Praca oryginalna Original paper
Association between GGA1 gene polymorphisms
and occurrence of mammary mixed tumors and aging
in domestic bitches
1)
WIESŁAWA KRANC*, ADRIAN CHACHUŁA**, KATARZYNA WOJTANOWICZ-MARKIEWICZ***, KATARZYNA ZAORSKA**, EDYTA OCIEPA***, ADAM PIOTROWSKI*, DOROTA BUKOWSKA***,
SYLWIA CIESIÓŁKA**, SYLWIA BORYS****, HANNA PIOTROWSKA****, AGNIESZKA SKOWROŃSKA*****, MARCIN NOWAK******, PAWEŁ ANTOSIK***, KLAUS-PETER BRÜSSOW***, BARTOSZ KEMPISTY*, **, MAŁGORZATA BRUSKA*,
MICHAŁ NOWICKI**, MACIEJ ZABEL**, *******
*Department of Anatomy, **Department of Histology and Embryology, Medicine Faculty I, Poznan University of Medical Sciences, Swiecickiego 6 St., 60-781 Poznan, Poland
***Institute of Veterinary Sciences, Faculty of Animal Breeding and Biology, Poznan University of Life Sciences, Wolynska 35 St., 60-637 Poznan, Poland ****Department of Toxicology, Faculty of Farmacy, Poznan University of Medical Sciences,
Dojazd 30 St., 60-631 Poznan, Poland
*****Department of Human Physiology, Faculty of Medical Sciences, University of Warmia and Mazury in Olsztyn, Olsztyn, Poland
******Department of Pathology, Faculty of Veterinary Medicine, Wroclaw University of Life Sciences, C. K. Norwida 31 St., 50-375 Wrocław, Poland
*******Department of Histology and Embryology, Wroclaw Medical University, 6a Chalubinskiego St., 50-368, Wroclaw, Poland
Received 13.07.2015 Accepted 03.11.2015
1) Supported by the Polish Ministry of Scientific Research and Higher Education (Grant No. 5279/B/P01/2011/40).
Kranc W., Chachuła A., Wojtanowicz-Markiewicz K., Zaorska K., Ociepa E., Piotrowski A., Bukowska D., Ciesiółka S., Borys S., Piotrowska H., Skowrońska A., Nowak M., Antosik P.,
Brüssow K.-P., Kempisty B., Bruska M., Nowicki M., Zabel M.
Association between GGA1 gene polymorphisms and occurrence of mammary mixed tumours and aging in domestic bitches
Summary
In recent years the number of malignant mammary gland tumor occurrences in domestic bitches has increased. This may be related to advanced diagnostic technologies, availability of a quick diagnosis, as well as genomic changes. Still the main genetic reasons for tumor development are frequently occurring gene mutations and/ or polymorphisms. The mutations in target genes often lead to amino acid changes in the structure of proteins, which may be the reason for uncontrolled cells proliferation, and induction and growth. Therefore, in this article we described the analysis of mutation/polymorphisms frequency of the GGA1 gene in association with canine malignant mammary gland tumor occurrence and aging. In this study, blood samples were obtained from 22 female dogs diagnosed with mammary tumors. Moreover, blood samples from geriatric (> 5 to 10 years old; n = 15), mature adult (> 2 to 5 years old; n = 10) and young (from 1 to 2 years old; n = 11) dogs were also collected. 36 bitches diagnosed on account of other reasons served as controls. After the Sanger sequencing analysis, 14 single nucleotide variations were identified, of which 3 were already known polymorphisms and 11 novel variations. We observed differences in frequencies for 4 polymorphisms (g.A-172T, c.T24C, c.A692G, c.C1185T) between cases and controls. Moreover, we found increased prevalence of heterozygotes and alternative alleles in 3 polymorphisms (g.A-172T, c.A692G, c.C1185T) in the tumor diagnosed group as compared to the control. Although the results were not statistically significant, it is worth mentioning the slightly different pattern of genetic segregation of alleles in the abovementioned polymorphisms in control and tumor patients.
