Molecular Genetic Testing Research Articles

Genetic Predisposition to Hematologic Malignancies in Childhood and Adolescence.

Advances in molecular biology and genetic testing have greatly improved our understanding of the genetic basis of hematologic malignancies and have enabled the identification of new cancer predisposition syndromes. Recognizing a germline mutation in a patient affected by a hematologic malignancy allows for a tailored treatment approach to minimize toxicities. It informs the donor selection, the timing, and the conditioning strategy for hematopoietic stem cell transplantation, as well as the comorbidities evaluation and surveillance strategies. This review provides an overview of germline mutations that predispose to hematologic malignancies, focusing on those most common during childhood and adolescence, based on the new International Consensus Classification of Myeloid and Lymphoid Neoplasms.

Aspects of Stage-by-Stage Diagnosis of Pulmonary Mycobacteriosis in Real Clinical Practice

The objective: to analyze the effectiveness of etiological verification of pulmonary mycobacteriosis when using various biological materials at different diagnostic stages.Subjects and Methods. In Center for Diagnosis and Rehabilitation of Respiratory Diseases, CTRI, the results of stage-by-stage examinations of 142 patients (from 2018 to 2022) were analyzed. A retrospective, prospective, cohort, open study was conducted. Inclusion criteria: pulmonary mycobacteriosis etiologically verified by comprehensive microbiological examinations (molecular genetic and cultural tests) (with retrospective analysis after all stages of examination). Exclusion criteria: HIV infection. 115 women and 27 men were enrolled in the study, the age ranged from 21 to 86 years, the mean age was 57,9 years.All patients referred to CTRI for examination due to newly detected changes in the lungs (bronchiectasis, focal changes, and cavities). For etiological verification of the diagnosis, microbiological sputum testing was performed (Stage 1 of the examination); when negative results were obtained, bronchoscopy was prescribed and bronchobiopsies were used for microbiological and molecular genetic diagnostics – Stage 2. In case of negative results of Stage 2, the patient was recommended to undergo a diagnostic resection of the lung – Stage 3 of the examination. At all stages, specimens were sent for comprehensive microbiological testing: fluorescent microscopy to detect acid-fact mycobacteria, real-time polymerase chain reaction (PCR-RV) to detect NTBM DNA. Identification of NTBMB was carried out by molecular genetic methods (PCR for the presence of MTB DNA/NTBM (SYNTOL, Russia), and on DNA strips (GenoType Mycobacterium CM\AS Hain Livescience, Germany)). Specimens were cultured on a liquid medium in the ВАСТЕС MGIT960 (BD, USA).The diagnosis of mycobacteriosis was established in accordance with the diagnostic criteria proposed in the guidelines of the American Thoracic Society (ATS) and the Infectious Diseases Society of America (IDSA) (2020): detection of NTBM in 2 sputum samples or in 1 sample of bronchobiopsy specimens or in 1 surgical specimens [15].The studied data were entered to the Microsoft Office Excel sheet (Microsoft, USA) for statistical processing; the level of statistical significance of differences was taken as 0.05.Results. It was found that slow-growing NTBM were identified as etiologically significant in 93.0%, of which 78.1% was M. avium, fast-growing NTBM were represented only by M. abscessus, 7% of observations. During X-ray examination, the bronchiectatic form – 52.1% (74/142) was detected more often compared to the abdominal – 30.9% (44/142) and focal forms – 16.9% (24/142) (p<0.001). When verifying mycobacteriosis in patients with a negative sputum test – Stage 2 of the examination – the effectiveness was 91.0% (80/88). Regardless of the radiological form of the disease and dissemination of the disease, bronchoscopy revealed pathology of the tracheobronchial tree in 85.2% of cases, mainly in the form of bronchitis with purulent secretions.

