The Women4Health cohort: a unique cohort to study women-specific mechanisms of cardio-metabolic regulation.

Aims: Epidemiological research has shown relevant differences between sexes in clinical manifestations, severity, and progression of cardiovascular and metabolic disorders. To date, the mechanisms underlying these differences remain unknown. Given the rising incidence of such diseases, gender-specific research on established and emerging risk factors, such as dysfunction of glycaemic and/or lipid metabolism, of sex hormones and of gut microbiome, is of paramount importance. The relationships between sex hormones, gut microbiome, and host glycaemic and/or lipid metabolism are largely unknown even in the homoeostasis status. Yet this knowledge gap would be pivotal to pinpoint to key mechanisms that are likely to be disrupted in disease context. Methods and results: Here we present the Women4Health (W4H) cohort, a unique cohort comprising up to 300 healthy women followed up during a natural menstrual cycle, set up with the primary goal to investigate the combined role of sex hormones and gut microbiota variations in regulating host lipid and glucose metabolism during homoeostasis, using a multi-omics strategy. Additionally, the W4H cohort will take into consideration another ecosystem that is unique to women, the vaginal microbiome, investigating its interaction with gut microbiome and exploring-for the first time-its role in cardiometabolic disorders. Conclusion: The W4H cohort study lays a foundation for improving current knowledge of women-specific mechanisms in cardiometabolic regulation. It aspires to transform insights on host-microbiota interactions into prevention and therapeutic approaches for personalized health care.

Which Came First? When Usher Syndrome Type 1 Couples with Neuropsychiatric Disorders.

Usher syndrome (USH) is an autosomal recessive disorder characterized by sensorineural hearing loss (HL), retinopathy, and vestibular areflexia, with variable severity. Although a high prevalence of behavioural and mental disorders in USH patients has been reported, few studies on these psychiatric and psychological issues have been conducted. This work describes the case of a 16-year-old boy affected by congenital bilateral sensorineural HL, presenting a suddenly altered behaviour concomitant with a decrease in visual acuity. To establish a molecular diagnosis, Whole-Exome Sequencing analysis was performed, detecting a pathogenetic homozygous variant (c. 5985C>A, p.(Tyr1995*)) within the CDH23 gene. CDH23 is a known USH type 1 causative gene, recently associated with schizophrenia-like symptoms and bipolar disorders. To date, no studies have provided evidence of a direct genotype-phenotype correlation between USH patients carrying CDH23 variants and mental/behavioural issues; however, considering the multiple biological functions of CDH23, it can be hypothesised that it could have a pleiotropic effect. Overall, this study highlights the relevance of a continuous clinical evaluation of USH patients, to monitor not only the disease progression, but to early detect any psychological or behavioural alterations, thus allowing a rapid implementation of therapeutic strategies aimed at improving their quality of life and well-being.

Puzzling Out the Genetic Architecture of Endometriosis: Whole-Exome Sequencing and Novel Candidate Gene Identification in a Deeply Clinically Characterised Cohort.

Endometriosis (EM) is a common multifactorial gynaecological disorder. Although Genome-Wide Association Studies have largely been employed, the current knowledge of the genetic mechanisms underlying EM is far from complete, and other approaches are needed. To this purpose, whole-exome sequencing (WES) was performed on a deeply characterised cohort of 80 EM patients aimed at the identification of rare and damaging variants within 46 EM-associated genes and novel candidates. WES analysis detected 63 rare, predicted, and damaging heterozygous variants within 24 genes in 63% of the EM patients. In particular, (1) a total of 43% of patients carried variants within 13 recurrent genes (FCRL3, LAMA5, SYNE1, SYNE2, GREB1, MAP3K4, C3, MMP3, MMP9, TYK2, VEGFA, VEZT, RHOJ); (2) a total of 8.8% carried private variants within eight genes (KAZN, IL18, WT1, CYP19A1, IL1A, IL2RB, LILRB2, ZNF366); (3) a total of 24% carried variants within three novel candidates (ABCA13, NEB, CSMD1). Finally, to deepen the polygenic architecture of EM, a comprehensive evaluation of the analysed genes was performed, revealing a higher burden (p < 0.05) of genes harbouring rare and damaging variants in the EM patients than in the controls. These results highlight new insights into EM genetics, allowing for the definition of novel genotype-phenotype correlations, thereby contributing, in a long-term perspective, to the development of personalised care for EM patients.

