Omjer S-adenozilmetionina i S-adenozilhomocisteina i polimorfizmi gena za S-adenozilhomocistein hidrolazu u novorođenčadi s prirođenim srčanim greškama i njihovih majki [Concentration of S-adenosylmethionine, S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase gene polymorphisms in newborns with congenital heart diseases and their mothers]

The congenital heart disease is the consequence of complex interaction of more or less known embryological and genetic factors, the environmental factor in the periconceptional period and the mother's lifestyle habits. One of the possible mechanisms is derangement in methylation pathway, both in mothers and in children. This thesis supports the fact that the low aviability of folic acid or vitamin B12 in the periconceptional period and consequent mother’s hyperhomocysteinemia are associated with the risk of having a child with congenital heart disease. The question is whether homocysteine itself is a risk factor or changes in methylation biomarkers S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) and methylation potential are responsible for a higher incidence of congenital heart disease. The first hypothesis of this dissertation was that increased plasma concentrations of AdoMet is associated with an increased risk of congenital heart disease, another hypothesis that increased plasma concentrations of AdoHcy is associated with an increased risk of congenital heart disease. The third hypothesis was that newborns with congenital heart disease and their mothers would have decreased methylation ratio compared to referral group. The fourth hypothesis was that newborns with congenital heart disease and their mothers would have a different prevalence of the AHCY rs13043752 and rs41301825 polymorphisms, compared to healthy subjects. In order to reach goals of this study, we included 127 newborns with congenital heart disease and 103 mothers of newborns with congenital heart disease and measured AdoMet and AdoHcy in their plasma by high performance liquid chromatography tandem mass spectrometry and then calculate their methylation potential. The prevalences of polymorphisms rs13043752 and rs41301825 of the AHCY gene were studied in examined group by sequencing AHCY gene exons 2, 3 and 4. In the group of newborns with congenital heart disease we have found statistically significant increased AdoMet compared to the referral group. There was no statistically significant difference in the concentration of AdoHcy. Methylation potential in this group was consequently statistically significantly higher. In the group of mothers of newborn with congenital heart disease we have found statistically significant increased of AdoHcy compared to the referral group, but no statistically significant difference in the concentration of AdoMet. Methylation potential was statistically significantly lower, according to the hypothesis. Increased concentrations of AdoMet or AdoHcy measured in our groups of examinees and statistically significant differences in the methylation potential values in our groups of examinees compared to the reference values point to changed methylation processes which can contribute to the pathogenesis of congenital heart disease. It is explained in the dissertation how changes in methylation potential and the increased concentration of AdoMet and AdoHcy may affect the methylation processes within the cell and contribute to increased risk of congenital heart disease. There was no statistically significant difference in the prevalence of polymorphisms rs13043752 and rs41301825 of the AHCY genes in the study groups compared to healthy subjects, suggesting that they are not associated with increased risk of congenital heart disease. The results of this study indicate that methylation disorders are involved in pathogenesis of congenital heart diseases. Further studies are needed to elucidate the exact mechanism by which disturbed methylation leads to changes in epigenetic programming of genes and other recipients of the methyl group and consequently to higher risk of congenital heart disease and changes in the programming of methylation processes in the fetus

Management of metabolic crisis in three-dayold newborn

Urea cycle disorders are a group of metabolic disorders, caused by deficiency of one of the enzymes in the urea cycle, presenting with hyperammonemia triggered by catabolism or protein overload. Newborns with severe mutations in the urea cycle, unless treated, rapidly develop cerebral edema, coma and death

Appendicitis within inguinal hernia – case report of a premature newborn with Amyand’s hernia

Upala crvuljka unutar preponske kile ekstremno je rijetka pojava u nedonoščadi s procijenjenom incidencijom od oko 0,08-0,13%. Dijagnoza ovog izuzetnog entiteta, nazvanog Amyandova kila prema autoru koji ga je prvi opisao, najčešće je slučajna i obično se postavlja tek operativnim zahvatom. U prikazanog muškog nedonoščeta rođenog u 28. tjednu gestacije znaci ukliještenja desnostrane preponske kile javili su se tridesetog dana života. Kirurški zahvat je bio i dijagnostička i terapijska metoda. Nalazom neperforiranog, gangrenozno promijenjenog crvuljka unutar preponske kile postavljena je dijagnoza Amyandove kile. Nakon operativnog zahvata i uz dugotrajno antibiotsko liječenje uslijedio je potpuni oporavak. Prikaz bolesnika je podsjetnik na to da unatoč rijetkoj pojavnosti na Amyandovu kilu treba pomisliti u diferencijalnoj dijagnozi ukliještene desnostrane preponske kile u nedonoščadi.Acute appendicitis within an inguinal hernia is an extremely rare condition among premature newborns with estimated incidence ranging from 0.08% to 0.13%. The diagnosis of this extraordinary entity, known as Amyand’s hernia according to the author who fi rst described it, is often accidental and usually established intraoperatively. We report a case of a boy born at 28 weeks of gestation, who presented with incarcerated right inguinal hernia on his thirty day of life. Surgical intervention was both a diagnostic and therapeutic procedure. Intraoperative diagnosis of Amyand’s hernia was established based on the fi nding of non-perforated, gangrenous appendicitis within the inguinal hernial sac. Appendectomy and hernia repair followed by long-term antibiotic treatment led to complete infant’s recovery. This case report reminds that regardless of its rarity, clinicians should be aware of Amyand’s hernia in the evaluation of incarcerated right side inguinal hernia in preterm newborns

