IDS gene-pseudogene exchange responsible for an intragenic deletion in a hunter patient

Hunter disease or mucopolysaccharidosis type II is an X-linked disease caused by the deficiency of the lysosomal enzyme iduronate-2-sulfatase (IDS). The IDS gene (24 kb) contains nine exons and has been completely sequenced. A pseudogene (IDS-2 locus) distal to the functional IDS gene has recently been identified. This work reports the characterization of IDS gene alterations in two severely affected patients. Patient 1 has a partial deletion that removes exons I to VI and extends about 200 kb upstream of the IDS gene. Patient 2 has an internal deletion of exons IV, V, VI, and VII, which results from an IDS gene-pseudogene exchange between highly homologous regions. In the rearranged gene, the junction intron contains pseudogene intron 3- and intron 7-related sequences. An interchromosomal recombination is probably the cause of this rearranged X chromosome. © 1996 Wiley-Liss, Inc.     

Production of therapeutic iduronate-2-sulfatase enzyme with a novel single-stranded RNA virus vector

The Sendai virus vector has received a lot of attention due to its broad tropism for mammalian cells. As a result of efforts for genetic studies based on a mutant virus, we can now express more than 10 genes of up to 13.5 kilo nucleotides in a single vector with high protein expression efficiency. To prove this benefit, we examined the efficacy of the novel ribonucleic acid (RNA) virus vector harboring the human iduronate-2-sulfatase (IDS) gene with 1,653 base pairs, a causative gene for mucopolysaccharidosis type II, also known as a disorder of lysosomal storage disorders. As expected, this novel RNA vector with the human IDS gene exhibited its marked expression as determined by the expression of enhanced green fluorescent protein and IDS enzyme activity. While these cells exhibited a normal growth rate, the BHK-21 transformant cells stably expressing the human IDS gene persistently generated an active human IDS enzyme extracellularly. The human IDS protein produced failed to be incorporated into the lysosome when cells were pretreated with mannose-6-phosphate, demonstrating that this human IDS enzyme has potential for therapeutic use by cross-correction. These results suggest that our novel RNA vector may be applicable for further clinical settings.     

Volumetric navigated MEGA‐SPECIAL for real‐time motion and shim corrected GABA editing

Mescher–Garwood (MEGA) editing with spin echo full intensity acquired localization (MEGA‐SPECIAL, MSpc) is a technique to acquire γ‐aminobutyric acid (GABA) without macromolecule (MM) contamination at a T_E of 68 ms. However, due to the requirement of multiple shot‐localization, it is often susceptible to subject motion and B₀ inhomogeneity. A method is presented for real‐time shim and motion correction (ShMoCo) using volumetric navigators to correct for motion and motion‐related B₀ inhomogeneity during MSpc acquisition. A phantom experiment demonstrates that ShMoCo restores the GABA peak and improves spectral quality in the presence of motion and zero‐ and first‐order shim changes. The ShMoCo scans were validated in three subjects who performed up–down and left–right head rotations. Qualitative assessment of these scans indicates effective reduction of subtraction artefacts and well edited GABA peaks, while quantitative analysis indicates superior fitting and spectral quality relative to scans with no correction. Copyright © 2015 John Wiley & Sons, Ltd.     

The in vivo J‐difference editing MEGA‐PRESS technique for the detection of n‐3 fatty acids

In this study, we present a method for the detection of n‐3 fatty acid (n‐3 FA) signals using MRS in adipose tissue in vivo. This method (called oMEGA‐PRESS) is based on the selective detection of the CH₃ signal of n‐3 FA using the MEGA‐PRESS (MEshcher–GArwood Point‐RESolved Spectroscopy) J‐difference editing technique. We optimized the envelope shape and frequency of spectral editing pulses to minimize the spurious co‐editing and incomplete subtraction of the CH₃ signal of other FAs, which normally obscure the n‐3 FA CH₃ signal in MR spectra acquired using standard PRESS techniques. The post‐processing of the individual data scans with the phase and frequency correction before data subtraction and averaging was implemented to further improve the quality of in vivo spectra. The technique was optimized in vitro on lipid phantoms using various concentrations of n‐3 FA and examined in vivo at 3 T on 15 healthy volunteers. The proportion of n‐3 FA estimated by the oMEGA‐PRESS method in phantoms showed a highly significant linear correlation with the n‐3 FA content determined by gas chromatography. The signal attributed to n‐3 FA was observed in all subjects. Comparisons with the standard PRESS technique revealed an enhanced identification of the n‐3 FA signal using oMEGA‐PRESS. The presented method may be useful for the non‐invasive quantification of n‐3 FA in adipose tissue, and could aid in obtaining a better understanding of various aspects of n‐3 FA metabolism. Copyright © 2014 John Wiley & Sons, Ltd.     

