Unravelling undiagnosed rare disease cases by HiFi long-read genome sequencing.

Solve-RD is a pan-European rare disease (RD) research program that aims to identify disease-causing genetic variants in previously undiagnosed RD families. We utilised 10-fold coverage HiFi long-read sequencing (LRS) for detecting causative structural variants (SVs), single nucleotide variants (SNVs), insertion-deletions (InDels), and short tandem repeat (STR) expansions in extensively studied RD families without clear molecular diagnoses. Our cohort includes 293 individuals from 114 genetically undiagnosed RD families selected by European Rare Disease Network (ERN) experts. Of these, 21 families were affected by so-called 'unsolvable' syndromes for which genetic causes remain unknown, and 93 families with at least one individual affected by a rare neurological, neuromuscular, or epilepsy disorder without genetic diagnosis despite extensive prior testing. Clinical interpretation and orthogonal validation of variants in known disease genes yielded thirteen novel genetic diagnoses due to de novo and rare inherited SNVs, InDels, SVs, and STR expansions. In an additional four families, we identified a candidate disease-causing SV affecting several genes including an MCF2 / FGF13 fusion and PSMA3 deletion. However, no common genetic cause was identified in any of the 'unsolvable' syndromes. Taken together, we found (likely) disease-causing genetic variants in 13.0% of previously unsolved families and additional candidate disease-causing SVs in another 4.3% of these families. In conclusion, our results demonstrate the added value of HiFi long-read genome sequencing in undiagnosed rare diseases.

Cardiac conduction disorders in young adults: Clinical characteristics and genetic background of an underestimated population.

BACKGROUND: Cardiac conduction disorder (CCD) in patients <50 years old is a rare and mostly unknown condition. OBJECTIVE: We aimed to assess clinical characteristics and genetic background of patients <50 years old with CCD of unknown origin. METHODS: We retrospectively reviewed a consecutive series of patients with a diagnosis of CCD before the age of 50 years referred to our center between January 2019 and December 2021. Patients underwent complete clinical examination and genetic evaluation. RESULTS: We enrolled 39 patients with a median age of 40 years (28-47 years) at the onset of symptoms. A cardiac implantable electronic device was implanted in 69% of the patients. In 15 of 39 CCD index patients (38%), we found a total of 13 different gene variations (3 pathogenic, 6 likely pathogenic, and 4 variants of uncertain significance), mostly in 3 genes (SCN5A, TRPM4, and LMNA). In our cohort, genetic testing led to the decision to implant an implantable cardioverter-defibrillator in 2 patients for the increased risk of sudden cardiac death. CONCLUSION: Patients with the occurrence of CCD before the age of 50 years present with a high rate of pathologic gene variations, mostly in 3 genes (SCN5A, TRPM4, and LMNA). The presence of pathogenic variations may add information about the prognosis and lead to an individualized therapeutic approach.

SCN5A mutation is associated with a higher Shanghai Score in patients with type 1 Brugada ECG pattern.

AIMS: Brugada syndrome (BrS) is an inherited arrhythmic disease characterized by a coved ST-segment elevation in the right precordial electrocardiogram leads (type 1 ECG pattern) and is associated with a risk of malignant ventricular arrhythmias and sudden cardiac death. In order to assess the predictive value of the Shanghai Score System for the presence of a SCN5A mutation in clinical practice, we studied a cohort of 125 patients with spontaneous or fever/drug-induced BrS type 1 ECG pattern, variably associated with symptoms and a positive family history. METHODS: The Shanghai Score System items were collected for each patient and PR and QRS complex intervals were measured. Patients were genotyped through a next-generation sequencing (NGS) custom panel for the presence of SCN5A mutations and the common SCN5A polymorphism (H558R). RESULTS: The total Shanghai Score was higher in SCN5A+ patients than in SCN5A- patients. The 81% of SCN5A+ patients and the 100% of patients with a SCN5A truncating variant exhibit a spontaneous type 1 ECG pattern. A significant increase in PR (P = 0.006) and QRS (P = 0.02) was detected in the SCN5A+ group. The presence of the common H558R polymorphism did not significantly correlate with any of the items of the Shanghai Score, nor with the total score of the system. CONCLUSION: Data from our study suggest the usefulness of Shanghai Score collection in clinical practice in order to maximize genetic test appropriateness. Our data further highlight SCN5A mutations as a cause of conduction impairment in BrS patients.

