Somatic Reversion Research Articles

Shared VH1-46 antibody gene usage in pemphigus vulgaris predicts antibody cross-reactivity to desmoglein 3 and rotavirus VP6 (HUM1P.271)

Abstract Pemphigus vulgaris (PV) is a potentially fatal skin blistering disease caused by autoantibodies (autoAbs) to desmoglein 3 (Dsg3). Previously, we identified shared VH1-46 gene usage in the anti-Dsg3 repertoire among 4 PV patients via high throughput B cell receptor (BCR) cloning. Somatic mutation reversion studies indicated that VH1-46 BCRs require few to no acidic amino acid mutations to bind Dsg3, which may favor their early selection in the immune response. Interestingly, shared VH1-46 gene usage also occurs in the B cell response to rotavirus protein VP6, and similarly, few somatic mutations are required to bind VP6. We thus investigated whether Dsg3-reactive VH1-46 BCRs cross-react to VP6 which may explain the tolerance of these autoreactive clones. We screened ~108 combinatorial IgM clones from a PV patient, which identified 13 heavy chain CDR3 clonotypic families that cross-react to Dsg3 and VP6. 7/13 used VH1-46, indicating enrichment by Dsg3/VP6 antigen selection. ELISA and human skin immunofluorescence using purified soluble Abs confirmed VP6 and Dsg3 reactivity. Additionally, 3 of 5 VH1-46 anti-Dsg3 IgG clonotypes previously isolated from 3 different PV patients cross-reacted to VP6 in either their somatically mutated or unmutated form. Our data suggest that a VH1-46 B cell response to rotavirus may be an initiating event that develops into an autoimmune response to Dsg3 in susceptible individuals.

Frequent somatic reversion of KRT1 mutations in ichthyosis with confetti.

Widespread reversion of genetic disease is rare; however, such events are particularly evident in some skin disorders in which normal clones develop on a background of affected skin. We previously demonstrated that mutations in keratin 10 (KRT10) cause ichthyosis with confetti (IWC), a severe dominant disorder that is characterized by progressive development of hundreds of normal skin spots via revertant mosaicism. Here, we report on a clinical and histological IWC subtype in which affected subjects have red, scaly skin at birth, experience worsening palmoplantar keratoderma in childhood, and develop hundreds of normal skin spots, beginning at around 20 years of age, that increase in size and number over time. We identified a causal de novo mutation in keratin 1 (KRT1). Similar to IWC-causing KRT10 mutations, this mutation in KRT1 resulted in a C-terminal frameshift, replacing 22 C-terminal amino acids with an alternate 30-residue peptide. Mutant KRT1 caused partial collapse of the cytoplasmic intermediate filament network and mislocalized to the nucleus. As with KRT10 mutations causing IWC, reversion of KRT1 mutations occurred via mitotic recombination. Because reversion is not observed with other disease-causing keratin mutations, the results of this study implicate KRT1 and KRT10 C-terminal frameshift mutations in the high frequency of revertant mosaicism in IWC.

RAG1 Reversion Mosaicism in a Patient with Omenn Syndrome

To identify mechanisms of disease in a child born to consanguineous parents, who presented with Omenn syndrome (OS) and was found to carry a heterozygous RAG1 mutation in peripheral blood DNA. Mutation analysis was performed on whole blood and buccal swab DNA. Recombination activity of the mutant RAG1 protein and diversity of T cell repertoire were tested. Apparent heterozygosity for a novel, functionally null RAG1 mutation in peripheral blood DNA from a patient with OS was shown to be secondary to true somatic reversion. Analysis of T cell repertoire demonstrated expression of various TCRBV families, but an overall restricted pattern. This is the first case of true somatic reversion of a RAG1 mutation in a patient with OS. The reversion event likely occurred at a stage where only a limited pool of T cell progenitors capable of performing V(D)J recombination could be generated. This work emphasizes the importance of performing functional studies to investigate the significance of novel genetic variants, and to consider somatic reversion as a possible disease modifier in SCID.

