BACKGROUND: Primary ciliary dyskinesia (PCD) is a multigenic autosomal recessive condition affecting respiratory tract and other organs where ciliary motility is required. The extent of its genetic heterogeneity is remarkable. The aim of the study was to develop a cost-effective pipeline for genetic diagnostics using a combination of Sanger and next generation sequencing (NGS). MATERIALS AND METHODS: Data and samples of 33 families with 38 affected subjects with PCD diagnosed in childhood were collected over the territory of the Czech Republic. A panel of 18 PCD causative or candidate genes was implemented into an Illumina TruSeq Custom Amplicon NGS assay, and three ancestral mutations in SPAG1 were screened by conventional Sanger sequencing, which was also used for the confirmation of the NGS results and for the analysis of familial segregation. RESULTS: The causative gene was DNAH5 in 11/33 (33%) probands, SPAG1 in 8/33 (24%), and DNAI1, CCDC40, LRRC6 in one family each. If the high proportion of subjects with bi-allelic ancestral mutations in SPAG1 is corroborated in other Caucasian populations, a simple Sanger sequencing test for these three mutations may serve as an effective pre-screening step, being followed by an NGS panel for other, much larger, PCD genes. CONCLUSIONS: We present a combination of Sanger sequencing with an NGS panel for known and candidate PCD genes, implemented in a moderate-size national collection of patients. This strategy has proven to be cost-effective, rapid and reliable, and was able to detect the causative gene in two thirds of our PCD patients.

• MeSH
• alely MeSH
• antigeny povrchové genetika   MeSH
• dítě MeSH
• Kartagenerův syndrom diagnóza   genetika   MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mutace * MeSH
• předškolní dítě MeSH
• proteiny vázající GTP genetika   MeSH
• vysoce účinné nukleotidové sekvenování * MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Geografické názvy
• Česká republika MeSH

OBJECTIVE: This study used next-generation sequencing (NGS) technologies to characterize the gut virome at the onset of islet autoimmunity. RESEARCH DESIGN AND METHODS: We conducted a case-control study nested within the Finnish Diabetes Prediction and Prevention (DIPP) cohort. The stool virome in 19 case children, who turned islet autoantibody positive before the age of 2 years and later developed clinical type 1 diabetes, and 19 tightly matched control subjects was analyzed using NGS performed from stool samples collected 3, 6, and 9 months before the onset of islet autoimmunity. Human virus findings were verified using real-time PCR. RESULTS: One or more human viruses were present in 10.4% and bacteriophages were in 54% of the samples. The virome composition showed no association with islet autoimmunity. NGS was less sensitive and specific than real-time PCR. CONCLUSIONS: The present data suggest no dramatic changes in the gut virome shortly before the emergence of islet autoimmunity and emphasize the need of verification of mass sequencing results when viral exposure is assessed in association studies.

• MeSH
• autoimunita imunologie   MeSH
• autoprotilátky metabolismus   MeSH
• diabetes mellitus 1. typu imunologie   virologie   MeSH
• dítě MeSH
• feces virologie   MeSH
• genotyp MeSH
• hyperglykemie imunologie   virologie   MeSH
• kojenec MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• Langerhansovy ostrůvky imunologie   MeSH
• lidé MeSH
• předškolní dítě MeSH
• střeva virologie   MeSH
• studie případů a kontrol MeSH
• viry izolace a purifikace   MeSH
• Check Tag
• dítě MeSH
• kojenec MeSH
• lidé MeSH
• mužské pohlaví MeSH
• předškolní dítě MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH