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A 3′ splice site mutation of IDS gene in a Chinese family with mucopolysaccharidosis type II
Gene 528 (2013) 236–240
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Gene journal homepage: www.elsevier.com/locate/gene
A 3′ splice site mutation of IDS gene in a Chinese family with mucopolysaccharidosis type II Ping Jin a, Jing-Wen Hao a, Ke Chen a, Chang-sheng Dong b, You-Bo Yang a, Zhao-Hui Mo a,⁎ a b
Division of Endocrinology, 3nd Xiangya Hospital, Central South University, 410007 Changsha, Hunan, China The Affiliated Tumor Hospital of Xiangya Medical School, Central South University, 410007 Changsha, Hunan, China
a r t i c l e
i n f o
Article history: Accepted 24 June 2013 Available online 16 July 2013 Keywords: Mucopolysaccharidosis type II Splicing mutations Iduronate-2-sulfatase
a b s t r a c t The purpose of this study was to identify the underlying genetic cause in a four generation Chinese family diagnosed with mucopolysaccharidosis type II. Peripheral blood samples were collected from family members and the iduronate-2-sulfatase (IDS) gene was analyzed by DNA sequencing. The impact of IDS mutations on mRNA transcription was determined by quantitative real-time RT-PCR (qRT-PCR) in both patients as well as in healthy control samples. In addition, RT-PCR was performed to confirm the characteristics of a found mutation located in non-canonical splicing site. A 3′ splice site mutation c.880-8ANG (IVS 6–8ANG) was identified in two members of the analyzed MPS II family and sequencing of RT-PCR products showed that this mutation activates an upstream cryptic splice-site in intron 6, leads to the 7 nucleotide insertion in exon 7, which in turn results in an exon 7 frameshift introducing a premature stop codon and shorter peptide chain. In addition, qRT-PCR products from the two patients showed a reduced IDS mRNA expression (43.9% and 71.2%, respectively), when compared with the average IDS mRNA expression in healthy control samples, possibly as a result of nonsense-mediated mRNA decay. In conclusion, in this study, we have identified an IDS gene splice mutation which is associated with clinically attenuated MPS II phenotype. In addition, our study accentuates the importance of cDNA analysis in the detection of intronic mutations, since in the studies examining only gDNA, the link between genotype and phenotype may have been misinterpreted. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Mucopolysaccharidosis type II (MPS II) is a rare X-linked lysosomal storage disease caused by deficiency of iduronate-2-sulfatase (IDS) activity. This deficiency leads to accumulation of dermatan sulfate and heparan sulfate in connective tissue, liver, spleen and brain, with excretion in the urine. MPS II is a progressive disorder and is traditionally categorized into a severe and a mild form. Severely affected individuals are characterized by progressive mental retardation, physical disability, severe airway obstruction, skeletal deformities and usually die before 15 years of age. Individuals with mild forms of MPS II can have normal intelligence, short stature and survive into adulthood (Neufeld and Muenzer, 2001). The disease almost exclusively affects males, although a few female cases have also been reported (Tuschl et al., 2005). Abbreviations: MPS II, Mucopolysaccharidosis type II; IDS, Iduronate-2-sulfatase; PCR, Polymerase Chain Reaction; qRT-PCR, Quantitative Real-time Polymerase Chain Reaction; HGMD, Human Gene Mutation Database; PTCs, Premature Translation Stop Codons; NMD, Nonsense-mediated Decay. ⁎ Corresponding author at: Department of Endocrinology, 3nd Xiangya Hospital, Central South University, Tongzipo Road, Changsha, Hunan Province, China. Tel.: +86 731 886501865; fax: +86 731 88618006. E-mail address: [email protected] (Z.-H. Mo). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.06.084
IDS is the only gene related to MPS II and has a conclusive role in MPS II diagnosis. The human IDS gene (Xq28) spans approximately 24 kb and contains nine exons. Its 1650-bp open reading frame encodes a 550 amino-acid polypeptide (Wilson et al., 1993). The wide spectrum of clinical severity of MPSII is associated with a high level of molecular heterogeneity at the IDS gene locus. To date, more than 350 different IDS mutations have been reported, which include point mutations, small insertions, deletions and major structural alterations, such as complex rearrangements, and gross insertions/deletions (Stenson et al., 2003). In general, gross structural changes are associated with severe phenotypes, while small gene variations may result in severe to mild clinical presentations (Martin et al., 2008). In the current report, we present two patients with MPS II resulting from a 3′ splice site IDS mutation and, in addition, we explore the genotype–phenotype correlation by analyzing the impact of IDS mutations on mRNA transcription and expression. 2. Materials and methods 2.1. Patient data A four-generation consanguineous family, comprising twenty-two members, was examined in this study (Fig. 1). The study was approved
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5′-CACAAGTTCCACAAGGTCCA-3′, a 373-bp fragment was amplified. The amplification profile consisted of an initial denaturation step of 94 °C for 4 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 1 min and final extension at 72 °C for 5 min. The fragments were sequenced using the ABI 3730xl automated sequencer (Applied Biosystems, USA).
