Clinical implications of VUS reclassification in a single-centre series from application of ACMG/AMP classification rules specified for BRCA1/2.

BACKGROUND: BRCA1/2 testing is crucial to guide clinical decisions in patients with hereditary breast/ovarian cancer, but detection of variants of uncertain significance (VUSs) prevents proper management of carriers. The ENIGMA (Evidence-based Network for the Interpretation of Germline Mutant Alleles) BRCA1/2 Variant Curation Expert Panel (VCEP) has recently developed BRCA1/2 variant classification guidelines consistent with ClinGen processes, specified against the ACMG/AMP (American College of Medical Genetics and Genomics/Association for Molecular-Pathology) classification framework. METHODS: The ClinGen-approved BRCA1/2-specified ACMG/AMP classification guidelines were applied to BRCA1/2 VUSs identified from 2011 to 2022 in a series of patients, retrieving information from the VCEP documentation, public databases, literature and ENIGMA unpublished data. Then, we critically re-evaluated carrier families based on new results and checked consistency of updated classification with main sources for clinical interpretation of BRCA1/2 variants. RESULTS: Among 166 VUSs detected in 231 index cases, 135 (81.3%) found in 197 index cases were classified by applying BRCA1/2-specified ACMG/AMP criteria: 128 (94.8%) as Benign/Likely Benign and 7 (5.2%) as Pathogenic/Likely Pathogenic. The average time from the first report as 'VUS' to classification using this approach was 49.4 months. Considering that 15 of these variants found in 64 families had already been internally reclassified prior to this work, this study provided 121 new reclassifications among the 151 (80.1%) remaining VUSs, relevant to 133/167 (79.6%) families. CONCLUSIONS: These results demonstrated the effectiveness of new BRCA1/2 ACMG/AMP classification guidelines for VUS classification within a clinical cohort, and their important clinical impact. Furthermore, they suggested a cadence of no more than 3 years for regular review of VUSs, which however requires time, expertise and resources.

Interplay between WNT/PI3K-mTOR axis and the microbiota in APC-driven colorectal carcinogenesis: data from a pilot study and possible implications for CRC prevention.

BACKGROUND: Wnt/ß-catenin signalling impairment accounts for 85% of colorectal cancers (CRCs), including sporadic and familial adenomatous polyposis (FAP) settings. An altered PI3K/mTOR pathway and gut microbiota also contribute to CRC carcinogenesis. We studied the interplay between the two pathways and the microbiota composition within each step of CRC carcinogenesis. METHODS: Proteins and target genes of both pathways were analysed by RT-qPCR and IHC in tissues from healthy faecal immunochemical test positive (FIT+, n = 17), FAP (n = 17) and CRC (n = 15) subjects. CRC-related mutations were analysed through NGS and Sanger. Oral, faecal and mucosal microbiota was profiled by 16 S rRNA-sequencing. RESULTS: We found simultaneous hyperactivation of Wnt/ß-catenin and PI3K/mTOR pathways in FAP-lesions compared to CRCs. Wnt/ß-catenin molecular markers positively correlated with Clostridium_sensu_stricto_1 and negatively with Bacteroides in FAP faecal microbiota. Alistipes, Lachnospiraceae, and Ruminococcaceae were enriched in FAP stools and adenomas, the latter also showing an overabundance of Lachnoclostridium, which positively correlated with cMYC. In impaired-mTOR-mutated CRC tissues, p-S6R correlated with Fusobacterium and Dialister, the latter also confirmed in the faecal-ecosystem. CONCLUSIONS: Our study reveals an interplay between Wnt/ß-catenin and PI3K/mTOR, whose derangement correlates with specific microbiota signatures in FAP and CRC patients, and identifies new potential biomarkers and targets to improve CRC prevention, early adenoma detection and treatment.

Update of penetrance estimates in Birt-Hogg-Dubé syndrome.

