Modulate Disease Severity Research Articles

Development of a Murine Model for Aerosolized Ebolavirus Infection Using a Panel of Recombinant Inbred Mice

Countering aerosolized filovirus infection is a major priority of biodefense research. Aerosol models of filovirus infection have been developed in knock-out mice, guinea pigs and non-human primates; however, filovirus infection of immunocompetent mice by the aerosol route has not been reported. A murine model of aerosolized filovirus infection in mice should be useful for screening vaccine candidates and therapies. In this study, various strains of wild-type and immunocompromised mice were exposed to aerosolized wild-type (WT) or mouse-adapted (MA) Ebola virus (EBOV). Upon exposure to aerosolized WT-EBOV, BALB/c, C57BL/6 (B6), and DBA/2 (D2) mice were unaffected, but 100% of severe combined immunodeficiency (SCID) and 90% of signal transducers and activators of transcription (Stat1) knock-out (KO) mice became moribund between 7–9 days post-exposure (dpe). Exposure to MA-EBOV caused 15% body weight loss in BALB/c, but all mice recovered. In contrast, 10–30% lethality was observed in B6 and D2 mice exposed to aerosolized MA-EBOV, and 100% of SCID, Stat1 KO, interferon (IFN)-γ KO and Perforin KO mice became moribund between 7–14 dpe. In order to identify wild-type, inbred, mouse strains in which exposure to aerosolized MA-EBOV is uniformly lethal, 60 BXD (C57BL/6 crossed with DBA/2) recombinant inbred (RI) and advanced RI (ARI) mouse strains were exposed to aerosolized MA-EBOV, and monitored for disease severity. A complete spectrum of disease severity was observed. All BXD strains lost weight but many recovered. However, infection was uniformly lethal within 7 to 12 days post-exposure in five BXD strains. Aerosol exposure of these five BXD strains to 10-fold less MA-EBOV resulted in lethality ranging from 0% in two strains to 90–100% lethality in two strains. Analysis of post-mortem tissue from BXD strains that became moribund and were euthanized at the lower dose of MA-EBOV, showed liver damage in all mice as well as lung lesions in two of the three strains. The two BXD strains that exhibited 90–100% mortality, even at a low dose of airborne MA-EBOV will be useful mouse models for testing vaccines and therapies. Additionally, since disease susceptibility is affected by complex genetic traits, a systems genetics approach was used to identify preliminary gene loci modulating disease severity among the panel BXD strains. Preliminary quantitative trait loci (QTLs) were identified that are likely to harbor genes involved in modulating differential susceptibility to Ebola infection.

MBNL1 gene variants as modifiers of disease severity in myotonic dystrophy type 1

Myotonic dystrophy type 1 (DM1) is a multisystemic autosomal dominant disorder characterized by a highly variable phenotype and caused by an unstable CTG repeat expansion in the 3' untranslated region of the dystrophia myotonica protein kinase (DMPK) gene. Longer CTG repeat expansions often correlate with an anticipated age at onset and CTG repeat number may account for 45-60 % of the variance in disease severity. In order to search for candidate genes that could act as modifiers of disease severity, we studied the association between Muscleblind-like protein-1 (MBNL1) gene polymorphisms and the DM1 phenotype. In a group of 301 patients diagnosed with DM1 based on clinical symptoms, diagnosis was confirmed by molecular analysis of the DMPK gene. Patients were divided into four subtypes. The first subtype corresponded to asymptomatic patients or those with a mild phenotype, the second included those with a classic phenotype, the third concerned childhood onset, and the fourth corresponded to the congenital form of DM1. Three SNPs located in the MBNL1 gene promoter, rs323622, rs17283597, and rs17433672, were studied. Case-control analysis revealed that allele frequencies for the latter two were significantly associated with DM1 (p = 0.037 and p = 0.020). Multivariate linear regression analysis using phenotype as the dependent variable demonstrated that the TT genotype of the third SNP, rs323622, was associated with a more severe phenotype (p = 0.0034) and accounted for 1.88 % of the variance in disease severity. We report the association of several genetic variants of the MBNL1 gene with DM1 or with the severity of the disease.