2.4. Real-time RT-PCR analysis
Fig. 1. Pedigree of studied family. Square and circle indicate male and female, respectively. Shaded symbols indicate patients (proband indicated by arrow). Half shaded symbols indicate carriers.
by the local Institutional Review Ethics Board and informed consent was obtained from all subjects included in the study. 2.1.1. Proband A 36-year-old man who had been experiencing recurrent respiratory infections was brought to our clinic. Physical examination showed that he suffered from a coarse face, including a broad nose, short neck, enlarged head and tongue. He had normal intelligence and eyesight but he also had short stature, small stubby fingers, thickened pebbled skin, distended abdomen due to hepatosplenomegaly and inguinal hernia. In addition, X-rays of the chest showed oar-shaped ribs (Fig. 2A). 2.1.2. Patient 2 A 2.5 year old boy, a young nephew of the proband. He was 93 cm tall and weighed 19 kg, with a head circumference of 54 cm. He also suffered from a mild coarsening of facial features. Physical examination showed a slightly enlarged liver and spleen and joint rigidity. An X-ray of the hands showed bullet-shape change, including short and thick phalangeal and metarcarpal bones, and contractures of distal interphalangeal joints (Fig. 2B). To date, his growth development is normal for his age. The diagnosis of MPS II in these two patients was based on the high excretion of urinary heparin and dermatan sulfates, and very low residual IDS activity in peripheral blood leukocytes. The IDS activity of the proband and Patient 2 was 11.8 and 15.3 nmol/h/mg of cell protein, respectively (normal range is 19.6–52.8). The phenotypes of the two patients' parents were both normal and they were not of intermarriage.
The impact of IDS mutations on mRNA transcripts was determined by quantitative real-time RT-PCR (qRT-PCR). Total RNA from two patients and ten healthy male controls were extracted and reverse transcribed as described above. Real-time PCR was performed using QuantiTect SYBR green PCR master mix kit (ToYoBo, Japan) following the manufacturer's instructions on a mastercycler EP realplex RT-PCR instrument (Eppendorf, Germany). The reaction volume was 10 μL and contained 5 μL QuantiTect SYBR green PCR master mix, 0.5 μmol/L primers and 100 ng cDNA and RNase-free water. The primers were specific to exons 7 and 8 of the human IDS cDNA as previously described and were designed to yield products of 151 bp (Lualdi et al., 2006). The primer sequences for the human GAPDH gene which served as the reference gene were as follows: 5′-GGCTGAGAACGGGAAGCTTGTCAT-3′ and 5′-CAGCCTTCTCCATGGTGGTGAAGA-3′. The PCR conditions were: an initial denaturation at 95 °C for 1 min, followed by 40 PCR cycles. Each cycle consisted of 15 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C. The relative amount of mRNA was calculated using the 2−ΔΔCT method. Each individual sample was run in triplicate. The relative IDS mRNA expression was determined through the ratio of the normalized expressions of the patients and control samples. 3. Results 3.1. Identification of IDS mutations Bidirectional sequencing of the entire coding region and the exon– intron boundaries of the IDS gene resulted in a detection of one point mutation, c.880-8ANG (IVS 6–8ANG) transition at the eighth nucleotide upstream of the 3′ splice acceptor site in intron 6 in two analyzed patients (Fig. 3). Further genetic testing showed that their mother and grandmother were heterozygous for this mutation, which indicated that the mutation was of maternal origin. DNA analysis of unaffected family members and 50 unrelated samples revealed a normal sequence at this position, indicating that the detected sequence alteration was a mutation and not a polymorphism.
2.2. IDS genomic DNA analysis 3.2. Impact of the IDS c.880-8ANG mutation on IDS mRNA Blood samples were collected from patients, their parents, and unaffected relatives. Genomic DNAs were extracted from peripheral blood leukocytes by standard phenol-chloroform procedures. All nine IDS gene (NM_000202) exons and their flanking introns were amplified by polymerase chain reaction (PCR) as previously described (Lin et al., 2006). Primer sequences used for the PCR amplification and DNA sequencing of IDS gene exon 7 were: 5′-CTGAGAAAATCATTAAGGGC-3′ and 5′-TTCACAGGAAAGTTCAGATG-3′. Mutations were identified by direct sequencing of PCR products bidirectionally using an ABI 3730xl automated sequencer (Applied Biosystems, USA). 2.3. Total RNA preparation and IDS cDNA analysis Total RNA was isolated from the patients' leukocytes using the High Pure RNA Isolation Kit (Omega, USA). The isolated RNA was reverse transcribed using the cDNA synthesis kit (ToYoBo, Japan) according to the manufacturer's protocol. Using primers that specifically amplify IDS cDNA, 5′-CATCAGTGTGCCGTATGGTC-3′ and
Unlike the first and second positions of the splice acceptor sequence, the eighth is not invariant and the consequence of this change can only be tested by RNA analysis. To confirm the presence of aberrant RNA transcript splice products, we performed RT-PCR. Sequencing of amplified RT-PCR products showed that this mutation resulted in loss of the original splicing acceptor site and activation of upstream cryptic splice-site in intron 6, which resulted in 7 nucleotide insertions in the transcript (Fig. 4). This change in turn resulted in an exon 7 frameshift introducing a TGA stop codon after coding for 49 amino acids differing from the primary amino acid sequence. This change resulted in peptide chain shortening from the initial 550 to 342 amino acids. Based on the predicted presence of the exon 7–8 junction in the splice variants, we quantified the transcript by qRT-PCR. The qRTPCR products from the proband and Patient 2 showed a reduced IDS mRNA expression (43.9% and 71.2%, respectively), when compared with the average IDS mRNA expression of ten controls (Fig. 5).
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Fig. 2. (A) X-ray of the proband shows oar-shaped ribs. (B) Patient 2 at the age of 2.5 years, X-ray of the hands show bullet-shaped change.
