Molecular study of six families originating from the Middle-East and presenting with autosomal recessive osteopetrosis

European Journal of Medical Genetics 50 (2007) 188e199http://www.elsevier.com/locate/ejmg

+ MODEL

Original article

Molecular study of six families originating fromthe Middle-East and presenting with autosomal

recessive osteopetrosis

Noelle Souraty a, Peter Noun b, Claudia Djambas-Khayat c,Eliane Chouery a, Alessandra Pangrazio d, Anna Villa d,

Gerard Lefranc e, Annalisa Frattini d, Andre Megarbane a,*

a Unite de Genetique Medicale, Laboratoire de Biologie Moleculaire et Cytogenetique, Faculte de Medecine,

Universite Saint-Joseph, Beirut, Lebanonb Service de Pediatrie; Jeitawi Hospital, Beirut, Lebanon

c Service de Pediatrie; Hotel-Dieu de France Hospital, Beirut, Lebanond Institute of Biomedical Technologies; CNR, Milan, Italy

e CNRS UPR 1142, Institut de Genetique Humaine, et Universite Montpellier II, France

Received 4 October 2006; accepted 30 January 2007

Available online 21 February 2007

Abstract

Autosomal recessive osteopetrosis is a severe hereditary bone disease whose cellular basis is in the os-teoclast, but with heterogeneous molecular defects. We hereby report the clinical and the molecular studyof seven patients affected by the recessive form of osteopetrosis (ARO) from six families originating fromthe Middle-East: four from Lebanon and two from Syria. Parental consanguinity was found in five fam-ilies. The mean age of diagnosis was 3 months. Failure to thrive, prominent forehead, exophthalmia, opticatrophy, hepatosplenomegaly, neurological manifestations, anaemia, thrombocytopenia, hypocalcaemia,elevated hepatic enzymes and acid phosphatase, and an early fatal outcome were common. Macrocephaly,strabismus, and brain malformations were relatively less common. Mutations were identified in two genes:TCIRG1 and OSTM1. Phenotype-genotype correlation is discussed.� 2007 Elsevier Masson SAS. All rights reserved.

Keywords: Osteopetrosis; Neurological defects; OSTM1; TCIRG1

* Corresponding author. Unite de Genetique Medicale, Faculte de Medecine, Universite Saint-Joseph, 42, rue de

Grenelle, 75007 Paris, France. Fax: þ961 1 421 021.

E-mail address: [email protected] (A. Megarbane).

1769-7212/$ - see front matter � 2007 Elsevier Masson SAS. All rights reserved.

doi:10.1016/j.ejmg.2007.01.005

mailto:[email protected]

http://www.elsevier.com/locate/ejmg

189N. Souraty et al. / European Journal of Medical Genetics 50 (2007) 188e199

1. Introduction

Autosomal recessive osteopetrosis (ARO, MIM 259700) also called malignant or infantileosteopetrosis, is a severe hereditary bone disease. It presents at birth and shows radiologicallygeneralized increase in bone density with typically broadened diaphyses and metaphyses. Thereduced marrow space results in extramedullary haematopoiesis and hepatosplenomegaly.Nerve palsies due to compression from the expanding bone can cause complications such asdeafness and blindness. Death occurs in early childhood due to severe anaemia and major in-fections. It should be distinguished from the carbonic anhydrase II deficiency syndrome, whichis associated with renal tubular acidosis and less severe osteopetrosis.

Three genes have been reported so far to be responsible for ARO. Two of them, the T-cellimmuno-regulator-1 (TCIRG1) [5,7,11,12] and the Chloride channel 7 (ClCN7) [6,8] genes ac-count for about 52 and 11% of cases respectively. The latest one, the osteopetrosis associatedtransmembrane protein 1 gene (OSTM1) has been described so far in only five ARO patients[4,9,10].

In this study, we describe the clinical and molecular analysis performed in six families orig-inating from Middle-Eastern countries, and discuss the genotype-phenotype correlation of theseven patients (two of them were brothers) affected by ARO.

2. Methods

2.1. Clinical investigations

Six unrelated families, four from Lebanon and two from Syria, with children fulfilling theclinical and radiological criteria of osteopetrosis were investigated (Fig. 1). Most of the

Fig. 1. Pedigrees of the six families included in this study. Patients affected with ARO are indicated by darkened

symbols.

