Congenital hypotrichosis in mammalian species consists
of partial or complete absence of a hair coat at
birth. Affected individuals having a partial hair coat
at birth may loose it subsequently.The aim of this paper was to physically map the
ovine hr gene using fluorescence in situ hybridization
(FISH).A preliminary study on an internet data bank
(http://www.informatics.jax.org) showed that the regions
of the hr gene on murine chromosome 14 and
human chromosome 8 present homology with ovine
chromosome 2

Budelli E

Caroli A

Castiglioni B

FINOCCHIARO, Raffaella

Pagnacco G.

PORTOLANO, Baldassare

Van Kaam JBCHM

Hereditas

R. Finocchiaro

B. Castiglioni

E. Budelli

J. B. C. H. M. Van Kaam

B. Portolano

A. Caroli

G. Pagnacco

AIR Universita degli studi di Milano

Brief report : Chromosomal assignment of the ovine hairless (hr) gene by fluorescence in situ hybridization

Archivio istituzionale della ricerca - Università di Palermo

Brief ReportChromosomal assignment of the ovine hairless (hr) geneby fluorescence in situ hybridizationRAFFAELLA FINOCCHIARO1,2, BIANCA CASTIGLIONI3, ELENA BUDELLI1,JOHANNES B.C.H.M. VAN KAAM2,4, BALDASSARE PORTOLANO1, ANNA CAROLI5 andGIULIO PAGNACCO61Dipartimento S.En.Fi.Mi.Zo.-Sezione Produzioni Animali, Universita` degli Studi di Palermo, Palermo, Italy2Associazione Nazionale Allevatori Frisona Italiana (ANAFI), Via Bergamo, IT-292-26100 Cremona, Italy3Istituto di Biologia e Biotecnologia Agraria, Milano, Italy4Istituto Zooprofilattico Sperimentale della Sicilia ‘‘A. Mirri,’’ Palermo, Italy5Dipartimento di Scienze Biomediche e Biotecnologie, Universita` degli Studi di Brescia, Brescia, Italy6Dipartimento di Scienze e Tecnologie Veterinarie per la Sicurezza Alimentare, Universita` degli Studi di Milano,Milano, Italy.Finocchiaro, F., Castiglioni, B., Budelli, E., van Kaam, J.B.C.H.M., Portolano, B., Caroli, A., Pagnacco, G. 2008.Chromosomal assignment of the ovine hairless (hr) gene by fluorescence in situ hybridization *Hereditas 145: 258261.Lund, Sweden. eISSN 1601-5223. Received February 25, 2008. Accepted May 26, 2008E-mail: raffaellafinocchiaro@anafi.itCongenital hypotrichosis in mammalian species con-sists of partial or complete absence of a hair coat atbirth. Affected individuals having a partial hair coatat birth may loose it subsequently. Tactile hairsand eyelashes are generally present. Cases of hypo-trichosis have been found in cattle (MOHR andWRIEDT 1928; JULIAN 1963; BRACHO et al. 1984;DRO¨GEMU¨LLER et al. 2002), and in sheep (NEL 1964;DOLLING and BROOKER 1966; LAZOVSKII 1983;MACKIE and MCINTYRE 1992). Several genes havebeen found responsible for hypotrichosis in variousspecies (PANTELOURIS 1968; CACHON-GONZALEZet al. 1994). In humans, mice, and rats, the hairless(hr) gene is often responsible for this disorder and ishighly homologous suggesting high conservationamong mammals (AHMAD et al. 1998; PANTELEYEVet al. 1999). The hr gene codes for a protein which is atranscriptional co-repressor for thyroid hormone re-ceptors (POTTER et al. 2001).More recently, hypotrichosis was described in Sici-lian dairy sheep belonging to the Valle del Belice breed(FINOCCHIARO et al. 2003). Because the hypotrichoticphenotype is an undesirable defect for farmers,affected lambs are slaughtered soon after birth. There-fore information on the population frequency of thedisorder is missing. The disorder was found underrecessive genetic control (FINOCCHIARO et al. 2003).Hence the hypotrichotic phenotype is observed onlywhen the allele is present in the population withreasonable frequency, so that homozygous carriersappear. Often the tendency towards mating relatedanimals results in the