The Endosomal Protein-Sorting Receptor Sortilin Has a Role in Trafficking α-1 Antitrypsin

Up to 1 in 3000 individuals in the United States have α-1 antitrypsin deficiency, and the most common cause of this disease is homozygosity for the antitrypsin-Z variant (ATZ). ATZ is inefficiently secreted, resulting in protein deficiency in the lungs and toxic polymer accumulation in the liver. However, only a subset of patients suffer from liver disease, suggesting that genetic factors predispose individuals to liver disease. To identify candidate factors, we developed a yeast ATZ expression system that recapitulates key features of the disease-causing protein. We then adapted this system to screen the yeast deletion mutant collection to identify conserved genes that affect ATZ secretion and thus may modify the risk for developing liver disease. The results of the screen and associated assays indicate that ATZ is degraded in the vacuole after being routed from the Golgi. In fact, one of the strongest hits from our screen was Vps10, which can serve as a receptor for the delivery of aberrant proteins to the vacuole. Because genome-wide association studies implicate the human Vps10 homolog, sortilin, in cardiovascular disease, and because hepatic cell lines that stably express wild-type or mutant sortilin were recently established, we examined whether ATZ levels and secretion are affected by sortilin. As hypothesized, sortilin function impacts the levels of secreted ATZ in mammalian cells. This study represents the first genome-wide screen for factors that modulate ATZ secretion and has led to the identification of a gene that may modify disease severity or presentation in individuals with ATZ-associated liver disease.

Differing Roles for TCF4 and COL8A2 in Central Corneal Thickness and Fuchs Endothelial Corneal Dystrophy

Fuchs endothelial corneal dystrophy (FECD) is the most common late-onset, vision-threatening corneal dystrophy in the United States, affecting about 4% of the population. Advanced FECD involves a thickening of the cornea from stromal edema and changes in Descemet membrane. To understand the relationship between FECD and central corneal thickness (CCT), we characterized common genetic variation in COL8A2 and TCF4, genes previously implicated in CCT and/or FECD. Other genes previously associated with FECD (PITX2, ZEB1, SLC4A11), and genes only known to affect CCT (COL5A1, FOXO1, AVGR8, ZNF469) were also interrogated. FECD probands, relatives and controls were recruited from 32 clinical sites; a total of 532 cases and 204 controls were genotyped and tested for association of FECD case/control status, a 7-step FECD severity scale and CCT, adjusting for age and sex. Association of FECD grade with TCF4 was highly significant (OR = 6.01 at rs613872; p = 4.8×10−25), and remained significant when adjusted for changes in CCT (OR = 4.84; p = 2.2×10−16). Association of CCT with TCF4 was also significant (p = 6.1×10−7), but was abolished with adjustment for FECD grade (p = 0.92). After adjusting for FECD grade, markers in other genes examined were modestly associated (p ∼ 0.001) with FECD and/or CCT. Thus, common variants in TCF4 appear to influence FECD directly, and CCT secondarily via FECD. Additionally, changes in corneal thickness due to the effect of other loci may modify disease severity, age-at-onset, or other biomechanical characteristics.

T.P.33 A cutting-edge approach to Spinal Muscular Atrophy treatment using antisense oligonucleotides

