Minor Hypospadias: The “Tip of the Iceberg” of the Partial Androgen Insensitivity Syndrome

Background

Androgens are critical in male external genital development. Alterations in the androgen sensitivity pathway have been identified in severely undermasculinized boys, and mutations of the androgen receptor gene (AR) are usually found in partial or complete androgen insensitivity syndrome (AIS).

Objective

The aim of this study was to determine whether even the most minor forms of isolated hypospadias are associated with AR mutations and thus whether all types of hypospadias warrant molecular analysis of the AR.

Materials and Methods

Two hundred and ninety-two Caucasian children presenting with isolated hypospadias without micropenis or cryptorchidism and 345 controls were included prospectively. Mutational analysis of the AR through direct sequencing (exons 1–8) was performed. In silico and luciferase functional assays were performed for unreported variants.

Results

Five missense mutations of the AR were identified in 9 patients with glandular or penile anterior (n = 5), penile midshaft (n = 2) and penile posterior (n = 2) hypospadias, i.e., 3%: p.Q58L (c.173A>T), 4 cases of p.P392S (c.1174C>T), 2 cases of p.A475V (c.1424C>T), p.D551H (c.1651G>C) and p.Q799E (c.2395C>G). None of these mutations was present in the control group. One mutation has never been reported to date (p.D551H). It was predicted to be damaging based on 6 in silico models, and in vitro functional studies confirmed the lowered transactivation function of the mutated protein. Three mutations have never been reported in patients with genital malformation but only in isolated infertility: p.Q58L, p.P392S, and p.A475V. It is notable that micropenis, a cardinal sign of AIS, was not present in any patient.

Conclusion

AR mutations may play a role in the cause of isolated hypospadias, even in the most minor forms. Identification of this underlying genetic alteration may be important for proper diagnosis and longer follow-up is necessary to find out if the mutations cause differences in sexual function and fertility later in life.     

Functional Consequences of a Novel Variant of PCSK1

Background

Common single nucleotide polymorphisms (SNPs) in proprotein convertase subtilisin/kexin type 1 with modest effects on PC1/3 in vitro have been associated with obesity in five genome-wide association studies and with diabetes in one genome-wide association study. We here present a novel SNP and compare its biosynthesis, secretion and catalytic activity to wild-type enzyme and to SNPs that have been linked to obesity.

Methodology/Principal Findings

A novel PC1/3 variant introducing an Arg to Gln amino acid substitution at residue 80 (within the secondary cleavage site of the prodomain) (rs1799904) was studied. This novel variant was selected for analysis from the 1000 Genomes sequencing project based on its predicted deleterious effect on enzyme function and its comparatively more frequent allele frequency. The actual existence of the R80Q (rs1799904) variant was verified by Sanger sequencing. The effects of this novel variant on the biosynthesis, secretion, and catalytic activity were determined; the previously-described obesity risk SNPs N221D (rs6232), Q665E/S690T (rs6234/rs6235), and the Q665E and S690T SNPs (analyzed separately) were included for comparative purposes. The novel R80Q (rs1799904) variant described in this study resulted in significantly detrimental effects on both the maturation and in vitro catalytic activity of PC1/3.

Conclusion/Significance

Our findings that this novel R80Q (rs1799904) variant both exhibits adverse effects on PC1/3 activity and is prevalent in the population suggests that further biochemical and genetic analysis to assess its contribution to the risk of metabolic disease within the general population is warranted.     

Characterization of Two New CTX-M-25-Group Extended-Spectrum β-Lactamase Variants Identified in Escherichia coli Isolates from Israel

Objectives

We characterized two new CTX-M-type extended-spectrum β-lactamase (ESBL) variants in Escherichia coli isolates from stool samples of two elderly patients admitted at the Tel Aviv Sourasky Medical Center, Israel. Both patients underwent treatment with cephalosporins prior to isolation of the E. coli strains.

Methods

ESBLs were detected by the double-disk synergy test and PCR-sequencing of β-lactamase genes. The bla _CTX-M genes were cloned into the pCR-BluntII-TOPO vector in E. coli TOP10. The role of amino-acid substitutions V77A and D240G was analyzed by site-directed mutagenesis of the bla _CTX-M-94 and bla _CTX-M-100 genes and comparative characterization of the resulting E. coli recombinants. MICs of β-lactams were determined by Etest. Plasmid profiling, mating experiments, replicon typing and sequencing of bla _CTX-M flanking regions were performed to identify the genetic background of the new CTX-M variants.

