Bp Tandem Duplication Research Articles

Long-Term Effect of Aromatase Inhibition in Aromatase Excess Syndrome

Aromatase excess syndrome (AEXS) is a very rare disorder characterized by prepubertal gynecomastia, bone age acceleration and early growth arrest. Heterozygote submicroscopic rearrangements within the promotor of CYP19A1 result in overexpression of aromatase and enhanced aromatization of androgens. Long-term treatment effects of aromatase inhibitors are unknown. Retrospectively we collected data from file records of 7 boys (three sibling pairs and one sporadic case) with AEXS. Genetic analysis revealed upstream of CYP19A1 a 165,901 bp deletion in 4 German cousins, a 198,662 bp deletion in 2 Japanese brothers and a 387,622 bp tandem duplication in a Japanese boy. All boys developed prepubertal gynecomastia, at 9.0 yr of age (median; range: 7.0 - 11.0). Height was +1.20 SDS (-0.24 - +1.98); predicted adult height was -1.29 (-3.29 - +1.09 SDS). Four boys were treated with anastrozole 1.0 mg daily, while three reached adult height untreated. Treatment with anastrozole was stopped after 5.6 yr (4.0 - 6.8). Three treated boys exceeded height prognosis by 2.4, 6.9 and 8.1 cm; while one untreated fell below prognosis by 8.6 cm. One treated with a low dose and two untreated reached their prognosis. Adult heights were -0.91 SDS with anastrozole (-2.86 - -0.29) and -0.15 SDS without (-2.31 - -0.03). Distance to target height was -0.22 SDS with anastrozole (-1.72 - +0.52) and +0.54 SDS without treatment (+0.23 - +1.30). Spontaneous growth in AEXS varied, even in the same family. Our data suggest that early started, long-term inhibition by aromatase inhibitor anastrozole (1 mg daily) promotes adult height in boys with AEXS.

Catechol-O-methyltransferase and dopamine receptor D4 gene variants: Possible association with substance abuse in Bangladeshi male.

Genetic risk of substance abuse is encoded mainly by central neurochemical pathways(mostly dopaminergic system) related to reinforcement and reward. In this study a functionalpolymorphism in Catechol-O-methyltransferase (COMT) (Val158Met) and the Dopamine receptor D4 gene (DRD4) (120 bp tandem duplication) has been studied in substance abused subjects. The study was carried out with 183 substance abused subjects and 175 healthy persons with no history of substance abuse. DNA was extracted and polymorphisms were analyzed using allele-specific PCR. The impact of these two polymorphisms was also analyzed on addictive characteristics (age of starting abuse, a pattern of drug habit, and period of addiction). It was found that only the heterozygous variant of COMT polymorphism (Val/Met) (p<0.05, OR = 1.66, 95% CI = 1.044-2.658) and both homozygous (p<0.05, OR = 0.43, 95% CI = 0.193-0.937) and heterozygous (p<0.05, OR = 0.37, 95% CI = 0.172-0.826) derived variants of DRD4 120 bp tandem duplication were significantly associated with risk of substance abuse compared to controls. In case of association of these polymorphisms with an age of onset, no significant difference was found among three different genotypic groups of COMT polymorphism. Whereas, the homozygous derived variant (240 bp/240 bp) of DRD4 gene was found to have a later age of onset (20.5±0.8) for substance abuse compared to heterozygous (120 bp/240 bp) (19.1±0.8) and wild type homozygous variant (120 bp/120 bp) (16.0±0.5), which was statistically significant (p<0.05). Again, in the case of the pattern of drug habit, the frequency of the Val/Val genotype is higher in polysubstance abused (>2 drugs) subjects (p<0.05) compared to the heterozygous Val/Met containing variants. An association of period of addiction was analyzed with an individual type of substance abuse and found that heroin abused subjects have a significantly higher period of addiction (11.6±1.0) compared to other abusers (p<0.01). Further, it was found that Met/Met containing variants of COMT polymorphism has a more extended period of addiction than other genetic variants in heroin abused subjects. These results indicate that genetic variability may influence the susceptibility to the risk of substance abuse and addictive characteristics.

Long-term Effect of Aromatase Inhibition in Aromatase Excess Syndrome.

