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Background: Preimplantation genetic diagnosis (PGD) usually involves blastomere biopsy 3 days post-insemination (p.i.), followed by genetic analysis and transfer of unaffected embryos later on day 3 or 4. We evaluate a strategy involving embryo biopsy on day 3 p.i., genetic analysis on day 4 and, following culture in blastocyst sequential media, transfer of unaffected embryos on day 5 p.i.Methods: PGD cycles were initiated in 15 couples at risk of transmitting beta-thalassaemia major. Oocyte retrieval and ICSI were performed according to standard protocols. Embryo culture used blastocyst sequential media. Embryos were biopsied on day 3 p.i. using acid Tyrode's for zona drilling, and the single blastomeres were genotyped by a protocol involving nested polymerase chain reaction and denaturing gradient gel electrophoresis analysis.Results: Forty of 109 (37%) embryos biopsied on day 3 p.i. developed to blastocysts by day 5 p.i., with at least one blastocyst available for transfer in 12 cycles (80%). Genotype analysis characterized 51/109 (47%) embryos unaffected for beta-thalassaemia major, of which 28 were blastocysts. Transfer of 37 day 5 p.i. embryos (blastocysts and non blastocysts) initiated eight clinical pregnancies. Implantation rate per embryo transferred was 12/37 (32%).Conclusions: Embryo biopsy on day 3, followed by delayed transfer until day 5 p.i. offers a novel and effective strategy to overcome the time limit encountered when performing PGD, without compromising embryo implantation. [ABSTRACT FROM AUTHOR]

The development of high-throughput technologies has enabled Expanded Carrier screening (ECS) as a more comprehensive and extensive approach for high-risk populations. The available methods of ECS are population-targeted gene-panels according to ethnicity, however these panels should be planned according to a real-world data evaluation. In this study, we estimate the frequency of pathogenic variants for autosomal-recessive and X-linked conditions in Exome Sequencing-ES data for a 176 gene panel proposed from ACMG and ACOG in a Greek cohort. ES data from 1000 unrelated individuals was evaluated for pathogenic SNVs and CNVs. Variants were filtered using 5% Minor Frequency Allele (MAF), ClinVar submissions, and classification with ACMG criteria. For the at-risk couple rate, we hypothesized that both parents carried variants in the same gene. It is noted that many common conditions (hemoglobinopathies, SMA, Fragile-X) may escape NGS-based detection as they require alternative methods for optimal detection. Amongst 1000 participants, 32% were heterozygous for at least one disorder and 14% for two or more, whereby 393 unique pathogenic/likely pathogenic heterozygous variants were identified. We calculated that 1.6% of couples have a risk for at least one AR condition, which means that for 85,000 births per year, 1380 couples require genetic counseling. This study provides data confirming that the ACMG/ACOG ECS list of 176 genes is suitable for carrier screening in Greece, and aids counseling prospective parents for residual risk, however it should be supported by appropriate interpretation and reproductive options, as well as ancillary genetic testing methods., (© 2024. The Author(s), under exclusive licence to European Society of Human Genetics.)

Epidemiological studies report the association of diverse cardiovascular conditions with coronavirus disease 2019 (COVID-19), but the causality has remained to be established. Specific genetic factors and the extent to which they can explain variation in susceptibility or severity are largely elusive. The present study aimed to evaluate the link between 32 cardio-metabolic traits and COVID-19. A total of 60 participants were enrolled, who were categorized into the following 4 groups: A control group with no COVID-19 or any other underlying pathologies, a group of patients with a certain form of dyslipidemia and predisposition to atherosclerotic disease, a COVID-19 group with mild or no symptoms and a COVID-19 group with severe symptomatology hospitalized at the Intensive Care Unit of Sotiria Hospital (Athens, Greece). Demographic, clinical and laboratory data were recorded and genetic material was isolated, followed by simultaneous analysis of the genes related to dyslipidemia using a custom-made next-generation sequencing panel. In the COVID-19 group with mild or absent symptoms, the variant c.112C>T:p.P38S was detected in the sodium channel epithelial 1 subunit α (SCNN1A) gene, with a major allele frequency (Maf) of <0.01. In the COVID-19 group with severe symptoms, the variant c.786G>A:p.T262T was detected in the SCNN1B gene, which encodes for the β-subunit of the epithelial sodium channel ENaC, with a Maf <0.01. None of the two rare variants were detected in the control or dyslipidemia groups. In conclusion, the current study suggests that ENaC variants are likely associated with genetic susceptibility to COVID-19, supporting the rationale for the risk and protective genetic factors for the morbidity and mortality of COVID-19., Competing Interests: The authors declare that they have no competing interests., (Copyright: © 2024 Koniari et al.)

