Non-truncating Mutations Research Articles

Characterization of 19 novel gene mutation sites associated with autosome-dominant polycystic kidney disease

By analyzing the of genetic testing data of patients with renal polycystic kidney disease and their relatives, this study aims to identify unreported novel gene mutation sites associated with autosomal dominant polycystic kidney disease (ADPKD). Structural prediction software was employed to investigate protein structural changes before and after mutations, explore genotype-phenotype correlations, and enrich the ADPKD gene database. In this single-center retrospective study, patients with multiple renal cysts diagnosed from January 2019 to February 2023 at the Zhong Da Hospital Southeast University were included. Genetic and clinical data of patients and their families were collected. Unreported novel gene mutation sites associated with ADPKD were identified. The AlphaFold v2.3.1 software was used to predict protein structures. Changes in protein structure before and after mutations were compared to explore genotype-phenotype correlations and enrich the ADPKD gene database. Twelve mutated genes associated with renal cysts were detected in 52 families. Nineteen novel gene mutation sites associated with ADPKD were identified, including 17 mutations in the PKD1 gene (one splicing mutation, seven frameshift mutations, four nonsense mutations, one whole-codon insertion, and four missense mutations); one ALG9 missense mutation; and one chromosomal structural variation. Truncating mutations in the PKD1 gene were correlated with a more severe clinical phenotype, while non-truncating mutations were associated with greater clinical heterogeneity. Numerous novel gene mutation sites associated with ADPKD remain unreported. Therefore, it is essential to analyze the pathogenicity of these novel mutation sites, establish genotype-phenotype correlations, and enrich the ADPKD gene database.

E-Cadherin Mutational Landscape and Outcomes in Breast Invasive Lobular Carcinoma

Invasive lobular carcinomas (ILC) are characterized by the loss of E-cadherin expression and CDH1 gene inactivation. Diagnostic reproducibility for this tumor type is currently suboptimal and could be improved by a better understanding of its histomolecular and clinical heterogeneity. We have analyzed the relationship between the presence, type, or position of CDH1 mutations, E-cadherin expression, and clinicopathological features (including outcome) in a retrospective series of 251 primary ILC with a long follow-up (median: 9.5 years). The mutational status of E-cadherin gene (CDH1) was determined by RNA sequencing from frozen tumor samples. E-cadherin immunohistochemistry (IHC) was performed with antibodies directed against the intracellular domain (clone 4A2C7) and the extracellular domain (clone NCH38). IHC expression of p120 and β-catenin was also assessed in E-cadherin diffusely positive cases. Three major patterns of E-cadherin membrane expression were identified by IHC, with good agreement between the 2 clones (overall concordance: 83.8%, Kappa 0.67): null/focal expression (≤10%) (72.8% of cases for 4A2C7 and 83.8% for NCH38), heterogeneous expression (11%-89%) (19.2% of cases for 4A2C7 and 6.9% for NCH38), and diffuse expression (≥90%) (8% of cases for 4A2C7 and 9.3% for NCH38). E-cadherin membranous expression, when present, was abnormal (incomplete labeling and/or reduced intensity). ILC with diffuse E-cadherin expression showed abnormal β-catenin or p120-catenin staining in 21% of cases. Interestingly, these cases with diffusely expressed E-cadherin had a CDH1 mutation rate as high as the E-cadherin null/focal cases (∼70%) but were enriched in nontruncating mutations. Regarding CDH1 mutation location, intracytoplasmic domain mutations correlated with a divergent E-cadherin IHC phenotype between the 2 antibodies (4A2C7 ≤ 10%/NCH38 ≥ 10%). Clinico-pathological correlation analyses found that stromal amount (inversely correlated with tumor cellularity) and tumor-infiltrating lymphocytes were less abundant in ILC with E-cadherin null/focal cases. In addition, CDH1 truncating mutations were associated with radiohistologic size discordance and were identified in multivariate survival analysis as an independent poor prognosis factor in terms of metastasis risk and breast cancer-related mortality. Overall, our study highlights the importance of the precise mutational status of CDH1 in the clinical, radiological, histologic, and phenotypic expression of lobular carcinomas. These findings should be taken into account in future attempts to improve diagnostic criteria or methods for ILC, as well as for clinicobiological studies dedicated to this tumor type.

#1049 Genetic characteristics of autosomal dominant polycystic kidney disease in a Greek population

