Mutations In Regulatory Regions Research Articles

Studying the Pathogenic Effect of New Nucleotide Changes in the Promoter Region of the TERT Gene in Patients with Glioblastoma Brain Tumor

Introduction: Brain tumors are considered to be one of the most dangerous types of cancer, despite decades of research and treatment efforts. The activation of the telomerase enzyme is a critical factor in the immortality of these cells. Mutations in the promoter region of the TERT gene, particularly in the promoter hot spots, can influence the binding of activating transcription factors and inhibit the binding of negative regulatory factors of the TERT gene, ultimately regulating the gene's expression. This study aimed to identify and investigate the mutations of the TERT gene promoter region in glioblastoma, a malignant brain tumor, using bioinformatics. Methods: A study was conducted using the case-control method where 35 patients with glioblastoma multiform brain tumor were examined along with 40 people who were examined as control samples. In this research, the Touchdown PCR technique and direct DNA sequencing were used to detect mutations in the promoter region of the TERT gene in patients with glioblastoma. Furthermore, bioinformatic analyses were conducted to investigate the pathogenic effect of nucleotide changes in this gene region. Results: In the core region of the TERT gene promoter, three-point mutations were identified (c.*146C>T، c.*144C>G and c.*143G>C). Two of these mutations were novel and have been observed for the first time in glioblastoma multiform. The remaining mutation was located in the promoter core's hot spot of the gene. This mutation was known as c.*146C>T. Conclusion: In this research, the harmful impact of TERT promoter region mutations as a biomarker for glioblastoma was examined. These findings suggest that mutations in regulatory regions, as well as coding sequences, could be significant contributory factors in the process of carcinogenesis.

EPCO-51. FUNCTIONAL ANALYSIS OF REGULATORY MUTATIONS IN PEDIATRIC BRAIN TUMORS

Abstract Pediatric brain tumors are the primary cause of cancer related mortality in children and are characterized by low mutations burden. The known somatic drivers reside in the coding regions of the genome. The role of non-coding mutations in brain tumors is important yet underexplored. In this study we investigate the impact of regulatory region mutations in pediatric brain tumors. We studied 1.3 million open chromatin regions (OCRs) genome-wide from 9 studies including developing/adult brain, and cancer. Within these OCRs we identified recurring mutations in whole genome sequences from 1003 pediatric brain tumors from CBTN and PCAWG cohorts. A total of 567 OCRs with significantly recurring mutations were found, showing enrichment in brain-specific enhancers and in repressed polycomb regions in stem cells. The mutations in these OCRs resulted in motif disruption or creation of 217 transcription factor binding sites. Differential expression analysis of mutated vs nonmutated samples identified perturbations of gene or protein expression in 67 (RNA) and 16 (protein) OCR-gene pairs. Furthermore, 17 mutated OCRs were found to correlate with alterations in gene expression programs, indicating a potential regulatory role affecting multiple downstream genes. We further performed network analysis to explore clusters of OCR associated genes that share biological networks and pathways, and identified 24 clusters. Among these, OCR mutation-associated genes in 6 clusters showed a survival impact in patients. These 6 clusters mapped to pathways including mRNA decay and degradation, longevity regulating pathway, autophagy, p53 pathway, HIF2-alpha transcription factor network, pathways in cancer among others. In summary, we report functionally important regulatory elements in the genome that impact the biological processes in pediatric brain tumors.

Genetic fine-mapping reveals single nucleotide polymorphism mutations in the MC1R regulatory region associated with duck melanism.

