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EEG anomalies and epilepsy are a not so rare clinical manifestation in patients with Phosphatase and tensin homolog (PTEN) variants. The main aim of this study is to analyze the characteristics of EEG traces, neuroimaging findings and epilepsy to better define the neurological aspects in a set of patients with PTEN variants collected in four Italian Centres. As a secondary aim, we describe the neurodevelopmental profile and the psychiatric comorbidities of this cohort.Patients with PTEN variants, identified by Sanger sequencing or target resequencing, were enrolled. For each subjects, clinical data were retrospectively extracted from medical charts, with a focus on epilepsy and neuroimaging data.54 patients with PTEN variants were enrolled, with a mean age of 18.8 years. 72.2% have at least one psychiatric diagnosis, being Autism Spectrum Disorder and Intellectual Disability the most frequent diagnosis (29 and 25 cases, respectively). 22 subjects show an abnormal EEG and 8 received a diagnosis of epilepsy, mainly focal epilepsy (7/8), with a mean age at seizure onset of 3.8 years. 3/8 subjects have a drug resistant epilepsy, independently from the underlying neuroimaging pattern. The finding of a Focal cortical dysplasia is significantly associated with both an abnormal EEG (p = 0.02) and the occurrence of seizures (p = 0.002).EEG should be taken into consideration in the first-line diagnostic flowchart of subjects with PTEN variants. The onset of a focal epilepsy, independently from its response to antiepileptic drugs, highly recommends to carry out a neuroimaging exam.

DNA mismatch repair (MMR) is essential for correction of DNA replication errors. Germline mutations of the human MMR gene MLH1 are the major cause of Lynch syndrome, a heritable cancer predisposition. In the MLH1 protein, a non-conserved, intrinsically disordered region connects two conserved, catalytically active structured domains of MLH1. This region has as yet been regarded as a flexible spacer, and missense alterations in this region have been considered non-pathogenic. However, we have identified and investigated a small motif (ConMot) in this linker which is conserved in eukaryotes. Deletion of the ConMot or scrambling of the motif abolished mismatch repair activity. A mutation from a cancer family within the motif (p.Arg385Pro) also inactivated MMR, suggesting that ConMot alterations can be causative for Lynch syndrome. Intriguingly, the mismatch repair defect of the ConMot variants could be restored by addition of a ConMot peptide containing the deleted sequence. This is the first instance of a DNA mismatch repair defect conferred by a mutation that can be overcome by addition of a small molecule. Based on the experimental data and AlphaFold2 predictions, we suggest that the ConMot may bind close to the C-terminal MLH1-PMS2 endonuclease and modulate its activation during the MMR process.

Genotype; Cancer phenotypes; Genetic tumour Genotip; Fenotips del càncer; Tumor genètic Genotipo; Fenotipos del cáncer; Tumor genético Background Truncating pathogenic or likely pathogenic variants of CDH1 cause hereditary diffuse gastric cancer (HDGC), a tumour risk syndrome that predisposes carrier individuals to diffuse gastric and lobular breast cancer. Rare CDH1 missense variants are often classified as variants of unknown significance. We conducted a genotype–phenotype analysis in families carrying rare CDH1 variants, comparing cancer spectrum in carriers of pathogenic or likely pathogenic variants (PV/LPV; analysed jointly) or missense variants of unknown significance, assessing the frequency of families with lobular breast cancer among PV/LPV carrier families, and testing the performance of lobular breast cancer-expanded criteria for CDH1 testing. Methods This genotype-first study used retrospective diagnostic and clinical data from 854 carriers of 398 rare CDH1 variants and 1021 relatives, irrespective of HDGC clinical criteria, from 29 institutions in ten member-countries of the European Reference Network on Tumour Risk Syndromes (ERN GENTURIS). Data were collected from Oct 1, 2018, to Sept 20, 2022. Variants were classified by molecular type and clinical actionability with the American College of Medical Genetics and Association for Molecular Pathology CDH1 guidelines (version 2). Families were categorised by whether they fulfilled the 2015 and 2020 HDGC clinical criteria. Genotype–phenotype associations were analysed by Student's t test, Kruskal-Wallis, χ2, and multivariable logistic regression models. Performance of HDGC clinical criteria sets were assessed with an equivalence test and Youden index, and the areas under the receiver operating characteristic curves were compared by Z test. Findings From 1971 phenotypes (contributed by 854 probands and 1021 relatives aged 1–93 years), 460 had gastric and breast cancer histology available. CDH1 truncating PV/LPVs occurred in 176 (21%) of 854 families and missense variants of unknown significance in 169 (20%) families. Multivariable logistic regression comparing phenotypes occurring in families carrying PV/LPVs or missense variants of unknown significance showed that lobular breast cancer had the greatest positive association with the presence of PV/LPVs (odds ratio 12·39 [95% CI 2·66–57·74], p=0·0014), followed by diffuse gastric cancer (8·00 [2·18–29·39], p=0·0017) and gastric cancer (7·81 [2·03–29·96], p=0·0027). 136 (77%) of 176 families carrying PV/LPVs fulfilled the 2015 HDGC criteria. Of the remaining 40 (23%) families, who did not fulfil the 2015 criteria, 11 fulfilled the 2020 HDGC criteria, and 18 had lobular breast cancer only or lobular breast cancer and gastric cancer, but did not meet the 2020 criteria. No specific CDH1 variant was found to predispose individuals specifically to lobular breast cancer, although 12 (7%) of 176 PV/LPV carrier families had lobular breast cancer only. Addition of three new lobular breast cancer-centred criteria improved testing sensitivity while retaining high specificity. The probability of finding CDH1 PV/LPVs in patients fulfilling the lobular breast cancer-expanded criteria, compared with the 2020 criteria, increased significantly (AUC 0·92 vs 0·88; Z score 3·54; p=0·0004). Interpretation CDH1 PV/LPVs were positively associated with HDGC-related phenotypes (lobular breast cancer, diffuse gastric cancer, and gastric cancer), and no evidence for a positive association with these phenotypes was found for CDH1 missense variants of unknown significance. CDH1 PV/LPVs occurred often in families with lobular breast cancer who did not fulfil the 2020 HDGC criteria, supporting the expansion of lobular breast cancer-centred criteria. Funding European Reference Network on Genetic Tumour Risk Syndromes, European Regional Development Fund, Fundação para a Ciência e a Tecnologia (Portugal), Cancer Research UK, and European Union's Horizon 2020 research and innovation programme. European Reference Network on Genetic Tumour Risk Syndromes, European Regional Development Fund, Fundação para a Ciência e a Tecnologia (Portugal), Cancer Research UK, and European Union's Horizon 2020 research and innovation programme.

