Pair Frameshift Research Articles

Mouse model of severe recessive RYR1-related myopathy.

Ryanodine receptor type I (RYR1)-related myopathies (RYR1 RM) are a clinically and histopathologically heterogeneous group of conditions that represent the most common subtype of childhood onset non-dystrophic muscle disorders. There are no treatments for this severe group of diseases. A major barrier to therapy development is the lack of an animal model that mirrors the clinical severity of pediatric cases of the disease. To address this, we used CRISPR/Cas9 gene editing to generate a novel recessive mouse model of RYR1 RM. This mouse (Ryr1TM/Indel) possesses a patient-relevant point mutation (T4706M) engineered into 1 allele and a 16 base pair frameshift deletion engineered into the second allele. Ryr1TM/Indel mice exhibit an overt phenotype beginning at 14days of age that consists of reduced body/muscle mass and myofibre hypotrophy. Ryr1TM/Indel mice become progressively inactive from that point onward and die at a median age of 42days. Histopathological assessment shows myofibre hypotrophy, increased central nuclei and decreased triad number but no clear evidence of metabolic cores. Biochemical analysis reveals a marked decrease in RYR1 protein levels (20% of normal) as compared to only a 50% decrease in transcript. Functional studies at end stage show significantly reduced electrically evoked Ca2+ release and force production. In summary, Ryr1TM/Indel mice exhibit a post-natal lethal recessive form of RYR1 RM that pheno-copies the severe congenital clinical presentation seen in a subgroup of RYR1 RM children. Thus, Ryr1TM/Indel mice represent a powerful model for both establishing the pathomechanisms of recessive RYR1 RM and pre-clinical testing of therapies for efficacy.

Abstract A044: Mutated NPM1 as target for immunotherapy of acute myeloid leukemia

Abstract The most frequent subtype of acute myeloid leukemia (AML) is defined by mutations in the nucleophosmin (NPM1) gene. Mutated NPM1 is an attractive target for immunotherapy, since it is an essential driver gene and 4 base pair frameshift insertions in exon 12 occur in 30-35% of AML, resulting in a novel C-terminal alternative reading frame of 11 amino acids. By searching in the HLA class I ligandome of primary AML, we identified multiple peptides derived from mutated NPM1. For one of these peptides, i.e., HLA-A*02:01-presented CLAVEEVSL, we searched for specific T-cells in AML patients and healthy individuals using peptide-MHC tetramers. Tetramer-positive CD8 T-cell clones were isolated and analyzed for reactivity against primary AML with mutated NPM1. From one selected clone with superior antitumor reactivity, we isolated the T-cell receptor (TCR) and demonstrated specific recognition and lysis of HLA-A*02:01-positive AML with mutated NPM1 in vitro after retroviral transfer to CD8 and CD4 T-cells. In vivo antitumor efficacy of TCR-transduced CD8 and CD4 T-cells was confirmed in immunodeficient mice engrafted with a human AML cell line expressing mutated NPM1. These data show that mutated NPM1-derived peptides are presented on AML and that CLAVEEVSL is a neoantigen that can be efficiently targeted on AML with mutated NPM1 by TCR gene transfer in a co-receptor independent fashion. Immunotherapy targeting mutated NPM1 may therefore contribute to treatment of AML. Citation Format: Dyantha I. van der Lee, Rogier M. Reijmers, M. Willy Honders, Renate S. Hagedoorn, Rob. M. de Jong, Michel G.D. Kester, Dirk M. van der Steen, Arnoud H. de Ru, Christiaan Kweekel, Inge Jedema, Hendrik Veelken, Mirjam M. Heemskerk, Peter A. van Veelen, J.H. Frederik Falkenburg, Marieke Griffioen. Mutated NPM1 as target for immunotherapy of acute myeloid leukemia [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A044.

