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RESEARCH ARTICLE

Identification of a Premature Stop Codon Mutationin the PHGDH Gene in Severe Neu-LaxovaSyndrome—Evidence for Phenotypic Variability

Eduardo P. Mattos,1,4 Andre Anjos da Silva,1,4 Jose Antonio A Magalhaes,2,5 Julio Cesar L. Leite,1

Sandra Leistner-Segal,1 Rejane Gus-Kessler,1 Juliano Adams Perez,3 Leonardo M. Vedolin,3,5

Albertina Torreblanca-Zanca,6,7 Pablo Lapunzina,6,8 Victor L. Ruiz-Perez,7,8

and Maria Teresa V. Sanseverino1*1Medical Genetics Service, Hospital de Clınicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil2Fetal Medicine Group, Hospital de Clınicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil3Radiology Service, Hospital de Clınicas de Porto Alegre, Porto Alegre, Rio Grande do Sul, Brazil4Department of Genetics, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil5Internal Medicine Department, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil6INGEMM, Instituto de Genetica Medica Y Molecular, Hospital Universitario La Paz, Madrid, Spain7Instituto de Investigaciones Biomedicas, Consejo Superior de Investigaciones Cientıficas-Universidad Autonoma de Madrid, Madrid, Spain8Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), Instituto de Salud Carlos III (ISCIII), Madrid, Spain

Manuscript Received: 30 September 2014; Manuscript Accepted: 21 Novemb

er 2014

Conflict of interest: none

Grant sponsor: Spanish Ministry of Science and Innovation;

Grant number: SAF2013-43365-R; Grant sponsor: The CIBERER

Program of Research on Pediatric diseases (ACCI 2012);

Grant sponsor: The Brazilian National Council for Research and

Development (CNPq); Grant sponsor: Hospital de Clınicas de Porto

Alegre (FIPE-HCPA).�Correspondence to:

Maria Teresa Vieira Sanseverino, Servico de Genetica Medica, Hospital

de Clınicas de Porto Alegre, Rua Ramiro Barcelos, 2350, Porto Alegre,

RS, Brazil CEP 90035-903.

E-mail: msanseverino@hcpa.ufrgs.br

Article first published online in Wiley Online Library

How to Cite this Article:Mattos EP, Silva AA, Magalhaes JAA, Leite

JCL, Segal SL, Gus-Kessler R, Perez JA,

Vedolin LM, Torreblanca-Zanca A,

Lapunzina P, Ruiz-Perez VL, Sanseverino

MTV. 2015. Identification of a premature

stop codon mutation in the PHGDH gene

in severe Neu-Laxova syndrome—Evidence

for phenotypic variability.

Am J Med Genet Part A. 9999A:1–7.

In some cases Neu-Laxova syndrome (NLS) is linked to serine

deficiency due to mutations in the phosphoglycerate dehydro-

genase (PHGDH) gene. We describe the prenatal and postnatal

findings in a fetus with one of the most severe NLS phenotypes

described so far, caused by a homozygous nonsense mutation of

PHGDH. Serial ultrasound (US) andpre- andpostnatalmagnetic

resonance imaging (MRI) evaluations were performed. Prena-

tally, serial US evaluations suggested symmetric growth restric-

tion, microcephaly, hypoplasia of the cerebellar vermis,

micrognathia, hydrops, shortened limbs, arthrogryposis, and

talipes equinovarus. The prenatal MRI confirmed these findings

prompting a diagnosis of NLS. After birth, radiological imaging

did not detect any gross bone abnormalities. DNAwas extracted

from fetal and parental peripheral blood, all coding exons of

PHGDH were PCR-amplified and subjected to Sanger sequenc-

ing. Sequencing of PHGDH identified a homozygous premature

stop codonmutation (c.1297C>T; p.Gln433*) in fetalDNA, both

parents (first-cousins) being heterozygotes. Based on previous

associations of mutations in this gene with a milder NLS pheno-

type, as well as cases of serine deficiency, these observations lend

further support to a genotype-phenotype correlation between

the degree of PHGDH inactivation and disease severity.

