Hum Genet (1998) 103 : 60–64 © Springer-Verlag 1998

Abstract Progressive pseudorheumatoid dysplasia (PPD),MIM 208230, is an autosomal-recessive disorder, clinicallycharacterized by spondyloepiphyseal dysplasia and pro-gressive arthropathy. Linkage analysis of three families ofdifferent geographic and ethnic origin, including 11 affect-ed individuals, showed strong evidence for localization of agene for progressive pseudorheumatoid dysplasia to chro-mosome 6q with a maximum two-point lod score forD6S1647 of 8.34 at θ=0. Analysis of regions of homozy-gosity placed the gene in a 3-cM interval between D6S1594and D6S432. No significant shared haplotype was found formarkers of the linked interval in the three families ana-lyzed. Five genes encoding collagen and one encoding aspecific procollagen-processing enzyme that map near thisinterval represent good candidates for the PPD gene.

J. Fischer (✉) · S. Pavek · C. Vandiedonck · J.-F. Prud’hommeCNRS URA 1922, Généthon, 1, rue de l’Internationale, F-91000 Evry, Francee-mail: fischer@genethon.fr, Tel.: +33-147692800, Fax: +33-160778698

J. A. UrtizbereaInstitut de Myologie, Hôpital de la Salpêtrière, F-75013 Paris, France

S. Saker · Y. AlkatipTischreen Hospital, Damascus, Syria

J. Weissenbach · T. BrulsGenoscope CNS, BP191, F-91006 Evry, France

Introduction

Spondyloepiphyseal dysplasia (SED) represents a hetero-geneous group of skeletal disorders that are characterizedby various degrees of platyspondyly and deformities of thevertebrae and by abnormal shape and structure of the epi-physes. Progressive pseudorheumatoid dysplasia (PPD;MIM 208230) (OMIM 1997), also referred to as spon-dyloepiphyseal dysplasia tarda with progressive arthro-

ORIGINAL INVESTIGATION

Judith Fischer · Jon Andoni Urtizberea Sylvana Pavek · Claire Vandiedonck · Thomas BrulsSafa Saker · Yhia Alkatip · Jean-François Prud’hommeJean Weissenbach

Genetic linkage of progressive pseudorheumatoid dysplasia to a 3-cM interval of chromosome 6q22

Received: 12 February 1998 / Accepted: 10 March 1998

pathy and progressive pseudorheumatoid arthritis of child-hood, has been described as a specific autosomal-recessivesubtype of SED (Wynne-Davies et al. 1982; Spranger et al.1983a, b; Teebi and Al-Awadiet al. 1986). PPD differs fromthe other spondyloepiphyseal tarda syndromes in the age ofonset and the severity of the disease. The disorder resem-bles juvenile rheumatoid arthritis in that joint stiffness andpains begin in childhood and progressively involve hips,knees, wrists and fingers. Conversely, the presence of pla-tyspondyly in the absence of soft tissue involvement and bi-ological inflammation is a distinctive feature. Secondarymuscle weakness is sometimes present. Reduced mobilityof multiple joints, contractures and growth retardation arecommon and may result in severe disability. Radiologically,the bone changes are clearly dysplastic. Vertebral bodiesare usually flattened with anterior ossification defects. El-bows, knees and hips show a loss of the joint spaces with ir-regular articular surface and adjacent osteoporosis. Epiphy-ses of interphalangeal joints are also enlarged.

The prevalence in the general population is estimated tobe 1 per 100,000. It is particularly frequent in the Mediter-ranean countries (Al-Awadi et al. 1984; El-Shanti et al.1997a), where high consanguinity is common. To identifythe gene defect in PPD, we applied the method of homozy-gosity mapping (Lander and Botsteinet al. 1987) to largeconsanguineous families.

Materials and methods

Blood samples for DNA extraction were collected from 23 membersof the three families. Standard procedures for DNA extraction fromwhole blood were used.

