GABRA1-Related Disorders: From Genetic to Functional Pathways.

OBJECTIVE: Variants in GABRA1 have been associated with a broad epilepsy spectrum, ranging from genetic generalized epilepsies to developmental and epileptic encephalopathies. However, our understanding of what determines the phenotype severity and best treatment options remains inadequate. We therefore aimed to analyze the electroclinical features and the functional effects of GABRA1 variants to establish genotype-phenotype correlations. METHODS: Genetic and electroclinical data of 27 individuals (22 unrelated and 2 families) harboring 20 different GABRA1 variants were collected and accompanied by functional analysis of 19 variants. RESULTS: Individuals in this cohort could be assigned into different clinical subgroups based on the functional effect of their variant and its structural position within the GABRA1 subunit. A homogenous phenotype with mild cognitive impairment and infantile onset epilepsy (focal seizures, fever sensitivity, and electroencephalographic posterior epileptiform discharges) was described for variants in the extracellular domain and the small transmembrane loops. These variants displayed loss-of-function (LoF) effects, and the patients generally had a favorable outcome. A more severe phenotype was associated with variants in the pore-forming transmembrane helices. These variants displayed either gain-of-function (GoF) or LoF effects. GoF variants were associated with severe early onset neurodevelopmental disorders, including early infantile developmental and epileptic encephalopathy. INTERPRETATION: Our data expand the genetic and phenotypic spectrum of GABRA1 epilepsies and permit delineation of specific subphenotypes for LoF and GoF variants, through the heterogeneity of phenotypes and variants. Generally, variants in the transmembrane helices cause more severe phenotypes, in particular GoF variants. These findings establish the basis for a better understanding of the pathomechanism and a precision medicine approach in GABRA1-related disorders. Further studies in larger populations are needed to provide a conclusive genotype-phenotype correlation. ANN NEUROL 2023.

Clinical Characterization of Alagille Syndrome in Patients with Cholestatic Liver Disease.

Alagille syndrome (ALGS) is a multisystem condition characterized by cholestasis and bile duct paucity on liver biopsy and variable involvement of the heart, skeleton, eyes, kidneys, and face and caused by pathogenic variants in the JAG1 or NOTCH2 gene. The variable expressivity of the clinical phenotype and the lack of genotype-phenotype correlations lead to significant diagnostic difficulties. Here we present an analysis of 18 patients with cholestasis who were diagnosed with ALGS. We used an NGS panel targeting coding exons of 52 genes, including the JAG1 and NOTCH2 genes. Sanger sequencing was used to verify the mutation in the affected individuals and family members. The specific facial phenotype was seen in 16/18 (88.9%). Heart defects were seen in 8/18 (44.4%) patients (pulmonary stenosis in 7/8). Butterfly vertebrae were seen in 5/14 (35.7%) patients. Renal involvement was detected in 2/18 (11.1%) cases-one patient had renal cysts, and one had obstructive hydronephrosis. An ophthalmology examination was performed on 12 children, and only one had posterior embryotoxon (8.3%). A percutaneous liver biopsy was performed in nine cases. Bile duct paucity was detected in six/nine cases (66.7%). Two patients required liver transplantation because of cirrhosis. We identified nine novel variants in the JAG1 gene-eight frameshift variants (c.1619_1622dupGCTA (p.Tyr541X), c.1160delG (p.Gly387fs), c.964dupT (p.C322fs), c.120delG (p.L40fs), c.1984dupG (p.Ala662Glyfs), c.3168_3169delAG (p.R1056Sfs*51), c.2688delG (p.896CysfsTer49), c.164dupG (p.Cys55fs)) and one missense variant, c.2806T > G (p.Cys936Gly). None of the patients presented with NOTCH2 variants. In accordance with the classical criteria, only six patients could meet the diagnostic criteria in our cohort without genetic analysis. Genetic testing is important in the diagnosis of ALGS and can help differentiate it from other types of cholestasis.

Complex Diagnostics of Non-Specific Intellectual Developmental Disorder.

Intellectual development disorder (IDD) is characterized by a general deficit in intellectual and adaptive functioning. In recent years, there has been a growing interest in studying the genetic structure of IDD. Of particular difficulty are patients with non-specific IDD, for whom it is impossible to establish a clinical diagnosis without complex genetic diagnostics. We examined 198 patients with non-specific IDD from 171 families using whole-exome sequencing and chromosome microarray analysis. Hereditary forms of IDD account for at least 35.7% of non-specific IDD, of which 26.9% are monogenic forms. Variants in the genes associated with the BAF (SWI/SNF) complex were the most frequently identified. We were unable to identify phenotypic features that would allow differential diagnosis of monogenic and microstructural chromosomal rearrangements in non-specific IDD at the stage of clinical examination, but due to its higher efficiency, exome sequencing should be the diagnostic method of the highest priority study after the standard examination of patients with NIDD in Russia.

Biallelic variants in TMEM222 cause a new autosomal recessive neurodevelopmental disorder.

