Accepted Manuscript RFC1 and non syndromic cleft lip with or without cleft palate: an association based study in Italy Ambra Girardi, Ph.D. Marcella Martinelli, Ph.D. Francesca Cura, Annalisa Palmieri, Ph.D. Francesco Carinci, M.D. Enrico Sesenna, M.D. Luca Scapoli, Ph.D. PII:
S1010-5182(14)00144-9
DOI:
10.1016/j.jcms.2014.04.021
Reference:
YJCMS 1797
To appear in:
Journal of Cranio-Maxillo-Facial Surgery
Received Date: 2 December 2013 Revised Date:
7 March 2014
Accepted Date: 22 April 2014
Please cite this article as: Girardi A, Martinelli M, Cura F, Palmieri A, Carinci F, Sesenna E, Scapoli L, RFC1 and non syndromic cleft lip with or without cleft palate: an association based study in Italy, Journal of Cranio-Maxillofacial Surgery (2014), doi: 10.1016/j.jcms.2014.04.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT RFC1 and non syndromic cleft lip with or without cleft palate: an association based study in Italy
Ambra Girardi, Ph.D.1, Marcella Martinelli, Ph.D.1,*, Francesca Cura1, Annalisa
RI PT
Palmieri, Ph.D.1, Francesco Carinci, M.D.2, Enrico Sesenna, M.D.3 and Luca Scapoli, Ph.D.1
Department of Experimental, Diagnostic and Specialty Medicine, University di
Bologna, Via Belmeloro, 8 - 40126 Bologna, Italy
Department of Morphology, Surgery and Experimental Medicine, University of
M AN U
2
SC
1
Ferrara, Via Luigi Borsari, 46 - 44121 Ferrara, Italy 3
Head and Neck Department, University Hospital of Parma, via Gramsci, 14 - 43100
Parma, Italy
EP
TE D
Running title: RFC1 role in CL/P
AC C
*Corresponding author: Marcella Martinelli, Ph.D. Dept. of Experimental, Diagnostic and Specialty Medicine University of Bologna Via Belmeloro 8 40126 Bologna Italy Tel. +39.051.2094106 Fax. +39.051.2094110 E-mail: [email protected]
1
ACCEPTED MANUSCRIPT Summary The molecular basis of orofacial development are largely unknown and need to be unravelled. Non syndromic cleft lip with or without cleft palate (NSCL/P) is the most common craniofacial malformation, with an incidence of about 1/700 live births, although variable according to ethnicity. Being a
RI PT
multifactorial disease, it arises as a result of an interplay between genetic and environmental factors. Several approaches have been developed to identify susceptibility genes. Genes belonging to the
folate/homocysteine pathway are attracting increasing interest because folate supplementation before
SC
and during early pregnancy can reduce the risk of NSCL/P. We performed a family based association study in order to asses if a genetic variant of RFC1 could be involved in NSCL/P onset.
M AN U
We genotyped 404 unrelated probands and their relatives for three biallelic polymorphic variants (rs1051266, rs4818789 and rs3788205), that were selected because they produced conflicting results on previous investigations.
Evidence of association was found between the investigated polymorphisms and NSCL/P in our sample of the Italian population, albeit with weak significance levels.
TE D
Results from this investigation provided a support of previous studies suggesting a role of RFC1 in NSCL/P aetiology, reinforcing the concept that genetic predisposition to NSCL/P varies enormously
AC C
EP
within different ethnic groups.
Key words: RFC1; cleft lip with or without cleft palate; association study; polymorphism
1
ACCEPTED MANUSCRIPT Introduction Among birth defects, non-syndromic cleft lip with or without cleft palate (NSCL/P), represents one of the most common, important and disabling conditions, having both anatomic and social implications. Oral clefts, compromise a number of vital functions such as nutrition, speech and breathing. Moreover, it has
RI PT
psychological and educational implications that must be taken into account. Its incidence varies
appreciably according to ethnicity and geographical location but, on average, NSCL/P affects 1.7 per 1000 live born babies (Mossey et al., 2009).
