MC1R studies in dogs with melanistic mask or brindle patterns.

Black mask is a characteristic pattern in which red, yellow, tan, fawn, or brindle dogs exhibit a melanistic muzzle which may extend up onto the ears. Melanistic mask is inherited in several breeds as an autosomal dominant trait, and appears to be a fixed trait in a few breeds of dogs. A MC1R nonsense mutation, R306ter, has been shown to cause a completely red or yellow coat color in certain breeds such as Irish setters, yellow Labrador retrievers, and golden retrievers. The amino acid sequence for the melanocortin receptor 1 gene (MC1R) was examined in 17 dogs with melanistic masks from seven breeds, 19 dogs without melanistic masks, and 7 dogs in which their coat color made the mask difficult to distinguish. We also examined nine brindle dogs of four breeds, including three dogs who also had a black mask. No consistent amino acid change was observed in the brindle dogs. All dogs with a melanistic mask had at least one copy of a valine substitution for methionine at amino acid 264 (M264V) and none were homozygous for the premature stop codon (R306ter). These results suggest that black mask, but not brindle, is caused by a specific MC1R allele.

Exclusion of melanocortin-1 receptor (mc1r) and agouti as candidates for dominant black in dogs.

The domestic dog exhibits a variety of coat colors that encompass a wide range of variation among different breeds. Very little is known about the molecular biology of dog pigmentation; current understanding is based mostly on traditional breeding experiments, which in some cases have suggested genetic interactions that are different from those reported in other mammals. We have examined the molecular genetics of dominant black, a uniform coat color characteristic of black Labrador retrievers or Newfoundlands that has been proposed to be caused by either variation in the melanocortin-1 receptor gene (Mc1r) or by variation in the Agouti gene (A). We identified several coding polymorphisms within Mc1r and several simple sequence repeat polymorphisms closely linked to A, and examined their inheritance in a Labrador retriever x greyhound cross that segregates dominant black. No single Mc1r allele was found consistently in animals carrying dominant black, and neither Mc1r nor A cosegregated with dominant black. These results refine our understanding of mammalian coat color inheritance and suggest that dominant black coat color in dogs is caused by a gene not previously implicated in pigment type switching.

Antagonism of central melanocortin receptors in vitro and in vivo by agouti-related protein.

Expression of Agouti protein is normally limited to the skin where it affects pigmentation, but ubiquitous expression causes obesity. An expressed sequence tag was identified that encodes Agouti-related protein, whose RNA is normally expressed in the hypothalamus and whose levels were increased eightfold in ob/ob mice. Recombinant Agouti-related protein was a potent, selective antagonist of Mc3r and Mc4r, melanocortin receptor subtypes implicated in weight regulation. Ubiquitous expression of human AGRP complementary DNA in transgenic mice caused obesity without altering pigmentation. Thus, Agouti-related protein is a neuropeptide implicated in the normal control of body weight downstream of leptin signaling.

RBM3, a novel human gene in Xp11.23 with a putative RNA-binding domain.

Using cDNA selection with a YAC from the Xp11.2 region, we have identified a novel gene (RBM3) that encodes a polypeptide with high sequence similarity to a group of proteins that bind to RNA. On a YAC contig map, RBM3 is located between OATL1 and GATA1/TFE3 in sub-band Xp11.23, and gives rise to alternatively spliced transcripts in a variety of human tissues. The longest open reading frame encodes a 157 amino acid protein with a predicted molecular weight of 17 kDa. Its putative RNA-binding domain most closely resembles that of two previously characterized human RNA-binding proteins, YRRM, the gene for which has been implicated in azoospermia, and hnRNP G, a glycoprotein, also identified as an auto-antigen. The homology of RMB3 in both sequence and size to an RNA binding protein from maize, AAIP , suggests that it functions in a fundamental pathway conserved from plants to mammals.

The mouse homolog of the Wiskott-Aldrich syndrome protein (WASP) gene is highly conserved and maps near the scurfy (sf) mutation on the X chromosome.

The mouse WASP gene, the homolog of the gene mutated in Wiskott-Aldrich syndrome, has been isolated and sequenced. the predicted amino acid sequence is 86% identical to the human WASP sequence. A distinct feature of the mouse gene is an expanded polymorphic GGA trinucleotide repeat that codes for polyglycine and varies from 15 to 17 triplets in different Mus musculus strains. The genomic structure of the mouse WASP gene is expressed as an approximately 2.4-kb mRNA in thymus and spleen. Chromosomal mapping in an interspecific M. Musculus/M. spretus backcross placed the Wasp locus near the centromere of the mouse X chromosome, inseparable from Gata1, Tcfe3, and scurfy (sf). This localization makes Wasp a candidate for involvement in scurfy, a T cell-mediated fatal lymphoreticular disease of mice that has previously been proposed as a mouse homolog of Wiskott-Aldrich syndrome. Northern analysis of sf tissue samples indicated the presence of WASP mRNA in liver and skin, presumably as a consequence of lymphocytic infiltration, but non abnormalities in the amount or size of mRNA present.

WASP gene mutations in Wiskott-Aldrich syndrome and X-linked thrombocytopenia.

The WASP gene has been recently cloned from Xp11.23 and shown to be mutated in three patients with the Wiskott-Aldrich syndrome (WAS). We have developed a screening protocol for identifying WASP gene alterations in genomic DNA and have identified a spectrum of novel mutations in 12 additional unrelated families. These missense, nonsense and frameshift mutations involve eight of the 12 exons of the gene. Two mutations creating premature termination codons were associated with lack of detectable mRNA on Northern blots. Four amino acid substitutions, Leu27Phe, Thr48Ile, Val75Met and Arg477Lys, were found in patients with congenital thrombocytopenia and no clinically evident immune defect indicating that the WASP gene is the site for mutations in X-linked thrombocytopenia as well as in WAS. A T-cell line from a WAS patient contained two independent DNA alterations, a constitutional frameshift mutation, also present in peripheral blood leukocytes from the patient, and compensatory splice site mutation unique to the cell line. The distribution of eight missense mutations provides valuable information on amino acids which are essential for normal protein function, and suggests that sites in the first two exons are hot-spots for mutation.

Identification of a novel paternally expressed gene in the Prader-Willi syndrome region.

We have isolated a novel gene from the Prader-Willi syndrome (PWS) smallest region of deletion overlap in proximal human chromosome 15q. IPW (Imprinted gene in the Prader-Willi syndrome region) was isolated using the direct selection method and yeast artificial chromosomes localized to the deletion region. IPW is spliced and polyadenylated but its longest open reading frame codes for only 45 amino acids, suggesting that it functions as an RNA, similar to H19 and XIST. The RNA is widely expressed in adult and fetal tissues and is found in the cytoplasmic fraction of human cells, which is also the case for the H19 non-translated RNA, but differs from the XIST RNA which is found predominantly in the nucleus. Using a sequence polymorphism, exclusive expression from the paternal allele in lymphoblasts and fibroblasts was demonstrated; monoallelic expression was found in fetal tissues. IPW is located about 150 kb distal to SNRPN, the only other known gene in the deletion interval, and about 50 kb proximal to the breakpoint of a translocation which defines the distal end of the PWS region and the proximal end of the Angelman syndrome (AS) region. As is the case with SNRPN, PWS patients with 15q11-q13 deletions do not express IPW, whereas expression is normal in Angelman syndrome patients. Lack of expression of IPW may contribute to the PWS phenotype directly. Alternatively, the mRNA product of IPW may play a role in the imprinting process, acting either on genes located proximally in the PWS region or distally in the AS region.