This study addresses the contributions of gene conversion and a founder effect to the distribution of the two most frequent severe point mutations of the 21-hydroxylase (21OH) gene causing congenital adrenal hyperplasia: the 655G splicing mutation at intron 2, and Gln318Stop in a Spanish population. Direct and indirect analyses of segregated mutant and normal 21OH genes in 200 Spanish families (classic and nonclassic 21OH deficiency) were performed. Both mechanisms were found to contribute to different degrees to the defective investigated alleles. The 655G splicing mutation (62 alleles, 15.5%) seemed to be almost exclusively related to recent conversion events, whereas Gln318Stop (33 alleles, 8.3%) is more likely to be due to the dissemination of remotely generated mutant alleles. Other severe defective alleles, 8 bp-deletion (13 alleles, 3.3%), 306insT (5 alleles, 1.3%), and gene deletions (43 alleles, 11%), as well as the mild mutation Val281Leu (120 alleles, 30%), also appear to be strongly associated with particular D6S273 alleles. Although gene conversion contributes to the generation of severe 21OH alleles, the high frequency of some severe mutations in different geographic areas is consistent with a founder effect.

ACTH-stimulated 17-OHP may predict the risk of severe mutations in compound heterozygosity in children (maximum predictive value 93% sensitivity and 83% specificity for a cut-off at 151 nmol l(-1)), although a certain overlap in individual values is observed and performance of molecular analysis should never be obviated in the genetic counselling of these patients.

Aim: To investigate the association between levels of 17‐hydroxyprogesterone (17‐OHP) and the risk of being compound heterozygous for severe mutations in children with non‐classical 21‐hydroxylase deficiency (NC21OHD). Methods: In 86 Spanish NC21OHD children (75 families) an analysis of the 21‐hydroxylase (21‐OH) gene was performed by CYP21B‐specific polymerase chain reaction amplification, allele‐specific oligonucleotide hybridization and Southern blotting. Familial analysis established how the alleles segregated, and allowed the selection of 21‐OH‐genotyped normal and carrier children, which proved useful in determining a more precise definition of the cut‐off for diagnosis. Receiver operating characteristics (ROC) curve analyses were performed to determine the potential value of 17‐OHP in predicting compound heterozygosity for severe mutations. Results: Thirty‐four of the 86 children (39%) were found to carry one severe 21‐OH mutation (7.3% deletions or conversions, 2.7% 655G, 2.7% Q318X, 1.3% I172N, 1.3% R356W, and 3.3% double microconversions or small conversions involving single exons). The predominant mutation was V281L (56.7%). P453S and P30L were less frequent (3.3 and 2%). No patient showed two severe mutations. The degree of enzymic deficiency, as measured by basal or adrenocorticotropic hormone (ACTH)‐stimulated 17‐OHP levels in fully genotyped patients, but not clinical severity (age and number of symptoms at diagnosis), was found to be significantly greater in children with the severe/mild genotype. ROC curve analyses revealed a strong association between ACTH‐17‐OHP and genotype (area under the curve 0.908, SE 0.057). Conclusion: ACTH‐stimulated 17‐OHP may predict the risk of severe mutations in compound heterozygosity in children (maximum predictive value 93% sensitivity and 83% specificity for a cut‐off at 151 nmol 1‐1), although a certain overlap in individual values is observed and performance of molecular analysis should never be obviated in the genetic counselling of these patients.

Background: SHOX haplo-insufficiency is considered the molecular basis of short stature in patients with Turner's syndrome, and gives rise to the short stature with mesomelic dysplasia and Madelung deformity of patients with Leri-Weill syndrome. Objective: Analysis of the intragenic SHOX microsatellite to define its utility in detecting SHOX haplo-insufficiency in patients with short stature. Patients and Methods: 207 patients with short stature (57 girls with Turner's syndrome [TS] [24 mosaicisms]; 73 children with isolated short stature [ISS]; 77 patients with short stature and skeletal disproportion) and 30 control subjects. DNA extraction and PCR amplification of the intragenic SHOX microsatellite, at the 5'-untranslated region. SSCP and partial sequencing of the SHOX gene in one patient with Madelung deformity and two SHOX alleles. DXS1055 (Xp) and DXS1192 (Xq) microsatellites were also analyzed, together with DXS233 and DXS234 at 0 and 2 cM of the pseudoautosomal region (PAR), in patients with one SHOX allele. Results: 1. 93% of patients with TS had a single SHOX allele, and allele unbalance was detected in the remainder. 2. Patients with ISS were not different from the normal population with respect to SHOX heterozygosity (0.92 and 0.93, respectively; ρ = 0.997). 3. Patients with short stature and skeletal disproportion showed a higher frequency of SHOX homo/hemizygosity (0.27 vs 0.08; ρ = 0.027). 4. Five patients with short stature with SHOX haplo-insufficiency were detected: three had Madelung deformity (inherited Yq;Xp translocation, de novo PAR deletion, and ST/OX microdeletion), and two had de «ovo/inherited Xp partial monosomy. Conclusions:The SHOX intragenic microsatellite might be a useful molecular marker to detect TS (including Xp distal deletions). SHOX haploinsufficiency seems not to be an important contributor to ISS, but when skeletal disproportion is associated with short stature, a significant proportion of patients is found to have a single SHOX allele. Some of these patients were found to be SHOX haplo-insufficient upon molecular, cytogenetic and radiological examination. KEY WORDSSHOX intragenic microsatellite, Turner's syndrome, Leri-Weill syndrome, isolated short stature, SHOX haplo-insufficiency