with a contagious enthusiasm,

problem solving and point of view angles none else noticed. Thank you for being patient

with me when I was struggling with how to handle research, clinical work and life and

found ways to make it possible.

Britt Husberg my co-supervisor for your insightful creative conversations, always bringing

me new knowledge, and good spirit. You are always supportive and make time for me.

Tomas Wester my co-supervisor for always being so wise and always having a great

refence up your sleeve. You can see what I don’t see and help me see it too, and you have

always been so supportive in research as well as in the clinic.

I also would like to thank all my co-authors and my unofficial co-supervisors

Ulla Ullberg, for always being there when I need to discuss radiology or life and bringing

the laughter. As soon I am done with my thesis I will read a book about breathtaking huge

fungus thanks to you. Maria Pettersson for always being so kind and supportive and

always takes time for me when I need to discuss genetics, and explains it in the best way.

You patiently led me through your areas of expertise and taught me a lot.

61

Margareta Michanek for our road trips when meeting families and collecting blood and

for all the personal talks.

Bo-Michael Bellander, my mentor who I knew was there as a support if needed.

The wonderful malformations genetics group and tissue engineering group that has

changed a lot over the years, for example when we moved from CMM to BioClinicum. Jia

Cao, who have been such a good friend and so patient with me during lab work, taught me

to make my own mistakes, and then helped me to get on the right path again. Christina

Nyström, who taught me how to extract DNA from blood and had such nice conversations

at our lunches. Wonderful Clara Chamorro always putting a smile on my face, and

fantastic adventurous Magdalena Fossum always with new exciting stories. Also Anna

Skarin Nordenvall, Samara Arkani, Lottie Phillips and Nikolai Juul for contributing

with good spirits.

Everyone in Rare diseases and Clincal Genetics Research group for your open

welcoming arms while I ran back and forth between clinical work and the lab. Nina Jäntti

with your great humor, for all the lunch talks and who always has a solution. I would also

like to thank you for the times you held my hand in the lab. Johanna Lundin and Kristina

Lagerstedt who saved me when I struggled with genetics. Fulya Taylan for your great

knowledge and for sharing it. Ann Nordgren with great enthusiasm, knowledge and

positive spirit, as well as Gierdre Grigelioniene and Isabel Tapia Paez. All fellow PhD-

students, especially Cecilia Arthur, Dominyka Batkovskyte, Jakob Schuy and Sofia

Frisk, you are positive and kind and share the struggle. Jesper Eisfeldt, you bring

happiness and laughter and make the most difficult things sound easy. Raquel Vas, you

have the best zebra fish stories and are always being kind and supportive.

All my friends and colleagues at the department of Pediatrics ALB, including the

subspecialities. A special thank you to my boss Jakob Frie, for making it easy to ask for

help when I was struggling, and schemaläggare Agnar Andrésson who made it possible

for me to get here. Anna Undeman Asarnoj for looking after ST and Charlotte Löttiger

Jansten for making everything so smooth and always saving me. Pediatric resident

colleagues, you have great spirits and are inspiring as well as hard working, empathetic and

always fun to work with. My clinical supervisor Elena Palleri who is so supportive, always

making time and want me to move forward. Marco Bartocci who hired me as a junior

62

doctor and all Doctors and Nurses at Neo Danderyd who let me into the world of

neonatology, made everyone feel like family and who always had research and learning in

mind. Anna Ekesbo Freisinger and Wouk Stannervik for allowing me into the warm

arms of pediatrics.

All friends and former colleagues at the department of pediatric surgery ALB. You all

contributed to my great memories at the pediatric surgery department. Especially I would

like to thank Björn Frenckner and Nader Ghaffarpour for introducing me to pediatric

surgery and for all the talks. Carmen Mesas Burgos for letting me be your tail when I was

a student. Henrik Ehrén for sharing your knowledge and giving me confidence during

surgical procedures, Pär-Johan Svensson for your wisdom. Katarina Bitkover for being a

great friend and Jakob Stenman for your expertise and great ring tone “I just can’t get

enough” that always made my day.

All friends and former colleagues at the department of pediatric surgery at

Akademiska Sjukhuset in Uppsala. Gisela Reinfeldt Engberg, Anders Stenbäck,

Elisabet Gustafson, Johan Danielson and Rolf Christofferson and the whole fantastic

gang, I loved to experience pediatric surgery with you.

KI:s Forskarskolan i molekylärmedicin, providing me with such invaluable insights and

also great new friends in the field.

Magnus Nordenskjöld for your support, insightful comments, and spot on jokes. And for

having the patience to hear Agnetas and I´s conversations on face time in the background

all day long during the pandemic.

Rune Hedlund for believing in that I could become an orthopedic back surgeon many

years ago and introducing me to research.

My friends, who stay by my side no matter what and especially Camilla, Therese, Beda

and Caroline for always making me laugh in the darkest of times. Parto, you are the

bravest person I know, and Mojgan soon we will go and buy plants together again.

Shervin, Ellen, Despina, Eleftheria and Bushra you were my dream team during the

university years and Ewonne, you had my back since I was 2 years old. Christine, I am so

glad we found each other again.

63

My parents, Annette and Behzad for always helping out with the children, bringing large

amounts of homemade meatballs, giving supportive talks and joining me for long evening

walks. Also my sister Irene who shares my weird humor and who always believes in me

when I don’t.

Finally I want to thank the best of the best, David for sticking with me even though I have

been married to work, teaching me the best phrase ever “det löser sig”, and my children for

reminding me of all the other wonderful things in life and bringing me some perspective.

In memory of Tina Granholm, always enthusiastic and knowledgeable about research.

You awoke my interest in neonatology and you were always supportive.

Researchers, clinicians, co-authors, family and friends who have contributed to the projects

in this thesis, without you I would not be here, and for all your hard work I am forever

grateful.

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