Heterozygous Deficiency Research Articles

Quantitative fluorescence measures for determination of intracellular perforin content.

We present methodologic details and operating characteristics of a procedure with whole blood for the quantitative assessment of intracellular perforin within distinct lymphocyte subsets. Using this method, we analyzed 20 healthy controls and 2 individuals with an inherited deficiency of perforin. The mean +/- standard deviation perforin contents of natural killer (NK) cells and cytotoxic T cells of healthy controls were 3561 +/- 1157 and 500 +/- 779 relative number of molecules (rMol) of antiperforin antibody bound per cell, respectively. The NK cell perforin contents of individuals with heterozygous and homozygous perforin deficiency (familial hemophagocytic lymphohistiocytosis) were 2260 and 212 rMol of antiperforin antibodies per NK cell. While the homozygous deficiency was found to be associated with negligible antiperforin binding, the heterozygous condition was associated with a level of perforin binding that was below the 15th percentile for healthy individuals. Because 83% of this subject's NK cells were shown to bind to antiperforin antibodies by conventional flow cytometry (relative to the normal range of 81% +/- 25%), quantitative cytometry may be more sensitive than conventional cytometric methods in identifying cytolytic defects.

Remission of thrombotic thrombocytopenic purpura in a patient with compound heterozygous deficiency of von Willebrand factor‐cleaving protease by infusion of solvent/detergent plasma

Plasma exchange or plasma infusion is considered to be the therapy of choice in patients with thrombotic thrombocytopenic purpura (TTP) who are deficient in von Willebrand factor‐cleaving protease (VWF‐CP). Recently, mutations in the ADAMTS13 gene were identified as being responsible for VWF‐CP deficiency in patients with familial TTP (VWF‐CP deficiency in the absence of an inhibitor). Here we report on a girl who presented with recurrent thrombocytopenia and anaemia since birth, developing the full pentad of characteristic TTP at the age of 16y. Congenital TTP was confirmed on the basis of severe VWF‐CP deficiency in the absence of an acquired inhibitor. The patient was found to be compound heterozygous for two hitherto undescribed mutations in the ADAMTS13 gene: a truncating frame shift mutation, 4143insA in exon 29, and the nonsense mutation 3100A > T in exon 24 (R1034X). After infusion of solvent/detergent plasma, the patient went into remission and remained asymptomatic under regular plasma therapy at 2‐wk intervals for over two years. Conclusion: TTP in childhood may be mild and oligosymptomatic. Determination of VWF‐CP activity is helpful in the differential diagnosis of thrombocytopenia.

A strong candidate gene for the Papg1 locus on mouse chromosome 4 affecting lung tumor progression.

Lung cancer is the leading cause of cancer death among both men and women, accounting for more than 28% of all cancer deaths. In fact, more people die of lung cancer than of colon, breast, and prostate cancers combined. Although lung cancer is largely induced by smoking, there is strong evidence for genetic susceptibility and gene-environment interactions in the development of lung cancer. Inbred mouse models offer an effective means of identifying candidate lung cancer susceptibility loci since genetic heterogeneity and enormous variation in exposure levels to environmental agents make it difficult to identify lung cancer susceptibility loci in humans. Papg-1 (pulmonary adenoma progression 1) was previously mapped to a region on mouse chromosome 4. This locus contains a candidate gene, Cdkn2a also referred to as Ink4a/Arf, which dually encodes two established tumor suppressors p16(INK4a) and ARF. Cdkn2a became a primary candidate for Papg-1 for two reasons: (1) two haplotypes of mouse Cdkn2a were found to segregate with differential genetic susceptibility to lung tumor progression in mice; and (2) in vitro studies showed that the p16(INK4a) allele from the BALB/cJ mouse had a significantly decreased ability to bind and inhibit CDK6 and to suppress cell growth when compared with the p16(INK4a) allele from the A/J mouse. Here, we report that mice with a heterozygous deficiency for the A/J Cdkn2a allele were significantly more susceptible to lung tumor progression than mice with a heterozygous deficiency for a BALB/cJ Cdkn2a allele, when compared to their respective wild type mice. These results offer strong evidence that naturally occurring variation of p16(INK4a) influences susceptibility to enhance lung tumor progression making it a strong candidate for the lung tumor progression locus, Papg-1.