MORTALITY OF NEWBORNS IN REPUBLIC OF CROATIA IN THE YEAR 2008

Rani neonatalni mortalitet (RNM) je sastavnica perinatalnog mortaliteta, zadnjih godina je manji od fetalnog mortaliteta. RNM je u 2007. godini u Hrvatskoj bio 69% za skupinu novorođenčadi porodne težine (PT) 500–749 g, 30% za novorođenčad PT 750–999 g, dok je 2008. godine RNM za djecu PT ≤999 g bio 42%. Po 100-gramskim razredima RNM je bio za djecu PT <500 g 100%, za djecu PT 500–599 g 72,2%, za djecu PT 600–699 g 77,8%, za djecu PT 700–799 g 46,4%, za djecu PT 800–899 g 20% i za djecu PT 900–999 g RNM je bio 16,2%. Za novoro|enčad PT 1000–1249 g RNM je bio 9,6% i za novoro|enčad PT 1250–1499 g 2,4%. Mortalitet do otpusta iz bolnice (MOB) je za skupine novorođenčadi iste porodne težine bio: za djecu <500 g 100%, za djecu PT 500–599 g 94,4%, za djecu PT 600–699 g 77,8%, za djecu PT 700–799 g 57,1%, za djecu PT 800–899 g 42,9%, za djecu 900–999 g 21,6%, za djecu PT 1000–1249 g 12% i za djecu PT 1250–1499 g 2,4%. Sve su te vrijednosti niže od odgovarajućih za prethodno razdoblje. U skupinama novorođenčadi veće porodne težine RNM i MOB bili su još niži. RNM sve novorođenčadi bio je 2,7‰, neonatalni mortalitet (NM) je bio 3,1‰ i MOB je bio 3,5‰, što je tako|er manje nego prethodnih godina. RNM novorođenčadi PT ≥1000 g bio je 1,3‰, NM 1,5‰ i MOB 1,7‰, manje nego za 2007. godinu. RNM je za svu novorođ enčad činio prosječno 77,1% smrtnosti (118/153), dok je ostalih 35 djece (22,9%) umrlo nakon prvog tjedna života. To poka zuje da RNM nije podcijenjen na račun visokog kasnijeg mortaliteta, i da pedijatrijska-neonatalna služba ne ostvaruje smanjenje RNM na račun kasnijeg povišenja neonatalnog mortaliteta ili mortaliteta do otpusta iz bolnice. U razdoblju 2003.–2006. godine je tri četvrtine novorođenčadi PT 500–1499 g ro|eno u rodilištima III. razine. U 2007. godini su četiri petine ove djece rođene u rodilištima III. razine. U 2008. godini je 18,5% djece PT ≤1499 g rođeno u rodilištima izvan III. razine. Trend rađanja ove djece usmjeren je prema rodilištima III. razine, ali još ima mjesta povećanju broja poroda ove djece u rodilištima III. razine. Usporedbe RNM i NM s europskim zemljama ukazuju na postojanje daljnjih mogućnosti poboljšanja perinatalnih pokazatelja. Za vjerodostojnu analizu podataka ishoda novorođenčadi i djece planirana je izrada novih obrazaca perinatalnih zbivanja, uz prikupljanje podataka o postnatalnom transportu novorođenčadi i mjestu liječenja novorođenčeta. Potrebno je nastaviti prikupljati detaljne podatke o vitalnim događajima do otpusta iz bolnice. Ti podatci predstavljat će osnovu za planiranje potreba neonatološke službe, izradu smjernica za prenatalno i postnatalno usmjeravanje novorođenčadi i za davanje vjerodostojnijih prognoza roditeljima novorođenčadi najnižih porodnih težina.Early neonatal mortality (ENM) is one of components of perinatal mortality. In recent years ENM is smaller than fetal mortality. ENM was in 2008 in Croatia 69% for newborns of birth-weight (BW) 500–749 g; 30% for those 750–999 g, 16% for those 1000–1249 g, and 6% for newborns of BW 1250–1499 g. In newborns divided by 100-grams, ENM for infants BW <500 g was 100%, for those of BW 500–599 g was 72,2%, for infants BW PT 600–699 g 77,8%, for infants of BW 700–799 g 46,4%, for infants of BW 800–899 g 20% and for infants of BW 900–999 g ENM was 16,2%, respectively. For infants of BW 1000–1249 g ENM was 9,6% and for infants of BW 1250–1499 g was 2,4%, respectively. Mortality to discharge from hospital (MDH) for subgroups of infants of the same BW was 100% for infants of BW <500 g, for infants of BW 500–599 g 94,4%, for infants of BW 600–699 g 77,8%, for infants of BW 700–799 g 57,1%, for infants of BW 800–899 g 42,9%, for infants of BW 900–999 g 21,6%, for infants of BW 1000–1249 g 12%, and for infants of BW 1250–1499 g was 2,4%, respectively. All these values were lower than corresponding in previous period. In groups of infants of larger BW, ENM and MDH were lower. ENM of all newborns was 2,7‰, neonatal mortal-1,3‰, NM was 1,5‰, and MDH was 1,7‰, respectively, lower than in the year 2007. ENM was 77,1% (118/153) of all infants deaths, while the remained 35 infants (22,9%) died after the first week. Therefore, ENM was not underestimated instead of possible higher late neonatal mortality, pediatric-neonatal services didn’t reduce ENM on expenses of higher late neonatal mortality or MDH. In the years 2003–2006, three fourths of newborns of BW 500–1499 g were born in maternities of IIIrd level. In the year 2007 four fifths of these newborns were born in maternities of IIIrd level. In the year 2008 18,5% of infants of BW ≤1499 g were born in maternities outside of IIIrd level. Through the years the proportion of these infants born in maternities of IIIrd level is increasing, but that proportion can be even larger. Comparisons of ENM and NM with some European countries show the possibility of further improvement in perinatal markers. In the aim of the proper analysis of newborns’ outcome data, creation of new certificates of vital events is planned with details of postnatal transport and place of the treatment of newborn. It is necessary to continue to follow survival or mortality of all newborns up to discharge from hospital. These data will give us benchmark for planning of neonatal resources, development of recommendations in perinatology-neonatology for prenatal and postnatal transfer, and for more exact prognoses to parents of the smallest newborns

