De novo intrachromosomal gene conversion from OPN1MW to OPN1LW in the male germline results in Blue Cone Monochromacy

X-linked cone dysfunction disorders such as Blue Cone Monochromacy and X-linked Cone Dystrophy are characterized by complete loss (of) or reduced L- and M- cone function due to defects in the OPN1LW/OPN1MW gene cluster. Here we investigated 24 affected males from 16 families with either a structurally intact gene cluster or at least one intact single (hybrid) gene but harbouring rare combinations of common SNPs in exon 3 in single or multiple OPN1LW and OPN1MW gene copies. We assessed twelve different OPN1LW/MW exon 3 haplotypes by semi-quantitative minigene splicing assay. Nine haplotypes resulted in aberrant splicing of ≥20% of transcripts including the known pathogenic haplotypes (i.e. ‘LIAVA’, ‘LVAVA’) with absent or minute amounts of correctly spliced transcripts, respectively. De novo formation of the ‘LIAVA’ haplotype derived from an ancestral less deleterious ‘LIAVS’ haplotype was observed in one family with strikingly different phenotypes among affected family members. We could establish intrachromosomal gene conversion in the male germline as underlying mechanism. Gene conversion in the OPN1LW/OPN1MW genes has been postulated, however, we are first to demonstrate a de novo gene conversion within the lineage of a pedigree.

directly sequenced with internal primers or re-amplified with exon-specific primers prior to sequencing. PCR products were purified by ExoSAP-IT treatment (Affymetrix, Santa Clara, CA) and cycle-sequenced applying BigDye Terminator V1.1 chemistry (Life Technologies, Darmstadt, Germany). For the determination of exon 3 haplotypes we amplified a 400 bp fragment using primers BCM-Ex3-F (5'-TGGTGGAAAGAAAGATGTCG-3') and BCM-Ex3-R (5'-GCCCAGAGAAAGGAAGTGATT-3') that was subsequently sequenced directly or after an intermitting cloning step if multiple mixed base positions were observed.
For subjects with multiple gene copies, the total number of LW/MW opsin genes was determined by means of real-time quantitative PCR (qPCR) with genomic DNA as template. We used two different TaqMan assays that target different parts of the LW/MW genes: the HS_01912094 assay (Life Technologies) targeting exon 6, and a custom-designed TaqMan assay (employing RGCP_TQF [5'-CCCAACAGAAAGCTGAAAGC-3'] as forward and RGCP_TQR [5'-GTGCAAAACTTTCGGATTGG-3'] as reverse primers, respectively, and RGCP_TQP [5'-CAGCCCGAGTCCTGCCATTGG-3'] with 5'-FAM and 3'-BHQ1 modifications as probe primer) targeting a common segment of intron 1. In parallel, we performed qPCR reactions for a human genome single copy reference sequence (RNaseP TaqMan Copy Number Reference Assay; Life Technologies). We used a series of male controls with defined LW/MW copy number [n=1-6; (4)] to generate a copy number dependent Ct calibration curve. All three assays were performed in triplicate for each sample. Obtained Ct values were used to infer copy numbers from the calibration curves.

Transfection and RNA extraction
HEK293 cells were routinely maintained in Dulbecco´s modified Eagle´s medium, DMEM (Gibco, Life Technologies) supplemented with 10% fetal bovine serum (FBS; Gibco, Life Technologies), Penicillin-Streptomycin (Sigma-Aldrich Chemie GmbH) at 100 µg/ml, and Amphotericin B (Biochrom GmbH) at 2.5 µg/ml. Cells were seeded in 6-well plates in DMEM with 10% FBS and the following day, at 80-90% confluency, cells were transfected with 4 µg DNA of the minigene construct using 20 µl Lipofectamine 2000 per well and OptiMEM® supplemented with GlutaMAX TM (Life Technologies) as diluents and medium. After 6 h incubation, cells were harvested by trypsinization with 0.05% Trypsine-EDTA (Gibco, Life Technologies), centrifuged at 1500 rpm for 5 min and transferred to a 6 cm dish with DMEM supplemented with 10% FCS and antibiotics. 24 h post-transfection, cells were lysed and total RNA was extracted applying the peqGOLD Total RNA Kit (PEQLAB Biotechnologie GmbH).

