Introduction The deductive method: from karyotyping to aCGH and WES is an important aspect in the diagnosis and search for the causes of intellectual disability due to congenital brain anomalies. There is recommendation to exclude the presence of CNV or monogenic variants for patients with a normal karyotype, but with a clinical picture of syndromic disease. Objectives Improvement of diagnosis of intellectual disability. Methods aCGH with 60K Agilent microarrays, WES with SureSelect Human All Exon V8 Results Pathogenic or potentially pathogenic CNVs were excluded previously by aCGH for 10 families (total 32 people, 2 families had 2 children) with intellectual disability and congenital brain anomalies (for example, polymicrogyria, pachygyria, lissencephaly). The WES identified candidate variants for all families that can lead to impaired neurodevelopment, including 3 pathogenic variants in 3 families, 3 likely pathogenic in three other families, and 10 variants with uncertain clinical significance for 4 families. Almost all of these variants were identified de novo, except for one family, where the proband has been a compound heterozygous for two variants in the RELN gene. The first case of pathogenic mutation de novo was detected in a girl with agenesis of the corpus callosum. It was a missense mutation DYNC1H1 (NM_001376.5): c.4868G>A (p.Arg1623Gln), which leads to impaired intellectual development in autosomal dominant type 13 (OMIM 614563). The second variant was detected in a boy with corpus callosum agenesis, pontine hypogenesis, pachygyria in the frontal lobes. It was a missense variant MACF1 (ENST00000567887.5): c.21989A>G(p.Asp7330Gly), which leads to lissencephaly 9 with complex brainstem malformation (OMIM 614563). The third variant was found in a girl with epilepsy and impaired myelination of the white matter of the parietal-occipital areas of the cerebral hemispheres. It was a missense variant CDKL5 (NM_001323289.2):c.404-1G>A that leads to developmental and epileptic encephalopathy 2 (OMIM 300672). Conclusions Sixteen candidate variants potentially responsible for mental health were reported in this study. Most of these variants were missense changes in genes. All except one anomalies arisen de novo. Trio-based WES has been shown to be an important step in making a genetic diagnosis if other chromosomal and subchromosomal abnormalities had been excluded. The clinical description of the patient is the most important step for the correct interpretation of WES results, which allows to establish the exact genetic cause of the disease if several variants with unclear clinical significance were previously identified. This study was supported by the Russian Science Foundation, grant 21-65-00017, https://rscf.ru/project/21-65-00017/ Disclosure of Interest None Declared

Introduction aCGH determines pathogenic copy number variations (CNVs) in about 10% of patients with intellectual disability (ID). In another 20% of patients, probably pathogenic CNVs or variants with uncertain clinical significance are detected. It may be variants that do not fully explain the patient’s symptoms, aberrations with reduced penetrance or inherited from healthy parents. The use of a sequencing method for such cases is advisable. Objectives Improvement of diagnosis of intellectual disability. Methods aCGH with 60K Agilent microarrays, qPCR, targeted sequencing, whole exome sequencing (WES). Results Six patients with ID and inherited deletions/duplications detected by aCGH and their parents if available were further examined by sequencing. Four patients had maternal CNVs: (1) del1q41 ( SPATA17, LINC00210, RRP15 ), (2) del7q35 ( TCAF2 , exon 8), (3) dup8p22p21.3 ( PSD3, exons 1-11), and (4) del12p11.1 ( SYT10, exons 1-2). Two patients had paternal CNVs: (5) dup1q44 ( SMYD3 , exons 2-5) and (6) del15q11.2 ( TUBGCP5, CYFIP1, NIPA1, NIPA2, LOC283683 ). The severe phenotype of patient (5) with dup1q44 could not be explained by the paternally inherited disruption of the single SMYD3 gene. WES determined probably pathogenic SNV in the MID1 gene associated with Opitz GBBB syndrome (OMIM 300000), which corresponds better to the patient’s phenotype and is likely to be the cause of the disease. Although del1q41 is included in the region of chromosome 1q41-q42 deletion syndrome (OMIM 612530) the phenotype of the patient (1) is much milder; WES in the patient detected two pathogenic ( MPO, MAN2C1 ) and one probably pathogenic ( ARID1B ) SNVs. In patient (6) with del15q11.2 pat WES detected additional pathogenic SNV in exon 7 of the ARSE gene. In patient (3) with dup8p22p21.3 WES determined two SNVs with uncertain significance in the KIDINS220, FOXG1 genes. No SNVs were detected by WES in patient (2) with del7q35. For patient (4) with del12p11.1 targeted SYT10 sequencing revealed no pathogenic SNVs as well. Conclusions Sometimes aCGH-analysis is sufficient to identify the causes of ID, however, in the case of detection of CNVs with uncertain clinical significance and/or inherited from healthy parents, it may be necessary to further examine the patient using sequencing methods. So, the accurate diagnosis was made by WES for one patient of eight. For another two patients the combination of CNVs and SNPs should be considered. For the last three patients the described aberrations could not explain the phenotype and whole genome sequencing may be the solution.This study was supported by the Russian Science Foundation, grant 21-65-00017, https://rscf.ru/project/21-65-00017/ Disclosure of Interest None Declared

