Correlating single nucleotide polymorphisms in vitamin D metabolism-related genes to autism susceptibility and vitamin D treatment: study protocol of a non-randomized parallelcohort controlled trial

Ling Shan, Bing Wang, Xiao-lan Hu, Fei-yong Jia＊

Rehabilitation Center, First Hospital, Jilin University, Changchun, Jilin Province, China

Background

Autism spectrum disorder (ASD) refers to a group of pervasive developmental disorders characterized by persistent deficits in social communication and social interaction, and restricted, repetitive patterns of behavior or interest. The National Cancer Institute (NCI) Centers for Disease Control and Prevention (CDC) estimates that the incidence of ASD in the USA is approximately 14.7% i.e., one out of 68 children suffers from ASD (Developmental Disabilities Monitoring Network Surveillance Year 2010 Principal Investigators,2014). In contrast, the incidence of ASD in Asia and Europe is around 1% (Kim et al., 2011; Parner et al., 2011). No ef-fective drugs have been developed for the treatment of ASD(Aman et al., 2009). Furthermore, little is known about its etiological factors and pathological mechanisms, although it is likely to be caused by an interaction between genetic and environmental factors (Strömland et al., 1994; Hall et al.,2008; Christensen et al., 2013; Huguet et al., 2013).

Recently, vitamin D has been shown to protect the skeleton by regulating calcium and phosphorus metabolism(DeLuca, 1980). It also plays an important role in fetal and early postnatal brain development (Groves et al.,2014; Lardner, 2015). Moreover, there is evidence that peripheral blood levels of vitamin D are significantly lower in children with ASD than in healthy controls (Wang et al., 2015). This is consistent with our previous clinical findings (Jia et al., 2015), which suggest that vitamin D deficiency may be a leading factor for ASD development.The cause of vitamin D deficiency is likely to be related to environmental and/or genetic factors.

Vitamin D is sourced from food and self-synthesis in vivo, with the latter being the main source. The vitamin D precursor, 7-dehydrocholesterol, is converted to previtamin D3 in the skin by exposure to ultraviolet irradiation. Next, previtamin D3 is isomerized to vitamin D3, which is then released into the blood, where it binds to the vitamin D binding protein (DBP) for transport to the liver. In the liver, vitamin D3 is converted by the action of 25-hydroxylase (CYP2R1) into 25(OH)D3. After binding to DBP, it is subsequently transported into the kidney and converted into 1,25(OH)2D3by the action of 25-hydroxyvitamin D31-α-hydroxylase (CYP27B1).Consequently, 1,25(OH)2D3acts within cells or is deposited in adipocytes. Furthermore, 1,25(OH)D3and 1,25(OH)2D3can generate 25,24(OH)2D3by the action of 25-hydroxyvitamin D324-hydroxylase (CYP24A1),which is discharged outside the body via bile (Leroith,2010; Lange, 2012; Morris et al., 2014) (Figure 1). Here,the purpose of this trial is to correlate single nucleotide polymorphisms (SNP) of vitamin D metabolism-related key enzymes to autism susceptibility.

Compared to vitamin D, 1,25(OH)2D and 25(OH)D have longer half-life periods (Clements et al., 1992) and are present at higher concentrations. Therefore, they are considered the optimal index to evaluate vitamin D nutritional status (Clements et al., 1992). Determination of vitamin D levels in ASD patients is possible by measuring and comparing peripheral blood levels of 25(OH)D with healthy controls. Jia et al. (2015)has confirmed that vitamin D supplementation can effectively improve symptoms in children with ASD. Based on this, we hypothesize that vitamin D can improve ASD symptoms, and choose to validate our assumption via a non-randomized parallel cohort-controlled trial.

Methods/design

Study design

A non-randomized parallel-cohort controlled trial.

Study setting

Rehabilitation Center, First Hospital of Jilin University,China.

Ethical issues and informed consent

This trial involves gene sequencing. The participant’s guardians are informed of the trial protocol and potential benefits and risk. The genetic research results are limited to scientific research and will not be divulged to others.Moreover, information resulting from the genetic research will not be available to the participants or their guardians.Signed written informed consent should be obtained from each participant’s guardian. Written approval should be obtained from the Medical Ethical Committee, First Hospital, Jilin University, China (Approval No. 2013-192).The trial protocol will be performed in accordance with the Declaration of Helsinki.

Study participants

Sixty children ASD patients who will receive treatment at the Department of Pediatric Neurological Rehabilitation, First Hospital, Jilin University, China, and healthy controls that will have concurrently undergone a physical examination.

Inclusion criteria

ASD group:

● Fulfill the diagnostic criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition (DSM-V)(American Psychiatric Association, 2013)

● Children with autism or ASD diagnosed using the Autism Diagnostic Interview-Revised (ADIR) (Lord et al., 1994)and Autism Diagnostic Observation Schedule (ADOS)(Lord et al., 1989)

● Aged 2–11 years, either gender

Control group:

● Age- and gender-matched healthy children from the same district

Exclusion criteria

● Have diseases that possibly influence vitamin D metabolism in vivo, including abnormal kidney function,depression, and mental disorders

● Complicated by other metabolic diseases

● Poor compliance

Intervention measures

Figure 1: Metabolism of vitamin D in vivo.