Birds are among the most colourful terrestrial vertebrates, with various plumage colours and patterns. We conducted a genome-wide association study (GWAS) on an intercross F2 population of Pekin ducks and mallards (n=722) and identified a 1.57-Mb genetic region (Chr11: 20,176,480-21,750,101 bp) related to duck melanism. Fine mapping by linkage disequilibrium (LD) and FST analysis narrowed the final candidate region to a region of 22,500 bp (Chr11: 20,677,500-20,700,000 bp) including three coding genes, TCF25, MC1R and TUBB3. Combined with transcriptome and qRT-PCR analysis, MC1R was identified as the unique genetic locus responsible for black plumage in ducks, and it was significantly more highly expressed in the feather bulbs of black ducks. We also identified 52G > A (Chr11: 20,696,354G > A) and 376G > A (Chr11: 20,696,678G > A) mutations in the MC1R coding region that have been widely studied in ducks. In addition, structural variations (SVs) were screened by nanopore sequencing, and no significant SV was found to be associated with the duck black plumage trait. However, we identified four novel single nucleotide polymorphisms in the MC1R regulator region (Chr11: 20,678,412G > A, Chr11: 20,679,236G > A, Chr11: 20,692,496 A > G and Chr11: 20,692,791 A > G) that had a strong association with the black plumage phenotype of ducks and combined with potential changes in transcription binding affinities. The luciferase reporter gene assay demonstrated that Chr11: 20,678,412G > A and Chr11: 20,679,236G > A led to significant promoter activity changes. Our research emphasizes the importance of MC1R regulatory region mutation in determining the duck black plumage phenotype, and these results expand our understanding of the genetic mechanism underlying duck plumage colour.

Dr.Nod: computational framework for discovery of regulatory non-coding drivers in tissue-matched distal regulatory elements.

The discovery of cancer driver mutations is a fundamental goal in cancer research. While many cancer driver mutations have been discovered in the protein-coding genome, research into potential cancer drivers in the non-coding regions showed limited success so far. Here, we present a novel comprehensive framework Dr.Nod for detection of non-coding cis-regulatory candidate driver mutations that are associated with dysregulated gene expression using tissue-matched enhancer-gene annotations. Applying the framework to data from over 1500 tumours across eight tissues revealed a 4.4-fold enrichment of candidate driver mutations in regulatory regions of known cancer driver genes. An overarching conclusion that emerges is that the non-coding driver mutations contribute to cancer by significantly altering transcription factor binding sites, leading to upregulation of tissue-matched oncogenes and down-regulation of tumour-suppressor genes. Interestingly, more than half of the detected cancer-promoting non-coding regulatory driver mutations are over 20 kb distant from the cancer-associated genes they regulate. Our results show the importance of tissue-matched enhancer-gene maps, functional impact of mutations, and complex background mutagenesis model for the prediction of non-coding regulatory drivers. In conclusion, our study demonstrates that non-coding mutations in enhancers play a previously underappreciated role in cancer and dysregulation of clinically relevant target genes.

Diagnostic Approach to Patients with Low Serum Alkaline Phosphatase.

Increased serum levels of alkaline phosphatase (ALP) are widely recognized as a biochemical marker of many disorders affecting the liver or bone. However, the approach for patients with low ALP phosphatase is not well-established. Low serum ALP is an epiphenomenon of many severe acute injuries and diseases. Persistently low serum ALP may be secondary to drug therapy (including antiresorptives) or a variety of acquired disorders, such as malnutrition, vitamin and mineral deficiencies, endocrine disorders, etc. Hypophosphatasia, due to pathogenic variants of the ALPL gene, which encodes tissue non-specific ALP, is the most common genetic cause of low serum ALP. Marked bone hypomineralization is frequent in severe pediatric-onset cases. However, adult forms of hypophosphatasia usually present with milder manifestations, such as skeletal pain, chondrocalcinosis, calcific periarthritis, dental problems, and stress fractures. The diagnostic approach to these patients is discussed. Measuring several ALP substrates, such as pyrophosphate, pyridoxal phosphate, or phosphoethanolamine, may help to establish enzyme deficiency. Gene analysis showing a pathogenic variant in ALPL may confirm the diagnosis. However, a substantial proportion of patients show normal results after sequencing ALPL exons. It is still unknown if those patients carry unidentified mutations in regulatory regions of ALPL, epigenetic changes, or abnormalities in other genes.

Pain and health-related quality of life in patients with hypophosphatasemia with and without ALPL gene mutations.

