Reviews

Risk Factor Genes in Patients with Dystonia: A Comprehensive Review

Vasileios Siokas1, Athina-Maria Aloizou1, Zisis Tsouris1, Amalia Michalopoulou1, Alexios-Fotios A. Mentis2,3 & Efthimios Dardiotis1*

1Department of Neurology, Laboratory of Neurogenetics, University of Thessaly, University Hospital of Larissa, Larissa, GR, 2Department of Microbiology, University of Thessaly, University Hospital of Larissa, Larissa, GR, 3Public Health Laboratories, Hellenic Pasteur Institute, Athens, GR

Abstract

Background: Dystonia is a movement disorder with high heterogeneity regarding phenotypic appearance and etiology that occurs in both sporadic and familial forms. The etiology of the disease remains unknown. However, there is increasing evidence suggesting that a small number of gene alterations may lead to dystonia. Although pathogenic variants to the familial type of dystonia have been extensively reviewed and discussed, relatively little is known about the contribution of single-nucleotide polymorphisms (SNPs) to dystonia. This review focuses on the potential role of SNPs and other variants in dystonia susceptibility.

Methods: We searched the PubMed database for peer-reviewed articles published in English, from its inception through January 2018, that concerned human studies of dystonia and genetic variants. The following search terms were included: “dystonia” in combination with the following terms: 1) “polymorphisms” and 2) “SNPs” as free words.

Results: A total of 43 published studies regarding TOR1A, BDNF, DRD5, APOE, ARSG, NALC, OR4X2, COL4A1, TH, DDC, DBH, MAO, COMT, DAT, GCH1, PRKRA, MR-1, SGCE, ATP1A3, TAF1, THAP1, GNAL, DRD2, HLA-DRB, CBS, MTHFR, and MS genes, were included in the current review.

Discussion: To date, a few variants, which are possibly involved in several molecular pathways, have been related to dystonia. Large cohort studies are needed to determine robust associations between variants and dystonia with adjustment for other potential cofounders, in order to elucidate the pathogenic mechanisms of dystonia and the net effect of the genes.

Keywords: Dystonia, genetic polymorphism, single nucleotide polymorphism, variant, cervical dystonia, blepharospasm, movement disorders

Citation: Siokas V, Aloizou AM, Tsouris Z, Michalopoulou A, Mentis AFA, Dardiotis E. Risk factor genes in patients with dystonia: a comprehensive review. Tremor Other Hyperkinet Mov. 2018; 8. doi: 10.7916/D8H438GS

*To whom correspondence should be addressed. E-mail: edar@med.uth.gr

Editor: Elan D. Louis, Yale University, USA,

Received: March 12, 2018 Accepted: November 20, 2018 Published: January 9, 2019

Copyright: © 2018 Siokas et al. This is an open-access article distributed under the terms of the Creative Commons Attribution–Noncommercial–No Derivatives License, which permits the user to copy, distribute, and transmit the work provided that the original authors and source are credited; that no commercial use is made of the work; and that the work is not altered or transformed.

Funding: None.

Financial Disclosures: None.

Conflict of Interest: The authors report no conflict of interest.

Ethics Statement: Not applicable for this category of article.

Introduction

Dystonia is a movement disorder with high heterogeneity regarding phenotypic appearance and etiology.1 The prevalence of dystonia is estimated to be 16:100,000.2,3 In 2013, a new general definition and classification of dystonia were introduced by an international panel of dystonia experts.4 The two main axes of this classification are considered to be the etiology and the clinical features.4 However, the pathophysiology and cause of most dystonia cases remain largely unknown.5

A polymorphism is a variation in the DNA sequence that occurs in a population with a frequency of 1 % or higher.6,7 When a variation occurs in a single nucleotide, at a specific position in the genome, it is called an SNP (single-nucleotide polymorphism).8,9 SNPs can occur within coding sequences of genes, non-coding sequences, introns, or the regions between genes (also known as intergenic regions).10,11 An SNP across a coding sequence of a gene can be characterized as synonymous (when the protein sequence is not affected) and non-synonymous (when the amino acid sequence of the protein is altered).10,12 The non-synonymous SNPs are divided into missense (when they result in a different amino acid) and nonsense (when they result in a premature stop codon).10,12 Recently, it has been recommended that both terms, “mutation” and “polymorphism”, be replaced by the term “variant”.13,14 An additional modifier (e.g. pathogenic, benign) to the term “variant” should be used, in order for its pathogenic or benign effect to be declared.13,14

