icacgm18th International Colloquim of Animal Cytogenetics and Gene Mapping 2008

Session II - Clinical Cytogenetics


Clinical and molecular cytogenetics in domestic animals

A. Pinton, K. Massip, N. Bonnet, A. Calgaro, B. Billoux., V. Bacquie, N. Mary, A.M. Dudez, A. Bonnet-Garnier, M. Yerle and A. Ducos

UMR 444 Genetique Cellulaire, 23 chemin des Capelles, BP 87614, 31076 Toulouse Cedex 3 (France)

Clinical animal cytogenetics developed from the beginning of the 1960’s, almost at the same time than human cytogenetics. In the 70’s, the adaptation of some specialized chromosome banding techniques allowed the identification of various chromosomal abnormalities in several livestock species. Nevertheless, in some cases, classical cytogenetics techniques did not allow an accurate characterization of the rearrangements. This limit was circumvented by the development of molecular cytogenetics from the beginning of the nineties onwards.

Constitutional chromosomal rearrangements affect the reproductive performance of heterozygote carriers or of their mates, due to spermatogenesis impairments and/or to the production of unbalanced gametes responsible for an early embryonic mortality. For a long time, our laboratory carries out systematic chromosomal controls of young bulls and boars candidates for reproduction. These analyses allowed us to discover various chromosomal rearrangements (Robertsonian, reciprocal translocations, peri- and paracentric inversions, trisomy mosaicism...). At the same time, our group developed scientific programs aimed at estimating the impact of these rearrangements on the reproductive function of carrier individuals, and bringing new insights on the meiotic behaviour of the rearrangements. Indeed, studies in humans have strong technical (collection of samples) and ethical limitations. In these conditions, the use of animal models, including livestock species, could be a good alternative. In this way, meiotic analysis of reciprocal and Robertsonian translocations as well as inversions in males and females (in the pig and bovine species) were carried out in our laboratory and allowed us on the one hand to estimate their impact on reproduction and, on the other hand, to produce original data about the influence of sex on the meiotic segregation of chromosomal rearrangements. Finally, fluorescence immunodetection using specific antibodies against meiotic proteins allowed us to analyse accurately the early stages of meiosis and then to identify chromosomal pairing disorders potentially responsible for gene silencing.


Identification of a marker chromosome in a female Sumatran orang utan (Pongo pygmaeus abeli)

C. Schelling (1), H.W. Steinmetz (2), R.M. Brunne (3), T. Goldammer (3), A. Pieńkowska-Schelling (4)

(1) Division of Animal Genetics, Vetsuisse-Faculty, University of Zurich, Zurich (Switzerland)
(2) Clinic of Zoo Animals, Exotic Pets and Wildlife, Vetsuisse-Faculty, University of Zurich, Zurich (Switzerland)
(3) Department of Molecular Biology, Research Institute for the Biology of Farm Animals (FBN), Dummerstorf (Germany);
(4) Department of Animal Sciences, Federal Institute of Technology Zurich, Zurich (Switzerland)

A female Sumatran orang utan (Pongo pygmaeus abeli) of the zoo park Zurich showed distinct differences in the phenotype in comparison to its companions. The animal seems to age quicker and had periods of daydreaming. The presence of a trisomy for chromosome 22, which has been described for orang utans and chimpanzees (Pan troglodytes), was suspected and therefore, metaphase chromosomes were prepared using both heparin-treated whole blood samples and fibroblast cell lines. The karyotype was analyzed and a diploid chromosome number of 2n=48 was found. However, the presence of a so called small supernumerary marker chromosome (sSMC) was detected. In order to better characterize its origin several copies were microdissected using an inverted microscope and conventional glass needles attached to a micromanipulator. The scraped material was amplified and labeled by DOP-PCR. Subsequent FISH-hybridization with this chromosome painting probe on orang utan and human chromosomes revealed signals in centromeric regions of some autosomes. Similar to the situation in humans, it is very difficult to elucidate the connection between a sSMC and a phenotype.