BackgroundLow serum alkaline phosphatase levels are the hallmark of hypophosphatasia, a disorder due to pathogenic variants of the ALPL gene. However, some patients do not carry ALPL variants and the cause of low alkaline phosphatase remains unknown. We aimed to determine health-related quality of life in adults with low alkaline phosphatase and explore the differences between patients with and without ALPL mutations.MethodsWe studied 35 adult patients with persistently low alkaline phosphatase unrelated to secondary acquired causes who had ALPL sequenced, and 35 controls of similar age. Three questionnaires about body pain (Brief Pain Inventory, BPI), physical disability (Health Assessment Questionnaire Disability Index, HAQ-DI), and health-related quality of life (36-item Short-Form Health Survey, SF-36) were delivered by telephone interviews.ResultsThe mean BPI intensity and interference scores were higher in the patient group (p=0.04 and 0.004, respectively). All domains of the HAQ instrument tended to score better in the control group, with significant differences in the “reach” score (p=0.037) and the overall mean score (0.23 vs 0.09; p=0.029). Patients scored worse than controls in several SF-36 dimensions (Role physical, p=0.039; Bodily pain p=0.046; Role emotional, p=0.025). Patients with and without pathogenic variants scored similarly across all tests, without between-group significant differences.ConclusionsPatients with persistently low levels of alkaline phosphatase have significantly worse scores in body pain and other health-related quality of life dimensions, without differences between patients with and without pathogenic variants identified in ALPL gene. This is consistent with the latter ones carrying mutations in regulatory regions.

Mutational patterns and clonal evolution from diagnosis to relapse in pediatric acute lymphoblastic leukemia

The mechanisms driving clonal heterogeneity and evolution in relapsed pediatric acute lymphoblastic leukemia (ALL) are not fully understood. We performed whole genome sequencing of samples collected at diagnosis, relapse(s) and remission from 29 Nordic patients. Somatic point mutations and large-scale structural variants were called using individually matched remission samples as controls, and allelic expression of the mutations was assessed in ALL cells using RNA-sequencing. We observed an increased burden of somatic mutations at relapse, compared to diagnosis, and at second relapse compared to first relapse. In addition to 29 known ALL driver genes, of which nine genes carried recurrent protein-coding mutations in our sample set, we identified putative non-protein coding mutations in regulatory regions of seven additional genes that have not previously been described in ALL. Cluster analysis of hundreds of somatic mutations per sample revealed three distinct evolutionary trajectories during ALL progression from diagnosis to relapse. The evolutionary trajectories provide insight into the mutational mechanisms leading relapse in ALL and could offer biomarkers for improved risk prediction in individual patients.

Discovering single nucleotide variants and indels from bulk and single-cell ATAC-seq.

Genetic variants and de novo mutations in regulatory regions of the genome are typically discovered by whole-genome sequencing (WGS), however WGS is expensive and most WGS reads come from non-regulatory regions. The Assay for Transposase-Accessible Chromatin (ATAC-seq) generates reads from regulatory sequences and could potentially be used as a low-cost ‘capture’ method for regulatory variant discovery, but its use for this purpose has not been systematically evaluated. Here we apply seven variant callers to bulk and single-cell ATAC-seq data and evaluate their ability to identify single nucleotide variants (SNVs) and insertions/deletions (indels). In addition, we develop an ensemble classifier, VarCA, which combines features from individual variant callers to predict variants. The Genome Analysis Toolkit (GATK) is the best-performing individual caller with precision/recall on a bulk ATAC test dataset of 0.92/0.97 for SNVs and 0.87/0.82 for indels within ATAC-seq peak regions with at least 10 reads. On bulk ATAC-seq reads, VarCA achieves superior performance with precision/recall of 0.99/0.95 for SNVs and 0.93/0.80 for indels. On single-cell ATAC-seq reads, VarCA attains precision/recall of 0.98/0.94 for SNVs and 0.82/0.82 for indels. In summary, ATAC-seq reads can be used to accurately discover non-coding regulatory variants in the absence of whole-genome sequencing data and our ensemble method, VarCA, has the best overall performance.

Identification and Characterization of DAMs Mutations Associated With Early Blooming in Sweet Cherry, and Validation of DNA-Based Markers for Selection.

