Original Article Cytogenet Genome Res 2014;142:95–100 DOI: 10.1159/000356209
Accepted: August 29, 2013 by M. Schmid Published online: November 22, 2013
De novo Reciprocal Translocation t(5;6)(q13;q34) in Cattle: Cytogenetic and Molecular Characterization L. De Lorenzi a E. Rossi b S. Gimelli c P. Parma a Department of Agricultural and Environmental Sciences, Milan University, Milan, and b Department of Molecular Medicine, University of Pavia, Pavia, Italy; c Department of Genetic and Laboratory Medicine, Geneva University Hospital, Geneva, Switzerland
Key Words Cattle · CGH array · FISH · Reciprocal translocation
Abstract The cytogenetic analysis of a phenotypically normal bull from the Marchigiana breed revealed the presence of an abnormal karyotype due to the presence of a very long chromosome. This finding, identified in all the metaphases observed, was associated with the 2n = 60, XY karyotype, suggesting the presence of a reciprocal translocation. RBGbanding analyses identified a de novo reciprocal translocation involving BTA5 and BTA6, t(5; 6)(q13;q34), while FISH analyses using cattle-specific BACs as probes enabled the confirmation and narrowed down the breakpoint regions. Array-CGH analysis also established that neither deletions nor duplications were present in the regions including the breakpoints, nor were they present elsewhere in the genome, confirming the balanced state of the translocation. © 2013 S. Karger AG, Basel
Introduction
Robertsonian translocations involving chromosomes 1 and 29 represent the most frequently occurring chromosomal abnormality observed in cattle breeds intended © 2013 S. Karger AG, Basel 1424–8581/13/1422–0095$38.00/0 E-Mail [email protected] www.karger.com/cgr
for meat production. Although in varying percentages, this anomaly has been reported across the world [Popescu and Pech, 1991]. However, other chromosomal anomalies have also been reported. Reciprocal translocations (RCPs) are characterized by physical exchange of DNA portions between non-homologous chromosomes. In cattle, these anomalies are responsible for economic loss as they greatly interfere with fertility, one of the key parameters for milk and meat production. In cattle, RCPs are difficult to detect during routine cytogenetic screening and, therefore, their frequency is underestimated. Recently, it has been proposed that the percentage of cattle carriers of de novo RCPs is not less than 0.14%, a 5-times higher frequency than that shown by the de novo Robertsonian translocations [De Lorenzi et al., 2012]. The effects of the RCPs on bovine fertility are well known [Switonski et al., 2008]: carrier subjects produce unbalanced gametes that culminate in the formation of unbalanced embryos having a high probability of mortality during embryonic development. To date, in cattle, only 19 cases of RCPs have been reported among which 12 involve autosomal chromosomes [reviewed in De Lorenzi et al., 2012]. All the RCPs identified during routine screening programs (on normal subjects) have breakpoints preferentially localized at the centromere-proximal regions and in the telomeric regions, while those identified in hypo-ferPietro Parma Department of Agricultural and Environmental Sciences Milan University, Via Celoria 2 IT–20133 Milan (Italy) E-Mail pietro.parma @ unimi.it
Downloaded by: Siriraj Medical Library, Mahidol University 202.28.191.34 - 3/3/2015 8:22:19 AM
a
Table 1. BACs used in this study
BACa
ENDsb
Length, UMD_3.1 genome positiond bpc chromosome/ position, bp strand
hg19 human assemblye chromo- position, bp some
382A08 CR799208 CR799207
850 855
5/+ 5/–
6,111,540 – 6,112,389 6,278,833 – 6,279,693
HS12 HS12
77,106,114 – 77,106,263 77,262,662 – 77,267,144
808F01
CR821347 CR821348
808 885
5/+ 5/–
7,274,701 – 7,275,507 7,402,124 – 7,403,005
HS12 HS12
78,196,233 – 78,203,874 78,332,573 – 78,333,459
866F10
CR823128 CR823129
721 799
6/+ 6/–
95,165,160 – 95,165,880 95,273,407 – 95,274,204
n.a. HS4
79,668,961 – 79,669,738
752C12 CR818470 CR818471
785 693
6/+ 6/–
98,931,884 – 98,932,663 99,051,180 – 99,051,872
HS4 HS4
83,293,205 – 83,293,972 83,405,617 – 83,406,320
n.a. = Not available. a All BAC names belong to the INRA Bovine BAC library (male) produced by Andre Eggen as reported in Eggen et al. [2001]. b Accession number in the GSS NCBI database. c Length of the GSS sequence. d The position on the UMD cattle genome obtained by BLAT facilities available at the UCSC genome web browser, showing the BTA chromosome, the strand and the genome position obtained. e The homology regions in human genome (hg19 assembly) were obtained using the convert facilities available at the UCSC genome web browser.