The implementation of cancer molecular characterization in clinical practice has improved prognostic re-definition, extending the eligibility to a continuously increasing number of targeted treatments. Broad molecular profiling technologies better than organ-based approaches are believed to serve such dynamic purposes. We here present the workflow our institution adopted to run a comprehensive cancer genome profiling in clinical practice. This article describes the workflow designed to make a comprehensive cancer genome profiling program feasible and sustainable in a large-volume referral hospital.

BackgroundSpinal muscular atrophy (SMA) is due to the homozygous absence ofSMN1in around 97% of patients, independent of the severity (classically ranked into types I–III). The high genetic homogeneity, coupled with the excellent results of presymptomatic treatments of patients with each of the three disease-modifying therapies available, makes SMA one of the golden candidates to genetic newborn screening (NBS) (SMA-NBS). The implementation of SMA in NBS national programmes occurring in some countries is an arising new issue that the scientific community has to address. We report here the results of the first Italian SMA-NBS project and provide some proposals for updating the current molecular diagnostic scenario.MethodsThe screening test was performed by an in-house-developed qPCR assay, amplifyingSMN1andSMN2. Molecular prognosis was assessed on fresh blood samples.ResultsWe found 15 patients/90885 newborns (incidence 1:6059) having the followingSMN2genotypes: 1 (one patient), 2 (eight patients), 2+c.859G>C variant (one patient), 3 (three patients), 4 (one patient) or 6 copies (one patient). Six patients (40%) showed signs suggestive of SMA at birth. We also discuss some unusual cases we found.ConclusionThe molecular diagnosis of SMA needs to adapt to the new era of the disease with specific guidelines and standard operating procedures. In detail, SMA diagnosis should be felt as a true medical urgency due to therapeutic implications;SMN2copy assessment needs to be standardised; commercially available tests need to be improved for higherSMN2copies determination; and theSMN2splicing-modifier variants should be routinely tested in SMA-NBS.

Craniosynostosis are a heterogeneous group of genetic conditions characterized by the premature fusion of the skull bones. The most common forms of craniosynostosis are Crouzon, Apert and Pfeiffer syndromes. They differ from each other in various additional clinical manifestations, e.g., syndactyly is typical of Apert and rare in Pfeiffer syndrome. Their inheritance is autosomal dominant with incomplete penetrance and one of the main genes responsible for these syndromes is

International audience; Purpose: Juvenile polyposis syndrome (JPS) is a rare, autosomal-dominantly inherited cancer predisposition caused in approximately 50% of cases by pathogenic germline variants in SMAD4 and BMPR1A. We aimed to gather detailed clinical and molecular genetic information on JPS disease expression to provide a basis for management guidelines and establish open access variant databases.Methods: We performed a retrospective, questionnaire-based European multicenter survey on and established a cohort of SMAD4/BMPR1A pathogenic variant carriers from the medical literature.Results: We analyzed questionnaire-based data on 221 JPS patients (126 kindreds) from ten European centers and retrieved literature-based information on 473 patients. Compared with BMPR1A carriers, SMAD4 carriers displayed anemia twice as often (58% vs. 26%), and exclusively showed overlap symptoms with hemorrhagic telangiectasia (32%) and an increased prevalence (39% vs. 13%) of gastric juvenile polyps. Cancer, reported in 15% of JPS patients (median age 41 years), mainly occurred in the colorectum (overall: 62%, SMAD4: 58%, BMPR1A: 88%) and the stomach (overall: 21%; SMAD4: 27%, BMPR1A: 0%).Conclusion: This comprehensive retrospective study on genotype-phenotype correlations in 694 JPS patients corroborates previous observations on JPS in general and SMAD4 carriers in particular, facilitates recommendations for clinical management, and provides the basis for open access variant SMAD4 and BMPR1A databases.

