Monoallelic Mutations Research Articles

BIOL-18. NEWLY DEVELOPED REPLICATION REPAIR DEFICIENT (RRD) MOUSE MODELS PROVIDE INSIGHTS INTO MEDULLOBLASTOMA/GLIOMAGENESIS AND RESPONSE TO IMMUNOTHERAPY

Abstract Replication Repair Deficiency (RRD), caused by germline monoallelic (Lynch Syndrome) or biallelic (Constitutional Mismatch Repair Deficiency, CMMRD) mutations in MMR genes, is present in 5-10% of glioblastomas in children, adolescents, and young adults. RRD glioblastomas are chemoradiation-resistant, but respond favorably to immune checkpoint inhibition (ICI). Representative immunocompetent animal models are urgently needed for 3 recently identified subgroups based on specific somatically-acquired mutations, survival, and immunotherapy response (RRD1: MMRD with POLE mutations, RRD2: MMRD associated with TP53 mutations, and RRD3: MMRD harboring IDH1 mutations). Using germline mutations and brain-specific Cre-drivers, we genetically engineered mouse models that recapitulate each human RRD-subgroup. RRD1 (Nestin- and Olig2-Cre+/ Msh2LoxP/LoxP/PoleS459F/+ and LSL-PoleP286R/+): CMMRD-like Nestin-Cre-driven mice develop posterior-fossa glioma-like or medulloblastoma (MB)-like tumors at ~2.7 months. Olig2-Cre-driven mice display hemispheric gliomas at ~10 months, suggesting distinct cell-of-origin. RRD2 (Nestin-Cre+/Trp53LoxP/LoxP and Msh2LoxP/LoxP or Mlh1-/-): CMMRD-like tumors develop in heterogenous locations at ~4.5 months (p<0.0001), classifying primarily as MB-like in hindbrain, and glioma-like in other brain regions. Strikingly, germline Mlh1 tumors occur earlier than Nestin-Cre-driven RRD2 tumors, indicating early developmental mutation accumulation in CMMRD-patients. Lynch-like RRD1/2 mice succumb exclusively to gliomas >13 months (p<0.0001). RRD3 (Olig2-Cre+/Msh2LoxP/LoxP/Trp53LoxP/LoxP/LSL-Idh1R132H/+): brain tumors occur later and are hemispheric. These observations recapitulate human data, where CMMRD-patients develop glioblastoma/MB earlier than Lynch-patients (8.6 vs. 14-years; p<0.0001), and posterior-fossa glioblastoma/MB presents earlier than hemispheric gliomas (p=0.04). Additionally, tumor onset and location vary (RRD1: 7.6-years, RRD2: 8.3-years, hemispheric/posterior-fossa; RRD3: 12-years, hemispheric; p=0.005). In both mice and humans, RRD1 exhibits ultra-hypermutation, high immune infiltration, and response to ICI, whereas RRD2 harbors lower mutational burden, are immune-cold, and ICI-monotherapy resistant. Temporal dynamics of RRD tumor development is currently being tracked by serial MRI to define biologically relevant time points. Our models accurately mimic the human condition and provide unique insights into RRD tumorigenesis, allowing optimization of subgroup-tailored therapeutic approaches.

TCF3 haploinsufficiency defined by immune, clinical, gene-dosage, and murine studies

TCF3 is a transcription factor contributing to early lymphocyte differentiation. Germline monoallelic dominant negative and biallelic loss-of-function (LOF) null TCF3 mutations cause a fully penetrant severe immunodeficiency. We identified 8 individuals from 7 unrelated families with monoallelic LOF TCF3 variants presenting with immunodeficiency with incomplete clinical penetrance. We sought to define TCF3 haploinsufficiency (HI) biology and its association with immunodeficiency. Patient clinical data and blood samples were analyzed. Flow cytometry, Western blot analysis, plasmablast differentiation, immunoglobulin secretion, and transcriptional activity studies were conducted on individuals carrying TCF3 variants. Mice with a heterozygous Tcf3 deletion were analyzed for lymphocyte development and phenotyping. Individuals carrying monoallelic LOF TCF3 variants showed B-cell defects (eg, reduced total, class-switched memory, and/or plasmablasts) and reduced serum immunoglobulin levels; most but not all presented with recurrent but nonsevere infections. These TCF3 LOF variants were either not transcribed or translated, resulting in reduced wild-type TCF3 protein expression, strongly suggesting HI pathophysiology for the disease. Targeted RNA sequencing analysis of T-cell blasts from TCF3-null, dominant negative, or HI individuals clustered away from healthy donors, implying that 2 WT copies of TCF3 are needed to sustain a tightly regulated TCF3 gene-dosage effect. Murine TCF3 HI resulted in a reduction of circulating Bcells but overall normal humoral immune responses. Monoallelic LOF TCF3 mutations cause a gene-dosage-dependent reduction in wild-type protein expression, B-cell defects, and a dysregulated transcriptome, resulting in immunodeficiency. Tcf3+/- mice partially recapitulate the human phenotype, underscoring the differences between TCF3 in humans and mice.

