Glutamine Mutation Research Articles

Crotonylation of NAE1 Modulates Cardiac Hypertrophy via Gelsolin Neddylation.

Cardiac hypertrophy and its associated remodeling are among the leading causes of heart failure. Lysine crotonylation is a recently discovered posttranslational modification whose role in cardiac hypertrophy remains largely unknown. NAE1 (NEDD8 [neural precursor cell expressed developmentally downregulated protein 8]-activating enzyme E1 regulatory subunit) is mainly involved in the neddylation modification of protein targets. However, the function of crotonylated NAE1 has not been defined. This study aims to elucidate the effects and mechanisms of NAE1 crotonylation on cardiac hypertrophy. Crotonylation levels were detected in both human and mouse subjects with cardiac hypertrophy through immunoprecipitation and Western blot assays. Tandem mass tag (TMT)-labeled quantitative lysine crotonylome analysis was performed to identify the crotonylated proteins in a mouse cardiac hypertrophic model induced by transverse aortic constriction. We generated NAE1 knock-in mice carrying a crotonylation-defective K238R (lysine to arginine mutation at site 238) mutation (NAE1 K238R) and NAE1 knock-in mice expressing a crotonylation-mimicking K238Q (lysine to glutamine mutation at site 238) mutation (NAE1 K238Q) to assess the functional role of crotonylation of NAE1 at K238 in pathological cardiac hypertrophy. Furthermore, we combined coimmunoprecipitation, mass spectrometry, and dot blot analysis that was followed by multiple molecular biological methodologies to identify the target GSN (gelsolin) and corresponding molecular events contributing to the function of NAE1 K238 (lysine residue at site 238) crotonylation. The crotonylation level of NAE1 was increased in mice and patients with cardiac hypertrophy. Quantitative crotonylomics analysis revealed that K238 was the main crotonylation site of NAE1. Loss of K238 crotonylation in NAE1 K238R knock-in mice attenuated cardiac hypertrophy and restored the heart function, while hypercrotonylation mimic in NAE1 K238Q knock-in mice significantly enhanced transverse aortic constriction-induced pathological hypertrophic response, leading to impaired cardiac structure and function. The recombinant adenoviral vector carrying NAE1 K238R mutant attenuated, while the K238Q mutant aggravated Ang II (angiotensin II)-induced hypertrophy. Mechanistically, we identified GSN as a direct target of NAE1. K238 crotonylation of NAE1 promoted GSN neddylation and, thus, enhanced its protein stability and expression. NAE1 crotonylation-dependent increase of GSN promoted actin-severing activity, which resulted in adverse cytoskeletal remodeling and progression of pathological hypertrophy. Our findings provide new insights into the previously unrecognized role of crotonylation on nonhistone proteins during cardiac hypertrophy. We found that K238 crotonylation of NAE1 plays an essential role in mediating cardiac hypertrophy through GSN neddylation, which provides potential novel therapeutic targets for pathological hypertrophy and cardiac remodeling.

Arginine to glutamine mutation in the substrate binding region impaired the isopentenyl activity of Mycobacterium tuberculosis MiaA.

tRNAs act as adaptors during protein synthesis and are chemically modified post-transcriptionally for their structural stability as well as accuracy of the translation. Hypomodifications of tRNAs are known to cause various human diseases, including cancer. Studies in bacteria and yeasts showed that levels of tRNA modifications vary under different stress conditions, enabling the organism to modulate gene expression for survival. Isopentelylation of the base 37 (i6A37) in the anticodon stem-loop by tRNA isopentenyltransferase (MiaA) is well-conserved modification present in prokaryotes and eukaryotes. i6A37 modification increases both the speed and fidelity of translation. A homozygous p.Arg323Gln mutation in the tRNA binding region of tRNA isopentenyltransferase reduced i6A37 levels in humans, affecting mitochondrial translation and thereby causing neurodevelopmental disorder. In this study, we mutated the Arg residue at the conserved position to Gln in Mycobacterium tuberculosis (M. tb) MiaA and analyzed the i6A modification activity of the enzyme on its target tRNAs. We found that p.Arg274Gln mutant MiaA could not modify the target tRNAs, tRNALeuCAA, tRNAPheGAA, and tRNASerCGA from M. tb, confirming the role of Arg residue in tRNA binding.

