The role of 5-HTTLPR in autism spectrum disorder: New evidence and a meta-analysis of this polymorphism in Latin American population with psychiatric disorders.

The autism spectrum disorder (ASD) is a complex disorder encompassing a broad phenotypic and genotypic variability. The short (S)/long (L) 5-HTTLPR polymorphism has a functional role in the regulation of extracellular serotonin levels and both alleles have been associated to ASD. Most studies including European, American, and Asian populations have suggested an ethnical heterogeneity of this polymorphism; however, the short/long frequencies from Latin American population have been under-studied in recent meta-analysis. Here, we evaluated the 5-HTTLPR polymorphism in Colombian individuals with idiopathic ASD and reported a non-preferential S or L transmission and a non-association with ASD risk or symptom severity. Moreover, to recognize the allelic frequencies of an under-represented population we also recovered genetic studies from Latin American individuals and compared these frequencies with frequencies from other ethnicities. Results from meta-analysis suggest that short/long frequencies in Latin American are similar to those reported in Caucasian population but different to African and Asian regions.

Introduction

Autism Spectrum Disorder (ASD) is a neurodevelopmental condition characterized by three core symptoms: repetitive/restricted behaviors, impairment in social interaction and variable communication skills [1]. The twin concordance rate and a milder autism phenotype in relatives reflects a strong genetic component in the pathophysiology of ASD; nevertheless, the broad phenotypic variability of this disorder and the 60% of individuals who remain with unknown etiology suggest an interplay of several genetic factors [2-4]

Most of the genetic factors involved in ASD have a role in brain development and the excitatory/inhibitory synaptic balance [5-8]. The serotoninergic system participates in neurogenesis, axon guidance and cell migration, and also modulates GABA and glutamate neurotransmitter release in presynaptic terminals. Within the serotoninergic system, the serotonin re-uptake transporter (SERT) located in presynaptic terminals and encoded by the SLC6A4 gene (Solute carrier family 6 member 4, Gene ID: 6532) has been widely studied. The transcriptional efficiency of SLC6A4 is regulated by the well-known short (S)/long (L) 5HTTLPR (5-HTT gene-linked polymorphic region) polymorphism, a repetitive sequence present in the upstream regulatory region of this gene [9-14]. The short allele which reduces the transcriptional activity has been reported with greater frequency in African and Egyptian individuals with ASD [15,16], and has been also associated with other psychiatric disorders such as major depressive disorder [17-20], bipolar disorder [21] and depression risk in Parkinson disease [22] among others; however, conflicting results have been also reported [23-25].

Despite three meta-analysis suggesting that the short/long alleles are not risk factors for ASD [26-28], the ethnical heterogeneity among included studies has been proposed as a factor that may affect the overall result. A comprehensive transmission disequilibrium test (TDT) meta-analysis including eighteen studies from diverse populations, demonstrated a non-preferential S/L transmission at the global scale, but preferential transmission of the S and L alleles in the American and Asian populations, respectively [29]. Thus, based on the frequency differences of 5-HTTLPR polymorphism in the worl-wide population, here we present an attempt to understand the role of short/long allele in Colombian population with idiopathic ASD in terms of risk, preferential transmission and symptoms severity. Additionally, we performed a meta-analysis aiming to evaluate the heterogeneity of the S and L alleles in the under-represented and highly admixed Latin America population.

Materials and methods

The research protocol was approved by ethics committee of participating institutions (Universidad de Los Andes and Instituto Colombiano del Sistema Nervioso—Clínica Montserrat). Written informed consent was obtained by all participants under 16. Parent or legal guardian written consent was obtained for all participants 15 and under or unable to consent.

Subjects

Idiopathic ASD diagnosis confirmed in 105 individuals was performed using the Diagnostic and Statistical Manual of Mental Disorders V (DSM-V) criteria [1]. Clinical evaluation also included the completion of the ADOS [30,31] and ADI-R [32] instruments. For our case-control analysis we used genotypic information of 171 unrelated and unaffected Colombian individuals belonging to the same region as the ASD trios. Peripheral blood was obtained from all participants and parents. DNA was extracted using Flexigene® DNA kit (Quiagen, Inc).

