Diversity of ARSACS mutations in French-Canadians.

BACKGROUND: The growing number of spastic ataxia of Charlevoix-Saguenay (SACS) gene mutations reported worldwide has broadened the clinical phenotype of autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS). The identification of Quebec ARSACS cases without two known SACS mutation led to the development of a multi-modal genomic strategy to uncover mutations in this large gene and explore phenotype variability. METHODS: Search for SACS mutations by combining various methods on 20 cases with a classical French-Canadian ARSACS phenotype without two mutations and a group of 104 sporadic or recessive spastic ataxia cases of unknown cause. Western blot on lymphoblast protein from cases with different genotypes was probed to establish if they still expressed sacsin. RESULTS: A total of 12 mutations, including 7 novels, were uncovered in Quebec ARSACS cases. The screening of 104 spastic ataxia cases of unknown cause for 98 SACS mutations did not uncover carriers of two mutations. Compounds heterozygotes for one missense SACS mutation were found to minimally express sacsin. CONCLUSIONS: The large number of SACS mutations present even in Quebec suggests that the size of the gene alone may explain the great genotypic diversity. This study does not support an expanding ARSACS phenotype in the French-Canadian population. Most mutations lead to loss of function, though phenotypic variability in other populations may reflect partial loss of function with preservation of some sacsin expression. Our results also highlight the challenge of SACS mutation screening and the necessity to develop new generation sequencing methods to ensure low cost complete gene sequencing.

Intersecting Genetics of Frailty and Cardiovascular Disease.

OBJECTIVES: To determine the genetic correlates of physical frailty and sarcopenia, focusing on single nucleotide polymorphisms (SNPs) in genome-wide association studies (GWAS), and to explore the genetic overlap of frailty with cardiovascular disease (CVD) and its risk factors. METHODS: PubMed was systematically searched for GWAS studies investigating the association between SNPs and objective measures of physical frailty or sarcopenia. SNPs were retained if they were associated with one of the phenotypes of interest by a p-value of 5.0x10-8 or less. RESULTS: Ten studies were included, with a total of 237 SNPs in 181 genes being associated with physical frailty or sarcopenia; as measured by handgrip strength or lean (muscle) mass. These genes were cross-referenced in the GWAS Catalog, and many of them were found to be associated with CVD or metabolic syndrome. CONCLUSIONS: Evidence from GWAS has shown that frailty is associated with common genetic polymorphisms. Many of these polymorphisms have been implicated in CVD, supporting the hypothesis of a shared pathophysiology between these entities. Future studies are eagerly anticipated to map out the mechanistic links and discover therapeutic targets and novel biomarkers for frailty.

Mutation of the gene encoding protein kinase C 1 stimulates mitotic recombination in Saccharomyces cerevisiae.

We have isolated a recessive allele of the yeast protein kinase C gene (PKC1) which promotes an elevated rate of mitotic recombination and confers a temperature-sensitive growth defect. The rate of recombination was increased between genes in direct repeat and at a series of heteroalleles and was dependent upon the RAD52 gene product. The mutant pkc1 allele was sequenced and found to encode a single amino acid change within the catalytic domain. Osmotic stabilizing agents rescued the temperature-sensitive growth defect but not the hyperrecombination phenotype, indicating that the two traits are separable. This separability suggests that the PKC1 gene product (Pkc1p) regulates DNA metabolism by an alternate pathway to that used in the regulation of cell lysis. The regulation of recombination is a previously unidentified role for Pkc1p.

A 5'-3' exonuclease from Saccharomyces cerevisiae is required for in vitro recombination between linear DNA molecules with overlapping homology.

When two linear DNA molecules with overlapping, homologous ends were incubated with a yeast nuclear extract, they recombined at the region of homology to produce a joint molecule. We have identified a 5'-3' exonuclease in the extract that is likely to be responsible for the formation of the observed product. We propose that the exonuclease degrades each substrate to reveal regions of complementary sequence which anneal to form a recombinant product. Consistent with this model, we have partially purified the activity that promotes joint molecule formation and found it to cofractionate with a 5'-3' exonuclease activity through three consecutive chromatography steps. We have further characterized the reaction to determine the optimal length of homology. Substrates with homologous terminal overlaps of 29 to 958 bp were capable of product formation, whereas substrates with longer overlaps were not. Extracts prepared from a number of recombination-defective or nuclease-deficient strains revealed no defect in exonuclease activity, indicating that the reaction is likely to be dependent upon the product of an as yet unidentified gene.

