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Neurobiology of Aging

journal homepage: www.elsevier .com/locate/neuaging

The APOE ε2 allele may decrease the age at onset in patients withspinocerebellar ataxia type 3 or Machado-Joseph disease from theChinese Han population

Huirong Peng a,1, Chunrong Wang a,1, Zhao Chen a,1, Zhanfang Sun a, Bin Jiao a, Kai Li a,Fengzhen Huang a, Xuan Hou a, Junling Wang a,b, Lu Shen a,b,c, Kun Xia c, Beisha Tang a,b,c,Hong Jiang a,b,c,*

aDepartment of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan, P. R. ChinabKey Laboratory of Hunan Province in Neurodegenerative Disorders, Central South University, Changsha, P. R. Chinac State Key Lab of Medical Genetics, Central South University, Changsha, Hunan, P. R. China

a r t i c l e i n f o

Article history:Received 23 August 2013Received in revised form 16 February 2014Accepted 14 March 2014

Keywords:SCA3/MJDATXN3 geneAPOE geneAllelesGenotypes

* Corresponding author at: Department of NeuroloSouth University, Changsha, Hunan 410008, P. R. Chifax: þ86 731 84327332.

E-mail address: jianghong73868@126.com (H. Jian1 Huirong Peng, Chunrong Wang, and Zhao Chen

work.

0197-4580/$ e see front matter � 2014 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.neurobiolaging.2014.03.020

a b s t r a c t

Polymorphism of the apolipoprotein E (APOE) gene has been defined as a modifying factor for age atonset (AO) in neurodegenerative disorders. The AO of spinocerebellar ataxia type 3 or Machado-Josephdisease (SCA3 or MJD) is inversely correlated with expanded CAG repeat lengths in the ATXN3 gene;however, AO is only partially explained by the expanded CAG repeats. We performed a case-control studyto explore whether APOE genotypes play a role in AO of SCA3 or MJD from the Chinese Han population.The APOE genotypes were analyzed in an independent cohort of 155 patients with SCA3 or MJD and 191controls both from Mainland China. Our study demonstrated that SCA3 or MJD patients experienced anearlier onset if they were carriers of APOE ε2 allele, which decreased the AO by nearly 4 years. This studymay also reconfirm the effect of the APOE gene on SCA3 or MJD patients from different races andindicated that certain APOE alleles might be genetic modifiers for AO in SCA3 or MJD.

� 2014 Elsevier Inc. All rights reserved.

1. Introduction

Machado-Joseph disease (MJD), also known as spinocerebellarataxia type 3 (SCA3), is a group of highly heterogeneous neurode-generative disorders characterized by progressive cerebellarsymptoms of imbalance, gait and limb ataxia, and dysarthria (Costaand Paulson, 2012; Paulson, 2012). The most prevalent subtype inChina and other countries, SCA3 or MJD is caused by pathogenicCAG expansions in the ATXN3 gene (Jiang et al., 2005; Kawaguchiet al., 1994; Moseley et al., 1998; Schols et al., 1995; Tang et al.,2000; Wang et al., 2009). Wild-type ATXN3 alleles contain 12 to44 CAG repeats, whereas pathogenic alleles have more than 52repeats (Maciel et al., 2001; Tang et al., 2000; van Alfen et al., 2001).The mean age at onset (AO) is nearly 40 years; however, AO varieswidely, from 4 years to 75 years (Carvalho et al., 2008; Sequeirosand Coutinho, 1993). The CAG repeat lengths in SCA3 or MJD is

gy, Xiangya Hospital, Centralna. Tel.: þ86 731 84327216;

g).contributed equally to this

ll rights reserved.

inversely related to age of disease onset and directly related todisease severity (Costa and Paulson, 2012); however, only 50%e80%of onset variance has been attributed to CAG repeat lengths,implying that additional modulators may influence AO (Scholset al., 1996; van de Warrenburg et al., 2002, 2005).

Apolipoprotein E (APOE), located at chromosome 19q13.2, is animportant protein involved in lipid metabolism, hormone trans-port, proteolytic activity, coagulation factor regulation, and regen-eration mechanisms (Siest et al., 1995). The human APOE gene has 3common isoforms, E2, E3, and E4, which are encoded by 3 alleles,ε2, ε3, and ε4, respectively (Siest et al., 1995; Weisgraber et al.,1981). apoE is highly expressed in intracellular and extracellularsites and is synthesized and secreted by astrocytes in the centralnervous system (Boyles et al., 1985; Hatters et al., 2006). Themajor role of apoE produced in the brain may be in redistribution oflipids among cells to maintain cerebral cholesterol homeostasis(Handelmann et al., 1992; Siest et al., 1995). Additionally, apoE hasseveral other functions, such as immunoregulation (Cardin et al.,1988), nerve regeneration (Handelmann et al., 1992), and antioxi-dant activity (Miyata and Smith, 1996).