Quantification of GABA,glutamate and glutamine in a single measurement at 3 T using GABA‐edited MEGA‐PRESS

γ‐Aminobutyric acid (GABA) and glutamate (Glu), major neurotransmitters in the brain, are recycled through glutamine (Gln). All three metabolites can be measured by magnetic resonance spectroscopy in vivo, although GABA measurement at 3 T requires an extra editing acquisition, such as Mescher–Garwood point‐resolved spectroscopy (MEGA‐PRESS). In a GABA‐edited MEGA‐PRESS spectrum, Glu and Gln co‐edit with GABA, providing the possibility to measure all three in one acquisition. In this study, we investigated the reliability of the composite Glu + Gln (Glx) peak estimation and the possibility of Glu and Gln separation in GABA‐edited MEGA‐PRESS spectra. The data acquired in vivo were used to develop a quality assessment framework which identified MEGA‐PRESS spectra in which Glu and Gln could be estimated reliably. Phantoms containing Glu, Gln, GABA and N‐acetylaspartate (NAA) at different concentrations were scanned using GABA‐edited MEGA‐PRESS at 3 T. Fifty‐six sets of spectra in five brain regions were acquired from 36 healthy volunteers. Based on the Glu/Gln ratio, data were classified as either within or outside the physiological range. A peak‐by‐peak quality assessment was performed on all data to investigate whether quality metrics can discriminate between these two classes of spectra. The quality metrics were as follows: the GABA signal‐to‐noise ratio, the NAA linewidth and the Glx Cramer–Rao lower bound (CRLB). The Glu and Gln concentrations were estimated with precision across all phantoms with a linear relationship between the measured and true concentrations: R¹ = 0.95 for Glu and R¹ = 0.91 for Gln. A quality assessment framework was set based on the criteria necessary for a good GABA‐edited MEGA‐PRESS spectrum. Simultaneous criteria of NAA linewidth <8 Hz and Glx CRLB <16% were defined as optimum features for reliable Glu and Gln quantification. Glu and Gln can be reliably quantified from GABA‐edited MEGA‐PRESS acquisitions. However, this reliability should be controlled using the quality assessment methods suggested in this work.     

Clinical characteristics and genotypes of 201 patients with mucopolysaccharidosis type II in China: A retrospective,observational study

Mucopolysaccharidosis type II (MPS II) is an X-linked recessive lysosomal storage disease caused by a disease-associated variant in the IDS gene, which encodes iduronate 2-sulfatase (IDS). We aimed to characterize the clinical characteristics and genotypes of the largest cohort of Chinese patients with MPS II and so gain a deeper understanding of natural disease progression. Patients with confirmed MPS II and without treatment were included. The disease was classified as severe in patients with neurological impairment, and as attenuated in patients aged >6 years without neurological impairment. Of the 201 male patients, 78.1% had severe MPS II. Cognitive regression occurred before age 6 years in 94.3% of patients. Of 122 IDS variants identified, 37 were novel. Among the large gene alteration types identified, only the frequency of IDS-IDS2 recombination was significantly higher in severe versus attenuated MPS II (P = 0.032). Some identified point variants could inform the understanding of genotype–phenotype correlations. In conclusion, this study showed that classification of the disease as attenuated should only be made in patients aged >6 years. Our findings expand the understanding of the genotype–phenotype relationship, inform the diagnostic process, and provide an indication of the likely prognosis.     

A comparison of 2‐hydroxyglutarate detection at 3 and 7 T with long‐TE semi‐LASER

