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Wilson disease (WD), an inherited disorder associated with ATP7B gene, has a wide spectrum of genotypes and phenotypes. In this study, we developed a rapid multiplex PCR-MassArray method for detecting 110 mutant alleles of interest, and used it to examine genomic DNA from 1222 patients and 110 healthy controls. In patients not found to have any mutation in the 110 selected alleles, PCR-Sanger sequencing was used to examine the ATP7B gene. We identified 88 mutations, including 9 novel mutations. Our analyses revealed p. Arg778Leu, p. Arg919Gly and p. Thr935Met showed some correlations to phenotype. The p. Arg778Leu was related to younger onset age and lower levels of ceruloplasmin (Cp) and serum copper, while p. Arg919Gly and p. Thr935Met both indicated higher Cp levels. Besides, the p. Arg919Gly was related to neurological subtype, and p. Thr935Met showed significant difference in the percentage of combined neurological and visceral subtype. Moreover, for ATP7B mutations, the more severe impact on ATP7B protein was, the younger onset age and lower Cp level presented. The feasibility of presymptomatic DNA diagnosis and predicting clinical manifestation or severity of WD would be facilitated with identified mutations and genotype-phenotype correlation precisely revealed in the study. [ABSTRACT FROM AUTHOR]

Background Ichthyosis vulgaris (IV; OMIM 146700) is a very common inherited skin disorder. Loss-of-function mutations in the filaggrin gene ( FLG) have been identified as the cause of IV. In a previous study, we found that the percentage of FLG null mutations was lower in IV associated with atopic dermatitis (AD) than in IV not associated with AD (isolated IV). We speculated that some clinical manifestations of IV in patients with AD are not induced by FLG mutations. Objectives In order to clarify this issue, we collected 21 IV pedigrees, 33 patients with sporadic isolated IV and 116 patients with AD-associated IV to analyse FLG mutation frequency and filaggrin expression in isolated IV and AD-associated IV. Methods A comprehensive sequencing of the FLG gene in all patients was performed using an overlapping polymerase chain reaction (PCR) strategy. We also studied the immunohistochemistry of profilaggrin/filaggrin protein expression in the skin and measured the mRNA expression using real-time PCR in seven patients, including one patient with IV harbouring the mutation c.3321delA, two patients with AD-associated IV harbouring c.3321delA and c.6834del5, and four patients with AD-associated IV without FLG mutations. Results The percentage of mutations in the FLG gene was 74% and 43% in patients with isolated IV and patients with AD-associated IV, respectively. Immunohistochemical staining revealed that profilaggrin/filaggrin peptides were remarkably reduced in the epidermis of all the patients. All the patients with either AD or IV showed lower FLG mRNA expression compared with the normal control. Conclusions These results indicate that factors other than FLG gene mutations can downregulate profilaggrin/filaggrin expression, leading to the ichthyosiform phenotype in the context of AD. [ABSTRACT FROM AUTHOR]

Background Recently, we have reported filaggrin mutations ( FLG) of atopic dermatitis in southern China. However, there have been few detailed reports of FLG mutations of patients with AD in northern China by now. Objectives The present aim was to establish the mutation spectrum of FLG gene in AD patients in northern China. Methods A total of 339 cases met Hanifin and Rajka diagnostic criteria of AD were recruited. A comprehensive sequencing of the entire FLG coding region in these patients was conducted. All detected FLG null mutations were screened in a cohort of 301 normal controls. Results Seven novel mutations (478insA, Q1070X, 4026delT, Q1712X, Q2397X, 7145del4 and 8001del4) and eleven reported mutations (3222del4, 3321delA, 4271delAA, S1515X, Q1790X, 5757del4, 6834del5, Q2417X, E2422X, 7945delA and K4671X) in AD were identified. Mutations 3321delA and K4671X were two of the most common mutations in AD. FLG null mutations were present in 26.0% of AD patients. FLG null alleles (compound genotypes) were significantly higher in AD ( P < 0.001) than in the controls. The compound genotypes for all FLG variants were significantly associated with IV ( P < 0.001) and palmar hyperlinearity ( P < 0.001). The common mutation, K4671X, was significantly associated with AD-coexistent allergic rhinitis ( P = 0.005). Conclusions Our study increases the total number of FLG mutations. We clearly demonstrated that FLG loss-of-function mutations were significantly associated with AD in northern China. The FLG null mutations in the Chinese population differed not only from that in the European population but also from that in sub-populations of Asians outside of the Chinese mainland. [ABSTRACT FROM AUTHOR]

