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A 5-methylcytosine hotspot responsible for the prevalent HSD17B10 mutation
Gene 515 (2013) 380–384
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
A 5-methylcytosine hotspot responsible for the prevalent HSD17B10 mutation Song-Yu Yang a,⁎, Carl Dobkin b, Xue-Ying He a, Manfred Philipp c, W. Ted Brown b a b c
Department of Developmental Biochemistry, NYS Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314, USA Department of Human Genetics, NYS Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314, USA Department of Chemistry, Lehman College, City University of New York, Bronx, NY 10486, USA
a r t i c l e
i n f o
Article history: Accepted 3 December 2012 Available online 22 December 2012 Keywords: Inherited metabolic disease Gene mutation Methylation analysis Imbalance of neurosteroid metabolism Multifunctional enzyme
a b s t r a c t Approximately half of the cases of hydroxysteroid (17β) dehydrogenase X (HSD10) deficiency are due to a missense C>T mutation in exon 4 of the HSD17B10 gene. The resulting HSD10 (p.R130C) loses most or all catalytic functions, and the males with this mutation have a much more severe clinical phenotype than those carrying p.V65A, p.L122V, or p.E249Q mutations. We found that the mutated cytosine which is +2259 nucleotide from the ATG of the gene, is >90% methylated in both the active and inactive X chromosomes in two normal females as well as in the X chromosome of a normal male. Since 5-methylcytosine is prone to conversion to thymine by deamination, the methylation of this cytosine in normal X chromosomes provides an explanation for the prevalence of the p.R130C mutation among patients with HSD10 deficiency. The substitution of arginine for cysteine eliminates several hydrogen bonds and reduces the van der Waals interaction between HSD10 subunits. The resulting disruption of protein structure impairs some if not all of the catalytic and non-enzymatic functions of HSD10. A meta-analysis of residual HSD10 activity in eight patients with the p.R130C mutation showed an average 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) activity of only 6 (±5) % of the normal control level. This is significantly lower than in cells of patients with other, clinically milder mutations and suggests that the loss of HSD10/MHBD activity is a marker for the disorder. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Hydroxysteroid (17β) dehydrogenase X deficiency also known as 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency, is a relatively rare X linked neurological syndrome resulting from a missense mutation in the HSD17B10 gene (Ofman et al., 2003; Seaver et al., 2011; Yang et al., 2009). The urine organic acid profile of affected individuals is characterized by high levels of isoleucine metabolites, namely 2-methyl-3-hydroxy butyrate and tiglylglycine, which indicate a blockade of the isoleucine and branched-chain fatty acid degradation pathway (Cazorla et al., 2007; Ensenauer et al., 2002; GarcíaVilloria et al., 2009, 2010; He and Yang, 2006; Olpin et al., 2002; Perez-Cerda et al., 2005; Poll-The et al., 2004; Sass et al., 2004; Sutton et al., 2003; Zschocke et al., 2000). Although the accumulation of these isoleucine and branched-chain fatty acid metabolites (He and Yang, 2006) may not be completely benign (Rossa et al., 2005), it does not cause neurological symptoms in patients with a different blockade of such pathways, β-KT thiolase deficiency (Fukao et al., 2001) that also shows an accumulation of these metabolites. Thus, the pathophysiology of HSD10 deficiency must lie in the disruption of other
Abbreviations: HSD10, hydroxysteroid (17beta) dehydrogenase X; MHBD, 2-methyl-3hydroxybutyryl-CoA dehydrogenase. ⁎ Corresponding author. Tel.: +1 718 494 5317; fax: +1 718 698 7916. E-mail address: [email protected] (S.-Y. Yang). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.12.064
catalytic or non-catalytic HSD10 activities, e.g., an imbalance of neurosteroid metabolism (Yang et al., 2009, 2011). The diagnosis of this disease is sometime difficult because of subtle or intermittent metabolite secretion (especially in female) unless the HSD10/MHBD activity in patients' cells is determined to be lower than the normal level (García-Villoria et al., 2009). There are at least nine reported and clinically related missense mutations in the HSD17B10 but about half of the patients with HSD10 deficiency carry a potent c.388C>T transition. Male patients with this mutation have a severe clinical phenotype. This mutated gene generates a mutant protein HSD10(p.R130C), whose residual activity appears to be negligible (Ofman et al., 2003; Yang et al., 2009). One group, however, reported (Rauschenberger et al., 2010) that the recombinant mutant protein, HSD10(p.R130C), exhibits up to 64% of residual MHBD activity of the wild type enzyme, and proposed a non-enzymatic theory of HSD10 deficiency. They also suggested that the prevalence of the c.388C>T mutation among HSD10 deficiency patients is due to the lethality of other HSD10 mutations (Rauschenberger et al., 2010). Here we report that a 5-methylcytosine is present in both active and inactive X chromosomes at +2259 nucleotide from the initiation ATG of the HSD17B10 gene. The presence of this hypermutable nucleotide at this position explains the prevalence of the p.R130C mutation among HSD10 deficiency patients. Our analysis of results from different laboratories suggests that cells of HSD10 deficiency patients carrying the c.388C>T transition at the HSD17B10 gene, have minimal
S.-Y. Yang et al. / Gene 515 (2013) 380–384
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Fig. 1. Methylation analysis of the exon 4 of the HSD17B10 gene. The bisulfite sequencing of the chromosome DNA from a normal male is displayed in (A); and that from a normal female in (B). The ordinate shows the relative light unit detected after dispensation of each nucleotide substrate. The abscissa shows the dispensation order (“e” and “s” are controls). The “bisulfite” line shows the predicted sequence after modification. “Y” represents C or T. Lines connecting the dispensed nucleotide to the bisulfite sequence indicate how the light signal represents the modified sequence. The height of the signal is proportional to the number of nucleotides at that position.
residual HSD10 activity which is consistent with the highly disruptive nature of the amino acid substitution in the p.R130C mutant protein.
