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Loss of Heterozygosity (LOH)
1120
L ong-Pe riod Inter sper sion
indicate linkage (compared to the 50:1 probability that any random pair of loci will be unlinked). See also: Linkage
Long-Period Interspersion Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1899
Long-period interspersion is a genomic pattern in which long stretches of moderately repetitive and nonrepetitive DNA alternate. See also: Genome Organization
Long Terminal Repeats (LTRs) Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1901
Long terminal repeats (LTRs) are identical DNA sequences, several hundred nucleotides in length, found at the ends of transposons and retrovirusderived DNA. LTRs contain inverted repeats and are thought to play an essential role in the integration of the transposon or provirus into the host DNA. In proviruses the upstream LTR acts as a promoter and enhancer and the downstream LTR as a polyadenylation site. See also: Provirus; Retroviruses; Transposable Elements
Loss of Heterozygosity (LOH) P Rabbitts Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1590
The development of tumors is associated with genetic damage confined to the cells of the tumor. This genetic damage can be visualized by examination of the tumor karyotype. Solid tumors, particularly those of
epithelial origin, are characterized by highly aneuploid karyotypes with deletions as a common, frequently tumor-specific feature. If a patient's normal and tumor DNA are compared at a locus known to be heterozygous in that patient's normal DNA, it is possible to determine whether the tumor DNA has suffered genetic loss (deletion) encompassing that locus. If it has, only one of the two alleles will be detectable, and the locus will appear to be homozygous in the tumor and will show loss of heterozygosity (LOH).
Sources of Heterozygosity and their Detection Within the mammalian genome, the majority of DNA is not involved in coding for proteins. Lack of selection pressure on this noncoding DNA allows inconsequential mutations to accrue. A locus at which the two parental alleles differ because of mutation is described as heterozygous/polymorphic. Single-nucleotide polymorphisms (SNPs) which form part of the recognition site for restriction enzymes were the first source of heterozygosity to be exploited for LOH analysis: first by Southern blotting, comparing normal and tumor DNA digested with the appropriate restriction enzyme, and then using PCR to amplify the region flanking the polymorphism followed by digestion of the PCR product with the restriction enzyme. However, the source of polymorphism most often used now exploits the observation that repetitive DNA occurs frequently in mammalian genomes. This DNA is often arranged in tandem repeat units, ranging in size from 8 to 50 bp, referred to as variable number of tandem repeats (VNTRs) or minisatellites. Of most value for LOH analysis are the repeat units ranging from 2 to 6 bp called `microsatellites.' Human populations are highly polymorphous in the number of these repeats, such that the average rate of heterozygosity is more than 70%. Furthermore they are abundant and evenly distributed throughout the human genome, making them ideal genetic markers. They are detected by size fractionation after amplification by PCR using priming sites which flank the repeat region. Recently there has been renewed interest in SNPs other than those involved in restriction enzyme sites. These are widely and evenly distributed throughout the human genome. Their information content is not as high as microsatellites, since they are biallelic, but the single base-change difference is much more amenable to high-throughput detection than the size differences of microsatellites, and they are likely to be the markers of choice for future genetic analyses, including LOH.
L o t u s j a p o n i c u s 1121
LOH and Location of Tumor Suppressor Genes Tumor suppressor genes are recessive and require inactivation of both alleles for a phenotypic effect. Inactivation is frequently by mutation of one allele and loss, through chromosomal deletion, of the second. Chromosomal deletion is often first discovered by cytogenetic analysis of a few samples, usually of cell lines, and then confirmed by LOH analysis of paired tumor and normal DNA from a larger number of individual patients. This requires a group of polymorphic loci within and flanking the deleted region whose relative chromosomal positions are known. Many such loci have been identified and assigned a chromosomal location (D number in humans). By comparing the delineated stretch of LOH on the chromosome in individual patients in a large number of tumor/normal pairs, a common, minimally deleted region can be defined. This is sometimes small enough (less than 1 Mb) to allow the region to be investigated for genes which can be evaluated as tumor suppressor genes. This method of gene isolation, known as positional cloning, has been effective in the isolation or confirmation of a number of tumor suppressor genes. Some tumors appear to have multiple but distinct regions of LOH on the same chromosome arm. It is uncertain whether all these regions of LOH indicate different tumor suppressor genes involved in the development of that tumor or whether some of the deletions occur as a consequence of the primary damage to the chromosome.