The importance of genetic factors was unambiguously demonstrated with the identification of causative pathogenic variants in monogenic cases of familial dystonia under the autosomal dominant, autosomal recessive, or X-linked mode of inheritance.3 Furthermore, a few candidate gene association studies (CGASs) have suggested that the presence of specific genetic loci may confer susceptibility to dystonia.15 Genetic variations may affect dystonia’s phenotypic appearance, age at onset, and spread to adjacent body regions, and may also affect the penetrance of other pathogenic variants suspected for dystonia.15

Previous reviews have mainly discussed the genetics of dystonia in general its monogenic forms and its phenotypic divergence.3,1625 However, genetics of sporadic forms of dystonia with no clearly discernible family history, and results from case–control studies are relatively rarely discussed.15,18,25 Therefore, in the present review article, we discuss the current state of knowledge regarding genetics of dystonia, by emphasizing the CGASs that have linked single nucleotide polymorphisms and variants across genes that predispose to dystonia. Owing to the lack of a widely accepted nomenclature gene classification system for dystonia, we have used gene names for loci identification.26 The main aim of the current comprehensive review is to shed some light on which polymorphisms predispose for dystonia, and to what extent.

Methods: study identification and selection

In order for any potentially relevant study to be identified, we searched through the Pubmed database (https://www.ncbi.nlm.nih.gov/pubmed) for peer-reviewed articles published in English, from its inception to January 2018, that concerned human studies of dystonia and genetic polymorphisms. The following search terms were included: “dystonia” in combination with 1) “polymorphisms” and 2) “SNPs” as free words. The complete search algorithm is available in the S1 Appendix. The last literature search was performed on February 20, 2018. Additionally, reference lists of retrieved articles were examined in order to identify missing from the initial database search results. The flowchart presenting the selection procedure of the studies is presented in Figure 1. Published studies between 1996 and 2017 were included.

The following data were extracted from each study, when possible: author, year of publication, ethnicity of the studied population, numbers of cases and controls, age at disease onset, mean age and gender distribution, tested variants, family history of the participants, screening or not for the TOR1A ∆GAG pathogenic variant, correction for multiple comparisons, assessment of Hardy–Weinberg equilibrium, and the tested dystonia phenotypes.

Results and discussion

Published studies between August 2001 and September 2017 were included. Baseline characteristics from studies regarding TOR1A, BDNF, DRD5, APOE, ARSG, NALC, OR4X2, COL4A1, TH, DDC, DBH, MAO, COMT, DAT, GCH1, PRKRA, MR-1, SGCE, ATP1A3, TAF1, THAP1, GNAL, DRD2, HLA-DRB, CBS, MTHFR, and MS are presented in Supplementary Tables 1–5. GnomAD frequencies (http://gnomad.broadinstitute.org/) and the type of individual variants are available at the S2 Appendix.

TOR1A

The TOR1A gene is a five-exon gene that covers an 11-kb region in chromosome 9. The TOR1A protein, called TorsinA, belongs to the family of the AAA+ ATPases. It can be found in the endoplasmic reticulum and the nuclear envelope of most cells,8 including those of the central nervous system.18 The function of TorsinA and how TOR1A gene pathogenic variants lead to dystonia remains largely unknown.27 TorsinA acts mainly as a molecular chaperone.28 The molecular and cellular processes in which TorsinA is involved include the interactions between cytoskeleton and membrane, important functions of the endoplasmic reticulum and the nuclear envelope, and the regulation of cellular lipid metabolism.18,2931 It is known that TorsinA needs to bind to TOR1AIP2 (Torsin 1A Interacting Protein 2) or to Heat Shock Protein Family A (Hsp70) Member 8 (HSPA8) in order to be activated,32 a procedure that is impaired by the GAG deletion, as has been confirmed by crystallography.33,34

