Mutation of the ER retention receptor KDELR1 leads to cell-intrinsic lymphopenia and a failure to control chronic viral infection

Endoplasmic reticulum (ER)-resident proteins are continually retrieved from the Golgi and returned to the ER by Lys-Asp-Glu-Leu (KDEL) receptors, which bind to an eponymous tetrapeptide motif at their substrate's C terminus. Mice and humans possess three paralogous KDEL receptors, but little is known about their functional redundancy, or if their mutation can be physiologically tolerated. Here, we present a recessive mouse missense allele of the prototypical mammalian KDEL receptor, KDEL ER protein retention receptor 1 (KDELR1). Kdelr1 homozygous mutants were mildly lymphopenic, as were mice with a CRISPR/Cas9-engineered frameshift allele. Lymphopenia was cell intrinsic and, in the case of T cells, was associated with reduced expression of the T-cell receptor (TCR) and increased expression of CD44, and could be partially corrected by an MHC class I-restricted TCR transgene. Antiviral immunity was also compromised, with Kdelr1 mutant mice unable to clear an otherwise self-limiting viral infection. These data reveal a nonredundant cellular function for KDELR1, upon which lymphocytes distinctly depend.

VE1 immunohistochemistry predicts BRAF V600E mutation status and clinical outcome in colorectal cancer

AIM VE1 is a monoclonal antibody detecting mutant BRAFV600E protein by immunohistochemistry. Here we aim to determine the inter-observer agreement and concordance of VE1 with mutational status, investigate heterogeneity in colorectal cancers and metastases and determine the prognostic effect of VE1 in colorectal cancer patients. METHODS Concordance of VE1 with mutational status and inter-observer agreement were tested on a pilot cohort of colorectal cancers (n = 34), melanomas (n = 23) and thyroid cancers (n = 8). Two prognostic cohorts were evaluated (n = 259, Cohort 1 and n = 226, Cohort 2) by multiple-punch tissue microarrays. VE1 staining on preoperative biopsies (n = 118 patients) was compared to expression in resections. Primary tumors and metastases from 13 patients were tested for VE1 heterogeneity using a tissue microarray generated from all available blocks (n = 100 blocks). RESULTS Inter-observer agreement was 100% (kappa = 1.0). Concordance between VE1 and V600E mutation was 98.5%. Cohort 1: VE1 positivity (seen in 13.5%) was associated with older age (p = 0.0175) and MLH1 deficiency (p < 0.0001). Cohort 2: VE1 positivity (seen in 12.8%) was associated with female gender (p = 0.0016), right-sided tumor location (p < 0.0001), higher tumor grade (p < 0.0001) and mismatch repair (MMR)-deficiency (p < 0.0001). In survival analysis, MMR status and postoperative therapy were identified as possible confounding factors. Adjusting for these features, VE1 was an unfavorable prognostic factor. Preoperative biopsy staining matched resections in all cases except one. No heterogeneity was found across any primary/metastatic tumor blocks. CONCLUSION VE1 is highly concordant for V600E and homogeneously expressed suggesting staining can be analysed on resection specimens, preoperative biopsies, metastatic lesions and tissue microarrays.

Myocardial expression profiles of candidate molecules in patients with arrhythmogenic right ventricular cardiomyopathy/dysplasia compared to those with dilated cardiomyopathy and healthy controls.

BACKGROUND Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is mainly an autosomal dominant disease characterized by fibrofatty infiltration of the right ventricle, leading to ventricular arrhythmias. Mutations in desmosomal proteins can be identified in about half of the patients. The pathogenic mechanisms leading to disease expression remain unclear. OBJECTIVE The purpose of this study was to investigate myocardial expression profiles of candidate molecules involved in the pathogenesis of ARVC/D. METHODS Myocardial messenger RNA (mRNA) expression of 62 junctional molecules, 5 cardiac ion channel molecules, 8 structural molecules, 4 apoptotic molecules, and 6 adipogenic molecules was studied. The averaged expression of candidate mRNAs was compared between ARVC/D samples (n = 10), nonfamilial dilated cardiomyopathy (DCM) samples (n = 10), and healthy control samples (n = 8). Immunohistochemistry and quantitative protein expression analysis were performed. Genetic analysis using next generation sequencing was performed in all patients with ARVC/D. RESULTS Following mRNA levels were significantly increased in patients with ARVC/D compared to those with DCM and healthy controls: phospholamban (P ≤ .001 vs DCM; P ≤ .001 vs controls), healthy tumor protein 53 apoptosis effector (P = .001 vs DCM; P ≤ .001 vs controls), and carnitine palmitoyltransferase 1β (P ≤ .001 vs DCM; P = 0.008 vs controls). Plakophillin-2 (PKP-2) mRNA was downregulated in patients with ARVC/D with PKP-2 mutations compared with patients with ARVC/D without PKP-2 mutations (P = .04). Immunohistochemistry revealed significantly increased protein expression of phospholamban, tumor protein 53 apoptosis effector, and carnitine palmitoyltransferase 1β in patients with ARVC/D and decreased PKP-2 expression in patients with ARVC/D carrying a PKP-2 mutation. CONCLUSION Changes in the expression profiles of sarcolemmal calcium channel regulation, apoptosis, and adipogenesis suggest that these molecular pathways may play a critical role in the pathogenesis of ARVC/D, independent of the underlying genetic mutations.

