Chromoanagenesis of chromosome 22 in a subject with obesity and borderline cognitive performance.

Chromoanagenesis events consist of complex chromosome rearrangements with multiple breakpoints in one or few chromosomes. Mechanisms of chromoanagenesis are split into three major groups: chromothripsis, chromoanasynthesis and chromoplexy. This study aims to delineate a chromoanagenesis event at the level of chromosome 22 in an individual showing obesity and borderline cognitive performance as major disturbances. The proband and his parents were subjected to conventional karyotyping, CGH array and whole genomic sequencing (WGS). By conventional karyotyping a "de novo" pericentric inversion of chromosome 22 was identified. CGH array identified several imbalances (either deletions or duplications) in the long arm of chromosome 22; the largest is a 4.5 Mb duplication at 22q12.1-22q1.3. The detection of extensive duplications would suggest the occurrence of a chromoanasynthesis event. WGS, in addition to the structural alterations identified by karyotyping and CGH array, revealed two translocations from chromosome 22 to chromosomes 6 and 21 as well as a heterozygous pathogenetic variant of ALMS1 gene; the latter could have contributed to the obesity of our patient. The pericentric inversion induces loss of initial part of TCF20 gene including the 5' regulatory region and the first, noncoding, exon. Heterozygous loss-of-function mutations of TCF20 gene have been found in patients with autism spectrum disorder or intellectual disability, some of them presenting obesity. It is, therefore, possible that disruption of TCF20 gene structure would contribute to a fraction of the patient's phenotype.

Genome-Wide Interaction Analyses of Serum Calcium on Ventricular Repolarization Time in 125 393 Participants.

BACKGROUND: Ventricular repolarization time (ECG QT and JT intervals) is associated with malignant arrhythmia. Genome-wide association studies have identified 230 independent loci for QT and JT; however, 50% of their heritability remains unexplained. Previous work supports a causal effect of lower serum calcium concentrations on longer ventricular repolarization time. We hypothesized calcium interactions with QT and JT variant associations could explain a proportion of the missing heritability. METHODS AND RESULTS: We performed genome-wide calcium interaction analyses for QT and JT intervals. Participants were stratified by their calcium level relative to the study distribution (top or bottom 20%). We performed a 2-stage analysis (genome-wide discovery [N=62 532] and replication [N=59 861] of lead variants) and a single-stage genome-wide meta-analysis (N=122 393, [European ancestry N=117 581, African ancestry N=4812]). We also calculated 2-degrees of freedom joint main and interaction and 1-degree of freedom interaction P values. In 2-stage and single-stage analyses, 50 and 98 independent loci, respectively, were associated with either QT or JT intervals (2-degrees of freedom joint main and interaction P value <5×10-8). No lead variant had a significant interaction result after correcting for multiple testing and sensitivity analyses provided similar findings. Two loci in the single-stage meta-analysis were not reported previously (SPPL2B and RFX6). CONCLUSIONS: We have found limited support for an interaction effect of serum calcium on QT and JT variant associations despite sample sizes with suitable power to detect relevant effects. Therefore, such effects are unlikely to explain a meaningful proportion of the heritability of QT and JT, and factors including rare variation and other environmental interactions need to be considered.

TAS2R38 Genotype Does Not Affect SARS-CoV-2 Infection in Primary Ciliary Dyskinesia.