Genotyping

5-HTTLPR polymorphism was screened through PCR and gel electrophoresis according to Petri S et al., 1996 protocol [33]. PCR product was visualized with 3% MetaPhor agarose gel electrophoresis [Lonza Group Ltd., Basel, Switzerland].

Literature search

The Meta-analysis followed the Preferred Reporting Items for Systematic Reviews and Meta-analysis (PRISMA) criteria [34]. We screened without date restriction reports that evaluate the association of 5-HTTLPR and psychiatric disorders in PubMed, ScienceDirect and Scielo databases up to April 2020. To minimize the chance of missing relevant studies the search terms in PubMed and ScienceDirect databases were “HTTLPR” “SLC6A4” AND the name of each country encompassed in South and Central America (each separated by the Boolean operator OR). For the Scielo database, we searched published literature in the indexed journals from Latin American countries (S1 Fig). The inclusion criteria were (1) full text article in English or Spanish languages (2) case-control studies evaluating the polymorphism in psychiatric disorders (3) genotypic or allelic frequency provided and (4) control individuals not deviated from Hardy-Weinberg Equilibrium (HWE). We excluded reviews, studies evaluating trios, cases and no controls, population without a clinical diagnosis and studies that did not evaluate the Latin American population or the association of 5-HTTLPR with psychiatric disorders. For articles that did not have all the information available the authors were contacted. The data extraction was carried out by two investigators independently. The collected data from each study was categorized as reference, country of origin, evaluated trait, number of individuals and allelic/genotypic frequencies.

Statistical analysis

The test of association between ASD and 5-HTTLPR polymorphism was evaluated under the allelic (L vs S), genotypic (SS vs SL vs LL), dominant (LL/LS vs SS) and recessive (SS/SL vs LL) models using the genotypic information of 105 individuals with ASD and 171 unaffected/unrelated controls; corrections were conducted by the Bonferroni method. Linear regression and quantitative trait association modeled the severity of common ASD traits as a function of the 5-HTTLPR genotype. The family-based association for the 5-HTTLPR genotype and ASD was evaluated with the transmission disequilibrium test (TDT) using genotypic information of 105 trios. For the meta-analysis, the allelic and genotypic frequencies were included as a common measure to evaluate the association between 5-HTTLPR polymorphism and psychiatric disorders in Latin American population. The heterogeneity across studies was estimated by the Cochran’s Q test and the pooled Odds Ratio (OR) was evaluated under allelic (L vs S) and genotypic (SS vs LL+LS)—(LL vs SS+SL) models with fixed-effect or random-effect models according to I2 value (Fixed-effect for I2 <50% and Random-effect for I2 > 50%). The publication bias was assessed with funnel plots and quantitatively evaluated with Egger’s regression and Begg’s rank correlation. The trim and fill method was used to estimate potential missing studies and the sensitivity analysis removing each study for every meta-analysis was conducted to evaluate the stability of the results. All statistical analysis were performed with PLINK [35] and R studio program [36].

Results

The genotyping results of 105 trios and 171 unrelated controls are described in S1 and S2 Tables. The genotypic and allelic frequencies are summarized in Table 1 and full sample analysis including families and unrelated controls were consistent with frequencies predicted by Hardy-Weinberg equilibrium (p = 0.12).

Table 1

5-HTTLPR polymorphism in Colombian individuals.

Group	Genotype frequency				Allele frequency
	LL	SL	SS	Total	L	S
Father	19 [18%]	49 [47%]	37 [35%]	105	0.41	0.59
Mother	23 [22%]	42 [40%]	40 [38%]	105	0.42	0.58
Child with ASD	22 [21%]	42 [40%]	41 [39%]	105	0.41	0.59
Unrelated controls	38 [22%]	89 [52%]	44 [26%]	171	0.48	0.52

Genotypic and allelic frequencies in ASD trios and unaffected individuals

Although a significant association between 5-HTTLPR and ASD was observed only under dominant model (SS vs SL+LL, (p = 0.022)) (S3 Table), after Bonferroni’s correction non-significant result was observed. Additionally, the transmission disequilibrium test (TDT) indicates a non-preferential transmission of either the short or long allele (χ2 = 0.0989, p = 0.75) (S4 Table).

