MSL2 variants lead to a neurodevelopmental syndrome with lack of coordination, epilepsy, specific dysmorphisms, and a distinct episignature.

Epigenetic dysregulation has emerged as an important etiological mechanism of neurodevelopmental disorders (NDDs). Pathogenic variation in epigenetic regulators can impair deposition of histone post-translational modifications leading to aberrant spatiotemporal gene expression during neurodevelopment. The male-specific lethal (MSL) complex is a prominent multi-subunit epigenetic regulator of gene expression and is responsible for histone 4 lysine 16 acetylation (H4K16ac). Using exome sequencing, here we identify a cohort of 25 individuals with heterozygous de novo variants in MSL complex member MSL2. MSL2 variants were associated with NDD phenotypes including global developmental delay, intellectual disability, hypotonia, and motor issues such as coordination problems, feeding difficulties, and gait disturbance. Dysmorphisms and behavioral and/or psychiatric conditions, including autism spectrum disorder, and to a lesser extent, seizures, connective tissue disease signs, sleep disturbance, vision problems, and other organ anomalies, were observed in affected individuals. As a molecular biomarker, a sensitive and specific DNA methylation episignature has been established. Induced pluripotent stem cells (iPSCs) derived from three members of our cohort exhibited reduced MSL2 levels. Remarkably, while NDD-associated variants in two other members of the MSL complex (MOF and MSL3) result in reduced H4K16ac, global H4K16ac levels are unchanged in iPSCs with MSL2 variants. Regardless, MSL2 variants altered the expression of MSL2 targets in iPSCs and upon their differentiation to early germ layers. Our study defines an MSL2-related disorder as an NDD with distinguishable clinical features, a specific blood DNA episignature, and a distinct, MSL2-specific molecular etiology compared to other MSL complex-related disorders.

A Cautionary Tale of Hypertrophic Cardiomyopathy-From "Benign" Left Ventricular Hypertrophy to Stroke, Atrial Fibrillation, and Molecular Genetic Diagnostics: A Case Report and Review of Literature.

This case report concerns a 48-year-old man with a history of ischemic stroke at the age of 41 who reported cardiac hypertrophy, registered in his twenties when explained by increased physical activity. Family history was positive for a mother with permanent atrial fibrillation from her mid-thirties. At the age of 44, he had a first episode of persistent atrial fibrillation, accompanied by left atrial thrombosis while on a direct oral anticoagulant. He presented at our clinic at the age of 45 with another episode of persistent atrial fibrillation and decompensated heart failure. Echocardiography revealed a dilated left atrium, reduced left ventricular ejection fraction, and an asymmetric left ventricular hypertrophy. Cardiac magnetic resonance was positive for a cardiomyopathy with diffuse fibrosis, while slow-flow phenomenon was present on coronary angiography. Genetic testing by whole-exome sequencing revealed three variants in the patient, c.309C > A, p.His103Gln in the ACTC1 gene, c.116T > G, p.Leu39Ter in the PLN gene, and c.5827C > T, p.His1943Tyr in the SCN5A gene, the first two associated with hypertrophic cardiomyopathy and the latter possibly with familial atrial fibrillation. This case illustrates the need for advanced diagnostics in unexplained left ventricular hypertrophy, as hypertrophic cardiomyopathy is often overlooked, leading to potentially debilitating health consequences.

Novel insights on GTPBP3-associated hypertrophic cardiomyopathy.

About 100 genes have been associated with cardiomyopathies with genotype-phenotype correlations often hard to establish. Genetic testing may help to confirm the genetic diagnosis and assess the risk of inheritance in the family. A 25-year old male with hypertrophic cardiomyopathy and fasciculoventricular accessory pathway was referred for genetic testing by his cardiologist. Targeted PRKAG2 screening and whole-exome sequencing were performed, followed by Sanger sequencing segregation analysis in the family. The PRKAG2 gene screening was negative. Whole-exome sequencing revealed the following four variants in the patient: c.181G>C (p.Ala61Pro) and c.1199C>T (p.Thr400Met) in the GTPBP3 gene, as well as c.752C>T (p.Thr251Ile) and c.1760C>T (p.Pro587Leu) in the POLG gene. Family segregation analysis showed that the patient's mother is a carrier of variant c.181G>C and the patient's paternal grandmother is a carrier of variant c.1199C>T in the GTPBP3 gene, which is in accordance with an autosomal recessive model of inheritance of the disease. Both variants in the POLG are found paternally inherited in the patient's healthy half-brother, thus are not considered disease-causing. GTPBP3 variants have been reported in patients with hypertrophic cardiomyopathy, associated with combined oxidative phosphorylation deficiency 23. These novel variants represent the probable cause of the observed clinical symptoms in the patient.

