Vpliv folatov na molekularne osnove prirojenih nepravilnosti ploda

doktorska disertacija

Maša Vidmar (Author), Ksenija Geršak (Mentor), Irena Mlinarič-Raščan (Co-mentor)

Abstract

Z nezadostnim vnosom folatov ali njegovih analogov, ter pomanjkanjem drugih vitaminov pred in med nosečnostjo, je povezano povečano tveganje za nastanek prirojenih napak ploda. Prirojene razvojne nepravilnosti in neželeni izidi nosečnosti predstavljajo velik javno-zdravstveni problem, saj imajo dolgotrajen vpliv na zdravje in kvaliteto življenja prizadetih otrok in njihovih staršev. Motnje in spremembe v presnovni poti folatov, ki so pogosto posledica prisotnih genskih polimorfizmov, so najpogostejši potencialni dejavnik tveganja za najpogostejše prirojene razvojne nepravilnosti, kot so prirojene srčne napake (CHD), orofacialne shize (OFC) in napake nevralne cevi (NTD). Poglavitni namen raziskovalnega dela v sklopu doktorske disertacije je bil odgovoriti na dve ključni temi oziroma znana problema, to sta zahtevna analitika folatov v bioloških vzorcih in vpliv genotipov na folatni status, ter tako doprinesti k razumevanju vpliva folatov na molekularne osnove prirojenih nepravilnosti ploda. Pri raziskovanju pomena folata je potrebno spremljati njegove ravni, vendar pa je analitika spričo slabe stabilnosti in izredno majhne količine folata v bioloških vzorcih zahtevna. V prvem delu doktorske naloge smo tako razvili in validirali metodo, ki nam je v nadaljevanju omogočala kvantifikacijo folne kisline (FA), biološko aktivne oblike folata, 5-metil-tetrahidrofolata (5-Me-THF) in homocisteina (Hcy). Razvili smo občutljivo, selektivno in hitro metodo tekočinske kromatografije visoke ločljivosti sklopljene s tandemsko masno spektrometrijo (HPLC-MS/MS) za sočasno določanje FA, 5-Me-THF in Hcy v lizatih humanih celic limfoblastoidne celične linije LCL. V nadaljevanju smo z raziskavami in vitro na celicah LCL in s študijo družin z otrokom s prirojeno razvojno nepravilnostjo in neprizadetim otrokom iste matere ugotavljali vpliv genotipa na folatni status. Zaradi izjemno pomembne vloge folatov so FA in njeni derivati najpogosteje uporabljeni prehranski dodatki. V Sloveniji z namenom zmanjševanja tveganja za pojav NTD že več kot 20 let porodničarji in ginekologi vsem nosečnicam v prvem trimesečju nosečnosti redno predpisujejo dodatke folatov. Ženskam, pri katerih se je v prejšnji nosečnosti pri plodu pojavila NTD, v naslednji nosečnosti predpišejo večje odmerke FA, medtem ko je uporabnost FA pri preprečevanju najpogostejših prirojenih razvojnih nepravilnosti, OFC in CHD, še premalo raziskana za uvedbo v klinično prakso. Na slovenskem tržišču so trenutno ii komercialno dostopne tri oblike folatov: FA in 5-Me-THF v obliki kalcijeve soli ali soli glukozamina. Splošno sprejeto dejstvo, ki ga promovirajo tudi proizvajalci pripravkov s 5-Me-THF je, da je 5-Me-THF bolj učinkovit v primerjavi s FA, vendar pa ta trditev ni mehanistično in klinično dokazana, prav tako pa tudi ni znano, kako genetske spremembe v folatnem in metioninskem ciklu modulirajo učinkovitost teh oblik folatov. Za odkrivanje vpliva različnih oblik folatov na folatni status glede na genetsko pogojene razlike v folatno metioninskem ciklu smo uporabili model LCL. Celice LCL smo z uporabo hibridizirajočih (LightSNiP) in hidrolizirajočih (TaqMan) sond genotipizirali za polimorfizme v genih, ki sodelujejo v folatnem ciklu % FPGS (rs1544105), DHFR (rs1677693), MTHFR (rs1801133 in rs1801131), MTRR (rs1801394), MTHFD1 (rs2236225), BHMT (rs3733890), GNMT (rs10948059), DNMT3B (rs2424913) in SLC19A1 (rs1051266). Nato smo celice en teden gojili