Abstract Spinal Muscular Atrophy (SMA), a severe neuromuscular disorder characterised by the loss of motor neurons, is typically caused by deletion of the Survival Motor Neuron (SMN) 1 gene. The loss of the SMN1 gene product is incompatible with life, however in humans the presence of one or more copies of the near-identical SMN2 modifies disease severity. A single C>T base change in exon 7 of SMN2 results in abnormal splicing with the majority of transcripts missing exon 7 that encodes a crucial oligomerisation domain, and therefore results in a non-functional protein. The C>T variation simultaneously abrogates an exonic splicing enhancer, and creates a silencing element that leads to the exclusion of this exon from the mature gene transcript. Hence, redirecting splicing of SMN2 pre-mRNA has become a potential therapeutic target with the aim of re-enforcing exon 7 recognition and inclusion to increase levels of the full-length transcript and functional protein. Antisense oligomers (AOs) can interfere with splice site recognition by the spliceosome, and depending upon design can promote exon skipping or exon inclusion in the mature transcript. In treating SMA, efforts have focused on either enhancing exon 7 recognition, or masking splice silencing motifs located in introns 6 or 7. We present a third strategy where targeting the acceptor site of exon 8 with AOs induced exon and intron 7 inclusion in the transcript. The retention of intron 7 occurs after the normal stop codon and results in a longer than normal 3′ untranslated region. Western blot analysis demonstrated increased SMN expression in various SMA patient fibroblasts transfected with these AOs. “GEMs” are intranuclear aggregates of SMN and are barely quantifiable in fibroblasts from SMA patients. Immunofluorescent GEM staining showed substantial increases in abundance of GEMs in the AO treated SMA fibroblasts, indicating this strategy has considerable therapeutic potential.

IKAP expression levels modulate disease severity in a mouse model of familial dysautonomia

Hereditary sensory and autonomic neuropathies (HSANs) encompass a group of genetically inherited disorders characterized by sensory and autonomic dysfunctions. Familial dysautonomia (FD), also known as HSAN type III, is an autosomal recessive disorder that affects 1/3600 live births in the Ashkenazi Jewish population. The disease is caused by abnormal development and progressive degeneration of the sensory and autonomic nervous systems and is inevitably fatal, with only 50% of patients reaching the age of 40. FD is caused by a mutation in intron 20 of the Ikbkap gene that results in severe reduction in the expression of its encoded protein, inhibitor of kappaB kinase complex-associated protein (IKAP). Although the mutation that causes FD was identified in 2001, so far there is no appropriate animal model that recapitulates the disorder. Here, we report the generation and characterization of the first mouse models for FD that recapitulate the molecular and pathological features of the disease. Important for therapeutic interventions is also our finding that a slight increase in IKAP levels is enough to ameliorate the phenotype and increase the life span. Understanding the mechanisms underlying FD will provide insights for potential new therapeutic interventions not only for FD, but also for other peripheral neuropathies.

Alternative Splicing and Tissue-specific Elastin Misassembly Act as Biological Modifiers of Human Elastin Gene Frameshift Mutations Associated with Dominant Cutis Laxa

Elastin is the extracellular matrix protein in vertebrates that provides elastic recoil to blood vessels, the lung, and skin. Because the elastin gene has undergone significant changes in the primate lineage, modeling elastin diseases in non-human animals can be problematic. To investigate the pathophysiology underlying a class of elastin gene mutations leading to autosomal dominant cutis laxa, we engineered a cutis laxa mutation (single base deletion) into the human elastin gene contained in a bacterial artificial chromosome. When expressed as a transgene in mice, mutant elastin was incorporated into elastic fibers in the skin and lung with adverse effects on tissue function. In contrast, only low levels of mutant protein incorporated into aortic elastin, which explains why the vasculature is relatively unaffected in this disease. RNA stability studies found that alternative exon splicing acts as a modifier of disease severity by influencing the spectrum of mutant transcripts that survive nonsense-mediated decay. Our results confirm the critical role of the C-terminal region of tropoelastin in elastic fiber assembly and suggest tissue-specific differences in the elastin assembly pathway.

