Mutation analysis of 11 Chinese patients with attenuated mucopolysaccharidosis type Ⅰ

Objective Mucopolysaccharidosis type Ⅰ (MPS Ⅰ ) is an autosomal recessive diseaseresulting from the deficiency in the lysosomal enzyme α-L-iduronidase (IDUA). The present study was conducted to identify IDUA gene mutations in attenuated (MPS Ⅰ H/S and MPS Ⅰ S) patients with MPS Ⅰin northern China. Methods Fourteen exons with adjacent intronic sequences of the IDUA gene in 11 MPS Ⅰ patients were amplified by polymerase chain reaction (PCR), and the PCR products were sequenced directly and origin analysis was conducted. Results Seven mutations were detected in the 11 MPS Ⅰ patients, i.e., c. 236 C＞T(p. A79V), c. 266 G＞A(p. R89Q), c. 265 C＞T(p. R89W), c. 532G＞A(p.E178K), c. 589G＞A(p. G197S), c. 1037T＞G(p. L346R), and c. 1877 G＞A(p. W626X). All of them were known mutations. Six patients were homozygotes and 1 was heterozygote with nonsense mutation. In addition, 9 reported single nucleotide polymorphism (SNP) were detected, i. e., p. A8, p. A20, p. H33Q,p. R105Q, p. A314, p. A361T, p. T388, p. T410 and p. V454I. Conclusion The mutation spectrum of the IDUA gene in attenuated MPS Ⅰ Chinese patients may be different from that in patients from other countries.     

11例中国人粘多糖贮积症Ⅰ型轻型患者的基因突变检测

目的 对经酶学确诊的粘多糖贮积症Ⅰ型患者进行α-L-艾杜糖苷酸酶(α-L-iduronidase,IDUA)基因突变检测,了解中国北方地区粘多糖贮积症Ⅰ型轻型患者基因突变特点.方法 应用PCR扩增技术及直接测序技术对11例中国北方地区经酶学确诊的粘多糖贮积症Ⅰ型患者IDUA基因14个外显子及相邻区域进行突变筛查,同时进行亲本来源分析.结果 11例患者中检测出7种基因突变:A79V、R89Q、R89w、E178K、G197S、L346R和W626X.7种突变均为已报道过子,11例中6例患者为纯合改变,1例为无义突变杂合子;发现了9种已知多态性位点.结论 中国北方地区粘多糖贮积症Ⅰ型轻型患者IDUA基因的突变特点可能不同于其他国家和地区.

Abstract：

Objective Mucopolysaccharidosis type Ⅰ (MPS Ⅰ ) is an autosomal recessive diseaseresulting from the deficiency in the lysosomal enzyme α-L-iduronidase (IDUA). The present study was conducted to identify IDUA gene mutations in attenuated (MPS Ⅰ H/S and MPS Ⅰ S) patients with MPS Ⅰin northern China. Methods Fourteen exons with adjacent intronic sequences of the IDUA gene in 11 MPS Ⅰ patients were amplified by polymerase chain reaction (PCR), and the PCR products were sequenced directly and origin analysis was conducted. Results Seven mutations were detected in the 11 MPS Ⅰ patients, i.e., c. 236 C＞T(p. A79V), c. 266 G＞A(p. R89Q), c. 265 C＞T(p. R89W), c. 532G＞A(p.E178K), c. 589G＞A(p. G197S), c. 1037T＞G(p. L346R), and c. 1877 G＞A(p. W626X). All of them were known mutations. Six patients were homozygotes and 1 was heterozygote with nonsense mutation. In addition, 9 reported single nucleotide polymorphism (SNP) were detected, i. e., p. A8, p. A20, p. H33Q,p. R105Q, p. A314, p. A361T, p. T388, p. T410 and p. V454I. Conclusion The mutation spectrum of the IDUA gene in attenuated MPS Ⅰ Chinese patients may be different from that in patients from other countries.     

