Glioma Association and Balancing Selection of ZFPM2

Abstract

ZFPM2, encoding a zinc finger protein and abundantly expressed in the brain, uterus and smooth muscles, plays important roles in cardiac and gonadal development. Abnormal expression of ZFPM2 in ovarian tumors and neuroblastoma has been reported but hitherto its genetic association with cancer and effects on gliomas have not been studied. In the present study, the hexamer insertion-deletion polymorphism rs71305152, located within a large haplotype block spanning intron 1 to intron 3 of ZFPM2, was genotyped in Chinese cohorts of glioma (n = 350), non-glioma cancer (n = 354) and healthy control (n = 463) by direct sequencing and length polymorphism in gel electrophoresis, and ZFPM2 expression in glioma tissues (n = 69) of different grades was quantified by real-time RT-PCR. Moreover, potential natural selection pressure acting on the gene was investigated. Disease-association analysis showed that the overall genotype of rs71305152 was significantly associated with gliomas (P = 0.016), and the heterozygous genotype compared to the combined homozygous genotypes was less frequent in gliomas than in controls (P = 0.005) or non-glioma cancers (P = 0.020). ZFPM2 mRNA expression was negatively correlated with the grades of gliomas (P = 0.002), with higher expression levels in the low-grade gliomas. In the astrocytoma subtype, higher ZFPM2 expression was also correlated with the rs71305152 heterozygous genotype (P = 0.028). In addition, summary statistics tests gave highly positive values, demonstrating that the gene is under the influence of balancing selection. These findings suggest that ZFPM2 is a glioma susceptibility gene, its genotype and expression showing associations with incidence and severity, respectively. Moreover, the balancing selection acting on ZFPM2 may be related to the important roles it has to play in multiple organ development or associated disease etiology.

Fig 1. Linkage disequilibrium (LD) plot generated using Haploview.

The plot depicts the haplotype block containing the rs71305152 indel for the HapMap CHB (Han Chinese in Beijing) population. The rs numbers of featured SNPs are given above the plot and the red arrow indicates the location of the rs71305152 indel. The LD plot for the whole ZFPM2 gene is given in the inset and the box within the inset indicates the depicted, enlarged region. The standard Haploview LD color scheme based on D’ and LOD (log of the likelihood odds ratio) is used and the value of 100 x D’ for each SNP pair is given in its respective tile unless D’ = 1.

Fig 2. The hexamer insertion-deletion polymorphism rs71305152 in ZFPM2.

(A) Schematic diagram showing that rs71305152 is located within intron 2 of ZFPM2, between two neighboring head to tail oriented AluSx and AluSp elements. The distance of rs71305152 to each exon and Alu element is indicated. (B) and (C) show the three genotypes of homozygous insertion (II), heterozygous (ID) and homozygous deletion (DD) identified by direct DNA sequencing and gel electrophoresis, respectively. M denotes 5-bp DNA size marker.

Fig 3. Association of rs71305152 genotypes with gliomas.

In part (A), the heterozygous genotype was significantly decreased in the glioma cohort compared to either the control cohort (P = 0.005), or the non-glioma cancer cohort (P = 0.020). In part (B), the heterozygous genotype was only significantly decreased in the male glioma cohort (P = 0.020) but not in the female glioma cohort (P = 0.148) compared to control. Red bars represent the combined homozygous deletion (upper portion) and homozygous insertion (lower portion) genotypes, and green bars represent the heterozygous genotype. The sample size of each genotype group is shown within the bars.

Fig 4. Correlation of ZFPM2 expression with glioma grades and rs71305152 genotypes.

(A) Gel electrophoresis of real-time qRT-PCR products of ZFPM2 and the two housekeeping genes HPRT1 and TBP. M denotes 100-bp DNA size marker. (B) Strong negative correlation between the ZFPM2 expression and the three glioma grades (P = 0.006). (C) Correlation of ZFPM2 expression with rs71305152 genotypes for astrocytomas (Upper panel; P = 0.028), and oligodendroglial tumors (Lower panel; P = 0.347), respectively. In parts B and C, the horizontal line in each box indicates the median; the upper and lower bounds of the box represent the 75th and 25th percentiles, respectively; the whiskers for each box mark either the values 1.5 times the interquartile range from the upper and lower edge of the box or the maximum and minimum values, whichever is the smaller; and the circles indicate outliers.

Fig 5. Correlation of ZFPM2 genotype and expression with survival duration.

(A) Survival duration profiles of different genotype groups for GBM. Black line, dashed line and grey dotted line represent homozygous insertion (II) genotype (n = 8, Male/Female = 1.66), heterozygous (ID) genotype (n = 12, Male/Female = 0.71) and homozygous deletion (DD) genotype (n = 27, Male/Female = 3.50) of rs71350512, respectively. The median survival time of the above three genotypes was 43 (range 11–149) weeks, 33 (range 2–133) weeks, and 39 (range 2–282) weeks, respectively. The P value based on the log rank test of Kaplan-Meier analysis was 0.339. (B) Correlation of ZFPM2 expression with survival duration. The Cox regression plot for 15 glioma patients with ZFPM2 expression level as covariate (exp(B) = 0.949; P = 0.077).

Fig 6. Summary statistics for ZFPM2 are plotted for the 11 HapMap populations.

The Tajima’s D (red circles), Fu and Li’s D* (blue circles) and Fu and Li’s F* (grey columns) values were obtained using HapMap Phase III data. The populations are ordered according to increasing Fu and Li’s F* values. All values significantly deviated from neutrality (P ≤ 0.01); * and ** depicts Tajima’s D values for which coalescent simulation gave P ≤ 0.01 and P < 0.001, respectively. The HapMap population descriptors are as follows: ASW: African ancestry in Southwest USA, CEU: Utah residents with Northern and Western European ancestry from the CEPH collection, CHB: Han Chinese in Beijing, China, CHD: Chinese in Metropolitan Denver, Colorado, GIH: Gujarati Indians in Houston, Texas, JPT: Japanese in Tokyo, Japan, LWK: Luhya in Webuye, Kenya, MEX: Mexican ancestry in Los Angeles, California, MKK: Maasai in Kinyawa, Kenya, TSI: Tuscan in Italy, YRI: Yoruban in Ibadan, Nigeria.