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Abstract
Background: Dyschromatosis symmetrica hereditaria (DSH) is a rare autosomal dominant skin disease. The mutation of the ADAR1 gene is the pathogenesis of this disorder. Aims: This study aimed to identify the mutations of the ADAR1 gene in two Chinese families with DSH. Methods and Materials: Eight patients from two Chinese families were diagnosed with DSH clinically. Blood samples were collected from the patients and unaffected individuals. Sanger sequencing for all polymerase chain reaction products of the whole coding regions of the ADAR1 gene was performed to identify the mutations. Mutation Taster software was used to predict the impact of the variant on the resultant protein. Results: The c.3358-3359insT (p.L1053fs-1092X) mutation in exon 12 was found in affected members of the pedigree1. In pedigree2, the c.3820-3821insG (p.G1207fs-1213X) mutation in exon 15 was found. These two mutations were not found in 100 unrelated healthy people. In this study, both mutations were damaged by the Mutation Taster software. Conclusions: We identified two novel frameshift mutations in the ADAR1 gene. Our study expands the database of ADAR1 gene mutations in DSH.
Keywords: ADAR1 gene, dyschromatosis symmetrica hereditaria, genodermatose
Introduction 
Dyschromatosis symmetrica hereditaria (DSH; Mendelian Inheritance in Man no. 127400) is sometimes called reticulate acropigmentation of Dohi.[1] It is an autosomal dominant skin disease with high penetrance, but some patients with sporadic DSH have been reported. It is thought to be a rare genodermatosis though the exact incidence of DSH is unknown. Because patients with DSH usually have only skin symptoms and some patients do not mind the skin lesions, it leads to underreporting.
DSH is characterized by mixed hyperpigmented and hypopigmented macules of various sizes on the dorsal side of the extremities, which usually appear in infancy or early childhood.[2] The symptoms always stop developing in puberty and last a lifetime. Sometimes the freckle-like pigmented macules can involve the face. There are no changes in color or distribution, once the lesions are fully established. The dermoscopic features of DSH include reticulate pigmented, hyperpigmented, or hypopigmented spots, diffuse pigmentation with hyperpigmented spots and monotonous pigmented or hypopigmented spots.[3]
The mutation of the ADAR1 gene (GenBank accession no. NM_001111.3, http://www.ncbi.nlm.nih.gov/genbank/) is the pathogenesis of this disorder.[4] The ADAR1 protein catalyses the transformation of adenosine to inosine in double-stranded RNA (dsRNA) substrates and is involved in various activities, such as viral inactivation, structural change of the protein and the resultant cell survival. However, its function in the skin and the mechanisms by which mutations in ADAR1 cause DSH are still mysterious. In this study, we performed a mutational analysis of the ADAR1 gene in two Chinese pedigrees with DSH.
Subjects and Methods Two pedigrees from the Shaanxi Province of China. This study was approved by the medical ethics committee of Xi'an Jiaotong University. After informed consent, 5 ml of peripheral blood samples were collected from the patients and unaffected individuals of the pedigree1 and pedigree2. Blood samples from 100 unrelated healthy Chinese population are considered as controls. Genomic DNA was extracted from peripheral blood lymphocytes by standard methods. The sequences of the primers were as previously described.[5] Sanger sequencing was applied to the mutation analysed. All 15 exons and the flanking intron sequences of ADAR1 were analysed by direct sequencing from polymerase chain reaction (PCR) amplicons. PCR was performed in 50 ul reaction volume containing 25 ng of genomic DNA, 0.3 mM dNTPs, 0.3 mM of each primer, 3.0 mM MgCL and 0.1 U of Taq DNA polymerase. The PCR conditions were as follows: Taq was activated at 95°C for 5 min, followed by 35 cycles, each cycle was denatured at 95°C for 30 s, annealed at 56°C for 60 s and extended at 72°C or 2 min, and the final extension was 72°C for 5 min. After amplification, the products were purified using a QIAquick PCR Purification Kit (Qiagen). We sequenced the ADAR1 gene using ABI PRISM 3730 automated sequencer (Applied Biosystems). Sequence comparisons and analysis were performed by the Phred-Phrap-Consed Version 12.0 program. Mutation Taster software (https://www.mutationtaster.org) was used to predict the impact of the variant on the resultant protein.
Results As shown in [Figure 1], pedigree 1 consists of two affected and one unaffected individuals, the proband was a 6-year-old boy. He was born with hyperpigmented and hypopigmented macules on the extensor aspect of his fingers and toes and then extended to the dorsal aspect of his hands and feet. Freckles appeared on his face when he was 2 years old. These lesions were irregular in shape and size and asymptomatic. His father also had the same clinical manifestations in his childhood. The skin lesions became more pronounced after exposure to sunlight. Pedigree 2 includes 6 affected and 16 unaffected individuals. The proband was a 12-year-old girl. He had asymptomatic hyperpigmented and hypopigmented macules on the back of the hands and feet at the age of 2 years and then gradually became prominent. The skin lesions became more pronounced when exposed to the sun. Other affected individuals in the pedigree2 showed similar skin changes. All the characteristics support the diagnosis of DSH in clinical.
