Familial hypercholesterolemia (FH) is an autosomal codominant disorder characterized by impaired clearance of low-density lipoproteins from the bloodstream, markedly elevated plasma total cholesterol and low density lipoprotein cholesterol levels, and early onset of atherosclerosis and cardiovascular disease. FH is one of the most common monogenic disorders in humans. The majority of FH cases are caused by pathogenic variants in three genes:
LDLR
(OMIM: 143890),
APOB
(OMIM: 107730), and
PCSK9
(OMIM: 607786). More than 80 % of FH cases are associated with mutations in the
LDLR
gene, located on chromosome 19. In 25–75 % of patients with a clinical FH phenotype, no pathogenic variant is identified in these genes. Whole-exome sequencing and targeted gene panel sequencing of the
LDLR
,
APOB
,
PCSK9
, and
LDLRAP1
genes were performed in patients with an FH phenotype, followed by confirmation of the identified variants by Sanger sequencing. Three unrelated probands were found to carry intronic
LDLR
variants for which functional evidence was previously unavailable. The aim of this study was to functionally assess
in vitro
the effects of three intronic
LDLR
variants (NM_000527.5: c.940+3_940+6del, c.941-3C>G, and c.2389+5G>A) on pre-mRNA splicing using a minigene assay. Minigene constructs encompassing target exons with flanking intronic sequences were generated and transfected into the HEK293 and HeLa cell lines. The deleterious effect of all three variants on pre-mRNA splicing was confirmed. The fournucleotide deletion c.940+3_940+6del resulted in two aberrant transcripts: inclusion of six nucleotides from intron 6 and complete retention of the minigene intronic sequence. The c.941-3C>G variant caused loss of the canonical acceptor splice site and activation of a cryptic site, with inclusion of intronic nucleotides from intron 6. The c.2389+5G>A variant resulted in exon 16 skipping. Functional
in vitro
analysis is a key tool for the molecular verification of intronic variants of uncertain clinical significance and, in conjunction with clinical data, supports the establishment of a definitive molecular diagnosis.
V. Danilchenko, D. Ivanoshchuk, O. Timoshchenko et al.· Vavilov Journal of Genetics...· 0 citations
Pathogenic variants in the
SLC26A4
gene (solute carrier family 26, member 4) are a common cause of inherited hearing loss. The
SLC26A4
gene encodes the transmembrane protein pendrin, a member of the SLC26 anion transporter family, with predominant expression in the inner ear, thyroid, and kidney tissues. Pathogenic
SLC26A4
variants cause nonsyndromic recessive hearing loss (DFNB4) and Pendred syndrome (sensorineural hearing loss and thyroid dysfunction). In many studies analyzing the
SLC26A4
gene, along with patients who have two recessive pathogenic
SLC26A4
variants (M2 patients) and an accurate molecular genetic diagnosis can be made, a group of patients with only one pathogenic
SLC26A4
variant (M1 patients) is often identified, which creates a diagnostic problem. The presence of M1 patients may reflect copy number variations (CNVs, deletions/duplications) in the
SLC26A4
gene that are not detected by routine diagnostic methods. The aim of the study is to search for deletions/duplications in the
SLC26A4
gene using the MLPA (Multiplex Ligation-dependent Probe Amplification) method in thirteen patients belonging to the indigenous people of the Tyva Republic (Southern Siberia), in whom only one pathogenic
SLC26A4
variant (M1 patients) was identified during the study of the etiology of hereditary hearing loss. As a result of MLPA analysis and molecular cloning in DNA samples of three M1 patients from two related Tuvinian families, a novel variant was revealed – tandem duplication of twelve nucleotides (c.1185_1196dup) in exon 10 of the
SLC26A4
gene. This variant is an in-frame insertion resulting in the inclusion of four additional amino acid residues Gly-Phe-Phe-Ser (p.(Gly396_Ser399dup)) in a highly conserved region of the pendrin protein. Most predictive programs predict the damaging effect of the c.1185_1196dup variant on the structure and function of the pendrin protein. The pathogenicity of the c.1185_1196dup variant is supported by its segregation with hearing pathology in the pedigree of patients in whom it was found in a compound heterozygous state with pathogenic
SLC26A4
variants, as well as its absence in a control sample of Tuvinians and in the world genomic databases. Functional
in vitro
studies are needed to confirm the potentially deleterious effects of the novel c.1185_1196dup (p.(Gly396_Ser399dup)) variant and its association with hearing pathology.
M. V. Zytsar, V. Danilchenko, A. Bondar et al.· Vavilov Journal of Genetics...· 0 citations
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