Phenotypic heterogeneity in individuals with MECOM variants in 2 families

MECOM encodes the transcriptional regulators, EVI1 and MDS1-EVI1, from two distinct transcription start sites. EVI1 plays important roles in hematopoiesis and stem cell self-renewal. Recently, our group and others revealed that individuals with MECOM variants present diverse hematological and skeletal defects, including radioulnar synostosis (RUS). In the present study, we analyzed two families suspected with MECOM-associated syndrome. In family 1, a MECOM splicing variant (c.2285+1G>A) was identified in an individual with bone marrow failure (TRS4) without RUS and her mother, who had mild leukocytopenia, thrombocytopenia, and bilateral RUS. A copy neutral loss of heterozygosity decreasing the variant allele frequency was observed in the bone marrow of TRS4 and the peripheral blood leukocytes of her mother. However, TRS4 remained transfusion-dependent. In family 2, a MECOM variant (c.2208-4A>G), which was predicted to cause a cryptic acceptor site that results in a 3-base insertion (an insertion of Ser) in the mRNA, was identified in the proband, with bone marrow failure; this variant was also observed in her brother and father, both of whom have skeletal malformations, but no cytopenia. RT-PCR using leukocytes revealed a transcript with a 3-bp insertion in the proband, her brother, and the father, suggesting that the transcript variant with a 3-bp insertion is independent of blood phenotype. Collectively, these results suggest the presence of intrafamilial clinical heterogeneity in both families with MECOM splicing variants. Somatic genetic event may complicate the understanding of clinical variability among family members.

Applying Four-Step Characteristic Ion Filtering with HPLC-Q-Exactive MS/MS Spectrometer Approach for Rapid Compound Structures Characterization and Major Representative Components Quantification in Modified Tabusen-2 Decoction

Modified Tabusen-2 decoction (MTBD) is traditional Chinese Mongolia medicine, mainly used to treat osteoporosis. However, the precise material basis of this prescription is not yet fully elucidated. Herein, we establish an HPLC-Q-Exactive MS/MS spectrometer method with four-step characteristic ion filtering (FSCIF) strategy to quickly and effectively identify the structural features of MTBD and determine the representative compounds content. The FSCIF strategy included database establishment, characteristic ions summarization, neutral loss fragments screening, and secondary mass spectrum fragment matching four steps. By using this strategy, a total of 143 compounds were unambiguously or tentatively annotated, including 5 compounds which were first reported in MTBD. Nineteen representative components were simultaneously quantified with the HPLC-Q-Exactive MS/MS spectrometer, and it is suitable for eight batches of MTBD. Methodology analysis showed that the assay method had good repeatability, accuracy, and stability. The method established above was successfully applied to assess the quality of MTBD extracts. Collectively, our findings enhance our molecular understanding of the MTBD formulation and will allow us to control its quality in a better way. At the same time, this study can promote the development and utilization of ethnic medicine.

In vitro Metabolic Stability of Drugs and Applications of LC-MS in Metabolite Profiling

Metabolic stability of a compound is an important factor to be considered during the early stages of drug discovery. If the compound has poor metabolic stability, it never becomes a drug even though it has promising pharmacological characteristics. For example, a drug is quickly metabolized in the body; it does not have sufficient in vivo exposure levels and leads to the production of toxic, non-active or active metabolites. A drug is slowly metabolized in the body it could remain longer periods in the body and lead to unwanted adverse reactions, toxicity or may cause drug interactions. Metabolic stability assay is performed to understand the susceptibility of the compound to undergo biotransformation in the body. Intrinsic clearance of the compound is measured by metabolic stability assays. Different in vitro test systems including liver microsomes, hepatocytes, S9 fractions, cytosol, recombinant expressed enzymes, and cell lines are used to investigate the metabolic stability of drugs. Metabolite profiling is a vital part of the drug discovery process and LC–MS plays a vital role. The development of high-resolution (HR) MS technologies with improved mass accuracy, in conjunction with novel data processing techniques, has significantly improved the metabolite detection and identification process. HR-MS based data acquisition (ion intensity-dependent acquisition, accurate-mass inclusion list-dependent acquisition, isotope pattern-dependent acquisition, pseudo neutral loss-dependent acquisition, and mass defect-dependent acquisition) and data mining techniques (extracted ion chromatogram, product ion filter, mass defect filter, isotope pattern filter, neutral loss filter, background subtraction, and control sample comparison) facilitate the drug metabolite identification process.