The median lifespan of dogs varies as a function of breed, with larger breeds typically having shorter lifespans than smaller breeds (7). The lifespan of dogs is also affected by diet and environmental conditions in which the dogs are kept. These same factors influ-ence the development of breast mixed tumors. In recent years, different types of tumor have been diagnosed in domestic bitches, and mixed tumors are the pre-dominating cause of mortality and morbidity of female dogs (13). Mammary gland tumor is the most common neoplasma diagnosed in females dog, however, the knowledge on the molecular carcinogenesis of canine mammary tumor (CMT) is not yet fully recognized (12, 13). There are differences and similarities between canine and human mammary tumors at the molecular level. The most important thing was to develop and to define the canine genome map, which enabled the identification of new molecular markers for CMT induction, invasion, and/or progression (15).
The GGA1 belongs to GGAs family. This is a ubiq-uitous coat protein, which belongs to the large pro-tein family of mammalian GGAs. Each of the three mammalian GGA proteins have a modular structure consisting of: (a) an NH2-terminal VHS domain, (b) a region of homology to the ear of the γ1-adaption submint of AP-1 related to the γ2-adaption, followed by a GAT domain, a variable domain (1, 2, 8). The γ-adaptin ear homology domain contributes to GGA functioning but is not necessary for full functioning that can be restored by replacing the GGA ear domain with the γ-adaptin ear domain. Deleting the γ-adaptin gene together with the two GGA genes exacerbates the phenotype in yeast, suggesting that they function on parallel regulatory pathways.
In mammalian cells, the association of GGAs with the membrane is extremely unstable and it is therefore not associated with purified clathrin-coated vesicles or with any kinds of the other components of AP-1 complex (1). The GGA1 is not associated with GGA2, but they are co-localized on perinuclear membranes, which correspond to trans elements of the Golgi stack and the trans-Golgi network. Moreover, the AP-1 contains bound casein kinase-2 that phos-phorylated GGA1 and GGA3, thereby caus-ing autoinhibition. It was demonstrated that this autoinhibition could induce the directed transfer of mannose 6-phosphate receptors from the GGAs to AP-1 (2). Doray et al. (2) concluded that GGAs and the AP-1 complex interact to package mannose 6-phosphate receptors into AP-1-containing coated ves-icles, because mannose 6-phosphate recep-tors, defective in binding to GGAs, were poorly incorporated into adaptor protein complex containing clathrin coated vesicles.
Despite the fact that this gene and its mutations have been intensively studied in humans and animals, in the case of other tumors, mutations/polymorphisms of this gene in bitches with mammary gland mixed tumor in relation to aging of the organism have not been studied. The goal of this study was to investigate polymorphisms and mutations of GGA1 gene.
Material and methods
Patients and samples collection. Blood samples were obtained from 22 female mongrels, mature adult 2 to 5-years-old dogs diagnosed with mammary mixed tumors during the surgery in the Small Animal Clinic, University of Life Sciences, Poznan, Poland. Blood was collected during standard surgery procedures. 36 bitches served as controls; in the good condition, clinically healthy, lab tests in physiological norm. They were divided into 3 subgroups of differing age according to the classification by Jugdutt et al (11): geriatric (> 5 to 10 years old; n = 15), mature adult (> 2 to 5 years old; n = 10) and young (from 1 to 2 years old; n = 11). Blood was collected from the saphenous vein/ jugular vein in vials with EDTA and frozen in –80°C until further analyses. Material (tumor) was fixed in parafin and formed into 60 blocks. Blocks were cut into 5 µm slices on automatic rotary microtome (Leica RM 2255). Each slice was stuck on a SuperFrost Plus glass slide (Thermo Scien-tific) and heated to 60°C for 90 minutes. After heating glass slides were incubated overnight in 37°C. After incubation glass slides were stained in an automatic slide stainer (Robot Stainer HMS 740, Microm) with standard H + E method, i.e.: Mayer hematoxylin for 25 min., tap water for 8 min and 0.1% eosin solution for 45 min. Stained slides were dried with alcohol and covered with transparent Histofluid resin and cover slip in automatic coverslipper (Leica CV5030).
