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.

Haemophilia in the developing world: successes, frustrations and opportunities.

There is a need to bridge key gaps between high- and low-income countries and individuals; between health policy and clinical practice; and between producers and users of healthcare technology and scientific evidence. The objective of this report was to perform a diagnosis of the situation in the developing world through a survey. This survey was conducted to gather specific information on various aspects related to haemophilia. Countries were chosen by their comparability in infant and adult mortality rates according to the regionalization proposed by the World Health Organization. These indicators are very sensitive to socioeconomic conditions, and have been widely used to study health inequalities. All regions, except Africa and the East Mediterranean, were represented. Africa was excluded because its indicators were not comparable. The East Mediterranean was not represented because of difficulties in contacting investigators. Twenty-one country representatives were contacted, and 11 answered the questionnaire. Successes obtained by developing countries are based more on the skills and creativity of the local professionals than on the availability of state-of-the-art technology. Frustrations were related to disease underregistration and the limited availability of treatment products. Haemophilia care in the developing world is not as fair as we would like it to be. Governments do not always cover treatment costs, and a very small percentage of the national health budgets is allotted to haemophilia care. The role of the World Federation of Hemophilia was considered crucial by all the investigators surveyed. Training programmes and supply of factor concentrates were the main contributions identified by the respondents.

Necrosis avascular de cabeza y cuello de fémur en un paciente con sida / Avascular osteonecrosis of femoral head and neck in an AIDS patient

Avascular osteonecrosis (AON) has increased in the last few years in patients infected with the human immunodeficiency virus type-1 (HIV-1). The most commonly affected bone is the femoral head and neck. Frequently these bilateral and clinical findings include moderate to severe pain and functional impotence of the affected joints. The etiology is multifactorial and highly active antiretroviral therapy (HAART) with protease inhibitors (PI) is probably related to its development. In the evolution, a total hip replacement may be needed. We present an hemophilic patient with AIDS, who developed a bilateral AON of the femoral head and neck during HAART.(AU)

La osteonecrosis avascular (ONA) es una complicación que se describe con frecuencia creciente en pacientes infectados por el virus de la inmunodeficiencia humana tipo-1 (HIV-1). En su localización más común compromete la cabeza y cuello del fémur con dolor e impotencia funcional, en una o ambas caderas. Su etiología es multifactorial y la terapia antirretroviral de alta eficacia (HAART) con inhibidoresde proteasa (IP) puede estar relacionada con la patogenia. En su evolución puede requerir el reemplazo total de la cadera con la colocación de una prótesis. Se presenta un paciente hemofílico, HIV-1 seropositivo, quedesarrolló una ONA bilateral de cabeza y cuello de fémur mientras se encontraba bajo HAART.(AU)

Necrosis avascular de cabeza y cuello de fémur en un paciente con sida / Avascular osteonecrosis of femoral head and neck in an AIDS patient

Avascular osteonecrosis (AON) has increased in the last few years in patients infected with the human immunodeficiency virus type-1 (HIV-1). The most commonly affected bone is the femoral head and neck. Frequently these bilateral and clinical findings include moderate to severe pain and functional impotence of the affected joints. The etiology is multifactorial and highly active antiretroviral therapy (HAART) with protease inhibitors (PI) is probably related to its development. In the evolution, a total hip replacement may be needed. We present an hemophilic patient with AIDS, who developed a bilateral AON of the femoral head and neck during HAART.

La osteonecrosis avascular (ONA) es una complicación que se describe con frecuencia creciente en pacientes infectados por el virus de la inmunodeficiencia humana tipo-1 (HIV-1). En su localización más común compromete la cabeza y cuello del fémur con dolor e impotencia funcional, en una o ambas caderas. Su etiología es multifactorial y la terapia antirretroviral de alta eficacia (HAART) con inhibidoresde proteasa (IP) puede estar relacionada con la patogenia. En su evolución puede requerir el reemplazo total de la cadera con la colocación de una prótesis. Se presenta un paciente hemofílico, HIV-1 seropositivo, quedesarrolló una ONA bilateral de cabeza y cuello de fémur mientras se encontraba bajo HAART.

HCV recovery from peripheral blood mononuclear cell culture supernatants derived from HCV-HIV co-infected haemophilic patients with undetectable HCV viraemia.

Hepatitis C viraemia, in 38 human immunodeficiency virus positive (HIV+)/hepatitis C virus positive (HCV+) patients, was determined in haemophilic patients during the 4 years since initiation of highly active antiretroviral therapy (HAART). Six of 38 patients had persistently HCV-negative viraemia for more than 2 years. No correlation between HCV-negative viraemia and CD4+ T-cell counts, HIV viral load, age, type or severity of haemophilia could be established. Reduced levels of HIV viral load and the immune reconstitution that follows the initiation of HAART were not enough to explain the disappearance of HCV from plasma. Individuals who cleared plasma HCV had significantly higher CD8+ T-cell counts (P=0.0013) (mean +/- SE: 1153 +/- 117.8 cells microL(-1)) than those with HCV-positive viraemia (819.1 +/- 40.72 cells microL(-1)). Because HCV could maintain a low replication level in peripheral blood mononuclear cells (PBMC), we cultured PBMC of five of six patients with undetectable HCV viraemia. We found four of five HCV RNA-positive cultures. The presence of HCV RNA in our cultures proved that these cells may be an important viral reservoir that could contribute to HCV recurrence in plasma even after long periods of negative viraemia. In summary, our results indicate that in spite of prolonged HCV-negative plasma viraemia, HCV patients that are co-infected with HIV may harbour replication-competent HCV in their PBMC. Therefore, true clearance of HCV infection is difficult to achieve in these patients.

