Pathogenic and likely pathogenic variants in PALB2, CHEK2, and other known breast cancer susceptibility genes among 1054 BRCA-negative Hispanics with breast cancer.

BACKGROUND: Breast cancer (BC) is the most common cancer and related cause of mortality among Hispanics, yet susceptibility has been understudied. BRCA1 and BRCA2 (BRCA) mutations explain less than one-half of hereditary BC, and the proportion associated with other BC susceptibility genes is unknown. METHODS: Germline DNA from 1054 BRCA-mutation-negative Hispanic women with hereditary BC (BC diagnosed at age <51 years, bilateral BC, breast and ovarian cancer, or BC diagnosed at ages 51-70 years with ≥2 first-degree or second-degree relatives who had BC diagnosed at age <70 years), 312 local controls, and 887 multiethnic cohort controls was sequenced and analyzed for 12 known and suspected, high-penetrance and moderate-penetrance cancer susceptibility genes (ataxia telangiectasia mutated [ATM], breast cancer 1 interacting protein C-terminal helicase 1 [BRIP1], cadherin 1 [CDH1], checkpoint kinase 2 [CHEK2], nibrin [NBN], neurofibromatosis type 1 [NF1], partner and localizer of BRCA2 [PALB2], phosphatase and tensin homolog [PTEN], RAD51 paralog 3 [RAD51C], RAD51D, serine/threonine kinase 11 [STK11], and TP53). RESULTS: Forty-nine (4.6%) pathogenic or likely pathogenic variants (PVs) in 47 of 1054 participants (4.5%), including 21 truncating frameshift, 20 missense, 5 nonsense, and 4 splice variants, were identified in CHEK2 (n = 20), PALB2 (n = 18), ATM (n = 5), TP53 (n = 3), BRIP1 (n = 2), and CDH1 and NF1 (both n = 1) and none were identified in NBN, PTEN, STK11, RAD51C, or RAD51D. Nine participants carried the PALB2 c.2167_2168del PV (0.85%), and 14 carried the CHEK2 c.707T>C PV (1.32%). CONCLUSIONS: Of 1054 BRCA-negative, high-risk Hispanic women, 4.5% carried a PV in a cancer susceptibility gene, increasing understanding of hereditary BC in this population. Recurrent PVs in PALB2 and CHEK2 represented 47% (23 of 49) of the total, suggesting a founder effect. Accurate classification of variants was enabled by carefully controlling for ancestry and the increased identification of at-risk Hispanics for screening and prevention.

Haplotype analyses of the c.1027C>T and c.2167_2168delAT recurrent truncating mutations in the breast cancer-predisposing gene PALB2.

PURPOSE: Breast cancer-predisposing mutations PALB2 c.1027C>T (p.Gln343*) and PALB2 c.2167_2168delAT have each been observed multiple times in breast cancer families of Italian ancestry. More recently, the c2167_2168delAT mutation was identified in unrelated breast cancer cases of various ancestries. For each mutation, we investigated whether the origin was multiple mutational events (a "hot-spot") or a single event (a founder allele). METHODS: We genotyped and reconstructed haplotypes for 36 participants of Italian, Italian-American, Hispanic, and Nigerian ancestries, using seven short tandem repeat (STR) markers that covered 3 Megabases within and flanking PALB2 on chromosome 16. RESULTS: For PALB2 c.1027C>T, a shared haplotype with a minimum size of 150 kb was shared by all 19 carriers investigated, all of Italian ancestry. This result suggests that this allele arose as a single event in a shared ancestor. For PALB2 c.2167_2168delAT, all 12 carriers from American-Italian and Italian families shared a 1-Mb haplotype, the 3 Hispanic carriers shared a different haplotype of size 2 Mb, and the Nigerian carrier had different alleles at all 7 STR markers. These results suggest that PALB2 c.2167_2168delAT arose multiple times, but that within each population, PALB2 c.2167_2168delAT likely represents a single mutational event. CONCLUSION: We identified two PALB2 mutations that are founder alleles in Italian families, one of which is, independently, also a founder mutation in American-Hispanic breast cancers.

Arabidopsis calmodulin-binding protein IQ67-domain 1 localizes to microtubules and interacts with kinesin light chain-related protein-1.