Abnormally high levels of the ‘oncometabolite’ 2‐hydroxyglutarate (2‐HG) occur in many grade II and III gliomas, and correlate with mutations in the genes of isocitrate dehydrogenase (IDH) isoforms. In vivo measurement of 2‐HG in patients, using magnetic resonance spectroscopy (MRS), has largely been carried out at 3 T, yet signal overlap continues to pose a challenge for 2‐HG detection. To combat this, several groups have proposed MRS methods at ultra‐high field (≥7 T) where theoretical increases in signal‐to‐noise ratio and spectral resolution could improve 2‐HG detection. Long echo time (long‐TE) semi‐localization by adiabatic selective refocusing (semi‐LASER) (TE = 110 ms) is a promising method for improved 2‐HG detection in vivo at either 3 or 7 T owing to the use of broad‐band adiabatic localization. Using previously published semi‐LASER methods at 3 and 7 T, this study directly compares the detectability of 2‐HG in phantoms and in vivo across nine patients. Cramér–Rao lower bounds (CRLBs) of 2‐HG fitting were found to be significantly lower at 7 T (6 ± 2%) relative to 3 T (15 ± 7%) (p = 0.0019), yet were larger at 7 T in an IDH wild‐type patient. Although no increase in SNR was detected at 7 T (77 ± 26) relative to 3 T (77 ± 30), the detection of 2‐HG was greatly enhanced through an improved spectral profile and increased resolution at 7 T. 7 T had a large effect on pairwise fitting correlations between γ‐aminobutyric acid (GABA) and 2‐HG (p = 0.004), and resulted in smaller coefficients. The increased sensitivity for 2‐HG detection using long‐TE acquisition at 7 T may allow for more rapid estimation of 2‐HG (within a few spectral averages) together with other associated metabolic markers in glioma.     

Mucolipidosis II‐Related Mutations Inhibit the Exit from the Endoplasmic Reticulum and Proteolytic Cleavage of GlcNAc‐1‐Phosphotransferase Precursor Protein (GNPTAB)

Mucolipidosis (ML) II and MLIII alpha/beta are two pediatric lysosomal storage disorders caused by mutations in the GNPTAB gene, which encodes an α/β‐subunit precursor protein of GlcNAc‐1‐phosphotransferase. Considerable variations in the onset and severity of the clinical phenotype in these diseases are observed. We report here on expression studies of two missense mutations c.242G>T (p.Trp81Leu) and c.2956C>T (p.Arg986Cys) and two frameshift mutations c.3503_3504delTC (p.Leu1168GlnfsX5) and c.3145insC (p.Gly1049ArgfsX16) present in severely affected MLII patients, as well as two missense mutations c.1196C>T (p.Ser399Phe) and c.3707A>T (p.Lys1236Met) reported in more mild affected individuals. We generated a novel α‐subunit‐specific monoclonal antibody, allowing the analysis of the expression, subcellular localization, and proteolytic activation of wild‐type and mutant α/β‐subunit precursor proteins by Western blotting and immunofluorescence microscopy. In general, we found that both missense and frameshift mutations that are associated with a severe clinical phenotype cause retention of the encoded protein in the endoplasmic reticulum and failure to cleave the α/β‐subunit precursor protein are associated with a severe clinical phenotype with the exception of p.Ser399Phe found in MLIII alpha/beta. Our data provide new insights into structural requirements for localization and activity of GlcNAc‐1‐phosphotransferase that may help to explain the clinical phenotype of MLII patients.     

In vivo estimation of gamma‐aminobutyric acid levels in the neonatal brain

Gamma‐aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain, and plays a key role in brain development. However, the in vivo levels of brain GABA in early life are unknown. Using edited MRS, in vivo GABA can be detected as GABA+ signal with contamination of macromolecule signals. GABA+ is evaluated as the peak ratio of GABA+/reference compound, for which creatine (Cr) or water is typically used. However, the concentrations and T₁ and T₂ relaxation times of these references change during development. Thus, the peak ratio comparison between neonates and children may be inaccurate. The aim of this study was to measure in vivo neonatal brain GABA+ levels, and to investigate the dependency of GABA levels on brain region and age. The basal ganglia and cerebellum of 38 neonates and 12 children were measured using GABA‐edited MRS. Two different approaches were used to obtain GABA+ levels: (i) multiplying the GABA/water ratio by the water concentration; and (ii) multiplying the GABA+/Cr by the Cr concentration. Neonates exhibited significantly lower GABA+ levels compared with children in both regions, regardless of the approach employed, consistent with previous ex vivo data. A similar finding of lower GABA+/water and GABA+/Cr in neonates compared with children was observed, except for GABA+/Cr in the cerebellum. This contrasting finding resulted from significantly lower Cr concentrations in the neonate cerebellum, which were approximately 52% of those of children. In conclusion, care should be taken to consider Cr concentrations when comparing GABA+/Cr levels between different‐aged subjects.     

