Alternative splicing in the coding region of Ppo-A1 directly influences the polyphenol oxidase activity in common wheat (Triticum aestivum L.)

Polyphenol oxidase (PPO) plays a crucial role in browning reactions in fresh and processed fruits and vegetables, as well as products made from cereal grains. Common wheat (Triticum aestivum L.) has a large genome, representing an interesting system to advance our understanding of plant PPO gene expression, regulation and function. In the present study, we characterized the expression of Ppo-A1, a major PPO gene located on wheat chromosome 2A, using DNA sequencing, semi-quantitative RT-PCR, PPO activity assays and whole-grain staining methods during grain development. The results indicated that the expression of the Ppo-A1b allele was regulated by alternative splicing of pre-mRNAs, resulting from a 191-bp insertion in intron 1 and one C/G SNP in exon 2. Eight mRNA isoforms were identified in developing grains based on alignments between cDNA and genomic DNA sequences. Only the constitutively spliced isoform b encodes a putative full-length PPO protein based on its coding sequence whereas the other seven spliced isoforms, a, c, d, e, f, g and h, have premature termination codons resulting in potential nonsense-mediated mRNA decay. The differences in expression of Ppo-A1a and Ppo-A1b were confirmed by PPO activity assays and whole grain staining, providing direct evidence for the influence of alternative splicing in the coding region of Ppo-A1 on polyphenol oxidase activity in common wheat grains.     

A Post-GWAS Replication Study Confirming the PTK2 Gene Associated with Milk Production Traits in Chinese Holstein

Our initial genome-wide association study (GWAS) demonstrated that two SNPs (ARS-BFGL-NGS-33248, UA-IFASA-9288) within the protein tyrosine kinase 2 (PTK2) gene were significantly associated with milk production traits in Chinese Holstein dairy cattle. To further validate if the statistical evidence provided in GWAS were true-positive findings, a replication study was performed herein through genotype-phenotype associations. The two tested SNPs were found to show significant associations with milk production traits, which confirmed the associations observed in the original study. Specifically, SNPs lying in the PTK2 gene were also detected by sequencing 14 unrelated sires in Chinese Holsteins and a total of thirty-three novel SNPs were identified. Thirteen out of these identified SNPs were genotyped and tested for association with milk production traits in an independent resource population. After Bonferroni correction for multiple testing, twelve SNPs were statistically significant for more than two milk production traits. Analyses of pairwise D’ measures of linkage disequilibrium (LD) between all SNPs were also explored. Two haplotype blocks were inferred and the association study at haplotype level revealed similar effects on milk production traits. In addition, the RNA expression analyses revealed that a non-synonymous coding SNP (g.4061098T>G) was involved in the regulation of gene expression. Thus the findings presented here provide strong evidence for associations of PTK2 variants with dairy production traits and may be applied in Chinese Holstein breeding program.     

Polyphenol oxidase genes as integral part of the evolutionary history of domesticated tetraploid wheat

Studying and understanding the genetic basis of polyphenol oxidases (PPO)-related traits plays a crucial role in genetic improvement of crops.A tetraploid wheat collection (T. turgidum ssp., TWC) was analyzed using the 90K wheat SNP iSelect assay and phenotyped for PPO activity. A total of 21,347 polymorphic SNPs were used to perform genome-wide association analysis (GWA) in TWC and durum wheat sub-groups, detecting 23 and 85 marker-trait associations (MTA). In addition, candidate genes responsible for PPO activity were predicted. Based on the 23 MTAs detected in TWC, two haplotypes associated with low and high PPO activity were identified. Four SNPs were developed and validated providing one reliable marker (IWB75732) for marker assisted selection. The 23 MTAs were used to evaluate the genetic divergence (F_ST > 0.25) between the T. turgidum subspecies, providing new information important for understanding the domestication process of Triticum turgidum ssp. and in particular of ssp. carthlicum.     

