A recurrent splice-site mutation in EPHA2 causing congenital posterior nuclear cataract

Intoduction: Inherited cataract, opacification of the lens, is the most common worldwide cause of blindness in children. We aimed to identify the genetic cause of autosomal dominant (AD) posterior nuclear cataract in a four generation British family. METHODS: Whole genome sequence (WGS) was performed on two affected and one unaffected individual of the family and further validated by direct sequencing. Haplotype analysis was performed via genotying. RESULTS: A splice-site mutation c.2826-9G>A in the gene EPHA2, encoding EPH receptor A2 was identified and found to co-segregate with disease. CONCLUSIONS: We have identified a recurrent splice-site mutation c.2826-9G>A in EPHA2 causing isolated posterior nuclear cataract, providing evidence of further phenotypic heterogeneity associated with this variant

Genomic variation and its impact on gene expression in Drosophila melanogaster

Understanding the relationship between genetic and phenotypic variation is one of the great outstanding challenges in biology. To meet this challenge, comprehensive genomic variation maps of human as well as of model organism populations are required. Here, we present a nucleotide resolution catalog of single-nucleotide, multi-nucleotide, and structural variants in 39 Drosophila melanogaster Genetic Reference Panel inbred lines. Using an integrative, local assembly-based approach for variant discovery, we identify more than 3.6 million distinct variants, among which were more than 800,000 unique insertions, deletions (indels), and complex variants (1 to 6,000 bp). While the SNP density is higher near other variants, we find that variants themselves are not mutagenic, nor are regions with high variant density particularly mutation-prone. Rather, our data suggest that the elevated SNP density around variants is mainly due to population-level processes. We also provide insights into the regulatory architecture of gene expression variation in adult flies by mapping cis-expression quantitative trait loci (cis-eQTLs) for more than 2,000 genes. Indels comprise around 10% of all cis-eQTLs and show larger effects than SNP cis-eQTLs. In addition, we identified two-fold more gene associations in males as compared to females and found that most cis-eQTLs are sex-specific, revealing a partial decoupling of the genomic architecture between the sexes as well as the importance of genetic factors in mediating sex-biased gene expression. Finally, we performed RNA-seq-based allelic expression imbalance analyses in the offspring of crosses between sequenced lines, which revealed that the majority of strong cis-eQTLs can be validated in heterozygous individuals

Genomic variation and its impact on gene expression in Drosophila melanogaster

Understanding the relationship between genetic and phenotypic variation is one of the great outstanding challenges in biology. To meet this challenge, comprehensive genomic variation maps of human as well as of model organism populations are required. Here, we present a nucleotide resolution catalog of single-nucleotide, multi-nucleotide, and structural variants in 39 Drosophila melanogaster Genetic Reference Panel inbred lines. Using an integrative, local assembly-based approach for variant discovery, we identify more than 3.6 million distinct variants, among which were more than 800,000 unique insertions, deletions (indels), and complex variants (1 to 6,000 bp). While the SNP density is higher near other variants, we find that variants themselves are not mutagenic, nor are regions with high variant density particularly mutation-prone. Rather, our data suggest that the elevated SNP density around variants is mainly due to population-level processes. We also provide insights into the regulatory architecture of gene expression variation in adult flies by mapping cis-expression quantitative trait loci (cis-eQTLs) for more than 2,000 genes. Indels comprise around 10% of all cis-eQTLs and show larger effects than SNP cis-eQTLs. In addition, we identified two-fold more gene associations in males as compared to females and found that most cis-eQTLs are sex-specific, revealing a partial decoupling of the genomic architecture between the sexes as well as the importance of genetic factors in mediating sex-biased gene expression. Finally, we performed RNA-seq-based allelic expression imbalance analyses in the offspring of crosses between sequenced lines, which revealed that the majority of strong cis-eQTLs can be validated in heterozygous individuals

Experience of a multidisciplinary task force with exome sequencing for Mendelian disorders

BACKGROUND: In order to optimally integrate the use of high-throughput sequencing (HTS) as a tool in clinical diagnostics of likely monogenic disorders, we have created a multidisciplinary “Genome Clinic Task Force” at the University Hospitals of Geneva, which is composed of clinical and molecular geneticists, bioinformaticians, technicians, bioethicists, and a coordinator. METHODS AND RESULTS: We have implemented whole exome sequencing (WES) with subsequent targeted bioinformatics analysis of gene lists for specific disorders. Clinical cases of heterogeneous Mendelian disorders that could potentially benefit from HTS are presented and discussed during the sessions of the task force. Debate concerning the interpretation of identified variants and the content of the final report constitutes a major part of the task force’s work. Furthermore, issues related to bioethics, genetic counseling, quality control, and reimbursement are also addressed. CONCLUSIONS: This multidisciplinary task force has enabled us to create a platform for regular exchanges between all involved experts in order to deal with the multiple complex issues related to HTS in clinical practice and to continuously improve the diagnostic use of HTS. In addition, this task force was instrumental to formally approve the reimbursement of HTS for molecular diagnosis of Mendelian disorders in Switzerland

Overview of variants.