Results

The novel CTX-M β-lactamases, CTX-M-94 and -100, belonged to the CTX-M-25-group. Both variants differed from CTX-M-25 by the substitution V77A, and from CTX-M-39 by D240G. CTX-M-94 differed from all CTX-M-25-group enzymes by the substitution F119L. Glycine-240 was associated with reduced susceptibility to ceftazidime and leucine-119 with increased resistance to ceftriaxone. bla _CTX-M-94 and bla _CTX-M-100 were located within ISEcp1 transposition units inserted into ∼93 kb non-conjugative IncFI and ∼130 kb conjugative IncA/C plasmids, respectively. The plasmids carried also different class 1 integrons.

Conclusions

This is the first report on CTX-M-94 and -100 ESBLs, novel members of the CTX-M-25-group.     

The diagnostic application of targeted re-sequencing in Korean patients with retinitis pigmentosa

Background

Identification of the causative genes of retinitis pigmentosa (RP) is important for the clinical care of patients with RP. However, a comprehensive genetic study has not been performed in Korean RP patients. Moreover, the genetic heterogeneity found in sensorineural genetic disorders makes identification of pathogenic mutations challenging. Therefore, high throughput genetic testing using massively parallel sequencing is needed.

Results

Sixty-two Korean patients with nonsyndromic RP (46 patients from 18 families and 16 simplex cases) who consented to molecular genetic testing were recruited in this study and targeted exome sequencing was applied on 53 RP-related genes. Causal variants were characterised by selecting exonic and splicing variants, selecting variants with low allele frequency (below 1 %), and discarding the remaining variants with quality below 20. The variants were additionally confirmed by an inheritance pattern and cosegregation test of the families, and the rest of the variants were prioritised using in-silico prediction tools. Finally, causal variants were detected from 10 of 18 familial cases (55.5 %) and 7 of 16 simplex cases (43.7 %) in total. Novel variants were detected in 13 of 20 (65 %) candidate variants. Compound heterozygous variants were found in four of 7 simplex cases.

Conclusion

Panel-based targeted re-sequencing can be used as an effective molecular diagnostic tool for RP.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1723-x) contains supplementary material, which is available to authorized users.     

Predictors of Appropriate ICD Therapy in Patients with Arrhythmogenic Right Ventricular Cardiomyopathy: Long Term Experience of a Tertiary Care Center

Introduction

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a rare genetically transmitted disease prone to ventricular arrhythmias. We therefore investigated the clinical, echocardiographical and electrophysiological predictors of appropriate implantable cardioverter defibrillator (ICD) therapy in patients with ARVC.

Methods

A retrospective analysis was performed in 26 patients (median age of 40 years at diagnosis, 21 males and 5 females) with ARVC who underwent ICD implantation.

Results

Over a median (range) follow-up period of 10 (2.7, 37) years, appropriate ICD therapy for ventricular arrhythmias was documented in 12 (46%) out of 26 patients. In all patients with appropriate ICD therapy the ICD was originally inserted for secondary prevention. Median time from ICD implantation to ICD therapy was 9 months (range 3.6, 54 months). History of heart failure was a significant predictor of appropriate ICD therapy (p = 0.033). Left ventricular disease involvement (p = 0.059) and age at implantation (p = 0.063) were borderline significant predictors. Patients with syncope at time of diagnosis were significantly less likely to receive ICD therapy (p = 0.02). Invasive electrophysiological testing was not significantly associated with appropriate ICD therapy.

Conclusion

In our cohort of patients with ARVC, history of heart failure was a significant predictor of appropriate ICD therapy, whereas left ventricular involvement and age at time of ICD implantation were of borderline significance. These predictors should be tested in larger prospective cohorts to optimize ICD therapy in this rare cardiomyopathy.     

Complex Analysis of Urate Transporters SLC2A9, SLC22A12 and Functional Characterization of Non-Synonymous Allelic Variants of GLUT9 in the Czech Population: No Evidence of Effect on Hyperuricemia and Gout

Objective

Using European descent Czech populations, we performed a study of SLC2A9 and SLC22A12 genes previously identified as being associated with serum uric acid concentrations and gout. This is the first study of the impact of non-synonymous allelic variants on the function of GLUT9 except for patients suffering from renal hypouricemia type 2.