Report on the work of the Reference center for pediatric cardiology Ministry of Health of the Republic of Croatia

Cilj je ovog izvješća u proteklom trogodišnjem razdoblju (2019–2022) prikazati: 1) aktivnosti Referentnog centra, 2) postignute stručne rezultate i primjenu novih metoda, postupaka i unaprjeđenje struke, i 3) znanstvenu i stručnu suradnju s inozemnim ustanovama visoke razine. Rezultati: Referentni je centar jedino mjesto u Republici Hrvatskoj koje kontinuirano zbrinjava populaciju najugroženijih i najtežih bolesnika pedijatrijske dobi sa srčanom patologijom. Ima kontinuirano, 24 sata dostupnu kardiološku, kardiokiruršku, anesteziološku, neonatalnu i intenzivnu skrb za djecu sa složenim prirođenim i stečenim srčanim bolestima. Također su dostupne metode nadomještanja funkcije organa u zatajivanju (ECMO potpora, LVAD-BiVAD, Berlin-Heart pumpa, hemodijaliza, program transplantacije srca / drugih organa), a sve zahvaljujući timskom radu i suradnji tima nekoliko Zavoda. Izvode se složene kardiokirurške operacije u djece s prirođenim srčanim greškama uz jasan trend povećanja broja i složenosti operacija, te uz i dalje prihvatljivo nisku smrtnost. Danas smo u mogućnosti samostalno liječiti gotovo sve srčane bolesti u djece. Godišnje se izvede oko 200 kateterizacija srca u djece. Više od 40% čine intervencijske procedure, a više od 50% tih intervencija izvodi se u dojenačkom periodu. Tijekom protekle tri godine uvedeno je pet novih perkutanih intervencijskih metoda: liječenje nativne koarktacije i rekoarktacije umetanjem stenta, liječenje stenoze pulmonalnih grana umetanjem stenta, perkutano umetanje valvule na pulmonalnu poziciju, dilatacija postojećeg stenta te zatvaranje aortopulmonalnih kolaterala u djece s univentrikulskim srcem. Navedeni iskoraci učinjeni su kontinuiranim zalaganjem članova tima uz potporu i mentorstvo, odnosno kontinuiranu suradnju s inozemnim stručnjacima iz triju inozemnih ustanova (DeutschesHerzZentrum Muenchen, KinderherzZentrum Linz, Kids Heart Center Budapest). Zaključak: Naš centar stoji uz bok rijetkih centara u Europi koji su u mogućnosti izvesti navedene procedure. Navedene su aktivnosti rezultirale unaprjeđenjem kvalitete skrbi na razini RH i temelj su za daljnji planirani rast i razvoj struke u okvirima naše zemlje.The aim of this report is to show in the past three-year period (2019–2022): 1)activities of the Reference Center, 2)achieved professional results and the application of new methods, procedures, and improvement of the profession, and 3)scientific and professional cooperation with high-level foreign institutions. Results: The reference center is the only place in the Republic of Croatia that continuously cares for the population of the most vulnerable children with cardiac pathology. It has continuous, 24-hour cardiology, cardiac surgery, anesthesiology, neonatal and intensive care for children with complex congenital and acquired heart diseases. Organ function replacement are also available (ECMO support, LVAD-BiVAD, Berlin-Heart pump, hemodialysis, heart/other organ transplant program) thanks to the teamwork and cooperation of different Departments. Complex cardiac surgeries are performed in children with a clear trend of increasing the number and complexity of surgeries, with low mortality. Today, we can independently treat almost all congenital heart defects in children. About 200 cardiac catheterizations are performed in children annually. More than 40% are interventional procedures with more than 50% of these interventions performed in infancy. In the past three years, five new percutaneous intervention methods have been introduced: stent insertion in native coarctation and in recoarctation, stent insertion in stenosis of the pulmonary branches, percutaneous valve insertion in the pulmonary position, dilatation of the existing stent, and closure of aortopulmonary collaterals in children with a univentricular heart. The steps were made by the continuous efforts of team members with support, mentoring, and continuous cooperation with foreign experts from three foreign institutions (DeutschesHerzZentrum Muenchen, KinderherzZentrum Linz, KidsHeart- Center Budapest). Conclusion: Our center stands alongside the rare centers in Europe that can perform the abovementioned procedures. The activities resulted in the improvement of the quality of care and form the basis for further development of the profession within the framework of our country

