Splice Donor Site Mutation Research Articles

Decoding 17-Beta-hydroxysteroid Dehydrogenase 13: A Multifaceted Perspective on Its Role in Hepatic Steatosis and Associated Disorders.

Chronic liver disease (CLD) represents a significant global health burden, with hepatic steatosis-associated disorders-such as metabolic dysfunction-associated steatohepatitis (MASH), alcoholic liver disease, and hepatitis C virus infection-being major contributors. Recent genome-wide association studies have identified the rs72613567:TA variant in the 17-beta-hydroxysteroid dehydrogenase 13 (HSD17B13) gene as a protective factor against the development and progression of these conditions. In this review, we summarized the current evidence surrounding the HSD17B13 rs72613567 variant, aiming to elucidate its impact on CLD risk and outcomes, and to explore the potential mechanisms behind its hepatoprotective effects. The rs72613567:TA variant induces a splice donor site mutation, resulting in a truncated, non-functional HSD17B13 protein. Numerous studies have demonstrated that this loss-of-function mutation confers protection against the development of cirrhosis and hepatocellular carcinoma (HCC) in patients with MASH, alcoholic liver disease, and hepatitis C virus infection. Moreover, the rs72613567:TA variant has been associated with reduced liver enzyme levels and improved survival in HCC patients. Integrating this variant into genetic risk scores has shown promise in predicting the progression of fatty liver disease to cirrhosis and HCC. Furthermore, inhibiting HSD17B13 expression through RNA interference and small molecule inhibitors has emerged as a potential therapeutic strategy for MASH. However, the precise molecular mechanisms underlying the hepatoprotective effects of the HSD17B13 rs72613567 variant remain to be fully elucidated. Future research should focus on clarifying the structure-function relationship of HSD17B13 and its role in liver pathophysiology to facilitate the development of targeted therapies for CLD associated with hepatic steatosis.

Three HPS5 mutations associated with depigmentation in diverse horse breeds

Depigmentation is a common phenotype in Equus caballus (horse); over 50 identified variants cause white spotting on the skin and coat. HPS5 may influence melanocyte proliferation and mutations in this gene cause depigmentation in humans, mice, and zebrafish. Here we report three equine HPS5 mutations, found in most major breed groups and associated with white spotting. We used a standardized method to quantitatively grade depigmentation, creating a White Score for each horse. We identified associations to each of three variants in HPS5 by comparing the average white score for carriers to that of 41 control horses possessing none of the studied variants. We estimated the functional impact of each mutation using SIFT, GenScan, I-TASSER and cross-species conservation. The first mutation, S1013C termed Eden White 1 (EDXW1), is a missense mutation found to increase average depigmentation by 5.56 points (p = 6.47E-6, n = 70, t-test). Eden White 2 (EDXW2), a missense mutation L560F, increased depigmentation by 9.5 points for individuals with one copy of the variant, but we were unable to phenotype homozygotes (p = 5.55E-16, n = 47, t-test). Eden White 3 (EDXW3), a putative donor splice site mutation at exon 4 + 4, increased white by 5.59 points on average (p = 9.56E-6, n = 101). EDXW1 and EDXW3 are epistatically influenced by MC1R, the gene that controls black vs red coat color in horses. Individuals possessing the dominant E allele produce black pigment, and this allele was associated with higher levels of depigmentation than individuals with the recessive chestnut genotype (e/e) (p = 1.50E-5, p = 1.26E-5 respectively). We tested for epistatic interactions between EDXW1 and Dominant White 20 and found that horses with one copy of each allele had increased average white spotting by 8 points with respect to EDXW1/n or W20/n horses. Compound heterozygotes were significantly more white than horses with one copy of either mutation (p = 7.33E-5), but interactions between other white spotting alleles and EDXW alleles remain to be tested. We propose the convention Eden White (EDXW) for HPS5 mutations in the domestic horse.

A novel pathogenic splicing mutation of RPGR in a Chinese family with X-linked retinitis pigmentosa verified by minigene splicing assay.

To report a novel splicing mutation in the RPGR gene (encoding retinitis pigmentosa GTPase regulator) in a three-generation Chinese family with X-linked retinitis pigmentosa (XLRP). Comprehensive ophthalmic examinations including best corrected visual acuity, fundus photography, vision field, and pattern-visual evoked potential were performed to identify the disease phenotype of a six-year-old boy from the family (proband). Genomic DNA was extracted from peripheral blood of five available members of the pedigree. Whole-exome sequencing (WES), Sanger sequencing, and pSPL3-based exon trapping were used to investigate the aberrant splicing of RPGR. Human Splice Finder v3.1 and NNSPLICE v0.9 were used for in silico prediction of splice site variants. The proband was diagnosed as having retinitis pigmentosa (RP). He had severe symptoms with early onset. A novel splicing mutation, c.619+1G>C in RPGR was identified in the proband by WES and in four family members by Sanger sequencing. Minigene splicing assays verified that c.619+1G>C in RPGR would result in the formation of a damaging alternative transcript in which the last 91 bp of exon 6 were skipped, leading to the subsequent deletion of 623 correct amino acids (c.529_619del p.Val177Glnfs*16). We identify a novel splice donor site mutation causing aberrant splicing of RPGR. Our findings add to the catalog of pathological mutations of RPGR and further emphasize the functional importance of RPGR in RP pathogenesis and its complex clinical phenotypes.