2. Materials and methods 2.1. Chromosomal DNA Chromosomal DNA was isolated from blood samples of normal individuals (one male and two females) with the FlexiGene kit (Qiagen, Volencia) and used as the template for the HSD17B10 gene-specific methylation analysis. This study was approved by the Institutional Review Board of NYS Institute for Basic Research in Developmental Disabilities and the human DNA samples were obtained in conformance with their guidelines and included the acquisition of written informed consent for genetic testing.
2.2. Bisulfite sequencing Bisulfite modification of human chromosomal DNA and pyrosequencing analysis (Sheridan et al., 2011; Tost et al., 2003) of a 118 bp segment of the exon 4 of the HSD17B10 gene (AF037438) was done by the EpigenDx, Inc. (http://www.epigendx.com) using a pair of primers designed by EpigenDx, Inc., ADS2501FP and biotin-labeled ADS2501RPB, and the PSQ™ 96HS system. Pyrosequencing dispensation order was ATCGCTGTGCTG.1
2.3. Enzymatic data analysis Enzymatic activity data were gathered and analyzed by reference to kinetic monographs (Segel, 1993). The statistic evaluation was accomplished by use of the Wilcoxon rank-sum test (Conover and Iman, 1981). 1 For experimental details the ASSAY DESIGN REPORT of the EpigenDx Inc. will be provided upon request.
2.4. Tertiary structural model of HSD10(p.R130C) Structural differences between the HSD10(p.R130C) mutant and the wild-type HSD10 were ascertained by bioinformatics analysis. The published crystal structure of human HSD10 was employed as the template structure (Kissinger et al., 2004). Data were extracted from a pdb file (1U7T) of the X-ray coordinates available from the RCSB Protein Data bank (www.resb.org) using DeepView/Swiss-pdb Viewer 4.04 (Guex and Peitsch, 1997). Substitution of the mutant amino acid was made with the “Mutating Amino-Acids” function in DeepView/Swiss-pdb Viewer 4.04. 3. Results 3.1. 5-Methylcytosine at nucleotide + 2259 from ATG Because the cytosine (C) at nucleotide + 2259 from the initiation ATG of the HSD17B10gene (He et al., 1998) has a 3′ flanking guanine (G), it is a potential target for methylation. Surprisingly we found that it is virtually completely (93 ± 5%) methylated not only on the inactive X chromosome of the two female DNAs, but also on the active X chromosome as well. DNA from a normal male also showed >90% methylation of this cytosine (Fig. 1). 3.2. Structural changes due to the substitution of cysteine for arginine at residue 130 Residue 130 is located in the α-helix E2 of HSD10 (Kissinger et al., 2004). Since arginine 130 was present at the interface of subunits A and B as well as that of subunits C and D, its side chain takes part in the stabilization of the dimer (Fig. 2 upper panel). Its guanidinium group forms hydrogen bonds with the side chain of asparagine 127 in the same α-helix. This positively charged group also forms a hydrogen-bonded ion-pair to the side chain of glutamate 68 in α-helix D and a hydrogen bond with the carbonyl oxygen of histidine 109 of the neighboring subunit. The p.R130C mutation eliminates three hydrogen bonds and the volume of the substituted cysteine side chain is much smaller than that of arginine (Fig. 2 lower panel).
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Fig. 2. Structural changes resulted from the substitution of cysteine for arginine at residue 130 of HSD10. The upper image showed that the arginine 130 (greenish blue) of subunit A (yellow) is located at the interface of subunits A and B (blue). A bound NAD+ was in black. The lower image showed that the guanidinium group of arginine 130 of subunit A forms a number of intra- and inter-subunit hydrogen bonding (dashed green line) to carboxyl or carbonyl oxygens (A), and that the mutation would eliminate several hydrogen bonds and the side chain of cystein (surfur in yellow) is much smaller than that of arginine (B). In the lower image nitrogen was shown in blue, hydrogen in greenish blue, and oxygen in red. For clarity, some amino acid residues in the protein have been rendered invisible.