A case of X0/XX/XXX mosaicism in a masculinized Spanish breed mare

M. Moreno Millán

Molecular and Applied Animal Cytogenetic Laboratory, Department of Genetic, Faculty of Veterinary, University of Córdoba, (Spain)

The cytogenetic analysis of one six year old Spanish breed mare with masculine behaviour showed an X0/XX/XXX mosaicism in its leucocitary cells. The percentage of each cell line was 25, 30 and 45% respectively. The external phenotype of this animal is clearly masculinized. However the external and internal sexual organs are normal. One histopathologycal first study of a uterine biopsy was negative. Its oestrus is absolutely normal but none gestation was obtained because the mare doesn’t accept the male and even it try to copulate with other mares. We continue analyzing this animal with the aim of study from all points of view, reproductive, histological and genetically, this interesting case.


Leucocitary chimerism 59,XX t(1;29)/59,XY t(1;29) in a case of heterosexual twins in Retinta cattle breed

M. Moreno Millán and E.R. Genero

Molecular and Applied Animal Cytogenetic Laboratory, Department of Genetic, Faculty of Veterinary, University of Córdoba, (Spain)

In the rutinary cytogenetic screening inside of the official Breeding Selection Scheme of the Retinta cattle breed a case of leucocitary chimerism XX/XY with the presence of the robertsonian translocation 1;29 in both cell lines were observed and analyzed in a heterosexual twin birth. Both animals showed the alteration. The female co-twin was a case of freemartin syndrome with typical physiological manifestations. This animal showed masculinization of its internal genitalia, being infantilized, whereas the external genitalia were not excessive modified.


Increased frequencies of chromosome abnormalities in Mediterranean Italian buffaloes affected by limb malformation (transversal hemimelia)

F. Ciotola (1), S. Albarella (2), V. Maisto (2), V. Barbieri (2), D. Di Berardino (3), L. Iannuzzi (4), V. Peretti (2)

(1) Department of Experimental and Clinical Medicine, University Magna Graecia, Catanzaro (Italy);
(2) Department of Animal Science and Food Inspection, Faculty of Veterinary Medicine, University Federico II, Naples (Italy);
(3) Department of Soil, Plant and Environment Science, University Federico II, Portici (Italy);
(4) CNR-ISPAAM, Laboratory of Animal Cytogenetics and Gene Mapping, Naples (Italy).

In the last few years some buffalo farms in Campania have reported the birth of calves with limb malformations, especially with transversal hemimelia, a congenital abnormality characterized by the absence of a portion of distal limb structures. This study was carried out on 20 Mediterranean Italian buffaloes (8 males and 12 females) from one day to six months of age, of which 10 were affected by transversal hemimelia and 10 were healthy controls. The following clinical and radiological patterns were observed in the malformed animals: hind limbs amputated, the right amputated off the second tarsus bones and the left amputated off the proximal epiphysis metatarsus, and the right thoracic limb hypoplasic (1 female); left hind limb amputated off the proximal epiphysis metatarsus (2 females and 1 male); left hind limb amputated off the third tarsus bones (1 female); left hind limb amputated off the tibia (1 female and 1 male); left hind limb amputated off the distal epiphysis metatarsus (1 female); left hind limb amputated off the first phalanx (1 male); right hind limb amputated off the proximal epiphysis metatarsus (1 male). In their malformed limbs all the animals presented more or less developed outlines of claws. The mean number of cells with structural aberrations (gaps, chromatid breaks and chromosome breaks) per cell was higher (P < 0.001) in animals with transversal hemimelia (6.95 ± 1.95) than that (2.91 ± 1.32) found in the control. Statistical differences were found also comparing the frequencies of aneuploid cells of animals with transversal hemimelia (13.5%) and control (8.8%). These results confirm those reported in a precedent study which revealed higher levels (P < 0.001) of SCEs in calves with transversal hemimelia (8.80 ± 3.19) than those observed in the control (6.61 ± 2.73).


Cattle Rcp 9;11: Break-point Identification

L. De Lorenzi (1), M. Zannotti (1), C. Denis (2), A. Eggen (2), L. Molteni (1), P. Parma (1)

(1) Dept. of Animal Science, Milan (Italy);
(2) INRA - UR339, Department of Animal Genetics, Jouy-en-Josas (France)

Recently we have identified and characterized a new reciprocal translocation in cattle: this new anomaly involves BTA9 and BTA11. The results show that the break-point on BTA9 is close by the telomere and the break-point on BTA11 is close by the centromere.