Tunicate pregnane X receptor (PXR) orthologs: Transcript characterization and natural variation

Vertebrate pregnane X receptor (PXR, NR1I2), a ligand-activated nuclear receptor (NR), regulates expression of detoxification genes. Vertebrate PXR orthologs may adaptively evolve to bind deleterious/toxic xenobiotics typically encountered by organisms from their diet. Tunicates (phylum Chordata) are marine filter-feeders that form a sister clade to the Vertebrata. Genomes of two tunicate taxa, Ciona intestinalis and Botryllus schlosseri, encode at least two PXR orthologs (abbreviated VDR/PXRα and β). Here we report characterization of the transcript structures and sequence variation of three tunicate PXR orthologs: C. intestinalis VDR/PXRα and β, and B. schlosseri VDR/PXRα. The three predicted proteins consist of both DNA-binding (DBD) and ligand-binding (LBD) domains typical of NRs. The C. intestinalis VDR/PXRβ LBD may be significantly larger than that of the VDR/PXRα orthologs. In both tunicate taxa, the mRNAs were characterized by high frequencies of single nucleotide polymorphisms (SNPs, ca. 3 SNPs/100 base pairs). The majority of SNPs were synonymous and standard tests (Tajima's D, dN/dS ratios) indicated strong purifying selection. However, one base pair frameshift allelic variants were found in the C. intestinalis VDR/PXRα and β genes. The predicted proteins consisted of a DBD but lacked an LBD. The persistence of these variants may possibly reflect constitutive expression of detoxification genes as a selective advantage in the marine environment. These results provide a foundation for further investigations into the molecular evolution, population genetics and functioning of tunicate receptors involved in detection of marine bioactive compounds.

Identification of an HMGB3 frameshift mutation in a family with an X-linked colobomatous microphthalmia syndrome using whole-genome and X-exome sequencing.

Microphthalmias are rare disorders whose genetic bases are not fully understood. HMGB3 is a new candidate gene for X-linked forms of this disease. To identify the causative gene in a pedigree with an X-linked colobomatous microphthalmos phenotype. Whole-genome sequencing and chromosome X-exome-targeted sequencing were performed at the High Throughput Sequencing Laboratory of the Genetic Resources Core Facility at the Johns Hopkins University School of Medicine on the DNA of the male proband and informatically filtered to identify rare variants. Polymerase chain reaction and Sanger sequencing were used to confirm the variant in the proband and the carrier status of his mother. Thirteen unrelated male patients with a similar phenotype were also screened. Whole-genome and X-exome sequencing to identify a frameshift variant in HMGB3. A 2-base pair frameshift insertion (c.477_478insTA, coding for p.Lys161Ilefs*54) in the HGMB3 gene was found in the proband and his carrier mother but not in the unrelated patients. The mutation, confirmed by 3 orthogonal methods, alters an evolutionarily conserved region of the HMGB3 protein from a negatively charged polyglutamic acid tract to a positively charged arginine-rich motif that is likely to interfere with normal protein function. In this family, microphthalmia, microcephaly, intellectual disability, and short stature are associated with a mutation on the X chromosome in the HMGB3 gene. HMGB3 should be considered when performing genetic studies of patients with similar phenotypes.

A versatile microsatellite instability reporter system in human cells

Here, we report the investigation of microsatellite instability (MSI) in human cells with a newly developed reporter system based on fluorescence. We composed a vector into which microsatellites of different lengths and nucleotide composition can be introduced between a functional copy of the fluorescent protein mCherry and an out-of-frame copy of EGFP; in vivo frameshifting will lead to EGFP expression, which can be quantified by fluorescence activated cell sorting (FACS). Via targeted recombineering, single copy reporters were introduced in HEK293 and MCF-7 cells. We found predominantly −1 and +1 base pair frameshifts, the levels of which are kept in tune by mismatch repair. We show that tract length and composition greatly influences MSI. In contrast, a tracts’ potential to form a G-quadruplex structure, its strand orientation or its transcriptional status is not affecting MSI. We further validated the functionality of the reporter system for screening microsatellite mutagenicity of compounds and for identifying modifiers of MSI: using a retroviral miRNA expression library, we identified miR-21, which targets MSH2, as a miRNA that induces MSI when overexpressed. Our data also provide proof of principle for the strategy of combining fluorescent reporters with next-generation sequencing technology to identify genetic factors in specific pathways.

