Huntington's Disease Collaborative Research
Huntington’s disease is an autosomal dominant neurodegenerative disorder characterized by an abnormally high number of CAG repeats (polyglutamine) in the huntingtin (HTT) gene (also known as IT15), found in the short arm of chromosome 4, specifically, p16.3 (Buetow et al. 1991). Symptoms of this disorder include chorea (involuntary jerking movements), cognitive deficits, motor deficits, and changes in behavior. This disease gets progressively worse over time, starting with subtle behavioral, cognitive, and physical changes, progressing to severe memory decline, substantial personality changes, and debilitating motor and physiological deficits, and ending in death due to the incapacitating symptoms (Zuccato et al. 2010). Patients are generally
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diagnosed in their 30s or 40s and succumb to the disease roughly 15-20 years following diagnosis; the larger the polyglutamine repeat, the earlier the symptoms start and the more devastating they are (Zuccato et al. 2010).
Mapping the HTT gene was a collaborative effort of a team of 58 scientists from all over the world known collectively as the Huntington Disease Collaborative Research Group.
It started in 1983 when Gusella et al. mapped the HTT gene using restriction fragment length polymorphisms (RFLPs). Two families were used for the linkage analysis, an American family and a Venezuelan family, and a total of 12 DNA probes were used to detect RFLPs, one of which, G8, suggested linkage to the gene. The LOD score for this locus was 1.81 for the American family and 6.72 for the Venezuelan family with a recombination frequency (훳) of 0, suggesting that two polymorphic sites in the G8 locus are closely linked. Using somatic cell hybridization, Gusella et al. mapped the G8 sequence to chromosome 4 (1983). In 1987, Gilliam et al. used RFLPs and DNA probes to map the gene using two American families and one Venezuelan family. The LOD score of the HD gene and D4S43, a marker closely linked to another HD marker (D4S10), was 1.85 in one of the American families, 5.28 in the other American family, and 37.62 in the Venezualan family, with a recombination frequency of 0.00., thus, HD was mapped to 4p16.3, where these markers resided. MacDonald et al. (1992) ultimately mapped the genetic defect to an area between D4S182 and D4S180 of 4p16.3 using haplotype analysis of two Western European Descent
families.
In 1993, the Huntington’s Disease Collaborative Research Group targeted the proximal area between D4S180 and D4S182. Using polymerase chain reaction (PCR) to amplify exons, the group cloned what is now known as the IT15 gene, and found that this sequence contains a CAG trinucleotide repeat. In the non-HD population, CAG repeats of 11 to 34 were seen, whereas, those with HD had CAG trinucleotide repeats that ranged from 42 to over 66; in some cases, the repeats were so long that they failed to yield a product. The group studied 75 HD families from various ethnic backgrounds and found similar results among all, suggesting that this aberrant trinucleotide repeat, found near the 5’ end of the causative gene, likely causes HD (The Huntington’s Disease Collaborative Research Group 1993).
With the clone of the gene, Mangiarini et al. developed a transgenic mouse model of HD (1996). To do this, her and her colleagues microinjected a fragment of DNA containing the first exon and part of the first intron of an expanded HTT gene (containing about 130 CAG repeats) into the embryos of wild-type mice. One male, known as R6, of the 29 newborn mouse was found to be transgenic. R6 was bred to wild-type females, and progeny were genotyped using PCR and Southern blots to determine CAG repeat size. One of the lines, known as R6/2, containing 141 to 157 CAG repeats, showed extremely early HD phenotypes most frequently between nine and eleven weeks. Mangiarini et al. (1996) observed that the transgenic mice died between 10 and 13 weeks, and they observed a progressive neurological phenotype, similar to human HD. The phenotypes observed included movements similar to chorea, resting tremors, clasping, loss of balance when standing on hind limbs, progressive weight loss, and in some cases, epileptic seizures. Mice with the transgene had on average 19% smaller brains, with uniform reduction in sizes of all CNS structures, most notably the striatum (Mangiarini et al. 1996).
