Rapid classification of a novel ALS-causing I149S variant in superoxide dismutase-1.

Variants of the oxygen free radical scavenging enzyme superoxide dismutase-1 (SOD1) are associated with the neurodegenerative disease amyotrophic lateral sclerosis (ALS). These variants occur in roughly 20% of familial ALS cases, and 1% of sporadic ALS cases. Here, we identified a novel SOD1 variant in a patient in their 50s who presented with movement deficiencies and neuropsychiatric features. The variant was heterozygous and resulted in the isoleucine at position 149 being substituted with a serine (I149S). In silico analysis predicted the variant to be destabilizing to the SOD1 protein structure. Expression of the SOD1I149S variant with a C-terminal EGFP tag in neuronal-like NSC-34 cells resulted in extensive inclusion formation and reduced cell viability. Immunoblotting revealed that the intramolecular disulphide between Cys57 and Cys146 was fully reduced for SOD1I149S. Furthermore, SOD1I149S was highly susceptible to proteolytic digestion, suggesting a large degree of instability to the protein fold. Finally, fluorescence correlation spectroscopy and native-PAGE of cell lysates showed that SOD1I149S was monomeric in solution in comparison to the dimeric SOD1WT. This experimental data was obtained within 3 months and resulted in the rapid re-classification of the variant from a variant of unknown significance (VUS) to a clinically actionable likely pathogenic variant.

Optimising clinical care through CDH1-specific germline variant curation: improvement of clinical assertions and updated curation guidelines.

BACKGROUND: Germline pathogenic variants in CDH1 are associated with increased risk of diffuse gastric cancer and lobular breast cancer. Risk reduction strategies include consideration of prophylactic surgery, thereby making accurate interpretation of germline CDH1 variants critical for physicians deciding on these procedures. The Clinical Genome Resource (ClinGen) CDH1 Variant Curation Expert Panel (VCEP) developed specifications for CDH1 variant curation with a goal to resolve variants of uncertain significance (VUS) and with ClinVar conflicting interpretations and continues to update these specifications. METHODS: CDH1 variant classification specifications were modified based on updated genetic testing clinical criteria, new recommendations from ClinGen and expert knowledge from ongoing CDH1 variant curations. The CDH1 VCEP reviewed 273 variants using updated CDH1 specifications and incorporated published and unpublished data provided by diagnostic laboratories. RESULTS: Updated CDH1-specific interpretation guidelines include 11 major modifications since the initial specifications from 2018. Using the refined guidelines, 97% (36 of 37) of variants with ClinVar conflicting interpretations were resolved to benign, likely benign, likely pathogenic or pathogenic, and 35% (15 of 43) of VUS were resolved to benign or likely benign. Overall, 88% (239 of 273) of curated variants had non-VUS classifications. To date, variants classified as pathogenic are either nonsense, frameshift, splicing, or affecting the translation initiation codon, and the only missense variants classified as pathogenic or likely pathogenic have been shown to affect splicing. CONCLUSIONS: The development and evolution of CDH1-specific criteria by the expert panel resulted in decreased uncertain and conflicting interpretations of variants in this clinically actionable gene, which can ultimately lead to more effective clinical management recommendations.

Clinical impact of whole-genome sequencing in patients with early-onset dementia.

BACKGROUND: In the clinical setting, identification of the genetic cause in patients with early-onset dementia (EOD) is challenging due to multiple types of genetic tests required to arrive at a diagnosis. Whole-genome sequencing (WGS) has the potential to serve as a single diagnostic platform, due to its superior ability to detect common, rare and structural genetic variation. METHODS: WGS analysis was performed in 50 patients with EOD. Point mutations, small insertions/deletions, as well as structural variants (SVs) and short tandem repeats (STRs), were analysed. An Alzheimer's disease (AD)-related polygenic risk score (PRS) was calculated in patients with AD. RESULTS: Clinical genetic diagnosis was achieved in 7 of 50 (14%) of the patients, with a further 8 patients (16%) found to have established risk factors which may have contributed to their EOD. Two pathogenic variants were identified through SV analysis. No expanded STRs were found in this study cohort, but a blinded analysis with a positive control identified a C9orf72 expansion accurately. Approximately 37% (7 of 19) of patients with AD had a PRS equivalent to >90th percentile risk. DISCUSSION: WGS acts as a single genetic test to identify different types of clinically relevant genetic variations in patients with EOD. WGS, if used as a first-line clinical diagnostic test, has the potential to increase the diagnostic yield and reduce time to diagnosis for EOD.

