Genome-wide association study on 13 167 individuals identifies regulators of blood CD34+cell levels.

Stem cell transplantation is a cornerstone in the treatment of blood malignancies. The most common method to harvest stem cells for transplantation is by leukapheresis, requiring mobilization of CD34+ hematopoietic stem and progenitor cells (HSPCs) from the bone marrow into the blood. Identifying the genetic factors that control blood CD34+ cell levels could reveal new drug targets for HSPC mobilization. Here we report the first large-scale, genome-wide association study on blood CD34+ cell levels. Across 13 167 individuals, we identify 9 significant and 2 suggestive associations, accounted for by 8 loci (PPM1H, CXCR4, ENO1-RERE, ITGA9, ARHGAP45, CEBPA, TERT, and MYC). Notably, 4 of the identified associations map to CXCR4, showing that bona fide regulators of blood CD34+ cell levels can be identified through genetic variation. Further, the most significant association maps to PPM1H, encoding a serine/threonine phosphatase never previously implicated in HSPC biology. PPM1H is expressed in HSPCs, and the allele that confers higher blood CD34+ cell levels downregulates PPM1H. Through functional fine-mapping, we find that this downregulation is caused by the variant rs772557-A, which abrogates an MYB transcription factor-binding site in PPM1H intron 1 that is active in specific HSPC subpopulations, including hematopoietic stem cells, and interacts with the promoter by chromatin looping. Furthermore, PPM1H knockdown increases the proportion of CD34+ and CD34+90+ cells in cord blood assays. Our results provide the first large-scale analysis of the genetic architecture of blood CD34+ cell levels and warrant further investigation of PPM1H as a potential inhibition target for stem cell mobilization.

Clinical characteristics and outcome of 318 families with familial monoclonal gammopathy: A multicenter Intergroupe Francophone du Myélome study.

Familial forms of monoclonal gammopathy, defined as multiple myeloma (MM) or Monoclonal Gammopathy of Undetermined Significance (MGUS), are relatively infrequent and most series reported in the literature describe a limited number of families. MM rarely occurs in a familial context. MGUS is observed much more commonly, which can in some cases evolve toward full-blown MM. Although recurrent cytogenetic abnormalities have been described in tumor cells of sporadic cases of MM, the pathogenesis of familial MM remains largely unexplained. In order to identify genetic factors predisposing to familial monoclonal gammopathy, the Intergroupe Francophone du Myélome identified 318 families with at least two confirmed cases of monoclonal gammopathy. There were 169 families with parent/child pairs and 164 families with cases in at least two siblings, compatible with an autosomal transmission. These familial cases were compared with sporadic cases who were matched for age at diagnosis, sex and immunoglobulin isotype, with 10 sporadic cases for each familial case. The gender distribution, age and immunoglobulin subtypes of familial cases were unremarkable in comparison to sporadic cases. With a median follow-up of 7.4 years after diagnosis, the percentage of MGUS cases having evolved to MM was 3%. The median overall survival of the 148 familial MM cases was longer than that of matched sporadic cases, with projected values of 7.6 and 16.1 years in patients older and younger than 65 years, respectively. These data suggest that familial cases of monoclonal gammopathy are similar to sporadic cases in terms of clinical presentation and carry a better prognosis.

No evidence that protein truncating variants in BRIP1 are associated with breast cancer risk: implications for gene panel testing.

BACKGROUND: BRCA1 interacting protein C-terminal helicase 1 (BRIP1) is one of the Fanconi Anaemia Complementation (FANC) group family of DNA repair proteins. Biallelic mutations in BRIP1 are responsible for FANC group J, and previous studies have also suggested that rare protein truncating variants in BRIP1 are associated with an increased risk of breast cancer. These studies have led to inclusion of BRIP1 on targeted sequencing panels for breast cancer risk prediction. METHODS: We evaluated a truncating variant, p.Arg798Ter (rs137852986), and 10 missense variants of BRIP1, in 48 144 cases and 43 607 controls of European origin, drawn from 41 studies participating in the Breast Cancer Association Consortium (BCAC). Additionally, we sequenced the coding regions of BRIP1 in 13 213 cases and 5242 controls from the UK, 1313 cases and 1123 controls from three population-based studies as part of the Breast Cancer Family Registry, and 1853 familial cases and 2001 controls from Australia. RESULTS: The rare truncating allele of rs137852986 was observed in 23 cases and 18 controls in Europeans in BCAC (OR 1.09, 95% CI 0.58 to 2.03, p=0.79). Truncating variants were found in the sequencing studies in 34 cases (0.21%) and 19 controls (0.23%) (combined OR 0.90, 95% CI 0.48 to 1.70, p=0.75). CONCLUSIONS: These results suggest that truncating variants in BRIP1, and in particular p.Arg798Ter, are not associated with a substantial increase in breast cancer risk. Such observations have important implications for the reporting of results from breast cancer screening panels.

