This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mechica, J. B. Articles by Latronico, A. C. Search for Related Content PubMed PubMed Citation Articles by Mechica, J. B. Articles by Latronico, A. C.

A Novel Nonsense Mutation in the First Zinc Finger of the Vitamin D Receptor Causing Hereditary 1,25-Dihydroxyvitamin D₃-Resistant Rickets

Division of Endocrinology, Hospital das Clínicas, University of Sao Paulo, Sao Paulo, Brazil

Address all correspondence and requests for reprints to: Dr. Ana C. Latronico, Divisão de Endocrinologia, Hospital das Clínicas da Universidade de Sao Paulo, Caixa Postal 3671, CEP: 01060–970, Sao Paulo, Brazil.

	Abstract

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Hereditary 1,25-dihydroxyvitamin D₃ [1,25-(OH)₂D₃]-resistant rickets (HVDRR) is a rare autosomal recessive disorder resulting in target organ resistance to the active form of vitamin D [1,25-(OH)₂D₃]. Point mutations in the vitamin D receptor (VDR) gene have been identified in HVDRR. We investigated the molecular basis of HVDRR in a Brazilian family with two affected siblings. The propositus is a 12-yr-old boy born to first cousin parents who exhibited the classical pattern of the HVDRR, including early-onset rickets, total alopecia, convulsions, hypocalcemia, secondary hyperparathyroidism, and elevated 1,25-(OH)₂D₃ serum levels. His younger sister also developed clinical and biochemical features of HVDRR at 1 month of age and died at 4 yr of age. Genomic DNA was isolated from peripheral blood of the boy and from dried umbilical cord tissue of his affected sister. We amplified exons 2 and 3 of the VDR gene, which encode the zinc finger DNA-binding domain by PCR. Direct sequencing of the PCR products revealed a homozygous substitution of cytosine for thymine at nucleotide position 88 in exon 2 of the VDR gene in both affected siblings. This point mutation determined the substitution of a stop codon (TGA) for arginine (CGA) at amino acid position 30 at the first zinc finger of the DNA-binding domain of the VDR. This substitution generated a truncated receptor missing 397 residues. The parents and a normal sister were heterozygous for this mutation. In conclusion, we describe a novel nonsense mutation in the first zinc finger of the VDR that generated a severely truncated form of this receptor.

	Introduction

Top Abstract Introduction Subjects and Methods Results Discussion References

 
HEREDITARY 1,25-dihydroxyvitamin D₃ [1,25-(OH)-D₃]-resistant rickets (HVDRR), also known as vitamin D-dependent rickets type II, is a rare autosomal recessive disorder due to target organ resistance to the active form of vitamin D [1,25-(OH)₂D₃]. The clinical manifestations of the syndrome include early-onset rickets, alopecia, hypocalcemia, secondary hyperparathyroidism, and elevated levels of 1,25-(OH)₂D₃ (1, 2, 3). Several studies have demonstrated that the unresponsiveness to 1,25-(OH)₂D₃ is caused by point mutations in the vitamin D receptor (VDR) gene (4, 5, 6, 7, 8, 9). However, a patient with HVDRR and normal complementary DNA sequence has been reported (10).

The VDR, a 48-kDa protein, belongs to the nuclear hormone receptor superfamily, which includes steroid, thyroid, retinoid, and several orphan receptors (11). These receptors have a common structural organization, comprising two main functional domains: a ligand-binding domain and a DNA-binding domain. The DNA-binding domain presents the highest degree of homology among steroid/thyroid/retinoid nuclear receptors. This region folds into two loops or zinc fingers essential for interaction with DNA (11, 12, 13).

The gene encoding the human VDR is contained within approximately 45 kilobases of genomic DNA in the human chromosome 12q 14, and its complex structure consists of nine coding exon sequences interrupted by intronic sequences ranging in size from 0.2–13 kilobases (7, 9, 11). The majority of the mutations found in the human VDR gene were evidenced in exons 2 and 3, which encode the DNA-binding domain (13). Here we report the identification of a novel premature stop mutation in the first zinc finger of the DNA-binding domain of the VDR in a Brazilian family with classical features of HVDRR.