IL26 gene inactivation in Equidae

Interleukin-26 (IL26) is a member of the IL10 cytokine family. The IL26 gene is located between two other well-known cytokines genes of this family encoding interferon-gamma (IFNG) and IL22 in an evolutionary conserved gene cluster. In contrast to humans and most other mammals, mice lack a functional Il26 gene. We analyzed the genome sequences of other vertebrates for the presence or absence of functional IL26 orthologs and found that the IL26 gene has also become inactivated in several equid species. We detected a one-base pair frameshift deletion in exon 2 of the IL26 gene in the domestic horse (Equus caballus), Przewalski horse (Equus przewalskii) and donkey (Equus asinus). The remnant IL26 gene in the horse is still transcribed and gives rise to at least five alternative transcripts. None of these transcripts share a conserved open reading frame with the human IL26 gene. A comparative analysis across diverse vertebrates revealed that the IL26 gene has also independently been inactivated in a few other mammals, including the African elephant and the European hedgehog. The IL26 gene thus appears to be highly variable, and the conserved open reading frame has been lost several times during mammalian evolution.

Novel and Recurrent Mutations of WISP3 in Two Chinese Families with Progressive Pseudorheumatoid Dysplasia

BackgroundThe WNT1-inducible signaling pathway protein 3 (WISP3), which belongs to the CCN (cysteine-rich protein 61, connective tissue growth factor, nephroblastoma overexpressed) family, is a secreted cysteine-rich matricellular protein that is involved in chondrogenesis, osteogenesis and tumorigenesis. WISP3 gene mutations are associated with progressive pseudorheumatoid dysplasia (PPD, OMIM208230), an autosomal recessive genetic disease that is characterized by the swelling of multiple joints and disproportionate dwarfism.Methodology/Principal FindingsFour PPD patients from two unrelated Chinese families were recruited for this study. The clinical diagnosis was confirmed by medical history, physical examinations, laboratory results and radiological abnormalities. WISP3 mutations were detected by direct DNA sequence analysis. In total, four different mutations were identified, which consisted of two missense mutations, one deletion and one insertion that spanned exons 3, 5 and 6 of the WISP3 gene. One of the missense mutations (c.342T>G/p.C114W) and a seven-base pair frameshift deletion (c.716_722del/p.E239fs*16) were novel. The other missense mutation (c.1000T>C/p. S334P) and the insertion mutation (c.866_867insA/p.Q289fs*31) had previously been identified in Chinese patients. All four cases had a compound heterozygous status, and their parents were heterozygous carriers of these mutations.Conclusions/SignificanceThe results of our study expand the spectrum of WISP3 mutations that are associated with PPD and further elucidate the function of WISP3.

