No abstract

Osteogenesis imperfecta (OI) is a rare genetic disorder demonstrating considerable phenotypic and genetic heterogeneity. The extensively studied genotype–phenotype correlation is a crucial issue for a reliable counseling, as the disease is recognized at increasingly earlier stages of life, including prenatal period. Based on population studies, clusters in COL1A1 and COL1A2 genes associated with the presence of glycine substitutions leading to fatal outcome have been distinguished and named as “lethal regions.” Their localization corresponds to the ligand-binding sites responsible for extracellular interactions of collagen molecules, which could explain high mortality associated with mutations mapping to these regions. Although a number of non-lethal cases have been identified from the variants located in lethal clusters, the mortality rate of mutations has not been updated. An next generation sequencing analysis, using a custom gene panel of known and candidate OI genes, was performed on a group of 166 OI patients and revealed seven individuals with a causative mutations located in the lethal regions. Patients’ age, ranging between 3 and 25 years, excluded the expected fatal outcome. The identification of non-lethal cases caused by mutations located in lethal domains prompted us to determine the actual mortality caused by glycine substitutions mapping to lethal clusters and evaluate the distribution of all lethal glycine mutations across collagen type I genes, based on records deposited in the OI Variant Database. Finally, we identified six glycine substitutions located in lethal regions of COL1A1 and COL1A2 genes, of which four are novel. The review of all mutations in the dedicated OI database, revealed 33 distinct glycine substitutions in two lethal domains of COL1A1, 26 of which have been associated with a fatal outcome. Similarly, 109 glycine substitutions have been identified in eight lethal clusters of COL1A2, of which 51 have been associated with a fatal manifestation. An analysis of all glycine substitutions leading to fatal phenotype, showed that their distribution along collagen type I genes is not regular, with 17% (26 out of 154) of mutations reported in COL1A1 and 64% (51 out of 80) in COL1A2 corresponding to localization of the lethal regions.

Familial hypercholesterolemia (FH) is an inherited, autosomal dominant metabolic disorder mostly associated with disease-causing variant in LDLR, APOB or PCSK9. Although the dominant changes are small-scale missense, frameshift and splicing variants, approximately 10% of molecularly defined FH cases are due to copy number variations (CNVs). The first-line strategy is to identify possible pathogenic SNVs (single nucleotide variants) using multiple PCR, Sanger sequencing, or with more comprehensive approaches, such as NGS (next-generation sequencing), WES (whole-exome sequencing) or WGS (whole-genome sequencing). The gold standard for CNV detection in genetic diagnostics are MLPA (multiplex ligation-dependent amplification) or aCGH (array-based comparative genome hybridization). However, faster and simpler analyses are needed. Therefore, it has been proposed that NGS data can be searched to analyze CNV variants. The aim of the study was to identify novel CNV changes in FH patients without detected pathogenic SNVs using targeted sequencing and evaluation of CNV calling tool (DECoN) working on gene panel NGS data; the study also assesses its suitability as a screening step in genetic diagnostics. A group of 136 adult and child patients were recruited for the present study. The inclusion criteria comprised at least “possible FH” according to the Simon Broome diagnostic criteria in children and the DLCN (Dutch Lipid Clinical Network) criteria in adults. NGS analysis revealed potentially pathogenic SNVs in 57 patients. Thirty selected patients without a positive finding from NGS were subjected to MLPA analysis; ten of these revealed possibly pathogenic CNVs. Nine patients were found to harbor exons 4–8 duplication, two harbored exons 6–8 deletion and one demonstrated exon 9–10 deletion in LDLR. To test the DECoN program, the whole study group was referred for bioinformatic analysis. The DECoN program detected duplication of exons 4–8 in the LDLR gene in two patients, whose genetic analysis was stopped after the NGS step. The integration of the two methods proved to be particularly valuable in a five-year-old girl presenting with extreme hypercholesterolemia, with both a pathogenic missense variant (c.1747C>T) and exons 9–10 deletion in LDLR. This is the first report of a heterozygous deletion of exons 9 and 10 co-occurring with SNV. Our results suggest that the NGS-based approach has the potential to identify large-scale variation in the LDLR gene and could be further applied to extend CNV screening in other FH-related genes. Nevertheless, the outcomes from the bioinformatic approach still need to be confirmed by MLPA; hence, the latter remains the reference method for assessing CNV in FH patients.

