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Microsatellites Within the Feline Androgen Receptor Are JFM0010.1177/1098612X16634386Journal of Feline Medicine and SurgeryFarwick et al 634386research-article2016 Original Article Journal of Feline Medicine and Surgery 1 –7 Microsatellites within the feline © The Author(s) 2016 Reprints and permissions: androgen receptor are suitable for X sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1098612X16634386 chromosome-linked clonality testing jfms.com in archival material Nadine M Farwick1, Robert Klopfleisch1, Achim D Gruber1 and Alexander Th A Weiss2 Abstract Objectives A hallmark of neoplasms is their origin from a single cell; that is, clonality. Many techniques have been developed in human medicine to utilise this feature of tumours for diagnostic purposes. One approach is X chromosome-linked clonality testing using polymorphisms of genes encoded by genes on the X chromosome. The aim of this study was to determine if the feline androgen receptor gene was suitable for X chromosome-linked clonality testing. Methods The feline androgen receptor gene, was characterised and used to test clonality of feline lymphomas by PCR and polyacrylamide gel electrophoresis, using archival formalin-fixed, paraffin-embedded material. Results Clonality of the feline lymphomas under study was confirmed and the gene locus was shown to represent a suitable target in clonality testing. Conclusions and relevance Because there are some pitfalls using X chromosome-linked clonality testing, further studies are necessary to establish this technique in the cat. Accepted: 26 January 2016 Introduction Most tumours arise from a single cell of origin and are To date, there is no routinely implementable clonality therefore considered clonal proliferations.1–3 Clonality test for non-lymphoid neoplasms in cats.23 The analysis testing has developed into a significant accessory tech- of clonal inactivation of genes located on the X chromo- nique in human cancer diagnostics.4 Clonality testing is some in females (X-linked clonality testing) has never- especially important for the diagnosis of haematological theless been applied to many human tumours.24,25 This malignancies such as lymphoma and leukaemia because approach is based on the fact that females carry two cop- lymphatic neoplasia and hyperplasia are not always eas- ies of the genes located on the X chromosome, of which ily differentiated histologically.5,6 one is inactivated to avoid an ‘overdose’ of gene expres- To improve lymphoma diagnostics in veterinary sion. The inactivated allele of X-chromosomal genes is medicine several techniques have been applied to ana- randomly selected during early embryogenesis and lyse clonal antigen receptor rearrangement, including inactivation is based on methylation of cytosine residues Southern blotting 7–10 and PCR.7,9–16 Further techniques have been used for clonality testing in veterinary medi- 1 cine and targeted clonally integrated feline leukaemia Department of Veterinary Pathology, Free University of Berlin, Berlin, Germany 17 virus (FeLV) provirus within the host’s genome, muta- 2 Chemical and Veterinary Analytical Institute Muensterland- tions within c-kit,18 clonal polymorphisms within mito- Emscher-Lippe, Muenster, Germany chondrial DNA,19 and polymorphisms of microsatellites and mitochondrial DNA in the transmissible venereal Corresponding author: 20 Alexander Weiss DVM, PhD, Chemical and Veterinary Analytical tumour. Clonality testing involving X-linked genes has Institute Muensterland-Emscher-Lippe, Joseph-Koenig-Str 40, 21,22 also been used in dogs and cats, both employing the Muenster, Germany androgen receptor gene. Email: [email protected] 2 Journal of Feline Medicine and Surgery within CpG-rich islands.26 To test for clonality, genomic the Gentra Puregene Tissue Kit (Qiagen), including DNA is digested by methylation-sensitive endonucle- RNase treatment as described.30 ases, such as HpaII or HhaI. In the following PCR only Primers spanning both CAG repeats and primers for the inactivated allele of the corresponding gene can be the first CAG region within exon 1 of the feline androgen amplified.27 One gene with such a polymorphism located receptor were newly designed according to a publicly next to a CpG island that is heterozygous in a large frac- available sequence (Table 1; GenBank Accession: AJ893545) tion of individuals is the human androgen receptor. This using GeneFisher.33 Primers for the second CAG stretch of gene features a polymorphic short tandem repeat of the feline androgen receptor were adapted from primers CAG,27 which has been used to demonstrate clonality of designed for the canine androgen receptor.32 tumours in women.28 This test has been termed the PCR was performed as described elsewhere.21 In 28 HUMARA assay. short, the master mix consisted of 2.0 mM MgCl2, The androgen receptor gene of cats has already been 0.2 mM of each dNTP, 0.2 µM of each primer and 0.024 sequenced.29 Similar to its canine counterpart it includes U/µl Taq DNA polymerase (GoTaq; Promega). Cycling two stretches of CAG