The molecular complexity of COL2A1 splicing variants and their significance in phenotype severity.

Pathogenic single nucleotide variants (SNVs) found in the COL2A1 gene are associated with a broad range of skeletal dysplasias due to their impact on the structure and function of the Col2a1 protein. However, the molecular mechanisms of some nucleotide variants detected during diagnostic testing remain unclear. The interpretation of missense and splicing variants caused by SNVs poses a significant challenge for clinicians. In this work, we analyzed 22 splicing variants in the COL2A1 gene which have been found in patients with COL2A1-associated skeletal dysplasias. Using a minigene system, we investigated the impact of these SNVs on splicing and gained insights into their molecular mechanisms and genotype-phenotype correlations for each patient. The results of our study are very useful for improving the accuracy of diagnosis and the management of patients with skeletal dysplasias caused by SNVs in the COL2A1 gene.

Molecular basis and genetics of hypohidrotic ectodermal dysplasias.

Ectodermal dysplasia (ED) is a heterogeneous group of hereditary diseases of the skin and its appendages, which are characterized by impaired development and/or homeostasis of two or more ectoderm derivatives, including: hair, teeth, nails, sweat glands and their modifications (mammary glands, for instance). The overall prevalence of ectodermal dysplasia remains precisely unknown not only in Russia, but also in the world, nor is known the contribution of individual genes to its structure. This complicates the DNA diagnosis establishment of this disease due to the lack of an accurate diagnostic algorithm and a universal cost-effective method of analysis. To date, the most highly-researched genes involved in the development of anhydrous or hypohidrotic forms of ED are EDA, EDAR, EDARADD and WNT10A. The ectodysplasin A (EDA) gene is the cause of the most common X-linked form of ED, a gene from the Wnt family (WNT10A) is responsible for the autosomal recessive form of the disease, and two other genes (EDAR and EDARADD) can cause both autosomal recessive and autosomal dominant forms. This review provides the characteristics of the genes involved in ED, their mutation spectra, the level of their expression in human tissues, as well as the interrelation of the aforementioned genes. The domain structures of the corresponding proteins are considered, as well as the molecular genetic pathways in which they are involved. Animal models for studying this disorder are also taken into consideration. Due to the cross-species genes conservation, their mutations cause the disruption of the development of ectoderm derivatives not only in humans, but also in mice, cows, dogs, and even fish. It can be exploited for a better understanding of the etiopathogenesis of ectodermal dysplasias. Moreover, this article brings up the possibility of recurrent mutations in the EDA and WNT10A genes. The review also presents data on promising approaches for intrauterine ED treatment.

[Early-onset familial Alzheimer's disease with spastic paraparesis associated with PSEN1 gene]. / Rannyaya semeinaya bolezn' Al'tsgeimera so spasticheskim paraparezom, svyazannaya s genom PSEN1.

A familial case of a rare autosomal dominant Alzheimer's disease (AD), related to PSEN1 gene (AD3, OMIM 607822), differing from common multifactorial form by earlier onset and, in part of cases, by accompanying neurological signs, spastic paraparesis particularly, is presented. The first sign in a female proband and in her son was paraparesis manifested at the age of 29 and 21 years, respectively. Cognitive disturbances developed soon; the former diagnosis was hereditary spastic paraplegia with cognitive impairment, In the proband examined in 2008 at 33 years old the diagnosis was not established. In the son examined in 2022 at 27 years old whole-exome sequencing detected a novel PSEN1 missense mutation p.Thr421Ala. The mutation was confirmed by Sanger sequencing in him, found out in the proband (who was severely disabled by that time) and excluded in her unaffected mother. Except for different age of onset, AD3 in two patients was similar, though in whole it is variable, also in relatives. The variability and rareness of the disease hampers clinical diagnostics. Massive parallel sequencing is a most reliable diagnostic method.

[A case of spastic paraplegia with SPG4 and SPG3 associated mutations]. / Sluchai spasticheskoi paraplegii s mutatsiyami v genakh dvukh form: SPG4 i SPG3.

