Mutations in MITF and PAX3 cause "splashed white" and other white spotting phenotypes in horses

During fetal development neural-crest-derived melanoblasts migrate across the entire body surface and differentiate into melanocytes, the pigment-producing cells. Alterations in this precisely regulated process can lead to white spotting patterns. White spotting patterns in horses are a complex trait with a large phenotypic variance ranging from minimal white markings up to completely white horses. The "splashed white" pattern is primarily characterized by an extremely large blaze, often accompanied by extended white markings at the distal limbs and blue eyes. Some, but not all, splashed white horses are deaf. We analyzed a Quarter Horse family segregating for the splashed white coat color. Genome-wide linkage analysis in 31 horses gave a positive LOD score of 1.6 in a region on chromosome 6 containing the PAX3 gene. However, the linkage data were not in agreement with a monogenic inheritance of a single fully penetrant mutation. We sequenced the PAX3 gene and identified a missense mutation in some, but not all, splashed white Quarter Horses. Genome-wide association analysis indicated a potential second signal near MITF. We therefore sequenced the MITF gene and found a 10 bp insertion in the melanocyte-specific promoter. The MITF promoter variant was present in some splashed white Quarter Horses from the studied family, but also in splashed white horses from other horse breeds. Finally, we identified two additional non-synonymous mutations in the MITF gene in unrelated horses with white spotting phenotypes. Thus, several independent mutations in MITF and PAX3 together with known variants in the EDNRB and KIT genes explain a large proportion of horses with the more extreme white spotting phenotypes.     

A 19‐Mb de novo deletion on BTA 22 including MITF leads to microphthalmia and the absence of pigmentation in a Holstein calf

Mutations in MITF lead to a large variety of phenotypes in human, mice and other species. They mostly affect pigmentation and hearing, whereas in mice, they may additionally cause microphthalmia and osteopetrosis. In this study, we report a single case of a Holstein calf with lack of pigmentation and microphthalmia born to healthy parents. Mendelian analysis of high‐density SNP genotypes reveals a large number of parentage errors showing missing paternal alleles in the offspring, indicating a deletion encompassing 19 Mb on BTA 22. The genomic deletion affects the paternal allele and includes MITF and 131 other annotated genes. As the calf shows only one copy of the BTA 22 segment, the observed phenotype is probably caused by haploinsufficiency of the genes in that genomic region. Both the observed lack of skin pigmentation and reduced eye size can most likely be explained by a lack of MITF function.     

A Mutation in the SUV39H2 Gene in Labrador Retrievers with Hereditary Nasal Parakeratosis (HNPK) Provides Insights into the Epigenetics of Keratinocyte Differentiation

Hereditary nasal parakeratosis (HNPK), an inherited monogenic autosomal recessive skin disorder, leads to crusts and fissures on the nasal planum of Labrador Retrievers. We performed a genome-wide association study (GWAS) using 13 HNPK cases and 23 controls. We obtained a single strong association signal on chromosome 2 (p_raw = 4.4×10⁻¹⁴). The analysis of shared haplotypes among the 13 cases defined a critical interval of 1.6 Mb with 25 predicted genes. We re-sequenced the genome of one case at 38× coverage and detected 3 non-synonymous variants in the critical interval with respect to the reference genome assembly. We genotyped these variants in larger cohorts of dogs and only one was perfectly associated with the HNPK phenotype in a cohort of more than 500 dogs. This candidate causative variant is a missense variant in the SUV39H2 gene encoding a histone 3 lysine 9 (H3K9) methyltransferase, which mediates chromatin silencing. The variant c.972T>G is predicted to change an evolutionary conserved asparagine into a lysine in the catalytically active domain of the enzyme (p.N324K). We further studied the histopathological alterations in the epidermis in vivo. Our data suggest that the HNPK phenotype is not caused by hyperproliferation, but rather delayed terminal differentiation of keratinocytes. Thus, our data provide evidence that SUV39H2 is involved in the epigenetic regulation of keratinocyte differentiation ensuring proper stratification and tight sealing of the mammalian epidermis.     