Diagnostic, grading and prognostic role of a restricted miRNAs signature in primary and metastatic brain tumours. Discussion on their therapeutic perspectives.

At present, brain tumours remain one of the "hard-to-treat" malignancies with minimal improvement in patients' survival. Recently, miRNAs have been shown to correlate with oncogenesis and metastasis and have been investigated as potential biomarkers for diagnosis, prognosis and therapy prediction in different brain malignancies. The aim of the current study was to select an accurate and affordable brain tumour detection and grading approach. In the present study, we analysed the applicability of a restricted miRNA signature that could differentiate among patients with primary as well as metastatic brain tumours. Fresh tumour tissues were collected from Bulgarian patients (n = 38), including high-grade gliomas (n = 23), low-grade gliomas (n = 10) and brain metastases (n = 5) from lung cancer. Total RNAs enriched with microRNAs were isolated and differentially expressed miRNAs were analyzed by RT-qPCR using TaqMan Advanced miRNA assay. We selected a signature of miR-21, miR-10b, miR-7, miR-491 that showed good diagnostic potential in high-grade gliomas, low-grade gliomas and brain metastases compared with normal brain tissues. Our results showed that miR-10b could reliably differentiate brain metastases from high-grade gliomas, while miR-491 could distinguish low-grade from high-grade gliomas and brain metastases from low-grade gliomas. We observed that miR-21 and miR-7 correlated with disease recurrence, survival status and the Karnofsky Performance Status. The selected signature of miR-7, miR-21, miR-10b and miR-491 could be used as a highly accurate diagnostic, grading and prognostic biomarker in differentiating various types of brain tumours. Our data suggest that the 4-miRNAs signature could be further analysed for predicting treatment response and for future miRs-based targeted therapy. The ongoing studies on miRs-based targeted therapy related to our selected miRNA signature are also reviewed.

Human cytomegalovirus DNA detection in a recurrent glioblastoma multiforme tumour, but not in whole blood: a case report and discussion about the HCMV latency and therapy perspectives.

In the current study, a 58-year-old male patient presented with recurrent glioblastoma multiforme (GBM). The patient underwent surgical resection, 4 months earlier, followed by radiotherapy and chemotherapy. During the second surgical intervention, tumour tissue and whole blood were sampled and analysed for human cytomegalovirus (HCMV) DNA, immediate early (IE) mRNA and pp65 mRNA. HCMV DNA was detected only in the recurrent tumour tissue but not in the whole blood. Neither IE mRNA nor pp65 mRNA was expressed. Our result suggests HCMV latency in the brain tumour with detectable level of viral DNA. More data are needed to understand the HCMV infection chronology in the brain tumours but our data could be important for further studies of HCMV antigens on the tumour surface and anti-GBM therapy.

First case of Roma ethnic origin with Andermann syndrome: A novel frameshift mutation in exon 20 of SLC12A6 gene.

Andermann syndrome (AS) is caused by mutation of SLC12A6 gene. It comprises severe progressive sensory and motor neuropathy with early onset, varying degree of agenesis of corpus callosum (ACC) and mental retardation. AS occurs occasionally among population outside the northeastern Quebec-Saguenay-Lac- St-Jean and Charlevoix regions, inhabited by French Canadians. None of the described patients were of Roma ethnic origin. We present an 8-month-old infant of Roma ethnic origin with AS, caused by a novel frame shift mutation c.2604delT,p.(Asp868GlufsTer11) in exon 20 of SLC12A6 gene. Our case presented with several atypical findings: clinical presentation resembling "spinal muscular atrophy plus" syndrome; tongue fasciculations, which are not reported in the literature; early contractures of the wrists; normal motor action potentials and preserved sensory action potentials. Our patient is the first of Roma origin from nonconsanguineous parents, which suggests that this mutation might be widespread in the Roma population, although screening for this mutation in 140 alleles from Roma individuals originating from the same geographic region did not reveal further carriers, implying the mutation is rare. We recommend that Roma patients presenting with the clinical phenotype of AS should be tested for this mutation primarily.

