RNA therapeutics for the treatment of blood disorders.

Blood disorders are defined as diseases related to the structure, function, and formation of blood cells. These diseases lead to increased years of life loss, reduced quality of life, and increased financial burden for social security systems around the world. Common blood disorder treatments such as using chemical drugs, organ transplants, or stem cell therapy have not yet approached the best goals, and treatment costs are also very high. RNA with a research history dating back several decades has emerged as a potential method to treat hematological diseases. A number of clinical trials have been conducted to pave the way for the use of RNA molecules to cure blood disorders. This novel approach takes advantage of regulatory mechanisms and the versatility of RNA-based oligonucleotides to target genes and cellular pathways involved in the pathogenesis of specific diseases. Despite positive results, currently, there is no RNA drug to treat blood-related diseases approved or marketed. Before the clinical adoption of RNA-based therapies, challenges such as safe delivery of RNA molecules to the target site and off-target effects of injected RNA in the body need to be addressed. In brief, RNA-based therapies open novel avenues for the treatment of hematological diseases, and clinical trials for approval and practical use of RNA-targeted are crucial.

Assessment of a complete and classified platelet proteome from genome-wide transcripts of human platelets and megakaryocytes covering platelet functions.

Novel platelet and megakaryocyte transcriptome analysis allows prediction of the full or theoretical proteome of a representative human platelet. Here, we integrated the established platelet proteomes from six cohorts of healthy subjects, encompassing 5.2 k proteins, with two novel genome-wide transcriptomes (57.8 k mRNAs). For 14.8 k protein-coding transcripts, we assigned the proteins to 21 UniProt-based classes, based on their preferential intracellular localization and presumed function. This classified transcriptome-proteome profile of platelets revealed: (i) Absence of 37.2 k genome-wide transcripts. (ii) High quantitative similarity of platelet and megakaryocyte transcriptomes (R = 0.75) for 14.8 k protein-coding genes, but not for 3.8 k RNA genes or 1.9 k pseudogenes (R = 0.43-0.54), suggesting redistribution of mRNAs upon platelet shedding from megakaryocytes. (iii) Copy numbers of 3.5 k proteins that were restricted in size by the corresponding transcript levels (iv) Near complete coverage of identified proteins in the relevant transcriptome (log2fpkm > 0.20) except for plasma-derived secretory proteins, pointing to adhesion and uptake of such proteins. (v) Underrepresentation in the identified proteome of nuclear-related, membrane and signaling proteins, as well proteins with low-level transcripts. We then constructed a prediction model, based on protein function, transcript level and (peri)nuclear localization, and calculated the achievable proteome at ~ 10 k proteins. Model validation identified 1.0 k additional proteins in the predicted classes. Network and database analysis revealed the presence of 2.4 k proteins with a possible role in thrombosis and hemostasis, and 138 proteins linked to platelet-related disorders. This genome-wide platelet transcriptome and (non)identified proteome database thus provides a scaffold for discovering the roles of unknown platelet proteins in health and disease.

The defining DNA methylation signature of Kabuki syndrome enables functional assessment of genetic variants of unknown clinical significance.

Kabuki syndrome (KS) is caused by mutations in KMT2D, which is a histone methyltransferase involved in methylation of H3K4, a histone marker associated with DNA methylation. Analysis of >450,000 CpGs in 24 KS patients with pathogenic mutations in KMT2D and 216 controls, identified 24 genomic regions, along with 1,504 CpG sites with significant DNA methylation changes including a number of Hox genes and the MYO1F gene. Using the most differentiating and significant probes and regions we developed a "methylation variant pathogenicity (MVP) score," which enables 100% sensitive and specific identification of individuals with KS, which was confirmed using multiple public and internal patient DNA methylation databases. We also demonstrated the ability of the MVP score to accurately reclassify variants of unknown significance in subjects with apparent clinical features of KS, enabling its potential use in molecular diagnostics. These findings provide novel insights into the molecular etiology of KS and illustrate that DNA methylation patterns can be interpreted as 'epigenetic echoes' in certain clinical disorders.

