Lessons learned from 5 years of newborn screening for congenital adrenal hyperplasia in the Czech Republic: 17-hydroxyprogesterone, genotypes, and screening performance.

The aims were to summarize the experience and to determine the performance metrics of newborn screening (NBS) for congenital adrenal hyperplasia (CAH) in the Czech Republic. 17-Hydroxyprogesterone (17OHP) was measured in NBS samples prospectively in 545,026 newborns and retrospectively in 31 CAH patients born outside the study period. A total of 2,811 screened newborns had abnormal 17OHP; CAH was confirmed in 46 probands. One patient with a severe-moderate genotype of CAH had 17OHP below the cut-off and was diagnosed clinically. This corresponds to a screening sensitivity of 98% and a false positive rate (FPR) of 0.51%. The median of 17OHP in the most severe genotypes was 484 nmol/L (n = 21); in severe/moderate, 321 nmol/L (n = 30); in moderate, 61 nmol/L (n = 20); and in mild genotypes, 31 nmol/L (n = 7). NBS is efficient to detect severe CAH but may fail to detect milder variants. However, the FPR is too high but could be improved by application of a second tier test.

Spectrum of low density lipoprotein receptor mutations in Czech hypercholesterolemic patients.

The aim of our study was to define mutations causing familial hypercholesterolemia (FH) phenotype in Czech hypercholesterolemic individuals. A combination of heteroduplex analysis, SSCP, DGGE, DNA sequencing and PCR/restriction analysis was used for this purpose. Molecular searching in the promoter region and coding sequence of the low density lipoprotein receptor (LDLR) gene in 130 patients from 68 unrelated families resulted in the identification of 37 sequence variations. Thirty of them are most likely disease causing mutations. Nineteen mutations were novel (two nonsense, five missense, six nucleotide(s) insertions and six nucleotide(s) deletions). Their pathological effect can be predicted on the basis of their position with respect to previously reported mutations with an estimated reduction of the receptor activity and/or premature termination of translation. These results expand our knowledge of mutations responsible for FH. Seven nucleotide variations were characterized as silent polymorphisms.

Genetic diversity within the R408W phenylketonuria mutation lineages in Europe.

The R408W phenylketonuria mutation in Europe has arisen by recurrent mutation in the human phenylalanine hydroxylase (PAH) locus and is associated with two major PAH haplotypes. R408W-2.3 exhibits a west-to-east cline of relative frequency reaching its maximum in the Balto-Slavic region, while R408W-1.8 exhibits an east-to-west cline peaking in Connacht, the most westerly province of Ireland. Spatial autocorrelation analysis has demonstrated that the R408W-2.3 cline, like that of R408W-1.8, is consistent with a pattern likely to have been established by human dispersal. Genetic diversity within wild-type and R408W chromosomes in Europe was assessed through variable number tandem repeat (VNTR) nucleotide sequence variation and tetranucleotide short tandem repeat (STR) allelic associations. Wild-type VNTR-8 chromosomes exhibited two major cassette sequence organizations: (a1)5-b3-b2-c1 and (a1)5-b5-b2-c1. R408W-1.8 was predominantly associated with (a1)5-B5-B2-C1. Both wild-type vntr-3 and r408w-2.3 chromosomes exhibited a single invariant cassette sequence organization, a2-b2-c1. STR allele distributions associated with the cassette variants were consistent with greater diversity in the wild-type VNTR-8 lineage and were suggestive of different levels of diversity between R408W-1.8 and R408W-2.3. The finding of greater genetic diversity within the wild-type VNTR-8 lineage compared to VNTR-3 suggests that VNTR-8 may be older within the European population. However, in the absence of a more extensive STR data-set, no such conclusions are possible for the respective R408W mutant lineages.

Chimeric CYP21A1P/CYP21A2 genes identified in Czech patients with congenital adrenal hyperplasia.

Congenital adrenal hyperplasia (CAH) comprises a group of autosomal recessive disorders caused by an enzymatic deficiency which impairs the biosynthesis of cortisol and, in the majority of severe cases, also the biosynthesis of aldosterone. Approximately 95% of all CAH cases are caused by mutations in the steroid 21-hydroxylase gene (CYP21A2). The CYP21A2 gene and its inactive pseudogene (CYP21A1P) are located within the HLA class III region of the major histocompatibility complex (MHC) locus on chromosome 6p21.3. In this study, we describe chimeric CYP21A1P/CYP21A2 genes detected in our patients with 21-hydroxylase deficiency (21OHD). Chimeric CYP21A1P/CYP21A2 genes were present in 171 out of 508 mutated CYP21A2 alleles (33.8%). We detected four types of chimeric CYP21A1P/CYP21A2 genes: three of them have been described previously as CH-1, CH-3, CH-4, and one type is novel. The novel chimeric gene, termed CH-7, was detected in 21.4% of the mutant alleles. Possible causes of CYP21A1P/CYP21A2 formation are associated with 1) high recombination rate in the MHC locus, 2) high recombination rate between highly homologous genes and pseudogenes in the CYP21 gene area, and 3) the existence of chi-like sequences and repetitive minisatellite consensus sequences in CYP21A2 and CYP21A1P which play a role in promoting genetic recombination.

