Genomic rearrangements in trypanosomatids: an alternative to the "one gene" evolutionary hypotheses?

Most molecular trees of trypanosomatids are based on point mutations within DNA sequences. In contrast, there are very few evolutionary studies considering DNA (re) arrangement as genetic characters. Waiting for the completion of the various parasite genome projects, first information may already be obtained from chromosome size-polymorphism, using the appropriate algorithms for data processing. Three illustrative models are presented here. First, the case of Leishmania (Viannia) braziliensis/L. (V.) peruviana is described. Thanks to a fast evolution rate (due essentially to amplification/deletion of tandemly repeated genes), molecular karyotyping seems particularly appropriate for studying recent evolutionary divergence, including eco-geographical diversification. Secondly, karyotype evolution is considered at the level of whole genus Leishmania. Despite the fast chromosome evolution rate, there is qualitative congruence with MLEE- and RAPD-based evolutionary hypotheses. Significant differences may be observed between major lineages, likely corresponding to major and less frequent rearrangements (fusion/fission, translocation). Thirdly, comparison is made with Trypanosoma cruzi. Again congruence is observed with other hypotheses and major lineages are delineated by significant chromosome rearrangements. The level of karyotype polymorphism within that "species" is similar to the one observed in "genus" Leishmania. The relativity of the species concept among these two groups of parasites is discussed.

Relevance of biallelic versus monoallelic TNFRSF13B mutations in distinguishing disease-causing from risk-increasing TNFRSF13B variants in antibody deficiency syndromes.

TNFRSF13B encodes transmembrane activator and calcium modulator and cyclophilin ligand interactor (TACI), a B cell- specific tumor necrosis factor (TNF) receptor superfamily member. Both biallelic and monoallelic TNFRSF13B mutations were identified in patients with common variable immunodeficiency disorders. The genetic complexity and variable clinical presentation of TACI deficiency prompted us to evaluate the genetic, immunologic, and clinical condition in 50 individuals with TNFRSF13B alterations, following screening of 564 unrelated patients with hypogammaglobulinemia. We identified 13 new sequence variants. The most frequent TNFRSF13B variants (C104R and A181E; n=39; 6.9%) were also present in a heterozygous state in 2% of 675 controls. All patients with biallelic mutations had hypogammaglobulinemia and nearly all showed impaired binding to a proliferation-inducing ligand (APRIL). However, the majority (n=41; 82%) of the pa-tients carried monoallelic changes in TNFRSF13B. Presence of a heterozygous mutation was associated with antibody deficiency (P< .001, relative risk 3.6). Heterozygosity for the most common mutation, C104R, was associated with disease (P< .001, relative risk 4.2). Furthermore, heterozygosity for C104R was associated with low numbers of IgD(-)CD27(+) B cells (P= .019), benign lymphoproliferation (P< .001), and autoimmune complications (P= .001). These associations indicate that C104R heterozygosity increases the risk for common variable immunodeficiency disorders and influences clinical presentation.

Congenital Myasthenic Syndromes with COLQ mutations: Long term follow-up

Congenital myasthenic syndromes (CMS) are clinically and genetically heterogeneous inherited disorders characterized by impaired neuromuscular transmission. Mutations in the acetylcholinesterase (AChE) collagenlike tail subunit gene (ColQ) cause recessive forms of synaptic CMS with end plate AChE deficiency. We report the time course of clinical manifestations in 15 COLQ-mutated patients followed from 1987 to 2010. All patients suffered from a muscle weakness with onset at birth or in childhood. Ocular and bulbar signs were found in 60% of the patients and delayed pupillary light response in 20% of our patients. EMG study demonstrated a decrement on repetitive nerve stimulation and repetitive compound muscle action potential in all patients. Clinical symptoms strongly fluctuated daily, weekly, monthly or even yearly. Severe relapses were characterized by a general motor weakness associated with pain which resolved spontaneously after a few months whereas the relapses with these symptoms and bulbar signs could last up to several years. Genetic analyses identified 16 different mutations including 9 novel ones. There was no genotype-phenotype correlation. Our study confirms the predominance of oculobulbar signs and the frequency of respiratory distress in COLQrelated CMS. At the end of the follow up of 23 years, interesting findings were (i) the spontaneous reversibility of severe relapses, some of them lasting for up to 5 years (ii) the good prognosis of COLQ-related CMS, since at the end of the follow-up 80% of patients were ambulant and 87% of patients had no respiratory trouble (iii) the efficacy of Ephedrine and, to a lesser extend, of 3-4 DAP. The triggering factors of relapses were esterase inhibitors, effort, puberty, pregnancy and delivery highlighting the importance of hormonal factors in CMS. In conclusion, patients diagnosed with unknown congenital myopathy should undergo an electrophysiological study of neuromuscular junction to identify ColQ-related CMS.

