Major histocompatibility complex class I associations in iron overload: evidence for a new link between the HFE H63D mutation, HLA-A29, and non-classical forms of hemochromatosis

 The present study is an analysis of the frequencies of HFE mutations in patients with different forms of iron overload compared with the frequencies found in healthy subjects from the same region. The frequencies of HLA-A and -B antigens and HLA haplotypes were also analyzed in the same subjects. The study population included: 71 healthy individuals; 39 genetically and clinically well-characterized patients with genetic hemochromatosis (HH); and 25 patients with non-classical forms of iron overload (NCH), excluding secondary hemochromatosis. All subjects were HLA-typed and HFE-genotyped by the oligonucleotide ligation assay (OLA). The gene frequencies found for the C282Y and H63D mutations of HFE were respectively: 0.03 and 0.23 in healthy individuals, 0.86 and 0.04 in HH patients, and 0.08 and 0.48 in NCH patients. An expected significant association between HH and HLA-A3 was observed, which was found to be in linkage disequilibrium with the C282Y mutation. A new association was seen, however, between HLA-A29 and NCH, in linkage disequilibrium with the H63D mutation. Again as expected, the HLA-B antigen B7 was associated with HH in linkage disequilibrium with HLA-A3. In addition, the HLA-B antigen B44 was found to be associated with NCH but not in linkage disequilibrium with either A29 or the H63D mutation. In conclusion, a new association of the HFE H63D mutation with forms of hemochromatosis other than HH and a new association between the HLA phenotype A29 and the HFE H63D mutation were found in the same patients. These findings reinforce evidence for the involvement of the major histocompatibility class I in iron metabolism, supporting the notion of a physiological role for the immunological system in the regulation of iron load. Received: 11 June 1997 / Revised: 29 October 1997     

Serum Iron Levels and the Risk of Parkinson Disease: A Mendelian Randomization Study

Background

Although levels of iron are known to be increased in the brains of patients with Parkinson disease (PD), epidemiological evidence on a possible effect of iron blood levels on PD risk is inconclusive, with effects reported in opposite directions. Epidemiological studies suffer from problems of confounding and reverse causation, and mendelian randomization (MR) represents an alternative approach to provide unconfounded estimates of the effects of biomarkers on disease. We performed a MR study where genes known to modify iron levels were used as instruments to estimate the effect of iron on PD risk, based on estimates of the genetic effects on both iron and PD obtained from the largest sample meta-analyzed to date.

Methods and Findings

We used as instrumental variables three genetic variants influencing iron levels, HFE rs1800562, HFE rs1799945, and TMPRSS6 rs855791. Estimates of their effect on serum iron were based on a recent genome-wide meta-analysis of 21,567 individuals, while estimates of their effect on PD risk were obtained through meta-analysis of genome-wide and candidate gene studies with 20,809 PD cases and 88,892 controls. Separate MR estimates of the effect of iron on PD were obtained for each variant and pooled by meta-analysis. We investigated heterogeneity across the three estimates as an indication of possible pleiotropy and found no evidence of it. The combined MR estimate showed a statistically significant protective effect of iron, with a relative risk reduction for PD of 3% (95% CI 1%–6%; p = 0.001) per 10 µg/dl increase in serum iron.

Conclusions

Our study suggests that increased iron levels are causally associated with a decreased risk of developing PD. Further studies are needed to understand the pathophysiological mechanism of action of serum iron on PD risk before recommendations can be made. Please see later in the article for the Editors'' Summary     

Expression of hereditary hemochromatosis C282Y HFE protein in HEK293 cells activates specific endoplasmic reticulum stress responses

Background  

Hereditary Hemochromatosis (HH) is a genetic disease associated with iron overload, in which individuals homozygous for the mutant C282Y HFE associated allele are at risk for the development of a range of disorders particularly liver disease. Conformational diseases are a class of disorders associated with the expression of misfolded protein. HFE C282Y is a mutant protein that does not fold correctly and consequently is retained in the Endoplasmic Reticulum (ER). In this context, we sought to identify ER stress signals associated with mutant C282Y HFE protein expression, which may have a role in the molecular pathogenesis of HH.     

