Fanconi's anemia: what have we learned from the genes so far?

Fanconi's anemia (FA) is a rare genetic disorder affecting children at an early age; patients suffer from progressive bone marrow failure and, in many cases, from congenital malformations. As cells from FA patients have an increased sensitivity to DNA-crosslinking agents, FA has been included among the group of DNA repair disorders. However, identification of a specific DNA repair defect in FA has not been firmly established. None the less, this cellular phenotype has allowed the classification of FA patients into eight complementation groups defining eight possible FA genes. Two of these genes have now been cloned and, although they have raised more questions than they have answered, are facilitating the identification of cellular processes implicated in the pathophysiology of FA, and the design of new therapies.     

Learning from a paradox: recent insights into Fanconi anaemia through studying mouse models

Fanconi anaemia (FA) is a rare autosomal recessive or X-linked inherited disease characterised by an increased incidence of bone marrow failure (BMF), haematological malignancies and solid tumours. Cells from individuals with FA show a pronounced sensitivity to DNA interstrand crosslink (ICL)-inducing agents, which manifests as G2-M arrest, chromosomal aberrations and reduced cellular survival. To date, mutations in at least 15 different genes have been identified that cause FA; the products of all of these genes are thought to function together in the FA pathway, which is essential for ICL repair. Rapidly following the discovery of FA genes, mutant mice were generated to study the disease and the affected pathway. These mutant mice all show the characteristic cellular ICL-inducing agent sensitivity, but only partially recapitulate the developmental abnormalities, anaemia and cancer predisposition seen in individuals with FA. Therefore, the usefulness of modelling FA in mice has been questioned. In this Review, we argue that such scepticism is unjustified. We outline that haematopoietic defects and cancer predisposition are manifestations of FA gene defects in mice, albeit only in certain genetic backgrounds and under certain conditions. Most importantly, recent work has shown that developmental defects in FA mice also arise with concomitant inactivation of acetaldehyde metabolism, giving a strong clue about the nature of the endogenous lesion that must be repaired by the functional FA pathway. This body of work provides an excellent example of a paradox in FA research: that the dissimilarity, rather than the similarity, between mice and humans can provide insight into human disease. We expect that further study of mouse models of FA will help to uncover the mechanistic background of FA, ultimately leading to better treatment options for the disease.     

Recent advances in the manipulation of murine gene expression and its utility for the study of human neurological disease

Transgenic mouse models have vastly contributed to our knowledge of the genetic and molecular pathways underlying the pathogenesis of neurological disorders that affect millions of people worldwide. Not only have they allowed the generation of disease models mimicking the human pathological state but they have also permitted the exploration of the pathological role of specific genes through the generation of knock-out and knock-in models. Classical constitutive transgenic mice have several limitations however, due to behavioral adaptation process occurring and conditional mouse models are time-consuming and often lack extensive spatial or temporal control of gene manipulation. These limitations could be overcome by means of innovative methods that are now available such as RNAi, viral vectors and large cloning DNA vectors. These tools have been extensively used for the generation of mouse models and are characterized by the superior control of transgene expression that has been proven invaluable in the assessment of novel treatments for neurological diseases and to further investigate the molecular processes underlying the etiopathology of neurological disorders. Furthermore, in association with classical transgenic mouse models, they have allowed the validation of innovative therapeutic strategies for the treatment of human neurological disorders. This review describes how these tools have overcome the limitations of classical transgenic mouse models and how they have been of value for the study of human neurological diseases.     

