Microsoft Word - 70-436-1-Galley.docx Genetic analysis of congenital hemimelia in buffaloes from Southern Italy Simona Tafuri, Luigi M. Pavone*, Dionea Santoro, Sara Albarella, Silviana Rea, Valeria De Pasquale, Rossella Della Morte, Vincenzo Peretti . All Res. J. Biol., 2013, 4, 2-6 The publication cost of this article might be covered by external sponsors. More info for sponsors at: sponsors@arjournals.com ARTICLE                                                                                                                    Issue 1, Vol 4, 2013, 2-6 Genetic analysis of congenital hemimelia in buffaloes from Southern Italy Simona Tafuria, Luigi M. Pavoneb,*, Dionea Santorob, Sara Albarellaa, Silviana Reab, Valeria De Pasqualeb, Rossella Della Mortea, Vincenzo Perettia aDepartment of Veterinary Medicine and Animal Productions, University of Naples Federico II, Via F. Delpino 1, 80137 Naples, Italy; bDepartment of Molecular Medicine and Medical Biotechnology, University of Naples Federico II, Via S. Pansini 5, 80131 Naples, Italy; *Corresponding author: luigimichele.pavone@unina.it Abstract: Hemimelia is a common congenital limb abnormality found in water buffaloes from Southern Italy. In humans, such a defect has been associated with mutations in WNT7A and ESCO2 genes. These two candidate genes were analyzed by polymerase chain reaction in the genomic DNA extracted from the blood of buffaloes, and cows for control. No differences in WNT7A and ESCO2 sequences between affected and healthy buffaloes were identified. However, comparing sequences of control cows and buffaloes, WNT7A showed simple species polymorphisms, and ESCO2 showed seven base-pair substitutions. These results demonstrate that limb malformations in buffaloes are not related to congenital defects in WNT7A gene. Interestingly, our findings highlight for the first time differences in the sequences of WNT7A and ESCO2 genes between buffaloes and cows. Keywords: Buffaloes, Cows, Hemimelia, ESCO2, WNT7A. Hemimelia is a congenital malformation characterized by the absence of a limb portion. It is anatomically classified into two types: transversal hemimelia, characterized by complete absence of distal portion of the limb, and paraxial hemimelia characterized by aplasia of either radius or ulna, or tibia and fibula. In humans, these malformations are sporadic or very rare, with an incidence of approximately 1 per 1 million of live births. Some genetic mutations that cause limb deficiencies are associated with an autosomal dominant inheritance; other genetic causes include an autosomal recessive inheritance and chromosomal aberrations. However, different teratogenic agents and drugs have been also related to hemimelia1. Many case reports of hemimelia in cattle, sheep, dog, cat and goat have been reported2-4. Recent studies highlighted the major molecular components that coordinate limb outgrowth along three axes: fibroblast growth factors (FGFs) control the proximodistal axis, the sonic hedgehog (SHH) the anteroposterior axis, while the dorsoventral axis is regulated by bone morphogenetic proteins (BMPs), engrailed-1 (EN1), and wingless-type member 7A protein (WNT7A)5. The activities of these genes are mutually dependent. The WNT7A gene encodes a secreted signalling molecule that plays several roles in vertebrate development, and it is expressed in limbs, central nervous system, and urogenital tract. Functional studies have demonstrated that WNT7A is needed for normal patterning of the limb buds6. Homozyguos missense mutations in WNT7A cause two distinct limb-malformation disorders in humans: the Fuhrmann syndrome and the Al-Awadi/Raas- Rothschild phocomelia syndrome7. However, the WNT7A G204S mutation results to be associated with both Al-Awadi- Raas Rothschild syndrome and Fuhrmann syndrome phenotypes8. A phenotype similar to Fuhrmann syndrome was detected in WNT7A knockout mice9. Furthermore, the autosomal recessive human disorder Roberts syndrome, characterized by craniofacial anomalies, tetraphocomelia, and loss of cohesion at heterochromatic regions of centromeres and Y chromosome, has been associated to mutations in ESCO2 gene10. This gene encodes a protein belonging to the highly conserved Eco1/Ctf7 family of acetyl-transferases, and it is involved in the regulation of sister chromatid cohesion11. In humans, most ESCO2 mutations cause premature stop codons that may result in truncated proteins or mRNA instability due to nonsense- mediated mRNA decay12. 