Single-nucleotide Polymorphism rs2275294 in ZNF512B is not Associated with Susceptibility to Amyotrophic Lateral Sclerosis in a Large Chinese Cohort.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that primarily affects motor neurons and has no effective treatment. Recently, Iida et al. identified a single-nucleotide polymorphism (SNP) rs2275294 in the ZNF512B gene that is significantly associated with susceptibility to ALS in the Japanese population. Here, we performed a case-control study examining the possible association of rs2275294 with risk of sporadic ALS (SALS) in a large Chinese cohort.
METHODS: To assess this association, we performed a replication study in 953 SALS patients and 1039 age- and gender-matched healthy control subjects, who were recruited from Peking University Third Hospital and the First Affiliated Hospital of Anhui Medical University from January 2004 to December 2013 throughout China. We genotyped the rs2275294 SNP using polymerase chain reaction and direct sequencing.
RESULTS: The allele frequency of rs2275294 in ZNF512B was different between Japanese and Chinese. The association in Chinese between ALS patients and controls did not reach statistical significance (P = 0.54; odds ratio = 0.94; 95% confidence interval = 0.76-1.15).
CONCLUSIONS: The SNP rs2275294 in ZNF512B is not considered to be associated with ALS susceptibility in the Chinese population. Our study highlights genetic heterogeneity in ALS susceptibility in different population. Given our negative results, further replication study involving larger and more homogeneous samples in different ethnicities should be performed in the future.

INTRODUCTION

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder caused by selective loss of motor neurons in the spinal cord, brainstem, and motor cortex, which is characterized by progressive wasting and weaknesses of the limb, bulbar, and respiratory muscles. In the absence of effective treatment, most patients die of respiratory failure within 3–5 years after onset.[1] Worldwide, familial ALS (FALS) accounts for 10% of all ALS cases with the remainder comprising sporadic ALS (SALS), which may result from combinations of genetic and environmental factors.[2] Eighteen different genes associated with FALS have been identified to date, including the superoxide dismutase 1 (SOD1), TARDNA-binding protein 43, fused in sarcoma/translocated in liposarcoma (FUS/TLS), and hexanucleotide repeat expansion in C9ORF72.[345] Increasing numbers of ALS susceptibility and modifier loci have been suggested, and there has been a great deal of research progress regarding the genes and molecular mechanisms related to ALS.[367] However, many remain unknown about the causes and mechanisms of ALS.

Although dozens of genes involved in FALS have been identified, little is known about the genetic contributions to SALS. Candidate gene studies have reported possible ALS risk genes, but they have been difficult to replicate. Some genome-wide association studies (GWASs) identified single-nucleotide polymorphisms (SNPs) in the FGGY, ITPR2, DPP6, KIFAP3, and UNC13A genes associated with SALS susceptibility.[8910111213] These results are promising, but remain slightly controversial.[1415161718192021] The association of the 9p21.2 locus has been independently replicated in four studies,[1920212223] but is not found in all population, including those from Japan to China.[2223] Our GWAS study identified two new susceptibility loci and suggested new pathogenic mechanisms of ALS.[24] Therefore, more studies are necessary to evaluate and confirm these previously reported ALS susceptibility genes in the Chinese population.

Recently, Iida et al. performed a large-scale case–control association study using gene-based tag (SNPs) and identified a new SNP (rs2275294) in the ZNF512B gene that is significantly associated with an increased risk of SALS in the Japanese population.[25] ZNF512B was originally identified as KIAA1196 in the Kazusa human cDNA sequencing project and was shown to be involved in cell differentiation and embryonic development. Similar to other transcription factors, ZNF512B was expressed in many tissues, including the brain and spinal cord.[2627] Tetsuka et al. reported that the ZNF512B gene is a prognostic factor in patients with ALS.[28] In addition, ZNF512B was suggested to be an important positive regulator of transforming growth factor β (TGF-β) signaling with decreased ZNF512B expression related to increased susceptibility to ALS. However, our previous GWAS suggested genetic heterogeneity in ALS susceptibility in Han Chinese and European population.[24] In this study, we checked the replication of the association by examining the rs2275294 SNP in 953 SALS patients and 1039 age- and sex-matched healthy controls in Han Chinese population.