Partial penetrance and phenotypic variability of aplasia of lacrimal and salivary glands caused by a novel FGF10 donor splice-site mutation.

Thirteen affected individuals of six generations of a single kindred presented with epiphora evident from infancy. Physical exam and Schirmer test revealed variable expression of tear deficiency, congenital punctal atresia, and dry mouth with multiple caries, without concomitant abnormalities of the ears or digits, commensurate with a diagnosis of aplasia of the lacrimal and salivary glands (ALSG). Reconstruction of the upper lacrimal drainage system was performed in some of the affected individuals. Genetic analysis, testing six affected individuals and three non-affected family members, identified a single novel heterozygous splice-site variant, c.429 + 1, G > T in fibroblast growth factor 10 (FGF10) (NM_004465.1), segregating throughout the family as expected for dominant heredity. RT-PCR assays of HEK-293 cells transfected with wild type or mutant FGF10 demonstrated that the variant causes skipping of Exon 2. Notably, individuals sharing the same variant exhibited phenotypic variability, with unilateral or bilateral epiphora, as well as variable expression of dry mouth and caries. Moreover, one of the variant carriers had no ALSG-related clinical findings, demonstrating incomplete penetrance. While coding mutations in FGF10 are known to cause malformations in the nasolacrimal system, this is the second FGF10 splice-site variant and the first donor-site variant reported to cause ALSG. Thus, our study of a unique large kindred with multiple affected individuals heterozygous for the same FGF10 variant highlights intronic splice-site mutations and phenotypic variability/partial penetrance in ALSG.

A splice site mutation in the FvePHP gene is associated with leaf development and flowering time in woodland strawberry.

Leaves and flowers are crucial for the growth and development of higher plants. In this study we identified a mutant with narrow leaflets and early flowering (nlef) in an ethyl methanesulfonate-mutagenized population of woodland strawberry (Fragaria vesca) and aimed to identify the candidate gene. Genetic analysis revealed that a single recessive gene, nlef, controlled the mutant phenotype. We found that FvH4_1g25470, which encodes a putative DNA polymerase α with a polymerase and histidinol phosphatase domain (PHP), might be the candidate gene, using bulked segregant analysis with whole-genome sequencing, molecular markers, and cloning analyses. A splice donor site mutation (C to T) at the 5' end of the second intron led to an erroneous splice event that reduced the expression level of the full-length transcript of FvePHP in mutant plants. FvePHP was localized in the nucleus and was highly expressed in leaves. Silencing of FvePHP using the virus-induced gene silencing method resulted in partial developmental defects in strawberry leaves. Overexpression of the FvePHP gene can largely restore the mutant phenotype. The expression levels of FveSEP1, FveSEP3, FveAP1, FveFUL, and FveFT were higher in the mutants than those in 'Yellow Wonder' plants, probably contributing to the early flowering phenotype in mutant plants. Our results indicate that mutation in FvePHP is associated with multiple developmental pathways. These results aid in understanding the role of DNA polymerase in strawberry development.

Loss-of-function mutations in SGCE found in Japanese patients with myoclonus-dystonia.

SGCE myoclonus-dystonia is a monogenic form of dystonia with an autosomal dominant mode of inheritance that co-occurs with a myoclonic jerk. In this study, we present 12 Japanese patients from nine families with this disease. Targeted next-generation sequencing covering major causative genes for monogenic dystonias identified nine distinct SGCE mutations from each of the families: three nonsense, two frameshift, two missense, one in-frame 15 bp deletion, and one splice donor site mutations, of which four were previously unreported. One missense mutation (c.662G>T, p.Gly221Val) was located at the 3' end of exon 5 (NM_001099400), which was predicted to cause aberrant splicing according to in silico predictions. Minigene assays performed together with the c.825+1G>C mutation demonstrated complete skipping of exon 5 and 6, respectively, in their transcripts. The other missense (c.1345A>G, p.Met449Val) and 15 bp deletion (c.168_182del, p.Phe58_Leu62del) mutations showed a significant reduction in cell membrane expression via HiBiT bioluminescence assay. Therefore, we concluded that all the detected mutations were disease-causing. Unlike the other detected mutations, p.Met449Val affects only isoform 3 (NP_001092870 encoded by NM_001099400) among the variously known isoforms of SGCE. This isoform is brain-specific and is mostly expressed in the cerebellum, which supports recent studies showing that cerebellar dysfunction is a key element in the pathophysiology of SGCE myoclonus-dystonia.