BACKGROUND: Birt-Hogg-Dubé (BHD) syndrome is a rare genetic syndrome caused by pathogenic or likely pathogenic germline variants in the FLCN gene. Patients with BHD syndrome have an increased risk of fibrofolliculomas, pulmonary cysts, pneumothorax and renal cell carcinoma. There is debate regarding whether colonic polyps should be added to the criteria. Previous risk estimates have mostly been based on small clinical case series. METHODS: A comprehensive review was conducted to identify studies that had recruited families carrying pathogenic or likely pathogenic variants in FLCN. Pedigree data were requested from these studies and pooled. Segregation analysis was used to estimate the cumulative risk of each manifestation for carriers of FLCN pathogenic variants. RESULTS: Our final dataset contained 204 families that were informative for at least one manifestation of BHD (67 families informative for skin manifestations, 63 for lung, 88 for renal carcinoma and 29 for polyps). By age 70 years, male carriers of the FLCN variant have an estimated 19% (95% CI 12% to 31%) risk of renal tumours, 87% (95% CI 80% to 92%) of lung involvement and 87% (95% CI 78% to 93%) of skin lesions, while female carriers had an estimated 21% (95% CI 13% to 32%) risk of renal tumours, 82% (95% CI 73% to 88%) of lung involvement and 78% (95% CI 67% to 85%) of skin lesions. The cumulative risk of colonic polyps by age 70 years old was 21% (95% CI 8% to 45%) for male carriers and 32% (95% CI 16% to 53%) for female carriers. CONCLUSIONS: These updated penetrance estimates, based on a large number of families, are important for the genetic counselling and clinical management of BHD syndrome.

Multi-Gene Next-Generation Sequencing Panel for Analysis of BRCA1/BRCA2 and Homologous Recombination Repair Genes Alterations Metastatic Castration-Resistant Prostate Cancer.

Despite significant therapeutic advances, metastatic CRPC (mCRPC) remains a lethal disease. Mutations in homologous recombination repair (HRR) genes are frequent in mCRPC, and tumors harboring these mutations are known to be sensitive to PARP inhibitors. The aim of this study was to verify the technical effectiveness of this panel in the analysis of mCRPC, the frequency and type of mutations in the BRCA1/BRCA2 genes, as well as in the homologous recombination repair (HRR) genes. A total of 50 mCRPC cases were analyzed using a multi-gene next-generation sequencing panel evaluating a total of 1360 amplicons in 24 HRR genes. Of the 50 cases, 23 specimens (46.0%) had an mCRPC harboring a pathogenic variant or a variant of uncertain significance (VUS), whereas in 27 mCRPCs (54.0%), no mutations were detected (wild-type tumors). BRCA2 was the most commonly mutated gene (14.0% of samples), followed by ATM (12.0%), and BRCA1 (6.0%). In conclusion, we have set up an NGS multi-gene panel that is capable of analyzing BRCA1/BRCA2 and HRR alterations in mCRPC. Moreover, our clinical algorithm is currently being used in clinical practice for the management of patients with mCRPC.

Chromoendoscopy Is Not Superior to White Light Endoscopy in Improving Adenoma Detection in Lynch Syndrome Cohort Undergoing Surveillance with High-Resolution Colonoscopy: A Real-World Evidence Study.

BACKGROUND: Endoscopic surveillance in patients with Lynch syndrome (LS) is crucial due to a genetically based high risk of colorectal cancer (CRC). We aimed to compare the adenoma detection rate (ADR) between high-resolution white light endoscopy (WLE) alone and WLE plus dye chromoendoscopy (CE) in a cohort of LS patients. METHODS: In a context of real-world data, we retrospectively enrolled 50 LS patients who had non-randomly undergone WLE versus CE surveillance examinations from 2007 to 2019. The 2 groups were compared at baseline (BL) in terms of the rate of patients with lesions and the number of lesions, and at follow-up (FU), to evaluate a possible enhanced detection rate. Longitudinal analysis of the effect of the endoscopy type on the main outcomes was performed by generalized linear mixed models. RESULTS: Forty-two patients had undergone at least one diagnostic colonoscopy. At BL and at FU analysis, we found no significant differences in detection rates and clinical-pathological features between WLE and CE groups. At the longitudinal analysis, an increase in the endoscopy rank (i.e., the position of each colonoscopy for all the colonoscopies that a patient had undergone) was associated with an increase in polyp detection rate (p = 0.006) and ADR (p = 0.005), while a trend toward significance (p = 0.069) was found for endoscopy type (CE vs. WLE) in the detection of serrated lesions. CONCLUSIONS: CE is not superior to high-resolution WLE in increasing the ADR. Even under standard WLE, an active and careful endoscopic surveillance of LS patients can prevent CRC.