Common variants at the 9q22.33, 14q13.3 and ATM loci, and risk of differentiated thyroid cancer in the Cuban population.

BACKGROUND: The incidence of differentiated thyroid carcinoma (DTC) in Cuba is low and the contribution of host genetic factors to DTC in this population has not been investigated so far. Our goal was to assess the role of known risk polymorphisms in DTC cases living in Havana. We genotyped five polymorphisms located at the DTC susceptibility loci on chromosome 14q13.3 near NK2 homeobox 1 (NKX2-1), on chromosome 9q22.33 near Forkhead factor E1 (FOXE1) and within the DNA repair gene Ataxia-Telangiectasia Mutated (ATM) in 203 cases and 212 age- and sex- matched controls. Potential interactions between these polymorphisms and other DTC risk factors such as body surface area, body mass index, size, ethnicity, and, for women, the parity were also examined. RESULTS: Significant association with DTC risk was found for rs944289 near NKX2-1 (OR per A allele = 1.6, 95% CI: 1.2-2.1), and three polymorphisms near or within FOXE1, namely rs965513 (OR per A allele = 1.7, 95% CI: 1.2-2.3), rs1867277 in the promoter region of the gene (OR per A allele = 1.5, 95% CI: 1.1-1.9) and the poly-alanine tract expansion polymorphism rs71369530 (OR per Long Allele = 1.8, 95% CI: 1.3-2.5), only the 2 latter remaining significant when correcting for multiple tests. Overall, no association between DTC and the coding SNP D1853N (rs1801516) in ATM (OR per A Allele = 1.1, 95% CI: 0.7-1.7) was seen. Nevertheless women who had 2 or more pregnancies had a 3.5-fold increase in risk of DTC if they carried the A allele (OR 3.5, 95% CI: 3.2-9.8) as compared to 0.8 (OR 0.8, 95% CI: 0.4-1.6) in those who had fewer than 2. CONCLUSIONS: We confirmed in the Cuban population the role of the loci previously associated with DTC susceptibility in European and Japanese populations through genome-wide association studies. Our results on ATM and the number of pregnancies raise interesting questions on the mechanisms by which oestrogens, or other hormones, alter the DNA damage response and DNA repair through the regulation of key effector proteins such as ATM. Due to the small size of our study and to multiple tests, all these results warrant further investigation.

Rare key functional domain missense substitutions in MRE11A, RAD50, and NBN contribute to breast cancer susceptibility: results from a Breast Cancer Family Registry case-control mutation-screening study.

INTRODUCTION: The MRE11A-RAD50-Nibrin (MRN) complex plays several critical roles related to repair of DNA double-strand breaks. Inherited mutations in the three components predispose to genetic instability disorders and the MRN genes have been implicated in breast cancer susceptibility, but the underlying data are not entirely convincing. Here, we address two related questions: (1) are some rare MRN variants intermediate-risk breast cancer susceptibility alleles, and if so (2) do the MRN genes follow a BRCA1/BRCA2 pattern wherein most susceptibility alleles are protein-truncating variants, or do they follow an ATM/CHEK2 pattern wherein half or more of the susceptibility alleles are missense substitutions? METHODS: Using high-resolution melt curve analysis followed by Sanger sequencing, we mutation screened the coding exons and proximal splice junction regions of the MRN genes in 1,313 early-onset breast cancer cases and 1,123 population controls. Rare variants in the three genes were pooled using bioinformatics methods similar to those previously applied to ATM, BRCA1, BRCA2, and CHEK2, and then assessed by logistic regression. RESULTS: Re-analysis of our ATM, BRCA1, and BRCA2 mutation screening data revealed that these genes do not harbor pathogenic alleles (other than modest-risk SNPs) with minor allele frequencies>0.1% in Caucasian Americans, African Americans, or East Asians. Limiting our MRN analyses to variants with allele frequencies of <0.1% and combining protein-truncating variants, likely spliceogenic variants, and key functional domain rare missense substitutions, we found significant evidence that the MRN genes are indeed intermediate-risk breast cancer susceptibility genes (odds ratio (OR)=2.88, P=0.0090). Key domain missense substitutions were more frequent than the truncating variants (24 versus 12 observations) and conferred a slightly higher OR (3.07 versus 2.61) with a lower P value (0.029 versus 0.14). CONCLUSIONS: These data establish that MRE11A, RAD50, and NBN are intermediate-risk breast cancer susceptibility genes. Like ATM and CHEK2, their spectrum of pathogenic variants includes a relatively high proportion of missense substitutions. However, the data neither establish whether variants in each of the three genes are best evaluated under the same analysis model nor achieve clinically actionable classification of individual variants observed in this study.

