[Analysis of ADAR1 gene mutation in a pedigree affected with dyschromatosis symmetrical hereditaria].

OBJECTIVE: To detect mutation of adenosine deaminase acting on RNA1 (ADAR1) gene in a pedigree affected with dyschromatosis symmetrical hereditaria (DSH). METHODS: Clinical data and peripheral blood samples of the patients from the pedigree were collected. Potential mutations of the ADAR1 gene were screened among 2 patients, 2 unaffected individual from the pedigree as well as 50 unrelated healthy controls by PCR amplification and direct sequencing. RESULTS: A c.3463C>T (p.R1155W) missense mutation of the ADAR gene was identified in the 2 patients, which was absent in the 2 healthy relatives and 50 unrelated controls. The mutation has been previously identified among 5 Chinese families and was the most common mutation site. CONCLUSION: The c.3463C>T missense mutation of the ADAR gene probably underlies the disease in this pedigree.

CRISPR/Cas9-mediated mutation of tyrosinase (Tyr) 3' UTR induce graying in rabbit.

The 3' untranslated regions (UTRs), located at the end of mRNA molecules, are believed to play a role in RNA replication and/or protein translation. Mutations in the tyrosinase (Tyr) gene are known to cause recessive albinism in humans and other species. In this study, to test whether the CRISPR/Cas9 system works on the mutation of the UTRs regulatory region in rabbit, the 3' UTR of the rabbit Tyr gene was deleted by a dual sgRNA directed CRISPR/Cas9 system. As expected, gray coat color and reduced melanin in hair follicles and irises was found in the mutated rabbit, thus increasing confidence in the association of the mutation of the Tyr 3' UTR with graying in rabbit. The graying phenotype was also found in the F1 generation, suggesting that the mutated allele can be stably inherited by the offspring. Thus, we provide the first evidence that reduced melanin and graying can be caused by deletion of the Tyr 3' UTR in rabbits. Additionally, CRISPR/Cas9-mediated large fragment deletions can facilitate genotype to phenotype studies of UTRs or non-coding RNAs in future.

[Two novel mutations of the ADAR1 gene associated with dyschromatosis symmetrica hereditaria].

OBJECTIVE: To identify potential mutation of the ADAR1 gene in a Chinese family and a sporadic case affected with dyschromatosis symmetrica hereditaria(DSH). METHODS: Clinical data and peripheral blood samples from the pedigree and the sporadic patient were collected. Following extraction of genomic DNA, all 15 exons and exon-intron flanking sequences of the ADAR1 gene were amplified by polymerase chain reaction and subjected to direct sequencing. RESULTS: A novel frame-shift mutation c.2638delG (p.Asp880ThrfsX15) from the patients of the pedigree was detected in exon 8 of the ADAR1 gene. And a novel nonsense mutation c.2867C>A (p.Ser956X) was detected in exon 10 of the ADAR1 gene from the sporadic case. Neither mutation was identified among the unaffected family members nor 100 unrelated healthy controls. CONCLUSION: The frame-shift mutation c.2638delG (p.Asp880ThrfsX15) and the nonsense mutation c.2867C>A (p.Ser956X) in the ADAR1 gene probably underlie the DSH in our patients.

[Detection of ADAR1 gene mutation in a family with dyschromatosis symmetrica hereditaria].

OBJECTIVE: To detect mutation of ADAR1 gene in a family affected with dyschromatosis symmetrica hereditaria. METHODS: Clinical data and blood samples of the family were collected. Potential mutation of the ADAR1 gene were scanned in 3 patients and 3 unaffected members by PCR amplification and direct sequencing. The coding sequences of the ADAR1 were also screened in 50 normal controls. RESULTS: A frameshift mutation (c.2252insG) of the ADAR1 gene was identified in all of the 3 patients. The same mutation was not found in the 3 unaffected members and 50 normal cases. CONCLUSION: The frameshift mutation of ADAR1 gene (c.2252insG) is probably responsible for the disease in this family.

The adenosine deaminase acting on RNA 1 p150 isoform is involved in the pathogenesis of dyschromatosis symmetrica hereditaria.