2 All Res. J.Biol, 2013, 4, 2-6     In livestock, several congenital malformations such as amelia, polymelia, ectromelia and hemimelia have been associated with genomic instability4,13. In this study, we screened WNT7A and ESCO2 genes in water buffaloes affected by limb defects in order to check whether mutations in these genes could be associated with their hemimelia phenotype. Bovine and human WNT7A genes differ for one additional exon present in the human genome, and bovine ESCO2 includes 10 exons whereas human ESCO2 comprises 11 exons spanning 30.3 kb, with the start codon in exon 2 and the stop codon in exon 11. We analyzed twenty-six Mediterranean Italian buffaloes from one day to six month old, 13 of which were affected by hemimelia (Figure 1), and 13 were healthy. Thirteen healthy cows were also studied controls. Figure 1. Italian Mediterranean buffalo calf with left hind limb amputated off proximal epiphysis metatarsus. The clinical and radiological patterns observed in the malformed animals are reported in Table 1. In their malformed limbs, all the animals showed more or less developed outlines of claws. Materials and Methods Genomic DNA was extracted from peripheral blood samples (1 ml) of all the animals using a PureLink genomic DNA mini kit (Invitrogen, Milan, Italy) according to manufacturer’s instructions. Using genomic DNA templates, polymerase chain reaction (PCR) was performed to amplify the three exons of the bovine WNT7A gene and exon 2 of bovine ESCO2 gene. This ESCO2 exon was selected because of its high susceptibility to mutations in humans. Primers were selected from bovine WNT7A and ESCO2 gene sequences (Ensembl Genome Browser) using the Primer3 Input 0.4.0 program, because buffalo genome has not been sequenced yet. PCR mix contained in a final volume of 25 µl: 60 ng of genomic DNA, 1 µM primer, 1.5 mM MgCl2, 1 U Taq polymerase (Eppendorf, Milan, Italy), and 0.2 mM dNTPs. PCR to amplify WNT7A exons was performed using a Gene Amp Mj Mini (BioRad Laboratories, Rome, Italy) as it follows: 1X (94°C for 4 min) and 38X (94°C for 45 s, 58.2°C (exon 3) and 63°C (exon 1 and 2) for 30 s, 72°C for 1 min). PCR to amplify ESCO2 exon 2 from genomic DNA was performed as it follows: after an initial 5 min denaturation step at 95°C, 35 amplification cycles (94°C for 40 s, 60°C for 30 s, 72°C for 1 min) were carried out followed by a 10 min incubation at 72°C. The oligonucleotide primer sequences, and PCR product size for each exon are reported in Table 2. Table 1. Clinical and radiological patterns observed in malformed animals. 1 female 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 2 females 1 male Left hind limb amputated off the proximal epiphysis metatarsus 1 female Left hind limb amputated off the third tarsus bones 1 female 1 male Left hind limb amputated off the tibia 1 female Left hind limb amputated off the distal epiphysis metatarsus 1 male Left hind limb amputated off the first phalanx 1 male Right hind limb amputated off the proximal epiphysis metatarsus 1 female Left hind limb amputated off the proximal epiphysis tibia 2 males Right hind limb amputated off the proximal epiphysis tibia 3 All Res. J.Biol, 2013, 4, 2-6     Table 2. Primers for amplification and sequencing of WNT7A exons and ESCO2 exon 2. Forward primer Reverse primer bp WNT7A Exon 1 5’- GTCTGCAGGCT GTGCCCCGC-3’ 5’- CCACTTTGAGC TCCTTGCCG-3’ 298 WNT7A Exon 2 5’- GGAGCCGGGA GGCCGCCTTC- 3’ 5’- CTTCCGGCCTG CCTCATTAT-3’ 272 WNT7A Exon 3 5’- ATCCTGGAGG AAAACATGAA- 3’ 5’- TCACTTGCACG TGTAGACCT-3 480 ESCO2 Exon2part1 5’- ATCAATGGAC TGTTTCCTTT- 3’ 5’- GGCTTAGAAC TCGAGGAGCA- 3 579 ESCO2 Exon2part2 5’- TGCAAGGAAA ACCAGTCTGC- 3’ 5’- TTAGAAGCTAT GAATTTCCA-3 504 Results and Discussion The PCR products were separated by electrophoresis to verify the expected length of amplified fragments. Figure 2 shows the PCR products of the three WNT7A exons and ESCO2 exon 2 from cows, healthy and malformed buffaloes. No length differences were observed between the amplified products from cows, healthy and malformed buffaloes. PCR products were purified and sequenced, and the sequence of each exon was analyzed using CodonCode Aligner software. PCR amplifications and sequencing were performed in triplicate. Table 3 summarizes the nucleotide differences between WNT7A sequences of cows, healthy and malformed buffaloes. The base-pair substitutions between cows and healthy buffaloes encode for the same amino acid, thus suggesting the occurrence of polymorphisms. No mutations were observed in WNT7A coding sequences of malformed buffaloes compared to healthy animals Table 4 reports the specie specific nucleotide differences observed between ESCO2 exon 2 sequences of cows and healthy