- Email: [email protected]
Normal HPRT coding region in a male with gout due to HPRT deficiency
Molecular Genetics and Metabolism 85 (2005) 78–80 www.elsevier.com/locate/ymgme
Brief communication
Normal HPRT coding region in a male with gout due to HPRT deWciency Paul A. Dawsona,b,¤, Ross B. Gordona, Dianne T. Keougha,c, Bryan T. Emmersona a
Department of Medicine, University of Queensland, Princess Alexandra Hospital, Brisbane, Qld 4102, Australia b School of Biomedical Sciences, University of Queensland, Brisbane, Qld 4072, Australia c Department of Biochemistry and Molecular Biology, University of Queensland, Brisbane, Qld 4072, Australia Received 4 November 2004; received in revised form 10 January 2005; accepted 11 January 2005 Available onlline 16 February 2005
Abstract A deWciency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT; EC 2.4.2.8) is associated with a spectrum of disease that ranges from gouty arthritis (OMIM 300323) to the more severe Lesch–Nyhan syndrome (OMIM 300322). To date, all cases of HPRT deWciency have shown a mutation within the HPRT cDNA. In the present study of an individual with gout due to HPRT deWciency, we found a normal HPRT cDNA sequence. This is the Wrst study to provide an example of HPRT deWciency which appears to be due to a defect in the regulation of the gene. 2005 Elsevier Inc. All rights reserved. Keywords: Hypoxanthine-guanine phosphoribosyltransferase; Lesch–Nyhan syndrome; Urate over-production; Gout; Allopurinol
Introduction Reduced levels of HPRT activity lead to urate overproduction with its common sequelae of hyperuricaemia, gout, and renal insuYciency [1]. This may occur with or without neurological dysfunction and, when this is present, it is referred to as the Lesch–Nyhan syndrome which shows the neurological features of mental retardation, spasticity, choreo-athetosis, and self-mutilation from early childhood [2]. The precise mechanism whereby very low levels of this enzyme activity produce neurological dysfunction has yet to be determined. The human HPRT gene is located on the X-chromosome at position q26-27 and consists of nine exons with a coding sequence of 654 bp [3]. To date, more than 270 disease-associated mutations have been found in the HPRT gene, including deletions, insertions, duplications,
¤
Corresponding author. Fax: +61 7 3365 1766. E-mail address: [email protected] (P.A. Dawson).
1096-7192/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2005.01.005
and a spectrum of single point mutations (reviewed in [4]). All patients with HPRT deWciency have shown a mutation within their HPRT cDNA.
Case report The propositus, a 20-year-old male business manager, developed acute podagra after activity during football practice. During the next three years, he suVered several attacks of gout. He was both neurologically and intellectually normal. His serum urate concentration was 0.70 mmol/L, compared with the reference range of 0.20– 0.42 mmol/L. A 24 h urine urate excretion was 11 mmol on a normal diet and fell to 9.9 mmol on a low purine diet. This indicated a great increase in urate production, since the urine urate on a low purine diet does not normally exceed 3.6 mmol/24 h. These Wndings suggested low levels of HPRT activity as a cause of his urate overproduction, and the activity of HPRT in both his erythrocytes and lymphoblasts was found to be
P.A. Dawson et al. / Molecular Genetics and Metabolism 85 (2005) 78–80
approximately 3% of controls [9]. Renal function was mildly impaired with a creatinine clearance of 1.4 ml/s. There was no family history of gout and none of his four siblings had ever suVered from gout. With allopurinol treatment, the serum urate was restored to normal and acute attacks of gout became rare. HPRT in this patient’s red blood cells and lymphoblasts have the same Km values for guanine and 5-phospho--D-ribosyl-1-pyrophosphate as for the wild type enzyme. This suggested that the enzyme from this patient functioned normally in vitro. Electrophoretic migration indicated that the enzyme protein was of normal size, suggesting that there were no major deletions in the gene. This was all consistent with his normal neurological function, suggesting that the HPRT activity in the developing brain had been suYcient to allow for normal cerebral development. Taken together, these Wndings prompted us to investigate the nature of the molecular abnormality in this patient, denoted HPRTMELBOURNE, as we had done in eight other Australian families with HPRT deWciency (reviewed in [5]).
