Phenotype-genotype correlations in hemophilia A carriers are consistent with the binary role of the phase between F8 and X-chromosome inactivation.

BACKGROUND: The recessive X-linked disorder hemophilia A (HA) is rarely expressed in female carriers, most of whom express about half of normal factor VIII activity ( FVIII: C). OBJECTIVE: To propose an integrative assessment model for the binary role of the phase between the mutated F8 and the active X-chromosome (Xa) in FVIII: C in HA carriers. METHODS: We studied 67 females at risk of severe HA, comprising five symptomatic females ( FVIII: C < 1.5 IU dL(-1) ) and 14 controls. A correlation study between FVIII: C (observed vs. expected) and X-chromosome inactivation (XCI) patterns (XIPs; androgen receptor gene [AR] system) in blood leukocyte DNA was performed in carriers, by comparison of a model correlating FVIII: C and XIP with arbitrary models devoid of biological significance, and with FVIII: C levels in non-carriers (mean model) as a proxy from background data dispersion not influenced by XIP. RESULTS: We provide proof-of-concept example from a family presenting with extremely skewed XIPs in which the severe HA phenotype appeared in a heterozygous carrier of a crossover between AR and F8 loci that phased the mutated F8 with the maternally inherited Xa. Furthermore, four cases of severe HA affected women who had a combination of a heterozygous F8 mutation and extremely skewed XIPs in leukocytes or oral mucosa are presented. Correlation analyses between FVIII: C levels and XIPs in carriers (n = 38) but not in non-carriers (n = 20) showed highly significant differences between the proposed correlation model and models without biological significance. The data support a binary influence of XCI, either increasing or decreasing the FVIII: C, subject to the underlying phase set between the F8 mutation and XCI. CONCLUSIONS: Our evidence suggests that the phase between XCI and mutated F8 acts as a molecular switch conditioning FVIII: C levels and HA expression in carriers.

Informativeness of a novel multiallelic marker-set comprising an F8 intron 21 and three tightly linked loci for haemophilia A carriership analysis.

The extraordinary heterogeneous nature of the deleterious mutations in the F8 gene that lead to functional deficiency of clotting factor VIII in haemophilia A makes routine direct mutation profiling difficult. When direct mutation analysis cannot be performed or a causative/candidate mutation is not found, a second-line approach to track the defective F8 gene within at-risk families is linkage genetic analysis with, tried-and-tested, F8-intragenic and/or extragenic non-recombining multiallelic short tandem repeats (STR). Although several typing STR loci within and around F8 have been described, there is need for improving assessment, because the combined informativeness of available assays rarely reaches 100%. Here, we characterized a newly identified 0.28 cM-resolution marker-set, consisting of a dinucleotide STR located on F8 intron 21 (F8Int21; [AC](n)) and three extragenic tetranucleotide STR located on GAB3 intron 1 (GAB3Int1; [TAAA](n)) and TMLHE intron 1 (TMLHEInt1.1; [GAAA](n) and TMLHEInt1.3; [ATTC](n)). Heterozygosity rates determined in 100 unrelated females ranged from 0.25 (GAB3Int1) to 0.63 (F8Int21). The set rendered a combined informativeness of 0.91 for at least one marker and 0.60 for a minimum of two loci, with at least one F8-intragenic. Multiallelic interlocus non-random association analysis revealed that GAB3Int1 is not in significant gametic disequilibrium (GD) with F8Int21, F8Int9.2, TMLHEInt1.3 or TMLHEInt1.1. Gametic disequilibrium breakdown attests historical recombination between GAB3Int1 and the F8 gene. Through computational analysis of reference assembly sequence data, we note in the GD breakdown region and in the F8 gene a higher than average density of the 13-mer CCNCCNTNNCCNC consensus motif, commonly associated with recombination hotspots.

BCG-itis in two antiretroviral-treated HIV-infected infants.

Although vaccination with Bacille Calmette-Guérin (BCG) is considered safe, adverse regional (BCG-itis) and disseminated (BCG-osis) diseases preferentially occur in the immunocompromised host. The infection with human immunodeficiency virus (HIV) by mother-to-child transmission leads to impaired cellular immune responses, a situation that poses a great challenge regarding the universal use of BCG vaccine. World Health Organization recommends that children who are known to be HIV-infected, even if asymptomatic, should no longer be immunized with BCG. Many of the complications of BCG vaccination occur in severely immunosuppressed HIV-infected children and are related to late institution of antiretroviral and antimycobacterial therapy. We report two cases of BCG-itis in HIV-infected infants, who fulfilled clinical criteria of immune reconstitution inflammatory syndrome: axillary adenitis, one with suppuration, and both temporally associated with precocious restoration of immunity elicited by the use of antiretroviral therapy. Isoniazid (10 mg/kg/day) was offered until regression of adenopathies, and lesions were not handled.

