First ever characterisation of the effects of short telomeres in a Singapore interstitial lung disease cohort.

BACKGROUND: Differences in disease behaviour and genotypes are described in Asian and Western interstitial lung disease (ILD) cohorts. Short leukocyte telomere length (LTL) correlates with poor outcomes in Western ILD cohorts but its significance in Asian populations is unknown. We aim to characterise the burden and clinical implications of short LTL in Singaporean ILD patients. METHODS: Patients diagnosed with ILD at Singapore General Hospital were prospectively recruited and compared against 36 healthy controls. The primary outcome was transplant-free survival. Genomic DNA from peripheral blood was extracted and LTL measured using quantitative polymerase chain reaction assay (qPCR). RESULTS: Amongst 165 patients, 37% had short LTL. There was a higher proportion of combined pulmonary fibrosis and emphysema (CPFE) patients with short LTL (n = 21, 34.4% vs n = 16, 15.4%; p < 0.001). Short LTL patients had reduced survival at 12-, 24- and 36-months and median survival of 24 months (p < 0.001) which remained significant following adjustment for smoking, GAP Stage and radiological UIP pattern (Hazard Ratio (HR), 2.74; 95%CI:1.46, 5.11; p = 0.002). They had increased respiratory-related mortality and acute exacerbation incidences. Despite similar baseline lung function, short LTL patients had a faster decline in absolute forced vital capacity (FVC) of -105.3 (95% CI: 151.4, -59.1) mL/year compared to -58.2 (95% CI: 82.9, -33.6) mL/year (p < 0.001) in normal LTL patients. CONCLUSION: Short LTL correlated with increased mortality and faster lung function decline in our Singaporean ILD cohort with a magnitude similar to that in Western ILD cohorts. Further research is needed to integrate LTL assessment into clinical practice.

Single-cell landscape of idiopathic multicentric Castleman disease in identical twins.

ABSTRACT: Idiopathic multicentric Castleman disease (iMCD) is a rare cytokine-driven disorder characterized by systemic inflammation, generalized lymphadenopathy, and organ dysfunction. Here, we present an unusual occurrence of iMCD in identical twins and examined the immune milieu within the affected lymphoid organs and the host circulation using multiomic high-dimensional profiling. Using spatial enhanced resolution omics sequencing (Stereo-seq) transcriptomic profiling, we performed unsupervised spatially constrained clustering to identify different anatomic structures, mapping the follicles and interfollicular regions. After a cell segmentation approach, interleukin 6 (IL-6) pathway genes significantly colocalized with endothelial cells and fibroblastic reticular cells, confirming observations using a single-cell sequencing approach (10× Chromium). Furthermore, single-cell sequencing of peripheral blood mononuclear cells revealed an "inflammatory" peripheral monocytosis enriched for the expression of S100A family genes in both twins. In summary, we provided evidence of the putative cell-of-origin of IL-6 signals in iMCD and described a distinct monocytic host immune response phenotype through a unique identical twin model.

ENIGMA CHEK2gether Project: A Comprehensive Study Identifies Functionally Impaired CHEK2 Germline Missense Variants Associated with Increased Breast Cancer Risk.

PURPOSE: Germline pathogenic variants in CHEK2 confer moderately elevated breast cancer risk (odds ratio, OR ∼ 2.5), qualifying carriers for enhanced breast cancer screening. Besides pathogenic variants, dozens of missense CHEK2 variants of uncertain significance (VUS) have been identified, hampering the clinical utility of germline genetic testing (GGT). EXPERIMENTAL DESIGN: We collected 460 CHEK2 missense VUS identified by the ENIGMA consortium in 15 countries. Their functional characterization was performed using CHEK2-complementation assays quantifying KAP1 phosphorylation and CHK2 autophosphorylation in human RPE1-CHEK2-knockout cells. Concordant results in both functional assays were used to categorize CHEK2 VUS from 12 ENIGMA case-control datasets, including 73,048 female patients with breast cancer and 88,658 ethnicity-matched controls. RESULTS: A total of 430/460 VUS were successfully analyzed, of which 340 (79.1%) were concordant in both functional assays and categorized as functionally impaired (N = 102), functionally intermediate (N = 12), or functionally wild-type (WT)-like (N = 226). We then examined their association with breast cancer risk in the case-control analysis. The OR and 95% CI (confidence intervals) for carriers of functionally impaired, intermediate, and WT-like variants were 2.83 (95% CI, 2.35-3.41), 1.57 (95% CI, 1.41-1.75), and 1.19 (95% CI, 1.08-1.31), respectively. The meta-analysis of population-specific datasets showed similar results. CONCLUSIONS: We determined the functional consequences for the majority of CHEK2 missense VUS found in patients with breast cancer (3,660/4,436; 82.5%). Carriers of functionally impaired missense variants accounted for 0.5% of patients with breast cancer and were associated with a moderate risk similar to that of truncating CHEK2 variants. In contrast, 2.2% of all patients with breast cancer carried functionally wild-type/intermediate missense variants with no clinically relevant breast cancer risk in heterozygous carriers.