v mediju brez FA, ter dokazali, da pomanjkanje folata povzroči zastoj celic v S fazi celičnega cikla. Po dodatku folata, se delež celic v S fazi zmanjša, dodatek pa povzroči večjo metabolno aktivnost celic LCL. Višja metabolna aktivnost je bila opažena pri celicah LCL, tretiranih z višjimi koncentracijami folata v primerjavi z nižjimi koncentracijami in pri celicah z dodano biološko aktivno obliko folata (5-Me-THF) v primerjavi s FA. Prav tako pa smo dokazali, da tudi polimorfizma v genu MTHFR, rs1801133 in rs1801131, vplivata na razlike v metabolni aktivnosti celic, ki so tretirane ali s FA ali s 5-Me-THF. S predhodno razvito metodo smo kvantificirali znotrajcelične koncentracije metabolitov folatnega cikla ter dokazali, da tako FA, kot 5-Me-THF povečata znotrajcelično koncentracijo biološko aktivnega folata, le-da 5-Me-THF v precej večjem obsegu. Pri upoštevanju klinično pomembnih polimorfizmov MTHFR C677T in A1298C smo pokazali, da so polimorfizmi z vplivom na aktivnost encima MTHFR povezani z znotrajcelično koncentracijo metilirane oblike folatov. V predhodnih raziskavah naše raziskovalne skupine smo primerjali pogoste variante v genih folatnega cikla in okoljske dejavnike tveganja med otroci z OFC, CHD ter njihovimi materami. V tej doktorski nalogi, pa smo študijo nadaljevali in jo nadgradili z analizo zdravih sorojencev otrok z OFC in CHD. Potrdili smo ugotovitev, da povišana telesna temperatura v nosečnosti predstavlja tveganje za pojav OFC, ter dokazali, da tveganje za nastanek OFC, poleg že potrjenega polimorfizma MTHFR 677, v slovenski populaciji poveča tudi polimorfizem rs1801131 v genu MTHFR. Na pojavnost CHD vpliva materina starost in spol otroka, kar je skladno s predhodnjimi študijami. Odkrili iii smo tudi nov genetski dejavnik tveganja, ki v slovenski populaciji prispeva k tveganju za nastanek bolezni CHD; polimorfizem rs1544105 v genu FPGS. Na folatni status ali folatni cikel pa preko različnih mehanizmov vplivajo tudi številna zdravila, ki imajo anti-folatno delovanje. Poraba zdravil v zadnjih desetletjih narašča in čeprav ni točnih podatkov, se ocenjuje, da več kot polovica žensk med nosečnostjo zaužije vsaj eno zdravilo. Ob upoštevanju dejstva, da je približno polovica nosečnosti nenačrtovanih, mnoge ženske svojega nerojenega otroka izpostavijo takim zdravilom. V zadnjem delu doktorske naloge smo tako raziskali vpliv anti-folatnih učinkovin fenitoina, metformina in trimetoprima ter dodatkov folata. Pokazali smo, da nekatere anti-folatne učinkovine, kot so metformin in trimetoprim vplivajo na preživetje celic in celični cikel celic LCL, medtem ko fenitoin ne vpliva. Prav tako tudi dodatki folata različno vplivajo na anti-folatni učinek izbranih učinkovin. Dodatki folata nimajo vpliva na zdravilo metformin, ima pa biološko aktivna oblika folata, 5-Me-THF kalcijeva sol, vpliv na zdravilo trimetoprim. Dodatek 5-Me-THF namreč poveča preživetje celic, ki so izpostavljene trimetoprimu, vpliva na porazdelitev celic v različnih fazah celičnega cikla, ter poveča znotrajcelično koncentracijo aktivnega folata in S-adenozil metionina. Citotoksične učinke metformina pa lahko le delno preprečimo z dodatkom predhodnikov nukleotidov. Rezultati tega doktorskega dela podajajo pomembne izsledke o MTHFR in folatnih dodatkih, ter njihov vpliv na folatni status. Z razvojem občutljive in specifične metode smo doprinesli k pravilni diagnostiki folatnega statusa na celicah LCL, z dodatnimi validacijami pa bi jo lahko prenesli tudi v klinične laboratorije, ter tako prispevali k boljšemu spremljanju nosečnic, pa tudi otrok in bolnikov na terapiji z anti-folati.

