Double Heterozygosity for Rare Deleterious Variants in the BRCA1 and BRCA2 Genes in a Hungarian Patient with Breast Cancer.

Hereditary breast cancer is most commonly attributed to germline BRCA1 and BRCA2 gene variants. The vast majority of BRCA1 and BRCA2 mutation carriers are single heterozygotes, and double heterozygosity (DH) is a very rare finding. Here, we describe the case of a BRCA1/BRCA2 double heterozygous female proband diagnosed with breast cancer. Genetic testing for hereditary breast and ovarian cancer revealed two pathogenic variants in the BRCA1 (c.5095C>T, p.(Arg1699Trp)) and in BRCA2 genes (c.658_659delGT, p.(Val220Ilefs*4)) in heterozygous form. None of the variants were founder Jewish mutations; to our knowledge, these rare deleterious variants have not been previously described in DH patients in the literature. The patient had triple-negative unilateral breast cancer at the age of 36 and 44 years. Based on family studies, the BRCA1 variant was maternally inherited.

[Genetic revision of the Hungarian Cystic Fibrosis Registry]. / A magyar Cystás Fibrosis Regiszter genetikai revíziója.

INTRODUCTION: Cystic fibrosis (CF) is one of the most common monogenic diseases. Genetic testing is becoming increasingly reasoned to establish or confirm the diagnosis by detecting abnormal mutations. OBJECTIVE: In order to develop a diagnostic strategy for cystic fibrosis and to facilitate mutation-specific treatments, the genetic revision of the Hungarian Cystic Fibrosis Registry was performed. METHOD: 582 patients' data and samples were used for the revision (528 originally included in the register and 54 received during the revision). First we reviewed the patients' existing genetic findings. Wherever necessary, a comprehensive three-level genetic analysis of the CFTR gene was done. RESULTS: According to our study, of the 528 patients present in the Registry, 395 (74.8%) had 2 pathogenic CFTR mutations. We completed and corrected 94 patients' previously incomplete genetic status. 73 different pathogenic variants were described, in which 1 aberration was not previously reported (c.3130G>A). The 5 most common mutations were: F508del (68.4%); CFTRdele2,3 (3.7%); G542X (3.2%); 2184insA (2.7%); W1282X (2.3%). Based on genotype and age, in Hungary 211 patients are eligible for the available lumacaftor-ivacaftor combination therapy, and 361 patients for the ivacaftor-tezacaftor-elexacaftor therapy. CONCLUSION: Due to the revision, we could identify the patients who can benefit from mutation-specific drugs instead of symptomatic therapy. In addition, the data obtained have been used to map the Hungarian distribution of mutations in the CFTR gene, which will help to develop a diagnostic strategy. Orv Hetil. 2022; 163(51): 2052-2059.

Helsmoortel-Van der Aa Syndrome-Cardiothoracic and Ectodermal Manifestations in Two Patients as Further Support of a Previous Observation on Phenotypic Overlap with RASopathies.

The ADNP-gene-related neurodevelopmental disorder Helsmoortel-Van der Aa syndrome is a rare syndromic-intellectual disability-an autism spectrum disorder first described by Helsmoortel and Van der Aa in 2014. Recently, a large cohort including 78 patients and their detailed phenotypes were presented by Van Dijck et al., 2019, who reported developmental delay, speech delay and autism spectrum disorder as nearly constant findings with or without variable cardiological, gastroenterological, urogenital, endocrine and neurological manifestations. Among cardiac malformations, atrial septal defect, patent ductus arteriosus, patent foramen ovale and mitral valve prolapse were the most common findings, but other unspecified defects, such as mild pulmonary valve stenosis, were also described. We present two patients with pathogenic ADNP variants and unusual cardiothoracic manifestations-Bland-White-Garland syndrome, pectus carinatum superiorly along the costochondral junctions and pectus excavatum inferiorly in one patient, and Kawasaki syndrome with pericardiac effusion, coronary artery dilatation and aneurysm in the other-who were successfully treated with intravenous immunoglobulin, corticosteroid and aspirin. Both patients had ectodermal and/or skeletal features overlapping those seen in RASopathies, supporting the observations of Alkhunaizi et al. 2018. on the clinical overlap between Helsmoortel-Van der Aa syndrome and Noonan syndrome. We observed a morphological overlap with the Noonan-like disorder with anagen hair in our patients.

