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Six novel mutations of the ADAR1 gene in Chinese patients with dyschromatosis symmetrica hereditaria
Journal of Dermatological Science (2008) 50, 109—114
www.intl.elsevierhealth.com/journals/jods
Six novel mutations of the ADAR1 gene in Chinese patients with dyschromatosis symmetrica hereditaria Furen Zhang a,1,*, Hong Liu a, Deke Jiang b, Hongqing Tian a, Changyuan Wang a, Long Yu b,1 a
Shandong Provincial Institute of Dermatovenereology, 57 Jiyan Lu, Jinan 250022, Shandong Province, PR China b State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, PR China Received 16 August 2007; received in revised form 13 November 2007; accepted 20 November 2007
KEYWORDS Dyschromatosis symmetrica hereditaria; Mutation analysis; ADAR1
Summary Background: Dyschromatosis symmetrica hereditaria (DSH) is a rare autosomal dominantly inherited dermatosis and characterized by a mixture of hyperpigmented and hypopigmented macules on the back of hands and feet. The DSH locus was mapped to chromosome 1q21 and subsequently pathogenic mutations were identified in the adenosine deaminase acting on RNA1 (ADAR1) gene in 2003. Objective: In this study, we performed a mutation analysis of the ADAR1 gene in eight Chinese families and one sporadic patient with typical DSH. Methods: PCR and direct sequencing of the ADAR1 gene were performed to identify and confirm the mutations in the eight families and the sporadic patient. Results: Six novel and one known mutations were identified, including four missense mutations (p.K1105N, p.G1047R, p.F1099L, p.G1068R), two frameshift mutations (p.Q779fs-792x, p.P441fs-463x) and one nonsense mutation (p.R1096x). Conclusion: Six novel mutations were found in five unrelated families and one sporadic case, which have further improved our understanding on the role of ADAR1 in DSH. Interestingly, we failed to detect any mutations of ADAR1 in two families. # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Abbreviations: DSH, dyschromatosis symmetrica hereditaria; ADAR1, adenosine deaminase acting on RNA1; PCR, polymerase chain reaction. * Corresponding author. Tel.: +86 531 87298801; fax: +86 531 87984734. E-mail address: [email protected] (FR. Zhang). 1 These two authors contributed equally to the paper. 0923-1811/$30.00 # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2007.11.011
110
FR. Zhang et al. we failed to detect any mutations of ADAR1 in two families.
1. Introduction Dyschromatosis symmetrica hereditaria (DSH, OMIM 127400), or symmetric dyschromatosis of the extremities, was first reported by Toyama in 1910 and formally named as a clinical entity in 1929 [1]. It is a pigmentary genodermatosis characterized by a mixture of hypopigmented and hyperpigmented macules distributed on the dorsa of hands and feet. Many patients with DSH also have small freckle-like pigmented macules on their faces. The lesions usually appear in infancy or early childhood, commonly stop spreading before adolescence, and last for life [2]. DSH has been reported mainly in Japan and China populations and also in many other ethnic groups [2]. DSH generally shows an autosomal dominant pattern of inheritance with high penetrance, but sporadic cases have also been reported. The DSH locus has been mapped to chromosome 1q21 by Chinese and Japanese groups, independently [3,4]. Pathogenic mutations were identified in the double-stranded RNA-specific adenosine deaminase (ADAR1) gene encoding double-stranded RNA-specific adenosine deaminase [4]. ADAR1 protein catalyzes the deamination of adenosine to inosine in double-stranded RNA substrates [5], which results in the creation of alternative splicing sites or alternations of codon and thus lead to functional changes in the protein. The ADAR1 gene is expressed ubiquitously, but its target gene(s) in the skin still remain unknown. The molecular pathogenesis of DSH has not been clarified yet. In this study, we performed a mutation analysis of the ADAR1 gene in eight Chinese families and one sporadic patient with typical DSH and identified six novel and one known mutations. Interestingly,