R6/2 transgenic mice were further studied in 1997 by Davies et al. This study further characterized the neuropathology, specifically neuronal intranuclear inclusions (NIIs), seen in these mice using immunohistochemistry (IHC). The authors used multiple antibodies against huntingtin to characterize NIIs; an antibody that binds to ubiquitin, a protein that tags other proteins for degradation; antibodies to Fos B and NGIF-A, both transcription factors (TFs); and, antibodies to tau and beta-amyloid, again, to characterize neuronal intranuclear inclusions. The results of IHC were as follows: htt was found in all regions of the CNS; htt as well as ubiquitin was found within the NIIs; tau nor beta-amyloid were seen within the NIIs; similarly, Fos B and NGFI-A were not detected in the NIIs. The authors did note that the largest inclusions were seen in the cerebral cortex, the cerebellum, and most severely, the striatum. This led Davies et al. to conclude that the molecular mechanism associated with the neurological dysfunction seen in R6/2 mice is likely due to these NIIs, specifically in the striatum (1997).
More recently, Yang et al. (2008) developed a transgenic HD model in rhesus macaques. Macaque oocytes were injected with lentiviruses containing exon 1 of an expanded HTT gene as well as the green fluorescent protein (GFP) reporter gene and introduced to surrogate mothers. Of the 130 oocytes, only five resulted in live births. To ensure integration of the transgene, PCR and southern blot analysis for each of the five transgenic animals was carried out. Confirmation that the transgene was being expressed was confirmed by western blot and IHC. PCR and southern blot results showed variations in the number of CAG repeats, ranging from 27 to 88. Both western blot analysis and IHC confirmed the presence of the expanded HTT gene. IHC results also showed aggregation of HTT in the striatum and cortex of some of the transgenic animals. In addition, Yang et al. observed motor deficits such as chorea and irregular movement coordination, and noticed that the severity of symptoms worsened in correlation with a higher number of CAG repeats (2008).
Since HD is a genetic disorder that is inherited in a certain pattern, autosomal dominant, it is easy to predict the likelihood that a child of a parent with HD will inherit the disorder. And with the ability to sequence DNA, it is easy to genetically test newborns, children, or adults who have a family history of HD. However, predictive testing was carried out before technology allowed scientists and doctors to look at the DNA sequence of humans. In 1988, Hayden et al. published their study detailing the use of three DNA markers linked closely to the causative HD gene, D4S10, D4S62, and D4S95 to predict likelihood of inheriting the gene for HD. These markers were cloned and hybridized against the DNA of 41 patients with HD ancestry. Results given to patients estimated risk of inheriting the disease, either a decrease in risk or a decrease in risk (Hayden et al. 1988).
diagnosed in their 30s or 40s and succumb to the disease roughly 15-20 years following diagnosis; the larger the polyglutamine repeat, the earlier the symptoms start and the more devastating they are (Zuccato et al. 2010).
Mapping the HTT gene was a collaborative effort of a team of 58 scientists from all over the world known collectively as the Huntington Disease Collaborative Research Group.
It started in 1983 when Gusella et al. mapped the HTT gene using restriction fragment length polymorphisms (RFLPs). Two families were used for the linkage analysis, an American family and a Venezuelan family, and a total of 12 DNA probes were used to detect RFLPs, one of which, G8, suggested linkage to the gene. The LOD score for this locus was 1.81 for the American family and 6.72 for the Venezuelan family with a recombination frequency (훳) of 0, suggesting that two polymorphic sites in the G8 locus are closely linked. Using somatic cell hybridization, Gusella et al. mapped the G8 sequence to chromosome 4 (1983). In 1987, Gilliam et al. used RFLPs and DNA probes to map the gene using two American families and one Venezuelan family. The LOD score of the HD gene and D4S43, a marker closely linked to another HD marker (D4S10), was 1.85 in one of the American families, 5.28 in the other American family, and 37.62 in the Venezualan family, with a recombination frequency of 0.00., thus, HD was mapped to 4p16.3, where these markers resided. MacDonald et al. (1992) ultimately mapped the genetic defect to an area between D4S182 and D4S180 of 4p16.3 using haplotype analysis of two Western European Descent
families.
