Nonconservative Amino Acid Change Research Articles

PO-01-158 MODELING AN ATRIAL FIBRILLATION-CAUSING TITIN MUTATION USING HUMAN IPSC-DERIVED ATRIAL CARDIOMYOCYTES

Atrial fibrillation (AF) affects more than 33 million people worldwide and, if left untreated, can lead to increased morbidity and mortality. Although AF has been classified as an ‘ion channelopathy’, next-generation sequencing has identified mutations in sarcomeric proteins like TTN, the gene encoding titin, that are associated with AF. Despite progress in understanding the genetic basis of AF, the translation of these discoveries to the bedside care of patients has been limited due to challenges in understanding the myocardial substrate for AF, especially that develops from the mutation in the structural protein. As human atrial tissue is rarely available and animal models are mechanistically limited, our goal is to model AF using mature human induced pluripotent stem cell-derived atrial cardiomyocytes (hiPSC-aCMs) by a CRISPR-Cas9-mediated single amino acid substitution of a rare missense ethnic-specific mutation (TTN_T32756I) in an immunoglobulin (Ig)-like domain of the A-band of titin that is enriched in AF-causing pathogenic variants. Isogenic TTN_T32756I hiPSC-aCM mutants were generated using the CRISPR-Cas9 technique. Cells were matured either with metabolic conditioning or co-culturing with atrial fibroblasts in a micropatterned platform. We used confocal microscopy to assess sarcomeric integrity, MuscleMotion to assess contractility, and optical voltage mapping to assess the electrophysiological properties of TTN_T32756I hiPSC-aCMs. T32756I results in a non-conservative amino acid change, which in-silico tools have previously predicted results in an impairment in titin function. Here, we show that TTN_T32756I hiPSC-aCM mutants display aberrant contractility (Fig. 1A-C) and Ca2+ homeostasis (Fig. 1D) leading to abnormal electrophysiology (Fig. 1E-F) at the cellular level without compromising the sarcomeric integrity of the atrial cardiomyocytes (Fig. 1G-H). Our results suggest that a single amino acid change could have a profound effect on titin function, further establishing titin as a major signaling hub for contractility. Ongoing studies will not only identify the key pathways and transcription factors involved in the pathogenesis of TTN_ T32756I mutant but also uncover the impact of the mutation on atrial action potential and cardiac ion channel activity. Our study will shed new light on the mechanisms by which TTN mutation cause AF and identify novel therapeutic targets for patients carrying AF-associated rare variants in the structural genes.

Molecular evolution and population genetics of glutamate decarboxylase acid resistance pathway in lactic acid bacteria.

Glutamate decarboxylase (GAD) pathway (GDP) is a major acid resistance mechanism enabling microorganisms' survival in low pH environments. We aimed to study the molecular evolution and population genetics of GDP in Lactic Acid Bacteria (LAB) to understand evolutionary processes shaping adaptation to acidic environments comparing species where the GDP genes are organized in an operon structure (Levilactobacillus brevis) versus lack of an operon structure (Lactiplantibacillus plantarum). Within species molecular population genetic analyses of GDP genes in L. brevis and L. plantarum sampled from diverse fermented food and other environments showed abundant synonymous and non-synonymous nucleotide diversity, mostly driven by low frequency changes, distributed throughout the coding regions for all genes in both species. GAD genes showed higher level of replacement polymorphism compared to transporter genes (gadC and YjeM) for both species, and GAD genes that are outside of an operon structure showed even higher level of replacement polymorphism. Population genetic tests suggest negative selection against replacement changes in all genes. Molecular structure and amino acid characteristics analyses showed that in none of the GDP genes replacement changes alter 3D structure or charge distribution supporting negative selection against non-conservative amino acid changes. Phylogenetic and between species divergence analyses suggested adaptive protein evolution on GDP genes comparing phylogenetically distant species, but conservative evolution comparing closely related species. GDP genes within an operon structure showed slower molecular evolution and higher conservation. All GAD and transporter genes showed high codon usage bias in examined LAB species suggesting high expression and utilization of acid resistance genes. Substantial discordances between species, GAD, and transporter gene tree topologies were observed suggesting molecular evolution of GDP genes do not follow speciation events. Distribution of operon structure on the species tree suggested multiple independent gain or loss of operon structure in LABs. In conclusion, GDP genes in LABs exhibit a dynamic molecular evolutionary history shaped by gene loss, gene transfer, negative and positive selection to maintain its active role in acid resistance mechanism, and enable organisms to thrive in acidic environments.

Impact of the Types and Relative Quantities of IGHV Gene Mutations in Predicting Prognosis of Patients With Chronic Lymphocytic Leukemia.