A novel mutation in BCS1L associated with deafness, tubulopathy, growth retardation and microcephaly

We report a novel homozygous missense mutation in the ubiquinol-cytochrome c reductase synthesis-like (BCS1L) gene in two consanguineous Turkish families associated with deafness, Fanconi syndrome (tubulopathy), microcephaly, mental and growth retardation. All three patients presented with transitory metabolic acidosis in the neonatal period and development of persistent renal de Toni-Debré-Fanconi-type tubulopathy, with subsequent rachitis, short stature, microcephaly, sensorineural hearing impairment, mild mental retardation and liver dysfunction. The novel missense mutation c.142A>G (p.M48V) in BCS1L is located at a highly conserved region associated with sorting to the mitochondria. Biochemical analysis revealed an isolated complex III deficiency in skeletal muscle not detected in fibroblasts. Native polyacrylamide gel electrophoresis (PAGE) revealed normal super complex formation, but a shift in mobility of complex III most likely caused by the absence of the BCS1L-mediated insertion of Rieske Fe/S protein into complex III. These findings expand the phenotypic spectrum of BCS1L mutations, highlight the importance of biochemical analysis of different primary affected tissue and underline that neonatal lactic acidosis with multi-organ involvement may resolve after the newborn period with a relatively spared neurological outcome and survival into adulthood. CONCLUSION Mutation screening for BCS1L should be considered in the differential diagnosis of severe (proximal) tubulopathy in the newborn period. What is Known: • Mutations in BCS1L cause mitochondrial complex III deficiencies. • Phenotypic presentations of defective BCS1L range from Bjornstad to neonatal GRACILE syndrome. What is New: • Description of a novel homozygous mutation in BCS1L with transient neonatal acidosis and persistent de Toni-Debré-Fanconi-type tubulopathy. • The long survival of patients with phenotypic presentation of severe complex III deficiency is uncommon.

IGHD II: A Novel GH-1 Gene Mutation (GH-L76P) Severely Affects GH Folding, Stability, and Secretion

CONTEXT The autosomal dominant form of GH deficiency (IGHD II) is characterized by markedly reduced GH secretion combined with low concentrations of IGF-1 leading to short stature. OBJECTIVE Structure-function analysis of a missense mutation in the GH-1 gene converting codon 76 from leucine (L) to proline (P) yielding a mutant GH-L76P peptide. DESIGN, SETTINGS, AND PATIENTS Heterozygosity for GH-L76P/wt-GH was identified in a nonconsanguineous Spanish family. The index patients, two siblings, a boy and a girl, were referred for assessment of their short stature (-3.2 and -3.8 SD). Their grandmother, father, and aunt were also carrying the same mutation and showed severe short stature; therefore, IGHD II was diagnosed. INTERVENTIONS AND RESULTS AtT-20 cells coexpressing both wt-GH and GH-L76P showed a reduced GH secretion (P < .001) after forskolin stimulation when compared with the cells expressing only wt-GH. In silico mutagenesis and molecular dynamics simulations presented alterations of correct folding and mutant stability compared with wt-GH. Therefore, further structural analysis of the GH-L76P mutant was performed using expressed and purified proteins in Escherichia coli by thermofluor assay and fast degradation proteolysis assay. Both assays revealed that the GH-L76P mutant is unstable and misfolded compared to wt-GH confirming the bioinformatic model prediction. CONCLUSIONS This is the first report of a family suffering from short stature caused by IGHD II, which severely affects intracellular GH folding and stability as well as secretion, highlighting the necessity of functional analysis of any GH variant for defining new mechanisms as a cause for IGHD II.