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is the predominant monogenic renal genetic disorder, and the fourth leading cause of end stage kidney disease. Confirmatory genetic testing defines the disease, provides prognostic information and may support therapeutic management. This study aims to investigate the genetic characteristics of a substantial cohort of ADPKD patients from a single Greek center. Method Ninety-three patients (46 females, 47 males with a mean age of 34 ± 15 years) were enrolled from 59 distinct families attending the outpatient ADPKD clinic at the Nephrology Department of the General Hospital of Athens “G. Gennimatas.” Genetic analysis included targeted next-generation sequencing (tNGS) of over 600 clinically relevant genes, Sanger sequencing, and Multiplex ligation-dependent probe amplification (MLPA). Whole exome and whole genome sequencing (WES and WGS) studies were conducted in 3 patients with negative results. Magnetic Resonance Imaging (MRI) was performed in 74 out of 93 patients, revealing more than 10 renal cysts in all but one patient. Children with a positive family history (FH) displayed at least one cyst in echography, while two without FH had multiple renal cysts. Results Genetic diagnosis was established in 88 of 93 patients (94.5%). Seventy patients (75%), demonstrated single nucleotide variants (SNVs) and large deletions (4 patients) in the PKD1 gene, whereas SNVs and large deletions (5 patients) in the PKD2 gene were identified in 15 (16%) patients. Additionally, mutations in genes other than PKD1 and PKD2 were identified in 3 patients (3.5%). Two cases, involving a father and son, revealed the presence of a SEC61B variant; the father exhibited more than 10 renal cysts and liver cysts in the MRI, while the son displayed only liver cysts. Additionally, a third patient, a woman with nephrolithiasis and over 10 renal cysts in the MRI, exhibited a SLC3A1 variant. Notably, 5 patients (5.5%) with ADPKD phenotype had negative genetic analyses, despite the application of both WES and WGS in three of these patients. Truncating mutations in PKD1 were observed in 43 of 70 cases, (61%), while the rest (39%) had non-truncating mutations. Detected variants were classified as Pathogenic (45.5%), Likely Pathogenic (36%), and Variants of Uncertain Significance (VUS-13.5%, 12 cases), according to the guidelines of the American College of Medical Genetics and Genomics. Reclassification to Likely Pathogenic could be proposed for 6 out of 12 cases with VUS variants for which family segregation was noted. Finally, 52% and 82% of the detected variants have not been previously reported in ClinVar and gnomAD databases, respectively. Conclusion In this ethnically homogenous population, truncating mutations in the PKD1 gene were the predominant genetic cause of ADPKD, followed by PKD1 non-truncating mutations, PKD2 gene variants and other rare or undefined genetic etiologies.

#662 Characterization of novel gene mutation sites associated with autosomal dominant polycystic kidney disease on 19 chromosomes

Abstract Background and Aims Autosomal dominant polycystic kidney disease (ADPKD) is the most common single-gene inherited kidney disorder, affecting approximately 1 in 500-1000 newborns and often leading to end-stage renal disease. It involves cyst formation in various organs, with renal manifestations including cysts, hematuria, kidney stones, proteinuria, and hypertension. Liver, pancreas, seminal vesicles, spleen, and intracranial aneurysms may also be affected. Genetic diagnosis, particularly for patients without a family history or with atypical imaging, is considered the gold standard. ADPKD is associated with several pathogenic genes, such as PKD1 and PKD2, as well as others like HNF-1B, GANAB, DNAJB11, SEC63, ALG8, ALG9, PRKCSH, LRP5, SEC61B, and SEC63. Understanding the genetic basis is crucial for early diagnosis and identifying high-risk individuals. This study, based on genetic data from patients with renal polycystic kidney disease at Zhong Da Hospital Southeast University, identified novel ADPKD gene mutations. It explored structural changes in proteins, predicting effects on phenotype, to contribute to the enrichment of the ADPKD gene database. Method In a single-center cross-sectional retrospective study, patients with renal multiple cysts diagnosed from January 2019 to February 2023 at the Zhong Da Hospital Southeast University were included. Genetic and clinical data of patients and their families were collected. Unreported novel gene mutation sites associated with ADPKD were identified, and AlphaFold v2.3.1 was employed to predict protein structures. Changes in protein structure before and after mutations were compared to explore genotype-phenotype correlations and enrich the ADPKD gene database. Results The study included 96 patients with renal multiple cysts and their relatives from 62 families. Twelve mutated genes associated with renal cysts were detected in 52 families. Nineteen novel gene mutation sites associated with ADPKD were identified (Table 1, Fig. 1) , including 17 mutations in the PKD1 gene (1 splicing mutation, 7 frameshift mutations, 4 nonsense mutations, 1 whole-codon insertion, and 4 missense mutations), 1 ALG9 missense mutation, and 1 chromosomal structural variation. Truncating mutations in the PKD1 gene were correlated with a more severe clinical phenotype, while non-truncating mutations were associated with greater clinical heterogeneity. Conclusion Numerous novel gene mutation sites associated with ADPKD remain unreported. It is essential to analyze the pathogenicity of these novel mutation sites, establish genotype-phenotype correlations, and enrich the ADPKD gene database.

#1040 Correlation of genotype to disease onset age and hypertension diagnosis in autosomal dominant polycystic kidney disease

