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High-throughput mutation analysis in patients with a nephronophthisis-associated ciliopathy applying multiplexed
Jan Halbritter1, Katrina Diaz, Moumita Chaki
1Department of Pediatrics, University of Michigan Health System, 8220A MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-5646, USA.
Journal of Medical Genetics
|November 29, 2012
Summary
This study developed a cost-effective method using next-generation sequencing to identify mutations in nephronophthisis-associated ciliopathy genes. The approach successfully detected pathogenic mutations in 18% of patients, aiding in genetic diagnosis.
Area of Science:
- Genetics
- Molecular Biology
- Medical Diagnostics
Background:
- Nephronophthisis-associated ciliopathy is a group of genetic disorders affecting kidney function.
- Identifying causative mutations is crucial for diagnosis and genetic counseling.
- Existing diagnostic methods can be costly and time-consuming.
Purpose of the Study:
- To develop a low-cost, high-throughput method for identifying disease-causing mutations in 11 known NPHP genes.
- To analyze a cohort of 192 patients with suspected nephronophthisis-associated ciliopathy.
- To evaluate the efficiency and sensitivity of the developed mutation detection approach.
Main Methods:
- Utilized PCR-based 48.48 Access Array microfluidic technology for multiplexed amplification of 475 amplicons (251 exons) from 48 DNA samples simultaneously.
- Employed next-generation sequencing (Illumina HiSeq2000) after indexing 192 patient-derived products.
- Confirmed potential mutations using Sanger sequencing and segregation analysis.
Main Results:
- Achieved sufficient sequencing coverage (≥30×) for 87.5% of samples and 93.2% of targeted coding exons.
- Successfully identified 90% of known mutations in a proof-of-principle analysis.
- Detected pathogenic mutations in 18% of patients, including 23 novel mutations across several NPHP genes, and identified 40 variants of unknown significance.
Conclusions:
- The combined approach of array-based multiplexed PCR and next-generation sequencing is highly cost-efficient.
- This method significantly facilitates diagnostic mutation analysis for heterogeneous Mendelian disorders like nephronophthisis-associated ciliopathy.
- The study demonstrates a powerful tool for genetic diagnostics in rare diseases.