Unedited in vivo detection and quantification of γ‐aminobutyric acid in the occipital cortex using short‐TE MRS at 3 T

Short‐TE MRS has been proposed recently as a method for the in vivo detection and quantification of γ‐aminobutyric acid (GABA) in the human brain at 3 T. In this study, we investigated the accuracy and reproducibility of short‐TE MRS measurements of GABA at 3 T using both simulations and experiments. LCModel analysis was performed on a large number of simulated spectra with known metabolite input concentrations. Simulated spectra were generated using a range of spectral linewidths and signal‐to‐noise ratios to investigate the effect of varying experimental conditions, and analyses were performed using two different baseline models to investigate the effect of an inaccurate baseline model on GABA quantification. The results of these analyses indicated that, under experimental conditions corresponding to those typically observed in the occipital cortex, GABA concentration estimates are reproducible (mean reproducibility error, <20%), even when an incorrect baseline model is used. However, simulations indicate that the accuracy of GABA concentration estimates depends strongly on the experimental conditions (linewidth and signal‐to‐noise ratio). In addition to simulations, in vivo GABA measurements were performed using both spectral editing and short‐TE MRS in the occipital cortex of 14 healthy volunteers. Short‐TE MRS measurements of GABA exhibited a significant positive correlation with edited GABA measurements (R = 0.58, p < 0.05), suggesting that short‐TE measurements of GABA correspond well with measurements made using spectral editing techniques. Finally, within‐session reproducibility was assessed in the same 14 subjects using four consecutive short‐TE GABA measurements in the occipital cortex. Across all subjects, the average coefficient of variation of these four GABA measurements was 8.7 ± 4.9%. This study demonstrates that, under some experimental conditions, short‐TE MRS can be employed for the reproducible detection of GABA at 3 T, but that the technique should be used with caution, as the results are dependent on the experimental conditions. Copyright © 2013 John Wiley & Sons, Ltd.     

TLR2 activation in corneal stromal cells by Staphylococcus aureus‐induced keratitis

The Toll‐Like Receptor 2 (TLR2) plays an active and important role in Staphylococcus aureus‐induced chronic ocular inflammation. The aim of this study was to investigate the expression and function of TLR2 of corneal stromal cells in ex vivo rabbit model of S. aureus keratitis. Corneal buttons with sclera rims placed in an ex vivo air‐interface organ culture were assigned to two groups: corneas with epithelial and stromal abrasions. Each group was then divided into two sub‐groups exposed to UV‐killed S. aureus ATCC 6538P and S. aureus ATCC 29213, respectively. TLR2 and IL‐8 mRNA expressions were analyzed by quantitative real‐time RT‐PCR. TLR2 localization was visualized by immunofluorescence analysis. The results demonstrated that TLR2 and IL‐8 mRNA were significantly expressed in the stromal cells of the groups exposed to S. aureus strains. Moreover, it has been demonstrated that, after corneal injury, keratocytes differentiated into myofibroblasts became able to express TLR2 only when exposed to S. aureus. Identification of mechanisms regulation of corneal TLRs may lead to development of therapeutic interventions aimed at controlling corneal inflammation. This ex vivo model can be used to clarify the molecular events of bacterial‐corneal tissue interactions and their inflammatory consequences.     

c‐Jun promotes cell migration and drives expression of the motility factor ENPP2 in soft tissue sarcomas

Genomic amplification of the c‐Jun proto‐oncogene has been identified in ～30% of dedifferentiated liposarcomas (DDLPS), but the functional contribution of c‐Jun to the progression of DDLPS remains poorly understood. In previous work we showed that knock‐down of c‐Jun by RNA interference impaired the in vitro proliferation and in vivo growth of a DDLPS cell line (LP6) with genomic amplification of the c‐Jun locus. Here, we used gene expression analysis and functional studies in a broad panel of cell lines to further define the role of c‐Jun in DDLPS and other soft tissue sarcomas. We show that c‐Jun knock‐down impairs transition through the G₁ phase of the cell cycle in multiple DDLPS cell lines. We also found that high levels of c‐Jun expression are both necessary and sufficient to promote DDLPS cell migration and invasion in vitro. Our data suggest that high levels of c‐Jun enhance motility in part by driving the expression of ENPP2/Autotaxin. c‐Jun over‐expression has minimal effects on in vitro proliferation but substantially enhances the in vivo growth of weakly tumourigenic DDLPS cell lines. Finally, we provide evidence that c‐Jun genomic amplification and over‐expression may have similar functional consequences in other types of soft tissue sarcoma. Our data suggest a model in which relatively low levels of c‐Jun are sufficient for in vitro proliferation, but high levels of c‐Jun enhance invasiveness and capacity for in vivo tumour growth. These observations provide an explanation for the selective advantage provided by c‐Jun genomic amplification in vivo and suggest that sarcomas with elevated c‐Jun levels are likely to have a particularly high malignant potential. Data from exon array and RNA‐Seq experiments have been deposited in the GEO database (Accession No. GSE57531). Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd     

