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.

Singapore Undiagnosed Disease Program: Genomic Analysis aids Diagnosis and Clinical Management.

OBJECTIVE: Use next-generation sequencing (NGS) technology to improve our diagnostic yield in patients with suspected genetic disorders in the Asian setting. DESIGN: A diagnostic study conducted between 2014 and 2019 (and ongoing) under the Singapore Undiagnosed Disease Program. Date of last analysis was 1 July 2019. SETTING: Inpatient and outpatient genetics service at two large academic centres in Singapore. PATIENTS: Inclusion criteria: patients suspected of genetic disorders, based on abnormal antenatal ultrasound, multiple congenital anomalies and developmental delay. EXCLUSION CRITERIA: patients with known genetic disorders, either after clinical assessment or investigations (such as karyotype or chromosomal microarray). INTERVENTIONS: Use of NGS technology-whole exome sequencing (WES) or whole genome sequencing (WGS). MAIN OUTCOME MEASURES: (1) Diagnostic yield by sequencing type, (2) diagnostic yield by phenotypical categories, (3) reduction in time to diagnosis and (4) change in clinical outcomes and management. RESULTS: We demonstrate a 37.8% diagnostic yield for WES (n=172) and a 33.3% yield for WGS (n=24). The yield was higher when sequencing was conducted on trios (40.2%), as well as for certain phenotypes (neuromuscular, 54%, and skeletal dysplasia, 50%). In addition to aiding genetic counselling in 100% of the families, a positive result led to a change in treatment in 27% of patients. CONCLUSION: Genomic sequencing is an effective method for diagnosing rare disease or previous 'undiagnosed' disease. The clinical utility of WES/WGS is seen in the shortened time to diagnosis and the discovery of novel variants. Additionally, reaching a diagnosis significantly impacts families and leads to alteration in management of these patients.

Evaluation of three polygenic risk score models for the prediction of breast cancer risk in Singapore Chinese.

Genome-wide association studies (GWAS) have proven highly successful in identifying single nucleotide polymorphisms (SNPs) associated with breast cancer (BC) risk. The majority of these studies are on European populations, with limited SNP association data in other populations. We genotyped 51 GWAS-identified SNPs in two independent cohorts of Singaporean Chinese. Cohort 1 comprised 1294 BC cases and 885 controls and was used to determine odds ratios (ORs); Cohort 2 had 301 BC cases and 243 controls for deriving polygenic risk scores (PRS). After age-adjustment, 11 SNPs were found to be significantly associated with BC risk. Five SNPs were present in <1% of Cohort 1 and were excluded from further PRS analysis. To assess the cumulative effect of the remaining 46 SNPs on BC risk, we generated three PRS models: Model-1 included 46 SNPs; Model-2 included 11 statistically significant SNPs; and Model-3 included the SNPs in Model-2 but excluded SNPs that were in strong linkage disequilibrium with the others. Across Models-1, -2 and -3, women in the highest PRS quartile had the greatest ORs of 1.894 (95% CI = 1.157-3.100), 2.013 (95% CI = 1.227-3.302) and 1.751 (95% CI = 1.073-2.856) respectively, suggesting a direct correlation between PRS and BC risk. Given the potential of PRS in BC risk stratification, our findings suggest the need to tailor the selection of SNPs to be included in an ethnic-specific PRS model.

Identification of Novel Breast Cancer Risk Loci.

It has been estimated that >1,000 genetic loci have yet to be identified for breast cancer risk. Here we report the first study utilizing targeted next-generation sequencing to identify single-nucleotide polymorphisms (SNP) associated with breast cancer risk. Targeted sequencing of 283 genes was performed in 240 women with early-onset breast cancer (≤40 years) or a family history of breast and/or ovarian cancer. Common coding variants with minor allele frequencies (MAF) >1% that were identified were presumed initially to be SNPs, but further database inspections revealed variants had MAF of ≤1% in the general population. Through prioritization and stringent selection criteria, we selected 24 SNPs for further genotyping in 1,516 breast cancer cases and 1,189 noncancer controls. Overall, we identified the JAK2 SNP rs56118985 to be significantly associated with overall breast cancer risk. Subtype analysis performed for patient subgroups defined by ER, PR, and HER2 status suggested additional associations of the NOTCH3 SNP rs200504060 and the HIF1A SNP rs142179458 with breast cancer risk. In silico analysis indicated that coding amino acids encoded at these three SNP sites were conserved evolutionarily and associated with decreased protein stability, suggesting a likely impact on protein function. Our results offer proof of concept for identifying novel cancer risk loci from next-generation sequencing data, with iterative data analysis from targeted, whole-exome, or whole-genome sequencing a wellspring to identify new SNPs associated with cancer risk. Cancer Res; 77(19); 5428-37. ©2017 AACR.

Systematic study on genetic and epimutational profile of a cohort of Amsterdam criteria-defined Lynch Syndrome in Singapore.