Figure 2: Trial protocol flow chart.

Table 1: Timing of outcome evaluation

Vitamin D therapy: in the ASD group, children patients receive alfacalcidol (0.5 μg/d, oral, 30 successive days) or alfacalcidol (0.5 μg/d, oral, 30 successive days) + vitamin D3 (150,000 units/month).

A flow chart of the trial is shown in Figure 2.

Blinding

All responsible physicians, patients, and guardians are informed of the patient’s condition and intervention measures.However, outcome assessors are blinded to the groups to avoid evaluation bias.

Outcome measures

Primary outcomes

● SNP allele frequencies of DHCR7, CYP2R1, CYP24A1,and GC genes (rs2282679, rs7041, and rs4588)

● Autism Behavior Checklist (ABC) score (Volkmar et al.,1988) and Childhood Autism Rating Scale (CARS) score(Schopler et al., 1980)

● Gesell Developmental Schedules (GDS) score (Gesell et al., 1940)

● Clinical Global Impression Scale-(CGI) for severity of illness (SI), global improvement (GI), and efficacy index(EI) scores

Secondary outcome

● Peripheral blood levels of 25(OH)D

Timing of the outcome evaluation is shown in Table 1.

Safety assessment

During the trial, any adverse events and other unexpected reactions should be recorded, evaluated, reported, and investigated. Corresponding solutions will be proposed and disposal outcomes recorded. The nature, severity, and drug correlations of all adverse events will be identified.

Data collection, management and statistical analysis

The above-mentioned outcomes are recorded. According to the Good Clinical Practices document, all trial records are kept in a secure, locked storage area by a designated person.All records will be preserved for at least 5 years. All data are statistically processed using SPSS17.0 software (SPSS,Chicago, IL, USA). Genotype and allele frequency are expressed as n(%), and compared between ASD and control groups. Vitamin D levels in ASD patients prior to and after vitamin D supplementation, and related scale scores are expressed as the mean ± SD. Paired sample t-tests are used for comparison between ASD and control groups. A level of P ＜ 0.05 is considered statistically significant.

DISCUSSION

Autism is a social issue that needs to be addressed due to its worldwide prevalence in children. Vitamin D plays an important role in brain development (Groves et al., 2014;Lardner, 2015), and children with ASD have an obvious disorder of brain development (Stoner et al., 2014; Zielinski et al., 2014). Cannell first proposed the hypothesis that vitamin D deficiency can cause ASD in 2008 (Cannell et al., 2008).Our previous findings have confirmed that vitamin D can improve ASD symptoms in children, including the persistent deficits in social communication and social interaction,restricted, repetitive patterns of behavior or interest, and also emotional and sleep disorders (Jia et al., 2015). If our randomized parallel-cohort controlled trial identifies a correlation between SNPs from vitamin D metabolism-related key enzymes and autism susceptibility, and also confirms that vitamin D improves ASD symptoms, then our findings will lay the foundation for scientific research regarding the causes of vitamin D deficiency in children with ASD. Our findings will also be beneficial to the underlying pathological mechanism of ASD, and provide clinical evidence for ASD treatment.

Trial status

The curative effects of vitamin D on autism was completed in April 2015 (article has been in the typesetting stage). The rest of the research is ongoing.

Preliminary trial data

In January 2015, FYJ and his colleagues treated one 32-month-old male ASD patient who presented with vitamin D3deficiency. After 2 months of vitamin D3supplementation, decreased ABC, CARS, and CGI scores (from 80, 35,and 6 to 39, 28, and 4, respectively) were observed. These findings indicate that the main symptoms of autism had improved, and suggest that vitamin D3plays an important role in autism pathology. In addition, clinical detection of blood vitamin D3levels, and in time, vitamin D3supplementation may be of importance for autism. In January 2015, FYJ and his colleagues also performed high performance liquid chromatogram-tandem mass spectrometry to detect 25(OH)D levels in blood collected from 117 children with ASD and 109 healthy controls. Blood 25(OH)D levels in ASD patients were significantly lower than in healthy controls.Accordingly, the proportion of vitamin D deficiency was approximately 89.7% in ASD patients, which was significantly higher than in healthy controls. These results suggest that children with ASD exhibit vitamin D deficiency. Moreover,vitamin D deficiency is likely to be an environmental/genetic factor of ASD.

Conflicts of interest

None declared.

Author contributions

LS and FYJ conceived and designed this trial protocol,wrote the article, and approved the final version. BW and XLH were responsible for screening autism using autismrelated scales.

Plagiarism check

This paper was screened twice using CrossCheck to verify originality before publication.

Peer review

This paper was double-blinded and stringently reviewed by international expert reviewers.

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