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Wednesday, July 27, 2011
Homozygosity Mapping and LOD Score
1. HOMOZYGOSITY MAPPING
Homozygosity is the state of possessing two identical forms of a particular gene (alleles), one inherited from each parent.
Homozygosity mapping is a method for mapping the human genome, used to detect genes that cause disease only when both copies in an individual are mutated (the genes are homozygous, or the same). This technique works for genetic disorders that are inherited from both parents, since inheriting a pair of heterozygous (different) genes results in expression of a non-mutated version from one parent, and the absence of disease symptoms. It is the way to map human recessive traits with the DNA of inbred children.
2. HOW IS HOMOZYGOSITY MAPPING DONE
Homozygosity mapping can be done by either of the following methods
1. SNPArrays
2. RFLP
3. Microsatellite markers
2.1. SNP ARRAYS FOR HOMOZYGOSITY MAPPING
It was proposed that a set of SNPs evenly spread across the human genome could be used to screen two populations (typically populations with and without a disorder) and that some SNPs would associate more with the disease group, thus implicating the SNP, or a DNA sequence close by, in the disease state. A massive technological effort followed and whole genome scans, using tens of thousands of SNPs, were made a reality with the advent of array-based technologies.
One recent advance is the development of high-density SNP microarrays for genotyping. The SNP arrays overcome low marker informativity by using a large number of markers to achieve greater coverage at finer resolution.
BARDET-BIEDL SYNDROME
We used SNP microarray genotyping for homozygosity mapping in a small consanguineous Israeli Bedouin family with autosomal recessive Bardet-Biedl syndrome (BBS; obesity, pigmentary retinopathy, polydactyly, hypogonadism, renal and cardiac abnormalities, and cognitive impairment) in which previous linkage studies using short tandem repeat (STR) polymorphisms failed to identify a disease locus.
SNP genotyping revealed a homozygous candidate region. Mutation analysis in the region of homozygosity identified a conserved homozygous mis-sense mutation in the TRIM32 gene, a gene coding for an E3 ubiquitin ligase. Functional analysis of this gene in zebra-fish and expression correlation analyses among other BBS genes in an expression quantitative trait loci data set demonstrate that TRIM32 is a BBS gene. This study shows the value of high-density SNP genotyping for homozygosity mapping and the use of expression correlation data for evaluation of candidate genes and identifies the proteosome degradation pathway as a pathway involved in BBS.
Positional cloning is a powerful approach to identify genes mutated in human and animal models monogenic (single gene case) diseases. The genomic DNA region (and the embedded polymorphisms) harboring the disease-causing mutations segregates with the disease in analyzed pedigrees. However, positional cloning relayed until recently on the availability of large pedigrees to reach a significant linkage.
This protocol describes the use of whole genome genotyping on sporadic consanguineous patients to identify potential disease loci and subsequent positional candidate genes, by homozygosity mapping (autozygosity). It takes advantage of high density single nucleotide polymorphism (SNP) genotyping arrays, and of the assumption that unrelated patients from several consanguineous families are mutated in the same gene.
2.2. RFLP (RESTRICTION FRAGMENT LENGTH POLYMORPHISM)
RFLP is a method used by molecular biologists to follow a particular sequence of DNA as it is passed on to other cells. It is technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites. By cutting two different DNA molecules with the same restriction enzyme, scientists can compare the lengths of the fragments; two identical molecules will have identical fragments, while two similar molecules may be largely alike, with perhaps a few differences in fragment size. These differences in restriction fragment lengths are called polymorphisms and are used in all types of DNA typing.
RFLP PRODUCTION
RFLP methodology involves
• Cutting a particular region of DNA with known variability, with restriction enzymes
• Separating the DNA fragments by agarose gel electrophoresis
• Determining the number of fragments and relative sizes
The pattern of fragment sizes will differ for each individual tested.
APPLICATIONS
RFLP technique has many applications like
DNA fingerprinting in forensic science
Tracing ancestry
Studying evolution and migration of wild life
Detection and diagnosis of certain diseases
Genetic mapping (to calculate the genetic distance between two loci)
2.3. MICROSATELLITE MARKERS
Microsatellites are simple sequence tandem repeats (SSTRs). The repeat units are generally di-, tri- tetra- or pentanucleotides. For example, a common repeat motif in birds is ACn, where the two nucleotides A and C are repeated in bead-like fashion a variable number of times (n could range from 8 to 50). They tend to occur in non-coding regions of the DNA (this should be fairly obvious for long dinucleotide repeats) although a few human genetic disorders are caused by (trinucleotide) microsatellite regions in coding regions. On each side of the repeat unit are flanking regions that consist of "unordered" DNA. The flanking regions are critical because they allow us to develop locus-specific primers to amplify the microsatellites with PCR (polymerase chain reaction). That is, given a stretch of unordered DNA 30-50 base pairs (bp) long, the probability of finding that particular stretch more than once in the genome becomes vanishingly small. In contrast, a given repeat unit (say AC19) may occur in thousands of places in the genome. We use this combination of widely occurring repeat units and locus-specific flanking regions as part of our strategy for finding and developing microsatellite primers. The primers for PCR will be sequences from these unique flanking regions. By having a forward and a reverse primer on each side of the microsatellite, we will be able to amplify a fairly short (100 to 500 bp, where bp means base pairs) locus-specific microsatellite region.
P.S SEE USES IN PRESENTATION.
3. HOMOZYGOSITY MAPPER
It is a web based approach for homozygosity mapping with a store of marker data in a database into which users can upload their SNP genotype files. Database analyses the data in a few minutes, detects homozygous portions (alleles) and provides agraphical interface of the results. Software also provides the option to zoom into single chromosomes anduser-defined chromosomal regions. It is integrated with a gene search engine GeneDistiller which enables users to determine most promising gene. Users can restrict access or make their uploaded sequences public. Hence homozygosity mapper can be used as a data repository for homozygosity mapping based researches.
4. GENETIC LINKAGE
Genetic linkage means that certain genes tend to be inherited together, because they are on the same chromosome. Thus parental combinations of characters are found more frequently in offspring than non parental. Genetic loci that are physically close to one another on the same chromosome tend to stay together during meiosis, and are thus genetically linked.
DISCOVERY
In 1905 the three geneticists William Bateson, Edith Rebecca Saunders, and Reginald C.Punnett discovered an apparent exception to one of Mendel's foundational proposals: the principle of independent assortment.
In their work with pea plants, these researchers noticed that not all of their crosses yielded results that reflected the principle of independent assortment specifically, some phenotypes appeared far more frequently than traditional Mendelian genetics would predict. Based on these findings, they proposed that certain alleles must somehow be coupled with one another, although they weren't sure how this linkage occurred. The answer to this question came just seven years later, when Thomas Hunt Morgan used fruit flies to demonstrate that linked genes must be real physical objects that are located in close proximity on the same chromosome.
In 1910, Morgan discovered a fly with mutant white eyes while normally fruit flies have red eyes, not white eyes. Morgan crossed this white eyed male fly to its red eyed sisters. Later he inbred the heterozygous F1 red-eyed flies, the traits of the F2 progeny did not assort independently. Morgan expected a 1:1:1:1 ratio of red-eyed females, red-eyed males, white-eyed males, and white-eyed females. Instead, he observed the following phenotypes in his F2 generation:
2,459 red-eyed females
1,011 red-eyed males
782 white-eyed males
There were no white-eyed females, and Morgan wondered whether this was because the trait was sex-limited and only expressed in male flies. To test whether this trait was sex limited he completed a second cross between the original white-eyed male fly and some of his F1 daughters. These crosses produced an F2 generation with the following phenotypes:
129 red-eyed females
132 red-eyed males
88 white-eyed females
86 white-eyed males
Thus, the results of this cross did produce white-eyed females, and the groups had approximately equal numbers. Morgan therefore hypothesized that the eye color trait was connected with the sex factor. This in turn led to the idea of genetic linkage, which means that when two genes are closely associated on the same chromosome, they do not assort independently.
