FLUJO DE OPERACIÓN DEL REACTOR BIOLÓGICO AEROBIO

Once the mode of inheritance for ITD had been established, it became possible to undertake linkage analysis to attempt to map the gene/s involved. Linkage analysis on single large pedigrees is desirable as a first step, so that the effect of locus heterogeneity will not mask the possibility of detecting linkage. A large pedigree of ITD (family R) originally described by Johnson et al (1962) was recruited to form the basis of a systematic linkage study at the Department of Neurology, Columbia University, New York. The family comprised 57

individuals of whom 14 had been diagnosed as having dystonia. Initial studies typed the family for genetic systems, including red cell enzymes, serum proteins, and human leucocyte antigens (HLA) (Falk et al, 1988). No evidence was found for linkage in any system tested, though small positive lod scores were obtained with the ABO locus on chromosome 9q (Zmax 0.591 at 0 = 0 .2 0 ). Further data excluded large regions of chromosomes l i p , 13q, and 21q by multipoint linkage analysis (Kramer et al, 1987). The family studied was further investigated and expanded to include 165 members, and continued linkage analysis performed utilising the advances in DNA technology such as the emergence of highly polymorphic variable number tandem repeat (VNTR) sequences. Approximately 60% of the genome had been excluded before linkage was found. Due to the positive two-point lod score for the ABO locus on chromosome 9q further markers in this area were studied (Ozelius et al, 1989). Highest lod scores were obtained with a cDNA probe for the gelsolin locus (GSN) which maps centromeric to ABL (Abelson oncogene) on 9q32-34. The dystonia locus (termed D Y Tl) and GSN locus showed significant evidence for linkage, with a maximum lod score at 0 = 0.0 of Z = 3.51. No obligate recombination events between GSN and DYTl were observed in 67 meioses, although most were not fully informative. Multi-point analysis of four markers in this region suggested that both GSN and DYTl lay between D9S26 and ABO (figure 3.3). A possible candidate gene noted in this area was the gene for dopamine-8-hydroxylase (DBH), which converts dopamine to noradrenaline. Abnormal DBH levels have been claimed in ITD (Ziegler et al, 1976), and an enzyme involved in the maintenance of catecholaminergic and cholinergic balance in the basal ganglia is an obvious suspect. Another possible candidate was gelsolin itself, which is an actin binding protein.

Kramer et al (1990) then looked at linkage analysis using DNA polymorphisms in this region in 12 Ashkenazi Jewish families with ITD, which it was felt, represented a homogeneous genetic group. Highest two-point lod scores (using the LIPED program) were obtained with the probe for the argininosuccinate synthetase (ASS) locus, with a maximum likelihood estimate of theta (0 ) at 0.03 with a lod score of 3.49. Positive two-point lod scores were also obtained for D9S29, D9S26, GSN, and AKl (adenylate kinase). Multi-point analysis (using the LINKMAP program) put the most likely position of the dystonia locus as midway between AKl and ASS (figure 3.3). These findings suggested that the same gene was responsible for susceptibility to ITD in both Jewish and non-Jewish populations, but the difference in penetrance of the dominant allele (0.75 in the non-Jewish family, and 0.30 in the Jewish families) was thought to imply allelic mutations.

8cM 7cM 7cM IcM IcM IcM IcM IcM 5cM IcM D9S26 GSN D9S61, AKl D9S65 D9S62, D9S63 ASS ABL D9S113 D9S64 D9S179 ABO DBH, D9S10 qter

Figure 3.3 Sex averaged genetic consensus map for chromosome 9q32-qter. Genetic distance shown in centiMorgans between markers. AKl - adenylate kinase, ASS - argininosuccinate synthetase, ABL - Abelson oncogene, ABO - ABO blood group, DBH - dopamine B hydroxylase, GSN - Gelsolin. Map taken from 4th International Chromosome 9 workshop (1996).