两个新RUNX2基因突变引起家族性锁骨颅骨发育不全

Objective To identify the RUNX2 gene mutation in two unrelated Chinese families with cleidocranial dysplasia (CCD), and to assess the feasibility of gene diagnosis for patients with CCD. Methods Genomic DNA was isolated from peripheral blood samples of 4 patients and 4 healthy members in the two pedigrees as well as 102 unrelated healthy controls. All 7 coding exons and their flanking intronic sequences of the RUNX2 gene were amplified by PCR, then the PCR products were sequenced bi-directionally. The sequencing results were compared with normal sequences in GenBank to identify the mutation. The mutation was confirmed by RFLP with restriction endonuclease. Results In one family, a novel heterozygous missense mutation c. 346T＞A (W116R) in exon 1 of the RUNX2 gene was detected in the two affected individuals, and the mutation was further confirmed with Bsr Ⅰ restriction endonuclease digestion. In the other family, a novel nonsense mutation c. 610A＞T (K204X) was identified in the two patients. No above sequence change was found in the 102 healthy controls. Conclusion Two novel RUNX2 mutations were found in two unrelated Chinese families with cleidoeranial dysplasia. The identification of these mutations further extended the mutation spectrum of RUNX2 gene and will facilitate prenatal diagnosis and gene diagnosis of CCD.     

两个新RUNX2基因突变引起家族性锁骨颅骨发育不全

Objective To identify the RUNX2 gene mutation in two unrelated Chinese families with cleidocranial dysplasia (CCD), and to assess the feasibility of gene diagnosis for patients with CCD. Methods Genomic DNA was isolated from peripheral blood samples of 4 patients and 4 healthy members in the two pedigrees as well as 102 unrelated healthy controls. All 7 coding exons and their flanking intronic sequences of the RUNX2 gene were amplified by PCR, then the PCR products were sequenced bi-directionally. The sequencing results were compared with normal sequences in GenBank to identify the mutation. The mutation was confirmed by RFLP with restriction endonuclease. Results In one family, a novel heterozygous missense mutation c. 346T＞A (W116R) in exon 1 of the RUNX2 gene was detected in the two affected individuals, and the mutation was further confirmed with Bsr Ⅰ restriction endonuclease digestion. In the other family, a novel nonsense mutation c. 610A＞T (K204X) was identified in the two patients. No above sequence change was found in the 102 healthy controls. Conclusion Two novel RUNX2 mutations were found in two unrelated Chinese families with cleidoeranial dysplasia. The identification of these mutations further extended the mutation spectrum of RUNX2 gene and will facilitate prenatal diagnosis and gene diagnosis of CCD.     

Mutation screening of the F Ⅷ gene in 10 hemophilia A families

Objective To identify the F Ⅷ gene mutations of patients and suspected female carriers in 10 Hemophilia A (HA) families, and to guide the prenatal diagnosis. Methods PCR, denaturing high performance liquid chromatogramphy(DHPLC) and DNA sequencing technologies were applied to screen the FⅧ gene of 8 HA patients and 12 suspected female carriers in the 10 families. Linkage analysis was performed by using St 14 (DXS 52), intron 13 (CA)n and EX18/Bcl Ⅰ of the FⅧ gene in the HA families.In prenatal diagnosis, we screened the same mutation found in the patients. PCR-restriction fragment length polymorphism was applied to detect the new missense mutations of F Ⅷ gene in 100 unrelated healthy individuals to exclude the possibility of polymorphism. Results (1) Five missense mutations, 3 frameshift mutations, 2 nonsense mutations and 2 single nucleotide polymorphism(SNP) were identified in 10 the HA families. Among them, c. 878A＞G, c. 1015A＞G, c. 6870G＞T, c. 1282delA, c. 3072_3073insT, c. 4880_4881insA and c. 5000G＞A were novel mutations or polymorphism. No missense mutations c. 878A＞G, c.1015A＞G and c. 6870G＞T, were found in the 100 healthy unrelated controls. (2) Nine suspected female carriers were confirmed at the gene level. (3) X risk chromosome could be determined in 4 HA families by genetic linkage analysis. (4) Among the four fetuses for prenatal diagnosis, 2 were normal, 1 was carrier and the remaining 1 was a patient. Conclusion Six novel mutations, i. e. , c. 878A＞G, c. 1015A＞G, c.6870G＞T, c. 1282delA, c. 3072_3073insT and c. 4880_4881insA, were identified in this study. PCR,DHPLC and DNA sequencing could be used to screen the gene mutations of HA patients, to carry out carrier detection and prenatal diagnosis of HA families efficiently, by combining with restriction endonuclease analysis and genetic linkage analysis.     