TOR1A remains the most extensively studied gene in both monogenic and sporadic forms of dystonia.15 However, results from case–control studies yielded conflicting results, with the association being affected by body distribution, ethnicity, and other phenotypic manifestations. A number of case–control studies have been conducted so far3552 and quite a few TOR1A variants have also been investigated (rs1801968, rs2296793, rs1182, rs3842225, rs13283584, rs11787741, rs13297609, rs2287367, rs1043186, and rs35153737). Apart from case–control studies, a number of variants have been identified through mutational screening (rs766483672, rs80358233, rs75881350, rs1183, rs563498119, rs573629050, rs1045441, rs144572721).53 Additionally, three meta-analyses have been conducted so far examining the effects of TOR1A gene variants on dystonia.15,39,40 The most recent evidence stemming from a meta-analysis, reveals a significant association of the rs1182 (allele frequency [AF] = 0.1666) and the rs1801968 (AF = 0.1236 for the G allele and AF = 4.061e-6 for the C allele) TOR1A variants with the development of focal dystonia (FD) and writer’s cramp (WC) respectively.15 Moreover, variants within 3′-UTR (untranslated region) encoded by exon 5 represent an additional functional genetic locus of TOR1A, though it may be under synergistic action with other TOR1A genetic variants.15 This comes in accordance with a recent case–control study, suggesting an association of the rs35153737 in the 3′-UTR of TOR1A with dystonia; a result, though, that has been attributed to functional variants that are in high linkage disequilibrium (LD).52

From a functional aspect, loci containing the aforementioned variants appear to have consequences; variants across exon 4 and 3′-UTR encoded by exon 5, in particular, appear to overall influence the function of the TOR1A gene.15 More specifically, rs1801968 was confirmed to be associated with reduced penetrance of the ∆GAG pathogenic variant in humans.54,55 Regarding the 3′-UTR of exon 5, there is only some indication that specific variants across this region may have some functional consequences under synergistic action.15,52 Interestingly, based on the results regarding frequencies, computational analyses and function experiments, rs563498119 in the 3′-UTR of TOR1A was reported to change the expression of the TOR1A gene.53 The regulation of TOR1A expression, by mutating the conserved region of the binding site of the human microRNA (hsa-miR-494), where rs563498119 is located, hints towards hsa-miR-494 being a possible therapeutic target.53

BDNF and APOE

Among the major mechanisms in dystonia, the reduced inhibition of the motor system and the increased plasticity are included.56 In greater detail, increased plasticity in the hand representation area of the motor cortex has been observed in focal hand dystonia, blepharospasm (BSP), and cervical dystonia (CD) using high-resolution transcranial stimulation.57 Consequently, abnormal plasticity within certain motor cortical circuits may represent a lineament of adult-onset dystonia forms.57,58

Synaptic plasticity is influenced by the brain-derived neurotrophic factor (BDNF). A common SNP across the BDNF gene within the prodomain region is the rs6265 (G/A) (AF = 0.1896) and it results in the substitution of Val in amino acid position 66 with Met (Val→Met), which may influence synaptic plasticity5961 and is possibly involved in dystonia development. Healthy carriers of the val66met appear to have differences in brain structure and abnormal motor cortex plasticity as well.62,63 Rs6265 has been found to be associated with quite a few diseases such as Parkinson’s disease, Alzheimer disease (AD), schizophrenia, bipolar disease, depressive disorder, and panic disorders, although strong evidence has yet to be presented.6469

The studies that have been conducted so far regarding the role of the rs6265 on dystonia have yielded conflicting results. More precisely, rs6265 has been reported to be associated with CD and BSP in multiethnic and Chinese cohorts respectively.70,71 Additionally, higher frequency of bilateral postural arm tremor in CD patients with the BDNF Met66Met variant than inVal66Met and Val66Val carriers has also been observed.72 However, these results have not been replicated in Serbian, Chinese, Italian, or Caucasian dystonia cohorts.58,7375 To date, two meta-analyses have evaluated the effects of rs6265 variant on dystonia.75,76 The most recent reports a statistically significant overall effect of the AA genotype on the development of idiopathic dystonia.76 However, the lack of reproducibility of the positive results could be attributed, among others, to the culture of null hypothesis significance testing,77 the possible influence of epigenetic mechanisms in the gene function (such as DNA methylation),78 and to the fact that additional variants across the BDNF may regulate the level of serum BDNF and its function.79 BDNF could be considered a potential therapeutic target in dystonia, as in neurological and psychiatric disorders.8082

Apolipoprotein E is the product of the APOE gene, which connects to lipids in order to form lipoproteins. There are at least three alleles (e2, e3, and e4) of the APOE gene, with the commonest one being the e3. The main function of lipoproteins is to package cholesterol and other fats, and transport them throughout tissues including the central nervous system.83 The e4 allele is associated with an increased risk of AD compared with the e3 allele, whereas the e2 allele is associated with decreased risk.84 Like BDNF, APOE may also influence neural plasticity and remodeling.71,85 In a Japanese cohort, E4 carriers were shown to develop dystonia on average approximately 10 years earlier than e4 non-carriers.58 TorsinA is also involved in cellular lipid metabolism.29 Therefore, variants that influence lipid biology may contribute to dystonia. Matsumoto et al.85 suggested that the e4 allele may severely affect neuronal reorganization and this impairment of neuronal repair may contribute to an earlier age of dystonia onset. Consequently, it is possible that variants within TOR1A, BDNF, APOE or even other genes under synergistic action, influence the phenotypic manifestation of dystonia.

