Alleviating misclassified germline variants in underrepresented populations: A strategy using popmax.

PURPOSE: Germline variant interpretation often depends on population-matched control cohorts. This is not feasible for population groups that are underrepresented in current population reference databases. METHODS: We classify germline variants with population-matched controls for 2 ancestrally diverse cohorts of patients: 132 early-onset or familial colorectal carcinoma patients from Singapore and 100 early-onset colorectal carcinoma patients from the United States. The effects of using a population-mismatched control cohort are simulated by swapping the control cohorts used for each patient cohort, with or without the popmax computational strategy. RESULTS: Population-matched classifications revealed a combined 62 pathogenic or likely pathogenic (P/LP) variants in 34 genes across both cohorts. Using a population-mismatched control cohort resulted in misclassification of non-P/LP variants as P/LP, driven by the absence of ancestry-specific rare variants in the control cohort. Popmax was more effective in alleviating misclassifications for the Singapore cohort than the US cohort. CONCLUSION: Underrepresented population groups can suffer from higher rates of false-positive P/LP results. Popmax can partially alleviate these misclassifications, but its efficacy still depends on the degree with which the population groups are represented in the control cohort.

Landscape of germline pathogenic variants in patients with dual primary breast and lung cancer.

BACKGROUND: Cancer predisposition is most often studied in the context of single cancers. However, inherited cancer predispositions can also give rise to multiple primary cancers. Yet, there is a paucity of studies on genetic predisposition in multiple primary cancers, especially those outside of well-defined cancer predisposition syndromes. This study aimed to identify germline variants associated with dual primary cancers of the breast and lung. METHODS: Exome sequencing was performed on germline DNA from 55 Singapore patients (52 [95%] never-smokers) with dual primaries in the breast and lung, confirmed by histopathology. Using two large control cohorts: the local SG10K_Health (n = 9770) and gnomAD non-cancer East Asians (n = 9626); and two additional local case cohorts of early-onset or familial breast cancer (n = 290), and lung cancer (n = 209), variants were assessed for pathogenicity in accordance with ACMG/AMP guidelines. In particular, comparisons were made with known pathogenic or likely pathogenic variants in the ClinVar database, pathogenicity predictions were obtained from in silico prediction software, and case-control association analyses were performed. RESULTS: Altogether, we identified 19 pathogenic or likely pathogenic variants from 16 genes, detected in 17 of 55 (31%) patients. Six of the 19 variants were identified using ClinVar, while 13 variants were classified pathogenic or likely pathogenic using ACMG/AMP guidelines. The 16 genes include well-known cancer predisposition genes such as BRCA2, TP53, and RAD51D; but also lesser known cancer genes EXT2, WWOX, GATA2, and GPC3. Most of these genes are involved in DNA damage repair, reaffirming the role of impaired DNA repair mechanisms in the development of multiple malignancies. These variants warrant further investigations in additional populations. CONCLUSIONS: We have identified both known and novel variants significantly enriched in patients with primary breast and lung malignancies, expanding the body of known cancer predisposition variants for both breast and lung cancer. These variants are mostly from genes involved in DNA repair, affirming the role of impaired DNA repair in the predisposition and development of multiple cancers.

Whole-exome sequencing of BRCA-negative breast cancer patients and case-control analyses identify variants associated with breast cancer susceptibility.