Calcium (Ca(2+)) is a key second messenger in eukaryotes and regulates diverse cellular processes, most notably via calmodulin (CaM). In Arabidopsis thaliana, IQD1 (IQ67 domain 1) is the founding member of the IQD family of putative CaM targets. The 33 predicted IQD proteins share a conserved domain of 67 amino acids that is characterized by a unique arrangement of multiple CaM recruitment motifs, including so-called IQ motifs. Whereas IQD1 has been implicated in the regulation of defense metabolism, the biochemical functions of IQD proteins remain to be elucidated. In this study we show that IQD1 binds to multiple Arabidopsis CaM and CaM-like (CML) proteins in vitro and in yeast two-hybrid interaction assays. CaM overlay assays revealed moderate affinity of IQD1 to CaM2 (K(d) ∼ 0.6 µm). Deletion mapping of IQD1 demonstrated the importance of the IQ67 domain for CaM2 binding in vitro, which is corroborated by interaction of the shortest IQD member, IQD20, with Arabidopsis CaM/CMLs in yeast. A genetic screen of a cDNA library identified Arabidopsis kinesin light chain-related protein-1 (KLCR1) as an IQD1 interactor. The subcellular localization of GFP-tagged IQD1 proteins to microtubules and the cell nucleus in transiently and stably transformed plant tissues (tobacco leaves and Arabidopsis seedlings) suggests direct interaction of IQD1 and KLCR1 in planta that is supported by GFP∼IQD1-dependent recruitment of RFP∼KLCR1 and RFP∼CaM2 to microtubules. Collectively, the prospect arises that IQD1 and related proteins provide Ca(2+)/CaM-regulated scaffolds for facilitating cellular transport of specific cargo along microtubular tracks via kinesin motor proteins.

Variable number tandem repeats (VNTRs) as modifiers of breast cancer risk in carriers of BRCA1 185delAG.

Despite substantial efforts in identifying both rare and common variants affecting disease risk, in the majority of diseases, a large proportion of unexplained genetic risk remains. We propose that variable number tandem repeats (VNTRs) may explain a proportion of the missing genetic risk. Herein, in a pilot study with a retrospective cohort design, we tested whether VNTRs are causal modifiers of breast cancer risk in 347 female carriers of the BRCA1 185delAG pathogenic variant, an important group given their high risk of developing breast cancer. We performed targeted-capture to sequence VNTRs, called genotypes with adVNTR, tested the association of VNTRs and breast cancer risk using Cox regression models, and estimated the effect size using a retrospective likelihood approach. Of 303 VNTRs that passed quality control checks, 4 VNTRs were significantly associated with risk to develop breast cancer at false discovery rate [FDR] < 0.05 and an additional 4 VNTRs had FDR < 0.25. After determining the specific risk alleles, there was a significantly earlier age at diagnosis of breast cancer in carriers of the risk alleles compared to those without the risk alleles for seven of eight VNTRs. One example is a VNTR in exon 2 of LINC01973 with a per-allele hazard ratio of 1.58 (1.07-2.33) and 5.28 (2.79-9.99) for the homozygous risk-allele genotype. Results from this first systematic study of VNTRs demonstrate that VNTRs may explain a proportion of the unexplained genetic risk for breast cancer.

Whole exome sequencing and replication for breast cancer among Hispanic/Latino women identifies FANCM as a susceptibility gene for estrogen-receptor-negative breast cancer.

Introduction: Breast cancer (BC) is one of the most common cancers globally. Genetic testing can facilitate screening and risk-reducing recommendations, and inform use of targeted treatments. However, genes included in testing panels are from studies of European-ancestry participants. We sequenced Hispanic/Latina (H/L) women to identify BC susceptibility genes. Methods: We conducted a pooled BC case-control analysis in H/L women from the San Francisco Bay area, Los Angeles County, and Mexico (4,178 cases and 4,344 controls). Whole exome sequencing was conducted on 1,043 cases and 1,188 controls and a targeted 857-gene panel on the remaining samples. Using ancestry-adjusted SKAT-O analyses, we tested the association of loss of function (LoF) variants with overall, estrogen receptor (ER)-positive, and ER-negative BC risk. We calculated odds ratios (OR) for BC using ancestry-adjusted logistic regression models. We also tested the association of single variants with BC risk. Results: We saw a strong association of LoF variants in FANCM with ER-negative BC (p=4.1×10-7, OR [CI]: 6.7 [2.9-15.6]) and a nominal association with overall BC risk. Among known susceptibility genes, BRCA1 (p=2.3×10-10, OR [CI]: 24.9 [6.1-102.5]), BRCA2 (p=8.4×10-10, OR [CI]: 7.0 [3.5-14.0]), and PALB2 (p=1.8×10-8, OR [CI]: 6.5 [3.2-13.1]) were strongly associated with BC. There were nominally significant associations with CHEK2, RAD51D, and TP53. Conclusion: In H/L women, LoF variants in FANCM were strongly associated with ER-negative breast cancer risk. It previously was proposed as a possible susceptibility gene for ER-negative BC, but is not routinely tested in clinical practice. Our results demonstrate that FANCM should be added to BC gene panels.