Aromatase excess syndrome (AEXS) is a very rare disorder characterized by prepubertal gynecomastia, bone age acceleration, and early growth arrest. Heterozygote submicroscopic rearrangements within the promotor of CYP19A1 result in overexpression of aromatase and enhanced aromatization of androgens. The objective was to study long-term treatment effects of an aromatase inhibitor. Data from 7 boys with AEXS were retrospectively collected. Genetic analysis revealed upstream of CYP19A1 a 165 901 bp deletion in 4 German cousins, a 198 662 bp deletion in 2 Japanese brothers, and a 387 622 bp tandem duplication in a Japanese boy. All boys developed prepubertal gynecomastia, at median 9.0 years of age (range: 7.0-11.0). Height was +1.20 standard deviation score (SDS) (-0.24 to +1.98); predicted adult height was -1.29 SDS (-3.29 to +1.09). Four boys were treated with 1.0 mg of anastrozole daily, while 3 reached adult height untreated. Treatment with anastrozole was stopped after 5.6 years (4.0-6.8). Three treated boys exceeded their prognosis by 2.4, 6.9, and 8.1 cm, while 1 untreated boy fell below the prognosis by 8.6 cm. One treated with a low dose and 2 untreated reached their prognosis. Adult heights were -0.91 SDS with anastrozole (-2.86 to -0.29) and -0.15 SDS without (-2.31 to -0.03). Distance to target height was -0.22 SDS with anastrozole (-1.72 to +0.52) and +0.54 SDS without (+0.23 to +1.30). Spontaneous growth in AEXS varied, even in the same family. Our data suggest that early started, long-term inhibition by anastrozole promotes adult height in boys with AEXS.

Joining the club: First detection of African swine fever in wild boar in Germany.

African swine fever (ASF) has spread across many countries in Europe since the introduction into Georgia in 2007. We report here on the first cases of ASF in wild boar detected in Germany close to the border with Poland. In addition to the constant risk of ASF virus (ASFV) spread through human activities, movements of infected wild boar also represent a route of introduction. Since ASF emerged in Western Poland in November 2019, surveillance efforts, in particular examination of wild boar found dead, were intensified in the regions of Germany bordering with Poland. The first case of ASF in wild boar in Germany was therefore detected by passive surveillance and confirmed on 10 September 2020. By 24 September 2020, 32 cases were recorded. Testing of samples from tissues of carcasses in different stages of decomposition yielded cycle threshold values from 18 to 36 in the OIE-recommended PCR, which were comparable between the regional and national reference laboratory. Blood swabs yielded reliable results, indicating that the method is suitable also under outbreak conditions. Phylogenetic analysis of the ASFV whole-genome sequence generated from material of the first carcass detected in Germany, revealed that it groups with ASFV genotype II including all sequences from Eastern Europe, Asia and Belgium. However, some genetic markers including a 14bp tandem repeat duplication in the O174L gene were confirmed that have so far been detected only in sequences from Poland (including Western Poland). Epidemiological investigations that include estimated postmortem intervals of wild boar carcasses of infected animals suggest that ASFV had been introduced into Germany in the first half of July 2020 or even earlier.

Hereditary β-mannosidosis in a dog: Clinicopathological and molecular genetic characterization

Hereditary β-mannosidosis causing progressive lysosomal neuropathy and other clinical signs, has been previously described in humans, Nubian goats, and Salers cattle. Here we report the clinicopathological, metabolic, and molecular genetic features of canine beta-mannosidase (MANBA, EC 3.2.1.25) deficiency. A 1-year-old male mix-breed dog from St. Kitts was presented with progressive stumbling, weakness, and regurgitation. Vacuolated lymphocytes were observed on the blood film. Postmortem findings included marked enlargement of nerves, megaesophagus, and internal hydrocephalus. Vacuolated macrophages, neurons, and secretory epithelial cells suggested an oligosaccharide storage disease. Plasma concentration of the β-mannosidosis specific oligosaccharide was approximately 75 fold that of controls. The plasma beta-mannosidase activity was severely reduced to ~5% of controls; five other lysosomal acid hydrolase activities were increased or within their normal reference interval. Genomic sequencing of this dog's MANBA gene identified a homozygous exonic five bp tandem duplication in the penultimate exon of the MANBA gene (c.2377_2381dupTATCA) which results in a reading frame shift, altering the subsequent amino acid sequence and creating a premature stop codon. The truncated beta-mannosidase enzyme is expected to be dysfunctional. This enzyme deficiency causes the accumulation of un-degraded oligosaccharides in cells, which affect the myelination of the peripheral and central nervous systems. This insertion was not encountered in 121 and 80-screened samples from dogs on St. Kitts (all were homozygous for wild-type) and Philadelphia region (wild-type), respectively. In conclusion, canine β-mannosidosis has similar clinicopathological features with some human patients, but milder signs than in ruminants and more severe than in knockout mice. Hence, dogs with β-mannosidosis could become a valuable disease model for the human disease.

