Alternative polyadenylation dysregulation contributes to the differentiation block of acute myeloid leukemia.

Posttranscriptional regulation has emerged as a driver for leukemia development and an avenue for therapeutic targeting. Among posttranscriptional processes, alternative polyadenylation (APA) is globally dysregulated across cancer types. However, limited studies have focused on the prevalence and role of APA in myeloid leukemia. Furthermore, it is poorly understood how altered poly(A) site usage of individual genes contributes to malignancy or whether targeting global APA patterns might alter oncogenic potential. In this study, we examined global APA dysregulation in patients with acute myeloid leukemia (AML) by performing 3' region extraction and deep sequencing (3'READS) on a subset of AML patient samples along with healthy hematopoietic stem and progenitor cells (HSPCs) and by analyzing publicly available data from a broad AML patient cohort. We show that patient cells exhibit global 3' untranslated region (UTR) shortening and coding sequence lengthening due to differences in poly(A) site (PAS) usage. Among APA regulators, expression of FIP1L1, one of the core cleavage and polyadenylation factors, correlated with the degree of APA dysregulation in our 3'READS data set. Targeting global APA by FIP1L1 knockdown reversed the global trends seen in patients. Importantly, FIP1L1 knockdown induced differentiation of t(8;21) cells by promoting 3'UTR lengthening and downregulation of the fusion oncoprotein AML1-ETO. In non-t(8;21) cells, FIP1L1 knockdown also promoted differentiation by attenuating mechanistic target of rapamycin complex 1 (mTORC1) signaling and reducing MYC protein levels. Our study provides mechanistic insights into the role of APA in AML pathogenesis and indicates that targeting global APA patterns can overcome the differentiation block in patients with AML.

On-Chip Circularly Polarized Circular Loop Antennas Utilizing 4H-SiC and GaAs Substrates in the Q/V Band.

This paper presents a comprehensive assessment of the performance of on-chip circularly polarized (CP) circular loop antennas that have been designed and fabricated to operate in the Q/V frequency band. The proposed antenna design incorporates two concentric loops, with the outer loop as the active element and the inner loop enhancing the CP bandwidth. The study utilizes gallium arsenide (GaAs) and silicon carbide (4H-SiC) semiconductor wafer substrates. The measured results highlight the successful achievement of impedance matching at 40 GHz and 44 GHz for the 4H-SiC and GaAs substrates, respectively. Furthermore, both cases yield an axial ratio (AR) of less than 3 dB, with variations in bandwidths and frequency bands contingent upon the dielectric constant of the respective substrate material. Moreover, the outcomes confirm that utilizing 4H-SiC substrates results in a significantly higher radiation efficiency of 95%, owing to lower substrate losses. In pursuit of these findings, a 4-element circularly polarized loop array antenna has been fabricated for operation at 40 GHz, employing a 4H-SiC wafer as a low-loss substrate. The results underscore the antenna's remarkable performance, exemplified by a broadside gain of approximately 9.7 dBic and a total efficiency of circa 92%. A close agreement has been achieved between simulated and measured results.

Profiling human pathogenic repeat expansion regions by synergistic and multi-level impacts on molecular connections.

Whilst DNA repeat expansions cause numerous heritable human disorders, their origins and underlying pathological mechanisms are often unclear. We collated a dataset comprising 224 human repeat expansions encompassing 203 different genes, and performed a systematic analysis with respect to key topological features at the DNA, RNA and protein levels. Comparison with controls without known pathogenicity and genomic regions lacking repeats, allowed the construction of the first tool to discriminate repeat regions harboring pathogenic repeat expansions (DPREx). At the DNA level, pathogenic repeat expansions exhibited stronger signals for DNA regulatory factors (e.g. H3K4me3, transcription factor-binding sites) in exons, promoters, 5'UTRs and 5'genes but were not significantly different from controls in introns, 3'UTRs and 3'genes. Additionally, pathogenic repeat expansions were also found to be enriched in non-B DNA structures. At the RNA level, pathogenic repeat expansions were characterized by lower free energy for forming RNA secondary structure and were closer to splice sites in introns, exons, promoters and 5'genes than controls. At the protein level, pathogenic repeat expansions exhibited a preference to form coil rather than other types of secondary structure, and tended to encode surface-located protein domains. Guided by these features, DPREx ( http://biomed.nscc-gz.cn/zhaolab/geneprediction/# ) achieved an Area Under the Curve (AUC) value of 0.88 in a test on an independent dataset. Pathogenic repeat expansions are thus located such that they exert a synergistic influence on the gene expression pathway involving inter-molecular connections at the DNA, RNA and protein levels.

