Fluorescent in situ hybridization ( FISH ) on the veterinary diagnostic field

Proceeding deals with  of genomic changes  detectable by FISH . The DSD syndrom in Yorkshire terrier  78,XY t (Y-;6p+) was observed by the use  of X and Y FISH WCP probes. Following results indicated numerous genomic changes in cancers. Using comparative genomic hybridization numerous chromosomal rearrangements were detected, which indicated the heterogeneity in tumour growth. In Bernese Mountain Dog bitch,8  loses on  chromosomes and gains on 18 different of chromosomes were detected. The last study was focused on   chromosomal position and nucleotide sequencing of the LCA5L exons. Those  were analysed in cattle of BTA1q44,  sheep  OAR1q43  and of  CHI1q44 in the goats. Keywords: fluorescent in situ hybridization, FISH, veterinary diagnostics References ALARM, M. R. et al. (2007)  Male pseudohermafroditism in dogs: three case reports. Veterinarni Medicina 52 pp.74- 78. Hallermann, C . et al. (2004) Chromosomal Aberration Patterns Differ in Subtypes of Primary Cutaneous B Cell Lymphomas. J. Invest. Dermatol. 122 pp. 1495–1502.  D’Haese, J . G. et al .(2005) Chromosomal aberrations in follicular non-Hodgkin lymphomas of Japanese patients, detected with comparative genomic hybridization and polymerase chain reaction analysis. Cancer Genet. Cytogenet. 162 pp. 107–114. Di MEO, G.P. et al. ( 2006) Mapping of 11 genes by FISH to BTA2, BBU2q, OAR2q and CHI2, and comparison with HSA2q. Anim. Genet. 2006; 37 pp. 299-300. NAFSTRoM K . et al .(2007). Effective treatment for the canine RPE65null mutation, a hereditary retinal dystrophy comparable to human Leber’s congenital amaurosis. In:  Retinal degenerations: biology, diagnostics, and therapeutics. 1st edn. Humana Press Inc., Totowa, New Jersey, pp. 415-431. Schibler L . et al. (2009) Molecular cytogenetics and comparative mapping in goats (Capra hircus, 2n=60). Cytogenet. Genome Res .126 pp 77-85. Streubel, B . et al. (2003) T(14;18)(q32;q21) involving IGH andMALT1 is a frequent chromosomal aberration in MALT lymphoma. Blood 101 pp . 2335–2339 .


Introduction
Fluorescence in situ hybridization (FISH) is a cytogenetic technique a that uses fluorescent probes that bind to only those parts of the chromosome with a high degree of sequence complementarity.. Diagnostic of diseases bases on FISH technique is applied in different areas i.e. fertility errors, developmental defects, genotoxic effects (mutagenicity and/or carcinogenity) and at the gene positional mapping, as well.
Numerous disorders of sexual development (DSD) are classified as a hereditary and chromosomal dependent deffects in human and animals, including dog (Alarm et al., 2007).
Lymphomas (malignant lymphomas or lymphosarcomas) are among the most frequently described tumours in mammals; it is known that they are a result of malignant transformation of developed lymphocytes.Numerous chromosomal aberrations were documented in human lymphomas.Using comparative genomic hybridization according to D´Haese et al. (2005) recorded partly chromosomal imbalances and partly the translocation t(14;18) after sequencing in BCL2 gene in patients with follicular non-Hodgkin lymphomas (Streubel et al., 2004).
FISH method allows the detection of the position of the gene on the chromosom and arrangement the conserved segments among species (Di Meo, 2006;Schibler, 2009).Leber congenital amaurosis 5-like) has been studied in humans.The clinical symptoms of LCA have not yet been described in ruminants.Similar clinical symptoms of the disease resulting from RPE65 gene mutation have been described in dogs and humans i.e.night blindness, visual deficits and nystagmus (Nafström, 2007).

Disorder of sexual development in dog
A 9-month-old female like Yorkshire terrier was examined due to references about abnormal rutting like behavior and visible clitoral hypertrophy.Peripheral blood was collected for the detection of sex hormones concentration b-estradiol, testosterone and progesterone by standard RIA tests (Beckman Coulter, ISO 9001); and analysis of sex chromosomes constitution.Second examination one month later was completed with laparotomy to obtain the material for standard histopathological examination of gonads and genital ridges.For FISH the orange-red labelled whole chromosome painting (WCP) probe, specific for canine chromosome X (CFA X) and green labelled whole chromosome painting probe, specific for canine chromosome Y, VeterinaryResearch Institute (VRI) Brno, were used for precise detection of sex chromosomes.

