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.
- MeSH
- genomika * MeSH
- genová knihovna MeSH
- hybridizace nukleových kyselin MeSH
- lidé MeSH
- vysoce účinné nukleotidové sekvenování * MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
OBJECTIVE: : Arterial hypertension is a common complication in patients with autosomal recessive polycystic kidney disease (ARPKD), occurring in 33-75% of children when measured by office blood pressure (OBP). Ambulatory blood pressure monitoring (ABPM) is a superior tool for investigating blood pressure relative to OBP. The aim of our study was to investigate the prevalence and control of hypertension in children with ARPKD based on ABPM. METHODS: This retrospective study evaluated 36 children with ARPKD and at least one ABPM performed in two our tertiary paediatric nephrology centres and 29 children with at least two ABPM. Ambulatory hypertension was defined as mean daytime or night-time BP at least 95th percentile or use of antihypertensives and controlled hypertension as normal ambulatory BP in children on antihypertensive drugs. RESULTS: The first ABPM study revealed ambulatory hypertension in 94% of children. Untreated or uncontrolled ambulatory hypertension was diagnosed in 67% and controlled hypertension in only 28%. Masked hypertension was found in 5.5% and white-coat hypertension in 14%. The last ABPM study revealed ambulatory hypertension in 86% (all 86% hypertensive children on drugs, i.e. no untreated hypertension), the prevalence of controlled hypertension increased to 59%. Masked hypertension was detected in 8.3% and white-coat hypertension in 10%. Ambulatory blood pressure correlated neither with kidney length nor with glomerular filtration rate. Echocardiography demonstrated left ventricular hypertrophy (LVH) in 27% of children at the time of their first ABPM. CONCLUSION: The prevalence of ambulatory hypertension is very high in children with ARPKD, while the control of hypertension improves over time.
- MeSH
- ambulantní monitorování krevního tlaku MeSH
- dítě MeSH
- hypertenze * komplikace farmakoterapie epidemiologie MeSH
- krevní tlak MeSH
- lidé MeSH
- polycystické ledviny autozomálně recesivní * komplikace epidemiologie MeSH
- retrospektivní studie MeSH
- Check Tag
- dítě MeSH
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
Cystic kidney diseases are a very heterogeneous group of chronic kidney diseases. The diagnosis is usually based on clinical and ultrasound characteristics and the final diagnosis is often difficult to be made. Next-generation sequencing (NGS) may help the clinicians to find the correct final diagnosis. The aim of our study was to test the diagnostic yield of NGS and its ability to improve the diagnosis precision in a heterogeneous group of children with cystic kidney diseases. Next-generation sequencing of genes responsible for the formation of cystic kidneys was performed in 31 unrelated patients with various clinically diagnosed cystic kidney diseases gathered at the Department of Pediatrics of Motol University Hospital in Prague between 2013 and 2018. The underlying pathogenic variants were detected in 71% of patients (n = 22), no or only one (in case of autosomal recessive inheritance) pathogenic variant was found in 29% of patients (n = 9). The result of NGS correlated with the clinical diagnosis made before the NGS in 55% of patients (n = 17), in the remaining 14 children (45%) the result of NGS revealed another type of cystic kidney disease that was suspected clinically before or did not find causal mutation in suspected genes. The most common unexpected findings were variants in nephronophthisis (NPHP) genes in children with clinically suspected autosomal recessive polycystic kidney disease (ARPKD, n = 4). Overall, 24 pathogenic or probably pathogenic variants were detected in the PKHD1 gene, 8 variants in the TMEM67 gene, 4 variants in the PKD1 gene, 2 variants in the HNF1B gene and 2 variants in BBS1 and NPHP1 genes, respectively. NGS is a valuable tool in the diagnostics of various forms of cystic kidney diseases. Its results changed the clinically based diagnoses in 16% (n = 5) of the children.