LOH Analysis and Clinical Research Where tumor karyotyping is difficult, tumor DNA samples can be assessed for regions of allele loss by performing LOH analysis using evenly distributed markers for all chromosomes: `allelotyping.' Different tumor types have regions of LOH in common, indicating a common defective gene in their etiology. This has been confirmed on isolation and mutation analysis of a gene within a deletion common to a variety of tumors. Despite this overlap, there are distinct patterns of LOH, sometimes associated with tumor progression, and thus loss of particular regions can have prognostic significance. The overall pattern of allele loss as determined by LOH analysis (together with any detected point mutations) can serve as a signature of an individual patient's tumor. The pattern of allele loss displayed by a tumor can be detected in material exfoliated from the tumor and sometimes in the patient's blood. This pattern, the signature, can be used as a means of following the course of disease
during treatment and can indicate relapse before obvious clinical symptoms appear.
References
Mao L (2000) Microsatellite analysis. Annals of the New York Academy of Sciences 906: 55± 62. Human SNP Database: http://www- enome.wi.mit.edu/SNP/ human/index.html Wistuba II, Behvens C, Virmani AK et al. (2000) High resolution chromosome 3 allelotyping of human lung cancer and preneoplastic/preinvasive bronchial epithelium reveals multiple discontinuous sites of 3p allele loss and three regions of frequent breakpoints. Cancer Research 60: 1949±1960.
See also: Chromosome Aberrations; Single Nucleotide Polymorphisms (SNPs); Tumor Suppressor Genes
Lotus japonicus J Stougaard Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1665
Lotus japonicus is a model plant for the legumes. The Leguminosae (or Fabaceae) family is represented by approximately 18 000 species and is the third largest family of angiosperms. With around 700 genera divided into three subfamilies, Papilionoideae, Caesalpinioideae and Mimosoideae, the Leguminosae present a wealth of diversity. Several legumes, for example pea (Pisum sativum), soybean (Glycine max), peanut (Arachis hypogaea), and beans (Phaseolus vulgaris) are well-known and important crop plants. Others are cultivated as ornamentals, vegetables, pulses, or for production of protein, oil, and pharmaceuticals. Lotus japonicus originates from East Asia and the species is distributed over the Japanese islands, the Korean peninsula, and east and central parts of China and has been reported from northern India, Pakistan, and Afghanistan. Two ecotypes `Gifu' and `Miyakojima' have been chosen for model studies. Lotus japonicus is a close relative of the tannin-containing tetraploid forage legume L. corniculatus (birdsfoot trefoil) cultivated for its antibloating properties. Phylogenetically, L. japonicus belongs to the tribe Loteae in Papilionoideae, the largest subfamily of the Leguminosae. Many cultivated legumes like pea and soybean have complex genomes or are, for other reasons, not amenable to modern molecular genetic methods. Its
L ong-Pe riod Inter sper sion
indicate linkage (compared to the 50:1 probability that any random pair of loci will be unlinked). See also: Linkage
Long-Period Interspersion Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1899
Long-period interspersion is a genomic pattern in which long stretches of moderately repetitive and nonrepetitive DNA alternate. See also: Genome Organization
Long Terminal Repeats (LTRs) Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1901
Long terminal repeats (LTRs) are identical DNA sequences, several hundred nucleotides in length, found at the ends of transposons and retrovirusderived DNA. LTRs contain inverted repeats and are thought to play an essential role in the integration of the transposon or provirus into the host DNA. In proviruses the upstream LTR acts as a promoter and enhancer and the downstream LTR as a polyadenylation site. See also: Provirus; Retroviruses; Transposable Elements
Loss of Heterozygosity (LOH) P Rabbitts Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1590
The development of tumors is associated with genetic damage confined to the cells of the tumor. This genetic damage can be visualized by examination of the tumor karyotype. Solid tumors, particularly those of
epithelial origin, are characterized by highly aneuploid karyotypes with deletions as a common, frequently tumor-specific feature. If a patient's normal and tumor DNA are compared at a locus known to be heterozygous in that patient's normal DNA, it is possible to determine whether the tumor DNA has suffered genetic loss (deletion) encompassing that locus. If it has, only one of the two alleles will be detectable, and the locus will appear to be homozygous in the tumor and will show loss of heterozygosity (LOH).
Sources of Heterozygosity and their Detection Within the mammalian genome, the majority of DNA is not involved in coding for proteins. Lack of selection pressure on this noncoding DNA allows inconsequential mutations to accrue. A locus at which the two parental alleles differ because of mutation is described as heterozygous/polymorphic. Single-nucleotide polymorphisms (SNPs) which form part of the recognition site for restriction enzymes were the first source of heterozygosity to be exploited for LOH analysis: first by Southern blotting, comparing normal and tumor DNA digested with the appropriate restriction enzyme, and then using PCR to amplify the region flanking the polymorphism followed by digestion of the PCR product with the restriction enzyme. However, the source of polymorphism most often used now exploits the observation that repetitive DNA occurs frequently in mammalian genomes. This DNA is often arranged in tandem repeat units, ranging in size from 8 to 50 bp, referred to as variable number of tandem repeats (VNTRs) or minisatellites. Of most value for LOH analysis are the repeat units ranging from 2 to 6 bp called `microsatellites.' Human populations are highly polymorphous in the number of these repeats, such that the average rate of heterozygosity is more than 70%. Furthermore they are abundant and evenly distributed throughout the human genome, making them ideal genetic markers. They are detected by size fractionation after amplification by PCR using priming sites which flank the repeat region. Recently there has been renewed interest in SNPs other than those involved in restriction enzyme sites. These are widely and evenly distributed throughout the human genome. Their information content is not as high as microsatellites, since they are biallelic, but the single base-change difference is much more amenable to high-throughput detection than the size differences of microsatellites, and they are likely to be the markers of choice for future genetic analyses, including LOH.
