De-novo Germline Research Articles

De Novo Germline DHX38 Variant Associated with Alternative Splicing of Multiple Transcripts in Iron-Related Pathways in a Patient with Atypical Congenital Dyserythropoietic Anemia with Ring Sideroblasts

Congenital Dyserythropoietic Anemias (CDAs) are a heterogeneous group of rare hereditary anemias caused by ineffective and dyspoietic erythropoiesis, with the pathognomonic feature of binuclear and/or multinuclear erythroid precursors in the bone marrow. The clinical diagnosis of CDA is based on identifying the characteristic morphology of erythroblasts in the bone marrow of patients presenting with chronic anemia, frequently with evidence of hemolysis but suboptimal reticulocytosis, and iron overload. CDAR (ClinicalTrials.gov identifier: NCT02964494), a registry for patients with CDA in North America, has been established with the goal of providing a longitudinal database and associated biorepository to facilitate natural history studies and research on the pathogenetic mechanisms in this heterogenous disease. A 2-year-old female, enrolled in CDAR, had transfusion-dependent anemia since birth. She demonstrated accelerated iron overload and had failure to thrive despite regular transfusions maintaining trough hemoglobin higher than 9.5 g/dL. Bone marrow evaluation revealed dyserythropoiesis along with ring sideroblasts (RS), resembling acquired myelodysplastic syndrome (MDS) with the characteristics of Refractory Anemia with RS (RARS). Whole-exome sequencing for the proband and her parents in trio analysis identified a de novo, heterozygous germline DHX38 missense variant c.3310A>C, p.Thr1104Pro in the proband. In silico prediction programs suggest this rare variant is deleterious. DEAH box RNA helicase DHX38 is an RNA helicase, homolog to the yeast Prp16 that has been shown to play a dual role in the spliceosome complex: an ATP-independent role in the first catalytic step mediating proofreading of the branchpoint sequence, and an ATP-dependent role in the second step, mediating removal of the lariat and joining the exons to complete the splicing (Tseng et al, RNA 2011). DHX38 is highly expressed during terminal erythropoiesis similarly to SF3B1, the splicing factor gene most frequently mutated in MDS-RARS. RNA-seq analysis of CD71+ reticulocytes isolated from the proband at three different time points compared to three different normal control samples, using AltAnalyze software, revealed approximately 3,000 differentially expressed genes. More than 1,500 differentially spliced events were identified, affecting over 200 genes, including SLC25A37, coding for an iron importer localized in the mitochondrial inner membrane and PPOX, coding for protoporphyrinogen oxidase (Figure 1A), both essential for the synthesis of mitochondrial heme. Mis-splicing of the same genes is a characteristic finding in MDS-RARS associated with SF3B1 pathogenic variants, most commonly p.Lys700Glu, and may contribute to RS formation. Moreover, DHX38 is known to interact with centromeric noncoding RNA, regulating chromosome segregation during mitosis, and its depletion in vitro causes abnormal grape-shaped nuclear morphology (Nishimura et al, Genes Cells 2019). Review of the patient's bone marrow studies revealed several erythroblasts with grape-shaped nuclei (Figure 1B). Erythropoiesis cultures from patient-derived iPSCs were utilized to phenocopy the disease in vitro. Cytospins of the generated erythroblasts revealed dyserythropoiesis that was verified quantitatively by increased frequency of nuclear abnormalities in erythroblasts produced in vitro from patient-derived iPSCs analyzed by imaging flow cytometry. Extra-hematopoietic abnormalities of the nucleus and mitotic defects were explored in fibroblasts generated from the patient's skin biopsy, which also tested positive for DHX38 p.Thr1104Pro. Grape-shaped nuclei were detected in cultures of patient's fibroblasts indicating improper mitotic exit due to aberrant chromosome segregation. Our findings indicate that DHX38 is likely the causative gene for this CDA with a RARS phenotype, causing mis-splicing of a multitude of genes critical for iron metabolism and erythropoiesis as well as defects in chromosome segregation and cell division. Out of approximately 250,000 alleles in the gnomAD database, the DHX38 p.Thr1104Pro variant has been detected in 4 alleles; however, SF3B1 p.Lys700Glu is also detected in 22. Therefore, the rare presence of this DHX38 variant in "healthy" population may indicate that acquired DHX38 pathogenic variants exist and could be responsible for SF3B1-negative MDS-RARS. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Correction to: De Novo Germline and Somatic Variants Convergently Promote Endothelial-to-Mesenchymal Transition in Simplex Brain Arteriovenous Malformation.

HomeCirculation ResearchVol. 130, No. 1Correction to: De Novo Germline and Somatic Variants Convergently Promote Endothelial-to-Mesenchymal Transition in Simplex Brain Arteriovenous Malformation Free AccessCorrectionPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessCorrectionPDF/EPUBCorrection to: De Novo Germline and Somatic Variants Convergently Promote Endothelial-to-Mesenchymal Transition in Simplex Brain Arteriovenous Malformation Originally published7 Jan 2022https://doi.org/10.1161/RES.0000000000000524Circulation Research. 2022;130:234This article corrects the followingDe Novo Germline and Somatic Variants Convergently Promote Endothelial-to-Mesenchymal Transition in Simplex Brain Arteriovenous MalformationIn the article by Li et al, “De Novo Germline and Somatic Variants Convergently Promote Endothelial-to-Mesenchymal Transition in Simplex Brain Arteriovenous Malformation,” which published in the October 15, 2021 issue of the journal (Circ Res. 2021;129:825–839. doi: 10.1161/CIRCRESAHA.121.319004), a correction was needed.There was an error in the Abstract and Introduction: The definition of AVM was incorrectly displayed as “arteriovenous-malformation-like” when it should have been “arteriovenous-malformation”.The journal apologizes for the error.The corrected version of this article is available at www.ahajournals.org/doi/10.1161/CIRCRESAHA.121.319004. Previous Back to top Next FiguresReferencesRelatedDetailsRelated articlesDe Novo Germline and Somatic Variants Convergently Promote Endothelial-to-Mesenchymal Transition in Simplex Brain Arteriovenous MalformationHao Li, et al. Circulation Research. 2021;129:825-839 January 7, 2022Vol 130, Issue 1 Advertisement Article InformationMetrics © 2021 American Heart Association, Inc.https://doi.org/10.1161/RES.0000000000000524PMID: 34995139 Originally publishedJanuary 7, 2022 PDF download Advertisement

De Novo Germline and Somatic Variants Convergently Promote Endothelial-to-Mesenchymal Transition in Simplex Brain Arteriovenous Malformation.

[Figure: see text].

Polymorphisms and rare variants identified by next-generation sequencing confer risk for lung cancer in han Chinese population

BackgroundLung cancer is one of the leading causes of cancer death worldwide, and genetic risk factors account for a large part of its carcinogenesis. The low economic requirements and high efficiency of next-generation sequencing (NGS) make it widely used in detecting genetic alterations in pathogenesis. MethodsWe performed targeted panel sequencing in 780 Han Chinese lung cancer patients using a commercial probe, and the correlations between dozens of susceptible sites were verified in 1113 healthy controls. This study used Fisher's exact test and Benjamini-Hochberg FDR correction to analyze the mutual exclusion between mutated genes, and Pearson's p was used to verify the correlations between mutations and lung cancer susceptibility. ResultsOur results determined the mutation spectrum and showed that each lung cancer patient carried at least one DNA mutation. The most frequently mutated gene was BRCA2 (mutation rate,10.6 %.). The co-occurrence and mutual exclusion analysis of DNA damage related genes showed that gene ATM was mutually exclusive from MSH6. We conducted a further case-control study in different subtypes of lung cancer and the results described 14 mutations associated with adenocarcinoma, 9 with squamous cell carcinoma, and 4 with small cell lung cancer. These variants were novel de-novo germline mutations in lung cancer. Particularly, rs3864017 in FANCD2 showed a protective effect of lung adenocarcinoma for carriers (OR = 0.146, 95 % CI = 0.052∼0.405, Padjusted = 3.37 × 10−4). Conclusions18 candidate mutations might alter the risk of lung cancer in the Han Chinese population, including polymorphisms rs3864017(FANCD2), rs55740729(MSH6) and 16 rare variants. The underlying mechanisms of candidate genes in lung cancer remain unclear and we suggest more functional studies on exploring how these genes affect the risk of lung cancer.

De Novo Germline Mutations in SEMA5A Associated With Infantile Spasms.