THAP 1

Around a hundred missense, nonsense, and frameshift pathogenic variants, throughout most part of the coding region of the “thanatos associated protein domain containing, apoptosis associated protein 1” (THAP1) gene, have been associated with dystonia3,86,87 in a genetically diverse population.18 The THAP1 gene encodes the transcription factor THAP1, a zinc finger protein with an amino-terminal THAP domain, a proline-rich region, and a carboxy-terminal nuclear domain as well.88 THAP1 is thought to regulate the transcription of several key genes, TOR1A included.18,89

Case–control studies regarding THAP1 variants are limited35,40 because of the variety and the rarity of THAP1 variants. Therefore, most findings derive from mutational screening and the comparison between dystonia cases and healthy individuals.86,87,9096 However, there is an indication that the frequency of the C allele of the c.71+126T>C pathogenic variant was elevated in British dystonia patients.90 -237_236GA>TT was also over-represented in dystonia when compared with controls in a European cohort94 but these results could not be replicated.90,91,97 Furthermore, the IVS2-87 A>G (rs11989331, AF = 0.003428) was over-represented in dystonia in an Indian study.95 The MAF of rs200209986 was also found to be significantly higher in dystonic patients (MAF = 0.359%) than in the controls (MAF = 0.0318%, p<0.05) in the Vemula et al.96 study and the 1000 Genomes project (MAF = 0.0916%, p<0.05), but not when compared with the EVS database (MAF = 0.199%, p = 0.13).

The large amount of THAP1 pathogenic variants linked to dystonia may suggest an interplay between environmental and genetic factors.98 Further, the type of work or the exposure to environmental factors, such as pesticides, may possibly predispose to dystonia development in pathogenic variant carriers.21,86,87

GNAL, TAF1, GCH1, MR-1 (PNKD), SGCE, ATP1A3, PRKRA, HLA-DRB, CBS, MTHFR, and MS

GNAL (guanine nucleotide-binding protein subunit alpha L) has been identified as responsible for adult-onset dystonia, which is primarily cervical or cranial.99 A few GNAL variants (rs9303742, rs9675415, rs1895689, rs8095592, rs72865259, rs1647556, rs200508915, rs138151459, rs2071140, rs2071141, rs199571902) have been examined for association with generalized, multifocal, segmental, and focal dystonia.100 Despite the fact that no strong evidence for association with dystonia was found, novel variants are constantly reported in single dystonia patients with various phenotypes,100 leading to approximately 30 different GNAL variants in dystonia patients.3GNAL encodes guanine nucleotide-binding protein G subunit alpha [Gα(olf)]. Gα(olf)is involved in both the direct and indirect pathway to the activation of adenylate cyclase, by coupling dopamine type 1 receptors and the adenosine A2A receptors in medium spiny neurons, respectively.99 In fact, the involvement in the indirect pathway of the activation leads to the activation of adenylyl cyclase type 5 (AC5). AC5 is encoded by the adenylyl cyclase 5 (ADCY5) gene, which was recently reported to be a co-founder of dystonia.101 It is possible that epistasis phenomenon with ADCY5 influences the causative effect of GNAL variants.

