Quantification of free fetal DNA in multiple pregnancies and relationship with chorionicity.

OBJECTIVE: Free fetal DNA (ffDNA) in the maternal plasma appears to originate mainly from the trophoblast. We tested the hypothesis that ffDNA concentration is increased in multiple pregnancies where trophoblastic mass has been shown to be increased. METHODS: Quantitative real-time PCR was used to measure the plasma concentration of DYS14 in singleton and twin pregnancies with one or two male fetuses. Royston and Wright's regression method was used to relate ffDNA to gestational age in singleton controls; z-scores were calculated for the multiple pregnancy subgroups. RESULTS: Fifty-five singleton and 65 twin pregnancies (36 with one and 29 with two male fetuses) were analysed. There was significantly higher ffDNA concentration in twin pregnancies with two male fetuses compared with pregnancies with one male fetus. In cases with two male fetuses, there was no statistically significant difference between monochorionic and dichorionic pregnancies. CONCLUSIONS: There is higher ffDNA concentration in multiple pregnancies, and this must be taken into account for future quantitative ffDNA applications.

Heterogeneous breakpoints in patients with acute lymphoblastic leukemia and the dic(9;20)(p11-13;q11) show recurrent involvement of genes at 20q11.21.

The dic(9;20)(p11-13;q11) is a recurrent chromosomal abnormality in patients with acute lymphoblastic leukemia. Although it results in loss of material from 9p and 20q, the molecular targets on both chromosomes have not been fully elucidated. From an initial cohort of 58 with acute lymphoblastic leukemia patients with this translocation, breakpoint mapping with fluorescence in situ hybridization on 26 of them revealed breakpoint heterogeneity of both chromosomes. PAX5 has been proposed to be the target gene on 9p, while for 20q, FISH analysis implicated the involvement of the ASXL1 gene, either by a breakpoint within (n=4) or centromeric (deletion, n=12) of the gene. Molecular copy-number counting, long-distance inverse PCR and direct sequence analysis identified six dic(9;20) breakpoint sequences. In addition to the three previously reported: PAX5-ASXL1, PAX5-C20ORF112 and PAX5-KIF3B; we identified three new ones in this study: sequences 3' of PAX5 disrupting ASXL1, and ZCCHC7 disrupted by sequences 3' of FRG1B and LOC1499503. This study provides insight into the breakpoint complexity underlying dicentric chromosomal formation in acute lymphoblastic leukemia and highlights putative target gene loci.

Evaluation of 13q14 status in patients with chronic lymphocytic leukemia using single nucleotide polymorphism-based techniques.

Deletions of chromosome 13q14 are common in chronic lymphocytic leukemia and other cancers, demonstrating the importance of this region in tumorigenesis. We report the use of two single-nucleotide polymorphism (SNP)-based techniques to determine 13q loss of heterozygosity (LOH) status in 15 patients with CLL: (i) digital SNP (dSNP), where analysis of heterozygous SNPs detects allelic imbalances, and (ii) DNA sequencing, where LOH is identified by comparison of allelic peak heights in normal and neoplastic cells. The SNP-based techniques were compared with established molecular techniques, fluorescence in situ hybridization and multiplex ligation-dependent probe amplification, to determine their utility and relative sensitivity. dSNP proved to be the most sensitive technique, identifying 13q14 LOH in 11 of 13 (85%) patients (95% CI: 55%, 98%) without the need for neoplastic cell enrichment. Three cases showed evidence of LOH by dSNP that was not apparent by other techniques. In 8 of 13 (62%) cases, partial or interstitial patterns of LOH were observed by dSNP. Our findings demonstrate that dSNP represents a useful, sensitive technique for the analysis of chromosomal aberrations that result in LOH. It may have applications for the analysis of other malignancies that are difficult to assess by conventional molecular techniques.

Methylation of tumour suppressor gene promoters in the presence and absence of transcriptional silencing in high hyperdiploid acute lymphoblastic leukaemia.

Promoter methylation is a common phenomenon in tumours, including haematological malignancies. In the present study, we investigated 36 cases of high hyperdiploid (>50 chromosomes) acute lymphoblastic leukaemia (ALL) with methylation-specific multiplex ligase-dependent probe amplification to determine the extent of aberrant methylation in this subgroup. The analysis, which comprised the promoters of 35 known tumour suppressor genes, showed that 16 genes displayed abnormal methylation in at least one case each. The highest number of methylated gene promoters seen in a single case was thirteen, with all but one case displaying methylation for at least one gene. The most common targets were ESR1 (29/36 cases; 81%), CADM1 (IGSF4, TSLC1; 25/36 cases; 69%), FHIT (24/36 cases; 67%) and RARB (22/36 cases; 61%). Interestingly, quantitative reverse transcription-polymerase chain reaction showed that although methylation of the CADM1 and RARB promoters resulted in the expected pattern of downregulation of the respective genes, no difference could be detected in FHIT expression between methylation-positive and -negative cases. Furthermore, TIMP3 was not expressed regardless of methylation status, showing that aberrant methylation does not always lead to gene expression changes. Taken together, our findings suggest that aberrant methylation of tumour suppressor gene promoters is a common phenomenon in high hyperdiploid ALL.