Materials and Methods Case Description A young bull from the Marchigiana breed underwent routine cytogenetic analysis as required for all male subjects selected to enter the reproduction center. At the time of analysis, the bull
96
Cytogenet Genome Res 2014;142:95–100 DOI: 10.1159/000356209
was 6-month-old with a normal external phenotype corresponding to the standard of the Marchigiana breed. Parental origin was confirmed by official parentage test performed with microsatellites. Cell Cultures, RBG-Banding Peripheral blood lymphocyte cultures were performed following the standard procedures [De Grouchy et al., 1964]. RBG-banding was performed as reported [De Lorenzi et al., 2010]. Chromosomes were classified following the most recent standard nomenclature [Cribiu et al., 2001]. Array-CGH Analysis Array-CGH was performed using a custom Agilent Bovine Genome CGH Microarray 180K (Agilent Technologies, Santa Clara, Calif., USA) and processed as reported in De Lorenzi et al. [2010]. FISH Analysis For probes, BACs obtained from the INRA library [Eggen et al., 2001] were used (table 1). They were grown overnight at 37 ° C in LB medium supplemented with chloramphenicol. DNA was extracted according to the method described on the CHORI website (http://bacpac.chori.org/). Then, 300 ng of DNA was labeled as a probe, and FISH analysis was performed as reported by De Lorenzi et al. [2010].
Cattle and Human Genome Information Cattle and Human genome information was obtained from Genome Browser Gateway (http://genome.ucsc.edu/cgi-bin/hgGateway, Bos taurus UMD_3.1 genome, Human assembly 19).
De Lorenzi /Rossi /Gimelli /Parma
Downloaded by: Siriraj Medical Library, Mahidol University 202.28.191.34 - 3/3/2015 8:22:19 AM
tile subjects show breakpoints distributed more evenly along the entire chromosomal length. This difference in the distribution of breakpoints along entire chromosomes is not linked to the phenotype observed but is a result of the detailed investigation that hypo-fertile subjects undergo [De Lorenzi et al., 2012]. These subjects are always studied with banding techniques that allow for highlighting RCPs even without the presence of an abnormal sized chromosome. In this study, we report a new case of a de novo RCP involving chromosomes 5 and 6, t(5;6)(q13;q33), identified in a young bull of the Marchigiana breed. The subject, chosen to enter an artificial insemination center, showed a normal phenotype. The chromosome rearrangement has been characterized using RBG-banding and FISH techniques as well as array-CGH technology. This report confirms once again the importance of cytogenetic analysis, at least for those individuals chosen for artificial insemination, to prevent the spreading of chromosomal anomalies (as rob(1;29)).
b
c
d
Fig. 1. Characterization of the chromosomes involved in RCP. a Giemsa-stained metaphase. b RBG-banded metaphase. c, d RBG-banding on the chromosomes involved. The blue arrows
indicate the sex chromosomes and the red arrows indicate the abnormal chromosome, further identified as der6.
Results and Discussion
Cytogenetic Analysis Cytogenetic analysis revealed an abnormal chromosomal constitution (fig. 1a) due to the presence of an over-sized chromosome. Consequently, the bull was excluded from the breeding program and, instead, intended for meat production. The parent animals, which had been analyzed, had each presented a normal karyotype. Therefore, we can establish a de novo origin of this anomaly (data not shown). RBG-banding revealed that the over-sized chromosome was derived from an RCP event involving BTA5 and BTA6 (fig. 1b–d). Using the banding techniques, a comparison of the abnormal chromosomes against the standard karyogram enabled the definition of the cytogenetic bands involved in the anomaly: 5q13 and 6q34. Once again, the 2 breakpoints were found to be located in the telomeric and centromeric regions as expected in this case [De Lorenzi et al., 2012]. Considering the results reported by De Lorenzi et al. [2012], theoretically only onehalf of the RCPs involving BTA5 and BTA6 would produce a derivative longer than BTA1 or shorter than Cattle 5;6 Reciprocal Translocation
BTA29. On the other hand, considering that the human eye is incapable of detecting small changes in the chromosome length, this value decreases to about a quarter (26.3%) even with an experienced operator. FISH Analysis Cattle-specific BAC probes were used both to confirm the involvement of BTA5 and BTA6 and to localize the breakpoints more specifically and linked to the available cattle genome. The breakpoints were identified between BAC 382A08 and 808F01 on BTA5 and between BAC 866F10 and 752C12 on BTA6, respectively (fig. 2a–c). Considering the position of these BACs (obtained with ENDs blast on the cattle genome), we can define the regions involved in the RCP event. They are 995 kb (from bp 6,279,693–7,274,701) and 3.6 Mb (from bp 95,274,204– 98,931,884) long for BTA5 and BTA6, respectively. The region of BTA5 involved in the breakpoint (online suppl. fig. 1a, www.karger.com/doi/10.1159/000356209) does not contain any gene or other element that can suggest the presence of a gene (mRNA, EST). However, 3 genes are present in the flanking genomic regions: ZDHHC17, CSRP2, and NAV3. Their 5′-3′ orientations suggest that the