Interlocus non-random association of multiallelic polymorphisms spanning the coagulation factor VIII gene on human chromosome distalmost Xq28.

The most common severe hereditary bleeding disorder phenotype in humans, the coagulation factor VIII (F8) deficiency haemophilia A (HEMA), maps on Xq28 band, a region that comprises 11.7% of genes and 14.2% of phenotypes on X chromosome. Information about the distribution and extent of gametic disequilibrium (GD) covering the F8 gene is scarce, despite its relevance for linkage and association studies. The aim of this study was to determine the patterns, by frequency and strength, of non-random multiallelic interallelic associations between two-locus combinations of seven microsatellite loci (REN90833, F8Int25.2, F8Int22, F8Int13.2, HEMA154311.3, TMLHEInt5 and HEMA154507.3, in that physical order) spanning 0.813 Mb on distalmost Xq28. We measured sign-based interallelic D' coefficients in 106 men and in 100 women drawn from a single unrelated Brazilian population. Significance and patterns of GD using haploid and phased diploid sample probabilities were close to conformity. Only 9.18% of the variance of D' could be accounted for by changes in length, indicating that GD is not a monotonically decreasing function of length. We defined two regions of overlapping long-range GD extending 698 735 base pairs (bp) (REN90833/TMLHEInt5 block) and 689 900 bp (F8Int13.2/HEMA154507.3 block) The extent of GD overlap is 575 637 bp (F8Int13.2/TMLHEInt5 interstice). Extended haplotype homozygosity analysis centred at the F8 intronic loci revealed that the most frequent core haplotypes decay the least in the flanking GD. The F8 intronic loci attend distinct non-random association forces; F8Int13.2 serves at maintenance of the long-range overlapping pattern of GD, whereas F8Int25.2 and F8Int22 serve at lessening it in force or effect.

Double aneuploidy (48,XXY,+21) of maternal origin in a child born to a 13-year-old mother: evaluation of the maternal folate metabolism.

The occurrence of non-mosaic double trisomy is exceptional in newborns. In this paper, a 48,XXY,+21 child, the parental origin of the extra chromosomes and the evaluation of the maternal folate metabolism are presented. The infant was born to a 13-year-old mother and presented with the typical clinical features of Down syndrome (DS). The origin of the additional chromosomes was maternal and most likely resulted from errors during the first meiotic division. Molecular analysis of 12 genetic polymorphisms involved in the folate metabolism revealed that the mother is heterozygous for the MTHFR C677T and TC2 A67G polymorphisms, and homozygous for the mutant MTRR A66G polymorphism. The maternal homocysteine concentration was 4.7 miromol/L, a value close to the one considered as a risk factor for DS in our previous study. Plasma methylmalonic acid and serum folate concentrations were 0.17 micromol/L and 18.4 ng/mL, respectively. It is possible that the presence of allelic variants for the folate metabolism and Hey concentration might have favored errors in chromosomal disjunction during gametogenesis in this young mother. To our knowledge, this is the first patient with non-mosaic Down-Klinefelter born to a teenage mother, resulting from a rare fertilization event combining an abnormal 25,XX,+21 oocyte and a 23,Y spermatozoon.

Improved criterion-referenced assessment in indirect tracking of haemophilia A using a 0.23 cM-resolution dense polymorphic marker set.

