Lethal Complications and Complex Genotypes in Shwachman Diamond Syndrome: Report of a Family with Recurrent Neonatal Deaths and a Case-Based Brief Review of the Literature.

Shwachman Diamond Syndrome (SDS) is a multi-system disease characterized by exocrine pancreatic insufficiency with malabsorption, infantile neutropenia and aplastic anemia. Life-threatening complications include progression to acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS), critical deep-tissue infections and asphyxiating thoracic dystrophy. In most patients, SDS results from biallelic pathogenic variants in the SBDS gene, different combinations of which contribute to heterogenous clinical presentations. Null variants are not well tolerated, supporting the theory that the loss of SBDS expression is likely lethal in both mice and humans. A novel complex genotype (SBDS:c.[242C>G;258+2T>C];[460-1G>A]/WFS1:c.[2327A>T];[1371G>T]) was detected in a family with recurrent neonatal deaths. A female neonate died three hours after birth with hemolytic anemia, and a male neonate with severe anemia, thrombocytopenia and neutropenia succumbed on day 40 after Staphylococcus epidermidis infection. A subsequent review of the literature focused on fatal complications, complex SBDS genotypes and/or unusual clinical presentations and disclosed rare cases, of which some had unexpected combinations of genetic and clinical findings. The impact of pathogenic variants and associated phenotypes is discussed in the context of data sharing towards expanding scientific expert networks, consolidating knowledge and advancing an understanding of novel underlying genotypes and complex phenotypes, facilitating informed clinical decisions and disease management.

SCN1A Channels a Wide Range of Epileptic Phenotypes: Report of Novel and Known Variants with Variable Presentations.

SCN1A, the gene encoding for the Nav1.1 channel, exhibits dominant interneuron-specific expression, whereby variants disrupting the channel's function affect the initiation and propagation of action potentials and neuronal excitability causing various types of epilepsy. Dravet syndrome (DS), the first described clinical presentation of SCN1A channelopathy, is characterized by severe myoclonic epilepsy in infancy (SMEI). Variants' characteristics and other genetic or epigenetic factors lead to extreme clinical heterogeneity, ranging from non-epileptic conditions to developmental and epileptic encephalopathy (DEE). This current study reports on findings from 343 patients referred by physicians in hospitals and tertiary care centers in Greece between 2017 and 2023. Positive family history for specific neurologic disorders was disclosed in 89 cases and the one common clinical feature was the onset of seizures, at a mean age of 17 months (range from birth to 15 years old). Most patients were specifically referred for SCN1A investigation (Sanger Sequencing and MLPA) and only five for next generation sequencing. Twenty-six SCN1A variants were detected, including nine novel causative variants (c.4567A>Τ, c.5564C>A, c.2176+2T>C, c.3646G>C, c.4331C>A, c.1130_1131delGAinsAC, c.1574_1580delCTGAGGA, c.4620A>G and c.5462A>C), and are herein presented, along with subsequent genotype-phenotype associations. The identification of novel variants complements SCN1A databases extending our expertise on genetic counseling and patient and family management including gene-based personalized interventions.

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.

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.

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.

A Greek National Cross-Sectional Study on Myotonic Dystrophies.

Myotonic Dystrophies (DM, Dystrophia Myotonia) are autosomal dominant inherited myopathies with a high prevalence across different ethnic regions. Despite some differences, mainly due to the pattern of muscle involvement and the age of onset, both forms, DM1 and DM2, share many clinical and genetic similarities. In this study, we retrospectively analyzed the medical record files of 561 Greek patients, 434 with DM1 and 127 with DM2 diagnosed in two large academic centers between 1994-2020. The mean age at onset of symptoms was 26.2 ± 15.3 years in DM1 versus 44.4 ± 17.0 years in DM2 patients, while the delay of diagnosis was 10 and 7 years for DM1 and DM2 patients, respectively. Muscle weakness was the first symptom in both types, while myotonia was more frequent in DM1 patients. Multisystemic involvement was detected in the great majority of patients, with cataracts being one of the most common extramuscular manifestations, even in the early stages of disease expression. In conclusion, the present work, despite some limitations arising from the retrospective collection of data, is the first record of a large number of Greek patients with myotonic dystrophy and emphasizes the need for specialized neuromuscular centers that can provide genetic counseling and a multidisciplinary approach.

Orofacial Manifestations Associated with Muscular Dystrophies: A Review.

