Odborna praâce ORTODONCIE

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Sekvenace cÏaÂsti genu pro PAX9 a mozÏna spojitost

nalezenyÂch polymorfizmuÊ s agenezõÂ zubuÊ

Sequencing of the region of the PAX9 gene and possible

connection between discovered polymorphisms

and tooth agenesis

*Mgr. Alena HlousÏkovaÂ, *, **Mgr. Ing. OndrÏej Bonczek, *, **doc. RNDr. Omar SÏeryÂ, Ph.D.,

*Mgr. Jan Lochman, Ph.D., ***prof. MUDr. JirÏõÂ VaneÏk, CSc., ***doc. MUDr. PavlõÂna CÏernochovaÂ, Ph.D., **,***MUDr. Jan SÏtembõÂrek, Ph.D., ****MUDr. PrÏemysl KrejcÏõÂ, Ph.D., **prof. MVDr. Ivan MõÂsÏek, CSc.,

*LaboratorÏ DNA diagnostiky, UÂstav biochemie, PrÏõÂrodoveÏdecka fakulta, Masarykova univerzita, Brno *Laboratory of DNA diagnostic, Department of Biochemistry, Faculty of Science, Masaryk University, Brno **LaboratorÏ embryologie zÏivocÏichuÊ, UÂstav zÏivocÏisÏne fyziologie agenetiky, AV CÏR v.v.i., Brno

**Laboratory of Animal Embryology, Institute of Animal Physiology and Genetics, v.v.i., AS CR Brno ***Stomatologicka klinika, LeÂka rÏska fakulta, Masarykova univerzita, Brno

***Department of Stomatology, Faculty of Medicine, Masaryk University, Brno ****KlinikazubnõÂho leÂka rÏstvõÂ, LeÂka rÏska fakulta, Univerzita PalackeÂho, Olomouc ****Institute of Dental Medicine, Medical Faculty, Palacky University Olomouc

Souhrn

Agenezõ zubuÊ rozumõÂme vrozeny stav, kdy v dentici chybõ jeden nebo võÂce zubuÊ. Tato porucha muÊzÏe mõÂt celou rÏadu prÏõÂcÏin - od puÊsobenõ lokaÂlnõÂch zmeÏn v obdobõ zaklaÂdaÂnõ zubnõÂch zaÂrodkuÊ azÏ po vliv prÏedevsÏõÂm genetickyÂch faktoruÊ. Polymorfizmus v genu, ktery koÂduje protein zapojeny do odontogeneze, muÊzÏe mõÂt za naÂsledek poruchu jeho funkce, cozÏ muÊzÏe vyÂvoj zubu negativneÏ ovlivnit a v konecÏneÂm duÊsledku uÂplneÏ zastavit. Mezi nejvõÂce studo-vane geny spojene se vznikem zubnõÂch agenezõ patrÏõ geny pro PAX9, MSX1, AXIN2, WNT10a a EDA.

CõÂlem teÂto studie bylo najõÂt mozÏny vztah mezi polymorfizmy v genu pro PAX9 a agenezõ zubuÊ na vzorku cÏeske populace. AnalyÂza spocÏõÂvala v DNA sekvenaci vybranyÂch oblastõ genu pro PAX9 a v naÂsledneÂm porovnaÂnõ vyÂsled-nyÂch sekvencõ s referencÏnõ sekvencõ z internetove databaÂze GenBank (NCBI).

Z vyÂsledkuÊ naÂmi provedene studie na cÏeske populaci se jevõ jako nejvyÂznamneÏjsÏõ inzerce 99-101insC

(rs138135767, rs11373281) se soucÏasnou zaÂmeÏnou 272C>G (rs4904155; heterozygotnõÂ i homozygotnõÂ) v exonu

1 a varianty -54A>G (rs12882923), -41A>G (rs12883049) a 605C>T (Gly203Gly, rs61754301) v exonu 3, ktereÂ

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Abstract

Under the term tooth agenesis we understand congenital absence of one or more teeth in the dentition. This disorder can have a variety of causes - from the influence of local causes during the formation of tooth germs primarily to the influence of genetic factors. Polymorphism in a gene, that encodes a protein involved in odonto-genesis, may cause its malfunction, which may adversely affect the tooth development and eventually stop it completely. Among the most studied genes associated with the dental agenesis belong PAX9, MSX1, AXIN2, WNT10a and EDA genes.

The aim of this study was to examine the possible relationship between the PAX9 gene polymorphisms and tooth agenesis in Czech population. The analysis was based on DNA sequencing of selected regions of the PAX9 gene and consequent comparison of obtained sequences with the reference sequence from GenBank online database (NCBI).

The most important results of our study on Czech population seem to be insertion 99-101insC (rs138135767,

rs11373281) with simultaneous substitution 272C>G (rs4904155; heterozygous and homozygous) in exon 1 and

variants -54A>G (rs12882923), -41A>G (rs12883049) and 605C>T (Gly203Gly, rs61754301) in exon 3, that will be

studied further for their possible effect on tooth agenesis in Czech population(Ortodoncie 2014, 23, No. 1, p. 44-51).