Genomic and Metabolic Disposition of Non-Obese Type 2 Diabetic Rats to Increased Myocardial Fatty Acid Metabolism

Lipotoxicity of the heart has been implicated as a leading cause of morbidity in Type 2 Diabetes Mellitus (T2DM). While numerous reports have demonstrated increased myocardial fatty acid (FA) utilization in obese T2DM animal models, this diabetic phenotype has yet to be demonstrated in non-obese animal models of T2DM. Therefore, the present study investigates functional, metabolic, and genomic differences in myocardial FA metabolism in non-obese type 2 diabetic rats. The study utilized Goto-Kakizaki (GK) rats at the age of 24 weeks. Each rat was imaged with small animal positron emission tomography (PET) to estimate myocardial blood flow (MBF) and myocardial FA metabolism. Echocardiograms (ECHOs) were performed to assess cardiac function. Levels of triglycerides (TG) and non-esterified fatty acids (NEFA) were measured in both plasma and cardiac tissues. Finally, expression profiles for 168 genes that have been implicated in diabetes and FA metabolism were measured using quantitative PCR (qPCR) arrays. GK rats exhibited increased NEFA and TG in both plasma and cardiac tissue. Quantitative PET imaging suggests that GK rats have increased FA metabolism. ECHO data indicates that GK rats have a significant increase in left ventricle mass index (LVMI) and decrease in peak early diastolic mitral annular velocity (E’) compared to Wistar rats, suggesting structural remodeling and impaired diastolic function. Of the 84 genes in each the diabetes and FA metabolism arrays, 17 genes in the diabetes array and 41 genes in the FA metabolism array were significantly up-regulated in GK rats. Our data suggest that GK rats’ exhibit increased genomic disposition to FA and TG metabolism independent of obesity.     

Cloning and characterization of murine Fanconi anemia group A gene: Fanca protein is expressed in lymphoid tissues, testis, and ovary

Fanconi anemia (FA) is an autosomal recessive disorder in humans characterized by bone marrow failure, cancer predisposition, and cellular hypersensitivity to cross-linking agents such as mitomycin C and diepoxybutane. FA genes display a caretaker function essential for maintenance of genomic integrity. We have cloned the murine homolog of FANCA, the gene mutated in the major FA complementation group (FA-A). The full-length mouse Fanca cDNA consists of 4503 bp and encodes a protein with a predicted molecular weight of 161 kDa. The deduced Fanca mouse protein shares 81% amino acid sequence similarity and 66% identity with the human protein. The nuclear localization signal and partial leucine zipper consensus motifs found in the human FANCA protein were also present in the murine homolog. In spite of the species difference, the murine Fanca cDNA was capable of correcting the cross-linker sensitive phenotype of human FA-A cells, suggesting functional conservation. Based on Northern as well as Western blots, Fanca was mainly expressed in lymphoid tissues, testis, and ovary. This expression pattern correlates with some of the clinical symptoms observed in FA patients. The availability of the murine Fanca cDNA now allows the gene to be studied in experimental mouse models. Received: 5 September 1999 / Accepted: 3 December 1999     

范可尼贫血基因在卵泡发育中的调节作用

范可尼贫血(Fanconi anemia, FA)是一种罕见的常染色体或X染色体连锁的隐性遗传病,其发生源于范可尼贫血基因(FA基因)突变。FA基因是一组在DNA交联损伤中起同源重组修复作用的基因。FA女性患者常见早发性卵巢功能衰退(premature ovarian insufficient, POI)的特征,而FA小鼠也表现出生殖细胞严重缺乏,这些结果提示FA基因在哺乳动物卵泡发育中起重要作用。研究显示FA基因在促进原始生殖细胞增生,维持正常卵母细胞减数分裂,参与卵泡发育的促性腺激素调节以及卵母细胞与颗粒细胞生长过程中的相互调节等方面调节卵泡发育。本文综述了FA基因在卵泡发育中的作用和分子机制方面的研究进展,为POI的病因学解析提供遗传基础。     

The mouse ascending: perspectives for human-disease models

The laboratory mouse is widely considered the model organism of choice for studying the diseases of humans, with whom they share 99% of their genes. A distinguished history of mouse genetic experimentation has been further advanced by the development of powerful new tools to manipulate the mouse genome. The recent launch of several international initiatives to analyse the function of all mouse genes through mutagenesis, molecular analysis and phenotyping underscores the utility of the mouse for translating the information stored in the human genome into increasingly accurate models of human disease.     

