Molecular analysis of new mutations for Huntington's disease: intermediate alleles and sex of origin effects.

Huntington's disease (HD) is associated with expansion of a CAG repeat in a novel gene. We have assessed 21 sporadic cases of HD to investigate sequential events underlying HD. We show the existence of an intermediate allele (IA) in parental alleles of 30-38 CAG repeats in the HD gene which is greater than usually seen in the general population but below the range seen in patients with HD. These IAs are meiotically unstable and in the sporadic cases, expand to the full mutation associated with the phenotype of HD. This expansion has been shown to occur only during transmission through the male germline and is associated with advanced paternal age. These findings suggest that new mutations for HD are more frequent than prior estimates and indicate a previously unrecognized risk of inheriting HD to siblings of sporadic cases of HD and their children.

Somatic and gonadal mosaicism of the Huntington disease gene CAG repeat in brain and sperm.

Huntington disease is associated with an unstable and expanded (CAG) trinucleotide repeat. We have analysed the CAG expansion in different tissues from 12 affected individuals. All tissues examined were found to display some repeat mosaicism, with the greatest levels detected in brain and sperm. Regions within the brain showing most obvious neuropathology, such as the basal ganglia and the cerebral cortex, displayed the greatest mosaicism, whereas the cerebellar cortex, which is seldom involved, displayed the lowest degree of CAG instability. In two cases of childhood onset disease we detected differences of 8 and 13 trinucleotides between the cerebellum and other regions of the brain. Our results provide evidence for tissue specific instability of the CAG repeat, with the largest CAG repeat lengths in affected regions of the brain.

The relationship between trinucleotide (CAG) repeat length and clinical features of Huntington's disease.

Huntington's disease (HD) is associated with the expansion of a CAG trinucleotide repeat in a novel gene. We have assessed 360 HD individuals from 259 unrelated families and found a highly significant correlation (r = 0.70, p = 10(-7)) between the age of onset and the repeat length, which accounts for approximately 50% of the variation in the age of onset. Significant associations were also found between repeat length and age of death and onset of other clinical features. Sib pair and parent-child analysis revealed that the CAG repeat demonstrates only mild instability. Affected HD siblings had significant correlations for trinucleotide expansion (r = 0.66, p < 0.001) which was not apparent for affected parent-child pairs.

Msh2 deficiency leads to chromosomal abnormalities, centrosome amplification, and telomere capping defect.

Msh2 is a key mammalian DNA mismatch repair (MMR) gene and mutations or deficiencies in mammalian Msh2 gene result in microsatellite instability (MSI+) and the development of cancer. Here, we report that primary mouse embryonic fibroblasts (MEFs) deficient in the murine MMR gene Msh2 (Msh2(-/-)) showed a significant increase in chromosome aneuploidy, centrosome amplification, and defective mitotic spindle organization and unequal chromosome segregation. Although Msh2(-/-) mouse tissues or primary MEFs had no apparent change in telomerase activity, telomere length, or recombination at telomeres, Msh2(-/-) MEFs showed an increase in chromosome end-to-end fusions or chromosome ends without detectable telomeric DNA. These data suggest that MSH2 helps to maintain genomic stability through the regulation of the centrosome and normal telomere capping in vivo and that defects in MMR can contribute to oncogenesis through multiple pathways.

Role of DNA mismatch repair in the cytotoxicity of ionizing radiation.

The DNA mismatch repair (MMR) system in mammalian cells not only serves to correct base mispairs and other replication errors, but it also influences the cellular response to certain forms of DNA damage. Cells that are deficient in MMR are relatively resistant to alkylation damage because, in wild-type cells, the MMR system is thought to promote toxicity via futile repair of alkylated mispairs. Conversely, MMR-deficient cells are sensitive to UV light, possibly due to the requirement for MMR factors in transcription-coupled repair of active genes. MMR deficiency has been associated with familial and sporadic carcinomas of the colon and other sites, and so, we sought to determine the influence of MMR status on cellular response to ionizing radiation, an agent commonly used for cancer therapy. Fibroblast cell lines were established from transgenic mice carrying targeted disruptions of one of three MMR genes in mammalian cells: Pms2, Mlh1, or Msh2. In comparison to wild-type cell lines from related mice, the Pms2-, Mlh1-, or Msh2-nullizygous cell lines were found to exhibit higher levels of clonogenic survival following exposure to ionizing radiation. Because ionizing radiation generates a variety of lesions in DNA, the differences in survival may reflect a role for MMR in processing a subset of these lesions, such as damaged bases. These results both identify a new class of DNA-damaging agents whose effects are modulated by the MMR system and may help to elucidate pathways of radiation response in cancer cells.