Figure 1: DSH pedigrees of the two families. (a) Pedigree of family 1 and (b) Pedigree of family 2. Affected members are represented by black symbols. The proband is indicated by the arrowThe results of the sequence analysis are shown in [Figure 2]. A frameshift mutation c.3358-3359insT (p.L1053fs-1092X) in exon 12 was found in the proband and his affected father of the pedigrees1, but not found in healthy individuals or the 100 unrelated controls. The T insert caused a frameshift mutation with the change of amino acids from 1053 to 1091 and introduced a new terminating TGA codon at position 1092. In pedigrees2, a frameshift mutation c.3820-3821insG (p.G1207fs-1213X) in exon 15 was found in patients and other affected individuals, but not found in healthy members. The G insert caused a frameshift mutation with the change of amino acids from 1207 to 1212 and introduced a new terminating TAG codon at position 1213. This mutation was not found in 100 unrelated controls. All these mutations were not included in the NCBI SNP database and it is suggested that the novel mutations may be a pathological mutation of DSH. Both these mutations in this study were damaged by the Mutation Taster software.
Figure 2: Mutations of the ADAR1 gene in two families. (a) c.3358-3359insT mutation in exon 12 of the ADAR1 gene in the proband of family 1. (b) Wild-type allele of exon 12 of the ADAR1 gene. (c) c.3820-3821insG mutation in exon 15 of the ADAR1 gene in the proband of family 2. (d) Wild-type allele of exon 15 of the ADAR1 gene Discussion DSH is a rare autosomal dominant pigmentary genodermatosis, but it has been reported to be transmitted in an autosomal recessive form. DSH mainly occurs in East Asian countries, such as Japan, China (including Taiwan) and Korea, with a few cases described among Indians, Europeans and South Americans. It reported that 77.6% of patients had a family history and 22.4% with no family history. Besides skin lesions, it may be accompanied by other diseases, such as bilateral striatal necrosis, chilblains, congenital heart disease and hemangioma disease.[6],[7],[8]
Clinically, it is not difficult to diagnose DSH, but it always needs to be differentiated from other hereditary pigmentary disorders. The hyperpigmented and hypopigmented macules of dyschromatosis universalis hereditaria (DUH) present are distributed all over the body, about half of the patients with freckle-like pigmentation on face, and the lesions do not change with seasons.
The rash characteristics of DUH are similar to DSH, but no ADAR1 mutations have been identified in patients with DUH, indicating that these two entities are genetically distinct.[9] Research findings showed that ABCB6 mutations may be the pathogenic gene associated with DUH.[10]
Reticularis Acropigmentatio Kitamura (RAK) presents as reticular pigmentation on the dorsa of hands and feet. The difference with DSH is that RAK has no depigmentation lesions, and reticular pigmentation is slightly concave and atrophy. Whole-exome sequencing-identified ADAM10 gene mutations was the pathogenesis of RAK.[11]
Xeroderma pigmentosum (XP) is another condition in the differential diagnosis of DSH. The difference between XP and DSH lies in that XP-present freckle-like lesions, which occur mainly in sun exposure sites, xerosis, symptoms of keratin, development of skin tumors (mostly basal cell carcinoma and squamous cell carcinoma) before the age of 20, and 80% of the patients with the damage of eyes. Many patients are mentally retarded and have poor development. The conditions are also genetically distinct.
Sometimes DSH should be differentiated from vitiligo. Vitiligo is distinguished from DSH by the development of sharply demarcated milky depigmented macules, which occur in any part of the body and are always the response to treatment.
There is no etiologic treatment in the clinic, but the combination of miniature punch grafting and excimer light to treat hypopigmented macules showed effectively.[12] For lesions on the exposed area, sun-protective clothing, umbrellas and sunscreen lotions are effective to some extent.[13]
ADAR1 gene is the pathogenesis of this disorder.[4] However, no mutations of the ADAR1 gene have been detected in some DSH families. There may be other genes involved in this disease. ADAR1 was located on chromosome 1q11-q12 and identified as the gene responsible for DSH.[4],[14] ADAR1 spans up to 40 kb, contains 15 exons and consists of 1226 amino acid residues. It has two Zalpha (Z-DNA-binding domain in adenosine Deaminases in the N-terminal region), three DSRM (double-stranded RNA binding motif) and one ADEAMc domain in the C-terminal region, corresponding to exon 2, exons 2–7 and exons 9–15, respectively.[15] Including our two new mutations, more than 60% of the mutations are located in the ADEAMc domain, so it is believed that the mutations within the ADEAMc domain play an important role in the onset of DSH, and this domain may be the hot spot for ADAR1 mutations, but further research is needed.
In this study, we detected two mutations, c.3358-3359insT (p.L1053fs-1092X) in exon 12 and c.3820-3821insG (p.G1207fs-1213X) in exon 15. These two mutations lead to frameshift and premature translation termination, making the truncated protein with no dsRNA-binding domains and deaminase catalytic domain. But still, there is a need to detect the ADAR1 protein expression in the future to support this speculation. The function of ADAR1 in the development of DSH is still unknown. Recent studies show that the ADAR1 p150 isoform is the determinant of DSH, and 5'UTR c.-60A>G variant of ADAR1 may lead to DSH.[16],[17] But it still remains unknown why all these different mutations and different mutation styles could cause the same phenotype and why the same mutation can lead to different phenotypes.
This study expands the database of ADAR1 gene mutations in DSH. More findings of novel mutations will help to further clarify the pathogenesis, reveal the correlation between phenotypes and genotypes of DSH and then provide more information on genetic counselling and molecular testing for the child-bearing couple. Further investigations should focus on the potential signaling pathway.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
This work was supported by National Natural Science Foundation of China (No. 81703129).
Conflicts of interest
There are no conflicts of interest.
References 
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