Cost-Effective Design of Polarization and Bandwidth Reconfigurable Millimeter-Wave Loop Antenna.

A singly fed reconfigurable circular loop antenna is proposed for millimeter-wave (mmWave) communication systems. This antenna's distinctive feature lies in its capacity to adjust both polarization and bandwidth characteristics, a capability made possible by the strategic integration of two PIN diodes. These diodes are engineered to function in various modes, allowing for three distinct polarization states and accommodating two distinct bandwidths. A meticulous alignment of these PIN diodes enables the utilization of a single DC bias network as a highly effective RF choke, which simplifies the design and reduces the associated losses. Additionally, a planar biasing network that consists of coplanar strip-lines (CPS) has been employed eliminating the need for lumped elements. The simple and totally planar configuration offers a choice of right-hand circularly polarized (RHCP) radiation or left-hand circularly polarized (LHCP) radiation at 28 GHz. This is accompanied by impedance matching and axial ratio (AR) bandwidths of 12.9% and 8%, respectively, over the same frequency range with a gain of 7.5 dBic. Moreover, when the PIN diodes are unbiased, the antenna offers linear polarization (LP) over two narrow bandwidths at 27 GHz and 29 GHz featuring a maximum gain of 7.2 dBic. Therefore, the proposed configuration offers three operating modes: wide-band RHCP, wide-band-LHCP, and LP over dual narrow bands. Significantly, simulated results closely align with the measured outcomes, affirming the robustness and accuracy of this design.

Distinct sequence features underlie microdeletions and gross deletions in the human genome.

Microdeletions and gross deletions are important causes (~20%) of human inherited disease and their genomic locations are strongly influenced by the local DNA sequence environment. This notwithstanding, no study has systematically examined their underlying generative mechanisms. Here, we obtained 42,098 pathogenic microdeletions and gross deletions from the Human Gene Mutation Database (HGMD) that together form a continuum of germline deletions ranging in size from 1 to 28,394,429 bp. We analyzed the DNA sequence within 1 kb of the breakpoint junctions and found that the frequencies of non-B DNA-forming repeats, GC-content, and the presence of seven of 78 specific sequence motifs in the vicinity of pathogenic deletions correlated with deletion length for deletions of length ≤30 bp. Further, we found that the presence of DR, GQ, and STR repeats is important for the formation of longer deletions (>30 bp) but not for the formation of shorter deletions (≤30 bp) while significantly (χ2 , p < 2E-16) more microhomologies were identified flanking short deletions than long deletions (length >30 bp). We provide evidence to support a functional distinction between microdeletions and gross deletions. Finally, we propose that a deletion length cut-off of 25-30 bp may serve as an objective means to functionally distinguish microdeletions from gross deletions.

A multicenter phase 1 study of nivolumab for relapsed hematologic malignancies after allogeneic transplantation.

Programmed cell death-1 (PD-1)/programmed death ligand-1 blockade may potentially augment graft-vs-tumor effects following allogeneic hematopoietic cell transplantation (alloHCT), but retrospective studies of anti-PD-1 therapy reported substantial toxicity from graft-versus-host-disease (GVHD). Here, we report the results of a prospective clinical trial of PD-1 blockade for relapsed hematologic malignancies (HMs) after alloHCT (NCT01822509). The primary objective in this phase 1 multicenter, investigator-initiated study was to determine maximum tolerated dose and safety. Secondary objectives were to assess efficacy and immunologic activity. Patients with relapsed HMs following alloHCT were eligible. Nivolumab was administered every 2 weeks until progression or unacceptable toxicity, starting with a 1-mg/kg cohort, with planned deescalation based on toxicity to a 0.5-mg/kg cohort. Twenty-eight patients were treated (n = 19 myeloid, n = 9 lymphoid). Median age was 57 years (range 27-76), and median time from alloHCT to enrollment was 21 months (range 5.6-108.5). Two of 6 patients treated at 1 mg/kg experienced dose-limiting toxicity (DLT) from immune-related adverse events (irAEs). Twenty-two patients were treated at 0.5 mg/kg, and 4 DLTs occurred, including 2 irAEs and 2 with fatal GVHD. The overall response rate in efficacy-evaluable patients was 32% (8/25). With a median follow-up of 11 months, the 1-year progression-free survival and overall survival were 23% and 56%, respectively. In this first prospective clinical trial of an anti-PD-1 antibody for post-alloHCT relapse, GVHD and irAEs occurred, requiring dose deescalation, with only modest antitumor activity. Further studies of anti-PD-1 therapy post-alloHCT may require specific toxicity mitigation strategies. This trial was registered at www.clinicaltrials.gov as #NCT01822509.

