Revisiting the Security of the ALRED Design and Two of Its Variants: Marvin and LetterSoup

The ALRED construction is a lightweight strategy for constructing message authentication algorithms from an underlying iterated block cipher. Even though this construction's original analyses show that it is secure against some attacks, the absence of formal security proofs in a strong security model still brings uncertainty on its robustness. In this paper, aiming to give a better understanding of the security level provided by different authentication algorithms based on this design strategy, we formally analyze two ALRED variants-the MARVIN message authentication code and the LETTERSOUP authenticated-encryption scheme,-bounding their security as a function of the attacker's resources and of the underlying cipher's characteristics.

Effect of Genetic Variants Related to Lipid Metabolism as Risk Factors for Cholelithiasis After Bariatric Surgery in Brazilian Population

The manifestation of cholelithiasis after bariatric surgery may depend on genetic factors related to lipid metabolism, including apolipoprotein E (APOE) and cholesteryl ester transfer protein (CETP) gene polymorphisms. We investigated the association between APOE HhaI and CETP TaqIB polymorphisms [PCR-RFLP] and occurrence of cholelithiasis over up to 8 months of follow-up after gastroplasty to Roux-en-Y gastric bypass in 220 patients distributed in Group 1 (G1) 114 with cholelithiasis postoperatively and Group 2 (G2) 106 without cholelithiasis, including biochemical and anthropometric profiles analyses. In our series, the allelic and genotypic distributions of CETP TaqIB and APOE HhaI polymorphisms were similar in both groups (P > 0.05). The subgroup analysis evidenced that 54% of the patients from G1, APOE*4 allele carriers compared with APOE*3/3 carriers, presented altered low-density lipoprotein cholesterol (LDL cholesterol) serum levels (P = 0.022) before bariatric surgery. The B1 allele for CETP was associated to more quickly elevation of HDL cholesterol levels just in individuals without cholelitiasis (P < 0.0001). The multivariate logistic regression analysis demonstrates correlation between APOE*4 allele, higher total cholesterol (TC) serum levels and prediposition to cholelitiasis in preoperative period. However, the presence of postoperative cholelithiasis was not associated with altered lipid profile. The CETP TaqIB and APOE HhaI polymorphisms do not seem to have association with gallstones in the late postoperative bariatric surgery, considering that these genetic variants do not differ subgroups of patients who are eligible to routine prophylactic cholecystectomy, at least in Brazilian population.

Functional matrix metalloproteinase (MMP)-9 genetic variants modify the effects of hemodialysis on circulating MMP-9 levels

Background: Altered levels of matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), are involved in cardiovascular alterations associated with end stage kidney disease (ESKD). Genetic polymorphisms in MMP-9 gene affect MMP-9 levels. We examined how MMP-9 polymorphisms and haplotypes affect the changes in plasma MMP-9 and TIMP-1 levels found in patients with ESKD undergoing hemodialysis. Methods: We studied 94 ESKD patients undergoing hemodialysis for at least 3 months. MMP-9 and TIMP-1 were measured by ELISA in plasma from blood samples collected before and after a session of hemodialysis. Genotypes for three MMP-9 polymorphisms (C-1562T, rs3918242; -90 (CA)(14-24), rs2234681; and Q279R, rs17576) were determined by Taqman (R) Allele Discrimination Assay and real-time polymerase chain reaction. Haplotype frequencies were determined with the software program PHASE 2.1. Results: Hemodialysis increased MMP-9 and TIMP-1 levels (P<0.05). Genotypes had no effects on baseline MMP-9 and TIMP-1 levels (P>0.05). Hemodialysis increased MMP-9 and TIMP-1 levels in subjects with the CC (but not CT or TT) genotype for the C-1562T polymorphism (P<0.05), and increased MMP-9 levels in subjects with the QQ (but not QR or RR) genotype for the Q279R polymorphism (P<0.05), whereas the CA(n)(14-24) polymorphism had no major effects. While MMP-9 haplotypes had no effects on baseline MMP-9 levels (P>0.05), hemodialysis increased MMP-9 levels and MMP-9/TIMP-1 ratios in subjects carrying the CLQ haplotype (P = 0.0012 and P = 0.0045, respectively). Conclusion: ESKD patients with the QQ genotype for the Q279R polymorphism or with the CLQ haplotype are exposed to more severe increases in MMP-9 levels after hemodialysis. Such patients may benefit from the use of MMP inhibitors. (C) 2012 Elsevier B.V. All rights reserved.