Болезнь Вильсона-Коновалова - редкое аутосомно-рецессивное заболевание, которое характеризуется патологическим накоплением меди в печени, головном мозге и других тканях. Дифференциальная диагностика болезни Вильсона-Коновалова представляет собой сложную задачу вследствие выраженной гетерогенности клинических проявлений. Это подчеркивает важность разработки как новых методов диагностики, так и усовершенствования существующих. В рамках настоящего исследования было проведено сравнение клинической диагностики заболевания с результатами молекулярно-генетических исследований. Проанализировано 42 пациента с подозрением на болезнь Вильсона-Коновалова. Произведена оценка значения биохимических показателей метаболизма меди (концентрация церулоплазмина, щелочной фосфатазы, общего билирубина, АСТ, АЛТ сыворотки крови, содержание меди в печени, экскреция меди с мочой) согласно Лейпцигской количественной шкале. Для молекулярно-генетического анализа использовали геномную ДНК. Обогащение интересуемых регионов генома проводилось с помощью ПЦР длинных фрагментов. Для подготовки ДНК библиотек был использован набор Nextera DNA Flex (Illumina, США). Секвенирование проводилось на приборе Illumina MiSeq (Illumina, США). В результате исследования в 62,5% случаев у пациентов, направленных на подтверждение диагноза (по Лейпцигской количественной шкале), были найдены мутации в гене ATP7B, что подтверждает ценность комплексной диагностики по Лейпцигской количественной шкале с учетом клинической симптоматики и лабораторных показателей метаболизма меди. Wilson’s disease is a rare autosomal recessive disorder characterized by abnormal accumulation of copper in the liver, brain, and other tissues. Wilson’s disease differential diagnosis is a difficult task due to the pronounced clinical heterogeneity. This emphasizes the importance of developing both new diagnostic methods and improving existing ones. As part of this study, we compared clinical diagnostics with the results of molecular genetic studies. We analyzed 42 patients with suspected Wilson’s disease. The biochemical parameters copper metabolism values were assessed (serum ceruloplasmin concentration, liver copper content, urinary copper excretion, alkaline phosphatase, total bilirubin, AST, ALT) according to the Leipzig quantitative scale. We used genomic DNA for molecular genetic analysis. Regions of interest in the genome was enriched using long-range PCR. The Nextera DNA Flex kit (Illumina, USA) was used to prepare DNA libraries. Sequencing was performed on an Illumina MiSeq device (Illumina, USA). As a result of the study, in 62.5% of cases in patients aimed at confirming the diagnosis (according to the Leipzig quantitative scale), we found mutations in the ATP7B gene, which confirms the value of a comprehensive diagnosis according to the Leipzig quantitative scale, taking into account the clinical symptoms and copper metabolism laboratory parameters.

Using massively parallel sequencing, the COL1A1 and COL1A2 genes were analyzed in 16 patients from 10 families with osteogenesis imperfecta types I, III, and IV. To analyze the mutations in these genes, a panel of primers was developed for sequencing the complete gene sequence. As a result of the work, 10 mutations were revealed: six of them are in the COL1A1 gene, four mutations in the COL1A2 gene. All mutations, except one, were previously described in the literature and were found in patients with various types of osteogenesis imperfecta. Missense mutations were identified in five families, nonsense mutations in two families, splice site mutations in two cases, and a frameshift mutation in one patient. Unique mutations that were not repeated in unrelated patients were found in all families. The revealed high heterogeneity of the spectrum of mutations in the COL1A1 and COL1A2 genes in osteogenesis imperfecta indicates the effectiveness of using MPS-based methods for the diagnosis of this pathology.

Болезнь Вильсона-Коновалова (БВК) - аутосомно-рецессивное заболевание, развивающееся вследствие накопления меди в организме при повреждениях гена АТР7В. В настоящем исследовании проводился поиск мутаций в этом гене методом массового параллельного секвенирования у больных с БВК. Для целевого обогащения интересуемых регионов была разработана панель праймеров для ПЦР длинных фрагментов. У 6 пациентов из 12 проанализированных выявлены патогенные и вероятно патогенные варианты нуклеотидной последовательности гена АТР7В. Полученные результаты указывают на то, что разработанный метод таргетного массового параллельного секвенирования позволяет эффективно выявлять мутации в гене ATP7B. Wilson’s disease is an autosomal recessive disease that develops as a result of the accumulation of copper in the organism when the ATP7B gene is damaged. The present study searched for mutations in this gene using massively parallel sequencing in patients with Wilson’s disease. For targeted enrichment of the regions of interest, a primer panel for PCR of long fragments was developed. In 6 patients out of 12 analyzed, pathogenic and probably pathogenic variants of the nucleotide sequence of the ATP7B gene were identified. The obtained results indicate that the developed method of targeted massively parallel sequencing allows efficient detection of mutations in the ATP7B gene.