Paradoxical Effects of Rapamycin on Experimental House Dust Mite-Induced Asthma

The mammalian target of rapamycin (mTOR) modulates immune responses and cellular proliferation. The objective of this study was to assess whether inhibition of mTOR with rapamycin modifies disease severity in two experimental murine models of house dust mite (HDM)-induced asthma. In an induction model, rapamycin was administered to BALB/c mice coincident with nasal HDM challenges for 3 weeks. In a treatment model, nasal HDM challenges were performed for 6 weeks and rapamycin treatment was administered during weeks 4 through 6. In the induction model, rapamycin significantly attenuated airway inflammation, airway hyperreactivity (AHR) and goblet cell hyperplasia. In contrast, treatment of established HDM-induced asthma with rapamycin exacerbated AHR and airway inflammation, whereas goblet cell hyperplasia was not modified. Phosphorylation of the S6 ribosomal protein, which is downstream of mTORC1, was increased after 3 weeks, but not 6 weeks of HDM-challenge. Rapamycin reduced S6 phosphorylation in HDM-challenged mice in both the induction and treatment models. Thus, the paradoxical effects of rapamycin on asthma severity paralleled the activation of mTOR signaling. Lastly, mediastinal lymph node re-stimulation experiments showed that treatment of rapamycin-naive T cells with ex vivo rapamycin decreased antigen-specific Th2 cytokine production, whereas prior exposure to in vivo rapamycin rendered T cells refractory to the suppressive effects of ex vivo rapamycin. We conclude that rapamycin had paradoxical effects on the pathogenesis of experimental HDM-induced asthma. Thus, consistent with the context-dependent effects of rapamycin on inflammation, the timing of mTOR inhibition may be an important determinant of efficacy and toxicity in HDM-induced asthma.

Somatic instability of the expanded CTG triplet repeat in myotonic dystrophy type 1 is a heritable quantitative trait and modifier of disease severity

Deciphering the contribution of genetic instability in somatic cells is critical to our understanding of many human disorders. Myotonic dystrophy type 1 (DM1) is one such disorder that is caused by the expansion of a CTG repeat that shows extremely high levels of somatic instability. This somatic instability has compromised attempts to measure intergenerational repeat dynamics and infer genotype-phenotype relationships. Using single-molecule PCR, we have characterized more than 17 000 de novo somatic mutations from a large cohort of DM1 patients. These data reveal that the estimated progenitor allele length is the major modifier of age of onset. We find no evidence for a threshold above which repeat length does not contribute toward age at onset, suggesting pathogenesis is not constrained to a simple molecular switch such as nuclear retention of the DMPK transcript or haploinsufficiency for DMPK and/or SIX5. Importantly, we also show that age at onset is further modified by the level of somatic instability; patients in whom the repeat expands more rapidly, develop the symptoms earlier. These data establish a primary role for somatic instability in DM1 severity, further highlighting it as a therapeutic target. In addition, we show that the level of instability is highly heritable, implying a role for individual-specific trans-acting genetic modifiers. Identifying these trans-acting genetic modifiers will facilitate the formulation of novel therapies that curtail the accumulation of somatic expansions and may provide clues to the role these factors play in the development of cancer, aging and inherited disease in the general population.

MCLCA3 Does Not Contribute to Calcium-Activated Chloride Conductance in Murine Airways

Ca(2+)-activated Cl(-) channels (CaCCs) contribute to airway Cl(-) and fluid secretion, and were implicated in the modulation of disease severity and as a therapeutic target in cystic fibrosis (CF). Previous in vitro studies suggested that members of the CLCA gene family, including the murine mCLCA3, contribute to CaCCs. However, the role of mCLCA3 in ion transport in native airway epithelia has not been studied, to the best of our knowledge. In this study, we used mCLCA3-deficient mice and determined bioelectric properties in freshly excised tracheal tissue, airway morphology, and gene expression studies, to determine the role of mCLCA3 in airway ion transport and airway structure. Bioelectric measurements did not detect any differences in basal short-circuit current, amiloride-sensitive Na(+) absorption, cyclic adenosine monophosphate-dependent Cl(-) secretion, and activation of Ca(2+)-activated (uridine-5'-triphosphate-mediated) Cl(-) secretion in mCLCA3-deficient mice compared with wild-type mice. Moreover, no histological changes were observed in the respiratory tract or any other tissues of mCLCA3-deficient mice when compared with wild-type control mice. The intratracheal instillation of IL-13 produced an approximately 30-fold up-regulation of mCLCA3 transcripts without inducing CaCC activity in wild-type airways, and induced goblet-cell hyperplasia and mucin gene expression to similar levels in both genotypes. Further, multiple specific reverse-transcriptase quantitative PCR assays for other CaCC candidates, including mCLCA1, mCLCA2, mCLCA4, mCLCA5, mCLCA6, mCLCA7, mBEST1, mBEST2, mCLC4, mTTYH3, and mTMEM16A, failed to identify the differential expression of genes in the respiratory tract that may compensate for a lack of mCLCA3 function. Together, these findings argue against a role of mCLCA3 in CaCC-mediated Cl(-) secretion in murine respiratory epithelia.