2. Materials and methods All patients analyzed in this study were admitted, diagnosed and treated at Shandong Provincial Institute of Dermatovenereology. Eight DSH pedigrees were recruited, all showing an autosomal dominant pattern of inheritance. A sporadic patient was also recruited. All the patients have typical macules distributed on the back of hands and feet. Table 1 summarizes the clinical and molecular findings of all the patients analyzed in this study. The study protocol was approved by Ethical Committee of both Fudan University and Shandong Provincial Institute of Dermatovenereology. Written informed consents were obtained from all the patients and their family members. DNA samples were extracted from peripheral blood samples using Takara DNA Mini Kit (Takara Biotechnology CO. LTD, Dalan, China). Control samples were obtained from the DNA collection at the State Key Laboratory of Genetic Engineering, Fudan University. All the 15 exons of ADAR1 genes and their flanking intronic sequences of 200 bps were amplified by PCR. The primers for amplifying exon 1 and exon 3 were designed in this study, and the rest primers were described in Zhang’s study [6]. PCR were carried out using a standard PCR reaction system, containing 18.7 mL of ddH2O, 2.5 mL of 10 PCR buffer, 2 mL of 1 dNTP, 0.5 mL of each primers, 0.3 mL of Taq DNA polymerase and 1 mL of genomic DNA. After an initial denaturation step at 94 degree for 5 min, 28 cycles of amplification consisting of 45 s at 94
Table 1 Clinical and molecular findings in this study Patient
Incidence
Affected individuals
Unaffected individuals
Part of lesion
Mutation
Exon
Mutation type
1 I
Sporadic Familial
— 2
— 16
p.K1105N p.G1047R
13 12
Missense Missense
II III
Familial Familial
2 4
4 6
p.R1096x p.Q779fs
13 7
Nonsense Frameshift
IV
Familial
3
4
p.P441fs
2
Frameshift
V
Familial
4
2
p.F1099L
13
Missense
VI VII
Familial Familial
4 6
5 14
p.G1068R —
12 —
Missense —
VIII
Familial
5
9
Back of hands and feet face Back of hands and feet face, calves, knees Back of hands and feet face Back of hands and feet face, neck Back of hands and feet Face, forearms Back of hands, back of feet (faint) Back of hands and feet forearms Back of hands and feet face, neck Back of hands and feet face, knees, ankles
—
—
—
Six novel mutations of the ADAR1 gene in Chinese patients with dyschromatosis symmetrica hereditaria degree, 30 s at annealing degree and 45 s at 72 degree were performed. Direct sequencing was performed using a DNA sequencing system (model 3730; ABI). Mutations were identified by comparing with the reported cDNA reference sequence (GenBank accession number: NM_001111). All the identified mutations were verified by the subsequent two-direction sequencing. Both the patients and control samples were analyzed using the same protocol.
3. Results The entire coding and flanking intronic sequences of ADAR1 were screened for mutations in eight families and one sporadic case of DSH. Sequencing analysis identified six novel and one known mutation, including four missense mutations (p.K1105N, p.G1047R, p.F1099L, p.G1068R), two frameshift mutations (p.Q779fs-792x, p.P441fs-463x) and one nonsense mutation (p.R1096x) (Fig. 1). We failed to detect any ADAR1 mutations in family VII and family VIII.
3.1. Missense mutations Four missense mutations were identified in the family I, V, VI and the sporadic case. Two base substitutions was 3139G > C and 3202G > C were identified in exon 12 in the family I and VI, causing Gly1047Arg and Gly1068Arg missense mutations respectively. Another two base substitutions 3295T > C and 3315G > T were identified in exon 13 in the family V and the sporadic case, causing Phe1099Leu and Lys1105Asn missense mutations.
3.2. Frameshift mutations Two different single-base deletions were identified and both lead to a shift in the reading frame and thus cause premature termination codons (PTCs). One deletion c.2337delA was identified in exon7 in the family III; and the other deletion c.1323delC was found in exon 2 in the family IV. The two mutations generated pre-terminating codon (PTC) at 13 and 22 codons downstream of deletion site respectively, resulting in truncated proteins likely without any functional activity.
3.3. Nonsense mutations One nonsense mutation c.3286C > T (p.R1096x) was detected in exon 13 in the family II, which has been reported previously [6]. The resulted truncated protein will lack 130 amino acids.
111
All the seven mutations were not detected in the unaffected family members and 96 unrelated healthy controls.