79
products. DNA fragments containing the HPRT minimal promoter [7] or the polyadenylation signal (Fig. 1F) of control and HPRTMELBOURNE were ampliWed using PCR, denatured and reannealed, and analysed by temperature gradient gel electrophoresis (TGGE) [8].
Results and discussion Markedly reduced HPRT mRNA levels were found in cultured lymphocytes derived from the aVected
Methods Analysis of RNA and cDNA Total RNA was isolated from cultured lymphoblasts, as previously described [6]. For Northern blot analysis, 20 g of total RNA were separated on a 1% agarose formaldehyde gel and transferred to Hybond-C membrane (Amersham). For dot blot analysis, total RNA (3.3, 10, and 33 g) was spotted onto Hybond-C. Membranes were hybridised with a full-length (950 bp) human HPRT cDNA probe [6]. Poly(A)+-RNA was reverse transcribed by using poly(dT) and murine Moloney leukemia virus reverse transcriptase (BRL). cDNA was ampliWed by PCR and used for direct sequencing of the entire HPRT coding region [6]. Sequence analyses were performed on both DNA strands from at least three separate RT-PCRs using RNA isolated from diVerent batches of cultured cells. Analysis of genomic DNA For Southern blotting, 10 g of genomic DNA were digested with BamHI, EcoRI, PstI, and TaqI, transferred onto Hybond-C membrane and hybridised with a full-length HPRT cDNA probe [6]. Each of the nine exons of HPRT, including the splice donor and acceptor sites, were ampliWed using PCR [6]. PCR products were sequenced using Sequenase V2.0 DNA sequencing kit (Amersham) and primers located within the HPRT introns and exons. Sequence analysis was performed on both strands from at least three independent PCR
Fig. 1. Analysis of HPRT mRNA and DNA derived from control (c) and HPRTMELBOURNE (p) lymphoblasts. (A) Northern blot analysis of total RNA, showing the 1.6 kb HPRT transcript (top panel) and ethidium bromide stained 18S and 28S ribosomal RNAs (bottom panel). (B) Total RNA (3.3, 10, and 33 g) spotted onto Hybond-C membrane and hybridised with a full-length HPRT cDNA probe. (C) Densitometric analysis of the dot blot signals in (B), representative of two other experiments with similar data. 䊉 , Control; 䊊, HPRTMELBOURNE. (D) Multiplex PCR ampliWcation of HPRT exons 2–6, 9 (lane 1) and 1, 7–8 (lane 2) from HPRTMELBOURNE. M is molecular size markers (phage DNA cut with HindIII and X174 DNA cut with HaeIII). (E) Southern analysis of BamHI-digested genomic DNA. The full-length HPRT cDNA probe detected 25, 22, and 14.5 kb fragments in control and 22, 18, and 14.5 kb fragments in HPRTMELBOURNE. (F) TGGE analysis of reannealed control and HPRTMELBOURNE PCR products. A 627 bp fragment containing exon 1 and the minimal promoter region (left panel) and a 951 bp fragment containing the 3⬘-untranslated region and polyadenylation signal (right panel) were subjected to electrophoresis at 80 mAmp for 40 and 80 min using a temperature gradient of 20–80 °C. The 951 bp fragment separated into heteroduplex (Ht) and homoduplex (Hm) products, which are indicated on the right.