LINKAGE MAP
A linkage map is a genetic map of a species or experimental population that shows the position of its known genes or genetic markers relative to each other in terms of recombination frequency, rather than as specific physical distance along each chromosome. Linkage mapping is critical for identifying the location of genes that cause genetic diseases.
A genetic map is a map based on the frequencies of recombination between markers during crossover of homologous chromosomes. The greater the frequency of recombination (segregation) between two genetic markers, the farther apart they are assumed to be. Conversely, the lower the frequency of recombination between the markers, the smaller the physical distance between them. Historically, the markers originally used were detectable phenotypes derived from coding DNA sequences; eventually, confirmed or assumed non-coding DNA sequences such as microsatellites or those generating restriction fragment length polymorphisms (RFLPs) have been used.
Genetic maps help researchers to locate other markers, such as other genes by testing for genetic linkage of the already known markers.
A genetic map is not a physical map (such as a radiation reduced hybrid map) or gene map.
A map of the genes on a chromosome based on linkage analysis. A linkage map does not show the physical distances between genes but rather their relative positions, as determined by how often two gene loci are inherited together. The closer two genes are (the more tightly they are linked), the more often they will be inherited together.
Linkage distance is measured in centimorgans (cM).
CONSTRUCTING A GENETIC LINKAGE MAP
Genetic linkage maps of each chromosome are made by determining how frequently two markers are passed together from parent to child. Because genetic material is sometimes exchanged during the production of sperm and egg cells, groups of traits (or markers) originally together on one chromosome may not be inherited together. Closely linked markers are less likely to be separated by spontaneous chromosome rearrangements. In this diagram, the vertical lines represent chromosome 4 pairs for each individual in a family. The father has two traits that can be detected in any child who inherits them: a short known DNA sequence used as a genetic marker (M) and Huntingtons disease (HD). The fact that one child received only a single trait (M) from that particular chromosome indicates that the fathers genetic material recombined during the process of sperm production. The frequency of this event helps determine the distance between the two DNA sequences on a genetic map.
5. LOD SCORE METHOD FOR ESTIMATING RECOMBINATION FREQUENCY
The LOD score (logarithm (base 10) of odds), developed by Newton E. Morton, is a statistical test often used for linkage analysis in human, animal, and plant populations. The LOD score compares the likelihood of obtaining the test data if the two loci are indeed linked, to the likelihood of observing the same data purely by chance. Positive LOD scores favor the presence of linkage, whereas negative LOD scores indicate that linkage is less likely. Computerized LOD score analysis is a simple way to analyze complex family pedigrees in order to determine the linkage between Mendelian traits (or between a trait and a marker, or two markers).
The method is described in greater detail by Strachan and Read. Briefly, it works as follows:
Establish a pedigree
Make a number of estimates of recombination frequency
Calculate a LOD score for each estimate
The estimate with the highest LOD score will be considered the best estimate
The LOD score is calculated as follows:
Where:
NR denotes the number of non-recombinant offspring,
R denotes the number of recombinant offspring.
Theta is the recombinant fraction, it is equal to R / (NR + R)
The reason 0.5 is used in the denominator is that any alleles that are completely unlinked (e.g. alleles on separate chromosomes) have a 50% chance of recombination, due to independent assortment.
In practice, LOD scores are looked up in a table which lists LOD scores for various standard pedigrees and various values of recombination frequency.
By convention, a LOD score greater than 3.0 is considered evidence for linkage. A LOD score of +3 indicates 1000 to 1 odds that the linkage being observed did not occur by chance. On the other hand, a LOD score less than -2.0 is considered evidence to exclude linkage. Although it is very unlikely that a LOD score of 3 would be obtained from a single pedigree, the mathematical properties of the test allow data from a number of pedigrees to be combined by summing the LOD scores. It is important to keep in mind that this traditional cutoff of LOD>+3 is an arbitrary one and that the difference between certain types of linkage studies, particularly analyses of complex genetic traits with hundreds of markers, these criteria should probably be modified to a somewhat higher cutoff.
5.1. MAPPING GENES WITH THE LOD SCORE METHOD
The dilemma of mapping genes can be overcome through the "lod score method" which involves the estimation of genetic distances in the situations other than simple testcrosses. The data obtained from the pedigree is used to calculate the map distances from the recombination frequencies. It is one of the basic and fundamental human genetics methods used today. The use of spread sheet programs i.e. Lotus 1-2-3 or Microsoft Excel makes the solution of this predicament effortless.
WHAT IS NEEDED?
The fundamental aim of this problem is to determine R, the recombinant fraction (fraction of gametes that are recombinant), using data from relatively small families. R can vary from 0 (2 genes completely linked) to 0.50 (2 genes unlinked).
STEPS INVOLVED
There are 4 basic steps in the method:-
(1) Determine the expected frequencies of F2 phenotypes for every value of R from 0.01 to 0.50
(2) Determine the "likelihood" (L) that the family data observed resulted from a given R value: the maximum likelihood is the best estimate of R for the given data
(3) Determine the Odds Ratio and the logarithm of the odds ratio (lod score) by comparing the Likelihood for each value of R to the Likelihood for unlinked genes (R = 0.50)
(4) Add LOD scores from different families to achieve an acceptably high lod score so a specific most likely R can be assigned.
The following example used for consideration comprises of:-
Two genes showing the complete dominancethe heterozygote is indistinguishable from the dominant homozygote
STEP 1: CALCULATE THE EXPECTED FREQUENCY OF OFFSPRING FOR VALUES OF R FROM 0 TO 0.50
The expected offspring numbers are calculated as follows:
Determine the frequency of each gamete produced by the F1's. For example, if R= 0.20, then 20% of the gametes produced by either parent will be recombinant. Since there are two types of recombinant gamete, A b and a B, the frequency of each will be 0.10. Since 80% of the gametes will be parental, the frequency of the parental types A B and a b will be 0.40 each.
Use a Punnett square to determine the offspring being formed from the union of the gametes. Multiply the gamete frequencies to get the offspring frequency. For instance, one cell of the Punnett square has the A B gamete from the father combining with the A b gamete from the mother. The frequency of the A B gamete is 0.40 and the frequency of the A b gamete is 0.10. Thus the frequency of the offspring in this cell is 0.40 x 0.10 = 0.04.
Determine the phenotype for each cell in the Punnett square and add up the frequencies to get the total frequency for each offspring phenotype.
A B
0.40 A b
0.10 a B
0.10 a b
0.40
A B
0.40 A B/A B
0.16 A b/A B
0.04 a B/A B
0.04 a b/A B
0.16
A b
0.10 A B/A b
0.04 A b/A b
0.01 a B/A b
0.01 a b/A b
0.04
a B
0.10 A B/a B
0.04 A b/a B
0.01 a B/a B
0.01 a b/a B
0.04
a b
0.40 A B/a b
0.16 A b/a b
0.04 a B/a b
0.04 a b/a b
0.16
F2 Phenotype Cell Sums Expected Freq
A_ B_ 0.16+.04+.04+.16+.04+.01+.04+.01+0.16 0.66
A_ bb 0.01 + 0.04 + 0.04 0.09
aa B_ 0.01 + 0.04 + 0.04 0.09
aa bb 0.16 0.16
Using a Punnett square to determine the genotypes and multiplying the frequencies of the two gametes that go into each type of offspring, then adding up offspring that have the same phenotype.
STEP 2: EXAMINE THE OBSERVED FAMILY DATA IN LIGHT OF THE EXPECTED DISTRIBUTION OF OFFSPRING FOR EACH R VALUE
This is done by determining the likelihood (L) of the observed family for each value of R. The likelihood is simply the probability of the observed family, as determined using the multinomial theorem, an extension of the binomial theorem.