KlõÂcÏova slova:odontogeneze, ageneze zubuÊ, PAX9, sekvenace Key words:odontogenesis, tooth agenesis, PAX9 gene, sequencing

UÂvod

Odontogeneze je velmi slozÏity akomplexnõ proces, naktereÂm se podõÂlõ prÏes 350 dosud identifikovanyÂch proteinuÊ, jezÏ spolu navzaÂjem reciprocÏneÏ interagujõ [1]. TranskripcÏnõÂa ruÊstove faktory, signaÂlnõÂmolekuly aj. jsou teÏsneÏ spjaty - regulujõ se navzaÂjem avytva rÏõ spletitou molekulaÂrnõ sõÂt', ktera kontroluje arÏõÂdõ cely proces odon-togeneze [2]. I velmi mala zmeÏnav proteinu nebo regu-lacÏnõÂm procesu, ktery se uÂcÏastnõ cÏasoprostorove pro-vaÂzanosti jednotlivyÂch proteinovyÂch interakcõÂ, muÊzÏe mõÂt zanaÂsledek nezvratnou poruchu vyÂvoje zubu [3].

Rozvoj molekulaÂrneÏ-biologickyÂch metod napomohl k naÂruÊstu pocÏtu studiõ molekulaÂrnõÂch mechanizmuÊ zaÂ-sadnõÂch pro vyÂvoj dentice. Tyto studie jsou provaÂdeÏny prÏedevsÏõÂm namysÏõÂch. VyÂzkum vyÂrazneÏprÏispeÏl k zõÂskaÂnõ znalostõ o cÏasove lokalizaci exprese velkeÂho mnozÏstvõ genuÊ zapojenyÂch do odontogeneze [1]. VyÂvoj probõÂha i v oblasti genetiky a zaÂrovenÏ roste mozÏnost jak identi-fikovat v lidskeÂm genomu geny nezbytne pro spraÂvny pruÊbeÏh odontogeneze, jejichzÏ polymorfizmy by mohly mõÂt zanaÂsledek agenezi zubuÊ [4, 5].

PAX9

Gen pro PAX9 (Paired box 9) se nachaÂzõ na14. chro-mozoÂmu (14q-12-q13), obsahuje peÏt exonuÊ a koÂduje protein Pax9, ktery funguje jako transkripcÏnõ faktor beÏ-hem embryogeneze, kdy se uÂcÏastnõ diferenciace audrzÏovaÂnõ pluripotence buneÏcÏnyÂch populacõÂ. BeÏhem samotne odontogeneze je klõÂcÏovy pro postupnou avzaÂjemnou interakci mezi uÂstnõÂm epitelem amezen-chymem, ktery je tvorÏen bunÏkami, jezÏ vycestovaly z ne-uraÂlnõ lisÏty [6, 7, 8].

Protein Pax9 je slozÏen z 341 aminokyselin, obsa-huje DNA-vazebnou domeÂnu naN-konci, transkripcÏneÏ regulacÏnõ domeÂnu naC-konci, kteraÂje bohata na ala-nin (13,7 %), serin (13,2 %), prolin (12,7 %) aglycin

Introduction

Odontogenesis is very complicated and complex process involving over 350 so far identified proteins that interact each other [1]. Transcription factors and growth factors, signalling molecules and others are closely lin-ked - they regulate each other and create a complex sy-stem of regulatory linkages which monitors and con-trols the entire process of odontogenesis [2]. Even avery small change in the protein or regulatory process that is involved in spatio-temporally cohesion between the in-dividual protein interactions, can result in irreversible di-sorder of tooth development [3].

The development of molecular-biological techni-ques has led to increase of number of studies of mole-cular mechanisms involved in the development of den-tition. Studies are carried out mainly on mouse models. This research significantly contributed to the know-ledge of temporal localization of the expression of large number of genes involved in odontogenesis [1]. Development is in progress in the field of genetics too and simultaneously increases the possibility to identify genes in the human genome that are neces-sary for the correct progress of odontogenesis whose polymorphisms could result in tooth agenesis [4, 5].

PAX9

PAX9 (Paired box 9) gene is located on the 14th chromosome (14q-12-q13), it contains 5 exons and encodes Pax9 protein. Pax9 protein acts as a transc-ription factor important for embryogenesis, during it Pax9 participates in differentiation and maintenance of pluripotence of cell populations. During odontoge-nesis Pax9 is essential for progressive and reciprocal interactions between oral epithelium and mesen-chyme, which is made up of cells derived from the ne-ural crest [6, 7, 8].

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Pax9 protein is composed of 341 amino acids, it contains the DNA-binding domain at the N-terminus, transcriptional regulatory domain at the C-terminus, that is rich in alanine (13,7 %), serine (13,2 %), proline (12,7 %) and glycine (8,3 %), and the octapeptide mo-tive. DNA-binding domain mediates recognition and binding on the sequence-specific DNA motives, which allows to activate or inhibit the expression of target ge-nes. This domain is composed of two subdomains, the N-terminal subdomain and the C-terminal subdomain, connected with ashort segment. Each of the subdo-mains contains threea-helices (twob-chains are pres-ent in the case of the N-subdomain additionally) that allow DNA binding [9] (Fig. 1).

