Conclusiones y Recomendaciones

Eighteen families with ADCA type II who had been classified according to the criteria

suggested by Enevoldson et al. (Enevoldson et al, 1994) were analyzed for the SCA7

mutation. These criteria are as follows:

1. A family history compatible with an autosomal dominant mode o f inheritance.

2. At least one member of the pedigree with a documented combination o f a progressive central nervous system disorder, in which cerebellar ataxia was the predominant feature, and gradually progressive visual failure associated with pigmentary retinopathy.

3. A similar central nervous system disorder and / or pigmentary retinopathy in at least one other non-sibling member o f the pedigree.

These families have different ethnic origins: 1 Brazilian, 1 Italian, 1 Indian, 1 Philippine, 1 South African, and 13 British. Seven families with ADCA type 1,26 families with ADCA type III, 56 patients with ILOCA, and 1 patient with early-onset maculopathy and

cerebellar ataxia were also screened. It was confirmed that no patient had the SCAl, SCA2, SCA3, or SCA6 mutation.

In addition to patients with cerebellar ataxia, 348 control DNA samples were analysed for the SCA7 mutation. These samples were from subjects referred to the Institute of

Neurology and were a heterogeneous group consisting of patients with other neurological disorders, both genetic and non-genetic, spouses o f these patients and o f patients with cerebellar ataxia.

7.3.2 Genotyping

DNA was extracted from lymphocytes using the methods described in chapter 2. The SCAT mutation was identified by use of PCR with oligonucleotide primers 4U1024

(fluorescendy labelled) and 4U716 (David et al.^ 1997) (see appendix 2).

PCR was performed in a final volume of 50 |ol containing 100 ng o f DNA in 1.5 mM MgC12, 60 mM KCl, 200 pM of each dNTP, 10% o f dimethyl sulfoxide, 12 pmol of each

primer, and 1.25 U o f Taq polymerase. After an initial dénaturation at 95°C for 5 min,

dénaturation, annealing, and extension were done at 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s for 27 cycles and one final extension for 2 min. The PCR products were checked on 3.2% agarose gels before analysis on an ABI 373 and 377 DNA sequencer by means of GENESCAN software (ABI).

Haplotype reconstruction was possible in 11 families by use o f six microsatellite markers flanking the SCA7 gene and spanning 8 cM: D3S3566—3698-1600- (CAG)«-1287-3635-

3644 (David et al.^ 1996; Krols et al.^ 1997). Microsatellite markers were amplified and

analyzed as described in chapter 2 and alleles were ascribed the same numbers as described

previously (David et al.^ 1996).

For statistical analysis, means were compared with Student's t test or F test for mean

distribution. Results are mean ± SD, unless otherwise indicated. Correlation analysis was done for CAG-repeat number with age at onset, age at death, and disease duration.

7.4 RESULTS

T h e analysis o f the SCA7 C A G expansion show ed th at 17 o f 18 families w ith A D C A type II had the novel triplet m utation, b u t the expansion was n o t fou n d in any o f the 7 families w ith A D C A type I, 26 families w ith A D C A type III, o r 56 subjects w ith IL O C A . A de novo m utatio n was identified in a subject w ith n o family history w h o presen ted w ith

m aculopathy at age 14 years (family V N , figure 7.4-1)

7.4.1 Distribution of Normal, Interm ediate, and Pathological Alleles

T h e distribution o f the non-pathological SCA7 C A G repeats in o u r series o f 941 in d ep en d e n t chrom osom es is show n in figure 7.4-2. 696 o f these are from controls and spouses o f family m em bers w ith A D C A type II and 62 from the n orm al chro m o so m es o f affected o r at-risk subjects, w ith the rem aining derived from the 56 subjects w ith IL O C A , 33 subjects w ith A D C A type I o r A D C A type III and on e w ith early-onset m aculopathy and cerebellar ataxia. T w o distinct allele ranges are evident. T h e first, 7 -1 9 repeats, contains the m ajority o f the aUeles found in norm al controls and unaffected at-risk

Fam ily VN Fam ily PT

Fam ily Wl

i I I i

F ig u re 7.4-1 P ed ig rees of F am ilies V N , P T an d W I. Affected status is denoted by filled symbols. The pedigrees has been altered by changing birth order, and by substituting diamond symbols for some individuals o f both sexes. Haplotypes for chromosom e 3p markers are shown. Boxes are surrounding haplotypes cosegregating with the expanded and intermediate CAG repeats. Dashes are used when alleles could not be typed or deduced.

subjects. The most common allele, with 10 repeats, accounted for 72% of these normal alleles and was found in a homozygous state in 55% of normal controls, in agreement with

a previous report (David et al.^ 1998). Three independent alleles in the second range of 28-

35 CAG repeats were found. The three alleles were found in one affected individual (family WI; 11:4), seven unaffected at-risk relatives and one unaffected spouse from three families. The seven at-risk subjects, whose mean age was 58.3 ± 17.4 years (range, 39-85), shared the same haplotype with their affected relatives. Therefore it is possible and indeed likely that this group represents intermediate alleles with a propensity for pathological expansion (see section 7.4.2), as is clearly shown in family VN (figure 7.4-1), whereas the first group, with alleles in the range of 7-19 repeats, represents the true normal range. Repeat-size distribution for 55 affected subjects and the 7 at-risk carriers is shown in figure 7.4-3. These pathological alleles had a range of 37 to ~220 repeats (median 48 ± 29.3), the largest of which were 180 and ~220 repeats and were transmitted by affected fathers with 39 and 54 repeats, respectively. 700 - 150 'Ù E o o E 2 100 o o o z 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 No of CAG repeats

F ig u re 7.4-2 D istrib u tio n o f SCA7 C A G -repeat n u m b e rs in 941 in d e p e n d e n t norm al c h ro m o so m es from control su b jects, spouses, a n d affected su b jects. Two ranges are evident: the first, 7—19, represents the true normal range, and the second, 28—35, represents intermediate alleles. Note that only 3 representative intermediate alleles are illustrated here but these alleles were found in eight individuals from the three families VN, PT and WI (see text).

10

I e

U-llllu ll III

ill II I

U

No. of CAG re p e a ts

F ig u re 7.4-3 D istrib u tio n o f SCA7 re p eat n u m b e rs o f 55 ch ro m o so m es from p a tie n ts and 7 from at-risk su b jects

Alleles with such a large number of CAG repeats have not been reported in other CAG- repeat diseases (SBMA, HD, DRPLA, SCAl, SCA2, or SCA3). There was no significant

difference in the distribution of the expanded repeats between male (» = 32) and female (»

= 30) subjects.

7.4.2 Sex of the transm itting parent and changes in repeat size

The change in size of the pathological allele in 31 parent-child transmissions is shown in figure 7.4-4. No decreases were observed. The changes in repeat size were as follows: in 17 father-child pairs, the median increase was 6 ± 48.9 (mean=23.9) compared to 3 ± 4.5 (mean=4) in 14 mother-child pairs. The combined median was 4 ± 37.2 (mean=14.9) in the 31 parent-child pairs. The difference between the paternal and maternal increases in repeat size was statistically significant (F test, p<0.0001). Increases in size >10 repeats, although less common, occurred more frequendy during paternal transmissions.

IILU