The molecular basis of hereditary 1,25-dihydroxyvitamin D3 resistant rickets in seven related families.

Abstract

Introduction Hereditary 1,25-dihydroxyvitamin D3 I1,25(OH)2D31 resistant rickets (HVDRR) is an autosomal recessive disease caused by target organ resistance to the action of 1,25(OH)1D3, the active form of the hormone. The defect in target cells is heterogenous and commonly appears to be a mutation in the gene encoding the vitamin D receptor (VDR). We have studied cultured skin fibroblasts and Epstein-Barr virus transformed lymphoblasts of seven family branches of an extended kindred having eight children affected with HVDRR. We have previously shown that cells from three affected children in this group contain an "ochre" nonsense mutation coding for a premature stop codon in exon 7 within the steroid-binding domain of the VDR gene.In The human VDR is a 50-kD protein and the cDNA has recently been cloned and sequenced (17) and found to belong to the steroid-thyroid-retinoic acid receptor superfamily of genes (18). Like the other members of this receptor family, VDR is comprised of at least two functional domains, a steroid hormone-binding domain and a DNA-binding domain (17-23). The steroid-binding domain stretches from amino acid 182 to the COOH-terminus at amino acid 427 and provides the molecule with high affinity, steroid-specific recognition of the 1,25(OH)2D3 metabolite. The DNA-binding domain, comprising amino acids 24-89, is a cysteine-rich region that contains two putative "zinc-fingers" (17). 1,25(OH)2D3 binding to the steroid-binding domain of the receptor "activates" the VDR to a form with high affinity for DNA. The DNA-binding domain of the VDR then binds to a vitamin D response element in the regulatory region of target genes to modulate transcription of responsive genes and thereby mediates hormone action (17)(18)(19)(20)(21)(22)(23).Progress to elucidate the molecular basis of HVDRR began with the demonstration that rodent skin possessed VDR (24, 25) and that cultured human dermal fibroblasts derived from skin biopsies could be used as a model system for the study of the VDR from patients (26). It was soon demonstrated that defects in the VDR were the likely cause of the HVDRR syndrome (5) and that different families exhibited different defects (5-16). An additional useful finding was the demonstration that 1,25(OH)2D3 could induce the enzyme 25-hydroxyvitamin D-24-hydroxylase (24-hydroxylase) in multiple target tissues by a receptor-mediated process (27). It was subsequently found that 24-hydroxylase activity could be induced in cultured skin fibroblasts so that induction of 24-hydroxylase activity could be employed as a bioassay ofthe ability of cultured fibroblasts to respond to 1,25(OH)2D3 (5). Using this bioresponse marker, cells from a variety of patients with HVDRR

Changes in bone metabolic parameters following oral calcium supplementation in an adult patient with vitamin D-dependent rickets type 2A

“…It has been reported that some nonsense mutations result in the total absence or greatly reduced amounts of VDR mRNA [25], but northern blot analysis has shown a normal amount of VDR mRNA in patients with R30X mutations [18]. We confirmed the presence of VDR mRNA in our patient by RT-PCR and real-time PCR using total RNA extracted from peripheral blood.…”

A unique insertion/duplication in the VDR gene that truncates the VDR causing hereditary 1,25-dihydroxyvitamin D-resistant rickets without alopecia

“…In contrast, a truncated form of the VDR was not detected in fibroblasts from a patient with HVDRR due to a premature stop signal at amino acid 295 (Y295X) (lane 3). We have previously demonstrated that the Y295X mutant VDR is undetectable in patient's fibroblasts most likely due to nonsense-mediated decay of the messenger RNA [28].…”

“…Cell debris was removed by centrifugation at 210,000 × g for 30 min at 4°C. The crude cell extracts were incubated with [ 3 H]1,25(OH) 2 D 3 with or without 250-fold excess of radioinert 1,25(OH) 2 D 3 and hydroxylapatite was used to separate bound and free hormone as previously described [28]. For western blotting, samples were denatured in lithium-dodecylsulfate sample buffer for 10 min at 70°C and electrophoresed on 10% NuPAGE gels in MOPS-SDS running buffer (Invitrogen).…”

Los niños del Cauca. I. Descripción de un foco de raquitismo dependiente de la vitamina D, Tipo II.

“…En algunos de los casos publicados en la literatura (17)(18)(19)(20)(21) se encontraron mutaciones puntuales en el gen del RVD que explica su hipofuncionalidad debida a defectos estructurales del RVD.…”

“…Se informó que éste contenía al menos 10 exones cuyos tamaños van desde aproximadamente 29 a3.595 nucleótidos, los cuales junto con los intrones hacen que el gen alcance un tamaño entre 60 y 70 kilobases (18). Varias mutaciones puntuales en el gen del RVD han sido detectadas en pacientes con RDVD II, en las cuales un cambio en un aminoácido altera la estructura terciaria de esta proteína, tanto en el dominio que se liga al DNA, como en el dominio que se liga a la hormona (17)(18)(19)(20)(21). Sin embargo, en un paciente con este tipo de raquitismo no se encontró ninguna alteración en la secuencia de nucleótidos del cDNA del RVD (21).…”