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A novel mutation of IDS gene in a Chinese patient with mucopolysaccharidosis II by next-generation sequencing
Clinica Chimica Acta 412 (2011) 2340–2342
Contents lists available at SciVerse ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Short communication
A novel mutation of IDS gene in a Chinese patient with mucopolysaccharidosis II by next-generation sequencing Xiaoming Wei a, 1, Fan Jin b, 1, Yinghui Ye b, Chenming Xu b, Ning Qu a, Xiangchun Ju a, Xin Yi a,⁎ a b
Beijing Genomics Institute at Shenzhen, Shenzhen 518083, China Zhejiang University School of Medicine Women's Hospital, Hangzhou 310058, Zhejiang, China
a r t i c l e
i n f o
Article history: Received 15 August 2011 Received in revised form 19 August 2011 Accepted 30 August 2011 Available online 3 September 2011 Keywords: Iduronate 2-sulfatase gene Mucopolysaccharidosis II Target sequence capture Next-generation sequencing
a b s t r a c t Background: Mucopolysaccharidosis (MPS) is induced by the absence or malfunctioning of lysosomal enzymes. MPS I and MPS II are similar in phenotypes but they are different in genotypes, which are caused by the deficiencies of alpha-L-iduronidase gene (IDUA) and iduronate 2-sulfatase gene (IDS) respectively. In this work, a 5-year-old Chinese young male with manifestations of MPS in a family with unaffected parents was described. Methods: 12 kb of all the targeted exon sequences plus flanking sequences chromosomal DNA of IDS and IDUA genes from the proband and 20 other case-unrelated controls were captured and sequenced by using next-generation sequencing technology. Results: One single-nucleotide deletion variant (c.1270delG) resulting in frameshift and premature truncation of I2S enzyme was detected, out of 20 controls, only in the proband, and which was further verified by Sanger sequencing. The proband's mother was also proved carrying c.1270delG by Sanger method but not for his father. Conclusions: The novel variant (c.1270delG) is a candidate disease-causing mutation predicted to affect the normal structure and function of the enzyme. Target sequence capture and next-generation sequencing technology can be effective for the gene testing of MPS II disorder. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The mucopolysaccharidosis (MPS) is a group of inherited metabolic disorders caused by deficiency of specific lysosomal enzymes involved in the processing of complex carbohydrates called mucopolysaccharide. Totally, 11 main types of MPS are recorded in OMIM database, and their clinical manifestations vary with different types of lysosomal enzymes affecting mucopolysaccharide. Mucopolysaccharidosis I (MPS I) is by far the most common type and classified into three major subtypes based on symptom severity. All three types of MPS I are caused by mutation in the gene encoding alpha-L-iduronidase (IDUA, MIM# 252800). The cytogenetic location of IDUA gene is 4p16.3, and the entire chromosome interval is 19 kb length containing 14 exons. IDUA enzyme can hydrolyze the terminal alpha-L-iduronic acid residues of the glycosaminoglycans dermatan sulfate and heparan sulfate and play important roles in the pathology of MPS II [1]. Mucopolysaccharidosis II (MPS II, also known as Hunter syndrome, MIM# 309900) is a rare X-linked recessive disorder caused by deficiency
⁎ Corresponding author at: Beijing Genomics Institute at Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen 518083, China. Tel.: + 86 755 25273461; fax: + 86 755 25273620. E-mail address: [email protected] (X. Yi). 1 Xiaoming Wei and Fan Jin contributed equally to this work. 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.08.031
of I2S activity, which leads to progressive accumulation of GAGs in multisystem [2]. MPS II is less frequent than MPS I but has no corneal clouding and is similar, but milder, to MPS I. MPS II is expected to be found mainly in males. In severely affected patients, death usually occurs before adulthood, but a high degree of clinical variability exists with many individuals surviving well into adulthood, which is thought to be related to different mutations of IDS gene [3]. The human iduronate 2sulfatase gene (IDS, MIM# 300823) is located in chromosome Xq28 and contains 9 exons spanning approximately 24 kb chromosome region. IDS gene encodes a lysomal exo-enzyme called iduronate 2-sulfatase (I2S), which can give rise to a breakdown of large sugar molecular glycosaminoglygans (GAGs) including heparin sulfate and dermatan sulfate [4]. Urine GAGs and skeletal survey are important factors in establishing the presence of MPS, but these detective methods are not specific to MPS II. The gold standard for diagnosing MPS II is to detect the deficiency of I2S activity. IDS is the only gene in which mutations are known to be related to MPS II and that has a conclusive role on the diagnosis for MPS II. More than 350 mutations in the IDS gene have been found to be associated with MPS II according to the record stored in the Human Gene Mutation Database (HGMD, http://www.hgmd.org/). All mutations inducing MPS II reduce or completely disrupt the biochemical activity of I2S enzyme and the mutations causing rearrangement, nonsense variation or complete absence of IDS lead to the more severe forms of MPS II
