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Mutation analysis of GLT8D1 and ARPP21 genes in amyotrophic lateral sclerosis patients from mainland China
Neurobiology of Aging xxx (2019) 1.e1e1.e4
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Negative Results
Mutation analysis of GLT8D1 and ARPP21 genes in amyotrophic lateral sclerosis patients from mainland China Wanzhen Li a, Zhen Liu a, Weining Sun a, Yanchun Yuan a, Yiting Hu a, Jie Ni a, Bin Jiao a, Liangjuan Fang a, b, c, d, Jinchen Li b, Lu Shen a, b, c, d, Beisha Tang a, b, c, d, Junling Wang a, b, c, d, * a
Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China National Clinical Research Center for Geriatric Diseases, Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China Key Laboratory of Hunan Province in Neurodegenerative Disorders, Central South University, Changsha, Hunan, People’s Republic of China d Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, People’s Republic of China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 May 2019 Received in revised form 17 September 2019 Accepted 20 September 2019
Variants in exon 4 of gene encoding GLT8D1 (glycosyltransferase 8 domain containing 1) gene have recently been suggested as a novel cause of amyotrophic lateral sclerosis (ALS). In addition, there is a synergism between GLT8D1 and ARPP21 (cAMP Regulated Phosphoprotein 21) variants for ALS. However, this observation has not been validated in other ALS cohorts. In this study, we analyzed the rare pathogenic variants in GLT8D1 and ARPP21 genes in a cohort of 512 ALS patients and 3210 healthy controls from mainland China. A total of 25 rare variants in ARPP21 were identified in the patients and controls, but we did not find rare variants in exon 4 of GLT8D1 in the patients. By using Fisher’s exact test, we did not find significant association between ALS and GLT8D1 or ARPP21. Therefore, GLT8D1 and ARPP21 are not likely the causative genes for ALS in mainland China. Ó 2019 Elsevier Inc. All rights reserved.
Keywords: Amyotrophic lateral sclerosis Whole exome sequencing GLT8D1 ARPP21
1. Introduction Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects upper and lower motor neurons and results in muscular weakness and atrophy. Patients eventually die of respiratory failure within 2e5 years. The incidence of ALS is estimated to be 2e3 per 100,000 people in Europe and 0.7e0.8 per 100,000 people in Asia (Mathis et al., 2019). The mean age of onset is 65 years (Mathis et al., 2019). Most cases of ALS are sporadic (sALS), while approximately 10% of the patients have a familial history of ALS (Chia et al., 2018). To date, mutations in more than 40 genes have been associated with the pathogenesis of ALS (Chia et al., 2018; Corcia et al., 2019; Mathis et al., 2019; Nicolas et al., 2018; Ozoguz et al., 2015; Praline et al., 2010; Renton et al., 2014). Recently, variants in genes encoding GLT8D1 (glycosyltransferase 8 domain containing 1) and ARPP21 (cAMP Regulated Phosphoprotein 21) have been identified in ALS patients of European ancestry. In an autosomal-dominant ALS pedigree, the authors identified p.R92C mutations in GLT8D1, which co-segregate with * Corresponding author at: Xiangya Hospital, Central South University, 87 Xiangya Rd, Changsha, Hunan, People’s Republic of China. Tel.: þ86 13548563641; fax: 0731-84327332. E-mail address: [email protected] (J. Wang). 0197-4580/$ e see front matter Ó 2019 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.neurobiolaging.2019.09.013
disease (Cooper-Knock et al., 2019). They further identified 4 possible pathogenic mutations in sALS cases. Interestingly, all 5 mutations are localized in the exon 4 of GLT8D1 gene. Moreover, they found that GLT8D1 p.R92C mutation and ARPP21 p.P529L may have a synergistic effect for ALS. However, this observation has not been validated in other ALS cohorts. In the current study, we sought to investigate the potential contribution of GLT8D1 and ARPP21 variants to ALS in mainland China.