Monocyte differentiation and HIV replication after prolonged culture of peripheral blood mononuclear cells from HIV-infected individuals.

Primary cultures of peripheral blood mononuclear cells (PBMC) from 51 HIV+ hemophiliac patients (HIV+ PBMC) were set up, allowing undisturbed cellular interaction in the absence of any exogenous stimuli. The optimum time for p24 detection was between 12 and 25 days. Infective virus was recovered from the culture supernatants (HIV+ SN) and the amount of p24 released ranged from 25 to 5300 pg/ml. Cells of the monocyte/macrophage (M/M) lineage were the main source of HIV in the HIV+ SN, as judged by intracellular staining of permeabilized cells with anti-p24 (KC57 monoclonal antibody) and flow cytometry analysis. M/M activation, differentiation, and proliferation occurred along the culture before the peak of in vitro HIV replication. Release of HIV p24 was highest in patients with >200 CD4+ T lymphocytes/mm3 who did not receive highly active antiretroviral therapy (HAART), but it was still detectable in 60-90% of patients who had responded to 1-2 years of HAART, reducing their plasma viral load to undetectable levels. It is proposed that this simple experimental system can be used to assess ongoing HIV infection of M/M with the patient's own viral variants.

[Cerebrospinal fluid viral load in HIV-1 positive hemophilic patients treated with HAART]. / Carga viral en líquido cefalorraquídeo de pacientes hemofilicos HIV-1 positivos bajo tratamiento con HAART.

As HIV seropositive patients with undetectable CSF viral load have a lower likelihood of developing neurologic disease, the determination of CSF viral load levels may be useful to evaluate the efficacy of HAART. We compared plasma viral load levels with HIV-1 RNA CSF levels in 18 hemophilic patients without neurocognitive involvement under HAART. We detected a significant correlation between plasma viral load levels and CSF viral load levels. Fourteen patients with undetectable plasma viral load had undetectable RNA HIV-1 CSF levels as well. Four patients with detectable plasma viral load had detectable HIV-RNA in CSF, but the latter were significantly lower. Viral load is usually lower in non-blood fluids and HAART decreases the viral load in CSF as well as in blood.

Carga viral en líquido cefalorraquídeo de pacientes hemofilicos HIV-1 positivos bajo tratamiento con HAART. / [Cerebrospinal fluid viral load in HIV-1 positive hemophilic patients treated with HAART]

As HIV seropositive patients with undetectable CSF viral load have a lower likelihood of developing neurologic disease, the determination of CSF viral load levels may be useful to evaluate the efficacy of HAART. We compared plasma viral load levels with HIV-1 RNA CSF levels in 18 hemophilic patients without neurocognitive involvement under HAART. We detected a significant correlation between plasma viral load levels and CSF viral load levels. Fourteen patients with undetectable plasma viral load had undetectable RNA HIV-1 CSF levels as well. Four patients with detectable plasma viral load had detectable HIV-RNA in CSF, but the latter were significantly lower. Viral load is usually lower in non-blood fluids and HAART decreases the viral load in CSF as well as in blood.

[Cavum lymphoma in a hemophilic patient with AIDS]. / Linfoma de cavum en un paciente hemofílico con SIDA.

Intermediate and highly malignant non-Hodgkin and primary central nervous system lymphomas are marker diseases for AIDS. Cavum and oropharynx involvement by these tumors is uncommon. Although there are few cases reported in the literature, these may be primary localizations of the tumor. We present a hemophilic HIV+ patient with non-Hodgkin lymphoma of the cavum. The histologic diagnosis was high-grade, pleomorphic, centroblastic lymphoma. The patient was treated with chemotherapy plus intrathecal chemotherapy and highly active antiretroviral therapy (HAART). His evolution has been excellent. One year after diagnosis, the patient is asymptomatic with no evidence of residual tumor, and responding well to HAART.

Intron 22 factor VIII gene inversions in Argentine families with severe haemophilia A.

Intron 22 factor VIII gene inversion (Inv22) is the most common mutation causing severe haemophilia A (SHA). We studied Inv22 in 34 SHA affected families by Southern blotting. Data from the familial history of the disease and the inhibitor status were also included. We found Inv22 in 41 % of SHA Argentine families (35 % with type 1 and 6 % with type 2), in close agreement with previously reported series. No significant correlation between the inheritance (familiar or sporadic disease) and the presence of inversions was found. Our population showed 24 % of families included at least one hemophiliac with inhibitor. In families positive for Inv22, 29 % of patients developed inhibitor but this increased frequency was not statistically significant. In conclusion, analysis of Inv22 in SHA patients should be used as a first line method because it provides useful and secure information for carrier detection and prenatal diagnosis in a high percentage of cases.

