Alessandra Balboni

A His-155 to Tyr Polymorphism Confers Gain-of-Function to the Human P2X7 Receptor of Human Leukemic Lymphocytes

The P2X7R is an ATP-gated cation channel expressed in hemopoietic cells that participates in both cell proliferation and apoptosis. Expression and function of the P2X7R have been associated with the clinical course of patients affected by chronic lymphocytic leukemia (CLL). Functional variants causing loss-of-function of the P2X7R have been identified, namely, polymorphisms 1513A>C (E496A), 1729T>A (I568N), and 946G>A (R307Q). Here we investigated other nonsynonymous polymorphisms located either in the extracellular portion of the receptor, such as the 489C>T (H155Y) variant, or in the long cytoplasmic tail of the receptor, such as the 1068G>A (A348T), 1096C>G (T357S), and 1405A>G (Q460R) variants. P2X7R function was monitored by measuring ATP-induced Ca2+ influx in PBL of patients affected by CLL and in recombinant human embryonic kidney (HEK) 293 cells stably transfected with each single P2X7 allelic variant. Ca2+ influx was markedly reduced in association with the 1513C allele, whereas variants located in the same intracellular domain, such as the 1068A, 1096G, or 1405G variants, were associated with a minor functional decrease. Significant Ca2+ flux increase was observed in lymphocytes from CLL patients bearing the 489C/T and 489T/T genotypes in association with the 1513A/A genotype. Functional analysis in recombinant HEK293 cells expressing P2X7R confirmed an increased ATP-dependent activation of the P2X7 489T mutant with respect to the wild type receptor, as assessed by both by [Ca2+]i influx and ethidium uptake experiments. These data identify the 489C>T as a gain-of-function polymorphism of the P2X7R.

Custom CGH array profiling of copy number variations (CNVs) on chromosome 6p21.32 (HLA locus) in patients with venous malformations associated with multiple sclerosis

BackgroundMultiple sclerosis (MS) is a complex disorder thought to result from an interaction between environmental and genetic predisposing factors which have not yet been characterised, although it is known to be associated with the HLA region on 6p21.32. Recently, a picture of chronic cerebrospinal venous insufficiency (CCSVI), consequent to stenosing venous malformation of the main extra-cranial outflow routes (VM), has been described in patients affected with MS, introducing an additional phenotype with possible pathogenic significance.MethodsIn order to explore the presence of copy number variations (CNVs) within the HLA locus, a custom CGH array was designed to cover 7 Mb of the HLA locus region (6,899,999 bp; chr6:29,900,001-36,800,000). Genomic DNA of the 15 patients with CCSVI/VM and MS was hybridised in duplicate.ResultsIn total, 322 CNVs, of which 225 were extragenic and 97 intragenic, were identified in 15 patients. 234 known polymorphic CNVs were detected, the majority of these being situated in non-coding or extragenic regions. The overall number of CNVs (both extra- and intragenic) showed a robust and significant correlation with the number of stenosing VMs (Spearman: r = 0.6590, p = 0.0104; linear regression analysis r = 0.6577, p = 0.0106).The region we analysed contains 211 known genes. By using pathway analysis focused on angiogenesis and venous development, MS, and immunity, we tentatively highlight several genes as possible susceptibility factor candidates involved in this peculiar phenotype.ConclusionsThe CNVs contained in the HLA locus region in patients with the novel phenotype of CCSVI/VM and MS were mapped in detail, demonstrating a significant correlation between the number of known CNVs found in the HLA region and the number of CCSVI-VMs identified in patients. Pathway analysis revealed common routes of interaction of several of the genes involved in angiogenesis and immunity contained within this region. Despite the small sample size in this pilot study, it does suggest that the number of multiple polymorphic CNVs in the HLA locus deserves further study, owing to their possible involvement in susceptibility to this novel MS/VM plus phenotype, and perhaps even other types of the disease.

Decreased production of human leukocyte antigen G molecules in sinonasal polyposis.

Background Sinonasal polyposis (SNP) is a chronic inflammatory pathology of nasal and paranasal cavities. Human leukocyte antigen (HLA) G molecules are nonclassic class I antigens with anti-inflammatory and tolerogenic properties. As most theories consider polyps to be the manifestation of chronic inflammation, there could be a possible implication of HLA-G molecules in SNP. The purpose of this study was to investigate the possible correlation between SNP and the production of soluble HLA-G (sHLA-G) by peripheral blood mononuclear cells (PBMCs). Methods The study involved 22 SNP patients (11 with no evidence of disease [NED] after surgery and 11 with relapse [RE]) and 20 healthy subjects. The presence of sHLA-G in PBMC lipopolysaccharide (LPS)-stimulated culture supernatants was analyzed. The levels of interleukin (IL) 10, one of the main up-regulators of sHLA-G production, were determined. Exogenous IL-10 was added to the SNP PBMC cultures to reconstitute the impairment in sHLA-G production. Results Increased IL-10 levels in LPS-activated PBMC culture supernatants were found in NED patients in comparison with healthy subjects (p = 0.0184). No sHLA-G production was observed in either of the patient subgroup supernatants (p < 0.0001). The addition of exogenous IL-10 showed the reconstitution of sHLA-G production in NED and in a lower amount in RE patients. Conclusion The results show a defect in sHLA-G production in SNP patients mainly related to the IL-10/HLA-G pathway. Given the anti-inflammatory functions of HLA-G molecules, this impairment could increase the susceptibility to the disease. The different sHLA-G production after exogenous IL-10 addition between NED and RE SNP could represent a marker of disease severity.

