Biallelic Changes Research Articles

Variation in floral form of CRISPR knock-outs of the poplar homologs of LEAFY and AGAMOUS after FT heat-induced early flowering.

Plant migration and gene flow from genetically modified or exotic trees to nearby lands or by crossing with wild relatives is a major public and regulatory concern. Many genetic strategies exist to mitigate potential gene flow; however, the long delay in onset of flowering is a severe constraint to research progress. We used heat-induced FT overexpression to speed assessment of the expected floral phenotypes after CRISPR knockout of poplar homologs of the key floral genes, LEAFY and AGAMOUS. We selected events with previously characterized CRISPR-Cas9 induced biallelic changes then re-transformed them with the Arabidopsis thaliana FLOWERING LOCUS T (AtFT) gene under control of either a strong constitutive promoter or a heat-inducible promoter. We successfully obtained flowering in both a male and female clones of poplar, observing a wide range of inflorescence and floral forms among flowers, ramets, and insertion events. Overall, flowers obtained from the selected LFY and AG targeted events were consistent with what would be predicted for loss-of-function of these genes. LFY-targeted events showed small catkins with leaf-like organs, AG-targeted events had nested floral organs consistent with reduction in floral determinacy and absence of well-formed carpels or anthers. These findings demonstrate the great developmental plasticity of Populus flowers during genetically accelerated flowering, which may be of horticultural value. They also provide an informative early view of floral phenotypes and apparent sterility from knockouts of both these gene targets.

Phenotypic Assessment of Pathogenic Variants in GNAO1 and Response to Caffeine in C. elegans Models of the Disease.

De novo mutations affecting the G protein α o subunit (Gαo)-encoding gene (GNAO1) cause childhood-onset developmental delay, hyperkinetic movement disorders, and epilepsy. Recently, we established Caenorhabditis elegans as an informative experimental model for deciphering pathogenic mechanisms associated with GNAO1 defects and identifying new therapies. In this study, we generated two additional gene-edited strains that harbor pathogenic variants which affect residues Glu246 and Arg209-two mutational hotspots in Gαo. In line with previous findings, biallelic changes displayed a variable hypomorphic effect on Gαo-mediated signaling that led to the excessive release of neurotransmitters by different classes of neurons, which, in turn, caused hyperactive egg laying and locomotion. Of note, heterozygous variants showed a cell-specific dominant-negative behavior, which was strictly dependent on the affected residue. As with previously generated mutants (S47G and A221D), caffeine was effective in attenuating the hyperkinetic behavior of R209H and E246K animals, indicating that its efficacy is mutation-independent. Conversely, istradefylline, a selective adenosine A2A receptor antagonist, was effective in R209H animals but not in E246K worms, suggesting that caffeine acts through both adenosine receptor-dependent and receptor-independent mechanisms. Overall, our findings provide new insights into disease mechanisms and further support the potential efficacy of caffeine in controlling dyskinesia associated with pathogenic GNAO1 mutations.

CRISPR/Cas9-induced modification of the conservative promoter region of VRN-A1 alters the heading time of hexaploid bread wheat.

In cereals, the vernalization-related gene network plays an important role in regulating the transition from the vegetative to the reproductive phase to ensure optimal reproduction in a temperate climate. In hexaploid bread wheat (Triticum aestivum L.), the spring growth habit is associated with the presence of at least one dominant locus of VERNALIZATION 1 gene (VRN-1), which usually differs from recessive alleles due to mutations in the regulatory sequences of the promoter or/and the first intron. VRN-1 gene is a key regulator of floral initiation; various combinations of dominant and recessive alleles, especially VRN-A1 homeologs, determine the differences in the timing of wheat heading/flowering. In the present study, we attempt to expand the types of VRN-A1 alleles using CRISPR/Cas9 targeted modification of the promoter sequence. Several mono- and biallelic changes were achieved within the 125-117 bp upstream sequence of the start codon of the recessive vrn-A1 gene in plants of semi-winter cv. 'Chinese Spring'. New mutations stably inherited in subsequent progenies and transgene-free homozygous plants carrying novel VRN-A1 variants were generated. Minor changes in the promoter sequence, such as 1-4 nucleotide insertions/deletions, had no effect on the heading time of plants, whereas the CRISPR/Cas9-mediated 8 bp deletion between -125 and -117 bp of the vrn-A1 promoter shortened the time of head emergence by up to 2-3 days. Such a growth habit was consistently observed in homozygous mutant plants under nonvernalized cultivation using different long day regimes (16, 18, or 22h), whereas the cold treatment (from two weeks and more) completely leveled the effect of the 8 bp deletion. Importantly, comparison with wild-type plants showed that the implemented alteration has no negative effects on main yield characteristics. Our results demonstrate the potential to manipulate the heading time of wheat through targeted editing of the VRN-A1 gene promoter sequence on an otherwise unchanged genetic background.