mRNA in situ hybridization exhibits unbalanced nuclear/cytoplasmic dystrophin transcript repartition in Duchenne myogenic cells and skeletal muscle biopsies.

To gain insight on dystrophin (DMD) gene transcription dynamics and spatial localization, we assayed the DMD mRNA amount and defined its compartmentalization in myoblasts, myotubes, and skeletal muscle biopsies of Duchenne muscular dystrophy (DMD) patients. Using droplet digital PCR, Real-time PCR, and RNAscope in situ hybridization, we showed that the DMD transcript amount is extremely reduced in both DMD patients' cells and muscle biopsies and that mutation-related differences occur. We also found that, compared to controls, DMD transcript is dramatically reduced in the cytoplasm, as up to 90% of it is localized in nuclei, preferentially at the perinuclear region. Using RNA/protein colocalization experiments, we showed that about 40% of nuclear DMD mRNA is localized in the nucleoli in both control and DMD myogenic cells. Our results clearly show that mutant DMD mRNA quantity is strongly reduced in the patients' myogenic cells and muscle biopsies. Furthermore, mutant DMD mRNA compartmentalization is spatially unbalanced due to a shift in its localization towards the nuclei. This abnormal transcript repartition contributes to the poor abundance and availability of the dystrophin messenger in cytoplasm. This novel finding also has important repercussions for RNA-targeted therapies.

Brugada Syndrome: More than a Monogenic Channelopathy.

Brugada syndrome (BrS) is an inherited cardiac channelopathy first diagnosed in 1992 but still considered a challenging disease in terms of diagnosis, arrhythmia risk prediction, pathophysiology and management. Despite about 20% of individuals carrying pathogenic variants in the SCN5A gene, the identification of a polygenic origin for BrS and the potential role of common genetic variants provide the basis for applying polygenic risk scores for individual risk prediction. The pathophysiological mechanisms are still unclear, and the initial thinking of this syndrome as a primary electrical disease is evolving towards a partly structural disease. This review focuses on the main scientific advancements in the identification of biomarkers for diagnosis, risk stratification, pathophysiology and therapy of BrS. A comprehensive model that integrates clinical and genetic factors, comorbidities, age and gender, and perhaps environmental influences may provide the opportunity to enhance patients' quality of life and improve the therapeutic approach.

DMD deletions underlining mild dystrophinopathies: literature review highlights phenotype-related mutation clusters and provides insights about genetic mechanisms and prognosis.

DMD gene pathogenic variations cause a spectrum of phenotypes, ranging from severe Duchenne muscular dystrophy, the Becker milder cases, the intermediate or very mild muscle phenotypes invariably characterized by high CK, and the ultrarare fully-asymptomatic cases. Besides these phenotypes, X-linked dilated cardiomyopathy is also caused by DMD mutations. Males carrying DMD deletions with absent or very mild phenotypes have been sparsely described. We performed a horizon scan on public datasets to enroll males with the above phenotypes and carrying DMD deletions to delineate myopathic genotype-phenotype relationships. We inventoried 81 males, who were divided into the following clinical categorization: fully-asymptomatic males aged >43 years (A, N = 22); isolated hyperCKemia (CK, N = 35); and mild weakness (any age) with or without high CK (WCK, N = 24). In all cases, deleted intervals were exons 2 to 55, and no downstream exons were ever involved, apart from an exon 78 deletion in a WCK patient. All deletions were in-frame apart from the known exception to the rule of exon 2 and exon 78. We correlated the mild phenotypes (A and CK) to deleted exons, intronic breakpoints, exon-exon junctions, 3' isoforms rule, and protein epitopes, and we found that some genetic profiles are exclusively/mainly occurring in A/CK phenotypes, suggesting they are compatible with a quasi-normal muscular performance. We discussed diverse pathogenic mechanisms that may contribute to mild dystrophinopathic phenotypes, and we tried to address some "critical" genetic configurations or exon content needed to preserve a semi-functional DMD gene.