BACKGROUND: Pathogenic PTEN germline variants cause PTEN Hamartoma Tumor Syndrome (PHTS), a rare disease with a variable genotype and phenotype. Knowledge about these spectra and genotype-phenotype associations could help diagnostics and potentially lead to personalized care. Therefore, we assessed the PHTS genotype and phenotype spectrum in a large cohort study. METHODS: Information was collected of 510 index patients with pathogenic or likely pathogenic (LP/P) PTEN variants (n = 467) or variants of uncertain significance. Genotype-phenotype associations were assessed using logistic regression analyses adjusted for sex and age. RESULTS: At time of genetic testing, the majority of children (n = 229) had macrocephaly (81%) or developmental delay (DD, 61%), and about half of the adults (n = 238) had cancer (51%), macrocephaly (61%), or cutaneous pathology (49%). Across PTEN, 268 LP/P variants were identified, with exon 5 as hotspot. Missense variants (n = 161) were mainly located in the phosphatase domain (PD, 90%) and truncating variants (n = 306) across all domains. A trend towards 2 times more often truncating variants was observed in adults (OR = 2.3, 95%CI = 1.5-3.4) and patients with cutaneous pathology (OR = 1.6, 95%CI = 1.1-2.5) or benign thyroid pathology (OR = 2.0, 95%CI = 1.1-3.5), with trends up to 2-4 times more variants in PD. Whereas patients with DD (OR = 0.5, 95%CI = 0.3-0.9) or macrocephaly (OR = 0.6, 95%CI = 0.4-0.9) had about 2 times less often truncating variants compared to missense variants. In DD patients these missense variants were often located in domain C2. CONCLUSION: The PHTS phenotypic diversity may partly be explained by the PTEN variant coding effect and the combination of coding effect and domain. PHTS patients with early-onset disease often had missense variants, and those with later-onset disease often truncating variants. ispartof: EUROPEAN JOURNAL OF MEDICAL GENETICS vol:65 issue:12 ispartof: location:Netherlands status: published

Costello syndrome (CS) is a rare disorder affecting development and growth characterized by cancer predisposition and caused by mutations in HRAS proto-oncogene. Somatic HRAS mutations drive bladder carcinogenesis. The aim of this study was to analyze prevalence and histological characterization of bladder cancer (BC) in a cohort of patients with CS to help clinicians plan effective management strategies. This study included 13 patients above 10 years of age with molecular diagnosis of CS. Screening cystoscopies (31 total procedures) were performed to exclude BC. Any lesion was analyzed through cold-cup biopsy or trans-urethral resection of the bladder. According to histology, patients were followed-up with urinalysis and abdominal ultrasound yearly, and cystoscopies every 12-24 months. During study enrollment, bladder lesions (often multifocal) were detected in 11/13 patients. Histological analysis documented premalignant lesions in 90% of cystoscopies performed, epithelial dysplasia in 71%, and papillary urothelial neoplasm of low-malignant potential in 19%. BC G1/low grade (Ta) were removed in 10%. Overall, 76% of patients showed a bladder lesion at first cystoscopy. The present findings document that individuals with CS aged 10 years and older have high prevalence of bladder lesions (premalignant/malignant), highlighting the importance of personalized screening protocols.

The development of innovative technologies and the advances in the genetics and genomics, have offered new opportunities for personalized treatment in oncology. Although the selection of the patient based on the molecular characteristics of the neoplasm has the potential to revolutionize the therapeutic scenario of oncology, this approach is extremely challenging. The access, homogeneity, and economic sustainability of the required genomic tests should be warranted in the clinical practice, as well as the specific scientific and clinical expertise for the choice of medical therapies. All these elements make essential the collaboration of different specialists within the Molecular Tumor Boards (MTBs). In this position paper, based on experts' opinion, the AIOM-SIAPEC/IAP-SIBioC-SIC-SIF-SIGU-SIRM Italian Scientific Societies critically discuss the available molecular profiling technologies, the proposed criteria for the selection of patients candidate for evaluation by the MTB, the criteria for the selection and analysis of biological samples, and the regulatory and pharmaco-economic issues.