Purpose: This study aims to comprehensively delineate the phenotypic spectrum of ACTL6B-related disorders, previously associated with both autosomal recessive and autosomal dominant neurodevelopmental disorders. Molecularly, the role of the nucleolar protein ACTL6B in contributing to the disease has remained unclear., Methods: We identified 105 affected individuals, including 39 previously reported cases, and systematically analysed detailed clinical and genetic data for all individuals. Additionally, we conducted knockdown experiments in neuronal cells to investigate the role of ACTL6B in ribosome biogenesis., Results: Biallelic variants in ACTL6B are associated with severe-to-profound global developmental delay/intellectual disability (GDD/ID), infantile intractable seizures, absent speech, autistic features, dystonia, and increased lethality. De novo monoallelic variants result in moderate-to-severe GDD/ID, absent speech, and autistic features, while seizures and dystonia were less frequently observed. Dysmorphic facial features and brain abnormalities, including hypoplastic corpus callosum, parenchymal volume loss/atrophy, are common findings in both groups. We reveal that in the nucleolus, ACTL6B plays a crucial role in ribosome biogenesis, in particular in pre-rRNA processing., Conclusion: This study provides a comprehensive characterization of the clinical spectrum of both autosomal recessive and dominant forms of ACTL6B-associated disorders. It offers a comparative analysis of their respective phenotypes provides a plausible molecular explanation and suggests their inclusion within the expanding category of 'ribosomopathies'., (Copyright © 2024. Published by Elsevier Inc.)

Introduction: Preimplantation Genetic Testing (PGT) is a cutting-edge test used to detect genetic abnormalities in embryos fertilized through Medically Assisted Reproduction (MAR). PGT aims to ensure that embryos selected for transfer are free of specific genetic conditions or chromosome abnormalities, thereby reducing chances for unsuccessful MAR cycles, complicated pregnancies, and genetic diseases in future children., Areas Covered: In PGT, genetics, embryology, and technology progress and evolve together. Biological and technological limitations are described and addressed to highlight complexity and knowledge constraints and draw attention to concerns regarding safety of procedures, clinical validity, and utility, extent of applications and overall ethical implications for future families and society., Expert Opinion: Understanding the genetic basis of diseases along with advanced technologies applied in embryology and genetics contribute to faster, cost-effective, and more efficient PGT. Next Generation Sequencing-based techniques, enhanced by improved bioinformatics, are expected to upgrade diagnostic accuracy. Complicating findings such as mosaicism, mt-DNA variants, variants of unknown significance, or variants related to late-onset or polygenic diseases will however need further appraisal. Emphasis on monitoring such emerging data is crucial for evidence-based counseling while standardized protocols and guidelines are essential to ensure clinical value and respect of Ethical, Legal and Societal Issues.

It is well known that modifiers play a role in ameliorating or exacerbating disease phenotypes in patients and carriers of recessively inherited disorders such as sickle cell disease and thalassemia. Here, we give an overview of the literature concerning a recently described association in carriers of SUPT5H Loss-of-Function variants with a beta-thalassemia-like phenotype including the characteristic elevated levels of HbA 2 . That SUPT5H acts as modifier in beta-thalassemia carriers became evident from three reported cases in whom combined heterozygosity of SUPT5H and HBB gene variants was observed to resemble a mild beta-thalassemia intermedia phenotype. The different SUPT5H variants and hematologic parameters reported are collected and reviewed to provide insight into the possible effects on hematologic expression, as well as potential disease mechanisms in carriers and patients.

