Johann-Christoph Jann

Accurate quantification of chromosomal lesions via short tandem repeat analysis using minimal amounts of DNA

Background Cytogenetic aberrations such as deletion of chromosome 5q (del(5q)) represent key elements in routine clinical diagnostics of haematological malignancies. Currently established methods such as metaphase cytogenetics, FISH or array-based approaches have limitations due to their dependency on viable cells, high costs or semi-quantitative nature. Importantly, they cannot be used on low abundance DNA. We therefore aimed to establish a robust and quantitative technique that overcomes these shortcomings. Methods For precise determination of del(5q) cell fractions, we developed an inexpensive multiplex-PCR assay requiring only nanograms of DNA that simultaneously measures allelic imbalances of 12 independent short tandem repeat markers. Results Application of this method to n=1142 samples from n=260 individuals revealed strong intermarker concordance (R²=0.77–0.97) and reproducibility (mean SD: 1.7%). Notably, the assay showed accurate quantification via standard curve assessment (R²>0.99) and high concordance with paired FISH measurements (R²=0.92) even with subnanogram amounts of DNA. Moreover, cytogenetic response was reliably confirmed in del(5q) patients with myelodysplastic syndromes treated with lenalidomide. While the assay demonstrated good diagnostic accuracy in receiver operating characteristic analysis (area under the curve: 0.97), we further observed robust correlation between bone marrow and peripheral blood samples (R²=0.79), suggesting its potential suitability for less-invasive clonal monitoring. Conclusions In conclusion, we present an adaptable tool for quantification of chromosomal aberrations, particularly in problematic samples, which should be easily applicable to further tumour entities.

Next-Generation Bisulfite Sequencing Of Cacna1c With Illumina Miseq

Background The complex etiology of psychiatric illness involves both genetic and environmental factors. The latter may act via epigenetic processes such as DNA methylation. Studies suggest that epigenetic regulation of gene expression may mediate the effects of early life experiences on adult behavior and susceptibility to psychiatric illness. CACNA1C is an epigenetically regulated gene implicated in the etiology of psychiatric illness. Here, the third CpG island located in intron 3 is of particular interest since: (1) differential methylation is reported between bipolar patients and controls, with higher methylation levels being observed in patients; (2) CpG sites in this CpG island show blood/brain correlation; (3) individual methylation level predict more of the variance than tissue methylation level; and (4) it is a significant meQTL, as it is located in close proximity (5kb) to rs1006737, which shows a genome wide significant association to bipolar disorder, schizophrenia, and major depression. The aim of the present study was to test the feasibility of next generation bisulfite sequencing in the methylation analysis of CpG sites of the third CACNA1C CpG island. Methods Next generation sequencing (NGS) analysis of the CACNA1C region of Intron 3 was performed. A total of 63 CpG sites from three bisulfite converted whole blood DNA samples were analyzed. Both single- and multiplex-PCRs were used. Results Singleplex-PCR generated 8,000 to 36,000 sequence reads, with a sequence identity of 99.0 to 99.9%. The standard deviation of the methylation level per CpG of the three samples ranged from 0.3 to 6.4%. Multiplex-PCR generated variable methylation values, with lower sequence identity and fewer sequence reads. Discussion This is the first comprehensive analysis of methylation levels of the CACNA1C third intronic region to use NGS. The results indicate that multiplex PCR is not an appropriate method for methylation analysis of bisulfite converted DNA. Now it is planed to perform simultaneous sequencing of 384 samples using singleplex-PCR.

Concomitant MDS with isolated 5q deletion and MGUS: case report and review of molecular aspects

Patients with monoclonal gammopathy of undetermined significance (MGUS) have a higher risk for the development of concomitant primary cancers such as multiple myeloma (MM) and myelodysplastic syndrome (MDS). We report the case of patient initially suffering from MGUS of the IgG lambda subtype for more than 10 yr, which evolved to MM and MDS with deletion (5q) with severe pancytopenia. Due to pancytopenia, he received dose‐reduced treatment with lenalidomide and dexamethasone. He achieved an ongoing transfusion independency after about 1 month of treatment. Bone marrow taken 14 months after start of treatment showed a complete cytogenetic response of the del(5q) clone and a plasma cell infiltration below 5%. In contrast to the development of MM in MGUS patients, the subsequent occurrence of MDS after diagnosis of MGUS is infrequent. Moreover, the biological association of MDS with MGUS is not sufficiently understood, but the non‐treatment‐related occurrence supports the pathogenetic role of pre‐existing alterations of stem cells. Here, we summarize data on concomitant MDS and MGUS/MM with particular emphasis on molecular aspects.

Clonal hierarchies and dynamic evolution in Myelodysplastic Syndromes (MDS) upon therapy

T cell stimulation with different cytokines results in distinct phenotypes and cytotoxic activity of CD19-specific CART cells

O-013 Mesenchymal stromal cells support significant engraftment of low-risk myelodysplastic syndromes (MDS) in a murine xenograft model

chondrial DNA (mtDNA) mutations. These mitochondrial-associated phenotypes suggest that mitochondrial dysfunction plays a possible role in the pathophysiology of MDS. Introduction: Next generation sequencing (NGS) is ideal to study mtDNA mutations because it allows for the detection of low-level heteroplasmy between flow-sorted samples. These changes would be challenging to detect by other methods. We hypothesized that in MDS, mtDNA changes would be found in genes or tRNAs involved in oxidative phosphorylation (OXPHOS) as had previously been shown in a wide range of cancers, including leukemia. Purpose: Using NGS, we sequenced mtDNA from hematopoietic progenitor cells of six MDS patients, flow-sorted by cell surface markers CD3+ (T-cell), CD33+ (myeloid) and CD34+ (hematopoietic progenitor). We analyzed the changes in mtDNA observed in the sorted hematopoietic progenitor and myeloid cells and compared them to T-cells. Materials and Methods: Bone marrow cells were flow-sorted prior to DNA extraction. mtDNA was enriched by long-range PCR (LRPCR). The LRPCR amplicons were equimolarly pooled and libraries constructed prior to Illumina NGS sequencing. Paired-end sequences were aligned to NC_012920 and PCR duplicates removed using BWA/Samtools and CLCBio. Variants were called using quality based metrics. Heteroplasmy levels were calculated using allele counts at a given m. position. Results: Three of the 6 samples had no sequence discordants between cell-sorted populations. One sample acquired two variants in CD34+ cells, a novel MT-TY m.5850T>C, and MT-ATP8 m.8412T>C p.Met16Thr previously reported as a polymorphism. Another sample had a novel variant with increasing heteroplasmy between CD33+ and CD34+ cells (23% to 92.8% alternate allele, respectfully) in MTCO2, m.7605T>C p.Val7Ala. The last sample had three novel variants in CD34+ cells, MT-ND1 m.3810A>G p.Thr168Thr; MT-CO2 m.7983T>A p.Leu133Gln and MT-CO3 m.9817A>G p.His204Arg. This patient also had a homoplasmic reversion back towild type compared with CD33+ cells in MT-C03 m.9477G p.Val61. Of the seven variants detected in 3/6 patients, five were novel. These novel variants are associated with OXPHOS complexes I, IV, and V. Conclusions: As most information regarding mtDNA variants is from mitochondrial disorder research, the identification of multiple novel variants from these MDS samples may be significant. Further NGS analysis and functional studies of novel mtDNA variants associated with MDSmay improve our understanding of the disease biology and possibly bear relevance to prognosis and treatment.