Examining Human Meiotic Recombination Biology Essay

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The DNA sequence of every human being is unique. This is caused by mutation and recombination events that take place when gametes are formed. Mutations induce changes at a low frequency, whereas genetic recombination generates new variations in the sequence that will get inherited to the future generations. Meiotic recombination plays a key role in maintaining sequence diversity in humans. Influence of meiotic recombination on the sequence diversity will help in understanding the evolution of human DNA and human populations overall.

Meiotic recombination occurs between homologous chromosomes that pair and form visible chiasmata during Prophase I. This process is essential for accurate disjunction of chromosomes. Reduced recombination rates can result in aneuploidy (leading to miscarriages and disorders such as Down syndrome) (Lamb et al. 1996), whereas ectopic exchange (unequal crossover or homologous recombination between non-allelic copies) can lead to chromosomal rearrangements for example, individuals with glucocorticoid-remediable aldosteronism possess gene duplications that arise from ectopic recombination that causes hereditary hypertension (Lifton et al. 1992).

Meiotic recombination can also cause rearrangements in the genome and hence pave way for genomic disorders. This was first elucidated through locus-specific studies of common autosomal dominant peripheral neuropathies such as Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy with liability to pressure palsy (HNPP). Many other common and complex disorders are being shown to be due to CNV in some fraction of patients. Thus, genomic disorders encompass not only rare multiple congenital anomaly and mental retardation syndromes, but also common and complex traits, such as autism and schizophrenia, as well as other neurobehavioral phenotypes (Lupski 2009).

Examining Human Meiotic Recombination

The rate of recombination per nucleotide is calculated by comparing genetic and physical maps (Yu et al. 2001). Human physical maps have been built by means of cytogenetics, overlapping DNA clones and radiation hybrids. But the most accurate physical map is the nucleotide sequence itself. Genetic maps are used to estimate the distances between DNA sequences that vary between parental homologues (Yu et al. 2001). The primary unit of distance is centiMorgan (cM) and is equivalent to 1% recombination.

Genome-wide recombination rates were first compared by Yu et al. in 2001. They identified the fundamental features of the human genome by analyzing 188 meioses (Broman et al. 1998). They reported an overall female recombination rate of 1.7 cM/Mb and an overall male rate of 0.9 cM/Mb. Additionally, they reported the existence of 12 recombination "jungles" (regions of very high recombination, >3.0 cM/Mb) up to 6 Mb in length and 19 recombination "deserts" (regions of very low recombination, <0.3 cM/Mb) up to 5 Mb in length. They found out that as the distance away from the centromere increased, so did the male recombination rate. They were unable to find any such trend with the female recombination rate.

Human genome sequences are 99.9% identical to each other (Pääbo 2003). The variations are grouped together in regions called haplotype blocks along the length of the chromosome. These blocks are formed when recombination tends to occur at a higher rate in specific regions within the chromosome (Patil et al. 2001, Jeffreys et al. 2001, Daly et al. 2001).

Meiotic crossovers can be studied indirectly from population data by examining associations between alleles at adjacent loci. In the presence of high rates of recombination, there is free association between such alleles, in contrast in the absence of recombination, alleles at adjacent markers will have non-random association or show Linkage Disequilibrium (LD) that would reflect the long term history of a specific genome. Therefore, blocks of LD suggest that the region has been recombinationally inactive in the past. Recombination rates can be determined using several approaches. Cytogenetic and linkage studies have provided low resolution crossover data and spotted the presence of recombination hot domains. Cytogenetic analyses reveal that there has to be a minimum of one crossover per chromosome to ensure proper chromosome segregation - a phenomenon called obligate chiasmata. Molecular assays like sperm analysis are widely used to identify hotspots in a sequence (Ting et al. 2009).

Human Recombination Hotspots

In mammals, regions with a higher recombination rate than the flanking regions of the genomic DNA are termed "hotspots". In other terms, hotspots are regions where there is a local clustering of recombination showing a higher frequency of exchange than the flanking regions of DNA (May et al. 2007). A single variation within a hotspot can directly influence the initiation rates at double strand breaks and cause polymorphism in crossover activity (Neumann & Jeffreys 2006).

