Panel-based next-generation sequencing identifies novel mutations in Bulgarian patients with inherited retinal dystrophies

Abstract

Background: Next-generation sequencing (NGS)-based method is being used broadly for genetic testing especially for clinically and genetically heterogeneous disorders, such as inherited retinal degenerations (IRDs) but still not routinely used for molecular diagnostics in Bulgaria. Consequently, the purpose of this study was to evaluate the effectiveness of a molecular diagnostic approach, based on targeted NGS for the identification of the disease-causing mutations in 16 Bulgarian patients with different IRDs.

Methods: We applied a customized NGS panel, including 125 genes associated with retinal and other eye diseases to the patients with hereditary retinopathies.

Results: Systematic filtering approach coupled with copy number variation analysis and segregation study lead to the identification of 16 pathogenic and likely pathogenic variants in 12/16 (75%) of IRD patients, 2 of which novel (12.5%): ABCA4-c.668delA (p.K223Rfs18) and RР1-c.2015dupA (p.K673Efs*25). Mutations in the ABCA4, PRPH2, USH2A, BEST1, RР1, CDHR1, and RHO genes were detected reaching a diagnostic yield between 42.9% for Retinitis pigmentosa cases and 100% for macular degeneration, Usher syndrome, and cone-rod dystrophy patients.

Conclusion: Our results confirm the usefulness of targeted NGS approach based on frequently mutated genes as a comprehensive and successful genetic diagnostic tool for IRDs with significant impact on patients counseling.

Keywords: inherited retinal degeneration; molecular diagnostics; novel mutations; targeted next generation sequencing.

FIGURE 1

Pedigrees of IRD families and segregation analysis of identified variants. Individuals are identified by pedigree number. Squares indicate males, circles indicate females, slashed symbols indicate deceased, solid symbols indicate affected individuals, open symbols indicate unaffected individuals, black arrow indicates the proband. Consanguinity is marked by a double horizontal line. Sequencing chromatograms showing mutation segregation in each pedigree except RD57 where three‐exon deletion in USH2A (NM_206933.4) was found.

FIGURE 2

Fundus photographs of both eyes of patients (a) RD30‐II:1 (31 years), (e) RD30‐II:1 (31 years), (k) RD59‐II:1 (13 years), (l) RD62‐II:1 (36 years), and (n) RD70‐II:1 (25 years) showing bilateral optic disc pallor, narrowed retinal vessels, and macular pigmentary deposits characterized by different ABCA4‐mutations; (b) RD36‐II:1 (46 years) carrying PRPH2‐mutation resulted in fleck‐shaped subretinal yellowish deposits and a RPE defect in the macula; patients with various syndromic and non‐syndromic RP phenotypes classical changes including attenuation of the retinal vessels, waxy pallor of the optic disc, retinal atrophy, and pigmentary deposits resembling bone spicules in peripheral retina seen in (c) RD50‐II:1 (31 years) carrying USH2A‐mutations; (d) RD51‐II:1 (28 years); (f) RD53‐II:1 (38 years); (g) RD54‐II:1 (26 years); (j) RD58‐II:1 (40 years); (i) RD57‐II:1 (36 years) carrying USH2A homozygous deletion; (m) severe rod‐cone dystrophy signs with macular involvement in patient RD64‐II:1 (29 years) carrying RP1 homozygous mutation; (o) 11‐year‐old male patient RD71‐II:1 with RHO‐associated RP showing bone spicule pigmentation in the mid‐periphery, vessel attenuation; and (h) fundus photograph of a 33‐year‐old female patient RD56‐III:1 with BEST1‐associated MD showing vitelliform lesions in a typical “egg yolk” shape in the center of the macula. RE: Right eye, LE: Left eye.

FIGURE 3

Sequencing chromatograms showing (a) DNA sequences of normal control (left) and the heterozygous deletion of one nucleotide A, c.668delA, in exon 6 of ABCA4 (NM_000350.3) resulting in a loss‐of‐function allele p.K223Rfs18 in patient RD30‐II:1 (right); (b) DNA sequences of normal control (left), heterozygous (middle), and homozygous (right) insertion of one nucleotide A, c.2015dupA, in exon 4 of RP1 (NM_006269.2) resulting in a homozygous loss‐of‐function allele p.K673EfsX25 in patient RD64‐II:1; (c) CNVs analysis and MLPA profile for the exon 22–24 deletion of USH2A (NM_206933.4) found in patient RD57‐II:1. Ratios below 0.7 were considered deletions; those above 1.2 were considered to be duplications. Deletion of exon 23 is shown on the first pane (MLPA probemix P361) and deletion of exons 22 and 24 is shown on the second pane (MLPA probemix P362).

FIGURE 4

Representative photographs of probands (a) RD59‐II:1 (13 years) and (b) RD70‐II:1 (25 years) both affected by STGD. For each scan, left pane top: Fundus photographs showing a central atrophic lesion with flecks around the macula; left pane bottom: FA images showing fluorescence blocking caused by the pigment mottling in the macular region, hyperfluorescent flecks extended to the midperipheral retina; middle pane top and bottom: Macular OCT scans showing a reduced thickness of the attenuated retina and an altered reflectivity in the choroid, RPE, and the outer segments of the photoreceptors in both eyes; right pane top: Macular thickness significance map, the central innermost 1‐mm‐diameter circle represents the central subfield; inner superior, inner nasal, inner inferior, and inner temporal areas bounded by the 3‐mm‐diameter circle form the inner macula; outer superior, outer nasal, outer inferior, and outer temporal areas bounded by the 6‐mm‐diameter circle form the outer macula; right pane bottom: 3‐D surface maps: The ILM‐RPE, displaying the retinal thickness in three dimensions. ILM: Inner limiting membrane; RPE: Retinal pigment epithelium; RE: Right eye, LE: Left eye.