Classification and genetic features of neonatalhaemochromatosis: a study of 27 aVectedpedigrees and molecular analysis of genesimplicated in iron metabolism

Alison L Kelly, Peter W Lunt, Fernanda Rodrigues, P J Berry, Diana M Flynn,Patrick J McKiernan, Deirdre A Kelly, Giorgina Mieli-Vergani, Timothy M Cox

AbstractNeonatal haemochromatosis (NH) is asevere and newly recognised syndrome ofuncertain aetiology, characterised by con-genital cirrhosis or fulminant hepatitisand widespread tissue iron deposition. NHoccurs in the context of maternal diseaseincluding viral infection, as a complica-tion of metabolic disease in the fetus, andsporadically or recurrently, without overtcause, in sibs. Although an underlyinggenetic basis for NH has been suspected,no test is available for predictive analysisin at risk pregnancies.

As a first step towards an understandingof the putative genetic basis for neonatalhaemochromatosis, we have conducted asystematic study of the mode of transmis-sion of this disorder in a total of 40 infantsborn to 27 families. We have moreovercarried out a molecular analysis of candi-date genes (â2-microglobulin, HFE, andhaem oxygenases 1 and 2) implicated iniron metabolism. No pathogenic muta-tions in these genes were identified thatsegregate consistently with the diseasephenotype in multiplex pedigrees. How-ever, excluding four pedigrees with clearevidence of maternal infection associatedwith NH, a pedigree showing transmissionof maternal antinuclear factor and ribo-nucleoprotein antibodies to the aVectedinfants, and two families with possiblematrilineal inheritance of disease in ma-ternal half sibs, a large subgroup of theaVected pedigrees point to the inheritanceof an autosomal recessive trait. Thisincluded 14 pedigrees with aVected andunaVected infants and a single pedigreewhere all four aVected infants were thesole oVspring of consanguineous but oth-erwise healthy parents.

We thus report three distinct patterns ofdisease transmission in neonatal haemo-chromatosis. In the diVerentiation of alarge subgroup showing transmission ofdisease in a manner suggesting autosomalrecessive inheritance, we also provide thebasis for further genome wide studies todefine chromosomal determinants of ironstorage disease in the newborn.(J Med Genet 2001;38:599–610)

Keywords: iron storage; haemochromatosis; neonatal;liver

Neonatal haemochromatosis (NH, OnlineMendelian Inheritance in Man, OMIM231100) is a newly recognised and raresyndrome in which congenital cirrhosis or ful-minant hepatitis in early infancy is associatedwith marked iron deposition in the liver andextrahepatic tissues.1–3 Although the presenta-tion of neonatal haemochromatosis with he-patic failure usually preceded by oligohydram-nios, placental oedema, and intrauterinegrowth retardation or stillbirth is stereotypical,the cause of the condition is ill understood.4–8

The liver is generally shrunken and bile stainedwith extensive fibrosis and nodular regenera-tion; there is massive loss of hepatocytes butsurviving cells show giant cell or pseudoglan-dular transformation with focal nodular regen-eration and varying degrees of cholestasis. Lit-tle inflammation is usually present and most ofthe iron deposition is found in the hepatocytes.Extrahepatic iron is seen in the acinar cells ofthe pancreas and minor salivary glands as wellas the proximal renal tubule, adrenal cortex,thyroid, and myocardium.9–11

Although the concentration of iron in theliver is greatly increased in neonatal haemo-chromatosis, in rare patients who spontane-ously recover and in those patients treated byorthotopic liver transplantation, the excess ironis apparently redistributed.12–15 Since increasedliver iron and extrahepatic haemosiderosis mayrarely result from other causes of liver diseaseduring fetal life,1 3 4 6 8 it has been suggestedthat the iron deposited in neonatal haemochro-matosis may not be directly responsible for thetissue injury.9 However, in one noteworthycase, severe systemic iron overload with fatalcardiomyopathy and recurrent iron depositionin the implanted liver occurred rapidly afterhepatic transplantation.16 It has been furthersuggested that the extrahepatic parenchymalsiderosis resembles shunt siderosis in adultswhere reduced transferrin concentrations withincreased iron transferrin saturation leads tosystemic redistribution of iron; in the infantwith cirrhosis a natural portocaval shunt maydevelop as a result of the patent ductusvenosus.3 17

At present the diagnosis of neonatal haemo-chromatosis is dependent on the identificationof severe liver disease of intrauterine onsetassociated with both hepatic and extrahepaticiron deposition that spares cells of the mono-nuclear phagocyte system.8 18 The diagnosis

J Med Genet 2001;38:599–610 599

Department ofMedicine, Universityof Cambridge, Level 5,Box 157,Addenbrooke’sHospital, CambridgeCB2 2QQ, UKA L KellyT M Cox

Clinical GeneticsService, Institute ofChild Health, BristolRoyal Hospital for SickChildren, St Michael’sHill, Bristol BS2 8BJ,UKP W Lunt

University of Bristol,Department ofPaediatric Pathology,St Michael’s Hospital,Southwell Street,Bristol BS2 8EG, UKP J Berry

Paediatric LiverService, King’s CollegeHospital, DenmarkHill, London SE5 9RS,UKF RodriguesG Mieli-Vergani

The Liver Unit, TheBirminghamChildren’s HospitalNHS Trust, SteelhouseLane, BirminghamB4 6NH, UKD M FlynnP J McKiernanD A Kelly

Correspondence to:Professor T M Cox,jbg@medschl.cam.ac.uk

Revised version received15 June 2001Accepted for publication18 June 2001

www.jmedgenet.com

should also exclude primary structural abnor-malities of the liver, internal exposure to toxins,blood group antigen incompatibility, othermetabolic disorders including bile acid synthe-sis defects and tyrosinaemia, infection associ-ated liver disease including cytomegalovirus(CMV), echovirus, and other agents, as well asDown’s syndrome.19–23 Neonatal haemochro-matosis has also been associated with birthdefects24 and with distinct syndromes involvingthe renal tubule, hair growth, and intestinalfunction as well as an acidotic syndromerecently reported from Finland.25–27 Neonatalhaemochromatosis has been noted in twofamilies where it aVected maternal half sibs,thus raising the possibility of causation by amaternal factor.28 Evidence for one such factorhas been the description in one family of neo-natal haemochromatosis as part of the neonatallupus erythematosus syndrome associated withmaternal anti-Ro/SS-A and anti-La/SS-B au-toantibodies.29