Alport syndrome (AS) is a hereditary glomerulopathy due to pathogenic variants in COL4A3 , COL4A4 , and COL4A5. Treatment with Renin-Angiotensin-Aldosterone System (RAAS) inhibitors can delay progression to end stage renal disease (ESRD). From 2018 until today, we performed Whole Exome Sequencing (WES) in 19 patients with AS phenotype with or without positive family history. Fourteen of these patients were children. Genetic testing was extended to family members at risk. All patients received a genetic diagnosis of AS: five X-linked AS (XLAS) males, five X-linked AS (XLAS) females, six autosomal dominant AS (ADAS), and one autosomal recessive AS (ARAS). After cascade screening four XLAS males and eight XLAS females, six ADAS and three ARAS heterozygotes were added to our initial results. Fifteen patients were eligible to start treatment with RAAS inhibitors after their diagnosis. All XLAS female patients, ARAS heterozygotes, and ADAS have been advised to be followed up, so that therapeutic intervention can begin in the presence of microalbuminuria. Genetic diagnosis of AS ensures early therapeutic intervention and appropriate follow up to delay progression to chronic kidney disease, especially in thet pediatric population.

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., (© 2024 The Author(s). Muscle & Nerve published by Wiley Periodicals LLC.)

Bryant-Li-Bhoj syndrome (BLBS), which became OMIM-classified in 2022 (OMIM: 619720, 619721), is caused by germline variants in the two genes that encode histone H3.3 (H3-3A/H3F3A and H3-3B/H3F3B) [1-4]. This syndrome is characterized by developmental delay/intellectual disability, craniofacial anomalies, hyper/hypotonia, and abnormal neuroimaging [1, 5]. BLBS was initially categorized as a progressive neurodegenerative syndrome caused by de novo heterozygous variants in either H3-3A or H3-3B [1-4]. Here, we analyze the data of the 58 previously published individuals along 38 unpublished, unrelated individuals. In this larger cohort of 96 people, we identify causative missense, synonymous, and stop-loss variants. We also expand upon the phenotypic characterization by elaborating on the neurodevelopmental component of BLBS. Notably, phenotypic heterogeneity was present even amongst individuals harboring the same variant. To explore the complex phenotypic variation in this expanded cohort, the relationships between syndromic phenotypes with three variables of interest were interrogated: sex, gene containing the causative variant, and variant location in the H3.3 protein. While specific genotype-phenotype correlations have not been conclusively delineated, the results presented here suggest that the location of the variants within the H3.3 protein and the affected gene (H3-3A or H3-3B) contribute more to the severity of distinct phenotypes than sex. Since these variables do not account for all BLBS phenotypic variability, these findings suggest that additional factors may play a role in modifying the phenotypes of affected individuals. Histones are poised at the interface of genetics and epigenetics, highlighting the potential role for gene-environment interactions and the importance of future research., (© 2024. The Author(s).)

Transmembrane protein 184B (TMEM184B) is an endosomal 7-pass transmembrane protein with evolutionarily conserved roles in synaptic structure and axon degeneration. We report six pediatric patients who have de novo heterozygous variants in TMEM184B . All individuals harbor rare missense or mRNA splicing changes and have neurodevelopmental deficits including intellectual disability, corpus callosum hypoplasia, seizures, and/or microcephaly. TMEM184B is predicted to contain a pore domain, wherein many human disease-associated variants cluster. Structural modeling suggests that all missense variants alter TMEM184B protein stability. To understand the contribution of TMEM184B to neural development in vivo , we suppressed the TMEM184B ortholog in zebrafish and observed microcephaly and reduced anterior commissural neurons, aligning with patient symptoms. Ectopic TMEM184B expression resulted in dominant effects for K184E and G162R. However, in vivo complementation studies demonstrate that all other variants tested result in diminished protein function and indicate a haploinsufficiency basis for disease. Expression of K184E and other variants increased apoptosis in cell lines and altered nuclear localization of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis, suggesting disrupted nutrient signaling pathways. Together, our data indicate that TMEM184B variants cause cellular metabolic disruption likely through divergent molecular effects that all result in abnormal neural development.

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 , 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&#95;1131delGAinsAC, c.1574&#95;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.