Patients with CLL with mutated IGHV genes (M-CLL) have better outcomes than patients with unmutated IGHVs (U-CLL). Since U-CLL usually express immunoglobulins (IGs) that are more autoreactive and more effectively transduce signals to leukemic B cells, B-cell receptor (BCR) signaling is likely at the heart of the worse outcomes of CLL cases without/few IGHV mutations. A corollary of this conclusion is that M-CLL follow less aggressive clinical courses because somatic IGHV mutations have altered BCR structures and no longer bind stimulatory (auto)antigens and so cannot deliver trophic signals to leukemic B cells. However, the latter assumption has not been confirmed in a large patient cohort. We tried to address the latter by measuring the relative numbers of replacement (R) mutations that lead to non-conservative amino acid changes (Rnc) to the combined numbers of conservative (Rc) and silent (S) amino acid R mutations that likely do not or cannot change amino acids, “(S+Rc) to Rnc IGHV mutation ratio”. When comparing time-to-first-treatment (TTFT) of patients with (S+Rc)/Rnc ≤ 1 and >1, TTFTs were similar, even after matching groups for equal numbers of samples and identical numbers of mutations per sample. Thus, BCR structural change might not be the main reason for better outcomes for M-CLL. Since the total number of IGHV mutations associated better with longer TTFT, better clinical courses appear due to the biologic state of a B cell having undergone many stimulatory events leading to IGHV mutations. Analyses of larger patient cohorts will be needed to definitively answer this question.

Substitution Trends in the Mitochondrial Cytochrome B Protein Coding Gene and the Corresponding Changes in its Amino Acid Sequence in Catfish Species

The study describes the divergence trends in the partial segment cytochrome b gene (cyt. b) in six catfish species on the basis of the nucleotide substitutions and their location in the triplet codons of the resulting amino acid sequence. The nucleotide sequences of all six species were found A+T rich with average nucleotide frequencies as A: 28.6%; T: 29.3%; G: 13.6%; C: 28.5%, showing an antiguanine bias which was found strongest in R. rita. The transition substitution rate was found highest at 3rd codon position in all the species. The evolutionary divergence follows the trend as highest at 3rd codon position followed by 1st codon position and the least on 2 nd codon position among all species. When amino acid sequences of cytochrome b were aligned, only 12 sites were found with a total 29 amino acid substitutions. which is a resultant of the substitution trend of three codon positions. Majority of the amino acid substitutions were found synonymous and conservative. Three different variability trends are observed in the cyt. b gene which run simultaneously on three different codon positions of triplet codons. Nucleotide substitutions at three codon positions follows the order 3 rd (87) >1st (32) >2nd (03) the frequency of amino acid substitution follows the order 1 st (11) >3rd (04) >2 nd (02); and the frequency of non-conservative amino acid changes found as 2 nd (1:2) >1st (5:11) >3rd (1:4). The study has provided an in-depth understanding of the relative variability and substitution trends in the mitochondrial cytochrome b gene in catfish species at both DNA and protein level.

Human PAR4 Is a More Potent Receptor for Activating Platelets Than Mouse PAR4

PAR4 is a protease-activated receptor with major roles in both platelet aggregation and platelet procoagulant function, contributing to both hemostasis and thrombosis in vivo. It is a major target for anti-thrombotic agents in current development. There are notable differences in the amino acid sequences between human (hu) PAR4 and mouse (mu) PAR4 in domains associated with the mechanisms of receptor action. These include the second transmembrane domain, which has a Valine at position 120 in muPAR4 while it is Alanine or Threonine in huPAR4 (Edelstein, Nature Med 2014). Other differences include 4 non-conservative amino acid changes in extracellular loop 2 and a major non-conservative change (mu = Cysteine, hu = Glutamine) in helix 8 in the cytosolic carboxy terminal (Ramachandran, Mol Pharm 2017). We generated a unique set of mice which enable us to compare for the first time the potential differences in platelet activation between huPAR4 and muPAR4 in the platelet context, rather than in heterologous cells. We generated and characterized 5 independent lines of mice transgenic for human PAR4, using an approach with a large genomic clone which we have implemented successfully in the past. Each of these huPAR4 transgenic lines has been bred to the mouse PAR4 knockout mice generated by Coughlin and colleagues (generously provided by S. Kunapuli, Temple University). The mice are referred to as PAR4 tgKO mice; 3 express the hu Thr120 allele and 2 the Ala120 allele. The level of huPAR4 expression in the tgKO platelets is equivalent to that of muPAR4 in wild-type mouse platelets. Washed platelets from wild-type mice, PAR4 tgKO mice, and muPAR4 KO mice were stimulated with a range of concentrations of PAR4 activating peptide (PAR4-AP, AYPGKF) and the activation of αIIbβ3 and expression of P-selectin on the surface were determined with flow cytometry. As expected, muPAR4 knockout mice showed no response to the treatment, but reacted normally to other agonists. While we observed small differences between the hu Ala120 and Thr120 tgKO mice, consistent with prior reports by Bray, Edelstein and colleagues for human platelets, we observed large and statistically significant differences between all tgKO mouse platelets tested and wild-type mouse platelets. In summary, all other things being equal (i.e. the same platelet context), human PAR4 is a more potent receptor for platelet activation than mouse PAR4. Studies are in progress to elucidate the contribution of the different functional domains and the roles of heterotrimeric G proteins, calcium and other signaling intermediates. Figure Disclosures No relevant conflicts of interest to declare.