About 100 cases of neonatal haemochroma-tosis have been reported and these show apanethnic distribution with an equal sexincidence.3 8 Although the syndrome may onoccasion represent the common outcome ofseveral diseases that aVect the fetal liver inutero, the disorder also occurs both sporadi-cally and recurrently in sibs in a manner that isconsistent with transmission as an autosomalrecessive trait.2 5 12 However, at present nogenetic test is available to identify at risk preg-nancies or provide appropriate counselling foraZicted families. An understanding of theputative genetic basis for neonatal haemochro-matosis in some families would not onlyimprove the outlook for predictive diagnosisbut would provide key information as to itscause, especially in relation to the molecularphysiology of iron in the fetus.

We have analysed a total of 40 infantsdiagnosed with neonatal haemochromatosisand who were members of 27 families. We haveaccordingly conducted a systematic analysis ofthe mode of transmission of this condition andhave categorised possible patterns of inherit-ance; we have moreover conducted a molecularanalysis of candidate genes implicated in disor-ders of iron metabolism.

PatientsTwo index pedigrees form the nucleus of thisstudy (fig 1).

FAMILY 1

The proband was the first child of a healthy 29year old woman who developed hypochromicanaemia because of iron deficiency in preg-nancy and received oral iron therapy. An emer-gency caesarian section was carried out at theonset of spontaneous labour at 36 weeks’ asso-ciated with oligohydramnios. The infant had anApgar score of 1 at one minute and 2 at 10minutes. The infant weighed 1450 g (3rd cen-tile indicating intrauterine growth retardation)and scalp bleeding and bruising was noted, aswell as marked hepatomegaly. Haemoglobinwas 7.5 g/dl, platelet count 79 × 109/l, and shehad abnormal blood coagulation. Peripheral

oedema and anuria developed. There was littlerespiratory eVort or spontaneous movement,hypotension occurred, and death followed at 3days. At necropsy there was moderate jaundiceand oedema with an otherwise normal externalappearance. The liver had a wrinkled surfacewith multiple brown-purple nodules up to 1cm in diameter and the spleen was enlarged.There was mesenteric haemorrhage but nor-mal bone marrow appearances. In the lungsthere was focal intra-alveolar haemorrhage andthe placenta showed villus oedema. A provi-sional diagnosis of neonatal hepatitis with con-genital cirrhosis was made. Examination of thehepatic histology showed fibrosis with cirrho-sis, giant cell transformation, and hepatocellu-lar necrosis with cholestasis and marked irondeposition in hepatocytes.

The second case in this family was the resultof the third pregnancy of the mother, at thattime 34 years of age. During this pregnancyand unlike those in which she gave birth tohealthy infants, she again required oral ironsupplements throughout pregnancy for irondeficiency anaemia (haemoglobin <10.5 g/dl).The female infant was delivered by emergencycaesarian section for fetal distress at 32 weeks’gestation. The Apgar score was one at 1minute, 7 at five minutes, and 8 at 10 minutes,but the infant required ventilatory support.There was facial bruising and a widespread

Figure 1 (A) Pedigree of index family aVected byneonatal haemochromatosis. Haplotypes are displayed inthe boxes, representing from top to bottom, alleles ofchromosome 6p loci in strong linkage with the HFE gene(HLA-F and D6S105) and HFE genotypes (C282Y andH63D) that are associated with adult haemochromatosis.ND indicates not done. (B) Pedigree of family withneonatal haemochromatosis showing segregation ofchromosome 6p marker alleles as shown in (A).

A (Family 1)

1

128128

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2

3 4

1 2

3 4

5 6

B (Family 2)

I

II

I

II

132128

–+

126128

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132128

–+

ND120

––

ND126

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ND122

––

ND116

––

ND122

––

ND126

–+

NDND––

NDND–+

126128

––

128128

––

NDND––

NDND––

600 Kelly, Lunt, Rodrigues, et al

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petechial eruption. Serological tests for hepati-tis A and B, acute Epstein-Barr virus infection,cytomegalovirus, and herpes simplex virusinfection were negative.

The infant had a prolonged bleeding time,thrombocytopenia, a prolonged prothrombintime, and anaemia. Hypoglycaemia was alsonoted and she was treated with intravenousglucose and received transfusions of platelets,blood, and fresh frozen plasma. Renal failurewith progressive hepatic failure occurred.Serum iron was 11.8 µmol/l, TIBC 12.3, andsaturation 96%. A normal karyotype wasobtained.

In the light of the findings in the previouschild, a gingival biopsy was conducted. Thisshowed extensive iron deposition in the epithe-lium of minor salivary glands and a diagnosis ofneonatal haemochromatosis was made. Activesupport was subsequently withdrawn after dis-cussion with the parents. The infant died at 6days of age. At necropsy the infant weighed1944 g (50th centile), there was jaundice,petechial eruption, generalised oedema, andskin bruising, but otherwise normal externalappearances.

The liver was shrunken and, as in the previ-ous infant, there were multiple nodules up to0.8 cm in diameter evident on the surface (fig2A). The cut surface was reddish brown. Theappearance of the bone marrow was normal.Microscopy showed oedematous placental villiand hepatic cirrhosis with marked iron excessin hepatocytes (fig 2B-D). Iron was also abun-dant in the exocrine pancreas (fig 2E), thyroid,the zona glomerulosa of the adrenal cortex,bronchial mucous glands, renal tubules, andpulmonary macrophages.

As depicted in fig 1A, the parents have hadfour children. Their second daughter and thefourth child (a son) were healthy at birth;serum tests subsequently carried out in the

parents and surviving family members showednormal transferrin iron saturation and serumferritin determinations.

