Naslov (srp)

Polimorfizmi u genu za fenilalanin hidroksilazu čoveka kao regulatori genske ekspresije, modulatori fenotipa i populaciono-genetički markeri

Autor

Stojiljković, Maja

Doprinosi

Pavlović, Sonja
Matić, Gordana
Radojković, Dragica

Opis (eng)

Phenylketonuria (PKU) is the most common inborn metabolic disease in Caucasians. Its incidence is 1/10000 in European populations and 1/12300 in Serbian population. PKU is transmitted in autosomal-recessive pattern, and it is caused by mutations in the phenylalanine hydroxylase gene (PAH) which change structure and decrease activity of the enzyme (PAH) involved in the conversion of phenylalanine to tyrosine in the liver. Even though PKU is monogenic disease, the wide spectrum of different phenotypes was described. Since the most severe form of PKU phenotype (mental retardation) is developed as a consequence of the genotype-environment interaction (phenylalanine in the food), the early diagnosis and restriction of a dietary phenylalanine intake are essential. Although the neonatal screening of PKU in Serbia is conducted since 1980. we present the first complete study on molecular characterization of mutations and polymorphisms in the PAH gene. Implemented molecular diagnostics has application in precise diagnostics, genetic counseling and creates possibility for selection of patients that could benefit from tetrahydrobiopterin therapy.34 unrelated patients were included in this study. According to pretreatment serum phenylalanine level, they were assigned to phenotypic categories: classical PKU (65%), mild PKU (35%). We designed the strategy and optimized methods for detection of mutations in the PAH gene of the patients with PKU from Serbia. By combining methods for indirect and direct mutation detection (multiplex 'broad range' DGGE for all 13 exons and their flanking intron regions, PCR-RFLP, PCR-ACRS and DNA sequencing analysis for selected regions) we reached mutation detection rate of 97%. We identified 19 disease-causing mutations (13 missense, 3 nonsense, 2 splice and 1 small deletion). The most frequent mutations were: L48S (21%), R408W (18%), P281L (9%), E390G (7%) and R261Q (6%), accounting for 60% of all mutant alleles. Interesting, L48S mutation, which was detected in many European populations, has the highest frequency in the Serbian population. The remaining mutations detected in the Serbian population (R158Q, 1306V, IVS12+1G>A, Q20X, R111X, V177L, P225T, R261X, L15/S16fsCTdel, S231F, R252Q, R297H, IVS10-llG>A and R413P) occurred at frequency less then 5%. Additionally, we found some common silent mutations (Q232Q, V245V and L385L) and intron polymorphisms (IVS2-19T>C, IVS5-54A>G, IVS12-35C>T). The mutation data were incorporated into The Serbian National Mutation Frequency Database (www.goldenhelix.org/serbian/).For the first time, the allele heterogeneity of the PAH locus in the Serbian population was analyzed. The homozygosity value of the PAH locus (0.10), as well as the genotypic homozygosity (8.82%) were low, indicating numerous historically documented migrations, co-existence of different populations in this part of the Balkan Peninsula, as well as confirmation that the gene flow occurred between populations.We made preliminary estimation of the potential benefit of tetrahydrobiopterin supplementation therapy in Serbia. Total frequency of mutations (L48S, E390G, R261Q, R158Q. and R413P) which could have positive response to tetrahydobiopterin is 40.2% for the Serbian population.On the basis of the genotype-phenotype correlation, studied in the homozygous and functionally hemizygous patients, we analyzed the effect of 9 mutations in the PAH gene. For three rare mutations (P225T, R413P, R297H), the in vivo effect was described for the first time. The effects of the majority of the remaining mutations (R252Q, R261Q, E390G, 1306V, V177L) were in concordance with the previous data. However, for the L48S mutation, previously described as mutation that causes different effects in patients, we observed consistently severe effect in the Serbian population. Among the mutations found in compound L48S/Mut heterozygotes associated with classical PKU, only the effect of S231F mutation was not previously analyzed. Therefore, we characterized S231F PAH protein in prokaryotic (E. coli) and eukaryotic (hepatoma cells) in vitro expression systems. The enzyme activity assay for S231F PAH, showed that the residual enzyme activity was 1% and activation fold (0.94). The GroEL/GroES did not increase amount or activity of S231F PAH in prokaryotic system, whiletetrahydrobiopterin slightly increased amount of S231F PAH in eukaryotic system. On the basis of presented results, S231F mutation was characterized as a functionally null one. This finding was applied for completing the genotype-phenotype correlation in patients with classical PKU in whom one of the mutations was L48S. Thus, the consistently severe effect of L48S mutation in the Serbian population was confirmed.In order to elucidate the prominence and phenotypic consistency of L48S detected in the Serbian population, we conducted the haplotype study on the patients with classical PKU, carriers of L48S (one homozygote and 8 compound heterozygotes). At the same time, the haplotype background on which a mutation had arosen, was investigated for the first time in Serbian population. We performed PCR- RFLP on six polymorphic sites {Bglll, Pvulla, Pvullb, EcoRI, Mspl and Xmnl) and identified VNTRs by PCR analysis. We found that Bglll, Pvulla and Pvullb polymorphic sites had low (0.08), while EcoRI, Mspl and Xmnl had high (0.47-0.5) expected heterozygosity. We found that L48S mutation was associated with haplotype 4.3 (70%) and haplotype 28.3 (20%), while in the 10% of the cases, haplotype could not be defined. Since, two different, previously described haplotypes were detected in the Serbian population, we concluded that L48S did not arise de novo in the Serbian population. We suggest that L48S was imported from populations with different genetic backgrounds and experienced the propagation afterwards by mechanisms of founder effect or genetic drift.Considering the lack of the data about the effect of the regulation of transcription on PKU phenotypic variability, we analyzed intronic parts of the PAH gene that include RFLP polymorphisms. In silica analysis was conducted in order to select regions which were consequently analyzed by methods for identification of DNA/protein interaction (EMSA and South-Western blot). Functional characterization of the region was conducted in eukaryotic (hepatoma cells) in vitro expression system by using CAT assay and beta-galactosidase assay. We showed that a region within PAH gene intron 8 contains transcription regulatory element which binds GATA-1 transcription factor and acts as an enhancer. This was the first time that a region in the PAH intron was characterized as a regulatory transcription element.In this study, different but complementary methods were used in order to identify the mutations in the human PAH gene and analyze their effects on phenotype modulation. Polymorphisms that do not influence phenotype, were used as a tool for analysis of molecular basis of PKU in the Serbian population. We pointed out the role of intronic regions on the expression of the PAH gene as additional, uncharacterized factors that have influence on phenylketonuria complex phenotype.