Base transitions dominate the mutational spectrum of a transgenic reporter gene in MSH2 deficient mice.

Tumors derived from individuals with hereditary nonpolyposis colorectal cancer syndrome frequently demonstrate mutations in both alleles of hMSH2, a key gene in DNA mismatch repair (MMR). Sporadic tumors also frequently exhibit MMR deficiency. In keeping with the role of MMR in the maintenance of genome integrity, mice deficient in MSH2 via gene targeting demonstrate a high incidence of thymic lymphomas and small intestinal adenocarcinomas. To investigate the effects of MSH2 deficiency in normal tissues, mice containing a retrievable transgenic lacI reporter gene for mutation detection were crossed with MSH2-/- mice. Mice homozygous for MSH2 deficiency revealed 4.8, 11.0 and 15.2-fold elevations in spontaneous mutation frequency in DNA obtained from brain, small intestine, and thymus, respectively, as compared to heterozygous or wild-type mice. Mutations most frequently recovered from MSH2-/- mice were single base substitutions (77%), particularly base transitions (64%). Frameshifts occurred less frequently (19%) and fell within very short (3-5 bp) mononucleotide runs. Thus the number of key growth control genes potentially impacted by MMR deficiency extends beyond those containing repetitive sequences. These results highlight the capacity for MSH2 deficiency to serve as a potent driving force during the multi-step evolution of tumors.

NPM-ALK mediates phosphorylation of MSH2 at tyrosine 238, creating a functional deficiency in MSH2 and the loss of mismatch repair.

The vast majority of anaplastic lymphoma kinase-positive anaplastic large cell lymphoma (ALK+ALCL) tumors express the characteristic oncogenic fusion protein NPM-ALK, which mediates tumorigenesis by exerting its constitutive tyrosine kinase activity on various substrates. We recently identified MSH2, a protein central to DNA mismatch repair (MMR), as a novel binding partner and phosphorylation substrate of NPM-ALK. Here, using liquid chromatography-mass spectrometry, we report for the first time that MSH2 is phosphorylated by NPM-ALK at a specific residue, tyrosine 238. Using GP293 cells transfected with NPM-ALK, we confirmed that the MSH2(Y238F) mutant is not tyrosine phosphorylated. Furthermore, transfection of MSH2(Y238F) into these cells substantially decreased the tyrosine phosphorylation of endogenous MSH2. Importantly, gene transfection of MSH2(Y238F) abrogated the binding of NPM-ALK with endogenous MSH2, re-established the dimerization of MSH2:MSH6 and restored the sensitivity to DNA mismatch-inducing drugs, indicative of MMR return. Parallel findings were observed in two ALK+ALCL cell lines, Karpas 299 and SUP-M2. In addition, we found that enforced expression of MSH2(Y238F) into ALK+ALCL cells alone was sufficient to induce spontaneous apoptosis. In conclusion, our findings have identified NPM-ALK-induced phosphorylation of MSH2 at Y238 as a crucial event in suppressing MMR. Our studies have provided novel insights into the mechanism by which oncogenic tyrosine kinases disrupt MMR.

DNA instability and human disease.