The association of APOE genotype with several neurodegenera-tive disorders has previously been investigated. The APOE ε4 allele

H. Peng et al. / Neurobiology of Aging xxx (2014) 1e42

was identified as a risk factor in AD, which decreased the AO ofsymptoms and increased risk of cognitive impairment (Boardmanet al., 2012; Sando et al., 2008; Schipper, 2011; Wierenga et al.,2012) and is associated with molecular pathways that promoteLate-onset Alzheimer’s disease (LOAD) (Rhinn et al., 2013), whereasthe APOE ε2 is associated only with delayed onset of symptoms(Schipper, 2011). Similarly, several studies found evidence forsignificantly earlier AO in PD patients with the APOE ε2/ε3 genotype(Inzelberg et al., 1998; Maraganore et al., 2000; Zareparsi et al.,2002), indicating the variable AO of complex diseases influencedby the APOE gene. As for polyglutamine diseases, the association ofthe APOE gene with onset of HD is controversial. One study foundthat the APOE ε2/ε3 genotype was associated with earlier onset inmales (Kehoe et al., 1999), whereas another found that the APOEgenotype did not significantly affect the course of HD (Saft et al.,2004). In SCA3 or MJD patients, there has been only one report(from Azores, Mainland Portugal, and Brazil) that the APOE ε2 alleleincreased the risk of earlier AO fromAzores, Mainland Portugal, andBrazil, suggesting that polymorphisms of the APOE gene have roleas genetic modifiers of SCA3 or MJD genotypes (Bettencourt et al.,2011). Until now, no analysis of APOE polymorphisms in ChineseSCA3 or MJD patients has been reported. Here, we performed acase-control study to analyze the roles of APOE gene on the phe-notypes of SCA3 or MJD in Mainland China.

2. Methods

2.1. Subjects and materials

A total of 155 unrelated Chinese SCA3 or MJD patients (91 malesand 64 females) from the outpatient neurology clinic of XiangyaHospital, Central South University were enrolled in our study.Clinical data were as follows: mean age of 42.84 � 11.03 years(range 19e75 years). This case-control study included 191 controlsmatched for age, gender, ethnicity, and area of residence. Informedconsent was obtained from all subjects for their participation in thisstudy.

All SCA3 or MJD patients were analyzed for CAG repeat lengthsand AO. Recombinant DNA technology, including T-vector cloning,followed by direct DNA sequencing was used to evaluate the CAGrepeat lengths in ATXN3 gene. The AO was defined as the age atwhich the patient, or a close person, noticed the first symptoms(usually gait unbalance). Patients with long disease duration wereasked for the AO of the mentioned symptoms. Additional infor-mation from previous records and the scores of internationalcooperative ataxia rating scale from 2 experienced neurologistswere also taken into account to get a more accurate AO. None of thesubjects had cardiac disease, tumors, or other neurologic disease.

Table 1The distribution of APOE alleles and genotypes in SCA3/MJD and control groups

Group Genotypes (n, %)

Total ε2/2 ε2/3 ε2/4

Patients 155 3 (1.94) 14 (9.03) 1 (0.65)SexMale 91 1 (1.10) 7 (7.69) 1 (1.10)Female 64 2 (3.13) 7 (10.94) 0 (0)

Controls 191 0 (0) 32 (16.75) 2 (1.05)SexMale 104 0 (0) 17 (16.35) 1 (0.96)Female 87 0 (0) 15 (17.24) 1 (1.15)

The numbers in parentheses of alleles and genotypes represent frequency (%). No ε4/4 gPatients compared with controls by genotype: odd ratio ¼ 0.836, 95% CI: (0.656, 1.064),Patients compared with controls by allele: odd ratio ¼ 0.802, 95% CI: (0.563, 1.142), p ¼Key: APOE, apolipoprotein E; CI, confidence interval; MJD, Machado-Joseph disease; SCA

All subjects including SCA3 or MJD patients and controls wereanalyzed for their APOE genotypes. Genomic DNA was extractedfrom peripheral blood leukocytes following standard extractionmethods (Miller et al., 1988). The amplification reaction contained1 mL genomic DNA (50 ng/mL), 0.1 mL LA Taq DNA Polymerase(TaKaRa, Japan), 5 mL 2� GC Buffer I (TaKaRa, Japan), 0.4 mL dNTPs,and 0.2 mL of each primer and sterile water to volume. Primers weredesigned according to a previous study: 50-ACGCGGGCACGGCTGTCCAAGGA-30 (forward) and 50-GCGGGCCCCGGCCTGGTACAC-30

(reverse) (Wierenga et al., 2012). The polymerase chain reactionwas performed in thermocyclers (Perkin-Elmer, Inc, Foster City, CA,USA) using the following amplification conditions: initial denatur-ation at 94.0 �C for 3 minutes followed by 30 cycles of 94.0 �C for30 seconds and 72.0 �C for 60 seconds with a final extension at72.0 �C for 4 minutes. Purified polymerase chain reaction productswere used for DNA sequencing on an ABI 3730XL DNA analyzer(Applied Biosystems, Foster City, CA, USA).