In 1993, the Huntington’s Disease Collaborative Research Group targeted the proximal area between D4S180 and D4S182. Using polymerase chain reaction (PCR) to amplify exons, the group cloned what is now known as the IT15 gene, and found that this sequence contains a CAG trinucleotide repeat. In the non-HD population, CAG repeats of 11 to 34 were seen, whereas, those with HD had CAG trinucleotide repeats that ranged from 42 to over 66; in some cases, the repeats were so long that they failed to yield a product. The group studied 75 HD families from various ethnic backgrounds and found similar results among all, suggesting that this aberrant trinucleotide repeat, found near the 5’ end of the causative gene, likely causes HD (The Huntington’s Disease Collaborative Research Group 1993).
With the clone of the gene, Mangiarini et al. developed a transgenic mouse model of HD (1996). To do this, her and her colleagues microinjected a fragment of DNA containing the first exon and part of the first intron of an expanded HTT gene (containing about 130 CAG repeats) into the embryos of wild-type mice. One male, known as R6, of the 29 newborn mouse was found to be transgenic. R6 was bred to wild-type females, and progeny were genotyped using PCR and Southern blots to determine CAG repeat size. One of the lines, known as R6/2, containing 141 to 157 CAG repeats, showed extremely early HD phenotypes most frequently between nine and eleven weeks. Mangiarini et al. (1996) observed that the transgenic mice died between 10 and 13 weeks, and they observed a progressive neurological phenotype, similar to human HD. The phenotypes observed included movements similar to chorea, resting tremors, clasping, loss of balance when standing on hind limbs, progressive weight loss, and in some cases, epileptic seizures. Mice with the transgene had on average 19% smaller brains, with uniform reduction in sizes of all CNS structures, most notably the striatum (Mangiarini et al. 1996).
R6/2 transgenic mice were further studied in 1997 by Davies et al. This study further characterized the neuropathology, specifically neuronal intranuclear inclusions (NIIs), seen in these mice using immunohistochemistry (IHC). The authors used multiple antibodies against huntingtin to characterize NIIs; an antibody that binds to ubiquitin, a protein that tags other proteins for degradation; antibodies to Fos B and NGIF-A, both transcription factors (TFs); and, antibodies to tau and beta-amyloid, again, to characterize neuronal intranuclear inclusions. The results of IHC were as follows: htt was found in all regions of the CNS; htt as well as ubiquitin was found within the NIIs; tau nor beta-amyloid were seen within the NIIs; similarly, Fos B and NGFI-A were not detected in the NIIs. The authors did note that the largest inclusions were seen in the cerebral cortex, the cerebellum, and most severely, the striatum. This led Davies et al. to conclude that the molecular mechanism associated with the neurological dysfunction seen in R6/2 mice is likely due to these NIIs, specifically in the striatum (1997).
More recently, Yang et al. (2008) developed a transgenic HD model in rhesus macaques. Macaque oocytes were injected with lentiviruses containing exon 1 of an expanded HTT gene as well as the green fluorescent protein (GFP) reporter gene and introduced to surrogate mothers. Of the 130 oocytes, only five resulted in live births. To ensure integration of the transgene, PCR and southern blot analysis for each of the five transgenic animals was carried out. Confirmation that the transgene was being expressed was confirmed by western blot and IHC. PCR and southern blot results showed variations in the number of CAG repeats, ranging from 27 to 88. Both western blot analysis and IHC confirmed the presence of the expanded HTT gene. IHC results also showed aggregation of HTT in the striatum and cortex of some of the transgenic animals. In addition, Yang et al. observed motor deficits such as chorea and irregular movement coordination, and noticed that the severity of symptoms worsened in correlation with a higher number of CAG repeats (2008).
Since HD is a genetic disorder that is inherited in a certain pattern, autosomal dominant, it is easy to predict the likelihood that a child of a parent with HD will inherit the disorder. And with the ability to sequence DNA, it is easy to genetically test newborns, children, or adults who have a family history of HD. However, predictive testing was carried out before technology allowed scientists and doctors to look at the DNA sequence of humans. In 1988, Hayden et al. published their study detailing the use of three DNA markers linked closely to the causative HD gene, D4S10, D4S62, and D4S95 to predict likelihood of inheriting the gene for HD. These markers were cloned and hybridized against the DNA of 41 patients with HD ancestry. Results given to patients estimated risk of inheriting the disease, either a decrease in risk or a decrease in risk (Hayden et al. 1988).