Comprehensive evaluation and efficient classification of BRCA1 RING domain missense substitutions.

BRCA1 is a high-risk susceptibility gene for breast and ovarian cancer. Pathogenic protein-truncating variants are scattered across the open reading frame, but all known missense substitutions that are pathogenic because of missense dysfunction are located in either the amino-terminal RING domain or the carboxy-terminal BRCT domain. Heterodimerization of the BRCA1 and BARD1 RING domains is a molecularly defined obligate activity. Hence, we tested every BRCA1 RING domain missense substitution that can be created by a single nucleotide change for heterodimerization with BARD1 in a mammalian two-hybrid assay. Downstream of the laboratory assay, we addressed three additional challenges: assay calibration, validation thereof, and integration of the calibrated results with other available data, such as computational evidence and patient/population observational data to achieve clinically applicable classification. Overall, we found that 15%-20% of BRCA1 RING domain missense substitutions are pathogenic. Using a Bayesian point system for data integration and variant classification, we achieved clinical classification of 89% of observed missense substitutions. Moreover, among missense substitutions not present in the human observational data used here, we find an additional 45 with concordant computational and functional assay evidence in favor of pathogenicity plus 223 with concordant evidence in favor of benignity; these are particularly likely to be classified as likely pathogenic and likely benign, respectively, once human observational data become available.

Rivaroxaban in the treatment of TEK-related venous malformation.

Low-flow vascular malformations are rare congenital anomalies due to errors in vascular development and may be associated with known pathogenic genetic variants. Slow flow through the blood vessels can lead to localized intralesional thromboses, which can cause debilitating pain and impair quality of life. We present a case of venous malformation due to a variant in the TEK gene in a 38-year-old woman in whom treatment with low dose rivaroxaban was successful in controlling symptoms of chronic localized intravascular coagulation.

Predictive functional assay-based classification of PMS2 variants in Lynch syndrome.

The large majority of germline alterations identified in the DNA mismatch repair (MMR) gene PMS2, a low-penetrance gene for the cancer predisposition Lynch syndrome, represent variants of uncertain significance (VUS). The inability to classify most VUS interferes with personalized healthcare. The complete in vitro MMR activity (CIMRA) assay, that only requires sequence information on the VUS, provides a functional analysis-based quantitative tool to improve the classification of VUS in MMR proteins. To derive a formula that translates CIMRA assay results into the odds of pathogenicity (OddsPath) for VUS in PMS2 we used a set of clinically classified PMS2 variants supplemented by inactivating variants that were generated by an in cellulo genetic screen, as proxies for cancer-predisposing variants. Validation of this OddsPath revealed high predictive values for benign and predisposing PMS2 VUS. We conclude that the OddsPath provides an integral metric that, following the other, higher penetrance, MMR proteins MSH2, MSH6 and MLH1 can be incorporated as strong evidence type into the upcoming criteria for MMR gene VUS classification of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP).

Real world outcomes and implementation pathways of exome sequencing in an adult genetic department.

PURPOSE: This study aimed to correlate the indications and diagnostic yield of exome sequencing (ES) in adult patients across various clinical settings. The secondary aim was to examine the clinical utility of ES in adult patients. METHODS: Data on demographics, clinical indications, results, management changes, and cascade testing were collected for 250 consecutive patients who underwent ES through an adult genetics department between 2016 and 2021. Data were analyzed using descriptive and inferential statistics. Testing in which traditional gene panels were in standard use, such as in heritable cancers, was excluded. RESULTS: The average age at testing was 43 years (range = 17-80 years). A molecular diagnosis was identified in 29% of patients. Older age at symptom onset did not pre-exclude a substantial diagnostic yield. Patients with syndromic intellectual disability and multiple system disorders had the highest yield. In >50% of patients with an exome diagnosis, the results changed management. Cascade testing occured in at least one family member for 30% of patients with a diagnosis. Diagnostic results had reproductive implications for 26% of patients and 31% of patients' relatives. CONCLUSION: ES has a robust diagnostic yield and clear clinical utility in adult patients across a range of ages and phenotypes.

TRACEBACK: Testing of Historical Tubo-Ovarian Cancer Patients for Hereditary Risk Genes as a Cancer Prevention Strategy in Family Members.