4. Discussion DSH shows an autosomal dominant pattern of inheritance with high penetrance. The genetic basis of DSH has been elucidated. In 2003, Miyamura et al. confirmed that mutations of the ADAR1 gene are responsible for DSH [4]. The human ADAR1 gene spans 30 kb sequences and contains 15 exons, which encodes RNA-specific adenosine deaminase composed of 1226 amino acid residues. The enzyme has two Z-alpha domains (Z-alpha), three dsRNAbinding domains (DSRM) and the putative deaminase domain (ADEAMc), corresponding to exon 2, exons 2—7 and exons 9—15 of ADAR1, respectively. So far, a total of 70 different mutations of the ADAR1 gene have been reported in Japanese and Chinese patients with DSH [6—15] (Fig. 2). All of the four novel missense mutations identified in this study are located at amino acid residues conserved among pufferfish, zebrafish, frog, rat, mouse, cow, and human within the deaminase domain of the ADAR1 protein. Homodimerization has been demonstrated to be essential for the enzyme activity of the ADAR1 encoded protein [16]. The nonfunctional mutant monomer with a missense mutation in the deaminase domain, like the four missense mutations identified in this study, could not form a normal homodimer with a wildtype monomer, which results in defective activity. So far, 30 of the 70 mutations (42.9%) reported are missense mutations within the deaminase domain of the ADAR1 protein. Hou et al. speculated that the deaminase domain might be a hot spot for mutations [9]. In addition to the four missense mutations, we found two novel single-based deletions in exon 2 and 7, both causing frameshift and thus premature translation termination. We also detected a known nonsense mutation in exon 13 located within the deaminase domain of the ADAR1 protein. This mutation, detected most often so far, has also been reported in another three unrelated families [6,9]. We speculate the mutation might be a hotspot that needs to be further validated. All of the two novel frameshift mutations and one nonsense mutation will lead to truncated proteins without the full deaminase domain and thus inactive enzymes of ADAR1. It is surprising that we could not identify any ADAR1 mutations in the family VII and family VIII after sequencing the entire coding region of ADAR1,
112
FR. Zhang et al.
Fig. 1 Direct automated sequencing of ADAR1 gene in one sporadic case and six families. The black arrows point at the mutated sites. (A): a 3315G > T missense mutation was detected in exon 13. (B): a 3139G > C missense mutation was
Six novel mutations of the ADAR1 gene in Chinese patients with dyschromatosis symmetrica hereditaria
113
Fig. 2 Mutations of the ADAR1 gene reported in patients with DSH. Mutations above the horizontal are nonsense, frameshift, and splicing mutations; those below are missense mutations. The mutations in panes indicate the mutations in this study. Za and Zb, Z-DNA-bingding domains; dsRBD, double-stranded RNA-binding domains; deaminase, deaminase domain.
including the exon-intron boundaries. All of patients of these two families have typical features of DSH. It is possible that the pathogenic mutations in these two families are regulatory ones within the promoter region, 30 and 50 untranlated regions and introns that influence the transcriptional regulation of ADAR1 expression. Such mutations will be missed in the current study where only the coding sequences and the exon-intron boundary sequences were targeted. It is also possible that a large genomic rearrangement could not be detectable by the mutation detection strategy used. In addition, we failed to find any relationship between the phenotypes and genotypes as other groups have reported. More mutation analyses of the ADAR1 gene are needed to clarify the correlation between the phenotypes and genotypes of DSH.
In conclusion, our results provide a significant addition to the DSH mutation database and will contribute further to the understanding of DSH genotype/phenotype correlations and to the pathogenesis of this disease.
Acknowledgments This work was funded by a grant from the ‘‘Research project of Shandong Provincial Science and Technology’’ (2006GG2202060). We are most grateful to all the families with DSH who have so willingly participated and encouraged us with these studies. Our thanks are also given to Dr. Liu jianjun, Genome Institute of Singapore, who took pains to make the paper perfect.
found in the proband of family I. (C): One heterozygous nonsense mutation c.3286C > T in exon 13 was detected in family II. (D): A deletion mutation (2337delA) was found, resulting in frameshift and premature termination codon (PTC) in the proband of family III. (E): Another deletion mutation (1323delC) was detected in the proband of family IV. (F): Base substitutions 3295T > C were identified in family V. (G): A 3202G > C missense mutation was detected in exon 12 in family VI.
114
FR. Zhang et al.
