Reported in vivo splice-site mutations in the factor IX gene: Severity of splicing defects and a hypothesis for predicting deleterious splice donor mutations

Abstract

Small consensus sequences have been defined for RNA splicing, but questions about splicing in humans remain unanswered. Analysis of germline mutations in the factor IX gene offers a highly advantageous system for studying the mutational process in humans. In a sample of 860 families with hemophilia B, 9% of independent mutations are likely to disrupt splicing as their primary mode of action. This includes 26 splicing mutations reported herein. When combined with the factor IX splice mutations reported by others, at least 104 independent mutations have been observed, 80 of which are single base substitutions within the splice donor and splice acceptor consensus sequences. After analysis of these mutations, the following inferences emerge: (1) the susceptibility of a splice donor sequence to deleterious mutation depends on the degree of similarity with the donor consensus sequence, suggesting a simple "5-6 hypothesis" for predicting deleterious vs. neutral mutations; (2) the great majority of mutations that disrupt the splice donor or splice acceptor sequences result in at least a 100-fold decrement in factor IX coagulant activity, indicating that the mutations at these sites generally function as an on/off switch; (3) mutations that create cryptic splice junctions or that shorten but do not interrupt the polypyrimidine tract in the splice acceptor sequence can reduce splicing by a variable amount; and (4) there are thousands of potential donor-acceptor consensus sequence combinations in the 38-kb factor IX gene region apparently not reduced by evolutionary selective pressure, presenting an apparent paradox; i.e., mutations in the donor and acceptor consensus sequences at intron/exon splice junctions can dramatically alter normal splicing, yet, appropriately spaced, good matches to the consensus sequences do not predispose to significant amounts of alternative splicing.

Comparative analysis detects dependencies among the 5′ splice-site positions

“…The 5Јss in metazoans provides nine potential positions for U1 snRNA:5Јss base-pairing. However, mutation analysis of the factor IX gene suggests that U1 snRNA:5Јss basepairing requires a minimal number of 5-6 Watson-Crick base pairs with U1 snRNA for mRNA splicing (Ketterling et al 1999). The idea of a minimal number of base pairs was also derived from another study (Zhuang and Weiner 1986).…”

U1-like snRNAs lacking complementarity to canonical 5′ splice sites

“…1D). This is in agreement with previous studies, which show that splicing requires the minimal number of six base pairs between U1A snRNA and the 59SS (Zhuang and Weiner 1986;Ketterling et al 1999;Carmel et al 2004;Freund et al 2005). Next, we used the same data set of human canonical introns to investigate possible base pairing interactions between the U1A5, U1A6, and U1A7 snRNA variants and 59SS sequences (Fig.…”

The human factor IX gene as germline mutagen test: Samples from mainland China have the putatively endogenous pattern of mutation

“…Although reliable clinical data on disease severity were generally not available for the Chinese sample, the presence of severe disease could be inferred from genotypic data. Mutations causing premature stop codons, frameshifts, certain mutations at splice junctions [Ketterling et al, 1999], and the four common missense mutations at CpG sites (R-4W, R-4Q, R-4L, and R180W) were classified as mutations known to cause severe disease. Based on these criteria, 41 of the 66 independent mutations were classified as severe mutations.…”