Deciphering the impact of coding and non-coding SCN1A gene variants on RNA splicing.

Variants that disrupt normal pre-mRNA splicing are increasingly being recognized as a major cause of monogenic disorders. The SCN1A gene, a key epilepsy gene that is linked to various epilepsy phenotypes, is no exception. Approximately 10% of all reported variants in the SCN1A gene are designated as splicing variants, with many located outside of the canonical donor and acceptor splice sites, and most have not been functionally investigated. However, given its restricted expression pattern, functional analysis of splicing variants in the SCN1A gene could not be routinely performed. In this study, we conducted a comprehensive analysis of all reported SCN1A variants and their potential to impact SCN1A splicing and conclude that splicing variants are substantially misannotated and under-represented. We created a splicing reporter system consisting of 18 splicing vectors covering all 26 protein-coding exons with different genomic contexts and several promoters of varying strengths in order to reproduce the wild-type splicing pattern of the SCN1A gene, revealing cis-regulatory elements essential for proper recognition of SCN1A exons. Functional analysis of 95 SCN1A variants was carried out, including all 68 intronic variants reported in the literature, located outside of the splice sites canonical dinucleotides; 21 exonic variants of different classes (synonymous, missense, nonsense and in-frame deletion) and six variants observed in patients with epilepsy. Interestingly, almost 20% of tested intronic variants had no influence on SCN1A splicing, despite being reported as causative in the literature. Moreover, we confirmed that the majority of predicted exonic variants affect splicing unravelling their true molecular mechanism. We used functional data to perform genotype-phenotype correlation, revealing distinct distribution patterns for missense and splice-affecting 'missense' variants and observed no difference in the phenotype severity of variants leading to in-frame and out-of-frame isoforms, indicating that the Nav1.1 protein is highly intolerant to structural variations. Our work demonstrates the importance of functional analysis in proper variant annotation and provides a tool for high-throughput delineation of splice-affecting variants in SCN1A in a whole-gene manner.

Phenotype and natural history of mitochondrial membrane protein-associated neurodegeneration.

Mitochondrial membrane protein-associated neurodegeneration (MPAN) is an ultraorphan neurogenetic disease from the group of neurodegeneration with brain iron accumulation (NBIA) disorders. Here we report cross-sectional and longitudinal data to define the phenotype, to assess disease progression and to estimate sample sizes for clinical trials. We enrolled patients with genetically confirmed MPAN from the Treat Iron-Related Childhood-Onset Neurodegeneration (TIRCON) registry and cohort study, and from additional sites. Linear mixed-effect modelling (LMEM) was used to calculate annual progression rates for the Unified Parkinson's Disease Rating Scale (UPDRS), Barry-Albright Dystonia (BAD) scale, Schwab and England Activities of Daily Living (SE-ADL) scale and the Pediatric Quality of Life Inventory (PedsQL). We investigated 85 MPAN patients cross-sectionally, with functional outcome data collected in 45. Median age at onset was 9 years and the median diagnostic delay was 5 years. The most common findings were gait disturbance (99%), pyramidal involvement (95%), dysarthria (90%), vision disturbances (82%), with all but dysarthria presenting early in the disease course. After 16 years with the disease, 50% of patients were wheelchair dependent. LMEM showed an annual progression rate of 4.5 points in total UPDRS. The total BAD scale score showed no significant progression over time. The SE-ADL scale and the patient- and parent-reported PedsQL showed a decline of 3.9%, 2.14 and 2.05 points, respectively. No patient subpopulations were identified based on longitudinal trajectories. Our cross-sectional results define the order of onset and frequency of symptoms in MPAN, which will inform the diagnostic process, help to shorten diagnostic delay and aid in counselling patients, parents and caregivers. Our longitudinal findings define the natural history of MPAN, reveal the most responsive outcomes and highlight the need for an MPAN-specific rating approach. Our sample size estimations inform the design of upcoming clinical trials.

De novo missense variants in FBXO11 alter its protein expression and subcellular localization.