Contribution of ATM and FOXE1 (TTF2) to risk of papillary thyroid carcinoma in Belarusian children exposed to radiation.

A dramatic increase in the incidence of papillary thyroid carcinoma (PTC) after childhood exposure to ionizing radiation from the Chernobyl nuclear accident has been described as the largest number of tumors of one type due to one cause that have ever occurred. inter-individual variations in response to radiation have been documented and the role of genetics in sporadic PTC is well established, suggesting that genetic factors may also affect the risk of radiation-related PTC. To investigate how environmental and host factors interplay to modify PTC risk, we genotyped 83 cases and 324 matched controls sampled from children living in the area contaminated by fallout from the Chernobyl power plant accident for 19 polymorphisms previously associated with PTC, thyroid biology or radiation-induced second primary tumors. Significant association with PTC was found for rs1801516 (D1853N) in ATM (odds ratio (OR) = 0.34, 95% confidence interval (CI) 0.16, 0.73) and rs1867277 in the promoter region of FOXE1 (OR = 1.55, 95% CI 1.03, 2.34). Analysis of additional polymorphisms confirmed the association between these two genes and PTC. Our findings suggest that both DNA double-strand break repair pathway and thyroid morphogenesis pathway or dysregulation of thyroid differentiated state maintenance are involved in the etiology of PTC, and that the studied genetic polymorphisms and radiation dose appear to act as independent multiplicative risk factors for PTC.

Mosaic chromosomal alterations in hematopoietic cells and clinical outcomes in patients with multiple myeloma.

Mosaic chromosomal alterations (mCAs) in hematopoietic cells increase mortality and risk of hematological cancers and infections. We investigated the landscape of mCAs and their clinical consequences in 976 patients with multiple myeloma undergoing high-dose chemotherapy and autologous stem cell support (ASCT) with median 6.4 years of follow-up. mCAs were detected in the stem cell harvest product of 158 patients (16.2%). Autosomal aberrations were found in 60 patients (6.1%) and affected all chromosomes. Loss of chromosome X was found in 51 females (12.7%) and loss of chromosome Y in 55 males (9.6%). Overall survival and progression were similar between carriers of autosomal mCAs and non-carriers. In contrast, female patients with loss of the X chromosome had longer overall survival (age-adjusted[a.a.] HR 0.54, 95% CI 0.32-0.93, p = 0.02), lower risk of progression (a.a. HR 0.55, 95% CI 0.35-0.87; p = 0.01), and better post-transplant response (higher degree of complete response (CR) or very good partial response (VGPR)). The reason for this substantial effect is unknown. Additionally, myeloma clones in the stem cell product was confirmed by mCA analysis in the few patients with multiple mCAs (n = 12 patients). Multiple mCAs conferred inferior overall survival (a.a. HR 2.0, 95% CI 1.02-3.84; p = 0.04) and higher risk of myeloma progression (a.a. HR 3.36, 95% CI 1.67-6.81; p < 0.001), which is presumed to be driven by suspected myeloma contaminants.

Deciphering the genetics and mechanisms of predisposition to multiple myeloma.