190 N. Souraty et al. / European Journal of Medical Genetics 50 (2007) 188e199

patients, except the ones from Syria, underwent an extensive work-up including a thoroughclinical evaluation, radiological investigations (total body X-rays and brain MRI or CT scan)and routine blood tests (complete blood count, serum electrolytes, blood glucose levels, triglyc-erides, cholesterol, thyroid, liver and renal function tests, calcium serum levels, and alkalinephosphatase).

2.2. Molecular studies

Given that clinical presentation, radiological abnormalities and pedigree analysis were sug-gestive of autosomal recessive osteopetrosis in all ascertained families, a molecular study of theTCIRG1, ClCN7 and OSTM1 genes was undertaken. EDTA blood samples were collected forgenetic studies and DNA was extracted from lymphocytes by standard methods. Informed con-sent was obtained from each family, before blood sampling and DNA analyses. Altogether, 20DNA samples, including six affected individuals, were investigated.

2.2.1. PCR amplificationPrimers used for the amplification of all coding exons, introneexon boundaries and 50 and 30

UTRs were deduced from genomic sequences of the TCIRG1 (accession number AF033033),the ClCN7 (accession number AL031600) and the OSTM1 (accession number Z98200) genes aspreviously described [5,6,9,13]. Automated sequencing was performed directly on the purifiedPCR products.

2.2.2. RNA analysisTo evaluate the consequences of the OSTM1 splicing defect found in family 3, total RNA

was isolated from PBMC of the proband and cDNA synthesis was performed as previously de-scribed [13]. The OSTM1 cDNA encompassing exons 3, 4, 5 and 6 was amplified using a for-ward primer within exon 3 (50-CCT GAC CTG CTT TGA ACA TAA CC-30) and a reverseprimer within exon 6 (50-TGT CTT CCA CCA TTC ATT CAC G-30), located 29 bp downstreamof the stop codon in the 30 UTR, yielding a 469-bp fragment in normal cells. PCR conditionswere as previously described [4] with a 60 �C annealing temperature.

3. Results

3.1. Clinical findings

Of the six families included in the study, five are consanguineous. Clinical, radiological, andlaboratory findings of the seven patients (two of them were siblings) are summarized in Table 1.

3.1.1. Family 1The proband (Fig. 1, V.1) was the only son of consanguineous Lebanese parents. He was

born full-term after an uneventful pregnancy with normal foetal movements and normal deliv-ery. Birth weight, length and head circumference were within normal limits. At age 2 months,the parents’ attention was drawn by persistent infections and respiratory problems.

He was evaluated by us at the age of 6 months. His OFC was 43.3 cm (25th centile), hisweight 6200 g (3rd centile), and his length 65.5 cm (15th centile). He had a round face,wide open and tended anterior fontanel, prominent forehead, mild exophthalmia, a strabismus,a long philtrum, a retrognathism, and a hepatosplenomegaly. Eye examinations revealed

www.ncbi.nlm.nih.gov



Table 1

5 6

Syrian Syrian

þ �

M F

3.5 months 13 months �

48 cm NA 47

2700 NA 3200

NA NA 37

1 week 3 months 10 months 2 years

48 48.5 54 78

2400 2850 NA 9200

31 NA 47.3

þ þ þ þ� � þ �� NA �þ þ þ �� þ þ� mild þ þ

þ þþ þ þ þmild mild NA NA

þþþ þþþ þ þþ þ NA NA

þ þ NA NA

� þþ � þþ þ þ þþ þ NA þþ NA �

TCIRG1 TCIRG1

S474X S474X

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Clinical, radiological, laboratory and mutational findings