A rare case of autosomal dominant spastic paraplegia in a 36-year-old female with two reported earlier mutations of most common spastic paraplegia forms: SPG4 (mutation p.Cys28Leufs*20 in SPAST gene) and SPG3 (mutation p.Val405Met in ATL1 gene) is presented. The mutations detected by massively parallel sequencing (MPS) panel were inherited from affected mother and clinically unaffected father, respectively. The proband, her 61-year-old mother and deceased grandfather had 'uncomplicated' paraplegia with onset in 4th decade. The 67-year-old father had no even minimal subclinical signs of the disease and no affected relatives, detection of his low-penetrating ATL1 mutation was unexpected. MPS methods are the most informative for identifying a patient and/or family members with a combined hereditary neurological pathology, especially a combination of similar forms of heterogeneous groups, such as spastic paraplegia.

Influence of human peripheral blood samples preprocessing on the quality of Hi-C libraries.

The genome-wide variant of the chromatin conformation capture technique (Hi-C) is a powerful tool for revealing patterns of genome spatial organization, as well as for understanding the effects of their disturbance on disease development. In addition, Hi-C can be used to detect chromosomal rearrangements, including balanced translocations and inversions. The use of the Hi-C method for the detection of chromosomal rearrangements is becoming more widespread. Modern high-throughput methods of genome analysis can effectively reveal point mutations and unbalanced chromosomal rearrangements. However, their sensitivity for determining translocations and inversions remains rather low. The storage of whole blood samples can affect the amount and integrity of genomic DNA, and it can distort the results of subsequent analyses if the storage was not under proper conditions. The Hi-C method is extremely demanding on the input material. The necessary condition for successfully applying Hi-C and obtaining high-quality data is the preservation of the spatial chromatin organization within the nucleus. The purpose of this study was to determine the optimal storage conditions of blood samples for subsequent Hi-C analysis. We selected 10 different conditions for blood storage and sample processing. For each condition, we prepared and sequenced Hi-C libraries. The quality of the obtained data was compared. As a result of the work, we formulated the requirements for the storage and processing of samples to obtain high-quality Hi-C data. We have established the minimum volume of blood sufficient for conducting Hi-C analysis. In addition, we have identified the most suitable methods for isolation of peripheral blood mononuclear cells and their long-term storage. The main requirement we have formulated is not to freeze whole blood.

Early audiological phenotype in patients with mutations in the USH2A gene.

INTRODUCTION: Nowadays, due to universal newborn hearing screening (UNHS) the number of children with mild-to-moderate hearing loss diagnosed in the first year of life has increased significantly. Aside from that, identification of the genetic cause improves the genetic counselling of the families and allows to reveal possible comorbidities which may need a special approach. OBJECTIVE: To present the characteristics of the early audiologic phenotype in hearing impaired patients with biallelic mutations in the USH2A gene based on systematic analysis of the audiological data. PATIENTS AND METHODS: 13 patients with mutations in the USH2A gene underwent audiological examination. Most of them were found among a large group of infants with bilateral nonsyndromic sensorineural hearing loss (SNHL) examined under 12 months. RESULTS: Eight out of eleven children failed UNHS and were initially diagnosed as having bilateral nonsyndromic SNHL. Seven children underwent an audiological assessment before the age of 9 months. The earliest audiological examination was carried out at 1 and 3 months. The children with pathogenic variants in the USH2A gene in our examined group were identified in the first year of life via UNHS. The hearing threshold levels (HTL) for the USH2A group are compactly distributed between 51.25 dB and 66.25 dB, quartiles are 54 dB and 63.4 dB, with a median of 60 dB. The audiological profile of patients with biallelic USH2A mutations differs from audiograms of patients who had STRC-related hearing loss. We have not found any significant elevation in hearing thresholds in the first decade of life. We also estimated the prevalence of the USH2A and STRC mutations among GJB2-negative infants with bilateral nonsyndromic SNHL examined under 12 months, and it was 7.5% and 16.1%, respectively. CONCLUSION: According to our results, the early hearing phenotype in pediatric patients with biallelic mutations in the USH2A- gene is characterized by nonsyndromic mild-to-moderate SNHL in the first decade of life. Our results indicate that the presence of mutations in the USH2A or STRC genes can be expected in a child with congenital mild-to-moderate nonsyndromic SNHL. This information is of practical importance for parents, as they have to know the prognosis of hearing loss for their child from the very beginning. Post-screening follow-up should include adequate clinical, genetic, and social support for children and their parents.