appearance of the disorder. Thehr gene was chosen as a functional candidate gene forthe hypotrichotic disorder in the Valle del Belice dairysheep (FINOCCHIARO et al. 2003). After sequencingthe hr gene in Valle del Belice carriers, a C0T pointmutation was found in exon 3 at 1312 bp resulting in aTAG stop codon. Successively a PCR-SSCP test wasdeveloped (FINOCCHIARO et al. 2003). All affectedanimals, genotyped with the PCR-SSCP test turnedout to be homozygous carriers of the mutation,whereas heterozygous carriers and non-carriers werenot affected. The hr gene therefore was found to be thegene responsible for the congenital hypotrichosis inValle del Belice dairy sheep.The aim of this paper was to physically map theovine hr gene using fluorescence in situ hybridization(FISH).The probe for the FISH was directly labelled by PCRin the presence of digoxigenin 11-dUTP (Roche Diag-nostics) with the primer sequences and PCR procedure,as reported in RICHARD et al. (1994). The primersequences were 5?-GCCCAGCTGCCAGCCCGCAA(HR3Forward) and 5?-ACAGTGGCTGGGAGT-AGTGGGC (HR4a Reverse).The PCR reaction was as follows: initial denatura-tion at 928C for 2 min, 35 cycles with denaturation at948C for 1 min, annealing at 658C for 40 s andelongation at 728C for 5 min, followed by a finaldenaturation at 728C for 5 min. Chromosome pre-parations were arranged from fibroblast cultures bystandard procedures (MEZZELANI et al. 1995), exceptfor the hypotonic treatment, with 0.02 M KCl, at 378Cfor 13 min. After a few days at 208C, the slides wereHereditas 145: 258261 (2008)DOI: 10.1111/j.1601-5223.2008.02062.xFig. 1. Chromosomal assignment to OAR 2p21-22 for the ovine hairless (hr) geneusing FISH with labelling of the mutation.Hereditas 145 (2008) Chromosomal assignment of the ovine hairless 259stained with 0.005% Quinacrine Mustard for 10 s(QFQ-banding), and well-spread metaphases, withdistinctive banding, were imaged on a Leitz Aristoplanmicroscope connected to a CCD-camera (Photo-metrics) controlled by an Apple Macintosh Quadra950 computer. The probe was then hybridized in situto metaphase chromosomes at a final concentration of0.5 ng ml1 (5 ng slide1) in the presence of 3 mg ofCot-1 DNA (Sigma-Aldrich). The probes were de-tected via antidigoxigenin-rhodamine (Roche Diag-nostics). DAPI was used to counterstain thechromosomes. Digitized images were taken separatelyfor each fluorochrome and merged with the computerprograms IPLab Spectrum MultiProbe (Signal Analy-tics) and Gene Join (Office of Cooperative Research,Yale University). Chromosome identification andbanding followed the latest chromosome standardnomenclature (ISCNDB 2000, CRIBIU et al. 2001).RESULTS AND DISCUSSIONThe results of the hybridization of the hr gene on ovinechromosomes are shown in Fig. 1. Fifty metaphasespreads were examined, with 50% showing a specificsignal on at least one of the homologous chromo-somes.A preliminary study on an internet data bank(http://www.informatics.jax.org) showed that the re-gions of the hr gene on murine chromosome 14 andhuman chromosome 8 present homology with ovinechromosome 2. Using FISH labelling the mutation thechromosomal assignment for the ovine hr gene wasOAR 2p21-22. The regional localization of the probewas established by determining the FLcen values onsheep chromosomes (0.5890.07), as described byLICHTER et al. (1990). This result confirmed themost likely location of the hr gene on ovine chromo-some 2.In human, the hairless gene has been localised onchromosome 8p21.2 by means of a radiation hybridpanel (CICHON et al. 1998). In pig, the HR gene hasbeen localised on chromosome 14 by means of linkagemapping (FERNANDEZ et al. 2003). In cattle, a