In haemophilia A, linkage analysis with coagulation factor VIII (F8) intragenic and/or neighbouring extragenic short tandem repeats (STRs) enables indirect tracking F8 pathogenic allelic variant-carriers. Even where DNA sequencing is available, linkage analysis still has a role if no causative or candidate mutation is unveiled. The cumulative heterozygosity rate of the available multiplexed STRs haplotyping assays rarely reaches 100%. This means that in a proportion of women these loci are uninformative. The norm-referenced assessment is based on at least one informative marker criterion. We reasoned that by typing a dense market set, spanning a small fraction of recombination, we should be able to improve assessment. The aim of this study was to improve criterion-referenced assessment in polymorphism segregation analyses using a low-recombination fraction and dense informative STRs set. The multiplex quantitative fluorescence PCR assay comprises four novel tetranucleotide and pentanucleotide STRs distant < or = 0.15 cM from the F8 gene, and three F8 intragenic dinucleotide STRs, mapped to a 0.23 cM interval spanning the F8 on human chromosome band Xq28. We determined heterozygosity rates and allele frequencies from 100 unrelated healthy females. To investigate about segregation stability, we typed 50 true trios (mother, daughter and father) and 50 true mother-and-son duos from the general population. The heterozygosity rates for the extragenic markers ranged 0.49-0.76. The 0.23 cM-resolution heptaplex rendered a cumulative heterozygosity of 0.89 for a minimum of two informative markers, with at least one F8 intragenic. The heptaplex assay enabled improving the criterion-referenced assessment in indirect carrier-detection.

High-resolution combined linkage physical map of short tandem repeat loci on human chromosome band Xq28 for indirect haemophilia A carrier detection.

Haemophilia A, the most common severe hereditary bleeding disorder in humans, is chiefly caused by mutations in the coagulation factor VIII F8 gene, which maps on chromosome band Xq28. Linkage analysis with F8 intragenic and/or closely located extragenic short tandem repeat (STR) elements is an effective method for indirect tracing of F8 pathogenic allelic variants in at-risk families. STR profiling is currently limited to 14 markers, 11 of which are dinucleotide elements. The aim of this study was to define novel polymorphic STR loci for haemophilia A carrier screening. The combined linkage physical map was restricted to a 2.4-Mb region on Xq28 that hosts 81 annotated genes, F8 inclusive. The inventory was in silico through comparative analyses with three X chromosome reference sequences, using microsatellite mining and validation computer software. Genetic distances for unmapped markers were interpolated on the Rutgers map of the human genome. The effort yielded 94 STR loci: 53 extragenic and 41 intragenic (14 STR elements map on the F8 gene; the other 27 on 19 further genes). The distribution per repeat period size was 61.7% di-, 5.3% tri-, 26.6% tetra- and 6.4% pentanucleotide loci. The success rate of validation of polymorphism for the new STR loci was 56.3%. For STR elements 0.78 Mb equidistant of the F8 gene, the interpolated downstream genetic length is 3.27 times the upstream genetic length. The inventory represents a 5.7-fold increase in polymorphic STR loci useful in carrier detection. Genotyping with the upstream extragenic tetra- and pentanucleotide markers is thus apprized.

Egg yolk anti-BfpA antibodies as a tool for recognizing and identifying enteropathogenic Escherichia coli.

Enteropathogenic Escherichia coli (EPEC) is a major aetiological agent of childhood diarrhoea in developing countries. The structural repeating protein A subunit, BfpA, found in the bundle-forming pilus, is one of the virulent factors for EPEC pathogenesis. Recombinant BfpA in laying hens elicited sustained and vigorous antibody production. Immunoglobulin Y (IgY) anti-BfpA antibodies were recovered from egg yolk, purified and characterized. Immunoadsorption with whole extracts of the isogenic E. coli EPEC adherence factor (EAF) strain that lacks BfpA rendered the resulting IgY preparations capable of: (a) recognizing purified or recombinant BfpA proteins in a dose-dependent fashion; (b) blocking the colonization of HeLa cells by EPEC EAF+, in vitro; (c) specifically identifying E. coli bearing EAF+; and (d) inhibiting the growth of E. coli EAF+ but not the EAF strain. IgY anti-BfpA is potentially useful as a specific, low-cost immunobiological reagent to screen human faecal specimens for the presence of EPEC.

RNAIII inhibiting peptide (RIP) inhibits agr-regulated toxin production.

Staphylococcal enterotoxins (SEs) are emetic toxins that cause food poisoning. SEs also function as powerful pyrogenic toxin superantigens that stimulate non-specific T-cell proliferation. Together with the hemolysins, SEs have been largely implicated as virulence factors in multiple infection models. Recent biochemical and genetic analyses have demonstrated that production of some of these toxins is partially regulated by quorum sensing mechanisms where proteins and peptides activate the accessory gene regulator (agr). Because toxin production is central to bacterial pathogenesis, therapeutic strategies alternative to antibiotics, and based on rational interference of the quorum sensing systems involved, are currently being developed. This approach would lead to repression of toxin production and, thus, to disease prevention. Here we provide evidence to conclude that synthetic analogs of the RNAIII inhibiting peptide (RIP) and antibodies to its target molecule TRAP function in vitro as efficient suppressors of agr-regulated exotoxin production by Staphylococcus aureus.