COVID-19 vaccination uptake and safety profile among germline BRCA1 and BRCA2 pathogenic variant carriers in Singapore.

BACKGROUND: Although Singapore is one of the highest vaccinated countries in the world, vaccine hesitancy remains in a subpopulation, including individuals with cancer predisposition syndromes. At the Cancer Genetics Service National Cancer Centre Singapore, we see patients with germline genetic alterations, most being BRCA1/2 pathogenic/likely pathogenic variant (PV/LPV) carriers. While reported safe for cancer patients, there are limited studies addressing the safety profile and outcomes of COVID-19 vaccination among individuals with germline PV/LPV in cancer predisposition genes such as BRCA1/2. This study aims to evaluate the outcomes of COVID-19 vaccination among germline PV/LPV carriers in BRCA1/2. METHODS: We conducted a phone call survey of COVID-19 vaccination uptake and toxicity in a prospective cohort of 189 participants with germline BRCA1/2 PV/LPV between 1st Sept 2021 and 30th Sept 2021. We collected demographics data including gender, race, age, history of cancer, types of cancer, and number of cancers. Statistical difference in baseline demographics between responders with history of cancer and those without were assessed using Chi-square, Fisher's exact and independent t-test analysis. Logistic regression was used to evaluate effect of demographics on the occurrence of post-vaccination side effects. RESULTS: Among 189 BRCA1/2 PV/LPV carriers responded, 97 carried PV/LPV in BRCA1 and 92 in BRCA2. Majority were vaccinated (89.5%) and had completed the two-dose vaccine schedule, with 7 (4.1%) received only one dose. The most common post-vaccination side effects was myalgia (56.5%) followed by fever (40.2%), headache (16.3%) and fatigue (11.2%). There were no major severe side events. Evaluation by logistic regression showed that the occurrence of side effects was not affected by PV/LPV gene (BRCA1 or BRCA2), gender, race, age or history of cancer. CONCLUSION: The post-vaccination side effects profile among individuals with germline PV/LPV in BRCA1/2 is consistent with the Singaporean general population, hence recommendations for COVID-19 vaccination for these individuals should not differ from non-carriers and should be encouraged by their healthcare providers.

Functional analysis of germline RAD51C missense variants highlight the role of RAD51C in replication fork protection.

Monoallelic or biallelic RAD51C germline mutations results in chromosome instability disorders such as Fanconi anemia and cancers. The bona fide function of RAD51C is to assist RAD51 nucleoprotein filament onto single-strand DNA to complete homologous recombination (HR) repair. In addition to HR repair, the role of RAD51C in DNA replication is emerging when replication forks are transiently or irreversibly stalled. We identified novel RAD51C variants of uncertain significance (VUS) from breast, ovarian, pancreatic and gastric cancer patients and functionally characterized the effect of these variants in replication fork protection and double-strand breaks (DSB's) repair. In RAD51C-deficient Chinese hamster CL-V4B cells, expression of RAD51C F164S, A87E, L134S and E49K variants heightened sensitivity to mitomycin C (MMC), etoposide and PARP inhibition. Differently, expression of subset of RAD51C variants R24L, R24W and R212H displayed mild sensitivity to MMC, etoposide and PARP inhibition. Further functional characterization of a subset of variants revealed that Rad51C F164S, A87E, L134S and E49K variants displayed reduced RAD51 foci formation and increased overall nuclear single strand DNA levels in the presence of replication stress. Additionally, DNA fiber assay revealed that RAD51C F164S, A87E, L134S and E49K variants displayed defective replication fork protection upon prolonged fork stalling. Investigations using patient-derived lymphoblastoid cell line carrying heterozygous RAD51C L134S variant showed an impairment in RAD51 chromatin association and replication fork protection, suggestive of deleteriousness of this VUS variant. Overall, our findings provide more insights into molecular roles of RAD51C in replication fork integrity maintenance and in DSB repair.