Identification of novel compound heterozygous mutation in Niemann-Pick disease type C gene (P14-11.006)

<h3>Objective:</h3> To report a novel compound heterozygous mutation in two siblings with Niemann-Pick disease type C who displayed distinct phenotypic presentation <h3>Background:</h3> Niemann–Pick disease type C (NPC) is a rare autosomal recessive lysosomal storage disease with heterogenous neurological and systemic manifestations. The vast majority of cases (95%) are caused by mutations in the Niemann–Pick C1 protein (NPC1), whereas the reminder of cases are attributed to mutations in Niemann–Pick C2 protein (NPC2). <h3>Design/Methods:</h3> Case report <h3>Results:</h3> <h3>Case 1:</h3> A 57-year-old female presented with a 5-year history of progressive slowness, poor coordination, and cognitive decline. This was followed by generalized stiffness and recurrent ballistic movements within two years of onset. Her poor postural stability lead to frequent falls. Initial neurological evaluation revealed severe dysarthric speech, moderate vertical eye movement limitation, spasticity and exaggerated deep tendon reflexes, along with dystonic movements. She progressed rapidly, developing generalized dystonia which made her bedbound. <h3>Case 2:</h3> the young sibling of case 1, presented at the age of 56 with 8-year history of poor balance. Symptoms were slowly progressive, noted initially with changes in his balance, slurred speech, and poor coordination. He was also noticed to have mild cognitive changes. Examination revealed cerebellar dysarthria, with failure to generate saccades from primary position downwards. Deep tendon reflexes were brisk, with no evidence of muscle wasting, or spasticity. He showed signs of mild axial and appendicular ataxia. The progression was slow, and he maintained his mobility without requiring walking aids. Molecular genetic testing revealed compound heterozygous mutation in the NPC1 gene; c.180G&gt;T (p.Gln60His) and c.3173C&gt;A (p.Ala1058Asp). All biochemical results were within normal range. Brain MRI showed generalized cerebral and cerebellar volume loss few years into the onset of symptoms in both patients. <h3>Conclusions:</h3> Our report highlights the high intrafamilial heterogeneity and poor genotype-phenotype correlation in the case of Niemann–Pick disease type C. <b>Disclosure:</b> Dr. Abusrair has nothing to disclose. Dr. Amorelli has nothing to disclose. Justyna Sarna has nothing to disclose.

The Roles of Fibroblast Growth Factor Receptor 3 (FGFR3) in the Spectrum of Skeletal Dysplasia

The fibroblast growth factor receptor 3 (FGFR3) gene mutations were identified to be involved in the pathogenesis of most chondropathies. The FGFR3 gene encodes the FGFR3 receptor and is involved in the regulation of bone growth by limiting the ossification of long bones. Mutations of the FGFR3 gene result in abnormal cell proliferation and improper cartilage development. We aim to provide an overview of the roles of FGFR3 in skeletal dysplasia by highlighting the pathogenesis, clinical variants of skeletal dysplasia, diagnosis, and their management.&#x0D; Achondroplasia is the most common form of chondropathies, occurring in approximately 1 in 20,000-30,000 live births, and it is the most common form of genetic dwarfism. In over 80% of cases of achondroplasia, the mutation is sporadic, and only 20% are inherited autosomal dominant. Achondroplasia results from a point mutation in the gene encoding the transmembrane portion of FGFR3. Two viable base substitutions are identified in achondroplasia, including a point mutation of guanine substituted for adenine (c.1138G&gt;A); this is identified in approximately 98% of the affected individuals, and transversion of guanine to cytosine (c.1138G&gt;C).&#x0D; Hypochondroplasia is a milder form of chondropathies with an incidence between 1 in 33,000 and 1 in 47,000 live births. Missense mutations of FGFR3 (p.Asn540Lys) are isolated in tyrosine kinase domain I occurring in approximately 60% of cases, and missense mutation of FGFR3 (p.Lys650Asn) identified in the tyrosine kinase domain II of FGFR3. Thanatophoric dysplasia is the most lethal form of chondropathies, with neonatal fetal death secondary to pulmonary hypoplasia. In thanatophoric dysplasia, there is Lys650Met substitution in FGFR3 (Type I) with impairment of endochondral bone growth and a pathogenic variant of p.Lys650Glu substitution in FGFR3 (Type II). In addition, specific amino acid substitution in the FGFR3 gene (G380R) was identified to be associated with severe achondroplasia with developmental delay and acanthosis nigricans (SADDAN).&#x0D; The diagnosis of achondroplasias is routinely made from clinical presentations and radiological findings. FGFR3 molecular genetic testing is performed in children with atypical presentations. In addition, a pre-implantation genetic diagnosis should be available for parents pursuing in-vitro fertilization and embryo implantation procedures. Management of chondropathies includes symptomatic treatment with drugs, surgical intervention, and lifelong follow-up care. Different pharmacological options have been used, including those that directly block FGFR3 activation or regulate signalling pathways controlling chondrocyte proliferation and differentiation.