4. Discussion MPS II is the predominant form of mucopolysaccharidoses among northeast Asians and Chinese (Kato et al., 2005; Kim et al., 2003; Lin
A
B
C
et al., 2009). The IDS gene is the only gene associated with MPS II, thus identifying IDS mutations is important for genetic counseling, prenatal diagnostics, as well as early prevention and treatment of this disease. A large number of IDS gene mutations have been identified in
Intron 6
Exon 7
Intron 6
Intron 6
Exon 7
Exon 7
Fig. 3. Chromatograms of the IDS mutation, in comparison with patient (A), normal sequence (B) and heterozygous IDS mutation carrier (mother C). Arrow indicates the substitution ANG transition in intron 6 at position –8.
P. Jin et al. / Gene 528 (2013) 236–240
A
Exon 6
B
Exon 6
Exon 7
Exon 7
Fig. 4. Sequence analysis of RT-PCR products of patient with the 7 bp insertion “TTTGCAG” (A) and normal control (B).
patients with Hunter disease, but the relationship between genotype and phenotype has not yet been fully established (Froissart et al., 2002). According to the classification (Neufeld and Muenzer, 2001), two patients in our study presented with a mild phenotype. Upon sequencing of all coding exons and exon–intron boundaries of the IDS gene, we identified a mutation in the 3′-splice acceptor site of intron 6 (c.880-8ANG) in these two patients. Upon the sequencing analysis of mother and grandmother of these two patients a carrier status was found which indicated that the mutation was of maternal origin and these findings were consistent with the inheritance characteristics of X-linked disorders. A search in the HGMD (Human Gene Mutation Database, http://www.hgmd.org) showed that this mutation had been previously reported in a Portuguese population (Alves et al., 2006), but this was the first report of this mutation in a Chinese population. The IDS gene has high genetic heterogeneity, and the mutation patterns of patients from different races and nations are very diverse (Hopwood et al., 1993; Rathmann et al., 1996). According to the IDS Control
(%)
Control
100 Patient 2
80 60
Proband
40 20 0 Fig. 5. Real-time RT-PCR quantification of mRNA transcripts in patients and normal control samples. Proband and Patient 2 are represented by white bars and control samples by gray bars.
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gene mutation data for Chinese patients with Hunter syndrome (Guo et al., 2005; Lau and Lam, 2008; Wei et al., 2011; Zhang et al., 2011), in which the mutation frequency for exon 7 is the highest, followed by exon 9, the next most mutation-prone, compared to the German population, in which exons 3, 8 and 9, are the most mutated exons (Rathmann et al., 1996). Therefore our results are in line with the findings that exon 7 of the IDS gene is a frequent hot spot in Chinese MPS II patients. It is estimated that about 15% of point mutations associated with human genetic diseases occur in splicing signals (Krawczak et al., 1992). As reported, the alternative splicing at the IDS gene results in the normal production of at least seven different transcripts (VEGA: http://vega.sanger.ac.uk/). The presence of several transcripts reflects a complex system of intron splicing and strongly suggests that the IDS gene is susceptible to splicing mutations. Recently, Alves et al. (2006) and Lualdi et al. (2006) confirmed that the IDS gene has a high splicing site mutation rate, but the effect of splice mutations in the IDS gene was experimentally examined in only a few cases (Froissart et al., 2002; Li et al., 1999; Moreira da Silva et al., 2001). Until our study, the c.880-8ANG substitution had been described only at the gDNA level. With respect to intronic splice mutations, molecular assays restricted to gDNA can lead to false interpretation as classic splice defects. Therefore, an accurate characterization of splicing mutations requires the screening of cDNA. As reported, there are two main mechanisms of novel splicing induced by mutations. In one of them, mutations of the splicing site affect classic motifs that include the invariant GU and AG di-nucleotides at the donor and acceptor splice sites, which results in skipping of the neighboring exon. The other mechanism includes the less-conserved variant preceding the 3′AG and the branch site that lies 18–40 bp upstream of the splice site, which in turn leads to the activation of a cryptic splice site or creation of another ectopic splice site (Logsdon, 1998). In our study, we found the c.880-8ANG substitution which activates an upstream cryptic splice-site in intron 6, leads to the 7 nucleotide insertion in exon 7, which in turn results in the premature stop codon and shorter peptide chain. As reported, the IDS gene encodes a 550-amino acid precursor of iduronate 2-sulfatase that is cleaved into two kinds of polypeptides at 42- and 14-kDa, respectively by an internal proteolysis in the human liver (Wilson et al., 1990). The 42-kDa polypeptide contains 455 N-terminal amino acids and the remaining 14-kDa polypeptide contains 95 C-terminal amino acids. Therefore, this candidate mutation results in an exon 7 frameshift introducing a premature stop codon and the ultimate peptide chain is shortened from 550 to 342 amino acids, which leads to a C-terminal 49-amino acid change and loss of 208 amino acids. We speculate that this mutation remarkably alters the primary and tertiary structure of IDS protein and potentially induces deficient activity of the 42-kDa polypeptide and complete loss of activity of the 14-kDa polypeptide, which in turn lowers the IDS enzymatic activity. Based on these premises, this substitution might therefore be responsible for the observed patients' phenotype. It is known that mutations introducing premature translation stop codons (PTCs) at a position more than 50–55 nucleotides upstream of a splicing-generated exon–exon junction trigger the mRNA surveillance pathway and nonsense-mediated decay (NMD) (Maquat, 2005). This mechanism is aimed at eliminating mRNAs containing PTCs and thus helps limit the synthesis of abnormal proteins. In our study, the intronic mutation c.880-8ANG resulted in a premature stop codon and the corresponding mRNA could thus possibly be subjected to degradation through the NMD. To evaluate the possible mRNA reduction, we performed a qRT-PCR IDS mRNA expression analysis and we found that the total IDS mRNA expression in the proband and Patient 2 was reduced by 56.1% and 28.8% respectively, in comparison with the IDS mRNA expression in control samples, which indicated that the abnormally spliced IDS mRNA was probably sensitive to nonsense-mediated mRNA decay.