Abstract Background and Aims Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the predominant genetic disease leading to End Stage Kidney Disease (ESKD). Genetic identity could correlate to the disease profile. Correlations between genetic and phenotype characteristics in a cohort of ADPKD patients are investigated. Method Genetic analysis was conducted in 85 patients with an ADPKD phenotype, employing targeted Next Generation (tNGS) or Sanger Sequencing and Multiplex ligation-dependent probe amplification (MLPA). Medical history, clinical and laboratory data, including Renal Magnetic Resonance Imaging (MRI) for Total Kidney Volume (TKV) measurement (in 66 patients), were recorded and subsequently correlated to the respective genotypes. Results Eighty-five patients (43 females, 42 males, 33 ± 16 years old) were included in the study. The mean age at diagnosis of the disease was at 21.5 ± 13 (0.6-69) years. Fifty-eight out of 85 patients (68%) had a familial history of ADPKD. The mean age at which the affected parent reached ESKD was 53.0 ± 9 years. Hypertension was diagnosed in 60 individuals (71%) within the study cohort. The mean age at hypertension diagnosis was 31 ± 11 years. In the assessment based on height-adjusted TKV and age, the Mayo Clinic Imaging Categories revealed that 9% of patients were classified as 1A, 20% as 1B, 29% as 1C, 29% as 1D, and 13% as 1E. Mutations in PKD1 gene were detected in 70 patients (82%), with 61% exhibiting truncating mutations and 39% non-truncating mutations, while PKD2 mutations were identified in 15 patients (18%). The average age of ADPKD diagnosis differed among genetic subgroups. Patients carrying PKD1-truncating mutations were diagnosed at 17.6 ± 11 years, those with PKD1-non-truncating mutations at 24.5 ± 12 years, and those with PKD2 mutations at 27.3 ± 16 years (p = 0.01). Moreover, the mean age at which the affected parent reached ESKD was 50 ± 8 years, 55 ± 10 and 60 ± 7 years in PKD1-truncating, PKD1-non-truncating and PKD2 patients respectively (p = 0.04). Additionally, the mean age of hypertension diagnosis was 29 ± 12 years in PKD1-truncating, 30 ± 10 years in PKD1-non-truncating and 40 ± 7 years in PKD2 patients (p = 0.05). Conclusion Mutation type correlates with the age of ADPKD diagnosis, the renal survival within families and the hypertension onset. Patients with PKD1-truncating mutations manifest an earlier onset of both the disease and hypertension compared to those with PKD2 gene mutations.

PKD1 Truncating Mutations Accelerate eGFR Decline in Autosomal Dominant Polycystic Kidney Disease Patients

Introduction: Autosomal dominant polycystic kidney disease (ADPKD) is a monogenic disease characterized by the accumulation of fluid-filled cysts in the kidneys, leading to renal volume enlargement and progressive kidney function impairment. Disease severity, though, may vary due to allelic and genetic heterogeneity. This study aimed to determine genotype-phenotype correlations between PKD1 truncating and non-truncating mutations and kidney function decline in ADPKD patients. Methods: We established a single-center retrospective cohort study in Kuwait where we followed every patient with a confirmed PKD1-ADPKD diagnosis clinically and genetically. Renal function tests were performed annually. We fitted generalized additive mixed effects models with random intercepts for each individual to analyze repeated measures of kidney function across mutation type. We then calculated survival time to kidney failure in a cox proportional hazards model. Models were adjusted for sex, age at visit, and birth year. Results: The study included 22 truncating and 20 non-truncating (42 total) patients followed for an average of 6.6 years (range: 1–12 years). Those with PKD1 truncating mutations had a more rapid rate of eGFR decline (−4.7 mL/min/1.73 m2 per year; 95% CI: −5.0, −4.4) compared to patients with PKD1 non-truncating mutations (−3.5 mL/min/1.73 m2 per year; 95% CI: −4.0, −3.1) (p for interaction <0.001). Kaplan-Meier survival analysis of time to kidney failure showed that patients with PKD1 truncating mutations had a shorter renal survival time (median 51 years) compared to those with non-truncating mutations (median 56 years) (P for log-rank = 0.008). Conclusion: In longitudinal and survival analyses, patients with PKD1 truncating mutations showed a faster decline in kidney function compared to patients PKD1 non-truncating mutations. Early identification of patients with PKD1 truncating mutations can, at best, inform early clinical interventions or, at least, help suggest aggressive monitoring.

Hearing and Hearing Loss Progression in Patients with GJB2 Gene Mutations: A Long-Term Follow-Up.

We aimed to investigate whether the degree of hearing loss with GJB2 mutations could be predicted by distinguishing between truncating and non-truncating mutations and whether the genotype could predict the hearing loss level. Additionally, we examined the progression of hearing loss in individuals monitored for over 2 years for an average of 6.9 years. The proportion of truncating mutations was higher in patients with profound and severe hearing loss, but it was not accurate enough to predict the degree of hearing loss. Via genotype analysis, mutations of the p.Arg143Trp variants were associated with profound hearing loss, while mutations of the p.Leu79Cysfs*3 allele exhibited a wide range of hearing loss, suggesting that specific genotypes can predict the hearing loss level. Notably, there were only three cases of progression in four ears, all of which involved the p.Leu79Cysfs*3 mutation. Over the long-term follow-up, 4000 Hz was significant, and there was a trend of progression at 250 Hz, suggesting that close monitoring at these frequencies during follow-up may be crucial to confirm progression. The progression of hearing loss was observed in moderate or severe hearing loss cases at the time of the initial diagnosis, emphasizing that children with this level of hearing loss need regular follow-ups.