Mouse models of type 1 and type 2 diabetes derived from the same closed colony: genetic susceptibility shared between two types of diabetes

Except for rare subtypes of diabetes, both type 1 and type 2 diabetes are multifactorial diseases in which genetic factors consisting of multiple susceptibility genes and environmental factors contribute to the disease development. Due to complex interaction among multiple susceptibility genes and between genetic and environmental factors, genetic analysis of multifactorial diseases is difficult in humans. Inbred animal models, in which the genetic background is homogeneous and environmental factors can be controlled, are therefore valuable in genetic dissection of multifactorial diseases. We are fortunate to have excellent animal models for both type 1 and type 2 diabetes--the nonobese diabetic (NOD) mouse and the Nagoya-Shibata-Yasuda (NSY) mouse, respectively. Congenic mapping of susceptibility genes for type 1 diabetes in the NOD mouse has revealed that susceptibility initially mapped as a single locus often consists of multiple components on the same chromosome, indicating the importance of congenic mapping in defining genes responsible for polygenic diseases. The NSY mouse is an inbred animal model of type 2 diabetes established from Jcl:ICR, from which the NOD mouse was also derived. We have recently mapped three major loci contributing to type 2 diabetes in the NSY mouse. Interestingly, support intervals where type 2 diabetes susceptibility genes were mapped in the NSY mouse overlapped the regions where type 1 diabetes susceptibility genes have been mapped in the NOD mouse. Although additional evidence is needed, it may be possible that some of the genes predisposing to diabetes are derived from a common ancestor contained in the original closed colony, contributing to type 1 diabetes in the NOD mouse and type 2 diabetes in the NSY mouse. Such genes, if they exist, will provide valuable information on etiological pathways common to both forms of diabetes, for the establishment of effective methods for prediction, prevention, and intervention in both type 1 and type 2 diabetes.     

Linking susceptibility genes and pathogenesis mechanisms using mouse models of systemic lupus erythematosus

Systemic lupus erythematosus (SLE) represents a challenging autoimmune disease from a clinical perspective because of its varied forms of presentation. Although broad-spectrum steroids remain the standard treatment for SLE, they have many side effects and only provide temporary relief from the symptoms of the disease. Thus, gaining a deeper understanding of the genetic traits and biological pathways that confer susceptibility to SLE will help in the design of more targeted and effective therapeutics. Both human genome-wide association studies (GWAS) and investigations using a variety of mouse models of SLE have been valuable for the identification of the genes and pathways involved in pathogenesis. In this Review, we link human susceptibility genes for SLE with biological pathways characterized in mouse models of lupus, and discuss how the mechanistic insights gained could advance drug discovery for the disease.KEY WORDS: Lupus, SLE, Human genetics, Mouse models, Susceptibility genes     

Identifying novel genes for atherosclerosis through mouse-human comparative genetics

Susceptibility to atherosclerosis is determined by both environmental and genetic factors. Its genetic determinants have been studied by use of quantitative-trait-locus (QTL) analysis. So far, 21 atherosclerosis QTLs have been identified in the mouse: 7 in a high-fat-diet model only, 9 in a sensitized model (apolipoprotein E- or LDL [low-density lipoprotein] receptor-deficient mice) only, and 5 in both models, suggesting that different gene sets operate in each model and that a subset operates in both. Among the 27 human atherosclerosis QTLs reported, 17 (63%) are located in regions homologous (concordant) to mouse QTLs, suggesting that these mouse and human atherosclerosis QTLs have the same underlying genes. Therefore, genes regulating human atherosclerosis will be found most efficiently by first finding their orthologs in concordant mouse QTLs. Novel mouse QTL genes will be found most efficiently by using a combination of the following strategies: identifying QTLs in new crosses performed with previously unused parental strains; inducing mutations in large-scale, high-throughput mutagenesis screens; and using new genomic and bioinformatics tools. Once QTL genes are identified in mice, they can be tested in human association studies for their relevance in human atherosclerotic disease.     