Methods

The cohort consisted of 250 individuals (150 controls, 54 nonspecific hyperuricemics and 46 primary gout and/or hyperuricemia subjects). We analyzed 13 exons of SLC2A9 (GLUT9 variant 1 and GLUT9 variant 2) and 10 exons of SLC22A12 by PCR amplification and sequenced directly. Allelic variants were prepared and their urate uptake and subcellular localization were studied by Xenopus oocytes expression system. The functional studies were analyzed using the non-parametric Wilcoxon and Kruskall-Wallis tests; the association study used the Fisher exact test and linear regression approach.

Results

We identified a total of 52 sequence variants (12 unpublished). Eight non-synonymous allelic variants were found only in SLC2A9: rs6820230, rs2276961, rs144196049, rs112404957, rs73225891, rs16890979, rs3733591 and rs2280205. None of these variants showed any significant difference in the expression of GLUT9 and in urate transport. In the association study, eight variants showed a possible association with hyperuricemia. However, seven of these were in introns and the one exon located variant, rs7932775, did not show a statistically significant association with serum uric acid concentration.

Conclusion

Our results did not confirm any effect of SLC22A12 and SLC2A9 variants on serum uric acid concentration. Our complex approach using association analysis together with functional and immunohistochemical characterization of non-synonymous allelic variants did not show any influence on expression, subcellular localization and urate uptake of GLUT9.     

Dynamic Conduction and Repolarisation Changes in Early Arrhythmogenic Right Ventricular Cardiomyopathy versus Benign Outflow Tract Ectopy Demonstrated by High Density Mapping & Paced Surface ECG Analysis

Aims

The concealed phase of arrhythmogenic right ventricular cardiomyopathy (ARVC) may initially manifest electrophysiologically. No studies have examined dynamic conduction/repolarization kinetics to distinguish benign right ventricular outflow tract ectopy (RVOT ectopy) from ARVC''s early phase. We investigated dynamic endocardial electrophysiological changes that differentiate early ARVC disease expression from RVOT ectopy.

Methods

22 ARVC (12 definite based upon family history and mutation carrier status, 10 probable) patients without right ventricular structural anomalies underwent high-density non-contact mapping of the right ventricle. These were compared to data from 14 RVOT ectopy and 12 patients with supraventricular tachycardias and normal hearts. Endocardial & surface ECG conduction and repolarization parameters were assessed during a standard S₁-S₂ restitution protocol.

Results

Definite ARVC without RV structural disease could not be clearly distinguished from RVOT ectopy during sinus rhythm or during steady state pacing. Delay in Activation Times at coupling intervals just above the ventricular effective refractory period (VERP) increased in definite ARVC (43±20 ms) more than RVOT ectopy patients (36±14 ms, p = 0.03) or Normals (25±16 ms, p = 0.008) and a progressive separation of the repolarisation time curves between groups existed. Repolarization time increases in the RVOT were also greatest in ARVC (definite ARVC: 18±20 ms; RVOT ectopy: 5±14, Normal: 1±18, p<0.05). Surface ECG correlates of these intracardiac measurements demonstrated an increase of greater than 48 ms in stimulus to surface ECG J-point pre-ERP versus steady state, with an 88% specificity and 68% sensitivity in distinguishing definite ARVC from the other groups. This technique could not distinguish patients with genetic predisposition to ARVC only (probable ARVC) from controls.

Conclusions

Significant changes in dynamic conduction and repolarization are apparent in early ARVC before detectable RV structural abnormalities, and were present to a lesser degree in probable ARVC patients. Investigation of dynamic electrophysiological parameters may be useful to identify concealed ARVC in patients without disease pedigrees by using endocardial electrogram or paced ECG parameters.     

CTAG-Containing Cleavage Site Profiling to Delineate Salmonella into Natural Clusters

Background

The bacterial genus Salmonella contains thousands of serotypes that infect humans or other hosts, causing mild gastroenteritis to potentially fatal systemic infections in humans. Pathogenically distinct Salmonella serotypes have been classified as individual species or as serological variants of merely one or two species, causing considerable confusion in both research and clinical settings. This situation reflects a long unanswered question regarding whether the Salmonella serotypes exist as discrete genetic clusters (natural species) of organisms or as phenotypic (e.g. pathogenic) variants of a single (or two) natural species with a continuous spectrum of genetic divergence among them. Our recent work, based on genomic sequence divergence analysis, has demonstrated that genetic boundaries exist among Salmonella serotypes, circumscribing them into clear-cut genetic clusters of bacteria.

