MLH1-methylated endometrial cancer under 60 years of age as the “sentinel” cancer in female carriers of high-risk constitutional MLH1 epimutation

Objective. Universal screening of endometrial carcinoma (EC) for mismatch repair deficiency (MMRd) and Lynch syndrome uses presence of MLH1 methylation to omit common sporadic cases from follow-up germline testing. However, this overlooks rare cases with high-risk constitutional MLH1 methylation (epimutation), a poorly-recognized mechanism that predisposes to Lynch-type cancers with MLH1 methylation. We aimed to de-termine the role and frequency of constitutional MLH1 methylation among EC cases with MMRd, MLH1- methylated tumors.Methods. We screened blood for constitutional MLH1 methylation using pyrosequencing and real-time methylation-specific PCR in patients with MMRd, MLH1-methylated EC ascertained from (i) cancer clinics (n = 4, <60 years), and (ii) two population-based cohorts; Columbus-area (n = 68, all ages) and Ohio Colo-rectal Cancer Prevention Initiative (OCCPI) (n = 24, <60 years).Results. Constitutional MLH1 methylation was identified in three out of four patients diagnosed between 36 and 59 years from cancer clinics. Two had mono-/hemiallelic epimutation (similar to 50% alleles methylated). One with multiple primaries had low-level mosaicism in normal tissues and somatic second-hits affecting the unmethylated allele in all tumors, demonstrating causation. In the population-based cohorts, all 68 cases from the Columbus-area cohort were negative and low-level mosaic constitutional MLH1 methylation was identified in one patient aged 36 years out of 24 from the OCCPI cohort, representing one of six (similar to 17%) patients <50 years and one of 45 patients (similar to 2%) <60 years in the combined cohorts. EC was the first/dual-first cancer in three pa-tients with underlying constitutional MLH1 methylation.Conclusions. A correct diagnosis at first presentation of cancer is important as it will significantly alter clinical management. Screening for constitutional MLH1 methylation is warranted in patients with early-onset EC or syn-chronous/metachronous tumors (any age) displaying MLH1 methylation.(c) 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http:// creativecommons.org/licenses/by-nc-nd/4.0/)

Large Cancer Pedigree Involving Multiple Cancer Genes including Likely Digenic MSH2 and MSH6 Lynch Syndrome (LS) and an Instance of Recombinational Rescue from LS

Funding Information: This research was funded in part by a Cedars-Sinai Medical Center Precision Health Initiative Award to Megan P. Hitchins and Andrew Hendifar. Publisher Copyright: © 2022 by the authors.Lynch syndrome (LS), caused by heterozygous pathogenic variants affecting one of the mismatch repair (MMR) genes (MSH2, MLH1, MSH6, PMS2), confers moderate to high risks for colorectal, endometrial, and other cancers. We describe a four-generation, 13-branched pedigree in which multiple LS branches carry the MSH2 pathogenic variant c.2006G>T (p.Gly669Val), one branch has this and an additional novel MSH6 variant c.3936_4001+8dup (intronic), and other non-LS branches carry variants within other cancer-relevant genes (NBN, MC1R, PTPRJ). Both MSH2 c.2006G>T and MSH6 c.3936_4001+8dup caused aberrant RNA splicing in carriers, including out-of-frame exon-skipping, providing functional evidence of their pathogenicity. MSH2 and MSH6 are co-located on Chr2p21, but the two variants segregated independently (mapped in trans) within the digenic branch, with carriers of either or both variants. Thus, MSH2 c.2006G>T and MSH6 c.3936_4001+8dup independently confer LS with differing cancer risks among family members in the same branch. Carriers of both variants have near 100% risk of transmitting either one to offspring. Nevertheless, a female carrier of both variants did not transmit either to one son, due to a germline recombination within the intervening region. Genetic diagnosis, risk stratification, and counseling for cancer and inheritance were highly individualized in this family. The finding of multiple cancer-associated variants in this pedigree illustrates a need to consider offering multicancer gene panel testing, as opposed to targeted cascade testing, as additional cancer variants may be uncovered in relatives.Peer reviewe

Identification and characterisation of genes from the Angelman and Prader-Willi syndrome region of chromosome 15q11-13