Establishing the Mutational Spectrum of Hungarian Patients with Familial Hypercholesterolemia.

Familial hypercholesterolemia (FH) is one of the most common autosomal, dominantly inherited diseases affecting cholesterol metabolism, which, in the absence of treatment, leads to the development of cardiovascular complications. The disease is still underdiagnosed, even though an early diagnosis would be of great importance for the patient to receive proper treatment and to prevent further complications. No studies are available describing the genetic background of Hungarian FH patients. In this work, we present the clinical and molecular data of 44 unrelated individuals with suspected FH. Sequencing of five FH-causing genes (LDLR, APOB, PCSK9, LDLRAP1 and STAP1) has been performed by next-generation sequencing (NGS). In cases where a copy number variation (CNV) has been detected by NGS, confirmation by multiplex ligation-dependent probe amplification (MLPA) has also been performed. We identified 47 causal or potentially causal (including variants of uncertain significance) LDLR and APOB variants in 44 index patients. The most common variant in the APOB gene was the c.10580G>A p.(Arg3527Gln) missense alteration, this being in accordance with literature data. Several missense variants in the LDLR gene were detected in more than one index patient. LDLR variants in the Hungarian population largely overlap with variants detected in neighboring countries.

A Comprehensive Analysis of Hungarian MODY Patients-Part I: Gene Panel Sequencing Reveals Pathogenic Mutations in HNF1A, HNF1B, HNF4A, ABCC8 and INS Genes.

Maturity-onset diabetes of the young (MODY) has about a dozen known causal genes to date, the most common ones being HNF1A, HNF4A, HNF1B and GCK. The phenotype of this clinically and genetically heterogeneous form of diabetes depends on the gene in which the patient has the mutation. We have tested 450 Hungarian index patients with suspected MODY diagnosis with Sanger sequencing and next-generation sequencing and found a roughly 30% positivity rate. More than 70% of disease-causing mutations were found in the GCK gene, about 20% in the HNF1A gene and less than 10% in other MODY-causing genes. We found 8 pathogenic and 9 likely pathogenic mutations in the HNF1A gene in a total of 48 patients and family members. In the case of HNF1A-MODY, the recommended first-line treatment is low dose sulfonylurea but according to our data, the majority of our patients had been on unnecessary insulin therapy at the time of requesting their genetic testing. Our data highlights the importance of genetic testing in the diagnosis of MODY and the establishment of the MODY subtype in order to choose the most appropriate treatment.

A Comprehensive Analysis of Hungarian MODY Patients-Part II: Glucokinase MODY Is the Most Prevalent Subtype Responsible for about 70% of Confirmed Cases.

MODY2 is caused by heterozygous inactivating mutations in the glucokinase (GCK) gene that result in persistent, stable and mild fasting hyperglycaemia (5.6-8.0 mmol/L, glycosylated haemoglobin range of 5.6-7.3%). Patients with GCK mutations usually do not require any drug treatment, except during pregnancy. The GCK gene is considered to be responsible for about 20% of all MODY cases, transcription factors for 67% and other genes for 13% of the cases. Based on our findings, GCK and HNF1A mutations together are responsible for about 90% of the cases in Hungary, this ratio being higher than the 70% reported in the literature. More than 70% of these patients have a mutation in the GCK gene, this means that GCK-MODY is the most prevalent form of MODY in Hungary. In the 91 index patients and their 72 family members examined, we have identified a total of 65 different pathogenic (18) and likely pathogenic (47) GCK mutations of which 28 were novel. In two families, de novo GCK mutations were detected. About 30% of the GCK-MODY patients examined were receiving unnecessary OAD or insulin therapy at the time of requesting their genetic testing, therefore the importance of having a molecular genetic diagnosis can lead to a major improvement in their quality of life.

Novel RB1 and MET Gene Mutations in a Case with Bilateral Retinoblastoma Followed by Multiple Metastatic Osteosarcoma.