Computational analysis of the metal selectivity of matrix metalloproteinase 8.

Matrix metalloproteinase (MMP) is a class of metalloenzyme that cleaves peptide bonds in extracellular matrices. Their functions are important in both health and disease of animals. Here using quantum mechanics simulations of the MMP8 protein, the coordination chemistry of different metal cofactors is examined. Structural comparisons reveal that Jhan-Teller effects induced by Cu(II) coordination distorts the wild-type MMP8 active site corresponding to a significant reduction in activity observed in previous experiments. In addition, further analysis suggests that a histidine to glutamine mutation at residue number 197 can potentially allow the MMP8 protein to utilize Cu(II) in reactions. Simulations also demonstrates the requirement of a conformational change in the ligand before enzymatic cleavage. The insights provided here will assist future protein engineering efforts utilizing the MMP8 protein.

Establishing a new human hypertrophic cardiomyopathy-specific model using human embryonic stem cells

Symptom of ventricular hypertrophy caused by cardiac troponin T (TNNT2) mutations is mild, while patients often showed high incidence of sudden cardiac death. The 92nd arginine to glutamine mutation (R92Q) of cTnT was one of the mutant hotspots in hypertrophic cardiomyopathy (HCM). However, there are no such human disease models yet. To solve this problem, we generated TNNT2 R92Q mutant hESC cell lines (heterozygote or homozygote) using TALEN mediated homologous recombination in this study. After directed cardiac differentiation, we found a relative larger cell size in both heterozygous and homozygous TNNT2 R92Q hESC-cardiomyocytes. Expression of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and sarcoplasmic reticulum Ca2+-ATPase2a (SERCA2a) were downregulated, while myocyte specific enhancer factor 2c (MEF2c) and the ratio of beta myosin to alpha myosin heavy chain (MYH7/MYH6) were increased in heterozygous TNNT2 R92Q hESC-cardiomyocytes. TNNT2 R92Q mutant cardiomyocytes exhibited efficient responses to heart-related pharmaceutical agents. We also found TNNT2 R92Q heterozygous mutant cardiomyocytes showed increased calcium sensitivity and contractility. Further, engineered heart tissues (EHTs) prepared by combining rat decellularized heart extracellular matrices with heterozygous R92Q mutant cardiomyocytes showed similar drug responses as to HCM patients and increased sensitivity to caspofungin-induced cardiotoxicity. Using RNA-sequencing of TNNT2 R92Q heterozygous mutant cardiomyocytes, we found dysregulation of calcium might participated in the early development of hypertrophy. Our hESC-derived TNNT2 R92Q mutant cardiomyocytes and EHTs are good in vitro human disease models for future disease studies and drug screening.

Interaction between γC87 and γR242 residues participates in energy coupling between catalysis and proton translocation in Escherichia coli ATP synthase

Functioning as a nanomotor, ATP synthase plays a vital role in the cellular energy metabolism. Interactions at the rotor and stator interface are critical to the energy transmission in ATP synthase. From mutational studies, we found that the γC87K mutation impairs energy coupling between proton translocation and nucleotide synthesis/hydrolysis. An additional glutamine mutation at γR242 (γR242Q) can restore efficient energy coupling to the γC87K mutant. Arrhenius plots and molecular dynamics simulations suggest that an extra hydrogen bond could form between the side chains of γC87K and βTPE381 in the γC87K mutant, thus impeding the free rotation of the rotor complex. In the enzyme with γC87K/γR242Q double mutations, the polar moiety of γR242Q side chain can form a hydrogen bond with γC87K, so that the amine group in the side chain of γC87K will not hydrogen-bond with βE381. As a conclusion, the intra-subunit interaction between positions γC87 and γR242 modulates the energy transmission in ATP synthase. This study should provide more information of residue interactions at the rotor and stator interface in order to further elucidate the energetic mechanism of ATP synthase.