PURPOSE: To elucidate the novel molecular cause in families with a new autosomal recessive neurodevelopmental disorder. METHODS: A combination of exome sequencing and gene matching tools was used to identify pathogenic variants in 17 individuals. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and subcellular localization studies were used to characterize gene expression profile and localization. RESULTS: Biallelic variants in the TMEM222 gene were identified in 17 individuals from nine unrelated families, presenting with intellectual disability and variable other features, such as aggressive behavior, shy character, body tremors, decreased muscle mass in the lower extremities, and mild hypotonia. We found relatively high TMEM222 expression levels in the human brain, especially in the parietal and occipital cortex. Additionally, subcellular localization analysis in human neurons derived from induced pluripotent stem cells (iPSCs) revealed that TMEM222 localizes to early endosomes in the synapses of mature iPSC-derived neurons. CONCLUSION: Our findings support a role for TMEM222 in brain development and function and adds variants in the gene TMEM222 as a novel underlying cause of an autosomal recessive neurodevelopmental disorder.

Spectrum of Mutations in PTPN11 in Russian Cohort.

Noonan syndrome is a group of diseases with a similar clinical picture, consisting of 16 diseases caused by mutations in 15 genes. According to the literature, approximately half of all cases are attributed to Noonan syndrome type 1, NSML, caused by mutations in the PTPN11 gene. We analyzed 456 unrelated probands using a gene panel NGS, and in 206 cases, the cause of the disease was identified. Approximately half of the cases (107) were caused by variants in the PTPN11 gene, including three previously undescribed variants, one of which was classified as VOUS, and the other two as LP causative complex alleles. Frequent variants of the PTPN11 gene characteristics for Russian patients were identified, accounting for more than 38% (c.922A>G p.Asn308Asp, c.417G>C p.Glu139Asp, c.1403C>T p.Thr468Met) of all cases with mutations in the PTPN11 gene. A comparative characterization of frequent variants of the PTPN11 gene in different populations is shown. The most common features of Noonan syndrome in the studied sample were facial dysmorphisms and cardiovascular system abnormalities. A lower representation of patients with growth delay was observed compared to previously described samples.

Genetic spectrum of sarcoglycanopathies in a cohort of Russian patients.

Sarcoglycanopathies encompass four distinct forms of limb-girdle muscular dystrophies (LGMD), denoted as LGMD R3-R6, arising from mutations within the SGCA, SGCB, SGCG, and SGCD genes. The global prevalence of sarcoglycanopathies is low, making it challenging to study these diseases. The principal objective of this study was to explore the spectrum of mutations in a cohort of Russian patients with sarcoglycanopathies and to ascertain the frequency of these conditions in the Russian Federation. We conducted a retrospective analysis of clinical and molecular genetic data from 49 Russian patients with sarcoglycan genes variants. The results indicated that variants in the SGCA gene were found in 71.4% of cases, with SGCB and SGCG genes each exhibiting variants in 12.2 % of patients. SGCD gene variants were detected in 4.1% of cases. Bi-allelic pathogenic and likely pathogenic variants were identified in 46 of the 49 cases of sarcoglycanopathies: LGMD R3 (n = 34), LGMD R4 (n = 4), LGMD R5 (n = 6), and LGMD R6 (n = 2). A total of 31 distinct variants were identified, comprising 25 previously reported and 6 novel variants. Two major variants, c.229C>T and c.271G>A, were detected within the SGCA, constituting 61.4% of all mutant alleles in Russian patients with LGMD R3. Both LGMD R6 cases were caused by the homozygous nonsense variant c.493C>T p.(Arg165Ter) in the SGCD gene. The incidence of sarcoglycanopathies in the Russian Federation was estimated to be at least 1 in 4,115,039, which is lower than the reported incidence in other populations.

mRNA analysis revealed a novel pathogenic EIF2S3 variant causing MEHMO syndrome.

EIF2S3 pathogenic variants have been shown to cause MEHMO syndrome - a rare X-linked intellectual disability syndrome. In most cases, DNA diagnostics of MEHMO syndrome is performed using exome sequencing. We describe two cousins with profound intellectual disability, severe microcephaly, microgenitalism, hypoglycemia, epileptic seizures, and hypertrichosis, whose clinical symptoms allowed us to suspect MEHMO syndrome. To confirm this diagnosis, we designed an mRNA analysis for the EIF2S3 gene. It is a cost-effective method to detect coding sequence variants in multi-exonic genes, as well as splicing defects and allelic imbalance. Our mRNA sequence analysis revealed a novel EIF2S3 variant c.820C>G in both cousins. We also found the same variant in female family members in the heterozygous state. To investigate the pathogenicity of the c.820C>G variant, we performed expression analysis, which showed that the DDIT3 transcript level was significantly increased in the patient relative to the controls. We, thus, demonstrate that mRNA analysis is an efficient tool for performing genetic testing in patients with distinct phenotypic features.