Identification of llama KRTAP7-1 and KRTAP8-1 fiber genes and polymorphism screening

Keratin-associated proteins (KAP) are one of the main structural components of hair fiber. Within this protein group, high glycine tyrosine (HGT)-KAP play a crucial role in the definition of their physical-mechanical properties. Polymorphisms in HGT genes have been associated with the variation of different wool traits in sheeps and goats. The Argentine llama is a fiber-producing animal which is valued by the textile industry. However, the genes encoding for fiber proteins have not yet been identified in this species. Here, we focus on studying the HGT-KRTAP7-1 and KRTAP8-1 genes and their variation using the High Resolution Melting (HRM) technique in a sample of 117 llamas. Four single nucleotide polymorphisms (SNPs) were detected in KRTAP7-1, two of which were non-synonymous substitutions leading to amino acid changes in the protein. Of the 5 polymorphisms identified in KRTAP8-1, c.1-5A > G was located in the Kozak sequence, known to regulate protein synthesis level. The other four, two SNPs and one double nucleotide polymorphism (DNP), were found in the coding region and produced three amino acid replacement: c.43 T > C and c.45C > A (p.Y15Q), c.46 G > T (p.G16W) and c.173 A > G (p.Y58C). In summary, most of the polymorphisms found in both KRTAP7-1 and KRTAP8-1 genes produce non-conservative amino acid changes involving tyrosine and glycine residues, which are essential to maintain HGT protein properties. Therefore, these mutations as well as the regulatory SNP here identified could modify the fiber characteristics. We discuss the possible impact of these polymorphisms on KAP7-1 and KAP8-1 structure and/or interaction with other fiber proteins.

Functional implications of single nucleotide polymorphisms rs662 and rs854860 on the antioxidative activity of paraoxonase1 (PON1) in patients with rheumatoid arthritis

BackgroundAtherosclerosis leading to cardiovascular disease (CVD) is the main cause of mortality and morbidity in patients with rheumatoid arthritis (RA). Paraoxonase1 (PON1) is the best understood member of plasma paraoxonases with anti-atherogenic properties.Patients and methodsSpanish RA (n = 549) consecutively recruited from 1 single center and 477 ethnically matched healthy controls were included in a case-control study. The concentration of PON1 was evaluated by means of an enzyme-linked immunosorbent sssay (ELISA). An arylesterase/paraoxonase assay kit was used to evaluate PON1 activity. Sample genotyping was performed by using TaqMan assays-on-demand. All results were expressed as medians ± interquartile range. One-way ANOVA comparisons were done using a nonparametric Kruskall-Wallis test. P values under 0.05 were considered to be significant.ResultsThe concentration of PON1 in the RA group was higher than in control group (p = 0.0003), although the differences were not significant when PON1 activities were compared between both groups. No significant differences were found related to distributions of rs662 genotypes in RA patients compared to healthy controls. Among rs854860 polymorphisms, overall genotype was widely distributed between RA patients and controls. Overall PON1 concentration in plasma was not significantly different between individuals carrying any of rs662 (p = 0.8501) or rs854860 (p = 0.2741) polymorphisms. Although PON1 levels were not associated with any of the SNPs in the study, differences appear when enzyme activities are compared for each SNP separately. CVD in RA patients correlate with increased PON1 levels and lower PON1 activity.ConclusionsAlthough protective role of PON1 against oxidative damage in vivo could be related to other activities, in our study arylesterase activity was useful to identify phenotypic differences with emphasis placed on two SNPs coding for nonconservative amino acid changes in the functional protein.

Natural variation in genes potentially involved in plant architecture and adaptation in switchgrass (Panicum virgatum L.)