<p>SNPs are shown in black/grey, insertions in red, deletions in blue, and complex variants in orange. (A) Number of base pairs affected by variants discovered per line, with lines ordered by depth of coverage (green dotted line). The line “Berkeley” is the reference line. (B) Number of unique variants by size (note that variants longer than 1,000 bp are grouped in a single x-coordinate). (C) Representation of variant density (0–10 SNPs/kb, 0–5 indels/kb, 0–5 complex variants/kb) across the euchromatic genome (concentric circles) in 50 kb bins. Large variants (>100 bp) mapping against a close homologous sequence (>90% sequence identify) are linked in the center with green lines representing intra-chromosomal- and black lines inter-chromosomal duplications. (D) Number of unique variants by number of lines.</p

Potential functional indels and complex variants in genes.

<p>Number in brackets indicates the number of genes affected.</p

Validation of <i>cis</i>-associations in F1.

<p>(A) Allelic imbalance measured for ∼7,100 transcripts in F1 (362/765 and reciprocal) with RNA-seq. Dots represent the fold-change (log2) between allele-specific reads counts. Red dots indicate transcripts with significant allelic imbalance in both crosses at a false discovery rate of 10%. Circles mark transcripts that demonstrate significant allelic imbalance and that were found to be associated with <i>cis</i>-eQTL in females (note that only <i>cis</i>-eQTLs were considered when the allele between both parental lines was not the same). (B) The proportion of <i>cis</i>-eQTL-associated transcripts that show allelic expression imbalance in F1s scales with the strength of the <i>cis</i>-eQTL (P<0.001, permutation-based, see Methods for details).</p

<i>Cis-</i>associations of variants with gene expression.

<p>(A) and (B) Variant density (blue) and significance of allele associations (red), in males around (A) the transcription start site (TSS) and (B) the transcription end site (TES) averaged out over all transcripts in a 10 kb window. The solid lines are cubic smoothing splines, fit to the data. Transcripts on both strands are orientated such that transcription takes place in the positive direction of the x-axis. The inlet in (A) corresponds to a 100 kb window length. (C) <i>cis-</i>eQTLs discovered in males, females, or both sexes (FDR<10%). (D) Breakdown of <i>cis-</i>eQTL-associated genes by sex. (E) Breakdown of <i>cis</i>-eQTL associated genes, discovered in males or females, by type of variant (<i>i.e.</i>, SNP and non-SNP).</p

Variants in genomic context.

<p>(A) Density of SNPs around variant breakpoints by variant type. The dashed lines show the SNP density at the same loci but in DGRP lines that do not have the variant. (B) and (C) Density of SNPs near indels with minor allele count 2 to 4 (B) and 11 to 19 (C). The dashed lines show the SNP density at the same locus for DGRP lines without the indel. If indels were mutagenic, one would expect enrichment for low allele count SNPs near the high allele count indels; instead, the allele count of the neighboring SNPs closely matches that of the indel. (D) Density of variants (reference bases affected per Mb) in selected genomic regions. (E) Number of indels in coding regions by indel size. Insertions are in red and deletions in blue. Bars representing indel sizes that are a multiple of three are coloured dark red and blue, respectively.</p

Examples of <i>cis-</i>eQTLs and their associated genes.

<p>DGRP lines in (A) and (B) are grouped by their allele. Male and female expression levels are depicted in blue and dark pink, respectively. (A) Sex-biased <i>cis</i>-eQTL. A SNP (3R:8,875,391) is associated with higher gene expression levels in females only. (B) Indel-based <i>cis</i>-eQTLs associated with gene expression. Two insertions (7 bp, 3L:332,512; 1 bp, 3L:332,594, r² = 0.20) are associated with markedly different expression levels in males and females. (C) <i>cis</i>-association overview. Plot illustrating the variant and association data for a single gene (<i>mthl9</i>) on a rolling window basis. The gene is shown on the top track, with UTRs in grey and coding regions in black. Significant <i>cis</i>-eQTLs are drawn below and color-coded by significance for each sex separately (red most significant). Linkage (r²>0.5) is shown by arcs, color-coded according to r², with higher values in red. Rows represent all 39 DGRP lines and the left column shows gene expression levels for each line and sex separately (red indicates the highest expression level and green the lowest). The grid contains a representation of variants in rolling 50 bp windows (successive windows overlapping by 45 bp) with net insertions in red, net deletions in blue, and variants not affecting the sequence size (mostly SNPs) in black. The height of each variant indicates the net size of variants with the window, up to 20 bp. The two shaded vertical bars mark the <i>cis</i>-eQTLs shown in (B).</p