Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia.

Familial platelet disorder with predisposition to acute myelogenous leukaemia (FPD/AML, MIM 601399) is an autosomal dominant disorder characterized by qualitative and quantitative platelet defects, and propensity to develop acute myelogenous leukaemia (AML). Informative recombination events in 6 FPD/AML pedigrees with evidence of linkage to markers on chromosome 21q identified an 880-kb interval containing the disease gene. Mutational analysis of regional candidate genes showed nonsense mutations or intragenic deletion of one allele of the haematopoietic transcription factor CBFA2 (formerly AML1) that co-segregated with the disease in four FPD/AML pedigrees. We identified heterozygous CBFA2 missense mutations that co-segregated with the disease in the remaining two FPD/AML pedigrees at phylogenetically conserved amino acids R166 and R201, respectively. Analysis of bone marrow or peripheral blood cells from affected FPD/AML individuals showed a decrement in megakaryocyte colony formation, demonstrating that CBFA2 dosage affects megakaryopoiesis. Our findings support a model for FPD/AML in which haploinsufficiency of CBFA2 causes an autosomal dominant congenital platelet defect and predisposes to the acquisition of additional mutations that cause leukaemia.

Correction: Imatinib mesylate inhibits STAT5 phosphorylation in response to IL-7 and promotes T cell lymphopenia in chronic myelogenous leukemia patients.

A correction to this paper has been published and can be accessed via a link at the top of the paper.

Differential expression of SMAD3 transcripts is not regulated by cis-acting genetic elements but has a gender specificity.

As a key component of the transforming growth factor-beta (TGF-beta) pathway, SMAD3 plays an essential role in development and maintenance of self-tolerance. Furthermore, a recent study based on gene-expression profiling in donors of allogeneic hematopoietic cell grafts revealed that the level of expression of several components of the TGF-beta pathway can predict the occurrence of graft-versus-host disease (GVHD) in recipients. The gene with the best GVHD predictive accuracy was SMAD3: no recipients suffered from GVHD when their donor cells expressed high levels of SMAD3 transcripts. The present study had two specific aims: to validate differential expression of SMAD3 transcripts in an independent and larger cohort of subjects and to determine whether interindividual differences were dictated by cis-acting genetic elements. In a cohort of 397 subjects, we found that SMAD3 transcript levels varied over a sixfold range. Analyses of SMAD3 single nucleotide polymorphisms and of SMAD3 promoter methylation patterns provide compelling evidence that interindividual differences in SMAD3 transcript levels do not result from in-cis genetic variations. Of note, part of the variance in SMAD3 expression was gender related as women expressed lower levels of SMAD3 transcripts than men.

Non-traumatic necrosis of bone (osteonecrosis) is associated with endothelial cell activation but not thrombophilia.

OBJECTIVE: The pathophysiology of non-traumatic osteonecrosis (ON) or avascular necrosis (AVN) of the femoral head remains poorly understood. Some studies have suggested the contribution of underlying thrombophilia as a mechanism; however, no specific thrombophilic factor has been consistently found in association with the disease. We are presenting data suggesting a role for endothelial cell activation rather than thrombophilia in ON. METHODS: A prospective consecutive cohort of 49 patients with a diagnosis of ON. The disease was considered idiopathic in five and secondary in 44 patients. The investigation included a coagulation and thrombophilia profile, endothelial cell activation and non-specific inflammatory markers as well as a biochemical profile. Statistical analysis using Fisher's exact test was obtained to assess correlation between endothelial cell markers and variables of inflammation. RESULTS: Patients with non-traumatic ON were not found to have a specific underlying thrombophilic factor compared with the general population. Out of 49 patients,19 had elevation of at least one endothelial cell markers. We found that activation of endothelial cell markers was independently correlated to ON but not correlated to the presence of inflammation (P = 1.0000). CONCLUSION: These results suggest that non-traumatic ON is not associated with a specific thrombophilic abnormality in those affected. This study demonstrates a potential association between regional endothelial dysfunction and ON. More studies are needed at a molecular level to further investigate the specific role of endothelium in the physiopathology of ON. A better understanding of the underlying mechanism could lead to potential preventive and therapeutic strategies of this devastating disease.

Demonstration of an osteoblast defect in two cases of human malignant osteopetrosis. Correction of the phenotype after bone marrow transplant.

