Bruce Poppe

Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes

BackgroundGene-expression analysis is increasingly important in biological research, with real-time reverse transcription PCR (RT-PCR) becoming the method of choice for high-throughput and accurate expression profiling of selected genes. Given the increased sensitivity, reproducibility and large dynamic range of this methodology, the requirements for a proper internal control gene for normalization have become increasingly stringent. Although housekeeping gene expression has been reported to vary considerably, no systematic survey has properly determined the errors related to the common practice of using only one control gene, nor presented an adequate way of working around this problem.ResultsWe outline a robust and innovative strategy to identify the most stably expressed control genes in a given set of tissues, and to determine the minimum number of genes required to calculate a reliable normalization factor. We have evaluated ten housekeeping genes from different abundance and functional classes in various human tissues, and demonstrated that the conventional use of a single gene for normalization leads to relatively large errors in a significant proportion of samples tested. The geometric mean of multiple carefully selected housekeeping genes was validated as an accurate normalization factor by analyzing publicly available microarray data.ConclusionsThe normalization strategy presented here is a prerequisite for accurate RT-PCR expression profiling, which, among other things, opens up the possibility of studying the biological relevance of small expression differences.

Breast-Cancer Risk in Families with Mutations in PALB2

BACKGROUND Germline loss-of-function mutations in PALB2 are known to confer a predisposition to breast cancer. However, the lifetime risk of breast cancer that is conferred by such mutations remains unknown. METHODS We analyzed the risk of breast cancer among 362 members of 154 families who had deleterious truncating, splice, or deletion mutations in PALB2. The age-specific breast-cancer risk for mutation carriers was estimated with the use of a modified segregation-analysis approach that allowed for the effects of PALB2 genotype and residual familial aggregation. RESULTS The risk of breast cancer for female PALB2 mutation carriers, as compared with the general population, was eight to nine times as high among those younger than 40 years of age, six to eight times as high among those 40 to 60 years of age, and five times as high among those older than 60 years of age. The estimated cumulative risk of breast cancer among female mutation carriers was 14% (95% confidence interval [CI], 9 to 20) by 50 years of age and 35% (95% CI, 26 to 46) by 70 years of age. Breast-cancer risk was also significantly influenced by birth cohort (P<0.001) and by other familial factors (P=0.04). The absolute breast-cancer risk for PALB2 female mutation carriers by 70 years of age ranged from 33% (95% CI, 25 to 44) for those with no family history of breast cancer to 58% (95% CI, 50 to 66) for those with two or more first-degree relatives with breast cancer at 50 years of age. CONCLUSIONS Loss-of-function mutations in PALB2 are an important cause of hereditary breast cancer, with respect both to the frequency of cancer-predisposing mutations and to the risk associated with them. Our data suggest the breast-cancer risk for PALB2 mutation carriers may overlap with that for BRCA2 mutation carriers. (Funded by the European Research Council and others.).

PHF6 mutations in T-cell acute lymphoblastic leukemia

Tumor suppressor genes on the X chromosome may skew the gender distribution of specific types of cancer. T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with an increased incidence in males. In this study, we report the identification of inactivating mutations and deletions in the X-linked plant homeodomain finger 6 (PHF6) gene in 16% of pediatric and 38% of adult primary T-ALL samples. Notably, PHF6 mutations are almost exclusively found in T-ALL samples from male subjects. Mutational loss of PHF6 is importantly associated with leukemias driven by aberrant expression of the homeobox transcription factor oncogenes TLX1 and TLX3. Overall, these results identify PHF6 as a new X-linked tumor suppressor in T-ALL and point to a strong genetic interaction between PHF6 loss and aberrant expression of TLX transcription factors in the pathogenesis of this disease.

Chromosomal radiosensitivity in breast cancer patients with a known or putative genetic predisposition.

The chromosomal radiosensitivity of breast cancer patients with a known or putative genetic predisposition was investigated and compared to a group of healthy women. The chromosomal radiosensitivity was assessed with the G2 and the G0-micronucleus assay. For the G2 assay lymphocytes were irradiated in vitro with a dose of 0.4 Gy 60Co γ-rays after 71 h incubation, and chromatid breaks were scored in 50 metaphases. For the micronucleus assay lymphocytes were exposed in vitro to 3.5 Gy 60Co γ-rays at a high dose rate or low dose rate. 70 h post-irradiation cultures were arrested and micronuclei were scored in 1000 binucleate cells. The results demonstrated that the group of breast cancer patients with a known or putative genetic predisposition was on the average more radiosensitive than a population of healthy women, and this with the G2 as well as with the high dose rate and low dose rate micronucleus assay. With the G2 assay 43% of the patients were found to be radiosensitive. A higher proportion of the patients were radiosensitive with the micronucleus assay (45% with high dose rate and 61% with low dose rate). No correlation was found between the G2 and the G0-micronucleus chromosomal radiosensitivity. Out of the different subgroups considered, the group of the young breast cancer patients without family history showed the highest percentage of radiosensitive cases in the G2 (50%) as well as in the micronucleus assay (75–78%).

