Complete Exon Research Articles

SLC12A1 variant c.1684+1 G>A causes Bartter syndrome type 1 by promoting exon 13 skipping.

Bartter syndrome type 1, an autosomal recessive genetic disorder, is caused by pathogenic loss-of-function variants in the SLC12A1 gene. It is characterized by metabolic alkalosis and prenatal-onset polyuria leading to polyhydramnios. We identified pathogenic gene in a 12-day-old newborn boy with Bartter syndrome type 1 using whole-exome sequencing. Sanger sequencing validated the identified variants. A minigene assay was performed to investigate the effect of a novel splice site variant on pre-mRNA splicing. We found a compound heterozygous variants in the SLC12A1 gene, consisting of a known pathogenic missense mutation (NM_000338: c.769 G>A; p.Gly257Ser) and a novel splice site variant (c.1684+1 G>A). In silico predictions and an in vitro minigene splicing assay demonstrated that the splicing variant c.1684+1 G>A abolished a consensus splice donor site of SLC12A1 intron 13, resulting in complete exon 13 skipping, translational frameshift, and premature termination codon, ultimately leading to loss of SLC12A1 function. Using a cell-based in vitro assay, we revealed the aberrant effect of the pathogenic splicing variant SLC12A1 c.1684+1 G>A on pre-mRNA splicing. Our findings expand the gene mutation spectrum of Bartter syndrome type 1, providing a basis for genetic diagnosis and the development of genetic medicines.

Functional characterization vs in silico prediction for TBX5 missense and splice variants in Holt-Oram syndrome

PurposePredicting effects of genomic variants has become a real challenge in the diagnosis of rare human diseases. Holt-Oram syndrome is an autosomal condition characterized by the association of radial and heart defects, due to variants in TBX5. Most variants are predicted to be truncating and result in haploinsufficiency. The pathogenicity of missense or splice variants is harder to demonstrate. MethodsFourteen TBX5 variants of uncertain significance (5 missense, 9 splice) and 6 likely pathogenic missense variants were selected for functional testing, depending on the variant-type (immunolocalization, western blot, reporter assays, minigene splice assays, and RT-PCR). Results were compared with in silico predictions. ResultsFunctional tests allowed to reclassify 9/14 variants of uncertain significance in TBX5 as likely pathogenic, confirming their role in Holt-Oram syndrome. We demonstrated loss of function (n = 8) or gain of function (n = 1) for 9 of the 11 missense variants, whereas no functional impact was shown for the 2 variants: p.(Gly195Ala) and p.(Ser261Cys), as suggested by contradictory predictions of in silico approaches. Of 9 splice variants predicted to affect splicing by SpliceAI, we observed partial or complete exon skipping (n = 6), intron retention (n = 2) or exon shortening (n = 1), inducing frame shifting with premature stop codons. ConclusionBioinformatic and biological approaches are complementary, together with a good knowledge of clinical conditions, for accurate American College of Medical Genetics and Genomics classification in human rare diseases.

A novel splice-altering TNC variant (c.5247A > T, p.Gly1749Gly) in an Chinese family with autosomal dominant non-syndromic hearing loss

BackgroundThis study aims to analyze the pathogenic gene in a Chinese family with non-syndromic hearing loss and identify a novel mutation site in the TNC gene.MethodsA five-generation Chinese family from Anhui Province, presenting with autosomal dominant non-syndromic hearing loss, was recruited for this study. By analyzing the family history, conducting clinical examinations, and performing genetic analysis, we have thoroughly investigated potential pathogenic factors in this family. The peripheral blood samples were obtained from 20 family members, and the pathogenic genes were identified through whole exome sequencing. Subsequently, the mutation of gene locus was confirmed using Sanger sequencing. The conservation of TNC mutation sites was assessed using Clustal Omega software. We utilized functional prediction software including dbscSNV_AdaBoost, dbscSNV_RandomForest, NNSplice, NetGene2, and Mutation Taster to accurately predict the pathogenicity of these mutations. Furthermore, exon deletions were validated through RT-PCR analysis.ResultsThe family exhibited autosomal dominant, progressive, post-lingual, non-syndromic hearing loss. A novel synonymous variant (c.5247A > T, p.Gly1749Gly) in TNC was identified in affected members. This variant is situated at the exon–intron junction boundary towards the end of exon 18. Notably, glycine residue at position 1749 is highly conserved across various species. Bioinformatics analysis indicates that this synonymous mutation leads to the disruption of the 5' end donor splicing site in the 18th intron of the TNC gene. Meanwhile, verification experiments have demonstrated that this synonymous mutation disrupts the splicing process of exon 18, leading to complete exon 18 skipping and direct splicing between exons 17 and 19.ConclusionThis novel splice-altering variant (c.5247A > T, p.Gly1749Gly) in exon 18 of the TNC gene disrupts normal gene splicing and causes hearing loss among HBD families.

A pan-cetacean MHC amplicon sequencing panel developed and evaluated in combination with genome assemblies.