� 2015 Wiley Periodicals, Inc.

Key words: Neu-Laxova syndrome; prenatal diagnosis; mag-

netic resonance imaging; phosphoglycerate dehydrogenase; serine

metabolism

(wileyonlinelibrary.com): 00 Month 2014

DOI 10.1002/ajmg.a.36930

2015 Wiley Periodicals, Inc. 1

2 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

INTRODUCTION

Neu-Laxova syndrome (NLS) is a lethal genetic condition charac-

terized by multiple congenital anomalies and ectodermal abnor-

malities [Neu et al., 1971; Laxova et al., 1972]. The diagnostic

findings of NLS are characteristic and include intrauterine growth

restrictionwith reduced fetalmobility and arthrogryposis, ichthyo-

sis, microcephaly, short neck, and hypoplastic lungs [Manar &

Asma, 2010]. Other neurologic and facial findings are suggestive of

a NLS diagnosis, such as lissencephaly, cerebellar hypoplasia,

corpus callosum abnormalities, severe proptosis with ectropion

and hypertelorism, micrognathia, flat nose and malformed ears.

Most NLS patients die shortly after birth [Manning et al., 2004].

Cerebro-oculo-facio-skeletal, Miller-Dieker, Roberts, Refsum,

Pena-Shokeir and Smith-Lemli-Opitz syndromes, congenital ich-

thyosis and some chromosomal abnormalities (trisomy 12 and 4p

deletion) should be considered in the differential diagnosis.

As it is the case for most rare genetic disorders, there are no

reliable prevalence estimates for NLS; however, it appears to occur

more commonly among inbred populations, and at least a dozen

families with multiple gestations affected by NLS have been de-

scribed [Neu et al., 1971; Laxova et al., 1972; Shved et al., 1985;

Ejeckam et al., 1986; Karimi-Nejad et al., 1987; Tolmie et al., 1987;

Ostrovskaya & Lazjuk, 1988; Naveed et al., 1990; Abdel Meguid &

Temtamy, 1991; Rode et al., 2001; Acuna-Hidalgo et al., 2014;

Shaheen et al., 2014]. Accordingly, autosomal recessive inheritance

FIG. 1. Appearance of two sibs with Neu-Laxova syndrome born to a con

at 31–32 weeks of gestation and weighted 450 g. Note: marked symmet

digital hypoplasia and club feet. Note the ichthyotic, stretched skin with

proptosis with ectropion, depressed nasal bridge, and micrognathia are a

fetus at 28–29 weeks of gestation, with a phenotype very similar to the

forNLSwas always suspected, and recently confirmed and shown to

be linked to deficiency of serine metabolism [Acuna-Hidalgo et al.,

2014; Shaheen et al., 2014].

A consanguineous (first-cousin) couple was referred to our

Institution during their fourth pregnancy due to the recurrence

of a severe, likely autosomal recessive condition.Here, we report on

the prenatal and postnatal application of magnetic resonance

imaging (MRI) and complete evaluation to establish the diagnosis

of NLS. A homozygous, premature stop codon mutation in the

phosphoglycerate dehydrogenase (PHGDH) gene was identified in

this patient, thus providing further support to the involvement of

this locus in the pathogenesis of NLS, as recently suggested [Acuna-

Hidalgo et al., 2014; Shaheen et al., 2014].

MATERIALS AND METHODS

Serial morphologic US evaluations were performed from 12 to

25 weeks of gestation. Upon establishment of a lethal condition,

standard MRI scanning was performed. Postnatally, whole-body

radiography (babygram) and an MRI were also done.