Sudden shift to remote genetic counseling during the COVID-19 pandemic: Experiences of genetics professionals in Italy.

The 2020 COVID-19 pandemic has rendered in-person provision of genetic counseling impossible for prolonged periods in many countries, mandating a sudden shift to remote delivery. We used qualitative thematic analysis to explore Italian genetics professionals' experience with remote genetic counseling. Fourteen group and four individual interviews were conducted after participants had delivered one or more remote sessions via videoconference or on the telephone. Data were coded and grouped under themes. Three main themes were identified as follows: (a) technical and logistical issues, (b) communication issues, and (c) clinical content and outcome of the session. The participants acknowledged that not having to travel to the clinic saves consultands time and expense. They also highlighted that not sharing a physical space with consultands and having to rely on technology can negatively impact on effective communication, building trusting relationships, and performing accurate psychosocial assessments. Regarding the clinical content of sessions, remote genetic counseling was perceived to favor greater focus and succinct, to the point communication. However, participants also felt uncomfortable not being able to use visual aids to support the explanation of complex concepts. Moreover, demographics and the socio-cultural status of consultands emerged as factors influencing the outcome of remote genetic counseling sessions. Finally, participants reported feeling that more experience with this novel approach would improve their confidence and their ability to adapt their counseling skills as appropriate. Based on these findings, we suggest that effective, equitable provision of remote genetic counseling will require an infrastructure that is able to support video counseling, sharing of clinical documents and visual aids, and connect with a wide range of devices. Moreover, the structure of sessions should be tailored to the specific requirements of remote genetic counseling and suitable training efforts should be promoted to enhance professionals' communication skills.

Preferences of Italian patients for return of secondary findings from clinical genome/exome sequencing.

Exome/genome sequencing (ES/GS) is increasingly becoming routine in clinical genetic diagnosis, yet issues regarding how to disclose and manage secondary findings (SFs) remain to be addressed, and limited evidence is available on patients' preferences. We carried out semi-structured interviews with 307 individuals undergoing clinical genetic testing to explore their preferences for return of SFs in the hypothetical scenario that their test would be performed using ES/GS. Participants were 254 females (82.7%) and 53 males (17.3%), aged 18-86 years; 73.9% (81.1% of those with lower education levels) reported no prior knowledge of ES/GS. Prior knowledge of ES/GS was more common among patients tested for Mendelian conditions (34.5%), compared to those undergoing cancer genetic testing (22.3%) or carrier screening (7.4%). Despite this reported lack of knowledge, most participants (213, 69.6%) stated they would prefer to be informed of all possible results. Reasons in favor of disclosure included wanting to be aware of any risks (168; 83.6%) and to help relatives (23; 11.4%), but also hope that preventive measures might become available in the future (10, 5%). Conversely, potential negative impact on quality of life was the commonest motivation against disclosure. Among 179 participants seen for cancer genetic counseling who were interviewed again after test disclosure, 81.9% had not heard about ES/GS in the meantime; however, the proportion of participants opting for disclosure of any variants was lower (116; 64.8%), with 36 (20.1%) changing opinion compared to the first interview. Based on these findings, we conclude that genetic counseling for ES/GS should involve enhanced education and decision-making support to enable informed consent to SFs disclosure.

Detecting Variants in the NBN Gene While Testing for Hereditary Breast Cancer: What to Do Next?