In vitro induction of neoantigen-specific T cells in myelodysplastic syndrome, a disease with low mutational burden.

Therapies that utilize immune checkpoint inhibition work by leveraging mutation-derived neoantigens and have shown greater clinical efficacy in tumors with higher mutational burden. Whether tumors with a low mutational burden are susceptible to neoantigen-targeted therapy has not been fully addressed. To examine the feasibility of neoantigen-specific adoptive T-cell therapy, the authors studied the T-cell response against somatic variants in five patients with myelodysplastic syndrome (MDS), a malignancy with a very low tumor mutational burden. DNA and RNA from tumor (CD34+) and normal (CD3+) cells isolated from the patients' blood were sequenced to predict patient-specific MDS neopeptides. Neopeptides representing the somatic variants were used to induce and expand autologous T cells ex vivo, and these were systematically tested in killing assays to determine the proportion of neopeptides yielding neoantigen-specific T cells. The authors identified a total of 32 somatic variants (four to eight per patient) and found that 21 (66%) induced a peptide-specific T-cell response and 19 (59%) induced a T-cell response capable of killing autologous tumor cells. Of the 32 somatic variants, 11 (34%) induced a CD4+ response and 11 (34%) induced a CD8+ response that killed the tumor. These results indicate that in vitro induction of neoantigen-specific T cells is feasible for tumors with very low mutational burden and that this approach warrants investigation as a therapeutic option for such patients.

The Human Gene Mutation Database (HGMD®): optimizing its use in a clinical diagnostic or research setting.

The Human Gene Mutation Database (HGMD®) constitutes a comprehensive collection of published germline mutations in nuclear genes that are thought to underlie, or are closely associated with human inherited disease. At the time of writing (June 2020), the database contains in excess of 289,000 different gene lesions identified in over 11,100 genes manually curated from 72,987 articles published in over 3100 peer-reviewed journals. There are primarily two main groups of users who utilise HGMD on a regular basis; research scientists and clinical diagnosticians. This review aims to highlight how to make the most out of HGMD data in each setting.

Higher Risks of Toxicity and Incomplete Recovery in 13- to 17-Year-Old Females after Marrow Donation: RDSafe Peds Results.

Although donation of bone marrow (BM) or peripheral blood stem cells (PBSCs) from children to family members undergoing allogeneic transplantation are well-established procedures, studies detailing levels of pain, symptoms, and long-term recovery are lacking. To address this lack, we prospectively enrolled 294 donors age <18 years at 25 pediatric transplantation centers in North America, assessing them predonation, peridonation, and at 1 month, 6 months, and 1 year postdonation. We noted that 71% of children reported pain and 59% reported other symptoms peridonation, with resolution to 14% and 12% at 1 month postdonation. Both older age (age 13 to 17 years versus younger) and female sex were associated with higher levels of pain peridonation, with the highest rates in older females (57% with grade 2-4 pain and 17% with grade 3-4 pain). Multivariate analyses showed a 4-fold increase in risk for older females compared with males age <13 years (P <.001). At 1 year, 11% of 13- to 17-year-old females reported grade 2-4 pain, compared with 3% of males age 13 to 17 years, 0% of females age <13 years, and 1% of males age <13 years (P = .01). Males and females age 13 to 17 years failed to return to predonation pain levels at 1 year 22% and 23% of the time, respectively, compared with 3% and 10% in males and females age <13 years (P = .002). Our data show that females age 13 to 17 years are at increased risk of grade 2-4 pain at 1 year and >20% of females and males age 13 to 17 years do not return to baseline pain levels by 1 year after BM donation. Studies aimed at decreasing symptoms and improving recovery in older children are warranted.