A Mosaic Mutation in the LAMA2 Gene in a Case of Merosin-deficient Congenital Muscular Dystrophy.

Merosine deficient congenital muscular dystrophy is one of the most common forms of congenital muscular dystrophy. This disease is caused by a primary deficiency or a functionally inactive form of the protein merosin in muscle tissue. The type of inheritance of this disease is autosomal recessive. De novo variants with this type of inheritance are rare, and it is quite possible that the de novo variant may hide a mosaic form in the parent of an affected child. We present a birth family with two affected children who inherited a previously undescribed pathogenic variant c.1755del from their mother and a previously described pathogenic variant c.9253C > T in the LAMA2 gene from their mosaic father. LAMA2 gene mutation analysis was performed by mass parallel sequencing and direct sequencing of genomic DNAs.

[Autosomal dominant spastic paraplegias]. / Autosomno-dominantnye spasticheskie paraplegii.

OBJECTIVE: To estimate the proportion and spectrum of infrequent autosomal dominant spastic paraplegias in a group of families with DNA-confirmed diagnosis and to investigate their molecular and clinical characteristics. MATERIAL AND METHODS: Ten families with 6 AD-SPG: SPG6 (n=1), SPG8 (n=2), SPG9A (n=1), SPG12 (n=1), SPG17 (n=3), SPG31 (n=2) were studied using clinical, genealogical, molecular-genetic (massive parallel sequencing, spastic paraplegia panel, whole-exome sequencing, multiplex ligation-dependent amplification, Sanger sequencing) and bioinformatic methods. RESULTS AND CONCLUSION: Nine heterozygous mutations were detected in 6 genes, including the common de novo mutation p.Gly106Arg in NIPA1 (SPG6), the earlier reported mutation p.Val626Phe in WASHC5 (SPG8) in isolated case and the novel p.Val695Ala in WASHC5 (SPG8) in a family with 4 patients, the novel mutation p.Thr301Arg in RTN2 (SPG12) in a family with 2 patients, the novel mutation c.105+4A>G in REEP1 (SPG31) in a family with 4 patients and the reported earlier p.Lys101Lys in REEP1 (SPG31) in a family with 3 patients, the known de novo mutation p.Arg252Gln in ALDH18A1 (SPG9A) in two monozygous twins; the common mutation p.Ser90Leu in BSCL2 (SPG17) in a family with 3 patients and in isolated case, reported mutation p.Leu363Pro in a family with 2 patients. SPG6, SPG8, SPG12 and SPG31 presented 'pure' phenotypes, SPG31 had most benign course. Age of onset varied in SPG31 family and was atypically early in SPG6 case. Patients with SPG9A and SPG17 had 'complicated' paraplegias; amyotrophy of hands typical for SPG17 was absent in a child and in an adolescent from 2 families, but may develop later.

[AP4-assocated hereditary spastic paraplegias]. / AP4-svyazannye nasledstvennye spasticheskie paraplegii.