BACKGROUND: Dyschromatosis symmetrica hereditaria (DSH) is characterized by the presence of hyperpigmented and hypopigmented macules mostly on the dorsal aspects of the extremities. Mutations in the adenosine deaminase acting on RNA1 (ADAR1) gene have been revealed as the cause of DSH. ADAR1 is known to give rise to two protein isoforms (p150 and p110) that differ by the 295 N-terminal amino acids, but the specific roles of its two isoforms in the pathogenesis of DSH are poorly understood. OBJECTIVES: A Chinese family with typical DSH was screened for mutation of ADAR1, and we aimed to investigate the functional significance of the identified mutation. METHODS: All exons and adjacent exon-intron sequences were amplified and sequenced. The possible influence of the identified mutation on the functionality of p150 and p110 was analysed using the minigene strategy and dual-luciferase reporter assay, respectively. RESULTS: We identified a novel two-base-pair deletion of AG (c.271_272delAG) in exon 2 of ADAR1. The AG deletion caused a frameshift mutation in the p150 isoform, and the mutant p150 transcripts were subjected to nonsense-mediated mRNA decay. However, the deletion mutation did not alter the encoded amino acid sequence of the p110 protein due its position in the 5'-untranslated region of the p110 isoform, and had no significant influence on the expression of p110. CONCLUSIONS: The results represent the first evidence that the ADAR1 p150 isoform is the determinant of DSH and may give insight into the currently unknown mechanisms involved in the development of DSH.

A novel mutation of the DSRAD gene in a Chinese family with dyschromatosis symmetrica hereditaria.

Dyschromatosis symmetrica hereditaria (DSH) is an autosomal dominant cutaneous disorder, characterized by a mixture of hyperpigmented and hypopigmented macules mostly on the dorsal portions of the extremities. Pathogenic mutations have been identified in the double-stranded RNA-specific adenosine deaminase (DSRAD) gene. We studied a Chinese family that included four affected individuals with DSH phenotypes. PCR and direct sequencing were carried out to detect the entire coding region and exon-intron boundaries of the DSRAD gene. A novel nucleotide c.3002G>T missense mutation in the exon 11 of the DSRAD gene was detected in the proband and his father. This information expands the database on DSRAD gene mutations associated with DSH.

[The c.3463C>T mutation of the ADAR1 gene in patients with dyschromatosis symmetrical hereditaria].

OBJECTIVE: To analyze the mutation of the adenosine deaminase acting on RNA 1 (ADAR1) gene in a pedigree with dyschromatosis symmetrical hereditaria (DSH). METHODS: Mutation analysis of the ADAR1 gene was carried out by PCR and direct DNA sequencing in the DSH family, as well as in 50 unrelated healthy controls. RESULTS: A missense mutation of c.3463C>T, which results in p.R1155W in the ADAR1 protein, was found in the 2 patients, but was absent in the 2 healthy members in the family and 50 unrelated individuals. CONCLUSION: A missense mutation of c.3463C>T in the ADAR1 gene was detected in the DSH family, which is likely responsible for the pathogenesis of the disease.

Recent progresses in understanding pigmentation.

Human pigmentation involves production and dispersion of melanin by epidermal melanocytes to neighboring keratinocytes. Melanin synthesis or melanogenesis occurs within the specialized organelle termed melanosomes where the amino acid L-tyrosine serves as the starting precursor. Melanocytes from individuals of different pigmentary phenotypes differ in their rate and types of melanin synthesis, as well as in the rate and manner of melanosome transfer. Modern molecular biology methods and the use of transgenic animals have greatly advanced our understanding in the molecular and cellular mechanisms regulating human pigmentation and its disorders. This review examines recent advances in the regulation of human pigmentation and their implication in treatments for the pigmentary disorders.

Screening of Nepalese crude drugs traditionally used to treat hyperpigmentation: in vitro tyrosinase inhibition.