Recently, others and we identified de novo FBXO11 (F-Box only protein 11) variants as causative for a variable neurodevelopmental disorder (NDD). We now assembled clinical and mutational information on 23 additional individuals. The phenotypic spectrum remains highly variable, with developmental delay and/or intellectual disability as the core feature and behavioral anomalies, hypotonia and various facial dysmorphism as frequent aspects. The mutational spectrum includes intragenic deletions, likely gene disrupting and missense variants distributed across the protein. To further characterize the functional consequences of FBXO11 missense variants, we analyzed their effects on protein expression and localization by overexpression of 17 different mutant constructs in HEK293 and HeLa cells. We found that the majority of missense variants resulted in subcellular mislocalization and/or reduced FBXO11 protein expression levels. For instance, variants located in the nuclear localization signal and the N-terminal F-Box domain lead to altered subcellular localization with exclusion from the nucleus or the formation of cytoplasmic aggregates and to reduced protein levels in western blot. In contrast, variants localized in the C-terminal Zn-finger UBR domain lead to an accumulation in the cytoplasm without alteration of protein levels. Together with the mutational data, our functional results suggest that most missense variants likely lead to a loss of the original FBXO11 function and thereby highlight haploinsufficiency as the most likely disease mechanism for FBXO11-associated NDDs.

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.

A Rare Coincidence of Three Inherited Diseases in a Family with Cardiomyopathy and Multiple Extracardiac Abnormalities.

A genetic diagnosis of primary cardiomyopathies can be a long-unmet need in patients with complex phenotypes. We investigated a three-generation family with cardiomyopathy and various extracardiac abnormalities that had long sought a precise diagnosis. The 41-year-old proband had hypertrophic cardiomyopathy (HCM), left ventricular noncompaction, myocardial fibrosis, arrhythmias, and a short stature. His sister showed HCM, myocardial hypertrabeculation and fibrosis, sensorineural deafness, and congenital genitourinary malformations. Their father had left ventricular hypertrophy (LVH). The proband's eldest daughter demonstrated developmental delay and seizures. We performed a clinical examination and whole-exome sequencing for all available family members. All patients with HCM/LVH shared a c.4411-2A>C variant in ALPK3, a recently known HCM-causative gene. Functional studies confirmed that this variant alters ALPK3 canonical splicing. Due to extracardiac symptoms in the female patients, we continued the search and found two additional single-gene disorders. The proband's sister had a p.Trp329Gly missense in GATA3, linked to hypoparathyroidism, sensorineural deafness, and renal dysplasia; his daughter had a p.Ser251del in WDR45, associated with beta-propeller protein-associated neurodegeneration. This unique case of three monogenic disorders in one family shows how a comprehensive approach with thorough phenotyping and extensive genetic testing of all symptomatic individuals provides precise diagnoses and appropriate follow-up, embodying the concept of personalized medicine. We also present the first example of a splicing functional study for ALPK3 and describe the genotype-phenotype correlations in cardiomyopathy.

Masks of Albinism: Clinical Spectrum of Hermansky-Pudlak Syndrome.

Hermansky-Pudlak syndrome (HPS) is a rare disease inherited in the autosomal recessive mode, including 11 clinical genetic subtypes. They are associated with impaired function of the BLOC protein complex (Biogenesis of Lysosome-related Organelles Complexes), and the subunits of the AP-3 complex (adaptor protein complex). Each has its own clinical features, but they are all characterized by albinism, bleeding disorder, and visual abnormalities. Eleven patients from eight unrelated families with an incoming diagnosis of albinism were examined and novel and previously described genetic variants in HPS1, HPS6, and BLOC1S6 genes (types HPS1, HPS6, and HPS9) were found. To determine the optimal therapy and recommendations for further follow up, it is necessary to consider the entire clinical spectrum and genetic polymorphism of the disease. An interdisciplinary approach, combined with the use of non-routine diagnostic techniques such as RNA analysis, is essential for achieving accurate diagnoses in certain complex cases.

Functional investigation of SCN1A deep-intronic variants activating poison exons inclusion.