	Subjects and Methods

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Subjects

The propositus of our study is a white 12-yr-old boy who was referred to Hospital das Clínicas, University of Sao Paulo (Sao Paulo, Brazil), at the age of 8 months with a history of classical features of tissue resistance to 1,25-(OH)₂D₃. He developed total alopecia at 1 month of age, and convulsions and rickets at 6 months of age. Biochemical analysis revealed hypocalcemia, slight hypophosphatemia, secondary hyperparathyroidism, and elevated circulating 1,25-(OH)₂D₃ (Table 1). The serum 25-hydroxyvitamin D₃ (25OHD₃) level was elevated; however, it was measured after oral administration of calciferol (100,000 IU/day). Bone x-rays showed classical findings of rickets. The patient showed improvement in the clinical, biochemical, and radiological findings after treatment with daily doses ranging from 3–6 µg 1,25-(OH)₂D₃, 600,000 IU calciferol, and 3.0 g elemental calcium. He presented no more convulsions, and the bone deformities did not develop after treatment. The alopecia remained. His height is 132 cm, 2 SD below the mean for the chronological age, and he is at prepubertal stage. Serum calcium has increased and stabilized around 7.9 mg/dL. His serum levels of alkaline phosphatase and PTH have reached levels close to normal.

View larger version (14K): [in this window] [in a new window]   Figure 1. Family pedigree. Solid symbols indicate homozygotes with clinical disease phenotype. Half-solid symbols indicate heterozygotes. Roman numerals represent generations, and Arabic numerals represent subjects within the generations. A double line indicates a consanguineous marriage. An arrow indicates the propositus. A symbol with a slash indicates the deceased sibling.

The study was approved by the ethics committee of Hospital das Clinicas (Sao Paulo, Brazil), and informed consent was obtained from the parents. Genomic DNA was isolated from peripheral blood from all family members, with the exception of the affected deceased sister, whose DNA was isolated from dried umbilical cord using DNA extraction kits (Nucleon II, Scotlab, Strathclyde, UK). Her umbilical cord had been kept in a handkerchief at room temperature for 11 yr. PCR was used to amplify exons 2 and 3 of the VDR gene. The following pairs of intron flanking primers were used: 2a sense, 5'-AGCTGGCCCTGGCACTGACTCTGCTCT-3'; 2b antisense, 5'-ATGGAAACACC TTGCTTCTTCTCCCTC-3'; 3a sense, 5'-AGGGTGAA GGAGCCGGAAGTTCAGTG AC-3'; and 3b antisense, 5'-CTTTCCCTGACTCCACTTCA GGCCCAA-3' (4). The amino acid numbering system for the VDR used in this report is according to the sequence described by Baker et al. (14).

Symmetric PCR products were used to produce single stranded DNA, which was purified by filtration through a Millipore membrane (Ultrafree-MC Filters, Millipore Corp., Bedford, MA). Intron flanking primers for exons 2 and 3 were used for sequencing by the dideoxy nucleotide chain termination method (Sequenase version 2.0 DNA Polymerase, U.S. Biochemical, Cleveland, OH) in the presence of [³⁵S]deoxy-ATP. The reaction products were run on a 6% polyacrylamide gel (15).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Exons 2 and 3 of the VDR gene were successfully amplified by PCR in all DNA samples studied and exhibited the expected sizes of 265 and 220 bp, respectively, on a 2% agarose gel. Direct sequencing of the PCR product revealed a homozygous substitution of cytosine by thymine at nucleotide position 88 in exon 2 of the open reading frame of the VDR gene from both affected siblings (Fig. 2). This point mutation substituted a stop codon (TGA) for arginine (CGA) at amino acid position 30, resulting in a truncated receptor. The parents and a normal sister were heterozygous for this mutation (Fig. 1). Direct sequencing of exon 3 was entirely normal.

