Genetic susceptibility of vitamin D receptor gene polymorphisms on autosomal recessive primary microcephaly patients in Pakistani population: a case-control and in-silico study

Autosomal recessive primary microcephaly (MCPH) is a rare genetic disorder that leads to reduced cerebral cortex caused by a mutation in corticogenesis. The expression of the Vitamin D receptor (VDR) gene is involved in the proliferation and differentiation of neural stem cells, and VDR polymorphisms have been associated with various neurological disorders. However, their relationship with MCPH has not been explored. This study aimed to investigate the association of VDR polymorphisms with MCPH due to its role in Wnt signaling pathway and its In-silico analysis. Blood samples of 64 MCPH patients and 52 controls were collected to genotype VDR SNPs (TaqI (rs731236), FokI (rs2228570) and BsmI (rs1544410). In-silico tools were also used to assess the effects of exonic SNPs on mRNA and protein structure and pathogenicity of exonic and intronic SNPs. The study found that serum 25-OH vitamin D3 levels were significantly different in MCPH patients and healthy controls (P = 0.000). The genetic analysis showed that VDR polymorphisms of FokI and BsmI were seven times more frequent in MCPH patients than in controls (P < 0.05) and the recessive model for TaqI and dominant model for BsmI polymorphisms were also associated with the pathogenesis of MCPH. In-silico analysis showed that the pathogenicity effects of rs2228570 and rs1544410 are neutral while rs731236 causes a silent mutation which has no effect on VDR protein. VDR polymorphisms of FokI and BsmI are associated with the risk of MCPH. These findings suggest that VDR polymorphisms play a role in MCPH, which could provide important insights for understanding the molecular mechanisms of the disease.


Introduction
Autosomal recessive primary microcephaly (MCPH) is a rare neurodevelopment condition in which the affected born with less than 3 standard deviation (SD) smaller head circumference (HC) than expected compared to other infants of the same gestational age, sex, and ethnic background [1].MCPH is associated with mild to moderate mental retardation and no obvious structural abnormalities in the brain [2].Due to consanguinity, the prevalence of primary microcephaly is 1 in 10,000 in Pakistan, which is higher than the other non-consanguineous populations whereas it is estimated around 1 in 250,000 [3].Both genetic and environmental factors involves in the progression of this disease [3,4].MCPH is caused by thirty genes (MCPH1-30) till date (MIM: PS251200).Some most prevalent genes include MCPH1, WDR62, CDK5RAP2, CASC5 (CASC6), ASPM, CENPJ, STIL, CEP135, CEP152, ZNF335, PHC1, CDK4, CENPE, SASS6 and MFSD2A, [3].The proteins encoded by MCPH genes involved in cell division of cell cycle by acting on centrosomes and decrease in neurogenesis process and low cerebral cortex is associated with the mutations in these genes [1][2][3][4].
1,25-Dihydroxyvitamin D3 (Vitamin D3(1,25(OH)2D3)) is primarily synthesized in the skin in an inactivated form (Provitamin D3) in the exposure of sun light (Fig. 1a) [5].Calcidiol (25(OH)D) is converted to the activated calcitriol(1,25(OH)2D3) for proper metabolism and interacts with vitamin D receptor (VDR) and regulate the process of transcription of genes involved in many different functions (Fig. 1a) [5][6][7].It is recognized as a central nervous system neurosteroid and it is significant for its role in neuroprotection, neurodevelopmental and neurogenesis [8].VDR is an intracellular hormone receptor that mediates the actions of vitamin D and the existence of the VDR in the brains of animal models and humans was reported [8].The expression of VDR plays a potential role for vitamin D in neural stem cell proliferation and differentiation [6][7][8].VDR gene is mapped to chromosome 12q12-q14 with 14 exons [9].VDR gene is around 75 kb long and encodes a protein with 48.3 kD of molecular mass and comprised on 427 amino acids [9].
Vitamin D potentially act as a co-activator of Wnt-signaling pathway and it has been observed to preserve the neurogenesis in different neurogeneic disorders such as Alzheimer's and schizophrenia (Fig. 1a) [6,10].Wnt pathway plays a vital role in the establishment of forebrain anterior-posterior axis and it is also majorly involved in the proliferation of neural progenitors, synaptogenesis, dedritogenesis and establishment and guidance of axons (Fig. 1a) [6,7].ASPM gene (MCPH5) promotes the signaling activity of Wnt and reduction of ASPM gene is associated with the dysregulation of Wnt signal transducer β-catenin and it results in neurogeneic disorders [7].
Several VDR gene polymorphisms have been identified in VDR gene for the susceptibility of neurodegenerative disorders including TaqI, FokI, BsmI, ApaI and Cdx2 [11].In neurological disorders, three prevalent single nucleotide polymorphisms (SNPs) of the VDR gene namely TaqI, FokI and BsmI have found to be significantly associated [12].TaqI is located at exon 9 and due to this polymorphism the binding specificity of vitamin D is altered results from a change in protein structure of VDR [13].TaqI (rs731236) polymorphism was significantly associated with autism disease confirmed in Polish population [14].Turkish population and Iranian populations were not associated with TaqI (rs731236) for Parkinson disease [15,16].FokI is positioned at the start codon of exon 2 and intronic site is altered in the result of this polymorphism which is ultimately generate two proteins of different size [13].Parkinson disease in Italian population and Chinese population was found to be associated with FokI (rs2228570) polymorphism [17,18].FokI (rs2228570) polymorphism with temporal lobe epilepsy was also studied in Chinese population and revealed its neuroprotective roles [19].BsmI is located at intron 8 which affects gene expression through the regulation of mRNA stability [20].BsmI (rs1544410) polymorphism was also significantly associated in Alzheimer disease in Silesian Population [21].
Due to the predominant role of VDR in Wnt signaling pathway and causing the etiology of neurological disorder, the aim of the present study was to investigate possible correlations between the VDR gene polymorphisms, specifically in TaqI (rs731236), FokI (rs2228570) and BsmI (rs1544410) and the Primary microcephaly in Pakistani population and to elucidate the impact of these SNPs on the structure and function of VDR along with their pathogenic effects.