Infantile spasm (IS) is an early-onset epileptic encephalopathy that usually presents with hypsarrhythmia on an electroencephalogram with developmental impairment or regression. In this study, whole-exome sequencing was performed to detect potential pathogenic de novo mutations, and finally we identified a novel damaging de novo mutation in SEMA5A and a compound heterozygous mutation in CLTCL1 in three sporadic trios with IS. The expression profiling of SEMA5A in the human brain showed that it was mainly highly expressed in the cerebral cortex, during the early brain development stage (8 to 9 post-conception weeks and 0 to 5 months after birth). In addition, we identified a close protein-protein interaction network between SEMA5A and candidate genes associated with epilepsy, autism spectrum disorder (ASD) or intellectual disability. Gene enrichment and function analysis demonstrated that genes interacting with SEMA5A were significantly enriched in several brain regions across early fetal development, including the cortex, cerebellum, striatum and thalamus (q < 0.05), and were involved in axonal, neuronal and synapse-associated processes. Furthermore, SEMA5A and its interacting genes were associated with ASD, epilepsy syndrome and developmental disorders of mental health. Our results provide insightful information indicating that SEMA5A may contribute to the development of the brain and is associated with IS. However, further genetic studies are still needed to evaluate the role of SEMA5A in IS to definitively establish the role of SEMA5A in this disorder.

Congenital X-Linked Myelodysplasia with Tetraploidy Is Associated with De Novo Germline C-Terminal Mutation of SEPT6, a Septin Filament Protein

Private germline mutations affecting hematopoiesis can cause progressive myelodysplasia and thus constitute pre-leukemic states. These can remain undetected, progressively transform and reveal themselves as infant leukemias, which can be linked to translocations involving the mixed lineage-leukemia (MLL) gene. Septin proteins play key roles in mammalian cell division and cytokinesis and are found as fusion partners of MLL in infant and early childhood acute myeloid leukemia (AML). We identified and describe a human germline disorder of septins in a newborn with myelodysplasia who required early hematopoietic stem cell transplant (HSCT) to prevent progressive disease.Materials & Methods: A Caucasian newborn male with no birth defects/malformations or suspicious family history presented with severe progressive neutropenia and was found to have bone marrow (BM) dysplasia with tetraploidy of myeloid progenitors. The patient developed unfavorable clonal aberrations (trisomy 7,8,9) and increased tetraploidy. Due to his progressive cytopenias and concern about risk of leukemic transformation, he underwent an allogeneic busulfan-cyclophosphamide/ATG conditioned DQ-mismatched unrelated HSCT at age 1 yo. He is currently 8 years post-HSCT with normal trilineage hematopoiesis (full donor chimerism), no graft versus host disease or any other non-hematological phenotype. To investigate the genetic etiology of this unique phenotype, we performed family trio germline exome/whole genome next-generation sequencing (NGS), and somatic pre-HSCT BM NGS for the index case. An established algorithm filtered for significant candidates following a de novo germline model. Immunohistochemical (IHC) staining of the pre- and post-HSCT BM biopsies for the candidate protein was performed. To validate the germline origin of the candidate mutation, we generated patient and control skin fibroblasts and induced pluripotent stem cells (iPSCs) that underwent fidelity testing by murine injection teratoma assays and 16-marker immunofluorescence (IF) staining. We then studied hematopoietic progenitor cells (HPCs) derived from iPSC-embryoid bodies (EB) in methylcellulose assays. To further determine the pathogenic nature of the mutation, we generated CRISPR/Cas9 knock-out of the human erythroleukemic cell line (TF-1) and studies these cells by cytomorphology, DNA and cell cycle assays. In-silico protein analysis of the candidate mutation and its effects on septin filament formation was performed.Results: Family trio and disease-tissue NGS identified a novel, germline C-terminal mutation in SEPT6, which was acquired de novo in the patient, and was not found in any database of common polymorphisms. IHC of pre-HSCT patient BM showed reduced Septin-6 staining in megakaryocyte and granulocyte precursors compared to post-HSCT and controls. Patient-derived iPSCs carried the mutation, were cytogenetically normal and bona-fide pluripotent by IF and teratoma assays. EB-derived HPCs from these cells recapitulated the patient's phenotype as they differentially failed to produce granulocyte vs erythroid colonies (fold-reductions CFU-M:8, CFU-G:36, CFU-GM:46, BFU-E:6, see Figure). Despite multiple approaches, SEPT6 CRISPR/Cas9 knock-out/in of the patient's mutation was not tolerated in iPSCs and human myeloid (granulo-/myelocytic) cell lines (HL-60, Molm-13, K562), and only tolerated in erythroid TF-1 cells. Analysis of SEPT6 knock-out TF-1 single-clone lines revealed a propensity to poly-nuclearity/lobation, as observed in the patient's BM. SEPT6 knock-in of C-terminal mutations caused cell death, consistent with existing literature. In silico protein analysis (incl. previously published crystallographic data) suggests that the mutation a) most likely modulates the key role of the coiled-coil SEPT6 domain in septin filament stabilization/bundling/bending, and thus deleteriously impacts cytokinesis, and b) perturbs the equilibrium of splice variants, possibly conferring tissue specificity.Conclusions: Mutation of the C-terminus of human SEPT6 causes aberrant cytokinesis in HPCs leading to a severe congenital neutropenia with tetraploidy and progressive myelodysplasia and cytogenetic aberrations. This report implicates a human germline disorder of SEPT6, and further investigations are required to elucidate the role septins in normal and disordered myelopoiesis. DisclosuresWilliams:Bluebird Bio: Research Funding.