Mutation screening of the F Ⅷ gene in 10 hemophilia A families

Objective To identify the F Ⅷ gene mutations of patients and suspected female carriers in 10 Hemophilia A (HA) families, and to guide the prenatal diagnosis. Methods PCR, denaturing high performance liquid chromatogramphy(DHPLC) and DNA sequencing technologies were applied to screen the FⅧ gene of 8 HA patients and 12 suspected female carriers in the 10 families. Linkage analysis was performed by using St 14 (DXS 52), intron 13 (CA)n and EX18/Bcl Ⅰ of the FⅧ gene in the HA families.In prenatal diagnosis, we screened the same mutation found in the patients. PCR-restriction fragment length polymorphism was applied to detect the new missense mutations of F Ⅷ gene in 100 unrelated healthy individuals to exclude the possibility of polymorphism. Results (1) Five missense mutations, 3 frameshift mutations, 2 nonsense mutations and 2 single nucleotide polymorphism(SNP) were identified in 10 the HA families. Among them, c. 878A＞G, c. 1015A＞G, c. 6870G＞T, c. 1282delA, c. 3072_3073insT, c. 4880_4881insA and c. 5000G＞A were novel mutations or polymorphism. No missense mutations c. 878A＞G, c.1015A＞G and c. 6870G＞T, were found in the 100 healthy unrelated controls. (2) Nine suspected female carriers were confirmed at the gene level. (3) X risk chromosome could be determined in 4 HA families by genetic linkage analysis. (4) Among the four fetuses for prenatal diagnosis, 2 were normal, 1 was carrier and the remaining 1 was a patient. Conclusion Six novel mutations, i. e. , c. 878A＞G, c. 1015A＞G, c.6870G＞T, c. 1282delA, c. 3072_3073insT and c. 4880_4881insA, were identified in this study. PCR,DHPLC and DNA sequencing could be used to screen the gene mutations of HA patients, to carry out carrier detection and prenatal diagnosis of HA families efficiently, by combining with restriction endonuclease analysis and genetic linkage analysis.     

中国Rett综合征患儿突变基因的亲源分析

Objective To identify the parental origin of methyl-CpG-binding protein 2 (MECP2)gene mutations in Chinese patients with Rett syndrome. Methods Single nucleotide polymorphisms (SNPs) in intron 3 of the MECP2 gene were analyzed by PCR and sequencing in 115 patients with Rett syndrome.Then sequencing of the SNP region was performed for the fathers of the patients who had at least one SNP,to determine which allele was from the father. Then allele-specific PCR was performed and the products were sequenced to see whether the allele from father or mother harbored the mutation. Results Seventy-six of the 115 patients had at least one SNP. Three hot SNPs were found in these patients. They were: IVS3+22C＞G, IVS3+266C＞T and IVS3+683C＞T. Among the 76 cases, 73 had a paternal origin of MECP2 mutations, and the other 3 had a maternal origin. There were multiple types of MECP2 mutation of the paternal origin, including 4 frame shift, 2 deletion and 67 point (56 C＞T, 6 C＞G, 2 A＞G, 2 G＞T and 1 A＞T) mutations. The mutation types of the 3 ptients with maternal origin included 2 frame shift and 1 point (C＞T) mutation. Conclusion In Chinese RTT patients, the MECP2 mutations are mostly of paternal origin.     

中国Rett综合征患儿突变基因的亲源分析

Objective To identify the parental origin of methyl-CpG-binding protein 2 (MECP2)gene mutations in Chinese patients with Rett syndrome. Methods Single nucleotide polymorphisms (SNPs) in intron 3 of the MECP2 gene were analyzed by PCR and sequencing in 115 patients with Rett syndrome.Then sequencing of the SNP region was performed for the fathers of the patients who had at least one SNP,to determine which allele was from the father. Then allele-specific PCR was performed and the products were sequenced to see whether the allele from father or mother harbored the mutation. Results Seventy-six of the 115 patients had at least one SNP. Three hot SNPs were found in these patients. They were: IVS3+22C＞G, IVS3+266C＞T and IVS3+683C＞T. Among the 76 cases, 73 had a paternal origin of MECP2 mutations, and the other 3 had a maternal origin. There were multiple types of MECP2 mutation of the paternal origin, including 4 frame shift, 2 deletion and 67 point (56 C＞T, 6 C＞G, 2 A＞G, 2 G＞T and 1 A＞T) mutations. The mutation types of the 3 ptients with maternal origin included 2 frame shift and 1 point (C＞T) mutation. Conclusion In Chinese RTT patients, the MECP2 mutations are mostly of paternal origin.     

