FAMILIAL ADENOMATOUS POLYPOSIS: ANALYSIS OF GENETIC INSTABILITY OF MICROSATELLITES LOCI AND GENETIC ALTERNATIONS OF TUMOR SUPPRESSOR GENES

Familial adenomatous polyposis (FAP) is an autosomal dominant illness with the highest risk for appearance of colorectal cancer's disease. In our study, we have used Bethesda criteria that defi ne colorectal cancers which can be tested on microsatellite instability. Th e aim of our study is make an analysis of microsatellite instability (MSI), appearance of RER+ phenotype, genetic alteration of tumor suppressor genes as like as one of responsible factor for genesis of adenomatous polyposis. Th e base for this study were shown families with clinical diagnosed FAP. In this study two families with clinical diagnosed adenomatous polyposis were involved. Our study of both families showed that three tumor tissues belonged to RER negative phenotype, but only one belonged to RER positive phenotype. Microsatellite analysis showed instability of mononucleotide marker Bat  at  samples and Bat  at  samples, but Bat  and in  sample. Dinucleotide marker TP  did no show any microsatellite alterations. Genetic alteration of tumor suppressor gene APC appeared at  samples, p at  samples, RB at  samples and NM only at  sample, but tumor suppressor genes DCC and DCC were homozygote. Our results are agree with results of earlier studies and also the got results confi rm the fact that loss of heterozygosity of tumor suppressor gene APC and p are responsible for genesis of adenomatous polypose and it also represents the characteristic of genetic changes FAP’s patients in our region.


Introduction
Familial adenomatous polyposis (FAP) is an autosomal dominant illness with the highest risk for appearance of colorectal cancer's disease (); which is appeared in over  of unoperated cases.This illness is characteristic of forming with over , usually about  polyps of colorectal cancer and often with stomach's adenoma as like as small intestine's stomach.Th e biggest number of persons with FAP illness get the colorectal cancer before their  year old.The illness frequency is ,:  .The illness is hereditary on descendants in  of its cases, but it can get sick and more generations in a family.Oftentimes FAP is caused by mutation of APC gene.Persons with hereditary mutated APC gene have a high risk for developing of adenoma in their childhood, so that estimated risk is over .Some cancers show p mutation before APC mutation.Usually, loss of heterozygosity which is appeared at colorectal cancers through the FAP's frame is appeared on hromosomes , , ,  and .Th is point has showed localization of tumor suppressor genes.It seems that the illness of males is some often that the illness of females ( : ).There is no positive history of the illness in  of diagnosed cases.Defect of MMR gene leads to high level of microsatelites instability (MSI-H) in tumor tissue.So that the microsatellites instability can be found in early stage of the adenoma.It is known that a full develop of a microsatelites mutator phenotype can depend and on accumulation of secondary mutations ().The microsatellite instability is established in  adenoma of hereditary non-polyposis colorectal cancer, and even , belongs to MSI-H ().Th at investigation in this group of MSI-L levels show that there is a relation commonly with loss of hMLH or hMSH expression, and what is diff erent to situation with MSI-L of sporadic colorectal cancers.According to authors, this phenomenon that MSI-L status is appeared perhaps in earlier phase is in relation with dinucleotide markers.Authors also conclude that adenomas of MSI-H status must pass through phase of MSI-L stage or how it is cited perhaps MSI-H phenotype can give a ''de novo'', and what is unknown in this moment yet.It seems that MSI-L can be appeared in evolution of MSI-H earlier at HNPCC or MSI-L, and MSI-H can be with split phenotypes as like as at sporadic colorectal cancers.Authors also concluded that there are no a signifi cant relation among MSI status, sex, years and cancer location ().Other authors show in own research works that MSI status is in r ank of - (-).Usage of these  dinucleotide markers gave results in appearance of low percent detected mutation in microsatellite loci at benigne adenoma than at adenomas which have had alteration on malignancy ().Th ere is relation among MSI-H adenomas and loss of MMR expression at colorectal cancers.It seems that MSI-H adenomas have more tendency to developing of cancers ().In our study, we have used Bethesda criteria that define colorectal cancers which can be tested on microsatellite instability ().According to these criterias, there is a cancers classifi cation on microsatellite instability as like as following: • -MSI -L -microsatellite instability-low (MSI in locus less than - ); • -MSI-H -microsatellite instability-high (MSI in locus more than  and replication positive error (RER+) and • -MSS cancers-microsatellite stable The aim of our study is make an analysis of microsatellite instability (MSI), appearance of RER+ phenotype, genetic alteration of tumor suppressor genes as like as one of responsible factor for genesis of adenomatous polyposis.Th e base for this study were shown families with clinical diagnosed FAP.

