OBJECTIVE: To investigate the transcriptome profile of genital tubercles (GTs) in male SD rats and explore the mechanism of hypospadias induced by Di (2-ethylhexyl) phthalate (DEHP).
METHODS: Forty time-pregnant SD rats were randomly divided into 4 equal groups, namely GD16 group and GD19 group (in which the male GTs were collected on gestation day[GD]16 and GD19 for RNA-seq, respectively), control group and DEHP exposure group (with administration of oil and 750 mg/kg DEHP by gavage from GD12 to GD19, respectively).In the control and DEHP exposure groups, the GTs were collected from the male fetuses on GD19.5, and scanning electron microscopy and HE staining were used to observe the morphological changes.The differentially expressed genes (DEGs) in the GTs were screened using lllumina HiSeq 2000 followed by GO and KEGG enrichment analyses to characterize the transcriptome profile.Immunofluorescence assay was performed to verify the DEGs (Mafb) identified by RNA-seq results.Immunofluorescence assay and Western blotting were used to examine the expression levels of Mafb in the penile tissue.
RESULTS: A total of 1360 DEGs were detected in the GTs between GD16 group and GD19 group by RNA-seq.Among these genes, 797 were up-regulated and 563 were down-regulated.These DEGs were mainly enriched in the cell adhesion plaque signaling pathway, axon guidance signaling pathway, and extracellular matrix receptor signaling pathway.Compared with that in GD16 group, Mafb was significantly up-regulated in GD19 group, which was consistent with the sequencing results.Mafb and β-catenin were significantly down-regulated in DEHP-exposed group compared with the control group (P < 0.01).
CONCLUSIONS: Mafb expression increases progressively with the development of GTs in male SD rats.DEHP exposure causes significant down-regulation of Mafb and β-catenin, suggesting that β-catenin signaling pathway that affects Mafb is related to DEHP-induced hypospadias in SD rats.

BACKGROUND: Non-syndromic cleft lip with or without cleft palate (NSCL/P) are the most common human congenital birth defects with a complex etiology. MAFB has been reported as a candidate gene involved in the pathogenesis of NSCL/P from genome-wide association study (GWAS) findings, and no replication studies have been performed in Western Han Chinese.
OBJECTIVE: The aim of this study was to investigate the associations of MAFB among NSCL/P trios in Western Han Chinese.
METHODS: We selected 6 single nucleotide polymorphisms (SNPs) (rs6072081, rs6065259, rs17820943, rs13041247, rs11698025 and rs6102085) near MAFB based on previous GWAS findings and recruited 298 case-parents trios with NSCL/P from Western Han Chinese population, while genotypes were done by SNPscan technology.
RESULTS: Strong evidence of an association was found at rs17820943 (p = 0.0023; odds ratio - ORtranmission = 0.7 and 95% confidence interval [CI]: 0.55-0.88) and rs13041247 (p = 0.0023; ORtranmission = 0.7 and 95% CI: 0.55-0.88) among NSCL/P; genotypic transmission-disequilibrium test (TDT) analysis further confirmed this. C/C homozygote at rs17820943 (z = 3.44 and p = 0.00058) and T/T homozygote at rs13041247 (z = 3.14 and p = 0.0017) was over-transmitted among NSCL/P, which indicated they could increase the risk of having an affected baby. Sliding window haplotype analysis showed that haplotypes consisting of C allele at rs17820943 and T allele at rs13041247 were still over-transmitted among NSCL/P (lowest p = 0.0021).
CONCLUSIONS: This study further confirmed that the targeted SNPs at MAFB were associated with NSCL/P trios from Western Han Chinese population, which provides more scientific evidence for the future research and genetic counseling.

Non-syndromic cleft lip with or without cleft palate (NSCLP) is a common complex birth defect. MAFB (v-maf musculoaponeurotic fibrosarcoma oncogene homolog B) is a new gene that may be involved in susceptibility to cleft lip with or without cleft palate (CL/P). To further assess its role in NSCLP, we investigated 3 identified single nucleotide polymorphisms in MAFB (rs13041247, rs6065259, and rs11696257) and examined them for association with NSCLP in 344 patients and 324 healthy controls in a northern Chinese Han population with a high incidence of the syndrome. Peripheral blood samples were taken when patients enrolled in the study and DNA samples were extracted from the blood. The 3 single nucleotide polymorphisms were genotyped using a mini-sequencing method (Snapshot(®) Multiplex System for SNP genotyping, Life Technologies Ltd, Paisley, UK). We found that rs6065259 was the most important single nucleotide polymorphism in MAFB (OR6065259-AA=0.45; 95% CI: 0.28 to 0.71; p=0.0027), followed by rs13041247; however, no association was found between rs11696257 and NSCLP. Our study provides further evidence regarding the role of MAFB variations in the development of NSCLP in this northern Chinese Han population.