Newman at al.40 in 2012, apart from TOR1A and THAP1, which are described in the above sections, genotyped several variants of other genes as well (TAF1, GCH1, MR-1 (PNKD), SGCE, ATP1A3, and PRKRA).40 The results were negative regarding quite a few variants across TAF1, MR-1 (PNKD), SGCE, ATP1A3, and PRKRA, yet weak associations were observed for the rs12147422 (AF = 0.217), rs3759664 (AF = 0.2353), and rs10483639 (AF = 0.2539) of GCH1 (GTP cyclohydrolase 1) variants when the entire, non-homogeneous phenotypic, dystonia group was compared with controls. The lack of reproducibility of these associations could be explained by the low prevalence of dystonia, suggesting the need of collaborative studies.102 Nevertheless, GCH1 belongs to the confirmed causative genes of dopa-responsive dystonia. Additionally, the penetrance of GCH1 pathogenic variants appears to be considerably higher in females than in males.103 The GCH1 gene encodes the rate-limiting enzyme in the biosynthesis of dopamine via the biopterin pathway. GTP cyclohydrolase1 is involved in tetrahydrobiopterin neo-synthesis from GTP, as it catalyzes the first step of this reaction.18 Variants of GHC1 influence enzyme activity, leading to a deficiency in dopamine and serotonin.104 Therefore, a possible role of GCH1 in non-monogenic forms of dystonia should not be dismissed, as scientific reasoning could not be substituted by statistical analysis.105

Finally there is no strong evidence for the association between HLA-DRB variants or variants in the homocysteine pathway (cystathionine β-synthase [CBS], methionine tetrahydrofolate reductase [MTHFR], methionine synthase [MS] genes) with dystonia.50

Dopamine pathway genes (DAT1, DRD1, DRD2, DRD3, DRD4, DRD5, COMT, DAT, TH, MAO-A and -B, DDC, and DBH)

Dystonic movements are considered the result of impaired function and abnormalities of dopaminergic neurotransmission and signaling in the basal ganglia.106 The involvement of the dopaminergic system in the pathophysiology of dystonia has also been enhanced via the mutated genes of the dopamine pathway in monogenic forms of dystonia (GCH1).107 Allele 2 of the DRD5 has been associated with increased risk of CD and BSP in British cohorts.108,109 Allele 6 and allele 4 of the DRD5 have been associated with CD in British and Italian cohorts respectively,109,110 thus strongly supporting the involvement of the DRD5 gene in dystonia.111 However, DRD5 has not been associated with dystonia in Italian, US, and German studies.49,50 Dopamine receptor genes regulate neurotransmission in response to dopamine.112 Dopamine receptors are divided into two families, based on either the activation (D1-like receptors: DRD1 and DRD5) or the inhibition (D2-like receptors: DRD2, DRD3, and DRD4) of adenylate cyclase.113 Although the negative results in genes of dopamine signaling pathway (dopamine receptors) are few,49,114,115 Groen et al.115 suggested that changes in dopamine levels may be secondary during the dystonia course and that rare single nucleotide variants of dopamine genes are possibly associated with dystonia.116

ARSG, NALC, OR4X2, COL4A1

To date, only two genome-wide association studies (GWASs) have been performed in order to identify variants that may predispose to dystonia.117,118 According to their results, there is a preliminary indication that arylsulfatase G (ARSG) and sodium leak channel (NALCN) variants play that role.117,118

In a GWAS executed by Lohmann et al.,117 it was suggested that the intronic rs11655081 (AF = 0.181) of the ARSG gene was associated with musician’s dystonia and writer’s cramp. The missense rs61999318 (AF = 0.002619) was significantly higher in the group of writer’s cramp patients than in European Americans in the EVS database (p = 0.0013).119 Functional analysis suggested that rs61999318 may represent a functional variant, as the underlying amino acid substitution of isoleucine at position 493 with threonine (p.I493T) appears to be disease causing.119 ARSG is the protein encoded by ARSG; it hydrolyzes sulfate esters and is therefore implicated in cell signaling, synthesis of hormones, and protein degradation.120 Moreover, it may be involved in neuronal ceroid lipofuscinosis,121 which can present itself as dystonia.117 In view of the former considerations, ARSG could be targeted as a gene for further study mainly in task-specific dystonias.

According to the GWAS from Mok et al.,118 the cluster of variants near exon 1 of NALCN was found nearest to the significance threshold in a British population with CD. The most statistically significant variants were NALCN (rs61973742, rs1338051, rs9518385, rs9518384, rs1338041 rs3916908), COL4A1 (rs619152), RGL1 (rs12132318), OR4X2 3 (rs67863238), intergenic (rs1249277, rs1249281, rs9416795), KIAA1715 (rs10930717), OR4B1 (rs35875350).118 However, a replication of this GWAS case–control study did not report any association of NALCN, OR4X2, COL4A1, and intergenic variants,122 and the results for NALC (rs1338041, rs61973742) were also not reproduced in a Chinese population.123 NALCN is a voltage-independent and cation-non-selective channel. Its main function is the leaky sodium transport across neuronal membranes and the regulation of neuronal excitability.124 In general, variants in genes, whose protein also acts like an ion channel, are crucial components and may be additional factors for dystonia development.117ANO3 is among the confirmed genes that cause a monogenic form of late-onset craniocervical dystonia, with a possible effect on the calcium-activated chloride channel.3,125