Materials & methods
The study was started after taking approval from Ethical Review Board (ERB) of Department of Biotechnology of Lahore College for Women, University Lahore (No. REF/ NO/LCWU/BIOT/304; Dated 03-05-2021).After the approval of the study, 116 participants were recruited including 52 unrelated healthy controls and 64 patients who were affected with autosomal recessive primary microcephaly according to the Declaration of Helsinki.The sample size was calculated by carefully following the assumptions of proposed power of study (80%) and the level of confidence (95%) (https://zzz.bwh.harvard.edu/)and most importantly assumed number of cases and controls from similar studies [22][23][24].
After the identification of participants, detailed consent was obtained.As the affected individual with MCPH are unable to communicate and understand due to intellectual disability so their parents or guardians signed the consent form for the study whereas control group signed for themselves.
The inclusion criteria for the enrolment of the patients was OFC at birth <-3 S.D below the mean for gender, age and ethnicity, and various degrees of mental retardation whereas the inclusion criteria for unrelated healthy controls was the absence of all the associated features such as seizures, epilepsy, short stature, awkward gait, hearing problems, diabetes mellitus, pigmented skin, bone issues and eye disorders with MCPH.The exclusion criteria for the MCPH patients was to select the individuals who have OFC − 2 to +2 S.D and normal mental health whereas the exclusion criteria for the unrelated healthy controls was the individuals who had diagnosed with other genetic disorders and features associated with MCPH which may affect the interpretation of the results were excluded.