Heterozygous cellular glutathione peroxidase deficiency in the mouse: abnormalities in vascular and cardiac function and structure.

Oxidant stress has been implicated in the pathogenesis of atherothrombosis and other vascular disorders accompanied by endothelial dysfunction. Glutathione peroxidases (GPx) play an important role in the cellular defense against oxidant stress by utilizing glutathione (GSH) to reduce lipid hydroperoxides and hydrogen peroxide to their corresponding alcohols. Cellular GPx (GPx-1) is the principal intracellular isoform of GPx. We hypothesized that GPx-1 deficiency per se induces endothelial dysfunction and structural vascular abnormalities through increased oxidant stress. A murine model of heterozygous deficiency of GPx-1 (GPx(+/-)) was investigated to examine this hypothesis. Mesenteric arterioles in GPx-1(+/-) mice demonstrated vasoconstriction to acetylcholine compared with vasodilation in wild-type mice (maximal change in vessel diameter, -13.0+/-2.8% versus 13.2+/-2.8%, P<0.0001). We also noted an increase in the plasma and aortic levels of the isoprostane iPF(2alpha)-III, a marker of oxidant stress, in GPx-1(+/-) mice compared with wild-type mice (170.4+/-23 pg/mL plasma versus 98.7+/-7.1 pg/mL plasma, P<0.03; 11.7+/-0.87 pg/mg aortic tissue versus 8.2+/-0.55 pg/mg aortic tissue, P<0.01). Histological sections from the coronary vasculature of GPx-1(+/-) mice show increased perivascular matrix deposition, an increase in the number of adventitial fibroblasts, and intimal thickening. These structural abnormalities in the myocardial vasculature were accompanied by diastolic dysfunction after ischemia-reperfusion. These findings demonstrate that heterozygous deficiency of GPx-1 leads to endothelial dysfunction, possibly associated with increased oxidant stress, and to significant structural vascular and cardiac abnormalities. These data illustrate the importance of this key antioxidant enzyme in functional and structural responses of the mammalian cardiovascular system.

Anesthetic Considerations for a Patient with Compound Heterozygous Medium-Chain Acyl-CoA Dehydrogenase Deficiency

Anesthetic Considerations for a Patient with Compound Heterozygous Medium-Chain Acyl-CoA Dehydrogenase Deficiency

Lung function of school children with low levels of alpha1-antitrypsin and tobacco smoke exposure.

Exposure to environmental tobacco smoke (ETS) and other air pollutants has been associated with small decrements in lung function. The susceptibility to pollution exposure may, however, vary substantially between individuals. Children with an impaired protease-antiprotease balance may be particularly vulnerable. Therefore this study aimed to investigate the effects of ETS exposure on children with reduced levels of alpha1-antitrypsin (alpha1-AT). Random samples of school children (aged 9-11 yrs) (n=3,526) were studied according to the International Study of Asthma and Allergies in Childhood (ISAAC) phase II protocol, including parental questionnaires, pulmonary function and allergy testing. Blood samples were obtained to measure plasma levels of alpha1-AT and to genotype for pleomorphic protein inhibitor (Pi)Z and PiS alleles. Children with low levels of alpha1-AT (< or = 116 mg x dL(-1)) showed significant, albeit small decrements in baseline lung function. When exposed to ETS, pronounced decrements of pulmonary function, particularly in measures of mid- to end-expiratory flow rates, were seen in these children as compared to exposed children with normal levels of alpha1-AT. The mean levels of % predicted+/-SE in both groups were: maximum expiratory flow at 50% of vital capacity 79.4+/-7.2 versus 99.0+/-1.5, maximum expiratory flow at 25% of vital capacity 67.4+/-10.0 versus 100.3+/-2.1, maximal midexpiratory flow 73.7+/-8.6 versus 99.9+/-1.7. These findings suggest that school children with low levels of alpha1-antitrypsin are at risk of developing pronounced decrements in pulmonary function, particularly if they are exposed to environmental tobacco smoke. Parents of children with heterozygous alpha1-antitrypsin deficiency resulting in significantly reduced blood concentrations should be advised to prevent their children from being exposed to environmental tobacco smoke and dissuade them from taking up smoking.

