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PubMed2000

Candidate gene analysis of thyroid hormone receptors in metamorphosing vs. nonmetamorphosing salamanders.

We used two different experimental approaches to test the hypothesis that thyroid hormone receptor (TR) variation is associated with alternate life cycles modes in ambystomatid salamanders. In the first experiment, the inheritance of TRalpha and TRbeta genotypes was determined for metamorphic and non metamorphic offspring from backcrosses between Ambystoma mexicanum (an obligate metamorphic-failure species) and metamorphic F1 hybrids (A. mexicanum x A. tigrinum tigrinum). The segregation of TR genotype was independent of the expression of life cycle mode phenotype, and neither TR locus was linked to DNA markers that flank a major-effect locus for life cycle mode. In the second experiment, a portion of the ligand-binding domain of TRalpha and TRbeta was cloned and sequenced for DNA samples from 14 different ambystomatid salamander populations, including obligate metamorphic, facultative metamorphic, and obligate metamorphic-failure taxa. Nucleotide sequence variation was found for both TRalpha and TRbeta, with several nonsynonomous substitutions that presumably code for nonconservative amino acid replacements. However, no general relationship was found between TR allelic variation and life cycle mode among populations or species. These data do not implicate TRs as candidate loci involved in the current maintenance or past evolution of alternate life cycle modes in members of the tiger salamander complex.

Alleles↗
PubMed1995

The Wee1 protein kinase regulates T14 phosphorylation of fission yeast Cdc2.

The Cdc2 protein kinase is a key regulator of the G1-S and G2-M cell cycle transitions in the fission yeast Schizosaccharomyces pombe. The activation of Cdc2 at the G2-M transition is triggered by dephosphorylation at a conserved tyrosine residue Y15. The level of Y15 phosphorylation is controlled by the Wee1 and Mik1 protein kinases acting in opposition to the Cdc25 protein phosphatase. Here, we demonstrate that Wee1 overexpression leads to a high stoichiometry of phosphorylation at a previously undetected site in S. pombe Cdc2, T14. T14 phosphorylation was also detected in certain cell cycle mutants blocked in progression through S phase, indicating that T14 phosphorylation might normally occur at low stoichiometry during DNA replication or early G2. Strains in which the chromosomal copy of cdc2 was replaced with either a T14A or a T14S mutant allele were generated and the phenotypes of these strains are consistent with T14 phosphorylation playing an inhibitory role in the activation of Cdc2 as it does in higher eukaryotes. We have also obtained evidence that Wee1 but not Mik1 or Chk1 is required for phosphorylation at this site, that the Mik1 and Chk1 protein kinases are unable to drive T14 phosphorylation in vivo, that residue 14 phosphorylation requires previous phosphorylation at Y15, and that the T14A mutant, unlike Y15F, is recessive to wild-type Cdc2 activity. Finally, the normal duration of G2 delay after irradiation or hydroxyurea treatment in a T14A mutant strain indicates that T14 phosphorylation is not required for the DNA damage or replication checkpoint controls.

Base Sequence↗
PubMed2005

Dictyostelium LIS1 is a centrosomal protein required for microtubule/cell cortex interactions, nucleus/centrosome linkage, and actin dynamics.

The widespread LIS1-proteins were originally identified as the target for sporadic mutations causing lissencephaly in humans. Dictyostelium LIS1 (DdLIS1) is a microtubule-associated protein exhibiting 53% identity to human LIS1. It colocalizes with dynein at isolated, microtubule-free centrosomes, suggesting that both are integral centrosomal components. Replacement of the DdLIS1 gene by the hypomorphic D327H allele or overexpression of an MBP-DdLIS1 fusion disrupted various dynein-associated functions. Microtubules lost contact with the cell cortex and were dragged behind an unusually motile centrosome. Previously, this phenotype was observed in cells overexpressing fragments of dynein or the XMAP215-homologue DdCP224. DdLIS1 was coprecipitated with DdCP224, suggesting that both act together in dynein-mediated cortical attachment of microtubules. Furthermore, DdLIS1-D327H mutants showed Golgi dispersal and reduced centrosome/nucleus association. Defects in DdLIS1 function also altered actin dynamics characterized by traveling waves of actin polymerization correlated with a reduced F-actin content. DdLIS1 could be involved in actin dynamics through Rho-GTPases, because DdLIS1 interacted directly with Rac1A in vitro. Our results show that DdLIS1 is required for maintenance of the microtubule cytoskeleton, Golgi apparatus and nucleus/centrosome association, and they suggest that LIS1-dependent alterations of actin dynamics could also contribute to defects in neuronal migration in lissencephaly patients.

