- Created by: rkerri200
- Created on: 10-01-21 15:23
BOD - molecular pathology
It is used to explain clinicsal phenotypes in terms of causative genetic lesions.
Different types of mutations have different impacts on how a protein functions. The main classes of mutation are:
Deletions, Insertions, Frameshifts, Single base pair substitutions (missense, nonsense, splice site), Dynamic mutations (triplet repeats), Imprinting Two main effects of mutations are: Loss of function (LoF); Gain of function (GoF)
Understanding molecular pathology can help a scientist to work out the molecular causes of a newly identified disease, and ultimately understanding the cause can lead to treatment(s). Lots of different types of mutations can lead to the loss of function phenotype of an allele. These affect all aspects of the transcription and translation of a gene. Deletions and insertions have severe effects because they cause frameshifts (compare the phenotype of the milder Becker MD to DMD). Nonsense mutations destabilise mRNA (fibrillin mutations). Substitutions in coding regions may alter localisation signals [in cystic fibrosis (CF) the deltaF508 mutation, affects localisation of the CFTR protein]. Changes in non coding regions may cause adverse effect by altering correct splicing (PKU ) or altering promoter function (Fragile X). A mutation is unlikely to give a gene product a radically new function. Most GoF mutations affect the way in which a gene or its product reacts to regulatory signals. Not exhaustive list of mutations, just examples. Expressed inappropriately (wrong time, wrong amount, in response to wrong signal, unable to respond to negative regulatory signals). Abnormal and pathogenic interaction with other cellular components. Gain of function mutation is Serine protease inhibitors cleave target proteins at specific sites: α1-antitrypsin (α1-AT), protects cells from high levels of elastase (elastase is produced by neutrophils during inflammatory responses and breaks down elastin in connective tissue). Elastase cleaves α1-AT at Met358Ser359 → conformational change of α1-AT → elastase inhibition. Missense mutation in α1-AT Met358 → Arg358 changes specificity of α1-AT , so now thrombin binds α1-AT → thrombin inhibition → lethal bleeding disorder (since thrombin is an enzyme that catalyses a crucial step in clot formation)
BOD - LOF mutation ATM
The ataxia-telangiectasia mutated (ATM) gene on chromosome 11q23 encodes a protein 3056 amino acids long that is involved in the detection of DNA damage.
Different patients show a wide variety of ATM gene mutations, mostly small frameshifting insertions or deletions, but also nonsense and splice site mutations, some missense changes, and occasional large deletions
Clearly, the cause of ataxia-telangiectasia is a loss of function of both copies of the ATM gene.
BOD - GOF mutation
Achondroplasia is a genetic disorder whose primary feature is dwarfism and can be inherited by autosomal dominance.
Achondroplasia is caused by a mutation in the fibroblast growth factor receptor 3 (FGFR3) gene. Two mutations in the FGFR3 gene (both at nucleotide c.1138, most commonly a G-to-A transition with a less frequent G-to-C transversion) cause a p.Gly380Arg substitution and are thought to account for the majority of ACH cases.
BOD - fragile X
Fragile X is an X-linked dominant condition causing mental retardation (it has variable penetrance, meaning that some people with the mutation do not develop features of the disease). Caused by (CGG)n near the promoter of the FMR1 gene. This gene is expressed in the brain and needed for normal cognitive development, involved in RNA processing and transport.
The FMR1 gene encodes a 4.8kb mRNA. It contains a CGG repeat 250bp downstream from a CpG island in an exon (5’UTR) and which is expanded from ~5-44 copies in healthy people to > 200 in patients. Expansion is thought to alter chromatin structure, leading to hyper -methylation of the CpG island and therefore gene silencing. Individuals with normal repeats express the FMR1 gene while those with pathological expansion do not express FMR1.