Profiling the Somatic Mutational Landscape of Breast Tumors from Hispanic/Latina Women Reveals Conserved and Unique Characteristics.

Somatic mutational profiling is increasingly being used to identify potential targets for breast cancer. However, limited tumor-sequencing data from Hispanic/Latinas (H/L) are available to guide treatment. To address this gap, we performed whole-exome sequencing (WES) and RNA sequencing on 146 tumors and WES of matched germline DNA from 140 H/L women in California. Tumor intrinsic subtype, somatic mutations, copy-number alterations, and expression profiles of the tumors were characterized and compared with data from tumors of non-Hispanic White (White) women in The Cancer Genome Atlas (TCGA). Eight genes were significantly mutated in the H/L tumors including PIK3CA, TP53, GATA3, MAP3K1, CDH1, CBFB, PTEN, and RUNX1; the prevalence of mutations in these genes was similar to that observed in White women in TCGA. Four previously reported Catalogue of Somatic Mutations in Cancer (COSMIC) mutation signatures (1, 2, 3, 13) were found in the H/L dataset, along with signature 16 that has not been previously reported in other breast cancer datasets. Recurrent amplifications were observed in breast cancer drivers including MYC, FGFR1, CCND1, and ERBB2, as well as a recurrent amplification in 17q11.2 associated with high KIAA0100 gene expression that has been implicated in breast cancer aggressiveness. In conclusion, this study identified a higher prevalence of COSMIC signature 16 and a recurrent copy-number amplification affecting expression of KIAA0100 in breast tumors from H/L compared with White women. These results highlight the necessity of studying underrepresented populations. SIGNIFICANCE: Comprehensive characterization of genomic and transcriptomic alterations in breast tumors from Hispanic/Latina patients reveals distinct genetic alterations and signatures, demonstrating the importance of inclusive studies to ensure equitable care for patients. See related commentary by Schmit et al., p. 2443.

ER-resident proteins PDR2 and LPR1 mediate the developmental response of root meristems to phosphate availability.

Inadequate availability of inorganic phosphate (Pi) in the rhizosphere is a common challenge to plants, which activate metabolic and developmental responses to maximize Pi acquisition. The sensory mechanisms that monitor environmental Pi status and regulate root growth via altered meristem activity are unknown. Here, we show that PHOSPHATE DEFICIENCY RESPONSE 2 (PDR2) encodes the single P(5)-type ATPase of Arabidopsis thaliana. PDR2 functions in the endoplasmic reticulum (ER) and is required for proper expression of SCARECROW (SCR), a key regulator of root patterning, and for stem-cell maintenance in Pi-deprived roots. We further show that the multicopper oxidase encoded by LOW PHOSPHATE ROOT 1 (LPR1) is targeted to the ER and that LPR1 and PDR2 interact genetically. Because the expression domains of both genes overlap in the stem-cell niche and distal root meristem, we propose that PDR2 and LPR1 function together in an ER-resident pathway that adjusts root meristem activity to external Pi. Our data indicate that the Pi-conditional root phenotype of pdr2 is not caused by increased Fe availability in low Pi; however, Fe homeostasis modifies the developmental response of root meristems to Pi availability.

Alternative mRNA splicing produces a novel biologically active short isoform of PGC-1alpha.

