Contribution of Whole-Genome Sequencing and Transcript Analysis to Decipher Retinal Diseases Associated with MFSD8 Variants

Abstract

Biallelic gene defects in MFSD8 are not only a cause of the late-infantile form of neuronal ceroid lipofuscinosis, but also of rare isolated retinal degeneration. We report clinical and genetic data of seven patients compound heterozygous or homozygous for variants in MFSD8, issued from a French cohort with inherited retinal degeneration, and two additional patients retrieved from a Swiss cohort. Next-generation sequencing of large panels combined with whole-genome sequencing allowed for the identification of twelve variants from which seven were novel. Among them were one deep intronic variant c.998+1669A>G, one large deletion encompassing exon 9 and 10, and a silent change c.750A>G. Transcript analysis performed on patients’ lymphoblastoid cell lines revealed the creation of a donor splice site by c.998+1669A>G, resulting in a 140 bp pseudoexon insertion in intron 10. Variant c.750A>G produced exon 8 skipping. In silico and in cellulo studies of these variants allowed us to assign the pathogenic effect, and showed that the combination of at least one severe variant with a moderate one leads to isolated retinal dystrophy, whereas the combination in trans of two severe variants is responsible for early onset severe retinal dystrophy in the context of late-infantile neuronal ceroid lipofuscinosis.

Keywords: MFSD8 gene; deep intronic variant; isolated macular dystrophy; neuronal ceroid lipofuscinosis; transcript analysis.

Figure 1

Ocular findings in isolated retinal dystrophy linked to MFSD8 variants. Fundus photography, SW-FAF, IRR and HD-OCT are shown in successive rows. Top, teenage-onset maculopathy/COD/CORD. Optic disc pallor and a punched-out atrophic foveal lesion with slightly hyperpigmented borders. Foveal lesion looked dark with hyperautofluorescent edges on SW-FAF. SD-OCT found either a temporary aspect of foveal cavitation in outer reflective layers (L-08031428, VV-1595021) or a larger destruction of outer layers (IM-190703) in the macula. Slow progression of macular lesions at follow-up, progression to a more widespread retinal lesions with mid-peripheral involvement in patient L-08031428. Bottom, adult-onset maculopathy/COD. Bull’s eye maculopathy. Dark fovea with hyperautofluorescent edges on SW-FAF corresponding to a more or less large interruption of outer reflective layers on SD-OCT. Slow progression of macular lesion at follow-up.

Figure 2

Ocular findings in patient with late-infantile neuronal ceroid lipofuscinosis (MFSD8-LINCL). (A) Waxy pallor of optic disc, severe vascular attenuation and whitish, depigmented appearance of posterior pole and midperipheral retina. Cellophane light reflex from the macula. (B) SW-FAF showed a large area of increased autofluorescence with indistinct border in the posterior pole and the second more narrow annulus of increased autofluorescence around the fovea. Fovea was irregularly hypoautofluorescent. Peripheral retina was isoautofluorescent. (C) SD-OCT. Retina was thin with indistinct lamination and widespread disappearance of outer layers (ONL, EZ and RPE). (D) ffERG. Unrecordable responses under dark- and light-adapted conditions.

Figure 3

MD-associated MFSD8 variants. (a) Loss-of-function variants reported in macular dystrophy. MFSD8 gene is represented with its 12 exons. Variants are annotated according to the cDNA nomenclature. In red are variants from the literature and in green are novel variants identified in this study. (b) Missense variants reported in macular dystrophy. CLN7 is composed of 12 transmembrane domains and 11 extracellular or cytoplasmic loops. The 518 amino acids of CLN7 protein are depicted as white circles. In red are variants from the literature; in green are novel variants identified in this study; underscored are the already reported variants found in our study.

Figure 4

Functional tests results for MFSD8 variants c.998+1669A>G and c.750A>G in LCLs Lymphoblastic cell lines from patients HD-OPH1206, L-08031428 and L-20021807 were used to analyse MFSD8 cDNA expression. (a) Overlapping primers located at the exons 9–10 and 11–12, respectively were used to show the inclusion of a 140 bp pseudoexon due to the c.998+1669A>G variant. On the right, electropherograms obtained from the upper and lower bands separated on agarose gel. (b) The exon 8 skipping due to c.750A>G variant was confirmed using primers located at exons 6–7 and 9–10 junctions. The exon 8 skipping is observed in control at a very low level, suggesting exon 8 as an alternative cassette, normally expressed, and enhanced by the presence of this variant. Semi-quantification of the bands (using primers located in exon 7 and 9) has been performed on cells not treated by puromycin. Ctl—control; MD—macular dystrophy; NCL—neuronal ceroid lipofushinose.