Chances of inheriting Fragile X depends on whether full mutation or pre-mutation is present in the parent, and if inherited from mother or father. WT number is 6-54 but in a premutation found in normal transmitting males this increases to 52-200. Females inheriting the premutation can expand the repeat number during meiosis to 250-4000 (full pathogenic mutation). Variable penetrance due to X chromosome inactivation which modifies phenotype in females. Affects 1:4000 males and 1:8000 females (UK) and has a wide range of characteristics, most common is learning disabilities
There are rare occasions where the disease has been attributed to a deletion of the gene or to point mutations, indicating that FRX caused by loss of function
BOD - position independent approaches - biochemist
Haemophilia is a rare genetic disorder that prevents blood from clotting properly. This can lead to internal ad external bleeding that can occur randomly or due to injury, bruising, surgery. Internal bleeding can be incredibly dangerous for those with haemophilia. It can lead to permanent disability e.g. bleeding inside joints or even death e.g. bleeding in organs. The genes used to create clotting factors are defective and cannot create blood clots to prevent bleeding. There are 12 clotting factors in the body. Haemophilia A is due to a mutation in the gene that creates clotting factor VIII. It is an X-linked recessive disorder Haemophilia B is caused by a mutation in the gene responsible for clotting factor IX. The most common tretamnet is clotting factor replacement therapy. Gene therapy could provide a one-time only treatment. A working copy of the defective gene can be delivered into the cell to produce the factor that isnt being made. This is done using a viral vector. To treat haemophilia this vector would be targetted to liver cells via infusion. This effect could wear off in children as the liver grows as we age so this treatment is being trialed in adults.
The biochemical approach was to isolate enough factor 8 from pigs blood to generate a partial amino acid sequence. If we know thepartial aa sequence of a protein then the DNA (gene) sequence can be inferred. Hopefully you know that the amino acid code is degenerate, eg. some amino acids are coded for by multiple codons and therefore it is not possible to completely accurately know the DNA sequence and that the predicted gene sequence is also degenerate in certain areas. But it can be used to generate degenerate oligos which can used to probe a cDNA library to find the gene of interest. In this example only one amino acid is degenerate (AA1 = AAT or AAA or AAC). And so 3 versions of the probe are made, one for each version of the amino acid.
In the case of factor 8, degenerate probe cocktails were made to correspond to partial aa sequence of porcine factor VIII and used to screen and porcine cDNA library to recover full length cDNA.
Porcine genomic clone was then used to recover human gene, which could then be used to make recombinant F8 for future therapy for patients with haemophilia A.
BOD - position independent approaches - biochemist
cDNA libraries can also be expressed in host cells, so that the host cell (normally bacteria) expresses protein. In this case you can identify the cDNA of interest by using the purified protein (of the unknown gene), if it is availableto raise an antibody thatrecognises the protein of interest. The antibody is then used to screen the proteins expressed from host cells (each host cell will express a different cDNA). Each host cell is located in a particular region on a plate, then the proteins are transferred onto a filter which can then be screened with the antibody. Antibody will identify the host cells that express cDNA related to the gene of interest. These host cells can be identified using a variety of approaches, including the one shown above, which uses autoradiography to identify 2 cells that express the cDNA of interest.
This method was used to identify the gene that causes phenylketonuria (PKU). PKU is a rare metabolic disease that can lead to severe brain disorders. It causes the build up of phenylalanine in the bood to toxic levels. Phenylalanine is an essential amino acid acquired through protein. Phenylalanine is coverted to tyrosine by the enzyme phenylalanine hydroxylase. Tyrosine is converted into neurotransmitters important for brain development and function. PKU is an autosomal recessive disorder that effect s phenylalanine hydroxylase. The gene for this enzyme is located on chromosome 12 and over 600 mutations have been associated. Phenylalanine uses the same transporters as tryptophan (for serotonin) and tyrosine (for dopamine and norepinephrine) so when these levels rise the block all receptors that allow passage across the blood brain barrier leading to abnormal development and intellectual disability.
BOD - position independent approaches - animal mod
The gene causing deafness in a large family was known to be located on chromosome 17 (more on this later) and in Shaker mice it mapped to a region on chromosome 11. Likely that the human and mouse gene are related (orthologues). Bacterial artificial chromosomes (BACs) were obtained that contained fragments of DNA from the chromosome 11 region. Each BAC was injected into shaker mice and the resulting offspring phenotype observed. One mouse had their phenotype corrected and so the sequence of the BAC insert was determined then compared to the same sequence in Shaker mice. Shaker mice had a mutation in the gene (myo15) contained in the BAC (which had a wild type version of the same gene). The related human gene could then be identified (MYO15) and sequenced in patients, and mutations in this gene were then identified.
Can also be done in mammalian cell lines (tumour suppressor genes), yeast (transcription factors) or using NIH 3T3 (oncogenes).
