Lentiviral Gene Therapy for Mucopolysaccharidosis II with Tagged Iduronate 2-Sulfatase Prevents Life-Threatening Pathology in Peripheral Tissues But Fails to Correct Cartilage.

Deficiency of iduronate 2-sulfatase (IDS) causes Mucopolysaccharidosis type II (MPS II), a lysosomal storage disorder characterized by systemic accumulation of glycosaminoglycans (GAGs), leading to a devastating cognitive decline and life-threatening respiratory and cardiac complications. We previously found that hematopoietic stem and progenitor cell-mediated lentiviral gene therapy (HSPC-LVGT) employing tagged IDS with insulin-like growth factor 2 (IGF2) or ApoE2, but not receptor-associated protein minimal peptide (RAP12x2), efficiently prevented brain pathology in a murine model of MPS II. In this study, we report on the effects of HSPC-LVGT on peripheral pathology and we analyzed IDS biodistribution. We found that HSPC-LVGT with all vectors completely corrected GAG accumulation and lysosomal pathology in liver, spleen, kidney, tracheal mucosa, and heart valves. Full correction of tunica media of the great heart vessels was achieved only with IDS.IGF2co gene therapy, while the other vectors provided near complete (IDS.ApoE2co) or no (IDSco and IDS.RAP12x2co) correction. In contrast, tracheal, epiphyseal, and articular cartilage remained largely uncorrected by all vectors tested. These efficacies were closely matched by IDS protein levels following HSPC-LVGT. Our results demonstrate the capability of HSPC-LVGT to correct pathology in tissues of high clinical relevance, including those of the heart and respiratory system, while challenges remain for the correction of cartilage pathology.

Tagged IDS causes efficient and engraftment-independent prevention of brain pathology during lentiviral gene therapy for Mucopolysaccharidosis type II.

Mucopolysaccharidosis type II (OMIM 309900) is a lysosomal storage disorder caused by iduronate 2-sulfatase (IDS) deficiency and accumulation of glycosaminoglycans, leading to progressive neurodegeneration. As intravenously infused enzyme replacement therapy cannot cross the blood-brain barrier (BBB), it fails to treat brain pathology, highlighting the unmet medical need to develop alternative therapies. Here, we test modified versions of hematopoietic stem and progenitor cell (HSPC)-mediated lentiviral gene therapy (LVGT) using IDS tagging in combination with the ubiquitous MND promoter to optimize efficacy in brain and to investigate its mechanism of action. We find that IDS tagging with IGF2 or ApoE2, but not RAP12x2, improves correction of brain heparan sulfate and neuroinflammation at clinically relevant vector copy numbers. HSPC-derived cells engrafted in brain show efficiencies highest in perivascular areas, lower in choroid plexus and meninges, and lowest in parenchyma. Importantly, the efficacy of correction was independent of the number of brain-engrafted cells. These results indicate that tagged versions of IDS can outperform untagged IDS in HSPC-LVGT for the correction of brain pathology in MPS II, and they imply both cell-mediated and tag-mediated correction mechanisms, including passage across the BBB and increased uptake, highlighting their potential for clinical translation.

A combinatorial panel for flow cytometry-based isolation of enteric nervous system cells from human intestine.

Efficient isolation of neurons and glia from the human enteric nervous system (ENS) is challenging because of their rare and fragile nature. Here, we describe a staining panel to enrich ENS cells from the human intestine by fluorescence-activated cell sorting (FACS). We find that CD56/CD90/CD24 co-expression labels ENS cells with higher specificity and resolution than previous methods. Surprisingly, neuronal (CD24, TUBB3) and glial (SOX10) selective markers appear co-expressed by all ENS cells. We demonstrate that this contradictory staining pattern is mainly driven by neuronal fragments, either free or attached to glial cells, which are the most abundant cell types. Live neurons can be enriched by the highest CD24 and CD90 levels. By applying our protocol to isolate ENS cells for single-cell RNA sequencing, we show that these cells can be obtained with high quality, enabling interrogation of the human ENS transcriptome. Taken together, we present a selective FACS protocol that allows enrichment and discrimination of human ENS cells, opening up new avenues to study this complex system in health and disease.

Lysosomal glycogen accumulation in Pompe disease results in disturbed cytoplasmic glycogen metabolism.