X. Wei et al. / Clinica Chimica Acta 412 (2011) 2340–2342
disorder [5,6]. The gold criterion for diagnosis on MPS II is detecting activity of I2S enzyme, while the clinical finding has shown limitation for phenotypic diversity depending on disease severity. Genetic testing for IDS gene shows a critical role in distinguishing different kind of clinical entities. Recently, the targeted next-generation sequencing has shown considerable potential in the gene testing for genetic diseases and compensated for the inability of Sanger sequencing in variation discovery [7–9]. Here, we reported a novel single-nucleotide-deletion variant as the candidate disease-causing mutation identified by targeted nextgeneration sequencing from a young male with MPS II disorder. 2. Materials and methods 2.1. Blood samples collection Written informed consent was obtained from blood donors who participated in the study before the collection of 5–10 ml of their peripheral blood for gene testing. Genomic DNA was extracted from peripheral blood samples according to the manufacturer's standard procedure using the QIAamp DNA BloodMiNi Kit (Qiagen, Hilden, Germany). 2.2. Targeted next-generation sequencing Genomic DNA from the proband's and controls' peripheral blood were fragmented, and 12 kb of the targeted exon sequences plus flanking sequences (all exons plus 100 bp on each side of a exon) of IDS and IDUA genes were specifically captured and enriched using array-based hybridization chip (NimbleGen, Madison, USA) followed by HiSeq2000 (Illumina, San Diego, USA) sequencing to generate paired end reads (90 bps at each end), adhering to the standard operating protocols. Short reads mapping, alignment were performed using BWA software (Burrows Wheeler Aligner). SNPs and indels were detected using the SOAPsnp software and GATK Indel Genotyper (http://www.broadinstitute.org/gsa/ wiki/index.php/, The Genome Analysis Toolkit) respectively. All reference sequences were based on the NCBI37/hg19 assembly of the human genome. 2.3. Candidate mutation confirmed by Sanger sequencing The potential mutated base and flanking sequence of IDS gene were amplified by polymerase chain reaction (PCR) and sequenced by Sanger sequencing (Forward primer 5′-TGAGGTGCCGAGGTGGTGTT and reverse primer 5′-ATAGTCTATGGTGCGTATGG AATAGCC). The sequencing result was compared with annotated IDS gene reference sequence (NG011900.1) to confirm the candidate nucleotide changes. 3. Results 3.1. Proband descriptions The proband was a five-year-old Chinese young male. He was 1.05 m in height with a head circumference of 55 cm and weighed 22 kg. He suffered from a coarse face with protruding forehead, coarse eyebrows, big nostrils, full lips. And the following characteristics also appeared: clawing of hands, funnel chest, and so on. The proband's roentgenograms did not show any obvious enlargement in sella turcica, and condyle was slightly planus. Thick and short clavicle, thick ribs, and partly lace-shaped ribs were observed. Right humerus, ulna, radius, tibia, fibula, and right metacarpus and phalange were thick and short. Metacarpus proximal end turned cuspidal. Right ulna and radius showed a V-shaped structure. Skeletal lag was found. Thoracolumbar vertebra was biconvex lens-like. Lower part of front edge of lumbar vertebral body had a beaklike protrusion. At the joint of thoracolumbar vertebra, slight hyboma was found. Ala ossis ilii turned wide and abducent/double coxa valga were found. Plasticity of upper and lower extremity long bone was suboptimal, and diaphysis did not show normal contraction changes. CT
2341
