Biochemical Analysis of Four Missense Mutations in the HSD17B3 Gene Associated with 46, XY Disorders of Sex Development in Egyptian Patients

Abstract

Background Mutations in the HSD17B3 gene are associated with a 46,XY disorder of sexual development (46,XY DSD) as a result of low testosterone production during embryogenesis. Aim To elucidate the molecular basis of the disorder by chemically analyzing four missense mutations in HSD17B3 (T54A, M164T, L194P, G289S) from Egyptian patients with 46,XY DSD. Methods Expression plasmids for wild-type 17β-hydroxysteroid hydrogenase type 3 (17β-HSD3) and mutant enzymes generated by site-directed mutagenesis were transiently transfected into human HEK-293 cells. Protein expression was verified by western blotting and activity was determined by measuring the conversion of radiolabeled Δ4-androstene-3,17-dione to testosterone. Application of a homology model provided an explanation for the observed effects of the mutations. Outcomes Testosterone formation by wild-type and mutant 17β-HSD3 enzymes was compared. Results Mutations T54A and L194P, despite normal protein expression, completely abolished 17β-HSD3 activity, explaining their severe 46,XY DSD phenotype. Mutant M164T could still produce testosterone, albeit with significantly lower activity compared with wild-type 17β-HSD3, resulting in ambiguous genitalia or a microphallus at birth. The substitution G289S represented a polymorphism exhibiting comparable activity to wild-type 17β-HSD3. Sequencing of the SRD5A2 gene in three siblings bearing the HSD17B3 G289S polymorphism disclosed the homozygous Y91H mutation in the former gene, thus explaining the 46,XY DSD presentations. Molecular modeling analyses supported the biochemical observations and predicted a disruption of cofactor binding by mutations T54A and M164T and of substrate binding by L196P, resulting in the loss of enzyme activity. In contrast, the G289S substitution was predicted to disturb neither the three-dimensional structure nor enzyme activity. Clinical Translation Biochemical analysis of mutant 17β-HSD3 enzymes is necessary to understand genotype-phenotype relationships. Strengths and Limitations Biochemical analysis combined with molecular modeling provides insight into disease mechanism. However, the stability of mutant proteins in vivo cannot be predicted by this approach. Conclusion The 17β-HSD3 G289S substitution, previously reported in other patients with 46,XY DSD, is a polymorphism that does not cause the disorder; thus, further sequence analysis was required and disclosed a mutation in SRD5A2, explaining the cause of 46,XY DSD in these patients.

Analyses of Molecular Characteristics and Enzymatic Activities of Ovine HSD17B3

“…In addition to DSD, it was reported that G289S is associated with the risks of prostate cancer and hypospadias [33,45]. However, consistent with our results, the enzymatic activity of these mutant proteins associated with this mutation was reported to be similar to that of wild-type protein in multiple previous studies [7,46,47]. Furthermore, the amino acid residue at this position is serine in almost all mammalian species except for primates (Figure 1A), although no studies have shown the associated susceptibility to prostate cancer and hypospadias in particular animal species.…”

17β hydroxysteroid dehydrogenase 3 deficiency in 46,XY disorders of sex development: Our experience and a gender role‐focused systematic review

“…Nevertheless, we propose a similar trend of androgen profile in the intrauterine period with higher T and T/A leading to frequent atypical genitalia and hence, initial male sex of rearing. The residual 17βHSD3 enzyme activity can explain the higher serum T and T/A in these patients 17 . However, many patients harbouring molecular variants causing severe deficiency of the 17βHSD3 enzyme also had higher T/A, suggesting a possible role of other isoenzymes like 17βHSD5.…”

“…The residual 17βHSD3 enzyme activity can explain the higher serum T and T/A in these patients. 17 However, many patients harbouring molecular variants causing severe deficiency of the 17βHSD3 enzyme also had higher T/A, suggesting a possible role of other isoenzymes like 17βHSD5. Notably, the initial male sex of rearing was significantly more frequent in Asians but less frequent in Europeans.…”

Identification of a Novel Mutation in an Iranian Family With 17-β Hydroxysteroid Dehydrogenase Type 3 Deficiency: A Case Series

“…This novel alteration has not been reported earlier in the literature for the HSD17B3 gene. So far, more than 30 different mutations in this gene have been identified, including insertion, exonic deletion, missense, and nonsense mutations (6) with the p. Arg80Gln alteration being the most common one in the Arab population (2). In the Turkish population, c655-1;G-A, p.Ala188Val, and c.777-783del_GATAACC mutations have previously been identified (11).…”

“…Affected individuals have female external genitalia with male normal wolffian ducts at birth (4). The incidence of this disorder is estimated at 1 T in 147,000 newborns (5) and is higher in the Arab population of Gaza, and its prevalence is approximately 1 in every 200 to 300 people, which is due to the high rate of consanguineous marriages (6). Pathogenic mutations in the HSD17B3 gene (MIM# 264300) are associated with the impaired sexual development of the 46, XY fetuses.…”