GWAS loci associated with Chagas cardiomyopathy influences DNA methylation levels

A recent genome-wide association study (GWAS) identified a locus in chromosome 11 associated with the chronic cardiac form of Chagas disease. Here we aimed to elucidate the potential functional mechanism underlying this genetic association by analyzing the correlation among single nucleotide polymorphisms (SNPs) and DNA methylation (DNAm) levels as cis methylation quantitative trait loci (cis-mQTL) within this region. A total of 2,611 SNPs were tested against 2,647 DNAm sites, in a subset of 37 chronic Chagas cardiomyopathy patients and 20 asymptomatic individuals from the GWAS. We identified 6,958 significant cis-mQTLs (False Discovery Rate [FDR]<0.05) at 1 Mb each side of the GWAS leading variant, where six of them potentially modulate the expression of the SAC3D1 gene, the reported gene in the previous GWAS. In addition, a total of 268 cis-mQTLs showed differential methylation between chronic Chagas cardiomyopathy patients and asymptomatic individuals. The most significant cis-mQTLs mapped in the gene bodies of POLA2 (FDR = 1.04x10^-11), PLAAT3 (FDR = 7.22x10^-03), and CCDC88B (FDR = 1.89x10^-02) that have been associated with cardiovascular and hematological traits in previous studies. One of the most relevant interactions correlated with hypermethylation of CCDC88B. This gene is involved in the inflammatory response, and its methylation and expression levels have been previously reported in Chagas cardiomyopathy. Our findings support the functional relevance of the previously associated genomic region, highlighting the regulation of novel genes that could play a role in the chronic cardiac form of the disease.     

Alzheimer’s Disease Risk Polymorphisms Regulate Gene Expression in the ZCWPW1 and the CELF1 Loci

Late onset Alzheimer’s disease (LOAD) is a genetically complex and clinically heterogeneous disease. Recent large-scale genome wide association studies (GWAS) have identified more than twenty loci that modify risk for AD. Despite the identification of these loci, little progress has been made in identifying the functional variants that explain the association with AD risk. Thus, we sought to determine whether the novel LOAD GWAS single nucleotide polymorphisms (SNPs) alter expression of LOAD GWAS genes and whether expression of these genes is altered in AD brains. The majority of LOAD GWAS SNPs occur in gene dense regions under large linkage disequilibrium (LD) blocks, making it unclear which gene(s) are modified by the SNP. Thus, we tested for brain expression quantitative trait loci (eQTLs) between LOAD GWAS SNPs and SNPs in high LD with the LOAD GWAS SNPs in all of the genes within the GWAS loci. We found a significant eQTL between rs1476679 and PILRB and GATS, which occurs within the ZCWPW1 locus. PILRB and GATS expression levels, within the ZCWPW1 locus, were also associated with AD status. Rs7120548 was associated with MTCH2 expression, which occurs within the CELF1 locus. Additionally, expression of several genes within the CELF1 locus, including MTCH2, were highly correlated with one another and were associated with AD status. We further demonstrate that PILRB, as well as other genes within the GWAS loci, are most highly expressed in microglia. These findings together with the function of PILRB as a DAP12 receptor supports the critical role of microglia and neuroinflammation in AD risk.     

Combination Testing Using a Single MSH5 Variant alongside HLA Haplotypes Improves the Sensitivity of Predicting Coeliac Disease Risk in the Polish Population

Assessment of non-HLA variants alongside standard HLA testing was previously shown to improve the identification of potential coeliac disease (CD) patients. We intended to identify new genetic variants associated with CD in the Polish population that would improve CD risk prediction when used alongside HLA haplotype analysis. DNA samples of 336 CD and 264 unrelated healthy controls were used to create DNA pools for a genome wide association study (GWAS). GWAS findings were validated with individual HLA tag single nucleotide polymorphism (SNP) typing of 473 patients and 714 healthy controls. Association analysis using four HLA-tagging SNPs showed that, as was found in other populations, positive predicting genotypes (HLA-DQ2.5/DQ2.5, HLA-DQ2.5/DQ2.2, and HLA-DQ2.5/DQ8) were found at higher frequencies in CD patients than in healthy control individuals in the Polish population. Both CD-associated SNPs discovered by GWAS were found in the CD susceptibility region, confirming the previously-determined association of the major histocompatibility (MHC) region with CD pathogenesis. The two most significant SNPs from the GWAS were rs9272346 (HLA-dependent; localized within 1 Kb of DQA1) and rs3130484 (HLA-independent; mapped to MSH5). Specificity of CD prediction using the four HLA-tagging SNPs achieved 92.9%, but sensitivity was only 45.5%. However, when a testing combination of the HLA-tagging SNPs and the MSH5 SNP was used, specificity decreased to 80%, and sensitivity increased to 74%. This study confirmed that improvement of CD risk prediction sensitivity could be achieved by including non-HLA SNPs alongside HLA SNPs in genetic testing.     