Progression and transformation of chronic lymphocytic leukemia/small lymphocytic lymphoma and B-cell prolymphocytic leukemia: Report from the 2021 SH/EAHP Workshop.

Session 3 of the 2021 Workshop of the Society for Hematopathology/European Association for Haematopathology examined progression and transformation of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) and B-cell prolymphocytic leukemia (B-PLL). Thirty-one cases were reviewed by the panel. Additional studies such as immunohistochemistry and molecular genetic testing, including whole-exome sequencing and expression profiling, were performed in select cases. Session 3 included 27 CLL/SLL cases and miscellaneous associated proliferations, 3 cases of B-PLL, and 1 case of small B-cell lymphoma. The criteria for -accelerated CLL/SLL are established for lymph nodes, but extranodal disease can be diagnostically challenging. Richter transformation (RT) is a broad term and includes true transformation from original CLL/SLL clone(s) and clonally unrelated neoplasms. The morphologic, immunophenotypic, and genetic spectrum is diverse with classical and highly unusual examples. T-cell proliferations can also be encountered in CLL/SLL. B-cell prolymphocytic leukemia is a rare, diagnostically challenging disease due to its overlaps with other lymphoid neoplasms. The workshop highlighted complexity of progression and transformation in CLL/SLL and B-PLL, as well as diagnostic caveats accompanying heterogeneous presentations of RT and other manifestations of disease progression. Molecular genetic studies are pivotal for diagnosis and determination of clonal relationship, and to predict response to treatment and identify resistance to targeted therapy.

Hereditary lung diseases and modern possibilities of genetic testing

The European Respiratory Society website gives the following criterion for the disease to be classified as rare (orphan) - the disease occurs in 1 person per 2 000. One of the well-studied rare lung diseases is cystic fibrosis (CF), which is often considered a medical care model for patients with other orphan diseases. However, effective diagnostics and therapies have not yet been developed for many other rare diseases. Moreover, their true prevalence remains unknown because these diseases often go undiagnosed. One of the problems in diagnosing rare diseases is the lack of knowledge among physicians.The aim of this review is to provide a brief clinical and genetic description of rare hereditary lung diseases and to show modern genetic diagnostics to raise awareness among physicians. Data from 95 articles on hereditary lung diseases were used.Results. The results of the analysis of lung diseases associated with bronchiectasis, fibrosis, pneumothorax, and hereditary storage diseases are presented. Genetics and diagnostics, including the three-step molecular genetic testing for cystic fibrosis, are considered in detail. The diagnosis has been developed for both neonatal screening and clinical manifestations. The emergence of targeted therapy based on genetic diagnosis makes neonatal screening even more relevant and leads to an increase in life expectancy. A patient registry was established within 10 years. A detailed analysis of the diagnosis of primary ciliary dyskinesia (PCD) is given, taking into account the absence of a single “golden” standard for the diagnosis of PCD. The genetic basis of the most common hereditary diseases and modern possibilities of their diagnosis are discussed, including sequencing of genes responsible for the development of orphan diseases using standard Sanger sequencing methods and next-generation sequencing, and creating multigene panels.Conclusion. New molecular diagnostic methods will help to understand the nature of orphan lung diseases, study their epidemiology, and develop new diagnostic algorithms. The study of the genetic causes of rare diseases may serve as a basis for the development of targeted therapy.