Genetic association between the DRD4 promoter polymorphism and clozapine-induced sialorrhea.

The use of clozapine, an effective antipsychotic drug used in treatment-resistant schizophrenia, is associated with adverse effects. Sialorrhea is one such effect, which can be distressing for many patients. Studies on the pharmacogenetics of the adverse effects of clozapine are limited. The aim of the present study was to determine whether clozapine-induced sialorrhea is associated with a 120 base-pairs (bp) tandem duplication polymorphism in the dopamine receptor subtype D4 (DRD4) gene. Ninety-five patients, mean age 35.43±9.43 years, with treatment-resistant schizophrenia and on clozapine were included in the study. Development of sialorrhea in response to the drug, as manifested by drooling of saliva, was documented in 45 (47.4%) patients. Genotyping of the patients was carried out to detect the presence of the polymorphism of interest. Clozapine-induced sialorrhea was found to be associated significantly with the 120-bp duplication in DRD4. The association was found to fit a log-additive model with an odds ratio of 2.95 (95% confidence interval 1.51-5.75; P=0.0006). Thus, the presence of the 120-bp duplication in DRD4 appears to confer a risk for sialorrhea in response to clozapine therapy. The underlying pathophysiology and clinical significance of this phenomenon warrant further investigation.

Aromatase excess syndromeas a model for genomic disorder: identification of molecular bases and phenotypic determinants

Aromatase excess syndrome (AEXS) is a rare autosomal dominant disorder characterized by gynecomastia. Although chromosomal inversions leading to abnormal fusion between CYP19A1 coding exons and non-coding exons of neighboring genes have been identified in a few patients with AEXS, its molecular basis and clinical spectrum remain largely unknown. We studied 18 affected males from six unrelated families A–F, and found a heterozygous 79,156 bp tandem duplication involving seven of 11 non-coding CYP19A1 exons 1 in families A and B, a heterozygous 211,631 bp deletion involving exons 2–43 of DMXL2 and exons 5–10 of GLDN in family C, and a heterozygous 165,901 bp deletion involving exons 2–43 of DMXL2 in families D–F. Analysis of transcripts revealed that duplicated exons 1 at the distal non-physiological position can also function as transcription start sites, and that the two deletions produced the same chimeric mRNA constituted by DMXL2 exon 1 and CYP19A1 exons 2–10. Clinical features such as gynecomastia and elevated estradiol/testosterone ratios were milder in patients with duplications and deletions than in those with inversions. Furthermore, genotype-phenotype correlations in patients with duplications, deletions, and inversions implies that phenotypic severity of AEXS is primarily determined by the expression pattern of CYP19A1 and the chimeric genes and by the structural property of the fused exons with a promoter function (i.e., the presence or absence of a natural translation start codon). The present study expands the genetic mechanism and phenotypic spectrum of AEXS, and provides novel models for genomic disorder leading to gain-of-function mutations. We also discuss on the effect of estradiol on the hypothalamic-pituitary gonadotropin control.

Phylogeographical patterns in the widespread arctic–alpine plant Bistorta vivipara (Polygonaceae) with emphasis on western North America

AbstractAimWe investigated genetic variation in Bistorta vivipara, a widespread Northern Hemisphere tundra species, to infer patterns of migration and where it may have survived during the Last Glacial Maximum (LGM).LocationSamples came primarily from western North America, with a few from the Arctic and Eurasia.MethodsWe sequenced two chloroplast DNA spacer regions, trnH–psbA and trnS–G, in individuals from 199 populations and mapped haplotype distributions and their relationships using a haplotype network. We calculated genetic and molecular diversity statistics for the seven geographical regions from which we obtained samples.ResultsFifteen haplotypes were detected, with very little divergence among them. The haplotypes are separated into two main groups by the presence or absence of a 22 bp tandem duplication. Four haplotypes are common, widespread and with substantial range overlap; 11 are rare and mostly unique to one region. Two rare haplotypes were found only in British Columbia (BC). Western North America and Asia have the highest levels of genetic and molecular diversity. Northern and southern BC have different haplotype complements.Main conclusionsBistorta vivipara has relatively low genetic diversity, with much less genetic structure than we expected for such a widespread species. We expected significant geographical structure due to the combined effects of genetic drift and geographical isolation. The asexual reproductive mode of B. vivipara may facilitate relatively rapid population establishment and spread compared with sexual reproduction by seed. Bistorta vivipara probably originated in Asia and spread to North America and Europe prior to the LGM. In western North America it spread to its modern distribution from Beringia and the western USA following the LGM. Populations in northern and southern BC may have different histories, possibly related to the timing and extent of glaciation. The occurrence of two unique haplotypes within BC suggests that some individuals may have survived in full glacial refugia within BC.