South-East Asian population is daily exposed to strong sunlight. As a result, the majority of population will have darker, ethnic skin. Moreover, many people suffer from dark spots, hyperpigmentation, which is considered to be a skin disorder and causes psychological disturbance. To treat dark spots, most of the population will still rely on traditionally used crude drugs, knowledge about which is transferred from generation to generation. Fifty-two crude drugs were selected based on the survey performed among local healers and beauticians of different ethnic origin. These crude drugs were screened for mushroom tyrosinase inhibitory activity, as tyrosinase inhibitors are becoming increasingly important as cosmetic and medicinal products, primarily to control hyperpigmentation. Among the tested crude drugs, methanolic extracts of Glycyrrhiza glabra, Morus alba, Syzygium aromaticum, Citrus aurantifolia, Cypreae moneta, Punica granatum and Citrus aurantium, at the final concentration of 50 microg mL(-1), showed mushroom tyrosinase inhibitory activity of 78.9%, 71.0%, 69.4%, 59.0%, 56.0%, 53.4 and 51.9%, respectively, with 91.4% inhibitory activity of kojic acid taken as positive control. To our knowledge, this is the first report that extracts of Cypreae moneta shell and Syzygium aromaticum flowering bud have tyrosinase inhibitory activity. These potent extracts were further evaluated at different concentration. The final concentration of the extracts in reaction mixtures was 50, 25 and 5 microg mL(-1) for the initial concentration of 1000, 500 and 100 microg mL(-1), respectively. They showed concentration-dependent inhibition of mushroom tyrosinase. Those extracts expressing relatively weak tyrosinase inhibitory activity may act through different inhibition pathway which is not based on tyrosinase activity. Further evaluation of the most potent tyrosinase inhibitors in in vivo conditions would be recommended.

Six novel mutations of the ADAR1 gene in Chinese patients with dyschromatosis symmetrica hereditaria.

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.

PRKAR1A mutations in primary pigmented nodular adrenocortical disease.

Primary Pigmented Nodular Adrenocortical Disease (PPNAD) is a rare primary bilateral adrenal defect causing corticotropin-independent Cushing's syndrome. It occurs mainly in children and young adults. Macroscopic appearance of the adrenals is characteristic with small pigmented micronodules observed in the cortex. PPNAD is most often diagnosed in patients with Carney complex (CNC), but it can also be observed in patients without other manifestations or familial history (isolated PPNAD). The CNC is an autosomal dominant multiple neoplasia syndrome characterized by the association of myxoma, spotty skin pigmentation and endocrine overactivity. One of the putative CNC genes has been identified as the gene of the regulatory R1A subunit of protein kinase A (PRKAR1A), located at 17q22-24. Germline heterozygous inactivating mutations of PRKAR1A have been reported in about 45% of patients with CNC, and up to 80% of CNC patients with Cushing's syndrome due to PPNAD. Interestingly, such inactivating germline PRKAR1A mutations have also been found in patients with isolated PPNAD. The hot spot PRKAR1A mutation termed c.709[-7-2]del6 predisposes mostly to isolated PPNAD, and is the first clear genotype/phenotype correlation described for this gene. Somatic inactivating mutations of PRKAR1A have been observed in macronodules of PPNAD and in sporadic cortisol secreting adrenal adenomas. Isolated PPNAD is a genetic heterogenous disease, and recently inactivating mutations of the gene of the phosphodiesterase 11A4 (PDE11A4) located at 2q31-2q35 have been identified in patients without PRKAR1A mutations. Interestingly, both PRKAR1A and PDE11A gene products control the cAMP signaling pathway, which can be altered at various levels in endocrine tumors.

Two novel mutations and evidence for haploinsufficiency of the ADAR gene in dyschromatosis symmetrica hereditaria.