References [1] Toyama I. Dyschromatosis symmetrica hereditaria (in Japanese). Jpn J Dermatol 1929;27:95—6. [2] Oyama M, Shimizu H, Ohata Y, et al. Dschromatosis symmetrica hereditaria: report of a Japanese family with the condition and a literature review of 185 cases. Br J Dermatol 1999;140:491—6. [3] Zhang XJ, Gao M, Li M, et al. Identification of a locus for dyschromatosis symmetrica hereditaria at chromosome 1q11—1q21. J Invest Dermatol 2003;120(5):776—80. [4] Miyamura Y, Suzuki T, Kono M, et al. Mutations of the RNASpecific Adenosine Deaminase Gene (DSRAD) are involved in dyschromatosis symmetrica hereditaria. Am J Hum Genet 2003;73(3):693—9. [5] Bass BL, Weintraub H. An unwinding activity that covalently modifies its double-stranded RNA substrate. Cell 1988;55:1089—98. [6] Zhang XJ, He PP, Li M, et al. Seven novel mutations of the ADAR gene in Chinese families and sporadic patients with dyschromtosis symmetrica hereditaria (DSH). Hum Mutat 2004;23(6):629—30. [7] Li CR, Li M, Ma HJ, et al. A new arginine substitution mutation of DSRAD gene in a Chinese family with dyschromatosis symmetrica hereditaria. J Dermatol Sci 2005;37(2):95—9. [8] Liu Q, Jiang L, Liu WL, et al. Two novel mutations and evidence for haploinsufficiency of the ADAR gene in dyschromatosis symmetrica hereditaria. Br J Dermatol 2006;154(4):636—42.
[9] Hou Y, Chen J, Gao M, et al. Five novel mutations of RNAspecific adenosine deaminase gene with dyschromatosis symmetrica hereditaria. Acta Derm Venereol 2007;87(1): 18—21. [10] Li M, Yang LJ, Shi YX, et al. A novel missense mutation in DSRAD in a family with dyschromatosis symmetrica hereditaria. Arch Dermatol Res 2007;299(5—6):273—5. [11] Liu Y, Xiao S, Peng Z, et al. A new mutation of the doublestranded RNA-specific adenosine deaminase gene in a family with dyschromatosis symmetrica hereditaria. Dermatology 2006;213(3):200—3. [12] Suzuki N, Suzuki T, Inagaki K, et al. Ten novel mutations of the ADAR1 gene in Japanese patients with dyschromatosis symmetrica hereditaria. J Invest Dermatol 2007;127(2): 309—11. [13] Lu J, Liao Z, Chen J, et al. Identification of two novel DSRAD mutations in two Chinese families with dyschromatosis symmetrica hereditaria. Arch Dermatol Res 2007;298(7): 357—60. [14] Liu Y, Xiao SX, Peng ZH, et al. Two frameshift mutations of the double-stranded RNA-specific adenosine deaminase gene in Chinese pedigrees with dyschromatosis symmetrica hereditaria. Br J Dermatol 2006;155(2): 473—6. [15] Yang Y, Li S, Zhu XJ, et al. DSRAD gene mutations in three families with dyschromatosis symmetrica hereditaria. Beijing Da Xue Bao 2004;36(5):466—8. [16] Cho DS, Yang W, Lee JT, et al. Requirement of dimerization for RNA editing activity of adenosine deaminase acting on RNA. J Biol Chem 2003;278:17093—102.
Available online at www.sciencedirect.com
www.intl.elsevierhealth.com/journals/jods
Six novel mutations of the ADAR1 gene in Chinese patients with dyschromatosis symmetrica hereditaria Furen Zhang a,1,*, Hong Liu a, Deke Jiang b, Hongqing Tian a, Changyuan Wang a, Long Yu b,1 a
Shandong Provincial Institute of Dermatovenereology, 57 Jiyan Lu, Jinan 250022, Shandong Province, PR China b State Key Laboratory of Genetic Engineering, Institute of Genetics, School of Life Science, Fudan University, Shanghai 200433, PR China Received 16 August 2007; received in revised form 13 November 2007; accepted 20 November 2007
KEYWORDS Dyschromatosis symmetrica hereditaria; Mutation analysis; ADAR1
Summary Background: Dyschromatosis symmetrica hereditaria (DSH) is a rare autosomal dominantly inherited dermatosis and characterized by a mixture of hyperpigmented and hypopigmented macules on the back of hands and feet. The DSH locus was mapped to chromosome 1q21 and subsequently pathogenic mutations were identified in the adenosine deaminase acting on RNA1 (ADAR1) gene in 2003. Objective: In this study, we performed a mutation analysis of the ADAR1 gene in eight Chinese families and one sporadic patient with typical DSH. Methods: PCR and direct sequencing of the ADAR1 gene were performed to identify and confirm the mutations in the eight families and the sporadic patient. Results: Six novel and one known mutations were identified, including four missense mutations (p.K1105N, p.G1047R, p.F1099L, p.G1068R), two frameshift mutations (p.Q779fs-792x, p.P441fs-463x) and one nonsense mutation (p.R1096x). Conclusion: Six novel mutations were found in five unrelated families and one sporadic case, which have further improved our understanding on the role of ADAR1 in DSH. Interestingly, we failed to detect any mutations of ADAR1 in two families. # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.