SC
Being a multifactorial disease, NSCL/P is the result interplay of both environmental and genetic factors. Until now, a great number of candidate genes and loci, and some metabolic pathways have been
M AN U
investigated and correlated to the malformation (Kohli & Kohli, 2012). Several studies in particular have highlighted the role of folate pathway in determining correct embryonic development, thus preventing birth defects (Taparia et al., 2007). Low folate status, as the consequence of inadequate intake or defective metabolization was associated with an increased risk of oral cleft (Blanton et al., 2011). The importance of folic acid and its derivatives is due to their role in cell cycle regulation, DNA
TE D
methylation, homocysteine remethylation and “one carbon unit” transfer to purines and pyrimidines during DNA biosynthesis (Crider et al., 2012). Some of these processes reflect the essential role of folic acid in correct embryological development.
EP
Dietary folate consists of a polyglutamate compound that must be converted into a biologically and metabolically active form. This is achieved in stages which include conversion from polyglutamate to
AC C
monoglutamate, intestinal absorption, transport through the bloodstream, cellular uptake and, finally, “one carbon unit” metabolism. Each step is catalyzed by a specific enzyme, whose activity is essential for the successful processing of folate, thus, in preventing hyperhomocysteinemia, DNA damage, hypomethylation and several other disorders (Blanton et al., 2011). As confirmation of the importance of the genes that play a role in the folate pathway in human embryo development, our research group highlighted a significant degree of association between NSCL/P and polymorphisms mapping in transcobalamine 2 (TCN2) (Martinelli et al., 2006), methylenetetrahydrofolate reductase (MTHFR) (Pezzetti et al., 2004), cystathionine beta-synthase genes (CBS) (Martinelli et al., 2011).
2
ACCEPTED MANUSCRIPT Intracellular uptake of folate is a crucial step that involves a carrier- or receptor- mediated transport. The integral membrane protein performing this function is coded by the reduced folate carrier 1 (RFC1) gene, also known as the solute carrier family 19 (SLC19A1) gene, mapping into 21q22.2–q22.3. Specifically speaking, its role is to deliver the metabolically active form of folate, the 5-
RI PT
methyltetrahydrofolate, into a great variety of cells (Nguyen et al., 1997). Conflicting results have been published as regard of allelic association between RFC1 G80A polymorphism and NSCL/P (Vieira et al., 2005, Shaw et al., 2003, Mostowska et al., 2006, Pei et al., 2006), in diverse populations. The important
SC
role played by this folate transporter, on the one hand, and the lack of European population data on the other, prompted our group to verify its involvement in NSCL/P onset in our sizeable homogeneous
M AN U
Italian sample study.
Materials and methods Sample study
For the present study, 404 Italian trios, composed of probands with nonsyndromic CL/P, and their
TE D
parents, were investigated. Among these patients, 147 cases of CL/P were considered sporadic or nonfamilial, as no other relative shared the same malformation. Patients presenting with clinical congenital features in addition to CL/P were excluded from the study.