BACKGROUND: For the majority of individuals with early-onset or familial breast cancer referred for genetic testing, the genetic basis of their familial breast cancer remains unexplained. To identify novel germline variants associated with breast cancer predisposition, whole-exome sequencing (WES) was performed. METHODS: WES on 290 BRCA1/BRCA2-negative Singaporeans with early-onset breast cancer and/or a family history of breast cancer was done. Case-control analysis against the East-Asian subpopulation (EAS) from the Genome Aggregation Database (gnomAD) identified variants enriched in cases, which were further selected by occurrence in cancer gene databases. Variants were further evaluated in repeated case-control analyses using a second case cohort from the database of Genotypes and Phenotypes (dbGaP) comprising 466 early-onset breast cancer patients from the United States, and a Singapore SG10K_Health control cohort. RESULTS: Forty-nine breast cancer-associated germline pathogenic variants in 37 genes were identified in Singapore cases versus gnomAD (EAS). Compared against SG10K_Health controls, 13 of 49 variants remain significantly enriched (False Discovery Rate (FDR)-adjusted p < 0.05). Comparing these 49 variants in dbGaP cases against gnomAD (EAS) and SG10K_Health controls revealed 23 concordant variants that were significantly enriched (FDR-adjusted p < 0.05). Fourteen variants were consistently enriched in breast cancer cases across all comparisons (FDR-adjusted p < 0.05). Seven variants in GPRIN2, NRG1, MYO5A, CLIP1, CUX1, GNAS and MGA were confirmed by Sanger sequencing. CONCLUSIONS: In conclusion, we have identified pathogenic variants in genes associated with breast cancer predisposition. Importantly, many of these variants were significant in a second case cohort from dbGaP, suggesting that the strategy of using case-control analysis to select variants could potentially be utilized for identifying variants associated with cancer susceptibility.

Development and validation of a circulating microRNA panel for the early detection of breast cancer.

BACKGROUND: Mammography is widely used for breast cancer screening but suffers from a high false-positive rate. Here, we perform the largest comprehensive, multi-center study to date involving diverse ethnic groups, for the identification of circulating miRNAs for breast cancer screening. METHODS: This study had a discovery phase (n = 289) and two validation phases (n = 374 and n = 379). Quantitative PCR profiling of 324 miRNAs was performed on serum samples from breast cancer (all stages) and healthy subjects to identify miRNA biomarkers. Two-fold cross-validation was used for building and optimising breast cancer-associated miRNA panels. An optimal panel was validated in cohorts with Caucasian and Asian samples. Diagnostic ability was evaluated using area under the curve (AUC) analysis. RESULTS: The study identified and validated 30 miRNAs dysregulated in breast cancer. An optimised eight-miRNA panel showed consistent performance in all cohorts and was successfully validated with AUC, accuracy, sensitivity, and specificity of 0.915, 82.3%, 72.2% and 91.5%, respectively. The prediction model detected breast cancer in both Caucasian and Asian populations with AUCs ranging from 0.880 to 0.973, including pre-malignant lesions (stage 0; AUC of 0.831) and early-stage (stages I-II) cancers (AUC of 0.916). CONCLUSIONS: Our panel can potentially be used for breast cancer screening, in conjunction with mammography.

Breast cancer in women with neurofibromatosis type 1 (NF1): a comprehensive case series with molecular insights into its aggressive phenotype.

PURPOSE: The purpose of the study was to improve the understanding of NF1-associated breast cancer, given the increased risk of breast cancer in this tumour predisposition syndrome and the limited data. METHODS: We identified 18 women with NF1 and breast cancer at our institution. Clinical and pathologic characteristics of NF1-associated breast cancers were compared with 7132 breast cancers in patients without NF1 from our institutional database. Next generation sequencing was performed on DNA from blood and breast cancer specimens available. Blood specimens negative for NF1 mutation were subjected to multiplex ligation-dependent probe amplification (MLPA) to identify complete/partial deletions or duplications. Expression of neurofibromin in the NF1-associated breast cancers was evaluated using immunohistochemistry. RESULTS: There was a higher frequency of grade 3 (83.3% vs 45.4%, p = 0.005), oestrogen receptor (ER) negative (66.7% vs 26.3%, p < 0.001) and human epidermal growth factor receptor 2 (HER2)-positive (66.7% vs 23.4%, p < 0.001) tumours among NF1 patients compared to non-NF1 breast cancers. Overall survival was inferior in NF1 patients in multivariable analysis (hazard ratio 2.25, 95% CI 1.11-4.60; p = 0.025). Apart from germline NF1 mutations (11/16; 69%), somatic mutations in TP53 (8/10; 80%), second-hit NF1 (2/10; 20%), KMT2C (4/10; 40%), KMT2D (2/10; 20%), and PIK3CA (2/10; 20%) were observed. Immunohistochemical expression of neurofibromin was seen in the nuclei and/or cytoplasm of all specimens, but without any consistent pattern in the intensity or extent. CONCLUSIONS: This comprehensive series of NF1-associated breast cancers suggests that their aggressive features are related to germline NF1 mutations in cooperation with somatic mutations in TP53, KMT2C and other genes.