Somatic reversion in dedicator of cytokinesis 8 immunodeficiency modulates disease phenotype

Autosomal recessive loss-of-function mutations in dedicator of cytokinesis 8 (DOCK8) cause a combined immunodeficiency characterized by atopy, recurrent infections, and cancer susceptibility. A genotype-phenotype explanation for the variable disease expression is lacking. We investigated whether reversions contributed to the variable disease expression. Patients followed at the National Institutes of Health's Clinical Center were studied. We performed detailed genetic analyses and intracellular flow cytometry to detect DOCK8 protein expression within lymphocyte subsets. We identified 17 of 34 DOCK8-deficient patients who had germline mutations with variable degrees of reversion caused by somatic repair. Somatic repair of the DOCK8 mutations resulted from second-site mutation, original-site mutation, gene conversion, and intragenic crossover. Higher degrees of reversion were associated with recombination-mediated repair. DOCK8 expression was restored primarily within antigen-experienced T cells or natural killer cells but less so in naive T or B cells. Several patients exhibited multiple different repair events. Patients who had reversions were older and had less severe allergic disease, although infection susceptibility persisted. No patients were cured without hematopoietic cell transplantation. In patients with DOCK8 deficiency, only certain combinations of germline mutations supported secondary somatic repair. Those patients had an ameliorated disease course with longer survival but still had fatal complications or required hematopoietic cell transplantation. These observations support the concept that some DOCK8-immunodeficient patients have mutable mosaic genomes that can modulate disease phenotype over time.

2013 Victor A. McKusick Leadership Award Introduction: Kurt and Rochelle Hirschhorn

2013 Victor A. McKusick Leadership Award Introduction: Kurt and Rochelle Hirschhorn

Abstract IA19: Exploiting DNA repair deficiencies in breast cancer using PARP inhibitors

Abstract Epithelial cancers arise through acquisition of mutation in multiple genes causing either gain or loss of functions that give a phenotypic advantage to the affected cell and acquisition of key features of malignancy commonly described as the “hallmarks of cancer”. This acquisition of sufficient mutational burden to acquire these “hallmarks” may be accelerated by deficiency in one or more of number of partially redundant DNA repair mechanisms that have evolved to maintain genome stability. Many forms of chemotherapy and radiation therapy have long exploited tumor's relative deficiencies in the DNA damage response and in DNA repair specifically. Recently more specific inhibitors of DNA repair and DNA damage response effectors have been developed and tested in preclinical models and early phase clinical trials. These have shown proof of concept for the principle of tumor specific “synthetic lethality” where the synergistic combination of a therapeutically induced deficiency in one form of genome stability mechanism with a tumor specific constitutive defect in a mutually dependant from DNA repair leads to high specific tumor cell killing. This principle has been found to apply to the combination of small molecule induced inhibition of PARP1 and PARP2 in combination with tumor cell intrinsic defects in the homologous recombination (HR) DNA repair mechanism. A number of genes involved in HR, including BRCA1, BRCA2, ATM and PALB2 lead to elevated risk of breast cancer when mutated in the germline. The tumors that develop in these contexts have, as a result, functional defects in HR. Recent evidence has also suggested that a significant proportion of breast cancers show distinctive patterns of genome instability and changes in DNA damage induced repair protein expression phenotypes that indicate loss of function of HR DNA repair. Recent correlative biology studies in breast cancer and ovarian cancer trials show correlation between these emerging biomarkers with efficacy of platinum based chemotherapy, also known to target deficiency in HR. The further investigation of PARP inhibitors in breast cancer has been slowed by an initial focus of interest in the larger sporadic breast cancer population and the difficulties in identifying a likely maximum effect population in this wider population without robust assays for defects in the DNA damage response. Progress has also been hampered by the difficulty of finding tolerable combinations of these agents with chemotherapy. Differences have been uncovered between PARP inhibitor agents both in terms of potency and mechanisms of action related to presence or absence of trapping of PARP in complex with DNA that may differentially effect efficacy in targeting HR DNA repair defects or potentiation of DNA damaging chemotherapeutics. The small molecule Iniparib has been found to have no significant PARP inhibitory effect and in consequence the results with this agent in breast cancer should not be regarded as having tested any role for PARP inhibition in this context. Focus in late phase clinical trials of PARP inhibitors in breast cancer is currently returning to potent PARP inhibitors and the original proof of concept germline mutation carrier population. Here crucial questions may still remain around prevalence of mechanisms of acquired resistance demonstrated to occur in preclinical models through somatic reversion of mutations in BRCA1 and BRCA2, selection for loss of function of genes such as 53BP1 that stimulate alternative use of Non-homologous end-joining (NHEJ) repair and overexpression of the P-glycoprotein drug efflux transporter. In this lecture I will review the preclinical and emerging biomarker data described above and their relevance to current and imminent clinical trials exploring PARP inhibition in breast cancer. Citation Format: Andrew Tutt. Exploiting DNA repair deficiencies in breast cancer using PARP inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research: Genetics, Biology, and Clinical Applications; Oct 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2013;11(10 Suppl):Abstract nr IA19.

