Using the proposed algorithm, all alleles were elucidated. False-positive results in MLPA occurred when mutations or polymorphisms were located close to the probe-binding regions. These difficulties were overcome by the association of MLPA with ASO-PCR and paternal segregation. Using these approaches, we can successfully use MLPA in a cost-effective laboratory routine for the molecular diagnosis of CAH-21OHD.

SHH mutations or copy number variations are not a common cause of congenital hypopituitarism in patients without complex midline cerebral defects. GLI2 variants are found in some patients with congenital hypopituitarism without complex midline cerebral defects or septo-optic dysplasia. However, functional analyses of these variants are needed to strengthen genotype-phenotype relationship.

There is a clinical spectrum of non-classical congenital adrenal hyperplasia due to 21-hydroxylase deficiency (NC-CAH). In addition, CYP21A2 gene mutations analysis present in homozygosis or as compound heterozygosis. Herein we hypothesize that allele combination associates with specific biochemical profiles. Objectives : To evaluate the relationship between the genotype and biochemical profiles in the diagnosis of NC-CAH. Patients and Methods : Clinical, hormonal and molecular data at diagnosis of 63 patients (78% female; 43 children, 20 adults) with NC-CAH followed from 1989 to 2019 were analyzed. Results: At the diagnosis, most children (75% of girls and 71% of boys), presented with premature pubarche. Clitoromegaly was present in 10% of the girls. Among adult patients, hirsutism (75%), menstrual abnormalities (50%), premature pubarche (42%), acne (35%) and clitoromegaly (10%) were observed. Basal and ACTH-stimulated 17OHP mean levels (ng/dL) were 1,329 ± 1,232 (97-5,560) and 4,364 ± 2,340 (1,115-10,648), respectively. The most frequent mutation was p.V281L (60% of alleles) being 31% in homozygosis. Fifty percent of the patients were compound heterozygotes for one classic (C) and one non-classical mutation (NC). C alleles were p.I172N, IVS2-13A/C>G, Δ8, CL6, p.Q318X, p.R356W and LGC. Basal and ACTH-stimulated 17OHP values were higher in patients carrying the C/NC genotypes compared to NC/NC genotypes (1,768±1,333 vs 835±923; p=0.0002 and 5,392±498 vs 3,551±400 ng/dl; p=0.005, respectively). Similarly, basal and ACTH-stimulated 17OHP to cortisol ratios were higher in patients carrying the C/NC compared to NC/NC genotypes (134 ±76 vs 53±34; p=0.0003 and 309±48 vs 199±27; p=0.04, respectively). Moreover, ROC curve analyses showed that the basal and ACTH-stimulated 17OHP levels of 610 and 3,913 ng/dl were the best cutoffs to identify NC-CAH patients carrying compound heterozygosis with C alleles. According to the severity of the mutation, basal 17OHP (ng/dL) was progressively higher p.V281L/p.V281L vs p.V281L/IVS2-13A/C>G (927 vs 3,094; p<0.01) and p.V281L/p.I172N vs p.V281L/IVS2-13A/C>G (1,011 vs 3,094; p<0.05). This was also observed in ACTH-stimulated 17OHP between p.V281L/p.V281L vs p.V281L/p.R356W (3,265 vs 5,817; p<0.01). There was a trend to early appearance of premature pubarche (5.7±1.8 vs 6.7±1.6 years; p=0.07) in C/NC genotype group compared to NC/NC. There were no differences in age, height, weight, androstenedione, DHEAS and testosterone levels between the C/NC and NC/NC groups at diagnosis. Conclusion : The molecular variability in NC-CAH associates with graded severities of adrenal biosynthetic defect. Unless otherwise noted, all abstracts presented at ENDO ...

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