Abbreviations: DSH, dyschromatosis symmetrica hereditaria; ADAR1, adenosine deaminase acting on RNA1; PCR, polymerase chain reaction. * Corresponding author. Tel.: +86 531 87298801; fax: +86 531 87984734. E-mail address: [email protected] (FR. Zhang). 1 These two authors contributed equally to the paper. 0923-1811/$30.00 # 2007 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.jdermsci.2007.11.011
110
FR. Zhang et al. we failed to detect any mutations of ADAR1 in two families.
1. Introduction Dyschromatosis symmetrica hereditaria (DSH, OMIM 127400), or symmetric dyschromatosis of the extremities, was first reported by Toyama in 1910 and formally named as a clinical entity in 1929 [1]. It is a pigmentary genodermatosis characterized by a mixture of hypopigmented and hyperpigmented macules distributed on the dorsa of hands and feet. Many patients with DSH also have small freckle-like pigmented macules on their faces. The lesions usually appear in infancy or early childhood, commonly stop spreading before adolescence, and last for life [2]. DSH has been reported mainly in Japan and China populations and also in many other ethnic groups [2]. DSH generally shows an autosomal dominant pattern of inheritance with high penetrance, but sporadic cases have also been reported. The DSH locus has been mapped to chromosome 1q21 by Chinese and Japanese groups, independently [3,4]. Pathogenic mutations were identified in the double-stranded RNA-specific adenosine deaminase (ADAR1) gene encoding double-stranded RNA-specific adenosine deaminase [4]. ADAR1 protein catalyzes the deamination of adenosine to inosine in double-stranded RNA substrates [5], which results in the creation of alternative splicing sites or alternations of codon and thus lead to functional changes in the protein. The ADAR1 gene is expressed ubiquitously, but its target gene(s) in the skin still remain unknown. The molecular pathogenesis of DSH has not been clarified yet. In this study, we performed a mutation analysis of the ADAR1 gene in eight Chinese families and one sporadic patient with typical DSH and identified six novel and one known mutations. Interestingly,
2. Materials and methods All patients analyzed in this study were admitted, diagnosed and treated at Shandong Provincial Institute of Dermatovenereology. Eight DSH pedigrees were recruited, all showing an autosomal dominant pattern of inheritance. A sporadic patient was also recruited. All the patients have typical macules distributed on the back of hands and feet. Table 1 summarizes the clinical and molecular findings of all the patients analyzed in this study. The study protocol was approved by Ethical Committee of both Fudan University and Shandong Provincial Institute of Dermatovenereology. Written informed consents were obtained from all the patients and their family members. DNA samples were extracted from peripheral blood samples using Takara DNA Mini Kit (Takara Biotechnology CO. LTD, Dalan, China). Control samples were obtained from the DNA collection at the State Key Laboratory of Genetic Engineering, Fudan University. All the 15 exons of ADAR1 genes and their flanking intronic sequences of 200 bps were amplified by PCR. The primers for amplifying exon 1 and exon 3 were designed in this study, and the rest primers were described in Zhang’s study [6]. PCR were carried out using a standard PCR reaction system, containing 18.7 mL of ddH2O, 2.5 mL of 10 PCR buffer, 2 mL of 1 dNTP, 0.5 mL of each primers, 0.3 mL of Taq DNA polymerase and 1 mL of genomic DNA. After an initial denaturation step at 94 degree for 5 min, 28 cycles of amplification consisting of 45 s at 94
Table 1 Clinical and molecular findings in this study Patient
Incidence
Affected individuals
Unaffected individuals
Part of lesion
Mutation
Exon
Mutation type
1 I
Sporadic Familial
— 2
— 16
p.K1105N p.G1047R
13 12
Missense Missense
II III
Familial Familial
2 4
4 6
p.R1096x p.Q779fs
13 7
Nonsense Frameshift
IV
Familial
3
4
p.P441fs
2
Frameshift
V
Familial
4
2
p.F1099L
13
Missense
VI VII
Familial Familial
4 6
5 14
p.G1068R —
12 —
Missense —
VIII
Familial
5
9
Back of hands and feet face Back of hands and feet face, calves, knees Back of hands and feet face Back of hands and feet face, neck Back of hands and feet Face, forearms Back of hands, back of feet (faint) Back of hands and feet forearms Back of hands and feet face, neck Back of hands and feet face, knees, ankles
—
—
—
Six novel mutations of the ADAR1 gene in Chinese patients with dyschromatosis symmetrica hereditaria degree, 30 s at annealing degree and 45 s at 72 degree were performed. Direct sequencing was performed using a DNA sequencing system (model 3730; ABI). Mutations were identified by comparing with the reported cDNA reference sequence (GenBank accession number: NM_001111). All the identified mutations were verified by the subsequent two-direction sequencing. Both the patients and control samples were analyzed using the same protocol.