2. Methods 2.1. Population A total of 512 ALS patients were recruited in this study (433 sALS cases and 79 probands of familial history of ALS). All patients were enrolled in the Department of Neurology, Xiangya Hospital, Central South University. All patients were diagnosed by at least 2 experienced senior neurologists. According to the revised El Escorial criteria (Ludolph et al., 2015), all patients were diagnosed as clinically definite, probable, or probable laboratory supported ALS. In total, 3210 healthy controls were recruited from Xiangya Hospital Health Center. This study was approved by the Ethics Committee of Xiangya Hospital, Central South University (equivalent to an
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W. Li et al. / Neurobiology of Aging xxx (2019) 1.e1e1.e4
Table 1 GLT8D1 and ARPP21 variants in 512 ALS patients No.
Gene
Inheritance
Chromosome
Position
Location
cDNA changea
Protein
Mutation type
MAF gnomAD
dbSNP
Functional predictions: pathogenic (total)b
M26080 M26080 A0095 A0095 A0182 M36116 M36389 M36978
GLT8D1 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21
S S S S S S S S
3 3 3 3 3 3 3 3
52,728,870 35,780,951 35,785,431 35,834,003 35,785,389 35,780,951 35,779,806 35,780,852
Exon Exon Exon Exon Exon Exon Exon Exon
c.1106dupA c.C1790G c.A2009G c.C2165G c.G1967A c.C1790G c.C1643T c.G1691A
p.N369fs p.S597C p.Q670R p.P722R p.R656Q p.S597C p.P548L p.R564Q
Frameshift Missense Missense Missense Missense Missense Missense Missense
e e 0.000406% 0.000406% 0.010462% e e 0.002167%
e e e e rs761270285 e e rs144656036
e 9 (11) 6 (11) 9 (11) 8 (11) 9 (11) 5 (9) 7 (11)
10 16 17 18 17 16 15 16
Key: ALS, amyotrophic lateral sclerosis; cDNA, complementary deoxyribonucleic acid; dbSNP, database of single nucleotide polymorphism; gnomAD, genome aggregation database; MAF, minor allele frequency; S, sporadic. a Transcript NM_001267619 has been used for ARPP21 variants nomenclature. Transcript NM_018446 has been used for GLT8D1 variants nomenclature. b The silico tools for predicting variants were (1) PolyPhen2 HDIV (polymorphism phenotyping version 2 human diversity), (2) PolyPhen2 HVAR (polymorphism phenotyping version 2 human variation), (3) SIFT (sorting intolerant from tolerant), (4) PROVEAN (Protein Variation Effect Analyzer), (5) LR (logistic regression), (6) CADD (combined annotation dependent depletion), (7) LRT (likelihood ratio test), (8) FATHMM (functional analysis through hidden Markov models), (9) M-CAP (Mendelian clinically applicable pathogenicity), (10) MutationTaster, and (11) MutationAssessor.
Institutional Review Board). Written informed consent was obtained from all subjects.
in silico; and (4) pathogenicity, defined as being predicted as pathogenic by at least 5 of 11 in silico tools (Quadri et al., 2018).
2.2. Mutation analysis
2.3. Statistical analysis
All ALS patients and 1039 healthy controls underwent whole exome sequencing, and 2171 healthy controls underwent whole genome sequencing by using a previously described method (Wang et al., 2011; Zeng et al., 2019). Variants that fulfilled the following criteria were included for further analysis: (1) the variant being present in the heterozygous state; (2) rarity, defined as a minor allele frequency less than 0.1% by the Exome Aggregation Consortium, the genome aggregation database; (3) exonic and nonsynonymous, insertions, deletions, or predicted to affect splicing