Osteopetrosis is an inherited disorder characterized by bone sclerosis due to reduced bone resorption. Here we report that human osteopetrotic osteoblast-like (Ob) cells express a defective phenotype in primary cultures in vitro, and that bone marrow transplant (BMT) corrects osteoblast function. DNA analysis at polymorphic short-tandem repeat loci from donor, recipient, and primary Ob-like cells pre-BMT and 2 yr post-BMT revealed that Ob were still of recipient origin post-BMT. Osteopetrotic Ob-like cells obtained pre-BMT showed normal and abnormal 1,25(OH)2D3-induced alkaline phosphatase (ALPase) and osteocalcin production, respectively, and failed to produce macrophage colony-stimulating factor (M-CSF) in response to IL-1a and TNF-alpha. These parameters were all normalized in primary Ob-like cells prepared 2 yr post-BMT. X-linked clonality analysis at the human androgen receptor (HUMARA) locus revealed that osteoblasts showed a polyclonal and an oligoclonal derivation pre- and post-BMT respectively, indicating that a limited number of progenitor reconstituted this population. Because osteoblasts were still of recipient origin post-BMT, this suggests that functional osteoclasts, due to the replacement of hematopoeitic cells, provided a local microenvironment in vivo triggering the differentiation and/or recruitment of a limited number of functional osteoblasts.

Imatinib mesylate inhibits STAT5 phosphorylation in response to IL-7 and promotes T cell lymphopenia in chronic myelogenous leukemia patients.

Imatinib mesylate (IM) therapy has been shown to induce lower T cell counts in chronic myelogenous leukemia (CML) patients and an interference of IM with T cell receptor (TCR) signaling has been invoked to explain this observation. However, IL-7 and TCR signaling are both essential for lymphocyte survival. This study was undertaken to determine whether IM interferes with IL-7 or TCR signaling to explain lower T cell counts in patients. At diagnosis, CML patients have typically lower CD4+ counts in their blood, yet CD8+ counts are normal or even increased in some. Following the initiation of IM treatment, CD4+ counts were further diminished and CD8+ T lymphocytes were dramatically decreased. In vitro studies confirmed IM interference with TCR signaling through the inhibition of ERK phosphorylation and we showed a similar effect on IL-7 signaling and STAT5 phosphorylation (STAT5-p). Importantly however, using an in vivo mouse model, we demonstrated that IM impaired T cell survival through the inhibition of IL-7 and STAT5-p but not TCR signaling which remained unaffected during IM therapy. Thus, off-target inhibitory effects of IM on IL-7 and STAT5-p explain how T cell lymphopenia occurs in patients treated with IM.

Analysis of meningiomas by methylation- and transcription-based clonality assays.

The clonal derivation of tumors can be determined by X chromosome inactivation analysis based on differential expression of genes or differential methylation of cytosine residues in CpG islands near polymorphic loci. In this report, we compared a transcription-based RNA analysis with a methylation-based DNA assay to determine clonality of meningiomas. Both clonality assays use PCR-based analysis at the hunan androgen-receptor gene (HUMARA) on the X chromosome. Among 23 meningiomas from female patients, 19 were informative heterozygotes at this locus (83%). The patterns of X chromosome inactivation in four patients were extremely skewed towards one allele in blood (unequal Lyonization), which precluded clonality determination in the tumor samples. Concordant clonality results with methylation- and transcription-based clonality assays were demonstrated in 9 of 13 informative tumors expressing the androgen receptor. Seven meningiomas were monoclonal, but surprisingly, two pathologically documented cases of meningiomas were polyclonal. There was disparity in 4 of 13 tumor specimens that were polyclonal by the methylation-based assay but monoclonal by the transcription assay. Clonality examination of these tumors by the methylation-based phosphoglycerate kinase assay provided identical results to the methylation-based analysis at the HUMARA locus. In addition, loss of heterozygosity (LOH) studies of chromosome 22, which is frequently deleted in meningiomas, showed that four of four informative samples of the six polyclonal tumors had partial LOH in tumor tissues. However, complete LOH was observed in primary cultured cells, which were also monoclonal by the methylation assay. Taken together, these data suggest that the disparity of the two assays in these four cases may be due to differences in the level of expression of the androgen receptor gene in tumors. Therefore, we conclude that: (a) clonal derivation of meningiomas determined by both transcription- and methylation-based clonality assays are in full agreement in many (9 of 13) but not all cases (4 of 13); and (b) most meningiomas (9 of 15) are monoclonal in origin, whereas some meningioma samples (6 of 15) are polyclonal or may contain heterogeneous components.

Favorable long-term outcome of patients with multiple myeloma using a frontline tandem approach with autologous and non-myeloablative allogeneic transplantation.