Mutation Update of the CLCN5 Gene Responsible for Dent Disease 1

Dent disease is a rare X-linked tubulopathy characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis and/or nephrolithiasis, progressive renal failure, and variable manifestations of other proximal tubule dysfunctions. It often progresses over a few decades to chronic renal insufficiency, and therefore molecular characterization is important to allow appropriate genetic counseling. Two genetic subtypes have been described to date: Dent disease 1 is caused by mutations of the CLCN5 gene, coding for the chloride/proton exchanger ClC-5; and Dent disease 2 by mutations of the OCRL gene, coding for the inositol polyphosphate 5-phosphatase OCRL-1. Herein, we review previously reported mutations (n = 192) and their associated phenotype in 377 male patients with Dent disease 1 and describe phenotype and novel (n = 42) and recurrent mutations (n = 24) in a large cohort of 117 Dent disease 1 patients belonging to 90 families. The novel missense and in-frame mutations described were mapped onto a three-dimensional homology model of the ClC-5 protein. This analysis suggests that these mutations affect the dimerization process, helix stability, or transport. The phenotype of our cohort patients supports and extends the phenotype that has been reported in smaller studies.

Complex genetic background in a large family with Brugada syndrome.

The Brugada syndrome (BrS) is an inherited arrhythmia characterized by ST-segment elevation in V1-V3 leads and negative T wave on standard ECG. BrS patients are at risk of sudden cardiac death (SCD) due to ventricular tachyarrhythmia. At least 17 genes have been proposed to be linked to BrS, although recent findings suggested a polygenic background. Mutations in SCN5A, the gene coding for the cardiac sodium channel Nav1.5, have been found in 15-30% of index cases. Here, we present the results of clinical, genetic, and expression studies of a large Iranian family with BrS carrying a novel genetic variant (p.P1506S) in SCN5A. By performing whole-cell patch-clamp experiments using HEK293 cells expressing wild-type (WT) or p.P1506S Nav1.5 channels, hyperpolarizing shift of the availability curve, depolarizing shift of the activation curve, and hastening of the fast inactivation process were observed. These mutant-induced alterations lead to a loss of function of Nav1.5 and thus suggest that the p.P1506S variant is pathogenic. In addition, cascade familial screening found a family member with BrS who did not carry the p.P1506S mutation. Additional next generation sequencing analyses revealed the p.R25W mutation in KCNH2 gene in SCN5A-negative BrS patients. These findings illustrate the complex genetic background of BrS found in this family and the possible pathogenic role of a new SCN5A genetic variant.

STUDIES ON THE PATTERNS OF NUCLEOTIDE AND AMINO ACID SUBSTITUTION (ELECTROPHORESIS, MUTATION RATE, SELECTION, BASE, COMPOSITION)

Theoretical and empirical studies were conducted on the pattern of nucleotide and amino acid substitution in evolution, taking into account the effects of mutation at the nucleotide level and purifying selection at the amino acid level. A theoretical model for predicting the evolutionary change in electrophoretic mobility of a protein was also developed by using information on the pattern of amino acid substitution. The specific problems studied and the main results obtained are as follows: (1) Estimation of the pattern of nucleotide substitution in DNA nuclear genomes. The pattern of point mutations and nucleotide substitutions among the four different nucleotides are inferred from the evolutionary changes of pseudogenes and functional genes, respectively. Both patterns are non-random, the rate of change varying considerably with nucleotide pair, and that in both cases transitions occur somewhat more frequently than transversions. In protein evolution, substitution occurs more often between amino acids with similar physico-chemical properties than between dissimilar amino acids. (2) Estimation of the pattern of nucleotide substitution in RNA genomes. The majority of mutations in retroviruses accumulate at the reverse transcription stage. Selection at the amino acid level is very weak, and almost non-existent between synonymous codons. The pattern of mutation is very different from that in DNA genomes. Nevertheless, the pattern of purifying selection at the amino acid level is similar to that in DNA genomes, although selection intensity is much weaker. (3) Evaluation of the determinants of molecular evolutionary rates in protein-coding genes. Based on rates of nucleotide substitution for mammalian genes, the rate of amino acid substitution of a protein is determined by its amino acid composition. The content of glycine is shown to correlate strongly and negatively with the rate of substitution. Empirical formulae, called indices of mutability, are developed in order to predict the rate of molecular evolution of a protein from data on its amino acid sequence. (4) Studies on the evolutionary patterns of electrophoretic mobility of proteins. A theoretical model was constructed that predicts the electric charge of a protein at any given pH and its isoelectric point from data on its primary and quaternary structures. Using this model, the evolutionary change in electrophoretic mobilities of different proteins and the expected amount of electrophoretically hidden genetic variation were studied. In the absence of selection for the pI value, proteins will on the average evolve toward a mildly basic pI. (Abstract shortened with permission of author.) ^