Angelman syndrome (AS) and Prader-Willi syndrome (PWS) are clinically distinct neurobehavioural disorders which map to 15ql 1.2-12. AS is characterised by mental retardation, seizures, ataxia, absent speech, excessive laughter, and distinctive EEG. PWS involves poor suck reflex and hypotonia in infancy, short stature, microgenitalia, and hyperphagia leading to obesity. Chromosome 15ql 1.2-12 is subject to genomic imprinting whereby certain genes are functionally active or silent depending on their parental origin of inheritance. AS and PWS are caused by various mechanisms including cytogenetic deletion, uniparental disomy and altered epigenetic modification, with the common factor being lack of a maternal contribution to 15ql 1.2-12 in AS, and loss of expression of genes on the paternally derived allele in PWS. Approximately 25% of AS patients, including familial cases, are due to mutations of an imprinted gene within 15ql 1.2-12, which is expressed from the maternal allele. The UBE3A gene has been implicated in AS. The lack of a similar category of patients in PWS suggests that this is a contiguous gene disorder. In order to identify genes which may be involved in the pathogenesis of either AS or PWS, positional cloning studies were performed in the AS and distal portion of the PWS candidate regions. UBE3A was located to the AS region, and a novel expressed sequence identified through direct cDNA selection was shown to represent an extended 3' untranslated region of UBE3A. No additional genes were identified in the AS region. USES A was screened for mutations in cases of familial and sporadic AS in which the genetic aetiology of the disease remained unidentified. Pathogenic mutations were identified in 3/5 familial and 4/21 sporadic cases, indicating that UBE3A mutations cause AS in a proportion of patients. Two novel transcripts, '395-H22' and '123-E19', were identified from the PWS region. 395- H22 was found to be expressed solely from the paternal allele in an in vitro imprinting assay system, indicating that it may represent an additional gene involved in the PWS phenotype

Favoritism in DNA methylation

This perspective on Candiloro and Dobrovic (beginning on p. 862 in this issue of the journal) highlights the interplay between epigenetic aberrations and underlying DNA sequence changes and illustrates how these alterations may predispose individuals to cancer. Candiloro and Dobrovic clearly show that particular genotypes of the MGMT gene are associated with its methylation in healthy individuals. Aberrant MGMT methylation may identify individuals who could be targeted for cancer screening and chemoprevention strategies

Epigenetic changes in childhood asthma (commentary)

Childhood asthma is linked strongly to atopy and is characterised by a T helper 2 (Th2)-polarised immunological response. Epidemiological studies implicate severe lower respiratory tract viral infections, especially in early childhood, and repeated inhalational exposure to allergens as important synergistic factors in the development of asthma. The way in which these and other environmental factors induce stable alterations in phenotype is poorly understood, but may be explained on the basis of epigenetic changes, which are now recognised to underlie the establishment and maintenance of a Th2 response. Furthermore, ongoing asthmatic inflammation of the airways may be driven by alterations in the expression profile of regulatory microRNA genes, to which epigenetic mechanisms may also contribute. Thus, an understanding of epigenetic mechanisms in asthma has the potential to reveal new approaches for primary prevention or therapeutic intervention in childhood asthma

A systematic review and meta-analysis : the diagnostic accuracy of methylated SEPTIN9 for the detection of hepatocellular carcinoma and the clinical evaluation of its use in combination with other surveillance modalities