Statistical analysis was performed using SPSS 22.0. Fisher’s exact test was used to determine the association between ARPP21 and GLT8D1 variants and ALS. The threshold of statistical significance was set at p < 0.05. 3. Results We did not identify pathogenic mutation in exon 4 of GLT8D1 in the ALS patients. However, we identified a variant in exon 10 in an
Table 2 GLT8D1 and ARPP21 variants in 3210 controls Gene
Chromosome
Position
Location
cDNA changea
Protein
Mutation type
MAF gnomAD
Functional predictions: pathogenic (total)b
GLT8D1 GLT8D1 GLT8D1 GLT8D1 GLT8D1 GLT8D1 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
52,731,943 52,728,947 52,730,631 52,734,466 52,728,971 52,729,561 35,779,809 35,758,789 35,731,574 35,730,799 35,758,843 35,763,303 35,780,951 35,729,284 35,723,353 35,732,388 35,763,166 35,835,302 35,785,431 35,731,630 35,770,800 35,778,706 35,785,389 35,763,135 35,770,819
Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon
c.122dupG c.1030G>C c.374T>C c.11G>A c.1006T>C c.688C>T c.1646T>C c.834-1G>T c.487G>C c.407A>T c.887T>G c.1100G>A c.1790C>G c.315A>C c.110A>G c.577T>A c.963G>C c.2294T>G c.2009A>G c.543C>G c.1129G>A c.1394T>C c.1967G>A c.932G>A c.1148G>A
p.I42Nfs*32 p.D344H p.I125T p.R4H p.W336R p.R230C p.M549T e p.D163H p.D136V p.L296R p.S367N p.S597C p.K105N p.E37G p.S193T p.W321C p.V765G p.Q670R p.N181K p.E377K p.I465T p.R656Q p.R311Q p.G383D
Frameshift Missense Missense Missense Missense Missense Splicing Splicing Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense
e e 0.0004063% 0.001637% 0.002437% 0.001219% 0.002259% e 0.001234% e e e e e e e e 0.0008123% 0.0004062% 0.0004115% 0.0004082% 0.002453% 0.01% 0.004076% 0.009766%
e 6 8 6 11 8 e e 9 9 6 6 9 7 8 8 6 9 7 5 6 7 9 8 8
4 10 5 2 10 8 15 11 7 6 11 12 16 5 2 8 12 19 17 7 13 14 17 12 13
(11) (11) (11) (11) (11)
(11) (11) (11) (11) (11) (11) (11) (11) (11) (11) (11) (11) (11) (11) (11) (11) (11)
Key: cDNA, complementary deoxyribonucleic acid; gnomAD, genome aggregation database; MAF, minor allele frequency. a Transcript NM_001267619 has been used for ARPP21 variants nomenclature. Transcript NM_018446 has been used for GLT8D1 variants nomenclature. b The silico tools for predicting variants were (1) PolyPhen2 HDIV (polymorphism phenotyping version 2 human diversity), (2) PolyPhen2 HVAR (polymorphism phenotyping version 2 human variation), (3) SIFT (sorting intolerant from tolerant), (4) PROVEAN (Protein Variation Effect Analyzer), (5) LR (logistic regression), (6) CADD (combined annotation dependent depletion), (7) LRT (likelihood ratio test), (8) FATHMM (functional analysis through hidden Markov models), (9) M-CAP (Mendelian clinically applicable pathogenicity), (10) MutationTaster, and (11) MutationAssessor.
W. Li et al. / Neurobiology of Aging xxx (2019) 1.e1e1.e4
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Table 3 GLT8D1 and ARPP21 variants in 512 ALS patients and 3210 controls Level of comparison
Entire gene Exons Exon 4 is excluded
GLT8D1
p value
Cases (n ¼ 512)
Controls (n ¼ 3210)
1 (0.19%)a
6 (0.19%)b
a
b
1 (0.19%)
5 (0.16%)
ARPP21
p value
Cases (n ¼ 512)
Controls (n ¼ 3210)
1.000
6 (1.17%)a
21 (0.65%)b
0.254
0.589
a
b
0.254
6 (1.17%)
21 (0.65%)
Key: ALS, amyotrophic lateral sclerosis. a The frequency of variants in cases. b The frequency of variants in controls.
