Clinical interpretation of variants identified in RNU4ATAC, a non-coding spliceosomal gene

Abstract

Biallelic variants in RNU4ATAC , a non-coding gene transcribed into the minor spliceosome component U4atac snRNA, are responsible for three rare recessive developmental diseases, namely Taybi-Linder/MOPD1, Roifman and Lowry-Wood syndromes. Next-generation sequencing of clinically heterogeneous cohorts (children with either a suspected genetic disorder or a congenital microcephaly) recently identified mutations in this gene, illustrating how profoundly these technologies are modifying genetic testing and assessment. As RNU4ATAC has a single non-coding exon, the bioinformatic prediction algorithms assessing the effect of sequence variants on splicing or protein function are irrelevant, which makes variant interpretation challenging to molecular diagnostic laboratories. In order to facilitate and improve clinical diagnostic assessment and genetic counseling, we present i) an update of the previously reported RNU4ATAC mutations and an analysis of the genetic variations affecting this gene using the Genome Aggregation Database (gnomAD) resource; ii) the pathogenicity prediction performances of scores computed based on an RNA structure prediction tool and of those produced by the Combined Annotation Dependent Depletion tool for the 285 RNU4ATAC variants identified in patients or in large-scale sequencing projects; iii) a method, based on a cellular assay, that allows to measure the effect of RNU4ATAC variants on splicing efficiency of a minor (U12-type) reporter intron. Lastly, the concordance of bioinformatic predictions and cellular assay results was investigated.

Deficiency of the minor spliceosome component U4atac snRNA secondarily results in ciliary defects

“…Some of the resulting domains such as Stem II and 5’ Stem-Loop bind essential splicing proteins. Most TALS patients carry homozygous or compound heterozygous mutations in the 5’ Stem-Loop, while all RFMN patients carry one of their two variants in Stem II, never found mutated in TALS (Benoit-Pilven et al , 2020). The physiopathological links between RNU4ATAC variants and TALS/RFMN/LWS symptoms remain largely unknown.…”

“…was used as a negative control. For rescue experiments, constructions of human U4atac snRNA of WT or mutated (c.16G> A, c.51G> A and c.55G> A) sequences were previously described(Benoit-Pilven et al, 2020). snRNA molecules were synthesized using a PCR amplicon as template (forward primer containing the T7 promoter (in italics): 5'-TAATACGACTCACTATAGGGAACCATCCTTTTCTTGGGGTTG-3' and the mMESSAGE mMACHINE™ T7 Transcription Kit (ThermoFisher Scientific), followed by a purification using RNA Clean and Concentrator kit (Zymo Research).…”

Inhibition of minor intron splicing reduces Na+ and Ca2+ channel expression and function in cardiomyocytes

“…So far, no cardiac disease has been linked directly to minor intron splicing. Mutations causing loss-of-function of the minor spliceosome are responsible for rare and complex syndromes, mainly affecting growth and the nervous system ( Benoit-Pilven et al, 2020 ; Doggett et al, 2018 ; Merico et al, 2015 ; Verma et al, 2018 ). However, some MOPD1/TALS and Roifman syndrome patients (caused by mutations in RNU4ATAC ) present with cardiac defects, including coarctation of aorta, Tetralogy of Fallot, atrial septal defect, ventricular septal defect and non-compaction of the heart.…”

Whole genome sequencing identifies pathogenic RNU4ATAC variants in a child with recurrent encephalitis, microcephaly, and normal stature

“…Of note, RNU4ATAC is located at 2q14.2, corresponding to the 11 Mb region of homozygosity identified by chromosomal microarray, but was not pursued given the absence of short stature or evidence of a skeletal dysplasia (Figure 2). RNU4ATAC (NR_023343.1) n.55G>A has been reported in multiple affected individuals in the literature in both the compound heterozygous and homozygous state (Benoit‐Pilven et al, 2020; Pierce & Morse, 2012). This variant was rare, seen in 0.007% of ostensibly healthy controls in gnomAD, and occurred at a conserved nucleotide in the 5′ stem loop of the RNA encoding gene (GERP 5.04).…”

“…Epilepsy and neuroendocrine dysfunction have also been documented (Pierce & Morse, 2012). Early death, within the first 2 years of life, has been reported in 70% of published cases (Benoit‐Pilven et al, 2020), with several patients reported to show profound clinical decompensation at the time of an intercurrent, febrile illness (Abdel‐Salam et al, 2012, 2013). In two cases, encephalitis was diagnosed (Abdel‐Salam et al, 2012); however, the acute findings on MR imaging were not characterized.…”