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.

A Large Case-Control Study Performed in Spanish Population Suggests That RECQL5 Is the Only RECQ Helicase Involved in Breast Cancer Susceptibility.

Around 50% of the familial breast cancer (BC) cases are estimated to be caused by germline variants in known low-, moderate-, and high-risk susceptibility genes, while the other half is of unknown genetic origin. In the present study, we wanted to evaluate the role of the RECQ helicases, some of which have been studied in the past as candidates, with unclear results about their role in the disease. Using next-generation sequencing (NGS) technology, we analyzed the whole coding sequence of BLM, RECQL1, RECQL4, RECQL5, and WRN in almost 2000 index cases from BC Spanish families that had previously tested negative for the known BC susceptibility genes (BRCAX) and compared the results with the controls extracted from gnomAD. Our results suggest that BLM, RECQL1, RECQL4, and WRN do not play a major role in BC susceptibility. However, in the combined analysis, joining the present results with those previously reported in a series of 1334 BC Spanish patients and controls, we found a statistically significant association between Loss of Function (LoF) variants in RECQL5 and BC risk, with an OR of 2.56 (p = 0.009; 95% CI, 1.18-4.98). Our findings support our previous work and places the RECQL5 gene as a new moderate-risk BC gene.

POLRMT as a Novel Susceptibility Gene for Cardiotoxicity in Epirubicin Treatment of Breast Cancer Patients.

Anthracyclines are among the most used chemotherapeutic agents in breast cancer (BC). However their use is hampered by anthracycline-induced cardiotoxicity (AIC). The currently known clinical and genetic risk factors do not fully explain the observed inter-individual variability and only have a limited ability to predict which patients are more likely to develop this severe toxicity. To identify novel predictive genes, we conducted a two-stage genome-wide association study in epirubicin-treated BC patients. In the discovery phase, we genotyped over 700,000 single nucleotide variants in a cohort of 227 patients. The most interesting finding was rs62134260, located 4kb upstream of POLRMT (OR = 5.76, P = 2.23 × 10-5). We replicated this association in a validation cohort of 123 patients (P = 0.021). This variant regulates the expression of POLRMT, a gene that encodes a mitochondrial DNA-directed RNA polymerase, responsible for mitochondrial gene expression. Individuals harbouring the risk allele had a decreased expression of POLRMT in heart tissue that may cause an impaired capacity to maintain a healthy mitochondrial population in cardiomyocytes under stressful conditions, as is treatment with epirubicin. This finding suggests a novel molecular mechanism involved in the development of AIC and may improve our ability to predict patients who are at risk.

Breast Cancer Risk Genes - Association Analysis in More than 113,000 Women.

BACKGROUND: Genetic testing for breast cancer susceptibility is widely used, but for many genes, evidence of an association with breast cancer is weak, underlying risk estimates are imprecise, and reliable subtype-specific risk estimates are lacking. METHODS: We used a panel of 34 putative susceptibility genes to perform sequencing on samples from 60,466 women with breast cancer and 53,461 controls. In separate analyses for protein-truncating variants and rare missense variants in these genes, we estimated odds ratios for breast cancer overall and tumor subtypes. We evaluated missense-variant associations according to domain and classification of pathogenicity. RESULTS: Protein-truncating variants in 5 genes (ATM, BRCA1, BRCA2, CHEK2, and PALB2) were associated with a risk of breast cancer overall with a P value of less than 0.0001. Protein-truncating variants in 4 other genes (BARD1, RAD51C, RAD51D, and TP53) were associated with a risk of breast cancer overall with a P value of less than 0.05 and a Bayesian false-discovery probability of less than 0.05. For protein-truncating variants in 19 of the remaining 25 genes, the upper limit of the 95% confidence interval of the odds ratio for breast cancer overall was less than 2.0. For protein-truncating variants in ATM and CHEK2, odds ratios were higher for estrogen receptor (ER)-positive disease than for ER-negative disease; for protein-truncating variants in BARD1, BRCA1, BRCA2, PALB2, RAD51C, and RAD51D, odds ratios were higher for ER-negative disease than for ER-positive disease. Rare missense variants (in aggregate) in ATM, CHEK2, and TP53 were associated with a risk of breast cancer overall with a P value of less than 0.001. For BRCA1, BRCA2, and TP53, missense variants (in aggregate) that would be classified as pathogenic according to standard criteria were associated with a risk of breast cancer overall, with the risk being similar to that of protein-truncating variants. CONCLUSIONS: The results of this study define the genes that are most clinically useful for inclusion on panels for the prediction of breast cancer risk, as well as provide estimates of the risks associated with protein-truncating variants, to guide genetic counseling. (Funded by European Union Horizon 2020 programs and others.).