WFS1 autosomal dominant variants linked with hearing loss: update on structural analysis and cochlear implant outcome

BackgroundWolfram syndrome type 1 gene (WFS1), which encodes a transmembrane structural protein (wolframin), is essential for several biological processes, including proper inner ear function. Unlike the recessively inherited Wolfram syndrome, WFS1 heterozygous variants cause DFNA6/14/38 and wolfram-like syndrome, characterized by autosomal dominant nonsyndromic hearing loss, optic atrophy, and diabetes mellitus. Here, we identified two WFS1 heterozygous variants in three DFNA6/14/38 families using exome sequencing. We reveal the pathogenicity of the WFS1 variants based on three-dimensional (3D) modeling and structural analysis. Furthermore, we present cochlear implantation (CI) outcomes in WFS1-associated DFNA6/14/38 and suggest a genotype-phenotype correlation based on our results and a systematic review.MethodsWe performed molecular genetic test and evaluated clinical phenotypes of three WFS1-associated DFNA6/14/38 families. A putative WFS1–NCS1 interaction model was generated, and the impacts of WFS1 variants on stability were predicted by comparing intramolecular interactions. A total of 62 WFS1 variants associated with DFNA6/14/38 were included in a systematic review.ResultsOne variant is a known mutational hotspot variant in the endoplasmic reticulum (ER)-luminal domain WFS1(NM_006005.3) (c.2051 C > T:p.Ala684Val), and the other is a novel frameshift variant in transmembrane domain 6 (c.1544_1545insA:p.Phe515LeufsTer28). The two variants were pathogenic, based on the ACMG/AMP guidelines. Three-dimensional modeling and structural analysis show that non-polar, hydrophobic substitution of Ala684 (p.Ala684Val) destabilizes the alpha helix and contributes to the loss of WFS1-NCS1 interaction. Also, the p.Phe515LeufsTer28 variant truncates transmembrane domain 7–9 and the ER-luminal domain, possibly impairing membrane localization and C-terminal signal transduction. The systematic review demonstrates favorable outcomes of CI. Remarkably, p.Ala684Val in WFS1 is associated with early-onset severe-to-profound deafness, revealing a strong candidate variant for CI.ConclusionsWe expanded the genotypic spectrum of WFS1 heterozygous variants underlying DFNA6/14/38 and revealed the pathogenicity of mutant WFS1, providing a theoretical basis for WFS1-NCS1 interactions. We presented a range of phenotypic traits for WFS1 heterozygous variants and demonstrated favorable functional CI outcomes, proposing p.Ala684Val a strong potential marker for CI candidates.

A fluidic platform for mobility evaluation of zebrafish with gene deficiency.

Zebrafish is a suitable animal model for molecular genetic tests and drug discovery due to its characteristics including optical transparency, genetic manipulability, genetic similarity to humans, and cost-effectiveness. Mobility of the zebrafish reflects pathological conditions leading to brain disorders, disrupted motor functions, and sensitivity to environmental challenges. However, it remains technologically challenging to quantitively assess zebrafish's mobility in a flowing environment and simultaneously monitor cellular behavior in vivo. We herein developed a facile fluidic device using mechanical vibration to controllably generate various flow patterns in a droplet housing single zebrafish, which mimics its dynamically flowing habitats. We observe that in the four recirculating flow patterns, there are two equilibrium stagnation positions for zebrafish constrained in the droplet, i.e., the "source" with the outward flow and the "sink" with the inward flow. Wild-type zebrafish, whose mobility remains intact, tend to swim against the flow and fight to stay at the source point. A slight deviation from streamline leads to an increased torque pushing the zebrafish further away, whereas zebrafish with motor neuron dysfunction caused by lipin-1 deficiency are forced to stay in the "sink," where both their head and tail align with the flow direction. Deviation angle from the source point can, therefore, be used to quantify the mobility of zebrafish under flowing environmental conditions. Moreover, in a droplet of comparable size, single zebrafish can be effectively restrained for high-resolution imaging. Using the proposed methodology, zebrafish mobility reflecting pathological symptoms can be quantitively investigated and directly linked to cellular behavior in vivo.

