The NF1 Gene Contains Hotspots for L1 Endonuclease-Dependent De Novo Insertion

Abstract

Long interspersed (L1) and Alu elements are actively amplified in the human genome through retrotransposition of their RNA intermediates by the ∼100 still retrotranspositionally fully competent L1 elements. Retrotransposition can cause inherited disease if such an element is inserted near or within a functional gene. Using direct cDNA sequencing as the primary assay for comprehensive NF1 mutation analysis, we uncovered in 18 unrelated index patients splicing alterations not readily explained at the genomic level by an underlying point-mutation or deletion. Improved PCR protocols avoiding allelic drop-out of the mutant alleles uncovered insertions of fourteen Alu elements, three L1 elements, and one poly(T) stretch to cause these splicing defects. Taken together, the 18 pathogenic L1 endonuclease-mediated de novo insertions represent the largest number of this type of mutations characterized in a single human gene. Our findings show that retrotransposon insertions account for as many as ∼0.4% of all NF1 mutations. Since altered splicing was the main effect of the inserted elements, the current finding was facilitated by the use of RNA–based mutation analysis protocols, resulting in improved detection compared to gDNA–based approaches. Six different insertions clustered in a relatively small 1.5-kb region (NF1 exons 21(16)–23(18)) within the 280-kb NF1 gene. Furthermore, three different specific integration sites, one of them located in this cluster region, were each used twice, i.e. NM_000267.3(NF1):c.1642-1_1642 in intron 14(10c), NM_000267.3(NF1):c.2835_2836 in exon 21(16), and NM_000267.3(NF1):c.4319_4320 in exon 33(25). Identification of three loci that each served twice as integration site for independent retrotransposition events as well as 1.5-kb cluster region harboring six independent insertions supports the notion of non-random insertion of retrotransposons in the human genome. Currently, little is known about which features make sites particularly vulnerable to L1 EN-mediated insertions. The here identified integration sites may serve to elucidate these features in future studies.

Identification of Cryptic Novel α-Galactosidase A Gene Mutations: Abnormal mRNA Splicing and Large Deletions

“…This overlapped 15 nucleotide element is known as a target site duplication (TSD) sequence. An exonic/intronic L1 insertion mutation has also been found in some other genetic diseases (Awano et al 2010;Kagawa et al 2014), immunity disorders (Brouha et al 2002), cancers (Wimmer et al 2011), and neurologic diseases (Martínez-Garay et al 2003). The L1 retrotransposon leads to splicing out of the exon 4 element in pre-mRNA maturation.…”

Intact piRNA pathway prevents L1 mobilization in male meiosis

“…In a few cases, specific retrotransposition events (i.e., insertions) have been determined to drive tumorigenesis (8,9). In the germline, retrotransposon insertions are responsible for sporadic cases of human genetic diseases, including hemophilia A (10) and neurofibromatosis 1 (11).…”

The Minimal Active Human SVA Retrotransposon Requires Only the 5′-Hexamer and Alu-Like Domains

“…These active L1 loci are the source for new L1 insertions and can provide the L1 proteins required to mobilize other RNAs (Alu [7,19], SVA elements [37,65,67], U6 [9,27,31], and processed pseudogenes [23,82]) in trans. Retrotransposition is ongoing in the human population (4,6,24,(41)(42)(43)46), with almost 100 cases of single-gene disease (5,11,39,83) caused by L1 (47), Alu (80), SVA (50, 65), or poly(A) insertions. L1-mediated insertions may be deleterious by disrupting mRNA expression (32, 76) of a specific gene (ϳ25 examples [39,78]) or mitigating nonallelic homologous recombination (18,35,71).…”