Phenotypic features and comorbidities in individuals with ASD were analyzed according to genotype (S5 Table). Aggressive behaviors were the most common comorbidity reported followed by intellectual disability and epilepsy. The sex ratio was 6:1 (male-to-female) with mean age of 10,8 ± 7,8 years old. For reasons of statistical power, the analysis between 5-HTTLPR and autism severity was restricted to the three core ASD symptoms (S1 and S2 Tables). The mean score for each trait was similar among the three genotypes and statistical test confirmed a non-association (Table 2).

Table 2

Phenotypic traits.

ADOS score in investigated dimension [Mean ± SD]	SS	SL	LL	p-value
ADOS Social communication	5,6 ± 1,9	5,9 ± 1,8	5 ± 1,8	0.4936
ADOS Restricted/Repetitive behaviors	2,9 ± 1,6	2,8 ± 1,3	2,9 ± 1,6	0.964
ADOS Social interaction	8,4 ± 3	9 ± 2,7	8,2 ± 2,4	0.8907

Mean score and standard deviation to each trait is presented according to genotype.

A total of 52 publications with the keywords provided in PubMed database were initially filtered, but only 13 case-control studies met the inclusion criteria: two from Colombia, three from Mexico, one from Argentina and seven from Brazil. In ScienceDirect database we found an additional study from Brazil and in Scielo database we found four additional studies: two from Colombia, one from Mexico and one from Brazil. No response was obtained from the authors that were contacted. Thus, from a total of 112 filtered reports, only 18 fulfilled the inclusion criteria (S1 Fig), and the allelic frequencies of each study were separately registered for cases and controls (Table 3).

Table 3

Allelic frequencies reported in Latin American population.

Country	Trait	Cases			Controls			Reference
		Sample size	S	L	Sample size	S	L
Colombia	ASD	105	0.59	0.41	171	0.52	0.48	Present study
Colombia	Bipolar disorder	103	0.49	0.51	112	0.53	0.47	[Ospina-Duque et al., 2000] [37]
Colombia	MDD	68	0.49	0.51	68	0.45	0.55	[Pérez-Olmos, et al., 2016] [38]
Colombia	Bipolar disorder	133	0.55	0.45	120	0.59	0.41	[Ramos, et al., 2012] [39]
Colombia	MDD	59	0.53	0.47	59	0.44	0.56	[Escobar, et al., 2011] [40]
Mexico	Obsessive‐compulsive disorder	115	0.58	0.42	136	0.52	0.48	[Camarena et al., 2001] [41]
Mexico	MDD	104	0.52	0.48	335	0.60	0.40	[Peralta-Leal et al., 2012] [42]
Mexico	ADHD	78	0.43	0.57	56	0.55	0.45	[Durán-González et al., 2018] [43]
Mexico	MDD and suicide attempt	200	0.63	0.37	233	0.51	0.49	[Sarmiento-Hernandez et al., 2019] [44]
Argentina	MDD	95	0.51	0.49	107	0.47	0.53	[Cajal et al., 2012] [45]
Brazil	Bipolar Disorder	167	0.37	0.63	184	0.36	0.64	[Neves et al., 2008] [46]
Brazil	Schizophrenia*	39	0.42	0.58	98	0.38	0.62	[Mendes De Oliveira et al., 1998] [47]
Brazil	Bipolar Disorder*	47	0.40	0.60	98	0.38	0.62	[Mendes De Oliveira et al., 1998] [47]
Brazil	Dysthimia~	62	0.43	0.57	197	0.38	0.62	[Oliveira et al., 2000] [48]
Brazil	Bipolar disorder~	64	0.38	0.62	197	0.38	0.62	[Oliveira et al., 2000] [48]
Brazil	MDD~	66	0.42	0.58	197	0.38	0.62	[Oliveira et al., 2000] [48]
Brazil	Anxiety	129	0.43	0.57	96	0.43	0.57	[Bortoluzzi et al., 2014] [49]
Brazil	Suicide in depressed patients	84	0.48	0.52	152	0.44	0.56	[Segal, et al, 2006] [50]
Brazil	Epilepsy	175	0.47	0.53	155	0.45	0.55	[Schenkel et al., 2011] [51]
Brazil	ASD	151	0.44	0.56	179	0.45	0.55	[Longo, et al, 2009] [52]
Brazil	Schizophrenia—Bipolar disorder	99	0.40	0.60	60	0.47	0.53	[Krelling et al., 2008] [53]
Brazil	Obsessive‐compulsive disorder	78	0.54	0.46	202	0.52	0.48	[Meira-Lima et al., 2004] [54]

