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.

Spectrum of Genes for Non-GJB2-Related Non-Syndromic Hearing Loss in the Russian Population Revealed by a Targeted Deafness Gene Panel.

Hearing loss is one of the most genetically heterogeneous disorders known. Over 120 genes are reportedly associated with non-syndromic hearing loss (NSHL). To date, in Russia, there have been relatively few studies that apply massive parallel sequencing (MPS) methods to elucidate the genetic factors underlying non-GJB2-related hearing loss cases. The current study is intended to provide an understanding of the mutation spectrum in non-GJB2-related hearing loss in a cohort of Russian sensorineural NSHL patients and establish the best diagnostic algorithm. Genetic testing using an MPS panel, which included 33 NSHL and syndromic hearing loss (SHL) genes that might be misdiagnosed as NSHL genes, was completed on 226 sequentially accrued and unrelated patients. As a result, the molecular basis of deafness was found in 21% of the non-GJB2 NSHL cases. The total contribution pathogenic, and likely pathogenic, variants in the genes studied among all hereditary NSHL Russian patients was 12%. STRC pathogenic and likely pathogenic, variants accounted for 30% of diagnoses in GJB2-negative patients, providing the most common diagnosis. The majority of causative mutations in STRC involved large copy number variants (CNVs) (80%). Among the point mutations, the most common were c.11864G>A (p.Trp3955*) in the USH2A gene, c.2171_2174delTTTG (p.Val724Glyfs*6) in the STRC gene, and c.107A>C (p.His36Pro) and c.1001G>T (p.Gly334Val) in the SLC26A4 gene. Pathogenic variants in genes involved in SHL accounted for almost half of the cases with an established molecular genetic diagnosis, which were 10% of the total cohort of patients with non-GJB2-related hearing loss.

Identification of novel variants in the LDLR gene in Russian patients with familial hypercholesterolemia using targeted sequencing.

Familial hypercholesterolemia (FH) is caused by mutations in various genes, including the LDLR, APOB and PSCK9 genes; however, the spectrum of these mutations in Russian individuals has not been fully investigated. In the present study, mutation screening was performed on the LDLR gene and other FH-associated genes in patients with definite or possible FH, using next-generation sequencing. In total, 59 unrelated patients were recruited and sorted into two separate groups depending on their age: Adult (n=31; median age, 49; age range, 23-70) and children/adolescent (n=28; median age, 11; age range, 2-21). FH-associated variants were identified in 18 adults and 25 children, demonstrating mutation detection rates of 58 and 89% for the adult and children/adolescent groups, respectively. In the adult group, 13 patients had FH-associated mutations in the LDLR gene, including two novel variants [NM_000527.4: c.433_434dupG p.(Val145Glyfs*35) and c.1186G>C p.(Gly396Arg)], 3 patients had APOB mutations and two had ABCG5/G8 mutations. In the children/adolescent group, 21 patients had FH-causing mutations in the LDLR gene, including five novel variants [NM_000527.4: c.325T>G p.(Cys109Gly), c.401G>C p.(Cys134Ser), c.616A>C p.(Ser206Arg), c.1684_1691delTGGCCCAA p.(Pro563Hisfs*14) and c.940+1_c.940+4delGTGA], and 2 patients had APOB mutations, as well as ABCG8 and LIPA mutations, being found in different patients. The present study reported seven novel LDLR variants considered to be pathogenic or likely pathogenic. Among them, four missense variants were located in the coding regions, which corresponded to functional protein domains, and two frameshifts were identified that produced truncated proteins. These variants were observed only once in different patients, whereas a splicing variant in intron 6 (c.940+1_c.940+4delGTGA) was detected in four unrelated individuals. Previously reported variants in the LDLR, APOB, ABCG5/8 and LIPA genes were observed in 33 patients. The LDLR p.(Gly592Glu) variant was detected in 6 patients, representing 10% of the FH cases reported in the present study, thus it may be a major variant present in the Russian population. In conclusion, the present study identified seven novel variants of the LDLR gene and broadens the spectrum of mutations in FH-related genes in the Russian Federation.

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.