Biallelic mutations in PLA2G5, encoding group V phospholipase A2, cause benign fleck retina

Abstract

Flecked-retina syndromes, including fundus flavimaculatus, fundus albipunctatus, and benign fleck retina, comprise a group of disorders with widespread or limited distribution of yellow-white retinal lesions of various sizes and configurations. Three siblings who have benign fleck retina and were born to consanguineous parents are the basis of this report. A combination of homozygosity mapping and exome sequencing helped to identify a homozygous missense mutation, c.133G>T (p.Gly45Cys), in PLA2G5, a gene encoding a secreted phospholipase (group V phospholipase A(2)). A screen of a further four unrelated individuals with benign fleck retina detected biallelic variants in the same gene in three patients. In contrast, no loss of function or common (minor-allele frequency>0.05%) nonsynonymous PLA2G5 variants have been previously reported (EVS, dbSNP, 1000 Genomes Project) or were detected in an internal database of 224 exomes (from subjects with adult onset neurodegenerative disease and without a diagnosis of ophthalmic disease). All seven affected individuals had fundoscopic features compatible with those previously described in benign fleck retina and no visual or electrophysiological deficits. No medical history of major illness was reported. Levels of low-density lipoprotein were mildly elevated in two patients. Optical coherence tomography and fundus autofluorescence findings suggest that group V phospholipase A(2) plays a role in the phagocytosis of photoreceptor outer-segment discs by the retinal pigment epithelium. Surprisingly, immunohistochemical staining of human retinal tissue revealed localization of the protein predominantly in the inner and outer plexiform layers.

Figure 1

Identification of PLA2G5 Mutations in Individuals from Two Families with Benign Fleck Retina Pedigrees of families J and K are shown. Homozygosity mapping with DNA from subject K-2 revealed a 12 cM region on 1p (flanked by rs10796459 and rs12407356). DNA samples from subjects J-1, J-2, J-3, J-4, J-5, and J-6 were also genotyped, and a 5 cM region (flanked by rs3738122 and rs1832047) was found to be homozygous in all affected individuals and was found to be consistent with disease segregation. RefSeq genes contained in this shared region between families K and J are shown. Exome sequencing with DNA from subject J-6 revealed a homozygous missense change, c.133G>T (p.Gly45Cys) in PLA2G5. Gene structure of PLA2G5, coverage depth distribution of the mapped reads along its five exons (Savant Genome Browser), and sequencing reads corresponding to this variant are presented (IGV viewer; 34 reads total: 10 forward and 24 reverse,100% thymine). Subsequently, bidirectional Sanger sequencing confirmed segregation of the p.Gly45Cys change in family J and identified a homozygous nonsense mutation (c.185G>A [p.Trp62X]) in individual K-2. Electropherograms of DNA sequences surrounding these two variants are shown. Both sequences are displayed in the forward orientation.

Figure 2

Retinal Imaging of Individuals with Benign Fleck Retina and PLA2G5 Mutations (A) Color photographs and corresponding fundus autofluorescence (FAF) images of the left fundi of subjects K-2 (aged 12), J-4 (aged 12), and M-1 (aged 39). On fundus photography, multiple yellow-white flecks of various sizes are observed. FAF reveals hyperautofluorecent lesions corresponding in location with the flecks. The macula is relatively spared in subjects J-4 and M-1 but not in K-2, in whom only the fovea appears to be unaffected. This might reflect a more detrimental effect of the c.185G>A (p.Trp62X) mutation in the homozygous state (subject K-2) as opposed to homozygous missense (c.133G>T [p.Gly45Cys] in subject J-4) or compound heterozygous (p.Gly45Cys and c.383delA, p.Gln128ArgfsX88 in M-1) mutations. (B) FAF imaging and linear spectral domain optical coherence tomography (OCT) scan of the left retina of subject K-2. Deep, discrete, hyper-reflective deposits, more obvious at the edge of the foveal scan, are observed. The panel with an enlarged image of the boxed region shows the outer retina and RPE in detail. The lesions are located posterior to the hyperreflective band corresponding to the photoreceptor inner/outer segment junction and do not disrupt it. An overlay of OCT with FAF is also presented. Deposits are spatially associated with the hyperautofluorescent lesions and thus correspond to the flecks. (C) Functional assessment of the central retina in subject K-2. Static-perimetry testing (threshold sensitivities from 0 to 20 dB, test spot size Goldmann III) results overlaid with FAF are presented. Retinal sensitivity was normal. (D) Longitudinal data showing evolution of fleck-like lesions over time. Magnified view of fundus photographs from the left eye (vascular arcades) of subject K-2 at ages 6 and 12. Flecks increase in number and size and become more confluent.