Molecular-genetic diagnostics of von Hippel-Lindau syndrome (VHL) in Bulgaria: first complex mutation event in the VHL gene.

Von Hippel-Lindau syndrome is an autosomal-dominant disease characterized by the formation of various tumours and cysts in many different parts of the body. Von Hippel-Lindau syndrome is caused by VHL gene mutations leading to production of impaired tumor suppressor Von Hippel-Lindau syndrome protein or its complete absence. PURPOSE: To study five patients with clinically suspected Von Hippel-Lindau syndrome, who were referred for molecular genetic testing. METHODS: Sanger sequencing of the coding regions of the VHL gene. RESULTS: Five clinically relevant germline mutations were detected. One of the pathogenic variants has not been previously reported. This novel mutation is a complex mutation event combining a duplication and an indel, rearranging exon 3 of the VHL gene - c. [516_517dupGTCAAGCCT; 532_542delCTGGACATCGTinsATTA], p. (Glu173Serfs*4). CONCLUSION: Overall, our results showed that the diagnosis of Von Hippel-Lindau syndrome in our country is difficult most probably because of its heterogeneous clinical manifestation and insufficient knowledge on the diagnostic criteria for the disease. From genetic point of view our results add some novel data on the mutation profile of the VHL gene. In order to prove or revise the diagnosis, early genetic testing is strongly recommended in affected patients and their family members to ensure appropriate follow-up and treatment of the malignancies.

Males with Paternally Inherited MKRN3 Mutations May Be Asymptomatic.

Ten girls with sporadic central precocious puberty were screened for mutations in the maternally imprinted gene MKRN3. We detected 1 novel frameshift mutation (p.Arg351Serfs*44) and a previously described mutation (p.Pro161Argfs*10). In the course of investigating the family, genetic analysis found 2 asymptomatic males with paternally inherited MKRN3 mutations, which has not been reported in previous studies.

Clinical Spectrum and Genetic Variability in Bulgarian Patients with Niemann-Pick Disease Type C.

BACKGROUND: Niemann-Pick disease type C (NP-C) is a rare autosomal-recessive lysosomal storage disorder caused by mutations in either the NPC1 (in 95% of cases) or the NPC2 gene. METHODS: In a prospective, observational cohort study, all Bulgarian patients diagnosed with NP-C to date (since 2010) underwent detailed neurological examination and neuro-ophthalmological, neuropsychological and psychiatric evaluations, as well as brain MRI, abdominal ultrasound and hearing tests. Plasma chitotriosidase was also measured, when possible. RESULTS: The Bulgarian national NP-C cohort comprised 11 patients who were diagnosed based on molecular genetic analysis (n = 9) and/or filipin staining of skin fibroblasts (n = 3). The mean age at onset was 14.4 (SD 8.3). Diagnoses were achieved 1-23 years after initial clinical presentation. All patients who underwent genetic mutation analysis were compound heterozygotes: a total of 12 NPC1 mutations were recorded, 5 of which were novel. Two patients had late-infantile onset, 4 had juvenile onset, and the remaining 5 had the adult-onset form of NP-C. Initial symptoms were neurological in 9 patients, visceral in one, and predominantly psychiatric in another. Vertical gaze palsy was present in all patients. Dysarthria, pyramidal involvement, cognitive impairment, and organomegaly with varied severity were observed in 10 of them. Ataxia was present in 9 and dystonia in 7. Four patients had epileptic seizures, and gelastic cataplexy was reported in 5. Brain MRI revealed hyperintense white matter lesions in 5 patients and cortical and/or cerebellar atrophy in 4. CONCLUSIONS: This Bulgarian NP-C cohort showed wide variability in terms of NPC1 mutations and predominant forms of neurological involvement. Diagnosing NP-C is challenging, and it was often delayed in this cohort due to the heterogeneity of patients' clinical signs and symptoms.

First cases of pyridoxine-dependent epilepsy in Bulgaria: novel mutation in the ALDH7A1 gene.

