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.

Factor VIII genotype characterization of haemophilia A affected patients with transient and permanent inhibitors: a comprehensive Argentine study of inhibitor risks.

Inhibitor development against exogenous factor VIII is a severe impairment of replacement therapy affecting 18% of Argentine patients with severe haemophilia A (HA). To study the molecular predisposition for inhibitor development, we genotyped 260 HA patients with and without inhibitors, countrywide. The inhibitor-positive population (19 transients, 15 low responders, LR and 70 high responders, HR) of 104 severe-HA patients showed 59 Inv22 (intron 22 inversions), 18 small ins/del-frameshifts, 12 gross deletions, 12 nonsense, one splicing defect and two missense, p.Arg531Pro and p.Leu575Pro, both LR and thought to impair FVIII A2 domain secondary structure. In addition, a patient with mild HA and HR showed the missense p.Glu1704Lys associated with two neutral intronic substitutions potentially affecting the A3 domain. A case/control study (84/143) permitted estimation of F8 genotype-specific inhibitor risks [OR; prevalence (CI)] in severe-HA patients classifying a high-risk group including multi-exon deletions [3.66; 55% (19-100)], Inv22 [1.8; 24% (19-100)] and nonsense in FVIII-LCh [1.2; 21% (7-59)]; an average risk group including single-exon deletions, indel frameshifts and nonsense-HCh; and a low-risk group represented by missense defects [0.14; 3% (0.6-11)]. Analysis of inhibitor concordance/discordance in related patients indicated additional genetic factors other than F8 genotype for inhibitor formation. No significant inhibitor-predisposing factors related to FVIII product exposure were found in age- and F8 genotype-stratified populations of severe-HA patients. In conclusion, the Argentine HA patient series presents similar global and mutation-specific inhibitor risks than the HA database and other published series. This case-specific information will help in designing fitted therapies and follow-up protocols in Argentina.

Developing a new generation of tests for genotyping hemophilia-causative rearrangements involving int22h and int1h hotspots in the factor VIII gene.

BACKGROUND: Inversions of F8-intron 22 (Inv22) and F8-intron 1 (Inv1) are responsible for 45-50% of severe hemophilia A cases. OBJECTIVE: In order to improve the molecular diagnosis of Inv22 and Inv1, and to enable rapid discrimination of Inv22-type 1 and Inv22-type 2 patterns, int22h-mediated deletions (Del22) and duplications (Dup22), we developed a genotyping system based on a novel inverse shifting-polymerase chain reaction (IS-PCR) approach. METHODS: IS-PCR involved BclI restriction, followed by self-ligation to create 'BclI circles', and finally PCR analysis. Three PCR analysis tests were developed: (i) Inv22-diagnostic for a pattern-sensitive detection of deleterious mutations (Inv22 and Del22) from non-deleterious variants (Dup22 and normal); (ii) Inv1-diagnostic; and (iii) Inv22-complementary for discrimination between Inv22 and Del22, and between Dup22 and normal. For rapid molecular analysis of F8, the Inv22 and Inv1 diagnostic tests can be performed simultaneously. The optional Inv22-complementary test need only be used for specific purposes. RESULTS AND CONCLUSIONS: Diagnostic tests were validated using previously studied samples. IS-PCR evaluated carrier mosaicisms and performed robustly over wide ranges of DNA qualities and procedural conditions. IS-PCR improved the molecular diagnosis of hemophilia A. This genotyping strategy may potentially be adapted to virtually all known rearrangements in the human genome.

Comparative genomic hybridization study of de novo myeloid neoplasia.

Comparative genomic hybridization (CGH) was used to detect chromosomal imbalances in 20 patients with a diagnosis of myelodysplastic syndrome (MDS) and acute myeloid leukaemia (AML). The results obtained were compared with G-banding analysis. This last methodology showed 50% of cases with clonal abnormalities whereas CGH detected 70% of cases with copy number changes. Gains were more frequent than losses and constituted 66% of total changes detected. The most common gains included chromosomes 21 and chromosome region 18p for AML and chromosome 17 and region 1p33p35 for MDS. Losses represent 34% of changes and the regions involved were 5q31q32, 7q22, 7p12 and 13q21q22. CGH revealed additional chromosome imbalances in 12 of 20 cases (60%) which were not detected by traditional cytogenetic studies, demonstrating complex karyotype in 50% (6/12). Combination of CGH and G-banding provides an efficient method to identify critical regions present in the malignant clone, which is of great value in the prognosis and outcome of myeloid neoplasias.

