Exome sequencing identifies breast cancer susceptibility genes and defines the contribution of coding variants to breast cancer risk.

Linkage and candidate gene studies have identified several breast cancer susceptibility genes, but the overall contribution of coding variation to breast cancer is unclear. To evaluate the role of rare coding variants more comprehensively, we performed a meta-analysis across three large whole-exome sequencing datasets, containing 26,368 female cases and 217,673 female controls. Burden tests were performed for protein-truncating and rare missense variants in 15,616 and 18,601 genes, respectively. Associations between protein-truncating variants and breast cancer were identified for the following six genes at exome-wide significance (P < 2.5 × 10-6): the five known susceptibility genes ATM, BRCA1, BRCA2, CHEK2 and PALB2, together with MAP3K1. Associations were also observed for LZTR1, ATR and BARD1 with P < 1 × 10-4. Associations between predicted deleterious rare missense or protein-truncating variants and breast cancer were additionally identified for CDKN2A at exome-wide significance. The overall contribution of coding variants in genes beyond the previously known genes is estimated to be small.

Uncovering the Contribution of Moderate-Penetrance Susceptibility Genes to Breast Cancer by Whole-Exome Sequencing and Targeted Enrichment Sequencing of Candidate Genes in Women of European Ancestry.

Rare variants in at least 10 genes, including BRCA1, BRCA2, PALB2, ATM, and CHEK2, are associated with increased risk of breast cancer; however, these variants, in combination with common variants identified through genome-wide association studies, explain only a fraction of the familial aggregation of the disease. To identify further susceptibility genes, we performed a two-stage whole-exome sequencing study. In the discovery stage, samples from 1528 breast cancer cases enriched for breast cancer susceptibility and 3733 geographically matched unaffected controls were sequenced. Using five different filtering and gene prioritization strategies, 198 genes were selected for further validation. These genes, and a panel of 32 known or suspected breast cancer susceptibility genes, were assessed in a validation set of 6211 cases and 6019 controls for their association with risk of breast cancer overall, and by estrogen receptor (ER) disease subtypes, using gene burden tests applied to loss-of-function and rare missense variants. Twenty genes showed nominal evidence of association (p-value < 0.05) with either overall or subtype-specific breast cancer. Our study had the statistical power to detect susceptibility genes with effect sizes similar to ATM, CHEK2, and PALB2, however, it was underpowered to identify genes in which susceptibility variants are rarer or confer smaller effect sizes. Larger sample sizes would be required in order to identify such genes.

Functional Analysis of Promoter Variants in Genes Involved in Sex Steroid Action, DNA Repair and Cell Cycle Control.

Genetic variants affecting the regulation of gene expression are among the main causes of human diversity. The potential importance of regulatory polymorphisms is underscored by results from Genome Wide Association Studies, which have already implicated such polymorphisms in the susceptibility to complex diseases such as breast cancer. In this study, we re-sequenced the promoter regions of 24 genes involved in pathways related to breast cancer including sex steroid action, DNA repair, and cell cycle control in 60 unrelated Caucasian individuals. We constructed haplotypes and assessed the functional impact of promoter variants using gene reporter assays and electrophoretic mobility shift assays. We identified putative functional variants within the promoter regions of estrogen receptor 1 (ESR1), ESR2, forkhead box A1 (FOXA1), ubiquitin interaction motif containing 1 (UIMC1) and cell division cycle 7 (CDC7). The functional polymorphism on CDC7, rs13447455, influences CDC7 transcriptional activity in an allele-specific manner and alters DNA⁻protein complex formation in breast cancer cell lines. Moreover, results from the Breast Cancer Association Consortium show a marginal association between rs13447455 and breast cancer risk (p=9.3x10-5), thus warranting further investigation. Furthermore, our study has helped provide methodological solutions to some technical difficulties that were encountered with gene reporter assays, particularly regarding inter-clone variability and statistical consistency.

Exome sequencing identifies mutations in the gene TTC7A in French-Canadian cases with hereditary multiple intestinal atresia.