First define the terms for the observed family:
a = number of A_ B_ offspring
b = number of A_ bb offspring
c = number of aa B_ offspring
d = number of aa bb offspring
n = total offspring (= a + b + c + d)
Then define the terms for the expected family proportions (obtained from step 1 above):
p = expected proportion of A _ B _ offspring
q = expected proportion of A_ bb offspring
r = expected proportion of aa B_ offspring
s = expected proportion of aa bb offspring
The term of the multinomial equation that describes the actual family is: pa qb rc sd multiplied by a coefficient.
The coefficient is: n! /(a! b! c! d!), where ! means "factorial".
This is very similar to the coefficient for the binomial.
Thus, the likelihood equation is: L = [n! /(a! b! c! d!)]pa qb rc sd
Above calculated the expected phenotype proportions for R = 0.20 (20 map units between A and B). They are: A_ B_ = 0.66; A_ bb = 0.09; aa B_ = 0.09; aa bb = 0.16. A family of 5 children has 2 with the A_ B_ phenotype, 1 with aa B_, and 2 with aa bb.
L = (5!/2! 0! 1! 2!)(.66)2(.09)0(.09)1(.16)2
L = 30(.4356)(.09)(.0256)
L = 0.0301
The likelihood (L) needs to be calculated for all values of R between 0.01 and 0.50. Note that the coefficient will be the same for all values of R; the coefficient only depends on the observed data. When this is done, the value of R with the highest likelihood is the best estimate of R that can be obtained with data from this particular family.
STEPS 3 AND 4: COMBINING DATA FROM SEVERAL FAMILIES
The data needs to be compared and added from several different families, to get a good estimate of R. To do this, the L values must be standardized by calculating the Odds Ratio (OR), which is the ratio of the L for each R value divided by the L for R = 0.50 (unlinked). Then, the logarithm of the Odds ratio is taken; this is the LOD score. LOD scores from different families can be added. (This is equivalent to multiplying the Odds Ratios, as in the AND rule for two events--family 1 AND family 2--both occurring.) A total LOD score for some R value of 3.0 is considered proof of linkage between the two genes.
For R = 0.20, the Odds Ratio = L0.20 / L0.50. We calculated L0.20 = 0.0301 above; L0.50 = 0.00695. The Odds ratio is thus 4.331 and the LOD score is the base 10 logarithm of this, 0.637. Clearly it would take several families of this size to reach a LOD score of 3.0.
REFRENCES
Discovery and Types of Genetic Linkage By: Ingrid Lobo, Ph.D. (Write Science Right) & Kenna Shaw, Ph.D. (Executive Editor, Nature Education) © 2008 Nature Education Citation: Lobo, I. & Shaw, K. (2008) Discovery and types of genetic linkage. Nature Education 1(1)
http://www.bios.niu.edu/johns/lodprob.htm
http://www.ornl.gov/sci/techresources/Human_Genome/publicat/primer/fig8.html
http://www.bio.davidson.edu/courses/genomics/method/RFLP.html
http://biotech.about.com/od/glossary/g/RFLPdef.htm
http://www.nlm.nih.gov/visibleproofs/education/dna/rflp.pdf
http://www.biology-online.org/dictionary/Genetic_linkage
European Journal of Human Genetics (2007) 15, 362–368. doi:10.1038/sj.ejhg.5201761.
Protocol Exchange (2007) doi:10.1038/nprot.2007.343.
Homozygosity is the state of possessing two identical forms of a particular gene (alleles), one inherited from each parent.
Homozygosity mapping is a method for mapping the human genome, used to detect genes that cause disease only when both copies in an individual are mutated (the genes are homozygous, or the same). This technique works for genetic disorders that are inherited from both parents, since inheriting a pair of heterozygous (different) genes results in expression of a non-mutated version from one parent, and the absence of disease symptoms. It is the way to map human recessive traits with the DNA of inbred children.
2. HOW IS HOMOZYGOSITY MAPPING DONE
Homozygosity mapping can be done by either of the following methods
1. SNPArrays
2. RFLP
3. Microsatellite markers
2.1. SNP ARRAYS FOR HOMOZYGOSITY MAPPING
It was proposed that a set of SNPs evenly spread across the human genome could be used to screen two populations (typically populations with and without a disorder) and that some SNPs would associate more with the disease group, thus implicating the SNP, or a DNA sequence close by, in the disease state. A massive technological effort followed and whole genome scans, using tens of thousands of SNPs, were made a reality with the advent of array-based technologies.
One recent advance is the development of high-density SNP microarrays for genotyping. The SNP arrays overcome low marker informativity by using a large number of markers to achieve greater coverage at finer resolution.
BARDET-BIEDL SYNDROME
We used SNP microarray genotyping for homozygosity mapping in a small consanguineous Israeli Bedouin family with autosomal recessive Bardet-Biedl syndrome (BBS; obesity, pigmentary retinopathy, polydactyly, hypogonadism, renal and cardiac abnormalities, and cognitive impairment) in which previous linkage studies using short tandem repeat (STR) polymorphisms failed to identify a disease locus.
SNP genotyping revealed a homozygous candidate region. Mutation analysis in the region of homozygosity identified a conserved homozygous mis-sense mutation in the TRIM32 gene, a gene coding for an E3 ubiquitin ligase. Functional analysis of this gene in zebra-fish and expression correlation analyses among other BBS genes in an expression quantitative trait loci data set demonstrate that TRIM32 is a BBS gene. This study shows the value of high-density SNP genotyping for homozygosity mapping and the use of expression correlation data for evaluation of candidate genes and identifies the proteosome degradation pathway as a pathway involved in BBS.
Positional cloning is a powerful approach to identify genes mutated in human and animal models monogenic (single gene case) diseases. The genomic DNA region (and the embedded polymorphisms) harboring the disease-causing mutations segregates with the disease in analyzed pedigrees. However, positional cloning relayed until recently on the availability of large pedigrees to reach a significant linkage.
This protocol describes the use of whole genome genotyping on sporadic consanguineous patients to identify potential disease loci and subsequent positional candidate genes, by homozygosity mapping (autozygosity). It takes advantage of high density single nucleotide polymorphism (SNP) genotyping arrays, and of the assumption that unrelated patients from several consanguineous families are mutated in the same gene.
2.2. RFLP (RESTRICTION FRAGMENT LENGTH POLYMORPHISM)
RFLP is a method used by molecular biologists to follow a particular sequence of DNA as it is passed on to other cells. It is technique that exploits variations in homologous DNA sequences. It refers to a difference between samples of homologous DNA molecules that come from differing locations of restriction enzyme sites. By cutting two different DNA molecules with the same restriction enzyme, scientists can compare the lengths of the fragments; two identical molecules will have identical fragments, while two similar molecules may be largely alike, with perhaps a few differences in fragment size. These differences in restriction fragment lengths are called polymorphisms and are used in all types of DNA typing.
RFLP PRODUCTION
RFLP methodology involves
• Cutting a particular region of DNA with known variability, with restriction enzymes
• Separating the DNA fragments by agarose gel electrophoresis
• Determining the number of fragments and relative sizes
The pattern of fragment sizes will differ for each individual tested.
APPLICATIONS
RFLP technique has many applications like
DNA fingerprinting in forensic science
Tracing ancestry
Studying evolution and migration of wild life
Detection and diagnosis of certain diseases
Genetic mapping (to calculate the genetic distance between two loci)
2.3. MICROSATELLITE MARKERS
Microsatellites are simple sequence tandem repeats (SSTRs). The repeat units are generally di-, tri- tetra- or pentanucleotides. For example, a common repeat motif in birds is ACn, where the two nucleotides A and C are repeated in bead-like fashion a variable number of times (n could range from 8 to 50). They tend to occur in non-coding regions of the DNA (this should be fairly obvious for long dinucleotide repeats) although a few human genetic disorders are caused by (trinucleotide) microsatellite regions in coding regions. On each side of the repeat unit are flanking regions that consist of "unordered" DNA. The flanking regions are critical because they allow us to develop locus-specific primers to amplify the microsatellites with PCR (polymerase chain reaction). That is, given a stretch of unordered DNA 30-50 base pairs (bp) long, the probability of finding that particular stretch more than once in the genome becomes vanishingly small. In contrast, a given repeat unit (say AC19) may occur in thousands of places in the genome. We use this combination of widely occurring repeat units and locus-specific flanking regions as part of our strategy for finding and developing microsatellite primers. The primers for PCR will be sequences from these unique flanking regions. By having a forward and a reverse primer on each side of the microsatellite, we will be able to amplify a fairly short (100 to 500 bp, where bp means base pairs) locus-specific microsatellite region.