Pompe disease is an inherited metabolic myopathy caused by deficiency of acid alpha-glucosidase (GAA), resulting in lysosomal glycogen accumulation. Residual GAA enzyme activity affects disease onset and severity, although other factors, including dysregulation of cytoplasmic glycogen metabolism, are suspected to modulate the disease course. In this study, performed in mice and patient biopsies, we found elevated protein levels of enzymes involved in glucose uptake and cytoplasmic glycogen synthesis in skeletal muscle from mice with Pompe disease, including glycogenin (GYG1), glycogen synthase (GYS1), glucose transporter 4 (GLUT4), glycogen branching enzyme 1 (GBE1), and UDP-glucose pyrophosphorylase (UGP2). Expression levels were elevated before the loss of muscle mass and function. For first time, quantitative mass spectrometry in skeletal muscle biopsies from five adult patients with Pompe disease showed increased expression of GBE1 protein relative to healthy controls at the group level. Paired analysis of individual patients who responded well to treatment with enzyme replacement therapy (ERT) showed reduction of GYS1, GYG1, and GBE1 in all patients after start of ERT compared to baseline. These results indicate that metabolic changes precede muscle wasting in Pompe disease, and imply a positive feedforward loop in Pompe disease, in which lysosomal glycogen accumulation promotes cytoplasmic glycogen synthesis and glucose uptake, resulting in aggravation of the disease phenotype.

Cellular Origin(s) of Congenital Diaphragmatic Hernia.

Congenital diaphragmatic hernia (CDH) is a structural birth defect characterized by a diaphragmatic defect, lung hypoplasia and structural vascular defects. In spite of recent developments, the pathogenesis of CDH is still poorly understood. CDH is a complex congenital disorder with multifactorial etiology consisting of genetic, cellular and mechanical factors. This review explores the cellular origin of CDH pathogenesis in the diaphragm and lungs and describes recent developments in basic and translational CDH research.

An in vitro assay to quantify satellite cell activation using isolated mouse myofibers.

Isolated myofibers offer the possibility of in vitro study of satellite cells in their niche. We describe a mouse myofiber isolation assay to assess satellite cell activation by quantifying myofiber-derived satellite cell progeny. The assay allows isolation of myofibers from a mouse using standard equipment and reagents. It can be used to compare satellite cells across different mouse models or to evaluate their response to treatments, offering a valuable complementary tool for in vitro experimentation.

Cell Surface Glycoprotein CD24 Marks Bone Marrow-Derived Human Mesenchymal Stem/Stromal Cells with Reduced Proliferative and Differentiation Capacity In Vitro.

Bone marrow-derived mesenchymal stem/stromal cells (BMSCs) are fundamental to bone regenerative therapies, tissue engineering, and postmenopausal osteoporosis. Donor variation among patients, cell heterogeneity, and unpredictable capacity for differentiation reduce effectiveness of BMSCs for regenerative cell therapies. The cell surface glycoprotein CD24 exhibits the most prominent differential expression during osteogenic versus adipogenic differentiation of human BMSCs. Therefore, CD24 may represent a selective biomarker for subpopulations of BMSCs with increased osteoblastic potential. In undifferentiated human BMSCs, CD24 cell surface expression is variable among donors (range: 2%-10%) and increased by two to fourfold upon osteogenic differentiation. Strikingly, FACS sorted CD24pos cells exhibit delayed mineralization and reduced capacity for adipocyte differentiation. RNAseq analysis of CD24pos and CD24neg BMSCs identified a limited number of genes with increased expression in CD24pos cells that are associated with cell adhesion, motility, and extracellular matrix. Downregulated genes are associated with cell cycle regulation, and biological assays revealed that CD24pos cells have reduced proliferation. Hence, expression of the cell surface glycoprotein CD24 identifies a subpopulation of human BMSCs with reduced capacity for proliferation and extracellular matrix mineralization. Functional specialization among BMSCs populations may support their regenerative potential and therapeutic success by accommodating cell activities that promote skeletal tissue formation, homeostasis, and repair.

Zebrafish macrophage developmental arrest underlies depletion of microglia and reveals Csf1r-independent metaphocytes.