The Enigmatic Genetic Landscape of Hereditary Hearing Loss: A Multistep Diagnostic Strategy in the Italian Population.

Hearing loss is the most frequent sensorineural disorder, affecting approximately 1:1000 newborns. Hereditary forms (HHL) represent 50-60% of cases, highlighting the relevance of genetic testing in deaf patients. HHL is classified as non-syndromic (NSHL-70% of cases) or syndromic (SHL-30% of cases). In this study, a multistep and integrative approach aimed at identifying the molecular cause of HHL in 102 patients, whose GJB2 analysis already showed a negative result, is described. In NSHL patients, multiplex ligation probe amplification and long-range PCR analyses of the STRC gene solved 13 cases, while whole exome sequencing (WES) identified the genetic diagnosis in 26 additional ones, with a total detection rate of 47.6%. Concerning SHL, WES detected the molecular cause in 55% of cases. Peculiar findings are represented by the identification of four subjects displaying a dual molecular diagnosis and eight affected by non-syndromic mimics, five of them presenting Usher syndrome type 2. Overall, this study provides a detailed characterisation of the genetic causes of HHL in the Italian population. Furthermore, we highlighted the frequency of Usher syndrome type 2 carriers in the Italian population to pave the way for a more effective implementation of diagnostic and follow-up strategies for this disease.

Whole-exome sequencing: Clinical characterization of pediatric and adult Italian patients affected by different forms of hereditary cardiovascular diseases.

BACKGROUND: Hereditary cardiovascular diseases comprise several different entities. In this study, we focused on cardiomyopathies (i.e., hypertrophic, dilated, arrhythmogenic, and left ventricular non-compaction), channelopathies (i.e., Brugada syndrome and long QT syndrome), and aortopathies and pulmonary arterial hypertension (i.e., thoracic/abdominal aortic aneurysm and pulmonary arterial hypertension), and genetically characterized 200 Italian patients affected by these diseases. METHODS: We employed whole-exome sequencing (WES), focused on four in silico gene panels, and the MLPA method for hypertrophic and arrhythmogenic right ventricular cardiomyopathy cases. RESULTS: Cardiomyopathies affected 87.5% of analyzed patients, channelopathies 7%, and aortopathies and pulmonary arterial hypertension 5.5%. The molecular diagnosis was confirmed for 21.5% of cases with a higher detection rate in familial forms (34%) than sporadic ones (14%). We highlighted the importance of family segregation to better understand the pathogenic role of the identified variants and their involvement in the clinical phenotype. Negative results could be ascribed to the high genetic and clinical heterogeneity of hereditary cardiovascular diseases; clinical follow-up and revaluation of WES data will be essential. CONCLUSION: This study highlights the importance of a multi-step approach (WES and MLPA) to characterize hereditary cardiovascular diseases, provides crucial information for clinical management and recurrence risk estimation, and lays the foundation for future personalized therapies.

Prognostic Prediction of Genotype vs Phenotype in Genetic Cardiomyopathies.

