Cristina Barlassina

Loss of Mismatched HLA in Leukemia after Stem-Cell Transplantation

BACKGROUND Transplantation of hematopoietic stem cells from partially matched family donors is a promising therapy for patients who have a hematologic cancer and are at high risk for relapse. The donor T-cell infusions associated with such transplantation can promote post-transplantation immune reconstitution and control residual disease. METHODS We identified 43 patients who underwent haploidentical transplantation and infusion of donor T cells for acute myeloid leukemia or myelodysplastic syndrome and conducted post-transplantation studies that included morphologic examination of bone marrow, assessment of hematopoietic chimerism with the use of short-tandem-repeat amplification, and HLA typing. The genomic rearrangements in mutant variants of leukemia were studied with the use of genomic HLA typing, microsatellite mapping, and single-nucleotide-polymorphism arrays. The post-transplantation immune responses against the original cells and the mutated leukemic cells were analyzed with the use of mixed lymphocyte cultures. RESULTS In 5 of 17 patients with leukemia relapse after haploidentical transplantation and infusion of donor T cells, we identified mutant variants of the original leukemic cells. In the mutant leukemic cells, the HLA haplotype that differed from the donors haplotype had been lost because of acquired uniparental disomy of chromosome 6p. T cells from the donor and the patient after transplantation did not recognize the mutant leukemic cells, whereas the original leukemic cells taken at the time of diagnosis were efficiently recognized and killed. CONCLUSIONS After transplantation of haploidentical hematopoietic stem cells and infusion of donor T cells, leukemic cells can escape from the donors antileukemic T cells through the loss of the mismatched HLA haplotype. This event leads to relapse.

Association of the α-Adducin Locus With Essential Hypertension

Abstract Previous studies on genetic rat hypertension have shown that polymorphism within the α-adducin gene may regulate blood pressure. Adducin is a cytoskeletal protein that may be involved in cellular signal transduction and interacts with other membrane-skeleton proteins that affect ion transport across the cell membrane. There is a high homology between rat and human adducin and pathophysiological similarities between the Milan hypertensive rat strain and a subgroup of patients with essential hypertension. Thus, we designed a case-control study to test the possible association between the α-adducin locus and hypertension. One hundred ninety primary hypertensive patients were compared with 126 control subjects. All subjects were white and unrelated. Four multiallelic markers surrounding the α-adducin locus located in 4p16.3 were selected: D4S125 and D4S95 mapping at 680 and 20 kb centromeric, and D4S43 and D4S228/E24 mapping at 660 and 2500 kb telomeric. Alleles for each marker were pooled into groups. Comparisons between control subjects and hypertensive patients were carried out by testing the allele-disease association relative to the marker genotype. The maximal association occurred for D4S95 (χ12 13.33), which maps closest to α-adducin. These data suggest that a polymorphism within the α-adducin gene may affect blood pressure in humans.

ACE and α-Adducin Polymorphism as Markers of Individual Response to Diuretic Therapy

Abstract—Renin-angiotensin system reactivity and the constitutive capacity of the renal tubule to reabsorb sodium play a role in the individual response to diuretic therapy; therefore we evaluated the blood pressure (BP) response to hydrochlorothiazide in 87 never-treated individuals with mild essential hypertension, according to ACE gene I/D and &agr;-adducin Gly460Trp polymorphism. These genotypes where chosen because previous data showed their interaction in determining the BP response to salt probably was the result of their involvement in the activation of the renin-angiotensin system (ACE) and in the constitutive capacity of the kidney to reabsorb sodium (&agr;-adducin) (treatment for 2 months). BP was measured after 3 run-in visits and after the first and second months of treatment by means of a standardized procedure. Data were analyzed by ANOVA, t test, and multivariate ANOVA for repeated measures (covarying for gender, age, and body mass index). Although basal mean BP (MBP) was similar in the different ACE and &agr;-adducin genotypes, patients carrying at least one I allele of ACE and one 460Trp allele of &agr;-adducin had the largest MBP decrease with treatment (12.7±1.9 mm Hg), the effect of the combination of genotypes being additive but not epistatic. These patients had an odds ratio of 15.75 of being a responder to hydrochlorothiazide compared with patients with Gly460Gly+DD, with the least MBP decrease (3.4±1.7 mm Hg). &agr;-Adducin and ACE I/D polymorphism may be useful to predict the interindividual degree of response to hydrochlorothiazide; the analysis of the combination of the 2 genotypes increases the accuracy of the prediction of response to the drug.