images have indicated no abnormality. His parents' phenotypes were normal and they were not of intermarriage. 3.2. Identification and characterization of candidate mutation Approximately 7.63 kb of exons and adjacent intronic regions of IDS gene for the proband were captured and sequenced in our study. For IDS gene, the average coverage for each base pair is approximately 114×, and the average median-depth across nine exons is 119×. On average 98.3% of base pairs with N20× coverage were successfully detected indicating high capability for variants identification. Only two variants were detected in coding region of exon 9 and intron 3 of IDS gene respectively. The single-nucleotide-insertion variation (IVS3-131_-132insC, NM_ 000202.5) was detected in the intronic non-coding region in all of 44 reads that spanned this position. The variants in the coding region of exon 9 (c.1270delG, NM_000202.5) were detected in all of 148 reads covering the changed nucleotide (Fig. 1A), indicating a complete disruption of IDS gene in the X-chromosome for the proband. Considering the existence of the intronic insertion change in 14 of these controls, we classified the intronic variant as the single nucleotide polymorphism (SNP). The deletion variant was detected only in the proband and predicted as the candidate mutation causing MPS II. Considering the similarity of MPS II and MPS I in phenotype, we also detected variations in MPS I-causing gene IDUA via the same pipeline as described for IDS gene. For IDUA gene encoding alpha-Liduronidase, two single-nucleotide variants (c.352 C N T and c.300– 246 C N T) were found. Both of the two variants were detected in 8 of these controls and identified to be SNPs with the frequency of 0.235 and 0.226 respectively based on the data in dbSNP database. To confirm the deletion variant (IDS, c.1270delG) and uncover the inheritance background of MPS II for the proband, Sanger sequencing was employed for the proband and his parents, as well as a case-unrelated control (Fig. 1B). The deletion variant of X-located IDS gene was identified in this proband. As detected by Sanger sequencing, the target base for proband's father is same with reference sequence but not for proband's mother: a carrier status was identified for proband's mother. This overall family status is coincident with the classical manner of Mendel inheritance (Fig. 1C). This variant was not detected in the genome of the control. In translational level, the candidate IDS mutation (c.1270delG) was predicted to result in 127-amino acid changes including 112-amino acid deletion and 15-residues variation in I2S precursor (p.V424FfsX16) analyzed by Mutalyzer 2.0β-8 (http://www.lovd.nl/mutalyzer/) (Fig. 1D). 4. Discussion MPS I and MPS II are similar in phenotypes but different in genotypes. In our work, we captured and sequenced all the target regions of IDS and IDUA genes using targeted next-generation sequencing. The candidate mutation causing MPS for the proband was focused on IDS gene and the disease happened in the proband was further confirmed as type II MPS disorder. MPS II is an X-linked recessive, lysosomal storage disease caused by the abnormality of I2S enzyme, which is the only one among the MPS in which a son can alone inherit the defective gene from the mother. Because of the phenotype diversity, GAGs assay has shown deficiency in clarifying and diagnosing different types of MPS disease. Among the 351 mutations stored in HGMD database, approximately 10% of IDS gene mutations are nonsense and nearly 30% of mutations are described as small or gross deletion/insertion described at genomic or cDNA level indicating that massive amino acids change in the peptide chain plays much more essential roles than missense mutations on the pathology of MPS II. We report here that a candidate single-nucleotide deletion (IDS, c.1270delG) causing MPS II in a proband by targeted nextgeneration sequencing. Then, the mutation site from proband's parents was analyzed and we found the carrier status for his mother. This candidate mutation was not stored in any human disease databases such as
2342
X. Wei et al. / Clinica Chimica Acta 412 (2011) 2340–2342
Fig. 1. Detection of a novel candidate mutation (IDS, c.1270delG) causing MPS II in a male proband. A. A small number of reads spanning the mutation site are expanded to show their DNA sequence and the C in positive genomic strand which was detected to be deleted in all mapped reads displayed, indicating an entire deficiency of IDS gene in X chromosome of the male proband. Arrow, changed base. B. DNA sequencing assay of the mutation in the proband's family confirmed a single-nucleotide deletion in proband and proband's mother, but not in proband's father and the control female individual. The sequencing assay was performed on the negative genomic strand. Arrow, changed base. C. Illustration of carrier status in the proband's family. The candidate mutation causing MPS II was inherited from proband's mother coincident with X-linked inheritance. Arrow, proband. D. The panel presents the wild-type and truncating protein sequence (p.V424FfsX16) expressed by IDS gene. Detail peptide sequence around the mutation site was expanded. Arrow, the first amino acid changed.