Aims: Daptomycin, one of the A21978C factors produced by Streptomyces roseosporus, is an acidic cyclic lipopeptide antibiotic with potent activity against a variety of Gram-positive pathogens. To increase the titre of this extensively used and clinically important antibiotic, we applied a reported-guided rpsL mutation selection system to generate strains producing high levels of A21978C. Methods and Results: In the reporter design, dptE was chosen as the overexpressing target, and neo-encoding neomycin phosphotransferase as the reporter. Using this reporter-guided selection system, 20% of the selected, streptomycin-resistant mutants produced greater amounts of A21978C than the starting strain. The selection system increased the screening efficiency about 10-fold with a frequency of 1·7% A21978C overproducing strains among str r mutants. A21978C production was increased approximately 2·2-fold in the rpsL K43N mutant. Conclusions: The combination of ribosome engineering and reporter-guided mutant selection generated an A21978C overproducing strain that produced about twice as much A21978C as the parental strain. Significance and Impact of the Study: The strategies presented here, which integrated the advantages of both ribosome engineering and reporter-guided mutation selection, could be applied to other bacteria to improve their yield of secondary metabolites. [ABSTRACT FROM AUTHOR]

SUMMARY The prognosis of esophageal squamous cell carcinoma (ESCC) is poor. It is urgent to improve this situation. Epidermal growth factor receptor (EGFR)-targeted therapy possesses a promising clinical efficacy. Mutations of EGFR and V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog (KRAS) have been identified in esophageal carcinoma, but corresponding Chinese data are limited. So we investigated the mutation status of EGFR and KRAS in Chinese patients with ESCC, and explored their correlations with clinicopathological features. Formalin-fixed paraffin-embedded surgically resected tumor samples were obtained from 50 randomly selected Chinese patients with ESCC. EGFR mutations in exons 18-21 were detected by Scorpions amplification refractory mutation system technology. KRAS mutations in codons 12, 13 were detected by direct sequencing of polymerase chain reaction products. The correlations between clinicopathological features and the mutation status of EGFR and KRAS were analyzed using the Statistical Package for the Social Sciences. In the present study, EGFR mutations were found in 7 (14%) out of 50 patients, including G719X missense mutation ( n= 1), in-frame deletion ( n= 2), and L858R missense mutation ( n= 5). Six (12%) out of 50 patients had KRAS mutations in codon 12. Concurrent EGFR and KRAS mutations were detected in one sample. The presences of EGFR and KRAS mutations were not associated with gender, age, smoking history, cell differentiation, or cancer stage. In conclusion, the incidence of EGFR mutations in Chinese patients with ESCC was higher than that of previous reports, and the incidence of KRAS mutations was not low. EGFR and KRAS mutations were mainly located in exons 19 and 21 and codon 12, respectively. Unlike in NSCLC, concurrent EGFR and KRAS mutations existed. [ABSTRACT FROM AUTHOR]