BackgroundAdvances in genomic technologies have expanded our ability to accurately and exhaustively detect natural genomic variants that can be applied in crop improvement and to increase our knowledge of plant evolution and adaptation. Switchgrass (Panicum virgatum L.), an allotetraploid (2n = 4× = 36) perennial C4 grass (Poaceae family) native to North America and a feedstock crop for cellulosic biofuel production, has a large potential for genetic improvement due to its high genotypic and phenotypic variation. In this study, we analyzed single nucleotide polymorphism (SNP) variation in 372 switchgrass genotypes belonging to 36 accessions for 12 genes putatively involved in biomass production to investigate signatures of selection that could have led to ecotype differentiation and to population adaptation to geographic zones.ResultsA total of 11,682 SNPs were mined from ~ 15 Gb of sequence data, out of which 251 SNPs were retained after filtering. Population structure analysis largely grouped upland accessions into one subpopulation and lowland accessions into two additional subpopulations. The most frequent SNPs were in homozygous state within accessions. Sixty percent of the exonic SNPs were non-synonymous and, of these, 45% led to non-conservative amino acid changes. The non-conservative SNPs were largely in linkage disequilibrium with one haplotype being predominantly present in upland accessions while the other haplotype was commonly present in lowland accessions. Tajima’s test of neutrality indicated that PHYB, a gene involved in photoperiod response, was under positive selection in the switchgrass population. PHYB carried a SNP leading to a non-conservative amino acid change in the PAS domain, a region that acts as a sensor for light and oxygen in signal transduction.ConclusionsSeveral non-conservative SNPs in genes potentially involved in plant architecture and adaptation have been identified and led to population structure and genetic differentiation of ecotypes in switchgrass. We suggest here that PHYB is a key gene involved in switchgrass natural selection. Further analyses are needed to determine whether any of the non-conservative SNPs identified play a role in the differential adaptation of upland and lowland switchgrass.

The Calcium-Sensing Receptor Gene Polymorphism rs1801725 and Calcium-Related Phenotypes in Hemodialysis Patients

Background/Aims: The calcium-sensing receptor gene (CASR) rs1801725 variant is responsible for a non-conservative amino-acid change (A986S) in the calcium-sensing receptor cytoplasmic tail. We hypothesized that rs1801725 polymorphism might be helpful in understanding Ca-related abnormalities in HD patients. Methods: In 1215 subjects (245 on cinacalcet), we determined the associations of rs1801725 with secondary hyperparathyroidism (sHPT)-related laboratory parameters, PTH-decreasing effect of cinacalcet hydrochloride, coronary artery disease (CAD), myocardial infarction (MI), nephrolithiasis-related ESRD, and mortality. CASR rs7652589(A<G)_rs1801725(G>T) haplotypes and rs1801725 epistatic interactions with vitamin D signaling pathway genes were examined for associations with selected phenotypes. Results: The rs1801725 variant allele showed an increasing independent effect on plasma PTH (P_corrected = 0.009). CASR rs7652589_rs1801725 AT haplotype was associated with 1.7-fold higher frequency of PTH levels over 437 pg/mL than the reference haplotype GG (P = 0.001). CASR rs7652589_rs1801725 AG haplotype was 1.5-fold more frequent in nephrolithiasis-related ESRD than the GG haplotype (P = 0.004). There were no significant associations between rs1801725, CAD, MI, and response to cinacalcet. Variant homozygosity of rs1801725 correlated independently with higher infection-related mortality compared with heterozygosity (HR 7.95, 95%CI 2.15 – 29.37, P = 0.003) and major homozygosity (HR 5.89, 95%CI 1.69 – 20.55, P = 0.040). CASR rs1801725 did not show epistatic interactions with vitamin D signaling pathway genes concerning tested associations. Conclusion: The variant allele of CASR rs1801725 solely and together with the variant allele of rs7652589 increases risk of more advanced sHPT. Homozygosity of the rs1801725 variant allele contributes to infection-related mortality in HD patients.

Excess of non-conservative amino acid changes in marine bacterioplankton lineages with reduced genomes.

Surface ocean waters are dominated by planktonic bacterial lineages with highly reduced genomes. The best examples are the cyanobacterial genus Prochlorococcus, the alphaproteobacterial clade SAR11 and the gammaproteobacterial clade SAR86, which together represent over 50% of the cells in surface oceans. Several studies have identified signatures of selection on these lineages in today's ocean and have postulated selection as the primary force throughout their evolutionary history. However, massive loss of genomic DNA in these lineages often occurred in the distant past, and the selective pressures underlying these ancient events have not been assessed. Here, we probe ancient selective pressures by computing %GC-corrected rates of conservative and radical nonsynonymous nucleotide substitutions. Surprisingly, we found an excess of radical changes in several of these lineages in comparison to their relatives with larger genomes. Furthermore, analyses of allelic genome sequences of several populations within these lineages consistently supported that radical replacements are more likely to be deleterious than conservative changes. Our results suggest coincidence of massive genomic DNA losses and increased power of genetic drift, but we also suggest that additional evidence independent of the nucleotide substitution analyses is needed to support a primary role of genetic drift driving ancient genome reduction of marine bacterioplankton lineages.

