Publisher Correction: Therapies for rare diseases: therapeutic modalities, progress and challenges ahead.

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Therapies for rare diseases: therapeutic modalities, progress and challenges ahead.

Most rare diseases still lack approved treatments despite major advances in research providing the tools to understand their molecular basis, as well as legislation providing regulatory and economic incentives to catalyse the development of specific therapies. Addressing this translational gap is a multifaceted challenge, for which a key aspect is the selection of the optimal therapeutic modality for translating advances in rare disease knowledge into potential medicines, known as orphan drugs. With this in mind, we discuss here the technological basis and rare disease applicability of the main therapeutic modalities, including small molecules, monoclonal antibodies, protein replacement therapies, oligonucleotides and gene and cell therapies, as well as drug repurposing. For each modality, we consider its strengths and limitations as a platform for rare disease therapy development and describe clinical progress so far in developing drugs based on it. We also discuss selected overarching topics in the development of therapies for rare diseases, such as approval statistics, engagement of patients in the process, regulatory pathways and digital tools.

Recovery from anemia and leukocytopenia after abstinence in Japanese alcoholic men and their genetic polymorphisms of alcohol dehydrogenase-1B and aldehyde dehydrogenase-2.

BACKGROUND: The combination of the fast-metabolizing alcohol dehydrogenase-1B (ADH1B*2 allele) and inactive heterozygous aldehyde dehydrogenase-2 (ALDH2*1/*2) increases susceptibility to macrocytic anemia and leukocytopenia in alcoholics due to severe acetaldehydemia. More than half of Japanese drinkers with esophageal cancer have this genotype combination. METHODS: To assess the recovery of hematologic abnormalities after drinking cessation, changes in blood erythrocyte indices and leukocyte count during 8-week hospital stay were evaluated in 925 Japanese alcoholic men. We used four categories in ascending order for high blood acetaldehyde exposure from drinking: A, ADH1B*1/*1 plus ALDH2*1/*1; B, ADH1B*2 plus ALDH2*1/*1; C, ADH1B*1/*1 plus ALDH2*1/*2; and D, ADH1B*2 plus ALDH2*1/*2. RESULTS: Mean values of hemoglobin and hematocrit were the lowest, and those of mean corpuscular volume (MCV) were markedly the highest in the D group on admission, and returning toward normal after abstinence, but the inter-group differences remained significant throughout the 8 weeks. The mean leukocyte count was the lowest in the D group on admission, but increased during 4-week abstinence when the inter-group differences were no longer significant. Frequencies of MCV ≥110 fl (50.5%), hemoglobin levels <11.5 g/dL (32.7%), hemoglobin levels <10.0 g/dL (9.9%) and leukocytopenia <4000/µL (22.8%) were the highest in the D group on the admission day and decreased at the 4-week abstinence (28.7%, 18.8%, 4.0% and 7.9%, respectively). The inter-group differences in frequencies of the severe anemia and leukocytopenia disappeared after 4-week abstinence. CONCLUSIONS: Drinking cessation before surgery and/or chemoradiation treatment for esophageal cancer may be effective for recovery from anemia and leukocytopenia in drinkers belonging to the D group.

The cyclopurine deoxynucleosides: DNA repair, biological effects, mechanistic insights, and unanswered questions.

Patients with the genetic disease xeroderma pigmentosum (XP) who lack the capacity to carry out nucleotides excision repair (NER) have a dramatically elevated risk of skin cancer on sun exposed areas of the body. NER is the DNA repair mechanism responsible for the removal of DNA lesions resulting from ultraviolet light. In addition, a subset of XP patients develop a progressive neurodegenerative disease, referred to as XP neurologic disease, which is thought to be the result of accumulation of endogenous DNA lesions that are repaired by NER but not other repair pathways. The 8,5-cyclopurine deoxynucleotides (cyPu) have emerged as leading candidates for such lesions, in that they result from the reaction of the hydroxyl radical with DNA, are strong blocks to transcription in human cells, and are repaired by NER but not base excision repair. Here I present a focused perspective on progress into understating the repair and biological effects of these lesions. In doing so, I emphasize the role of Tomas Lindahl and his laboratory in stimulating cyPu research. I also include a critical evaluation of the evidence supporting a role for cyPu lesions in XP neurologic disease, with a focus on outstanding questions, and conceptual and technologic challenges.

Blood Leukocyte Counts and Genetic Polymorphisms of Alcohol Dehydrogenase-1B and Aldehyde Dehydrogenase-2 in Japanese Alcoholic Men.