Fine mapping of the polled locus to a 1-Mb region on bovine Chromosome 1q12

The absence of horns in Bos taurus is under genetic control of the autosomal dominant polled locus which has been genetically mapped to the centromeric region of cattle Chromosome 1. Recently a 4-Mb BAC contig of this chromosomal region has been constructed. Toward positional cloning of the bovine polled locus, we identified 20 additional microsatellite markers spread over the contig map by random sequencing of bacterial artificial chromosome (BAC) subclones. A total of 26 markers were genotyped in 30 two-generation half-sib families of six different German cattle breeds segregating for the hornless phenotype including 336 informative meioses for the polled character. Our fine-mapping study involving 19 recombinant haplotypes allowed us to narrow the critical region for the bovine polled locus to a 1-Mb segment with a centromeric boundary at RP42-218J17_MS1 and a telomeric boundary at BM6438. For marker-assisted selection purposes, the first evidence of informative flanking markers helps to predict polled genotypes with a higher degree of accuracy within families until testing of the causative mutation is available.     

P63 immunoreactivity distinguishes upper urinary tract transitional-cell carcinoma and renal-cell carcinoma even in poorly differentiated tumors.

P63 is essential for the differentiation of normal urothelium and is also expressed in transitional cell carcinoma (TCC) of the bladder. We investigated p63 immunoreactivity in upper urinary tract TCC (n=53) and in renal-cell carcinoma (RCC; n=188) using a tissue microarray technique. P63 expression was detected in 51/53 (96.2%) TCCs, showing decreased expression in high-stage (pT1 and pT2 100%; pT3 90.9%) and poorly differentiated (G1 and G2 100%; G3 92%) tumors. All RCCs were negative for p63. P63 proved to be a helpful tool, even in poorly differentiated and undifferentiated renal malignancies, to distinguish TCC from RCC.     

Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention

The contamination of cell cultures by mycoplasmas remains a major problem in cell culture. Mycoplasmas can produce a virtually unlimited variety of effects in the cultures they infect. These organisms are resistant to most antibiotics commonly employed in cell cultures. Here we provide a concise overview of the current knowledge on: (1) the incidence and sources of mycoplasma contamination in cell cultures, the mycoplasma species most commonly detected in cell cultures, and the effects of mycoplasmas on the function and activities of infected cell cultures; (2) the various techniques available for the detection of mycoplasmas with particular emphasis on the most reliable detection methods; (3) the various methods available for the elimination of mycoplasmas highlighting antibiotic treatment; and (4) the recommended procedures and working protocols for the detection, elimination and prevention of mycoplasma contamination. The availability of accurate, sensitive and reliable detection methods and the application of robust and successful elimination methods provide powerful means for overcoming the problem of mycoplasma contamination in cell cultures. This revised version was published online in August 2006 with corrections to the Cover Date.     

Molecular characterization and SNP development for the porcine IL6 and IL10 genes

Different cytokines are secreted in response to specific microbial molecules referred to as pathogen associated molecular patterns (PAMPs). Interleukin 6 (IL6) and interleukin 10 (IL10), both secreted by macrophages and lymphocytes, play a central role in the immunological response. In this work we obtained the genomic structure and complete DNA sequence of the porcine IL6 and IL10 genes and identified polymorphisms in the genomic sequences of these genes on a panel of ten different pig breeds. Comparative intra- and interbreed sequence analysis revealed a total of eight polymorphisms in the porcine IL6 gene and 21 in the porcine IL10 gene, which include single nucleotide polymorphisms (SNPs) and insertion deletion polymorphisms (indels). Additionally, the chromosomal localization of the IL10 gene was determined by FISH and RH mapping.     

Identification of a missense mutation in the bovine ATP2A1 gene in congenital pseudomyotonia of Chianina cattle: an animal model of human Brody disease

Congenital pseudomyotonia in Chianina cattle is a muscle function disorder very similar to that of Brody disease in humans. Mutations in the human ATP2A1 gene, encoding SERCA1, cause Brody myopathy. The analysis of the collected Chianina pedigree data suggested monogenic autosomal recessive inheritance and revealed that all 17 affected individuals traced back to a single founder. A deficiency of SERCA1 function in skeletal muscle of pseudomyotonia affected Chianina cattle was observed as SERCA1 activity in affected animals was decreased by about 70%. Linkage analysis showed that the mutation was located in the ATP2A1 gene region on BTA25 and subsequent mutation analysis of the ATP2A1 exons revealed a perfectly associated missense mutation in exon 6 (c.491G > A) leading to a p.Arg164His substitution. Arg164 represents a functionally important and strongly conserved residue of SERCA1. This study provides a suitable large animal model for human Brody disease.