RNA-seq in DMD urinary stem cells recognized muscle-related transcription signatures and addressed the identification of atypical mutations by whole-genome sequencing.

Urinary stem cells (USCs) are a non-invasive, simple, and affordable cell source to study human diseases. Here we show that USCs are a versatile tool for studying Duchenne muscular dystrophy (DMD), since they are able to address RNA signatures and atypical mutation identification. Gene expression profiling of DMD individuals' USCs revealed a profound deregulation of inflammation, muscle development, and metabolic pathways that mirrors the known transcriptional landscape of DMD muscle and worsens following USCs' myogenic transformation. This pathogenic transcription signature was reverted by an exon-skipping corrective approach, suggesting the utility of USCs in monitoring DMD antisense therapy. The full DMD transcript profile performed in USCs from three undiagnosed DMD individuals addressed three splicing abnormalities, which were decrypted and confirmed as pathogenic variations by whole-genome sequencing (WGS). This combined genomic approach allowed the identification of three atypical and complex DMD mutations due to a deep intronic variation and two large inversions, respectively. All three mutations affect DMD gene splicing and cause a lack of dystrophin protein production, and one of these also generates unique fusion genes and transcripts. Further characterization of USCs using a novel cell-sorting technology (Celector) highlighted cell-type variability and the representation of cell-specific DMD isoforms. Our comprehensive approach to USCs unraveled RNA, DNA, and cell-specific features and demonstrated that USCs are a robust tool for studying and diagnosing DMD.

Urine-Derived Stem Cells Express 571 Neuromuscular Disorders Causing Genes, Making Them a Potential in vitro Model for Rare Genetic Diseases.

Background: Neuromuscular disorders (NMDs) are a heterogeneous group of genetic diseases, caused by mutations in genes involved in spinal cord, peripheral nerve, neuromuscular junction, and muscle functions. To advance the knowledge of the pathological mechanisms underlying NMDs and to eventually identify new potential drugs paving the way for personalized medicine, limitations regarding the availability of neuromuscular disease-related biological samples, rarely accessible from patients, are a major challenge. Aim: We characterized urinary stem cells (USCs) by in-depth transcriptome and protein profiling to evaluate whether this easily accessible source of patient-derived cells is suitable to study neuromuscular genetic diseases, focusing especially on those currently involved in clinical trials. Methods: The global transcriptomics of either native or MyoD transformed USCs obtained from control individuals was performed by RNA-seq. The expression of 610 genes belonging to 16 groups of disorders (http://www.musclegenetable.fr/) whose mutations cause neuromuscular diseases, was investigated on the RNA-seq output. In addition, protein expression of 11 genes related to NMDs including COL6A, EMD, LMNA, SMN, UBA1, DYNC1H1, SOD1, C9orf72, DYSF, DAG1, and HTT was analyzed in native USCs by immunofluorescence and/or Western blot (WB). Results: RNA-seq profile of control USCs shows that 571 out of 610 genes known to be involved in NMDs, are expressed in USCs. Interestingly, the expression levels of the majority of NMD genes remain unmodified following USCs MyoD transformation. Most genes involved in the pathogenesis of all 16 groups of NMDs are well represented except for channelopathies and malignant hyperthermia related genes. All tested proteins showed high expression values, suggesting consistency between transcription and protein representation in USCs. Conclusion: Our data suggest that USCs are human cells, obtainable by non-invasive means, which might be used as a patient-specific cell model to study neuromuscular disease-causing genes and that they can be likely adopted for a variety of in vitro functional studies such as mutation characterization, pathway identification, and drug screening.