Blood sampling and DNA isolation
The blood samples were collected with a sterile syringe by puncturing the vein in purple capped disodium ethylene diamine tetra acetic acid (EDTA) containing tube for genotype analysis and yellow capped gel and clot activator tube to collect serum for biochemical analysis.The DNA was isolated using phenol-chloroform method (organic method) [25].Extracted DNA was quantified using NanoDrop Polymorphisms (TaqI (rs731236) and FokI (rs2228570) in their exonic regions.Furthermore, BsmI (rs1544410), located within Intron 8 of VDR gene, was also assessed through in silico analysis.The nucleotide sequence for VDR genes (accession# NM_001364085) was extracted from the National Center for Biotechnology Information (NCBI) data bank.For subsequent applications, the coding sequence domain of VDR for exonic region SNPs (rs731236 and rs2228570) with both wild and mutant alleles were translated using ExPASy server (https://web.expasy.org/translate/).Mode 2 of RNAsnp (https://rth.dk/resources/rnasnp/) was used to assess the effects of exonic SNPs on VDR mRNA structures.Physical and chemical properties of translated sequences were assessed using Prot-Param tool (https://web.expasy.org/protparam/)available through ExPASy server.To assess the effects of VDR SNPs (rs731236 and rs2228570) on protein secondary structure, Chou-Farman method (https://web.expasy.org/protscale/) was used, which measures relative frequencies for each amino acid within alpha helices, beta sheets, and turns.In order to determine the hydrophobic pattern of both normal and mutant proteins, the Kyte and Doolittle scale (https:// web.expasy.org/protscale/)provided by ExPaSy server was used.On Kyte-Doolittle's hydropathy plot, each amino acid was given a hydrophobicity score to indicate its hydrophobic nature.To evaluate the effect of VDR SNPs (rs731236 and rs2228570) on protein function, SNAP (Screening for Non-Acceptable Polymorphisms) (https://rostlab.org/services/snap/) and PredictProtein (https://predictprotein.org/) web servers were used.Furthermore, three-dimensional structures for mutant and normal proteins were predicted using Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2/ html/page.cgi?id=index) which will be utilized in other subsequent applications.

Statistical analysis
The statistical analysis was performed using SPSS statistical software (version 20.0).Independent T-test was used to perform the comparison of means according to normal distribution and its result give the frequency, percentage, mean and SD for both control and cases population.The genotyping frequencies of VDR gene polymorphism in patients with MCPH and controls was determined using Hardy-Weinberg equillibrium (HWE).Binary logistic regression was used to analyzed the odd ratios (OR) and 95% confidence intervals (CI) to compare both allele frequencies in MCPH patients and control groups.OR and CI were also calculated for all the genetic models including allelic, dominant, recessive, over-dominant, co-dominant and additive model ≤ 0.05 p-value considered statistically significant.Akaike Information Criterion (AIC) was used to choose the most effective genetic model with the smallest AIC value.

In-silico analysis
An In-Silico analysis was performed to evaluate the potential biological impacts of VDR Single Nucleotide with Hardy Weinberg's law at the level of significance 0.05 for our study population.
The frequencies of the wild-type (CC), heterozygous (CT) and homozygous (TT) at VDR polymorphism site TaqI (rs731236) were 31.9%,39.7% and 28.4% respectively and the allele frequency of TaqI C > T allele was 80.8%.For TaqI polymorphism, the CC genotype resulted to be protective against MCPH as it was present in 53.8% in normal controls whereas 14.1% of MCPH cases have CC genotype.On the contrary, the other genotype TT is said to be a risky genotype for MCPH susceptibility as the frequency of TT in MCPH is 45.3% which is highly greater than the normal controls (7.7%).In particular, the results for allelic (C/T) genetic model suggest that T-allele is strongly associated with MCPH as it appeared almost 5 times more often in MCPH patients (OR 95%CI = 5.182 (2.941-9.130),P-value= (0.000)) (Table 2).In genotype analysis, there was only a significant association at recessive (CC + CT/ TT) genetic model (OR (95%CI) = 9.943(3.204-30.858),P-value= (0.000), AIC = 12.095) whereas dominant, overdominant, co-dominant and additive model had no association with MCPH disorder (Table 2).
At the FokI (rs2228570) VDR gene polymorphism site the frequency of all three genotypes (CC, CT and TT) were observed in MCPH patients and controls.Among 116 participants, the frequency of wild-type CC was 58.6%, heterozygous CT was 21.6% and homozygous TT was 19.8%.The allele frequency of FokI C > T allele was 80.8%.The results for FokI polymorphism suggest that CC genotype was acting as protective against primary microcephaly due to its greater frequency (67.3%) in normal controls as compared to MCPH cases which has 51.5% CC genotype.On the other side, TT genotype was resulted to be risk for MCPH as it was present 31.3% in MCPH patients than normal controls only have 5.5%.Moreover, the results for T-allele suggested a strong association for MCPH susceptibility as it appeared almost 2 times more in MCPH cases than normal control group (OR (95%CI) = 2.782 (1.523-5.082)P-value = 0.001)) (Table 2).In the genotype analysis, there were no significant associations at the dominant, recessive, over-dominant, co-dominant and additive genetic models (Table 2).
At BsmI (rs1544410), the three GG, GA, AA genotypes were observed in both MCPH and healthy control group.Out of 116 participants, 55.2% had genotype GG, 19.8% had GA and 25.0% had AA and said to be wild-type, heterozygous and homozygous respectively.The allele frequency of BsmI G > A allele was 79.8%.In particular, the AA genotype resulted to be a risk factor for MCPH disorder as it was present in 39.1% of MCPH cases but only in 7.7% in normal control group (OR (95% CI) = 7.543 (2.354-24.172),P-value = 0.001)) whereas the other GG genotype appeared base tool was used to analyze the functional consequences of this SNP for the non-coding variant.RegulomeDB 2.0.3 (https://regulomedb.org/) was used to identify the DNA features and regulatory elements in FokI SNP rs1544410.Genemania database (https://genemania.org/)was utilized to examine gene-gene interaction between VDR and MCPH genes.

