Extensive double humanization of both liver and hematopoiesis in FRGN mice.

Preclinical research in animals often fails to adequately predict the outcomes observed in human patients. Chimeric animals bearing individual human tissues have been developed to provide improved models of human-specific cellular processes. Mice transplanted with human hematopoietic stem cells can be used to study human immune responses, infections of blood cells and processes of hematopoiesis. Animals with humanized livers are useful for modeling hepatotropic infections as well as drug metabolism and hepatotoxicity. However, many pathophysiologic processes involve both the liver and the hematolymphoid system. Examples include hepatitis C/HIV co-infection, immune mediated liver diseases, liver injuries with inflammation such as steatohepatitis and alcoholic liver disease. We developed a robust protocol enabling the concurrent double-humanization of mice with mature hepatocytes and human blood. Immune-deficient, fumarylacetoacetate hydrolase (Fah(-/-)), Rag2(-/-) and Il2rg(-/-) deficient animals on the NOD-strain background (FRGN) were simultaneously co-transplanted with adult human hepatocytes and hematopoietic stem cells after busulfan and Ad:uPA pre-conditioning. Four months after transplantation the average human liver repopulation exceeded 80% and hematopoietic chimerism also was high (40-80% in bone marrow). Importantly, human macrophages (Kupffer cells) were present in the chimeric livers. Double-chimeric FRGN mice will serve as a new model for disease processes that involve interactions between hepatocytes and hematolymphoid cells.

Ochronosis in a murine model of alkaptonuria is synonymous to that in the human condition.

OBJECTIVE: Alkaptonuria (AKU) is a rare genetic disease which results in severe early onset osteoarthropathy. It has recently been shown that the subchondral interface is of key significance in disease pathogenesis. Human surgical tissues are often beyond this initial stage and there is no published murine model of pathogenesis, to study the natural history of the disease. The murine genotype exists but it has been reported not to demonstrate ochronotic osteoarthropathy consistent with the human disease. Recent anecdotal evidence of macroscopic renal ochronosis in a mouse model of tyrosinaemia led us to perform histological analysis of tissues of these mice that are known to be affected in human AKU. DESIGN: The homogentisate 1,2-dioxygenase Hgd(+/)(-)Fah(-)(/)(-) mouse can model either hereditary tyrosinaemia type I (HT1) or AKU depending on selection conditions. Mice having undergone Hgd reversion were sacrificed at various time points, and their tissues taken for histological analysis. Sections were stained with haematoxylin eosin (H&E) and Schmorl's reagent. RESULTS: Early time point observations at 8 months showed no sign of macroscopic ochronosis of tissues. Macroscopic examination at 13 months revealed ochronosis of the kidneys. Microscopic analysis of the kidneys revealed large pigmented nodules displaying distinct ochre colouration. Close microscopic examination of the distal femur and proximal fibula at the subchondral junctions revealed the presence of numerous pigmented chondrocytes. CONCLUSIONS: Here we present the first data showing ochronosis of tissues in a murine model of AKU. These preliminary histological observations provide a stimulus for further studies into the natural history of the disease to provide a greater understanding of this class of arthropathy.

Transcriptomes of the major human pancreatic cell types.

AIMS/HYPOTHESIS: We sought to determine the mRNA transcriptome of all major human pancreatic endocrine and exocrine cell subtypes, including human alpha, beta, duct and acinar cells. In addition, we identified the cell type-specific distribution of transcription factors, signalling ligands and their receptors. METHODS: Islet samples from healthy human donors were enzymatically dispersed to single cells and labelled with cell type-specific surface-reactive antibodies. Live endocrine and exocrine cell subpopulations were isolated by FACS and gene expression analyses were performed using microarray analysis and quantitative RT-PCR. Computational tools were used to evaluate receptor-ligand representation in these populations. RESULTS: Analysis of the transcriptomes of alpha, beta, large duct, small duct and acinar cells revealed previously unrecognised gene expression patterns in these cell types, including transcriptional regulators HOPX and HDAC9 in the human beta cell population. The abundance of some regulatory proteins was different from that reported in mouse tissue. For example, v-maf musculoaponeurotic fibrosarcoma oncogene homologue B (avian) (MAFB) was detected at equal levels in adult human alpha and beta cells, but is absent from adult mouse beta cells. Analysis of ligand-receptor interactions suggested that EPH receptor-ephrin communication between exocrine and endocrine cells contributes to pancreatic function. CONCLUSIONS/INTERPRETATION: This is the first comprehensive analysis of the transcriptomes of human exocrine and endocrine pancreatic cell types-including beta cells-and provides a useful resource for diabetes research. In addition, paracrine signalling pathways within the pancreas are shown. These results will help guide efforts to specify human beta cell fate by embryonic stem cell or induced pluripotent stem cell differentiation or genetic reprogramming.