3.3. Reduction of most HSD10 activity by the mutation p.R130C The residual HSD10/MHBD activity in cultured cells from HSD10 deficiency patients as measured in a number of laboratories is listed in
Table 1. Quantitative data are currently available for eight patients carrying the c.388C>T mutation in the HSD17B10 gene (Ensenauer et al., 2002; García-Villoria et al., 2009; Ofman et al., 2003; Perez-Cerda et al., 2005; Sass et al., 2004; Sutton et al., 2003; Yang et al., 2009; Zschocke
Table 1 Residual HSD10/MHBD activity in cells of HSD10 deficiency patients. Patient
Sex
Mutation
Residual activity (%)
Substrates
1 2 3 4 5 6 7 8 9 10 11
M M F M M M M M M M M
p.R130C p.R130C p.R130C p.R130C p.R130C p.R130C p.R130C p.R130C p.L122V p.E249Q p.V65A
Undetectable 8.5 14 8.5 Absent 11 3.6 3.6 25.4 12 50.5
2-Methyl-3-hydroxybutyryl-CoA; 2-Methylacetoacetyl-CoA; NADH 2-Methylacetoacetyl-CoA; NADH 2-Methylacetoacetyl-CoA; NADH 2-Methyl-3-hydroxybutyryl-CoA; 2-Methylacetoacetyl-CoA; NADH 2-Methyl-3-hydroxybutyryl-CoA; 2-Methyl-3-hydroxybutyryl-CoA; 2-Methylacetoacetyl-CoA; NADH 2-Methylacetoacetyl-CoA; NADH 2-Methyl-3-hydroxybutyryl-CoA;
Ref. NAD+
NAD+ NAD+ NAD+
NAD+
Sutton et al. (2003); Yang et al. (2009) Ofman et al. (2003); Zschocke et al. (2000) Ensenauer et al. (2002); Ofman et al. (2003) Ensenauer et al. (2002); Ofman et al. (2003) Sass et al. (2004) Perez-Cerda et al. (2005) Cazorla et al. (2007) García-Villoria et al. (2009) Ofman et al. (2003) Olpin et al. (2002); Yang et al. (2009) Seaver et al. (2011)
S.-Y. Yang et al. / Gene 515 (2013) 380–384
et al., 2000). The cultured patient cells were either fibroblasts or lymphoblastoid cells, and the enzymatic assays were performed in either forward or backward direction depending on the substrate used by the particular laboratory. Based on the data in Table 1, the average level of residual HSD10/MHBD activity in patients' cells carrying mutation p.R130C is estimated at 6 ± 5% of the normal control. This is significantly lower than the residual HSD10/MHBD activity reported for HSD10 deficiency patients with p.V65A, p.L122V or p.E249Q mutation (pb 0.05) (see Table 1) all of which are associated with a milder clinical phenotype (Ofman et al., 2003; Seaver et al., 2011; Yang et al., 2009).
4. Discussion The identification of a 5-methylated CpG in the exon 4 of the HSD17B10 gene (see Fig. 1) explains the high prevalence of the p.R130C mutation among HSD10 deficiency patients. 5-Methylcytosine (5mC) is produced by post-synthetic modification of cytosine residue, and it is a hypermutable site because it can undergo spontaneous or enzyme catalyzing deamination to thymine (Holliday and Grigg, 1993; Morgan et al., 2004). Deamination of unmethylated cytosine produces uracil which is recognized and removed efficiently by uracil DNA glycosylase (Int. Hum. Genome Seq. Cons., 2001). However, 5mC deaminates at a higher rate and the product of this reaction is a thymine. The repair of G–T mismatch is not highly efficient. Thus, the 5mC>T transition occurs about 10 times more frequently than other transitions (Holliday and Grigg, 1993). For this reason a 5mC>T transition at this nucleotide will occur more frequently than other mutations in this gene. The idea that “most other mutations in the HSD17B10 gene are not observed because they are incompatible with life” (Rauschenberger et al., 2010) is unlikely to be the explanation for the prevalence of p.R130C mutation, especially since patients carrying mutations p.V65A, p.L122V or p.E249Q have a milder clinical phenotype and higher residual HSD10 activity (Ofman et al., 2003; Seaver et al., 2011; Yang et al., 2009). A missense mutation in the HSD17B10 gene resulted in HSD10 deficiency (OMIM#300438) whereas a silent mutation caused the mental retardation, X-linked, and abnormal behavior (MRXS10) (OMIM#300220) (Lenski et al., 2007; Reyniers et al., 1999; Yang et al., 2011) (Fig. 3). To date 27 cases of HSD10 deficiency have been described. The manifestation of most female patients is mild intellectual disability or borderline learning disability (García-Villoria et al., 2009, 2010) because females have two X chromosomes. The data of enzymatic activity are not available for most female patients. Also, the mosaicism of the X-inactivation of HSD17B10 gene (He et al., 2011) makes the situation more complicated. At the present time it is not
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feasible to include such cases in any discussions of the genotype– phenotype relationship. Male patients especially those with the c.388C>T mutation show a severe clinical phenotype (Ensenauer et al., 2002; García-Villoria et al., 2009; Ofman et al., 2003; Perez-Cerda et al., 2005; Sass et al., 2004; Sutton et al., 2003; Yang et al., 2009; Zschocke et al., 2000). This mutation results in a mutant HSD10 (p.R130C), in which arginine 130 is replaced by cysteine. As a result, the enzyme structure is seriously disturbed (Fig. 2). Since the side chain of cysteine is much smaller than that of arginine, the van der Waals interaction between neighboring subunits is greatly reduced at that location. HSD10, the product of the HSD17B10 gene, is a multifunctional enzyme (Yang et al., 2005). It has not only multiple enzymatic activities but also non-enzymatic functions, e.g., its ability to form a complex with (guanine-9-)methyltransferase or estrogen receptor α (ERα) (Yang et al., 2011). The observations from the rescue experiments in Xenopus embryos (Rauschenberger et al., 2010) suggest that enzymatic or non-enzymatic functions of HSD10 are essential to cell survival, growth, and differentiation. HSD10 deficiency, an inherited metabolic disease, was considered an inborn error of isoleucine and branched-chain fatty acids metabolism (García-Villoria et al., 2009; Korman, 2006). The HSD10/MHBD activity in patients' cells was diminished by mutations in the HSD17B10 gene (see Table 1). It was later realized that a blockade of the isoleucine degradation pathway does not play a major role in the pathogenesis of this disease, and it was proposed that an imbalance of neurosteroid metabolism may affect the brain function (Yang et al., 2009). Although the MHBD activity may not be directly related with the severity of the disease, an MHBD assay is still required for the clinical diagnosis in combination with the urine organic acid profile, clinical manifestation (e.g., intellectual disability, learning disability and/or microcephaly) and the identification of a mutation in the HSD17B10 gene (Ofman et al., 2003; Seaver et al., 2011; Yang et al., 2009). Conflict of interest The authors declare no conflict of interest. Acknowledgments This study was partially supported by the New York State Office for People with Developmental Disabilities. We also thank Dr. Yu-Xiao Yang for important advice in the statistical evaluation. References
Fig. 3. Missense mutations of HSD10 result in HSD10 deficiency (OMIM#300438) whereas a silent mutation causes MRXS10 (OMIM#300220). Female cases are indicated by a red bar while male cases are in blue. The bar height is approximately proportional to the number of cases. The N and C represent the N- and C-terminals of HSD10, respectively.
Cazorla, M.R., Verdu, A., Perez-Cerda, C., Ribes, A., 2007. Neuroimage findings in 2-methy3-hydroxybutyryl-CoA dehydrogenase deficiency. Pediatr. Neurol. 36, 264–267. Conover, W.J., Iman, R.L., 1981. Rank transformations as a bridge between parametric and nonparametric statistics. Am. Stat. 35, 124–129. Ensenauer, R., et al., 2002. Clinical variability in 3-hydroxy-2-methylbutyryl-CoA dehydrogenase deficiency. Ann. Neurol. 51, 656–659. Fukao, T., Scriver, C.R., Kondo, N., 2001. The clinical phenotype and outcome of mitochondrial acetoacetyl-CoA thiolase deficiency (β-ketothiolase or T2 deficiency) in 26 enzymatically proved and mutation-defined patients. Mol. Genet. Metab. 72, 109–114. García-Villoria, J., et al., 2009. Study of patients and carriers with 2-methyl-3hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency: difficulties in the diagnosis. Clin. Biochem. 1–2, 27–33. Garcia-Villoria, J., et al., 2010. X-inactivation of HSD17B10 revealed by cDNA analysis in two female patients with 17β-hydroxysteroid dehydrogenase 10 deficiency. Eur. J. Hum. Genet. 18, 1353–1355. Guex, N., Peitsch, M.C., 1997. SWISS-MODEL and the swiss-Pdb viewer: an environment for comparative protein modeling. Electrophoresis 18, 2714–2723. He, X.Y., Yang, S.Y., 2006. Roles of type 10 17beta-hydroxysteroid dehydrogenase in intracrinology and metabolism of isoleucine and fatty acids. Endocrinol. Metab. Immun. Dis. Drug Targets 6, 95–102. He, X.Y., Schulz, H., Yang, S.Y., 1998. A human brain L-3-hydroxyacyl-CoA dehydrogenase is identical with an amyloid β-peptide binding protein involved in Alzheimer's disease. J. Biol. Chem. 273, 10741–10746.
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He, X.Y., Dobkin, C., Yang, S.Y., 2011. Does the HSD17B10 gene escape from X-inactivation? Eur. J. Hum. Genet. 19, 123–124. Holliday, R., Grigg, G., 1993. DNA methylation and mutation. Mutat. Res. 285, 61–69. International Human Genome Sequencing Consortium, 2001. Initial sequencing and analysis of the human genome. Nature 409, 860–921. Kissinger, C.R., et al., 2004. Crystal structure of human ABAD/HSD10 with a bound inhibitor: implications for design of Alzheimer's disease therapeutics. J. Mol. Biol. 342, 943–952. Korman, S.H., 2006. Inborn errors of isoleucine degradation: a review. Mol. Genet. Metab. 89, 289–299. Lenski, C., et al., 2007. The reduced expression of the HADH2 protein causes X-linked mental retardation, choreoathetosis, and abnormal behavior. Am. J. Hum. Genet. 80, 372–377. Morgan, H.D., Dean, W., Coker, H.A., Reik, W., Petersen-Mahrt, K., 2004. Activationinduced cytidine deaminase deaminates 5-methylcytosine in DNA and is expressed in pluripotent tissues. Implications for epigenetic reprogramming. J. Biol. Chem. 279, 52353–52360. Ofman, R., et al., 2003. 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency is caused by mutations in the HADH2 gene. Am. J. Hum. Genet. 72, 1300–1307. Olpin, S.E., et al., 2002. 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency in a 23-year-old man. J. Inherit. Metab. Dis. 25, 477–482. Perez-Cerda, C., et al., 2005. 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) deficiency: an X-linked inborn error of isoleucine metabolism that may mimic a mitochondrial disease. Pediatr. Res. 58, 488–491. Poll-The, B.T., et al., 2004. Spastic diplegia and periventricular white matter abnormalities in 2-methy-3-hydroxybutyryl-CoA dehydrogenase deficiency, a defect of isoleucine metabolism: differential diagnosis with hypoxic-ischemic brain diseases. Mol. Genet. Metab. 81, 295–299. Rauschenberger, K., et al., 2010. A non-enzymatic function of 17β-hydroxysteroid dehydrogenase type 10 is required for mitochondrial integrity and cell survival. EMBO Mol. Med. 2, 1–12.