It is now well established that non-Mendelian examples of DNA instability are associated with human disease. Most malignancies are associated with various chromosomal instabilities, such as aneuploidy, gene amplification, and chromosomal deletion. Furthermore, widespread microsatellite instability (MSI) is associated with a variety of tumors, and instability at specific dynamic repeat expansions underlies a family of neurologic disorders. Inactivation of DNA mismatch repair genes results in genomic instabilities affecting microsatellite regions. Mutations in genes involved in DNA polymerization or Okazaki fragment processing are also associated with MSI. Such instabilities convey a 'mutator' phenotype which is pathogenic. The mechanisms controlling trinucleotide repeat expansions are less well understood. Why this type of genomic instability is particularly pathogenic to neurons is also not clear. An understanding of what normally maintains stability is the first step towards preventing such loss of control and maintaining health.

MutS interaction with mismatch and alkylated base containing DNA molecules detected by optical biosensor.

An optical biosensor was used to monitor interactions between the Escherichia coli DNA mismatch repair molecule MutS and various immobilized oligonucleotides. While associating poorly with single-stranded DNA, MutS was capable of rapid association/dissociation from homoduplex DNA. The interaction of MutS with oligonucleotide 30-mers containing single site mismatches demonstrated that during the dissociation phase, MutS binding was greatest to a G-G mismatch, followed by G-T > A-A > C-T, A-C. Binding to A-G, T-T and C-C mispairs was marginally higher than that seen between MutS and homoduplex DNA. The ability of MutS to interact with 30-mers containing alkylated bases was also tested. While binding to O6-methyl-G-C, or to O4-methyl-T-A base pairs was similar to that of homoduplex DNA, strong binding was seen to a O6-methyl-G-T mispair. O4-methyl-T-G, however, was poorly recognized by MutS, with relative binding affinity similar to homoduplex DNA, predicting poor in vivo recognition of O4-methyl-T-G by MutS. Interestingly, MutS demonstrated a relatively high affinity for an 1,N6-etheno-A-T containing homoduplex. Thus, in allowing rapid evaluation of interactions between such molecules, the biosensor will be useful to structure-function analyses.

Comparison of selectable and plaque assay systems to detect menadione- and UV-induced lacI mutations in mammalian cells.

We have compared the spontaneous mutation frequency and spectrum of lacI genes recovered from a rat embryonic fibroblast line transfected with a lambda-phage shuttle vector (Rat2lambdalacI) using both the traditional plaque assay as well as a positive selection assay. In addition, mutation frequencies and spectrum were determined after treatment of the cells with either the intracellular superoxide-generating compound, menadione, or UVC light. The differences in mutation frequency between the two systems suggested that the selectable assay was better at discerning relatively small mutation frequency increases, more rapidly and at lower cost, than the plaque assay method. Some novel lacI mutations were observed in mutants derived from the selectable assay. This indicates that the selectable assay system may be a useful tool for assessing the mutagenic potential of different agents.

A novel lacI transgenic mutation-detection system and its application to establish baseline mutation frequencies in the scid mouse.

To assess DNA mutations in vivo, we have established a new transgenic mouse line, BC-1, carrying a lacI target gene for mutation detection within a bacteriophage shuttle-vector. The lacI gene was positioned within sequences derived from a rearranged murine immunoglobulin gene locus, a feature that distinguishes the BC-1 transgene from other shuttle vector systems. As mutations in lacI transgenes likely reflect mutations occurring throughout the genome, these systems have been successfully used to investigate spontaneous and induced mutations in a variety of tissues. An important additional application of the transgenic systems is the characterization of lacI mutations occurring in murine strains having specific DNA repair defects. For this study, scid (severe combined immunodeficiency) mice were selected as animals with this mutation have a defect in double-strand DNA break repair. To determine what impact the scid mutation might have on spontaneous mutation frequencies within DNA recovered from various tissues, these mice were crossed with the BC-1 line. Interestingly, mutation frequencies within BC-1/scid mouse DNA were not significantly different from those of BC-1 control mice. Furthermore, spontaneous lacI mutations obtained from BC-1 and from BC-1/scid liver DNA were similar in spectrum. As spontaneous BC-1 liver mutations were similar to those reported previously for other lacI systems, such as the Big Blue transgenic line, this suggested that the nature of the DNA sequences flanking the reporter gene did not modify lacI mutation rate or character.

Equine ulcerative keratomycosis: visual outcome and ocular survival in 39 cases (1987-1996).