Lack of transglutaminase 2 diminished T‐cell responses in mice

Transglutaminase 2 (TG2) has been reported to play a role in dendritic cell activation and B‐cell differentiation after immunization. Its presence and role in T cells, however, has not been explored. In the present study, we determined the expression of TG2 on mouse T cells, and evaluated its role by comparing the behaviours of wild‐type and TG2^?/? T cells after activation. In our results, naive T cells minimally expressed TG2, expression of which was increased after activation. T‐cell proliferation, expression of activation markers such as CD69 and CD25, and secretions of interleukin‐2 and interferon‐γ were suppressed in the absence of TG2, presumably due, in part, to diminished nuclear factor‐κB activation. These effects on T cells seemed to be reflected in the in vivo immune response, the contact hypersensitivity reaction elicited by 2,4‐dinitro‐1‐fluorobenzene, with lowered peak responses in the TG2^?/? mice. When splenic T cells from mice immunized with tumour lysate‐loaded wild‐type dendritic cells were re‐challenged ex vivo with the same antigen, the profile of surface markers including CD44, CD62L, and CD127 strongly indicated lesser generation of memory CD8⁺ T cells in TG2^?/? mice. In the TG2^?/? CD8⁺ T cells, moreover, Eomes expression was markedly decreased. These results indicate possible roles of TG2 in CD8⁺ T‐cell activation and CD8⁺ memory T‐cell generation.     

HHQ and PQS,two Pseudomonas aeruginosa quorum‐sensing molecules,down‐regulate the innate immune responses through the nuclear factor‐κB pathway

To explore whether bacterial secreted 4‐hydroxy‐2‐alkylquinolines (HAQs) can regulate host innate immune responses, we used the extracts of bacterial culture supernatants from a wild‐type (PA14) and two mutants of Pseudomonas aeruginosa that have defects in making HAQs. Surprisingly, the extract of supernatants from the P. aeruginosa pqsA mutant that does not make HAQs showed strong stimulating activity for the production of innate cytokines such as tumour necrosis factor‐α and interleukin‐6 in the J774A.1 mouse monocyte/macrophage cell line, whereas the extract from the wild‐type did not. The addition of 4‐hydroxy‐2‐heptylquinoline (HHQ) or 2‐heptyl‐3,4‐dihydroxyquinoline (PQS, Pseudomonas quinolone signal) to mammalian cell culture media abolished this stimulating activity of the extracts of supernatants from the pqsA mutant on the expression of innate cytokines in J774A.1 cells and in the primary bronchoalveolar lavage cells from C57BL/6 mice, suggesting that HHQ and PQS can suppress the host innate immune responses. The pqsA mutant showed reduced dissemination in the lung tissue compared with the wild‐type strain in a mouse in vivo intranasal infection model, suggesting that HHQ and PQS may play a role in the pathogenicity of P. aeruginosa. HHQ and PQS reduced the nuclear factor‐κB (NF‐κB) binding to its binding sites and the expression of NF‐κB target genes, and PQS delayed inhibitor of κB degradation, indicating that the effect of HHQ and PQS was mediated through the NF‐κB pathway. Our results suggest that HHQ and PQS produced by P. aeruginosa actively suppress host innate immune responses.     

Dominant CD4‐dependent RNA‐dependent RNA polymerase‐specific T‐cell responses in children acutely infected with human enterovirus 71 and healthy adult controls