Reyniers, E., et al., 1999. A new neurological syndrome with mental retardation, choreoathetosis, and abnormal behavior maps to chromosome Xp11. Am. J. Hum. Genet. 65, 1406–1412. Rossa, R.B., et al., 2005. Inhibition of energy metabolism by 2-methylacetoacetate and 2-methyl-3-hydroxybutyrate in cerebral cortex of developing rats. J. Inherit. Metab. Dis. 28, 501–515. Sass, J.O., Forstner, R., Sperl, W., 2004. 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency: impaired catabolism of isoleucine presenting as neurodegenerative disease. Brain Dev. 26, 12–14. Seaver, L.H., et al., 2011. A novel mutation in the HSD17B10 gene of a 10-year-old boy with refractory epilepsy, choreoathetosis and learning disability. PLoS One 6, e27348. Segel, I.H., 1993. Enzyme kinetics, behavior and analysis of rapid equilibrium and steadystate enzyme systems. Wiley Classics Library ed. Wiley Interscience, New York. Sheridan, S.D., et al., 2011. Epigenetic characterization of the FMR1 gene and aberrant neurodevelopment in human induced pluripotent stem cell models of fragile X syndrome. PLoS One 6, e26203. Sutton, V.R., O'Brien, W.E., Clark, G.D., Kim, J., Wanders, R.J., 2003. 3-Hydroxy-2methylbutyryl-CoA dehydrogenase deficiency. J. Inherit. Metab. Dis. 26, 69–71. Tost, J., Dunker, J., Gut, I.G., 2003. Analysis and quantification of multiple methylation variable positions in CpG islands by pyrosequencing. Biotechniques 35, 152–156. Yang, S.Y., He, X.Y., Schulz, H., 2005. Multiple functions of type 10 17betahydroxysteroid dehydrogenase. Trends Endocrinol. Metab. 16, 167–175. Yang, S.Y., et al., 2009. Mental retardation linked to mutations in the HSD17B10 gene interfering with neurosteroid and isoleucine metabolism. Proc. Natl. Acad. Sci. U.S.A. 106, 14820–14824. Yang, S.Y., He, X.Y., Miller, D., 2011. Hydroxysteroid (17β) dehydrogenase X in human health and disease. Mol. Cell. Endocrinol. 343, 1–6. Zschocke, J., et al., 2000. Progressive infantile neurodegeneration caused by 2-methyl-3hydroxybutyryl-CoA dehydrogenase deficiency: a novel inborn error of branchedchain fatty acid and isoleucine metabolism. Pediatr. Res. 48, 852–855.
Contents lists available at SciVerse ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
Short Communication
A 5-methylcytosine hotspot responsible for the prevalent HSD17B10 mutation Song-Yu Yang a,⁎, Carl Dobkin b, Xue-Ying He a, Manfred Philipp c, W. Ted Brown b a b c
Department of Developmental Biochemistry, NYS Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314, USA Department of Human Genetics, NYS Institute for Basic Research in Developmental Disabilities, Staten Island, NY 10314, USA Department of Chemistry, Lehman College, City University of New York, Bronx, NY 10486, USA
a r t i c l e
i n f o
Article history: Accepted 3 December 2012 Available online 22 December 2012 Keywords: Inherited metabolic disease Gene mutation Methylation analysis Imbalance of neurosteroid metabolism Multifunctional enzyme
a b s t r a c t Approximately half of the cases of hydroxysteroid (17β) dehydrogenase X (HSD10) deficiency are due to a missense C>T mutation in exon 4 of the HSD17B10 gene. The resulting HSD10 (p.R130C) loses most or all catalytic functions, and the males with this mutation have a much more severe clinical phenotype than those carrying p.V65A, p.L122V, or p.E249Q mutations. We found that the mutated cytosine which is +2259 nucleotide from the ATG of the gene, is >90% methylated in both the active and inactive X chromosomes in two normal females as well as in the X chromosome of a normal male. Since 5-methylcytosine is prone to conversion to thymine by deamination, the methylation of this cytosine in normal X chromosomes provides an explanation for the prevalence of the p.R130C mutation among patients with HSD10 deficiency. The substitution of arginine for cysteine eliminates several hydrogen bonds and reduces the van der Waals interaction between HSD10 subunits. The resulting disruption of protein structure impairs some if not all of the catalytic and non-enzymatic functions of HSD10. A meta-analysis of residual HSD10 activity in eight patients with the p.R130C mutation showed an average 2-methyl-3-hydroxybutyryl-CoA dehydrogenase (MHBD) activity of only 6 (±5) % of the normal control level. This is significantly lower than in cells of patients with other, clinically milder mutations and suggests that the loss of HSD10/MHBD activity is a marker for the disorder. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Hydroxysteroid (17β) dehydrogenase X deficiency also known as 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency, is a relatively rare X linked neurological syndrome resulting from a missense mutation in the HSD17B10 gene (Ofman et al., 2003; Seaver et al., 2011; Yang et al., 2009). The urine organic acid profile of affected individuals is characterized by high levels of isoleucine metabolites, namely 2-methyl-3-hydroxy butyrate and tiglylglycine, which indicate a blockade of the isoleucine and branched-chain fatty acid degradation pathway (Cazorla et al., 2007; Ensenauer et al., 2002; GarcíaVilloria et al., 2009, 2010; He and Yang, 2006; Olpin et al., 2002; Perez-Cerda et al., 2005; Poll-The et al., 2004; Sass et al., 2004; Sutton et al., 2003; Zschocke et al., 2000). Although the accumulation of these isoleucine and branched-chain fatty acid metabolites (He and Yang, 2006) may not be completely benign (Rossa et al., 2005), it does not cause neurological symptoms in patients with a different blockade of such pathways, β-KT thiolase deficiency (Fukao et al., 2001) that also shows an accumulation of these metabolites. Thus, the pathophysiology of HSD10 deficiency must lie in the disruption of other
Abbreviations: HSD10, hydroxysteroid (17beta) dehydrogenase X; MHBD, 2-methyl-3hydroxybutyryl-CoA dehydrogenase. ⁎ Corresponding author. Tel.: +1 718 494 5317; fax: +1 718 698 7916. E-mail address: [email protected] (S.-Y. Yang). 0378-1119/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2012.12.064
catalytic or non-catalytic HSD10 activities, e.g., an imbalance of neurosteroid metabolism (Yang et al., 2009, 2011). The diagnosis of this disease is sometime difficult because of subtle or intermittent metabolite secretion (especially in female) unless the HSD10/MHBD activity in patients' cells is determined to be lower than the normal level (García-Villoria et al., 2009). There are at least nine reported and clinically related missense mutations in the HSD17B10 but about half of the patients with HSD10 deficiency carry a potent c.388C>T transition. Male patients with this mutation have a severe clinical phenotype. This mutated gene generates a mutant protein HSD10(p.R130C), whose residual activity appears to be negligible (Ofman et al., 2003; Yang et al., 2009). One group, however, reported (Rauschenberger et al., 2010) that the recombinant mutant protein, HSD10(p.R130C), exhibits up to 64% of residual MHBD activity of the wild type enzyme, and proposed a non-enzymatic theory of HSD10 deficiency. They also suggested that the prevalence of the c.388C>T mutation among HSD10 deficiency patients is due to the lethality of other HSD10 mutations (Rauschenberger et al., 2010). Here we report that a 5-methylcytosine is present in both active and inactive X chromosomes at +2259 nucleotide from the initiation ATG of the HSD17B10 gene. The presence of this hypermutable nucleotide at this position explains the prevalence of the p.R130C mutation among HSD10 deficiency patients. Our analysis of results from different laboratories suggests that cells of HSD10 deficiency patients carrying the c.388C>T transition at the HSD17B10 gene, have minimal
S.-Y. Yang et al. / Gene 515 (2013) 380–384
381
Fig. 1. Methylation analysis of the exon 4 of the HSD17B10 gene. The bisulfite sequencing of the chromosome DNA from a normal male is displayed in (A); and that from a normal female in (B). The ordinate shows the relative light unit detected after dispensation of each nucleotide substrate. The abscissa shows the dispensation order (“e” and “s” are controls). The “bisulfite” line shows the predicted sequence after modification. “Y” represents C or T. Lines connecting the dispensed nucleotide to the bisulfite sequence indicate how the light signal represents the modified sequence. The height of the signal is proportional to the number of nucleotides at that position.
residual HSD10 activity which is consistent with the highly disruptive nature of the amino acid substitution in the p.R130C mutant protein.
2. Materials and methods 2.1. Chromosomal DNA Chromosomal DNA was isolated from blood samples of normal individuals (one male and two females) with the FlexiGene kit (Qiagen, Volencia) and used as the template for the HSD17B10 gene-specific methylation analysis. This study was approved by the Institutional Review Board of NYS Institute for Basic Research in Developmental Disabilities and the human DNA samples were obtained in conformance with their guidelines and included the acquisition of written informed consent for genetic testing.
2.2. Bisulfite sequencing Bisulfite modification of human chromosomal DNA and pyrosequencing analysis (Sheridan et al., 2011; Tost et al., 2003) of a 118 bp segment of the exon 4 of the HSD17B10 gene (AF037438) was done by the EpigenDx, Inc. (http://www.epigendx.com) using a pair of primers designed by EpigenDx, Inc., ADS2501FP and biotin-labeled ADS2501RPB, and the PSQ™ 96HS system. Pyrosequencing dispensation order was ATCGCTGTGCTG.1
2.3. Enzymatic data analysis Enzymatic activity data were gathered and analyzed by reference to kinetic monographs (Segel, 1993). The statistic evaluation was accomplished by use of the Wilcoxon rank-sum test (Conover and Iman, 1981). 1 For experimental details the ASSAY DESIGN REPORT of the EpigenDx Inc. will be provided upon request.
