A new mutation (1062 del 16) of iduronate-2-sulfatase gene from a Chinese patient with Hunter syndrome

A new mutation (1062 del 16) of iduronate-2-sulfatase gene from a Chinese patient with Hunter syndrome 1 first-author †E-mail:guoyibin@mail.sysu.edu.cngyblym@yahoo.com.cn †E-mail:guoyibin@mail.sysu.edu.cngyblym@yahoo.com.cn guoyibin@mail.sysu.edu.cn 2 1 Department of Medical Genetics, Sun Yat-sen Medical College, Sun Yat-sen University, Guangzhou 510080, China Department of Medical Genetics, Sun Yat-sen Medical College, Sun Yat-sen University, Guangzhou 510080, China Department of Internal Medicine, the Second Affiliated Hospital, Fujian University of Medical Science, Quanzhou 362000, China Department of Internal Medicine, the Second Affiliated Hospital, Fujian University of Medical Science, Quanzhou 362000, China Objective: 2 To identify the mutations of iduronate-2-sulfatase ( IDS ) gene, to reveal its mutation features, and to establish a basis for genetic counseling and prenatal gene diagnosis of Hunter syndrome. Methods: 2 Urine glycosaminoglycans (GAGs) assay, PCR and DNA sequencing were performed to detect mutation of IDS gene of the patient and his parents. Results: 2 The result showed that the patient was: DS(++), HS(++), KS(−), CS(−), and that both of his parents were negative. A frame-shift deletion mutation (1062 del 16) was identified in exon 7 of the patient’s IDS gene. His parents’ genotypes were normal. Conclusion: 2 The patient’s mutation was not inherited by his parents but a novel one. The mutation probably altered the primary structure and tertiary structure of IDS enzyme protein remarkably and lowered the activity of IDS enzyme greatly. Therefore it is supposed to be the direct cause of the disorder. Hunter syndrome Mucopolysaccharidosis type II (MPS II) Glycosaminoglycan (GAG) Iduronate-2-sulfatase (IDS) Mutation Gene diagnosis DNA sequencing INTRODUCTION 1 Hunter syndrome (MIM 309900), also called mucopolysaccharidosis type II (MPS II), is worldwide distributed and a severe disabling, fatal monogenic X-linked recessively inherited disease. The morbidity is 1/132 000 males (Du and Liu, 1992 1992 ). It is the basic cause of Hunter syndrome that iduronate-2-sulfatase ( IDS ) gene mutation lowers the activity of IDS enzyme (EC 3.1.6.13) and influences the degradation of heparan sulfate (HS) and dermatan sulfate (DS). Investigation of the IDS gene mutation is the key to diagnose the disorder and the necessary premise for genetic counseling, prenatal gene diagnosis and early prevention and cure. The new mutation rate of IDS gene is high (Hopwood et al., 1993 1993 ) and IDS gene has high genetic heterogeneity. The mutation patterns of patients from different races, nations and areas are diversified, and mutation hot spots also are various. In order to investigate the mutations of Chinese patients with Hunter syndrome, search for mutation hot spots and identify new mutation, we have collected and further studied the IDS gene of a typical Hunter syndrome pedigree from Guangdong recently. We identified a novel mutation which had not been reported, and the following is the detail. MATERIALS AND METHODS 1 Proband 2 He was 5 years old and suffered from short stature, mental retardation, a coarse face, stiff joints, swollen abdomen and navel hernia. Physical examination 2 He was 103 cm tall and 18 kg, with a head circumference of 54 cm. He suffered from a coarse face including protruding forehead, coarse eyebrows, low nose bridge, big tongue, wide distance between two eyes, full tips, sparse teeth and short neck. He also suffered from remarkable mental retardation, hearing decline, obscured pronounciation, clawing of hands, ulna and radius stiff, funnel chest, asthma, swollen abdomen, navel hernia and so on. But his eyesight was normal and his corneas were not turbid. His parents’ phenotypes were normal and they were non-consanguineously married. Laboratory examination 2 Urinary glycosaminoglycans (GAGs) collected from middle stage of urination of the patient and his parents were tested with methods of agarose gel electrophoresis and toluidine blue established by ourselves with standard dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS) and chondroitin sulfate (CS) as positive controls and with normal people as negative controls (Xu et al., 2005 2005 ; Huang et al., 2007 2007 ). Two to three millilitres EDTA anticoagulated blood of patient and his parents was extracted respectively to be used for preparing DNA samples. Thereafter, exons 1~9 of IDS gene were tested with methods of PCR and DNA bidirectional sequencing (Guo et al., 2005 2005 ; 2006 2006 ; Guo and Du, 2006a 2006a ; 2006b 2006b ). 