RESUMO
Cell fate decisions during early B cell activation determine the outcome of responses to pathogens and vaccines. We examined the early B cell response to T-dependent antigen in mice by single-cell RNA sequencing. Early after immunization, a homogeneous population of activated precursors (APs) gave rise to a transient wave of plasmablasts (PBs), followed a day later by the emergence of germinal center B cells (GCBCs). Most APs rapidly exited the cell cycle, giving rise to non-GC-derived early memory B cells (eMBCs) that retained an AP-like transcriptional profile. Rapid decline of antigen availability controlled these events; provision of excess antigen precluded cell cycle exit and induced a new wave of PBs. Fate mapping revealed a prominent contribution of eMBCs to the MBC pool. Quiescent cells with an MBC phenotype dominated the early response to immunization in primates. A reservoir of APs/eMBCs may enable rapid readjustment of the immune response when failure to contain a threat is manifested by increased antigen availability.
Assuntos
Linfócitos B/imunologia , Centro Germinativo/imunologia , Imunidade Humoral/imunologia , Memória Imunológica/imunologia , Ativação Linfocitária/imunologia , Animais , Apresentação de Antígeno/imunologia , Diferenciação Celular/imunologia , Camundongos , Plasmócitos/imunologia , Células Precursoras de Linfócitos B/imunologiaRESUMO
Longitudinal bone growth in children is sustained by growth plates, narrow discs of cartilage that provide a continuous supply of chondrocytes for endochondral ossification1. However, it remains unknown how this supply is maintained throughout childhood growth. Chondroprogenitors in the resting zone are thought to be gradually consumed as they supply cells for longitudinal growth1,2, but this model has never been proved. Here, using clonal genetic tracing with multicolour reporters and functional perturbations, we demonstrate that longitudinal growth during the fetal and neonatal periods involves depletion of chondroprogenitors, whereas later in life, coinciding with the formation of the secondary ossification centre, chondroprogenitors acquire the capacity for self-renewal, resulting in the formation of large, stable monoclonal columns of chondrocytes. Simultaneously, chondroprogenitors begin to express stem cell markers and undergo symmetric cell division. Regulation of the pool of self-renewing progenitors involves the hedgehog and mammalian target of rapamycin complex 1 (mTORC1) signalling pathways. Our findings indicate that a stem cell niche develops postnatally in the epiphyseal growth plate, which provides a continuous supply of chondrocytes over a prolonged period.
Assuntos
Condrócitos/citologia , Células Clonais/citologia , Lâmina de Crescimento/citologia , Nicho de Células-Tronco/fisiologia , Envelhecimento , Animais , Cartilagem/citologia , Autorrenovação Celular , Células Clonais/metabolismo , Feminino , Lâmina de Crescimento/metabolismo , Masculino , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , CamundongosRESUMO
Immature multipotent embryonic peripheral glial cells, the Schwann cell precursors (SCPs), differentiate into melanocytes, parasympathetic neurons, chromaffin cells, and dental mesenchymal populations. Here, genetic lineage tracing revealed that, during murine embryonic development, some SCPs detach from nerve fibers to become mesenchymal cells, which differentiate further into chondrocytes and mature osteocytes. This occurred only during embryonic development, producing numerous craniofacial and trunk skeletal elements, without contributing to development of the appendicular skeleton. Formation of chondrocytes from SCPs also occurred in zebrafish, indicating evolutionary conservation. Our findings reveal multipotency of SCPs, providing a developmental link between the nervous system and skeleton.