A new polymorphism in the human factor VIII gene: implications for linkage analysis in haemophilia A and for the evolution of int22h sequences.

A new polymorphism in the human factor VIII gene has been localized and characterized. It is a biallelic, single nucleotide polymorphism located in intron 22 of the gene, within the 9.5 kb int22h-1 segment. The allelic forms are G (frequency 0.65) and A (frequency 0.35), giving a predicted rate of heterozygosity of 0.46. The polymorphism occurs within a CG dinucleotide and affects an MspI site (CCGG). Int22h-1 is duplicated twice extragenically at Xq28; both extragenic copies (int22h-2 and -3) are also polymorphic with respect to MspI. Investigation of 156 MspI [-] alleles, comprising 30 intragenic and 126 extragenic sites, indicated that all were due to A alleles and none had arisen by C to T transition within the CG dinucleotide. The intragenic MspI site (designated MspI A) is located 737 bases downstream of a previously described XbaI restriction fragment length polymorphism. Despite their close proximity, the polymorphisms are not in complete linkage disequilibrium; haplotype analysis in 85 factor VIII genes from a Caucasian population predicts an informativity of approximately 60% in linkage studies using both, compared with an informativity of approximately 47% in studies using either on its own.

Specific analysis of the intron 22 XbaI polymorphism of the human factor VIII gene using long-distance PCR.

A rapid, non-radioactive, PCR-based method to genotype the XbaI restriction fragment length polymorphism of the human factor VIII gene is described. The method uses long-distance PCR followed by XbaI restriction digestion and agarose gel electrophoresis. The 6.6 kb amplification product includes a constant XbaI site, which provides a digestion control. The specificity of the method was challenged by a blind experiment with 16 genomic DNA samples previously genotyped by Southern blot analysis: a perfect correlation was obtained between genotypes determined using Southern blot and PCR.

Promyelocytic blast crisis of chronic myelogenous leukaemia with translocations (9;22) and (15;17).

The promyelocytic blast crisis is a rare form of transformation during the evolution of chronic myeloid leukaemia (CML). We report a case of promyelocytic blast crisis with t(15;17) in addition to t(9;22). The morphology and immunophenotype of the blasts were similar to those seen in acute promyelocytic leukaemia (APL). The t(15;17) was confirmed by FISH. The patient had evidence of coagulopathy with clinical and laboratory findings of disseminated intravascular coagulation (DIC). This report highlights the importance of correlating the results of multiple diagnostic methods in order to establish a correct diagnosis of the promyelocytic blast crisis of CML.

Clinical relevance of the breakpoint sites within the M-BCR in 50 patients from Argentina with chronic myeloid leukemia.

Fifty patients from Argentina with chronic myeloid leukemia (CML) were studied in order to characterize the breakpoint site within the major breakpoint cluster region (M-BCR) and its relationship with the duration of the chronic phase (CP). The DNA digestion with the restriction enzymes: Bgl II, BAM HI and Hind III and hybridization with the 1.2Kb Hind III-Bgl II bcr probe showed that 56% of cases had the breakpoint in 5'M-bcr region and the remaining 44% in 3'M-bcr region. The duration of chronic phase from diagnosis to the onset of the blast crisis (BC) was correlated with the location of the breakpoint within the M-bcr and no statistical differences were observed between the 5' and the 3' groups. These data indicate that the breakpoint site within the bcr gene is not a prognostic indicator of the duration of CP of the disease.

[Molecular genetics of hemophilia A]. / Genética molecular de la hemofilia A.

