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Published on: January 16, 2019
Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa
Thomas Rio Frio1, Sylwia Panek, Christian Iseli
1Department of Medical Genetics, University of Lausanne, Lausanne, Switzerland.
Purpose:
Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28.
Methods:
With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages.
Results:
Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease.
Conclusions:
The MDH1 gene is not the cause of RP28-linked arRP. Our experimental strategy shows that long-range genomic PCR followed by UHTs provides an excellent system to perform a thorough screening of candidate genes for hereditary retinal degeneration.
Insights
The MDH1 gene does not cause RP28-linked autosomal recessive retinitis pigmentosa (arRP). This study demonstrates ultra-high-throughput sequencing is effective for screening candidate genes in inherited retinal degeneration.
Area of Science:
- Genetics
- Ophthalmology
- Molecular Biology
Background:
- Mutations in IDH3B cause autosomal recessive retinitis pigmentosa (arRP).
- The MDH1 gene is located within the RP28 arRP linkage interval and is functionally related to IDH3B.
- Candidate gene screening is crucial for diagnosing hereditary retinal diseases.
Purpose of the Study:
- To investigate the MDH1 gene as a potential cause of RP28-linked arRP.
- To validate ultra-high-throughput sequencing (UHTs) for routine candidate gene screening.
- To analyze MDH1 in patients from two families with arRP.
Main Methods:
- Genomic long-range PCR was used to amplify all introns and exons of the MDH1 gene.
- Short-read UHTs were employed for sequencing the amplified PCR products.
- Multiple independent software packages were used for read mapping and mutation detection.
Main Results:
- All algorithms successfully mapped and analyzed the complex human genome sequences.
- Both patients were homozygous for identified DNA variants, confirming linkage and homozygosity mapping.
- No DNA changes in MDH1 were associated with the disease in the studied patients.
Conclusions:
- The MDH1 gene is not responsible for RP28-linked arRP.
- The combination of long-range PCR and UHTs is an effective strategy for screening candidate genes in hereditary retinal degeneration.
- Genetic or allelic heterogeneity may explain the RP28 linkage in these families.