Sanger sequencing in exonic regions of STK11 gene uncovers a novel de-novo germline mutation (c.962_963delCC) associated with Peutz-Jeghers syndrome and elevated cancer risk: case report of a Chinese patient

BackgroundPeutz-Jeghers syndrome (PJS) is caused by mutations in the tumor suppressor gene, STK11, and is characterized by gastrointestinal hamartomas, melanin spots on the lips and the extremities, and an increased risk of developing cancer.Case presentationWe reported an isolated PJS patient who died of colon cancer, whose blood sample was collected together with all the available family members’. The entire coding region of the STK11 gene was amplified by PCR and analyzed by Sanger sequencing, through which, a novel mutation, c.962_963delCC in exon 8 was identified in this patient. This mutation causes a frameshift mutation and a premature termination at codon 358. Protein structure prediction by Swiss-Model indicated a dramatic change and partial loss of the C-terminal domain. We did not observe this mutation in both parents of the proband. Therefore, it is considered a novel de-novo mutation. Furthermore, the mutation was not found in 50 unrelated healthy people.ConclusionsThe novel mutation we reported here had not been recorded in databases or literature, and the patient who possessed it suffered from PJS and colon cancer. So our results enlarge the spectrum of STK11 variants in PJS patients. This mutation is most likely responsible for development of the PJS phenotype, especially the cancer occurrence.

Paternal Age Explains a Major Portion of De Novo Germline Mutation Rate Variability in Healthy Individuals.

De novo mutations (DNM) are an important source of rare variants and are increasingly being linked to the development of many diseases. Recently, the paternal age effect has been the focus of a number of studies that attempt to explain the observation that increasing paternal age increases the risk for a number of diseases. Using disease-free familial quartets we show that there is a strong positive correlation between paternal age and germline DNM in healthy subjects. We also observed that germline CNVs do not follow the same trend, suggesting a different mechanism. Finally, we observed that DNM were not evenly distributed across the genome, which adds support to the existence of DNM hotspots.

Exome Sequencing Reveals De Novo Germline Mutation of the Mammalian Target of Rapamycin (MTOR) in a Patient with Megalencephaly and Intractable Seizures

A de novo somatic mutation in the mammalian target of rapamycin (MTOR) has previously been described in one patient with hemimegalencephaly and epilepsy. Here, we present a case of a young girl with megalencephaly and intractable seizures who was found to have an MTOR mutation in multiple cell lineages (p.Cys1483Phe) and, therefore, presumed to be of germline origin. The mutation was detected in peripheral blood DNA by exome sequencing of the patient and her parents, substantiating the utility of this approach for detection of clinically relevant de novo variations.

Whole-Genome Sequencing in Autism Identifies Hot Spots for De Novo Germline Mutation

De novo mutation plays an important role in autism spectrum disorders (ASDs). Notably, pathogenic copy number variants (CNVs) are characterized by high mutation rates. We hypothesize that hypermutability is a property of ASD genes and may also include nucleotide-substitution hot spots. We investigated global patterns of germline mutation by whole-genome sequencing of monozygotic twins concordant for ASD and their parents. Mutation rates varied widely throughout the genome (by 100-fold) and could be explained by intrinsic characteristics of DNA sequence and chromatin structure. Dense clusters of mutations within individual genomes were attributable to compound mutation or gene conversion. Hypermutability was a characteristic of genes involved in ASD and other diseases. In addition, genes impacted by mutations in this study were associated with ASD in independent exome-sequencing data sets. Our findings suggest that regional hypermutation is a significant factor shaping patterns of genetic variation and disease risk in humans.