Identification of cytogenetic subclasses and recurring chromosomal aberrations in AML and MDS with complex karyotypes using m-FISH

Complex chromosomal aberrations (CCAs) can be detected in a substantial proportion of AML and MDS patients, de novo as well as secondary or therapy‐related, and are associated with an adverse prognosis. Comprehensive analysis of the chromosomal rearrangements in these complex karyotypes has been hampered by the limitations of conventional cytogenetics. As a result, our knowledge concerning the cytogenetics of these malignancies is sparse. Here we describe a multiplex‐FISH (M‐FISH) study of CCAs in 36 patients with AML and MDS. M‐FISH generated a genome‐wide analysis of chromosomal aberrations in CCAs, establishing several cytogenetic subgroups. ‐5/5q‐ was demonstrated in the majority of patients (86%). Other rearrangements (present with or without ‐5/5q‐) included: deletion of 7q (47%), 3q rearrangements (19%), and MLL copy gain or amplification (17%). These genetic subgroups seem to display biological heterogeneity: MLL copy gain or amplification in association with 5q‐ was detected only in AML patients and was significantly associated with extremely short survival (median overall survival: 30 days, P = 0.0102). A partially cryptic t(4;5)(q31;q31), a balanced t(1;8)(p31;q22), and an unbalanced der(7)t(7;14)(q21;q13) were detected as possible new recurrent rearrangements in association with CCAs. Novel reciprocal translocations included t(5;11)(q33;p15)del(5)(q13q31) and t(3;6)(q26;q25). We conclude that AML and MDS with CCAs can be subdivided into molecular cytogenetic subclasses, which could reflect different clinical behavior and prognosis, and that three recurrent chromosomal aberrations are associated with karyotype complexity.

A new recurrent inversion, inv(7)(p15q34), leads to transcriptional activation of HOXA10 and HOXA11 in a subset of T-cell acute lymphoblastic leukemias

Chromosomal translocations with breakpoints in T-cell receptor (TCR) genes are recurrent in T-cell malignancies. These translocations involve the TCRαδ gene (14q11), the TCRβ gene (7q34) and to a lesser extent the TCRγ gene at chromosomal band 7p14 and juxtapose T-cell oncogenes next to TCR regulatory sequences leading to deregulated expression of those oncogenes. Here, we describe a new recurrent chromosomal inversion of chromosome 7, inv(7)(p15q34), in a subset of patients with T-cell acute lymphoblastic leukemia characterized by CD2 negative and CD4 positive, CD8 negative blasts. This rearrangement juxtaposes the distal part of the HOXA gene cluster on 7p15 to the TCRβ locus on 7q34. Real time quantitative PCR analysis for all HOXA genes revealed high levels of HOXA10 and HOXA11 expression in all inv(7) positive cases. This is the first report of a recurrent chromosome rearrangement targeting the HOXA gene cluster in T-cell malignancies resulting in deregulated HOXA gene expression (particularly HOXA10 and HOXA11) and is in keeping with a previous report suggesting HOXA deregulation in MLL-rearranged T- and B cell lymphoblastic leukemia as the key factor in leukaemic transformation. Finally, our observation also supports the previous suggested role of HOXA10 and HOXA11 in normal thymocyte development.

Molecular cytogenetic and clinical findings in ETV6/ABL1-positive leukemia

Rearrangements of 12p, resulting from deletions or translocations, are common findings in hematologic malignancies. In many cases, these rearrangements target the ETV6 gene (previously called TEL) located at 12p13. Various partner genes have been implicated in the formation of fusion genes with ETV6. These include PDGFRB, JAK2, NTRK3, ABL2, and ABL1, each of which encodes for proteins with tyrosine kinase activity. To date, ETV6/ABL1 transcripts have been detected in only four patients with a leukemic disorder. Here, we describe one adult with chronic myeloid leukemia and a child with T‐cell acute lymphocytic leukemia with ETV6/ABL1. Molecular cytogenetic analysis confirmed that formation of an ETV6/ABL1 fusion in these patients required at least three chromosomal breaks and showed that each of these translocations is the result of a complex chromosomal rearrangement. Molecular analysis showed the presence of two fusion transcripts in both patients as the result of alternative splicing, questioning the suggested role of these transcripts in the lineage specificity. Clinical findings of these patients were compared to those of previously reported cases, and the possible clinical and biological similarities between ETV6/ABL1 and other fusion genes leading to increased tyrosine kinase activity are discussed.© 2000 Wiley‐Liss, Inc.