repeats. However, possible poly- conditions encompassed an initial denaturing at 94°C for morphic microsatellites within exon 1 have not yet been 5 mins followed by 40 cycles of DNA melting at 92°C for reported. Therefore, we investigated exon 1 of the feline 30 s, annealing for 15 s, amplification at 72°C for 80 s in androgen receptor for potentially useful microsatellites the case of the long amplicons (>600 base pairs) and 20 s and applied them in clonality tests. We then designed in the case of the short ones. This was followed by a final the assay such that archival formalin-fixed and paraffin amplification at 72°C for 5 mins. Annealing tempera- embedded material could be tested. tures for specific primer combinations are summarised in Table 1. Materials and methods Using a semi-nested PCR protocol, products of the Case selection first round of PCR were diluted 1:500 with 5 mM Tris To assess the presence of microsatellites within the feline buffer. Cycling conditions were modified in the second androgen receptor we used the DNA of 42 cats from a round of PCR by using only 30 cycles, 59°C as the primer previous study.30 The newly developed assay was then annealing temperature and 15 s elongation time. After applied to 50 feline lymphomas and lymph nodes with amplification, PCR products were screened by agarose reactive lymphocytic hyperplasia that had already been gel electrophoresis using 2% gels impregnated with eth- thoroughly investigated.15,31 idium bromide. To detect deactivated alleles of the canine androgen Nucleic acid extraction, PCR amplification of the feline receptor genomic DNA was digested prior to PCR ampli- androgen receptor exon 1 and polyacrylamide gel fication with the methylation-sensitive endonuclease electrophoresis HpaII (restriction site: CCGG) (Fermentas). Genomic Genomic DNA was extracted from macro-dissected, for- DNA (1 μg) was digested with 10 U endonuclease over malin-fixed and paraffin-embedded tissue samples with night at 37°C in a final volume of 20 µl. Table 1 Primer sequences, melting temperatures, amplicon sizes and positions No Identification Primer sequence Temperature (°C) Amplicon size (bp) in AJ893545 Outer primers 1 FeARof1* 5’-CAAGACCTATCGAGGAGCTTTC-3’ 60–55† 645 2 FeARor1* 5’-GTCGAACTGCCACCTAGGTAAC-3’ First polymorphic CAG repeat – FELARA1 3 FELARAf1 5’- TATTCCAGAGCGTGCGCGAAG -3’ 57 245 4 FELARAr1 5’- GCTGTTGTGAAGGCTGCTGTTC -3’ Second polymorphic CAG repeat – FELARA232 5 FELARAf2* 5’-GACTCAGCTGCCCCATCCAC-3’ 57 250 6 FELARAr2* 5’-GGTAACTGTCCTTGGAGGAGG-3’ Additional combinations used 3/6 FELARAf1 and FELARAr2 59 606 1/4 FeARof1 and FELARAr1 57 269 5/2 FELARAf2 and FeARor1 57 265 *Designed for the canine androgen receptor but also works with the feline counterpart †A touch-down protocol was applied starting at 60°C, reducing the temperature by 0.5°C per cycle for 10 cycles followed by 30 cycles at 55°C bp = base pairs Farwick et al 3 To test complete digestion, a male sample in which canine gene, but it follows one position downstream. only one active (ie, digestion-sensitive) allele is present Both carnivores featured a stretch of different triplets – was also digested and later amplified. Polyacrylamide three in the cat and four in the dog – within this micros- gel electrophoresis (PAGE) was carried out as described atellite when compared with the human sequence. before.21 In contrast, sequences of the second microsatellite revealed distinct length polymorphisms in cats. While Sequence analysis dogs had 10–13 consecutive tandem repeats within this To analyse the sequence and exact repeat number of the region, cats had 16–20 CAG repeats (see Figures 1b and 2, feline androgen receptor gene PCR products were puri- as well as Figure 3 for a schematic; see also Table 2). In fied as described and submitted to a commercial labora- dogs the consecutive tandem repeats of ‘CAG’ are inter- tory (SeqLab, Göttingen, Germany) for sequencing.15 rupted by ‘CAA’ triplets; the feline sequences lacked Sequence analysis was carried out using Blast Search interspersed ‘CAA’ triplets (see Figure 1b). Higher (NCBI) and ClustalW (EMBL-EBI). GeneDoc 2.6.003 was numbers of tandem repeats (especially 18 and 19) were used to create the multiple sequence alignments. more frequent in our data sets than the lower numbers (Table 2). Results The first microsatellite that is highly polymorphic in First, microsatellites within the feline androgen receptor humans is shorter in dogs and cats (Figure 1a). In con- of 56 male cats (‘myocardium’ and ‘lymphoma’ groups) trast, the second microsatellite, which is not polymor- were studied because they only feature one allele. phic in humans, is much longer in cats and especially in Analysis of sequences obtained from PCR products of dogs (Figure 1b). the first potential microsatellite within the feline andro- Of all 32 female animals (‘myocardium’ and ‘lym- gen receptor revealed that, in contrast to the situation in phoma’ groups) tested in this study, 12 were heterozy- dogs,21 no length polymorphism was present in the cats gous (37.5%) (Table 2, Figure 4).
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