Expression of PAX9 gene is a marker of early sta-dium of odontogenesis and it occures before the expression of other genes [10]. In the bud stage and during the transition to the next cap stage too, Pax9 in-teracts with Msx1 (Muscle segment homeobox 1) and Bmp4 (Bone morphogenetic protein 4) [11] During the cap stage Pax9 is required for dental mesenchyme condensation, around which the oral epithelium over-grows. If the Pax9 function is impaired, the expression of key odontogenic molecules decreases in the me-senchyme and tooth development stops at the bud stage [12]. High level of expression of Pax9 thus per-sists in the dental mesenchyme during the bud stage and the cup stage, then it decreases during the bell stage [10].

Given that Pax9 acts as a transcription factor, poly-morphisms in this gene may affect many functions and processes, such as the ability of DNA binding, transc-riptional activity or synergistic interactions with co-ac-tivators, such as Msx1 mentioned above [9]. Disrup-tion of DNA-binding funcDisrup-tion is thus considered as the main pathological event [13]. To date over 30 poly-morphisms in the gene for PAX9 have been presented that have been associated with tooth agenesis.

Material and methods

The aim of this study was to find which polymor-fisms and mutations are located in critical regions of the PAX9 gene in individuals with tooth agenesis in the Czech population. We selected first three exons and adjacent intronic region to determine the presence of polymorphisms in the PAX9 gene because of the lar-gest published frequency of mutations associated with tooth agenesis in these critical areas. The exon 1 of PAX9 gene is not translated (5'-UTR region), but it is supossed, that exon 1 may influence its regulatory function during translation. Exon 2 is the next part of the 5'-UTR region and at the end of this region there are four bases which are translated (ie, an initiation co-don and the first base of the next encoding triplet). (8,3 %), aoktapeptidovy motiv. DNA-vazebnaÂ

do-meÂnazprostrÏedkovaÂva rozpoznaÂnõ avazbu nase-kvencÏneÏ specificke motivy DNA, cozÏ umozÏnÏuje aktivo-vat nebo potlacÏoaktivo-vat expresi cõÂlovyÂch genuÊ. Tato do-meÂnaje slozÏenaze dvou subdomeÂn, N-koncove a C-koncoveÂ, spojenyÂch kraÂtkyÂm segmentem. KazÏda ze subdomeÂn obsahuje trÏi a-sÏroubovice (v prÏõÂpadeÏ N-subdomeÂny jsou prÏõÂtomny navõÂc jesÏteÏ dvab-rÏeteÏzce), ktere umozÏnÏujõ vazbu na DNA [9] (Obr. 1).

Exprese Pax9 je markerem cÏasne faÂze odontoge-neze aprÏedchaÂzõÂexpresi dalsÏõÂch genuÊ [10]. Ve faÂzi zub-nõÂho pupenu i beÏhem prÏechodu do naÂsledujõÂcõÂho stadia zubnõ cÏepicÏky interaguje s Msx1 (Muscle segment ho-meobox 1) aBmp4 (Bone morphogenetic protein 4) [11]. BeÏhem staÂdiacÏepicÏky je Pax9 potrÏebny pro kon-denzaci zubnõÂho mezenchymu, okolo neÏhozÏ obruÊsta uÂstnõ epitel. Pokud je funkce Pax9 narusÏena, dochaÂzõ v mezenchymu ke snõÂzÏenõ exprese klõÂcÏovyÂch odonto-gennõÂch molekul avyÂvoj zubu se zastavõ ve staÂdiu pu-penu [12]. Vysoka uÂrovenÏ exprese Pax9 tedy prÏetrvaÂva v zubnõÂm mezenchymu beÏhem staÂdiapupenu apo-haÂrku, pote se beÏhem staÂdiazubnõÂho zvonku snizÏuje [10].

Vzhledem k tomu, zÏe Pax9 puÊsobõ jako transkripcÏnõ faktor, mohou polymorfizmy v tomto genu ovlivnit mnohe funkce a procesy, jako je schopnost vazby na DNA, transkripcÏnõ aktivita nebo synergisticka inte-rakce s koaktivaÂtory, jakyÂm je naprÏõÂklad vyÂsÏe zmõÂneÏny Msx1 [9]. Za hlavnõ patologickou udaÂlost je poklaÂdaÂno praÂveÏ narusÏenõ DNA-vazebne funkce [13]. Dosud bylo publikovaÂno prÏes 30 polymorfizmuÊ v genu pro PAX9, ktere byly asociovaÂny s agenezemi zubuÊ.