The medical records of 39 horses treated for ulcerative keratomycosis over a 10 year period were reviewed. Records were evaluated to determine the medical and/or surgical treatment protocol, visual outcome, globe survival and whether the outcome was influenced by the fungal species isolated. Stromal abscesses and iris prolapses caused by fungi were not included. Twenty of the horses underwent medical treatment only, and 19 horses had combined medical and surgical treatment. Most horses had been treated with topical antibiotics (n = 32) and atropine sulphate (n = 23) prior to referral; topical antifungals had been employed less frequently (n = 14). Fungi were identified by cytology (n = 31), culture (n = 33) and/or surgical histopathology (n = 6). Aspergillus (n = 13) and Fusarium (n = 10) were the most commonly isolated fungi. Miconazole (n = 35) was the most common topical antifungal medication utilised. Median duration of treatment was 48 days (range 31-192 days). Associated bacterial infection (n = 13) was frequently encountered. Visual outcome was favourable in 36/39 (92.3%) eyes. All eyes (20/20) retained vision following medical management only, and 16/19 (84%) retained vision following combined medical and surgical therapy. All medically treated horses (20/20), and 17/19 (89%) of those treated medically and surgically retained their globes. Overall ocular survival was favourable in 37/39 (94.9 %) eyes. Aggressive therapy can result in successful results for equine ulcerative keratomycosis.

Ocular manifestations of feline herpesvirus.

Feline herpesvirus-1 (FHV-1) infection is ubiquitous in the domestic cat population worldwide. The most common clinical ocular manifestations of infection with FHV-1 are conjunctivitis and keratitis. This paper reviews the pathogenesis of feline herpesvirus-1 and discusses the various clinical ocular manifestations, diagnostic techniques and treatment of FHV-1-induced diseases. Ocular manifestations include: conjunctivitis, keratitis, stromal keratitis, keratoconjunctivitis sicca, ophthalmia neonatorium, symblepharon, corneal sequestrum, eosinophilic keratitis and anterior uveitis. Diagnostic techniques discussed include: virus isolation, fluorescent antibody testing, serum neutralising titers, ELISA and polymerase chain reaction. Various therapies are also discussed.

Orbital neurofibrosarcoma in a dog.

A young dog was presented with rapidly progressive, unilateral, exophthalmos. Ultrasound-guided fine-needle aspiration of the retrobulbar mass resulted in a diagnosis of fibrosarcoma. Magnetic resonance imagery revealed tumor invasion into the brain, and palliative therapy was elected. The dog was euthanized 4 weeks following diagnosis due to progressive neurological signs. The final diagnosis was neurofibrosarcoma involving the pons, brainstem, left orbit and left trigeminal nerve.

Comparison of Optisol-GS and neomycin-polymyxin B-gramicidin ophthalmic solution for corneal storage in the dog.

The objective of the research was to compare the efficacy of Optisol-GS (OGS, Bausch & Lomb Surgical, Irvine, CA, USA) with triple antibiotic ophthalmic solution (neomycin-polymyxin B-gramicidin, NPG; Bausch & Lomb, Tampa, FL, USA) in preserving the viability of corneal endothelial cells. The study subjects were thirty young to middle-aged dogs with no gross corneal pathology that had been euthanized by pentobarbital overdose for reasons unrelated to this project. Corneal tissues were harvested, analyzed, and randomly assigned to treatment groups: one of two media (OGS or NPG), and one of five storage times (1, 7, 14, 21, or 35 days). Six corneas were stored in each medium for each time period. Corneal endothelial cell viability was evaluated pre- and poststorage by vital staining (trypan blue and alizarin red S), and endothelial cell morphology was evaluated with scanning electron microscopy. Storage in NPG caused significant loss (100%) of endothelial cells after all storage times. OGS storage maintained a high level of endothelial cell viability up to 21 days (98.9% +/- 1.3% viability). A significant decrease in percentage viability was also found for OGS-stored corneas between 21 and 35 days, when endothelial cell viability decreased to 61.4% +/- 45.9%. The conclusions are that NPG storage at -20 degrees C is a very poor choice of media for corneal tissue banking if graft clarity is the goal. Storage in Optisol-GS at 4 degrees C for up to 21 days resulted in significantly higher percentages of viable endothelial cells. Optisol-GS storage should facilitate corneal preservation for canine keratoplasty patients.