BOD - position dependent approaches - LINKAGE
Linkage analysis uses genetic information from families to identify the likely location of novel disease CAUSING genes. One fundamental principle underlies linkage analysis: genes located closely together on a DNA molecule are co-inherited: Recombination occurs during meiosis; The further apart two alleles are on a chromosome, the more likely they are to be separated by recombination during meiosis. Genetic markers from across the genome are genotyped in family members from different generations and used to track the inheritance of DNA regions. If a disease is known to run in a Mendelian manner in families, then linkage analysis can be used to work out what gene causes the disease, through genetic analysis of multi generations.
Humans have 23 pairs of homologos chromosomes. There are lots of genes on each chromosome so it is likely that some of these genes are inherited together. It is known that jomologous chromosomes cross-over during the prophase 1 of meiosis making recombinant chromosomes. This means that not all of the alleles from a chromosome will be passed on in a fertilised gamete. Sturtevant concluded that the farther apart two genes are on a chromosome the more liely it is that crossing over will occur between them allowing for recombination. This is the recombination frequency. y using quatitative data he was able to create a linkage map by calculating how far appart different genes are on a chromosome. This was an early example and was used by Sturtevant to map the genes of fruit fly.
BOD - position dependent approaches - LINKAGE EOAD
Linkage analysis carried out using microsatellite markers. Led to the identification of 3 causal genes containing mutations that lead to increased levels of Aβ42 peptide (found in plaques): Amyloid precursor protein (APP); Presenilin 1 (PS1); Presenilin 2 (PS2). One of the first linkage analysis results in EOAD families identified a locus on chromosome 21 that segregated with disease. The existence of recombinant people helped to identify a smaller region where candidate gene may be found. This led to identification of APP. Linkage analysis of families who did not have mutations in APP were looked at in many other studies and two further genes for early onset familial AD were found. Presenilin 1, found on chromosome 14. Mutations in this gene cause the most aggressive type of AD and the age at onset (AAO) can be as young as 16. Discovey of PS1 led to the identification of PS2 (gene with a 63% amino acid identity to PS1, both have 7 transmembrane spanning domains). More PS1 mutations than PS2, which are relatively rare. Through identification of the genetic causes of EOAD, we have learned a lot about what these proteins do and can propose hypotheses for the underlying molecular causes of Alzheimer’s disease. APP, PS1 and PS2 involved are in a process called amyloid processing. APP is a transmembrane protein and it contains the Aβ peptide. The Aβ peptide is sequentially cleaved out of APP by the action of secretases; β, γ,ε. At its most simplest, amyloid plaques contain more Aβ42 than Aβ40 and this is thought to be toxic to neurons. AD mutations somehow affect APP processing leading to more Aβ42 compared to Aβ40.
BOD - position dependent approaches - ASSOCIATION
You want to have a large number of cases and controls, and preferably you want the same number (or more) of controls. You want to make sure your individuals with disease have the same disease (homogeneous, eg. There are different types of dementia, not all caused by Alzheimer’s disease – if you mix diseases then it will make it harder to find disease genes, even if disease is related). Make sure the average age of control and case populations are similar. Age at onset should be similar (homogenous disease). Similar number of males and females and also make sure ethnicity is matched – different genetic variatns can cause disease in individuals of different ethnic background. To compare the genetic make up of all individuals in your study you need to genotype markers across either the genome or a specific region of interest. This is normally done with SNPs but other markers can be used (microsatellites). Once you have genotype data for all markers of interest and all idnividuals you then need to carry out some quality control. One of the main things to do is to check the HWE of your control population – if a marker is not in HWE then it has to be removed from the study. Remaining markers are analysed statistically to determine whether it is more or less frequent in the disease population. HWE occurs in an idealised large population where mating occurs randomly and allele frequencies remain constant over time. A marker not in HWE implies there is a selection pressure on that allele/gene (irrespective of it causing disease). HWE is calculated using online tools, but essentially what they do is to carry out a chi squared test to see whether observed (O) genotype or allele frequencies are different to what might be expected (E) (if mating happened randomly) in the CONTROL population. A p value is the probability that a tested hypothesis is TRUE (eg. that the marker tested is NOT associated with disease under study). If p = 0.05, indicates that if you did a test 20 times, you would only get that result once (1/20=0.05). A P ≤ 0.05 implies that the tested hypothesis is unlikely to be true and therefore