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Deep sequencing of the Nicastrin gene in pooled DNA, the identification of genetic variants that affect risk of Alzheimer's disease.

ABSTRACT: Nicastrin is an obligatory component of the ?-secretase; the enzyme complex that leads to the production of A? fragments critically central to the pathogenesis of Alzheimer's disease (AD). Analyses of the effects of common variation in this gene on risk for late onset AD have been inconclusive. We investigated the effect of rare variation in the coding regions of the Nicastrin gene in a cohort of AD patients and matched controls using an innovative pooling approach and next generation sequencing. Five SNPs were identified and validated by individual genotyping from 311 cases and 360 controls. Association analysis identified a non-synonymous rare SNP (N417Y) with a statistically higher frequency in cases compared to controls in the Greek population (OR 3.994, CI 1.105-14.439, p = 0.035). This finding warrants further investigation in a larger cohort and adds weight to the hypothesis that rare variation explains some of genetic heritability still to be identified in Alzheimer's disease.

SUBMITTER: Lupton MK

PROVIDER: S-EPMC3045431 | biostudies-literature | 2011 Feb

REPOSITORIES: biostudies-literature

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Deep sequencing of the Nicastrin gene in pooled DNA, the identification of genetic variants that affect risk of Alzheimer's disease.

Lupton Michelle K MK Proitsi Petroula P Danillidou Makrina M Tsolaki Magda M Hamilton Gillian G Wroe Richard R Pritchard Megan M Lord Kathryn K Martin Belinda M BM Kloszewska Iwona I Soininen Hilkka H Mecocci Patrizia P Vellas Bruno B Harold Denise D Hollingworth Paul P Lovestone Simon S Powell John F JF

PloS one 20110225 2

Nicastrin is an obligatory component of the γ-secretase; the enzyme complex that leads to the production of Aβ fragments critically central to the pathogenesis of Alzheimer's disease (AD). Analyses of the effects of common variation in this gene on risk for late onset AD have been inconclusive. We investigated the effect of rare variation in the coding regions of the Nicastrin gene in a cohort of AD patients and matched controls using an innovative pooling approach and next generation sequencing ...[more]

PMID: 21364883

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Project description:BackgroundRNA-sequencing (RNA-seq) is a powerful technique for the identification of genetic variants that affect gene-expression levels, either through expression quantitative trait locus (eQTL) mapping or through allele-specific expression (ASE) analysis. Given increasing numbers of RNA-seq samples in the public domain, we here studied to what extent eQTLs and ASE effects can be identified when using public RNA-seq data while deriving the genotypes from the RNA-sequencing reads themselves.MethodsWe downloaded the raw reads for all available human RNA-seq datasets. Using these reads we performed gene expression quantification. All samples were jointly normalized and subjected to a strict quality control. We also derived genotypes using the RNA-seq reads and used imputation to infer non-coding variants. This allowed us to perform eQTL mapping and ASE analyses jointly on all samples that passed quality control. Our results were validated using samples for which DNA-seq genotypes were available.Results4,978 public human RNA-seq runs, representing many different tissues and cell-types, passed quality control. Even though these data originated from many different laboratories, samples reflecting the same cell type clustered together, suggesting that technical biases due to different sequencing protocols are limited. In a joint analysis on the 1,262 samples with high quality genotypes, we identified cis-eQTLs effects for 8,034 unique genes (at a false discovery rate ≤0.05). eQTL mapping on individual tissues revealed that a limited number of samples already suffice to identify tissue-specific eQTLs for known disease-associated genetic variants. Additionally, we observed strong ASE effects for 34 rare pathogenic variants, corroborating previously observed effects on the corresponding protein levels.ConclusionsBy deriving and imputing genotypes from RNA-seq data, it is possible to identify both eQTLs and ASE effects. Given the exponential growth of the number of publicly available RNA-seq samples, we expect this approach will become especially relevant for studying the effects of tissue-specific and rare pathogenic genetic variants to aid clinical interpretation of exome and genome sequencing.

Project description:Muscle pathology in inclusion body myositis (IBM) typically includes inflammatory cell infiltration, muscle fibers with rimmed vacuoles and cytochrome c oxidase (COX)-deficient fibers. Previous studies have revealed clonal expansion of large mitochondrial DNA (mtDNA) deletions in the COX-deficient muscle fibers. Technical limitations have prevented complete investigations of the mtDNA deletions and other mtDNA variants. Detailed characterization by deep sequencing of mtDNA in muscle samples from 21 IBM patients and 10 age-matched controls was performed after whole genome sequencing with a mean depth of mtDNA coverage of 46,000x. Multiple large mtDNA deletions and duplications were identified in all IBM and control muscle samples. In general, the IBM muscles demonstrated a larger number of deletions and duplications with a mean heteroplasmy level of 10% (range 1%-35%) compared to controls (1%, range 0.2%-3%). There was also a small increase in the number of somatic single nucleotide variants in IBM muscle. More than 200 rearrangements were recurrent in at least two or more IBM muscles while 26 were found in both IBM and control muscles. The deletions and duplications, with a high recurrence rate, were mainly observed in three mtDNA regions, m.534-4429, m.6330-13993, and m.8636-16072, where some were flanked by repetitive sequences. The mtDNA copy number in IBM muscle was reduced to 42% of controls. Immunohistochemical and western blot analyses of IBM muscle revealed combined complex I and complex IV deficiency affecting the COX-deficient fibers. In conclusion, deep sequencing and quantitation of mtDNA variants revealed that IBM muscles had markedly increased levels of large deletions and duplications, and there were also indications of increased somatic single nucleotide variants and reduced mtDNA copy numbers compared to age-matched controls. The distribution and type of variants were similar in IBM muscle and controls indicating an accelerated aging process in IBM muscle, possibly associated with chronic inflammation.

Dataset Information

Deep sequencing of the Nicastrin gene in pooled DNA, the identification of genetic variants that affect risk of Alzheimer's disease.

Publications

Deep sequencing of the Nicastrin gene in pooled DNA, the identification of genetic variants that affect risk of Alzheimer's disease.

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