Fluorescent genomic mapping

Fluorescent polymerase chain reaction (PCR) primers (Dib et al.1996) for genome-wide screening, spaced at an average distance of 15cM over the whole genome, were used. Microsatellite primers werelabeled with either 6-Fam, Hex or Tet phosphoramides. PCRs wereperformed in 192-well microtiter plates (Falcon) in a final volume of15 µl containing 30 µg/ml DNA, 0.16 mM dNTPs, 1 × NBL buffer,

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Fig. 1 Three consanguineouspedigrees, in which the parentsare either first cousins (familyA) or first cousins once removed(families B and C), are shown.The disease-associatedhaplotypes are boxedand thecommon linked interval is inbold. Affected individuals arerepresented by black symbolsand nonaffected family membersby open symbols. Individualswho were not available are notshown

0.33 µM of each primer and 0.03 IU Taqpolymerase. After a hot-startprocedure (enzyme added at 94°C after denaturation for 5 min at96°C) (Vignal et al. 1993) 26 cycles (40 s 94°C, 30 s 55°C) were per-formed. The rest of the procedure was similar to that described byReed et al. (1994).

Nonfluorescent genotyping

Genotyping for the other microsatellite markers using unlabeledprimers was carried out for the region of interest. PCRs were per-formed in 96-well microtiter plates (Falcon) in a final volume of 50 µlcontaining 40 ng genomic DNA, 125 µM dNTPs, 1 µM of each prim-er and 0.05 IU Taqpolymerase. The amplification products from thesame DNA sample generated with different primer sets were pooledand allowed to comigrate in a single lane of a 6% polyacrylamide gel.After transfer to Hybond N+ (Amersham) membranes, hybridizationwas performed with either peroxidase-labeled PCR primers, or apoly(AC) probe (Vignal et al. 1993).

Linkage analysis

Linkage analysis was performed using the LINKAGE 5.1 program as-suming autosomal-recessive inheritance, a penetrance of 95% and adisease frequency of 1 per 100,000. Pairwise lod scores were calculat-ed with the MLINK program (Lathrop et al. 1985). Consanguineousloops were incorporated in the pedigree files. Allelic frequencies usedwere based on unrelated individuals from eight CEPH families (Dib etal. 1996).

Results

Clinical data

Three consanguineous families of different geographic andethnic origin in which PPD had been diagnosed were stud-ied. Of the 23 family members examined by the same phy-sician (J.-A. U.), 11 individuals were affected and 12 non-affected. The first family originates from Syria andcomprises four affected sisters and four healthy brothersborn to two healthy first-cousin parents (Fig. 1, family A).

The first symptoms occurred at about the age of 3 years andincluded difficulties in walking and progressive stiffeningof joints. Growth retardation, enlarged finger joints, andtypical radiological findings were found on examination ofthe patients. The second family originates from Senegal butlives in France. Five out of seven siblings are affected withtypical symptoms of PPD (Fig. 1, family B). The parentsare first cousins once removed and are healthy. Clinical fea-tures are less severe than in family 1, as joint deformitiesand growth retardation are milder. In the third family, whichis of Moroccan origin, the healthy parents are also firstcousins once removed. There are three children, two ofwhom are affected (Fig. 1, family C). The onset of the dis-ease was at age 4 years in the oldest sister who becamewheelchair bound when she was 12 years old owing to per-sistent muscle weakness and joint deformities. The young-er sister who had similar symptoms and radiological find-ings also had moderate mental retardation and lostambulation when she was 11 years old.

Linkage analysis

Since there was no candidate genomic region responsiblefor PPD and no significant clinical heterogeneity in thethree families, a genome-wide scan of family A was per-formed. A 25-cM region encompassing each marker foundto be homozygous was tested with additional markers toconfirm or exclude a larger homozygous region.

After data for 100 markers had been analyzed, evidencefor linkage to chromosome 6q22 was obtained. This local-ization was confirmed using the two additional families (Band C). The pedigrees and haplotypes of markers from thecentral part of this region are shown in Fig. 1. The two larg-est families, A and B, each showed a lod score above 3. Themaximum pairwise lod score for the three families formarker D6S1647 on chromosome 6q22 was 8.34 for θ=0(Table 1).

The smallest cosegregating region in the three informa-tive families was established using haplotype analysis (Fig. 2). Recombination events were defined by loss of homozygosity or recombination in affected patients. Thehaplotypes were constructed assuming the most parsimoni-ous linkage phase. The largest homozygous regions were21 cM for family B, 18 cM for family C, and 7 cM for fam-ily A. The telomeric limit is defined by marker D6S1706 infamily A and the centromeric limit by marker D6S1635 infamily B. The interval between the two markers is approxi-mately 3 cM.

Discussion

The analysis of three families from different geographicand ethnic backgrounds showed positive linkage to locus6q22, providing strong evidence that in our PPD popula-tion, in spite of the different degrees of clinical severity, asingle gene is responsible for the disease.

