Positive Selection of CAG Repeats of the ATXN2 Gene in Chinese Ethnic Groups

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ScienceDirect Journal of Genetics and Genomics 40 (2013) 543e548

JGG LETTER TO THE EDITOR

Positive Selection of CAG Repeats of the ATXN2 Gene in Chinese Ethnic Groups The ataxin-2 (ATXN2) gene is located on human chromosome 12q24.1. In normal individuals, the coding region in exon 1 of this gene has fewer than 31 CAG repeats (Yu et al., 2005; Laffita-Mesa et al., 2012). However, an abnormal expansion of CAG trinucleotide repeats results in the aggregation of polyglutamine (polyQ), which causes spinocerebellar ataxia type 2 (SCA2) (Pulst et al., 1996). The expanded alleles have more than 32 repeats in the affected individuals, and generally there is an inverse correlation between CAG repeat length and age of onset (Pulst et al., 1996). SCA2 is an autosomal dominant inheritance neurodegenerative disease, whose major clinical feature is progressive cerebellar ataxia. Atrophies of the brainstem and frontal lobe have been frequently detected by magnetic resonance imaging (MRI) (Yamamoto-Watanabe et al., 2010). This disease has the strong effect on sensory and motor control. Previous studies indicated that the CAG repeat expansion in the ATXN2 gene has undergone positive selection in a European population, while no sign of selection was observed in two Asian groups, Han Chinese and Japanese (Yu et al., 2005). It was suggested that the geographic location and living environment of different populations might be the driving factors of the selection. In order to elucidate whether this locus is under positive selection in other populations in China, we investigated CAG repeats in the ATXN2 gene and eight single nucleotide polymorphisms (SNPs) surrounding this locus in six different ethnic groups, and carried out neutral tests in this study. Among these groups, there are two Mongolian groups from Baotou, Inner Mongolia Autonomous Region in the north of China (IM) and from Yuxi, Yunnan Province in the southwest of China (YN), respectively, with similar genetic background but with different geographic locations and living environments. These two groups were used to analyze the impact of environmental factors upon natural selection. Dai, Hani and Yi ethnic groups live close to the Mongolian group in Yunnan Province (YN), which help to understand whether the various genetic backgrounds contribute to selection. The natural condition of the Hui ethnic group was quite different from the others. This group was selected to analyze the effect of

natural selection on CAG repeats under complicated genetic background. The research was approved by the Ethics Committee at the Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College. All of the 291 individuals signed informed consents. To study positive selection of CAG repeats in Chinese ethnic groups, all of the samples from the six ethnic groups were first genotyped and sequenced. The results of microsatellite genotyping were completely consistent with their corresponding (CAG)n sequences. Based on the sequence polymorphism, we found 20 different alleles (AL-1 to AL-20). (CAG)8CAA(CAG)4CAA(CAG)8 (AL-11) was the most prevalent allele in the six ethnic groups, followed by (CAG)13CAA(CAG)8 (AL-10). The frequencies of the common alleles AL-10 and AL-11 were higher than the other alleles, ranging from 91% to 98% in the six ethnic groups. Furthermore, the six ethnic groups had their own particular rare alleles and the different rare allele distributions (Table 1). Eight SNPs were surveyed according to the previous description (Yu et al., 2005), which constructed a strong linkage disequilibrium (LD) core region under positive selection. The whole region spans approximately 71 kb surrounding exon 1 of the ATXN2 gene (Fig. S1). The haplotypes constructed by eight SNPs were diverse in the six ethnic groups, and the CAACCCGC (CH-1) was the most frequent core haplotype (71%e87%) (Table S1), which was different from the European population reported previously. The pairwise LD test was also performed using the data of the eight SNPs in the six groups. The result of this test was represented as D0 , which indicates the level of LD. In this study, a strong LD was shown among these SNPs spanning a 71 kb region, except for rs593226 in Yi, and rs653178 in Yi, Dai, Hani and Mongolian (YN) with less polymorphism than the others (Fig. 1). Tajima’s D test, Fu and Li’s D* test and F* test are classical neutral tests based on frequency spectrum of variation. The selection on the sequences of CAG repeats in this study was detected by performing these three tests (Tajima, 1989; Fu and Li, 1993). Different from Han Chinese and Japanese (Yu et al.,