Novel mutation in the CHST6 gene causes macular corneal dystrophy in a black South African family

BackgroundMacular corneal dystrophy (MCD) is a rare autosomal recessive disorder that is characterized by progressive corneal opacity that starts in early childhood and ultimately progresses to blindness in early adulthood. The aim of this study was to identify the cause of MCD in a black South African family with two affected sisters.MethodsA multigenerational South African Sotho-speaking family with type I MCD was studied using whole exome sequencing. Variant filtering to identify the MCD-causal mutation included the disease inheritance pattern, variant minor allele frequency and potential functional impact.ResultsOphthalmologic evaluation of the cases revealed a typical MCD phenotype and none of the other family members were affected. An average of 127 713 variants per individual was identified following exome sequencing and approximately 1.2 % were not present in any of the investigated public databases. Variant filtering identified a homozygous E71Q mutation in CHST6, a known MCD-causing gene encoding corneal N-acetyl glucosamine-6-O-sulfotransferase. This E71Q mutation results in a non-conservative amino acid change in a highly conserved functional domain of the human CHST6 that is essential for enzyme activity.ConclusionWe identified a novel E71Q mutation in CHST6 as the MCD-causal mutation in a black South African family with type I MCD. This is the first description of MCD in a black Sub-Saharan African family and therefore contributes valuable insights into the genetic aetiology of this disease, while improving genetic counselling for this and potentially other MCD families.Electronic supplementary materialThe online version of this article (doi:10.1186/s12881-016-0308-0) contains supplementary material, which is available to authorized users.

Abstract B12: Targeted sequencing of ovarian clear cell carcinomas reveals intertumor heterogeneity and distinct clustering related to mutational load.

Abstract Objective: Ovarian clear cell carcinomas (OCCCs) constitute a rare ovarian cancer subtype. Despite relatively clear clinical features, OCCCs can be difficult to distinguish morphologically from other epithelial ovarian cancers. OCCC gene expression signatures capturing not only the morphology but also the biology of this tumor subtype have been described, and a previous study using comparative genomic hybridization showed that OCCCs are indeed heterogeneous. Still, only a few genes have been analyzed using gene sequencing techniques. In the present study we aimed to further characterize OCCCs and their histotype specific genetic changes using targeted sequencing of 60 well-established cancer genes. Increased knowledge about genetic aberrations in OCCCs may help to guide future development of targeted treatments and improve the outcome for these patients. Methods: Formalin fixed, paraffin-embedded (FFPE) tumor tissue was collected from 11 primary OCCCs, diagnosed at the Skane University Hospital, Lund, Sweden (1998-2013). One sample was excluded due to poor DNA quality. For the remaining 10 samples, 600 ng genomic DNA/sample was used for single strand targeted DNA sequencing using the SureSeq™ solid tumor panel consisting of 60 key genes involved in cancer evolution, including ovarian cancer. The panel covers all coding exons in the included genes, and is validated for research use on FFPE samples. The publicly available ENSEMBL database was used to classify the mutations according to severity across the genes. Serious mutations, defined as major non-conservative amino acid changes, splice or frameshift variants or changes in stop codons, affecting single nucleotide variations (SNVs) or insertions or deletions of DNA bases (indels) were used for further analyses. The mutation call cut-off was set to 20% and the reading depth ≥300x, and unsupervised hierarchical clustering of the tumor samples was performed. Results: &amp;gt;99% of the target bases had a 30x coverage across all samples, and the mean target coverage was 534x. Using the 20% cut-off, approximately 300 mutations were detected in the 60 genes across the 10 tumor samples. Unsupervised hierarchical clustering revealed two distinct clusters related to mutational load. Mutations in genes previously reported to be affected in OCCCs, such as ARID1A and ERBB2, were identified. Whereas ARID1A was mainly affected in one cluster, mutations in genes signifying a highly malignant phenotype, such as BRCA1 and BRCA2 dominated in the other. Genes in signaling pathways often affected in malignant tumors, as e.g. the RAS pathway, however, were unaffected. Other mutations detected in this dataset were ZFHX3 and KMT2C. KMT2C was the only highly mutated gene across both clusters. The clustering pattern was not related to age of FFPE blocks or tumor cell content. Conclusions: In the present study we show that OCCCs are genetically heterogeneous. The variation in mutation rate is in line with a previous publication, and points to differences in tumor biology that may be important for tumor behavior and thus patient outcome. The highly mutated genes may help in guiding trials of targeted treatments; the methyl transferase gene KMT2C, also known as MLL3, may potentially be one such target. Rearrangements of this gene are well-described in acute leukemias. Interestingly, KMT2C/MLL3 is also reported to be a mutational cancer driver gene in renal clear cell carcinomas according to the TCGA. Likewise, ZFHX3 has recently been reported in endometrial clear cell carcinomas. Despite the small size of this study it points to the well-established fact that ovarian cancer is not one single disease, and also confirms that even the rare OCCCs seem to harbor different biological features which may be of potential clinical importance. Citation Format: Jenny-Maria Jönsson, Nicolai Skovbjerg Arildsen, Anna Ebbesson, Sofia Westbom-Fremer, Anna Måsbäck, Susanne Malander, Mef Nilbert, Ingrid Hedenfalk. Targeted sequencing of ovarian clear cell carcinomas reveals intertumor heterogeneity and distinct clustering related to mutational load. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B12.