Comprehensive spectrum of BRCA1 and BRCA2 deleterious mutations in breast cancer in Asian countries.

Approximately 5%-10% of breast cancers are due to genetic predisposition caused by germline mutations; the most commonly tested genes are BRCA1 and BRCA2 mutations. Some mutations are unique to one family and others are recurrent; the spectrum of BRCA1/BRCA2 mutations varies depending on the geographical origins, populations or ethnic groups. In this review, we compiled data from 11 participating Asian countries (Bangladesh, Mainland China, Hong Kong SAR, Indonesia, Japan, Korea, Malaysia, Philippines, Singapore, Thailand and Vietnam), and from ethnic Asians residing in Canada and the USA. We have additionally conducted a literature review to include other Asian countries mainly in Central and Western Asia. We present the current pathogenic mutation spectrum of BRCA1/BRCA2 genes in patients with breast cancer in various Asian populations. Understanding BRCA1/BRCA2 mutations in Asians will help provide better risk assessment and clinical management of breast cancer.

Machine learning unveils an immune-related DNA methylation profile in germline DNA from breast cancer patients.

BACKGROUND: There is an unmet need for precise biomarkers for early non-invasive breast cancer detection. Here, we aimed to identify blood-based DNA methylation biomarkers that are associated with breast cancer. METHODS: DNA methylation profiling was performed for 524 Asian Chinese individuals, comprising 256 breast cancer patients and 268 age-matched healthy controls, using the Infinium MethylationEPIC array. Feature selection was applied to 649,688 CpG sites in the training set. Predictive models were built by training three machine learning models, with performance evaluated on an independent test set. Enrichment analysis to identify transcription factors binding to regions associated with the selected CpG sites and pathway analysis for genes located nearby were conducted. RESULTS: A methylation profile comprising 51 CpGs was identified that effectively distinguishes breast cancer patients from healthy controls achieving an AUC of 0.823 on an independent test set. Notably, it outperformed all four previously reported breast cancer-associated methylation profiles. Enrichment analysis revealed enrichment of genomic loci associated with the binding of immune modulating AP-1 transcription factors, while pathway analysis of nearby genes showed an overrepresentation of immune-related pathways. CONCLUSION: This study has identified a breast cancer-associated methylation profile that is immune-related to potential for early cancer detection.

Degradation of methylation signals in cryopreserved DNA.

BACKGROUND: Blood-based DNA methylation has shown great promise as a biomarker in a wide variety of diseases. Studies of DNA methylation in blood often utilize samples which have been cryopreserved for years or even decades. Therefore, changes in DNA methylation associated with long-term cryopreservation can introduce biases or otherwise mislead methylation analyses of cryopreserved DNA. However, previous studies have presented conflicting results with studies reporting hypomethylation, no effect, or even hypermethylation of DNA following long-term cryopreservation. These studies may have been limited by insufficient sample sizes, or by their profiling of methylation only on an aggregate global scale, or profiling of only a few CpGs. RESULTS: We analyzed two large prospective cohorts: a discovery (n = 126) and a validation (n = 136) cohort, where DNA was cryopreserved for up to four years. In both cohorts there was no detectable change in mean global methylation across increasing storage durations as DNA. However, when analysis was performed on the level of individual CpG methylation both cohorts exhibited a greater number of hypomethylated than hypermethylated CpGs at q-value < 0.05 (4049 hypomethylated but only 50 hypermethylated CpGs in discovery, and 63 hypomethylated but only 6 hypermethylated CpGs in validation). The results were the same even after controlling for age, storage duration as buffy coat prior to DNA extraction, and estimated cell type composition. Furthermore, we find that in both cohorts, CpGs have a greater likelihood to be hypomethylated the closer they are to a CpG island; except for CpGs at the CpG islands themselves which are less likely to be hypomethylated. CONCLUSION: Cryopreservation of DNA after a few years results in a detectable bias toward hypomethylation at the level of individual CpG methylation, though when analyzed in aggregate there is no detectable change in mean global methylation. Studies profiling methylation in cryopreserved DNA should be mindful of this hypomethylation bias, and more attention should be directed at developing more stable methods of DNA cryopreservation for biomedical research or clinical use.

