Entropy of human leukocyte antigen and killer-cell immunoglobulin-like receptor systems in immune-mediated disorders: A pilot study on multiple sclerosis.

BACKGROUND: Entropy is a thermodynamic variable statistically correlated with the disorder of a system. The hypothesis that entropy can be used to identify potentially unhealthy conditions was first suggested by Schrödinger, one of the founding fathers of quantum mechanics. Shannon later defined entropy as the quantity of information stored in a system. Shannon's entropy has the advantage of being adaptable across a variety of disciplines, including genetic studies on complex immunogenetic systems such as the human leukocyte antigen (HLA) and killer-cell immunoglobulin-like receptor (KIR) systems. METHODS: In our study, entropy associated to the HLA and KIR systems was compared between a cohort of 619 Sardinian healthy controls and a group of 270 patients affected by multiple sclerosis (MS), the latter stratified into 81 patients with primary progressive multiple sclerosis (PPMS) and 189 patients with relapsing remitting multiple sclerosis (RRMS). RESULTS: The entropy associated to HLA four-loci haplotypes (A, B, C, DR) and combinations of two inhibitory KIR genes was significantly higher in patients affected by RRMS than in healthy controls. No significant differences were observed for patients with PPMS. By calculating the total HLA and KIR entropy ratio in each subject, it was possible to determine the individual risk of developing MS, particularly RRMS. CONCLUSIONS: In addition to the standard statistical methods used to evaluate immunogenetic parameters associated to immune-mediated disease, the analysis of entropy measures the global disorder status deriving from these parameters. This innovative approach may represent a useful complementary tool to the risk assessment of immune-mediated disorders. Improved risk assessment is particularly important for family members of patients with MS. However, further investigation is warranted to confirm our findings and to evaluate the validity of the entropy-based method in other types of immune-mediated disorders.

KIR and their HLA Class I ligands: Two more pieces towards completing the puzzle of chronic rejection and graft loss in kidney transplantation.

BACKGROUND: Kidney transplantation is a life-saving treatment for patients with end-stage renal disease. However, despite progress in surgical techniques and patient management, immunological rejection continues to have a negative impact on graft function and overall survival. Incompatibility between donors and recipients for human leukocyte antigens (HLA) of the major histocompatibility complex (MHC) generates a series of complex cellular and humoral immune response mechanisms that are largely responsible for rejection and loss of graft function. Within this context, a growing amount of evidence shows that alloreactive natural killer (NK) cells play a critical role in the immune response mechanisms elicited by the allograft. Killer immunoglobulin-like receptors (KIRs) are prominent mediators of NK cell alloreactivity. METHODS AND FINDINGS: A cohort of 174 first cadaveric kidney allograft recipients and their donors were selected from a total cohort of 657 transplanted patients for retrospective immunogenetic analyses. Patients with HLA Class II mismatches were excluded. HLA Class I allele frequencies were compared among patients with chronic rejection, patients with stable graft function and a group of 2388 healthy controls. Activating and inhibitory KIR gene frequencies, KIR haplotypes, KIR-HLA ligand matches/mismatches and combinations of recipient KIRs and donor HLA Class I ligands were compared among patients with and without chronic rejection and a group of 221 healthy controls. Patients transplanted from donors homozygous for HLA-C1 antigens had a significantly higher risk for chronic rejection than patients transplanted from donors homozygous or heterozygous for HLA-C2 antigens or with epitopes belonging to the HLA-Bw4 ligand group. The Kaplan-Meier curves obtained by dividing the patients into 3 groups according to the presence or absence of one or both of the combinations of recipient KIRs and donor HLA ligands (rKIR2DL1/dHLA-C2 and rKIR3DL1/dHLA-Bw4) showed a significantly higher cumulative incidence of chronic rejection in the group of patients completely lacking these functional units. These patients showed a progressively stronger decline in modification of diet in renal disease-estimated glomerular filtration rate. CONCLUSIONS: KIR genotyping should be performed at the time of enrolment of patients on the waiting list for organ transplantation. In our study, a significantly higher risk of chronic rejection after kidney transplantation was observed when recipient (r) and donor (d) pairs completely lacked the two functional rKIR-dHLA ligand combinations rKIR2DL1/dHLA-C2 and rKIR3DL1/dHLA-Bw4. This immunogenetic profile corresponds to low levels of NK cell inhibition. Therefore, patients with this high risk profile could benefit from immunosuppressive therapy aimed at reducing NK-cell cytotoxicity.

