Gene duplications in 21-hydroxylase deficiency: the importance of accurate molecular diagnosis in carrier detection and prenatal diagnosis

Abstract

The proposed method discriminated between the severe Q318X mutation and the normal Q318X variant in gene duplication, and could be a useful complementary tool in prenatal diagnosis and carrier detection.

Monogenic and Polygenic Models Detected in Steroid 21-Hydroxylase Deficiency-Related Paediatric Hyperandrogenism

“…Only patients carrying a severe mutation (about 40% of NC21OHD [compound heterozygous with severe mutations]) and about 10% of hyperandrogenic carriers are at risk. Our group has recently stressed the importance of adequately detecting 21OHD carriers in the partners of NC21OHD patients [24, 32]. …”

“…It should be remembered that the adequate segregation of mutations (double microconversions, small conversions including more than one mutation) and a complete characterization of alleles, including the investigation of gene deletions and duplications (i.e. normal alleles carrying gene duplications with the Gln318Stop mutation [24] or PCR amplifiable deletion hybrid genes resulting from a break point upstream of exon 6, including 655G and 8 bp deletion mutations [11]), is required in this type of analysis.…”

“…The carrier frequency in siblings used by other authors [16] was not considered adequate for this purpose. The partners of the 21OHD-carriers who had been analyzed for genetic counseling purposes [24] (see table 4, last two columns) were considered a nonbiased general population. As a whole, both severe and mild mutations were more frequent in the hyperandrogenic carriers.…”

“…Differential length 5′-extensions of the specific oligonucleotides (table 1) were used. The primer extension protocol used was essentially that previously described [24]. 1 µl of pooled primers (1 pmol of PON1, TNFR2 and IRS1, and 0.1 pmol CAPN and 0.4 pmol IGF2) was used along with 1 µl of the PCR multiplex.…”

“…Native acrylamide gels were used to detect the common 8-bp deletion at exon 3, and Southern blotting and semiquantitative primer extension [24] to detect gene deletions and/or duplications, respectively. Complementary direct sequencing (coding regions and intron-exon boundaries) was performed as previously described [25] in nonclassical patients who were not characterized after basic screening, and for whom confirmatory clinical and biochemical information regarding their deficiency was available.…”