2.2. Statistics

Frequencies of APOE alleles and genotypes between SCA3 or MJDpatients and controls were calculated and tested for statisticalsignificance using the c2 test. We used multivariate linear regres-sion analyses to test the effect of several potential variables on AO,including the CAG repeat lengths, the presence or the absence ofthe APOE ε2 allele, and gender. The difference of the distributionof CAG repeat lengths in the 2 groups, the presence or the absenceof the APOE ε2 allele was analyzed using the ManneWhitney test.To further explore whether the presence of APOE ε2 influence AO, acovariance analysis model was used to adjust the effect of expendedCAG repeats. All analyses were performed using the SPSS Statisticssoftware (version 17.0). A p-value �0.05 was considered significant.

3. Results

The frequencies of alleles and genotypes are summarized(Table 1). Five genotypes (ε2/ε2, ε2/ε3, ε2/ε4, ε3/ε4, and ε3/ε3)appeared in SCA3 or MJD patients group, and the APOE ε4/ε4 ge-notype was not detected in any subject (Fig. 1). The frequency ofAPOE genotypewas not significantly different between SCA3 orMJDpatient and control groups (odd ratio¼ 0.836, 95% CI: [0.656,1.064],p¼ 0.145). In addition, therewas no significant difference regardingthe frequency of APOE alleles between the 2 groups (odd ratio ¼0.802, 95% CI: [0.563, 1.142], p ¼ 0.221).

Additionally, AO of SCA3 or MJD was negatively correlated withthe length of CAG repeat in the expanded ATXN3 gene (presentseries, r ¼ �0.645, p ¼ 0.000) (Fig. 2). No significant difference wasdetected in the distribution of the CAG repeat lengths between 2

Alleles (%)

ε3/4 ε3/3 ε2 ε3 ε4

34 (21.94) 103 (66.45) 6.77 81.94 11.29

21 (23.08) 61 (67.03) 5.49 82.42 11.5413 (20.31) 42 (65.62) 8.59 81.25 10.1634 (17.80) 123 (64.40) 8.90 81.68 9.42

19 (18.27) 67 (64.42) 8.65 81.73 9.6215 (17.24) 56 (64.37) 9.20 81.61 9.20

enotype was detected in any subject.p ¼ 0.145.0.221.3, spinocerebellar ataxia type 3.

Fig. 2. The association of expanded CAG repeats with age at onset (AO) in patients withSCA3/MJD. The X-axis denotes the expanded CAG repeat lengths and the Y-axis in-dicates age at onset in years. AO of SCA3/MJD was negatively correlated with the lengthof CAG repeat in the expanded ATXN3 gene (r ¼ �0.645, p ¼ 0.000). Abbreviations:MJD, Machado-Joseph disease; SCA3, spinocerebellar ataxia type 3.

Fig. 1. Frequencies of APOE genotypes in patients with SCA3/MJD. The 3 most frequentAPOE genotypes were (in decreasing order): ε3/3, ε3/4, and ε2/3. The APOE ε4/4 ge-notype was not detected in any subject. Abbreviations: APOE, apolipoprotein E; MJD,Machado-Joseph disease; SCA3, spinocerebellar ataxia type 3.

H. Peng et al. / Neurobiology of Aging xxx (2014) 1e4 3

groups (the presence and absence of the APOE ε2 allele) (p¼ 0.588).Based on one previous report that APOE ε2 allele might be a riskfactor for earlier onset (Bettencourt et al., 2011), the role of thepresence or the absence of the APOE ε2 allele in AO of SCA3 or MJDpatients was analyzed. When APOE ε2 status was taken intoconsideration (to dissociate the effect of the ε2 from that of the ε4allele, the patient with ε2/ε4 genotype was excluded), the per-centage of explanation of the onset variance increased from 44.6%to 45.9% (F ¼ 21.83, p ¼ 0.000). When gender was included as avariable, the model was not significantly improved (data notshown). Subsequently, in the covariance analysis model, SCA3 orMJD patients were divided into 2 groups (the presence or theabsence of the APOE ε2 allele) to investigate the effect of ε2 on AOand the expended CAG repeats as the covariate. Remarkably, afteradjusting for the size of expended CAG repeats, the presence ofε2 allele decreased the AO by approximately 4 years (F ¼ 4.48,p ¼ 0.036) (Table 2).