Biallelic ZNFX1 variants are associated with a spectrum of immuno-hematological abnormalities.

Biallelic changes in the ZNFX1 gene have been recently reported to cause severe familial immunodeficiency. Through a search of our bio/databank with information from genetic testing of >55 000 individuals, we identified nine additional patients from seven families with six novel homozygous ZNFX1 variants. Consistent with the previously described phenotype, our patients suffered from monocytosis, thrombocytopenia, hepatosplenomegaly, recurrent infections, and lymphadenopathy. The two most severely affected probands also had renal involvement and clinical presentations compatible with hemophagocytic lymphohistiocytosis. The disease was less lethal among our patients than previously reported. We identified two missense changes, two variants predicted to result in complete protein loss through nonsense-mediated decay, and two frameshift changes that likely introduce a truncation. Our findings (i) independently confirm the role of ZNFX1 in primary genetic immunodeficiency, (ii) expand the genetic and clinical spectrum of ZNFX1-related disease, and (iii) illustrate the utility of large, well-curated, and continually updated genotype-phenotype databases in resolving molecular diagnoses of patients with initially negative genetic testing findings.

Molecular Profiling of Relapsed and Refractory Myeloma Identifies Frequent Bi-Allelic Driver Events — Interim Results of the ‘Myeloma UK Seven’ Biomarker Trial

IntroductionPomalidomide and low-dose dexamethasone (PomDex) are approved for relapsed and refractory myeloma (RRMM) with Overall Response Rates of about 30%, including durable remissions. Combining PomDex with biologic agents can improve response, but there are currently no markers to predict which patients benefit from the addition of drugs to PomDex alone. Myeloma UK (MUK) seven is a multi-center UK phase II trial that is designed to generate evidence for improved outcomes and treatment stratification in RRMM by: 1) building a bio-repository for molecular characterization of RRMM and discovery of predictive markers for response 2) patient randomisation to Cyclophosphamide-PomDex (CyPomDex) or PomDex to investigate if the cost-effective CyPomDex combination increases progression free survival compared to PomDex 3) a flexible design that allows addition of trial arms with other promising PomDex combinations. We report high patient and investigator bio-sampling adherence and first comprehensive genetic profiling results in RRMM using a novel molecular assay platform.MethodsMUKseven (NCT02406222) will in total recruit 250 RRMM patients who are currently randomised 1:1 to CyPomDex or PomDex therapy. Registered patients undergo bone marrow sampling at screening, Cycle 1 Day 14, Cycle 4 Day 14 and at disease progression, and peripheral blood sampling on Cycle 1 Day 1. Patient consent includes mandatory bio-sampling at screening. Material is shipped to central laboratories for processing and storage of CD138-purified (>95%) MM tumor and non-tumor bone marrow microenvironment cells. Participants are currently recruited from over 30 NHS Hospitals in the MUK Early Phase Clinical Network.Digital multiplex ligation-dependent probe amplification (dMLPA®; MRC Holland) is a novel assay for high-throughput genome-wide copy number aberration (CNA) profiling. The MM specific D006 assay contains 160 probes that interrogate regions frequently affected by CNA in MM, including hotspots CDKN2C, CKS1B, MYC and regions encoding for therapeutic targets such