Journal of Heredity Advance Access published online on December 28, 2007
Journal of Heredity, doi:10.1093/jhered/esm101
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Brief Communications |
Characterization of Bovine TGM1 and Exclusion as Candidate Gene for Ichthyosis in Chianina
From the Dipartimento di Scienze Animali, Sezione di Zootecnica Veterinaria, Università degli Studi di Milano, 20133 Milano, Italy (Dardano, Gandolfi, Polli, Bighignoli, Strillacci, Cozzi, and Longeri); and the Dipartimento di Scienze Animali, Sezione di Zootecnia Agraria, Università degli studi di Milano, 20133 Milano, Italy (Parma and Molteni)
Address correspondence to M. Longeri at the address above, or e-mail: maria.longeri{at}unimi.it.
Ichthyosis is a heterogeneous group of keratinization disorders reported both in human and animals. Two rare, inherited forms have been reported in cattle, both characterized by autosomal recessive transmission. Because mutations of transglutaminase 1 (TGM1) gene are associated with autosomal recessive ichthyosis in people, this gene was investigated as a candidate for the diseases in cattle. Three different polymorphisms were identified in 5' end region of cattle TGM1. Marker homozygosity was not found among affected calves. Linkage analysis excluded (logarithmic odds [LOD] score –2.0) TGM1 as the cause for ichthyosis phenotype in the analyzed Chianina cases.
Hereditary ichthyosis has been reported in humans (for a review see, Richard 2004 and www.ncbi.nih.gov/sites/entrez?db=Omim), mice (Jensen and Esterly 1977), chickens (Renden and Abbot 1980), and cattle. Whereas cases of congenital and supposed inherited ichthyosis were reported in dogs (Alhaidari et al. 1994) and kudu (Chittick et al. 2002). The human ichthyosis is a heterogeneous group of dermatosis characterized by hyperkeratosis with excessive cutaneous scale formation causing the skin to resemble a fish.
In cattle, two inherited forms of ichthyosis were reported, both caused by single autosomal recessive genes. The most severe and lethal form (fetal ichthyosis) was described in Norwegian Red Poll, Friesian, and Brown Swiss calves. Congenital ichthyosis is less severe, and lesions are more localized. It was reported in Jersey, Pinzgauer, Chianina, and Holstein–Friesians (Baker and Ward 1985; Raoofi et al. 2001; Testoni et al. 2006). Several cases of congenital ichtyosis were recently found in the Chianina breed in Italy. Affected animals showed deep fissures separating keratinized plaques and eversion of mucocutaneous junctions consistent with the more severe autosomal recessive forms in man (Molteni et al. 2006).
In order to find the causative mutation, we analyzed related Chianina animals segregating for ichthyosis. Transglutaminase 1 (TGM1) was chosen as candidate gene because mutations of this gene are responsible for a homologous condition in people.
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Materials and Methods |
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Animals
- 1) For sequencing and linkage analyses, a family with 11 affected cattle out of 49 individuals was tested as described by Molteni et al. (2006).
- 2) A total of 13 unrelated healthy animals within Italian Holstein–Friesian, Piedmont, Grey Alpine, Rendena, and Aosta Chestnut breeds were analyzed for TGM1 polymorphism.
- 2) A total of 13 unrelated healthy animals within Italian Holstein–Friesian, Piedmont, Grey Alpine, Rendena, and Aosta Chestnut breeds were analyzed for TGM1 polymorphism.
TGM1 Sequencing
Total RNA was isolated from skin biopsies using Trizol Reagent (Invitrogen, San Diego, CA), and reverse transcription was performed using oligo-dT primer provided in a Superscript First-Strand Synthesis System for reverse transcriptase–polymerase chain reaction (Invitrogen). To amplify the bovine TGM1 from cDNA, specific primers were designed on bovine expressed sequence tag (EST) selected by alignment among amino acidic sequence of human TGase1 (GenBank accession no. NP_000350
[GenBank]
) and bovine National Center for Biotechnology Information EST database (http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). cDNA 5' and 3' ends were determinated using the RACE System for rapid amplification of cDNA ends (Invitrogen). Intron 1 region was also sequenced on genomic DNA by specific primers used for reverse transcription reactions. The polymerase chain reaction (PCR) products were electrophoresed through 1.5% agarose gels and visualized with ethidium bromide. The PCR products of appropriate lengths were purified and directly sequenced both strands using Big Dye Terminator chemistry on an ABI 310 Genetic Analyzer (Applied Biosystems, Foster City, CA). Sequence data were aligned using ClustalX or BLAST and manipulated by BioEdit (http://www.mbio.ncsu.edu/BioEdit/bioedit.html).