Treatment phase stratified outcomes of core binding factor acute myeloid leukemia.

e19055 Background: Mutations in core binding factor (CBF) are associated with a favorable prognosis in acute myeloid leukemia (AML). However, between 30-40% of patients with CBF-AML relapse, and relative outcomes with each subsequent line of therapy remain unclear. Additionally, the overall survival of CBF-AML compared to other favorable risk molecular aberrations — such as NPM1mut and CEBPA — is unknown in light of the addition of monoallelic CEBPA in ELN 2022. Therefore, the purpose of this study was to clarify the outcomes of CBF-AML relative to other favorable risk aberrations and to determine the relative overall survival associated with each subsequent relapse. Methods: We retrospectively analyzed 46 consecutive patients with CBF-AML treated from January 2013 to January 2022 at VCU Massey Cancer Center that underwent intensive chemotherapy (IC) with or without gemtuzumab ozogamicin. During the same study period, we analyzed a comparison cohort of 272 consecutive patients: 103 ELN 2017 intermediate-risk patients, 111 adverse-risk patients, eight patients with mono- or biallelic mutations in CEBPA, and 50 with ELN 2017 favorable-risk NPM1mut. All patients were treated with front-line IC. We analyzed survival by the Kaplan-Meier method and compared groups with the log-rank test. Results: We analyzed 46 patients with CBF-AML across 136 treatment phases. Patients with CBF-AML had superior overall survival compared to patients with NPM1mut (median overall survival not reached at a median follow-up time of 5.0 years versus 21.0 m., p = 0.028). There were no differences in survival between CBF-AML and AML with monoallelic or biallelic mutations in CEBPA at a median follow-up time of 3.7 years (not reached, p = 0.976). Expectedly, the overall survival for CBF-AML was superior to ELN 2017 intermediate (30.1 m., p = 0.011) and adverse (10.9 m., p < 0.0001) risk disease. Next, we analyzed survival trends in the relapsed or refractory setting for CBF-AML by lines of therapy, using ELN 2017 intermediate and adverse risk cohorts as a comparison. During the first relapse, CBF-AML was associated with a significantly superior overall survival compared to the adverse cohort (19.1 m. versus 10.0 m., p = 0.024) but not the intermediate cohort (14.5 m., p = 0.734). The absence of KITmut was not associated with improved survival compared to the ELN 2017 intermediate category at the first relapse ( p = 0.610). At the second relapse, the survival disparity disappeared between the CBF-AML and adverse risk groups (7.7 m. vs 4.0 m., p = 0.964). Conclusions: CBF-AML was associated with similar survival to mono- or biallelic mutations in CEBPA, and significantly prolonged overall survival compared to NPM1mut. Relative to a matched cohort of adverse-risk AML at the time of the first relapse, outcomes of relapsed CBF-AML remain favorable. The relative overall survival benefit of CBF-AML disappeared at the time of the second relapse.

Gene Supplementation in Mice Heterozygous for the D477G RPE65 Variant Implicated in Autosomal Dominant Retinitis Pigmentosa.

The use of AAV-RPE65 vectors for gene supplementation has achieved spectacular success as a treatment for individuals with autosomal recessive retinal disease caused by biallelic mutations in the visual cycle gene RPE65. However, the efficacy of this approach in treating autosomal dominant retinitis pigmentosa (adRP) associated with a monoallelic mutation encoding a rare D477G RPE65 variant has not been studied. Although lacking a severe phenotype, we now find that knock-in mice heterozygous for D477G RPE65 (D477G KI mice) can be used to evaluate outcomes of AAV-RPE65 gene supplementation. Total RPE65 protein levels, which are decreased in heterozygous D477G KI mice, were doubled following subretinal delivery of rAAV2/5.hRPE65p.hRPE65. In addition, rates of recovery of the chromophore 11-cis retinal after bleaching were significantly increased in eyes that received AAV-RPE65, consistent with increased RPE65 isomerase activity. While dark-adapted chromophore levels and a-wave amplitudes were not affected, b-wave recovery rates were modestly improved. The present findings establish that gene supplementation enhances 11-cis retinal synthesis in heterozygous D477G KI mice and complement previous studies showing that chromophore therapy results in improved vision in individuals with adRP associated with D477G RPE65.