Structure-function analysis of the Saccharomyces cerevisiae G1 cyclin Cln2.

We have generated 50 new alleles of the yeast CLN2 gene by using site-directed mutagenesis. With the recently obtained crystal structure of cyclin A as a guide, a peptide linker sequence was inserted at 13 sites within the cyclin box of Cln2 to determine if the architecture of Cln2 is similar to that of cyclin A. Linkers inserted in what are predicted to be helices 1, 2, 3, and 5 of the cyclin box resulted in nonfunctional Cln2 molecules. Linkers inserted between these putative helix sites and in the region believed to contain a fourth helix did not have significant effects upon Cln2 function. A series of deletions in the region between the third and fifth helices indicate that the putative fourth helix may lie at the C-terminal end of this region yet is not essential for function. Two residues that are predicted to form a buried salt bridge important for interaction of two helices of the cyclin box were also mutated, and an additional set of 31 mutant alleles was generated by clustered-charge-to-alanine scanning mutagenesis. All of the mutant CLN2 alleles made in this study were tested in a variety of genetic and functional assays previously demonstrated to differentiate specific cyclin functions. Some alleles demonstrated restricted patterns of defects, suggesting that these mutations may interfere with specific aspects of Cln2 function.

Exonuclease I of Saccharomyces cerevisiae functions in mitotic recombination in vivo and in vitro.

We previously described a 5'-3' exonuclease required for recombination in vitro between linear DNA molecules with overlapping homologous ends. This exonuclease, referred to as exonuclease I (Exo I), has been purified more than 300-fold from vegetatively grown cells and copurifies with a 42-kDa polypeptide. The activity is nonprocessive and acts preferentially on double-stranded DNA. The biochemical properties are quite similar to those of Schizosaccharomyces pombe Exo I. Extracts prepared from cells containing a mutation of the Saccharomyces cerevisiae EXO1 gene, a homolog of S. pombe exo1, had decreased in vitro recombination activity and when fractionated were found to lack the peak of activity corresponding to the 5'-3' exonuclease. The role of EXO1 on recombination in vivo was determined by measuring the rate of recombination in an exo1 strain containing a direct duplication of mutant ade2 genes and was reduced sixfold. These results indicate that EXO1 is required for recombination in vivo and in vitro in addition to its previously identified role in mismatch repair.

An accurate air temperature measurement system based on an envelope pulsed ultrasonic time-of-flight technique.

A new microcomputer based air temperature measurement system is presented. An accurate temperature measurement is derived from the measurement of sound velocity by using an ultrasonic time-of-flight (TOF) technique. The study proposes a novel algorithm that combines both amplitude modulation (AM) and phase modulation (PM) to get the TOF measurement. The proposed system uses the AM and PM envelope square waveform (APESW) to reduce the error caused by inertia delay. The APESW ultrasonic driving waveform causes an envelope zero and phase inversion phenomenon in the relative waveform of the receiver. To accurately achieve a TOF measurement, the phase inversion phenomenon was used to sufficiently identify the measurement pulse in the received waveform. Additionally, a counter clock technique was combined to compute the phase shifts of the last incomplete cycle for TOF. The presented system can obtain 0.1% TOF resolution for the period corresponding to the 40 kHz frequency ultrasonic wave. Consequently, with the integration of a humidity compensation algorithm, a highly accurate and high resolution temperature measurement can be achieved using the accurate TOF measurement. Experimental results indicate that the combined standard uncertainty of the temperature measurement is approximately 0.39 degrees C. The main advantages of this system are high resolution measurements, narrow bandwidth requirements, and ease of implementation.

Envelope pulsed ultrasonic distance measurement system based upon amplitude modulation and phase modulation.