In this short review, we presented and discussed studies on the expression of globin genes in β-thalassemia, focusing on the impact of α-globin gene expression and α-globin modifiers on the phenotype and clinical severity of β-thalassemia. We first discussed the impact of the excess of free α-globin on the phenotype of β-thalassemia. We then reviewed studies focusing on the expression of α-globin-stabilizing protein (AHSP), as a potential strategy of counteracting the effects of the excess of free α-globin on erythroid cells. Alternative processes controlling α-globin excess were also considered, including the activation of autophagy by β-thalassemia erythroid cells. Altogether, the studies reviewed herein are expected to have a potential impact on the management of patients with β-thalassemia and other hemoglobinopathies for which reduction in α-globin excess is clinically beneficial.

Purpose: RNF213, encoding a giant E3 ubiquitin ligase, has been recognized for its role as a key susceptibility gene for moyamoya disease. Case reports have also implicated specific variants in RNF213 with an early-onset form of moyamoya disease with full penetrance. We aimed to expand the phenotypic spectrum of monogenic RNF213-related disease and to evaluate genotype-phenotype correlations., Methods: Patients were identified through reanalysis of exome sequencing data of an unselected cohort of unsolved pediatric cases and through GeneMatcher or ClinVar. Functional characterization was done by proteomics analysis and oxidative phosphorylation enzyme activities using patient-derived fibroblasts., Results: We identified 14 individuals from 13 unrelated families with (de novo) missense variants in RNF213 clustering within or around the Really Interesting New Gene (RING) domain. Individuals presented either with early-onset stroke (n = 11) or with Leigh syndrome (n = 3). No genotype-phenotype correlation could be established. Proteomics using patient-derived fibroblasts revealed no significant differences between clinical subgroups. 3D modeling revealed a clustering of missense variants in the tertiary structure of RNF213 potentially affecting zinc-binding suggesting a gain-of-function or dominant negative effect., Conclusion: De novo missense variants in RNF213 clustering in the E3 RING or other regions affecting zinc-binding lead to an early-onset syndrome characterized by stroke or Leigh syndrome., Competing Interests: Conflict of Interest A.S. is supported by the BONFOR program of the Medical Faculty, University of Bonn (O-149.0134). All other authors declare no conflicts of interest., (Copyright © 2023 American College of Medical Genetics and Genomics. Published by Elsevier Inc. All rights reserved.)

Background: Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder typified by various combination of numerous Café-au-lait macules, cutaneous and plexiform neurofibromas, freckling of inguinal or axillary region, optic glioma, Lisch nodules and osseous lesions. Cherubism is a rare genetic syndrome described by progressive swelling of the lower and/or upper jaw due to replacement of bone by fibrous connective tissue. Patients are reported in the literature with NF1 and cherubism-like phenotype due to the NF1 osseous lesions in the jaws. The purpose of this case report is the description of a young male genetically diagnosed with both NF1 and cherubism., Methods and Results: A 9 years and six month old patient with clinical findings of NF1 and cherubism in whom both diseases were genetically confirmed, is presented. The patient was evaluated by a pediatrician, a pediatric endocrinologist, an ophthalmologist, and an oral and maxillofacial surgeon. A laboratory and hormonal screening, a histological examination, a chest X-ray, a magnetic resonance imaging (MRI) of the orbit and a digital panoramic radiography were performed. Genetic testing applying Whole Exome Sequencing was conducted., Conclusions: A novel and an already reported pathogenic variants were detected in NF1 and SH3BP2 genes, respectively. This is the first described patient with coexistence of NF1 and cherubism. The contribution of Next Generation Sequencing (NGS) in gene variant identification as well as the importance of close collaboration between laboratory scientists and clinicians, is highlighted. Both are essential for optimizing the diagnostic approach of patients with a complex phenotype., (© 2024. The Author(s).)