1. PCR amplification 3 Primer pairs were designed according to IDS gene sequence mapped on Xq27.3~q28 (ACCESSION: NC_000023; REGION: complement 148371 663~148394841; SPAN: 23179 bp). The PCR reactions were carried out in a reaction volume of 30 µl which contained 3 µl 10×Taq buffer, 3 µl dNTPs (2 mmol/L), 1 µl each primer (10 µmol/L), 1.5 µl (300 ng) genomic DNA, 2.5 U Taq DNA polymerase and 19 µl ddH 2 O. PCR was performed using the Hema type 480 PCR cycler (Guo et al., 2005 2005 ; 2006 2006 ; Guo and Du, 2006a 2006a ; 2006b 2006b ). 2. DNA sequencing 3 Sequencing was performed using the ABI PRISM 377 or 3730 DNA sequencer by Shanghai Invitrogen Company. RESULTS 1 1. The result of patient’s urine GAGs assay was: DS(++), HS(++), KS(−), CS(−), and both of his parents’ were: DS(−), HS(−), KS(−), CS(−). 2. In the encoding region of patient’s exon 7, there is a frame-shift deletion mutation (1062 del 16, cDNA 1062~1077 bp, codon 313th~318th) shown in Figs. 1d 1d and 2d 2d ), while his parents were normal shown in Figs. 1b, 1c 1b, 1c , 2b and 2c 2b and 2c ). The sequence of exon 7 was mapped on X chromosome 148379697~148380006, spaning 310 bp. (a) Normal control; (b) Father; (c) Mother; (d) Patient. The underline shows the normal sequence which contains 16 base pairs. The sequences of patient’s father and mother are the same as the normal control’s, while the patient’s sequence deletes 16 bp of “GCCTCTTGAGTGCTTT” (cDNA 1062~1077 bp) (a) Normal control; (b) Father; (c) Mother; (d) Patient. The underline shows the normal sequence which contains 16 base pairs. The sequences of patient’s father and mother are the same as the normal control’s, while the patient’s sequence deletes 16 bp of “AAAGCACTCAAGAGGC” (cDNA 1077~1062 bp) DISCUSSION 1 Hunter syndrome is a lysosomal storage disease caused by the deficiency of IDS enzyme or lowered activity and incomplete degradation or lysosomal accumulation of HS and DS. The surplus GAGs can be drained from urethra, so urinary GAGs qualitative assay can be used for preliminary diagnosis. The types of MPS are various and MPS can be classified into three types (MPS I, II, VII) only by the difference of DS and HS upraising (Luo, 2000 2000 ). Therefore, urinary GAGs assay cannot be used for classification, diagnosis, and prenatal diagnosis. Gene diagnosis is essential to diagnose the disease. According to the evidence that Hunter syndrome is X-linked recessively inherited and patients always were male whose corneas were not turbid, we firstly examined patient’s IDS gene. Thereafter, a novel deletion mutation (1062 del 16) in the encoding region of exon 7 was identified. This mutation resulted in loss of 6 amino acids of RLLSAL in position 313th~318th of IDS enzyme and the base sequence after mutation spot occurred dislocation. It caused elongation of the amino acid chain to a terminal codon TAG at position 334, thus the peptide chain was shortened from 550 to 333 amino acids and lost 217 amino acids. By searching HGMD database ( http://www.hgmd.org http://www.hgmd.org ) and document research, we confirmed that this mutation was a novel one that was discovered firstly. The parents’ genotypes were normal, which suggested that the mutation was not inherited by his parents but was changed at the first time. Early in 1991, some cases had been reported abroad (Machill et al., 1991 1991 ). This mutation altered the primary structure and tertiary structure of IDS enzyme protein remarkably and lowered the activity of IDS greatly. Therefore it is supposed to be the direct cause of the patient with Hunter syndrome. We have discovered that 1062 del 16 is the ninth kind of mutation from Chinese patients with Hunter syndrome in recent years. The detection of this mutation is helpful for demonstrating that IDS gene has a high new mutation rate and remarkable hot spots (Rathmann et al., 1996 1996 ). Recently, Alves et al.( 2006 2006 ) confirmed that IDS gene has a high mutation rate of splicing site. The study of Lualdi et al.( 2006 2006 ) revealed that point mutation of IDS gene can activate multiple larvaceous splicing sites, leading to mis-splicing RNA. Their results verified again that IDS gene had a high new mutation rate and remarkable hot spots. 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