Sequence and Copy Number Analyses of HEXB Gene in Patients Affected by Sandhoff Disease: Functional Characterization of 9 Novel Sequence Variants

Sandhoff disease (SD) is a lysosomal disorder caused by mutations in the HEXB gene. To date, 43 mutations of HEXB have been described, including 3 large deletions. Here, we have characterized 14 unrelated SD patients and developed a Multiplex Ligation-dependent Probe Amplification (MLPA) assay to investigate the presence of large HEXB deletions. Overall, we identified 16 alleles, 9 of which were novel, including 4 sequence variation leading to aminoacid changes [c.626C>T (p.T209I), c.634C>A (p.H212N), c.926G>T (p.C309F), c.1451G>A (p.G484E)] 3 intronic mutations (c.1082+5G>A, c.1242+1G>A, c.1169+5G>A), 1 nonsense mutation c.146C>A (p.S49X) and 1 small in-frame deletion c.1260_1265delAGTTGA (p.V421_E422del). Using the new MLPA assay, 2 previously described deletions were identified. In vitro expression studies showed that proteins bearing aminoacid changes p.T209I and p.G484E presented a very low or absent activity, while proteins bearing the p.H212N and p.C309F changes retained a significant residual activity. The detrimental effect of the 3 novel intronic mutations on the HEXB mRNA processing was demonstrated using a minigene assay. Unprecedentedly, minigene studies revealed the presence of a novel alternative spliced HEXB mRNA variant also present in normal cells. In conclusion, we provided new insights into the molecular basis of SD and validated an MLPA assay for detecting large HEXB deletions.

A new portal for the analysis of human splicing variants

Understanding alternative splicing is crucial to elucidate the mechanisms behind several biological phenomena, including diseases. The huge amount of expressed sequences available nowadays represents an opportunity and a challenge to catalog and display alternative splicing events (ASEs). Although several groups have faced this challenge with relative success, we still lack a computational tool that uses a simple and straightforward method to retrieve, name and present ASEs. Here we present SPLOOCE, a portal for the analysis of human splicing variants. SPLOOCE uses a method based on regular expressions for retrieval of ASEs. We propose a simple syntax that is able to capture the complexity of ASEs.

Protective Effect of Human Endogenous Retrovirus K dUTPase Variants on Psoriasis Susceptibility

Previous genetic and functional studies have implicated the human endogenous retrovirus K (HERV-K) dUTPase located within the PSORS1 locus in the major histocompatibility complex region as a candidate psoriasis gene. Here, we describe a variant discovery and case-control association study of HERV-K dUTPase variants in 708 psoriasis cases and 349 healthy controls. Five common HERV-K dUTPase variants were found to be highly associated with psoriasis, with the strongest association occurring at the missense single-nucleotide polymorphism (SNP) rs3134774 (K158R, P=3.28 x 10(-15), odds ratio = 2.36 (95% confidence interval: 1.91-2.92)). After adjusting the association of the HERV-K dUTPase variants for the potential confounding effects of HLA alleles associated with psoriasis, the HERV-K SNPs rs9264082 and rs3134774 remained significantly associated. Haplotype analysis revealed that HERV-K haplotypes containing the non-risk alleles for rs3134774 and rs9264082 significantly reduced the risk of psoriasis. Functional testing showed higher antibody responses against recombinant HERV-K dUTPase in psoriasis patients compared with controls (P<0.05), as well as higher T-cell responses against a single HERV-K dUTPase peptide (P<0.05). Our data support an independent role for the HERV-K dUTPase on psoriasis susceptibility, and suggest the need for additional studies to clarify the role of this dUTPase in the pathogenesis of psoriasis. Journal of Investigative Dermatology (2012) 132, 1833-1840; doi:10.1038/jid.2012.69; published online 22 March 2012

Growth hormone pharmacogenetics: the interactive effect of a microsatellite in the IGF1 promoter region with the GHR-exon 3 and-202 A/C IGFBP3 variants on treatment outcomes of children with severe GH deficiency