FAMILY 2

The proband was the first born male infant ofa 17 year old woman of Italian origin and aTurkish father (fig 1B). Intrauterine growthretardation was noted and oligohydramnioswas detected by ultrasound at 36 weeks of ges-tation. No atypical antibodies or rubellaimmunity was detected. The membranesruptured four hours before delivery withmeconium stained liquor. The infant weighed2260 g (3rd centile), the crown-rump lengthwas 49 cm (15th centile), and head circumfer-ence 34 cm (50th centile). Cord blood pH was7.25 at birth and oedema was noted.

Shortly after delivery, hypoglycaemia wasrecorded and the infant was treated with intra-venous glucose and four hourly enteral feedingas well as parenteral antimicrobials. By the sec-ond day of life, tachypnoea and jaundice wereevident as was a metabolic acidosis. Serumbilirubin was 140 µmol/l and plasma sodiumwas 122 mmol/l. The acidosis was treated withintravenous bicarbonate but oliguria associatedwith worsening jaundice and a rising serumcreatinine occurred by day 3 and there was aprolonged prothrombin time. Total bilirubinrose to 168 µmol/l (20 µmol/l conjugated) andthe serum albumin decreased to 20 g/l. A pre-sumptive diagnosis of herpes simplex encepha-litis was made and acyclovir was administered.A diagnosis of hepatorenal failure in relation toa possible metabolic disorder was also consid-ered.

The patient was noted to have an enlargedliver and spleen but on the fourth day of lifedeveloped a massive haematemesis andmelaena requiring exchange blood transfusions

Figure 2 Pathological features in recurrent fatal neonatal haemochromatosis. (A) Macroscopic appearances of abdominal contents at necropsy showingenlarged liver with multiple dark surface nodules; generalised oedema and ascites were also present. This infant died on the sixth day of life. (B, C)Microscopic features of disrupted liver architecture showing nodule formation and pigmented and hypertrophic hepatocytes with cirrhosis (B, silver reticulinstain, × 100; C, haematoxylin and eosin stain, × 400). (D) Liver section stained to show massive deposition of iron in hepatocytes (Perls’s stain, × 400).(E) Deposition of iron in pancreatic tissue; note heavy staining of glandular acini of exocrine pancreas and also isolated punctate staining with islet cells(lower left of section) (Perls’s stain, × 100).

Classification and genetic features of neonatal haemochromatosis 601

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Tabl

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Clin

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deta

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602 Kelly, Lunt, Rodrigues, et al

www.jmedgenet.com

and albumin infusions together with intra-venous glucose. Ultrasound examinationshowed a shrunken liver with normal bile ductsand extensive ascites, with a patent portal vein.Serological investigation showed negative HIV1 and hepatitis serology, a negative directCoomb’s test, normal G6PD activity, and anormal bone marrow (showing reactivechanges only). Urine succinyl acetone was notdetected, thus excluding hereditary tyrosinae-mia. The activity of enzymes for galactosaemiawas normal and á1-antitrypsin phenotypingwas normal. Serological tests for toxoplasma,herpes simplex, and CMV titres were negative.Hyperammonaemia was shown together withprogressive renal failure and prolonged bloodcoagulation parameters. The infant was trans-ferred to Addenbrooke’s Hospital for hepatictransplantation. Further tests showed no re-ducing substances in the urine but generalisedraised plasma amino acids compatible withhepatic failure. Orthotopic transplantation ofthe liver was carried out from a 3 year olddonor but the patient suVered the conse-quences of hepatic arterial thrombosis inrelation to diYcult anastomotic proceduresand died on the 19th day of life.

At necropsy, the child was oedematous withextensive bruising; the explanted liver weighed43 g and showed widespread nodules and bilestaining with evidence of regeneration. Micros-copy showed vacuolated hepatocytes, giant celltransformation, metaplastic bile ductules withbile plugs, and widespread haemosiderin inhepatocytes and ductular cells with a smallamount of fibrosis.

Extensive iron deposits were also identifiedin the myocardium, acinar cells of the pancreas,gastric crypts, tracheal and bronchial glands,proximal renal tubules, adrenal cortex, thymus,thyroid, and salivary glands, as well as pulmo-nary alveolar macrophages. No excess iron wasidentified in the spleen, testes, or bone marrow.On the basis of these findings, a diagnosis ofneonatal haemochromatosis was made. Subse-quently the mother had another pregnancywhich terminated in a spontaneous abortion at30 weeks of gestation with an infant withgrowth retardation, jaundice, and other signs ofneonatal hepatitis.

A further 20 families (3-22) were recruitedfrom paediatric liver units in the United King-dom in a nationwide collaborative study (table1). Also included in this study were establishedfibroblast cell lines representing five families(23-27) from Corriell Cell Repositories, Cam-den, New Jersey. Twenty-two families are fromreferral centres within the United Kingdom.Examining information supplied about all 27aVected pedigrees with a total of 68 children,40 have been diagnosed as having neonatalhaemochromatosis. The male to female ratio ofthese neonatal haemochromatosis patients(16:24) does not strictly follow the 1:1 ratiopreviously reported but is not significantly dif-ferent from that expected to arise by chance(÷2=2.67, 1 df, p>0.05). In the 28 unaVectedchildren, there is a slight but statistically insig-nificant bias towards females rather than males(8:14), but at the time of writing the gender ofTa

ble

1co

ntin

ued

Fam

ilyPa

tient

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ex)

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t(a

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Classification and genetic features of neonatal haemochromatosis 603

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six children is not available. Thirty of the 40neonatal haemochromatosis patients have died,four of whom had undergone hepatic trans-plantation. A further six patients survived afterorthotopic liver transplantation and are cur-rently making good progress without evidenceof continuing tissue injury or organ failure.Four neonatal haemochromatosis patients didnot receive hepatic transplantation but made aspontaneous recovery and remain well.

These patients were diagnosed principally onthe basis of liver biopsy and for that reason couldnot meet the strict criteria required for showingextrahepatic iron deposits; they are howeverincluded in this study for completeness.