The first human crossover hotspot was identified at the MS32 minisatellite and this lead to the fact that human meiotic crossovers generally lie in very narrow hotspots (1-2kb) that constitute a very small part of the genome (Jeffreys et al. 1998). All human crossover hotspots have similar features - sperm crossovers are normally distributed throughout the region of the hotspot. Hotspots contain a particular 13-bp DNA motif (called Myer's motif), CCNCCNTNNCCNC, which is seen to be over represented in the region of the hotspot. This relates to crossover events that occur in at least 40% of the historical hotspots in humans, identified via analysis of the Phase 2 HapMap data (Myers et al. 2008). Prior to identifying this motif, Myers et al. identified a 7-mer motif, CCTCCCT from Phase I data (Myers et al. 2005).

Major turnover of hotspots has occurred within the last six million years ever since the human evolutionary lineage diverged from that of chimpanzees. These hotspot turnovers, gives rise to the possibility of hotspot polymorphisms within existing populations that arise from newly evolved hotspots and from hotspots that are on the verge of extinction. Population data from European Americans and African Americans suggest that the number of hotspots vary between populations (Crawford et al. 2004). These data, however, cannot detect polymorphism activity among individuals predicted from hotspot turnover. So, how can hotspot polymorphisms be detected?

Methods such as Sperm Typing help draw a line about the evolution of hotspot with the help of detecting crossover rate variation among men. Polymorphisms can be detected by two methods:

Detecting crossover frequency in sperm

Reciprocal crossover asymmetry test (Jeffreys & Neumann 2002) by which the organisation of exchange points can be studied and consequently test if they are dispersed in a different way across a hotspot. Till date, polymorphism has been depicted in about 8 of 19 hotspots analysed (Jeffreys et al. 1998, Neumann & Jeffreys 2006, Jeffreys & Neumann 2002, Jeffreys & Neumann 2005, Jeffreys et al. 2005).

A single base variation in a hotspot can cause polymorphism in crossover activity. For example, in the hotspot DNA2, an A/G polymorphism is located approximately 5 bp from the centre. The G variant is seen to be transmitted to 75% of heterozygous progeny. Homozygotes do not show asymmetry but the GG homozygotes exhibit lower crossover rates than AA homozygotes. Additionally, the G variant disrupts the CCTCCCT hotspot motif (Myers et al. 2005).

So, how are hotspots formed? A study was performed by observing a local area of hotspots. It was found that as one human hotspot dies out, the recombination rates of the hotspots nearby increases (Pineda-Krch & Redfield 2005). PC4-1a and PC4-1b are two hotspots that are present in chromosome 21, 3kb apart. Tiemann-Boege et al. analysed three men, out of whom, two were seen to have PC4-1a active and PC4-1b repressed. The third person had an active PC4-1b and a repressed PC4-1a. Neumann and Jeffreys, in 2006, identified two other hotspots that showed dissimilar effects to those of Tiemann-Boege et al. The hotspots - MSTM1a and MSTMS1b, being 2 kb apart from each other, showed very a unique occurrence in men. MSTM1b was seen to be active in all men tested; whereas MSTM1a was active in only 10% of men and inactive in the others tested. This hotspot thus gives a clear picture of the presence or absence of polymorphism in humans.

Human crossover hotspots are also active in gene conversion (Jeffreys & May 2004). This suggests that hotspots mark sites of recombination initiation, where initiation occurs due to double-strand DNA breaks, arising within a restricted zone at the centre of the hotspot (Jeffreys & May 2004, Qin et al. 2004).

PRDM9 Specifies Hotspots

The Myer's motif is characteristic of a zinc finger binding site (Myers et al. 2010). Computational analysis of zinc-finger binding proteins yielded PRDM9 as the most stable binding partner (Baudat et al. 2010). Bernard de Massy and Kenneth Paigen showed a correlation between Prdm9 alleles and hotspots in Mice. They identified a region on chromosome 17 of mice that functioned in trans to activate hotspots located far-away from each other. The focus was then shifted to a region containing four genes, one of which, Prdm9, was hypothesized to be involved in regulation of mammalian hotspots. They showed that differences in hotspots can be explained by differences in Prdm9 (Baudat et al. 2010, Parvanov et al. 2010). In the absence of Prdm9, meiotic arrest is seen to occur in mice (Hayashi et al. 2005). This is not due to the loss of hotspot activity suggesting that Prdm9 has a more intricate role in the control of meiotic crossover formation. This correlation holds true in humans as well, proving that the gene is a central regulator during mammalian crossover (Hochwagen & Marais 2010).