Tab. 1. PCR primer pairs with thermal conditions used for the amplifi-cation of GGA gene exons
Exon Sequences Annealing temp./elongation exon 1 F 5’-TAATTTTGCCCGAATGGCTA-3’ R 5’-TTCTGTGGGCACAACCATAG-3’ 58°C/45 sec exon 2 F 5’-GAATATGCTTGTGGGACTGGA-3’ R 5’-TCAGATCAAAGAAGGCCAAG-3’ 58°C/30 sec exon 3 F 5’-ACTCTTTTCCCCTTCGCTTC-3’ R 5’-TCAATTATGCCACAAAACAAACA-3’ 58°C/30 sec exon 4 F 5’-TCCAAATGTGGTTTCTGGTG-3’ R 5’-GGGAAGACAGCATCTTCTCAT-3’ 57°C/30 sec exon 5 F 5’-TGTTGAGCTTTAAACCATACTTCC-3’ R 5’-GCAAGCATCTGATCCTCCAT-3’ 61°C/30 sec exon 6 F 5’-CACTTAAGGGTTCATGGATGC-3’ R 5’-AGCACTCATCAGTCAATTGAAAA-3’ 62°C/30 sec exon 7 F 5’-CACATGTGTGTTTTGTAATGCTG-3’ R 5’-GGGAAAGTAAATGGCACGAA-3’ 58°C/30 sec exon 8, exon 9 F 5’-ACCAGGCAAATGAATTCCAC-3’
R 5’-TTGATTTTTCTGTAATGAGGCTTT-3’ 58°C/30 sec exon 10 F 5’-TGCATAAAAACAGCAGTGTCAG-3’ R 5’-AAGGCAAAATAATCAATAGCCACT-3’ 59°C/30 sec exon 11 F 5’-TGAGTATAACCCATTGCAGAGAAA-3’ R 5’-TTCGTGCTGCTTTTGTTGAC-3’ 58°C/45 sec exon 12 F 5’-GCACTAGTGGATTTTGTAAATCATCA-3’ R 5’-TCTCCAAGGTTTGGAAGGAA-3’ 58°C/30 sec
Molecular analysis. Genomic DNA was extracted from whole peripheral blood using QIAamp DNA Blood Mini Kit (Qiagen), according to the manufacturer’s instructions. DNA was resuspended in 100 µl of Qiagen elution buffer and stored in –20°C. 12 exons of the GGA gene (exon 1 – exon 12) were amplified in the polymerase chain reac-tion (PCR) including up to 70-bp flanking regions of every exon. The sequences of the primers used with thermal and time conditions are listed in Tab. 1. The reactions were car-ried out in a total volume of 12.5 µl containing: 10 × Taq DNA Polymerase buffer with MgCl2, 5 × GC-rich solu-tion, 0.24 mM dNTPs, 0.5 µM of the primers, 1 unit of Taq Polymerase (Roche) and 40-60 ng of genomic DNA. The PCR cycle conditions were: an initial denaturation at 94°C for 4 min, followed by 35 cycles of denaturation at 95°C for 30 sec, annealing at temperatures shown in Table 1 for 1 min and elongation at 72°C for 30/45 sec, with a final extension at 72°C for 7 min. PCR products were purified using membrane plates (Millipore) and used as templates in PCR-sequencing reamplification. The latter reaction was performed in Veriti 96 well Thermal Cycler using BigDye Terminator v3.1 Cycle Sequencing Kit (Life Technologies) and one of the specific primers (Forward or Reverse). Ream-plification products were purified with EDTA and ethanol precipitation and separated by electrophoresis using ABI 3130 sequencer (Applied Biosystems).