Assuntos
Osso e Ossos/citologia , Linhagem da Célula/genética , Condrócitos/citologia , Células-Tronco Mesenquimais/citologia , Tecido Nervoso/citologia , Células de Schwann/citologia , Animais , Biomarcadores/metabolismo , Osso e Ossos/embriologia , Osso e Ossos/metabolismo , Diferenciação Celular , Condrócitos/metabolismo , Células Cromafins/citologia , Células Cromafins/metabolismo , Embrião de Mamíferos , Embrião não Mamífero , Desenvolvimento Embrionário , Expressão Gênica , Melanócitos/citologia , Melanócitos/metabolismo , Células-Tronco Mesenquimais/metabolismo , Camundongos , Células-Tronco Multipotentes/citologia , Células-Tronco Multipotentes/metabolismo , Proteína Proteolipídica de Mielina/genética , Proteína Proteolipídica de Mielina/metabolismo , Fibras Nervosas/metabolismo , Tecido Nervoso/embriologia , Tecido Nervoso/metabolismo , Crista Neural/citologia , Crista Neural/crescimento & desenvolvimento , Crista Neural/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Osteócitos/citologia , Osteócitos/metabolismo , Fatores de Transcrição SOXE/genética , Fatores de Transcrição SOXE/metabolismo , Células de Schwann/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Topoisomerase inhibitors are widely used in cancer chemotherapy. However, one of the potential long-term adverse effects of such therapy is acute leukemia. A key feature of such therapy-induced acute myeloid leukemia (t-AML) is recurrent chromosomal translocations involving AML1 (RUNX1) or MLL (KMT2A) genes. The formation of chromosomal translocation depends on the spatial proximity of translocation partners and the mobility of the DNA ends. It is unclear which of these two factors might be decisive for recurrent t-AML translocations. Here, we used fluorescence in situ hybridization (FISH) and chromosome conformation capture followed by sequencing (4C-seq) to investigate double-strand DNA break formation and the mobility of broken ends upon etoposide treatment, as well as contacts between translocation partner genes. We detected the separation of the parts of the broken AML1 gene, as well as the increased mobility of these separated parts. 4C-seq analysis showed no evident contacts of AML1 and MLL with loci, implicated in recurrent t-AML translocations, either before or after etoposide treatment. We suggest that separation of the break ends and their increased non-targeted mobility-but not spatial predisposition of the rearrangement partners-plays a major role in the formation of these translocations.
Assuntos
Leucemia Mieloide Aguda , Translocação Genética , DNA , Quebras de DNA de Cadeia Dupla , Etoposídeo/efeitos adversos , Humanos , Hibridização in Situ Fluorescente , Leucemia Mieloide Aguda/genética , Inibidores da Topoisomerase II/efeitos adversosRESUMO
BACKGROUND: DNA methylation is involved in the regulation of gene expression. Although bisulfite-sequencing based methods profile DNA methylation at a single CpG resolution, methylation levels are usually averaged over genomic regions in the downstream bioinformatic analysis. RESULTS: We demonstrate that on the genome level a single CpG methylation can serve as a more accurate predictor of gene expression than an average promoter / gene body methylation. We define CpG traffic lights (CpG TL) as CpG dinucleotides with a significant correlation between methylation and expression of a gene nearby. CpG TL are enriched in all regulatory regions. Among all promoters, CpG TL are especially enriched in poised ones, suggesting involvement of DNA methylation in their regulation. Yet, binding of only a handful of transcription factors, such as NRF1, ETS, STAT and IRF-family members, could be regulated by direct methylation of transcription factor binding sites (TFBS) or its close proximity. For the majority of TF, an alternative scenario is more likely: methylation and inactivation of the whole regulatory element indirectly represses functional TF binding with a CpG TL being a reliable marker of such inactivation. CONCLUSIONS: CpG TL provide a promising insight into mechanisms of enhancer activity and gene regulation linking methylation of single CpG to gene expression. CpG TL methylation can be used as reliable markers of enhancer activity and gene expression in applications, e.g. in clinic where measuring DNA methylation is easier compared to directly measuring gene expression due to more stable nature of DNA.