The homologous genes GTPBP1 and GTPBP2 encode GTP-binding proteins 1 and 2, which are involved in ribosomal homeostasis. Pathogenic variants in GTPBP2 were recently shown to be an ultra-rare cause of neurodegenerative or neurodevelopmental disorders (NDDs). Until now, no human phenotype has been linked to GTPBP1. Here, we describe individuals carrying bi-allelic GTPBP1 variants that display an identical phenotype with GTPBP2 and characterize the overall spectrum of GTP-binding protein (1/2)-related disorders. In this study, 20 individuals from 16 families with distinct NDDs and syndromic facial features were investigated by whole-exome (WES) or whole-genome (WGS) sequencing. To assess the functional impact of the identified genetic variants, semi-quantitative PCR, western blot, and ribosome profiling assays were performed in fibroblasts from affected individuals. We also investigated the effect of reducing expression of CG2017, an ortholog of human GTPBP1/2, in the fruit fly Drosophila melanogaster. Individuals with bi-allelic GTPBP1 or GTPBP2 variants presented with microcephaly, profound neurodevelopmental impairment, pathognomonic craniofacial features, and ectodermal defects. Abnormal vision and/or hearing, progressive spasticity, choreoathetoid movements, refractory epilepsy, and brain atrophy were part of the core phenotype of this syndrome. Cell line studies identified a loss-of-function (LoF) impact of the disease-associated variants but no significant abnormalities on ribosome profiling. Reduced expression of CG2017 isoforms was associated with locomotor impairment in Drosophila. In conclusion, bi-allelic GTPBP1 and GTPBP2 LoF variants cause an identical, distinct neurodevelopmental syndrome. Mutant CG2017 knockout flies display motor impairment, highlighting the conserved role for GTP-binding proteins in CNS development across species., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Purpose: IGSF1 deficiency syndrome (immunoglobulin superfamily member 1) is considered the most common sex-linked cause of secondary congenital hypothyroidism and is characterized by a wide variety of other clinical and biochemical features, including hypoprolactinemia, transient and partial growth hormone deficiency, early/normal timing of testicular enlargement but delayed testosterone rise in puberty, and adult macro-orchidism. Congenital central hypothyroidism is a rare disease (1:65,000 births); the detection of which may be delayed and missed by neonatal screening programs since most neonatal screening programs are based on TSH determination in dried blood spots only. Untreated hypothyroidism may cause abnormal liver biochemistry and non-alcoholic fatty liver disease. Our aim is to report a case of secondary hypothyroidism in an infant with an uncommon initial presentation., Case Presentation (methods/results): A 3-month-old male baby was referred to our hospital due to elevated alpha-fetoprotein levels, hypercholesterolemia, and macrosomia. Initial investigations revealed enlarged fatty liver and central hypothyroidism. Pituitary insufficiency was biochemically excluded and a pituitary MRI showed normal findings. Upon genetic analysis, a hemizygous variant NM&#95;001170961.1:c.2422dup, p.(His808Profs*14), in IGSF1 gene was detected, establishing the diagnosis of the IGSF1 deficiency syndrome. In our patient, no other clinical findings were identified. Treatment with levothyroxine led to the remission of liver disease., Conclusion: Liver disease may be the initial presentation of secondary hypothyroidism in neonates and infants. Macrosomia in patients with isolated secondary central hypothyroidism is a strong indicator of IGSF1 syndrome., (© 2023. The Author(s), under exclusive licence to Hellenic Endocrine Society.)

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., Competing Interests: The authors declare no conflict of interest.

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.

Objective: Genomics Quality Assessment has provided external quality assessments (EQAs) for preimplantation genetic testing (PGT) for 12 years for eight monogenic diseases to identify sub-optimal PGT strategies, testing and reporting of results, which can be shared with the genomics community to aid optimised standards of PGT services for couples., Method: The EQAs were provided in two stages to mimic end-to-end protocols. Stage 1 involved DNA feasibility testing of a couple undergoing PGT and affected proband. Participants were required to report genotyping results and outline their embryo testing strategy. Lymphoblasts were distributed for mock embryo testing for stage 2. Submitted clinical reports and haplotyping results were assessed against peer-ratified criteria. Performance was monitored to identify poor performance., Results: The most common testing methodology was short tandem repeat linkage analysis (59%); however, the adoption of single nucleotide polymorphism-based platforms was observed and a move from blastomere to trophectoderm testing. There was a variation in testing strategies, assigning marker informativity and understanding test limitations, some clinically unsafe. Critical errors were reported for genotyping and interpretation., Conclusion: EQA provides an overview of the standard of preimplantation genetic testing-M clinical testing and identifies areas of improvement for accurate detection of high-risk embryos., (© 2022 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd.)

Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset, X-linked, neurodegenerative disorder that affects premutation carriers of the FMR1 gene. FXTAS is often misdiagnosed as spinocerebellar ataxia (SCA) or Parkinson's disease (PD). Herein, we sought to investigate the frequency, genotypic and phenotypic profile of FXTAS in two cohorts of Greek patients with late-onset movement disorders, one with cerebellar ataxia and the other with PD. In total, 90 index patients with late-onset cerebellar ataxia and 171 with PD were selected. None of the cases had male-to-male transmission. Genetic screening for the FMR1 premutation was performed using standard methodology. The FMR1 premutation was detected in two ataxia patients (2.2%) and two PD patients (1.2%). Additional clinical features in FXTAS patients from the ataxia cohort included neuropathy, mild parkinsonism, cognitive impairment and pyramidal signs. The FXTAS patients from the PD cohort had typical PD. We conclude that, in the Greek population, the FMR1 premutation is an important, albeit rare, cause of late-onset movement disorders. Routine premutation screening should be considered in SCA panel-negative late-onset ataxia cases. Directed premutation screening should be considered in all ataxia and PD cases with additional features suggestive of FXTAS. Our study highlights the importance of FMR1 genetic testing in the diagnosis of late-onset movement disorders., Competing Interests: Declaration of competing interest The authors have no conflict of interest to report., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

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.

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.

Purpose: Although ACTH is considered a secondary regulator of aldosterone production, patients with apparent essential hypertension have been treated with mineralocorticoid receptor antagonists (MRAs). In this study, we aimed to identify potentially damaging variants that might be implicated in the phenotype of a well-characterized cohort of 21 hypertensive patients without PA but with stress-induced aldosterone hypersecretion. The patients' blood pressure was normalized though MRA administration., Methods: Genetic screening was performed through whole-exome sequencing (WES), and variants in PA-associated or in ion-channels of aldosterone-regulating genes were prioritized. Variants with population frequency < 0.01, predicted to alter protein structure and classified as likely pathogenic by in silico tools, were retained., Results: Qualifying variants were identified in nine of the 21 patients screened. Seven patients were carriers of six potentially damaging variants in six genes associated with PA (KCNK9, KCNK5, ATP13A3, SLC26A2, CACNA1H, and CACNA1D). A novel variant in the KCNK9 gene (p.V221M) is reported. Our analysis revealed two variants in two novel susceptibility genes for aldosterone hypersecretion, namely, KCNK16 (p.P255H) and CACNA2D3 (p.V557I)., Conclusion: WES revealed potentially damaging germline variants in genes participating in aldosterone synthesis/regulating pathways in 9/21 patients of our cohort. The variants identified might play a role in aldosterone hypersecretion under conditions of stress. The potential pathogenicity of these variants should be examined in future functional studies., (© 2022. The Author(s), under exclusive licence to Hellenic Endocrine Society.)

ATP6V1B2 pathogenic variants are linked with variable phenotypes, such as dominant deafness-onychodystrophy syndrome (DDOD), autosomal dominant Zimmermann-Laband syndrome type 2 (ZLS2), and some cases of DOORS (deafness, onychodystrophy, osteodystrophy, intellectual disability [ID], and seizures). Epilepsy was first linked to ATP6V1B2, when the p.(Glu374Gln) missense variant was detected in a patient with ID and seizures, but without characteristic features of DDOD or ZLS2 syndromes. We herein report a novel pathogenic ATP6V1B2:p.Glu374Gly variant detected in an adult patient with ID and myoclonic-atonic seizures. The (re)occurrence of different variants affecting the same highly conserved hydrophilic glutamic acid on position 374 of the V-proton ATPase subunit B, indicates a potential novel pathogenic hotspot and a critical role for the specific residue in the development of epilepsy. ATP6V1B2 gene defects should be considered when analyzing patients with epilepsy, even in the absence of most cardinal features of DDOD, DOORS, or ZLS such as deafness, onychodystrophy, and osteodystrophy., (© 2022 Wiley Periodicals LLC.)