80
P.A. Dawson et al. / Molecular Genetics and Metabolism 85 (2005) 78–80
individual using Northern blot (Fig. 1A) and dot blot (Fig. 1B) analyses. Quantitation of the dot blot signals (Fig. 1C) indicated that HPRTMELBOURNE had t10% of the control HPRT mRNA level, which is consistent with the low HPRT protein activity (t3% of control [9]) in this patient’s lymphoblasts and red blood cells. These features were associated with a normal HPRT cDNA sequence, together with no changes to the molecular size (Fig. 1D) and genomic DNA sequence of the nine individual HPRT exons and their intron splice donor/acceptor sites (data not shown). Southern analysis of HPRTMELBOURNE genomic DNA (Fig. 1E) identiWed a common 22/18 kb BamHI restriction fragment length polymorphism [10], whereas no changes were detected using EcoRI, PstI, and TaqI restriction endonucleases. TGGE detected a sequence variant in a 951 bp DNA fragment, containing the 3⬘-untranslated region (UTR) and polyadenylation signal of HPRTMELBOURNE (Fig. 1F). Sequence analysis of this DNA fragment identiWed a C to T transition, located 364 nucleotides downstream of the polyadenylation signal. Since this sequence variant is located outside of the HPRT cDNA and is reported as a single nucleotide polymorphism in the NCBI database (http:/ /www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs D 6634995), it would not be expected that this variant would aVect HPRT gene expression. In addition, TGGE (Fig. 1F) and sequence analysis revealed no changes in the 5⬘-UTR and minimal promoter region [7] of HPRTMELBOURNE. Taken together, the present study provides an example of HPRT deWciency which appears to result from a defect in the transcriptional regulation of the gene rather than the previously described deletions or mutations in the coding region. There are a number of possibilities which could result in low levels of HPRT mRNA and activity (reviewed in [7]) in the lymphoblasts from this patient. To our knowledge, this is the Wrst report of a normal HPRT cDNA sequence in a patient with gout due to HPRT deWciency.
Acknowledgments We thank Prof. Martin B. Van Der Weyden (Haematology Department, Alfred Hospital) for assistance with clinical information. This work was supported in part by the Princess Alexandra Hospital Research Foundation and the National Health and Medical Research Council of Australia. References [1] W.N. Kelley, F.M. Rosenbloom, J.F. Henderson, J.E. Seegmiller, A speciWc enzyme defect in gout associated with overproduction of uric acid, Proc. Natl. Acad. Sci. USA 57 (1967) 1735–1739. [2] M. Lesch, W.L. Nyhan, A familial disorder of uric acid metabolism and central nervous system function, Am. J. Med. 36 (1964) 561–570. [3] J.T. Stout, C.T. Caskey, HPRT: Gene structure, expression, and mutation, Annu. Rev. Genet. 19 (1985) 127–148. [4] H.A. Jinnah, L. De Gregorio, J.C. Harris, W.L. Nyhan, J.P. O’Neill, The spectrum of inherited mutations causing HPRT deWciency: 75 new cases and a review of 196 previously reported cases, Mutat. Res. 463 (2000) 309–326. [5] D.G. Sculley, P.A. Dawson, B.T. Emmerson, R.B. Gordon, A review of the molecular basis of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deWciency, Hum. Genet. 90 (1992) 195–207. [6] R.B. Gordon, P.A. Dawson, D.G. Sculley, B.T. Emmerson, C.T. Caskey, R.A. Gibbs, The molecular characterisation of HPRT CHERMSIDE and HPRT COORPAROO: Two Lesch–Nyhan patients with reduced amounts of mRNA, Gene 108 (1991) 299–304. [7] S. Jiralerspong, P.I. Patel, Regulation of the hypoxanthine phosphoribosyltransferase gene: In vitro and in vivo approaches, Proc. Soc. Exp. Biol. Med. 212 (1996) 116–127. [8] P.A. Dawson, D.A. Cochran, B.T. Emmerson, J.P. Kraus, N.P. Dudman, R.B. Gordon, Variable hyperhomocysteinaemia phenotype in heterozygotes for the Gly307Ser mutation in cystathionine beta-synthase, Aust. N.Z.J. Med. 26 (1996) 180–185. [9] D.T. Keough, L.A. McConachie, R.B. Gordon, J. de Jersey, B.T. Emmerson, Human hypoxanthine-guanine phosphoribosyltransferase. Development of a spectrophotometric assay and its use in detection and characterization of mutant forms, Clin. Chim. Acta 163 (1987) 301–308. [10] R.L. Nussbaum, W.E. Crowder, W.L. Nyhan, C.T. Caskey, A three-allele restriction-fragment-length polymorphism at the hypoxanthine phosphoribosyltransferase locus in man, Proc. Natl. Acad. Sci. USA 80 (1983) 4035–4039.