Regulatory CDH4 Genetic Variants Associate With Risk to Develop Capecitabine-Induced Hand-Foot Syndrome.

Capecitabine-induced hand-foot syndrome (CiHFS) is a common dermatological adverse reaction affecting around 30% of patients with capecitabine-treated cancer, and the main cause of dose reductions and chemotherapy delays. To identify novel genetic factors associated with CiHFS in patients with cancer, we carried out an extreme-phenotype genomewide association study in 166 patients with breast and colorectal capecitabine-treated cancer with replication in a second cohort of 85 patients. We discovered and replicated a cluster of four highly correlated single-nucleotide polymorphisms associated with susceptibility to CiHFS at 20q13.33 locus (top hit = rs6129058, hazard ratio = 2.40, 95% confidence interval = 1.78-3.20; P = 1.2 × 10-8 ). Using circular chromosome conformation capture sequencing, we identified a chromatin contact between the locus containing the risk alleles and the promoter of CDH4, located 90 kilobases away. The risk haplotype was associated with decreased levels of CDH4 mRNA and the protein it encodes, R-cadherin (RCAD), which mainly localizes in the granular layer of the epidermis. In human keratinocytes, CDH4 downregulation resulted in reduced expression of involucrin, a protein of the cornified envelope, an essential structure for skin barrier function. Immunohistochemical analyses revealed that skin from patients with severe CiHFS exhibited low levels of RCAD and involucrin before capecitabine treatment. Our results uncover a novel mechanism underlying individual genetic susceptibility to CiHFS with implications for clinically relevant risk prediction.

Association Between ABCB1 Genetic Variants and Persistent Chemotherapy-Induced Alopecia in Women With Breast Cancer.

Importance: Persistent chemotherapy-induced alopecia (pCIA) has been recently described in patients with breast cancer and in its most severe form occurs in up to 10% of these patients. Genetic risk factors associated with pCIA have not been adequately explored. Objective: To identify genetic variants associated with pCIA. Design, Setting, and Participants: In this genetic association study, 215 women with breast cancer treated with docetaxel-based chemotherapy with a follow-up of 1.5 to 10 years after the end of the treatment were recruited retrospectively through 3 hospital oncology units across Spain between 2005 and 2018. Severe pCIA was defined as lack of scalp hair recovery (Common Terminology Criteria for Adverse Events, version 3.0, grade 2) 18 months or more after the end of treatment. Patients with grade 2 pCIA were selected as cases, and those with no sign of residual alopecia 12 months after the end of docetaxel treatment were selected as controls. A genome-wide association study in a discovery phase was conducted, and logistic regression was used to identify variants associated with the risk to develop this adverse effect. The validity of the association was addressed through a replication phase. Exposures: Docetaxel-based chemotherapy. Main Outcomes and Measures: Genotypes of single-nucleotide variants associated with pCIA. Results: In total, 215 women with breast cancer (median age, 51.6 years; interquartile range, 44-60 years) were recruited (173 patients for the discovery phase and 42 patients for the replication phase). In the discovery phase, ABCB1 genetic variants were associated with risk to develop pCIA. In particular, single-nucleotide variation rs1202179, a regulatory variant located in an enhancer element that interacts with the ABCB1 promoter, was associated with the occurrence of pCIA. This finding was validated in the replication cohort (combined odds ratio, 4.05; 95% CI, 2.46-6.67; P = 3.946 × 10-8). This variant is associated with ABCB1 mRNA expression, and the risk allele was associated with decreased ABCB1 expression levels (P = 1.64 × 10-20). Conclusions and Relevance: This is the first study, to our knowledge, that identifies an association between a regulatory variant in the ABCB1 gene and the occurrence of pCIA in patients with breast cancer who were treated with docetaxel-based therapies. This finding suggests an important insight into the biological mechanisms underlying pCIA and opens the opportunity to explore personalized treatment of these patients.

BRAF V600E Detection in Liquid Biopsies from Pediatric Central Nervous System Tumors.