BACKGROUND: Diverse genetic backgrounds often lead to phenotypic heterogeneity in cardiomyopathies (CMPs). Previous genotype-phenotype studies have primarily focused on the analysis of a single phenotype, and the diagnostic and prognostic features of the CMP genotype across different phenotypic expressions remain poorly understood. OBJECTIVES: We sought to define differences in outcome prediction when stratifying patients based on phenotype at presentation compared with genotype in a large cohort of patients with CMPs and positive genetic testing. METHODS: Dilated cardiomyopathy (DCM), arrhythmogenic right ventricular cardiomyopathy, left-dominant arrhythmogenic cardiomyopathy, and biventricular arrhythmogenic cardiomyopathy were examined in this study. A total of 281 patients (80% DCM) with pathogenic or likely pathogenic variants were included. The primary and secondary outcomes were: 1) all-cause mortality (D)/heart transplant (HT); 2) sudden cardiac death/major ventricular arrhythmias (SCD/MVA); and 3) heart failure-related death (DHF)/HT/left ventricular assist device implantation (LVAD). RESULTS: Survival analysis revealed that SCD/MVA events occurred more frequently in patients without a DCM phenotype and in carriers of DSP, PKP2, LMNA, and FLNC variants. However, after adjustment for age and sex, genotype-based classification, but not phenotype-based classification, was predictive of SCD/MVA. LMNA showed the worst trends in terms of D/HT and DHF/HT/LVAD. CONCLUSIONS: Genotypes were associated with significant phenotypic heterogeneity in genetic cardiomyopathies. Nevertheless, in our study, genotypic-based classification showed higher precision in predicting the outcome of patients with CMP than phenotype-based classification. These findings add to our current understanding of inherited CMPs and contribute to the risk stratification of patients with positive genetic testing.

[The Regional Registry of Sudden Cardiac Death of Friuli Venezia Giulia. Protocols, best practices and results of a multidisciplinary project]. / Il Registro Regionale delle Morti Cardiache Improvvise in età giovanile del Friuli Venezia Giulia. Protocolli operativi e risultati di un progetto multidisciplinare.

With the regional law n. 26 of December 30, 2020, the Friuli Venezia Giulia Region wanted to promote the establishment of the Regional Register of Sudden Cardiac Death, with the aim of favoring the study of all those deaths that occurred suddenly and unexpectedly under the age of 50 years in which it is not possible to trace the cause of death with certainty. Such dramatic events, difficult to quantify considering the complexity of data collection, are often accepted with resignation without any further investigation of the possible causes. The Regional Register of Sudden Cardiac Deaths of Friuli Venezia Giulia was born from this premise and from the awareness of the importance of going back with a rigorous scientific methodology and through a multidisciplinary approach, to the diagnosis of hereditary heart diseases which, when determined, allow the enrollment of relatives in a cardiological screening process and, therefore, primary prevention of potentially fatal events. The authors describe the operating procedures feeding the Regional Register and present the results of the first year of activity on 26 cases.

Long QT syndrome and left ventricular non-compaction in a family with KCNH2 mutation: A case report.

Background: Left ventricular non-compaction (LVNC) is an abnormality of the myocardium, characterized by prominent left ventricular trabeculae and deep inter-trabecular recesses. Long QT syndrome (LQTS) is a cardiac ion channelopathy presenting with a prolonged QT interval on resting electrocardiogram and is associated with increased susceptibility to sudden death. The association between LVNC and LQTS is uncommon. Case presentation: We report an Italian family with a novel pathogenic KCNH2 variant who presented with clinical features of LVNC and LQTS. The proband came to our attention after two syncopal episodes without prodromal symptoms. His ECG showed QTc prolongation and deep T wave inversion in anterior leads, and the echocardiogram fulfilled LVNC criteria. After that, also his sister was found to have LQTS and LVNC, while his father only presented LQTS. Conclusions: Physicians should be aware of the possible association between LVNC and LQTS. Even if this association is rare, patients with LVNC should be investigated for LQTS to prevent possible severe or even life-threatening arrhythmic episodes.

Pendred Syndrome, or Not Pendred Syndrome? That Is the Question.

Pendred syndrome (PDS) is the most common form of syndromic Hearing Loss (HL), characterized by sensorineural HL, inner ear malformations, and goiter, with or without hypothyroidism. SLC26A4 is the major gene involved, even though ~50% of the patients carry only one pathogenic mutation. This study aims to define the molecular diagnosis for a cohort of 24 suspected-PDS patients characterized by a deep radiological and audiological evaluation. Whole-Exome Sequencing (WES), the analysis of twelve variants upstream of SLC26A4, constituting the "CEVA haplotype" and Multiplex Ligation Probe Amplification (MLPA) searching for deletions/duplications in SLC26A4 gene have been carried out. In five patients (20.8%) homozygous/compound heterozygous SLC26A4 mutations, or pathogenic mutation in trans with the CEVA haplotype have been identified, while five subjects (20.8%) resulted heterozygous for a single variant. In silico protein modeling supported the pathogenicity of the detected variants, suggesting an effect on the protein stabilization/function. Interestingly, we identified a genotype-phenotype correlation among those patients carrying SLC26A4 mutations, whose audiograms presented a characteristic slope at the medium and high frequencies, providing new insights into PDS. Finally, an interesting homozygous variant in MYO5C has been identified in one patient negative to SLC26A4 gene, suggesting the identification of a new HL candidate gene.