Absence of Prdm9 results in sterility. PRDM9 contains a conserved domain with H3K4 methyltransferase activity (Grey et al. 2009).

DNA diversity comparisons in humans and chimpanzees showed deviating LD landscapes at orthologous loci in spite of the 99% sequence identity (Winckler et al. 2005, Wall & Pritchard 2003, Ptak et al. 2005, Ptak et al. 2004). The subset of homologous regions with a conserved motif in both species that show elevated recombination rates in humans have normal recombination rates in chimps (Myers et al. 2010). Sequence analysis of PRDM9 in other mammals shows that zinc-finger domain numbers and sequences vary a lot among species (Oliver et al. 2009).

The PRDM9 gene is highly polymorphic. The polymorphic forms of PRDM9 bind to different DNA sequences and therefore initiate crossovers at several chromosomal sites. PRDM9 was identified through mouse and human studies and by computational analyses.

The PRDM9 gene encloses a carboxy-terminal domain, which comprises a set of C2H2-type zinc-finger repeats. If the zinc-finger domains of PRDM9 identify hotspots, then every PRDM9 allele should bind to hotspot motifs in a unique manner, and hence individuals should show unique recombination activity across the genome (Neale 2010).

Baudat et al. identified PRDM9 as the candidate gene and studied a human population. They found three allelic forms of Prdm9: A, B, and I on human chromosome 5. The most common form was A, comprising 94% of the alleles. The I allele was found to encode a protein that does not recognize the 13-mer hotspot motif. (Baudat et al. 2010).

The Human Pseudoautosomal Regions:

Recombination differs between sexes in humans, especially with respect to the sex chromosomes. In human females, both X chromosomes are identical physically and genetically and hence recombination can occur anywhere along the length of the chromosome. Whereas, in males, due to the presence of different chromosomes (X and Y), recombination seems to occur at very small regions of sequence identity that are located at the tip of short and long arms, the so called Pseudoautosomal regions or PARs (Fig.1). PAR1 is 2.6 Mb long and represents the single largest region of homology that remains between X and Y chromosomes and PAR2 covers 0.4 Mb in the chromosome's long arm and represents recent acquired homology between X and Y chromosomes via interspersed repeats that were present in non-homologous stretches of the sex chromosomes.

The PARs behave like autosomes but have a much higher recombination rate than the adjacent regions during male meiosis (Blaschke & Rappold 2006, Rappold 1993).

The recombination activity in PAR1 is extremely different between sexes. In men, it exhibits the highest recombination frequencies of the genome (Flaquer et al. 2008) - 20 fold greater than the genome average (Rappold 1993). The female recombination activity is within the autosomal range for PAR1 and PAR2. The PAR1 region at the tip of chromosomes Xp/Yp undergoes an obligate crossover at male meiosis, vital for pairing and correct disjunction. As a result, it serves as a male-specific recombination hot domain. The rate of male-specific recombination in PAR2 is 6 fold greater than the average (Blaschke & Rappold 2006).

Fig.1: Structure of human male sex chromosomes and the location of the PAR regions. The PAR1 lies on the tip of the short arms and is 2.6Mb long. PAR2 lies on the tip of the long arms and is 0.4Mb long.

A total of 28 genes have been identified within the PARs till date - 24 in PAR1 and 4 in PAR2. In the case of the PAR2 region, 4 genes have been identified to date: SPRY3, SYBL1, IL9R and CXYorf1. Of these, only SYBL1 and IL9R have a known function.

Recombination In PAR:

In mammals, pairing between the X and Y chromosomes was initially observed in the rat (Koller PC 1934). Studies on sex chromosomes during pre-metaphasic phase revealed regions of homology in the tips of the short arms of the X and Y chromosome (Müller & Schempp 1982). Arguments that a single crossover within these regions should exist between the chromosomes aroused and it was later found out that these regions would behave in a similar manner to an autosomal segment. Hence, these regions were called "Pseudoautosomal regions". Molecular studies of the sex chromosomes in humans and mice have subsequently proved the existence of certain markers that recombine between the sex chromosomes (Rappold 1993).