Five mutations in the GABA A alpha6 gene 5' flanking region are associated with a reduced basal and ethanol-induced alpha6 upregulation in mutated Sardinian alcohol non-preferring rats.

The presence of four nucleotide changes and a three base-pair deletion in the GABA A alpha6-subunit promoter is described in Sardinian alcohol non-preferring rats, selectively bred for their ethanol aversion. These mutations are associated with the R100Q alpha6 intragenic mutation that was previously characterized in the same animals. The possibility that these mutated nucleotides alter the ethanol-induced upregulation of the alpha6 gene was investigated by measuring cerebellar alpha6 mRNA levels after a chronic ethanol liquid diet in sNP rat. Real-time quantitative PCR showed an increased alpha6 gene expression after ethanol ingestion in normal and mutated rats. However, lower amounts of alpha6 mRNA levels were detected both in control and in ethanol-treated sNP rats carrying the five promoter and the intragenic mutations in a homozygous state. Using the electromobility shift assay, specific DNA binding sites were found in cerebellar extracts of the alpha6 regions comprising the five mutations. These results suggest that one or more of the mutated binding sites that were found in the 5' flanking alpha6 region may be a consensus sequence for regulatory factors which are responsible for both basal and ethanol-induced alpha6 gene expression.

Molecular characterization of new polymorphisms at the beta2, alpha1, gamma2 GABA(A) receptor subunit genes associated to a rat nonpreferring ethanol phenotype.

Recent preclinical and clinical studies have indicated a possible involvement of the genes encoding for the GABA(A) receptor subunits alpha6, beta2, alpha1 and gamma2 in the genetic susceptibility to alcohol abuse. We have recently found an (R) to (Q) mutation in codon 100 of the alpha6 GABA(A) subunit, that segregated in a rat line selectively bred for its voluntary ethanol aversion, Sardinian alcohol nonpreferring (sNP), but not in their Sardinian alcohol preferring (sP) counterpart, selected for its ethanol preference. In the present study the molecular composition of other GABA(A) subunits (beta2, alpha1 and gamma2) were analyzed in order to further investigate the involvement of the GABA(A) receptors in the genetic predisposition to voluntary alcohol intake. Automated sequencing analysis indicated the presence of six new silent substitutions (289 T-->C in the beta2 gene; 115 G-->A in the alpha1 gene; 157 G-->A, 174 C-->T, 347 A-->G and 385 A-->T in the gamma2 gene), in sNP but not in sP rats. These polymorphisms were linked to the alpha6 R100Q mutation previously described in sNP rats. The strict association between the alpha6 point mutation and the new polymorphisms found in the beta2, alpha1 and gamma2 genes, demonstrate that such genes belong to the same cluster and are inherited together in the rat. These results sustain the synteny for these clusters between the rodent and human genomes, and suggest that mutated GABA(A) beta2, alpha6, alpha1 and gamma2 subunit genes might contribute to the expression of an ethanol nonpreferring phenotype in a rat line that voluntarily avoids alcoholic solutions.

Haloperidol versus risperidone on rat "early onset" vacuous chewing.

Similarly to acute rat catalepsy, "early onset" vacuous chewing movements (VCMs) induced by subchronic treatment with antipsychotic have recently been proposed as a model of human extrapyramidal symptoms. In the present study, the propensities of haloperidol and risperidone in inducing rat "early onset" VCMs were compared using doses of the two antipsychotics that acutely induce similar catalepsy. Comparable rat catalepsy states were observed when the effects produced by 0.1, 0.5, and 1mg/kg of haloperidol were compared with those induced by 1, 4, and 10mg/kg of risperidone, respectively. These doses of the two antipsychotics were then administered twice a day for 4 weeks and VCMs scored after 12h, 5 days, or 3 weeks of drug withdrawal. Among the haloperidol-treated groups, only those rats injected with 0.5 and 1mg/kg showed high levels of VCMs after 12h and 5 days of drug withdrawal when compared to vehicle-treated rats, while basal levels of VCMs were reached after 3 weeks from the last injection. High VCMs levels were observed in risperidone-treated rats only at the dose of 10mg/kg and after 12h of drug withdrawal, but not after 5 days or 3 weeks. The present results indicated that haloperidol possessed a much higher propensity to induce rat "early onset" VCMs than risperidone.

The cerebellar GABAA alpha6 subunit is differentially modulated by chronic ethanol exposure in normal (R100R) and mutated (Q100Q) sNP rats.