Microsatellite Marker Genotyping
TGM1 orthologous region was identified in cattle by data mining as a comparative mapping between human and cattle using the current Hereford breed Btau_2.0 whole-genome shotgun sequences assembly (http://www.hgsc.bcm.tmc.edu/projects/bovine/) and BLAST searches. Two microsatellite markers, BMS2635 and BL1035, were found about 10 cM in the flanking TGM1-DIK4897 orthologous region, according to United States Department of Agriculture Genetic Map (http://www.marc.usda.gov/genome/genome.html). Cattle were typed by automatic sequencer, and genotypes were assigned using GENESCAN and GENOTYPER softwares.
Linkage Analysis
Exclusion linkage analysis was performed using the MLINK program from the LINKAGE package software, according to Radi et al. (2005), and including available pedigree information and segregation data for the disease phenotype, 2 flanking microsatellite markers, and 3 polymorphisms found within the cattle TGM1 gene (Molteni et al. 2006).
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Bovine TGM1 Gene Characterization
Complete bovine TGM1 cDNA sequence (GenBank accession no. AY676606) showed a total length of 2770 bp. The bovine coding region had 89% identity to human TGM1. The deduced bovine TGM1 protein was 846 amino acids long and 87%, 86%, 85% identical to dog, mouse, and human TGM1, respectively. The exon–intron boundaries were established by comparison with structural organization of the human gene (GenBank accession no. NT026437) and by BLAST alignment with BTA 10 whole-genome shotgun sequences reference assembly (GenBank accession no. NC_007308). The exon–intron boundaries and the exon length were conserved between cattle and man except Bovine exon 2 that showed a longer sequence in comparison with man. The ATG initiation codon for bovine TGM1 was located in exon 1.
Bovine TGM1 Gene Polymorphisms
Polymorphisms within Chianina cattle samples segregating for ichthyosis were found at TGM1 5' end as follows: one indel (I1-3 InDelGGT) at 3' end of intron 1, one single-nucleotide polymorphism (SNP) (E2 18 T/C) and one 24-bp repeated motif (E2 271(24)rep) in exon 2. These polymorphisms were linked in 2 haplotypes; however, none were associated with the disease. The same polymorphisms and one additional SNP in exon 2 (E2 356 A/C) were also identified in healthy and unrelated animals from 5 different Italian breeds.
Linkage Analysis
Linkage analyses showed a logarithmic odds [LOD] score less than –2.0 for TGM1 genetic markers segregating within the Chianina family with ichthyosis at 
< of their distance to TGM1 (Table 1).
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In the present work, some calves affected by an autosomal recessive form of ichthyosis and related to a single founder sire widely used in Italy in the recent past were analyzed in order to identify the causative mutations (Molteni et al. 2006). TGM1 was chosen as a candidate gene by comparative analyses with human autosomal recessive forms of ichthyosis. TGM1 is expressed in the upper differentiated layers of epidermis, and it is a crucial enzyme that catalyses the calcium-dependent
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-glutamyl) lysine cross-linking reaction to facilitate insoluble protein envelope formation. TGM1 in man maps on chromosome 14q11.2–13 and mutations in coding region lead to reduced or absent enzyme activity and to the disease (Pigg et al. 1998; Akiyama et al. 2003). We sequenced and characterized bovine TGM1-coding region in affected and unaffected cattle. Bovine sequence obtained (GenBank accession no. AY676606) showed differences compared with other species. The coding region started in exon 1, whereas the initiation codon was located in exon 2 in man, rat, and dog.
Exon 2 showed a longer sequence in bovine than in man and the highest divergence between the other species TGM1 sequences (Polakowska et al. 1992). Moreover only TGM1 intron 1–exon 2 region showed polymorphism both in the pedigree subjects and in the 13 cattle belonging to 5 Italian breeds (3 SNPs and one 24-bp repeated motif). The TGM1 5' end polymorphisms described in cattle confirm literature data that record the 5' end—exon 2 region shows the highest divergence between species and suggest that this region confers a specialized and/or species-specific function to the enzyme (Credille et al. 2001). However, linkage analysis clearly excluded TGM1 as the gene responsible for this trait.
In cattle, the defect could be determined by other orthologous causal loci mutations. At least other 4 genes are implicated in severe autosomal recessive forms in humans, including lipoxygenase (ALOX12B and ALOXE3), loricrin, and ABCA12 genes (Kelsell et al. 2005).
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University of Milan First Fund (n.12-1-5172222-129).
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Acknowledgments |
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The authors thank F. Filippini, (Associazione Nazionale Allevatori Bovini Italiani de Carne, Perugia, Italy) for the helpful and qualified support in samples and data collection.
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Footnotes |
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Corresponding Editor: Ernest Bailey
Received June 14, 2007
Accepted October 7, 2007
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