The NBN gene has been included in breast cancer (BC) multigene panels based on early studies suggesting an increased BC risk for carriers, though not confirmed by recent research. To evaluate the impact of NBN analysis, we assessed the results of NBN sequencing in 116 BRCA-negative BC patients and reviewed the literature. Three patients (2.6%) carried potentially relevant variants: two, apparently unrelated, carried the frameshift variant c.156_157delTT and another one the c.628G>T variant. The latter was subsequently found in 4/1390 (0.3%) BC cases and 8/1580 (0.5%) controls in an independent sample, which, together with in silico predictions, provided evidence against its pathogenicity. Conversely, the rare c.156_157delTT variant was absent in the case-control set; moreover, a 50% reduction of NBN expression was demonstrated in one carrier. However, in one family it failed to co-segregate with BC, while the other carrier was found to harbor also a probably pathogenic TP53 variant that may explain her phenotype. Therefore, the c.156_157delTT, although functionally deleterious, was not supported as a cancer-predisposing defect. Pathogenic/likely pathogenic NBN variants were detected by multigene panels in 31/12314 (0.25%) patients included in 15 studies. The risk of misinterpretation of such findings is substantial and supports the exclusion of NBN from multigene panels.

Primary malignant pericardial tumour in Lynch syndrome.

BACKGROUND: This case represents the first report of malignant primary cardiac tumour in a patient with Lynch Syndrome associated with MSH2 pathogenic variant. CASE PRESENTATION: A 57-year-old woman with previous ovarian cystadenocarcinoma was admitted to the emergency room for hematic pericardial effusion. Multimodal diagnostic imaging revealed two solid pericardial vascularized masses. After pericardiectomy, the final histological diagnosis was poorly differentiated pleomorphic sarcomatoid carcinoma. During follow-up she developed an ampulla of Vater adenocarcinoma. Genetic analysis identified an MSH2 pathogenic variant. CONCLUSION: This case contributes to expand the tumour spectrum of Lynch syndrome, suggesting that MSH2 pathogenic variants cause a more complex multi-tumour cancer syndrome than the classic Lynch Syndrome. In MSH2 variant carriers, symptoms such as dyspnoea and chest discomfort might alert for rare tumours and a focused cardiac evaluation should be considered.

An Analysis of Clinical, Surgical, Pathological and Molecular Characteristics of Endometrial Cancer According to Mismatch Repair Status. A Multidisciplinary Approach.

Since 2016, our hospital has applied tumor testing with immunohistochemistry (IHC) in endometrial cancer in order to detect mutations of mismatch repair genes (MMR). All cases with MMR deficiency proteins expression are sent for genetic testing, except those with MLH1 protein deficiency, in which case genetic testing is performed if negative for promoter hypermethylation. The primary aim of this study was to investigate the ability of our algorithm to identify Lynch syndrome (LS). The Secondary aims were to investigate the relationship between MMR status and clinicopathological features and prognosis of primary endometrial cancer (EC). From January 2016 to December 2018, 239 patients with EC were retrospectively analyzed and subdivided according to MMR status. Patients were divided in three groups: MMR proficient, LS and Lynch-like cancer (LLC). LS was characterized by a lower age and BMI, more use of contraceptive and less use of hormonal replacement therapy, nulliparity and a trend versus a better prognosis. LLC appeared more related to MMR proficient than LS and exhibited a more aggressive behavior. Our multidisciplinary approach permitted a correct diagnosis of germline mutation in patients with newly diagnosis EC and it confirmed clinicopathologic and prognostic characteristics of LS.

The Gen-Equip Project: evaluation and impact of genetics e-learning resources for primary care in six European languages.

PURPOSE: Genetic advances mean patients at risk of genetic conditions can be helped through testing, clinical screening, and preventive treatment, but they must first be identified to benefit. Ensuring quality of genetic care for patients requires genetic expertise in all health services, including primary care. To address an educational shortfall, a series of e-learning resources was developed in six languages to equip primary care professionals with genetic skills relevant for practice. The purpose of the study was to evaluate these resources using Kirkpatrick's framework for educational outcomes. METHODS: Mixed methods (qualitative and quantitative) were used over four phases of the study. RESULTS: A high level of satisfaction with the resources was reported. Knowledge and skills improved significantly after using the education material. Participants reported changes in confidence and practice behavior, including family history taking, seeking advice from specialists and referring patients. The resources helped users to learn how to explain genetics. Many visited the resources repeatedly and some used them to educate colleagues or students. CONCLUSION: Gen-Equip modules are effective in improving genetic knowledge, skills, and attitudes for primary care professionals. They provide both continuing professional development and just-in-time learning for a potentially large global audience at a practical level.

