Genotyping of CYP21, Linked Chromosome 6p Markers, and a Sex-Specific Gene in Neonatal Screening for Congenital Adrenal Hyperplasia

Abstract

We investigated the feasibility and diagnostic utility of genotyping 9 CYP21 mutations, linked chromosome 6p markers, and a dimorphic X-Y marker from neonatal screening samples. Blood-impregnated filter papers (Guthrie cards) from 603 randomly chosen New Zealand neonates were genotyped blind to 17-hydroxyprogesterone (17-OHP) levels. Another 50 samples from Swiss and North American infants with correlative hormonal data were also genotyped. DNA was extracted, and gene-specific PCR was performed. CYP21 PCR products were subjected to ligase detection reaction, simultaneously analyzing 9 CYP21 mutations; PCR products of other genes were subjected to direct gel analysis. CYP21 genotyping indicated a heterozygote rate of 2.8% for classic mutations (excluding CYP21 deletions), and 2.0% for nonclassic mutations in New Zealanders. Ten full-term affected neonates showed a wide range of 17-OHP levels (15-1400 nmol/L). Sick or preterm infants or infants screened on the first day of life with high 17-OHP proved genetically unaffected. Genetic linkage disequilibrium was found between two CYP21 mutations and chromosome 6p markers. Guthrie cards can be used to accurately genotype CYP21 and other relevant markers, potentially enhancing the specificity and sensitivity of congenital adrenal hyperplasia screening. CYP21 heterozygote frequency for classic mutations is higher than expected based on genotype compared with that predicted by hormonal newborn screening.

Hormonal evaluation and mutation screening for steroid 21-hydroxylase deficiency in patients with unilateral and bilateral adrenal incidentalomas

“…Based on plasma 17-OHP responses to ACTH stimulation, heterozygous carrier frequencies have been estimated as one in 16 for Caucasians (42). Molecular genetic screening of normal newborn infants in New Zealand showed that one in 20 were heterozygous for CYP21B mutations, which implies a disease frequency similar to that estimated in the Caucasian population (43). In our study, the detection of one homozygous and three heterozygous germline mutations in 19 patients with bilateral (21.1%), and five heterozygous germline mutations in 31 patients with unilateral incidentalomas (16.1%) suggests a higher frequency of mutations in both groups of adrenal tumors compared with that found in the general European population.…”

Triagem neonatal para hiperplasia adrenal congênita: experiência do estado do Rio de Janeiro

“…(26) recomenda que a coleta de sangue para triagem para HAC-D21OH seja feita entre 48 e 72h após o nascimento e no máximo até o final da primeira semana de vida. Os recentes trabalhos que utilizam a genotipagem de CYP21B como complemento à triagem para HAC-D21OH trouxeram dados mais objetivos entre a relação dos valores de 17OHP, as formas clínicas e o genótipo apresentado (13,(27)(28)(29)(30)(31). A maior utilização da análise molecular, entretanto, refere-se à elucidação dos casos duvidosos, evitando, assim, o tratamento desnecessário.…”

Bases Moleculares da Hiperplasia Adrenal Congênita

“…Também pode ocorrer falha de amplificação de um alelo (allelic dropout) normal ou mutante durante a PCR. Para minimizar este problema, o estudo de microssatélites altamente polimórficos que flanqueiam o gene da 21-hidroxilase tem sido utilizado no diagnóstico pré-natal em adjunto ao estudo molecular do gene CYP21 (39).…”