Multiple myeloma (MM) is an incurable malignancy of plasma cells. Epidemiological studies indicate a substantial heritable component, but the underlying mechanisms remain unclear. Here, in a genome-wide association study totaling 10,906 cases and 366,221 controls, we identify 35 MM risk loci, 12 of which are novel. Through functional fine-mapping and Mendelian randomization, we uncover two causal mechanisms for inherited MM risk: longer telomeres; and elevated levels of B-cell maturation antigen (BCMA) and interleukin-5 receptor alpha (IL5RA) in plasma. The largest increase in BCMA and IL5RA levels is mediated by the risk variant rs34562254-A at TNFRSF13B. While individuals with loss-of-function variants in TNFRSF13B develop B-cell immunodeficiency, rs34562254-A exerts a gain-of-function effect, increasing MM risk through amplified B-cell responses. Our results represent an analysis of genetic MM predisposition, highlighting causal mechanisms contributing to MM development.

Functional and molecular profiling of hematopoietic stem cells during regeneration.

Hematopoietic stem cells (HSCs) enable hematopoietic stem cell transplantation (HCT) through their ability to replenish the entire blood system. Proliferation of HSCs is linked to decreased reconstitution potential, and a precise regulation of actively dividing HSCs is thus essential to ensure long-term functionality. This regulation becomes important in the transplantation setting where HSCs undergo proliferation followed by a gradual transition to quiescence and homeostasis. Although mouse HSCs have been well studied under homeostatic conditions, the mechanisms regulating HSC activation under stress remain unclear. Here, we analyzed the different phases of regeneration after transplantation. We isolated bone marrow from mice at 8 time points after transplantation and examined the reconstitution dynamics and transcriptional profiles of stem and progenitor populations. We found that regenerating HSCs initially produced rapidly expanding progenitors and displayed distinct changes in fatty acid metabolism and glycolysis. Moreover, we observed molecular changes in cell cycle, MYC and mTOR signaling in both HSCs, and progenitor subsets. We used a decay rate model to fit the temporal transcription profiles of regenerating HSCs and identified genes with progressively decreased or increased expression after transplantation. These genes overlapped to a large extent with published gene sets associated with key aspects of HSC function, demonstrating the potential of this data set as a resource for identification of novel HSC regulators. Taken together, our study provides a detailed functional and molecular characterization of HSCs at different phases of regeneration and identifies a gene set associated with the transition from proliferation to quiescence.

Prevalence of BRCA1 mutations among 403 women with triple-negative breast cancer: implications for genetic screening selection criteria: a Hellenic Cooperative Oncology Group Study.

In spite the close association of the triple-negative breast cancer immunophenotype with hereditary breast cancers and the BRCA1 pathway, there is a lack of population studies that determine the frequency of BRCA1 mutations among triple-negative breast cancer patients. To address this, we have screened a large sample of 403 women diagnosed with triple-negative invasive breast cancer, independently of their age or family history, for germline BRCA1 mutations. Median age at diagnosis was 50 years (range 20-83). The overall prevalence of triple-negative cases among the initial patient group with invasive breast cancer was 8%. BRCA1 was screened by direct DNA sequencing in all patients, including all exons where a mutation was previously found in the Greek population (exons 5, 11, 12, 16, 20, 21, 22, 23, 24-77% of the BRCA1 coding region), including diagnostic PCRs to detect the three Greek founder large genomic rearrangements. Sixty-five deleterious BRCA1 mutations were identified among the 403 triple-negative breast cancer patients (16%). Median age of onset for mutation carriers was 39 years. Among a total of 106 women with early-onset triple-negative breast cancer (<40 years), 38 (36%) had a BRCA1 mutation, while 27% of women with triple-negative breast cancer diagnosed before 50 years (56/208) had a BRCA1 mutation. A mutation was found in 48% (50/105) of the triple-negative breast cancer patients with family history of breast or ovarian cancer. It is noteworthy, however, that of the 65 carriers, 15 (23%) had no reported family history of related cancers. All but one of the carriers had grade III tumors (98%). These results indicate that women with early-onset triple-negative breast cancer, and ideally all triple-negative breast cancer patients, are candidates for BRCA1 genetic testing even in the absence of a family history of breast or ovarian cancer.

Functional dissection of inherited non-coding variation influencing multiple myeloma risk.

