Functional characterization of novel MFSD8 pathogenic variants anticipates neurological involvement in juvenile isolated maculopathy

Abstract Biallelic MFSD8 variants are an established cause of severe late‐infantile subtype of neuronal ceroid lipofuscinosis (v‐LINCL), a severe lysosomal storage disorder, but have also been associated with nonsyndromic adult‐onset maculopathy. Here, we functionally characterized two novel MFSD8 variants found in a child with juvenile isolated maculopathy, in order to establish a refined prognosis. ABCA4 locus resequencing was followed by the analysis of other inherited retinal disease genes by whole exome sequencing (WES). Minigene assays and cDNA sequencing were used to assess the effect of a novel MFSD8 splice variant. MFSD8 expression was quantified with qPCR and overexpression studies were analyzed by immunoblotting. Transmission electron microscopy (TEM) was performed on a skin biopsy and ophthalmological and neurological re‐examinations were conducted. WES revealed two novel MFSD8 variants: c.[590del];[439+3A>C] p.[Gly197Valfs*2];[Ile67Glufs*3]. Characterization of the c.439+3A>C variant via splice assays showed exon‐skipping (p.Ile67Glufs*3), while overexpression studies of the corresponding protein indicated expression of a truncated polypeptide. In addition, a significantly reduced MFSD8 RNA expression was noted in patient's lymphocytes. TEM of a skin biopsy revealed typical v‐LINCL lipopigment inclusions while neurological imaging of the proband displayed subtle cerebellar atrophy. Functional characterization demonstrated the pathogenicity of two novel MFSD8 variants, found in a child with an initial diagnosis of juvenile isolated maculopathy but likely evolving to v‐LINCL with a protracted disease course. Our study allowed a refined neurological prognosis in the proband and expands the natural history of MFSD8‐associated disease.

lipopigment inclusions while neurological imaging of the proband displayed subtle cerebellar atrophy. Functional characterization demonstrated the pathogenicity of two novel MFSD8 variants, found in a child with an initial diagnosis of juvenile isolated maculopathy but likely evolving to v-LINCL with a protracted disease course. Our study allowed a refined neurological prognosis in the proband and expands the natural history of MFSD8-associated disease. (v-LINCL, CLN7, NCL7), which is a severe lysosomal storage disorder leading to neurodegeneration. [5][6][7] The first NCL symptoms usually arise between 2 and 5 years of age and are characterized by epileptic seizures and developmental regression. 8 Ultimately ataxia, myoclonus, and visual impairment are seen, which are typical features of a progressive NCL disease leading to premature death. As in other NCL subtypes, accumulation of autofluorescent storage material in neurons and in other cell types can sometimes be observed, ranging from fingerprint and curvilinear structures to rectilinear profiles. 9,10 Here, the female proband presented with an isolated maculopathy initially diagnosed as atypical Stargardt disease at age 5 and underwent genetic testing of the entire ABCA4 gene, followed by whole

| Subjects
Legal parental consent was obtained for the study, which was approved by the institutional ethical committee (B670201525349 and B670201734438). The proband is a currently 12-year-old Caucasian female, with nonconsanguineous parents. She was first noted to have visual problems at age 5, and was originally diagnosed with atypical Stargardt disease.

| Clinical assessment of the proband
Ophthalmic examination of the female proband at age 5 included best-corrected visual acuity (BCVA) measurements, fundus photography, and electroretinography (ERG) according to the International Society for Clinical Electrophysiology of Vision (ISCEV) standards. In addition, spectral-domain optical coherence tomography (SD-OCT) and blue light (488 nm) autofluorescence imaging (BAF), Heidelberg Spectralis HRA+OCT (Heidelberg Engineering, Heidelberg, Germany) were performed. Ophthalmologic reassessments were undertaken at ages 8 and 10. In addition, a neurological assessment was performed, consisting of a clinical neurological examination, electroencephalogram (EEG) and brain magnetic resonance imaging (MRI).

| Molecular genetic analyses
EDTA blood samples were obtained from the proband, the unaffected parents and five healthy controls. DNA was isolated from peripheral blood lymphocytes by standard procedures. In addition, short-term lymphocyte cultures were established for all samples.