rupture of chromosome 5 could interfere with the promoter regions limited to the last 2 genes. Interestingly, 1 EST (EE891129) is probably a 5′ longer version of the NAV3 gene, and this mRNA is located inside the breakpoint region. In humans, the NAV3 gene (Neuron navigator 3) was first partially described by Nagase et al. [1999] by screening a fetal brain cDNA library. It was cloned and reported as POMFIL1 [Coy et al., 2002]. This gene is mainly expressed in the fetal and adult brains, and the protein is localized at the outer nuclear membrane of the neuron. No data for the knockout phenotype is available. In addition, the 5′ longer version observed in cattle (EE891129 EST) is not described in humans. CRSP2 is a member of the CRP family belonging to the LIM domain proteins [Weiskirchen et al., 2001]. Knockout mice exhibit problems in the cardiovascular system and homeostasis. The BTA5 portion involved corresponds to the 12q21.2 region (at about 77.2 Mb) in humans. In the human region another gene, E2F7, is found to be present. Looking for the E2F7 gene in the database of the bovine genome, a high homology is observed with BTA5 6,426,803– 6,466,792 bp, in a position clearly within the region containing the breakpoint. The concept that this gene is present, even if not characterized as yet, is supported by the presence of some ESTs (DV851513, EE242572, BE757648, CN433474, and EE363700). E2F7 is a transcription factor Cytogenet Genome Res 2014;142:95–100 DOI: 10.1159/000356209
97
Downloaded by: Siriraj Medical Library, Mahidol University 202.28.191.34 - 3/3/2015 8:22:19 AM
a
a
b
a
c
b
involved in the regulation of the progression of the cell cycle [Di Stefano et al., 2003]. Mice homozygous for a knockout allele develop normally, grow, and live to old age [Li et al., 2008]. As the genomic region of BTA6 (online suppl. fig. 1b), in which the breakpoint is present, is at least 3 times larger than that of chromosome 5, there is a greater probability that some genes are present. The cattle genome region contains 11 genes: PAQR3, NAA11, PRKG2, ANTXR2, PRDM8, FGF5, C6H3orf22, BMP3, PRKG2, RASGEF1B, and HNRNPD. This region is homologous to the human 4q21q22. With the exception of 3 genes, all human genes 98
Cytogenet Genome Res 2014;142:95–100 DOI: 10.1159/000356209
Fig. 3. The CGH array results: BTA5 (a) and BTA6 (b) CGH array profiles. For each BTA, the whole chromosome is reported on the left, while an enlargement of the breakpoint regions (from 6.1 to 7.4 Mb on BTA5 and from 95.3 to 99.0 Mb on BTA6), showing absence of unbalance on both chromosomes, is reported on the right.
in the region are also found in the bovine genome: 2 LOC sequences (LOC100505875 and LOC100506035) and the GDEP gene. Using the BLAT pairing software, we identified 3 high homology regions in the bovine genome, at about 95.9 Mb (as expected). However, no cattle ESTs were present. The 11 genes span about 1.1 MB, equal to 31% of the length of the region probably containing the breakpoint. Consequently, further analyses are warranted to conclude whether the chromosomal breakage ocDe Lorenzi /Rossi /Gimelli /Parma
Downloaded by: Siriraj Medical Library, Mahidol University 202.28.191.34 - 3/3/2015 8:22:19 AM
Fig. 2. Characterization of the RCP by FISH analysis. a, b FISH localization of the BACs used for localization of the breakpoints. c R-banded ideograms of normal BTA5 and BTA6 as well as der(6) and der(5). The dotted line indicates the breakpoints responsible for RCP formation, while the localization of the BACs used are also reported.
curred outside or inside a genetic factor, as this second possibility is neither a negligible nor a remote one. Not all these genes possess knockout mice information; therefore, no comments can be made regarding the phenotypic changes caused by the alteration of some of these genes. Knockout mice homozygous for a null allele of ANTXR2 (anthrax toxin receptor 2 isoform 1 precursor) exhibit female infertility, while mice homozygous for a second allele denote premature embryo death and failure of parturition (MGI http://genome.ucsc.edu/ cgi-bin/hgTracks?db=hg19&position=chr12:7710611477106263). Mutations in the FGF5 gene lead to a longhaired phenotype [Hébert et al., 1994], while a homozygous mutation of BMP3 leads to an increased bone density [Daluiski et al., 2001]. Finally, PRKG2 knockout mice exhibit dwarfism with abnormal skull morphology and short limbs and vertebrae [Chikuda et al., 2004]. The bull carrying the RCP 5;6 does not express any of these clinical features. Therefore, we assume that no gene is altered by the breakage or in other words, the heterozygous state does not affect the phenotype. The function of a gene may be disrupted by an RCP essentially in 2 ways: either by direct rupture of the coding sequence or because of the interference in the regulatory regions. In human beings the first example of this event was reported in 1987 where the dystrophin (DMD) gene is interrupted in a Duchenne muscular dystrophy patient carrying the Xp21/autosome RCP [Kenwrick et al., 1987]. An example of how the RCP can cause an abnormal phenotype by interfering with the regulatory regions comes from case of RCP associated with Campomelic Dysplasia where regulatory regions of the SOX9 gene are perturbed [Wagner et al., 1994].
