ABSTRACT
Ichthyosis follicularis, atrichia, and photophobia syndrome (IFAP syndrome) is a rare, X-linked disorder caused by pathogenic variants in membrane-bound transcription factor protease, site 2 (MBTPS2). Pathogenic MBTPS2 variants also cause BRESHECK syndrome, characterized by the IFAP triad plus intellectual disability and multiple congenital anomalies. Here we present a patient with ichthyosis, sparse hair, pulmonic stenosis, kidney dysplasia, hypospadias, growth failure, thrombocytopenia, anemia, bone marrow fibrosis, and chronic diarrhea found by research-based exome sequencing to harbor a novel, maternally inherited MBTPS2 missense variant (c.766 G>A; (p.Val256Leu)). In vitro modeling supports variant pathogenicity, with impaired cell growth in cholesterol-depleted media, attenuated activation of the sterol regulatory element-binding protein pathway, and failure to activate the endoplasmic reticulum stress response pathway. Our case expands both the genetic and phenotypic spectrum of BRESHECK syndrome to include a novel MBTPS2 variant and cytopenias, bone marrow fibrosis, and chronic diarrhea.
Subject(s)
Intellectual Disability , Alopecia/genetics , Brain/abnormalities , Congenital Abnormalities , Ear/abnormalities , Ectodermal Dysplasia , Endoplasmic Reticulum Stress/genetics , Genetic Diseases, X-Linked , Hirschsprung Disease , Humans , Intellectual Disability/genetics , Kidney/abnormalities , Male , Metalloendopeptidases/genetics , Peptide Hydrolases , Sterols , Transcription FactorsABSTRACT
Twelvepatients without therapy-related leukemia were studied after completing TOP2 poison chemotherapy in a high-risk neuroblastoma regimen. One patient harbored an inv(11) that was a KMT2A rearrangement. The KMT2A-MAML2 transcript was expressed at low level. The patient was prospectively followed. The inv(11) was undetectable in ensuing samples. Leukemia never developed after a 12.8-year follow-up period. Enriched etoposide-induced TOP2A cleavage in the relevant MAML2 genomic region supports a TOP2A DNA damage mechanism. After completing TOP2 poison chemotherapies, covert KMT2A-R clones may occur in a small minority of patients; however, not all KMT2A rearrangements herald a therapy-related leukemia diagnosis.
Subject(s)
Histone-Lysine N-Methyltransferase , Leukemia , Myeloid-Lymphoid Leukemia Protein , Neuroblastoma , Trans-Activators , Etoposide/administration & dosage , Follow-Up Studies , Gene Rearrangement , Humans , Leukemia/genetics , Myeloid-Lymphoid Leukemia Protein/genetics , Neuroblastoma/drug therapy , Neuroblastoma/genetics , Transcription Factors/geneticsABSTRACT
Ring-finger protein 213 (RNF213) encodes a protein of unknown function believed to play a role in cellular metabolism and angiogenesis. Gene variants are associated with susceptibility to moyamoya disease. Here, we describe two children with moyamoya disease who also demonstrated kidney disease, elevated aminotransferases, and recurrent skin lesions found by exome sequencing to have de novo missense variants in RNF213. These cases highlight the ability of RNF213 to cause Mendelian moyamoya disease in addition to acting as a genetic susceptibility locus. The cases also suggest a new, multi-organ RNF213-spectrum disease characterized by liver, skin, and kidney pathology in addition to severe moyamoya disease caused by heterozygous, de novo C-terminal RNF213 missense variants.
Subject(s)
Adenosine Triphosphatases/genetics , Kidney Diseases/genetics , Moyamoya Disease/genetics , Skin Diseases/genetics , Ubiquitin-Protein Ligases/genetics , Female , Genetic Predisposition to Disease , Humans , Infant , Infant, Newborn , Kidney Diseases/complications , Kidney Diseases/pathology , Male , Moyamoya Disease/complications , Moyamoya Disease/pathology , Neovascularization, Physiologic/genetics , Skin Diseases/complications , Skin Diseases/pathology , Transaminases/genetics , Exome SequencingABSTRACT
BACKGROUND: Peripheral blood mononuclear cells (PBMCs) are widely used as a model in the study of different human diseases. There is often a time delay from blood collection to PBMC isolation during the sampling process, which can result in an experimental bias, particularly when performing single cell RNA-seq (scRNAseq) studies. METHODS: This study examined the impact of different time periods from blood draw to PBMC isolation on the subsequent transcriptome profiling of different cell types in PBMCs by scRNAseq using the 10X Chromium Single Cell Gene Expression assay. RESULTS: Examining the five major cell types constituting the PBMC cell population, i.e., CD4+ T cells, CD8+ T cells, NK cells, monocytes, and B cells, both common changes and cell-type-specific changes were observed in the single cell transcriptome profiling over time. In particular, the upregulation of genes regulated by NF-kB in response to TNF was observed in all five cell types. Significant changes in key genes involved in AP-1 signaling were also observed. RBC contamination was a major issue in stored blood, whereas RBC adherence had no direct impact on the cell transcriptome. CONCLUSIONS: Significant transcriptome changes were observed across different PBMC cell types as a factor of time from blood draw to PBMC isolation and as a consequence of blood storage. This should be kept in mind when interpreting experimental results.