Brief communication
Normal HPRT coding region in a male with gout due to HPRT deWciency Paul A. Dawsona,b,¤, Ross B. Gordona, Dianne T. Keougha,c, Bryan T. Emmersona a
Department of Medicine, University of Queensland, Princess Alexandra Hospital, Brisbane, Qld 4102, Australia b School of Biomedical Sciences, University of Queensland, Brisbane, Qld 4072, Australia c Department of Biochemistry and Molecular Biology, University of Queensland, Brisbane, Qld 4072, Australia Received 4 November 2004; received in revised form 10 January 2005; accepted 11 January 2005 Available onlline 16 February 2005
Abstract A deWciency of the enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT; EC 2.4.2.8) is associated with a spectrum of disease that ranges from gouty arthritis (OMIM 300323) to the more severe Lesch–Nyhan syndrome (OMIM 300322). To date, all cases of HPRT deWciency have shown a mutation within the HPRT cDNA. In the present study of an individual with gout due to HPRT deWciency, we found a normal HPRT cDNA sequence. This is the Wrst study to provide an example of HPRT deWciency which appears to be due to a defect in the regulation of the gene. 2005 Elsevier Inc. All rights reserved. Keywords: Hypoxanthine-guanine phosphoribosyltransferase; Lesch–Nyhan syndrome; Urate over-production; Gout; Allopurinol
Introduction Reduced levels of HPRT activity lead to urate overproduction with its common sequelae of hyperuricaemia, gout, and renal insuYciency [1]. This may occur with or without neurological dysfunction and, when this is present, it is referred to as the Lesch–Nyhan syndrome which shows the neurological features of mental retardation, spasticity, choreo-athetosis, and self-mutilation from early childhood [2]. The precise mechanism whereby very low levels of this enzyme activity produce neurological dysfunction has yet to be determined. The human HPRT gene is located on the X-chromosome at position q26-27 and consists of nine exons with a coding sequence of 654 bp [3]. To date, more than 270 disease-associated mutations have been found in the HPRT gene, including deletions, insertions, duplications,
¤
Corresponding author. Fax: +61 7 3365 1766. E-mail address: [email protected] (P.A. Dawson).
1096-7192/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2005.01.005
and a spectrum of single point mutations (reviewed in [4]). All patients with HPRT deWciency have shown a mutation within their HPRT cDNA.
Case report The propositus, a 20-year-old male business manager, developed acute podagra after activity during football practice. During the next three years, he suVered several attacks of gout. He was both neurologically and intellectually normal. His serum urate concentration was 0.70 mmol/L, compared with the reference range of 0.20– 0.42 mmol/L. A 24 h urine urate excretion was 11 mmol on a normal diet and fell to 9.9 mmol on a low purine diet. This indicated a great increase in urate production, since the urine urate on a low purine diet does not normally exceed 3.6 mmol/24 h. These Wndings suggested low levels of HPRT activity as a cause of his urate overproduction, and the activity of HPRT in both his erythrocytes and lymphoblasts was found to be
P.A. Dawson et al. / Molecular Genetics and Metabolism 85 (2005) 78–80
approximately 3% of controls [9]. Renal function was mildly impaired with a creatinine clearance of 1.4 ml/s. There was no family history of gout and none of his four siblings had ever suVered from gout. With allopurinol treatment, the serum urate was restored to normal and acute attacks of gout became rare. HPRT in this patient’s red blood cells and lymphoblasts have the same Km values for guanine and 5-phospho--D-ribosyl-1-pyrophosphate as for the wild type enzyme. This suggested that the enzyme from this patient functioned normally in vitro. Electrophoretic migration indicated that the enzyme protein was of normal size, suggesting that there were no major deletions in the gene. This was all consistent with his normal neurological function, suggesting that the HPRT activity in the developing brain had been suYcient to allow for normal cerebral development. Taken together, these Wndings prompted us to investigate the nature of the molecular abnormality in this patient, denoted HPRTMELBOURNE, as we had done in eight other Australian families with HPRT deWciency (reviewed in [5]).