Altering Stomatal Density for Manipulating Transpiration and Photosynthetic Traits in Rice through CRISPR/Cas9 Mutagenesis.

Stomata regulates conductance, transpiration and photosynthetic traits in plants. Increased stomatal density may contribute to enhanced water loss and thereby help improve the transpirational cooling process and mitigate the high temperature-induced yield losses. However, genetic manipulation of stomatal traits through conventional breeding still remains a challenge due to problems involved in phenotyping and the lack of suitable genetic materials. Recent advances in functional genomics in rice identified major effect genes determining stomatal traits, including its number and size. Widespread applications of CRISPR/Cas9 in creating targeted mutations paved the way for fine tuning the stomatal traits for enhancing climate resilience in crops. In the current study, attempts were made to create novel alleles of OsEPF1 (Epidermal Patterning Factor), a negative regulator of stomatal frequency/density in a popular rice variety, ASD 16, using the CRISPR/Cas9 approach. Evaluation of 17 T0 progenies identified varying mutations (seven multiallelic, seven biallelic and three monoallelic mutations). T0 mutant lines showed a 3.7-44.3% increase in the stomatal density, and all the mutations were successfully inherited into the T1 generation. Evaluation of T1 progenies through sequencing identified three homozygous mutants for one bp insertion. Overall, T1 plants showed 54-95% increased stomatal density. The homozygous T1 lines (# E1-1-4, # E1-1-9 and # E1-1-11) showed significant increase in the stomatal conductance (60-65%), photosynthetic rate (14-31%) and the transpiration rate (58-62%) compared to the nontransgenic ASD 16. Results demonstrated that the genetic alterations in OsEPF1 altered the stomatal density, stomatal conductance and photosynthetic efficiency in rice. Further experiments are needed to associate this technology with canopy cooling and high temperature tolerance.

Abstract 4458: Single cell landscape of multicentric Castleman disease in identical twins

Abstract Idiopathic Multicentric Castleman Disease (iMCD) is a rare IL-6-driven hematological disorder characterized by systemic lymphadenopathy, elevated immunoglobulin levels, and prominent plasmacytosis in the bone marrow and lymph nodes. An unusual occurrence of iMCD in identical twins provided a unique opportunity to answer genetic and molecular features of this disease, including the cell-of-origin of IL-6 signals, and the immune milieu within affected lymphoid organs and in circulation. Germline whole genome sequencing of the affected twins identified pathogenic homozygous mutations of NCOA4 c.G1322A and monoallelic mutations of TRAF3 c.G1504A - both genes recently implicated in IL-6 signaling and B-cell regulation. Their unaffected sister was heterozygous mutant for NCOA4 and homozygous wildtype for TRAF3 loci. Via single cell sequencing of 63,519 cells from bone marrow, lymph node, and peripheral blood mononuclear cells, we identified nodal endothelial cells and fibroblastic reticular cells (FRC) as the source of IL-6 signals. The latter are composed of mainly T-cell zone FRCs (CCL19+/CCL21+/IL7+/PDPN+/MADCAM1-) and a minor population of follicular dendritic cells (FDCs) (CD21+/CD35+/CXCL13+). An “inflammatory” peripheral monocytosis enriched for the expression of S100A family genes was evident in both twins, as well as a group of monocytes expressing cytotoxic gene signatures in the affected twin with milder clinical manifestations. Their unaffected sister mainly carried monocytes enriched for expression of major histocompatibility complex (MHC) class II genes. In conclusion, we provided evidence of a genetic cause of iMCD, identified the putative cell-of-origin of IL-6 signals in this rare disease, and described a distinct monocytic immune response phenotype. Citation Format: Jason Yongsheng Chan, Jui Wan Loh, Jing Quan Lim, Herty Liany, Elizabeth Chun Yong Lee, Jing Yi Lee, Bavani Kannan, Boon Yee Lim, Kerry Lim, Jeslin Chian Hung Ha, Cedric Chuan-Young Ng, Tun Kiat Ko, Dachuan Huang, Dominique Yuan Bin Seow, Chee Leong Cheng, Sock Hoai Chan, Joanne Ngeow, Bin Tean Teh, Soon Thye Lim, Choon Kiat Ong. Single cell landscape of multicentric Castleman disease in identical twins. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4458.