MORTALITY OF NEWBORNS IN CROATIA IN 2005

Rani neonatalni mortalitet (RNM) je sastavnica perinatalnog mortaliteta, i zadnjih godina je manji od fetalnog mortaliteta. RNM je u 2005. g. u Hrvatskoj bio 71% za skupinu novorođenčadi porodne težine (PT) 500–749 g, 32% za novorođenčad PT 750–999 g, 16% za novorođenčad PT 1000–1249 g, i 9% za novorođenčad PT 1250–1499 g. Mortalitet do otpusta iz bolnice (MOB) je za skupine novorođenčadi iste porodne težine bio 84%, potom 48%, zatim 20% i 10%. U skupinama novorođenčadi veće porodne težine RNM i MOB bili su još niži. RNM sve novorođenčadi >500 g bio je 3,4‰, neonatalni mortalitet (NM) je bio 4,1‰ i MOB je bio 4,4‰. RNM novorođenčadi PT >1000 g bio je 2,2‰, NM je bio 2,6‰ i MOB je bio 2,9‰. RNM je za svu novorođenčad PT >500 g činio prosječno 76,7% smrtnosti (145/189), dok je ostalih 44 djece (23,3%) umrlo nakon prvog tjedna života. To ukazuje da RNM nije podcijenjen na račun visokog kasnijeg moraliteta, i da pedijatrijska-neonatalna služba ne ostvaruje smanjenje RNM na račun kasnijeg povišenja ¬mortaliteta ili MOB. U 2005. godini je kao i u 2003. i 2004. tri četvrtine novorođenčadi PT 500–1499 g rođeno u rodilištima III. razine. Za vjerodostojnu analizu podataka ishoda novorođenčadi i djece planirana je izrada novih obrazaca perinatalnih zbivanja, uz prikupljanje podataka o postnatalnom transportu novorođenčadi i mjestu liječenja novorođenčeta. Potrebno je nastaviti prikupljati detaljne podatke o vitalnim događajima do otpusta iz bolnice. Ti podatci predstavljat će osnovu za planiranje potreba neonatološke službe, izradu smjernica za prenatalno i postnatalno usmjeravanje novorođenčadi i za davanje vjerodostojnijih prognoza roditeljima novorođenčadi najnižih porodnih težina.Early neonatal mortality (ENM) is one of components of perinatal mortality. In recent years ENM is smaller than fetal mortality. ENM was in 2004 in Croatia 71% for newborns of birth-weight (BW) 500–749 g; 32% for those 750–999 g, 16% for those 1000–1249 g, and 9% for newborns of BW 1250–1499 g. Mortality to discharge from hospital (MDH) for newborns in these birth-weight groups was 84%, 48%, 20% and 10%, respectively. In groups of newborns with larger BW over 1500 g ENM and MDH were even lower. ENM for all newborns BW >500 g was 3,4‰, neonatal mortality (NM) was 4,1‰, and MDH was 4,4‰, respectively. ENM for newborns of BW > 1000 g was 2,2‰, NM was 2,6‰, and MDH was 2,9‰, respectively. ENM made 76,7% mortality of all newborns (BW >500 g) (145/189), while the rest of 44 newborns (23,3%) died after the first week of life. Therefore, ENM was not underestimated due to possible higher late neonatal mortality, pediatric-neonatal services didn\u27t reduce ENM on expenses of higher late neonatal mortality or MDH. In the year 2005, as in 2003 and 2004, three fourths of newborns of BW 500–1499 grams were born in maternities of 3rd level. Within the aims of the proper analysis of newborns’ outcome data, is creation of new certificates of vital events with details of postnatal transport and place of treatment of newborn. It is necessary to continue to follow survival or mortality of all newborns to discharge from the hospital. These data will give us benchmark for planning of neonatal resources, development of recommendations in perinatology-neonatology for prenatal and postnatal transfer, and for more exact prognoses of the smallest newborns in the process of decision making

Concentration of S-adenosylmethionine, S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase gene polymorphisms in newborns with congenital heart diseases and their mothers