Identification of De Novo Germline Mutations in the HRPT2 Gene in Two Apparently Sporadic Cases with Challenging Parathyroid Tumor Diagnoses

The diagnosis of parathyroid carcinomas is often difficult. HRPT2 mutations have been identified in familial [hyperparathyroidism-jaw tumor (HPT-JT) syndrome] and sporadic parathyroid carcinomas, supporting that HRPT2 mutations may confer a malignant potential to parathyroid tumors. In this study, we report the clinical, histopathological, and genetic investigation of two unrelated cases, whom had apparently sporadic malignant parathyroid tumors, initially diagnosed as adenomas. In one case, the differential diagnosis was complicated by cervical seeding of parathyroid tumor cells. Genetic studies identified de novo HRPT2 germline mutations in cases 1 (c.518_521delTGTC [p.Ser174LysfsX27]) and 2 (c.226C > T [p.Arg76X]), unveiling the hereditary HPT-JT syndrome in both patients. Furthermore, the identification of somatic mutations in the patients‟ parathyroid tumors provided evidence for complete inactivation of the HRPT2 gene, which was consistent with the tumor malignant features. The sensitivity of parafibromin immunostaining to detect HRPT2 mutations was limited. The present data suggests that patients with apparently sporadic parathyroid carcinomas, or parathyroid tumors with atypical histological features, should undergo molecular genetic testing, as it may detect germline HRPT2 mutations. Establishing the diagnosis of hereditary HPT-JT syndrome is relevant for clinical counseling and management of the carriers and their relatives.

Report on de-novo mutation in the MSH2 gene as a rare event in hereditary nonpolyposis colorectal cancer

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant predisposition for early-onset colorectal cancer and associated tumour types caused by mutations in DNA mismatch repair genes, mainly MLH1 and MSH2. The Amsterdam criteria, stressing familial tumour events, as well as the less stringent Bethesda criteria, help to raise suspicion of HNPCC. Mutation screening in a colon cancer patient of young age but negative family history revealed the MSH2 splice site mutation c.2006-2A>G. This mutation was present in all three germ cell layers in the patient, but absent in her biological parents, reporting the second case of de-novo germline mutation in MSH2. Our findings suggest that at least in approximately 1% of mutation-positive cases, a de-novo event might be involved. The identification of a newly evolved rare mutation calls for alertness and encourages the application of the Bethesda guidelines and certain clinical and histopathological features to define candidates for HNPCC gene mutation screening despite a negative family history.

Association of germline mutation in the PTEN tumour suppressor gene and Proteus and Proteus-like syndromes

The molecular aetiology of Proteus syndrome (PS) remains elusive. Germline mutations in PTEN cause Cowden syndrome and Bannayan-Riley-Ruvalcaba syndrome, which are hereditary hamartoma syndromes. Some features-eg, macrocephaly, lipomatosis, and vascular malformations-can be seen in all three syndromes. We examined PTEN in patients with PS and undefined Proteus-like syndromes (PS-like) and identified de-novo germline mutations in two of nine patients with PS and three of five patients with PS-like. Germline PTEN mutation analysis should be done in individuals with PS and PS-like because of its association with increased risk of cancer development and potential of germline-mutation transmission.

Recent progress in the genetics of incontinentia pigmenti (Bloch-Sulzberger syndrome).

Incontinentia pigmenti (IP) is a rare disorder which affects organs and tissues of ectodermal and mesodermal origin. It is characterized by swirled patterns of hyperpigmentation. In some cases, the condition is also associated with malformations of the teeth, nails, skeleton, hair, eyes, and the central nervous system. The disorder is inherited as an X-linked dominant trait and mostly affects females. However, there have been several cases of IP in males that survived to birth. While IP in females could be caused by a skewed pattern of X-inactivation, three mechanisms: namely, the half-chromatid hypothesis, unstable premutation, and a higher rate of de-novo germline mutations, have been proposed to explain the survival of affected male patients. Cytogenetic studies in several sporadic cases with signs similar to IP exhibited an X/autosomal translocation involving a breakpoint at Xp11, suggesting a gene locus on Xp11 (IP1). Linkage analysis of familial IP, on the other hand, has identified a second locus, in the Xq28 region (IP2). Molecular genetic analysis of two candidate genes located at Xp11 and Xq28, as well as the human homologue of the murine Str gene, failed to reveal any disease-causing mutations. Although heterozygous female mice deficient for the IKKgamma/NEMO gene exhibited dermatopathy similar to that in human IP, studies of the gene in human IP have not yet been available. In an effort to isolate the genes causing IP, cosmid clones containing the translocation breakpoint located at Xp11 and the transcriptional map of the Xq28 region were constructed. These maps could be invaluable tools in the identification of genes in the near future.