Germline Variants Associated with Nasopharyngeal Carcinoma Predisposition Identified through Whole-Exome Sequencing.

The current understanding of genetic susceptibility factors for nasopharyngeal carcinoma (NPC) is still incomplete. To identify novel germline variants associated with NPC predisposition, we analysed whole-exome sequencing data from 119 NPC patients from Singapore with a family history of NPC and/or with early-onset NPC, together with 1337 Singaporean participants without NPC. Variants were prioritised and filtered by selecting variants with minor allele frequencies of <1% in both local control (n = 1337) and gnomAD non-cancer (EAS) (n = 9626) cohorts and a high pathogenicity prediction (CADD score > 20). Using single-variant testing, we identified 17 rare pathogenic variants in 17 genes that were associated with NPC. Consistent evidence of enrichment in NPC patients was observed for five of these variants (in JAK2, PRDM16, LRP1B, NIN, and NKX2-1) from an independent case-control comparison of 156 NPC patients and 9770 unaffected individuals. In a family with five siblings, a FANCE variant (p. P445S) was detected in two affected members, but not in three unaffected members. Gene-based burden testing recapitulated variants in NKX2-1 and FANCE as being associated with NPC risk. Using pathway analysis, endocytosis and immune-modulating pathways were found to be enriched for mutation burden. This study has identified NPC-predisposing variants and genes which could shed new insights into the genetic predisposition of NPC.

Rapid detection of rifampicin- and isoniazid-resistant Mycobacterium tuberculosis by high-resolution melting analysis.

We have developed a high-resolution melting (HRM) assay to scan for mutations in the rpoB, inhA, ahpC, and katG genes and/or promoter regions for the detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis. For assay development, 23 drug-resistant isolates of M. tuberculosis having 29 different mutations, together with 40 drug-susceptible isolates, were utilized. All 29 mutations were accurately detected by our assay. We further validated the assay with a series of 59 samples tested in a blind manner. All sequence alterations that were within the regions targeted by the HRM assay were correctly identified. Compared against results of DNA sequencing, the sensitivity and specificity of our HRM assay were 100%. For the blinded samples, the specificities and sensitivities were 89.3% and 100%, respectively, for detecting rifampin resistance and 98.1% and 83.3%, respectively, for detecting isoniazid resistance, as isolates with mutations in regions not encompassed by our assay were not detected. A C-to-T sequence alteration at position -15 of the ahpC regulatory region, which was previously reported to be associated with isoniazid resistance, may possibly be a polymorphism, as it was detected in an isoniazid-susceptible M. tuberculosis isolate. HRM is a rapid, accurate, simple, closed-tube, and low-cost method. It is thus an ideal assay to be used in countries with a high prevalence of drug-resistant M. tuberculosis and where cost-effectiveness is essential. As a mutation-scanning assay for detecting drug-resistant M. tuberculosis, it can potentially lead to better treatment outcomes resulting from earlier treatment with the appropriate antibiotics.

Contribution of dfrA and inhA mutations to the detection of isoniazid-resistant Mycobacterium tuberculosis isolates.

Screening of 127 isoniazid (INH)-resistant Mycobacterium tuberculosis isolates from Singapore for mutations within the dfrA and inhA genes revealed mutations in 0 and 5 (3.9%) isolates respectively, implying that mutations in dfrA do not contribute to the detection of INH-resistant M. tuberculosis and that mutations within inhA are rare. Thirty-seven (29%) of the 127 isolates had no mutations in any of the genes implicated in INH resistance (katG, kasA, and ndh; inhA and ahpC promoters), suggesting that there are new INH targets yet to be discovered.