Different Effects of the RNASEL R462Q Mutation on the Risk of Developing Prostate and Cervical Cancer in Latin American Subjects: A Meta-Analysis

Background: Prostate and cervical tissues are highly susceptible to carcinogenesis. Furthermore, some reports suggest that alterations in RNASEL have been associated with augmented risk of developing cancer, specifically the arginine to glutamine mutation at position 462 (R462Q). However, with conflicting results of the R462Q mutation on cancer risk, our goal was to determine what effect this mutation had on prostate and cervical cancers in Latin Americans. Methods: PubMed, EBSCO, SCOUPS, Wiley and OVID databases, and study bibliographies were systematically searched for case-control studies that examined for the R462Q mutation until June 2015. Odds ratios (ORs) and 95%CI were calculated from the genotype data. The pooled ORs were obtained by the Peto method for the heterozygous, the homozygous, the dominant, the recessive, and the allelic genetic models. Heterogeneity was assessed by the Q-test and I2-test. Publication bias was assessed by the Begg and Mazumdar’s test and the Egger’s test. The sensitivity was determined by reevaluation of the pooled OR after removal of one study. Results: From the 153 retrieved studies, four studies met the inclusion criteria (n=808 subjects). The pooled results did not indicate any association between R462Q and overall cancer risk for any of the genetic model assessed. However, when stratified by type of cancer, the homozygous and the recessive genetic models demonstrated a significant association between prostate cancer (OR=2.26, 95%CI=1.15-4.44, p<0.05 and OR=2.18, 95%CI=1.12-4.23, p<0.05, respectively) and cervical cancer (OR=0.32, 95%CI=0.13-0.74, p<0.01 and OR=0.35, 95%CI=0.16-0.77, p<0.01, respectively). Furthermore, the risk associated with this mutation for prostate cancer and cervical cancer was different (p<0.01). Conclusion: Here we denote, for Latin Americans, the different effects the RNASEL R462Q mutation has for prostate (increased risk) and cervical (decreased risk) cancers.

Neutron structure of the cyclic glucose-bound xylose isomerase E186Q mutant

Ketol-isomerases catalyze the reversible isomerization between aldoses and ketoses. D-Xylose isomerase carries out the first reaction in the catabolism of D-xylose, but is also able to convert D-glucose to D-fructose. The first step of the reaction is an enzyme-catalyzed ring opening of the cyclic substrate. The active-site amino-acid acid/base pair involved in ring opening has long been investigated and several models have been proposed. Here, the structure of the xylose isomerase E186Q mutant with cyclic glucose bound at the active site, refined against joint X-ray and neutron diffraction data, is reported. Detailed analysis of the hydrogen-bond networks at the active site of the enzyme suggests that His54, which is doubly protonated, is poised to protonate the glucose O5 position, while Lys289, which is neutral, promotes deprotonation of the glucose O1H hydroxyl group via an activated water molecule. The structure also reveals an extended hydrogen-bonding network that connects the conserved residues Lys289 and Lys183 through three structurally conserved water molecules and residue 186, which is a glutamic acid to glutamine mutation.

Mitochondrial Localized Stat3 Promotes Breast Cancer Growth via Phosphorylation of Serine 727

Signal transducer and activator of transcription 3 (Stat3) is a key mediator in the development of many cancers. For 20 years, it has been assumed that Stat3 mediates its biological activities as a nuclear localized transcription factor activated by many cytokines. However, recent studies from this laboratory and others indicate that Stat3 has an independent function in the mitochondria (mitoStat3) where it controls the activity of the electron transport chain (ETC) and mediates Ras-induced transformation of mouse embryo fibroblasts. The actions of mitoStat3 in controlling respiration and Ras transformation are mediated by the phosphorylation state of serine 727. To address the role of mitoStat3 in the pathogenesis of cells that are transformed, we used 4T1 breast cancer cells, which form tumors that metastasize in immunocompetent mice. Substitution of Ser-727 for an alanine or aspartate in Stat3 that has a mitochondrial localization sequence, MLS-Stat3, has profound effects on tumor growth, complex I activity of the ETC, and accumulation of reactive oxygen species (ROS). Cells expressing MLS-Stat3(S727A) display slower tumor growth, decreased complex I activity of the ETC, and increased ROS accumulation under hypoxia compared with cells expressing MLS-Stat3. In contrast, cells expressing MLS-Stat3(S727D) show enhanced tumor growth and complex I activity and decreased production of ROS. These results highlight the importance of serine 727 of mitoStat3 in breast cancer and suggest a novel role for mitoStat3 in regulation of ROS concentrations through its action on the ETC.