The aim of this review is to evaluate the developmental, functional, and morphological aspects of the craniofacial complex in patients with myotonic dystrophy type 1 (DM1), Facioscapulohumeral muscular dystrophy (FSHD), and Duchenne muscular dystrophy (DMD). The degree of disease onset and severity varied from patient to patient, and most parameters indicated a greater degree of deterioration in older patients. It was found that all the muscular dystrophies studied showed altered craniofacial morphology, with malocclusion as the most consistent clinical characteristic. Particularly DM1 patients, who are the most studied, showed significant vertical aberration and post-normal occlusion. DMD patients are reported mainly with altered dental arch dimensions which influence functional capacities. Data for FSHD patients are very limited, but facial asymmetry and muscular weakness appear to be the most prominent findings. Patients with muscular dystrophies present deviations in growth and development as well as in orofacial morphology. Increased prevalence of malocclusions, of both skeletal and dental origins, characterize patients with muscular dystrophies. Different dentofacial characteristics are reported among patients with different types of muscular dystrophies. Further research is needed to clarify the orofacial phenotypic expression of muscular dystrophies.

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.

Homozygosity of the Z-2 polymorphic variant in the aldose reductase gene promoter confers increased risk for neuropathy in children and adolescents with Type 1 diabetes.

BACKGROUND: Diabetic neuropathy (DN) is the least recognized complication of diabetes mellitus and may start early in the course of the disease. Aldose reductase (AKR1B1) gene promoter Z-2/Z-2 polymorphism increases the expression of AKR1B1 enzyme and may contribute to DN. SUBJECTS: We evaluated 108 Type 1 diabetes (T1D) children and adolescents (mean ± SD age: 13.5 ± 3.46 years, disease duration: 5.3 ± 3.4 years) and 150 healthy controls (age: 11.9 ± 2.7 years). METHODS: In both groups, pupillary dilation (PD) in darkness, postural blood pressure test (PBPT), and vibration sensation thresholds (VST) in upper and lower limbs were estimated as indices of autonomic and peripheral neuropathy, respectively. Nerve conduction studies (NCS) were performed in patients as peripheral neuropathy index. The polymorphisms of AKR1B1 gene were evaluated using microsatellite (AC)n sequence Z. RESULTS: PBPT, PD, and VST impairments were more frequent in patient group compared with controls, while 38.6% of patients exhibited NCS abnormality. Gender, age, pubertal status, height, body mass index, diabetes duration, HbA1c, and anti-GAD titers were associated with neuropathy indices in patients. There was a strong correlation between PD and NCS in patients, while homozygous patients for Z-2 AKR1B1 gene polymorphism had higher prevalence of abnormal NCS (83.3% vs. 34.6%), PD (62.5% vs. 31.5%), and PBPT values compared with heterozygous or negative patients. Homozygous AKR1B1 status predicted PD, NCS, and PBPT variance, while PD, VST, NCS, and PBPT parameters accurately discriminated homozygous AKR1B1 patients. CONCLUSIONS: Impaired indices of peripheral and autonomic DN were present in a significant proportion of young T1D patients. PD, VST, NCS, and PBPT parameters were simultaneously associated with homozygous state of AKR1B1 Z-2 gene polymorphism, implicating polyol metabolism with both autonomic and peripheral neuropathies.

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.

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.

Clinical Outcomes in Duchenne Muscular Dystrophy: A Study of 5345 Patients from the TREAT-NMD DMD Global Database.

BACKGROUND: Recent short-term clinical trials in patients with Duchenne Muscular Dystrophy (DMD) have indicated greater disease variability in terms of progression than expected. In addition, as average life-expectancy increases, reliable data is required on clinical progression in the older DMD population. OBJECTIVE: To determine the effects of corticosteroids on major clinical outcomes of DMD in a large multinational cohort of genetically confirmed DMD patients. METHODS: In this cross-sectional study we analysed clinical data from 5345 genetically confirmed DMD patients from 31 countries held within the TREAT-NMD global DMD database. For analysis patients were categorised by corticosteroid background and further stratified by age. RESULTS: Loss of ambulation in non-steroid treated patients was 10 years and in corticosteroid treated patients 13 years old (p = 0.0001). Corticosteroid treated patients were less likely to need scoliosis surgery (p < 0.001) or ventilatory support (p < 0.001) and there was a mild cardioprotective effect of corticosteroids in the patient population aged 20 years and older (p = 0.0035). Patients with a single deletion of exon 45 showed an increased survival in contrast to other single exon deletions. CONCLUSIONS: This study provides data on clinical outcomes of DMD across many healthcare settings and including a sizeable cohort of older patients. Our data confirm the benefits of corticosteroid treatment on ambulation, need for scoliosis surgery, ventilation and, to a lesser extent, cardiomyopathy. This study underlines the importance of data collection via patient registries and the critical role of multi-centre collaboration in the rare disease field.