The clinical and laboratory evaluation of patients with suspected hypocellular marrow failure

The overlap in clinical presentation and bone marrow features of acquired and inherited causes of hypocellular marrow failure poses a significant diagnostic challenge in real case scenarios, particularly in nonsevere disease. The distinction between acquired aplastic anemia (aAA), hypocellular myelodysplastic syndrome (MDS), and inherited bone marrow failure syndromes presenting with marrow hypocellularity is critical to inform appropriate care. Here, we review the workup of hypocellular marrow failure in adolescents through adults. Given the limitations of relying on clinical stigmata or family history to identify patients with inherited etiologies, we outline a diagnostic approach incorporating comprehensive genetic testing in patients with hypocellular marrow failure that does not require immediate therapy and thus allows time to complete the evaluation. We also review the clinical utility of marrow array to detect acquired 6p copy number-neutral loss of heterozygosity to support a diagnosis of aAA, the complexities of telomere length testing in patients with aAA, short telomere syndromes, and other inherited bone marrow failure syndromes, as well as the limitations of somatic mutation testing for mutations in myeloid malignancy genes for discriminating between the various diagnostic possibilities.

Hematopoietic mosaic chromosomal alterations in the New England Centenarian Study

Mosaic chromosomal alterations (mCAs) are structural alterations that include deletions, duplications, or copy-neutral loss of heterozygosity. mCAs are reported to be associated with survival, age, cancer, and cardiovascular disease. Previous studies of mCAs in large population-based cohorts (UK Biobank, MGBB, BioBank Japan, and FinnGen) have demonstrated a steady increase of mCAs as people age. The distribution of mCAs in centenarians and their offspring is not well characterized. We applied MOsaic CHromosomal Alteration (MoChA) caller on 2298 genome-wide genotype samples of 1582 centenarians, 443 centenarians’ offspring, and 273 unrelated controls from the New England Centenarian Study (NECS). Integrating Log R ratio and B-allele frequency (BAF) intensities with genotype phase information, MoChA employs a Hidden Markov Model to detect mCA-induced deviations in allelic balance at heterozygous sites consistent with genotype phase in the DNA microarray data. We analyzed mCAs spanning over 100 k base pairs, with an estimated cell fraction less than 50%, within samples with genome-wide BAF phase concordance across phased heterozygous sites less than 0.51, and with LOD score of more than 10 for the model based on BAF and genotype phase. Our analysis showed that somatic mCAs increase with older age up to approximately 102 years, but the prevalence of the subjects with mCAs tend to decrease after that age, thus suggesting that accumulation of mCAs is less prevalent in long-lived individuals. We also used Poisson regression to show that centenarians and their offspring tend to accumulate less mCA (RR = 0.63, p=0.045) compared to the controls.

Reversion Mosaicism in Primary Immunodeficiency Diseases

Reversion mosaicism has been reported in an increasing number of genetic disorders including primary immunodeficiency diseases. Several mechanisms can mediate somatic reversion of inherited mutations. Back mutations restore wild-type sequences, whereas second-site mutations result in compensatory changes. In addition, intragenic recombination, chromosomal deletions, and copy-neutral loss of heterozygosity have been demonstrated in mosaic individuals. Revertant cells that have regained wild-type function may be associated with milder disease phenotypes in some immunodeficient patients with reversion mosaicism. Revertant cells can also be responsible for immune dysregulation. Studies identifying a large variety of genetic changes in the same individual further support a frequent occurrence of reversion mosaicism in primary immunodeficiency diseases. This phenomenon also provides unique opportunities to evaluate the biological effects of restored gene expression in different cell lineages. In this paper, we review the recent findings of reversion mosaicism in primary immunodeficiency diseases and discuss its clinical implications.

Copy-Neutral Loss-of-Heterozygosity Adds Diagnostic and Prognostic Information to the Molecular Profiling of Hematological Malignancies