Sardinian alcohol non-preferring (sNP) rats carry a point mutation (R100Q) in the cerebellar expressed GABAA receptor alpha6 subunit gene, leading to a higher sensitivity to ethanol and diazepam. The role of the alpha6 subunit gene cluster in the ethanol non-preferring phenotype was here investigated by measuring the levels of alpha1, alpha6 and gamma2 peptide in the cerebellum of normal (RR) and mutated (QQ) sNP rats after 2 weeks of chronic ethanol administration. Western blot analysis revealed that the alpha6 subunit is increased in RR sNP rats after chronic ethanol exposure (25.44%+/-8.69 versus control), while it remained unchanged in mutated QQ sNP rats. Interestingly, chronic ethanol administration decreased alpha1 peptide levels in the cerebellum of both rat lines to a similar extent (30.99%+/-6.74 and 27.12%+/-9.83 in RR and QQ rats, respectively), while gamma2 peptide levels remained unchanged. To further correlate the genetic and biochemical difference of the normal and mutated sNP rats with their aversive phenotype, we exposed sNP rats to a protocol of acquisition and maintenance of ethanol drinking. QQ sNP rats drank less ethanol than RR rats during the acquisition phase, but such difference was lost during the maintenance phase. These data may contribute to elucidating the mechanisms of alcohol avoidance in rat lines selected for this behavior when exposed to ethanol solution.

Characterization of wild-type (R100R) and mutated (Q100Q) GABAA alpha 6 subunit in Sardinian alcohol non-preferring rats (sNP).

Sardinian alcohol non-preferring (sNP) rats, selected for their low ethanol preference and consumption, carry a point mutation (R100Q) in the gene coding for GABA(A) receptor alpha(6) subunit, which becomes more sensitive to diazepam-evoked GABA currents. We performed binding studies in the cerebellum of normal (RR) and mutated (QQ) sNP rats using [3H]Ro 15-4513, an inverse agonist for the benzodiazepine site which binds both diazepam insensitive and diazepam sensitive sites. Saturation curves performed on cerebellar membrane from genotyped rats indicated an higher affinity of [3H]Ro 15-4513 for GABA(A) receptors in QQ with respect to RR rats (K(d) values 4.0+/-0.67 and 6.24+/-0.95 nM, respectively), with similar B(max) values (3.5+/-0.25 and 3.9+/-0.39 pmol/mg protein, respectively). Diazepam displacement curves showed a two component model for both genotypes, with similar K(i1) values for QQ and RR (3.6+/-0.62 and 4.9+/-0.33 nM, respectively). In QQ rats diazepam is able to completely displace [3H]Ro 15-4513 (K(i2)=1.48+/-0.27 microM), while in RR rats the diazepam sensitive sites are still present (K(i2)>10 microM). The basal mRNA and protein expression level of the alpha(6) subunit were similar in RR and QQ rats. The electrophysiological profile of oocytes of Xenopus laevis injected with cerebellar synaptosomes showed that ethanol positively modulated GABA-evoked currents significantly more in QQ than in RR rats. These data contribute to the characterization of the function of GABA(A) alpha(6) subunit and its involvement in determining alcohol related behavior.

Multiplex genotyping of CYP3A4, CYP3A5, CYP2C9 and CYP2C19 SNPs using MALDI-TOF mass spectrometry.

BACKGROUND: Pharmacogenetics is the study of genetic variations that cause alterations in drug level, drug response and adverse drug reactions. SNPs found in CYP450 genes have the greatest genetic influences on interindividual variability in drug bioavailability. The polymorphic nature of these genes may modulate several enzyme levels that affect individual responses to pharmacological treatment. Among them, CYP3A4, CYP3A5, CYP2C9 and CYP2C19 isoforms of CYP450 enzymes are involved in the metabolism of many commonly prescribed drugs. AIMS: In this study, we would like to develop a CYP450 genotyping platform that could lead a complete definition of a patient's metabolic genotype in order to improve the clinical outcome of some drug treatments. MATERIALS & METHODS: We used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Sequenom) to develop a SNP genotyping method. RESULTS: This MALDI-TOF-based multiplexing system allows the simultaneous and efficient genotyping of a set of CYP450 gene polymorphisms. CONCLUSION: The multiple CYP450 gene testing achieved with this application can be used to develop diagnostic tests to predict drug responses and clinical outcomes.