α-Synuclein Locus Triplication Causes Parkinson's Disease

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Science  31 Oct 2003:
Vol. 302, Issue 5646, pp. 841
DOI: 10.1126/science.1090278

Mutations in the α-synuclein gene (SNCA) in the Contursi kindred (1) implicated this gene in Parkinson's disease (PD). Subsequently, α-synuclein was identified as the major component of Lewy bodies, the pathological hallmark of PD, and of glial cell cytoplasmic inclusions (2).

We examined a large family with autosomal dominant PD (average age of onset, 34 years), ranging clinically from dementia with Lewy bodies to typical PD (3). Neuropathological examination of affected members revealed profound pathology including extensive Lewy bodies and some glial cell cytoplasmic inclusions. Screening this family for mutations in, or linkage to, SNCA was negative. Linkage analysis revealed a chromosome 4p15 haplotype segregating with parkinsonism and essential tremor, with suggestive evidence for linkage to PARK4 [multipoint logarithm of odds (LOD) = 2.64 at D4S1609] (4). However, an unaffected individual who did not share the 4p15 haplotype became ill. This prompted a second genome-wide search at higher resolution, which revealed a haplotype co-segregating with disease over 26 cM (D4S2367–D4S1560), with a multipoint LOD of 3.50 at D4S2460. The SNCA genotypes were inconsistent with previous data, leading to initial exclusion; re-evaluation of the original linkage revealed a sample swap. Resequencing of SNCA failed to reveal pathogenic mutations.

The heterozygous single nucleotide polymorphisms in the SNCA promoter and in intron 5 suggested that deletion of this region was unlikely. Reverse transcriptase–polymerase chain reaction (RT-PCR) amplification of SNCA revealed only transcripts of normal length and sequence in affected family members. Analysis of intragenic markers MG4S2 and MG4S5 at the SNCA locus showed apparent examples of non-mendelian inheritance, which could be interpreted as multiple alleles. Quantitative real-time PCR amplification of SNCA exons yielded results consistent with whole gene triplication (Fig. 1). To confirm SNCA triplication, we performed fluorescent in situ hybridization (FISH) of chromosomes from Epstein-Barr virus (EBV)-immortalized lymphocytes from an affected family member (9-77). SNCA triplication in this family segregates with parkinsonism but not postural tremor (fig. S1).

Fig. 1.

(A) Gene dosage analysis of SNCA and flanking genes from affected family member 9-77. Results are the mean of six replicates and expressed as 2-ΔΔCt ± SD. (B) FISH analysis of interphase (i and iii) and metaphase (ii and iv) chromosomes from EBV-transformed lymphocytes of patient 9-77 showing SNCA triplication. Red, labeled PAC 27M7 marker spanning SNCA; green, 11-kb fragment corresponding to the promoter region and first 3 exons of SNCA. Tight apposition of the two metaphase chromatids precludes resolution of double minutes.

Using quantitative PCR of flanking annotated genes in the human sequence, we determined the extent of triplication. The telomeric end of the triplication occurs within the model gene KIAA1680, between exons 1 and 3, a distance of 181 kb; the centromeric end occurs between exon 23 of the cyclin E binding protein gene (CEB1) and exon 7 of hypothetical protein DKFZp761G058, an interval of 243 kb (fig. S2). The triplicated region is between 1.61 Mb and 2.04 Mb in size and contains 17 annotated or putative genes (table S1). This region includes SNCA, at least 1.2 Mb flanking the centromeric sequence, and more than 280 kb flanking the telomeric sequence. Carriers of the SNCA triplication are predicted to have four fully functional copies of SNCA, with doubling of the effective load of an estimated 17 genes. It is possible that one or more of these genes rather than SNCA is responsible for disease pathology or that structural mutation in a gene at the end of the triplication is the pathogenic event. Parsimony argues, however, that increased dosage of SNCA is the cause of PD in this family.

These results are consistent with haplotype data suggesting that genetic variability in the SNCA promoter contributes to the risk of developing PD (5). They support evidence that mutant α-synuclein behaves differently from the wild-type protein in a quantitative rather than qualitative manner. Finally, the disease process in this family may resemble the etiology of Alzheimer's disease in Down syndrome, where overexpression of the APP gene due to chromosome 21 trisomy is the key event.

Supporting Online Material

Materials and Methods

Figs. S1 and S2

Table S1

References and Notes

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