Niti jedna prirođena srčana greška, neovisno o složenosti, nije poseban entitet, već je rezultat složene interakcije više ili manje poznatih embrioloških i genskih čimbenika, čimbenika okoline u perikoncepcijskom razdoblju te majčinih životnih navika. Jedan od mogućih mehanizama nastanka jest poremećaj u metilaciji kako u majke, tako i u potomaka. U prilog toj tezi govori činjenica da su manjak folne kiseline i niže koncentracije vitamina B12 u plazmi majke u perikoncepcijskom razdoblju te posljedična majčina hiperhomocisteinemija čimbenici rizika za pojavu prirođene srčane greške u potomaka. Postavlja se pitanje je li homocistein sam po sebi čimbenik rizika ili su promjene koncentracije njegovih prethodnika u ciklusu metionina AdoMet-a i AdoHcy-a te promjene njihovog međusobnog omjera (metilacijskog potencijala) odgovorni za veću pojavnost prirođenih srčanih grešaka. U ovoj je disertaciji prva hipoteza bila da će povišena koncentracija AdoMet-a u plazmi biti čimbenik rizika za pojavu prirođene srčane greške, druga hipoteza da će povišena koncentracija AdoHcy-a u plazmi biti čimbenik rizika za pojavu prirođene srčane greške. Treća hipoteza je bila da će novorođenčad s prirođenom srčanom greškom i njihove majke imati snižen metilacijski potencijal u odnosu na referentnu skupinu. Četvrta hipoteza je bila da će novorođenčad s prirođenom srčanom greškom i njihove majke imati različitu učestalost polimorfizama rs13043752 i rs41301825 gena AHCY u odnosu na zdrave ispitanike. Da bismo provjerili hipoteze i ispunili ciljeve istraživanja testirali smo 127 novorođenčadi s prirođenom srčanom greškom i 103 majke novorođenčadi s prirođenom srčanom greškom i u njih u plazmi metodom tekućinske kromatografije visoke djelotvornosti i tandemske spektometrije masa izmjerili AdoMet i AdoHcy te potom izračunali metilacijski potencijal. Učestalost polimorfizama rs13043752 i rs41301825 gena AHCY ispitali smo sekvenciranje egzona 2, 3 i 4 gena AHCY u 116 novorođenčadi s prirođenom srčanom greškom i u 94 majki novorođenčadi s prirođenom srčanom greškom. U skupini ispitanika novorođenčadi s prirođenom srčanom greškom dokazali smo statistički značajno više koncentracije AdoMet-a u odnosu na referentnu skupinu. Nije bilo statistički značajne razlike u koncentraciji AdoHcy-a. Metilacijski potencijal u ovoj skupini bio je posljedično statistički značajno viši. U skupini ispitanica majki novorođenčadi s prirođenom srčanom greškom dokazali smo statistički značajno povišene koncentracije AdoHcy-a u odnosu na referentnu skupinu, a nije bilo statistički značajne razlike u koncentraciji AdoMet-a. Metilacijski potencijal je u skladu s hipotezom bio statistički značajno niži. Povišene koncentracije AdoMet-a odnosno AdoHcy-a koje su izmjerene u ispitivanim skupinama te statistički značajne razlike u vrijednostima metilacijskog potencijala u ispitivanim skupinama u odnosu na referentne vrijednosti upućuju na narušene procese metilacije što može pridonijeti patogenezi prirođenih srčanih grešaka. U disertaciji je obrazloženo na koji način promijenjen metilacijski potencijal te povišene koncentracije AdoMet-a i AdoHcy-a mogu utjecati na metilacijske procese unutar stanice i pridonositi povećanom riziku pojave srčanih grešaka. Nije nađena statistički značajna razlika u prevalenciji polimorfizama rs13043752 i rs41301825 gena AHCY u ispitivanim skupinama u odnosu na zdrave ispitanike, što sugerira da nisu udruženi s povećanim rizikom za pojavu prirođenih srčanih grešaka. Rezultati ovog istraživanja upućuju da poremećaji metilacije sudjeluju u patogenezi prirođenih srčanih grešaka. Daljnja istraživanja su potrebna da se utvrde točni patološki mehanizmi te stupanj poremećaja metilacije koji dovode do promjene u epigenetskom programiranju gena i drugih primatelja metilne skupine i posljedično do pojave prirođene srčane greške i promjene u programiranju metilacijskih procesa u fetusa.The congenital heart disease is the consequence of complex interaction of more or less known embryological and genetic factors, the environmental factor in the periconceptional period and the mother's lifestyle habits. One of the possible mechanisms is derangement in methylation pathway, both in mothers and in children. This thesis supports the fact that the low aviability of folic acid or vitamin B12 in the periconceptional period and consequent mother’s hyperhomocysteinemia are associated with the risk of having a child with congenital heart disease. The question is whether homocysteine itself is a risk factor or changes in methylation biomarkers S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) and methylation potential are responsible for a higher incidence of congenital heart disease. The first hypothesis of this dissertation was that increased plasma concentrations of AdoMet is associated with an increased risk of congenital heart disease, another hypothesis that increased plasma concentrations of AdoHcy is associated with an increased risk of congenital heart disease. The third hypothesis was that newborns with congenital heart disease and their mothers would have decreased methylation ratio compared to referral group. The fourth hypothesis was that newborns with congenital heart disease and their mothers would have a different prevalence of the AHCY rs13043752 and rs41301825 polymorphisms, compared to healthy subjects. In order to reach goals of this study, we included 127 newborns with congenital heart disease and 103 mothers of newborns with congenital heart disease and measured AdoMet and AdoHcy in their plasma by high performance liquid chromatography tandem mass spectrometry and then calculate their methylation potential. The prevalences of polymorphisms rs13043752 and rs41301825 of the AHCY gene were studied in examined group by sequencing AHCY gene exons 2, 3 and 4. In the group of newborns with congenital heart disease we have found statistically significant increased AdoMet compared to the referral group. There was no statistically significant difference in the concentration of AdoHcy. Methylation potential in this group was consequently statistically significantly higher. In the group of mothers of newborn with congenital heart disease we have found statistically significant increased of AdoHcy compared to the referral group, but no statistically significant difference in the concentration of AdoMet. Methylation potential was statistically significantly lower, according to the hypothesis. Increased concentrations of AdoMet or AdoHcy measured in our groups of examinees and statistically significant differences in the methylation potential values in our groups of examinees compared to the reference values point to changed methylation processes which can contribute to the pathogenesis of congenital heart disease. It is explained in the dissertation how changes in methylation potential and the increased concentration of AdoMet and AdoHcy may affect the methylation processes within the cell and contribute to increased risk of congenital heart disease. There was no statistically significant difference in the prevalence of polymorphisms rs13043752 and rs41301825 of the AHCY genes in the study groups compared to healthy subjects, suggesting that they are not associated with increased risk of congenital heart disease. The results of this study indicate that methylation disorders are involved in pathogenesis of congenital heart diseases. Further studies are needed to elucidate the exact mechanism by which disturbed methylation leads to changes in epigenetic programming of genes and other recipients of the methyl group and consequently to higher risk of congenital heart disease and changes in the programming of methylation processes in the fetus