Actins↗
PubMed1996

Azorhizobium caulinodans uses both cytochrome bd (quinol) and cytochrome cbb3 (cytochrome c) terminal oxidases for symbiotic N2 fixation.

Azorhizobium caulinodans employs both cytochrome bd (cytbd; quinol oxidase) and cytcbb3 (cytc oxidase) as terminal oxidases in environments with very low O2 concentrations. To investigate physiological roles of these two terminal oxidases both in microaerobic culture and in symbiosis, knockout mutants were constructed. As evidenced by visible absorbance spectra taken from mutant bacteria carrying perfect gene replacements, both the cytbd- and cytcbb3- mutations were null alleles. In aerobic culture under 2% O2 atmosphere, Azorhizobium cytbd- and cytcbb3- single mutants both fixed N2 at 70 to 90% of wild-type rates; in root nodule symbiosis, both single mutants fixed N2 at 50% of wild-type rates. In contrast, Azorhizobium cytbd- cytcbb3-double mutants, which carry both null alleles, completely lacked symbiotic N2 fixation activity. Therefore, both Azorhizobium cytbd and cytcbb3 oxidases drive respiration in environments with nanomolar O2 concentrations during symbiotic N2 fixation. In culture under a 2% O2 atmosphere, Azorhizobium cytbd- cytcbb3- double mutants fixed N2 at 70% of wild-type rates, presumably reflecting cytaa3 and cytbo (and other) terminal oxidase activities. In microaerobic continuous cultures in rich medium, Azorhizobium cytbd- and cytcbb3- single mutants were compared for their ability to deplete a limiting-O2 sparge; cytbd oxidase activity maintained dissolved O2 at 3.6 microM steady state, whereas cytcbb3 oxidase activity depleted O2 to submicromolar levels. Growth rates reflected this difference; cytcbb3 oxidase activity disproportionately supported microaerobic growth. Paradoxically, in O2 limited continuous culture, Azorhizobium cytbd oxidase is inactive below 3.6 microM dissolved O2 whereas in Sesbania rostrata symbiotic nodules, in which physiological, dissolved O2 is maintained at 10 to 20 nM, both Azorhizobium cytbd and cytcbb3 seem to contribute equally as respiratory terminal oxidases.

Aerobiosis↗
PubMed1981

Homothallic mating type switching generates lethal chromosome breaks in rad52 strains of Saccharomyces cerevisiae.

In homothallic cells of Saccharomyces cerevisiae, a or alpha mating type information at the mating type locus (MAT) is replaced by the transposition of the opposite mating type allele from HML alpha or HMRa. The rad52-1 mutation, which reduces mitotic and abolishes meiotic recombination, also affects homothallic switching (Malone and Esposito, Proc. Natl. Acad. Sci. U.S.A. 77:503-507, 1980). We have found that both HO rad52 MATa and HO rad52 MAT alpha cells die. This lethality is suppressed by mutations that substantially reduce but do not eliminate homothallic conversions. These mutations map at or near the MAT locus (MAT alpha inc, MATa-inc, MATa stk1) or are unlinked to MAT (HO-1 and swi1). These results suggest that the switching event itself is involved in the lethality. With the exception of swi1, HO rad52 strains carrying one of the above mutations cannot convert mating type at all. MAT alpha rad52 HO swi1 strains apparently can switch MAT alpha to MATa. However, when we analyzed these a maters, we found that few, if any, of them were bona fide MATa cells. These a-like cells were instead either deleted for part of chromosome III distal to and including MAT or had lost the entire third chromosome. Approximately 30% of the time, an a-like cell could be repaired to a normal MATa genotype if the cell was mated to a RAD52 MAT alpha-inc strain. The effects of rad52 were also studied in mata/MAT alpha-inc rad52/rad52 ho/HO diploids. When this diploid attempted to switch mata to MATa, an unstable broken chromosome was generated in nearly every cell. These studies suggest that homothallic switching involves the formation of a double-stranded deoxyribonucleic acid break or a structure which is labile in rad52 cells and results in a broken chromosome. We propose that the production of a double-stranded deoxyribonucleic acid break is the lethal event in rad52 HO cells.

