The oxytocin receptor gene polymorphism rs2268491 and serum oxytocin alterations are indicative of autism spectrum disorder: A case-control paediatric study in Iraq with personalized medicine implications

Zainab Al-Ali; Akeel Abed Yasseen; Arafat Al-Dujailli; Ahmed Jafar Al-Karaqully; Katherine Ann McAllister; Alaa Salah Jumaah

doi:10.1371/journal.pone.0265217

. 2022 Mar 22;17(3):e0265217. doi: 10.1371/journal.pone.0265217

The oxytocin receptor gene polymorphism rs2268491 and serum oxytocin alterations are indicative of autism spectrum disorder: A case-control paediatric study in Iraq with personalized medicine implications

Zainab Al-Ali ¹, Akeel Abed Yasseen ², Arafat Al-Dujailli ³, Ahmed Jafar Al-Karaqully ⁴, Katherine Ann McAllister ^5,^‡,^*, Alaa Salah Jumaah ^2,^‡

Editor: Elsayed Abdelkreem⁶

PMCID: PMC8939799 PMID: 35316293

Abstract

Background

Impairment of social functioning skills is a key hallmark of autism. The neuropeptide oxytocin (OXT) is a blood-based biomarker of social functioning, and a candidate for individualized treatment of ASD. The effects of OXT on the social brain are mediated by the OXT receptor (OXTR). This study assessed the clinical utility of blood OXT serum levels and the OXT receptor (OXTR) genotype as biomarkers of autism and its severity in a pediatric population in Iraq.

Methods

Blood samples were collected from patients with a clinical diagnosis of ASD (n = 60) and corresponding age and gender matched healthy controls (n = 60). All clinical samples were processed at the Department of Pathology and Forensic Medicine, Faculty of Medicine, University of Kufa in Iraq. Blood serum was assayed for OXT by sandwich ELISA. Receiver operator analysis (ROC) determined area under the curve (AUC), cutoff values, and sensitivity and specificity of OXT values for accuracy of diagnosis of ASD. Isolated genomic DNA was genotyped for the OXTR gene rs2268491(C/T) SNP using allele-specific PCR. The significance of genotype (CC, CT, and TT) and allele (C and T) distributions in different patient groups was assessed using odd ratios (OR) with 95% confidence intervals (CI) and the Chi-square test. All statistical analysis was performed used SPSS software.

Results

Study characteristics in the ASD population revealed a high level of consanguinity (36.66%), and ASD recurrence rate (11.66%) and family history (28.33%). OXT levels in patients with ASD (157.58±28.81 pg/ml) were significantly higher (p = 0.003) compared to controls (75.03±6.38 pg/ml). Within stratified ASD severity groups—OXT levels were significantly different (P = 0.032). ROC analysis determined similar AUC values for overall ASD (0.807), and stratified mild (0.793), moderate (0.889), and severe categories (0.795). The best cutoff for diagnosis of ASD was 83.8 pg/ml OXT with a sensitivity and specificity of 80% and 72.1% respectively. OXTR gene rs2268491(C/T) genotyping found that ASD patients have significantly lower (p = 0.021) genotype CC frequency and a significantly higher (p = 0.04) occurrence of the heterozygous CT genotype relative to controls. ASD subjects produced highest OXT levels with the TT genotype. T allele distribution was higher in ASD males. ASD males had significantly lower distribution of the CC genotype (48.89%) compared to females (80%) (Chi-square test: χ2 = 4.43, df = 1, p = 0.035). Whereas distribution of the CT genotype was significantly higher in autistic males (44.45%) compared to females (13.33%) (Chi-square test: χ2 = 4.68, df = 1, p = 0.03).

Conclusion

Peripheral OXT levels and OXTR genetic alterations are potential biomarkers of social functioning in the ASD patient setting. The stratification of patients with ASD into severity categories shows significant differences both in OXT levels and OXTR (rs2268491, C/T) genotype and allele distributions, that can be sex dependent. OXT based therapies will require personalized medicine tactics to correctly identify patients with ASD who require neuropeptide boosting in social settings.

Introduction

Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by early age social communication deficits, language delay and repetitive sensory motor behaviors. The clinical presentation of autism is usually lifelong, while severity ranges from very mild in some people to a severe occurrence of developmental disability in others [1]. Autism is not a single clinical disorder because of this variable phenotypic spectrum. Substantial care is required to support individuals with ASD who cannot function independently. This causes an economic burden for society. The worldwide elevation in autism stresses the need for collective global efforts to investigate this problem to support autistic children and their families. The prevalence of autism has increased in developed countries [2], with recent estimates of 1.70% and 1.85% in US children aged 4 and 8 years respectively [3]. The pathogenesis of ASD is still poorly understood, and is believed to entail genetic and environmental risk factors [4].

The incidence of ASD in Middle East countries has increased. Both the volatile environment in parts of the Middle East and genetic factors could exacerbate the development of autism. However there is a paucity of genetic and molecular biology studies among these populations to provide adequate diagnostic methods, patient support and guidance [5]. Twin and family research studies provide compelling evidence for the role of genetics in the occurrence of autism. Family studies reveal an eight to ten percent recurrence risk of autism in the siblings of affected probands [6]. Blood-related marriages may increase the risk of producing offspring with ASD [7]. While consanguinity occurs in 10.4% of the global population [8], Arab countries have the highest rates approaching 60% [9]. Therefore evaluation and diagnosis of ASD in these communities should entail screening for consanguinity.

Oxytocin is a potential diagnostic metric for the social core symptoms of ASD. The plasma levels of OXT are positively correlated with autistic diagnostic interview (ADI) reciprocal interaction and communication scores [10]. However studies report contradictory blood levels of OXT in autistic individuals that compromise its clinical utility. Lower OXT levels have been reported in European (Slovakia), US, and Middle East (Iraq) populations of children with ASD in comparison with normal healthy controls [10–12]. One study in Germany [13] reported no significant difference in OXT levels between ASD and healthy control patients (p = 0.132). Whereas other studies report elevated plasma OXT level among US and European children and adults with ASD [14, 15] and Chinese Han children with ASD [16] relative to healthy controls. The association between OXT levels and social function in ASD is not straightforward and requires further investigation to determine whether high or low serum OXT levels are a biomarker of ASD.

Single nucleotide polymorphisms (SNPs) in the OXT receptor (OXTR) gene are associated with autism, and include rs2268491(C/T), rs2254298 (G/A) and rs53576 (G/A). Recently brain activity was found markedly reduced in adolescent autistic females with the rs2268491 genotype who responded to an emotion recognition task in comparison to normal controls [17]. Furthermore the OXTR SNPs rs2254298 (G/A) and rs53576 (G/A) are found associated with autism in the Chinese Han population [18] and rs2254298 in Caucasian patients [19]. Conversely meta-analysis suggests no association of both rs2254298 (G/A) and rs53576 (G/A) SNPs with human social behaviour [20].

Building upon all the above arguments, we investigated the OXTR serum levels and OXTR SNP RS2268491 in a pediatric population with ASD in Iraq. Our objectives were to (1) determine if serum OXT concentration could be used as an ASD biomarker (2) determine genotype and allele distributions of rs2268491 in patients with ASD (3) explore associations between OXT protein and the OXTR polymorphism.

Materials and methods

Study population and sample size calculation

The study was conducted at the Department of Pathology and Forensic Medicine, Faculty of Medicine, University of Kufa. During a one-year period (December 2019 to December 2020) paediatric patients (median age of 7, range 3–15 years) with a clinical diagnosis of ASD by the study consultant psychiatrist were recruited.

A sample size of N = 36 was calculated as the minimal requirement to determine serum oxytocin alterations in the study. The study variable of interest (peripheral oxytocin alterations) was identified as dichotomous (proportion). The sample size was calculated using the following equation: N = 4 * Zα2 * p (1 − p)/w². Where Zα is the confidence level, W is the width of the confidence interval (equal to twice the margin of error) and P is the study estimate of the proportion to be measured [21]. The proportion of ASD patients who present with oxytocin alterations was estimated at 90% compared to controls (S1 Appendix), with a selected confidence interval of ± 10 [21]. N = 4 * 1.96 * 0.9 (1 − 0.9)/0.2² = 17.64 (18 cases of ASD)

Minimum sample size for 1:1 case-control study: N = 36

A final total of 60 cases of ASD were recruited for the study along with age and gender matched healthy controls (n = 60) for comparison. All the cases were recruited from the teaching hospitals scattered throughout the Middle and South Euphrates regions in Iraq.