Haemoglobinopathies are the commonest monogenic diseases worldwide and are caused by variants in the globin gene clusters. With over 2400 variants detected to date, their interpretation using the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) guidelines is challenging and computational evidence can provide valuable input about their functional annotation. While many in silico predictors have already been developed, their performance varies for different genes and diseases. In this study, we evaluate 31 in silico predictors using a dataset of 1627 variants in HBA1 , HBA2, and HBB . By varying the decision threshold for each tool, we analyse their performance (a) as binary classifiers of pathogenicity and (b) by using different non-overlapping pathogenic and benign thresholds for their optimal use in the ACMG/AMP framework. Our results show that CADD, Eigen-PC, and REVEL are the overall top performers, with the former reaching moderate strength level for pathogenic prediction. Eigen-PC and REVEL achieve the highest accuracies for missense variants, while CADD is also a reliable predictor of non-missense variants. Moreover, SpliceAI is the top performing splicing predictor, reaching strong level of evidence, while GERP++ and phyloP are the most accurate conservation tools. This study provides evidence about the optimal use of computational tools in globin gene clusters under the ACMG/AMP framework., Competing Interests: ST, MX, AM, CS, CH, CB, JT, IF, NY, FA, EE, HH, BZ, AK, MK, PK No competing interests declared, (© 2022, Tamana, Xenophontos et al.)

Preimplantation Genetic Testing (PGT) aims to reduce the chance of an affected pregnancy or improve success in an assisted reproduction cycle. Since the first established pregnancies in 1990, methodological approaches have greatly evolved, combined with significant advances in the embryological laboratory. The application of preimplantation testing has expanded, while the accuracy and reliability of monogenic and chromosomal analysis have improved. The procedure traditionally employs an invasive approach to assess the nucleic acid content of embryos. All biopsy procedures require high technical skill, and costly equipment, and may impact both the accuracy of genetic testing and embryo viability. To overcome these limitations, many researchers have focused on the analysis of cell-free DNA (cfDNA) at the preimplantation stage, sampled either from the blastocoel or embryo culture media, to determine the genetic status of the embryo non-invasively. Studies have assessed the origin of cfDNA and its application in non-invasive testing for monogenic disease and chromosomal aneuploidies. Herein, we discuss the state-of-the-art for modern non-invasive embryonic genetic material assessment in the context of PGT. The results are difficult to integrate due to numerous methodological differences between the studies, while further work is required to assess the suitability of cfDNA analysis for clinical application., (© 2022 Bentham Science Publishers.)

Frasier syndrome (FS) is a rare condition, caused by splice-site mutations of intron 9 in the Wilms' tumor suppressor gene 1 (WT1 gene). The WT1 protein is essential for urogenital development and patients with 46XY karyotype present with female (FS type 1) or male phenotype, gonadal dysgenesis, progressive glomerulopathy, and high risk of gonadoblastoma. We describe a female patient with an IVS9+4C>T donor splice-site mutation, who underwent a preventive gonadectomy at the age of 6 years due to imaging findings of dysplastic gonads. The biopsy revealed bilateral gonadoblastoma, emphasizing the need for early gonadectomy in 46XY FS patients., Competing Interests: The authors declare no conflict of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Hemoglobinopathies constitute some of the most common inherited disorders worldwide. Manifestations are very severe, patient management is difficult and treatment is not easily accessible. Preimplantation genetic testing for monogenic disorders (PGT-M) is a valuable reproductive option for hemoglobinopathy carrier-couples as it precludes the initiation of an affected pregnancy. PGT-M is performed on embryos generated by assisted reproductive technologies and only those found to be free of the monogenic disorder are transferred to the uterus. PGT-M has been applied for 30 years now and β-thalassemia is one of the most common indications. PGT may also be applied for human leukocyte antigen typing to identify embryos that are unaffected and also compatible with an affected sibling in need of hemopoietic stem cell transplantation. PGT-M protocols have evolved from PCR amplification-based, where a small number of loci were analysed, to whole genome amplification-based, the latter increasing diagnostic accuracy, enabling the development of more generic strategies and facilitating multiple diagnoses in one embryo. Currently, numerous PGT-M cycles are performed for the simultaneous diagnosis of hemoglobinopathies and screening for chromosomal abnormalities in the embryo in an attempt to further improve success rates and increase deliveries of unaffected babies., (© 2022 John Wiley & Sons Ltd.)