OBJECTIVE: In the course of studies of spastic paraplegias in Russian patients to detect AP4-associated forms, estimate their proportion in the total SPG group and analyze clinical and molecular characteristics. MATERIAL AND METHODS: Five families of Russian ethnicity: four with SPG47, one with SPG51 (4 girls and a boy aged 2.5-9 years) were studied. Clinical and genealogical methods, whole-exome sequencing (WES) and verification by familial Sanger sequencing were used. RESULTS: In our total group, including 118 families with 21 different forms, SPG AP4-associated forms accounted for 4.2% owing mainly to SPG47 (3.4%, 5th place in SPG structure; 20% and 2nd place in AE subgroup.) In non-consanguineous, unrelated SPG47 families three patients had identical genotypes: homozygosity for an earlier reported mutation c.1160_1161 delCA (p.Thr387ArgfsTer30) in AP4B1 exon 6; the 4th patient was compound-heterozygous for the same mutation and novel c.1240C>T (p.Gln414Ter) in exon 7. Frequency of c.1160_1161 delCA may be caused by founder effect in Slavic populations though the idea needs additional studies. The SPG51 patient was compound heterozygous for novel AP4E1 mutations c.2604delA (p.Ser868fs) and c.3346A>G (p.Arg1116Gly). Parent's heterozygosity in all cases was confirmed by Sanger sequencing. Phenotypes were typical: early development delay, muscle hypotony transforming into sever spasticity, mental deficiency, microceplaly (in all SPG47 cases), epilepsy (in 3 SPG47 and SPG51 cases), MRI changes, mainly hydrocephalus and/or hypoplasia of corpus callosum (in 3 SPG47 cases) and few extraneural signs. CONCLUSION: AP4-associated SPG should be taken into consideration in patients with early-onset severe nervous diseases mimicking non-genetic organic CNS disorders and massive exome sequencing (WES or other variants) should be performed.

KIF1A-related autosomal dominant spastic paraplegias (SPG30) in Russian families.

BACKGROUND: Spastic paraplegia type 30 (SPG30) caused by KIF1A mutations was first reported in 2011 and was initially considered a very rare autosomal recessive (AR) form. In the last years, thanks to the development of massive parallel sequencing, SPG30 proved to be a rather common autosomal dominant (AD) form of familial or sporadic spastic paraplegia (SPG),, with a wide range of phenotypes: pure and complicated. The aim of our study is to detect AD SPG30 cases and to examine their molecular and clinical characteristics for the first time in the Russian population. METHODS: Clinical, genealogical and molecular methods were used. Molecular methods included massive parallel sequencing (MPS) of custom panel 'spastic paraplegias' with 62 target genes complemented by familial Sanger sequencing. One case was detected by the whole -exome sequencing. RESULTS: AD SPG30 was detected in 10 unrelated families, making it the 3rd (8.4%) most common SPG form in the cohort of 118 families. No AR SPG30 cases were detected. In total, 9 heterozygous KIF1A mutations were detected, with 4 novel and 5 known mutations. All the mutations were located within KIF1A motor domain. Six cases had pure phenotypes, of which 5 were familial, where 2 familial cases demonstrated incomplete penetrance, early onset and slow relatively benign SPG course. All 4 complicated cases were caused by novel mutations without familial history. The phenotypes varied from severe in two patients (e.g. lack of walking, pronounced mental retardation) to relatively mild non-disabling symptoms in two others. CONCLUSION: AD SPG30 is one of the most common forms of SPG in Russia, the disorder has pronounced clinical variability while pure familial cases represent a significant part.

[Spastic ataxia of Charlevoix-Saguenay: the first Russian case report and literature review]. / Spasticheskaia ataksiia Sharlevua-Sagené: pervoe rossiiskoe nabliudenie i obzor literatury.

Spastic ataxia of Charlevoix-Saguenay (ARSACS) is a rare autosomal recessive neurodegenerative disease related to SACS gene and characterized by cerebellar, pyramidal and some other signs. The disease was delineated in Quebec, where it cumulates due to founder effect and has similar phenotype with very early onset. ARSACS in other populations is more variable. The first Russian case of ARSACS in a 37-year-old woman, an only patient in a Lak (one of Dagestan ethnicities) family, is presented. Along with main typical features, she had atypical late disease onset (in 32 years) and moderate cognitive decline. MPS-panel 'hereditary paraplegias' detected an earlier reported homo- or hemizygous mutation c.72276C>T (p.Arg2426Stop) in SACS exon 10.

[Hereditary spastic paraplegia type 4 (SPG4) in Russian patients]. / Nasledstvennaia spasticheskaia paraplegiia 4-go tipa u rossiiskikh bol'nykh.