genesimilar to hairless (accession number XR_028305) hasbeen recently predicted on chromosome 8 by auto-mated computational analysis derived from an anno-tated genomic sequence (NW_001495463) using thegene prediction method GNOMON.As reported in ISCNDB 2000, the InternationalSystem for Chromosome Nomenclature of DomesticBovids the bovine chromosome 8 is homologous toovine chromosome 2p and to human chromosomes 8pand 9 (CRIBIU et al. 2001). Comparative status ofporcine, ovine, bovine and human chromosomesobtained by chromosome painting and gene mappingrevealed the correspondence among SSC 14q1.2-q1.4,OAR 2, BTA 8q2.1-q2.3 and HAS 8p (FRONICKE andWIENBERG 2001).Furthermore, we used the Oxford grid comparative-mapping tool available on the http://oxgrid.angis.org.au/sheep/ website, which is able to move from a regionof interest on a particular chromosome of a species tothe orthologous regions of any other species, linkingup with relevant sequence data when available. In thisway, the comparitive predictions of orthologues be-tween sheep and cattle and between sheep and humanshowed the correspondence among a region of sheepchromosome 2 with a part of bovine chromosome 8and of human chromosome 8. In particular, tworeference loci in this region are LPL and BMP1.Using the Entrez Gene tool, the LPL gene is localizedon OAR 2, on HSA8p21.3, on BTA8q and on SSC14q12-q14, whereas BMP1 is mapped on HSA8p21and BTA8q21.Acknowledgements  This work was supported by PIR-Zootecnia/6F 2004 funding of the Assessorato Agriculturae Foreste della Regione Sicilia.REFERENCESAhmad, W., Haque, M. F., Brancolini, V. et al. 1998.Alopecia universalis associated with a mutation in thehuman hairless gene.  Science 279: 720724.Bracho, G. A., Beeman, K., Johnson, J. L. et al. 1984.Further studies of congenital hypotrichosis in Herefordcattle.  Zentralbl. Veterinarmed. A. 31: 7280.Cachon-Gonzalez, M., Fenner, S., Coffin, J. et al. 1994.Structure and expression of the hairless gene of mice. Proc. Natl Acad. Sci. USA 1: 77177721.Cichon, S., Anker, M., Vogt, I. R. et al. 1998. Cloning,genomic organization, alternative transcripts and muta-tional analysis of the gene responsible for autosomalrecessive universal congenital alopecia.  Hum. Mol.Genet. 7: 16711679.Cribiu, E. P., Di Berardino, D., Di Meo, G. P. et al. 2001.ISCNDB 2000, International System for ChromosomeNomenclature of Domestic Bovids.  Cytogenet. CellGenet. 92: 283299.Dolling, C. and Brooker, M. 1966. A viable hypotrichosis inPoll Dorset sheep.  J. Hered. 57: 8790.Dro¨gemu¨ller, C., Kupier, H., Peters, M. et al. 2002.Congenital hypotrichosis with anodontia in cattle: agenetic, clinical and histological analysis.  Vet. Derma-tol. 13: 307313.Fernandez, A., Silio, L., Noguera, J. L. et al. 2003. Linkagemapping of the porcine hairless gene (HR) to chromo-some 14.  Anim. Genet. 34: 317318.Fronicke, L. and Wienberg, J. 2001. Comparative chromo-some painting defines the high rate of karyotype changesbetween pigs and bovids. Mamm. 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Chromosomal assignment of the ovine hairless (hr) gene by fluorescence in situ hybridization.

Raffaella  Finocchiaro

Bianca Castiglioni

Elena Budelli

Johannes B.C.H.M. Van Kaam

Baldassare Portolano

Anna Caroli

Giulio Pagnacco

Crossref

Chromosomal assignment of the ovine hairless (hr) gene by fluorescence insitu hybridization

https://iris.unipa.it/retrieve/handle/10447/104228/142699/Chromosomal%20assignment%20of%20the%20ovine%20hairless%20%28hr%29%20gene.pdf

Chromosomal assignment of the ovine hairless (hr) gene by fluorescence in situ hybridization.

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