as MCL1 or SLAMF7. In addition, 219 probes are distributed over all chromosomal arms and inform a virtual karyogram. The targeted design allows for analysis of over 80 patient samples in a single MiSeq (Illumina) run, using 10-20 ng DNA input material.ResultsBetween March 2016 and July 2017 a total of 83 patients were randomised to MUKseven with a median follow up of 6 cycles (1-13). Bone marrow material was received by central laboratories for all patients (100%) at screening, 69/79 (87.3%) at Cycle 1 day 14, 47/54 (87.0%) at Cycle 4 day 14 and 13/29 (44.8%) at disease progression. Peripheral blood was received for 73/79 (92.4%) on Cycle 1 Day 1. These return rates are higher than those reported for recent commercial trials. Of importance, for 52% of patients, matched bone marrow samples from earlier disease stages collected as part of UK academic research studies were available.We profiled the first 81 RRMM samples from trial entry with dMLPA and compared CNA frequency to a recently published newly diagnosed MM (NDMM) cohort which was assessed with MLPA (Shah V, Leukemia 2017). We found significantly more focal bi-allelic CNA of driver lesions in RRMM than in NDMM (P = 0.002). These included homozygous deletions of CDKN2C (6.2 vs 1.9%) and RB1 (3.7 vs 0.8%), amplifications of MYC (8.6 vs 2.3%), and heterozygous losses of TP53 (16.0 vs 9.6%). There were significantly more RRMM patients with one or more of the following bi-allelic changes: CDKN2C, RB1 or CYLD homozygous loss, or 1q amplification (18.5 vs 8.2%; P = 0.007), and 3.7% of patients in the RRMM cohort showed more than one bi-allelic change. We identified frequent homozygous deletions in NFκB pathway genes such as CYLD (4.9%), and recurrent amplifications of therapeutic targets like MCL1 and SLAMF7 on chromosome 1 (8.6% amplifications). The trial is recruiting and profiling of additional cases as well as longitudinal profiling of cases with material from earlier disease stages is ongoing.ConclusionWe demonstrate highly successful conduct of a multi-center biomarker discovery trial by the Myeloma UK Clinical Trial Network and the UK Myeloma Research Alliance. Our results demonstrate that bi-allelic CNA increase significantly in RRMM and some of these may help guide therapy. Recruitment and analyses of additional cases enrolled into MUKseven are ongoing and updated data will be presented at the meeting. DisclosuresPawlyn:Takeda: Honoraria, Other: Travel support; Janssen: Other: Travel support; Celgene: Honoraria, Other: Travel support. Atanesyan:MRC-Holland: Employment. Savola:MRC-Holland: Employment. Thakurta:Celgene Corporation: Employment, Equity Ownership. Garg:Janssen: Other: travel support, Research Funding, Speakers Bureau; Takeda: Other: travel support; Novartis: Other: travel support, Research Funding. Cook:Celgene: Consultancy, Honoraria, Research Funding, Speakers Bureau; Janssen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Sanofi: Consultancy, Honoraria, Speakers Bureau; Bristol-Myers Squibb: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Research Funding, Speakers Bureau; Glycomimetcs: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau. Boyd:Janssen: Honoraria, Other: Travel support; Amgen: Consultancy, Honoraria; Novartis: Consultancy, Honoraria; Celgene: Consultancy, Honoraria. Kaiser:Takeda: Consultancy; BMS: Consultancy, Other: Travel expenses; Janssen: Honoraria; Chugai: Consultancy; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Genetics of Wilson disease and Wilson-like phenotype in a clinical series from eastern Spain.