Cross-species coherence in effects and modes of action in support of causality determinations in the U.S. Environmental Protection Agency's Integrated Science Assessment for Lead.

The peer-reviewed literature on the health and ecological effects of lead (Pb) indicates common effects and underlying modes of action across multiple organisms for several endpoints. Based on such observations, the United States (U.S.) Environmental Protection Agency (EPA) applied a cross-species approach in the 2013 Integrated Science Assessment (ISA) for Lead for evaluating the causality of relationships between Pb exposure and specific endpoints that are shared by humans, laboratory animals, and ecological receptors (i.e., hematological effects, reproductive and developmental effects, and nervous system effects). Other effects of Pb (i.e., cardiovascular, renal, and inflammatory responses) are less commonly assessed in aquatic and terrestrial wildlife limiting the application of cross-species comparisons. Determinations of causality in ISAs are guided by a framework for classifying the weight of evidence across scientific disciplines and across related effects by considering aspects such as biological plausibility and coherence. As illustrated for effects of Pb where evidence across species exists, the integration of coherent effects and common underlying modes of action can serve as a means to substantiate conclusions regarding the causal nature of the health and ecological effects of environmental toxicants.

Immunologic assessment and KMT2D mutation detection in Kabuki syndrome.

Kabuki or Niikawa-Kuroki syndrome (KS) is a rare disorder with multiple malformations and recurrent infections, especially otitis media. This study aimed to investigate the genetic defects in Kabuki syndrome and determine if immune status is related to recurrent otitis media. Fourteen patients from 12 unrelated families were enrolled in the 9-year study period (2005-2013). All had Kabuki faces, cleft palate, developmental delay, mental retardation, and the short fifth finger. Recurrent otitis media (12/14) and hearing impairment (8/14) were also more common features. Immunologic analysis revealed lower memory CD19+ cells (11/13), lower memory CD4+ cells (8/13), undetectable anti-HBs antibodies (7/13), and antibody deficiency (7/13), including lower IgA (4), IgG (2), and IgG2 (1). Naïve emigrant lymphocytes, lymphocyte proliferation function, complement activity, and superoxide production in polymorphonuclear cells were all normal. All the patients had KMT2D mutations and 10 novel mutations of R1252X, R1757X,Y1998C, P2550R fs2604X, Q4013X, G5379X, E5425K, R5432X, R5432W, and R5500W. Resembling the phenotype of common variable immunodeficiency, KS patients with antibody deficiency, decreased memory cells, and poor vaccine response increased susceptibility to recurrent otitis media. Large-scale prospective studies are warranted to determine if regular immunoglobulin supplementation decreases the frequency of otitis media and severity of hearing impairment.

Miniaturized FISH for screening of onco-hematological malignancies.

Fluorescence in situ hybridization (FISH) represents a major step in the analysis of chromosomal aberrations in cancer. It allows the precise detection of specific rearrangements, both for diagnostic and prognostic purposes. Here we present a miniaturized FISH method performed on fresh and fixed hematological samples. This procedure has been developed together with a microfluidic device that integrates cluster-assembled nanostructured TiO2 (ns-TiO2) as a nanomaterial promoting hematopoietic cell immobilization in conditions of shear stress. As a result of miniaturization, FISH can be performed with at least a 10-fold reduction in probe usage and minimal cell requirements, creating the possibility of using FISH in genetic screening applications. We developed the protocol on tumor cells and bone marrow (BM) from a normal donor using commercially sex-specific and onco-hematology probes. The procedure was then validated using either BM or peripheral blood (PB) from six patients with hematological diseases, each associated with different genetic lesions. Miniaturized FISH demonstrated comparable performance to standard FISH, indicating that it is suitable for genetic screenings, in research, and in clinical settings for the diagnosis of samples from onco-hematological malignancies.