Variants in the 3′ untranslated region of the KCNQ1-encoded Kv7.1 potassium channel modify disease severity in patients with type 1 long QT syndrome in an allele-specific manner

AimsHeterozygous mutations in KCNQ1 cause type 1 long QT syndrome (LQT1), a disease characterized by prolonged heart rate-corrected QT interval (QTc) and life-threatening arrhythmias. It is unknown why disease penetrance and expressivity is so variable between individuals hosting identical mutations. We aimed to study whether this can be explained by single nucleotide polymorphisms (SNPs) in KCNQ1's 3′ untranslated region (3′UTR).Methods and resultsThis study was performed in 84 LQT1 patients from the Academic Medical Center in Amsterdam and validated in 84 LQT1 patients from the Mayo Clinic in Rochester. All patients were genotyped for SNPs in KCNQ1's 3′UTR, and six SNPs were found. Single nucleotide polymorphisms rs2519184, rs8234, and rs10798 were associated in an allele-specific manner with QTc and symptom occurrence. Patients with the derived SNP variants on their mutated KCNQ1 allele had shorter QTc and fewer symptoms, while the opposite was also true: patients with the derived SNP variants on their normal KCNQ1 allele had significantly longer QTc and more symptoms. Luciferase reporter assays showed that the expression of KCNQ1's 3′UTR with the derived SNP variants was lower than the expression of the 3′UTR with the ancestral SNP variants.ConclusionOur data indicate that 3′UTR SNPs potently modify disease severity in LQT1. The allele-specific effects of the SNPs on disease severity and gene expression strongly suggest that they are functional variants that directly alter the expression of the allele on which they reside, and thereby influence the balance between proteins stemming from either the normal or the mutant KCNQ1 allele.

A High Through-Put Screen Identifies MCP-1 (CCL2) As a Novel Regulator of Iron Homeostasis and a Modifier of Hereditary Hemochromatosis Disease Severity

Abstract 685To identify genes that modify the severity of human iron disorders we pre-selected 74 genes from gene expression profiles of cells and tissues with altered iron levels and assessed whether siRNA-mediated knock-down of these genes affects uptake of transferrin, a key cellular process to acquire iron. This screen identifies the monocyte chemoattractant protein-1 (MCP-1), also known as CCL2, as a critical suppressor of transferrin receptor mRNA expression in human cells. We next analyzed CCL2-deficient mice and demonstrate profound alterations of parameters of systemic iron homeostasis. Specifically, CCL2 knock-out mice show decreased serum iron levels and transferrin saturation, strong iron-overload in the spleen and duodenum as well as mild iron accumulation in the liver. Iron imbalance in CCL2−/− mice is unlikely explained by an impairment of the major control system of systemic iron homeostasis, the hepcidin/ferroportin regulatory system: hepcidin mRNA expression is unaltered and splenic ferroportin protein expression is strongly increased in CCL2−/− mice, as would be expected as a consequence of splenic iron overload. We speculate that increased iron absorption from the plasma, possibly mediated by inappropriately high levels of TfR1 in the spleen, duodenum and liver, may be responsible for tissue iron overload.It is of note that CCL2 levels are strongly decreased in Hfe-deficient mice and patients with Hfe-associated Hereditary Hemochromatosis (HH). We therefore asked whether CCL2 levels could modify disease severity of HH. Analysis of 51 HH patients, all homozygous for the C282Y HFE mutation, confirms significantly lower MCP-1 levels in the serum compared to a group of 23 sex- and age-matched normal controls. Importantly, CCL2 levels in HH patients show a significant negative correlation with liver iron overload at the time point of diagnosis. Furthermore, low CCL2 concentrations are significantly associated with the HLA-A3 genotype and the CD8+ T lymphocyte phenotype, both traits previously shown to correlate with iron overload in HH patients. These patient data and the data from CCL2-deficient mice suggest that appropriate CCL2 expression is required to prevent iron overload.Taken together our data demonstrate the power of siRNA screens to identify novel regulators of iron metabolism in human cells that are critically involved in maintaining systemic iron homeostasis in the mouse and that play a role in modifying hepatic iron overload in the frequent iron overload disorder Hereditary Hemochromatosis. Disclosures:No relevant conflicts of interest to declare.

