Identification of novel sarcoma risk genes using a two-stage genome wide DNA sequencing strategy in cancer cluster families and population case and control cohorts.

BACKGROUND: Although familial clustering of cancers is relatively common, only a small proportion of familial cancer risk can be explained by known cancer predisposition genes. METHODS: In this study we employed a two-stage approach to identify candidate sarcoma risk genes. First, we conducted whole exome sequencing in three multigenerational cancer families ascertained through a sarcoma proband (n = 19) in order to prioritize candidate genes for validation in an independent case-control cohort of sarcoma patients using family-based association and segregation analysis. The second stage employed a burden analysis of rare variants within prioritized candidate genes identified from stage one in 560 sarcoma cases and 1144 healthy ageing controls, for which whole genome sequence was available. RESULTS: Variants from eight genes were identified in stage one. Following gene-based burden testing and after correction for multiple testing, two of these genes, ABCB5 and C16orf96, were determined to show statistically significant association with cancer. The ABCB5 gene was found to have a higher burden of putative regulatory variants (OR = 4.9, p-value = 0.007, q-value = 0.04) based on allele counts in sarcoma cases compared to controls. C16orf96, was found to have a significantly lower burden (OR = 0.58, p-value = 0.0004, q-value = 0.003) of regulatory variants in controls compared to sarcoma cases. CONCLUSIONS: Based on these genetic association data we propose that ABCB5 and C16orf96 are novel candidate risk genes for sarcoma. Although neither of these two genes have been previously associated with sarcoma, ABCB5 has been shown to share clinical drug resistance associations with melanoma and leukaemia and C16orf96 shares regulatory elements with genes that are involved with TNF-alpha mediated apoptosis in a p53/TP53-dependent manner. Future genetic studies in other family and population cohorts will be required for further validation of these novel findings.

Whole-exome sequencing in multiplex preeclampsia families identifies novel candidate susceptibility genes.

OBJECTIVE: Preeclampsia is a common and serious heritable disorder of human pregnancy. Although there have been notable successes in identification of maternal susceptibility genes a large proportion of the heritability of preeclampsia remains unaccounted for. It is has been postulated that rare variation may account for some of this missing heritability. In this study, we performed whole-exome sequencing (WES) in multiplex families to identify rare exonic risk variants. METHODS: We conducted WES in 244 individuals from 34 Australian/New Zealand multiplex preeclampsia families. Variants were tested for association with preeclampsia using a threshold model and logistic regression. RESULTS: We found significant association for two moderately rare missense variants, rs145743393 (Padj = 0.0032, minor allele frequency = 0.016) in the chromosome 1 open reading frame 35 (C1orf35) gene, and rs34270076 (Padj = 0.0128, minor allele frequency = 0.024) in the pyroglutamylated RFamide peptide receptor (QRFPR) gene. To replicate these associations we performed imputation in our Australian genome wide association scan for preeclampsia and found no significant exonic variants in either C1orf35 or QRFPR. However, 11 variants demonstrating nominal significance (P < 0.05) in the genomic region between QRFPR and annexin A5 (ANXA5) were identified. We further leveraged publicly available genome-wide available summary data from the UK Biobank to investigate association of these two variants with the underlying clinical phenotypes of preeclampsia and detected nominal association of the QRFPR variant (rs34270076, P = 0.03) with protein levels in females. CONCLUSION: The study represents the first to use WES in multiplex families for preeclampsia and identifies two novel genes (QRFPR and C1orf35) not previously associated with preeclampsia and find nominal association of rs34270076 with protein levels, a key clinical feature of preeclampsia. We find further support for ANXA5 previously associated with pregnancy complications, including preeclampsia.

Analysis of tandem E-box motifs within human Complement receptor 2 (CR2/CD21) promoter reveals cell specific roles for RP58, E2A, USF and localized chromatin accessibility.

