DNA isolation protocol effects on nuclear DNA analysis by microarrays, droplet digital PCR, and whole genome sequencing, and on mitochondrial DNA copy number estimation.

Potential bias introduced during DNA isolation is inadequately explored, although it could have significant impact on downstream analysis. To investigate this in human brain, we isolated DNA from cerebellum and frontal cortex using spin columns under different conditions, and salting-out. We first analysed DNA using array CGH, which revealed a striking wave pattern suggesting primarily GC-rich cerebellar losses, even against matched frontal cortex DNA, with a similar pattern on a SNP array. The aCGH changes varied with the isolation protocol. Droplet digital PCR of two genes also showed protocol-dependent losses. Whole genome sequencing showed GC-dependent variation in coverage with spin column isolation from cerebellum. We also extracted and sequenced DNA from substantia nigra using salting-out and phenol / chloroform. The mtDNA copy number, assessed by reads mapping to the mitochondrial genome, was higher in substantia nigra when using phenol / chloroform. We thus provide evidence for significant method-dependent bias in DNA isolation from human brain, as reported in rat tissues. This may contribute to array "waves", and could affect copy number determination, particularly if mosaicism is being sought, and sequencing coverage. Variations in isolation protocol may also affect apparent mtDNA abundance

A 6.4 Mb Duplication of the α-Synuclein Locus Causing Frontotemporal Dementia and Parkinsonism:Phenotype-Genotype Correlations

IMPORTANCE: SNCA locus duplications are associated with variable clinical features and reduced penetrance but the reasons underlying this variability are unknown. OBJECTIVE: 1) To report a novel family carrying a heterozygous 6.4Mb duplication of the SNCA locus with an atypical clinical presentation strongly reminiscent of frontotemporal dementia (FTD) and late-onset pallidopyramidal syndromes. 2) To study phenotype-genotype correlations in SNCA locus duplications. DESIGN, SETTING, PARTICIPANTS AND DATA SOURCES: We report the clinical and neuropathologic features of a family carrying a 6.4Mb duplication of the SNCA locus. To identify candidate disease modifiers, we undertake a genetic analysis in the family and conduct statistical analysis on previously published cases carrying SNCA locus duplication using regression modelling with robust standard errors to account for clustering at the family level. MAIN OUTCOME MEASURES: To assess whether length of the SNCA locus duplication influences disease penetrance and severity, and whether extra-duplication factors have a disease-modifying role. RESULTS: We identified a large 6.4Mb duplication of the SNCA locus in this family. Neuropathological analysis showed extensive α-synuclein pathology with minimal phospho-tau pathology. Genetic analysis showed an increased burden of PD-related risk factors and the disease-predisposing H1/H1 MAPT haplotype. Statistical analysis of previously published cases suggested that there is a trend towards increasing disease severity and disease penetrance with increasing duplication size. The corresponding odds ratios (95% CI) from the univariate analyses were 1.17 (0.81 to 1.68) and 1.34 (0.78 to 2.31) respectively. Gender was significantly associated with both disease risk and severity; males compared to females had increased disease risk and severity and the corresponding odds ratios (95% CI) from the univariate analyses were 8.36 (1.97 to 35.42) and 5.55 (1.39 to 22.22) respectively. CONCLUSIONS AND RELEVANCE: These findings further expand the phenotypic spectrum of SNCA locus duplications. Increased dosage of genes located within the duplicated region probably cannot increase disease risk and disease severity without the contribution of additional risk factors. Identification of disease modifiers accounting for the substantial phenotypic heterogeneity of patients with SNCA locus duplications could provide insight into molecular events involved in α-synuclein aggregation

Whole genome sequencing coverage in relation to GC content.

<p>The mean normalized coverage per 100 kb window of PD3 cerebellum is shown, after 25 mg overnight SC isolation and Puregene isolation.</p

Demographic details of individuals whose brains were used.

<p>Demographic details of individuals whose brains were used.</p

Effect of DNA isolation on copy number determination by ddPCR for cerebellar samples.

<p>(A) <i>EIF2C1</i> and (B) <i>TSC2</i>. The medians and interquartile ranges from the original results, and repeats after overnight SC extractions from 25 mg and 5 mg starting material, and Puregene, are shown. n = 6, except 5 mg SC, where n = 4.</p

Chromosome 1 in aCGH.

<p>The 10 Mb moving average and the aberration calls by ADM2 (after raising threshold to 12, with FZ off) are plotted for each sample. Losses are green, gains are red. </p><p></p><p></p><p>(A) Brain DNA hybridised against PBL reference DNA. Cerebellar samples are orange, and FC green. The moving average of a male to female DNA reference hybridisation is also shown (dark blue).</p><p></p><p></p><p>(B) Genome isochores. GC content range for each 100 kb isochore is 30–65% (blue to orange).</p><p></p><p></p><p>(C) Cerebellar DNA hybridised against FC DNA of the same brain for three PD cases with overnight SC extraction. PD1 = purple, PD2 = black, PD4 = green. Data for PD2 are derived after combining the dye-flip hybridisation pair.</p><p></p><p></p><p>(D) Hybridisations between DNA from the same brain as follows.</p><p>(1–3) Hybridisations of SC-isolated cerebellar DNA, with Puregene-isolated DNA from same cerebellum as reference. (1) PD3, 5 mg SC; (2) PD3, 25mg SC; (3) PD4, 25 mg SC.</p><p>(4) PD1, Puregene-isolated DNA, cerebellar (test) with FC as reference. Note the absence of waves and losses. This sample combination, but with spin column extraction, had led to waves and losses (PD1 in panel C).</p><p></p><p></p><p></p> <p>(A) Brain DNA hybridised against PBL reference DNA. Cerebellar samples are orange, and FC green. The moving average of a male to female DNA reference hybridisation is also shown (dark blue).</p> <p>(B) Genome isochores. GC content range for each 100 kb isochore is 30–65% (blue to orange).</p> <p>(C) Cerebellar DNA hybridised against FC DNA of the same brain for three PD cases with overnight SC extraction. PD1 = purple, PD2 = black, PD4 = green. Data for PD2 are derived after combining the dye-flip hybridisation pair.</p> <p>(D) Hybridisations between DNA from the same brain as follows.</p> <p>(1–3) Hybridisations of SC-isolated cerebellar DNA, with Puregene-isolated DNA from same cerebellum as reference. (1) PD3, 5 mg SC; (2) PD3, 25mg SC; (3) PD4, 25 mg SC.</p> <p>(4) PD1, Puregene-isolated DNA, cerebellar (test) with FC as reference. Note the absence of waves and losses. This sample combination, but with spin column extraction, had led to waves and losses (PD1 in panel C).</p