The transcriptional co-activator PGC-1alpha regulates functional plasticity in adipose tissue by linking sympathetic input to the transcriptional program of adaptive thermogenesis. We report here a novel truncated form of PGC-1alpha (NT-PGC-1alpha) produced by alternative 3' splicing that introduces an in-frame stop codon into PGC-1alpha mRNA. The expressed protein includes the first 267 amino acids of PGC-1alpha and 3 additional amino acids from the splicing insert. NT-PGC-1alpha contains the transactivation and nuclear receptor interaction domains but is missing key domains involved in nuclear localization, interaction with other transcription factors, and protein degradation. Expression and subcellular localization of NT-PGC-1alpha are dynamically regulated in the context of physiological signals that regulate full-length PGC-1alpha, but the truncated domain structure conveys unique properties with respect to protein-protein interactions, protein stability, and recruitment to target gene promoters. Therefore, NT-PGC-1alpha is a co-expressed, previously unrecognized form of PGC-1alpha with functions that are both unique from and complementary to PGC-1alpha.

The RAD52 S346X variant reduces risk of developing breast cancer in carriers of pathogenic germline BRCA2 mutations.

Women who carry pathogenic mutations in BRCA1 and BRCA2 have a lifetime risk of developing breast cancer of up to 80%. However, risk estimates vary in part due to genetic modifiers. We investigated the association of the RAD52 S346X variant as a modifier of the risk of developing breast and ovarian cancers in BRCA1 and BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2. The RAD52 S346X allele was associated with a reduced risk of developing breast cancer in BRCA2 carriers [per-allele hazard ratio (HR) = 0.69, 95% confidence interval (CI) 0.56-0.86; P = 0.0008] and to a lesser extent in BRCA1 carriers (per-allele HR = 0.78, 95% CI 0.64-0.97, P = 0.02). We examined how this variant affected DNA repair. Using a reporter system that measures repair of DNA double-strand breaks (DSBs) by single-strand annealing (SSA), expression of hRAD52 suppressed the loss of this repair in Rad52-/- mouse embryonic stem cells. When hRAD52 S346X was expressed in these cells, there was a significantly reduced frequency of SSA. Interestingly, expression of hRAD52 S346X also reduced the stimulation of SSA observed upon depletion of BRCA2, demonstrating the reciprocal roles for RAD52 and BRCA2 in the control of DSB repair by SSA. From an immunofluorescence analysis, we observed little nuclear localization of the mutant protein as compared to the wild-type; it is likely that the reduced nuclear levels of RAD52 S346X explain the diminished DSB repair by SSA. Altogether, we identified a genetic modifier that protects against breast cancer in women who carry pathogenic mutations in BRCA2 (P = 0.0008) and to a lesser extent BRCA1 (P = 0.02). This RAD52 mutation causes a reduction in DSB repair by SSA, suggesting that defects in RAD52-dependent DSB repair are linked to reduced tumor risk in BRCA2-mutation carriers.

Variants of the human RAD52 gene confer defects in ionizing radiation resistance and homologous recombination repair in budding yeast.

RAD52 is a structurally and functionally conserved component of the DNA double-strand break (DSB) repair apparatus from budding yeast to humans. We recently showed that expressing the human gene, HsRAD52 in rad52 mutant budding yeast cells can suppress both their ionizing radiation (IR) sensitivity and homologous recombination repair (HRR) defects. Intriguingly, we observed that HsRAD52 supports DSB repair by a mechanism of HRR that conserves genome structure and is independent of the canonical HR machinery. In this study we report that naturally occurring variants of HsRAD52, one of which suppresses the pathogenicity of BRCA2 mutations, were unable to suppress the IR sensitivity and HRR defects of rad52 mutant yeast cells, but fully suppressed a defect in DSB repair by single-strand annealing (SSA). This failure to suppress both IR sensitivity and the HRR defect correlated with an inability of HsRAD52 protein to associate with and drive an interaction between genomic sequences during DSB repair by HRR. These results suggest that HsRAD52 supports multiple, distinct DSB repair apparatuses in budding yeast cells and help further define its mechanism of action in HRR. They also imply that disruption of HsRAD52-dependent HRR in BRCA2-defective human cells may contribute to protection against tumorigenesis and provide a target for killing BRCA2-defective cancers.

Polyunsaturated fatty acid suppression of fatty acid synthase (FASN): evidence for dietary modulation of NF-Y binding to the Fasn promoter by SREBP-1c.