Array-CGH Analysis To date, all RCPs discovered in cattle have been defined as ‘balanced’. By contrast, over the last few years, due to the introduction of array-CGH analysis, it has been shown that 40% of apparently balanced human rearrangements associated with phenotypic anomalies are in reality characterized by the presence of cryptic imbalances responsible for the abnormal phenotype [De Gregori et al., 2007]. To exclude the presence of an imbalance, we investigated the bull DNA using a specific cattle arrayCGH. The CGH analysis excluded a deletion or a duplication around the breakpoints (fig. 3a, b) and elsewhere in the genome. Thus, this RCP is really balanced as the only one previously analyzed using this technique: RCP 4; 7 [De Lorenzi et al., 2010]. In conclusion, we report a de novo reciprocal translocation in cattle, and we characterize it by several cytogenetic approaches, viz., chromosome banding, FISH, and array-CGH. The cytogenetic analysis of the subjects intended for entry into a reproduction program represents a key step in the prevention of the diffusion of genetic abnormalities that have a clear, well-known negative impact on the fertility of the carriers. Moreover, further development of analytical technologies capable of detecting a higher percentage of reciprocal translocations is also warranted, because, with the techniques existent until now, only about 16% of these anomalies can be identified during routine screening. Acknowledgments We are grateful to A.N.A.B.I.C for providing us with fresh blood and animal data. We thank the CRB GADIE (INRA Jouy-en-Josas, France) staff for providing us with cattle BACs.
References
Cattle 5;6 Reciprocal Translocation
Daluiski A, Engstrand T, Bahamonde ME, Gamer LW, Agius E, et al: Bone morphogenetic protein-3 is a negative regulator of bone density. Nat Genet 27:84–88 (2001). De Gregori M, Ciccone R, Magini P, Pramparo T, Gimelli S, et al: Cryptic deletions are a common finding in ‘balanced’ reciprocal and complex chromosome rearrangements: a study of 59 patients. J Med Genet 44:750–762 (2007). De Grouchy J, Roubin M, Passage E: Microtechnique pour l’étude des chromosomes humains a partir d’une culture de leucocytes sanguins. Ann Génét 7:45–46 (1964).
De Lorenzi L, Kopecna O, Gimelli S, Cernohorska H, Zannotti M, et al: Reciprocal translocation t(4;7)(q14;q28) in cattle: molecular characterization. Cytogenet Genome Res 129:298–304 (2010). De Lorenzi L, Morando P, Planas J, Zannotti M, Molteni L, Parma P: Reciprocal translocations in cattle: frequency estimation. J Anim Breed Genet 129:409–416 (2012). Di Stefano L, Jensen MR, Helin K: E2F7, a novel E2F featuring DP-independent repression of a subset of E2F-regulated genes. EMBO J 22: 6289–6298 (2003).
Cytogenet Genome Res 2014;142:95–100 DOI: 10.1159/000356209
99
Downloaded by: Siriraj Medical Library, Mahidol University 202.28.191.34 - 3/3/2015 8:22:19 AM
Chikuda H, Kugimiya F, Hoshi K, Ikeda T, Ogasawara T, et al: Cyclic GMP-dependent protein kinase II is a molecular switch from proliferation to hypertrophic differentiation of chondrocytes. Genes Dev 18:2418–2429 (2004). Coy JF, Wiemann S, Bechmann I, Bächner D, Nitsch R, et al: Pore membrane and/or filament interacting like protein 1 (POMFIL1) is predominantly expressed in the nervous system and encodes different protein isoforms. Gene 290:73–94 (2002). Cribiu EP, Di Berardino D, Di Meo GP, Eggen A, Gallagher DS, et al: International System for Chromosome Nomenclature of Domestic Bovids (ISCNDB 2000). Cytogenet Cell Genet 92:283–299 (2001).
100
Li J, Ran C, Li E, Gordon F, Comstock G, et al: Synergistic function of E2F7 and E2F8 is essential for cell survival and embryonic development. Dev Cell 14:62–75 (2008). Nagase T, Ishikawa K, Suyama M, Kikuno R, Hirosawa M, et al: Prediction of the coding sequences of unidentified human genes. XIII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA Res 6:63–70 (1999). Popescu CP, Pech A: Une bibliographie sur la translocation 1/29 de bovins dans le monde (1964–1990). Ann Zootec 40:271–305 (1991). Switonski M, Andersson M, Nowacka-Woszuk J, Szczerbal I, Sosnowski J, et al: Identification of a new reciprocal translocation in an AI bull by synaptonemal complex analysis, followed by chromosome painting. Cytogenet Genome Res 121:245–248 (2008).
Cytogenet Genome Res 2014;142:95–100 DOI: 10.1159/000356209
Wagner T, Wirth J, Meyer J, Zabel B, Held M, et al: Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell 796: 1111–1120 (1994). Weiskirchen R, Moser M, Weiskirchen S, Erdel M, Dahmen S, et al: LIM-domain protein cysteine- and glycine-rich protein 2 (CRP2) is a novel marker of hepatic stellate cells and binding partner of the protein inhibitor of activated STAT1. Biochem J 359: 485–496 (2001).
De Lorenzi /Rossi /Gimelli /Parma
Downloaded by: Siriraj Medical Library, Mahidol University 202.28.191.34 - 3/3/2015 8:22:19 AM
Eggen A, Gautier M, Billaut A, Petit E, Hayes H, et al: Construction and characterization of a bovine BAC library with four genome-equivalent coverage. Genet Sel Evol 33: 543–548 (2001). Hébert JM, Rosenquist T, Götz J, Martin GR: FGF5 as a regulator of the hair growth cycle: evidence from targeted and spontaneous mutations. Cell 78:1017–1025 (1994). Kenwrick S, Patterson M, Speer A, Fischbeck K, Davies K: Molecular analysis of the Duchenne muscular dystrophy region using pulsed field gel electrophoresis. Cell 48:351–357 (1987).