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Although MPS II treatment with stem cell transplantation had been proposed in the 1980s, the results were not considered satisfactory (Wraith et al., 2008). Since 2006, enzyme replacement therapy with recombinant IDS has been commonly used to treat MPS II (Anon, 2006a,b). Most of the clinical trials studied the Elaprase effect on patients with relatively attenuated symptoms (Muenzer et al., 2006). Nevertheless, the high price of Elaprase limits its use in China and MPS II still remains an important social and economic concern, which requires the coordination of several medical specialties. In summary, we have identified an IDS gene splice mutation which is associated with the clinically attenuated MPS II phenotype. This mutation contributes to further understanding of IDS genotype–phenotype correlations and the pathogenesis of this disease. Moreover, our study stresses the importance of cDNA analysis in the detection of intronic mutations, since in the studies examining only the gDNA, the link between genotype and phenotype may have been misinterpreted. Conflicts of Interest The authors declare no conflicts of interest. Acknowledgments We are grateful to all family members included in this study for their invaluable participation and cooperation. This study was supported by the National Science Foundation for Young Scholars of China (81100583, 81001464), the Natural Science Foundation of China Hunan Province (12JJ4083), the Social Development Supporting Plan Program of Science and Technology Bureau of Hunan Province (2011SK3242), and the Freedom to Explore Program of Central South University (2011QNZT191). References Alves, S., et al., 2006. Molecular characterization of Portuguese patients with mucopolysaccharidosis type II shows evidence that the IDS gene is prone to splicing mutations. J. Inherit. Metab. Dis. 29 (6), 743–754. Anon, 2006a. First treatment for Hunter syndrome. FDA Consum. 40 (6), 5. Anon, 2006b. Treatment for Hunter syndrome approved. FDA Consum. 40 (5), 4. Froissart, R., Moreira da Silva, I., Guffon, N., Bozon, D., Maire, I., 2002. Mucopolysaccharidos is type II-genotype/phenotype aspects. Acta Paediatr. Suppl. 91, 82–87. Guo, Y.B., Pan, J.X., Du, C.S., 2005. Detection of a new mutation (1467-A) for the pedigree with mucopolysaccharidosis type II from a Chinese family. Chin. Sci. Bull. 50 (21), 2534–2536. Hopwood, J.J., et al., 1993. Molecular basis of mucopolysaccharidosis type II: mutations in the iduronate-2-sulphatase gene. Hum. Mutat. 2 (6), 435–442.
Kato, T., et al., 2005. Mutational and structural analysis of Japanese patients with mucopolysaccharidosis type II. J. Hum. Genet. 50 (8), 395–402. Kim, C.H., et al., 2003. Mutational spectrum of the iduronate 2 sulfatase gene in 25 unrelated Korean Hunter syndrome patients: identification of 13 novel mutations. Hum. Mutat. 21 (4), 449–450. Krawczak, M., Reiss, J., Cooper, D.N., 1992. The mutational spectrum of single base-pair substitutions in mRNA splice junctions of human genes: causes and consequences. Hum. Genet. 90, 41–54. Lau, K.C., Lam, C.W., 2008. Molecular investigations of a novel iduronate-2-sulfatase mutant in a Chinese patient. Clin. Chim. Acta 392 (1–2), 8–10. Li, P., Bellows, A.B., Thompson, J.N., 1999. Molecular basis of iduronate-2- sulphatase gene mutations in patients with mucopolysaccharidosis type II (Hunter syndrome). J. Med. Genet. 36, 21–27. Lin, S.P., Chang, J.H., Lee-Chen, G.J., Lin, D.S., Lin, H.Y., Chuang, C.K., 2006. Detection of Hunter syndrome (mucopolysaccharidosis type II) in Taiwanese: biochemical and linkage studies of the iduronate-2-sulfatase gene defects in MPS II patients and carriers. Clin. Chim. Acta 369 (1), 29–34. Lin, H.Y., et al., 2009. Incidence of the mucopolysaccharidoses in Taiwan, 1984–2004. Am. J. Med. Genet. A 149 A, 960–964. Logsdon, J.M., 1998. The recent origins of spliceosomal introns revisited. Curr. Opin. Genet. Dev. 8 (6), 637–648. Lualdi, S., et al., 2006. Multiple cryptic splice sites can be activated by IDS point mutations generating misspliced transcripts. J. Mol. Med. 84 (8), 692–700. Maquat, L.E., 2005. Nonsense-mediated mRNA decay in mammals. J. Cell Sci. 118, 1773–1776. Martin, R., et al., 2008. Recognition and diagnosis of mucopolysaccharidosis II (Hunter syndrome). Pediatrics 121 (2), e377–e386. Moreira da Silva, I., Froissart, R., Marques dos Santos, H., Caseiro, C., Maire, I., Bozon, D., 2001. Molecular basis of mucopolysaccharidosis type II in Portugal: identification of four novel mutations. Clin. Genet. 