EP
The study was approved by the ethical committee "Comitato Etico Unico per la Provincia di Parma" and complied with the Helsinki Declaration's Ethical Principles for Medical Research Involving Human
AC C
Subjects. After obtaining informed consent, peripheral venous blood samples were collected and DNA extraction was performed, as previously described (Carinci et al., 1995). Polymorphic variants
The missense variation rs1051266 (G80A) in the exon 2 was selected because it was previously proposed as a risk factor for NSCL/P (Vieira et al., 2008). In addition, we selected two others flanking single nucleotide polymorphisms: rs4818789 and rs3788205 using the “Life Technologies SNPbrowser” software (Life Technologies, Foster City, CA) in order to maximize information content of genetic markers. The rs4818789 was within an intron, 8.9 kb upstream of the non-synonymous polymorphism, while the rs3788205 mapped near the 5'-UTR region, 6.5 kb downstream of the rs1051266. Genotypes
3
ACCEPTED MANUSCRIPT were collected using the ABI PRISM 7500 Real-Time PCR System and TaqMan chemistry, in accordance with the manufacturer's protocol (Life Technologies, Monza, Italy). Statistical analysis Allelic association analysis was performed using the FBAT program v1.7.3 (Horvath et al., 2001). The
RI PT
additive genetic model, which examines the transmission of marker alleles from parents to affected
offspring, was used for the allelic association tests. In this study, the FBAT multi-marker test (FBAT-MM) was used to deal with the multiple comparison issue. The FBAT-MM simultaneously tests H0 (null
SC
hypothesis): no linkage or association between any marker and any disease susceptibility locus
underlying the trait (Rakovski et al., 2007). This method was developed to test markers in linkage
M AN U
disequilibrium, since a simple Bonferroni-correction for multiple comparisons would probably have been too conservative to adapt for the complicated multiple testing. Subsequently, each marker contribution was tested with the null hypothesis of no linkage and no association between the markers and the underlying causal locus. The Monte Carlo procedure with 10,000 permutations was performed to calculate the empirical P values for allelic association. The Haploview program was used to check for
TE D
Hardy-Weinberg equilibrium and to examine linkage disequilibrium between SNPs (Barrett et al., 2005). The haplotype version of FBAT (HBAT) was applied to test the association between phenotypes and haplotypes (Horvath et al., 2004). The ‘‘mode a’’ command was used to obtain both biallelic and
AC C
permutations.
EP
multiallelic tests. For the haplotype analyses, the Monte Carlo procedure was performed with 10,000
Results
Three polymorphic loci were analysed to test association between alleles and CL/P. The genotype frequencies observed in parents met those expected according with the alleles frequency and the Hardy-Weinberg law. Linkage disequilibrium, which measures the degree of non-random allelic association between neighbouring polymorphisms, was consistent with data reported by the Hapmap 2
Consortium. Observed indices of LD were, in fact, 0.5
S1010-5182(14)00144-9
DOI:
10.1016/j.jcms.2014.04.021
Reference:
YJCMS 1797
To appear in:
Journal of Cranio-Maxillo-Facial Surgery
Received Date: 2 December 2013 Revised Date:
7 March 2014
Accepted Date: 22 April 2014
Please cite this article as: Girardi A, Martinelli M, Cura F, Palmieri A, Carinci F, Sesenna E, Scapoli L, RFC1 and non syndromic cleft lip with or without cleft palate: an association based study in Italy, Journal of Cranio-Maxillofacial Surgery (2014), doi: 10.1016/j.jcms.2014.04.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT RFC1 and non syndromic cleft lip with or without cleft palate: an association based study in Italy
Ambra Girardi, Ph.D.1, Marcella Martinelli, Ph.D.1,*, Francesca Cura1, Annalisa
RI PT
Palmieri, Ph.D.1, Francesco Carinci, M.D.2, Enrico Sesenna, M.D.3 and Luca Scapoli, Ph.D.1
Department of Experimental, Diagnostic and Specialty Medicine, University di
Bologna, Via Belmeloro, 8 - 40126 Bologna, Italy
Department of Morphology, Surgery and Experimental Medicine, University of
M AN U
2
SC
1
Ferrara, Via Luigi Borsari, 46 - 44121 Ferrara, Italy 3
Head and Neck Department, University Hospital of Parma, via Gramsci, 14 - 43100
Parma, Italy
EP
TE D
Running title: RFC1 role in CL/P
AC C
*Corresponding author: Marcella Martinelli, Ph.D. Dept. of Experimental, Diagnostic and Specialty Medicine University of Bologna Via Belmeloro 8 40126 Bologna Italy Tel. +39.051.2094106 Fax. +39.051.2094110 E-mail: [email protected]
1
ACCEPTED MANUSCRIPT Summary The molecular basis of orofacial development are largely unknown and need to be unravelled. Non syndromic cleft lip with or without cleft palate (NSCL/P) is the most common craniofacial malformation, with an incidence of about 1/700 live births, although variable according to ethnicity. Being a
RI PT
multifactorial disease, it arises as a result of an interplay between genetic and environmental factors. Several approaches have been developed to identify susceptibility genes. Genes belonging to the
folate/homocysteine pathway are attracting increasing interest because folate supplementation before
SC
and during early pregnancy can reduce the risk of NSCL/P. We performed a family based association study in order to asses if a genetic variant of RFC1 could be involved in NSCL/P onset.