High prevalence of Arginine to Glutamine Substitution at 98, 141 and 162 positions in Troponin I (TNNI3) associated with hypertrophic cardiomyopathy among Indians

BackgroundTroponin I (TNNI3) is the inhibitory subunit of the thin filament regulatory complex Troponin, which confers calcium-sensitivity to striated muscle actomyosin ATPase activity. Mutations (2-7%) in this gene had been reported in hypertrophic cardiomyopathy patients (HCM). However, the frequencies of mutations and associated clinical presentation have not been established in cardiomyopathy patients of Indian origin, hence we have undertaken this study.MethodsWe have sequenced all the exons, including the exon-intron boundaries of TNNI3 gene in 101 hypertrophic cardiomyopathy patients (HCM), along with 160 healthy controls, inhabited in the same geographical region of southern India.ResultsOur study revealed a total of 16 mutations. Interestingly, we have observed Arginine to Glutamine (R to Q) mutation at 3 positions 98, 141 and 162, exclusively in HCM patients with family history of sudden cardiac death. The novel R98Q was observed in a severe hypertrophic obstructive cardiomyopathy patient (HOCM). The R141Q mutation was observed in two familial cases of severe asymmetric septal hypertrophy (ASH++). The R162Q mutation was observed in a ASH++ patient with mean septal thickness of 29 mm, and have also consists of allelic heterogeneity by means of having one more synonymous (E179E) mutation at g.4797: G → A: in the same exon 7, which replaces a very frequent codon (GAG: 85%) with a rare codon (GAA: 14%). Screening for R162Q mutation in all the available family members revealed its presence in 9 individuals, including 7 with allelic heterogeneity (R162Q and E179E) of which 4 were severely affected. We also found 2 novel SNPs, (g.2653; G → A and g.4003 C → T) exclusively in HCM, and in silico analysis of these SNPs have predicted to cause defect in recognition/binding sites for proteins responsible for proper splicing.ConclusionOur study has provided valuable information regarding the prevalence of TNNI3 mutations in Indian HCM patients and its risk assessment, these will help in genetic counseling and to adopt appropriate treatment strategies.

STAT1 Signaling Is Not Regulated by a Phosphorylation-Acetylation Switch

The treatment of cells with histone deacetylase inhibitors (HDACi) was reported to reveal the acetylation of STAT1 at lysine 410 and lysine 413 (O. H. Krämer et al., Genes Dev. 20:473-485, 2006). STAT1 acetylation was proposed to regulate apoptosis by facilitating binding to NF-κB and to control immune responses by suppressing STAT1 tyrosine phosphorylation, suggesting that STAT1 acetylation is a central mechanism by which histone deacetylase inhibitors ameliorate inflammatory diseases (O. H. Krämer et al., Genes Dev. 23:223-235, 2009). Here, we show that the inhibition of deacetylases had no bearing on STAT1 acetylation and did not diminish STAT1 tyrosine phosphorylation. The glutamine mutation of the alleged acetylation sites, claimed to mimic acetylated STAT1, similarly did not diminish the tyrosine phosphorylation of STAT1 but precluded its DNA binding and nuclear import. The defective transcription activity of this mutant therefore cannot be attributed to STAT1 acetylation but rather to the inactivation of the STAT1 DNA binding domain and its nuclear import signal. Experiments with respective cDNAs provided by the authors of the studies mentioned above confirmed the results reported here, further questioning the validity of the previous data. We conclude that the effects and potential clinical benefits associated with histone deacetylase inhibition cannot be explained by promoting the acetylation of STAT1 at lysines 410 and 413.