Concentration of S-adenosylmethionine, S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase gene polymorphisms in newborns with congenital heart diseases and their mothers

Niti jedna prirođena srčana greška, neovisno o složenosti, nije poseban entitet, već je rezultat složene interakcije više ili manje poznatih embrioloških i genskih čimbenika, čimbenika okoline u perikoncepcijskom razdoblju te majčinih životnih navika. Jedan od mogućih mehanizama nastanka jest poremećaj u metilaciji kako u majke, tako i u potomaka. U prilog toj tezi govori činjenica da su manjak folne kiseline i niže koncentracije vitamina B12 u plazmi majke u perikoncepcijskom razdoblju te posljedična majčina hiperhomocisteinemija čimbenici rizika za pojavu prirođene srčane greške u potomaka. Postavlja se pitanje je li homocistein sam po sebi čimbenik rizika ili su promjene koncentracije njegovih prethodnika u ciklusu metionina AdoMet-a i AdoHcy-a te promjene njihovog međusobnog omjera (metilacijskog potencijala) odgovorni za veću pojavnost prirođenih srčanih grešaka. U ovoj je disertaciji prva hipoteza bila da će povišena koncentracija AdoMet-a u plazmi biti čimbenik rizika za pojavu prirođene srčane greške, druga hipoteza da će povišena koncentracija AdoHcy-a u plazmi biti čimbenik rizika za pojavu prirođene srčane greške. Treća hipoteza je bila da će novorođenčad s prirođenom srčanom greškom i njihove majke imati snižen metilacijski potencijal u odnosu na referentnu skupinu. Četvrta hipoteza je bila da će novorođenčad s prirođenom srčanom greškom i njihove majke imati različitu učestalost polimorfizama rs13043752 i rs41301825 gena AHCY u odnosu na zdrave ispitanike. Da bismo provjerili hipoteze i ispunili ciljeve istraživanja testirali smo 127 novorođenčadi s prirođenom srčanom greškom i 103 majke novorođenčadi s prirođenom srčanom greškom i u njih u plazmi metodom tekućinske kromatografije visoke djelotvornosti i tandemske spektometrije masa izmjerili AdoMet i AdoHcy te potom izračunali metilacijski potencijal. Učestalost polimorfizama rs13043752 i rs41301825 gena AHCY ispitali smo sekvenciranje egzona 2, 3 i 4 gena AHCY u 116 novorođenčadi s prirođenom srčanom greškom i u 94 majki novorođenčadi s prirođenom srčanom greškom. U skupini ispitanika novorođenčadi s prirođenom srčanom greškom dokazali smo statistički značajno više koncentracije AdoMet-a u odnosu na referentnu skupinu. Nije bilo statistički značajne razlike u koncentraciji AdoHcy-a. Metilacijski potencijal u ovoj skupini bio je posljedično statistički značajno viši. U skupini ispitanica majki novorođenčadi s prirođenom srčanom greškom dokazali smo statistički značajno povišene koncentracije AdoHcy-a u odnosu na referentnu skupinu, a nije bilo statistički značajne razlike u koncentraciji AdoMet-a. Metilacijski potencijal je u skladu s hipotezom bio statistički značajno niži. Povišene koncentracije AdoMet-a odnosno AdoHcy-a koje su izmjerene u ispitivanim skupinama te statistički značajne razlike u vrijednostima metilacijskog potencijala u ispitivanim skupinama u odnosu na referentne vrijednosti upućuju na narušene procese metilacije što može pridonijeti patogenezi prirođenih srčanih grešaka. U disertaciji je obrazloženo na koji način promijenjen metilacijski potencijal te povišene koncentracije AdoMet-a i AdoHcy-a mogu utjecati na metilacijske procese unutar stanice i pridonositi povećanom riziku pojave srčanih grešaka. Nije nađena statistički značajna razlika u prevalenciji polimorfizama rs13043752 i rs41301825 gena AHCY u ispitivanim skupinama u odnosu na zdrave ispitanike, što sugerira da nisu udruženi s povećanim rizikom za pojavu prirođenih srčanih grešaka. Rezultati ovog istraživanja upućuju da poremećaji metilacije sudjeluju u patogenezi prirođenih srčanih grešaka. Daljnja istraživanja su potrebna da se utvrde točni patološki mehanizmi te stupanj poremećaja metilacije koji dovode do promjene u epigenetskom programiranju gena i drugih primatelja metilne skupine i posljedično do pojave prirođene srčane greške i promjene u programiranju metilacijskih procesa u fetusa.The congenital heart disease is the consequence of complex interaction of more or less known embryological and genetic factors, the environmental factor in the periconceptional period and the mother's lifestyle habits. One of the possible mechanisms is derangement in methylation pathway, both in mothers and in children. This thesis supports the fact that the low aviability of folic acid or vitamin B12 in the periconceptional period and consequent mother’s hyperhomocysteinemia are associated with the risk of having a child with congenital heart disease. The question is whether homocysteine itself is a risk factor or changes in methylation biomarkers S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) and methylation potential are responsible for a higher incidence of congenital heart disease. The first hypothesis of this dissertation was that increased plasma concentrations of AdoMet is associated with an increased risk of congenital heart disease, another hypothesis that increased plasma concentrations of AdoHcy is associated with an increased risk of congenital heart disease. The third hypothesis was that newborns with congenital heart disease and their mothers would have decreased methylation ratio compared to referral group. The fourth hypothesis was that newborns with congenital heart disease and their mothers would have a different prevalence of the AHCY rs13043752 and rs41301825 polymorphisms, compared to healthy subjects. In order to reach goals of this study, we included 127 newborns with congenital heart disease and 103 mothers of newborns with congenital heart disease and measured AdoMet and AdoHcy in their plasma by high performance liquid chromatography tandem mass spectrometry and then calculate their methylation potential. The prevalences of polymorphisms rs13043752 and rs41301825 of the AHCY gene were studied in examined group by sequencing AHCY gene exons 2, 3 and 4. In the group of newborns with congenital heart disease we have found statistically significant increased AdoMet compared to the referral group. There was no statistically significant difference in the concentration of AdoHcy. Methylation potential in this group was consequently statistically significantly higher. In the group of mothers of newborn with congenital heart disease we have found statistically significant increased of AdoHcy compared to the referral group, but no statistically significant difference in the concentration of AdoMet. Methylation potential was statistically significantly lower, according to the hypothesis. Increased concentrations of AdoMet or AdoHcy measured in our groups of examinees and statistically significant differences in the methylation potential values in our groups of examinees compared to the reference values point to changed methylation processes which can contribute to the pathogenesis of congenital heart disease. It is explained in the dissertation how changes in methylation potential and the increased concentration of AdoMet and AdoHcy may affect the methylation processes within the cell and contribute to increased risk of congenital heart disease. There was no statistically significant difference in the prevalence of polymorphisms rs13043752 and rs41301825 of the AHCY genes in the study groups compared to healthy subjects, suggesting that they are not associated with increased risk of congenital heart disease. The results of this study indicate that methylation disorders are involved in pathogenesis of congenital heart diseases. Further studies are needed to elucidate the exact mechanism by which disturbed methylation leads to changes in epigenetic programming of genes and other recipients of the methyl group and consequently to higher risk of congenital heart disease and changes in the programming of methylation processes in the fetus

