ABSTRACT Loss-of-function germline variants of _MLH1_ cause Lynch syndrome. Here, we present the case of a 43-year-old male patient diagnosed with cecal and transverse colon adenocarcinomas.

The characteristics of the case met the revised Bethesda guidelines, and the tumors demonstrated a high frequency of microsatellite instability. Genetic testing for mismatch repair genes

(indicative of Lynch syndrome) revealed a novel heterozygous germline pathogenic variant, NM_000249.4:c.856A>T/NP_000240.1:p.(Lys286Ter), in _MLH1_. SIMILAR CONTENT BEING VIEWED BY OTHERS

_AN MSH6_ GERMLINE PATHOGENIC VARIANT P.GLY162TER ASSOCIATED WITH LYNCH SYNDROME Article Open access 26 October 2022 GERMLINE DELETION OF CHROMOSOME 2P16-21 ASSOCIATED WITH LYNCH SYNDROME

extracolonic malignancies, such as endometrial, ovarian, stomach, small bowel, hepatobiliary, and urothelial cancers1. LS is caused either by germline loss-of-function (LoF) pathogenic (P)

and likely pathogenic (LP) variants in one of the four DNA mismatch repair (MMR) genes (_MLH1_, _MSH2_, _MSH6_, and _PMS2_) or by germline deletions in the epithelial cell adhesion molecule

(_EPCAM_) gene leading to epigenetic silencing of the adjacent _MSH2_1,2. The microsatellite instability (MSI) phenotype is a hallmark of LS-associated tumors caused by MMR system deficiency

(dMMR)2. For the definitive diagnosis of LS, genetic testing of these MMR genes is currently used in clinical practice. Therefore, accumulated knowledge regarding germline variants in MMR

genes is necessary for the accurate diagnosis of LS. Genetic identification of LS patients not only alerts the probands to their own life and health risks but also warns their relatives of

their own cancer risk and enables subsequent genetic testing, with significant benefits in terms of the timing, cost, and effectiveness of surveillance, early detection, and reduced cancer

mortality. _MLH1_ and _MSH2_ are the major pathogenic genes for LS2. Additionally, the majority of variants of _MLH1_ and _MSH2_ reported in one of the disease-related databases (InSiGHT

variant database, https://www.insight-group.org/variants/databases/) are truncated (predominantly nonsense or frameshift variants)2, frequently leading to the LoF of these genes. Here, we

report a novel _MLH1_ nonsense variant, NM_000249.4:c.856A>T/NP_000240.1:p.(Lys286Ter), associated with LS and classified as LP according to the joint consensus guidelines of the American

College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP)3. A 43-year-old Japanese male (III-2, Fig. 1) was admitted with sudden abdominal pain. The

patient had no significant medical history. Through imaging evaluation via abdominal computed tomography (CT), the patient was diagnosed with perforation of the cecal and transverse colon

and panperitonitis. On the same day, the patient underwent an emergency right hemicolectomy. Pathological examination revealed pT3N3 adenocarcinoma on the basis of the TNM classification4

for both cecal and transverse colon cancers. Detailed imaging examinations via contrast-enhanced CT and magnetic resonance imaging before postoperative chemotherapy revealed multiple

metastases in the liver. The results of MSI testing of the resected CRC tumors demonstrated a high frequency of MSI (MSI-H); however, the tumors were negative for the BRAF V600E variant.

Additionally, the characteristics of the case met the criteria of the revised Bethesda guidelines5; therefore, the patient was referred to the Clinical Genetics Department of our hospital

for hereditary tumor risk assessment, although the characteristics of the family members did not meet the Amsterdam Criteria II for LS6 (Fig. 1). Several commercially available genetic tests

have been proposed by clinical geneticists for the definitive diagnosis of hereditary CRC. Following pretest genetic counseling and the acquisition of informed consent, the patient opted

for and underwent MMR gene testing for the evaluation of the _MLH1_, _MSH2_, _MSH6_, _PMS2_, and _EPCAM_ genes via very long amplicon sequencing (vLAS), an optimized long-range polymerase

chain reaction (PCR)-based next-generation sequencing method (Center for Clinical Genomics, Kanazawa Medical University Hospital, Uchinada, Japan)7. Using vLAS technology, single-nucleotide

variants (SNVs), small insertions or deletions (indels), large indels, and structural variants, including exon-level copy number variants (CNVs) within the regions covered by long-range PCR,

can be detected. The identified variants were interpreted on the basis of ACMG/AMP Guidelines3. The heterozygous nonsense variant NM_000249.4:c.856A>T/NP_000240.1:p.(Lys286Ter)

