Aneuploidy PDF

**L1: Aneuploidy** #### **Miscarriage Rates and Risk Factors** - - - - - - - - - #### **Chromosome Abnormalities and Miscarriage** - - - #### **Meiosis and Chromosome Segregation** - - - - - #### **Molecular Mechanisms in Meiosis** - - - - -...

**L1: Aneuploidy** #### **Miscarriage Rates and Risk Factors** - - - - - - - - - #### **Chromosome Abnormalities and Miscarriage** - - - #### **Meiosis and Chromosome Segregation** - - - - - #### **Molecular Mechanisms in Meiosis** - - - - - - #### **Errors in Chromosome Segregation** - - - - - - - #### **Aneuploidy and Associated Syndromes** - - - - - - - #### **Mechanisms Underlying Aneuploidy** - - - - - - #### **Advanced Maternal Age and Aneuploidy** - - - - - - - #### **Screening and Detection** - - - - - - - - - Approximately 15% of all pregnancies are expected to spontaneously abort or miscarry. This figure is slightly lower for women aged under 30 (approximately 10%), and slightly higher in women aged between 35 to 39 (roughly 20%). For women aged over 45 - expected that approximately 50% of pregnancies will result in miscarriage. Many factors have been linked to pregnancy loss. Some of these factors are environmental such as smoking and drug abuse. Obesity has also been linked. Other factors include maternal chronic health conditions such as diabetes, high blood pressure and infection. However perhaps one of the biggest factors that contributes to pregnancy loss, particularly during the first trimester, is the inheritance of an abnormal complement of chromosomes. In fact, it is estimated that 50% of all spontaneous abortions have an abnormal complement of chromosomes. We will look at this issue in depth as it's important to understand the molecular basis of this for the module and the exam. The vast majority of abnormal chromosome complements in aborted pregnancies result from the abnormal inheritance of chromosomes, this is not always the case. Before we can visualise and discuss what's going wrong in these individuals, we must first understand what happens under normal circumstances. You must understand how chromosomes are segregated in meiosis - during gametogenesis. Meiosis is a specialized type of cell division. In humans meiosis reduces the diploid complement of chromosomes, by half -- 2n to 1n. Meiosis achieves this via a single round of DNA replication (which occurs during interphase), followed by 2 successive rounds of chromosome segregation. The events leading to and including the the first segregation are referred to as Meiosis I or MI for short, which is subdivided into discrete stages called prophase I (which consists of a highly conserved sequence of events named leptotene, zygotene, pachytene and dictyotene) followed by metaphase I and finally anaphase I when homologous chromosomes part company from one another as they are drawn to opposite poles of the cell by the spindle complex. A cell entering meiosis has 2 copies of each chromosome, one inherited from each parent. In the slide, maternally inherited chromosomes are shown in red, paternally inherited chromosomes are shown in green. When the cell entered S-phase, these chromosomes were replicated to generate sister chromatids, which start to become visible in prophase and remain visible through to metaphase. Sister chromatids are visible in some human G-banded preps as shown in the slide. The G-Banded metaphase cell in the slide was harvested from an amniocyte nucleus (sampled via amniocentesis) from a real PND patient. It's possible to see sister chromatids in some of the metaphase chromosomes. Like the 7 for example, and the 11s at the bottom of the spread. If you look carefully you should be able to see a faint grey line running along the axis of each chromosome -- which is more obvious in the pale bands of the long arm of each chromosome The "glue" that holds sister chromatids together along their length is called sister chromatid cohesion -- represented in the figure at the top of the slide as small dark blue circles between the chromatids. Sister chromatid cohesion is composed of ring like protein complexes shown one the following slide..... These protein complexes and the genes that encode them are highly conserved in eukaryotes. You can see in this figure how the structure is able to physically link the DNA duplexes of 2 sister chromatids. The complex links sister chromatids in mitosis and meiosis -- though there are subtle differences between these ring structures -- the major one being the replacement of Scc1 with Rec8 in meiotic complexes. Cohesin complexes are laid down behind replication forks, which allows sister chromatids to be zipped up as they are created. Cohesin links are not the only thing that are formed as replication proceeds. As DNA is replicated (in meiosis) a protein called Spo11 is recruited to sites of replication. This topoisomerase deliberately damages newly replicated DNA duplexes, by initiating double strand breaks -- which act as nucleation points and substrates for homologous recombination -- which stimulates the pairing of homologous chromosomes and the formation of the synaptonemal complex, which is the protein complex that holds homologous chromosomes together. This is the focus of the next slide.. The early stages of homologous recombination take place within the synaptonemal complex. Consider a homologous chromosome in prophase I. The first step of homologous recombination involves just one sister chromatid (which is a single DNA duplex -- that has just been synthesised by semi conservative replication). The duplex is broken by Spo11, which initiates DNA end resection to form single stranded DNA tracts which are able to invade a nearby DNA duplex with homologous sequence. There are a