The most common form of inherited lipid disorders is familial hypercholesterolemia (FH). It is characterized primarily by high concentrations of the clinical triad of low-density lipoprotein cholesterol, tendon xanthomas and premature CVD. The well-known genetic background are mutations in LDLR, APOB and PCSK9 gene. Causative mutations can be found in 60–80% of definite FH patients and 20–30% of those with possible FH. Their occurrence could be attributed to the activity of minor candidate genes, whose causal mechanism has not been fully discovered. The aim of the conducted study was to identify disease-causing mutations in FH-related and candidate genes in pediatric patients from Poland using next generation sequencing (NGS). An NGS custom panel was designed to cover 21 causative and candidate genes linked to primary dyslipidemia. Recruitment was performed using Simon Broome diagnostic criteria. Targeted next generation sequencing was performed on a MiniSeq sequencer (Illumina, San Diego, CA, USA) using a 2 × 150 bp paired-end read module. Sequencing data analysis revealed pathogenic and possibly pathogenic variants in 33 out of 57 studied children. The affected genes were LDLR, APOB, ABCG5 and LPL. A novel pathogenic 7bp frameshift deletion c.373_379delCAGTTCG in the exon 4 of the LDLR gene was found. Our findings are the first to identify the c.373_379delCAGTTCG mutation in the LDLR gene. Furthermore, the double heterozygous carrier of frameshift insertion c.2416dupG in the LDLR gene and missense variant c.10708C>T in the APOB gene was identified. The c.2416dupG variant was defined as pathogenic, as confirmed by its cosegregation with hypercholesterolemia in the proband’s family. Although the APOB c.10708C>T variant was previously detected in hypercholesterolemic patients, our data seem to demonstrate no clinical impact. Two missense variants in the LPL gene associated with elevated triglyceride plasma level (c.106G>A and c.953A>G) were also identified. The custom NGS panel proved to be an effective research tool for identifying new causative aberrations in a genetically heterogeneous disease as familial hypercholesterolemia (FH). Our findings expand the spectrum of variants associated with the FH loci and will be of value in genetic counseling among patients with the disease.

Introduction: Congenital hypogonadotropic hypogonadism results from a dysfunction of the hypothalamic-pituitary-gonadal axis, which is essential for the development and function of the reproductive system. It may be associated with anosmia, referred to as Kallmann syndrome, or a normal sense of smell. Numerous studies have proven that hypogonadotropic hypogonadism is not simply a monogenic Mendelian disease, but that more than one gene may be involved in its pathogenesis in a single patient. The oligogenic complex architecture underlying the disease is still largely unknown. Material and methods: Targeted next-generation sequencing (NGS) was used to screen for DNA variants in a cohort of 47 patients with congenital hypogonadotropic hypogonadism. The NGS panel consists of over 50 wellknown and candidate genes, associated with hypogonadotropic state. Results: Here we report the identification of new oligogenic variants in SPRY4/SEMA3A, SRA1/SEMA7A, CHD7/SEMA7A, CCDC141/POLR3B/POLR3B, and PROKR2/SPRY4/NSMF. These genes are known to contribute to the phenotype of hypogonadotropic hypogonadism, yet our results point to potential new "partners" underlying digenic and trigenic patterns. Conclusions: The finding supports the importance of oligogenic inheritance and demonstrates the complexity of genetic architecture in hypogonadotropic hypogonadism. It also underlines the necessity for developing finetuned guidelines to provide a tool for adequate and precise sequence variant classification in non-Mendelian conditions.