P.S SEE USES IN PRESENTATION.
3. HOMOZYGOSITY MAPPER
It is a web based approach for homozygosity mapping with a store of marker data in a database into which users can upload their SNP genotype files. Database analyses the data in a few minutes, detects homozygous portions (alleles) and provides agraphical interface of the results. Software also provides the option to zoom into single chromosomes anduser-defined chromosomal regions. It is integrated with a gene search engine GeneDistiller which enables users to determine most promising gene. Users can restrict access or make their uploaded sequences public. Hence homozygosity mapper can be used as a data repository for homozygosity mapping based researches.
4. GENETIC LINKAGE
Genetic linkage means that certain genes tend to be inherited together, because they are on the same chromosome. Thus parental combinations of characters are found more frequently in offspring than non parental. Genetic loci that are physically close to one another on the same chromosome tend to stay together during meiosis, and are thus genetically linked.
DISCOVERY
In 1905 the three geneticists William Bateson, Edith Rebecca Saunders, and Reginald C.Punnett discovered an apparent exception to one of Mendel's foundational proposals: the principle of independent assortment.
In their work with pea plants, these researchers noticed that not all of their crosses yielded results that reflected the principle of independent assortment specifically, some phenotypes appeared far more frequently than traditional Mendelian genetics would predict. Based on these findings, they proposed that certain alleles must somehow be coupled with one another, although they weren't sure how this linkage occurred. The answer to this question came just seven years later, when Thomas Hunt Morgan used fruit flies to demonstrate that linked genes must be real physical objects that are located in close proximity on the same chromosome.
In 1910, Morgan discovered a fly with mutant white eyes while normally fruit flies have red eyes, not white eyes. Morgan crossed this white eyed male fly to its red eyed sisters. Later he inbred the heterozygous F1 red-eyed flies, the traits of the F2 progeny did not assort independently. Morgan expected a 1:1:1:1 ratio of red-eyed females, red-eyed males, white-eyed males, and white-eyed females. Instead, he observed the following phenotypes in his F2 generation:
2,459 red-eyed females
1,011 red-eyed males
782 white-eyed males
There were no white-eyed females, and Morgan wondered whether this was because the trait was sex-limited and only expressed in male flies. To test whether this trait was sex limited he completed a second cross between the original white-eyed male fly and some of his F1 daughters. These crosses produced an F2 generation with the following phenotypes:
129 red-eyed females
132 red-eyed males
88 white-eyed females
86 white-eyed males
Thus, the results of this cross did produce white-eyed females, and the groups had approximately equal numbers. Morgan therefore hypothesized that the eye color trait was connected with the sex factor. This in turn led to the idea of genetic linkage, which means that when two genes are closely associated on the same chromosome, they do not assort independently.
LINKAGE MAP
A linkage map is a genetic map of a species or experimental population that shows the position of its known genes or genetic markers relative to each other in terms of recombination frequency, rather than as specific physical distance along each chromosome. Linkage mapping is critical for identifying the location of genes that cause genetic diseases.
A genetic map is a map based on the frequencies of recombination between markers during crossover of homologous chromosomes. The greater the frequency of recombination (segregation) between two genetic markers, the farther apart they are assumed to be. Conversely, the lower the frequency of recombination between the markers, the smaller the physical distance between them. Historically, the markers originally used were detectable phenotypes derived from coding DNA sequences; eventually, confirmed or assumed non-coding DNA sequences such as microsatellites or those generating restriction fragment length polymorphisms (RFLPs) have been used.
Genetic maps help researchers to locate other markers, such as other genes by testing for genetic linkage of the already known markers.
A genetic map is not a physical map (such as a radiation reduced hybrid map) or gene map.
A map of the genes on a chromosome based on linkage analysis. A linkage map does not show the physical distances between genes but rather their relative positions, as determined by how often two gene loci are inherited together. The closer two genes are (the more tightly they are linked), the more often they will be inherited together.
Linkage distance is measured in centimorgans (cM).
CONSTRUCTING A GENETIC LINKAGE MAP
Genetic linkage maps of each chromosome are made by determining how frequently two markers are passed together from parent to child. Because genetic material is sometimes exchanged during the production of sperm and egg cells, groups of traits (or markers) originally together on one chromosome may not be inherited together. Closely linked markers are less likely to be separated by spontaneous chromosome rearrangements. In this diagram, the vertical lines represent chromosome 4 pairs for each individual in a family. The father has two traits that can be detected in any child who inherits them: a short known DNA sequence used as a genetic marker (M) and Huntingtons disease (HD). The fact that one child received only a single trait (M) from that particular chromosome indicates that the fathers genetic material recombined during the process of sperm production. The frequency of this event helps determine the distance between the two DNA sequences on a genetic map.
5. LOD SCORE METHOD FOR ESTIMATING RECOMBINATION FREQUENCY
The LOD score (logarithm (base 10) of odds), developed by Newton E. Morton, is a statistical test often used for linkage analysis in human, animal, and plant populations. The LOD score compares the likelihood of obtaining the test data if the two loci are indeed linked, to the likelihood of observing the same data purely by chance. Positive LOD scores favor the presence of linkage, whereas negative LOD scores indicate that linkage is less likely. Computerized LOD score analysis is a simple way to analyze complex family pedigrees in order to determine the linkage between Mendelian traits (or between a trait and a marker, or two markers).
The method is described in greater detail by Strachan and Read. Briefly, it works as follows:
Establish a pedigree
Make a number of estimates of recombination frequency
Calculate a LOD score for each estimate
The estimate with the highest LOD score will be considered the best estimate
The LOD score is calculated as follows:
Where:
NR denotes the number of non-recombinant offspring,
R denotes the number of recombinant offspring.
Theta is the recombinant fraction, it is equal to R / (NR + R)
The reason 0.5 is used in the denominator is that any alleles that are completely unlinked (e.g. alleles on separate chromosomes) have a 50% chance of recombination, due to independent assortment.
In practice, LOD scores are looked up in a table which lists LOD scores for various standard pedigrees and various values of recombination frequency.
By convention, a LOD score greater than 3.0 is considered evidence for linkage. A LOD score of +3 indicates 1000 to 1 odds that the linkage being observed did not occur by chance. On the other hand, a LOD score less than -2.0 is considered evidence to exclude linkage. Although it is very unlikely that a LOD score of 3 would be obtained from a single pedigree, the mathematical properties of the test allow data from a number of pedigrees to be combined by summing the LOD scores. It is important to keep in mind that this traditional cutoff of LOD>+3 is an arbitrary one and that the difference between certain types of linkage studies, particularly analyses of complex genetic traits with hundreds of markers, these criteria should probably be modified to a somewhat higher cutoff.
5.1. MAPPING GENES WITH THE LOD SCORE METHOD
The dilemma of mapping genes can be overcome through the "lod score method" which involves the estimation of genetic distances in the situations other than simple testcrosses. The data obtained from the pedigree is used to calculate the map distances from the recombination frequencies. It is one of the basic and fundamental human genetics methods used today. The use of spread sheet programs i.e. Lotus 1-2-3 or Microsoft Excel makes the solution of this predicament effortless.
WHAT IS NEEDED?
The fundamental aim of this problem is to determine R, the recombinant fraction (fraction of gametes that are recombinant), using data from relatively small families. R can vary from 0 (2 genes completely linked) to 0.50 (2 genes unlinked).
