Saturation editing of RNU4-2 reveals distinct dominant and recessive disorders | Nature

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Subjects

- Disease genetics

- Mutagenesis

- Neurodevelopmental disorders

- RNA splicing

- Small RNAs

Abstract

Recently, de novo variants in an 18-nucleotide region in the centre of RNU4-2 were shown to cause ReNU syndrome, a syndromic neurodevelopmental disorder that is predicted to affect tens of thousands of individuals worldwide1,2. RNU4-2 is a non-protein-coding gene that is transcribed into the U4 small nuclear RNA component of the major spliceosome3. ReNU syndrome variants disrupt spliceosome function and alter 5′ splice site selection1,4. Here we performed saturation genome editing (SGE) of RNU4-2 to identify the functional and clinical impact of variants across the entire gene. The resulting SGE function scores, derived from variants’ effects on cell fitness, discriminate ReNU syndrome variants from those observed in the population and markedly outperform in silico variant effect prediction. Using these data, we redefine the ReNU syndrome critical region at single-nucleotide resolution, resolve variant pathogenicity for variants of uncertain significance and show that SGE function scores delineate variants by phenotypic severity and the extent of observed splicing disruption. Furthermore, we identify variants affecting function in regions of RNU4-2 that are critical for interactions with other spliceosome components. We show that these variants cause a new recessive neurodevelopmental disorder that is distinct from ReNU syndrome. Together, this work defines the landscape of variant function across RNU4-2, providing critical insights for both diagnosis and therapeutic development.

Main

The spliceosome is a large ribonucleoprotein complex that mediates RNA splicing. De novo variants in a gene encoding one of the small nuclear RNA (snRNA) components of the spliceosome, RNU4-2, were recently shown to cause ReNU syndrome, a prevalent neurodevelopmental disorder (NDD)1,2. ReNU syndrome is a complex multi-system disorder characterized by moderate to severe global developmental delay, intellectual disability, hypotonia, acquired microcephaly, speech and motor difficulties, low bone density and often seizures1,4.

RNU4-2 encodes the U4 snRNA, which is a critical component of the major spliceosome. In particular, U4 is tightly bound with the U6 snRNA in the U4/U6.U5 tri-small-nuclear ribonucleoprotein and the U4/U6 duplex needs to be unwound for activation of splicing3. Variants identified in individuals with ReNU syndrome cluster in an 18-nucleotide (nt) region in the centre of RNU4-2 that is depleted of variants in population datasets (the ‘critical region’, or CR)1. This region is known to accurately position U6 for recognition of the 5′ splice site. Consistent with this, variants causing ReNU syndrome have been shown to alter 5′ splice site usage1, with this disruption correlating with phenotype severity4. Similarly, variants in two distinct structures within the 18-nt CR (the T-loop and Stem III) have been proposed to differ in clinical severity4.

The precise relationship between genetic variation in RNU4-2 and clinical impact remains incompletely characterized. The variants initially characterized in individuals with ReNU syndrome are all within the 18-nt CR; however, more recent work has proposed a role for variants outside this region, in the 5′ stem loop5. It is unclear which, if any, variants outside the CR could also cause NDD. This is particularly important as the increased mutation rate of RNU4-2 and other snRNA genes means that there will be many chance occurrences of variants among sequenced individuals with syndromic NDD6. Up to 75% of individuals with ReNU syndrome have the same single-nucleotide insertion (n.64_65insT). Whether the high recurrence of this particular variant is due to ascertainment bias, germline selection and/or an increased mutation rate is at present unknown. Furthermore, it is unclear whether available variant effect predictors (for example, CADD7) can effectively distinguish between pathogenic and benign variants in RNU4-2.

Resolving these questions will be critical to ensure accurate, comprehensive diagnoses of individuals affected by ReNU syndrome. One approach to clarifying variant impact is through the generation of functional data of variant effect, which can mechanistically inform why specific variants cause disease and improve clinical interpretation of rare variants8. However, no experimental assay has yet been established to evaluate variants in RNU4-2, owing to its recent association with NDD.

Saturation genome editing (SGE) is a powerful approach to delineate genotype–phenotype relationships9. Crucially, it does not rely on variants being observed in an individual with or without disease. Instead, every possible variant across a gene or region can be engineered and the relative functional effects of each determined through a cellular readout. SGE experiments have been performed across numerous protein-coding genes, including BRCA110,