BACKGROUND: Roughly 40% of East Asians have inactive aldehyde dehydrogenase-2 (ALDH2) encoded by the ALDH2*2 allele, and 90% have highly active alcohol dehydrogenase-1B (ADH1B) encoded by the ADH1B*2 allele. Macrocytosis and macrocytic anemia in alcoholics have been associated with ADH1B and ALDH2 gene variants which increase acetaldehyde (AcH) levels. METHODS: We investigated the relationship between ADH1B*2, ALDH2*2, and leukocyte counts of Japanese alcoholic men (N = 1,661). RESULTS: After adjusting for age, drinking habits, smoking habits, body mass index, presence of liver cirrhosis, and serum levels of C-reactive protein, we found that total and differential leukocyte counts were lower in the presence of the ALDH2*1/*2 genotype (vs. ALDH2*1/*1 genotype). ALDH2*2/*2 carriers were not found in our study population. Leukocyte, granulocyte, and monocyte counts were also lower in the presence of ADH1B*2 (vs. ADH1B*1/*1 genotype), but the lymphocyte count was higher. The ALDH2*1/*2 genotype was associated with leukocytopenia (<4,000/µl; adjusted odds ratio [95% confidence interval] = 1.89 [1.27 to 2.80]), granulocytopenia (<2,000/µl; 1.86 [1.22 to 2.82]), monocytopenia (<250/µl; 2.22 [1.49 to 3.29]), and lymphocytopenia (<1,000/µl; 1.93 [1.32 to 2.83]). In contrast, the ADH1B*2 had the opposite effect on lymphocytopenia (0.65 [0.46 to 0.93]). Considering genotype effects under conditions of immune stimulation, we observed suppressive effects of ADH1B*2 allele on leukocytosis (≥9,000/µl; 0.69 [0.50 to 0.97]), granulocytosis (≥6,500/µl; 0.66 [0.47 to 0.93]), and monocytosis (≥750/µl; 0.56 [0.39 to 0.79]). The ADH1B*2 plus ALDH2*1/*2 combination had the greatest suppressive effects on the leukocyte, granulocyte, and monocyte counts. CONCLUSIONS: The total and differential blood leukocyte counts of Japanese alcoholics were strongly affected by their ADH1B and ALDH2 gene variants. High AcH exposure levels probably play a critical role in the suppression of blood leukocyte counts in alcoholics.

The NIH Extracellular RNA Communication Consortium.

The Extracellular RNA (exRNA) Communication Consortium, funded as an initiative of the NIH Common Fund, represents a consortium of investigators assembled to address the critical issues in the exRNA research arena. The overarching goal is to generate a multi-component community resource for sharing fundamental scientific discoveries, protocols, and innovative tools and technologies. The key initiatives include (a) generating a reference catalogue of exRNAs present in body fluids of normal healthy individuals that would facilitate disease diagnosis and therapies, (b) defining the fundamental principles of exRNA biogenesis, distribution, uptake, and function, as well as development of molecular tools, technologies, and imaging modalities to enable these studies,

Mechanism of RNA polymerase II bypass of oxidative cyclopurine DNA lesions.

In human cells, the oxidative DNA lesion 8,5'-cyclo-2'-deoxyadenosine (CydA) induces prolonged stalling of RNA polymerase II (Pol II) followed by transcriptional bypass, generating both error-free and mutant transcripts with AMP misincorporated immediately downstream from the lesion. Here, we present biochemical and crystallographic evidence for the mechanism of CydA recognition. Pol II stalling results from impaired loading of the template base (5') next to CydA into the active site, leading to preferential AMP misincorporation. Such predominant AMP insertion, which also occurs at an abasic site, is unaffected by the identity of the 5'-templating base, indicating that it derives from nontemplated synthesis according to an A rule known for DNA polymerases and recently identified for Pol II bypass of pyrimidine dimers. Subsequent to AMP misincorporation, Pol II encounters a major translocation block that is slowly overcome. Thus, the translocation block combined with the poor extension of the dA.rA mispair reduce transcriptional mutagenesis. Moreover, increasing the active-site flexibility by mutation in the trigger loop, which increases the ability of Pol II to accommodate the bulky lesion, and addition of transacting factor TFIIF facilitate CydA bypass. Thus, blocking lesion entry to the active site, translesion A rule synthesis, and translocation block are common features of transcription across different bulky DNA lesions.

Implications of acetaldehyde-derived DNA adducts for understanding alcohol-related carcinogenesis.