Retinoblastoma (Rb) is a malignant tumor of the developing retina that affects children before the age of five years in association with inherited or early germline mutations of the RB1 gene. The genetic predisposition is also a driver for other primary malignancies, which have become the leading cause of death in retinoblastoma survivors. Other malignancies can occur as a consequence of radiotherapy. We describe a patient with retinoblastoma in which we detected a novel RB1 c.2548C > T, p.(Gln850Ter) and a synchronous MET c.3029C > T, p.(Thr1010Ile) mutation as well. After presenting with bilateral retinoblastoma, the patient developed at least four different manifestations of two independent osteosarcomas. Our goal was to identify all germline and somatic genetic alterations in available tissue samples from different time periods and to reconstruct their clonal relations using next generation sequencing (NGS). We also used structural and functional prediction of the mutant RB and MET proteins to find interactions between the defected proteins with potential causative role in the development of this unique form of retinoblastoma. Both histopathology and NGS findings supported the independent nature of a chondroblastic osteosarcoma of the irradiated facial bone followed by an osteoblastic sarcoma of the leg (tibia).

A Rare Double Heterozygous Mutation in Low-Density Lipoprotein Receptor and Apolipoprotein B-100 Genes in a Severely Affected Familial Hypercholesterolaemia Patient.

Familial hypercholesterolaemia (FH) is characterized by high plasma low-density lipoprotein cholesterol (LDL-C) levels and premature cardiovascular disease risk. Mutations in the genes that encode proteins involved in LDL uptake and catabolism, including LDL-receptor (LDLR) and apolipoprotein-B (APOB), are known to cause FH. We present the case of a severely affected FH proband with two mutations in two different causing genes and characterize her first-degree blood relatives. The proband was a 54-year-old woman with a severe FH phenotype with treated LDL-C of 8.3 mmol/L, total cholesterol (TC) level of 11.6 mmol/L, peripheral artery disease, early myocardial infarction, aortic stenosis, and carotid artery disease. Exons of the LDLR and APOB genes were amplified by polymerase chain reactions (PCR). PCR products were examined by pyrosequencing and proven by bidirectional DNA sequencing. The proband was heterozygous for both the LDLR c.420G>C (p.Glu140Asp) mutation known to be pathogenic and a rare APOB c.10708C>T (p.His3570Tyr) mutation with unproven pathogenicity. Cascade testing has been performed in her 15 first-degree blood relatives. Her daughter carries only the LDLR c.420 G>C mutation with a TC of 8.4 mmol/L. Her two sisters carry only the APOB c.10708C>T with a TC of 5.7 and 6.2 mmol/L. This case provides evidence that the rare APOB c.10708C>T mutation alone is not pathogenic, but has a synergic effect on LDLR mutation. The finding is important for understanding the genotype-phenotype correlation and highlights the need to consider the presence of additional mutations in FH families where relatives have varying phenotypes.

A DNA pool of FLT3-ITD positive DNA samples can be used efficiently for analytical evaluation of NGS-based FLT3-ITD quantitation - Testing several different ITD sequences and rates, simultaneously.

Internal tandem duplication (ITD) in the fms-like tyrosine kinase 3 (FLT3) gene is one of the most frequent genetic alteration in acute myeloid leukemia (AML), and it is associated with worse clinical outcome. Not only the presence but also the size, localization and the rate of this variant or the presence of multiple ITDs has prognostic information. The traditional PCR based diagnostic methods cannot provide information about all of these parameters in one assay, however the application of next generation sequencing (NGS) technique can be a reliable solution for this diagnostic problem. In order to evaluate the analytical properties of an NGS-based FLT3-ITD detection assay a quality control sample was prepared from DNA of AML patients containing 19 different FLT3-ITD variants identified by NGS. The higher the total read count was in a certain sample of the NGS run, the more ITD variant types could be detected. The maximal sensitivity of FLT3-ITD detection by NGS technique was as low as 0.007% FLT3-ITD/total allele rate, however, below 0.1% rate, the reproducibility of the quantitation was poor (CV > 25%). DNA pools with several FLT3-ITDs can be used efficiently for analytical evaluation of NGS-based FLT3-ITD quantitation testing several different ITD sequences and rates, simultaneously.

