title: New COL6A6 variant detected by whole-exome sequencing is linked to break points in intron 4 and 3'-UTR, deleting exon 5 of RHO, and causing adRP.
abstract: OBJECTIVE
  This study aimed to test a newly devised cost-effective multiplex PCR assay for the molecular diagnosis of autosomal dominant retinitis pigmentosa (adRP), as well as the use of whole-exome sequencing (WES) to detect disease-causing mutations in adRP.
  
  
  METHODS
  Genomic DNA was extracted from peripheral blood lymphocytes of index patients with adRP and their affected and unaffected family members. We used a newly devised multiplex PCR assay capable of amplifying the genetic loci of RHO, PRPH2, RP1, PRPF3, PRPF8, PRPF31, IMPDH1, NRL, CRX, KLHL7, and NR2E3 to molecularly diagnose 18 index patients with adRP. We also performed WES in affected and unaffected members of four families with adRP in whom a disease-causing mutation was previously not found.
  
  
  RESULTS
  We identified five previously reported mutations (p.Arg677X in the RP1 gene, p.Asp133Val and p.Arg195Leu in the PRPH2 gene, and p.Pro171Leu and p.Pro215Leu in the RHO gene) and one novel mutation (p.Val345Gly in the RHO gene) representing 33% detection of causative mutations in our adRP cohort. Comparative WES analysis showed a new variant (p.Gly103Arg in the COL6A6 gene) that segregated with the disease in one family with adRP. As this variant was linked with the RHO locus, we sequenced the complete RHO gene, which revealed a deletion in intron 4 that encompassed all of exon 5 and 28 bp of the 3'-untranslated region (UTR).
  
  
  CONCLUSIONS
  The novel multiplex PCR assay with next-generation sequencing (NGS) proved effective for detecting most of the adRP-causing mutations. A WES approach led to identification of a deletion in RHO through detection of a new linked variant in COL6A6. No pathogenic variants were identified in the remaining three families. Moreover, NGS and WES were inefficient for detecting the complete deletion of exon 5 in the RHO gene in one family with adRP. Carriers of this deletion showed variable clinical status, and two of these carriers had not previously been diagnosed with RP.
authors: Molecular Genetics Unit, Hospital of Terrassa, Barcelona, Spain.
authors: Miguel
authors: M
authors: de Sousa Dias
authors: Molecular Genetics Unit, Hospital of Terrassa, Barcelona, Spain.
authors: Imma
authors: I
authors: Hernan
authors: Ophthalmology Service, Hospital of Terrassa Barcelona, Spain.
authors: Barbara
authors: B
authors: Delás
authors: Molecular Genetics Unit, Hospital of Terrassa, Barcelona, Spain.
authors: Beatriz
authors: B
authors: Pascual
authors: Molecular Genetics Unit, Hospital of Terrassa, Barcelona, Spain.
authors: Emma
authors: E
authors: Borràs
authors: Molecular Genetics Unit, Hospital of Terrassa, Barcelona, Spain.
authors: Maria José
authors: MJ
authors: Gamundi
authors: Molecular Genetics Unit, Hospital of Terrassa, Barcelona, Spain.
authors: Begoña
authors: B
authors: Mañé
authors: Department of Genetics, IIS-Jimenez Diaz Foundation (IIS-FJD), Madrid, Spain ; Centre for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Madrid, Spain.
authors: Patricia
authors: P
authors: Fernández-San José
authors: Department of Genetics, IIS-Jimenez Diaz Foundation (IIS-FJD), Madrid, Spain ; Centre for Biomedical Network Research on Rare Diseases (CIBERER), ISCIII, Madrid, Spain.
authors: Carmen
authors: C
authors: Ayuso
authors: Molecular Genetics Unit, Hospital of Terrassa, Barcelona, Spain.
authors: Miguel
authors: M
authors: Carballo
mesh_terms: D020413:3' Untranslated Regions; D024142:Collagen Type VI / Q000235:genetics*; D053960:DNA Breaks; D004252:DNA Mutational Analysis; D059472:Exome; D005091:Exons; D005260:Female; D005799:Genes, Dominant; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007438:Introns; D008297:Male; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods; D010375:Pedigree; D012174:Retinitis Pigmentosa / Q000235:genetics*; D012243:Rhodopsin / Q000235:genetics*; D017384:Sequence Deletion
chemical_list: D020413:3' Untranslated Regions; C555240:COL6A6 protein, human; D024142:Collagen Type VI; D012243:Rhodopsin