Assuntos
Ilhas de CpG , Metilação de DNA , Regulação da Expressão Gênica , Genoma Humano , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo , Humanos , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Transcrição GênicaRESUMO
The three-spined stickleback (Gasterosteus aculeatus) represents a convenient model to study microevolution-adaptation to a freshwater environment. Although genetic adaptations to freshwater environments are well-studied, epigenetic adaptations have attracted little attention. In this work, we investigated the role of DNA methylation in the adaptation of the marine stickleback population to freshwater conditions. DNA methylation profiling was performed in marine and freshwater populations of sticklebacks, as well as in marine sticklebacks placed into a freshwater environment and freshwater sticklebacks placed into seawater. We showed that the DNA methylation profile after placing a marine stickleback into fresh water partially converged to that of a freshwater stickleback. For six genes including ATP4A ion pump and NELL1, believed to be involved in skeletal ossification, we demonstrated similar changes in DNA methylation in both evolutionary and short-term adaptation. This suggested that an immediate epigenetic response to freshwater conditions can be maintained in freshwater population. Interestingly, we observed enhanced epigenetic plasticity in freshwater sticklebacks that may serve as a compensatory regulatory mechanism for the lack of genetic variation in the freshwater population. For the first time, we demonstrated that genes encoding ion channels KCND3, CACNA1FB, and ATP4A were differentially methylated between the marine and the freshwater populations. Other genes encoding ion channels were previously reported to be under selection in freshwater populations. Nevertheless, the genes that harbor genetic and epigenetic changes were not the same, suggesting that epigenetic adaptation is a complementary mechanism to selection of genetic variants favorable for freshwater environment.
Assuntos
Adaptação Fisiológica/genética , Epigênese Genética/genética , Smegmamorpha/genética , Aclimatação/genética , Amilopectina , Animais , Evolução Biológica , Metilação de DNA/genética , Evolução Molecular , Água Doce , Variação Genética/genética , Estudo de Associação Genômica Ampla , Modelos Genéticos , Água do Mar , Seleção Genética/genéticaRESUMO
Rapidly developing next-generation sequencing (NGS) technologies produce a large amount of data across the whole human genome and allow a large number of DNA samples to be analyzed simultaneously. Screening cell-free fetal DNA (cffDNA) obtained from maternal blood using NGS technologies has provided new opportunities for non-invasive prenatal diagnosis (NIPD) of fetal aneuploidies. One of the major challenges to the analysis of fetal abnormalities is the development of accurate and reliable algorithms capable of analyzing large numbers of short sequence reads. Several such algorithms have recently been developed. Here, we provide a review of recent NGS-based NIPD methods as well as the available algorithms for short-read sequence analysis. We furthermore introduce the practical application of these algorithms for the detection of different types of fetal aneuploidies, and compare the performance, cost and complexity of each approach for clinical deployment. Our review identifies several main technologies and trends in NGS-based NIPD. The main considerations for clinical development for NIPD and screening tests using DNA sequencing are: accuracy, intellectual property, cost and the ability to screen for a wide range of chromosomal abnormalities and genetic defects. The cost of the diagnostic test depends on the sequencing method, diagnostic algorithm and volume of the tests. If the cost of sequencing equipment and reagents remains at or around current levels, targeted approaches for sequencing-based aneuploidy testing and SNP-based methods are preferred.
Assuntos
Aneuploidia , DNA/sangue , Testes Genéticos/métodos , Diagnóstico Pré-Natal/métodos , Análise de Sequência de DNA/métodos , Algoritmos , Feminino , Humanos , GravidezRESUMO
Glial cells have been proposed as a source of neural progenitors, but the mechanisms underpinning the neurogenic potential of adult glia are not known. Using single cell transcriptomic profiling, we show that enteric glial cells represent a cell state attained by autonomic neural crest cells as they transition along a linear differentiation trajectory that allows them to retain neurogenic potential while acquiring mature glial functions. Key neurogenic loci in early enteric nervous system progenitors remain in open chromatin configuration in mature enteric glia, thus facilitating neuronal differentiation under appropriate conditions. Molecular profiling and gene targeting of enteric glial cells in a cell culture model of enteric neurogenesis and a gut injury model demonstrate that neuronal differentiation of glia is driven by transcriptional programs employed in vivo by early progenitors. Our work provides mechanistic insight into the regulatory landscape underpinning the development of intestinal neural circuits and generates a platform for advancing glial cells as therapeutic agents for the treatment of neural deficits.
