Objective: To analyze the mutational spectrum of steroid 21-hydroxylase (CYP21) and the genotypephenotype correlation in patients with congenital adrenal hyperplasia (CAH) registered in the Middle European Society for Pediatric Endocrinology CAH database, and to design a reliable and rational approach for CYP21 mutation detection in Middle European populations. Design and methods: Molecular analysis of the CYP21 gene was performed in 432 CAH patients and 298 family members. Low-resolution genotyping was performed to detect the eight most common point mutations. High-resolution genotyping, including Southern blotting and sequencing was performed to detect CYP21 gene deletions, conversions, point mutations or other sequence changes. Results: CYP21 gene deletion and In2 and Ile172Asn mutation accounted for 72.7% of the affected alleles in the whole study group. A good genotype -phenotype correlation was observed, with the exception of Ile172Asn and Pro30Leu mutations. In 37% of patients low resolution genotyping could not identify the causative mutation or distinguish homozygosity from hemizygosity. Using high-resolution genotyping, the causative mutations could be identified in 341 out of 348 analyzed patients. A novel mutation Gln315Stop was found in one simple virilising CAH (SV-CAH) patient from Austria. In the remaining seven patients polymorphisms were identified as the leading sequence alteration. The presence of elevated basal and ACTH-stimulated 17-hydroxyprogesterone, premature pubarche, advanced bone age and clitoral hypertrophy directly implicated Asn493Ser polymorphism in the manifestation of nonclassical-(NC) and even SV-CAH. Conclusions: By genotyping for the most common point mutations, CYP21 gene deletion/conversion and the 8 bp deletion in exon 3, it should be possible to identify the mutation in 94 -99% of the diseased alleles in any investigated Middle European population. In patients with a mild form of the disease and no detectable mutation CYP21 gene polymorphisms should be considered as a plausible disease-causing mutation.European Journal of Endocrinology 153 99-106

Objective: To analyse the mutational spectrum, the associated haplotypes and the genotype -phenotype correlation, and to design a reliable and rational approach for CYP21 mutation detection in Slovenian congenital adrenal hyperplasia (CAH) patients. Design: Molecular analysis of the CYP21 gene was performed in 36 CAH patients and 79 family members. Methods: Southern blotting, sequence-specific PCR amplification (PCR-SSP), sequence-specific oligonucleotide hybridisation (PCR-SSO) and sequencing were used to detect CYP21 gene deletions, conversions and point mutations. Results: CYP21 gene deletion was the most frequent mutation (36.4%). Large gene conversions detectable only by Southern blotting represented 12.1%, and gene conversions involving the promoter region represented 7.6% of the mutated alleles. The most frequent point mutations were: intron 2 splice mutation 16.7%, Ile172Asn mutation 7.6%, Gln318Stop 7.5% and Pro30Leu 12.2% of alleles. A correlation between the genotype and the clinical phenotype similar to those described for large populations was observed. The finding of Pro30Leu mutation linked to a gene conversion could explain the simple virilising (SV) phenotype in compound heterozygotes for the Pro30Leu and a severe mutation. In two siblings with a salt wasting form of CAH (SW-CAH), a novel mutation Ala15Thr was found on the allele characterised by Pro30Leu mutation and gene conversion involving the promoter region. Conclusions: Our genotyping approach allowed reliable diagnosis of CAH in the Slovenian population. The high frequency of CYP21 gene aberrations on Pro30Leu positive alleles justified systematic searching for a gene conversion in the promoter region using the PCR-SSP reaction.

A combination of specific HLA class II antigens and the presence of type 1 diabetes (T1D)-related antibodies has a high positive predictive value for T1D but low sensitivity. The aim of the present study was to determine the frequencies of HLA-DRB-DQB deduced haplotypes associated with susceptibility and protection in Slovenian patients with established T1D, to evaluate the relationship between the HLA-DRB1-QBP-DQB1 haplotypes and the presence of insulin autoantibodies (IAA) and glutamic acid decarboxylase antibodies (GADA), and to access the possible impact of polymorphic QBP promoters on this relationship. A cohort of 135 patients with T1D (age 17.5 +/- 7.0 years, duration of T1D 9.14 +/- 6.3 years) was investigated. HLA-DRB1 and DQB1 alleles were typed using the polymerase chain reaction (PCR)-reverse line blot method. QBP promoter region alleles were determined using PCR-sequence-specific oligonucleotide hybridization (SSO) and PCR-sequence-specific primers (SSP). IAA and GADA antibodies were determined by enzyme-linked immunosorbent assay (ELISA). The chi-square test with Yates' correction was used for statistical analysis. Deduced haplotypes DRB1*0301-DQB1*0201 (P = 0.0001, OR = 3.4), DRB1*0401-DQB1*0302 (P = 0.0001, OR = 29.8), and DRB1*0402-DQB1*0302 (P = 0.008, OR = 4.7) were significantly more common, and DRB1*1501-DQB1*0602 (P = 0.0001, OR = 0.03) significantly less common in the investigated cohort than in a Slovenian control group. The highest risk and the strongest protective HLA-DR-DQ haplotypes found in Slovenian patients with T1D did not differ from those found in other Caucasian populations. While the DRB1*0301-QBP2.1-DQB1*0201 haplotype, where QBP2.1 did not help to further distinguish DQB1*0201-possessing haplotypes in IAA-positive and IAA-negative patients, was strongly associated with the presence of IAA, the DRB1*0101-QBP5.12-DQB1*0501 haplotype, although not protective compared to the control population, was associated with an absence of IAA in the investigated cohort. It is suggested that there may be a combined influence of the QBP5.12 promoter and the DQB1*0501 functional molecule on reduced IAA production.

Barth syndrome is an X-linked recessive disorder characterised by dilated cardiomyopathy and a variable expression of skeletal myopathy, short statue and neutropenia. Molecular genetic analysis is currently the most reliable diagnostic method. A kindred with a novel 535delC mutation in the G4.5 (TAZ) gene responsible for Barth syndrome is presented. Beside the patient, the same mutation was detected in patient's mother and grandmother. In contrast to the so far reported patients with mutations in the same region of G4.5 (TAZ) gene, the patient described here has only a mild and transitory clinical presentation. This could be attributed to alternative splicing of G4.5 (TAZ) gene, since mRNA lacking exon 6 (with 535delC mutation) was detected. Genetic analysis of the G4.5 (TAZ) gene was helpful for establishing the precise diagnosis of Barth syndrome and for adequate genetic counselling. Predicting the phenotype on the basis of mutations is unreliable especially if mutations are localised in alternatively spliced exons of the G4.5 (TAZ) gene which may result in a milder clinical presentation than expected.