The association of Dihydrofolate reductase (DHFR) expression and 10q25.3 with non-syndromic cleft lip with or without left palate in children residing in Northern China: a case-control study

Background: Non-syndromic cleft lip with or without cleft palate (NSCL/P) is one of the most common congenital deformities and notable for significant lifelong morbidity and complex etiology. Previous research demonstrated that genetic factors play a key role in the development of NSCL/P; however, no causative genes have been identified thus far. The aim of this study was to investigate the association between dihydrofolate reductase (DHFR) gene expression, the 10q25.3 chromosome, and NSLC with or without cleft palate. Methods: In total, we recruited 220 and 180 healthy controls to investigate the association between DHFR gene expression, the 10q25.3 chromosome, and NSLC with or without cleft palate in the northern Chinese population. In particular, we attempted to detect genetic variations by analyzing single nucleotide polymorphism (SNPs). Genomic DNA was extracted from peripheral blood samples and then analyzed by polymerase chain reaction (PCR) to determine target gene expression. PCR-restriction fragment length polymorphism (PCR-RFLP) was then used to identify the genotypes that were present in the experimental group. Finally, we performed case-controlled family analysis. Results: The gene frequency in the case and control groups all conformed to the Hardy-Weinberg (HW) equilibrium. Analysis revealed that there were no significant differences in the frequency distributions of genotypes or alleles in the experimental group and control group for two genes. The TT (GA) genotype of rs11742688 and rs7078160 was normal in the experimental groups (DHFR rs11742688, p > 0.05; 10q25.3 rs7078160, p > 0.05, respectively). Transmission disequilibrium testing further revealed that these were transmitted in equilibrium by rs11742688 and rs7078160. Conclusions: rs11742688 and rs7078160 polymorphism is not related to the development of NSCL/P in the population of north China. There is no significant difference in the C/T (G/A) genotype when compared between the experimental and control groups.

Non-syndromic cleft lip with or without cleft palate (NSCL/P); DHFR gene polymorphism; Hardy-Weinberg equilibrium; Transmission disequilibrium test

[1] Alamoudi NM, Sabbagh HJ, Innes NP, El Derwi D, Hanno AZ, Al-Aama JY, et al. Prevalence and characteristics of non-syndromic orofacial clefts and the influence of consanguinity. Journal of Clinical Pediatric Dentistry. 2014; 38: 241–246.

[2] Sousa SB, Pina R, Ramos L, Pereira N, Krahn M, Borozdin W, et al. Tetra-amelia and lunghypo/aplasia, syndrome: new case report and review. American Journal of Medical Genetics Part A. 2008; 146A: 2799–2803

[3] Wang Y, Li X, Zhu WL, Guo JZ, Song XM, Li SQ, et al. Genome-wide and interaction linkage scan for nonsyndromic cleft lip with or without cleft palate in two multiplex family in Shenyang, China. Biomedical and Environmental Sciences. 2010; 23: 363–370.

[4] Cheng X, Du F, Long X, Huang J. Genetic inheritance models of non-syndromic cleft lip with or without palate: from monogenic to polygenic. Genes. 2023; 14: 1859.

[5] Nikopensius T, Birnbaum S, Ludwig KU, Jagomägi T, Saag M, Herms S, et al. Susceptibility locus for non-syndromic cleft lip with or without cleft palate on chromosome 10q25 confers risk in Estonina patients. European Journal of Oral Sciences. 2010; 118: 317–319.

[6] Rojas-Martinez A, Reutter H, Chacon-Camacho O, Leon-Cachon RB, Munoz-Jimenez SG, Nowak S, et al. Genetic risk factors for nonsyndromic cleft lip with or without cleft palate in a Mesoamerican population: evidence for IRF6 and variants at 8q24 and 10q25. Birth Defects Research Part A: Clinical and Molecular Teratology. 2010; 88: 535–537.

[7] Martinelli M, Girardi A, Cura F, Carinci F, Morselli PG, Scapoli L. Evidence of the involvement of the DHFR gene in nonsyndromic cleft lip with or without cleft palate. European Journal of Medical Genetics. 2014; 57: 1–4.

[8] Beaty TH, Murray JC, Marazita ML, Munger RG, Ruczinski I, Hetmanski JB, et al. A genome-wide association study of cleft lip with and without cleft palate identifies risk variants near MAFB and ABCA4. Nature Genetics. 2010; 42: 525–529.

[9] Mangold E, Ludwig KU, Birnbaum S, Baluardo C, Ferrian M, Herms S, et al. Genome-wide association study identifies two susceptibility loci for nonsyndromic cleft lip with or without cleft palate. Nature Genetics. 2010; 42: 24–26.

[10] Wyszynski DF, Sarkozi A, Vargha P, Czeizel AE. Birth weight and gestational age of newborns with cleft lip with or without cleft palate and with isolated cleft palate. Journal of Clinical Pediatric Dentistry. 2003; 27: 185–190.

[11] Mishima K, Shiraishi M, Umeda H. Bilateral cleft lip and palate accompanied by 13q-syndrome with deficiencies of FVII and FX: a case report. Journal of Clinical Pediatric Dentistry. 2019; 43: 288–291.

[12] Won HJ, Kim JW, Won HS, Shin JO. Gene regulatory networks and signaling pathways in palatogenesis and cleft palate: a comprehensive review. Cells. 2023; 12: 1954.

[13] Askarian S, Gholami M, Khalili-Tanha G, Tehrani NC, Joudi M, Khazaei M, et al. The genetic factors contributing to the risk of cleft lip-cleft palate and their clinical utility. Maxillofacial Surgery. 2023; 27: 177–186.

[14] Alvizi L, Nani D, Brito LA, Kobayashi GS, Passos-Bueno MR, Mayor R. Neural crest e-cadherin loss drives cleft lip/palate by epigenetic modulation via pro-inflammatory gene-environment interaction. Nature Communications. 2023; 14: 2868.

[15] Sinha SP, Bajracharya M, Huang CS, Ko EW. Does cleft lip and palate affect the severity of malocclusion? Clinical Oral Investigations. 2023; 27: 7557–7567.

[16] Menderes A, Ateşşahin FB, Babahan T, Terzi M, Atalmiş SE, Çağli HB. Median cleft lip. Journal of Craniofacial Surgery. 2023; 34: e780–e781.

[17] Babai A, Irving M. Orofacial clefts: genetics of cleft lip and palate. Genes. 2023; 14: 1603.

[18] De-Regil LM, Fernandez-Gaxiola AC, Dowswell T. Effects and safety of periconceptional folate supplementation for preventing birth defects. Cochrane Database of Systematic Reviews. 2010; 6: CD007950.

[19] Nasroen SL, Tammama T, Darwis RS, Adil A, Rahmutia S, Maskoen AM, et al. The IRF6 rs2013162 and MTHFR A1298C rs1801131 gene polymorphisms related to non-syndromic cleft lip and palate among Deutero-Malay in Indonesia. The Cleft Palate-Craniofacial Journal. 2023; 2: 10556656231191003.

[20] Petersen B, Strassbrug HM, Feichtinger W, Kress W, Schmid M. Terminal deletion of the long arm of chromosome 10: a new case with breakpoint in 10q25. American Journal of Medical Genetics. 1998; 77: 60–62.