The major histocompatibility complex (MHC) is a highly polymorphic gene family that is crucial in immunity, and its diversity can be effectively used as a fitness marker for populations. Despite this, MHC remains poorly characterised in non-model species (e.g., cetaceans: whales, dolphins and porpoises) as high gene copy number variation, especially in the fast-evolving class I region, makes analyses of genomic sequences difficult. To date, only small sections of class I and IIa genes have been used to assess functional diversity in cetacean populations. Here, we undertook a systematic characterisation of the MHC class I and IIa regions in available cetacean genomes. We extracted full-length gene sequences to design pan-cetacean primers that amplified the complete exon 2 from MHC class I and IIa genes in one combined sequencing panel. We validated this panel in 19 cetacean species and described 354 alleles for both classes. Furthermore, we identified likely assembly artefacts for many MHC class I assemblies based on the presence of class I genes in the amplicon data compared to missing genes from genomes. Finally, we investigated MHC diversity using the panel in 25 humpback and 30 southern right whales, including four paternity trios for humpback whales. This revealed copy-number variable class I haplotypes in humpback whales, which is likely a common phenomenon across cetaceans. These MHC alleles will form the basis for a cetacean branch of the Immuno-Polymorphism Database (IPD-MHC), a curated resource intended to aid in the systematic compilation of MHC alleles across several species, to support conservation initiatives.

Three exonic variants in the COL4A5 gene alter RNA splicing in a minigene assay.

X-linked Alport syndrome (XLAS) is an inherited renal disease caused by rare variants of COL4A5 on chromosome Xq22. Many studies have indicated that single nucleotide variants (SNVs) in exons can disrupt normal splicing process of the pre-mRNA by altering various splicing regulatory signals. The male patients with XLAS have a strong genotype-phenotype correlation. Confirming the effect of variants on splicing can help to predict kidney prognosis. This study aimed to investigate whether single nucleotide substitutions, located within three bases at the 5' end of the exons or internal position of the exons in COL4A5 gene, cause aberrant splicing process. We analyzed 401 SNVs previously presumed missense and nonsense variants in COL4A5 gene by bioinformatics programs and identified candidate variants that may affect the splicing of pre-mRNA via minigene assays. Our study indicated three of eight candidate variants induced complete or partial exon skipping. Variants c.2678G>C and c.2918G>A probably disturb classic splice sites leading to corresponding exon skipping. Variant c.3700C>T may disrupt splicing enhancer motifs accompanying with generation of splicing silencer sequences resulting in the skipping of exon 41. Our study revealed that two missense variants positioned the first nucleotides of the 5' end of COL4A5 exons and one internal exonic nonsense variant caused aberrant splicing. Importantly, this study emphasized the necessity of assessing the effects of SNVs at the mRNA level.

Splice-Switching Antisense Oligonucleotides Targeting Extra- and Intracellular Domains of Epidermal Growth Factor Receptor in Cancer Cells.

Cancer is one of the leading causes of death globally. Epidermal growth factor receptor is one of the proteins involved in cancer cell proliferation, differentiation, and invasion. Antisense oligonucleotides are chemical nucleic acids that bind to target messenger ribonucleic acid and modulate its expression. Herein, we demonstrate the efficacy of splice-modulating antisense oligonucleotides to target specific exons in the extracellular (exon 3) and intracellular (exon 18, 21) domains of epidermal growth factor receptor. These antisense oligonucleotides were synthesized as 25mer 2'-O methyl phosphorothioate-modified ribonucleic acids that bind to complementary specific regions in respective exons. We found that PNAT524, PNAT525, PNAT576, and PNAT578 effectively skipped exon 3, exon 18, and exon 21 in glioblastoma, liver cancer, and breast cancer cell lines. PNAT578 treatment also skipped partial exon 19, complete exon 20, and partial exon 21 in addition to complete exon 21 skipping. We also found that a cocktail of PNAT576 and PNAT578 antisense oligonucleotides performed better than their individual counterparts. The migration potential of glioblastoma cancer cells was reduced to a greater extent after treatment with these antisense oligonucleotides. We firmly believe that using these splice-modulating antisense oligonucleotides in combination with existing EGFR-targeted therapies could improve therapeutic outcomes.

Partner Gene Associated Trinary Fusion and Orchestration of Multiple Molecular Events in Acute Promyelocytic Leukemia with Variant Rara Fusion Genes

