UGT1A1 Variants c.864+5G>T and c.996+2_996+5del of a Crigler-Najjar Patient Induce Aberrant Splicing in Minigene Assays

Abstract

A large fraction of DNA variants impairs pre-mRNA splicing in human hereditary disorders. Crigler-Najjar syndrome (CNS) is characterized by a severe unconjugated hyperbilirubinemia caused by variants in the UGT1A1 gene. We previously reported one CNS-type II patient with two splice-site variants in trans (c.864+5G>T and c.996+2_996+5del). According to MaxEntScan, both disrupt their corresponding donor sites (c.864+5G>T: 6.99 → 2.28; c.996+2_996+5del: 5.96 → −11.02), so they were selected for subsequent functional tests. Given the unavailability of patient RNA, we constructed an UGT1A1 splicing-reporter minigene with exons 1-4 to characterize the underlying splicing anomaly. The variant c.996+2_996+5del generated two aberrant transcripts, (E2) (exon 2 skipping/64%) and (E2q135) (intron retention of 135nt/36%), which lead to the loss of 18 conserved amino-acids and the gain of 45 new ones of a critical functional domain, respectively. The c.864+5G>T variant mainly produced the aberrant transcript (E1q141) (141-nt deletion/70.4%) and the full-length isoform (29.6%). (E1q141) would provoke the loss of 47 amino-acids of the N-terminal domain that encodes for substrate specificity. Thus, the three anomalous transcripts are likely to inactivate UGT1A1. Moreover, this patient is also homozygous for the promoter variant A(TA)7TAA that decreases UGT1A1 expression by 70%, so the fulllength transcript produced by c.864+5G>T would be even more reduced (<9%), thus supporting the diagnosis of CNS-type II. Therefore, minigenes represent valuable tools for the functional and clinical classifications of genetic variants.

Comprehensive Functional Characterization and Clinical Interpretation of 20 Splice-Site Variants of the RAD51C Gene

“…The functional study of variants provides critical data that may increase the number of families that may benefit from preventive or therapeutic measures. In this regard pSAD-derived minigenes have been proven as robust and high capacity approaches for the primary characterization of variant-associated defective splicing, since they replicate splicing results of patient RNA, as we have shown in RAD51C and other disease genes [57,[77][78][79]. By these means and the application of ACMG-AMP-based criteria, we have classified 15 RAD51C variants as pathogenic or likely pathogenic, which constitute the largest number of spliceogenic variants of this gene reported so far.…”

Modeling a Novel Variant of Glycogenosis IXa Using a Clonal Inducible Reprogramming System to Generate “Diseased” Hepatocytes for Accurate Diagnosis

“…The splice donor site consensus sequence is GT(A/G)AG. Intronic mutations of the type "+5G > T", such as the one described here, have been reported for various diseases [19][20][21][22][23] and have always been associated with aberrant splicing.…”

“…Mutations in these sequences are potential pathogenic variants that can disrupt splicing and consequently lead to disease. Despite the implementation of novel algorithms and deep-learning methods, predicting the effect of the variants, even for canonical splice sites, remains limited [ 19 ]. Therefore, RT-PCR of patient RNA samples is the most direct method to test for splicing.…”

Splicing Analysis of 16 PALB2 ClinVar Variants by Minigene Assays: Identification of Six Likely Pathogenic Variants

“…Finally, minigene assays allowed assessing more than 600 variants of the main breast cancer susceptibility genes up to now, demonstrating their high simplicity and robustness. Furthermore, this tool has been used to successfully assay variants at other disease genes, such as UGT1A1 (Crigler–Najjar syndrome) [ 51 ], CHD7 (Charge syndrome) [ 52 ], or TRPM4 (colorectal cancer) [ 53 ], among others ( , accessed on 13 July 2022).…”