Inherited human sex reversal due to impaired nucleocytoplasmic trafficking of SRY defines a male transcriptional threshold

Human testis determination is initiated by SRY (sex determining region on Y chromosome). Mutations in SRY cause gonadal dysgenesis with female somatic phenotype. Two subtle variants (V60L and I90M in the high-mobility group box) define inherited alleles shared by an XY sterile daughter and fertile father. Whereas specific DNA binding and bending are unaffected in a rat embryonic pre-Sertoli cell line, the variants exhibited selective defects in nucleocytoplasmic shuttling due to impaired nuclear import (V60L; mediated by Exportin-4) or export (I90M; mediated by chromosome region maintenance 1). Decreased shuttling limits nuclear accumulation of phosphorylated (activated) SRY, in turn reducing occupancy of DNA sites regulating Sertoli-cell differentiation [the testis-specific SRY-box 9 (Sox9) enhancer]. Despite distinct patterns of biochemical and cell-biological perturbations, V60L and I90M each attenuated Sox9 expression in transient transfection assays by twofold. Such attenuation was also observed in studies of V60A, a clinical variant associated with ovotestes and hence ambiguity between divergent cell fates. This shared twofold threshold is reminiscent of autosomal syndromes of transcription-factor haploinsufficiency, including XY sex reversal associated with mutations in SOX9. Our results demonstrate that nucleocytoplasmic shuttling of SRY is necessary for robust initiation of testicular development. Although also characteristic of ungulate orthologs, such shuttling is not conserved among rodents wherein impaired nuclear export of the high-mobility group box and import-dependent phosphorylation are compensated by a microsatellite-associated transcriptional activation domain. Human sex reversal due to subtle defects in the nucleocytoplasmic shuttling of SRY suggests that its transcriptional activity lies near the edge of developmental ambiguity.

Three distinct mutational mechanisms acting on a single gene underpin the origin of yellow flesh in peach

Peach flesh color (white or yellow) is among the most popular commercial criteria for peach classification, and has implications for consumer acceptance and fruit nutritional quality. Despite the increasing interest in improving cultivars of both flesh types, little is known about the genetic basis for the carotenoid content diversity in peach. Here we describe the association between genotypes at a locus encoding the carotenoid cleavage dioxygenase 4 (PpCCD4), localized in pseudomolecule 1 of the Prunus persica reference genome sequence, and the flesh color for 37 peach varieties, including two somatic revertants, and three ancestral relatives of peach, providing definitive evidence that this locus is responsible for flesh color phenotype. We show that yellow peach alleles have arisen from various ancestral haplotypes by at least three independent mutational events involving nucleotide substitutions, small insertions and transposable element insertions, and that these mutations, despite being located within the transcribed portion of the gene, also result in marked differences in transcript levels, presumably as a consequence of differential transcript stability involving nonsense-mediated mRNA decay. The PpCCD4 gene provides a unique example of a gene for which humans, in their quest to diversify phenotypic appearance and qualitative characteristics of a fruit, have been able to select and exploit multiple mutations resulting from a variety of mechanisms.