View larger version (34K): [in this window] [in a new window]   Figure 2. VDR nucleotide sequence analysis. Direct sequencing of the complementary DNA of the VDR gene revealed the homozygous substitution of cytosine for thymine at position 88 and resulted in the exchange of Arg30 (CGA) with stop codon (TGA) in the first zinc finger of the DNA domain. The parents and a normal sister were heterozygous for this mutation.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

Serum levels of 1,25-(OH)₂D₃ were above the normal range in the parents, suggesting a mild form of hormone resistance in the obligate heterozygous parents. A similar finding has been reported previously (16, 17).

It is unclear if the position and/or the type of the mutations in VDR gene of patients with HVDRR determine variable phenotypic features. Nonsense and missense mutations located in different domains of VDR have been identified in patients with similar clinical and biochemical features of HVDRR. It is significant that 6 of 15 mutations known to cause HVDRR, including that reported here, involve the amino acid arginine (CGA) (7, 13). The apparent trend for deleterious mutations that occur at arginine residues in VDR was previously suggested to be a consequence of deamination of methylcytosine or cytosine at CpG in genomic DNA, resulting in a mutational hot spot (7).

Despite the molecular evidence of a nonfunctional VDR in these two Brazilian children, the boy with HVDRR had a satisfactory clinical course after conventional therapy. The mechanisms that could explain this phenomenon are not clear at this time. We speculate that an increase in the diffusion process of calcium absorption, the actions of multiple calciferol metabolites, the presence of different isoforms of VDR, and the nongenomic effects of vitamin D on calcium transport (transcaltachia) could represent alternative pathways of calcium transport and 1,25-(OH)₂D₃ actions (11).

In conclusion, we describe a novel nonsense mutation in the first zinc finger of the VDR that generated a severely truncated form of this receptor. The replacement of Arg³⁰ by stop codon in the first zinc finger would affect the capacity of the receptor to bind to the hormone, to bind to the VDR elements properly, and to activate transcription of target genes, representing the molecular basis of HVDRR in this Brazilian family.

	Acknowledgments

 
We thank Miriam Y. Nishi and Maria A. Medeiros for the technical support.

	Footnotes

 
¹ Supported by the FAPESP (96/2020–1).

Received March 11, 1997.

Revised July 31, 1997.

Accepted August 5, 1997.

	References

Top Abstract Introduction Subjects and Methods Results Discussion References

 