HGMD, LSMD, etc. and also not a literature-annotated disease mutation, so the mutation (IDS, c.1270delG) was firstly discovered in this study. IDS gene comprises nine exons, and the exon 9 is the longest one of 4.5 kb coding 158 amino acids and one of the hot spots in IDS gene [10]. As reported, IDS gene encodes a 550-amino acid precursor of iduronate 2-sulfatase that is cleaved into two kinds of polypeptides in 42- and 14kDa respectively by an internal proteolysis in human liver [11,12]. The 14-kDa polypeptide contains 95 amino acids C-terminal and the 42kDa polypeptide contains 455 amino acids N-terminal. The candidate mutation resulted in elongation of the amino acid chain to a terminal codon TGA at position 439, thus the ultimate peptide was shortened from 550 to 438 amino acids. Therefore this candidate mutation led to a 15-amino acids change C-terminal and a 112-amino acids loss. We speculated that eventually amino acids variations induced the potential activity deficiency of the 42-kDa polypeptide but completely activity loss of the 14-kDa polypeptide encoded in the hot spot-exon 9. In this study, we employed targeted sequence capture and nextgeneration sequencing to detect candidate mutation from IDS gene for the proband with MPS II disorder. At the meantime, we also excluded the possibility of MPS I disorder by detecting variations in IDUA gene. All the base pairs of target chromosome intervals were successfully sequenced, and a novel candidate mutation resulting in frameshift and premature truncation of I2S protein was identified. Targeted nextgeneration sequencing, especially exons of hot spots, has considerable potential for both clinic and research use. The sequence capture followed by HiSeq2000 sequencing was specifically used in our pipeline. Combined applications of this platform and bioinformatics are effective methods for MPS II-causing gene testing. Acknowledgments We thank all the blood donors for their invaluable contribution to this study. We thank Ping Yu from Zhejiang University for her kindly
help in special knowledge. We gratefully acknowledge support from the National High Technology Research and Development Program of China (863 Program, 2006AA02A302), the Shenzhen Development and Reform Commission project, and the project of International Innovation Team on the Cancer Genome Research of Guangdong Province.
References [1] Neufeld EF, Muenzer J. The mucopolysaccharidoses. In: Scriver CR, Beaudet AL, Sly WS, Valle D, editors. The Metabolic & Molecular Bases of Inherited Disease, 8th ed., Vol. 2. New York: McGraw-Hill; 2001. [2] Wraith JE, Scarpa M, Beck M, et al. Mucopolysaccharidosis type II (Hunter syndrome): a clinical review and recommendations for treatment in the era of enzyme replacement therapy. Eur J Pediatr 2008;167:267–77. [3] Wraith JE, Cooper A, Thornley M, et al. The clinical phenotype of two patients with a complete deletion of the iduronate-2-sulphatase gene (mucopolysaccharidosis II — Hunter syndrome). Hum Genet 1991;87:205–6. [4] Bielicki J, Freeman C, Clements PR, et al. Human liver iduronate-2-sulphatase. Purification, characterization and catalytic properties. Biochem J 1990;271:75–86. [5] Flomen RH, Green PM, Bentley DR, et al. Detection of point mutations and a gross deletion in six Hunter syndrome