A Stable dw3 Allele in Sorghum and a Molecular Marker to Facilitate Selection

ABSTRACTDwarfism plays a key role in adapting crops to high‐input production systems by contributing to improved lodging resistance and fertilizer efficiency. In sorghum [Sorghum bicolor (L.) Moench], four dwarfing mutations have been found that profoundly reduce the stalk height. Notable among these mutations is dwarf‐3 (dw3), even though the phenotype it confers is unstable. The objectives of this research were to identify and characterize a stable mutant allele of the dw3 locus. Polymerase chain reaction (PCR) was used to amplify a region of dw3 containing the 882 bp tandem duplication that is diagnostic for the unstable allele. Analyses of PCR fragments from dwarf sorghum accessions that lacked the duplicated region led to the identification of Tx2737 as a possible carrier of a novel mutant dw3 allele. Sequence analysis confirmed and revealed the presence of a 6 bp deletion in exon 5 of dw3 that eliminates two highly conserved amino acids, Q1275 and R1276, from the enzyme. The frequency of this allele in sorghum germplasm appears to be very low with only 1% of the accessions in the diversity panel exhibiting this deletion. Field trials demonstrated that this allele of dw3 produces a stable dwarf phenotype with no height mutants found in Tx2737 and KS19. The new allele has been coined dw3‐sd2. The discovery of dw3‐sd2 and a DNA marker assay to facilitate selection will provide an opportunity to replace the unstable dw3 allele currently used in most commercial sorghum hybrids through introgression of dw3‐sd2 into elite parent lines.

Partial tandem duplication of mtDNA–tRNA Phe impairs mtDNA translation in late-onset mitochondrial myopathy

An 80-year-old woman (PI) has been suffering of late onset progressive weakness and wasting of lower-limb muscles, accompanied by high creatine kinase levels in blood. A muscle biopsy, performed at 63 years, showed myopathic features with partial deficiency of cytochrome c oxidase. A second biopsy taken 7 years later confirmed the presence of a mitochondrial myopathy but also of vacuolar degeneration and other morphological features resembling inclusion body myopathy. Her 46-year-old daughter (PII) and 50-year-old son (PIII) are clinically normal, but the creatine kinase levels were moderately elevated and the EMG was consistently myopathic in both. Analysis of mitochondrial DNA sequence revealed in all three patients a novel, homoplasmic 15 bp tandem duplication adjacent to the 5′ end of mitochondrial tRNA Phe gene, encompassing the first 11 nucleotides of this gene and the four terminal nucleotides of the adjacent D-loop region. Both mutant fibroblasts and cybrids showed low oxygen consumption rate, reduced mitochondrial protein synthesis, and decreased mitochondrial tRNA Phe amount. These findings are consistent with an unconventional pathogenic mechanism causing the tandem duplication to interfere with the maturation of the mitochondrial tRNA Phe transcript.

Evolutionary dynamics and sites of illegitimate recombination revealed in the interspersion and sequence junctions of two nonhomologous satellite DNAs in cactophilic Drosophila species

Satellite DNA (satDNA) is a major component of genomes but relatively little is known about the fine-scale organization of unrelated satDNAs residing at the same chromosome location, and the sequence structure and dynamics of satDNA junctions. We studied the organization and sequence junctions of two nonhomologous satDNAs, pBuM and DBC-150, in three species from the neotropical Drosophila buzzatii cluster (repleta group). In situ hybridization to microchromosomes, interphase nuclei and extended DNA fibers showed frequent interspersion of the two satellites in D. gouveai, D. antonietae and, to a lesser extent, D. seriema. We isolated by PCR six pBuM x DBC-150 junctions: four are exclusive to D. gouveai and two are exclusive to D. antonietae. The six junction breakpoints occur at different positions within monomers, suggesting independent origin. Four junctions showed abrupt transitions between the two satellites, whereas two junctions showed a distinct 10 bp tandem duplication before the junction. Unlike pBuM, DBC-150 junction repeats are more variable than randomly cloned monomers and showed diagnostic features in common to a 3-monomer higher-order repeat seen in the sister species D. serido. The high levels of interspersion between pBuM and DBC-150 repeats suggest extensive rearrangements between the two satellites, maybe favored by specific features of the microchromosomes. Our interpretation is that the junctions evolved by multiples events of illegitimate recombination between nonhomologous satDNA repeats, with subsequent rounds of unequal crossing-over expanding the copy number of some of the junctions.

