Long-Range PCR-Based NGS Applications to Diagnose Mendelian Retinal Diseases

Int J Mol Sci. 2021 Feb 3;22(4):1508. doi: 10.3390/ijms22041508.


The purpose of this study was to develop a flexible, cost-efficient, next-generation sequencing (NGS) protocol for genetic testing. Long-range polymerase chain reaction (PCR) amplicons of up to 20 kb in size were designed to amplify entire genomic regions for a panel (n = 35) of inherited retinal disease (IRD)-associated loci. Amplicons were pooled and sequenced by NGS. The analysis was applied to 227 probands diagnosed with IRD: (A) 108 previously molecularly diagnosed, (B) 94 without previous genetic testing, and (C) 25 undiagnosed after whole-exome sequencing (WES). The method was validated with 100% sensitivity on cohort A. Long-range PCR-based sequencing revealed likely causative variant(s) in 51% and 24% of proband from cohorts B and C, respectively. Breakpoints of 3 copy number variants (CNVs) could be characterized. Long-range PCR libraries spike-in extended coverage of WES. Read phasing confirmed compound heterozygosity in 5 probands. The proposed sequencing protocol provided deep coverage of the entire gene, including intronic and promoter regions. Our method can be used (i) as a first-tier assay to reduce genetic testing costs, (ii) to elucidate missing heritability cases, (iii) to characterize breakpoints of CNVs at nucleotide resolution, (iv) to extend WES data to non-coding regions by spiking-in long-range PCR libraries, and (v) to help with phasing of candidate variants.

Keywords: ABCA4; BEST1; CNV; NGS; PRPH2; diagnostics; genetic testing; long-range PCR; missing heritability; phasing; retinal diseases; sequencing.

MeSH terms

  • ATP-Binding Cassette Transporters / genetics*
  • Adolescent
  • Adult
  • Aged
  • Bestrophins / genetics*
  • Child
  • Child, Preschool
  • Cyclic Nucleotide-Gated Cation Channels / genetics
  • DNA Copy Number Variations
  • Eye Proteins / genetics
  • Female
  • High-Throughput Nucleotide Sequencing*
  • Humans
  • Infant
  • Infant, Newborn
  • Male
  • Membrane Proteins / genetics
  • Microtubule-Associated Proteins / genetics
  • Middle Aged
  • Mutation
  • Nerve Tissue Proteins / genetics
  • Peripherins / genetics*
  • Polymerase Chain Reaction
  • Retinal Diseases / congenital
  • Retinal Diseases / diagnosis
  • Retinal Diseases / genetics*
  • Sequence Analysis, DNA*
  • Young Adult


  • ABCA4 protein, human
  • ATP-Binding Cassette Transporters
  • BEST1 protein, human
  • Bestrophins
  • CNGB3 protein, human
  • CRB1 protein, human
  • Cyclic Nucleotide-Gated Cation Channels
  • Eye Proteins
  • Membrane Proteins
  • Microtubule-Associated Proteins
  • Nerve Tissue Proteins
  • PRPH2 protein, human
  • Peripherins
  • RP1 protein, human
  • RPGR protein, human

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