Assuntos
Neurogênese , Neuroglia , Adulto , Humanos , Neurogênese/genética , Diferenciação Celular , Sistema Nervoso Autônomo , Técnicas de Cultura de CélulasRESUMO
In this study we use comparative genomics to uncover a gene with uncharacterized function (1700011H14Rik/C14orf105/CCDC198), which we hereby name FAME (Factor Associated with Metabolism and Energy). We observe that FAME shows an unusually high evolutionary divergence in birds and mammals. Through the comparison of single nucleotide polymorphisms, we identify gene flow of FAME from Neandertals into modern humans. We conduct knockout experiments on animals and observe altered body weight and decreased energy expenditure in Fame knockout animals, corresponding to genome-wide association studies linking FAME with higher body mass index in humans. Gene expression and subcellular localization analyses reveal that FAME is a membrane-bound protein enriched in the kidneys. Although the gene knockout results in structurally normal kidneys, we detect higher albumin in urine and lowered ferritin in the blood. Through experimental validation, we confirm interactions between FAME and ferritin and show co-localization in vesicular and plasma membranes.
Assuntos
Metabolismo Energético , Estudo de Associação Genômica Ampla , Animais , Humanos , Peso Corporal , Metabolismo Energético/genética , Ferritinas/genética , Rim , Homem de NeandertalRESUMO
The generation of high-affinity antibodies against pathogens and vaccines requires the germinal center (GC) reaction, which relies on a complex interplay between specialized effector B and CD4 T lymphocytes, the GC B cells and T follicular helper (TFH) cells. Intriguingly, several positive key regulators of the GC reaction are common for both cell types. Here, we report that the transcription factor Bhlhe40 is a crucial cell-intrinsic negative regulator affecting both the B and T cell sides of the GC reaction. In activated CD4 T cells, Bhlhe40 was required to restrain proliferation, thus limiting the number of TFH cells. In B cells, Bhlhe40 executed its function in the first days after immunization by selectively restricting the generation of the earliest GC B cells but not of early memory B cells or plasmablasts. Bhlhe40-deficient mice with progressing age succumbed to a B cell lymphoma characterized by the accumulation of monoclonal GC B-like cells and polyclonal TFH cells in various tissues.
Assuntos
Linfócitos B/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Suscetibilidade a Doenças , Centro Germinativo/imunologia , Centro Germinativo/metabolismo , Proteínas de Homeodomínio/genética , Ativação Linfocitária/imunologia , Células T Auxiliares Foliculares/metabolismo , Animais , Linfócitos B/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biomarcadores , Diferenciação Celular/genética , Modelos Animais de Doenças , Regulação da Expressão Gênica , Proteínas de Homeodomínio/metabolismo , Imunofenotipagem , Ativação Linfocitária/genética , Linfoma de Células B/etiologia , Linfoma de Células B/metabolismo , Linfoma de Células B/patologia , Camundongos , Camundongos Knockout , Células T Auxiliares Foliculares/imunologiaRESUMO
Hypermethylation of tumour suppressors and other aberrations of DNA methylation in tumours play a significant role in cancer progression. DNA methylation can be affected by various environmental conditions, including hypoxia. The response to hypoxia is mainly achieved through activation of the transcriptional program associated with HIF1A transcription factor. Inactivation of Von Hippel-Lindau Tumour Suppressor gene (VHL) by genetic or epigenetic events, which also induces aberrant activation of HIF1A, is the most common driver event for renal cancer. With whole-genome bisulphite sequencing and LC-MS, we demonstrated that VHL inactivation induced global genome hypermethylation in human kidney cancer cells under normoxic conditions. This effect was reverted by exogenous expression of wild-type VHL. We showed that global genome hypermethylation in VHL mutants can be explained by transcriptional changes in MDH and L2HGDH genes that cause the accumulation of 2-hydroxyglutarate - a metabolite that inhibits DNA demethylation by TET enzymes. Unlike the known cases of DNA hypermethylation in cancer, 2-hydroxyglutarate was accumulated in the cells with the wild-type isocitrate dehydrogenases.