A Carboxy-terminal Smarcb1 Point Mutation Induces Hydrocephalus Formation and Affects AP-1 and Neuronal Signalling Pathways in Mice

The BAF (BRG1/BRM-associated factor) chromatin remodelling complex is essential for the regulation of DNA accessibility and gene expression during neuronal differentiation. Mutations of its core subunit SMARCB1 result in a broad spectrum of pathologies, including aggressive rhabdoid tumours or neurodevelopmental disorders. Other mouse models have addressed the influence of a homo- or heterozygous loss of Smarcb1, yet the impact of specific non-truncating mutations remains poorly understood. Here, we have established a new mouse model for the carboxy-terminal Smarcb1 c.1148del point mutation, which leads to the synthesis of elongated SMARCB1 proteins. We have investigated its impact on brain development in mice using magnetic resonance imaging, histology, and single-cell RNA sequencing. During adolescence, Smarcb11148del/1148del mice demonstrated rather slow weight gain and frequently developed hydrocephalus including enlarged lateral ventricles. In embryonic and neonatal stages, mutant brains did not differ anatomically and histologically from wild-type controls. Single-cell RNA sequencing of brains from newborn mutant mice revealed that a complete brain including all cell types of a physiologic mouse brain is formed despite the SMARCB1 mutation. However, neuronal signalling appeared disturbed in newborn mice, since genes of the AP-1 transcription factor family and neurite outgrowth-related transcripts were downregulated. These findings support the important role of SMARCB1 in neurodevelopment and extend the knowledge of different Smarcb1 mutations and their associated phenotypes.

Immunological Role of TP53 Somatic Mutation Classification in Human Cancers.

TP53 is a very common tumor suppressor gene and has implicated in various cancers. A systematic immunological analysis of TP53 somatic mutation classification in multiple cancers is still lacking. To assess the immunological value of TP53 somatic mutation classification in various cancers, we integrated a series of bioinformatics methods to analyze the role of TP53 gene across the public databases, such as UCSC Xena, Cancer Cell Line Encyclopedia (CCLE), Ensembl, and Genotype-Tissue Expression (GTEx). The results revealed that the TP53 expression level had significant difference in tumor tissues and normal tissues, and it had a high expression level in most malignant tumors. Moreover, the missense mutation is the most common type of TP53 mutation in most cancers. In addition, the Cox proportional hazards model analysis and Kaplan-Meier (KM) survival analysis demonstrated that the TP53 expression is a high-risk factor in brain lower-grade glioma (LGG), prostate adenocarcinoma (PRAD), and uterine carcinosarcoma (UCS), which is opposite in uterine corpus endometrial carcinoma (UCEC). Besides, compared to the TP53 nontruncating mutation classification samples, we found that TP53 truncating mutation samples had lower TP53 expression levels in certain types of cancer. Notably, TP53 was associated with the mismatch repair (MMR) gene in some cancers which contained truncating or nontruncating mutation. Based on the classification of truncating or nontruncating mutation, we also discovered that TP53 expression was positively or negatively correlated with the immune score, stromal score, and the levels of immune cell infiltration in different cancers. Our research reveals an overarching landscape of immunological value on TP53 status in various malignant tumors. According to our results, we demonstrate that TP53 also plays an immunological role in various cancers.

Plasma Level of Pyrophosphate Is Low in Pseudoxanthoma Elasticum Owing to Mutations in the ABCC6 Gene, but It Does Not Correlate with ABCC6 Genotype.

Pseudoxanthoma elasticum (PXE), a monogenic disorder resulting in calcification affecting the skin, eyes and peripheral arteries, is caused by mutations in the ABCC6 gene, and is associated with low plasma inorganic pyrophosphate (PPi). It is unknown how ABCC6 genotype affects plasma PPi. We studied the association of ABCC6 genotype (192 patients with biallelic pathogenic ABCC6 mutations) and PPi levels, and its association with the severity of arterial and ophthalmological phenotypes. ABCC6 variants were classified as truncating or non-truncating, and three groups of the 192 patients were formed: those with truncating mutations on both chromosomes (n = 121), those with two non-truncating mutations (n = 10), and a group who had one truncating and one non-truncating ABCC6 mutation (n = 61). The hypothesis formulated before this study was that there was a negative association between PPi level and disease severity. Our findings confirm low PPi in PXE compared with healthy controls (0.53 ± 0.15 vs. 1.13 ± 0.29 µM, p < 0.01). The PPi of patients correlated with increasing age (β: 0.05 µM, 95% CI: 0.03-0.06 per 10 years) and was higher in females (0.55 ± 0.17 vs. 0.51 ± 0.13 µM in males, p = 0.03). However, no association between PPi and PXE phenotypes was found. When adjusted for age and sex, no association between PPi and ABCC6 genotype was found. Our data suggest that the relationship between ABCC6 mutations and reduced plasma PPi may not be as direct as previously thought. PPi levels varied widely, even in patients with the same ABCC6 mutations, further suggesting a lack of direct correlation between them, even though the ABCC6 protein-mediated pathway is responsible for ~60% of this metabolite in the circulation. We discuss potential factors that may perturb the expected associations between ABCC6 genotype and PPi and between PPi and disease severity. Our findings support the argument that predictions of pathogenicity made on the basis of mutations (or on the structure of the mutated protein) could be misleading.