Apoptosis as a target for gene therapy in rheumatoid arthritis

Rheumatoid arthritis (RA) is characterized by chronic inflammation of the synovial joints resulting from hyperplasia of synovial fibroblasts and infiltration of lymphocytes, macrophages and plasma cells, all of which manifest signs of activation. All these cells proliferate abnormally, invade bone and cartilage, produce an elevated amount of pro-inflammatory cytokines, metalloproteinases and trigger osteoclast formation and activation. Some of the pathophysiological consequences of the disease may be explained by the inadequate apoptosis, which may promote the survival of autoreactive T cells, macrophages or synovial fibroblasts. Although RA does not result from single genetic mutations, elucidation of the molecular mechanisms implicated in joint destruction has revealed novel targets for gene therapy. Gene transfer strategies include inhibition of pro-inflammatory cytokines, blockade of cartilage-degrading metalloproteinases, inhibition of synovial cell activation and manipulation of the Th1-Th2 cytokine balance. Recent findings have iluminated the idea that induction of apoptosis in the rheumatoid joint can be also used to gain therapeutic advantage in the disease. In the present review we will discuss different strategies used for gene transfer in RA and chronic inflammation. Particularly, we will highlight the importance of programmed cell death as a novel target for gene therapy using endogenous biological mediators, such as galectin-1, a beta-galactoside-binding protein that induces apoptosis of activated T cells and immature thymocytes.

Characterization of the Aspergillus nidulans biotin biosynthetic gene cluster and use of the bioDA gene as a new transformation marker.

The genes involved in the biosynthesis of biotin were identified in the hyphal fungus Aspergillus nidulans through homology searches and complementation of Escherichia coli biotin-auxotrophic mutants. Whereas the 7,8-diaminopelargonic acid synthase and dethiobiotin synthetase are encoded by distinct genes in bacteria and the yeast Saccharomyces cerevisiae, both activities are performed in A. nidulans by a single enzyme, encoded by the bifunctional gene bioDA. Such a bifunctional bioDA gene is a genetic feature common to numerous members of the ascomycete filamentous fungi and basidiomycetes, as well as in plants and oömycota. However, unlike in other eukaryota, the three bio genes contributing to the four enzymatic steps from pimeloyl-CoA to biotin are organized in a gene cluster in pezizomycotina. The A. nidulans auxotrophic mutants biA1, biA2 and biA3 were all found to have mutations in the 7,8-diaminopelargonic acid synthase domain of the bioDA gene. Although biotin auxotrophy is an inconvenient marker in classical genetic manipulations due to cross-feeding of biotin, transformation of the biA1 mutant with the bioDA gene from either A. nidulans or Aspergillus fumigatus led to the recovery of well-defined biotin-prototrophic colonies. The usefulness of bioDA gene as a novel and robust transformation marker was demonstrated in co-transformation experiments with a green fluorescent protein reporter, and in the efficient deletion of the laccase (yA) gene via homologous recombination in a mutant lacking non-homologous end-joining activity.

Clinical manifestations in patients with SOS1 mutations range from Noonan syndrome to CFC syndrome.

Noonan syndrome (NS) and cardio-facio-cutaneous (CFC) syndrome are autosomal dominant disorders characterized by heart defects, facial dysmorphism, ectodermal abnormalities, and mental retardation. There is a significant clinical overlap between NS and CFC syndrome, but ectodermal abnormalities and mental retardation are more frequent in CFC syndrome. Mutations in PTPN11 and KRAS have been identified in patients with NS and those in KRAS, BRAF and MAP2K1/2 have been identified in patients with CFC syndrome, establishing a new role of the RAS/MAPK pathway in human development. Recently, mutations in the son of sevenless gene (SOS1) have also been identified in patients with NS. To clarify the clinical spectrum of patients with SOS1 mutations, we analyzed 24 patients with NS, including 3 patients in a three-generation family, and 30 patients with CFC syndrome without PTPN11, KRAS, HRAS, BRAF, and MAP2K1/2 (MEK1/2) mutations. We identified two SOS1 mutations in four NS patients, including three patients in the above-mentioned three-generation family. In the patients with a CFC phenotype, three mutations, including a novel three amino-acid insertion, were identified in one CFC patient and two patients with both NS and CFC phenotypes. These three patients exhibited ectodermal abnormalities, such as curly hair, sparse eyebrows, and dry skin, and two of them showed mental retardation. Our results suggest that patients with SOS1 mutations range from NS to CFC syndrome.