PURPOSE: Tubo-ovarian cancer (TOC) is a sentinel cancer for BRCA1 and BRCA2 pathogenic variants (PVs). Identification of a PV in the first member of a family at increased genetic risk (the proband) provides opportunities for cancer prevention in other at-risk family members. Although Australian testing rates are now high, PVs in patients with TOC whose diagnosis predated revised testing guidelines might have been missed. We assessed the feasibility of detecting PVs in this population to enable genetic risk reduction in relatives. PATIENTS AND METHODS: In this pilot study, deceased probands were ascertained from research cohort studies, identification by a relative, and gynecologic oncology clinics. DNA was extracted from archival tissue or stored blood for panel sequencing of 10 risk-associated genes. Testing of deceased probands ascertained through clinic records was performed with a consent waiver. RESULTS: We identified 85 PVs in 84 of 787 (11%) probands. Familial contacts of 39 of 60 (65%) deceased probands with an identified recipient (60 of 84; 71%) have received a written notification of results, with follow-up verbal contact made in 85% (33 of 39). A minority of families (n = 4) were already aware of the PV. For many (29 of 33; 88%), the genetic result provided new information and referral to a genetic service was accepted in most cases (66%; 19 of 29). Those who declined referral (4 of 29) were all male next of kin whose family member had died more than 10 years before. CONCLUSION: We overcame ethical and logistic challenges to demonstrate that retrospective genetic testing to identify PVs in previously untested deceased probands with TOC is feasible. Understanding reasons for a family member's decision to accept or decline a referral will be important for guiding future TRACEBACK projects.

Arrhythmic Phenotypes Are a Defining Feature of Dilated Cardiomyopathy-Associated SCN5A Variants: A Systematic Review.

BACKGROUND: Variants in the SCN5A gene, that encodes the cardiac sodium channel, Nav1.5, are associated with a highly arrhythmogenic form of dilated cardiomyopathy (DCM). Our aim was to review the phenotypes, natural history, functional effects, and treatment outcomes of DCM-associated rare SCN5A variants. METHODS: A systematic review of reported DCM-associated rare SCN5A variants was undertaken using PubMed and Embase. RESULTS: Eighteen SCN5A rare variants in 29 families with DCM (173 affected individuals) were identified. Eleven variants had undergone experimental evaluation, with 7 of these resulting in increased sustained current flow during the action potential (eg, increased window current) and at resting membrane potentials (eg, creation of a new gating pore current). These variants were located in transmembrane voltage-sensing domains and had a consistent phenotype characterized by frequent multifocal narrow and broad complex ventricular premature beats (VPB; 72% of affected relatives), ventricular arrhythmias (33%), atrial arrhythmias (32%), sudden cardiac death (13%), and DCM (56%). This VPB-predominant phenotype was not seen with 1 variant that increased late sodium current, or with variants that reduced peak current density or had mixed effects. In the latter groups, affected individuals mainly showed sinus node dysfunction, conduction defects, and atrial arrhythmias, with infrequent VPB and ventricular arrhythmias. DCM did not occur in the absence of arrhythmias for any variant. Twelve studies (23 total patients) reported treatment success in the VPB-predominant cardiomyopathy using sodium channel-blocking drug therapy. CONCLUSIONS: SCN5A variants can present with a diverse spectrum of primary arrhythmic features. A majority of DCM-associated variants cause a multifocal VPB-predominant cardiomyopathy that is reversible with sodium channel blocking drug therapy. Early recognition of the distinctive phenotype and prompt genetic testing to identify variant carriers are needed. Our findings have implications for interpretation and management of SCN5A variants found in DCM patients with and without arrhythmias.

Scaling national and international improvement in virtual gene panel curation via a collaborative approach to discordance resolution.

Clinical validity assessments of gene-disease associations underpin analysis and reporting in diagnostic genomics, and yet wide variability exists in practice, particularly in use of these assessments for virtual gene panel design and maintenance. Harmonization efforts are hampered by the lack of agreed terminology, agreed gene curation standards, and platforms that can be used to identify and resolve discrepancies at scale. We undertook a systematic comparison of the content of 80 virtual gene panels used in two healthcare systems by multiple diagnostic providers in the United Kingdom and Australia. The process was enabled by a shared curation platform, PanelApp, and resulted in the identification and review of 2,144 discordant gene ratings, demonstrating the utility of sharing structured gene-disease validity assessments and collaborative discordance resolution in establishing national and international consensus.