It was evident early in the course of thisstudy that neonatal haemochromatosis indeedaVects multiple subjects within given sibships.However, further scrutiny of the aVected pedi-grees showed three clear patterns of diseasetransmission. These are shown in the addi-tional pedigrees (fig 3).

(1) Neonatal haemochromatosis transmittedas a result of maternal disease (infection)acquired during pregnancy (fig 3A). In family5, infection was Coxsackie viral infection; infamily 7 the infection was E coli bacteraemiaand candidiasis; in family 9 it was Staphylococ-cus aureus; and in family 11 it was Herpes sim-plex virus and candidiasis. In the aVectedinfants of these families, excess iron was foundonly in the liver.

(2) Neonatal haemochromatosis acquired asa result of a maternal factor (fig 3B).

(3) Neonatal haemochromatosis transmittedin a manner compatible with or indicating anautosomal recessive trait (fig 3C).

MethodsgDNA STUDIES

Genomic DNA was isolated from 3-5 ml ofperipheral blood from families 1-22, twohealthy controls, and from established fibro-blasts cell lines of families 23-27.30

cDNA STUDIES

Peripheral blood mononuclear cells were iso-lated from venous blood using Ficoll-paque(Amersham, Pharmacia Biotech) densitygradient centrifugation from all members offamily 1 except subject 3 where fibroblastswere used and one control sample. Total RNAisolation from these leucocytes was carried outusing Trizol reagent (Life Technologies). Firststrand cDNA was then synthesised using thePro StarTM RT-PCR kit (Stratagene).

DNA SEQUENCING: HFE GENE

To analyse the complete HFE gene in families1 and 2 and in two healthy controls, oligonucle-otide primers were designed from the humanHFE genomic sequence (Gene Bank accessionno Z92910) and used to amplify genomicDNA sequences. Purification of amplified PCRproducts and direct automated sequencing wascarried out as described previously.30

CLASS I MHC AND RELATED GENOTYPING

MHC class I genotypes for the HLA-F locusand additional genotyping at the D6S105 locustelomeric to the class I region on chromosome6p but showing strong linkage association withadult haemochromatosis was carried out aspreviously described.31

â2-MICROGLOBULIN GENE

The â2-microglobulin gene was fully sequencedin families 1 and 2, as well as two healthy con-trols, using primers and PCR conditions previ-ously described and using genomic DNA as atemplate.32

HAEM OXYGENASE GENES

Haem oxygenase 1 (HMOX1)This gene was amplified from genomic DNAisolated from members of families 1 and 2,aVected subjects only from families 23 to 25,and two normal subjects, as two long range PCRproducts using the ExpandTM Long TemplatePCR system (Boehringer Mannheim). Twopairs of primers were designed from the humanH01 sequence (Gene Bank accession NoXO6985) to amplify the two long range PCRproducts and are as follows: HMOX1-1, agcgtcctcagcgcagccgc; HMOX1-4, ctgactcgggagtcatctcca; HMOX1-2, catggccctgggagccagcat;and HMOX1-3, agaagagctgcaccgcaaggc. Ampli-fication was carried out according to the manu-facturers’ instructions which included tworounds of amplification and elongation in thesecond cycle increased by 20 seconds per cycle.The long range PCR products were purifiedusing an equal volume of 6% polyethylene glycol

Figure 3 Patterns of disease transmission in pedigrees aVected by neonatalhaemochromatosis. (A) Neonatal haemochromatosis associated with maternal infections. (B)Neonatal haemochromatosis acquired as a result of (non-infectious) maternal factor(s).

A

Family 5

1 2

3 4

1 2 3

4 5

1 2 3

4 65

1 2 3 4

5 6 7 8 9 10

1 2

3 4 5

21 3

4 5

2 31

54

Family 9

Family 3 Family 19 Family 20

Family 7 Family 11

I

II

I

II

B

I

II

604 Kelly, Lunt, Rodrigues, et al

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8000 solution at room temperature for 20 min-utes followed by bench centrifugation at 13 000× g for 30 minutes. After the supernatant hadbeen removed, the pellet was washed twice withcold 70% ethanol and washed by centrifugationfor 20 minutes. The pellet was then allowed todry in air before being resuspended in steriledistilled water and used as a DNA template forautomated sequencing. The above primers(HMO1X-1 and HMOX1-4) and an additionalseven primers (HMOX1-5-11) were used toensure that the sequences included all fiveexons, spliced sites, and lariat spliced junctionregions of the HMOX1 gene. The sequences ofHMOX1 primers 5-11 are as follows:HMOX1-5 ggcagagaatgctgagttcatg; HMOX1-6aagccgtctcgggtcacctg; HMOX1-7 ttgaggagttgcaggagctg; HMOX1-8 tccttggtgcatgggtcag;HMOX1-9 acacccgctacctgggtgac; HMOX1-10

catgccccaggatttgtcag; and HMOX1-11 gtcacccaggtagcgggtgt.

Haem oxygenase 2 (HMOX2)A pair of primers, HMOX2-A, tgactggaggctggcggaca and HMOX2-B, ttagtcagggctggagaaag, were designed from the human HO2mRNA sequence (Gene Bank Accession NoS34389) to amplify the full length HMOX-2cDNA. Complementary cDNA was isolatedfrom peripheral blood monocytes from mem-bers of family 1 and a healthy control. RT-PCRwas carried out using 5 µl of template cDNAfrom all members of family 1. An annealingtemperature of 58°C was used with a total of 35cycles. One µl of the amplified RT-PCRproduct was then used as a template in a nestedPCR reaction using two sets of primers,HMOX2-A and HMOX2-C, cacattctcaaacatgand HMOX2-B and HMOX2-D, tctacctgtttgagaatgtg, resulting in two products encompass-ing the full length HMOX2 cDNA sequence.The nested PCR products were purified andsequenced directly as described for the HFEgene.30

CONFIRMATION OF BASE CHANGES

The base changes from the wild type (Wt)sequence identified in the HFE gene were con-firmed by restriction enzyme digestion for sitesthat either created or removed a recognitionsequence for a restriction endonuclease (table2). The base diVerences identified in theHMOX1 gene were confirmed by sequencingthe opposite strand using the primers G425Ar(tggcatcttggcatgtcgcct), A198Gf (tacagctca-gacctaattgc), and A226Gr (ttgccagtgagctga-gatcg).