sALS patient (0.19%) and 6 variants in exons 2, 4, 5, 8, and 10 in 6 healthy controls (0.19%). For the ARPP21 gene, we identified 6 variants in 6 sALS patients (1.17%) and 19 variants in 21 healthy controls (0.65%) (Tables 1e3). There was no significant association between ALS and variants in ARPP21 or GLT8D1 (Fisher’s exact test: p ¼ 0.254, ARPP21; p ¼ 1.000, GLT8D1). 4. Discussion Cooper-Knock et al. recently identified GLT8D1 as a risk gene for ALS, and demonstrated that ALS-related GLT8D1 mutations impair its glycosyltransferase activity and negatively impact ganglioside signaling. They also found that ARPP21 variants might be an independent risk factor that acted synergistically with GLT8D1 for the pathogenesis of ALS (Cooper-Knock et al., 2019). However, we did not find association between ALS and GLT8D1 or ARPP21 in the current study. The discrepancy between these 2 studies may arise from different ethnic backgrounds. This phenomenon is not unprecedented. For instance, the hexanucleotide repeat expansion in C9orf72 has been reported as the most common cause for ALS in Caucasian populations but is very rare in mainland China and other Asian countries (Jiao et al., 2014; Nishiyama et al., 2017; Zou et al., 2013). Nevertheless, the present study cannot rule out the possible pathogenic role of GLT8D1 and ARPP21 variants in ALS due to the limited sample size. In conclusion, our study does not support GLT8D1 or ARPP21 mutation as a risk factor for ALS in mainland China. Further studies with larger sample size are needed to validate the potential contribution of GLT8D1 and ARPP21 variants to ALS. Disclosure The authors have no actual or potential conflicts of interests. Acknowledgements We thank Professor Jiada Li for the language help on the writing and editing of the manuscript. We are grateful to the participating patients for their involvement. This study was funded by the National Key Research and Development Program of China, China (#2018YFC1312003); the Program of National Natural Science Foundation of China, China (#81671120, 81300981); and the Clinical Scientific program of Xiangya Hospital, Central South University, China (#2015105). References Chia, R., Chio, A., Traynor, B.J., 2018. Novel genes associated with amyotrophic lateral sclerosis: diagnostic and clinical implications. Lancet Neurol. 17, 94e102. Cooper-Knock, J., Moll, T., Ramesh, T., Castelli, L., Beer, A., Robins, H., Fox, I., Niedermoser, I., Van Damme, P., Moisse, M., Robberecht, W., Hardiman, O., Panades, M.P., Assialioui, A., Mora, J.S., Basak, A.N., Morrison, K.E., Shaw, C.E., AlChalabi, A., Landers, J.E., Wyles, M., Heath, P.R., Higginbottom, A., Walsh, T., Kazoka, M., Mcdermott, C.J., Hautbergue, G.M., Kirby, J., Shaw, P.J., 2019.
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Negative Results
Mutation analysis of GLT8D1 and ARPP21 genes in amyotrophic lateral sclerosis patients from mainland China Wanzhen Li a, Zhen Liu a, Weining Sun a, Yanchun Yuan a, Yiting Hu a, Jie Ni a, Bin Jiao a, Liangjuan Fang a, b, c, d, Jinchen Li b, Lu Shen a, b, c, d, Beisha Tang a, b, c, d, Junling Wang a, b, c, d, * a
Department of Neurology, Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China National Clinical Research Center for Geriatric Diseases, Xiangya Hospital, Central South University, Changsha, Hunan, People’s Republic of China Key Laboratory of Hunan Province in Neurodegenerative Disorders, Central South University, Changsha, Hunan, People’s Republic of China d Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, People’s Republic of China b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 May 2019 Received in revised form 17 September 2019 Accepted 20 September 2019
Variants in exon 4 of gene encoding GLT8D1 (glycosyltransferase 8 domain containing 1) gene have recently been suggested as a novel cause of amyotrophic lateral sclerosis (ALS). In addition, there is a synergism between GLT8D1 and ARPP21 (cAMP Regulated Phosphoprotein 21) variants for ALS. However, this observation has not been validated in other ALS cohorts. In this study, we analyzed the rare pathogenic variants in GLT8D1 and ARPP21 genes in a cohort of 512 ALS patients and 3210 healthy controls from mainland China. A total of 25 rare variants in ARPP21 were identified in the patients and controls, but we did not find rare variants in exon 4 of GLT8D1 in the patients. By using Fisher’s exact test, we did not find significant association between ALS and GLT8D1 or ARPP21. Therefore, GLT8D1 and ARPP21 are not likely the causative genes for ALS in mainland China. Ó 2019 Elsevier Inc. All rights reserved.