Exact moment calculations for genetic models with migration, mutation, and drift

Using properties of moment stationarity we develop exact expressions for the mean and covariance of allele frequencies at a single locus for a set of populations subject to drift, mutation, and migration. Some general results can be obtained even for arbitrary mutation and migration matrices, for example: (1) Under quite general conditions, the mean vector depends only on mutation rates, not on migration rates or the number of populations. (2) Allele frequencies covary among all pairs of populations connected by migration. As a result, the drift, mutation, migration process is not ergodic when any finite number of populations is exchanging genes. in addition, we provide closed form expressions for the mean and covariance of allele frequencies in Wright's finite-island model of migration under several simple models of mutation, and we show that the correlation in allele frequencies among populations can be very large for realistic rates of mutation unless an enormous number of populations are exchanging genes. As a result, the traditional diffusion approximation provides a poor approximation of the stationary distribution of allele frequencies among populations. Finally, we discuss some implications of our results for measures of population structure based on Wright's F-statistics.

Role of FbpA and FbpB in the pathogenesis and mycolyltransferase activity of Mycobacterium tuberculosis

Mycobacterium tuberculosis infects more people worldwide each year than any other single organism. The Antigen 85 Complex, a family of fibronectin-binding proteins (Fbps) found in several species of mycobacteria and possibly involved in host interaction, is considered among the putative virulence factors of M. tuberculosis. These proteins are implicated in the production of trehalose dimycolate (TDM) and arabinogalactan-mycolate (AG-M), two prominent components of the mycobacterium cell wall and potent modulators of the immune system during infection. For these reasons, the principal members of the complex, FbpA and FbpB, were the focus of these studies. The genes encoding these proteins, fbpA and fbpB, were each disrupted by insertion of a kanamycin resistance cassette in a pathogenic strain of M. tuberculosis, H37Rv. Neither mutation affected growth in routine broth culture. Thin layer chromatography analysis of TDM and AG-M showed no difference in content between the parent strain H37Rv and the FbpA- and FbpB-deficient mutants grown under two different culture conditions. However, metabolic radiolabeling of the strains showed that the production of TDM (but not its precursor TMM) was delayed in the FbpA- and FbpB-deficient mutants compared to the parent H37Rv. During this same labeling period, FbpA-deficient mutant LAa1 failed to produce AG-M and in the FpbB-deficient mutant LAb1 production was decreased. In macrophage tissue culture assay, LAa1 failed to multiply when bacteria in early log phase were used to infect monolayers while LAb1 grew like the parent strain. The growth deficiency of LAa1 as well as the deficiencies in TDM and AG-M production were restored by complementing LAa1 with a functional fbpA gene. These results suggest that the FbpA and FbpB proteins are involved in synthesis of TDM (but not its precursor TMM) as well as AG-M. Other members of the complex appear to compensate for defects in synthesis caused by mutation of single genes in the complex over time. Mutation of the FbpA gene causes greater in vivo effect than mutation of the FbpB gene despite very similar deficiencies in the rate of production of mycolate containing molecules on the cell surface. ^

A coalescent analysis for modeling the mutation process in colorectal cancer

Colorectal cancer is the forth most common diagnosed cancer in the United States. Every year about a hundred forty-seven thousand people will be diagnosed with colorectal cancer and fifty-six thousand people lose their lives due to this disease. Most of the hereditary nonpolyposis colorectal cancer (HNPCC) and 12% of the sporadic colorectal cancer show microsatellite instability. Colorectal cancer is a multistep progressive disease. It starts from a mutation in a normal colorectal cell and grows into a clone of cells that further accumulates mutations and finally develops into a malignant tumor. In terms of molecular evolution, the process of colorectal tumor progression represents the acquisition of sequential mutations. ^ Clinical studies use biomarkers such as microsatellite or single nucleotide polymorphisms (SNPs) to study mutation frequencies in colorectal cancer. Microsatellite data obtained from single genome equivalent PCR or small pool PCR can be used to infer tumor progression. Since tumor progression is similar to population evolution, we used an approach known as coalescent, which is well established in population genetics, to analyze this type of data. Coalescent theory has been known to infer the sample's evolutionary path through the analysis of microsatellite data. ^ The simulation results indicate that the constant population size pattern and the rapid tumor growth pattern have different genetic polymorphic patterns. The simulation results were compared with experimental data collected from HNPCC patients. The preliminary result shows the mutation rate in 6 HNPCC patients range from 0.001 to 0.01. The patients' polymorphic patterns are similar to the constant population size pattern which implies the tumor progression is through multilineage persistence instead of clonal sequential evolution. The results should be further verified using a larger dataset. ^