Subject(s)
Leukocytes, Mononuclear , Single-Cell Gene Expression Analysis , Humans , Leukocytes, Mononuclear/metabolism , Gene Expression Profiling , Transcriptome , Killer Cells, NaturalABSTRACT
Central conducting lymphatic anomaly (CCLA) due to congenital maldevelopment of the lymphatics can result in debilitating and life-threatening disease with limited treatment options. We identified 4 individuals with CCLA, lymphedema, and microcystic lymphatic malformation due to pathogenic, mosaic variants in KRAS. To determine the functional impact of these variants and identify a targeted therapy for these individuals, we used primary human dermal lymphatic endothelial cells (HDLECs) and zebrafish larvae to model the lymphatic dysplasia. Expression of the p.Gly12Asp and p.Gly13Asp variants in HDLECs in a 2dimensional (2D) model and 3D organoid model led to increased ERK phosphorylation, demonstrating these variants activate the RAS/MAPK pathway. Expression of activating KRAS variants in the venous and lymphatic endothelium in zebrafish resulted in lymphatic dysplasia and edema similar to the individuals in the study. Treatment with MEK inhibition significantly reduced the phenotypes in both the organoid and the zebrafish model systems. In conclusion, we present the molecular characterization of the observed lymphatic anomalies due to pathogenic, somatic, activating KRAS variants in humans. Our preclinical studies suggest that MEK inhibition should be studied in future clinical trials for CCLA due to activating KRAS pathogenic variants.
Subject(s)
Proto-Oncogene Proteins p21(ras) , Zebrafish , Animals , Humans , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Endothelial Cells/metabolism , Phosphorylation , Mitogen-Activated Protein Kinase Kinases/metabolism , Zebrafish Proteins/genetics , Zebrafish Proteins/metabolismABSTRACT
We examined the MLL translocation in two cases of infant AML with X chromosome disruption. The G-banded karyotype in the first case suggested t(X;3)(q22;p21)ins(X;11)(q22;q13q25). Southern blot analysis showed one MLL rearrangement. Panhandle PCR approaches were used to identify the MLL fusion transcript and MLL genomic breakpoint junction. SEPTIN6 from chromosome band Xq24 was the partner gene of MLL. MLL exon 7 was joined in-frame to SEPTIN6 exon 2 in the fusion transcript. The MLL genomic breakpoint was in intron 7; the SEPTIN6 genomic breakpoint was in intron 1. Spectral karyotyping revealed a complex rearrangement disrupting band 11q23. FISH with a probe for MLL confirmed MLL involvement and showed that the MLL-SEPTIN6 junction was on the der(X). The MLL genomic breakpoint was a functional DNA topoisomerase II cleavage site in an in vitro assay. In the second case, the karyotype revealed t(X;11)(q22;q23). Southern blot analysis showed two MLL rearrangements. cDNA panhandle PCR detected a transcript fusing MLL exon 8 in-frame to SEPTIN6 exon 2. MLL and SEPTIN6 are vulnerable to damage to form recurrent translocations in infant AML. Identification of SEPTIN6 and the SEPTIN family members hCDCrel and MSF as partner genes of MLL suggests a common pathway to leukaemogenesis.