Demographic and clinical data characteristics of recruited subjects
The current study included 116 participants from different areas of Pakistan.The demographic and clinical data was collected from all the patients affected with MCPH (male (57.8%), female (42.2%)).The mean ± SD age, HC, weight, height and body mass index (BMI) of patients were measured significantly different from the normal healthy controls.(Table 1).The significant difference was also observed in vitamin D level in the MCPH patients and healthy controls (Table 1).The percentage of consanguinity in the data is 75.0%.The clinical parameters such as varies degree of mental retardation, cognitive delay, motor delay, seizures, absent speech, aggression and awkward gait were also recorded in recruited samples of affected individuals with MCPH (Fig. 1e).

VDR polymorphisms genotyping
The genotype frequencies of TaqI (rs731236), FokI (rs2228570) and BsmI (rs1544410) polymorphismsin VDR gene were studied in 64 affected individuals with MCPH and 52 controls.The data of the frequencies of TaqI alleles was in Hardy-Weinberg equilibrium (HWE) with p-value > 0.05 whereas FokI and BsmI were not consistent value of 6.08.In a normal protein, there were 60 negatively charged residues (Asp and Glu) and 53 positively charged residues (Arg and Lys).This resulted in an aliphatic index of 77.87 for this particular VDR peptide.Furthermore, the stability index for normal VDR protein was 53.26 which indicates it's unstable.The Grand Average of Hydropathicity (GRAVY) for normal VDR was calculated at -0.410 and its estimated half-life was 30 h in mammalian reticulocytes in vitro.These parameters remain unchanged when considering at I352I phenotype as VDR rs731236 polymorphism is an A-G substitution in nucleotide 1058 of VDR, which does not alter any amino acid sequence (p.Ile352Ile), and making this mutation silent in nature.Whereas for M1T, the physiochemical properties are summarized in Table 3.
Predicting the effects of VDR rs731236A > G and rs2228570T > C on local VDR RNA secondary structure revealed that neither SNP made any fundamental modifications to mRNA's secondary structure with p = 0.9456 and 1.000 respectively, as the p-values are greater than 0.2.Chou and Doolittle scored beta sheets at 352 for residue I, with no score for M at position 1 (rs2228570), while Kyte and Doolittle hydrophobic score for VDR at position 352 to protective against MCPH cases as it was present in 67.3% of normal control group but only in 45.3% of MCPH cases.Notably, the results suggested an association between the presence of A-allele at BsmI position and the presence of MCPH.The A allele appeared almost 3 times more in MCPH patients (OR (95%CI) = 3.487 (1.930-6.300),P-value = 0.000) than in normal control group (Table 2).In the genotype analysis, there were a significant associations at dominant model for MCPH patients in Pakistani population (OR (95%CI) = 2.485 (1.162-5314), P-value = 0.019, AIC = 12.896) whereas recessive, overdominant, codominant and additive genetic models were not showed association (Table 2).

In-Silico analysis of VDR polymorphisms
For In silico analysis the retrieved VDR sequence was translated to get the protein sequence for other applications.ProtParam was used to determine the physical and chemical properties of VDR protein; predicted molecular formula: C 2091 H 3342 N 596 O 648 S 34 with a molecular weight: 48289.18Da encompassing 6711 atoms and an isoelectric point (pI) indicates that the effect of this SNP is neutral or benign as the score is below 0.5.RegulomeDB give the predicting score as 0.41198 which means this variant is most likely to be a regulatory variant and it can be predicted as transcription binding factor, any motif, DNase footprint and DNase peak due to its rank score of 2b.
From the results of GENEMANIA database it is predicted that VDR gene has an interaction with MCPH genes such as CEP152, LMNB1 and BUB1 and some non-microcephalic genes including HR and CEP170 through co-expression, genetic interaction and physical interaction (Fig. 3c).These genes are widely involves in chromosomal organization in mitotic cycle of neurogenesis.

