Hemoglobinopathies are among the most common autosomal-recessive disorders worldwide. A comprehensive next-generation sequencing (NGS) test would greatly facilitate screening and diagnosis of these disorders. An NGS panel targeting the coding regions of hemoglobin genes and four modifier genes was designed. We validated the assay by using 2522 subjects affected with hemoglobinopathies and applied it to carrier testing in a cohort of 10,111 couples who were also screened through traditional methods. In the clinical genotyping analysis of 1182 β-thalassemia subjects, we identified a group of additional variants that can be used for accurate diagnosis. In the molecular screening analysis of the 10,111 couples, we detected 4180 individuals in total who carried 4840 mutant alleles, and identified 186 couples at risk of having affected offspring. 12.1% of the pathogenic or likely pathogenic variants identified by our NGS assay, which were undetectable by traditional methods. Compared with the traditional methods, our assay identified an additional at-risk 35 couples. We describe a comprehensive NGS-based test that offers advantages over the traditional screening/molecular testing methods. To our knowledge, this is among the first large-scale population study to systematically evaluate the application of an NGS technique in carrier screening and molecular diagnosis of hemoglobinopathies.

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Background: This study aimed to identify new genes associated with CRC in patients with normal mismatch repair (MMR) protein expression. Method: Whole-genome sequencing (WGS) was performed in seven early-age-onset Malay CRC patients. Potential germline genetic variants, including single-nucleotide variations and insertions and deletions (indels), were prioritized using functional and predictive algorithms. Results: An average of 3.2 million single-nucleotide variations (SNVs) and over 800 indels were identified. Three potential candidate variants in three genes—IFNE, PTCH2 and SEMA3D—which were predicted to affect protein function, were identified in three Malay CRC patients. In addition, 19 candidate genes—ANKDD1B, CENPM, CLDN5, MAGEB16, MAP3K14, MOB3C, MS4A12, MUC19, OR2L8, OR51Q1, OR51AR1, PDE4DIP, PKD1L3, PRIM2, PRM3, SEC22B, TPTE, USP29 and ZNF117—harbouring nonsense variants were prioritised. These genes are suggested to play a role in cancer predisposition and to be associated with cancer risk. Pathway enrichment analysis indicated significant enrichment in the olfactory signalling pathway. Conclusion: This study provides a new spectrum of insights into the potential genes, variants and pathways associated with CRC in Malay patients.

BACKGROUND Hereditary nonpolyposis colorectal cancer, or Lynch syndrome, caused by germline mutations or genetic defects in mismatch repair (MMR) genes (MLH1, MSH2, PMS2, MSH6, and epithelial cellular adhesion molecule), is an autosomal dominant condition accounting for 2–5% of all colorectal carcinomas (CRCs). Reports on MMR loss in many populations are available; however, there are no reports on the frequency of MMR protein expression in Nepalese cohorts. Therefore, this study was aimed to assess the expression profiles of MLH1 and MSH2 protein by immunohistochemistry (IHC) in Nepalese CRC patients. METHODS This retrospective study used archived formalin-fixed paraffin-embedded tissue blocks from 43 Nepalese CRC patients. IHC staining was performed using MLH1 and MSH2 antibodies. IHC scoring analysis was assessed using semiquantitative scoring. RESULTS Of the 43 CRC patients, 8 (18.6%) showed loss of staining for MLH1 antibody, 5 (11.6%) showed loss of staining for MSH2 antibody, and 4 (9.3%) showed loss of staining for both MLH1 and MSH2 antibodies. CONCLUSIONS IHC is a potential screening method of determining the MMR expression profile of Nepalese CRC patients. IHC can be performed in local clinical laboratories to find MMR protein defects in selected cases prior to expensive molecular tests.

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