Despite survival improvement with novel agents and use of autologous hematopoietic stem cell transplantation (HSCT), cure of patients with multiple myeloma (MM) remains anecdotal. Initial observations suggested that chronic GvHD was accompanied by an anti-myeloma effect after myeloablative HSCT, but unfortunately this procedure was hampered by high non-relapse mortality (NRM). To maximize the anti-myeloma effect and minimize NRM, we developed a non-myeloablative (NMA) regimen associated with a high incidence of chronic GvHD and tested its efficacy on patient survival and disease eradication. From 2001 to 2010, 92 patients aged ⩽ 65 years with a compatible sibling donor received autologous HSCT followed by an outpatient NMA allogeneic HSCT using a conditioning of fludarabine and cyclophosphamide. Patient median age was 52 years and 97% presented Durie-Salmon stages II-III disease. After a median follow-up of 8.8 years, probability of 10-year progression free and overall survival were 41% and 62%, respectively. Although the cumulative incidence of extensive chronic GvHD was high (at 79%), the majority of long-term survivors were off immunosuppressive drugs by year 5 and NRM was low (at 10%). Together, our results suggest that potential MM cure can be achieved with NMA transplantation regimens that maximize graft-versus-myeloma effect and minimize NRM.

Maintaining high autopsy rates in a Canadian blood and marrow transplant program: preserving a diagnostic and research tool.

Autopsy series have revealed patterns of injury in graft-versus-host disease and provided insight into infectious and toxic complications following hematopoietic stem cell transplantation (HSCT). Overall autopsy rates have declined significantly in recent decades including specialized services such as neonatal medicine and cardiac care. However, rates of post-mortem exams at HSCT centers have not been specifically documented. We reviewed hospital records between 1992 and 2002 to determine overall autopsy rates at our hospital and within the HSCT program. Although the overall autopsy rate declined steadily from 24% in 1992 to 9% in 2002, rates of post-mortem exams in the HSCT program remained relatively stable at 32% (24-46%). Autopsy rates were not significantly different for recipients of allogeneic vs autologous transplants and no clear difference was observed for the proportion of autopsies requested on weekdays compared with weekends. Autopsies confirmed major clinical diagnoses and/or suspected causes of death in 45 of 61 autopsies (74%) and yielded major or minor disagreements in clinical diagnosis in 10 cases (16%) and seven cases (11%), respectively. The preservation of high rates of autopsy within our HSCT program demonstrates that specialized programs are able to maintain elevated rates of post-mortem examinations despite overall declining rates.

Quantitative assessment of hematopoietic chimerism after allogeneic bone marrow transplantation has predictive value for the occurrence of irreversible graft failure and graft-vs.-host disease.

Primary graft failure, secondary to either host-vs.-graft reaction or delayed engraftment, and graft-vs.-host disease (GVHD) are among the most difficult clinical problems to manage in the field of allogeneic bone marrow transplantation (BMT). Early diagnosis of both conditions would greatly improve their outcome. Using fluorescence in situ hybridization (FISH) with an X- and Y-probe mixture, we sequentially monitored chimerism of neutrophils and lymphoid cells from day 1 to 100 in 28 consecutive recipients of sex-mismatched unmanipulated bone marrow grafts. The objective was to quantitatively assess the evolution of chimerism during this crucial time interval and to determine whether chimerism patterns would be predictive of engraftment and GVHD. In recipients with primary graft failure (n=7), the presence of donor-type neutrophils and NK cells as well as the predominance of donor-type T cells distinguished patients who responded to G-CSF (n=5) from nonresponders (n=2). Furthermore, the clearance of host CD3+CD56- cells during days 5-10 posttransplantation was significantly hastened in patients who subsequently developed acute (delta=80%) or chronic (delta=81%) GVHD compared with patients without GVHD (delta=17%). Thus, our data suggest that molecular monitoring of the fate of host/donor hematopoietic cells in the early posttransplantation period could be useful in differentiating patients with delayed engraftment from those with irreversible rejection and in predicting the occurrence of GVHD as soon as day 10. This investigational approach may provide an appropriate basis on which to select adequate treatment for primary graft failure and high-risk candidates that could benefit from novel preemptive therapies for GVHD.

Human pituitary adenomas show no loss of heterozygosity at the retinoblastoma gene locus.