PS-C26-10: THE PROPKD SCORE IS ASSOCIATED WITH THE PROGRESSION OF RENAL INVOLVEMENT IN PATIENTS WITH POLYCYSTIC KIDNEY DISEASE TREATED WITH TOLVAPTAN

Background: The PROPKD score has been proposed to evaluate the risk of the progression to end stage renal failure in patients with autosomal dominant polycystic kidney disease (ADPKD), based on gender (male: 1point), hypertension under 35 years old (2 points), urological event under 35 years old (2 points), and gene mutation (PKD1 truncating mutation: 4 points, PKD1 non-truncating mutation: 2 points, PKD2: 0 point). Although tolvaptan has benefits for renal involvement, whether PROPKD score could predict future renal involvement in patients with ADPKD treated with tolvaptan is unknown. Thus, we explored the effects of tolvaptan on the annual changes in total kidney volume (%TKV) and estimated glomerular filtration rate (%eGFR) according to the PROPKD score in these patients. Methods: 32 ADPKD patients treated tolvaptan for at least a year were retrospectively analyzed (the mean observation period: 3.6 years). We examined the decline in renal function, %TKV by computed tomography, and gene mutation. The association between %eGFR and %TKV change before and after tolvaptan treatment, annual TKV change (%TKV) and PROPKD score was analyzed. Results: We identified gene mutations in ADPKD patients: PKD1 truncating mutation (n = 15), PKD1 non-truncating mutation (n = 5), PKD2 mutation (n = 6), and mutation not found (n = 6). Mean %TKV and eGFR were 1843 ± 986 ml and 54.7 ± 21.0 ml/min/1.73 m2, respectively. The mean PROPKD score was 3.3 ± 2.4 points and showed a significant negative correlation with %eGFR before and after tolvaptan treatment (p = 0.044/0.016), and no significant correlation with %TKV before and after tolvaptan treatment. Conclusion: The higher PROPKD score, the faster the decrease in %eGFR during tolvaptan therapy, suggesting that the PROPKD score may be useful as a predictor of the risk of progression to renal failure after tolvaptan introduction.

Predicting the Risk of Progression in Indian ADPKD Cohort using PROPKD Score - A Single-Center Retrospective Study.

With the variable genotype-phenotype expression of autosomal dominant polycystic kidney disease (ADPKD) and availability of novel targeted therapies, it is important to find predictors for rapid progression. The PROPKD score, consisting of genetic and clinical parameters like sex, hypertension, and urological events, is a useful tool in predicting the risk of progression. This study was aimed to determine the risk of ADPKD progression in Indian patients using the PROPKD score. A retrospective study was done from 2006 to 2021. ADPKD patients with ESRD were included in the study. Scoring was done as per the PROPKD score as follows: male sex: 1, onset of hypertension before 35 years: 2, first urological event before 35 years: 2, PKD1 truncating mutation: 4, PKD1 non-truncating mutation: 2, and PKD2 mutation: 0. Two types of risk classifications were done as follows: (a) considering the clinical variables in all 73 patients (male sex, onset of hypertension before 35 years, and first urological event before 35 years), they were classified into three risk groups: low-risk group (0-1), intermediate-risk group (2-3), and high-risk group (4-5) and (b) considering the clinical variables and type of mutation in 39 patients, they were classified into three risk groups: low-risk group (0-3), intermediate-risk group (4-6), and high-risk group (7-9). Total number of patients included was 73, with the median age at ESRD being 54 years. High-risk group of clinical variables with hazard ratio (HR) of 4.570 (2.302-9.075, P < 0.001) and high-risk group of the PROPKD score with HR of 6.594 (1.868-23.284, P = 0.003) were associated with early ESRD. High-risk groups of both classifications were associated with early ESRD. High-risk groups based on the PROPKD scoring and clinical variables were associated with early progression to ESRD.

Role of TNFRSF17 and GPRC5D Structural and Point Mutations in Resistance to Targeted Immunotherapies in Multiple Myeloma (MM)