Macrophages derive from multiple sources of hematopoietic progenitors. Most macrophages require colony-stimulating factor 1 receptor (CSF1R), but some macrophages persist in the absence of CSF1R. Here, we analyzed mpeg1:GFP-expressing macrophages in csf1r-deficient zebrafish and report that embryonic macrophages emerge followed by their developmental arrest. In larvae, mpeg1+ cell numbers then increased showing two distinct types in the skin: branched, putative Langerhans cells, and amoeboid cells. In contrast, although numbers also increased in csf1r-mutants, exclusively amoeboid mpeg1+ cells were present, which we showed by genetic lineage tracing to have a non-hematopoietic origin. They expressed macrophage-associated genes, but also showed decreased phagocytic gene expression and increased epithelial-associated gene expression, characteristic of metaphocytes, recently discovered ectoderm-derived cells. We further demonstrated that juvenile csf1r-deficient zebrafish exhibit systemic macrophage depletion. Thus, csf1r deficiency disrupts embryonic to adult macrophage development. Zebrafish deficient for csf1r are viable and permit analyzing the consequences of macrophage loss throughout life.

Immune cells called macrophages are found in all organs in the body. These cells are highly effective at eating and digesting large particles including dead cells and debris, and microorganisms such as bacteria. Macrophages are also instrumental in shaping developing organs and repairing tissues during life. Macrophages were, until recently, thought to be constantly replenished from cells circulating in the bloodstream. However, it turns out that separate populations of macrophages become established in most tissues during embryonic development and are maintained throughout life without further input. Previous studies of zebrafish, rodents and humans have shown that, when a gene called CSF1R is non-functional, macrophages are absent from many organs including the brain. However, some tissue-specific macrophages still persist, and it was not clear why these cells do not rely on the CSF1R gene while others do. Kuil et al. set out to decipher the precise requirement for the CSF1R gene in macrophage development in living zebrafish. The experiments used zebrafish that make a green fluorescent protein in their macrophages. As these fish are transparent, this meant that Kuil et al. could observe the cells within the living fish and isolate them to determine which genes are switched on and off. This approach revealed that zebrafish with a mutated version of the CSF1R gene make macrophages as embryos but that these cells then fail to multiply and migrate into the developing organs. This results in fewer macrophages in the zebrafish's tissues, and an absence of these cells in the brain. Kuil et al. went on to show that new macrophages did emerge in zebrafish that were about two to three weeks old. However, unexpectedly, these new cells were not regular macrophages. Instead, they were a new recently identified cell-type called metaphocytes, which share similarities with macrophages but have a completely different origin, move faster and do not eat particles. Zebrafish lacking the CSF1R gene thus lose nearly all their macrophages but retain metaphocytes. These macrophage-free mutant zebrafish constitute an unprecedented tool for further studies looking to discriminate the different roles of macrophages and metaphocytes.

Comparison of the functional and structural characteristics of rare TSC2 variants with clinical and genetic findings.

The TSC1 and TSC2 gene products interact to form the tuberous sclerosis complex (TSC), an important negative regulator of the mechanistic target of rapamycin complex 1 (TORC1). Inactivating mutations in TSC1 or TSC2 cause TSC, and the identification of a pathogenic TSC1 or TSC2 variant helps establish a diagnosis of TSC. However, it is not always clear whether TSC1 and TSC2 variants are inactivating. To determine whether TSC1 and TSC2 variants of uncertain clinical significance affect TSC complex function and cause TSC, in vitro assays of TORC1 activity can be employed. Here we combine genetic, functional, and structural approaches to try and classify a series of 15 TSC2 VUS. We investigated the effects of the variants on the formation of the TSC complex, on TORC1 activity and on TSC2 pre-mRNA splicing. In 13 cases (87%), the functional data supported the hypothesis that the identified TSC2 variant caused TSC. Our results illustrate the benefits and limitations of functional testing for TSC.

Restoring the regenerative balance in neuromuscular disorders: satellite cell activation as therapeutic target in Pompe disease.

Skeletal muscle is capable of efficiently regenerating after damage in a process mediated by tissue-resident stem cells called satellite cells. This regenerative potential is often compromised under muscle-degenerative conditions. Consequently, the damage produced during degeneration is not efficiently repaired and the balance between repair and damage is lost. Here we review recent progress on the role of satellite cell-mediated repair in neuromuscular disorders with a focus on Pompe disease, an inherited metabolic myopathy caused by deficiency of the lysosomal enzyme acid alpha glucosidase (GAA). Studies performed in patient biopsies as well as in Pompe disease mouse models demonstrate that muscle regeneration activity is compromised despite progressing muscle damage. We describe disease-specific mechanisms of satellite cell dysfunction to highlight the differences between Pompe disease and muscle dystrophies. The mechanisms involved provide possible targets for therapy, such as modulation of autophagy, muscle exercise, and pharmacological modulation of satellite cell activation. Most of these approaches are still experimental, although promising in animal models, still warrant caution with respect to their safety and efficiency profile.