SPP1 genotype is a determinant of disease severity in Duchenne muscular dystrophy

# {#article-title-2} To the Editor: Variability in disease severity in Duchenne muscular dystrophy (DMD) is considered a biological phenomenon, possibly due to genetic or epigenetic mechanisms. It was exciting to read the article by Pegoraro et al.1 describing the discovery of osteopontin as a possible modifier gene. As discussed in the accompanying editorial by Drs. Nelson and Griggs,2 utrophin and myostatin are 2 other proteins modifying disease severity in the mdx mouse. However, human data supporting an important role for utrophin were not mentioned. Kleopas et al.3 demonstrated that utrophin expression in the initial diagnostic muscle biopsies of …

Human apolipoprotein E genotypes differentially modify house dust mite-induced airway disease in mice

Apolipoprotein E (apoE) is an endogenous negative regulator of airway hyperreactivity (AHR) and mucous cell metaplasia in experimental models of house dust mite (HDM)-induced airway disease. The gene encoding human apoE is polymorphic, with three common alleles (ε2, ε3, and ε4) reflecting single amino acid substitutions at amino acids 112 and 158. The objective of this study was to assess whether the human apoE alleles modify airway responses to repeated nasal HDM challenges. Mice expressing the human apoE ε2 (huApoE2), ε3 (huApoE3), or ε4 (huApoE4) alleles received nasal HDM challenges, and airway responses were compared with mice expressing the endogenous murine apoE gene (muApoE). huApoE3 mice displayed significant reductions in AHR, mucous cell metaplasia, and airway inflammation compared with muApoE mice. The attenuated severity of airway inflammation in huApoE3 mice was associated with reductions in lung mRNA levels of Th2 and Th17 cytokines, as well as chemokines (CCL7, CCL11, CCL24). huApoE4 mice had an intermediate phenotype, with attenuated AHR and IgE production, compared with muApoE mice, whereas airway inflammation and mucous cell metaplasia were not reduced. In contrast, HDM-induced airway responses were not modified in mice expressing the huApoE2 allele. We conclude that the polymorphic huApoE alleles differentially modulate HDM-induced airway disease, which can be stratified, in rank order of increasing disease severity, ε3 < ε4 < ε2. These results raise the possibility that the polymorphic apoE alleles may modify disease severity in human asthma.

Chemical Chaperones Protect Epidermolysis Bullosa Simplex Keratinocytes from Heat Stress–Induced Keratin Aggregation: Involvement of Heat Shock Proteins and MAP Kinases

Epidermolysis bullosa simplex (EBS) is a blistering skin disease caused by mutations in keratin genes (KRT5 or KRT14), with no existing therapies. Aggregates of misfolded mutant keratins are seen in cultured keratinocytes from severe EBS patients. In other protein-folding disorders, involvement of molecular chaperones and the ubiquitin-proteasome system may modify disease severity. In this study, the effects of heat stress on keratin aggregation in immortalized cells from two patients with EBS (KRT5) and a healthy control were examined with and without addition of various test compounds. Heat-induced (43 °C, 30 minutes) aggregates were observed in all cell lines, the amount of which correlated with the donor phenotype. In EBS cells pre-exposed to proteasome inhibitor, MG132, and p38-mitogen-activated protein kinase (MAPK) inhibitor, SB203580, the proportion of aggregate-positive cells increased, suggesting a role of proteasomes and phosphorylation in removing mutated keratin. In contrast, aggregates were reduced by pretreatment with two chemical chaperones, trimethylamine N-oxide (TMAO) and 4-phenylbutyrate (4-PBA). TMAO also modulated stress-induced p38/c-jun N-terminal kinase (JNK) activation and expression of heat shock protein (HSPA1A), the latter of which colocalized with phosphorylated keratin 5 in EBS cells. Taken together, our findings suggest therapeutic targets for EBS and other keratinopathies.

