Pathogenic neurofibromatosis type 1 (NF1) RNA splicing resolved by targeted RNAseq.

Neurofibromatosis type 1 (NF1) is caused by loss-of-function variants in the NF1 gene. Approximately 10% of these variants affect RNA splicing and are either missed by conventional DNA diagnostics or are misinterpreted by in silico splicing predictions. Therefore, a targeted RNAseq-based approach was designed to detect pathogenic RNA splicing and associated pathogenic DNA variants. For this method RNA was extracted from lymphocytes, followed by targeted RNAseq. Next, an in-house developed tool (QURNAs) was used to calculate the enrichment score (ERS) for each splicing event. This method was thoroughly tested using two different patient cohorts with known pathogenic splice-variants in NF1. In both cohorts all 56 normal reference transcript exon splice junctions, 24 previously described and 45 novel non-reference splicing events were detected. Additionally, all expected pathogenic splice-variants were detected. Eleven patients with NF1 symptoms were subsequently tested, three of which have a known NF1 DNA variant with a putative effect on RNA splicing. This effect could be confirmed for all 3. The other eight patients were previously without any molecular confirmation of their NF1-diagnosis. A deep-intronic pathogenic splice variant could now be identified for two of them (25%). These results suggest that targeted RNAseq can be successfully used to detect pathogenic RNA splicing variants in NF1.

Congenital methaemoglobinaemia in a 61-year-old patient with normal haemoglobin levels.

A 61-year-old Ghanaian woman presented with dizziness and low oxygen saturations whereupon a methaemoglobin level of 24.9% was obtained. Initially it was thought to be caused by an unknown toxin. However, failure to normalise spontaneously and a short recurrence following administration of methylene blue suggested a congenital cause. Subsequently a novel variant in the CYB5R3 gene, coding for Cytochrome b5 reductase, was demonstrated. Absence of polycythaemia prompted additional analysis for a concomitant haemoglobinopathy.

De novo and inherited mutations in the X-linked gene CLCN4 are associated with syndromic intellectual disability and behavior and seizure disorders in males and females.

Variants in CLCN4, which encodes the chloride/hydrogen ion exchanger CIC-4 prominently expressed in brain, were recently described to cause X-linked intellectual disability and epilepsy. We present detailed phenotypic information on 52 individuals from 16 families with CLCN4-related disorder: 5 affected females and 2 affected males with a de novo variant in CLCN4 (6 individuals previously unreported) and 27 affected males, 3 affected females and 15 asymptomatic female carriers from 9 families with inherited CLCN4 variants (4 families previously unreported). Intellectual disability ranged from borderline to profound. Behavioral and psychiatric disorders were common in both child- and adulthood, and included autistic features, mood disorders, obsessive-compulsive behaviors and hetero- and autoaggression. Epilepsy was common, with severity ranging from epileptic encephalopathy to well-controlled seizures. Several affected individuals showed white matter changes on cerebral neuroimaging and progressive neurological symptoms, including movement disorders and spasticity. Heterozygous females can be as severely affected as males. The variability of symptoms in females is not correlated with the X inactivation pattern studied in their blood. The mutation spectrum includes frameshift, missense and splice site variants and one single-exon deletion. All missense variants were predicted to affect CLCN4's function based on in silico tools and either segregated with the phenotype in the family or were de novo. Pathogenicity of all previously unreported missense variants was further supported by electrophysiological studies in Xenopus laevis oocytes. We compare CLCN4-related disorder with conditions related to dysfunction of other members of the CLC family.