Pairing and synapsis between homologous chromosomes result due to physical contact during meiotic prophase, and result in the formation of a synaptenomal complex. Pairing of human chromosomes is seen to initiate at sites very close to the telomere (Laurie & Hultén 1985). Pairing of the sex chromosomes in male meiosis is initiated between the Xp and Yp regions,

but occasionally between Xq and Yq (Speed & Chandley 1990, Chandley et al. 1984).

Indeed in such cases, after synapsis of XpYp homologous segments (PAR1), the long arm of the X chromosome condenses and folds down to reach the tip of the long arm of the Y chromosome. However, such XqYq associations are usually transient, though it was discovered through linkage maps that exchanges occur in these regions (Armstrong et al. 1994).

Very little is known about the fine scale distribution of recombination in PAR2. We consider the PAR2 region in this project. Data from the International HapMap Project showed the presence of two peaks of historical recombination activity within the 330kb long PAR2 region. The most proximal is already being studied in the lab and by sperm analysis it has been shown to correspond to an active hotspot ~1.5kb wide in the vicinity of the HSPRY gene (Sarbajna & May, unpublished). The second peak identified by HapMap, which is the subject of project, is much broader than a typical single hotspot. The primary aim of this project is therefore to refine the population analysis using a higher density of SNP markers to see if the region resolves into two or more closely located historical recombination hotspots. A two-tier strategy for analysis of meiotic crossover hotspots in human DNA will be employed (Fig.2).

Fig: 2. Two-tier strategy for high resolution analysis of meiotic crossovers in human DNA. (a) A chosen target region is subjected to high-density SNP genotyping samples from 96 unrelated semen donor DNAs. The patterns of LD thus obtained will provide information about recombination events that have occurred in the past. (b) Semen samples are subjected to nested allele-specific PCR to selectively amplify recombinant molecules from the DNA. (Figure adapted from May, C.A., Slingsby, M.T. & Jeffreys, A.J. Human recombination hotspots: before & after the HapMap project. in "Genome Dynamics and Stability vol 3: Recombination and meiosis" eds. D. Lankenau & R. Egel, Springer Verlag (2007).

The first step involves SNP genotyping in the region of interest referring to dbSNP to identify additional markers in order to carry out a high resolution association analysis. Work will be carried out on 96 North-European semen donors. Whole genome amplification is carried out by using multiple displacement amplification (MDA) method (Dean et al. 2002) the products of which are used to genotype the sperm DNA by PCR amplifying the target region and subsequently hybridizing with allele-specific oligonucleotides (ASO). The PCR products are obtained by designing amplicons that overlap in regions containing high density dbSNP markers, and employing a nested PCR to achieve high yields from MDA DNA of the semen donors (Fig.3).

Fig 3: Allele-specific PCR design. The whole region is split into regions or amplicons and a nested PCR is done in such a way that regions containing high density dbSNP markers overlap, to make sure all markers are typed. The PCR products thus obtained are then dot-blotted and hybridised to enable genotyping.

Next, association analysis is carried out to detect the presence of Linkage Disequilibrium (LD) from which LD blocks are identified. Certain pair wise methods like D' and coalescent based methods like LDhot and metric linkage disequilibrium mapping will be performed. These blocks help in recognising the putative region of the hotspot. Three outcomes are possible. Firstly, LD may decrease uniformly denoting the random distribution of recombination events. Secondly, LD may be present between markers denoting extreme suppression of meiotic recombination. Thirdly, there may be separate blocks of LD freely associated with each other. These intervals may denote the presence of putative hotspots (Fig.4)

Fig.4: LD Analysis. Linkage Disequilibrium is the non random association of alleles at closely linked loci. LD analysis may result in the following: (i) The LD may decrease uniformly denoting the random distribution of recombination events. (ii) The LD may be present between markers denoting extreme suppression of meiotic recombination. (iii) There may be separate blocks of LD freely associated with each other. These intervals may denote the presence of putative hotspots.

The second step involves recovering of crossover molecules from the sperm DNA. Heterozygous SNP sites are targeted in an allele-specific PCR to selectively amplify recombinant molecules from the sperm DNA and crossover points are mapped by internally typing the SNPs again by ASO hybridization (Fig.2).

From the above approach (sperm crossover), the activity of the region within the male germ line can be ascertained. If it is found to be active, then the pattern of variation between the haplotypes of men remains a mystery. It can be assumed that variation may occur on the basis of polymorphisms within the haplotypes.