Triphalangeal thumb-polysyndactyly syndrome and syndactyly type IV are caused by genomic duplications involving the long range, limb-specific SHH enhancer.

BACKGROUND: The Sonic hedgehog (SHH) protein produced in the zone of polarising activity (ZPA) is a major determinant of the identity and numbers of digits in early limb development. Preaxial polydactyly types II (PPD2) and III (PPD3) have been mapped to a critical region at 7q36, and subsequently shown to be caused by point mutations in the ZPA regulatory sequence (ZRS), a long range cis-regulator for the SHH gene. Triphalangeal thumb-polysyndactyly syndrome (TPTPS) and syndactyly type IV (SD4) were also mapped to the 7q36 region but pathogenic mutations in ZRS have not yet been affirmed. METHODS AND RESULTS: We performed linkage and haplotype analysis in six Han Chinese families with TPTPS and/or SD4, and refined the disease locus to an interval of 646 kb containing ZRS. In all families, the affected individuals heterozygous at rs10254391 (a single nucleotide polymorphism within ZRS) revealed a remarkable allele imbalance on sequence chromatogram. Using real-time quantitative polymerase chain reaction (qPCR), we identified duplication of ZRS and found that this duplication segregated with the limb phenotypes in all families but was not detected in unaffected family members or in unrelated control individuals. The duplication was also confirmed by interphase fluorescence in situ hybridisation (FISH) in an affected individual. We designed 17 additional qPCR assays and defined the minimum duplications in all six families, ranging from 131kb to 398kb. CONCLUSION: Both TPTPS and SD4 are due to duplications involving ZRS, the limb specific SHH enhancer. Point mutations in the ZRS and duplications encompassing the ZRS cause distinctive limb phenotypes.

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.