M AN U
We genotyped 404 unrelated probands and their relatives for three biallelic polymorphic variants (rs1051266, rs4818789 and rs3788205), that were selected because they produced conflicting results on previous investigations.
Evidence of association was found between the investigated polymorphisms and NSCL/P in our sample of the Italian population, albeit with weak significance levels.
TE D
Results from this investigation provided a support of previous studies suggesting a role of RFC1 in NSCL/P aetiology, reinforcing the concept that genetic predisposition to NSCL/P varies enormously
AC C
EP
within different ethnic groups.
Key words: RFC1; cleft lip with or without cleft palate; association study; polymorphism
1
ACCEPTED MANUSCRIPT Introduction Among birth defects, non-syndromic cleft lip with or without cleft palate (NSCL/P), represents one of the most common, important and disabling conditions, having both anatomic and social implications. Oral clefts, compromise a number of vital functions such as nutrition, speech and breathing. Moreover, it has
RI PT
psychological and educational implications that must be taken into account. Its incidence varies
appreciably according to ethnicity and geographical location but, on average, NSCL/P affects 1.7 per 1000 live born babies (Mossey et al., 2009).
SC
Being a multifactorial disease, NSCL/P is the result interplay of both environmental and genetic factors. Until now, a great number of candidate genes and loci, and some metabolic pathways have been
M AN U
investigated and correlated to the malformation (Kohli & Kohli, 2012). Several studies in particular have highlighted the role of folate pathway in determining correct embryonic development, thus preventing birth defects (Taparia et al., 2007). Low folate status, as the consequence of inadequate intake or defective metabolization was associated with an increased risk of oral cleft (Blanton et al., 2011). The importance of folic acid and its derivatives is due to their role in cell cycle regulation, DNA
TE D
methylation, homocysteine remethylation and “one carbon unit” transfer to purines and pyrimidines during DNA biosynthesis (Crider et al., 2012). Some of these processes reflect the essential role of folic acid in correct embryological development.
EP
Dietary folate consists of a polyglutamate compound that must be converted into a biologically and metabolically active form. This is achieved in stages which include conversion from polyglutamate to
AC C
monoglutamate, intestinal absorption, transport through the bloodstream, cellular uptake and, finally, “one carbon unit” metabolism. Each step is catalyzed by a specific enzyme, whose activity is essential for the successful processing of folate, thus, in preventing hyperhomocysteinemia, DNA damage, hypomethylation and several other disorders (Blanton et al., 2011). As confirmation of the importance of the genes that play a role in the folate pathway in human embryo development, our research group highlighted a significant degree of association between NSCL/P and polymorphisms mapping in transcobalamine 2 (TCN2) (Martinelli et al., 2006), methylenetetrahydrofolate reductase (MTHFR) (Pezzetti et al., 2004), cystathionine beta-synthase genes (CBS) (Martinelli et al., 2011).