Among various potential mechanisms that could explain alcohol carcinogenicity, the metabolism of ethanol to acetaldehyde represents an obvious possible mechanism, at least in some tissues. The fundamental principle of genotoxic carcinogenesis is the formation of mutagenic DNA adducts in proliferating cells. If not repaired, these adducts can result in mutations during DNA replication, which are passed on to cells during mitosis. Consistent with a genotoxic mechanism, acetaldehyde does react with DNA to form a variety of different types of DNA adducts. In this chapter we will focus more specifically on N2-ethylidene-deoxyguanosine (N2-ethylidene-dG), the major DNA adduct formed from the reaction of acetaldehyde with DNA and specifically highlight recent data on the measurement of this DNA adduct in the human body after alcohol exposure. Because results are of particular biological relevance for alcohol-related cancer of the upper aerodigestive tract (UADT), we will also discuss the histology and cytology of the UADT, with the goal of placing the adduct data in the relevant cellular context for mechanistic interpretation. Furthermore, we will discuss the sources and concentrations of acetaldehyde and ethanol in different cell types during alcohol consumption in humans. Finally, in the last part of the chapter, we will critically evaluate the concept of carcinogenic levels of acetaldehyde, which has been raised in the literature, and discuss how data from acetaldehyde genotoxicity are and can be utilized in physiologically based models to evaluate exposure risk.

Dysregulation of gene expression as a cause of Cockayne syndrome neurological disease.

Cockayne syndrome (CS) is a multisystem disorder with severe neurological symptoms. The majority of CS patients carry mutations in Cockayne syndrome group B (CSB), best known for its role in transcription-coupled nucleotide excision repair. Indeed, because various repair pathways are compromised in patient cells, CS is widely considered a genome instability syndrome. Here, we investigate the connection between the neuropathology of CS and dysregulation of gene expression. Transcriptome analysis of human fibroblasts revealed that even in the absence of DNA damage, CSB affects the expression of thousands of genes, many of which are neuronal genes. CSB is present in a significant subset of these genes, suggesting that regulation is direct, at the level of transcription. Importantly, reprogramming of CS fibroblasts to neuron-like cells is defective unless an exogenous CSB gene is introduced. Moreover, neuroblastoma cells from which CSB is depleted show defects in gene expression programs required for neuronal differentiation, and fail to differentiate and extend neurites. Likewise, neuron-like cells cannot be maintained without CSB. Finally, a number of disease symptoms may be explained by marked gene expression changes in the brain of patients with CS. Together, these data point to dysregulation of gene regulatory networks as a cause of the neurological symptoms in CS.

Macrocytosis, macrocytic anemia, and genetic polymorphisms of alcohol dehydrogenase-1B and aldehyde dehydrogenase-2 in Japanese alcoholic men.

BACKGROUND: Oxidation of ethanol by alcohol dehydrogenase (ADH) generates acetaldehyde (AcH), which is converted to acetate by aldehyde dehydrogenase-2 (ALDH2). Roughly 40% of East Asians are ALDH2-deficient due to an inactive enzyme encoded by the ALDH2*2 allele. ALDH2-deficient individuals have a dramatically elevated risk of esophageal cancer from alcohol consumption. METHODS: We investigated the relationship between ALDH2*2, ADH1B*2 (encoding a highly active ADH) and erythrocyte abnormalities, in a population of Japanese alcoholic men (N = 1,238). RESULTS: Macrocytosis (mean corpuscular volume [MCV] ≥100 fl) and macrocytic anemia (MCV ≥100 fl and hemoglobin <13.5 g/dl) were found in 62.4 and 24.1% of the subjects, respectively. Age-adjusted daily alcohol consumption did not differ according to ADH1B and ALDH2 genotypes. However, macrocytosis and macrocytic anemia were strongly associated with the ALDH2*1/*2 genotype multivariate odds ratios (ORs; 95% confidence interval [CI] = 2.85 [1.95 to 4.18] and 3.68 [2.64 to 5.15], respectively, versus ALDH2*1/*1). In comparison with the ADH1B*1/*1 and ALDH2*1/*1 genotype combination, the ADH1B*1/*1 and ALDH2*1/*2 genotype combination and the ADH1B*2 allele and ALDH2*1/*2 genotype combination increased stepwise the ORs (95% CI) for macrocytosis (1.65 [0.92 to 2.94] and 4.07 [2.33 to 7.11], respectively, p for difference in OR = 0.015) and macrocytic anemia (2.80 [1.52 to 5.15] and 5.32 [3.29 to 8.62], respectively, p for difference in OR = 0.045). Genotype effects were more prominent on the risks of the more advanced erythrocyte abnormalities. Older age, cigarette smoking, and low body mass index independently increased the risks of the erythrocyte abnormalities. Consumption of beer, which contains folate, decreased the risks, whereas consumption of alcoholic beverages lacking folate did not. CONCLUSIONS: These results suggest that the erythrocyte abnormalities in alcoholics are attributable to high AcH exposure as well as to nutritional deficiencies and may be prevented by folate.