MateriaÂl a metodika

CõÂlem teÂto studie bylo nalezenõ polymorfizmuÊ amu-tacõ lokalizovanyÂch v kritickyÂch oblastech genu pro PAX9 u osob s agenezõ zubuÊ z cÏeske populace. Pro stanovenõ prÏõÂtomnosti polymorfizmuÊ v genu pro PAX9 byly vybraÂny prvnõ trÏi exony aprÏilehle intronove oblasti, z duÊvodu nejveÏtsÏõ cÏetnosti polymorfizmuÊ amutacõ sou-visejõÂcõÂch s agenezõ zubuÊ doposud publikovanyÂch v li-teraturÏe. Exon 1 je cely netranslatovaÂn (5'-UTR oblast), ale prÏedpoklaÂda se, zÏe muÊzÏe beÏhem translace uplatnÏo-vat regulacÏnõ funkci. Exon 2 obsahuje zbyÂvajõÂcõ 5'-UTR oblast, pouze na jeho konci se nachaÂzejõ cÏtyrÏi baÂze, ktere jsou translatovaÂny (tj. iniciacÏnõ kodon aprvnõÂ

Obr. 1.ScheÂmaticke znaÂzorneÏnõ proteinove struktury PAX9 Fig. 1.Schematic illustration of PAX9 protein structure

NSD = N-terminal subdomain; CSD = C-terminal subdomain; num-bers show amino acid sequence

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Entire exon 3 is translated and encodes a DNA-binding domain. Remaining exon 4 and exon 5 have not yet been analyzed in this study (Fig. 2).

Intronic regions, that are adjacent the coding re-gions, were analyzed too. Intronic polymorphisms are usually not of major importance because they are cut out during the post-transcriptional modifications. Lately, however, was found that introns may have a sig-nificant influence during the regulation of gene expres-sion through ncRNA (non-coding RNA). Intronic poly-morphisms can control gene expression through ncRNA influence and therefore it is advisable to pay at-tention to these areas [4].

The sample consisted of 97 patients with tooth age-nesis and 6 subjects with complete dentition as the control group. Diagnosis of dental agenesis was per-formed using X-ray images. Samples were sent from dental clinics in Brno (doc. MUDr. CÏernochova P., Ph.D.), Ostrava (MUDr. SÏtembõÂrek J., Ph.D.) and Olo-mouc (prim. MUDr. KrejcÏõ P., Ph.D.).

DNA of patients was isolated from blood or buccal swabs using isolating kit UltraClean BloodSpin DNA (MoBio). Exons 1 and 3 were amplified with primers specific to these areas by KAPA2G Fast HotStart Rea-dymix (Kapa Biosystems). The PCR products for exon 2 were prepared using a modified polymerase KA-PA2G Robust HotStart (Kapa Biosystems) containing DMSO due to the high content of GC regions in the DNA sequence. Special sequencing primers were de-signed to ensure specificity. The amplicons were se-quenced using a genetic analyzer ABI PRISM 3130 (Life Technologies). Obtained sequences were com-pared with the standard sequence of PAX9 gene [14] in the BioEdit v.7.0.8.0 software [15] and polymorp-hisms have been described from the poin of view of their possible effects on protein structure.

Results

The most frequently missing teeth were third mo-lars, followed by second lower premolars and second upper incisors in the group of 97 patients. The group comprised of 59 women and 38 men.

We found the heterozygous/homozygous insertion of cytosine at the nucleotide position 99-101insC (rs138135767, rs11373281) in exon 1 in control sub-jects with complete dentition and in group of patients. We did not accurately determined the position of inser-tion of cytosine due to the occurrence of two adjacent cytosines.

The heterozygous substitution 272C>G

(rs4904155) occurred simultaneously with the inser-tion 99-101insC in 4 samples and the combinainser-tion of the homozygous insertion 99-101insC occurred si-multaneously with the homozygous substitution at po-baÂze z naÂsledujõÂcõÂho koÂdujõÂcõÂho tripletu). Exon 3 je celyÂ

prÏeklaÂdaÂn, tato oblast koÂduje DNA-vazebnou do-meÂnu. ZbyÂvajõÂcõÂ 4. a 5. exon prozatõÂm nebyly v nasÏõÂ stu-dii analyzovaÂny (Obr. 2).

Spolu s koÂdujõÂcõÂmi oblastmi byly analyzovaÂny prÏile-hle intronove oblasti. PolymorfizmuÊm v intronech ne-byÂva prÏiklaÂdaÂn velky vyÂznam z toho duÊvodu, zÏe jsou beÏhemposttranskripcÏnõÂchuÂpravvystrÏizÏeny. Vposlednõ dobeÏ bylo ovsÏem zjisÏteÏno, zÏe introny (nc-RNA) mohou mõÂt vyÂznamny vliv nasestrÏih aprÏõÂtomne polymorfizmy mohou tento proces pozmeÏnit, proto je vhodne teÏmto oblastem veÏnovat pozornost [4].

Zkoumany soubor tvorÏilo 97 pacientuÊ s agenezõ zubuÊ a6 osob s kompletnõÂdenticõÂ, jezÏ slouzÏily jako kon-trolnõ vzorky. DiagnoÂzaageneze zubuÊ bylaprovedena pomocõ rentgenovyÂch snõÂmkuÊ. Vzorky byly zasõÂlaÂny ze zubnõÂch klinik v BrneÏ (doc. MUDr. CÏernochova P., Ph.D.), OstraveÏ (MUDr. SÏtembõÂrek J., Ph.D.) aOlo-mouci (prim. MUDr. KrejcÏõ P., Ph.D.).