- MeSH
- adaptorové proteiny signální transdukční genetika MeSH
- cystická onemocnění ledvin diagnóza genetika MeSH
- cytoskeletální proteiny genetika MeSH
- dítě MeSH
- genetická predispozice k nemoci genetika MeSH
- hepatocytární jaderný faktor 1-beta genetika MeSH
- kationtové kanály TRPP genetika MeSH
- kojenec MeSH
- lidé MeSH
- mutace * MeSH
- novorozenec MeSH
- polycystické ledviny autozomálně recesivní diagnóza genetika MeSH
- předškolní dítě MeSH
- proteiny asociované s mikrotubuly genetika MeSH
- receptory buněčného povrchu genetika MeSH
- vysoce účinné nukleotidové sekvenování metody MeSH
- Check Tag
- dítě MeSH
- kojenec MeSH
- lidé MeSH
- mužské pohlaví MeSH
- novorozenec MeSH
- předškolní dítě MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
During standard molecular diagnostic procedure, two Czech families with APC (Adenomatous polyposis coli gene) mosaicism have been detected. A woman with attenuated familial adenomatous polyposis (AFAP, OMIM #175100) was recently inspected by next generation sequencing. Standard bioinformatics pipeline, restricted to variants with at least 20% of reads (for germline variants) would miss mutation p.G1412X (NM_000038.5) present in 17% of reads. This novel variant was not present in any of her two children. Another woman with a clinical manifestation of attenuated FAP was tested 16 years ago without conclusive APC mutation found when denaturing gradient gel electrophoresis (DGGE), protein truncation test (PTT), multiplex ligation probe amplification (MLPA) and direct Sanger sequencing were applied. Recent inspection of her son showed clear mutation p.Q1062X (NM_000038.5, NP_000029.2) leading to premature stop codon. This finding led to re-evaluation of this protein position in his mother and detection of mosaicism (11% of allele, 22% of heterozygous cells in blood), which was primarily overlooked. Mutations in both patients were confirmed by allele-specific real time PCR (AS qPCR). In both index patients it was possible to detect and quantify the mosaic allele in biological samples of polyps, adjacent colonic mucosa and buccal swabs. In cases of sporadic appearance of FAP, besides blood we plan to preferably inspect also other samples, where mosaic fraction might be under detection limit of bioinformatics pipelines (<3%). For our future routine NGS sequencing analysis we will apply our in-house somatic variant detection pipeline to minimize the false negative calls when genes with high level of de-novo mutations are analyzed.
- MeSH
- familiární adenomatózní polypóza genetika MeSH
- geny APC * MeSH
- lidé MeSH
- mozaicismus * MeSH
- mutace MeSH
- mutační analýza DNA MeSH
- Check Tag
- lidé MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- Geografické názvy
- Česká republika MeSH
BACKGROUND: Mutations in INF2 are frequently responsible for focal segmental glomerulosclerosis (FSGS), which is a common cause of end stage renal disease (ESRD); additionally, they are also connected with Charcot-Marie-Tooth neuropathy. INF2 encodes for inverted formin 2. This protein participates in regulation of the dynamics of the actin cytoskeleton, involving not only the polymerisation, but also the depolymerisation of filaments. The present study is the first mutational analysis of INF2 done in the Czech Republic. METHODS: Mutational analysis of INF2 was performed on 109 patients (mean age at onset 41.44 ± 18.91 years) with FSGS or minimal change disease (MCD); and also in 6 patients without renal biopsy who had already developed chronic kidney disease (CKD)/ESRD at the time of diagnosis. We used high resolution melting method (HRM), with subsequent Sanger sequencing, in suspect samples from HRM analysis. The HRM method is an effective method for the screening of large cohorts of patients. RESULTS: Two pathogenic mutations (p.Arg214His and p.Arg218Gln) were detected in INF2. The first (p.Arg214His) was identified in the FSGS patient with a positive family history. The second mutation (p.Arg218Gln) was found in two brothers with ESRD of unknown etiology. The most frequent sequence change was the substitution p.P35P, the incidence of which corresponded with the frequencies available in the ExAC Browser and gnomAD Browser databases. This analysis also detected different exonic and intronic changes that probably did not influence the phenotype of the included patients. CONCLUSIONS: The INF2 mutational screening is useful in familial FSGS cases as well as in patients with an unknown cause for their ESRD, but with a positive family history. INF2 seems to be not only the cause of FSGS, but also of ESRD of unknown etiology. Our study has confirmed that the HRM analysis is a very useful method for the identification of single nucleotide substitutions.
- MeSH
- Charcotova-Marieova-Toothova nemoc genetika metabolismus MeSH
- chronické selhání ledvin genetika metabolismus MeSH
- dospělí MeSH
- exony genetika MeSH
- fenotyp MeSH
- fokálně segmentální glomeruloskleróza genetika metabolismus MeSH
- introny genetika MeSH
- kohortové studie MeSH
- lidé MeSH
- mikrofilamentové proteiny genetika metabolismus MeSH
- mutace genetika MeSH
- mutační analýza DNA metody MeSH
- Check Tag
- dospělí MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
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.
- MeSH
- dospělí MeSH
- genetická variace genetika MeSH
- kationtové kanály TRPP genetika MeSH
- lidé středního věku MeSH
- lidé MeSH
- missense mutace genetika MeSH
- polycystické ledviny autozomálně dominantní diagnóza genetika MeSH
- rodokmen MeSH
- senioři nad 80 let MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- senioři nad 80 let MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika MeSH
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.
- MeSH
- duplikace genu genetika MeSH
- fenotyp MeSH
- genetické testování MeSH
- homeodoménové proteiny genetika MeSH
- lidé MeSH
- multiplexová polymerázová řetězová reakce MeSH
- mutační analýza DNA metody MeSH
- nanismus genetika MeSH
- osteochondrodysplazie diagnóza genetika MeSH
- poruchy růstu diagnóza genetika MeSH
- retrospektivní studie MeSH
- sekvenční delece genetika MeSH
- techniky amplifikace nukleových kyselin MeSH
- tělesná výška genetika MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
- Publikační typ
- abstrakt z konference MeSH