(NC_000003.12:g.37017571A>T) in _MLH1_ was identified and confirmed via Sanger sequencing (Fig. 2). To our knowledge (4 June 2024 date last accessed), this SNV has never been reported in

disease-related databases, including the Human Gene Mutation Database (HGMD) Professional (https://my.qiagendigitalinsights.com/bbp/view/hgmd/pro/start.php), Leiden Open Variation Database

(LOVD) v3.0 (https://www.lovd.nl/), InSiGHT variant database, or ClinVar (https://www.ncbi.nlm.nih.gov/clinvar/), and has rarely been reported in population databases (PM2 ACMG/AMP variant

criterion3), including gnomAD v4.1.0 (https://gnomad.broadinstitute.org/, allele frequency = 6.842 × e−7) and 54KJPN-SNV/INDEL (https://jmorp.megabank.tohoku.ac.jp/, allele frequency = 0).

This SNV is predicted to generate a stop codon, possibly leading to a premature termination codon and causing a LoF (PVS1 ACMG/AMP variant criterion3) via nonsense-mediated mRNA decay (NMD).

According to the ACMG/AMP guidelines3, this SNV was classified as LP on the basis of the PM2 and PVS1 criteria. No other variants that could be responsible for LS were detected in any of

the tested genes. Therefore, the patient was diagnosed with LS due to a novel germline nonsense variant of _MLH1_. Following the diagnosis of the proband, _MLH1_ genetic testing of at-risk

family members, especially unaffected first-degree relatives, was suggested for genetic counseling but has not yet been performed. At the same nucleotide position, NM_000249.4:c.856, two

other nucleotide changes, A > C and A > G, which cause missense substitutions of amino acids at codon 286, p.(Lys286Gln) and p.(Lys286Glu), respectively, have been reported in

disease-related databases as variants of uncertain significance or as benign or likely benign variants. Because nonsense variants in neighboring codons, such as codon 284, have been reported

as P or LP in the ClinVar and InSiGHT databases, it is reasonable to predict that c.856A>T is a null variant causing NMD to lead to LS, although the nonsense SNV in codon 286 has not

been reported previously. Identifying a pathogenic MMR variant in the proband is essential to confirm the genetic predisposition to LS in the proband and enable the presymptomatic diagnosis

of variant carriers in family members. Therefore, reporting novel pathogenic variants responsible for LS will help in the accurate diagnosis of LS. HGV DATBASE The relevant data from this

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  Download references ACKNOWLEDGEMENTS The authors thank all individuals who participated in this study. This work was supported in part by the Aichi Cancer Research Foundation (N. Takaiso)

and the Japan Agency for Medical Research and Development (AMED); grant numbers JP22kk0305020 and JP23ck0106872; (I. Imoto). FUNDING This work was supported in part by the Aichi Cancer

Research Foundation (N. Takaiso) and the Japan Agency for Medical Research and Development (AMED); grant numbers JP22kk0305020 and JP23ck0106872; (I. Imoto). AUTHOR INFORMATION Author notes

* These authors contributed equally: Nobue Takaiso, Issei Imoto. AUTHORS AND AFFILIATIONS * Risk Assessment Unit, Aichi Cancer Center Hospital, Nagoya, Japan Nobue Takaiso, Issei Imoto &

 Akiyo Yoshimura * Aichi Cancer Center Research Institute, Nagoya, Japan Issei Imoto * Department of Medical Oncology, Ichinomiyanishi Hospital, Ichinomiya, Japan Toshihiko Matsumoto *

Department of Breast Oncology, Aichi Cancer Center Hospital, Nagoya, Japan Akiyo Yoshimura Authors * Nobue Takaiso View author publications You can also search for this author inPubMed 

Google Scholar * Issei Imoto View author publications You can also search for this author inPubMed Google Scholar * Toshihiko Matsumoto View author publications You can also search for this

author inPubMed Google Scholar * Akiyo Yoshimura View author publications You can also search for this author inPubMed Google Scholar CORRESPONDING AUTHOR Correspondence to Issei Imoto.

ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ETHICS This study was approved by the Institutional Review Board of Aichi Cancer Center (No. S06002).

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_MLH1_ nonsense variant in a patient with suspected Lynch syndrome. _Hum Genome Var_ 11, 36 (2024). https://doi.org/10.1038/s41439-024-00294-9 Download citation * Received: 19 July 2024 *

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