American Journal of Medical Genetics 37:272-276 (1990)

Spondyloepiphyseal Dysplasia, Mild Autosomal Dominant Type Is not Due to Primary Defects of Type I1 Collagen Ilse J. Anderson, Petros Tsipouras, Carron Scher, Raj S. Ramesar, Robert W. Martell, and Peter Beighton Department of Pediatrics, University of Connecticut Health Center, Farmington, Connecticut (I.J.A., P.T.); Provincial Laboratory for Tissue Immunology (R.W.M.) and Department of H u m a n Genetics, University of Cape Town (C.S., R.S.R., P.B.), Cape Town, South Africa

A mild autosomal dominantform of spondyloepiphyseal dysplasia (SED)is present in several generations of a South African family of English stock. This phenotype differs from that of any other previously described. Although type I1 collagen defects have been found in some families with SED congenita, the phenotype in our family showed discordant segregation with COL2Al gene associated restriction fragment length polymorphisms (RFLPs), the markers for the structural locus of type I1 collagen. It is evident that the SED group of disorders is heterogeneous.

KEY WORDS: COLBAI, heterogeneity, phenotype INTRODUCTION Spondyloepiphyseal dysplasia (SED) is a heterogeneous disorder in which stunted growth and premature degenerative arthropathy of the hip joints are major manifestations. With the aid of histopathological, biochemical, and molecular techniques, abnormalities of type I1 collagen and the gene coding for it (COLBAl) have been detected in some individuals with the autosoma1 dominant form of SED congenita [Horton et al., 1985; Murray et al., 1989; Lee et al., 19891. Linkage analysis in a 4 generation family with SED congenita using COL2Al gene associated restriction fragment length polymorphism (RFLPs) as markers established genetic linkage of the phenotype to the molecular markers [Anderson e t al., 19901. We performed genetic linkage analysis in 11individReceived for publication November 20, 1989; revision received February 15, 1990. Address reprint requests to Professor P Beighton, Department of Human Genetics, University of Cape Town Medical School, Observatory 7925.

o 1990 Wiley-Liss, Inc.

uals in 4 generations of a South African family of British stock (7 of whom are affected), in which short stature and severe premature degenerative arthropathy was transmitted a s a n autosomal dominant trait. The radiographic changes did not correspond exactly to those of any established skeletal dysplasia but the disorder could best be regarded a s a mild form of SED. Here we describe the new phenotype and show discordant segregation of the phenotype to the COL2Al gene associated RFLPs.

MATERIALS AND METHODS Patients and Family Studies The family (Fig. 1)was ascertained a t a Cape Town genetic clinic, when the propositus (11-3)sought counseling concerning the medical and genetic prognosis for affected relatives. The earliest known affected progenitor, a woman, was born in Sussex, England, about 1860. Her husband and one son were tall, while 3 other sons were dwarfed. The youngest affected son (1-2)emigrated to South Africa and married twice, producing a n affected daughter (11-2)and a n affected son (11-3, the propositus) in successive marriages to normal women. Thereafter, the condition was passed to 2 further generations, with male to male transmission. The general phenotype of the affected persons was short stature, with a stocky habitus (Fig. 2). Seven individuals in 3 generations were found to be affected. The height of all affected individuals was below the 5th centile for age. There was no significant disproportion between trunk and limb length (Table I). The upper to lower segment ratio in all affected individuals ranged from (-11 to ( + I 1 S.D. The height of affected men ranged from 141 cm (11-3)to 162 cm (IV-2) and of the women from 138 cm (11-2)to 149 cm (111-2).There was a 20 cm difference in the height of a pair of adult brothers, (normal IV-1 and affected IV-2); their unaffected father was also tall (180 cm). Severe progressive generalized osteoarthropathy was a major complication which caused a variety of mechanical problems in adulthood. There was no inflammatory component to this complication.