Invasive lobular carcinoma with extracellular mucin (ILCEM): clinicopathologic and molecular characterization of a rare entity

AbstractInvasive lobular carcinoma with extracellular mucin (ILCEM) is a rare histologic subtype of breast cancer. Little is known about the pathologic or genomic signatures that distinguish ILCEM from classic invasive lobular carcinoma (ILC) or mucinous carcinoma. We studied 17 breast cancers with lobular morphology and extracellular mucin. Thirteen tumors with sufficient tissue for DNA extraction were analyzed by a next generation sequencing (NGS) assay that interrogates 447 genes for mutations and copy number variations (CNVs). Median patient age was 66 yrs (range: 31–77 yrs). Sixteen patients presented with masses, 7 of which were >2 cm. Seven patients had lymph node metastases. The cases of ILCEM were moderately (n = 13) or poorly differentiated (n = 4), frequently exhibiting variant morphology that has not been previously described or emphasized, including grade 3 nuclei (n = 11), diffuse signet ring cells (n = 10), solid growth (n = 4), tumor necrosis (n = 3) or apocrine features (n = 2). All tumors showed absent or reduced membranous E-cadherin expression. Concurrent lobular carcinoma in situ (LCIS) was seen in 11/17 cases, 1 of which was a striking example of signet ring cell LCIS with extracellular mucin. Receptor profiles were ER+/HER2− (n = 15) and ER+/HER2+ (n = 2). With a median follow-up of 83.5 months (range: 3–171 months) in 12 patients with available information, 8 patients had recurrences resulting in 4 cancer-related deaths. The most common CNVs were 16q loss (n = 11) and 1q gain (n = 9). CDH1 gene-level alterations were detected in all but one case, including frameshift (n = 7), nonsense (n = 2), and donor splice site (n = 1) mutations and indels (n = 2). Recurrent mutations were also seen in PIK3CA (n = 3), POLQ (n = 3), TP53 (n = 3), ERBB3 (n = 3), ERBB2 (n = 2), and RUNX1 (n = 2). Genes with recurrent amplifications included GATA3 (n = 4), FOXA1 (n = 3), CCND1 (n = 2). Our data highlights ILCEM as a distinct variant of ILC that often presents with higher-grade and variant morphologic features and is associated with an aggressive clinical course. NGS data support an overall lobular-type molecular profile and reveal potentially targetable alterations in a subset of cases with recurrence.

A novel germline mutation of the <i>PALB</i> gene in a young Yakut breast cancer woman

Background. Breast cancer (BC) is the most common female malignancy worldwide. partner and localizer of BRCA2 gene (PALB2) is directly involved in DNA damage response. germline mutation in PALB2 has been identified in breast cancer and familial pancreatic cancer cases, accounting for approximately 1–2% and 3–4%, respectively. the goal of this report was to describe new PALB2 mutation in a young Yakut breast cancer patient with family history of cancer. Material and methods. Genomic DNA were isolated from blood samples and used to prepare libraries using a capture-based target enrichment kit, Hereditary Cancer Solution™ (SOPHIA GENETICS, Switzerland), covering 27 genes (ATM, APC, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, FAM175A, MLH1, MRE11A, MSH2, MSH6, MUTYH, NBN, PALB2, PIK3CA, PMS2, PMS2CL, PTEN, RAD50, RAD51C, RAD51D, STK11, TP53 and XRCC2). paired-end sequencing (2 × 150 bp) was conducted using NextSeq 500 system (Illumina, USA). Results. Here we describe a case of a never-before-reported mutation in the PALB gene that led to the early onset breast cancer. We report the case of a 39-year-old breast cancer Yakut woman with a family history of pancreatic cancer. Bioinformatics analysis of the NGS data revealed the presence of the new PALB2 gene germinal frameshift deletion (NM_024675:exon1:c.47dela:p.K16fs). in accordance with dbPubMed ClinVar, new mutation is located in codon of the PALB2 gene, where the likely pathogenic donor splice site mutation (NM_024675.3:c.48+1delG) associated with hereditary cancer-predisposing syndrome has been earlier described. Conclusion. We found a new never-before-reported mutation in PALB2 gene, which probably associated with early onset breast cancer in Yakut indigenous women with a family history of pancreatic cancer.

Adult T-cell leukemia-lymphoma acquires resistance to DNA demethylating agents through dysregulation of enzymes involved in pyrimidine metabolism.