Dormancy release and bloom time of sweet cherry cultivars depend on the environment and the genotype. The knowledge of these traits is essential for cultivar adaptation to different growing areas, and to ensure fruit set in the current climate change scenario. In this work, the major sweet cherry bloom time QTL qP-BT1.1m (327 Kbs; Chromosome 1) was scanned for candidate genes in the Regina cv genome. Six MADS-box genes (PavDAMs), orthologs to peach and Japanese apricot DAMs, were identified as candidate genes for bloom time regulation. The complete curated genomic structure annotation of these genes is reported. To characterize PavDAMs intra-specific variation, genome sequences of cultivars with contrasting chilling requirements and bloom times (N = 13), were then mapped to the ‘Regina’ genome. A high protein sequence conservation (98.8–100%) was observed. A higher amino acid variability and several structural mutations were identified in the low-chilling and extra-early blooming cv Cristobalina. Specifically, a large deletion (694 bp) upstream of PavDAM1, and various INDELs and SNPs in contiguous PavDAM4 and -5 UTRs were identified. PavDAM1 upstream deletion in ‘Cristobalina’ revealed the absence of several cis-acting motifs, potentially involved in PavDAMs expression. Also, due to this deletion, a non-coding gene expressed in late-blooming ‘Regina’ seems truncated in ‘Cristobalina’. Additionally, PavDAM4 and -5 UTRs mutations revealed different splicing variants between ‘Regina’ and ‘Cristobalina’ PavDAM5. The results indicate that the regulation of PavDAMs expression and post-transcriptional regulation in ‘Cristobalina’ may be altered due to structural mutations in regulatory regions. Previous transcriptomic studies show differential expression of PavDAM genes during dormancy in this cultivar. The results indicate that ‘Cristobalina’ show significant amino acid differences, and structural mutations in PavDAMs, that correlate with low-chilling and early blooming, but the direct implication of these mutations remains to be determined. To complete the work, PCR markers designed for the detection of ‘Cristobalina’ structural mutations in PavDAMs, were validated in an F2 population and a set of cultivars. These PCR markers are useful for marker-assisted selection of early blooming seedlings, and probably low-chilling, from ‘Cristobalina’, which is a unique breeding source for these traits.

Insights into Hematopoiesis in GATA2 Deficiency Using Single-Cell Transcriptomics

GATA2 is an essential transcription factor in hematopoiesis. Germline GATA2 mutations have been identified as the cause of familial syndromes with immunodeficiency and predisposition to myeloid malignancies. GATA2 deficiency results in varying clinical outcomes manifesting at either young or older age. Reason for the variation of the disease phenotype preceding hematological malignancy is not known. GATA2 mutations appear to cause loss of function of the mutated allele resulting in haploinsufficiency. Several different types of mutations in GATA2 have been identified; truncating, missense, large deletions, and regulatory region mutations. The most direct GATA2 control pathway is the effect it has on hematopoietic stem cells (HSCs). Through the progeny of the hematopoietic stem cells that function under GATA2 control, GATA2 indirectly has a great effect on immune system, both protection against external infection and internal tumor formation. We aimed at deciphering how do GATA2-mutated cells differ from wild type (wt) and what are the differences between GATA2 wt vs. GATA2-mutated cells' progeny and downstream signaling? One of the genes positively regulated by GATA2 is NFE2 . NFE2 encodes a component of the NF-E2 complex essential for regulating erythroid and megakaryocytic maturation and differentiation. As GATA2-positive cells are scarce in PB we focused in our analyses on NFE2-positive cells.We conducted single-cell transcriptomic analysis using 10x Genomics Chromium Single Cell 3′ Solution (Zheng et al . Nature Communications, 2017) on peripheral blood (PB) samples from five GATA2 mutation carriers and two healthy controls. The studied GATA2 variants were p.Q328X (two cases), p.T354M, p.G237D, and a sample where a GATA2 mutation has not been identified, but only one allele GATA2 is expressed i.e. haploinsufficiency. Erythrocytes were lysed prior to washing, filtering and diluting the white blood cells in suitable concentration (1x106 cells/mL) for the analysis. The gel beads in emulsion (GEM) generation was performed using the Chromium Single Cell 3‘ v2 Reagent Kit (10x Genomics) aiming for a 3000-cell capture per sample. The Illumina ready sequencing libraries were prepared according to the 10x Genomics instructions and the sample libraries were sequenced in Illumina HiSeq 2500 using Rapid mode. The Cell Ranger v1.3 analysis pipelines (10x Genomics) were used to demultiplex and convert Chromium single cell 3‘ RNA-sequencing barcode and read data to FASTQ files and to generate align reads and gene-cell matrices.Significant gene expression differences in NFE2 expressing cells between patients carrying different variants in GATA2 were observed. Intriguingly, significant expression changes were noted e.g. in CD74, CFD, CTSS, CLEC1B, and CMTM5, all linked to cell signaling and immunological responses. Comparison of GATA2 -mutated vs. controls in NFE2 expressing cells revealed significant changes in expression patterns in hemoglobin genes HBA1, HBA2, and HBB, as well as in e.g. other erythrocyte-associated genes ALAS2 and AHSP. Further analyses also revealed significantly distinct expression patterns in PB eosinophils between study patients and controls in e.g. CEBPD, VCAN, and RGS2 . Also, when comparing the eosinophils from individual patients with controls and each other, we identified distinct expression patterns in e.g. CAMP, and NKG7, which play important roles in the immune system.Following germline GATA2 mutation discovery in acute myeloid leukemia / myelodysplastic syndrome / immunodeficiency patients, we conducted whole transcriptome analysis to decipher the GATA germline mutation defects on single-cell level. Single-cell transcriptomics enables analyzing the role of e.g. GATA2 in a selected cell type and its' progeny. Phenotype of GATA2-deficient patients is multi-faced but generally manifested by challenges in immune functions and autoinflammation. Connection between our findings and patients' clinical manifestations is interesting and triggers us to investigate the possible link further. We will furthermore study the bone marrow sample single-cell transcriptomes to deepen the understanding of GATA2 deficiency on stem cell level and validate our transcriptome analysis findings. All this will hopefully increase our understanding on GATA2 deficiency disease mechanisms and improve patient (family) care. DisclosuresNo relevant conflicts of interest to declare.