The First Russian Patient with Native American Myopathy.

Congenital myopathy associated with pathogenic variants in the STAC3 gene has long been considered native American myopathy (NAM). In 2017, the first case of a non-Amerindian patient with this myopathy was described. Here, we report the first Russian patient with NAM. The patient is a 17-year-old female with compound-heterozygous single nucleotide variants in the STAC3 gene: c.862A>T, p.(Lys288Ter) and c.93del, p.(Lys32ArgfsTer78). She has a milder phenotype than the earlier described patients. To our knowledge, this is the first case of a patient who had both nonsense and frameshift variants. It is assumed that the frameshift variant with premature stop codon lead to nonsense-mediated RNA decay. However, there are two additional coding isoforms of the STAC3 gene, which are not affected by this frameshift variant. We can speculate that these isoforms may partially carry out the function, and possibly explain the milder phenotype of our patient.

Case report: Unusual episodic myopathy in a patient with novel homozygous deletion of first coding exon of MICU1 gene.

We present a patient with unusual episodes of muscular weakness due to homozygous deletion of exon 2 in the MICU1 gene. Forty-three patients from 33 families were previously described with homozygous and compound heterozygous, predominantly loss of function (LoF) variants in the MICU1 gene that lead to autosomal recessive myopathy with extrapyramidal signs. Most described patients developed muscle weakness and elevated CK levels, and half of the patients had progressive extrapyramidal signs and learning disabilities. Our patient had a few severe acute episodes of muscle weakness with minimal myopathy features between episodes and a strongly elevated Creatinine Kinase (CK). Whole exome sequencing (WES) was performed and the homozygous deletion of exon 2 was suspected. To validate the diagnosis, we performed an RNA analysis of all family members. To investigate the possible impact of this deletion on the phenotype, we predicted a new Kozak sequence in exon 4 that could lead to the formation of a truncated MICU1 protein that could partly interact with MCU protein in a mitochondrial Ca2+ complex. We suspect that this unusual phenotype of the proband with MICU1-related myopathy could be explained by the presence of the truncated but partly functional protein. This work helps to define the clinical polymorphism of MICU1 deficiency better.

A Two-Year Clinical Description of a Patient with a Rare Type of Low-GGT Cholestasis Caused by a Novel Variant of USP53.

Here, we report a novel truncating mutation in the ubiquitin-specific peptidase gene (USP53) causing low-γ-GT (GGT) cholestasis. Genetic testing was carried out, including clinical exome sequencing for the proband and Sanger sequencing for the proband and his parents. The proband harbored a novel c.1017_1057del (p.(Cys339TrpfsTer7)) mutation in the ubiquitin carboxyl-terminal hydrolase (UCH) domain of USP53; we describe the clinical and laboratory features of the patient with a rare type of low-GGT cholestasis caused by this variant. The clinical presentation was found to be similar to that of phenotypes described in previous studies. However, there was an unusual presence of liver hemangiomas observed in our patient. Thus, our report reinforces the link between USP53 mutations and cholestasis. With this report, we confirm USP53 as the gene for low-GGT cholestasis and describe liver hemangiomas as a possible additional symptom of the phenotype spectrum. The inclusion of USP53 in the OMIM database and liver gene panels can further increase the effectiveness of molecular genetic studies.

Retrospective analysis of 17 patients with mitochondrial membrane protein-associated neurodegeneration diagnosed in Russia.

INTRODUCTION: Mitochondrial membrane protein-associated neurodegeneration (MPAN) is a rare neurological syndrome caused by pathogenic variants in the C19orf12 and is characterized by iron deposition in the basal ganglia and substantia nigra. Only a limited number of cohort studies were published to date and the prevalence of MPAN remains uncertain. METHODS: Recruited subjects with MPAN in Russia were diagnosed by whole-exome sequencing or Sanger sequencing of the C19orf12 gene. Data of over 14000 whole exome sequencing analyses was used to calculate the estimated disease frequency. RNA analysis was performed by RT-PCR. QSVanalyzer software was used to quantify the allelic disbalance. RESULTS: We describe the clinical and molecular characterizations of 17 patients with MPAN. DNA analysis detected three previously undescribed pathogenic/likely pathogenic variants in the C19orf12 gene. The estimated disease frequency was calculated to be 1:619150. We describe unusual clinical observations in several cases. One patient showed severe neurogenic muscle weakness along with a lack of marked spasticity or optic nerve atrophy. In another mild clinical case with the NM_001031726.3:c.204_214del (p.(Gly69Argfs*10)) variant in a heterozygous state, a marked allelic disbalance was observed on the RNA level with reduced expression level of the wild-type allele. Thus, this case became the first one of a possible regulatory variant causing MPAN. CONCLUSION: We reported a detailed clinical and molecular characterization of the third-largest MPAN cohort. We expanded the mutational and clinical spectrum of MPAN. Moreover, we calculated the estimated MPAN frequency in the Russian population for the first time.