Pediatric Central Nervous System (CNS) tumors are the most fatal cancer diseases in childhood. Due to their localization and infiltrative nature, some tumor resections or biopsies are not feasible. In those cases, the use of minimally invasive methods as diagnostic, molecular marker detection, prognostic or monitoring therapies are emerging. The analysis of liquid biopsies which contain genetic information from the tumor has been much more widely explored in adults than in children. We compare the detection of BRAF V600E targetable mutation by digital-PCR from cell-free-DNA and EV-derived DNA (ctDNA) in serum, plasma and cerebrospinal fluid (CSF) isolated from a cohort of 29 CNS pediatric patients. Here we demonstrate that ctDNA isolated from serum and plasma could be successfully analyzed to obtain tumor genetic information which could be used to guide critical treatment decisions.

Contributions of PHOX2B in the pathogenesis of Hirschsprung disease.

Hirschsprung disease (HSCR) is a congenital malformation of the hindgut resulting from a disruption of neural crest cell migration during embryonic development. It has a complex genetic aetiology with several genes involved in its pathogenesis. PHOX2B plays a key function in the development of neural crest derivatives, and heterozygous mutations cause a complex dysautonomia associating HSCR, Congenital Central Hypoventilation Syndrome (CCHS) and neuroblastoma (NB) in various combinations. In order to determine the role of PHOX2B in isolated HSCR, we performed a mutational screening in a cohort of 207 Spanish HSCR patients. Our most relevant finding has been the identification of a de novo and novel deletion (c.393_410del18) in a patient with HSCR. Results of in silico and functional assays support its pathogenic effect related to HSCR. Therefore our results support that PHOX2B loss-of-function is a rare cause of HSCR phenotype.

Comprehensive analysis of NRG1 common and rare variants in Hirschsprung patients.

Hirschsprung disease (HSCR, OMIM 142623) is a developmental disorder characterized by the absence of ganglion cells along variable lengths of the distal gastrointestinal tract, which results in tonic contraction of the aganglionic gut segment and functional intestinal obstruction. The RET proto-oncogene is the major gene for HSCR with differential contributions of its rare and common, coding and noncoding mutations to the multifactorial nature of this pathology. Many other genes have been described to be associated with the pathology, as NRG1 gene (8p12), encoding neuregulin 1, which is implicated in the development of the enteric nervous system (ENS), and seems to contribute by both common and rare variants. Here we present the results of a comprehensive analysis of the NRG1 gene in the context of the disease in a series of 207 Spanish HSCR patients, by both mutational screening of its coding sequence and evaluation of 3 common tag SNPs as low penetrance susceptibility factors, finding some potentially damaging variants which we have functionally characterized. All of them were found to be associated with a significant reduction of the normal NRG1 protein levels. The fact that those mutations analyzed alter NRG1 protein would suggest that they would be related with HSCR disease not only in Chinese but also in a Caucasian population, which reinforces the implication of NRG1 gene in this pathology.

Association of X-linked hydrocephalus and Hirschsprung disease: report of a new patient with a mutation in the L1CAM gene.

X-linked hydrocephalus (XLH) has an incidence of 1/30,000 male births and is characterized by intellectual disability, spastic paraplegia, adducted thumbs, and agenesis of corpus callosum, and/or corticospinal tract. The great proportion of cases is ascribed to loss of function mutations of L1CAM gene. Hirschsprung disease (HSCR) is characterized by the absence of ganglion cells in the submucosal and myenteric plexuses along a variable portion of the intestinal tract and has incidence of about 1/5,000. Although with several genes involved in its pathogenesis, the major HSCR gene is the RET proto-oncogene. To date only a few patients have been reported with both phenotypes and mutations in the L1CAM gene. In this report, we describe a new patient with concurrent XLH and HSCR. L1CAM mutational screening showed the presence of the G698R hemizygous mutation, which is a deleterious substitution affecting a key residue necessary for the correct folding of the protein. Moreover, the patient also carried the transcriptional enhancer RET mutation (c.73 + 9277T > C) in heterozygosis. We speculate that both the RET enhancer variant, and the L1CAM mutation may act in combination to produce the enteric phenotype, probably with the participation of other still unidentified molecular events. While it is obvious that additional studies are necessary to further delineate the association between XLH and HSCR in the presence of L1CAM mutations, the documentation of this new patient reinforces the role of this gene acting either in a direct or indirect way in the pathogenesis of Hirschsprung disease.