Topo IIIalpha and BLM act within the Fanconi anemia pathway in response to DNA-crosslinking agents.

The Bloom protein (BLM) and Topoisomerase IIIalpha are found in association with proteins of the Fanconi anemia (FA) pathway, a disorder manifesting increased cellular sensitivity to DNA crosslinking agents. In order to determine if the association reflects a functional interaction for the maintenance of genome stability, we have analyzed the effects of siRNA-mediated depletion of the proteins in human cells. Depletion of Topoisomerase IIIalpha or BLM leads to increased radial formation, as is seen in FA. BLM and Topoisomerase IIIalpha are epistatic to the FA pathway for suppression of radial formation in response to DNA interstrand crosslinks since depletion of either of them in FA cells does not increase radial formation. Depletion of Topoisomerase IIIalpha or BLM also causes an increase in sister chromatid exchanges, as is seen in Bloom syndrome cells. Human Fanconi anemia cells, however, do not demonstrate increased sister chromatid exchanges, separating this response from radial formation. Primary cell lines from mice defective in both Blm and Fancd2 have the same interstrand crosslink-induced genome instability as cells from mice deficient in the Fancd2 protein alone. These observations demonstrate that the association of BLM and Topoisomerase IIIalpha with Fanconi proteins is a functional one, delineating a BLM-Topoisomerase IIIalpha-Fanconi pathway that is critical for suppression of chromosome radial formation.

The clinical picture of the Börjeson-Forssman-Lehmann syndrome in males and heterozygous females with PHF6 mutations.

The usual description of the Börjeson-Forssman-Lehmann syndrome (BFLS) is that of a rare, X-linked, partially dominant condition with severe intellectual disability, epilepsy, microcephaly, coarse facial features, long ears, short stature, obesity, gynecomastia, tapering fingers, and shortened toes. Recently, mutations have been identified in the PHF6 gene in nine families with this syndrome. The clinical history and physical findings in the affected males reveal that the phenotype is milder and more variable than previously described and evolves with age. Generally, in the first year, the babies are floppy, with failure to thrive, big ears, and small external genitalia. As schoolboys, the picture is one of learning problems, moderate short stature, with emerging truncal obesity and gynecomastia. Head circumferences are usually normal, and macrocephaly may be seen. Big ears and small genitalia remain. The toes are short and fingers tapered and malleable. In late adolescence and adult life, the classically described heavy facial appearance emerges. Some heterozygous females show milder clinical features such as tapering fingers and shortened toes. Twenty percent have significant learning problems, and 95% have skewed X inactivation. We conclude that this syndrome may be underdiagnosed in males in their early years and missed altogether in isolated heterozygous females.

Focal neurometabolic alterations in mice deficient for succinate semialdehyde dehydrogenase.

Metabolite profiling in succinate semialdehyde dehydrogenase (SSADH; Aldh5a1-/-) deficient mice previously revealed elevated gamma-hydroxybutyrate (GHB) and total GABA in urine and total brain and liver extracts. In this study, we extend our metabolic characterization of these mutant mice by documenting elevated GHB and total GABA in homogenates of mutant kidney, pancreas and heart. We quantified beta-alanine (a GABA homolog and putative neurotransmitter) to address its potential role in pathophysiology. We found normal levels of beta-alanine in urine and total homogenates of mutant brain, heart and pancreas, but elevated concentrations in mutant kidney and liver extracts. Amino acid analysis in mutant total brain homogenates revealed no abnormalities except for significantly decreased glutamine, which was normal in mutant liver and kidney extracts. Regional amino acid analysis (frontal cortex, parietal cortex, hippocampus and cerebellum) in mutant mice confirmed glutamine results. Glutamine synthetase protein and mRNA levels in homogenates of mutant mouse brain were normal. We profiled organic acid patterns in mutant brain homogenates to assess brain oxidative metabolism and found normal concentrations of Kreb's cycle intermediates but increased 4,5-dihydroxyhexanoic acid (a postulated derivative of succinic semialdehyde) levels. We conclude that SSADH-deficient mice represent a valid metabolic model of human SSADH deficiency, manifesting focal neurometabolic abnormalities which could provide key insights into pathophysiologic mechanisms.

Long-term therapy with NTBC and tyrosine-restricted diet in a murine model of hereditary tyrosinemia type I.

