Improvement of Diagnostic Yield by an Additional Amplicon Module to Hybridization-Based Next-Generation Sequencing Panels.

Many approaches aimed at improving next-generation sequencing output for clinical purposes exist. However, sequencing gaps or misalignments for regions that are difficult to cover because of their low complexity or high homology still exist. Our aim was to improve the yield of sequencing data. A hybridization-based next-generation sequencing library was pooled with custom add-on amplicon-based libraries processed by the same commercial test and run in parallel and sequenced simultaneously. Formulas and steps for proper amplicon pooling (250 to 7000 bp) and final library merging are presented. The novel strategy was tested on selected archetypal situations: diagnostics of a gene with many pseudogenes, a genomic region surrounded by Alu repeats, simple one-time addition of an extra gene, and mosaicism detection. The sequence of all supplemented genomic regions was traced with reasonable coverage at the background of a hybridization captured library. The flexible add-on module expands the possibilities of routine diagnostics. The technical solution makes it possible to mix amplicons that differ significantly in size and process them in one tube simultaneously with samples of the hybridization-based panel. The proposed approach reduces turnaround time and increases diagnostic yield.

Evidence for the Association between the Intronic Haplotypes of Ionotropic Glutamate Receptors and First-Episode Schizophrenia.

The heritable component of schizophrenia (SCH) as a polygenic trait is represented by numerous variants from a heterogeneous group of genes each contributing a relatively small effect. Various SNPs have already been found and analyzed in genes encoding the NMDAR subunits. However, less is known about genetic variations of genes encoding the AMPA and kainate receptor subunits. We analyzed sixteen iGluR genes in full length to determine the sequence variability of iGluR genes. Our aim was to describe the rate of genetic variability, its distribution, and the co-occurrence of variants and to identify new candidate risk variants or haplotypes. The cumulative effect of genetic risk was then estimated using a simple scoring model. GRIN2A-B, GRIN3A-B, and GRIK4 genes showed significantly increased genetic variation in SCH patients. The fixation index statistic revealed eight intronic haplotypes and an additional four intronic SNPs within the sequences of iGluR genes associated with SCH (p < 0.05). The haplotypes were used in the proposed simple scoring model and moreover as a test for genetic predisposition to schizophrenia. The positive likelihood ratio for the scoring model test reached 7.11. We also observed 41 protein-altering variants (38 missense variants, four frameshifts, and one nonsense variant) that were not significantly associated with SCH. Our data suggest that some intronic regulatory regions of iGluR genes and their common variability are among the components from which the genetic predisposition to SCH is composed.

Bilineal inheritance of pathogenic PKD1 and PKD2 variants in a Czech family with autosomal dominant polycystic kidney disease - a case report.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary renal disorder, leading to end stage renal failure and kidney transplantation in its most serious form. The severity of the disease's manifestation depends on the genetic determination of ADPKD. The huge variability of different phenotypes (even within a single family) is not only modulated by the two main ADPKD genes (PKD1 and PKD2) but also by modifier genes and the whole genetic background. CASE PRESENTATION: This is a report of an ADPKD family with co-inheritance of PKD1 and PKD2 pathogenic variants. The proband, with an extremely serious manifestation of ADPKD (the man was diagnosed in early childhood, and with end stage renal disease aged 23), underwent genetic analysis of PKD1 and PKD2, which revealed the presence of pathogenic mutations in both of these genes. The missense PKD2 mutation p.Arg420Gly came from the proband's father, with a mild ADPKD phenotype. The same mutation of the PKD2 gene and similar mild disease presentation were found in the proband's aunt (father's sister) and her son. The nonsense mutation p.Gln2196* within the PKD1 gene was probably inherited from the proband's mother, who died at the age of 45. It was only discovered post mortem, that the real cause of her death was kidney failure as a consequence of untreated ADPKD. Unfortunately, neither the DNA of the proband's mother nor the DNA of any other family members from this side of the pedigree were available for further examination. The proband underwent successful cadaveric kidney transplantation at the age of 24, and this replacement therapy lasted for the next 15 years. CONCLUSIONS: Here, we present a first case of bilineal ADPKD inheritance in the Czech Republic. This report highlights the significant role of modifier genes in genetic determination of ADPKD, especially in connection with seriously deteriorated disease phenotypes. In our case, the modifying role is probably mediated by the PKD2 gene.