few such duplexes available, including the other sister chromatid of the same replicated-chromosome, and the 2 sister chromatids which make up the homolog. The synaptonemal complex, which is a unique structure to meiotic cells, encourages single stranded DNA tracts to invade a duplex in the homologous chromosome -- and this is an essential event for proper chromosome segregation. What happens next is still incompletely understood. The strand invasion intermediate (shown at the bottom of the slide) needs to undergo further processing to repair the damage created by Spo11. If this damage was not repaired- the cell would die, as a DNA double strand break is a lethal genetic insult. The repair pathway therefore splits into two streams. One stream resolves the repair intermediate at the bottom of the slide in a manner that leaves the chromatids of homologous chromosomes free of entanglements. This means that at the end of the pathway, sister chromatid cohesion links the sister chromatids of the paternal chromosome together -- and the same is true for the maternal sister chromatids. The other stream however repairs the break by covalently linking the chromatid that was first broken by Spo11 (shown in black) to the invaded chromatid of the homolog (shown in red). This creates a crossover, which means that sister chromatid cohesion from both homologs now hold the 2 homologous chromosomes together This provides a mechanism to physically link homologous chromosomes together in meiosis. Crossovers are essential for faithful segregation (aka disjunction), which in turn helps to ensure that gametes receive the correct complement of chromosomes and that the next generation of progeny are viable. If homologous chromosomes could not interact then life as we know it could not exist. Crossing over also permits genetic diversity of a species. Under certain experimental conditions the visible consequences of cross over events may be observed under the microscope -- these structures are called chiasmata - shown at the bottom of the slide. Look at this image carefully. You can see the axes of two homologous chromosomes quite clearly, each composed of 2 sister chromatids. I have used red and green arrows to keep the colour scheme consistent with the figure at the top of the slide. You also need to bear in mind that sister chromatid cohesion (the ringed links) are still holding the sisters together. Just take a moment to think about the forces at play here and what they are doing. Sister chromatids of the maternal homolog are held together by cohesin links along the length of the chromosome. The same is true for the paternal chromosome. The maternal and paternal chromosomes are therefore linked sister chromatid cohesion -- permitted by crossing-over. Without sister chromatid cohesion -- homologous chromosomes could simply untangle from one another and move independently of each other in the cell. Without crossing over there'd be no interaction between the homologs -- and again the maternal and paternal chromosomes would be free of one another, meaning they could segregate randomly. Dictyotene is the final stage of prophase I. For chromosomes to be segregated to opposite poles of the cell in a coordinated manner in anaphase I, the bivalents must be aligned on the metaphase plate to establish tension across the meiotic spindle. Only once tension has been achieved (which of course is dependent on the attachment of the spindle microtubule filaments to the kinetochores of chromosomes -- located at the centromere, as well as sister chromatid cohesion), can the homologous chromosomes be pulled to opposite poles of the cell. This coordinated segregation occurs by the destruction of a key component of the cohesin complex (Rec8) by the enzyme separase along the length of chromosome arms. This cleavage event opens the ringed clamp, which allows the chiasmata to resolve. The 2 homologous chromosomes untangle from one another and migrate to opposite poles of the cell. Meiosis then completes via a pathway that is very similar to mitosis -- called meiosis II. This process of chromosome segregation is also vulnerable to error -- and thus contributes to the production of unbalanced gametes but in a far more diminished capacity in comparison to MI errors (at least for maternal meiosis) -- and we'll look at why this is later. What you do need to be aware of now - is that faithful segregation of chromosomes in MII is dependent on two major factors. 1\. Sister chromatid cohesion around the centromere remains intact after MI. 2\. The appropriate number and positioning of crossovers must occur in MI, for MII to complete normally. Aneuploidy for every human chromosome has now been recorded, but only a small number of aneuploidies are viable. Trisomy for chromosome 21 is the most common viable human aneuploidy. This trisomy is of course seen in Down Syndrome, though the disease is actually caused by the gain of a critical region on 21q. This means that [trisomy] is not the only cause of down syndrome, but it does account for more than 95% of all cases. Down syndrome is detected in 1 in every 700 births approximately and is characterised by intellectual and developmental problems that range in severity from mild to severe (severity is heavily influenced by mosaicism -- the type of tissue affected and level of mosaicism). Apart from the typical morphological features of the disease that include a short neck, flattened face, protruding tongue, palpebral fissures, short stature and others -- the disease is also characterised by serious physiological complications including heart defects, obesity, and increased risk of dementia and autoimmune disorders. The disease also predisposes to a particular subtype of acute