Dietary PUFAs (polyunsaturated fatty acids) co-ordinately suppress transcription of a group of hepatic genes encoding glycolytic and lipogenic enzymes. Suppression of Fasn (fatty acid synthase) transcription involves two PUFA-responsive regions, but the majority of PUFA sensitivity maps to a region within the proximal promoter containing binding sites for NF-Y (nuclear factor-Y), Sp1 (stimulatory protein 1), SREBP (sterol-regulatory-elementbinding protein), and USF (upstream stimulatory factor). Promoter activation assays indicate that altered NF-Y is the key component in regulation of Fasn promoter activity by PUFA. Using electrophoretic mobility-shift assay and chromatin immunoprecipitation analysis, we demonstrate for the first time that PUFAs decrease in vivo binding of NF-Y and SREBP-1c to the proximal promoter of the hepatic Fasn gene and the promoters of three additional genes, spot 14, stearoyl-CoA desaturase and farnesyl diphosphate synthase that are also down-regulated by PUFA. The comparable 50% decrease in NF-Y and SREBP-1c binding to the promoters of the respective PUFA-sensitive genes occurred despite no change in nuclear NF-Y content and a 4-fold decrease in SREBP-1c. Together, these findings support a mechanism whereby PUFA reciprocally regulates the binding of NF-Y and SREBP-1c to a subset of genes which share similar contiguous arrangements of sterol regulatory elements and NF-Y response elements within their promoters. PUFA-dependent regulation of SREBP-1c and NF-Y binding to this unique configuration of response elements may represent a nutrient-sensitive motif through which PUFA selectively and co-ordinately targets subsets of hepatic genes involved in lipid metabolism.

Methylator-induced, mismatch repair-dependent G2 arrest is activated through Chk1 and Chk2.

SN1 DNA methylating agents such as the nitrosourea N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) elicit a G2/M checkpoint response via a mismatch repair (MMR) system-dependent mechanism; however, the exact nature of the mechanism governing MNNG-induced G2/M arrest and how MMR mechanistically participates in this process are unknown. Here, we show that MNNG exposure results in activation of the cell cycle checkpoint kinases ATM, Chk1, and Chk2, each of which has been implicated in the triggering of the G2/M checkpoint response. We document that MNNG induces a robust, dose-dependent G2 arrest in MMR and ATM-proficient cells, whereas this response is abrogated in MMR-deficient cells and attenuated in ATM-deficient cells treated with moderate doses of MNNG. Pharmacological and RNA interference approaches indicated that Chk1 and Chk2 are both required components for normal MNNG-induced G2 arrest. MNNG-induced nuclear exclusion of the cell cycle regulatory phosphatase Cdc25C occurred in an MMR-dependent manner and was compromised in cells lacking ATM. Finally, both Chk1 and Chk2 interact with the MMR protein MSH2, and this interaction is enhanced after MNNG exposure, supporting the notion that the MMR system functions as a molecular scaffold at the sites of DNA damage that facilitates activation of these kinases.

Discovery of mutations in homologous recombination genes in African-American women with breast cancer.

African-American women are more likely to develop aggressive breast cancer at younger ages and experience poorer cancer prognoses than non-Hispanic Caucasians. Deficiency in repair of DNA by homologous recombination (HR) is associated with cancer development, suggesting that mutations in genes that affect this process may cause breast cancer. Inherited pathogenic mutations have been identified in genes involved in repairing DNA damage, but few studies have focused on African-Americans. We screened for germline mutations in seven HR repair pathway genes in DNA of 181 African-American women with breast cancer, evaluated the potential effects of identified missense variants using in silico prediction software, and functionally characterized a set of missense variants by yeast two-hybrid assays. We identified five likely-damaging variants, including two PALB2 truncating variants (Q151X and W1038X) and three novel missense variants (RAD51C C135R, and XRCC3 L297P and V337E) that abolish protein-protein interactions in yeast two-hybrid assays. Our results add to evidence that HR gene mutations account for a proportion of the genetic risk for developing breast cancer in African-Americans. Identifying additional mutations that diminish HR may provide a tool for better assessing breast cancer risk and improving approaches for targeted treatment.

The spectrum of genetic variants in hereditary pancreatic cancer includes Fanconi anemia genes.

