MUTATION IN BRIEF

HUMAN MUTATION Mutation in Brief #717 (2004) Online

© 2004 WILEY-LISS, INC. DOI: 10.1002/humu.9246

Received 30 October 2003; accepted revised manuscript 4 March 2004.

Seven Novel Mutations of the ADAR Gene in Chinese Families and Sporadic Patients with Dyschromatosis Symmetrica Hereditaria (DSH) Xue-Jun Zhang1,4*, Ping-Ping He1,2,4, Ming Li1,4, Chun-Di He3, Kai-Lin Yan1,4, Yong Cui1,4, Sen Yang1,4, Kai-Yue Zhang2, Min Gao1,4, Jian-Jun Chen1,4, Cheng-Rang Li1,4, Lin Jin2, Hong-Duo Chen3, Shi-Jie Xu2, and Wei Huang2*

1Institute of Dermatology; Department of Dermatology at No. 1 Hospital, Anhui Medical University, Hefei, Anhui, China; 2Chinese National Human Genome Center at Shanghai, Shanghai, China; 3Department of Dermatology, No.1 Hospital of China Medical University, Shenyang, Liaoning, China; 4Key Laboratory of Genome Research at Anhui, Hefei, Anhui, China

*Correspondence to: Professor Xue-Jun Zhang, Institute of Dermatology, Anhui Medical University, 69 Meishan Road, Hefei, Anhui 230032, PR China; Tel.: +86 551 5161002; Fax: +86 551 5161016; E-mail: ayzxj@mail.hf.ah.cn or Dr. Wei Huang, Chinese National Human Genome Center at Shanghai, 250 Bi Bo Road, Shanghai 201203, PR China; Tel.: +86 21 50801795; Fax: +86 21 50801922; E-mail: Huangwei@chgc.sh.cn Communicated by Haig Kazazian

Dyschromatosis symmetrica hereditaria (DSH) is an autosomal dominant pigmentary genodermatosis characterized by hyperpigmented and hypopigmented macules of on the extremities and caused by the mutations in the ADAR gene(also called DSRAD) encoding for RNA-specific adenosine deaminase. Here we reported clinical and molecular findings of 6 Chinese multi-generation families and 2 sporadic patients with DSH. We found that the same mutation could lead to different phenotypes even in the same family and we did not establish a clear correlation between genotypes and phenotypes. Seven novel heterozygous mutations of ADAR were identified, which were c.2433_2434delAG (p.T811fs), c.2197G>T (p.E733X), c.3286C>T (p.R1096X), c.2897G>T (p.C966F), c.2797C>T (p.Q933X), c.2375delT (p.L792fs) and c.3203-2A>G respectively. Our data add new variants to the repertoire of ADAR mutations in DSH. © 2004 Wiley-Liss, Inc.

KEY WORDS: dyschromatosis symmetrica hereditaria; DSH; ADAR; Chinese; genotype-phenotype

INTRODUCTION

Dyschromatosis symmetrica hereditaria (DSH, MIM# 127400), also called reticulate acropigmentation of Dohi (Ostlere et al., 1995) is a pigmentary genodermatosis characterized by a mixture of hyperpigmented and hypopigmented macules of various sized on the dorsal aspects of the extremities and freckle-like macules on the face. DSH has been reported mainly in Japan and China, although a few cases were described among Koreans, Indians, Chinese, Europeans and South Americans (Oyama et al., 1999; He et al., 2003). DSH generally shows an autosomal dominant pattern of inheritance with high penetrance, but some patients with sporadic DSH have been

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reported (Oyama et al., 1999). Our previous study has mapped the DSH gene on chromosome 1q11-q21 (Zhang et al., 2003). When we are on

the way to clone the DSH gene, Miyamura et al. (2003) identified 4 heterozygous mutations of c.1420C>T (p.R474X), c.2768T>C (p.L923P), c.2854A>T (p.K952X) and c.3494T>C (p.F1165S ) in the RNA-Specific Adenosine Deaminase Gene (ADAR, MIM# 601059) responsible for DSH among Japanese families recently. The ADAR gene encodes the enzyme responsible for RNA editing by site-specific deamination of adenosines. ADAR protein catalyzes the deamination of adenosine to inosine in dsRNA substrates, induces translation within the nucleus, possibly at the surface of the nucleolus (Herbert et al., 2002). We directly performed mutation detection of the ADAR gene in 6 Chinese families and 2 sporadic cases with DSH by sequencing. Our purpose was to identify the spectrum of mutations in ADAR and verify if genotype-phenotype correlations could be established.