ASD: Autism Spectrum Disorder, MDD: Major Depressive Disorder, ADHD: Attention Deficit Hyperactivity Disorder.

*Two traits evaluated independently but using the same control group,

~Three traits evaluated independently but using the same control group

The Latin American meta-analysis performed under three models (S vs L), (SS vs SL+LL) and (LL vs SL+SS) reflected a heterogeneity of 33.1%, 19.5% and 13.7%, respectively (Table 4, Fig 1 and S2 Fig). Fixed-effect model was selected to estimate the pooled OR based on heterogeneity results. The overall OR in either of the three models failed to find significant association (Table 4) suggesting that the 5-HTTLPR polymorphism does not increase the risk for psychiatric disorders in Latin American population (Fig 1 and S2 Fig). For publication bias, the funnel plots suggested absence of a bias (S3 Fig) and both Egger´s and Begg’s test confirmed non significance (Table 4). The trim and fill method did not identified missing studies for each model (S4 Fig) and the recalculated OR did not yield different conclusions (Table 4) just like the sensitivity analysis (S6 Table).

Table 4

Results of meta-analysis.

Genetic model	Meta-analysis		Heterogeneity		Bias		Recalculated OR with trim and fill method
	Pooled OR (95% CI)	p Value	I2	p Value	Egger p value	Begg p value	Pooled OR (95% CI)	p Value
S vs L	1.0698 [0.9880; 1.1584]	0.0962	33.1%	0.0673	0.5125	0.9775	1.0625 [0.9593; 1.1769]	0.2306
SS vs SL+LL	1.1008 [0.9644; 1.2565]	0.1548	19,5%	0.2034	0.5359	0.7997	1.0913 [0.9355; 1.2730]	0.2513
LL vs SL+SS	0.9146 [0.8066; 1.0371]	0.1641	13,7%	0.2766	0.636	0.8435	0.9218 [0.7975; 1.0654]	0.2550

Fig 1

Meta-analysis evaluating 5-HTTLPR polymorphism in Latin American population.

Forest plot for the allelic model. The trait and country for each study are found in Table 3. The forest plots for other two models are found in S2 Fig.

Discussion

The serotonin re-uptake transporter (SERT) located in presynaptic neuron removes this neurotransmitter from the synaptic cleft, regulates the serotonin concentration in the synapse and controls the magnitude or duration of post-synaptic transmission. Single nucleotide, Indel and VNTR polymorphisms in the SLC6A4 gene have been implicated in the re-uptake efficiency; for example, the LL genotype of 5HTTLPR polymorphism is related with increased SERT concentration becoming the S carrier variants as a risk factor for psychiatric disorders [16-21,55].

The role of 5-HTTLPR polymorphism in the ASD pathophysiology evaluated through case-control studies and family-based association have reflected conflicting results. For instance, two studies suggested S allele as risk variant for ASD [15,16], while three meta-analysis did not find association [26-28]. We assessed this polymorphism through a case-control approach failing to find association between 5-HTTLPR and idiopathic ASD, and also through a family-based assessment that did not find a preferential transmission of either S or L allele. A previous evaluation of this polymorphism in Colombian individuals with ASD also failed to find a significant association [56].