the OPPOSITE IS TRUE and it implies that the marker IS ASSOCIATED WITH TESTED DISEASE. There are inherent problems in the design of association studies. It can be difficult to detect genes with small effect if the sample size is not large enough (this can also be affected by too much genetic heterogeneity, and hence important to ensure matching of cases and controls and homogeneity of disease). Another factor hindering gene discovery is that in complex disease the environment can be involved and sometimes a genetic variant by itself does not increase risk unless an environmental factor is also considered. The same is true for multiple genetic variants. Individually two genes may not increase disease risk but when considered together they do. This is epistasis and needs to be considered in analysis of GWAS data. Another consideration is the level of significant used, if you set too low a p-value threshold, then there is an increased chance of identifying false positives (markers that show positive association with disease but which only give that result because the threshold is too low). Associated genes are only susceptibility factors and are neither necessary nor sufficient to cause disease. They Increase likelihood of disease and can be different in different ethnic groups
BOD - position dependent approaches - ASSOCIATION
The first risk factor for AD was discovered by an initial linkage study followed up by association studies. In this graph, you have along the x axis the position of markers along chromosome 19. The y axis is the LOD score (probability) that each marker is linked to Alzheimer’s disease. Remember that a LOD score of > 3 indicates probability of a disease gene located in the area. Chromosome 19 contains a risk gene for AD. The APOE gene was selected as a candidate for testing based on a number of known factors. Association analysis was carried out looking at 3 haplotypes of the APOE gene. Individuals with AD, and control individuals, were genotyped to determine what APOE haplotype they carried. There are 3 alleles, epsilon 2/3/4. Individual genotypes can be e2/e2, e2/e3, e2/e4, e3/e3, e3/e4, e4/e4. By comparing the frequency of the alleles and carrying out a statistical comparison it was calculated that individuals with AD were more likely than control to have an E4 allele (56% AD patients had an E4 allele, only 14-16% control individuals had an E4 allele). The APOE gene has 3 different haplotypes. This haplotype is defined by two SNPs, one that changes an amino acid at position 112 and one that changes an amino acid at position 158. Because of the SNPs position 112 can be Cysteine or Arginine, position 158 can be cysteine or arginine. You can find haplotypes of CysCys, CysArg or Arg Arg. APOE E1 (ArgCys) is very rare. The e4 allele of APOE is associated with increased risk of AD. E2 carriers are less likely to develop AD. Since 50% of LOAD patients do not have an E4 APOE allele then it was clear more genes to be found. Development of larger association studies, GWAS. Once a stage 1 study has been carried out, it is then important to replicate it in a second independent set of samples. This is an attempt to prevent negative findings, which blighted early association studies. The replication study does not look at all SNPs, it follows up the positive associations from stage 1. Stage 2 confirmed that: The CLU SNP and the PICALM SNP was still associated with risk of AD. From the association data another measure can be calculated called the Odds Ratio (OR). This describes the effect of the allele that is associated. Association does not always mean that the allele associated increases disease risk.
BOD - position dependent approaches - CHROMOSOMAL
Fluorescent in situ hybridisation is a technique that uses fluorescently labelled probes to identify regions on chromosomes, for example if you want to know where the APOE gene is then you can synthesise a small piece of DNA from the APOE sequence, label it with a probe, then hybridise this to a chromosome. Visualisation of this using a fluorescent microscope enables you to see which chromosome the probe binds to and at which part. If a disease has been cause by a translocation then it might be because a gene has been disrupted. The exact location of this disruption can be identified through FISH. In the example here, the green probe binds to 3 chromosomes rather than the expected 2. This is because the gene is split across the middle two chromosomes and part of the probe can bind one and the other part can bind the second. Another method, which was again covered in MD is aCGH, so I won’t go into much detail here. Red line along middle implies same amount of chromosome from control and test sample. A value of -1.0 implies they are not equal and one sample has some chromosomal sequence missing. Both of these methods identify a region on a chromosome that is likely to contain a gene that is disrupted in disease. aCGH uses SNP markers and so could be more precise than FISH.
BOD - position dependent approaches - MOUSE MODEL
My third example in this section is to show that you can also compare human chromosomes to mouse chromosomes to identify disease genes.
Many mouse models of disease have been created as it is relatively easy to systematically knock out mouse genes and create novel mutant lines. Resulting phenotypes in the mouse can be analysed and some may be similar to human.