Opis (srp)

Fenilketonurija (PKU, phenylketonuria) je najčešća nasledna metabolička bolest u populaciji belaca. Prosečna učestalost PKU je u evropskim populacijama 1/10000, a u srpskoj 1/12300. Fenilketonurija se nasleđuje autozomalno-recesivno, a uzrok su mutacije u genu za fenilalanin hidroksilazu (PAH, phenylalanine hydroxylase) koje dovode do promenjene strukture i smanjene efikasnosti enzima (PAH) čija je uloga u konverziji fenilalanina u tirozin u jetri. lako je fenilketonurija rrionogenetska bolest, opisan je čitav spektar različitih fenotipova. S obzirom da se najteži oblik PKU fenotipa (mentalna retardacija), razvija tek kao posledica interakcije genotipa i uslova sredine (fenilalanina u hrani), rana dijagnoza i lečenje dijetalnom restrikcijom fenilalanina su od presudne važnosti. lako se neonatalni skrining na fenilketonuriju u Srbiji sprovodi od 1980. godine, ovaj rad predstavlja prvu kompletnu studiju o molekularnoj karakterizaciji mutacija i polimorfizama u genu za PAH. Uvedena molekularna dijagnostika ima značaj za postavljanje preciznije dijagnoze, genetičko savetovanje i otvara mogućnost za odabir pacijenata kojima bi koristila terapija preparatom tetrahidrobiopterina.U studiju su bila uključena 34 nesrodna pacijenta, koji su na osnovu nivoa fenilalanina pre lečenja, svrstani u fenotipske kategorije: klasičnu PKU (65%) i blagu PKU (35%). Osmišljena je strategija i optimizovane su metode za detekciju mutacija u genu za fenilalanin hidroksilazu pacijenata obolelih od fenilketonurije u Srbiji. Kombinacijom metoda za indirektnu i direktnu detekciju mutacija (multipleks DGGE širokog spektra za analizu 13 egzona i njihovih okolnih intronskih regiona, PCR-RFLP, PCR-ACRS i DNK sekvenciranje odabranih regiona) postignut je nivo detekcije od 97%. Identifikovano je 19 mutacija u genu za PAH koje dovode do bolesti (13 "missense" tipa, 3 "nonsense" tipa, 2 splajsing mutacije i 1 mala delecija). Najčešće mutacije su L48S (21%), R408W (18%), P281L (9%), E390G (7%) and R261Q(6%), koje predstavljaju 60% od mutiranih alela. Interesantno je da mutacija L48S, koja je detektovana u mnogim evropskim populacijama, u srpskoj populaciji ima najvišu relativnu učestalost. Ostale mutacije detektovane u srpskoj populaciji (R158Q, I3O6V, 1VS12+1G>A, Q20X, R111X, V177L, P225T, R261X, L15/S16fsCTdel, S231F, R252Q, R297H, IVS10-llG>A i R413P) imaju učestalost manju od 5%. Takode, identifikovane su i neke česte tihe mutacije (Q232Q, V245V and L385L) kao i intronski polimorfizmi (IVS2-19T>C, IVS5-54A>G, IVS12-35OT). Podaci o mutacijama uneti su u nacionalnu bazu podataka o učestalosti mutacija (www.goldenhelix.org/serbian/).Po prvi put je analizirana alelska heterogenost PAH lokusa u srpskoj populaciji. Homozigotna vrednost PAH lokusa (0.10), kao i genotipska homozigotnost (8.82%) su niske i ukazuju na heterogenost molekularne osnove fenilketonurije u srpskoj populaciji, što oslikava brojne istorijski dokumentovane migracije, suživot različitih populacija u ovom delu balkanskog poluostrva, kao i potvrdu da se protok gena dešavao izmedu populacija.Data je prva procena dobrobiti od primene terapije sa preparatom tetrahidrobiopterina u Srbiji. Ukupna učestalost mutacija (L48S, E390G, R261Q, R158Q. i R413P) koje bi potencijalno pozitivno reagovale na tetrahidrobiopterin iznosi 40.2% u srpskoj populaciji.Na osnovu korelacije genotipa i fenotipa u homozigotnim i funkcionalno hemizigotnim pacijentima, analiziran je efekat 9 mutacija u genu za PAH. Za tri retke mutacije (P225T, R413P, R297H) je po prvi put opisan in vivo efekat, a analiza ostalih mutacija (R252Q, R261Q, E390G, 1306V, V177L) je u većini slučajeva pokazala slaganje sa predhodnim podacima. U slučaju L48S mutacije, koja je predhodno opisana kao mutacija koja dovodi do