ResultsFrom the information obtained from 27families aZicted by neonatal haemochromato-sis (table 1, figs 1A, B and 3A, B, C), 12 hadmore than one aVected infant. In five of these12 families (Nos 2, 19, 20, 22, and 23) all theinfants born were aVected by neonatal haemo-chromatosis; in the remaining seven families(Nos 1, 3, 6, 15, 16, 21, and 24) children notaVected by the disease were also born.

In relation to birth order, in three of theseseven families (Nos 1, 6, and 24), unaVectedoVspring born to the same parents either alter-nated with or followed infants aVected by neo-natal haemochromatosis. In the remaining fourfamilies (Nos 3, 15, 16, and 21), it was the firstborn child of the parents who was unaVected;in these families the healthy child was followedby the birth of two or more aVected sibs.

In 15 out of the total of 27 families, only onechild was aVected by neonatal haemochroma-tosis; in three (Nos 8, 26, and 27) no morechildren were born, leaving a residue of 12families (Nos 4, 5, 7, 9-14, 17, 18, and 25) whohad also had oVspring unaVected by thedisease.

Table 2 Restriction endonuclease digestion confirming base substitutions in the HFE gene

Polymorphism Restriction enzymeRestriction enzymesite

Restriction siteWild type

Restriction siteBase change

G→A C282Y RsaI GT▼AC Absent PresentCA▲TG

C→G H63D BclI T▼GATCA Present AbsentACTAG▲T

A→T S65C HinfI G▼ANTC Present AbsentCTNA▲G

T→C 4T→C RsaI GT▼AC Absent PresentCA▲TG

T→C 116T→C Sau96I G▼GNCC Absent PresentCCNG▲G

C

Family 6

1 2

3 4 5 6

I

II

Family 14

1 2

3 4 5 6 7

Family 10

1 2

3 4 5 6

Family 13

1 2

3 4 5

I

II

Family 16

1 2

3 4 5 6

Family 15

1 2

3 4 5

Family 12

1 2

3 4 5

I

II

Family 17

1 2

3 4

Family 18

1 2

3 4

Family 21

1 2

3 4 5 6 7 8

I

II

Family 22

1 2

3 4

Family 4

1 2 3

4 5

Figure 3 (C) Neonatal haemochromatosis transmitted as a putative autosomal recessivetrait.

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MODE OF DISEASE TRANSMISSION

Infection acquired in pregnancyIn four families there was clear evidence ofperinatal infection associated with haemochro-matosis (families 5, 7, 9, and 11) in whichCoxsackie B virus, bacterial sepsis with E coliand S aureus, and Herpes simplex virus (HSV)type 1 infections, respectively, were associatedwith the disease. All had a single aVected infantand one or more unaVected sibs or half sibs. Infamily 5, a male infant (second born) presentedwith liver failure at 3 days of age. The diagno-sis was confirmed on liver biopsy and increasediron deposition was identified by magneticresonance imaging (MRI) of the abdomen.Acute Coxsackie B virus infection was shownserologically in the mother and the infant wastreated with an antioxidant cocktail leading to aresolution of the symptoms with survival of thismale child for at least 21⁄2 years at the time ofwriting. It is not possible to determine whetherin family 5 extrahepatic iron deposits werepresent; in family 7 and family 8 necropsyshowed extensive hepatic iron deposits only. Infamily 11, a gingival biopsy was conducted andno increased iron was detected; liver biopsyshowed hepatic iron deposits in this infant whorecovered. AVected patients in all four pedi-grees presented within the first 10 days of birth.

In family 7, a female infant who subse-quently had an unaVected paternal half sibpresented with liver failure at the age of 4 days.E coli sepsis was found together with candidia-sis on blood cultures. Antibody tests showed noevidence of maternal ribonuclear protein anti-bodies or antinuclear factor and the infant wastreated with an antioxidant cocktail but died.Necropsy showed the typical features ofneonatal haemochromatosis with greatly in-creased iron deposition in the liver. Staphylo-coccal sepsis occurred in the mother of family9 and the infant died within two weeks of birthwith fulminant hepatitis and septicaemia com-plicated by myocarditis and liver abscessformation.

In family 11, where there are two unaVectedolder maternal half sibs, the index casepresented with fulminant hepatic failure. Therewas serological evidence of an acute Herpessimplex virus infection in the mother. Siderosisand neonatal hepatitis were diagnosed on liverbiopsy and the patient was treated initially withan antioxidant cocktail.33 She failed to improveand later an orthotopic liver transplant wascarried out from which she has made a goodrecovery and is now alive two years later. Adiagnosis of neonatal haemochromatosis wasconfirmed by examination of the explantedliver.

Maternal transmission of diseaseWe have observed two distinct patterns of dis-ease transmission in neonatal haemochromato-sis that suggest the influence of a maternal fac-tor. In family 3, with the second and third bornaVected, in a sibship of three there was clearevidence of maternal antibodies to ribonuclearproteins; anti-Ro and anti-La antibodies werealso detected in the aVected infants. The

association of full blown neonatal haemochro-matosis with the appearance of maternalantinuclear factor and ribonuclear antibodieshas been reported,29 but the presence ofantibody in plasma from the aVected infantsappears not to be obligatory. The occurrence ofneonatal haemochromatosis in the context ofmaternal antibodies may indicate a favourableoutcome, since in the two infants reported herein family 3 both have survived 41⁄2 and 6 yearsafter diagnosis and remain in good health. Inthis particular family, one infant received anhepatic allograft but the other responded totreatment with antioxidants.33

In the remaining subgroup, neonatal haemo-chromatosis has appeared in oVspring of eithersex who are maternal half sibs (that is, families19 and 20). The occurrence of disease in halfsibs has been previously noted,28 although inthese instances the presence of maternalantinuclear or ribonuclear protein antibodieswas not formally excluded. Clearly in theseinstances, the occurrence of disease could alsoresult from either maternal transmission ofmitochondria harbouring pathogenic muta-tions in the organellar genome, or from gonadalmosaicism for a new dominant gene mutationin the mother. Evidence for the possibility ofgonadal mosaicism might be suggested in onefamily (family 9) in which twin half sibs whodied of unknown causes may have beenaVected by neonatal haemochromatosis; thesesibs were paternal rather than maternal halfsibs.