Statistical analysis. Genotypes obtained in the study were aligned with the reference sequences from the Ensembl database. Single nucleotide variations were assessed and calculation of the chi-square test for deviation from the Hardy-Weinberg equilibrium (HWE) was performed. The genotype and allele frequencies were evaluated and com-pared between study and control groups using the Fisher’s exact test. Odds ratio values (OR) were also evaluated and p < 0.05 was considered statistically significant. Addition-ally, we used Haploview 3.2 software to obtain the GGA gene structure. Linkage disequilibrium values (LD) were calculated as R2 value and the Gabriel et al algorithm was
used.
Results and discussion
In total, Sanger sequencing of 12 exons (exon 1 to exon 12) with approximately 70-bp non-coding splicing regions of the GGA gene in 58 subjects was performed. Upon alignment of obtained and reference sequences 14 single nucleotide variations were identified, of which 3 were already known polymorphisms with determined rs numbers (rs8932443 in 5’UTR region, rs8602105 in exon 6, rs23382375 in exon 12) and 11 novel varia-tions. Of those 11, 2 were localized in the 5’UTR region, 4 in exons (6, 8) and 5 in introns and splicing regions (2 variations in intron 5 and 8, 1 in intron 7). The num-bering of nucleotides was carried with reference to the first nt in the first coding triplet AUG in exon 1. All single nucleotide variations identified in the study are listed in Tab. 2.
Eleven of the nucleotide changes were substitutions and were biallelic and 1 was in/del type of variation. Of all 6 variations in coding regions, 5 were synonymous and did not change the amino acid sequence and 1 was
non-synonymous and changed the amino acid sequence of the protein.
Genotype and allele frequencies determined fre-quencies of alleles and genotypes for all 14 variants. Distribution of all GGA genotypes was consistent with HWE. Frequencies and OR values for chosen varia-tions are shown in Tab. 3. There were differences in the genotype and allele frequencies between the tumor and control group for heterozygote, alternative homo-zygote and for alternative allele for 4 polymorphisms (g.A-172T, c.T24C, c.A692G, c.C1185T). Although they were not statistically significant or were on the bound-ary of statistical significance, they could suggest an inclination to risk or protective variants with reference to a larger group of subjects. Much higher prevalence of heterozygotes and alternative alleles was observed in 3 polymorphisms (g.A-172T, c.A692G, c.C1185T) in the tumor group in comparison with the control group. The odds ratio values were quite high (see Tab. 3); however the results for all of the variations were not statistically significant or were on the boundary of significance. Very high OR values for both heterozygote AT and for the alternative allele T was observed in polymorphism g.A-172T for the tumor group in comparison with the control group (OR = 8.9 for the AT genotype, p = 0.1647; OR = 8.5 for the T allele, p = 0.1698). There were no differences in the genotype and allele frequencies among all 3 control subgroups, as the wild allele A comprised all genotypes in that group. Therefore, the more than 8-fold higher incidence of the T allele in tumor patients could indicate a possible risk allele for tumor presence.