Alleles↗
PubMed2004

Association between the Thr715Pro P-selectin gene polymorphism and soluble P-selectin levels in a multiethnic population in South London.

The aim was to investigate whether the Thr715Pro P-selectin polymorphism is associated with soluble P-selectin (sP-selectin) levels in individuals from different ethnic groups. Plasma sP-selectin and Thr715Pro (A/C) P-selectin gene polymorphism were measured in 237 white (106 females), 177 black African origin (92 females) and 201 South Asian (94 females) individuals living in England. All were free from coronary heart disease (CHD), stroke and other cardiovascular disease, diabetes, drug therapy for hypertension or high lipids, hormone replacement therapy or oral contraceptive pill. The Thr715Pro C allele was rare in blacks (0.8%) and intermediate in South Asians (3.0%) compared to whites (11.2%; p <0.001). sP-selectin levels were significantly lower in the individuals with the AC or CC compared to the AA genotype in both whites (-25% (95% C.I. -33.3 to -16.9); p <0.001) and South Asians (-25.2% (-40.5 to -6.1); p <0.012). There was insufficient power for this analysis in blacks. In conclusion, in whites and South Asians the C allele of the Thr715Pro P-selectin polymorphism is associated with lower sP-selectin levels. Lower levels of sP-selectin were not accounted for by this polymorphism in blacks, in whom the C allele was very rare.

Age Factors↗
PubMed2004

Identification of variant ABC-transporter genes among Onchocerca volvulus collected from ivermectin-treated and untreated patients in Ghana, West Africa.

Treatment with ivermectin (IVM) is known to cause a loss of polymorphism at certain loci of the beta-tubulin, gamma-aminobutyric-acid-receptor, glutamate-gated-chloride-channel and ATP-binding-cassette (ABC) transporter genes of IVM-resistant Haemonchus contortus. The genetic variation of four ABC-transporter homologues from Onchocerca volvulus was therefore investigated, to determine if any change in genetic polymorphism occurs in these genes following repeated treatment with IVM. Samples were collected in the Northern, Brong-Ahafo and Volta regions of Ghana, in 1999 and 2002; nodules containing adult O. volvulus were removed from subjects who had either received multiple IVM treatments or never taken IVM. The ATP-binding domains of four ABC-transporter genes (OvMDR-1, OvMDR-3, OvABC-1 and OvABC-6) were amplified from individual O. volvulus and examined for polymorphism, using single-strand-conformation-polymorphism (SSCP) analysis. In the samples collected in 1999, OvMDR-1 and OvABC-1 showed significant reduction in polymorphism following IVM treatment whereas OvABC-6 and OvMDR-3 were not found to be polymorphic. The samples collected in 2002 also showed a reduction in polymorphism for both OvMDR-1 and OvABC-1. Several single-nucleotide polymorphisms, which resulted in either amino-acid-replacement substitutions or nonsense mutations, were identified in the alleles of OvMDR-1 and OvABC-1.

ATP-Binding Cassette Transporters↗
PubMed2006

Genetically augmenting Abeta42 levels in skeletal muscle exacerbates inclusion body myositis-like pathology and motor deficits in transgenic mice.