Discussion
Vitamin D is involved in neurogenesis and there is developing evidence that low levels of vitamin D may increase the risk of developing neurodegenerative disorders like multiple sclerosis and Alzheimer's [27].The pathogenesis of MCPH results from the mutation in any of the MCPH gene or environmental factor [3].This is the first study to our knowledge to analyze the effect of VDR polymorphisms on Pakistani MCPH patients.The selection of VDR variants in our study was based on several considerations.First, previous research has suggested a potential link between VDR and neurogenic disorder.Studies have indicated that vitamin D signaling pathway (Wnt Pathway) plays a crucial role in was 0.856 (Isoleucine), with no score at position 1 (Fig. 2a).Furthermore, PredictProtein revealed I352 is buried in the structure while M1 is exposed by solvent accessibility analysis; SNAPserver showed no effect of I352I substitution on protein structure (Score: -93; Expected accuracy: 97%) as well as M1T substitution on protein structure (Score: 70; Expected accuracy: 85%), which are both neutral effects (Score: -93; Expected accuracy: 97%) (Fig. 2b).
ConSurf was used for conservation analysis of M1 and I352 residues, which revealed that both amino acids are highly conserved with their conservation score being 8 (Fig. 3a).On the basis of sequence homology and physiochemical similarity between amino acids, PolyPhen-2, SIFT, Mutation Taster and PhD-SNP were interpreted to be neutral for M1T mutation pathogenicity; I-Mutant predicted an increase in stability due to this mutation (Table 4).Project HOPE's results revealed that the mutant residue is smaller than its wild-type counterpart, is more hydrophobic, and might lead to loss of interactions.Furthermore, while the Wild Type residue remains conserved, its mutant counterpart shared some properties with it; therefore, this mutation might take place without damaging the protein (Fig. 3b).PSIPRED predicted the coils at M1 position; however, when this initiated methionine was lost the M4 act as start codons and the coil was once again at its initial position.No significant alteration in VDR structure due to M1T mutation was observed (Fig. 2c).
FATHMM gives 0.15081 score for rs1544410 SNP of VDR at position chromosome 12: 48,239,835 which  The effect of M1T substitution on protein functions evaluated by SNAP Server, (c) Secondary structure of VDR for rs2228570T > C SNP predicting coils at M1T residue generated using PSIPRED with high confidence of prediction reported e.g.92.9% and 94.3% respectively while, epilepsy frequency (28.0%) is reported in Pakistani MCPH patients.Many studies also gives evidence of the high frequency of cognitive and motor impairment and frequent epilepsy associated with MCPH [30].Our results are consistent with the previous reports as the lobes of cerebral cortex are fails to develop normal and play a major role in causing motor delay, aggression and difficulty in processing the information in MCPH (MIM: PS251200).
Biochemical analysis revealed that MCPH patients were detected with low serum 25-OH vitamin D3 (below normal 30-100 ng/ml) as compared to normal cases e.g.20.48 ± 2.00 ng/ml and it is found to significantly different (p-value 0.000) in recruited controls and MCPH of Pakistani population.In Korean and Swedish population, hypovitamin D level has been found to be associated with Parkinson disease and ADHD, schizophrenia and autism respectively [27].The absence or depletion of VDR leads to the progression of microcephaly, seizures, Alzheimer and intellectual disability due to its role in Wnt signaling pathway as it is necessary for the proper process of proliferation of neuronal progenitors, dendritogenesis and forebrain establishment [5].The purpose of this research study was to investigate the association of VDR polymorphisms (TaqI, FokI and BsmI) with autosomal recessive primary microcephaly in Pakistani population.VDR polymorphisms have been potentially associated with the progression of several neurological disorders such as Parkinson, autism, Alzheimer and epilepsy disorders [11,14,16,21,22].
In the current study the average mean head circumference of primary microcephaly patient is reported as -7.25 ± 2.32 S.D which is slightly below the mean that less − 3 S.D in Asian population [1].The reduction in HC in recruited samples is may be due to consanguinity which may leads to more frequency of severe mentally retarded cases 43.8% (28/64 cases) than mild and moderate level hence the size of skull would be more small.However, other clinical features such as cognitive and motor delay are  In order to explore the genotype phenotype association between VDR polymorphisms and MCPH, there are different genotypic test models were predicted, i.e. dominant, recessive, and over-dominant, co-dominant and additive.The results for model prediction reveals that recessive model for TaqI polymorphism and dominant model for BsmI polymorphism were found to be associated with the 9-fold and 2-fold increase with risk of the susceptibility of MCPH disorder in this finding (OR (95%CI) = 9.943 (3.204-30.858)and (OR (95%CI) = 2.485 (31.162-5.314))with 0.000 and 0.019 p-value respectively whereas the dominant, over-dominant, co-dominant and additive models for TaqI polymorphism and all the models for FokI and recessive, over-dominant, co-dominant and additive models for BsmI polymorphism at VDR site were not associated with the disease risk.In Southeastern European Caucasian population, the dominant model of TaqI was associated with Alzheimer's disease [28].In another review conducted in Chinese population BsmI and FokI showed significantly association with Parkinson's disease [18].The reason for controversial results may be the difference in ethnicity and different disease group.Moreover, more studies are suggestive to confirm these results in Asian populations specifically in Pakistan.