60, 316–318. Muenzer, J., et al., 2006. A phase II/III clinical study of enzyme replacement therapy with idursulfase in mucopolysaccharidosis II (Hunter syndrome). Genet. Med. 8 (8), 465–473. Neufeld, E.F., Muenzer, J., 2001. The mucopolysaccharidoses, In: Scriver, C.R., Beaudet, A.L., Sly, W.S., Valle, D. (Eds.), The Metabolic and Molecular Bases of Inherited Disease, 8th edition. McGraw-Hill Press, New York, pp. 3421–3452. Rathmann, M., Bunge, S., Beck, M., Kresse, H., Tylki-Szymanska, A., Gal, A., 1996. Mucopolysaccharidosis type II (Hunter syndrome): mutation “hot spots” in the iduronate-2-sulfatase gene. Am. J. Hum. Genet. 59 (6), 1202–1209. Stenson, P.D., et al., 2003. Human Gene Mutation Database (HGMD): 2003 update. Hum. Mutat. 21 (6), 577–581. Tuschl, K., Gal, A., Paschke, E., Kircher, S., Bodamer, O.A., 2005. Mucopolysaccharidosis, type II in females: case report and review of literature. Pediatr. Neurol. 32 (4), 270–272. Wei, X., et al., 2011. A novel mutation of IDS gene in a Chinese patient with mucopolysaccharidosis II by next-generation sequencing. Clin. Chim. Acta. 412 (23–24), 2340–2342 (20). Wilson, P.J., et al., 1990. Hunter syndrome: isolation of an iduronate-2- sulfatase cDNA clone and analysis of patient DNA. Proc. Natl. Acad. Sci. 87, 8531–8535. Wilson, P.J., Meaney, C.A., Hopwood, J.J., Morris, C.P., 1993. Sequence of the human iduronate2-sulfatase (IDS) gene. Genomics 17 (3), 773–775. Wraith, J.E., et al., 2008. Mucopolysaccharidosis type II (Hunter syndrome): a clinical review and recommendations for treatment in the era of enzyme replacement therapy. Eur. J. Pediatr. 167 (267-7). Zhang, H., et al., 2011. Analysis of the IDS gene in 38 patients with Hunter syndrome: the c.879GNA (p.Gln293Gln) synonymous variation in a female create exonic splicing. PLoS One 6, e22951.
Contents lists available at ScienceDirect
Gene journal homepage: www.elsevier.com/locate/gene
A 3′ splice site mutation of IDS gene in a Chinese family with mucopolysaccharidosis type II Ping Jin a, Jing-Wen Hao a, Ke Chen a, Chang-sheng Dong b, You-Bo Yang a, Zhao-Hui Mo a,⁎ a b
Division of Endocrinology, 3nd Xiangya Hospital, Central South University, 410007 Changsha, Hunan, China The Affiliated Tumor Hospital of Xiangya Medical School, Central South University, 410007 Changsha, Hunan, China
a r t i c l e
i n f o
Article history: Accepted 24 June 2013 Available online 16 July 2013 Keywords: Mucopolysaccharidosis type II Splicing mutations Iduronate-2-sulfatase
a b s t r a c t The purpose of this study was to identify the underlying genetic cause in a four generation Chinese family diagnosed with mucopolysaccharidosis type II. Peripheral blood samples were collected from family members and the iduronate-2-sulfatase (IDS) gene was analyzed by DNA sequencing. The impact of IDS mutations on mRNA transcription was determined by quantitative real-time RT-PCR (qRT-PCR) in both patients as well as in healthy control samples. In addition, RT-PCR was performed to confirm the characteristics of a found mutation located in non-canonical splicing site. A 3′ splice site mutation c.880-8ANG (IVS 6–8ANG) was identified in two members of the analyzed MPS II family and sequencing of RT-PCR products showed that this mutation activates an upstream cryptic splice-site in intron 6, leads to the 7 nucleotide insertion in exon 7, which in turn results in an exon 7 frameshift introducing a premature stop codon and shorter peptide chain. In addition, qRT-PCR products from the two patients showed a reduced IDS mRNA expression (43.9% and 71.2%, respectively), when compared with the average IDS mRNA expression in healthy control samples, possibly as a result of nonsense-mediated mRNA decay. In conclusion, in this study, we have identified an IDS gene splice mutation which is associated with clinically attenuated MPS II phenotype. In addition, our study accentuates the importance of cDNA analysis in the detection of intronic mutations, since in the studies examining only gDNA, the link between genotype and phenotype may have been misinterpreted. © 2013 Elsevier B.V. All rights reserved.