Concluding remarks

Genetic factors confer susceptibility to dystonia development. More precisely, based on our review, exon 4 and the 3′-UTR of exon 5 represent loci that appear to have a strong influence on the function of the TOR1A gene, and their pathogenic variants may be associated with sporadic forms of dystonia, specifically with focal distribution. Moreover, rs6265 of BDNF appears to be strongly associated with dystonia as well. As the function of the BDNF gene may be influenced by other variants, additional loci across it may be worth examining. Further analysis of the ARSG gene, notably the rs61999318 in focal task-specific dystonia cohorts and the DRD5 gene in focal dystonia, is warranted. Additional studies of GCH1 may be required. Owing to their rarity, THAP1 gene variants are insecure targets for future case–control studies. The continuing identification of pathogenic variants that cause monogenic forms of dystonia will lead us to new possible targets for case–control studies.1,3

Next-generation sequencing has led to the identification of new dystonia genes on a monthly basis.3,125,126 Therefore, a large amount of common and rare genetic variants that may predispose to dystonia have been identified. Also, a few identified variants may affect penetrance, age at onset, spread to adjacent body, or the phenotype of dystonia.15 However, it is not prudent to assume that all these genes truly lead to dystonia, and therefore results need to be interpreted with caution. Therefore, applying the CGASs approach to next-generation data could possibly shed some light on the mechanisms of the complex traits.127

The understanding of the genetic basis of monogenic and sporadic forms of dystonia will permit the identification and deeper knowledge of dystonia’s pathogenesis. This will provide physicians with more personalized tools to manage dystonia in the future, even from the time of diagnosis, and they may also be used for assessing the biological progression of the disease and guide the treatment decisions.128 Implications, even at the DNA and/or RNA level, are already considered as new possible targeted therapeutic approaches.53,80 The stronger grasp on dystonia’s genetic susceptibility will also improve genetic testing and counseling.

The lack of validation reproducibility of the positive results could be attributed to several factors; firstly, the culture of null hypothesis significance testing.77 Moreover, low power CGASs because of relatively small sample sizes is a common phenomenon, as the effective population should ideally be very large (∼10,000 individuals) in order for a modest genetic effect to be identified.129 The interplay between environmental (e.g. pesticides)86,98 and genetic factors, as well as among genetic factors,1 may variably determine the penetrance of pathogenic variants and the phenotype.18,20,86,98,130,131 Furthermore, the phenotypic divergence of dystonia and the possible classification bias should be considered, as the majority of the studies were performed before the new dystonia classification.4,15 Finally, epigenetic mechanisms may represent an additional explanation for the lack of result validation.78

Therefore, it is of great necessity that more collaborative studies132,133 with adjustment for other potential cofounders (e.g. gene–environment interactions with adjustment for pesticide exposure,86 air pollution,134 diseases of the anterior segment of the eye, preceded injury, trauma, surgical intervention or sore throat,130 time spent handwriting per day and the writing time before dystonia onset,135 genome methylation status) and a supportive functional analysis be conducted in the future. In this way, the pathogenic mechanisms of dystonia and the net effect of the genes could be elucidated and, consequently, the inherent limitations of association studies will be avoided.136

Certain limitations of the present review need to be acknowledged. Firstly, supportive data regarding functional analysis of variants would give more robustness to our conclusions. Moreover, we included relevant studies regardless of the sample power and without any prior quality assessment. Therefore, a possible confounding by population stratification or technical factors cannot totally be excluded. Finally, based on our search strategy procedure, it is possible that some eligible studies might not have been identified. However, this is an inherent limitation of such studies, and the inclusion of a large number of studies does not affect the major conclusions of our results.

We should bear in mind that positive results from genetic association studies require biological and functional evidence that the risk variant is actually involved in the pathophysiology and the pathogenesis of the relevant disease. Pathway-based analysis could facilitate more robust analysis even of GWAS and provide additional biological insights on the mechanisms of disease complex traits.137,138 Therefore, the scientific reasoning could not be replaced by any single statistical value, index, or test.105,139 As a consequence, by the correct interpretation of statistical values, the misinterpretation of results could be avoided.140

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