| Targeted next-generation sequencing of the coding region of ABCA4
The coding region of ABCA4 was enriched by PCR amplification of all coding exons and flanking splice-site sequences, followed by targeted next-generation sequencing (NGS) as described (MiSeq, Illumina, San Diego, California). 11
Keywords used for filtering were Stargardt disease, blindness, and macular degeneration. Sanger sequencing was used to confirm and assess segregation of the filtered variants, in both the proband and the parents (Tables S1 and S2).

| Expression analysis
Quantitative polymerase chain reaction (qPCR; LC480, Roche, Basel, Switzerland) was used to compare RNA expression levels of MFSD8 between the proband, the carrier parents and five healthy controls.
Primers can be found in Table S4. qBasePlus (Biogazelle, Zwijnaarde, Belgium) was used for data-analysis, normalization was performed using two reference genes (YWAZ and GAPDH).

| In vitro splice assays
A genomic segment of MFSD8 spanning exon 5 (hg19, chr4:128864292-128865864) was amplified from patient DNA using primers with NotI and BamHI recognition sequences (Table S5) Table S6. PCR products were separated and purified from agarose gels, cleaved with restriction enzymes and cloned into the p3xFLAG-CMV10 expression vector (Sigma-Aldrich, St. Louis, Missouri) generating the mutant 3xFLAG p.Ile67Glufs*3.

| Western blot analysis
HEK293T cells were transiently transfected using Jet Pei reagent (Polyplus Transfection, Illkrich, France) with either 3xFLAG-CLN7, 3xFLAG-p.Ile67Glufs*3 or a mock construct as control and harvested 24 hours after the start of transfection. Preparation of postnuclear supernatants and total membrane fractions was performed as described previously. 13 Membrane pellets were homogenized in extraction buffer containing 50 mM Tris-HCl, pH 7.5, 1% TX-100 (v/v), 1 mM EDTA and inhibitor cocktail and incubated on ice for 30 minutes. Immunoblot analyses were performed using anti-FLAG and anti-α-tubulin antibodies (Sigma, St. Louis, Missouri).

| Transmission electron microscopy
A skin biopsy from the patient was explanted and incubated at 37 C in a humidified atmosphere containing 5% CO 2 . Part of this biopsy was fixed and processed for transmission electron microscopy (TEM), using standard techniques: fixation in 2.5% glutaraldehyde and 4% formaldehyde, postfixation in osmium tetroxide, embedding in araldite, staining with uranyl acetate and lead citrate. An FEI CM10 transmission electron microscope was used at 60 kV.

| Identification of biallelic novel MFSD8 variants
Given the initial diagnosis of atypical Stargardt disease in the female proband at age 5, targeted testing of ABCA4 was conducted. Sequencing of the coding region of ABCA4 and of the entire ABCA4 locus revealed one heterozygous ABCA4 variant c.3113C>T; p.(Ala1038Val).
No other (likely) pathogenic coding or noncoding ABCA4 variants including copy number variants were identified. Subsequently, WES was performed in the proband and data were filtered in order to select variants in known retinopathy genes, assuming an autosomal recessive inheritance mode (

| Overexpression studies
In order to study the effect of the c.439+3A>C variant at the protein level, overexpression studies with the truncated CLN7 protein fused to an N-terminal triple FLAG tag (3xFLAG p.Ile67Glufs*3) were performed in HEK293T cells, followed by FLAG immunoblotting of membrane fractions (Figure 3). In 3xFLAG CLN7 expressing HEK293T F I G U R E 2 A, Lymphocyte cDNA derived from both patient (P) and parents (M: mother and F: father) confirms the exon-skip in the patient and carrier mother. This was seen with (+) and without puromycin (−) supplementation. The normal cDNA PCR product, containing exon 5, is 341 bp in size, while the exon 5 loss created a product that is 241 bp smaller. B, The normalized expression profiles of patient (P) and both parents (F and M) are severely reduced when compared to controls (C1-C5). C, MFSD8 mRNA expression levels in the patient are higher with puromycin (+) addition. C, negative control, NTC, notemplate control; SEM, standard error of the mean

| Ultrastructural features
Transmission electron microscopy revealed characteristic membrane-  In contrast to the patient described here, in whom the first visual problems were noticed at age 5, the reported onset of the symptoms in these IRD patients occurred in late adolescence or adulthood (15-46 years) and had a slower progression.

| Neurological assessment
As the majority of the pathogenic variants in the MFSD8 gene have been associated with the syndromic v-LINCL phenotype, we were prompted to determine the functional impact and severity of these novel MFSD8 variants. Based on the predictions at the protein level, the c.590del variant p.(Gly197Valfs*2), can be considered a severe mutation. Using a minigene assay and transcript analysis of lymphocyte RNA, we could establish that the other allele of interest c.439+3A>C induced skipping of exon 5 -although other aberrant splicing cannot be ruled out-and is predicted to result in a truncated protein p. (Ile67Glufs*3). Overexpression studies showed that the truncated protein was still expressed, but based on previously published data it is likely to be retained in the endoplasmic reticulum and does not reach the lysosomes. 14 In addition, the proband has a reduced mRNA MFSD8 expression profile when compared to healthy controls and her carrier parents. report on known biallelic MFSD8 variants previously described in v-LINCL but occurring in patients with an isolated maculopathy in this study raises even more questions and points to possible cis-or transacting modifiers. 4 There is no apparent link between the positions of the affected amino acids in the CLN7 protein (NP_689991.1) and the presenting symptom or disease progression, although in the latter case, these v-LINCL-associated variants are located in the last two exons of the gene. Localization studies have shown that none of the investigated missense variants interfere with mislocalization of the protein, but most likely are pathogenic by affecting the correct functioning of the protein, for example by aberrant proteolysis (Thr294Lys located in luminal loop 7 and Pro412Leu located in luminal loop 9). 14 Although CLN7 is ubiquitously expressed, high expression levels have been seen in specific neuronal cell types and retinal cells, which is in line with the most severely affected tissues in v-LINCL. 13,[18][19][20] MFSD8 transcripts that lack exon 2, exon 7, exons 7 and 8, and exon 11 have been described, but only the 5 kb transcript, consisting of 13 exons seems to be expressed in the brain. 5 There is no indication In case of residual functional CLN7 protein however, slower disease progression or even an atypical disease course is seen, with the macula being first affected and eventually leading to a more widespread retinopathy or even neuronal loss in line with the severity of the MFSD8 pathogenic variants.
In numerous other NCL disorders, similar clinical heterogeneity was observed. 8 A first example is CLN1 disease, were milder CLN1 mutations, giving rise to a phenotype of later onset (late infantile, juvenile, and adult) compared to the classic infantile disease. [21][22][23][24] The correlation between the severity of CLN1 mutations and onset of disease however is not fully understood, as some patients homozygous for an apparent milder variant have an earlier onset than patients who are compound heterozygous for a severe and a mild allele. 8 There is also a spectrum of CLN3-associated phenotypes, ranging from the syndromic NCL to isolated late onset RP (Wang et al) and protracted NCL with autophagic myopathy. [25][26][27][28][29][30][31] Finally, KCTD7 (CLN14) mutations were reported to underlie progressive myoclonic epilepsy and opsoclonus-myoclonus ataxia-like syndrome. 32 Surprisingly, a homozygous missense variant in KCTD7 associated with infantile onset NCL with vision loss and cognitive and motor regression has also been reported. [32][33][34] In conclusion, WES-based testing revealed two novel likely pathogenic MFSD8 variants in a 5-year-old patient manifesting only visual symptoms at the time of genetic testing. Seven years later, she does not display clinically apparent neurological symptoms, which is atypical for classical v-LINCL disease. Apart from the nonsyndromic MFSD8-associated retinopathies, an atypical disease course of neurological MFSD8-associated disease has been described once. Based on the young age of the patient, the functional characterization of the variants and the typical NCL storage in a skin biopsy, however, we anticipate progression to a neurodegenerative NCL-like disorder. Finally, this study illustrates the power of integrated genomic and functional characterization for precision medicine of a heterogeneous disease, to refine clinical diagnoses and to anticipate disease progression.