Insulin-like growth factor type 1 (IGF1) is a mediator of growth hormone (GH) action, and therefore, IGF1 is a candidate gene for recombinant human GH (rhGH) pharmacogenetics. Lower serum IGF1 levels were found in adults homozygous for 19 cytosine-adenosine (CA) repeats in the IGF1 promoter. The aim of this study was to evaluate the influence of (CA)n IGF1 polymorphism, alone or in combination with GH receptor (GHR)-exon 3 and -202 A/C insulin-like growth factor binding protein-3 (IGFBP3) polymorphisms, on the growth response to rhGH therapy in GH-deficient (GHD) patients. Eighty-four severe GHD patients were genotyped for (CA) n IGF1, -202 A/C IGFBP3 and GHR-exon 3 polymorphisms. Multiple linear regressions were performed to estimate the effect of each genotype, after adjustment for other influential factors. We assessed the influence of genotypes on the first year growth velocity (1st y GV) (n = 84) and adult height standard deviation score (SDS) adjusted for target-height SDS (AH-TH SDS) after rhGH therapy (n = 37). Homozygosity for the IGF1 19CA repeat allele was negatively correlated with 1st y GV (P = 0.03) and AH-TH SDS (P = 0.002) in multiple linear regression analysis. In conjunction with clinical factors, IGF1 and IGFBP3 genotypes explain 29% of the 1st y GV variability, whereas IGF1 and GHR polymorphisms explain 59% of final height-target-height SDS variability. We conclude that homozygosity for IGF1 (CA) 19 allele is associated with less favorable short-and long-term growth outcomes after rhGH treatment in patients with severe GHD. Furthermore, this polymorphism exhibits a non-additive interaction with -202 A/C IGFBP3 genotype on the 1st y GV and with GHR-exon 3 genotype on adult height. The Pharmacogenomics Journal (2012) 12, 439-445; doi:10.1038/tpj.2011.13; published online 5 April 2011

Structural studies of Helicase NS3 variants from Hepatitis C virus genotype 3 in virological sustained responder and non-responder patients

Abstract Background About 130 million people are infected with the hepatitis C virus (HCV) worldwide, but effective treatment options are not yet available. One of the most promising targets for antiviral therapy is nonstructural protein 3 (NS3). To identify possible changes in the structure of NS3 associated with virological sustained response or non-response of patients, a model was constructed for each helicase NS3 protein coding sequence. From this, the goal was to verify the interaction between helicases variants and their ligands. Findings Evidence was found that the NS3 helicase portion of non-responder patients contained substitutions in its ATP and RNA binding sites. K210E substitution can cause an imbalance in the distribution of loads, leading to a decrease in the number of ligations between the essential amino acids required for the hydrolysis of ATP. W501R substitution causes an imbalance in the distribution of loads, leading and forcing the RNA to interact with the amino acid Thr269, but not preventing binding of ribavirin inhibitor. Conclusions Useful information is provided on the genetic profiling of the HCV genotype 3, specifically the coding region of the NS3 protein, improving our understanding of the viral genome and the regions of its protein catalytic site.

Association of genetic variants in the promoter region of genes encoding p22phox (CYBA) and glutamate cysteine ligase catalytic subunit (GCLC) and renal disease in patients with type 1 diabetes mellitus

Background Oxidative stress is recognized as a major pathogenic factor of cellular damage caused by hyperglycemia. NOX/NADPH oxidases generate reactive oxygen species and NOX1, NOX2 and NOX4 isoforms are expressed in kidney and require association with subunit p22phox (encoded by the CYBA gene). Increased expression of p22phox was described in animal models of diabetic nephropathy. In the opposite direction, glutathione is one of the main endogenous antioxidants whose plasmatic concentrations were reported to be reduced in diabetes patients. The aim of the present investigation was to test whether functional single nucleotide polymorphisms (SNPs) in genes involved in the generation of NADPH-dependent O2•- (-675 T → A in CYBA, unregistered) and in glutathione metabolism (-129 C → T in GCLC [rs17883901] and -65 T → C in GPX3 [rs8177412]) confer susceptibility to renal disease in type 1 diabetes patients. Methods 401 patients were sorted into two groups according to the presence (n = 104) or absence (n = 196) of overt diabetic nephropathy or according to glomerular filtration rate (GFR) estimated by Modification of Diet in Renal Disease (MDRD) equation: ≥ 60 mL (n = 265) or < 60 mL/min/1.73 m2 (n = 136) and were genotyped. Results No differences were found in the frequency of genotypes between diabetic and non-diabetic subjects. The frequency of GFR < 60 mL/min was significantly lower in the group of patients carrying CYBA genotypes T/A+A/A (18.7%) than in the group carrying the T/T genotype (35.3%) (P = 0.0143) and the frequency of GFR < 60 mL/min was significantly higher in the group of patients carrying GCLC genotypes C/T+T/T (47.1%) than in the group carrying the C/C genotype (31.1%) (p = 0.0082). Logistic regression analysis identified the presence of at least one A allele of the CYBA SNP as an independent protection factor against decreased GFR (OR = 0.38, CI95% 0.14-0.88, p = 0.0354) and the presence of at least one T allele of the GCLC rs17883901 SNP as an independent risk factor for decreased GFR (OR = 2.40, CI95% 1.27-4.56, p = 0.0068). Conclusions The functional SNPs CYBA -675 T → A and GCLC rs17883901, probably associated with cellular redox imbalances, modulate the risk for renal disease in the studied population of type 1 diabetes patients and require validation in additional cohorts.

