Mutations In NKX2 Research Articles

7775 Clinical Features of NKX2-1 Gene-Related Diseases

Abstract Disclosure: S. Fukui: None. E. Tanimoto: None. N. Ujita: None. H. Miyagi: None. K. Yoshii: None. N. Yasuhiro: None. R. Horikawa: None. Background: Congenital hypothyroidism (CH) is the most common congenital endocrine disorder. The utilization of molecular genetic techniques has facilitated the detection of CH resulting from various single-gene mutations; NKX2-1 gene-related disease stands out as one etiology of symptomatic CH. We report the clinical course of three cases with CH caused by NKX2-1 pathogenic variants. Case 1: An 8-year-old boy. He was born at 40 weeks and six days of gestation, weighing 2845g, and was presented to our department by high TSH (76.1 µIU/mL) at newborn screening (NBS), and LT4 treatment was initiated. He showed motor developmental delay, and choreiform movements were exhibited at two years and 11 months without respiratory symptoms. Case 2: An 18-year-old female. She was born at 40 weeks and five days gestation, weighing 2680g, and was admitted to her previous hospital following poor weight gain at approximately six months. During hospitalization, she was initiated LT4 treatment with a TSH elevation(14.7μIU/mL). After receiving a diagnosis of idiopathic interstitial pneumonia, she was subsequently referred to our hospital. At the age of 11, an NKX2-1 mutation was identified. Case 3: An 18-year-old female. She was born at 41 weeks and three days of gestation, weighing 2364g.TSH elevation (27.9 µIU/ml) was detected at NBS, and LT4 treatment was started. Thyroid hormone levels were maintained within normal range. However, wheezing and tracheomalacia were noted around five months. Hypotonia and delayed motor development were evident, and an NKX2-1 mutation was identified at the age of 12 years. Discussion: Although NKX2-1 gene-related disorders can be readily suspected when typical symptoms of central nervous system, pulmonary, and thyroid dysfunction manifest concurrently, diagnosis is sometimes established later because of the diversity of the occurrence of these symptoms. Despite reports of choreiform movements being inevitable, they did not appear in Case 2.3 until the age of 18 years. Conversely, muscle hypotonia and delayed motor development were observed in all our cases. When atypical neurological symptoms combined with CH are observed, NKX2-1 gene-related disease should be considered since the disease is known to develop malignant tumors such as lung cancer and thyroid cancer, which can significantly affect the prognosis of patients. Conclusion: When muscle hypotonia and delayed motor development are observed in addition to hypothyroidism and respiratory symptoms, NKX2-1 gene-related disease should be suspected, and follow-up for complications, including malignancy, is essential after diagnosis. Presentation: 6/2/2024

Brain abnormalities in young mice harboring combined NKX2-1 and PAX8 gene mutations

Brain abnormalities in young mice harboring combined NKX2-1 and PAX8 gene mutations

NKX2.5 coding exons sequencing reveals novel non-synonymous mutations in patients with sporadic congenital heart diseases among the Tanzanian population