The PTEN hamartoma tumor syndrome (PHTS) is a heterogeneous set of multisystem disorders caused by germline pathogenic variants in the PTEN tumor suppressor gene. Manifestations include developmental anomalies and proliferative lesions. Evidence of involvement of the GI tract has accrued over time, leading to the incorporation of GI manifestations (multiple hamartomas, glycogenic acanthosis and colorectal cancer) into the diagnostic criteria. Polyps of the upper and lower GI tract are found in most adult patients and in a significant fraction of children. Polyps tend to be of mixed histology, with a predominance of hamartomas and ganglioneuromas. PHTS patients are also at increased risk of colorectal cancer, and surveillance by colonoscopy is advised starting at the age of 35-40 years. A number of additional manifestations, including eosinophilic gastrointestinal disorders, have been observed in few or single cases, and their association with PHTS has yet to be determined.

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and autism caused by the instability of a CGG trinucleotide repeat in exon 1 of the FMR1 gene. The co-occurrence of FXS with other genetic disorders has only been occasionally reported. Here, we describe three independent cases of FXS co-segregation with three different genetic conditions, consisting of Duchenne muscular dystrophy (DMD), PPP2R5D--related neurodevelopmental disorder, and 2p25.3 deletion. The co-occurrence of DMD and FXS has been reported only once in a young boy, while in an independent family two affected boys were described, the elder diagnosed with FXS and the younger with DMD. This represents the second case in which both conditions coexist in a 5-year-old boy, inherited from his heterozygous mother. The next double diagnosis had never been reported before: through exome sequencing, a girl with FXS who was of 7 years of age with macrocephaly and severe psychomotor delay was found to carry a de novo variant in the PPP2R5D gene. Finally, a maternally inherited 2p25.3 deletion associated with a decreased level of the MYT1L transcript, only in the patient, was observed in a 33-year-old FXS male with severe seizures compared to his mother and two sex- and age-matched controls. All of these patients represent very rare instances of genetic conditions with clinical features that can be modified by FXS and vice versa.

Findings 5585 families with Lynch syndrome from 22 countries were eligible for the analysis. Of these, there were insufficient numbers to estimate penetrance for Asia and South America, and for those with EPCAM variants. Therefore, we used data (collected between July 11, 2014, and Dec 31, 2018) from 5255 families (1829 MLH1, 2179 MSH2, 798 MSH6, and 449 PMS2), comprising 79 809 relatives, recruited in 15 countries in North America, Europe, and Australasia. There was strong evidence of the existence of unknown familial risk factors modifying colorectal cancer risk for Lynch syndrome carriers (p 0 center dot 0001 for each of the three three continents). These familial risk factors resulted in a wide within-gene variation in the risk of colorectal cancer for men and women from each continent who all carried pathogenic variants in the same gene or the MSH2 c.942+3A T variant. The variation was especially prominent for MLH1 and MSH2 variant carriers, depending on gene, sex and continent, with 7-56% of carriers having a colorectal cancer penetrance of less than 20%, 9-44% having a penetrance of more than 80%, and onlyBackground Existing clinical practice guidelines for carriers of pathogenic variants of DNA mismatch repair genes (Lynch syndrome) are based on the mean age-specific cumulative risk (penetrance) of colorectal cancer for all carriers of pathogenic variants in the same gene. We aimed to estimate the variation in the penetrance of colorectal cancer between carriers of pathogenic variants in the same gene by sex and continent of residence. Methods In this retrospective cohort study, we sourced data from the International Mismatch Repair Consortium, which comprises 273 members from 122 research centres or clinics in 32 countries from six continents who are involved in Lynch syndrome research. Families with at least three members and at least one confirmed carrier of a pathogenic or likely pathogenic variant in a DNA mismatch repair gene (MLH1, MSH2, MSH6, or PMS2) were included. The families of probands with known de-novo pathogenic variants were excluded. Data were collected on the method of ascertainment of the family, sex, carrier status, cancer diagnoses, and ages at the time of pedigree collection and at last contact or death. We used a segregation analysis conditioned on ascertainment to estimate the mean penetrance of colorectal cancer and modelled unmeasured polygenic factors to estimate the variation in penetrance. The existence of unknown familial risk factors modifying colorectal cancer risk for Lynch syndrome carriers was tested by use of a Wald p value for the null hypothesis that the polygenic SD is zero. Findings 5585 families with Lynch syndrome from 22 countries were eligible for the analysis. Of these, there were insufficient numbers to estimate penetrance for Asia and South America, and for those with EPCAM variants. Therefore, we used data (collected between July 11, 2014, and Dec 31, 2018) from 5255 families (1829 MLH1, 2179 MSH2, 798 MSH6, and 449 PMS2), comprising 79 809 relatives, recruited in 15 countries in North America, Europe, and Australasia. There was strong evidence of the existence of unknown familial risk factors modifying colorectal cancer risk for Lynch syndrome carriers (pT variant. The variation was especially prominent for MLH1 and MSH2 variant carriers, depending on gene, sex and continent, with 7-56% of carriers having a colorectal cancer penetrance of less than 20%, 9-44% having a penetrance of more than 80%, and only 10-19% having a penetrance of 40-60%. Interpretation Our study findings highlight the important role of risk modifiers, which could lead to personalised risk assessments for precision prevention and early detection of colorectal cancer for people with Lynch syndrome. Funding National Health and Medical Research Council, Australia. Copyright (c) 2021 Elsevier Ltd. All rights reserved.Methods In this retrospective cohort study, we sourced data from the International Mismatch Repair Consortium, which comprises 273 members from 122 research centres or clinics in 32 countries from six continents who are involved in Lynch syndrome research. Families with at least three members and at least one confirmed carrier of a pathogenic or likely pathogenic variant in a DNA mismatch repair gene (MLH1, MSH2, MSH6, or PMS2) were included. The families of probands with known de-novo pathogenic variants were excluded. Data were collected on the method of ascertainment of the family, sex, carrier status, cancer diagnoses, and ages at the time of pedigree collection and at last contact or death. We used a segregation analysis conditioned on ascertainment to estimate the mean penetrance of colorectal cancer and modelled unmeasured polygenic factors to estimate the variation in penetrance. The existence of unknown familial risk factors modifying colorectal cancer risk for Lynch syndrome carriers was tested by use of a Wald p value for the null hypothesis that the polygenic SD is zero.