Quantitative trait locus mapping for atherosclerosis susceptibility

PURPOSE OF REVIEW: Atherosclerosis is a complex trait with both environmental and genetic aspects. Although some progress has been made in defining genes associated with atherosclerosis in humans, animal models have been useful in learning about pathways and genes involved in atherogenesis. This review describes an unbiased genetic mapping method called quantitative trait locus mapping and progress in using this method to identify genes that alter atherosclerosis susceptibility in mice. RECENT FINDINGS: Approximately 10 well defined genetic loci have been described that are associated with lesion severity in diet-induced or gene knockout mouse models of atherosclerosis. Recently, two of these genetic loci were narrowed considerably by analysis of genetic recombinants within these loci. In addition, a computational method to discover quantitative trait loci has been applied to atherosclerosis. However, none of the genes responsible for these atherosclerosis quantitative trait loci has been definitively identified. The recent completion of the mouse draft genome should facilitate the task of identifying these genes. SUMMARY: Quantitative trait locus mapping studies in mouse models of atherosclerosis have defined genetic regions that alter lesion severity. The identification of the responsible genes may lead to insights into the pathogenesis of atherosclerosis as well as to candidates for human genetic association studies.     

Using genomewide mutagenesis screens to identify the genes required for neural tube closure in the mouse

BACKGROUND: Neural tube closure is a critical embryological process that requires the coordination of many molecular and cellular events. Only recently has the molecular basis of the cell movements that drive neural tube closure begun to be elucidated. This has been accomplished in part due to the analysis of a growing number of genetically targeted and naturally occurring mouse mutant strains that have neural tube defects (NTDs). Currently there are more than 100 genes that when mutated result in NTDs in the mouse. Yet only approximately 10% of genes in the mouse genome have been mutated and their gross phenotype analyzed, suggesting that only a small percentage of the genes that can cause NTDs have been identified. METHODS: In order to more systematically and fully understand the genetic basis of neural tube closure and to begin to define the molecular pathways that direct this key embryonic event, our laboratories have undertaken a forward genetic screen in mice. From this we hope to gain a better understanding of the regulation of this complex morphogenic processes. CONCLUSIONS: The mouse provides a good model for human neural tube closure, and therefore the information gained from generating novel mouse models of NTDs will help to predict the genes responsible for human NTDs and provide experimental evidence for how they function.     

Werner and Hutchinson-Gilford progeria syndromes: mechanistic basis of human progeroid diseases

Progeroid syndromes have been the focus of intense research in part because they might provide a window into the pathology of normal ageing. Werner syndrome and Hutchinson-Gilford progeria syndrome are two of the best characterized human progeroid diseases. Mutated genes that are associated with these syndromes have been identified, mouse models of disease have been developed, and molecular studies have implicated decreased cell proliferation and altered DNA-damage responses as common causal mechanisms in the pathogenesis of both diseases.     

Cloning and analysis of the mouse Fanconi anemia group A cDNA and an overlapping penta zinc finger cDNA

Despite the cloning of four disease-associated genes for Fanconi anemia (FA), the molecular pathogenesis of FA remains largely unknown. To study FA complementation group A using the mouse as a model system, we cloned and characterized the mouse homolog of the human FANCA cDNA. The mouse cDNA (Fanca) encodes a 161-kDa protein that shares 65% amino acid sequence identity with human FANCA. Fanca is located at the distal region of mouse chromosome 8 and has a ubiquitous pattern of expression in embryonic and adult tissues. Expression of the mouse cDNA in human FA-A cells restores the cellular drug sensitivity to normal levels. Thus, the expression pattern, protein structure, chromosomal location, and function of FANCA are conserved in the mouse. We also isolated a novel zinc finger protein, Zfp276, which has five C(2)H(2) domains. Interestingly, Zfp276 is situated in the Fanca locus, and the 3'UTR of its cDNA overlaps with the last four exons of Fanca in a tail-to-tail manner. Zfp276 is expressed in the same tissues as Fanca, but does not complement the mitomycin C (MMC)-sensitive phenotype of FA-A cells. The overlapping genomic organization between Zfp276 and Fanca may have relevance to the disease phenotype of FA.     