DNA pacientuÊ bylaizolovaÂnaz krve nebo bukaÂlnõÂch steÏruÊ pomocõ izolacÏnõÂho kitu UltraClean BloodSpin DNA (Mo-Bio). Exony 1 a3 byly amplifikovaÂny specific-kyÂmi primery k teÏmto oblastem pomocõ KAPA2G Fast HotStart ReadyMix (Kappa Biosystems). Produkty PCR pro exon 2 byly prÏipraveny pomocõ modifikovane polymeraÂzy Kapa 2G Robust Hot Start (Kapa Biosy-stems) s prÏõÂdavkem DMSO vzhledem k vysokeÂmu podõÂlu GC oblastõ v DNA sekvenci. Pro zarucÏenõ speci-fity byly navrhnuty sekvenacÏnõÂprimery. Amplikony byly sekvenovaÂny pomocõ genetickeÂho analyzaÂtoru ABI 3130 Prism (Life Technologies). VyÂsledne sekvence byly porovnaÂny se standardnõ sekvencõ genu pro PAX9 [14] v programu BioEdit v.7.0.8.0 [15] a polymor-fizmy byly popsaÂny z hlediskajejich mozÏneÂho vlivu na proteinovou strukturu.

VyÂsledky

V celkoveÂm souboru 97 pacientuÊ byly nejcÏasteÏji chybeÏjõÂcõÂm zubem trÏetõ molaÂry, naÂsledovane druhyÂmi dolnõÂmi premolaÂry apostrannõÂmi hornõÂmi rÏezaÂky. Sou-bor se sklaÂdal z 59 zÏen a38 muzÏuÊ.

V exonu 1 se u kontrolnõÂch osob s plnou denticõ i u pacientuÊ vyskytovala heterozygotnõÂ/homozygotnõ inzerce cytosinu v nukleotidove pozici 99-101insC (rs138135767, rs11373281). KvuÊli dveÏmacytosinuÊm

Obr. 2.ScheÂmaticke znaÂzorneÏnõ genu pro PAX9 Fig. 2.Schematic illustration of the PAX9 gene

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sition 272C>G in 19 samples. Third molars in the first quadrant, followed by agenesis of third molars in the remaining quadrants, were the most commonly mis-sing teeth in this group of 19 subjects. Mandibular se-cond molars and mandibular sese-cond premolars in the third and fourth quadrant were other frequently mis-sing teeth.

The insertion 99-101insC occurred with the poly-morphism -40A­C together in 3 people. The gous insertion 99-101insC occurred with the heterozy-gous substitution 272C>G and -40A>C, but only in a few sporadic cases.

We found no polymorphism in exon 2 in patients and controls, just the standard sequences.

The polymorphism 54A>G (rs12882923) and -41A>G (rs12883049), both individually and in combi-nations, both in patients and in control subjects, were the most commonly polymorphisms in exon 3. Both polymorphisms are located in the noncoding region before exon 3 at position distant 54, respectively 41 bp from the first nucleotide of exon 3. In 3 patients, in-cluding 2 related, we discovered the polymorphism 605C>T (rs61754301), located at the coding nucleo-tide position 605, the coded 203th amino acid remains glycine. Other sequences were standard.

Conclusion and discussion

The homozygous combination of insertion cytosine 99-101insC with the homozygous substitution 272C>G were the most frequently found polymorp-hisms in group of patients (19 of 97 cases). Both poly-morphisms are located in the 5'-untranslated region (5'-UTR), which may play a role in regulation of gene expression, such as to influence the transcription, post-transcriptional modifications of RNA or transla-tion [16, 17]. Most patients suffered from agenesis of third molars, mandibular second molars and notewor-thy mandibular second premolars agenesis were other frequently missing teeth. We found the heterozygous variant of these two simultaneously located polymorp-hisms. We cannot consider if these polymorphisms they comprise just interindividual variance without sig-nificant consequence, because these polymorphisms occurred in patients and in persons with complete dentition. It is interesting, that these two polymorp-hisms always occur in the sequence of PAX9 gene si-multaneously in all cases. In patient samples, except the above-mentioned polymorphisms, we found other intronic substitutions and combinations, that occur but only in a few sporadic cases, and their possible re-lationship with tooth agenesis we could check in fur-ther analyzes on a larger group of patients.

During the study of literary sources we were able to determine that no study concerned with the exon 1 of vedle sebe jsme prÏesneÏ nestanovili polohu inzerce

cy-tosinu.

Heterozygotnõ zaÂmeÏna272C>G (rs4904155) se vy-skytovala soucÏasneÏ s inzercõ 99-101insC u cÏtyrÏ vzorkuÊ akombinace homozygotnõ inzerce 99-101insC se vy-skytovala spolecÏneÏ s homozygotnõ substitucõ v pozici 272C>G u devatenaÂcti vzorkuÊ. NejcÏasteÏji chybeÏjõÂcõÂmi zuby u teÏchto 19 jedincuÊ byly trÏetõ molaÂry v prvnõÂm kvadrantu, naÂsledovane agenezõÂtrÏetõÂch molaÂruÊ ve zbyÂ-vajõÂcõÂch kvadrantech. DalsÏõÂmi chybeÏjõÂcõÂmi zuby byly dolnõ druhe molaÂry adolnõ druhe premolaÂry ve trÏetõÂm a cÏtvrteÂm kvadrantu.

