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

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

Principle of OI array analysis.

<p>Eight probes (four each for sense and antisense strands) associated with every queried site were used. Each probe consisted of a 25-base oligonucleotide, while the 13th base was different among the probes to cover all potential nucleotide mutations.</p

Variants identification, selection, filtering.

<p>(A)The name of the fragment, the position of the altered nucleotides and the reference nucleotides are signed on the left side. The altered nucleotides of the samples are itemized. The sign N (blue) corresponds to the intensity of the signal which did not allow for a specific base call. The signs y, r, k, etc. (orange) indicate a nucleotide change in the heterozygous state. Signs a, t, c or g (green) denote a nucleotide change to homozygous A, T, C or G. (B) Sequence output files of samples. Part of the sequence containing nucleotide alterations is shown. Reference sequence and positions of nucleotides are shown in red at the top. The signs are identical to those in image (A).</p

Mutations identified in OI patients.

<p>* new mutation found in the present study</p><p>Mutations identified in OI patients.</p