FBN1 gene mutations in 26 Hungarian patients with suspected Marfan syndrome or related fibrillinopathies.

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder mainly affecting the cardiovascular, ocular and musculo-skeletal systems. FBN1 gene mutations lead to MFS and related connective tissue disorders. In this work we described clinical and molecular data of 26 unrelated individuals with suspected MFS who were referred for FBN1 mutation analysis. FBN1 gene sequencing was performed by next generation sequencing and Sanger sequencing methods. We identified 23 causal or potentially causal (including variants of uncertain significance) FBN1 variants, seven of them was novel (˜30%). About 30% of the cases were sporadic. FBN1 mutations were associated with MFS in the majority of the patients, in two cases with severe and early onset manifestation of the syndrome. Missense mutations were detected in 69.6% (16/23), the majority of them were located in one of the cbEGF motifs and ˜70% of them substituted conserved cystein residues. Small deletions/duplications were identified in 13% of the cases (3/23), while splice site variants were detected in 17.4% (4/23). In three unrelated patients a low frequency recurrent silent variant (c.3294C > T (p.Asp1098=) was identified. FBN1 mRNA analysis showed that the mutation does not lead to aberrant splicing, based on available data the mutation was classified as benign.

Comprehensive genetic testing in children with a clinical diagnosis of ARPKD identifies phenocopies.

BACKGROUND: Autosomal recessive polycystic kidney disease (ARPKD) is genetically one of the least heterogeneous ciliopathies, resulting primarily from mutations of PKHD1. Nevertheless, 13-20% of patients diagnosed with ARPKD are found not to carry PKHD1 mutations by sequencing. Here, we assess whether PKHD1 copy number variations or second locus mutations explain these cases. METHODS: Thirty-six unrelated patients with the clinical diagnosis of ARPKD were screened for PKHD1 point mutations and copy number variations. Patients without biallelic mutations were re-evaluated and screened for second locus mutations targeted by the phenotype, followed, if negative, by clinical exome sequencing. RESULTS: Twenty-eight patients (78%) carried PKHD1 point mutations, three of whom on only one allele. Two of the three patients harbored in trans either a duplication of exons 33-35 or a large deletion involving exons 1-55. All eight patients without PKHD1 mutations (22%) harbored mutations in other genes (PKD1 (n = 2), HNF1B (n = 3), NPHP1, TMEM67, PKD1/TSC2). Perinatal respiratory failure, a kidney length > +4SD and early-onset hypertension increase the likelihood of PKHD1-associated ARPKD. A patient compound heterozygous for a second and a last exon truncating PKHD1 mutation (p.Gly4013Alafs*25) presented with a moderate phenotype, indicating that fibrocystin is partially functional in the absence of its C-terminal 62 amino acids. CONCLUSIONS: We found all ARPKD cases without PKHD1 point mutations to be phenocopies, and none to be explained by biallelic PKHD1 copy number variations. Screening for copy number variations is recommended in patients with a heterozygous point mutation.

Interfering effect of maternal cell contamination on invasive prenatal molecular genetic testing.

OBJECTIVE: Fetal samples obtained by invasive techniques are prone to maternal cell contamination (MCC), which may lead to false genotyping results. Our aim was to determine 3 molecular genetic tests' sensitivity to MCC. METHOD: By mixing experiments, 1%, 5%, 10%, 20%, 30%, and 40% MCC was simulated, and significant MCC levels were determined for Sanger DNA sequencing, multiplex ligation-dependent probe amplification (MLPA), and pyrosequencing, a next-generation sequencing method. RESULTS: For Sanger sequencing, the limit of sensitivity to MCC was 5% to 30%. For MLPA, a higher proportion of MCC (≥40%) was shown to lead to diagnostic uncertainty. In contrast, pyrosequencing proved to be very sensitive to MCC, detecting a proportion as low as 1%. CONCLUSION: In the case of Sanger sequencing, sensitivity to MCC was variable, while for MLPA, only high levels of MCC proved to be significant. Although the next-generation sequencing method was sensitive to low-level MCC, if MCC level is determined in parallel, accurate quantification of allelic ratios can help to interpret the diagnostic results. Knowledge of significant MCC levels allows correct prenatal diagnosis even if samples are not purely of fetal origin and repeated sampling can be avoided in many of the cases.