Inclusion and exclusion criteria, CARS evaluation of pediatric ASD

Patients who fulfilled the criteria of being a typical ASD subject according to the diagnostic criteria of the Diagnostic and Statistical Manual of Mental Disorders, (American Psychiatric Association) were included [22]. The diagnosis of ASD was confirmed by a consultant psychiatrist who evaluated the child in person, in collaboration with the research team. The patients with ASD were stratified according to severity into the following sub-groups [22]: mild ASD (n = 39), moderate ASD (n = 13) and severe ASD (n = 8) according to the Childhood Autism Rating Scale (CARS) [23]. The CARS is a diagnostic assessment method that rated individuals on a scale ranging from normal to severe and yields a composite score ranging from non-autistic to mildly autistic, moderately autistic, or severely autistic. The scale was used to observe and subjectively rate the following fifteen items in both ASD and case control subjects. Each of the following 15-items were scored in patients according to seven levels of severity by interview:

relationship to people
imitation
emotional response
body
object use
adaptation to change
visual response
listening response
taste-smell-touch response and use
fear and nervousness
verbal communication
non-verbal communication
activity level
level and consistency of intellectual response
general impressions

The controls were normal healthy children, unrelated to the autistic subjects and without any of the following exclusion criteria:

Any medical condition likely to be etiological for ASD (e.g. Rett syndrome, focal epilepsy).
Any neurologic disorder involving pathology above the brain stem, other than uncomplicated non-focal epilepsy.
Contemporaneous evidence, or unequivocal retrospective evidence, of probable neonatal brain damage.
Any genetic syndrome involving the CNS, even if the link with autism is uncertain.
Clinically significant visual or auditory impairment, even after correction.
Any circumstances that might possibly account for the picture of autism (e.g. severe nutritional or psychological deprivation).
Active treatment with pharmacological or other agents.

Blood sample collection and preparation

A venous blood sample was collected from patients with ASD and healthy controls in parallel at the University of Kufa teaching hospital, Department of Clinical Laboratories. Both groups were matched in both gender and age. A 3.0 mL whole blood volume sample was transferred to a sterilize serum collection tube and allowed to clot for 20 minutes at room temperature. This sample was centrifuged at 2000–3000 RPM for 20 minutes. The separated serum was divided into small aliquots and stored at -20°C for later OXT measurements. A further 2.5 mL of whole blood was transferred into a EDTA tube for DNA extraction and PCR genotyping. All laboratory work was performed at the Department of Pathology and Forensic Medicine, Faculty of Medicine, University of Kufa.

Isolation of genomic DNA for PCR genotyping

Instruments and equipment, along with chemicals and reagents for DNA extraction and PCR are available in S1 and S2 Tables.

Genomic DNA was isolated from 2.5 mL whole blood samples using the Column-pure blood Genomic DNA Mini Kit, Applied Biological Materials (Anatolia Turkey) according to the manufacturer instructions under sterile conditions (S3 Table). Genomic DNA concentrations were measured using the Nano-drop spectrophotometer. The DNA concentrations were determined by measuring the absorbance at 260 nm wavelength (A260) and 280 nm wavelength (A280). Purity was determined by calculating the ratio of absorbance at 260 nm and the absorbance at 280 nm (A260/A280). Absorbance scans showed a symmetric peak at 260 nm confirming high purity. The purity of DNA -was considered acceptable if it was in the range of 1.8–2.0.

DNA quality was assessed by gel electrophoresis (S3 Table). The electrophoresis process was conducted at 5–8 voltage/cm for 45 min. After termination of electrophoresis, the agarose gel was visualized using a UV-transilluminator (Cleaver Scientific Co., UK) [24]. Agarose gel electrophoresis was used in this study to fulfill two purposes: (1) evaluate the quality of genomic DNA samples enrolled in the study prior to allele specific PCR and (2) check the presence of PCR products at the expected sizes after termination of the allele specific PCR.

Allele specific PCR primer design and synthesis

The study cohort DNA samples were genotyped for OXTR SNP accession numbers rs2268491 using the allele specific PCR technique.

All allele specific PCR primers used in this study were designed manually. The SNP was retrieved from the database dbSNP (contains human SNP variations, microsatellites, small scale-insertions, and deletions, https://www.ncbi.nlm.nih.gov/snp/). Mainly nucleotide sequences of 1000 bp or 500bp containing the SNP were retrieved from the database. Then, the allele specific primers were designed manually and were computationally checked regarding 3’ complementarity, 3’self -complementarity, GC content, and melting temperature using the primer-blast online program, localized at the server (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) from NCBI (National Center for Biotechnology and Information). All designed primers in this study were synthesized in Integrated DNA Biotechnology (IDT Co., Canada). The sequences of allele specific primers for the oxytocin receptor SNP gene rs2268491, and full PCR cycling condition are outlined in S4 Table.

Effective SNP discrimination by allele-specific PCR was performed using standard PCR conditions. Each DNA sample (1 μg/PCR reaction) was processed through two alleles specific PCR reactions. Each allele specific PCR reaction was directed with a primer set: forward primer carrying the allele SNP base at 3’ prime end and a reverse primer. The control reaction was established like the allele specific PCR reaction, except that the forward allele primer was replaced by control forward primer (primer set: control forward and common reverse primer). Two allele-specific PCR products were generated of different lengths and separated by agarose gel electrophoresis for direct visualization of the genotyping result.

Enzyme-Linked Immunosorbent Assay (ELISA) oxytocin measurements

The ELISA quantitative immunoassay sandwich kit measured human OXT in serum (manufacturer, Bioassay Technology laboratory). The assay range was 2 pg/ml to 600 pg/ml. Each serum sample was added in duplicate to a pre-coated human OXT antibody plate. Biotinylated human OT Antibody was added to bind to OXT in the sample followed by streptavidin-HRP. After incubation, unbound Streptavidin-HRP is washed away. The substrate solution was added to enable color development in proportion to the amount of human OXT. The reaction was terminated using acidic stop solution and absorbance measured at 450 nm with a plate reader (Bioassay Technology laboratory) and serum OXT concentration assessed via the standard curve method.

Statistical analysis

The Statistical Package of Social Sciences version 27 (SPSS Inc.; Chicago, IL, USA) computer program was used for results analysis. For each variable the values were presented as mean ± SD. The student t test determined the statistical difference between two groups. One-Way ANOVA was performed to evaluate the differences among multiple groups. For statistical comparison between different groups, the statistical significance was defined as p≤0.05, while a p-value of >0.05 was not significant. A p-value of <0.001 was considered highly significant. In the studied groups, the representativeness of alleles and genotypes was estimated by the Hardy-Weinberg equilibrium (HWE) by comparing the observed and expected frequencies of genetic variants. The Chi-square test was applied to assess genotype and allele frequencies between patients and controls. The genotype and allele distributions were determined in each group, and odds ratios (OR) with 95% confidence intervals (95% CI) were calculated. A p-value of <0.05 was considered statistically significant at a confidence interval (CI) of 95%. Receiver operating characteristic (ROC) analysis assessed the accuracy of OXT as a biomarker for autism. The area under the curve (AUC) indicates an excellent diagnostic and predictive marker when close to one, a curve that lies close to the diagonal (AUC = 0.5) has no diagnostic significance. Normality testing of OXT levels in the patient serum samples was assessed using the Kolmogorov-Smirnov test.

Ethical approval

The present study was approved by the Institutional Review Board of the University of Kufa, Faculty of Medicine, in accordance with the 1964 Helsinki declaration and its later amendments. “The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.” All patients were informed about the purpose of the work and written parental consent and questionnaire information was obtained from the father of each patient.

Results

Study patient characteristics

The patients with ASD had a mean age of 7.08± 2.54 compared to the control group 6.95±1.94 (Table 1). There were no significant differences in age between both groups (p = 0.708). The rate of consanguinity among the Arabic study populations was high at 36.66% and 40% in the ASD and healthy control groups. Within the stratified population of ASD subjects, the consanguinity rates occurred at 35.89%, 30.76% and 50% in mild, moderate, and severe cases. Recurrence of ASD within the same family occurred in 7 cases (11.66%) out of the total population (n = 60). Furthermore 28.33% of the ASD population had a family history of the condition, that increased in the most severe cases to 37.50%. Eight patients (13.3%) out of the population with ASD suffered from fit episodes, that occurred at higher frequency in the severe cases (37.5%).

Table 1. Patient ethnicity, consanguinity, recurrence, and family history of ASD.

