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

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.

Epigenetic inactivation of heparan sulfate (glucosamine) 3-O-sulfotransferase 2 in lung cancer and its role in tumorigenesis.

BACKGROUND: This study was aimed at investigating the functional significance of heparan sulfate (glucosamine) 3-O-sulfotransferase 2 (HS3ST2) hypermethylation in non-small cell lung cancer (NSCLC). METHODOLOGY/ PRINCIPAL FINDINGS: HS3ST2 hypermethylation was characterized in six lung cancer cell lines, and its clinical significance was analyzed using 298 formalin-fixed paraffin-embedded tissues and 26 fresh-frozen tissues from 324 NSCLC patients. MS-HRM (methylation-specific high-resolution melting) and EpiTYPER(TM) assays showed substantial hypermethylation of CpG island at the promoter region of HS3ST2 in six lung cancer cell lines. The silenced gene was demethylated and re-expressed by treatment with 5-aza-2'-deoxycytidine (5-Aza-dC). A promoter assay also showed the core promoter activity of HS3ST2 was regulated by methylation. Exogenous expression of HS3ST2 in lung cancer cells H460 and H23 inhibited cell migration, invasion, cell proliferation and whereas knockdown of HS3ST2 in NHBE cells induced cell migration, invasion, and cell proliferation in vitro. A negative correlation was observed between mRNA and methylation levels of HS3ST2 in 26 fresh-frozen tumors tissues (ρ = -0.51, P = 0.009; Spearman's rank correlation). HS3ST2 hypermethylation was found in 95 (32%) of 298 primary NSCLCs. Patients with HS3ST2 hypermethylation in 193 node-negative stage I-II NSCLCs with a median follow-up period of 5.8 years had poor overall survival (hazard ratio = 2.12, 95% confidence interval = 1.25-3.58, P = 0.005) compared to those without HS3ST2 hypermethylation, after adjusting for age, sex, tumor size, adjuvant therapy, recurrence, and differentiation. CONCLUSIONS/ SIGNIFICANCE: The present study suggests that HS3ST2 hypermethylation may be an independent prognostic indicator for overall survival in node-negative stage I-II NSCLC.

Synthesis of heparan sulfate with cyclophilin B-binding properties is determined by cell type-specific expression of sulfotransferases.

Cyclophilin B (CyPB) induces migration and adhesion of T lymphocytes via a mechanism that requires interaction with 3-O-sulfated heparan sulfate (HS). HS biosynthesis is a complex process with many sulfotransferases involved. N-Deacetylases/N-sulfotransferases are responsible for N-sulfation, which is essential for subsequent modification steps, whereas 3-O-sulfotransferases (3-OSTs) catalyze the least abundant modification. These enzymes are represented by several isoforms, which differ in term of distribution pattern, suggesting their involvement in making tissue-specific HS. To elucidate how the specificity of CyPB binding is determined, we explored the relationships between the expression of these sulfotransferases and the generation of HS motifs with CyPB-binding properties. We demonstrated that high N-sulfate density and the presence of 2-O- and 3-O-sulfates determine binding of CyPB, as evidenced by competitive experiments with heparin derivatives, soluble HS, and anti-HS antibodies. We then showed that target cells, i.e. CD4+ lymphocyte subsets, monocytes/macrophages, and related cell lines, specifically expressed high levels of NDST2 and 3-OST3 isoforms. Silencing the expression of NDST1, NDST2, 2-OST, and 3-OST3 by RNA interference efficiently decreased binding and activity of CyPB, thus confirming their involvement in the biosynthesis of binding sequences for CyPB. Moreover, we demonstrated that NDST1 was able to partially sulfate exogenous substrate in the absence of NDST2 but not vice versa, suggesting that both isoenzymes do not have redundant activities but do have rather complementary activities in making N-sulfated sequences with CyPB-binding properties. Altogether, these results suggest a regulatory mechanism in which cell type-specific expression of certain HS sulfotransferases determines the specific binding of CyPB to target cells.

Female infertility in grt mice is caused by thyroid hormone deficiency, not by insufficient TPST2 activity in the reproductive organs.

The growth-retarded (grt) mouse has an autosomal recessive hypothyroidism and the female shows lifelong infertility. We previously reported that these mutant phenotypes are caused by a deficiency in the enzymatic activity of tyrosylprotein sulfotransferase-2 (TPST2), and severe thyroid hypogenesis and consequent dwarfism are mainly due to the impairment of the tyrosine sulfation of thyroid-stimulating hormone receptor (TSHR) by TPST2. Although TPST2 is ubiquitously expressed and many proteins are predicted to be tyrosine sulfated and involved in many biological processes, the functional roles of tyrosine sulfation in the reproductive organs remain unclear. These findings tempted us to hypothesize two possible mechanisms underlying the infertility; a deficiency in TPST2 activity in the reproductive organs might cause the infertility in grt mice, or a significant decrease in serum thyroid hormones might impair the normal development of reproductive organs. When mutant female mice were fed a diet supplemented with sufficient thyroid powder to correct their growth retardation, the rate of copulation, pregnancy, and parturition was completely restored. Therefore, we concluded that the infertility in grt female is due to a thyroid hormone deficiency.

Increased hippocampal and cortical beta oscillations in mice deficient for the HNK-1 sulfotransferase.

The HNK-1 carbohydrate is detectable in perineuronal nets around inhibitory neurons in the hippocampus and neocortex. To address the functional contribution of HNK-1 to interneuron function in the adult brain, we recorded EEG and auditory-evoked potential in freely moving mice deficient for HNK-1 sulfotransferase (ST-/- mice) and in wild-type littermates. While ST-/- mice displayed normal theta oscillations, both cortical and hippocampal oscillations within the beta range were enhanced, and gamma oscillations showed an opposite trend. ST-/- mice had amplitudes of auditory-evoked potentials similar to control mice, but the latencies of their hippocampal responses were shorter. Morphological analysis revealed a decreased density of parvalbumin-positive interneurons in the hippocampal CA3 subfield of ST-/- mice, which may contribute to the observed changes in networks oscillations. These findings reveal alterations in ST-/- mice that differ from EEG abnormalities of mice deficient in the HNK-1 carrier molecule tenascin-R.

Aberrant cholesterol transport and impaired steroidogenesis in Leydig cells lacking estrogen sulfotransferase.

Estrogen sulfotransferase (EST) is a cytosolic enzyme that catalyzes the sulfoconjugation and inactivation of estrogens. It is expressed abundantly in the mammalian testes in which it may modulate the activity of locally produced estrogen. We demonstrate here that testicular Leydig cells from mice rendered deficient in EST expression by targeted gene deletion acquire a phenotype of increased cholesterol ester accumulation and impaired steroidogenesis with natural aging or in response to estrogen challenge. Abnormal accumulation of cholesterol ester in the mutant Leydig cells correlated with induced expression of the scavenger receptor type B class I, and cultured EST-deficient but not wild-type Leydig cells avidly uptook high-density lipoprotein cholesterol ester ex vivo. EST-deficient Leydig cells in culture produced 50-70% less testosterone than wild-type cells. This deficiency was reversed by androstenedione but not progesterone supplementation, indicating that reduced activities of 17-alpha-hydroxylase-17, 20-lyase were responsible. This conclusion was corroborated by decreased expression levels of 17-alpha-hydroxylase-17, 20-lyase but not of other key steroidogenic enzymes in the mutant cells. These results suggest that EST plays a physiologic role in protecting Leydig cells from estrogen-induced biochemical lesions and provide an example of critical regulation of tissue estrogen sensitivity by a ligand-transformation enzyme rather than through estrogen receptors.