Several chronic respiratory diseases could be risk factors for acquiring SARS-CoV-2 infection: among them, Primary Ciliary Dyskinesia (PCD) is a rare (about 1:10.000) inherited ciliopathy (MIM 242650) characterized by recurrent upper and lower respiratory tract infections due to a dysfunction of the respiratory cilia. In this study, we aimed to investigate whether PCD subjects are more susceptible to infection by SARS-CoV-2 and whether some polymorphisms of the TAS2R38 bitter taste receptor correlate with an increased prevalence of SARS-CoV-2 infection and severity of symptoms. Patients answered several questions about possible SARS-CoV-2 infection, experienced symptoms, and vaccinations; in the case of infection, they also filled out a SNOT-22 questionnaire and ARTIQ. Forty PCD adult patients (mean age, 36.6 ± 16.7 years; 23 females, 17 males) participated in this study, out of which 30% had tested positive for COVID-19 during the last four years; most of them reported a mildly symptomatic disease. We found no differences in age or sex, but a statistically significant difference (p = 0.03) was observed in body mass index (BMI), which was higher in the COVID-acquired group (23.2 ± 3.3 vs. 20.1 ± 4.1 kg/m2). Genotyping for TAS2R38 polymorphisms showed a prevalence of 28.6% PAV/PAV, 48.6% PAV/AVI, and 22.8% AVI/AVI individuals in our cohort. In contrast to our hypothesis, we did not observe a protective role of the PAV allele towards SARS-CoV-2 infection. Conclusions: Our findings suggest that subjects with PCD may not be at increased risk of severe outcomes from COVID-19 and the TAS2R38 bitter taste receptor genotype does not affect SARS-CoV-2 infection.

Proangiogenic properties of complement protein C1q can contribute to endometriosis.

Endometriosis (EM) is defined as the engraftment and proliferation of functional endometrial-like tissue outside the uterine cavity, leading to a chronic inflammatory condition. While the precise etiology of EM remains elusive, recent studies have highlighted the crucial involvement of a dysregulated immune system. The complement system is one of the predominantly altered immune pathways in EM. Owing to its involvement in the process of angiogenesis, here, we have examined the possible role of the first recognition molecule of the complement classical pathway, C1q. C1q plays seminal roles in several physiological and pathological processes independent of complement activation, including tumor growth, placentation, wound healing, and angiogenesis. Gene expression analysis using the publicly available data revealed that C1q is expressed at higher levels in EM lesions compared to their healthy counterparts. Immunohistochemical analysis confirmed the presence of C1q protein, being localized around the blood vessels in the EM lesions. CD68+ macrophages are the likely producer of C1q in the EM lesions since cultured EM cells did not produce C1q in vitro. To explore the underlying reasons for increased C1q expression in EM, we focused on its established pro-angiogenic role. Employing various angiogenesis assays on primary endothelial endometriotic cells, such as migration, proliferation, and tube formation assays, we observed a robust proangiogenic effect induced by C1q on endothelial cells in the context of EM. C1q promoted angiogenesis in endothelial cells isolated from EM lesions (as well as healthy ovary that is also rich in C1q). Interestingly, endothelial cells from EM lesions seem to overexpress the receptor for the globular heads of C1q (gC1qR), a putative C1q receptor. Experiments with siRNA to silence gC1qR resulted in diminished capacity of C1q to perform its angiogenic functions, suggesting that C1q is likely to engage gC1qR in the pathophysiology of EM. gC1qR can be a potential therapeutic target in EM patients that will disrupt C1q-mediated proangiogenic activities in EM.

Understanding the genetic complexity of puberty timing across the allele frequency spectrum.

Pubertal timing varies considerably and is associated with later health outcomes. We performed multi-ancestry genetic analyses on ~800,000 women, identifying 1,080 signals for age at menarche. Collectively, these explained 11% of trait variance in an independent sample. Women at the top and bottom 1% of polygenic risk exhibited ~11 and ~14-fold higher risks of delayed and precocious puberty, respectively. We identified several genes harboring rare loss-of-function variants in ~200,000 women, including variants in ZNF483, which abolished the impact of polygenic risk. Variant-to-gene mapping approaches and mouse gonadotropin-releasing hormone neuron RNA sequencing implicated 665 genes, including an uncharacterized G-protein-coupled receptor, GPR83, which amplified the signaling of MC3R, a key nutritional sensor. Shared signals with menopause timing at genes involved in DNA damage response suggest that the ovarian reserve might signal centrally to trigger puberty. We also highlight body size-dependent and independent mechanisms that potentially link reproductive timing to later life disease.

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.

Clenching the Strings of Bruxism Etiopathogenesis: Association Analyses on Genetics and Environmental Risk Factors in a Deeply Characterized Italian Cohort.