The evolution of bat nucleic acid-sensing Toll-like receptors.

We characterized the nucleic acid-sensing Toll-like receptors (TLR) of a New World bat species, the common vampire bat (Desmodus rotundus), and through a comparative molecular evolutionary approach searched for general adaptation patterns among the nucleic acid-sensing TLRs of eight different bats species belonging to three families (Pteropodidae, Vespertilionidae and Phyllostomidae). We found that the bat TLRs are evolving slowly and mostly under purifying selection and that the divergence pattern of such receptors is overall congruent with the species tree, consistent with the evolution of many other mammalian nuclear genes. However, the chiropteran TLRs exhibited unique mutations fixed in ligand-binding sites, some of which involved nonconservative amino acid changes and/or targets of positive selection. Such changes could potentially modify protein function and ligand-binding properties, as some changes were predicted to alter nucleic acid binding motifs in TLR 9. Moreover, evidence for episodic diversifying selection acting specifically upon the bat lineage and sublineages was detected. Thus, the long-term adaptation of chiropterans to a wide variety of environments and ecological niches with different pathogen profiles is likely to have shaped the evolution of the bat TLRs in an order-specific manner. The observed evolutionary patterns provide evidence for potential functional differences between bat and other mammalian TLRs in terms of resistance to specific pathogens or recognition of nucleic acids in general.

Intermediate MCAD Deficiency Associated with a Novel Mutation of the ACADM Gene: c.1052C>T.

Medium-chain acyl-CoA dehydrogenase deficiency (MCADD) is an autosomal recessive disorder that leads to a defect in fatty acid oxidation. ACADM is the only candidate gene causing MCAD deficiency. A single nucleotide change, c.985A>G, occurring at exon 11 of the ACADM gene, is the most prevalent mutation. In this study, we report a Caucasian family with multiple MCADD individuals. DNA sequence analysis of the ACADM gene performed in this family revealed that two family members showing mild MCADD symptoms share the same novel change in exon 11, c.1052C>T, resulting in a threonine-to-isoleucine change. The replacement is a nonconservative amino acid change that occurs in the C-terminal all-alpha domain of the MCAD protein. Here we report the finding of a novel missense mutation, c.1052C>T (p.Thr326Ile), in the ACADM gene. To our knowledge, c.1052C>T has not been previously reported in the literature or in any of the current databases we utilize. We hypothesize that this particular mutation in combination with p.Lys304Glu results in an intermediate clinical phenotype of MCADD.

Mutation in the protease cleavage site of GDF9 increases ovulation rate and litter size in heterozygous ewes and causes infertility in homozygous ewes.

Litter size (LS) in sheep is determined mainly by ovulation rate (OR). Several polymorphisms have been identified in the growth differentiation factor 9 (GDF9) gene that result in an increase in OR and prolificacy of sheep. Screening the databank of the Brazilian Sheep Breeders Association for triplet delivery, we identified flocks of prolific Ile de France ewes. After resequencing of GDF9, a point mutation (c.943C>T) was identified, resulting in a non-conservative amino acid change (p.Arg315Cys) in the cleavage site of the propeptide. This new allele was called Vacaria (FecG(v) ). A flock of half-sib ewes was evaluated for OR in the first three breeding seasons, and Vacaria heterozygotes had higher OR (P<0.001), averaging 2.1±0.1 when compared to 1.2±0.1 in wild-type ewes. The OR was also influenced by age, increasing in the second and third breeding seasons (P<0.001). In flocks segregating this allele, the LS was higher in mutant sheep (P<0.001), averaging 1.61±0.07 in heterozygotes and 1.29±0.03 in wild-type ewes. Analysis of homozygote reproductive tract morphology revealed uterine and ovarian hypoplasia. Ovarian follicles continue to develop up to small antral stages, although with abnormal oocyte morphology and altered arrangement of granulosa cells. After the collapse of the oocyte in most follicles, the remaining cells formed clusters that persisted in the ovary. This SNP is useful to improve selection for dam prolificacy and also as a model to investigate GDF9 post-translation processing and the fate of the follicular cells that remain after the oocyte demise.