Pyridoxine-dependent epilepsy (PDE) is a rare autosomal recessive disorder characterized by intractable seizures in neonates and infants. The seizures cannot be controlled with antiepileptic medications but respond both clinically and electrographically to large daily supplements of pyridoxine (vitamin B6). PDE is caused by mutations in the ALDH7A1 gene. Molecular genetic analysis of the ALDH7A1 gene was performed in seven patients, referred with clinical diagnosis of PDE. Mutations were detected in a dizygotic twin pair and a non-related boy with classical form of PDE. Direct sequencing of the ALDH7A1 gene revealed one novel (c.297delG, p.Trp99*) and two already reported (c.328C>T, p.Arg110*; c.584A>G, p.Asn195Ser) mutations. Here, we report the first genetically proven cases of PDE in Bulgaria.

Molecular and clinico-histological data in aggressive prostate cancer patients from Bulgaria.

PURPOSE: Metastatic prostate cancer (PCa) is one of the leading causes of death in men worldwide. We report Bulgarian patients with strongly aggressive, castration-resistant PCa. METHODS: PCA3 overexpression, GSTP1 promoter hyper-methylation, TMPRSS2-ERG gene fusions, IVS1-27G>A in the KLF6 gene and mutations in androgen receptor (AR) gene, for diagnostic purposes were assessed. PCR, real-time PCR (RT-PCR), sequencing, and bisulfite conversion of DNA were applied. We correlated the molecular data to the histological and clinical findings. RESULTS: The obtained molecular profile in 11 PCa Bulgarian patients coincided with the clinico-histological data of strongly aggressive PCa. Association was detected between the tumor stage (assessed by TNM as T3 and T4) and the detected molecular profile of aggressive cancer behavior with one exception, assessed as T2. None of our patients had positive family history of prostate cancer and no somatic mutations were detected in the AR gene. All patients showed normal genotype with respect to the KLF6 IVS1- 27G>A polymorphism. The rest of the markers were positive in fresh prostatic tissues and biopsies from all patients, whereas only one blood sample showed triple positive result. CONCLUSIONS: The appearance of PCa-specific markers in blood was considered as a predictor for a PCa (micro) dissemination into the circulation. The GSTP1 promoter hypermethylation is the earliest epigenetic alteration, which indicates cancerous changes and the first and long-lasting marker that is detectable in blood circulation. The molecular profile needs to be strictly monitored during treatment, which is of great help in determining the patient's individual response to therapy.

Genetic Modifiers of ALS: The Impact of Chromogranin B P413L in a Bulgarian ALS Cohort.

This study investigated the role of the CHGB P413L variant (rs742710) in sporadic amyotrophic lateral sclerosis (sALS) within the Bulgarian population. We analyzed 150 patients with sALS (85 male and 65 female) for the presence of this variant, its potential impact on disease susceptibility, and age of onset. Genotyping was performed using PCR amplification and direct Sanger sequencing. Statistical analyses included comparisons with control data from GnomAD v2.1.1, one-way ANOVA, and Kaplan-Meier survival analysis. Results revealed a higher frequency of the minor T allele in patients with sALS compared to all control groups and a statistically significant increase in carrier genotypes compared to non-Finnish Europeans (χ2 = 15.4572, p = 0.000440). However, the impact on age of onset was less clear, with no statistically significant differences observed across genotypes or between carriers and non-carriers of the T allele. Kaplan-Meier analysis suggested a potential 2.5-year-earlier onset in T allele carriers, but the small sample size of carriers limits the reliability of this finding. Our study provides evidence for an association between the CHGB P413L variant and sALS susceptibility in the Bulgarian population, while its effect on age of onset remains uncertain, highlighting the need for further research in larger, diverse cohorts.

CYP21A2 Intron 2 Genetic Variants Might Be Associated with the Clinical Characteristics of Women with PCOS.