Correction: The Gen-Equip Project: evaluation and impact of genetics e-learning resources for primary care in six European languages.

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Primary constitutional MLH1 epimutations: a focal epigenetic event.

BACKGROUND: Constitutional MLH1 epimutations are characterised by monoallelic methylation of the MLH1 promoter throughout normal tissues, accompanied by allele-specific silencing. The mechanism underlying primary MLH1 epimutations is currently unknown. The aim of this study was to perform an in-depth characterisation of constitutional MLH1 epimutations targeting the aberrantly methylated region around MLH1 and other genomic loci. METHODS: Twelve MLH1 epimutation carriers, 61 Lynch syndrome patients, and 41 healthy controls, were analysed by Infinium 450 K array. Targeted molecular techniques were used to characterise the MLH1 epimutation carriers and their inheritance pattern. RESULTS: No nucleotide or structural variants were identified in-cis on the epimutated allele in 10 carriers, in which inter-generational methylation erasure was demonstrated in two, suggesting primary type of epimutation. CNVs outside the MLH1 locus were found in two cases. EPM2AIP1-MLH1 CpG island was identified as the sole differentially methylated region in MLH1 epimutation carriers compared to controls. CONCLUSION: Primary constitutional MLH1 epimutations arise as a focal epigenetic event at the EPM2AIP1-MLH1 CpG island in the absence of cis-acting genetic variants. Further molecular characterisation is needed to elucidate the mechanistic basis of MLH1 epimutations and their heritability/reversibility.

Assessment of the InSiGHT Interpretation Criteria for the Clinical Classification of 24 MLH1 and MSH2 Gene Variants.

Pathogenicity assessment of DNA variants in disease genes to explain their clinical consequences is an integral component of diagnostic molecular testing. The International Society for Gastrointestinal Hereditary Tumors (InSiGHT) has developed specific criteria for the interpretation of mismatch repair (MMR) gene variants. Here, we performed a systematic investigation of 24 MLH1 and MSH2 variants. The assessments were done by analyzing population frequency, segregation, tumor molecular characteristics, RNA effects, protein expression levels, and in vitro MMR activity. Classifications were confirmed for 15 variants and changed for three, and for the first time determined for six novel variants. Overall, based on our results, we propose the introduction of some refinements to the InSiGHT classification rules. The proposed changes have the advantage of homogenizing the InSIGHT interpretation criteria with those set out by the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium for the BRCA1/BRCA2 genes. We also observed that the addition of only few clinical data was sufficient to obtain a more stable classification for variants considered as "likely pathogenic" or "likely nonpathogenic." This shows the importance of obtaining as many as possible points of evidence for variant interpretation, especially from the clinical setting.

Type and frequency of MUTYH variants in Italian patients with suspected MAP: a retrospective multicenter study.

To determine prevalence, spectrum and genotype-phenotype correlations of MUTYH variants in Italian patients with suspected MAP (MUTYH-associated polyposis), a retrospective analysis was conducted to identify patients who had undergone MUTYH genetic testing from September 2002 to February 2014. Results of genetic testing and patient clinical characteristics were collected (gender, number of polyps, age at polyp diagnosis, presence of colorectal cancer (CRC) and/or other cancers, family data). The presence of large rearrangements of the MUTYH gene was evaluated by Multiplex Ligation-dependent Probe Amplification analysis. In all, 299 patients with colorectal neoplasia were evaluated: 61.2% were males, the median age at polyps or cancer diagnosis was 50 years (16-80 years), 65.2% had <100 polyps and 51.8% had CRC. A total of 36 different MUTYH variants were identified: 13 (36.1%) were classified as pathogenetic, whereas 23 (63.9%) were variants of unknown significance (VUS). Two pathogenetic variants were observed in 78 patients (26.1%). A large homozygous deletion of exon 15 was found in one patient (<1.0%). MAP patients were younger than those with negative MUTYH testing at polyps diagnosis (P<0.0001) and at first cancer diagnosis (P=0.007). MAP patients carrying the p.Glu480del variant presented with a younger age at polyp diagnosis as compared to patients carrying p.Gly396Asp and p.Tyr179Cys variants. A high heterogeneity of MUTYH variants and a high rate of VUS were identified in a cohort of Italian patients with suspected MAP. Genotype-phenotype analysis suggests that the p.Glu480del variant is associated with a severe phenotype.