MATERIALS AND METHODS

Subjects

Six multi-generation DSH families including 53 affected and 58 unaffected individuals were identified through probands from Anhui and Liaoning provinces in China respectively. Two sporadic DSH cases that had no positive family histories were also collected. All affected individuals had typical hyperpigmented and hypopigmented macules on the extremities, and skin lesions even spread on neck, chest or face (freckle-like macules) in some patients. Phenotypes of all individuals were confirmed by experienced dermatologists. Informed consent was obtained from all available patients and relatives for clinical and genetic investigation.

Mutation analysis

We designed primers flanking all 15 coding exons and intron-exon boundaries of the ADAR gene using the web-based version of the Primer 3.0 program (htttp://www.genome.wi.mit.edu/cgi-bin/primer/primer3_www.cgi). Primer sequences were available on request. PCR was performed in 15-µl reaction volume containing 20 ng of genomic DNA, 0.3 mM dNTPs, 0.3 µM of each primer, 3.0 mM MgCl2 and 0.1 units of Hotstar®Taq DNA polymerase (Qiagen). The PCR conditions were: Hotstar®Taq activation at 95? for 15 min, followed by 40 cycles, each having denaturation at 94? for 40 s, annealing at 58? for 60 s and extension at 72? for 55 s, except that in the first 10 cycles the annealing temperature decreased from 63? to 58? by 0.5? per cycle, and the final extension was 72? for 10 min. After the amplification, products were purified using a QIAquick PCR Purification Kit (Qiagen) and directly sequenced on ABI PRISM® 3730 automated sequencer (Applied Biosystems). Sequence comparisons and analysis were performed using Phred-Phrap-Consed Version 12.0 program. In addition, Samples from 100 unrelated, population-match controls were sequenced for missense and splicing mutations to exclude the possibility that these are polymorphism in the ADAR gene. ADAR GenBank sequences used: NM_001111.2 (mRNA); NP_001102.1 (protein).

RESULTS

Tables 1 and 2 summarized clinical and molecular findings of all families and sporadic patients studied respectively. A total of 7 novel ADAR mutations (one missense, three nonsense, two frameshift and one splice acceptor) were identified, all of which were heterozygous (Figure 1).

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Figure 1. The map for functional domains of ADAR mutations responsible for DSH. The three functional

domains (z-alpha, Adenosine deaminase z-alpha domain; DSAM, Double-stranded RNA binding motif; and ADEAMc, tRNA-specific and double-stranded RNA adenosine deaminase) are indicated in the figure. Previously reported mutations are indicated by black arrows and the new mutations identified in this study are indicated by red arrows.

The c.2433_2434delAG (p.T811fs) mutation was found in all patients but not in the healthy individuals from

Family 1. The c.2375delT (p.L792fs) was found in sporadic patient 1 but not in his healthy parents and a healthy sister. These two mutations lead to frameshift and premature translation termination within exon 7. The truncated proteins with no functional activity would be synthesized from the gene with these two frameshift mutations.

The c.2197G>T (p.E733X) mutation was identified in the Family 2. The predicted protein lacks 494 amid acids, including part of the double-stranded RNA binding motif (DSRM) and tRNA-specific and double-stranded RNA adenosine deaminase (ADEAMc) domains. The c.3286C>T (p.R1096X) was carried by all patients from both Family 3 and Family 4. The predicted protein lacks 131 amid acids, which are part of the ADEAMc domain. The c.2797C>T (p.Q933X) mutation in Family 6 is also located in the putative deaminase domain and the predicted protein lacks 294 amid acids.

The missense c.2897G>T (p.C966F) mutation in exon 11 was found in Family 5. This mutation replaces a highly conserved cysteine residue with phenylalanine. The amino acid residue at 966 in exon 11 is located in the putative deaminase domain. This mutation was not detected in the 100 unrelated, population-match control individuals by sequencing.

The c.3203-2A>G mutation was identified in sporadic patient 2 who hadn’t a positive family history of DSH. It alters the canonical splice acceptor sequence of IVS12 and thereby should prevent proper splicing of the transcript. This mutation was not also detected in the 100 normal individuals.

We carefully evaluated the clinical features in all familial and sporadic patients (Table 1), and compared them with the different mutations identified. Among Families 1,3,4,5 and 6, some patients have hyperpigmented and hypopigmented macules only distributed on the extremities, and the other patients have skin lesions not only on the extremities but also on the chest, neck or face (freckle-like macules). Among all patients from Family 2 and 2 sporadic cases, skin lesions are only distributed on the extremities. Although DSH generally appears in infancy or childhood, the detail age of disease onset in these patients is also different (range from infancy to 15 years). The same mutation will lead to different distribution of skin lesions and age of onset, even in the same family. We did not establish a clear correlation between clinical features and genotypes.

