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� 2008 Wiley-Liss, Inc. American Journal of Medical Genetics Part A 146A:1865–1870 (2008)

Clinical Report

A Multiplex Family With Possible MetaphysealSpahr-Type Dysplasia and Exclusion of RMRP and

COL10A1 as Candidate Genes

Andre Megarbane,1* Eliane Chouery,1 and Ismat Ghanem2

1Unite de Genetique Medicale, Faculte de Medecine, Universite Saint Joseph, Beirut, Lebanon2Service d’Orthopedie, Hotel-Dieu de France, Beirut, Lebanon

Received 29 January 2008; Accepted 9 April 2008

We report a consanguineous Lebanese family where sixindividuals had disproportionate short stature, short limbs,and bilateral genu varum that were apparent after 2 years ofage. Major radiographic features in infancy included flared,cupped, and ragged metaphyses of all long bones, delayedmaturation of the scaphoid, trapezoid, and trapezium bones,wide and slightly irregular ribs, short femoral necks, flatfemoral heads, and irregularity of the iliac crest. Radiographsof an affected adult showed diminished metaphysealchanges and slightly short femoral necks, when compared

to the younger patients. We suggest that this family isaffected with the Spahr type of metaphyseal chondrodys-plasia. Sequencing of RMRP, and a haplotype analysis usinghighly informative markers around the COL10A1 excludedboth genes from being pathogenic in this family. We areaware of only two previous reports of families with clinicalfeatures similar to the Spahr type of metaphyseal chondro-dysplasia. � 2008 Wiley-Liss, Inc.

Key words: dysplasia; metaphysis; genu varum; short stature

How to cite this article: Megarbane A, Chouery E, Ghanem I. 2008. A multiplex family with possiblemetaphyseal Spahr-type dysplasia and exclusion of RMRP and COL10A1 as candidate genes.

Am J Med Genet Part A 146A:1865–1870.

INTRODUCTION

The metaphyseal dysplasias are a clinically hetero-geneous group of disorders characterized by meta-physeal irregularities and, usually, with short stature.They can be distinguished by clinical and radio-graphic findings and genetic testing. We recentlyevaluated a consanguineous Lebanese family wheresix individuals of different ages presented withpostnatal disproportionate short stature, short limbs,genu varum, and metaphyseal abnormalities. Clin-ical analysis and candidate gene testing were used toevaluate the family.

MATERIALS AND METHODS

Members of this kindred (Fig. 1) originate fromBeirut, capital of Lebanon, and belong to the SunniMuslim community. Three patients have beenexamined. The patients underwent an extensivework-up including a thorough clinical evaluation,total body X-rays, and routine blood tests (completeblood count, serum electrolytes, blood glucoselevels, white blood cells enzyme assays screening,triglycerides, cholesterol, thyroid, liver and renalfunction tests, PTH and phosphatase alkaline levels).

Peripheral blood was obtained after informedconsent was signed from individuals IV-4 and IV-5and their three sibs (Fig. 1). Genomic DNA wasextracted from leucocytes by standard salt-precip-itation methods.

Genomic and cDNA sequences of RMRP(AF334829, M29916.1) were obtained from NCBIdatabase. The entire transcribed region and�500 bppromoter region were amplified by polymerasechain reaction from genomic DNA, and werescreened for mutations through direct sequencing.Primers sequences were chosen according to Naka-shima et al. [2003]. Mutations were numbered fromthe putative translational start site of M29916.1.

PCR was performed using Taq DNA polymerase(Invitrogen Life Technologies, Carlsbad, CA). Theamplification conditions for each PCR were 958C for5 min, followed by 35 cycles of 958C for 60 sec, 508C

*Correspondence to: Andre Megarbane, M.D., Ph.D., Unite deGenetique Medicale, Faculte de Medecine, Universite Saint Joseph deBeyrouth, 42, rue de Grenelle, 75007 Paris, France.E-mail: [email protected]

DOI 10.1002/ajmg.a.32390

for 60 sec and 728C for 60 sec, with a final extensionof 10 min at 728C. The amplified PCR fragments wereanalyzed on 0.8% agarose gels.

PCR products from genomic DNA were purifiedusing the Ultra PCR Clean-Up Kit (Abgene, Surrey,UK), and the products were sequenced for bothstrands using the BigDye1 Terminator v1.1 CycleSequencing Kit (Applied Biosystems, Foster City,CA). A total volume of 7.5 ml sequencing reactionsincluding 2 ml Big Dye, 10–23 ng of purified DNAtemplate, and 1.6 pmol of either forward or reverseprimerswas incubated for 25 cycles at 968C for 10 sec,508C for 5 sec, and 608C for 4 min. Sequencereactions were purified on sephadex G50 (Amer-sham Pharmacia Biotech, Foster City, CA). Thelabeled products were dissolved in HiDi formamideprior to loading into an ABI 3100 system. The sameprimers as for the DNA amplification were used forsequencing. Electrophoregrams were analyzedusing Sequence Analysis Software version 5.2(Applied Biosystems) and compared to referencesequences using ChromasPro version 1.22 (Techne-lysium, Queensland, Australia).

