Development of a High-Throughput Resequencing Array for the Detection of Pathogenic Mutations in Osteogenesis Imperfecta

<div><p>Objective</p><p>Osteogenesis imperfecta (OI) is a rare inherited skeletal disease, characterized by bone fragility and low bone density. The mutations in this disorder have been widely reported to be on various exonal hotspots of the candidate genes, including <i>COL1A1</i>, <i>COL1A2</i>, <i>CRTAP</i>, <i>LEPRE1</i>, and <i>FKBP10</i>, thus creating a great demand for precise genetic tests. However, large genome sizes make the process daunting and the analyses, inefficient and expensive. Therefore, we aimed at developing a fast, accurate, efficient, and cheaper sequencing platform for OI diagnosis; and to this end, use of an advanced array-based technique was proposed.</p><p>Method</p><p>A CustomSeq Affymetrix Resequencing Array was established for high-throughput sequencing of five genes simultaneously. Genomic DNA extraction from 13 OI patients and 85 normal controls and amplification using long-range PCR (LR-PCR) were followed by DNA fragmentation and chip hybridization, according to standard Affymetrix protocols. Hybridization signals were determined using GeneChip Sequence Analysis Software (GSEQ). To examine the feasibility, the outcome from new resequencing approach was validated by conventional capillary sequencing method.</p><p>Result</p><p>Overall call rates using resequencing array was 96–98% and the agreement between microarray and capillary sequencing was 99.99%. 11 out of 13 OI patients with pathogenic mutations were successfully detected by the chip analysis without adjustment, and one mutation could also be identified using manual visual inspection.</p><p>Conclusion</p><p>A high-throughput resequencing array was developed that detects the disease-associated mutations in OI, providing a potential tool to facilitate large-scale genetic screening for OI patients. Through this method, a novel mutation was also found.</p></div

It is a reverse sequencing for the insertion GAT by direct sequencing.

<p>It is a reverse sequencing for the insertion GAT by direct sequencing.</p

Dose response curves of fluorescence intensity and concentrations of human IgG and IgM.

<p>A. The fluorescence signals scanned at different concentrations of human IgG 5, 10, 25, 50, 100 µg/ml; B. The fluorescence signals scanned at different concentrations of human IgM 2.5, 5, 10, 25, 50,100 µg/ml. The equation and the coefficient of determination (R²) are indicated for each curve.</p

Comparison of ELISAs and antigen array for serum IgG reactivity.

<p>A. Comparison of commercial ELISA and antigen array; B. Comparison of in-house ELISA and antigen array *indicated P<0.05.</p

Comparison of ELISA and antigen array for CSF IgG reactivity.

<p>n. not calculated.</p

Calibration curves of fluorescence intensity and concentrations of four recombinant viral antigens.

<p>A. The fluorescence signals scanned at different concentrations of antigen HSV1-gG from 1.56, 3.125, 6.25, 12.5, 25 µg/ml to 50 µg/ml; B. The fluorescence signals scanned at different concentrations of antigen HSV2-gG from 1.56, 3.125, 6.25, 12.5, 25 µg/ml to 50 µg/ml; C. The fluorescence signals scanned at different concentrations of antigen CMV-pp150 from 1.56, 3.125, 6.25, 12.5, 25 µg/ml to 50 µg/ml; D. The fluorescence signals scanned at different concentrations of antigen RV-EP from 0.31, 0.625, 1.25 2.5, 5, 10 µg/ml; the equation and the coefficient of determination (R²) are indicated for each curve.</p

The insertion GAT verified by direct sequencing in patient 8^#.

<p>It is a reverse sequencing, and the frame shifted to move right with the duplication GAT.</p

Comparison of ELISA and antigen array for serum IgM reactivity.

<p>n. not calculated.</p

Determination of the cutoff value for IgG reactivity against HSV1, HSV2, CMV and RV.

<p>Scatter plots of IgG reactivities for different specific antigens of HSV1-gG, HSV2-gG, CMV-pp150, RV-EP compared to other unspecific antigens and blank control when positive reference sera of HSV1, HSV2, CMV and RV were used.</p

Repeatability of the recombinant antigen array.

<p>Fluorescence images and Q-Q plot analysis after hybridization with the positive serum of HSV1 and CMV on two different batches of array slides.</p