Background All-trans retinoic acid (ATRA) has been used with great success in acute promyelocytic leukemia (APL) cases with PML::RARA fusion gene (FG). There are still PML::RARA negative cases manifest as APL. So far, 19 RARA fusion partner genes have been reported, with all reported as 5′ partners ( X::RARA). Intriguingly, a significant proportion of X::RARA cases were resistant to ATRA, and mechanisms are yet to be elucidated. Here we report the finding of a novel type of trinary fusion and the orchestrating of multiple molecular events that confer leukemogenesis and ATRA resistance in X::RARA-APL. Methods A total of 23 cases (Fig. 1A) with RNA-seq and WGS data from 10 centers were enrolled. We used Arriba for routine FG calling and manually investigated the nonconventional fusion sequence. RT-PCR, Sanger sequencing, and optical genome mapping (OGM) were used to validate the FGs and to determine whether the RARA 5′ and 3′ fusion events were located on the same cistron. The responsiveness of the fusion proteins to ATRA was evaluated using an optimized UAS/GAL4 reporter system. Results Among the cases enrolled, there were 17 males and 6 females, aged 3-66 (median 32) years. Morphological and immunophenotype analyses all showed features of APL. We first identified the STAT3::RARA fusion event in an ATRA-resistant case (the index case, #A1). Moreover, we noticed another fusion event of truncated RARA exon 9 and a 3′ partner sequence that did not map to any annotated protein-coding genes. Further analysis determined it was the RC sequence of the transposable element (TE) LTR40a with the annotated loci at 17p11.2. Both OGM (at the genome level) and RT-PCR (Fig. 1A) confirmed that the two RARA fusion events were in the same cistron. Thus, the fusion transcript being STAT3::RARA::LTR40a-RC spliced in tandem, but not separate transcripts of STAT3::RARA and RARA::LTR40a-RC. We named this novel form of tandem splicingtrinary fusion. We, therefore, recruit X::RARA-APL cases to investigate the recurrence of RARA 3′ fusion. We identified 9 RARA 5′ partner genes in 23 cases (Fig. 1B), with TTMV -ORF2, STAT5B, STAT3, IRF2BP1, THRAP3, and TNRC18 in multiple cases. Ten (43.5%) cases also harbor RARA 3′ fusion partners. The partner of case #A1_8 were TEs, and the partner of case #A9_10 was the same gene as their 5′ one. Thirteen cases did not harbor 3′ fusions. Notably, both TNRC18::RARA cases (#15_16) carry mutations on their RARA ligand binding domain (LBD) at the primary onset. Gene expression analysis confirmed the aberrant activation of the involved TEs. All TEs involved in the RARA 3' fusions confer a poly_A signal sequence, which was essential for a mRNA. In-depth analysis indicated that TEs participate in forming RARA-FGs through a transposition mechanism rather than a translocation mechanism. The molecular events addition to RARA 5′ splicing showed a 5′ partner associated pattern: all of the 4 STAT3::RARA and 4 STAT5B::RARA cases harbor TEs as their 3′ partner, both TNRC18::RARA cases carry LBD mutation, while all 7 TTMV -ORF2::RARA cases did not harbor 3′ splicing or LBD mutation. Remarkably, although the RARA 3' splicing sites varied, they all resulted in partial or complete exon 9 truncation. RARA exon 9 encodes helix 11_12 (H11_12) of its LBD, which is pivotal in allosteric response to ATRA. The RARA trinary fusion will result in LBD-H12 or H11_12 castration due to the 3' splicing, then lead to losing responsiveness to ATRA via an allosteric disability mechanism (Fig. 1C, 1D). Experiments confirmed that cells transfected with STAT3::RARA fusion with intact LBD responded well to ATRA, while both the fusion in case #A1 and artificial fusion with LBD-H12 castration showed no response to physiological and pharmacological concentrations of ATRA (Fig. 1E). Conclusions This is the first report of pathological trinary fusion, the TEs and transposition mechanism participating in composing pathological fusion genes, and the RARA LBD castration confers ATRA resistance in X::RARA-APL. Also, there might be another synergistic mechanism of RARA 5' fusion and LBD mutation. The formation of trinary fusion and gain of LBD mutation, in addition to X::RARA fusion, requires more genome splicing or transposition or mutation events, which explains the rarity of X::RARA-APL. This study highlights mechanisms of partner gene-associated trinary fusion and the orchestrating of multiple molecular events in the leukemogenesis in X::RARA-APL cases.

Genome-wide detection of human intronic AG-gain variants located between splicing branchpoints and canonical splice acceptor sites.

Human genetic variants that introduce an AG into the intronic region between the branchpoint (BP) and the canonical splice acceptor site (ACC) of protein-coding genes can disrupt pre-mRNA splicing. Using our genome-wide BP database, we delineated the BP-ACC segments of all human introns and found extreme depletion of AG/YAG in the [BP+8, ACC-4] high-risk region. We developed AGAIN as a genome-wide computational approach to systematically and precisely pinpoint intronic AG-gain variants within the BP-ACC regions. AGAIN identified 350 AG-gain variants from the Human Gene Mutation Database, all of which alter splicing and cause disease. Among them, 74% created new acceptor sites, whereas 31% resulted in complete exon skipping. AGAIN also predicts the protein-level products resulting from these two consequences. We performed AGAIN on our exome/genomes database of patients with severe infectious diseases but without known genetic etiology and identified a private homozygous intronic AG-gain variant in the antimycobacterial gene SPPL2A in a patient with mycobacterial disease. AGAIN also predicts a retention of six intronic nucleotides that encode an in-frame stop codon, turning AG-gain into stop-gain. This allele was then confirmed experimentally to lead to loss of function by disrupting splicing. We further showed that AG-gain variants inside the high-risk region led to misspliced products, while those outside the region did not, by two case studies in genes STAT1 and IRF7. We finally evaluated AGAIN on our 14 paired exome-RNAseq samples and found that 82% of AG-gain variants in high-risk regions showed evidence of missplicing. AGAIN is publicly available from https://hgidsoft.rockefeller.edu/AGAIN and https://github.com/casanova-lab/AGAIN.