Clinical and genetic characteristics of 9 rare cases with coexistence of dual genetic diagnoses

Objective: To analyze the clinical and genetic characteristics of pediatric patients with dual genetic diagnoses (DGD). Methods: Clinical and genetic data of pediatric patients with DGD from January 2021 to February 2022 in Peking University First Hospital were collected and analyzed retrospectively. Results: Among the 9 children, 6 were boys and 3 were girls. The age of last visit or follow-up was 5.0 (2.7,6.8) years. The main clinical manifestations included motor retardation, mental retardation, multiple malformations, and skeletal deformity. Cases 1-4 were all all boys, showed myopathic gait, poor running and jumping, and significantly increased level of serum creatine kinase. Disease-causing variations in Duchenne muscular dystrophy (DMD) gene were confirmed by genetic testing. The 4 children were diagnosed with DMD or Becker muscular dystrophy combined with a second genetic disease, including hypertrophic osteoarthropathy, spinal muscular atrophy, fragile X syndrome, and cerebral cavernous malformations type 3, respectively. Cases 5-9 were clinically and genetically diagnosed as COL9A1 gene-related multiple epiphyseal dysplasia type 6 combined with NF1 gene-related neurofibromatosis type 1, COL6A3 gene-related Bethlem myopathy with WNT1 gene-related osteogenesis imperfecta type XV, Turner syndrome (45, X0/46, XX chimera) with TH gene-related Segawa syndrome, Chromosome 22q11.2 microduplication syndrome with DYNC1H1 gene-related autosomal dominant lower extremity-predominant spinal muscular atrophy-1, and ANKRD11 gene-related KBG syndrome combined with IRF2BPL gene-related neurodevelopmental disorder with regression, abnormal movement, language loss and epilepsy. DMD was the most common, and there were 6 autosomal dominant diseases caused by de novo heterozygous pathogenic variations. Conclusions: Pediatric patients with coexistence of double genetic diagnoses show complex phenotypes. When the clinical manifestations and progression are not fully consistent with the diagnosed rare genetic disease, a second rare genetic disease should be considered, and autosomal dominant diseases caused by de novo heterozygous pathogenic variation should be paid attention to. Trio-based whole-exome sequencing combining a variety of molecular genetic tests would be helpful for precise diagnosis.

A SPART missense mutation causes Troyer syndrome in two siblings

Abstract Troyer syndrome, a rare autosomal-recessive disorder, manifests with complex spastic paraplegia. In Ohio, Amish families were the first to be reported with this syndrome. The neurological and musculoskeletal symptoms include short stature, dysarthria, distal amyotrophy, and spastic paralysis. A mutation in the SPGP20 gene, located on chromosome 13q13, results in the loss of the Spartin proteins, the source of pathogenicity. Spartin is a multifunctional protein thought to be crucial for neuron viability. Here, we report the clinical and molecular diagnostic findings of Troyer syndrome in two siblings from a consanguineous family in Saudi Arabia. The clinical phenotypes of the patients showed short stature and intellectual disability. The molecular genetic test, including whole-exome sequencing, was performed to identify and confirm the causative variant. A homozygous missense mutation in the SPART variant c.988A&gt;G p.(Met330Val) was identified through molecular analysis. According to HGMD Professional 2021.1, this variant has previously been described as a disease-causing intellectual disability and Troyer syndrome.

DYGGVE-MELCHIOR-CLAUSEN SYNDROME-A CASE REPORT

We report a case of short stature and bony deformities. The child wasevaluated and differential diagnosis of Pseudoachondroplasia, Morquio and Dyggve- Melchior- Clausen syndrome were considered. Conrmation of thediagnosis was done by molecular genetic testing and required conrmation in the index case for planning further conception.This casereport highlights the utility of molecular genetic testing in denitive diagnosis ofthe proband.

The Complex Cyto-Molecular Landscape of Thyroid Nodules in Pediatrics.

Thyroid fine-needle aspiration (FNA) is a commonly used diagnostic cytological procedure in pediatric patients for the evaluation of thyroid nodules, triaging them for the detection of thyroid cancer. In recent years, greater attention has been paid to thyroid FNA in this setting, including the use of updated ultrasound score algorithms to improve accuracy and yield, especially considering the theoretically higher risk of malignancy of these lesions compared with the adult population, as well as to minimize patient discomfort. Moreover, molecular genetic testing for thyroid disease is an expanding field of research that could aid in distinguishing benign from cancerous nodules and assist in determining their clinical management. Finally, artificial intelligence tools can help in this task by performing a comprehensive analysis of all the obtained data. These advancements have led to greater reliance on FNA as a first-line diagnostic tool for pediatric thyroid disease. This review article provides an overview of these recent developments and their impact on the diagnosis and management of thyroid nodules in children.