The development of highly polymorphic (GT)n microsatellites within the genes for GSN, ABL, and ASS (Kwiatkowski and Perman, 1991; Kwiatkowski, 1991; Kwiatkowski et al, 1991a) allowed further refinement of the genetic map of this region using Family R and the 12 Ashkenazi Jewish families (Kwiatkowski et al, 1991b). Family R exhibited a broad region of positive lod scores at 0% recombination (GSN to ASS), indicating that there were no crossover events over this entire interval. In the Jewish families the gene causing dystonia could be localised to an l l c M interval between AKl and D9S10, and the most likely position was midway between AKl and ASS (about 6cM apart). These studies also allowed the exclusion of GSN as a candidate in the Jewish families, as five likely crossovers between the dystonia trait and GSN were identified. DBH was also excluded as a candidate in families with both Jewish and non-Jewish ITD, dopa-responsive dystonia (DRD), and myoclonic dystonia by analysis of RFLPs near the DBH gene (Schuback et al, 1991). Further study of a large kindred with DRD with the ASS (GT)n repeat and other 9q32-34 markers excluded loci in this region as a cause for this form of dystonia (Kwiatkowski et al, 1991a).

Ozelius et al (1992a and b) typed further Ashkenazi kindreds and defined obligate recombination events which delineated the region containing DYTl to the 6cM between AKl and ASS. They also found highly significant linkage disequilibrium between a particular extended haplotype of ASS-ABL loci and DYTl in 52 unrelated affected Ashkenazi Jewish individuals. Association was shown for a haplotype comprising the ABL allele 4( 141 bp), a combination of two ASS RFLPs (ASSGl and G3) designated A, and the ASS allele 12 (106 bp). This 4/A 12 haplotype was found to be present in 69% of disease bearing chromosomes, compared to only 1% of control Jewish chromosomes (p <0.001), suggesting that D Y T l, ASS, and ABL probably lay within l-2cM of each other, with DYTl centromeric to ASS. Study of this extended haplotype in sporadic Jewish ITD cases showed the proportion of those potentially carrying the haplotype (as they were single cases, phase and disease chromosome status could not be designated) was similar to the proportion of familial cases with the haplotype, suggesting many sporadic cases were in fact hereditary. This meant that the disease gene frequency must be higher, and the penetrance lower than previously reported in this population. It was felt that this 4/A 12 haplotype would be of predictive value for testing carrier status in Jewish individuals (de Leon et al, 1994).

Subsequent study of the presence or absence of the 4/A 12 haplotype in 144 Ashkenazi Jewish ITD cases found that 79/144 (44 families) were carriers. Carriers had significantly earlier age of onset of ITD than non-carriers; 95% of all cases with onset before 10 years carried the haplotype whilst no cases with onset over 44 years did. In addition, onset in a limb (arm or leg)

was strongly associated with carriage of the haplotype (Bressman et al, 1994a). These data support the idea originally proposed by Risch et al (1990) that the majority of early limb onset ITD in the Ashkenazim arose from a single mutation of the DYTl gene. This allelic association was further studied by Risch et al (1995) and is discussed in section 3.8.2.a.

The extent of genetic heterogeneity in ITD is uncertain. Locus heterogeneity has been reported in one large non-Jewish Australian kindred with ITD (Ahmad et al, 1993). The family, first reported by Parker (1985), is phenotypically unusual in that dystonia first involved speech in 7 of the 10 affected individuals and eight of the cases progressed to generalised dystonia. Two individuals also had biochemically and pathologically proven W ilson’s disease. Their neurological features were indistinguishable from those of other affected family members with normal copper studies, despite penicillamine therapy. Linkage analysis excluded a locus for ITD in chromosome 9q32-34 (GSN to D9S10). A dystonia locus close to 13ql4.2-q21, the region containing the Wilson’s disease gene, was also excluded. This study demonstrated the presence of at least one other ITD locus. A linkage study of this family to attempt to identify this second autosomal ITD locus is described in section 3.5.