Assuntos
Carcinoma de Células Renais , Neoplasias Renais , Oxirredutases do Álcool/genética , Oxirredutases do Álcool/metabolismo , Carcinoma de Células Renais/genética , DNA/metabolismo , Metilação de DNA , Humanos , Hipóxia/genética , Isocitrato Desidrogenase , Neoplasias Renais/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
Adrenal glands are the major organs releasing catecholamines and regulating our stress response. The mechanisms balancing generation of adrenergic chromaffin cells and protecting against neuroblastoma tumors are still enigmatic. Here we revealed that serotonin (5HT) controls the numbers of chromaffin cells by acting upon their immediate progenitor "bridge" cells via 5-hydroxytryptamine receptor 3A (HTR3A), and the aggressive HTR3Ahigh human neuroblastoma cell lines reduce proliferation in response to HTR3A-specific agonists. In embryos (in vivo), the physiological increase of 5HT caused a prolongation of the cell cycle in "bridge" progenitors leading to a smaller chromaffin population and changing the balance of hormones and behavioral patterns in adulthood. These behavioral effects and smaller adrenals were mirrored in the progeny of pregnant female mice subjected to experimental stress, suggesting a maternal-fetal link that controls developmental adaptations. Finally, these results corresponded to a size-distribution of adrenals found in wild rodents with different coping strategies.
Assuntos
Células Cromafins , Neuroblastoma , Glândulas Suprarrenais/metabolismo , Animais , Catecolaminas/metabolismo , Células Cromafins/metabolismo , Feminino , Camundongos , Neuroblastoma/metabolismo , Gravidez , Serotonina/metabolismoRESUMO
Characterization of the progression of cellular states during human embryogenesis can provide insights into the origin of pediatric diseases. We examined the transcriptional states of neural crest- and mesoderm-derived lineages differentiating into adrenal glands, kidneys, endothelium and hematopoietic tissue between post-conception weeks 6 and 14 of human development. Our results reveal transitions connecting the intermediate mesoderm and progenitors of organ primordia, the hematopoietic system and endothelial subtypes. Unexpectedly, by using a combination of single-cell transcriptomics and lineage tracing, we found that intra-adrenal sympathoblasts at that stage are directly derived from nerve-associated Schwann cell precursors, similarly to local chromaffin cells, whereas the majority of extra-adrenal sympathoblasts arise from the migratory neural crest. In humans, this process persists during several weeks of development within the large intra-adrenal ganglia-like structures, which may also serve as reservoirs of originating cells in neuroblastoma.
Assuntos
Linhagem da Célula , Embrião de Mamíferos/metabolismo , Neuroblastoma/embriologia , Neuroblastoma/genética , Análise de Célula Única , Sistema Simpático-Suprarrenal/embriologia , Transcriptoma/genética , Animais , Células Cromafins/metabolismo , Células Cromafins/patologia , Análise por Conglomerados , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Regulação Neoplásica da Expressão Gênica , Humanos , Lactente , Camundongos , Células-Tronco Neurais/metabolismo , Neuroblastoma/patologia , Células de Schwann/metabolismo , Células de Schwann/patologia , Microambiente TumoralRESUMO
[This corrects the article DOI: 10.18632/oncoscience.395.].
RESUMO
VHL inactivation is a key oncogenic event for renal carcinomas. In normoxia, VHL suppresses HIF1a-mediated transcriptional response, which is characteristic to hypoxia. It has previously been shown that hypoxic conditions inhibit TET-dependent hydroxymethylation of cytosines and cause DNA hypermethylation at gene promoters. In this work, we performed VHL inactivation by CRISPR/Cas9 and studied its effects on gene expression and DNA methylation. We showed that even without hypoxia, VHL inactivation leads to hypermethylation of the genome. Hypermethylated cytosines were evenly distributed throughout the genome with a slight preference for AP-1 (JUN and FOS) binding sites. Hypermethylated cytosines tended to be enriched within the binding sites of transcription factors that showed increased gene expression after VHL inactivation. We also observed promoter hypermethylation associated with decreased gene expression for several regulators of transcription and DNA methylation including SALL3.