Characteristics	Control patients	ASD patients	Mild ASD	Moderate ASD	Severe ASD
N	60	60	39	13	8
Ethnicity	Arab	Arab	Arab	Arab	Arab
Mean Age (+/-SD)	^a6.95 ± 1.94	7.08± 2.54	6.89±2.19	7.23±3.81	7.78±3.67
Age Range, Median	3–15, 7	3–15, 7	3–15, 7	3–15, 10	4–15, 8
Consanguanity	24 (40.0)	22 (36.66)	14 (35.89)	4 (30.76)	4 (50)
OR	-	1	0.97	0.77	1.73
P value	-	1	0.94	0.69	0.47
95% CI	-	0.48–2.10	0.42–2.24	0.21–2.79	0.39–7.60
Recurrent (Same family)	0 (0)	7 (11.66)	6 (15.38)	1 (7.69)	0 (0)
ASD Family History	0 (0)	17 (28.33)	12 (30.76)	3 (23.07)	3 (37.50)
^bFit	0 (0)	8 (13.33)	2 (5.12)	3 (23.07)	3 (37.5)

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^aNo significant differences in age between control and ASD groups (p = 0.708).

^bFit is a convulsion or seizure and a clinical sign that occurs when there is a sudden burst of electrical activity in the brain temporarily interfering with the normal messaging processes.

Serum OXT levels in ASD and control subjects and ROC curve analysis

Table 2 shows the ELISA measurements of serum OXT among the ASD patients (n = 60) and the healthy control group (n = 60) in the present study. The normal distribution of the results was checked using Kolmogorov-Smirnov testing (p>0.05). Serum OXT levels in the patients with ASD (157.58±28.81 pg/ml) were significantly higher (p = 0.003) in comparison to the healthy control group (75.03±6.38 pg/ml). The serum OXT levels in the patients with ASD were stratified into severity categories of mild (n = 39), moderate (n = 13), and severe (n = 8). The ANOVA test found statistically significant difference (p = 0.032) in OXT levels among the ASD severity groups, as shown in Table 2. The greatest significance difference in OXT occurred between mild and severe ASD patients (p = 0.001). The correlation of OXT with age in ASD patients was not significant (P = 0.396, R = -0.140, R² = 0.0196; S1 Fig). There was no also significant difference (p>0.05) in OXT levels in children from related parents compared to unrelated (S5 Table). Fig 1A shows the elevated OXT levels in the overall ASD population and stratified ASD severe subtype in comparison to lower levels in healthy controls.

Table 2. ELISA serum OXT levels among study participants.

Group	N	^aSerum OXT (pg/mL)	P-value OXT
Control Patients	60	75.03±6.38	0.003^*
ASD Patients	60	157.58±28.81
Mild ASD	39	165.02±28.03	^c0.032^*
Moderate ASD	13	152.78±27.86
Severe ASD	8	129.09±11.04

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^aNormality tested using Kolmogorov-Smirnov test: controls (0.055), ASD (0.072), mild ASD (0.21), moderate ASD (0.2), severe ASD (0.2).

^bNo significant differences in age between control and ASD groups (p = 0.708).

^cANOVA test compared OXT levels among the ASD severity groups of mild, moderate, and severe (p = 0.032*). Significance was also compared between specific ASD severity groups: mild versus moderate (p = 0.178), mild versus severe (p = 0.001*), and moderate versus severe (0.034*).

*Denotes a significant difference (P<0.05).

Table 3 show the ROC analysis of OXT serum data presented as AUC, best cut off values, specificity, and sensitivity in patient groups. Fig 1B shows the ROC curve for all ASD cases, while Fig 1C–1E shows the mild, moderate, and severe groups independently. The OXT exhibited AUC values higher than 0.8 in the overall ASD patient population (AUC = 0.807), and similar values in mild (AUC = 0.793) moderate (AUC = 0.889), and severe autistic patients (AUC = 0.795). The best cutoff in pediatric serum samples of 83.8 pg/mL OXT offers a diagnostic accuracy of ASD at sensitivity and specificity levels of 80% and 72.1% respectively. Within the stratified population of ASD, the cutoff was highest in mild patients (87.2 pg/mL) and lowest in severe cases (71.4 pg/mL). The highest test accuracy occurred in patient with moderate ASD according to sensitivity and specificity indicators.

Table 3. ROC analysis.

Groups	Area under curve	Best cutoff (pg/mL)	Sensitivity	Specificity
ASD patients	0.807	83.8	80	72.1
Mild	0.793	87.2	76	68.2
Moderate	0.889	78.3	86	80.6
Severe	0.795	71.4	75	70.4

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OXTR gene rs2268491 polymorphism in study population

Table 4 reports the allele and genotype distributions of the OXTR SNP rs2268491 (C, T) in the study population. ASD patients had a significantly lower (p = 0.021) frequency of the wild-type genotype CC (56.67% versus 76.66%) and a significantly higher (p = 0.04) occurrence of the heterozygous CT genotype (36.67% versus 20%) relative to healthy controls. The odd ratio (OR) for both the CT and the TT genotype favored the ASD population at 2.315 (95%CI; 1.0178–5.2692) and 2.071 (95%CI; 0.3648–11.762) respectively. Whereas likelihood of the wild-type CC homozygous genotype is reduced in the ASD population with an OR of 0.398 (95% CI; 0.1813–0.8739) and favors the healthy population. The C allele distribution was higher in healthy controls while the T allele distribution was higher in the ASD population.

Table 4. Genotype distribution (CC, CT, TT) and alleles (C, T) of rs2268491 in patients with ASD (N = 60) and healthy controls (N = 60).

Genotype / Allele	Patients		Control		P value	OR	95% CI
Genotype / Allele	No.	%	No.	%	P value	OR	95% CI
Genotype
CC	34	56.67	46	76.66	0.021^*	0.398	0.1813 to 0.8739
CT	22	36.67	12	20	0.04^*	2.315	1.0178 to 5.2692
TT	4	6.66	2	3.33	0.411	2.071	0.3648 to 11.762
Alleles
C	90	75	104	86.67	0.866	1	0.5418 to 1.8457
T	30	25	16	13.33	0.866	1	0.5418 to 1.8457

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*Denotes a significant difference (P<0.05).

The distribution of the OXTR SNP rs2254298 genotypes according to the Hardy-Weinberg equilibrium found consistency in both groups (χ2 = 0.03, df = 1, p >0.05 in ASD subjects; χ2 = 0.01, df = 1, p >0.05 in healthy controls).

OXTR gene rs2268491 polymorphism in ASD males and females

Table 5 reports the allele and genotype distributions of the OXTR SNP rs2268491 (C, T) in males and females of the ASD study population. The distribution of the CC genotype was significantly lower in males (48.89%) compared to females (80%) according to the Chi-square test (χ2 = 4.43, df = 1, p = 0.035). Whereas distribution of the CT genotype was significantly higher in autistic males (44.45%) compared to females (13.33%) using the Chi-square test (χ2 = 4.68, df = 1, p = 0.03). No significant difference was evidenced in the distribution of the genotype TT among the male and female autistic patients. Overall the C allele distribution was higher in females while the T allele distribution was higher in males.

Table 5. OXTR polymorphism rs2268491 in ASD males (n = 45) and females (n = 15).

Genotype / allele	Male		Female		DF	X ²	P-value
Genotype / allele	No.	%	No.	%	DF	X ²	P-value
Genotype
CC	22	48.89	12	80	1	4.43	0.035^*
CT	20	44.45	2	13.33	1	4.68	0.03^*
TT	3	6.66	1	6.66	1	0	1
Alleles
C	64	71.12	26	86.67	1	2.904	0.088
T	26	28.88	4	13.33	1	2.904	0.088

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*Significant association (P<0.05).

OXTR gene rs2268491 polymorphism in ASD severity categories

Table 6 reports the allele and genotype distributions of the OXTR SNP rs2268491 (C, T) in the ASD mild (n = 39), moderate (n = 13) and severe (n = 8) cases. There are no significant findings in the genotype or allele frequencies in any of the severity categories.

Table 6. OXTR polymorphism rs2268491 in ASD severity categories.

Genotype / Allele	Mild		Moderate			Severe			DF	X ²	P -value
Genotype / Allele	No.	%	No.		%	No.		%	DF	X ²	P -value
Genotype
CC	20	51.29	8		61.5	6	75		2	1.68	0.431
CT	16	41.0	4		30.7	2	25		2	0.98	0.612
TT	3	7.69	1		7.69	0	0		2	0.65	0.719
Alleles
C	56	71.7	20	76.9		14	87.5		2	1.81	0.404
T	22	28.2	6	23.07		2	12.5		2	1.81	0.404

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OXT levels according to genotype distributions in stratified autism patients and healthy controls

The OXT levels in stratified autistic and control groups are presented according to genotype distribution of rs2268491 in Table 7 and Fig 2. A marked trend for higher OXT was observed in the three genotypes of CC, CT, and TT in ASD patients relative to control. The healthy controls produced highest OXT protein (85.45±8.01 pg/mL) in the normal homozygous CC genotype, however there are no significant differences between the three control genotypes (CC, CT, TT). ASD subjects produced the highest OXT in the TT genotype (169.52±8.42 pg/mL). The ASD subjects exhibited a significant association between serum oxytocin levels and the three OXTR SNP rs2254298 genotypes (p = 0.045). In stratified ASD patients, serum oxytocin levels were highest in the mild category with the polymorphism genotype TT (175.02±13.05). The moderate ASD cases displayed significant differences among the three genotypes in serum OXT levels (p = 0.038). The ASD severe stratified population had lower numbers (n = 8) reducing power of interpreting observations.