The Q283P amino-acid change in HFE leads to structural and functional consequences similar to those described for the mutated 282Y HFE protein

In Caucasians, 4–35% of hemochromatosis patients carry at least one chromosome without a common HFE mutation (i.e. C282Y, H63D and S65C). Several studies have now shown that iron overload phenotypes in such patients can be associated with uncommon HFE mutations. We previously supported implication of the C282Y/Q283P compound heterozygous genotype in hemochromatosis phenotypes and, based on molecular dynamics simulations, proposed that the Q283P substitution prevents normal folding of the HFE ₃-domain. In the current work, we have used HeLa cells carrying wild-type or Q283P-mutant HFE cDNA under the control of a tetracycline-sensitive promoter to functionally characterise the Q283P mutation. Experiments using cells over-expressing wild-type HFE confirm the existence of ₂microglobulin(₂m)/HFE and HFE/transferrin receptor 1 (TfR1) interactions, as well as the capacity of HFE to reduce transferrin-mediated iron uptake. In contrast, neither ₂m/HFE nor HFE/TfR1 complex formation was detected in cells over-expressing the mutated form of HFE. Moreover, the 283P HFE protein was found to have a very limited effect on the major cellular iron uptake pathway. Combined, our results indicate that the Q283P mutation leads to structural and functional consequences similar to those described for the main hereditary hemochromatosis mutation. As a consequence, our study has implications for the screening of hemochromatosis patients that have one or two copies of HFE which lack the main mutations. It also highlights that protein structure prediction methods could be more generally used to better interpret relationships between rare genotypes and molecular diagnosis of a human inherited disorder.     

Meta-analyses of HFE variants in coronary heart disease

Aim

HFE gene variants can cause hereditary hemochromatosis (HH) that often comes along with an increased risk of coronary heart disease (CHD). The goal of our study is to assess the contribution of four HFE gene variants to the risk of CHD.

Methods and results

We conducted four meta-analyses of the studies examining the association between four HFE gene variants and the risk of CHD. A systematic search was conducted using MEDLINE, EMBASE, Web of Science and China National Knowledge Infrastructure (CNKI), Wanfang Chinese Periodical.

Results

Meta-analyses showed that HFE rs1799945-G allele was associated with a 6% increased risk of CHD (P = 0.02, odds ratio (OR) = 1.06, 95% confidence interval (CI) = 1.01–1.11). However, no association between the other three HFE gene variants (rs1800562, rs1800730, and rs9366637) and CHD risk was observed by the meta-analyses (all P values > 0.05). In addition, the results of our case–control study indicated that rs1800562 and rs1800730 were monomorphic, and that rs1799945 and rs9366637 were not associated with CHD in Han Chinese.

Conclusions

Our meta-analysis suggested that a significant association existed between rs1799945 mutation and CHD, although this mutation was rare in Han Chinese.     

Clinical grade mesenchymal stem cells transdifferentiated under xenofree conditions alleviates motor deficiencies in a rat model of Parkinson's disease

Bone marrow derived mesenchymal stem cells (BMMSCs) is a valid, definitive candidate for repair of damaged tissues in degenerative disorders in general and neurological diseases in particular. We have standardized the processing conditions for proliferation of BMMSCs using xenofree medium and checked their in vitro and in vivo neurogenic potential.

Method

The proliferative potential of BMMSCs was analyzed using xenofree media and functionality checked by transplantation into Parkinson's disease (PD) animal models. In vitro neuronal differentiation was investigated by neuronal induction media supplemented with growth factors. Differentiated cells were characterized at cellular and molecular levels. In vitro functionality estimated by dopamine secretion.

Results

A pure population of BMMSCs showing an 8–10 fold expansion was obtained using xenofree media. On differentiation to neuronal lineage, they exhibited neuronal morphology. Detectable levels of dopamine (1.93 ng/ml) were secreted into the culture media of differentiated cells. There was a significant behavioural improvement in PD models 3 months post transplantation.

Conclusion

Our study demonstrates that BMMSCs can be transdifferentiated efficiently into functional dopaminergic neurons both in vitro and in vivo. This holds immense clinical potential as a replacement therapy for PD and other neurodegenerative diseases.     

Saliva levels of Abeta1-42 as potential biomarker of Alzheimer's disease: a pilot study

Background  

Simple, non-invasive tests for early detection of degenerative dementia by use of biomarkers are urgently required. However, up to the present, no validated extracerebral diagnostic markers for the early diagnosis of Alzheimer disease (AD) are available. The clinical diagnosis of probable AD is made with around 90% accuracy using modern clinical, neuropsychological and imaging methods. A biochemical marker that would support the clinical diagnosis and distinguish AD from other causes of dementia would therefore be of great value as a screening test. A total of 126 samples were obtained from subjects with AD, and age-sex-matched controls. Additionally, 51 Parkinson's disease (PD) patients were used as an example of another neurodegenerative disorder. We analyzed saliva and plasma levels of β amyloid (Aβ) using a highly sensitive ELISA kit.     