Analysis of clinical manifestation and genetic mutations in two patients with Cornelia de Lange syndromeCSCD

Objective: To detect potential mutations in two neonates suspected for Cornelia de Lange syndrome (CdLS). Methods: Peripheral blood samples from the neonates and their parents were collected and analyzed for CdLS-related genes using targeted sequence capture and next-generation sequencing. Suspected mutations were confirmed by direct Sanger sequencing. Results: The neonates were found to respectively carry mutations c. 7219C>T and p. D2339Lfs ∗ 4 of the NIPBL gene, among which the p. D2339Lfs ∗ 4 mutation has not been reported previously. No pathogenic mutation was found in other CdLS-related genes including NIPBL, SMC1A, SMC3, RAD21 and HDAC8. Conclusion: The c. 7219C>T and p. D2339Lfs ∗ 4 mutations of the NIPBL gene probably account for the disease in both patients. © 2018 MeDitorial Ltd. All rights reserved.     

河南地区苯丙酮尿症患者苯丙氨酸羟化酶基因突变研究

Objective To study the characteristics of the phenylalanine hydroxylase gene (PAH)mutations in patients with phenylketonuria (PKU) in Henan province, in order to provide basic information for genetic counseling and prenatal diagnosis. Methods Mutations of the PAH gene were detected in exons 1-13 with flanking introns of PAH gene by PCR and DNA sequencing in 47 families with PKU. Results A total of 25 different mutations were detected in 83 out of 94 PAH alleles (88. 3%). Among them,E79fX13, H271R and D415Y have not been reported previously. It was the first time that IVS10-14C＞Gmutation was reported in Chinese PKU population. The mutations p. R243Q, EX6-96A＞G, p. Y356X,IVS4-1G＞A, p. R111X, p. V399V and p. R413P, were the prevalent mutations with relative frequencies of 20. 5 %, 12.0%, 9.6%, 9. 6%, 8. 4%, 8. 4% and 7.2% respectively. Conclusion The mutations of the PAH gene in patients with classical phenylketonuria in Henan province were similar to that in other areas of China. Prenatal gene diagnosis for PKU by PAH gene sequencing is efficient for most PKU families.     

河南地区苯丙酮尿症患者苯丙氨酸羟化酶基因突变研究

Objective To study the characteristics of the phenylalanine hydroxylase gene (PAH)mutations in patients with phenylketonuria (PKU) in Henan province, in order to provide basic information for genetic counseling and prenatal diagnosis. Methods Mutations of the PAH gene were detected in exons 1-13 with flanking introns of PAH gene by PCR and DNA sequencing in 47 families with PKU. Results A total of 25 different mutations were detected in 83 out of 94 PAH alleles (88. 3%). Among them,E79fX13, H271R and D415Y have not been reported previously. It was the first time that IVS10-14C＞Gmutation was reported in Chinese PKU population. The mutations p. R243Q, EX6-96A＞G, p. Y356X,IVS4-1G＞A, p. R111X, p. V399V and p. R413P, were the prevalent mutations with relative frequencies of 20. 5 %, 12.0%, 9.6%, 9. 6%, 8. 4%, 8. 4% and 7.2% respectively. Conclusion The mutations of the PAH gene in patients with classical phenylketonuria in Henan province were similar to that in other areas of China. Prenatal gene diagnosis for PKU by PAH gene sequencing is efficient for most PKU families.     