myeloid leukaemia, which has its own world health organization classification -- that acknowledges the unique origins of the malignancy. The relatively high incidence of Down Syndrome is reflected in the lethality of the genetic imbalance. Chromosome 21 is of course the smallest human autosome, which is likely to be a major factor towards the viability of the trisomy (in addition to the function of specific genes on the chromosome). In fact just over 1 fifth of clinically recognised pregnancies affected with +21, result in live birth. Trisomy for other autosomal chromosomes are far more lethal. For example 95% of conceptions affected with trisomy for chromosome 18 are expected to spontaneously abort. When this disease comes to term, individuals are affected by Edwards' Syndrome and the majority of newborns are only expected to survive for 24 hours or so. 99% of pregnancies affected with trisomy 13 are expected to spontaneously abort. The few pregnancies that come to term suffer from Patau Syndrome -- that limits the median life expectancy of individuals to only a few days. Due to a relatively gene poor nature of the Y chromosome, and X chromosome inactivation -- aneuploidy of the sex chromosomes also results in live birth. In fact the only viable human monosomy is even more prevalent in the population than some autosomal trisomies (autosomal monosomy is lethal in humans). Turner Syndrome (45,X) is the disease in question here and is detected in approximately 1 in every 2,000 births. Disomy/Trisomy for sex chromosomes results in genomic imbalance that is rather more tolerable than gains of autosomal chromosomes -- and this is reflected in the proportion of pregnancies affected by such an aneuploidy. Examples of this type of imbalance include Klinefelter Syndrome and Triple X Syndrome -- and these disorders are associated rather low levels of spontaneous abortion as you can see in the slide -- the vast majority of pregnancies with a sex chromosome disomy/(trisomy in the case of triple X) are likely to result in live birth. A study involving 10,000 clinically recognised pregnancies evaluated that around 30% of miscarriages were due to the gain and loss of the important chromosomes shown in the slide (21, 18, 13, X and Y). That's why pregnant women are offered screening for these common aneuploidies between 10 and 14 weeks gestation. The screening programme is called the combined test, which generates a numerical risk for a pregnancy being affected by a common aneuploidy, based on the nuchal translucency, which if you remember is the diameter of the fluid filled sac at the back of the neck of a foetus which is measured during routine ultrasound scans. And a blood test that measures the levels of 2 important hormones -- Pregnancy associated plasma protein A (PAPP-A) and free B-human chorionic gonadotropin. A risk of 1 in 150 or greater is defined as a positive screening result -- and this triggers further investigations that often include a genetic screen for the common aneuploidies. The precise nature of this test varies depending on where you live. Many labs offer free foetal DNA qPCR, others offer FISH, and most still use karyotyping to look for other abnormalities too. Aneuploidy and other genetic abnormalities are also picked later on in pregnancy -- after around 20 weeks following the observation of abnormal morphological features in routine ultrasound scans. Markers of interest include abnormal cardiac activity, kidney and bowel features and morphological abnormalities of the face, limbs and hands. There is an 11 point checklist that an ultrasonographer will work through during a typical scan. Specific abnormal markers include characteristic clenched fist (described as the first and 4^th^ fingers overlapping the middle 2 fingers) and rocker bottom feet are both markers of trisomy 18. Cyclopia and polydactyly (extra fingers) are common in +13 affected pregnancies A short thigh, arm and nose bones, are seen in prenatal cases of Down syndrome. These soft markers are risk factors for genetic disease. Some confer higher risks than others. However perhaps one of the best know risk factors linked to aneuploidy in pregnancy is advanced maternal age. Data backing up this observation is shown here. Most, human trisomies, are affected by increasing maternal age. The precise relationship varies slightly between chromosomes -- but as a general rule this is a key take home message for the module. The magnitude of the effect is shown in the graph. For women under the age of 25 years \~2% of all clinically recognized pregnancies are trisomic. But for women aged 40 or over the proportion of pregnancies affected by trisomy increases to around 30%. For aneuploid pregnancies -- it's possible to identify the origin of a chromosomal imbalance, by characterising highly polymorphic regions of the genome on the chromosome in question, and comparing the identify of these loci, with parental homologs -- represented by the diagram on the right of the slide. The numbers represent the identity of a high polymorphic region of the chromosome. If we were now to perform the same analysis on maternal and paternal chromosomes -- we are able to identify the parental origin of the extra chromosome.. which in this case was the mother. This chart shows the parental origin of the extra chromosome 21, in live born cases of down syndrome. You can see that in almost all cases, the extra chromosome was inherited maternally (shown by the red and green series in the chart). The paternal contribution is shown in blue. There are 2 other things in this graph that I'd like you to take away. Firstly -- the maternal age trend is clearly evident, as the rate of Down syndrome increases with age. But it's