Phenotypic heterogeneity in ASD has been also studied in relation with the 5-HTTLPR polymorphism, two reports suggested an association of the S allele with higher severity in social communication impairment [57,58], while another group associated the severity of this trait with the LL genotype for 5-HTTLPR and the AA genotype for rs25531 [59]. Brune et al., 2006 also reported the LL genotype associated with severity of repetitive behaviors [58]. In our study, we did not find an association between 5-HTTLPR genotype and severity in the three core ASD symptoms (repetitive/restricted behaviors, impairment in social interaction and variable communication skills) just like other three reports [52,60,61]. An association between aggressive behaviors and the serotoninergic system has been also suggested [13,60,62,63]; in our study we observed that self-injury behaviors were mainly reported in individuals with the SS genotype (15/29), and although we did not find a significant association (p>0.05) our sample size does not have the statistical power to reveal this association. A summary of these conflicting results is presented in S7 Table.

Replication of results across ethnicities has been postulated as a good strategy to discover true risk variants or to understand if the lack of replication reflects the complexity in the genetic architecture of some complex disorders as ASD [64,65]. Although two meta-analyses recovering case-control studies mainly from European, American, and Asian population did not find association of this polymorphism with ASD, they also confirmed high heterogeneity in the included studies probably explained by population ethnicity [27,28]. These results suggest that the 5-HTTLPR polymorphism varies across ethnicities and the genetic background of each population may have a role in the risk conferred by this polymorphism [64,65].

Recognizing the under-representation of Latin American population in studies aimed to understand the role of 5-HTTLPR on worldwide [26,27,29], here we present a meta-analysis including articles which evaluate this polymorphism in Latin American individuals with psychiatric disorders as ASD, bipolar disorder, major depressive disorder, obsessive compulsive disorder, attention deficit hyperactive disorder, schizophrenia, dysthymia, anxiety disorder and suicide [37-54]. Although the S allele has been associated with increased risk for psychiatric disorders [16-21,55], our meta-analysis revealed no significant heterogeneity among studies, no publication bias and failed to find an association between 5-HTTLPR and a risk for psychiatric disorders. Comparing our frequencies with frequencies reported in other continents, the Latin American frequencies are more similar to those reported in Caucasian population [62,66-68], while S allele has been greater in Asian population [69,70] and L allele greater in African population [70,71]. Nevertheless, none of Latin American studies had a strict genetic control for population substructure between cases and controls and according to previous reports the Brazilian population should have an over-representation of African component in some regions [72-74]. Even though Latin America presents a diverse population substructure, most of the regions presented in the meta-analysis are likely to have an elevated Caucasian component compared to native American and African backgrounds [70,75,76]; however, slight differences may not be discarded in some small or separate regions according to demographic history of these admixed countries [72-76].

In summary, our study supports the absence of association between ASD and 5-HTTLPR polymorphism in a homogeneous cohort of Colombian individuals with idiopathic ASD. Additionally, a meta-analysis evaluating this polymorphism in Latin American regions suggests that frequencies of short/long alleles in this under-represented population are relatively homogeneous to frequencies reported in Caucasian populations.

Genotyping data for each trio and ADOS score for each individual with idiopathic ASD.

(DOCX)

Click here for additional data file.

Genotyping data for each unrelated control.

(DOCX)

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Results of case-control association test between ASD and 5-HTTLPR polymorphism.

(DOCX)

Click here for additional data file.

Transmission disequilibrium test in Colombian trios with ASD.

TDT was performed with families having heterozygotes parents (49 fathers and 42 mothers).

(DOCX)

Click here for additional data file.

Phenotypic traits in individuals with ASD according to 5-HTTLPR genotypes.