Connecting the Dots: Potential of Data Integration to Identify Regulatory SNPs in Late-Onset Alzheimer's Disease GWAS Findings

Late-onset Alzheimer''s disease (LOAD) is a multifactorial disorder with over twenty loci associated with disease risk. Given the number of genome-wide significant variants that fall outside of coding regions, it is possible that some of these variants alter some function of gene expression rather than tagging coding variants that alter protein structure and/or function. RegulomeDB is a database that annotates regulatory functions of genetic variants. In this study, we utilized RegulomeDB to investigate potential regulatory functions of lead single nucleotide polymorphisms (SNPs) identified in five genome-wide association studies (GWAS) of risk and age-at onset (AAO) of LOAD, as well as SNPs in LD (r²≥0.80) with the lead GWAS SNPs. Of a total 614 SNPs examined, 394 returned RegulomeDB scores of 1–6. Of those 394 variants, 34 showed strong evidence of regulatory function (RegulomeDB score <3), and only 3 of them were genome-wide significant SNPs (ZCWPW1/rs1476679, CLU/rs1532278 and ABCA7/rs3764650). This study further supports the assumption that some of the non-coding GWAS SNPs are true associations rather than tagged associations and demonstrates the application of RegulomeDB to GWAS data.     

Genome-wide association study identifies genetic variation in neurocan as a susceptibility factor for bipolar disorder

We conducted a genome-wide association study (GWAS) and a follow-up study of bipolar disorder (BD), a common neuropsychiatric disorder. In the GWAS, we investigated 499,494 autosomal and 12,484 X-chromosomal SNPs in 682 patients with BD and in 1300 controls. In the first follow-up step, we tested the most significant 48 SNPs in 1729 patients with BD and in 2313 controls. Eight SNPs showed nominally significant association with BD and were introduced to a meta-analysis of the GWAS and the first follow-up samples. Genetic variation in the neurocan gene (NCAN) showed genome-wide significant association with BD in 2411 patients and 3613 controls (rs1064395, p = 3.02 × 10⁻⁸; odds ratio = 1.31). In a second follow-up step, we replicated this finding in independent samples of BD, totaling 6030 patients and 31,749 controls (p = 2.74 × 10⁻⁴; odds ratio = 1.12). The combined analysis of all study samples yielded a p value of 2.14 × 10⁻⁹ (odds ratio = 1.17). Our results provide evidence that rs1064395 is a common risk factor for BD. NCAN encodes neurocan, an extracellular matrix glycoprotein, which is thought to be involved in cell adhesion and migration. We found that expression in mice is localized within cortical and hippocampal areas. These areas are involved in cognition and emotion regulation and have previously been implicated in BD by neuropsychological, neuroimaging, and postmortem studies.     

Development and validation of a new Ppo-A1 marker useful for marker-assisted selection in tetraploid wheats

Polyphenol oxidase (PPO) activity is a major cause of undesirable brown color of semolina. In tetraploid wheat, the Ppo-A1 gene is significantly involved in the phenotypic expression of PPO activity. The main goal of this study was to develop and validate a more efficient marker for Ppo-A1 to facilitate marker-assisted selection for low PPO activity in tetraploid wheat breeding programs. A large tetraploid wheat collection, including durum cultivars, domesticated and wild accessions, was used to evaluate the PPO activity. The heritability values indicated that the phenotypic expression of PPO activity was mainly due to genotypic effect. PPO18, and a new marker named MG18, were used to study the Ppo-A1 allelic variation in a tetraploid wheat collection. PPO18 analysis detected four alleles (Ppo-A1b, Ppo-A1e, Ppo-A1f and Ppo-A1g). The high frequency of Ppo-A1g (no PCR product) detected in the tetraploid wheat collection, led to the development of a new genome-specific Ppo-A1 marker (MG18). MG18 analysis identified the same alleles as PPO18 which were associated with low or high PPO activity. The new MG18 marker was more efficient than PPO18 in detecting the four different alleles of Ppo-A1 in the tetraploid wheat collection. Indeed, the accessions assigned to the Ppo-A1g group, according to PPO18, when tested with MG18, were better classified in the four alleles of the Ppo-A1 gene. The MG18 analysis proved that the PPO18 marker overestimated the number of accessions with Ppo-A1g. Therefore, MG18 can be applied to large-scale marker-assisted selection for PPO activity in durum breeding programs.     