Variable breakpoints target PAX5 in patients with dicentric chromosomes: a model for the basis of unbalanced translocations in cancer.

The search for target genes involved in unbalanced acquired chromosomal abnormalities has been largely unsuccessful, because the breakpoints of these rearrangements are too variable. Here, we use the example of dicentric chromosomes in B cell precursor acute lymphoblastic leukemia to show that, despite this heterogeneity, single genes are targeted through a variety of mechanisms. FISH showed that, although they were heterogeneous, breakpoints on 9p resulted in the partial or complete deletion of PAX5. Molecular copy number counting further delineated the breakpoints and facilitated cloning with long-distance inverse PCR. This approach identified 5 fusion gene partners with PAX5: LOC392027 (7p12.1), SLCO1B3 (12p12), ASXL1 (20q11.1), KIF3B (20q11.21), and C20orf112 (20q11.1). In each predicted fusion protein, the DNA-binding paired domain of PAX5 was present. Using quantitative PCR, we demonstrated that both the deletion and gene fusion events resulted in the same underexpression of PAX5, which extended to the differential expression of the PAX5 target genes, EBF1, ALDH1A1, ATP9A, and FLT3. Further molecular analysis showed deletion and mutation of the homologous PAX5 allele, providing further support for the key role of PAX5. Here, we show that specific gene loci may be the target of heterogeneous translocation breakpoints in human cancer, acting through a variety of mechanisms. This approach indicates an application for the identification of cancer genes in solid tumours, where unbalanced chromosomal rearrangements are particularly prevalent and few genes have been identified. It can be extrapolated that this strategy will reveal that the same mechanisms operate in cancer pathogenesis in general.

The complex genomic profile of ETV6-RUNX1 positive acute lymphoblastic leukemia highlights a recurrent deletion of TBL1XR1.

The ETV6-RUNX1 fusion is the molecular consequence of the t(12;21)(p13;q22) seen in approximately 25% of children with acute lymphoblastic leukemia (ALL). Studies have shown that the fusion alone is insufficient for the initiation of leukemia; additional genetic changes are required. Genomic profiling identified copy number alterations at high frequencies in these patients. Focal deletions of TBL1XR1 were observed in 15% of cases; 3 patients exhibited deletions distal to the gene. Fluorescence in situ hybridization confirmed these deletions and quantitative RT-PCR showed that the TBL1XR1 gene was significantly under-expressed. TBL1XR1 is a key component of the SMRT and N-CoR compressor complexes, which control hormone-receptor mediated gene expression. Differential expression of the retinoic acid target genes, RARB, CRABP1, and CRABP2, indicated that deletion of TBL1XR1 compromised the function of SMRT/N-CoR in the appropriate control of gene expression. This study identifies deletions of TBL1XR1 as a recurrent abnormality in ETV6-RUNX1 positive ALL. We provide evidence that implicates this deletion in the inappropriate control of gene expression in these patients. The target of the interaction between TBL1XR1 and the signaling pathways described here may be exploited in cancer therapy.

Five members of the CEBP transcription factor family are targeted by recurrent IGH translocations in B-cell precursor acute lymphoblastic leukemia (BCP-ALL).

CCAAT enhancer-binding protein (CEBP) transcription factors play pivotal roles in proliferation and differentiation, including suppression of myeloid leukemogenesis. Mutations of CEBPA are found in a subset of acute myeloid leukemia (AML) and in some cases of familial AML. Here, using cytogenetics, fluorescence in situ hybridization (FISH), and molecular cloning, we show that 5 CEBP gene family members are targeted by recurrent IGH chromosomal translocations in BCP-ALL. Ten patients with t(8;14)(q11;q32) involved CEBPD on chromosome 8, and 9 patients with t(14;19)(q32;q13) involved CEBPA, while a further patient involved CEBPG, located 71 kb telomeric of CEBPA in chromosome band 19q13; 4 patients with inv(14)(q11q32)/t(14;14)(q11;q32) involved CEBPE and 3 patients with t(14;20)(q32;q13) involved CEBPB. In 16 patients the translocation breakpoints were cloned using long-distance inverse-polymerase chain reaction (LDI-PCR). With the exception of CEBPD breakpoints, which were scattered within a 43-kb region centromeric of CEBPD, translocation breakpoints were clustered immediately 5' or 3' of the involved CEBP gene. Except in 1 patient with t(14;14)(q11;q32), the involved CEBP genes retained germ-line sequences. Quantitative reverse transcription (RT)-PCR showed overexpression of the translocated CEBP gene. Our findings implicate the CEBP gene family as novel oncogenes in BCP-ALL, and suggest opposing functions of CEBP dysregulation in myeloid and lymphoid leukemogenesis.