Pediatric-type high-grade neuroepithelial tumors with CIC gene fusion share a common DNA methylation signature

Pediatric neoplasms in the central nervous system (CNS) show extensive clinical and molecular heterogeneity and are fundamentally different from those occurring in adults. Molecular genetic testing contributes to accurate diagnosis and enables an optimal clinical management of affected children. Here, we investigated a rare, molecularly distinct type of pediatric high-grade neuroepithelial tumor (n = 18), that was identified through unsupervised visualization of genome-wide DNA methylation array data, together with copy number profiling, targeted next-generation DNA sequencing, and RNA transcriptome sequencing. DNA and/or RNA sequencing revealed recurrent fusions involving the capicua transcriptional repressor (CIC) gene in 10/10 tumor samples analyzed, with the most common fusion being CIC::LEUTX (n = 9). In addition, a CIC::NUTM1 fusion was detected in one of the tumors. Apart from the detected fusion events, no additional oncogenic alteration was identified in these tumors. The histopathological review demonstrated a morphologically heterogeneous group of high-grade neuroepithelial tumors with positive immunostaining for markers of glial differentiation in combination with weak and focal expression of synaptophysin, CD56 and CD99. All tumors were located in the supratentorial compartment, occurred during childhood (median age 8.5 years) and typically showed early relapses. In summary, we expand the spectrum of pediatric-type tumors of the CNS by reporting a previously uncharacterized group of rare high-grade neuroepithelial tumors that share a common DNA methylation signature and recurrent gene fusions involving the transcriptional repressor CIC. Downstream functional consequences of the fusion protein CIC::LEUTX and potential therapeutic implications need to be further investigated.

Magnetic-resonance imaging semiology of cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) in subjects from Russian Federation

Introduction. Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is common variant of small vessel disease. Determination of neuroimaging markers have already contributed at an early stage to the more timely diagnosis of the CADASIL.Objective. The aim of the study was to determine probable CADASIL magnetic resonance imaging (MRI) biomarkers for patients from the Russian population.Material and methods. The study included 16 patients. Diagnosis of CADASIL was suspected based on clinical data and an MRI and confi rmed by a molecular genetic study of the NOTCH3 gene. Brain MRI data from 16 patients and spinal cord MRI data from 5 patients were collected and analyzed. Cerebrospinal fl uid (CSF) analysis was performed for diff erential diagnosis in 2 subjects.Results. White matter hyperintensity was observed in 100% of cases. The involvement of the anterior temporal lobes and external capsules was revealed in 62 and 68% of subjects respectively. Multiple lacunar infarcts are characteristic for CADASIL. Microbleeds were detected in 27% of patients. Brain atrophy was observed in 56% of cases. Lesions in corpus callosum and cervical spinal cord were found in 2 patients.Conclusion. Detection of white matters hyperintensities in frontal and parietal lobes, multiple lacunar infarcts with suspected CADASIL makes it possible to select the group of patients for further molecular genetic testing in order to detect mutations in NOTCH3 gene.

Development and characterization of reference materials for EGFR, KRAS, NRAS, BRAF, PIK3CA, ALK, and MET genetic testing.

Along with the dramatic development of molecular diagnostic testing for the detection of oncogene variations, reference materials (RMs) have become increasingly important in performance evaluation of genetic testing. In this study, we built a set of RMs for genetic testing based on next-generation sequencing (NGS). Solid tumor tissues were selected as the samples of RMs for preparation. NGS was used to determine and validate the variants and the mutation frequency in DNA samples. Digital PCR was used to determine the copy numbers of RNA samples. The performance of the RMs was validated by six laboratories. Thirty common genetic alterations were designed based on these RMs. RMs consisted of a positive reference, a limit of detection reference, and a negative reference. The validation results confirmed the performance of the RMs. These RMs may be an attractive tool for the development, validation, and quality monitoring of molecular genetic testing.

CDNA sequencing increases the molecular diagnostic yield in Chediak-Higashi syndrome.