Bruxism is a worldwide oral health problem. Although there is a consensus about its multifactorial nature, its precise etiopathogenetic mechanisms are unclear. This study, taking advantage of a deeply characterized cohort of 769 individuals (aged 6-89 years) coming from Northern Italy's genetically isolated populations, aims to epidemiologically describe environmental risk factors for bruxism development and identify genes potentially involved through a Genome-Wide Association Study (GWAS) approach. Logistic mixed models adjusted for age and sex were performed to evaluate associations between bruxism and possible risk factors, e.g., anxiety, smoking, and alcohol and caffeine intake. A case-control GWAS (135 cases, 523 controls), adjusted for age, sex, and anxiety, was conducted to identify new candidate genes. The GTEx data analysis was performed to evaluate the identified gene expression in human body tissues. Statistical analyses determined anxiety as a bruxism risk factor (OR = 2.54; 95% CI: 1.20-5.38; p-value = 0.015), and GWAS highlighted three novel genes potentially associated with bruxism: NLGN1 (topSNP = rs2046718; p-value = 2.63 × 10-7), RIMBP2 (topSNP = rs571497947; p-value = 4.68 × 10-7), and LHFP (topSNP = rs2324342; p-value = 7.47 × 10-6). The GTEx data analysis showed their expression in brain tissues. Overall, this work provided a deeper understanding of bruxism etiopathogenesis with the long-term perspective of developing personalized therapeutic approaches for improving affected individuals' quality of life.

The Bittersweet Symphony of COVID-19: Associations between TAS1Rs and TAS2R38 Genetic Variations and COVID-19 Symptoms.

The innate immune system is crucial in fighting SARS-CoV-2 infection, which is responsible for coronavirus disease 2019 (COVID-19). Therefore, deepening our understanding of the underlying immune response mechanisms is fundamental for the development of novel therapeutic strategies. The role of extra-oral bitter (TAS2Rs) and sweet (TAS1Rs) taste receptors in immune response regulation has yet to be fully understood. However, a few studies have investigated the association between taste receptor genes and COVID-19 symptom severity, with controversial results. Therefore, this study aims to deepen the relationship between COVID-19 symptom presence/severity and TAS1R and TAS2R38 (TAS2Rs member) genetic variations in a cohort of 196 COVID-19 patients. Statistical analyses detected significant associations between rs307355 of the TAS1R3 gene and the following COVID-19-related symptoms: chest pain and shortness of breath. Specifically, homozygous C/C patients are exposed to an increased risk of manifesting severe forms of chest pain (OR 8.11, 95% CI 2.26-51.99) and shortness of breath (OR 4.83, 95% CI 1.71-17.32) in comparison with T/C carriers. Finally, no significant associations between the TAS2R38 haplotype and the presence/severity of COVID-19 symptoms were detected. This study, taking advantage of a clinically and genetically characterised cohort of COVID-19 patients, revealed TAS1R3 gene involvement in determining COVID-19 symptom severity independently of TAS2R38 activity, thus providing novel insights into the role of TAS1Rs in regulating the immune response to viral infections.

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.

Understanding the genetic complexity of puberty timing across the allele frequency spectrum.

Pubertal timing varies considerably and has been associated with a range of health outcomes in later life. To elucidate the underlying biological mechanisms, we performed multi-ancestry genetic analyses in ~800,000 women, identifying 1,080 independent signals associated with age at menarche. Collectively these loci explained 11% of the trait variance in an independent sample, with women at the top and bottom 1% of polygenic risk exhibiting a ~11 and ~14-fold higher risk of delayed and precocious pubertal development, respectively. These common variant analyses were supported by exome sequence analysis of ~220,000 women, identifying several genes, including rare loss of function variants in ZNF483 which abolished the impact of polygenic risk. Next, we implicated 660 genes in pubertal development using a combination of in silico variant-to-gene mapping approaches and integration with dynamic gene expression data from mouse embryonic GnRH neurons. This included an uncharacterized G-protein coupled receptor GPR83, which we demonstrate amplifies signaling of MC3R, a key sensor of nutritional status. Finally, we identified several genes, including ovary-expressed genes involved in DNA damage response that co-localize with signals associated with menopause timing, leading us to hypothesize that the ovarian reserve might signal centrally to trigger puberty. Collectively these findings extend our understanding of the biological complexity of puberty timing and highlight body size dependent and independent mechanisms that potentially link reproductive timing to later life disease.