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Table 1 Sequencing results and neutral tests of (CAG)n repeats in the ATXN2 gene in six Chinese ethnic groups Allele

Repeat

Sequence

Allele frequency in six groups (n) Hui

13

(CAG)13

18

(CAG)15(CCG)2CAG

AL-3

19

(CAG)10CAA(CAG)8

AL-4

19

(CAG)13CAA(CAG)5

AL-5

20

(CAG)8CAA(CAG)2CAA(CAG)8

AL-6

21

(CAG)12CAA(CAG)8

AL-7

21

(CAG)15CAA(CAG)5

AL-8

22

(CAG)22

AL-9

22

(CAG)8CAA(CAG)13

Mongolian (YN)

Mongolian (IM)

Yi

Dai

0.03 (3) 0.01 (1) 0.01 (1) 0.06 (6)

0.02 (2) 0.02 (2)

0.01 (1) 0.01 (1) 0.01 (1) 0.02 (2)

0.01 (1)

AL-10

22

(CAG)13CAA(CAG)8

0.33 (33)

0.12 (10)

0.26 (25)

0.30 (31)

0.30 (28)

0.34 (34)

AL-11

22

(CAG)8CAA(CAG)4CAA(CAG)8

0.58 (58)

0.82 (72)

0.69 (66)

0.61 (64)

0.65 (61)

0.64 (64)

AL-12

23

(CAG)14CAA(CAG)8

0.02 (2)

0.01 (1)

AL-13

24

(CAG)21CCG(CAG)2

AL-14

25

(CAG)16CAA(CAG)8

0.02 (2)

0.01 (1)

AL-15

25

(CAG)25

AL-16

26

(CAG)13CAA(CAG)8CAA(CAG)3

0.01 (1)

0.01 (1) 0.02 (2)

AL-17

27

(CAG)13CAA(CAG)13

0.01 (1)

AL-18

29

(CAG)29

0.01 (1)

AL-19

30

(CAG)21CAA(CAG)8

0.01 (1)

AL-20

31

(CAG)22CAA(CAG)8

0.01 (1)

Total

0.01 (1)

1 (100)

1 (88)

1 (96)

1 (104)

1 (94)

1 (100)

Tajima’s D

1.776

L2.052

L2.240a

L2.224a

L2.429a

L1.951

Fu and Li’s D*

0.463

0.066

0.492

L3.190

L3.563

Fu and Li’s F*

1.187

0.897

0.712

L3.359

L3.731

0.411

Tajima’s D of coalescent simulation

N.A.

1.626

1.384

1.603

1.680

1.609

Neutral tests 1.711

Tajima’s D test, Fu and Li’s D* test and F* test with significant P values (<0.05 level, two tails) were highlighted in bold. Based on historical records, different demographic scenarios were assigned to each group in the coalescent simulations. The simulated parameters of five ethnic groups were listed in Table S2. The coalescent simulations were performed 10,000 times to generate samples under these assumptions. N.A. means not analyzed. Furthermore, in multiple testing correction, Bonferroni correction was applied to these statistical tests. a means significant after Bonferroni correction (corrected P value cutoff ¼ 0.017).

Letter to the Editor / Journal of Genetics and Genomics 40 (2013) 543e548

AL-1 AL-2

Hani

Letter to the Editor / Journal of Genetics and Genomics 40 (2013) 543e548

545

Fig. 1. LD of eight adjacent SNPs in the ATXN2 gene in six ethnic groups. A: Dai. B: Hani. C: Hui. D: Mongolian (IM). E: Yi. F: Mongolian (YN). The SNPs are rs593226, rs616513, rs653178, rs695872, rs695871, rs3809274, rs1544396 and rs9300319. For rs653178, no polymorphism was observed in Dai, Hani, Yi, and less in Mongolian (YN). The red color indicates strong LD (D0 > 0.8).