L o t u s j a p o n i c u s 1121
LOH and Location of Tumor Suppressor Genes Tumor suppressor genes are recessive and require inactivation of both alleles for a phenotypic effect. Inactivation is frequently by mutation of one allele and loss, through chromosomal deletion, of the second. Chromosomal deletion is often first discovered by cytogenetic analysis of a few samples, usually of cell lines, and then confirmed by LOH analysis of paired tumor and normal DNA from a larger number of individual patients. This requires a group of polymorphic loci within and flanking the deleted region whose relative chromosomal positions are known. Many such loci have been identified and assigned a chromosomal location (D number in humans). By comparing the delineated stretch of LOH on the chromosome in individual patients in a large number of tumor/normal pairs, a common, minimally deleted region can be defined. This is sometimes small enough (less than 1 Mb) to allow the region to be investigated for genes which can be evaluated as tumor suppressor genes. This method of gene isolation, known as positional cloning, has been effective in the isolation or confirmation of a number of tumor suppressor genes. Some tumors appear to have multiple but distinct regions of LOH on the same chromosome arm. It is uncertain whether all these regions of LOH indicate different tumor suppressor genes involved in the development of that tumor or whether some of the deletions occur as a consequence of the primary damage to the chromosome.
LOH Analysis and Clinical Research Where tumor karyotyping is difficult, tumor DNA samples can be assessed for regions of allele loss by performing LOH analysis using evenly distributed markers for all chromosomes: `allelotyping.' Different tumor types have regions of LOH in common, indicating a common defective gene in their etiology. This has been confirmed on isolation and mutation analysis of a gene within a deletion common to a variety of tumors. Despite this overlap, there are distinct patterns of LOH, sometimes associated with tumor progression, and thus loss of particular regions can have prognostic significance. The overall pattern of allele loss as determined by LOH analysis (together with any detected point mutations) can serve as a signature of an individual patient's tumor. The pattern of allele loss displayed by a tumor can be detected in material exfoliated from the tumor and sometimes in the patient's blood. This pattern, the signature, can be used as a means of following the course of disease
during treatment and can indicate relapse before obvious clinical symptoms appear.
References
Mao L (2000) Microsatellite analysis. Annals of the New York Academy of Sciences 906: 55± 62. Human SNP Database: http://www- enome.wi.mit.edu/SNP/ human/index.html Wistuba II, Behvens C, Virmani AK et al. (2000) High resolution chromosome 3 allelotyping of human lung cancer and preneoplastic/preinvasive bronchial epithelium reveals multiple discontinuous sites of 3p allele loss and three regions of frequent breakpoints. Cancer Research 60: 1949±1960.
See also: Chromosome Aberrations; Single Nucleotide Polymorphisms (SNPs); Tumor Suppressor Genes
Lotus japonicus J Stougaard Copyright ß 2001 Academic Press doi: 10.1006/rwgn.2001.1665
Lotus japonicus is a model plant for the legumes. The Leguminosae (or Fabaceae) family is represented by approximately 18 000 species and is the third largest family of angiosperms. With around 700 genera divided into three subfamilies, Papilionoideae, Caesalpinioideae and Mimosoideae, the Leguminosae present a wealth of diversity. Several legumes, for example pea (Pisum sativum), soybean (Glycine max), peanut (Arachis hypogaea), and beans (Phaseolus vulgaris) are well-known and important crop plants. Others are cultivated as ornamentals, vegetables, pulses, or for production of protein, oil, and pharmaceuticals. Lotus japonicus originates from East Asia and the species is distributed over the Japanese islands, the Korean peninsula, and east and central parts of China and has been reported from northern India, Pakistan, and Afghanistan. Two ecotypes `Gifu' and `Miyakojima' have been chosen for model studies. Lotus japonicus is a close relative of the tannin-containing tetraploid forage legume L. corniculatus (birdsfoot trefoil) cultivated for its antibloating properties. Phylogenetically, L. japonicus belongs to the tribe Loteae in Papilionoideae, the largest subfamily of the Leguminosae. Many cultivated legumes like pea and soybean have complex genomes or are, for other reasons, not amenable to modern molecular genetic methods. Its