Dravet syndrome is a devastating epileptic syndrome characterized by intractable epilepsy with an early age of onset, regression of developmental milestones, ataxia, and motor deficits. Loss-of-function pathogenic variants in the SCN1A gene are found in the majority of patients with Dravet syndrome; however, a significant number of patients remain undiagnosed even after comprehensive genetic testing. Previously, it was shown that intronic elements in the SCN1A gene called poison exons can incorporate into SCN1A mRNA, leading to haploinsufficiency and potentially causing Dravet syndrome. Here, we developed a splicing reporter assay for all described poison exons of the SCN1A gene and validated it using previously reported and artificially introduced variants. Overall, we tested 18 deep-intronic single nucleotide variants and one complex allele in the SCN1A gene. Our approach is capable of evaluating the effect of both variants affecting cis-regulatory sequences and splice-site variants, with the potential to functionally annotate every possible variant within these elements. Moreover, using antisense-modified uridine-rich U7 small nuclear RNAs, we were able to block poison exon incorporation in mutant constructs, an approach that could be used as a promising therapeutic intervention in Dravet syndrome patients with deep-intronic variants.

TRA2B Gene Splice Variant Linked to Seizures and Neurodevelopmental Delay: A Second Case Study.

In this study, we report a novel splice variant in the TRA2B gene identified in a patient presenting with seizures and neurodevelopmental delay. This paper represents the second investigation of pathogenic variants in the TRA2B gene in humans, reaffirming the conclusions of the initial study and underscoring the importance of this research. Comprehensive genetic testing, including whole genome sequencing, Sanger sequencing, and mRNA analysis, was performed on the proband and her parents. The proband harbored a de novo c.170+1G>A variant in the RS1 domain of Tra2ß, which was confirmed to be pathogenic through mRNA analysis, resulting in exon 2 deletion and a frameshift (p.Glu13Valfs*2). The clinical presentation of the patient was consistent with phenotypes described in one of the previous studies. These findings contribute to the dissemination and reinforcement of prior discoveries in the context of TRA2B-related syndrome and highlight the need for further investigation into the functional consequences and underlying pathogenic mechanisms associated with TRA2B mutations.

Complex Chromosomal Rearrangement Involving Chromosomes 10 and 11, Accompanied by Two Adjacent 11p14.1p13 and 11p13p12 Deletions, Identified in a Patient with WAGR Syndrome.

Three years ago, our patient, at that time a 16-month-old boy, was discovered to have bilateral kidney lesions with a giant tumor in the right kidney. Chemotherapy and bilateral nephron-sparing surgery (NSS) for Wilms tumor with nephroblastomatosis was carried out. The patient also had eye affection, including glaucoma, eye enlargement, megalocornea, severe corneal swelling and opacity, complete aniridia, and nystagmus. The diagnosis of WAGR syndrome was suspected. De novo complex chromosomal rearrangement with balanced translocation t(10,11)(p15;p13) and a pericentric inversion inv(11)(p13q12), accompanied by two adjacent 11p14.1p13 and 11p13p12 deletions, were identified. Deletions are raised through the complex molecular mechanism of two subsequent rearrangements affecting chromosomes 11 and 10. WAGR syndrome diagnosis was clinically and molecularly confirmed, highlighting the necessity of comprehensive genetic testing in patients with congenital aniridia and/or WAGR syndrome.

Specificities of the DMD Gene Mutation Spectrum in Russian Patients.

Duchenne/Becker muscular dystrophy (DMD/BMD) is the most common form of muscular dystrophy, accounting for over 50% of all cases. In this regard, in Russia we carry out a program of selective screening for DMD/BMD, which mainly involves male patients. The main inclusion criteria are an increase in the level of creatine phosphokinase (>2000 U/L) or an established clinical diagnosis. At the first stage of screening, patients are scanned for extended deletions and duplications in the DMD gene using multiplex ligase-dependent probe amplification (MLPA SALSA P034 and P035 DMD probemix, MRC-Holland). The second stage is the search for small mutations using a custom NGS panel, which includes 31 genes responsible for various forms of limb-girdle muscular dystrophy. In a screening of 1025 families with a referral Duchenne/Becker diagnosis, pathogenic and likely pathogenic variants in the DMD gene were found in 788 families (in 76.9% of cases). In the current study, we analyzed the mutation spectrum of the DMD gene in Russian patients and noted certain differences between the examined cohort and the multi-ethnic cohort. The analysis of the DMD gene mutation spectrum is essential for patients with DMD/BMD because the exact mutation type determines the application of a specific therapeutic method.