Concentration of S-adenosylmethionine, S-adenosylhomocysteine and S-adenosylhomocysteine hydrolase gene polymorphisms in newborns with congenital heart diseases and their mothers

Niti jedna prirođena srčana greška, neovisno o složenosti, nije poseban entitet, već je rezultat složene interakcije više ili manje poznatih embrioloških i genskih čimbenika, čimbenika okoline u perikoncepcijskom razdoblju te majčinih životnih navika. Jedan od mogućih mehanizama nastanka jest poremećaj u metilaciji kako u majke, tako i u potomaka. U prilog toj tezi govori činjenica da su manjak folne kiseline i niže koncentracije vitamina B12 u plazmi majke u perikoncepcijskom razdoblju te posljedična majčina hiperhomocisteinemija čimbenici rizika za pojavu prirođene srčane greške u potomaka. Postavlja se pitanje je li homocistein sam po sebi čimbenik rizika ili su promjene koncentracije njegovih prethodnika u ciklusu metionina AdoMet-a i AdoHcy-a te promjene njihovog međusobnog omjera (metilacijskog potencijala) odgovorni za veću pojavnost prirođenih srčanih grešaka. U ovoj je disertaciji prva hipoteza bila da će povišena koncentracija AdoMet-a u plazmi biti čimbenik rizika za pojavu prirođene srčane greške, druga hipoteza da će povišena koncentracija AdoHcy-a u plazmi biti čimbenik rizika za pojavu prirođene srčane greške. Treća hipoteza je bila da će novorođenčad s prirođenom srčanom greškom i njihove majke imati snižen metilacijski potencijal u odnosu na referentnu skupinu. Četvrta hipoteza je bila da će novorođenčad s prirođenom srčanom greškom i njihove majke imati različitu učestalost polimorfizama rs13043752 i rs41301825 gena AHCY u odnosu na zdrave ispitanike. Da bismo provjerili hipoteze i ispunili ciljeve istraživanja testirali smo 127 novorođenčadi s prirođenom srčanom greškom i 103 majke novorođenčadi s prirođenom srčanom greškom i u njih u plazmi metodom tekućinske kromatografije visoke djelotvornosti i tandemske spektometrije masa izmjerili AdoMet i AdoHcy te potom izračunali metilacijski potencijal. Učestalost polimorfizama rs13043752 i rs41301825 gena AHCY ispitali smo sekvenciranje egzona 2, 3 i 4 gena AHCY u 116 novorođenčadi s prirođenom srčanom greškom i u 94 majki novorođenčadi s prirođenom srčanom greškom. U skupini ispitanika novorođenčadi s prirođenom srčanom greškom dokazali smo statistički značajno više koncentracije AdoMet-a u odnosu na referentnu skupinu. Nije bilo statistički značajne razlike u koncentraciji AdoHcy-a. Metilacijski potencijal u ovoj skupini bio je posljedično statistički značajno viši. U skupini ispitanica majki novorođenčadi s prirođenom srčanom greškom dokazali smo statistički značajno povišene koncentracije AdoHcy-a u odnosu na referentnu skupinu, a nije bilo statistički značajne razlike u koncentraciji AdoMet-a. Metilacijski potencijal je u skladu s hipotezom bio statistički značajno niži. Povišene koncentracije AdoMet-a odnosno AdoHcy-a koje su izmjerene u ispitivanim skupinama te statistički značajne razlike u vrijednostima metilacijskog potencijala u ispitivanim skupinama u odnosu na referentne vrijednosti upućuju na narušene procese metilacije što može pridonijeti patogenezi prirođenih srčanih grešaka. U disertaciji je obrazloženo na koji način promijenjen metilacijski potencijal te povišene koncentracije AdoMet-a i AdoHcy-a mogu utjecati na metilacijske procese unutar stanice i pridonositi povećanom riziku pojave srčanih grešaka. Nije nađena statistički značajna razlika u prevalenciji polimorfizama rs13043752 i rs41301825 gena AHCY u ispitivanim skupinama u odnosu na zdrave ispitanike, što sugerira da nisu udruženi s povećanim rizikom za pojavu prirođenih srčanih grešaka. Rezultati ovog istraživanja upućuju da poremećaji metilacije sudjeluju u patogenezi prirođenih srčanih grešaka. Daljnja istraživanja su potrebna da se utvrde točni patološki mehanizmi te stupanj poremećaja metilacije koji dovode do promjene u epigenetskom programiranju gena i drugih