There were high OR values for heterozygote or alter-native homozygote and alteralter-native allele in the other two putative risk variants for the tumor group in comparison with the control group, and differences were also seen in control subgroups. In polymorphism c.A692G there
Tab. 2. Single nucleotide variations identified in the GGA gene in the studied subjects
Name of variation Localization nt change aa change 1 rs8932443 5’UTR T/– – 2 g.A-172T 5’UTR A > T – 3 g.G-50T 5’UTR G > T – 4 c.T24C exon 1 T > C syn: G8G (GGT > GGC) 5 c.T522C exon 5 T > C syn: C174C (TTT > TTC) 6 g.A43540G intron 5 A > G – 7 g.T43580A intron 5 T > A –
8 rs8602105 exon 6 C > T syn: H208H (CAC > CAT) 9 c.A692G exon 7 A > G non: E231G (GAA > GGA) 10 g.T171934C intron 7 T > C –
11 g.A172092C intron 8 A > C – 12 g.T172127C intron 8 T > C –
13 c.C1185T exon 11 C > T syn: D395D (GAC > GAT) 14 rs23382375 exon 12 G > A syn: Q427Q (GAG > GAA)
Explanations: (g. = genomic; c. = coding; syn = synonymous; nt = nucleotide; aa = amino acid)
Tab. 3. Frequencies and odds ratio values for chosen single nucleotide variations for the GGA gene
Variation Tumor patients Controls p value OR (95% CI) g.A-172T Genotype frequency (n = 22) (n = 36)
AA 20 (0.91) 36 (1.0) AT 2 (0.09) 0 T vs. C 0.1647 8.9 (0.4-194.5) subgroup Y (n = 11) AA 11 (1.0) subgroup A (n = 10) AA 10 (1.0) subgroup G (n = 15) AA 15 (1.0) Allele frequency A 42 (0.95) 72 (1.0) T 2 (0.05) 0 T vs. C 0.1698 8.5 (0.4-182) subgroup Y A 22 (1.0) subgroup A A 20 (1.0) subgroup G A 30 (1.0) c.T24C Genotype frequency (n = 22) (n = 36) TT 12 (0.54) 15 (0.42) CT 7 (0.32) 15 (0.42) C vs. T 0.4544 1.5 (0.5-4.7) CC 3 (0.14) 6 (0.16) subgroup Y (n = 11) TT 3 (0.27) CT 6 (0.55) CC 2 (0.18) subgroup A (n = 10) TT 6 (0.6) CT 3 (0.3) Y vs. A 0.2621 2.8 (0.5-16.9) CC 1 (0.1) Y vs. A 0.5974 2 (0.2-26.2) subgroup G (n = 15) TT 6 (0.4) CT 6 (0.4) Y vs. G 0.464 1.8 (0.4-8.7) CC 3 (0.2) Allele frequency T 31 (0.7) 45 (0.625) C 13 (0.3) 27 (0.375) C vs. T 0.3828 1.4 (0.6-3.2) subgroup Y T 12 (0.55) C 10 (0.45) subgroup A T 15 (0.75) C 5 (0.25) Y vs. A 0.172 2.5 (0.7-9.3) subgroup G T 18 (0.6) C 12 (0.4)
Variation Tumor patients Controls p value OR (95% CI) c.A692G Genotype frequency (n = 22) (n = 36)
AA 19 (0.86) 33 (0.92) AG 2 (0.09) 3 (0.08) T vs. C 0.9206 1.1 (0.2-7.2) GG 1 (0.05) 0 T vs. C 0.3254 5.1 (0.2-130.6) subgroup Y (n = 11) AA 11 (1.0) subgroup A (n = 10) AA 9 (0.9) AG 1 (0.1) A vs. Y 0.4456 3.6 (0.1-99.9) subgroup G (n = 15) AA 13 (0.87) AG 2 (0.13) G vs. Y 0.3652 4.3 (0.2-98.1) Allele frequency A 40 (0.91) 69 (0.96) G 4 (0.09) 3 (0.04) T vs. C 0.2912 2.3 (0.5-10.8) subgroup Y A 22 (1.0) subgroup A A 19 (0.95) G 1 (0.05) A vs. Y 0.455 3.5 (0.1-90) subgroup G A 28 (0.93) G 2 (0.07) G vs. Y 0.3832 4 (0.2-86.4) c.C1185T Genotype frequency (n = 22) (n = 36) CC 18 (0.82) 35 (0.97) CT 4 (0.18) 1 (0.03) T vs. C 0.0757 7.8 (0.8-74.8) subgroup Y (n = 11) CC 11 (1.0) CT 0 G vs. Y 0.6059 2.4 (0.1-64.1) subgroup A (n = 10) CC 10 (1.0) CT 0 G vs. A 0.6447 2.2 (0.1-58.8) subgroup G (n = 15) CC 14 (0.93) CT 1 (0.07) Allele frequency C 40 (0.91) 71 (0.99) T 4 (0.09) 1 (0.01) T vs. C 0.0843 7.1 (0.8-65.7) subgroup Y C 22 (1.0) T 0 G vs. Y 0.6174 2.3 (0.1-58.6) subgroup A C 20 (1.0) T 0 G vs. A 0.6577 2.1 (0.1-53.8) subgroup G C 29 (0.97) T 1 (0.03)
Explanations: (Y = young; A = adult; G = geriatric)