METHODS

Participants

ALS patients diagnosed as having probable or definite ALS according to the El Escorial revised criteria and without family history (sporadic) were included in the study.[29] A total of 953 ALS patients and 1039 healthy control subjects were recruited from Peking University Third Hospital and the First Affiliated Hospital of Anhui Medical University between January 2004 and December 2013 throughout China. All ALS and control subjects provided written informed consent to participate in the study in accordance with the process approved by the Institutional Ethics Committee of Peking University Third Hospital. Patients with mutations in SOD1, TARDBP, and FUS determined by sequencing of their genomic DNA were not included in the study. A total of 1039 age- and sex-matched healthy control subjects with no previous personal or family history of neurodegenerative disease were also included as controls.

Genotyping

Genomic DNA was extracted from whole blood using standard protocols (Qiagen, Valencia, CA, USA). We genotyped the rs2275294 SNP using polymerase chain reaction (PCR) and direct sequencing.[26] PCR was performed using 20 ng genomic DNA-derived from ALS and control subjects, and amplification was performed in the GeneAmp PCR System 9700 (PE Applied Biosystems, Foster City, CA, USA) under the following conditions: Initial denaturation at 95°C for 2 min, followed by 35 cycles of denaturation at 96°C for 30 s, annealing at 60–65°C for 30 s, extension at 72°C for 2 min, and postextension at 72°C for 7 min. The PCR products served as templates for direct sequencing using the fluorescent dye-terminator cycle sequencing method. Meta-analysis was performed by an inverse variance method.

Statistical analysis

We used exact methods to assess cases and controls separately for Hardy–Weinberg equilibrium (HWE). Values were expressed as n or mean ± standard deviation (SD). Differences in genotype frequencies between the patients and controls were compared through a chi-squared test. Minor allele frequencies and odds ratios (ORs) with 95% confidence interval (CI) were estimated to determine the role of the SNP. Ages between the two groups were analyzed using Student's t-test. A two-tailed P ≤ 0.05 was considered statistically significant. Statistical analyses were performed using the SPSS version 16.0 software package (SPSS Inc., Chicago, Illinois, USA).

RESULTS

The demographic information of the samples (953 SALS and 1039 controls) is shown in Table 1. The study population consisted of 953 patients, including 617 male patients (64.74%) and 336 female patients (35.26%). The male-to-female ratio for the cohort was 1.84:1. Limb onset occurred in 820 cases, while bulbar onset was observed in the others. The mean age at onset of the ALS patients was 50.91 ± 12.00 years, similar to that of the controls (50.24 ± 14.96 years) [Table 1].

Table 1

Demographic description of SALS patients and control subjects in Han Chinese

Items	SALS (n = 953)	Control (n = 1039)
Male, n (%)	617 (64.74)	659 (63.43)
Female, n (%)	336 (35.26)	380 (36.57)
Age on set (years), mean±SD	50.91 ± 12.00	50.24 ± 14.96
Spinal onset, n (%)	820 (86.04)	–
Bulbar onset, n (%)	133 (13.96)	–

–: Data not applicable; SALS: Sporadic amyotrophic lateral sclerosis; SD: Standard deviation.

The respective genotype distributions in ALS patients and controls did not significantly deviate from HWE. Table 2 summarizes the association of rs2275294 with ALS in Chinese compared to Japanese. However, the allele frequency of rs2275294 in ZNF512B was different between Chinese and Japanese population. There is no significant difference in the prevalence of this SNP (rs2275294) between ALS patients and controls (P = 0.54; OR = 0.94; 95% CI = 0.76–1.15). Based on the negative result of this replication study, we considered no association to exist between the rs2275294 SNP in ZNF512B and SALS susceptibility in the Chinese population.

Table 2

Association of rs2275294 with ALS in Chinese compared to Japanese

Population	Samples (n)		Risk allele frequency		P	OR (95% CI)
	ALS	Controls	ALS	Controls
Japanese (by Iida et al.)
Discovery	454	958	0.49	0.42	6.3×10−4	1.32 (1.13–1.55)
Set 1	249	1030	0.51	0.43	1.80×10−3	1.37 (1.12–1.66)
Set 2	602	2256	0.48	0.42	5.60×10−5	1.30 (1.14–1.48)
Combined	1305	4244			9.30×10−10	1.32 (1.21–1.44)
Chinese (by Yang et al.)
Replication	301	457	0.52	0.46	0.035	1.25 (1.02–1.53)
Chinese (in our study)
Replication	953	1039	0.50	0.51	0.54	0.94 (0.76–1.15)

ALS: Amyotrophic lateral sclerosis; OR: Odds ratio; CI: Confidence interval.