Background: Copy-neutral loss-of-heterozygosity (CN-LOH) - not detectable by chromosome banding analysis - is gaining importance as a prognostic factor and can either cause the duplication of an activating mutation in an oncogene, the deletion of a tumor suppressor gene or the gain/loss of specific methylated regions. However, examination for possible CN-LOH in hematological diagnostics is at present not routinely performed and, hence, data regarding the occurrence of CN-LOH across different entities as well as the association of relevant genes is limited. Aim: (1) Frequency assessment of CN-LOH by target enrichment sequencing (TES) in a diagnostic setting, (2) evaluation of whole genome sequencing (WGS) data to estimate the prevalence of CN-LOH in a larger cohort, to pinpoint relevant genes for CN-LOHs with so far unknown associations, and to determine cross-entity variability. Patients and Methods: 1196 patients (507 female, 689 male, median age: 66 years), sent between 04/2021-07/2021 for diagnostic work-up, were analyzed by TES with a median coverage of 1765x for the gene panel and 52x for the CNV spike-in panel (IDT, Coralville, IA). Amplification-free WGS libraries of 3851 different patients were sequenced with a median coverage of 102x. Reads were aligned to the human reference genome (GRCh37, Ensembl annotation, Isaac aligner). Cnvkit (v 0.9.9) was used to call copy number variations (CNVs) and CN-LOH for TES and HadoopCNV (Yang et al. 2017) was used to call CN-LOH for WGS. Results: 1196 patients were analyzed by TES. For 10% of the patients at least one CN-LOH event was detected without any association to age or gender but a slightly higher incidence in myeloid compared to lymphoid neoplasms (10% vs 6%). In 14 patients, CN-LOH affected more than one chromosome arm. CN-LOH occurred most frequently in 4q (n = 15), 7q (n = 16), 9p (n = 25) and 11q (n = 10). As expected, 4q CN-LOH co-occurred with high variant allele frequencies (VAF) of TET2. Based on WGS data, 4q CN-LOH occurred predominately in AML (35%), CMML (22%), and MDS (20%). In rare cases, 4q CN-LOH was associated with FBXW7 variants in T-ALL. 7q CN-LOH occurred nearly exclusively in myeloid neoplasms (95%) and was associated with high VAFs in EZH2 in 69% of TES and 82% of WGS cases. CUX1 variants with high VAFs were detected in 80% (TES) and 45% (WGS) of the remaining cases, respectively. The well-known 9p CN-LOH led to JAK2V617F homozygosity in all myeloid neoplasms and occurred most often in MPNs. In T-ALL, regions of 9p CN-LOH harbored CDKN2A/B deletions. 11q CN-LOH occurred more often in myeloid than lymphoid neoplasms (79% vs 21%) and was associated with CBL variants in 61% and KMT2A-PTD in 19% of the cases. In contrast, ATM was the relevant gene in all lymphoid cases with 11q CN-LOH. CN-LOH in 11p was detected less frequently and only in 25% of cases an association with WT1 variants could be identified. Our WGS data confirmed the known associations between 1p CN-LOH and high allele burden in MPL, CSF3R and NRAS, 2p CN-LOH and DNMT3A variants, 13q CN-LOH and FLT3-ITD, the near exclusive occurrence of 16p CN-LOH in follicular lymphoma (FL, 98%) with high CREBBP-mutant allele burden , 17p CN-LOH and TP53 homozygosity, and the exclusive occurrence of 21q CN-LOH in AML and its association with RUNX1 mutations. Besides, 12q CN-LOH was associated with KMT2D in FL, with SH2B3 in MDS/MPN overlaps and in rare cases with KDM2B. For 17q CN-LOH the relevant gene was not unequivocally identifiable with high mutant allele variants in SRSF2, STAT5B, and NF1. 18q CN-LOH was a very rare event but consistently associated with a high VAF of MBD2, which presumably influences cell proliferation (Cheng et al. 2018). 19q CN-LOH was mostly (63%) associated with a high VAF of CEBPA variants, except for patients with hairy cell leukemia: in these cases nonsense mutations in CIC (VAF > 90%) were detected. CN-LOH in 22q was more common in myeloid malignancies (65% vs 35%) and associated with PRR14L mutations in the majority of myeloid cases (62%). Of note, this association occurred neither in AML samples nor in lymphoid neoplasms. No recurrent mutations were found for 6p and 14q CN-LOHs. For all other chromosomes, CN-LOH events were very rare. Conclusions: By using a CNV spike-in panel, TES adds additional diagnostic and prognostic information by enabling simultaneous detection of selected gene mutations and genome-wide CNVs, as well as CN-LOH, without increase in sequencing costs and turn-around times. Figure 1 Figure 1. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.