Table 7. OXTR polymorphism rs2268491 in ASD severity categories.

Group	Genotype OXT levels (mean pg/ml ±SD)			P value
Group	CC	CT	TT	P value
Control	85.45±8.01	70.73±9.18	68.92±7.24	0.056
ASD	145.21±9.21	132.51±5.08	169.52±8.42	0.045^*
Mild	161.74±12.04	158.12±14.2	175.02±13.05	0.118
Moderate	139.34±8.21	114.41±4.8	164.01±9.05	0.038^*
Severe	134.54±4.28	125.02±8.72	--------	0.069

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*Significant association (P<0.05).

Discussion

This study aimed to develop a framework to improve diagnostic testing of children and adolescents with ASD in Iraq to help improve their outlook as adults. There is a currently a paucity of genetic and molecular biology studies of these specific populations to provide adequate diagnosis, to support patients and parents and issue guidance for the future [4]. Our study characteristics revealed a high level of consanguinity (36.66%), recurrence rate (11.66%) and family history (28.33%) among the ASD population. Given that the consanguinity rate worldwide is 10.4% [8], the rates observed in both the ASD and control study populations are at least three fold higher. Therefore it is likely that specific gene pools are the root cause of some of the ASD cases in the study population of Arabs. The ASD candidate genes need to be identified so that a personalized medicine treatment plan can be developed for future pediatric ASD cases to improve their prognosis and social functioning to achieve independency within society as adults.

Autism is a lifelong neurodevelopmental disorder. However studies show that early diagnosis and intervention leads to significantly improved outcomes. Ignoring early and accurate diagnosis of this disorder might lead to secondary disorders such as depression and anxiety [25]. Currently there is no gold standard treatment to improve social functioning skills in autism, apart from medications that help to relieve the comorbid anxiety and panic disorders associated with the condition. Since the neuropeptide OXT and its receptor OXTR regulate social functioning in animals and humans, it is the front-runner among candidate social treatments for ASD. Plasma OXT concentrations and OXTR polymorphisms predict social impairments in children with and without ASD [26]. Furthermore intranasal OXT administration improves social abilities in children with ASD [26]. The probability that innovative uses of OXT will improve the lives of autism patients is twofold because of its dual therapeutic and diagnostic potential.

Our study found that serum OXT levels are significantly elevated (p = 0.003) in the pediatric patients with ASD in comparison to the matched healthy control group. The gold standard statistical tool of ROC analysis was used to demonstrate the potential of OXT as a blood-based biomarker of ASD. OXT had excellent AUC values higher than 0.8 in the overall ASD patient population (AUC = 0.807), and along with mild (AUC = 0.793) moderate (AUC = 0.889), and severe autistic patients (AUC = 0.795) and had good accuracy with specificity and sensitivity values. These results prove the potential role of OXT serum measurements in the diagnosis of ASD at optimal cut off values, assessment of severity, and, for prognostic purposes in this specific ethnic population.

The trend for elevated OXT in this study agrees with other studies [14–16]. However, the use of OXT as a biomarker of ASD is not straightforward, as these results are contradictory with those reporting lower OXT in ASD patients [10–12]. However, within the stratified ASD population in this study, the highest OXT levels occurred in the mild subgroup, while the lowest OXT levels occurred in the severe group. Our study found that ASD severity categories had statistically significant (p = 0.032) difference in OXT levels. This trend does agree with the previous study of ASD children in Iraq [12]. Here OXT level were highest in mild autistic patients with a significant decrease (p<0.05) in moderate autistic patients, and a highly significant decrease (p<0.01) in severe autistic patients compared with control. Interestingly, this variability in OXT levels in stratified ASD patients reported both Iraqi studies may relate to a key finding of the Stanford study [27]. The US investigators noted that pretreatment blood OXT concentrations also predicted response to intranasal OXT treatment [27]. Those individuals with the lowest pretreatment OXT concentrations showed the greatest social improvement [27]. Therefore, the ASD severe patients with lowest OXT in our study could be suitable candidates for individualized intranasal therapy with OXT to boost their social functioning. However, much wider study populations and statistical analysis is required to reach any conclusion before OXT concentrations are used as hallmarks for ASD diagnosis and treatments.

Emerging evidence also links alterations in OXT signaling pathways and its receptor -the OXTR, in the etiology of ASD. The effects of OXT on the social brain are mediated by the OXTR. A recent study used in vivo arterial spin labelling to identify changes in cerebral blood flow following intranasal administration of OXT that implicated many target areas within the brain [28]. The knockout of OXT receptors in mouse models showed autistic-like deficits in social interaction [29].

The OXTR SNP rs2268491 has been significantly associated with ASD by meta-analysis [30]. In particular, T allele carriers of the SNP are strongly linked to ASD related social behaviors [30]. Brain imaging also shows that activity is markedly reduced in adolescent autistic T carriers of the rs2268491 genotype when given a social decision task in comparison to normal control T carriers [17]. Our SNP genotyping study (rs2268491) discovered that ASD patients had a significantly lower (p = 0.021) frequency of the genotype CC and a significantly higher (p = 0.04) occurrence of the heterozygous CT genotype relative to healthy controls. This finding again suggests that the T allele is contributing to ASD.

Our study also found that ASD subjects with the TT genotype had the highest OXT levels. The elevated OXT levels noted among these patients are likely a reflection of genetic changes in the OXTR rs2254298 SNP. The ASD subjects exhibited a significant association between serum oxytocin levels and the three OXTR SNP rs2254298 genotypes (p = 0.045). In stratified ASD patients, serum OXT levels were highest in the mild category with the polymorphism genotype TT. This also suggests that patients with milder ASD symptoms and the TT genotype may have evolved an ability to upregulate OXT levels to compensate for the cerebral social response skills deficiency caused by the OXTR polymorphism. The higher amounts of OXT will help to improve facial processing and human interpersonal contact, causing milder symptoms of ASD. A recent mouse ASD model study has demonstrated that targeting OXTR-expressing neurons in the lateral septum restores social skills [31]. Both our current study and previous research [27, 31] suggest that OXT is adaptive and can restore the homeostasis in the body.

Autism also has a sex bias with up to five times higher frequency amongst boys compared to girls [32]. There are also known sex differences in the phenotypic presentation of ASD in girls compared to boys. Our study found that males with ASD had a significantly lower distribution of the normal homozygous CC genotype (48.89%) compared to females (80%) (Chi-square test: χ2 = 4.43, df = 1, p = 0.035). Whereas distribution of the CT genotype was significantly higher in autistic males (44.45%) compared to females (13.33%) (Chi-square test: χ2 = 4.68, df = 1, p = 0.03). The T allele distribution was more than twice as high in male subjects. Given that the T allele carriers have compromised OXT mediated brain activity for social responses, this could provide a genetic explanation for why ASD affects some of the girls less frequently and possibly less severely in phenotypic presentation than boys. There may be an underlying OXT related mechanism impaired more so in the boys that gives rise to greater preponderance of ASD.

The implications of this study are that measurement of OXT levels and genetic alterations in its receptor could serve as a simple blood-based biomarker test to gauge social functioning in the ASD patient for diagnostic, monitoring and support purposes. Genetic and protein biomarkers in peripheral blood samples are easier and less expensive to analyze with a high throughput potential in the laboratory for ASD diagnostics, in comparison with genome-wide sequencing or brain imaging approaches.

Study limitations

Ideally the conclusions presented in this study should be generalizable to the wider Iraqi Arab population. Therefore the study samples must be representative of the population and adequate in number [33]. Feasibility issues in the Middle and South Euphrates population region limited recruitment of large sample sizes in the current study. Although the estimated study sample size was sufficient for the serum OXT analysis, numbers are still inadequate to generalize to the entire population. For example, consanguinity was high among the ASD populations as expected, but also the control group. The sample size might explain why the consanguinity proportions are higher among control patients. Sample size also affects the association between SNP markers and disease. Testing a single SNP marker in a 1:1 case control study requires 248 cases to achieve 80% statistical power according to the allelic genetic model, under certain assumptions [34]. Insufficient study numbers are a limitation of the current investigation of SNP rs2254298.