Two sub-categories were well-thought-out, patients who loss the acquired skills at 2 years old were classified as “ASD with regressive development” and patients with ASD and higher cognitive skills were classified as “High functioning ASD”. Intellectual disability was evaluated during clinical consultation without standardized test. Aggressive behaviors were analyzed according to ADIR reports.

(DOCX)

Click here for additional data file.

Sensitivity analysis.

(DOCX)

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5-HTTLPR polymorphism and ASD severity.

This table summarizes some results of studies evaluating severity of ASD symptoms and the serotoninergic system.

(DOCX)

Click here for additional data file.

Flowchart for selection of studies in the meta-analysis.

(DOCX)

Click here for additional data file.

Meta-analysis evaluating 5-HTTLPR in Latin American population.

The trait and country for each study and are in Table 3. a. Forest plot for SS vs SL+LL model, b. Forest plot for LL vs SL+SS model.

(TIF)

Click here for additional data file.

Funnel plots to evaluate publication bias.

a. Funnel plot for S vs L model b. Funnel plot for SS vs SL+LL model, c. Funnel plot for LL vs SL+SS model.

(TIF)

Click here for additional data file.

Trim and fill funnel plots.

Funnel plots without missing studies a. for S vs L model, b. for SS vs SL+LL model and c. for LL vs SL+SS model.

(TIF)

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PRISMA checklist.

(DOC)

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12 May 2020

PONE-D-20-10849

The role of 5-HTTLPR polymorphism in Autism Spectrum Disorder and other psychiatric disorders in the Latin American population

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Reviewer #1: In this paper, in light of the controversial evidence on the association between the 5-HTTLPR short variant (S) and 5-HTTLPR long variant (L) and ASD, the authors carried out a meta-analysis on the topic, focusing on the Colombian population.

The two variants fall into the promoter sequence of the SLC6A4 gene that codes for a transporter and regulator of serotonin levels, located in the presynaptic membrane.

The individuals were divided into two groups: the cases group consisting of 105 ASD trios, selected on the basis of the DSM-V, ADOS and ADI-R scale criteria; the control group of 171 apparently healthy unrelated individuals, from the same geographical area. After genotyping, the authors investigated the association between ASD and polymorphisms based on allelic, genotypic, dominant and recessive models. A linear regression analysis and association with quantitative traits was conducted to evaluate the association with severity for common traits for ASD. Subsequently, the results were compared with a meta-analysis on the data present in literature. In conclusion, the authors did not find a significant association between ASD and 5-HTTLPR polymorphisms in their case-control study. In addition, their meta-analysis highlighted a homogeneity of the frequency of the two variants in the Colombian population, which appears to be similar to that of the Caucasian population, but different from the African and Asian ones.

The article, despite its simplicity, is interesting and well-focused in the descriptive part. It would be appropriate to adapt the Discussion section accordingly, avoiding elements of repetitiveness, in order to make the reading more smooth and pleasant.

According to the Preferred Reporting Items for Systematic Review and Meta-analysis statement (PRISMA checklist 2009) we suggest revising the title to introduce a statement regarding meta-analysis work.

It might be useful to describe the information sources including databases’ dates of coverage and eventual contact with study authors. Aiming for a stronger significance of the results, we suggest to include any assessment of publication bias and to describe the methods used to investigate the presence of missing studies. We also recommend specifying whether duplicates have been found. If so, the authors might want to insert the duplicates exclusion step in the flow chart (S1 Figure) in the screening section.

In the text line 119, the information in the first parenthesis “(S vs )” seems to be incomplete.

In the text lines 130-133, we recommend the substitution of the term “synapsis” (that is the pairing of homologous chromosomes during the first prophase of meiosis) with the term “synapse”. We also recommend to rephrase the sentence from text line 132 to 133.

The main points are consistent with the analysis carried out. The tables are easy to interpret and the discussion does not highlight major issues.

However, the cohort under examination is reduced in number, therefore it cannot be considered statistically strong, as reported by the authors. The results could also be affected by stratification in the population under examination.