Pathogenicity analysis and splicing rescue of a classical splice site variant (c.1343+1G>T) of CNOT1 gene associated with neurodevelopmental disorders.

Mutations in the CNOT1 gene lead to an incurable rare neurological disorder mainly manifested as a clinical spectrum of intellectual disability, developmental delay, seizures, and behavioral problems. In this study, we investigated a classical splice site variant of CNOT1 (c.1343+1G>T) associated with neurodevelopmental disorders, which was a master regulator, orchestrating gene expression, RNA deadenylation, and protein ubiquitination. To link CNOT1 dysfunction with the neurodevelopmental phenotype observed in a patient, in vitro minigene assay was used to verify the effect of CNOT1 gene splice site variant c.1343+1G>T on mRNA splicing. We also explored the impact of transient transfection introducing modified U1 snRNA on correcting the splicing variant. Through minigene expression in mammalian cells, we demonstrated that the variant induced complete exon 12 skipping, which explained the patient's clinical condition and provided additional genetic diagnosis evidence for the clinical significance of the variant. Moreover, we confirmed that the aberrant splice pattern could be partially corrected by the modified U1 snRNA at the mRNA level, which provided strong evidence for the therapeutic potential of modified U1 snRNA in neutralizing the hazardous effect of incorrect splicing patterns.

Unique Haplotypes in OPN1LW as a Common Cause of High Myopia With or Without Protanopia: A Potential Window Into Myopic Mechanism.

Specific haplotypes (LVAVA, LIVVA, and LIAVA) formed by five polymorphisms (p.L153M, p.V171I, p.A174V, p.I178V, and p.S180A in exon 3 of OPN1LW) that cause partial or complete exon skipping have been reported as unique genetic causes of high myopia with or without colorblindness. This study aimed to identify the contribution of OPN1LW to early-onset high myopia (eoHM) and the molecular basis underlying eoHM with or without colorblindness. Comparative analysis of exome sequencing data was conducted for 1226 families with eoHM and 9304 families with other eye conditions. OPN1LW variants detected by targeted or whole exome sequencing were confirmed by long-range amplification and Sanger sequencing, together with segregation analysis. The clinical data were thoroughly analyzed. Unique haplotypes and truncation variants in OPN1LW were detected exclusively in 68 of 1226 families with eoHM but in none of the 9304 families with other visual diseases (P = 1.63 × 10-63). Four classes of variants were identified: haplotypes causing partial splicing defects in OPN1LW (LVAVA or LIVVA in 31 families), LVAVA in OPN1LW-OPN1MW hybrid gene (in 3 families), LIAVA in OPN1LW (in 29 families), and truncations in OPN1LW (in 5 families). The first class causes partial loss of red photopigments, whereas the latter three result in complete loss of red photopigments. This is different from the replacement of red with green owing to unequal re-arrangement causing red-green colorblindness alone. Of the 68 families, 42 affected male patients (31 families) with the first class of variants (LVAVA or LIVVA in OPN1LW) had eoHM alone, whereas 37 male patients with the latter 3 classes had eoHM with protanopia. Adaptive optics retinal imaging demonstrated reduced cone regularity and density in men with eoHM caused by OPN1LW variants compared to those patients with eoHM and without OPN1LW variants. Based on the 68 families with unique variants in OPN1LW, our study provides firm evidence that the two different phenotypes (eoHM with or without colorblindness) are caused by two different classes of variants (partial splicing-effect haplotypes or complete splicing-effect haplotypes/truncation variants, respectively). The contribution of OPN1LW to eoHM (isolated and syndromic) was characterized by OPN1LW variants found in 5.5% (68/1226) of the eoHM families, making it the second most common cause of monogenic eoHM alone (2.4%) and a frequent cause of syndromic monogenic eoHM with colorblindness. Such haplotypes, in which each individual variant alone is considered a benign polymorphism, are potential candidates for other hereditary diseases with causes of missing genetic defects.

Abstract 6109: Multi-site evaluation of novel BRCA1/2 reference materials including large genomic rearrangements