1. Introduction Mucopolysaccharidosis type II (MPS II) is a rare X-linked lysosomal storage disease caused by deficiency of iduronate-2-sulfatase (IDS) activity. This deficiency leads to accumulation of dermatan sulfate and heparan sulfate in connective tissue, liver, spleen and brain, with excretion in the urine. MPS II is a progressive disorder and is traditionally categorized into a severe and a mild form. Severely affected individuals are characterized by progressive mental retardation, physical disability, severe airway obstruction, skeletal deformities and usually die before 15 years of age. Individuals with mild forms of MPS II can have normal intelligence, short stature and survive into adulthood (Neufeld and Muenzer, 2001). The disease almost exclusively affects males, although a few female cases have also been reported (Tuschl et al., 2005). Abbreviations: MPS II, Mucopolysaccharidosis type II; IDS, Iduronate-2-sulfatase; PCR, Polymerase Chain Reaction; qRT-PCR, Quantitative Real-time Polymerase Chain Reaction; HGMD, Human Gene Mutation Database; PTCs, Premature Translation Stop Codons; NMD, Nonsense-mediated Decay. ⁎ Corresponding author at: Department of Endocrinology, 3nd Xiangya Hospital, Central South University, Tongzipo Road, Changsha, Hunan Province, China. Tel.: +86 731 886501865; fax: +86 731 88618006. E-mail address: [email protected] (Z.-H. Mo). 0378-1119/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.gene.2013.06.084
IDS is the only gene related to MPS II and has a conclusive role in MPS II diagnosis. The human IDS gene (Xq28) spans approximately 24 kb and contains nine exons. Its 1650-bp open reading frame encodes a 550 amino-acid polypeptide (Wilson et al., 1993). The wide spectrum of clinical severity of MPSII is associated with a high level of molecular heterogeneity at the IDS gene locus. To date, more than 350 different IDS mutations have been reported, which include point mutations, small insertions, deletions and major structural alterations, such as complex rearrangements, and gross insertions/deletions (Stenson et al., 2003). In general, gross structural changes are associated with severe phenotypes, while small gene variations may result in severe to mild clinical presentations (Martin et al., 2008). In the current report, we present two patients with MPS II resulting from a 3′ splice site IDS mutation and, in addition, we explore the genotype–phenotype correlation by analyzing the impact of IDS mutations on mRNA transcription and expression. 2. Materials and methods 2.1. Patient data A four-generation consanguineous family, comprising twenty-two members, was examined in this study (Fig. 1). The study was approved
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5′-CACAAGTTCCACAAGGTCCA-3′, a 373-bp fragment was amplified. The amplification profile consisted of an initial denaturation step of 94 °C for 4 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C for 1 min and final extension at 72 °C for 5 min. The fragments were sequenced using the ABI 3730xl automated sequencer (Applied Biosystems, USA).
2.4. Real-time RT-PCR analysis
Fig. 1. Pedigree of studied family. Square and circle indicate male and female, respectively. Shaded symbols indicate patients (proband indicated by arrow). Half shaded symbols indicate carriers.
by the local Institutional Review Ethics Board and informed consent was obtained from all subjects included in the study. 2.1.1. Proband A 36-year-old man who had been experiencing recurrent respiratory infections was brought to our clinic. Physical examination showed that he suffered from a coarse face, including a broad nose, short neck, enlarged head and tongue. He had normal intelligence and eyesight but he also had short stature, small stubby fingers, thickened pebbled skin, distended abdomen due to hepatosplenomegaly and inguinal hernia. In addition, X-rays of the chest showed oar-shaped ribs (Fig. 2A). 2.1.2. Patient 2 A 2.5 year old boy, a young nephew of the proband. He was 93 cm tall and weighed 19 kg, with a head circumference of 54 cm. He also suffered from a mild coarsening of facial features. Physical examination showed a slightly enlarged liver and spleen and joint rigidity. An X-ray of the hands showed bullet-shape change, including short and thick phalangeal and metarcarpal bones, and contractures of distal interphalangeal joints (Fig. 2B). To date, his growth development is normal for his age. The diagnosis of MPS II in these two patients was based on the high excretion of urinary heparin and dermatan sulfates, and very low residual IDS activity in peripheral blood leukocytes. The IDS activity of the proband and Patient 2 was 11.8 and 15.3 nmol/h/mg of cell protein, respectively (normal range is 19.6–52.8). The phenotypes of the two patients' parents were both normal and they were not of intermarriage.
The impact of IDS mutations on mRNA transcripts was determined by quantitative real-time RT-PCR (qRT-PCR). Total RNA from two patients and ten healthy male controls were extracted and reverse transcribed as described above. Real-time PCR was performed using QuantiTect SYBR green PCR master mix kit (ToYoBo, Japan) following the manufacturer's instructions on a mastercycler EP realplex RT-PCR instrument (Eppendorf, Germany). The reaction volume was 10 μL and contained 5 μL QuantiTect SYBR green PCR master mix, 0.5 μmol/L primers and 100 ng cDNA and RNase-free water. The primers were specific to exons 7 and 8 of the human IDS cDNA as previously described and were designed to yield products of 151 bp (Lualdi et al., 2006). The primer sequences for the human GAPDH gene which served as the reference gene were as follows: 5′-GGCTGAGAACGGGAAGCTTGTCAT-3′ and 5′-CAGCCTTCTCCATGGTGGTGAAGA-3′. The PCR conditions were: an initial denaturation at 95 °C for 1 min, followed by 40 PCR cycles. Each cycle consisted of 15 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C. The relative amount of mRNA was calculated using the 2−ΔΔCT method. Each individual sample was run in triplicate. The relative IDS mRNA expression was determined through the ratio of the normalized expressions of the patients and control samples. 3. Results 3.1. Identification of IDS mutations Bidirectional sequencing of the entire coding region and the exon– intron boundaries of the IDS gene resulted in a detection of one point mutation, c.880-8ANG (IVS 6–8ANG) transition at the eighth nucleotide upstream of the 3′ splice acceptor site in intron 6 in two analyzed patients (Fig. 3). Further genetic testing showed that their mother and grandmother were heterozygous for this mutation, which indicated that the mutation was of maternal origin. DNA analysis of unaffected family members and 50 unrelated samples revealed a normal sequence at this position, indicating that the detected sequence alteration was a mutation and not a polymorphism.