The pathogenic basis of inclusion body myositis (IBM), the leading muscle degenerative disease afflicting the elderly, is unknown, although the histopathological features are remarkably similar to those observed in Alzheimer's disease. One leading hypothesis is that the buildup of amyloid-beta (Abeta) peptide within selective skeletal muscle fibers contributes to the degenerative phenotype. Abeta is a small peptide derived via endoproteolysis of the amyloid precursor protein (APP). To determine the pathogenic effect of augmenting Abeta42 levels in skeletal muscle, we used a genetic approach to replace the endogenous wild-type presenilin-1 (PS1) allele with the PS1(M146V) allele in MCK-APP mice. Although APP transgene expression was unaltered, Abeta levels, particularly Abeta42, were elevated in skeletal muscle of the double transgenic (MCK-APP/PS1) mice compared to the parental MCK-APP line. Elevated phospho-tau accumulation was found in the MCK-APP/PS1 mice, and the greater activation of GSK-3beta and cdk5 were observed. Other IBM-like pathological features, such as inclusion bodies and inflammatory infiltrates, were more severe and prominent in the MCK-APP/PS1 mice. Motor coordination and balance were more adversely affected and manifested at an earlier age in the MCK-APP/PS1 mice. The data presented here provide experimental evidence that Abeta42 plays a proximal and critical role in the muscle degenerative process.

Amyloid beta-Peptides↗
PubMed2001

[The multifunctional fructose-specific component of the phosphoenolpyruvate-dependent phosphotransferase system of Escherichia coli K12--fruA gene product].

Mutational damage of the ptsH gene leads to pleiotropic disturbance of sugar utilization in Escherichia coli K12. A fruS mutation suppresses the defect because of a constitutional expression of the fruB and fruA genes. FruB protein possessing a pseudo-HPr activity replaces the HPr. It was shown that wild type allele fruS+ dominates over the fruS1156 mutation in heterozygous merodiploid. The existence of thermosensitive mutations (fruS4 and fruS12) which repair the ptsH damage was also demonstrated. The fruS mutations were located in the fru operon. Fructose utilization was not disturbed in fruS1156 mutant, but fruS2 and fruS12 mutants were unable to utilize fructose. Spontaneous mutations (fruS6 and fruS13) possessing the same phenotype at any temperature similar to the thermosensitive ones under nonpermissive conditions were isolated. They were mapped using the P1vir transduction. The fruS mutations were found in the structural gene of the fructose operon. Presumably it is the fruA gene that cods for the fructose-specific multidomain protein IIB'Bc of the phosphoenolpyruvate-dependent phosphotransferase system.

Escherichia coli↗
PubMed2006

Conversion of Lactococcus lactis cell envelope proteinase specificity by partial allele exchange.

AIMS: To determine whether conversion of lactocepin substrate binding regions by gene replacement can alter lactocepin specificity in Lactococcus lactis starter bacteria without affecting other important strain properties. METHODS AND RESULTS: We utilized two-step gene replacement to convert substrate-binding determinants in the L. lactis prtP genes encoding group h (bitter) lactocepin in two industrial strains into the corresponding group b (nonbitter) variant. Analysis of lactocepin activity toward alpha(s1)-casein (f 1-23) by reversed-phase high-pressure liquid chromatography demonstrated enzyme specificity among isogenic derivatives had been altered in a manner that was consistent with predicted amino acid substitutions in substrate binding regions. Milk acidification properties of some mutants were not statistically different (P > 0.05) from wild-type parent strains, and strain propensity for autolysis was also not significantly (P > 0.05) changed. CONCLUSIONS: Conversion of lactocepin substrate binding regions by allele exchange can effectively alter lactocepin specificity in industrial strains of L. lactis without significantly affecting other important strain properties. SIGNIFICANCE AND IMPACT OF THE STUDY: Methodology outlined in this study can be used to alter lactocepin specificity in commercial starter cultures with a propensity for bitter flavour defect, and prtP derivatives developed by this approach should be suitable for commercial application.