Lights and Shadows in the Genetics of Syndromic and Non-Syndromic Hearing Loss in the Italian Population.

Hearing loss (HL), both syndromic (SHL) and non-syndromic (NSHL), is the most common sensory disorder, affecting ~460 million people worldwide. More than 50% of the congenital/childhood cases are attributable to genetic causes, highlighting the importance of genetic testing in this class of disorders. Here we applied a multi-step strategy for the molecular diagnosis of HL in 125 patients, which included: (1) an accurate clinical evaluation, (2) the analysis of GJB2, GJB6, and MT-RNR1 genes, (3) the evaluation STRC-CATSPER2 and OTOA deletions via Multiplex Ligation Probe Amplification (MLPA), (4) Whole Exome Sequencing (WES) in patients negative to steps 2 and 3. Our approach led to the characterization of 50% of the NSHL cases, confirming both the relevant role of the GJB2 (20% of cases) and STRC deletions (6% of cases), and the high genetic heterogeneity of NSHL. Moreover, due to the genetic findings, 4% of apparent NSHL patients have been re-diagnosed as SHL. Finally, WES characterized 86% of SHL patients, supporting the role of already know disease-genes. Overall, our approach proved to be efficient in identifying the molecular cause of HL, providing essential information for the patients' future management.

Next generation sequencing study in a cohort of Italian patients with syndromic hearing loss.

Hearing loss (HL), one of the most common congenital disorder, affects about one child in 1000. Among the genetic forms of HL, ∼30% of the cases are associated with other signs or symptoms, leading to Syndromic Hearing Loss (SHL) with about 700 different forms described so far. In this report, we refer the clinical and molecular data of 38 Italian SHL unrelated patients, and their relatives, affected by the most common syndromes associated with HL (i.e., Usher, Pendred, Charge, Waardenburg, Alport, Stickler, Branchiootorenal and Microdeletions syndromes). Patients have been analysed using next-generation sequencing (NGS) and High Density (HD)-SNP array technologies. Data analysis led to the identification of nine novel and 27 known causative mutations in 12 genes and two microdeletions in chromosomes 1 and 10, respectively. In particular, as regards to Usher syndrome, that affects 32% of our patients, we were able to reach a molecular diagnosis in 83% of the cases and to identify in Northern Eastern Italy a very common USH2A gene mutation (39%) (c.11864G > A, p.(Trp3955*) which can be defined "Central-Eastern European allele." As regards to Alport syndrome, we were able to potentially reclassify a pathogenic allele in the COL4A3 gene, previously associated only with benign familial hematuria. In all the other cases, the genomic analysis allowed us to confirm the role of known causative genes and to identify several novel and known alleles. Overall, our results highlight the effectiveness of combining an accurate clinical characterization with the use of genomic technologies (NGS and SNP arrays) for the molecular diagnosis of SHL, with a clear positive impact in the management and treatment of all the patients.

Genomic Studies in a Large Cohort of Hearing Impaired Italian Patients Revealed Several New Alleles, a Rare Case of Uniparental Disomy (UPD) and the Importance to Search for Copy Number Variations.