Identification of CYP21A2 mutant alleles in Czech patients with 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) is comprised of a group of autosomal recessive disorders caused by an enzymatic deficiency which impairs the biosynthesis of cortisol and, in most of the severe cases, also the biosynthesis of aldosterone. Approximately 90-95% of all the CAH cases are due to mutations in the steroid 21-hydroxylase gene (CYP21A2). In this study, the molecular genetic analysis of CYP21A2 was performed in 267 Czech probands suspected of 21-hydroxylase deficiency (21OHD). 21OHD was confirmed in 241 probands (2 mutations were detected). In 26 probands, a mutation was found only in 1 CYP21A2 allele. A set of 30 different mutant alleles was determined. We describe i) mutated CYP21A2 alleles carrying novel point mutations (p.Thr168Asn, p.Ser169X and p.Pro386Arg), ii) mutated CYP21A2 alleles carrying the novel chimeric gene designated as CH-7, which was detected in 21.4% of the mutant alleles, iii) an unusual genotype with a combination of the CYP21A2 duplication, 2 point mutations and the CYP21A2 large-scale gene conversion on the second allele, and (iv) a detailed analysis of the chimeric CYP21A1P/CYP21A2 genes. In conclusion, our genotyping approach allowed for the accurate identification of the CYP21A2 gene mutations in 21OHD patients and their families and provided some useful information on diagnosis and genetic counselling.

Identification and characterization of large deletions in the phenylalanine hydroxylase (PAH) gene by MLPA: evidence for both homologous and non-homologous mechanisms of rearrangement.

Large gene deletions and duplications were analyzed in 59 unrelated phenylketonuria (PKU) patients negative for phenylalanine hydroxylase (PAH) mutations on one or both alleles from previous exon by exon analysis. Using the novel multiplex ligation-dependent probe amplification (MLPA) method, a total of 31 partial PAH deletions involving single exons were identified in 31 PKU patients. Nineteen cases exhibited deletion of exon 5, and 12 cases provided evidence for the deletion of exon 3. Subsequently, using restriction enzyme digestion and DNA sequencing, three different large deletions, EX3del4765 (12 cases), EX5del955 (2 cases) and EX5del4232ins268 (17 cases) were identified and confirmed by long-range PCR and by the analysis of aberrant transcripts. Altogether, the 31 large deletions presented account for 3% of all PAH mutant alleles investigated in Czech PKU patients. Bioinformatic analysis of three breakpoints showed that the mutation EX3del4765 had arisen through an Alu-Alu homologous recombination, whereas two other mutations-the EX5del955 and EX5del4232ins268, had been created by a non-homologous end joining (NHEJ). We conclude that MLPA is a convenient, rapid and reliable method for detection of intragenic deletions in the PAH gene and that a relatively high number of alleles with large deletions are present in the Slavic PKU population.

Molecular genetic analysis of SLC3A1 and SLC7A9 genes in Czech and Slovak cystinuric patients.

Cystinuria is a frequently inherited metabolic disorder in the Czech population (frequency 1/5,600) caused by a defect in the renal transport of cystine and dibasic amino acids (arginine, lysine and ornithine). The disease is characterized by increased urinary excretion of the amino acids and often leads to recurrent nephrolithiasis. Cystinuria is classified into two subtypes (type I and type non-I). Type I is caused predominantly by mutations in the SLC3A1 gene (2p16.3), encoding heavy subunit (rBAT) of the heterodimeric transporter. Cystinuria non-I type is caused by mutations in the SLC7A9 gene (19q13.1). In this study, we present results of molecular genetic analysis of the SLC3A1 and the SLC7A9 genes in 24 unrelated cystinuria families. Individual exons of the SLC3A1 and SLC7A9 genes were analyzed by direct sequencing. We found ten different mutations in the SLC3A1 gene including six novel ones: three missense mutations (G140R), D179Y and R365P), one splice site mutation (1137-2A>G), one deletion (1515_1516delAA), and one nonsense mutation (Q119X). The most frequent mutation, M467T; was detected in 36% of all type I classified alleles. In the SLC7A9 gene we found six mutations including three new ones: one missense mutation (G319R), one insertion (611_612insA) and one deletion (205_206delTG). One patient was compound heterozygote for one SLC3A1 and one SLC7A9 mutation. Our results confirm that cystinuria is a heterogeneous disorder at the molecular level, and contribute to the understanding of the distribution and frequency of mutations causing cystinuria in the Caucasian population.