Comparison of SHOX and associated elements duplications distribution between patients (Leri-Weill dyschondrosteosis/idiopathic short stature) and population sample.

The effect of heterozygous duplications of SHOX and associated elements on Leri-Weill dyschondrosteosis (LWD) and idiopathic short stature (ISS) development is less distinct when compared to reciprocal deletions. The aim of our study was to compare frequency and distribution of duplications within SHOX and associated elements between population sample and LWD (ISS) patients. A preliminary analysis conducted on Czech population sample of 250 individuals compared to our previously reported sample of 352 ISS/LWD Czech patients indicated that rather than the difference in frequency of duplications it is the difference in their distribution. Particularly, there was an increased frequency of duplications residing to the CNE-9 enhancer in our LWD/ISS sample. To see whether the obtained data are consistent across published studies we made a literature survey to get published cases with SHOX or associated elements duplication and formed the merged LWD, the merged ISS, and the merged population samples. Relative frequency of particular region duplication in each of those merged samples were calculated. There was a significant difference in the relative frequency of CNE-9 enhancer duplications (11 vs. 3) and complete SHOX (exon1-6b) duplications (4 vs. 24) (p-value 0.0139 and p-value 0.000014, respectively) between the merged LWD sample and the merged population sample. We thus propose that partial SHOX duplications and small duplications encompassing CNE-9 enhancer could be highly penetrant alleles associated with ISS and LWD development.

Detection of SHOX gene aberrations in routine diagnostic practice and evaluation of phenotype scoring form effectiveness.

Heterozygous aberrations of SHOX gene have been reported to be responsible for Léri-Weill dyschondrosteosis (LWD) and small portion of idiopathic short stature. The study was established to assess effectiveness of using phenotype 'scoring form' in patients indicated for SHOX gene defect analysis. The submitted study is based on a retrospective group of 352 unrelated patients enrolled as a part of the routine diagnostic practice and analyzed for aberrations affecting the SHOX gene. All participants were scanned for deletion/duplication within the main pseudoautosomal region (PAR1) using the multiplex ligation-dependent probe amplification (MLPA) method. The phenotype 'scoring form' is used in our laboratory practice to preselect patients for subsequent mutation analysis of SHOX gene-coding sequences. The overall detection rate was 11.1% but there was a significant increase in frequency of SHOX gene defect positive with increasing achieved score (P<0.0001). The most frequent aberration was a causal deletion within PAR1. In three probands, MLPA analysis indicated a more complex rearrangement. Madelung deformity or co-occurrence of disproportionate short stature, short forearm and muscular hypertrophy had represented the most potent markers to determine the likelihood of SHOX gene defect detection. We conclude that appliance of phenotype 'scoring form' had saved excessive sample analysis and enabled effective routine diagnostic testing.

Mutation analysis of TRPS1 gene including core promoter, 5'UTR, and 3'UTR regulatory sequences with insight into their organization.

The TRPS1 protein is a potent regulator of proliferation, differentiation, and apoptosis. The TRPS1 gene aberrations are strongly associated with rare trichorhinophalangeal syndrome (TRPS) development. We have conducted MLPA analysis to capture deletion within the crucial 8q24.1 chromosomal region in combination with mutation analysis of TRPS1 gene including core promoter, 5'UTR, and 3'UTR sequences in nine TRPS patients. Low complexity or extent of untranslated regulatory sequences avoided them from analysis in previous studies. Amplicon based next generation sequencing used in our study bridge over these technical limitations. Finally, we have made extended in silico analysis of TRPS1 gene regulatory sequences organization. Single contiguous deletion and an intragenic deletion intervening several exons were detected. Mutation analysis revealed five TRPS1 gene aberrations (two structural rearrangements, two nonsense mutations, and one missense substitution) reaching the overall detection rate of 78%. Several polymorphic variants were detected within the analysed regulatory sequences but without proposed pathogenic effect. In silico analysis suggested alternative promoter usage and diverse expression effectivity for different TRPS1 transcripts. Haploinsufficiency of TRPS1 gene was responsible for most of the TRPS phenotype. Structure of TRPS1 gene regulatory sequences is indicative of generally low single allele expression and its tight control.