Heterogeneous clinical phenotypes and cerebral malformations reflected by rotatin cellular dynamics.

Recessive mutations in RTTN, encoding the protein rotatin, were originally identified as cause of polymicrogyria, a cortical malformation. With time, a wide variety of other brain malformations has been ascribed to RTTN mutations, including primary microcephaly. Rotatin is a centrosomal protein possibly involved in centriolar elongation and ciliogenesis. However, the function of rotatin in brain development is largely unknown and the molecular disease mechanism underlying cortical malformations has not yet been elucidated. We performed both clinical and cell biological studies, aimed at clarifying rotatin function and pathogenesis. Review of the 23 published and five unpublished clinical cases and genomic mutations, including the effect of novel deep intronic pathogenic mutations on RTTN transcripts, allowed us to extrapolate the core phenotype, consisting of intellectual disability, short stature, microcephaly, lissencephaly, periventricular heterotopia, polymicrogyria and other malformations. We show that the severity of the phenotype is related to residual function of the protein, not only the level of mRNA expression. Skin fibroblasts from eight affected individuals were studied by high resolution immunomicroscopy and flow cytometry, in parallel with in vitro expression of RTTN in HEK293T cells. We demonstrate that rotatin regulates different phases of the cell cycle and is mislocalized in affected individuals. Mutant cells showed consistent and severe mitotic failure with centrosome amplification and multipolar spindle formation, leading to aneuploidy and apoptosis, which could relate to depletion of neuronal progenitors often observed in microcephaly. We confirmed the role of rotatin in functional and structural maintenance of primary cilia and determined that the protein localized not only to the basal body, but also to the axoneme, proving the functional interconnectivity between ciliogenesis and cell cycle progression. Proteomics analysis of both native and exogenous rotatin uncovered that rotatin interacts with the neuronal (non-muscle) myosin heavy chain subunits, motors of nucleokinesis during neuronal migration, and in human induced pluripotent stem cell-derived bipolar mature neurons rotatin localizes at the centrosome in the leading edge. This illustrates the role of rotatin in neuronal migration. These different functions of rotatin explain why RTTN mutations can lead to heterogeneous cerebral malformations, both related to proliferation and migration defects.

Satellite cells maintain regenerative capacity but fail to repair disease-associated muscle damage in mice with Pompe disease.

Pompe disease is a metabolic myopathy that is caused by glycogen accumulation as a result of deficiency of the lysosomal enzyme acid alpha glucosidase (GAA). Previously, we showed that adult muscle stem cells termed satellite cells are present at normal levels in muscle from patients with Pompe disease, but that these are insufficiently activated to repair the severe muscle pathology. Here we characterized the muscle regenerative response during disease progression in a mouse model of Pompe disease and investigated the intrinsic capacity of Gaa-/- satellite cells to regenerate muscle damage. Gaa-/- mice showed progressive muscle pathology from 15 weeks of age as reflected by increased lysosomal size, decreased fiber diameter and reduced muscle wet weight. Only during the first 15 weeks of life but not thereafter, we detected a gradual increase in centrally nucleated fibers and proliferating satellite cells in Gaa-/- muscle, indicating a mild regenerative response. The levels of Pax7-positive satellite cells were increased in Gaa-/- mice at all ages, most likely as result of enhanced satellite cell activation in young Gaa-/- animals. Surprisingly, both young and old Gaa-/- mice regenerated experimentally-induced muscle injury efficiently as judged by rapid satellite cell activation and complete restoration of muscle histology. In response to serial injury, Gaa-/- mice also regenerated muscle efficiently and maintained the satellite cell pool. These findings suggest that, similar to human patients, Gaa-/- mice have insufficient satellite cell activation and muscle regeneration during disease progression. The initial endogenous satellite cell response in Gaa-/- mice may contribute to the delayed onset of muscle wasting compared to human patients. The rapid and efficient regeneration after experimental muscle injury suggest that Gaa-/- satellite cells are functional stem cells, opening avenues for developing muscle regenerative therapies for Pompe disease.

Large-Scale Expansion of Human iPSC-Derived Skeletal Muscle Cells for Disease Modeling and Cell-Based Therapeutic Strategies.