Autoimmunity and immunodeficiency associated with monoallelic LIG4 mutations via haploinsufficiency

Biallelic mutations in LIG4 encoding DNA-ligase 4 cause a rare immunodeficiency syndrome manifesting as infant-onset life-threatening and/or opportunistic infections, skeletal malformations, radiosensitivity and neoplasia. LIG4 is pivotal during DNA repair and during V(D)J recombination as it performs the final DNA-break sealing step. This study explored whether monoallelic LIG4 missense mutations may underlie immunodeficiency and autoimmunity with autosomal dominant inheritance. Extensive flow-cytometric immune-phenotyping was performed. Rare variants of immune system genes were analyzed by whole exome sequencing. DNA repair functionality and T-cell-intrinsic DNA damage tolerance was tested with an ensemble of invitro and in silico tools. Antigen-receptor diversity and autoimmune features were characterized by high-throughput sequencing and autoantibody arrays. Reconstitution of wild-type versus mutant LIG4 were performed in LIG4 knockout Jurkat T cells, and DNA damage tolerance was subsequently assessed. A novel heterozygous LIG4 loss-of-function mutation (p.R580Q), associated with a dominantly inherited familial immune-dysregulation consisting of autoimmune cytopenias, and in the index patient with lymphoproliferation, agammaglobulinemia, and adaptive immune cell infiltration into nonlymphoid organs. Immunophenotyping revealed reduced naive CD4+ T cells and low TCR-Vα7.2+ T cells, while T-/B-cell receptor repertoires showed only mild alterations. Cohort screening identified 2 other nonrelated patients with the monoallelic LIG4 mutation p.A842D recapitulating clinical and immune-phenotypic dysregulations observed in the index family and displaying T-cell-intrinsic DNA damage intolerance. Reconstitution experiments and molecular dynamics simulations categorize both missense mutations as loss-of-function and haploinsufficient. This study provides evidence that certain monoallelic LIG4 mutations may cause human immune dysregulation via haploinsufficiency.

Severe Microcytic Anemia Caused by Complex Hereditary Spherocytosis and X-Linked Sideroblastic Anemia with Mutations in SPTB and ALAS2 Genes

We report a case of severe anemia caused by complex hereditary spherocytosis (HS) and X-linked sideroblastic anemia (XLSA) with two mutations in the spectrin beta (SPTB) and 5-aminolevulinic acid synthase (ALAS2) genes. The proband was a 16-year-old male with severe jaundice and microcytic hypochromic anemia since his childhood. He had more severe anemia requiring erythrocyte transfusion, and had no response to vitamin B6 treatment. Next-generation sequencing (NGS) revealed double heterozygous mutations, one in exon 19 (c.3936G > A:p.W1312X) of the SPTB gene and another in exon 2 (c.37A > G:p.K13E) of the ALAS2 gene, and confirmed again by Sanger sequencing. The mutation of ALAS2 (c.37A > G) is inherited from his asymptomatic heterozygous mother, causing amino acid p.K13E, and the mutation has not yet been reported. The mutation of SPTB (c.3936G > A) is a nonsense mutation, leading to a premature termination codon in exon 19, and the mutation in the SPTB gene is not found in any of his relatives, which indicates a de novo monoallelic mutation. Conclusions: The double heterozygous mutations in the SPTB and ALAS2 genes lead to the joint occurrence of HS and XLSA in this patient, and are implicated in the more severe clinical phenotypes.

TMEM151A variants associated with paroxysmal kinesigenic dyskinesia.

TMEM151A, located at 11q13.2 and encoding transmembrane protein 151A, was recently reported as causative for autosomal dominant paroxysmal kinesigenic dyskinesia (PKD). Here, through comprehensive analysis of sporadic and familial cases, we expand the clinical and mutation spectrum of PKD. In doing so, we clarify the clinical and genetic features of Chinese PKD patients harboring TMEM151A variants and further explore the relationship between TMEM151A mutations and PKD. Whole exome sequencing was performed on 26 sporadic PKD patients and nine familial PKD pedigrees without PRRT2 variants. Quantitative real-time PCR was used to assess the gene expression of frameshift mutant TMEM151A in a PKD patient. TMEM151A variants reported to date were reviewed. Four TMEM151A variants were detected in four unrelated families with 12 individuals, including a frameshift mutation [c.606_607insA (p.Val203fs)], two missense mutations [c.166G > A (p.Gly56Arg) and c.791T > C (p.Val264Ala)], and a non-pathogenic variant [c.994G > A (p.Gly332Arg)]. The monoallelic frameshift mutation [c.606_607insA (p.Val203fs)] may cause TMEM151A mRNA decay, suggesting a potential pathogenic mechanism of haploinsufficiency. Patients with TMEM151A variants had short-duration attacks and presented with dystonia. Our study provides a detailed clinical description of PKD patients with TMEM151A mutations and reports a new disease-causing mutation, expanding the known phenotypes caused by TMEM151A mutations and providing further detail about the pathoetiology of PKD.