Identifying missing pieces in color vision defects: a genome-wide association study in Silk Road populations.

Introduction: Color vision defects (CVDs) are conditions characterized by the alteration of normal trichromatic vision. CVDs can arise as the result of alterations in three genes (OPN1LW, OPN1MW, OPN1SW) or as a combination of genetic predisposition and environmental factors. To date, apart from Mendelian CVDs forms, nothing is known about multifactorial CVDs forms. Materials and Methods: Five hundred and twenty individuals from Silk Road isolated communities were genotyped and phenotypically characterized for CVDs using the Farnsworth D-15 color test. The CVDs traits Deutan-Protan (DP) and Tritan (TR) were analysed. Genome Wide Association Study for both traits was performed, and results were corrected with a False Discovery Rate linkage-based approach (FDR-p). Gene expression of final candidates was investigated using a published human eye dataset, and pathway analysis was performed. Results: Concerning DP, three genes: PIWIL4 (FDR-p: 9.01*10-9), MBD2 (FDR-p: 4.97*10-8) and NTN1 (FDR-p: 4.98*10-8), stood out as promising candidates. PIWIL4 is involved in the preservation of Retinal Pigmented Epithelium (RPE) homeostasis while MBD2 and NTN1 are both involved in visual signal transmission. With regards to TR, four genes: VPS54 (FDR-p: 4.09*10-9), IQGAP (FDR-p: 6,52*10-10), NMB (FDR-p: 8.34*10-11), and MC5R (FDR-p: 2.10*10-8), were considered promising candidates. VPS54 is reported to be associated with Retinitis pigmentosa; IQGAP1 is reported to regulate choroidal vascularization in Age-Related Macular Degeneration; NMB is involved in RPE homeostasis regulation; MC5R is reported to regulate lacrimal gland function. Discussion: Overall, these results provide novel insights regarding a complex phenotype (i.e., CVDs) in an underrepresented population such as Silk Road isolated communities.

Genetic Evaluation of Prelingual Hearing Impairment: Recommendations of an European Network for Genetic Hearing Impairment.

The cause of childhood hearing impairment (excluding infectious pathology of the middle ear) can be extrinsic (embryofoetopathy, meningitis, trauma, drug ototoxicity, noise trauma, etc [...].

Odontostomatological Traits in North-Eastern Italy's Isolated Populations: An Epidemiological Cross-Sectional Study.

Malocclusions and temporomandibular disorders (TMDs) are oral health problems that are spread worldwide. To date, few studies focused on their prevalence and associated risk factors are available. This study aims to define the prevalence and distribution of odontostomatological traits and evaluate specific risk factors in isolated villages in north-eastern Italy, taking advantage of their environmental homogeneity. Nine hundred and forty-four participants aged six to eighty-nine years were enrolled. Thirty-one odontostomatological phenotypes, classified into five domains (airways, bad habits, extraoral and intraoral parameters, TMDs, and teeth), were evaluated. A descriptive statistical analysis was performed; mixed logistic models were used to test the relationships among the traits. According to the study's findings, Angle's class I was prevalent (65.3%) followed by class II malocclusion (24.3%); class III and reversed overjet were the least frequent malocclusions (10.4% and 1.8%, respectively). Temporomandibular joint (TMJ) click/noise was prevalent among TMDs (34.7%). The statistically significant (p-value < 0.05) risk factors were ankyloglossia for phonetic issues (OR 1.90) and bruxism for TMJ click/noise (OR 1.70) and pain (OR 2.20). Overall, this work provides a picture of the prevalence of malocclusions and TMDs in a large Italian sample and reveals risk factors to take into account in the development of preventive strategies and treatments.

Genome-wide analysis identifies genetic effects on reproductive success and ongoing natural selection at the FADS locus.