Investigation of LINC00493/SMIM26 Gene Suggests Its Dual Functioning at mRNA and Protein Level.

The amount of human long noncoding RNA (lncRNA) genes is comparable to protein-coding; however, only a small number of lncRNAs are functionally annotated. Previously, it was shown that lncRNAs can participate in many key cellular processes, including regulation of gene expression at transcriptional and post-transcriptional levels. The lncRNA genes can contain small open reading frames (sORFs), and recent studies demonstrated that some of the resulting short proteins could play an important biological role. In the present study, we investigate the widely expressed lncRNA LINC00493. We determine the structure of the LINC00493 transcript, its cell localization and influence on cell physiology. Our data demonstrate that LINC00493 has an influence on cell viability in a cell-type-specific manner. Furthermore, it was recently shown that LINC00493 has a sORF that is translated into small protein SMIM26. The results of our knockdown and overexpression experiments suggest that both LINC00493/SMIM26 transcript and protein affect cell viability, but in the opposite manner.

Recessive myotonia congenita caused by a homozygous splice site variant in CLCN1 gene: a case report.

BACKGROUND: Myotonia congenita is a rare neuromuscular disease, which is characterized by a delay in muscle relaxation after evoked or voluntary contraction. Myotonia congenita can be inherited in a dominant (Thomsen disease) and recessive form (Becker disease) and both are caused by pathogenic variants in the CLCN1 gene. Noncanonical splice site variants are often classified as variants of uncertain significance, due to insufficient accuracy of splice-predicting tools. Functional analysis using minigene plasmids is widely used in such cases. Moreover, functional analysis is very useful in investigation of the disease pathogenesis, which is necessary for development of future therapeutic approaches. To our knowledge only one noncanonical splice site variant in the CLCN1 gene was functionally characterized to date. We further contribute to this field by evaluation the molecular mechanism of splicing alteration caused by the c.1582 + 5G > A in a homozygous state. CASE PRESENTATION: We report a clinical case of an affected 6-y.o boy with athletic appearance due to muscle hypertrophy, calf muscle stiffness, cramping and various myotonic signs in a consanguineous family with no history of neuromuscular disorders. The neurological examination showed percussion-activated myotonia in the hands and legs. Plasma creatine kinase enzyme and transaminases levels were normal. Electromyography at the time of examination shows myotonic runs in the upper and lower extremities. CONCLUSIONS: Functional analysis of the variant in a minigene system showed alteration of splicing leading to loss of function, thereby confirming that the variant is pathogenic.

Noncompaction cardiomyopathy is caused by a novel in-frame desmin (DES) deletion mutation within the 1A coiled-coil rod segment leading to a severe filament assembly defect.

Mutations in DES, encoding desmin protein, are associated with different kinds of skeletal and/or cardiac myopathies. However, it is unknown, whether DES mutations are associated with left ventricular hypertrabeculation (LVHT). Here, we performed a clinical examination and subsequent genetic analysis in a family, with two individuals presenting LVHT with conduction disease and skeletal myopathy. The genetic analysis revealed a novel small in-frame deletion within the DES gene, p.Q113_L115del, affecting the α-helical rod domain. Immunohistochemistry analysis of explanted myocardial tissue from the index patient revealed an abnormal cytoplasmic accumulation of desmin and a degraded sarcomeric structure. Cell transfection experiments with wild-type and mutant desmin verified the cytoplasmic aggregation and accumulation of mutant desmin. Cotransfection experiments were performed to model the heterozygous state of the patients and revealed a dominant negative effect of the mutant desmin on filament assembly. DES:p.Q113_L115del is classified as a pathogenic mutation associated with dilated cardiomyopathy with prominent LVHT.