Background Aim of this work was to describe a rare inheritance pattern of Primary Failure of Eruption (PFE) in a small family with incomplete penetrance of PFE and a novel nonsense PTH1R variant. Case presentation The proband, a 26 year-old man with a significant bilateral open-bite, was diagnosed with PFE using clinical and radiographic characteristics. DNA was extracted from the proband and his immediate family using buccal swabs and the entire PTH1R coding sequence was analyzed, revealing a novel heterozygous nonsense variant in exon 7 of PTH1R (c.505G > T). This variant introduces a premature stop codon in position 169, predicted to result in the production of a truncated and non-functional protein. This variant has never been reported in association with PFE and is not present in the Genome Aggregation Database (gnomAD). Interestingly, the c.505G > T variant has also been identified in the unaffected mother of our proband, suggesting incomplete penetrance of PFE. Conclusions In this study, we report a new PTH1R variant that segregates in an autosomal dominant pattern and causes PFE with incomplete penetrance. This underlines the diagnostic value of a thorough clinical and genetic analysis of all family members in order to estimate accurate recurrence risks, identify subtle clinical manifestations and provide proper management of PFE patients.

Item does not contain fulltext Hereditary breast and ovarian cancer (HBOC) is a syndrome defined by an increased risk of developing breast and/or ovarian cancer most commonly due to germline disease-causing variants in the BRCA1 and BRCA2 genes, but also other causative genes such as PALB2, ATM and CHEK2. As genetic testing becomes more prevalent and new clinical data emerge, updates of national guidelines are required to incorporate these advances in our knowledge. The aim of this work is to review the guidelines for HBOC genetic testing and clinical surveillance across European countries, mostly affiliated to the European Reference Network (ERN) for Genetic Tumor Risk Syndroms (GENTURIS). Young onset breast cancer (BC), triple negative phenotype, or bilateral BC are considered as criteria for genetic testing in all, with differences in age limits. Testing of invasive epithelial non-mucinous ovarian cancer is also universally accepted. While breast magnetic resonance imaging (MRI) is consistently recommended in high-risk individuals, age of onset for mammograms differ between 30 and 40 years. Risk-reducing mastectomy is commonly offered as an option, while risk-reducing salpingo-oophorectomy is universally recommended. The largest differences are observed with respect to ovarian surveillance prior to risk-reducing salpingo-oophorectomy and in breast surveillance for carriers of non-BRCA1/2 genes. These differences in national guidelines reflect the variations in clinical consensus that may be reached in the absence of consistent evidence for some recommendations.

Genomic testing expansion is accompanied by an increasing need for genetic counselling and intrafamilial communication. Genetic counselling can play an important role in facilitating intrafamilial communication and relationships. We conducted a cross-sectional, multicenter study including 252 Italian women, using a questionnaire divided in two sections, the first one to be filled after the pre-test counselling and the second after receiving BRCA test results. We assessed the factors influencing intrafamilial disclosure of genetic information for hereditary breast and ovarian cancer, family members with whom probands are more prone to share genetic information, and the perceived understanding of information received by counselees during genetic counselling. Women were accompanied to the counselling more often by their husband/partner. Among those with a positive BRCA test result, 49% intended to communicate it to their offspring and 27% to their husband/partner. Younger women, those living with their husband/partner, and those who described family communication as open/profound and spontaneous/sincere had a higher probability of being accompanied during genetic counselling and discuss about it with relatives. Spontaneous/sincere or open/profound family communication and joyful/happy familial relationships were associated with the decision to undergo genetic testing as a responsibility towards relatives. Women had a good understanding of counselling contents (mean score 9.27 in a scale 1–10). Genetic counselling providers should consider that genetic information disclosure does not depend only on the clarity of the information provided, but also on pre-existing intrafamilial communication and relationships, family structure and marital status, indicating the need for a personalised approach accounting for these factors.