Role of the H1 haplotype of microtubule-associated protein tau (MAPT) gene in Greek patients with Parkinson's disease

Background  

The extended tau haplotype (H1) that covers the entire human microtubule-associated protein tau (MAPT) gene has been implicated in Parkinson's disease (PD). Nevertheless, controversial results, such as two studies in Greek populations with opposite effects, have been reported. Therefore, we set out to determine whether the H1 haplotype and additional single nucleotide polymorphisms (SNPs) included in H1 are associated with PD in a sample of Greek patients.     

Linkage disequilibrium and extended haplotypes in the HLA-A to D6S105 region: implications for mapping the hemochromatosis gene (HFE)

The hemochromatosis gene (HFE) maps to 6p21.3, in close linkage with the HLA Class I genes. Linkage disequilibrium (LD) studies were designed to narrow down the most likely candidate region for HFE, as an alternative to traditional linkage analysis. However, both the HLA-A and D6S105 subregions, which are situated 2–3 cM and approximately 3 Mb apart, have been suggested to contain HFE. The present report extends our previous study based upon the analysis of a large number of HFE and normal chromosomes from 66families of Breton ancestry. In addition to the previously used RFLP markers spanning the 400-kb surrounding HLA-A, we examined three microsatellites: D6S510, HLA-F, and D6S105. Our combined data not only confirm a peak of LD at D6S105, but also reveal a complex pattern of LD over the i82 to D6S105 interval. Within our ethnically well-defined population of Brittany, the association of HFE with D6S105 is as great as that with HLA-A, while the internal markers display a lower LD. Fine haplotype analysis enabled us to identify two categories of haplotypes segregating with HFE. In contrast to the vast majority of normal haplotypes, 50% of HFE haplotypes are completely conserved over the HLA-A to D6S105 interval. These haplotypes could have been conserved through recombination suppression, selective forces and/or other evolutionary factors. This particular haplotypic configuration might account for the apparent inconsistencies between genetic linkage and LD data, and additionally greatly complicates positional cloning of HFE through disequilibrium mapping.The authors contributed equally to this work     

The quality of care delivered to Parkinson's disease patients in the U.S. Pacific Northwest Veterans Health System

Background  

Parkinson's disease (PD) is the second most common chronic neurological disorder of the elderly. Despite the fact that a comprehensive review of general health care in the United States showed that the quality of care delivered to patients usually falls below professional standards, there is limited data on the quality of care for patients with PD.     

Anonymous Marker Loci within 400 kb of HLA-A Generate Haplotypes in Linkage Disequilibrium with the Hemochromatosis Gene (HFE)

The hemochromatosis gene (HFE) maps to 6p21.3 and is less than 1 cM from the HLA class I genes; however, the precise physical location of the gene has remained elusive and controversial. The unambiguous identification of a crossover event within hemochromatosis families is very difficult; it is particularly hampered by the variability of the phenotypic expression as well as by the sex- and age-related penetrance of the disease. For these practical considerations, traditional linkage analysis could prove of limited value in further refining the extrapolated physical position of HFE. We therefore embarked upon a linkage-disequilibrium analysis of HFE and normal chromosomes from the Brittany population. In the present report, 66 hemochromatosis families yielding 151 hemochromatosis chromosomes and 182 normal chromosomes were RFLP-typed with a battery of probes, including two newly derived polymorphic markers from the 6.7 and HLA-F loci located 150 and 250 kb telomeric to HLA-A, respectively. The results suggest a strong peak of existing linkage disequilibrium focused within the i82-to-6.7 interval (approximately 250 kb). The zone of linkage disequilibrium is flanked by the i97 locus, positioned 30 kb proximal to i82, and the HLA-F gene, found 250 kb distal to HLA-A, markers of which display no significant association with HFE. These data support the possibility that HFE resides within the 400-kb expanse of DNA between i97 and HLA-F. Alternatively, the very tight association of HLA-A3 and allele 1 of the 6.7 locus, both of which are comprised by the major ancestral or founder HFE haplotype in Brittany, supports the possibility that the disease gene may reside immediately telomeric to the 6.7 locus within the linkage-disequilibrium zone. Additionally, hemochromatosis haplotypes possessing HLA-A11 and the low-frequency HLA-F polymorphism (allele 2) are supportive of a separate founder chromosome containing a second, independently arising mutant allele. Overall, the establishment of a likely “hemochromatosis critical region” centromeric boundary and the identification of a linkage-disequilibrium zone both significantly contribute to a reduction in the amount of DNA required to be searched for novel coding sequences constituting the HFE defect.     