Disease of the equine posterior segment.

The appearance of the equine fundus is reviewed from the perspective of differentiating normal variations from disease, and the descriptions have been updated to include recently published ocular fundic abnormalities. Most pathological lesions are identified near the optic nerve head, and typically involve depigmentation or hyperpigmentation. Depending upon configuration and appearance, linear pigmented bands may reflect the course of the vortex veins, the transition from tapetal to nontapetal fundus, or indicate chorioretinitis or equine motor neuron disease. Choroidal vasculature is readily apparent in color-dilute (subalbinotic) horses and must be differentiated from hemorrhage. Retinal hemorrhages in foals are common and may occur independently to hypoxic ischemic encephalopathy. Retinal cysts may signal more significant disease in the eye such as anterior segment dysgenesis. Prominence of gray or tan-colored material on or near the optic nerve head may represent traumatic optic neuropathy, benign optic neuropathy, proliferative optic neuropathy or actual neoplasia.

Rose bengal positive epithelial microerosions as a manifestation of equine keratomycosis.

Purpose To describe the clinical appearance of corneal epithelial cell microerosions associated with keratomycosis in the horse. METHODS: Retrospective clinical study. RESULTS: Multifocal, punctate, superficial corneal opacities with positive rose bengal retention were noted in six horses with presumed 'viral keratitis'. Faint fluorescein staining was also present in three cases. Equine herpesvirus tissue culture inoculation was negative for a cytopathic effect in three cases. Aspergillus (n = 3), Curvularia (n = 1), and an unidentified fungus (n = 1) were cultured in five horses, and hyphae found on corneal cytology from the sixth. Mixed bacterial infections were present in three eyes. The eyes of two horses with Aspergillus progressed to deep melting corneal ulcers that required surgical therapy. The microerosions remained superficial, but persistent in the other four eyes. Natamycin was utilized topically in all six horses. Transmission electron microscopy from case 6 revealed mucin layer disruption, an intact corneal epithelial cell layer, and fungal attachment to degenerating epithelial cells. The visual outcome was positive in all six horses, although healing was prolonged (48.5 +/- 14.5 days on average in the horses with no surgery; 62 days on average in the two horses that required surgery). CONCLUSIONS: Complete removal or full-thickness penetration of the corneal epithelial cell barrier may not be necessary to allow fungal adherence and initiation of keratomycosis in the horse. Prior to colonization and invasion of the horse cornea, fungi may induce changes in the mucin layer of the tear film that result in or are associated with rose bengal positive microerosions of the superficial corneal epithelium. Horses with painful eyes, and eyes with superficial, multifocal corneal opacities should have their corneas stained with both fluorescein and rose bengal as fungal microerosions may stain weakly, or not at all, with fluorescein, and may thus be mistaken for presumed 'viral keratitis' of the horse.

Posterior lamellar keratoplasty for treatment of deep stromal absesses in nine horses.

OBJECTIVE: To describe and evaluate the use of posterior lamellar keratoplasty as a surgical treatment for deep corneal stromal abscesses in horses. Animals studied Nine horses of various breeds and ages that presented with corneal stromal abscesses located in the posterior one-third of the cornea. Procedure Retrospective medical record study. RESULTS: Nine horses had deep corneal stromal abscesses that were treated with posterior lamellar keratoplasty. Median patient age was 3 years. Six patients were females and three were geldings. Medical therapy alone had been attempted prior to surgery in all nine animals. Corneal abscess culture and histopathology were performed in 8/9 horses. Cultures were positive for an infectious etiology in 4/8 (50%). Histopathology was positive for an infectious etiology in 5/8 (62.5%). Mean surgical time was 71.0 +/- 18.8 min and the average healing time was 23.7 +/- 5.2 days. Visual outcome was positive in 8/9 cases. Conclusion Posterior lamellar keratoplasty is a promising procedure for treatment of deep corneal stromal abscesses in horses. The procedure resulted in considerable shorter surgery time and healing time than had been observed with full-thickness penetrating keratoplasty. Scar formation with this procedure was not significantly different than with penetrating keratoplasty.