RT-PCR and relative quantification
First strand cDNA synthesis was performed using 2 µg of total RNA and random hexamer primers, according to manufacturer's instructions (Transcriptor First Strand cDNA Synthesis Kit, Roche).
Subsequent PCR was performed with a 5' FAM (6-carboxyfluorescein) labeled forward primer, FEO35: 5'-ACCATGAAGTTCAAGAAGCT-3', and O4-104-Rv:5'-AGCAGGTGACCATGAGGA-3' as reverse primer and using the QIAGEN Multiplex PCR Kit reagent chemistry (Qiagen) including 1/10 volume of Qsolution. Cycling conditions were 95°C for 15s, 40 cycles of 94°C for 30s, 60°C for 90s and 72°C for 45 to 60s, and a final extension at 72°C for 7 min. PCR products were separated by electrophoresis on a 2% agarose gel. FAM-labeled RT-PCR products were diluted 1:10 in water and mixed with 1 µl of GeneScan ROX500 size standard (Life Technologies) and 8 µl of Hi-Di Formamide (Life Technologies) in a total volume of 10 µl. Mixes were separated by capillary electrophoresis on an ABI 3130XL Genetic Analyzer instrument (Life Technologies). The area-under-the-curve (AUC) was calculated with GeneMapper 5 (Life Technologies) software. Ratios of splicing products were determined as the AUC for individual peaks divided by the sum of AUC of all differentially spliced products.

Microsatellite analysis
Centromeric (DXS8011, DXS8103, DXS1356, DXS8087) and telomeric (L441TA, L441CA, AF277A, AF277B and DXS1073) markers to the LW/MW cluster were used to genotype the three BCM72 family members (see Figure. 4A, Supplementary Table S2 and Supplementary Fig. S1). Primers were labeled either with TET, HEX or FAM. Standard PCR reactions were performed with 50-100 ng of genomic DNA. PCR products were diluted 2-10 fold with water, mixed with GeneScan ROX500 size standard and Hi-Di Formamide, and separated by capillary electrophoresis. GeneMapper 5 Software was used for allele calling. Markers for which the mother BCM72-II:1 was heterozygous were used to reconstruct haplotypes.

Mapping of the gene conversion event
LD-PCRs were performed for all three members of BCM72 using primers LCR1F and RGCP2-5R for the proximal gene copy, and E1G-F and RGCP2-5 for the distal copies. Upon AvrII-KpnI digestion, the resulting 7494 bp and 6211 bp fragments from LW (intron 1 to intron 5) and MW (intron 1 to intron 4) respectively, were purified with CHROMA SPIN+TE-1000 columns (Takara Bio Europe) and ligated to XbaI-KpnI double-digested pGEM3Zf(+). We selected two independent clones of the following gene copies for further analysis: (1) LW-derived clones bearing the 'LIAVA' haplotype from subjects BCM72-II:1 and BCM72-III:1, (2) MW-derived clones bearing the 'LIAVA' haplotype from subject BCM72-I:1, and (3) LWderived clones bearing the 'LIAVS' haplotype from subject BCM72-I:1. We sequenced exon 3 and flanking introns in these clones by means of primer walking.

Supplementary Tables and Figures
Supplementary  Figure S2: Mapping of the outermost borders of the gene conversion event in family BCM72. The maximal converted tract in family BCM72 is delimited by SNPs rs3788802 (c.409+949G>A) in intron 2 and rs69018729 (c.578+91G>A) in intron 3, for which the 'LIAVA' bearing MW gene copy in BCM72-I:1 differs from the LW gene copies in subjects BCM72-I:1, BCM72-II:2 and BCM72-III:1. Note that the gene conversion event converts the 'LIAVS' bearing LW gene copy in BCM72-I:1 into a 'LIAVA' bearing LW copy that is transmitted to the mother and the grandson.