Myotonia congenita in a Greek cohort: Genotype spectrum and impact of the CLCN1:c.501C > G variant as a genetic modifier.

INTRODUCTION/AIMS: Myotonia congenita (MC) is the most common hereditary channelopathy in humans. Characterized by muscle stiffness, MC may be transmitted as either an autosomal dominant (Thomsen) or a recessive (Becker) disorder. MC is caused by variants in the voltage-gated chloride channel 1 (CLCN1) gene, important for the normal repolarization of the muscle action potential. More than 250 disease-causing variants in the CLCN1 gene have been reported. This study provides an MC genotype-phenotype spectrum in a large cohort of Greek patients and focuses on novel variants and disease epidemiology, including additional insights for the variant CLCN1:c.501C > G. METHODS: Sanger sequencing for the entire coding region of the CLCN1 gene was performed. Targeted segregation analysis of likely candidate variants in additional family members was performed. Variant classification was based on American College of Medical Genetics (ACMG) guidelines. RESULTS: Sixty-one patients from 47 unrelated families were identified, consisting of 51 probands with Becker MC (84%) and 10 with Thomsen MC (16%). Among the different variants detected, 11 were novel and 16 were previously reported. The three most prevalent variants were c.501C > G, c.2680C > T, and c.1649C > G. Additionally, c.501C > G was detected in seven Becker cases in-cis with the c.1649C > G. DISCUSSION: The large number of patients in whom a diagnosis was established allowed the characterization of genotype-phenotype correlations with respect to both previously reported and novel findings. For the c.501C > G (p.Phe167Leu) variant a likely nonpathogenic property is suggested, as it only seems to act as an aggravating modifying factor in cases in which a pathogenic variant triggers phenotypic expression.

Phenotype-driven variant filtration strategy in exome sequencing toward a high diagnostic yield and identification of 85 novel variants in 400 patients with rare Mendelian disorders.

About 6000 to 7000 different rare disorders with suspected genetic etiologies have been described and almost 4500 causative gene(s) have been identified. The advent of next-generation sequencing (NGS) technologies has revolutionized genomic research and diagnostics, representing a major advance in the identification of pathogenic genetic variations. This study presents a 3-year experience from an academic genetics center, where 400 patients were referred for genetic analysis of disorders with unknown etiology. A phenotype-driven proband-only exome sequencing (ES) strategy was applied for the investigation of rare disorders, in the context of optimizing ES diagnostic yield and minimizing costs and time to definitive diagnosis. Overall molecular diagnostic yield reached 53% and characterized 243 pathogenic variants in 210 cases, 85 of which were novel and 148 known, contributing information to the community of disease and variant databases. ES provides an opportunity to resolve the genetic etiology of disorders and support appropriate medical management and genetic counseling. In cases with complex phenotypes, the identification of complex genotypes may contribute to more comprehensive clinical management. In the context of effective multidisciplinary collaboration between clinicians and laboratories, ES provides an efficient and appropriate tool for first-tier genomic analysis.

Single amino acid loss in the dystrophin protein associated with a mild clinical phenotype.

INTRODUCTION: The dystrophinopathies include a spectrum of muscle diseases caused by mutations in the dystrophin (DMD) gene. The clinical phenotype ranges from severe Duchenne muscular dystrophy to a mild phenotype with elevated creatine kinase (CK). METHODS: Clinical and molecular assessment of 7 patients carrying a single amino acid loss in the dystrophin protein (p.His1690del) caused by a c.5068_5070delCAC tri-nucleotide deletion in exon 36 of the DMD gene. RESULTS: All patients were asymptomatic or oligosymptomatic and had elevated CK levels. Febrile illness, but not exercise, induced muscle symptoms in some patients. None had evidence of cardiomyopathy. Analysis of the short tandem repeat (STR)45 locus and sequencing of exon 36 of the DMD gene indicates that c.5068_5070delCAC is a founder mutation. CONCLUSIONS: The c.5068_5070delCAC locus in the DMD gene is associated with a very mild phenotype. Further study is needed to evaluate disease progression in these patients. Muscle Nerve 55: 46-50, 2017.