Impact of antithrombin deficiency in thrombogenesis: lipopolysaccharide and stress-induced thrombus formation in heterozygous antithrombin-deficient mice

Antithrombin (AT) deficiency is an autosomal disorder associated with venous thromboembolism. However, a diagnosis of homozygous AT deficiency is seldom made. Most patients are heterozygous and have approximately 50% AT activities, and they are at higher risk for the development of thromboembolism. Through gene targeting we generated AT-deficient mice and previously reported that completely AT-deficient mice could not survive the prenatal period because of extensive thrombosis in the myocardium and liver sinusoids. In contrast, heterozygous AT-deficient mice with 50% AT activities have not shown spontaneous thromboembolic episodes. To demonstrate a thrombotic tendency in heterozygous AT deficiency, we challenged heterozygous AT-deficient mice (AT+/- mice) with the administration of lipopolysaccharide (LPS) or with restraint stress by immobilization. LPS injection markedly induced fibrin deposition in the kidney glomeruli, myocardium, and liver sinusoids in AT+/- mice compared with wild-type mice (AT+/+ mice). Restraint stress tests were performed by placing mice in 50-mL conical centrifuge tubes for 20 hours. Fibrin deposition was observed in the kidney of AT+/+ and AT+/- mice, but AT+/- mice exhibited more extensive fibrin deposition than AT+/+ mice. After prophylactic administration of human AT concentrates to increase plasma AT activities of AT+/- mice, LPS-induced fibrin deposition was effectively prevented. These results suggest that heterozygous AT deficiency is significantly associated with a tendency toward thrombosis formation in the kidney. The AT+/- mouse thus is a useful model for studying the effect of environmental or genetic risk factors on thrombogenesis.

Assay procedure optimization of a rapid, reusable protein C immunosensor for physiological samples.

A fiber-optic immunosensor for quantifying the protein C (PC) amount in a plasma sample is being developed to provide accurate, fast, cost-effective diagnosis of heterozygous PC deficiency (0.5-2.5 microg ml(-1)). As a progress report on the sensor development, this paper focuses on optimizations of vanous assay steps. These include: (1) the primary antibody concentration; (2) the effect of the primary antibody leaching on the sensitivity; (3) the fluorophore to secondary antibody ratio; and (4) the sample and secondary antibody incubation times. The optimal primary antibody concentration for the PC sensor was determined to be 65 microg ml(-1); its leaching was minor, stabilized within 3 days, and the sensor sensitivity change after 30 days of storage was minor; sample and secondary antibody incubation times were reduced from 10 to 5 and from 5 to 3 min, respectively, while maintaining a reasonable signal-to-noise level. The immunosensor was also tested with (5) human serum albumin and human plasma and (6) with and without convective flow. High viscosity of human plasma decreased signal intensity, but clear signal discrimination was possible in the concentration range of interest. (7) Application of convective flow increased the signal intensity by increasing PC mass transport rate to the sensor surface. The optimized PC immunosensor provides a smaller (100 microl sample chamber) and faster (10-15 min) tool for PC detection in human plasma.

Putative tumor-suppressor gene regions responsible for radiation lymphomagenesis in F1 mice with different p53 status.