Accepted: August 29, 2013 by M. Schmid Published online: November 22, 2013
De novo Reciprocal Translocation t(5;6)(q13;q34) in Cattle: Cytogenetic and Molecular Characterization L. De Lorenzi a E. Rossi b S. Gimelli c P. Parma a Department of Agricultural and Environmental Sciences, Milan University, Milan, and b Department of Molecular Medicine, University of Pavia, Pavia, Italy; c Department of Genetic and Laboratory Medicine, Geneva University Hospital, Geneva, Switzerland
Key Words Cattle · CGH array · FISH · Reciprocal translocation
Abstract The cytogenetic analysis of a phenotypically normal bull from the Marchigiana breed revealed the presence of an abnormal karyotype due to the presence of a very long chromosome. This finding, identified in all the metaphases observed, was associated with the 2n = 60, XY karyotype, suggesting the presence of a reciprocal translocation. RBGbanding analyses identified a de novo reciprocal translocation involving BTA5 and BTA6, t(5; 6)(q13;q34), while FISH analyses using cattle-specific BACs as probes enabled the confirmation and narrowed down the breakpoint regions. Array-CGH analysis also established that neither deletions nor duplications were present in the regions including the breakpoints, nor were they present elsewhere in the genome, confirming the balanced state of the translocation. © 2013 S. Karger AG, Basel
Introduction
Robertsonian translocations involving chromosomes 1 and 29 represent the most frequently occurring chromosomal abnormality observed in cattle breeds intended © 2013 S. Karger AG, Basel 1424–8581/13/1422–0095$38.00/0 E-Mail [email protected] www.karger.com/cgr
for meat production. Although in varying percentages, this anomaly has been reported across the world [Popescu and Pech, 1991]. However, other chromosomal anomalies have also been reported. Reciprocal translocations (RCPs) are characterized by physical exchange of DNA portions between non-homologous chromosomes. In cattle, these anomalies are responsible for economic loss as they greatly interfere with fertility, one of the key parameters for milk and meat production. In cattle, RCPs are difficult to detect during routine cytogenetic screening and, therefore, their frequency is underestimated. Recently, it has been proposed that the percentage of cattle carriers of de novo RCPs is not less than 0.14%, a 5-times higher frequency than that shown by the de novo Robertsonian translocations [De Lorenzi et al., 2012]. The effects of the RCPs on bovine fertility are well known [Switonski et al., 2008]: carrier subjects produce unbalanced gametes that culminate in the formation of unbalanced embryos having a high probability of mortality during embryonic development. To date, in cattle, only 19 cases of RCPs have been reported among which 12 involve autosomal chromosomes [reviewed in De Lorenzi et al., 2012]. All the RCPs identified during routine screening programs (on normal subjects) have breakpoints preferentially localized at the centromere-proximal regions and in the telomeric regions, while those identified in hypo-ferPietro Parma Department of Agricultural and Environmental Sciences Milan University, Via Celoria 2 IT–20133 Milan (Italy) E-Mail pietro.parma @ unimi.it
Downloaded by: Siriraj Medical Library, Mahidol University 202.28.191.34 - 3/3/2015 8:22:19 AM
a
Table 1. BACs used in this study
BACa
ENDsb
Length, UMD_3.1 genome positiond bpc chromosome/ position, bp strand
hg19 human assemblye chromo- position, bp some
382A08 CR799208 CR799207
850 855
5/+ 5/–
6,111,540 – 6,112,389 6,278,833 – 6,279,693
HS12 HS12
77,106,114 – 77,106,263 77,262,662 – 77,267,144
808F01
CR821347 CR821348
808 885
5/+ 5/–
7,274,701 – 7,275,507 7,402,124 – 7,403,005
HS12 HS12
78,196,233 – 78,203,874 78,332,573 – 78,333,459
866F10
CR823128 CR823129
721 799
6/+ 6/–
95,165,160 – 95,165,880 95,273,407 – 95,274,204
n.a. HS4
79,668,961 – 79,669,738
752C12 CR818470 CR818471
785 693
6/+ 6/–
98,931,884 – 98,932,663 99,051,180 – 99,051,872
HS4 HS4
83,293,205 – 83,293,972 83,405,617 – 83,406,320
n.a. = Not available. a All BAC names belong to the INRA Bovine BAC library (male) produced by Andre Eggen as reported in Eggen et al. [2001]. b Accession number in the GSS NCBI database. c Length of the GSS sequence. d The position on the UMD cattle genome obtained by BLAT facilities available at the UCSC genome web browser, showing the BTA chromosome, the strand and the genome position obtained. e The homology regions in human genome (hg19 assembly) were obtained using the convert facilities available at the UCSC genome web browser.