The image here depicts human chromosomes, and the different colours depicts the mouse chromosomes that matches each region. IF a mouse mutant is available that causes a certain phenotype related to a human disease then the mouse gene location can be used to determine the likely area of a specific human chromosome that is affected in the human disease. Human genes in the area can then be examined.
Humans 22 autosomes, 1 pair sex chromosomes
Mouse 19 autosomes, 1 pair sex chromosomes
BOD - position dependent approaches - CONFIRMATION
It may be that from carrying out analysis of your patients, they all have mutations in the same gene. This provides powerful confirmation that the correct gene has been identified. Regardless of what your initial experiment shows (linked markers, associated markers, genes located in chromosomal breakpoints etc). If we start with genes identified through linkage or association studies as an example, you can identify genes in the linkage or associated area using genome browsers. For Mendelian conditions, once a candidate gene had been identified the next step would be to look for mutations in that gene within a cohort of unrelated affected patients. Assuming the mutation affects the coding sequence (and hence the amino acid) the usual approach is to amplify exon fragments using PCR which can then be sequenced to identify any mutations. If there are a number of genes to be sequenced, for example an area of association contains many relevant candidate genes, then it can be a challenge to sequence all of them. This might also be the case even if only one gene, some genes can contain many exons and/or be very long. Initial screening techniques are often used to identify whether a particular sequence might contain a mutation that can then be investigated further. dHPLC compares DNA samples based on their melting temperature (melting temperature (Tm) is the temperature required to denature a nucleotide sequence - different nucleotide sequences denature at different temperatures). Samples are PCRd over a specific sequence (incorporating a fluorescent dye that binds dsDNA), then they are analysed on a dHPLC machine, generating a chromatogram. Different chromatograms indicates potentially different DNA sequences and these specific regions can be sequenced, as opposed to sequencing all. Once a mutation has been identified in patients then it is good practise to check whether this mutation can also be found in healthy individuals. We want to identify pathogenic mutations as opposed to neutral non-pathogenic mutations. If a mutation is also found in healthy individuals then it is unlikely to be disease-causing. You should be aware that some mutations may not be revealed when using certain analytical methods. For example, gene inversions cannot be detected by sequencing. The sequence is all still present in the genome albeit inverted, so unless the primers used to amplify the sequence have been affected by the inversion then it won’t be identified. So bear in mind that even if no mutations are found after dHPLC or sequencing there may well still be a DNA change, but a different method is required to identify it (a FISH experiment might be useful if you were certain the gene is disease-causing).
In addition, epigenetic changes such as DNA methylation may also cause disease but with no change to the DNA sequence (eg. cancer). However, there is not much evidence for epigenetic changes as primary causes of hereditary disease in humans. Identifying a mutation in a candidate gene is the first step in determining if that gene actually does contribute to the disease under study. Once a mutation has been identified and it is thought to causes disease (eg. is not found in healthy people), then functional experiments are required to confirm the mutation is pathogenic. Many different approaches can be used (note this slide is not exhaustive). And the method used is likely to depend on whether the suspected pathogenic mutation has resulted in a loss or gain of function. One of the easiest ways to examine mutant phenotypes is to use cultured cells. For a loss of function mutation, then RNA interference (RNAi) can be used to knock-down expression of the candidate gene in the cell then some phenotype is measured, for example the effect of knocking down this gene on cell migration, or perhaps the effect of knockdown on expression of another protein that is known to be involved in the disease (using western blot). Level of knockdown can be variable and RNAi can have off-target effects. A complete knock-out can be generated in cells using CRISPR, and as above the affect on phenotypes determined. CRISPR can be time-consuming. Conversely, once you have a knock-out cell line with a phenotype, then you can do the reciprocal experiment and look at whether the phenotype can be rescued by addition of the wild-type gene or perhaps different versions of the gene. This can be achieved by cloning the gene of interest into an expression vector then transfecting this into the cells. Gene cloning is mostly quite simple and quick to do. For a suspected gain of function mutation, CRISPR can also be used to generate knock-in mutations, so modification of one SNP version to another eg C to a T base at a specific location, and as above the affect on phenotypes determined. In addition, to investigate overexpression of a gene (perhaps due to a gain of function