Evidence consistent with human L1 retrotransposition in maternal meiosis I

Abstract

We have used a unique polymorphic 3' transduction to show that a human L1, or LINE-1 (long interspersed nucleotide element-1), retrotransposition event most likely occurred in the maternal primary oocyte during meiosis I. We characterized a truncated L1 retrotransposon with a 3' transduction that was inserted, in a Dutch male patient, into the X-linked gene CYBB, thereby causing chronic granulomatous disease. We used the unique flanking sequence to localize the precursor L1 locus, LRE3, to chromosome 2q24.1. In a cell culture assay, the retrotransposition frequency of LRE3 is greater than that for any other element that has been tested to date. The patient's mother had two LRE3 alleles that differed slightly in the 3'-flanking genomic DNA. The patient had a single LRE3 allele that was identical to one of the maternal alleles; however, the patient's insertion matched the maternal LRE3 allele that he did not inherit. Other data indicate that there is only a small chance that the father (unavailable for analysis) carries the precursor LRE3 allele. In addition, paternal origin of the insertion would have required that an LRE3 mRNA transcribed before meiosis II be carried separately from its precursor LRE3 allele in the fertilizing sperm. Since the mother carries a potential precursor allele and the insertion was on the patient's maternal X chromosome, it is highly likely that the insertion originated during maternal meiosis I.

Figure 1

Insertion analyses. A, CYBB-specific Southern blot with EcoRI-digested DNA—from an unrelated control individual, the patient's mother, and the patient—demonstrating the shift, in the patient's sample, of the exon 4–containing fragment. The sizes of the wild-type bands are given; the arrow (←) indicates the aberrant band. The probe was constructed with coding cDNA of CYBB as template. B, Expand Long Template PCR of exon 4, in the patient, the patient’s mother, and an unrelated control. The arrows (→) indicate markers in the ladder.

Figure 2

Insertion sequence analyses. A, Schematic representation of the CYBB insertion. PT = important sequence of a PCR product from the patient’s exon 4. B, CYBB-insertion sequence alignments. CYBB = GenBank sequence at the point of insertion, into exon 4, of CYBB; PT = sequence of a PCR product from the patient’s CYBB exon 4 (which includes the disease-causing insertion); LRE3 = sequence of a PCR product from the patient’s chromosome 2q24.1; L1_RP = GenBank sequence of the most active element known (the nucleotide number of the adjacent base is given in parentheses). A possible 2-bp TSD in the disease-causing insertion is underlined; differences between sequences are shaded; leader dots indicate sequence that is consistent with the L1_RP sequence.

Figure 3

Expand Long Template PCR with flanking primers, demonstrating the presence or absence of LRE3 in the patient's mother, in the patient, and in an unrelated heterozygote. All bands were sequenced, to confirm findings.

Figure 4

LRE3 sequence analyses. A, Schematic representation of the LRE3 insertion into chromosome 2q24.1. LRE3 = important sequence from LRE3. Potential TSDs are underlined; asterisks (*) are placed at locations where differences exist between LRE3 and L1_RP. The larger asterisk indicates an amino acid change. B, LRE3 sequence alignments. CHR2 = GenBank sequence's “empty site” at the LRE3 locus; LRE3 = sequence of a PCR product from both the patient’s chromosome 2q24.1 and the patient's mother’s chromosome 2q24.1; L1_RP = GenBank sequence of the most active element known (the nucleotide number of the adjacent base is given in parentheses). A possible 28-bp TSD of LRE3 is underlined; nucleotide differences are shaded; leader dots indicate sequence that is consistent with the L1_RP sequence.

Figure 5

Example of flow-cytometry results. A live/dead gate was set by use of forward and sideward scatter of cells, and ∼10,000 live cells were assayed for fluorescence. Cells within the M1 marker were counted as positive. The M1 marker was zeroed using a single JM111 clone (L1_RP, with two disabling ORF1 point mutations) and a single L1_RP clone (not shown) that lacked the 3,832-bp AflII fragment. Both negative controls were assayed in triplicate; one L1_RP clone was assayed in triplicate, as a positive control; two LRE3 clones, one derived from maternal genomic DNA and another derived from patient genomic DNA, were assayed in triplicate. Numbers give the average number, over three assays, of positive cells for each clone ± 1 SD.

Figure 6

Assay and gel analyses. A, Schematic representation of the polymorphism assay that was used to determine the absence or presence of an element. B, Sample gel, showing results from 20 individuals of diverse ethnic backgrounds.