Introduction: Chediak-Higashi syndrome (CHS) is rare autosomal recessive disorder caused by bi-allelic variants in the Lysosomal Trafficking Regulator (LYST) gene. Diagnosis is established by the detection of pathogenic variants in LYST in combination with clinical evidence of disease. Conventional molecular genetic testing of LYST by genomic DNA (gDNA) Sanger sequencing detects the majority of pathogenic variants, but some remain undetected for several individuals clinically diagnosed with CHS. In this study, cDNA Sanger sequencing was pursued as a complementary method to identify variant alleles that are undetected by gDNA Sanger sequencing and to increase molecular diagnostic yield. Methods: Six unrelated individuals with CHS were clinically evaluated and included in this study. gDNA Sanger sequencing and cDNA Sanger sequencing were performed to identify pathogenic LYST variants. Results: Ten novel LYST alleles were identified, including eight nonsense or frameshift variants and two in-frame deletions. Six of these were identified by conventional gDNA Sanger sequencing; cDNA Sanger sequencing was required to identify the remaining variant alleles. Conclusion: By utilizing cDNA sequencing as a complementary technique to identify LYST variants, a complete molecular diagnosis was obtained for all six CHS patients. In this small CHS cohort, the molecular diagnostic yield was increased, and canonical splice site variants identified from gDNA Sanger sequencing were validated by cDNA sequencing. The identification of novel LYST alleles will aid in diagnosing patients and these molecular diagnoses will also lead to genetic counseling, access to services and treatments and clinical trials in the future.

Adult-Onset Genetic Leukoencephalopathies With Movement Disorders

Genetic leukoencephalopathies (GLEs) are a group of white matter abnormalities with heterogeneous radiological and phenotypic features. Although these conditions have mostly been described in children, adult-onset cases are increasingly recognized owing to the widespread use of neuroimaging and advances in molecular genetic testing. The disease course is often progressive with a varied spectrum of presentations, trapping neurologists in the dilemma of differential diagnosis. Movement disorders are among the most common symptoms, and their diversity makes diagnosis challenging. In this review, we focus on adult-onset GLEs with movement disorders and offer a step-by-step diagnostic approach by clarifying the phenomenology of movement, advising investigations for acquired causes, describing the clinical and radiological clues to each disease, emphasizing the limitations of advanced molecular testing, and discussing the future application of artificial intelligence. We provide a list summarizing the leukoencephalopathies associated with different categories of movement disorders. In addition to guiding clinicians on how to narrow the list of differential diagnoses with the tools currently available, another aim of this review is to emphasize the inevitable trend toward applying advanced technology in diagnosing these difficult diseases.

Molecular genetic basis of epidermolysis bullosa.

Epidermolysis bullosa (EB) is an inherited disorder of skin fragility, caused by mutations in a large number of genes associated with skin integrity and dermal-epidermal adhesion. Skin fragility is manifested by a decrease in resistance to external mechanical influences, the clinical signs of which are the formation of blisters, erosions and wounds on the skin and mucous membranes. EB is a multisystemic disease and characterized by a wide phenotypic spectrum with extracutaneous complications in severe types, besides the skin and mucous membranes, with high mortality. More than 30 clinical subtypes have been identified, which are grouped into four main types: simplex EB, junctional EB, dystrophic EB and Kindler syndrome. To date, pathogenic variants in 16 different genes are associated with EB and encode proteins that are part of the skin anchoring structures or are signaling proteins. Genetic mutations cause dysfunction of cellular structures, differentiation, proliferation and apoptosis of cells, leading to mechanical instability of the skin. The formation of reduced proteins or decrease in their level leads mainly to functional disorders, forming mild or intermediate severe phenotypes. Absent protein expression is a result of null genetic variants and leads to structural abnormalities, causing a severe clinical phenotype. For most of the genes involved in the pathogenesis of EB, certain relationships have been established between the type and position of genetic variant and the severity of the clinical manifestations of the disease. Establishing an accurate diagnosis depends on the correlation of clinical, genealogical and immunohistological data in combination with molecular genetic testing. In general, the study of clinical, genetic and ultrastructural changes in EB has significantly expanded the understanding of the natural history of the disease and supplemented the data on genotype-phenotype correlations, promotes the search and study of epigenetic and non-genetic disease modifier factors, and also allows developing approaches to radical treatment of the disease. New advances of sequencing technologies have made it possible to describe new phenotypes and study their genetic and molecular mechanisms. This article describes the pathogenetic aspects and genes that cause main and rare syndromic subtypes of EB.