Genomic Risk Prediction for Breast Cancer in Older Women.

Genomic risk prediction models for breast cancer (BC) have been predominantly developed with data from women aged 40-69 years. Prospective studies of older women aged ≥70 years have been limited. We assessed the effect of a 313-variant polygenic risk score (PRS) for BC in 6339 older women aged ≥70 years (mean age 75 years) enrolled into the ASPREE trial, a randomized double-blind placebo-controlled clinical trial investigating the effect of daily 100 mg aspirin on disability-free survival. We evaluated incident BC diagnoses over a median follow-up time of 4.7 years. A multivariable Cox regression model including conventional BC risk factors was applied to prospective data, and re-evaluated after adding the PRS. We also assessed the association of rare pathogenic variants (PVs) in BC susceptibility genes (BRCA1/BRCA2/PALB2/CHEK2/ATM). The PRS, as a continuous variable, was an independent predictor of incident BC (hazard ratio (HR) per standard deviation (SD) = 1.4, 95% confidence interval (CI) 1.3-1.6) and hormone receptor (ER/PR)-positive disease (HR = 1.5 (CI 1.2-1.9)). Women in the top quintile of the PRS distribution had over two-fold higher risk of BC than women in the lowest quintile (HR = 2.2 (CI 1.2-3.9)). The concordance index of the model without the PRS was 0.62 (95% CI 0.56-0.68), which improved after addition of the PRS to 0.65 (95% CI 0.59-0.71). Among 41 (0.6%) carriers of PVs in BC susceptibility genes, we observed no incident BC diagnoses. Our study demonstrates that a PRS predicts incident BC risk in women aged 70 years and older, suggesting potential clinical utility extends to this older age group.

Genetic Dominant Variants in STUB1, Segregating in Families with SCA48, Display In Vitro Functional Impairments Indistinctive from Recessive Variants Associated with SCAR16.

Variants in STUB1 cause both autosomal recessive (SCAR16) and dominant (SCA48) spinocerebellar ataxia. Reports from 18 STUB1 variants causing SCA48 show that the clinical picture includes later-onset ataxia with a cerebellar cognitive affective syndrome and varying clinical overlap with SCAR16. However, little is known about the molecular properties of dominant STUB1 variants. Here, we describe three SCA48 families with novel, dominantly inherited STUB1 variants (p.Arg51_Ile53delinsProAla, p.Lys143_Trp147del, and p.Gly249Val). All the patients developed symptoms from 30 years of age or later, all had cerebellar atrophy, and 4 had cognitive/psychiatric phenotypes. Investigation of the structural and functional consequences of the recombinant C-terminus of HSC70-interacting protein (CHIP) variants was performed in vitro using ubiquitin ligase activity assay, circular dichroism assay and native polyacrylamide gel electrophoresis. These studies revealed that dominantly and recessively inherited STUB1 variants showed similar biochemical defects, including impaired ubiquitin ligase activity and altered oligomerization properties of the CHIP. Our findings expand the molecular understanding of SCA48 but also mean that assumptions concerning unaffected carriers of recessive STUB1 variants in SCAR16 families must be re-evaluated. More investigations are needed to verify the disease status of SCAR16 heterozygotes and elucidate the molecular relationship between SCA48 and SCAR16 diseases.

Genetic variants associated with inherited cardiovascular disorders among 13,131 asymptomatic older adults of European descent.

Genetic testing is used to optimise the management of inherited cardiovascular disorders that can cause sudden cardiac death. Yet more genotype-phenotype correlation studies from populations not ascertained on clinical symptoms or family history of disease are required to improve understanding of gene penetrance. We performed targeted sequencing of 25 genes used routinely in clinical genetic testing for inherited cardiovascular disorders in a population of 13,131 asymptomatic older individuals (mean age 75 years) enrolled in the ASPREE trial. Participants had no prior history of cardiovascular disease events, dementia or physical disability at enrolment. Variants were classified following ACMG/AMP standards. Sudden and rapid cardiac deaths were clinically adjudicated as ASPREE trial endpoints, and assessed during mean 4.7 years of follow-up. In total, 119 participants had pathogenic/deleterious variants in one of the 25 genes analysed (carrier rate of 1 in 110 or 0.9%). Participants carried variants associated with hypertrophic cardiomyopathy (N = 24), dilated cardiomyopathy (N = 29), arrhythmogenic right-ventricular cardiomyopathy (N = 22), catecholaminergic polymorphic ventricular tachycardia (N = 4), aortopathies (N = 1), and long-QT syndrome (N = 39). Among 119 carriers, two died from presumed sudden/rapid cardiac deaths during follow-up (1.7%); both with pathogenic variants in long-QT syndrome genes (KCNQ1, SCN5A). Among non-carriers, the rate of sudden/rapid cardiac deaths was significantly lower (0.08%, 11/12936, p < 0.001). Variants associated with inherited cardiovascular disorders are found in asymptomatic individuals aged 70 years and older without a history of cardiovascular disease.