Mis‐expression of a PISTILLATA‐like MADS box gene prevents fruit development in grapevine

The FLESHLESS BERRY (Flb) somatic variant identified in the grapevine cultivar Ugni Blanc develops grape berries without flesh, suggesting a role for the altered gene in differentiation of flesh cells. Here we describe identification of the molecular defect responsible for this phenotype. Using a combination of genetic and transcriptomic approaches, we detected the insertion of a miniature inverted-repeat transposable element in the promoter region of the PISTILLATA-like (VvPI) gene, the grapevine homologue of Arabidopsis PISTILLATA. The transposon insertion causes specific ectopic expression of the corresponding VvPI allele during early fruit development, causing expression of genes specific for petal and stamen development within the fruit. A causal relationship between the insertion and the phenotype was demonstrated by phenotypic and molecular analyses of somatic revertants showing that ectopic expression and mutant phenotype were always linked to the presence of the transposon insertion. The various phenotypic effects of the flb mutation on ovary morphology, fruit set and fruit development, depending on the cell lineage affected, are presented for each phenotype, offering new insights into floral and fleshly fruit development. The results highlight the importance of VvPI repression after fertilization to achieve normal fleshy fruit development, and the complex genetic, genomic and cellular interactions required for the flower to fruit transition in grapevine.

Genomic Contingency and Protein Evolution: Accelerated Divergence of a DNA-Binding Module due to an Adjoining Micro-Satellite Invasion

The sex-determining region of the mammalian Y chromosome encodes an architectural transcription factor containing a conserved sequence-specific high-mobility-group (HMG) box. Designated SRY, transient expression of this gene in the fetal gonadal ridge leads to Sertoli-cell differentiation and in turn testis formation. Mutations in human SRY are associated with 46, XY pure gonadal dysgenesis and somatic sex reversal. We have investigated the evolutionary history of SRY and its protein product SRY among placental mammals as a model for the conservation and divergence of a sequence-specific DNA-binding and DNA-bending module (the HMG box). Remarkably, the rate of divergence among mammalian SRY genes is markedly more rapid in Rodentia than among other Orders of placental mammals. Evidence will be provided that this accelerated evolution is a consequence of the in-frame insertion of a CAG triplet-expansion micro-satellite element, encoding a poly-glutamine or poly-alanine domain with autonomous biochemical properties. A model is proposed wherein relaxation of selective pressure on the HMG box has led to accumulation of deleterious amino-acid substitutions. Thus, a contingency of genomic dynamics (micro-satellite invasion) has led to a change in the pace of the mutational clock governing the evolution of a DNA-binding module.

First reported case of Omenn syndrome in a patient with reticular dysgenesis

First reported case of Omenn syndrome in a patient with reticular dysgenesis

Expansion of somatically reverted memory CD8+ T cells in patients with X-linked lymphoproliferative disease caused by selective pressure from Epstein-Barr virus

Patients with the primary immunodeficiency X-linked lymphoproliferative disease (XLP), which is caused by mutations in SH2D1A, are highly susceptible to Epstein-Barr virus (EBV) infection. Nonetheless, some XLP patients demonstrate less severe clinical manifestations after primary infection. SH2D1A encodes the adaptor molecule SLAM-associated protein (SAP), which is expressed in T and natural killer cells and is required for cytotoxicity against B cells, the reservoir for EBV. It is not known why the clinical presentation of XLP is so variable. In this study, we report for the first time the occurrence of somatic reversion in XLP. Reverted SAP-expressing cells resided exclusively within the CD8(+) T cell subset, displayed a CD45RA(-)CCR7(-) effector memory phenotype, and were maintained at a stable level over time. Importantly, revertant CD8(+) SAP(+) T cells, but not SAP(-) cells, proliferated in response to EBV and killed EBV-infected B cells. As somatic reversion correlated with EBV infection, we propose that the virus exerts a selective pressure on the reverted cells, resulting in their expansion in vivo and host protection against ongoing infection.

Multiple Reversions of an IL2RG Mutation Restore T cell Function in an X-linked Severe Combined Immunodeficiency Patient

Reversion mosaicism is increasingly being reported in primary immunodeficiency diseases, but there have been few cases with clinically improved immune function. Here, a case is reported of X-linked severe combined immunodeficiency (SCID-X1) with multiple somatic reversions in T cells, which restored sufficient cell-mediated immunity to overcome viral infection. Lineage-specific analysis revealed multiple reversions in T cell receptor (TCR) αβ+ and TCRγδ+ T cells. Diversity of the TCRVβ repertoire was comparable to normal and, furthermore, mitogen-induced proliferation of the patient's T cells was minimally impaired compared to healthy controls. In vivo and in vitro varicella antigen-specific T cell responses were comparable to those of healthy controls, although a reduced level of T cell receptor excision circles suggested that recent thymic output was low. During long-term evaluation of the patient's immunologic status, both the number of CD4+ and CD8+ T cells and T cell proliferation responses were stable and the patient remained healthy. This case demonstrates that multiple but restricted somatic reversions in T cell progenitors can improve the clinical phenotype of SCID-X1.