STEPS INVOLVED
There are 4 basic steps in the method:-
(1) Determine the expected frequencies of F2 phenotypes for every value of R from 0.01 to 0.50
(2) Determine the "likelihood" (L) that the family data observed resulted from a given R value: the maximum likelihood is the best estimate of R for the given data
(3) Determine the Odds Ratio and the logarithm of the odds ratio (lod score) by comparing the Likelihood for each value of R to the Likelihood for unlinked genes (R = 0.50)
(4) Add LOD scores from different families to achieve an acceptably high lod score so a specific most likely R can be assigned.
The following example used for consideration comprises of:-
Two genes showing the complete dominancethe heterozygote is indistinguishable from the dominant homozygote
STEP 1: CALCULATE THE EXPECTED FREQUENCY OF OFFSPRING FOR VALUES OF R FROM 0 TO 0.50
The expected offspring numbers are calculated as follows:
Determine the frequency of each gamete produced by the F1's. For example, if R= 0.20, then 20% of the gametes produced by either parent will be recombinant. Since there are two types of recombinant gamete, A b and a B, the frequency of each will be 0.10. Since 80% of the gametes will be parental, the frequency of the parental types A B and a b will be 0.40 each.
Use a Punnett square to determine the offspring being formed from the union of the gametes. Multiply the gamete frequencies to get the offspring frequency. For instance, one cell of the Punnett square has the A B gamete from the father combining with the A b gamete from the mother. The frequency of the A B gamete is 0.40 and the frequency of the A b gamete is 0.10. Thus the frequency of the offspring in this cell is 0.40 x 0.10 = 0.04.
Determine the phenotype for each cell in the Punnett square and add up the frequencies to get the total frequency for each offspring phenotype.
A B
0.40 A b
0.10 a B
0.10 a b
0.40
A B
0.40 A B/A B
0.16 A b/A B
0.04 a B/A B
0.04 a b/A B
0.16
A b
0.10 A B/A b
0.04 A b/A b
0.01 a B/A b
0.01 a b/A b
0.04
a B
0.10 A B/a B
0.04 A b/a B
0.01 a B/a B
0.01 a b/a B
0.04
a b
0.40 A B/a b
0.16 A b/a b
0.04 a B/a b
0.04 a b/a b
0.16
F2 Phenotype Cell Sums Expected Freq
A_ B_ 0.16+.04+.04+.16+.04+.01+.04+.01+0.16 0.66
A_ bb 0.01 + 0.04 + 0.04 0.09
aa B_ 0.01 + 0.04 + 0.04 0.09
aa bb 0.16 0.16
Using a Punnett square to determine the genotypes and multiplying the frequencies of the two gametes that go into each type of offspring, then adding up offspring that have the same phenotype.
STEP 2: EXAMINE THE OBSERVED FAMILY DATA IN LIGHT OF THE EXPECTED DISTRIBUTION OF OFFSPRING FOR EACH R VALUE
This is done by determining the likelihood (L) of the observed family for each value of R. The likelihood is simply the probability of the observed family, as determined using the multinomial theorem, an extension of the binomial theorem.
First define the terms for the observed family:
a = number of A_ B_ offspring
b = number of A_ bb offspring
c = number of aa B_ offspring
d = number of aa bb offspring
n = total offspring (= a + b + c + d)
Then define the terms for the expected family proportions (obtained from step 1 above):
p = expected proportion of A _ B _ offspring
q = expected proportion of A_ bb offspring
r = expected proportion of aa B_ offspring
s = expected proportion of aa bb offspring
The term of the multinomial equation that describes the actual family is: pa qb rc sd multiplied by a coefficient.
The coefficient is: n! /(a! b! c! d!), where ! means "factorial".
This is very similar to the coefficient for the binomial.
Thus, the likelihood equation is: L = [n! /(a! b! c! d!)]pa qb rc sd
Above calculated the expected phenotype proportions for R = 0.20 (20 map units between A and B). They are: A_ B_ = 0.66; A_ bb = 0.09; aa B_ = 0.09; aa bb = 0.16. A family of 5 children has 2 with the A_ B_ phenotype, 1 with aa B_, and 2 with aa bb.
L = (5!/2! 0! 1! 2!)(.66)2(.09)0(.09)1(.16)2
L = 30(.4356)(.09)(.0256)
L = 0.0301
The likelihood (L) needs to be calculated for all values of R between 0.01 and 0.50. Note that the coefficient will be the same for all values of R; the coefficient only depends on the observed data. When this is done, the value of R with the highest likelihood is the best estimate of R that can be obtained with data from this particular family.
STEPS 3 AND 4: COMBINING DATA FROM SEVERAL FAMILIES
The data needs to be compared and added from several different families, to get a good estimate of R. To do this, the L values must be standardized by calculating the Odds Ratio (OR), which is the ratio of the L for each R value divided by the L for R = 0.50 (unlinked). Then, the logarithm of the Odds ratio is taken; this is the LOD score. LOD scores from different families can be added. (This is equivalent to multiplying the Odds Ratios, as in the AND rule for two events--family 1 AND family 2--both occurring.) A total LOD score for some R value of 3.0 is considered proof of linkage between the two genes.
For R = 0.20, the Odds Ratio = L0.20 / L0.50. We calculated L0.20 = 0.0301 above; L0.50 = 0.00695. The Odds ratio is thus 4.331 and the LOD score is the base 10 logarithm of this, 0.637. Clearly it would take several families of this size to reach a LOD score of 3.0.
REFRENCES
Discovery and Types of Genetic Linkage By: Ingrid Lobo, Ph.D. (Write Science Right) & Kenna Shaw, Ph.D. (Executive Editor, Nature Education) © 2008 Nature Education Citation: Lobo, I. & Shaw, K. (2008) Discovery and types of genetic linkage. Nature Education 1(1)
http://www.bios.niu.edu/johns/lodprob.htm
http://www.ornl.gov/sci/techresources/Human_Genome/publicat/primer/fig8.html
http://www.bio.davidson.edu/courses/genomics/method/RFLP.html
http://biotech.about.com/od/glossary/g/RFLPdef.htm
http://www.nlm.nih.gov/visibleproofs/education/dna/rflp.pdf
http://www.biology-online.org/dictionary/Genetic_linkage
European Journal of Human Genetics (2007) 15, 362–368. doi:10.1038/sj.ejhg.5201761.
Protocol Exchange (2007) doi:10.1038/nprot.2007.343.
my project on RIBOZYMES
RIBOZYMES__________________________________________
The structural molecular biology of ribozymes took another great leap forward during the past two years. Before ribozymes were discovered in the early 1980s, all enzymes were thought to be proteins. No detailed structural information on ribozymes became available until 1994. Now, within the past two years, near atomic resolution crystal structures are available for almost all of the known ribozymes. The latest additions include ribonuclease P, group I intron structures, the ribosome (the peptidyl transferase appears to be a ribozyme) and several smaller ribozymes, including a Diels–Alderase, the glmS ribozyme and a new hammerhead ribozyme structure that reconciles 12 years of discord. Although not all ribozymes are metalloenzymes, acid-base catalysis appears to be a universal property shared by all ribozymes as well as many of their protein cousins
Introduction
Ribozymes are enzymes whose catalytic centers are composed entirely of RNA and therefore do not require proteins for catalysis (although many exist naturally as RNA–protein complexes).
All ribozymes were believed originally to be metalloenzymes, requiringMg2+ or other divalentmetal ions for both folding and catalysis. A ‘two-metal mechanism’ had been proposed in which hydratedMg2+ ions played the roles of general acids and bases. This prediction appears to have been correct for the group I intron. Acid-base catalysis appears to be a catalytic strategy so fundamental that it occurs in both protein and RNA enzymes; in many cases, it seems that the RNA itself, rather than acting as a passive scaffold for metal ion binding, is an active participant in acid-base catalysis in the sense that nucleotide functional groups, rather than metal complexes, often mimic the roles that amino acids play in the active sites of protein enzymes. Several of the small self-cleaving RNAs as a consequence do not strictly require divalent metal ions for catalysis and no divalent metal ions have yet been observed in the
active site of the peptidyltransferase, the ribozyme that is embedded in the ribosome.