Background Genomic testing expansion for hereditary breast and ovarian cancer (HBOC) comes with an increased need for genetic counseling and intra-familial communication. Genetic counseling can play an important role in facilitating intra-familial communication and relationships, thus avoiding misunderstandings within the family. Methods We conducted a cross-sectional, multicenter study including 252 Italian women, using a questionnaire divided in two sections; the first to be filled after the pre-test counseling and the second after receiving the genetic test results. This aimed at assessing the factors influencing the disclosure of genetic information for HBOC, family members that probands share genetic information with, and the degree of understanding of the information received by the counselees during genetic counseling. Results Women were accompanied to the counseling more by their husbands/partners. Among those with a positive test result, 49% shared it with their offspring and 27 % with their husband/partner. Younger women, those living with their husbands/partners and those who described family communication as open/deep had a higher probability of being accompanied at the genetic counseling and discuss about it with relatives. Being alone at the genetic counseling was significantly associated to non-existing familial relationship (p = 0.003) and problematic intra-familial communication (p = 0.005). Sincere or joyful familial relations were associated with the decision to undergo genetic testing as a responsibility towards relatives. Women had a good understanding of the counseling (mean score 9.2 in a scale1-10). Discussion Genetic counseling providers should consider that the disclosure of genetic information does not depend only on the clarity of the information provided, but also on pre-existing intra-familial communication and relationship, family structure and marital status, indicating the need of a personalized approach based on these factors. Key messages The disclosure of genetic information from women to their relatives in the case of HBOC is a complex relational process, which poses practical and ethical problems for individuals and families. Genetic counseling providers should be able to advice their patients on the importance of genetic information disclosure, considering, also, the pre-existing intra-familial communication dynamics.

Background- Identification of variants of uncertain significance (VUSs) poses relevant challenges in counseling and managing patients. They have an unknown impact on health, making the genetic tests clinically irrelevant. Recent studies demonstrate that a routine reclassification analysis enables to reclassify from 20% to 80% of this type of variant, improving risk stratification. Purpose- We investigated whether, in the context of inherited cardiac conditions, a review of the updated literature, including new functional data, allele frequency (GnomAD) and segregation analysis may help in the variant reclassification. Methods- Retrospective review of all VUSs in genes associated with inherited cardiac conditions identified in our cardiogenetic clinic between 2016 and 2018. Results- Thirty-one VUSs, classified using ACMG guidelines, were identified in 26 cases with a confirmed or suspected diagnosis of inherited cardiovascular diseases, including Long QT syndrome, Brugada syndrome, Arrhythmogenic Cardiomyopathy and Hypertrophic Cardiomyopathy). Twentyfour variants were identified in well-defined causative genes (SCN5A, KCNQ1, KCNH2, KCNE1, DSP, DSG2, MYH7, TPM1, TNNI3, TNNT2, CACNA1C, MYL3) while the remaining variants were identified in minor genes with limited evidence to support their disease causation as, ANK2 and AKAP9 gene. Preliminary results of the reclassification analysis showed that two variants were downgraded to likely benign (LB) applying the BS1 criterion (allele frequency) and 4 variants were upgraded to likely pathogenic (LP) according to novel published data and family segregation studies. Moreover, further studies to assess cosegregation in other variants are still ongoing. Conclusion- Based on our experience, 25% of variants of uncertain significance in well-defined causative genes, identified in patients with a confirmed or suspected diagnosis of inherited cardiovascular disease, were reclassified. These findings suggest that re-evaluation of genetic test results should be performed routinely in all diagnostic labs, in order to improve risk stratification and identification of family members at high risk.

Fragile X syndrome (FXS) is mostly due to the expansion and subsequent methylation of a polymorphic CGG repeat in the 5' UTR of the FMR1 gene. Full mutation alleles (FM) have more than 200 repeats and result in FMR1 gene silencing and FXS. FMs arise from maternal premutations (PM) that have 56-200 CGGs, contractions of a maternal PM or FM are rare. Here, we describe two unaffected boys in two independent FXS families who inherited a non-mosaic allele in the normal and intermediate range, respectively, from their mothers who are carriers of an expanded CGG allele. The first boy inherited a 51 CGG allele (without AGG interruptions) from his mother, who carries a PM allele with 72 CGGs. The other boy inherited from his FM mother an unusual allele with 19 CGGs resulting from a deletion, removing 85 bp upstream of the CGG repeat. Given that transcription of the deleted allele was found to be preserved, we assume that the binding sites for FMR1 transcription factors are excluded from the deletion. Such unusual cases resulting in non-mosaic reduction of maternal CGG expansions may help to clarify the molecular mechanisms underlying the instability of the FMR1 gene.