Keywords

folatni cikel;5,10-metilentetrahidrofolat reduktaza (MTHFR);folna kislina (FA);5-metil-tetrahidrofolat (5-Me-THF);prirojene razvojne nepravilnosti;anti-folatne učinkovine;

Data

Language:	Slovenian
Year of publishing:	2020
Typology:	2.08 - Doctoral Dissertation
Organization:	UL FFA - Faculty of Pharmacy
Publisher:	[M. Vidmar Golja]
UDC:	577.16:618.2:615(043.3)
COBISS:	47865859
Views:	6
Downloads:	0
Average score:	0 (0 votes)
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Other data

Secondary language:	English
Secondary title:	ǂThe ǂinfluence of folates on the molecular basis of fetal congenital malformations
Secondary abstract:	Inadequate intake of folate or its analogues, and the lack of other vitamins before and during pregnancy, are associated with an increased risk of birth defects. Congenital malformations and adverse pregnancy outcomes are a major public health problem as they have a lasting impact on the health and quality of life of the affected children and their parents. Disorders and changes in folate metabolic pathway, which are often the result of genetic polymorphisms, are the most common potential risk factor for the most common congenital malformations, such as congenital heart defects (CHD), orofacial clefts (OFC) and neural tube defects (NTD). The overall goal of the research presented in this thesis was to answer to key topics or known problems, namely the complex analytics of folates in biological samples and the influence of genotypes on folate status, thus contributing to the understanding of the influence of folates on the molecular basis of fetal congenital malformations. To address the importance of folate, its levels need to be monitored, although poor stability and extremely small amounts of folate in biological samples are factors that make the analysis of folate challenging. In the first part of the doctoral thesis, we developed and validated a method that subsequently allowed us to quantify folic acid (FA), the biologically active form of folate, 5-methyltetrahydrofolate (5-Me-THF) and homocysteine (Hcy). We developed a simple, sensitive and fast high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method for the simultaneous quantification of intracellular concentrations of FA, 5-Me-THF and Hcy in lysates of human lymphoblastoid cell line cells (LCLs). Next, we performed in vitro studies on LCL cells and studies of families with a child with congenital malformation and an unaffected child to determine the effect of genotype on folate status. Due to the extremely important role of folates, FA and its derivatives are the most commonly used dietary supplements. Slovenian obstetricians and gynecologists have regularly prescribed folate supplements to all pregnant women during the first trimester of pregnancy for more than 20 years, with the goal of reducing the risk of NTD. Higher doses of periconceptional FA supplements are prescribed in women who have had a previous pregnancy affected by a NTD, while the usefulness of FA supplements in preventing the most common congenital malformations, OFC and CHD, is still under-researched for clinical practice. Three forms of folate are vi commercially available in Slovenian market: FA and 5-Me-THF in the form of a calcium or glucosamine salt. A commonly accepted fact, which is also promoted by manufacturers of 5-Me-THF formulations is that 5-Me-THF is more effective compared to FA, but this claim is not mechanistically and clinically proven nor is it known how genetic changes in the folate and methionine cycle modulate the efficacy of these folate forms. A lymphoblastoid cell line (LCL) model was used to study the influence of different folate forms on the folate status with the respect to genetic variations in folate and methionine cycle. LCL cells were genotyped for genes involved in the folate cycle using hybridization (LightSNiP) and hydrolysis (TaqMan) probes % FPGS (rs1544105), DHFR (rs1677693), MTHFR (rs1801133 in rs1801131), MTRR (rs1801394), MTHFD1 (rs2236225), BHMT (rs3733890), GNMT (rs10948059), DNMT3B (rs2424913) and SLC19A1 (rs1051266). Cells were one week cultured in the medium without FA and we demonstrated that folate deficiency causes cell arrest in the S phase of cell cycle. After folate supplementation, the proportion of cells in the S phase of cell cycle decreases, and the addition results in increased metabolic activity of LCL cells. Higher metabolic activity was observed in LCLs treated with higher versus lower concentrations of folates and in cells treated with biologically active folate (5-Me-THF) versus FA. We also showed that polymorphisms in the MTHFR gene, rs1801133 and rs1801131, influence the metabolic activity of cells treated with either FA or 5-Me-THF. With the previously developed method, we quantified the intracellular concentrations of folate metabolites and demonstrated that both FA and 5-Me-THF increase the intracellular concentration of biologically active folate, although 5-Me-THF to a much larger extent. Considering the clinically relevant MTHFR polymorphisms, C677T and A1298C, we have shown that polymorphisms influencing the enzyme activity of MTHFR are associated with intracellular concentrations of the methylated forms of folates. In the previous studies from our research group, we compared common variants in folate cycle genes and environmental risk factors among children with OFC, CHD, and their mothers. In this doctoral thesis, we continued the study and upgraded it by analyzing healthy siblings of children with OFC and CHD. We confirmed the finding that fever in pregnancy increase a risk of OFC and proved that besides already confirmed polymorphism rs1801133 in the MTHFR gene, also polymorphism rs1801131 in the MTHFR gene increased the risk of OFC in Slovenian population. The incidence of CHD is influenced by the maternal age at conception and child gender, vii which is consistent with previous studies. However, we detected a new genetic risk factor that contributes to the risk of developing CHD in Slovenian population; rs1544105 in FPGS gene. Several medications, especially agents that impair the action of folate, disturb folate status or folate cycle through a variety of mechanisms. Nowadays we use more prescription medicines than a decade ago, and although there is no exact data, it is estimated that more than fifty percents of pregnant women take at least one medicine. Moreover, since approximately half of the pregnancies are unplanned, many women exposed their unborn child to medicines before they know they are pregnant. In the final part of the doctoral dissertation, we evaluated the influence of anti-folate effects of phenytoin, metformin and trimethoprim, and folate supplements. We showed that anti-folate agents have different effects on cell survival and cell cycle of LCL cells. Also, the addition of folate supplements to cells cultured with anti-folates, have different effects % in some cases folate supplements improved the cell viability, altered the cell distribution at different phases of cell cycle, and altered intracellular levels of folate metabolites, while in some cases, they have no effects. The results of this doctoral thesis provide important findings on MTHFR and folate supplements, and their influence on folate status. By developing a new sensitive and specific method we have contrubuted to the correct diagnosis of folate status in in vito system of LCLs. With additional validations, the method could be transferred to clinical laboratories, thus contributing to better monitoring of pregnant women, as well as children and patients on anti-folate therapy.
Secondary keywords:	Vitamini;Nosečnost;Zdravila;
Type (COBISS):	Dissertation
Thesis comment:	Univ. v Ljubljani, Fak. za farmacijo
Pages:	X, 247 str.
ID:	15504143