BACKGROUND: Congenital multiple intestinal atresia (MIA) is a severe, fatal neonatal disorder, involving the occurrence of obstructions in the small and large intestines ultimately leading to organ failure. Surgical interventions are palliative but do not provide long-term survival. Severe immunodeficiency may be associated with the phenotype. A genetic basis for MIA is likely. We had previously ascertained a cohort of patients of French-Canadian origin, most of whom were deceased as infants or in utero. The goal of the study was to identify the molecular basis for the disease in the patients of this cohort. METHODS: We performed whole exome sequencing on samples from five patients of four families. Validation of mutations and familial segregation was performed using standard Sanger sequencing in these and three additional families with deceased cases. Exon skipping was assessed by reverse transcription-PCR and Sanger sequencing. RESULTS: Five patients from four different families were each homozygous for a four base intronic deletion in the gene TTC7A, immediately adjacent to a consensus GT splice donor site. The deletion was demonstrated to have deleterious effects on splicing causing the skipping of the attendant upstream coding exon, thereby leading to a predicted severe protein truncation. Parents were heterozygous carriers of the deletion in these families and in two additional families segregating affected cases. In a seventh family, an affected case was compound heterozygous for the same 4bp deletion and a second missense mutation p.L823P, also predicted as pathogenic. No other sequenced genes possessed deleterious variants explanatory for all patients in the cohort. Neither mutation was seen in a large set of control chromosomes. CONCLUSIONS: Based on our genetic results, TTC7A is the likely causal gene for MIA.

Co-variation of STI1 and WDR36/UTP21 alters cell proliferation in a glaucoma model.

PURPOSE: To investigate the role of multigenic variation in primary open-angle glaucoma (POAG) involving the rRNA processing gene WD repeat domain 36 (WDR36). METHODS: We examined the heat shock protein 70/90 (HSP70/90)-organizing co-chaperone stress-induced-phosphoprotein 1 (STI1) as a potential co-modifying gene in glaucoma patients found to harbor WDR36 amino acid variation. The STI1 gene was sequenced and its POAG-associated amino acid variant K434R, as well as the single nucleotide polymorphism (SNP) P173T, were tested for functional defects in a yeast model system previously used to characterize WDR36 variants (using the homologous yeast gene U3 protein 21 [UTP21]). RESULTS: A POAG patient heterozygous for the WDR36 variant L25P was discovered to also carry the STI1 variant K434R in a heterozygous state. Variant K434R, located at an evolutionarily-conserved site, was not found in a pool of clinically-examined individuals lacking WDR36 variation which included 55 normal controls and 20 patients with normal tension glaucoma (NTG). STI1 (K434R) and the homologous yeast variant K470R were able to rescue yeast growth-inhibition by the HSP90-inhibitor radicicol. Double mutant haploid strains expressing human STI1 (K434R) and recombinant yeast UTP21 variants did not have significantly different levels of 18S rRNA from the corresponding hSTI1 (WT) strains. However, specific double mutant K434R strains exhibited significantly slower culture growth at 37 °C. Double mutant P173T strains also displayed altered growth rates at 37 °C. CONCLUSIONS: STI1 variation does not play a significant direct role in the genetics of POAG. However, as previously found for the STI1 null allele, non-synonymous variants of human STI1 confer growth dysregulation in the context of specific yeast UTP21 mutations and heat stress. Based on the genetic association of two co-heterozygous STI1 and WDR36 variants in a POAG patient and the functional analyses performed in a model system for basic eukaryotic cellular processes, these experiments point to a conserved molecular pathway involving STI1 and WDR36.

How to translate therapeutic recommendations in clinical practice guidelines into rules for critiquing physician prescriptions? Methods and application to five guidelines.