Incorporating the amino acid properties to predict the significance of missense mutations.

Determining if missense mutations are deleterious is critical for the analysis of genes implicated in disease. However, the mutational effects of many missense mutations in databases like the Breast Cancer Information Core are unclassified. Several approaches have emerged recently to determine such mutational effects but none have utilized amino acid property indices. We modified a previously described phylogenetic approach by first classifying benign substitutions based on the assumption that missense mutations that are maintained in orthologs are unlikely to affect function. A consensus conservation score based on 16 amino acid properties was used to characterize the remaining substitutions. This approach was evaluated with experimentally verified T4 lysozyme missense mutations and is shown to be able to sieve out putative biochemical and structurally important residues. The use of amino acid properties can enhance the prediction of biochemical and structurally important residues and thus also predict the significance of missense mutations.

The dimer-dependent catalytic activity of RAF family kinases is revealed through characterizing their oncogenic mutants.

Although extensively studied for three decades, the molecular mechanisms that regulate the RAF/MEK/ERK kinase cascade remain ambiguous. Recent studies identified the dimerization of RAF as a key event in the activation of this cascade. Here, we show that in-frame deletions in the ß3-αC loop activate ARAF as well as BRAF and other oncogenic kinases by enforcing homodimerization. By characterizing these RAF mutants, we find that ARAF has less allosteric and catalytic activity than the other two RAF isoforms, which arises from its non-canonical APE motif. Further, these RAF mutants exhibit a strong oncogenic potential, and a differential inhibitor resistance that correlates with their dimer affinity. Using these unique mutants, we demonstrate that active RAFs, including the BRAF(V600E) mutant, phosphorylate MEK in a dimer-dependent manner. This study characterizes a special category of oncogenic kinase mutations, and elucidates the molecular basis that underlies the differential ability of RAF isoforms to stimulate MEK-ERK pathway. Further, this study reveals a unique catalytic feature of RAF family kinases that can be exploited to control their activities for cancer therapies.

BRCA1 and BRCA2 mutations in an Asian clinic-based population detected using a comprehensive strategy.

BACKGROUND AND OBJECTIVE: Genetic testing for germ line mutations in the BRCA1 and BRCA2 genes for some families at high risk for breast and/or ovarian cancer may yield negative results due to unidentified mutations or mutations with unknown clinical significance. We aimed to accurately determine the prevalence of mutations in these genes in an Asian clinic-based population by using a comprehensive testing strategy. MATERIALS AND METHODS: Ninety-four subjects from 90 families were accrued from risk assessment clinics. In addition to conventional mutational screening of BRCA1 and BRCA2, multiplex ligation-dependent probe amplification for the detection of large genomic rearrangements, evaluation of splice site alterations using transcript analysis and SpliceSiteFinder prediction, and analysis of missense mutations of unknown significance by multiple sequence alignment, PolyPhen analysis, and comparison of Protein Data Bank structures were incorporated into our testing strategy. RESULTS: The prevalence rates for clearly deleterious BRCA1 and BRCA2 mutations were 6.7% (6 of 90) and 8.9% (8 of 90), respectively, or 7.8% (7 of 90) and 11.1% (10 of 90), respectively, by including missense mutations predicted to be deleterious by computational analysis. In contrast to observations from European and American populations, deleterious mutations in BRCA2 (10 families) were more common than for BRCA1 (7 families). Overall, the frequency of mutations was 12.2% (n=11) by conventional screening. However, by including deleterious mutations detected using multiplex ligation-dependent probe amplification (n=1), transcript analysis (n=2), and computational evaluation of missense mutations (n=3), the frequency increased substantially to 18.9%. This suggests that the comprehensive strategy used is effective for identifying deleterious mutations in Asian individuals at high risk for breast and/or ovarian cancer.

EGFR Intron 1 polymorphism in Asian Populations and its correlation with EGFR gene expression and amplification in breast tumor tissues.