Family 1 2 3 4

Ethnic origin Lebanese Lebanese Lebanese Lebanese

Consanguinity þ þ þ þPatients’ general data

Gender M F M M

Age at death � 9 months 9 months 6 months

Birth history

Birth length (cm) 47 45.5 47 48

Birth weight (g) 3000 3000 2750 3100

Head circumference at birth (cm) 35 NA 33 NA

Clinical report

Age when seen at consultation 6 months 3 months 40 days 6 months 40 days

Length (cm) 65.5 54 3800 6000 45

Weight (g) 6200 3500 51 cm 57 <2800

Head circumference (cm) 43.3 37 37 43.8

Growth retardation þ þ � þ þMacrocephaly � � � þ �Hydrocephaly/large ventricules � � � � �Prominent forehead þ þ � þ þStrabismus þ � � � �Exophthalmia þ þ � þ �Optic atrophy þ � þ þHepatosplenomegaly þ þ � � þHypocalcaemia þ þ þ þ þAnaemia/thrombocytopenia þ þ þ � þþþElevated hepatic enzymes þ þ � � þElevated PA NA þ þ þ þMultiple fractures � þ � � þþOsteopetrosis þ þ þ þ þWide metaphysis � þ þ þ þBrain MRI abnormalities þ � � þ

Genetic analysis

Genes TCIRG1 TCIRG1 OSTM1 OSTM1

Mutations I235fs278X I235fs278X IVS5 þ 5 � exon skipping C12X


a bilateral optic atrophy. X-rays showed a diffuse increase in bone density with evidence of‘‘bone within bone’’ appearance, and sclerosis of the basis of the skull with increased densityof the orbits. Brain MRI revealed sclerosis and hypertrophy of the basis of the skull, stenosis ofthe optical channels and a mild bilateral fronto-temporal cortical atrophy. Laboratory examsrevealed a hypochromic anaemia and pancytopenia.

Now he is 2 years old and has a significant developmental delay.

3.1.2. Family 2The proband (Fig. 1, VI.2) was born full-term after an uneventful pregnancy with normal

foetal movements and normal delivery. Birth weight, length and head circumference werewithin normal limits. At age 3 months, she was evaluated because of growth delay. Her OFCwas 37 cm (25th centile), her weight 3500 g (10th centile), and her length 54 cm (10th centile).She had a prominent forehead, mild exophthalmia, blue sclerae, and hepatosplenomegaly(Fig. 2). Total body X-rays showed increased bone density with loss of cortico-medullary dif-ferentiation, thin ribs with a fracture in the first two, irregularly shaped metaphyses of the longbones, and bilateral proximal humeral and pubic fractures. MRI of the brain was unremarkable.She died at the age of 9 months after an episode of infection.

In the family history, an older sister of the proband died at age 7 months from the samedisease and eight nephews and nieces from both parent sides died most probably from thepathology.

Fig. 2. Photograph of patient VI.2 (family 2) at the age of 3 months showing the prominent forehead and mild

exophthalmia.


3.1.3. Family 3The proband (Fig. 1, V.1) was the only son of consanguineous Lebanese parents. He was

born at full-term after an uneventful pregnancy with normal foetal movements and normaldelivery. Birth weight, length and head circumference were within normal limits. The parents’attention was drawn at the age of 40 days to the child’s hypotonia and poor sucking capacity. Atexamination, the boy was pale and hypotonic. Length and weight were within normal limits. Hehad a triangular face, a prominent forehead, a microretrognathia, and slightly low set ears. Oph-thalmological examination revealed a bilateral optic atrophy. Biological examination showedthe presence of hypocalcaemia, anaemia, thrombocytopenia, positive anti-nuclear antibody(ANA) (present in his mother), and a low bone marrow megakaryocytic content. CT scan ofthe brain was normal. Total body X-rays showed increased bone density with loss of cortico-medullary differentiation, and irregularly shaped metaphyses of the long bones.

At age 3 months, weight was 4250 g (10th centile), length 53 cm (3rd centile), and OFC40.5 cm (50th centile). The baby still had poor sucking and important axial hypotonia. The ante-rior fontanel was bulging, and exophthalmia was apparent. There was no anaemia or thrombocy-topenia, but hypocalcaemia was still present. A brain MRI revealed the presence of diffuse corticosub-cortical atrophy, enlargement of the sub-arachnoid spaces, and thin optical channels (Fig. 3).

At age 4.5 months he presented episodes of seizures. At age 6 months, length was largelybelow the 3rd centile, while OFC was in the normal limits. He was deteriorating neurologically.He had poor eye contact, axial hypotonia, a bulging anterior fontanel, and exophthalmia. Hedied suddenly at age 9 months.