BACKGROUND: Clinical practice guidelines give recommendations about what to do in various medical situations, including therapeutical recommendations for drug prescription. An effective way to computerize these recommendations is to design critiquing decision support systems, i.e. systems that criticize the physician's prescription when it does not conform to the guidelines. These systems are commonly based on a list of "if conditions then criticism" rules. However, writing these rules from the guidelines is not a trivial task. The objective of this article is to propose methods that (1) simplify the implementation of guidelines' therapeutical recommendations in critiquing systems by automatically translating structured therapeutical recommendations into a list of "if conditions then criticize" rules, and (2) can generate an appropriate textual label to explain to the physician why his/her prescription is not recommended. METHODS: We worked on the therapeutic recommendations in five clinical practice guidelines concerning chronic diseases related to the management of cardiovascular risk. We evaluated the system using a test base of more than 2000 cases. RESULTS: Algorithms for automatically translating therapeutical recommendations into "if conditions then criticize" rules are presented. Eight generic recommendations are also proposed; they are guideline-independent, and can be used as default behaviour for handling various situations that are usually implicit in the guidelines, such as decreasing the dose of a poorly tolerated drug. Finally, we provide models and methods for generating a human-readable textual critique. The system was successfully evaluated on the test base. CONCLUSION: We show that it is possible to criticize physicians' prescriptions starting from a structured clinical guideline, and to provide clear explanations. We are now planning a randomized clinical trial to evaluate the impact of the system on practices.

The novel Y371D myocilin mutation causes an aggressive form of juvenile open-angle glaucoma in a Caucasian family from the Middle-East.

PURPOSE: To search for the genetic cause of juvenile open-angle glaucoma (JOAG) in a Caucasian family and to perform genotype/phenotype correlation studies in the kindred. METHODS: Six members of a three-generation family originating from Uzbekistan and now living in the Middle East were recruited from one large clinic in Israel. Ophthalmologic investigations comprised of visual field assessments, intraocular pressure measurements, optic disc evaluation, and gonioscopy. Medical charts were obtained to date the onset of glaucoma and to evaluate aggressivity of the trait. We screened the myocilin gene (MYOC, OMIM 601652) by direct genomic sequencing of its three exons in all family members. RESULTS: JOAG segregated as an autosomal dominant trait in four members of the family. The proband, a 14-year-old girl, had been diagnosed with juvenile open-angle glaucoma at 12 years old. Her mother, maternal aunt, and maternal grandfather all had JOAG that started at an early age. The disorder progressed rapidly even under optimal medical treatment, and all four patients had to undergo trabeculectomy. One missense mutation, Y371D (1111t-->g, Tyr [Y] 371 Asp [D]), was identified. This mutation cosegregated with the disorder in all affected members and was absent in 200 Caucasian controls. The Y371D MYOC mutation has not been reported before. One cousin of the proband was a silent heterozygotic carrier of the mutation and was still asymptomatic at nine years of age. CONCLUSIONS: We identified a novel mutation (Y371D) in MYOC from a Caucasian family who presented with an aggressive form of JOAG that required early trabeculectomy. Genetic screening of the MYOC mutation was beneficial in predicting one asymptomatic heterozygotic carrier.

Glaucoma-associated WDR36 variants encode functional defects in a yeast model system.

Primary open-angle glaucoma (POAG) is a leading cause of blindness worldwide. POAG is associated with a characteristic progression of changes to ocular morphology and degeneration at the optic nerve head with the loss of visual fields. Physical mapping efforts identified genomic loci in which to search for causative POAG gene mutations. WDR36, at locus GLC1G, was initially identified as a gene with a low frequency of non-synonymous sequence variations that were exclusive to adult-onset POAG patients. It has since been shown that rare WDR36 sequence variants are also present in the normal population at similarly low frequencies. The lack of a consistent genotype:phenotype correlation prompted us to investigate the functional consequences of WDR36 sequence variations. WDR36 is involved in rRNA processing, a critical step in ribosome biogenesis, and is very similar to yeast Utp21p which is a member of the small subunit (SSU) processome complex responsible for maturation of 18S rRNA. We, therefore, developed a yeast model system to test the functional and phenotypic consequences of POAG-associated sequence variants introduced into UTP21. Alone, the POAG variants did not produce any significant defects in cell viability or rRNA processing. However, when combined with disruption of STI1 (which synthetically interacts with UTP21), 5 of the 11 tested variants had increased or decreased cell viability which corresponded to reduced or elevated levels of pre-rRNA, respectively. These results demonstrate that, in the correct genetic background, WDR36 sequence variants can lead to an altered cellular phenotype, supporting the theory that WDR36 participates in polygenic forms of glaucoma.