U trÏõÂ osob se spolecÏneÏ s inzercõÂ 99-101insC vyskytl polymorfizmus -40A>C. Objevovala se i heterozygotnõÂ inzerce 99-101insC se soucÏasnou heterozygotnõÂ zaÂ-meÏnou 272C>G a-40A>C, ovsÏem pouze v neÏkolika ojedineÏlyÂch prÏõÂpadech.

V exonu 2 nebyl nalezen zÏaÂdny polymorfizmus, pou-ze standardnõ sekvence u pacientuÊ i kontrolnõÂch vzorkuÊ.

Ve trÏetõÂm exonu byly nejcÏasteÏji nalezeny polymorfi-zmy -54A>G (rs12882923) a-41A>G (rs12883049), ato jak samostatneÏ, tak take v kombinacõÂch, a to u pacientuÊ i kontrolnõÂch osob. Obapolymorfizmy se nacha zejõ v nekoÂdujõÂcõ oblasti prÏedchaÂzejõÂcõ exonu 3 napozici vzdaÂlene 54, resp. 41 bp od prvnõÂho nukleotidu exonu 3. U trÏõ pacientuÊ, z toho dvou prÏõÂbuznyÂch, byl nalezen polymorfizmus 605C>T (rs61754301), ktery se nachaÂzõ nanukleotidove pozici 605, koÂdovanou 203. aminoky-selinou vsÏa k zuÊstaÂva glycin. Ostatnõ sekvence byly standardnõÂ.

ZaÂveÏr a diskuse

NejcÏetneÏji naleÂzanyÂmi polymorfizmy nasouboru pacientuÊ (19 osob z 97) byly kombinace homozygotnõ inzerce cytosinu 99-101insC s homozygotnõ zaÂmeÏnou 272C>G. Obapolymorfizmy se nachaÂzõ v 5'-netransla-tujõÂcõ se oblasti (5'-UTR), ktera muÊzÏe hraÂt roli v regulaci genove exprese, naprÏ. ovlivnÏovat transkripci, post-transkripcÏnõ uÂpravy RNA nebo translaci [16, 17]. U veÏt-sÏiny pacientuÊ se vyskytovala ageneze trÏetõÂch molaÂruÊ, dalsÏõÂmi cÏasto chybeÏjõÂcõÂmi zuby byly dolnõ druhe molaÂry azazmõÂnku stojõ i ageneze dolnõÂch druhyÂch premolaÂruÊ. Byla nalezena i heterozygotnõ varianta teÏchto dvou soucÏasneÏ se nachaÂzejõÂcõÂch polymorfizmuÊ.

ProtozÏe se tyto polymorfizmy vyskytovaly u pa-cientuÊ i osob s kompletnõ denticõÂ, nemuÊzÏeme urcÏit, zda majõÂvliv na vznik agenezõ zubuÊ nebo tvorÏõ pouze in-terindividuaÂlnõ rozdõÂl bez zaÂva zÏnyÂch naÂsledkuÊ. ZajõÂma-vostõ je, zÏe se tyto dveÏ odchylky v sekvenci genu pro PAX9 vyskytujõ vzÏdy soucÏasneÏ ato ve vsÏech nasÏich zji-sÏteÏnyÂch prÏõÂpadech. U vzorkuÊ pacientuÊ byly kromeÏ vyÂsÏe zmõÂneÏnyÂch polymorfizmuÊ nalezeny i dalsÏõ intronove zaÂ-meÏny akombinace,ktere se ale vyskytujõÂpouze v

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neÏko-PAX9 gene, so our partial results could contribute for further research. Thanks to analyzing of results of va-rious publications we found incorrect numbering of exons in these publications, when there exon 2 means the third exon in the fact.

As the most interesting result we consider discove-ring of the polymorphism 605C>T (Gly203Gly) in the coding region in 2 patients with agenesis of the third molars in two quadrants and 1 patient with agenesis of the third molars in all quadrants and second premo-lars in two quadrants.

The intron substitutions -41A>G and -54A>G were other identified polymorphisms in exon 3, but these polymorphisms occurred even in the control samples. Polish study published by Pawlowska et al. [18] asso-ciate these two polymorphisms with possible influence on tooth agenesis. In our study we also found these polymorphisms in persons with complete dentition, so it is possible to exclude direct effect of these poly-morphisms on the dental agenesis.

When comparing our results with other international studies carried out on PAX9 gene, we did not reach any significant agreement. Special attention is given to exon 3 because DNA-binding domain is coded in this area and this domain allows to act the protein as a transcription factor and disruption of its function is generally regarded as the main pathological cause of tooth agenesis.