Wilson's disease (WD) is an autosomal recessive disorder caused by ATP7B mutations. Subjects with only one mutation may show clinical signs and individuals with biallelic changes may remain asymptomatic. We aimed to achieve a conclusive genetic diagnosis for 34 patients clinically diagnosed of WD. Genetic analysis comprised from analysis of exons to WES (whole exome sequencing), including promoter, introns, UTRs (untranslated regions), besides of study of large deletions/duplications by MLPA (multiplex ligation-dependent probe amplification). Biallelic ATP7B mutations were identified in 30 patients, so that four patients were analyzed using WES. Two affected siblings resulted to be compound heterozygous for mutations in CCDC115, which is involved in a form of congenital disorder of glycosylation. In sum, the majority of patients with a WD phenotype carry ATP7B mutations. However, if genetic diagnosis is not achieved, additional genes should be considered because other disorders may mimic WD.

Mutation Profile of PPM1D and TP53 in Patients with Myelodysplastic Syndromes and Complex Chromosome Abnormalities

Introduction: Complex (≥3) abnormalities (cA) are associated with an inferior outcome in myelodysplastic syndromes (MDS). About 50% of MDS with cA show mutations in TP53 that might contribute to the formation of the cA and worsen prognosis (Haase et al., Leukemia, 2019). In former single nucleotid polymorphism (SNP) analysis we found chromosome 17q being affected in several patients with cA with a higher incidence as by chance. In just this region is a gene called PPM1D located which already has been observed as one of the most frequently mutated genes in pts./individuals with clonal hematopoiesis with indetermined significance (CHIP). PPM1D is encoding for a protein named Wip1. This protein acts as an inhibitor of p53. About 5% of MDS with 5q deletions show mutations in PPM1D (Panagiota et al., ASH 2017). Mutations in PPM1D are even more common among pts with therapy-related MDS (15%, Lindsley et al., 2017). The aim of our study was to determine the frequency of PPM1D mutations in MDS with cA and to shed light upon their possible contribution to the formation of cA. Methods and patients: We included 100 patients characterized by conventional cytogenetics in our analysis (67x MDS; 30x secondary acute myeloid leukemia, AML; 3x chronic myelomonocytic leukemia, CMML). 20 pts had a therapy-related MDS. All the included pts had cA with a median number of aberrations of 8 (range: 3-50). The median age at first diagnosis of MDS with cA was 72 (range 29-95). A deletion of 5q was found in 71 patients (71%). The TP53 status was known for all pts by fluorescence in situ hybridization (FISH) and/or molecular karyotyping (TP53 deletion status) and sequencing (TP53 mutation status). 68 of 100 pts had an alteration on TP53 (68%, 4 deletions, 34 mutations, 30 biallelic changes). All pts were subjected to next generation sequencing of PPM1D. Amplicons for exons 1 to 6 were generated by multiplex polymerase chain reaction (PCR). The pooled amplicons were processed using the Nextera XT2 sample preparation kit (Illumina, San Diego, Ca, USA) followed by sequencing on a MiniSeq platform (Illumina, San Diego, Ca, USA). We used our local bioinformatics pipeline to identify single-nucleotide variants (SNVs) and indels. Results: In ten pts (10%) we found single-nucleotide variants of PPM1D. The median number of aberrations was 8 (range: 5-15). Six of those PPM1D variants have already been described as very rare SNPs. Three of them were located in the 3'UTR (untranslated region), the other three seem to be silent mutations. The other four are not listed in common databases. Three of those four are potential missense mutations, one is a potential nonsense mutation. Two variants are located at the same -previously undescribed- position (c.230A>C, p.D77A). Two of those four patients showed an additional TP53 mutation, one of them biallelic. A deletion of 5q was identified in two of them. One pt had therapy related MDS. At a clone size of the complex karyotype of 94% and 90%, the VAF of three of the recurrent mutation was just 7% and 8%, indicating that the PPM1D mutation arised in a subclone in these pts. In one pt the VAF was 33,6%. The VAF of 30-38% in the other cases implies PPM1D being an ancestral or co-dominant mutation. Conclusion: We were able to show that PPM1D is mutated in MDS with cA in a relevant fraction of pts. In our cohort, 10% of MDS pts with cA are affected. 4% may have a deleterious mutation of PPM1D. Although PPM1D mutations were described to preferentially occur in therapy related diseases (Lindsley et al., 2017), in our cohort three of four patients with potential PPM1D mutation had no known prior chemo-/radiation therapy. Mutations in PPM1D might contribute to the formation or toleration of cA alternatively to TP53 mutations as two of four patients with PPM1D mutations did not show TP53 mutations and the PPM1D mutations could be the ancestral or co-dominant mutation in two of four cases. Our data imply that also mutations in PPM1D may be important for prognosis and therapy decisions in MDS patients with cA. We will continue observing our patients in order to enlarge the database and to find out which impact mutations in PPM1D may have on overall survival and whether they can affect the prognosis of patients with cA. Disclosures Germing: Jazz Pharmaceuticals: Honoraria; Novartis: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria. Hertenstein:RS Media: Research Funding. Platzbecker:Novartis: Consultancy, Honoraria, Research Funding; Abbvie: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Generation and characterization of a human iPSC line (UAMi005-A) from a patient with nonketotic hyperglycinemia due to mutations in the GLDC gene

A human induced pluripotent stem cell (iPSC) line was generated from fibroblasts of a patient with nonketotic hyperglycinemia bearing the biallelic changes c.1742C > G (p.Pro581Arg) and c.2368C > T (p.Arg790Trp) in the GLDC gene. Reprogramming factors OCT3/4, SOX2, KLF4 and c-MYC were delivered using a non-integrative method based on the Sendai virus. Once established, iPSCs have shown full pluripotency, differentiation capacity and genetic stability. This cellular model provides a good resource for disease modeling and drug discovery.