Adaptive T cell therapy using chimeric antigen receptor (CAR) T cells and T cell engagers (TCE) targeting BCMA and GPRC5D have demonstrated very encouraging responses in heavily pre-treated multiple myeloma (MM). Recent studies interrogating T cells repertoires and fitness correlated resistance to these immune based therapies with exhausted T cell states. While in lymphoma, antigen escape with CD19 loss is observed in up to 30% of patients with acquired resistance to adaptive therapies, in MM TNFRSF17 or GPRC5D structural mutations are thought to be less prevalent in recipients of BCMA or GPRC5D-targeting CARs or TCEs . Bi-allelic loss (n=2) and a missense truncating mutation (n=1) of TNFRSF17 with CAR T cells, as well as one case of biallelic loss in TNFRSF17 with TCE are reported to date with no reported cases of bi-allelic loss of GPRC5D. Further studies of the role TNFRSF17 or GPRC5D structural or point mutations to the immune escape to these adaptive therapies are needed. Serial BM aspirates were collected from patients treated with BCMA or GPRC5D-targeting TCE or CARs prior to therapy and at relapse. MM cells were profiled with single cell mRNA (scRNAseq), single cell copy number (scCNVseq) using the 10x Genomics GemCode system and whole genome sequencing (WGS). Sequenced reads were aligned to hg38. Single cell reads were processed with CellRanger and downstream analyses with Seurat R package. In order to define the prevalence of TNFRSF17 or GRPC5D structural variants and mutations in MM, we performed WGS (n=19, 14 pre-CAR/TCE, 5 post-CAR/TCE) as well as scCNVseq (n=33, 16 RRMM, 7 pre-CAR/TCE, 8 post-CAR/TCE) on sorted CD138+ cells from relapsed MM patients. WGS (100x coverage) identified at baseline a subclonal deletion at the TNFRSF17 gene locus in only 1 patient; this small subclone subsequently drove the progression post BCMA CAR T cell therapy with an acquired biallelic loss of TNFRSF17. Mono-allelic large and focal deletions of GPRC5D (chr.12p) were detected by WGS in 7 cases (5 deletions, 2 copy neutral LOH). scCNVseq studies identified more frequent subclonal monoallelic copy number losses of TNFRSF17 in 11.2% of the cells (27884 MM cells) prior to anti-BCMA therapy exposure with the deletions ranging from 200 kb to 2.3 Mb. A homozygous deletion of TNFRSF17 was noted in 2 patients at the time of disease progression, one post CAR T and the other post BCMAxCD3 TCE. Structural mutations at the GPRC5D locus were also identified by scCNVseq with monoallelic and biallelic deletions in 11.9% and 2.7% of the cells, respectively. Clonal biallelic loss of GPRC5D post GPRC5DxCD3 TCE was detected in one patient who harbored a monoallelic loss in 79.2% of the cells pre-TCE. With regard to point mutations, in one patient progressing from a durable CR on BCMA TCE, we observed an acquired mono-allelic loss of TNFRSF17 (scCNV) coupled with de novo mutation in TNFRSF17 extracellular domain c.81G>C [p.(R27P)]. This point R27P mutation is predicted to be damaging to BCMA (Polyphen) with disruption of several H-bonds between R27 and C24, Q25, S29, S30 and T32 of BCMA extracellular aa residues. Importantly, molecular dynamic simulations of WT vs. mutant R27P using publicly deposited crystal structural of BCMA bound to anti-BCMA Ab (J22.9-xi) (PBD ID 4ZFO) demonstrated that this mutation disrupts all H-bonds between BCMA and the light chain of chimeric mouse/human anti-BCMA Ab (J22.9-xi). Functionally, primary CD138+ cells with mutant BCMA [p.(R27P)] as well as K562 cells transduced with mutant BCMA [p.(R27P)] were completely resistant to BCMAxCD3 cytotoxicity with teclistamab and elranatamab compared to cells with WT BCMA. While structural alterations of TNFRSF17 and GPRC5D remain relatively rare, we have identified biallelic loss of TNFRSF17 post BCMA CAR T and BCMAxCD3 TCE therapy and herein reported the first case of biallelic loss of GPRC5D post GPRC5DxCD3 TCE treatment. Importantly, we also identified a monoallelic loss of TNFRSF17 (del16p) coupled with BCMA extracellular domain point mutation [p.(R27P)] that confers resistance to two BCMAxCD3 TCEs in MM. Therefore, in addition to structural alterations (biallelic loss of TNFRSF17 or GPRC5D), antigen escape resulting from non-truncating missense point mutations in BCMA extracellular domain also mediate resistance to targeted-immunotherapies.

XBP1 Activation Reduces Severity of Polycystic Kidney Disease due to a Nontruncating Polycystin-1 Mutation in Mice.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in Pkd1 and Pkd2. They encode the polytopic integral membrane proteins polycystin-1 (PC1) and polycystin-2 (PC2), respectively, which are expressed on primary cilia. Formation of kidney cysts in ADPKD starts when a somatic second hit mechanism inactivates the wild-type Pkd allele. Approximately one quarter of families with ADPDK due to Pkd1 have germline nonsynonymous amino acid substitution (missense) mutations. A subset of these mutations is hypomorphic, retaining some residual PC1 function. Previous studies have shown that the highly conserved Ire1 α -XBP1 pathway of the unfolded protein response can modulate levels of functional PC1 in the presence of mutations in genes required for post-translational maturation of integral membrane proteins. We examine how activity of the endoplasmic reticulum chaperone-inducing transcription factor XBP1 affects ADPKD in a murine model with missense Pkd1 . We engineered a Pkd1 REJ domain missense murine model, Pkd1 R2216W , on the basis of the orthologous human hypomorphic allele Pkd1 R2220W , and examined the effects of transgenic activation of XBP1 on ADPKD progression. Expression of active XBP1 in cultured cells bearing PC1 R2216W mutations increased levels and ciliary trafficking of PC1 R2216W . Mice homozygous for Pkd1 R2216W or heterozygous for Pkd1 R2216Win trans with a conditional Pkd1 fl allele exhibit severe ADPKD following inactivation in neonates or adults. Transgenic expression of spliced XBP1 in tubule segments destined to form cysts reduced cell proliferation and improved Pkd progression, according to structural and functional parameters. Modulating ER chaperone function through XBP1 activity improved Pkd in a murine model of PC1, suggesting therapeutic targeting of hypomorphic mutations.