BackgroundThe evolutionally conserved homeobox transcription factor NKX2-5 has been at the forefront in the field of cardiac biology, providing molecular insights into the mechanisms of cardiac development and disease. This homodomain transcription factor is a central regulator of cardiac development and is expressed in both the first and second heart fields (FHF and SHF). Mutations in the NKX2-5 gene have been linked to sporadic cases of congenital heart disease (CHD), making it a significant target for research and study. While several studies have been conducted on Caucasian populations, there is a dearth of knowledge on the effects of NKX2-5 gene mutations in other settings, underscoring the need for further investigation. Due to differences in geographical and ancestral origin, we hypothesize that mutations may vary across different populations. Understanding the genetic factors that cause CHD is essential for providing effective genetic counseling and developing strategies for risk reduction. Additionally, identification of NKX2-5 mutations in individuals with CHDs is crucial because patients with CHDs are at a higher risk of progressive conduction disease and sudden cardiac death, and genetic information is taken into consideration while making decisions regarding pacemakers and implantable cardiac defibrillators. To determine the risk of congenital heart disease among infants, we conducted a study where we sequenced the exon 1 and exon 2 of NKX 2.5 in patients with sporadic CHDs, with the aim of identifying mutations in the NKX2.5 gene.ResultsIn this study, a novel frame-shift disease-causing mutation was discovered in patients with atrial-ventricular septal defect. The mutation, identified as c95_95 del A; cDNA.369–369 delA; g 369–369 delA, resulted in the substitution of phenylalanine to leucine (F295L), which in turn caused a truncated NKX2.5 protein. In addition, a non-synonymous mutation, g 316C > T; cDNA 316C > T leucine to arginine (L37R) substitution, was found in a patient with the tetralogy of Fallot, affecting protein function. Furthermore, a novel non-synonymous mutation identified as g 2295–2298; cDNA 755–758 delins AGGG, was predicted by mutation taster to be disease-causing in a ventricular septal defect. It is worth noting that none of these mutations were found among the control subjects, highlighting their potential significance in the pathogenesis of these cardiac defects.ConclusionMutations in the NKX2.5 gene are associated with congenital heart diseases and provide molecular insight into the pathogenesis of congenital heart diseases. We recommend that patients with NKX2.5 mutations have periodic screening for cardiac conduction abnormalities and be evaluated for potential implanted cardiac defibrillators and pacemakers.

A Pediatric Case of Dilated Cardiomyopathy Due to NKX2-5 Mutation: Autopsy and Postmortem Molecular Diagnosis.

A Pediatric Case of Dilated Cardiomyopathy Due to NKX2-5 Mutation: Autopsy and Postmortem Molecular Diagnosis.

Human Genetics of Tetralogy of Fallot and Double-Outlet Right Ventricle.

Tetralogy of Fallot (TOF) and double-outlet right ventricle (DORV) are conotruncal defects resulting from disturbances of the second heart field and the neural crest, which can occur as isolated malformations or as part of multiorgan syndromes. Their etiology is multifactorial and characterized by overlapping genetic causes. In this chapter, we present the different genetic alterations underlying the two diseases, which range from chromosomal abnormalities like aneuploidies and structural mutations to rare singlenucleotide variations affecting distinct genes. For example, mutations in the cardiac transcription factors NKX2-5, GATA4, and HAND2 have been identified in isolated TOF cases, while mutations of TBX5 and 22q11 deletion, leading to haploinsufficiency of TBX1, cause Holt-Oram and DiGeorge syndrome, respectively. Moreover, genes involved in signaling pathways, laterality determination, and epigenetic mechanisms have also been found mutated in TOF and/or DORV patients. Finally, genome-wide association studies identified common singlenucleotide polymorphisms associated with the risk for TOF.

Abstract 19018: Unraveling the Regulatory Landscape of NKX2-5 in Human Left Atrial Tissue: Implications for Atrial Fibrillation

Cardiac transcription factors are emerging as crucial modulators of atrial fibrillation (AF), with rare and common variation implicated in AF. Mutations in NKX2-5 and more recently, NFATC1 were reported in families with autosomal dominant young-onset AF. Here, we explore the landscape of NKX2-5 and NFATC1 in human left atrium, using Chromatin Immunoprecipitation Sequencing (ChIP-seq), DAVID functional annotation and HOMER motif analyses. Post-mortem human left atrial tissue samples (n=5) were obtained from the National Disease Research Interchange (NDRI) within a recovery interval of < 4 hours. Preserved microstructural integrity and normal staining patters for TNNT2 and vimentin were verified using confocal microscopy. ChIP-seq analysis revealed an average of 2.62k NKX2-5 binding peaks, with 118 genes bound in all samples. Functional analysis identified significant enrichment in gene ontology categories and pathways (Table 1), including adrenergic and PPAR signaling pathways known to participate in AF susceptibility. Using HOMER, we mapped 55 NFATC1 consensus binding sites to the NKX2-5 ChIP peaks. Two NFATC1 consensus sites localized to an NKX2-5 binding peak located upstream from MEF2A (Figure 1). Published HI-C data from human right atrium indicates the NFATC1 binding sites are adjacent to the promoter of MEF2A in 3D space, suggesting that NKX2-5 and NFATC1 co-regulate MEF2A. In murine atria, mef2A regulates genes involved in fibrosis and adhesion, processes that are crucial in the pathogenesis of atrial fibrillation. Our data, together with published literature, provide a framework for understanding transcriptional networks that may predispose to human AF.