Approximately 5-10% of all pancreatic cancer patients carry a predisposing mutation in a known susceptibility gene. Since >90% of patients present with late stage disease, it is crucial to identify high risk individuals who may be amenable to early detection or other prevention. To explore the spectrum of hereditary pancreatic cancer susceptibility, we evaluated germline DNA from pancreatic cancer participants (n = 53) from a large hereditary cancer registry. For those without a known predisposition mutation gene (n = 49), germline next generation sequencing was completed using targeted capture for 706 candidate genes. We identified 16 of 53 participants (30%) with a pathogenic (P) or likely pathogenic (LP) variant that may be related to their hereditary pancreatic cancer predisposition; seven had mutations in genes associated with well-known cancer syndromes (13%) [ATM (2), BRCA2 (3), MSH2 (1), MSH6 (1)]. Many had mutations in Fanconi anemia complex genes [BRCA2 (3 participants), FANCF, FANCM]. Eight participants had rare protein truncating variants of uncertain significance with no other P or LP variants. Earlier age of pancreatic cancer diagnosis (57.5 vs 64.8 years) was indicative of possessing a P or LP variant, as was cancer family history (p values <0.0001). Our multigene panel approach for identifying known cancer predisposing genetic susceptibility in those at risk for hereditary pancreatic cancer may have direct applicability to clinical practice in cases with mutations in actionable genes. Future pancreatic cancer predisposition studies should include evaluation of the Fanconi anemia genes.

Hepatocyte nuclear factor-4alpha contributes to carbohydrate-induced transcriptional activation of hepatic fatty acid synthase.

Refeeding a carbohydrate-rich meal after a fast produces a co-ordinated induction of key glycolytic and lipogenic genes in the liver. The transcriptional response is mediated by insulin and increased glucose oxidation, and both signals are necessary for optimal induction of FAS (fatty acid synthase). The glucose-regulated component of FAS promoter activation is mediated in part by ChREBP [ChoRE (carbohydrate response element)-binding protein], which binds to a ChoRE between -7300 and -7000 base-pairs in a carbohydrate-dependent manner. Using in vivo footprinting with nuclei from fasted and refed rats, we identify an imperfect DR-1 (direct repeat-1) element between -7110 and -7090 bp that is protected upon carbohydrate refeeding. Electrophoretic mobility-shift assays establish that this DR-1 element binds HNF-4alpha (hepatocyte nuclear factor 4alpha), and chromatin immunoprecipitation establishes that HNF-4alpha binding to this site is increased approx. 3-fold by glucose refeeding. HNF-4alpha transactivates reporter constructs containing the distal FAS promoter in a DR-1-dependent manner, and this DR-1 is required for full glucose induction of the FAS promoter in primary hepatocytes. In addition, a 3-fold knockdown of hepatocyte HNF-4alpha by small interfering RNA produces a corresponding decrease in FAS gene induction by glucose. Co-immunoprecipitation experiments demonstrate a physical interaction between HNF-4alpha and ChREBP in primary hepatocytes, further supporting an important complementary role for HNF-4alpha in glucose-induced activation of FAS transcription. Taken together, these observations establish for the first time that HNF-4alpha functions in vivo through a DR-1 element in the distal FAS promoter to enhance gene transcription following refeeding of glucose to fasted rats. The findings support the broader view that HNF-4alpha is an integral component of the hepatic nutrient sensing system that co-ordinates transcriptional responses to transitions between nutritional states.

Genetic Gastric Cancer Susceptibility in the International Clinical Cancer Genomics Community Research Network.

Few susceptibility genes for gastric cancer have been identified. We sought to identify germline susceptibility genes from participants with gastric cancer from an international hereditary cancer research network. Adults with gastric cancer of any histology, and with a germline DNA sample (n = 51), were retrospectively selected. For those without previously identified germline mutations (n = 43), sequencing was performed for 706 candidate genes. Twenty pathogenic or likely pathogenic variants were identified among 18 participants. Eight of the 18 participants had previous positive clinical testing, including six with CDH1 pathogenic or likely pathogenic variants, and two with pathogenic MSH2 and TP53 variants. Of the remaining 10, six were in BRCA1 DNA damage response pathway genes (ATM, ATR, BRCA2, BRIP1, FANCC, TP53), other variants were identified in CTNNA1, FLCN, SBDS, and GNAS. Participants identified with pathogenic or likely pathogenic variants were younger at gastric cancer diagnosis than those without, 39.1 versus 48.0 years, and over 50% had a close family member with gastric cancer (p-values < 0.0001). In conclusion, many participants were identified with mutations in clinically-actionable genes. Age of onset and family history of gastric cancer were mutation status predictors. Our findings support multigene panels in identifying gastric cancer predisposition.