Prevention of diseases caused by Staphylococcus aureus using the peptide RIP.

Staphylococcus aureus causes many diseases including cellulitis, keratitis, osteomyelitis, septic arthritis and mastitis. The heptapeptide RIP has been shown to prevent cellulitis in mice, which was induced by S. aureus strain Smith diffuse. Here we show that RIP can also significantly reduce the overall pathology and delay the onset of disease symptoms in several other models of S. aureus infections, including: keratitis (tested in rabbits against S. aureus 8325-4), osteomyelitis (tested in rabbits against S. aureus MS), mastitis (tested in cows against S. aureus Newbould 305, AE-1, and environmental infections) and septic arthritis (tested in mice against S. aureus LS-1). These findings substantiate that RIP is not strain specific in its inhibitory activity and that RIP is an effective inhibitor of bacterial pathology at multiple body sites following diverse routes and doses of administration. These findings strongly evidence the potential value of RIP as a chemotherapeutic agent.

Genetic data showing evolutionary links between Leishmania and Endotrypanum.

Striking similarities at the morphological, molecular and biological levels exist between many trypanosomatids isolated from sylvatic insects and/or vertebrate reservoir hosts that make the identification of medically important parasites demanding. Some molecular data have pointed to the relationship between some Leishmania species and Endotrypanum, which has an important epidemiological significance and can be helpful to understand the evolution of those parasites. In this study, we have demonstrated a close genetic relationship between Endotrypanum and two new leishmanial species, L. (V.) colombiensis and L. (V.) equatorensis. We have used (a) numerical zymotaxonomy and (b) the variability of the internal transcribed spacers of the rRNA genes to examine relationships in this group. The evolutionary trees obtained revealed high genetic similarity between L. (V.) colombiensis, L. (V.) equatorensis and Endotrypanum, forming a tight cluster of parasites. Based on further results of (c) minicircle kDNA heterogeneity analysis and (d) measurement of the sialidase activity these parasites were also grouped together.

Trypanosoma cruzi in the opossum Didelphis marsupialis: a study of the correlations and kinetics of the systemic and scent gland infections in naturally and experimentally infected animals.

The genus Didelphis (Marsupialia, Didelphidae) has the unique capacity of supporting both multiplication cycles of Trypanosoma cruzi simultaneously; besides the intracellular forms, the epimastigotes can be found multiplying and differentiating abundantly in the lumen of the scent glands. The biological significance of the life cycle of T. cruzi within the scent glands of Didelphis marsupialis, as well as its contribution to the epidemiology of the disease, is presently unclear. In order to clarify the mechanisms involved in the colonization of this singular habitat by T. cruzi, as well as to understand its biological role, we have carried out a serological and parasitological follow-up of both natural and experimental infections of young and adult opossums. Although all natural infections were stable and long lasting, no infected scent glands were found, indicating that the stability of the systemic infections does not depend on the presence of flagellates in the scent gland. In 84% of the experimentally infected animals the colonization of the scent glands was preceded by a period of patent parasitemia. Parasitism of the scent glands was essentially permanent and bilateral, and its maintenance was independent of circulating parasites. Moreover, the course of the infection differed depending on the source (scent glands versus axenic culture-derived) of the metacyclic forms. Our results suggest that parasitism of the SG of D. marsupialis is most likely a secondary acquisition, a step toward independence from the insect vector, similarly to what is accepted for Trypanosoma equiperdum.

Trans-sialidase and sialidase activities discriminate between morphologically indistinguishable trypanosomatids.