Thirteen strains of Chlamydia trachomatis were exposed to subinhibitory concentrations of erythromycin (0.5 μg ml−1), azithromycin (0.5 μg ml−1) and josamycin (0.04 μg ml−1) to select macrolide-resistant mutants with serial passages. The C. trachomatis mutants presented with low-level resistance to erythromycin, azithromycin and josamycin for which a 16-fold increase, a 16-fold increase and an 8-fold increase respectively in the minimal inhibitory concentration (MICs) for the mutant strains compared with the MIC for the susceptible strains were found. The results of chemosensitivity showed that josamycin had the highest susceptibility rate compared with erythromycin and azithromycin in the treatment of C. trachomatis. The ribosomal protein L4 and 23S rRNA genes of the susceptible and resistant strains of C. trachomatis were partially sequenced. A double mutation was found in ribosomal protein L4 of the mutants, leading to Pro109(CCG)→Leu(CTG), and Pro151(CCG)→Ala(GCC) ( Escherichia coli numbering) in the corresponding protein, but these mutations were also found in parent strains. An investigation into the sequences of 23S rRNAs in the mutants revealed point mutations of A2057G, A2059G and T2611C ( E. coli numbering). These results suggest that point mutations located in 23S rRNA were associated with macrolide resistance in C. trachomatis. [ABSTRACT FROM AUTHOR]

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 Ill (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 (S04) were also mapped to the 7q36 region but pathogenic mutations in ZAS 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 realtime 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 ZAS, the limb specific SHH enhancer. Point mutations in the ZRS and duplications encompassing the ZRS cause distinctive limb phenotypes. [ABSTRACT FROM AUTHOR]

Darier disease (DD) is a rare autosomal dominant skin disorder with characteristic abnormal keratinization and acantholysis. The causative gene, ATP2A2, is located on chromosome 12, and encodes a sarco/endoplasmic reticulum calcium pump ATPase (SERCA2). Two Chinese patients with sporadic DD participated in this study. Genomic sequence analysis identified two novel missense mutations (c.742C>A and c.2098A>G) in the ATP2A2 gene. Our findings provide an additional ATP2A2 mutation causative for DD development, and new lines of evidences for the understanding of genotype-phenotype correlations. [ABSTRACT FROM AUTHOR]

The article discusses the medical condition of Chinese families with autosomal dominant disorder Hailey-Hailey disease (HHD) caused by mutations in adenosine triphosphatase (ATPase), Ca++ transporting, type 2C, member 1 (ATP2C1) gene. It discusses the linkage between HHD cases and condyloma acuminata (CA) in two Chinese families and its effect on the human secretory pathway Ca2+/ Mn2 ± ATPase protein 1. It also presents three missense mutations in the ATP2C1 gene.

Contents The polymorphism of several genes has been shown to affect the milk composition traits in dairy cattle, including DGAT1-exon8 K232A, GH-intron3 MspI, GH-exon5 AluI, GHR-exon8 F279Y, PRL-exon3 RsaI and PRLR-exon3 S18N. However, the polymorphism and effects of these genes on the milk traits of water buffalo are still unclear. In this study, four DNA pooling samples from Murrah, Nili-ravi, Murrah-Nili-Swamp crossbreed and Chinese swamp buffalo were constructed, respectively, and polymorphism of these sites was investigated using PCR-Single-strand conformation polymorphism and sequencing. Twenty-eight inter-specific single-nucleotide polymorphism (SNPs) were found in these six assayed gene fragments between buffalo and dairy cattle, including nine intra-specific SNPs among buffalo groups. All buffalo fixed a K allele genotype in DGAT1-exon8, MspI+ restriction site(c nucleotide) and AluI+ site(c nucleotide) at intron3 and exon5 of GH gene, F allele genotype of F279Y mutation in GHR gene, RsaI− restriction site at PRL-exon3/exon4 and N allele genotype of S18N mutation at PRLR-exon3. It provides an indirect evidence that water buffalo have fixed alleles with genotypes reported in dairy cattle, which is thought to be responsible for high milk fat, high protein content and low milk yield. Moreover, three new intra-specific SNPs were found including 275th bp (c/t) in DGAT1 of Murrah buffalo, 109th bp (t/a) in PRL-exon3/exon4 and 43rd bp (c/t) in PRLR-exon3 of Chinese swamp buffalo. Information provided in this study will be useful in further studies to improve buffalo breeding for better lactation performances. [ABSTRACT FROM AUTHOR]