SED, Mild AD Type 2

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Fig. 1. The pedigree of the kindred showing discordant inheritance of SED and COL2A1 alleles. The affected grandchildren IV-3, IV-4 inherited different COL2Al alleles than their affected grandfather. Restriction site polymorphisms are indicated for PvuII (row l),HindIII (row 21, and HinfI (row 3).PvuII and HindIII RFLPs are dimorphisms, while the HinfI RFLP is a three allele system.

childhood, achieving a n adult height of 141 cm. His general health had been good. During his youth, he had Arm Lower Upper played rugby football but his sporting activities had Age Height span segment segment been curtailed by degenerative changes in knees and Patient Sex (yr) (cm) (cm) (cm) (cm) hips. Bilateral prosthetic hip joint replacement, which 138 11-2 F 74 142 71 67 had been undertaken at age 58 years, had given good 141 11-3 149 71 70 M 62 results but persistent pain in the knees caused disabil149 111-2 F 50 153 76 73 ity. Backache in the lumbar region had also been in144 111-4 147 71 73 F 35 143 146 72 71 F 28 111-6 creasingly troublesome during the past 2 decades. Es162 82 80 162 M 28 IV-2 sential hypertension of moderate severity, was managed 143 IV-3 F 15 145 71 72 with conventional therapy. His 2 daughters (111-4 and 136 140 68 68 IV-4 F 13 111-6)were short. His half-sister (11-2)was also short, as 112 113 57 55 IV-5 M 7 was her daughter (111-2). When examined in 1988,he was found to be short and to have a thickset, muscular physique. His face, intelligence, and skin were normal. The range of joint moveIn contrast to the marked shortness of stature, radio- ment was generally consistent with age, but movements graphic skeletal survey demonstrated surprisingly of the lumbar spine were diminished and painful. Crepminor changes. All affected adults had widespread os- itus was elicited in knee joints. He had mild left genu teoarthropathy, which was especially evident in the varus and right genu valgus, with a left hallux valgus weight bearingjoints. In the vertebrae, end plate irregu- and a n associated bunion. No abnormalities were delarity and sclerosis of bodies was prominent. During tected in any other system. On radiographs the skeleton was robust and the skull childhood there was some delay in bone age and a t a later stage mild variable changes were evident in some was normal. Marked sclerosis and irregularity were eviepiphyses (Fig. 3). The skull, limb girdles, metaphyses, dent in the end plates of the vertebral bodies, and anteand diaphyses of the long bones, together with the tubu- rior osteophytes, minor platyspondyly and vertebral lar bones of the limbs, were normal. Essential details of wedging were present (Fig. 4).The limb girdles were the propositus are outlined below. The propositus (11-3) normal as were the tubular bones and limbs, but severe had been substantially shorter than his peers from early degenerative osteoarthropathy was present in all joints. TABLE I. Body Measurements of the Affected Individuals

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Fig. 3. Anterioriposterior view ofhips and pelvis ofIV-5, age 7 years. The femoral capital epiphyses are flattened in their medial portions.

Fig. 2. Patient 11-2 age 75 years (left) and her daughter, 111-2 (right) age 50 years with a normal female. Both are dwarfed with squat, thickset physiques.

Chromosome studies, including high resolution banding, yielded normal results. There were no abnormal findings in routine hematological and biochemical investigations.

DNA Analysis After informed consent was obtained, peripheral blood was collected in EDTA tubes from all available relatives. Nuclear DNA was extracted from lymphocytes as described previously [Kunkel et al., 19771. Genotyping for the various RFLPs was undertaken in the USA (IJA, PT) and in Cape Town, SA (CS,RSR,). Five micrograms of DNA were digested to completion with various endonucleases under conditions recommended Fig. 4. Lateral radiographic view of the spine of the propositus (11-3). by the manufacturer. Resulting DNA fragments were bodies are flattened with gross irregularities of their end separated by electrophoresis on 1%(pVu11, ~ i ~ d 1 1 1 The , vertebral plates and anterior osteophytosis. EcoRI) or 2%(Hinff) agarose gels a t 20-25 V for 24-36 hours, denatured, and transferred to nylon membranes (Zetabind) following established protocols. The DNA probes were labeled with 32Pusing the random oli-