3.1.4. Family 4The proband (Fig. 1, IV.3) has already been described [9]. Briefly, he had a squared face,

mild exophthalmia, a long philtrum, marked hepatosplenomegaly, diffuse increase in bone

Fig. 3. MRI coronal T2 view of patient V.1 (family 3) at the age of 3 months showing the diffuse cortico sub-cortical

atrophy, the enlargement of the sub-arachnoid spaces, and the thin optical channels.


density with loss of cortico-medullary differentiation especially in skull bones, upper limbs,pelvis and femora, bilateral congenital hip sub-luxation, fractures of the upper left humerusand of the right femur (Fig. 4), and hypochromic anaemia and pancytopenia. Bone marrowanalysis showed a defect of central origin involving the megakaryocyte and granulocyte line-age. The boy died at age 5 months from diffuse bleeding.

Two years after the proband’s birth, the mother delivered twin babies, a boy and a girl. Clin-ical investigations showed that the boy (Fig. 1, IV.5) had the same disease. At 1 week of age, hisOFC was 31 cm (5th centile), his weight 2400 g (25th centile), and his length 42 cm (10thcentile). He had a prominent forehead, hepatosplenomegaly, increased bone density, widemetaphyses of the long bones, mild hypocalcaemia, elevated hepatic enzymes and alkalinephosphatase, and severe anaemia and thrombocytopenia. An MRI of the brain performed atage 2 months did not show specific abnormalities. At 3 months, he had severe growth retarda-tion with weight and length below the 3rd centile, and in addition to his latest exam, exophthal-mia and multiple fractures. He died at age 3.5 months from diffuse bleeding.

Fig. 4. (a)e(c) X-rays of patient IV.3 (family 4) showing a diffuse increase in bone density with loss of cortico-

medullary differentiation, bilateral congenital hip sub-luxation, and a fracture of the upper left humerus.


In the family history, an older sister died at 6 months of age and the family description sug-gests osteopetrosis as the most likely cause of death.

3.1.5. Family 5This family originates from Western Syria and the parents of the proband are first cousins.

Their first affected daughter (Fig. 1, IV.2) was born full-term after an uneventful pregnancywith normal foetal movements and normal delivery. Birth weight, length and head circumfer-ence were not recorded. At age 2 months, she was evaluated because of growth delay, fastgrowing head and strabismus. A diagnosis of osteopetrosis was suspected because of the aspectof increased bone density. At age 10 months, her length was 54 cm (<3rd centile). She had se-vere anaemia and thrombocytopenia and died at age 13 months. Another daughter (Fig. 1, IV.3)was born a year after, and died at age 8 months. According to the parents, she had the sameaffection and also had a severe growth delay with a relative macrocephaly. No other informa-tion is available.

3.1.6. Family 6This family denied any consanguinity but both parents originate from Western Syria. Their

affected daughter (Fig. 1, III.1) was born full-term after an uneventful pregnancy with normalfoetal movements and normal delivery by caesarean section. Birth weight, length and head cir-cumference were within normal limits. At age 2 months, she was evaluated because of growthdelay, exophthalmia and anaemia. The different analyses led to the diagnosis of osteopetrosis.At age 2 years, she was evaluated by us. Her OFC was 47.3 cm (35th centile), her weight9200 g (3rd centile), and her length 78 cm (5th centile). She had a prominent occiput, exoph-thalmia, a flat nasal bridge, a retrognathia, a short neck, and small teeth. Ophthalmological ex-amination revealed the presence of a bilateral optic atrophy, strabismus, and a horizontalnystagmus. Total body X-rays showed a generalized increase in bone density with loss of cor-tico-medullary differentiation, and a fracture in the right femur. Blood examination revealedsevere anaemia and thrombocytopenia.

3.2. Molecular findings

The exploration of the entire coding sequence of OSTM1, TCIRG1 and ClCN7 allowed theidentification of the causative mutation in all six families (Table 1).