ND4L gene concurrent 10609T>C and 10663T>C mutations are associated with Leber's hereditary optic neuropathy in a large pedigree from Kuwait

BackgroundLeber's hereditary optic neuropathy (LHON) is a condition characterised by a rapid bilateral central vision loss due to death of the retinal ganglion cells, leading to visual impairment commonly occurring...

Family-based association of the brain-derived neurotrophic factor gene in attention-deficit hyperactivity disorder

Attention-deficit hyperactivity disorder (ADHD), a highly heritable neurodevelopmental disorder, is characterized by inattention and hyperactivity. The brain-derived neurotrophic factor (BDNF), a member of the neurotrophic factor family, is important for normal neuronal development. It has been proposed that BDNF may play a role in the therapeutic action and pathogenesis of ADHD (Tsai, 2003). A common single-nucleotide polymorphism (SNP) consisting of a missense change (G196A) that produces a nonconservative amino acid change (valine to methionine) has been identified in the coding exon of the BDNF gene at position 66 (Val66Met, rs6265). The replacement of Val66 by Met66 disrupts cellular processing, trafficking, and activity-dependent secretion of BDNF (Egan et al., 2003). Several association studies have tested the possible involvement of this BDNF Val66Met polymorphism in ADHD, although the results are controversial (Forero et al., 2009; Sanchez-Mora et al., 2010). To further clarify the role of BDNF in ADHD, we tested the hypothesis that the BDNF gene was associated with ADHD in Taiwanese families, using transmission disequilibrium analysis.

Novel mutation in the ATM gene in a Malian family with ataxia telangiectasia

Dear Sirs, Ataxia telangiectasia (A-T) is a rare, autosomal recessive disorder of childhood characterized by progressive cerebellar ataxia, telangiectasia, and immune defects, and is caused by mutations in the ataxia-telangiectasia mutated (ATM) gene [1]. The incidence of the disease is about 1 in 40,000 to 100,000 births [2]. A-T has been reported worldwide [3], but reports of this disease in Africa are rare and generally limited to clinical description. We describe a Malian family with parental consanguinity (Fig. 1a) and three of ten children presenting with cerebellar symptoms in early childhood. Two patients, 14- and 10-year-old boys, had normal births and development until age 2, when they presented with progressive gait difficulty, including difficulty stopping when running and falls. They both later developed slurred speech, weakness, and decreased coordination of the upper extremities. No sensory or bladder difficulty was noted. Family history was remarkable for a grandfather who died at age 75 and had balance problems since he was a teenager. Fig. 1 Pedigree and genetic features of the family. a Pedigree of the family showing consanguinity, and unaffected (white) and affected (black) individuals. b Sequencing shows a homozygous T7985A mutation (asterisk) in ATM. c ATM protein alignment in various ... Neurological examination of both patients showed an ataxic gait, markedly reduced hand coordination, and nystagmus on fixation and lateral gaze. They had slight distal weakness and atrophy in the lower legs. Reflexes were normal to reduced, and the Babinski sign was absent. The older brother had scoliosis. Cardiologic examination and testing were normal. Brain CT scan showed cerebellar atrophy with prominent cisterna magna. Vitamin E and beta and gamma tocopherol serum levels were normal. Genetic testing for Friedreich's ataxia was negative. A follow-up clinical assessment showed oculomotor apraxia, ocular telangiectasia, and square wave jerks. The parents noted that the patients had recurrent diarrhea and upper respiratory infections. These new findings were in favor of ataxia telangiectasia (Table 1). Additional blood testing showed high alphafetoprotein (AFP) levels and low IgA, IgE, and IgG2 levels. Also, aspartate and alanine aminotransferases (AST, ALT), and C-reactive protein (CRP) were elevated in the two patients, suggesting liver dysfunction. Genetic analysis of the ATM gene identified a novel homozygous single-nucleotide substitution at position c.7985T > A (Fig. 1b), predicting the amino acid substitution V2662D. Five available unaffected siblings did not have this sequence variant. The V2662 residue lies in a predicted ATP binding domain [3] and is conserved across a broad range of vertebrate species (Fig. 1c). In addition, this non-conservative amino acid change yields a score of –3 according to the BLOSUM 62 substitution matrix [4], and was not found in 100 ethnically matched controls, suggesting that the mutation found here is likely deleterious. Table 1 Summary of clinical and laboratory findings in patients More recently, the parents noticed that their 2-year-old son also had an ataxic gait. His AFP levels were elevated, but he had no telangiectasias. This highlights the usefulness of AFP testing in the diagnosis of A-T, as previously discussed [5]. Recurrent upper respiratory infections due to immune deficiency [6] occur in ataxia telangiectasia, but the two older patients also presented with frequent diarrhea, which may represent an associated infectious disease specific to the region in Mali where the patients live. Increased cancer susceptibility has been associated with A-T [7], however, hemato-oncological examination showed no signs of malignancy in our patients. In addition, abdominal and inguinal echography showed no tumors. Although cases of A-T have been reported in populations with African ancestry [3, 8] and in North Africa [9–11], reports of this disease in sub-Saharan Africa have been limited to clinical characterization [12, 13]. We report here genetically confirmed A-T with a novel mutation in this region, and add to the global spectrum of this disease. Our study shows that hereditary neurological diseases may not be uncommon in this region of Africa, although limited expertise and lack of diagnostic tools might lead to their underestimation and neglect.

