Candidate Gene Screening for Attention Deficit/Hyperactive Disorder (ADHD)
Condition: Gene Product Sequence Variation
MeSH terms: Attention Deficit Disorder with Hyperactivity
Study type: Observational

Summary: 
      Source: Sample bank of Xijing Hospital and Children's Hospital Affiliated to Soochow
      University; Sample form: Whole blood; Estimated number of samples: 100 patients with ADHD and
      age, sex matched healthy controls; Case exclusion criteria: all kinds of neuropsychiatric
      disorders, IQ value of less than 70;

      Study protocol:

        1. Using Qiagen kit to extract the genomic DNA of 200 microliters of blood.

        2. UV spectrophotometer test DNA purity of 260/280 close to 1.8 (1.8 ± 0.05), the
           concentration of 100ng/μL or more before the next sequencing.

        3. The extracted genomic DNA will be sent to Sangon Biology Engineering Limited Company
           (Shanghai) and sequenced to find candidate mutations related to ADHD risk sequence.
           According to NIH gene database, the longest transcript of NDRG2 (ID: 57447 gene,
           https://www.ncbi.nlm.nih.gov/nuccore/NC_000014.9?
           Report=genbank&from=21016763&to=21070872&strand=true) (a total of 17 exons and 16
           introns and the gene 5 'UTR and 3' UTR region) will be alignmented sequences to find
           potential mutations.

        4. Using the chi square analysis and other statistical methods to determine the
           relationship between the mutations and susceptibility to ADHD.
    

Detailed description: 
      Source: Sample bank of Xijing Hospital and Children's Hospital Affiliated to Soochow
      University; Sample form: Whole blood; Estimated number of samples: 100 patients with ADHD and
      age, sex matched healthy controls; Case exclusion criteria: all kinds of neuropsychiatric
      disorders, IQ value of less than 70;

      Study protocol:

        1. Using Qiagen kit to extract the genomic DNA of 200 microliters of blood. (1) melt frozen
           blood at room temperature; (2) take the blood of 0.2 ml into a sterile anti coagulation
           centrifuge tube, add an equal volume of PBS phosphate buffer, after fully mixing
           12000rpm centrifugation 5min, discard supernatant; (3) 66.7 L STE 2.4 L, 20% SDS, 37 DEG
           C water bath 1h; (4) protease K plus 1 20mg/ml l mix, 55 DEG C water bath digestion
           overnight (10 ~ 14h); (5) the digested samples treated with Tris saturated phenol, shake
           well, 12000rpm centrifugal 10min; (6) the upper aqueous phase to a sterile centrifuge
           tube; adding volume of Tris saturated phenol, shake well, 12000rpm centrifugal 10min;
           (7) the upper aqueous phase was transferred to another sterile centrifuge tube, and an
           equal volume of phenol was added: chloroform: isoamyl alcohol (25: 24: 1). The
           oscillation of the vortex was fully mixed, and the 12000rpm was centrifuged to 10min;
           (8) the upper aqueous phase was transferred to another sterile centrifuge tube, and an
           equal volume of chloroform was added: iso amyl alcohol (24: 1), which was fully vibrated
           and mixed with 12000rpm centrifugation 10min; (9) transfer the supernatant to another
           sterile centrifuge tube; adding 2 times volume ethanol, the volume level of sodium
           acetate 1/10 shake, until the flocculent precipitation of DNA visible; (10) the sample
           is placed at -20 DEG C refrigerator freezer 30min or ice bath for 15 ~ 20min, after
           removal of 12000rpm centrifugal 10min again, so that DNA precipitation; (11) the gun
           head will pick DNA to another sterile centrifugal tube, with the amount of 70% ethanol
           washing and shaking, to wash out impurities DNA; (12) out of ethanol, DNA by vacuum
           drying or dry naturally, TE buffer adding dissolution, -20 stored at standby.

        2. UV spectrophotometer test DNA purity of 260/280 close to 1.8 (1.8 ± 0.05), the
           concentration of 100ng/μL or more before the next sequencing.

        3. The extracted genomic DNA will be sent to Sangon Biology Engineering Limited Company
           (Shanghai) and sequenced to find candidate mutations related to ADHD risk sequence.
           According to NIH gene database, the longest transcript of NDRG2 (ID: 57447 gene,
           https://www.ncbi.nlm.nih.gov/nuccore/NC_000014.9?
           Report=genbank&from=21016763&to=21070872&strand=true) (a total of 17 exons and 16
           introns and the gene 5 'UTR and 3' UTR region) will be alignmented sequences to find
           potential mutations.

        4. Using the chi square analysis and other statistical methods to determine the
           relationship between the mutations and susceptibility to ADHD.
    

Observational model: Case-Control
Time perspective: Retrospective
Sampling method: Probability Sample
Study population: 
        Diagnoses of the children with ADHD were made in Xijing Hospital and Children's Hospital
        Affiliated to Soochow University according to criteria described in the Diagnostic and
        Statistical Manual of Mental Disorders. Children with ADHD had an IQ score above 70.
      
Biospecimen description: 
      Whole blood