Rapid detection of VHL exon deletions using real-time quantitative PCR

Various types of mutations exist that exert an effect on the normal function of a gene. Among these, exon/gene deletions often remain unnoticed in initial mutation screening. Until recently, no fast and efficient methods were available to detect this type of mutation. Molecular detection methods for gene copy number changes included Southern blot (SB) and fluorescence in situ hybridisation, both with their own intrinsic limitations. In this paper, we report the development and application of a fast, sensitive and high-resolution method for the detection of single exon or larger deletions in the VHL gene based on real-time quantitative PCR (Q-PCR). These deletions account for approximately one-fifth of all patients with the von Hippel–Lindau syndrome, a dominantly inherited highly penetrant familial cancer syndrome predisposing to specific malignancies including phaeochromocytomas and haemangioblastomas. Our VHL exon quantification strategy is based on SYBR Green I detection and normalisation using two reference genes with a normal copy number, that is, ZNF80 (3q13.31) and GPR15 (3q12.1). Choice of primer sequences and the use of two reference genes appears to be critical for accurate discrimination between 1 and 2 exon copies. In a blind Q-PCR study of 29 samples, all 14 deletions were detected, which is in perfect agreement with previously determined SB results. We propose Q-PCR as the method of choice for fast (within 3.5 h), accurate and sensitive (ng amount of input DNA) exon deletion screening in routine DNA diagnosis of VHL disease. Similar assays can be designed for deletion screening in other genetic disorders.

The H3K27me3 demethylase UTX is a gender-specific tumor suppressor in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive form of leukemia that is mainly diagnosed in children and shows a skewed gender distribution toward males. In this study, we report somatic loss-of-function mutations in the X-linked histone H3K27me3 demethylase ubiquitously transcribed X (UTX) chromosome, in human T-ALL. Interestingly, UTX mutations were exclusively present in male T-ALL patients and allelic expression analysis revealed that UTX escapes X-inactivation in female T-ALL lymphoblasts and normal T cells. Notably, we demonstrate in vitro and in vivo that the H3K27me3 demethylase UTX functions as a bona fide tumor suppressor in T-ALL. Moreover, T-ALL driven by UTX inactivation exhibits collateral sensitivity to pharmacologic H3K27me3 inhibition. All together, our results show how a gender-specific and therapeutically relevant defect in balancing H3K27 methylation contributes to T-cell leukemogenesis.

ABT-199 mediated inhibition of BCL-2 as a novel therapeutic strategy in T-cell acute lymphoblastic leukemia

T-cell acute lymphoblastic leukemia (T-ALL) is a high-risk subtype of acute lymphoblastic leukemia (ALL) with gradually improved survival through introduction of intensified chemotherapy. However, therapy-resistant or refractory T-ALL remains a major clinical challenge. Here, we evaluated B-cell lymphoma (BCL)-2 inhibition by the BH3 mimetic ABT-199 as a new therapeutic strategy in human T-ALL. The T-ALL cell line LOUCY, which shows a transcriptional program related to immature T-ALL, exhibited high in vitro and in vivo sensitivity for ABT-199 in correspondence with high levels of BCL-2. In addition, ABT-199 showed synergistic therapeutic effects with different chemotherapeutic agents including doxorubicin, l-asparaginase, and dexamethasone. Furthermore, in vitro analysis of primary patient samples indicated that some immature, TLX3- or HOXA-positive primary T-ALLs are highly sensitive to BCL-2 inhibition, whereas TAL1 driven tumors mostly showed poor ABT-199 responses. Because BCL-2 shows high expression in early T-cell precursors and gradually decreases during normal T-cell differentiation, differences in ABT-199 sensitivity could partially be mediated by distinct stages of differentiation arrest between different molecular genetic subtypes of human T-ALL. In conclusion, our study highlights BCL-2 as an attractive molecular target in specific subtypes of human T-ALL that could be exploited by ABT-199.