TP53 Mutations, Deletions, and CN-LOH: Comparison of TP53 single Hit and Double Hit Events and Their Impact on Prognosis in Hematological Malignancies

Background: TP53 is altered in ~50% of human cancers. Alterations mainly include mutations and/or deletions, but also copy-neutral loss of heterozygosity (CN-LOH) was reported. Frequently, both TP53 alleles are altered (by mutation + deletion, mutation + CN-LOH or ≥2 mutations), leading to a "double hit" event. Aim: Analysis of TP53 aberrations using WGS in 4646 cases with 29 different hematological malignancies, comparing (1) the frequencies of TP53 alterations, (2) occurrence of single hit vs. double hit, (3) correlation with complex karyotype and (4) impact on survival. Methods: Whole-genome sequencing (WGS) was performed for all 4,646 patients (median coverage 100x). 151bp paired-end reads were generated on NovaSeq 6000 and HiSeqX machines (Illumina, San Diego, CA). As no sample specific normal tissue was available, a so-called Tumor/Unmatched normal (TUN) workflow was used to reduce technical artefacts and germline calls. All reported p-values are two-sided and were considered significant at p<0.05. Results: In the total cohort of 4,646 cases, in 582 (13%) at least one alteration (alt) involving TP53 was detected (comprising mutations (mut), deletions (del) and CN-LOH (LOH); Fig 1A,B). Cases were categorized as follows: cases with (1) 1 TP53 mut only (n=166), (2) del only (n=100), (3) LOH only (n=15), constituting the single hit events. Further, (4) cases with mut+del (might include 1 or more mut, n=211), cases with mut+LOH (≥1 mut, n=41), cases with ≥2 mut only (without del or LOH, n=49), resulting in double hit events (Fig 1B). Regarding the respective entities, high frequencies of TP53 alt were mainly detected in lymphoid malignancies (e.g. HGBL, MPAL, vHZL, MZL, MCL), whereas they were infrequent or absent in many myeloid malignancies (e.g. aCML, MPN, CMML, CML, MLN_eo; Fig 1A). For further analysis, only entities in which >10 cases showed TP53 alt events were used. Comparison of single hit vs. double hit revealed that T-NHL, MM, MPN and MDS predominantly showed a single hit, whereas the double hit was frequent in MPAL, MZL, MDS/MPN-U, CLL and MCL cases (Fig 1A). However, the type of double hit differed between myeloid and lymphoid malignancies, as myeloid neoplasms showed a high frequency of cases with ≥2 mut only, whereas in many lymphoid malignancies the double hit was predominantly generated by mut+del (Fig 1A,C). All TP53-associated events (mut, del, LOH and the respective combinations) were found to be associated with a complex karyotype in the total cohort (LOH: 14% complex karyotype in cases without TP53 alt vs. 59% in cases with TP53 alt, p<0.001). This association was also detected for most of the selected entities (exceptions: MZL, T-NHL). Regarding overall survival (OS), in the total cohort, all events involving TP53 impact on OS (TP53 alt: 22 months vs. 84 months, p<0.001; TP53 mut: 20 vs. 82 months, p<0.001; TP53 del: 20 vs. 79 months, p<0.001; TP53 LOH: 20 vs. 75 months, p<0.001). Moreover, although the single hit already impacts on OS, the double hit leads to an even inferior outcome (no hit vs. single hit vs. double hit: 84 vs. 39 vs. 14 months, p<0.001). In the selected entities, an influence of TP53 alt on OS was detected for all malignancies except HGBL, MZL and T-NHL, for which also the presence of a double hit did not show an effect on OS. For the majority of the other entities, the double hit leads to a shorter OS than the single hit (as observed for the total cohort), with the exceptions of MCL and MPAL: in these entities, the single hit did not impact on OS, but only a double hit is associated with inferior outcome. Conclusions: (1) Frequency of TP53 alterations and of double hit vs. single hit differs markedly between entities. (2) The kind of TP53 complexity differs between both lineages (double hit in myeloid neoplasms: often ≥2 mut only; in lymphoid malignancies: predominantly mut+del). (3) In 7% (41/582) of cases with TP53 alt, CN-LOH transforms a single hit into a double hit. (4) In the total cohort and in the majority of selected entities (except MZL and T-NHL), TP53 alt are associated with complex karyotype. (5) In the total cohort, all events involving TP53 impact on OS; cases with double hit show an inferior outcome compared to single hit. (6) Regarding OS, the selected entities can be divided into three categories: (i) no influence of TP53 alt (HGBL, MZL, T-NHL); (ii) double hit required for impact on OS (MCL, MPAL); (iii) influence of both single hit and double hit with inferior outcome of double hit (all other). Figure 1 Figure 1. Disclosures Kern: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership. Haferlach: MLL Munich Leukemia Laboratory: Other: Part ownership.