Protein-tyrosine phosphatase, nonreceptor type 11 mutation analysis and clinical assessment in 45 patients with Noonan syndrome.

We report on PTPN11 (protein-tyrosine phosphatase, nonreceptor type 11) mutation analysis and clinical assessment in 45 patients with Noonan syndrome. Sequence analysis was performed for all of the coding exons 1-15 of PTPN11, revealing a novel 3-bp deletion mutation and 10 recurrent missense mutations in 18 patients. Clinical assessment showed that 1) the growth pattern was similar in mutation-positive and mutation-negative patients, with no significant difference in birth length [-0.6 +/- 2.2 sd (n = 10) vs. -0.6 +/- 1.4 sd (n = 21); P = 0.95], childhood height [-2.6 +/- 1.1 sd (n = 14) vs. -2.1 +/- 1.6 sd (n = 23); P = 0.28], or target height [-0.4 +/- 0.9 sd (n = 14) vs. -0.2 +/- 0.7 sd (n = 17); P = 0.52]; 2) pulmonary valve stenosis was more frequent in mutation-positive patients than in mutation-negative patients (10 of 18 vs. 6 of 27; P = 0.02), as was atrial septal defect (10 of 18 vs. 4 of 27; P = 0.005), whereas hypertrophic cardiomyopathy was present in five mutation-negative patients only; and 3) other features were grossly similar in the prevalence between mutation-positive and mutation-negative patients, but hematological abnormalities, such as bleeding diathesis and juvenile myelomonocytic leukemia, were exclusively present in mutation-positive patients (5 of 18 vs. 0 of 27; P = 0.007). The results suggest that PTPN11 mutations account for approximately 40% of Noonan syndrome patients, as has been reported previously. Furthermore, assessment of clinical features, in conjunction with data reported previously, implies that the type of cardiovascular lesions and the occurrence of hematological abnormalities are different in mutation-positive and mutation-negative patients, whereas the remaining findings are similar in the two groups of patients.

Structural genomics efforts at the Chinese Academy of Sciences and Peking University.

Structural genomics efforts at the Chinese Academy of Sciences and Peking University are reported in this article. The major targets for the structural genomics project are targeted proteins expressed in human hematopoietic stem/progenitor cells, proteins related to blood diseases and other human proteins. Up to now 328 target genes have been constructed in expression vectors. Among them, more than 50% genes have been expressed in Escherichia coli, approximately 25% of the resulting proteins are soluble, and 35 proteins have been purified. Crystallization, data collection and structure determination are continuing. Experiences accumulated during this initial stage are useful for designing and applying high-throughput approaches in structural genomics.

Mothers at risk of alloimmunization to the Rh(D) antigen and availability of gamma-globulin at the Mexican Institute of Social Security

Hemolytic disease of the newborn develops mainly when an Rh negative (D-) mother becomes sensitized and produces anti-Rh possitve (anti-D) antibodies capable of hemolysing D+ fetal erythrocytes. Maternal alloimmunization can be prevented by the administration of anti-D gamma-globulin immediately after the birth of each Rh positive child. In order to identify the frequency of prevention of alloimmunization at the Instituto Mexicano del Seguro Social(IMSS), the amount of mothers at risk of sensitization from 1985 to 1995 was estimated from Rh and ABO blood group frequencies and with the number of deliveries and abortions at the Medical Institutions. Also, information in regard to the dose of gamma-globulin units purchased by the Institute of Social Security from 1985 to 1993 was obtained. The number of mothers at risk stedily increased from 16,616 in 1985 to 21,071 in 1995, amounting to a total of 203,203 in the 10-year period, while only 120,800 gamma-globulin units were purchased in that same period. The findings in this study suggest the need to define reasonable policies for the acquisition of gamma-globulin lots to prevent alloisoimmunization of mothers at risk