Peripheral blood samples were collected after delivery from the

fetus and both parents for DNA analysis. Since the publication by

Shaheen et al. [2014] identified homozygous mutations in

PHGDH in three unrelated Saudi Arabian patients, the whole

gene was amplified by polymerase chain reaction (PCR) in the

parent-fetus trio and subjected to conventional Sanger sequenc-

sanguineous Brazilian couple. A: Present case. The fetus was stillborn

ric growth restriction, contractions of upper and lower limbs with

generalized subcutaneous edema. Microcephaly, malformed ears,

lso present. B: Previous gestation of the couple, depicting a stillborn

case presented here.

FIG. 2. Prenatal magnetic resonance imaging of the fetus at 25–26 weeks of gestation confirms the ultrasonographic diagnostic hypothesis

of Neu-Laxova syndrome. A: Fetal profile shows proportionate growth restriction and arthrogryposis with contracted upper limbs and club feet

(RA: right arm; RF: right foot; LF: left foot). B: Sagittal section of the fetus shows microcephaly, micrognathia, and a hypoplastic cerebellum,

but no neural tube defects. C and D: Coronal sections of the fetus depicting a hypersignal with thickening of the cranial subcutaneous

tissues, absence of the corpus callosum and lissencephaly.

MATTOS ET AL. 3

ing (primers and PCR conditions available upon request). The

nomenclature and position of the identified mutation is in

accordance to the PHGDH reference messenger RNA sequence

NM_006623.3, obtained from the National Center for Biotech-

nology Information. Functional prediction of the identified

mutation was performed using the MutationTaster algorithm

[Schwarz et al., 2010].

Informed consent for the anonymous disclosure of patient data

and pictures was obtained from the family reported here.

RESULTS

A consanguineous couple (first-degree cousins; 29-year-old moth-

er and 43-year-old father) was referred for evaluation during their

fourth pregnancy due to detection of a fetus with multiple anoma-

lies with familial recurrence. Their first pregnancy ended in spon-

taneous abortion at 8–9 weeks of gestation, while the second

pregnancy gave rise to a healthy daughter, who is now 8 years

old. In the third gestation, the fetus presented with severe growth

restriction and multiple abnormalities. Preterm labor occurred at

28 weeks of gestation and the baby died shortly after birth, but,

unfortunately, the patient was not further evaluated and remained

undiagnosed. Retrospectively, clinical pictures taken by the father

were reviewed and compared to the prospective case, fitting the

diagnosis of NLS (Fig. 1).

In the present case, an ultrasound (US) evaluation at 19 weeks of

gestation detected symmetric growth restriction, hypoplasia of the

cerebellar vermis, micrognathia, hydrops, possible spina bifida,

shortened limbs, articular rigidity, and club feet. These abnormali-

ties were confirmed in a subsequent US examination at 22 weeks of

gestation. Supplementary Table I shows the body measurements

obtained in the sequential US investigations, evidencing marked

growth retardation with several data points falling below the first

centile for gestational age.

Amniocentesis showed a normal female chromosome constitu-

tion (46, XX). Considering the severity of the case, magnetic

resonance imaging (MRI) was elected at 25–26 weeks of gestation

in an attempt to better characterize the abnormal findings evi-

denced by the US evaluations (Fig. 2). It showed marked fetal

growth restriction, microcephaly, a flat nose, micrognathia, talipes

equinovarus, and arthrogryposis of the upper limbs. There was also

a hypersignal with thickening of cranial subcutaneous tissues, but

no evidence of myelomeningocele. Collectively, these findings led

to the prenatal diagnostic hypothesis of NLS, confirmed

postnatally.

Preterm labor followed by spontaneous delivery occurred at 31–

32weeks of gestation. The stillborn infantweighted 450 g (below1st

centile), and had anthropometric measures compatible with 22–24

weeks of gestation, severe growth restriction, along with contrac-

tures of upper and lower limbswithhypoplasiaof digits, “stretched”

skin with mild ichthyosis and generalized subcutaneous edema,

short neck, scoliosis, club feet, and female external genitalia (Fig. 1).