Altered proliferative kinetics in PHA‐activated human T‐lymphocytes treated with the anti‐HLA class I monoclonal antibody 01.65

Abstract. Anti‐HLA class I monoclonal antibody (mAb) 01.65 inhibited phyto‐haemagglutinin (PHA)‐induced human lymphocyte proliferation. The inhibitory effect was inversely correlated to the strength of the proliferative response. It was increased when lymphocytes were stimulated with suboptimal doses of PHA but it disappeared with supraoptimal doses. Proliferation inhibition was achieved by prolonging the cell cycle time and by slowing down its recruitment rate. The former effect was not restricted to the G1‐phase but also included the S phase. These results support the idea that HLA class I molecules are important in the PHA‐induced proliferation of human T‐lymphocytes.

Identification of a novel protein kinase C inhibitor in microsomes from phytohaemagglutinin activated human peripheral blood mononuclear cells.

A peptide inhibiting either corpuscolate or purified PKC has been identified from microsomes of PHA‐activated human PBMC but it is not detectable in microsomes of resting PBMC. The peptide was obtained from a microsomal preparation in an oligomeric form that could be transformed into a monomeric form by β‐MSH. The active peptide (IN) was retained on a PC‐11 chromatographic column and could be eluted with Nad. IN is ineffective on PKC‐dependent protamine phosphorylation of protamine and on Ca2+ and phospholipid‐independent activity generated by mild hydrolysis with trypsin of PK‐C. Ca2+ binding is permissive for IN activity. IN inhibits particulate PK.C in PHA‐activated PBMC, but is ineffective after TPA activation. All these data indicate that IN acts at the regulatory domain of PKC.

Impairment of capping in lymphoblastoid cell lines of Duchenne patients indicates an intrinsic cellular defect

SummaryDuchenne muscular dystrophy (DMD) is a lethal sex-linked degenerative disorder of the muscle in man. Generalized cell membrane abnormalities seem to be involved in the pathogenesis of the disease; in particular, the impairment of lymphocyte capping capacities has been repeatedly confimed. To clarify whether capping impairment is a consequence of factors related to the activity of the disease or an expression of an intrinsic cellular defect, we have investigated the capping capacities of DMD EBV-transformed cell lines. The results indicate a significant impairment of capping capacity in cultured cell lines, providing evidence for an intrinsic cell deficiency in DMD.

Genetic counseling for women referred for advanced maternal age: a telegenetic approach.

To the Editor: Medical genetics and genetic counseling represent vital tools for communicating with patients about genetic risk, reproductive options, prenatal testing, and novel therapies. Medical geneticists and genetic counselors are the professionals in charge of genetic diagnosis and risk evaluation, representing the most important resource for communicating to patients all issues related to genetic diseases (both rare and common). There is a general consensus, promoted by the Eurogentest Network of Excellence (http://www.eurogentest.org), that genetic counseling should be a mandatory accompaniment to all medical genetics interventions. This becomes particularly relevant with the advent of high-throughput diagnosis, including noninvasive prenatal testing. Indeed, despite the potential for rapid results, the need for counseling delivered by expert geneticists must not be overlooked, and the issues surrounding data filtering and validation need to be addressed. This entails providing access to genetic counseling at various points within the health-care system, which will inevitably impact both national and local policy making. In the current economic climate, the medical geneticist is a relatively marginal figure, often restricted to a few specialist centers, even in countries of the European Union. Patients who are affected by rare diseases have the right to benefit from expert assistance in their clinical management, diagnosis, and care, no matter where they are or the rareness of their disease. Through the adoption of telemedical technology in genetics (telegenetics), as encouraged by various EU initiatives, the gap between specialists and patients may more easily be bridged. Multimedia telegenetics tools will enable us to provide interactive genetic consultations from a distance—feasibly to people in other countries—thereby increasing the accessibility of this invaluable service and helping to meet the needs of centers of excellence for rare diseases. According to the World Health Organization, telemedicine is “the delivery of health-care services, where distance is a critical factor, by all health-care professionals using information and communication technologies for the exchange of valid information for diagnosis, treatment and prevention of disease and injuries, research and evaluation, and for the continuing education of health care providers, all in the interests of advancing the health of individuals and their communities.”1 With this in mind, we produced a short digital film on telegenetic counseling for pregnant women of advanced maternal age. Reasoning that the increasing number of women and couples requiring genetic testing, many of whom will not have easy access to counseling centers, need a standardized, reliable approach, we explain the genetic risks associated with advanced maternal age, as well as possible preventive measures, prenatal testing procedures (both invasive and noninvasive), laboratory assessment methods (stating the specificity and accuracy of the available tests), and best-practice communication of results. We also explain any possible causes of test failure and stress the role of the medical geneticist and the importance of effective communication during genetic counseling. This video is freely available in both English and Italian (www.ospfe.it/geneticamedica). It is our hope that this initiative will facilitate communication among geneticists, obstetricians, pregnant women, and all stakeholders involved in prenatal care and diagnosis.

Anchored anti-HLA class I monoclonal antibody fails to induce inhibition of PHA- activated lymphocytes proliferation

It is known that anti-HLA Class I antibodies inhibit the proliferative response of PHA-activated T-lymphocytes. We found that plastic- or sepharose-linked anti-HLA Class I monoclonal antibody 01.65 does not inhibit either [3H]Thymidine incorporation or recruitment in the cell cycle, nor does it reduce the expression of c-myc mRNA and the membrane expression of Interleukin-2 Receptor and Transferrin Receptor. Furthermore, particulate Protein Kinase C is not affected by anchored anti-HLA Class I monoclonal antibody 01.65. We suggest that anti-HLA Class I monoclonal antibody may act through crosslinking or internalization of HLA Class I antigens.