Methodologies/Principal Findings

To further test the genetic boundary concept for delineating Salmonella into clearly defined natural lineages (e.g., species), we sampled a small subset of conserved genomic DNA sequences, i.e., the endonuclease cleavage sites that contain the highly conserved CTAG sequence such as TCTAGA for XbaI. We found that the CTAG-containing cleavage sequence profiles could be used to resolve the genetic boundaries as reliably and efficiently as whole genome sequence comparisons but with enormously reduced requirements for time and resources.

Conclusions

Profiling of CTAG sequence subsets reflects genetic boundaries among Salmonella lineages and can delineate these bacteria into discrete natural clusters.     

Rational design of a plasmid origin that replicates efficiently in both gram-positive and gram-negative bacteria

Background

Most plasmids replicate only within a particular genus or family.

Methodology/Principal Findings

Here we describe an engineered high copy number expression vector, pBAV1K-T5, that produces varying quantities of active reporter proteins in Escherichia coli, Acinetobacter baylyi ADP1, Agrobacterium tumefaciens, (all Gram-negative), Streptococcus pneumoniae, Leifsonia shinshuensis, Peanibacillus sp. S18-36 and Bacillus subtilis (Gram-positive).

Conclusions/Significance

Our results demonstrate the efficiency of pBAV1K-T5 replication in different bacterial species, thereby facilitating the study of proteins that don''t fold well in E. coli and pathogens not amenable to existing genetic tools.     

DRD2/CHRNA5 Interaction on Prefrontal Biology and Physiology during Working Memory

Background

Prefrontal behavior and activity in humans are heritable. Studies in animals demonstrate an interaction between dopamine D2 receptors and nicotinic acetylcholine receptors on prefrontal behavior but evidence in humans is weak. Therefore, we hypothesize that genetic variation regulating dopamine D2 and nicotinic acetylcholine receptor signaling impact prefrontal cortex activity and related cognition. To test this hypothesis in humans, we explored the interaction between functional genetic variants in the D2 receptor gene (DRD2, rs1076560) and in the nicotinic receptor α5 gene (CHRNA5, rs16969968) on both dorsolateral prefrontal cortex mediated behavior and physiology during working memory and on prefrontal gray matter volume.

Methods

A large sample of healthy subjects was compared for genotypic differences for DRD2 rs1076560 (G>T) and CHNRA5 rs16969968 (G>A) on prefrontal phenotypes, including cognitive performance at the N-Back task, prefrontal physiology with BOLD fMRI during performance of the 2-Back working memory task, and prefrontal morphometry with structural MRI.

Results

We found that DRD2 rs1076560 and CHNRA5 rs16969968 interact to modulate cognitive function, prefrontal physiology during working memory, and prefrontal gray matter volume. More specifically, CHRNA5-AA/DRD2-GT subjects had greater behavioral performance, more efficient prefrontal cortex activity at 2Back working memory task, and greater prefrontal gray matter volume than the other genotype groups.

Conclusions

The present data extend previous studies in animals and enhance our understanding of dopamine and acetylcholine signaling in the human prefrontal cortex, demonstrating interactions elicited by working memory that are modulated by genetic variants in DRD2 and CHRNA5.     

A Novel Enediynyl Peptide Inhibitor of Furin That Blocks Processing of proPDGF-A,B and proVEGF-C

Background

Furin represents a crucial member of secretory mammalian subtilase, the Proprotein Convertase (PC) or Proprotein Convertase Subtilisin/Kexin (PCSK) superfamily. It has been linked to cancer, tumorgenesis, viral and bacterial pathogenesis. As a result it is considered a major target for intervention of these diseases.

Methodology/Principal Findings

Herein, we report, for the first time, the synthesis and biological evaluation of a newly designed potent furin inhibitor that contains a highly reactive beta-turn inducing and radical generating “enediynyl amino acid” (Eda) moiety. “Eda” was inserted between P1 and P1′ residues of hfurin^98–112 peptide, derived from the primary cleavage site of furin''s own prodomain. The resulting hexadecapeptide derivative inhibited furin in vitro with IC₅₀ ∼40 nM when measured against the fluorogenic substrate Boc-RVRR-MCA. It also inhibited furin-mediated cleavage of a fluorogenic peptide derived from hSARS-CoV spike protein with IC₅₀ ∼193 nM. Additionally it also blocked furin-processing of growth factors proPDGF-A, B and VEGF-C that are linked to tumor genesis and cancer. Circular dichroism study showed that this inhibitor displayed a predominantly beta-turn structure while western blots confirmed its ability to protect furin protein from self degradation.