A dynamic trinucleotide repeat (TNR) expansion in the DMD gene.

Dystrophinopathies are allelic X-linked myopathies caused by large deletions/duplications or small lesions along the DMD gene. An unexpected dynamic trinucleotide (GAA) expansion, ranging from ∼59 to 82 pure GAA repeats, within the DMD intron 62, was revealed to segregate through three family generations. From the pedigree, two female patients were referred for DMD investigation due to chronic myopathy and a muscle biopsy compatible with dystrophinopathy. As the size of the GAA repeat is limited to 11-33 within the general population our findings may provide a novel insight towards a Trinucleotide Repeat Expansion. Whether this TNR has an impact on the reported phenotype remains to be resolved.

Germline CNV Detection through Whole-Exome Sequencing (WES) Data Analysis Enhances Resolution of Rare Genetic Diseases.

Whole-Exome Sequencing (WES) has proven valuable in the characterization of underlying genetic defects in most rare diseases (RDs). Copy Number Variants (CNVs) were initially thought to escape detection. Recent technological advances enabled CNV calling from WES data with the use of accurate and highly sensitive bioinformatic tools. Amongst 920 patients referred for WES, 454 unresolved cases were further analysed using the ExomeDepth algorithm. CNVs were called, evaluated and categorized according to ACMG/ClinGen recommendations. Causative CNVs were identified in 40 patients, increasing the diagnostic yield of WES from 50.7% (466/920) to 55% (506/920). Twenty-two CNVs were available for validation and were all confirmed; of these, five were novel. Implementation of the ExomeDepth tool promoted effective identification of phenotype-relevant and/or novel CNVs. Among the advantages of calling CNVs from WES data, characterization of complex genotypes comprising both CNVs and SNVs minimizes cost and time to final diagnosis, while allowing differentiation between true or false homozygosity, as well as compound heterozygosity of variants in AR genes. The use of a specific algorithm for calling CNVs from WES data enables ancillary detection of different types of causative genetic variants, making WES a critical first-tier diagnostic test for patients with RDs.

Retrospective analysis of persistent HyperCKemia with or without muscle weakness in a case series from Greece highlights vast DMD variant heterogeneity.

BACKGROUND: Persistent hyperCKemia results from muscle dysfunction often attributed to genetic alterations of muscle-related genes, such as the dystrophin gene (DMD). Retrospective assessment of findings from DMD analysis, in association with persistent HyperCKemia, was conducted. PATIENTS AND METHODS: Evaluation of medical records from 1354 unrelated cases referred during the period 1996-2021. Assessment of data concerning the detection of DMD gene rearrangements and nucleotide variants. RESULTS: A total of 730 individuals (657 cases, 569 of Greek and 88 of Albanian origins) were identified, allowing an overall estimation of dystrophinopathy incidence at ~1:3800 live male births. The heterogeneous spectrum of 275 distinct DMD alterations comprised exon(s) deletions/duplications, nucleotide variants, and rare events, such as chromosome translocation {t(X;20)}, contiguous gene deletions, and a fused gene involving the DMD and the DOCK8 genes. Ethnic-specific findings include a common founder variant in exon 36 ('Hellenic' variant). CONCLUSIONS: Some 50% of hyperCKemia cases were characterized as dystrophinopathies, highlighting that DMD variants may be considered the most common cause of hyperCKemia in Greece. Delineation of the broad genetic and clinical heterogeneity is fundamental for actionable public health decisions and theragnosis, as well as the establishment of guidelines addressing ethical considerations, especially related to the mild asymptomatic patient subgroup.

Combined exome analysis and exome depth assessment achieve a high diagnostic yield in an epilepsy case series, revealing significant genomic heterogeneity and novel mechanisms.