Thousands of non-coding variants have been associated with increased risk of human diseases, yet the causal variants and their mechanisms-of-action remain obscure. In an integrative study combining massively parallel reporter assays (MPRA), expression analyses (eQTL, meQTL, PCHiC) and chromatin accessibility analyses in primary cells (caQTL), we investigate 1,039 variants associated with multiple myeloma (MM). We demonstrate that MM susceptibility is mediated by gene-regulatory changes in plasma cells and B-cells, and identify putative causal variants at six risk loci (SMARCD3, WAC, ELL2, CDCA7L, CEP120, and PREX1). Notably, three of these variants co-localize with significant plasma cell caQTLs, signaling the presence of causal activity at these precise genomic positions in an endogenous chromosomal context in vivo. Our results provide a systematic functional dissection of risk loci for a hematologic malignancy.

Common breast cancer susceptibility alleles are associated with tumour subtypes in BRCA1 and BRCA2 mutation carriers: results from the Consortium of Investigators of Modifiers of BRCA1/2.

INTRODUCTION: Previous studies have demonstrated that common breast cancer susceptibility alleles are differentially associated with breast cancer risk for BRCA1 and/or BRCA2 mutation carriers. It is currently unknown how these alleles are associated with different breast cancer subtypes in BRCA1 and BRCA2 mutation carriers defined by estrogen (ER) or progesterone receptor (PR) status of the tumour. METHODS: We used genotype data on up to 11,421 BRCA1 and 7,080 BRCA2 carriers, of whom 4,310 had been affected with breast cancer and had information on either ER or PR status of the tumour, to assess the associations of 12 loci with breast cancer tumour characteristics. Associations were evaluated using a retrospective cohort approach. RESULTS: The results suggested stronger associations with ER-positive breast cancer than ER-negative for 11 loci in both BRCA1 and BRCA2 carriers. Among BRCA1 carriers, single nucleotide polymorphism (SNP) rs2981582 (FGFR2) exhibited the biggest difference based on ER status (per-allele hazard ratio (HR) for ER-positive = 1.35, 95% CI: 1.17 to 1.56 vs HR = 0.91, 95% CI: 0.85 to 0.98 for ER-negative, P-heterogeneity = 6.5 × 10-6). In contrast, SNP rs2046210 at 6q25.1 near ESR1 was primarily associated with ER-negative breast cancer risk for both BRCA1 and BRCA2 carriers. In BRCA2 carriers, SNPs in FGFR2, TOX3, LSP1, SLC4A7/NEK10, 5p12, 2q35, and 1p11.2 were significantly associated with ER-positive but not ER-negative disease. Similar results were observed when differentiating breast cancer cases by PR status. CONCLUSIONS: The associations of the 12 SNPs with risk for BRCA1 and BRCA2 carriers differ by ER-positive or ER-negative breast cancer status. The apparent differences in SNP associations between BRCA1 and BRCA2 carriers, and non-carriers, may be explicable by differences in the prevalence of tumour subtypes. As more risk modifying variants are identified, incorporating these associations into breast cancer subtype-specific risk models may improve clinical management for mutation carriers.

International distribution and age estimation of the Portuguese BRCA2 c.156_157insAlu founder mutation.

The c.156_157insAlu BRCA2 mutation has so far only been reported in hereditary breast/ovarian cancer (HBOC) families of Portuguese origin. Since this mutation is not detectable using the commonly used screening methodologies and must be specifically sought, we screened for this rearrangement in a total of 5,443 suspected HBOC families from several countries. Whereas the c.156_157insAlu BRCA2 mutation was detected in 11 of 149 suspected HBOC families from Portugal, representing 37.9% of all deleterious mutations, in other countries it was detected only in one proband living in France and in four individuals requesting predictive testing living in France and in the USA, all being Portuguese immigrants. After performing an extensive haplotype study in carrier families, we estimate that this founder mutation occurred 558 ± 215 years ago. We further demonstrate significant quantitative differences regarding the production of the BRCA2 full length RNA and the transcript lacking exon 3 in c.156_157insAlu BRCA2 mutation carriers and in controls. The cumulative incidence of breast cancer in carriers did not differ from that of other BRCA2 and BRCA1 pathogenic mutations. We recommend that all suspected HBOC families from Portugal or with Portuguese ancestry are specifically tested for this rearrangement.