Neurotherapeutic effects of novel HO‐1 inhibitors in vitro and in a transgenic mouse model of Alzheimer's disease

Heme oxygenase‐1 (HO‐1) encoded by the HMOX1 gene is a 32‐kDa stress protein that catabolizes heme to biliverdin, free iron, and carbon monoxide (CO). Glial HO‐1 is over‐expressed in the CNS of subjects with Alzheimer's disease (AD), Parkinson's disease (PD), and multiple sclerosis (MS). The HMOX1 gene is exquisitely sensitive to oxidative stress and is induced in brain and other tissues in various models of disease and trauma. Induction of the glial HMOX1 gene may lead to pathological brain iron deposition, intracellular oxidative damage, and bioenergetic failure in AD and other human CNS disorders such as PD and MS. Therefore, targeted suppression of glial HO‐1 hyperactivity may prove to be a rational and effective therapeutic intervention in AD and related neurodegenerative disorders. In this study, we report the effects of QC‐47, QC‐56, and OB‐28, novel azole‐based competitive and reversible inhibitors of HO‐1, on oxidative damage to whole‐cell and mitochondrial compartments in rat astrocytes transfected with the HMOX1 gene. We also report the effect of OB‐28 on the behavior and neuropathology of APP_swe/PS1_?E9 mice. OB‐28 was found to reduce oxidative damage to whole‐cell and mitochondrial compartments in rat astrocytes transfected with the HMOX1 gene. Moreover, OB‐28 was found to significantly counter behavioral deficits and neuropathological alterations in APP_swe/PS1_?E9 mice. Attenuation of AD‐associated behavioral deficits and neuropathological changes suggests that HO‐1 may be a promising target for neuroprotective intervention in AD and other neurodegenerative diseases.

     

An exploratory study on CLU, CR1 and PICALM and Parkinson disease

Background

Recent GWAS and subsequent confirmation studies reported several single-nucleotide polymorphisms (SNPs) at the CLU, CR1 and PICALM loci in association with late-onset Alzheimer''s disease (AD). Parkinson disease (PD) shares several clinical and pathologic characteristics with AD; we therefore explored whether these SNPs were also associated with PD risk.

Methodology/Principal Findings

791 non-Hispanic Whites cases and 1,580 matched controls were included in the study. Odds ratios (OR) and 95% confidence intervals (CI) were obtained from logistic regression models. rs11136000 at the CLU locus was associated with PD risk under the recessive model (comparing TT versus CC+CT: OR = 0.71, 95% CI: 0.55-0.92, p = 0.008) after adjusting for year of birth, gender, smoking, and caffeine intake. Further adjustment for family history of PD and ApoE ε4 status did not change the result. In addition, we did not find evidence for effect modification by ApoE or known PD risk factors. The association, however, appeared to be stronger for PD with dementia (OR = 0.49, 95% CI: 0.27-0.91) than for PD without dementia (OR = 0.81, 95% CI: 0.61-1.06). The two other SNPs, rs6656401 from CR1, and rs3851179 from PICALM region were not associated with PD (p>0.05).

Conclusion

Our exploratory analysis suggests an association of CLU with PD. This exploratory finding and the role of dementia in explaining this finding needs further investigation.     

Atopic dermatitis‐mitigating effects of new Lactobacillus strain,Lactobacillus sakei probio 65 isolated from Kimchi

Aims

Atopic dermatitis (AD) is an inflammatory skin disease. Probiotics have been reported to modulate immune responses and thus are now being suggested as potential treatments for allergies. In this study, we investigated the inhibitory effects of Lactobacillus sakei probio 65 isolated from Kimchi on artificially inducing AD in NC/Nga mice.