Conclusion

In conclusion the stratification of patients with ASD into severity categories shows statistically significant (p = 0.032) differences in OXT levels. The elevated OXT levels noted among patients with ASD are likely a reflection of genetic changes in the OXTR, especially when certain genotype or alleles are more predominant in younger patients with ASD. Certain OXTR genotypes such as the T carriers of rs2268491 may have upregulated levels of peripheral OXT to compensate for ineffective processing of OXT at its receptor for social functioning in ASD. Undoubtably, the successful application of OXT based therapies for ASD patients will necessitate personalized medicine tactics to stratify patients who require neuropeptide boosting in social settings.

Supporting information

S1 Fig. Nonsignificant correlation of serum OCT levels and age of ASD patient cohort (P = 0.396, R = -0.140, R² = 0.0196).

(TIF)

Click here for additional data file.^{(71.9KB, tif)}

S1 Table. Instruments and equipment used in the study.

(DOCX)

Click here for additional data file.^{(16.2KB, docx)}

S2 Table. Chemicals and reagents for DNA extraction and polymerase chain reaction.

(DOCX)

Click here for additional data file.^{(14.1KB, docx)}

S3 Table. Lab protocols.

(DOCX)

Click here for additional data file.^{(16.7KB, docx)}

S4 Table. The sequences of allele specific primers of oxytocin receptor diallelic gene rs2268491 (C/T) and PCR information.

(DOCX)

Click here for additional data file.^{(16KB, docx)}

S5 Table. OXT levels of control and autistic groups with related and unrelated parents.

(DOCX)

Click here for additional data file.^{(15.7KB, docx)}

S1 Appendix. Study datasheet.

(XLSX)

Click here for additional data file.^{(16.6KB, xlsx)}

Acknowledgments

The study participant children and their parents who supported this study are gratefully acknowledged by all the study authors.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The author(s) received no specific funding for this work.

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PLoS One. doi: 10.1371/journal.pone.0265217.r001

Decision Letter 0

Elsayed Abdelkreem

29 Nov 2021

PONE-D-21-31116The oxytocin receptor gene polymorphism rs2268491 and serum oxytocin alterations are indicative of autism spectrum disorder:

A case-control paediatric study in Iraq with personalized medicine implicationsPLOS ONE

Dear Dr. McAllister,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. Specifically, the authors should address the following points:

Criterion 3. “Experiments, statistics, and other analyses are performed to a high technical standard and are described in sufficient detail”. Experiments must have been conducted rigorously, with appropriate controls and replication. Sample sizes must be large enough to produce robust results, where applicable. Methods and reagents must be described in sufficient detail for another researcher to reproduce the experiments described. (1) Authors should clarify sampling method and justify sample size. (2) Define severity of ASD. (3) Provide required details for all used kits and reagents, such as manufacturer, city, and country. (4) Experimental details on Isolation of genomic DNA and Agarose gel electrophoresis are commonly used techniques so that they should be summarized in the main text and the technical details can be better provided as supplementary. (5) Did the authors test data for normality? (6) Did the author consider confounders “e.g., the Median age of control is lower than that of ASD, which may affect serum OXT level”. (7) In table 2, authors should describe which statistical tests were used; moreover, data are presented as mean (SD) but some appear to be not normally distributed. (8) In addition “The ANOVA test found statistically significant difference (p=0.032) in OXT levels according to severity of ASD, as shown in Table 2”. Please, specify the difference? (9) In table 2, it seems that Oxytocin levels are lower in severe ASD (higher in mild ASD), which may be associated with older age. (10) It is interesting to see that healthy controls had highest OXT levels in the CC genotype, while ASD subjects had the highest OXT in the TT genotype; it would be meaningful to explain/discuss such findings.
Criterion 4. “Conclusions are presented in an appropriate fashion and are supported by the data.” Authors have to revise the conclusion of their study to be based on study findings (e.g., “The measurement of peripheral OXT levels and OXTR genetic alterations provide a simple dual-biomarker test to gauge social functioning in the ASD patient for diagnostic, monitoring and support purposes”; however, this study didn’t investigate monitoring and support aspects). Other suggested implications/recommendations based on study findings/conclusion should be separated from the conclusion itself.
Criterion 5. “The article is presented in an intelligible fashion and is written in standard English.” (1) Table 1: better to show ASD and controls in columns and variables as rows. Please, provide other important data, such as gender and comorbid conditions. (2) The article contain several errors related to English language, spelling, and grammar. PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. We may reject papers that do not meet these standards. If the language of a paper is difficult to understand or includes many errors, we may recommend that authors seek independent editorial help before submitting a revision.
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Criterion 7. “The article adheres to appropriate reporting guidelines and community standards for data availability.” (1) In the abstract and discussion, authors seem to selectively report some findings in an inappropriate manner, such as “study characteristics in the ASD population revealed a high level of consanguinity (36.66%)” although the percentage of consanguinity was higher among control group (40%). (2) The authors stated that “all data are fully available without restriction” but they did not provide unidentified patients’ data. Authors are required to make all data underlying the findings described fully available, without restriction, and from the time of publication. PLOS allows rare exceptions to address legal and ethical concerns, but this has to be completely explained. Please, review PLOS Data Policy.

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Reviewer #1: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: I Don't Know

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Reviewer #1: Yes

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Reviewer #1: This is an important study from clinical point of view. The whole article seemed to be written in readers friendly language, well articulated and intelligently structured. It is publishable, though I would like to suggest for further evaluation of statistical issues by an expert in this field.

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PLoS One. 2022 Mar 22;17(3):e0265217. doi: 10.1371/journal.pone.0265217.r002

Author response to Decision Letter 0

19 Jan 2022

1. Criterion 3:

a. (1) Authors should clarify sampling method and justify sample size.

Answer: (lines 126 paragraph manuscript) literature searches were performed to assess the prevalence of autism spectrum disorder. An estimated 1 % of the world’s population has autism spectrum disorder, with recent figures of 1.5%. Since the prevalence of the disease is a relatively uncommon event at 1 out of 100 cases, a case control study was selected as the most efficient sampling method to investigate autism among the Iraqi Arab population. A study sample size of N=120 (60 ASD and 60 controls) was feasible to manage in the face of opposition to the study from local tribal and religious restrictions. Our study had a 3:1 male to female ratio in the ASD and control populations. The DSM-5 states that “autism spectrum disorder is diagnosed four times more often in males than in females.”(American Psychiatric Association 2013), and most recent research suggest that it is closer to 3:1 (Loomes, Hull, and Mandy 2017). Therefore the study population is reflective of the gender ratio in ASD and provides a useful model to explore specific genetic associations.

b. (2) Define severity of ASD.

Answer: This was delineated in the PLOS PDF lines 145-182 as follow:

The severity was defined according to the Childhood Autism Rating Scale as follow:

The patients with ASD were stratified according to severity into the following sub-groups (20): mild ASD (n=39), moderate ASD (n=13) and severe ASD (n=8) according to the Childhood Autism Rating Scale

(CARS) (21). The CARS is a diagnostic assessment method that rated individuals on a scale ranging from normal to severe and yields a composite score ranging from non-autistic to mildly autistic, moderately autistic, or severely autistic. The scale was used to observe and subjectively rate the following fifteen items in both ASD and case control subjects. Each of the following 15-items were scored in patients according to seven levels of severity by interview:

1. Relationship to people

2. Imitation

3. Emotional response

4. Body

5. Object use

6. Adaptation to change

7. Visual response

8. Listening response

9. Taste-smell-touch response and use

10. Fear and nervousness

11. Verbal communication

12. Non-verbal communication

13. Activity level

14. Level and consistency of intellectual response

15. General impressions

c. (3) Provide required details for all used kits and reagents, such as manufacturer, city, and country.

Answer: done. These were delineated in supplementary Table S1, S2

S1 Table. Instruments and Equipment used in the study.

S2 Table. Chemicals and reagents for DNA extraction and polymerase chain reaction.

d. (4) Experimental details on Isolation of genomic DNA and Agarose gel electrophoresis are commonly used techniques so that they should be summarized in the main text and the technical details can be better provided as supplementary.

Answer:

The experimental details for both isolation of genomic DNA and preparation of gels for electrophoresis are now summarized in the main manuscript with full lab protocols described in supplementary table 3.

e. (5) Did the authors test data for normality?

Answers: The Kolmogorov-Smirnov test was used to test normality, which showed a normal distribution, methods (statistical analysis): paragraph 270. The test for all variables was greater than 0.05 so that samples are normally distributed (Table 2).

f. Did the author consider confounders “e.g., the Median age of control is lower than that of ASD, which may affect serum OXT level”.

Answer. Ages were matched between the ASD and control group. There were no significant differences in age distribution between studied and control group P=0.708 (Table 2).

g. (7) In table 2, authors should describe which statistical tests were used; moreover, data are presented as mean (SD) but some appear to be not normally distributed.