Comprehensive analysis of RET common and rare variants in a series of Spanish Hirschsprung patients confirms a synergistic effect of both kinds of events.

BACKGROUND: RET is the major gene associated to Hirschsprung disease (HSCR) with differential contributions of its rare and common, coding and noncoding mutations to the multifactorial nature of this pathology. In the present study, we have performed a comprehensive study of our HSCR series evaluating the involvement of both RET rare variants (RVs) and common variants (CVs) in the context of the disease. METHODS: RET mutational screening was performed by dHPLC and direct sequencing for the identification of RVs. In addition Taqman technology was applied for the genotyping of 3 RET CVs previously associated to HSCR, including a variant lying in an enhancer domain within RET intron 1 (rs2435357). Statistical analyses were performed using the SPSS v.17.0 to analyze the distribution of the variants. RESULTS: Our results confirm the strongest association to HSCR for the "enhancer" variant, and demonstrate a significantly higher impact of it in male versus female patients. Integration of the RET RVs and CVs analysis showed that in 91.66% of cases with both kinds of mutational events, the enhancer allele is in trans with the allele bearing the RET RV. CONCLUSIONS: A gender effect exists on both the transmission and distribution of rare coding and common HSCR causing mutations. In addition, these RET CVs and RVs seem to act in a synergistic way leading to HSCR phenotype.

Expression of PROKR1 and PROKR2 in human enteric neural precursor cells and identification of sequence variants suggest a role in HSCR.

BACKGROUND: The enteric nervous system (ENS) is entirely derived from neural crest and its normal development is regulated by specific molecular pathways. Failure in complete ENS formation results in aganglionic gut conditions such as Hirschsprung's disease (HSCR). Recently, PROKR1 expression has been demonstrated in mouse enteric neural crest derived cells and Prok-1 was shown to work coordinately with GDNF in the development of the ENS. PRINCIPAL FINDINGS: In the present report, ENS progenitors were isolated and characterized from the ganglionic gut from children diagnosed with and without HSCR, and the expression of prokineticin receptors was examined. Immunocytochemical analysis of neurosphere-forming cells demonstrated that both PROKR1 and PROKR2 were present in human enteric neural crest cells. In addition, we also performed a mutational analysis of PROKR1, PROKR2, PROK1 and PROK2 genes in a cohort of HSCR patients, evaluating them for the first time as susceptibility genes for the disease. Several missense variants were detected, most of them affecting highly conserved amino acid residues of the protein and located in functional domains of both receptors, which suggests a possible deleterious effect in their biological function. CONCLUSIONS: Our results suggest that not only PROKR1, but also PROKR2 might mediate a complementary signalling to the RET/GFRα1/GDNF pathway supporting proliferation/survival and differentiation of precursor cells during ENS development. These findings, together with the detection of sequence variants in PROKR1, PROK1 and PROKR2 genes associated to HSCR and, in some cases in combination with RET or GDNF mutations, provide the first evidence to consider them as susceptibility genes for HSCR.

A novel study of copy number variations in Hirschsprung disease using the multiple ligation-dependent probe amplification (MLPA) technique.

BACKGROUND: Hirschsprung disease (HSCR) is a congenital malformation of the hindgut produced by a disruption in neural crest cell migration during embryonic development. HSCR has a complex genetic etiology and mutations in several genes, mainly the RET proto-oncogene, have been related to the disease. There is a clear predominance of missense/nonsense mutations in these genes whereas copy number variations (CNVs) have been seldom described, probably due to the limitations of conventional techniques usually employed for mutational analysis. METHODS: In this study we have aimed to analyze the presence of CNVs in some HSCR genes (RET, EDN3, GDNF and ZFHX1B) using the Multiple Ligation-dependent Probe Amplification (MLPA) approach. RESULTS: Two alterations in the MLPA profiles of RET and EDN3 were detected, but a detailed inspection showed that the decrease in the corresponding dosages were due to point mutations affecting the hybridization probes regions. CONCLUSION: Our results indicate that CNVs of the gene coding regions analyzed here are not a common molecular cause of Hirschsprung disease. However, further studies are required to determine the presence of CNVs affecting non-coding regulatory regions, as well as other candidate genes.