BACKGROUND: Dyschromatosis symmetrica hereditaria (DSH, MIM 127400) is a dominantly inherited skin disease associated with mutations in ADAR, the gene that encodes a double-stranded RNA-specific adenosine deaminase. We previously reported two novel ADAR mutations (p.Q513X and p.R916W) and confirmed the role of ADAR in Chinese patients with DSH. Both haploinsufficiency and a dominant-negative effect have been suggested as the potential mechanism by which ADAR mutations cause DSH. OBJECTIVES: To identify ADAR mutations in two additional Chinese DSH families and to obtain insight into the pathogenic mechanism of heterozygous ADAR mutations. METHODS: For mutation detection, all ADAR exons and their flanking intronic sequences were amplified and sequenced. Mutations were further confirmed by restriction analysis. Direct sequencing of cDNA fragments produced by reverse transcription-polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR were used to examine the expression of ADAR in peripheral lymphocytes isolated from affected individuals. RESULTS: A small deletion, c.1555delT (p.C519fs), and a missense mutation, c.3116A>G (p.K1039R), were found in families A and B, respectively. In individuals carrying p.Q513X or p.C519fs, sequencing of cDNA fragments indicated almost total loss of mRNA expression from the mutant alleles, and real-time quantitative RT-PCR showed an approximately 50% reduction of ADAR expression. However, equal abundance of the wild-type and mutant cDNA sequences without reduction of ADAR expression was found in a patient with the missense p.R916W mutation. These results suggest that both the nonsense p.Q513X and frameshift p.C519fs mutations have generated null alleles probably by nonsense-mediated mRNA decay. CONCLUSIONS: Two novel ADAR mutations were found in Chinese patients with DSH. Evidence for ADAR haploinsufficiency as a mechanism underlying the molecular pathogenesis of DSH was obtained.

[DSRAD gene mutations in three families with dyschromatosis symmetrica hereditaria].

OBJECTIVE: To identify the DSRAD gene; mutations in three Chinese families with dyschromatosis symmetrica hereditaria. METHODS: All exons of DSRAD gene were analyzed in each person of these families with PCR-DNA sequencing. DNA samples from 100 unrelated, normally pigmented adult individuals were also included as control. RESULTS: We identified a missense mutation of C3220T (R1074C) in DSRAD gene in family A, and another missense mutation of G3325T (D1109Y) in DSRAD gene in family B and C. No same mutation was found in unaffected individuals in the families and the controls. CONCLUSION: We found two special missense mutations in DSRAD gene in three families of dyschromatosis symmetrica hereditaria. These mutations may impair DSRAD protein function, and as a consequence, cause skin dyschromatosis.

Seven novel mutations of the ADAR gene in Chinese families and sporadic patients with dyschromatosis symmetrica hereditaria (DSH).

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.

Mutations of the RNA-specific adenosine deaminase gene (DSRAD) are involved in dyschromatosis symmetrica hereditaria.

Dyschromatosis symmetrica hereditaria (DSH) (also called "reticulate acropigmentation of Dohi") is a pigmentary genodermatosis of autosomal dominant inheritance characterized by a mixture of hyperpigmented and hypopigmented macules distributed on the dorsal aspects of the hands and feet. To determine the gene responsible for this disease, we performed a genomewide search in three families with DSH and mapped the DSH locus to chromosome 1q21.3. The mutations involved in causing DSH have been identified in the gene that encodes double-stranded RNA-specific adenosine deaminase (DSRAD) as the disease gene.

Modulation of melanogenesis by aloesin: a competitive inhibitor of tyrosinase.

Aloesin, [2-acetonyl-8-beta-d-glucopyranosyl-7-hydroxy-5-methylchromone], a compound isolated from the Aloe plant, is shown in these studies to modulate melanogenesis via competitive inhibition of tyrosinase. Aloesin inhibits purified tyrosinase enzyme and specifically inhibits melanin production in vitro. Enzyme kinetics studies using normal human melanocyte cell lysates and cell-based melanin production demonstrated that aloesin is a competitive inhibitor of tyrosinase from mushroom, human and murine sources. Tyrosine hydroxylase and 3,4-dihydroxyphenylalanine (DOPA) oxidase activities of tyrosinase from normal human melanocyte cell lysates were inhibited by aloesin in a dose dependent manner. In a percutaneous absorption study a finite dose of aloesin penetrated the skin slowly and was recovered primarily in the surface wash. Aloesin shows promise as a pigmentation-altering agent for cosmetic or therapeutic applications.