DISCUSSION

The current report is a large-scale study of the genetic susceptibility to ALS, which we presented an SNP analysis of the ZNF512B gene in a Chinese cohort comprising 953 ALS cases and 1039 controls. This study confirms that the rs2275294 SNP in ZNF512B is negatively associated with susceptibility to ALS in Chinese (P = 0.54; OR = 0.94; 95% CI = 0.76–1.15). Meanwhile, we could not confirm whether the ZNF512B gene is a prognostic factor in Chinese ALS patients. However, our results help improve knowledge regarding ZNF512B in the Chinese population.

A previous study by Iida et al.[25] suggested that a new SNP rs2275294 is significantly associated with an increased risk of ALS in Japanese subjects. This locus is located in an enhancer region of ZNF512B, which is a transcription factor regulating the TGF-β signaling pathway. This risk allele can decrease ZNF512B expression and consequently increase susceptibility to ALS. In addition, it was reported that ZNF512B is widely expressed in the spinal cord of ALS patients, and ZNF512B showed significantly increased expression in the motor neurons of ALS patients compared with controls. Iida et al.[25] performed luciferase assays and electrophoretic mobility shift assays to determine allelic differences in the ZNF512B expression level and showed that ZNF512B expression level was lower in patients with susceptibility alleles compared with those with nonsusceptibility alleles. The expression of susceptibility alleles led to reduced ZNF512B enhancer activity, which in turn led to increased ZNF512B insufficiency, eventually resulting in insufficient TGF-β signaling activation and in reduced potential for motor neuron survival and functional recovery.

In the absence of effective treatments, most ALS patients die of respiratory failure within 3–5 years after onset. Tetsuka et al.[28] used Kaplan–Meier survival curves to evaluate the prognosis of ALS in comparison with the survival time of patients without the risk allele. The results indicated that the mean survival time of patients without the risk allele was approximately 12 months longer than that of patients with the risk allele. In addition, Tetsuka et al.[28] found that the ZNF512B gene was a new prognostic factor for ALS, independent of gender, age, time from onset to diagnosis, site of symptom onset (bulbar/spinal cord), and treatment with riluzole. However, the study was based on a small sample of 176 patients, and they obtained different results from the original research of Iida et al. included 1305 ALS patients. The small sample size might have influenced the results. Although we also conducted a study in 953 patients and performed statistical analyses and related methods, we obtained a negative result regarding the association between rs2275294 and SALS susceptibility in Chinese subjects. Therefore, our results were not similar to studies of Iida et al.[25] and Tetsuka et al.[28]

During our manuscript under revised, Yang et al. reported that the CC genotype and C allele at rs2275294 in ZNF512B are associated with increased risk of ALS in Han Chinese, particularly females, while no significant differences were identified in patients with PD.[30] The study included 301 patients with ALS and 457 controls, as well as 555 patients with PD and 473 controls. The results agree with similar studies in Japan, suggesting that rs2275294 affects risk of ALS in East Asian population.

However, our study did not show any evidence of an association between SALS and the rs2275294 variant in the Chinese population. Similar to other controversial causative SNPs of SALS,[14151617181920212223] there are conflicting results regarding rs2275294. There were several possible reasons for the negative association found in the replication analysis. First, ALS may be a more genetically and clinically heterogeneous disease among different population than previously recognized.[24] Second, due to reciprocities of unknown specific gene–gene or gene–environment interactions in the different onset age, the influences of some risk alleles identified by GWAS may be population-specific. Due to the different environmental and occupational factors, the age of onset of ALS patients from Guangzhou is younger than those from Shanghai.[31] Third, sample size is very important, and a small sample size can result in insufficient power, thus bringing about type 1 errors in the results.[33233] Our sample size including 953 ALS and 1039 controls is much higher than the study conducted by Yang et al. (including 301 ALS and 457 controls). Therefore, different investigation may yield different results, and reliable identification of the risk alleles of ALS will require studies using much larger sample sizes. This emphasizes the importance of identifying and replicating risk loci of SALS in independent cohorts. Hence, the public availability of raw genotype data will be helpful for increasing the reliability of future study.

In summary, our replication findings did not provide evidence of an association between the variant rs2275294 in the ZNF512B gene and an increased risk of SALS in a Chinese cohort. Our study also highlights the genetic heterogeneity in ALS susceptibility in different population. Given our negative results, future research would benefit from increased sample sizes in different ethnicities and to understand the more genetic heterogeneity fully.

Financial support and sponsorship

The research was supported by grants from the National Natural Science Foundation of China (No. 81473042, No. 31171048, No. 81072374).

Conflicts of interest

There are no conflicts of interest.