Cryptic chromosomal rearrangements in children with idiopathic mental retardation in the Czech population.

AIMS: The aim of our study was to scan for cryptic rearrangements using the multiplex ligation probe amplification method in a cohort of 64 probands with mental retardation or developmental delays in combination with at least one of the following symptoms: hypotonia after birth, congenital anomalies, or face dysmorphisms; but without a positive cytogenetic finding. The study contributes to the knowledge of microdeletion syndromes and helps disclose their natural phenotypic variability. RESULTS: In total, 10 positives (16%) were detected, particularly 3 duplications (Xpter-p22.32; 17p11.2; 22q11) and 6 different deletions (1p36; 7q11.23; 10p15; 15q11-q13; 17p11.2; 17p13.3), 1 of these in 2 probands. Besides the well-characterized syndromes, less-often described rearrangements with ambiguous phenotype associations were also detected. CONCLUSIONS: Some rearrangements, particularly duplications, are associated with vague phenotypes; and their frequency could be underestimated.

Impact of apolipoprotein A5 variants on statin treatment efficacy.

AIMS: Despite the fact that statin treatment efficacy is very high, there are substantial differences in treatment effectiveness among individuals. It is supposed that genetic predisposition plays an important role in these differences, but the contribution of individual polymorphisms is poorly understood. So far, more than 30 genes have been examined with ambiguous results. Apolipoprotein A5 is an important determinant of plasma lipid concentrations and its genetic variation could account for some of the observed differences in the response to statin therapy. However, this has not been analyzed before. MATERIALS AND METHODS: We examined the putative association between APOA5 SNPs (c.-1131T>C, c.56C>G and c.457G>A) and efficacy during 3 months of statin treatment in 187 adult Caucasians. Patients were treated with low-dose (10 or 20 mg per day) simvastatin (46.3%), atorvastatin (40.5%) and lovastatin (13.2%). RESULTS: The decrease in cholesterol was not significantly associated with the type or dose of statin. Carriers of the APOA5 genotype TT-1131 (n = 154) benefited more from statin treatment when compared with the C-1131 allele carriers (n = 33) (Delta low-density lipoprotein cholesterol: -36.3 +/- 15.1% vs Delta low-density lipoprotein cholesterol: -29.9 +/- 12.5%; p < 0.005, Mann-Whitney test). This result was independent of sex, age, BMI and APOE polymorphism. CONCLUSION: Our results suggest that the APOA5 gene variants may play an important role in the pharmacogenetics of statin treatment.

The apo(a) gene (TTTTA)n promoter polymorphism and its association with variability in exons of the kringle IV types 8 to 10.

BACKGROUND: A large portion of lipoprotein (a) concentration is explained by the variable number of kringle domains at the apo(a) gene. The (TTTTA)(n) repetition from the promoter of the apo(a) gene is supposed to be a relevant candidate for the additional regulation of lipoprotein (a) levels. METHODS: We scanned the kringle IV types 8-10 of the apo(a) gene for candidate variants that could be at least partly responsible for the (TTTTA)(n) repetition effect. The selected sample group comprises of 160 unrelated individuals designed to cover a representative amount even of the rare TTTTA(n) allele carriers. The high resolution melting method was used to scan exons of selected kringles for alteration. Linkage disequilibrium and haplotype frequencies were assigned using Arlequine software. RESULTS: Overall eight different sequence variants were detected from which all but two were rare (c.66A-->C; c.74C-->T; c.133G-->A; c.154C-->T;+1G-->A 5'splice site; c.16G-->T; c.61G-->A; c.63C-->T). However the TTTTA(n) repetition was in allelic association with all five tested polymorphic loci (P<0.0023). CONCLUSIONS: The sequence variants from the coding portion of kringle IV types 8-10 of the LPA locus are significantly associated with the TTTTA(n) repetition but still are not able to markedly account for the TTTTA(n) locus effect.