Single step multiple genotyping by MALDI-TOF mass spectrometry, for evaluation of minor histocompatibility antigens in patients submitted to allogeneic stem cell transplantation from HLA-matched related and unrelated donor

The outcome of patients underwent to allogeneic stem cell transplantation (allo- SCT) is closely related to graft versus host disease (GvHD) and graft versus leukemia (GvL) effects which can be mediated by mHAgs. 23 mHAgs have been identified and reported to be differently correlated with GVHD or GVL and the aim of this work was develop a method to genotype the mHAgs described so far. For this study we used MALDI-TOF iPLEX Gold Mass Array technology. We tested 46 donor/recipient matched pairs that underwent allo-SCT because of Philadelphia positive (Ph+) chronic myeloid leukemia (n=29) or Ph+ acute lymphoblastic leukemia (n=17). Our data show that sibling pairs had a lesser number of mHAgs mismatches compared to MUD pairs. Notably, donor/recipient genomic mismatch on DPH1 was correlated with an increased risk of acute GvHD and LB-ADIR-1R mismatch on graft versus host direction was correlated with a better RFS with no increase of GvHD risk. Our work provides a simple, accurate and highly automatable method for mHAgs genotyping and suggest the role of mHAgs in addressing the immune reaction between donor and host

Complex interplay between neutral and adaptive evolution shaped differential genomic background and disease susceptibility along the Italian peninsula

The Italian peninsula has long represented a natural hub for human migrations across the Mediterranean area, being involved in several prehistoric and historical population movements. Coupled with a patchy environmental landscape entailing different ecological/cultural selective pressures, this might have produced peculiar patterns of population structure and local adaptations responsible for heterogeneous genomic background of present-day Italians. To disentangle this complex scenario, genome-wide data from 780 Italian individuals were generated and set into the context of European/Mediterranean genomic diversity by comparison with genotypes from 50 populations. To maximize possibility of pinpointing functional genomic regions that have played adaptive roles during Italian natural history, our survey included also ∼250,000 exomic markers and ∼20,000 coding/regulatory variants with well-established clinical relevance. This enabled fine-grained dissection of Italian population structure through the identification of clusters of genetically homogeneous provinces and of genomic regions underlying their local adaptations. Description of such patterns disclosed crucial implications for understanding differential susceptibility to some inflammatory/autoimmune disorders, coronary artery disease and type 2 diabetes of diverse Italian subpopulations, suggesting the evolutionary causes that made some of them particularly exposed to the metabolic and immune challenges imposed by dietary and lifestyle shifts that involved western societies in the last centuries

Elicitation of resistance to bacterial canker of stone fruits by humic and fulvic acids (glucohumates): a cDNA-AFLP-dHPLC approach

Bacterial leaf/fruit spot and canker of stone fruits, caused by Xanthomonas arboricola pv. pruni is a challenging disease in Italian orchards. In recent years, novel molecules such as biostimulants or/and resistance-inducers have been developed and used to implement an effective Integrated Pest Management. An extensive study in vitro, in glasshouse and in a peach orchard was carried out to verify the efficacy of commercial glucohumates to control bacterial spot/canker outbreaks and to understand their mode of action. A transcriptomic approach was implemented to study the complex transcriptional changes that these biomolecules may possibly elicit in the plant-pathogen interaction. The cDNA-AFLP analysis of differential gene expression in treated plant tissue was made. Discrimination of differentially expressed sequences was made with denaturing High Performance Liquid Chromatography (dHPLC), and functional annotation of such transcripts was assigned based on similarity search on public genome databases. The results highlighted the activity of glucohumates in controlling Xanthomonas arboricola pv. pruni both in vitro and in vivo. Beneficial effects of humic substances towards this pathogen were confirmed by a significant disease reduction (up to 78%) in the commercial orchard. On the other hand, cDNA-AFLPdHPLC analysis allowed the collection of fourteen up-regulated transcript-derived fragments belonging to peach genes and putatively involved in the defence response. In particular, the activation of these genes within 24 h after treatment supposedly triggered the early-induced resistance, notoriously involved in maintaining a protection state in plants against biotic stresses

Identification and validation of loss of function variants in clinical contexts

The choice of an appropriate variant calling pipeline for exome sequencing data is becoming increasingly more important in translational medicine projects and clinical contexts. Within GOSgene, which facilitates genetic analysis as part of a joint effort of the University College London and the Great Ormond Street Hospital, we aimed to optimize a variant calling pipeline suitable for our clini- cal context. We implemented the GATK/Queue framework and evaluated the performance of its two callers: the classical UnifiedGenotyper and the new vari- ant discovery tool HaplotypeCaller. We performed an experimental validation of the loss-of-function (LoF) variants called by the two methods using Seque- nom technology. UnifiedGenotyper showed a total validation rate of 97.6% for LoF single-nucleotide polymorphisms (SNPs) and 92.0% for insertions or dele- tions (INDELs), whereas HaplotypeCaller was 91.7% for SNPs and 55.9% for INDELs. We confirm that GATK/Queue is a reliable pipeline in translational medicine and clinical context. We conclude that in our working environment, UnifiedGenotyper is the caller of choice, being an accurate method, with a high validation rate of error-prone calls like LoF variants. We finally highlight the importance of experimental validation, especially for INDELs, as part of a stan- dard pipeline in clinical environments

An optimized cDNA-AFLP protocol for the identification of TDFs involved in the malus-venturia inaequalis interaction.