2225P FOX1 (rs965513), NKX2-1 (rs944289) and BRAF (V600E) gene mutations and their role in development of papillary thyroid cancer

2225P FOX1 (rs965513), NKX2-1 (rs944289) and BRAF (V600E) gene mutations and their role in development of papillary thyroid cancer

An Anterior Second Heart Field Enhancer Regulates the Gene Regulatory Network of the Cardiac Outflow Tract.

Conotruncal defects due to developmental abnormalities of the outflow tract (OFT) are an important cause of cyanotic congenital heart disease. Dysregulation of transcriptional programs tuned by NKX2-5 (NK2 homeobox 5), GATA6 (GATA binding protein 6), and TBX1 (T-box transcription factor 1) have been implicated in abnormal OFT morphogenesis. However, there remains no consensus on how these transcriptional programs function in a unified gene regulatory network within the OFT. We generated mice harboring a 226-nucleotide deletion of a highly conserved cardiac enhancer containing 2 GATA-binding sites located ≈9.4 kb upstream of the transcription start site of Nkx2-5 (Nkx2-5∆enh) using CRISPR-Cas9 gene editing and assessed phenotypes. Cardiac defects in Nkx2-5∆enh/∆enh mice were structurally characterized using histology and scanning electron microscopy, and physiologically assessed using electrocardiography, echocardiography, and optical mapping. Transcriptome analyses were performed using RNA sequencing and single-cell RNA sequencing data sets. Endogenous GATA6 interaction with and activity on the NKX2-5 enhancer was studied using chromatin immunoprecipitation sequencing and transposase-accessible chromatin sequencing in human induced pluripotent stem cell-derived cardiomyocytes. Nkx2-5∆enh/∆enh mice recapitulated cyanotic conotruncal defects seen in patients with NKX2-5, GATA6, and TBX1 mutations. Nkx2-5∆enh/∆enh mice also exhibited defects in right Purkinje fiber network formation, resulting in right bundle-branch block. Enhancer deletion reduced embryonic Nkx2-5 expression selectively in the right ventricle and OFT of mutant hearts, indicating that enhancer activity is localized to the anterior second heart field. Transcriptional profiling of the mutant OFT revealed downregulation of important genes involved in OFT rotation and septation, such as Tbx1, Pitx2, and Sema3c. Endogenous GATA6 interacted with the highly conserved enhancer in human induced pluripotent stem cell-derived cardiomyocytes and in wild-type mouse hearts. We found critical dose dependency of cardiac enhancer accessibility on GATA6 gene dosage in human induced pluripotent stem cell-derived cardiomyocytes. Our results using human and mouse models reveal an essential gene regulatory network of the OFT that requires an anterior second heart field enhancer to link GATA6 with NKX2-5-dependent rotation and septation gene programs.

Novel missense mutation in NKX2.6 gene (c.389 G > C, Arg130Pro) as a potentially pathogenic variant in pediatric patients with congenital heart disease

Congenital heart diseases (CHD) are the most common anomalies that occur during heart embryogenesis and are associated with significant mortality and morbidity in infants. It has been found that genomic alterations in cardiac-specific transcription factor genes play an important role in the pathogenesis of CHD. However, the prevalence of point mutations in these genes has not yet been determined. The aim of this research was to investigate the relationship of NKX2.6 gene mutations as a candidate gene in patients with congenital heart diseases. In the present study, two coding exons and flanking introns of the NKX2.6 gene, encoding a homeodomain-containing transcription factor, were sequenced in a cohort of patients. Five heterozygous NKX2.6 missense mutations, Ser37Cys, Glu54Val, Ala129Glu, Arg130Pro, Ser232Thr, and a synonymous variation, Asp68Asp, were identified in patients. They were not observed in controls, and the altered amino acids were evolutionarily highly conserved across species. The structural and functional consequences of missense mutations were assessed using various prediction servers. The consensus results showed that the Arg130Pro mutation is pathogenic/deleterious and likely to significantly affect protein function. To our knowledge, this is the first study on the association of NKX2.6 missense mutations with susceptibility to CHD in Iranian patients; our results present the spectrum of NKX2.6 mutations associated with patients and suggest that these missense mutations could be important as an uncommon cause of CHD.

