A. Holubová

Gross genomic rearrangements involving deletions in the CFTR gene: characterization of six new events from a large cohort of hitherto unidentified cystic fibrosis chromosomes and meta-analysis of the underlying mechanisms

Gross genomic rearrangements involving deletions in the CFTR gene have recently been found to account for ∼20% of unidentified cystic fibrosis (CF) chromosomes in both French and Italian patients. Using QMPSF and walking quantitative DHPLC, six novel mutations (three simple deletions, two complex deletions with short insertions of 3–6 bp, and a complex deletion with a 182 bp inverted downstream sequence) were characterized by screening 274 unidentified CF chromosomes from 10 different countries. These lesions increase the total number of fully characterized large CFTR genomic rearrangements involving deletions to 21. Systematic analysis of the 42 associated breakpoints indicated that all 21 events were caused by nonhomologous recombination. Whole gene complexity analysis revealed a significant correlation between regions of low sequence complexity and the locations of the deletion breakpoints. Known recombination-promoting motifs were noted in the vicinity of the breakpoints. A total of 11 simple deletions were potentially explicable in terms of the classical model of replication slippage. However, the complex deletions appear to have arisen via multiple mechanisms; three of the five complex deletions with short insertions and both examples of large inverted insertions (299 and 182 bp, respectively) can be explained by either a model of serial replication slippage in cis (SRScis) or SRS in trans (SRStrans). Finally, the nature and distribution of large genomic rearrangements in the CFTR gene were compared and contrasted with those of two other genes, DMD and MSH2, with a view to gaining a broader understanding of DNA sequence context in mediating the diverse underlying mutational mechanisms.

Pilot newborn screening project for cystic fibrosis in the Czech Republic: defining role of the delay in its symptomatic diagnosis and influence of ultrasound-based prenatal diagnosis on the incidence of the disease.

The objective need for cystic fibrosis (CF) newborn screening (NBS) in the Czech Republic has recently been substantiated by a significant delay of its symptomatic diagnosis. This trend most likely resulted from the process of decentralisation of health care which led to the deterioration of care for patients who need specialised approaches. Applied newborn screening model (IRT/DNA/IRT) was efficacious enough to detect CF cases with median age at diagnosis of 37 days. The incidence of CF (1 in 6946 live births) ascertained in this project was lower than that established previously by epidemiological studies (1 in 2700-1 in 3300). However, adjustment for broadly applied ultrasound-based prenatal diagnosis (PND) in the 2nd trimester of pregnancy, that was performed within the period of the project (1/2/2005-2/11/2006), rendered an incidence estimate of 1 in 4023. This value is closer to that observed in other CF NBS programmes and reflects influence of PND on the incidence of CF.

Molecular genetic analysis in 14 Czech Kabuki syndrome patients is confirming the utility of phenotypic scoring

Kabuki syndrome (KS) is a dominantly inherited disorder mainly due to de novo pathogenic variation in KMT2D or KDM6A genes. Initially, a representative cohort of 14 Czech cases with clinical features suggestive of KS was analyzed by experienced clinical geneticists in collaboration with other specialties, and observed disease features were evaluated according to the ‘MLL2‐Kabuki score’ defined by Makrythanasis et al. Subsequently, the aforementioned genes were Sanger sequenced and copy number variation analysis was performed by MLPA, followed by genome‐wide array CGH testing. Pathogenic variants in KMT2D resulting in protein truncation in 43% (6/14; of which 3 are novel) of all cases were detected, while analysis of KDM6A was negative. MLPA analysis was negative in all instances. One female patient bears a 6.6 Mb duplication of the Xp21.2–Xp21.3 region that is probably disease causing. Subjective KS phenotyping identified predictive clinical features associated with the presence of a pathogenic variant in KMT2D. We provide additional evidence that this scoring approach fosters prioritization of patients prior to KMT2D sequencing. We conclude that KMT2D sequencing followed by array CGH is a diagnostic strategy with the highest diagnostic yield.

Neonatal screening in the Czech Republic: increased prevalence of selected diseases in low birthweight neonates

Neonates with low birthweight (LBW) represent a vulnerable population. This retrospective study analyzed the birth frequency of diseases detected by neonatal screening (NBS) in normal and LBW neonates in the Czech Republic. Between years 2002 and 2016, the number of screened disorders in the Czech Republic gradually increased from two to 13. Prevalence of screened diseases was calculated for cohorts ranging from 777,100 to 1,277,283 neonates stratified by birthweight. Odds ratio of the association of LBW with each disease was calculated and statistical significance was evaluated using the chi-square test or Fisher’s exact test, as appropriate. Three diseases were associated with higher risk of prevalence in LBW neonates, namely congenital hypothyroidism (OR 2.50, CI 1.92; 3.25), cystic fibrosis (OR 2.44, CI 1.51; 3.94), and long chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) (OR 7.74, CI 2.18; 27.42).Conclusion: Although the underlying mechanisms are not well understood, results can be hypothesized that LBW (respectively prematurity) may lead to the secondary and often transitory hypothyroidism while cystic fibrosis and LCHADD may manifest already prenatally and result into preterm birth and LBW.What is Known:• The percentage of low birthweight (LBW) neonates in the Czech Republic has been increasing.• Previously published studies reported positive association between LBW and congenital hypothyroidism and cystic fibrosis.What is New:• The association between LCHADD and LBW has not yet been described.• LBW can be the first manifestation of cystic fibrosis and LCHADD.