Ulcerative keratitis caused by beta-hemolytic Streptococcus equi in 11 horses.

Purpose To describe 11 clinical cases of ulcerative keratitis in horses associated with beta-hemolytic Streptococcus equi in Florida, USA. METHODS: Retrospective clinical study (1996-99). RESULTS: Beta-hemolytic Streptococcus equi was cultured from 11 horses with deep ulcers, descemetoceles or iris prolapse (n = 8), a suture abscess found with a penetrating keratoplasty for a stromal abscess (n = 1), and ulceration that developed following keratectomy/irradiation for corneal squamous cell carcinoma (n = 2). Beta-hemolytic Streptococcus equi subspecies zooepidemicus was found in 10 eyes and subspecies equi in one. Marked signs of uveitis including miosis and hypopyon were present in 8/11 (72.7%) eyes. Keratomalacia was severe in all eyes. The mean diameter of the ulcers associated with beta-hemolytic Streptococcus was 10.2 +/- 6.1 mm. Eight of the eyes required conjunctival flap surgery (four grafts dehisced) and one eye corneal transplantation. Two eyes were treated with medication only. Isolate sensitivity to antibiotics included ampicillin (6/11), bacitracin (11/11), cephalothin (11/11), chloramphenicol (11/11), gentamicin (5/11), polymyxin B (2/11), and tobramycin (1/11). All isolates were resistant to neomycin. The average healing time was 44.7 +/- 26.7 days. The visual outcome was positive in 8/11 eyes, and the globe retained in 9/11 eyes. CONCLUSIONS: Although Gram-positive bacteria predominate in the normal conjunctival microflora of horses throughout the world, Gram-negative bacteria and fungi are more often isolated from equine ulcers. Beta-hemolytic Streptococcus spp. are associated with a very aggressive ulcerative keratitis with the capability to digest conjunctival graft tissue. Clinical signs are pronounced. Aggressive surgical and intensive medical therapy with topical antibiotics and protease inhibitors is indicated.

Evaluation of tear film proteinases in horses with ulcerative keratitis.

Ulcerative keratitis is a common and potentially blinding ocular disease of horses, capable of progressing to corneal perforation in as little as 24 h. This rapid stromal degeneration is mediated in part by exogenous and endogenous proteinases. We measured and compared the concentrations of two matrix metalloproteinases (MMP-2 and MMP-9) and a serine proteinase (neutrophil elastase) present in the precorneal tear film of normal horses and horses with rapidly progressing ulcerative keratitis. Precorneal tear film samples were collected from 23 ulcerated and 21 unaffected eyes of 23 horses with unilateral ulcerative keratitis, and from 33 normal eyes of 17 control horses. MMP-2, MMP-9, and neutrophil elastase were identified by casein and gelatin zymography and quantified by computerized image analysis. Median MMP-9 levels were significantly higher in the precorneal tear film of young control horses vs. older control horses (P = 0.005). Median MMP-2, MMP-9, and neutrophil elastase levels were significantly higher in the precorneal tear film of ulcerated eyes when compared to age-matched normal controls (P = 0.004, P = 0.001, and P = 0.012, respectively). Median MMP-2 levels were also significantly higher in the precorneal tear film of contralateral eyes of affected horses when compared to age-matched normal controls (P = 0.004). No significant differences in median proteinase levels were detected between 'sterile' ulcers and those from which bacteria or mixed infections (bacteria and fungi) were isolated. However, median MMP-2 and neutrophil elastase levels were significantly higher in the precorneal tear film of eyes with 'sterile' ulcers when compared with ulcerated eyes from which fungi were isolated (P < 0.05). The results of this study support the use of topical antiproteinase therapy which targets both MMPs and serine proteinases in progressive equine ulcerative keratitis.