Single Nucleotide Polymorphisms Alter the Promoter Activity of Bovine MIF

Macrophage migration inhibitory factor (MIF) is a unique pro-inflammatory cytokine whose chief functions include modulating TLR4 expression, and suppressing the anti-inflammatory effects of glucocorticoids. Not surprisingly, MIF is involved in a number of inflammatory diseases and single nucleotide polymorphisms (SNPs) have been implicated in modulating disease severity. The objective of the present study was to determine if SNPs in 5′ region of bovine MIF affects its promoter activity. Three SNPs were identified, -1078A>G, -395A>G, and -400G>C, all of which fall within predicted transcription factor binding regions. Reporter gene assays indicate that the identified SNPs have a significant effect of modulating MIF promoter activity. Finally, gene association analysis suggests a significant relationship of -395A>G with the susceptibility to Mycobacterium avium ssp. paratuberculosis infection, the causative agent of Johne's disease. Given the relationships revealed in the current study, it is clear that the role of MIF in bovine diseases such as Johne's disease merits further investigation.

FBN1 isoform expression varies in a tissue and development-specific fashion

Mutations in FBN1 cause Marfan syndrome, a heritable disorder of connective tissue. FBN1 encodes the extracellular matrix protein, fibrillin. Our objective was to elucidate the extent that variation in RNA splicing contributes to FBN1 isoforms. To identify FBN1 splice variants, we scanned each of its 64 internal exons in a set of pooled human brain cDNA samples. FBN1 splicing is generally efficient as we identified only two variants. Neither variant has previously been reported in the literature and include (i) an isoform which contains a cryptic 105 basepair exon between exons 54 and 55 (54A-FBN1) and (ii) an isoform which contains a cryptic 62 basepair exon between exons 57 and 58 (57A-FBN1). We compared 57A-FBN1 and FBN1 expression in multiple human tissues, including adult skeletal muscle and brain, as well as fetal skeletal muscle, brain, liver, aorta, lung, skin, and heart. 57A-FBN1 represents 8–44% of FBN1 mRNA and varies in a tissue- and development-specific fashion. In adult brain, 57A-FBN1 represented 39±3 (%, mean±SD) of total FBN1 expression. In contrast, 57A-FBN1 represented 19±2 (%, mean±SD) of FBN1 expression in skeletal muscle. In fetal tissue, the 57A-FBN1 proportion was highest in brain (27%) and low elsewhere, e.g., skin, aorta and lung (9–13%). In summary, a significant proportion of FBN1 is expressed as 57A-FBN1 and this proportion varies in a tissue- and development-specific fashion. Since the 57A insertion creates a premature stop codon that mimics Marfan-associated mutations, the protein encoded by 57A-FBN1 is likely to not be functional. These results suggest that altered splicing may modulate disease severity, regulate FBN1 expression, and potentially represent a therapeutic target.

Polyparasitism Is Associated with Increased Disease Severity in Toxoplasma gondii-Infected Marine Sentinel Species

In 1995, one of the largest outbreaks of human toxoplasmosis occurred in the Pacific Northwest region of North America. Genetic typing identified a novel Toxoplasma gondii strain linked to the outbreak, in which a wide spectrum of human disease was observed. For this globally-distributed, water-borne zoonosis, strain type is one variable influencing disease, but the inability of strain type to consistently explain variations in disease severity suggests that parasite genotype alone does not determine the outcome of infection. We investigated polyparasitism (infection with multiple parasite species) as a modulator of disease severity by examining the association of concomitant infection of T. gondii and the related parasite Sarcocystis neurona with protozoal disease in wild marine mammals from the Pacific Northwest. These hosts ostensibly serve as sentinels for the detection of terrestrial parasites implicated in water-borne epidemics of humans and wildlife in this endemic region. Marine mammals (151 stranded and 10 healthy individuals) sampled over 6 years were assessed for protozoal infection using multi-locus PCR-DNA sequencing directly from host tissues. Genetic analyses uncovered a high prevalence and diversity of protozoa, with 147/161 (91%) of our sampled population infected. From 2004 to 2009, the relative frequency of S. neurona infections increased dramatically, surpassing that of T. gondii. The majority of T. gondii infections were by genotypes bearing Type I lineage alleles, though strain genotype was not associated with disease severity. Significantly, polyparasitism with S. neurona and T. gondii was common (42%) and was associated with higher mortality and more severe protozoal encephalitis. Our finding of widespread polyparasitism among marine mammals indicates pervasive contamination of waterways by zoonotic agents. Furthermore, the significant association of concomitant infection with mortality and protozoal encephalitis identifies polyparasitism as an important factor contributing to disease severity in marine mammals.

