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ID naloge: 35 Letnik: 1998 Predmet: genetika
Zgodnje odkrivanje genetske hemokromatoze
Avtor: Jelena Berger
Mentor: doc. dr. Saša Markovic, dr. med
IZHODIŠCE: Genetska hemokromatoza je najpogostejša avtosomno recesivno dedna bolezen presnove. Pojavlja se pretežno v populaciji severno evropskega porekla, pri potomcih Keltov. Heterozigotnih nosilcev gena je 1:10. Zboli vsak 200-ti. Doslej sta opisani dve tockovni mutaciji HFE- gena na kromosomu 6. To sta C282Y in H63D. Za genetsko hemokromatozo zbolevajo C282Y homozigoti in mešani heterozigoti (compound), z obema mutacijama. Zaradi povecane absorpcije železa se slednje nalaga v številne organe in povzroca njihove okvare. Razvita bolezen se pokaže z jetrno cirozo v kateri se kasneje razvije jetrno celicni rak, s sladkorno boleznijo, kardiomiopatijo in motnjami žlez z notranjim izlocanjem. Ko je bolezen polno razvita je neozdravljiva. Z zgodnjim odkrivanjem in preventivnimi venepunkcijami, jo lahko preprecimo. Zato je potrebno, da jo odkrijemo v zgodnji asimptomatski fazi.
NAMEN: Poglavitni namen je zgodnje odkrivanje GH pri sorodnikih bolnikov z GH. Drugi namen naše raziskave je bil ugotoviti korelacijo med fenotipom in genotipom bolnikov z genetsko hemokromatozo.
PREISKOVANCI IN METODE: V preiskavo smo vkljucili 22 slovenskih bolnikov z GH. Na podlagi družinskih debel smo k raziskavi povabili tudi 74 najožjih družinskih clanov bolnikov. Bolnikom z že razvitimi bolezenskimi znaki dolocimo feno in genotip. Svojce smo testirali fenotipsko in genotipsko. Za genotipizacijo smo uporabili preprost genetski test, specificen za mutacijo C282Y. Temelji na izolaciji DNK iz nekoagulirane krvi, pomnoževanja izbranega odseka genoma z polimerazno verižno reakcijo (PCR) in selektivni hibridizaciji z alelsko specificnimi DNK lovkami. Na dnu encimske mikrotitracijske plošcice potece imunska reakcija, na podlagi katere dolocimo genotip.
REZULTATI: Ugotovili smo prisotnost mutacije C282Y pri 85% preiskovanih oseb. Med 22 bolniki s fenotipsko izraženo GH smo ugotovili prisotnost mutacije C282Y pri 14 (63,5%), pri osmih (36,4%) pa te mutacije ni bilo. Dvanajst bolnikov (54,5%) je bilo homozigotov, dva (9,1%) pa C282Y heterozigotov. Med družinskimi clani smo testirali 33 sorodnikov homozigotnih ali heterozigotnih bolnikov. Med 33 preiskovanimi osebami smo odkrili C282Y mutacijo pri 28 (84,8%), pri 5 (15,2%) pa te mutacije ni bilo. Štirje preiskovanci (12,1%) so bili homozigoti, 24 (72,7%) pa C282Y heterozigoti.
ZAKLJUCEK: Genetska analiza C282Y mutacije je zaneslivejša kot fenotipska analiza pri predsimptomatskem odkrivanju bolnikov z GH. Pri bolnikih z razvito boleznijo, fenotipsko opredeljenih kot GH pa je ugotovljeno število homozigotov najnižje doslej opisano
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[Abstract / English version]
Early diagnosis in genetic hemochromatosis
Author: Jelena Berger
Mentor: doc. dr. Saša Markovic, dr. med
INTRODUCTION: Genetic hemochromatosis (GH) is the most common autosomal recesive disorder of iron metabolism among individuals of Northern European discent of Celtic origin. The frequency of affected homozigotes is 1 in 200 with a heterozygous carrier rate of 1 in 10. The disease results in multiorgan dysfunction caused by excessive iron absorption and deposition with severe conseqences. Liver cirrhosis, hepatocelular carcinoma (HCC), diabetes mellitus, cardiomiopathy and hipogonadism are among the most common diseases associated with GH. Detection of the disease in the presymptomatic stage is important since removal of the excess iron by phlebotomy can prevent organ damage. More then 85% GH patients carry the point mutation - cysteine to tyrosine substituton at codon 282 (C282Y).
AIM: A genotipization was done in GH patients and in first degree relatives. Siblings were tested for early diagnose in asymptomatic stage. Patients genotip was used for a study of genotip- fenotip correlation. We tried to evaluate a genetic test as a screening test for early diagnose in GH.
PATIENTS AND METHODS: Twenty-two (22) probands with GH and 74 siblings from 16 unrelated slovenian famiels were included in the study. The diagnosis in probands was established using established clinical critera. For genotypisation we have used a simple genetic test specific for C282Y mutation. The test is based on DNA isolation from anticoagulated blood then in vitro amplification of relevant gene sequences with polymeraze chain reaction (PCR), PCR produkcts are selectively hybridized to allele specific oligonucreotide probes and in separate cavities of a microwells detected by immunoreation.
RESULTS: The overall prevalence of C282Y mutation in the investigated group was 85%. Among 22 phenotypic GH probands we have found C282Y mutation in 14 patients (63,5%) and 8 individuals (36,4%) homozygous for wild type allel. 12 probands (54,5%) were CV282Y homozygotes, whereas 2 (9,1%) were C282Y heterozygotes. Among siblings, 33 individuals of homozygous or heterozygous probands were tested for C282Y mutation. Among 33 tested, 28 (84,8%) were carrying C282Y mutation, while 5 (15,2%) were homozygous for wilde type allel. Through screening, 4 (12,1%) were identified as C282Y homozygotes whereas 24 (72,7%) were C282Y heterozygotes.
CONCLUSION: Genetic test is more reliable in presymptomatic screening in famylies with GH. In patients with phenotipic expression of GH we have found the lowest noumber of C282Y homozygotes poblished as far.
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