Clearly, if maternal transmission of neonatalhaemochromatosis either as an acquired traitor as a result of mitochondrial mutations is apossibility, this has profound implications forgenetic counselling. In at least two instances inthe series reported here, mothers who hadborne children aVected by the disease had hadfurther aVected pregnancies as a result ofinsemination from sperm donors, which hadbeen arranged after genetic counselling on themistaken assumption of the operation of reces-sive inheritance in all cases of neonatal haemo-chromatosis.

Transmission suggesting autosomal recessiveinheritanceAs noted above, in 15 of the 27 families onlyone child was aVected by neonatal haemochro-matosis; in three of 15 (families 8, 26, and 27)no further children were born. Thus in 12families (Nos 4,5, 7, 9-14, 17, 18, and 25)unaVected children were also born. Owing touncertainties of ascertainment, it is of limitedvalue to estimate recurrence figures for the sib-ships as a whole or afterborn sibs alone. How-ever, based on the 13 families with full sibshipsof two or more, in which there was no evidenceof infection, maternal antibody, maternaltransmission, or mosaicism (families 1, 2, 6, 10,12-18, 21-25), there were 18 full sibs born afterthe proband; this includes two unaVected sibsin the multiple births that occurred in family14. Of these, 10 of the 18 were aVected by neo-natal haemochromatosis, which, even allowingfor exaggerated ascertainment bias, exceeds theexpected (approximately four) aVected sibs

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predicted for the 1 in 4 recurrence risk associ-ated with inheritance of an autosomal recessivetrait. However, since it may be assumed thatthe likelihood of ascertainment of any givensibship is proportional to the number ofaVected sibs,34 then by excluding the probandthe ratio of aVected to unaVected sibs can becalculated as the segregation ratio. In thisinstance, for pedigrees 1, 2, 6, 10, 12-18,21-25, and excluding stillbirths, there were 11aVected sibs and 20 unaVected sibs observedwhich is not significantly diVerent from theexpected 7.75 aVected sibs out of the total of31. The trend of this proportion is thus to befound biased in the expected direction ofascertainment with respect to the present studyof multiply aVected sibships, in which ascer-tainment would be expected to be enhancedover the likelihood of identifying haemochro-matosis in sibs where this was proportionalsimply to the number of sibs aVected.

Consanguinity was observed in only onefamily (family 6) which was of Asian descentbut produced all four oVspring aVected (onestillbirth, three neonatal deaths). Investigationsshowed no evidence of infection and a normalbile acid profile. However, among 27 families,one consanguineous family of Asian descentwould not be exceptional and cannot alone beregarded as decisive evidence of inheritance ofan autosomal recessive trait. Similarly, al-though recessive inheritance could provide analternative explanation for the aVected paternalhalf sibs in family 9, this would require bothmothers to be heterozygous carriers whichwould be exceptional, unless they were in factrelated by blood.

In another family (family 14), one maleinfant of five children born to the parents wasaVected by neonatal haemochromatosis. Thisinfant was one of non-identical triplets anddied at 57 days after having undergone twoorthotopic liver transplant procedures; in thisinstance, transplantation surgery was under-taken after the administration of the parenteralantioxidants had proved to be unsuccessful. Infamily 15, one infant died and one recoveredwithout the benefit of transplantation. We donot consider that this excludes a genetic traitwhich could predispose to disease under

conditions of extreme stress, for example, dur-ing the neonatal period when haemolysis ismost active. The occurrence of disease in onlyone of three non-identical newborn tripletsprovides little support for mitochondrial inher-itance or for the operation of other maternalfactors including infection in this family, butclearly cannot formally exclude de novo muta-tion.

Overall it is clear that acquisition of neonatalhaemochromatosis as a result of maternaltransmission, mitochondrial inheritance, denovo dominant mutation but with a significantchance of gonadal mosaicism all need consid-eration in any given pedigree, especially wheregenetic counselling is being sought. However, itcan be seen that in an important subgroup offamilies aVected by neonatal haemochromato-sis, inheritance of an autosomal recessive traitcannot be excluded.

DIRECT SEQUENCE ANALYSIS OF CANDIDATE

GENES

HFE geneThe principal mutation, C282Y, in the HFEgene responsible for adult haemochromatosiswas not present in any of the six families exam-ined by direct sequencing and/or restrictionendonuclease digestion (table 3). However, theH63D mutation segregated in families 1, 2,and 25 but not in association with neonatalhaemochromatosis, since only the father andfirst unaVected child in family 1 carried a sin-gle copy of H63D. The frequency of the H63Dallele of HFE in the European population isabout 0.2534 with a predicted frequency ofhomozygotes of about 0.02. The 193A→Tsubstitution in the second exon of the HFEgene, which leads to the S65C substitution35

that has been shown to be associated with amild form of adult haemochromatosis, onlysegregated in family 1 and was not present in108 HFE alleles from 54 control samples.Complete sequencing of the HFE gene infamilies 1 and 2 identified two intronic basechanges, intron 2 4T→C and intron 4116T→C,36 37 the allele frequencies being 48%and 19%, respectively. Thus, no additionalcausative mutations were identified in any ofthe eight subjects aVected by neonatal haemo-chromatosis that we examined.