A homozygous truncating variant in GDF9 in siblings with primary ovarian insufficiency.

Premature or primary ovarian insufficiency (POI) affects approximately 1% of women and can be due to a variety of causes. Genetic causes include syndromic and non-syndromic POI. There are several promising candidate genes for whom a clear Mendelian association with non-syndromic POI has not yet been conclusively established, including GDF9. GDF9 is an oocyte-secreted factor and is part of the TGF-beta superfamily of morphogens. It has an important role in follicular development and granulosa cell maturation. We report the case of two siblings with primary ovarian insufficiency (POI) and a homozygous truncating variant in GDF9 (c.604C>T; p.(Gln202*). This report helps establish a clear gene-disease association between GDF9 and POI and argues for routine evaluation for GDF9 variants in patients undergoing genomic investigation for POI.

FANCM c5791C>T stopgain mutation (rs144567652) is a familial colorectal cancer risk factor.

PURPOSE: While familial aggregation of colorectal cancer (CRC) is recognized, the majority of the germline predisposition factors remain unidentified, and many high-risk CRC pedigrees remain unexplained by known risk variants. Fanconi Anemia genes have been recognized to be associated with cancer risk. Notably, FANCM (OMIM 609644) variants have been reported to confer risk for CRC and breast cancer. METHODS: Exome sequencing of CRC-affected cousins in a set of 47 independent extended high-risk CRC pedigrees identified a candidate set of rare, shared variants. Variants were tested for association with risk in 744 Utah CRC cases and 1525 controls, and for segregation with CRC in affected relatives. RESULTS: A FANCM stopgain variant was observed in two CRC-affected cousin pairs, each from an independent Utah high-risk pedigree, and yielded a nonsignificant, but elevated OR = 2.05 in a set of Utah cases and controls. Segregation of the variant to other related CRC-affected cases was observed in the two extended pedigrees. CONCLUSION: A rare stopgain variant in FANCM (rs144567652) that is recognized as a breast cancer predisposition variant, and that has previously been proposed, but not confirmed, as a CRC predisposition variant, is validated here as a risk factor for familial CRC.

Contribution of mRNA Splicing to Mismatch Repair Gene Sequence Variant Interpretation.

Functional assays that assess mRNA splicing can be used in interpretation of the clinical significance of sequence variants, including the Lynch syndrome-associated mismatch repair (MMR) genes. The purpose of this study was to investigate the contribution of splicing assay data to the classification of MMR gene sequence variants. We assayed mRNA splicing for 24 sequence variants in MLH1, MSH2, and MSH6, including 12 missense variants that were also assessed using a cell-free in vitro MMR activity (CIMRA) assay. Multifactorial likelihood analysis was conducted for each variant, combining CIMRA outputs and clinical data where available. We collated these results with existing public data to provide a dataset of splicing assay results for a total of 671 MMR gene sequence variants (328 missense/in-frame indel), and published and unpublished repair activity measurements for 154 of these variants. There were 241 variants for which a splicing aberration was detected: 92 complete impact, 33 incomplete impact, and 116 where it was not possible to determine complete versus incomplete splicing impact. Splicing results mostly aided in the interpretation of intronic (72%) and silent (92%) variants and were the least useful for missense substitutions/in-frame indels (10%). MMR protein functional activity assays were more useful in the analysis of these exonic variants but by design they were not able to detect clinically important splicing aberrations identified by parallel mRNA assays. The development of high throughput assays that can quantitatively assess impact on mRNA transcript expression and protein function in parallel will streamline classification of MMR gene sequence variants.

Medically actionable pathogenic variants in a population of 13,131 healthy elderly individuals.