Primary hyperoxaluria I, II, III types in children (review of literature)

This review presents the latest data on the classification, pathogenesis, clinical and genetic features, and therapy of primary hyperoxaluria types I, II, and III in children with autosomal recessive inheritance. ORPHA portal of orphan diseases presents genes responsible for primary hyperoxaluria type I AGXT (93598); type II and type II GRHPR (93599), type III HOGA1 (93600). Worldwide genetic studies have established the pathogenesis, clinical phenotype and genotype features of primary hyperoxaluria. The pathogenesis of primary hyperoxaluria in children is based on impaired hepatic glyoxylate metabolism. The enzyme AGT catalyzes the conversion of L-alanine and glyoxylate to pyruvate and glycine, with vitamin B6 (pyridoxine) serving as a coenzyme for this reaction. Increased production of endogenous oxalate leads to increased blood oxalate concentrations and urinary oxalate excretion with the formation of renal calcium oxalate crystals and radiopaque concrements (calcium oxalate monohydrate – vevelite, calcium oxalate dihydrate – vedellite). High risk of progression to chronic kidney disease in primary hyperoxaluria in children of types I and II. Systemic oxalosis develops with increasing serum oxalate levels and the formation of calcium oxalate crystals with deposition in many organs and tissues. Therapy for primary hyperoxaluria in children includes: hydration (3l/m2/day) and citrates 100–150 mg/kg/day (potassium citrate 0.3–0.5 mmol/kg/day), pyridoxine at a dose of 5 to 20 mg/kg/day for vitamin B6 sensitive type I primary hyperoxaluria. Administration of oxalobacter formigenes and diet is effective. Combined liver and then kidney transplantation or simultaneous liver and kidney transplantation in patients with type I PH in B6-insensitive and isolated liver transplantation in B6-sensitive variants are performed. Timely molecular genetic testing in children with nephrocalcinosis makes it possible to establish a clinical and genetic diagnosis of type I, II, III PH, to carry out a personalised approach to treatment and to predict future health status.

Structural analysis of pathogenic TMPRSS3 variants and their cochlear implantation outcomes of sensorineural hearing loss

TMPRSS3, a type II transmembrane serine protease, is involved in various biological processes including the development and maintenance of the inner ear. Biallelic variants in TMPRSS3 typically result in altered protease activity, causing autosomal recessive non-syndromic hearing loss (ARNSHL). Structural modeling has been conducted to predict the pathogenicity of TMPRSS3 variants and to gain a better understanding of their prognostic correlation. The mutant-driven changes in TMPRSS3 had substantial impacts on neighboring residues, and the pathogenicity of variants was predicted based on their distance from the active site. However, a more in-depth analysis of other factors, such as intramolecular interactions and protein stability, which affect proteolytic activities is yet to be conducted for TMPRSS3 variants. Among 620 probands who provided genomic DNA for molecular genetic testing, eight families with biallelic TMPRSS3 variants that were segregated in a trans configuration were included. Seven different mutant alleles, either homozygous or compound heterozygous, contributed to TMPRSS3-associated ARNSHL, expanding the genotypic spectrum of disease-causing TMPRSS3 variants. Through three-dimensional modeling and structural analysis, TMPRSS3 variants compromise protein stability by altering intramolecular interactions, and each mutant differently interacts with the serine protease active site. Furthermore, the changes in intramolecular interactions leading to regional instability correlate with the results of functional assay and residual hearing function, but overall stability predictions do not. Our findings also build on previous evidence indicating that most recipients with TMPRSS3 variants have favorable cochlear implantation (CI) outcomes. We found that age at CI was significantly correlated with speech performance outcomes; genotype was not correlated with these outcomes. Collectively, the results of this study contribute to a more structural understanding of the underlying mechanisms of ARNSHL caused by TMPRSS3 variants.