NRAS and BRAF mutations in primary cutaneous melanoma: A comparison of mutation rates between radial and vertical growth phases in individual tumors

Primary cutaneous melanoma is a cancer arising from melanocytes in the skin. In recent decades the incidence of this malignancy has increased significantly. Mortality rates are high for patients with tumors measuring over a few millimeters in thickness. Response rates to conventional radiation and chemotherapy are very low in patients with metastatic melanoma. New therapies targeting melanoma’s aberrant cell signaling pathways such as the MAP Kinase pathway are being developed. Mutations of NRAS and BRAF genes are quite common in cutaneous melanoma and lead to constitutive activation of the MAP Kinase pathway. This study tests the hypothesis that NRAS and BRAF mutations increase as a tumor progresses from the noninvasive radial growth phase (RGP) to the invasive vertical growth phase (VGP). Laser capture microdissection was used to obtain separate, pure tumor DNA samples from the RGP and VGP of thirty primary cutaneous melanomas. PCR was used to amplify NRAS exon 2 and BRAF exon 15 tumor DNA. The amplified DNA was sequenced and analyzed for mutations. An overall mutation rate of 74% was obtained for the twenty-three melanomas in which there were complete sequence results. With the exception of one melanoma NRAS and BRAF mutations were mutually exclusive. All seven NRAS exon 2 mutations involved codon 61. Three of these melanomas had mutations in both the RGP and VGP. The remaining four tumors were wild type for NRAS exon 2 in the RGP but mutated in the VGP. Of the fifteen BRAF exon 15 mutated melanomas all but one involved codon 600. Twelve of the fifteen BRAF exon 15 mutations were the T1799A type. Nine of the fifteen BRAF mutated tumors had the same mutation in both the RGP and VGP. Five of fifteen melanomas had wild type RGP DNA and BRAF exon 15 mutated VGP DNA. A single melanoma had BRAF exon 15 mutated DNA in the RGP and wild type DNA in the VGP. Overall, these results suggest a trend toward the acquisition of NRAS and BRAF mutations as cutaneous melanomas change from a noninvasive to an invasive, potentially deadly cancer.^

Antibody chemical reactivity: Beneficial and pathogenic roles

Antibodies (Abs) to autoantigens and foreign antigens (Ags) mediate, respectively, various pathogenic and beneficial effects. Abs express enzyme-like nucleophiles that react covalently with electrophiles. A subpopulation of nucleophilic Abs expresses proteolytic activity, which can inactivate the Ag permanently. This thesis shows how the nucleophilicity can be exploited to inhibit harmful Abs or potentially protect against a virus. ^ Inactivation of pathogenic Abs from Hemophilia A (HA) patients by means of nucleophile-electrophile pairing was studied. Deficient factor VIII (FVIII) in HA subjects impairs blood coagulation. FVIII replacement therapy fails in 20-30% of HA patients due to production of anti-FVIII Abs. FVIII analogs containing electrophilic phosphonate group (E-FVIII and E-C2) were hypothesized to inactivate the Abs by reacting specifically and covalently with nucleophilic sites. Anti-FVIII IgGs from HA patients formed immune complexes with E-FVIII and E-C2 that remained irreversibly associated under conditions that disrupt noncovalent Ab-Ag complexes. The reaction induced irreversible loss of Ab anti-coagulant activity. E-FVIII alone displayed limited interference with coagulation. E-FVIII is a prototype reagent suitable for further development as a selective inactivator of pathogenic anti-FVIII Abs. ^ The beneficial function of Abs to human immunodeficiency virus type 1 (HIV-1) was analyzed. HIV-1 eludes the immune system by rapidly changing its coat protein structure. IgAs from noninfected subjects hydrolyzed gp120 and neutralized HIV-1 with modest potency by recognizing the gp120 421-433 epitope, a conserved B cell superantigenic region that is also essential for HIV-1 attachment to host cell CD4 receptors. An adaptive immune response to superantigens is generally prohibited due to their ability to downregulate B cells. IgAs from subjects with prolonged HIV-1 infection displayed improved catalytic hydrolysis of gp120 and exceptionally potent and broad neutralization of diverse CCR5-dependent primary HIV isolates attributable to recognition of the 421-433 epitope. This indicates that slow immunological bypass of the superantigenic character of gp120 is possible, opening the path to effective HIV vaccination. ^ My research reveals a novel route to inactivate pathogenic nucleophilic Abs using electrophilic antigens. Conversely, naturally occurring nucleophilic Abs may help impede HIV infection, and the Abs could be developed for passive immunotherapy of HIV infected subjects. ^