Alleles↗
PubMed2003

A common methylenetetrahydrofolate reductase (C677T) polymorphism is associated with low bone mineral density and increased fracture incidence after menopause: longitudinal data from the Danish osteoporosis prevention study.

A polymorphism in the gene encoding methylenetetrahydrofolate reductase (MTHFR) has recently been associated with bone mineral density (BMD) in postmenopausal Japanese women. It is not known whether this effect is also present in European populations and whether it is caused by lower peak bone mass or accelerated postmenopausal bone loss. MTHFR genotyping was done in 1748 healthy postmenopausal Danish women participating in a prospective study of risk factors for osteoporosis. At the time of enrollment, 3-24 months after last menstrual period, the less prevalent genotype (TT, 8.7% of the population) was associated with significantly lower BMD at the femoral neck (ANOVA, p < 0.05), total hip (p < 0.01), and spine (p < 0.05 adjusted for lifestyle covariates, p = 0.06 without adjustment). The mean difference was between 0.1 and 0.3 SD, depending on measurement site. MTHFR genotype added significantly to prediction of BMD by weight and age. Fracture incidence was increased more than 2-fold in subjects with the TT genotype (risk ratio [RR], 2.6; 95% CI 1.2-5.6). This remained significant when the Cox analysis was controlled for BMD (RR, 2.4; 95% CI 1.1-5.2). No differences in serum osteocalcin, bone-specific alkaline phosphatase, and 25-OH-vitamin D were found between genotypes. The response to hormone replacement therapy (HRT) did not differ, but the association of the TT genotype with reduced BMD was maintained at the total hip after 5 years of HRT. The MTHFR TT genotype is associated with low BMD and increased fracture incidence in early postmenopausal women.

Alleles↗
PubMed1996

Gene-targeted deletion and replacement mutations of the T-cell receptor beta-chain enhancer: the role of enhancer elements in controlling V(D)J recombination accessibility.

To assess the role of transcriptional enhancers in regulating accessibility of the T-cell receptor beta-chain (TCRbeta) locus, we generated embryonic stem cell lines in which a single allelic copy of the endogenous TCRbeta enhancer (Ebeta) was either deleted or replaced with the immunoglobulin heavy-chain intronic enhancer. We assayed the effects of these mutations on activation of the TCRbeta locus in normal T- and B-lineage cells by RAG-2 (recombination-activating gene 2)-deficient blastocyst complementation. We found that Ebeta is required for rearrangement and germ-line transcription of the TCRbeta locus in T-lineage cells. In the absence of Ebeta, the heavy-chain intronic enhancer partially supported joining region beta-chain rearrangement in T- but not in B-lineage cells. However, ability of the heavy-chain intronic enhancer to induce rearrangements was blocked by linkage to an expressed neomycin-resistance gene (neo(r)). These results demonstrate a critical role for Ebeta in promoting accessibility of the TCRbeta locus and suggest that additional negative elements may cooperate to further modulate this process.

Alleles↗
PubMed2000

Allelic polymorphisms of human fcgamma receptor IIa and Fcgamma receptor IIIb among distinct groups in Brazil.