Hereditary hearing loss (HHL) is a common disorder characterized by a huge genetic heterogeneity. The definition of a correct molecular diagnosis is essential for proper genetic counseling, recurrence risk estimation, and therapeutic options. From 20 to 40% of patients carry mutations in GJB2 gene, thus, in more than half of cases it is necessary to look for causative variants in the other genes so far identified (~100). In this light, the use of next-generation sequencing technologies has proved to be the best solution for mutational screening, even though it is not always conclusive. Here we describe a combined approach, based on targeted re-sequencing (TRS) of 96 HHL genes followed by high-density SNP arrays, aimed at the identification of the molecular causes of non-syndromic HHL (NSHL). This strategy has been applied to study 103 Italian unrelated cases, negative for mutations in GJB2, and led to the characterization of 31% of them (i.e., 37% of familial and 26.3% of sporadic cases). In particular, TRS revealed TECTA and ACTG1 genes as major players in the Italian population. Furthermore, two de novo missense variants in ACTG1 have been identified and investigated through protein modeling and molecular dynamics simulations, confirming their likely pathogenic effect. Among the selected patients analyzed by SNP arrays (negative to TRS, or with a single variant in a recessive gene) a molecular diagnosis was reached in ~36% of cases, highlighting the importance to look for large insertions/deletions. Moreover, copy number variants analysis led to the identification of the first case of uniparental disomy involving LOXHD1 gene. Overall, taking into account the contribution of GJB2, plus the results from TRS and SNP arrays, it was possible to reach a molecular diagnosis in ~51% of NSHL cases. These data proved the usefulness of a combined approach for the analysis of NSHL and for the definition of the epidemiological picture of HHL in the Italian population.

Genome-Wide Meta-Analysis Unravels Interactions between Magnesium Homeostasis and Metabolic Phenotypes.

Magnesium (Mg2+) homeostasis is critical for metabolism. However, the genetic determinants of the renal handling of Mg2+, which is crucial for Mg2+ homeostasis, and the potential influence on metabolic traits in the general population are unknown. We obtained plasma and urine parameters from 9099 individuals from seven cohorts, and conducted a genome-wide meta-analysis of Mg2+ homeostasis. We identified two loci associated with urinary magnesium (uMg), rs3824347 (P=4.4×10-13) near TRPM6, which encodes an epithelial Mg2+ channel, and rs35929 (P=2.1×10-11), a variant of ARL15, which encodes a GTP-binding protein. Together, these loci account for 2.3% of the variation in 24-hour uMg excretion. In human kidney cells, ARL15 regulated TRPM6-mediated currents. In zebrafish, dietary Mg2+ regulated the expression of the highly conserved ARL15 ortholog arl15b, and arl15b knockdown resulted in renal Mg2+ wasting and metabolic disturbances. Finally, ARL15 rs35929 modified the association of uMg with fasting insulin and fat mass in a general population. In conclusion, this combined observational and experimental approach uncovered a gene-environment interaction linking Mg2+ deficiency to insulin resistance and obesity.

Common variants in CLDN14 are associated with differential excretion of magnesium over calcium in urine.

The nature and importance of genetic factors regulating the differential handling of Ca2+ and Mg2+ by the renal tubule in the general population are poorly defined. We conducted a genome-wide meta-analysis of urinary magnesium-to-calcium ratio to identify associated common genetic variants. We included 9320 adults of European descent from four genetic isolates and three urban cohorts. Urinary magnesium and calcium concentrations were measured centrally in spot urine, and each study conducted linear regression analysis of urinary magnesium-to-calcium ratio on ~2.5 million single-nucleotide polymorphisms (SNPs) using an additive model. We investigated, in mouse, the renal expression profile of the top candidate gene and its variation upon changes in dietary magnesium. The genome-wide analysis evidenced a top locus (rs172639, p = 1.7 × 10-12), encompassing CLDN14, the gene coding for claudin-14, that was genome-wide significant when using urinary magnesium-to-calcium ratio, but not either one taken separately. In mouse, claudin-14 is expressed in the distal nephron segments specifically handling magnesium, and its expression is regulated by chronic changes in dietary magnesium content. A genome-wide approach identified common variants in the CLDN14 gene exerting a robust influence on the differential excretion of Mg2+ over Ca2+ in urine. These data highlight the power of urinary electrolyte ratios to unravel genetic determinants of renal tubular function. Coupled with mouse experiments, these results support a major role for claudin-14, a gene associated with kidney stones, in the differential paracellular handling of divalent cations by the renal tubule.