Letter to the Editor

18 June, 2009 Dear Editor, DUODENAL ATRESIA: CONSIDER FEINGOLD SYNDROME Duodenal atresia affects one in 6000 births and is usually isolated. Feingold syndrome is an autosomal dominant cause of upper intestinal atresia (oesophagus or duodenum), digital anomalies and microcephaly. Infants with either atresia should be assessed for short middle phalanges (brachymesophalangy) of the 2nd and 5th fingers, cutaneous toe syndactyly and microcephaly. The presence of these signs should prompt further evaluation with hand X-rays, echocardiography, renal imaging, audiology and genetics consultation. We highlight this recommendation with the description of a neonate initially thought to have isolated duodenal atresia ultimately diagnosed with Feingold syndrome, confirmed by MYCN sequence analysis. The term female neonate with birthweight 3.16 kg (10th percentile), length 50 cm (50–90th percentile) and head circumference 32 cm (10th percentile) developed bilious vomiting after birth. Abdominal X-ray suggested duodenal atresia confirmed and repaired by laparotomy on day 3. As she had mild microcephaly, genetic consultation was requested (Fig. 1). Subtle digital anomalies including brachymesophalangy and bilateral clinodactyly of the 2nd and 5th fingers, broad feet, short toes and bilateral 4th/5th toe syndactyly were noted. Echocardiography, renal ultrasound and karyotype were normal. Hand X-rays confirmed hypoplasia of the middle phalynx of the index and little fingers (Fig. 2). Parental hands, feet and head circumference were all normal. Microcephaly and subtle dysmorphic features. Plain X-ray of the left hand demonstrating hypoplasia of the middle phalanx of the 2nd and 5th digits. MYCN sequence analysis detected a ‘de novo’ heterozygous 6 base pair frameshift deletion in exon 3, confirming the clinical diagnosis of Feingold syndrome. On follow-up at 13 months, she was developmentally appropriate. Her height was on the 10–25th percentile, weight 25–50th percentile and head circumference below the 2nd percentile. The commonest syndrome association with duodenal atresia is trisomy 21, present in 24% of affected neonates1 in one series. A less common association is Feingold Syndrome (OMIM #146280), first described in 1975 as a clinically variable autosomal dominant condition characterised by microcephaly, digital anomalies and intestinal atresia2, 3 caused by heterozygous mutations in MYCN gene.4 The combination of mild-moderate microcephaly and digital anomalies is highly suggestive of Feingold syndrome.3 The most consistent clinical features, present in almost all affected cases, are brachymesophalangy of the second and fifth fingers and cutaneous toe syndactyly, typically of fourth and fifth toes.3, 5 Ninety percent of affected patients have a head circumference less than the 10th percentile and up to 55% have gastrointestinal atresia, typically of the oesophageus or duodenum.5 Approximately half have intellectual disability, usually mild-moderate. Ten percent have hearing loss. Cardiac and renal anomalies may also be present. Suggestive facial features include short palpebral fissures and micrognathia. VACTERL association is often a consideration. Feingold syndrome has complete penetrance, but variable expressivity, so may be unrecognised in the parent of an affected child.5 A family history of gastrointestinal atresia should be sought and the parents should be carefully assessed with measurement of head circumference and examination of the hands and feet. A MYCN gene mutation is identified in 75% of patients with clinically suspected Feingold syndrome, suggesting other genes may be involved in this syndrome. All mutations result in haploinsufficiency, suggesting that gene dosage is important in embryogenesis and post-natal head growth,4 although the exact mechanism is not understood. MYCN mutation detection confirms the diagnosis and allows for accurate genetic counselling about recurrence. In summary, Feingold syndrome is a rare cause of upper intestinal atresia. Infants with intestinal atresia should be routinely evaluated for digital anomalies and microcephaly. Although rare, recognition of this condition is important for genetic counselling and ongoing management. The authors acknowledge Dr EJ Kamsteeg and Dr FA Hol at the DNA laboratory KGCN (Klinisch- Genetisch Centrum Nijmegen) for performing MYNC sequence analysis. We would also like to thank the parents for their consent to publish this article. Answer: This baby has staphylococcal scalded skin syndrome. It is caused by toxin-producing strains of Staphylococcus aureus, which can be either sensitive strains or methicillin-resistant.

Prenyldiphosphate synthase, subunit 1 (PDSS1) and OH-benzoate polyprenyltransferase (COQ2) mutations in ubiquinone deficiency and oxidative phosphorylation disorders