Using cattle BACs from three different banks (INRA Library, RP-42 and CH-240) we shorted the two break-point containing regions as follow:
a) On BTA9 the break-point is present in a region of around 40 kb, as it is included in two overlapping BAC sequences: 245I24 and 156I12.
b) On BTA11 the break-point is on the first 2,5 Mb from the centromere.
Further analyses will be performed in the aim to identify the two break-points at molecular level (e.g. DNA sequences).


Mouse Lymphomas and Multicolour FISH - an overview

R. Banerjee, N. Conte, O. Dovey, B. L. Ng, F. Yang, N. P. Carter and A. Bradley (UK)

Mouse lymphomas (MLs) were derived from T cells of KO Blm -/- gamma irradiated mice. Genome wide analysis of copy number gains and losses was carried out using array CGH. Howevr, as this method is unable to detect balanced rearrangements, we complemented this approach using Multicolour FISH (fluorescence in situ hybridization) with the aim of identifying whether recurrent translocation is important in the generation of lymphomas.

For multicolour FISH we developed a set of three chromosome painting probe pools each containing 7 different chromosomes labelled in distinctive colours. Thus, in three hybridizations every mouse chromosome was painted. Each chromosome type was labelled with a unique combination of modified nucleotides by DOP - PCR i.e. FITC-dUTP, Biotin- dUTP and Digoxigenin -dUTP. Biotin labeling was detected with Avidin Texas Red and Digoxigening labeling with Cy5.5

14 out of 40 mouse lymphomas showed a recurrent translocation involving chromosome 12 with other chromosomes (i.e. chromosomes 1, 5, 14, 15, 16 and 17). Only two of these lymphomas showed reciprocal, apparently balanced translocations. The remaining 12 were all non-reciprocal, unbalanced translocations. A further 3 MLs showed a rearrangement involving chromosome 12 only i.e. one intrachromosomal amplification and two cases of heterozygous deletion.

The rearranged chromosomes from the two reciprocal translocations were flow sorted, labelled and array painted (i.e. hybridized to a mouse BAC tilepath array). The breakpoint spanning BAC identified this way was identical for both MLs. This was confirmed by FISH’ing the spanning BAC onto metaphase spreads. However, different breakpoints were found in the 4 non-reciprocal translocation MLs studied so far with the breakpoint region identified from the two reciprocal translocations being either heterozygously or homozygously deleted.
This study highlights the importance of complementing genome- wide array- CGH studies with structural analysis of chromosomes using FISH.


Molecular Cytogenetic characterization of a Cat Mammary Tumour: disclosing candidate genes by in silico analysis

S Santos (1), A Borges (1), F Gärtner (2,3), H Guedes-Pinto (1), J Wienberg (4), R Chaves (1)

(1) Institute for Biotechnology and Bioengineering, Centre of Genetics and Biotechnology of the University of Trás-os-Montes and Alto Douro (IBB/CGB-UTAD), 5001-801 Vila Real, (Portugal).
(2) Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, 4099-003 Porto, (Portugal)
(3) Institute of Pathology and Immunology (IPATIMUP), University of Porto, 4200-465 Porto, (Portugal)
(4) University of Munich, Department of Biology II, Munich, (Germany)

Animal cancer cytogenetics has been a challenge due to the difficulties involving chromosome preparation and karyotype heterogeneity. Despite the relatively frequent occurrence of mammary tumours in veterinary medicine, particularly in cats (Felis catus), cytogenetic banding and in situ hybridization data, are extremely scarce. The detection of segmental chromosome gains and losses or structural chromosome alterations such as translocations, amplifications, insertions, and deletions, in different types of mammary tumours will be important for veterinary and comparative tumour research.