Conclusion/Significance

These findings imply its potential as a therapeutic agent for intervention of cancer and other furin-associated diseases.     

Analysis of Ultra-Deep Pyrosequencing and Cloning Based Sequencing of the Basic Core Promoter/Precore/Core Region of Hepatitis B Virus Using Newly Developed Bioinformatics Tools

Aims

The aims of this study were to develop bioinformatics tools to explore ultra-deep pyrosequencing (UDPS) data, to test these tools, and to use them to determine the optimum error threshold, and to compare results from UDPS and cloning based sequencing (CBS).

Methods

Four serum samples, infected with either genotype D or E, from HBeAg-positive and HBeAg-negative patients were randomly selected. UDPS and CBS were used to sequence the basic core promoter/precore region of HBV. Two online bioinformatics tools, the “Deep Threshold Tool” and the “Rosetta Tool” (http://hvdr.bioinf.wits.ac.za/tools/), were built to test and analyze the generated data.

Results

A total of 10952 reads were generated by UDPS on the 454 GS Junior platform. In the four samples, substitutions, detected at 0.5% threshold or above, were identified at 39 unique positions, 25 of which were non-synonymous mutations. Sample #2 (HBeAg-negative, genotype D) had substitutions in 26 positions, followed by sample #1 (HBeAg-negative, genotype E) in 12 positions, sample #3 (HBeAg-positive, genotype D) in 7 positions and sample #4 (HBeAg-positive, genotype E) in only four positions. The ratio of nucleotide substitutions between isolates from HBeAg-negative and HBeAg-positive patients was 3.5∶1. Compared to genotype E isolates, genotype D isolates showed greater variation in the X, basic core promoter/precore and core regions. Only 18 of the 39 positions identified by UDPS were detected by CBS, which detected 14 of the 25 non-synonymous mutations detected by UDPS.

Conclusion

UDPS data should be approached with caution. Appropriate curation of read data is required prior to analysis, in order to clean the data and eliminate artefacts. CBS detected fewer than 50% of the substitutions detected by UDPS. Furthermore it is important that the appropriate consensus (reference) sequence is used in order to identify variants correctly.     

Cleavage of the moaX-encoded fused molybdopterin synthase from Mycobacterium tuberculosis is necessary for activity

Background

Molybdopterin cofactor (MoCo) biosynthesis in Mycobacterium tuberculosis is associated with a multiplicity of genes encoding several enzymes in the pathway, including the molybdopterin (MPT) synthase, a hetero tetramer comprising two MoaD and two MoaE subunits. In addition to moaD1, moaD2, moaE1, moaE2, the M. tuberculosis genome also contains a moaX gene which encodes an MPT-synthase in which the MoaD and MoaE domains are located on a single polypeptide. In this study, we assessed the requirement for post-translational cleavage of MoaX for functionality of this novel, fused MPT synthase and attempted to establish a functional hierarchy for the various MPT-synthase encoding genes in M. tuberculosis.

Results

Using a heterologous Mycobacterium smegmatis host and the activity of the MoCo-dependent nitrate reductase, we confirmed that moaD2 and moaE2 from M. tuberculosis together encode a functional MPT synthase. In contrast, moaD1 displayed no functionality in this system, even in the presence of the MoeBR sulphurtransferase, which contains the rhodansese-like domain, predicted to activate MoaD subunits. We demonstrated that cleavage of MoaX into its constituent MoaD and MoaE subunits was required for MPT synthase activity and confirmed that cleavage occurs between the Gly82 and Ser83 residues in MoaX. Further analysis of the Gly81-Gly82 motif confirmed that both of these residues are necessary for catalysis and that the Gly81 was required for recognition/cleavage of MoaX by an as yet unidentified protease. In addition, the MoaE component of MoaX was able to function in conjunction with M. smegmatis MoaD2 suggesting that cleavage of MoaX renders functionally interchangeable subunits. Expression of MoaX in E. coli revealed that incorrect post-translational processing is responsible for the lack of activity of MoaX in this heterologous host.