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.

A novel point mutation affecting Asn76 of dystrophin protein leads to dystrophinopathy.

Mutations in the DMD gene lead to Duchenne and Becker muscular dystrophy (DMD/BMD). Missense mutations are rare cause of DMD/BMD. A six-month-old male patient presented with mild generalized muscle weakness, hypotonia, and delayed motor development. Dystrophinopathy was suspected because of highly elevated serum creatine kinase level (1497 U/L) and tiered DMD gene analysis was performed. Multiplex ligation-dependent probe amplification (MLPA) assay showed deletion of exon 4, which could not be confirmed by another method. Sequencing of exon 4 revealed a novel de novo point mutation (c.227A>T, p.Asn76Ile) in the N-terminal actin-binding domain (N-ABD) of dystrophin protein. The false positive MLPA result was explained by the fact that the affected nucleotide lies directly at the 3' ligation site of the MLPA probe. Sequencing of the whole coding region of DMD gene proved c.227A>T to be the sole variant being potentially pathogenic. According to in silico analyses the mutation was predicted to be highly destabilizing on N-ABD structure possibly leading to protein malfunction. Muscle biopsy was performed and dystrophin immunohistochemistry results were suggestive of BMD. Our results highlight the importance of confirmatory testing of single-exon deletions detected by MLPA and we describe a novel, destabilizing missense mutation in the DMD gene.

Congenital Hyperinsulinism Caused by a De Novo Mutation in the ABCC8 Gene - A Case Report.

Congenital hyperinsulinism (CHI) is a rare genetic disorder characterized by inappropriate insulin secretion and severe hypoglycaemia. There are two histological subtypes: diffuse and focal form. Diffuse form is most common in autosomal recessive mutations in ABCC8/KCNJ11 gene, while focal CHI is caused a paternally inherited mutation and a somatic maternal allele loss. Here we report a case of a term male infant presented with severe hyperinsulinaemic hypoglycaemia. Gene panel testing was performed to give rapid genetic diagnosis. We detected the c.4415-13G>A heterozygous mutation in the ABCC8 gene. Targeted genetic testing of the parents proved the de novo origin of the mutation. The mutation has been previously described. The infant received octreotide treatment and is prepared for 18-fluoro-dopa PET-CT examination in order to localize the lesion. Rapid genetic testing might be crucial in the clinical management strategy, with decision algorithms taking into account of the genetic status of the patient.

Different phenotypes in identical twins with cerebrotendinous xanthomatosis: case series.

Cerebrotendinous xanthomatosis (CTX) is a rare, genetically determined error of metabolism. The characteristic clinical symptoms are diarrhea, juvenile cataracts, tendon xanthomas and neuropsychiatric alterations. The aim of this study is to present a pair of identical adult twins with considerable differences in the severity of phenotype. With regards to neuropsychiatric symptoms, the predominant features were severe Parkinsonism and moderate cognitive dysfunctions in the more-affected individual, whereas these alterations in the less-affected patient were only very mild and mild, respectively. The characteristic increase in the concentrations of serum cholestanol and the lesion volumes in dentate nuclei in the brain assessed with magnetic resonance imaging were quite similar in both cases. The lifestyle conditions, including eating habits of the twin pair, were quite similar as well; therefore, currently unknown genetic modifiers or certain epigenetic factors may be responsible for the differences in severity of phenotype. This case series serves as the first description of an identical twin pair with CTX presenting heterogeneous clinical features.

CpG methylation in human papillomavirus (HPV) type 31 long control region (LCR) in cervical infections associated with cytological abnormalities.

The mechanisms that regulate papillomavirus gene expression include DNA methylation. The transcription of papillomavirus oncogenes E6 and E7 is controlled by certain regulatory elements in the LCR, which include binding sites for the E2 protein, a viral regulator of oncogene expression. In HPV-31-infected exfoliated cervical cells, the CpG methylation of the entire LCR was determined by next-generation sequencing after bisulfite modification. Six of the 22 cases had methylated CpG sites in the HPV-31 LCR, including position 7479 and/or 7485, at the promoter distal E2 binding site, thus suggesting a potential regulatory mechanism for papillomavirus transcription.