Monitoring the Dynamics of Monomer Exchange Using Electrospray Mass Spectrometry: The Case of the Dimeric Glucosamine-6-Phosphate Synthase

Escherichia coli glucosamine-6-phosphate synthase (GlmS) is a dimeric enzyme from the glutamine-dependent amidotransferases family, which catalyses the conversion of D-fructose-6-phosphate (Fru6P) and glutamine (Gln) into D-glucosamine-6-phosphate (GlcN6P) and glutamate, respectively. Extensive X-ray crystallography investigations have been reported, highlighting the importance of the dimeric association to form the sugar active site as well as significant conformational changes of the protein upon substrate and product binding. In the present work, an approach based on time-resolved noncovalent mass spectrometry has been developed to study the dynamics of GlmS subunit exchange. Using (14)N versus (15)N labeled proteins, the kinetics of GlmS subunit exchange was monitored with the wild-type enzyme in the presence of different substrates and products as well as with the protein bearing a key amino acid mutation specially designed to weaken the dimer interface. Determination of rate constants of subunit exchange revealed important modifications of the protein dynamics: while glutamine, glutamate, and K603A mutation accelerates subunit exchange, Fru6P and GlcN6P totally prevent it. These results are described in light of the available structural information, providing additional useful data for both the characterization of GlmS catalytic process and the design of new GlmS inhibitors. Finally, time-resolved noncovalent MS can be proposed as an additional biophysical technique for real-time monitoring of protein dynamics.

The effects of histone H4 tail acetylations on cation-induced chromatin folding and self-association

Understanding the molecular mechanisms behind regulation of chromatin folding through covalent modifications of the histone N-terminal tails is hampered by a lack of accessible chromatin containing precisely modified histones. We study the internal folding and intermolecular self-association of a chromatin system consisting of saturated 12-mer nucleosome arrays containing various combinations of completely acetylated lysines at positions 5, 8, 12 and 16 of histone H4, induced by the cations Na+, K+, Mg2+, Ca2+, cobalt-hexammine3+, spermidine3+ and spermine4+. Histones were prepared using a novel semi-synthetic approach with native chemical ligation. Acetylation of H4-K16, but not its glutamine mutation, drastically reduces cation-induced folding of the array. Neither acetylations nor mutations of all the sites K5, K8 and K12 can induce a similar degree of array unfolding. The ubiquitous K+, (as well as Rb+ and Cs+) showed an unfolding effect on unmodified arrays almost similar to that of H4-K16 acetylation. We propose that K+ (and Rb+/Cs+) binding to a site on the H2B histone (R96-L99) disrupts H4K16 ε-amino group binding to this specific site, thereby deranging H4 tail-mediated nucleosome–nucleosome stacking and that a similar mechanism operates in the case of H4-K16 acetylation. Inter-array self-association follows electrostatic behavior and is largely insensitive to the position or nature of the H4 tail charge modification.

A novel alanine or threonine 789 to proline mutation causing type 2N von Willebrand’s disease when inherited homozygously or heterozygously with arginine 854 to glutamine mutation

A novel alanine or threonine 789 to proline mutation causing type 2N von Willebrand’s disease when inherited homozygously or heterozygously with arginine 854 to glutamine mutation

SIRT1 Regulates UV-Induced DNA Repair through Deacetylating XPA

SIRT1, a NAD(+)-dependent histone deacetylase, plays crucial roles in multiple biological processes including gene transcription, cellular metabolism, stress response, and tumorigenesis. Xeroderma pigmentosum group A (XPA) is a core nucleotide excision repair (NER) factor essential for NER process. Here we show that SIRT1 plays an important role in the regulation of NER pathway. Downregulation of SIRT1 significantly sensitizes cells to UV irradiation. SIRT1 interacts with XPA, and the interaction is enhanced after UV irradiation. XPA can be acetylated at lysines 63 and 67. SIRT1 deacetylates XPA both in vitro and in cells. Importantly, SIRT1-mediated deacetylation of XPA is required for optimal NER pathway since XPA-deficient cells complemented with XPA-K6367Q, which mimics hyperacetylated XPA, display significantly higher UV sensitivity compared with the XPA cells complemented with wild-type XPA. Furthermore, SIRT1-mediated XPA deacetylation enhances its interaction with RPA32. Our results demonstrate that SIRT1 regulates NER pathway through modulation of XPA acetylation status.