Tissue specificity of oncogenic BRAF targeted to lung and thyroid through a shared lineage factor

Cells of origin in cancer determine tumor phenotypes, but whether lineage-defining transcription factors might influence tissue specificity of tumorigenesis among organs with similar developmental traits are unknown. We demonstrate here that tumor development and progression markedly differ in lung and thyroid targeted by Braf mutation in Nkx2.1CreERT2 mice heterozygous for Nkx2-1. In absence of tamoxifen, non-induced Nkx2.1CreERT2;BrafCA/+ mutants developed multiple full-blown lung adenocarcinomas with a latency of 1-3months whereas thyroid tumors were rare and constrained, although minute BrafCA activation documented by variant allele sequencing was similar in both tissues. Induced oncogene activation accelerated neoplastic growth only in the lungs. By contrast, NKX2-1+ progenitor cells were equally responsive to constitutive expression of mutant Braf during lung and thyroid development. Both lung and thyroid cells transiently downregulated NKX2-1 in early tumor stages. These results indicate that BRAFV600E-induced tumorigenesis obey organ-specific traits that might be differentially modified by a shared lineage factor.

Nav1-8, a new marker of Purkinje fibers, provides insight into defective conduction system maturation in a mouse model of congenital cardiomyopathy

The transcription factor Nkx2-5 is known to be a key regulator of cardiac development and NKX2-5 mutations have been found in numerous cardiac diseases associated with conduction defects. This factor is required for the differentiation of Purkinje fibers (PF), which have been implicated in triggering ventricular arrhythmias. A better understanding of the development of the Purkinje system in normal and Nkx2-5 mutant hearts could provide insight into their involvement in conduction defects in cardiac diseases. However, analysis of the Purkinje network development is hampered by the lack of specific lineage tracers. Recently, the voltage-gated sodium channel Nav1-8 short transcript has been shown to be expressed in the ventricular conduction system (VCS). This study aims to characterize the expression domain of Nav1-8 in the murine VCS to determine whether it is a suitable tool for tracing conductive cells. This would also help to identify defects in conductive recruitment in congenital disease. Nav1-8-Flp mice were crossed with a GFP reporter line for genetic tracing analyses in WT and Nkx2-5 heterozygous mice. The specificity and efficiency of Nav1-8 labelling was measured from P1 to P21 based on the number of GFP-positive (Nav1-8 derivatives), Cntn2-positive (VCS) and double positive cells (Nav1-8 derivatives in the VCS). GFP reporter expression starts at birth and is restricted to the PF at the distal part of the VCS. The specificity of Nav1-8 as a marker of PF increased over time between P1 and P21, suggesting that Nav1.8 is expressed in PF progenitor cells prior to their recruitment to the conduction system. However, its efficiency is limited to 20% of PF being marked at a mature stage of development (P21). In Nkx2-5 mutant hearts, GFP labelling within the left ventricle delimits the PF scaffold and is almost absent in the complex ramifications of the PF network. This anatomical distribution is consistent with the hypoplastic Purkinje system of these mice. However, unlike in control hearts, additional GFP+ cells are found in the bundle branches at the proximal part of the VCS in Nkx2-5 mutants. Our study shows that the sodium channel Nav1-8 represents a unique and highly specific lineage tracer of the peripheral PF in mature hearts. In addition, we show an enlargement of the Nav1-8 expression domain to the bundle branches in Nkx2-5 mutants. This indicates that Nkx2-5 may influence cell fate choice within the VCS.

Nkx2-5 Loss of Function in the His-Purkinje System Hampers Its Maturation and Leads to Mechanical Dysfunction.