Quantitative and in situ detection of oxidatively generated DNA damage 8,5'-cyclo-2'-deoxyadenosine using an immunoassay with a novel monoclonal antibody.

Xeroderma pigmentosum (XP) is a genetic disorder associated with defects in nucleotide excision repair, which eliminates a wide variety of helix-distorting types of DNA damage including sunlight-induced pyrimidine dimers. In addition to skin disease, approximately 30% of XP patients develop progressive neurological disease, which has been hypothesized to be associated with the accumulation of a particular type of oxidatively generated DNA damage called purine 8,5'-cyclo-2'-deoxynucleosides (purine cyclonucleosides). However, there are no currently available methods to detect purine cyclonucleosides in DNA without the need for DNA hydrolysis. In this study, we generated a novel monoclonal antibody (CdA-1) specific for purine cyclonucleosides in single-stranded DNA that recognizes 8,5'-cyclo-2'-deoxyadenosine (cyclo-dA). An immunoassay using CdA-1 revealed a linear dose response between known amounts of cyclo-dA in oligonucleotides and the antibody binding to them. The quantitative immunoassay revealed that treatment with Fenton-type reagents (CuCl(2)/H(2)O(2)/ascorbate) efficiently produces cyclo-dA in DNA in a dose-dependent manner. Moreover, immunofluorescent analysis using CdA-1 enabled the visualization of cyclo-dA in human osteosarcoma cells, which had been transfected with oligonucleotides containing cyclo-dA. Thus, the CdA-1 antibody is a valuable tool for the detection and quantification of cyclo-dA in DNA, and may be useful for characterizing the mechanism(s) underlying the development of XP neurological disease.

Acetaldehyde and the genome: beyond nuclear DNA adducts and carcinogenesis.

The designation of acetaldehyde associated with the consumption of alcoholic beverages as "carcinogenic to humans" (Group 1) by the International Agency for Research on Cancer (IARC) has brought renewed attention to the biological effects of acetaldehyde, as the primary oxidative metabolite of alcohol. Therefore, the overall focus of this review is on acetaldehyde and its direct and indirect effects on the nuclear and mitochondrial genome. We first consider different acetaldehyde-DNA adducts, including a critical assessment of the evidence supporting a role for acetaldehyde-DNA adducts in alcohol related carcinogenesis, and consideration of additional data needed to make a conclusion. We also review recent data on the role of the Fanconi anemia DNA repair pathway in protecting against acetaldehyde genotoxicity and carcinogenicity, as well as teratogenicity. We also review evidence from the older literature that acetaldehyde may impact the genome indirectly, via the formation of adducts with proteins that are themselves critically involved in the maintenance of genetic and epigenetic stability. Finally, we note the lack of information regarding acetaldehyde effects on the mitochondrial genome, which is notable since aldehyde dehydrogenase 2 (ALDH2), the primary acetaldehyde metabolic enzyme, is located in the mitochondrion, and roughly 30% of East Asian individuals are deficient in ALDH2 activity due to a genetic variant in the ALDH2 gene. In summary, a comprehensive understanding of all of the mechanisms by which acetaldehyde impacts the function of the genome has implications not only for alcohol and cancer, but types of alcohol related pathologies as well.

Insight into mechanisms of 3'-5' exonuclease activity and removal of bulky 8,5'-cyclopurine adducts by apurinic/apyrimidinic endonucleases.