Accelerating target deconvolution for therapeutic antibody candidates using highly parallelized genome editing.

Therapeutic antibodies are transforming the treatment of cancer and autoimmune diseases. Today, a key challenge is finding antibodies against new targets. Phenotypic discovery promises to achieve this by enabling discovery of antibodies with therapeutic potential without specifying the molecular target a priori. Yet, deconvoluting the targets of phenotypically discovered antibodies remains a bottleneck; efficient deconvolution methods are needed for phenotypic discovery to reach its full potential. Here, we report a comprehensive investigation of a target deconvolution approach based on pooled CRISPR/Cas9. Applying this approach within three real-world phenotypic discovery programs, we rapidly deconvolute the targets of 38 of 39 test antibodies (97%), a success rate far higher than with existing approaches. Moreover, the approach scales well, requires much less work, and robustly identifies antibodies against the major histocompatibility complex. Our data establish CRISPR/Cas9 as a highly efficient target deconvolution approach, with immediate implications for the development of antibody-based drugs.

Germline variants at SOHLH2 influence multiple myeloma risk.

Multiple myeloma (MM) is caused by the uncontrolled, clonal expansion of plasma cells. While there is epidemiological evidence for inherited susceptibility, the molecular basis remains incompletely understood. We report a genome-wide association study totalling 5,320 cases and 422,289 controls from four Nordic populations, and find a novel MM risk variant at SOHLH2 at 13q13.3 (risk allele frequency = 3.5%; odds ratio = 1.38; P = 2.2 × 10-14). This gene encodes a transcription factor involved in gametogenesis that is normally only weakly expressed in plasma cells. The association is represented by 14 variants in linkage disequilibrium. Among these, rs75712673 maps to a genomic region with open chromatin in plasma cells, and upregulates SOHLH2 in this cell type. Moreover, rs75712673 influences transcriptional activity in luciferase assays, and shows a chromatin looping interaction with the SOHLH2 promoter. Our work provides novel insight into MM susceptibility.

Genetic predisposition for multiple myeloma.

Multiple myeloma (MM) is the second most common blood malignancy. Epidemiological family studies going back to the 1920s have provided evidence for familial aggregation, suggesting a subset of cases have an inherited genetic background. Recently, studies aimed at explaining this phenomenon have begun to provide direct evidence for genetic predisposition to MM. Genome-wide association studies have identified common risk alleles at 24 independent loci. Sequencing studies of familial cases and kindreds have begun to identify promising candidate genes where variants with strong effects on MM risk might reside. Finally, functional studies are starting to give insight into how identified risk alleles promote the development of MM. Here, we review recent findings in MM predisposition field, and highlight open questions and future directions.

Correction: S100A6 is a critical regulator of hematopoietic stem cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

S100A6 is a critical regulator of hematopoietic stem cells.

The fate options of hematopoietic stem cells (HSCs) include self-renewal, differentiation, migration, and apoptosis. HSCs self-renewal divisions in stem cells are required for rapid regeneration during tissue damage and stress, but how precisely intracellular calcium signals are regulated to maintain fate options in normal hematopoiesis is unclear. S100A6 knockout (KO) HSCs have reduced total cell numbers in the HSC compartment, decreased myeloid output, and increased apoptotic HSC numbers in steady state. S100A6KO HSCs had impaired self-renewal and regenerative capacity, not responding to 5-Fluorouracil. Our transcriptomic and proteomic profiling suggested that S100A6 is a critical HSC regulator. Intriguingly, S100A6KO HSCs showed decreased levels of phosphorylated Akt (p-Akt) and Hsp90, with an impairment of mitochondrial respiratory capacity and a reduction of mitochondrial calcium levels. We showed that S100A6 regulates intracellular and mitochondria calcium buffering of HSC upon cytokine stimulation and have demonstrated that Akt activator SC79 reverts the levels of intracellular and mitochondrial calcium in HSC. Hematopoietic colony-forming activity and the Hsp90 activity of S100A6KO are restored through activation of the Akt pathway. We show that p-Akt is the prime downstream mechanism of S100A6 in the regulation of HSC self-renewal by specifically governing mitochondrial metabolic function and Hsp90 protein quality.