primatelja metilne skupine i posljedično do pojave prirođene srčane greške i promjene u programiranju metilacijskih procesa u fetusa.The congenital heart disease is the consequence of complex interaction of more or less known embryological and genetic factors, the environmental factor in the periconceptional period and the mother's lifestyle habits. One of the possible mechanisms is derangement in methylation pathway, both in mothers and in children. This thesis supports the fact that the low aviability of folic acid or vitamin B12 in the periconceptional period and consequent mother’s hyperhomocysteinemia are associated with the risk of having a child with congenital heart disease. The question is whether homocysteine itself is a risk factor or changes in methylation biomarkers S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) and methylation potential are responsible for a higher incidence of congenital heart disease. The first hypothesis of this dissertation was that increased plasma concentrations of AdoMet is associated with an increased risk of congenital heart disease, another hypothesis that increased plasma concentrations of AdoHcy is associated with an increased risk of congenital heart disease. The third hypothesis was that newborns with congenital heart disease and their mothers would have decreased methylation ratio compared to referral group. The fourth hypothesis was that newborns with congenital heart disease and their mothers would have a different prevalence of the AHCY rs13043752 and rs41301825 polymorphisms, compared to healthy subjects. In order to reach goals of this study, we included 127 newborns with congenital heart disease and 103 mothers of newborns with congenital heart disease and measured AdoMet and AdoHcy in their plasma by high performance liquid chromatography tandem mass spectrometry and then calculate their methylation potential. The prevalences of polymorphisms rs13043752 and rs41301825 of the AHCY gene were studied in examined group by sequencing AHCY gene exons 2, 3 and 4. In the group of newborns with congenital heart disease we have found statistically significant increased AdoMet compared to the referral group. There was no statistically significant difference in the concentration of AdoHcy. Methylation potential in this group was consequently statistically significantly higher. In the group of mothers of newborn with congenital heart disease we have found statistically significant increased of AdoHcy compared to the referral group, but no statistically significant difference in the concentration of AdoMet. Methylation potential was statistically significantly lower, according to the hypothesis. Increased concentrations of AdoMet or AdoHcy measured in our groups of examinees and statistically significant differences in the methylation potential values in our groups of examinees compared to the reference values point to changed methylation processes which can contribute to the pathogenesis of congenital heart disease. It is explained in the dissertation how changes in methylation potential and the increased concentration of AdoMet and AdoHcy may affect the methylation processes within the cell and contribute to increased risk of congenital heart disease. There was no statistically significant difference in the prevalence of polymorphisms rs13043752 and rs41301825 of the AHCY genes in the study groups compared to healthy subjects, suggesting that they are not associated with increased risk of congenital heart disease. The results of this study indicate that methylation disorders are involved in pathogenesis of congenital heart diseases. Further studies are needed to elucidate the exact mechanism by which disturbed methylation leads to changes in epigenetic programming of genes and other recipients of the methyl group and consequently to higher risk of congenital heart disease and changes in the programming of methylation processes in the fetus