1. Brooks MH, Bell NH, Love L, et al. 1978 Vitamin-D dependent rickets type II: resistance of target organs to 1,25-dihydroxyvitamin D. N Engl J Med. 298:996–999.[Abstract]
2. Marx SJ, Spiegel AM, Brown EM, et al. 1978 A familial syndrome of decrease in sensitivity to 1,25-dihydroxyvitamin D. J Clin Endocrinol Metab. 47:1303–1310.[Abstract]
3. Tsuchiya Y, Nobutake M, Cho H, et al. 1980 An unusual form of vitamin D dependent rickets in a child: alopecia and marked end-organ hyposensitivity to biologically active vitamin D. J Clin Endocrinol Metab. 51:685–690.[Abstract]
4. Hughes MR, Malloy PJ, Kieback DE, et al. 1988 Point mutations in the human vitamin D receptor gene associated with hypocalcemic rickets. Science. 242:1702–1705.[Abstract/Free Full Text]
5. Ritchie HH, Hughes MR, Thompson ET, et al. 1989 An ochre mutation in the vitamin D receptor gene causes hereditary 1,25-dihydroxyvitamin D₃-resistant rickets in three families. Proc Natl Acad Sci USA. 86:9783–9787.[Abstract/Free Full Text]
6. Kristjansson K, Rut AR, Hewison M, O’Riordan JLH, Hughes MR. 1993 Two mutations in the hormone binding domain of the vitamin D receptor cause tissue resistance to 1,25 dihydroxyvitamin D₃. J Clin Invest. 92:12–16.
7. Rut AR, Hewison M, Kristjansson K, Luisi B, Hughes MR, O’Riordan JLH. 1994 Two mutations causing vitamin D resistant rickets: modelling on the basis of steroid hormone receptor DNA-binding domain crystal structures. Clin Endocrinol (Oxf). 41:581–590.[Medline]
8. Malloy PJ, Weisman Y, Feldman D. 1994 Hereditary 1,25-dihydroxyvitamin D resistant rickets resulting from a mutation in the vitamin D receptor deoxyribonucleic acid-binding domain. J Clin Endocrinol Metab. 78:313–316.[Abstract]
9. Lin N, Malloy PJ, Sakati N, Al-Ashwal A, Feldman D. 1996 A novel mutation in the deoxiribonucleic acid-binding domain of the vitamin D receptor causes hereditary 1,25-dihydroxyvitamin D-resistant rickets. J Clin Endocrinol Metab. 81:2564–2569.[Abstract]
10. Hewison M, Kristjansson K, Rut AR, et al. 1993 Tissue resistance to 1,25-dihydroxyvitamin D without a mutation of the vitamin D receptor gene. Clin Endocrinol (Oxf). 39:663–670.[Medline]
11. Bouillon R, Okamura WH, Norman AW. 1995 Structure-function relationships in the vitamin D endocrine system. Endocr Rev. 16:200–257.[CrossRef][Medline]
12. Jameson JL. 1996 Steroid/thyroid hormone receptors. Syllabus 1996: introduction to molecular and cellular research. 33–43.
13. Bilezikian JP, Raisz LG, Rodan GA. 1996 Principles of bone biology. San Diego: Academic Press; 1398.
14. Baker AR, McDonnell DP, Hughes M, et al. 1988 Cloning and expression of full-length cDNA encoding human vitamin D receptor. Proc Natl Acad Sci USA. 85:3294–3298.[Abstract/Free Full Text]
15. Kadowaki T, Kadowaki H, Taylor SI. 1990 A nonsense mutation causing decreased levels of insulin receptor mRNA: detection by a simplified technique for direct sequencing of genomic DNA amplified by polymerase chain reaction. Proc Natl Acad Sci USA. 87:658–663.[Abstract/Free Full Text]
16. Yokota I, Takeda E, Ito M, Kabashi H, Saijo T, Kureda Y. 1991 Clinical and biochemical findings in parents of children with vitamin D-dependents rickets type II. J Inherit Metab Dis. 14:231–240.[CrossRef][Medline]
17. Malloy PJ, Eccleshall R, Gross C, Maldergen LV, Bouillon R, Feldman D. 1997 Hereditary vitamin D resistent rickets caused by a novel mutation in the vitamin D receptor that results in decreased affinity for hormone and cellular hyporesponsiveness. J Clin Invest. 99:297–304.[Medline]

This article has been cited by other articles:

				A. Zlotogorski, Z.'e. Hochberg, P. Mirmirani, A. Metzker, D. Ben-Amitai, A. Martinez-Mir, A. A. Panteleyev, and A. M. Christiano Clinical and Pathologic Correlations in Genetically Distinct Forms of Atrichia Arch Dermatol, December 1, 2003; 139(12): 1591 - 1596. [Abstract] [Full Text] [PDF]

				P. J. Malloy, J. W. Pike, and D. Feldman The Vitamin D Receptor and the Syndrome of Hereditary 1,25-Dihydroxyvitamin D-Resistant Rickets Endocr. Rev., April 1, 1999; 20(2): 156 - 188. [Abstract] [Full Text]

This Article Abstract Full Text (PDF) Submit a related Letter to the Editor Purchase Article View Shopping Cart Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Copyright Permission Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Mechica, J. B. Articles by Latronico, A. C. Search for Related Content PubMed PubMed Citation Articles by Mechica, J. B. Articles by Latronico, A. C.