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.

Caveolinopathies in Greece.

INTRODUCTION: Mutations in the CAV3 gene are usually inherited in an autosomal dominant manner and lead to distinct disorders including limb-girdle muscular dystrophy 1C, rippling muscle disease, and isolated creatine kinase elevation. PATIENTS AND METHODS: The features of the first patients with caveolin-3 deficiency from Greece are presented. Patients' phenotypes ranged from asymptomatic creatine kinase elevation to severe weakness of lower extremities. Clinical evaluation disclosed muscle hypertrophy in 2 patients, whereas percussion-induced muscle mounding was a consistent finding in all of them. Muscle histopathology was variable and unrelated with disease severity. The diagnosis was based on the immunohistochemical study of caveolin-3 expression and molecular analysis of the caveolin-3 gene. CONCLUSIONS: Clinical manifestations and histochemical findings in caveolinopathy patients may be mild or nonspecific or overlapping with features of other muscular dystrophies. Immunohistochemical study of caveolin-3 expression on muscle biopsy should be routinely performed when investigating isolated hyperCKemia or undetermined myopathy especially in the presence of percussion-induced muscle mounding.

The TREAT-NMD DMD Global Database: analysis of more than 7,000 Duchenne muscular dystrophy mutations.

Analyzing the type and frequency of patient-specific mutations that give rise to Duchenne muscular dystrophy (DMD) is an invaluable tool for diagnostics, basic scientific research, trial planning, and improved clinical care. Locus-specific databases allow for the collection, organization, storage, and analysis of genetic variants of disease. Here, we describe the development and analysis of the TREAT-NMD DMD Global database (http://umd.be/TREAT_DMD/). We analyzed genetic data for 7,149 DMD mutations held within the database. A total of 5,682 large mutations were observed (80% of total mutations), of which 4,894 (86%) were deletions (1 exon or larger) and 784 (14%) were duplications (1 exon or larger). There were 1,445 small mutations (smaller than 1 exon, 20% of all mutations), of which 358 (25%) were small deletions and 132 (9%) small insertions and 199 (14%) affected the splice sites. Point mutations totalled 756 (52% of small mutations) with 726 (50%) nonsense mutations and 30 (2%) missense mutations. Finally, 22 (0.3%) mid-intronic mutations were observed. In addition, mutations were identified within the database that would potentially benefit from novel genetic therapies for DMD including stop codon read-through therapies (10% of total mutations) and exon skipping therapy (80% of deletions and 55% of total mutations).

A simplified approach for FSHD molecular testing.

BACKGROUND: Facioscapulohumeral muscular dystrophy (FSHD) is characterized by complex genetics linked to DNA rearrangements in a polymorphic genomic region of tandemly repeated D4Z4 segments. A panel of FSHD biomarkers including contracted D4Z4 array repeat combined with the 4qA(159/161/168)PAS haplotype has been proposed as molecular signature for defining alleles causally related to FSHD. The aim of the present study was to develop a simple approach for FSHD molecular testing in order to extend studies related to the applicability of FSHD molecular signature in Greek population. METHODS AND RESULTS: The method comprises: (i) visual genotyping of the common 4qA and 10qA subtelomeric haplotypes by a multiplex assay in a dipstick format. (ii) Detection of 4qA161 haplotype in D4Z4 contracted alleles by tri-primer PCR. (iii) Detection of PAS SNP in PLAM region and G>C SNP in the first proximal D4Z4 unit by tri-primer PCR. The method was evaluated by analysing DNA from monoallelic sources representing common 4q and 10q haplotypes, samples from 3 FSHD families, 36 unrelated probands and 38 control individuals of Greek origin. CONCLUSIONS: The proposed method could be a very useful tool for FSHD testing making it more accessible to clinical diagnostic laboratories and the wider FSHD community.