2.2. IDS genomic DNA analysis 3.2. Impact of the IDS c.880-8ANG mutation on IDS mRNA Blood samples were collected from patients, their parents, and unaffected relatives. Genomic DNAs were extracted from peripheral blood leukocytes by standard phenol-chloroform procedures. All nine IDS gene (NM_000202) exons and their flanking introns were amplified by polymerase chain reaction (PCR) as previously described (Lin et al., 2006). Primer sequences used for the PCR amplification and DNA sequencing of IDS gene exon 7 were: 5′-CTGAGAAAATCATTAAGGGC-3′ and 5′-TTCACAGGAAAGTTCAGATG-3′. Mutations were identified by direct sequencing of PCR products bidirectionally using an ABI 3730xl automated sequencer (Applied Biosystems, USA). 2.3. Total RNA preparation and IDS cDNA analysis Total RNA was isolated from the patients' leukocytes using the High Pure RNA Isolation Kit (Omega, USA). The isolated RNA was reverse transcribed using the cDNA synthesis kit (ToYoBo, Japan) according to the manufacturer's protocol. Using primers that specifically amplify IDS cDNA, 5′-CATCAGTGTGCCGTATGGTC-3′ and
Unlike the first and second positions of the splice acceptor sequence, the eighth is not invariant and the consequence of this change can only be tested by RNA analysis. To confirm the presence of aberrant RNA transcript splice products, we performed RT-PCR. Sequencing of amplified RT-PCR products showed that this mutation resulted in loss of the original splicing acceptor site and activation of upstream cryptic splice-site in intron 6, which resulted in 7 nucleotide insertions in the transcript (Fig. 4). This change in turn resulted in an exon 7 frameshift introducing a TGA stop codon after coding for 49 amino acids differing from the primary amino acid sequence. This change resulted in peptide chain shortening from the initial 550 to 342 amino acids. Based on the predicted presence of the exon 7–8 junction in the splice variants, we quantified the transcript by qRT-PCR. The qRTPCR products from the proband and Patient 2 showed a reduced IDS mRNA expression (43.9% and 71.2%, respectively), when compared with the average IDS mRNA expression of ten controls (Fig. 5).
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Fig. 2. (A) X-ray of the proband shows oar-shaped ribs. (B) Patient 2 at the age of 2.5 years, X-ray of the hands show bullet-shaped change.
4. Discussion MPS II is the predominant form of mucopolysaccharidoses among northeast Asians and Chinese (Kato et al., 2005; Kim et al., 2003; Lin
A
B
C
et al., 2009). The IDS gene is the only gene associated with MPS II, thus identifying IDS mutations is important for genetic counseling, prenatal diagnostics, as well as early prevention and treatment of this disease. A large number of IDS gene mutations have been identified in
Intron 6
Exon 7
Intron 6
Intron 6
Exon 7
Exon 7
Fig. 3. Chromatograms of the IDS mutation, in comparison with patient (A), normal sequence (B) and heterozygous IDS mutation carrier (mother C). Arrow indicates the substitution ANG transition in intron 6 at position –8.
P. Jin et al. / Gene 528 (2013) 236–240
A
Exon 6
B
Exon 6
Exon 7
Exon 7
Fig. 4. Sequence analysis of RT-PCR products of patient with the 7 bp insertion “TTTGCAG” (A) and normal control (B).
patients with Hunter disease, but the relationship between genotype and phenotype has not yet been fully established (Froissart et al., 2002). According to the classification (Neufeld and Muenzer, 2001), two patients in our study presented with a mild phenotype. Upon sequencing of all coding exons and exon–intron boundaries of the IDS gene, we identified a mutation in the 3′-splice acceptor site of intron 6 (c.880-8ANG) in these two patients. Upon the sequencing analysis of mother and grandmother of these two patients a carrier status was found which indicated that the mutation was of maternal origin and these findings were consistent with the inheritance characteristics of X-linked disorders. A search in the HGMD (Human Gene Mutation Database, http://www.hgmd.org) showed that this mutation had been previously reported in a Portuguese population (Alves et al., 2006), but this was the first report of this mutation in a Chinese population. The IDS gene has high genetic heterogeneity, and the mutation patterns of patients from different races and nations are very diverse (Hopwood et al., 1993; Rathmann et al., 1996). According to the IDS Control
(%)
Control
100 Patient 2
80 60
Proband
40 20 0 Fig. 5. Real-time RT-PCR quantification of mRNA transcripts in patients and normal control samples. Proband and Patient 2 are represented by white bars and control samples by gray bars.