Foggy D-shaped zone of inhibition in Staphylococcus aureus owing to a dual character of both inducible and constitutive resistance to macrolide–lincosamide–streptogramin B

We identified Staphylococcus aureus strains having blunt inhibitory zones around the clindamycin or josamycin discs proximal to the erythromycin disc filled with slight bacterial growth. This ambiguous phenotype was termed as 'macrolide-lincosamide-streptogramin B antimicrobial (MLS(B)) resistance having a foggy D-shaped inhibitory zone' (fMLS(B)), and we tried to analyse its molecular mechanisms. Forty-one clinically isolated strains of fMLS(B) S. aureus were studied. The regulatory region of the erm(A) gene, which was found to be the only molecular mechanism of fMLS(B), was sequenced. Then, beta-galactosidase assays were performed to observe their expression patterns through the nucleotide sequential alteration. According to the sequencing electropherogram, a grouping was made of a homogeneous nucleotide sequence group (73.2%) and a heterogeneous nucleotide sequence group (26.8%). The former group was composed of a 25 bp tandem duplication type and a 25 bp tandem triplication type. Their beta-galactosidase activity was similar to that of constitutive MLS(B) resistance due to its high basal level. Nevertheless, the predicted mRNA secondary structure of the regulatory region maintains the stem-loops of inducible wild-type erm(A), and thereby its inducible character might be expected in vivo. Strains in the latter group were proven to have two different erm(A) genes, and then dual effect of expression was observed. The ambiguous phenotype of fMLS(B) is due to its possessing the dual character of inducible and constitutive expression of erm(A). The dual character is due to having one erm(A) gene of dual character or coexistence of two characterized erm(A) genes simultaneously.

A novel duplication in theFMR1gene: implications for molecular analysis in fragile X syndrome and repeat instability

We have observed a 49 bp tandem duplication adjacent to the triplet repeat of the FMR1 gene and have shown it to occur as a variant in Finland. It affects the primers commonly used in molecular analysis of fragile X syndrome by polymerase chain reaction (PCR) methods. One concern is that females with the full mutation and variant alleles might be missed because of the two PCR products generated by the variant. We suggest that the duplication has arisen by a misalignment of the proximal end of the repeat tract and the non-adjacent GGCGGCGGCGG-sequence located 37 bp upstream and may indicate a mutation hot spot. The discovery of this duplication and the previous observations on deletions associated with full mutations in FMR1 indicate that realignment between the repeat tract and dispersed non-adjacent homologous repetitive sequences may also play a role in repeat instability in fragile X.

A Follow-up Study in a Taiwanese Family with Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis and Stroke-like Episodes Syndrome

MELAS (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes) syndrome is often associated with A3243G point mutation of mitochondrial DNA (mtDNA). We previously described a MELAS family characterized by harboring an additional approximately 260 bp tandem duplication in the D-loop and a novel C3093G point mutation in the 16S rRNA gene of mtDNA in the proband. We studied the clinical progression and fluctuation of mtDNA mutations in this Taiwanese MELAS family. We followed up the clinical course in all members of this family (1 proband, her mother and 3 sons) for 12 years. Mutations of mtDNA in serial muscle biopsies of the proband and blood samples and hair follicles taken at different time points from the members of this family were analyzed. The proband developed repeated stroke-like episodes, chronic intestinal pseudo-obstruction, polyneuropathy, progressive renal failure and dilated cardiomyopathy with heart failure. During the follow-up period, the mother and one of the siblings of the proband developed stroke-like episodes at age 62 and 12, respectively. There was no significant difference in the proportions of mtDNA with A3243G mutation among five serial muscle biopsies of the proband. In one carrier (I-2), the proportion of A3243G mutated mtDNA in blood cells was slightly increased with disease progression. This study underlines the importance of early detection of extraneuromuscular symptoms in the members of a family with MELAS syndrome by adequate follow-up. The age of onset of stroke-like episode in MELAS syndrome may be as late as 62 years. We suggest that the manifestations of MELAS syndrome in this family might be associated with the additional approximately 260 bp tandem duplication in the D-loop region and the coexistence of C3093G mutation in the 16S rRNA gene with the A3243G mutation of mtDNA.