Novel case of neurodegeneration with brain iron accumulation 4 (NBIA4) caused by a pathogenic variant affecting splicing.

Neurodegeneration with brain iron accumulation type 4 (NBIA4) also known as MPAN (mitochondria protein-associated neurodegeneration) is a rare neurological disorder which main feature is brain iron accumulation most frequently in the globus pallidus and substantia nigra. Whole exome sequencing (WES) in a 12-year-old patient revealed 2 variants in the C19orf12 gene, a previously reported common 11 bp deletion c.204_214del11, p.(Gly69Argfs*10) and a novel splicing variant c.193+5G>A. Functional analysis of novel variant showed skipping of the second exon, resulting in a formation of a truncated nonfunctional protein. This is the first functionally annotated pathogenic splicing variant in NBIA4.

Case Report: Phenotype-Driven Diagnosis of Atypical Dravet-Like Syndrome Caused by a Novel Splicing Variant in the SCN2A Gene.

Febrile-associated epileptic encephalopathy is a large genetically heterogeneous group that is associated with pathogenic variants in SCN1A, PCDH19, SCN2A, SCN8A, and other genes. The disease onset ranges from neonatal or early-onset epileptic encephalopathy to late-onset epilepsy after 18 months. Some etiology-specific epileptic encephalopathies have target therapy which can serve as a clue for the correct genetic diagnosis. We present genetic, clinical, electroencephalographic, and behavioral features of a 4-year-old girl with epileptic encephalopathy related to a de novo intronic variant in the SCN2A gene. Initial NGS analysis revealed a frameshift variant in the KDM6A gene and a previously reported missense variant in SCN1A. Due to lack of typical clinical signs of Kabuki syndrome, we performed X-chromosome inactivation that revealed nearly complete skewed inactivation. Segregation analysis showed that the SCN1A variant was inherited from a healthy father. The proband had resistance to multiple antiseizure medications but responded well to sodium channel inhibitor Carbamazepine. Reanalysis of NGS data by a neurogeneticist revealed a previously uncharacterized heterozygous variant c.1035-7A>G in the SCN2A gene. Minigene assay showed that the c.1035-7A>G variant activates a cryptic intronic acceptor site which leads to 6-nucleotide extension of exon 9 (NP_066287.2:p.(Gly345_Gln346insTyrSer). SCN2A encephalopathy is a recognizable severe phenotype. Its electro-clinical and treatment response features can serve as a hallmark. In such a patient, reanalysis of genetic data is strongly recommended in case of negative or conflicting results of DNA analysis.

Genetic and Clinical Spectrum of GNE Myopathy in Russia.

GNE myopathy (GNEM) is a rare hereditary disease, but at the same time, it is the most common distal myopathy in several countries due to a founder effect of some pathogenic variants in the GNE gene. We collected the largest cohort of patients with GNEM from Russia and analyzed their mutational spectrum and clinical data. In our cohort, 10 novel variants were found, including 2 frameshift variants and 2 large deletions. One novel missense variant c.169_170delGCinsTT (p.(Ala57Phe)) was detected in 4 families in a homozygous state and in 3 unrelated patients in a compound heterozygous state. It was the second most frequent variant in our cohort. All families with this novel frequent variant were non-consanguineous and originated from the 3 neighboring areas in the European part of Russia. The clinical picture of the patients carrying this novel variant was typical, but the severity of clinical manifestation differed significantly. In our study, we reported two atypical cases expanding the phenotypic spectrum of GNEM. One female patient had severe quadriceps atrophy, hand joint contractures, keloid scars, and non-classical pattern on leg muscle magnetic resonance imaging, which was more similar to atypical collagenopathy rather than GNEM. Another patient initially had been observed with spinal muscular atrophy due to asymmetric atrophy of hand muscles and results of electromyography. The peculiar pattern of muscle involvement on magnetic resonance imaging consisted of pronounced changes in the posterior thigh muscle group with relatively spared muscles of the lower legs, apart from the soleus muscles. Different variants in the GNE gene were found in both atypical cases. Thus, our data expand the mutational and clinical spectrum of GNEM.