Coenzyme Q10 (CoQ10) plays a pivotal role in oxidative phosphorylation (OXPHOS), as it distributes electrons among the various dehydrogenases and the cytochrome segments of the respiratory chain. We have identified 2 novel inborn errors of CoQ10 biosynthesis in 2 distinct families. In both cases, enzymologic studies showed that quinone-dependent OXPHOS activities were in the range of the lowest control values, while OXPHOS enzyme activities were normal. CoQ10 deficiency was confirmed by restoration of normal OXPHOS activities after addition of quinone. A genome-wide search for homozygosity in family 1 identified a region of chromosome 10 encompassing the gene prenyldiphosphate synthase, subunit 1 (PDSS1), which encodes the human ortholog of the yeast COQ1 gene, a key enzyme of CoQ10 synthesis. Sequencing of PDSS1 identified a homozygous nucleotide substitution modifying a conserved amino acid of the protein (D308E). In the second family, direct sequencing of OH-benzoate polyprenyltransferase (COQ2), the human ortholog of the yeast COQ2 gene, identified a single base pair frameshift deletion resulting in a premature stop codon (c.1198delT, N401fsX415). Transformation of yeast Deltacoq1 and Deltacoq2 strains by mutant yeast COQ1 and mutant human COQ2 genes, respectively, resulted in defective growth on respiratory medium, indicating that these mutations are indeed the cause of OXPHOS deficiency.

A trinucleotide deletion together with a base duplication event at codon 439 in the human tyrosinase gene identifies a mutational hotspot

Molecular analysis of the human tyrosinage gene in two patients suffering from a temperature-sensitive form of albinism has identified a thymine triplet deletion at codon 439 which is accompanied by a duplication of the immediately preceding cytosine residue. This results in a two base pair frame shift leading to premature termination at codon 448, giving a truncated protein. Its relationship to other mutations in tyrosinase and the possible cause are discussed. The temperature-sensitive phenotype is due to the guanine to adenine mutation at codon 422, known to generate a temperature-sensitive enzyme. The CTTT at F 439 in tyrosinase is also present at F 508 in CFTR, the main mutation causing cystic fibrosis.

Prokaryotic ribosomes recode the HIV-1 gag-pol-1 frameshift sequence by an E/P site post-translocation simultaneous slippage mechanism.

The mechanism favoured for -1 frameshifting at typical retroviral sites is a pre-translocation simultaneous slippage model. An alternative post-translocation mechanism would also generate the same protein sequence across the frameshift site and therefore in this study the strategic placement of a stop codon has been used to distinguish between the two mechanisms. A 26 base pair frameshift sequence from the HIV-1 gag-pol overlap has been modified to include a stop codon immediately 3' to the heptanucleotide frameshift signal, where it often occurs naturally in retroviral recoding sites. Stop codons at the 3'-end of the heptanucleotide sequence decreased the frame-shifting efficiency on prokaryote ribosomes and the recording event was further depressed when the levels of the release factors in vivo were increased. In the presence of elevated levels of a defective release factor 2, frameshifting efficiency in vivo was increased in the constructs containing the stop codons recognized specifically by that release factor. These results are consistent with the last six nucleotides of the heptanucleotide slippery sequence occupying the ribosomal E and P sites, rather than the P and A sites, with the next codon occupying the A site and therefore with a post-translocation rather than a pre-translocation -1 slippage model.

The complexity of nitrosoguanidine mutagenesis increases with size: observations of the mutational specificity of N-propyl-N'-nitro-N-nitrosoguanidine.

The mutational specificity of the monofunctional alkylating agent N-propyl-N'-nitro-N-nitrosoguanidine (PNNG) has been determined through the DNA sequence characterization of 109 LacI- mutations of Escherichia coli. The predominant mutation induced was the G:C----A:T transition (73%), presumably the result of O6-propylguanine damage. Transversions constituted 18% of the mutants, almost entirely due to G:C----T:A (9%) and A:T----C:G (8%) events. Two identical deletions, one single base pair frameshift and a tandem double base change were also recovered. In contrast, N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) was previously found to induce only transitions, again predominantly G:C----A:T events (98%). Moreover, the site specificity observed for PNNG-induced G:C----A:T transitions is quite distinct from that induced by MNNG. G:C----A:T transitions recovered following PNNG treatment do not appear to be influenced by neighbouring base sequence to the extent seen for MNNG.