2.4. Tertiary structural model of HSD10(p.R130C) Structural differences between the HSD10(p.R130C) mutant and the wild-type HSD10 were ascertained by bioinformatics analysis. The published crystal structure of human HSD10 was employed as the template structure (Kissinger et al., 2004). Data were extracted from a pdb file (1U7T) of the X-ray coordinates available from the RCSB Protein Data bank (www.resb.org) using DeepView/Swiss-pdb Viewer 4.04 (Guex and Peitsch, 1997). Substitution of the mutant amino acid was made with the “Mutating Amino-Acids” function in DeepView/Swiss-pdb Viewer 4.04. 3. Results 3.1. 5-Methylcytosine at nucleotide + 2259 from ATG Because the cytosine (C) at nucleotide + 2259 from the initiation ATG of the HSD17B10gene (He et al., 1998) has a 3′ flanking guanine (G), it is a potential target for methylation. Surprisingly we found that it is virtually completely (93 ± 5%) methylated not only on the inactive X chromosome of the two female DNAs, but also on the active X chromosome as well. DNA from a normal male also showed >90% methylation of this cytosine (Fig. 1). 3.2. Structural changes due to the substitution of cysteine for arginine at residue 130 Residue 130 is located in the α-helix E2 of HSD10 (Kissinger et al., 2004). Since arginine 130 was present at the interface of subunits A and B as well as that of subunits C and D, its side chain takes part in the stabilization of the dimer (Fig. 2 upper panel). Its guanidinium group forms hydrogen bonds with the side chain of asparagine 127 in the same α-helix. This positively charged group also forms a hydrogen-bonded ion-pair to the side chain of glutamate 68 in α-helix D and a hydrogen bond with the carbonyl oxygen of histidine 109 of the neighboring subunit. The p.R130C mutation eliminates three hydrogen bonds and the volume of the substituted cysteine side chain is much smaller than that of arginine (Fig. 2 lower panel).
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Fig. 2. Structural changes resulted from the substitution of cysteine for arginine at residue 130 of HSD10. The upper image showed that the arginine 130 (greenish blue) of subunit A (yellow) is located at the interface of subunits A and B (blue). A bound NAD+ was in black. The lower image showed that the guanidinium group of arginine 130 of subunit A forms a number of intra- and inter-subunit hydrogen bonding (dashed green line) to carboxyl or carbonyl oxygens (A), and that the mutation would eliminate several hydrogen bonds and the side chain of cystein (surfur in yellow) is much smaller than that of arginine (B). In the lower image nitrogen was shown in blue, hydrogen in greenish blue, and oxygen in red. For clarity, some amino acid residues in the protein have been rendered invisible.
3.3. Reduction of most HSD10 activity by the mutation p.R130C The residual HSD10/MHBD activity in cultured cells from HSD10 deficiency patients as measured in a number of laboratories is listed in
Table 1. Quantitative data are currently available for eight patients carrying the c.388C>T mutation in the HSD17B10 gene (Ensenauer et al., 2002; García-Villoria et al., 2009; Ofman et al., 2003; Perez-Cerda et al., 2005; Sass et al., 2004; Sutton et al., 2003; Yang et al., 2009; Zschocke
Table 1 Residual HSD10/MHBD activity in cells of HSD10 deficiency patients. Patient
Sex
Mutation
Residual activity (%)
Substrates
1 2 3 4 5 6 7 8 9 10 11
M M F M M M M M M M M
p.R130C p.R130C p.R130C p.R130C p.R130C p.R130C p.R130C p.R130C p.L122V p.E249Q p.V65A
Undetectable 8.5 14 8.5 Absent 11 3.6 3.6 25.4 12 50.5
2-Methyl-3-hydroxybutyryl-CoA; 2-Methylacetoacetyl-CoA; NADH 2-Methylacetoacetyl-CoA; NADH 2-Methylacetoacetyl-CoA; NADH 2-Methyl-3-hydroxybutyryl-CoA; 2-Methylacetoacetyl-CoA; NADH 2-Methyl-3-hydroxybutyryl-CoA; 2-Methyl-3-hydroxybutyryl-CoA; 2-Methylacetoacetyl-CoA; NADH 2-Methylacetoacetyl-CoA; NADH 2-Methyl-3-hydroxybutyryl-CoA;
Ref. NAD+
NAD+ NAD+ NAD+
NAD+
Sutton et al. (2003); Yang et al. (2009) Ofman et al. (2003); Zschocke et al. (2000) Ensenauer et al. (2002); Ofman et al. (2003) Ensenauer et al. (2002); Ofman et al. (2003) Sass et al. (2004) Perez-Cerda et al. (2005) Cazorla et al. (2007) García-Villoria et al. (2009) Ofman et al. (2003) Olpin et al. (2002); Yang et al. (2009) Seaver et al. (2011)
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et al., 2000). The cultured patient cells were either fibroblasts or lymphoblastoid cells, and the enzymatic assays were performed in either forward or backward direction depending on the substrate used by the particular laboratory. Based on the data in Table 1, the average level of residual HSD10/MHBD activity in patients' cells carrying mutation p.R130C is estimated at 6 ± 5% of the normal control. This is significantly lower than the residual HSD10/MHBD activity reported for HSD10 deficiency patients with p.V65A, p.L122V or p.E249Q mutation (pb 0.05) (see Table 1) all of which are associated with a milder clinical phenotype (Ofman et al., 2003; Seaver et al., 2011; Yang et al., 2009).