Although skeletal muscle cells can be generated from human induced pluripotent stem cells (iPSCs), transgene-free protocols include only limited options for their purification and expansion. In this study, we found that fluorescence-activated cell sorting-purified myogenic progenitors generated from healthy controls and Pompe disease iPSCs can be robustly expanded as much as 5 × 1011-fold. At all steps during expansion, cells could be cryopreserved or differentiated into myotubes with a high fusion index. In vitro, cells were amenable to maturation into striated and contractile myofibers. Insertion of acid α-glucosidase cDNA into the AAVS1 locus in iPSCs using CRISPR/Cas9 prevented glycogen accumulation in myotubes generated from a patient with classic infantile Pompe disease. In vivo, the expression of human-specific nuclear and sarcolemmar antigens indicated that myogenic progenitors engraft into murine muscle to form human myofibers. This protocol is useful for modeling of skeletal muscle disorders and for using patient-derived, gene-corrected cells to develop cell-based strategies.

Elevated Plasma Cardiac Troponin T Levels Caused by Skeletal Muscle Damage in Pompe Disease.

BACKGROUND: Elevated plasma cardiac troponin T (cTnT) levels in patients with neuromuscular disorders may erroneously lead to the diagnosis of acute myocardial infarction or myocardial injury. METHODS AND RESULTS: In 122 patients with Pompe disease, the relationship between cTnT, cardiac troponin I, creatine kinase (CK), CK-myocardial band levels, and skeletal muscle damage was assessed. ECG and echocardiography were used to evaluate possible cardiac disease. Patients were divided into classic infantile, childhood-onset, and adult-onset patients. cTnT levels were elevated in 82% of patients (median 27 ng/L, normal values <14 ng/L). Cardiac troponin I levels were normal in all patients, whereas CK-myocardial band levels were increased in 59% of patients. cTnT levels correlated with CK levels in all 3 subgroups (P<0.001). None of the abnormal ECGs recorded in 21 patients were indicative of acute myocardial infarction, and there were no differences in cTnT levels between patients with and without (n=90) abnormalities on ECG (median 28 ng/L in both groups). The median left ventricular mass index measured with echocardiography was normal in all the 3 subgroups. cTnT mRNA expression in skeletal muscle was not detectable in controls but was strongly induced in patients with Pompe disease. cTnT protein was identified by mass spectrometry in patient-derived skeletal muscle tissue. CONCLUSIONS: Elevated plasma cTnT levels in patients with Pompe disease are associated with skeletal muscle damage, rather than acute myocardial injury. Increased cTnT levels in Pompe disease and likely other neuromuscular disorders should be interpreted with caution to avoid unnecessary cardiac interventions.

Lack of robust satellite cell activation and muscle regeneration during the progression of Pompe disease.

INTRODUCTION: Muscle stem cells termed satellite cells are essential for muscle regeneration. A central question in many neuromuscular disorders is why satellite cells are unable to prevent progressive muscle wasting. We have analyzed muscle fiber pathology and the satellite cell response in Pompe disease, a metabolic myopathy caused by acid alpha-glucosidase deficiency and lysosomal glycogen accumulation. Pathology included muscle fiber vacuolization, loss of cross striation, and immune cell infiltration. RESULTS: The total number of Pax7-positive satellite cells in muscle biopsies from infantile, childhood onset and adult patients (with different ages and disease severities) were indistinguishable from controls, indicating that the satellite cell pool is not exhausted in Pompe disease. Pax7/Ki67 double stainings showed low levels of satellite cell proliferation similar to controls, while MyoD and Myogenin stainings showed undetectable satellite cell differentiation. Muscle regenerative activity monitored with expression of embryonic Myosin Heavy Chain was weak in the rapidly progressing classic infantile form and undetectable in the more slowly progressive childhood and adult onset disease including in severely affected patients. CONCLUSIONS: These results imply that ongoing muscle wasting in Pompe disease may be explained by insufficient satellite cell activation and muscle regeneration. The preservation of the satellite cell pool may offer a venue for the development of novel treatment strategies directed towards the activation of endogenous satellite cells.

Silencing of SPRY1 triggers complete regression of rhabdomyosarcoma tumors carrying a mutated RAS gene.