Different approaches for dealing with rare variants in family-based genetic studies: application of a Genetic Analysis Workshop 17 problem

Rare variants are becoming the new candidates in the search for genetic variants that predispose individuals to a phenotype of interest. Their low prevalence in a population requires the development of dedicated detection and analytical methods. A family-based approach could greatly enhance their detection and interpretation because rare variants are nearly family specific. In this report, we test several distinct approaches for analyzing the information provided by rare and common variants and how they can be effectively used to pinpoint putative candidate genes for follow-up studies. The analyses were performed on the mini-exome data set provided by Genetic Analysis Workshop 17. Eight approaches were tested, four using the trait’s heritability estimates and four using QTDT models. These methods had their sensitivity, specificity, and positive and negative predictive values compared in light of the simulation parameters. Our results highlight important limitations of current methods to deal with rare and common variants, all methods presented a reduced specificity and, consequently, prone to false positive associations. Methods analyzing common variants information showed an enhanced sensibility when compared to rare variants methods. Furthermore, our limited knowledge of the use of biological databases for gene annotations, possibly for use as covariates in regression models, imposes a barrier to further research.

Influence of genetic variants and post transcriptional factors on imatinib transporters as determinants of the pharmacological response

Introduction – Although imatinib (IM) is a recognized gold standard in chronic myeloid leukemia (CML) therapy, resistance has emerged in a significant proportion of patients. Aim – The aim of this study was: (1) to investigate the role of genetic variants in genes encoding for IM transporters, as candidate of IM responsiveness and (2) to test the influence of miRNAs on IM response, focusing on efflux transporters. Methods – As a first step, a panel of polymorphisms (SNPs) was genotyped in a subgroup population of 189 patients enrolled in the Tyrosine Kinase Inhibitor Optimization and Selectivity (TOPS) trial. The association with cytogenetic response and molecular response (MR) was assessed for each SNP. As a second step, an in vitro IM-resistant model (K-562 CML cell line) was established. miRNAs profiles were analyzed using Taqman arrays and in silico search was performed for miRNAs deregulated after IM treatment. mRNA and protein expression were quantified using TaqMan realtime PCR and Western blotting, respectively. Results – (1) Among Caucasian patients, ABCB1 rs60023214 significantly correlated with complete MR (P = 0.005). Concerning SNPs combination in IM uptake transporters, the associations with treatment outcomes were statistically significant for both major and complete MR (P = 0.005 and P = 0.01, respectively). (2) ABCB1 protein was not expressed under any conditions of treatment, differently from ABCG2. Two deregulated miRNAs, namely miR-212 and miR-328, were identified to be inversely correlated with ABCG2 (r2= 0.57; p=0.03 and r2=0.47; p=0.06, respectively). Experiments of loss and gain of function confirmed the functional influence of these miRNAs on ABCG2. Conclusion – The multiple candidate gene approach identified single and combination of SNPs that can be proposed as predictor of IM response. The in vitro study suggested that IM resistance could be mediated by miRNA-dependent mechanism. Further studies are needed to validate these preliminary findings.