Subject(s)
Chromosomes, Human, Pair 11/genetics , DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/genetics , GTP-Binding Proteins/genetics , Leukemia, Myeloid/genetics , Proto-Oncogenes , Transcription Factors , Translocation, Genetic/genetics , X Chromosome/genetics , Acute Disease , Base Sequence , Chromosome Breakage/genetics , Chromosome Mapping , Chromosomes, Human, Pair 3/genetics , Cytoskeletal Proteins , Histone-Lysine N-Methyltransferase , Humans , In Situ Hybridization, Fluorescence , Infant , Leukemia, Myelomonocytic, Acute/genetics , Molecular Sequence Data , Myeloid-Lymphoid Leukemia Protein , SeptinsABSTRACT
Few t(9;11) translocations in DNA topoisomerase II inhibitor-related leukemias have been studied in detail and the DNA damage mechanism remains controversial. We characterized the der(11) and der(9) genomic breakpoint junctions in a case of AML following etoposide and doxorubicin. Etoposide-, etoposide metabolite- and doxorubicin-induced DNA topoisomerase II cleavage was examined in normal homologues of the MLL and AF-9 breakpoint sequences using an in vitro assay. Induction of DNA topoisomerase II cleavage complexes in CEM and K562 cell lines was investigated using an in vivo complex of enzyme assay. The translocation occurred between identical 5'-TATTA-3' sequences in MLL intron 8 and AF-9 intron 5 without the gain or loss of bases. The 5'-TATTA-3' sequences were reciprocally cleaved by DNA topoisomerase II in the presence of etoposide, etoposide catechol or etoposide quinone, creating homologous 4-base 5' overhangs that would anneal to form both breakpoint junctions without any processing. der(11) and der(4) translocation breakpoints in a treatment-related ALL at the same site in MLL are consistent with a damage hotspot. Etoposide and both etoposide metabolites induced DNA topoisomerase II cleavage complexes in the hematopoietic cell lines. These results favor the model in which the chromosomal breakage leading to MLL translocations in DNA topoisomerase II inhibitor-related leukemias is a consequence of DNA topoisomerase II cleavage.
Subject(s)
DNA Topoisomerases, Type II/metabolism , DNA-Binding Proteins/metabolism , Leukemia, Myeloid, Acute/metabolism , Nuclear Proteins/metabolism , Protein Processing, Post-Translational , Proto-Oncogenes , Transcription Factors , Adolescent , DNA Topoisomerases, Type II/genetics , DNA-Binding Proteins/genetics , Histone-Lysine N-Methyltransferase , Humans , In Situ Hybridization, Fluorescence , Leukemia, Myeloid, Acute/genetics , Male , Molecular Sequence Data , Myeloid-Lymphoid Leukemia Protein , Nuclear Proteins/genetics , Recombination, Genetic , Translocation, GeneticABSTRACT
PURPOSE: Although p53 mutations occur in alkylating agent-related leukemias, their frequency and spectrum in leukemias after ovarian cancer have not been addressed. The purpose of this study was to examine p53 mutations in leukemias after ovarian cancer, for which treatment with platinum analogues was widely used. EXPERIMENTAL DESIGN: Adequate leukemic or dysplastic cells were available in 17 of 82 cases of leukemia or myelodysplastic syndrome that occurred in a multicenter, population-based cohort of 23,170 women with ovarian cancer. Eleven of the 17 received platinum compounds and other alkylating agents with or without DNA topoisomerase II inhibitors and/or radiation. Six received other alkylating agents, in one case, with radiation. Genomic DNA was extracted and p53 exons 5, 6, 7, and 8 were amplified by PCR. Mutations and loss of heterozygosity were analyzed on the WAVE instrument (Transgenomic) followed by selected analysis by sequencing. RESULTS: Eleven p53 mutations involving all four exons studied and one polymorphism were identified. Genomic DNA analyses were consistent with loss of heterozygosity for four of the mutations. The 11 mutations occurred in 9 cases, such that 6 of 11 leukemias after platinum-based regimens (55%) and 3 of 6 leukemias after other treatments (50%) contained p53 mutations. Two leukemias that occurred after treatment with platinum analogues contained two mutations. Among eight mutations in leukemias after treatment with platinum analogues, there were four G-to-A transitions and one G-to-C transversion. CONCLUSIONS: p53 mutations are common in leukemia and myelodysplastic syndrome after multiagent therapy for ovarian cancer. The propensity for G-to-A transitions may reflect specific DNA damage in leukemias after treatment with platinum analogues.