Automatic DNA replication tract measurement to assess replication and repair dynamics at the single-molecule level.

MOTIVATION: DNA fibre assay has a potential application in genomic medicine, cancer and stem cell research at the single-molecule level. A major challenge for the clinical and research implementation of DNA fibre assays is the slow speed in which manual analysis takes place as it limits the clinical actionability. While automatic detection of DNA fibres speeds up this process considerably, current publicly available software have limited features in terms of their user interface for manual correction of results, which in turn limit their accuracy and ability to account for atypical structures that may be important in diagnosis or investigative studies. We recognize that core improvements can be made to the GUI to allow for direct interaction with automatic results to preserve accuracy as well as enhance the versatility of automatic DNA fibre detection for use in variety of situations. RESULTS: To address the unmet needs of diverse DNA fibre analysis investigations, we propose DNA Stranding, an open-source software that is able to perform accurate fibre length quantification (13.22% mean relative error) and fibre pattern recognition (R > 0.93) with up to six fibre patterns supported. With the graphical interface, we developed, user can conduct semi-automatic analyses which benefits from the advantages of both automatic and manual processes to improve workflow efficiency without compromising accuracy. AVAILABILITY AND IMPLEMENTATION: The software package is available at https://github.com/lgole/DNAStranding. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

CDKN2A germline alterations and the relevance of genotype-phenotype associations in cancer predisposition.

Although CDKN2A is well-known as a susceptibility gene for melanoma and pancreatic cancer, germline variants have also been anecdotally associated with a broader range of neoplasms including neural system tumors, head and neck squamous cell carcinomas, breast carcinomas, as well as sarcomas. The CDKN2A gene encodes for two distinct tumor suppressor proteins, p16INK4A and p14ARF, however, the independent association of germline alterations affecting these two proteins with cancer is under-appreciated. Here, we reviewed CDKN2A germline alterations reported among individuals and families with cancer in the literature, specifically addressing the cancer phenotypes in relation to the molecular consequence on p16INK4A and p14ARF. While melanoma is observed to associate with variants affecting both p16INK4A and p14ARF transcripts, it is noted that variants affecting p14ARF are more frequently observed with a heterogenous range of cancers. Finally, we reflected on the implications of this inferred genotype-phenotype association in clinical practice and proposed that clinical management of CDKN2A germline variant carriers should involve dedicated cancer genetics services, with multidisciplinary input from various healthcare professionals.

Family history assessment significantly enhances delivery of precision medicine in the genomics era.

BACKGROUND: Family history has traditionally been an essential part of clinical care to assess health risks. However, declining sequencing costs have precipitated a shift towards genomics-first approaches in population screening programs rendering the value of family history unknown. We evaluated the utility of incorporating family history information for genomic sequencing selection. METHODS: To ascertain the relationship between family histories on such population-level initiatives, we analysed whole genome sequences of 1750 research participants with no known pre-existing conditions, of which half received comprehensive family history assessment of up to four generations, focusing on 95 cancer genes. RESULTS: Amongst the 1750 participants, 866 (49.5%) had high-quality standardised family history available. Within this group, 73 (8.4%) participants had an increased family history risk of cancer (increased FH risk cohort) and 1 in 7 participants (n = 10/73) carried a clinically actionable variant inferring a sixfold increase compared with 1 in 47 participants (n = 17/793) assessed at average family history cancer risk (average FH risk cohort) (p = 0.00001) and a sevenfold increase compared to 1 in 52 participants (n = 17/884) where family history was not available (FH not available cohort) (p = 0.00001). The enrichment was further pronounced (up to 18-fold) when assessing only the 25 cancer genes in the American College of Medical Genetics (ACMG) Secondary Findings (SF) genes. Furthermore, 63 (7.3%) participants had an increased family history cancer risk in the absence of an apparent clinically actionable variant. CONCLUSIONS: These findings demonstrate that the collection and analysis of comprehensive family history and genomic data are complementary and in combination can prioritise individuals for genomic analysis. Thus, family history remains a critical component of health risk assessment, providing important actionable data when implementing genomics screening programs. TRIAL REGISTRATION: ClinicalTrials.gov NCT02791152 . Retrospectively registered on May 31, 2016.