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.

WS17.4 Unexpected findings in the broadly used Elucigene CF-EU2 CFTR genotyping assay

Objectives The commercial assay Elucigene CF-EU2 Tm (Elucigene, U.K.) has been used at our Department since VII/2010 for routine DNA testing in patients with clinical suspicion of CF, partners of CF carriers, male infertility and in CF newborn screening (NBS). DNA analysis of 50 European mutations present in this assay renders a relatively high mutation detection rate of cca. 91%. However, in some instances interpretation of the results may be difficult since this assay may also be affected by intra- CFTR insertions and/or deletions in amplified target sequences or presence of variants under proprietary primer annealing sites. Methods Manufacturer-recommended protocols for the Elucigene CF-EU2™ assay and the internal diagnostic database were utilised throughout the study. Results During approx. 4.5 years of using this assay we found four atypical findings: 3 during the course of CF NBS and one in an infertile male. All three newborns were heterozygous for the F508del mutation, while in the infertile case no mutation was detected by the Elucigene assay. Nonetheless, due to additional “non-standard” observations in the assay electrophoretograms (e.g. absence of wild type peak in I507del or extra peaks in mixture B) we performed target DNA Sanger sequencing of corresponding exons/introns and found a second CFTR mutation in two children (G509D, 2789+2insA), including a non-CF-causing variant (F508C). Conclusion The Elucigene assay is highly reliable, but targeted DNA sequencing of the CFTR gene should be considered in instances when atypical electrophoretograms are being observed. Supported by 00064203, CZ.2.16/3.1.00/24022OPPK and NT/13770.

25 Cystic fibrosis (CF) prevalence derived from CF newborn screening (CFNBS) in the Czech Republic: comparison of previous epidemiological and current CFNBS-based disease prevalence data

Objectives: This study reviews parental views on how they were informed of the probable CF diagnosis, following neonatal screening, and explores which method parent’s found most acceptable. Methods: Retrospective questionnaires were used to find parents’ views on how they were told that their child had a probable diagnosis of CF, who told them initially, were they told at home or by the GP followed by hospital, how acceptable was the mode of information sharing? Results: 40 parents of children with CF were studied. Most parents (9 out of 12) who had been informed by their GP recall receiving inaccurate information about the condition. All parents (28 out of 28) who had received a home visit to be told about the CF diagnosis felt that the information received had been accurate and found it easier to accept. Conclusion: Our qualitative study confirms that parents who were given accurate information by CF professionals during a home visit found this an acceptable means of being informed of the probable diagnosis of CF. Parents reported less negative recollections of that time than parents who were informed by their GP and met CF professionals in hospital. We consider that this is a superior method of sharing the information about the diagnosis of CF.

17 Cystic fibrosis newborn screening (CF NBS): 4 year experience of the Prague Centre with the IRT/DNA/IRT protocol

Background: Cystic Fibrosis Newborn screening (CFNBS) as a pilot study started at the Institute of Mother and Child (IMC) Centre in 1999 and 444 063 newborns were examined until 2003. Current CFNBS has started gradually in Poland in 2006, covering the whole country in 2009 and is ongoing. 582 693 children were screened until the end of 2011. During CFNBS different protocol’s strategies were used. Aim: Impact of the implementation of NBS on the age of CF diagnosis. Methods: The study involved children diagnosed and treated only in IMC CF Centre (from 1999 until the end of 2011). Three groups were formed: 1. Pilot group (p-NBS) − 56 children, according to protocol strategy IRT/IRT and IRT/IRT/DNA, only F508del mutation was assessed; 2. Current group (c-NBS) − 92 children, IRT/IRT/DNA and IRT/DNA protocol, with expanded DNA analysis panel; 3. Patient diagnosed clinically (non-NBS) − 56 children. Moreover, DNA analysis in c-NBS group contains also frequent mutation in Polish population: 3849+10kbC>T, which is combined with low sweat test values. All patients underwent sweat tests. Results: The age of diagnosis was significantly lower (both p T than p-NBS and non-NBS group (both 0.9%). Conclusion: IRT/DNA strategy with extended DNA analysis provides an opportunity of earlier CF diagnosis even in children with normal sweat test values.