The ventricular conduction or His-Purkinje system (VCS) mediates the rapid propagation and precise delivery of electrical activity essential for the synchronization of heartbeats. Mutations in the transcription factor Nkx2-5 have been implicated in a high prevalence of developing ventricular conduction defects or arrhythmias with age. Nkx2-5 heterozygous mutant mice reproduce human phenotypes associated with a hypoplastic His-Purkinje system resulting from defective patterning of the Purkinje fiber network during development. Here, we investigated the role of Nkx2-5 in the mature VCS and the consequences of its loss on cardiac function. Neonatal deletion of Nkx2-5 in the VCS using a Cx40-CreERT2 mouse line provoked apical hypoplasia and maturation defects of the Purkinje fiber network. Genetic tracing analysis demonstrated that neonatal Cx40-positive cells fail to maintain a conductive phenotype after Nkx2-5 deletion. Moreover, we observed a progressive loss of expression of fast-conduction markers in persistent Purkinje fibers. Consequently, Nkx2-5-deleted mice developed conduction defects with progressively reduced QRS amplitude and RSR' complex associated with higher duration. Cardiac function recorded by MRI revealed a reduction in the ejection fraction in the absence of morphological changes. With age, these mice develop a ventricular diastolic dysfunction associated with dyssynchrony and wall-motion abnormalities without indication of fibrosis. These results highlight the requirement of postnatal expression of Nkx2-5 in the maturation and maintenance of a functional Purkinje fiber network to preserve contraction synchrony and cardiac function.

Mutational Assessment in NKX2-5 and ACTC1 Genes in Patients with Congenital Cardiac Septal Defect (CCSD) from Ethnic Kashmiri Population.

(1) Background globe. The etiology of CHDs is complex and involves both genetic and non-genetic factors. Although, significant progress has been made in deciphering the genetic components involved in CHDs, recent reports have revealed that mutations in Nk2 homeobox5 (NKX2-5) and actin alpha cardiac muscle1 (ACTC1) genes play a key role in CHDs such as atrial and ventricular septum defects. Therefore, the present study evaluates the role of key hotspot mutations in NKX2-5 and ACTC1 genes of congenital cardiac septal defect (CCSD) in ethnic Kashmiri population. (2) Methods: A total of 112 confirmed CHD patients were included in the current study, of which 30 patients were evaluated for mutational analysis for hotspot mutations of NKX2-5 and ACTC1 genes. The total genomic DNA was extracted from the samples (cardiac tissue/blood) and were subjected to amplification for NKX2-5 (exon 1 and 2), and ACTC1 (exon 2) genes by using PCR specific primers to analyze the hotspot mutations in respective exons. The amplified products obtained were sent to Macrogen Korea for sequencing by Sanger’s method. (3) Results: Our results confirmed that not a single mutation was found in either hotspot exon 1 and 2 of NKX2-5 and exon 2 of ACTC1 in the patients included in the current study. Interestingly, a novel synonymous nucleotide variation leading to G > C transversion (GCG > GCC) was found in exon 2 of NKX2-5 gene of CCSD patient. (4) Conclusions: The current findings demonstrated the role of NKX2-5 and ACTC1 in cardiac development. The study will provide an insight in understanding the genetic etiology and highlights the role of newly identified mutations in patients with CDS’s in ethnic Kashmiri population. In silico findings revealed amino acid changes, splice site variation and the creation of new site. Furthermore, the study warrants complete screening of genes involved in CCSDs.

A Novel Splicing Mutation c.335–1 G > A in the Cardiac Transcription Factor NKX2-5 Leads to Familial Atrial Septal Defect Through miR-19 and PYK2

Mutations of NKX2-5 largely contribute to congenital heart diseases (CHDs), especially atrial septal defect (ASD). We identified a novel heterozygous splicing mutation c.335-1G > A in NKX2-5 gene in an ASD family via whole exome sequencing (WES) and linkage analysis. Utilizing the human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) as a disease model, we showed that haploinsufficiency of NKX2-5 contributed to aberrant orchestration of apoptosis and proliferation in ASD patient-derived hiPSC-CMs. RNA-seq profiling and dual-luciferase reporter assay revealed that NKX2-5 acts upstream of PYK2 via miR-19a and miR-19b (miR-19a/b) to regulate cardiomyocyte apoptosis. Meanwhile, miR-19a/b are also downstream mediators of NKX2-5 during cardiomyocyte proliferation. The novel splicing mutation c.335-1G > A in NKX2-5 and its potential pathogenic roles in ASD were demonstrated. Our work provides clues not only for deep understanding of NKX2-5 in cardia development, but also for better knowledge in the molecular mechanisms of CHDs.