was a 5-fold higher incidence of the alternative homo-zygote GG in the tumor group (OR = 5.1, p = 0.3254) and also higher prevalence of the heterozygote AG in more advanced age subgroups: adult and geriatric sub-groups in comparison with the young subgroup (OR = 3.6, p = 0.4456 for adults vs. young and OR = 4.3, p = 0.3652 for geriatrics vs. young). The results for the allele frequency were similar. High OR values for the G allele were observed both for the tumor group in comparison with controls (OR = 2.3, p = 0.2912) and in adults and geriatrics in comparison with young subjects (OR = 3.5, p = 0.4550 for adults vs. young and OR = 4, p = 0.3832 for geriatrics vs. young). Moreover, single nucleotide substitution in this variation changed the amino acid sequence of the protein from polar glutamic acid into non-polar hydrophobic glycine, which could change the protein activity. That could strongly indicate not only the alternative allele G as a risk variant but also higher incidence of tumor presence with reference to the age of a subject.
Moreover, differences in allele and genotype frequen-cies in polymorphism c.C1185T between study groups suggest it to be a risk variant for tumor incidence. Very high OR values for both heterozygote CT and alternative allele T were observed for the tumor group in comparison with the control group (OR = 7.8, p = 0.0757 for the CT genotype and OR = 7.1, p = 0.0843 for the T allele). Moreover, there was over a 2-fold higher prevalence of heterozygote CT and the T allele in Geriatric subjects in comparison with two younger age subgroups. The nucleotide substitution did not change the amino acid sequence of the protein; however, the nucleotide change itself could influence the folding of the mRNA and its biological interactions with other molecules, e.g. splicing factors. Therefore, similarly to polymorphism c. A692G, the presence of the alternative allele T in variation c.C1185T could suggest it to be a risk allele for tumor presence and higher incidence of tumorigenesis with age.
In contrast, slightly higher prevalence of the hetero-zygote CT and alternative allele C were observed in the control group in comparison with tumor patients. Also, there were higher OR values for the heterozygote and C allele prevalence in young patients with reference to older age subgroups. Although the results were not statistically significant and the OR values were lower than in the case of the putative risk variants described above, the higher incidence of the C allele in the control group could suggest a putative protective variant with reference to the larger study group.
The results in polymorphism g.G-50T were inconclu-sive. There was higher prevalence of the heterozygote GT and the alternative allele T in young subjects in comparison with older age subgroups (OR = 5.5 for the GT genotype, p = 0.2891 and OR = 5 for the T allele, p = 0.3085 for young vs. adults and OR = 3.1 for the GT genotype, p = 0.3816 and OR = 2.9 for the T allele, p = 0.3976 for young vs. geriatrics; data not shown). However, we did not observe any differences in
geno-type and allele frequency between tumor and control groups. Moreover, the OR values for the rest of identified variations did not differ between the study groups and were close to the neutral ‚1’ value.