79
products. DNA fragments containing the HPRT minimal promoter [7] or the polyadenylation signal (Fig. 1F) of control and HPRTMELBOURNE were ampliWed using PCR, denatured and reannealed, and analysed by temperature gradient gel electrophoresis (TGGE) [8].
Results and discussion Markedly reduced HPRT mRNA levels were found in cultured lymphocytes derived from the aVected
Methods Analysis of RNA and cDNA Total RNA was isolated from cultured lymphoblasts, as previously described [6]. For Northern blot analysis, 20 g of total RNA were separated on a 1% agarose formaldehyde gel and transferred to Hybond-C membrane (Amersham). For dot blot analysis, total RNA (3.3, 10, and 33 g) was spotted onto Hybond-C. Membranes were hybridised with a full-length (950 bp) human HPRT cDNA probe [6]. Poly(A)+-RNA was reverse transcribed by using poly(dT) and murine Moloney leukemia virus reverse transcriptase (BRL). cDNA was ampliWed by PCR and used for direct sequencing of the entire HPRT coding region [6]. Sequence analyses were performed on both DNA strands from at least three separate RT-PCRs using RNA isolated from diVerent batches of cultured cells. Analysis of genomic DNA For Southern blotting, 10 g of genomic DNA were digested with BamHI, EcoRI, PstI, and TaqI, transferred onto Hybond-C membrane and hybridised with a full-length HPRT cDNA probe [6]. Each of the nine exons of HPRT, including the splice donor and acceptor sites, were ampliWed using PCR [6]. PCR products were sequenced using Sequenase V2.0 DNA sequencing kit (Amersham) and primers located within the HPRT introns and exons. Sequence analysis was performed on both strands from at least three independent PCR
Fig. 1. Analysis of HPRT mRNA and DNA derived from control (c) and HPRTMELBOURNE (p) lymphoblasts. (A) Northern blot analysis of total RNA, showing the 1.6 kb HPRT transcript (top panel) and ethidium bromide stained 18S and 28S ribosomal RNAs (bottom panel). (B) Total RNA (3.3, 10, and 33 g) spotted onto Hybond-C membrane and hybridised with a full-length HPRT cDNA probe. (C) Densitometric analysis of the dot blot signals in (B), representative of two other experiments with similar data. 䊉 , Control; 䊊, HPRTMELBOURNE. (D) Multiplex PCR ampliWcation of HPRT exons 2–6, 9 (lane 1) and 1, 7–8 (lane 2) from HPRTMELBOURNE. M is molecular size markers (phage DNA cut with HindIII and X174 DNA cut with HaeIII). (E) Southern analysis of BamHI-digested genomic DNA. The full-length HPRT cDNA probe detected 25, 22, and 14.5 kb fragments in control and 22, 18, and 14.5 kb fragments in HPRTMELBOURNE. (F) TGGE analysis of reannealed control and HPRTMELBOURNE PCR products. A 627 bp fragment containing exon 1 and the minimal promoter region (left panel) and a 951 bp fragment containing the 3⬘-untranslated region and polyadenylation signal (right panel) were subjected to electrophoresis at 80 mAmp for 40 and 80 min using a temperature gradient of 20–80 °C. The 951 bp fragment separated into heteroduplex (Ht) and homoduplex (Hm) products, which are indicated on the right.