991P EGFR exon 20 insertions in non-small cell lung cancer (NSCLC): Impact of TP53 mutation status and value of immune checkpoint blockade (ICB)

About 4% of all EGFR mutations account as exon 20 insertions, which convey resistance to the first three generations of EGFR-tyrosine kinase inhibitors (TKIs). Recent research has revealed new drugs to target these mutations specifically. However, little is known about the impact of immune-checkpoint blockade (ICB) in this subgroup, especially in patients with co-occurring TP53 mutations. We analyzed co-occurring mutations in patients with EGFR exon 20 insertions and their influence on the efficacy of systemic treatment. The main focus was impact of ICB on overall survival (OS) in these patients with or without TP53 mutations. Co-mutational status was assessed using next-generation-sequencing (NGS) panels. TP53 mutations were further divided into truncating vs. non-truncating mutations. Real-world-data (RWD) of 159 advanced NSCLC patients with EGFR exon 20 insertion in the timeframe of 2014-2020 were analyzed. TP53 mutation was the most common co-occurring mutation in 69 (43.6%) patients. OS of TP53 wildtype patients was significantly longer with 851 vs. 444 days (p=0.003, Log Rank). No difference of OS referring to the subtype of TP53 mutations was observed (p=0.703). In total, 66 pts were treated with ICB in one of the treatment lines and trended towards longer OS of 824 vs. 479 days, without reaching significance (p=0.402). In patients with co-occurring TP53, patients who received ICB (n=33) had longer OS than those without ICB exposure (507 vs. 369 days, p=0.086). Lack of significance might be explainable by a median follow-up time of 1498 days (4.1 years). Regardless of subtype, TP53 co-occurring mutation seems to be a strong negative prognostic factor in NSCLC patients with EGFR exon 20 insertions. However, these patients seem to benefit from the ICB, especially at the beginning of their treatment pathway.

Abstract 2281: Molecular profiling of the RUNX1 RUNT domain in myeloid disorders

Abstract Background: AML with RUNX1 mutation is a provisional entity in the WHO classification. RUNX1 variants generally consist of truncating mutations throughout the gene, as well as SNVs, insertions, and deletions in the RUNT domain. In the germline setting, these mutations may lead to familial platelet disorders and have profound implications in donor selection for allogeneic stem cell transplant. Approximately half of mutations in RUNX1 are classified as variants of unknown clinical significance (VOUS), underscoring the unmet needs of this patient population. Methods: Bone marrow, peripheral blood, or FFPE tissue samples from 10,118 patients with suspected myeloid disease were sequenced using a 303 gene myeloid NGS panel. A total of 1209 RUNX1-mutated patients formed the cohort for this study. While pair-matched samples were not available and germline status not established, these patients were likely to be in the 40-60% variant allele frequency (VAF) range. Statistics were performed using Fishers exact test. Results: Among RUNX1-mutated patients, 277 (22.9%) had a RUNX1 variant at a subclonal VAF (&amp;lt;20%), 505 (41.7%), 352 (29.1%), &amp; 75 (6.2%) had variants at VAFs of 20-39%, 40-60%, &amp; &amp;gt;60%, respectively. While all truncating mutations were classified as pathogenic, 84.3% of non-truncating RUNT domain mutations were classified as VOUS. RUNT domain mutations included 272 non-truncating and 220 truncating mutations. There were 599 (49.5%) truncating mutations throughout the entire gene. Non-truncating RUNT domain-mutated patients most frequently harbored mutations in ASXL1 (31.6%), SRSF2 (31.3%), TET2 (29.4%), STAG2 (19.1%), RAS (18.3%), and BCOR/BCORL1 (17.3%). The frequency of RUNT domain co-mutations in the ASXL1 (36.8% [35] vs. 10.4% [5]; p=0.0007) and RAS families (28.4% [26] vs. 10.4% [5]; p=0.03) were significantly higher in the 40-60% VAF group compared to subclonal populations, while BCOR/BCORL1 mutations (10.5% [10] vs. 29.1% [14]; p=0.008) were significantly lower. These results were recapitulated in a cohort of VOUS-only RUNT domain patients with a VAF of 40-60% compared to subclonal populations: ASXL1 (36.8% [21] vs. 22.2% [10]), RAS family (24.5% [14] vs. 10.9% [5]), and BCOR/BCORL1 (8.8% [5] vs. 31.1% [14]; p=0.005). Among patients with truncating mutations in RUNX1, mutations in ASXL1 (34.4% [44] vs 20.3% [42]; p=0.0047) and RAS family members (25.8% [33] vs. 10.1% [21]; p=0.0002) were more frequent in patients with VAFs of 40-60% when compared to subclonal populations, and mutations in BCOR/BCORL1 were similar (28.1% [36] vs. 20.7% [42]). Conclusions: The majority of RUNX1 mutations clustered in the RUNT domain are VOUS. This study demonstrates that both truncations and RUNT domain mutations harbor molecular signatures that reflect similar oncogenic mechanisms. These molecular signatures are also present when filtering exclusively by VOUS in the RUNT domain. Citation Format: Frank J. Scarpa, Madhuri Paul, Rachel Daringer, Sally Agersborg, Vincent A. Funari, Forrest J. Blocker. Molecular profiling of the RUNX1 RUNT domain in myeloid disorders [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2281.

Establishment and Characterization of MUi027-A: A Novel Patient-Derived Cell Line of Polycystic Kidney Disease with PKD1 Mutation.