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gene mutation data for Chinese patients with Hunter syndrome (Guo et al., 2005; Lau and Lam, 2008; Wei et al., 2011; Zhang et al., 2011), in which the mutation frequency for exon 7 is the highest, followed by exon 9, the next most mutation-prone, compared to the German population, in which exons 3, 8 and 9, are the most mutated exons (Rathmann et al., 1996). Therefore our results are in line with the findings that exon 7 of the IDS gene is a frequent hot spot in Chinese MPS II patients. It is estimated that about 15% of point mutations associated with human genetic diseases occur in splicing signals (Krawczak et al., 1992). As reported, the alternative splicing at the IDS gene results in the normal production of at least seven different transcripts (VEGA: http://vega.sanger.ac.uk/). The presence of several transcripts reflects a complex system of intron splicing and strongly suggests that the IDS gene is susceptible to splicing mutations. Recently, Alves et al. (2006) and Lualdi et al. (2006) confirmed that the IDS gene has a high splicing site mutation rate, but the effect of splice mutations in the IDS gene was experimentally examined in only a few cases (Froissart et al., 2002; Li et al., 1999; Moreira da Silva et al., 2001). Until our study, the c.880-8ANG substitution had been described only at the gDNA level. With respect to intronic splice mutations, molecular assays restricted to gDNA can lead to false interpretation as classic splice defects. Therefore, an accurate characterization of splicing mutations requires the screening of cDNA. As reported, there are two main mechanisms of novel splicing induced by mutations. In one of them, mutations of the splicing site affect classic motifs that include the invariant GU and AG di-nucleotides at the donor and acceptor splice sites, which results in skipping of the neighboring exon. The other mechanism includes the less-conserved variant preceding the 3′AG and the branch site that lies 18–40 bp upstream of the splice site, which in turn leads to the activation of a cryptic splice site or creation of another ectopic splice site (Logsdon, 1998). In our study, we found the c.880-8ANG substitution which activates an upstream cryptic splice-site in intron 6, leads to the 7 nucleotide insertion in exon 7, which in turn results in the premature stop codon and shorter peptide chain. As reported, the IDS gene encodes a 550-amino acid precursor of iduronate 2-sulfatase that is cleaved into two kinds of polypeptides at 42- and 14-kDa, respectively by an internal proteolysis in the human liver (Wilson et al., 1990). The 42-kDa polypeptide contains 455 N-terminal amino acids and the remaining 14-kDa polypeptide contains 95 C-terminal amino acids. Therefore, this candidate mutation results in an exon 7 frameshift introducing a premature stop codon and the ultimate peptide chain is shortened from 550 to 342 amino acids, which leads to a C-terminal 49-amino acid change and loss of 208 amino acids. We speculate that this mutation remarkably alters the primary and tertiary structure of IDS protein and potentially induces deficient activity of the 42-kDa polypeptide and complete loss of activity of the 14-kDa polypeptide, which in turn lowers the IDS enzymatic activity. Based on these premises, this substitution might therefore be responsible for the observed patients' phenotype. It is known that mutations introducing premature translation stop codons (PTCs) at a position more than 50–55 nucleotides upstream of a splicing-generated exon–exon junction trigger the mRNA surveillance pathway and nonsense-mediated decay (NMD) (Maquat, 2005). This mechanism is aimed at eliminating mRNAs containing PTCs and thus helps limit the synthesis of abnormal proteins. In our study, the intronic mutation c.880-8ANG resulted in a premature stop codon and the corresponding mRNA could thus possibly be subjected to degradation through the NMD. To evaluate the possible mRNA reduction, we performed a qRT-PCR IDS mRNA expression analysis and we found that the total IDS mRNA expression in the proband and Patient 2 was reduced by 56.1% and 28.8% respectively, in comparison with the IDS mRNA expression in control samples, which indicated that the abnormally spliced IDS mRNA was probably sensitive to nonsense-mediated mRNA decay.
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Although MPS II treatment with stem cell transplantation had been proposed in the 1980s, the results were not considered satisfactory (Wraith et al., 2008). Since 2006, enzyme replacement therapy with recombinant IDS has been commonly used to treat MPS II (Anon, 2006a,b). Most of the clinical trials studied the Elaprase effect on patients with relatively attenuated symptoms (Muenzer et al., 2006). Nevertheless, the high price of Elaprase limits its use in China and MPS II still remains an important social and economic concern, which requires the coordination of several medical specialties. In summary, we have identified an IDS gene splice mutation which is associated with the clinically attenuated MPS II phenotype. This mutation contributes to further understanding of IDS genotype–phenotype correlations and the pathogenesis of this disease. Moreover, our study stresses the importance of cDNA analysis in the detection of intronic mutations, since in the studies examining only the gDNA, the link between genotype and phenotype may have been misinterpreted. Conflicts of Interest The authors declare no conflicts of interest. Acknowledgments We are grateful to all family members included in this study for their invaluable participation and cooperation. This study was supported by the National Science Foundation for Young Scholars of China (81100583, 81001464), the Natural Science Foundation of China Hunan Province (12JJ4083), the Social Development Supporting Plan Program of Science and Technology Bureau of Hunan Province (2011SK3242), and the Freedom to Explore Program of Central South University (2011QNZT191). References Alves, S., et al., 2006. Molecular characterization of Portuguese patients with mucopolysaccharidosis type II shows evidence that the IDS gene is prone to splicing mutations. J. Inherit. Metab. Dis. 29 (6), 743–754. Anon, 2006a. First treatment for Hunter syndrome. FDA Consum. 40 (6), 5. Anon, 2006b. Treatment for Hunter syndrome approved. FDA Consum. 40 (5), 4. Froissart, R., Moreira da Silva, I., Guffon, N., Bozon, D., Maire, I., 2002. Mucopolysaccharidos is type II-genotype/phenotype aspects. Acta Paediatr. Suppl. 91, 82–87. Guo, Y.B., Pan, J.X., Du, C.S., 2005. Detection of a new mutation (1467-A) for the pedigree with mucopolysaccharidosis type II from a Chinese family. Chin. Sci. Bull. 50 (21), 2534–2536. Hopwood, J.J., et al., 1993. Molecular basis of mucopolysaccharidosis type II: mutations in the iduronate-2-sulphatase gene. Hum. Mutat. 2 (6), 435–442.
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