buffaloes. In three cases, the base-pair substitutions encode for the same amino acid, whereas, in five cases, the base-pair substitutions encode for amino acids with similar chemical properties, and, in two cases, for amino acids with different chemical properties. Figure 2. PCR products of WNT7A exons and ESCO2 exon 2. A. PCR product of WNT7A exons from extracted DNA of control cow (lane 1), healthy buffaloes (lane 2) and malformed buffaloes (lane 3). Lane 4: negative control, SM: DNA ladder. B. PCR product of ESCO2 exon 2 part 1 and exon 2 part 2 from extracted DNA of control cows (lanes 1 and 4), healthy buffaloes (lanes 2 and 5) and malformed buffaloes (lanes 3 and 6). Lanes 7 and 8: negative controls, SM: DNA ladder. Arrows indicate the size of PCR products. Figure 3 shows the sequences of ESCO2 exon 2 that give rise to different amino acids between cows and healthy buffaloes. No differences were observed in the ESCO2 exon 2 sequences between healthy and malformed buffaloes (Table 4). 4 All Res. J.Biol, 2013, 4, 2-6     Table 3. WNT7A bp differences between control cows, healthy and malformed buffaloes. Control cow Healthy buffaloes Malformed buffaloes bp aa bp aa bp aa 48 g L 16 48 a L 16 48 a L 16 273 t T 91 273 c/t T 91 273 c/t T 91 372 c T 124 372 g/c T 124 372 g T 124 393 c C 131 393 t C 131 393 t C 131 474 c Y 158 474 t/c Y 158 474 t Y 158 519 a K 173 519 g K 173 519 g K 173 609 c H 203 609 t H 203 609 t H 203 633 g T 211 633 c T 211 633 c T 211 684 c L 228 684 t L 228 684 t L 228 798 t T 266 798 c T 266 798 c T 266 969 g Q 323 969 a Q 323 969 a Q 323 The bp numbers are referred to bos taurus WNT7A cDNA (ENSBTAT00000002188) Table 4. ESCO2 bp and aa differences between control cows, healthy and malformed buffaloes. Control cow Healthy buffaloes Malformed buffaloes bp aa bp aa bp aa 209 g R 70 209 a K 70 209 a K 70 262 g A 88 262 t S 88 262 t S 88 272 t V 91 272 c A 91 272 c A 91 313 t L 105 313 c L 105 313 c L 105 498 g V 166 498 a V 166 498 a V 166 566 a Y 189 566 c S 189 566 c S 189 571 g A 191 571 a T 191 571 a T 191 626 t V 209 626 c A 209 626 c A 209 673 t S 225 673 a T 225 673 a T 225 831 c N 277 831 t N 277 831 t N 277 The bp numbers are referred to bos taurus ESCO2 exon 2+exon 1 cDNA (ENSBTAT00000008606). In red highlighted species polymorphisms, in black highlighted conservative mutations and in green highlighted mutations for amino acids with different properties. Figure 3. Sequence analysis of the ESCO 2 exon 2 shows differences in the coding sequence between control cow and healthy buffaloes. In recent years, an increasing number of calves born in Southern Italy shows limb defects, and in particular, transversal hemimelia4. Genomic instability has been demonstrated in these animals as proved by the high rates of structural chromosomal aberrations and increased sister chromatid exchanges detected in affected calves4,13-14. Due to the economic and social impact of such a problem, molecular genetic studies, which allow identifying the genes responsible for these congenital defects, will help to find adequate strategies for the prevention of the disease. Here, we investigated for the first time the WNT7A and ESCO 2 genes that are the main candidate genes involved in human severe limb pathologies such as Fuhrmann syndrome and Roberts syndrome. Our results do not show genetic alterations in the WNT7A exons and ESCO2 exon 2 coding sequences of malformed buffaloes, although further studies on ESCO2 gene are needed to rule out its involvement in the pathogenesis of these congenital malformations. These findings suggest that the pathogenesis of hemimelia in buffaloes from Southern Italy could be probably related to genetic alterations in other genes involved in embryonic limb development. Interestingly, our findings highlight for the first time differences in the sequences of WNT7A and ESCO2 genes between buffaloes and cows. 5 All Res. J.Biol, 2013, 4, 2-6     Acknowledgments This work was supported by a grant “Analisi genetica dell'emimelia congenita nel bufalo” from Regione Campania, Italy. We thank Dr. E. Cirillo for administrative help. References 1. Sanders, D.D. and Stephens, T.D. (1991) Review of drug-induced limb defects in mammals. Teratology 44, 335-354. 2. Baum, K.H., Hull, B.L., Weisbrode, S.E. (1985). Radial agenesis and ulnar hypoplasia in two caprine kids. J. Am. Vet. Med. Association 186, 170-171. 3. Lapointe, J.M., Lachance, S., Steffen, D.J. (2000). Tibial hemimelia, meningocele, and abdominal hernia in Shorthorn cattle. Vet. Pathol. 37, 508-511. 4. 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