Evaluating the Impact of Non‐coding Variants in the Cardiac Transcription Factor NKX2‐5 Binding Sites

Transcription Factors (TFs) are DNA‐specific binding proteins that regulate gene expression and other biological processes like cell differentiation. For example, the cardiac TF NKX2‐5 has been proven to be a master regulator in the transcriptional network needed for heart development. Mutations in NKX2‐5 have been shown to cause congenital heart diseases (CHDs), the most common birth defect that result in structural abnormalities in the heart. However, ~95% of disease‐associated mutations occur within the non‐coding genome, such as promoters and enhancers. The molecular mechanism of these genetic variations and their role in many diseases has remained unexplored. We hypothesize that non‐coding variants within NKX2‐5 binding sites will affect DNA recognition and alter TF‐DNA binding. First, using ~84 million Single Nucleotide Polymorphisms (SNPs) reported in the 1000 Genomes Project, I identified 8,475 that are predicted to affect NKX2‐5 binding. After filtering these variants, I found 901 SNPs localized in cardiac enhancers, and 30 disease‐associated SNPs from the GWAS catalog predicted to affect NKX2‐5 binding sites. Using position weight matrix (PWM) DNA‐specificity models, binding scores for the identified mutations were calculated. The variants rs7350789 and rs7719885 were predicted to have the greatest impact in NKX2‐5 binding affinity with ΔPWM scores of 258 and ‐212 respectively. These variants were prioritized for in vitro validation through Electrophoretic Mobility Shift Assay (EMSA) using purified full‐length NKX2‐5 to evaluate TF‐DNA complex formation. After performing an EMSA, both variants resulted in an increase in TF‐DNA binding even though rs7719885 had a negative ΔPWM score which predicts a decrease in binding affinity. Although not as expected, two of the variants predicted to impact TF‐DNA binding were successfully validated.

Protein‐DNA Interactomes of NKX2‐5 and TBX5 Mutants Identified in Congenital Heart Defects

Congenital Heart Defects (CHDs) are the most common birth defect from children to adults in the United States. CHDs are characterized by malformations in the heart’s chambers, walls, and great vessels, sometimes leading to embryonic death. Cardiac Transcription Factors (cardiac TFs) such as NKX2‐5 and TBX5 play a critical role in developmental stages and physiological processes of the heart by regulating gene expression through DNA‐protein interactions. Genetic analyses have identified various non‐synonymous mutations within the homeodomain (HD) DNA‐binding domain (DBD) of NKX2‐5 and the (Tbox) DBD of TBX5 in associated with CHD. We hypothesize that non‐synonymous mutations in cardiac TFs DBD will alter their DNA‐binding properties ultimately affecting transcriptional pathways necessary for proper heart development. To address this hypothesis, we studied how the cardiac TFs DNA‐protein interactomes are affected by non‐synonymous mutations found in CHD patients. We expressed and purified the DBD of NKX2‐5 (HD), TBX5 (Tbox), also NKX2‐5 and TBX5 non‐synonymous mutations (A148E, E154G, R161P, T178M, Q181H, R190C, Y191C), and (I54T, M74V, G80R, I101F, R113K, R237Q, R237W) respectively. Functional analysis using Electrophoretic Mobility Shift Assay (EMSA) confirms HD and Tbox binding to cognate DNA binding sites. DNA‐binding activity as assessed by EMSA, shows retention of DNA activity in the case of NKX2‐5 and TBX5 mutants A148E, R161P, Y191C and I54T, R113K respectively. Mutants T178M, Q181H, R190C, M74V, G80R, I101F, R237Q, R237W showed complete loss in DNA binding activity towards their specific cognate sites. We measured the comprehensive protein‐DNA interactomes of NKX2‐5 and TBX5 non‐synonymous mutants using High‐Throughput Systematic Evolution of Ligands and Exponential Enrichment (SELEX‐seq) to identify changes in DNA targets between wild‐type and mutant TFs. Mutants A148E, R161P and Y191C bind to similar binding sites as NKX2‐5 (HD). Interestingly, mutant Y191C showed an altered DNA binding site significantly different from NKX2‐5 (HD). Mutants I54T, R113K bind to similar binding sites as TBX5 (Tbox). Mutants E154G, T178M, Q181H, R190C, M74V, G80R, I101F, R237Q and R237W showed no significant enrichment for specific DNA binding sites. Integrative analysis with available NKX2‐5 and TBX5 genomic binding sites data (ChIP‐seq), human and mouse cardiac enhancers and promoters catalogued in the literature will facilitate the identification of putative genes mutant cardiac TFs are regulating and how different are from their wild‐type TFs.