Effect of Aging and Predonation Comorbidities on the Related Peripheral Blood Stem Cell Donor Experience: Report from the Related Donor Safety Study.

The development of reduced-intensity approaches for allogeneic hematopoietic cell transplantation has resulted in growing numbers of older related donors (RDs) of peripheral blood stem cells (PBSCs). The effects of age on donation efficacy, toxicity, and long-term recovery in RDs are poorly understood. To address this we analyzed hematologic variables, pain, donation-related symptoms, and recovery in 1211 PBSC RDs aged 18 to 79 enrolled in the Related Donor Safety Study. RDs aged > 60 had a lower median CD34+ level before apheresis compared with younger RDs (age > 60, 59 × 106/L; age 41 to 60, 81 × 106/L; age 18 to 40, 121 × 106/L; P < .001). This resulted in older donors undergoing more apheresis procedures (49% versus 30% ≥ 2 collections, P < .001) and higher collection volumes (52% versus 32% > 24 L, P < .001), leading to high percentages of donors aged > 60 with postcollection thrombocytopenia <50 × 109/L (26% and 57% after 2 and 3days of collection, respectively). RDs aged 18 to 40 had a higher risk of grades 2 to 4 pain and symptoms pericollection, but donors over age 40 had more persistent pain at 1, 6, and 12 months (odds ratio [OR], 1.7; P = 0.02) and a higher rate of nonrecovery to predonation levels (OR, 1.7; P = .01). Donors reporting comorbidities increased significantly with age, and those with comorbidities that would have led to deferral by National Marrow Donor Program unrelated donor standards had an increased risk for persistent grades 2 to 4 pain (OR, 2.41; P < .001) and failure to recover to predonation baseline for other symptoms (OR, 2.34; P = .004). This information should be used in counseling RDs regarding risk and can assist in developing practice approaches aimed at improving the RD experience for high-risk individuals.

Ipilimumab for Patients with Relapse after Allogeneic Transplantation.

BACKGROUND: Loss of donor-mediated immune antitumor activity after allogeneic hematopoietic stem-cell transplantation (HSCT) permits relapse of hematologic cancers. We hypothesized that immune checkpoint blockade established by targeting cytotoxic T-lymphocyte-associated protein 4 with ipilimumab could restore antitumor reactivity through a graft-versus-tumor effect. METHODS: We conducted a phase 1/1b multicenter, investigator-initiated study to determine the safety and efficacy of ipilimumab in patients with relapsed hematologic cancer after allogeneic HSCT. Patients received induction therapy with ipilimumab at a dose of 3 or 10 mg per kilogram of body weight every 3 weeks for a total of 4 doses, with additional doses every 12 weeks for up to 60 weeks in patients who had a clinical benefit. RESULTS: A total of 28 patients were enrolled. Immune-related adverse events, including one death, were observed in 6 patients (21%), and graft-versus-host disease (GVHD) that precluded further administration of ipilimumab was observed in 4 patients (14%). No responses that met formal response criteria occurred in patients who received a dose of 3 mg per kilogram. Among 22 patients who received a dose of 10 mg per kilogram, 5 (23%) had a complete response, 2 (9%) had a partial response, and 6 (27%) had decreased tumor burden. Complete responses occurred in 4 patients with extramedullary acute myeloid leukemia and 1 patient with the myelodysplastic syndrome developing into acute myeloid leukemia. Four patients had a durable response for more than 1 year. Responses were associated with in situ infiltration of cytotoxic CD8+ T cells, decreased activation of regulatory T cells, and expansion of subpopulations of effector T cells in the blood. CONCLUSIONS: Our early-phase data showed that administration of ipilimumab was feasible in patients with recurrent hematologic cancers after allogeneic HSCT, although immune-mediated toxic effects and GVHD occurred. Durable responses were observed in association with several histologic subtypes of these cancers, including extramedullary acute myeloid leukemia. (Funded by the National Institutes of Health and others; ClinicalTrials.gov number, NCT01822509.).