Revertant Somatic Mosaicism by Mitotic Recombination in Dyskeratosis Congenita

Revertant mosaicism is an infrequently observed phenomenon caused by spontaneous correction of a pathogenic allele. We have observed such reversions caused by mitotic recombination of mutant TERC (telomerase RNA component) alleles in six patients from four families affected by dyskeratosis congenita (DC). DC is a multisystem disorder characterized by mucocutaneous abnormalities, dystrophic nails, bone-marrow failure, lung fibrosis, liver cirrhosis, and cancer. We identified a 4 nt deletion in TERC in a family with an autosomal-dominant form of DC. In two affected brothers without bone-marrow failure, sequence analysis revealed pronounced overrepresentation of the wild-type allele in blood cells, whereas no such skewing was observed in the other tissues tested. These observations suggest that this mosaic pattern might have resulted from somatic reversion of the mutated allele to the normal allele in blood-forming cells. SNP-microarray analysis on blood DNA from the two brothers indeed showed independent events of acquired segmental isodisomy of chromosome 3q, including TERC, indicating that the reversions must have resulted from mitotic recombination events. Subsequently, after developing a highly sensitive method of detecting mosaic homozygosity, we have found four additional cases with a mosaic-reversion pattern in blood cells; these four cases are part of a cohort of 17 individuals with germline TERC mutations. This shows that revertant mosaicism is a recurrent event in DC. This finding has important implications for improving diagnostic testing and understanding the variable phenotype of DC.

ZBTB1 is a determinant of lymphoid development

In this study, we describe a chemically induced mouse mutation that caused a complete and cell-intrinsic T cell deficiency. Development of other lymphoid lineages was also partially impaired and was severely compromised under competitive conditions. Positional cloning, retroviral transduction, and a somatic reversion event revealed that the causative mutation lay within Zbtb1 (zinc finger and BTB domain containing 1), a gene conserved throughout vertebrate evolution. Our data establish ZBTB1 as a critical determinant of T cell development and lymphopoiesis in general, most likely by acting as a transcriptional regulator.

Somatic Mosaicism Caused by Monoallelic Reversion of a Mutation in T Cells of a Patient with ADA‐SCID and the Effects of Enzyme Replacement Therapy on the Revertant Phenotype

Patients with adenosine deaminase (ADA) deficiency exhibit spontaneous and partial clinical remission associated with somatic reversion of inherited mutations. We report a child with severe combined immunodeficiency (T-B- SCID) due to ADA deficiency diagnosed at the age of 1 month, whose lymphocyte counts including CD4+ and CD8+ T and NK cells began to improve after several months with normalization of ADA activity in Peripheral blood lymphocytes (PBL), as a result of somatic mosaicism caused by monoallelic reversion of the causative mutation in the ADA gene. He was not eligible for haematopoietic stem cell transplantation (HSCT) or gene therapy (GT); therefore he was placed on enzyme replacement therapy (ERT) with bovine PEG-ADA. The follow-up of metabolic and immunologic responses to ERT included gradual improvement in ADA activity in erythrocytes and transient expansion of most lymphocyte subsets, followed by gradual stabilization of CD4+ and CD8+ T (with naïve phenotype) and NK cells, and sustained expansion of TCRγδ+ T cells. This was accompanied by the disappearance of the revertant T cells as shown by DNA sequencing from PBL. Although the patient's clinical condition improved marginally, he later developed a germinal cell tumour and eventually died at the age of 67 months from sepsis. This case adds to our current knowledge of spontaneous reversion of mutations in ADA deficiency and shows that the effects of the ERT may vary among these patients, suggesting that it could depend on the cell and type in which the somatic mosaicism is established upon reversion.