Materials and Methods Case Description A young bull from the Marchigiana breed underwent routine cytogenetic analysis as required for all male subjects selected to enter the reproduction center. At the time of analysis, the bull
96
Cytogenet Genome Res 2014;142:95–100 DOI: 10.1159/000356209
was 6-month-old with a normal external phenotype corresponding to the standard of the Marchigiana breed. Parental origin was confirmed by official parentage test performed with microsatellites. Cell Cultures, RBG-Banding Peripheral blood lymphocyte cultures were performed following the standard procedures [De Grouchy et al., 1964]. RBG-banding was performed as reported [De Lorenzi et al., 2010]. Chromosomes were classified following the most recent standard nomenclature [Cribiu et al., 2001]. Array-CGH Analysis Array-CGH was performed using a custom Agilent Bovine Genome CGH Microarray 180K (Agilent Technologies, Santa Clara, Calif., USA) and processed as reported in De Lorenzi et al. [2010]. FISH Analysis For probes, BACs obtained from the INRA library [Eggen et al., 2001] were used (table 1). They were grown overnight at 37 ° C in LB medium supplemented with chloramphenicol. DNA was extracted according to the method described on the CHORI website (http://bacpac.chori.org/). Then, 300 ng of DNA was labeled as a probe, and FISH analysis was performed as reported by De Lorenzi et al. [2010].
Cattle and Human Genome Information Cattle and Human genome information was obtained from Genome Browser Gateway (http://genome.ucsc.edu/cgi-bin/hgGateway, Bos taurus UMD_3.1 genome, Human assembly 19).
De Lorenzi /Rossi /Gimelli /Parma
Downloaded by: Siriraj Medical Library, Mahidol University 202.28.191.34 - 3/3/2015 8:22:19 AM
tile subjects show breakpoints distributed more evenly along the entire chromosomal length. This difference in the distribution of breakpoints along entire chromosomes is not linked to the phenotype observed but is a result of the detailed investigation that hypo-fertile subjects undergo [De Lorenzi et al., 2012]. These subjects are always studied with banding techniques that allow for highlighting RCPs even without the presence of an abnormal sized chromosome. In this study, we report a new case of a de novo RCP involving chromosomes 5 and 6, t(5;6)(q13;q33), identified in a young bull of the Marchigiana breed. The subject, chosen to enter an artificial insemination center, showed a normal phenotype. The chromosome rearrangement has been characterized using RBG-banding and FISH techniques as well as array-CGH technology. This report confirms once again the importance of cytogenetic analysis, at least for those individuals chosen for artificial insemination, to prevent the spreading of chromosomal anomalies (as rob(1;29)).
b
c
d
Fig. 1. Characterization of the chromosomes involved in RCP. a Giemsa-stained metaphase. b RBG-banded metaphase. c, d RBG-banding on the chromosomes involved. The blue arrows
indicate the sex chromosomes and the red arrows indicate the abnormal chromosome, further identified as der6.
Results and Discussion
Cytogenetic Analysis Cytogenetic analysis revealed an abnormal chromosomal constitution (fig. 1a) due to the presence of an over-sized chromosome. Consequently, the bull was excluded from the breeding program and, instead, intended for meat production. The parent animals, which had been analyzed, had each presented a normal karyotype. Therefore, we can establish a de novo origin of this anomaly (data not shown). RBG-banding revealed that the over-sized chromosome was derived from an RCP event involving BTA5 and BTA6 (fig. 1b–d). Using the banding techniques, a comparison of the abnormal chromosomes against the standard karyogram enabled the definition of the cytogenetic bands involved in the anomaly: 5q13 and 6q34. Once again, the 2 breakpoints were found to be located in the telomeric and centromeric regions as expected in this case [De Lorenzi et al., 2012]. Considering the results reported by De Lorenzi et al. [2012], theoretically only onehalf of the RCPs involving BTA5 and BTA6 would produce a derivative longer than BTA1 or shorter than Cattle 5;6 Reciprocal Translocation
BTA29. On the other hand, considering that the human eye is incapable of detecting small changes in the chromosome length, this value decreases to about a quarter (26.3%) even with an experienced operator. FISH Analysis Cattle-specific BAC probes were used both to confirm the involvement of BTA5 and BTA6 and to localize the breakpoints more specifically and linked to the available cattle genome. The breakpoints were identified between BAC 382A08 and 808F01 on BTA5 and between BAC 866F10 and 752C12 on BTA6, respectively (fig. 2a–c). Considering the position of these BACs (obtained with ENDs blast on the cattle genome), we can define the regions involved in the RCP event. They are 995 kb (from bp 6,279,693–7,274,701) and 3.6 Mb (from bp 95,274,204– 98,931,884) long for BTA5 and BTA6, respectively. The region of BTA5 involved in the breakpoint (online suppl. fig. 1a, www.karger.com/doi/10.1159/000356209) does not contain any gene or other element that can suggest the presence of a gene (mRNA, EST). However, 3 genes are present in the flanking genomic regions: ZDHHC17, CSRP2, and NAV3. Their 5′-3′ orientations suggest that the