Clinical and molecular genetic analysis for 7 patients from 5 pedigrees with 17α-hydroxylase/17, 20 lyase deficiency

Objective To investigate the clinical and genetic characteristics of 7 patients from 5 families with 17α-hydroxylase/17, 20 lyase deficiency (17OHD) and the CYP17A1 mutation in Chinese. Methods Clinical features and laboratory data were collected from 5 families with 17OHD. PCR direct sequencing was performed to screen the mutation of CYP17A1 gene of the patients. Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and sequencing were performed to screen the mutations of CYP17A1 gene in 288 healthy individuals from Shandong province. Results Seven patients (5 of them were 46, XX; 2 were 46, XY) had typical clinical presentation of sexual infantilism, hypertension and hypokalemia. Hormone profile indicated decreased plasma cotisol and sex hormones, and elevated blood adrenocorticotrophic hormone (ACTH). TAC329AA and H373L in exon 6 and D487_ F489del in exon 8 were identified from the patients. One heterozygote for D487_F489del was identified in 288 healthy controls. Conclusion The TAC329AA and D487_F489del of the CYP17A1 gene were the most frequent mutations in Chinese with 17OHD. There might be certain frequency of heterozygotes for D487_ F489del in Chinese population.     

Clinical and molecular genetic analysis for 7 patients from 5 pedigrees with 17α-hydroxylase/17, 20 lyase deficiency

Objective To investigate the clinical and genetic characteristics of 7 patients from 5 families with 17α-hydroxylase/17, 20 lyase deficiency (17OHD) and the CYP17A1 mutation in Chinese. Methods Clinical features and laboratory data were collected from 5 families with 17OHD. PCR direct sequencing was performed to screen the mutation of CYP17A1 gene of the patients. Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and sequencing were performed to screen the mutations of CYP17A1 gene in 288 healthy individuals from Shandong province. Results Seven patients (5 of them were 46, XX; 2 were 46, XY) had typical clinical presentation of sexual infantilism, hypertension and hypokalemia. Hormone profile indicated decreased plasma cotisol and sex hormones, and elevated blood adrenocorticotrophic hormone (ACTH). TAC329AA and H373L in exon 6 and D487_ F489del in exon 8 were identified from the patients. One heterozygote for D487_F489del was identified in 288 healthy controls. Conclusion The TAC329AA and D487_F489del of the CYP17A1 gene were the most frequent mutations in Chinese with 17OHD. There might be certain frequency of heterozygotes for D487_ F489del in Chinese population.     

Clinical and molecular genetic analysis for 7 patients from 5 pedigrees with 17α-hydroxylase/17, 20 lyase deficiency

Objective To investigate the clinical and genetic characteristics of 7 patients from 5 families with 17α-hydroxylase/17, 20 lyase deficiency (17OHD) and the CYP17A1 mutation in Chinese. Methods Clinical features and laboratory data were collected from 5 families with 17OHD. PCR direct sequencing was performed to screen the mutation of CYP17A1 gene of the patients. Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and sequencing were performed to screen the mutations of CYP17A1 gene in 288 healthy individuals from Shandong province. Results Seven patients (5 of them were 46, XX; 2 were 46, XY) had typical clinical presentation of sexual infantilism, hypertension and hypokalemia. Hormone profile indicated decreased plasma cotisol and sex hormones, and elevated blood adrenocorticotrophic hormone (ACTH). TAC329AA and H373L in exon 6 and D487_ F489del in exon 8 were identified from the patients. One heterozygote for D487_F489del was identified in 288 healthy controls. Conclusion The TAC329AA and D487_F489del of the CYP17A1 gene were the most frequent mutations in Chinese with 17OHD. There might be certain frequency of heterozygotes for D487_ F489del in Chinese population.     

Clinical and molecular genetic analysis for 7 patients from 5 pedigrees with 17α-hydroxylase/17, 20 lyase deficiency

Objective To investigate the clinical and genetic characteristics of 7 patients from 5 families with 17α-hydroxylase/17, 20 lyase deficiency (17OHD) and the CYP17A1 mutation in Chinese. Methods Clinical features and laboratory data were collected from 5 families with 17OHD. PCR direct sequencing was performed to screen the mutation of CYP17A1 gene of the patients. Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and sequencing were performed to screen the mutations of CYP17A1 gene in 288 healthy individuals from Shandong province. Results Seven patients (5 of them were 46, XX; 2 were 46, XY) had typical clinical presentation of sexual infantilism, hypertension and hypokalemia. Hormone profile indicated decreased plasma cotisol and sex hormones, and elevated blood adrenocorticotrophic hormone (ACTH). TAC329AA and H373L in exon 6 and D487_ F489del in exon 8 were identified from the patients. One heterozygote for D487_F489del was identified in 288 healthy controls. Conclusion The TAC329AA and D487_F489del of the CYP17A1 gene were the most frequent mutations in Chinese with 17OHD. There might be certain frequency of heterozygotes for D487_ F489del in Chinese population.     