Case Report: Functional Investigation of an Undescribed Missense Variant Affecting Splicing in a Patient With Dravet Syndrome.

Pathogenic variants in the SCN1A gene are associated with a spectrum of epileptic disorders ranging in severity from familial febrile seizures to Dravet syndrome. Large proportions of reported pathogenic variants in SCN1A are annotated as missense variants and are often classified as variants of uncertain significance when no functional data are available. Although loss-of-function variants are associated with a more severe phenotype in SCN1A, the molecular mechanism of single nucleotide variants is often not clear, and genotype-phenotype correlations in SCN1A-related epilepsy remain uncertain. Coding variants can affect splicing by creating novel cryptic splicing sites in exons or by disrupting exonic cis-regulation elements crucial for proper pre-mRNA splicing. Here, we report a novel case of Dravet syndrome caused by an undescribed missense variant, c.4852G>A (p.(Gly1618Ser)). By midigene splicing assay, we demonstrated that the identified variant is in fact splice-affecting. To our knowledge, this is the first report on the functional investigation of a missense variant affecting splicing in Dravet syndrome.

Clinical and genetic characterization of autosomal recessive stickler syndrome caused by novel compound heterozygous mutations in the COL9A3 gene.

BACKGROUND: Stickler syndrome (STL) is a clinically variable and genetically heterogeneous collagenopathy characterized by ophthalmic, auditory, skeletal, and orofacial abnormalities. STL is mainly inherited in an autosomal dominant pattern with mutations in the COL2A1, COL11A1, and COL11A2 genes. Autosomal recessive forms are rare. However, 19 patients have been reported to date, with STL caused by homozygous or compound heterozygous mutations in genes that encode for the three chains of type IX collagen: COL9A1, COL9A2, and COL9A3. METHODS: Genetic analysis was performed using the next-generation sequencing of 166 genes associated with skeletal disorders and sequenced on an Ion Torrent S5 system with a minimum coverage of 100X. The two variants in the COL9A3 gene identified in the proband and the parents were confirmed by Sanger sequencing on an ABI3130xl sequencer. RESULTS: We describe a novel case of autosomal recessive Stickler syndrome caused by two undescribed mutations in the COL9A3 gene: c.268C>T (p.Arg90Ter) and c.1729C>T (p.Arg577Ter). The clinical features included severe sensorineural hearing loss, high myopia, vitreoretinal degeneration, and early-onset arthropathy of the lower limbs. Radiography revealed mild spondyloepiphyseal dysplasia. CONCLUSION: This case further expands the mutational and phenotypic spectrum of COL9A-associated STL with a more severe presentation.

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.

Various haploinsufficiency mechanisms in Pitt-Hopkins syndrome.

Pitt-Hopkins syndrome is a rare neurodevelopment disorder caused by haploinsufficiency of the transcription factor 4 (TCF4). The main clinical symptoms of Pitt-Hopkins syndrome are severe development delay, intellectual disability, characteristic facial phenotype, and breathing abnormalities, including episodic hyperventilation. Different pathogenic variants can lead to Pitt-Hopkins syndrome. The most common are large deletions at 18q21 encompassing the TCF4 gene and frameshifting/nonsense single nucleotide variants. However, variants in noncoding regions can also lead to Pitt-Hopkins syndrome by disrupting the normal pre-mRNA splicing machinery. Here we describe three patients with Pitt-Hopkins syndrome caused by a large deletion in chromosome 18, a nonsense variant, and a novel variant located in intron 11 of TCF4 c.922+5G > A. Using RT-PCR analysis and minigene splicing assay we showed that this intronic variant leads to exon 11 skipping resulting in a formation of a premature stop codon. To our knowledge, this is the first functional annotation of a splicing variant in Pitt-Hopkins syndrome.