High burden of clonal hematopoiesis in first responders exposed to the World Trade Center disaster.

The terrorist attacks on the World Trade Center (WTC) created an unprecedented environmental exposure to aerosolized dust, gases and potential carcinogens. Clonal hematopoiesis (CH) is defined as the acquisition of somatic mutations in blood cells and is associated with smoking and exposure to genotoxic stimuli. Here we show that deep targeted sequencing of blood samples identified a significantly higher proportion of WTC-exposed first responders with CH (10%; 48 out of 481) when compared with non-WTC-exposed firefighters (6.7%; 17 out of 255; odds ratio, 3.14; 95% confidence interval, 1.64-6.03; P = 0.0006) after controlling for age, sex and race/ethnicity. The frequency of somatic mutations in WTC-exposed first responders showed an age-related increase and predominantly affected DNMT3A, TET2 and other CH-associated genes. Exposure of lymphoblastoid cells to WTC particulate matter led to dysregulation of DNA replication at common fragile sites in vitro. Moreover, mice treated with WTC particulate matter developed an increased burden of mutations in hematopoietic stem and progenitor cell compartments. In summary, the high burden of CH in WTC-exposed first responders provides a rationale for enhanced screening and preventative efforts in this population.

Photoaffinity labeling of the Plasmodium falciparum chloroquine resistance transporter with a novel perfluorophenylazido chloroquine.

Several models describing how amino acid substitutions in the Plasmodium falciparum chloroquine resistance transporter (PfCRT) confer resistance to chloroquine (CQ) and other antimalarial drugs have been proposed. Further progress requires molecular analysis of interactions between purified reconstituted PfCRT protein and these drugs. We have thus designed and synthesized several perfluorophenyl azido (pfpa) CQ analogues for PfCRT photolabeling studies. One particularly useful probe (AzBCQ) places the pfpa group at the terminal aliphatic N of CQ via a flexible four-carbon ester linker and includes a convenient biotin tag. This probe photolabels PfCRT in situ with high specificity. Using reconstituted proteoliposomes harboring partially purified recombinant PfCRT, we analyze AzBCQ photolabeling versus competition with CQ and other drugs to probe the nature of the CQ binding site. We also inspect how pH, the chemoreversal agent verapamil (VPL), and various amino acid mutations in PfCRT that cause CQ resistance (CQR) affect the efficiency of AzBCQ photolabeling. Upon gel isolation of AzBCQ-labeled PfCRT followed by trypsin digestion and mass spectrometry analysis, we are able to define a single AzBCQ covalent attachment site lying within the digestive vacuolar-disposed loop between putative helices 9 and 10 of PfCRT. Taken together, the data provide important new insight into PfCRT function and, along with previous results, allow us to propose a model for a single CQ binding site in the PfCRT protein.

A single S1034C mutation confers altered drug sensitivity to PfMDR1 ATPase activity that is characteristic of the 7G8 isoform.

The mechanism behind how PfMDR1 may contribute to antimalarial drug resistance is unclear. Transfection studies suggest that PfMDR1 mutations may make small contributions to drug sensitivity in a strain-dependent fashion, whereas field data link over expression (not necessarily mutation) of the gene with clinical drug treatment failure. This study dissects the contribution of individual mutations of PfMDR1 that contribute to the unique behavior of the 7G8 PfMDR1 isoform. A single mutation in putative TM 11 (S1034C) is found to abolish drug stimulation of PfMDR1 ATPase activity.

TRAPPC11 and GOSR2 mutations associate with hypoglycosylation of α-dystroglycan and muscular dystrophy.