â2-microglobulin geneComplete sequencing of the â2-microglobulingene in families 1 and 2 identified diVerencesfrom the published sequence (gene bankM17986) in exon 1 (absence of G837 andCCT in place of GGC at nucleotides 940-942), exon 2 (substitution of A for a C atnucleotides 107, gene bank M17987, and theabsence of a previously reported base change atnucleotide 20031) and intron 1 (an additional cafter nucleotide 989 and an additional 21 basepairs). This latter additional base change989+C in intron 1 and codon substitutionQ70P (210C→A) in exon 2 were not observedin families 1 and 2.32 In the two families inves-tigated, the remaining â2-microglobulin codingsequence and splice sites corresponded to the

Table 3 HFE genotypes for families 1 and 2 and aVected patients from families 23, 24,25, and 27

Family SubjectExon 4C282Y

Exon 2H63D

Exon 2S65C

Intron 24T→C

Intron 4116T→C

1 1 (father) −/− +/− −/− +/− −/−1 2 (mother) −/− −/− +/− +/− +/−1 4 (unaVected) −/− +/− +/− +/+ +/−1 5 (aVected) −/− −/− +/− +/− +/−1 6 (unaVected) −/− −/− −/− −/− −/−

2 1 (father) −/− +/− −/− +/− −/−2 2 (mother) −/− −/− −/− −/− −/−2 3 (aVected) −/− +/− −/− +/− −/−2 4 (aVected) −/− +/− −/− +/− −/−

23 3 (aVected) −/− +/+ −/− −/− −/−23 4 (aVected) −/− +/+ −/− −/− −/−

24 4 (aVected) −/− +/+ −/− −/− −/−

25 4 (aVected) −/− +/− −/− +/− −/−

27 3 (aVected) −/− +/+ −/− −/− −/−

Classification and genetic features of neonatal haemochromatosis 607

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wild type sequence and we believe that the dif-ferences in intronic and exonic sequencesreported here are likely to be innocuous andthus of little relevance to the pathology of neo-natal haemochromatosis.

Haem oxygenase 1 gene (HMOX1)The HMOX1 gene was sequenced in itsentirety for all members of families 1 and 2 andthe aVected neonatal haemochromatosis pa-tients from families 23 to 25. No base substitu-tions were identified in the five exonic se-quences of HMOX1; however, three nucleotidechanges were found in the intronic sequences:intron 1 G→A at nucleotide 42 and A→G atnucleotides 198 and 226 in intron 4. Addition-ally, a frameshift was found in intron 3, an extrat at nucleotide 198 from the intron/exonboundary of exon 4, followed by a repeatedsequence (ccttcctt) which ensured no altera-tion in the exon 4 sequence (table 4).

Haem oxygenase 2 gene (HMOX2)Complete sequencing of the HMOX2 cDNA infamily 1 showed no base substitutions in thecoding sequence. Complementary cDNAanalysis provided no information on the flank-ing intron/exon sequences. However, on thenature of the intron/exon boundaries no splicevariants were identified.

DiscussionCriteria for the diagnosis of neonatal haemo-chromatosis have emerged only recently. Thecondition is defined as a rapidly progressivedisorder with death in utero or in the earlyneonatal period, with increased tissue irondeposits seen in the liver, pancreas, heart, andendocrine glands, but with relative sparing ofextrahepatic mononuclear phagocytes. Thediagnosis can only be considered in the absenceof haemolytic disease or other syndromes asso-ciated with haemosiderosis or exogenous ironoverload from transfusions.3 4 8 9

Neonatal haemochromatosis occurs as a partof neonatal lupus erythematosus syndromeassociated with maternal anti-Ro/SS-A andanti-Ro/SS-B autoantibodies.29 In this previoussingle case report, full blown neonatal haemo-chromatosis in which the criteria for extrahe-patic iron deposition were met was associated

with the maternal autoantibodies that wereidentified in the patients depicted in fig 3B.Increased deposition of hepatic iron (hepatic“siderosis”) has also been associated withgenetic defects of bile acid synthesis, hereditarytyrosinaemia, Zellweger syndrome, and lepre-chaunism.8 Hepatic pathology is accompaniedby a bleeding diathesis, hypoglycaemia, meta-bolic acidosis, and incipient renal failure and itseems likely that the giant cell hepatitis thatcharacterises the condition in the presence orabsence of defined viral infection in the motheris the end result of multiple insults.

We review here pedigree studies of a largeseries of patients and the molecular analysis ofcandidate genes implicated in iron metabolism.This includes genes expressed in the humanplacenta which were studied because themother represents the only source of additionaliron before birth. However, although MRIimaging and chemical analysis of tissue ironconcentrations indicate that neonatal haemo-chromatosis is indeed associated with excessiron in liver tissue4 38 and its redistribution toextrahepatic tissues,3 8 9 it is by no means clearthat all patients with neonatal haemochromato-sis harbour an increase in total body iron. Inthose patients who recovered from the condi-tion either as a result of, or despite administra-tion of antioxidant therapy or successful ortho-topic hepatic transplantation, the majorityshow no evidence of disordered iron metabo-lism. Moreover, no other iron storage diseasesare found in the parents or surviving sibs ofaVected subjects.13–15 39 40

The degree of iron overload in patients withhaemochromatosis may be suYcient to induce(as with the adult disease) malignant tumoursand there are three published reports in whichprimary hepatocellular carcinomas have oc-curred in neonatal haemochromatosis.3 It is ofgreat interest that in one of these patients, fur-ther iron accumulated in the donor liver and inan amount comparable to that present in theexplanted organ: the recipient eventually diedas a result of a cardiac arrythmia associatedwith marked cardiac siderosis and cardiomyo-pathy.16 In this patient at least, an iron loadingdisorder was present in utero and after birththat presumably aVected transplacental andpossibly also intestinal transport of iron.