Isolation of a cDNA representing the Fanconi anemia complementation group E gene

Fanconi anemia (FA) is an autosomal recessive chromosomal instability syndrome with at least seven different complementation groups. Four FA genes (FANCA, FANCC, FANCF, and FANCG) have been identified, and two other FA genes (FANCD and FANCE) have been mapped. Here we report the identification, by complementation cloning, of the gene mutated in FA complementation group E (FANCE). FANCE has 10 exons and encodes a novel 536-amino acid protein with two potential nuclear localization signals.     

腓骨肌萎缩症2型(CMT2)小鼠模型的研究进展

腓骨肌萎缩症(Charcot-Marie-Tooth disease, CMT)是人类最常见的遗传性运动和感觉神经疾病之一, 全球群体发病率约为1/2500。CMT主要分为脱髓鞘型(包括CMT1, CMT3, CMT4和CMTX1)和轴索型(CMT2)。迄今为止, 先后已有17个CMT2的致病基因被定位和克隆, 然而对这些基因的致病机制所知甚少。建立CMT2小鼠模型是从动物水平研究突变基因致病机制的有效手段。目前已成功构建了近10种CMT2的转基因小鼠、基因敲除小鼠或基因敲入小鼠模型, 其中尤以带有人源致病基因的转基因小鼠模型为多。文章简要介绍了CMT2小鼠模型构建策略, 着重阐述了CMT2小鼠模型的研究进展, 并对个别小鼠模型进行了剖析。     

A Protein Prioritization Approach Tailored for the FA/BRCA Pathway

Fanconi anemia (FA) is a heterogeneous recessive disorder associated with a markedly elevated risk to develop cancer. To date sixteen FA genes have been identified, three of which predispose heterozygous mutation carriers to breast cancer. The FA proteins work together in a genome maintenance pathway, the so-called FA/BRCA pathway which is important during the S phase of the cell cycle. Since not all FA patients can be linked to (one of) the sixteen known complementation groups, new FA genes remain to be identified. In addition the complex FA network remains to be further unravelled. One of the FA genes, FANCI, has been identified via a combination of bioinformatic techniques exploiting FA protein properties and genetic linkage. The aim of this study was to develop a prioritization approach for proteins of the entire human proteome that potentially interact with the FA/BRCA pathway or are novel candidate FA genes. To this end, we combined the original bioinformatics approach based on the properties of the first thirteen FA proteins identified with publicly available tools for protein-protein interactions, literature mining (Nermal) and a protein function prediction tool (FuncNet). Importantly, the three newest FA proteins FANCO/RAD51C, FANCP/SLX4, and XRCC2 displayed scores in the range of the already known FA proteins. Likewise, a prime candidate FA gene based on next generation sequencing and having a very low score was subsequently disproven by functional studies for the FA phenotype. Furthermore, the approach strongly enriches for GO terms such as DNA repair, response to DNA damage stimulus, and cell cycle-regulated genes. Additionally, overlaying the top 150 with a haploinsufficiency probability score, renders the approach more tailored for identifying breast cancer related genes. This approach may be useful for prioritization of putative novel FA or breast cancer genes from next generation sequencing efforts.     

The mouse secreted gel-forming mucin gene cluster

Using genomic cosmid and BAC clones and genome shotgun supercontigs available in GenBank, we determined the complete gene structure of the four mouse secreted gel-forming mucin genes Muc2, Muc5ac, Muc5b and Muc6 and the organization of the genomic locus harboring these genes. The mouse secreted gel-forming mucin gene is 215 kb on distal chromosome 7 to 69.0 cM from the centromere and organized as: Muc6-Muc2-Muc5ac-Muc5b with Muc2, Muc5ac and Muc5b arranged in the same orientation and Muc6 in opposite. Mouse mucin genes have highly similar genomic organization to each other and to their respective human homologues indicating that they have been well conserved through evolution. Deduced peptides showed striking sequence similarities in their N- and C-terminal regions whereas the threonine/serine/proline-rich central region is specific for each other and for species. Expression studies also showed that they have expression patterns similar to human mucin genes with Muc2 expressed in small and large intestines, Muc5ac and Muc6 in stomach, and Muc5b in laryngo-tracheal tract. These data constitute an important initial step for investigation of mucin gene regulation and mucin function through the use of animal models.