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DISCUSSION Diagnostic precision a t the clinical and radiological level is essential for meaningful correlations between the phenotype and the fundamental biomolecular defect. In the family which we report, short stature and severe degenerative arthropathy contrast with the inDNA Probes nocuous radiographic manifestations. In view of the preThe following COL2Al probes were used: dominant involvement of the vertebrae and epiphyses, 1. a 2.0 kb XhoIiKpnI subclone ofthe 8.0 kb EcoRI(E)/ with sparing of other components of the skeleton, it is BamHI fragment of Pis2 [Sangiorgi et al., 19851for the reasonable to regard the condition as a form of spondyloPvuII and HindIII RFLPs. The PvuII site polymorphism epiphyseal dysplasia (SED). However, it must be generated 2 bands, 3.3 kb and 1.7 kb. The HindIII site strongly emphasized, that this disorder is very different polymorphism also gave 2 bands, 13 kb and 7 kb in size. from the classical forms of SED, and that it is possibly a Both RFLPs are dimorphisms. private syndrome. 2. a 1.6 kb EcoRUBglII subclone of the 7.3 kb E/E Type I1 collagen, the major component of the fibrils fragment of Pis10 for the Hinf I RFLP [Strom, 1988; found in cartilage has long been suspected to be a candiAnderson et al., 19901. The HinfI site polymorphism date molecule in the etiology of SED. It is a homotrimer generated 3 alleles, 2.1 kb, 1.75 kb, and 1.15 kb + 0.6 composed of al(I1) chains which form a triple helix. The kb. proal(I1) chain is encoded in man a t a single locus COL2Al assigned to chromosome 12q14.3 [Law et al., Linkage Analysis 19861.Murray et al. [1989] reported biochemical studies Pairwise LOD scores were calculated using the com- performed on type I1 collagen extracted from the carputer program LIPED [Ott, 19741. The SED gene was tilage of individuals with spondyloepiphyseal and assumed to be autosomal dominant with a penetrance of spondyloepimetaphyseal dysplasia. Type I1 collagen 100% and with a n allele frequency of .001. Likelihoods from almost all patients studied exhibited a slower elecwere calculated a t various recombination fractions (0) trophoretic mobility. In addition, the retarded mobility from 0.0 to 0.5, and the results presented as LOD scores of many but not all of the CNBr peptides suggested a (Table 11). delay in helix formation and consequent overmodificaThe family was also typed for the following genetic tion of the al(I1) chain. The molecular defect in one markers: esterase D (ESD), phosphoglucomutase 1 family with SED congenita has recently been charac(PGMl), glyoxalase 1 (GLO1) complement component 3 terized. A single 390 base pair deletion was found ex(C3),vitamin D binding protein (DBP),properdin factor tending from the middle of intron 47 to the 5' splice site B (BF), haptoglobin (HP), ABO, MNS, Rhesus, and of intron 48 of the COL2Al gene [Lee et al., 19891. The Duffy blood groups in the Laboratories of the Blood deletion was predicted to result in the excision of 36 Transfusion Service, Cape Town (RWM). amino acids from the al(I1) chain. Genetic linkage of the COL2Al locus to the SED pheRESULTS notype in one family with SED congenita has also been A total of 11individuals was genotyped for previously established [Anderson et al., 19901. In contrast, linkage reported COL2Al RFLPs using restriction endo- of the COL2Al locus to the SED phenotype in the family nucleases PvuII, HindIII, and HinfI. There was clear which we studied was excluded indicating that the SED evidence of recombination between all 3 DNA markers mutation in this family is not within the COL2A1 locus. and the SED phenotype in several meioses. The LOD Cartilage contains a tissue-specific set of collagens. In score was less than - 2 a t 0 = 0.06 indicating that the addition to type 11, type XI collagen molecules form the SED phenotype and the COL2Al locus in this family are core of fibrils [Olsen, 1988; Mendler e t al., 19891. Colnot genetically linked (Table 11).In addition, all individ- lagen XI is a heterotrimer consisting of al(XI), a2(XI), uals were genotyped for previously reported COLllAl and a3(XI)chains. The a3(XI)chain has a CNBr peptide [Hudgins et al., 1990, Henry e t al., 19881 and COLllA2 pattern very similar to that of the al(I1) chain, suggest[Kimura et al., 19891 polymorphisms. The family was ing that, the 2 polypeptides might be derived from the not informative for either RFLP. Linkage studies using same gene. Thus, logical candidate loci included the the serum protein and cell surface antigen markers preC O L llA l and COL11A2. Unfortunately, because of hoviously referred to failed to establish genetic linkage mozygosity a t polymorphic restriction sites the family between the phenotype and the markers. was not informative. The cause of this condition remains unknown.