Regions of allelic loss on chromosomes in many tumors of human and some experimental animals are generally considered to harbor tumor-suppressor genes involved in tumorigenesis. Allelotype analyses have greatly improved our understanding of the molecular mechanism of radiation lymphomagenesis. Previously, we and others found frequent loss of heterozygosity (LOH) on chromosomes 4, 11, 12, 16 and 19 in radiation-induced lymphomas from several F1 hybrid mice. To examine possible contributions of individual tumor-suppressor genes to tumorigenesis in p53 heterozygous deficiency, we investigated the genome-wide distribution and status of LOH in radiation-induced lymphomas from F1 mice with different p53 status. In this study, we found frequent LOH (more than 20%) on chromosomes 4 and 12 and on chromosomes 11, 12, 16 and 19 in radiation-induced lymphomas from (STS/A X MSM/Ms)F1 mice and (STS/A X MSM/Ms)F1-p53KO/+ mice, respectively. Low incidences of LOH (10-20%) were also observed on chromosomes 11 in mice with wild-type p53, and chromosomes 1, 2, 9, 17 and X in p53 heterozygous-deficient mice. The frequency of LOH on chromosomes 9 and 11 increased in the (STS/A X MSM/Ms)F1-p53KO/+ mice. Preferential losses of the STS-derived allele on chromosome 9 and wild-type p53 allele on chromosome 11 were also found in the p53 heterozygous-deficient mice. Thus, the putative tumor-suppressor gene regions responsible for lymphomaganesis might considerably differ due to the p53 status.

The responses to low dose azathioprine in patients with heterozygous TPMT deficiency

The responses to low dose azathioprine in patients with heterozygous TPMT deficiency

Regulation of the hypothalamic-pituitary-adrenocortical system in mice deficient for CRH receptors 1 and 2.

Recent investigations in mouse lines either deficient for the CRH receptor 1 (CRHR1) or 2 (CRHR2) suggest that the CRH neuronal system may comprise two separate pathways that can be coordinately and inversely activated in stress-induced hypothalamic-pituitary-adrenal (HPA) response and anxiety-like behavior. We generated mice deficient for both CRHR1 (Crhr1(-/-)) and CRHR2 (Crhr2(-/-)) to investigate the HPA system regulation in the absence of known functionally active CRH receptors under basal conditions and in response to different ethologically relevant stressors. To elucidate possible gene dose effects on the action of both CRH receptors, our analysis included heterozygous and homozygous CRHR1- or CRHR2-deficient mice, mutants lacking both CRH receptors, compound mutants with homozygous and heterozygous deficiency for either of the receptors, and their wild-type littermates. Both male and female Crhr1(-/-)Crhr2(-/-) mutants were viable, fertile, and indistinguishable in size from wild-type littermates. We show that the endocrine phenotype of mice lacking both CRHRs is dominated by the functional loss of CRHR1. CRHR2 does not compensate for CRHR1 deficiency, nor does the lack of CRHR2 exacerbate the CRHR1-dependent impairment of the HPA system function. Within the intraadrenal CRH/ACTH system, our data suggest different roles for CRHR1 and CRHR2 in fine-tuning of adrenocortical corticosterone release.

A family-based study of hyperinsulinemia and hypertriglyceridemia in heterozygous lipoprotein lipase deficiency

Case report: A case is presented of predisposing a patient's father with obligate heterozygous lipoprotein lipase (LPL) deficiency to mild hypertriglyceridemia in Japanese I-family members ( n=8) with patient DI, who was a compound heterozygote for a novel missense mutation of G154V (G G 716C→G T C/Gly 154 Val) in exon 5 and a novel splice mutation (Int8/5′-dss/t(+2)c; a T-to-C transition in the invariant GT at position +2 of the 5′ donor splice site (dss)) in intron 8 of the LPL gene. Results: The patient's father and paternal grandmother were heterozygotes for the Int8/5′-dss/t(+2)c allele, while the patient's mother and maternal grandmother were heterozygotes for the G154V allele. These four heterozygous carriers with one defective LPL allele showed 45–57% of the mean LPL activity and mass in the post-heparin plasma (PHP) observed in normal individuals. Among the four heterozygous carriers, the patient's father, who was <40 years old, nonobese and hyperinsulinemia, manifested mild hypertriglyceridemia (type IV hyperlipoproteinemia). The remaining three healthy heterozygous carriers (two were >40 years old and the other was <40 years old) were all normolipidemic state. Conclusion: In this family, hyperinsulinemia as a marker of insulin resistance may be a strong determinant of hypertriglyceridemia in the carrier with heterozygous LPL deficiency.