Abstract Mutations in the tumor suppressor BRCA1 and BRCA2 genes can cause cell damage that significantly increases the risk of developing breast, ovarian, prostate, and pancreatic cancers. Large genomic rearrangements (LGRs) are defined as deletions, duplications or insertions; often involving a significant portion of an exon. Usually pathogenic, they have been reported to account for up to 27% of the BRCA1 and 5% of BRCA2 disease-causing mutations with a strong founder effect accounting for about 1/3 of all cancer diagnoses in some populations. Accurate detection of a BRCA1/2 pathogenic variants has immense impact on clinical management of disease including eligibility for PARP inhibitor therapy. However, these LGRs are frequently missed by PCR-based methods and NGS assays that do not detect partial or complete exon losses or gains. Given the difficulty in detecting LGRs, there is a need for improvement of BRCA1/2 testing algorithms including reference materials that incorporate challenging LGRs to support NGS assays that analyze for these mutations. Biosynthetic DNA constructs bearing clinically relevant BRCA1/2 variants including deletions up to 500 bp spanning 2 exons and duplications up to 170 bp, were mixed with purified genomic DNA from the GM24385 human reference cell line at 10% and 50% variant allele frequency (VAF) to represent somatic and inherited BRCA disease states, respectively. The same variants were engineered into GM24385 cells at the desired VAF (>5%) and the cells were formalin fixed and paraffin embedded using a proprietary method to mimic preserved tumor biopsies. In both materials, VAF was measured using digital PCR. The prepared purified gDNA and FFPE materials were sent to multiple clinical laboratories and analyzed using various NGS assays to assess variant detection performance and ensure that the product was compatible with clinical testing workflows. Digital PCR confirmed the presence of all 20 BRCA1 and BRCA2 variants within a 20% range of the target VAF in the purified gDNA material and between 11-23% in FFPE. NGS results were variable but confirmed the presence of most variants. A few variants were detected at very low levels but reported as not detected by the bioinformatic software due to limit of detection. We have successfully developed a reference material to support both BRCA1 and BRCA2 inherited and somatic genetic testing, providing laboratories and assay developers with a tool to challenge and optimize their assay’s detection and quantitation of pathogenic variants by NGS Provided as full process in FFPE format or in purified gDNA, these reference materials include 11 challenging LGRs, which are useful in evaluating the ability of different NGS platforms to detect such variants. This study has highlighted the need for reference materials that contain these challenging genomic alterations to improve genetic testing and ultimately, support PARP inhibitor treatment selection. Citation Format: Dana Ruminski Lowe, Benedicta Forson, Maria Cowen, Matthew G. Butler, Yves Konigshofer, Melissa Berenger, Krystyna Nahlik, Dianren Xia, Catherine Huang, Russell Garlick, Bharathi Anekella. Multi-site evaluation of novel BRCA1/2 reference materials including large genomic rearrangements. [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 6109.

A case of novel mutation in ANOS1 (KAL1) gene and review of Kallmann syndrome.

Kallmann syndrome (KS) is a genetically heterogeneous condition characterized by hypogonadotropic hypogonadism with coexisting anosmia or hyposmia along with potential other phenotypic abnormalities depending on the specific genetic mutation involved. Several genetic mutations have been described to cause KS. The ANOS1 (KAL1) gene is responsible for 8% of mutations causing KS. A 17-year-old male presented to our clinic with delayed puberty and hyposmia, along with a family history suggestive of hypogonadism in his maternal uncle. Genetic testing for KS revealed complete exon 3 deletion in the ANOS1 gene. To the best of our knowledge, this specific mutation has not been previously described in the literature. Missense and frameshift mutations in the KAL1 or ANOS1 gene located in the X chromosome are responsible for 8% of all known genetic mutations of Kallmann syndrome. Exon 3 deletion is one of the ANOS1 gene is a novel mutation, not reported before. Targeted gene sequencing for hypogonadotropic hypogonadism can be employed based on the phenotypic presentation.

Abstract P5-02-22: Circulating Tumor DNA Genotyping of Intrinsic and Acquired Gene Alterations in Patients With Advanced Breast Cancer Receiving the Cyclin-Dependent Kinase 4/6 Inhibitor Palbociclib: Biomarker Results from POLARIS

