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Free access

V Dolzan, M Stopar-Obreza, M Zerjav-Tansek, K Breskvar, C Krzisnik and T Battelino

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.

Open access

L Stavber, T Hovnik, P Kotnik, L Lovrečić, J Kovač, T Tesovnik, S Bertok, K Dovč, M Debeljak, T Battelino and M Avbelj Stefanija

Context

Defining the underlying etiology of idiopathic short stature (ISS) improves the overall management of an individual.

Objective

To assess the frequency of pathogenic ACAN variants in selected individuals.

Design

The single-center cohort study was conducted at a tertiary university children’s hospital. From 51 unrelated patients with ISS, the 16 probands aged between 3 and 18 years (12 females) with advanced bone age and/or autosomal dominant inheritance pattern of short stature were selected for the study. Fifteen family members of ACAN-positive probands were included. Exome sequencing was performed in all probands, and additional copy number variation (CNV) detection was applied in selected probands with a distinct ACAN-associated phenotype.

Results

Systematic phenotyping of the study cohort yielded 37.5% (6/16) ACAN-positive probands, with all novel pathogenic variants, including a 6.082 kb large intragenic deletion, detected by array comparative genomic hybridization (array CGH) and exome data analysis. All variants were co-segregated with short stature phenotype, except in one family member with the intragenic deletion who had an unexpected growth pattern within the normal range (−0.5 SDS). One patient presented with otosclerosis, a sign not previously associated with aggrecanopathy.

Conclusions

ACAN pathogenic variants presented a common cause of familial ISS. The selection criteria used in our study were suggested for a personalized approach to genetic testing of the ACAN gene in clinical practice. Our results expanded the number of pathogenic ACAN variants, including the first intragenic deletion, and suggested CNV evaluation in patients with typical clinical features of aggrecanopathy as reasonable. Intra-familial phenotypic variability in growth patterns should be considered.

Free access

V Dolž;an, J Sólyom, G Fekete, J Kovács, V Rakosnikova, F Votava, J Lebl, Z Pribilincova, SM Baumgartner-Parzer, S Riedl, F Waldhauser, H Frisch, M Stopar-Obreza, C Krž;išnik and T Battelino

Objective: To analyze the mutational spectrum of steroid 21-hydroxylase (CYP21) and the genotype– phenotype 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.