Haplotype analysis was performed using eighthighly informative markers (D6S302, D6S416,D6S418, D6S432, D6S304, D6S262, D6S292, andD6S308) flanking the COL10A1 gene. The positionof the gene and markers were determined by UCSCHuman Genome Browser (http://www.genome.ucsc.edu). Genotyping was performed as describedelsewhere [Delague et al., 2000].

CLINICAL REPORT

The proband (Fig. 1: V-5) was born at term to a 27-year-old mother and the 30-year-old father. Her birthweight was 3,500 g (�50th centile), and length 48 cm(�25th centile). Head circumference (OFC) was notrecorded but considered normal by the parents. At

the age of 4 months, the parents noticed the presenceof short limbs and genu varum. Developmentalmilestones were within the normal limits.

This girl was seen at the age of 7–8/12 years, whenshe was referred for familial short stature. She hadnormal school performance. Her height was 113 cm(�3rd centile), weight 23 kg (�50th centile), head

FIG. 1. Pedigree of the family. Affected patients are indicated by darkened symbols.

FIG. 2. Photograph of Patient V-5. Note the short stature and the genu varum.[Color figure can be viewed in the online issue, which is available atwww.interscience.wiley.com.]

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circumference 52 cm (>50th to <75th centile) andchest circumference 60 cm (50th centile). She had anormal facial appearance and normal joint mobilityalthough she complained of painful knees occasion-ally. Apparent shortening of the limbs, bilateralclinodactyly of the fifth fingers and genu varumwere observed (Fig. 2). Her upper arm length was23 cm (�3rd centile), forearm length was 18 cm(�5th centile), hand length was 11.4 cm (<3rdcentile), upper leg length was 24 cm (�3rd centile),and lower leg length was 26 cm (�25th centile).

Radiographic examination disclosed cupping,fraying, and sclerosis of the metaphyses of all longbones with some widened physeal plates withsclerosis on both sides of the epiphyseal plates insome regions. Delayed maturation of the scaphoids,trapezoids, and trapezia, wide and slightly irregularribs, coxa vara, flat femoral heads, and short femoralnecks were also noted. Her bone age was 7–10/12 years (Fig. 3A–D). Ophthalmological examina-

tion and echocardiography were normal. Laboratoryresults were all within normal limits. Karyotypeperformed at the 550–650 band level according tostandard methods was normal 46,XX.

Individual V-6 was born 6 years after her affectedsister. Her birth weight was 3,200 g (>25th to <50thcentile), and length 49 cm (�25th centile). She was1 year old at the time of the examination. Her lengthwas 73 cm (>25th to <50th centile), her weight was9,200 g (�50th centile), and her OFC was 45.4 cm(�50th centile). She had rhizomelic and acromelicshortenings.

A skeletal survey showed similar abnormalities tothose in individual V-5 with, in addition, iliac crestirregularity, and slightly irregular ribs at the costoen-chondral junction (Fig. 4A and B). Bone age was6 months.

The mother of these sisters (IV-4) had similarclinical features. She had a genu varum, which hadbeen operated at the age of 12 years, and short limbs.

FIG. 3. Radiographs of Patient V-5 at age 7–8/12 years showing. A: Irregular sclerotic metaphyses, coxa vara, slightly flat femoral heads, and short femoral necks.B: Cupping and flaring of metaphyses, and sclerosis of the epiphyseal side of the growth plate.C: Hand radiographs showing irregular metaphyses, wide physeal plates,and delayed maturation of the scaphoids, trapezoids, and trapezia. D: Chest and spine radiographs showing the wide and slightly irregular ribs.

METAPHYSEAL SPAHR-TYPE DYSPLASIA 1867


At examination, her age was 35 years. Her height was140 cm (<3rd centile), upper arm length was 29 cm(�25th centile), forearm length was 23.8 cm (>50thto <75th centile), hand length was 16 cm (<3rdcentile), upper leg length was 38 cm (�15th centile),and lower leg length was 36 cm (10th centile).Radiographs showed mildly shallow acetabulae,slightly short femoral necks, but no evidence ofprevious metaphyseal changes (Fig. 5).

Three other individuals in the family were mostprobably affected as well (Fig. 1). They all had a shortstature and a genu varum. No radiographs wereavailable for further analysis.