Hyaline fibromatosis syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors.

Hyaline Fibromatosis Syndrome (HFS) is a human genetic disease caused by mutations in the anthrax toxin receptor 2 (or cmg2) gene, which encodes a membrane protein thought to be involved in the homeostasis of the extracellular matrix. Little is known about the structure and function of the protein or the genotype-phenotype relationship of the disease. Through the analysis of four patients, we identify three novel mutants and determine their effects at the cellular level. Altogether, we show that missense mutations that map to the extracellular von Willebrand domain or the here characterized Ig-like domain of CMG2 lead to folding defects and thereby to retention of the mutated protein in the endoplasmic reticulum (ER). Mutations in the Ig-like domain prevent proper disulphide bond formation and are more efficiently targeted to ER-associated degradation. Finally, we show that mutant CMG2 can be rescued in fibroblasts of some patients by treatment with proteasome inhibitors and that CMG2 is then properly transported to the plasma membrane and signalling competent, identifying the ER folding and degradation pathway components as promising drug targets for HFS.

Five new CYLD mutations in skin appendage tumors and evidence that aspartic acid 681 in CYLD is essential for deubiquitinase activity.

Brooke-Spiegler syndrome, familial cylindromatosis, and familial trichoepithelioma are autosomal-dominant genetic predispositions for benign tumors of skin appendages caused by mutations in the CYLD gene localized on chromosome 16q12-q13. The encoded protein functions as ubiquitin-specific protease (UBP), which negatively regulates NF-kappaB and c-Jun N-terminal kinase (JNK) signaling. We investigated five families affected with these skin neoplasms and identified four premature stop codons and the novel missense mutation D681G in a family in which 11 of 12 investigated tumors were trichoepitheliomas. CYLD protein harboring this missense mutation had a significant reduced ability to inhibit TNF receptor-associated factor (TRAF)2- and TRAF6-mediated NF-kappaB activation, tumor necrosis factor-alpha (TNFalpha)-induced JNK signaling, and to deubiquitinate TRAF2. CYLD-D681G was coimmunoprecipitated by TRAF2, but was unable to cleave K63-linked polyubiquitin chains. Aspartic acid 681 is highly conserved in CYLD homologues and other members of the UBP family, but does not belong to the Cys and His boxes providing the CYLD catalytic triad (Cys601, His871, and Asp889). As reported previously, the homologous residue D295 of HAUSP/USP-7 forms a hydrogen bond with the C-terminal end of ubiquitin and is important for the enzymatic activity. These results underline that D681 in CYLD is required for cleavage of K63-linked polyubiquitin chains.

Mutations in Eml1 lead to ectopic progenitors and neuronal heterotopia in mouse and human.

Neuronal migration disorders such as lissencephaly and subcortical band heterotopia are associated with epilepsy and intellectual disability. DCX, PAFAH1B1 and TUBA1A are mutated in these disorders; however, corresponding mouse mutants do not show heterotopic neurons in the neocortex. In contrast, spontaneously arisen HeCo mice display this phenotype, and our study revealed that misplaced apical progenitors contribute to heterotopia formation. While HeCo neurons migrated at the same speed as wild type, abnormally distributed dividing progenitors were found throughout the cortical wall from embryonic day 13. We identified Eml1, encoding a microtubule-associated protein, as the gene mutated in HeCo mice. Full-length transcripts were lacking as a result of a retrotransposon insertion in an intron. Eml1 knockdown mimicked the HeCo progenitor phenotype and reexpression rescued it. We further found EML1 to be mutated in ribbon-like heterotopia in humans. Our data link abnormal spindle orientations, ectopic progenitors and severe heterotopia in mouse and human.

Use-dependent block of the voltage-gated Na+ channel by tetrodotoxin and saxitoxin: Effect of pore mutations that change ionic selectivity.