Genomic rearrangement at 10q24 in non-syndromic split-hand/split-foot malformation

Split-hand/split-foot malformation (SHFM) is a congenital limb malformation characterized by a median cleft of hand and/or foot due to the absence of central rays. Five loci for syndromic and non-syndromic SHFM, termed SHFM1-5, have been mapped to date. Recently, a 0.5 Mb tandem genomic duplication was found at chromosome 10q24 in SHFM3 families. To refine the minimum duplicated region and to further characterize the SHFM3 locus, we screened 28 non-syndromic SHFM families for tandem genomic duplication of 10q24 by Southern blot and sequence analysis of the dactylin gene. Of 28 families, only two showed genomic rearrangements. Representative patients from the two families exhibit typical SHFM, with symmetrically affected hands and feet. One patient is a familial case with a 511,661 bp tandem duplication, whereas the second is a sporadic case arising from a de novo, 447,338 bp duplication of maternal origin. The smaller duplication in the second patient contained the LBX1, BTRC, POLL, and DPCD genes and a disrupted extra copy of the dactylin gene, and was nearly identical to the smallest known duplicated region of SHFM3. Our results indicate that genomic rearrangement of SHFM3 is rare among non-syndromic SHFM patients and emphasize the importance of screening for genomic rearrangements even in sporadic cases of SHFM.

Pedigree disequilibrium test (PDT) replicates association and linkage between DRD4 and ADHD in multigenerational and extended pedigrees from a genetic isolate.

Association/linkage between dopamine D4 receptor (DRD4) polymorphisms and attention-deficit/hyperactivity disorder (ADHD) has been suggested by case-control- and nuclear-family-based studies. Here, we present a candidate gene analysis for DRD4 using 14 extended and multigenerational families segregating ADHD derived from the 'Paisa' community of Antioquia, Colombia, a genetic isolate. Two DRD4 polymorphisms (a 120 bp tandem duplication at the promoter and a 48 bp-VNTR at exon 3), reported associated to ADHD, were genotyped. Parametric and non-parametric linkage analyses, and a family-based association test (FBAT), the pedigree disequilibrium test (PDT), were applied to search for evidence of association/linkage. Two-point LOD scores were significantly negative, with values ranging from -3.21 (P=0.011158) to -7.66 (P=0.000091 at theta=0). Non-parametrical analysis resulted in nonsignificant evidence for linkage. The PDT showed a moderate trend toward significance of association/linkage between the 7-repeat (7R) allele at the 48 bp VNTR and ADHD (P=0.0578). Furthermore, the haplotype analysis shows a significant association/linkage of the 7R-240 bp haplotype (P=0.0467) with ADHD. Results suggest that either a moderate DRD4 genetic effect, or linkage disequilibrium of DRD4 with an ADHD disease locus in the vicinity or the linkage to a phenotypic component of the ADHD spectrum could be underlying this association/linkage. These results provide further evidence for the association of ADHD to genetic variation in or near to DRD4 and replicate the previously reported association between ADHD and the 7R allele.

Host plant-dependent phenotypic reversion of Ralstonia solanacearum from non-pathogenic to pathogenic forms via alterations in the phcA gene.

Ralstonia solanacearum is a plant pathogenic bacterium that undergoes a spontaneous phenotypic conversion (PC) from a wild-type pathogenic to a non-pathogenic form. PC is often associated with mutations in phcA, which is a key virulence regulatory gene. Until now, reversion to the wild-type pathogenic form has not been observed for PC variants and the biological significance of PC has been questioned. In this study, we characterized various alterations in phcA (eight IS element insertions, three tandem duplications, seven deletions and a base substitution) in 19 PC mutants from the model strain GMI1000. In five of these variants, reversion to the pathogenic form was observed in planta, while no reversion was ever noticed in vitro whatever culture media used. However, reversion was observed for a 64 bp tandem duplication in vitro in the presence of tomato root exudate. This is the first report showing a complete cycle of phenotypic conversion/reversion in a plant pathogenic bacterium.