Genetic Control of Individual Differences in Gene-Specific Methylation in Human Brain

We have observed extensive interindividual differences in DNA methylation of 8590 CpG sites of 6229 genes in 153 human adult cerebellum samples, enriched in CpG island “shores” and at further distances from CpG islands. To search for genetic factors that regulate this variation, we performed a genome-wide association study (GWAS) mapping of methylation quantitative trait loci (mQTLs) for the 8590 testable CpG sites. cis association refers to correlation of methylation with SNPs within 1 Mb of a CpG site. 736 CpG sites showed phenotype-wide significant cis association with 2878 SNPs (after permutation correction for all tested markers and methylation phenotypes). In trans analysis of methylation, which tests for distant regulation effects, associations of 12 CpG sites and 38 SNPs remained significant after phenotype-wide correction. To examine the functional effects of mQTLs, we analyzed 85 genes that were with genetically regulated methylation we observed and for which we had quality gene expression data. Ten genes showed SNP-methylation-expression three-way associations—the same SNP simultaneously showed significant association with both DNA methylation and gene expression, while DNA methylation was significantly correlated with gene expression. Thus, we demonstrated that DNA methylation is frequently a heritable continuous quantitatively variable trait in human brain. Unlike allele-specific methylation, genetic polymorphisms mark both cis- and trans-regulatory genetic sites at measurable distances from their CpG sites. Some of the genetically regulated DNA methylation is directly connected with genetically regulated gene expression variation.     

Atrial Fibrillation Associated Chromosome 4q25 Variants Are Not Associated with PITX2c Expression in Human Adult Left Atrial Appendages

Atrial Fibrillation (AF), the most common sustained arrhythmia, has a strong genetic component, but the mechanism by which common genetic variants lead to increased AF susceptibility is unknown. Genome-wide association studies (GWAS) have identified that the single nucleotide polymorphisms (SNPs) most strongly associated with AF are located on chromosome 4q25 in an intergenic region distal to the PITX2 gene. Our objective was to determine whether the AF-associated SNPs on chromosome 4q25 were associated with PITX2c expression in adult human left atrial appendages. Analysis of a lone AF GWAS identified four independent AF risk SNPs at chromosome 4q25. Human adult left atrial appendage tissue was obtained from 239 subjects of European Ancestry and used for SNP analysis of genomic DNA and determination of PITX2c RNA expression levels by quantitative PCR. Subjects were divided into three groups based on their history of AF and pre-operative rhythm. AF rhythm subjects had higher PITX2c expression than those with history of AF but in sinus rhythm. PITX2c expression was not associated with the AF risk SNPs in human adult left atrial appendages in all subjects combined or in each of the three subgroups. However, we identified seven SNPs modestly associated with PITX2c expression located in the introns of the ENPEP gene, ∼54 kb proximal to PITX2. PITX2c expression in human adult left atrial appendages is not associated with the chromosome 4q25 AF risk SNPs; thus, the mechanism by which these SNPs are associated with AF remains enigmatic.     

Genetic and Epigenetic Regulation of TOX3 Expression in Breast Cancer

Genome wide association studies (GWAS) have identified low penetrance and high frequency single nucleotide polymorphisms (SNPs) that contribute to genetic susceptibility of breast cancer. The SNPs at 16q12, close to the TOX3 and CASC16 genes, represent one of the susceptibility loci identified by GWAS, showing strong evidence for breast cancer association across various populations. To examine molecular mechanisms of TOX3 regulation in breast cancer, we investigated both genetic and epigenetic factors using cell lines and datasets derived from primary breast tumors available through The Cancer Genome Atlas (TCGA). TOX3 expression is highly up-regulated in luminal subtype tumors compared to normal breast tissues or basal-like tumors. Expression quantitative trait loci (eQTL) analyses revealed significant associations of rs3803662 and rs4784227 genotypes with TOX3 expression in breast tumors. Bisulfite sequencing of four CpG islands in the TOX3 promoter showed a clear difference between luminal and basal-like cancer cell lines. 5-Aza-2’-deoxycytidine treatment of a basal-like cancer cell line increased expression of TOX3. TCGA dataset verified significantly lower levels of methylation of the promoter in luminal breast tumors with an inverse correlation between methylation and expression of TOX3. Methylation QTL (mQTL) analyses showed a weak or no correlation of rs3803662 or rs4784227 with TOX3 promoter methylation in breast tumors, indicating an independent relationship between the genetic and epigenetic events. These data suggest a complex system of TOX3 regulation in breast tumors, driven by germline variants and somatic epigenetic modifications in a subtype specific manner.     