Methods and Results

Oral administration of viable or heat‐inactivated Lact. sakei probio 65 improved the condition of skin and reduced scratching frequency. Serum levels of IgE and cutaneous T‐cell‐attracting chemokine (CTACK) were significantly decreased by this therapy. Dead Lact. sakei probio 65 also decreased IL‐4 and IL‐6 serum concentrations. Moreover, both live and dead Lact. sakei probio 65 inhibited the expression of Thymus and activation‐regulated chemokine and CTACK in AD‐like skin lesions. The increased levels of Foxp3 expression in the lesional skin and ears were also suppressed by Lact. sakei probio 65. In addition, Lact. sakei probio 65 inhibited β‐hexosaminidase release and the secretion of IL‐4, TNF‐α and IL‐6 from RBL‐2H3 cells.

Conclusions

Oral treatment with both viable and heat‐inactivated Lact. sakei probio 65 inhibits skin inflammation and AD‐like skin lesions, as well as mast cell activation.

Significance and Impact of the Study

Lactobacillus sakei probio 65 has an inhibitory effect on atopic dermatitis‐like skin lesions and may represent an effective new anti‐inflammatory agent.     

Cingulate cortex hypoperfusion predicts Alzheimer's disease in mild cognitive impairment

Background  

Mild cognitive impairment (MCI) was recently described as a heterogeneous group with a variety of clinical outcomes and high risk to develop Alzheimer's disease (AD). Regional cerebral blood flow (rCBF) as measured by single photon emission computed tomography (SPECT) was used to study the heterogeneity of MCI and to look for predictors of future development of AD.     

A blood based 12-miRNA signature of Alzheimer disease patients

Background

Alzheimer disease (AD) is the most common form of dementia but the identification of reliable, early and non-invasive biomarkers remains a major challenge. We present a novel miRNA-based signature for detecting AD from blood samples.

Results

We apply next-generation sequencing to miRNAs from blood samples of 48 AD patients and 22 unaffected controls, yielding a total of 140 unique mature miRNAs with significantly changed expression levels. Of these, 82 have higher and 58 have lower abundance in AD patient samples. We selected a panel of 12 miRNAs for an RT-qPCR analysis on a larger cohort of 202 samples, comprising not only AD patients and healthy controls but also patients with other CNS illnesses. These included mild cognitive impairment, which is assumed to represent a transitional period before the development of AD, as well as multiple sclerosis, Parkinson disease, major depression, bipolar disorder and schizophrenia. miRNA target enrichment analysis of the selected 12 miRNAs indicates an involvement of miRNAs in nervous system development, neuron projection, neuron projection development and neuron projection morphogenesis. Using this 12-miRNA signature, we differentiate between AD and controls with an accuracy of 93%, a specificity of 95% and a sensitivity of 92%. The differentiation of AD from other neurological diseases is possible with accuracies between 74% and 78%. The differentiation of the other CNS disorders from controls yields even higher accuracies.

Conclusions

The data indicate that deregulated miRNAs in blood might be used as biomarkers in the diagnosis of AD or other neurological diseases.     

A Novel Immunological Assay for Hepcidin Quantification in Human Serum

Background

Hepcidin is a 25-aminoacid cysteine-rich iron regulating peptide. Increased hepcidin concentrations lead to iron sequestration in macrophages, contributing to the pathogenesis of anaemia of chronic disease whereas decreased hepcidin is observed in iron deficiency and primary iron overload diseases such as hereditary hemochromatosis. Hepcidin quantification in human blood or urine may provide further insights for the pathogenesis of disorders of iron homeostasis and might prove a valuable tool for clinicians for the differential diagnosis of anaemia. This study describes a specific and non-operator demanding immunoassay for hepcidin quantification in human sera.

Methods and Findings

An ELISA assay was developed for measuring hepcidin serum concentration using a recombinant hepcidin25-His peptide and a polyclonal antibody against this peptide, which was able to identify native hepcidin. The ELISA assay had a detection range of 10–1500 µg/L and a detection limit of 5.4 µg/L. The intra- and interassay coefficients of variance ranged from 8–15% and 5–16%, respectively. Mean linearity and recovery were 101% and 107%, respectively. Mean hepcidin levels were significantly lower in 7 patients with juvenile hemochromatosis (12.8 µg/L) and 10 patients with iron deficiency anemia (15.7 µg/L) and higher in 7 patients with Hodgkin lymphoma (116.7 µg/L) compared to 32 age-matched healthy controls (42.7 µg/L).

Conclusions

We describe a new simple ELISA assay for measuring hepcidin in human serum with sufficient accuracy and reproducibility.