A novel microcomputer-based ultrasonic distance measurement system is presented. This study proposes an efficient algorithm which combines both the amplitude modulation (AM) and the phase modulation (PM) of the pulse-echo technique. The proposed system can reduce error caused by inertia delay and amplitude attenuation effect when using the AM and PM envelope square wave form (APESW). The APESW ultrasonic driving wave form causes a phase inversion phenomenon in the relative wave form of the receiver. The phase inversion phenomenon sufficiently identifies the "measurement pulse" in the received wave forms, which can be used for accurate time-of-flight (TOF) measurement. In addition, combining a countertechnique to compute the phase shifts of the last cycle for TOF, the presented system can obtain distance resolution of 0.1% of the wavelength corresponding to the 40 kHz frequency of the ultrasonic wave. The standard uncertainty of the proposed distance measurement system is found to be 0.2 mm at a range of 50-500 mm. The APESW signal generator and phase detector of this measuring system are designed on a complex programmable logic device, which is used to govern the TOF measurement and send the data to a personal computer for distance calibration and examination. The main advantages of this APESW system are high resolution, low cost, narrow bandwidth requirement, and ease of implementation.

Suppressors of a Saccharomyces cerevisiae pkc1 mutation identify alleles of the phosphatase gene PTC1 and of a novel gene encoding a putative basic leucine zipper protein.

The PKC1 gene product, protein kinase C, regulates a mitogen-activated protein kinase (MAPK) cascade, which is implicated in cell wall metabolism. Previously, we identified the pkc1-4 allele in a screen for mutants with increased rates of recombination, indicating that PKC1 may also regulate DNA metabolism. The pkc1-4 allele also conferred a temperature-sensitive (ts) growth defect. Extragenic suppressors were isolated that suppress both the ts and hyperrecombination phenotypes conferred by the pkc1-4 mutation. Eight of these suppressors for into two complementation groups, designated KCS1 and KCS2. KCS1 was cloned and found to encode a novel protein with homology to the basic leucine zipper family of transcription factors. KCS2 is allelic with PTC1, a previously identified type 2C serine/threonine protein phosphatase. Although mutation of either KCS1 or PTC1 causes little apparent phenotype, the kcs1 delta ptc1 delta double mutant fails to grow at 30 degrees. Furthermore, the ptc1 deletion mutation is synthetically lethal in combination with a mutation in MPK1, which encodes a MAPK homologue proposed to act in the PKC1 pathway. Because PTC1 was initially isolated as a component of the Hog1p MAPK pathway, it appears that these two MAPK cascades share a common regulatory feature.

The 5HT2 receptor defines a family of structurally distinct but functionally conserved serotonin receptors.

Serotonin exerts its diverse physiological effects by interacting with multiple distinct receptor subtypes. We have isolated a rat brain 5HT2 serotonin receptor cDNA by virtue of its homology with the 5HT1c receptor. The 5HT2 receptor is a member of the family of receptors that are linked to guanine nucleotide-binding proteins and are predicted to span the lipid bilayer seven times. Overall sequence identity between the 5HT2 and 5HT1c receptors is 49%, but identity within the transmembrane domains is 80%. Expression of both the 5HT2 and 5HT1c receptors in transfected mouse fibroblasts activates phospholipase C signaling pathways and promotes cellular transformation. However, RNA blotting shows that these two receptor subtypes are differentially expressed in the central nervous system. In this manner, structurally and functionally homologous receptor subtypes may elicit distinct physiologic actions.

The serotonin receptor subtype 2 locus HTR2 is on human chromosome 13 near genes for esterase D and retinoblastoma-1 and on mouse chromosome 14.

Serotonin (5-hydroxytryptamine) functions as a neurotransmitter and a hormone. Its diverse actions are mediated by at least seven distinct cell surface receptor subtypes. The serotonin receptor subtype 2 (gene symbol HTR2) is a G-protein-coupled receptor, expressed primarily in the cerebral cortex, where upon stimulation it stimulates the hydrolysis of inositol phospholipids. We have mapped the HTR2 locus to human chromosome 13 and to mouse chromosome 14 by somatic cell hybrid analysis. Linkage studies in CEPH families, using a PvuII RFLP detected with the HTR2 probe, revealed tight linkage between HTR2 and ESD, the locus for esterase D. The most likely position for HTR2 is between ESD and RB1, the retinoblastoma-1 gene. The homologous loci in mouse, Rb-1 and Esd(Es-10) are on mouse chromosome 14, close to ag, agitans, a recessive neurological mutation. Having mapped Htr-2 to mouse chromosome 14, we predict that it falls into this known conserved gene cluster.