AIMS: Pathogenic variants in the CYP21A2 gene are related to the classic and non-classic forms of congenital adrenal hyperplasia (CAH). However, the role of CAH carrier status in the clinical presentation of polycystic ovarian syndrome (PCOS) is still unclear. Moreover, the possible associations of different CYP21A2 gene polymorphisms with metabolic and reproductive abnormalities in PCOS have not been investigated. Therefore, the present study aims to examine the prevalence of the most common CYP21A2 pathogenic variant IVS2-13A/C>G (c.293-13A/C>G) in Eastern European women with PCOS and to evaluate the associations between common intron 2 genetic polymorphisms and the clinical symptoms of the patients. METHODS: Sixty consecutively recruited women with PCOS were genotyped for the CYP21A2 intron 2 IVS2-13A/C>G genetic variant. Additionally, CYP21A2 intron 2 polymorphic variants rs6453 (c.293-44G>T), rs6451 (c.293-67C>A/G), rs369651496 (c.293-104del), and rs6474 (c.308G>A/p.R103L) were tested and described. The clinical and hormonal characteristics were compared in women with PCOS and with polymorphic and wild-type genotypes. RESULTS: The heterozygous CYP21A2 pathogenic variant IVS2-13A/C>G was found in one of the investigated PCOS patients (1.67%) with a non-hyperandrogenic type of PCOS. The presence of the rs6453 (c.293-44G>T) T-allele was associated with increased levels of DHEAS (15.18 vs. 9.14 µmol/L, p = 0.003) compared to the wild-type genotype in the investigated group. The rs6451 (c.293-67C>A/G) minor alleles were associated with an earlier age of menarche in the patients (12.0 vs. 13.0 years, p = 0.007). The polymorphic rs369651496 minor 6G allele was related to a better lipid profile in the women with PCOS, while the rs6474 variant modulated the blood pressure of the patients. CONCLUSIONS: The presence of CYP21A2 genetic minor alleles of rs6467 (IVS2-13A/C, c.293-13A/C), rs6453 (c.293-44G>T), rs6451 (c.293-67C>A/G), rs369651496 (c.293-104del), and rs6474 (c.308G>A/p.R103L) might modulate the adrenal androgens, age of menarche, and metabolic features in women with PCOS. Further studies on 21-hydroxylase genetic variants (pathogenic and polymorphisms) in different ethnic groups might help reveal the influence of adrenal steroidogenesis on PCOS development, clinical manifestations, and lifelong cardiovascular risks.

Clinical characteristics and multimodal imaging can help diagnosing and treating mild malformation of cortical development with oligodendroglial hyperplasia and epilepsy.

OBJECTIVE: Mild malformation of cortical development with oligodendroglial hyperplasia and epilepsy (MOGHE) is a recently described, histopathologically and molecularly defined (SLC35A2-mutated) type of cortical malformation. Although increasingly recognized, the diagnosis of MOGHE remains a challenge. We present the characteristics of the first six patients diagnosed in Bulgaria, with the aim to facilitate identification, proper presurgical evaluation, and surgical treatment approach in this disease. METHODS: Revision of histopathological specimens of 202 patients operated on for drug-resistant focal epilepsy identified four cases with MOGHE. Another two were suggested, based on clinical characteristics and subsequently, were histologically confirmed. Sanger SLC35A2 sequencing on paraffin-embedded or fresh-frozen brain tissue was performed. Analysis of seizure types, neuropsychological profiles, electroencephalographic (EEG), imaging features and epilepsy surgery outcomes was done. RESULTS: Three out of the six cases (50%) harbored pathogenic SLC35A2 mutations. One patient had a heterozygous somatic variant with uncertain significance. Clinical characteristics included epilepsy onset in infancy (in 100% under 3 years of age), multiple seizure types, and moderate or severe intellectual/developmental delay. Epileptic spasms with hypsarrhythmia on EEG were the initial seizure type in five patients. The subsequent seizure types resembled those in Lennox-Gastaut syndrome. The majority of the patients (n = 4) presented prominent and persisting autistic features. Magnetic resonance imaging (MRI) showed multilobar (n = 6) and bilateral (n = 3) lesions, affecting the frontal lobes (n = 5; bilaterally in three) and characterized by increased signal on T2/fluid-attenuated inversion recovery (FLAIR). Voxel-based morphometric MRI post-processing and positron emission tomography helped determining the localization and extent of the lesions and presumed epileptogenic zones. After surgery, four patients (66.7%) were seizure-free ≥2 years. Interestingly, all seizure-free patients carried somatic SLC35A2-alterations. SIGNIFICANCE: Epileptic spasms, early prominent neuropsychological disturbances, MRI-T2/FLAIR hyperintense lesions with cortico-subcortical blurring, frequently multilobar and especially frontal, can preoperatively help to suspect MOGHE. Epilepsy surgery is still the only successful treatment option in MOGHE.

MiRNA Signatures Related to Invasiveness and Recurrence in Patients With Non-Functioning Pituitary Neuroendocrine Tumors.