No mutations were found in the ClCN7 gene, while the analysis of the TCIRG1 gene allowedus to identify two different types of mutations in two Lebanese families (families 1 and 2) andin two Syrian family (families 5 and 6). In the probands of both Lebanese families a homozy-gous deletion of a single nucleotide was found in exon 7 at nucleotide 4668 of the TCIRG1 gene(Fig. 5A). The mutation leads to a frameshift starting at codon 235 and premature terminationafter 43 extraneous amino acids (I235fsX43). However, there was no evidence of relatedness inthese families.

In the proband of the Syrian family 6 a nonsense mutation in exon 12 (8788 C to A change, thatcauses a premature stop codon S474X), in homozygous state, was found (Fig. 5B). As no DNAwas available from the two affected daughters of the Syrian family 5, the molecular diagnosis wascarried out on the parents’ DNA. Both carried at the heterozygous state the same TCIRG1 muta-tion showed by the proband family 6 (III.1); again the two families were not known to be related.

The molecular analysis of the OSTM1 gene was performed in the probands of the Lebanesefamilies 3 and 4, on the basis of the severity and the early presentation of the pathology.


The proband of family 3 was homozygous for the G to C change at position þ5 of the donorsplice site of intron 5 (G105801C, IVS5 þ 5). A nucleotide change at this position has previ-ously been described [4], but in the present patient a different nucleotide change (G10581A)was found. To study the effect of this mutation on the OSTM1 transcript, an RT-PCR assayspanning the exon 5 was designed. RT-PCR on total RNA isolated from PBMC resulted inthe amplification of two bands (355 bp and 303 bp respectively); both smaller than the469 bp band which is detected in the normal control (Fig. 6). Direct sequencing of the smallerbands revealed that all lacked exon 5.

The sequence of the heavier band showed the presence of an additional 52 bp of intron 5,resulting from the use of a cryptic acceptor splice site, while the 303 bp form corresponded ex-actly to the skipping of the entire exon 5.

The proband of family 4, as previously reported [9], showed a single point mutation in exon1, leading to a nonsense mutation (80433T / A, C12X) in both the affected brothers.

4. Discussion

The exact frequency of ARO in the Middle-East remains unknown, although data from sev-eral countries within the Arabian Peninsula have indicated that it is relatively common [14]. InKuwait, Abdel-Al et al. [1] diagnosed 18 Arab children with ARO over a 5-year period in var-ious hospitals. In a survey of 2000 Palestinian Arab families, Zlotogora [15] diagnosed orstrongly suspected 601 individuals with autosomal recessive diseases, and found a high prev-alence of recessive osteopetrosis. Al-Rasheed et al. [3] observed, over a 10-year period, 28Arab children with autosomal recessive osteopetrosis in two hospitals in Riyadh (Saudi Arabia).Finally, in the United Arab Emirates, Al-Gazali et al. [2] reported in 2003 an incidence rate ofARO at 0.26/10,000 births. In Lebanon, a rapid assessment of children born in 2004 conductedwith all the reference genetic laboratories in Lebanon revealed four confirmed cases. In 2004,about 74,000 births were registered. Thus, a gross ARO incidence rate may be estimated at

Fig. 5. Chromatograms showing the mutations in the TCIRG1 gene in families 1, 2 and 5, 6.


about 5.4/100,000 births. No data are available from Syria, but most likely we can consider thesame incidence rate for ARO as that in Lebanon.

Overall, the phenotype in the patients of our series was in accordance with the literature. Fail-ure to thrive, prominent forehead, exophthalmia, optic atrophy, hepatosplenomegaly, neurolog-ical manifestations and severe neuro-degeneration, anaemia, thrombocytopenia, hypocalcaemia,elevated hepatic enzymes and acid phosphatase, and an early fatal outcome (5 out of 7) werecommon. Macrocephaly, strabismus, and brain malformations were relatively less common.The latter did not permit a tentative treatment by bone marrow transplantation. The moleculardefect was identified in OSTM1 or TCIRG1 genes in the tested families (Table 1).