The natural profilin from Russian thistle (Salsola kali ) contains a low IgE‐binding ability isoform – molecular and immunological characterization

Chenopodiaceae pollens such as those from Salsola kali and Chenopodium album are important causes of allergy in Mediterranean areas because of the progress of desertification in European countries. Among the various allergenic protein families, profilins constitute a group of pan-allergens that are involved in polysensitization and pollen-food allergy syndrome. Two-dimensional electrophoresis analysis of S. kali profilin highlighted a polymorphic pattern, with several isoforms that have different molecular features (isoelectric point and molecular mass) and immunological features. Two isoforms have been cloned and sequenced. Sal k 4.02 and Sal k 4.03 displayed non-conservative amino acid changes in critical positions of the IgE epitopes. Both isoforms were produced in Escherichia coli and structurally and spectroscopically characterized. Changes in the electrophoretic mobility and in their IgG and IgE immunological behavior were observed in comparison with Che a 2, their counterpart from C. album. The IgE-binding ability of Sal k 4.03 is similar to that of Che a 2, whereas Sal k 4.02 showed a 35% reduction in IgE binding in 86% of patients, suggesting a hypoallergenic character. Three-dimensional modeling allowed us to propose which amino acid residues are involved in those immunological changes based on epitope mapping studies previously performed in other profilins. These profilin isoforms constitute suitable candidates for specific immunotherapy with recombinant allergens.

Influence of evolutionary events on the Indian subcontinent on the phylogeography of dengue type 3 and 4 viruses

During 1960–80 dengue disease profile in India was mild despite circulation of all four serotypes of dengue virus (DENV). Increase in disease severity with a concomitant change in the population of DENV-1 and 2 have been reported since then. To determine population dynamics of DENV-3 and 4, the envelope (E) gene sequence was determined for 16 Indian isolates of DENV-3 and 11 of DENV-4 and analyzed together with 97 DENV-3 and 43 DENV-4 global sequences.All Indian DENV-3 isolates belonged to genotype III, lineages C, D, E and F. Lineage F was newly identified and represented non-circulating viruses. Three non-conservative amino acid changes in domain I, II & III were identified during the transition from lineages F/E, associated with mild disease, to A–D, associated with severe disease. For DENV-4, the current viruses clustered in genotype I, lineage C, whilst the isolates from 1960s formed the new genotype V. A 1979 Indian isolate of DENV-4 was found to be an inter-genotypic recombinant of Sri Lankan isolate (1978) of genotype I and Indian isolate (1961) of genotype V. The rates of nucleotide substitution and time to the most recent common ancestor (tMRCA) estimated for DENV-3 (1782–1934) and DENV-4 (1719–1931) were similar to earlier reports. However, the divergence time for genotype III of DENV-3, 1938–1963, was a more accurate estimate with the inclusion of Indian isolates from the 1960s. By phylogeographical analysis it was revealed that DENV-3 GIII viruses emerged from India and evolved through Sri Lanka whilst DENV-4 emerged and dispersed from India.The present study demonstrates the crucial role that India/Sri Lanka have played in the evolution and dispersion of the major genotypes, GIII of DENV-3 and GI of DENV-4 which are more virulent and show higher dissemination potential.