Keywords: Amyotrophic lateral sclerosis Whole exome sequencing GLT8D1 ARPP21
1. Introduction Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects upper and lower motor neurons and results in muscular weakness and atrophy. Patients eventually die of respiratory failure within 2e5 years. The incidence of ALS is estimated to be 2e3 per 100,000 people in Europe and 0.7e0.8 per 100,000 people in Asia (Mathis et al., 2019). The mean age of onset is 65 years (Mathis et al., 2019). Most cases of ALS are sporadic (sALS), while approximately 10% of the patients have a familial history of ALS (Chia et al., 2018). To date, mutations in more than 40 genes have been associated with the pathogenesis of ALS (Chia et al., 2018; Corcia et al., 2019; Mathis et al., 2019; Nicolas et al., 2018; Ozoguz et al., 2015; Praline et al., 2010; Renton et al., 2014). Recently, variants in genes encoding GLT8D1 (glycosyltransferase 8 domain containing 1) and ARPP21 (cAMP Regulated Phosphoprotein 21) have been identified in ALS patients of European ancestry. In an autosomal-dominant ALS pedigree, the authors identified p.R92C mutations in GLT8D1, which co-segregate with * Corresponding author at: Xiangya Hospital, Central South University, 87 Xiangya Rd, Changsha, Hunan, People’s Republic of China. Tel.: þ86 13548563641; fax: 0731-84327332. E-mail address: [email protected] (J. Wang). 0197-4580/$ e see front matter Ó 2019 Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.neurobiolaging.2019.09.013
disease (Cooper-Knock et al., 2019). They further identified 4 possible pathogenic mutations in sALS cases. Interestingly, all 5 mutations are localized in the exon 4 of GLT8D1 gene. Moreover, they found that GLT8D1 p.R92C mutation and ARPP21 p.P529L may have a synergistic effect for ALS. However, this observation has not been validated in other ALS cohorts. In the current study, we sought to investigate the potential contribution of GLT8D1 and ARPP21 variants to ALS in mainland China.
2. Methods 2.1. Population A total of 512 ALS patients were recruited in this study (433 sALS cases and 79 probands of familial history of ALS). All patients were enrolled in the Department of Neurology, Xiangya Hospital, Central South University. All patients were diagnosed by at least 2 experienced senior neurologists. According to the revised El Escorial criteria (Ludolph et al., 2015), all patients were diagnosed as clinically definite, probable, or probable laboratory supported ALS. In total, 3210 healthy controls were recruited from Xiangya Hospital Health Center. This study was approved by the Ethics Committee of Xiangya Hospital, Central South University (equivalent to an
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Table 1 GLT8D1 and ARPP21 variants in 512 ALS patients No.
Gene
Inheritance
Chromosome
Position
Location
cDNA changea
Protein
Mutation type
MAF gnomAD
dbSNP
Functional predictions: pathogenic (total)b
M26080 M26080 A0095 A0095 A0182 M36116 M36389 M36978
GLT8D1 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21
S S S S S S S S
3 3 3 3 3 3 3 3
52,728,870 35,780,951 35,785,431 35,834,003 35,785,389 35,780,951 35,779,806 35,780,852
Exon Exon Exon Exon Exon Exon Exon Exon
c.1106dupA c.C1790G c.A2009G c.C2165G c.G1967A c.C1790G c.C1643T c.G1691A
p.N369fs p.S597C p.Q670R p.P722R p.R656Q p.S597C p.P548L p.R564Q
Frameshift Missense Missense Missense Missense Missense Missense Missense
e e 0.000406% 0.000406% 0.010462% e e 0.002167%
e e e e rs761270285 e e rs144656036
e 9 (11) 6 (11) 9 (11) 8 (11) 9 (11) 5 (9) 7 (11)
10 16 17 18 17 16 15 16
Key: ALS, amyotrophic lateral sclerosis; cDNA, complementary deoxyribonucleic acid; dbSNP, database of single nucleotide polymorphism; gnomAD, genome aggregation database; MAF, minor allele frequency; S, sporadic. a Transcript NM_001267619 has been used for ARPP21 variants nomenclature. Transcript NM_018446 has been used for GLT8D1 variants nomenclature. b The silico tools for predicting variants were (1) PolyPhen2 HDIV (polymorphism phenotyping version 2 human diversity), (2) PolyPhen2 HVAR (polymorphism phenotyping version 2 human variation), (3) SIFT (sorting intolerant from tolerant), (4) PROVEAN (Protein Variation Effect Analyzer), (5) LR (logistic regression), (6) CADD (combined annotation dependent depletion), (7) LRT (likelihood ratio test), (8) FATHMM (functional analysis through hidden Markov models), (9) M-CAP (Mendelian clinically applicable pathogenicity), (10) MutationTaster, and (11) MutationAssessor.