The retinoblastoma tumor suppressor gene (RB1) is inactivated in hereditary and sporadic forms of retinoblastoma as well as in a number of other sporadic tumors. The majority of human pituitary tumors have been shown to be monoclonal neoplasms, suggesting that 1 or more somatic mutations are involved in the clonal expansion of a single progenitor cell. Recently, a high percentage of transgenic mice containing a disrupted RB1 allele have been shown to develop pituitary tumors. To investigate whether RB1 inactivation contributes to the development of human pituitary adenomas, we searched for loss of heterozygosity (LOH) within the RB1 gene locus in a variety of human pituitary adenomas. We screened 34 adenomas for LOH using a polymerase chain reaction (PCR)-based microsatellite polymorphic marker at the RB1 gene locus. In addition, a variable number of tandem repeat markers from within the RB1 gene was also used to search for LOH in 14 tumors. We found no LOH or microsatellite instability at the RB1 locus in any of the informative cases (30 of 34). Additionally, we showed that 4 representative adenomas from female patients are monoclonal in origin using a PCR-based clonality analysis assay. We conclude that the RB1 gene shows no LOH in a variety of human pituitary adenomas and that PCR-based microsatellite markers can serve as a useful tool for LOH analysis in human pituitary tumors.

Alloimmunization to platelet antigen HPA-1a (PIA1) is strongly associated with both HLA-DRB3*0101 and HLA-DQB1*0201.

Antibodies to the platelet HPA-1a antigen can elicit in the newborn a condition known as neonatal alloimmune thrombocytopenic purpura (NAITP). Previous studies based on RFLP analysis showed that 100% of HPA-1a-negative women who produced anti-HPA-1a antibodies (responders) were HLA-DRw52a (DRB3*0101). However, this specificity could also be found in some HPA-1a-negative women not producing anti-HPA-1a antibodies (nonresponders). We have analyzed in detail by PCR-SSOP the HLA-DR, -DQ, and -DP loci of 36 responders and 10 nonresponders. We found that while the allele DRB3*0101 was present in the vast majority of responders (91%), there were exceptions. Furthermore, the DQB1*0201 allele was found to be present in almost all responders (94%), but again was also found in nonresponders. The risk of alloimmunization to HPA-1a in an HPA-1b homozygous mother significantly increases with the presence of either allele, the odds ratio being 39.7 for DQB1*0201 and 24.9 for DRB3*0101. Sequencing of exon 2 of these two alleles from responders indicated no sequence difference when compared with the consensus sequences. This indicates that they do not represent variants when compared with the same alleles found in some nonresponders.

Clonality analysis of childhood ALL in remission: no evidence of clonal hematopoiesis.

We studied 98 female patients in remission (2-240 months) from childhood ALL to determine the clonality status of their hematopoiesis. Thirty-one (31.6%) were heterozygous at the PGK locus for the BstX1 endonuclease restriction site, permitting X-linked clonality assays to be performed. Two patients were in relapse at the time of study and were excluded. We used the PGK-PCR clonality assay (PPCA) to analyze DNA from PMN and mononuclear cells of the remaining 29 female patients. All (29/29) patients demonstrated polyclonal hematopoiesis. These data show that remission from childhood ALL involves reestablishment of polyclonally derived hematopoiesis in all patients studied.

Unrelated bone marrow transplantation for leukocyte adhesion deficiency.

The severe form of leukocyte adhesion deficiency type I (LAD-I) usually leads to death early in life. Allogeneic haematopoietic transplantation is the only cure. Unrelated transplantation has been reported only once. We describe three children with LAD-I transplanted with T cell non-depleted bone marrow from unrelated HLA-matched donors. All patients engrafted, one of them at second transplant. One patient developed grade I and one grade II acute GVHD. Two patients are alive, one of them with a decrease in CD11/CD18 expression. Early referral for HLA-matched unrelated BMT is a reasonable option for patients with LAD-I lacking an HLA-matched related donor.

Sequential analysis of early hematopoietic reconstitution following allogeneic bone marrow transplantation with fluorescence in situ hybridization (FISH).

Using in situ hybridization with an X and Y chromosome probe mixture, we have sequentially studied peripheral blood samples from 10 patients (four males/six females) in an HLA-matched allogeneic setting in order to monitor the kinetics of early hematopoietic reconstitution. Interphase cells from smears consisting of purified granulocytic and lymphocytic populations respectively were studied in three patients at 24, 48, 72 and 96 h post-transplant. This period was arbitrarily defined as the immediate post-transplant period. These three patients plus seven others were studied sequentially at days 5, 10, 15, 20, 25 and 50 post-transplant, defined as the intermediate post-transplant period. The X and Y probes were indirectly labelled with rhodamine and fluoresceine isothiocyanate, respectively. Donor neutrophils were detected as early as 24 h post marrow infusion followed by a significant expansion at 48 h. At 96 h post-transplant, the median percentage of donor neutrophils was > 90%. In the immediate post-transplant period, most of the lymphocytes were of recipient origin. However, we have documented a significant expansion in donor lymphocytes, starting at day 5 post-transplant in most patients. Almost complete chimerism for the myeloid and lymphoid lineages was established at days 10 and 25 post-transplant, respectively. All patients engrafted normally according to standard clinical criteria. Follow-up data for those surviving > or = 100 days (eight patients), showed persistence of this pattern of hematopoietic reconstitution in all but one patient. Molecular monitoring of early engraftment has enabled us to unravel a distinct biphasic pattern of myeloid and lymphoid engraftment.

