rs7590268 present on the 2p21 locus was identified to be associated with non-syndromic cleft lip with or without cleft palate (NSCL/P) in several populations, including the Chinese Han population, indicating that 2p21 was a susceptibility locus for NSCL/P. However, previous studies have only identified common single-nucleotide polymorphism (SNP) within the THADA gene, neglecting the rare variants and other genes in 2p21; thus, this study was designed to investigate additional variants and novel susceptibility genes in 2p21. A total of 159 NSCL/P patients and 542 controls were recruited in the discovery phase, whereas 1830 NSCL/P patients and 2,436 controls were recruited in the replication phase. After targeted region sequencing, we performed association and burden analyses for the common and rare variants, respectively. Furthermore, RNA-seq, proliferation assay and cell cycle analysis were performed to clarify the possible function of the candidate gene ZFP36L2. Association analysis showed that four SNPs were specifically associated with non-syndromic cleft lip only (NSCLO) and two SNPs were associated with both NSCLO and NSCL/P. Burden analysis indicated that ZFP36L2 was associated with NSCLO (p = .0489, OR = 2.41, 95% CI: 0.98–5.90). Moreover, SNPs in the ZFP36L2 targeted gene JUP were also associated with NSCLO. ZFP36L2 also inhibited cell proliferation and induced G2 phase arrest in the GMSM-K cell line. Therefore, we proposed that ZFP36L2 is a novel susceptibility gene of NSCLO in the 2p21 locus, which could lead to NSCLO by modulating cell proliferation and cycle.

Rs560426 at 1p22 was proved to be associated with NSCL/P (non-syndromic cleft lip with or without the palate) in several populations, including Han Chinese population. Here, we conducted a deep sequencing around rs560426 to locate more susceptibility variants in this region. In total, 2,293 NSCL/P cases and 3,235 normal controls were recruited. After sequencing, association analysis was performed. Western blot, RT-qPCR, HE, immunofluorescence staining, and RNA sequencing were conducted for functional analyses of the selected variants. Association analysis indicated that rs77179923 was the only SNP associated with NSCLP specifically (p = 4.70E-04, OR = 1.84), and rs12071152 was uniquely associated with LCLO (p = 4.00E-04, OR = 1.30, 95%CI: 1.12–1.51). Moreover, de novo harmful rare variant NM_004815.3, NP_004806.3; c.1652G>C, p.R551T in ARHGAP29 resulted in a decreased expression level of ARHGAP29, which in turn affected NSCL/P-related biological processes; however, no overt cleft palate (CP) phenotype was observed. In conclusion, rs12071152 was a new susceptible variant, which is specifically associated with LCLO among the Han Chinese population. Allele A of it could increase the risk of having a cleft baby. Rs77179923 and rare variant NM_004815.3, NP_004806.3; c.1652G>C, p.R551T at 1p22 were both associated with NSCLP among the Han Chinese population. However, this missense variation contributes to no overt CP phenotype due to dosage insufficiency or compensation from other genes.