Related peripheral blood stem cell donors experience more severe symptoms and less complete recovery at one year compared to unrelated donors.

Unlike unrelated donor registries, transplant centers lack uniform approaches to related donor assessment and deferral. To test whether related donors are at increased risk for donation-related toxicities, we conducted a prospective observational trial of 11,942 related and unrelated donors aged 18-60 years. Bone marrow (BM) was collected at 37 transplant and 78 National Marrow Donor Program centers, and peripheral blood stem cells (PBSC) were collected at 42 transplant and 87 unrelated donor centers in North America. Possible presence of medical comorbidities was verified prior to donation, and standardized pain and toxicity measures were assessed pre-donation, peri-donation, and one year following. Multivariate analyses showed similar experiences for BM collection in related and unrelated donors; however, related stem cell donors had increased risk of moderate [odds ratios (ORs) 1.42; P<0.001] and severe (OR 8.91; P<0.001) pain and toxicities (OR 1.84; P<0.001) with collection. Related stem cell donors were at increased risk of persistent toxicities (OR 1.56; P=0.021) and non-recovery from pain (OR 1.42; P=0.001) at one year. Related donors with more significant comorbidities were at especially high risk for grade 2-4 pain (OR 3.43; P<0.001) and non-recovery from toxicities (OR 3.71; P<0.001) at one year. Related donors with more significant comorbidities were at especially high risk for grade 2-4 pain (OR 3.43; P<0.001) and non-recovery from toxicities (OR 3.71; P<0.001) at one year. Related donors reporting grade ≥2 pain had significant decreases in Health-Related Quality of Life (HR-QoL) scores at one month and one year post donation (P=0.004). In conclusion, related PBSC donors with comorbidities are at increased risk for pain, toxicity, and non-recovery at one year after donation. Risk profiles described in this study should be used for donor education, planning studies to improve the related donor experience, and decisions regarding donor deferral. Registered at clinicaltrials.gov identifier:00948636.

Insights into hominid evolution from the gorilla genome sequence.

Gorillas are humans' closest living relatives after chimpanzees, and are of comparable importance for the study of human origins and evolution. Here we present the assembly and analysis of a genome sequence for the western lowland gorilla, and compare the whole genomes of all extant great ape genera. We propose a synthesis of genetic and fossil evidence consistent with placing the human-chimpanzee and human-chimpanzee-gorilla speciation events at approximately 6 and 10 million years ago. In 30% of the genome, gorilla is closer to human or chimpanzee than the latter are to each other; this is rarer around coding genes, indicating pervasive selection throughout great ape evolution, and has functional consequences in gene expression. A comparison of protein coding genes reveals approximately 500 genes showing accelerated evolution on each of the gorilla, human and chimpanzee lineages, and evidence for parallel acceleration, particularly of genes involved in hearing. We also compare the western and eastern gorilla species, estimating an average sequence divergence time 1.75 million years ago, but with evidence for more recent genetic exchange and a population bottleneck in the eastern species. The use of the genome sequence in these and future analyses will promote a deeper understanding of great ape biology and evolution.

Proinflammatory Dual Receptor T Cells in Chronic Graft-versus-Host Disease.

Defective post-transplantation thymopoiesis is associated with chronic graft-versus-host disease (GVHD), a multiorgan pathology affecting up to 80% of patients after allogeneic hematopoietic stem cell transplantation (HSCT). Previous work demonstrated that the subset of T cells expressing 2 T cell receptors (TCRs) is predisposed to alloreactivity, driving selective and disproportionate activity in acute GVHD in both mouse models and HSCT patients. Here we investigate a potential role for this pathogenic T cell subset in chronic GVHD (cGVHD). HSCT patients with cGVHD demonstrated increased numbers of dual TCR cells in circulation. These dual receptor cells had an activated phenotype, indicating an active role in cGVHD. Notably, single-cell RNA sequencing identified the increased dual TCR cells in cGVHD as predominantly expressing Tbet, indicative of a proinflammatory phenotype. These results identify dual TCR cells as specific mediators of pathogenic inflammation underlying cGVHD and highlight Tbet-driven T cell function as a potential pathway for potential therapeutic targeting.