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Letter to the Editor / Journal of Genetics and Genomics 40 (2013) 543e548

2005), significantly negative Tajima’s D values were observed in Dai, Hani, Yi, Mongolian (YN), and Mongolian (IM) groups. Also, significant Fu and Li’s D* and/or F* values were observed in Dai, Mongolian (IM), and Yi groups (Table 1). However, demographic expansion might also drive the results observed. To confirm the reliability of positive selection, Hudson’s MS program was used to accomplish coalescent simulations under a variety of assumptions (Hudson, 2002). The mutation rate for calculating parameter q was 1.16  108 per site per generation (20 years per generation) (Lynch, 2010). Different scenarios and parameters were set according to reliable ethnic historical records, effective population size and hypothesized East Asians origin event (Su et al., 1999) (Table S2). If demographic expansion has the impact on the alleles in these ethnic groups, significant values would be observed under our assumptions. Actually, there were no significant Tajima’s D values obtained from such simulations (Table 1), which means that demographic expansion might not influence the neutral tests on the CAG repeats of ATXN2, and implies that positive selection may act on the locus. Even after stringent Bonferroni correction, significant Tajima’s D values still existed in Mongolian (IM), Mongolian (YN) and Yi groups at a corrected significance level of 0.017 (Table 1). These results could not be generated by chance but by positive selection. From the long-range haplotype test, there were no significant results to support positive selection (Table S3). However, high frequency of CH-1 and strong LD were shown in the 71 kb core region, and the significances were observed in neutral tests. These results suggest the possibility of positive selection. CH-1 had the highest frequency in haplotypes, but other core haplotypes still showed genetic diversity in these groups. Our results indicate that the core region has not undergone a standard selective sweep. It is also possible that positive selection on this core region might be ongoing or not strong enough. Therefore, these factors can explain why significant evidence of long-range haplotype test was not detectable in these six groups. China is a unified multi-ethnic country with abundant population genetic resources, which provides many advantages on investigating natural selection in the human genome during evolutionary process. According to the history record, ancestors of two Mongolian groups were traced back to the Mongolian in Yuan Dynasty in China. In the year 1253 AD, about 100,000 Mongolians migrated to Yunnan and some of them settled there after 1283 AD. Since then, Mongolian (YN) has experienced significant changes in their environments, such as climate, living habits and even the challenge of pathogen pressure. It’s worth noting that positive selection signals of CAG repeats were observed in both of the two Mongolian groups, which suggests that some common features could be preserved in this segment and changes in the living environment of Mongolian (IM) and Mongolian (YN) were probably not the key factor in the differential selection on this locus. Furthermore, Dai, Yi and Hani in this study were also sampled from Yunnan, and these three ethnic groups and Mongolian (YN) are located in the similar geographic environment with initially observed significant negative values of

Tajima’s D test. It is thus clear that various genetic backgrounds have no effect on selection and may also be involved in the conservation of their primitive hereditary feature as a result of relative isolation. Besides, there was no significance of neutral tests in the Hui group. This population with complicated genetic background and living environment emerged from multiple groups of Muslims from Central Asia, West Asia, etc. The negative values observed could result from an excess of low-frequency (rare) alleles in Hui, and the detection of natural selection at this locus might be influenced by their complicated genetic background. The six ethnic groups studied here were different in genetic backgrounds, geographic origins, and living environments. Therefore, these ethnic resources could provide very valuable information to facilitate the analysis of nature selection in various genetic backgrounds and environments. Although this study didn’t include a larger sample size, it was less likely to have an impact on the interpretation of results from neutral test. However, a larger sample size would provide more complete genetic information. ATXN2 is the widely recognized pathogenic gene of SCA2, and it is very interesting that evolutionary forces have driven the selection for the ATXN2 gene in these ethnic groups. To disclose that the expansion in different ethnic groups would contribute to revealing clues of selection for the ATXN2 gene, we speculated two possible explanations to account for the observed phenomenon. The first one is that CAA interruption pattern could have advantages on hereditary stability of ATXN2. The common allele (CAG)8CAA (CAG)4CAA(CAG)8 (AL-11) with two CAA interruptions is the most frequent allele in the six ethnic groups, consistent with those of the European and Asian populations (Yu et al., 2005). This CAA interruption pattern could be correlated to hereditary stability, whereas the absence of CAA results in enhanced instability and the allele is prone to expanding in the transmission, thus easily leading to pathological expansion in the offspring (Choudhry et al., 2001; Charles et al., 2007). Furthermore, different hairpins can be formed because of the difference of RNA conformation. Slipped hairpin structure can be generated by uninterrupted CAG repeats, while the branched one can be formed by CAA interrupted expansions. Both of these two hairpin structures may have different abilities to interact with double-stranded RNA-binding proteins, which brings out the changes of mRNA folding and stability (Sobczak and Krzyzosiak, 2005; Charles et al., 2007). At mRNA level, CAA interruption could reduce the toxicity of abnormal ataxin-2 (Velazquez et al., 2009). We supposed that the AL-11 allele may be the predominant determinant of positive selection due to its stable conformation at DNA and mRNA levels as well as its less toxicity at protein level. Moreover, two sequence alternations (CAG / CCG) were observed in this study, i.e., (CAG)15(CCG)2CAG and (CAG)21CCG(CAG)2. The polymorphism is similar to that reported by Mizushima et al. (1999). The CCG interruption was stable in the sequence structure of (CAG)n(CCG)1e2CAG, and proline encoded by a CCG

