Analytical parameters and validation of homopolymer detection in a pyrosequencing-based next generation sequencing system.

BACKGROUND: Current technologies in next-generation sequencing are offering high throughput reads at low costs, but still suffer from various sequencing errors. Although pyro- and ion semiconductor sequencing both have the advantage of delivering long and high quality reads, problems might occur when sequencing homopolymer-containing regions, since the repeating identical bases are going to incorporate during the same synthesis cycle, which leads to uncertainty in base calling. The aim of this study was to evaluate the analytical performance of a pyrosequencing-based next-generation sequencing system in detecting homopolymer sequences using homopolymer-preintegrated plasmid constructs and human DNA samples originating from patients with cystic fibrosis. RESULTS: In the plasmid system average correct genotyping was 95.8% in 4-mers, 87.4% in 5-mers and 72.1% in 6-mers. Despite the experienced low genotyping accuracy in 5- and 6-mers, it was possible to generate amplicons with more than a 90% adequate detection rate in every homopolymer tract. When homopolymers in the CFTR gene were sequenced average accuracy was 89.3%, but varied in a wide range (52.2 - 99.1%). In all but one case, an optimal amplicon-sequencing primer combination could be identified. In that single case (7A tract in exon 14 (c.2046_2052)), none of the tested primer sets produced the required analytical performance. CONCLUSIONS: Our results show that pyrosequencing is the most reliable in case of 4-mers and as homopolymer length gradually increases, accuracy deteriorates. With careful primer selection, the NGS system was able to correctly genotype all but one of the homopolymers in the CFTR gene. In conclusion, we configured a plasmid test system that can be used to assess genotyping accuracy of NGS devices and developed an accurate NGS assay for the molecular diagnosis of CF using self-designed primers for amplification and sequencing.

Under the mask of Kabuki syndrome: Elucidation of genetic-and phenotypic heterogeneity in patients with Kabuki-like phenotype.

Kabuki syndrome is mainly caused by dominant de-novo pathogenic variants in the KMT2D and KDM6A genes. The clinical features of this syndrome are highly variable, making the diagnosis of Kabuki-like phenotypes difficult, even for experienced clinical geneticists. Herein we present molecular genetic findings of causal genetic variation using array comparative genome hybridization and a Mendeliome analysis, utilizing targeted exome analysis focusing on regions harboring rare disease-causing variants in Kabuki-like patients which remained KMT2D/KDM6A-negative. The aCGH analysis revealed a pathogenic CNV in the 14q11.2 region, while targeted exome sequencing revealed pathogenic variants in genes associated with intellectual disability (HUWE1, GRIN1), including a gene coding for mandibulofacial dysostosis with microcephaly (EFTUD2). Lower values of the MLL2-Kabuki phenotypic score are indicative of Kabuki-like phenotype (rather than true Kabuki syndrome), where aCGH and Mendeliome analyses have high diagnostic yield. Based on our findings we conclude that for new patients with Kabuki-like phenotypes it is possible to choose a specific molecular testing approach that has the highest detection rate for a given MLL2-Kabuki score, thus fostering more precise patient diagnosis and improved management in these genetically- and phenotypically heterogeneous clinical entities.

Cost-of-illness analysis and regression modeling in cystic fibrosis: a retrospective prevalence-based study.

BACKGROUND: Economic data pertaining to cystic fibrosis (CF), is limited in Europe generally, and completely lacking in Central and Eastern Europe. We performed an analysis of all direct costs associated with CF relative to key disease features and laboratory examinations. METHODS: A retrospective prevalence-based cost-of-illness (COI) study was performed in a representative cohort of 242 CF patients in the Czech Republic, which represents about 65 % of all Czech CF patients. Medical records and invoices to health insurance companies for reference year 2010 were analyzed. RESULTS: The mean total health care costs were €14,486 per patient, with the majority of the costs going towards medicinal products and devices (€10,321). Medical procedures (€2676) and inpatient care (€1829) represented a much smaller percentage of costs. A generalized linear model showed that the strongest cost drivers, for all cost categories, were associated with patient age and lung disease severity (assessed using the FEV1 spirometric parameter), when compounded by chronic Pseudomonas aeruginosa airway infections. Specifically, maximum total costs are around the age 16 years; a FEV1 increase of 1 % point represented a cost decrease of: 0.9 % (medicinal products), 1.7 % (total costs), 2.8 % (procedures) and 7.0 % (inpatient care). CONCLUSIONS: COI analysis and regression modeling using the most recent data available can provide a better understanding of the overall economic CF burden. A comparison of our results with other methodologically similar studies demonstrates that although overall costs may differ, FEV1 can nonetheless be utilized as a generally transferrable indicator of the relative economic impact of CF.

