Mutations in the U4 snRNA gene RNU4-2 cause one of the most prevalent monogenic neurodevelopmental disorders

Abstract

Most people with intellectual disability (ID) do not receive a molecular diagnosis following genetic testing. To identify new etiologies of ID, we performed a genetic association analysis comparing the burden of rare variants in 41,132 noncoding genes between 5,529 unrelated cases and 46,401 unrelated controls. RNU4-2, which encodes U4 small nuclear RNA, a critical component of the spliceosome, was the most strongly associated gene. We implicated de novo variants among 47 cases in two regions of RNU4-2 in the etiology of a syndrome characterized by ID, microcephaly, short stature, hypotonia, seizures and motor delay. We replicated this finding in three collections, bringing the number of unrelated cases to 73. Analysis of national genomic diagnostic data showed RNU4-2 to be a more common etiological gene for neurodevelopmental abnormality than any previously reported autosomal gene. Our findings add to the growing evidence of spliceosome dysfunction in the etiologies of neurological disorders.

Fig. 1. Discovery and replication of RNU4-2 as an etiological gene for a novel neurodevelopmental disorder.

a, BeviMed PPA between RNU4-2 and the 100KGP Specific Disease ID. All other noncoding genes had a PPA < 0.5. Three RNU4-2 variants had a conditional PPP > 0.5. b, Probability distribution for the number of unexplained cases among 34 randomly selected ID cases in the 100KGP. The actual number of unexplained ID cases (34) among the 34 ID cases with one of the three RNU4-2 variants having a PPP > 0.5 is indicated with a red line. c. Distribution of phenotypic homogeneity scores (Methods) for randomly selected sets of 34 participants chosen from the 4,468 HPO-coded, unexplained, unrelated ID cases. The actual score for the 34 ID cases with one of the three RNU4-2 variants having a PPP > 0.5 is indicated with a red line. d, For each of the 141 bases of the RNU4-2 gene (Seq.), the number of participants with a rare allele at that position on the cDNA, stratified by affection status and inheritance information of the carried rare allele. The bases corresponding to the three variants with a PPP > 0.5 are in bold. These three bases and adjacent bases for which no unaffected participants carry a rare allele are highlighted. At each base, the number of distinct rare alternate alleles (NAA) observed in the 100KGP is shown. Underneath, the presence of an alternate allele in gnomAD is indicated by a filled circle (GAD). e, Pedigrees for participants with a rare alternate allele at one of the highlighted bases. Pedigrees used for discovery have a ‘G’ prefix and are labeled in black. NBR, 100KGP Pilot and GMS pedigrees used for replication have an ‘N’, ‘P’ or ‘S’ prefix, respectively, and are labeled in blue. The affection status for pedigree members who were not study participants was obtained from pedigree tables, except where the proband was assigned to a Specific Disease that was unrelated to neurodevelopmental disorders despite having an NDA (this applies to the nine grayed out members of G9, G17, G20 and G36). One variant was called homozygous for the reference allele in a parent but found to be mosaic by inspection of aligned WGS reads (#).

Fig. 2. Phenotypic characterization and prevalence of a novel neurological disorder.

a, Graph showing the is-a relationships among HPO terms present in at least half of the 46 NDA-coded RNU4-2 cases identified or significantly enriched among these 46 cases relative to 9,112 other unrelated NDA-coded participants of the 100KGP. Terms are shortened to remove ‘Abnormality of (the)’ or ‘Abnormal’ for conciseness. The significantly overrepresented terms are highlighted. For each term, the number of cases with the term and the percentage that number represents out of 46 is shown. For each overrepresented term, the proportion of NDA-coded participants that are not RNU4-2 cases with the term and the proportion of NDA-coded RNU4-2 cases with the term are represented as the base and the head of an arrow, respectively. b, Of the 9,112 NDA-coded cases in the 100KGP, the number solved through P or LP variants in a gene, provided at least 11 cases were diagnosed. In the case of RNU4-2, the number of NDA-coded cases with a rare variant in the highlighted region of Fig. 1d is shown instead of the number solved with P or LP variants. c, Of the 5,527 NDA-coded cases in the GMS, the number solved through P or LP variants in a gene, provided at least eight cases were diagnosed. In the case of RNU4-2, the number of NDA-coded cases with a rare variant in the highlighted region of Fig. 1d is shown instead of the number solved with P or LP variants.

Extended Data Fig. 1. Organization of the U4/U6 snRNA duplex within the tri-snRNP U4/U6.U5.

Human U4 snRNA is stabilized by three intramolecular stem loop motifs (ribonucleotides 20–52, 85–117 and 127–144) and maintains an inhibitory interaction with U6 snRNA through extensive intermolecular pairings involving U4 ribonucleotides within stem I (56–61), stem II (1–16), stem III (73–79) and the U4 quasi-pseudoknot (62 and 64). This region is further stabilized by interactions between U4 ribonucleotides 68–70 and the RNA-binding protein 42 (RBM42)^,, and with more extensive contacts between this region and U4/U6.U5 tri-snRNP-associated protein 1 (SNUT1) and U4/U6.U5 snRNP 27 kDa protein (SNRNP-27K). The U4 stem III, quasi-pseudoknot and associated RNA binding proteins determine the orientation of the U6 snRNA ACAGAGA loop, ensuring its accessibility for interaction with the 5′ splice site of target introns that is necessary for the catalytic activation of the spliceosome complex. The variants associated with neurodevelopmental disorder lie in the central region of the U4/U6 snRNA duplex (yellow boxes; bolded ribonucleotides represent those harboring variants with a PPP > 0.5), are predicted to disrupt Watson-Crick interactions within U4 stem III (n.76 C > T and n.77_78insT) or to disrupt configuration of the U4 quasi-pseudoknot (n.63 T > G; n.64_65insG; n.64_65insT; n65A>G; n.65_66insT; n.67 A > G and n.68_69insA) thereby de-stabilizing U4 snRNA interactions with U6 snRNA and with RNA binding proteins that are necessary for correct spliceosome function. The image represents the predicted U4/U6 duplex secondary structure with ribonucleotide numbering according to the canonical RNU4-2 and RNU6-1 reference sequences ENST00000365668.1 and ENST00000383898.1 respectively.

Extended Data Fig. 2. Mosaicism analysis.

For each of the eight rare variants carried by 100KGP participants in the highlighted region of Fig. 1c, truncated bar charts showing the distribution of the number of reads supporting the alternate allele. The participants called heterozygous for a given RNU4-2 rare variant are represented in red, while other participants are represented in gray. The embedded window in the panel for variant n.65 A > G shows the read pileups at this position in the heterozygous proband of pedigree G38 and his mother, who had a homozygous reference genotype call but is likely mosaic. The reads supporting the reference allele are in blue and those supporting the variant allele are in gray.

Extended Data Fig. 3. Expression of the gene encoding the U4 snRNA, RNU4-2, across tissues.

Boxplot of RNU4-2 log expression across adult tissues subjected to RNA-seq by the GTEx Consortium. The number of samples corresponding to each tissue type is shown in brackets. Brain tissue types are highlighted. TPM: transcripts per million. The lower, center and upper edges of the boxes respectively indicate the lower quartile, median and upper quartiles. Whiskers are drawn up to the most extreme points that are less than 1.5 times the interquartile range away from the nearest quartile.