Wide morphological spectrums of renal neoplasms with TSC/mTOR, NF2, or monoallelic MUTYH germline mutation

Advances in molecular biology techniques have led to the recognition of more molecularly defined renal entities. However, some molecular-defined renal cell carcinomas (RCC) exhibit heterogeneous morphologies. In recent years, TSC/mTOR and NF2 alterations in renal cancers have attracted considerable attention from pathologists, including several renal tumors that have been validated as different entities. The monoallelic MUTYH germline mutation has also recently been demonstrated to be a driver of tumorigenesis, which is relatively enriched in renal cancers and may predict the risk of metastasis. In the current study, we presented seven cases with TSC/mTOR alterations (two with fibromyomatous stroma, one concomitant with ELOC mutation, three eosinophilic solid and cystic RCC, and two RCC, NOS), four cases with NF2 mutations (one concomitant with FH mutation, one biphasic hyalinizing psammomatous RCC, and two RCC, NOS), and two cases with monoallelic MUTYH germline mutations (one collecting duct carcinoma and one papillary RCC). We observed that renal cancers harboring these mutations had a wide morphological spectrum. Renal neoplasms with TSC/mTOR mutations may have an indolent outcome. NF2-mutated RCC appeared to display calcification and sclerotic stroma. The identification of these renal cancers can help reduce the number of tumors diagnosed as RCC, NOS. However, whether they can be grouped into TSC/mTOR, NF2, or MUTYH -associated RCC requires further validation.

Hereditary Cancer Syndrome in a Family with Double Mutation in BRIP1 and MUTYH Genes

Hereditary cancer syndromes predispose to several types of cancer due to inherited pathogenic variants in susceptibility genes. We describe the case of a 57-year-old woman, diagnosed with breast cancer, and her family. The proband belongs to a family with a suspected tumor syndrome, due to other cancer cases in her family from the paternal and maternal sides. After oncogenetic counseling, she was subjected to mutational analysis with an NGS panel analyzing 27 genes. The genetic analysis showed two monoallelic mutations in low penetrance genes, c.1187G>A (p.G396D) in MUTYH and c.55dup (p.Tyr19Leufs*2) in BRIP1. One of the mutations was inherited from the maternal side and the other from the paternal side, suggesting two different cancer syndrome types in the family. MUTYH mutation was related to the onset of cancers on the paternal side, as confirmed by the occurrence of the same mutation in the proband's cousin. BRIP1 mutation was found in the proband's mother, indicating that it was related to the cancer cases observed on the maternal side, including breast cancer and sarcoma. Advances in NGS technologies have allowed the identification of mutations in families with hereditary cancers in genes other than those related to a specific suspected syndrome. A complete oncogenetic counseling, together with molecular tests that enable a simultaneous analysis of multiple genes, is essential for the identification of a correct tumor syndrome and for clinical decision-making in a patient and his/her family. The detection of mutations in multiple susceptibility genes allows the initiation of early risk-reducing measures for identified mutation carriers among family members and to include them in a proper surveillance program for specific syndromes. Moreover, it may enable an adapted treatment for the affected patient, permitting personalized therapeutic options.

Acid ceramidase involved in pathogenic cascade leading to accumulation of α-synuclein in iPSC model of GBA1-associated Parkinson's disease.

Bi-allelic mutations in GBA1, the gene that encodes β-glucocerebrosidase (GCase), cause Gaucher disease (GD), whereas mono-allelic mutations do not cause overt pathology. Yet mono- or bi-allelic GBA1 mutations are the highest known risk factor for Parkinson's disease (PD). GCase deficiency results in the accumulation of glucosylceramide (GluCer) and its deacylated metabolite glucosylsphingosine (GluSph). Brains from patients with neuronopathic GD have high levels of GluSph, and elevation of this lipid in GBA1-associated PD has been reported. To uncover the mechanisms involved in GBA1-associated PD, we used human induced pluripotent stem cell-derived dopaminergic (DA) neurons from patients harboring heterozygote mutations in GBA1 (GBA1/PD-DA neurons). We found that compared with gene-edited isogenic controls, GBA1/PD-DA neurons exhibit mammalian target of rapamycin complex 1 (mTORC1) hyperactivity, a block in autophagy, an increase in the levels of phosphorylated α-synuclein (129) and α-synuclein aggregation. These alterations were prevented by incubation with mTOR inhibitors. Inhibition of acid ceramidase, the lysosomal enzyme that deacylates GluCer to GluSph, prevented mTOR hyperactivity, restored autophagic flux and lowered α-synuclein levels, suggesting that GluSph was responsible for these alterations. Incubation of gene-edited wild type (WT) controls with exogenous GluSph recapitulated the mTOR/α-synuclein abnormalities of GBA1/PD neurons, and these phenotypic alterations were prevented when GluSph treatment was in the presence of mTOR inhibitors. We conclude that GluSph causes an aberrant activation of mTORC1, suppressing normal lysosomal functions, including the clearance of pathogenic α-synuclein species. Our results implicate acid ceramidase in the pathogenesis of GBA1-associated PD, suggesting that this enzyme is a potential therapeutic target for treating synucleinopathies caused by GCase deficiency.