mutation) then the gene can be cloned into an expression vector with an active promoter, then transfected into cells to see how the phenotype is affected. Animal models can also be used and clearly are more physiologically relevant than cell culture. Some animal models might be better suited to understanding certain diseases and must be aware that genes and proteins may not always behave the same way as in humans. But they certainly have provided very useful information in many instances. In terms of how to investigate LoF and GoF in animal models, not all techniques possible in cell culture will work with animal studies, and there are many alternative methods available. For example. To investigate loss of function, 1) Knock-down zebrafish models can be created using morpholinos to target specific gene expression and 2) Knock-out transgenic mouse models can be created by injecting embryos with modified genes or using gene-editing methods such as CRISPR. Gene editing and creation of mouse model is very time-consuming and it can take up to 2 years to make a new mouse model. In contrast analysis of zebrafish using morpholinos is relatively quick. The MO is injected into the embryo at the 2-cell stage and depending on the phenotype may be observable after 48 hours. There are issues with morpholinos too as they don’t completely knock out a gene and the level of knockdown should be determined. They may also have off target effects. To investigate gain of function, 1) Over-expression of candidate gene in mice, for example using viral vectors. Gene therapy can use viral vectors such as HSV or AAV1. Working with viral vectors is becoming more commonly accepted and easy to do, the viruses are attenuated and only infect tissues under special circumstances. It can be quite challenging to create a viral vector expressing the gene of interest. 2) Creation of a knock-in transgenic mouse using gene-editing methods. As above, Gene editing and creation of mouse model is very time-consuming and it can take up to 2 years to make a new mouse model. 3) Over-expression of a candidate gene in zebrafish by injection RNA transcript. This is also time-consuming as requires the creation of an initial vector expressing the gene variant required so some cloning and editing may be needed. Then the vector is expressed and the RNA is collected ready for injection into 2-cell zebrafish embryos. The example shown at the bottom of this slide is taken from my current research in spinal muscular atrophy (SMA). SMA is caused by mutations in the SMN1 gene resulting in reduced expression of SMN protein. Therefore models with reduced expression of SMN are useful to study this disease further. A zebrafish model of spinal muscular atrophy can be made by using a morpholino approach. The MO knocks down expression of SMN gene and generated a motor neuron phenotype as would be expected.
BOD - chromatin structure
DNA contains a structure called chromatin, found in eukaryotes within the nucleus of the cells. Chromosomes are made from chromatin and makeup this DNA. Th arrangement of hromatin is responsible for the appearance of a chromosome. Only 30% of chromatin is DNA. The vast majority is histone protein that give structure and regulate gene expression (60%). There are 8 histones and accompanying DNA form a nucleosome. Necleosomes are the functional parts of chromatin. The histone has a beaded necklace look. The DNA that links each nucleosome together is called linker DNA. A factor in gene modification is histone alteration. Nucleosomes wrap into tight helices - around 30 nm in diameter. They fold repeated to form a chromatid. The remaining 10% of the chromatid is new RNA or RNA that is being synthesised. Euchromatin is active chromatin about to undergo transcription and is loosely packed. Heterochromatin refers to tightly packed chromatin that is not undergoing transcription.
BOD - Genomic imprinting
Some organisms reproduce asexually in pathenogenesis - plants, reptiles and birds. In mammals this does not occur because of genomic imprinting. This is because genes are differentially expressed depending on where the genes came from - mum or dad. For the majority of genes - 99% of protein coding genes- genes are passed down in equal measure but for the remaining 1 % only one copy is functonal and the other is silenced. This was developed by Helen Crouse in sciarid flies. There are 3 hypothesised ways of silencing the second copy. The first is methylating a nucleotide base group to turn the gene off and prevent access for transcription. The second method is changing the histones in the chromatin and therefore changing its function. This is also done via methylation. The third method is bringing in non-coding RNA to signal that this copy should either be active or silenced. The female has to modulate her contribution to the offspring in order to provide for other offspring in multiple pregnancies. For the male, however, to ensure the offspring has all the resources required for survival and to pass on his genes. With an embryo that has 2 male copies of the genome it causes an overgrowth in plancental tissue and no viable foetus. If an embryo has 2 maternal copies of the genome, an ovarian teratoma is formed with abnormal expression of hair, teeth and bone. However, parthenogenesis was successfully modelled in mice in the early 2000sin Japan. A mouse was created using 2 copies of maternal DNA.