gonucleotide primer method [Feinberg and Vogelstein, 19841 or nick translation [Rigby et al., 19771. Filters were hybridized according to standard methods, then washed a t 65°C in 0.1 x SSC, 0.2% SDS. Autoradiography was performed at -70°C for 1-10 days.

TABLE 11. LOD Scores for SED and COL2Al (Haplotype for PVuII. HindIII. and HinfI polymorphisms) ~

0.00

0.01

--a

-3.81

Recombination fraction 0 0.05 0.10 0.20

0.30

0.40

-0.39

-0.14

~~

Z

-2.26

-1.54

-0.80

ACKNOWLEDGMENTS The authors would like to thank Drs. T Kimura, F Ramirez, and W Upholt for their generous gift of COL2A1, COL11A1, and COL11A2 gene-specific probes and Drs. S Blanton and M Sarfarazi for assisting in the linkage analysis. We thank Gillian Shapley for typing the manuscript.

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This research was supported by grants from the South African Medical Research Council, the Mauerberger Foundation, the Harry Crossley Foundation, The University of Cape Town Staff Research Fund. P.T. was supported by research grants from the National Institutes of Health, Pol-HD22610, and the Coles Family Foundation.

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Kunkel LM, Smith KD, Boyer SH, Borgaonkar DS, Wachtel SS, Miller OJ, Breg WR, Jones HW Jr, Rary HM (1977):Analysis of human Y chromosome variants. Proc Natl Acad Sci USA 74:1245-1249. Law ML, Tung L, Morse HG, Berger R, Jones C, Cheah KSE, Solomon E (1986): The human type I1 collagen gene (COL2A1) assigned to 12q14.3. Ann Hum Genet 50:131-137. Lee B, Vissing H, Ramirez F, Rogers D, Rimoin D (1989):Identification of the molecular defect in a family with spondyloepiphyseal dysplasia. Science 244:978-980. Mendler M, Eich-Bender SG, Vaughan L, Winterhalter KH, Bruckner P (19891: Cartilage contains mixed fibrils of collagen types 11, IX and XI. J Cell Biol 108:191-197. Murray L, Bautista J, James PL, Rimoin DL (1989):Q p e I1 collagen defects in the chondrodysplasias: 1.Spondyloepiphyseal dysplasias. Am J Hum Genet 45:5-15. Olsen B (1988): Molecular biology of the cartilage collagens. Path01 Immunopathol Res 7:20-23. Ott J (1974):Estimation of the recombination fraction in human pedigrees: Efficient computation of the likelihood for human linkage studies. Am J Hum Genet 26588-597. Rigby PJW, Dieckmann M, Rhodes C, Berg P (1977): Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase. J Mol Biol 113:237-251. Sangiorgi FO, Benson Chanda V, de Wet WJ, Sobel ME, Tsipouras P, Ramirez F (1985): Isolation and partial characterization of the entire proal(I1) collagen gene. Nucleic Acid Res 13:2207-2224. Strom CM (1988): A three allele restriction length fragment polymorphism within the human COL2Al gene. Nucleic Acid Res 16:9077.