PURPOSE: This preliminary study aimed to analyze and identify differentially expressed miRNAs in Bulgarian patients with non-functioning pituitary neuroendocrine tumors (NFPitNET). The relationship between deregulated miRNAs and tumor invasiveness, recurrence, and size was determined. METHODS: Twenty patients with NFPitNET were selected and fresh pituitary tumor tissues were collected. RNA containing miRNAs were isolated using miRNAeasy mini kit and analyzed by quantitative real-time polymerase chain reaction (PCR) using LNA miRNA Cancer-Focus PCR Panel (Qiagen). RESULTS: Three miRNAs (miR-210-3p, miR-149-3p, and miR-29b-3p) were deregulated in invasive compared to non-invasive NFPitNETs. Differential expression of four-miRNA signatures - miRNA-17, miR-19, miR-106a, and miR-20, correlated with patient recurrence. CONCLUSION: This prospective pilot study selected a unique miRNA expression profile, that correlates with invasiveness and recurrence in non-functioning pituitary neuroendocrine tumors. Moreover, some of the selected miRNAs are reported for the first time in patients with this disease, shedding light on the molecular mechanisms involved in pituitary pathogenesis. The identified miRNAs demonstrate potential as biomarkers, deserving further investigation in a larger cohort to validate their clinical applicability.

Phenotypic Variability of LGMD 2C/R5 in a Genetically Homogenous Group of Bulgarian Muslim Roma.

Sarcoglycanopathies are among the most frequent and severe forms of autosomal recessive forms of limb-girdle muscular dystrophies (LGMDs) with childhood onset. Four subtypes are known: LGMDR3, LGMDR4, LGMDR5 and LGMDR6, which are caused, respectively, by mutations in the SGCA, SGCB, SGCG and SGCD genes. We present the clinical variability of LGMD 2C/R5 among a genetically homogeneous group of 57 patients, belonging to 35 pedigrees. Molecular genetic analysis showed that all 57 patients were homozygous for the C283Y variant. The muscles of the pelvic girdle and the trunk were affected early and were more severely affected, followed by the shoulder girdle. Macroglossia, hypertrophy of the calves, scapular winging and lumbar hyperlordosis were common in the ambulatory phase. A great intra and interfamilial variability in the clinical presentation of LGMD 2C/R5 was observed, despite having the same underlying molecular defect. Females demonstrated a relatively milder clinical course compared to males. Mean creatine phosphokinase (CK) CK levels were 20 times above normal values. Muscle computer tomography (CT) CT or MRIs showed earlier and more severe involvement of the flexor proximal limb muscles in comparison to extensor muscles.

Advancing the Understanding of Vesicle-Associated Membrane Protein 1-Related Congenital Myasthenic Syndrome: Phenotypic Insights, Favorable Response to 3,4-Diaminopyridine, and Clinical Characterization of Five New Cases.

BACKGROUND: Congenital myasthenic syndromes (CMS) are a group of inherited neuromuscular junction (NMJ) disorders arising from gene variants encoding diverse NMJ proteins. Recently, the VAMP1 gene, responsible for encoding the vesicle-associated membrane protein 1 (VAMP1), has been associated with CMS. METHODS: This study presents a characterization of five new individuals with VAMP1-related CMS, providing insights into the phenotype. RESULTS: The individuals with VAMP1-related CMS exhibited early disease onset, presenting symptoms prenatally or during the neonatal period, alongside severe respiratory involvement and feeding difficulties. Generalized weakness at birth was a common feature, and none of the individuals achieved independent walking ability. Notably, all cases exhibited scoliosis. The clinical course remained stable, without typical exacerbations seen in other CMS types. The response to anticholinesterase inhibitors and salbutamol was only partial, but the addition of 3,4-diaminopyridine (3,4-DAP) led to significant and substantial improvements, suggesting therapeutic benefits of 3,4-DAP for managing VAMP1-related CMS symptoms. Noteworthy is the identification of the VAMP1 (NM_014231.5): c.340delA; p.Ile114SerfsTer72 as a founder variant in the Iberian Peninsula and Latin America. CONCLUSIONS: This study contributes valuable insights into VAMP1-related CMS, emphasizing their early onset, arthrogryposis, facial and generalized weakness, respiratory involvement, and feeding difficulties. Furthermore, the potential efficacy of 3,4-DAP as a useful therapeutic option warrants further exploration. The findings have implications for clinical management and genetic counseling in affected individuals. Additional research is necessary to elucidate the long-term outcomes of VAMP1-related CMS.