In the present work, direct chromosome preparations from a cat mammary tumour were prepared. In order to identify the complex chromosomes rearranged, we used whole chromosome cat paint probes in Chromosome Painting trials. Several chromosomal rearrangements, as translocations and chromosomal gains were revealed. The databases GARField, Ensembl, NCBI allowed disclosing mammary tumours candidate genes in the rearranged chromosome regions.

This work was supported by the project POCI/CVT/62940/2004 and PhD grant SFRH/BD/23406/2005 from Science and Technology Foundation of Portugal-FCT.


Identification of a new reciprocal translocation - t(2;4)(q45;q34) in an AI bull of Ayrshire breed, with the use of synaptonemal complex technique and chromosome painting

M. Switonski (1), M. Andersson (2), J. Nowacka-Woszuk (1), I. Szczerbal (1), J. Sosnowski (1), C. Kopp (2), H. Cernohorska (3), J. Rubes (3)

(1) Department of Genetics and Animal Breeding, Agricultural University of Poznan (Poland);
(2) Department of Production Animal Medicine, University of Helsinki (Finland);
(3) Veterinary Research Institute in Brno (Czech Republic)

An AI Ayrshire bull was subjected for cytogenetic examination due to a lowered non-return rate at day 60 (NRR60 = 45,7%). Semen picture as well as libido of this bull were normal. Preliminary Giemsa staining revealed normal chromosome complement (60,XY) and G-banding did not allow to draw a conclusion concerning occurrence of a chromosomal rearrangement. At slaughter of the bull testicles were collected to conduct synaptonemal complex (SC) analysis under electron and light microscopes. A cross-shaped tetravalent configuration was observed in all pachytene spreads. No associations between the tetravalent and XY bivalent were observed. Length of the tetravalent arms suggested that two large chromosomes were involved in the translocation. Chromosome painting on metaphase spreads, with the use of bovine whole chromosome probes, conjugated with DAPI staining facilitated a detailed description of the translocation - t(2;4)(q45;q34).

This study shows that post mortem analysis of synaptonemal complexes can be a simple and useful tool for preliminary detection of the reciprocal translocation carriers. Further chromosome banding or/and painting may facilitate detailed designation of the aberration which is crucial for cytogenetic monitoring of the carrier relatives.


FA-SAT: A molecular physical inspection tool for chromosome rearrangements in cat tumours

A. Borges (1), S. Santos (1), F. Gärtner (2, 3), H. Guedes-Pinto (1), R. Chaves(1)

(1) Institute for Biotechnology and Bioengineering, Centre of Genetics and Biotechnology of the University of Trás-os-Montes and Alto Douro (IBB/CGB-UTAD), 5001-801 Vila Real, (Portugal).
(2) Department of Pathology and Molecular Immunology, Institute of Biomedical Sciences Abel Salazar (ICBAS), University of Porto, 4099-003 Porto, (Portugal).
(3) Institute of Pathology and Immunology (IPATIMUP), University of Porto, 4200-465 Porto, (Portugal).

FA-SAT is a major satellite DNA family of the cat (Felis catus) genome. Its physical location in cat chromosomes produces a distinct and characteristic in situ hybridization pattern that enables the identification of most chromosomes. Naturally occurring cat mammary tumours (CMT) offer a unique opportunity as models for human cancer biology and therapeutics. However, data regarding CMT cytogenetic characterization is very scarce, especially at the molecular level. Moreover, the cytogenetic analysis of solid tumours has been a challenge because of the difficulties in chromosome preparation and complexity of the karyotypes displayed.
In the present work we demonstrate how the FA-SAT in situ hybridization patterns can be used as molecular physical inspection tools to detect the chromosomes rearranged in CMT. Two cases are presented as example, and in both, the FA-SAT patterns disclosed the best candidate chromosomes involved in the numerical or structural chromosome alterations.

This work was supported by the project POCI/CVT/62940/2004 and a PhD
grant SFRH/BD/23406/2005 from Science and Technology Foundation of
Portugal (FCT).