Genomic characteristics of invasive mucinous adenocarcinoma of the lung with multiple pulmonary sites of involvement

Invasive mucinous adenocarcinoma (IMA) of the lung frequently presents with diffuse pneumonic-type features or multifocal lesions, which are regarded as a pattern of intrapulmonary metastases. However, the genomics of multifocal IMAs have not been well studied. We performed whole exome sequencing on samples taken from 2 to 5 regions in seven patients with synchronous multifocal IMAs of the lung (24 regions total). Early initiating driver events, such as KRAS, NKX2-1, TP53, or ARID1A mutations, are clonal mutations and were present in all multifocal IMAs in each patient. The tumor mutational burden of multifocal IMAs was low (mean: 1.13/mega base), but further analyses suggested intra-tumor heterogeneity. The mutational signature analysis found that IMAs were predominantly associated with endogenous mutational process (signature 1), APOBEC activity (signatures 2 and 13), and defective DNA mismatch repair (signature 6), but not related to smoking signature. IMAs synchronously located in the bilateral lower lobes of two patients with background usual interstitial pneumonia had different mutation types, suggesting that they were double primaries. In conclusion, genomic evidence found in this study indicated the clonal intrapulmonary spread of diffuse pneumonic-type or multifocal IMAs, although they can occur in multicentric origins in the background of usual interstitial pneumonia. IMAs exhibited a heterogeneous genomic landscape despite the low somatic mutation burden. Further studies are warranted to determine the clinical significance of the genomic characteristics of IMAs in expanded cohorts.