Brugada ECG pattern in hypertrophic cardiomyopathy: Brugada phenocopy or overlapping syndrome?

The term phenocopy indicates a condition that imitates one produced by a gene and is also used for acquired Brugada-like ECG manifestations. Cases of Brugada phenocopies are increasingly reported in literature and an international registry is ongoing. We describe two patients with Hypertrophic Cardiomyopathy (HCM) and Brugada ECG pattern. Both patients carried the same pathogenic splicing mutation in MYBPC3 gene (responsible for HCM) while no genetic mutation associated with Brugada Syndrome was identified. To the best of our knowledge, Brugada ECG pattern has been rarely reported in patients with HCM.

Identification of a New Mutation in RSK2, the Gene for Coffin-Lowry Syndrome (CLS), in Two Related Patients with Mild and Atypical Phenotypes.

BACKGROUND: Coffin-Lowry syndrome (CLS) is a syndromic form of X-linked intellectual disability, in which specific associated facial, hand, and skeletal abnormalities are diagnostic features. METHODS: In the present study, an unreported missense genetic variant of the ribosomal S6 kinase 2 (RSK2) gene has been identified, by next-generation sequencing, in two related males with two different phenotypes of intellectual disability (ID) and peculiar facial dysmorphisms. We performed functional studies on this variant and another one, already reported in the literature, involving the same amino acid residue but, to date, without an efficient characterization. RESULTS: Our study demonstrated that the two variants involving residue 189 significantly impaired its kinase activity. CONCLUSIONS: We detected a loss-of-function RSK2 mutation with loss in kinase activity in a three-generation family with an X-linked ID.

Functional Characterization of Two Novel Mutations in SCN5A Associated with Brugada Syndrome Identified in Italian Patients.

BACKGROUND: Brugada syndrome (BrS) is an autosomal dominantly inherited cardiac disease characterized by "coved type" ST-segment elevation in the right precordial leads, high susceptibility to ventricular arrhythmia and a family history of sudden cardiac death. The SCN5A gene, encoding for the cardiac voltage-gated sodium channel Nav1.5, accounts for ~20-30% of BrS cases and is considered clinically relevant. METHODS: Here, we describe the clinical findings of two Italian families affected by BrS and provide the functional characterization of two novel SCN5A mutations, the missense variant Pro1310Leu and the in-frame insertion Gly1687_Ile1688insGlyArg. RESULTS: Despite being clinically different, both patients have a family history of sudden cardiac death and had history of arrhythmic events. The Pro1310Leu mutation significantly reduced peak sodium current density without affecting channel membrane localization. Changes in the gating properties of expressed Pro1310Leu channel likely account for the loss-of-function phenotype. On the other hand, Gly1687_Ile1688insGlyArg channel, identified in a female patient, yielded a nearly undetectable sodium current. Following mexiletine incubation, the Gly1687_Ile1688insGlyArg channel showed detectable, albeit very small, currents and biophysical properties similar to those of the Nav1.5 wild-type channel. CONCLUSIONS: Overall, our results suggest that the degree of loss-of-function shown by the two Nav1.5 mutant channels correlates with the aggressive clinical phenotype of the two probands. This genotype-phenotype correlation is fundamental to set out appropriate therapeutical intervention.

Methylation of SERPINA1 gene promoter may predict chronic obstructive pulmonary disease in patients affected by acute coronary syndrome.