A novel In-silico analysis for TaqI (rs731236) and FokI (rs2228570) was conducted to evaluate the effects of these SNPs on the VDR structure and expression.The results of the bioinformatics tools showed that rs731236 is silent mutation and not affecting the structure and expression of VDR protein as current study also showed no association for Taq (rs731236) polymorphism with MCPH.This mutation was also found to be silent in the study conducted in Polish population [14].While rs2228570 is a missense mutation and results in the shifting of ATG hence the start Methionine is lost and resulted protein is shorter than the wild type protein [14].This change may influence the some changes in the secondary structure of VDR but the lost is very minor and the analysis of pathogenicity prediction reveals this mutation is benign in the result of most of the In-silico tools (Table 4).Bioinformatics analysis of rs1544410, an intronic SNP of VDR showed that this is the mutation of regulatory variant and it is also a benign mutation.It means the presence or absence of these variants of VDR gene would not lead to a significant change in the structure [14].
It is of the utmost necessity to broaden this investigation to uncover relevant variants at the genome-wide level in order to identify genetic variables associated with MCPH and comprehend its pathophysiology.Genotypeprotein correlation analyses would be its future prospect to strengthen the scientific impact and expand our knowledge of the genetic basis of the phenotype.In addition, the identification of genetic variations associated with MCPH has the the vitamin D supplementation to MCPH patients for the improvement in their cognitive and motor skills.
In order to validate the SNPs, experimental assays have been performed such as genotyping.This allowed the confirmation of the association of the identified SNPs with the target phenotype of MCPH.A detailed description of the experimental procedures have been provided, including the sample size, techniques employed, and statistical analysis methods used to validate the associations.TaqI polymorphism is involved in the alteration of binding specificity of vitamin D and ultimately play important role in the pathogenicity of neurological disorders [13].The current data shows that for TaqI polymorphism (rs731236), CC genotype was protective against MCPH as the frequency of CC genotype was found to be 53.8% in normal controls and 14.1% in MCPH cases.In Turkish.Hungarian, Polish, Silesian and Iranian populations CC genotype was also revealed as a protective role against of Parkinson, Autism and Alzheimer disease respectively [14][15][16][27][28][29].
FokI polymorphism (rs2228570) was found to be significantly associated with MCPH in Pakistani population as the results of this study showed that it was seven times more common in MCPH than in unrelated healthy controls (OR = 7.071 (95% CI: 1.921-26.032),p-value: 0.05).Turkish, Hungarian and Chinese populations have shown an association between autism, Parkinson's disease and temporal lobe epilepsy respectively [17,24,27].FokI polymorphism results in the alteration of intronic site and give two proteins of different sizes in result and involves in disease progression [13].Therefore, the study of this polymorphism is pivotal to understand its involvement in the etiology of MCPH.Expression studies on animal models will be helpful to uncover its function in MCPH.
For the association between BsmI polymorphism (rs1544410) of VDR gene and MCPH, GG genotype was found to be associated with neuroprotective role due to its highest frequency (67.3%) in control population whereas AA genotype was associated with risk of susceptibility of MCPH in this study as it is more than seven time increases the event of occurrence of MCPH in this case group (OR (95%CI) = 7.543 (2.354-24.172).In Turkish population AA genotype of BsmI polymorphism was also found to be associated with autism [29].However, studies conducted in Polish and Hungarian population showed that this polymorphism was not associated with childhood autism and Parkinson disease respectively [14,27].Findings of Polish and Hungarian population association analysis of BsmI polymorphism and neurological disorders is not homogeneous with this report.Gene expression studies will be helpful to understand its mechanism in MCPH pathogenicity as this polymorphism is involved in the change of stability of mRNA [20].potential to facilitate individualized carrier screening and genetic counseling for genetically at-risk people and families with MCPH.