BOD - X-inactivation
In normal cells, the femal has ** chromosomes and the male has Xy chromosomes. The X part contains not only sex traits but somatic traits as well. To prevent an imbalance in the sexes as females have 2 and males only have 1, one of the X chromosomes is inactivated. There is equal chance that the X chromosome from the mother or father will be inactivated. X-inactivation occurs in the embryonic stage at around 8 undifferentiated cells it begins. It prevents the cell from having twice as many genes. It was discovered by Mary Lyon and can be referred to a lyonisation. The inactivated X chromosome is highly methylated and condensed, becoming a Barr body or heterochromatin. This means that transcription will not occur.
BOD - Rett syndroms
Rett syndrom is a neurological condition resulting from a mutation in MECP2. The MECP2 protein is composed of 486 amino acids. A missence mutation on base 473 substitutes a C base for a T base. This changes the amino acid in the sequence. There is now a methionine instead of a threonine at the 158th amino acid. This mutation is referred to a T158M and creates a dysfunctional protein. A nonsense mutation can stop the protein from being transcribed after that point creating a stop codon. A frameshift mutation shifts the reading frame of the gene and results in different amino acids being made and non-functional. A cluster of Rett syndrome missence mutations prevent MECP2 from binding to an essential protein. Through investigation of these mutations it appears that the whole protein of importance and can sub-divide it into 2 sections the methyl CpG binding domain and the transcriptional resppression domain. Mutations in the MBD prevent DNA binding. The TRD domain is where MECP2 binds to an essential protein called NCoR. NCoR is important in gene silencing. MECP2 acts as a bridging molecule bewtween the NCoR and the methylated histones of DNA. Mutations prevent this function. It is hypothesised that the inability of MECP2 to bridge NCoR to histones is what underlies the pathology of Rett syndrome.
BOD - beckwith-wiedemann syndrome
This is an example of genomic imprinting. The area affected in beckwith-wiedemann syndrome is 11p15 which is responsible for insulin and IGF-2, oncogenes and tumour suppressor genes. This can cause overgrowth due to the growth factor dysregulation and hypoglycaemia. It is also associated with neoplasms due to cancer gene dyregulation.
BOD - prader-willi syndrome
This is a genetic disorder that causes poor feeding and low muscle tone in infants. It goes on to develop over-eating, intellectual dissability and low sex hormones in children. Genes on chromosome 15 are not expressed. A couple of these are SNRPN and snoRNAs which create proteins responsible for the modification of other RNAs. Maternal genes in this region are silenced normally and the male genes expressed. This is genetic imprinting. In prader-willi syndrome, the maternal genes are silence normally but the male genes are also not expressed. This can occur through deletion of the prader-willi region. Deletions also include the OCA2 gene causing loss of pigmentation in the eyes, hair and skin. Another way is paternal uniparental disomy. Both copies of chromosome 15 came from the mother. This results in trisomy 15 when the zygote develops. This would normally be a failed pregnancy unless a copy of chromosome 15 is lost in trisomy rescue. Paternal uniparental dismy 15 results if it is the paternal copy that gets lost. A third way is a mutation in the imprinting center. This region directs imprinting via sex-specific methylation. An epimutation can occur even if there is no defect in the imprinting center due to non-deletion of the methylation when the paternal sperm is being made. There could also be translocation moving the prader-willi genes away from the imprinting center. Having the same mutation of the maternal chromosome can affect the gene UBE3A, resulting in Angelman syndrome.
BOD - parkinsons disease
Parkinsons is the second most common neurological disease after Alzheimers. It causes slow movement, muscle rigidness and tremors at rest. It can also cause psychological symptoms, loss of smell, GL/bladder dysfunction and cardiovascular issues. Research is focused on the brain motor region - substantia nigra in the migrostriatal pathway. Dopaminergic neurons in this area start to die and are therefore treated with dopaminergic drugs but tolerance becomes a problem and the death of receptors also. It is characterised by clumps of misfolded proteins such as Lewy bodies. The misfolded protein is alpha-synuclein and creat fibrils which are toxic to neurons. Misfolded proteins are cleared by proteasomes and autophagosomes but could be overwhelmed by the quantity of alpha-synuclein. It is also thought that the neuronal mitochondria become impaired and can no longer function preventing energy from being made. They are cleared by similar mechanisms. Microglia are thought to take up the cellular debris causing an inflammatory response and release of cytokines. This activates more microglia and astrocytes and the chemicals released can damage the neurons. This can be treated by stem cell therapy.