Ribonuclease P
Ribonuclease P (RNase P) was the first true RNA enzyme identified. An RNA–protein complex, the catalytic subunit of bacterial RNase P is composed entirely of RNA (and it is thought that this is the case with the eukaryotic version as well). It processes precursor tRNAs and other RNAs required for cellular metabolism.
Group I intron
The folds of the various group I introns are quite similar, permitting comparisons between molecular species. The first Azoarcus structure was in a pre-catalytic state, in which both exons (the substrate of the reaction in which adjacent exons are spliced as the intron excises itself) were present. The Tetrahymena group I intron structure represents a state in which the 30 -terminal v-guanosine and a metal ion are present in the active site. The newer structures complement these two states with an enzyme– product complex, and a complex in which all substrate, ribozyme functional groups and predicted metal ions are present in the active site.
A Diels–Alderase ribozyme
The protein Diels–Alderase is a catalytic antibody whose structure is known. The structure of a Diels–Alder ribozyme is in both the unbound and enzyme–product complex states, revealing that the ribozyme uses a combination of proximity, spatial complementarity and electronic effects to activate stereoselective catalysis, reminiscent of the protein Diels–Alderase.
The glmS ribozyme
The glmS ribozyme is a recently discovered ribozyme that is unique in the world of naturally occurring ribozymes in two respects. First, it is a ribozyme that is also a riboswitch. Second, the regulatory effector of the ribozyme, glucosamine-6-phosphate (GlcN6P), is actually a functional group that binds to the ribozyme active site and participates in the acid-base catalysis of RNA self-cleavage. The glmS ribozyme is derived from a self-cleaving RNA sequence found in the 50 -untranslatedregion (50 - UTR) of the glmS message; it cleaves itself, inactivating the message, when the cofactor GlcN6P binds. GlcN6P production is thus regulated in many Gram-positive bacteria via this ribozyme-mediated negative-feedback mechanism.
The hammerhead ribozyme
Because it is small and has a simple cleavage mechanism, the hammerhead ribozyme is perhaps the best experimentally characterized RNA enzyme, and therefore, is a clear candidate for
Structural studies. The hammerhead motif consists of three base-paired stems flanking a central core of 15 conserved nucleotides. The conserved central bases are essential for ribozyme activity. Most of these conserved bases cannot form conventional Watson-Crick base pairs, but instead form more complex structures, which mediate RNA folding and catalysis. Substitution of any of the conserved bases with other naturally occurring bases, or sometimes even artificial alteration of their functional groups, results in diminished catalytic activity. In addition, two sets of base pairs in stem III and one pair in stem II are conserved; changing these to other base pairs either impairs or abolishes catalytic function.
The structural molecular biology of ribozymes took another great leap forward during the past two years. Before ribozymes were discovered in the early 1980s, all enzymes were thought to be proteins. No detailed structural information on ribozymes became available until 1994. Now, within the past two years, near atomic resolution crystal structures are available for almost all of the known ribozymes. The latest additions include ribonuclease P, group I intron structures, the ribosome (the peptidyl transferase appears to be a ribozyme) and several smaller ribozymes, including a Diels–Alderase, the glmS ribozyme and a new hammerhead ribozyme structure that reconciles 12 years of discord. Although not all ribozymes are metalloenzymes, acid-base catalysis appears to be a universal property shared by all ribozymes as well as many of their protein cousins
Introduction
Ribozymes are enzymes whose catalytic centers are composed entirely of RNA and therefore do not require proteins for catalysis (although many exist naturally as RNA–protein complexes).
All ribozymes were believed originally to be metalloenzymes, requiringMg2+ or other divalentmetal ions for both folding and catalysis. A ‘two-metal mechanism’ had been proposed in which hydratedMg2+ ions played the roles of general acids and bases. This prediction appears to have been correct for the group I intron. Acid-base catalysis appears to be a catalytic strategy so fundamental that it occurs in both protein and RNA enzymes; in many cases, it seems that the RNA itself, rather than acting as a passive scaffold for metal ion binding, is an active participant in acid-base catalysis in the sense that nucleotide functional groups, rather than metal complexes, often mimic the roles that amino acids play in the active sites of protein enzymes. Several of the small self-cleaving RNAs as a consequence do not strictly require divalent metal ions for catalysis and no divalent metal ions have yet been observed in the
active site of the peptidyltransferase, the ribozyme that is embedded in the ribosome.
Ribonuclease P
Ribonuclease P (RNase P) was the first true RNA enzyme identified. An RNA–protein complex, the catalytic subunit of bacterial RNase P is composed entirely of RNA (and it is thought that this is the case with the eukaryotic version as well). It processes precursor tRNAs and other RNAs required for cellular metabolism.
Group I intron
The folds of the various group I introns are quite similar, permitting comparisons between molecular species. The first Azoarcus structure was in a pre-catalytic state, in which both exons (the substrate of the reaction in which adjacent exons are spliced as the intron excises itself) were present. The Tetrahymena group I intron structure represents a state in which the 30 -terminal v-guanosine and a metal ion are present in the active site. The newer structures complement these two states with an enzyme– product complex, and a complex in which all substrate, ribozyme functional groups and predicted metal ions are present in the active site.
A Diels–Alderase ribozyme
The protein Diels–Alderase is a catalytic antibody whose structure is known. The structure of a Diels–Alder ribozyme is in both the unbound and enzyme–product complex states, revealing that the ribozyme uses a combination of proximity, spatial complementarity and electronic effects to activate stereoselective catalysis, reminiscent of the protein Diels–Alderase.
The glmS ribozyme
The glmS ribozyme is a recently discovered ribozyme that is unique in the world of naturally occurring ribozymes in two respects. First, it is a ribozyme that is also a riboswitch. Second, the regulatory effector of the ribozyme, glucosamine-6-phosphate (GlcN6P), is actually a functional group that binds to the ribozyme active site and participates in the acid-base catalysis of RNA self-cleavage. The glmS ribozyme is derived from a self-cleaving RNA sequence found in the 50 -untranslatedregion (50 - UTR) of the glmS message; it cleaves itself, inactivating the message, when the cofactor GlcN6P binds. GlcN6P production is thus regulated in many Gram-positive bacteria via this ribozyme-mediated negative-feedback mechanism.
The hammerhead ribozyme
Because it is small and has a simple cleavage mechanism, the hammerhead ribozyme is perhaps the best experimentally characterized RNA enzyme, and therefore, is a clear candidate for
Structural studies. The hammerhead motif consists of three base-paired stems flanking a central core of 15 conserved nucleotides. The conserved central bases are essential for ribozyme activity. Most of these conserved bases cannot form conventional Watson-Crick base pairs, but instead form more complex structures, which mediate RNA folding and catalysis. Substitution of any of the conserved bases with other naturally occurring bases, or sometimes even artificial alteration of their functional groups, results in diminished catalytic activity. In addition, two sets of base pairs in stem III and one pair in stem II are conserved; changing these to other base pairs either impairs or abolishes catalytic function.
my project on GENE THERAPY
GENE THERAPY_________________________________________
GENE THERAPY
Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease.
Gene therapy is an experimental technique that uses genes to treat or prevent disease. In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient’s cells instead of using drugs or surgery.
It is a technique for correcting defective genes that are responsible for disease development. The most common form of gene therapy involves the insertion of functional genes into an unspecified genomic location in order to replace a mutatedgene, but other forms involve directly correcting the mutation or modifying normal gene that enables a viral infection. Although the technology is still in its infancy, it has been used with some success.
APPROACHES OF GENE THERAPY
• Replacing a mutated gene that causes disease with a healthy copy of the gene.
• Inactivating, or “knocking out,” a mutated gene that is functioning improperly.
• Introducing a new gene into the body to help fight a disease.