BACKGROUND: Diagnostic biomarkers for detecting chronic obstructive pulmonary disease (COPD) in acute coronary syndrome (ACS) patients are not available. SERPINA1, coding for the most potent circulating anti-inflammatory protein in the lung, has been found to be differentially methylated in blood cells from COPD patients. This study aimed to investigate the methylation profile of SERPINA1 in blood cells from ACS patients, with (COPD+) or without COPD (COPD-). METHODS: Blood samples were from 115 ACS patients, including 30 COPD+ and 85 COPD- according to lung function phenotype, obtained with spirometry. DNA treated with sodium bisulfite was PCR-amplified at SERPINA1 promoter region. Methylation analysis was carried out by sequencing the PCR products. Lymphocytes count in ACS patients was recorded at hospital admission and discharge. RESULTS: SERPINA1 was hypermethylated in 24/30 (80%) COPD+ and 48/85 (56.5%) COPD- (p < 0.05). Interestingly, at hospital discharge, lymphocytes count was higher in COPD- patients carrying SERPINA1 hypermethylated (1.98 × 103 ± 0.6 cell/µl) than in COPD- carrying SERPINA1 hypomethylated (1.7 × 103 ± 0.48 cell/µl) (p < 0.05). CONCLUSIONS: SERPINA1 is hypermethylated in blood cells from COPD+ patients. COPD- carrying SERPINA1 hypermethylated and high lymphocytes count may be at risk of COPD development. Therefore, SERPINA1 hypermethylation may represent a potential biomarker for predicting COPD development in ACS patients.

Chitosan-Shelled Nanobubbles Irreversibly Encapsulate Morpholino Conjugate Antisense Oligonucleotides and Are Ineffective for Phosphorodiamidate Morpholino-Mediated Gene Silencing of DUX4.

Orphan drugs, including antisense oligonucleotides (AONs), siRNAs/miRNAs, Cas9 nuclease, and recombinant genes, have recently been made available for rare diseases. However, the main bottleneck for these new therapies is delivery. Drugs/synthetic genes need to reach the affected tissues with minimal off-target effects and immune reactions. AON molecules are currently delivered as backboned naked compounds or via viral vectors. Nanocarriers are considered promising vehicles, able to improve drug distribution by organ targeting and limiting safety issues. We tested perfluoropentane-based nanobubbles (NBs) as vehicles for loading phosphorodiamidate morpholino (PMO) AON to suppress DUX4 expression in a facioscapulohumeral muscular dystrophy cell model. In vitro cell-free analysis demonstrated a good loading capacity of PMO into NBs, while experiments in cell cultures showed lack of therapeutic effect since expression of DUX4 and its targets remained unmodified. We conclude that these types of chitosan-shelled NBs do not release PMO-AON and are therefore not ideal for PMO AON-related therapies.

Ethnicity-related DMD Genotype Landscapes in European and Non-European Countries.

OBJECTIVE: Genetic diagnosis and mutation identification are now compulsory for Duchenne (DMD) and Becker muscular dystrophies (BMD), which are due to dystrophin (DMD) gene mutations, either for disease prevention or personalized therapies. To evaluate the ethnic-related genetic assortments of DMD mutations, which may impact on DMD genetic diagnosis pipelines, we studied 328 patients with DMD and BMD from non-European countries. METHODS: We performed a full DMD mutation detection in 328 patients from 10 Eastern European countries (Poland, Hungary, Lithuania, Romania, Serbia, Croatia, Bosnia, Bulgaria, Ukraine, and Russia) and 2 non-European countries (Cyprus and Algeria). We used both conventional methods (multiplex ligation-dependent probe amplification [MLPA] followed by gene-specific sequencing) and whole-exome sequencing (WES) as a pivotal study ran in 28 patients where DMD mutations were already identified by standard techniques. WES output was also interrogated for DMD gene modifiers. RESULTS: We identified DMD gene mutations in 222 male patients. We identified a remarkable allele heterogeneity among different populations with a mutation landscape often country specific. We also showed that WES is effective for picking up all DMD deletions and small mutations and its adoption could allow a detection rate close to 90% of all occurring mutations. Gene modifiers haplotypes were identified with some ethnic-specific configurations. CONCLUSIONS: Our data provide unreported mutation landscapes in different countries, suggesting that ethnicity may orient genetic diagnosis flowchart, which can be adjusted depending on the mutation type frequency, with impact in drug eligibility.

