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The frequency of pathogenic/likely pathogenic (P/LP) germ line variants in patients with myelodysplastic syndrome (MDS) diagnosed at age 40 years or less is 15% to 20%. However, there are no comprehensive studies assessing the frequency of such variants across the age spectrum. We performed augmented whole-exome sequencing of peripheral blood samples from 404 patients with MDS and their related donors before allogeneic hematopoietic stem cell transplantation. Single-nucleotide and copy number variants in 233 genes were analyzed and interpreted. Germ line status was established by the presence of a variant in the patient and related donor or for those seen previously only as germ line alleles. We identified P/LP germ line variants in 28 of 404 patients with MDS (7%), present within all age deciles. Patients with P/LP variants were more likely to develop higher-grade MDS than those without (43% vs 25%; P = .04). There was no statistically significant difference in outcome parameters between patients with and without a germ line variant, but the analysis was underpowered. P/LP variants in bone marrow failure syndrome genes were found in 5 patients aged less than 40 years, whereas variants in DDX41 (n = 4), telomere biology disorder genes (n = 2), and general tumor predisposition genes (n = 17) were found in patients aged more than 40 years. If presumed germ line variants were included, the yield of P/LP variants would increase to 11%, and by adding suspicious variants of unknown significance, it would rise further to 12%. The high frequency of P/LP germ line variants in our study supports comprehensive germ line genetic testing for all patients with MDS regardless of their age at diagnosis.

Germline variants in RUNX1, GATA2 and DDX41 may confer a predisposition to hereditary haematopoietic malignancies (HHMs) such as MDS and AML yet have distinct age ranges of malignancy diagnosis and a highly variable overall risk for leukemogenesis. The increased awareness and identification of carriers of these germline variants, particularly before development of malignancy, has changed the way in which individuals and families need to be managed in the clinic. Individuals need lifelong monitoring and may also need modification to treatments when malignancy does develop, compared to sporadic counterparts. Gaps in understanding pre-malignant states in HHM syndromes have hampered efforts to design effective clinical surveillance regimes, provide personalized pre-emptive treatments, and appropriate counselling to patients.

Inherited myeloid malignancies are included as a provisional category in the current World Health Organization classification and within clinical testing guidelines of the National Comprehensive Cancer Network for myelodysplastic syndrome (MDS) and the European LeukemiaNet. The frequency of deleterious germline variants in MDS patients diagnosed at or younger than 40 years old ranges from 15-20%. To determine the frequency of germline predisposition in MDS patients of all ages, we accessed peripheral blood samples collected by the Center for International Blood and Marrow Transplant Research (CIBMTR) from all MDS patients who had undergone allogeneic hematopoietic stem cell transplantation (HSCT) using related donors (Table 1). We performed whole exome sequencing in 404 donor/recipient pairs augmented with spike-in probes covering non-coding regions known to contain inherited risk alleles. Single nucleotide variants (SNVs) and copy number variants (CNVs) were interpreted according to American College of Medical Genetics and Genomics (ACMG), Association for Molecular Pathology, and Clinical Genome Sequence Variant Interpretation Working Group guidelines. Germline status was confirmed by the presence of a variant in the MDS patient and related donor and for variants previously only seen as germline alleles with a variant allele frequency of 40-60%. We examined variants in 236 genes associated with hematopoietic malignancies, bone marrow failure syndromes, immunodeficiencies and congenital/acquired cytopenias. We identified pathogenic germline variants in 28 out of 404 MDS patients (7%): 23 with autosomal dominant and five with autosomal recessive inheritance. MDS patients with deleterious germline variants were found in all age deciles, from age 11 to 71 (Figure 2A), and were more likely to develop higher-grade MDS (43% vs. 25%, p=0.04). Deleterious variants in 5 bone marrow failure syndrome-related genes were found predominantly in younger people, whereas middle-aged and senior MDS patients had most often deleterious variants in DDX41 (n=4) and cancer predisposition genes (n= 16; Figure 2B). Seventy-one percent of MDS patients with deleterious autosomal dominant germline variants shared that variant with their related donor. However, there were no differences in HSCT outcomes for these individuals. The 7% overall frequency of deleterious germline variants is likely an underestimate, because we were constrained in calling germline variants as those detectable in both the MDS patient and related donor, since only peripheral blood samples were available from the CIBMTR. Thus, we expect that 50% of related donors would not carry the variant. Moreover, because some germline predisposition disorders lead to cytopenias or other clinical features, some relatives with those variants may have been excluded as HSCT donors. We also expect that some variants deemed variants of uncertain significance are pathogenic, but will require segregation or functional studies to upgrade them into deleterious categories. Finally, our HSCT outcome measurements are underpowered due to the small numbers of MDS patients with each disorder. Based on the ACMG's recommendation to test clinical germline predisposition when positive findings are anticipated at >5%, our data support comprehensive germline genetic testing for all MDS patients regardless of their age at diagnosis or family history. Testing should include analysis of both SNVs and CNVs in genes and non-coding regions known to contain cancer predisposition alleles. Our data also justify a larger study to evaluate clinical impact of pathogenic germline variants on transplant outcomes. Until we know the impact of deleterious germline variants on HSCT outcomes, we continue to recommend avoiding using HSCT donors with such variants whenever possible given the risk of poor graft function/failure and donor-derived leukemias in HSCT recipients. Figure 1 Figure 1. Disclosures Scott: Bristol Myers Squibb: Consultancy, Honoraria, Research Funding. Sobecks: CareDX: Membership on an entity's Board of Directors or advisory committees. Saber: Govt. COI: Other.