Transcriptional regulation of dosage compensation in Carica papaya

Sex chromosome evolution results in the disparity in gene content between heterogametic sex chromosomes and creates the need for dosage compensation to counteract the effects of gene dose imbalance of sex chromosomes in males and females. It is not known at which stage of sex chromosome evolution dosage compensation would evolve. We used global gene expression profiling in male and female papayas to assess gene expression patterns of sex-linked genes on the papaya sex chromosomes. By analyzing expression ratios of sex-linked genes to autosomal genes and sex-linked genes in males relative to females, our results showed that dosage compensation was regulated on a gene-by-gene level rather than whole sex-linked region in papaya. Seven genes on the papaya X chromosome exhibited dosage compensation. We further compared gene expression ratios in the two evolutionary strata. Y alleles in the older evolutionary stratum showed reduced expression compared to X alleles, while Y alleles in the younger evolutionary stratum showed elevated expression compared to X alleles. Reduced expression of Y alleles in the older evolutionary stratum might be caused by accumulation of deleterious mutations in regulatory regions or transposable element-mediated methylation spreading. Most X-hemizygous genes exhibited either no or very low expression, suggesting that gene silencing might play a role in maintaining transcriptional balance between females and males.

A novel cause of DKC1‐related bone marrow failure: Partial deletion of the 3′ untranslated region

Telomere biology disorders (TBDs), including dyskeratosis congenita (DC), are a group of rare inherited diseases characterized by very short telomeres. Mutations in the components of the enzyme telomerase can lead to insufficient telomere maintenance in hematopoietic stem cells, resulting in the bone marrow failure that is characteristic of these disorders. While an increasing number of genes are being linked to TBDs, the causative mutation remains unidentified in 30‐40% of patients with DC. There is therefore a need for whole genome sequencing (WGS) in these families to identify novel genes, or mutations in regulatory regions of known disease‐causing genes. Here we describe a family in which a partial deletion of the 3′ untranslated region (3′ UTR) of DKC1, encoding the protein dyskerin, was identified by WGS, despite being missed by whole exome sequencing. The deletion segregated with disease across the family and resulted in reduced levels of DKC1 mRNA in the proband. We demonstrate that the DKC1 3′ UTR contains two polyadenylation signals, both of which were removed by this deletion, likely causing mRNA instability. Consistent with the major function of dyskerin in stabilization of the RNA subunit of telomerase, hTR, the level of hTR was also reduced in the proband, providing a molecular basis for his very short telomeres. This study demonstrates that the terminal region of the 3′ UTR of the DKC1 gene is essential for gene function and illustrates the importance of analyzing regulatory regions of the genome for molecular diagnosis of inherited disease.