Subject(s)
Leukemia/genetics , Myelodysplastic Syndromes/genetics , Ovarian Neoplasms/drug therapy , Tumor Suppressor Protein p53/genetics , Adult , Aged , Antineoplastic Combined Chemotherapy Protocols/therapeutic use , Base Sequence , Cisplatin/administration & dosage , Combined Modality Therapy , DNA Mutational Analysis , DNA, Neoplasm/chemistry , DNA, Neoplasm/genetics , Female , Humans , Leukemia/complications , Middle Aged , Mutation , Myelodysplastic Syndromes/complications , Ovarian Neoplasms/complications , Ovarian Neoplasms/radiotherapyABSTRACT
Panhandle PCR techniques to amplify known sequence flanked by unknown sequence have been useful for MLL genomic breakpoint junctions and fusion transcripts because MLL has a large number of partner genes. However, genomic panhandle PCR approaches are impeded when the restriction fragment that contains the breakpoint junction is too large to amplify. We devised new panhandle PCR approaches for MLL genomic breakpoint junctions that create the template from BglII restriction fragments by attaching MLL sequence to a BglII site in the partner gene. This leads to the annealing of MLL and its complement in the handle and creates an intrastrand loop containing the breakpoint junction sequence for amplification with primers all from MLL. BglII panhandle PCR for der(11) breakpoint junctions was accomplished by ligating a phosphorylated oligonucleotide containing a BglII overhang and sequence complementary to MLL exon 7 to the 3' ends of BglII digested DNA, and forming the template from the sense strand of DNA. In BglII reverse panhandle PCR for der(other) breakpoint junctions, a phosphorylated oligonucleotide containing a BglII overhang and the complement of antisense sequence in MLL exon 10 was ligated to the 3' ends of BglII digested DNA, and the template was formed from the antisense strand of DNA. These approaches amplified 5'-MLL-MLLT4-3' and 5'-AFF1-MLL-3' breakpoint junctions. The former is significant because few t(6;11) genomic breakpoint junctions have been sequenced. BglII panhandle PCR approaches increase the possibilities for cloning MLL genomic breakpoint junctions where there is heterogeneity in partner genes and breakpoint locations.
Subject(s)
Bacterial Proteins , Chromosome Breakage , Chromosomes, Human, Pair 11 , Cloning, Molecular/methods , Deoxyribonucleases, Type II Site-Specific , Myeloid-Lymphoid Leukemia Protein/genetics , Polymerase Chain Reaction/methods , Translocation, Genetic , Adolescent , Base Sequence , Chromosomes, Human, Pair 4 , Chromosomes, Human, Pair 6 , Humans , Models, Biological , Models, Genetic , Molecular Sequence Data , Myeloid-Lymphoid Leukemia Protein/metabolism , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor Cell Lymphoblastic Leukemia-Lymphoma/metabolismABSTRACT
Rearrangements involving the MLL gene on chromosome band 11q23 are a hallmark of therapy-related acute myeloid leukemias following treatment with topoisomerase II poisons including etoposide. Therapy-related and de novo genomic translocation breakpoints cluster within a well-characterized 8.3-kb fragment of MLL. Repair of etoposide-stabilized DNA topoisomerase II covalent complexes may initiate MLL rearrangements observed in patients. We used a culture system of primary human hematopoietic CD34+ cells and inverse polymerase chain reaction to characterize the spectrum of stable genomic rearrangements promoted by etoposide exposure originating within an MLL translocation hotspot in therapy-related leukemia. Alterations to the region were observed at a readily detectable frequency in etoposide-treated cells. Illegitimate repair events after minimal repair included MLL tandem duplications and translocations, with minor populations of deletions or insertions. In stably repaired cells that proliferated for 10 to 14 days, the significant majority of illegitimate events were MLL tandem duplications, and several deletions, inversions, insertions, and translocations. Thus, etoposide promotes specific rearrangements of MLL consistent with the full spectrum of oncogenic events identified in leukemic samples. Although etoposide-initiated rearrangements are frequent, only a small subset of translocations occurs in cells that proliferate significantly.