Although more polymorphisms were described in exon 3, these data are not being confirmed on the sam-ple of Czech population and on the other hand, we de-tected polymorphisms that were not described in any of the studies. The explanation of this difference could be the fact that the population of people with tooth agenesis in Czech Republic differ from previously stu-died populations genetically. This finding highlights the importance to conduct research of genetic condi-tions of dental agenesis in different populacondi-tions.

Standard nucleotide sequences also occured in pa-tients with confirmed dental agenesis. Tooth develop-ment is controlled by expression of alarge number of genes that have not been analyzed yet. Therefore we suppose the influence of other genes on dental agene-sis in our group of patients.

In further research we will focus our attention on se-quence analysis of MSX1 and AXIN2 genes. We will enlarge the group of patients and control subjects in order to be able statistically evaluate possible associa-tions.

Familiar studies are one of effective methods for checking of association of newly identified mutations in relationship with teeth agenesis.

The most frequent absence of molars followed by premolars follows from our study from the perspective likaojedineÏlyÂch prÏõÂpadech a jejich mozÏny vztah s

age-nezõÂ zubuÊ by se mohl prokaÂza t prÏi dalsÏõÂch analyÂzaÂch na veÏtsÏõÂm souboru pacientuÊ.

PrÏi studiu literaÂrnõÂch zdrojuÊ se naÂm podarÏilo zjistit, zÏe exonem 1 genu pro PAX9 se zÏaÂdna studie dosud ne-zabyÂvala, a proto mohou i tyto dõÂlcÏõ vyÂsledky byÂt prÏõÂno-sem pro dalsÏõ vyÂzkum. DõÂky analyÂze vyÂsledkuÊ rozlicÏ-nyÂch zahranicÏnõÂch studiõ jsme zjistili, zÏe v publikacõÂch dochaÂzõ k nespraÂvneÂmu cÏõÂslovaÂnõ exonuÊ, kdy je exo-nem 2 mysÏlen ve skutecÏnosti exon 3.

NejvyÂrazneÏjsÏõÂm vyÂsledkem bylo nalezenõÂ polymorfi-zmu v koÂdujõÂcõÂoblasti 605C>T (Gly203Gly) u 2 pacientuÊ s agenezõÂ trÏetõÂch molaÂruÊ ve dvou kvadrantech a u 1 pa-cientas agenezõÂ trÏetõÂch molaÂruÊ ve vsÏech kvadrantech adruhyÂch premolaÂruÊ ve dvou kvadrantech.

DalsÏõÂmi identifikovanyÂmi polymorfizmy v exonu 3 byly intronove zaÂmeÏny -41A>G a-54A>G, ktere se vy-skytovaly i u kontrolnõÂch vzorkuÊ. Polska studie Pa-wlowskeÂ, et al. [18] asociovala tyto dva polymorfizmy s mozÏnyÂm vlivem naageneze zubuÊ. V na sÏõ studii jsme tyto polymorfizmy nalezly take u osob s kompletnõ denticõÂ, takzÏe lze vyloucÏit prÏõÂmy efekt teÏchto polymor-fizmuÊ nadentaÂlnõ agenezi.

PrÏi srovnaÂnõ nasÏich vyÂsledkuÊ s vyÂsledky ostatnõÂch zahranicÏnõÂch studiõ provedenyÂch nagenu pro PAX9 jsme nedosÏli k zÏaÂdne vyÂrazne shodeÏ. PraÂveÏ exonu 3 byÂva veÏnovaÂnazvyÂsÏena pozornost, protozÏe je v teÂto oblasti koÂdovaÂnaDNA-vazebna domeÂna, ktera umo-zÏnÏuje proteinu fungovat jako transkripcÏnõ faktor a naru-sÏenõ jejõ funkce je obecneÏ poklaÂdaÂno za hlavnõ patolo-gickou udaÂlost zpuÊsobujõÂcõ zubnõ ageneze.

AcÏkoliv bylo v oblasti exonu 3 popsaÂno veÏtsÏõ mnozÏ-stvõÂpolymorfizmuÊ, navzorku cÏeske populace jsme tato data prozatõÂm nepotvrdili a naopak, naÂmi odhalene va -rianty nebyly zatõÂm v zÏaÂdne studii popsaÂny. VysveÏtle-nõÂm tohoto rozporu by mohlabyÂt skutecÏnost, zÏe se po-pulace osob s agenezõ zubuÊ v CÏeske republice gene-ticky lisÏõ od doposud studovanyÂch populacõÂ. Tento naÂlez poukazuje na duÊlezÏitost provaÂdeÏnõ vyÂzkumu ge-neticke podmõÂneÏnosti vzniku zubnõÂch agenezõ v ruÊ-znyÂch populacõÂch.

I u pacientuÊ, u kteryÂch bylapotvrzenazubnõ age-neze, se vyskytovaly standardnõ nukleotidove se-kvence. VyÂvoj zubu je rÏõÂzen expresõÂgenuÊ velkeÂho pocÏtu proteinuÊ, ktere nebyly dosud analyzovaÂny. PrÏedpoklaÂ-daÂme tedy vliv dalsÏõÂch genuÊnaagenezi zubuÊ ve sku-pineÏ na sÏich pacientuÊ.