Hemophilia A (HemA), an X linked genetic disease, is the most common coagulation disorder with an incidence of about 1-2 in 10,000 males and is caused by mutations in the factor VIII (FVIII) coagulation gene. Firstly, some clinical aspects of the HemA are presented: the current methods to assess both the amount and activity of FVIII, the severity range observed and the presence of inhibitor antibodies against the therapeutic FVIII. Follows a discussion of the relationship of the structural domains of the FVIII protein (Figure 1), the aminoacid sequence and their functions. An activation-inactivation model of the successive peptide bonds cleavages of the FVIII is also presented (Figure 2). After the cloning of the FVIII gene in 1984, almost all types of HemA causing mutations have been characterized. However, the size and complexity of this gene prevented a screening of the full range of mutations for an accurate molecular diagnosis. Moreover, most of the patients with moderate and mild disease have missense mutations whereas approximately half of severe patients have nonsense, frameshift, and some missense mutations. There are also less frequently mutations such as deletions and insertions leading to severe phenotype and mutations affecting mRNA splicing and duplications causing both severe and mild HemA. In order to give genetic counselling in HemA families, studies at the DNA level using intragenic and/ or extragenic polymorphism analysis have been used. But this approach is not entirely satisfactory because it fails in several situations. Most of the causing mutations described above are private, and they have been found in only a few unrelated families. Recently, a common molecular inversion of the FVIII gene was identified in 50% of unrelated patients with severe HemA. The copies of a particular DNA sequence (termed F8A gene). One copy is located within intron 22 of the FVIII gene and the other two, 500 kb upstream. An homologous recombination mechanism was proposed for the inversion between an intragenic copy of the F8A gene and either the distal (80% of the inversion) or the proximal copy (20%). Both of these inversions lead to severe HemA because no intact FVIII is produced and can be easily diagnosed by Southern blot analysis. This inversion originates almost exclusively in male germ cells, because pairing Xq with its homologous in female meiosis would probably inhibit the proposed intrachromosome recombination. The molecular analysis of the inversion of intron 22 is now considered as the first line for families with severe HemA patients. In recent years the treatment of patients with hemophilia A and B has been intravenous injection of FVIII or FIX concentrates, respectively. This regimen of regular injection of plasmatic proteins bears a high risk of infection by contaminating viruses (HIV, HBV, etc). Future treatment for patients with hemophilia may include the use of either gene therapy or recombinant coagulation factors. Both strategies would completely avoid the infection risk offering a safe and effective treatment for the disease. Recombinant factors, obtained by genetic engineering methods, provide a renewable and unlimited source of FVIII or FIX. The clinical trials of recombinant factors have already started in mid-1995 giving positive results. On the other hand, gene therapy for hemophilia is now in the pre-clinical stage but offers the prospect of a cure for the disease, thus potentially freeing patients from regular injections of the lacking protein. However, experiments in animal models suggest that it may be difficult to obtain adequate therapeutic levels of factors for long periods of time. Recently, a retroviral-mediated gene delivery of human FVIII in mice has been reported using the ex vivo strategy of gene therapy. Therapeutic levels of FVIII in the circulation were obtained for > 1 week and it was also observed that the capacity of primary cells to deliver FVIII in blood was strongly dependent on

Chromosomal damage in workers occupationally exposed to chronic low level ionizing radiation.

Chromosomal aberrations were evaluated in cultures of peripheral lymphocytes from subjects working in diagnostic X-ray and nuclear medicine areas, exposed to electromagnetic ionizing radiation and particulate ionizing emissions, respectively. A 4-fold increase in the level of chromosomal aberrations was found between the exposed and control groups without qualitative or quantitative cytogenetic differences between X-rays and nuclear medicine-exposed workers. Results are discussed in view of the early damage detection from chronic exposures particularly related to biological controls, hygienic improvements and overwork in a developing country.

Chromosome instability in lymphocytes from patients with celiac disease.

Cytogenetic studies were performed in celiac disease (CD) patients to determine if the presence of chromosome instability is related to the predisposition to cancer. Chromosome aberrations (CA) and sister chromatid exchange (SCE) frequencies in peripheral blood lymphocyte cultures from untreated CD patients and healthy controls were analyzed. Patients showed aberrations in 23% of cells, while only 3% were detected in the control group (p < 0.0001). The mean frequencies of gaps, breaks and total CA were found to be higher in CD patients compared to controls (p < 0.0001). Breakpoint distribution was nonrandom among chromosomes from celiac patients (p = 0.01), but not among controls (p = 0.04). The frequency of SCE/cell showed a mean value of 6.9 +/- 0.6 in CD patients and 7.3 +/- 0.2 in controls. No statistical differences were found. Breakpoints involved in CD patients presented a strong coincidence with the location of fragile sites (78.6%) and sites of cancer chromosome rearrangements (57.1%), most of them (75%) associated with malignant non-Hodgkin lymphomas. These results suggest that CD is a condition with increased chromosome instability characterized by a high level of CA and normal SCE frequencies, probably related to the increased incidence of cancer.