Genomewide association study using a high-density single nucleotide polymorphism array and case-control design identifies a novel essential hypertension susceptibility locus in the promoter region of endothelial NO synthase

Essential hypertension is a multifactorial disorder and is the main risk factor for renal and cardiovascular complications. The research on the genetics of hypertension has been frustrated by the small predictive value of the discovered genetic variants. The HYPERGENES Project investigated associations between genetic variants and essential hypertension pursuing a 2-stage study by recruiting cases and controls from extensively characterized cohorts recruited over many years in different European regions. The discovery phase consisted of 1865 cases and 1750 controls genotyped with 1M Illumina array. Best hits were followed up in a validation panel of 1385 cases and 1246 controls that were genotyped with a custom array of 14 055 markers. We identified a new hypertension susceptibility locus (rs3918226) in the promoter region of the endothelial NO synthase gene (odds ratio: 1.54 [95% CI: 1.37–1.73]; combined P=2.58 · 10−13). A meta-analysis, using other in silico/de novo genotyping data for a total of 21 714 subjects, resulted in an overall odds ratio of 1.34 (95% CI: 1.25–1.44; P=1.032 · 10−14). The quantitative analysis on a population-based sample revealed an effect size of 1.91 (95% CI: 0.16–3.66) for systolic and 1.40 (95% CI: 0.25–2.55) for diastolic blood pressure. We identified in silico a potential binding site for ETS transcription factors directly next to rs3918226, suggesting a potential modulation of endothelial NO synthase expression. Biological evidence links endothelial NO synthase with hypertension, because it is a critical mediator of cardiovascular homeostasis and blood pressure control via vascular tone regulation. This finding supports the hypothesis that there may be a causal genetic variation at this locus.

Copy-Number Disorders Are a Common Cause of Congenital Kidney Malformations

We examined the burden of large, rare, copy-number variants (CNVs) in 192 individuals with renal hypodysplasia (RHD) and replicated findings in 330 RHD cases from two independent cohorts. CNV distribution was significantly skewed toward larger gene-disrupting events in RHD cases compared to 4,733 ethnicity-matched controls (p = 4.8 × 10(-11)). This excess was attributable to known and novel (i.e., not present in any database or in the literature) genomic disorders. All together, 55/522 (10.5%) RHD cases harbored 34 distinct known genomic disorders, which were detected in only 0.2% of 13,839 population controls (p = 1.2 × 10(-58)). Another 32 (6.1%) RHD cases harbored large gene-disrupting CNVs that were absent from or extremely rare in the 13,839 population controls, identifying 38 potential novel or rare genomic disorders for this trait. Deletions at the HNF1B locus and the DiGeorge/velocardiofacial locus were most frequent. However, the majority of disorders were detected in a single individual. Genomic disorders were detected in 22.5% of individuals with multiple malformations and 14.5% of individuals with isolated urinary-tract defects; 14 individuals harbored two or more diagnostic or rare CNVs. Strikingly, the majority of the known CNV disorders detected in the RHD cohort have previous associations with developmental delay or neuropsychiatric diseases. Up to 16.6% of individuals with kidney malformations had a molecular diagnosis attributable to a copy-number disorder, suggesting kidney malformations as a sentinel manifestation of pathogenic genomic imbalances. A search for pathogenic CNVs should be considered in this population for the diagnosis of their specific genomic disorders and for the evaluation of the potential for developmental delay.