Increased Foxp3+Helios+ Regulatory T Cells and Decreased Acute Graft-versus-Host Disease after Allogeneic Bone Marrow Transplantation in Patients Receiving Sirolimus and RGI-2001, an Activator of Invariant Natural Killer T Cells.

Regulatory T (Treg) cells play a central role in immune tolerance and prevention of aberrant immune responses. Several studies have suggested that the risk of graft-versus-host disease (GVHD) after allogeneic hematopoietic cell transplantation (HCT) can be ameliorated by increasing Tregs. We have developed an approach of in vivo expansion of Tregs with RGI-2001, a novel liposomal formulation of a synthetic derivative of alpha-galactosylceramide, a naturally occurring ligand that binds to CD1 and activates and expands invariant natural killer cells. In preclinical studies, a single intravenous infusion of RGI-2001 expanded Treg and could ameliorate GVHD in a mouse model of allogeneic HCT. To explore the role of RGI-2001 in clinical HCT, we initiated a phase 2A clinical trial (n = 29), testing 2 different doses of RGI-2001 administered as a single infusion on day 0 of allogeneic HCT. RGI-2001 was well tolerated and without infusion reactions or cytokine release syndrome. A subset of patients (8 of 29, 28%) responded to RGI-2001 by inducing a markedly increased number of cells with a Treg phenotype. The Treg had a high Ki-67 index and were almost exclusively Helios+ and Foxp3+, indicating that their accumulation was due to expansion of natural Treg. Notably, the incidence of grade 2 to 4 GVHD in the 8 patients who responded to RGI-2001 was 12.5%, compared with 52.4% in the 21 patients who did not respond. No grade 3 or 4 GVHD was observed in the responder group, compared with a 9.5% incidence among nonresponders. Immunosuppression with sirolimus was also associated with a low incidence of GVHD, suggesting that RGI-2001 may have synergized with sirolimus to promote Treg expansion.

The Human Gene Mutation Database: towards a comprehensive repository of inherited mutation data for medical research, genetic diagnosis and next-generation sequencing studies.

The Human Gene Mutation Database (HGMD®) constitutes a comprehensive collection of published germline mutations in nuclear genes that underlie, or are closely associated with human inherited disease. At the time of writing (March 2017), the database contained in excess of 203,000 different gene lesions identified in over 8000 genes manually curated from over 2600 journals. With new mutation entries currently accumulating at a rate exceeding 17,000 per annum, HGMD represents de facto the central unified gene/disease-oriented repository of heritable mutations causing human genetic disease used worldwide by researchers, clinicians, diagnostic laboratories and genetic counsellors, and is an essential tool for the annotation of next-generation sequencing data. The public version of HGMD ( http://www.hgmd.org ) is freely available to registered users from academic institutions and non-profit organisations whilst the subscription version (HGMD Professional) is available to academic, clinical and commercial users under license via QIAGEN Inc.

Guanine holes are prominent targets for mutation in cancer and inherited disease.

Single base substitutions constitute the most frequent type of human gene mutation and are a leading cause of cancer and inherited disease. These alterations occur non-randomly in DNA, being strongly influenced by the local nucleotide sequence context. However, the molecular mechanisms underlying such sequence context-dependent mutagenesis are not fully understood. Using bioinformatics, computational and molecular modeling analyses, we have determined the frequencies of mutation at G • C bp in the context of all 64 5'-NGNN-3' motifs that contain the mutation at the second position. Twenty-four datasets were employed, comprising >530,000 somatic single base substitutions from 21 cancer genomes, >77,000 germline single-base substitutions causing or associated with human inherited disease and 16.7 million benign germline single-nucleotide variants. In several cancer types, the number of mutated motifs correlated both with the free energies of base stacking and the energies required for abstracting an electron from the target guanines (ionization potentials). Similar correlations were also evident for the pathological missense and nonsense germline mutations, but only when the target guanines were located on the non-transcribed DNA strand. Likewise, pathogenic splicing mutations predominantly affected positions in which a purine was located on the non-transcribed DNA strand. Novel candidate driver mutations and tissue-specific mutational patterns were also identified in the cancer datasets. We conclude that electron transfer reactions within the DNA molecule contribute to sequence context-dependent mutagenesis, involving both somatic driver and passenger mutations in cancer, as well as germline alterations causing or associated with inherited disease.