BACKGROUND: Germline defects of mismatch repair (MMR) genes underlie Lynch Syndrome (LS). We aimed to gain comprehensive genetic and epigenetic profiles of LS families in Singapore, which will facilitate efficient molecular diagnosis of LS in Singapore and the region. METHODS: Fifty nine unrelated families were studied. Mutations in exons, splice-site junctions and promoters of five MMR genes were scanned by high resolution melting assay followed by DNA sequencing, large fragment deletions/duplications and promoter methylation in MLH1, MSH2, MSH6 and PMS2 were evaluated by multiplex ligation-dependent probe amplification. Tumor microsatellite instability (MSI) was assessed with five mononucleotide markers and immunohistochemical staining (IHC) was also performed. RESULTS: Pathogenic defects, all confined to MLH1 and MSH2, were identified in 17 out of 59 (28.8%) families. The mutational spectrum was highly heterogeneous and 28 novel variants were identified. One recurrent mutation in MLH1 (c.793C>T) was also observed. 92.9% sensitivity for indication of germline mutations conferred by IHC surpassed 64.3% sensitivity by MSI. Furthermore, 15.6% patients with MSS tumors harbored pathogenic mutations. CONCLUSIONS: Among major ethnic groups in Singapore, all pathogenic germline defects were confined to MLH1 and MSH2. Caution should be applied when the Amsterdam criteria and consensus microsatellite marker panel recommended in the revised Bethesda guidelines are applied to the local context. We recommend a screening strategy for the local LS by starting with tumor IHC and the hotspot mutation testing at MLH1 c.793C>T followed by comprehensive mutation scanning in MLH1 and MSH2 prior to proceeding to other MMR genes.

Rapid prenatal diagnosis of common beta-thalassemia mutations in Southeast Asia using pyrosequencing.

OBJECTIVE: Current methods of prenatal diagnosis to detect beta-thalassemia are Sanger sequencing and reverse dot blot. These methods are time-consuming and can prolong assay turnaround time. We aim to develop a sensitive and rapid method to detect 27 beta-thalassemia mutations using pyrosequencing. METHOD: Pyrosequencing primer pairs and sequencing primers were designed to detect 27 most common beta-thalassemia mutations found in Singapore. Pyrosequencing was performed on 191 DNA samples with known beta-thalassemia mutations isolated from 143 peripheral blood and 48 prenatal samples (seven chorionic villus biopsies, 26 cultured amniocytes, 15 uncultured amniocytes). All mutations were validated with Sanger sequencing. RESULTS: Pyrosequencing identified 210 alleles with beta-thalassemia mutations and 82 alleles without mutations with 100% sensitivity (lower 95% confidence interval [CI], 97.8%) and 100% specificity (lower 95% CI, 94.4%). All pyrosequences were concordant with Sanger-based sequences. Pyrosequencing was able to detect DNA concentrations as low as 2 ng, obviating the need for cell culture in volume-restricted samples. Sample receipt-to-report assay turnaround times were 16 to 18 h (Sanger sequencing) and 4 to 6 h (pyrosequencing). CONCLUSION: Pyrosequencing is a rapid and sensitive method to detect common beta-thalassemia mutations without the need for cell culture, thus reducing the assay turnaround time.

Global DNA hypermethylation in down syndrome placenta.

Down syndrome (DS), commonly caused by an extra copy of chromosome 21 (chr21), occurs in approximately one out of 700 live births. Precisely how an extra chr21 causes over 80 clinically defined phenotypes is not yet clear. Reduced representation bisulfite sequencing (RRBS) analysis at single base resolution revealed DNA hypermethylation in all autosomes in DS samples. We hypothesize that such global hypermethylation may be mediated by down-regulation of TET family genes involved in DNA demethylation, and down-regulation of REST/NRSF involved in transcriptional and epigenetic regulation. Genes located on chr21 were up-regulated by an average of 53% in DS compared to normal villi, while genes with promoter hypermethylation were modestly down-regulated. DNA methylation perturbation was conserved in DS placenta villi and in adult DS peripheral blood leukocytes, and enriched for genes known to be causally associated with DS phenotypes. Our data suggest that global epigenetic changes may occur early in development and contribute to DS phenotypes.

Ethnic differences in pain perception and patient-controlled analgesia usage for postoperative pain.

UNLABELLED: There are reports suggesting that sensitivity to and tolerance of both clinical and experimental pain differ among ethnic groups. We examined self-rated pain score and morphine usage in 1034 women who underwent elective lower cesarian section (LSCS) for their deliveries. Data on pain scores and amount of total morphine use according to patient-controlled analgesia were collected every 4 hours. Overall, lowest pain scores were recorded 12 hours after surgery and highest at 24 hours. Morphine consumption was highest within the first 4 hours and lowest between 12 and 16 hours. There were statistically significant ethnic group differences in pain scores (P = 1.7 x 10(-7)) and morphine usage (P = 2.8 x 10(-15)) between ethnic groups, with Indians having the highest mean pain score and using the highest amount of morphine. The ethnic differences in pain score and morphine self-administration persisted after controlling for age, body mass index, and duration of operation. PERSPECTIVE: Our findings of highly significant ethnic group difference in self-reported pain level and the amount of analgesia self-administered may have implications on optimal management of acute postoperative pain. Inadequate management of pain after cesarian deliveries might affect the emotional well-being and physical recovery of patients and affect mother-child bonding.