Allogeneic transplantation for multiple myeloma: further evidence for a GVHD-associated graft-versus-myeloma effect.

We report a series of 37 consecutive patients with multiple myeloma (MM) who received an allograft between 1990 and 2000 at our institution. Median age was 47 years, and nearly 70% of patients were Durie-Salmon stage III. A median of five cycles of chemotherapy were given before transplant, with a median interval between diagnosis and transplant of 9.3 months. We report a nonrelapse mortality rate of 22% with a median follow-up period of 40 months, whereas complete remission (CR) rate at 12 months is estimated at 57%. Treatment failure rate and overall survival at 40 months are estimated at 52% and 32%, respectively. The number of chemotherapy cycles prior to allotransplantation achieved borderline statistical significance as a poor prognosis factor for overall survival (P = 0.05), while the presence of chronic graft-versus-host disease (cGVHD) was significantly correlated with CR achievement (P = 0.036). Our study confirms that early allografting in MM can yield toxicity rates significantly lower than those associated with historical cohorts, and supports the hypothesis that cumulative chemotoxicity has a negative influence on mortality and survival rates. More importantly, our study clearly demonstrates an association between cGVHD and CR and brings further evidence in favor of a graft-versus-myeloma effect.

The PGK-PCR clonality assay (PPCA).

The PGK-PCR assay offers an accurate method for determination of clonal derivation of cells in females who are heterozygous for the PGK BstXI polymorphism. Techniques to screen for heterozygosity and determine clonality using polymerase chain reaction based techniques are provided.

Development of a highly polymorphic STR marker for identity testing purposes at the human androgen receptor gene (HUMARA).

We developed a non-isotopic method which improves the technical quality of the X-linked HUMARA locus typing process. The use of formamide and a low concentration of acrylamide increased resolution and sharpness of HUMARA alleles in silver-stained polyacrylamide gels. In addition, the construction of an allelic ladder containing amplified sequence of 9 alleles (even-numbered alleles) of the HUMARA locus, allows confident, rapid and precise assignment of discretely defined alleles. Allele and genotype frequencies for the HUMARA locus were determined in a French Canadian population sample. Observed genotype frequencies in females conformed to Hardy-Weinberg expectations. Furthermore, the HUMARA locus is highly polymorphic with 18 observed alleles and an heterozygosity value of 89.3%. Also, this locus has average powers of discrimination of 97.8% and 88.7% for testing samples of female and male origin, respectively. In the French Canadian population, the average probability of excluding a random man as the father in paternity analysis when both mother and daughter are tested for this locus is 88.0%. Together, the results indicate that the HUMARA locus provides a highly discriminatory system that is appropriate for the purposes of forensic identification and paternity testing involving a female child.

Analysis of allele distribution for six short tandem repeat loci in the French Canadian population of Québec.

Short tandem repeat (STR) loci represent a rich source of highly polymorphic markers in the human genome which are useful for the purposes of forensic identification and determination of biological relatedness of individuals. Here, as a part of an ongoing extensive study, we report the analysis of a multilocus genotype survey of 642 to 870 chromosomes in the French Canadian Caucasian population of Québec at six STR loci. The loci HUMCSF1PO, HUMTPOX, HUMTH01, HUMF13A01, HUMFESFPS, and HUMvWA were typed using two multiplex polymerase chain reactions (PCR). Amplified DNA samples were subsequently analyzed by polyacrylamide gel electrophoresis followed by silver staining. The heterozygote frequencies of the loci range from 0.614 to 0.820 (0.661 to 0.818 expected) and the number of alleles from 7 to 12 per locus. Although statistically significant deviation from Hardy-Weinberg expectations of genotype frequencies was noted at some loci by one or more tests, in general, the genotype frequencies are well estimated from the product of allele frequencies at all loci. The most frequent six-locus genotype is expected to occur in the French Canadian population with a frequency of 3.50 by 10(-5) and together, these six loci have an average probability of discrimination of 0.9999985. The study presented here indicates that these six STR loci are informative genetic markers for identity testing purposes in the French Canadian Caucasian population of Québec.