Leptin does not play a major role in platelet aggregation in obesity and leptin deficiency.

A recent study suggested that high concentrations of leptin enhance platelet aggregations. Therefore, the aim of this study was to investigate whether platelet aggregation is altered in patients with leptin gene mutations compared with obese subjects or controls. Four men (one homozygous man and his three heterozygous brothers) carrying a leptin gene mutation; 20 age-matched, healthy, unrelated men; and 18 age-matched obese men were enrolled in the study. Adenosine diphosphate (ADP)-, collagen-, and epinephrine-induced platelet aggregation were evaluated in all individuals. Our results show that patients with the leptin gene mutation (both the homozygous and heterozygous patients) had significantly higher ADP-induced (78.3 +/- 3.4% vs. 57.9 +/- 9.3%, p = 0.001), collagen-induced (78.1 +/- 2.9% vs. 56.7 +/- 9.3%, p = 0.007), and epinephrine-induced (76.5 +/- 9.2% vs. 59.5 +/- 7.70%, p = 0.003) platelet aggregation compared with controls. However, ADP-, collagen-, or epinephrine-induced platelet aggregations were similar to those in obese patients. Platelet aggregation responses to a combination of pretreatment with leptin at concentrations of 20, 50, 100, or 500 ng/mL for 5 minutes and ADP at concentrations of 2 micromol/liter also were evaluated. However, we did not find significant increases in platelet aggregation even at high concentrations of leptin (100 or 500 ng/mL) in leptin-deficient patients, obese subjects, or controls. Our data show that similar to findings in obese humans, homozygous or heterozygous leptin deficiency is associated with increased platelet aggregation compared with controls, and that higher concentrations of leptin do not increase platelet aggregation.

The mechanisms by which both heterozygous peroxisome proliferator-activated receptor gamma (PPARgamma) deficiency and PPARgamma agonist improve insulin resistance.

Peroxisome proliferator-activated receptor (PPAR) gamma is a ligand-activated transcription factor and a member of the nuclear hormone receptor superfamily that is thought to be the master regulator of fat storage; however, the relationship between PPARgamma and insulin sensitivity is highly controversial. We show here that supraphysiological activation of PPARgamma by PPARgamma agonist thiazolidinediones (TZD) markedly increases triglyceride (TG) content of white adipose tissue (WAT), thereby decreasing TG content of liver and muscle, leading to amelioration of insulin resistance at the expense of obesity. Moderate reduction of PPARgamma activity by heterozygous PPARgamma deficiency decreases TG content of WAT, skeletal muscle, and liver due to increased leptin expression and increase in fatty acid combustion and decrease in lipogenesis, thereby ameliorating high fat diet-induced obesity and insulin resistance. Moreover, although heterozygous PPARgamma deficiency and TZD have opposite effects on total WAT mass, heterozygous PPARgamma deficiency decreases lipogenesis in WAT, whereas TZD stimulate adipocyte differentiation and apoptosis, thereby both preventing adipocyte hypertrophy, which is associated with alleviation of insulin resistance presumably due to decreases in free fatty acids, and tumor necrosis factor alpha, and up-regulation of adiponectin, at least in part. We conclude that, although by different mechanisms, both heterozygous PPARgamma deficiency and PPARgamma agonist improve insulin resistance, which is associated with decreased TG content of muscle/liver and prevention of adipocyte hypertrophy.

Wildtype Kras2 can inhibit lung carcinogenesis in mice.