DOCK2 Mutation and Recurrent Hemophagocytic Lymphohistiocytosis

Hemophagocytic lymphohistiocytosis (HLH) is a syndrome resulting from uncontrolled hyper-inflammation, excessive immune system activation, and elevated levels of inflammatory cytokines. HLH can be caused by the inability to downregulate activated macrophages by natural killer (NK) and CD8 cytotoxic T cells through a process reliant on perforin and granzyme B to initiate apoptosis. Homozygous genetic mutations in this process result in primary HLH (pHLH), a disorder that can lead to multi-system organ failure and death in infancy. Heterozygous, dominant-negative, or monoallelic hypomorphic mutations in these same genes can cause a similar syndrome in the presence of an immune trigger, leading to secondary HLH (sHLH). A genetic mutation in a potential novel HLH-associated gene, dedicator of cytokinesis 2 (DOCK2), was identified in a patient with recurrent episodes of sHLH and hyperinflammation in the setting of frequent central line infections. He required baseline immune suppression for the prevention of sHLH, with increased anti-cytokine therapies and corticosteroids in response to flares and infections. Using a foamy-virus approach, the patient's DOCK2 mutation and wild-type (WT) control DOCK2 cDNA were separately transduced into a human NK-92 cell line. The NK-cell populations were stimulated with NK-sensitive K562 erythroleukemia target cells in vitro and degranulation and cytolysis were measured using CD107a expression and live/dead fixable cell dead reagent, respectively. Compared to WT, the patient's DOCK2 mutation was found to cause significantly decreased NK cell function, degranulation, and cytotoxicity. This study speaks to the importance of DOCK2 and similar genes in the pathogenesis of sHLH, with implications for its diagnosis and treatment.

Characterizing colorectal cancer (CRC) carriers of the recessive MUTYH founders (G396D/Y179C) and the low-penetrance APC founder APC-I1307K mutation.

210 Background: The clinical significance of CRC risk imparted by prevalent recessive mutations MUTYH-G396D and Y179C (Caucasian carrier rate 1/50) and the low-penetrance APC-I1307K mutation (Ashkenazi Jewish carrier rate ~1/15) is debated. Evidence supporting differential mutational spectrum, immune biology, and clinical outcomes in tumors that arise in carriers could lend support to their relevance. We characterized CRC carriers of MUTYH-G396D/Y179C and APC-I1307K, as well as those with concurrent second-hit mutations, in comparison to MUTYH or APC wild-type (WT) tumors. Methods: Retrospective review of patient samples (n = 13,896) submitted to a CLIA-certified laboratory (Caris Life Sciences, Phoenix, AZ) for next-generation sequencing (NGS) of DNA (592-gene or whole exome) and RNA (whole transcriptome). Deficient mismatch repair/high microsatellite instability (dMMR/MSI-H) was assessed by IHC/NGS. PD-L1 expression was tested by IHC (SP142; positive (+): ≥2+, ≥%5). Immune cell infiltration was estimated by RNA deconvolution using quanTIseq (Finotello, 2019). Presumed monoallelic (mMut) and biallelic mutations (bMut) included those with or without, respectively, a mutation or variant allele frequency ≥60%. Statistical significance determined by Fisher’s-Exact/Mann Whitney/X2 tests. *indicates raw p-value that was not significant after correction for multiple comparisons. Real world overall survival was obtained from insurance claims data, with Kaplan-Meier estimates used for comparison. Results: MUTYH-G396D/Y179C (119, 26 bMut) and APC-I1307K (11 mMut, ) alterations were identified, with all 3 founders being more common among male patients and associated with slightly increased median age at time of biopsy. MUTYH bMut had higher rates of genomic vs mMut 6.0%, p=0.02*; WT 11.0%, p=0.05*), and increased dendritic cell (p=0.04) and CD4 T-cell (p=0.007) proportions. APC bMut tumors were less frequently dMMR/MSI-H (2.6% vs mMut 18.%, p=0.11*; WT 15.5%, p=0.03*), TMB-H (2.6% vs mMut 27.3%, p=0.03*; WT 17.3%, p=0.02*), and PD-L1+ (0% vs mMut 18.2%, p=0.05*; WT 9.4%, p=0.04*), with enrichment of the canonical CMS2 subtype (44% vs 9% mMut, p<0.01; WT 11%, p<0.0001). In pMMR/MSS CRC, the prognosis of MUTYH founder carriers was superior to MUTYH WT tumors (HR 0.44, CI 0.25-0.78, p=0.004), with a non-significant trend toward greater sensitivity (HR 0.50, CI 0.21-1.2, p=0.). The prognosis of APC I1307K carriers was superior to APC WT tumors (HR 0.51, CI 0.34-0.76, p<0.001). Conclusions: Carrier status of MUTYH-G396D/Y179C and APC-I3017K founders may positively impact prognosis in patients with pMMR/MSS tumors. Differences may derive from distinct molecular signatures in tumors from MUTYH and APC founder mutations and warrant further investigation of these biomarkers in CRC.