Answer: done. Analysis of Variance (ANOVA) test were used for statistical analysis as the data were normally distributed which was ensured by doing Kolmogorov-Smirnov test (Table 2).

h. (8) In addition “The ANOVA test found statistically significant difference (p=0.032) in OXT levels according to severity of ASD, as shown in Table 2”. Please, specify the difference?

Answer: done. The oxytocin level was 165.02±28.03 pg/mL in mild cases, 152.78±27.86 pg/mL in moderate cases, and 129.09±11.04 pg/mL in severe cases. Furthermore, there were significant differences in oxytocin level when comparing mild and severe cases (p=0.001), moderate and severe cases (p=0.034). On other hand there were no significant differences in oxytocin level between mild and moderate cases (p=0.178) (Table 2, table footnotes).

i. (9) In table 2, it seems that Oxytocin levels are lower in severe ASD (higher in mild ASD), which may be associated with older age.

Answer. Done. Oxytocin level was higher in mild cases (165.02±28.03 pg/mL) than severe cases (129.09±11.04 pg/mL). However there was no significant correlation between age of the patient and oxytocin level as stated in the manuscript in lines 554 and Supplementary Figure 1 (P= 0.396, R=-0.140, R2=0.0196; Supplementary Fig 1).

j. (10) It is interesting to see that healthy controls had highest OXT levels in the CC genotype, while ASD subjects had the highest OXT in the TT genotype; it would be meaningful to explain/discuss such findings.

Answer. Done (Discussion, main manuscript). In stratified ASD patients, serum oxytocin levels were higher in the mild category with the polymorphism genotype TT. This suggests that patients with milder ASD symptoms and the TT genotype may have evolved an ability to upregulate OXT levels to help or try to compensate for the cerebral social response skills deficiency caused by the OXTR polymorphism. The higher amounts of OXT will help to improve facial processing and human interpersonal contact. As showed in previous study D LoParo et al 2015 showed oxytocin receptor gene rs2268491 (‘T’ allele is risk-inducing) in the meta-analysis, these SNPs were shown to be substantially related with ASD, implying that signals from these SNPs may represent a shared connection with ASD. Liu x, et al. 2010 showed two SNPs (rs2268491 and rs2254298), which were significantly associated with ASD in there study in which re 2268491 with allele C predominant.

2. Criterion 4. “Conclusions are presented in an appropriate fashion and are supported by the data.” Authors have to revise the conclusion of their study to be based on study findings (e.g., “The measurement of peripheral OXT levels and OXTR genetic alterations provide a simple dual-biomarker test to gauge social functioning in the ASD patient for diagnostic, monitoring and support purposes”; however, this study didn’t investigate monitoring and support aspects). Other suggested implications/recommendations based on study findings/conclusion should be separated from the conclusion itself.

Answer: Done. The final conclusion paragraph has been amended, the implications/recommendations of the study have been presented as such from lines 485

3. Criterion 5. “The article is presented in an intelligible fashion and is written in standard English.”

a. (1) Table 1: better to show ASD and controls in columns and variables as rows. Please, provide other important data, such as gender and comorbid conditions.

Answer. Done. Table 1 was corrected as requested by the reviewers.

b. (2) The article contain several errors related to English language, spelling, and grammar. PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. We may reject papers that do not meet these standards. If the language of a paper is difficult to understand or includes many errors, we may recommend that authors seek independent editorial help before submitting a revision.

Done.

4. Criterion 6. “The research meets all applicable standards for the ethics of experimentation and research integrity.”. The authors repeat the ethics statement twice, once under study populations “Formal parental consent was obtained from families who expressed interest in the study, who were provided with consent sheets and a questionnaire to complete” and under Ethical approval “All patients were informed about the purpose of the work, and a written consent obtained from each patient.”. Authors should provide this information once. Moreover, did they obtain consent from the patients “children?”, parents, or both?

Answer: In fact, written consent was taken from the father of the ASD child only who is in charge and responsible for any matter regarding the ASD patients according to the societal customs in Iraq. The ethics statement has been updated as recommended. Please refer to paragraph lines 272

Criterion 7. “The article adheres to appropriate reporting guidelines and community standards for data availability.”

a. (1) In the abstract and discussion, authors seem to selectively report some findings in an inappropriate manner, such as “study characteristics in the ASD population revealed a high level of consanguinity (36.66%)” although the percentage of consanguinity was higher among control group (40%).

Answer: The sample size might be behind this variation which shows that the percentage of consanguinity was higher among control group. Much larger sample sizes will facilitate a clear picture on these variations. The consanguinity findings are now fully reported in the results and discussion sections lines 282 and 403:

• Results: The rate of consanguinity among the Arabic study populations was high at 36.66% and 40% in the ASD and healthy control groups (Table 1).

• Discussion: Given that the consanguinity rate worldwide is 10.4% (7), the rates observed in both the ASD and control study populations are at least three fold higher.

b. (2) The authors stated that “all data are fully available without restriction” but they did not provide unidentified patients’ data. Authors are required to make all data underlying the findings described fully available, without restriction, and from the time of publication. PLOS allows rare exceptions to address legal and ethical concerns, but this has to be completely explained. Please, review PLOS Data Policy.

Done. All data is available as supplementary material as Supplementary S1 Appendix.

5. Data sharing statement.

Answer: All the data cannot be made publically available as a results of local institution policy. However, it can be made available on a reasonable request from the corresponding author or from the department of pathology and forensic medicine (third party).

Contact information of the department of pathology and forensic medicine is: Email: pathology.med@uokufa.edu.iq

(email included in the cover letter)

6. Please amend either the title on the online submission form (via Edit Submission) or the title in the manuscript so that they are identical.

Amended

7. PLOS PDF (phrase data not shown).

Done. This phrase has been removed, and the OXT and patient age Supplementary Fig 1 is now included.

Attachment

Submitted filename: answer to reviewers.docx

Click here for additional data file.^{(24.8KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0265217.r003

Decision Letter 1

Elsayed Abdelkreem

2 Feb 2022

PONE-D-21-31116R1The oxytocin receptor gene polymorphism rs2268491 and serum oxytocin alterations are indicative of autism spectrum disorder: A case-control paediatric study in Iraq with personalized medicine implications

PLOS ONE

Dear Dr. McAllister,

Besides the important reviewers' comments shown below, authors are requiried to address the following points:

(1) Previous comment: Authors should clarify sampling method and justify sample size.

Authors’ response: (lines 126 paragraph manuscript) literature searches were performed to assess the prevalence of autism spectrum disorder. An estimated 1 % of the world’s population has autism spectrum disorder, with recent figures of 1.5%. Since the prevalence of the disease is a relatively uncommon event at 1 out of 100 cases, a case control study was selected as the most efficient sampling method to investigate autism among the Iraqi Arab population. A study sample size of N=120 (60 ASD and 60 controls) was feasible to manage in the face of opposition to the study from local tribal and religious restrictions. Our study had a 3:1 male to female ratio in the ASD and control populations. The DSM-5 states that “autism spectrum disorder is diagnosed four times more often in males than in females.”(American Psychiatric Association 2013), and most recent research suggest that it is closer to 3:1 (Loomes, Hull, and Mandy 2017). Therefore the study population is reflective of the gender ratio in ASD and provides a useful model to explore specific genetic associations.

New comment: Please, provide calculation of the sample size that would be required for this study. If the number enrolled in this study satisfy the calculated sample size, this would be perfect. If not, you can acknowledge this as one of the study limitations and provide justifications, including feasibility issues.

(2) Previous comment: Did the author consider confounders “e.g., the Median age of control is lower than that of ASD, which may affect serum OXT level”.

Authors response. Ages were matched between the ASD and control group. There were no significant differences in age distribution between studied and control group P=0.708 (Table 2).

New comment: Thank you for your important clarification. However, as shown in Table 2, the mean age for controls is lower than that of patients, and the p-value may become significant if larger number was included. This underscores the importance of sample size calculation.

(3) Previous comment: It is interesting to see that healthy controls had highest OXT levels in the CC genotype, while ASD subjects had the highest OXT in the TT genotype; it would be meaningful to explain/discuss such findings.

Authors’ response. Done (Discussion, main manuscript). In stratified ASD patients, serum oxytocin levels were higher in the mild category with the polymorphism genotype TT. This suggests that patients with milder ASD symptoms and the TT genotype may have evolved an ability to upregulate OXT levels to help or try to compensate for the cerebral social response skills deficiency caused by the OXTR polymorphism. The higher amounts of OXT will help to improve facial processing and human interpersonal contact. As showed in previous study D LoParo et al 2015 showed oxytocin receptor gene rs2268491 (‘T’ allele is risk-inducing) in the meta-analysis, these SNPs were shown to be substantially related with ASD, implying that signals from these SNPs may represent a shared connection with ASD. Liu x, et al. 2010 showed two SNPs (rs2268491 and rs2254298), which were significantly associated with ASD in there study in which re 2268491 with allele C predominant.