[The future of oncohematology]. / El futuro de la oncohematología.

The last 40 years have witnessed important changes in the understanding and treatment of oncohematological affections. Palliative therapy was gradually replaced by chemotherapy (CT) which rapidly proved unexpectedly effective. In 1948, the first antifolic drugs, aminopterin and methotrexate, were discovered, followed in 1950 by the corticoids and in 1953 by antipurine agents. By 1967, a combination of these drugs yielded a survival index of 50% in acute lymphoblastic leukemia (ALL) with a progressive increase in all important cancer centers today, including in GATLA (Argentine Group for Acute Leukemia). As for acute myeloblastic leukemia (AML) the CT results were not as spectacular although now there is a 25% survival index which reaches 40-50% in young adults. As for allogeneic transplant in acute leukemia, its use must be evaluated for each patient and for each circumstance. Leukemias are genetic diseases for which gene therapy undoubtedly has potential value. However, the problems raised by the election of the right gene or gene marker and specially of the adequate vector have not yet been solved. In Hodgkin's disease, the results obtained with CT since the decade of the 60s have been spectacular and today different combinations of drugs have yielded a survival rate above 80%. Immunotherapy with or without CT has opened up a completely new and promising field. The route from basic research to clinical application has been long and arduous but the results obtained in leukemia and lymphomas have undoubtedly been life-saving and hopefully will open up even better possibilities in the near future.

Linfoma de cavum en un paciente hemofílico con SIDA. / [Cavum lymphoma in a hemophilic patient with AIDS]

Intermediate and highly malignant non-Hodgkin and primary central nervous system lymphomas are marker diseases for AIDS. Cavum and oropharynx involvement by these tumors is uncommon. Although there are few cases reported in the literature, these may be primary localizations of the tumor. We present a hemophilic HIV+ patient with non-Hodgkin lymphoma of the cavum. The histologic diagnosis was high-grade, pleomorphic, centroblastic lymphoma. The patient was treated with chemotherapy plus intrathecal chemotherapy and highly active antiretroviral therapy (HAART). His evolution has been excellent. One year after diagnosis, the patient is asymptomatic with no evidence of residual tumor, and responding well to HAART.

El futuro de la oncohematología. / [The future of oncohematology]

The last 40 years have witnessed important changes in the understanding and treatment of oncohematological affections. Palliative therapy was gradually replaced by chemotherapy (CT) which rapidly proved unexpectedly effective. In 1948, the first antifolic drugs, aminopterin and methotrexate, were discovered, followed in 1950 by the corticoids and in 1953 by antipurine agents. By 1967, a combination of these drugs yielded a survival index of 50

in acute lymphoblastic leukemia (ALL) with a progressive increase in all important cancer centers today, including in GATLA (Argentine Group for Acute Leukemia). As for acute myeloblastic leukemia (AML) the CT results were not as spectacular although now there is a 25

survival index which reaches 40-50

in young adults. As for allogeneic transplant in acute leukemia, its use must be evaluated for each patient and for each circumstance. Leukemias are genetic diseases for which gene therapy undoubtedly has potential value. However, the problems raised by the election of the right gene or gene marker and specially of the adequate vector have not yet been solved. In Hodgkins disease, the results obtained with CT since the decade of the 60s have been spectacular and today different combinations of drugs have yielded a survival rate above 80

. Immunotherapy with or without CT has opened up a completely new and promising field. The route from basic research to clinical application has been long and arduous but the results obtained in leukemia and lymphomas have undoubtedly been life-saving and hopefully will open up even better possibilities in the near future.

Extensor supracondylar femoral osteotomy as treatment for flexed haemophilic knee.

The experience with extensor supracondylar femoral osteotomy as treatment for the flexed haemophilic knee is presented with the description of 19 patients treated during a 30-year period (1968-98). The average age of the patients was 16 (8-35 years), and the average age follow-up was 13 years (3-30 years). Six patients had flexion fixed deformity while the rest presented 40 degrees average range of motion (10-75 degrees). In 13 patients a single osteotomy without internal fixation was performed, in one an osteosynthesis with a condylar plate and in five stabilization was achieved by means of Blount staples. Previous surgery was performed in two patients with patello-femoral ankylosis. The osteotomy site was consolidated in every patient and the deformity was corrected. Two bleeding complications were observed: one haemarthrosis and one psoas haematoma. Flexion relapsed in one patient who underwent another procedure after 12 years. One patient presented with a peroneal nerve paralysis; another one a genu recurvartum; which required flexor osteotomy. The extensor supracondylar femoral osteotomy is a procedure that aligns the limb with scarce modification of articular mobility.