Reviewer #2: This study is a case-control analysis whose purpose is to evaluate a possible association between S/L 5HTTLPR polymorphism and idiopathic ASD in Colombian population. Moreover, they also investigated if there is a preferential transmission of the S or L allele and if there is association between the 5HTTLPR genotype and the severity of ASD symptoms, considering three main phenotypic traits (social communication, Restricted/repetitive behaviors and Social interaction) evaluated through ADOS scale. For a deeper analysis in comparison with the current scientific findings on this topic, three databases were consulted for a literature search: PubMed, ScienceDirect and Scielo. Among the results, 17 case-control studies fitted the inclusion criteria.

5-HTTLPR polymorphism was screened through PCR in 105 individuals with diagnosis of Idiopathic ASD and 171 unrelated and unaffected individuals belonging to the same region. The polymorphism was evaluated under the allelic (L VS S), genotypic (SS vs LS vs LL), dominant (LL/LS vs SS) and recessive (SS vs LS/LL) models. No significant association was found between 5-HTTLPR and ASD (after Bonferroni’s correction) [table 1]. The family-based association between the genotype and ASD in the 105 trios was evaluated with TDT test, resulting in no preferential transmission of either the S or L allele.

Regarding the association between ASD core symptoms severity and 5-HTTLPR according to genotype, none was found using statistical tests [table 2].

They also conducted a meta-analysis evaluating 5-HTTLPR polymorphism in Latin American population and its relation with psychiatric disorders[table 3; figure 1], resulting in no significant association.

As pointed out by the author, there are already three meta-analysis that demonstrate that the S/L 5HTTLPR polymorphism is not a risk factor for ASD, however this work has also taken into account the ethnical heterogeneity as a factor of influence. Through the meta-analysis they showed also that the frequency of L/S alleles is in the Latin-American population is quite similar to the reported frequency for Caucasian population (thus quite homogeneous). Due to conflicting results in other studies, it is yet to be confirmed if 5-HTTLPR polymorphism actually varies between ethnicities and if the genetic background of each population may have a role in the risk conferred by this polymorphism [27;28;29].

I think this work is interesting and able to offer a more comprehensive point of view about the relation between ASD and 5-HTTLPR polymorphism, especially of course in Latin American population.

All the tables presented are clear and easy to comprehend.

The principal Flaw I personally found in the article, was how the “Discussion” section was constructed.

Some topics were actually proposed more than once, rather than being articulated cohesively. For example, while explaining the preferential transmission of L or S alleles: lines 140-144 vs lines 176-178 (in association with ethnicity).

Some sentences are a little bit redundant and could have been better formulated, like: “Comparing our frequencies with… of the L allele” (lines: 194-198).

At line 119 there is a letter missing: (S vs L*).

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Reviewer #1: No

Reviewer #2: No

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2 Jun 2020

We appreciate the careful reading and the provided feedback on the manuscript “The role of 5-HTTLPR polymorphism in Autism Spectrum Disorder and other psychiatric disorders in the Latin American population”. We changed the original title to “The role of 5-HTTLPR in autism spectrum disorder: New evidence and a Meta-Analysis of this polymorphism in Latin American population with psychiatric disorders”. We incorporated suggestions made by the reviewers and editor with track changes in the “Revised Manuscript with track changes” and answer them below:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming.

A/ We adjusted the revised manuscript file according to PlosOne requirements.

We also included new tables (S1, S2 and S3 Tables) to fulfill publicly available information belonging to this manuscript. S1 Table includes genotypic information of trios and ADIR and ADOS scores for each individual with ASD. S2 Table has the genotypic information of controls. The S3 table includes the statistical results for each model.

2. PLOS requires an ORCID D for the corresponding author in Editorial Manager on papers submitted after December 6th, 2016. Please ensure that you have an ORCID iD and that it is validated in Editorial Manager.