also possible to identify at which point in meiosis the error occurs. This can be done by looking at polymorphic regions very close to the centromere, where a crossover is unlikely to have occurred, which means that in these regions -- it's very likely that the allelic identity is preserved. This analysis clearly shows that the vast majority of chromosome segregation errors occurred in female meiosis I, followed by female meiosis II. Paternal chromosome mal-segregation is a relatively minor factor, so to is the contribution from mitotic non-disjunction, which refers to errors in chromosome segregation after fertilisation -- in the foetus. So what is going on -- what is causing these trends? Well if the same type of analysis that was used to identify paternal/maternal, MI and MII origin is applied to the entire length of chromosome arms -- it's possible to map how many crossovers occurred between chromosomes -- that data is shown on the following slide. This chart shows the genetic map of various trisomic chromosomes. The greater the number of crossovers between homologous chromosomes, the longer the map (measured in centimorgans) the taller the bar.... The "normal/standard" map is represented by the blue bars for chromosomes 15, 16, 18, and 21. The chart shows that extra chromosomes, that were inherited as a result of an MI non-disjunction -- the genetic map of the chromosomes is shorter than it should be in all cases. This data means that a reduction in the number of crossovers between homologous chromosomes in MI -- is likely to be a major cause of aneuploidy.. And this of course makes sense -- as crossovers are needed to hold homologous chromosomes together. What's also interesting is that too many crossovers in MI seems to explain MII non-disjunction errors. This data indicates that the number of crossovers in MI is very important for chromosomes to segregate correctly. But there are other observations too. In cases where there very few or perhaps only 1 crossover linking homologous chromosomes -- the POSITION of the crossover is very important. If the crossover is located too close to the telomere (i.e. distally to the centromere) that the two homologs are more likely to mal segregate in MI. If the position of crossovers are too close to the centromere -- then MII errors are more likely. So lets try to pull this data together and try to make sense of it. Consider chromosome 21 during meiosis I -- represented here. The rings represent sister chromatid cohesion. The chromosomes are paired at the metaphase plate and Spo11 induced DSBs have been repaired -- and 1 of them has resulted in a crossover. This crossover tethers one homolog to the other and this allows the mitotic spindle -- which has attached to the kinetochore of each homolog -- to exert tension across the bivalent. Think about this tension and where force is being exerted.. To do this imagine pulling the homologs apart from the centromere, as tension builds up the centromere begin to move apart until the force exerted by the meiotic spindle is equal to the force holding the bivalent together = sister chromatid between the crossover and the telomere. You can see clearly that if there was no crossover structures to hold the homologs together -- then it wouldn't be possible to segregate the chromosomes correctly, as each is free to move independently of the other -- meaning that there'd be a 50% chance of both segregating to the same pole, leading to a disomic gamete and most likely and aneuploid conception. Now look at the situation when a single distal crossover is present. This reduces the amount of sister chromatid cohesion that holds the chromatids together -- and this is likely to explain why these Crossovers seem to predispose to MI non-disjunction. With this model in mind it's now possible to provide a model to help explain why advanced maternal age is associated with aneuploidy. There are important sex-specific differences in the timing, duration and outcome of gametogenesis. In males, meiosis begins and ends in a single continuous process. However in females, meiotic processes being during foetal development, and just before birth the oocytes enter a period of extended arrest that lasts for many years. During this arrest, chromosomes are held together in bivalents, which of course are dependent on chiasmata and sister chromatid cohesion. It's likely that during this arrest, these structures become damaged and this leads to mal segregation events. Consider to the situation of the far right -- an abnormal bivalent -- which is abnormal with respect to 2 factors. Firstly there is only a single crossover holding the homologs together -- under normal circumstances it likely for homologs to be held by more than 1. This single crossover is also positioned abnormally in the bivalent, being too close to the telomere. This means that any damage or degradation to sister chromatid cohesion is likely to have a very large impact on the faithful segregation of the homologs and is therefore likely to result in the production of disomic and nullisomic gametes. A model is therefore proposed by researchers in this field. That at least two 'hits' are required for age-dependent aneuploidy. The first hit is the establishment of a susceptible bivalent (for example, a bivalent with a single, distally placed exchange as shown in the slide above). This component has been proposed to be age independent. The second hit involves abnormal processing of the susceptible bivalent during meiosis I, i.e. during the prolonged meiotic arrest -- and this is therefore the age dependent process. The longer the arrest, the older the individual would be, and the greater the chance is of malsegregation, i.e of something going wrong.