Abstract Background: POLARIS is a prospective, real-world study of palbociclib in patients with hormone receptor–positive (HR+)/human epidermal growth factor receptor 2–negative (HER2–) advanced breast cancer (ABC) in the United States and Canada. We present results from analyses of serial circulating tumor gene alteration profiles from patients treated with palbociclib in the first and second or later lines of ABC treatment to illustrate the potential mutagenic drivers of resistance (intrinsic/acquired) and carrier-mutations (intrinsic/maintained). Methods: The clinical database cutoff date was March 30, 2022. Patients in the biomarker analysis group provided consent for serial blood sample collection, received ≥1 dose of initial palbociclib combination treatment, and had ≥1 circulating tumor DNA (ctDNA) measurement available. The Guardant360 platform with somatic single-nucleotide variants in complete or critical exons of 73 genes was used. Tumor gene alteration profiles (at baseline, on-treatment at Cycle 2 Day 1 [C2D1], and at end of treatment [EOT]) were evaluated. Cox proportional hazard models were used to estimate hazard ratios and 95% CIs. Results: Patient samples (n=345) were analyzed and gene alterations were detected in 85% of baseline samples (n=337), 72% of on-treatment samples (n=280), and 85% of EOT samples (n=104). Most frequently altered genes were PIK3CA (38%), TP53 (28%), and ESR1 (15%) at baseline; TP53 (28%), PIK3CA (24%), and NF1 (10%) at C2D1; and TP53 (40%), PIK3CA (40%), and ESR1 (33%) at EOT. Most frequent gene amplifications (amp) were detected in CCND1 (8.3%), FGFR1 (7.7%), and EGFR (5.9%) at baseline; FGFR1 (5.0%), CCND1 (4.3%), and EGFR (3.2%) at C2D1; and CCND1 (13.5%), FGFR1 (9.6%), and EGFR (9.6%) at EOT. Baseline mutations of ESR1 and PIK3CA led to shorter real-world progression-free survival (rwPFS) than wild-type (hazard ratio [95% CI], 1.99 [1.38, 2.86] and 1.67 [1.24, 2.25], respectively). Baseline amp of CCND1 and FGFR1 also led to shorter rwPFS than wild-type (2.13 [1.36, 3.34] and 1.93 [1.20, 3.10]). Acquired mutations in ESR1, ATM, and RB1 were observed at EOT. Most frequently acquired ESR1 mutations at EOT were D538G, Y537N, and Y537S. Patients with all mutations cleared at C2D1, had longer rwPFS than those without (hazard ratio [95% CI], 0.58 [0.41, 0.83]). Conclusion: Patients with mutated ESR1 and PIK3CA or CCND1 and FGFR1 amp at baseline had shorter rwPFS than patients with wild-type genes. Genotyping analysis of progression ctDNA highlights the emergence of mutations in estrogen receptor and cell cycle pathways under selective therapeutic pressure and could guide monitoring and therapeutic sequencing for patients with HR+/HER2– MBC. ClinicalTrials.gov: NCT03280303 Citation Format: Debu Tripathy, Joanne L. Blum, Shibing Deng, Steven L. McCune, Kamal Patel, Yao Wang, Shailendra Lakhanpal, Meghan S. Karuturi, Zhe Zhang, Chetan Deshpande, Monica Z. Montelongo, Eric Gauthier, Yuan Liu, Gabrielle B. Rocque, Aditya Bardia. Circulating Tumor DNA Genotyping of Intrinsic and Acquired Gene Alterations in Patients With Advanced Breast Cancer Receiving the Cyclin-Dependent Kinase 4/6 Inhibitor Palbociclib: Biomarker Results from POLARIS [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-22.

Minigene splicing assays reveal new insights into exonic variants of the SLC12A3 gene in Gitelman syndrome.

Gitelman syndrome (GS) is a type of salt-losing tubular disease, most of which is caused by SLC12A3 gene variants, and missense variants account for the majority. Recently, the phenomenon of exon skipping, in which variants disrupt normal pre-mRNA splicing, has been related to a variety of diseases. Therefore, we hypothesize that a certain proportion of SLC12A3 variants can result in disease via interfering with the normal splicing process. We analyzed 342 previously presumed SLC12A3 missense variants using bioinformatics programs and identified candidate variants that may alter the splicing of pre-mRNA through minigene assays. Our study revealed that, among ten candidate variants, six variants (c.602G>A, c.602G>T, c.1667C>T, c.1925G>A, c.2548G>C, and c.2549G>C) led to complete or incomplete exon skipping by affecting exonic splicing regulatory elements and/or disturbing canonical splice sites. It is worth mentioning that this is the largest study on pre-mRNA splicing of SLC12A3 exonic variants. In addition, our study emphasizes the importance of detecting splicing function at the mRNA level in GS and indicates that minigene analysis is a valuable tool for splicing functional assays of variants in vitro.

Novel biallelic mutations in TMEM126B cause splicing defects and lead to Leigh-like syndrome with severe complex I deficiency

Leigh syndrome (LS)/Leigh-like syndrome (LLS) is one of the most common mitochondrial disease subtypes, caused by mutations in either the nuclear or mitochondrial genomes. Here, we identified a novel intronic mutation (c.82-2 A > G) and a novel exonic insertion mutation (c.290dupT) in TMEM126B from a Chinese patient with clinical manifestations of LLS. In silico predictions, minigene splicing assays and patients’ RNA analyses determined that the c.82-2 A > G mutation resulted in complete exon 2 skipping, and the c.290dupT mutation provoked partial and complete exon 3 skipping, leading to translational frameshifts and premature termination. Functional analysis revealed the impaired mitochondrial function in patient-derived lymphocytes due to severe complex I content and assembly defect. Altogether, this is the first report of LLS in a patient carrying mutations in TMEM126B. Our data uncovers the functional effect and the molecular mechanism of the pathogenic variants c.82-2 A > G and c.290dupT, which expands the gene mutation spectrum of LLS and clinical spectrum caused by TMEM126B mutations, and thus help to clinical diagnosis of TMEM126B mutation‐related mitochondrial diseases.

Exon skipping caused by splicing mutation in TNNT1 nemaline myopathy.

The TNNT1 gene encoding the slow skeletal muscle TnT has been identified as a causative gene for nemaline myopathy. TNNT1 nemaline myopathy is mainly characterized by neonatal-onset muscle weakness, pectus carinatum and respiratory insufficiency. Herein, we report on a Chinese girl with TNNT1 nemaline myopathy with mild clinical phenotypes without thoracic deformities or decreased respiratory function. Muscle biopsy showed moderate to marked type 1 fiber atrophy and nemaline rods. Next-generation sequencing identified the compound heterozygous c. 587dupA (p. D196Efs*41) and c. 387+5G>A mutations in the TNNT1 gene according to the transcript NM_003283.4. RNA sequencing revealed complete exon 9 skipping caused by the c. 387+5G>A mutation. Through quantitative PCR, we found that both the truncation c. 587dupA (p. D196Efs*41) and the splicing c. 387+5G>A mutations triggered nonsense-mediated mRNA decay (NMD). Western blotting showed the residual amount of the truncated TNNT1 protein by deletion of exon 9, which may ameliorate the disease to some extent.