Materials and Methods
Th rough the study we involved two families with clinical diagnosed adenomatous polyposis and samples were collected from Gastroenterological of University Clinical Center in Tuzla (Bosnia and Herzegovina).Tumor's and healthy surroundings tissue were fixed in formalin, and after that tissue was shaped in paraffi n blocks.Methods of isolation of genomic DNA is made on deparaffinization of tissue sections as like as on cell proteolises of tissue by proteinase K. () Fluoroscent chain synthesis of DNA is a method which has very broad application in tumor detection, and it is specially important in determination of microsatellite instability (MSI) and loss of heterozygosity (LOH) of tumor suppressor gene.We used mononucleotide and dinucleotide microsatellite markers in detection of microsatellite instability.We used in the group of mononucleotide markers following: BAT, BAT and BAT, but in the group of dinucleotide markers we used following: DS and TP . for detection of LOH, we used intragene markers for following tumor suppressor genes: NM, p, APC, RB, DCC and DCC.() PCR was performed using a PCR Th ermocycler  (Perkin-Elmer).The PCR conditions were as follows: after an initial  min denaturation step at °C,  amplifications cycles were performed, each consisting of a  s step at °C, a  s step at °C, and a  s elongation step at °C.Amplification was completed with a final incubation step at °C for  min.For separating of amplified PCR products, we used automatical sequencer  ABI PRISM, Genetic Analyser  (Perkin Elmer), which enable separating and quantification of DNA fragments according to principle of capillary electroforesis.Microsatellite analysis presents comparation of healthy and tumor tissue at the same patient by means of Genescan program package for analysis.Software program detects fluorescent peaks and shows them on electropherograme.Each fl uorescent peak is automatic quantifi ed in base size, height and peah fi eld.All samples were tested twice because result confi rmation.Loss of heterozygosity was calculated by matematics () for heterozygosity cases, allele ratio was calculated for each pair of normal and tumor tissue according to formula T:T/N:N , where T and N are valuable fi elds of shorter allele length; T and N are fi elds of value of longer allele for tumor and healthy samples.The result had rank from ,-,.If the result was lower or equal to ,.Then it is showed a significant loss of heterozigosity and on longer allele.Homozygosity cases cannot be used in calculation.

Results
Our study involved two families with clinical diagnosed adenomatous polyposis.We have made analysis of four members in Family ''A'' (two brothers and two sisters).
One of brothers (Patient No.) was operated of FAP with adenocarcinomas of rectosigmoid region in his  age.Adenomas were tubular and tubulovillous type.Other three members were phenotypely healthy.According to the history of illness of this family, we have concluded that mother of these four children had colorectal cancer.Microsatellite analysis at three members of the Family ''A'' did not fi nd any change, but at their sick member that had developed cancer are found changes of mononucleotide markers both Bat  and Bat .Loss of heterozygosity analysis has shown a genetic alteration of tumor suppressor genes both APC and RB, as like as homozygosity of DCC and DCC locus at sick patient (Table ).Family "B" has six members (fi ve brothers and one sister).One of brothers (Patient No.) was operated FAP adenocarcinomas fl exurae hepatalis in his  years old.Adenomas of this patient were pathohistological Adenoma tubulare and Adenoma tubulovilosum.Sister (Patient No.) was operated FAP adenocarcinomas colonis rectosygmoidei in her  years old.Polypes were also Adenoma tubulare and adenoma tubulovilosum.One of brothers (Patient No.) had diagnosed polype Adenoma tubulare.Other three brothers are phenotypely healthy.It has analyzed the familial history of their mother who died in her  years old from colorectal carcinoma.Mothers sister also died from cancer and one mothers brother was operated colorectal cancer and he is still alive.Microsatellite analysis of the Family ''B'' has showed that it three members have no alteration, but that alteration exists at other it three members.Th e microsatellite instability analysis of adenoma (Patient No.) has shown alteration of markers both Bat  and Bat , *MSI -microsatellite instability **N -without microsatellite instability ' AI -allele imbalance or loss of heterozygosity (LOH) "NAI -without loss of heterozygosity H -homozygous

Conclusion
Our results are agree with results of earlier studies and also the got results confi rm the fact that loss of heterozygosity of tumor suppressor gene APC and p are responsible for genesis of adenomatous polypose and it also represents the characteristic of genetic changes FAP's patients in our region.

TABLE 1 .
Microsatellite and genetic alteration of the Family '' A'' VESNA HADŽIAVDIĆ ET AL.: FAMILIAL ADENOMATOUS POLYPOSIS: ANALYSIS OF GENETIC INSTABILITY OF MICROSATELLITES LOCI AND GENETIC ALTERNATIONS OF TUMOR SUPPRESSOR GENES as like as LOH at NM, P and APC.One of brothers (Patient No.) getting sick FAP with developed cancer has found alteration both Bat  and Bat  as like as loss of heterozygosity P, APC and RB.Th e sister (Patient No.) getting sick FAP with developed cancer had alteration Bat , Bat  and DS , as like as LOH P and APC tumor suppressor gene.All members of family has showed homozygous locus' tumor suppressor gene DCC and DCC (Table ).Authors have found  germline mutations of APC locus.Many colon cancer syndromes have been characterized based upon their phenotype genetic changes.Among them, the most common and highly studied colon can familial adenomatous polyposis and hereditary nonpolyposis colorectal which are caused by mutations in APC and MMR genes, respectively ().Mutation in APC locus are considered as one the initiation and progression of colorectal cancer.Our study of both families showed that three tumor tissues belonged to RER negative phenotype, but only one belonged to RER positive phenotype.Microsatellite analysis showed instability of mononucleotide marker Bat  at  samples and Bat  at  samples, but Bat  and in  sample.Dinucleotide marker TP  did no show any microsatellite alterations.Genetic alteration of tumor suppressor gene APC appeared at  samples, p at  samples, RB at  samples and NM only at  sample, but tumor suppressor genes DCC and DCC were homozygote.Microsatellite analysis and genetic alteration all members of FAP's Families ''A'' and ''B'' with developed colorectal cancer have shown the identical changes as like as at the cancer.