Familial Aggregation of Cation Transport Abnormalities and Essential Hypertension

The maximal rate of activity of Na extrusion by the Na pump, Na-K outward cotransport, Na-Li countertransport and the rate constant for passive permeability to Na, K and Li were measured in the RBC of 24 normotensive subjects with both parents normotensive, 45 hypertensive subjects and 24 of their normotensive offspring. The Na extrusion by the Na pump and the passive permeability to Na, K and Li are equal. The hypertensives have significantly greater Na-Li countertransport and smaller Na-K cotransport when compared to the normotensives. Na-K cotransport and Na-Li countertransport are positively correlated, thus suggesting some relationship between the two systems. When arbitrary normal limits are set the hypertensives are divided in three groups: normal cotransport and countertransport (22.2%) high countertransport (31.1%) and low cotransport (44.4%). In nine hypertensive families studied if either alteration was observed in a hypertensive propositus, this was of the same kind as the one in case observed in any first degree relative, whether already hypertensive or young normotensive. The observed alterations are primitive to the development of hypertension and possibly related to its pathogenesis.

Role of the adducin family genes in human essential hypertension

Objective In both humans and rats, polymorphisms of the alpha adducin (ADD1) gene are involved in renal sodium handling, essential hypertension and some of its organ complications. Adducin functions within cells as a heterodimer composed of various combinations of three subunits that are coded by three genes (ADD1, 2, 3) each located on a different chromosome. Design These characteristics provide the biochemical basis for investigating epistatic interactions among these loci. Methods We examined the three adducin gene polymorphisms and their association with ambulatory blood pressure (ABPM) and with plasma levels of renin activity (PRA), endogenous ouabain (EO), in 512 newly discovered and never-treated hypertensive patients. Results Relative to carriers of the wild type (Gly/Gly) ADD1 gene, patients carrying the mutated Trp ADD1 allele had higher blood pressure (systolic blood pressure (SBP) 143.2 ± 1.0 versus 140.6 ± 0.6 mmHg P = 0.027 and diastolic blood pressure (DBP) 94.2 ± 0.77 versus 92.3 ± 0.5 mmHg, P = 0.03), lower PRA and EO, consistent with the hypothesis of the renal sodium retaining effect of the Trp allele. Polymorphisms in the ADD2 and ADD3 genes taken alone were not associated with these variables. However, the differences in SBP and DBP between the two ADD1 genotypes were greatest in carriers of the ADD3 G allele (around + 8 mmHg). The significance of the interaction between ADD1 and ADD3 ranged between P = 0.020 to P = 0.006 according to the genetic model applied. Conclusions The interaction of ADD1 and ADD3 gene variants in humans is statistically associated with variation in blood pressure, suggesting the presence of epistatic effects among these loci.

Genes involved in vasoconstriction and vasodilation system affect salt-sensitive hypertension.

The importance of excess salt intake in the pathogenesis of hypertension is widely recognized. Blood pressure is controlled primarily by salt and water balance because of the infinite gain property of the kidney to rapidly eliminate excess fluid and salt. Up to fifty percent of patients with essential hypertension are salt-sensitive, as manifested by a rise in blood pressure with salt loading. We conducted a two-stage genetic analysis in hypertensive patients very accurately phenotyped for their salt-sensitivity. All newly discovered never treated before, essential hypertensives underwent an acute salt load to monitor the simultaneous changes in blood pressure and renal sodium excretion. The first stage consisted in an association analysis of genotyping data derived from genome-wide array on 329 subjects. Principal Component Analysis demonstrated that this population was homogenous. Among the strongest results, we detected a cluster of SNPs located in the first introns of PRKG1 gene (rs7897633, p = 2.34E-05) associated with variation in diastolic blood pressure after acute salt load. We further focused on two genetic loci, SLC24A3 and SLC8A1 (plasma membrane sodium/calcium exchange proteins, NCKX3 and NCX1, respectively) with a functional relationship with the previous gene and associated to variations in systolic blood pressure (the imputed rs3790261, p = 4.55E-06; and rs434082, p = 4.7E-03). In stage 2, we characterized 159 more patients for the SNPs in PRKG1, SLC24A3 and SLC8A1. Combined analysis showed an epistatic interaction of SNPs in SLC24A3 and SLC8A1 on the pressure-natriuresis (p interaction = 1.55E-04, p model = 3.35E-05), supporting their pathophysiological link in cellular calcium homeostasis. In conclusions, these findings point to a clear association between body sodium-blood pressure relations and molecules modulating the contractile state of vascular cells through an increase in cytoplasmic calcium concentration.

