30th April 2014
By Sarah Lindsay and Jackie MacArthur
Many rare genetic disorders are caused by deletions or duplications of portions of the genome in sperm and eggs before they go on to produce a child. There are several mechanisms that can cause this, but one of the best understood is non-allelic homologous recombination, which occurs when highly similar portions of the genome wrongly recombine, deleting and sometimes duplicating a portion of the genome that lies between them.
What factors influence the rate of the deletion? Are these factors inherited? We set out to answer these questions in our study.
The rates of this type of mutation are known to be very small, perhaps one mutation in every 30,000 sperm, so it is challenging to study the rate directly. Fortunately, males produce sperm cells in large quantities; in any one ejaculate there is a range of 40 to 800 million sperm, which means that we should be able to directly detect even extremely infrequent deletion events in a sample from a healthy man.
However, in order to find out whether any difference in rate that we see between individual males is inherited or merely due to chance, we needed to study the sperm from identical males, or twins. If we found that twins with the same DNA had very different rates of deletion we could be confident that environmental rather than genetic factors influenced the rate. We approached the TWINS UK study which was fortunately able to persuade some of its members to send us semen samples for our study.
However, detecting this extremely rare event in a sample of so many cells presented a technical challenge. For each donor, we screened between 500,000 and 1 million single sperm molecules using Polymerase Chain Reaction, a process that enables us to focus on and copy segments of DNA. We looked at rearrangements at a location on the DNA that has been linked to a clinical condition (Charcot-Marie-Tooth type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsies (HNPP)).
We detected a surprisingly variable range of non-allelic homologous recombination events from 1 event in every 10,000 molecules to 1 event in every 100,000 molecules depending on the donor. Unlike point mutation rates, which cause the replacement of a single base nucleotide and can lead to cancers, these rates were not correlated with the age of the donor, although they were highly correlated between twin pairs.
These findings suggest that the mutation rate between these repeats is largely genetically determined. Intriguingly, most of our donors share the same allele at the most likely candidate for local regulation (PRDM9), as well as almost perfect sequence identity in the repeats themselves. So the genetic determinant currently remains unknown.
Why don’t we see a paternal age effect in the mutation rate, like we do in other mutation processes? This is likely to be because different types of mutation occur at different stages of reproduction, and while sperm undergo many DNA replications, leading to an accumulation of mutations with age, the cellular processes involved in non-allelic homologous recombination occur only twice, the last immediately before sperm production.
These findings have important clinical implications; the rate heritability and absence of age effect are likely to apply to other disease loci where the causative agents are similar. However, they also raise some thought-provoking questions; are the factors influencing the rates of non-allelic homologous recombination genome-wide or locus-specific? Is there any possibility that environmental exposures immediately prior to sperm generation could also influence non-allelic homologous recombination rates?
In the future, developments in single-cell technologies may help to answer these questions by enabling us to study recombination and haplotype patterns directly in individual sperm.
Jackie MacArthur is currently a Scientific Curator at EMBL-European Bioinformatics Institute, working on the NHGRI GWAS Catalog and Locus Reference Genomic projects.
- Macarthur JA, Spector TD, Lindsay SJ, Mangino M, Gill R, Small KS, Hurles ME. (2014) The Rate of Nonallelic Homologous Recombination in Males Is Highly Variable, Correlated between Monozygotic Twins and Independent of Age. PLOS Genetics. DOI: 10.1371/journal.pgen.1004195
- Cooper TG et al. (2014) World Health Organization reference values for human semen. Human Reproduction Update. DOI:10.1093/humupd/dmp048
- Turner DJ, Miretti M, Rajan D, Fiegler H, Carter NP, Blayney ML, Beck S, Hurles ME. (2008) Germline rates of de novo meiotic deletions and duplications causing several genomic disorders. Nature Genetics. DOI:10.1038/ng.2007.40