A newly discovered member of the fatty acid desaturase gene family: a non-coding, antisense RNA gene to delta5-desaturase.

The rate limiting steps in the conversion of 18-carbon unsaturated fatty acids to 20- and 22-carbon products are catalyzed by two desaturase enzymes (Delta5-desaturase and Delta6-desaturase) found within a lipid desaturase gene cluster. Careful examination of this cluster revealed the existence of a conventionally spliced (human) and an intronless (mouse and rat) non-coding RNA gene, reverse Delta5-desaturase, which is transcribed from the opposite strand of the Delta5-desaturase gene. The 654 bp human reverse Delta5-desaturase transcript contains 269 nucleotides that are complementary to exon 1 and intron 1 of the Delta5-desaturase transcript, and the 3'-end of this sequence contains a 143 nucleotide stretch that is 100% complementary to the 5'-end of the Delta5-desaturase. The rat and mouse transcripts are 1355 and 690 bp long and complementary to a portion of the first intron and the entire first exon of their respective Delta5-desaturases. All reverse Delta5-desaturase transcripts contain several stop codons in all frames suggesting that they do not encode a peptide. Reverse Delta5-desaturase RNA was detected in all rat tissues where Delta5-desaturase is expressed, and the transition between fasting and refeeding produced a significant increase in reverse Delta5-desaturase RNA relative to Delta5-desaturase mRNA. Transient expression of reverse Delta5-desaturase in CHO cells stably transformed with Delta5-desaturase produced a modest decrease in Delta5-desaturase mRNA (30%), but lowered Delta5-desaturase enzymatic activity by >70%. More importantly, a diet enriched in fish oil produced a reciprocal increase in reverse Delta5-desaturase mRNA and decrease in Delta5-desaturase mRNA that was accompanied by a 5-6-fold decrease in Delta5-desaturase enzyme activity. These findings support a significant role for reverse Delta5-desaturase as a natural antisense regulator of Delta5-desaturase.

Detailed comparison of two popular variant calling packages for exome and targeted exon studies.

The Genome Analysis Toolkit (GATK) is commonly used for variant calling of single nucleotide polymorphisms (SNPs) and small insertions and deletions (indels) from short-read sequencing data aligned against a reference genome. There have been a number of variant calling comparisons against GATK, but an equally comprehensive comparison for VarScan not yet been performed. More specifically, we compare (1) the effects of different pre-processing steps prior to variant calling with both GATK and VarScan, (2) VarScan variants called with increasingly conservative parameters, and (3) filtered and unfiltered GATK variant calls (for both the UnifiedGenotyper and the HaplotypeCaller). Variant calling was performed on three datasets (1 targeted exon dataset and 2 exome datasets), each with approximately a dozen subjects. In most cases, pre-processing steps (e.g., indel realignment and quality score base recalibration using GATK) had only a modest impact on the variant calls, but the importance of the pre-processing steps varied between datasets and variant callers. Based upon concordance statistics presented in this study, we recommend GATK users focus on "high-quality" GATK variants by filtering out variants flagged as low-quality. We also found that running VarScan with a conservative set of parameters (referred to as "VarScan-Cons") resulted in a reproducible list of variants, with high concordance (>97%) to high-quality variants called by the GATK UnifiedGenotyper and HaplotypeCaller. These conservative parameters result in decreased sensitivity, but the VarScan-Cons variant list could still recover 84-88% of the high-quality GATK SNPs in the exome datasets. This study also provides limited evidence that VarScan-Cons has a decreased false positive rate among novel variants (relative to high-quality GATK SNPs) and that the GATK HaplotypeCaller has an increased false positive rate for indels (relative to VarScan-Cons and high-quality GATK UnifiedGenotyper indels). More broadly, we believe the metrics used for comparison in this study can be useful in assessing the quality of variant calls in the context of a specific experimental design. As an example, a limited number of variant calling comparisons are also performed on two additional variant callers.