Fragile X syndrome (FXS) is mostly due to the expansion and subsequent methylation of a polymorphic CGG repeat in the 5’ UTR of the FMR1 gene. Full mutation alleles (FM) have more than 200 repeats and result in FMR1 gene silencing and FXS. FMs arise from maternal premutations (PM) that have 56–200 CGGs; contractions of a maternal PM or FM are rare. Here, we describe two unaffected boys in two independent FXS families who inherited a non-mosaic allele in the normal and intermediate range, respectively, from their mothers who are carriers of an expanded CGG allele. The first boy inherited a 51 CGG allele (without AGG interruptions) from his mother, who carries a PM allele with 72 CGGs. The other boy inherited from his FM mother an unusual allele with 19 CGGs resulting from a deletion, removing 85 bp upstream of the CGG repeat. Given that transcription of the deleted allele was found to be preserved, we assume that the binding sites for FMR1 transcription factors are excluded from the deletion. Such unusual cases resulting in non-mosaic reduction of maternal CGG expansions may help to clarify the molecular mechanisms underlying the instability of the FMR1 gene.

Background Glioblastoma (GBM) stemlike cells (GSCs) are thought to be responsible for the maintenance and aggressiveness of GBM, the most common primary brain tumor in adults. This study aims at elucidating the involvement of deregulations within the imprinted delta-like homolog 1 gene‒type III iodothyronine deiodinase gene (DLK-DIO3) region on chromosome 14q32 in GBM pathogenesis. Methods Real-time PCR analyses were performed on GSCs and GBM tissues. Methylation analyses, gene expression, and reverse-phase protein array profiles were used to investigate the tumor suppressor function of the maternally expressed 3 gene (MEG3). Results Loss of expression of genes and noncoding RNAs within the DLK1-DIO3 region was observed in GSCs and GBM tissues compared with normal brain. This downregulation is mainly mediated by epigenetic silencing. Kaplan–Meier analysis indicated that low expression of MEG3 and MEG8 long noncoding (lnc)RNAs significantly correlated with short survival in GBM patients. MEG3 restoration impairs tumorigenic abilities of GSCs in vitro by inhibiting cell growth, migration, and colony formation and decreases in vivo tumor growth, reducing infiltrative growth. These effects were associated with modulation of genes involved in cell adhesion and epithelial-to-mesenchymal transition (EMT). Conclusion In GBM, MEG3 acts as a tumor suppressor mainly regulating cell adhesion, EMT, and cell proliferation, thus providing a potential candidate for novel GBM therapies.

Fragile X-related disorders are due to a dynamic mutation of the CGG repeat at the 5? UTR of the FMR1 gene, coding for the RNA-binding protein FMRP. As the CGG sequence expands from premutation (PM, 56-200 CGGs) to full mutation (> 200 CGGs), FMRP synthesis decreases until it is practically abolished in fragile X syndrome (FXS) patients, mainly due to FMR1 methylation. Cells from rare individuals with no intellectual disability and carriers of an unmethylated full mutation (UFM) produce slightly elevated levels of FMR1-mRNA and relatively low levels of FMRP, like in PM carriers. With the aim of clarifying how UFM cells differ from CTRL and FXS cells, a comparative proteomic approach was undertaken, from which emerged an overexpression of SOD2 in UFM cells, also confirmed in PM but not in FXS. The SOD2-mRNA bound to FMRP in UFM more than in the other cell types. The high SOD2 levels in UFM and PM cells correlated with lower levels of superoxide and reactive oxygen species (ROS), and with morphological anomalies and depolarization of the mitochondrial membrane detected through confocal microscopy. The same effect was observed in CTRL and FXS after treatment with MC2791, causing SOD2 overexpression. These mitochondrial phenotypes reverted after knock-down with siRNA against SOD2-mRNA and FMR1-mRNA in UFM and PM. Overall, these data suggest that in PM and UFM carriers, which have high levels of FMR1 transcription and may develop FXTAS, SOD2 overexpression helps to maintain low levels of both superoxide and ROS with signs of mitochondrial degradation.

Ventricular arrhythmias (VAs) are the most common cause of death in athletes. The differences in the electroanatomic substrate in athletes and nonathletes with complex VA are unknown.The purpose of this study was to compare the electroanatomic substrate of complex VA in athletes vs nonathletes.The study prospectively enrolled young athletes and nonathletes with VA. Patients underwent 2-dimensional echocardiography, cardiac magnetic resonance (CMR) imaging, coronary angiography, 3-dimensional electroanatomic mapping (3D-EAM), and 3D-EAM-guided endomyocardial biopsy (EMB). Follow-up included 24-hour electrocardiographic Holter or implantable cardioverter-defibrillator/loop recorder interrogation for VA recurrence.Thirty-three patients were enrolled: 18 competitive athletes (56%) and 15 nonathletes (44%). Left ventricular and right ventricular (RV) findings by echocardiography and CMR did not show structural disease. Nine athletes (50%) were asymptomatic compared to 1 nonathlete (7%; P.05). Unifocal origin of VA was reported in 14 athletes (93%) and 17 nonathletes (94%). Athletes showed a larger RV unipolar than bipolar scar (18 ± 17 cmThis study showed the need for extensive diagnostic workup in apparently healthy young patients with complex VA in order to characterize concealed cardiomyopathies.

