New Approach for Detection of Normal Alternative Splicing Events and Aberrant Spliceogenic Transcripts with Long-Range PCR and Deep RNA Sequencing

RNA sequencing is a promising technique for detecting normal and aberrant RNA isoforms. Here, we present a new single-gene, straightforward 1-day hands-on protocol for detection of splicing alterations with deep RNA sequencing from blood. We have validated our method’s accuracy by detecting previously published normal splicing isoforms of STK11 gene. Additionally, the same technique was used to provide the first comprehensive catalogue of naturally occurring alternative splicing events of the NBN gene in blood. Furthermore, we demonstrate that our approach can be used for detection of splicing impairment caused by genetic variants. Therefore, we were able to reclassify three variants of uncertain significance: NBN:c.584G>A, STK11:c.863-5_863-3delCTC and STK11:c.615G>A. Due to the simplicity of our approach, it can be incorporated into any molecular diagnostics laboratory for determination of variant’s impact on splicing.

Detecting Variants in the NBN Gene While Testing for Hereditary Breast Cancer: What to Do Next?

The NBN gene has been included in breast cancer (BC) multigene panels based on early studies suggesting an increased BC risk for carriers, though not confirmed by recent research. To evaluate the impact of NBN analysis, we assessed the results of NBN sequencing in 116 BRCA-negative BC patients and reviewed the literature. Three patients (2.6%) carried potentially relevant variants: two, apparently unrelated, carried the frameshift variant c.156_157delTT and another one the c.628G>T variant. The latter was subsequently found in 4/1390 (0.3%) BC cases and 8/1580 (0.5%) controls in an independent sample, which, together with in silico predictions, provided evidence against its pathogenicity. Conversely, the rare c.156_157delTT variant was absent in the case-control set; moreover, a 50% reduction of NBN expression was demonstrated in one carrier. However, in one family it failed to co-segregate with BC, while the other carrier was found to harbor also a probably pathogenic TP53 variant that may explain her phenotype. Therefore, the c.156_157delTT, although functionally deleterious, was not supported as a cancer-predisposing defect. Pathogenic/likely pathogenic NBN variants were detected by multigene panels in 31/12314 (0.25%) patients included in 15 studies. The risk of misinterpretation of such findings is substantial and supports the exclusion of NBN from multigene panels.

Quick and easy: 1-day-protocol for detection of normal alternative splicing events and aberrant spliceogenic transcripts with deep RNA sequencing

RNA sequencing is a promising technique for detecting normal and aberrant RNA isoforms. Here, we present a new single-gene, straightforward 1-day hands-on protocol for detection of splicing alterations with deep RNA sequencing from blood. We have validated our method’s accuracy by detecting all normal splicing isoforms of STK11 gene that were previously published. Additionally, the same technique was used to provide the first comprehensive catalogue of naturally occurring alternative splicing events of the NBN gene in blood. Furthermore, we demonstrate that our approach can be used for detection of splicing impairment caused by genetics variants. Due to the simplicity of our approach it can be incorporated into any molecular diagnostics laboratory for determination of variant’s impact on splicing.

Involvement of NBN gene polymorphism in predisposition to dystropic diseases

Изучение генетических особенностей дистропных заболеваний (в том числе, астмы и туберкулеза), определяющих формирование взаимоисключающих фенотипов на клиническом уровне, важно для лучшего понимания их патогенеза. Цель настоящего исследования заключалась в оценке значимости генетического полиморфизма гена NBN в формировании предрасположенности к астме и туберкулезу. Изучена изменчивость rs1805800 и rs709816 гена NBN в выборках больных бронхиальной астмой (БА), туберкулезом (ТБ) и популяционной выборке г. Томска (ПВ), общая выборка составила 683 человека. Показано разнонаправленное изменение частот аллелей, генотипов, сочетаний генотипов по rs1805800 и rs709816 гена NBN между группами больных БА и ТБ по сравнению с популяционной выборкой. Для развития БА генотип CT rs1805800 обладает рисковым (OR=1,66, χ2=5,41, p=0,02); а генотипа СС - протективным (OR=0,57, χ2=5,57, p=0,018) эффектом. Статистически значимые различия между группами БА и ТБ выявлены также по частотам сочетаний генотипов rs1805800/rs709816 в гене NBN (χ2 = 12,88; p=0,045). The research of genetic features of dystropic diseases (including asthma and tuberculosis) that determine the formation of mutually exclusive phenotypes at the clinical level is important for a better understanding of their pathogenesis. The aim of this study was to assess the significance of the genetic polymorphism of the NBN gene in the formation of a predisposition to asthma and tuberculosis. The variability of rs1805800 and rs709816 of the NBN gene in the samples of patients with bronchial asthma (BA), tuberculosis (TB) and the population sample of Tomsk (PV) was studied, the total sample was 683 people. The different-directional change of frequencies of alleles, genotypes, combinations of genotypes by rs1805800 and rs709816 of the NBN gene between groups of patients with BA and TB compared to the population sample is shown. For the development of BA, the genotype CT rs1805800 is risky (OR=1.66, χ2=5.41, p=0.02); And the SS genotype is a protective (OR=0.57, χ2=5.57, p=0.018) effect. Statistically significant differences between BA and TB groups are also revealed by the frequencies of combinations of the genotypes rs1805800/rs709816 in the NBN gene (χ2=12.88; p=0,045).

A case of Nijmegen syndrome in a child with frequent relapses of a respiratory infection

The article provides clinical dynamic observation of a child with a primary immunodeficiency state, presents the stages of the differential diagnostic search for congenital genetic pathology. Nijmegen syndrome is an autosomal recessive disease caused by a mutation of the NBN gene, characterized by microcephaly, Bird facial features, delayed physical development, immunodeficiency, congenital malformations, increased sensitivity to x-ray radiation and a high susceptibility to cancer. This syndrome is relevant for Russian pediatricians due to the high frequency of carriage of the Slavic NBN gene mutation among the population of the Russian Federation. The presented clinical observation describes a patient with characteristic phenotypic features, frequent episodes of acute respiratory viral infections (up to 8 times a year) with the addition of a bacterial infection, fever up to febrile numbers. The duration of verification of the immunodeficiency state was 5 years. Despite the complicated course of infectious diseases, the general well-being of the child did not suffer significantly, the level of neuropsychic development corresponded to age. The diagnosis of Nijmegens syndrome was made on the basis of characteristic phenotypic features, anamnesis, and laboratory data, instrumental studies and confirmed by the results of molecular genetic analysis.