Spectrum of Germline Mutations Within Fanconi Anemia-Associated Genes Across Populations of Varying Ancestry.

BACKGROUND: Fanconi anemia (FA) is a rare genetic disorder associated with hematological disorders and solid tumor predisposition. Owing to phenotypic heterogeneity, some patients remain undetected until adulthood, usually following cancer diagnoses. The uneven prevalence of FA cases with different underlying FA gene mutations worldwide suggests variable genetic distribution across populations. Here, we aim to assess the genetic spectrum of FA-associated genes across populations of varying ancestries and explore potential genotype-phenotype associations in cancer. METHODS: Carrier frequency and variant spectrum of potentially pathogenic germline variants in 17 FA genes (excluding BRCA1/FANCS, BRCA2/FANCD1, BRIP1/FANCJ, PALB2/FANCN, RAD51C/FANCO) were evaluated in 3523 Singaporeans and 7 populations encompassing Asian, European, African, and admixed ancestries from the Genome Aggregation Database. Germline and somatic variants of 17 FA genes in 7 cancer cohorts from The Cancer Genome Atlas were assessed to explore genotype-phenotype associations. RESULTS: Germline variants in FANCA were consistently more frequent in all populations. Similar trends in carrier frequency and variant spectrum were detected in Singaporeans and East Asians, both distinct from other ancestry groups, particularly in the lack of recurrent variants. Our exploration of The Cancer Genome Atlas dataset suggested higher germline and somatic mutation burden between FANCA and FANCC with head and neck and lung squamous cell carcinomas as well as FANCI and SLX4/FANCP with uterine cancer, but the analysis was insufficiently powered to detect any statistical significance. CONCLUSION: Our findings highlight the diverse genetic spectrum of FA-associated genes across populations of varying ancestries, emphasizing the need to include all known FA-related genes for accurate molecular diagnosis of FA.

Impact of Variant Reclassification in Cancer Predisposition Genes on Clinical Care.

PURPOSE: Genetic testing has clinical utility in the management of patients with hereditary cancer syndromes. However, the increased likelihood of encountering a variant of uncertain significance in individuals of non-European descent such as Asians may be challenging to both clinicians and patients. This study aims to evaluate the impact of variant reclassification in an Asian country with variants of uncertain significance reported in cancer predisposition genes. METHODS: A retrospective analysis of patients seen at the Cancer Genetics Service at the National Cancer Centre Singapore between February 2014 and March 2020 was conducted. The frequency, direction, and time to variant reclassification were evaluated by comparing the reclassified report against the original report. RESULTS: A total of 1,412 variants of uncertain significance were reported in 49.9% (845 of 1,695) of patients. Over 6 years, 6.7% (94 of 1,412) of variants were reclassified. Most variants of uncertain significance (94.1%, 80 of 85) were downgraded to benign or likely benign variant, with a smaller proportion of variants of uncertain significance (5.9%, 5 of 85) upgraded to pathogenic or likely pathogenic variant. Actionable variants of uncertain significance upgrades and pathogenic or likely pathogenic variant downgrades, which resulted in management changes, happened in 31.0% (39 of 126) of patients. The median and mean time taken for reclassification were 1 and 1.62 year(s), respectively. CONCLUSION: We propose a clinical guideline to standardize management of patients reported to have variants of uncertain significance. Management should be based on the patient's personal history, family history, and variant interpretation. For clinically relevant or suspicious variants of uncertain significance, follow-up is recommended every 2 years, as actionable reclassifications may happen during this period.

Investigation into the origins of an ancient BRCA1 founder mutation identified among Chinese families in Singapore.

Identification of ancestry-specific pathogenic variants is imperative for diagnostic, treatment, management and prevention strategies, and to understand penetrance/modifiers on risk. Our study aimed to determine the clinical significance of a recurrent BRCA1 c.442-22_442-13del variant of unknown significance identified among 13 carriers from six Chinese families, all with a significant history of breast and/or ovarian cancer. We further aimed to establish whether this was due to a founder effect and explore its origins. Haplotype analysis, using nine microsatellite markers encompassing 2.5 megabase pairs around the BRCA1 locus, identified a common haploblock specific to the variant carriers, confirming a founder effect. Variant age was estimated to date back 77.9 generations to 69 bc using the Gamma approach. On principal component analysis using single nucleotide polymorphisms merged with 1000 Genomes dataset, variant carriers were observed to overlap predominantly with the southern Han Chinese population. To determine pathogenicity of the variant, we assessed the functional effect on RAD51 foci formation as well as replication fork stability upon induction of DNA damage and observed an impaired DNA repair response associated with the variant. In summary, we identified an ancient Chinese founder mutation dating back 77.9 generations, possibly common among individuals of southern Han Chinese descent. Using evidence from phenotypic/family history studies, segregation analysis and functional characterization, the BRCA1 variant was reclassified from uncertain significance to pathogenic.

Functional characterisation guides classification of novel BAP1 germline variants.

We have identified six patients harbouring distinct germline BAP1 mutations. In this study, we functionally characterise known BAP1 pathogenic and likely benign germline variants out of these six patients to aid in the evaluation and classification of unknown BAP1 germline variants. We found that pathogenic germline variants tend to encode truncated proteins, show diminished expression of epithelial-mesenchymal transition (EMT) markers, are localised in the cytosol and have reduced deubiquitinase capabilities. We show that these functional assays are useful for BAP1 variant curation and may be added in the American College of Medical Genetics and Genomics (ACMG) criteria for BAP1 variant classification. This will allow clinicians to distinguish between BAP1 pathogenic and likely benign variants reliably and may aid to quickly benchmark newly identified BAP1 germline variants. Classification of novel BAP1 germline variants allows clinicians to inform predisposed patients and relevant family members regarding potential cancer risks, with appropriate clinical interventions implemented if required.

Multiple neoplasia in a patient with Gitelman syndrome harboring germline monoallelic MUTYH mutation.

Gitelman syndrome is a rare, recessively inherited disease characterized by chronic hypokalemia and hypomagnesemia as a result of defective electrolyte co-transport at the level of the distal convoluted tubule of the kidney. Here, we present the first report of a patient with Gitelman syndrome who developed multiple neoplasia including colorectal polyposis, synchronous colorectal cancers, recurrent breast fibroadenomata and a desmoid tumor. Whole-exome sequencing confirmed germline compound heterozygous mutations of c.179C > T and c.1326C > G in SLC12A3, and in addition, identified a monoallelic germline c.934-2A > G splice site mutation in MUTYH. In vitro, magnesium deficiency potentiated oxidative DNA damage in lymphoblastoid cell lines derived from the same patient. We postulate that monoallelic MUTYH mutations may manifest in the presence of cooperative non-genetic mechanisms, in this case possibly magnesium deficiency from Gitelman syndrome.

Biallelic NF1 inactivation in high grade serous ovarian cancers from patients with neurofibromatosis type 1.

Neurofibromatosis type 1 (NF1) is a multisystem disorder caused by germline heterozygous NF1 loss-of-function variants. The NF1 gene encodes neurofibromin, a RAS GTPase-activating protein, which functions by down-regulating RAS/RAF/MAPK-signalling pathways. Somatic NF1 aberrations frequently occur in sporadic ovarian cancer (OC), but the incidence of OC in NF1 patients is rare. Here we report the germline and somatic findings for two unrelated patients with NF1 and high-grade serous OC. Germline testing revealed a heterozygous NF1 pathogenic variant in each patient, c.7096_7101del (p.Asn2366_Phe2367del) and c.964delA (p.Ile322Leufs*54), respectively. No germline variants in well-established OC predisposition genes were detected, including BRCA1 and BRCA2. Tumor loss-of-heterozygosity analysis demonstrated loss of the wild type NF1 allele for both patients. Biallelic NF1 inactivation occurs as part of OC pathogenesis in NF1 patients. Although the penetrance of NF1-associated OC is insufficient to warrant risk-reducing interventions, our findings highlight the potential for therapies targeting the RAS/RAF/MAPK-signalling pathway for these cases.