Investigating the role of Copy Number Variants in Specific Language Impairment and identification of new candidate genes

Specific language impairment (SLI) is a complex neurodevelopmental disorder defined as an unexpected failure to develop normal language abilities for no obvious reason. Copy number variants (CNVs) are an important source of variation in the susceptibility to neuropsychiatric disorders. Therefore, a CNV study within SLI families was performed to investigate the role of structural variants in SLI. Among the identified CNVs, we focused on CNVs on chromosome 15q11-q13, recurrently observed in neuropsychiatric conditions, and a homozygous exonic microdeletion in ZNF277. Since this microdeletion falls within the AUTS1 locus, a region linked to autism spectrum disorders (ASD), we investigated a potential role of ZNF277 in SLI and ASD. Frequency data and expression analysis of the ZNF277 microdeletion suggested that this variant may contribute to the risk of language impairments in a complex manner, that is independent of the autism risk previously described in this region. Moreover, we identified an affected individual with a dihydropyrimidine dehydrogenase (DPD) deficiency, caused by compound heterozygosity of two deleterious variants in the gene DPYD. Since DPYD represents a good candidate gene for both SLI and ASD, we investigated its involvement in the susceptibility to these two disorders, focusing on the splicing variant rs3918290, the most common mutation in the DPD deficiency. We observed a higher frequency of rs3918290 in SLI cases (1.2%), compared to controls (~0.6%), while no difference was observed in a large ASD cohort. DPYD mutation screening in 4 SLI and 7 ASD families carrying the splicing variant identified six known missense changes and a novel variant in the promoter region. These data suggest that the combined effect of the mutations identified in affected individuals may lead to an altered DPD activity and that rare variants in DPYD might contribute to a minority of cases, in conjunction with other genetic or non-genetic factors.

Analysis of Copy Number Variants identifies new candidate genes for Autism Spectrum Disorder and Intellectual Disability

Autism spectrum disorder (ASD) and Intellectual Disability (ID) are complex neuropsychiatric disorders characterized by extensive clinical and genetic heterogeneity and with overlapping risk factors. The aim of my project was to further investigate the role of Copy Numbers Variants (CNVs), identified through genome-wide studies performed by the Autism Geome Project (AGP) and the CHERISH consortium in large cohorts of ASD and ID cases, respectively. Specifically, I focused on four rare genic CNVs, selected on the basis of their impact on interesting ASD/ID candidate genes: a) a compound heterozygous deletion involving CTNNA3, predicted to cause the lack of functional protein; b) a 15q13.3 duplication containing CHRNA7; c) a 2q31.1 microdeletion encompassing KLHL23, SSB and METTL5; d) Lastly, I investigated the putative imprinting regulation of the CADPS2 gene, disrupted by a maternal deletion in two siblings with ASD and ID. This study provides further evidence for the role of CTNNA3, CHRNA7, KLHL23 and CADPS2 as ASD and/or ID susceptibility genes, and highlights that rare genetic variation contributes to disease risk in different ways: some rare mutations, such as those impacting CTNNA3, act in a recessive mode of inheritance, while other CNVs, such as those occurring in the 15q13.3 region, are implicated in multiple developmental and/or neurological disorders possibly interacting with other susceptibility variants elsewhere in the genome. On the other hand, the discovery of a tissue-specific monoallelic expression for the CADPS2 gene, implicates the involvement of epigenetic regulatory mechanisms as risk factors conferring susceptibility to ASD/ID.

Multiple LRRK2 variants modulate risk of Parkinson disease: a Chinese multicenter study

We and others found two polymorphic LRRK2 (leucine-rich repeat kinase 2) variants (rs34778348:G>A; p.G2385R and rs33949390:G>C; p.R1628P) associated with Parkinson disease (PD) among Chinese patients, but the common worldwide rs34637584:G>A; p.G2019S mutation, was absent. Focusing exclusively on Han Chinese, we first sequenced the coding regions in young onset and familial PD patients and identified 59 variants. We then examined these variants in 250 patients and 250 control subjects. Among the 17 polymorphic variants, five demonstrated different frequency in cases versus controls and were considered in a larger sample of 1,363 patients and 1,251 control subjects. The relative risk of an individual with both p.G2385R and p.R1628P is about 1.9, and this is reduced to 1.5-1.6 if the individual also carries rs7133914:G>C; p.R1398H or rs7308720:C>A: p.N551K. The risk of a carrier with p.R1628P is largely negated if the individual also carries p.R1398H or p.N551K. In dopaminergic neuronal lines, p.R1398H had significantly lower kinase activity, whereas p.G2385R and p.R1628P showed higher kinase activity than wild type. We provided the first evidence that multiple LRRK2 variants exert an individual effect and together modulate the risk of PD among Chinese.