TYPES OF GENE THERAPY_______________________________
Gene therapy may be classified into the two following types:
Germ line gene therapy
In the case of germ line gene therapy, germ cells, i.e., sperm or eggs are modified by the introduction of functional genes, which are integrated into their genomes. Therefore, the change due to therapy would be heritable and would be passed on to later generations. This new approach, theoretically, should be highly effective in counteracting genetic disorders and hereditary diseases. However, many jurisdictions prohibit this for application in human beings, at least for the present, for a variety of technical and ethical reasons.
Somatic gene therapy
In the case of somatic gene therapy, the therapeutic genes are transferred into the somatic cells of a patient. Any modifications and effects will be restricted to the individual patient only, and will not be inherited by the patient's offspring or later generations.
Somatic cells are non-reproductive. Somatic cell therapy is viewed as a more conservative, safer approach because it affects only the targeted cells in the patient, and is not passed on to future generations. In other words, the therapeutic effect ends with the individual who receives the therapy. This type of therapy presents unique problems of its own. Often the effects of somatic cell therapy are short-lived. Because the cells of most tissues ultimately die and are replaced by new cells, repeated treatments over the course of the individual's life span are required to maintain the therapeutic effect. Transporting the gene to the target cells or tissue is also problematic.
Regardless of these difficulties, however, somatic cell gene therapy is appropriate and acceptable for many disorders, including cystic fibrosis, muscular dystrophy, cancer, and certain infectious diseases. Clinicians can even perform this therapy in utero, potentially correcting or treating a life-threatening disorder that may significantly impair a baby's health or development if not treated before birth.
CATEGORIES OF SOMATIC GENE THERAPY________________________
Somatic gene therapy can be broadly split into two categories:
EX VIVO TECHNIQUE
Ex vivo, which means exterior (where cells are modified outside the body and then transplanted back in again). In some gene therapy clinical trials, cells from the patient’s blood or bone marrow are removed and grown in the laboratory. The cells are exposed to the virus that is carrying the desired gene. The virus enters the cells and inserts the desired gene into the cells’ DNA. The cells grow in the laboratory and are then returned to the patient by injection into a vein. This type of gene therapy is called ex vivo because the cells are treated outside the body.
IN VIVO TECHNIQUE
In vivo, which means interior (where genes are changed in cells still in the body). This form of gene therapy is called in vivo, because the gene is transferred to cells inside the patient’s body.
Uses of gene therapy
Gene therapy is being used in many ways. For example, to:
• Replace missing or defective genes;
• Deliver genes that speed the destruction of cancer cells;
• Supply genes that cause cancer cells to revert back to normal cells;
• Deliver bacterial or viral genes as a form of vaccination;
• Provide genes that promote or impede the growth of new tissue; and;
• Deliver genes that stimulate the healing of damaged tissue.
A large variety of genes are now being tested for use in gene therapy.
Examples : a gene for the treatment of cystic fibrosis (a gene called CFTR that regulates chloride); genes for factors VIII and IX, deficiency of which is responsible for classic hemophilia (hemophilia A) and another form of hemophilia (hemophilia B), respectively; genes called E1A and P53 that cause cancer cells to undergo cell death or revert to normal; AC6 gene which increases the ability of the heart to contract and may help in heart failure; and VEGF, a gene that induces the growth of new blood vessels (angiogenesis) of use in blood vessel disease.
A short synthetic piece of DNA (called an oligonucleotide) is being used by researchers to "pre-treat" veins used as grafts for heart bypass surgery. The piece of DNA seems to switch off certain genes in the grafted veins to prevent their cells from dividing and thereby prevent atherosclerosis.
Delivery of genes into cells
Genes can be carried into cells by viruses. Viral vectors or carriers take advantage of the natural ability of a virus to enter a cell and deliver genetic material to the nucleus of the cell that contains its DNA. In developing virus carriers, the DNA coding for some or all of the normal genes of the virus to be used as a carrier are removed and replaced with a treatment gene. Most of these virus carriers are engineered so that they are able to enter cells, but they cannot reproduce themselves and so are innocuous.
Genes can also be delivered within tiny synthetic "envelopes" of fat molecules. Cell membranes contain a very high concentration of fat molecules. The fat molecule "envelope" can carry the therapeutic gene into the cell by being admitted through the cell membrane as if it were one of its own molecules.
Genes can also gain entrance into cells when an electrical charge is applied to the cell to create tiny openings in the membrane that surrounds a cell. This technique is called electroporation.
Route of administration of gene therapy
The choice of route for gene therapy depends on the tissue to be treated and the mechanism by which the therapeutic gene exerts its effect. Gene therapy for cystic fibrosis, a disease which effects cells within the lung and airway, may be inhaled. Most genes designed to treat cancer are injected directly into the tumor. Proteins such as factor VIII or IX for hemophilia are also being introduced directly into target tissue (the liver).
PROBLEMS OF GENE THERAPY____________________________
Some of the problems of gene therapy include:
• Short-lived nature of gene therapy – Before gene therapy can become a permanent cure for any condition, the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy.
• Immune response – Anytime a foreign object is introduced into human tissues, the immune system has evolved to attack the invader. The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a possibility. Furthermore, the immune system's enhanced response to invaders that it has seen before makes it difficult for gene therapy to be repeated in patients.
• Problems with viral vectors – Viruses, the carrier of choice in most gene therapy studies, present a variety of potential problems to the patient —toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease.
• Multigene disorders – Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. Unfortunately, some of the most commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes. Multigene or multifactorial disorders such as these would be especially difficult to treat effectively using gene therapy.
• Chance of inducing a tumor (insertional mutagenesis) - If the DNA is integrated in the wrong place in the genome, for example in a tumor suppressor gene, it could induce a tumor. This has occurred in clinical trials for X-linked severe combined immunodeficiency (X-SCID) patients, in which hematopoietic stem cells were transduced with a corrective transgene using a retrovirus, and this led to the development of T cell leukemia in 3 of 20 patients.
APPLICATIONS OF GENE THERAPY________________________
A "bionic chip"
A new "bionic chip" has been developed to help gene therapists using electroporation to slip fragments of DNA into cells. Electroporation was originally a hit-or-miss technique because there was no way to determine how much of an electrical jolt it took to open the cell membrane.
The "bionic chip" solves this problem. It contains a single living cell embedded in a tiny silicon circuit. The cell acts as a diode, or electrical gate. When it is hit with just the right charge, the cell membrane opens, allowing the electricity to pass from the top to the bottom of the bionic chip. By recording what voltage caused this phenomenon to occur, it is now posssible to determine precisely how much electricity it takes to pry open different types of cells.
GENE THERAPY IN FUTURE______________________________
The potential of gene therapy is great but, compared to its promise, the results to date are still quite limited. However, the benefits of gene therapy are believed to be on the near horizon. Gene therapy is one of the hottest areas of medical research today.
The remarkable advances in genetics, including the human genome project, have opened new doors for the exploration of gene therapy. New technologies are needed to speed the progress of gene therapy. As these new technologies such as the "bionic chip" arrive, we believe that, without a doubt, gene therapy will play an increasingly important and prominent part in medicine in the decades to come.
GENE THERAPY
Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease.
Gene therapy is an experimental technique that uses genes to treat or prevent disease. In the future, this technique may allow doctors to treat a disorder by inserting a gene into a patient’s cells instead of using drugs or surgery.
It is a technique for correcting defective genes that are responsible for disease development. The most common form of gene therapy involves the insertion of functional genes into an unspecified genomic location in order to replace a mutatedgene, but other forms involve directly correcting the mutation or modifying normal gene that enables a viral infection. Although the technology is still in its infancy, it has been used with some success.
APPROACHES OF GENE THERAPY
• Replacing a mutated gene that causes disease with a healthy copy of the gene.
• Inactivating, or “knocking out,” a mutated gene that is functioning improperly.
• Introducing a new gene into the body to help fight a disease.
TYPES OF GENE THERAPY_______________________________
Gene therapy may be classified into the two following types:
Germ line gene therapy
In the case of germ line gene therapy, germ cells, i.e., sperm or eggs are modified by the introduction of functional genes, which are integrated into their genomes. Therefore, the change due to therapy would be heritable and would be passed on to later generations. This new approach, theoretically, should be highly effective in counteracting genetic disorders and hereditary diseases. However, many jurisdictions prohibit this for application in human beings, at least for the present, for a variety of technical and ethical reasons.