Assuntos
Carcinoma de Células Renais/genética , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Neoplasias Renais/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Sistemas CRISPR-Cas/genética , Carcinoma de Células Renais/patologia , Linhagem Celular Tumoral , Citosina/metabolismo , Inativação Gênica , Genoma Humano/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Neoplasias Renais/patologia , Regiões Promotoras Genéticas/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Hipóxia Tumoral , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
While many efforts have been made to pave the way toward human space colonization, little consideration has been given to the methods of protecting spacefarers against harsh cosmic and local radioactive environments and the high costs associated with protection from the deleterious physiological effects of exposure to high-Linear energy transfer (high-LET) radiation. Herein, we lay the foundations of a roadmap toward enhancing human radioresistance for the purposes of deep space colonization and exploration. We outline future research directions toward the goal of enhancing human radioresistance, including upregulation of endogenous repair and radioprotective mechanisms, possible leeways into gene therapy in order to enhance radioresistance via the translation of exogenous and engineered DNA repair and radioprotective mechanisms, the substitution of organic molecules with fortified isoforms, and methods of slowing metabolic activity while preserving cognitive function. We conclude by presenting the known associations between radioresistance and longevity, and articulating the position that enhancing human radioresistance is likely to extend the healthspan of human spacefarers as well.
RESUMO
EGFR-based targeted therapies have shown limited success in smokers. Identification of alternate signaling mechanism(s) leading to TKI resistance in smokers is critically important. We observed increased resistance to erlotinib in H358 NSCLC (non-small cell lung carcinoma) cells chronically exposed to cigarette smoke (H358-S) compared to parental cells. SILAC-based mass-spectrometry approach was used to study altered signaling in H358-S cell line. Importantly, among the top phosphosites in H358-S cells we observed hyperphosphorylation of EGFR (Y1197) and non-receptor tyrosine kinase FAK (Y576/577). Supporting these observations, a transcriptomic-based pathway activation analysis of TCGA NSCLC datasets revealed that FAK and EGFR internalization pathways were significantly upregulated in smoking patients, compared to the never-smokers and were associated with elevated PI3K signaling and lower level of caspase cascade and E-cadherin pathways activation. We show that inhibition of FAK led to decreased cellular proliferation and invasive ability of the smoke-exposed cells, and restored their dependency on EGFR signaling. Our data suggests that activation of focal adhesion pathway significantly contributes to erlotinib resistance, and that FAK is a potential therapeutic target for management of erlotinib resistance in smoke-induced NSCLC.
RESUMO
Signalling pathway activation analysis is a powerful approach for extracting biologically relevant features from large-scale transcriptomic and proteomic data. However, modern pathway-based methods often fail to provide stable pathway signatures of a specific phenotype or reliable disease biomarkers. In the present study, we introduce the in silico Pathway Activation Network Decomposition Analysis (iPANDA) as a scalable robust method for biomarker identification using gene expression data. The iPANDA method combines precalculated gene coexpression data with gene importance factors based on the degree of differential gene expression and pathway topology decomposition for obtaining pathway activation scores. Using Microarray Analysis Quality Control (MAQC) data sets and pretreatment data on Taxol-based neoadjuvant breast cancer therapy from multiple sources, we demonstrate that iPANDA provides significant noise reduction in transcriptomic data and identifies highly robust sets of biologically relevant pathway signatures. We successfully apply iPANDA for stratifying breast cancer patients according to their sensitivity to neoadjuvant therapy.
Assuntos
Algoritmos , Biomarcadores/metabolismo , Simulação por Computador , Área Sob a Curva , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Feminino , Perfilação da Expressão Gênica , Humanos , Modelos Biológicos , Paclitaxel/farmacologia , Paclitaxel/uso terapêutico , Curva ROC , Reprodutibilidade dos Testes , Transcriptoma/genéticaRESUMO
We report a family case of type II early-onset Alzheimer's disease (AD) inherited over three generations. None of the patients in the family had mutations in the genes believed to be the major risk factors for AD, such as APP, presenilin 1 or 2. Targeted exome sequencing of 249 genes that were previously reported to be associated with AD revealed a rare mutation in hemochromatosis (HFE) gene known to be associated with hemochromotosis. Compared to previous studies, we show that HFE mutation can possess the risk of AD in transferrin-, APOE- and APP-normal patients.