Prognostic impact of tumour mutational burden in resected stage I and II lung adenocarcinomas from a European Thoracic Oncology Platform Lungscape cohort

BackgroundThe primary objective of this study is to evaluate tumor mutational burden (TMB), its associations with selected clinicopathological and molecular characteristics as well as its clinical significance, in a retrospective cohort of surgically resected stage I-II lung adenocarcinomas, subset of the ETOP Lungscape cohort. MethodsTMB was evaluated on tumor DNA extracted from resected primary lung adenocarcinomas, based on FoundationOne®CDx (F1CDx) genomic profiling, centrally performed at the University Hospital Zurich. The F1CDx test sequences the complete exons of 324 cancer-related genes and detects substitutions, insertions and deletions (indels), copy number alterations and gene rearrangements. In addition, the genomic biomarkers TMB and microsatellite instability (MSI) are analyzed. ResultsIn the Lungscape cohort, TMB was assessed in 78 surgically resected lung adenocarcinomas from two Swiss centers (62 % males, 55 %/45 % stage I/II). Median TMB was 7.6 Muts/Mb, with TMB high (≥10 Muts/Mb) in 40 % of cases (95 %CI:29 %–52 %). The most frequently mutated genes were TP53/KRAS/EGFR/MLL2 detected in 58 %/38 %/33 %/30 % of samples, respectively.TMB was significantly higher among males (TMB high: 50 % vs 23 % in females, p = 0.032), as well as among current/former smokers (TMB high: 44 % vs 8 % in never smokers, p = 0.023). Furthermore, TMB was significantly higher in TP53 mutated than in non-mutated patients (TMB high: 60 % vs 12 %, p < 0.001), while it was higher in EGFR non-mutated patients compared to EGFR mutated (TMB high: 48 % vs 23 %, p = 0.049).At a median follow-up time of 56.1 months (IQR:38.8–72.0), none of the three outcome variables (OS, RFS, TTR) differed significantly by TMB status (all p-values > 5 %). This was also true when adjusting for clinicopathological characteristics. ConclusionsWhile presence of TP53 mutations and absence of EGFR mutations are associated with high TMB, increased TMB had no significant prognostic impact in patients with resected stage I/II lung adenocarcinoma beyond T and N classification, in both unadjusted and adjusted analyses.

Profiling of gene fusion involving targetable genes in Chinese gastric cancer.

BACKGROUNDApproximately half of all new cases of gastric cancer (GC) and related deaths occur in China. More than 80% of patients with GC are diagnosed at an advanced stage, which results in poor prognosis. Although HER2-directed therapy and immune checkpoint inhibitors have been somewhat successful, new drugs are still needed for the treatment of GC. Notably, several gene fusion-targeted drugs have been approved by the United States Food and Drug Administration for solid tumors, including GC, such as larotrectinib for NTRK fusion-positive cancers and zenocutuzumab for NRG1 fusion-positive cancers. However, gene fusions involving targetable genes have not been well characterized in Chinese patients with GC.AIMTo identify the profile of fusions involving targetable genes in Chinese patients with GC using clinical specimens and determine the distribution of patients with gene fusion variants among the molecular subtypes of GC.METHODSWe retrospectively analyzed gene fusion events in tumor tissue samples from 954 Chinese patients with GC. Clinicopathological characteristics were obtained from their medical records. Genetic alterations, such as single nucleotide variants, indels, amplifications, and gene fusions, were identified using a targeted sequencing panel containing 825 genes. Fusions were validated by fluorescence in situ hybridization (FISH) using break-apart probes. The microsatellite instability (MSI) status was evaluated using MSIsensor from the targeted sequencing panel data. Tumor mutational burden (TMB) was calculated using the total number of nonsynonymous mutations divided by the total genomic targeted region. Chi-square analysis was used to determine the enrichment of gene fusions associated with the molecular subtypes of GC.RESULTSWe found that 1.68% (16/954) of patients harbored 20 fusion events involving targetable genes. RARA fusions (n = 5) were the most common, followed by FGFR2, BRAF, MET, FGFR3, RET, ALK, EGFR, NTRK2, and NRG1 fusions. Two of the RARA fusions, EML4-ALK (E6:E20) and EGFR-SEPTIN14 (E7:E10), have been identified in other tumors but not in GC. Surprisingly, 18 gene fusion events were previously not reported in any cancer types. Twelve of the eighteen novel gene fusions included complete exons encoding functional domains of targetable genes, such as the tyrosine kinase domain of receptor tyrosine kinases and the DNA- and ligand-binding domains of RARA. Consistent with the results of detection using the targeted sequencing fusion panel, the results of FISH (fluorescence in situ hybridization) confirmed the rearrangement of FGFR2 and BRAF in tumors from patients 04 and 09, respectively. Genetic analysis indicated that the fusion genes were significantly enriched in patients with ERBB2 amplification (P = 0.02); however, there were no significant differences between fusion-positive and fusion-negative patients in age, sex, MSI status, and TMB.CONCLUSIONWe characterized the landscape of fusions involving targetable genes in a Chinese GC cohort and found that 1.68% of patients with GC harbor potential targetable gene fusions, which were enriched in patients with ERBB2 amplification. Gene fusion detection may provide a potential treatment strategy for patients with GC with disease progression following standard therapy.