RAD51C germline mutations in breast and ovarian cancer cases from high-risk families.

BRCA1 and BRCA2 are the most well-known breast cancer susceptibility genes. Additional genes involved in DNA repair have been identified as predisposing to breast cancer. One such gene, RAD51C, is essential for homologous recombination repair. Several likely pathogenic RAD51C mutations have been identified in BRCA1- and BRCA2-negative breast and ovarian cancer families. We performed complete sequencing of RAD51C in germline DNA of 286 female breast and/or ovarian cancer cases with a family history of breast and ovarian cancers, who had previously tested negative for mutations in BRCA1 and BRCA2. We screened 133 breast cancer cases, 119 ovarian cancer cases, and 34 with both breast and ovarian cancers. Fifteen DNA sequence variants were identified; including four intronic, one 5' UTR, one promoter, three synonymous, and six non-synonymous variants. None were truncating. The in-silico SIFT and Polyphen programs were used to predict possible pathogenicity of the six non-synonomous variants based on sequence conservation. G153D and T287A were predicted to be likely pathogenic. Two additional variants, A126T and R214C alter amino acids in important domains of the protein such that they could be pathogenic. Two-hybrid screening and immunoblot analyses were performed to assess the functionality of these four non-synonomous variants in yeast. The RAD51C-G153D protein displayed no detectable interaction with either XRCC3 or RAD51B, and RAD51C-R214C displayed significantly decreased interaction with both XRCC3 and RAD51B (p<0.001). Immunoblots of RAD51C-Gal4 activation domain fusion peptides showed protein levels of RAD51C-G153D and RAD51C-R214C that were 50% and 60% of the wild-type, respectively. Based on these data, the RAD51C-G153D variant is likely to be pathogenic, while the RAD51C- R214C variant is hypomorphic of uncertain pathogenicity. These results provide further support that RAD51C is a rare breast and ovarian cancer susceptibility gene.

Differential coupling of beta3A- and beta3B-adrenergic receptors to endogenous and chimeric Galphas and Galphai.

Chimeric G proteins made by replacing the COOH-terminal heptapeptide of G(alpha)q with the COOH-terminal heptapeptide of G(alpha)s or G(alpha)i were used to assess the relative coupling of beta(3)-adrenergic receptor (beta(3)-AR) splice variants (beta(3A) and beta(3B)) to G(alpha)s and G(alpha)i. The G(alpha)q/s and G(alpha)q/i chimeras transformed the response to receptor activation from regulation of adenylyl cyclase to mobilization of intracellular calcium (Ca(2+)(i)). Complementary high-throughput and single-cell approaches were used to evaluate agonist-induced coupling of the receptor to the G protein chimeras. In cells stably transformed with rat beta(3)-AR, transfected with the G protein chimeras, and evaluated using a scanning fluorometer, beta(3)-AR-induced coupling to G(alpha)q/s produced a rapid eightfold increase in Ca(2+)(i) followed by a slow decay to levels 25% above baseline. G(alpha)q/i also linked rat beta(3)-AR to mobilization of Ca(2+)(i) in a similar time- and agonist-dependent manner, but the net 2.5-fold increase in Ca(2+)(i) was only 30% of the response obtained with G(alpha)q/s. Activation of the rat beta(3)-AR also increased GTP binding to endogenous G(alpha)i threefold in membranes from CHO cells stably transformed with the receptor. A complementary single-cell imaging approach was used to assess the relative coupling of mouse beta(3A)- and beta(3B)-AR to G(alpha)i under conditions established to produce equivalent agonist-dependent coupling of the receptor splice variants to G(alpha)q/s and to increases in intracellular cAMP through endogenous G(alpha)s. The beta(3A)- and beta(3B)-AR coupled equivalently to G(alpha)q/i, but the temporal patterns of Ca(2+)(i) mobilization indicated that coupling was significantly less efficient than coupling to G(alpha)q/s. Collectively, these findings indicate less efficient but equivalent coupling of beta(3A)- and beta(3B)-AR to G(alpha)i vs. G(alpha)s and suggest that differential expression of the splice variants would not produce local differences in signaling networks linked to beta(3)-AR activation.