različitog efekta kod pacijenata, u srpskoj populaciji je zapaženkonzistentno težak efekat. Od mutacija koje su se našle u genotipu sa L48S mutacijom i dovele do klasične fenilketonurije, jedino efekat S231F mutacije, ranije nije bio proučavan u in vitro sistemima.Iz tog razloga, po prvi put je urađena analiza efekta S231F mutacije u prokariotskom (E. coli) i eukariotskom (hepatoma) ekspresionom in vitro sistemu. Na osnovu eseja za aktivnost PAH, odredeni su rezidualna aktivnost (1%) i nivo aktivacije supstratom (0.94) S231F PAH. Utvrdeno je da GroEL/GroES šaperoni ne dovode do povećanja količine i aktivnosti S231F PAH u prokariotskom sistemu, dok tetrahidrobiopterin dovodi do neznatnog povećanja količine S231F PAH u eukariotskom sistemu. Na osnovu ovih rezultata, S231F mutacija je okarakterisana kao funkcionalno „nulta", što je upotrebljeno za kompletiranje korelacije genotipa i fenotipa funkcionalno hemizigotnih pacijenata sa teškom kliničkom slikom kod kojih je jedna od mutacija L48S. Time je potvrden konzistentno težak efekat L48S mutacije u srpskoj populaciji.Da bi rasvetlili poreklo visoke relativne učestalosti i konzistentnog fenotipskog efekta L48S mutacije u srpskoj populaciji, sproveli smo haplotipsku studiju na pacijentima sa klasičnom fenilketonurijom, kod kojih je u genotipu prisutna L48S mutacija (jedan homozigot i 8 kombinovanih heterozigota). Istovremeno, po prvi put je u srpskoj populaciji ustanovljeno na kakvom hromozomskom okruženju se nalazi jedna mutacija u genu za PAH koja dovodi do fenilketonurije. Analizirali smo 6 bialelskih polimorfizama {Bglll, Pvulla, Pvullb, EcoRI, Mspl i Xmnl) primenom PCR-RFLP metode i jedno multialelski polimorfizam (VNTR) pomoću PCR metode. Uočena je niska očekivana heterozigotnost (0.08) za Bglll, Pvulla i Pvullb polimorfna mesta, dok je za EcoRI, Mspl i Xmnl polimorfna mesta, uočena visoka očekivana heterozigotnost (0.47-0.5). Utvrdeno je da se L48S mutacija kod pomenute grupe pacijenata nalazi na haplotipu 4.3 (70%) i haplotipu 28.3 (20%), dok u 10% slučajeva haplotip nije bilo moguće odrediti. S obzirom da su u srpskoj populaciji detektovana dva različita, predhodno opisana haplotipa, smatramo da L48S nije nastala de novo u srpskoj populaciji i da je u procesu migracija uneta u srpsku populaciju iz populacija sa različitim genetičkim osnovama posle čega je doživela ekspanziju (efekat osnivača ili genetički drift).S obzirom na nedostatak podataka o uticaju regulacije transkripcije na varijabilnost fenotipa PKU, analizirani su delovi introna gena za PAH koji sadrže RFLP polimorfizme. In silico analiza primenjena je za odabir regiona koji je zatim analiziran metodama za identifikaciju interakcije izmedu DNK i proteina (EMSA i South-Western blot). Funkcionalna karakterizacija regiona sprovedena je u eukariotskom (hepatoma) in vitro sistemu uz primenu CAT i beta galaktozidaznog eseja. Pokazano je da u osmom intronu gena za fenilalanin hidroksilazu postoji transkripcioni regulatorni element koji vezuje GATA-1 transkripcioni faktor i funkcioniše kao pojačivač. Ovo je prvi put da je za neki region u okviru PAH introna, pokazano da je regulatorni transkripcioni element.U ovom radu, primenom raznovrsnih all komplementarnih metoda identifikovane su mutacije u genu za fenilalanin hidroksilazu čoveka i analiziran je njihov efekat na modulaciju fenotipa. Polimorfizmi koji ne utiču na fenotipsku ekspresiju, iskorišćeni su kao sredstvo za analizu molekularne osnove PKU u srpskoj populaciji. Skrenuta je pažnja na ulogu intronskih regiona na ekspresiju gena za PAH, kao dodatnih, do sada neistraženih faktora koji utiču na kompleksan fenotip fenilketonurije.

Opis (srp)

Datum odbrane: 19.06.2009.

Jezik

srpski

Datum

2009

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