Identifying genetic determinants of reproductive success may highlight mechanisms underlying fertility and identify alleles under present-day selection. Using data in 785,604 individuals of European ancestry, we identified 43 genomic loci associated with either number of children ever born (NEB) or childlessness. These loci span diverse aspects of reproductive biology, including puberty timing, age at first birth, sex hormone regulation, endometriosis and age at menopause. Missense variants in ARHGAP27 were associated with higher NEB but shorter reproductive lifespan, suggesting a trade-off at this locus between reproductive ageing and intensity. Other genes implicated by coding variants include PIK3IP1, ZFP82 and LRP4, and our results suggest a new role for the melanocortin 1 receptor (MC1R) in reproductive biology. As NEB is one component of evolutionary fitness, our identified associations indicate loci under present-day natural selection. Integration with data from historical selection scans highlighted an allele in the FADS1/2 gene locus that has been under selection for thousands of years and remains so today. Collectively, our findings demonstrate that a broad range of biological mechanisms contribute to reproductive success.

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.

Haploinsufficiency as a Foreground Pathomechanism of Poirer-Bienvenu Syndrome and Novel Insights Underlying the Phenotypic Continuum of CSNK2B-Associated Disorders.

CSNK2B encodes for the regulatory subunit of the casein kinase II, a serine/threonine kinase that is highly expressed in the brain and implicated in development, neuritogenesis, synaptic transmission and plasticity. De novo variants in this gene have been identified as the cause of the Poirier-Bienvenu Neurodevelopmental Syndrome (POBINDS) characterized by seizures and variably impaired intellectual development. More than sixty mutations have been described so far. However, data clarifying their functional impact and the possible pathomechanism are still scarce. Recently, a subset of CSNK2B missense variants affecting the Asp32 in the KEN box-like domain were proposed as the cause of a new intellectual disability-craniodigital syndrome (IDCS). In this study, we combined predictive functional and structural analysis and in vitro experiments to investigate the effect of two CSNK2B mutations, p.Leu39Arg and p.Met132LeufsTer110, identified by WES in two children with POBINDS. Our data prove that loss of the CK2beta protein, due to the instability of mutant CSNK2B mRNA and protein, resulting in a reduced amount of CK2 complex and affecting its kinase activity, may underlie the POBINDS phenotype. In addition, the deep reverse phenotyping of the patient carrying p.Leu39Arg, with an analysis of the available literature for individuals with either POBINDS or IDCS and a mutation in the KEN box-like motif, might suggest the existence of a continuous spectrum of CSNK2B-associated phenotypes rather than a sharp distinction between them.

Whole genome sequencing for USH2A-associated disease reveals several pathogenic deep-intronic variants that are amenable to splice correction.

A significant number of individuals with a rare disorder such as Usher syndrome (USH) and (non-)syndromic autosomal recessive retinitis pigmentosa (arRP) remain genetically unexplained. Therefore, we assessed subjects suspected of USH2A-associated disease and no or mono-allelic USH2A variants using whole genome sequencing (WGS) followed by an improved pipeline for variant interpretation to provide a conclusive diagnosis. One hundred subjects were screened using WGS to identify causative variants in USH2A or other USH/arRP-associated genes. In addition to the existing variant interpretation pipeline, a particular focus was put on assessing splice-affecting properties of variants, both in silico and in vitro. Also structural variants were extensively addressed. For variants resulting in pseudoexon inclusion, we designed and evaluated antisense oligonucleotides (AONs) using minigene splice assays and patient-derived photoreceptor precursor cells. Biallelic variants were identified in 49 of 100 subjects, including novel splice-affecting variants and structural variants, in USH2A or arRP/USH-associated genes. Thirteen variants were shown to affect USH2A pre-mRNA splicing, including four deep-intronic USH2A variants resulting in pseudoexon inclusion, which could be corrected upon AON treatment. We have shown that WGS, combined with a thorough variant interpretation pipeline focused on assessing pre-mRNA splicing defects and structural variants, is a powerful method to provide subjects with a rare genetic condition, a (likely) conclusive genetic diagnosis. This is essential for the development of future personalized treatments and for patients to be eligible for such treatments.