8,5'-cyclo-2'-deoxyadenosine (cdA) and 8,5'-cyclo-2'-deoxyguanosine generated in DNA by both endogenous oxidative stress and ionizing radiation are helix-distorting lesions and strong blocks for DNA replication and transcription. In duplex DNA, these lesions are repaired in the nucleotide excision repair (NER) pathway. However, lesions at DNA strand breaks are most likely poor substrates for NER. Here we report that the apurinic/apyrimidinic (AP) endonucleases--Escherichia coli Xth and human APE1--can remove 5'S cdA (S-cdA) at 3' termini of duplex DNA. In contrast, E. coli Nfo and yeast Apn1 are unable to carry out this reaction. None of these enzymes can remove S-cdA adduct located at 1 or more nt away from the 3' end. To understand the structural basis of 3' repair activity, we determined a high-resolution crystal structure of E. coli Nfo-H69A mutant bound to a duplex DNA containing an α-anomeric 2'-deoxyadenosine:T base pair. Surprisingly, the structure reveals a bound nucleotide incision repair (NIR) product with an abortive 3'-terminal dC close to the scissile position in the enzyme active site, providing insight into the mechanism for Nfo-catalyzed 3'→5' exonuclease function and its inhibition by 3'-terminal S-cdA residue. This structure was used as a template to model 3'-terminal residues in the APE1 active site and to explain biochemical data on APE1-catalyzed 3' repair activities. We propose that Xth and APE1 may act as a complementary repair pathway to NER to remove S-cdA adducts from 3' DNA termini in E. coli and human cells, respectively.

Quantitative assessment of Tet-induced oxidation products of 5-methylcytosine in cellular and tissue DNA.

Recent studies showed that Ten-eleven translocation (Tet) family dioxygenases can oxidize 5-methyl-2'-deoxycytidine (5-mdC) in DNA to yield the 5-hydroxymethyl, 5-formyl and 5-carboxyl derivatives of 2'-deoxycytidine (5-HmdC, 5-FodC and 5-CadC). 5-HmdC in DNA may be enzymatically deaminated to yield 5-hydroxymethyl-2'-deoxyuridine (5-HmdU). After their formation at CpG dinucleotide sites, these oxidized pyrimidine nucleosides, particularly 5-FodC, 5-CadC, and 5-HmdU, may be cleaved from DNA by thymine DNA glycosylase, and subsequent action of base-excision repair machinery restores unmethylated cytosine. These processes are proposed to be important in active DNA cytosine demethylation in mammals. Here we used a reversed-phase HPLC coupled with tandem mass spectrometry (LC-MS/MS/MS) method, along with the use of stable isotope-labeled standards, for accurate measurements of 5-HmdC, 5-FodC, 5-CadC and 5-HmdU in genomic DNA of cultured human cells and multiple mammalian tissues. We found that overexpression of the catalytic domain of human Tet1 led to marked increases in the levels of 5-HmdC, 5-FodC and 5-CadC, but only a modest increase in 5-HmdU, in genomic DNA of HEK293T cells. Moreover, 5-HmdC is present at a level that is approximately 2-3 and 3-4 orders of magnitude greater than 5-FodC and 5-CadC, respectively, and 35-400 times greater than 5-HmdU in the mouse brain and skin, and human brain. The robust analytical method built a solid foundation for dissecting the molecular mechanisms of active cytosine demethylation, for measuring these 5-mdC derivatives and assessing their involvement in epigenetic regulation in other organisms and for examining whether these 5-mdC derivatives can be used as biomarkers for human diseases.

Gene therapy for rare diseases: summary of a National Institutes of Health workshop, September 13, 2012.

Gene therapy has shown clinical efficacy for several rare diseases, using different approaches and vectors. The Gene Therapy for Rare Diseases workshop, sponsored by the National Institutes of Health (NIH) Office of Biotechnology Activities and Office of Rare Diseases Research, brought together investigators from different disciplines to discuss the challenges and opportunities for advancing the field including means for enhancing data sharing for preclinical and clinical studies, development and utilization of available NIH resources, and interactions with the U.S. Food and Drug Administration.

Moderate alcohol consumption and breast cancer in women: from epidemiology to mechanisms and interventions.

Epidemiologic studies indicate that moderate alcohol consumption increases breast cancer risk in women. Understanding the mechanistic basis of this relationship has important implications for women's health and breast cancer prevention. In this commentary, we focus on some recent epidemiologic studies linking moderate alcohol consumption to breast cancer risk and place the results of those studies within the framework of our current understanding of the temporal and mechanistic basis of human carcinogenesis. This analysis supports the hypothesis that alcohol acts as a weak cumulative breast carcinogen and may also be a tumor promoter. We discuss the implications of these mechanisms for the prevention and treatment of alcohol-related breast cancer and present some considerations for future studies. Moderate alcohol consumption has been shown to benefit cardiovascular health and recently been associated with healthy aging. Therefore, a better understanding of how moderate alcohol consumption impacts breast cancer risk will allow women to make better informed decisions about the risks and benefits of alcohol consumption in the context of their overall health and at different stages of their life. Such mechanistic information is also important for the development of rational clinical interventions to reduce ethanol-related breast cancer mortality.