New comment: Thank you for these important discussions/thoughts . However, authors are encouraged to expand the discussion of this point in the main text (please, integrate the rest of your response, including the references, in the main text).

(4) Previous comment: In the abstract and discussion, authors seem to selectively report some findings in an inappropriate manner, such as “study characteristics in the ASD population revealed a high level of consanguinity (36.66%)” although the percentage of consanguinity was higher among control group (40%).

Authors’ response: The sample size might be behind this variation which shows that the percentage of consanguinity was higher among control group. Much larger sample sizes will facilitate a clear picture on these variations. The consanguinity findings are now fully reported in the results and discussion sections lines 282 and 403. •Results: The rate of consanguinity among the Arabic study populations was high at 36.66% and 40% in the ASD and healthy control groups (Table 1). •Discussion: Given that the consanguinity rate worldwide is 10.4% (7), the rates observed in both the ASD and control study populations are at least three fold higher

New comment: Thank you for your important clarifications. Please, integrate your comments regarding sample size in the main text.

(5)Other comments

Please, provide the age of study participants in table 1 (with other characteristics) rather than table 2.
It is not necessary to provide the p-value for the Kolmogorov-Smirnov test in table 2. Instead, just report this in the table footnote or results section.
Authors are encouraged to acknowledge and discuss study limitations and generalizability of findings.

Please submit your revised manuscript by Mar 19 2022 11:59PM. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

We look forward to receiving your revised manuscript.

Kind regards,

Elsayed Abdelkreem, MD, PhD

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #2: Title: The oxytocin receptor gene polymorphism rs2268491 and serum oxytocin alterations are indicative of autism spectrum disorder: A case-control paediatric study in Iraq with personalized medicine implications

The manuscript is precisely written and it presents an excellent point that might help in the suggestion of personalized precision intervention strategy. I recommend the acceptance but only after major revision. From my experience the number of samples is satisfactory, however,

- Most recent prevalence of ASD should be cited:

E.g.: Bougeard Clémence, Picarel-Blanchot Françoise, Schmid Ramona, Campbell Rosanne, Buitelaar Jan. Prevalence of Autism Spectrum Disorder and Co-morbidities in Children and Adolescents: A Systematic Literature Review, Frontiers in Psychiatry, 12, 2021 DOI=10.3389/fpsyt.2021.744709

- The significantly high oxytocin in mild ASD patients compared to moderate and severe should be explained and supported by other studies if possible.

- Individuals with autism do not outgrow autism (This sentence is not clear please clarify or delete)

“Ignoring early and accurate diagnosis of this disorder might lead to secondary disorders such as depression and anxiety (Reference is needed). Please see the reference below:

Hollocks MJ, Lerh JW, Magiati I, Meiser-Stedman R, Brugha TS. Anxiety and depression in adults with autism spectrum disorder: a systematic review and meta-analysis. Psychol Med. 2019 Mar;49(4):559-572. doi: 10.1017/S0033291718002283. Epub 2018 Sep 4. PMID: 30178724.

- “These results suggest the ROC curve could become the gold standard for the identification of parameters that are sensitive and specific enough to support ASD diagnosis”. ROC curves are already known as excellent statistical tool in the field of biomarkers, so this statement should be corrected.

- The authors stated that “ within the stratified ASD population in this study, the highest OXT levels occurred in the mild subgroup, while the lowest OXT levels occurred in the severe group. This should be explained and supported at least with the fact that nasal oxytocin is recommended to decrease the severity of ASD symptoms (Support is mandatory).

- The significant difference between mild, moderate and severe ASD should be clearly presented in the table not only in the text. Roc curves for mild, moderate and severe should be presented independently.

- The authors mentioned that “This suggests that patients with milder ASD symptoms and the TT genotype may have evolved an ability to upregulate OXT levels to help or try to compensate for the cerebral social response skills deficiency caused by the OXTR polymorphism. The higher amounts of OXT will help to improve facial processing and human interpersonal contact” (Again support your suggestion).

- Please go through the manuscript below it might help

https://www.nature.com/articles/s41598-020-79109-0.pdf

Horiai, M., Otsuka, A., Hidema, S. et al. Targeting oxytocin receptor (Oxtr)-expressing neurons in the lateral septum to restore social novelty in autism spectrum disorder mouse models. Sci Rep 10, 22173 (2020). https://doi.org/10.1038/s41598-020-79109-0

Gene expression analysis shows that Oxt mRNA is up-regulated in brain and bone which indicate that Oxt is adaptive and important in restoring the homeostasis of the body.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Afaf El-Ansary

PLoS One. 2022 Mar 22;17(3):e0265217. doi: 10.1371/journal.pone.0265217.r004

Author response to Decision Letter 1

7 Feb 2022

Response to Reviewers

PLOS Editor specific revisions:

The authors would like to thank the PLOS editor for these helpful manuscript suggestions.

• Previous comment: Authors should clarify sampling method and justify sample size.

New comment to address: Please, provide calculation of the sample size that would be required for this study. If the number enrolled in this study satisfy the calculated sample size, this would be perfect. If not, you can acknowledge this as one of the study limitations and provide justifications, including feasibility issues.

Response from authors:

Methods, Lines (131-144) A sample size of N=36 was calculated as the minimal requirement to determine serum oxytocin alterations in the study cohort. The study variable of interest (peripheral oxytocin alterations) was identified as dichotomous (proportion). The sample size was calculated using the equation: N = 4 * Zα2 * p (1 − p)/w2. Where Zα is the confidence level, W is the width of the confidence interval and P is the study estimate of the proportion to be measured. The proportion of ASD patients who present with oxytocin alterations compared to controls was estimated at about 90% (S1 Appendix) with a selected confidence interval of ± 10 [16]

N = 4 * 1.96 * 0.9 (1 − 0.9)/0.22 = 17.64 (18 cases of ASD)

Minimum sample size for 1:1 case-control study cohort: N=36

Please note that a bigger sample size is ideal for estimating a single OXT SNP in a population (about N=250), this is discussed in the new study limitation section of the discussion (lines 508 paragraph).

• Previous comment: Did the author consider confounders “e.g., the Median age of control is lower than that of ASD, which may affect serum OXT level”.

New comment to address: Thank you for your important clarification. However, as shown in Table 2, the mean age for controls is lower than that of patients, and the p-value may become significant if larger number was included. This underscores the importance of sample size calculation

Responses from authors: Thank you for raising the issue of confounders. The study is of a case control age and gender matched design which should reduce effects of these confounder variables. Although the mean age of patients with ASD is slightly higher - it is not significant (Table 1). The sample size calculations indicates that the study numbers are sufficient to investigate the variable of serum oxytocin.

• Previous comment: It is interesting to see that healthy controls had highest OXT levels in the CC genotype, while ASD subjects had the highest OXT in the TT genotype; it would be meaningful to explain/discuss such findings.

New comment: Thank you for these important discussions/thoughts. However, authors are encouraged to expand the discussion of this point in the main text (please, integrate the rest of your response, including the references, in the main text).

Responses from authors: The discussion of this key finding of the how OXT levels can differ in relation to OXTR genotype has been reworked and expanded in relation to social functioning with references (lines 465 to 488)

• New comment: Thank you for your important clarifications. Please, integrate your comments regarding sample size in the main text.

Response: Done. Refer to the study limitation section of the discussion (lines 508 paragraph).

• Please, provide the age of study participants in table 1 (with other characteristics) rather than table 2.

Response: Done

• It is not necessary to provide the p-value for the Kolmogorov-Smirnov test in table 2. Instead, just report this in the table footnote or results section.

Response: Done

• Authors are encouraged to acknowledge and discuss study limitations and generalizability of findings.

Response: done. (Lines 507-519) ‘Ideally the conclusions presented in this study should be generalizable to the wider Iraqi Arab population. Therefore the study samples must be representative of the population and adequate in number (33). Feasibility issues in the Middle and South Euphrates population region limited recruitment of large sample sizes in the current study. Although the estimated study sample size was sufficient for the serum OXT analysis, numbers are still inadequate to generalize to the entire population. For example, consanguinity was high among the ASD populations as expected, but also the control group. The sample size might explain why the consanguinity proportions are higher among control patients. Sample size also affects the association between SNP markers and disease. Testing a single SNP marker in a 1:1 case control study requires 248 cases to achieve 80% statistical power according to the allelic genetic model, under certain assumptions (34). Insufficient study numbers are a limitation of the current investigation of SNP rs2254298.