Letter to the Editor / Journal of Genetics and Genomics 40 (2013) 543e548

trinucleotide could inhibit structure expansion and toxicity of polyQ (Mizushima et al., 1999; Bhattacharyya et al., 2006). We found that this CCG may take the place of a CAA interruption in the sequence. It looks that alleles with stable conformation are prone to be favored by selection and to be fixed in the population. The second explanation is that the gene pleiotropy of ATXN2 may confer a selective advantage that outweighs the health risk. Recently, ATXN2 gene has been implicated as a susceptibility gene for amyotrophic lateral sclerosis (ALS), which is a devastating neurodegenerative disease (Elden et al., 2010). It was found that the longer polyQ expansion (29 CAG repeats) was strongly associated with ALS (Daoud et al., 2011). The most frequent allele (CAG)8CAA(CAG)4CAA (CAG)8 (AL-11) in this study encodes 22 glutamine expansion, which has a relatively stable structure and less tendency of expansion. Therefore, AL-11 may play a role in preventing ALS predisposition in populations. In another words, the protection mechanism against ALS might drive this selection on the CAG repeat locus. Furthermore, the study of another neurodegenerative disorder also suggested that ATXN2 might modulate the age of onset in hereditary spastic paraplegia patients (Nielsen et al., 2012). In addition, ataxin-2, the ATXN2 gene product, can activate its own transcription and regulate RNA metabolism (Nonhoff et al., 2007; Hallen et al., 2011). The SNP rs653178 at the upstream region of ATXN2 was shown to be associated with hypertension (Levy et al., 2009). It is possible that this variant might regulate the expression of ataxin-2 and thereby further effect the regulation of blood pressure. Although we still need more evidence to support the hypothesis, the pleiotropy of ATXN2 was the plausible explanation for the observed phenomenon. In this study, CAG repeats in the ATXN2 gene present possible signals of positive selection in Mongolian (IM), Mongolian (YN) and Yi ethnic groups, but not in reported Han Chinese and Japanese populations (Yu et al., 2005). Moreover, selection on the locus is not sensitive to environmental factors in two Mongolian groups with significant geographic and climatic differences. More thorough and extended studies of the selection mechanism need to be done, which will demonstrate the complex evolutionary force in these ethnic groups. Meanwhile, the current study indicates that CAG repeats in humans could be under positive selection at the nucleotide level (Schaefer et al., 2012). CAG repeat accumulation of some pathogenic genes such as ATXN1 and ATXN3 (Orr et al., 1993; Kawaguchi et al., 1994) could cause other neurodegenerative diseases, and whether these genes undergo positive selection is worth further investigation. ACKNOWLEDGEMENTS This study was supported by the National Natural Science Foundation of China (No. 30400264), the Natural Science Foundation of Yunnan Province, China (No. 2008ZC068M) and the Chinese National High Technology Research and Development Program (No. 2012AA021802).

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SUPPLEMENTARY DATA Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jgg.2013.08.003. Xiao-Chen Chena, Hao Suna, Chang-Jun Zhangb, Ying Zhangb, Ke-Qin Lina, Liang Yua, Lei Shia, Yu-Fen Taoa, Xiao-Qin Huanga, Jia-You Chua, Zhao-Qing Yanga,* a

Department of Medical Genetics, Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, 935 Jiaoling Road, Kunming 650118, Yunnan, China

b

Reproductive Medicine Center, Hubei University of Medicine, Shiyan 442000, China *Corresponding author. Tel: þ86 871 6833 4872, fax: þ86 871 6833 4483. E-mail address: [email protected] (Z.-Q. Yang)

Received 6 April 2013 Revised 28 August 2013 Accepted 30 August 2013 Available online 24 September 2013

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