Comparison of the ABC and ACMG systems for variant classification.

The ABC and ACMG variant classification systems were compared by asking mainly European clinical laboratories to classify variants in 10 challenging cases using both systems, and to state if the variant in question would be reported as a relevant result or not as a measure of clinical utility. In contrast to the ABC system, the ACMG system was not made to guide variant reporting but to determine the likelihood of pathogenicity. Nevertheless, this comparison is justified since the ACMG class determines variant reporting in many laboratories. Forty-three laboratories participated in the survey. In seven cases, the classification system used did not influence the reporting likelihood when variants labeled as "maybe report" after ACMG-based classification were included. In three cases of population frequent but disease-associated variants, there was a difference in favor of reporting after ABC classification. A possible reason is that ABC step C (standard variant comments) allows a variant to be reported in one clinical setting but not another, e.g., based on Bayesian-based likelihood calculation of clinical relevance. Finally, the selection of ACMG criteria was compared between 36 laboratories. When excluding criteria used by less than four laboratories (<10%), the average concordance rate was 46%. Taken together, ABC-based classification is more clear-cut than ACMG-based classification since molecular and clinical information is handled separately, and variant reporting can be adapted to the clinical question and phenotype. Furthermore, variants do not get a clinically inappropriate label, like pathogenic when not pathogenic in a clinical context, or variant of unknown significance when the significance is known.

Different methylation patterns in BWS/SRS cases clarified by MS-MLPA.

Molecular abnormalities in the 11p15.5 imprinted gene cluster lead to two different growth diseases: Beckwith-Wiedemann syndrome (BWS) and Silver-Russell syndrome (SRS). They are mainly caused by epigenetic alterations in one of the two imprinting 11p15 control regions (ICR1 and ICR2). These CpG-rich regions are differentially methylated on the maternally and paternally derived chromosomes. We report four different methylation patterns along the BWS/SRS critical region, clarified by methylation-specific multiplex ligation-dependent probe amplification. The mathematical processing of the data provides information about alterations in the methylation status: from hypo- to almost complete demethylation of KvDMR, hypo- and hypermethylation of H19DMR and combined results from both regions provide information on paternal uniparental disomy (patUPD). The study concerns two BWS cases with KvDMR hypomethylation and almost complete loss of methylation, respectively; two patUPD11p15 cases with H19DMR hypermethylation/KvDMR hypomethylation, and one SRS case with H19DMR demethylation. In some cases KvDMR hypomethylation in patUPD11p15 can be difficult to assess, which requires combination with STR analysis or alternative method. The STR analysis provides also information on complete or segmental coverage and iso- or heterodisomy. Following this systematic approach, the precise diagnosis can be clarified in a few days and different methylation patterns could be detected.

Novel variant c.92T > G (p.Val31Gly) in the PFN1 gene (ALS18) responsible for a specific phenotype in a large Bulgarian amyotrophic lateral sclerosis pedigree.

Objectives: Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive deterioration of motor function, disability, and death. Variants in the PFN1 gene, encoding the Profilin-1 protein, are related to ALS18. Methods: We present a pedigree consisting of 3 generations and 4 affected individuals, 3 of which carry a novel heterozygous variant: c.92T > G (p.Val31Gly) in the PFN1 gene. This variant was discovered through means of whole exome sequencing (WES) and targeted analysis of ALS-related genes. Results: The mean age of onset in our pedigree was 59.75 (±10.11 SD) years with a significant difference between the first two generations (females) and the third (male) of 22.33 (±3.4 SD) years. For this ALS form, we observed a longer disease progression of 4 (±1.87 SD) years (three of four affected are still alive). Clinical manifestations displayed predominant impairment of the lower motor neuron (LMN) in one limb, with gradual involvement of other limbs. A novel heterozygous missense variant c.92T > G, p. Val31Gly (NM_005022.4) in exon 1 in the PFN1 gene was discovered through means of whole exome sequencing (WES). Segregation analysis in the family showed that the detected variant was inherited from the affected mother, and the affected aunt also turned out to be a variant carrier. Conclusions: ALS18 is a very rare form of the disease. We report here a relatively large pedigree with a novel variant, leading to late onset (after 50 years), initial involvement of the lower limbs and relatively slow progression.