Human enterovirus 71 (EV71) is one of the major causes of hand, foot and mouth disease (HFMD), which leads to significant mortality in infected children. A prophylactic vaccine is urgently needed. However, little is known about the protective T‐cell immunity in individuals infected with the EV71 virus. In this study, we performed a comprehensive ex vivo interferon‐γ ELISPOT analysis in 31 children infected with EV71 as well as in 40 healthy adult controls of the CD4⁺ and CD8⁺ T‐cell responses to overlapping peptides spanning the VP1 structural protein and RNA‐dependent RNA polymerase (RdRp) non‐structural protein. EV71‐specific CD4 T‐cell responses were detected in most of the acute patients and were mostly CD4‐dependent RdRp‐specific responses. CD8‐dependent VP1 and RdRp‐specific responses were also detected in a small proportion of recently infected children. There was no significant association between the strength of the T‐cell responses and disease severity observed during the acute EV71 infection phase. Interestingly, an RdRp‐specific, but no VP1‐specific, CD4‐dependent T‐cell response was detected in 30% of the adult controls, and no T‐cell responses were detected in healthy children. In addition, 24 individual peptides containing potential T‐cell epitope regions were identified. The data suggest that CD4‐dependent RdRp‐specific T‐cell responses may play an important role in protective immunity, and the epitopes identified in this study should provide valuable information for future therapeutic and prophylactic vaccine design as well as basic research.     

Over‐expression of the LTC4 synthase gene in mice reproduces human aspirin‐induced asthma

Background The pathogenesis of aspirin‐induced asthma (AIA) is presumed to involve the aspirin/non‐steroidal anti‐inflammatory drug (NSAID)‐induced abnormal metabolism of arachidonic acid, resulting in an increase in 5‐lipoxygenase (5‐LO) metabolites, particularly leukotriene C₄ (LTC₄). However, the role of LTC₄ in the development of AIA has yet to be conclusively demonstrated. Objective The aim of this study was to evaluate the contribution of the lipid product LTC₄ secreted by the 5‐LO pathway to the pathogenesis of AIA. Methods To evaluate antigen‐induced airway inflammation, the concentrations of T‐helper type 2 cytokine in bronchoalveolar lavage fluid (BALF) obtained from LTC₄ synthase‐transgenic (Tg) and wild‐type (WT) mice after challenge with ovalbumin were measured. Subsequently, the ex vivo and in vivo effects of the NSAID sulpyrine were investigated in these Tg and WT mice by measuring the secretion of LTC₄ from sulpyrine‐treated BAL cells and the levels of LTC₄ in BALF following challenge with sulpyrine. Finally, the sulpyrine‐induced airway response by the administration of pranlukast, an antagonist of the cysteinyl (cs)‐LT1 receptor, was analysed. Results The concentrations of IL‐4, ‐5, and ‐13 in BALF from Tg mice were significantly higher than those in WT mice. In addition, sulpyrine augmented the secretion of LTC₄ in BALF and by BAL cells in Tg mice, but not in WT mice. Additionally, the increased airway resistance induced by sulpyrine could be reduced by treatment with pranlukast. Furthermore, the secretion of LTC₄ from mast cells, eosinophils, and macrophages was increased in the allergen‐stimulated LTC₄ synthase gene Tg mice, even in the absence of sulpyrine, as well as in BAL cells after sulpyrine. Conclusion and clinical relevance The over‐expression of the LTC₄ synthase in a mouse asthma model also replicates the key features of AIA. And our study supports that cys‐LTs play a major role in the pathogenesis of AIA in patients with chronic asthma. Cite this as: H. Hirata, M. Arima, Y. Fukushima, K. Honda, K. Sugiyama, T. Tokuhisa and T. Fukuda, Clinical & Experimental Allergy, 2011 (41) 1133–1142.     

Monomeric C‐reactive protein and inflammation in age‐related macular degeneration

Age‐related macular degeneration (AMD) is a devastating disease characterized by central vision loss in elderly individuals. Previous studies have suggested a link between elevated levels of total C‐reactive protein (CRP) in the choroid, CFH genotype, and AMD status; however, the structural form of CRP present in the choroid, its relationship to CFH genotype, and its functional consequences have not been assessed. In this report, we studied genotyped human donor eyes (n = 60) and found that eyes homozygous for the high‐risk CFH (Y402H) allele had elevated monomeric CRP (mCRP) within the choriocapillaris and Bruch's membrane, compared to those with the low‐risk genotype. Treatment of choroidal endothelial cells in vitro with mCRP increased migration rate and monolayer permeability compared to treatment with pentameric CRP (pCRP) or medium alone. Organ cultures treated with mCRP exhibited dramatically altered expression of inflammatory genes as assessed by RNA sequencing, including ICAM‐1 and CA4, both of which were confirmed at the protein level. Our data indicate that mCRP is the more abundant form of CRP in human choroid, and that mCRP levels are elevated in individuals with the high‐risk CFH genotype. Moreover, pro‐inflammatory mCRP significantly affects endothelial cell phenotypes in vitro and ex vivo, suggesting a role for mCRP in choroidal vascular dysfunction in AMD. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.