RAS oncogenes are among the most frequently mutated genes in human cancer, but effective strategies for therapeutic inhibition of the RAS pathway have been elusive. Sprouty1 (SPRY1) is an upstream antagonist of RAS that is activated by extracellular signal-related kinase (ERK), providing a negative feedback loop for RAS signaling, and other evidence suggests that SPRY1 may have a tumor suppressor function. Studies of RAS status in the human childhood tumor rhabdomyosarcoma (RMS) indicated mutations in approximately half of the tumors of the embryonal rhabdomyosarcoma subtype (ERMS) but not the alveolar subtype (ARMS). ERMS tumors also showed overexpression of SPRY1, which was indeed upregulated by mutant RAS. However, we found that, in the presence of mutant RAS, the function of SPRY1 was changed from an antagonist to an agonist of RAS signaling. Thus, SPRY1 supported formation of activated ERK and mitogen-activated protein/ERK kinase and was essential for ERMS cell proliferation and survival. Conversely, silencing of SPRY1 in ERMS cells (but not ARMS cells) abolished their tumorigenicity in mice. Moreover, silencing of SPRY1 caused regression of established ERMS tumors (but not ARMS tumors) formed in xenograft settings. Our findings argue that SPRY1 inhibition can offer a therapeutic strategy to treat childhood RMS and possibly other tumors carrying oncogenic RAS mutations.

Genomic imbalances in rhabdomyosarcoma cell lines affect expression of genes frequently altered in primary tumors: an approach to identify candidate genes involved in tumor development.

Rhabdomyosarcomas (RMS) are the most common pediatric soft tissue sarcomas. They resemble developing skeletal muscle and are histologically divided into two main subtypes; alveolar and embryonal RMS. Characteristic genomic aberrations, including the PAX3- and PAX7-FOXO1 fusion genes in alveolar cases, have led to increased understanding of their molecular biology. Here, we determined the effect of genomic copy number on gene expression levels through array comparative genomic hybridization (CGH) analysis of 13 RMS cell lines, confirmed by multiplex ligation-dependent probe amplification copy number analyses, combined with their corresponding expression profiles. Genes altered at the transcriptional level by genomic imbalances were identified and the effect on expression was proportional to the level of genomic imbalance. Extrapolating to a public expression profiling dataset for 132 primary RMS identified features common to the cell lines and primary samples and associations with subtypes and fusion gene status. Genes identified such as CDK4 and MYCN are known to be amplified, overexpressed, and involved in RMS tumorigenesis. Of the many genes identified, those with likely functional relevance included CENPF, DTL, MYC, EYA2, and FGFR1. Copy number and expression of FGFR1 was validated in additional primary material and found amplified in 6 out of 196 cases and overexpressed relative to skeletal muscle and myoblasts, with significantly higher expression levels in the embryonal compared with alveolar subtypes. This illustrates the ability to identify genes of potential significance in tumor development through combining genomic and transcriptomic profiles from representative cell lines with publicly available expression profiling data from primary tumors.

Statistical comparison of two or more SAGE libraries: one tag at a time.

Several statistical tests have been introduced for the comparison of serial analysis of gene expression (SAGE) libraries to quantitatively analyze the differential expression of genes. As each SAGE library is only one measurement, the necessary information on biological variation or experimental precision is lacking. Therefore, each test includes its own approach to derive such a variance measure from the data set or a theoretical distribution. Because the confidence in tag counts depends on the library size, a test between two or more libraries should be based on original tag counts. When groups of libraries are compared, the test should determine that the proportion of a specific tag in all libraries is the same (null hypothesis), but also offer the possibility to detect specific differences between individual libraries and groups of libraries. The Z-test and the G-test encompass these characteristics and are described for the comparison of two libraries and (two or more) groups of libraries, respectively.

Scaling of gene expression data allowing the comparison of different gene expression platforms.

Serial analysis of gene expression (SAGE) and microarrays have found a widespread application, but much ambiguity exists regarding the amalgamation of the data resulting from these technologies. Cross-platform utilization of gene expression data from the SAGE and microarray technology could reduce the need for duplicate experiments and facilitate a more extensive exchange of data within the research community. This requires a measure for the correspondence of the results from different gene expression platforms. To date, a number of cross-platform evaluations (including a few studies using SAGE and Affymetrix GeneChips) have been conducted showing a variable, but overall low, concordance using different overall correlation approaches, such as Up/Down classification, contingency tables, and correlation coefficients. Here, we demonstrate an approach to compare two platforms based on the calculation of the difference between expression ratios observed in each platform for each individual transcript. This approach results in a concordance measure per gene, as opposed to the commonly used overall concordance measures between platforms. This between-ratio difference is a filtering-independent measure for between-platform concordance. Moreover, the between-ratio difference per gene can be used to identify transcripts with similar regulation on both platforms.