The expression of trans-sialidase and sialidase activities in the kinetoplastid protozoa was explored as a potential marker to discriminate between the morphologically indistinguishable flagellates isolated from human, insects and vertebrate reservoir hosts. By virtue of the differences observed in the ratios of these enzyme activities, a collection of 52 species and strains comprising the major taxa of these parasites could be separated into four expression types. Type-I parasites express comparable levels of both trans-sialidase and sialidase activities (Endotrypanum species and Trypanosoma lewisi). Type-II parasites express predominantly trans-sialidase activity (Trypanosoma cruzi and Trypanosoma conorhini). Type-III parasites express sialidase activity exclusively (Trypanosoma rangeli and Trypanosoma leeuwenhoeki). Type-IV parasites do not express either activity (Leishmania species and Trypanoplasma borreli). The measurement of trans-sialidase and sialidase activities thus permits the differentiation of parasites frequently found in the same insect vectors that are difficult to distinguish, such as T. cruzi and T. rangeli, or in the same sylvatic vertebrate and invertebrate hosts, such as Leishmania and Endotrypanum.

Combined occurrence of trypanosomal sialidase/trans-sialidase activities and leishmanial metalloproteinase gene homologues in Endotrypanum sp.

Endotrypanum (order Kinetoplastida: family Trypanosomatidae) is a parasite of forest dwelling tree sloths (Edentata: genera Choleopus and Bradypus). Unique among the haemoflagellates, this protozoan has an intraerythrocytic phase in the mammalian host. Nevertheless, many striking similarities exist between Endotrypanum and the human pathogen Leishmania that make it a useful model for epidemiological and evolutionary aspects of the biology of trypanosomatids. Importantly, Endotrypanum species share both the insect vector and host reservoir with certain species of Leishmania (subgenus Viannia). Because mixed infections with Endotrypanum and Leishmania are common in sloths and, therefore, likely to occur in the sandfly vector, there is a need for adequate biochemical markers to distinguish Endotrypanum from Leishmania infections. In this paper we show that Endotrypanum promastigotes possess sialidase and trans-sialidase activities, which are absent from Leishmania, and which are not closely related to the previously described trypanosomal sialidase/trans-sialidase enzyme. We also document the occurrence in Endotrypanum of homologues of the leishmanial surface metalloproteinase gp63 genes. The combined occurrence of sialidase/trans-sialidase activities and gp63 gene homologues in a unique organism has important ramifications for both field and laboratory studies on the biology of trypanosomatids, especially those related to host infection and evolution.

Effective immunization against cutaneous leishmaniasis with recombinant bacille Calmette-Guérin expressing the Leishmania surface proteinase gp63.

Leishmania parasites cause a spectrum of diseases that afflict the populations of 86 countries in the world. The parasites can survive within the lysosomal compartments of the host's macrophages, unless those macrophages are appropriately activated. Despite the fact that protective immunity can be induced by vaccination with crude parasite preparations, little progress has been made toward a defined vaccine for humans. In this study the gene encoding the Leishmania surface proteinase gp63 was cloned and expressed as a cytoplasmic protein in a bacille Calmette-Guérin (BCG) vaccine strain. BALB/c and CBA/J mice were inoculated with a single dose of recombinant BCG and challenged with infective Leishmania major or Leishmania mexicana promastigotes. Significant protection was observed in both mouse strains against L. mexicana and in CBA/J against L. major, whereas only a delay in L. major growth was seen in BALB/c mice. Recombinant BCG also engendered a strong protective response against challenge with amastigotes of L. mexicana, demonstrating that the induced immune response recognized the intracellular form of the parasite. The results support the view that recombinant BCG expressing gp63 may prove a useful vaccine for inducing protective cell-mediated immune responses to Leishmania species causing American cutaneous leishmaniasis.

Rapid isolation of DNA from trypanosomatid protozoa using a simple 'mini-prep' procedure.

Although several methods for isolating genomic DNA from trypanosomatid protozoa exist, all are time-consuming and cumbersome. Faster, simpler and efficient protocols for preparation of DNA from these protozoa are needed to ease the screening of mutants and transfectants. We describe the use of a bacterial lysis method to isolate chromosomal DNA from a wide range of trypanosomatids. The method is based on the finding reported by He et al., who noticed that phenol/chloroform treatment of Escherichia coli cells in the presence of LiCl and Triton X-100 solubilizes plasmid DNA, while precipitating unwanted chromosomal DNA and denatured cellular proteins. In applying this lysis method to the isolation of episomal DNA from transfected trypanosomatids, we found that, unlike bacterial genomic DNA, chromosomal DNA of trypanosomatids was soluble in the phenol/chloroform/Triton/LiCl mixture. This observation prompted us to use the bacterial lysis method as a routine protocol for extraction of DNA from trypanosomatids.