Of particular note in the present series ofpatients is the evidence for a substantialsubgroup in which the transmission of neonatalhaemochromatosis is compatible with inherit-ance of an autosomal recessive trait. In at leastone family, family 1, that an inherited factordetermines net transfer of iron from the motherto the fetus was shown by the occurrence ofneonatal haemochromatosis in two of four sibswith the disease alternating with healthychildren. This was suggested also by the occur-rence of maternal iron deficiency anaemia,requiring iron supplementation, that developedonly during the aVected pregnancies. Studies ofthe segregation haplotypes in this pedigreeshowed that if there were to be recessive inher-itance of determinants of neonatal haemochro-matosis on the short arm of chromosome 6,there is no common background haplotype on

Table 4 HMOX1 genotypes for families 1 and 2 and aVected subjects from families 23–25

Family SubjectIntron 1425G→A

Intron 4198A→G

Intron 4226A→G

Intron 3frameshift, Tbase 194

1 1 (father) +/− −/− +/− Present1 2 (mother) −/− −/− −/− Wt1 4 (unaVected) +/− +/− +/− Present1 5 (aVected) −/− −/− −/− Wt1 6 (unaVected) +/− +/− −/− Present

2 1 (father) −/− −/− −/− Wt2 2 (mother) ND +/− −/− Present2 3 (aVected) ND +/− +/− Present2 4 (aVected) −/− −/− −/− Wt

23 3 (aVected) −/− +/+ −/− Present23 4 (aVected) −/− +/+ −/− Present

24 4 (aVected) −/− +/+ −/− Present

25 4 (aVected) −/− +/− −/− Present

608 Kelly, Lunt, Rodrigues, et al

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which the mutation resides, since the aVectedhaplotypes are diVerent between the two copiesof 6p within the aVected subjects and arediVerent between family 1 and family 2. It ishowever noteworthy that no mutation in theHFE gene was identified on direct sequencing,as described below.

To investigate the role of candidate genesimplicated in iron metabolism in the develop-ment of neonatal haemochromatosis, we con-ducted a molecular analysis of the human HFEgene41 and the related gene for â2-microglobulin,42 since their cognate proteinsare expressed in the placenta and disorderedexpression of HFE at cell surfaces mediated byinteractions with â2-microglobulin is associatedwith systemic iron overload.42–45 Since neonatalhaemochromatosis occurs at a time of rapidcatabolism of haem derived from fetal haemo-globin,46 we have also conducted a molecularanalysis of the genes encoding the principalhaem oxygenase isozymes that are responsiblefor the breakdown of haem to iron, biliverdinand carbon monoxide.47–49 Haem oxygenase 1(HMOX1) maps to human chromosome22q13.1 and is the main inducible form of theenzyme; the homologous isozyme haem oxyge-nase 2 (HMOX2) is located on chromosome16p13.3 and encodes the constitutive iso-form.50 51 Knockout mice lacking HMOX1show growth retardation, diminished resistanceto oxidative injury, and develop anaemia withtissue iron deposition.52 Mice lacking HMOX2show pulmonary haemosiderosis and are sensi-tive to hyperoxia induced tissue injury.53 Thefirst case of human haem oxygenase 1 defi-ciency has recently been described: the 26month old infant had hepatomegaly, asplenia,abnormal coagulation factors, and endothelialinjury in the kidney.54 Iron deposition wasnoted in renal and liver tissue, thus furtherimplicating mutations in HMOX1 andHMOX2 as potential causes of neonatalhaemochromatosis.

Since completion of this study, nonsensemutations, representing null alleles of thenewly identified transferrin receptor 2 (TfR 2)gene that maps to chromosome 7q22, havebeen discovered in adult patients with haemo-chromatosis otherwise clinically indistinguish-able from that resulting from mutations inHFE.55 56 The indolent nature of the iron stor-age and degree of tissue injury resulting fromthis disorder does not resemble neonatalhaemochromatosis, and thus TfR 2 is anunlikely candidate gene for the infantiledisease. We also describe a consanguineousfamily with four aVected members and theoccurrence of disease in one of three non-identical triplets as well as in maternal half sibs.Caution is required, however, in advising anindividual family about the pattern of inherit-ance and the likelihood of disease recurrence.Although in our series there was no report ofneonatal haemochromatosis in generationsprevious to those of the parents, the formalpossibility remains that some cases of thedisease could be the result of digenic inherit-ance in which a rare mutant allele only causesthe condition in combination with a much

more common polymorphic variant, not neces-sarily in the same gene. An example of thisphenomenon is given by erythropoietic pro-toporphyria which shows both recessive anddigenic inheritance patterns.57 58 In our currentseries, two families with neonatal haemochro-matosis recapitulate published reports of recur-rent disease in maternal half-sibs, in some casesoccurring after donor insemination that pro-ceeded after genetic counselling.28 Clearly thepossibility of recurrent disease in half sibs mustalways be considered and an assumption ofautosomal recessive inheritance of neonatalhaemochromatosis in any given pedigree couldwell prove to be erroneous and lead toinappropriate reproductive decisions. Here weprovide additional evidence for a maternalautoantibody factor in two pedigrees and amode of transmission suggesting matrilinealinheritance compatible with mitochondrialdefects as recently reported in two pedigrees.59

We also report here one family in whichneonatal haemochromatosis occurred in pater-nal half sibs.

The present study provides strong evidenceto exclude neonatal iron storage disease result-ing from mutations in genes encoding humanHFE, â2-microglobulin, and haem oxygenases1 and 2. To identify further putative disease lociresponsible for the inheritance of haemochro-matosis in those cases with transmissionsuggesting inheritance as an autosomal reces-sive trait, a genome wide scan conducted bymolecular analysis of DNA samples obtainedfrom first degree relatives and aVected subjectsdescribed here is under way. Given theburgeoning knowledge about genes indicatedin mammalian iron transport and storage aswell as emerging information from the humangenome project and comparative genomicstudies,45 we are confident that major loci thatpredispose to the development of neonatalhaemochromatosis will ultimately be identi-fied.

We are indebted to The Children’s Liver Disease Foundation,the Grocer’s Trust, the Peel Medical Research Trust, and theEuropean Union for research support. Dr David A Rhodeskindly carried out the class I MHC and chromosome 6p geno-typing. Mrs Joan Grantham provided expert secretarialassistance.

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