Validation of a reverse-hybridization StripAssay for the simultaneous analysis of common alpha-thalassemia point mutations and deletions.

BACKGROUND: alpha-Thalassemia is a worldwide disease and considered to be a major public health problem in countries within the so-called thalassemia belt. The complex genetics of alpha-thalassemias requires diagnostic methods with the capacity to screen rapidly and accurately for common causative mutations. METHODS: We developed and validated a reverse-hybridization assay (Alpha-Globin StripAssay) for the rapid and simultaneous detection of 21 alpha-globin mutations: two single gene deletions (-alpha(3.7); -alpha(4.2)), five double gene deletions [--(MED); --(SEA); --(THAI); --(FIL); -(alpha)(20.5)], alpha alpha alpha(anti-3.7) gene triplication, two point mutations in the alpha1 gene (cd 14 G>A; Hb Adana) and 11 point mutations in the alpha2 gene (initiation cd T>C; cd 19 -G; IVS1 -5nt; cd 59 G>A; Hb Quong Sze; Hb Constant Spring; Hb Icaria; Hb Pakse; Hb Koya Dora; polyA-1; polyA-2). RESULTS: Reliable genotyping of recombinant mutant clones and reference DNA samples was achieved by means of two corresponding test strips presenting parallel arrays of allele-specific oligonucleotides. The entire procedure from blood sampling to the identification of mutations required less than 6 h, and hybridization/detection was manual or automated. The diagnostic potential of this Alpha-Globin StripAssay was carefully evaluated on 272 pre-typed samples in a multicenter validation study. In 96.14% of the cases, StripAssay typing was completely concordant with the reference methods. CONCLUSIONS: The Alpha-Globin StripAssay proved to be a fast, easy-to-perform and reliable screening method to identify >90% of alpha-globin mutations in endemic areas worldwide.

An isothermal method for whole genome amplification of fresh and degraded DNA for comparative genomic hybridization, genotyping and mutation detection.

Molecular genotyping has important biomedical and forensic applications. However, limiting amounts of human biological material often yield genomic DNA (gDNA) in insufficient quantity and of poor quality for a reliable analysis. This motivated the development of an efficient whole genome amplification method with quantitatively unbiased representation usable on fresh and degraded gDNA. Amplification of fresh frozen, formalin-fixed paraffin-embedded (FFPE) and DNase-degraded DNA using degenerate oligonucleotide-primed PCR or primer extension amplification using a short primer sequence bioinformatically optimized for coverage of the human genome was compared with amplification using current primers by chromosome-based and BAC-array comparative genomic hybridization (CGH), genotyping at short tandem repeats (STRs) and single base mutation detection. Compared with current primers, genome amplification using the bioinformatically optimized primer was significantly less biased on CGH in self-self hybridizations, and replicated tumour genome copy number aberrations, even from FFPE tissue. STR genotyping could be performed on degraded gDNA amplified using our technique but failed with multiple displacement amplification. Of the 18 different single base mutations 16 (89.5%) were correctly identified by sequencing gDNA amplified from clinical samples using our technique. This simple and efficient isothermal method should be helpful for genetic research and clinical and forensic applications.

MS analysis of single-nucleotide differences in circulating nucleic acids: Application to noninvasive prenatal diagnosis.

The analysis of circulating nucleic acids has revealed applications in the noninvasive diagnosis, monitoring, and prognostication of many clinical conditions. Circulating fetal-specific sequences have been detected and constitute a fraction of the total DNA in maternal plasma. The diagnostic reliability of circulating DNA analysis depends on the fractional concentration of the targeted sequence, the analytical sensitivity, and the specificity. The robust discrimination of single-nucleotide differences between circulating DNA species is technically challenging and demands the adoption of highly sensitive and specific analytical systems. We have developed a method based on single-allele base extension reaction and MS, which allows for the reliable detection of fetal-specific alleles, including point mutations and single-nucleotide polymorphisms, in maternal plasma. The approach was applied to exclude the fetal inheritance of the four most common Southeast Asian beta-thalassemia mutations in at-risk pregnancies between weeks 7 and 21 of gestation. Fetal genotypes were correctly predicted in all cases studied. Fetal haplotype analysis based on a single-nucleotide polymorphism linked to the beta-globin locus, HBB, in maternal plasma also was achieved. Consequently, noninvasive prenatal diagnosis in a mother and father carrying identical beta-thalassemia mutations was accomplished. These advances will help in catalyzing the clinical applications of fetal nucleic acids in maternal plasma. This analytical approach also will have implications for many other applications of circulating nucleic acids in areas such as oncology and transplantation.