Haploview analysis. The GGA gene structure of the case-control association study revealed no haplotype blocks, according to the Gabriel et al. (4) algorithm (Fig. 1). There was a high linkage between variations: c.T522C and c.A692G (in 58% of all subjects studied) and between variations: g.T43580A and g.T172127C (in 100% of all subjects studied). Those values did not differ between control and tumor groups separately. Interestingly, 100% LD between g.T43580A and g.T172127C was seen only in the control group, as both variations were not differentiated and comprised only wild homozygotes in all tumor patients. It is worth mentioning that there was a 30% linkage between varia-tions: g.G-50T and rs8602105 and between c.T24C and rs8602105 in tumor patients, while no such association was seen in the control group (data not shown).
The GGA1 gene and its pathogenic influence on organisms has not been widely studied. Several stud-ies about GGA1 in humans were conducted, but till now only limited information has been available about its role in diseases (2, 19). A study was conducted about consensus coding sequences of the human breast and colorectal and determined the sequence of well-annotated human protein-coding genes in two common human tumor types (19). 189 genes were identified, which were mutated at significant frequency. Most of these genes were not known to be altered in tumors and are predicted to affect a wide range of cellular functions, including transcription, adhesion, and invasion (21). It has been reported that genes coding proteins responsible for intercellular trafficking like OTOF, LRBA, AEGP, PLEKHA8, LOC283849, SORL1, KTN1 and GGA1 are significantly mutated in the breast (20). It has been proposed to call these genes candidate genes, because their influence is not well-known (19). In the same year the study about GGA1 and its influence on pathogenesis
Fig. 1. Structure of GGA gene shown as a case-control asso-ciation
of Alzheimer Disease was conducted and it was reported that the GGA1 protein could be involved in AD patho-genesis (21). Whilst GGA1 in humans is analyzed in several studies, there is an incomparably lower amount of studies about GGA1 in animals.
Recently, a significant advance has been made in the understanding of molecular events of the mammary gland tumor in humans and dogs (7, 16, 17). This is cru-cial for the development of novel effective therapeutic agents and strategies. Grüntzig (5) in his own study tried to answer the Swiss canine registry and classified the dog according to the breed, age, sex, neuter status, and place of residence. Among all dogs diagnosed, 23.5% related to mammary gland tumors (5, 13). The most common group of dogs suffering are middle-age bitches (9, 18, 20). In recent years, an increasing number of canine molecular tools have been developed, not only on genomic, RNA and protein levels. These studies explain specific aspects of canine carcinogenesis, particularly of the mammary gland.
The GGAs family are proteins of monomeric clathrin adaptors primarily localized at the trans-Golgi network; moreover, they have been reported in late endosomes. GGAs contain four domains: VHS (an N-terminal Vps27, Hrs, STAM homology), a domain related to cargo proteins with protein receptors (cation-dependent mannose 6-phosphate receptors CD-MPR); GAT domain that binds Arfs, ubiquitin, and Tsg101 (GGA and Tom) is responsible for recruitment of GGA proteins to the membrane via interaction with GTP-bound Arf; a hinge region that recruits clathrin; and the last domain: GAE is a C-terminal γ-adaptin ear homology – this domain binds a few proteins, including rabaptin 5, epsinR, and γ-synergin (10). Joshi (10) in his own study on the ret-rovirus proved that GGA over-expression induces the formation of large swollen compartments that sequester Arf proteins.
In the current study, the association between the GGA1 and occurrence of malignant mammary tumors and aging in domestic bitches was examined. GGA1 and its properties are well-described in literature, but according to the discoveries of Sjöblom et al, Wahle et al. (19, 21) and our recent results we can assume that our knowledge about this protein is very narrow, as it is proven that GGA1 takes part in pathogenesis of severe diseases, such as breast cancer and Alzheimer Disease. It is very important to provide more studies about GGA1 to expand our knowledge of negative interactions of GGA1. The GGA1 gene is conserved among a number of species, e.g.: chimpanzee, Rhesus Monkey, rat, mouse and human, which is a big facilitation in research on the GGA1 gene and its products, as we can transfer research results from animals to humans.
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