Interchromosomal effect and reciprocal translocation in pigs

A. Bonnet-Garnier, S. Guardia, N. Bonnet, A. Calgaro, S. Billoux, V. Bacquie, N. Mary, A. Ducos, A. Pinton and M. Yerle

UMR 444 INRA-ENVT, Genetique cellulaire, Toulouse, (France)

Individuals with balanced reciprocal translocation have generally a normal development but a risk of producing chromosomally unbalanced gametes. The interchromosomal effect (ICE) refers to a disturbance by a rearrangement of the disjunction of chromosome pairs not involved in the rearrangement. The existence of ICE, demonstrated by an increase of aneuploid gametes, is still controversial regarding human data. Moreover some data argue that ICE is positively correlated with altered sperm parameters, with chromosome (not involved in the translocation) size or type (acrocentric...) and shape of the quadrivalent (symmetrical or asymmetrical). For pig with normal sperm parameters, we have investigated if some type of chromosomes is more prone to missegregation and if certain type of quadrivalent leads to an ICE. Probes for chromosomes 1 and 10, 11 and 18 and 13, X and Y were hybridized for dual or triple colour FISH on decondensed spermatozoa of three specimen: two boar carriers of a reciprocal translocation (t(12;14) and t(3;15)) and one normal boar. No significant difference were found between aneuploidy rates in carriers compared to control for all chromosomes except for chromosome 1 in t(3;15) carrier boar. This difference between rates (0.07% vs 0.15%) remains very low when compared with that found in the presence of a proved ICE (0.07 vs 0.73 - human data).


Significance for cytogenetic monitoring system of the domestic animals

M. Shirai (1), H. Miyazaki (2), and S. Muramatsu (3)

(1) Ibaraki Prefectural Institute of Public Health, Mito (Japan)
(2) Chiba Industrial Technology Research Institute, Chiba (Japan)
(3) Japan Livestock Technology Association, Tokyo (Japan)

Farm animals are continuously received the effects of various chemical and physical agents from environment. Cytogenetic monitoring is one of excellent check system for the evaluation of the health and genetic control of breeding farm animals. Since 1978, we are working on cytogenetic survey of farm animals (cattle, horse, swine, chickens, and so on) in Japan.
(Horse) Various breeds of racing and Japanese native horses were cytogenetically studied by in vitro lymphocyte culture, and one XX/XO female was found out in 300 analyzed animals. (Cattle) Eight-handreds and thirty-five Japanese Black cattle were cytogenetically analyzed : and 53 out of 835 animals showed abnormal karyotypes (6.3%) (51 Robertsonian translocation carriers(1/29, 7/21 heterozygotes and/or homozygotes), 1 XYY Syndrome, and 1 X delation subfertile female). (Swine) To test the possible mutagenic effects of long-continued feeding single cell protein (SCP : cell mass of yeast K and/or baeteria L) as protein source of feed, 193 Landrace swine were raised and fed a SCP containing feed throughout three successive generations. Chromosome aberrations were studied on aneuploidy, polyploidy, and structural abnormalities (gaps, breaks, fragments) as criteria ; however abnormalities observed were distributed within a spontaneous range which appeared naturally in Landrace.
Based on these studies, we are now building up the data base of the chromosome abnormalities observed in the farm animals to obtain the assessment for the sanitation and safety control of breeding farm animals.


Equine XY and XX sex-reversal syndrome in Sry positive and negative mares

DAF Villagómez (1), T L Lear (3), T Chenier (2), S Lee (2), J Cahill (4), WA King (1)

(1) Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, (Canada);
(2) Department of Population Medicine, University of Guelph, Guelph, Ontario, (Canada);
(3) Veterinary Science Department, M.H. Gluck Equine Research Center, University of Kentucky, Lexington, KY (USA);
(4)Equine Parentage and Animal Genetics Services Centre, Massey University, Palmerston North (New Zealand).