Whole-Exome Sequencing of Paroxysmal Nocturnal Hemoglobinuria

OBJECTIVE: In order to elucidate the possible mechanism on the proliferation of paroxysmal nocturnal hemoglobinuria (PNH) clone, and investigate the significance of gene mutation in PNH patients with thrombosis and provide a theoretical basis for related molecules markers and potential therapeutic targets in PNH, we performed the whole genome exon sequencing in PNH patients.METHODS: The peripheral blood and nail specimens of 7 PNH and 6 AA-PNH patients were obtained and we have used V2's NimbleGen capture probe to capturethe whole exon group (WES), and performed whole genome sequencing of exon in patients, meanwhile, processing and analysis of sequencing data were obtained and compared with the 1000 genome project database, to obtain high frequency variation genes and further gene enrichment pathway analysis in these patients. Using a combination of KEGG pathway analysis and GO pathway enrichment analysis methods to analyze the experimental data.RESULTS: We detected somatic PIGA mutations in the PNH fractions of 8 of 13 cases analyzed by WES. And we identified a total of 4 shear site, 4 non-synonymous, 4 truncation, and 6 frame-shift mutations in cases analyzed by WES. All mutations were grade one. We performed KEGG pathway enrichment analysis and GO gene annotation of 5717 mutated genes screened, the union as a target gene set, there were 25 target genes, SUZ12, CUX1, MLL2, MUC4, CTBP1, AXIN2, MLL, SDHA, CARD11, MLH1, RB1, SMO and NKX2-1 mutations were recurrent in PNH patients; FGFR3, ERCC5, MPL, FLT3, TSC2, POLE, TET2, MLL3, MSH6, FAT1, CIC and ZFHX3 mutations were recurrent in AA-PNH patients. Among them, SUZ12, FLT3, CUX1, MUC4, TET2, and RB1 gene are involved in the regulation of cell proliferation and differentiation, promote the growth, metastasis and invasion of tumor cells. In addition, the pathways enriched by mutant genes included pathways in cancer, central carbon metabolism in cancer signaling pathway and cell cycle signal pathways. GO analysis showed that the coding protein CDP has the functions of cell proliferation regulation, metabolism regulation, gene expression regulation, cell invasiveness, cell motility, cell apoptosis, extracellular matrix compositions, and so on. Furthermore, we analyzed the gene mutations in patients with thrombosis, which includes several genes with the higher deleterious mutation in protein structure, such as BMPR2, ZFPM2, F5, F8, F12, F13A1, MCL1, MMRN1, NBEAL2, NOS1and PC. The major functions of mutation sites were non synonymous single nucleotide variants and synonymous single nucleotide variants. The pathways enriched by mutant genes included the Notch signaling pathway, Ribosome biogenesis in eukaryotes signaling pathway, Wnt signaling pathway and Arachidonic acid metabolism signaling pathways, which may be related to the pathogenesis of PNH associated thrombosis.CONCLUSION: We identified additional somatic mutations that resulted in a complex hierarchical clonal architecture, similar to that observed in myeloid neoplasms and solid tumors. In addition to PIG-A mutations, mutations were found in genes known to be involved in myeloid neoplasm pathogenesis and solid tumors, including SUZ12, FLT3, CUX1, MUC4, TET2, and RB1. Our data indicate that in addition to PIG-A mutations, accessory genetic events are frequent in PNH, suggesting a stepwise clonal evolution derived from a singular stem cell clone. Meanwhile, several genes with higher deleterious mutation in PNH patients with thrombosis were found: F5, F8, F12, F13A1, THBD, ITGA2 and BMPR2, which enriched the Notch signaling pathway, Ribosome biogenesis in eukaryotes signaling pathway, Wnt signaling pathway and Arachidonic acid metabolism signaling pathways. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Gestational intermittent hyperoxia rescues murine genetic congenital heart disease in part

Cardiac development is a dynamic process, temporally and spatially. When disturbed, it leads to congenital cardiac anomalies that affect approximately 1% of live births. Genetic variants in several loci lead to anomalies, with the transcription factor NKX2-5 being one of the largest. However, there are also non-genetic factors that influence cardiac malformations. We examined the hypothesis that hyperoxia may be beneficial and can rescue genetic cardiac anomalies induced by an Nkx2-5 mutation. Intermittent mild hyperoxia (40% PO2) was applied for 10 h per day to normal wild-type female mice mated with heterozygous Nkx2-5 mutant males from gestational day 8.5 to birth. Hyperoxia therapy reduced excessive trabeculation in Nkx2-5 mutant mice compared to normoxic conditions (ratio of trabecular layer relative to compact layer area, normoxia 1.84 ± 0.07 vs. hyperoxia 1.51 ± 0.04) and frequency of muscular ventricular septal defects per heart (1.53 ± 0.32 vs. 0.68 ± 0.15); however, the incidence of membranous ventricular septal defects in Nkx2-5 mutant hearts was not changed. Nkx2-5 mutant embryonic hearts showed defective coronary vessel organization, which was improved by intermittent mild hyperoxia. The results of our study showed that mild gestational hyperoxia therapy rescued genetic cardiac malformation induced by Nkx2-5 mutation in part.

Congenital Hypothyroidism: A Review of 2020 European Society for Pediatric Endocrinology and the European Society for Endocrinology Consensus Guidelines

Congenital Hypothyroidism: A Review of 2020 European Society for Pediatric Endocrinology and the European Society for Endocrinology Consensus Guidelines