Conclusion
This is the first report on the potential correlation between VDR polymorphisms and MCPH in Pakistani population.Current study concluded that both FokI (rs2228570) and BsmI (rs1544410) polymorphisms of VDR gene are found genetically associated with primary microcephaly.While only recessive model for TaqI polymorphism (rs731236) and dominant model for BsmI polymorphism (rs1544410) are predicted to be associated for the pathogenesis of MCPH.Since serum 25-OH vitamin D3 was significantly different between MCPH and healthy controls this could indicate that the metabolism of Vitamin D might be affected when contributing to the pathogenicity of among MCPH patients.

Fig. 1
Fig. 1 (a) Regulation of gene expression through the activation of Wnt signaling pathway.Left panel: Wnt mediated activation results in establishment of forebrain, proliferation of neural progenitors, synaptogenesis and dendritogenesis, Right panel: Disregulation of the pathway due to absence of Wnt results in β-catenin degradation and results in microcephaly, intellectual disability and seizures (Modified from: [6]).(b) 2% Agarose gel electrophoresis of RFLP-PCR showing wild type, heterozygous and homozygous mutant variants for VDR SNPs TaqI enzyme, (c) VDR SNPs FokI enzyme, (d) VDR SNPs BsmI enzyme, (e) Graphical presentation of clinical data of MCPH patients

Fig. 2
Fig. 2 (a) Score for Secondary Structure Prediction by Chou and Fasman and Hydrophobicity score by Kyte and Doolittle (Red dot shows the score for 352I residue whereas M1T is not predicted by these tools), (b) The effect of M1T substitution on protein functions evaluated by SNAP Server, (c) Secondary structure of VDR for rs2228570T > C SNP predicting coils at M1T residue generated using PSIPRED with high confidence of prediction Vitamin D plays a crucial role in the development and functioning of brain.The low level of Vitamin D in serum may disturb the pathway of development of brain and ultimately the proper function of brain may altered in the result of subsequent change in the level.In Korean population, the supplementation of vitamin D helped in the prevention of deterioration of Hoehn & Yahr stage in Parkinson disease patients in clinical trials [27].Hence, it is suggestive to give brain development and neuronal differentiation, and alterations in these pathways can contribute to microcephaly.Given the involvement of VDR in mediating the effects of vitamin D, we deemed it relevant to explore the potential association between VDR variants and MCPH.

Fig. 3
Fig.3(a) Conservation analysis by ConSurf (black square showed the Methionine reside at position 1 with score 8 and red square showed the Isoleucine residue at position 352 with score 8, means highly conserved residues), (b) Schematic Structure for wild type Methionine (left) and mutated Threonine (right) residue for rs2228570T > C SNP of VDR generated using HOPE project, (c) Gene-Gene interaction network of VDR with other MCPH genes generated using GEN-EMANIA database

Table 1
Anthropometric and biochemical parameter data analysis by independent T-test in MCPH patients and healthy control subjects * S.D = Standard Deviation, MR = Mental Retardation **P < 0.05 indicates significant difference between MCPH patients and controls

Table 2
Allele frequency and models proposed for TaqI, FokI and BsmI polymorphisms between MCPH cases and normal controls in Pakistani

Table 3
Physiochemical properties of VDR SNPs rs731236A > G and rs2228570T > C using ProtParam Protein Phenotype

Table 4
In Silico Prediction of Pathogenicity