Different clinical conditions of the sex-reversal syndrome have been described in the horse. Four new clinical cases are reported here. Three mares and a filly with external genital ambiguity (penis-like enlarged clitoris) and male behavior were subjected to cytogenetic and molecular analysis for the Sry gene (PCR) and exploratory surgery or ultrasound. The first case, an American Quarter horse mare, demonstrated two inguinal hypoplastic testes, a normal G-banded male karyotype (2n= 64, XY) and presence of the Sry gene. Two mares (an Andalusian and Tennessee Walking mare) and one filly (Arabian), had upon R- or G-banding analysis a normal female equine karyotype (2n= 64, XX), and were Sry negative. Transrectal ultrasonography failed to demonstrate the presence of gonadal structures in one, although high testosterone levels (201.8pg), suggestive of testicular tissue was observed in another. Predominantly testicular tissue with small area resembling ovarian stroma was observed in both gonads of the filly. Genetic and phenotypic heterogeneity have been proposed as underlying factors leading to the range of expression in this syndrome. Androgen insensitivity due to a mutation of the androgen receptor gene may explain the presence of normal male karyotype and presence of Sry for our American Quarter horse case. Less is understood about sex reversal syndrome in pseudohermaphrodites with XX chromosome and Sry negative constitutions. Nevertheless, familial studies have proposed autosomal inheritance of that syndrome.


Identification of three new reciprocal chromosome exchanges in breeding boars

TA Quach (1), DAF Villagómez (1), C Gianfranco (1), A. Pinton (2), WA King (1)

(1) Department of Biomedical Sciences, University of Guelph, Guelph, Ontario, (Canada);
(2) UMR 444 INRA-ENVT, Genetique Cellulaire, Toulouse, (France).

One per 200 phenotypically normal boars of unproven fertility, are expected to be chromosome aberration carriers. We conducted a pilot cytogenetics survey of 78 breeding boars from four different farms to get an estimate of the frequency of chromosome abnormalities in the Canadian pig population. Three new autosomal reciprocal translocations were identified by means of the GTG-banding technique which, following the international standard nomenclature for the pig karyotype, were described as rcp(1;6)(p22,q12), rcp(10;13)(p13,q45) and rcp(9;14)(p24;q27). Subsequent application of FISH analysis corroborated the reciprocal nature of the exchanges in the 10/13. Additionally, C-banding on chromosome spreads of another boar revealed a polymorphic block of heterochromatin in one chromosome of pair SSC17. The rcp(1;6) was found in two related boars and the rcp(10;13) in another hypoprolific boar. When compared with boars in the same farm, their reproductive records showed a reduction of litter size of 40%, while the rates of repeat breeding and stillbirth were 28%, 38% and 0.3%, 0.6%, respectively. The rcp(9;14) boar carrier was from a sample of unproven young animals. These results suggest that chromosome abnormalities occur in a significant proportion of the Canadian pig populations.

Funded by Canada Research Chair Program (WAK); The Vietnamese Overseas Scholarship Program (TAQ) and the Organzation of Economic Cooperation and development fellowship program (AP).


Cytogenetic studies in Grey Steppe cattle

I. Nicolae (1), M. Sofronie (2), S. Creanga (3) , N. Isfan (3), R.Popa (3)

(1) Research and Development Institute for Bovine, Balotesti (Romania)
(2) Research and Development Station for Bovine, Dancu-Iasi (Romania)
(3) University of Agricultural Sciences Bucharest (Romania)

The Grey Steppe cattle is one of the native Romanian cattle breeds. Over the last years a drastic decrease of it occured. For this reason a special project concerning the preservation of the genetic resources of indigenous and disappearing breeds was developed. Taking into account that the cytogenetic studies represent an useful tool for the genetic evaluation, the purpose of this work was to obtain and describe the karyotype of a very small nucleus, reared in the Research and Development Station Dancu-Iasi.

Cytogenetic studies were carried out in 10 Grey Steppe cattle, 9 female and 1 male. Chromosomal investigations showed a high rate of chromosome instability, represented by chromosome or chromatid breaks and gaps. The most common abnormality observed was repeted mono- and bichromatidic Xq breaks and also mono- and bichromatidic autosomal breaks.Two of the investigated cows showed, not only the instability of heterosomes but a frequent presence of metaphases where the two XX had different size. It could be a structural abnormality, like deletion (del) of a chromatidic segment which make one X larger than its homologue but this will be elucidate by further investigations. The preliminary results and the observed configurations give us the reason to continue our investigations by using different techniques which will provide us more details to elucidate the real cytogenetic diagnostic.