Regulatory region mutations of TERT, PLEKHS1 and GPR126 genes as urinary biomarkers in upper tract urothelial carcinomas.

Background: The hotspot regulatory region mutations of the TERT, PLEKHS1 and GPR126 genes have been shown to occur frequently in urothelial bladder carcinoma (UBC). However, it is currently unclear whether these mutations are all present in upper tract urothelial carcinomas (UTUC) including renal pelvic carcinoma (RPC) and ureter carcinoma (UC), although TERT promoter mutations were previously observed in these malignancies.Methods: The hotspot mutations of TERT and PLEKHS1 promoters and GPR126 intron 6 (enhancer) in tumors derived from 164 patients with UTUC were determined using Sanger sequencing, and the obtained results were further compared with the mutation frequency in 106 UBCs. The mutations were also assessed in urine from patients with UTUC and UBC.Results: The mutation frequencies in UTUC tumors were 28%, 5.8% and 11% for TERT and PLEKHS1 promoters and GPR126 intron 6, respectively, which were lower than those (44.3%, 26.4%, and 31.4%, respectively) in UBCs. The total frequencies for the presence of any of these mutations were 50.8% and 34.4% for RPCs and UCs, respectively. All these mutated DNA sequences were detectable in urine from both UTUC and UBC patients and disappeared rapidly in most patients after surgery.Conclusions: This proof-of-concept study demonstrates that the hotspot mutations in the TERT, PLEKHS1 and GPR126 non-coding regions are present in UTUCs, and that urinary assays of these mutated sequences serve as potential biomarkers for UTUC diagnostics and disease monitoring.

Differential Allele-Specific Expression Uncovers Breast Cancer Genes Dysregulated by Cis Noncoding Mutations.

Identifying cancer-relevant mutations in noncoding regions is challenging due to the large numbers of such mutations, their low levels of recurrence, and difficulties in interpreting their functional impact. To uncover genes that are dysregulated due to somatic mutations in cis, we build upon the concept of differential allele-specific expression (ASE) and introduce methods to identify genes within an individual's cancer whose ASE differs from what is found in matched normal tissue. When applied to breast cancer tumor samples, our methods detect the known allele-specific effects of copy number variation and nonsense-mediated decay. Further, genes that are found to recurrently exhibit differential ASE across samples are cancer relevant. Genes with cis mutations are enriched for differential ASE, and we find 147 potentially functional noncoding mutations cis to genes that exhibit significant differential ASE. We conclude that differential ASE is a promising means for discovering gene dysregulation due to cis noncoding mutations.

The novel TP53 3'-end methylation pattern associated with its expression would be a potential biomarker for breast cancer detection.

Deficiency or silence of TP53 is an early event in breast tumorigenesis. Aberrant methylation and mutation in regulatory regions were considered as crucial regulators of gene expression. Using multiplex-PCR and next-generation sequencing technology, we analyzed TP53 mutation spectrum in its promoter region. Using PCR target sequence enrichment and next-generation bisulfite sequencing technology, we analyzed the methylation profile of the promoter and 3'-end regions of TP53 gene in paired breast tumor and normal tissues from 120 breast cancer patients. The expression of TP53 and the flanking gene ATP1B2 was explored with qPCR method in the same cohort. No promoter mutation of TP53 gene was found in the cohort of the 120 breast cancer patients. The 3'-end of TP53 gene was hyper-methylated (average 78.71%) compared with the promoter region (average less than 1%) in breast tumor tissues. TP53 was significantly lower expressed (P = 1.68E-15) and hyper-methylated in 3'-end (P = 1.82E-18) in tumor. Negative cis correlation was found between the TP53 expression and its 3'-end methylation (P = 9.02E-8, R = 0.337). TP53 expression was significantly associated with PR status (P = 0.0128), Ki67 level (P = 0.0091), and breast cancer subtypes (P = 0.0109). TP53 3'-end methylation and expression showed a good performance in discriminating breast cancer and normal tissues with an AUC of 0.930. The 3'-end methylation of TP53 might be a crucial regulator for its expression in breast cancer, suggesting that TP53 3'-end hyper-methylation associated with its lower expression could be a potential biomarker for breast cancer diagnosis.