AIM: The purpose of this study was to test the hypothesis if longer CA dinucleotide repeats are more common in the Asian population and also to gain insights into the interplay between the CA dinucleotide repeats and the frequencies of EGFR gene expression and amplifications as this might have therapeutic implications with regards to treatment with tyrosine kinase inhibitors. MATERIALS AND METHODS: The EGFR intron 1 polymorphism was analysed in three distinct healthy Asian subjects, namely, Chinese (N = 96), Malays (N = 98) and Indians (N = 100). Comparative genomic hybridisation was performed to investigate for changes in DNA copy number in relation to the polymorphic CA dinucleotide repeats in breast tumor tissues (N = 22). RESULTS: The frequency of short alleles with 14 and 15 CA repeats were most common in the Asian populations and significantly higher than those reported for Caucasians. The frequency of 20 CA repeats was 5%, almost 13-fold lower than previous reports. EGFR amplifications were detected in 23% and 11% of breast tumor tissues harboring short and long CA repeats, respectively. CONCLUSION: Our results show that the frequency of alleles encoding for short CA dinucleotide repeats is common in Asian populations. EGFR expression and amplification levels were also higher in Asian breast tumor tissues with short CA dinucleotide repeats. These findings suggest that the EGFR intron 1 polymorphism may influence response to treatment with tyrosine kinase inhibitors in breast cancer patients and further studies are warranted.

Inherited breast cancer predisposition in Asians: multigene panel testing outcomes from Singapore.

Genetic testing for germline mutations in breast cancer predisposition genes can potentially identify individuals at a high risk of developing breast and/or ovarian cancer. There is a paucity of such mutational information for Asians. Panel testing of 25 cancer susceptibility genes and BRCA1/2 deletion/duplication analysis was performed for 220 Asian breast cancer patients or their family members referred for genetics risk assessment. All 220 participants had at least one high-risk feature: having a family history of breast and/or ovarian cancer in first- and/or second-degree relatives; having breast and ovarian cancer in the same individual or bilateral breast cancer; having early-onset breast cancer or ovarian cancer (⩽40 years of age). We identified 67 pathogenic variants in 66 (30.0%) patients. Of these, 19 (28.3%) occurred in BRCA1, 16 (23.9%) in BRCA2, 7 (10.4%) in PALB2, 6 (9.0%) in TP53, 2 (3.0%) in PTEN, 2 (3.0%) in CDH1 and 15 (22.4%) in other predisposition genes. Notably, 47.8% of pathogenic variants were in non-BRCA1/2 genes. Of the 66 patients with pathogenic mutations, 63.6% (42/66) were under the age of 40 years. Family history of breast and/or ovarian cancer is enriched in patients with BRCA1/2 pathogenic variants but less predictive for non-BRCA1/2 related pathogenic variations. We detected a median of three variants of unknown significance (VUS) per gene (range 0-21). Custom gene panel testing is feasible and useful for the detection of pathogenic mutations and should be done in the setting of a formal clinical cancer genetics service given the rate of VUS.

microRNAs in breast cancer: regulatory roles governing the hallmarks of cancer.

A large number of etiological factors and the complexity of breast cancers present challenges for prevention and treatment. Recently, the emergence of microRNAs (miRNAs) as cancer biomarkers has added an extra dimension to the 'molecular signatures' of breast cancer. Bioinformatic analyses indicate that each miRNA can regulate hundreds of target genes and could serve functionally as 'oncogenes' or 'tumour suppressor' genes, and co-ordinate multiple cellular processes relevant to cancer progression. A number of studies have shown that miRNAs play important roles in breast tumorigenesis, metastasis, proliferation and differentiation of breast cancer cells. This review provides a comprehensive overview of miRNAs with established functional relevance in breast cancer, their established target genes and resulting cellular phenotype. The role and application of circulating miRNAs in breast cancer is also discussed. Furthermore, we summarize the role of miRNAs in the hallmarks of breast cancer, as well as the possibility of using miRNAs as potential biomarkers for detection of breast cancer.