patients. Genomics 1992;13:543–50. [6] Wilson PJ, Suthers GK, Callen DF, et al. Frequent deletions at Xq28 indicate genetic heterogeneity in Hunter syndrome. Hum Genet 1991;86:505–8. [7] Bell CJ, Dinwiddie DL, Miller NA, et al. Carrier testing for severe childhood recessive diseases by next-generation sequencing. Sci Transl Med 2011;3 65ra4. [8] Shen P, Wang W, Krishnakumar S, et al. High-quality DNA sequence capture of 524 disease candidate genes. Proc Natl Acad Sci 2011;108:6549–54. [9] Simpson DA, Clark GR, Alexander S, et al. Molecular diagnosis for heterogeneous genetic diseases with targeted high-throughput DNA sequencing applied to retinitis pigmentosa. J Med Genet 2011;48:145–51. [10] Guo YB, Du CS. Detection of a new mutation (1343-TT) in the iduronate-2sulfatase gene from a Chinese patient with mucopolysaccharidosis type II. Zhonghua Er Ke Za Zhi 2006;44:110–3. [11] Di NP, Ronsisvalle L. Identification and partial characterization of two enzyme forms of iduronate sulfatase from human placenta. Biochim Biophys Acta 1981;661:106–11. [12] Wilson PJ, Morris CP, Anson DS, et al. Hunter syndrome: isolation of an iduronate-2sulfatase cDNA clone and analysis of patient DNA. Proc Natl Acad Sci 1990;87: 8531–5.
Contents lists available at SciVerse ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Short communication
A novel mutation of IDS gene in a Chinese patient with mucopolysaccharidosis II by next-generation sequencing Xiaoming Wei a, 1, Fan Jin b, 1, Yinghui Ye b, Chenming Xu b, Ning Qu a, Xiangchun Ju a, Xin Yi a,⁎ a b
Beijing Genomics Institute at Shenzhen, Shenzhen 518083, China Zhejiang University School of Medicine Women's Hospital, Hangzhou 310058, Zhejiang, China
a r t i c l e
i n f o
Article history: Received 15 August 2011 Received in revised form 19 August 2011 Accepted 30 August 2011 Available online 3 September 2011 Keywords: Iduronate 2-sulfatase gene Mucopolysaccharidosis II Target sequence capture Next-generation sequencing
a b s t r a c t Background: Mucopolysaccharidosis (MPS) is induced by the absence or malfunctioning of lysosomal enzymes. MPS I and MPS II are similar in phenotypes but they are different in genotypes, which are caused by the deficiencies of alpha-L-iduronidase gene (IDUA) and iduronate 2-sulfatase gene (IDS) respectively. In this work, a 5-year-old Chinese young male with manifestations of MPS in a family with unaffected parents was described. Methods: 12 kb of all the targeted exon sequences plus flanking sequences chromosomal DNA of IDS and IDUA genes from the proband and 20 other case-unrelated controls were captured and sequenced by using next-generation sequencing technology. Results: One single-nucleotide deletion variant (c.1270delG) resulting in frameshift and premature truncation of I2S enzyme was detected, out of 20 controls, only in the proband, and which was further verified by Sanger sequencing. The proband's mother was also proved carrying c.1270delG by Sanger method but not for his father. Conclusions: The novel variant (c.1270delG) is a candidate disease-causing mutation predicted to affect the normal structure and function of the enzyme. Target sequence capture and next-generation sequencing technology can be effective for the gene testing of MPS II disorder. © 2011 Elsevier B.V. All rights reserved.
