Karen Crain

Zinc-finger Nuclease Editing of Human cxcr4 Promotes HIV-1 CD4+ T Cell Resistance and Enrichment

HIV-1-infected individuals can harbor viral isolates that can use CCR5, as well as CXCR4, for viral entry. To genetically engineer HIV-1 resistance in CD4(+) T cells, we assessed whether transient, adenovirus delivered zinc-finger nuclease (ZFN) disruption of genomic cxcr4 or stable lentiviral expression of short hairpin RNAs (shRNAs) targeting CXCR4 mRNAs provides durable resistance to HIV-1 challenge. ZFN-modification of cxcr4 in CD4(+) T cells was found to be superior to cell integrated lentivirus-expressing CXCR4 targeting shRNAs when CD4(+) T cells were challenged with HIV-1s that utilizes CXCR4 for entry. Cxcr4 disruption in CD4(+) T cells was found to be stable, conferred resistance, and provided for continued cell enrichment during HIV-1 infection in tissue culture and, in vivo, in peripheral blood mononuclear cell transplanted NSG mice. Moreover, HIV-1-infected mice with engrafted cxcr4 ZFN-modified CD4(+) T cells demonstrated lower viral levels in contrast to mice engrafted with unmodified CD4(+) T cells. These findings provide evidence that ZFN-mediated disruption of cxcr4 provides a selective advantage to CD4(+) T cells during HIV-1 infection.HIV-1-infected individuals can harbor viral isolates that can use CCR5, as well as CXCR4, for viral entry. To genetically engineer HIV-1 resistance in CD4+ T cells, we assessed whether transient, adenovirus delivered zinc-finger nuclease (ZFN) disruption of genomic cxcr4 or stable lentiviral expression of short hairpin RNAs (shRNAs) targeting CXCR4 mRNAs provides durable resistance to HIV-1 challenge. ZFN-modification of cxcr4 in CD4+ T cells was found to be superior to cell integrated lentivirus-expressing CXCR4 targeting shRNAs when CD4+ T cells were challenged with HIV-1s that utilizes CXCR4 for entry. Cxcr4 disruption in CD4+ T cells was found to be stable, conferred resistance, and provided for continued cell enrichment during HIV-1 infection in tissue culture and, in vivo, in peripheral blood mononuclear cell transplanted NSG mice. Moreover, HIV-1-infected mice with engrafted cxcr4 ZFN-modified CD4+ T cells demonstrated lower viral levels in contrast to mice engrafted with unmodified CD4+ T cells. These findings provide evidence that ZFN-mediated disruption of cxcr4 provides a selective advantage to CD4+ T cells during HIV-1 infection.

Polymorphisms and mutations of human TMPRSS6 in iron deficiency anemia.

Male subjects with iron deficiency from the general population were examined for polymorphisms or sporadic mutations in TMPRSS6 to identify genetic risk factors for iron deficiency anemia. Three uncommon non-synonymous polymorphisms were identified, G228D, R446W, and V795I (allele frequencies 0.0074, 0.023 and 0.0074 respectively), of which the R446W polymorphism appeared to be overrepresented in the anemic population. In addition, three children with iron refractory iron deficiency anemia, and one sibling with iron responsive iron deficiency anemia were also examined for polymorphisms or sporadic mutations in TMPRSS6. Two children (family 1) were compound heterozygotes for a L674F mutation and a previously described splicing defect predicted to cause skipping of exon 13 (IVS13+1 G>A). One child from the second family was homozygous for a deletion (497T) causing a frameshift (L166X+36) and premature termination. The sibling and mother from the second family were compound heterozygotes for the L166X mutation and the uncommon R446W polymorphism. Although in vitro expression studies demonstrated that the R446W isoform was biologically similar to wildtype Tmprss6, clinical data indicate that the R446W produces a milder disease when carried in trans with severe mutation in Tmprss6. The four children carrying mutations in TMPRSS6 all exhibited inappropriately high urinary hepcidin levels for the degree of iron deficiency.

A New Case of Human Atransferrinemia with a Previously Undescribed Mutation in the Transferrin Gene

Hereditary atransferrinemia is a very rare disorder characterized by microcytic anemia and iron overload. It has been reported in only 10 patients in 8 families. The molecular basis of atransferrinemia has been determined in only 3 human cases. We now report a new patient with this rare disorder, who is the first known case in Turkey, the 11th patient reported in the published literature and only the 4th case of human atransferrinemia characterized on a molecular basis. DNA analysis of the serum transferrin gene in the patient revealed a previously undescribed mutation in exon 4, a G→A transition at cDNA 410(Cys137Tyr). A number of previously known polymorphisms and a previously undescribed mutation at IVS10(–23)C→T, presumably a polymorphism, were also documented.

Genetic polymorphisms and susceptibility to lung disease

Susceptibility to infection by bacterium such as Bacillus anthracis has a genetic basis in mice and may also have a genetic basis in humans. In the limited human cases of inhalation anthrax, studies suggest that not all individuals exposed to anthrax spores were infected, but rather, individuals with underlying lung disease, particularly asthma, sarcoidosis and tuberculosis, might be more susceptible. In this study, we determined if polymorphisms in genes important in innate immunity are associated with increased susceptibility to infectious and non-infectious lung diseases, particularly tuberculosis and sarcoidosis, respectively, and therefore might be a risk factor for inhalation anthrax. Examination of 45 non-synonymous polymorphisms in ten genes: p47phox (NCF1), p67phox (NCF2), p40phox (NCF4), p22phox (CYBA), gp91phox (CYBB), DUOX1, DUOX2, TLR2, TLR9 and alpha 1-antitrypsin (AAT) in a cohort of 95 lung disease individuals and 95 control individuals did not show an association of these polymorphisms with increased susceptibility to lung disease.

Relative red blood cell enzyme levels as a clue to the diagnosis of pyruvate kinase deficiency

Evaluation of two patients with transfusion dependent anemia revealed RBC pyruvate kinase to be 33% and 41% of the mean normal value, with normal or high values of other RBC enzymes. Parental PK activities were just below normal in three of four of the parents. Subsequent DNA analysis revealed both patients to be compound heterozygotes for PKLR gene mutations, two of which are previously undescribed. Borderline low pyruvate kinase activities with increased in other RBC enzyme activities should prompt consideration of measurement of parental enzyme activities, and confirmation by DNA analysis if available. Pediatr Blood Cancer 2008;51:819–821.