Prdm9 Controls Activation of Mammalian Recombination Hotspots

Nearly all rhLOF genotypes were found within autozygous segments (94.9%), and the mean number of rhLOF per individual was proportional to autozygosity.

“Nearly all rhLOF genotypes were found within autozygous segments (94.9%)(5), and the mean number of rhLOF per individual was proportional to autozygosity (fig. 1B).”

Autozygous segments were observed across all exomic sites with a density distribution not significantly different from random (Shapiro-Wilks P=0.112).

“Autozygous segments were observed across all exomic sites with a density distribution not significantly different from random (5)(Shapiro-Wilks P=0.112).”

In 100,000 subjects with first-cousin related parents of the same genetic ancestry, at least one knockout is expected in about 9,000 of the approximately 20,000 human protein-coding genes.

“On the basis of these observations we predict that in 100,000 subjects with first-cousin related parents of the same genetic ancestry we would expect at least one knockout in ~9,000 of the ~20,000 human protein-coding genes (fig. S3)(5).”

107 rhLOF genes were common to all three datasets.

“In total, 107 rhLOF genes were common to all three datasets (5)”

Exome sequencing of 3,222 British Pakistani-heritage adults with high parental relatedness identified 1,111 rare homozygous predicted loss-of-function genotypes in 781 genes.

“We sequenced the exomes of 3,222 British Pakistani-heritage adults with high parental relatedness, discovering 1,111 rare-variant homozygous genotypes with predicted loss of gene function (knockouts) in 781 genes.”

Matching 16,708 rare annotated loss-of-function variants to randomly selected synonymous variants of the same allele frequency, 842 rare loss-of-function variants had at least one homozygous genotype versus an average of 975.5 rare synonymous variants with at least one homozygote, indicating a deficit of 13.7% (95% CI 8-20%) of variants with rhLOF genotypes.

“We matched each of the 16,708 rare annotated LOF (heterozygous and homozygous) variants to a randomly selected synonymous variant of the same allele frequency, and observed 842 rare LOF variants with >= 1 homozygous genotype versus an average of 975.5 rare synonymous variants with >= 1 homozygote, indicating a deficit of 13.7% (95% confidence interval 8–20%) of variants with rhLOF genotypes (fig.”

Using epidemiological data from 13,586 mothers from the Born In Bradford birth cohort, the estimated lethal equivalents resulting in miscarriage, stillbirth or infant mortality per individual is 0.5.

“Using epidemiological data from 13,586 mothers from the same Born In Bradford birth cohort studied in our genetic analysis, we estimated 0.5 lethal equivalents resulting in miscarriage, stillbirth or infant mortality per individual in our population(5).”

The average load of recessive-lethal-equivalent loss-of-function variants per adult in the sampled population is 1.6.

“implying an average load of 1.6 recessive-lethal-equivalent LOF variants per adult.”

Overall, 1,111 rhLOF genotypes at 847 variants in 781 different protein-coding genes were identified in 821 individuals.

“Overall we identified 1,111 rhLOF genotypes at 847 variants (575 annotated as LOF in all GENCODE-basic transcripts) in 781 different protein-coding genes (fig. 1C)(5) in 821 individuals.”

A healthy adult mother with a predicted rare homozygous loss-of-function mutation in PRDM9 was identified and confirmed experimentally.

“we identified a healthy PRDM9 knockout mother”

The observed number of homozygous knockout genotypes was 13.7% lower than expected.

“We observed 13.7% fewer than expected homozygous knockout genotypes”

Knockouts were not associated with clinical consultation rate or prescription rate.

“knockouts were not associated with clinical consultation or prescription rate.”

PRDM9 is the major known determinant of the genomic locations of meiotic recombination events in humans and mice through its DNA binding site zinc finger domain.

“PRDM9 is the major known determinant of the genomic locations of meiotic recombination events in humans and mice through its DNA binding site zinc finger domain (6, 18, 19).”

18.5% of crossovers in the PRDM9 knockout duo (CI 1%-42%) occurred in linkage disequilibrium based hotspots versus 75.7% in the control duo (CI 57%-89%).