Design of a decision support system for chronic diseases coupling generic therapeutic algorithms with guideline-based specific rules.

Clinical Decision Support Systems (DSS) help improve health care quality. They usually incorporate an Execution Engine (EE), defined for each disease. We have designed, and present here, a generic execution engine, coupled with guideline-based disease specific rules stored in knowledge base (KB) as part of the prescription-critiquing mode of the ASTI project. This system was designed using two national guidelines for type 2 diabetes and hypertension. It takes into account the patient's clinical data, the tolerance and effectiveness of previous and current treatments and the physician's prescription made at the time. The functioning of the system has been speeded up and its maintenance made easier by indexing the KB rules according to the type of treatment they are linked to (e.g. monotherapy, etc.) and by classifying them into four categories. The EE's design formalizes generic therapeutic algorithms, leading to treatment options for cases of bad tolerance or insufficient effectiveness of the current treatment. Its applicability to other diseases was shown by applying it to dyslipidemia.

A genome-wide scan maps a novel juvenile-onset primary open angle glaucoma locus to chromosome 5q.

PURPOSE: To map the disease-associated locus of a family with autosomal dominant juvenile-onset primary open angle glaucoma (JOAG) and to screen the novel glaucoma gene WD repeat domain 36 (WDR36). METHODS: Complete ophthalmic examination and genomic DNA were obtained from 27 family members, in which nine were confirmed JOAG patients. Myocilin (MYOC), optineurin (OPTN), and WDR36 were screened for mutations by polymerase chain reaction and direct sequencing. Genome-wide scanning was carried out using the ABI PRISM Linkage Mapping Set MD-10. Two-point and multipoint linkage analyses were performed with the MLINK, ILINK, and LINKMAP programs. For fine mapping, additional markers flanking the most promising region on chromosome 5q were also analyzed. The significance of LOD scores was tested with simulation analyses using FASTLINK. Haplotypes were constructed using Simwalk2. RESULTS: MYOC or OPTN mutations were excluded in all family members. A maximum LOD score value of 4.82 at theta=0.00 was obtained for the marker D5S2011. Markers D5S2065, D5S1384, D5S471, D5S503, D5S2098, and D5S638 had LOD score values over 4.0 at theta=0.00. Haplotype analysis and recombination mapping further confined this region to 5q22.1-q32 within a region of 36 Mb flanked by D5S2051 and D5S2090. Screening of the novel WDR36 glaucoma-associated gene, which lies centromeric to the disease interval, revealed no mutations within any of the 23 coding exons or splicing junctions. CONCLUSIONS: Our results provided the mapping of a novel locus for JOAG at 5q and excluded coding or splicing junctions mutations within the WDR36 gene.

Axenfeld-Rieger anomaly: a novel mutation in the forkhead box C1 (FOXC1) gene in a 4-generation family.

OBJECTIVE: To characterize DNA mutations in a pedigree of Axenfeld-Rieger anomaly (ARA) (Online Mendelian Inheritance of Man 601631), a clinically and genetically heterogeneous, autosomal dominantly inherited disorder associated with anterior chamber abnormalities and glaucoma. DESIGN: Observational case-control and DNA linkage and screening studies. PARTICIPANTS: Affected (10 cases) and unaffected (5 controls) members of a family with ARA. METHODS: Clinical characteristics of ARA were documented by history or physical examination of symptomatic individuals. With their informed consent, a blood sample was collected from each of 10 affected and 5 unaffected family members. DNA was tested for linkage to the IRID1 locus at chromosome 6p25, a known locus for ARA/Rieger syndrome. A candidate gene previously mapped at this locus, FOXC1, was screened for mutations in cases and controls. Main Outcome Measure Linkage of the ARA phenotype at the 6p25 locus and mutation detected in FOXC1. RESULTS: Direct sequencing of FOXC1 detected a new mutation, T272C, that segregated with the ARA phenotype in this family and was not detected in DNA from family members without ARA. This mutation, a T-->C transition, is predicted to result in a change of isoleucine to threonine (Ile9lThr) in a highly conserved location within the first helix of the forkhead domain. CONCLUSION: Characterization of the FOXC1 mutation in family members with ARA furthers our understanding of the molecular origin of developmental glaucoma and other anterior segment disorders.