Meta-analysis of two Chinese populations identifies an autoimmune disease risk allele in 22q11.21 as associated with systemic lupus erythematosus

IntroductionSystemic lupus erythematosus (SLE) is a heterogeneous disease with a diverse spectrum of clinical symptoms, ranging from skin rash to end-organ damage. 22q11.21 has been identified as a susceptibility region for several autoimmune diseases, including SLE. However, detailed information for SLE association and the underlying functional mechanism(s) is still lacking.MethodsThrough meta-analysis of two genome-wide association studies (GWAS) on Han Chinese populations, comprising a total of 1,659 cases and 3,398 controls matched geographically, we closely examined the 22q11.21 region, especially on the reported single-nucleotide polymorphisms (SNPs) associated with different autoimmune diseases and their relationships. We further replicated the most significant associations of SNPs with SLE using 2,612 cases and 2,323 controls of Asian ancestry.ResultsAll reported SNPs in the 22q11.21 region with different autoimmune diseases were examined using the two GWAS data and meta-analysis results, and supportive evidence of association with SLE was found (meta-analysis: P_meta ≤ 7.27E-05), which might require further investigation. SNP rs2298428 was identified as the most significant SNP associated with SLE in this region (P_meta =2.70E-09). It showed independent effects through both stepwise and conditional logistic regression, and there is no evidence of other independent association signals for SLE in this region. The association of rs2298428 was further replicated in three cohorts from Hong Kong, Anhui and Thailand comprising a total of 2,612 cases and 2,323 controls (joint analysis of GWAS and replication result: P_all =1.31E-11, odds ratio =1.23). SNP rs2298428 was shown to be an expression quantitative locus for UBE2L3 gene in different cell types, with the risk allele (T) being correlated with higher expression of UBE2L3. This is consistent with earlier reports on higher expression of UBE2L3 in patients with SLE.ConclusionsAssociation with distinct autoimmune diseases highlights the significance of this region in autoreactive responses and potentially shared functional mechanisms in these diseases.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0577-6) contains supplementary material, which is available to authorized users.     

Genome-wide association studies reveal the role of polymorphisms affecting factor H binding protein expression in host invasion by Neisseria meningitidis

Many invasive bacterial diseases are caused by organisms that are ordinarily harmless components of the human microbiome. Effective interventions against these microbes require an understanding of the processes whereby symbiotic or commensal relationships transition into pathology. Here, we describe bacterial genome-wide association studies (GWAS) of Neisseria meningitidis, a common commensal of the human respiratory tract that is nevertheless a leading cause of meningitis and sepsis. An initial GWAS discovered bacterial genetic variants, including single nucleotide polymorphisms (SNPs), associated with invasive meningococcal disease (IMD) versus carriage in several loci across the meningococcal genome, encoding antigens and other extracellular components, confirming the polygenic nature of the invasive phenotype. In particular, there was a significant peak of association around the fHbp locus, encoding factor H binding protein (fHbp), which promotes bacterial immune evasion of human complement by recruiting complement factor H (CFH) to the meningococcal surface. The association around fHbp with IMD was confirmed by a validation GWAS, and we found that the SNPs identified in the validation affected the 5’ region of fHbp mRNA, altering secondary RNA structures, thereby increasing fHbp expression and enhancing bacterial escape from complement-mediated killing. This finding is consistent with the known link between complement deficiencies and CFH variation with human susceptibility to IMD. These observations demonstrate the importance of human and bacterial genetic variation across the fHbp:CFH interface in determining IMD susceptibility, the transition from carriage to disease.     

Inferred expression regulator activities suggest genes mediating cardiometabolic genetic signals

Expression QTL (eQTL) analyses have suggested many genes mediating genome-wide association study (GWAS) signals but most GWAS signals still lack compelling explanatory genes. We have leveraged an adipose-specific gene regulatory network to infer expression regulator activities and phenotypic master regulators (MRs), which were used to detect activity QTLs (aQTLs) at cardiometabolic trait GWAS loci. Regulator activities were inferred with the VIPER algorithm that integrates enrichment of expected expression changes among a regulator’s target genes with confidence in their regulator-target network interactions and target overlap between different regulators (i.e., pleiotropy). Phenotypic MRs were identified as those regulators whose activities were most important in predicting their respective phenotypes using random forest modeling. While eQTLs were typically more significant than aQTLs in cis, the opposite was true among candidate MRs in trans. Several GWAS loci colocalized with MR trans-eQTLs/aQTLs in the absence of colocalized cis-QTLs. Intriguingly, at the 1p36.1 BMI GWAS locus the EPHB2 cis-aQTL was stronger than its cis-eQTL and colocalized with the GWAS signal and 35 BMI MR trans-aQTLs, suggesting the GWAS signal may be mediated by effects on EPHB2 activity and its downstream effects on a network of BMI MRs. These MR and aQTL analyses represent systems genetic methods that may be broadly applied to supplement standard eQTL analyses for suggesting molecular effects mediating GWAS signals.     