A/ We included the ORCID ID of the corresponding author as follows https://orcid.org/0000-0003-2113-9266

Additionally, while reviewing the prisma checklist we encountered an article that contained all the genetic data but was initially excluded because it did not contain Hardy-Weinberg information. With the information provided by the authors in the article it was possible for us to obtain the HW results and therefore include it in our study. The results with the new information did not change the overall results

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer # 1:

1. The article, despite its simplicity, is interesting and well-focused in the descriptive part. It would be appropriate to adapt the Discussion section accordingly, avoiding elements of repetitiveness, in order to make the reading more smooth and pleasant.

According to the Preferred Reporting Items for Systematic Review and Meta-analysis statement (PRISMA checklist 2009) we suggest revising the title to introduce a statement regarding meta-analysis work.

A/ We appreciate your comments, they are very helpful and aimed to improve the quality of our work. _The original tittle “The role of 5-HTTLPR polymorphism in Autism Spectrum Disorder and other psychiatric disorders in the Latin American population” was modified to “The role of 5-HTTLPR in autism spectrum disorder: New evidence and a Meta-Analysis of this polymorphism in Latin American population with psychiatric disorders” to include the meta-analysis performed

2. It might be useful to describe the information sources including databases’ dates of coverage and eventual contact with study authors. Aiming for a stronger significance of the results, we suggest to include any assessment of publication bias and to describe the methods used to investigate the presence of missing studies. We also recommend specifying whether duplicates have been found. If so, the authors might want to insert the duplicates exclusion step in the flow chart (S1 Figure) in the screening section.

A/ We included the PRISMA checklist 2009 that contains information regarding dates, number of duplicated studies and authors contact in the paragraph of “Literature search”. Moreover, the paragraph “Statistical analysis” was modified to include the methodology used to evaluate for publication bias (Funnel plots, Egger’s / Begg’s tests) and the presence of missing studies (Trim and fill method).

A sensitivity assessment was also included to evaluate a risk of bias within selected studies.

The section Literature Search and Statistical Analysis was modified to include all these aspects.

3.In the text line 119, the information in the first parenthesis “(S vs )” seems to be incomplete.

A/The L letter was added to correct this mistake.

4. In the text lines 130-133, we recommend the substitution of the term “synapsis” (that is the pairing of homologous chromosomes during the first prophase of meiosis) with the term “synapse”. We also recommend to rephrase the sentence from text line 132 to 133.

A/We substituted the “Synapsis” by “Synapse” term. Moreover, we rephrased the next sentence for better understanding.

Reviewer #2:

1. I think this work is interesting and able to offer a more comprehensive point of view about the relation between ASD and 5-HTTLPR polymorphism, especially of course in Latin American population. All the tables presented are clear and easy to comprehend.

2. The principal Flaw I personally found in the article, was how the “Discussion” section was constructed. Some topics were actually proposed more than once, rather than being articulated cohesively. For example, while explaining the preferential transmission of L or S alleles: lines 140-144 vs lines 176-178 (in association with ethnicity). Some sentences are a little bit redundant and could have been better formulated, like: “Comparing our frequencies with… of the L allele” (lines: 194-198).

A/We appreciate your comments, they are very helpful and aimed to improve the quality of our work. We removed repetitive information previously consigned in these paragraphs and rephrased sentences to provide a better formulation of this information.

3. At line 119 there is a letter missing: (S vs L*).

A/The L letter was added to correct this mistake.

Submitted filename: Response reviewer.docx

Click here for additional data file.

17 Jun 2020

The role of 5-HTTLPR in autism spectrum disorder: New evidence and a Meta-Analysis of this polymorphism in Latin American population with psychiatric disorders

PONE-D-20-10849R1

Dear Dr. Lattig,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Giuseppe Novelli

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PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

23 Jun 2020

PONE-D-20-10849R1

The role of 5-HTTLPR in autism spectrum disorder: New evidence and a Meta-Analysis of this polymorphism in Latin American population with psychiatric disorders

Dear Dr. Lattig:

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org.

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Thank you for submitting your work to PLOS ONE and supporting open access.

Kind regards,

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on behalf of

Prof. Giuseppe Novelli

Academic Editor

PLOS ONE