PMC7245606_01
Pedigree of the MOL1154 family. Filled symbols designate affected individuals. The hatched round symbol represents an affected case with a milder phenotype. A square with a question mark symbol represents an individual who participated in genetic analysis, but clinical examination was not performed due to their young age. The red arrow represents the index case. The individual number is depicted below each symbol. The identified variants are depicted below the individual number: M1: CFH - c.2247_2252del; p.(Leu750_Lys751del), M2: chr6:100040974A>C, +: wild-type allele.
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MOL1154 is a Georgian Jewish family consisting of six affected subjects from four generations (Figure 1) who underwent comprehensive ocular examinations.
OCT images of affected family members. Representative OCT scans of five affected individuals are shown. Individual numbers and the age in years are shown on the left. MOL1154 I:2 - preserved inner retinal layer combined with granular interdigitation layer. MOL1154 II:2 and II:4 - pseudodrusens with intact outer and inner retinal layers. MOL1154 III:1- OCT cross-sections show normal retinal structure. Note that drusen-like deposits were seen clinically but do not appear in the OCT sections. MOL1154 IV:2 - subfoveal RPE atrophy combined with thickening of the interdigitation layer, while the ellipsoid layer seems to be intact.
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SD-OCT showed overall preservation of the retinal layers except for subtle inter-digitation layer irregularity compatible with the drusen-like deposits (Figure 2), and small drusen-like lesions engirdling the fovea were evident in BE (Figure 3), resulting in a hyper autofluorescence dot-like pattern surrounding the fovea fixation test results in this case were within the low range of stable fixation. These were reflected by SD-OCT as a disruption of the inter-digitation layer combined with subfoveal pseudodrusens mainly in the RE (Figure 2 and Figure 3). The oldest subject was the 87 YO great grandmother (MOL1154 I:2) who had a previous medical history of atrial fibrillation treated with warfarin anticoagulation therapy.
Retinal imaging by fundoscopy of affected members. Representative color fundus photos of five affected individuals are shown. Individual numbers and the age in years are shown on the left. MOL1154 III:1, II:4, and II:2 - confluent foveal drusen-like clusters, while no significant findings are evident in the periphery. MOL1154 III:3 - advanced macular atrophy in both eyes with pigmentary dots and patches in RE and a yolk-like lesion surrounded by atrophic retina temporal to the fovea in LE. MOL1154 IV:2 - refractile drusen-like dots surrounding macular atrophy containing yellowish deposits. OD (oculus dextrus) - right eye; OS (oculus sinister) - left eye.
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SD-OCT showed overall preservation of the retinal layers except for subtle inter-digitation layer irregularity compatible with the drusen-like deposits (Figure 2), and small drusen-like lesions engirdling the fovea were evident in BE (Figure 3), resulting in a hyper autofluorescence dot-like pattern surrounding the fovea fixation test results in this case were within the low range of stable fixation. These were reflected by SD-OCT as a disruption of the inter-digitation layer combined with subfoveal pseudodrusens mainly in the RE (Figure 2 and Figure 3). The oldest subject was the 87 YO great grandmother (MOL1154 I:2) who had a previous medical history of atrial fibrillation treated with warfarin anticoagulation therapy.
Identified genetic variants. A: Domains of the CFH protein that is composed of 20 repetitive units of 60 amino acids called short consensus repeats (SCRs) or complement control protein modules (CCPs). The amino acid sequence of CCPs 12 (blue color) and 13 (red color) are depicted (690-804). The eight residues that are in the black box are located in the CCP 12 and 13 linker region. The pink colored box shows amino acid residues that were found to be deleted in our study. B: Chromatograms of a homozygous wild type (top) and a heterozygous affected individual (bottom) with the c.2247_2252del; p.(Leu750_Lys751del) CFH variant. Arrows represent the beginning and the end of the six deletion nucleotides. C: Illustration of reported variants in chromosome 6: chr6:100020205-100143306 duplication, 100,040,906 G>T, 100,040,987 G>C, and 100,041,040 C>T. The red arrow represents the location of the variant identified in the current study within the previously reported hotspot region- a DNase hypersensitivity site (blue), upstream of PRDM13 gene (green). Genomic localization is based on human reference sequence hg19 (GRCH37). D: Chromatograms of a homozygous wild-type (top) and a heterozygous affected individual (bottom) with the chr6: 100040974A>C variant.
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Finally, the possible sequence variants in genes that were previously reported to be associated with IRDs were studied, and a heterozygous variant, c.2247_2252del; p.(Leu750_Lys751del) that is absent from the gnomAD database was identified in exon 15 of the complement factor H (CFH) gene (Figure 4B).
Partial amino acid sequence alignment of the CFH protein. The red box represents the amino acids that were found to be deleted in the present study. Red dots denote the amino acids that are highly conserved among species. Different species (human - NP 000,177.2; chimpanzee - XP 001,136,531.1; Rhesus monkey - XP 001,111,875.1; dog - XP 536,110.2; cattle - NP 001,029,108.1; house mice - NP 034,018.2; and Norway rats - NP 569,093.2) were used to check the conserved amino acids.
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Multiple sequence alignment revealed that the two deleted amino acids are highly conserved among species (Figure 5).
Structure-based analysis of the effect of the L750-K751 deletion. CPP12, linker, and CPP13 colored in green, purple and cyan, respectively. The structure is shown in cartoon, with the linker side chain shown in sticks; residues in the two CPP domains that participate in the formation of the hydrophobic patch are shown in spheres. A: Structural model of wt after loop remodeling, highlighting the central position of L750. B: Corresponding model of the deletion mutant: in the shortened loop, D748 replaces L750, perturbing the hydrophobic character of this region and leading to destabilization. C, D: Different relative orientations adapted by the CPP domains in wt (C) and deletion mutant (D) indicate increased variability in orientation for the deletion mutant compared to the wt.
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Structural inspection of the NMR solved structure of the tandem CPP12-13 repeat (pdb ID 2KMS) revealed that the deletion is located in the connecting linker (residues 746-751 AIDKLK); L750 is positioned at the center of a small hydrophobic core formed by residues in CPP12 (T724, I726) and the linker (V745, I747, K749, L750), which stabilizes the relative orientation between CPP12 and CPP13 (Figure 6A). In the mutant, the shortened linker positions D748 at the same site, thereby changing the hydrophobic side chain character to a negative charge, which significantly destabilizes the structure (DeltaDeltaGWT,mut = -15.73 Rosetta energy units [REU]; Figures 6A,B). While the solved NMR structural models show some variation in orientation, this variation increases in the deletion mutant (Figures 6C,D).