Somatic gene therapy
In the case of somatic gene therapy, the therapeutic genes are transferred into the somatic cells of a patient. Any modifications and effects will be restricted to the individual patient only, and will not be inherited by the patient's offspring or later generations.
Somatic cells are non-reproductive. Somatic cell therapy is viewed as a more conservative, safer approach because it affects only the targeted cells in the patient, and is not passed on to future generations. In other words, the therapeutic effect ends with the individual who receives the therapy. This type of therapy presents unique problems of its own. Often the effects of somatic cell therapy are short-lived. Because the cells of most tissues ultimately die and are replaced by new cells, repeated treatments over the course of the individual's life span are required to maintain the therapeutic effect. Transporting the gene to the target cells or tissue is also problematic.
Regardless of these difficulties, however, somatic cell gene therapy is appropriate and acceptable for many disorders, including cystic fibrosis, muscular dystrophy, cancer, and certain infectious diseases. Clinicians can even perform this therapy in utero, potentially correcting or treating a life-threatening disorder that may significantly impair a baby's health or development if not treated before birth.
CATEGORIES OF SOMATIC GENE THERAPY________________________
Somatic gene therapy can be broadly split into two categories:
EX VIVO TECHNIQUE
Ex vivo, which means exterior (where cells are modified outside the body and then transplanted back in again). In some gene therapy clinical trials, cells from the patient’s blood or bone marrow are removed and grown in the laboratory. The cells are exposed to the virus that is carrying the desired gene. The virus enters the cells and inserts the desired gene into the cells’ DNA. The cells grow in the laboratory and are then returned to the patient by injection into a vein. This type of gene therapy is called ex vivo because the cells are treated outside the body.
IN VIVO TECHNIQUE
In vivo, which means interior (where genes are changed in cells still in the body). This form of gene therapy is called in vivo, because the gene is transferred to cells inside the patient’s body.
Uses of gene therapy
Gene therapy is being used in many ways. For example, to:
• Replace missing or defective genes;
• Deliver genes that speed the destruction of cancer cells;
• Supply genes that cause cancer cells to revert back to normal cells;
• Deliver bacterial or viral genes as a form of vaccination;
• Provide genes that promote or impede the growth of new tissue; and;
• Deliver genes that stimulate the healing of damaged tissue.
A large variety of genes are now being tested for use in gene therapy.
Examples : a gene for the treatment of cystic fibrosis (a gene called CFTR that regulates chloride); genes for factors VIII and IX, deficiency of which is responsible for classic hemophilia (hemophilia A) and another form of hemophilia (hemophilia B), respectively; genes called E1A and P53 that cause cancer cells to undergo cell death or revert to normal; AC6 gene which increases the ability of the heart to contract and may help in heart failure; and VEGF, a gene that induces the growth of new blood vessels (angiogenesis) of use in blood vessel disease.
A short synthetic piece of DNA (called an oligonucleotide) is being used by researchers to "pre-treat" veins used as grafts for heart bypass surgery. The piece of DNA seems to switch off certain genes in the grafted veins to prevent their cells from dividing and thereby prevent atherosclerosis.
Delivery of genes into cells
Genes can be carried into cells by viruses. Viral vectors or carriers take advantage of the natural ability of a virus to enter a cell and deliver genetic material to the nucleus of the cell that contains its DNA. In developing virus carriers, the DNA coding for some or all of the normal genes of the virus to be used as a carrier are removed and replaced with a treatment gene. Most of these virus carriers are engineered so that they are able to enter cells, but they cannot reproduce themselves and so are innocuous.
Genes can also be delivered within tiny synthetic "envelopes" of fat molecules. Cell membranes contain a very high concentration of fat molecules. The fat molecule "envelope" can carry the therapeutic gene into the cell by being admitted through the cell membrane as if it were one of its own molecules.
Genes can also gain entrance into cells when an electrical charge is applied to the cell to create tiny openings in the membrane that surrounds a cell. This technique is called electroporation.
Route of administration of gene therapy
The choice of route for gene therapy depends on the tissue to be treated and the mechanism by which the therapeutic gene exerts its effect. Gene therapy for cystic fibrosis, a disease which effects cells within the lung and airway, may be inhaled. Most genes designed to treat cancer are injected directly into the tumor. Proteins such as factor VIII or IX for hemophilia are also being introduced directly into target tissue (the liver).
PROBLEMS OF GENE THERAPY____________________________
Some of the problems of gene therapy include:
• Short-lived nature of gene therapy – Before gene therapy can become a permanent cure for any condition, the therapeutic DNA introduced into target cells must remain functional and the cells containing the therapeutic DNA must be long-lived and stable. Problems with integrating therapeutic DNA into the genome and the rapidly dividing nature of many cells prevent gene therapy from achieving any long-term benefits. Patients will have to undergo multiple rounds of gene therapy.
• Immune response – Anytime a foreign object is introduced into human tissues, the immune system has evolved to attack the invader. The risk of stimulating the immune system in a way that reduces gene therapy effectiveness is always a possibility. Furthermore, the immune system's enhanced response to invaders that it has seen before makes it difficult for gene therapy to be repeated in patients.
• Problems with viral vectors – Viruses, the carrier of choice in most gene therapy studies, present a variety of potential problems to the patient —toxicity, immune and inflammatory responses, and gene control and targeting issues. In addition, there is always the fear that the viral vector, once inside the patient, may recover its ability to cause disease.
• Multigene disorders – Conditions or disorders that arise from mutations in a single gene are the best candidates for gene therapy. Unfortunately, some of the most commonly occurring disorders, such as heart disease, high blood pressure, Alzheimer's disease, arthritis, and diabetes, are caused by the combined effects of variations in many genes. Multigene or multifactorial disorders such as these would be especially difficult to treat effectively using gene therapy.
• Chance of inducing a tumor (insertional mutagenesis) - If the DNA is integrated in the wrong place in the genome, for example in a tumor suppressor gene, it could induce a tumor. This has occurred in clinical trials for X-linked severe combined immunodeficiency (X-SCID) patients, in which hematopoietic stem cells were transduced with a corrective transgene using a retrovirus, and this led to the development of T cell leukemia in 3 of 20 patients.
APPLICATIONS OF GENE THERAPY________________________
A "bionic chip"
A new "bionic chip" has been developed to help gene therapists using electroporation to slip fragments of DNA into cells. Electroporation was originally a hit-or-miss technique because there was no way to determine how much of an electrical jolt it took to open the cell membrane.
The "bionic chip" solves this problem. It contains a single living cell embedded in a tiny silicon circuit. The cell acts as a diode, or electrical gate. When it is hit with just the right charge, the cell membrane opens, allowing the electricity to pass from the top to the bottom of the bionic chip. By recording what voltage caused this phenomenon to occur, it is now posssible to determine precisely how much electricity it takes to pry open different types of cells.
GENE THERAPY IN FUTURE______________________________
The potential of gene therapy is great but, compared to its promise, the results to date are still quite limited. However, the benefits of gene therapy are believed to be on the near horizon. Gene therapy is one of the hottest areas of medical research today.
The remarkable advances in genetics, including the human genome project, have opened new doors for the exploration of gene therapy. New technologies are needed to speed the progress of gene therapy. As these new technologies such as the "bionic chip" arrive, we believe that, without a doubt, gene therapy will play an increasingly important and prominent part in medicine in the decades to come.
Saturday, July 23, 2011
Developmental genetics
GO TO THIS LINK PLZ
http://www.slideshare.net/xulkifal/developmental-genetics-4294650
www.slideshare.net/xulkifal/developmental-genetics-4294650
http://www.slideshare.net/xulkifal/developmental-genetics-4294650
www.slideshare.net/xulkifal/developmental-genetics-4294650
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