Clinical and molecular genetic analysis for 7 patients from 5 pedigrees with 17α-hydroxylase/17, 20 lyase deficiency

Objective To investigate the clinical and genetic characteristics of 7 patients from 5 families with 17α-hydroxylase/17, 20 lyase deficiency (17OHD) and the CYP17A1 mutation in Chinese. Methods Clinical features and laboratory data were collected from 5 families with 17OHD. PCR direct sequencing was performed to screen the mutation of CYP17A1 gene of the patients. Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and sequencing were performed to screen the mutations of CYP17A1 gene in 288 healthy individuals from Shandong province. Results Seven patients (5 of them were 46, XX; 2 were 46, XY) had typical clinical presentation of sexual infantilism, hypertension and hypokalemia. Hormone profile indicated decreased plasma cotisol and sex hormones, and elevated blood adrenocorticotrophic hormone (ACTH). TAC329AA and H373L in exon 6 and D487_ F489del in exon 8 were identified from the patients. One heterozygote for D487_F489del was identified in 288 healthy controls. Conclusion The TAC329AA and D487_F489del of the CYP17A1 gene were the most frequent mutations in Chinese with 17OHD. There might be certain frequency of heterozygotes for D487_ F489del in Chinese population.     

Clinical and molecular genetic analysis for 7 patients from 5 pedigrees with 17α-hydroxylase/17, 20 lyase deficiency

Objective To investigate the clinical and genetic characteristics of 7 patients from 5 families with 17α-hydroxylase/17, 20 lyase deficiency (17OHD) and the CYP17A1 mutation in Chinese. Methods Clinical features and laboratory data were collected from 5 families with 17OHD. PCR direct sequencing was performed to screen the mutation of CYP17A1 gene of the patients. Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and sequencing were performed to screen the mutations of CYP17A1 gene in 288 healthy individuals from Shandong province. Results Seven patients (5 of them were 46, XX; 2 were 46, XY) had typical clinical presentation of sexual infantilism, hypertension and hypokalemia. Hormone profile indicated decreased plasma cotisol and sex hormones, and elevated blood adrenocorticotrophic hormone (ACTH). TAC329AA and H373L in exon 6 and D487_ F489del in exon 8 were identified from the patients. One heterozygote for D487_F489del was identified in 288 healthy controls. Conclusion The TAC329AA and D487_F489del of the CYP17A1 gene were the most frequent mutations in Chinese with 17OHD. There might be certain frequency of heterozygotes for D487_ F489del in Chinese population.     

Clinical and molecular genetic analysis for 7 patients from 5 pedigrees with 17α-hydroxylase/17, 20 lyase deficiency

Objective To investigate the clinical and genetic characteristics of 7 patients from 5 families with 17α-hydroxylase/17, 20 lyase deficiency (17OHD) and the CYP17A1 mutation in Chinese. Methods Clinical features and laboratory data were collected from 5 families with 17OHD. PCR direct sequencing was performed to screen the mutation of CYP17A1 gene of the patients. Polymerase chain reaction-restriction fragment length polymorphism(PCR-RFLP) and sequencing were performed to screen the mutations of CYP17A1 gene in 288 healthy individuals from Shandong province. Results Seven patients (5 of them were 46, XX; 2 were 46, XY) had typical clinical presentation of sexual infantilism, hypertension and hypokalemia. Hormone profile indicated decreased plasma cotisol and sex hormones, and elevated blood adrenocorticotrophic hormone (ACTH). TAC329AA and H373L in exon 6 and D487_ F489del in exon 8 were identified from the patients. One heterozygote for D487_F489del was identified in 288 healthy controls. Conclusion The TAC329AA and D487_F489del of the CYP17A1 gene were the most frequent mutations in Chinese with 17OHD. There might be certain frequency of heterozygotes for D487_ F489del in Chinese population.