Nitrate removal from culture media is widely used to enhance autofermentative hydrogen production in cyanobacteria during dark anaerobiosis. Here we have performed a systematic inventory of carbon and nitrogen metabolites, redox pools, and excreted product fluxes which show that addition of nitrate to cultures of Synechococcus sp. PCC 7002 has no influence on glycogen catabolic rate, but shifts the distribution of excreted products from predominantly lactate and H 2 to predominantly CO 2 and nitrite, while increasing the total consumption of intracellular reducing equivalents (mainly glycogen) by 3-fold. Together with LC–MS derived metabolite pool sizes these data show that glycogen catabolism is redirected from the upper-glycolytic (EMP) pathway to the oxidative pentose phosphate (OPP) pathway upon nitrate addition. This metabolic switch in carbon catabolism is shown to temporally correlate with the pyridine nucleotide redox-poise (NAD(P)H/NAD(P) + ) and demonstrates the reductant availability controls H 2 evolution in cyanobacteria.

ADP-glucose pyrophosphorylase, encoded by glgC, catalyzes the first step of glycogen and glucosylglycer(ol/ate) biosynthesis. Here we report the construction of the first glgC null mutant of a marine cyanobacterium (Synechococcus sp. PCC 7002) and investigate its impact on dark anoxic metabolism (autofermentation). The glgC mutant had 98% lower ADP-glucose, synthesized no glycogen and produced appreciably more soluble sugars (mainly sucrose) than wild type (WT). Some glucosylglycerol was still observed, which suggests that the mutant has another, inefficient ADP-glucose synthesis pathway. In contrast, hypersaline conditions (1M NaCl) were lethal to the mutant strain, indicating that, unlike other strains, the elevated sucrose does not compensate for the reduced GG as osmolyte. In contrast to WT, nitrate limitation did not cause bleaching of N-containing pigments or carbohydrate accumulation in the glgC mutant, indicating impaired recycling of nitrogen stores. Despite the 2-fold increase in osmolytes, both the respiration and autofermentation rates of the glgC mutant were appreciably slower (2-4-fold) and correlated quantitatively with the lower fraction of insoluble carbohydrates relative to WT (85% vs. 12%). However, the remaining insoluble carbohydrates still accounted for a high fraction of the carbohydrate catabolized (38%), indicating that insoluble carbohydrates rather than osmolytes were the preferred substrate for autofermentation.

Some aquatic microbial oxygenic photoautotrophs (AMOPs) make hydrogen (H 2 ), a carbon-neutral, renewable product derived from water, in low yields during autofermentation (anaerobic metabolism) of intracellular carbohydrates previously stored during aerobic photosynthesis. We have constructed a mutant (the ldhA mutant) of the cyanobacterium Synechococcus sp. strain PCC 7002 lacking the enzyme for the NADH-dependent reduction of pyruvate to d -lactate, the major fermentative reductant sink in this AMOP. Both nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) metabolomic methods have shown that autofermentation by the ldhA mutant resulted in no d -lactate production and higher concentrations of excreted acetate, alanine, succinate, and hydrogen (up to 5-fold) compared to that by the wild type. The measured intracellular NAD(P)(H) concentrations demonstrated that the NAD(P)H/NAD(P) + ratio increased appreciably during autofermentation in the ldhA strain; we propose this to be the principal source of the observed increase in H 2 production via an NADH-dependent, bidirectional [NiFe] hydrogenase. Despite the elevated NAD(P)H/NAD(P) + ratio, no decrease was found in the rate of anaerobic conversion of stored carbohydrates. The measured energy conversion efficiency (ECE) from biomass (as glucose equivalents) converted to hydrogen in the ldhA mutant is 12%. Together with the unimpaired photoautotrophic growth of the ldhA mutant, these attributes reveal that metabolic engineering is an effective strategy to enhance H 2 production in AMOPs without compromising viability.