Gene expression profile of the human trabecular meshwork: NEIBank sequence tag analysis.

PURPOSE: To characterize the gene expression pattern in the human trabecular meshwork (TM) and identify candidate genes for glaucoma by expressed sequence tag (EST) analysis as part of the NEIBank project. METHODS: RNA was extracted from dissected human TM and used to construct unamplified, un-normalized cDNA libraries in the pSPORT1 vector. More than 4000 clones were sequenced from the 5' end. Clones were clustered and identified using GRIST software. In addition, the expression patterns of genes encoding olfactomedin-domain proteins were analyzed by RT-PCR. RESULTS: After non-mRNA contaminants were removed, 3459 independent TM-expressed clones were obtained. These were grouped in 1888 clusters, potentially representing individual expressed genes. Transcripts for the myocilin gene, a locus for inherited glaucoma, formed the third most abundant cluster in the TM collection, and several other genes implicated in glaucoma (PITX2, CYP1B1, and optineurin) were also represented. One abundant TM transcript was from the gene for the angiopoietin-like factor CTD6, which is located at on the long arm of chromosome 1, area 36.2-36.1 in the region of the glaucoma locus GLC3B, whereas other transcripts were from genes close to known glaucoma loci. The TM collection contains cDNAs for genes that are preferentially expressed in the lymphatic endothelium (matrix Gla protein, apolipoprotein D precursor, and selenoprotein P precursor). In addition to EST profiling, RT- PCR was used to detect transcripts of the olfactomedin-domain proteins latrotoxin receptor Lec3 and optimedin in the TM. CONCLUSIONS: The TM libraries are a good source of molecular markers for TM and candidate genes for glaucoma. The abundance of myocilin cDNAs corresponds to the critical role of this gene in glaucoma and contrasts with libraries derived from cultured tissue. The expression profile raises the possibility that cells of the TM and Schlemm's canal may be more similar to lymphatic, rather than blood vascular endothelium.

Founder TIGR/myocilin mutations for glaucoma in the Québec population.

Primary open-angle glaucoma (POAG) is a complex disorder characterized by a progressive and treatable degeneration of the optic nerve. TIGR/myocilin (MYOC) gene mutations are found in approximately 4% of all POAG patients. Populations with frequent founder effects, such as the French-Canadians, offer unique advantages to implement genetic testing for the disorder. To assess molecular diagnosis for POAG in this population, we determined the prevalence of TIGR/MYOC mutations in 384 unrelated glaucoma patients, 38 ocular hypertensive subjects and 18 affected families (180 patients). We further analyzed the clinical features associated with these variations. Nine coding sequence variants were defined as mutations causing mostly, but not exclusively, POAG. Four families segregated distinct mutations (Gly367Arg, Gln368Stop, Lys423Glu and Pro481Leu), while 14 unrelated glaucoma patients harbored six known mutations (Thr293Lys, Glu352Lys, Gly367Arg, Gln368Stop, Lys423Glu and Ala445Val) and two novel (Ala427Thr and Arg126Trp). The frequencies of these mutations were respectively 3.8% and 22.2% in the unrelated and family studies. The Gly367Arg and Lys423Glu variants caused the earliest ages at onset. When achievable, assessment of relatives of unrelated mutation carriers showed the Arg126Trp and Gly367Arg to be familial. Characteristic allele signatures, indicative of specific founder effects, were observed for five of the six mutations conveyed by at least two patients. Recombination probability estimates suggested that the French-Canadian population had most probably inherited these six mutations from 7-10 Québec settlers. Our data demonstrated that genetic screening for TIGR/MYOC mutations should be offered to glaucoma families and to close relatives of unrelated patients aware of a family history for the disorder.