The interaction of apple genotypes and Venturia inaequalis, the causal agent of apple scab, is nowadays the most studied plant-pathogen interaction in a non-model woody plant. After the cloning of the apple scab resistance gene HcrVf2, the cascade of reactions induced after pathogen recognition is under investigation. To understand the gene networks that underlie plant defense responses, it is necessary to identify and characterize the genes responding to pathogen infection. Young leaf samples were collected from genetically modified \u2018Gala\u2019 plants carrying the HcrVf2 gene, at different times after inoculation with V. inaequalis. A cDNA-AFLP procedure, successfully applied to study plant-pathogen interactions, has been chosen in order to identify sequences (TDFs, transcript derived fragments) that are differentially expressed after pathogen inoculation. An optimized and highly reproducible cDNA-AFLP protocol was set up on PAGE, starting with an RNA extraction from apple leaves until gel band elution from polyacrylamide gels. The feasibility of this cDNA-AFLP protocol by the dHPLC for fragment separation in order to automatize all band elution steps will be discussed

Onset of type 1 diabetes mellitus in two patients with maturity onset diabetes of the young

The association between maturity onset diabetes of the young (MODY) and type 1 diabetes mellitus (T1DM) has been rarely described. We report two patients affected by MODY who developed T1DM. Case 1: a 4-yr-old girl referred for glycosuria presented hemoglobin A1c (HbA1c) of 6.6%. Islet cell antibodies (ICA) and anti-glutamic acid decarboxylase (GADA) were initially negative. As her father, uncle and grandmother showed mild hyperglycemia, they were screened for MODY 2. A novel mutation in glucokinase gene was found in the family. Few months later, her glycemic control worsened consistently and she required insulin treatment. A high titer of GADA and ICA was then detected. Six years afterwards insulin requirement is 0.8 U/kg and HbA1c 6.7%. Case 2: a 15-yr-old boy treated for growth hormone deficiency was found with a blood glucose level of 106 mg/dL. HbA1c was 7.2%, ICA and GADA were negative. Family history was positive for autoimmune diseases and type 2 diabetes mellitus. The patient was investigated for MODY 2 and MODY 3, and a mutation of hepatocyte nuclear factor-1 alpha gene was found. The same mutation was found in the mother who had never been referred for hyperglycemia. After 1 yr, due to an unjustified worsening of the metabolic control, autoimmunity was again investigated and a mild positivity was found. He then required insulin therapy and after 5 yr current HbA1c was 8.2%. The diagnosis of MODY does not exclude the risk of developing T1DM. Therefore autoimmunity should be investigated when ordinary treatments fail and metabolic control unexpectedly worsens

Prospective evaluation of aberrant p16 methylation in serum of patients before and after therapy for localized HCC

none7Abstract della presentazione di dati originali al 58th Annual Meeting of the American Association for the Study of Liver Diseases (AASLD), Boston, MA, November 2-6 2007, pubblicato sulla rivista ufficiale Hepatology.noneFerrara F.; Lodato F.; Chieco P.; Mantovani V.; Colecchia A.; Roda E.; Brillanti S.Ferrara F.; Lodato F.; Chieco P.; Mantovani V.; Colecchia A.; Roda E.; Brillanti S

First evidence of d-HPLC efficiency for an automated cDNA-AFLP in the apple scab resistance model.

Apple scab resistance is one of the most well-characterized plant-pathogen interactions in woody plant species. After the cloning of the apple scab resistance gene HcrVf2, we aimed at identifying the network of genes that are differentially expressed after pathogen challenge by cDNA-AFLP. As electrophoretic analyses are labour-intensive with only limited potential for automation and the recovery of DNA fragments from gels is cumbersome, we investigated the possibility of performing the cDNA-AFLP analysis by d-HPLC (denaturing high performance liquid chromatography) and automating the DNA fragments collection using the Transgenomic WAVE\uae System. Direct sequencing of the collected DNA fragments was also carried out and our preliminary results are reported

Bile salt export pump deficiency disease: Two novel, late onset, ABCB11 mutations identified by next generation sequencing

Progressive familial intrahepatic cholestasis (PFIC) is a heterogeneous group of autosomal recessive cholestatic diseases of childhood and represents the main indication for liver transplantation at this age; PFIC2 involves ABCB11 gene, that encodes the ATPdependent canalicular bile salt export pump (BSEP). Benign intrahepatic cholestasis (BRIC) identifies a group of diseases involving the same genes and characterized by intermittent attacks of cholestasis with no progression to liver cirrhosis. Diagnosis with standard sequencing techniques is expensive and available only at a few tertiary centers. We report the application of next generation sequencing (NGS) in the diagnosis of the familial intrahepatic cholestasis with a parallel sequencing of three causative genes. We identified the molecular defects in ABCB11 gene in two different probands who developed a severe cholestatic disease of unknown origin. In the first patient a compound heterozygosity for the novel frameshift mutation p.Ser1100GlnfsX38 and the missense variant p.Glu135Lys was detected. In the second patient, triggered by contraceptive therapy, we identified homozygosity for a novel missense variant p.Ala523Gly. In conclusion, these mutations seem to have a late onset and a less aggressive clinical impact, acting as an intermediate form between BRIC and PFIC