A product of immunoreactive trypsinogen and pancreatitis-associated protein as second-tier strategy in cystic fibrosis newborn screening.

BACKGROUND: In cystic fibrosis newborn screening (CFNBS), immunoreactive trypsinogen (IRT) and pancreatitis-associated protein (PAP) can be used as screening parameters. We evaluated the IRT×PAP product as second-tier parameter in CFNBS in newborns with elevated IRT. METHODS: Data on 410,111 screened newborns including 78 patients with classical cystic fibrosis (CF) from two European centers were retrospectively analyzed by discrimination analysis to identify a screening protocol with optimal cutoffs. We also studied differences in PAP measurement methods and the association of IRT and PAP with age. RESULTS: PAP values differed systematically between fluorometric and photometric assays. The IRT×PAP product showed better discrimination for classical CF than PAP only as second-tier screening parameter (p<0.001). In CF patients, IRT decreased while PAP values remained high over years. In newborns without CF, IRT decreased after birth over weeks while PAP increased within days. CONCLUSIONS: The IRT×PAP product performs well as second-tier cutoff parameter for CFNBS. Screening quality parameters depend on the analytic method and on age at blood collection.

Complex morphological and molecular genetic examination of amelogenesis imperfecta: a case presentation of two Czech siblings with a non-syndrome form of the disease.

Amelogenesis imperfecta (AI) is an overarching term for a group of rare inherited disorders of hard tooth tissues. It is characterized by various defects in proper enamel formation. AI is a severe disorder that affects both the aesthetics and function of the dentition, with affected teeth increasingly suffering from dental caries. Therefore, early diagnosis and lifelong stomatological interventions are important. Due to the complex nature of AI family history, stomatological, radiographic, and molecular genetic examinations should be part of the diagnostic portfolio. Additionally, we utilized new visualization methods for the assessment of teeth demineralization. We present a case report of two affected Czech sisters (6 and 8 years old) with clinically defined AI. These are the first Czech cases in which comprehensive clinical and genetic analysis had been carried out and reflect the complex clinical nature, positive treatment options, and limitations of candidate-gene molecular genetic testing.

Comparison of different IRT-PAP protocols to screen newborns for cystic fibrosis in three central European populations.

BACKGROUND: In recent years different IRT/PAP protocols have been evaluated, but the individual performance remains unclear. To optimize the IRT/PAP strategy we compared protocols from three regional CF newborn screening centers (Heidelberg, Dresden, and Prague). METHODS: We evaluated the effect of elevating the IRT-cut-off from 50 to 65 µg/l (~97.5th to ~99.0th percentile), the need of a failsafe protocol (FS, IRT ≥ 99.9th percentile) and the relative performance using either two IRT-dependent PAP-cut-offs or one PAP-cut-off. FINDINGS: Elevation of the IRT cut-off to 65 µg/l (~99.0th percentile) increased the PPV significantly (Dresden: 0.065 vs. 0.080, p < 0.0001, Prague: 0.052 vs. 0.074, p < 0.0001) without reducing sensitivity. All three IRT/PAP protocols showed a trend towards a higher sensitivity with FS than without and when using one PAP-cut-off instead of two IRT-dependent PAP-cut-offs. CONCLUSIONS: For best performance we suggest an IRT/PAP protocol with an IRT-cut-off close to the 99.0th percentile, FS, and a single PAP-cut-off.

A genome-wide analysis of populations from European Russia reveals a new pole of genetic diversity in northern Europe.

Several studies examined the fine-scale structure of human genetic variation in Europe. However, the European sets analyzed represent mainly northern, western, central, and southern Europe. Here, we report an analysis of approximately 166,000 single nucleotide polymorphisms in populations from eastern (northeastern) Europe: four Russian populations from European Russia, and three populations from the northernmost Finno-Ugric ethnicities (Veps and two contrast groups of Komi people). These were compared with several reference European samples, including Finns, Estonians, Latvians, Poles, Czechs, Germans, and Italians. The results obtained demonstrated genetic heterogeneity of populations living in the region studied. Russians from the central part of European Russia (Tver, Murom, and Kursk) exhibited similarities with populations from central-eastern Europe, and were distant from Russian sample from the northern Russia (Mezen district, Archangelsk region). Komi samples, especially Izhemski Komi, were significantly different from all other populations studied. These can be considered as a second pole of genetic diversity in northern Europe (in addition to the pole, occupied by Finns), as they had a distinct ancestry component. Russians from Mezen and the Finnic-speaking Veps were positioned between the two poles, but differed from each other in the proportions of Komi and Finnic ancestries. In general, our data provides a more complete genetic map of Europe accounting for the diversity in its most eastern (northeastern) populations.