Background: Hepatocellular carcinoma (HCC) lacks a suitable biomarker for minimally-invasive disease detection. Methylated SEPTIN9 (mSEPT9) is an emerging liquid biopsy test. We aimed to investigate recent studies that applied mSEPT9 for HCC diagnosis. Furthermore, we evaluated the combinations of other surveillance modalities for the detection of HCC. Methods: A systematic review was performed on the diagnostic accuracy of mSEPT9 for the detection of HCC. Using a bivariate model, the pooled sensitivity and specificity were calculated. Additionally, Fagan’s nomograms were used to calculate the pre-test and post-test probabilities of HCC for various combinations of surveillance modalities. Results: Six full texts were included in the meta-analysis. The pooled sensitivity and specificity of mSEPT9 for the detection of HCC, were 0.80 (95% CI, 0.67–0.89) and 0.90 (95% CI, 0.84–0.94). The area under the receiver operating curve was 0.92. The probability of having HCC for the combinations of mSEPT9+ ultrasound scan (USS) and mSEPT9+ Alpha fetoprotein (AFP) were 0.7% and 1.2% respectively if both tests were negative (in a population with 10% HCC prevalence). The combination of USS and AFP would miss relatively fewer cancers for 1000 patients in comparison to other combinations of two surveillance modalities. Conclusion: Test combinations have superior performance for the detection of HCC than any individual test. mSEPT9 has shown promise in the detection of HCC with higher estimates of performance accuracy. mSEPT9 has potential for use as an HCC surveillance modality in adjunct with other tests to improve detection rates. However, cost effectiveness of this approach needs further evaluation

Methylation of the 3p22 region encompassing MLH1 is representative of the CpG island methylator phenotype in colorectal cancer

Epigenetic silencing of cancer-related genes by promoter methylation is a frequent event in sporadic colorectal cancer. The CpG island methylator phenotype (CIMP), in which discrete genes throughout the genome are simultaneously methylated, and long-range epigenetic silencing, whereby multiple genes within contiguous chromosomal regions are methylated, have been described in subsets of colorectal cancer. We previously reported the concurrent methylation of the mismatch repair gene MLH1 with a cluster of flanking genes in chromosome region 3p22 in sporadic colorectal carcinoma exhibiting microsatellite instability and the BRAF V600E mutation. Herein, we aimed to determine whether methylation of MLH1 and neighbouring 3p22 genes, singly or concomitantly, correlate with the germline c.-93GA SNP within the MLH1 promoter, CIMP and other clinicopathological and molecular features of the tumours. By studying a cohort of 946 sporadic colorectal cancer cases, we show a strong association between concordant methylation of 3 of five 3p22 genes with CIMP and the BRAF V600E mutation (P0.001). These associations were independent of microsatellite instability, as concomitant methylation of 3p22 genes other than MLH1 was found in microsatellite stable cancers. These findings show that long-range epigenetic silencing across 3p22 occurs in the context of CIMP and the BRAF V600E mutation, and only gives rise to microsatellite instability when this process encompasses MLH1. Furthermore, the strong relationship between long-range epigenetic silencing of 3p22 and CIMP provides further evidence that these two purportedly distinct epigenetic phenotypes represent a single entity with a common aetiology. Low-level methylation of MLH1 and flanking 3p22 genes, as well as the BRAF V600E mutation, were detected in the apparently normal colonic mucosa of a small number of cases whose tumours showed a similar molecular profile, suggesting that these concurring genetic and epigenetic events can occur as a field defect in neoplastic development

Implementation of novel pyrosequencing assays to screen for common mutations of BRAF and KRAS in a cohort of sporadic colorectal cancers

Activating mutations of the BRAF and KRAS genes cause constitutive stimulation of an important cell-signaling pathway promoting tumorigenesis, and are increasingly recognized as determinants of response to targeted cancer therapies. The V600E mutation accounts for most of the BRAF mutations in cancer, and KRAS mutations are predominantly encoded by nucleotide substitutions within codons 12 and 13. We designed novel pyrosequencing assays for the detection of the common "hotspot" mutations in these genes, which demonstrated analytical sensitivities of ≤10% in titrations of mutant cell lines. The KRAS pyrosequencing assay has the ability to simultaneously identify all potential nucleotide changes within the mutation cluster at codons 12 and 13, with a sequence output in the sense direction to facilitate results interpretation. These assays were used to determine the mutation status in a prospective series of 1198 sporadic colorectal cancers. The BRAF V600E mutation was detected in 13.2% of the colorectal cancers. The frequency of KRAS mutations in our cohort was 32.4%, with G>A transitions at position 2 of codons 12 and 13 being most prevalent. Both assays proved highly sensitive and specific when applied to clinical specimens, and were applicable to both fresh-frozen and formalin-fixed paraffin-embedded archival tissues. These assays would serve as a suitable platform for large-scale mutation detection in cancer specimens where the facility for pyrosequencing is available