1. Introduction The mucopolysaccharidosis (MPS) is a group of inherited metabolic disorders caused by deficiency of specific lysosomal enzymes involved in the processing of complex carbohydrates called mucopolysaccharide. Totally, 11 main types of MPS are recorded in OMIM database, and their clinical manifestations vary with different types of lysosomal enzymes affecting mucopolysaccharide. Mucopolysaccharidosis I (MPS I) is by far the most common type and classified into three major subtypes based on symptom severity. All three types of MPS I are caused by mutation in the gene encoding alpha-L-iduronidase (IDUA, MIM# 252800). The cytogenetic location of IDUA gene is 4p16.3, and the entire chromosome interval is 19 kb length containing 14 exons. IDUA enzyme can hydrolyze the terminal alpha-L-iduronic acid residues of the glycosaminoglycans dermatan sulfate and heparan sulfate and play important roles in the pathology of MPS II [1]. Mucopolysaccharidosis II (MPS II, also known as Hunter syndrome, MIM# 309900) is a rare X-linked recessive disorder caused by deficiency
⁎ Corresponding author at: Beijing Genomics Institute at Shenzhen, Beishan Industrial Zone, Yantian District, Shenzhen 518083, China. Tel.: + 86 755 25273461; fax: + 86 755 25273620. E-mail address: [email protected] (X. Yi). 1 Xiaoming Wei and Fan Jin contributed equally to this work. 0009-8981/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2011.08.031
of I2S activity, which leads to progressive accumulation of GAGs in multisystem [2]. MPS II is less frequent than MPS I but has no corneal clouding and is similar, but milder, to MPS I. MPS II is expected to be found mainly in males. In severely affected patients, death usually occurs before adulthood, but a high degree of clinical variability exists with many individuals surviving well into adulthood, which is thought to be related to different mutations of IDS gene [3]. The human iduronate 2sulfatase gene (IDS, MIM# 300823) is located in chromosome Xq28 and contains 9 exons spanning approximately 24 kb chromosome region. IDS gene encodes a lysomal exo-enzyme called iduronate 2-sulfatase (I2S), which can give rise to a breakdown of large sugar molecular glycosaminoglygans (GAGs) including heparin sulfate and dermatan sulfate [4]. Urine GAGs and skeletal survey are important factors in establishing the presence of MPS, but these detective methods are not specific to MPS II. The gold standard for diagnosing MPS II is to detect the deficiency of I2S activity. IDS is the only gene in which mutations are known to be related to MPS II and that has a conclusive role on the diagnosis for MPS II. More than 350 mutations in the IDS gene have been found to be associated with MPS II according to the record stored in the Human Gene Mutation Database (HGMD, http://www.hgmd.org/). All mutations inducing MPS II reduce or completely disrupt the biochemical activity of I2S enzyme and the mutations causing rearrangement, nonsense variation or complete absence of IDS lead to the more severe forms of MPS II
X. Wei et al. / Clinica Chimica Acta 412 (2011) 2340–2342
disorder [5,6]. The gold criterion for diagnosis on MPS II is detecting activity of I2S enzyme, while the clinical finding has shown limitation for phenotypic diversity depending on disease severity. Genetic testing for IDS gene shows a critical role in distinguishing different kind of clinical entities. Recently, the targeted next-generation sequencing has shown considerable potential in the gene testing for genetic diseases and compensated for the inability of Sanger sequencing in variation discovery [7–9]. Here, we reported a novel single-nucleotide-deletion variant as the candidate disease-causing mutation identified by targeted nextgeneration sequencing from a young male with MPS II disorder. 2. Materials and methods 2.1. Blood samples collection Written informed consent was obtained from blood donors who participated in the study before the collection of 5–10 ml of their peripheral blood for gene testing. Genomic DNA was extracted from peripheral blood samples according to the manufacturer's standard procedure using the QIAamp DNA BloodMiNi Kit (Qiagen, Hilden, Germany). 2.2. Targeted next-generation sequencing Genomic DNA from the proband's and controls' peripheral blood were fragmented, and 12 kb of the targeted exon sequences plus flanking sequences (all exons plus 100 bp on each side of a exon) of IDS and IDUA genes were specifically captured and enriched using array-based hybridization chip (NimbleGen, Madison, USA) followed by HiSeq2000 (Illumina, San Diego, USA) sequencing to generate paired end reads (90 bps at each end), adhering to the standard operating protocols. Short reads mapping, alignment were performed using BWA software (Burrows Wheeler Aligner). SNPs and indels were detected using the SOAPsnp software and GATK Indel Genotyper (http://www.broadinstitute.org/gsa/ wiki/index.php/, The Genome Analysis Toolkit) respectively. All reference sequences were based on the NCBI37/hg19 assembly of the human genome. 