Evaluation of Parkinson Disease Risk Variants as Expression-QTLs

The recent Parkinson Disease GWAS Consortium meta-analysis and replication study reports association at several previously confirmed risk loci SNCA, MAPT, GAK/DGKQ, and HLA and identified a novel risk locus at RIT2. To further explore functional consequences of these associations, we investigated modification of gene expression in prefrontal cortex brain samples of pathologically confirmed PD cases (N = 26) and controls (N = 24) by 67 associated SNPs in these 5 loci. Association between the eSNPs and expression was evaluated using a 2-degrees of freedom test of both association and difference in association between cases and controls, adjusted for relevant covariates. SNPs at each of the 5 loci were tested for cis-acting effects on all probes within 250 kb of each locus. Trans-effects of the SNPs on the 39,122 probes passing all QC on the microarray were also examined. From the analysis of cis-acting SNP effects, several SNPs in the MAPT region show significant association to multiple nearby probes, including two strongly correlated probes targeting the gene LOC644246 and the duplicated genes LRRC37A and LRRC37A2, and a third uncorrelated probe targeting the gene DCAKD. Significant cis-associations were also observed between SNPs and two probes targeting genes in the HLA region on chromosome 6. Expanding the association study to examine trans effects revealed an additional 23 SNP-probe associations reaching statistical significance (p<2.8×10⁻⁸) including SNPs from the SNCA, MAPT and RIT2 regions. These findings provide additional context for the interpretation of PD associated SNPs identified in recent GWAS as well as potential insight into the mechanisms underlying the observed SNP associations.     

Investigation of Susceptibility Genes Triggering Lachrymal/Salivary Gland Lesion Complications in Japanese Patients with Type 1 Autoimmune Pancreatitis

Autoimmune pancreatitis (AIP) is a unique form of chronic pancreatitis characterized by high serum IgG4 concentration and a variety of complicating extra-pancreatic lesions. In particular, lachrymal/salivary gland lesions tend to manifest in a highly active AIP disease state, and several genes are speculated to be associated with the onset of this complication. We therefore searched for candidate susceptibility genes related to lachrymal/salivary gland lesions in a genome-wide association study (GWAS) with the GeneChip Human Mapping 500k Array Set (Affymetrix, CA) that was followed by fine mapping of additional single nucleotide polymorphisms (SNPs) in strongly significant genes with TaqMan assays. Venous blood samples were obtained from 50 type 1 AIP patients with lachrymal/salivary gland lesions (A group) and 53 type 1 AIP patients without (B group). The mean values of IgG and IG4 were both significantly different (P<0.05) between the groups. SNPs that showed a significant association with the A group at the genome-wide level (P<0.0001) were identified and subsequently used in fine SNP mapping of candidate genes. In total, five SNPs had a positive association with complicated AIP (most notably rs2284932 [P=0.0000021]) and five SNPs possessed a negative association (particularly rs9371942 [P=0.00000039]). Among them, KLF7, FRMD4B, LOC101928923, and MPPED2 were further examined for complication susceptibility using additional SNPs that were not included in the GWAS. Individual genotyping of KLF7 rs2284932 revealed that the frequency of the minor C allele was significantly increased (P=0.00062, Pc=0.0018, OR=2.98, 95%CI=1.58-5.65) in group A. The minor T allele of rs4473559 in FRMD4 demonstrated a significant association in the A group (P=0.00015, OR=3.38, 95%CI=1.77-7.65). In the LOC101928923 gene, the frequency of the minor C allele of rs4379306 was significantly decreased in group A in both TaqMan and GWAS analyses. Lastly, the minor C allele of MPPED2 rs514644 carried a significantly increased risk of complications. These four genes may be linked with the onset of lachrymal/salivary gland lesions in type 1 AIP patients and require further study.