title: Application of a high-throughput genotyping method for loci exclusion in non-consanguineous Australian pedigrees with autosomal recessive retinitis pigmentosa.
abstract: OBJECTIVE
  Retinitis pigmentosa (RP) is the most common form of inherited blindness, caused by progressive degeneration of photoreceptor cells in the retina, and affects approximately 1 in 3,000 people. Over the past decade, significant progress has been made in gene therapy for RP and related diseases, making genetic characterization increasingly important. Recently, high-throughput technologies have provided an option for reasonably fast, cost-effective genetic characterization of autosomal recessive RP (arRP). The current study used a single nucleotide polymorphism (SNP) genotyping method to exclude up to 28 possible disease-causing genes in 31 non-consanguineous Australian families affected by arRP.
  
  
  METHODS
  DNA samples were collected from 59 individuals affected with arRP and 74 unaffected family members from 31 Australian families. Five to six SNPs were genotyped for 28 genes known to cause arRP or the related disease Leber congenital amaurosis (LCA). Cosegregation analyses were used to exclude possible causative genes from each of the 31 families. Bidirectional sequencing was used to identify disease-causing mutations in prioritized genes that were not excluded with cosegregation analyses.
  
  
  RESULTS
  Two families were excluded from analysis due to identification of false paternity. An average of 28.9% of genes were excluded per family when only one affected individual was available, in contrast to an average of 71.4% or 89.8% of genes when either two, or three or more affected individuals were analyzed, respectively. A statistically significant relationship between the proportion of genes excluded and the number of affected individuals analyzed was identified using a multivariate regression model (p<0.0001). Subsequent DNA sequencing resulted in identification of the likely disease-causing gene as CRB1 in one family (c.2548 G>A) and USH2A in two families (c.2276 G>T).
  
  
  CONCLUSIONS
  This study has shown that SNP genotyping cosegregation analysis can be successfully used to refine and expedite the genetic characterization of arRP in a non-consanguineous population; however, this method is effective only when DNA samples are available from more than one affected individual.
authors.affiliation: Australian Inherited Retinal Disease Register & DNA Bank, Department of Medical Technology and Physics, Sir Charles Gairdner Hospital, Perth, Western Australia, Australia.
authors.forename: Rachel L
authors.initials: RL
authors.lastname: Paterson
authors.forename: John N
authors.initials: JN
authors.lastname: De Roach
authors.forename: Terri L
authors.initials: TL
authors.lastname: McLaren
authors.forename: Alex W
authors.initials: AW
authors.lastname: Hewitt
authors.forename: Ling
authors.initials: L
authors.lastname: Hoffmann
authors.forename: Tina M
authors.initials: TM
authors.lastname: Lamey
mesh_terms: D001315:Australia; D016022:Case-Control Studies; D016326:Extracellular Matrix Proteins / Q000235:genetics*; D005136:Eye Proteins / Q000235:genetics*; D005260:Female; D005808:Genes, Recessive; D008040:Genetic Linkage; D056426:Genetic Loci; D060005:Genotyping Techniques / Q000379:methods*; D006239:Haplotypes; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000235:genetics*; D015999:Multivariate Analysis; D009419:Nerve Tissue Proteins / Q000235:genetics*; D010375:Pedigree; D020641:Polymorphism, Single Nucleotide; D012174:Retinitis Pigmentosa / Q000175:diagnosis / Q000235:genetics*; D017422:Sequence Analysis, DNA
chemical_list: C448939:CRB1 protein, human; D016326:Extracellular Matrix Proteins; D005136:Eye Proteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; C112735:USH2A protein, human