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Table 1.Clinical Features of Six Chinese Families and Two Sporadic Patients with DSH

Table 2. Seven Novel Mutations of ADAR Identified in this Study

aGenBank Accession No. NM_001111.2, position 1 is A of the translation initiation codon. DSAM, Double-stranded RNA binding motif. ADEAMc, tRNA-specific and double-stranded RNA adenosine deaminase.

DISCUSSION

ADAR, also called DSRAD, is composed of 1226 amino acid residues, with a calculated molecular mass of 139 kDa (O’Connell et al., 1995). The enzyme converts adenosine to inosine in dsRNA, which destabilizes the dsRNA helix. Among 6 Chinese families and 2 sporadic patients with DSH, we detected 7 different heterozygous mutations of ADAR involved in this disorder, all of which were novel. Two frameshift mutations (p.T811fs,

Extremities involvement only

Involvements of Extremities + face or neck or chest

Family and sporadic patient ID

Affected individuals

Unaffected individuals

Disease onset in patients

M F M F

Family 1 17 16 4~10 yrs 8 4 2 3

Family 2 10 10 4~9 yrs 6 4 0 0 Family 3 9 11 8 mths ~10 yrs 2 2 2 3 Family 4 8 8 3~11 yrs 2 3 2 1 Family 5 4 7 4~15 yrs 1 0 2 1 Family 6 5 6 6 mths ~ 6 yrs 2 1 1 1 Sporadic patient 1 _ _ 10 yrs 1 _ 0 _ Sporadic patient 2 _ _ 8 yrs 1 _ 0 _

Family and sporadic patient ID

Nucleotide Substitutiona

Position Amid acid substitution

Mutation type

Protein domain

Family 1 c.2433_2434delAG Exon 7 Thr811fs (p.T811fs)

frameshift ADEAMc

Family 2 c.2197G>T Exon 6 Glu733Stop (p.E733X)

nonsense DSRM

Families 3 & 4 c.3286C>T Exon 13 Arg1096Stop (p.R1096X)

nonsense ADEAMc

Family 5 c.2897G>T Exon 11 Cys966Phe (p.C966F)

missense ADEAMc

Family 6 c.2797C>T Exon 10 Gln933Stop (p.Q933X)

nonsense ADEAMc

Sporadic patient 1 c.2375delT Exon 7 Leu792fs (p.L792fs)

frameshift DSRM

Sporadic patient 2 c.3203-2A>G

Intron 12 _ Splice acceptor

ADEAMc

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p.L792fs) and three nonsense mutations (p.E733X, p.Q933X, p.R1096X) will lead to premature translation termination, and the truncated proteins with no functional activity would be synthesized. The missense mutation (p.C966F) alters a highly conserved amino acid residue at 966 in exon 11, which is located in the putative deaminase domain, so the amino acid residue at 966 is suspected to play an important role in the conformation of the catalytic site of the enzyme, and the mutation at this position could probably influence enzyme activity. The c.3203-2A>G mutation is located in the acceptor splice site and could prevent proper splicing of the transcript. In the previous study, Miyamura et al. found two missense and two nonsense mutations among Japanese patients with DSH, all of which are different from these seven mutations detected among Chinese patients. These indicate mutations of the ADAR gene responsible for DSH varied considerably among populations. We compared the clinical features with the mutation identified in all familial and sporadic patients but we did not establish a clear correlation between genotypes and phenotypes. The same mutation will lead to different phenotype even in the same family, which suggested that some environmental factors, such as sun exposure could influence the phenotypes including the distribution of skin lesions and disease onset.

The ADAR gene spans 30 kb and contains 15 exons (Wang et al., 1995). Two Z-alpha domains, three dsRNA-binding domains and the putative deaminase domain are located in exon 2, exons 2-7 and exons 9-15, respectively (Wang et al., 1995; Schade et al., 1999). The heterozygosity for the ADAR knockout causes embryonic lethality in mice (Wang et al., 2000), whereas patients with DSH have a good prognosis of dyschromatosis, which is localized specifically on the backs of hands and on tops of the feet. Miyamura et al. (2003) speculated that when melanoblasts migrate from the neural crest to the skin during development, a greater reduction in ADAR activity might occur at anatomic sites distant from the neural crest. Failure of correct RNA editing may induce the differentiation of melanoblasts to hyperactive or hypoactive melanocytes, then colonizing in an irregular distribution in the skin lesions.

Here we identified 7 novel mutations of ADAR in Chinese DSH families and sporadic patients. The identification of ADAR as the disease-causing gene and the ongoing recognition of different mutations may give insight into the still unknown mechanism leading to DSH.

ACKNOWLEDGMENTS

This work was funded by grants from National Natural Science Foundation of China (30170529) and the Chinese High Tech Program (863) (2001AA227031, 2001AA224021, 2002BA711A10).

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