3. Results The entire coding and flanking intronic sequences of ADAR1 were screened for mutations in eight families and one sporadic case of DSH. Sequencing analysis identified six novel and one known mutation, including four missense mutations (p.K1105N, p.G1047R, p.F1099L, p.G1068R), two frameshift mutations (p.Q779fs-792x, p.P441fs-463x) and one nonsense mutation (p.R1096x) (Fig. 1). We failed to detect any ADAR1 mutations in family VII and family VIII.
3.1. Missense mutations Four missense mutations were identified in the family I, V, VI and the sporadic case. Two base substitutions was 3139G > C and 3202G > C were identified in exon 12 in the family I and VI, causing Gly1047Arg and Gly1068Arg missense mutations respectively. Another two base substitutions 3295T > C and 3315G > T were identified in exon 13 in the family V and the sporadic case, causing Phe1099Leu and Lys1105Asn missense mutations.
3.2. Frameshift mutations Two different single-base deletions were identified and both lead to a shift in the reading frame and thus cause premature termination codons (PTCs). One deletion c.2337delA was identified in exon7 in the family III; and the other deletion c.1323delC was found in exon 2 in the family IV. The two mutations generated pre-terminating codon (PTC) at 13 and 22 codons downstream of deletion site respectively, resulting in truncated proteins likely without any functional activity.
3.3. Nonsense mutations One nonsense mutation c.3286C > T (p.R1096x) was detected in exon 13 in the family II, which has been reported previously [6]. The resulted truncated protein will lack 130 amino acids.
111
All the seven mutations were not detected in the unaffected family members and 96 unrelated healthy controls.
4. Discussion DSH shows an autosomal dominant pattern of inheritance with high penetrance. The genetic basis of DSH has been elucidated. In 2003, Miyamura et al. confirmed that mutations of the ADAR1 gene are responsible for DSH [4]. The human ADAR1 gene spans 30 kb sequences and contains 15 exons, which encodes RNA-specific adenosine deaminase composed of 1226 amino acid residues. The enzyme has two Z-alpha domains (Z-alpha), three dsRNAbinding domains (DSRM) and the putative deaminase domain (ADEAMc), corresponding to exon 2, exons 2—7 and exons 9—15 of ADAR1, respectively. So far, a total of 70 different mutations of the ADAR1 gene have been reported in Japanese and Chinese patients with DSH [6—15] (Fig. 2). All of the four novel missense mutations identified in this study are located at amino acid residues conserved among pufferfish, zebrafish, frog, rat, mouse, cow, and human within the deaminase domain of the ADAR1 protein. Homodimerization has been demonstrated to be essential for the enzyme activity of the ADAR1 encoded protein [16]. The nonfunctional mutant monomer with a missense mutation in the deaminase domain, like the four missense mutations identified in this study, could not form a normal homodimer with a wildtype monomer, which results in defective activity. So far, 30 of the 70 mutations (42.9%) reported are missense mutations within the deaminase domain of the ADAR1 protein. Hou et al. speculated that the deaminase domain might be a hot spot for mutations [9]. In addition to the four missense mutations, we found two novel single-based deletions in exon 2 and 7, both causing frameshift and thus premature translation termination. We also detected a known nonsense mutation in exon 13 located within the deaminase domain of the ADAR1 protein. This mutation, detected most often so far, has also been reported in another three unrelated families [6,9]. We speculate the mutation might be a hotspot that needs to be further validated. All of the two novel frameshift mutations and one nonsense mutation will lead to truncated proteins without the full deaminase domain and thus inactive enzymes of ADAR1. It is surprising that we could not identify any ADAR1 mutations in the family VII and family VIII after sequencing the entire coding region of ADAR1,
112
FR. Zhang et al.