The effect of SLC26A4 gene mutations on long-term rehabilitative outcomes in cochlear implant patients

Background SLC26A4 gene mutations related to hearing loss patients can obtain good hearing and speech rehabilitation effects after cochlear implantation (CI). Objective To explore the long-term rehabilitative outcomes of CI in patients with different SLC26A4 mutation groups. Material and Methods Clinical data of 71 patients with SLC26A4 gene mutations who received CI in the Second Hospital of Lanzhou University from 2012 to 2015 were retrospectively reviewed. According to the genetic test results, use One-way ANOVA analysis to compare the differences in auditory results, categories of auditory performance (CAP) and speech intelligibility rating (SIR) index questionnaire scores and speech recognition rates among different groups in 4-5 years after CI. Result Compared with other genotypes of SLC26A4, the patients with homozygous mutation of c.919-2A > G in SLC26A4 had better hearing aid threshold at 500 Hz and better recognition rates of Yangyang words than other monoallelic mutation groups after CI (p < .05). Conclusions and Significance The most common hot spot mutation of SLC26A4 gene is c.919-2A > G. The patients with homozygous mutation of c.919-2A > G in SLC26A4 gene had partly better hearing and speech rehabilitation than other monoallelic mutation groups after CI.

Biallelic TET2 mutations confer sensitivity to 5'-azacitidine in acute myeloid leukemia.

Precision medicine can significantly improve outcomes for patients with cancer, but implementation requires comprehensive characterization of tumor cells to identify therapeutically exploitable vulnerabilities. Here, we describe somatic biallelic TET2 mutations in an elderly patient with acute myeloid leukemia (AML) that was chemoresistant to anthracycline and cytarabine but acutely sensitive to 5'-azacitidine (5'-Aza) hypomethylating monotherapy, resulting in long-term morphological remission. Given the role of TET2 as a regulator of genomic methylation, we hypothesized that mutant TET2 allele dosage affects response to 5'-Aza. Using an isogenic cell model system and an orthotopic mouse xenograft, we demonstrate that biallelic TET2 mutations confer sensitivity to 5'-Aza compared with cells with monoallelic mutations. Our data argue in favor of using hypomethylating agents for chemoresistant disease or as first-line therapy in patients with biallelic TET2-mutated AML and demonstrate the importance of considering mutant allele dosage in the implementation of precision medicine for patients with cancer.

Identification of novel variants in retinitis pigmentosa genes by whole-exome sequencing

Retinitis pigmentosa is an inherited degenerative disorder causing severe retinal dystrophy and visual impairment, mainly with onset in the first or second decades. The next-generation sequencing has become an efficient tool to identify disease-causing mutations in retinitis pigmentosa. The aim of this retrospective study was to investigate novel gene variants and evaluate the utility of whole-exome sequencing in patients with retinitis pigmentosa. The medical records of 20 patients with retinitis pigmentosa at Eskişehir City Hospital between September 2019 and February 2022 were analyzed retrospectively. Peripheral venous blood was obtained, followed by the extraction of genomic DNAs. The medical and ophthalmic histories were collected, and ophthalmological examinations were performed. Whole-exome sequencing was performed to determine the genetic etiology of the patients. The proportion of genetically solved cases was 75% (15/20) in the patients with retinitis pigmentosa. Molecular genetic testing identified 13 biallelic and 4 monoallelic mutations in known retinitis pigmentosa genes, including 11 novel variants. According to in silico prediction tools, nine variants were predicted as pathogenic or possibly pathogenic. We identified six previously reported mutations to be associated with retinitis pigmentosa. The age of onset of the patients ranged from 3 to 19, with a mean age of onset of 11.6. All patients had a loss of central vision. As the first study of the application of whole-exome sequencing among patients with retinitis pigmentosa in a Turkish cohort, our results may contribute to the characterization of the spectrum of variants related to retinitis pigmentosa in the Turkish population. Future population-based studies will enable us to reveal the detailed genetic epidemiology of retinitis pigmentosa.