V dalsÏõÂm vyÂzkumu bude nasÏe pozornost zameÏrÏenana sekvenacÏnõ analyÂzy genuÊ pro MSX1 aAXIN2. Bude zveÏt-sÏen soubor vzorkuÊ pacientuÊ i kontrolnõÂch vzorkuÊ, aby-chom mohli statisticky vyhodnocovat mozÏne asociace.

Rodinne studie se jevõ jako jedna z efektivnõÂch me-tod pro oveÏrÏovaÂnõÂvztahu noveÏ identifikovanyÂch mutacõ s agenezõ zubuÊ.

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of comparison of morphological class. In these results we are in agreement with the results of other studies.

In our group of patients bilateral absence of teeth appears very often, that also corresponds with the Czech and foreign studies, such as Symons et al. [19], ZaÂvadova [20], Heringova et CÏernochova [21].

The prevalence of agenesis differs by gender. Our results show 59 women and 38 men, in group of wo-men the incidence is 1,55 times higher based therefore on this study. When we compare our results with the known data, prevalence is reported higher in women too (ratio of female:male 3:2), incidence in women is re-ported 1,37 times higher than for men [12].

In conclusion, from the results of our study on Czech population, the most important result is the in-sertion 99-101insC with the simultaneously substitu-tion 272C­G (rs4904155, heterozygous and homozy-gous) in exon 1 and the polymorphisms -54A­G (rs12882923), -41A­G (rs12883049) and 605C­T (Gly203Gly, rs61754301) in exon 3 which will be further studied for their possible effect on tooth agenesis in Czech population.

The authors have no comercial, proprietary, or financial interests in the products or companies described in this article.

Acknowledgement

This project was supported by Internal grant agency of The Ministry of Health of The Czech Republic IGA MZ CÏR No. NT/11420-6/2010.

Z pohledu srovnaÂnõÂmorfologicke trÏõÂdy vyplyÂva z nasÏõ studie nejcÏasteÏjsÏõ absence molaÂruÊ, naÂsledovanyÂch premolaÂry. V tom se nasÏe vyÂsledky shodujõ s vyÂsledky jinyÂch studiõÂ.

Velmi cÏasto se v nasÏem souboru pacientuÊ objevuje bilateraÂlnõ absence zubuÊ, v cÏemzÏ se rovneÏzÏ shodujeme s cÏeskyÂmi i zahranicÏnõÂmi studiemi, naprÏ. Symons et al. [19], ZaÂvadova [20], Heringova et CÏernochova [21].

Prevalence ageneze se lisÏõÂ podle pohlavõÂ. NasÏe vyÂ-sledky uvaÂdeÏjõÂ 59 zÏen a38 muzÏuÊ, u zÏen je tedy nazaÂ-kladeÏ teÂto studie vyÂskyt 1,55x vysÏsÏõÂ. Pro srovnaÂnõÂ se znaÂmyÂmi daty, prevalence je uvaÂdeÏnatakteÂzÏ vysÏsÏõÂ u zÏen (v pomeÏru zÏeny:muzÏi 3:2), u zÏen je udaÂvaÂn vyÂskyt 1,37x vysÏsÏõÂ nezÏ u muzÏuÊ [12].

ZaÂveÏrem, z vyÂsledkuÊ naÂmi provedene studie nacÏe-ske populaci se tedy jevõ jako nejvyÂznamneÏjsÏõ inzerce 99-101insC se soucÏasnou zaÂmeÏnou 272C­G (rs4904155; heterozygotnõ i homozygotnõÂ) v exonu 1 a varianty -54A­G (rs12882923), -41A­G (rs12883049) a605C­T (Gly203Gly, rs61754301) v exonu 3, ktere bu-dou daÂle studovaÂny pro jejich mozÏny vliv naageneze zubuÊ v cÏeske populaci.

AutorÏi nemajõ komercÏnõÂ, vlastnicke nebo financÏnõ zaÂjmy na pro-duktech nebo spolecÏnostech popsanyÂch v tomto cÏlaÂnku.

PodeÏkovaÂnõÂ

Tento projekt byl financovaÂn InternõÂ grantovou agenturou Ministerstva zdravotnictvõÂ CÏR IGA MZ CÏR cÏõÂ-slo NT/11420-6/2010.

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14. [URL] http://www.ncbi.nlm.nih.gov/nuccore/NG_013357.1. Homo sapiens paired box 9 (PAX9), RefSeqGene on chromosome 14, NCBI. 2008. [cit. 19. 3. 2013].

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20. ZaÂvadovaÂ, A.: Ageneze dolnõÂch druhyÂch premolaÂruÊ CÏaÂst 1 - UÂvod do problematiky; epidemiologie a etiologie age-nezõÂ, diagnostika. Ortodoncie 2002, 11, cÏ. 2, s. 21-28. 21. HeringovaÂ, D.; CÏernochovaÂ, P.: Etiology of the agenesis of

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Doc. RNDr. Omar SÏeryÂ, Ph.D. LaboratorÏ embryologie zÏivocÏichuÊ UÂZÏFG AV CÏR, v.v.i.

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