Leber Congenital Amaurosis Associated with AIPL1: Challenges in Ascribing Disease Causation, Clinical Findings, and Implications for Gene Therapy

Leber Congenital Amaurosis (LCA) and Early Childhood Onset Severe Retinal Dystrophy are clinically and genetically heterogeneous retinal disorders characterised by visual impairment and nystagmus from birth or early infancy. We investigated the prevalence of sequence variants in AIPL1 in a large cohort of such patients (n = 392) and probed the likelihood of disease-causation of the identified variants, subsequently undertaking a detailed assessment of the phenotype of patients with disease-causing mutations. Genomic DNA samples were screened for known variants in the AIPL1 gene using a microarray LCA chip, with 153 of these cases then being directly sequenced. The assessment of disease-causation of identified AIPL1 variants included segregation testing, assessing evolutionary conservation and in silico predictions of pathogenicity. The chip identified AIPL1 variants in 12 patients. Sequencing of AIPL1 in 153 patients and 96 controls found a total of 46 variants, with 29 being novel. In silico analysis suggested that only 6 of these variants are likely to be disease-causing, indicating a previously unrecognized high degree of polymorphism. Seven patients were identified with biallelic changes in AIPL1 likely to be disease-causing. In the youngest subject, electroretinography revealed reduced cone photoreceptor function, but rod responses were within normal limits, with no measurable ERG in other patients. An increasing degree and extent of peripheral retinal pigmentation and degree of maculopathy was noted with increasing age in our series. AIPL1 is significantly polymorphic in both controls and patients, thereby complicating the establishment of disease-causation of identified variants. Despite the associated phenotype being characterised by early-onset severe visual loss in our patient series, there was some evidence of a degree of retinal structural and functional preservation, which was most marked in the youngest patient in our cohort. This data suggests that there are patients who have a reasonable window of opportunity for gene therapy in childhood.

Frequency of the Common MYH Mutations (G382D and Y165C) in MMR Mutation Positive and Negative HNPCC Patients

Recently mutations in the MYH gene have been associated with a milder form of adenomatous polyposis which is characterized by a variable level of colonic polyps ranging from a few to several hundred. In the context of HNPCC it is not unusual to identify patients with a smattering of polyps. The MYH gene product is involved in DNA repair and indeed the hMSH2/hMSH6 complex (both genes being essential elements of the DNA mismatch repair pathway) is required to stimulate MYH activity. We reasoned that because of the clinical similarity of a subset of HNPCC patients to those described with MYH mutations and the role of the hMSH2/hMSH6 complex in the activation of MYH protein that MYH mutations may account for a small proportion of HNPCC patients. In a study of 442 HNPCC patients we identified MYH mutations at the same frequency as that expected in the general population. Nevertheless, two HNPCC families were identified harbouring biallelic changes in MYH.

Homozygous deletions and point mutations of the Rit1/Bcl11b gene in γ-ray induced mouse thymic lymphomas

Allelic loss (LOH) mapping and sequence analysis were conducted for γ-ray induced mouse thymic lymphomas and a novel tumor suppressor gene, Rit1/Bcl11b, on chromosome 12 was isolated. Bi-allelic changes were found in 17 of the 66 p53-proficient lymphomas with Rit1 LOH but in only 2 of the 54 p53-deficient lymphomas. This suggests an association between the presence of functional p53 and inactivation of the Rit1 gene in the lymphoma development. Introduction of Rit1 into HeLa cells lacking Rit1 expression suppressed cell growth. These results indicate that loss-of-function mutations of Rit1 contribute to mouse lymphomagenesis and possibly to human cancer development.

Genetic mapping and allelic loss analysis in mouse thymic lymphomas of Helios and Aiolos belonging to the Ikaros gene family.

The Ikaros gene undergoes bi‐allelic changes at a high frequency in γ‐ray‐induced mouse thymic lymphomas, suggesting the relevance of Ikaros to the lymphoma development. Here we test whether Helios and Aiolos, two other members of the Ikaros gene family, are also involved in lymphomagenesis. Genetic mapping showed that Helios is located between D1Mit531 and D1Mit19 on chromosome 1 and Aiolos is between D11Mit222 and D11Mit332 on chromosome 11. Analysis using polymorphic markers around the two regions revealed that neither locus exhibited allelic loss in the 78 lymphomas that were induced in p53 wild‐type mice, whereas in 102 p53(KO/+) mouse‐derived lymphomas Helios and Aiolos loci showed allelic loss in 8% (8/102) and 33% (34/102), respectively. However, 33 of the 34 lymphomas showing allelic loss at Aiolos were/j53(KO/‐) and were accompanied by loss of the p53 wild‐type allele on the same chromosome. Homozygous deletion and mutation analyses failed to detect bi‐allelic alterations. These results do not suggest any obvious contribution of Helios or Aiolos to oncogenesis of the mouse thymic lymphomas.