Fragile X syndrome (FXS) is a very frequent cause of inherited intellectual disability (ID) and autism. Most FXS patients have an expansion over 200 repeats of (CGG)(n) sequence (“full mutation” (FM)) located in the 5′UTR of the FMR1 gene, resulting in local DNA methylation (methylated “full mutation” (MFM)) and epigenetic silencing. The absence of the FMRP protein is responsible for the clinical phenotype of FXS. FM arises from a smaller maternal allele with 56–200 CGG repeats (“premutation” (PM)) during maternal meiosis. Carriers of PM alleles, which are typically unmethylated, can manifest other clinical features (primary ovarian insufficiency (POI) or FXS-associated tremor-ataxia syndrome (FXTAS)), known as fragile X-related disorders. In FXS families, rare males who have inherited an unmethylated “full mutation” (UFM) have been described. These individuals produce enough FMRP to allow normal intellectual functioning. Here we report the rare case of three sisters with a completely methylated PM of around 140 CGGs and detail their neuropsychological function. X inactivation analysis confirmed that the three sisters have a random inactivation of the X chromosome, suggesting that the PM allele is always methylated also when residing on the active X. We propose that in exceptional cases, just as the FM may be unmethylated, also a PM allele may be fully methylated. To our knowledge, females with a methylated PM allele and a mild impairment have reported only once. The study of these atypical individuals demonstrates that the size of the CGG expansion is not as tightly coupled to methylation as previously thought.

DNA methylation in the human genome is largely programmed and shaped by transcription factor binding and interaction between DNA methyltransferases and histone marks during gamete and embryo development. Normal methylation profiles can be modified at single or multiple loci, more frequently as consequences of genetic variants acting in cis or in trans, or in some cases stochastically or through interaction with environmental factors. For many developmental disorders, specific methylation patterns or signatures can be detected in blood DNA. The recent use of high-throughput assays investigating the whole genome has largely increased the number of diseases for which DNA methylation analysis provides information for their diagnosis. Here, we review the methylation abnormalities that have been associated with mono/oligogenic diseases, their relationship with genotype and phenotype and relevance for diagnosis, as well as the limitations in their use and interpretation of results.

The contribution of recently established or candidate susceptibility genes to melanoma missing heritability has yet to be determined. Multigene panel testing could increase diagnostic yield and better define the role of candidate genes. We characterized 273 CDKN2A/ARF and CDK4-negative probands through a custom-designed targeted gene panel that included CDKN2A/ARF, CDK4, ACD, BAP1, MITF, POT1, TERF2IP, ATM, and PALB2. Co-segregation, loss of heterozygosity (LOH)/protein expression analysis, and splicing characterization were performed to improve variant classification. We identified 16 (5.9%) pathogenic and likely pathogenic variants in established high/medium penetrance cutaneous melanoma susceptibility genes (BAP1, POT1, ACD, MITF, and TERF2IP), including two novel variants in BAP1 and 4 in POT1. We also found four deleterious and five likely deleterious variants in ATM (3.3%). Thus, including potentially deleterious variants in ATM increased the diagnostic yield to about 9%. Inclusion of rare variants of uncertain significance would increase the overall detection yield to 14%. At least 10% of melanoma missing heritability may be explained through panel testing in our population. To our knowledge, this is the highest frequency of putative ATM deleterious variants reported in melanoma families, suggesting a possible role in melanoma susceptibility, which needs further investigation.

Since developments are global in the healthcare arena, more should be done to align EU and other big markets’ regulatory practices for rare disease patients. Notwithstanding efforts and cooperation between the US and EU aimed to harmonize their strategic plans in the field of orphan drugs, regulatory criteria and procedures to gain the designation, terms and classifications should be still harmonised. Aligning the criteria of prevalence and support to orphan medicines in the various jurisdictions internationally, would facilitate patient recruitment eventually at global level, so as to gain the data and the biological insights required to identify biomarkers and appropriate endpoints needed for progressing clinical development. A conducive regulatory environment can further support the development of medicines to treat rare diseases. Overall there is a need for joined-up regulatory process coordination. Better integration of regulatory pathways and better integration of regulatory systems, such as scientific tools and methods to generate evidence, would be helpful. There is a need to revise and agree the current frameworks to be improved which will take into account the considerations and challenges to diagnose and treat different rare diseases and improve quality of life. Deliberative processes with multi-stakeholders’ involvement for reimbursement should be considered. This paper explores the successes and limitation of both the regulation and its implementation mechanisms in the current regulatory context, and suggests some improvements that could maximise its benefits and boost rare disease research even further.