Craniofacial findings included microcephaly, hypertelorism and

malformed ears, proptosis with ectropion, depressed nasal bridge,

micrognathia, and complete posterior cleft palate. An autopsy was

performed and internal organ malformations consisted mainly of

pulmonary, thymic, and left ventricle hypoplasia. Skeletal evalua-

tion by radiography showed fusion of the bones of the cranial vault

and scoliosis; no signs of other major malformation or dysplasia

were detected (Fig. 3).

Postnatal MRI (Fig. 4) confirmed the severe involvement of the

cerebellum and agenesis of the corpus callosum, as indicated by the

diagnostic procedures performed inutero. Furthermore, absence of

cerebral gyri and sulci reinforced the prenatal finding of

lissencephaly.

By the time this study was initiated, there were yet no genetic loci

consistently associated to NLS, which led us to consider exome

sequencing of the parent-offspring trio, in an attempt to identify

FIG. 3. Whole-body radiography of the patient in antero-posterior (A) and lateral (B) views, showing scoliosis but no additional gross

abnormalities.

4 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

potentially pathogenic coding variants in the fetus. However, the

data subsequently published by Shaheen et al. [2014] prompted us

to focus the analysis on the PHGDH gene [Shaheen et al., 2014].

Indeed, a potentially pathogenic, homozygous single-nucleotide

substitution (c.1297C>T) was identified in the fetal DNA, both

parents being heterozygous (Fig. 5A and B). Structurally, this

mutation changes glutamine residue 433 for a premature stop

codon (p.Gln433*) at the C-terminal regulatory domain of

PHGDG, anticipating the deletion of the last 101 amino acids

(Fig. 5C). Although no functional studies were performed, this

mutation was predicted to behave as a null allele, preventing the

expression of PHGDH from both alleles due to surveillance by the

mRNA nonsense-mediated decay system.

DISCUSSION

Although NLS has been recognized as a distinct genetic entity for

over 40 years, its molecular basis remained elusive until recently,

when Shaheen et al. performed autozygosity mapping and gene

sequencing in three NLS patients from distinct consanguineous

Saudi Arabian families [Shaheen et al., 2014]. Prior to this study,

some authors suggested that NLS might belong to the neuro-

ectodermal dysplasia disease group [Lazjuk et al., 1979; Ejeckam

et al., 1986; Naveed et al., 1990; Bronshtein et al., 1993]. Others

hypothesized defects in one or more genes on the 6q and/or 9p

regions, based onmousemodels of restrictive dermopathies, which

resemble the “cocoon”-like aspect of the fetus [Manning et al.,

2004].Hence, the associationof thePHGDHlocuswithNLScameas

a surprise [Shaheen et al., 2014], since coding variants in this gene

were already known to cause PHGDH deficiency, a less severe

condition characterized by delayed development, microcephaly,

seizures and growth retardation [Jaeken et al., 1996; Van der

Crabben et al., 2013]. Interestingly, the conceptual link between

phenotype and an inborn error of metabolism in NLS was estab-

lished more than two decades after its initial proposal by Shapiro

et al. [1992], who argued that the abnormal fat deposits, the

FIG. 4. Postnatal magnetic resonance imaging of the fetus after delivery (31–32 weeks of gestation) confirms the prenatal detection of central nervous

system abnormalities. Coronal (A), sagittal (B), and transverse (C) sections of the fetus illustrate cerebellar hypoplasia and generalized lissencephaly.

MATTOS ET AL. 5

ichthyotic skin, and the eventual development of cataract seen in

NLS should relate to an underlyingmetabolic defect [Shapiro et al.,

1992].