BACKGROUND: The FcgammaRIIA gene is expressed in two polymorphic forms, R131 and H131, which differ by the replacement of histidine by arginine at position 131. The FCGR3B (FcgammaRIIIB) gene exists in two allelic isoforms, known as FCGR3B1 (FcgammaRIIIB-NA1) and FCGR3B2 (FcgammaRIIIB-NA2), which differ in nucleotides 141, 147, 227, 277, and 349. An additional polymorphism is the SH antigen that is associated with the FCGR3B3 (FcgammaRIIIB-SH) allele. STUDY DESIGN AND METHODS: By use of a PCR with allele-specific primers, the allelic polymorphisms of FcgammaRIIA and FcgammaRIIIB were determined among 263 unrelated Brazilian subjects, including Amazon Indians (n = 92), blood donors (n = 85), and patients with sickle cell disease (SCD) (n = 86). RESULTS: Amazon Indians had a significantly higher frequency of the R131 allele than did blood donors and SCD patients (0.91 vs. 0.55 vs. 0.55; p<0.001). NA1 and NA2 gene frequencies were found to be 0.67 and 0.21 for Amazon Indians, 0.58 and 0.42 for blood donors, and 0.61 and 0.39 for SCD patients, respectively. The FcgammaRIIIB-SH allele was absent from the Amazon Indians, but 9 (10.6%) blood donors and 10 (11.6%) SCD patients expressed this allele. CONCLUSION: Overall, the data indicate that the distribution of the FcgammaRIIIB alleles is significantly different in Amazon Indians from the distribution in Brazilian blood donors or African Brazilian patients with SCD, but that it is similar to the distributions reported in Asian populations. Moreover, the distribution of the FcgammaRIIA and FcgammaRIIIB alleles among Brazilian blood donors and SCD patients is comparable to the distributions reported in whites from the United States and Europe.

Alleles↗
PubMed2003

Stroke damage in mice after knocking the neutrophin-4 gene into the brain-derived neurotrophic factor locus.

Neurotrophins play a protective role during cerebral ischemia, and mice lacking both alleles for neurotrophin 4 (Nt4-/- ) or deficient in a single allele for brain-derived neurotrophic factor (Bdnf+/-) have increased susceptibility to cerebral ischemia. This study directly compared the biologic activities of brain-derived neurotrophic factor (BDNF) and NT4 by replacing the coding sequence with the Nt4 sequence (Bdnf +/nt4-ki ). Mice expressing one allele in place of develop 61% bigger lesions after 1-hour middle cerebral artery occlusion compared with wild-type littermates. Physiologic parameters did not contribute to ischemia susceptibility. In conclusion, NT4 is less potent than BDNF in promoting brain survival after stroke.

Animals↗
PubMed2002

Genetic modeling of estrogen metabolism as a risk factor of hormone-dependent disorders.

Estradiol is a pleiotropic hormone, involved in the etiology of a wide variety of diseases. Over the last decade individual genetic variability of the estradiol metabolism has been described as a significant contributor to disease susceptibility with variations depending on ethnic background. Among others, genetic variations of genes encoding cytochrome P450 (CYP) enzymes play an important role in this regard. Mutant alleles of the CYP 1A1 gene are major modulators of lung cancer risk among smokers, mediate gender differences in lung cancer susceptibility, and have been associated with an elevated risk for breast, prostate, colorectal, and oral squamous cell cancer. Variants of the CYP 1B1 gene modulate the risk for prostate, ovarian, lung, and breast cancer. Also, mutations in the CYP 1B1 gene are the major genetic determinant of congenital glaucoma. Mutant CYP 17 alleles are associated with serum and plasma levels of steroid hormones, use of hormone replacement therapy, and endometrial, prostate, and breast cancer. Available data indicate that the protective effect of a later age at menarche is limited to mutant CYP 17 allele carriers. Among women with the Polycystic Ovary (PCO) syndrome, mutant CYP 17 alleles are sufficient to aggravate the clinical presentation of the disease. Molecular variants of the CYP 19 gene are associated with an increased risk for breast cancer, advanced disease stage, and tumor aromatase production. Carriage of a mutant catechol-O-methyltransferase (COMT) allele is associated with breast cancer, neurologic disorders such as Parkinson's disease, and modulates behavior among patients with schizophrenia, alcoholics and the general population. In summary, the available evidence points to estrogen metabolising genes as strong hereditary determinants of the susceptibility to benign and malignant conditions.

Breast Neoplasms↗
PubMed1997

DNA polymorphism at the Pgi locus of a wild yam, Dioscorea tokoro.