In human patients with hereditary tyrosinemia type I (HT1) a combination therapy of 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3 cyclohexane dione (NTBC) and dietary restriction of phenylalanine and tyrosine is currently widely used. We previously reported that the use of NTBC in a murine model of HT1 abolished acute liver failure but did not prevent the development of hepatocellular carcinoma (HCC) in the setting of nonrestricted protein intake. Here we present the results obtained with higher doses of NTBC plus dietary tyrosine restriction on long-term follow up (>2 years). Liver function tests and succinylacetone levels were completely corrected with this regimen and cancer-free survival was improved when compared to historical controls. However, while no HT1 animals had HCC at age 13 months, the incidence was 2/16 (13%) at age 18 months and 1/6 (17%) after 24 months. Thus, even the most stringent therapy could not prevent the emergence of HCC in the mouse model of HT1, even when initiated prenatally.

The pathophysiology and treatment of hereditary tyrosinemia type 1.

The topic of this review is hepatorenal tyrosinemia (hereditary tyrosinemia type 1 [HT1], or fumarylacetoacetate hydrolase deficiency; OMIM# 276700). HT1 is the most serious and common of the genetic defects in tyrosine degradation. In addition, this disorder has importance as a model of spontaneous self-correction of liver disease, as a model of liver repopulation by transplanted cells and gene therapy, and as a genetic cause of hepatocarcinoma. However, other forms of hypertyrosinemia exist; hence, the differential diagnosis also will be described briefly. Recent years have seen much progress in our understanding of the molecular basis, the pathophysiology, and especially the treatment of HT1. The current intervention with 2-(2-nitro-4-trifluoro-methylbenzyol)-1,3 cyclohexanedione (NTBC) therapy has improved the outcome of this once devastating disorder. The successful repopulation of the HT1 liver with transplanted cells and positive results in the use of gene therapy in animal models may someday lead to therapy in humans that will obviate the need for life-long dietary and pharmacological therapy.

The 4N cell cycle delay in Fanconi anemia reflects growth arrest in late S phase.

Fanconi anemia (FA) is a human genetic disorder characterized by hypersensitivity to DNA crosslinking agents. Its cellular phenotypes include increased chromosome breakage and a marked cell-cycle delay with 4N DNA content after introduction of interstrand DNA crosslinks (ICL). To further understand the nature of this delay previously described as a G2/M arrest, we introduced ICL specifically during G2 and monitored the cells for passage into mitosis. Our results showed that, even at the highest doses, postreplication ICL produced neither G2/M arrest nor chromosome breakage in FA-A or FA-C cells. This suggests that, similar to wild-type cells, DNA replication is required to trigger both responses. Therefore, the 4N cell DNA content observed in FA cells after ICL treatment also represents incomplete DNA replication and arrest in late S phase. FA fibroblasts from complementation groups A and C were able to recover from the ICL-induced cell-cycle arrest, but took approximately 3 times longer than controls. These results indicate that the FA pathway is required for the efficient resolution of ICL-induced S-phase arrest.

Function of the Fanconi anemia pathway in Fanconi anemia complementation group F and D1 cells.

OBJECTIVE: Fanconi anemia (FA) is a human autosomal-recessive cancer susceptibility disorder characterized by multiple congenital abnormalities, progressive bone marrow failure, and cellular sensitivity to mitomycin C (MMC). FA has at least eight complementation groups (A, B, C, D1, D2, E, F, G), and six of the FA genes have been cloned. Several FA proteins, including FANCA, FANCC, FANCF, and FANCG, interact in a nuclear complex, and this complex is required for the activation (monoubiquitination) of the downstream FANCD2 protein. Activation of FANCD2 results in the assembly of FANCD2/BRCA1 foci. The aim of this study was to analyze the FA pathway in several FA patient-derived cell lines. MATERIALS AND METHODS: We generated an antibody to FANCF and analyzed FANCF expression in human lymphoblasts corresponding to all known FA subtypes. We systematically analyzed the FA pathway (FANCD2 monoubiquitination and assembly of FANCD2 nuclear foci) in patient-derived FA-F and FA-D1 cell lines. RESULTS: FANCF protein expression is normal in cells derived from all FA complementation groups except FA-F and does not vary during cell cycle progression. FANCF, but not FANCD2, is a component of the nuclear FA protein complex and appears to stabilize other subunits of the complex. FANCF is required for the monoubiquitination of the FANCD2 protein following ionizing radiation. FANCD2 is monoubiquitinated in FA-D1 cells, even though these cells are highly sensitive to MMC. CONCLUSIONS: The recently cloned FANCF protein is required for FANCD2 activation, and the yet uncloned FANCD1 protein functions further downstream or independently of the FA pathway.