Deleterious- and disease-allele prevalence in healthy individuals: insights from current predictions, mutation databases, and population-scale resequencing.

We have assessed the numbers of potentially deleterious variants in the genomes of apparently healthy humans by using (1) low-coverage whole-genome sequence data from 179 individuals in the 1000 Genomes Pilot Project and (2) current predictions and databases of deleterious variants. Each individual carried 281-515 missense substitutions, 40-85 of which were homozygous, predicted to be highly damaging. They also carried 40-110 variants classified by the Human Gene Mutation Database (HGMD) as disease-causing mutations (DMs), 3-24 variants in the homozygous state, and many polymorphisms putatively associated with disease. Whereas many of these DMs are likely to represent disease-allele-annotation errors, between 0 and 8 DMs (0-1 homozygous) per individual are predicted to be highly damaging, and some of them provide information of medical relevance. These analyses emphasize the need for improved annotation of disease alleles both in mutation databases and in the primary literature; some HGMD mutation data have been recategorized on the basis of the present findings, an iterative process that is both necessary and ongoing. Our estimates of deleterious-allele numbers are likely to be subject to both overcounting and undercounting. However, our current best mean estimates of ~400 damaging variants and ~2 bona fide disease mutations per individual are likely to increase rather than decrease as sequencing studies ascertain rare variants more effectively and as additional disease alleles are discovered.

Augmentation of autologous T cell reactivity with acute myeloid leukemia (AML) blasts by Toll-like receptor (TLR) agonists.

This study investigated whether TNF-α, Toll-like receptors (TLRs) 7/8 agonist resiquimod (R848), the TLR4 agonist lipopolysaccharide (LPS) and their combinations can enhance autologous AML-reactive T cell generation in an in vitro culture. AML peripheral blood or bone marrow mononuclear cells were cultured in medium supplemented with GM-CSF/IL-4 to induce dendritic cell (DC) differentiation of AML blasts (AML-DC). The impact of TNF-α, LPS, R848 and their combinations on AML-DC cultures was analyzed. Significantly enhanced CD80, CD40, CD83, CD54, HLA-DR and CD86 expression of AML cells was observed by addition of TNF-α, LPS, R848 alone or combinations. Induced CD80 expression of AML cells was significantly higher through the combination of TNF-α, LPS and R848 (T + L + R) than that by T alone. CTL induced from T + L + R, T + R, T + L, L + R and R, but not T, L alone stimulated cultures showed significantly higher IFN-γ release than the medium control in response to autologous AML cells. IFN-γ release by T + L + R was significantly higher than T or L alone, and T + R was significantly higher than T alone. CTL generated from T + L + R, T + L, T + R, L + R and L alone exerted significantly higher AML cell killing than medium control. AML cell killing by T + L + R and T + R was significantly higher than T or R alone. These results indicate that the combination of T + L + R induces a significantly enhanced antigen presentation effect of AML-DC. We speculate that the complementary effects of reagent combinations may better address the heterogeneity of responses to any single agent in AML cells from different patients.

The Human Gene Mutation Database: building a comprehensive mutation repository for clinical and molecular genetics, diagnostic testing and personalized genomic medicine.

The Human Gene Mutation Database (HGMD®) is a comprehensive collection of germline mutations in nuclear genes that underlie, or are associated with, human inherited disease. By June 2013, the database contained over 141,000 different lesions detected in over 5,700 different genes, with new mutation entries currently accumulating at a rate exceeding 10,000 per annum. HGMD was originally established in 1996 for the scientific study of mutational mechanisms in human genes. However, it has since acquired a much broader utility as a central unified disease-oriented mutation repository utilized by human molecular geneticists, genome scientists, molecular biologists, clinicians and genetic counsellors as well as by those specializing in biopharmaceuticals, bioinformatics and personalized genomics. The public version of HGMD (http://www.hgmd.org) is freely available to registered users from academic institutions/non-profit organizations whilst the subscription version (HGMD Professional) is available to academic, clinical and commercial users under license via BIOBASE GmbH.