title: Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa.
abstract: OBJECTIVE
  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.
authors.affiliation: Department of Medical Genetics, University of Lausanne, Lausanne, Switzerland.
authors.forename: Thomas
authors.initials: T
authors.lastname: Rio Frio
authors.forename: Sylwia
authors.initials: S
authors.lastname: Panek
authors.forename: Christian
authors.initials: C
authors.lastname: Iseli
authors.forename: Silvio Alessandro
authors.initials: SA
authors.lastname: Di Gioia
authors.forename: Arun
authors.initials: A
authors.lastname: Kumar
authors.forename: Andreas
authors.initials: A
authors.lastname: Gal
authors.forename: Carlo
authors.initials: C
authors.lastname: Rivolta
mesh_terms: D056426:Genetic Loci / Q000235:genetics*; D020022:Genetic Predisposition to Disease; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D008291:Malate Dehydrogenase / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D012174:Retinitis Pigmentosa / Q000201:enzymology* / Q000235:genetics*; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D008291:Malate Dehydrogenase