Frequency of a FAS ligand gene variant associated with inherited feline autoimmune lymphoproliferative syndrome in British shorthair cats in New Zealand.

AIMS To determine the frequency of the FAS-ligand gene (FASLG) variant associated with feline autoimmune lymphoproliferative syndrome (FALPS) and the proportion of carriers of the variant in three British shorthair (BSH) breeding catteries in New Zealand. METHODS Buccal swabs were collected from all cats in two BSH breeding catteries from the South Island and one from the North Island of New Zealand. DNA was extracted and was tested for the presence of the FASLG variant using PCR. Cats with the FASLG variant were identified and the frequency of the FASLG variant allele calculated. Pedigree analysis was performed and inbreeding coefficients were calculated for cats with the FASLG variant. RESULTS Of 32 BSH cats successfully tested for the presence of the FASLG variant, one kitten (3%) was homozygous (FALPS-affected), and seven (22%) cats were heterozygous (carriers) for the FASLG variant allele, and 24 (75%) cats were homozygous for the wild type allele. The overall frequency of the FASLG variant allele in these 32 cats was 0.14. Cats carrying the FASLG variant were from all three breeding catteries sampled, including two catteries that had not previously reported cases of FALPS. Pedigree analysis revealed common ancestry of FALPS-affected and carrier cats within six generations, as well as frequent inbreeding, with inbreeding coefficients >0.12 for five cats with the FASLG variant. CONCLUSIONS AND CLINICAL RELEVANCE There was a high frequency of the FASLG variant allele (0.14) in this small sample of BSH cats, with 22% of healthy cats identified as carriers of the FASLG variant. For an inherited disease, lethal at a young age, in a small population in which inbreeding is common, these results are significant. To prevent future cases of disease and stop further spread of the FASLG variant allele within the BSH population in New Zealand, it is recommended that all BSH and BSH-cross cats be tested for the presence of the FASLG variant before mating. Cats identified as carriers of the variant allele should be desexed and not used for breeding. Results support the need for further investigations of the true frequency of the FASLG variant allele and occurrence of FALPS in the wider population of BSH cats in New Zealand.

Multiple viral plaques with sebaceous differentiation associated with an unclassified papillomavirus type in a cat.

CASE HISTORY AND CLINICAL FINDINGS A 15-year-old neutered male domestic short-haired cat was presented due to multiple 0.5-2 cm-diameter crusting plaques in the left preauricular region, over the bridge of nose, and in the right periocular region. The plaques did not appear to cause discomfort. HISTOPATHOLOGICAL FINDINGS Biopsy samples of four plaques were examined histologically. Three plaques consisted of well-demarcated foci of mild epidermal hyperplasia overlying markedly hyperplastic sebaceous glands. Approximately 60% of the hyperplastic cells contained a large cytoplasmic vacuole that ranged from being clear to containing prominent grey-blue fibrillar material. The fourth plaque was composed solely of epidermal hyperplasia, consistent with previous descriptions of feline viral plaques. MOLECULAR BIOLOGY Papillomavirus DNA was amplified from all four plaques using PCR. A single DNA sequence was amplified from the plaques with sebaceous differentiation. This sequence was identical to the FdPV-MY sequence previously suggested to be from a putative unclassified papillomavirus type. Felis catus papillomavirus type 2 sequences were amplified from the plaque typical of feline viral plaques. Immunohistochemistry to detect p16CDKN2A protein (p16) showed marked immunostaining throughout the hyperplastic epidermis and adnexal structures within the plaques with sebaceous differentiation. DIAGNOSIS Multiple feline viral plaques with variable sebaceous differentiation. CLINICAL RELEVANCE Feline viral plaques with sebaceous differentiation have not been previously reported in cats. The presence of unique cell changes within these lesions, the detection of an unclassified papillomavirus type, and the p16 immunostaining within these plaques suggest that they may have been caused by the papillomavirus that contains the FdPV-MY sequence.

Wilms tumour histology is determined by distinct types of precursor lesions and not epigenetic changes.