Identification of Driver Mutations in MicroRNAs for Early Diagnosis of Lung Cancer

Over the past years, it has been discovered that microRNA possesses a critical role in lung cancer related deaths worldwide. Different research on molecular biology of lung cancer has improved our understanding that determines the behavior of malignant cells which has encouraged the discovery of driver mutations. The identification of driver mutations in lung cancer has led to a paradigm shift and provides us with new direction towards personalized therapies with improved survival rates. Therefore, it is of interest to explore microRNA driver mutations in this context. Hence, in this research different consistent point mutations are identified in candidate microRNAs at different sites and different numbers of transcription factors binding at these consistent mutations are examined. The highest consistency of mutation was 18 found in mir-23A at the genomic point (13880331) where total 5 transcription factors were binding. The second highest consistency was 12 found in different miRNAs at different genomic locals where 1-2 transcription factors were binding whereas; mir-25 is the only having consistency of 7 at genomic point (100089125) with 1 transcription factor attachment. The result of consistent mutation in regulatory regions indicates that these mutated sites may serve as potential biomarkers for lung cancer. Moreover, the comprehensive utilization of these microRNAs mutations, different drugs can be designed that mainly target these specified mutated loci and would be helpful in treating lung cancer.

Single-Cell RNA Sequencing Reveals a Distinct Transcriptome Signature of Hematopoiesis in GATA2 Deficiency

Constitutional GATA2 deficiency caused by heterozygous germline GATA2 mutations has a broad spectrum of clinical phenotypes including systemic infections, lymphedema, cytopenias, myelodysplasia, and a high risk of developing myeloid leukemias. GATA2 deficiency is recognized as a major MDS predisposition syndrome, germline GATA2 mutations are found in 7% of primary MDS cases in children and adolescents. Monosomy 7 and trisomy 8 are frequent chromosomal abnormalities in GATA2 deficiency and features of disease prognosis and malignant transformation. GATA2 mutations mostly affect two zinc finger domains and broadly classify into three major categories: missense, null, and regulatory region mutations. Profound monocytopenia, B-cell and NK-cell lymphopenias, and dendritic cell deficiency are specific for GATA2 deficiency. Absence of multi-lymphoid progenitor and decreased granulocyte -macrophage progenitors occur early in disease. There are limited studies of gene expression profiles in GATA2 deficiency and these usually in bulk populations, partly due to the paucity of cells in patients. How GATA2 deficiency negatively affects hematopoiesis, especially the preferential loss of specific lineages, is poorly understood. We performed single-cell RNA sequencing of sorted bone marrow CD34+ hematopoietic stem and progenitor cells (HSPCs) from eight GATA2 deficiency patients, who had various well characterized pathogenic GATA2 mutations and clinically manifest myelodysplasia (Fig A). We characterized transcriptomes in lineages, computationally defined cells with chromosomal abnormalities, and described gene expression of these cells. We first used gene expression signatures of single HSPCs from healthy donors to impute identity of stem cells or progenitors of erythroid/megakaryocytic (MEP), myeloid and lymphoid lineages, and then proceeded to reconstruct hematopoietic differentiation by pseudotemporal ordering. Mapping patients' cells onto normal hematopoiesis, we observed marked deficiency in granulocyte/monocyte/lymphocyte differentiation and relatively preserved MEP (Fig B, top). Based on signature genes of each differentiated lineage, we computed a score of gene expression to corresponding lineage in individual patient and found a significantly lower score for granulocyte/monocyte and B cell progenitors in patients, MEP scores tended to be higher in patients without significance (Fig B, bottom). Upregulated genes in patients' HSPCs related to erythroid differentiation, and cell cycle and proliferation (Fig C), while downregulated gene sets were highly enriched in immune responses (Fig D). Differential co-expressed genes were defined by weighted correlation network analysis; they were enriched in immune response and DNA repair (genes exhibited higher correlation in MTOR and T cell receptor signaling pathway in patients [Fig E]). We observed differential effects of GATA2 mutations in distinct lineages, which could only be resolved with the single cell method in rare cell populations. In HSCs, upregulated genes were related to apoptosis and downregulated genes were involved in maintenance of quiescence and cell cycle; there was increased expression of erythroid/megakaryocytic priming programs and decreased expression of lymphoid priming programs. We also observed lower GATA2 expression levels at initial stages of hematopoiesis, likely contributing to disturbed stem cell homeostasis in GATA2 deficiency. DNA repair genes were downregulated in trisomy 8 cells, possibly rendering these cells vulnerable to second-hit somatic mutations and additional chromosomal abnormalities. Cells with complex cytogenetics had defects in multi-lineage differentiation and cell cycle. In conclusion, germline GATA2 mutations modulate gene expression and change gene coexpression patterns. Distinct lineages show different transcriptional profiles resulting from GATA2 mutations. The prominent deficiency in myeloid/lymphoid lineages in GATA2 deficiency is partly due to expression of aberrant gene programs in HSCs prior to lineage commitment. Disclosures No relevant conflicts of interest to declare.