To study the origin and maintenance mechanisms of the PGI allozyme polymorphism of a wild plant, Dioscorea tokoro, DNA sequences of the entire coding region (1701 bp) and two intronic regions (total 2049 bp) of the Pgi gene as well as a part of the Adh gene (590 bp) were analyzed. Two replacement substitutions were revealed to be responsible for the differentiation of three allozymes alleles (Pgi-a, Pgi-b and Pgi-c) that occur in natural population in intermediate frequencies. Interspecific comparison of DNA sequences identified Pgi-b as the oldest allele, from which two other alleles were derived probably within the last 150,000 years. The level of DNA polymorphism at D. tokoro Pgi locus was low. No elevated level of DNA polymorphism was detected in the close vicinity of the two replacement sites differentiating the three allozymes. Departures from the neutral mutation hypothesis were detected by Fu and Li's and MK tests. The observed patterns of DNA polymorphism are explainable by both (1) the neutral mutation hypothesis with an assumption of small effective size of D. tokoro population, and (2) the positive selection hypothesis that the allele frequencies of Pgi-a and Pgi-c have increased in a short time by their selective advantages.

Alcohol Dehydrogenase↗
PubMed1999

Sequence diversity of flagellin (fliC) alleles in pathogenic Escherichia coli.

To study the molecular evolution of flagellin, the protein subunit specifying flagellar (H) antigens, the fliC genes from 15 pathogenic strains of Escherichia coli were amplified by PCR and sequenced. Comparison of fliC sequences of H6 and H7 strains revealed that alleles have a mosaic structure indicating the occurrence of past horizontal transfer of DNA segments between strains. The close similarity of H7 sequences also indicates the exchange of an entire fliC H7 allele between distant clonal lineages. In addition, the ratio of silent substitutions to amino acid replacements suggests that a short segment in the central region of fliC has been under positive selection in the divergence of H6 and H7 alleles. Phylogenetic analysis demonstrates that the fliC sequences of O157:H7 and O55:H7 serotypes are nearly identical and highly divergent from those of E. coli strains expressing H6 and H2 flagellar antigens. A nonmotile clone of sorbitol-fermenting O157 has rapidly accumulated multiple mutations in fliC, presumably as a result of the silencing of flagellin expression.

Alleles↗
PubMed2001

Heterologous expression and characterization of a rare Gaucher disease mutation (c.481C > T) from a Canadian aboriginal population using archival tissue samples.

Gaucher disease is an inherited sphingolipidosis resulting from deleterious mutations in the glucocerebrosidase gene. Through direct sequence analysis of genomic DNA from whole blood, fibroblast cultures, and formalin-fixed archival tissue samples, we have identified a rare homozygous C > T transition at cDNA nucleotide 481 of the glucocerebrosidase gene that results in a proline to serine amino acid substitution (p.P122S) in an aboriginal family of Cree descent in northern Alberta, Canada. A 13-month-old boy (JB) presented with severe visceral Gaucher disease and was treated with enzyme replacement. Currently, at 11 years he is developmentally delayed, with oculomotor apraxia. A cousin (MS) had previously died at age 7 from complications of severe Gaucher disease, before enzyme replacement therapy was available. She was also developmentally delayed. Heterologous expression of this allele using a baculovirus expression system revealed 19.2% of normal enzyme activity on the artificial substrate 4-methylumbelliferyl beta-d-glucopyranoside (4MUGP). Genotype/phenotype correlation is complicated by incomplete clinical details, enzyme replacement therapy, and the difficulty in excluding other genetic and environmental causes of developmental delay. However the development of oculomotor apraxia in JB suggests a Type 3 Gaucher phenotype. The only previous report of this mutation was also from a member of the Cree Nation, who has had a rather similar clinical course. A protocol is described for the isolation of genomic DNA from formalin-fixed bone marrow aspirate archival specimens obtained from the deceased for subsequent PCR-based sequence analysis and mutation detection. This technique will be applicable to the screening of this and other populations for the frequency of known Gaucher mutations where traditional DNA sources are unavailable.

Adolescent↗