Voltage-gated Na(+) channels (NaV channels) are specifically blocked by guanidinium toxins such as tetrodotoxin (TTX) and saxitoxin (STX) with nanomolar to micromolar affinity depending on key amino acid substitutions in the outer vestibule of the channel that vary with NaV gene isoforms. All NaV channels that have been studied exhibit a use-dependent enhancement of TTX/STX affinity when the channel is stimulated with brief repetitive voltage depolarizations from a hyperpolarized starting voltage. Two models have been proposed to explain the mechanism of TTX/STX use dependence: a conformational mechanism and a trapped ion mechanism. In this study, we used selectivity filter mutations (K1237R, K1237A, and K1237H) of the rat muscle NaV1.4 channel that are known to alter ionic selectivity and Ca(2+) permeability to test the trapped ion mechanism, which attributes use-dependent enhancement of toxin affinity to electrostatic repulsion between the bound toxin and Ca(2+) or Na(+) ions trapped inside the channel vestibule in the closed state. Our results indicate that TTX/STX use dependence is not relieved by mutations that enhance Ca(2+) permeability, suggesting that ion-toxin repulsion is not the primary factor that determines use dependence. Evidence now favors the idea that TTX/STX use dependence arises from conformational coupling of the voltage sensor domain or domains with residues in the toxin-binding site that are also involved in slow inactivation.

Germline mutations in retinoma patients: relevance to low-penetrance and low-expressivity molecular basis.

PURPOSE: To study phenotype-genotype correlation in patients who have retinoma, which is a benign tumor resembling the post irradiation regression pattern of retinoblastoma (RB). METHODS: We selected patients who had retinoma and positive family history for RB and patients who had retinoma in one eye and either retinoma or RB in the other eye. The study included 22 patients with available DNA: 18 from 11 families and four sporadic cases. DNA was extracted from peripheral blood leukocytes. The RB1 gene was screened by DHPLC and direct sequencing of the promoter and all the exons. RESULTS: We identified 17 occurrences of 11 distinct germline mutations in two sporadic and in 15 familial cases (nine families). The 11 identified mutations were located in exons 1, 10,11,13,14, and 19 to 23. Four of the identified mutations were not previously reported, including g.64407delT, g.153236A>T, g.156743delTCTG, and g.162078delA. Eight out the 11 mutations were truncating and three were nontruncating (missense). There was no correlation between the type of mutation and the number of tumor foci per eye (RB or retinomas). Highly heterogeneous intrafamilial expressivity was observed. CONCLUSIONS: To our knowledge, this study is the largest series of mutations of consecutive retinoma patients. The present data suggest that the type of inherited mutations underlying retinoma is undistinguishable from RB related ones, i.e., largely dominated by truncating mutants. This finding is in contrast with the RB1 genotypic spectrum of mutations associated with low-penetrance RB, i.e., nontruncating mutants. The molecular mechanism underlying low-penetrance and attenuated expressivity (retinomas) appeared to be distinct.

Human immunodeficiency virus type 1 (HIV-1) genotyping in Rio de Janeiro, Brazil: assessing subtype and drug-resistance associated mutations in HIV-1 infected individuals failing highly active antiretroviral therapy

In order to assess the human immunodeficiency virus type 1 (HIV-1) drug resistance mutation profiles and evaluate the distribution of the genetic subtypes in the state of Rio de Janeiro, Brazil, blood samples from 547 HIV-1 infected patients failing antiretroviral (ARV) therapy, were collected during the years 2002 and 2003 to perform the viral resistance genotyping at the Renageno Laboratory from Rio de Janeiro (Oswaldo Cruz Foundation). Viral resistance genotyping was performed using ViroSeqTM Genotyping System (Celera Diagnostic-Abbott, US). The HIV-1 subtyping based on polymerase (pol) gene sequences (protease and reverse transcriptase-RT regions) was as follows: subtype B (91.2%), subtype F (4.9%), and B/F viral recombinant forms (3.3%). The subtype C was identified in two patients (0.4%) and the recombinant CRF_02/AG virus was found infecting one patient (0.2%). The HIV-1 genotyping profile associated to the reverse transcriptase inhibitors has shown a high frequency of the M184V mutation followed by the timidine-associated mutations. The K103N mutation was the most prevalent to the non-nucleoside RT inhibitor and the resistance associated to protease inhibitor showed the minor mutations L63P, L10F/R, and A71V as the more prevalent. A large proportion of subtype B was observed in HIV-1 treated patients from Rio de Janeiro. In addition, we have identified the circulation of drug-resistant HIV-1 subtype C and are presenting the first report of the occurrence of an African recombinant CRF_02/AG virus in Rio de Janeiro, Brazil. A clear association between HIV-1 subtypes and protease resistance mutations was observed in this study. The maintenance of resistance genotyping programs for HIV-1 failing patients is important to the management of ARV therapies and to attempt and monitor the HIV-1 subtype prevalence in Brazil.