Conclusions

There is a degree of functional interchangeability between the MPT synthase subunits of M. tuberculosis. In the case of MoaX, post-translational cleavage at the Gly82 residue is required for function.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-015-0355-2) contains supplementary material, which is available to authorized users.     

Targeted exon sequencing fails to identify rare coding variants with large effect in rheumatoid arthritis

Introduction

Although it has been suggested that rare coding variants could explain the substantial missing heritability, very few sequencing studies have been performed in rheumatoid arthritis (RA). We aimed to identify novel functional variants with rare to low frequency using targeted exon sequencing of RA in Korea.

Methods

We analyzed targeted exon sequencing data of 398 genes selected from a multifaceted approach in Korean RA patients (n = 1,217) and controls (n = 717). We conducted a single-marker association test and a gene-based analysis of rare variants. For meta-analysis or enrichment tests, we also used ethnically matched independent samples of Korean genome-wide association studies (GWAS) (n = 4,799) or immunochip data (n = 4,722).

Results

After stringent quality control, we analyzed 10,588 variants of 398 genes from 1,934 Korean RA case controls. We identified 13 nonsynonymous variants with nominal association in single-variant association tests. In a meta-analysis, we did not find any novel variant with genome-wide significance for RA risk. Using a gene-based approach, we identified 17 genes with nominal burden signals. Among them, VSTM1 showed the greatest association with RA (P = 7.80 × 10⁻⁴). In the enrichment test using Korean GWAS, although the significant signal appeared to be driven by total genic variants, we found no evidence for enriched association of coding variants only with RA.

Conclusions

We were unable to identify rare coding variants with large effect to explain the missing heritability for RA in the current targeted resequencing study. Our study raises skepticism about exon sequencing of targeted genes for complex diseases like RA.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-014-0447-7) contains supplementary material, which is available to authorized users.     

Genome-wide next-generation DNA and RNA sequencing reveals a mutation that perturbs splicing of the phosphatidylinositol glycan anchor biosynthesis class H gene (PIGH) and causes arthrogryposis in Belgian Blue cattle

Background

Cattle populations are characterized by regular outburst of genetic defects as a result of the extensive use of elite sires. The causative genes and mutations can nowadays be rapidly identified by means of genome-wide association studies combined with next generation DNA sequencing, provided that the causative mutations are conventional loss-of-function variants. We show in this work how the combined use of next generation DNA and RNA sequencing allows for the rapid identification of otherwise difficult to identify splice-site variants.

Results

We report the use of haplotype-based association mapping to identify a locus on bovine chromosome 10 that underlies autosomal recessive arthrogryposis in Belgian Blue Cattle. We identify 31 candidate mutations by resequencing the genome of four cases and 15 controls at ~10-fold depth. By analyzing RNA-Seq data from a carrier fetus, we observe skipping of the second exon of the PIGH gene, which we confirm by RT-PCR to be fully penetrant in tissues from affected calves. We identify - amongst the 31 candidate variants - a C-to-G transversion in the first intron of the PIGH gene (c211-10C > G) that is predicted to affect its acceptor splice-site. The resulting PIGH protein is likely to be non-functional as it lacks essential domains, and hence to cause arthrogryposis.

Conclusions

This work illustrates how the growing arsenal of genome exploration tools continues to accelerate the identification of an even broader range of disease causing mutations, therefore improving the management and control of genetic defects in livestock.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1528-y) contains supplementary material, which is available to authorized users.     

Identification of low-frequency TRAF3IP2 coding variants in psoriatic arthritis patients and functional characterization

Introduction

In recent genome-wide association studies for psoriatic arthritis (PsA) and psoriasis vulgaris, common coding variants in the TRAF3IP2 gene were identified to contribute to susceptibility to both disease entities. The risk allele of p.Asp10Asn (rs33980500) proved to be most significantly associated and to encode a mutant protein with an almost completely disrupted binding property to TRAF6, supporting its impact as a main disease-causing variant and modulator of IL-17 signaling.

Methods

To identify further variants, exons 2-4 encoding both known TNF-receptor-associated factor (TRAF) binding domains were sequenced in 871 PsA patients. Seven missense variants and one three-base-pair insertion were identified in 0.06% to 1.02% of alleles. Five of these variants were also present in 931 control individuals at comparable frequency. Constructs containing full-length wild-type or mutant TRAF3IP2 were generated and used to analyze functionally all variants for TRAF6-binding in a mammalian two-hybrid assay.