Fanconi anemia and DNA repair.

Fanconi anemia (FA) is an autosomal recessive disorder caused by defects in at least eight distinct genes FANCA, B, C, D1, D2, E, F and G. The clinical phenotype of all FA complementation groups is similar and is characterized by progressive bone marrow failure, cancer proneness and typical birth defects. The principal cellular phenotype is hypersensitivity to DNA damage, particularly interstrand DNA crosslinks. The FA proteins constitute a multiprotein pathway whose precise biochemical function(s) remain unknown. Five of the FA proteins (FANCA, C, E, F and G) interact in a nuclear complex upstream of FANCD2. FANCB and FANCD1 have not yet been cloned, but it is likely that FANCB is part of the nuclear complex and that FANCD1 acts downstream of FANCD2. The FA nuclear complex regulates the mono-ubiquitination of FANCD2 in response to DNA damage, resulting in targeting of this protein into nuclear foci. These foci also contain BRCA1 and other DNA damage response proteins. In male meiosis, FANCD2 also co-localizes with BRCA1 at synaptonemal complexes. Together, these data suggest that the FA pathway functions primarily as a DNA damage response system, although its exact role (direct involvement in DNA repair versus indirect, facilitating role) has not yet been defined.

Pharmacologic rescue of lethal seizures in mice deficient in succinate semialdehyde dehydrogenase.

Succinate semialdehyde dehydrogenase (ALDH5A1, encoding SSADH deficiency is a defect of 4-aminobutyric acid (GABA) degradation that manifests in humans as 4-hydroxybutyric (gamma-hydroxybutyric, GHB) aciduria. It is characterized by a non-specific neurological disorder including psychomotor retardation, language delay, seizures, hypotonia and ataxia. The current therapy, vigabatrin (VGB), is not uniformly successful. Here we report the development of Aldh5a1-deficient mice. At postnatal day 16-22 Aldh5a1-/- mice display ataxia and develop generalized seizures leading to rapid death. We observed increased amounts of GHB and total GABA in urine, brain and liver homogenates and detected significant gliosis in the hippocampus of Aldh5a1-/- mice. We found therapeutic intervention with phenobarbital or phenytoin ineffective, whereas intervention with vigabatrin or the GABAB receptor antagonist CGP 35348 (ref. 2) prevented tonic-clonic convulsions and significantly enhanced survival of the mutant mice. Because neurologic deterioration coincided with weaning, we hypothesized the presence of a protective compound in breast milk. Indeed, treatment of mutant mice with the amino acid taurine rescued Aldh5a1-/- mice. These findings provide insight into pathomechanisms and may have therapeutic relevance for the human SSADH deficiency disease and GHB overdose and toxicity.

Loss of p27(Kip1) enhances the transplantation efficiency of hepatocytes transferred into diseased livers.

p27(Kip1) is an important regulator of cyclin-dependent kinases. Studies with p27 knockout mice have revealed abnormalities in proliferation and differentiation of multiple cell types. Here we show that primary hepatocytes isolated from livers of adult p27 knockout mice exhibit higher levels of DNA synthesis activity in culture than do wild-type cells. Interestingly, we found that, compared with control hepatocytes, p27 knockout hepatocytes proliferate better after transplantation into diseased livers to reverse liver failure. These results reveal an aspect of p27 that could be used to benefit cell-based therapy.

Functional analysis of patient-derived mutations in the Fanconi anemia gene, FANCG/XRCC9.

OBJECTIVE: Fanconi anemia (FA) is an autosomal-recessive cancer susceptibility syndrome with seven complementation groups. Six of the FA genes have been cloned (corresponding to subtypes A, C, D2, E, F, and G) and the encoded proteins interact in a common pathway. Patient-derived mutations in FA genes have been helpful in delineating functional domains of FA proteins. The purpose of this work was to subtype FA patient-derived cell lines in our repository and to identify FA gene mutations. METHODS: We subtyped 62 FA patients as type A, G, C, or non-ACG by using a combination of retroviral gene transfer and immunoblot analysis. Among these FA patients, we identified six FA-G patients for further analysis. We used a strategy involving amplification of FANCG/XRCC9 exons and direct sequencing to identify novel FANCG mutations in cell lines derived from these FA-G patients. We functionally analyzed FANCG mutant alleles by transducing the corresponding cDNAs into a known FA-G indicator cell line and scoring correction of MMC sensitivity. RESULTS: Our results demonstrate a wide range of mutations in the FANCG gene (splice, nonsense, and missense mutations). Based on this mutational screen, a carboxy terminal functional domain of the FANCG protein appears to be required for complementation of FA-G cells and for normal assembly of the FANCA/FANCG/FANCC protein complex. CONCLUSION: The identification of patient-derived mutant alleles of FA genes can provide important insights to the function of FA proteins. FA subtyping is also a necessary precondition for gene therapy.