Figure 3

Structure of PLA2G5 and Hypothetical Model of Human Group V Phospholipase A₂ Binding to a Phospholipid Membrane Surface (A) Exons are depicted with boxes in which the shaded areas denote the coding sequence and the unshaded areas denote the 5′ and 3′ untranslated regions. Numbers under the line correspond to intron size (kb), and arrows indicate the position of mutations identified in this study. The amino acid sequence of the signal peptide is shown in normal font; the sequence of the 118 amino acid mature enzyme after cleavage of the prepeptide is shown in bold font (Uniprot⁸). Cystine residues forming the six disulfide bridges maintaining the enzyme's rigid three-dimensional structure are italicized (Uniprot⁸). Amino acids responsible for interfacial binding (tryptophan 50) and catalytic activity (histidine 67 and aspartic acid 111) are underlined. (B) A homology model of human group V phospholipase A₂ (Protein Data Bank accession code 2ghn) after hypothetical association with a phospholipid membrane is presented. Structural features of the active site, conserved among secreted phospholipase A₂s, are highlighted; these features include a catalytic Ca²⁺ ion bound by a peptide loop (yellow) and a catalytic dyad formed by amino acids His67 and Asp111 (dark blue). The Ca²⁺ coordination includes carbonyl backbone interactions from Tyr47, Gly49, and Gly51, as well as a shared bidentate interaction from Asp68 (amino acids colored in yellow; Uniprot). Trp50, a key amino acid in the enzyme's interfacial binding surface (distinct from the active site) is highlighted in red; its indole chain contributes to the characteristic ability of group V phospholipase A₂ to bind to both zwitterionic and anionic phospholipid vesicles. Cationic residues that are also responsible for membrane binding at the carboxyl end of the protein are colored in purple. PyMOL (Delano Scientific, Portland, OR) was used for viewing the human group V phospholipase A₂ three-dimensional molecular structure (orthoscopic view, cartoon setting, cylindrical helices).

Figure 4

Localization of Group V Phospholipase A₂ within a Control Human Retinal Tissue Human retinal tissue from an 87-year-old male donor's eye was obtained from the eye bank at Moorfields Eye Hospital with the approval of Moorfields and Whittington Research Ethics Committee (06/Q0504/78) and embedded in an optimal-cutting-temperature compound. Cryostat sections were cut at 10 μm and thaw-mounted onto charged slides. Immunohistochemistry was performed at room temperature to reveal group V phospholipase A₂ localization via mouse anti-human PLA2G5 monoclonal antibody (LS-C11702, clone MCL-3G1, Lifespan Bioscience, Seattle, WA, USA) at a dilution factor of 1/20. An alkaline phosphatase-conjugated avidin-biotin complex kit (Vectastain ABC-AP Mouse IgG kit, Vector Laboratories, Burlingame, CA, USA) was used as a secondary detection method according to the manufacturer's guidelines. An additional quenching step was performed with 1% Levamisole for 30 min so that autofluorescence would be reduced. Abbreviations are as follows: Ch, Choroid; RPE, retinal pigment epithelium; OS, photoreceptor outer segments; IS photoreceptor inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; an dG ganglion cell layer. The scale bar represents 50 μm.