Distribution of CFTR mutations in the Czech population: positive impact of integrated clinical and laboratory expertise, detection of novel/de novo alleles and relevance for related/derived populations.

BACKGROUND: This two decade long study presents a comprehensive overview of the CFTR mutation distribution in a representative cohort of 600 Czech CF patients derived from all regions of the Czech Republic. METHODS: We examined the most common CF-causing mutations using the Elucigene CF-EU2v1™ assay, followed by MLPA, mutation scanning and/or sequencing of the entire CFTR coding region and splice site junctions. RESULTS: We identified 99.5% of all mutations (1194/1200 CFTR alleles) in the Czech CF population. Altogether 91 different CFTR mutations, of which 20 were novel, were detected. One case of de novo mutation and a novel polymorphism was revealed. CONCLUSION: The commercial assay achieved 90.7%, the MLPA added 1.0% and sequencing increased the detection rate by 7.8%. These comprehensive data provide a basis for the improvement of CF DNA diagnostics and/or newborn screening in our country. In addition, they are relevant to related Central European populations with lower mutation detection rates, as well as to the sizeable North American "Bohemian diaspora".

Genetic characterization of northeastern Italian population isolates in the context of broader European genetic diversity.

Population genetic studies on European populations have highlighted Italy as one of genetically most diverse regions. This is possibly due to the country's complex demographic history and large variability in terrain throughout the territory. This is the reason why Italy is enriched for population isolates, Sardinia being the best-known example. As the population isolates have a great potential in disease-causing genetic variants identification, we aimed to genetically characterize a region from northeastern Italy, which is known for isolated communities. Total of 1310 samples, collected from six geographically isolated villages, were genotyped at >145000 single-nucleotide polymorphism positions. Newly genotyped data were analyzed jointly with the available genome-wide data sets of individuals of European descent, including several population isolates. Despite the linguistic differences and geographical isolation the village populations still show the greatest genetic similarity to other Italian samples. The genetic isolation and small effective population size of the village populations is manifested by higher levels of genomic homozygosity and elevated linkage disequilibrium. These estimates become even more striking when the detected substructure is taken into account. The observed level of genetic isolation in Friuli-Venezia Giulia region is more extreme according to several measures of isolation compared with Sardinians, French Basques and northern Finns, thus proving the status of an isolate.

Prospective and parallel assessments of cystic fibrosis newborn screening protocols in the Czech Republic: IRT/DNA/IRT versus IRT/PAP and IRT/PAP/DNA.

UNLABELLED: Cystic fibrosis (CF) is a life-threatening disease for which early diagnosis following newborn screening (NBS) improves the prognosis. We performed a prospective assessment of the immunoreactive trypsinogen (IRT)/DNA/IRT protocol currently in use nationwide, versus the IRT/pancreatitis-associated protein (PAP) and IRT/PAP/DNA CF NBS protocols. Dried blood spots (DBS) from 106,522 Czech newborns were examined for IRT concentrations. In the IRT/DNA/IRT protocol, DNA-testing was performed for IRT ≥ 65 ng/mL. Newborns with IRT ≥ 200 ng/mL and no detected cystic fibrosis transmembrane conductance regulator gene (CFTR) mutations were recalled for a repeat IRT. In the same group of newborns, for both parallel protocols, PAP was measured in DBS with IRT ≥ 50 ng/mL. In PAP-positive newborns (i.e., ≥1.8 if IRT 50-99.9 or ≥1.0 if IRT ≥ 100, all in ng/mL), DNA-testing followed as part of the IRT/PAP/DNA protocol. Newborns with at least one CFTR mutation in the IRT/DNA/IRT and IRT/PAP/DNA protocols; a positive PAP in IRT/PAP; or a high repeat IRT in IRT/DNA/IRT were referred for sweat testing. CONCLUSION: the combined results of the utilized protocols led to the detection of 21 CF patients, 19 of which were identified using the IRT/DNA/IRT protocol, 16 using IRT/PAP, and 15 using IRT/PAP/DNA. Decreased cut-offs for PAP within the IRT/PAP protocol would lead to higher sensitivity but would increase false positives. Within the IRT/PAP/DNA protocol, decreased PAP cut-offs would result in high sensitivity, an acceptable number of false positives, and would reduce the number of DNA analyses. Thus, we concluded that the IRT/PAP/DNA protocol would represent the most suitable protocol in our conditions.