HS6ST1 Insufficiency Causes Self-Limited Delayed Puberty in Contrast With Other GnRH Deficiency Genes

Abstract

Context: Self-limited delayed puberty (DP) segregates in an autosomal-dominant pattern, but the genetic basis is largely unknown. Although DP is sometimes seen in relatives of patients with hypogonadotropic hypogonadism (HH), mutations in genes known to cause HH that segregate with the trait of familial self-limited DP have not yet been identified.

Objective: To assess the contribution of mutations in genes known to cause HH to the phenotype of self-limited DP.

Design, patients, and setting: We performed whole-exome sequencing in 67 probands and 93 relatives from a large cohort of familial self-limited DP, validated the pathogenicity of the identified gene variant in vitro, and examined the tissue expression and functional requirement of the mouse homolog in vivo.

Results: A potentially pathogenic gene variant segregating with DP was identified in 1 of 28 known HH genes examined. This pathogenic variant occurred in HS6ST1 in one pedigree and segregated with the trait in the six affected members with heterozygous transmission (P = 3.01 × 10-5). Biochemical analysis showed that this mutation reduced sulfotransferase activity in vitro. Hs6st1 mRNA was expressed in peripubertal wild-type mouse hypothalamus. GnRH neuron counts were similar in Hs6st1+/- and Hs6st1+/+ mice, but vaginal opening was delayed in Hs6st1+/- mice despite normal postnatal growth.

Conclusions: We have linked a deleterious mutation in HS6ST1 to familial self-limited DP and show that heterozygous Hs6st1 loss causes DP in mice. In this study, the observed overlap in potentially pathogenic mutations contributing to the phenotypes of self-limited DP and HH was limited to this one gene.

Figure 1.

Flowchart of WES filtering strategy to identify HS6ST1. WES was performed on DNA extracted from peripheral blood leukocytes of 160 individuals from our cohort (67 DP probands, 58 DP relatives, and 35 controls). The exome sequences were aligned to the University of California Santa Cruz hg19 reference genome. Picard tools and the Genome Analysis Toolkit were used to mark PCR duplicates, realign around indels, recalibrate quality scores, and call variants. Potential causal variants were identified using filters for quality control, predicted functional annotation, MAF, biological relevance (i.e., “HH gene” list), and segregation with trait (see Methods and Table 1 for further information on filtering criteria). Targeted exome sequencing using a Fluidigm array of one candidate gene was then performed in a further 42 families from the same cohort (288 individuals, 178 with DP and 110 controls). Variants identified via targeted resequencing were filtered using the same criteria as the WES data, with additional rare variant burden testing. Functional annotation of the variants is as described in “Methods.”

Figure 2.

Pedigree and clinical details of a patient with a p.Arg375His mutation in HS6ST1 that impairs sulfotransferase activity. (A) Pedigree of the proband with the p.Arg375His mutation. Squares indicate male family members; circles indicate female family members. Black symbols represent clinically affected family members, gray symbols represent family members with unknown phenotype, and clear symbols represent unaffected individuals. The arrow labeled “P” indicates the proband in the family. A horizontal black line above or adjacent to an individual’s symbol indicates that they are heterozygous for the p.Arg375His mutation as identified by either WES or Fluidigm array and verified by Sanger sequencing. (B) Clinical details of the proband with the p.Arg375His mutation. Height chart for the proband shows reduction in growth velocity from 12 y of age, with associated delayed bone age shown in red. The subject was prepubertal until 14.1 y of age. This was followed by spontaneous onset of puberty and subsequent development during 2 y to normal adult testosterone levels and testicular volume. (C) Sulfotransferase activity assay. The p.Arg375His mutation reduces HS6ST1 sulfotransferase activity. Relative specific activity (mean ± SD) of recombinant WT or mutant (p.Arg375His or p.Met404Val) HS6ST1 proteins are shown. All assays were done as three independent biological repeats (n = 3 for each experiment) using equal amounts of protein. ***P < 0.001. G, Tanner genital stage, Ph, Tanner pubic hair stage, T, testosterone; T vol, testicular volume.

Figure 3.

Hs6st1 mRNA expression in GnRH neuronal territories on P35. (A) Coronal sections of adult mouse OB were labeled for Hs6st1 by ISH. The squared box in the left image is shown at higher magnification to the right. (B) Contiguous coronal sections of adult mouse hypothalamus representing the MPOA (upper panels) and the ME (lower panels) were labeled for Gnrh and Hs6st1 by ISH; arrowheads indicate the Hs6st1 expression in the ARC and ME. (C) Hs6st1 ISH (green) of coronal sections from P35 MPOA followed by immunolabeling for GnRH (red) revealed no expression of Hs6st1 in GnRH-positive neurons (example of GnRH neuron is indicated with a solid arrowhead). Scale bars: 125 µm (A and B, upper panels), 50 µm (B, lower panels), 25 µm (C). 3v, third ventricle; GL, glomerular layer; GR, granular layer; MI, mitral layer.

Figure 4.

Hs6st1^+/− mice showed no defects in olfactory bulbs or in the number and projections of GnRH neurons. (A and B) Perfused adult Hs6st1^+/+ and Hs6st1^+/− brains were dissected, photographed, and showed similar olfactory bulb structure in both genotypes. (C and D) Coronal sections of adult mouse MPOA were immunostained for GnRH; arrowheads indicate examples of GnRH neurons that are normally present both in Hs6st1^+/− and WT mice. (E and F) Immunostaining for GnRH in coronal sections of adult mouse ME shows that GnRH neurons project similarly to the ME in Hs6st1^+/+ and Hs6st1^+/− mice. Scale bars: 3 mm (A and B), 125 µm (C and D), 50 µm (E and F). Arrows demonstrate representative GnRH cell bodies. 3v, third ventricle.

Figure 5.

Peripubertal female Hs6st1^+/− mice show delayed VO whereas young adult male Hs6st1^+/− mice showed normal testes morphology. (A) Age (left graph) and weight (right graph) at the time of the VO in female Hs6st1^+/+ (n = 12) and Hs6st1^+/− (n = 13) mice. Values for littermates are shown in the same color. *P < 0.05. (B and C) Hs6st1^+/+ and Hs6st1^+/− mice have similar testes size (B, upper panels), normal spermatogenesis after H&E staining of paraffin sections (B, lower panels), and similar number of seminiferous tubules (C). Scale bars: 3 mm (B, upper panels), 25 µm (B, lower panels).