The effect of DNA replication mutations on CAG tract stability in yeast.

CAG repeat tracts are unstable in yeast, leading to frequent contractions and infrequent expansions in repeat tract length. To compare CAG repeats to other simple repeats and palindromic sequences, we examined the effect of DNA replication mutations, including alleles of pol alpha, pol delta, pol epsilon, and PCNA (proliferating cell nuclear antigen), on tract stability. Among the polymerase mutations, the pol delta mutation (pol3-14) destabilizes tracts with either CAG or CTG as the lagging strand template. One pol alpha mutation, pol1-1, destabilizes the orientation with CAG as the lagging strand template, but it has little effect on the CTG orientation. In contrast, the pol1-17 mutation has no effect on either orientation. Similarly, mutations in the proofreading functions of pol delta and pol epsilon, as well as a temperature-sensitive pol epsilon mutation, pol2-18, have no effect on tract stability. Three PCNA mutations, pol30-52, pol30-79, and pol30-90, all have drastic effects on tract stability. Of the three, pol30-52 is unique in yielding small tract changes that are indicative of an impairment in mismatch repair. These results show that while CAG repeats are destabilized by many of the same mutations that destabilize other simple repeats, they also have some behaviors that are suggestive of their potential to form hairpin structures.

Meiotic alterations in CAG repeat tracts.

We have investigated meiotic changes in CAG repeat tracts embedded in a yeast chromosome. Repeat tracts undergo either conversion events between homologs or expansion and contraction events that appear to be confined to a single chromatid. We did not find evidence for conversion of tract interruptions or excess exchange of flanking markers.

Neurodegeneration in Niemann-Pick Type C disease and Huntington's disease: impact of defects in membrane trafficking.

Although neurodegenerative diseases exhibit distinct pathologies, such as affected neuronal cell population, age of onset, and pathological symptoms, overlapping characteristics can be observed at the cellular level. In particular, several neurodegenerative diseases display defects in intracellular vesicular trafficking. Here we discuss the range of cellular phenotypes observed in two rare neurodegenerative diseases, Niemann-Pick Type C and Huntington's Disease, both of which involve vesicular trafficking defects that may contribute to neuronal cell death. In NPC, the primary defect is cholesterol and glycosphingolipid accumulation, but NPC mutant cells display widespread trafficking alterations. In HD, protein aggregates are a hallmark feature, but HD cells also exhibit changes in vesicular traffic, including axonal transport and early endosomal trafficking, that likely impact neuronal cell viability. Here we discuss current therapies that seek to address cellular defects in NPC and HD and describe areas of investigation that may lead to new therapeutic treatment.

Destabilization of CAG trinucleotide repeat tracts by mismatch repair mutations in yeast.

To examine the genetic factors that affect the stability of disease-associated trinucleotide repeats, we have assessed the stability of CAG repeats in yeast strains with mutations in the mismatch repair system. We have found that both pms1 and msh2 mutations destabilize repeat tracts. Destabilization is evidenced both by the increased frequency of repeat length changes and in the pattern of changes that are observed. In wild-type cells repeats are relatively stable when CAG serves as the lagging strand template but relatively unstable when CTG serves as the lagging strand template. Large contractions in repeat length are the most common change. In pms1 and msh2 mutants the relatively stable tracts incur more tract length changes. In addition, many small deletions and some small additions, most often of one repeat unit, are frequent in repeats of the stable orientation. These small changes also are seen as a new class of events that occur in repeats in the unstable orientation. The results show that in yeast the mismatch repair system prevents small changes from occurring but cannot prevent larger changes from occurring.

Expansions of CAG repeat tracts are frequent in a yeast mutant defective in Okazaki fragment maturation.

To understand the causes of CAG repeat tract changes that occur in the passage of human disease alleles, we are studying the effect of replication and repair mutations on CAG repeat tracts embedded in a yeast chromosome. In this report, we examine the effect of a mutation in the RTH1/RAD27 gene encoding a deoxyribonuclease needed for removal of excess nucleotides at the 5'-end of Okazaki fragments. Deletion of the RTH1/RAD27 gene has two effects on CAG tracts. First, the rth1/rad27 mutation destabilizes CAG tracts. Second, although most tract length changes in wild-type yeast cells are tract contractions, approximately half of the changes that occur as a result of the rth1/rad27 mutation are expansions of one or more repeat units. These results support the hypothesis that tract expansions that occur during passage of human disease alleles bearing expanded CAG tracts result from excess DNA synthesis on the lagging strand of replication.

Chemistry profiles in "wellness programs": test selection and participant outcomes.

Insurers, employers, and individuals create demands for laboratory testing in "wellness programs." Tests chosen to identify cases deserving intervention included routine automated chemical tests plus high-density lipoprotein cholesterol, ferritin, and thyroid tests. Participants' unwarranted concerns were addressed with a personalized reporting schema. We tested 1338 individuals, identified 224 (16.7%) with significant abnormalities, and made phone contact follow-up with 193 (86%) of these six to 14 months later. Cholesterol results suggesting increased risk of heart disease were frequent, and were not studied. Interventions were initiated in 55 of the 193 followup cases (49 by physician and six by participants), including prescription of iron or thyroid hormone, counseling on dietary or alcohol intake, and repeat testing. For 58, there was medical advice without intervention; abnormal results were ignored by 79. Noteworthy participant anxiety was manifested in two of the 193 cases, both of whom were treated with iron. We conclude that 4% of the original 1338 participants potentially benefitted from intervention. Ferritin and thyroid tests initiated 33 (61%) of these 55 specific therapeutic interventions.