2.3. Candidate mutation confirmed by Sanger sequencing The potential mutated base and flanking sequence of IDS gene were amplified by polymerase chain reaction (PCR) and sequenced by Sanger sequencing (Forward primer 5′-TGAGGTGCCGAGGTGGTGTT and reverse primer 5′-ATAGTCTATGGTGCGTATGG AATAGCC). The sequencing result was compared with annotated IDS gene reference sequence (NG011900.1) to confirm the candidate nucleotide changes. 3. Results 3.1. Proband descriptions The proband was a five-year-old Chinese young male. He was 1.05 m in height with a head circumference of 55 cm and weighed 22 kg. He suffered from a coarse face with protruding forehead, coarse eyebrows, big nostrils, full lips. And the following characteristics also appeared: clawing of hands, funnel chest, and so on. The proband's roentgenograms did not show any obvious enlargement in sella turcica, and condyle was slightly planus. Thick and short clavicle, thick ribs, and partly lace-shaped ribs were observed. Right humerus, ulna, radius, tibia, fibula, and right metacarpus and phalange were thick and short. Metacarpus proximal end turned cuspidal. Right ulna and radius showed a V-shaped structure. Skeletal lag was found. Thoracolumbar vertebra was biconvex lens-like. Lower part of front edge of lumbar vertebral body had a beaklike protrusion. At the joint of thoracolumbar vertebra, slight hyboma was found. Ala ossis ilii turned wide and abducent/double coxa valga were found. Plasticity of upper and lower extremity long bone was suboptimal, and diaphysis did not show normal contraction changes. CT
2341
images have indicated no abnormality. His parents' phenotypes were normal and they were not of intermarriage. 3.2. Identification and characterization of candidate mutation Approximately 7.63 kb of exons and adjacent intronic regions of IDS gene for the proband were captured and sequenced in our study. For IDS gene, the average coverage for each base pair is approximately 114×, and the average median-depth across nine exons is 119×. On average 98.3% of base pairs with N20× coverage were successfully detected indicating high capability for variants identification. Only two variants were detected in coding region of exon 9 and intron 3 of IDS gene respectively. The single-nucleotide-insertion variation (IVS3-131_-132insC, NM_ 000202.5) was detected in the intronic non-coding region in all of 44 reads that spanned this position. The variants in the coding region of exon 9 (c.1270delG, NM_000202.5) were detected in all of 148 reads covering the changed nucleotide (Fig. 1A), indicating a complete disruption of IDS gene in the X-chromosome for the proband. Considering the existence of the intronic insertion change in 14 of these controls, we classified the intronic variant as the single nucleotide polymorphism (SNP). The deletion variant was detected only in the proband and predicted as the candidate mutation causing MPS II. Considering the similarity of MPS II and MPS I in phenotype, we also detected variations in MPS I-causing gene IDUA via the same pipeline as described for IDS gene. For IDUA gene encoding alpha-Liduronidase, two single-nucleotide variants (c.352 C N T and c.300– 246 C N T) were found. Both of the two variants were detected in 8 of these controls and identified to be SNPs with the frequency of 0.235 and 0.226 respectively based on the data in dbSNP database. To confirm the deletion variant (IDS, c.1270delG) and uncover the inheritance background of MPS II for the proband, Sanger sequencing was employed for the proband and his parents, as well as a case-unrelated control (Fig. 1B). The deletion variant of X-located IDS gene was identified in this proband. As detected by Sanger sequencing, the target base for proband's father is same with reference sequence but not for proband's mother: a carrier status was identified for proband's mother. This overall family status is coincident with the classical manner of Mendel inheritance (Fig. 1C). This variant was not detected in the genome of the control. In translational level, the candidate IDS mutation (c.1270delG) was predicted to result in 127-amino acid changes including 112-amino acid deletion and 15-residues variation in I2S precursor (p.V424FfsX16) analyzed by Mutalyzer 2.0β-8 (http://www.lovd.nl/mutalyzer/) (Fig. 1D). 