4. Discussion

In this study, we have investigated the frequencies of APOE al-leles and genotypes in SCA3 or MJD patients and controls but didnot find statistical significance between the 2 groups. Interestingly,we found an association between AO of SCA3 or MJD and APOEgenotypes. When the status of APOE ε2 was taken into account,explanation of AO variance improved 1%; however, in our study, theexpanded CAG repeats explained less than 50% AO variance, lowerthan recent a report (Bettencourt et al., 2011), which may bebecause of the differences in race and smaller sample size. Inaddition to the CAG repeat lengths in expanded alleles, AO of SCA3or MJD was significantly earlier in patients with the ε2/ε2 and ε2/ε3genotype than that with the ε3/ε3 and ε3/ε4 genotype. Further-more, similarly to previous research (Bettencourt et al., 2011), thepresence of APOE ε2 decreased the AO by nearly 4 years, whichshows that APOE may be a modifier for AO in MJD.

As mentioned earlier, variance of AO is only partially explainedby CAG repeat lengths. Taking into account the phenotypes affected

Table 2Age at onset in SCA3/MJD patients with APOE genotype

Characteristics APOE genotypes

ε2/ε2, ε2/ε3 (n ¼ 17)

Age at onset (y)Mean � SD (range) 35.59 � 7.37 (20e59)Adjusted, mean (SE)a 35.42 (1.74)

Number of CAG repeat lengthsExpanded, mean � SD (range) 73.88 � 4.27 (67e82)

Key: APOE, apolipoprotein E; MJD, Machado-Joseph disease; SCA3, spinocerebellar ataxia Adjusted for the mean size of expanded CAG repeats in the patients. One SCA3/MJD pa

of ε4.

by APOE gene, APOE genotype ubiquitously determines the efficacyof neuronal maintenance and repair in neurodegenerative diseases(Chapman et al., 2001; Handelmann et al., 1992; Mukherjee et al.,2003; Posse et al., 2000). In a rat model, hippocampal astrocytesrevealed a decreased affinity of apoE2 binding to cholesterolcompared with apoE3 or apoE4 (Rapp et al., 2006). This low affinityof apoE2 led to reduced cholesterol uptake in the hippocampalneurons and altered cholesterol homeostasis in the brain(Bettencourt et al., 2011). These findings imply that apoE may in-fluence neuronal function in the brain, which may be associatedwith the earlier manifestation of SCA3 or MJD in ε2 allele carriers.

Here, we analyzed the role of APOE alleles in patients with SCA3or MJD from the Chinese Han population, which may reconfirm theeffect of APOE gene on SCA3 or MJD patients from different racesand showed pairwise correlation between APOE ε2 and AO.Although our findings may be subjected to a relatively smallamount of samples and should be validated based upon largersamples, it is helpful to gain new insights into modifiers of SCA3 orMJD. Further studies of the relationship between SCA3 or MJD andAPOE alleles in different races using larger samples are necessary.Functional assays should be encouraged to elucidate the potentialrisk of APOE alleles in developing SCA3 or MJD. Additionally, thecorrelation between other polyglutamine diseases (including SCA1,SCA2, SCA6, SCA7, SCA17, HD, DRPLA, SBMA) and APOE alleles re-mains to be further investigated and would be beneficial to betterunderstanding of the relationship among causative genes, geneticmodifiers, and phenotypes.

ε3/ε3, ε3/ε4 (n ¼ 137) p-value

39.30 � 9.81 (16e62)39.32 (0.61) 0.036

74 � 3.85 (64e84)

a type 3; SD, standard deviation; SE, standard error.tient with ε2/ε4 genotype was removed to study the effect of ε2 dissociated from that

H. Peng et al. / Neurobiology of Aging xxx (2014) 1e44

Disclosure statement

The authors have reported no actual or potential conflicts ofinterest. Written informed consent was obtained from all in-dividuals. The study was approved by the Expert Committee ofXiangya Hospital of Central South University in China (equivalent toan Institutional Review Board).

Acknowledgements

The authors are grateful to all subjects for their participation inour study. The study was supported by the National Basic ResearchProgram (973 Program) (No. 2012CB944601, 2012CB517902,2011CB510002, to Hong Jiang), the New Century Excellent Talents inUniversity (No. NCET-10-0836, to Hong Jiang), the National NaturalScience Foundation of China (No. 81271260, 30971585 to HongJiang),HunanFunds forDistinguishedYoungScientists (No.14JJ1008toHong Jiang), XinjiangNatural Science Foundation (No.201318101-4 to Hong Jiang) and the Undergraduate Innovation Project of Cen-tral South University (No. CY12400 and YB13028 to Hong Jiang).

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