Results

None of the newly found alleles, though, encoded proteins with different binding properties to TRAF6, or to the cytoplasmic tail of the IL-17-receptor α-chain, suggesting that they do not contribute to susceptibility.

Conclusions

Thus, the TRAF3IP2-variant p.Asp10Asn is the only susceptibility allele with functional impact on TRAF6 binding, at least in the German population.     

Studies of the association of Arg72Pro of tumor suppressor protein p53 with type 2 diabetes in a combined analysis of 55,521 Europeans

Aims

A study of 222 candidate genes in type 2 diabetes reported association of variants in RAPGEF1, ENPP1, TP53, NRF1, SLC2A2, SLC2A4 and FOXC2 with type 2 diabetes in 4,805 Finnish individuals. We aimed to replicate these associations in a Danish case-control study and to substantiate any replicated associations in meta-analyses. Furthermore, we evaluated the impact on diabetes-related intermediate traits in a population-based sample of middle-aged Danes.

Methods

We genotyped nine lead variants in the seven genes in 4,973 glucose-tolerant and 3,612 type 2 diabetes Danish individuals. In meta-analyses we combined case-control data from the DIAGRAM+ Consortium (n = 47,117) and the present genotyping results. The quantitative trait studies involved 5,882 treatment-naive individuals from the Danish Inter99 study.

Results

None of the nine investigated variants were significantly associated with type 2 diabetes in the Danish samples. However, for all nine variants the estimate of increase in type 2 diabetes risk was observed for the same allele as previously reported. In a meta-analysis of published and online data including 55,521 Europeans the G-allele of rs1042522 in TP53 showed significant association with type 2 diabetes (OR = 1.06 95% CI 1.02–1.11, p = 0.0032). No substantial associations with diabetes-related intermediary phenotypes were found.

Conclusion

The G-allele of TP53 rs1042522 is associated with an increased prevalence of type 2 diabetes in a combined analysis of 55,521 Europeans.     

The First Human Epitope Map of the Alphaviral E1 and E2 Proteins Reveals a New E2 Epitope with Significant Virus Neutralizing Activity

Background

Venezuelan equine encephalitis virus (VEEV) is responsible for VEE epidemics that occur in South and Central America and the U.S. The VEEV envelope contains two glycoproteins E1 (mediates cell membrane fusion) and E2 (binds receptor and elicits virus neutralizing antibodies). Previously we constructed E1 and E2 epitope maps using murine monoclonal antibodies (mMAbs). Six E2 epitopes (E2^c,d,e,f,g,h) bound VEEV-neutralizing antibody and mapped to amino acids (aa) 182–207. Nothing is known about the human antibody repertoire to VEEV or epitopes that engage human virus-neutralizing antibodies. There is no specific treatment for VEE; however virus-neutralizing mMAbs are potent protective and therapeutic agents for mice challenged with VEEV by either peripheral or aerosol routes. Therefore, fully human MAbs (hMAbs) with virus-neutralizing activity should be useful for prevention or clinical treatment of human VEE.

Methods

We used phage-display to isolate VEEV-specific hFabs from human bone marrow donors. These hFabs were characterized by sequencing, specificity testing, VEEV subtype cross-reactivity using indirect ELISA, and in vitro virus neutralization capacity. One E2-specific neutralizing hFAb, F5n, was converted into IgG, and its binding site was identified using competitive ELISA with mMAbs and by preparing and sequencing antibody neutralization-escape variants.

Findings

Using 11 VEEV-reactive hFabs we constructed the first human epitope map for the alphaviral surface proteins E1 and E2. We identified an important neutralization-associated epitope unique to the human immune response, E2 aa115–119. Using a 9 Å resolution cryo-electron microscopy map of the Sindbis virus E2 protein, we showed the probable surface location of this human VEEV epitope.

Conclusions

The VEEV-neutralizing capacity of the hMAb F5 nIgG is similar to that exhibited by the humanized mMAb Hy4 IgG. The Hy4 IgG has been shown to limit VEEV infection in mice both prophylactically and therapeutically. Administration of a cocktail of F5n and Hy4 IgGs, which bind to different E2 epitopes, could provide enhanced prophylaxis or immunotherapy for VEEV, while reducing the possibility of generating possibly harmful virus neutralization-escape variants in vivo.