Efficient editing BMP15 in porcine oocytes through microinjection of CRISPR ctRNP

Bone morphogenetic protein 15 (BMP15) is an X-linked gene encoding an oocyte secreted factor, which plays varied functions in the female fertility between mono-ovulatory and poly-ovulatory mammalian species. We previously found that knockout of BMP15 completely blocked porcine follicular development at preantral stages. However, the specific function of BMP15 on porcine oocytes in vitro maturation remains largely unknown. Here, we injected the pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complex into the cytoplasm of germinal vesicle stage porcine oocytes to disrupt BMP15. The ctRNP composed of Cas9 nuclease and crRNA-tracrRNA complex at 1.2/1 content ratio. The tested crRNA-tracrRNA complex concentration ranging from 50 to 200 ng/μL, all presented effective editing of BMP15 in porcine oocytes, and the 125 ng/μL crRNA-tracrRNA complex presented the highest editing efficiency (39.23 ± 3.33%). Surprisingly, we found approximately 95% edited oocytes presented monoallelic mutations, and only 5% edited oocytes harbored biallelic mutations. Interestingly, the coinjected two crRNAs guided the ctRNP complex to concurrently cut within a 10 bp window of the PAM (protospacer adjacent motif), resulting in a precise deletion within BMP15 in 85.9% edited oocytes, and additional deletion happened in 14.1% edited oocytes, which resulted in large fragment deletions in BMP15. Most deletions caused frameshift and introduced premature stop codon in BMP15, resulting in the disruption of BMP15 protein expression, which was confirmed by the Western blot analysis showing the reduced BMP15 protein expression in ctRNP injected oocytes. The disruption of BMP15 attenuated the activation of SMAD1/5/8 signaling, and impaired cumulus expansion of porcine cumulus cell-oocyte complexes (COCs). Our study proved that delivering CRISPR ctRNP into porcine oocytes by microinjection was able to edit BMP15 efficiently, providing a new strategy to investigate the functions of oocyte-specific secreted factors in oocyte in vitro maturation.

A congenital hydrocephalus-causing mutation in Trim71 induces stem cell defects via inhibiting Lsd1mRNA translation.

Congenital hydrocephalus (CH) is a major cause of childhood morbidity. Mono-allelic mutations in Trim71, a conserved stem-cell-specific RNA-binding protein, cause CH; however, the molecular basis for pathogenesis mediated by these mutations remains unknown. Here, using mouse embryonic stem cells as a model, we reveal that the mouse R783H mutation (R796H in human) alters Trim71's mRNA substrate specificity and leads to accelerated stem-cell differentiation and neural lineage commitment. Mutant Trim71, but not wild-type Trim71, binds Lsd1 (Kdm1a) mRNA and represses its translation. Specific inhibition of this repression or a slight increase of Lsd1 in the mutant cells alleviates the defects in stem cell differentiation and neural lineage commitment. These results determine a functionally relevant target of the CH-causing Trim71 mutant that can potentially be a therapeutic target and provide molecular mechanistic insights into the pathogenesis of this disease.

Mitochondrial DNA heteroplasmy distinguishes disease manifestation in PINK1/PRKN-linked Parkinson's disease.

Biallelic mutations in PINK1/PRKN cause recessive Parkinson's disease. Given the established role of PINK1/Parkin in regulating mitochondrial dynamics, we explored mitochondrial DNA integrity and inflammation as disease modifiers in carriers of mutations in these genes. Mitochondrial DNA integrity was investigated in a large collection of biallelic (n = 84) and monoallelic (n = 170) carriers of PINK1/PRKN mutations, idiopathic Parkinson's disease patients (n = 67) and controls (n = 90). In addition, we studied global gene expression and serum cytokine levels in a subset. Affected and unaffected PINK1/PRKN monoallelic mutation carriers can be distinguished by heteroplasmic mitochondrial DNA variant load (area under the curve = 0.83, CI 0.74-0.93). Biallelic PINK1/PRKN mutation carriers harbour more heteroplasmic mitochondrial DNA variants in blood (P = 0.0006, Z = 3.63) compared to monoallelic mutation carriers. This enrichment was confirmed in induced pluripotent stem cell-derived (controls, n = 3; biallelic PRKN mutation carriers, n = 4) and post-mortem (control, n = 1; biallelic PRKN mutation carrier, n = 1) midbrain neurons. Last, the heteroplasmic mitochondrial DNA variant load correlated with IL6 levels in PINK1/PRKN mutation carriers (r = 0.57, P = 0.0074). PINK1/PRKN mutations predispose individuals to mitochondrial DNA variant accumulation in a dose- and disease-dependent manner.