Ethanol Sensitivity of GABAergic Currents in Cerebellar Granule Neurons Is Not Increased by a Single Amino Acid Change (R100Q) in the α6 GABAA Receptor Subunit

Cerebellar granule neurons (CGNs) extrasynaptically express GABA(A) receptors containing alpha(6)beta(x)delta subunits, which mediate tonic inhibitory currents. Although it has been shown that the function of these receptors is potently and directly enhanced by ethanol, this finding has not been reproducible across different laboratories. In outbred Sprague-Dawley rats, a naturally occurring arginine (R) to glutamine (Q) mutation in position 100 of the alpha(6) subunit was reported to increase the ethanol sensitivity of these receptors. However, we did not detect an action of this mutation in selectively bred rats (alcohol-tolerant and alcohol-nontolerant). Consequently, we reexamined the effect of the mutation on ethanol sensitivity in Sprague-Dawley rats. Using patch-clamp electrophysiological techniques in cerebellar vermis parasagittal slices, we found that 25 mM ethanol increases the tonic current amplitude, tonic current noise, and spontaneous inhibitory postsynaptic current (sIPSC) frequency to a similar extent in alpha(6)-100R/100R and alpha(6)-100Q/100Q CGNs. Exposure to 80 mM ethanol increased the tonic current amplitude to a significantly greater extent in alpha(6)-100R/100R than in alpha(6)-100Q/100Q CGNs; however, the effects of 80 mM ethanol on the tonic current noise and sIPSC frequency were not significantly different between these groups. In the presence of tetrodo-toxin, a non-N-methyl-d-aspartate receptor antagonist, exogenous GABA, and a GABA transporter inhibitor, neither 8 nor 40 mM ethanol consistently affected tonic current amplitude or noise in alpha(6)-100R/100R or alpha(6)-100Q/100Q CGNs. Thus, the alpha(6)-R100Q GABA(A) receptor subunit polymorphism does not in-crease the acute ethanol sensitivity of extrasynaptic receptors, lending further support to the hypothesis that ethanol modulates these currents indirectly via a presynaptic mechanism.

Identifying the CRAC Channel

Release of Ca 2+ from intracellular stores elicits Ca 2+ entry through plasma membrane Ca 2+ release-activated Ca 2+ (CRAC) channels. Stromal interaction molecule (Stim in Drosophila , STIM1 in mammals) and Orai (in Drosophila , Orai1 in humans, also known as CRACM1) have been implicated in mediating the CRAC current; however, the precise relation of Orai to the CRAC channel has been unclear. Two groups now use mutational analysis to identify Orai as forming the CRAC channel pore subunit. Yeromin et al . overexpressed wild-type or mutant Orai together with Stim in S1 cells. Store depletion promoted association of the wild-type proteins and elicited a large CRAC current; coexpression of Stim with Orai mutants altered CRAC current properties. A glutamate to aspartate mutation at position 180 (E180D) altered ion selectivity and rectification; a glutamate to alanine mutation at the same location acted as a dominant negative to block native CRAC current; and mutations of aspartate to alanine at 184 and 186 or asparagine to alanine at 188 reduced sensitivity to Gd 3+ block. Prakirya et al . used both biotinylation with a cell-impermeant reagent and immunocytochemical staining of tagged Orai1 expressed in human embryonic kidney (HEK) 293 cells to show that it was a plasma membrane protein. They found that a glutamate to alanine mutation at position 106 (E106D, corresponding to Yeromin et al .’s E180D mutation) altered ion selectivity of Orai1 expressed in T cells deficient in CRAC current and Ca 2+ -influx, as did a glutamate to glutamine mutation at position 190. Thus, both groups conclude that Orai forms the CRAC channel pore subunit. M. Prakriya, S. Feske, Y. Gwack, S. Srikanth, A. Rao, P. G. Hogan, Orai1 is an essential pore subunit of the CRAC channel. Nature 443 , 230-233 (2006). [PubMed] A. V. Yeromin, S. L. Zhang, W. Jiang, Y. Yu, O. Safrina, M. D. Cahalan, Molecular identification of the CRAC channel by altered ion selectivity in a mutant of Orai. Nature 443 , 226-229 (2006). [PubMed]

Asymptomatic Factor VII Deficiency in African Americans

African Americans with factor VII (FVII) deficiency, as defined by clinical laboratory values, are frequently asymptomatic. To date the genotypes underlying this FVII defect in asymptomatic African Americans have not been established. We show in 3 unrelated African-American patients that the defect is due to a G to A nucleotide change resulting in an arginine to glutamine mutation in Factor VII amino acid 304. This defect results in low FVII coagulant activity levels using rabbit brain thromboplastin but not using human thromboplastin. This report may aid transfusion and hematology specialists evaluate patient results and prevent unnecessary transfusions to treat patients with abnormal laboratory values.