4. Discussion MPS I and MPS II are similar in phenotypes but different in genotypes. In our work, we captured and sequenced all the target regions of IDS and IDUA genes using targeted next-generation sequencing. The candidate mutation causing MPS for the proband was focused on IDS gene and the disease happened in the proband was further confirmed as type II MPS disorder. MPS II is an X-linked recessive, lysosomal storage disease caused by the abnormality of I2S enzyme, which is the only one among the MPS in which a son can alone inherit the defective gene from the mother. Because of the phenotype diversity, GAGs assay has shown deficiency in clarifying and diagnosing different types of MPS disease. Among the 351 mutations stored in HGMD database, approximately 10% of IDS gene mutations are nonsense and nearly 30% of mutations are described as small or gross deletion/insertion described at genomic or cDNA level indicating that massive amino acids change in the peptide chain plays much more essential roles than missense mutations on the pathology of MPS II. We report here that a candidate single-nucleotide deletion (IDS, c.1270delG) causing MPS II in a proband by targeted nextgeneration sequencing. Then, the mutation site from proband's parents was analyzed and we found the carrier status for his mother. This candidate mutation was not stored in any human disease databases such as
2342
X. Wei et al. / Clinica Chimica Acta 412 (2011) 2340–2342
Fig. 1. Detection of a novel candidate mutation (IDS, c.1270delG) causing MPS II in a male proband. A. A small number of reads spanning the mutation site are expanded to show their DNA sequence and the C in positive genomic strand which was detected to be deleted in all mapped reads displayed, indicating an entire deficiency of IDS gene in X chromosome of the male proband. Arrow, changed base. B. DNA sequencing assay of the mutation in the proband's family confirmed a single-nucleotide deletion in proband and proband's mother, but not in proband's father and the control female individual. The sequencing assay was performed on the negative genomic strand. Arrow, changed base. C. Illustration of carrier status in the proband's family. The candidate mutation causing MPS II was inherited from proband's mother coincident with X-linked inheritance. Arrow, proband. D. The panel presents the wild-type and truncating protein sequence (p.V424FfsX16) expressed by IDS gene. Detail peptide sequence around the mutation site was expanded. Arrow, the first amino acid changed.
HGMD, LSMD, etc. and also not a literature-annotated disease mutation, so the mutation (IDS, c.1270delG) was firstly discovered in this study. IDS gene comprises nine exons, and the exon 9 is the longest one of 4.5 kb coding 158 amino acids and one of the hot spots in IDS gene [10]. As reported, IDS gene encodes a 550-amino acid precursor of iduronate 2-sulfatase that is cleaved into two kinds of polypeptides in 42- and 14kDa respectively by an internal proteolysis in human liver [11,12]. The 14-kDa polypeptide contains 95 amino acids C-terminal and the 42kDa polypeptide contains 455 amino acids N-terminal. The candidate mutation resulted in elongation of the amino acid chain to a terminal codon TGA at position 439, thus the ultimate peptide was shortened from 550 to 438 amino acids. Therefore this candidate mutation led to a 15-amino acids change C-terminal and a 112-amino acids loss. We speculated that eventually amino acids variations induced the potential activity deficiency of the 42-kDa polypeptide but completely activity loss of the 14-kDa polypeptide encoded in the hot spot-exon 9. In this study, we employed targeted sequence capture and nextgeneration sequencing to detect candidate mutation from IDS gene for the proband with MPS II disorder. At the meantime, we also excluded the possibility of MPS I disorder by detecting variations in IDUA gene. All the base pairs of target chromosome intervals were successfully sequenced, and a novel candidate mutation resulting in frameshift and premature truncation of I2S protein was identified. Targeted nextgeneration sequencing, especially exons of hot spots, has considerable potential for both clinic and research use. The sequence capture followed by HiSeq2000 sequencing was specifically used in our pipeline. Combined applications of this platform and bioinformatics are effective methods for MPS II-causing gene testing. Acknowledgments We thank all the blood donors for their invaluable contribution to this study. We thank Ping Yu from Zhejiang University for her kindly
help in special knowledge. We gratefully acknowledge support from the National High Technology Research and Development Program of China (863 Program, 2006AA02A302), the Shenzhen Development and Reform Commission project, and the project of International Innovation Team on the Cancer Genome Research of Guangdong Province.
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