4. Discussion The identification of a 5-methylated CpG in the exon 4 of the HSD17B10 gene (see Fig. 1) explains the high prevalence of the p.R130C mutation among HSD10 deficiency patients. 5-Methylcytosine (5mC) is produced by post-synthetic modification of cytosine residue, and it is a hypermutable site because it can undergo spontaneous or enzyme catalyzing deamination to thymine (Holliday and Grigg, 1993; Morgan et al., 2004). Deamination of unmethylated cytosine produces uracil which is recognized and removed efficiently by uracil DNA glycosylase (Int. Hum. Genome Seq. Cons., 2001). However, 5mC deaminates at a higher rate and the product of this reaction is a thymine. The repair of G–T mismatch is not highly efficient. Thus, the 5mC>T transition occurs about 10 times more frequently than other transitions (Holliday and Grigg, 1993). For this reason a 5mC>T transition at this nucleotide will occur more frequently than other mutations in this gene. The idea that “most other mutations in the HSD17B10 gene are not observed because they are incompatible with life” (Rauschenberger et al., 2010) is unlikely to be the explanation for the prevalence of p.R130C mutation, especially since patients carrying mutations p.V65A, p.L122V or p.E249Q have a milder clinical phenotype and higher residual HSD10 activity (Ofman et al., 2003; Seaver et al., 2011; Yang et al., 2009). A missense mutation in the HSD17B10 gene resulted in HSD10 deficiency (OMIM#300438) whereas a silent mutation caused the mental retardation, X-linked, and abnormal behavior (MRXS10) (OMIM#300220) (Lenski et al., 2007; Reyniers et al., 1999; Yang et al., 2011) (Fig. 3). To date 27 cases of HSD10 deficiency have been described. The manifestation of most female patients is mild intellectual disability or borderline learning disability (García-Villoria et al., 2009, 2010) because females have two X chromosomes. The data of enzymatic activity are not available for most female patients. Also, the mosaicism of the X-inactivation of HSD17B10 gene (He et al., 2011) makes the situation more complicated. At the present time it is not
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feasible to include such cases in any discussions of the genotype– phenotype relationship. Male patients especially those with the c.388C>T mutation show a severe clinical phenotype (Ensenauer et al., 2002; García-Villoria et al., 2009; Ofman et al., 2003; Perez-Cerda et al., 2005; Sass et al., 2004; Sutton et al., 2003; Yang et al., 2009; Zschocke et al., 2000). This mutation results in a mutant HSD10 (p.R130C), in which arginine 130 is replaced by cysteine. As a result, the enzyme structure is seriously disturbed (Fig. 2). Since the side chain of cysteine is much smaller than that of arginine, the van der Waals interaction between neighboring subunits is greatly reduced at that location. HSD10, the product of the HSD17B10 gene, is a multifunctional enzyme (Yang et al., 2005). It has not only multiple enzymatic activities but also non-enzymatic functions, e.g., its ability to form a complex with (guanine-9-)methyltransferase or estrogen receptor α (ERα) (Yang et al., 2011). The observations from the rescue experiments in Xenopus embryos (Rauschenberger et al., 2010) suggest that enzymatic or non-enzymatic functions of HSD10 are essential to cell survival, growth, and differentiation. HSD10 deficiency, an inherited metabolic disease, was considered an inborn error of isoleucine and branched-chain fatty acids metabolism (García-Villoria et al., 2009; Korman, 2006). The HSD10/MHBD activity in patients' cells was diminished by mutations in the HSD17B10 gene (see Table 1). It was later realized that a blockade of the isoleucine degradation pathway does not play a major role in the pathogenesis of this disease, and it was proposed that an imbalance of neurosteroid metabolism may affect the brain function (Yang et al., 2009). Although the MHBD activity may not be directly related with the severity of the disease, an MHBD assay is still required for the clinical diagnosis in combination with the urine organic acid profile, clinical manifestation (e.g., intellectual disability, learning disability and/or microcephaly) and the identification of a mutation in the HSD17B10 gene (Ofman et al., 2003; Seaver et al., 2011; Yang et al., 2009). Conflict of interest The authors declare no conflict of interest. Acknowledgments This study was partially supported by the New York State Office for People with Developmental Disabilities. We also thank Dr. Yu-Xiao Yang for important advice in the statistical evaluation. References
Fig. 3. Missense mutations of HSD10 result in HSD10 deficiency (OMIM#300438) whereas a silent mutation causes MRXS10 (OMIM#300220). Female cases are indicated by a red bar while male cases are in blue. The bar height is approximately proportional to the number of cases. The N and C represent the N- and C-terminals of HSD10, respectively.
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