We highlight a range of cryoprobe-assisted NMR methods for studying metabolite production by cyanobacteria, which should be valuable for a wide range of biological applications requiring ultrasensitivity and precise concentration determination over a large dynamic range. Cyroprobe-assisted 1H and 13C NMR have been applied to precise determination of metabolic products excreted during autofermentation in two cyanobacterial species: filamentous Arthrospira (Spirulina) maxima CS-328 and unicellular Synechococcus sp. PCC 7002. Several fermentative end products were identified and quantified in concentrations ranging from 50 to 3000 µM in cell-free media (a direct measurement of native-like samples) with less than 5.5% relative error in under 10 min of acquisition per sample with the assistance of an efficient water-suppression protocol. Relaxation times (T1) of these metabolites in aqueous (1H2O) solution were measured and found to vary by nearly threefold, necessitating generation of individual calibration curves for each species for highest precision. However, using a 4.5 × longer overall recycle delay between scans, the metabolite concentrations can be predicted within 25% error by calibrating only to a single calibration standard (succinate); other metabolites are then calculated on the basis of their signal integrals and known proton degeneracies. Precise ratios of concentrations of 13C-labeled versus unlabeled metabolites were determined from integral ratios of 1H peaks that exhibit 13C1H J-couplings and independently confirmed by direct measurement of areas of corresponding 13C resonances. 13C NMR was used to identify and quantify production of osmolytes, trehalose, and glucosylglycerol by A. maxima. Copyright © 2009 John Wiley & Sons, Ltd.

The nifJ gene codes for pyruvate:ferredoxin oxidoreductase (PFOR), which reduces ferredoxin during fermentative catabolism of pyruvate to acetyl-coenzyme A (acetyl-CoA). A nifJ knockout mutant was constructed that lacks one of two pathways for the oxidation of pyruvate in the cyanobacterium Synechococcus sp. strain PCC 7002. Remarkably, the photoautotrophic growth rate of this mutant increased by 20% relative to the wild-type (WT) rate under conditions of light-dark cycling. This result is attributed to an increase in the quantum yield of photosystem II (PSII) charge separation as measured by photosynthetic electron turnover efficiency determined using fast-repetition-rate fluorometry ( F v / F m ). During autofermentation, the excretion of acetate and lactate products by nifJ mutant cells decreased 2-fold and 1.2-fold, respectively. Although nifJ cells displayed higher in vitro hydrogenase activity than WT cells, H 2 production in vivo was 1.3-fold lower than the WT level. Inhibition of acetate-CoA ligase and pyruvate dehydrogenase complex by glycerol eliminated acetate production, with a resulting loss of reductant and a 3-fold decrease in H 2 production by nifJ cells compared to WT cells. Continuous electrochemical detection of dissolved H 2 revealed two temporally resolved phases of H 2 production during autofermentation, a minor first phase and a major second phase. The first phase was attributed to reduction of ferredoxin, because its level decreased 2-fold in nifJ cells. The second phase was attributed to glycolytic NADH production and decreased 20% in nifJ cells. Measurement of the intracellular NADH/NAD + ratio revealed that the reductant generated by PFOR contributing to the first phase of H 2 production was not in equilibrium with bulk NADH/NAD + and that the second phase corresponded to the equilibrium NADH-mediated process.

We highlight a range of cryoprobe-assisted NMR methods for studying metabolite production by cyanobacteria, which should be valuable for a wide range of biological applications requiring ultrasensitivity and precise concentration determination over a large dynamic range. Cyroprobe-assisted (1)H and (13)C NMR have been applied to precise determination of metabolic products excreted during autofermentation in two cyanobacterial species: filamentous Arthrospira (Spirulina) maxima CS-328 and unicellular Synechococcus sp. PCC 7002. Several fermentative end products were identified and quantified in concentrations ranging from 50 to 3000 microM in cell-free media (a direct measurement of native-like samples) with less than 5.5% relative error in under 10 min of acquisition per sample with the assistance of an efficient water-suppression protocol. Relaxation times (T1) of these metabolites in aqueous ((1)H(2)O) solution were measured and found to vary by nearly threefold, necessitating generation of individual calibration curves for each species for highest precision. However, using a 4.5 x longer overall recycle delay between scans, the metabolite concentrations can be predicted within 25% error by calibrating only to a single calibration standard (succinate); other metabolites are then calculated on the basis of their signal integrals and known proton degeneracies. Precise ratios of concentrations of (13)C-labeled versus unlabeled metabolites were determined from integral ratios of (1)H peaks that exhibit (13)C-(1)H J-couplings and independently confirmed by direct measurement of areas of corresponding (13)C resonances. (13)C NMR was used to identify and quantify production of osmolytes, trehalose, and glucosylglycerol by A. maxima.