SERPINA1 Gene Promoter Is Differentially Methylated in Peripheral Blood Mononuclear Cells of Pregnant Women.

SERine Protein INhibitor-A1 (SERPINA1) is an inducible blood cell gene coding for alpha1-antitrypsin (AAT), a plasma protease inhibitor whose circulating levels are raised during inflammation, infection and advanced pregnancy. DNA methylation has been suggested to play a role in SERPINA1 gene expression regulation in peripheral blood mononuclear cells (PBMCs). The methylation status of SERPINA1 in PBMCs is unknown. The aim of this study was to evaluate the methylation profile of the SERPINA1 promoter in PBMC. To this purpose PBMCs and serum were collected from healthy subjects (HS) (n = 75), including blood donors (BD) (n = 25), pregnant women at early pregnancy (EP) (n = 25), i.e., within the first trimester, and pregnant women at late pregnancy (LP) (n = 25), i.e., at the third trimester. DNA from PBMCs was treated with sodium bisulfite and PCR amplified for SERPINA1 gene promoter, followed by sequencing analyses. AAT serum levels were determined by ELISA test. SERPINA1 was found hypermethylated in 58.7% of HS. The prevalence of SERPINA1 hypermethylation was significantly higher in BD (68%) and EP (88%) than in LP (20%) (p < 0.01). The median serum AAT concentration was 1.07, 0.63, and 3.15 mg/ml in BD, EP, and LP, respectively (p < 0.05, BD and EP vs LP). This study indicates, for the first time, that SERPINA1 gene promoter is differentially methylated in PBMCs from HS. Likely, modulation of the methylation may be a novel epigenetic regulator mechanism of AAT expression in the PBMC of HS. Therefore, SERPINA1 gene promoter methylation may represent an epigenetic biomarker of PBMCs in healthy subjects.

Tumor Necrosis Factor Receptor SF10A (TNFRSF10A) SNPs Correlate With Corticosteroid Response in Duchenne Muscular Dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a rare and severe X-linked muscular dystrophy in which the standard of care with variable outcome, also due to different drug response, is chronic off-label treatment with corticosteroids (CS). In order to search for SNP biomarkers for corticosteroid responsiveness, we genotyped variants across 205 DMD-related genes in patients with differential response to steroid treatment. METHODS AND FINDINGS: We enrolled a total of 228 DMD patients with identified dystrophin mutations, 78 of these patients have been under corticosteroid treatment for at least 5 years. DMD patients were defined as high responders (HR) if they had maintained the ability to walk after 15 years of age and low responders (LR) for those who had lost ambulation before the age of 10 despite corticosteroid therapy. Based on interactome mapping, we prioritized 205 genes and sequenced them in 21 DMD patients (discovery cohort or DiC = 21). We identified 43 SNPs that discriminate between HR and LR. Discriminant Analysis of Principal Components (DAPC) prioritized 2 response-associated SNPs in the TNFRSF10A gene. Validation of this genotype was done in two additional larger cohorts composed of 46 DMD patients on corticosteroid therapy (validation cohorts or VaC1), and 150 non ambulant DMD patients and never treated with corticosteroids (VaC2). SNP analysis in all validation cohorts (N = 207) showed that the CT haplotype is significantly associated with HR DMDs confirming the discovery results. CONCLUSION: We have shown that TNFRSF10A CT haplotype correlates with corticosteroid response in DMD patients and propose it as an exploratory CS response biomarker.

POPDC2 a novel susceptibility gene for conduction disorders.