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most prevalent genetic diseases affecting the kidneys. A genetically specific mutation model is required to comprehend its pathophysiology and to develop a drug treatment. In this study, we successfully developed human induced pluripotent stem cells (hiPSCs) named MUi027-A from skin fibroblasts of a patient diagnosed with ADPKD and carrying the PKD1 frameshift mutation (c.7946_7947delCT). MUi027-A cells showed the same genetic fingerprints as the parental cells, including the presence of the PKD1 mutation. MUi027-A hiPSCs displayed embryonic stem cell-like characteristics with the capability of differentiating into the three germ layers. Upon directed differentiation, MUi027-A hiPSCs could be differentiated into tubular organoids with the expression of renal cell markers. Furthermore, we compared the efficiency of cyst formation in two human iPSC lines with different PKD1 mutations. When cyst formation was induced by either forskolin or blebbistatin, MUi027-A hiPSC-derived kidney organoids displayed higher frequencies of cyst formation when compared to organoids generated from an iPSC cell line with non-truncating PKD1 mutation genotype (c.5878C > T), suggesting the presence of physiological differences in the mechanism of cyst formation between different PKD1 mutants. Overall, we generated and characterized a novel human iPSC line with a specific PKD mutation and demonstrated its potential as a disease model to study the pathophysiology of genetic determinants in the development of ADPKD disease.

POS-439 The PROPKD score affect renal involvement in patients with tolvaptan-treated autosomal dominant polycystic kidney disease ?

The PROPKD score has been proposed to evaluate the risk of the progression to end stage renal failure in patients with autosomal dominant polycystic kidney disease (ADPKD), based on gender (male: 1point), hypertension under 35 years old (2 points), urological event under 35 years old (2 points), and gene mutation (PKD1 truncating mutation: 4 points, PKD1 non-truncating mutation: 2 points, PKD2: 0 point). Although tolvaptan has benefits for renal involvement, whether PROPKD score could predict future renal involvement in patients with ADPKD treated with tolvaptan is unknown.

Dissecting the genotype-phenotype correlation of COL4A5 gene mutation and its response to renin-angiotensin-aldosterone system blockers in Chinese male patients with Alport syndrome.

Alport syndrome (AS) is an inherited type IV collagen-related disorder with an irreversible tendency to progress to end-stage renal disease (ESRD). X-linked AS (XLAS) is caused by mutations in the COL4A5 gene. The aim of this study was to investigate the effects of underlying mutations on clinical manifestations and the response to therapy in XLAS. We conducted a retrospective cohort study of 187 Chinese male patients with XLAS confirmed by pathological examination and genetic analysis. The Kaplan-Meier method and Cox proportional hazards model were used to assess the age and risk of progression to ESRD under different genotypes and treatment conditions. A strong relationship between transcript type and renal outcome was observed, with the median age of ESRD onset being 22 years for truncating mutations and 39 years for non-truncating mutations. The response of affected patients to renin-angiotensin-aldosterone system (RAAS) blockers was genotype-associated. This therapy delayed the onset of ESRD by 16 years in patients with non-truncating mutations and 3 years in patients with truncating mutations. The efficacy of RAAS blockers functioned in a time-dependent manner, with a 7% reduction in the risk of progression to ESRD per each 6-month increase in treatment duration [hazard ratio 0.93 (95% confidence interval 0.89-0.96); P < 0.001]. Clinical features and response to RAAS blockers were observed to be strongly correlated with the genotypes of male XLAS patients. Genotyping of COL4A5 gene mutations is essential and is a useful tool to assess the prognosis of AS patients.

Detection of PKD1 and PKD2 Somatic Variants in Autosomal Dominant Polycystic Kidney Cyst Epithelial Cells by Whole-Genome Sequencing.

Autosomal dominant polycystic kidney disease (ADPKD) is a genetic disorder characterized by the development of multiple cysts in the kidneys. It is often caused by pathogenic mutations in PKD1 and PKD2 genes that encode polycystin proteins. Although the molecular mechanisms for cystogenesis are not established, concurrent inactivating germline and somatic mutations in PKD1 and PKD2 have been previously observed in renal tubular epithelium (RTE). To further investigate the cellular recessive mechanism of cystogenesis in RTE, we conducted whole-genome DNA sequencing analysis to identify germline variants and somatic alterations in RTE of 90 unique kidney cysts obtained during nephrectomy from 24 unrelated participants. Kidney cysts were overall genomically stable, with low burdens of somatic short mutations or large-scale structural alterations. Pathogenic somatic "second hit" alterations disrupting PKD1 or PKD2 were identified in 93% of the cysts. Of these, 77% of cysts acquired short mutations in PKD1 or PKD2 ; specifically, 60% resulted in protein truncations (nonsense, frameshift, or splice site) and 17% caused non-truncating mutations (missense, in-frame insertions, or deletions). Another 18% of cysts acquired somatic chromosomal loss of heterozygosity (LOH) events encompassing PKD1 or PKD2 ranging from 2.6 to 81.3 Mb. 14% of these cysts harbored copy number neutral LOH events, while the other 3% had hemizygous chromosomal deletions. LOH events frequently occurred at chromosomal fragile sites, or in regions comprising chromosome microdeletion diseases/syndromes. Almost all somatic "second hit" alterations occurred at the same germline mutated PKD1/2 gene. These findings further support a cellular recessive mechanism for cystogenesis in ADPKD primarily caused by inactivating germline and somatic variants of PKD1 or PKD2 genes in kidney cyst epithelium.