Missense PALB2 germline variant disrupts nuclear localization of PALB2 in a patient with breast cancer.

The PALB2 protein is essential to RAD51-mediated homologous recombination (HR) repair. Germline monoallelic PALB2 pathogenic variants confer significant risks for breast cancer. However, the majority of PALB2 variants remain classified as variants of unknown significance (VUS). We aim to functionally and mechanistically evaluate three novel PALB2 VUS. Patient-derived lymphoblastoid cell lines containing the VUS were analyzed for nuclear localization and foci formation of RAD51 as a measure of HR efficiency. To understand the mechanism underlying the HR deficiency, PALB2 nuclear localization was assessed using immunofluorescence studies. Among these VUS, c.3251C>T (p.Ser1084Leu) occurred in a patient with metastatic breast cancer while c.1054G>C (p.Glu352Gln) and c.1057A>G (p.Lys353Glu) were seen in patients with squamous cell carcinoma of skin and renal cell carcinoma respectively. Variant c.3251C>T was located within the WD40 domain which normally masked the nuclear export signal sequence responsible for nuclear delocalization of PALB2. Correspondingly, c.3251C>T displayed aberrant cytoplasmic localization of PALB2 which led to an impaired RAD51 nuclear localization and foci formation. On the other hand, both c.1054G>C and c.1057A>G showed intact HR functions and nuclear localization of PALB2, consistent with their locations within domains of no known function. Additionally, the prevalence of c.1054G>C was similar among healthy controls and patients with breast cancer (as seen in other studies), suggestive of its non-pathogenicity. In conclusion, our studies provided the functional evidence showing the deleterious effect of c.3251C>T, and non-deleterious effects of c.1054G>C and c.1057A>G. Using the ClinGen Pathogenicity calculator, c.3251C>T remains a VUS while c.1054G>C and c.1057A>G may be classified as likely benign variants.

Functional analysis of clinical BARD1 germline variants.

Germline pathogenic variants in BRCA1/2 account for one-third of familial breast cancers. The majority of BRCA1 function requires heterodimerization with BARD1. In contrast to BRCA1, BARD1 is a low-penetrance gene with an unclear clinical relevance, partly because of limited functional evidence. Using patient-derived lymphoblastoid cells, we functionally characterized two pathogenic variants (c.1833dupT, c.2099delG) and three variants of uncertain significance (VUSs) (c.73G>C, c.1217G>A, c.1918C>A). Three of these patients had breast cancers, whereas the remaining had colorectal cancers (n = 3). Both patients with pathogenic variants (c.1833dupT, c.2099delG) developed breast cancers with aggressive disease phenotypes such as triple-negative breast cancer and high cancer grades. As BARD1 encompasses multiple functional domains, including those of apoptosis and homologous recombination repair, we hypothesized that the function being impaired would correspond with the domain where the variant was located. Variants c.1918C>A, c.1833dupT, c.1217G>A, and c.2099delG, located within and proximal to apoptotic domains of ankyrin and BRCT, were associated with impaired apoptosis. Conversely, apoptosis function was preserved in c.73G>C, which was distant from the ankyrin domain. All variants displayed normal BRCA1 heterodimerization and RAD51 colocalization, consistent with their location being distal to BRCA1-and RAD51-binding domains. In view of deficient apoptosis, VUSs (c.1217G>A and c.1918C>A) may be pathogenic or likely pathogenic variants. In summary, functional analysis of BARD1 VUSs requires a combination of assays and, more importantly, the use of appropriate functional assays with consideration to the variant's location.

Clinical relevance of screening checklists for detecting cancer predisposition syndromes in Asian childhood tumours.