“Using similar methods we saw that 18.5% of crossovers observed in the PRDM9 knockout duo (confidence interval: 1% - 42%) occurred in linkage disequilibrium based hotspots versus 75.7% in the control duo (confidence interval 57%-89% consistent with a previously published estimate of an average of 60% of crossovers occurring at hotspots(18))(5).”

A similar reduction in molecular interactions was seen in the Icelandic data, in contrast to known pathogenic loss-of-function and gain-of-function variants which showed increased overall molecular interactions.

“We saw a similar reduction in the Icelandic data (table S4), in contrast to both known pathogenic LOF variants and pathogenic gain-of-function (GOF) variants reported in Orphanet, which showed increased overall molecular interactions (P=1.1 × 10−6, 2 × 10−12 respectively)(5).”

One subject was a healthy adult mother with a predicted rare homozygous loss-of-function mutation in PRDM9, confirmed experimentally.

“One of our subjects was a healthy adult mother with a predicted rare homozygous LOF mutation in PRDM9, which we confirmed experimentally(5)(fig. S7A, S7B).”

After quality control and annotation review, 16 of 54 rhLOF genotypes were considered possible genome annotation errors.

“We considered 16 of 54 rhLOF genotypes to be possible genome annotation errors (i.e. incorrectly described as LOF) (5)(table S2).”

Using DSB maps and a maximum likelihood model, only 5.9% (2 log unit CI 0-24%) of observed PRDM9 knockout duo maternal gamete crossovers matched DSB sites from wild type PRDM9-A allele homozygotes.

“Using double strand break (DSB) maps and a maximum likelihood model to account for variability in region size and hotspot density(18), we estimated that only 5.9% (2 log unit confidence interval: 0 - 24%) of the observed PRDM9 knockout duo maternal gamete crossovers matched DSB sites from wild type PRDM9-A allele homozygotes(5).”

By 10X Genomics long-range molecularly-phased whole-genome sequencing, 39 candidate crossovers were identified in the maternal gamete transmitted from the PRDM9 knockout mother to her child.

“We determined the locations of meiotic recombination in the maternal gamete transmitted from the mother to her child by 10X Genomics long-range molecularly-phased whole-genome sequencing and identified 39 candidate crossovers(5).”

Whether the mouse ortholog knockout is lethal is not associated with alteration of protein function, duplication or changes in gene expression.

“Whether or not the mouse ortholog knockout is lethal is not associated with alteration of protein function, duplication or changes in gene expression(5).”

rhLOF genes that are drug targets have an 11.4% phase I to approval rate versus 6.7% for all target-indication pairs, but there is no difference in the proportion of genes known or predicted to be druggable targets for rhLOF genes (15%) compared to all genes (13%).

“Furthermore rhLOF genes that are drug targets have 11.4% phase I to approval rate versus 6.7% for all target-indication pairs (chi-squared P=0.046), although we observed no difference in the proportion of genes known or predicted to be druggable targets(11) for rhLOF genes (15%) compared to all genes (13%, P=0.098)(5).”

The PRDM9-knockout duo did not show increased overlap of crossover intervals with promoters and their flanking regions or enrichment in GC content compared to the control duo.

“However we did not see an increased overlap in our PRDM9-knockout duo crossover intervals with promoters and their flanking regions or enrichment in GC content, compared to the control duo(5).”

Comparing individuals with one or more rhLOF (n=638) to those without rhLOF (n=1524), there was no association with prescription rate (OR 1.001, 95% CI 0.988-1.0144) or consultation rate (OR 1.017, 95% CI 0.996-1.038), nor any associations in rhLOF subgroups.

“We compared individuals with one or more rhLOF (n=638) to individuals without rhLOF (n=1524), and found no association with prescription rate (logistic regression, OR 1.001, 95% CI 0.988 - 1.0144) or consultation rate (OR 1.017, 95% CI 0.996 - 1.038), nor any associations in rhLOF subgroups (5).”