Asymptomatic Factor VII Deficiency in African Americans

African Americans with factor VII (FVII) deficiency, as defined by clinical laboratory values, are frequently asymptomatic. To date the genotypes underlying this FVII defect in asymptomatic African Americans have not been established. We show in 3 unrelated African-American patients that the defect is due to a G to A nucleotide change resulting in an arginine to glutamine mutation in Factor VII amino acid 304. This defect results in low FVII coagulant activity levels using rabbit brain thromboplastin but not using human thromboplastin. This report may aid transfusion and hematology specialists evaluate patient results and prevent unnecessary transfusions to treat patients with abnormal laboratory values.

Disruption of POF1B Binding to Nonmuscle Actin Filaments Is Associated with Premature Ovarian Failure

Premature ovarian failure (POF) is characterized by elevated gonadotropins and amenorrhea in women aged <40 years. In a Lebanese family with five sisters who received the diagnosis of POF, we established linkage to the long arm of the X chromosome (between Xq21.1 and Xq21.3.3), using whole-genome SNP typing and homozygosity-by-descent mapping. By sequencing one candidate gene within that region, POF1B, we identified a point mutation localized in exon 10. This substitution of a nucleotide (G-->A), at position 1123, results in an arginine-->glutamine mutation of the protein sequence at position 329 (mutation R329Q). All the affected family members were homozygous for the mutation, whereas the unaffected members were heterozygous. Because POF1B shares high homology with the tail portion of the human myosin, we assessed the ability of both wild-type and mutant POF1B proteins to bind nonmuscle actin filaments in vitro. We found that the capacity of the mutant protein to bind nonmuscle actin filaments was diminished fourfold compared with the wild type, suggesting a function of POF1B in germ-cell division. Our study suggests that a homozygous point mutation in POF1B influences the pathogenesis of POF by altering POF1B binding to nonmuscle actin filaments.

Asymptomatic Factor VII Padua in African Americans.

Background: Symptomatology in congenital human FVII deficiency with FVII:C (Factor VII Coagulant Activity) levels < 1% ranges from asymptomatic to severe hemorrhagic problems. Additionally, FVII:C differs markedly depending upon the source of tissue factor (TF) in the thromboplastin utilized for measuring FVII:C. FVII deficiency reported in 1978 in patients from Padua, Italy was later shown to be associated with an arginine (R) to glutamine (Q) mutation at FVII amino acid 304. A study by Triplett et al. from 1985 reported FVII deficiency in 26 patients including 16 Caucasians, 1 Hispanic and 9 blacks. Of these patients, all 9 black patients were asymptomatic whereas the other 17 patients had a clinically relevant bleeding disorder. Since that time there have been no reports describing the genetic cause of FVII deficiency in asymptomatic African American individuals.Methods: Plasma was obtained from patient blood samples obtained in 3.2% sodium citrate. FVII:C levels were performed using one-stage clotting assays with either rabbit brain thromboplastin (Simplastin Excel, Biomerieux, Durham, NC) or lyophilized recombinant human TF (Innovin, Dade Behring, Inc., Newark, DE). Factor VII antigen levels (VII:Ag) were measured using an enzyme-linked immunoabsorbent assay (American Bioproducts Co, Parsippany, NJ). A normal pool was constructed by mixing equal volumes of plasma from greater than 20 healthy control subjects.ResultsPatient #Prothrombin Time in secondsFVII:C Simplastin ExcelFVII:C InnovinFVII:AgGenotypePhenotype1-African American15.616%33%61%Heterozygote R304QMinor Nose Bleeds2- African AmericanProlonged26%54%67%Heterozygote R304QAsymptomatic for bleeding3- African American14.521%64%NAHeterozygote R304QAsymptomatic for bleedingConclusions: We show that FVII deficiency in three asymptomatic African American patients at our specialty laboratory was due to FVII-Padua (R304Q). An additional ten African American patients showed prolonged prothrombin times and FVII:C values consistent with the laboratory phenotype of FVII-Padua. This diagnosis prevented inappropriate blood transfusions prior to surgery in several of these patients.