Modeling mutant/wild-type interactions to ascertain pathogenicity of PROKR2 missense variants in patients with isolated GnRH deficiency.

A major challenge in human genetics is the validation of pathogenicity of heterozygous missense variants. This problem is well-illustrated by PROKR2 variants associated with Isolated GnRH Deficiency (IGD). Homozygous, loss of function variants in PROKR2 was initially implicated in autosomal recessive IGD; however, most IGD-associated PROKR2 variants are heterozygous. Moreover, while IGD patient cohorts are enriched for PROKR2 missense variants similar rare variants are also found in normal individuals. To elucidate the pathogenic mechanisms distinguishing IGD-associated PROKR2 variants from rare variants in controls, we assessed 59 variants using three approaches: (i) in silico prediction, (ii) traditional in vitro functional assays across three signaling pathways with mutant-alone transfections, and (iii) modified in vitro assays with mutant and wild-type expression constructs co-transfected to model in vivo heterozygosity. We found that neither in silico analyses nor traditional in vitro assessments of mutants transfected alone could distinguish IGD variants from control variants. However, in vitro co-transfections revealed that 15/34 IGD variants caused loss-of-function (LoF), including 3 novel dominant-negatives, while only 4/25 control variants caused LoF. Surprisingly, 19 IGD-associated variants were benign or exhibited LoF that could be rescued by WT co-transfection. Overall, variants that were LoF in ≥ 2 signaling assays under co-transfection conditions were more likely to be disease-associated than benign or 'rescuable' variants. Our findings suggest that in vitro modeling of WT/Mutant interactions increases the resolution for identifying causal variants, uncovers novel dominant negative mutations, and provides new insights into the pathogenic mechanisms underlying heterozygous PROKR2 variants.

Gender Identity and Sexual Function in 46,XX Patients with Congenital Adrenal Hyperplasia Raised as Males.

In individuals with congenital adrenal hyperplasia (CAH) and 46,XX karyotype, androgens produced by the adrenal glands during the intrauterine development promote virilization of the genitals, which may even result in the development of a well-formed penis. Some of these children with late diagnosis are registered as males after birth. After obtaining approval from the internal review board, we evaluated gender identity and sexual function in four 46,XX severely virilized patients with CAH, who were originally registered and raised as males, assisted in our Disorders of Sexual Development Clinic. The evaluation consisted of questionnaires to assess gender identity and sexual activity and interview with the multidisciplinary team that provides care for these patients. The patients underwent surgery to remove uterus, ovaries, and remaining vaginal structures, in addition to implantation of testicular prosthesis and correction of hypospadias, when necessary. All four patients have developed a clear male gender identity, and when evaluated for sexual activity, they have reported having erections, libido, orgasms, and sexual attraction to women only. Two of these 4 patients had satisfactory sexual intercourses when assessed using the International Index of Erectile Function questionnaire. The other two patients who never had sexual intercourse reported not having a partner for sexual activity; one is 18 years old, and the other is 14 years old. This study showed that this group of 46,XX severely virilized patients with CAH, registered and raised as males, adapted well to the assigned male gender, with satisfactory sexual function in patients who had sexual intercourse.

COX-2 rs20417 Polymorphism Is Associated with Stroke and White Matter Disease.

BACKGROUND: To investigate the effect of COX-2 polymorphism and its product, prostaglandin E2 (PGE2), on stroke risk in an endemic area for Chagas disease. In a separate cohort, to investigate the effect of COX-2 polymorphisms on the total burden of cerebral white matter disease. METHODS: Cases were outpatients with ischemic stroke; controls were stroke-free subjects from 2 outpatient clinics (heart failure and caregivers of a movement disorders clinic). We extracted DNA from total blood to investigate the rs20417 COX-2 polymorphism. Serologic tests (Enzime-linked immunosorbent assay) were performed to confirm Trypanosoma cruzi infection and to quantify PGE2 levels. In the Boston cohort, white matter hyperintensity volume (WMHv) was quantified on the admission brain magnetic resonance images of subjects with ischemic stroke, who also donated DNA for the COX-2 gene region analysis. RESULTS: We studied 44 patients with stroke and 96 controls (46 with heart failure and 50 caregivers) in the Brazilian cohort; and 788 stroke patients (302 cardioembolic and 486 noncardioembolic) in the Boston cohort. In the Brazilian cohort, rs20417 polymorphism was associated with both stroke (P = 5 × 10(-6)) and decreased PGE2 levels (P = 4 × 10(-5)); similarly, Chagas was associated with stroke (P = 4 × 10(-3)) and decreased PGE2 levels (P = 7 × 10(-3)). In the Boston cohort, rs20417 polymorphism was associated with increased WMHv among noncardioembolic (P = .037), but not among cardioembolic stroke patients. CONCLUSIONS: Variation in COX-2 gene is associated with both symptomatic and silent brain cerebrovascular disease. This candidate gene region should be tested in population-based samples.