Current models of Wilms tumour development propose that histological features of the tumours are programmed by the underlying molecular aberrations. For example, tumours associated with WT1 mutations arise from intralobar nephrogenic rests (ILNR), concur with CTNNB1 mutations and have distinct histology, whereas tumours with IGF2 loss of imprinting (LOI) often arise from perilobar nephrogenic rests (PLNR). Intriguingly, ILNR and PLNR are found simultaneously in Wilms tumours in children with overgrowth who have constitutional IGF2 LOI. We therefore examined whether the precursor lesions or early epigenetic changes are the primary determinant of Wilms tumour histology. We examined the histological features and gene expression profiles of IGF2 LOI tumours and WT1-mutant tumours which are associated with PLNR and/or ILNR. Two distinct types of IGF2 LOI tumours were identified: the first type had a blastemal-predominant histology associated with PLNR, while the second subtype had a myogenic histology, increased expression of mesenchymal lineage genes and an association with ILNR, similar to WT1-mutant tumours. These ILNR-associated IGF2 LOI tumours also showed signatures of activation of the WNT signalling pathway: differential expression of beta-catenin targets (MMP2, RARG, DKK1) and WNT antagonist genes (DKK1, WIF1, SFRP4). Unexpectedly, the majority of these tumours had CTNNB1 mutations, which are normally only seen in WT1-mutant tumours. The absence of WT1 mutations in tumours with IGF2 LOI indicated that CTNNB1 mutations occur predominantly in tumours arising from ILNR independent of the presence or absence of WT1 mutations. Thus, even though these two classes of tumours with IGF2 LOI have the same underlying predisposing epigenetic error, the tumour histology and the gene expression profiles are determined by the nature of the precursor cells within the nephrogenic rests and subsequent CTNNB1 mutations.

Imprinting, expression, and localisation of DLK1 in Wilms tumours.

BACKGROUND: Loss of imprinting (LOI) of the H19/IGF2 domain is a common feature of Wilms tumour. The GTL2/DLK1 domain is also imprinted and is structurally similar to H19/IGF2. The question arises as to whether DLK1 also undergoes LOI in Wilms tumour, or whether the LOI mechanism is restricted to the H19/IGF2 domain. AIM: To investigate the imprinting status of DLK1 in Wilms tumours with IGF2 LOI. The cellular localisation of DLK1 in the tumours was also examined. METHODS: DLK1 expression was measured by quantitative real time polymerase chain reaction (Q-PCR) in 30 Wilms tumours that had previously been classified according to whether they had IGF2 LOI, WT1 mutations, or 11p15.5 loss of heterozygosity. Allele specific expression of DLK1 was examined by direct sequencing using a DLK1 exon 5 polymorphism (rs1802710). Immunohistochemical analysis of DLK1 was performed on 13 tumours and two intralobar nephrogenic rests, in addition to two fetal kidneys and one fetal skeletal muscle sample. RESULTS: Ten of 30 tumours were heterozygous for rs1802710 and all tumours showed retention of imprinting of DLK1. Moderate to high expression of DLK1 was detected by Q-PCR in nine of 13 tumours with myogenic differentiation. Immunohistochemical expression of DLK1 was detected in the myogenic elements. CONCLUSION: LOI does not occur at the GTL2/DLK1 domain in Wilms tumour. This finding suggests that LOI at 11p15.5 does not reflect non-specific disruption of a shared imprinting mechanism. DLK1 expression in Wilms tumour might reflect the presence of myogenic differentiation, rather than an alteration of its imprinting status.

The physical maps for sequencing human chromosomes 1, 6, 9, 10, 13, 20 and X.

We constructed maps for eight chromosomes (1, 6, 9, 10, 13, 20, X and (previously) 22), representing one-third of the genome, by building landmark maps, isolating bacterial clones and assembling contigs. By this approach, we could establish the long-range organization of the maps early in the project, and all contig extension, gap closure and problem-solving was simplified by containment within local regions. The maps currently represent more than 94% of the euchromatic (gene-containing) regions of these chromosomes in 176 contigs, and contain 96% of the chromosome-specific markers in the human gene map. By measuring the remaining gaps, we can assess chromosome length and coverage in sequenced clones.

From long range mapping to sequence-ready contigs on human chromosome 6.

Our aim is to construct physical clone maps covering those regions of chromosome 6 that are not currently extensively mapped, and use these to determine the DNA sequence of the whole chromosome. The strategy we are following involves establishing a high density framework map of the order of 15 markers per Megabase using radiation hybrid (RH) mapping. The markers are then used to identify large-insert genomic bacterial clones covering the chromosome, which are assembled into sequence-ready contigs by restriction enzyme fingerprinting and sequence tagged site (STS) content analysis. Contig gap closure is performed by walking experiments using STSs developed from the end sequences of the clone inserts.

Physical mapping of chromosome 6: a strategy for the rapid generation of sequence-ready contigs.

The development of radiation hybrid (RH) mapping (Cox et al., 1990) and the availability of large numbers of STS markers, together with extensive bacterial clone resources provided a means to accelerate the process of mapping a human chromosome and preparing bacterial clone contigs ready to sequence. Our aim is to construct physical clone maps covering those regions of chromosome 6 that are not currently extensively mapped, and use these to determine the DNA sequence of the whole chromosome. We report here a strategy which initially involves establishing a high density framework map using RH mapping. The framework markers are then used for the identification of bacterial genomic clones covering the chromosome. The bacterial clones are analysed by restriction enzyme fingerprinting and STS-content analysis to identify sequence-ready contigs. Contig gap closure will also be performed by clone walking.