BACKGROUND: Transport protein particle (TRAPP) is a supramolecular protein complex that functions in localizing proteins to the Golgi compartment. The TRAPPC11 subunit has been implicated in muscle disease by virtue of homozygous and compound heterozygous deleterious mutations being identified in individuals with limb girdle muscular dystrophy and congenital muscular dystrophy. It remains unclear how this protein leads to muscle disease. Furthermore, a role for this protein, or any other membrane trafficking protein, in the etiology of the dystroglycanopathy group of muscular dystrophies has yet to be found. Here, using a multidisciplinary approach including genetics, immunofluorescence, western blotting, and live cell analysis, we implicate both TRAPPC11 and another membrane trafficking protein, GOSR2, in α-dystroglycan hypoglycosylation. CASE PRESENTATION: Subject 1 presented with severe epileptic episodes and subsequent developmental deterioration. Upon clinical evaluation she was found to have brain, eye, and liver abnormalities. Her serum aminotransferases and creatine kinase were abnormally high. Subjects 2 and 3 are siblings from a family unrelated to subject 1. Both siblings displayed hypotonia, muscle weakness, low muscle bulk, and elevated creatine kinase levels. Subject 3 also developed a seizure disorder. Muscle biopsies from subjects 1 and 3 were severely dystrophic with abnormal immunofluorescence and western blotting indicative of α-dystroglycan hypoglycosylation. Compound heterozygous mutations in TRAPPC11 were identified in subject 1: c.851A>C and c.965+5G>T. Cellular biological analyses on fibroblasts confirmed abnormal membrane trafficking. Subject 3 was found to have compound heterozygous mutations in GOSR2: c.430G>T and c.2T>G. Cellular biological analyses on fibroblasts from subject 3 using two different model cargo proteins did not reveal defects in protein transport. No mutations were found in any of the genes currently known to cause dystroglycanopathy in either individual. CONCLUSION: Recessive mutations in TRAPPC11 and GOSR2 are associated with congenital muscular dystrophy and hypoglycosylation of α-dystroglycan. This is the first report linking membrane trafficking proteins to dystroglycanopathy and suggests that these genes should be considered in the diagnostic evaluation of patients with congenital muscular dystrophy and dystroglycanopathy.

Leukemic Transdifferentiation of Follicular Lymphoma Into an Acute Histiocytic Leukemia in a 52-Year-Old Caucasian Woman.

We report an instructive case of acute myeloid leukemia with histiocytic differentiation (acute histiocytic leukemia) arising in a patient, a 52-year-old woman with a history of follicular lymphoma. The results of genetic studies proved a clonal relationship between the lymphoma and the leukemic cells. To our knowledge, this is the first report of leukemic transdifferentiation of follicular lymphoma into modified base 5-methylcytosine (M(5)c)-like acute histiocytic leukemia and the first reported karyotype on a transdifferentiated neoplasm.

Purified Plasmodium falciparum multi-drug resistance protein (PfMDR 1) binds a high affinity chloroquine analogue.

We utilize the recent successful overexpression of recombinant Plasmodium falciparum multi-drug resistance transporter, purification and reconstitution of the protein, and a novel high affinity chloroquine analogue to probe hypothesized interaction between the transporter and quinoline drugs. Results suggest that PfMDR1 binding sites for chloroquine, mefloquine, and quinine overlap, that P. falciparum chloroquine resistance transporter has intrinsically higher affinity for chloroquine relative to P. falciparum multi-drug resistance transporter, and that there is an isoform specific competition between the two transporters for binding of quinoline antimalarial drugs.

Heterologous expression and ATPase activity of mutant versus wild type PfMDR1 protein.

Mutation of the P. falciparum chloroquine resistance transporter (PfCRT) causes resistance to chloroquine (CQ) and other antimalarial drugs. Mutation and/or overexpression of one of the multidrug resistance protein homologues found in this malarial parasite (PfMDR1) may further modify or tailor the degree of multidrug resistance. However, considerable controversy surrounds the precise contribution of PfMDR1, in part because no direct biochemical studies of PfMDR1 have yet been possible. Using codon optimization and other principles, we have designed and constructed a yeast optimized version of the wild type pfmdr1 gene and have successfully overexpressed PfMDR1 protein in P. pastoris yeast. The protein is well expressed in either full length form or as two separate half transporters, is well localized to the yeast plasma membrane and is fully functional as evidenced by ATPase activity measurements. We have also expressed mutants that have previously been hypothesized to influence drug resistance in parasites. Using purified plasma membrane fractions, we have analyzed antimalarial drug effects on ATPase activity for wild type versus mutant proteins. Relative to other ABCB transporters involved in drug resistance, PfMDR1 is unusual. It has similar pH, [ATP], and Mg++ dependencies for ATP hydrolysis, yet relatively high Km and Vmax values for ATP hydrolysis, and ATPase activity is only mildly stimulated by antimalarial drugs. The largest measured drug effect is for CQ (to which PfMDR1 is not believed to confer resistance), and it is strongly inhibitory for WT PfMDR1. Drug resistance associated PfMDR1 mutants show either elevated (Dd2 allele encoded) or reduced (7G8 allele) basal ATPase activity and different patterns of drug stimulation or inhibition, relative to WT PfMDR1. The Dd2 PfMDR1 isoform also shows a slightly more alkaline pH optimum. Surprisingly, verapamil alone (1-300 microM) does not significantly affect WT ATPase activity but inhibits the Dd2 isoform at 1 microM. These data should assist ongoing analysis of the contribution of PfMDR1 to antimalarial drug resistance.