Despite recent progress in the understanding of cardiac ion channel function and its role in inherited forms of ventricular arrhythmias, the molecular basis of cardiac conduction disorders often remains unresolved. We aimed to elucidate the genetic background of familial atrioventricular block (AVB) using a whole exome sequencing (WES) approach. In monozygotic twins with a third-degree AVB and in another, unrelated family with first-degree AVB, we identified a heterozygous nonsense mutation in the POPDC2 gene causing a premature stop at position 188 (POPDC2W188⁎), deleting parts of its cAMP binding-domain. Popeye-domain containing (POPDC) proteins are predominantly expressed in the skeletal muscle and the heart, with particularly high expression of POPDC2 in the sinoatrial node of the mouse. We now show by quantitative PCR experiments that in the human heart the POPDC-modulated two-pore domain potassium (K2P) channel TREK-1 is preferentially expressed in the atrioventricular node. Co-expression studies in Xenopus oocytes revealed that POPDC2W188⁎ causes a loss-of-function with impaired TREK-1 modulation. Consistent with the high expression level of POPDC2 in the murine sinoatrial node, POPDC2W188⁎ knock-in mice displayed stress-induced sinus bradycardia and pauses, a phenotype that was previously also reported for POPDC2 and TREK-1 knock-out mice. We propose that the POPDC2W188⁎ loss-of-function mutation contributes to AVB pathogenesis by an aberrant modulation of TREK-1, highlighting that POPDC2 represents a novel arrhythmia gene for cardiac conduction disorders.

High Human Papillomavirus DNA loads in Inflammatory Middle Ear Diseases.

Background. Previous studies reported human papillomaviruses (HPVs) in middle ear tumors, whereas these viruses have been poorly investigated in chronic inflammatory middle ear diseases. We investigated HPVs in non-tumor middle ear diseases, including chronic otitis media (COM). Methods. COM specimens (n = 52), including chronic suppurative otitis media (CSOM) (n =38) and cholesteatoma (COMC) (n = 14), as well as normal middle ear (NME) specimens (n = 56) were analyzed. HPV sequences and DNA loads were analyzed by quantitative-PCR. HPV genotyping was performed by direct sequencing. Results. HPV DNA was detected in 23% (12/52) of COM and in 30.4% (17/56) of NME (p > 0.05). Specifically, HPV DNA sequences were found in 26.3% (10/38) of CSOM and in 14.3% (2/14) of COMC (p > 0.05). Interestingly, the HPV DNA load was higher in COMC (mean 7.47 copy/cell) than in CSOM (mean 1.02 copy/cell) and NME (mean 1.18 copy/cell) (P = 0.03 and P = 0.017 versus CSOM and NME, respectively). HPV16 and HPV18 were the main genotypes detected in COMC, CSOM and NME. Conclusions. These data suggest that HPV may infect the middle ear mucosa, whereas HPV-positive COMCs are associated with higher viral DNA loads as compared to NME.

The Genetic Landscape of Dystrophin Mutations in Italy: A Nationwide Study.

Dystrophinopathies are inherited diseases caused by mutations in the dystrophin (DMD) gene for which testing is mandatory for genetic diagnosis, reproductive choices and eligibility for personalized trials. We genotyped the DMD gene in our Italian cohort of 1902 patients (BMD n = 740, 39%; DMD n =1162, 61%) within a nationwide study involving 11 diagnostic centers in a 10-year window (2008-2017). In DMD patients, we found deletions in 57%, duplications in 11% and small mutations in 32%. In BMD, we found deletions in 78%, duplications in 9% and small mutations in 13%. In BMD, there are a higher number of deletions, and small mutations are more frequent than duplications. Among small mutations that are generally frequent in both phenotypes, 44% of DMD and 36% of BMD are nonsense, thus, eligible for stop codon read-through therapy; 63% of all out-of-frame deletions are eligible for single exon skipping. Patients were also assigned to Italian regions and showed interesting regional differences in mutation distribution. The full genetic characterization in this large, nationwide cohort has allowed us to draw several correlations between DMD/BMD genotype landscapes and mutation frequency, mutation types, mutation locations along the gene, exon/intron architecture, and relevant protein domain, with effects on population genetic characteristics and new personalized therapies.