Increased expression of 5q31 fragile site in a Bloom syndrome family.

In this work, we report spontaneous chromosomal breakpoints and fragile site expression induced by 5-fluorodeoxyuridine (FdUrd) and FdUrd plus caffeine in a family with Bloom's syndrome (BS) and 2 healthy donors. Standard and G-banded metaphases from each individual and each treatment were analyzed. Among the 59 common fragile sites (c-fra) identified in this work, only the frequency of 5q31 was significantly increased in the BS family with respect to healthy donors (P less than 0.005). A remarkable coincidence between the breakpoints involved in spontaneous chromosome aberrations and induced c-fra was found in BS homozygote patients. The importance of the interaction between fragile sites and chromosome rearrangements in cancer is discussed.

Genotoxic action of an aqueous extract of Heliotropium curassavicum var. argentinum.

Heliotropium curassavicum var. argentinum is widely employed in gout, rheumatism, neuralgias, arteriosclerotic disorders, muscular algias, phlebitis, varix and other illnesses. In order to analyze the genotoxic effect produced in vitro by this medicinal plant, chromosomal aberrations (CA), mitotic index (MI) and anaphase delay (AD) were studied in the CHO cell line, with and without the addition of S9 mix. Prepared according to the Argentine pharmacopeia 0.1, 1, 10 and 100 micrograms/ml plant decoction (aqueous extract) were assayed. One hundred cells per culture were studied for CA and AD, while MI was calculated for 2000 nuclei. The results revealed a significant increase in the percentage of abnormal metaphases (p less than 0.001) and in total aberrations (p less than 0.001). Both the MI and the AD affected the cell cycle. All results were enhanced by the addition of an S9 fraction. The toxic effect could be associated with pyrrolizidine alkaloids and their N-oxides, which through a process of in vitro metabolism become activated by microsomal oxidation and change into pyrrolic derivatives.

Lack of sensitivity of sister-chromatid exchange for lymphocyte chromosomal damage detection caused by antichagasic treatment.

No studies exist on sister-chromatid exchange (SCE) formation in chagasic patients therapeutically exposed to nifurtimox (NFX) or benznidazol (BZ). In the present study SCE was analyzed in cultures of peripheral lymphocytes of patients aged 11 months to 11 years treated with NFX 12-15 mg/kg/d for 60 days or with BZ 5 mg/kg/d for 30 days. Chagasic patients before treatment constituted a control group. A mean of 30 metaphases were examined for each individual. All treated patients compared with untreated controls did not show a significant increase in SCE frequency. Compared with the percentage of chromosomal aberrations in these patients and others belonging to the same epidemic protocol, SCE seems to be less sensitive in the detection of lymphocyte chromosomal damage caused by NFX or BZ.

[Induction of fragile sites by chemical and physical agents]. / Inducción de sitios frágiles por agentes químicos y físicos.

Many human malignancies are thought to be caused in large part by environmental agents. Studies indicate that ionizing radiation and many chemical mutagens/carcinogens appear to induce chromosomal mutations. Recently, it was suggested that chromosome aberrations (CA) might be produced by breakage at specific chromosomal points known as fragile sites (FS). FS induced by different chemical agents has been widely analyzed in lymphocytes cultures, but very little is known of the environmental factors that may influence on FS expression. No studies exist about FS expression induced with X-rays or about the interaction between radiation and chemical FS inducers. In this work FS expression induced by X-rays and 3 known FS inducers such as: BUDR, FUDR and aphidicolin, is analyzed. It was found that 17 chromosomal bands were significantly damaged (p < 0.001), thus being defined as FS. FS 3p14 and 16q23 were the more frequently observed. CA and FS levels from cultures exposed to X-rays and to FUDR plus X-rays were significantly increased (p < 0.01), suggesting that it would be convenient to use other radiosensitizer. These results suggest that FS could be induced by environmental factors such as chemical or physical agents. Finally, the correlation between FS and radiation induced CA, cancer CA and oncogene localization was established, showing the importance of the interaction between FS, CA and oncogenes in cancer development.