Association analysis of polymorphisms in the upstream region of the human dopamine D4 receptor gene in schizophrenia

Several studies have suggested that the transcriptional activity of the DRD4 gene may exert an important role in susceptibility to schizophrenia. To address this issue, we studied the association of schizophrenia and polymorphisms including −616C>G, −603T>del, −602G>del, 600G>C, −521C>T, −376C>T and a 120 bp tandem duplication polymorphism (120 bp repeat) in 1.2 kb upstream from the initiation codon in the promoter region of the DRD4 gene with 210 schizophrenic cases and 206 healthy controls. The results showed a significant excess of allele L of the 120 bp repeat in the schizophrenic patients compared to the controls ( X 2=8.585, df=1, P=0.003, OR=1.546, 95% CI=1.154–2.070). No significant difference was detected in the frequencies of genotype and allele of six other polymorphisms between the two groups. However, haplotypic distribution of 120 bp repeat, −616C>G, −602G>del, −521C>T and −376C>T was significantly different between case and control groups ( P=0.005). This might cause the alteration of the transcriptional regulation of the DRD4 gene, as the consensus sequences of binding sites for several known transcription factors are involved in this region.

Insertional polymorphism in introns 4 and 10 of the maize beta-glucosidase gene glu1.

The major beta-glucosidase isozyme Glu1 is encoded by a highly polymorphic gene (glu1) in maize. The glu1 gene comprises 12 exons and 11 introns. Two of these introns, introns 4 and 10, show insertional polymorphism: those in allele glu1-1 (represented by inbred line OH7B) are longer than those in other inbred genotypes and in two teosintes (Zea mexicana and Zea parviglumis) surveyed. Sequence data revealed that an increase in the length of intron 4 from 150 to 477 bp in OH7B is due to a short (11 bp) tandem duplication and a large insertion sequence of 313 bp plus a 4-bp (5' ATAG 3') direct repeat. The 313-bp insertion sequence (referred to as mzsTn-1) has all the features of a transposon, having a 25-bp well-conserved (3/25 mismatches) inverted repeat sequence at its termini flanked by a 4-bp direct repeat. The increase in length from 1041 to 1302 bp in intron 10 of OH7B is due to a 259-bp insertion sequence (referred to as mzsTn-2) plus a 2-bp (5' TA 3') direct repeat. The mzsTn-2 element also possesses all the hallmarks of a transposon: a 34-bp well-conserved (3/34 mismatches) inverted repeat sequence at its termini flanked by a 2-bp direct repeat. The mzsTn-1 element belongs to a new family of inverted repeat elements, while mzsTn-2 belongs to the Stowaway family of inverted repeat elements. Analysis of PCR products from amplifications off genomic-DNA templates, using primers derived from the inverted repeat termini, and Southern blotting data suggest that both small transposons are members of a multigene family. The occurrence of two different small transposons in introns of the same glu1 allele in inbred OH7B and their absence in other genotypes suggest that they have moved into this glu1 allele recently through mediation of their autonomous counterparts that are active in OH7B or in its ancestry.

Co-existence of high levels of a cytochrome b mutation and of a tandem 200 bp duplication in the D-loop of muscle human mitochondrial DNA

Previous studies have suggested that some patients with large-scale mitochondrial DNA (mtDNA) deletions also presented a heteroplasmic 260 bp tandem duplication in the mtDNA D-loop region. Such duplications were observed not only in patients with mitochondrial pathology but also in aged subjects. However, the percentage of duplicated mtDNA did not exceed a few per cent of the total mtDNA, except in one example where it reached 30%. We report here another type of 200 bp duplication in the mtDNA D-loop region that, instead of being associated with a large-scale deletion, is correlated to the presence of a point mutation in the cytochrome b gene. The 200 bp duplication concerned up to 95% of the total mtDNA of some muscle mitochondria and was absent from the patient lymphocyte DNA. The percentages of the 200 bp duplication and that of the cytochrome b mutation were relatively close in whole muscle as well as in single muscle fibres, suggesting a correlation between the mutation and the duplication. This duplication could also be detected by PCR in two other patients with mitochondrial disorders but without known deletion or mtDNA mutation. These data suggest that the accumulation of these small duplications in the mtDNA D-loop could be indicative of the presence of other defects of the mtDNA which would damage the respiratory chain function. These deficiencies would induce the generation of small duplications in the D-loop.