rupture of chromosome 5 could interfere with the promoter regions limited to the last 2 genes. Interestingly, 1 EST (EE891129) is probably a 5′ longer version of the NAV3 gene, and this mRNA is located inside the breakpoint region. In humans, the NAV3 gene (Neuron navigator 3) was first partially described by Nagase et al. [1999] by screening a fetal brain cDNA library. It was cloned and reported as POMFIL1 [Coy et al., 2002]. This gene is mainly expressed in the fetal and adult brains, and the protein is localized at the outer nuclear membrane of the neuron. No data for the knockout phenotype is available. In addition, the 5′ longer version observed in cattle (EE891129 EST) is not described in humans. CRSP2 is a member of the CRP family belonging to the LIM domain proteins [Weiskirchen et al., 2001]. Knockout mice exhibit problems in the cardiovascular system and homeostasis. The BTA5 portion involved corresponds to the 12q21.2 region (at about 77.2 Mb) in humans. In the human region another gene, E2F7, is found to be present. Looking for the E2F7 gene in the database of the bovine genome, a high homology is observed with BTA5 6,426,803– 6,466,792 bp, in a position clearly within the region containing the breakpoint. The concept that this gene is present, even if not characterized as yet, is supported by the presence of some ESTs (DV851513, EE242572, BE757648, CN433474, and EE363700). E2F7 is a transcription factor Cytogenet Genome Res 2014;142:95–100 DOI: 10.1159/000356209
97
Downloaded by: Siriraj Medical Library, Mahidol University 202.28.191.34 - 3/3/2015 8:22:19 AM
a
a
b
a
c
b
involved in the regulation of the progression of the cell cycle [Di Stefano et al., 2003]. Mice homozygous for a knockout allele develop normally, grow, and live to old age [Li et al., 2008]. As the genomic region of BTA6 (online suppl. fig. 1b), in which the breakpoint is present, is at least 3 times larger than that of chromosome 5, there is a greater probability that some genes are present. The cattle genome region contains 11 genes: PAQR3, NAA11, PRKG2, ANTXR2, PRDM8, FGF5, C6H3orf22, BMP3, PRKG2, RASGEF1B, and HNRNPD. This region is homologous to the human 4q21q22. With the exception of 3 genes, all human genes 98
Cytogenet Genome Res 2014;142:95–100 DOI: 10.1159/000356209
Fig. 3. The CGH array results: BTA5 (a) and BTA6 (b) CGH array profiles. For each BTA, the whole chromosome is reported on the left, while an enlargement of the breakpoint regions (from 6.1 to 7.4 Mb on BTA5 and from 95.3 to 99.0 Mb on BTA6), showing absence of unbalance on both chromosomes, is reported on the right.
in the region are also found in the bovine genome: 2 LOC sequences (LOC100505875 and LOC100506035) and the GDEP gene. Using the BLAT pairing software, we identified 3 high homology regions in the bovine genome, at about 95.9 Mb (as expected). However, no cattle ESTs were present. The 11 genes span about 1.1 MB, equal to 31% of the length of the region probably containing the breakpoint. Consequently, further analyses are warranted to conclude whether the chromosomal breakage ocDe Lorenzi /Rossi /Gimelli /Parma
Downloaded by: Siriraj Medical Library, Mahidol University 202.28.191.34 - 3/3/2015 8:22:19 AM
Fig. 2. Characterization of the RCP by FISH analysis. a, b FISH localization of the BACs used for localization of the breakpoints. c R-banded ideograms of normal BTA5 and BTA6 as well as der(6) and der(5). The dotted line indicates the breakpoints responsible for RCP formation, while the localization of the BACs used are also reported.
curred outside or inside a genetic factor, as this second possibility is neither a negligible nor a remote one. Not all these genes possess knockout mice information; therefore, no comments can be made regarding the phenotypic changes caused by the alteration of some of these genes. Knockout mice homozygous for a null allele of ANTXR2 (anthrax toxin receptor 2 isoform 1 precursor) exhibit female infertility, while mice homozygous for a second allele denote premature embryo death and failure of parturition (MGI http://genome.ucsc.edu/ cgi-bin/hgTracks?db=hg19&position=chr12:7710611477106263). Mutations in the FGF5 gene lead to a longhaired phenotype [Hébert et al., 1994], while a homozygous mutation of BMP3 leads to an increased bone density [Daluiski et al., 2001]. Finally, PRKG2 knockout mice exhibit dwarfism with abnormal skull morphology and short limbs and vertebrae [Chikuda et al., 2004]. The bull carrying the RCP 5;6 does not express any of these clinical features. Therefore, we assume that no gene is altered by the breakage or in other words, the heterozygous state does not affect the phenotype. The function of a gene may be disrupted by an RCP essentially in 2 ways: either by direct rupture of the coding sequence or because of the interference in the regulatory regions. In human beings the first example of this event was reported in 1987 where the dystrophin (DMD) gene is interrupted in a Duchenne muscular dystrophy patient carrying the Xp21/autosome RCP [Kenwrick et al., 1987]. An example of how the RCP can cause an abnormal phenotype by interfering with the regulatory regions comes from case of RCP associated with Campomelic Dysplasia where regulatory regions of the SOX9 gene are perturbed [Wagner et al., 1994].