Fig. 1 Direct automated sequencing of ADAR1 gene in one sporadic case and six families. The black arrows point at the mutated sites. (A): a 3315G > T missense mutation was detected in exon 13. (B): a 3139G > C missense mutation was
Six novel mutations of the ADAR1 gene in Chinese patients with dyschromatosis symmetrica hereditaria
113
Fig. 2 Mutations of the ADAR1 gene reported in patients with DSH. Mutations above the horizontal are nonsense, frameshift, and splicing mutations; those below are missense mutations. The mutations in panes indicate the mutations in this study. Za and Zb, Z-DNA-bingding domains; dsRBD, double-stranded RNA-binding domains; deaminase, deaminase domain.
including the exon-intron boundaries. All of patients of these two families have typical features of DSH. It is possible that the pathogenic mutations in these two families are regulatory ones within the promoter region, 30 and 50 untranlated regions and introns that influence the transcriptional regulation of ADAR1 expression. Such mutations will be missed in the current study where only the coding sequences and the exon-intron boundary sequences were targeted. It is also possible that a large genomic rearrangement could not be detectable by the mutation detection strategy used. In addition, we failed to find any relationship between the phenotypes and genotypes as other groups have reported. More mutation analyses of the ADAR1 gene are needed to clarify the correlation between the phenotypes and genotypes of DSH.
In conclusion, our results provide a significant addition to the DSH mutation database and will contribute further to the understanding of DSH genotype/phenotype correlations and to the pathogenesis of this disease.
Acknowledgments This work was funded by a grant from the ‘‘Research project of Shandong Provincial Science and Technology’’ (2006GG2202060). We are most grateful to all the families with DSH who have so willingly participated and encouraged us with these studies. Our thanks are also given to Dr. Liu jianjun, Genome Institute of Singapore, who took pains to make the paper perfect.
found in the proband of family I. (C): One heterozygous nonsense mutation c.3286C > T in exon 13 was detected in family II. (D): A deletion mutation (2337delA) was found, resulting in frameshift and premature termination codon (PTC) in the proband of family III. (E): Another deletion mutation (1323delC) was detected in the proband of family IV. (F): Base substitutions 3295T > C were identified in family V. (G): A 3202G > C missense mutation was detected in exon 12 in family VI.
114
FR. Zhang et al.
References [1] Toyama I. Dyschromatosis symmetrica hereditaria (in Japanese). Jpn J Dermatol 1929;27:95—6. [2] Oyama M, Shimizu H, Ohata Y, et al. Dschromatosis symmetrica hereditaria: report of a Japanese family with the condition and a literature review of 185 cases. Br J Dermatol 1999;140:491—6. [3] Zhang XJ, Gao M, Li M, et al. Identification of a locus for dyschromatosis symmetrica hereditaria at chromosome 1q11—1q21. J Invest Dermatol 2003;120(5):776—80. [4] Miyamura Y, Suzuki T, Kono M, et al. Mutations of the RNASpecific Adenosine Deaminase Gene (DSRAD) are involved in dyschromatosis symmetrica hereditaria. Am J Hum Genet 2003;73(3):693—9. [5] Bass BL, Weintraub H. An unwinding activity that covalently modifies its double-stranded RNA substrate. Cell 1988;55:1089—98. [6] Zhang XJ, He PP, Li M, et al. Seven novel mutations of the ADAR gene in Chinese families and sporadic patients with dyschromtosis symmetrica hereditaria (DSH). Hum Mutat 2004;23(6):629—30. [7] Li CR, Li M, Ma HJ, et al. A new arginine substitution mutation of DSRAD gene in a Chinese family with dyschromatosis symmetrica hereditaria. J Dermatol Sci 2005;37(2):95—9. [8] Liu Q, Jiang L, Liu WL, et al. Two novel mutations and evidence for haploinsufficiency of the ADAR gene in dyschromatosis symmetrica hereditaria. Br J Dermatol 2006;154(4):636—42.
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