Linear Diagnostic Procedure Elicited by Clinical Genetics and Validated by mRNA Analysis in Neuronal Ceroid Lipofuscinosis 7 Associated with a Novel Non-Canonical Splice Site Variant in MFSD8

Abstract

Neuronal ceroid lipofuscinoses (CNL) are lysosomal storage diseases that represent the most common cause of dementia in children. To date, 13 autosomal recessive (AR) and 1 autosomal dominant (AD) gene have been characterized. Biallelic variants in MFSD8 cause CLN7 type, with nearly 50 pathogenic variants, mainly truncating and missense, reported so far. Splice site variants require functional validation. We detected a novel homozygous non-canonical splice-site variant in MFSD8 in a 5-year-old girl who presented with progressive neurocognitive impairment and microcephaly. The diagnostic procedure was elicited by clinical genetics first, and then confirmed by cDNA sequencing and brain imaging. Inferred by the common geographic origin of the parents, an autosomal recessive inheritance was hypothesized, and SNP-array was performed as the first-line genetic test. Only three AR genes lying within the observed 24 Mb regions of homozygosity were consistent with the clinical phenotype, including EXOSC9, SPATA5 and MFSD8. The cerebral and cerebellar atrophy detected in the meantime by MRI, along with the suspicion of accumulation of ceroid lipopigment in neurons, prompted us to perform targeted MFSD8 sequencing. Following the detection of a splice site variant of uncertain significance, skipping of exon 8 was demonstrated by cDNA sequencing, and the variant was redefined as pathogenic.

Keywords: CNL7; MFSD8; clinical genetics; mRNA; neurodevelopmental disorders.

Figure 1

Sagittal (A) and axial (B) brain and spinal cord MRI showed diffuse and bilateral cerebellar atrophy and bilateral and symmetrical hyperintensity of the posterior periventricular white matter and mesial portions of the thalami, on T2 and FLAIR sequences; (C) EEG show focal epileptiform anomalies in the middle-posterior regions and diffuse anomalies.

Figure 2

Electropherograms of sequencing analyses: (A) exon 8–intron 8 junction of the MFSD8 gene (NM_001371596.2, forward strand) in our patient, presenting with a homozygous insertion of a thymine between the 2nd and 3rd position of the donor splice site (c.863 + 2dup); (B) exon 8–intron 8 junction of the MFDS8 gene (forward strand) in the patient’s mother showing the heterozygous variant; (C) MFSD8 cDNA analysis in our patient showing two overlapping sequences both skipping exon 8, with the shorter lacking exon 9 as well. Junctions between exons 7–9 (7–10 in the shorter fragment) and 9–10 are indicated; (D) MFSD8 cDNA analysis in our patient displaying the junction between exons 7 and 9 on the reverse strand; (E) MFSD8 cDNA analysis in a control subject displaying both the junctions between exons 7–8 and 8–9.