Subject(s)
DNA-Binding Proteins/genetics , Etoposide/adverse effects , Gene Rearrangement/drug effects , Hematopoietic Stem Cells/drug effects , Leukemia, Myeloid/chemically induced , Neoplasms, Second Primary/genetics , Proto-Oncogenes/genetics , Transcription Factors/genetics , Acute Disease , Antigens, CD34 , Cell Proliferation , Cells, Cultured , Clone Cells , Fetal Blood , Hematopoietic Stem Cells/pathology , Histone-Lysine N-Methyltransferase , Humans , Leukemia, Myeloid/genetics , Myeloid-Lymphoid Leukemia Protein , Translocation, GeneticABSTRACT
An inactivating polymorphism at position 609 in the NAD(P)H:quinone oxidoreductase 1 gene (NQO1 C609T) is associated with an increased risk of adult leukemia. A small British study suggested that NQO1 C609T was associated with an increased risk of infant leukemias with MLL translocations, especially infant acute lymphoblastic leukemia (ALL) with t(4;11). We explored NQO1 C609T as a genetic risk factor in 39 pediatric de novo and 18 pediatric treatment-related leukemias with MLL translocations in the United States. Children with de novo B-lineage ALL without MLL translocations and a calculation of the expected genotype distribution in an ethnically matched population of disease-free subjects served as the comparison groups. Patients with de novo leukemias with MLL translocations were significantly more likely to be heterozygous at NQO1 C609T (odds ratio [OR] = 2.77, 95% confidence intervals [CI] 1.17-6.57; P =.02), and significantly more likely to have low/null NQO1 activity than patients with de novo B-lineage ALL without MLL translocations (OR = 2.47, 95% CI 1.08-5.68; P =.033). They were also significantly more likely to have low/null NQO1 activity than expected in an ethnically matched population of disease-free subjects (OR = 2.50, P =.02). Infants younger than 12 months old at diagnosis of leukemia with t(4;11) were most likely to have low/null NQO1 activity (OR > 10.0). Conversely, the distribution of NQO1 genotypes among patients with treatment-related leukemias with MLL translocations was not statistically different than in the comparison groups. The inactivating NQO1 polymorphism is associated with an increased risk of de novo leukemia with MLL translocations in infants and children.
Subject(s)
Chromosomes, Human, Pair 11/ultrastructure , Chromosomes, Human, Pair 18/ultrastructure , DNA-Binding Proteins/genetics , Leukemia/enzymology , Mutation, Missense , NAD(P)H Dehydrogenase (Quinone)/deficiency , Neoplasm Proteins/deficiency , Point Mutation , Proto-Oncogenes , Transcription Factors , Translocation, Genetic , Acute Disease , Amino Acid Substitution , Child, Preschool , Ethnicity/genetics , Female , Genetic Predisposition to Disease , Histone-Lysine N-Methyltransferase , Humans , Infant , Infant, Newborn , Leukemia/epidemiology , Leukemia/genetics , Leukemia, Myeloid/enzymology , Leukemia, Myeloid/epidemiology , Leukemia, Myeloid/genetics , Male , Myeloid-Lymphoid Leukemia Protein , NAD(P)H Dehydrogenase (Quinone)/genetics , Neoplasm Proteins/genetics , Neoplastic Stem Cells/enzymology , Odds Ratio , Precursor Cell Lymphoblastic Leukemia-Lymphoma/enzymology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/epidemiology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/genetics , Risk FactorsABSTRACT
We used panhandle PCR to clone the der(11) genomic breakpoint junction in three leukemias with t(4;11) and devised reverse-panhandle PCR to clone the breakpoint junction of the other derivative chromosome. This work contributes two elements to knowledge on MLL translocations. First is reverse-panhandle PCR for cloning breakpoint junctions of the other derivative chromosomes, sequences of which are germane to understanding the MLL translocation process. The technique revealed duplicated sequences in one case of infant acute lymphoblastic leukemia (ALL) and small deletions in a case of treatment-related ALL. The second element is discovery of a three-way rearrangement of MLL, AF-4, and CDK6 in another case of infant ALL. Cytogenetic analysis was unsuccessful at diagnosis, but suggested t(4;11) and del(7)(q21q31) at relapse. Panhandle PCR analysis of the diagnostic marrow identified a breakpoint junction of MLL intron 8 and AF-4 intron 3. Reverse-panhandle PCR identified a breakpoint junction of CDK6 from band 7q21-q22 and MLL intron 9. CDK6 encodes a critical cell cycle regulator and is the first gene of this type disrupted by MLL translocation. Cdk6 is overexpressed or disrupted by translocation in many cancers. The in-frame CDK6-MLL transcript is provocative with respect to a potential contribution of the predicted Cdk6-MLL fusion protein in the genesis of the ALL, which also contains an in-frame MLL-AF4 transcript. The sequences in these three cases show additional MLL genomic breakpoint heterogeneity. Each breakpoint junction suggests nonhomologous end joining and is consistent with DNA damage and repair. CDK6-MLL is a new fusion of both genes.