Classification of NLS phenotypes was proposed by Curry

[1982], who divided NLS into three subgroups according to

FIG. 5. Molecular analysis identifies a homozygous premature termination c

Pedigree of the reported family. B: Chromatograms illustrating the sequencin

parents, who are heterozygous carriers for the mutation (C; Individuals I-1 a

PHGDH structure with depicted mutations identified so far in both serine defi

identified in this study is depicted in bold. SB, substrate binding domain; NA

selected clinical findings. Patients of group I would be those

who present mainly with arthrogryposis, partial syndactyly,

squamous skin and bone hypomineralization. Group II would

include patients with severe swelling of hands and feet—also

known as “glove-shaped” limbs—secondary to oedema and fat

odon mutation in the PHGDH gene in a patient with NLS syndrome. A:

g profile at the PHGDH locus of a normal control individual (N), both

nd I-2), and the patient (P; Proband II-4). C: Schematic representation of

ciency (lower notations) and NLS (upper notations). The variation

D(P), NADþ/NADPþ binding domain; RD, regulatory domain.

6 AMERICAN JOURNAL OF MEDICAL GENETICS PART A

deposition, pronounced ichthyosis and poor bone mineraliza-

tion. Patients with the most typical clinical findings of NLS

would be placed in this group [Manning et al., 2004]. Finally,

group III would be reserved for patients with hypoplastic digits,

short limbs, microtia, and severe ichthyosis similar to that

observed in harlequin ichthyosis. It is not known whether these

phenotypic differences are due to variable expressivity of a

single mutated gene, or mutations in different genes.

Besides variable expression in NLS, mutations in the phospho-

serine aminotransferase (PSAT1) and phosphoserine phosphatase

(PSPH) genes have also been reported, supporting the hypothesis of

allelic heterogeneity for NLS. These results also show that NLS

might arise frommutations in at least three different genes involved

in the L-serine biosynthesis pathway, probably reflecting severe

forms ofmilder inborn errors ofmetabolism [Acuna-Hidalgo et al.,

2014; Shaheen et al., 2014].

In light of our results and those presented by Shaheen and

colleagues and Acuna-Hidalgo and colleagues [Acuna-Hidalgo

et al., 2014; Shaheen et al., 2014], we propose that at least part of

the variable clinical presentations of NLS might be explained by

different PHGDH mutations that give rise to a variable of

disease. Accordingly, the NLS-associated variants described by

Shaheen et al., who reported on patients that seem phenotypically

less affected then the one we describe here, are missense sub-

stitutions not entirely different from other PHGDH missense

mutations observed in individuals with the inborn error of serine

metabolism known as PHGDH deficiency [Jaeken et al., 1996;

Shaheen et al., 2014]. Despite missense mutations located in

conserved catalytic residues of the enzyme [Shaheen et al., 2014],

PHGDHmight still have some degree of residual activity in those

cases (although functional studies remain to be conducted to

experimentally confirm this hypothesis). On the other hand, the

patient reported here was shown to encode a homozygous

premature termination codon mutation, which is predicted to

result in complete PHGDH absence, due to the activity of the

cellular nonsense mRNA-mediated decay system [Chang et al.,

2007], which in turn might result in the more severe phenotype

observed here.

In conclusion, we present a clinical characterization of a NLS

patient using MRI evaluations. A homozygous premature stop

codon mutation in the PHGDH gene was identified in the patient

reported here, which corroborates a recent association of this locus

with NLS [Acuna-Hidalgo et al., 2014; Shaheen et al., 2014]. The

identification of additional patients will likely shed light on the

phenotypic and genetic diversity of NLS, leading to a better

understanding of serine-deficiency diseases and its genotype-phe-

notype correlations.

ACKNOWLEDGMENTS

The authors are indebted to the family reported here, for their

support for publication. Part of this work was supported by the

Spanish Ministry of Science and Innovation SAF2013-43365-R,

the CIBERER Program of Research on Pediatric diseases (ACCI

2012), the Brazilian National Council for Research and Devel-

opment (CNPq), and Hospital de Clınicas de Porto Alegre (FIPE-

HCPA).

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