Assessment of cancer predisposition syndromes (CPS) in childhood tumours is challenging to paediatric oncologists due to inconsistent recognizable clinical phenotypes and family histories, especially in cohorts with unknown prevalence of germline mutations. Screening checklists were developed to facilitate CPS detection in paediatric patients; however, their clinical value have yet been validated. Our study aims to assess the utility of clinical screening checklists validated by genetic sequencing in an Asian cohort of childhood tumours. We evaluated 102 patients under age 18 years recruited over a period of 31 months. Patient records were reviewed against two published checklists and germline mutations in 100 cancer-associated genes were profiled through a combination of whole-exome sequencing and multiplex ligation-dependent probe amplification on blood-derived genomic DNA. Pathogenic germline mutations were identified in ten (10%) patients across six known cancer predisposition genes: TP53, DICER1, NF1, FH, SDHD and VHL. Fifty-four (53%) patients screened positive on both checklists, including all ten pathogenic germline carriers. TP53 was most frequently mutated, affecting five children with adrenocortical carcinoma, sarcomas and diffuse astrocytoma. Disparity in prevalence of germline mutations across tumour types suggested variable genetic susceptibility and implied potential contribution of novel susceptibility genes. Only five (50%) children with pathogenic germline mutations had a family history of cancer. We conclude that CPS screening checklists are adequately sensitive to detect at-risk children and are relevant for clinical application. In addition, our study showed that 10% of Asian paediatric solid tumours have a heritable component, consistent with other populations.

Germline Pathogenic Variants in Homologous Recombination and DNA Repair Genes in an Asian Cohort of Young-Onset Colorectal Cancer.

BACKGROUND: Growing evidence suggests a role for cancer susceptibility genes such as BRCA2 and PALB2 in young-onset colorectal cancers. Using a cohort of young colorectal cancer patients, we sought to identify and provide functional evidence for germline pathogenic variants of DNA repair genes not typically associated with colorectal cancer. METHODS: We recruited 88 patients with young-onset colorectal cancers seen at a general oncology center. Whole-exome sequencing was performed to identify variants in DNA repair and colorectal cancer predisposition genes. Pathogenic BRCA2 and PALB2 variants were analyzed using immunoblot and immunofluorescence on patient-derived lymphoblastoid cells. RESULTS: In general, our cohort displayed characteristic features of young-onset colorectal cancers. Most patients had left-sided tumors and were diagnosed at late stages. Four patients had familial adenomatous polyposis, as well as pathogenic APC variants. We identified 12 pathogenic variants evenly distributed between DNA repair and colorectal cancer predisposition genes. Six patients had pathogenic variants in colorectal cancer genes: APC (n = 4) and MUTYH monoallelic (n = 2). Another six had pathogenic variants in DNA repair genes: ATM (n = 1), BRCA2 (n = 1), PALB2 (n = 1), NTHL1 (n = 1), and WRN (n = 2). Pathogenic variants BRCA2 c.9154C>T and PALB2 c.1059delA showed deficient homologous recombination repair, evident from the impaired RAD51 nuclear localization and foci formation. CONCLUSION: A substantial portion of pathogenic variants in young-onset colorectal cancer was found in DNA repair genes not previously associated with colorectal cancer. This may have implications for the management of patients. Further studies are needed to ascertain the enrichment of pathogenic DNA repair gene variants in colorectal cancers.

Germline Mutations in Cancer Predisposition Genes are Frequent in Sporadic Sarcomas.

Associations of sarcoma with inherited cancer syndromes implicate genetic predisposition in sarcoma development. However, due to the apparently sporadic nature of sarcomas, little attention has been paid to the role genetic susceptibility in sporadic sarcoma. To address this, we performed targeted-genomic sequencing to investigate the prevalence of germline mutations in known cancer-associated genes within an Asian cohort of sporadic sarcoma patients younger than 50 years old. We observed 13.6% (n = 9) amongst 66 patients harbour at least one predicted pathogenic germline mutation in 10 cancer-associated genes including ATM, BRCA2, ERCC4, FANCC, FANCE, FANCI, MSH6, POLE, SDHA and TP53. The most frequently affected genes are involved in the DNA damage repair pathway, with a germline mutation prevalence of 10.6%. Our findings suggests that genetic predisposition plays a larger role than expected in our Asian cohort of sporadic sarcoma, therefore clinicians should be aware of the possibility that young sarcoma patients may be carriers of inherited mutations in cancer genes and should be considered for genetic testing, regardless of family history. The prevalence of germline mutations in DNA damage repair genes imply that therapeutic strategies exploiting the vulnerabilities resulting from impaired DNA repair may be promising areas for translational research.