Abstract P1-18-05: Early changes in circulating tumor DNA and its effect on clinical outcomes in patients with advanced breast cancer receiving the CDK4/6 inhibitor palbociclib: Genotyping results from POLARIS

Abstract Background: POLARIS is a prospective, real-world study of palbociclib in patients with hormone receptor-positive (HR+)/human epidermal growth factor receptor 2-negative (HER2–) advanced breast cancer (ABC) in the United States and Canada. These analyses evaluated the difference in tumor mutation profiles of patients receiving palbociclib in the first line versus second line or greater and examined the applicability of circulating tumor DNA (ctDNA) monitoring in a real-world setting. Methods: The clinical database cut-off date was March 16, 2021. Patients in the biomarker analysis group provided consent for serial blood sample collections, received ≥1 dose of initial palbociclib combination treatment, and had ≥1 ctDNA measurement available. The Guardant360 platform with somatic single-nucleotide variants in complete or critical exons of 73 genes was used. The association between early changes in ctDNA mutation allele fractions at Cycle 2 Day 1 (C2D1) and disease progression at Week 24 was evaluated using univariate and multivariable logistic regression analysis adjusting for line of therapy. Results: Among patients with HR+/HER2– ABC (N=347), 93.9% (n=326) had ctDNA measured at baseline, of which 85.9% (n=280) had ≥1 ctDNA alteration detected; 66.6% of patients received palbociclib treatment in the first line and 33.4% as second line or greater ABC setting. The frequencies of gene mutations at baseline were generally higher in patients receiving palbociclib as a second-line or greater therapy compared to those who received it as first-line therapy, particularly for ESR1 mutations (30% vs 15%) and FGFR1 mutations (13% vs 9%). With a median (range) follow-up duration of 18.5 (0.1-46.3) months, patients with total ctDNA increase at C2D1 (log ratio change &amp;gt;0) were 3.74 times more likely to have disease progression at Week 24 (odds ratio, 3.74 [95% CI, 1.71-8.17]; P=0.001) compared with those without change or with a decrease in ctDNA at C2D1 (log ratio change ≤0). The observed significant association remained after adjusting for line of therapy in the multivariable regression analysis (odds ratio, 2.62 [95% CI, 1.14-6.04]; P=0.023). Conclusions: Among patients with HR+/HER2- ABC receiving palbociclib, early changes in ctDNA mutations were significantly associated with disease progression. Further studies are needed to confirm these findings and to evaluate the clinical utility of ctDNA-guided “adaptive” early therapeutic interventions to improve outcomes in patients with metastatic breast cancer. Pfizer; NCT03280303 Citation Format: Debu Tripathy, Zhe Zhang, Joanne L. Blum, Meghan S. Karuturi, Steven L. McCune, Bijoy Telivala, Shailendra Lakhanpal, Kamal Patel, Richard C. Frank, Kit Lu, Chetan Deshpande, Yao Wang, Yuan Liu, Aditya Bardia. Early changes in circulating tumor DNA and its effect on clinical outcomes in patients with advanced breast cancer receiving the CDK4/6 inhibitor palbociclib: Genotyping results from POLARIS [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-18-05.

Post-mortem genetic investigation in sudden cardiac death victims: complete exon sequencing of forty genes using next-generation sequencing.

Sudden cardiac death (SCD) in young people is predominantly caused by genetic causes as cardiomyopathies. Hypertrophic cardiomyopathy is the most common genetic cardiovascular disease and is responsible for the major proportion of SCD in the young. The purpose of this study was to identify the genetic variants present in young SCD victims with HCM characteristics. From the Portuguese records of autopsies performed at the National Institute of Legal Medicine and Forensic Sciences, North Delegation, 16 young (16-50 years) SCD victims whose death was suspected to be a manifestation of HCM were selected. Using next-generation sequencing, the coding regions of 40 genes associated with HCM, candidates, or strongly related to HCM-phenocopies were investigated. The victims included in this study were all males, with a mean age of 33.4 ± 11.7 years, left ventricle mean thickness of 21.5 ± 6.28 mm, and the majority of deaths occurred during sleep (36%). A pathogenic or likely pathogenic variant was identified in six out of 16 (37.5%) victims, in the most common HCM genes (MYBPC3 and MYH7). Our results indicate that molecular autopsy of SCD victims contributes to a more precise identification of a cause of death, and this can be used in the prevention of SCD cases through family screening of first relatives who may carry the same pathogenic variant.