Although the ras genes have long been established as proto-oncogenes, the dominant role of activated ras in cell transformation has been questioned. Previous studies have shown frequent loss of the wildtype Kras2 allele in both mouse and human lung adenocarcinomas. To address the possible tumor suppressor role of wildtype Kras2 in lung tumorigenesis, we have carried out a lung tumor bioassay in heterozygous Kras2-deficient mice. Mice with a heterozygous Kras2 deficiency were highly susceptible to the chemical induction of lung tumors when compared to wildtype mice. Activating Kras2 mutations were detected in all chemically induced lung tumors obtained from both wildtype and heterozygous Kras2-deficient mice. Furthermore, wildtype Kras2 inhibited colony formation and tumor development by transformed NIH/3T3 cells and a mouse lung tumor cell line containing an activated Kras2 allele. Allelic loss of wildtype Kras2 was found in 67% to 100% of chemically induced mouse lung adenocarcinomas that harbor a mutant Kras2 allele. Finally, an inverse correlation between the level of wildtype Kras2 expression and extracellular signal-regulated kinase (ERK) activity was observed in these cells. These data strongly suggest that wildtype Kras2 has tumor suppressor activity and is frequently lost during lung tumor progression.

Deficiency of tissue factor pathway inhibitor promotes atherosclerosis and thrombosis in mice.

Tissue factor initiates blood coagulation after atherosclerotic plaque disruption. Tissue factor pathway inhibitor (TFPI) inhibits tissue factor activity and may reduce thrombus formation in this setting. We evaluated the effect of heterozygous TFPI deficiency on the development of atherosclerosis and thrombosis in atherosclerosis-prone mice. Mice with a combined heterozygous TFPI deficiency and homozygous apolipoprotein E deficiency (TFPI(+/-)/apoE(-/-)) were generated by crossbreeding, and they were analyzed for atherosclerosis throughout the vascular tree. Compared with mice with a normal TFPI genotype (TFPI(+/+)/apoE(-/-)), mice with a TFPI deficiency exhibited a greater atherosclerotic burden involving the carotid and common iliac arteries. Staining for active tissue factor within the plaque revealed more activity in TFPI(+/-)/apoE(-/-) mice compared with TFPI(+/+)/apoE(-/-) mice. Consistent with increased plaque tissue factor activity, the time to occlusive thrombosis after photochemical carotid plaque injury was significantly decreased in TFPI(+/-)/apoE(-/-) mice. These observations indicate that TFPI protects from atherosclerosis and is an important regulator of the thrombosis that occurs in the setting of atherosclerosis.

Responses to low dose azathioprine in patients with heterozygous thiopurine methyl transferase deficiency

Responses to low dose azathioprine in patients with heterozygous thiopurine methyl transferase deficiency

Responses to low dose azathioprine in patients with heterozygous thiopurine methyl transferase deficiency

Responses to low dose azathioprine in patients with heterozygous thiopurine methyl transferase deficiency

Rearrangements in the salivary gland chromosomes ofChironomus ripariusMg. (Diptera, Chironomidae) following exposure to lead

Abstract The effect of lead (Pb) nitrate on the structural and functional organization of the polytene chromosomes of Chironomus riparius larvae was examined. No specimens with standard polytene chromosomes were found in the material treated with different concentrations of Pb ions. The polytene chromosomes of all individuals exposed to Pb showed various somatic chromosome rearrangements (heterozygous inversions, deletions, duplications and deficiencies), which were not detected in the studied controls. Deletions in chromosome G occurred in a high frequency resulting in the formation of so called “pompons”. The activity of Balbiani rings (BRs) and nucleolar organizer (NOR) significantly decreased with increasing the concentrations of Pb ions. The number of BRc/BRb showing little or no activity (-/-) increased following exposure of the F2 generation to low and high concentrations of Pb ions. Sections of the polytenechromosomes where somatic aberrations were concentrated are considered as “weak” sites. It w...

Gene targeting in hemostasis. tissue factor pathway inhibitor.

Tissue Factor Pathway Inhibitor (TFPI) is a serine protease inhibitor of the Factor VIIa/Tissue Factor (FVIIa/TF)-initiated clotting cascade. Mice expressing a mutant form of TFPI, in which its Kunitz-1 domain has been deleted (TFPIKu1delta/delta), die prematurely in embryogenesis between E9.5dpc and birth. These results provide a rationale for the absence of TFPI-deficient patients. This early mortality can be ameliorated by an accompanying heterozygous or homozygous deficiency in FVII. Thus, diminishment of FVII activity precludes the requirement for TFPI-mediated inhibition of the FVIIa/TF pathway during embryogenesis.