PURPOSE: To measure the prevalence of medically actionable pathogenic variants (PVs) among a population of healthy elderly individuals. METHODS: We used targeted sequencing to detect pathogenic or likely pathogenic variants in 55 genes associated with autosomal dominant medically actionable conditions, among a population of 13,131 individuals aged 70 or older (mean age 75 years) enrolled in the ASPirin in Reducing Events in the Elderly (ASPREE) trial. Participants had no previous diagnosis or current symptoms of cardiovascular disease, physical disability or dementia, and no current diagnosis of life-threatening cancer. Variant curation followed American College of Medical Genetics and Genomics/Association for Molecular Pathology (ACMG/AMP) standards. RESULTS: One in 75 (1.3%) healthy elderly individuals carried a PV. This was lower than rates reported from population-based studies, which have ranged from 1.8% to 3.4%. We detected 20 PV carriers for Lynch syndrome (MSH6/MLH1/MSH2/PMS2) and 13 for familial hypercholesterolemia (LDLR/APOB/PCSK9). Among 7056 female participants, we detected 15 BRCA1/BRCA2 PV carriers (1 in 470 females). We detected 86 carriers of PVs in lower-penetrance genes associated with inherited cardiac disorders. CONCLUSION: Medically actionable PVs are carried in a healthy elderly population. Our findings raise questions about the actionability of lower-penetrance genes, especially when PVs are detected in the absence of symptoms and/or family history of disease.

A novel AFG3L2 mutation close to AAA domain leads to aberrant OMA1 and OPA1 processing in a family with optic atrophy.

Autosomal dominant optic atrophy (ADOA) is a neuro-ophthalmic condition characterized by bilateral degeneration of the optic nerves. Although heterozygous mutations in OPA1 represent the most common genetic cause of ADOA, a significant number of cases remain undiagnosed.Here, we describe a family with a strong ADOA history with most family members spanning three generation having childhood onset of visual symptoms. The proband, in addition to optic atrophy, had neurological symptoms consistent with relapsing remitting multiple sclerosis. Clinical exome analysis detected a novel mutation in the AFG3L2 gene (NM_006796.2:c.1010G > A; p.G337E), which segregated with optic atrophy in family members. AFG3L2 is a metalloprotease of the AAA subfamily which exerts quality control in the inner mitochondrial membrane. Interestingly, the identified mutation localizes close to the AAA domain of AFG3L2, while those localized in the proteolytic domain cause dominant spinocerebellar ataxia type 28 (SCA28) or recessive spastic ataxia with epilepsy (SPAX5). Functional studies in patient fibroblasts demonstrate that the p.G337E AFG3L2 mutation strongly destabilizes the long isoforms of OPA1 via OMA hyper-activation and leads to mitochondrial fragmentation, thus explaining the family phenotype. This study widens the clinical spectrum of neurodegenerative diseases caused by AFG3L2 mutations, which shall be considered as genetic cause of ADOA.

Comprehensive Constitutional Genetic and Epigenetic Characterization of Lynch-Like Individuals.

The causal mechanism for cancer predisposition in Lynch-like syndrome (LLS) remains unknown. Our aim was to elucidate the constitutional basis of mismatch repair (MMR) deficiency in LLS patients throughout a comprehensive (epi)genetic analysis. One hundred and fifteen LLS patients harboring MMR-deficient tumors and no germline MMR mutations were included. Mutational analysis of 26 colorectal cancer (CRC)-associated genes was performed. Pathogenicity of MMR variants was assessed by splicing and multifactorial likelihood analyses. Genome-wide methylome analysis was performed by the Infinium Human Methylation 450K Bead Chip. The multigene panel analysis revealed the presence of two MMR gene truncating mutations not previously found. Of a total of 15 additional MMR variants identified, five -present in 6 unrelated individuals- were reclassified as pathogenic. In addition, 13 predicted deleterious variants in other CRC-predisposing genes were found in 12 probands. Methylome analysis detected one constitutional MLH1 epimutation, but no additional differentially methylated regions were identified in LLS compared to LS patients or cancer-free individuals. In conclusion, the use of an ad-hoc designed gene panel combined with pathogenicity assessment of variants allowed the identification of deleterious MMR mutations as well as new LLS candidate causal genes. Constitutional epimutations in non-LS-associated genes are not responsible for LLS.