The fou2 gain-of-function allele and the wild-type allele of Two Pore Channel 1 contribute to different extents or by different mechanisms to defense gene expression in Arabidopsis.

The fatty acid oxygenation up-regulated 2 (fou2) mutant in Arabidopsis thaliana creates a gain-of-function allele in a non-selective cation channel encoded by the Two Pore Channel 1 (TPC1) gene. This mutant genetically implicates cation fluxes in the control of the positive feedback loop whereby jasmonic acid (JA) stimulates its own synthesis. In this study we observed extensive transcriptome reprogramming in healthy fou2 leaves closely resembling that induced by treatment with methyl jasmonate, biotic stresses and the potassium starvation response. Proteomic analysis of fou2 leaves identified increased levels of seven biotic stress- and JA-inducible proteins. In agreement with these analyses, epistasis studies performed by crossing fou2 with aos indicated that elevated levels of JA in fou2 are the major determinant of the mutant phenotype. In addition, generation of fou2 aba1-5, fou2 etr1-1 and fou2 npr1-1 double mutants showed that the fou2 phenotype was only weakly affected by ABA levels and unaffected by mutations in NPR1 and ETR1. The results now suggest possible mechanisms whereby fou2 could induce JA synthesis/signaling early in the wound response. In contrast to fou2, transcriptome analysis of a loss-of-function allele of TPC1, tpc1-2, revealed no differential expression of JA biosynthesis genes in resting leaves. However, the analysis disclosed reduced mRNA levels of the pathogenesis-related genes PDF1.2a and THI2.1 in healthy and diseased tpc1-2 leaves. The results suggest that wild-type TPC1 contributes to their expression by mechanisms somewhat different from those affecting their expression in fou2.

Patterns of hepatitis B virus infection in Brazilian human immunodeficiency virus infected patients: high prevalence of occult infection and low frequency of lamivudine resistant mutations

Hepatitis B virus (HBV) molecular profiles were determined for 44 patients who were infected with human immunodeficiency virus (HIV) type 1 and had antibodies to the hepatitis B core antigen (anti-HBc), with and without other HBV serological markers. In this population, 70% of the patients were under lamivudine treatment as a component of antiretroviral therapy. HBV DNA was detected in 14 (32%) patients. Eight out of 12 (67%) HBsAg positive samples, 3/10 (30%) anti-HBc only samples, and 3/22 (14%) anti-HBs positive samples were HBV DNA positive. HBV DNA loads, measured by real time polymerase chain reaction, were much higher in the HBsAg positive patients (mean, 2.5 × 10(9) copies/ml) than in the negative ones (HBV occult infection; mean, 2.7 × 10(5) copies/ml). Nine out of the 14 HBV DNA positive patients were under lamivudine treatment. Lamivudine resistant mutations in the polymerase gene were detected in only three patients, all of them belonging to the subgroup of five HBsAg positive, HBV DNA positive patients. A low mean HBV load (2.7 × 10(5) copies/ml) and an absence of lamivudine resistant mutations were observed among the cases of HBV occult infection.

Nonredundant Regulation of Rice Arbuscular Mycorrhizal Symbiosis by Two Members of the PHOSPHATE TRANSPORTER1 Gene Family.

Pi acquisition of crops via arbuscular mycorrhizal (AM) symbiosis is becoming increasingly important due to limited high-grade rock Pi reserves and a demand for environmentally sustainable agriculture. Here, we show that 70% of the overall Pi acquired by rice (Oryza sativa) is delivered via the symbiotic route. To better understand this pathway, we combined genetic, molecular, and physiological approaches to determine the specific functions of two symbiosis-specific members of the PHOSPHATE TRANSPORTER1 (PHT1) gene family from rice, ORYsa;PHT1;11 (PT11) and ORYsa;PHT1;13 (PT13). The PT11 lineage of proteins from mono- and dicotyledons is most closely related to homologs from the ancient moss, indicating an early evolutionary origin. By contrast, PT13 arose in the Poaceae, suggesting that grasses acquired a particular strategy for the acquisition of symbiotic Pi. Surprisingly, mutations in either PT11 or PT13 affected the development of the symbiosis, demonstrating that both genes are important for AM symbiosis. For symbiotic Pi uptake, however, only PT11 is necessary and sufficient. Consequently, our results demonstrate that mycorrhizal rice depends on the AM symbiosis to satisfy its Pi demands, which is mediated by a single functional Pi transporter, PT11.