Array-CGH Analysis To date, all RCPs discovered in cattle have been defined as ‘balanced’. By contrast, over the last few years, due to the introduction of array-CGH analysis, it has been shown that 40% of apparently balanced human rearrangements associated with phenotypic anomalies are in reality characterized by the presence of cryptic imbalances responsible for the abnormal phenotype [De Gregori et al., 2007]. To exclude the presence of an imbalance, we investigated the bull DNA using a specific cattle arrayCGH. The CGH analysis excluded a deletion or a duplication around the breakpoints (fig. 3a, b) and elsewhere in the genome. Thus, this RCP is really balanced as the only one previously analyzed using this technique: RCP 4; 7 [De Lorenzi et al., 2010]. In conclusion, we report a de novo reciprocal translocation in cattle, and we characterize it by several cytogenetic approaches, viz., chromosome banding, FISH, and array-CGH. The cytogenetic analysis of the subjects intended for entry into a reproduction program represents a key step in the prevention of the diffusion of genetic abnormalities that have a clear, well-known negative impact on the fertility of the carriers. Moreover, further development of analytical technologies capable of detecting a higher percentage of reciprocal translocations is also warranted, because, with the techniques existent until now, only about 16% of these anomalies can be identified during routine screening. Acknowledgments We are grateful to A.N.A.B.I.C for providing us with fresh blood and animal data. We thank the CRB GADIE (INRA Jouy-en-Josas, France) staff for providing us with cattle BACs.
References
Cattle 5;6 Reciprocal Translocation
Daluiski A, Engstrand T, Bahamonde ME, Gamer LW, Agius E, et al: Bone morphogenetic protein-3 is a negative regulator of bone density. Nat Genet 27:84–88 (2001). De Gregori M, Ciccone R, Magini P, Pramparo T, Gimelli S, et al: Cryptic deletions are a common finding in ‘balanced’ reciprocal and complex chromosome rearrangements: a study of 59 patients. J Med Genet 44:750–762 (2007). De Grouchy J, Roubin M, Passage E: Microtechnique pour l’étude des chromosomes humains a partir d’une culture de leucocytes sanguins. Ann Génét 7:45–46 (1964).
De Lorenzi L, Kopecna O, Gimelli S, Cernohorska H, Zannotti M, et al: Reciprocal translocation t(4;7)(q14;q28) in cattle: molecular characterization. Cytogenet Genome Res 129:298–304 (2010). De Lorenzi L, Morando P, Planas J, Zannotti M, Molteni L, Parma P: Reciprocal translocations in cattle: frequency estimation. J Anim Breed Genet 129:409–416 (2012). Di Stefano L, Jensen MR, Helin K: E2F7, a novel E2F featuring DP-independent repression of a subset of E2F-regulated genes. EMBO J 22: 6289–6298 (2003).
Cytogenet Genome Res 2014;142:95–100 DOI: 10.1159/000356209
99
Downloaded by: Siriraj Medical Library, Mahidol University 202.28.191.34 - 3/3/2015 8:22:19 AM
Chikuda H, Kugimiya F, Hoshi K, Ikeda T, Ogasawara T, et al: Cyclic GMP-dependent protein kinase II is a molecular switch from proliferation to hypertrophic differentiation of chondrocytes. Genes Dev 18:2418–2429 (2004). Coy JF, Wiemann S, Bechmann I, Bächner D, Nitsch R, et al: Pore membrane and/or filament interacting like protein 1 (POMFIL1) is predominantly expressed in the nervous system and encodes different protein isoforms. Gene 290:73–94 (2002). Cribiu EP, Di Berardino D, Di Meo GP, Eggen A, Gallagher DS, et al: International System for Chromosome Nomenclature of Domestic Bovids (ISCNDB 2000). Cytogenet Cell Genet 92:283–299 (2001).
100
Li J, Ran C, Li E, Gordon F, Comstock G, et al: Synergistic function of E2F7 and E2F8 is essential for cell survival and embryonic development. Dev Cell 14:62–75 (2008). Nagase T, Ishikawa K, Suyama M, Kikuno R, Hirosawa M, et al: Prediction of the coding sequences of unidentified human genes. XIII. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA Res 6:63–70 (1999). Popescu CP, Pech A: Une bibliographie sur la translocation 1/29 de bovins dans le monde (1964–1990). Ann Zootec 40:271–305 (1991). Switonski M, Andersson M, Nowacka-Woszuk J, Szczerbal I, Sosnowski J, et al: Identification of a new reciprocal translocation in an AI bull by synaptonemal complex analysis, followed by chromosome painting. Cytogenet Genome Res 121:245–248 (2008).
Cytogenet Genome Res 2014;142:95–100 DOI: 10.1159/000356209
Wagner T, Wirth J, Meyer J, Zabel B, Held M, et al: Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell 796: 1111–1120 (1994). Weiskirchen R, Moser M, Weiskirchen S, Erdel M, Dahmen S, et al: LIM-domain protein cysteine- and glycine-rich protein 2 (CRP2) is a novel marker of hepatic stellate cells and binding partner of the protein inhibitor of activated STAT1. Biochem J 359: 485–496 (2001).
De Lorenzi /Rossi /Gimelli /Parma
Downloaded by: Siriraj Medical Library, Mahidol University 202.28.191.34 - 3/3/2015 8:22:19 AM
Eggen A, Gautier M, Billaut A, Petit E, Hayes H, et al: Construction and characterization of a bovine BAC library with four genome-equivalent coverage. Genet Sel Evol 33: 543–548 (2001). Hébert JM, Rosenquist T, Götz J, Martin GR: FGF5 as a regulator of the hair growth cycle: evidence from targeted and spontaneous mutations. Cell 78:1017–1025 (1994). Kenwrick S, Patterson M, Speer A, Fischbeck K, Davies K: Molecular analysis of the Duchenne muscular dystrophy region using pulsed field gel electrophoresis. Cell 48:351–357 (1987).