Mutations of the gene encoding the protein kinase A type I-alpha regulatory subunit (PRKAR1A) in patients with the "complex of spotty skin pigmentation, myxomas, endocrine overactivity, and schwannomas" (Carney complex).

Carney complex (CNC) is a familial multiple neoplasia syndrome associated with abnormal skin and mucosal pigmentation. The complex has features overlapping those of McCune-Albright syndrome (MAS) and the other multiple endocrine neoplasias (MENs). CNC is inherited as an autosomal dominant trait, and the responsible genes have been mapped by linkage analysis to loci at 2p16 and 17q22-24. Because of its unusual biochemical features (e.g., paradoxical responses to various endocrine signals) and its clinical similarities to MAS, genes implicated in cyclic nucleotide-dependent signaling, including GNAS1 (which is responsible for MAS), had been considered likely candidates for causing CNC. The gene encoding the protein kinase A (PKA) type I-alpha regulatory subunit (RI alpha), PRKAR1A, had been mapped to 17q22-24; loss-of-heterozygosity (LOH) analysis using polymorphic markers from this region revealed consistent changes in tumors from patients with CNC, including those from one family previously mapped to 17q22-24. Investigation of a polymorphic site within the 5' of the PRKAR1A gene showed segregation with the disease and retention of the allele bearing the disease gene in CNC tumors. Mutations of the PRKAR1A gene were also found to have occurred de novo in sporadic cases of CNC; no mutations were found in kindreds mapping to 2p16. Thus, genetic heterogeneity in CNC was confirmed; in total, 41% of all patients with CNC had mutations in the PRKAR1A gene. All mutations were frameshifts, insertions, and deletions that led to nonsense mRNA and premature termination of the predicted peptide product. Functional studies in CNC tumors suggested that inactivating mutations of the PRKAR1A gene led to nonsense mRNA decay (the mutant peptide product was not present) and were associated with dysregulated PKA activity, increased responsiveness to cAMP, and excess of type-II PKA activity. We conclude that the PRKAR1A gene, coding for the RIalpha subunit of PKA, a critical cellular component of a number of cyclic nucleotide-dependent signaling pathways, is mutated in a subset of patients with CNC. In their tumors, there is LOH of the normal allele, suggesting that normal RI-alpha may have tumor suppression function in the tissues affected by CNC. An excess of type-II PKA activity was present in affected tissues, which may be responsible for the apparent tumorigenicity of PRKAR1A mutations in endocrine tissues.

The essential role of RI alpha in the maintenance of regulated PKA activity.

Cloning of the individual regulatory (R) and catalytic (C) subunits of the cAMP-dependent protein kinase (PKA) and expression of these subunits in cell culture have provided mechanistic answers about the rules for PKA holoenzyme assembly. One of the central findings of these studies is the essential role of the RI alpha regulatory subunit in maintaining the catalytic subunit under cAMP control. The role of RI alpha as the key compensatory regulatory subunit in this enzyme family was confirmed by gene knockouts of the three other regulatory subunits in mice. In each case, RI alpha has demonstrated the capacity for significant compensatory regulation of PKA activity in tissues where the other regulatory subunits are expressed, including brain, brown and white adipose tissue, skeletal muscle, and sperm. The essential requirement of the RI alpha regulatory subunit in maintaining cAMP control of PKA activity was further corroborated by the knockout of RI alpha in mice, which results in early embryonic lethality due to failed cardiac morphogenesis. Closer examination of RI alpha knockout embryos at even earlier stages of development revealed profound deficits in the morphogenesis of the mesodermal embryonic germ layer, which gives rise to essential structures including the embryonic heart tube. Failure of the mesodermal germ layer in RI alpha knockout embryos can be rescued by crossing RI alpha knockout mice to C alpha knockout mice, supporting the conclusion that inappropriately regulated PKA catalytic subunit activity is responsible for the phenotype. Isolation of primary embryonic fibroblasts from RI alpha knockout embryos reveals profound alterations in the actin-based cytoskeleton, which may account for the failure in mesoderm morphogenesis at gastrulation.