A mutation in PAK3 with a dual molecular effect deregulates the RAS/MAPK pathway and drives an X-linked syndromic phenotype.

Loss-of-function mutations in PAK3 contribute to non-syndromic X-linked intellectual disability (NS-XLID) by affecting dendritic spine density and morphology. Linkage analysis in a three-generation family with affected males showing ID, agenesis of corpus callosum, cerebellar hypoplasia, microcephaly and ichthyosis, revealed a candidate disease locus in Xq21.33q24 encompassing over 280 genes. Subsequent to sequencing all coding exons of the X chromosome, we identified a single novel variant within the linkage region, affecting a conserved codon of PAK3. Biochemical studies showed that, similar to previous NS-XLID-associated lesions, the predicted amino acid substitution (Lys389Asn) abolished the kinase activity of PAK3. In addition, the introduced residue conferred a dominant-negative function to the protein that drives the syndromic phenotype. Using a combination of in vitro and in vivo studies in zebrafish embryos, we show that PAK3(N389) escapes its physiologic degradation and is able to perturb MAPK signaling via an uncontrolled kinase-independent function, which in turn leads to alterations of cerebral and craniofacial structures in vivo. Our data expand the spectrum of phenotypes associated with PAK3 mutations, characterize a novel mechanism resulting in a dual molecular effect of the same mutation with a complex PAK3 functional deregulation and provide evidence for a direct functional impact of aberrant PAK3 function on MAPK signaling.

Impact of Genetic Counseling in Women with a Family History of Breast Cancer in Italy.

As the impact of breast cancer (BC) risk assessment in asymptomatic women with a family history of BC had never been explored in Italy, we performed a study on a retrospective series of women who had undergone BC risk assessment. To this aim, a semi-structured telephone interview was administered to 82 women. Most participants considered the information received as clear (96.2 %) and helpful (76.8 %). Thirty-eight (46.3 %) stated that their perceived risk of BC had changed after the counseling: for 40.2 % it had decreased, for 6.1 % increased; however, women highly overestimating their risk at the baseline (≥ 4-fold) failed to show improvements in risk perception accuracy. Sixty-six women (80.5 %) stated they had followed the recommended surveillance, while 19.5 % had not, mainly due to difficulties in arranging examinations. Most women (89.0 %) had shared the information with their relatives, with 57.3 % reporting other family members had undertaken the recommended surveillance. BC risk assessment was associated with high rates of satisfaction and had a favorable impact on risk perception in a subgroup of women. The impact on surveillance adhesion extended to relatives. Organized programs for identification and surveillance may help identify a larger fraction of at-risk women and overcome the reported difficulties in arranging surveillance.

A systematic review of interventions to provide genetics education for primary care.

BACKGROUND: At least 10 % of patients seen in primary care are said to have a condition in which genetics has an influence. However, patients at risk of genetic disease may not be recognised, while those who seek advice may not be referred or managed appropriately. Primary care practitioners lack knowledge of genetics and genetic testing relevant for daily practice and feel inadequate to deliver genetic services. The aim of this systematic review was to evaluate genetics educational interventions in the context of primary care. METHODS: Following the process for systematic reviews developed by the Centre for Reviews and Dissemination, we conducted a search of five relevant electronic databases. Primary research papers were eligible for inclusion if they included data on outcomes of interventions regarding genetics education for primary care practitioners. The results from each paper were coded and grouped under themes. RESULTS: Eleven studies were included in the review. The five major themes identified inductively (post hoc) were: prior experience, changes in confidence, changes in knowledge, changes in practice, satisfaction and feedback. In five of the studies, knowledge of practitioners was improved following the educational programmes, but this tended to be in specific topic areas, while practitioner confidence improved in six studies. However, there was little apparent change to practice. CONCLUSIONS: There are insufficient studies of relevant quality to inform educational interventions in genetics for primary care practitioners. Educational initiatives should be assessed using changes in practice, as well as in confidence and knowledge, to determine if they are effective in causing significant changes in practice in genetic risk assessment and appropriate management of patients.