Crossreactive influenza A virus M1- and EBV-BMLF-1-specific CD8 T cells modulate disease severity of Epstein-Barr Virus-induced infectious mononucleosis (49.20)

Abstract During EBV-associated infectious mononucleosis (IM) influenza A-specific crossreactive memory T cells are highly activated and play a role in disease severity. In HLA-A2+ IM patients, influenza M158 (IAV-M1)-specific CD8 memory T cell responses cross-reacted with two different EBV lytic epitopes, BRLF-1(190) (19/20) and BMLF-1(280) (17/29). Furthermore, 11/22 IM patients demonstrated some intra-viral cross-reactivity between EBV-BRLF-1 and -BMLF-1 responses. Disease severity of IM did not correlate with viral load, but instead directly correlated with the percentage of IAV-M1, EBV-BMLF-1 and crossreactive IAV-M1/EBV-BMLF-1-specific CD8 T cells. This was associated with altered TCR Vbeta usage. In all IM patients cross-reactive T cell responses were observed but different patterns of cross-reactivity were seen in each individual. A number of unique features of the TCR of the highly cross-reactive BRLF-1 epitope were noted. BRLF-1 T cell responses were of very high avidity. There was tremendous variation in the Vbeta usage between different individuals and even in the same individual over time. Cross-reactive BRLF-1 responses only became apparent at higher antigen doses in vitro, such as might occur during different phases of infectious mononucleosis.

Evidence for population variation in TSC1 and TSC2 gene expression

BackgroundTuberous sclerosis complex (TSC) is an autosomal dominant neurogenetic disorder caused by mutations in one of two genes, TSC1 or TSC2, which encode the proteins hamartin and tuberin, respectively [1-3]. Common features of TSC include intractable epilepsy, mental retardation, and autistic features. TSC is associated with specific brain lesions, including cortical tubers, subependymal nodules and subependymal giant cell astrocytomas. In addition, this disease frequently produces characteristic tumors, termed hamartomas, in the kidneys, heart, skin, retina, and lungs. Disease severity in TSC can be quite variable and is not determined by the primary mutation alone. In fact, there is often considerable variability in phenotype within single families, where all affected individuals carry the same mutation. Factors suspected to influence phenotype in TSC include the specific primary mutation, random occurrence of second-hit somatic mutations, mosaicism, "modifying genes", and environmental factors. In addition to these factors, we hypothesize that differences in mRNA expression from the non-mutated TSC allele, or possibly from the mutated allele, play a part in modifying disease severity. Common genetic variants that regulate mRNA expression have previously been shown to play important roles in human phenotypic variability, including disease susceptibility. A prediction based on this idea is that common regulatory variants that influence disease severity in TSC should be detectable in non-affected individuals.MethodsA PCR/primer extension assay was used to measure allele specific expression of TSC1 and TSC2 mRNAs in leukocytes isolated from normal volunteers. This assay can be used to measure "allelic expression imbalance" (AEI) in individuals by making use of heterozygous "marker" single nucleotide polymorphisms (SNPs) located within their mRNA.ResultsIn this study we show for the first time that TSC1 and TSC2 genes exhibit allele-specific differences in mRNA expression in blood leukocytes isolated from normal individuals.ConclusionsThese results support the possibility that allele-specific variation in TSC mRNA expression contributes to the variable severity of symptoms in TSC patients.