The analysis of the TCIRG1 gene showed an abnormality in this gene in four families, twofrom Lebanon and two from Syria. Interestingly, although both families denied any relation,both Lebanese families carried the same mutation (I235fs278X); likewise both Syrian probandsshared the same mutation (S474X), albeit different from the one found in Lebanese families.This suggests that both mutations derived from an ancestral mutation which was geographicallyrestricted. This hypothesis is strengthened by the fact that a previously described Lebanese pa-tient bore the same homozygous mutation (patient 55, Table 1, in ref. [13]). The Syrian muta-tion has previously been described (patient 49, Table 1, in ref. [13]). Looking into intragenicpolymorphisms, we found only one polymorphism in all the patients. This result does not

Fig. 6. Chromatogram and RT-PCR of patient V.1, family 3. (A) Chromatogram showing a homozygous G to C mutation

at position þ5 of the donor splice site in intron 5 of the OSTM1 gene. (B) RT-PCR amplification spanning exon 5 of the

OSTM1 gene. M, markers (1-kb ladder); P, patient V.1 family 3; WT, unrelated wild-type control; C,- PCR mix control.

Sizes of the PCR products and corresponding results of the skipping of exon 5 are marked.


support any hypothesis of founder effect since the haplotypes are identical in all the familiesprobably due to close populations.

The remaining two families had mutations in the OSTM1 gene. These Lebanese patientswith the OSTM1 mutations were more severely affected than the patients with mutations inthe TCIRG1 gene. They presented with severe bone sclerosis, growth failure, anaemia, throm-bocytopenia, visual impairment with optic atrophy, and severe primary central nervous systeminvolvement leading to a very poor prognosis. Brain lesions were apparent after the 1st monthof life. To date, a total of four distinct DNA mutations (three already reported and the new onefound in proband of family 3) have been identified in the OSTM1 gene. All the describedmutations were null mutations and were associated with similar clinical features and the most se-vere prognosis among ARO patients, probably due to central nervous system involvement [9]. Theonly discrepancy found was haematological in the proband of family 3. This boy was bornwith a major anaemia and thrombocytopenia but these features regressed spontaneouslywithin 1 month. It is suggested that these haematological findings may have been linked tothe ANA positive status of the boy, inherited from a maternal undiagnosed lupus ratherthan to the ARO. As ANA antibodies regressed, so did other associated haematological signs.

TCIRG1-dependent ARO patients have a severe homogeneous bone phenotype, due to a de-fect in bone resorption, and their nervous system involvement (hydrocephalus and cranial nervedefects) is secondary to the compression due to skull deformities and the haematological defectcan be rescued by HLA-matched haematopoietic stem cells transplantation (HSCT). On theother hand, there is increasing evidence that patients with recessive ClCN7 and OSTM1 [9] mu-tations, besides the bone manifestations, show a primary severe neurological defect (retinopa-thy and progressive cortical atrophy in addition to secondary neural defects) due to lysosomalstorage disease [16]. It has been reported that ClC-7 and OSTM1 proteins co-localize in lateendosomes and lysosomes of various cell types, as well as in the ruffled border of bone-resorb-ing osteoclasts and that ClC-7 and OSTM1 form a molecular complex in which OSTM1 couldbe a beta-subunit of ClC-7 and that mutations in both genes lead to lysosomal storage and neu-rodegeneration in addition to osteopetrosis. These finding could explain why the prognosis ofboth of these forms seems particularly poor due to the co-existence of this neurological defectwhich leads to death in spite of HSCT, but did not explain the origin of the neurologicalinvolvement.

In conclusion, we report the clinical and the molecular study of six families originating fromLebanon and Syria affected by ARO. From our study, we noticed that the clinical features andthe evolution varied within the same family, and between different families having the samemutation. Most probably, this observation may be due to the different environmental conditionsof the families. Nevertheless, we anticipate that other reports of similar cases in the Middle-East will enable further delineation of the clinical spectrum of ARO especially the one withthe OSTM1 gene mutation, help to find if other genetic and/or environmental factors playa role in modifying the severity of ARO, and confirm if we are dealing or not with a foundingeffect.

Acknowledgements

We are grateful to the families for their collaboration. This work was supported by grantsfrom the Saint Joseph University, and from the N.o.b.e.l. Project of Fondazione CARIPLOto Anna Villa.


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Documents

Molecular study of six families originating from the Middle-East and presenting with autosomal recessive osteopetrosis