Inducción de sitios frágiles por agentes químicos y físicos. / [Induction of fragile sites by chemical and physical agents]

Many human malignancies are thought to be caused in large part by environmental agents. Studies indicate that ionizing radiation and many chemical mutagens/carcinogens appear to induce chromosomal mutations. Recently, it was suggested that chromosome aberrations (CA) might be produced by breakage at specific chromosomal points known as fragile sites (FS). FS induced by different chemical agents has been widely analyzed in lymphocytes cultures, but very little is known of the environmental factors that may influence on FS expression. No studies exist about FS expression induced with X-rays or about the interaction between radiation and chemical FS inducers. In this work FS expression induced by X-rays and 3 known FS inducers such as: BUDR, FUDR and aphidicolin, is analyzed. It was found that 17 chromosomal bands were significantly damaged (p < 0.001), thus being defined as FS. FS 3p14 and 16q23 were the more frequently observed. CA and FS levels from cultures exposed to X-rays and to FUDR plus X-rays were significantly increased (p < 0.01), suggesting that it would be convenient to use other radiosensitizer. These results suggest that FS could be induced by environmental factors such as chemical or physical agents. Finally, the correlation between FS and radiation induced CA, cancer CA and oncogene localization was established, showing the importance of the interaction between FS, CA and oncogenes in cancer development.

Inducción de sitios frágiles por agentes químicos y físicos. / [Induction of fragile sites by chemical and physical agents]

Many human malignancies are thought to be caused in large part by environmental agents. Studies indicate that ionizing radiation and many chemical mutagens/carcinogens appear to induce chromosomal mutations. Recently, it was suggested that chromosome aberrations (CA) might be produced by breakage at specific chromosomal points known as fragile sites (FS). FS induced by different chemical agents has been widely analyzed in lymphocytes cultures, but very little is known of the environmental factors that may influence on FS expression. No studies exist about FS expression induced with X-rays or about the interaction between radiation and chemical FS inducers. In this work FS expression induced by X-rays and 3 known FS inducers such as: BUDR, FUDR and aphidicolin, is analyzed. It was found that 17 chromosomal bands were significantly damaged (p < 0.001), thus being defined as FS. FS 3p14 and 16q23 were the more frequently observed. CA and FS levels from cultures exposed to X-rays and to FUDR plus X-rays were significantly increased (p < 0.01), suggesting that it would be convenient to use other radiosensitizer. These results suggest that FS could be induced by environmental factors such as chemical or physical agents. Finally, the correlation between FS and radiation induced CA, cancer CA and oncogene localization was established, showing the importance of the interaction between FS, CA and oncogenes in cancer development.

[Expression of new fragile sites detected in lymphoproliferative processes]. / Expresión de sitios frágiles nuevos detectados en procesos linfoproliferativos.

Fragile sites are specific chromosomal sites prone to breakage and rearrangements and they are probably related with cancer development. Fragile sites expression induced by 5-fluorodeoxyuridine (FudR) or 5-bromodeoxyuridine (BrdU) was analyzed in 4 healthy individuals and 4 patients affected with lymphoproliferative disorders: one Hodgkin's disease, one mycosis fungoides and two chronic lymphoproliferative disorders. Three standard peripheral lymphocyte cultures with F-10 medium and 5% of fetal calf serum were set up for each individual. For fragile sites induction, 10 micrograms/mL FudR or 50 micrograms/mL BrdU were added 24 hr or 6 hr before harvest. Standard and sequential G banded metaphases were studied for each individual and treatment. Quantitative analysis showed a low incidence of acentric fragments, dicentric, tri- or quadriradials, while gaps and breaks were more frequently observed. Chromosome or chromatid type aberrations were compared, showing similar values in all non-treated cultures. Chromosome type aberrations were increased in patients and controls treated cultures. Patient cultures treated by FudR presented a threefold increase of chromosome type alterations respect to chromatid ones. Moreover, chromosome breaks showed a twofold increase in patient treated cultures respect to control ones. The high number of chromosome breaks detected in these cases could be associated with an increased chromosome instability in cancer patients. Twenty common fragile sites (c-fra) were identified with sequential G banding. Patients and controls individuals have expressed 14 c-fra. Eleven of them were induced, in different proportions, by both chemical agents, showing that fragile sites share a structural homology in DNA.(ABSTRACT TRUNCATED AT 250 WORDS)