Nonisotopic single strand conformation analysis of the 5 alpha-reductase type 2 gene for the diagnosis of 5 alpha-reductase deficiency.

Abstract

5 alpha-Reductase deficiency is a rare autosomal recessive disorder of defective virilization in karyotypic males due to reduced conversion of testosterone to dihydrotestosterone. The gene encoding the affected 5 alpha-reductase type 2 enzyme has recently been cloned, and mutations within the coding region have been discovered as the cause of this disease. We address the possibility of a rapid nonradioactive molecular genetic screening technique for initial diagnosis and report different point mutations in this gene in eight unrelated patients with clinical features of 5 alpha-reductase deficiency. For molecular genetic analysis, DNA from peripheral blood leukocytes was studied. The coding region of the 5 alpha-reductase type 2 gene was characterized by exon-specific PCR amplification, nonradioactive single strand conformation analysis, and direct sequencing. In seven patients, homozygous point mutations were identified (Leu55-Gln, delta Met157, Gly196-Ser, Arg227-Gln, Ala228-Thr, and His231-Arg). One individual was a compound heterozygote carrier of two mutations (Ile112-Asn and Gln126-Arg). We conclude that molecular genetic characterization of point mutations in the 5 alpha-reductase type 2 gene may be used as an additional valuable procedure for the diagnosis of this disorder.

Partial sequencing and tissue distribution of the canine isoforms of steroid 5?-Reductase type I and type II

“…In comparison, monkey isozymes appear to be functionally indistinguishable from human isozymes, as they showed the same sensitivity toward a panel of 30 tested inhibitors [24]. All but one of the 19 bases in the corresponding part of the human type II isozyme that were reported to lead to a nonfunctional enzyme when mutated [25][26][27][28][29] are conserved in the human, monkey, dog, and rat isozymes. This underlines the importance of these amino acids for the functionality of the 5␣-reductase isozymes.…”

“…Amino acids differing with the human isoform are shown in lowercase. Asterisks indicate amino-acid positions at which changes give rise to 5␣-reductase type II deficiency [25][26][27][28][29]. reductase, respectively, spanning 34% and 56% of the respective human coding sequences.…”

Effect of GH replacement therapy in two male siblings with combined X-linked hypophosphatemia and partial GH deficiency

“…Exons 1-22 of the PHEX gene were individually amplified by polymerase chain reaction with specific primers. Subsequently, amplified products were analyzed by non-isotopic single strand conformation analysis (SSCA) (10). The exon displaying an aberrant electrophoretic pattern on SSCA was directly sequenced employing CY5-labeled primers on an automated sequencer (ALF Express, Pharmacia, Freiburg, Germany) according to the specifications of the manufacturer (11).…”

Molecular Characterization of 6 Unrelated Italian Patients With 5α‐Reductase Type 2 Deficiency

“…Most affected 46,XY individuals are characterized at birth by predominantly female external genitalia, bilateral testes, absence of müllerian structures, and normal masculinized wolffian ducts ending in a vaginal pouch (Wilson et al, 1993; Imperato‐McGinley and Zhu, 2002). However, the clinical spectrum is heterogeneous, varying from a female to a fully male phenotype with hypospadias or only microphallus (Carpenter et al, 1990; Hiort et al, 1996a; Hackel et al, 2005). Some patients are sufficiently masculinized at birth to be raised as boys, whereas patients with predominantly normal external female structures are often raised as girls.…”