OBJECTIVES: Genetics of epilepsy are highly heterogeneous and complex. Lesions detected involve genes encoding various types of channels, transcription factors, and other proteins implicated in numerous cellular processes, such as synaptogenesis. Consequently, a wide spectrum of clinical presentations and overlapping phenotypes hinders differential diagnosis and highlights the need for molecular investigations toward delineation of underlying mechanisms and final diagnosis. Characterization of defects may also contribute valuable data on genetic landscapes and networks implicated in epileptogenesis. METHODS: This study reports on genetic findings from exome sequencing (ES) data of 107 patients with variable types of seizures, with or without additional symptoms, in the context of neurodevelopmental disorders. RESULTS: Multidisciplinary evaluation of ES, including ancillary detection of copy number variants (CNVs) with the ExomeDepth tool, supported a definite diagnosis in 59.8% of the patients, reflecting one of the highest diagnostic yields in epilepsy. CONCLUSION: Emerging advances of next-generation technologies and 'in silico' analysis tools offer the possibility to simultaneously detect several types of variations. Wide assessment of variable findings, specifically those found to be novel and least expected, reflects the ever-evolving genetic landscape of seizure development, potentially beneficial for increased opportunities for trial recruitment and enrollment, and optimized, even personalized, medical management.

Orofacial Muscle Weakening in Facioscapulohumeral Muscular Dystrophy (FSHD) Patients.

BACKGROUND: Facioscapulohumeral muscular dystrophy is the third most commonly found type of muscular dystrophy. The aim of this study was to correlate the D4Z4 repeat array fragment size to the orofacial muscle weakening exhibited in a group of patients with a genetically supported diagnosis of FSHD. METHODS: Molecular genetic analysis was performed for 52 patients (27 female and 25 male) from a group that consisted of 36 patients with autosomal dominant pedigrees and 16 patients with either sporadic or unknown family status. The patients were tested with the southern blotting technique, using EcoRI/Avrll double digestion, and fragments were detected by a p13E-11 telomeric probe. Spearman's correlation was used to compare the fragment size with the degree of muscle weakening found in the forehead, periocular and perioral muscles. RESULTS: A positive non-significant correlation between the DNA fragment size and severity of muscle weakness was found for the forehead (r = 0.27; p = 0187), the periocular (r = 0.24; p = 0.232) and the left and right perioral (r = 0.29; p = 0.122), (r = 0.32; p = 0.085) muscles. CONCLUSIONS: Although FSHD patients exhibited a decrease in muscular activity related to the forehead, perioral, and periocular muscles the genotype-phenotype associations confirmed a weak to moderate non-significant correlation between repeat size and the severity of muscle weakness. Orofacial muscle weakening and its association with a D4Z4 contraction alone may not have the significance to serve as a prognostic biomarker, due to the weak to moderate association. Further studies with larger sample sizes are needed to determine the degree of genetic involvement in the facial growth in FSHD patients.

Evaluation of Genotypes and Epidemiology of Spinal Muscular Atrophy in Greece: A Nationwide Study Spanning 24 Years.

BACKGROUND: Promising genetic treatments targeting the molecular defect of severe early-onset genetic conditions are expected to dramatically improve patients' quality of life and disease epidemiology. Spinal Muscular Atrophy (SMA), is one of these conditions and approved therapeutic approaches have recently become available to patients. OBJECTIVE: Analysis of genetic and clinical data from SMA patients referred to the single public-sector provider of genetic services for the disease throughout Greece followed by a retrospective assessment in the context of epidemiology and genotype-phenotype associations. METHODS: Molecular genetic analysis and retrospective evaluation of findings for 361 patients tested positive for SMA- and 862 apparently healthy subjects from the general population. Spearman rank test and generalized linear models were applied to evaluate secondary modifying factors with respect to their impact on clinical severity and age of onset. RESULTS: Causative variations- including 5 novel variants- were detected indicating a minimal incidence of about 1/12,000, and a prevalence of at least 1.5/100,000. For prognosis a minimal model pertaining disease onset before 18 months was proposed to include copy numbers of NAIP (OR = 9.9;95% CI, 4.7 to 21) and SMN2 (OR = 6.2;95% CI, 2.5-15.2) genes as well as gender (OR = 2.2;95% CI, 1.04 to 4.6). CONCLUSIONS: This long-term survey shares valuable information on the current status and practices for SMA diagnosis on a population basis and provides an important reference point for the future assessment of strategic advances towards disease prevention and health care planning.