Analysis of LDLR mRNA in patients with familial hypercholesterolemia revealed a novel mutation in intron 14, which activates a cryptic splice site

Abstract

Familial hypercholesterolemia (FH) is caused by a defective low-density lipoprotein receptor (LDLR), and 41000 mutations in LDLR have been identified. However, in some patients with clinically defined FH, no mutation can be detected within the exons and adjacent intronic segments of the LDLR. We have analyzed RNA extracted from blood samples of patients with clinically defined FH and identified an aberrantly spliced mRNA containing an 81-bp insert from intron 14. The aberrant splicing was caused by a novel intronic mutation, c.2140+86C4G, which activated a cryptic splice site. Although the cryptic splice site does not completely surpass the normal splice site, the mutation was found to cosegregate with high cholesterol levels in a family, which supports the notion that c.2140+86C4G causes FH. The insertion of 81 bp in LDLR mRNA encodes an in-frame insertion of 27 amino acids in the LDLR. However, the insertion was found to hamper LDLR activity by preventing the receptor from leaving the endoplasmic reticulum, probably because of misfolding of the protein. In patients with clinically defined hypercholesterolemia, despite normal results from sequencing of exonic regions of the LDLR gene, characterization of the LDLR mRNA might identify the underlying genetic defect.

The UCL low-density lipoprotein receptor gene variant database: pathogenicity update

“…Also, 11 variants within the first 12 bases of the intron/exon boundaries were found to be pathogenic by either inactivating the wild-type splice site resulting in exon skipping or by the creation of novel splice sites that were used preferentially. Furthermore, in vitro studies revealed that c.1359-31_1359-23delinsCGGCT resulted in the removal of the invariant adenine at the consensus splicing branch site in intron 9, causing retention of intron 9 and use of cryptic splice sites in exon 10,39 and c.2140+86C>G resulted in the creation and use of a novel splice site in intron 14 42. Thus, there is the potential for variants deep into the introns to be pathogenic, and as with the synonymous variants, some of these intronic variants may warrant further examination.…”

Alternative Spliced Variants as Biomarkers of Colorectal Cancer

“…For example, autosomal-dominant polycystic kidney disease (ADPKD), a heterogeneous genetic disorder, is caused by mutations in PKD1 [17] and PKD2 [18] genes triggering aberrant alternative splicing [19]. In fact, about 20-50% of disease-causing mutations in human genes affect pre-mRNA splicing [20,21], such as classical phenylketonuria (PKU) [22], duchenne muscular dystrophy [23], familial hypercholesterolemia [24], neurological disorders [25], and cancers, including multiple osteochondromas [26], breast cancer [27], prostate cancer [28], and other cancers. The connection between splicing aberrations and cancer risks is a hot spot in recent years, and atypical alternative splicing process or its products in cancer cells have a hopeful role in clinical treatment.…”

Low-density lipoprotein receptor mutational analysis in diagnosis of familial hypercholesterolemia

“…This alteration causes the insertion of 81 bp in LDLR mRNA and encodes for an in-frame insertion of 27 amino acids in the LDLR, preventing the receptor from leaving the endoplasmic reticulum, probably because of misfolding of the protein. This case highlights the importance of looking for mutations in sites not commonly studied specially in patients without an identifiable mutation in the three genes causing familial hypercholesterolaemia, but with a phenotype very suggestive of familial hypercholesterolaemia [39].…”

“…For instance, if only 20% or less of aberrant transcripts are detected, this alteration may be classified as benign, because at least 80% of LDLR activity is expected and 80% is the minimum considered to be necessary for normal LDL clearance [20 && ,42,43]. In fact, there are patients with splicing mutations who do present with a severe phenotype, whereas other carriers of such variants have mild phenotypes [39,40]. We postulate that the difference between these two different groups has to do with transcript quantification that can be determined by real-time PCR.…”