2
ACCEPTED MANUSCRIPT Intracellular uptake of folate is a crucial step that involves a carrier- or receptor- mediated transport. The integral membrane protein performing this function is coded by the reduced folate carrier 1 (RFC1) gene, also known as the solute carrier family 19 (SLC19A1) gene, mapping into 21q22.2–q22.3. Specifically speaking, its role is to deliver the metabolically active form of folate, the 5-
RI PT
methyltetrahydrofolate, into a great variety of cells (Nguyen et al., 1997). Conflicting results have been published as regard of allelic association between RFC1 G80A polymorphism and NSCL/P (Vieira et al., 2005, Shaw et al., 2003, Mostowska et al., 2006, Pei et al., 2006), in diverse populations. The important
SC
role played by this folate transporter, on the one hand, and the lack of European population data on the other, prompted our group to verify its involvement in NSCL/P onset in our sizeable homogeneous
M AN U
Italian sample study.
Materials and methods Sample study
For the present study, 404 Italian trios, composed of probands with nonsyndromic CL/P, and their
TE D
parents, were investigated. Among these patients, 147 cases of CL/P were considered sporadic or nonfamilial, as no other relative shared the same malformation. Patients presenting with clinical congenital features in addition to CL/P were excluded from the study.
EP
The study was approved by the ethical committee "Comitato Etico Unico per la Provincia di Parma" and complied with the Helsinki Declaration's Ethical Principles for Medical Research Involving Human
AC C
Subjects. After obtaining informed consent, peripheral venous blood samples were collected and DNA extraction was performed, as previously described (Carinci et al., 1995). Polymorphic variants
The missense variation rs1051266 (G80A) in the exon 2 was selected because it was previously proposed as a risk factor for NSCL/P (Vieira et al., 2008). In addition, we selected two others flanking single nucleotide polymorphisms: rs4818789 and rs3788205 using the “Life Technologies SNPbrowser” software (Life Technologies, Foster City, CA) in order to maximize information content of genetic markers. The rs4818789 was within an intron, 8.9 kb upstream of the non-synonymous polymorphism, while the rs3788205 mapped near the 5'-UTR region, 6.5 kb downstream of the rs1051266. Genotypes
3
ACCEPTED MANUSCRIPT were collected using the ABI PRISM 7500 Real-Time PCR System and TaqMan chemistry, in accordance with the manufacturer's protocol (Life Technologies, Monza, Italy). Statistical analysis Allelic association analysis was performed using the FBAT program v1.7.3 (Horvath et al., 2001). The
RI PT
additive genetic model, which examines the transmission of marker alleles from parents to affected
offspring, was used for the allelic association tests. In this study, the FBAT multi-marker test (FBAT-MM) was used to deal with the multiple comparison issue. The FBAT-MM simultaneously tests H0 (null
SC
hypothesis): no linkage or association between any marker and any disease susceptibility locus
underlying the trait (Rakovski et al., 2007). This method was developed to test markers in linkage
M AN U
disequilibrium, since a simple Bonferroni-correction for multiple comparisons would probably have been too conservative to adapt for the complicated multiple testing. Subsequently, each marker contribution was tested with the null hypothesis of no linkage and no association between the markers and the underlying causal locus. The Monte Carlo procedure with 10,000 permutations was performed to calculate the empirical P values for allelic association. The Haploview program was used to check for
TE D
Hardy-Weinberg equilibrium and to examine linkage disequilibrium between SNPs (Barrett et al., 2005). The haplotype version of FBAT (HBAT) was applied to test the association between phenotypes and haplotypes (Horvath et al., 2004). The ‘‘mode a’’ command was used to obtain both biallelic and
AC C
permutations.
EP
multiallelic tests. For the haplotype analyses, the Monte Carlo procedure was performed with 10,000
Results
Three polymorphic loci were analysed to test association between alleles and CL/P. The genotype frequencies observed in parents met those expected according with the alleles frequency and the Hardy-Weinberg law. Linkage disequilibrium, which measures the degree of non-random allelic association between neighbouring polymorphisms, was consistent with data reported by the Hapmap 2
Consortium. Observed indices of LD were, in fact, 0.5