Liver repopulation for the treatment of metabolic diseases.

Orthotopic liver transplantation is the treatment of choice for several inborn errors of metabolism. Unfortunately, the supply of donor organs is limiting and therefore many patients cannot benefit from this therapy. In contrast, hepatocyte transplantation could potentially overcome the shortage in donor livers by use of cells from a single donor for multiple recipients. In classic hepatocyte transplantation, however, only 1% of the liver mass or less can be replaced by donor cells. Recently, though, it has been shown in animal models that >90% of host hepatocytes can be replaced by a small number of transplanted donor cells in a process we term 'therapeutic liver repopulation'. This phenomenon is analogous to repopulation of the haematopoietic system after bone marrow transplantation. Liver repopulation occurs when transplanted cells have a growth advantage in the setting of damage to recipient liver cells. It has been discovered that transplanted cells from extrahepatic sources such as the adult pancreas or bone marrow can also be used for liver repopulation. Because bone marrow donors are widely available, this finding raises the hope of therapeutic application of these cells in the future. Here, the current knowledge regarding therapeutic liver repopulation and the hopeful implications for treatment of liver diseases will be discussed.

Positional cloning of a novel Fanconi anemia gene, FANCD2.

Fanconi anemia (FA) is a genetic disease with birth defects, bone marrow failure, and cancer susceptibility. To date, genes for five of the seven known complementation groups have been cloned. Complementation group D is heterogeneous, consisting of two distinct genes, FANCD1 and FANCD2. Here we report the positional cloning of FANCD2. The gene consists of 44 exons, encodes a novel 1451 amino acid nuclear protein, and has two protein isoforms. Similar to other FA proteins, the FANCD2 protein has no known functional domains, but unlike other known FA genes, FANCD2 is highly conserved in A. thaliana, C. elegans, and Drosophila. Retroviral transduction of the cloned FANCD2 cDNA into FA-D2 cells resulted in functional complementation of MMC sensitivity.

Interaction of the Fanconi anemia proteins and BRCA1 in a common pathway.

Fanconi anemia (FA) is a human autosomal recessive cancer susceptibility disorder characterized by cellular sensitivity to mitomycin C and ionizing radiation. Although six FA genes (for subtypes A, C, D2, E, F, and G) have been cloned, their relationship to DNA repair remains unknown. In the current study, we show that a nuclear complex containing the FANCA, FANCC, FANCF, and FANCG proteins is required for the activation of the FANCD2 protein to a monoubiquitinated isoform. In normal (non-FA) cells, FANCD2 is monoubiquitinated in response to DNA damage and is targeted to nuclear foci (dots). Activated FANCD2 protein colocalizes with the breast cancer susceptibility protein, BRCA1, in ionizing radiation-induced foci and in synaptonemal complexes of meiotic chromosomes. The FANCD2 protein, therefore, provides the missing link between the FA protein complex and the cellular BRCA1 repair machinery. Disruption of this pathway results in the cellular and clinical phenotype common to all FA subtypes.

Prenatal diagnosis of succinic semialdehyde dehydrogenase deficiency: increased accuracy employing DNA, enzyme, and metabolite analyses.

Inherited succinic semialdehyde dehydrogenase (SSADH; EC1.2.1.24; McKusick 271980) deficiency is a defect of GABA degradation which leads to accumulation of 4-hydroxybutyric acid (gamma-hydroxybutyric acid; GHB) in physiologic fluids of patients. Prenatal diagnosis (PND) was performed in three at-risk pregnancies employing combinations of: (1) reverse-transcription-polymerase chain reaction (RT-PCR) and genomic DNA amplification followed by sequencing using isolated leukocytes or cultured human lymphoblasts; (2) GHB quantitation in amniotic fluid; or (3) SSADH enzyme assay in chorionic villus (CV) and/or amniocytes. In two pregnancies, all analyses were concordant for prediction of disease status in the fetus. In the third case, enzyme activity in CV (deficient) and metabolite analysis in amniotic fluid (normal) were discordant. For clarification, mutation analysis was undertaken in CV, confirming heterozygosity for the mutation previously identified in the proband. We hypothesize that delayed transit time for shipment of CV between Greece and the United States (8 days) led to enhanced degradation of heterozygous SSADH enzyme activity. Our data demonstrate the importance of combined metabolite, enzyme, and DNA analysis for increased accuracy in the PND of SSADH deficiency.