Molecular Findings

Sequencing of RMRP for this family revealed thepresence of common polymorphisms (g.�56A!G,g.�48C!A, g.�6G!A, g.156G!C, andg.177C!T) already reported [Thiel et al., 2007]. Inaddition, two novel sequence variations around thetranscribed region of the RMRP gene: g.�231insG

and g.281G!T, were identified in a homozygousstate in all the tested members of the family. No othermutations in RMRP were identified. Haplotypeanalysis of COL10A1 flanking markers showed thatPatients V-5 and V-6 and their nonaffected sister V-4shared the same haplotypes (Fig. 6).

DISCUSSION

This family presents an apparently autosomalrecessive chondrodysplasia with postnatal dispro-portionate short stature, genu varum, and osseous

FIG. 4. Radiographs of Patient V-6 at age 1 year. A: Pelvic images showingmetaphyseal and iliac crest irregularity. B: Hand radiographs showing cuppingand flaring of the metaphyses and wide physeal plates.

FIG. 5. Radiograph of Patient 3 at age 35, showing mildly shortened femoralnecks.

FIG. 6. Haplotypes for eight microsatellite markers flanking the COL10A1gene. Genotyped markers are shown to the left, and individuals’ allele numbersfor each marker are given next to the bar.



malformations that consist mainly of early metaphy-seal changes with apparent regression. Because oneof the most striking features was the metaphysealdysplasia, differential diagnoses were thus consid-ered with skeletal dysplasias featuring predominantmetaphyseal involvement.

The overall pattern of abnormalities seen in thepatients described herein is distinct from themetaphyseal dysplasia Jansen type (OMIM 156400),the Vaandrager–Pina syndrome [Kozlowski andSikorska, 1970] (OMIM 250300), the metaphysealdysplasia Shwachman type [Makitie et al., 2004](OMIM 260400), the entity described by Castriota-Scanderbeg et al. [2001], and the child described byHoeffel et al. [1987]. The anadysplasia type I and II[Maroteaux et al., 1991; Le Merrer and Maroteaux,1998], and the entity reported by Wiedemann andSpranger [1970] were ruled out as possible diagnosesby the fact that there is only a mild decrease in finallength or a normal stature in those syndromes, and adifferent mode of inheritance.

The cartilage-hair-hypoplasia dysplasia (CHH)(OMIM 250250) is an autosomal recessive chondro-dysplasia characterized by metaphyseal dysplasia,short stature, and often, fine and sparse hair,transient macrocytic anemia, and immunodefi-ciency. It is differentiated from the patients reportedhere by the fact that in the latter the length is normalat birth, the hands are not pudgy, and there are noround epiphyses. Nevertheless, as carpal abnormal-ities had been reported in CHH and found in thepresent family, and because of the marked clinicalheterogeneity found in those with RMRP mutations[Ridanpaa et al., 2001], we sequenced this gene butno mutations were found.

The Schmid metaphyseal dysplasia (OMIM156500) is a well known type X collagen chondro-dysplasia. Its manifestations are similar to the presentfamily including anormal appearance at birth, a shortadult stature, bowed legs, and a coxa vara. Never-theless, it is an unlikely diagnosis for the presentfamily because it has an autosomal dominant modeof inheritance. In addition, in the Schmid type,metaphyseal irregularity is most pronounced at theknees, epiphyses are wide, and mild platyspondylyin infancy and early childhood, and mild handinvolvement such as shortening of tubular bonesand metaphyseal cupping of metacarpals andproximal phalanges can be seen [Lachman et al.,1988; Nishimura et al., 2001; Elliot et al., 2005].Haplotype analysis excluded linkage of COL10A1 tothis phenotype.

Spahr and Spahr-Hartmann [1961] described aconsanguineous family with an autosomal recessivemetaphyseal dysplasia very similar to Schmid meta-physeal dysplasia. Farag and Teebi [1990] reportedfour affected sibs who probably had the samemetaphyseal dysplasia. Unfortunately, in the Faragand Teebi report, the clinical history was not well

documented and the radiological photos were notinsufficient for further comparison. Similaritiesbetween the Spahr and Spahr–Hartmann reportand the patients reported here include a normalfacial appearance, a short stature that appearedprogressively after the age of 2 years with a finalheight at around 140 cm, a mild form of metaphysealdysplasia affecting the bones and that apparentlyimproved with age, no metaphyseal irregularity ofthe fingers, and normal joint mobility. The pheno-type is variable as the patients reported here hadadditional features such as carpal bone hypoplasia,abnormal ribs, slightly short femoral necks, and iliaccrest irregularity (patient V-6), but not the abnormalgait, lordosis, osteoporosis and large articulationsseen in the patients reported by Spahr and Spahr-Hartmann [1961]. Other similar reports and identi-fication of the gene will further characterize theclinical spectrum of this disorder and clarify itsetiology.

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