Complement receptor 2 (CR2/CD21) plays an important role in the generation of normal B cell immune responses. As transcription appears to be the prime mechanism via which surface CR2/CD21 expression is controlled, understanding transcriptional regulation of this gene will have broader implications to B cell biology. Here we report opposing, cell-context specific control of CR2/CD21 promoter activity by tandem E-box elements, spaced 22 bp apart and within 70 bp of the transcription initiation site. We have identified E2A and USF transcription factors as binding to the distal and proximal E-box sites respectively in CR2-positive B-cells, at a site that is hypersensitive to restriction enzyme digestion compared to non-expressing K562 cells. However, additional unidentified proteins have also been found to bind these functionally important elements. By utilizing a proteomics approach we have identified a repressor protein, RP58, binding the distal E-box motif. Co-transfection experiments using RP58 overexpression constructs demonstrated a specific 10-fold repression of CR2/CD21 transcriptional activity mediated through the distal E-box repressor element. Taken together, our results indicate that repression of the CR2/CD21 promoter can occur through one of the E-box motifs via recruitment of RP58 and other factors to bring about a silenced chromatin context within CR2/CD21 non-expressing cells.

Molecular prioritization strategies to identify functional genetic variants in the cardiovascular disease-associated expression QTL Vanin-1.

There is now good evidence that non-coding sequence variants are involved in the heritability of many common complex traits. The current 'gold standard' approach for assessing functionality is the in vitro reporter gene assay to assess allelic differences in transcriptional activity, usually followed by electrophoretic mobility shift assays to assess allelic differences in transcription factor binding. Although widely used, these assays have inherent limitations, including the lack of endogenous chromatin context. Here we present a more contemporary approach to assessing functionality of non-coding sequence variation within the Vanin-1 (VNN1) promoter. By combining 'gold standard' assays with in vivo assessments of chromatin accessibility, we greatly increase our confidence in the statistically assigned functional relevance. The standard assays revealed the -137 single nucleotide variant to be functional but the -587 variant to have no functional relevance. However, our in vivo tests show an allelic difference in chromatin accessibility surrounding the -587 variant supporting strong functional potential at both sites. Our approach advances the identification of functional variants by providing strong in vivo biological evidence for function.

Novel mutations in the calcium-sensing receptor gene associated with biochemical and functional differences in familial hypocalciuric hypercalcaemia.

OBJECTIVE: Heterozygous inactivating mutations of the calcium-sensing receptor (CaR) gene cause familial hypocalciuric hypercalcaemia (FHH), a generally benign disorder characterized by mild to moderate PTH-dependent hypercalcaemia. We aimed to identify the causative CaR mutations in three families with FHH and examine the correlation between type of mutation and biochemical and/or functional phenotypes. PATIENTS, DESIGN AND MEASUREMENTS: The CaR gene from FHH family members was assessed for mutations by direct DNA sequencing and mutations were confirmed by restriction enzyme analysis. Functional studies on two missense mutations were conducted by introducing them by site-directed mutagenesis into the CaR cloned into a mammalian expression vector, and assessing calcium responsiveness using an inositol phosphate (IP) assay in HEK293 cells. Biochemical data from patients heterozygous for each type of mutant were correlated with functionality. RESULTS: Two novel nonsense mutations (R25stop and K323stop) and one novel missense mutation (G778D) were identified. The G778D mutant receptor and another mutation identified in an earlier study (L174R) demonstrated a complete lack of Ca2+ responsiveness using the IP assay. When cotransfected with wild-type receptor, the mutant receptors demonstrated a dominant-negative effect on wild-type receptor response, with L174R having a more pronounced effect than G778D. Significantly more severe hypercalcaemia and a trend towards higher PTH levels were observed in patients heterozygous for CaR mutants with a stronger dominant-negative effect. CONCLUSIONS: Naturally occurring CaR mutations with differences in dominant-negative effect on wild-type receptor demonstrate differences in biochemical severity in FHH.