Heparan sulfate 6-O-sulfotransferase 1, a gene involved in extracellular sugar modifications, is mutated in patients with idiopathic hypogonadotrophic hypogonadism

Abstract

Neuronal development is the result of a multitude of neural migrations, which require extensive cell-cell communication. These processes are modulated by extracellular matrix components, such as heparan sulfate (HS) polysaccharides. HS is molecularly complex as a result of nonrandom modifications of the sugar moieties, including sulfations in specific positions. We report here mutations in HS 6-O-sulfotransferase 1 (HS6ST1) in families with idiopathic hypogonadotropic hypogonadism (IHH). IHH manifests as incomplete or absent puberty and infertility as a result of defects in gonadotropin-releasing hormone neuron development or function. IHH-associated HS6ST1 mutations display reduced activity in vitro and in vivo, suggesting that HS6ST1 and the complex modifications of extracellular sugars are critical for normal development in humans. Genetic experiments in Caenorhabditis elegans reveal that HS cell-specifically regulates neural branching in vivo in concert with other IHH-associated genes, including kal-1, the FGF receptor, and FGF. These findings are consistent with a model in which KAL1 can act as a modulatory coligand with FGF to activate the FGF receptor in an HS-dependent manner.

Fig. 1.

Function and sequence of human HS6ST1 and positions of amino acids mutated in patients with IHH. (A) Characteristic disaccharide of HS consisting of a hexuronic acid and a glucosamine residue. The positions within the HS sugars that can be modified by HS-modifying enzymes are indicated. Vertebrate genomes encode a single HS C-5 glucuronyl epimerase (GLCE) and HS 2-O-sulfotransferase (HS2ST) as well as at least three, four, or seven HS 6-O-sulfotransferases (HS6STs, indicated in red), N-deacetylase/sulfotransferases (NDSTs), or HS 3-O-sulfotransferases (HS3STs), respectively (5). (B) Schematic representation of the human HS6ST1 protein with the conserved sulfotransferase domain indicated in blue. 5′PBS and 3′PBS indicate the phosphoadenosyl-phosphosulfate (PAPS) cofactor binding sites. A multiple sequence alignment of two sections of the C terminus is shown with nonsynonymous changes indicated and amino acid positions denoted on the right. Amino acids shaded in black and gray indicate identical and similar residues, respectively.

Fig. 2.

Mutations in HS6ST1 reduce HS6ST1 activity in vitro and in vivo. (A) Relative specific activity of recombinant WT or mutant HS6ST1 variant. Enzymatic activity was determined with two different substrates. Acceptor substrates were used at three different concentrations (50, 250, and 500 μM) previously shown to cover the logarithmic nonsaturated range of HS6ST1 activity in this enzymatic assay (20). All experiments were done in duplicate using equal amounts of protein (Fig. S5). (B–D) Epifluorescent micrographs and schematics of the kal-1–dependent axonal branching phenotype in AIY interneurons. (B) WT morphology of AIY interneurons. (C) Animals overexpressing kal-1 in AIY interneurons display axon branching (indicated in red) (15). (D) Axonal branching is suppressed by a null mutation in the C. elegans hst-6 (15). (E) Transgenic introduction of the human HS6ST1 cDNA in a C. elegans hst-6 null mutant background restores the branches. (F) Quantification of rescue of kal-1–dependent axonal branching in AIY interneurons with human HS6ST1 variants as indicated. Shown are the numbers of transgenic lines for each construct that rescue the phenotype partially, fully, or not at all. Partial rescue was defined as ≥50% of activity and full rescue as ≥95% of activity compared with the mean of human HS6ST1 WT rescuing activity (n = 100–127 per transgenic line). Individual data of transgenic lines are presented in Fig. S2.

Fig. 3.

kal-1 function requires hst-6, FGFR/egl-15, and FGF/egl-17. (A) Schematic of the two FGFR/EGL-15 extracellular splice variants 5A and 5B, which differ by a short sequence between Ig domains 1 and 2 indicated in blue and red. The nonsense alleles n484 and n1456 produce premature stop codons in 5A-specific or all splice variants, respectively. The n1456 allele results in complete loss of function. (B) Quantification of kal-1–dependent axonal branching in AIY interneurons in different mutant backgrounds. Indicated are transgenic lines (#1 = otEx1262, #2 = otEx1266 for the “-5B” strains and #1 = otEx1254 for the “-5A” strain) that exclusively express the egl-15(5A) or egl-15(5B) splice variant, respectively, in an egl-15(n1456) null mutant background (26). The egl-15(n484) allele is an egl-15(5A)–specific null allele (43), egl-15(n1477) is a strong temperature-sensitive allele, and egl-17(n1377) is a null allele (27). (C) Ventral view of the pair of AFD sensory interneurons (anterior is to the left). A kal-1–dependent axonal branch (otIs83) (15) is indicated (Lower, arrowhead) that is not observed in WT animals (Upper). (D) kal-1–dependent branching phenotype in AFD sensory neurons (otIs83) is suppressed by loss of the HS C-5 epimerase (hse-5), hst-2, or hst-6. (E) The kal-1–dependent branching phenotype in AFD sensory neurons (otIs83) is suppressed by loss of the FGFR/egl-15(5A) variant or the FGF/egl-17 ligand [using three null alleles: egl-17(n1377), egl-17(ay6), and egl-17(ay8)] (27). Indicated are transgenic lines (#1 = otEx1262, #2 = otEx1266 for the “-5B” strains and #1 = otEx1254, #2 = otEx1255 for the “-5A” strain) that exclusively express the egl-15(5A) or egl-15(5B) splice variant, respectively, in an egl-15(n1456) null mutant background (26). (F) Suppression of the kal-1–dependent branching phenotype in AFD sensory neurons by loss of the FGF receptor egl-15 is rescued by expression of FGFR/egl-15 specifically in AFD neurons (dzEx480, using the gcy-8 promoter) (42) but not in the hypodermis (dzEx484, using the dpy-7 promoter) (41). (G) Suppression of kal-1–dependent branching in AFD sensory neurons by loss of the FGF ligand egl-17 is partially rescued by expression of FGF/egl-17 in the hypodermis (dzEx472, using the dpy-7 promoter) (41) or in AFD neurons (dzEx483, using the gcy-8 promoter) (41). Representative transgenic lines are shown in F and G (for additional transgenic lines see Fig. S3).

Fig. 4.

Complex inheritance of KS/nIHH in families with HS6ST1 mutations. Pedigrees of seven families with IHH (nIHH/KS). Note that 11 of 11 genotyped individuals with IHH (nIHH/KS) carry one of the loss-of-function mutations in HS6ST1 described here. Phenotypic symbols are listed in the key, and probands are indicated by a red circle and a unique number (compare Table S1 and SI Text for phenotypic details). Available genotypes are indicated below each individual. + denotes a WT allele. Numbers within symbols denote the number of additional siblings.