Molecular Analysis of Cystic Fibrosis Patients in Hungary - An Update to the Mutational Spectrum.

BACKGROUND: In this study the authors present an update to the CFTR mutation profile in Hungary, utilizing data from a selected cohort of 45 cystic fibrosis (CF) patients from different regions of the country. METHODS: Depending on the preceding analysis, four different mutation detection methods were used. A commercial assay targeting the most common CF-causing mutations was performed as the first test followed by an allele specific PCR for CFTRdele2,3(21kb), Sanger sequencing and MLPA analysis of the coding region of the CFTR gene. RESULTS: In our recent study 27 different mutations were detected, including 2 novel ones (c.1037_1038insA and c.1394C>T). Besides F508del (c.1521_1523delCTT), the following mutations were found at a frequency of ≥ 4.0%: W1282X (c.3846G>A), N1303K (c.3909C>G), CFTRdele2,3(21kb) (c.54-5940_273+10250del21kb) and 2184insA (c.2052_2053insA). In addition, four mutations (G542X, Y1092X, 621+1G>T, and 2143delT) were found in more than one allele. CONCLUSIONS: The updated database of Hungarian mutations not only enables to increase the efficiency of the existing diagnostic approach, but also provides a further refined basis for the introduction of the molecular newborn screening (NBS) program in Hungary.

[Laboratory diagnosis of a rare congenital neurodegenerative disease: cerebrotendinous xanthomatosis]. / Egy ritka, veleszületett neurodegeneratív betegség: a cerebrotendinosus xanthomatosis laboratóriumi diagnosztikája.

Cerebrotendinous xanthomatosis is a rare neurodegenerative disease characterized by the accumulation of cholesterol and cholestanol in the brain and the tendons caused by mutations of the gene encoding sterol 27-hydroxylase (CYP27A1), which is involved in bile acid synthesis. The diagnosis is often missed and delayed because of the variable clinical presentation of the disease. Blood testing for cerebrotendinous xanthomatosis is routinely performed using gas chromatography-mass spectrometry measurement of elevated cholestanol level, and the diagnosis is confirmed by molecular genetic analysis. Early recognition and initiation of chenodeoxycholic acid therapy with hydoxymethyl­glutaryl­Coenzyme-A reductase inhibitors is critical to prevent irreversible neurological damage and permanent disability. The authors summarize the current knowledge about the pathomechanism, laboratory diagnosis and therapeutic options of cerebrotendinous xanthomatosis.

Distribution of CFTR mutations in Eastern Hungarians: relevance to genetic testing and to the introduction of newborn screening for cystic fibrosis.

BACKGROUND: The aim of this study was characterization of an updated distribution of CFTR mutations in a representative cohort of 40 CF patients with the classical form of the disease drawn from Eastern Hungary. Due to the homogeneity of the Hungarian population our data are generally applicable to other regions of the country, including the sizeable diaspora. METHODS: We utilized the recommended "cascade" CFTR mutation screening approach, initially using a commercial assay, followed by examination of the common "Slavic" deletion CFTRdele2,3(21kb). Subsequently, the entire CFTR coding region of the CFTR gene was sequenced in patients with yet unidentified mutations. RESULTS: The Elucigene CF29(Tm) v2 assay detected 81.25% of all CF causing mutations. An addition of the CFTRdele2,3(21kb) increased the mutation detection rate to 86.25%. DNA sequencing enabled us to identify mutations on 79/80 CF alleles. Mutations [CFTRdele2,3(21kb), p.Gln685ThrfsX4 (2184insA) were found at an unusually high frequency, each comprising 5.00% of all CF alleles. CONCLUSION: We have identified common CF causing mutations in the Hungarian population with the most common mutations (p.Phe508del, p.Asn1303Lys, CFTRdele2,3(21kb), 2184insA, p.Gly542X, and p.Leu101X), comprising over 93.75% of all CF alleles. Obtained data are applicable to the improvement of DNA diagnostics in Hungary and beyond, and are the necessary prerequisite for the introduction of a nationwide "two tier" CF newborn screening program.