Reviewer 2:

The authors would like to thank reviewer 2 for these helpful revisions.

• Most recent prevalence of ASD should be cited:

Response: This is an excellent suggestion, we have updated our introductory prevalence of ASD literature, citing ‘recent estimates of 1.70 and 1.85% in US children aged 4 and 8 years respectively’ (lines 81-82)

• The significantly high oxytocin in mild ASD patients compared to moderate and severe should be explained and supported by other studies if possible.

Response: Done. Please refer to discussion lines 441- 457

• The trend for elevated OXT in this study agrees with other studies (14-16). However, the use of OXT as a biomarker of ASD is not straightforward, as these results are contradictory with those reporting lower OXT in ASD patients (10-12). However, within the stratified ASD population in this study, the highest OXT levels occurred in the mild subgroup, while the lowest OXT levels occurred in the severe group. Our study found that ASD severity categories had statistically significant (p=0.032) difference in OXT levels. This trend does agree with the previous study of ASD children in Iraq (12). Here OXT level were highest in mild autistic patients with a significant decrease (p<0.05) in moderate autistic patients, and a highly significant decrease (p<0.01) in severe autistic patients compared with control. Interestingly, this variability in OXT levels in stratified ASD patients reported both Iraqi studies may relate to a key finding of the Stanford study (27). The US investigators noted that pretreatment blood OXT concentrations also predicted response to intranasal OXT treatment(27). Those individuals with the lowest pretreatment OXT concentrations showed the greatest social improvement (27). Therefore, the ASD severe patients with lowest OXT in our study could be suitable candidates for individualized intranasal therapy with OXT to boost their social functioning. Individuals with autism do not outgrow autism (This sentence is not clear please clarify or delete)

Response: Done. Sentence is updated to: ‘Autism is a lifelong neurodevelopmental disorder’.

• “Ignoring early and accurate diagnosis of this disorder might lead to secondary disorders such as depression and anxiety (Reference is needed). Please see the reference below: Hollocks MJ, Lerh JW, Magiati I, Meiser-Stedman R, Brugha TS. Anxiety and depression in adults with autism spectrum disorder: a systematic review and meta-analysis. Psychol Med. 2019 Mar;49(4):559-572. doi: 10.1017/S0033291718002283. Epub 2018 Sep 4. PMID: 30178724.

Response: Done. Thank you for the reference suggestion.

• “These results suggest the ROC curve could become the gold standard for the identification of parameters that are sensitive and specific enough to support ASD diagnosis”. ROC curves are already known as excellent statistical tool in the field of biomarkers, so this statement should be corrected.

Response: Done. ROC curves are now discussed as an existing ‘gold standard statistical tool (lines 432)

• The authors stated that “within the stratified ASD population in this study, the highest OXT levels occurred in the mild subgroup, while the lowest OXT levels occurred in the severe group. This should be explained and supported at least with the fact that nasal oxytocin is recommended to decrease the severity of ASD symptoms (Support is mandatory).

Response: Done. (Lines 451-456). The US investigators noted that pretreatment blood OXT concentrations also predicted response to intranasal OXT treatment (27). Those individuals with the lowest pretreatment OXT concentrations showed the greatest social improvement (27). Therefore, the ASD severe patients with lowest OXT in our study could be suitable candidates for individualized intranasal therapy with OXT to boost their social functioning.

• The significant difference between mild, moderate and severe ASD should be clearly presented in the table not only in the text. Roc curves for mild, moderate and severe should be presented independently

Response: Done. Figure 1 has been updated to show all ROC curves independently, including mild, moderate and sever (Fig 1C-E).

• The authors mentioned that “This suggests that patients with milder ASD symptoms and the TT genotype may have evolved an ability to upregulate OXT levels to help or try to compensate for the cerebral social response skills deficiency caused by the OXTR polymorphism. The higher amounts of OXT will help to improve facial processing and human interpersonal contact” (Again support your suggestion).

Please go through the manuscript below it might help

https://www.nature.com/articles/s41598-020-79109-0.pdf

Gene expression analysis shows that Oxt mRNA is up-regulated in brain and bone which indicate that Oxt is adaptive and important in restoring the homeostasis of the body.

Response: Done. Suitable discussion material (lines 484-487) has been added from the suggested paper. ‘A recent mouse ASD model study demonstrated that targeting OXTR-expressing neurons in the lateral septum restores social skills (31). The results of the current study and previous research (27, 31) suggest that OXT is adaptive and can restore the homeostasis of the body.

Attachment

Submitted filename: response to reviewers 070222.docx

Click here for additional data file.^{(25.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0265217.r005

Decision Letter 2

Elsayed Abdelkreem

28 Feb 2022

The oxytocin receptor gene polymorphism rs2268491 and serum oxytocin alterations are indicative of autism spectrum disorder: A case-control paediatric study in Iraq with personalized medicine implications

PONE-D-21-31116R2

Dear Dr. McAllister,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. 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.

Kind regards,

Elsayed Abdelkreem, MD, PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

Reviewer #2: Yes

**********

6. Review Comments to the Author

Reviewer #2: Thanks for your response to my previous comments

The authors answered my comments and use the provided references to modify the discussion accordingly and I think the manuscript is suitable for publication.

**********

7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: Yes: Afaf Kamal El-Din El-Ansary

PLoS One. doi: 10.1371/journal.pone.0265217.r006

Acceptance letter

Elsayed Abdelkreem

7 Mar 2022

PONE-D-21-31116R2

Dear Dr. McAllister:

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.

If we can help with anything else, please email us at plosone@plos.org.

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

Kind regards,

PLOS ONE Editorial Office Staff

on behalf of

Dr. Elsayed Abdelkreem

Academic Editor

PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig. Nonsignificant correlation of serum OCT levels and age of ASD patient cohort (P = 0.396, R = -0.140, R² = 0.0196).

(TIF)

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S1 Table. Instruments and equipment used in the study.

(DOCX)

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S2 Table. Chemicals and reagents for DNA extraction and polymerase chain reaction.

(DOCX)

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S3 Table. Lab protocols.

(DOCX)

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S4 Table. The sequences of allele specific primers of oxytocin receptor diallelic gene rs2268491 (C/T) and PCR information.

(DOCX)

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S5 Table. OXT levels of control and autistic groups with related and unrelated parents.

(DOCX)

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S1 Appendix. Study datasheet.

(XLSX)

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Attachment

Submitted filename: answer to reviewers.docx

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Submitted filename: response to reviewers 070222.docx

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Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

The oxytocin receptor gene polymorphism rs2268491 and serum oxytocin alterations are indicative of autism spectrum disorder: A case-control paediatric study in Iraq with personalized medicine implications

Zainab Al-Ali

Akeel Abed Yasseen

Arafat Al-Dujailli

Ahmed Jafar Al-Karaqully

Katherine Ann McAllister

Alaa Salah Jumaah

Roles

Abstract

Background

Methods

Results

Conclusion

Introduction

Materials and methods

Study population and sample size calculation

Inclusion and exclusion criteria, CARS evaluation of pediatric ASD

Blood sample collection and preparation

Isolation of genomic DNA for PCR genotyping

Allele specific PCR primer design and synthesis

Enzyme-Linked Immunosorbent Assay (ELISA) oxytocin measurements

Statistical analysis

Ethical approval

Results

Study patient characteristics

Table 1. Patient ethnicity, consanguinity, recurrence, and family history of ASD.

Serum OXT levels in ASD and control subjects and ROC curve analysis

Table 2. ELISA serum OXT levels among study participants.

Fig 1.

Table 3. ROC analysis.

OXTR gene rs2268491 polymorphism in study population

Table 4. Genotype distribution (CC, CT, TT) and alleles (C, T) of rs2268491 in patients with ASD (N = 60) and healthy controls (N = 60).

OXTR gene rs2268491 polymorphism in ASD males and females

Table 5. OXTR polymorphism rs2268491 in ASD males (n = 45) and females (n = 15).

OXTR gene rs2268491 polymorphism in ASD severity categories

Table 6. OXTR polymorphism rs2268491 in ASD severity categories.

OXT levels according to genotype distributions in stratified autism patients and healthy controls

Table 7. OXTR polymorphism rs2268491 in ASD severity categories.

Fig 2. Bar chart of the oxytocin levels according to the three oxytocin gene polymorphism genotypes in autism patients and controls.

Discussion

Study limitations

Conclusion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Decision Letter 0

Elsayed Abdelkreem

Roles

Author response to Decision Letter 0

Decision Letter 1

Elsayed Abdelkreem

Roles

Author response to Decision Letter 1

Decision Letter 2

Elsayed Abdelkreem

Roles

Acceptance letter

Elsayed Abdelkreem

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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