Broadening the phenotypic and molecular spectrum of FINCA syndrome: Biallelic NHLRC2 variants in 15 novel individuals

FINCA syndrome [MIM: 618278] is an autosomal recessive multisystem disorder characterized by fibrosis, neurodegeneration and cerebral angiomatosis. To date, 13 patients from nine families with biallelic NHLRC2 variants have been published. In all of them, the recurrent missense variant p.(Asp148Tyr) was detected on at least one allele. Common manifestations included lung or muscle fibrosis, respiratory distress, developmental delay, neuromuscular symptoms and seizures often followed by early death due to rapid disease progression.Here, we present 15 individuals from 12 families with an overlapping phenotype associated with nine novel NHLRC2 variants identified by exome analysis. All patients described here presented with moderate to severe global developmental delay and variable disease progression. Seizures, truncal hypotonia and movement disorders were frequently observed. Notably, we also present the first eight cases in which the recurrent p.(Asp148Tyr) variant was not detected in either homozygous or compound heterozygous state.We cloned and expressed all novel and most previously published non-truncating variants in HEK293-cells. From the results of these functional studies, we propose a potential genotype-phenotype correlation, with a greater reduction in protein expression being associated with a more severe phenotype.Taken together, our findings broaden the known phenotypic and molecular spectrum and emphasize that NHLRC2-related disease should be considered in patients presenting with intellectual disability, movement disorders, neuroregression and epilepsy with or without pulmonary involvement

Dynamics of Co-Transcriptional Pre-mRNA Folding Influences the Induction of Dystrophin Exon Skipping by Antisense Oligonucleotides

Antisense oligonucleotides (AONs) mediated exon skipping offers potential therapy for Duchenne muscular dystrophy. However, the identification of effective AON target sites remains unsatisfactory for lack of a precise method to predict their binding accessibility. This study demonstrates the importance of co-transcriptional pre-mRNA folding in determining the accessibility of AON target sites for AON induction of selective exon skipping in DMD. Because transcription and splicing occur in tandem, AONs must bind to their target sites before splicing factors. Furthermore, co-transcriptional pre-mRNA folding forms transient secondary structures, which redistributes accessible binding sites. In our analysis, to approximate transcription elongation, a “window of analysis” that included the entire targeted exon was shifted one nucleotide at a time along the pre-mRNA. Possible co-transcriptional secondary structures were predicted using the sequence in each step of transcriptional analysis. A nucleotide was considered “engaged” if it formed a complementary base pairing in all predicted secondary structures of a particular step. Correlation of frequency and localisation of engaged nucleotides in AON target sites accounted for the performance (efficacy and efficiency) of 94% of 176 previously reported AONs. Four novel insights are inferred: (1) the lowest frequencies of engaged nucleotides are associated with the most efficient AONs; (2) engaged nucleotides at 3′ or 5′ ends of the target site attenuate AON performance more than at other sites; (3) the performance of longer AONs is less attenuated by engaged nucleotides at 3′ or 5′ ends of the target site compared to shorter AONs; (4) engaged nucleotides at 3′ end of a short target site attenuates AON efficiency more than at 5′ end

Identification and analysis of antisense RNA target regions of the human immunodeficiency virus type 1.

Antisense RNA, transcribed intracellularly from constitutive expression cassettes, inhibits the replication of the human immunodeficiency virus type 1 (HIV-1) as demonstrated by a quantitative microinjection assay in human SW480 cells. Infectious proviral HIV-1 DNA was co-microinjected together with a fivefold molar excess of plasmids expressing antisense RNA complementary to a set of ten different HIV-1 target regions. The most inhibitory antisense RNA expression plasmids were targeted against a 1 kb region within the gag open reading frame and against a 562 base region containing the coding sequences for the regulatory viral proteins tat and rev. Experimental evidence is presented that the antisense principle is the inhibitory mechanism in this assay system

In vitro selection of fast-hybridizing and effective antisense RNAs directed against the human immunodeficiency virus type 1.

The rate of double strand formation between procaryotic antisense RNA and complementary RNA in vitro is known to correlate with the effectiveness of antisense RNA in vivo. In this work, an in vitro assay for determining the hybridization rates of a large number of antisense RNA species was developed. A set of HIV-1-directed antisense RNAs with the same 5'-end but successively shortened 3'-ends was produced by alkaline hydrolysis of a 150 nt HIV-1-directed antisense transcript. This mixture was used to determine hybridization rates for individual chain lengths with a complementary HIV-1-derived RNA in vitro. The second order binding rate constants of individual antisense RNA species differed by more than a factor of 100, although in some cases, slow-hybridizing and fast-hybridizing antisense RNAs differed by only two or three 3'-terminally-located nucleotides. The results indicated that there was not a trivial dependence of binding rates on the chain length of antisense RNAs. Further, the binding rate constants determined in vitro for individual antisense RNA species correlated with the extent of inhibition of HIV-1 replication in vivo