Clinical expression of cystic fibrosis in a large cohort of Italian siblings

BackgroundA clinical heterogeneity was reported in patients with Cystic Fibrosis (CF) with the same CFTR genotype and between siblings with CF.MethodsWe investigated all clinical aspects in a cohort of 101 pairs of siblings with CF (including 6 triplets) followed since diagnosis.ResultsSevere lung disease had a 22.2% concordance in sib-pairs, occurred early and the FEV1% at 12 years was predictive of the severity of lung disease in the adulthood. Similarly, CF liver disease occurred early (median: 15 years) and showed a concordance of 27.8% in sib-pairs suggesting a scarce contribution of genetic factors; in fact, only 2/15 patients with liver disease in discordant sib-pairs had a deficiency of alpha-1-antitrypsin (a known modifier gene of CF liver phenotype). CF related diabetes was found in 22 pairs (in 6 in both the siblings). It occurred later (median: 32.5 years) and is strongly associated with liver disease. Colonization by P. aeruginosa and nasal polyposis that required surgery had a concordance > 50% in sib-pairs and were poorly correlated to other clinical parameters. The pancreatic status was highly concordant in pairs of siblings (i.e., 95.1%) but a different pancreatic status was observed in patients with the same CFTR mutations. This suggests a close relationship of the pancreatic status with the “whole” CFTR genotype, including mutations in regulatory regions that may modulate the levels of CFTR expression. Finally, a severe course of CF was evident in a number of patients with pancreatic sufficiency.ConclusionsPhysicians involved in care of patients with CF and in genetic counseling must be aware of the clinical heterogeneity of CF even in sib-pairs that, at the state of the art, is difficult to explain.

Dark Side of the Genome: Study Links Noncoding Mutations in Regulatory Regions of the Cancer Genome to Altered Gene Expression

Dark Side of the Genome: Study Links Noncoding Mutations in Regulatory Regions of the Cancer Genome to Altered Gene Expression

DBTSS/DBKERO for integrated analysis of transcriptional regulation.

DBTSS (Database of Transcriptional Start Sites)/DBKERO (Database of Kashiwa Encyclopedia for human genome mutations in Regulatory regions and their Omics contexts) is the database originally initiated with the information of transcriptional start sites and their upstream transcriptional regulatory regions. In recent years, we updated the database to assist users to elucidate biological relevance of the human genome variations or somatic mutations in cancers which may affect the transcriptional regulation. In this update, we facilitate interpretations of disease associated genomic variation, using the Japanese population as a model case. We enriched the genomic variation dataset consisting of the 13,368 individuals collected for various genome-wide association studies and the reference epigenome information in the surrounding regions using a total of 455 epigenome datasets (four tissue types from 67 healthy individuals) collected for the International Human Epigenome Consortium (IHEC). The data directly obtained from the clinical samples was associated with that obtained from various model systems, such as the drug perturbation datasets using cultured cancer cells. Furthermore, we incorporated the results obtained using the newly developed analytical methods, Nanopore/10x Genomics long-read sequencing of the human genome and single cell analyses. The database is made publicly accessible at the URL (http://dbtss.hgc.jp/).