Genetics of Essential Hypertension: From Families to Genes

Family studies demonstrated the contribution of genetic factors to the development of primary hypertension. However, the transition from this phenomenologic-biometric approach to the molecular-genetic one is more difficult. This last approach is mainly based on the Mendel paradigm; that is, the dissection of the poligenic complexity of hypertension is brought about on the assumption that the individual genetic variants underlying the development of hypertension must be more frequent in hypertensive patients than in controls and must cosegregate with hypertension in families. The validity of these assumptions was clearly demonstrated in the so-called monogenic form of hypertension. However, because of the network of the feedback mechanisms regulating BP, it is possible that that the same gene variant may have an opposite effect on BP according to the genetic and environmental backgrounds. Independent groups of observations (acute BP response to saline infusion, incidence of hypertension in a population follow-up of 9 yr, age-related changes on BP) discussed in this review suggest a positive answer to this question. Therefore the impact of a given genetic variant on BP level must be evaluated within the context of the appropriate genetic epistatic interactions. A negative finding or a minor genetic effect in a general population may become a major gene effect in a subset of people with the appropriate genetic and environmental backgrounds.

Alpha-adducin gene polymorphism and cardiovascular phenotypes in a general population.

Background Previous studies have shown that molecular variants of the cytoskeletal protein adducin may be involved in regulation of blood pressure both in genetic rat hypertension and in human essential hypertension. Objective To investigate the relationship of genetic polymorphism of α-adducin with blood pressure, cardiovascular structure, and some biochemical indexes of cardiovascular risk in a sample of general population. Design and methods A sample of 246 subjects (124 men and 122 women, aged 57.7 ± 3.7 years) was randomly chosen from a middle-aged population. Twenty-four-hour ambulatory blood pressure, as well as left ventricular mass (by echocardiographic methods) and carotid wall thickness (by B-mode ultrasound methods) were measured. DNA was extracted from peripheral blood samples; the Gly460Trp diallelic variant of human α-adducin was genotyped by polymerase chain reaction amplification and then allele-specific oligo hybridization. Results A trend toward higher 24 h ambulatory blood pressure values in subjects not treated with antihypertensive drugs was observed among carriers of Trp460 allele, although the differences did not attain statistical significance (at closest, P = 0.066 for a dominant effect of Trp460 on systolic blood pressure). When blood pressure was considered a dichotomous variable, allowing the inclusion of treated hypertensives), a higher prevalence of Trp460 allele among hypertensives was observed (0.188 versus 0.106 among normotensives, P = 0.02). There was no evidence of association either of left ventricular mass or of common carotid wall thickness with Gly460Trp polymorphism. Conclusions In this sample of a general population, the relationship of a genetic polymorphism of α-adducin with blood pressure values was rather weak. However, a population-based case–control analysis indicated that there was an association between Trp460 allele and hypertension, with a relative risk for subjects carrying at least one Trp460 allele of approximately 1.6. Further investigation of larger and different population samples in order to assess the role of adducin gene polymorphism as a marker of genetic predisposition to the development of hypertension is warranted.