Compatative genomic hybridization in malignant lymphoma
Tested genomic DNA was isolated from tumor tissue (lymphoma) of two dogs 10 and 12 years old bitches, and reference DNA from whole blood of healthy 7 years old male dog using DNeasy® Blood & Tissue Kit (Qiagen).Tested DNA was labeled by BioPrime® Array CGH Genomic Labeling System (Invitrogen) with Cyanine 3-dUTP (Enzo Life Sciences, red color) fluorescent dye; reference DNA was labeled with Green 500-dUTP (Enzo Life Sciences, green color).Comparative genomic hybridization has been performed, subsequently.After CGH chromosome metaphases were evaluated using an Olympus BX 60 fluorescence microscope and captured image was analyzed by CGH-ISIS software.Nine marker chromosomes (8-10 metaphases) were karyotyped and used for average red:green ratio calculation.A ratio of >1.25 : 1 indicated gain of genetic material, and ratio <0.75 : 1 loss.

FISH in comparative mapping of LCA5L gene in bovine family
Peripheral blood samples from two calves (Slovak indigenous cattle), two sheep (Walachian sheep) and two goats (White goats) were collected and cultured in a medium for 72 h at 37 °C.Chromosome preparations were prepared by standard cytogenetic methodone.BAC clone (BAC CH240-118J20) containing a partial sequence of LCA5L gene (VRI, Brno) was used for comparative FISH mapping.The digoxigenin labelled BAC probe was detected by sheep Anti-digoxigenin-rhodamine and slides were counterstained with DAPI/Antifade.Chromosomes with mapped loci were identified by computer DAPI reversed banding according to standard karyotype.

DSD syndrom
External genitalia i. e. a vagina and a vulva were stated in a normal configuration, except of the clitoral hypertrophy (2.5 cm in length).Hormonal profile indicated the low level of 17 bestradiol (<10.0 pg ml -1 ) contrary to blood testosterone with concentration of 9.1 ng ml -1 corresponding to the normal male values (ranges 3.0-10.0ng ml -1 ).
Histopathological examination confirmed that the primary gonads were testicles.The seminiferous tubules were well developed, but only Sertoli cells were present next to the baseline membranes.Significant accumulation of Leydig cells was detectable in interstitial space Well developed epididymis free of spermatozoa were visible in the cross-section of its head Bilateral deferent ducts were late up to next tubular structure.All of layers of the uterine wall were developed.Endometrium was hypoplastic, lacking corresponding glands.The rest parts of genital tract i.e. cervix uteri, vagina and vulva were classified as a standard female organs except the clitoral hypertrophy, as described above.
Conventional cytogenetic analysis (Giemsa and GTG) showed that the studied Yorkshire dog had a male chromosomal complement 78, XY in all metaphases, with normal size and morphology of both sex chromosomes.The results were confirmed by FISH with WCP probes specific for chromosome X and Y and by PCR amplification of the Y-linked fragment of SRY gene (271 bp).Telomeric parts of presumed autosome 6 (78, XY t (Y-; 6p+), as deduced from GTG banding pattern) were recognised by CFA Y probe as an additional mark, only.

FISH in comparative mapping of LCA5L gene in bovine family
Positional mapping of LCA5L gene in Bovids has not yet been made.In our study BAC clone CH240-118J20 containing the LCA5L gene was used to determine the chromosomal localization in Bos taurus, Ovis aries and Capra hircus In comparison with standard karyotypes the FISH-mapped locus was assigned to BTA1q44 in cattle, OAR1q43 in sheep, and CHI1q44 in goats.Our results confirmed the high conservation of autosomal chromosomes within the Bovids and the FISH locations of LCA5L approximate the positions reported in the sequence map.

Conclusions
As to our results we can conclude the fluorescence in situ hybridisation (FISH) method allows for: the detection of the position of the gene on the chromosome, identifying conserved segments among species; and defining the chromosomal rearrangements or abnormalities that may cause an abnormal phenotype.