80
P.A. Dawson et al. / Molecular Genetics and Metabolism 85 (2005) 78–80
individual using Northern blot (Fig. 1A) and dot blot (Fig. 1B) analyses. Quantitation of the dot blot signals (Fig. 1C) indicated that HPRTMELBOURNE had t10% of the control HPRT mRNA level, which is consistent with the low HPRT protein activity (t3% of control [9]) in this patient’s lymphoblasts and red blood cells. These features were associated with a normal HPRT cDNA sequence, together with no changes to the molecular size (Fig. 1D) and genomic DNA sequence of the nine individual HPRT exons and their intron splice donor/acceptor sites (data not shown). Southern analysis of HPRTMELBOURNE genomic DNA (Fig. 1E) identiWed a common 22/18 kb BamHI restriction fragment length polymorphism [10], whereas no changes were detected using EcoRI, PstI, and TaqI restriction endonucleases. TGGE detected a sequence variant in a 951 bp DNA fragment, containing the 3⬘-untranslated region (UTR) and polyadenylation signal of HPRTMELBOURNE (Fig. 1F). Sequence analysis of this DNA fragment identiWed a C to T transition, located 364 nucleotides downstream of the polyadenylation signal. Since this sequence variant is located outside of the HPRT cDNA and is reported as a single nucleotide polymorphism in the NCBI database (http:/ /www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?rs D 6634995), it would not be expected that this variant would aVect HPRT gene expression. In addition, TGGE (Fig. 1F) and sequence analysis revealed no changes in the 5⬘-UTR and minimal promoter region [7] of HPRTMELBOURNE. Taken together, the present study provides an example of HPRT deWciency which appears to result from a defect in the transcriptional regulation of the gene rather than the previously described deletions or mutations in the coding region. There are a number of possibilities which could result in low levels of HPRT mRNA and activity (reviewed in [7]) in the lymphoblasts from this patient. To our knowledge, this is the Wrst report of a normal HPRT cDNA sequence in a patient with gout due to HPRT deWciency.
Acknowledgments We thank Prof. Martin B. Van Der Weyden (Haematology Department, Alfred Hospital) for assistance with clinical information. This work was supported in part by the Princess Alexandra Hospital Research Foundation and the National Health and Medical Research Council of Australia. References [1] W.N. Kelley, F.M. Rosenbloom, J.F. Henderson, J.E. Seegmiller, A speciWc enzyme defect in gout associated with overproduction of uric acid, Proc. Natl. Acad. Sci. USA 57 (1967) 1735–1739. [2] M. Lesch, W.L. Nyhan, A familial disorder of uric acid metabolism and central nervous system function, Am. J. Med. 36 (1964) 561–570. [3] J.T. Stout, C.T. Caskey, HPRT: Gene structure, expression, and mutation, Annu. Rev. Genet. 19 (1985) 127–148. [4] H.A. Jinnah, L. De Gregorio, J.C. Harris, W.L. Nyhan, J.P. O’Neill, The spectrum of inherited mutations causing HPRT deWciency: 75 new cases and a review of 196 previously reported cases, Mutat. Res. 463 (2000) 309–326. [5] D.G. Sculley, P.A. Dawson, B.T. Emmerson, R.B. Gordon, A review of the molecular basis of hypoxanthine-guanine phosphoribosyltransferase (HPRT) deWciency, Hum. Genet. 90 (1992) 195–207. [6] R.B. Gordon, P.A. Dawson, D.G. Sculley, B.T. Emmerson, C.T. Caskey, R.A. Gibbs, The molecular characterisation of HPRT CHERMSIDE and HPRT COORPAROO: Two Lesch–Nyhan patients with reduced amounts of mRNA, Gene 108 (1991) 299–304. [7] S. Jiralerspong, P.I. Patel, Regulation of the hypoxanthine phosphoribosyltransferase gene: In vitro and in vivo approaches, Proc. Soc. Exp. Biol. Med. 212 (1996) 116–127. [8] P.A. Dawson, D.A. Cochran, B.T. Emmerson, J.P. Kraus, N.P. Dudman, R.B. Gordon, Variable hyperhomocysteinaemia phenotype in heterozygotes for the Gly307Ser mutation in cystathionine beta-synthase, Aust. N.Z.J. Med. 26 (1996) 180–185. [9] D.T. Keough, L.A. McConachie, R.B. Gordon, J. de Jersey, B.T. Emmerson, Human hypoxanthine-guanine phosphoribosyltransferase. Development of a spectrophotometric assay and its use in detection and characterization of mutant forms, Clin. Chim. Acta 163 (1987) 301–308. [10] R.L. Nussbaum, W.E. Crowder, W.L. Nyhan, C.T. Caskey, A three-allele restriction-fragment-length polymorphism at the hypoxanthine phosphoribosyltransferase locus in man, Proc. Natl. Acad. Sci. USA 80 (1983) 4035–4039.