AIM: To investigate molecular, clinical and genealogical characteristics of SPG4 in a first representative Russian group, to estimate SPG4 proportion among all DNA-diagnosed spastic paraplegias. MATERIAL AND METHODS: Fifty unrelated Russian families with SPG4 detected in the course of clinical and molecular studies of spastic paraplegias were studied. Clinical, genealogical and several molecular methods were used, i.e. Sanger sequencing of SPAST, massive parallel sequencing MPS (panel 'hereditary paraplegias') and multiplex ligation-dependent amplification MLPA. RESULTS: SPG4 proportion was 56% among all DNA verified SPG cases (90 families/14 forms) and 68% in subgroup of dominant SPG. In 50 families, 43 different SPAST mutations were detected, of which 21 were novel; percentage of large rearrangements was 30% (13 mutations in 15 families). Four mutations were detected in two families each, nonsense mutation c.1291C>T (p.Arg431*) in 4 unrelated families. Proportion of familial cases was 68%, pedigrees with 'missing' disease in elderly carriers pointed to incomplete penetrance. Age of onset varied from one year to 58 years, middle-age onset was common but the proportion of early-onset cases, particularly in male index cases, was also high. Onset age showed marked intrafamilial differences (more than 10 years in 14 pedigrees, up to 50 year in one) and between families with identical mutations. Insidious onset, slow development with most patients ambulant and 'uncomplicated' phenotype were typical. Cases with additional signs were: a family with ataxia in both patients, two families with epilepsy in one of SPG4 patients; three families with mild mental deficiency in one of SPG4 patients. A case described separately is a 29-year-old male patient with indeterminate myalgia and no SPG signs in whom SPAST previously reported mutation p.Ala430Thr de novo was an unexpected MPS finding. CONCLUSION: SPG4 substantially predomimates in SPG structure in Russian families as practically everywhere else. Half of 43 detected SPAST mutations are novel, the proportion of large rearrangements is 30% higher than in most of studies. Clinical inter- and intrafamilial variability concerns mostly age of onset. SPG4 is not exclusively adult-onset as was thought earlier.

Mutational Spectrum of Spast (Spg4) and Atl1 (Spg3a) Genes In Russian Patients With Hereditary Spastic Paraplegia.

Hereditary spastic paraplegia (HSP) comprises a heterogeneous group of neurodegenerative disorders, it share common symptom - of progressive lower spastic paraparesis. The most common autosomal dominant (AD) forms of HSP are SPG4 (SPAST gene) and SPG3 (ATL1 gene). In the current research we investigated for the first time the distribution of pathogenic mutations in SPAST and ATL1 genes within a large cohort of Russian HSP patients (122 probands; 69 famillial cases). We determined the frequencies of genetic abnormalities using Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and Next Generation Sequencing (NGS) of targeted gene panels. As a result, SPG4 was diagnosed in 30.3% (37/122) of HSP cases, where the familial cases represented 37.7% (26/69) of SPG4. In total 31 pathogenic and likely pathogenic variants were detected in SPAST, with 14 new mutations. Among all detected SPAST variants, 29% were gross deletions and duplications. The proportion of SPG3 variants in Russian cohort was 8.2% (10/122) that were all familial cases. All 10 detected ATL1 mutations were missense substitutions, most of which were in the mutational hot spots of 4, 7, 8, 12 exons, with 2 novel mutations. This work will be helpful for the populational genetics of HSP understanding.

The study of the full spectrum of variants leading to hyperphenylalaninemia have revealed 10 new variants in the PAH gene.

This study presents further research into the spectrum of variants in genes responsible for the development of phenylketonuria (PKU) and hyperphenylalaninemia (HPA) in patients in Russia. After a study of 25 frequent variants, 293 patients (327 chromosomes without detected variants) from among 1265 probands still had no confirmed diagnosis. A study involving methods of next generation sequencing (NGS) of PAH, PTS, GCH1, PCBD1, QDPR, SPR and DNAJC12 genes to search for point mutations and multiplex ligation-dependent probe amplification (MLPA) methods to search for gross deletions were conducted for these patients. Among 327 chromosomes without identified variants, variants in the PAH gene were found on 260 chromosomes, and variants in the PTS gene were found on 10 chromosomes. On 10 chromosomes gross deletions by the MLPA method were detected. 104 rare variants of the РАН gene, including 10 variants not previously described, and 6 variants of the PTS gene were revealed. The NGS method revealed additional РАН gene variants on 10.3% of chromosomes and PTS gene variants on 0.4%. Gross deletions of the РАН gene were revealed in 0.5% of chromosomes. Thus, the most complete understanding of the spectrum of variants leading to the development of the PKU and HPA in Russia with the use of all methods available today has been obtained. Such a detailed study of the spectrum of rare variants on the genetic material from Russia was undertaken for the first time.

