Four novel mutations of ADAR1 in Chinese patients with dyschromatosis symmetrica hereditaria

Introduction

Dyschromatosis symmetrica hereditaria (DSH; MIM # 127400) is an autosomal dominant inherited pigmentary genodermatosis characterized by intermingled hyperpigmented and hypopigmented macules on the dorsal aspect of the distal extremities, and freckle-like macules on the face. The lesions usually appear in infancy or early childhood, commonly stop spreading before adolescence, and last for life.^[1] The gene responsible for DSH was identified in 2003 as adenosine deaminase acting on RNA1 (ADAR1).^[2]

Methods

In this study, we studied four pedigrees of patients with DSH, four individual DSH patients whose family members refused to participate in our study or had died, and 100 unrelated normal individuals [Figure - 1]. All affected individuals had typical hyperpigmented and hypopigmented macules on their dorsal aspects of their hands and feet. The clinical features of the four probands of DSH pedigrees and four individual DSH patients are shown in [Figure - 2]. Phenotypes of all individuals were confirmed by experienced dermatologists on the basis of clinical features and family histories of DSH patients. The study was approved by the Ethics Committee of Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology and written informed consent was obtained from all subjects in the study.

Figure 1: Pedigrees of the family with dyschromatosis symmetrica hereditaria and genetic findings. (a) c.2722 G>T (arrow) in exon 9 in family 1. (b) c.2865_2866delGT (arrow) in exon 10 in family 2. (e) c.1657delA (arrow) in exon 3 in family 3. (f) c.3363_3364insT (arrow) in exon 14 in family 4. (i) c.2563_2564delCT (arrow) in exon 8 of ADAR1 in individual patient 1. (j) c.526T>G (arrow) in exon 2 of ADAR1 in individual patient 2. (m) c.1630C>T (arrow) in exon 3 of ADAR1 in individual patient 3. (n) c.2894C>T (arrow) in exon 11 of ADAR1 in individual patient 4. (c), (d), (g), (h), (k), (l), (o), (p) showed relative control DNA sequences of ADAR1 from control individuals, respectively

Figure 2: Typical skin lesions of the patients with dyschromatosis symmetrica hereditaria. (a) Intermingled hyper-pigmented and hypo-pigmented maculeson the dorsal aspect of the hands and freckle-like macules on the face of the DSH patient. (b) Intermingled hyper-pigmented and hypo-pigmented macules onthe dorsal aspect of the DSH patient's distal extremities

Genomic DNA was extracted from peripheral blood samples of 14 DSH patients, six clinically unafffected family members in the four pedigrees, and 100 control individuals. All 15 exons of ADAR1 and their flanking intron sequences were amplified by polymerase chain reaction (PCR) using specific primers designed by Primer Premier 5 [Table - 1]. PCR was performed as previously described.^[3] After the amplification, the products were purified using a QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) and sequenced using an ABI PRISM^® 3730 automated sequencer (Applied Biosystems, Foster City, CA, USA). Sequence comparisons and analyses were performed using the Basic BLAST program. Mutations were identified by comparison with the reported cDNA reference sequences for ADAR1 (GenBank accession number: NM_001111.4). Then all ADAR1 mutations previously reported were collected according to PubMed and Human Gene Mutation Database (HGMD). Eight different heterozygous mutations of ADAR1 were identified in this study [Table - 2].

Table 1: Polymerase chain reaction primers and reaction conditions for ADAR1 used in this study

Table 2: Eight mutations of ADAR1 identified in this study

Results

In family 1, a missense mutation, c.2722 G>T, p.(Asp908Tyr) in exon 9 of ADAR1 was identified in the proband, his mother and his daughter, but not in unaffected members of his family and 100 control individuals [Figure - 1]a and [Figure - 1]c. The same running procedure was performed and a deletion mutation, c.2865_2866delGT, p.(Val955fs) in exon 10 of ADAR1 was found in family 2 [Figure - 1]b and [Figure - 1]d. Family 3 had a deletion mutation c.1657delA, p.(Ser553fs) in exon 3 of ADAR1 [Figure - 1]e and [Figure - 1]g. Family 4 harbored an insertion mutation c.3363_3364insT, p.(Lys1122fs) in exon 14 of ADAR1 [Figure - 1]f and [Figure - 1]h. Individual patient 1 had a deletion mutation c.2563_2564delCT, p.(Leu855fs) in exon 8 of ADAR1 [Figure - 1]i and [Figure - 1]k. Individual patient 2 carried a missense mutation c.526T>G, p.(Leu176Val) in exon 2 of ADAR1 [Figure - 1]j and [Figure - 1]l. Individual patient 3 carried a nonsense mutation c.1630C>T, p.(Arg544X) in exon 3 of ADAR1 [Figure - 1]m and [Figure - 1]o. Individual patient 4 had a missense mutation c.2894C>T, p.(Pro965Leu) in exon 11 of ADAR1 [Figure - 1]n and [Figure - 1]p. Four mutations c.2865_2866delGT, p.(Val955fs), c.3363_3364insT, p.(Lys1122fs), c.1630C>T, p.(Arg544X) and c.2894C>T, p.(Pro965Leu) of ADAR1 have been reported previously.^{[4],[5],[6],[7]} All eight mutations were proved to be pathogenic by Mutation Taster, Provean, and SIFT.

Discussion

ADAR1 consists of 1,226 amino acid residues and contains at least six functional domains: Two copies of a Z-DNA-binding domain (Zα and Zβ), three copies of the double stranded ribonucleic acid (dsRNA)-binding domain (RI, RII, and RIII) and a transfer RNA (tRNA)-specific and dsRNA adenosine deaminase domain (ADEAMc).^[8] It has been discovered to possess different functions in RNA editing and RNA interference (RNAi) by the formation of either ADAR1/ADAR1 homodimer or Dicer/ADAR1 heterodimer complexes, respectively.^[9]

In this study, we identified four novel ADAR1 mutations in four pedigrees and four individual patients with DSH. To date, 196 different ADAR1 mutations have been found in patients with DSH around the world including our data. According to the functional domain of ADAR1, three (1.5%) mutations among them are located in the Zα domain, 5 (2.6%) in the Zβ domain, 13 (6.6%) in the RI domain, four (2.0%) in the RII domain, seven (3.6%) in the RIII domain and 103 (52.6%) within the ADEAMc domain. These results clearly suggest that the ADEAMc domain might be a hot spot for ADAR1 mutations associated with DSH. This is in accord with the finding reported by Li.^[10] The ADEAMc domain of ADAR1 has been verified to be highly conserved in different species. Any mutation in the domain would result in dysfunction of adenosine deaminase, but the exact mechanisms involved are still mysterious.

In summary, our findings expanded the spectrum of ADAR1 mutations associated with DSH and could be a great help for future clinical genetic counseling.

There were also some limitations in our research. We did not study the exact pathogenic mechanism of ADAR1 in DSH patients. So, further research is needed to clarify the function of ADAR1.

Acknowledgments

We would like to thank all the subjects that participated in this study.

Declaration of patient consent

The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient has given his consent for his images and other clinical information to be reported in the journal. The patient understand that name and initials will not be published and due efforts will be made to conceal identity, but anonymity cannot be guaranteed.

Financial support and sponsorship

This work was supported financially by Grants from National Natural Science Foundation of China (No. 81371728, No. 81472864).

Conflicts of interest

There are no conflicts of interest.