Institutional Review Board). Written informed consent was obtained from all subjects.
in silico; and (4) pathogenicity, defined as being predicted as pathogenic by at least 5 of 11 in silico tools (Quadri et al., 2018).
2.2. Mutation analysis
2.3. Statistical analysis
All ALS patients and 1039 healthy controls underwent whole exome sequencing, and 2171 healthy controls underwent whole genome sequencing by using a previously described method (Wang et al., 2011; Zeng et al., 2019). Variants that fulfilled the following criteria were included for further analysis: (1) the variant being present in the heterozygous state; (2) rarity, defined as a minor allele frequency less than 0.1% by the Exome Aggregation Consortium, the genome aggregation database; (3) exonic and nonsynonymous, insertions, deletions, or predicted to affect splicing
Statistical analysis was performed using SPSS 22.0. Fisher’s exact test was used to determine the association between ARPP21 and GLT8D1 variants and ALS. The threshold of statistical significance was set at p < 0.05. 3. Results We did not identify pathogenic mutation in exon 4 of GLT8D1 in the ALS patients. However, we identified a variant in exon 10 in an
Table 2 GLT8D1 and ARPP21 variants in 3210 controls Gene
Chromosome
Position
Location
cDNA changea
Protein
Mutation type
MAF gnomAD
Functional predictions: pathogenic (total)b
GLT8D1 GLT8D1 GLT8D1 GLT8D1 GLT8D1 GLT8D1 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21 ARPP21
3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3
52,731,943 52,728,947 52,730,631 52,734,466 52,728,971 52,729,561 35,779,809 35,758,789 35,731,574 35,730,799 35,758,843 35,763,303 35,780,951 35,729,284 35,723,353 35,732,388 35,763,166 35,835,302 35,785,431 35,731,630 35,770,800 35,778,706 35,785,389 35,763,135 35,770,819
Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon Exon
c.122dupG c.1030G>C c.374T>C c.11G>A c.1006T>C c.688C>T c.1646T>C c.834-1G>T c.487G>C c.407A>T c.887T>G c.1100G>A c.1790C>G c.315A>C c.110A>G c.577T>A c.963G>C c.2294T>G c.2009A>G c.543C>G c.1129G>A c.1394T>C c.1967G>A c.932G>A c.1148G>A
p.I42Nfs*32 p.D344H p.I125T p.R4H p.W336R p.R230C p.M549T e p.D163H p.D136V p.L296R p.S367N p.S597C p.K105N p.E37G p.S193T p.W321C p.V765G p.Q670R p.N181K p.E377K p.I465T p.R656Q p.R311Q p.G383D
Frameshift Missense Missense Missense Missense Missense Splicing Splicing Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense Missense
e e 0.0004063% 0.001637% 0.002437% 0.001219% 0.002259% e 0.001234% e e e e e e e e 0.0008123% 0.0004062% 0.0004115% 0.0004082% 0.002453% 0.01% 0.004076% 0.009766%
e 6 8 6 11 8 e e 9 9 6 6 9 7 8 8 6 9 7 5 6 7 9 8 8
4 10 5 2 10 8 15 11 7 6 11 12 16 5 2 8 12 19 17 7 13 14 17 12 13
(11) (11) (11) (11) (11)
(11) (11) (11) (11) (11) (11) (11) (11) (11) (11) (11) (11) (11) (11) (11) (11) (11)
Key: cDNA, complementary deoxyribonucleic acid; gnomAD, genome aggregation database; MAF, minor allele frequency. a Transcript NM_001267619 has been used for ARPP21 variants nomenclature. Transcript NM_018446 has been used for GLT8D1 variants nomenclature. b The silico tools for predicting variants were (1) PolyPhen2 HDIV (polymorphism phenotyping version 2 human diversity), (2) PolyPhen2 HVAR (polymorphism phenotyping version 2 human variation), (3) SIFT (sorting intolerant from tolerant), (4) PROVEAN (Protein Variation Effect Analyzer), (5) LR (logistic regression), (6) CADD (combined annotation dependent depletion), (7) LRT (likelihood ratio test), (8) FATHMM (functional analysis through hidden Markov models), (9) M-CAP (Mendelian clinically applicable pathogenicity), (10) MutationTaster, and (11) MutationAssessor.
