participated in a linkage study. Pedigrees are presented in Appendix, part II. Full clinical details of the families are also included in this section. A detailed examination of all the individuals were carried out by Dr Karen Temple and Dr Michael Barraitser. The diagnosis of Stickler syndrom e was based on manifestations of at least 2 of the 4 major clinical groups: 1) eye findings; 2)
arthropathy; 3) orofacial abnormalities; 4) deafness. Blood samples were obtained from 41 m embers of the 6 families. Chromosome studies were
perform ed on an affected individual from each family. All karyotypes were norm al except in family DN w here a t5;17(ql5:q23) w as found to segregate w ith the disease in 4 affected relatives.
2.2.2 Cell line inform ation
A somatic cell hybrid w as constructed at the Imperial Cancer Research Fund w ith individual II-3 from family DN (see Appendix, part II). The hybrid was constructed from a lymphoblastoid line w ith a t5:17(ql5:q23) karyotype and
the Chinese hamster line A23 TK-.
2.3.0 DATA ANALYSIS 2.3.1 Linkage studies
Data was prepared for analysis using LINKSYS and analyzed with two point linkage using LIPED. Two different m odes of inheritance and 3 different levels of penetrance w ere used in the analysis of the C L /P families.
Firstly, lod scores w ere calculated based on an autosomal dom inant m odel of inheritance w ith a gene frequency of 0.001 and 80% penetrance. These values appeared to best reflect the inheritance pattern observed in the 8 families w ith
C L /P (Winter, personal communication). Lod scores however w ere also calculated w ith this type of inheritance but w ith a level of penetrance set at 28%. This w as obtained by averaging the separate penetrances w hich w ere set for males and females in an analysis of American clefted families by Hecht et al, (1991a). The second model tested autosomal recessive inheritance with a gene frequency of 0.035 and an average penetrance of 35%. This reflected the model proposed by Chung et al, (1986).
Two point linkage analysis was also carried out on families affected with Stickler syndrom e. For this analysis, a gene frequency of 0.0001 was used with an autosomal dom inant pattern of inheritance and 1 0 0% penetrance.
M ultipoint linkage analysis was carried out using the LINKMAP section of LINKAGE version 5.04. This involves the sim ultaneous analysis of 3 or more loci to determ ine their order on the chromosome. Loci on chromosome 1 were analyzed in this m anner. The location of an unknow n locus for C L /P was placed in relation to m arkers pYNZ23 and pEKH7.4. The distance between the tw o markers w as quoted in Buetow et al, (1990) and converted into a recombination fraction using H aldane's m apping function.
LINKMAP generates likelihood scores of the location of m axim um likelihood for the un k n o w n locus. This inform ation is expressed in the form of - 2
likelihood score. To convert the likelihood score into a location score, it is necessary to subtract the - 2 likelihood scores generated at the various points
from the -2 likelihood score at 0.5 recom bination. The location score can then be converted into a lod score by dividing by 4.6.
2.3.2 A ssociation stu d ies
analysis w as used to assess allele and w here necessary, haplotype frequency differences betw een cases and controls. For m ost analyses, a 2 x 2 contingency table w as used, how ever occasionally this w as increased t o a 3 x 2 o r a 4 x 2 table. Results generated from analyses w ere com pared w ith values from a probability table derived by Fisher and Yates (Swinscow, 1985).
ûf/efe
/ '
2.4.0 PROBES/TANDEM REPEATS (loci and m ap position) pYNZ23 (D1S58, lq31-32)
pYNZ23 is contained in the vector pBR322. The insert size is 5kb w hich can be excised w ith the enzym e Clal. A two allele polym orphism is recognized by this probe at a Bgll restriction enzym e site. The band sizes generated are 5.0 and 4.2kb w ith respective frequencies of 0.49 and 0.51 (N akam ura et al, 1987).
pEKH7.4 (D1S65, lq31-32)
pEKH7.4 recognizes a two allele polym orphism at a Taql restriction enzym e site. The sizes of the bands are i) 5.0kb w ith a frequency of 0.53 and ii) 3.8kb w ith a frequency of 0.47. The 2.9kb insert is contained in the pUC18 vector and can be excised w ith a Pstl enzym e (Kumlin-Wolff et al, 1987).
TGFA (2pl3)
Two genomic subclones of TGFA were used. phT G Fl-10-925 is a 0.93kb insert while phTG Fl-10-3350 is a 3.5kb insert. Both inserts are contained in the pBR327 vector and can be excised w ith EcoRI. Three polymorphic fragments are associated w ith these probes after digesting w ith Taql, BamHÎ and Rsal (M urray et al, 1986; H ayw ard et al, 1987). These fragm ents are outlined in Table 1. Each fragm ent is represented in Figures 4, 5 and 6 respectively.
Probe Restriction enzym e Fragm ent (kb) Allele* Frequency phTGFl- 10-3350 Taql 3.0 2.7 C l C2 0.94 0.06 BamHl 9.0 3.7 A1 A2 0.19 0.81 phTGFl- 10-925 Rsal 1.5 1.2 B1 B2 0.29 0.71
Table 1. Polym orphism s at the TGFA locus.
Cl = 3.0kb-"
C2 = 2.7kb
Figure 4. Diallelic Taql RFLP of TGFA-3350. 60