Mammalian Testis-determining Factor SRY and the Enigma of Inherited Human Sex Reversal: FRUSTRATED INDUCED FIT IN A BENT PROTEIN-DNA COMPLEX

Mammalian testis-determining factor SRY contains a high mobility group box, a conserved eukaryotic motif of DNA bending. Mutations in SRY cause XY gonadal dysgenesis and somatic sex reversal. Although such mutations usually arise de novo in spermatogenesis, some are inherited and so specify male development in one genetic background (the father) but not another (the daughter). Here, we describe the biophysical properties of a representative inherited mutation, V60L, within the minor wing of the L-shaped domain (box position 5). Although the stability and DNA binding properties of the mutant domain are similar to those of wild type, studies of SRY-induced DNA bending by subnanosecond time-resolved fluorescence resonance energy transfer (FRET) revealed enhanced conformational fluctuations leading to long range variation in bend angle. (1)H NMR studies of the variant protein-DNA complex demonstrated only local perturbations near the mutation site. Because the minor wing of SRY folds on DNA binding, the inherited mutation presumably hinders induced fit. Stopped-flow FRET studies indicated that such frustrated packing leads to accelerated dissociation of the bent complex. Studies of SRY-directed transcriptional regulation in an embryonic gonadal cell line demonstrated partial activation of downstream target Sox9. Our results have demonstrated a nonlocal coupling between DNA-directed protein folding and protein-directed DNA bending. Perturbation of this coupling is associated with a genetic switch poised at the threshold of activity.

Instability of the Greens‐Type Phenotype in Poa annua L.

ABSTRACTThe turfgrass species Poa annua L. is most prevalent as an invasive, annual weed in managed turfs. Conversely, the dwarf perennial greens‐type biotype produces a high turf quality with great utility to the golf‐course industry. In the past 60 yr, several attempts have been made to breed a commercial cultivar of the greens‐type biotype with little sustained success. Here, we characterize the morphological traits of the greens‐type phenotype and investigate its inheritance and stability through genetic crosses. The results indicate that the greens‐type phenotype links single‐branching inflorescences with reductions in culm length, tiller length, leaf length, and panicle length to a single genetic mechanism. However, in advanced‐generation progeny, the segregation of the greens‐type phenotype does not conform to the disomic single‐gene inheritance model. Tetrasomic inheritance, gene complementation, and quantitative inheritance models are also presented. These results, along with the observation of somatic reversions, suggest that the greens‐type phenotype is unstable and may be regulated by an epigenetic mechanism.

Clinical and immunologic consequences of a somatic reversion in a patient with X-linked severe combined immunodeficiency

AbstractX-linked severe combined immunodeficiency is a life-threatening disorder caused by mutations in the gene encoding the interleukin-2 receptor gamma chain (IL2RG). Hypomorphic mutations and reversion of mutations in subpopulations of cells can result in variant clinical phenotypes, making diagnosis and treatment difficult. We describe a 5-year-old boy with mild susceptibility to infection who was investigated for a mutation in IL2RG due to persistent natural killer (NK)– and T-cell lymphopenia. A functionally relevant novel T466C point mutation was found in B, NK, and epithelial cells, whereas α/β and γ/δ T cells showed the normal gene sequence, suggesting reversion of the mutation in a common T-cell precursor. This genetic correction in T cells resulted in a diverse T-cell repertoire and significant immunity despite failure to produce specific antibodies linked to an intrinsic defect of mutant B cells. These observations confirm the potential of revertant T-cell precursors to reconstitute immune function, but questions remain on the longevity of revertant cells implicating the need for careful follow up and early consideration of hematopoietic stem cell transplantation (HSCT).

Unprecedented diversity of genotypic revertants in lymphocytes of a patient with Wiskott-Aldrich syndrome

AbstractSpontaneous somatic reversions of inherited mutations are poorly understood phenomena that are thought to occur uncommonly in a variety of genetic disorders. When molecularly characterized, revertant cells have rarely exhibited more than one revertant genotype per patient. We analyzed individual allospecific T-cell clones derived from a Wiskott-Aldrich syndrome (WAS) patient identified by flow cytometry to have 10% to 15% revertant, WAS protein–expressing lymphocytes in his blood. Genotypic analysis of the clones revealed a remarkable diversity of deletions and base substitutions resulting in at least 34 different revertant genotypes that restored expression of WASp. A large fraction of these revertant genotypes were also identified in primary T cells purified from peripheral blood. These data suggest that the use of sensitive methods may reveal the presence of wide arrays of individual genotypic revertants in WAS patients and offer opportunities for further understanding of their occurrence.