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Table 3 GLT8D1 and ARPP21 variants in 512 ALS patients and 3210 controls Level of comparison
Entire gene Exons Exon 4 is excluded
GLT8D1
p value
Cases (n ¼ 512)
Controls (n ¼ 3210)
1 (0.19%)a
6 (0.19%)b
a
b
1 (0.19%)
5 (0.16%)
ARPP21
p value
Cases (n ¼ 512)
Controls (n ¼ 3210)
1.000
6 (1.17%)a
21 (0.65%)b
0.254
0.589
a
b
0.254
6 (1.17%)
21 (0.65%)
Key: ALS, amyotrophic lateral sclerosis. a The frequency of variants in cases. b The frequency of variants in controls.
sALS patient (0.19%) and 6 variants in exons 2, 4, 5, 8, and 10 in 6 healthy controls (0.19%). For the ARPP21 gene, we identified 6 variants in 6 sALS patients (1.17%) and 19 variants in 21 healthy controls (0.65%) (Tables 1e3). There was no significant association between ALS and variants in ARPP21 or GLT8D1 (Fisher’s exact test: p ¼ 0.254, ARPP21; p ¼ 1.000, GLT8D1). 4. Discussion Cooper-Knock et al. recently identified GLT8D1 as a risk gene for ALS, and demonstrated that ALS-related GLT8D1 mutations impair its glycosyltransferase activity and negatively impact ganglioside signaling. They also found that ARPP21 variants might be an independent risk factor that acted synergistically with GLT8D1 for the pathogenesis of ALS (Cooper-Knock et al., 2019). However, we did not find association between ALS and GLT8D1 or ARPP21 in the current study. The discrepancy between these 2 studies may arise from different ethnic backgrounds. This phenomenon is not unprecedented. For instance, the hexanucleotide repeat expansion in C9orf72 has been reported as the most common cause for ALS in Caucasian populations but is very rare in mainland China and other Asian countries (Jiao et al., 2014; Nishiyama et al., 2017; Zou et al., 2013). Nevertheless, the present study cannot rule out the possible pathogenic role of GLT8D1 and ARPP21 variants in ALS due to the limited sample size. In conclusion, our study does not support GLT8D1 or ARPP21 mutation as a risk factor for ALS in mainland China. Further studies with larger sample size are needed to validate the potential contribution of GLT8D1 and ARPP21 variants to ALS. Disclosure The authors have no actual or potential conflicts of interests. Acknowledgements We thank Professor Jiada Li for the language help on the writing and editing of the manuscript. We are grateful to the participating patients for their involvement. This study was funded by the National Key Research and Development Program of China, China (#2018YFC1312003); the Program of National Natural Science Foundation of China, China (#81671120, 81300981); and the Clinical Scientific program of Xiangya Hospital, Central South University, China (#2015105). References Chia, R., Chio, A., Traynor, B.J., 2018. Novel genes associated with amyotrophic lateral sclerosis: diagnostic and clinical implications. Lancet Neurol. 17, 94e102. Cooper-Knock, J., Moll, T., Ramesh, T., Castelli, L., Beer, A., Robins, H., Fox, I., Niedermoser, I., Van Damme, P., Moisse, M., Robberecht, W., Hardiman, O., Panades, M.P., Assialioui, A., Mora, J.S., Basak, A.N., Morrison, K.E., Shaw, C.E., AlChalabi, A., Landers, J.E., Wyles, M., Heath, P.R., Higginbottom, A., Walsh, T., Kazoka, M., Mcdermott, C.J., Hautbergue, G.M., Kirby, J., Shaw, P.J., 2019.
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