[Common forms of hereditary spastic paraplegias]. / Rasprostranennye formy nasledstvennykh spasticheskikh paraplegii.

A group of hereditary spastic paraplegias includes about 80 spastic paraplegia genes (SPG): forms with identified (almost 70) or only mapped (about 10) genes. Methods of next generation sequencing (NGS), along with new SPG discovering, modify knowledge about earlier delineated SPG. Clinical and genetic characteristics of common autosomal dominant (SPG4, SPG3, SPG31) and autosomal recessive (SPG11, SPG7, SPG15) forms are presented.

[The third case report a patient with primary aldosteronism, seizures, and neurologic abnormalities (PASNA) syndrome de novo variant mutations in the CACNA1D gene]. / Tretii sluchai sindroma pervichnogo al'dosteronizma, sudorog i nevrologicheskikh narushenii (PASNA), obuslovlennogo variantom mutatsii de novo v gene CACNA1D.

Germline mutations in CACNA1D cause the primary aldosteronism, seizures, and neurologic abnormalities (PASNA) syndrome (OMIM# 615474) characterized by primary aldosteronism, seizures and neurological abnormalities. The authors present a case-report of a 1-year 3-month male patient with neurological symptoms such as seizures and global developmental delay with primary hyperaldosteronism. The heterozygosis disease-causing variant c.776T>A in CACNA1D gene was identified.

[Clinical-genetic analysis of limb-girdle muscular dystrophy 2 type I].

Seventy four patients with clinical presentation of LGMD and probably autosomal-recessive type of inheritance were examined. Five different mutations of FKRP gene responsible for LGMD2 type I were detected in eight (10.8%) patients. Two of them с.341C>G, c.826C>A were described before and three c.229C>T, с.265C>T, с.1078G>C were found for the first time. The significant clinical polymorphism due to the difference in age of manifestation and severity of clinical presentation was identified.

[Hygienic assessment of biologically rigid linear alkylbenzene sulfonates].

It has been found that there are both biologically rigid and biologically soft homologues in the homologous series of linear alkylbenzene sulfonates (LABS). It is shown that absorption of LABS molecules from aqueous to activated sludge phase may serve as a determinant that should be used to refer a homologue to as rigid or soft surfactants. The biodegradability, detergency, and toxicity of LABS were ascertained to be related to the size of molecular alkyl molecular substitute. It has established that the fractional compositions of linear alkobenzenes should be changed for the synthesis of LABS that have the maximum detergency, a high biodegradability rate, and a low toxicity.

[Clinical-genetic characteristics of limb girdle-muscular dystrophy type 2A].

We present the results of the molecular genetic study of 26 patients, aged from 12 to 60 years, from 24 unrelated families with limb girdle-muscular dystrophy (LGMD) type 2A. The disease duration varied from 6 months to 30 years. The diagnosis of LGMD 2capital A, Cyrillic was confirmed by molecular genetic methods basing on the presence of a CAPN3 mutation in homozygous, compound-heterozygous and heterozygous state. The Leyden-Moebius variant that is characterized by the primary affection of muscles of pelvic girdle and shin with the gradual progression of the pathological process in shoulder girdle muscles was the most frequent in the Russian population. Tip-toe walking or difficulties in walking upstairs and running were the first symptoms reported by patients. In contrast to criteria of the European Neuromuscular Center, the characteristic symptoms of the disease were early contractures of ankle joints and pseudohypertrophy of gastrocnemius muscles. The major c.550delA mutation in the CAPN3 gene was identified in 70% of Russian patients.