MOLECULAR

GENETIC ANALYSIS
USING TARGETED
NGS ANALYSIS OF 677
INDIVIDUALS WITH
RETINAL DYSTROPHY
Authors: Cathrine Jespersgaard, Mingyan Fang, Mette
Bertelsen, Xiao Dang, Hanne Jensen, Yulan Chen, Niels Bech,
Lanlan Dai, Thomas Rosenberg,

PRESENTED BY: Zubair Alam

SP21-RBI-009 © Sci Rep 9, 1219 (2019), Jespersgaard, C., Fang, M., Bertelsen, M. et
COMSATS UNIVERSITY ISLAMABAD, PAKISTAN al. Molecular genetic analysis using targeted NGS analysis of 677 individuals
OUTLINE

Introduction Aims and Methodology Results Conclusion

Objectives
INTRODUCTION
 Inherited retinal diseases (IRDs)

- Sequence variations in more than 150 genes

- Heterogeneous conditions
- Retinitis pigmentosa (RP) 1:4000
- Syndromic and non-syndromic
- Autosomal recessive (AR), autosomal dominant (AD),
X-linked (XL).
- Digenic - rare
 AIMS AND
OBJECTIVES
 Next generation sequencing (NGS)
 Molecular genetic diagnosis

- Genetic counselling and prognosis

- Gene therapy strategies
METHODOLOGY
 677 individuals (Denmark)

- Clinically diagnosed or Suspected

 Patients and clinical evaluation

- 4 to 100 years age (Mean 53.3, Median 55)

- 493 with no previous record – through DNA sample
 Gene selection and target enrichment

- RetNet database
- OMIM
- NimbleGen SeqCap Target Enrichment System, - coding exons, 5-/3-UTR
regions and 20 bp flanking intronic regions
- Size: 2.07 MB and Coverage: 95%
- Heterozygous form ABCA4 or USH2A - Primer set - followed by Sanger
sequencing.
 Next-generation sequencing and bioinformatics

- Genomic DNA - Peripheral blood

- Illumina HiSeq 2000 (494 patients) or HiSeq 4000 (184 patients)
- Raw sequencing image files and base-calling - Illumine Pipeline
- SOAPnuke software
- Remaining high-quality reads - Burrows–Wheeler Algorithm (BWA)
- Local realignment and base recalibration - GATK
 Interpretation and classification of variants

- Pathogenic (class 5)
- Likely-pathogenic (class 4)
- VUS (class 3)
- Likely benign (class 2)
- Benign (class 1)
 ACMG guidelines - Alamut Visual (Interactive Bio software, France)

- Human Splicing Finder.

 Deletion and duplication analysis

- R software package ExomeDepth or using the VarSeq Copy number variation (CNV) software.
- CNV analysis - 24 deletions and duplications
- CNVs were validated either by MLPA, qPCR or chromosome microarray
- Tree primer sets
- PCR - 7500 ABI SDS system
- Chromosome microarray - CytoScan HD array
- Data analysis - ChAS software
RESULTS
 Patient characteristics:

- Based on available clinical record - Biobank

- Divided into seven groups
- Generalized retinal dystrophies - RP and cone-rod dystrophies (72%)
- Macular dystrophy - Stargardt disease (17%)
© Sci Rep 9, 1219 (2019), Jespersgaard, C., Fang, M., Bertelsen, M. et
al. Molecular genetic analysis using targeted NGS analysis of 677 individuals
 NGS Panel:

- Out of 125 genes- Retinitis pigmentosa (41%), Bardet-Biedl syndrome (14%),cone

rod dystrophy (12%), Leber congenital amaurosis (10%), Usher syndrome
(7%) and congenital stationary night blindness (3%).
- 70% AR, 20% AD, 5% XL
- PRPH2 and ROM1 - Digenic inheritance
- 1156.22Mb per individual
- 7440 rare variants (4030 SNVs and 3410 indels) – Mean of 9.2 (range from 2 to 29).
- Reference sequence - GRCh37/hg19
 Sequence variations
 Disease causing and VUS (variant of unknown clinical
significance)
– Phenotype
 American College of Medical Genetics and Genomics
(ACMG) classification.
 A total of 806 variants - 532 unique variants, are
reported;
-187 novel
 Disease causing variants - 487 individuals
 7% (47/677)

© Sci Rep 9, 1219 (2019), Jespersgaard, C., Fang, M., Bertelsen, M. et

al. Molecular genetic analysis using targeted NGS analysis of 677 individuals
Sequence variations are distributed
across 81 genes:
- 59 genes with AR inheritance (n=183),
- 14 genes with AD inheritance (n=77),
- Four with XL inheritance (n=32) and
- Four genes known with both AR and AD
inheritance (n=31)

© Sci Rep 9, 1219 (2019), Jespersgaard, C., Fang, M., Bertelsen, M. et

al. Molecular genetic analysis using targeted NGS analysis of 677 individuals
Frequently mutated genes were:
USH2A (n=42, 6%), ABCA4 (n=40,
6%), EYS (n=29, 4%), RHO (n=21,
3%), BEST1 (n=18, 3%), RPGR (n=17,
2%) and RP1 (n=15, 2%).

- Solved cases (182 out of 323, 56%)

- Variants in 22 genes solved only one
or two cases each.
- 47 individuals - pathogenic or likely
pathogenic

© Sci Rep 9, 1219 (2019), Jespersgaard, C., Fang, M., Bertelsen, M. et al. Molecular
genetic analysis using targeted NGS analysis of 677 individuals with retinal dystrophy.
All rights reserved.
CONCLUSION
 Targeted NGS

 CNV analysis

 Genetic counseling

 Molecular genetic diagnosis

 Classification of variants

 Phenotype-genotype correlation