Clinically Focused Molecular Investigation of 1000 Consecutive Families with Inherited Retinal Disease

Abstract

Purpose: To devise a comprehensive multiplatform genetic testing strategy for inherited retinal disease and to describe its performance in 1000 consecutive families seen by a single clinician.

Design: Retrospective series.

Participants: One thousand consecutive families seen by a single clinician.

Methods: The clinical records of all patients seen by a single retina specialist between January 2010 and June 2016 were reviewed, and all patients who met the clinical criteria for a diagnosis of inherited retinal disease were included in the study. Each patient was assigned to 1 of 62 diagnostic categories, and this clinical diagnosis was used to define the scope and order of the molecular investigations that were performed. The number of nucleotides evaluated in a given subject ranged from 2 to nearly 900 000.

Main outcome measures: Sensitivity and false genotype rate.

Results: Disease-causing genotypes were identified in 760 families (76%). These genotypes were distributed across 104 different genes. More than 75% of these 104 genes have coding sequences small enough to be packaged efficiently into an adeno-associated virus. Mutations in ABCA4 were the most common cause of disease in this cohort (173 families), whereas mutations in 80 genes caused disease in 5 or fewer families (i.e., 0.5% or less). Disease-causing genotypes were identified in 576 of the families without next-generation sequencing (NGS). This included 23 families with mutations in the repetitive region of RPGR exon 15 that would have been missed by NGS. Whole-exome sequencing of the remaining 424 families revealed mutations in an additional 182 families, and whole-genome sequencing of 4 of the remaining 242 families revealed 2 additional genotypes that were invisible by the other methods. Performing the testing in a clinically focused tiered fashion would be 6.1% more sensitive and 17.7% less expensive and would have a significantly lower average false genotype rate than using whole-exome sequencing to assess more than 300 genes in all patients (7.1% vs. 128%; P < 0.001).

Conclusions: Genetic testing for inherited retinal disease is now more than 75% sensitive. A clinically directed tiered testing strategy can increase sensitivity and improve statistical significance without increasing cost.

Figure 1. Distribution of patients and molecular findings across all levels of the clinical classification system

The structure of the classification system is shown at left with the common clinical terms for each phenotypic group shown in the adjacent column. The Total column provides the number of probands assigned to each clinical group, while the Solved column shows the number of probands in each group with a disease-causing genotype identified. The Genes columns provide the number of genes that have been observed to cause the diseases of that clinical group in the published literature and/or at the University of Iowa. The false genotype rate (FGR) columns give the percentage of normal individuals that would be expected to harbor a plausible disease-causing complete genotype by chance in any of the genes assigned to each clinical category in the published literature and/or at the University of Iowa. PV is the average number of plausible disease-causing variants one would expect to observe in a normal individual by chance in any of the genes assigned to each clinical category in the published literature. The bar lengths represent the percent of solved cases for each clinical category while the alternating shades represent the proportional contributions of each gene in descending order. Gene names are given for any genes that cause at least 15% of the disease in a given category. Blue bars indicate categories with an FGR less than 5% while grey bars indicate categories with an FGR greater than or equal to 5%. Abbreviations: AD, Autosomal Dominant; ADNIV, Autosomal Dominant Neovascular Inflammatory Vitreoretinopathy; AR, Autosomal Recessive; CSNB, Congenital Stationary Night Blindness; CSSD, Congenital Stationary Synaptic Dysfunction; DDND, Developmental Delay and/or Neuromuscular Degeneration; ECORD, Early Childhood Onset Retinal Dystrophy; EV, Erosive Vitreoretinopathy; FEVR, Familial Exudative Vitreoretinopathy; HMA, Homocystinuria with Macular Atrophy; HPCD, Helicoid Peripapillary Chorioretinal Degeneration; LHON, Leber Hereditary Optic Neuropathy; MCLMR, Microcephaly Congenital Lymphedema and Chorioretinopathy; MIDD, Maternally Inherited Diabetes and Deafness; SECORD, Severe Early Childhood Onset Retinal Dystrophy; XL, X-linked.

Figure 2. Graphical depiction of the distribution of 1,000 consecutive probands among the larger diagnostic categories

The center chart indicates the proportion of probands assigned to each of the three main branches of the classification system. The outer charts show the fraction of probands assigned to the larger diagnostic categories within each branch.

Figure 3. Distribution of the number of probands per gene

Thirteen genes each caused disease in 1% or more of the probands in this study (left of dashed vertical line) while the other 91 each caused disease in less than 1%. These data are presented in more detail in Table 3.

Figure 4. Financial cost and diagnostic yield of tiered testing strategy

Patients are ordered from lowest cost to highest cost with colors representing the component costs our currently recommended series of genetic tests for each clinical category. A black bar beneath a patient indicates that a causative genotype was discovered in this individual. The horizontal lines highlight the higher cost of uniform whole exome sequencing (upper line) as compared to the average cost of clinically-focused individualized tests (lower line).

Figure 5. Statistical cost

The false genotype rate (FGR) is the average number of complete genotypes one would expect to observe by chance in a healthy individual in a specified genomic space, based on data from 60,000 normal individuals . The probands in this study are shown ordered according to the FGR associated with their clinical category (see Figure 1). The red line indicates the FGR associated with the genes observed to cause disease in this cohort (see also Supplemental Figure 2). The dashed line indicates an FGR of 5% (i.e., the threshold at which one in 20 people would be expected to harbor a plausibly pathogenic, complete genotype by chance). The black bars at the bottom of the figure indicate that a disease-causing genotype was identified in this proband. Assessing the coding sequences of all 301 non-mitochondrial genes in all probands (green line) would result in an average FGR of 128% (i.e., these probands would be expected to harbor an average of 1.28 plausible, complete genotypes by chance).

Figure 6. A 47-year-old male with RPGR-associated X-linked cone dystrophy. A

Fundus photograph of the right eye. B: Optical coherence tomogram of the right eye. C: Goldmann visual field of the right eye.

Figure 7. An 8-year-old male with choroideremia. A

Fundus photograph of the right eye. B: Goldmann visual field of the right eye.

Figure 8. A 48-year-old female with maternally inherited diabetes and deafness. A

Fundus photograph of the right eye. B: Fundus photograph of the left eye. C-D: Fundus autofluorescence images of both the right eye (C) and left eye (D).

Figure 9. A 10-year-old female with ABCA4-associated Stargardt disease. A

Fundus photograph of the right eye. B: Optical coherence tomogram of the right eye. C: Goldmann visual field of the right eye.

Figure 10. A 42-year-old male with ABCA4-associated Stargardt disease. A

Fundus photograph of the right eye. B: Optical coherence tomogram of the right eye. C: Goldmann visual field of the right eye.