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Table 1 Combined pairwise lod scores for progressive pseudo-rheumatoid dysplasia for three families (A, B, C) for markers in thecommon interval

Lod scores at recombination fraction θ

0.0 0.01 0.05 0.1 0.2 0.3 0.4

D6S1635 –0.63 1.91 2.25 2.10 1.52 0.86 0.28

D6S1594 7.65 7.50 6.89 6.12 4.51 2.86 1.22

D6S404 7.07 6.93 6.34 5.60 4.09 2.58 1.14

D6S416 2.65 2.57 2.31 1.99 1.38 0.82 0.34

D6S1647 8.34 8.13 7.48 6.64 4.91 3.15 1.42

D6S302 8.13 7.97 7.32 6.50 4.79 3.05 1.35

D6S432 5.92 5.79 5.27 4.61 3.27 1.96 0.75

D6S1706 –0.42 2.12 2.45 2.29 1.66 0.94 0.29

The smallest cosegregating region of 3 cM betweenmarkers D6S1635 and D6S1706 was defined by recombi-nation events or loss of homozygosity in these families. Nocommon haplotype was observed, but the existence of asmaller region with shared haplotypes cannot be excluded.

Over 150 distinct types of chondrodysplasias with eightmajor subtypes have been described in the literature(Spranger et al. 1992; Horton and Hechtet al. 1993). Theclassification is based on various criteria such as clinical,radiological and genetic features. A large number of tissue-

specific macromolecules are present in connective tissue.Most forms of chondrodysplasia are caused by mutations inthe genes for structural proteins of connective tissue andmany of these are in the genes for collagen. Frequently, dis-eases involving defects in structural proteins are inheritedas autosomal-dominant traits. Chondrodysplasias are rarelydue to mutations in genes for nonstructural proteins. Never-theless there are a few examples of mutations in genes forprocollagen-processing enzymes, such a lysyl hydroxylaseand prolyl hydroxylase, that are inherited in a autosomal-

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Fig. 2 Homozygosity regions(double-headed arrows) in thethree families and limits of thecommon interval (shaded). Thecentromeric limit (qCEN) is defined by marker D6S1635 infamily B and the telomeric limit(qTEL) by marker D6S1706 infamily A. The distances betweenmarkers are indicated in centim-organs on the left

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recessive manner. In Stickler syndrome type 2, a glycine toarginine mutation in the alpha-2 chain of type XIprocollagen causes an autosomal-recessive form of the dis-order, which is more severe than the autosomal-dominantvariant of this locus (Vikkula et al. 1995).

At least 20 different collagens have been identified indifferent tissues (Kuivaniemi et al. 1997). These are subdi-vided into two major groups: fibrillar and nonfibrillar. Fivedifferent collagens are found in the neighborhood of thelinked region on chromosome 6q: four of these code for thealpha-1 chains of collagen types IX, X, XII and XIX, andone codes for the alpha-2 chain of type XI. The fibrillartype XI collagen is an alpha-heterotrimer, wrapped in a tri-ple-helical conformation by glycine. Collagens of types IX,XII and XIX are fibril associated, with interrupted triple he-lices, and type X collagen is a member of the network-forming collagen group.

The involvement of collagen in the mechanism of patho-genesis of PPD seems likely. Two genes mapping to 6q22were suspected to be potential candidate genes for PPD:these were the COL10A1 gene and the gene encoding pro-lyl endopeptidase (PREP), a procollagen-processing en-zyme. Recently El-Shanti et al. (1997b) reported a localiza-tion for PPD to the long arm of chromosome 6 andexcluded the COL10A1 gene as cause of the disease. Thepositions of the COL10A1 gene and the PREP gene wereconfirmed in our laboratory using the Genebridge 4 panelof radiation hybrids (Gyapay et al. 1996); both genes werefound to be outside of the candidate region for PPD.

Acknowledgements We wish to thank the family members for theirparticipation and Louis Viollet and Thémar-Noël for identifying fam-ilies B and C. We are especially grateful to Susan Cure for help inwriting this manuscript and to Gabor Gyapay for providing informa-tion on the RH data. This study was supported by the AssociationFrançaise contre les Myopathies (AFM). We wish also to thank Rob-ert Manaranche and Gérard Peirano for supporting our work.

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