Adult T‐cell leukemia‐lymphoma (ATL) is an aggressive neoplasm derived from T‐cells transformed by human T‐cell lymphotropic virus‐1 (HTLV‐1). Recently, we reported that regional DNA hypermethylation in HTLV‐1‐infected T‐cells reflects the disease status of ATL and the anti‐ATL effects of DNA demethylating agents, including azacitidine (AZA), decitabine (DAC) and a new DAC prodrug, OR‐2100 (OR21), which we developed. Here, to better understand the mechanisms underlying drug resistance, we generated AZA‐, DAC‐ and OR21‐resistant (AZA‐R, DAC‐R and OR21‐R, respectively) cells from the ATL cell line TL‐Om1 and the HTLV‐1‐infected cell line MT‐2 via long‐term drug exposure. The efficacy of OR21 was almost the same as that of DAC, indicating that the pharmacodynamics of OR21 were due to release of DAC from OR21. Resistant cells did not show cellular responses observed in parental cells induced by treatment with drugs, including growth suppression, depletion of DNA methyltransferase DNMT1 and DNA hypomethylation. We also found that reduced expression of deoxycytidine kinase (DCK) correlated with lower susceptibility to DAC/OR21 and that reduced expression of uridine cytidine kinase2 (UCK2) correlated with reduced susceptibility to AZA. DCK and UCK2 catalyze phosphorylation of DAC and AZA, respectively; reconstitution of expression reversed the resistant phenotypes. A large homozygous deletion in DCK and a homozygous splice donor site mutation in UCK2 were identified in DAC‐R TL‐Om1 and AZA‐R TL‐Om1, respectively. Both genomic mutations might lead to loss of protein expression. Thus, inactivation of UCK2 and DCK might be a putative cause of phenotypes that are resistant to AZA and DAC/OR21, respectively.

Modeling Relapsed, Refractory Acute Lymphoblastic Leukemia from a Child with Neurofibromatosis

Neurofibromatosis type 1 (NF-1) is an autosomal dominant disorder affecting approximately 1:3000 individuals globally. While approximately 50% are familial, with over 3000 causative germline variants in the neurofibromatosis (NF1) gene identified, the remainder occur sporadically. These mutations lead to haploinsufficiency of NF1 and neurofibromin, a tumor suppressor and important negative regulator of RAS signaling. Children with NF-1 have a higher risk of developing juvenile myelomonocytic leukemia and acute myeloid leukemia, but rarely develop acute lymphoblastic leukemia (ALL).A 9-year-old male presented in 2015 with persistent migratory subcutaneous swellings and multiple bony aches with lytic lesions on bone imaging. He had a high white cell count with eosinophilia (WCC 43.4 x 10 9/L, eosinophils 23.87 x 10 9/L) with no circulating blasts, 10% marrow blasts (CD10+/CD19+/CD34+) and was CNS negative. Although previously undiagnosed, NF-1 was clinically suspected due to typical skin changes.He was diagnosed with iAMP21 ALL and NF-1 was confirmed with the identification of a germline NF1 donor splice site mutation (c.1845G>A:p.L615=). Bone marrow cells were sorted by flow cytometry on CD19 positivity and underwent transcriptomic sequencing. This revealed a P2RY8-CRLF2 gene fusion, with no other clinically relevant variants, while a custom Taqman low density array indicated high-risk B-ALL subtype Ph-like ALL. Multiplex ligation-dependent probe amplification (MLPA) confirmed iAMP21 and also identified IKZF1 exon 2-3 and BTG1 deletions.Treatment followed the high-risk B-ALL arm of the AEIOP-BFM ALL2009 protocol due to persistent end-consolidation MRD in addition to iAMP21 and the Ph-like phenotype. He relapsed three years later off treatment and was refractory to both salvage chemotherapy and blinatumomab. The iAMP21, P2RY8-CRLF2 gene fusion, IKZF1 exon 2-3 and BTG1 deletions remained detectable. Whole exome sequencing of CD19 positive samples from diagnosis, relapse and mesenchymal stem cells (germline control) was performed, identifying a NF1 c.7400dupT:p.L2467 frameshift (fs) mutation only at relapse.To understand the implications of NF1 p.L2467fs, the P2RY8-CRLF2 gene fusion was first transduced into the interleukin 3 (IL3) dependent murine pro-B cell line Ba/F3. P2RY8-CRLF2 alone is not transforming and is thought to be a secondary event in iAMP21 ALL, providing an ideal model to study the cumulative effect of the NF1fs. The NF1fs was then introduced to the P2RY8-CRLF2 cells by CRISPR/Cas9. A proliferation assay was performed without IL3 and demonstrated the P2RY8-CRLF2+NF1fs cell line was IL3 independent, indicative of leukemic transformation, whereas all other lines were not (vs Ba/F3, p = 0.001).Neurofibromin can be constitutively phosphorylated at the c-terminus, negatively regulating NF1-GAP activity, suppressing RAS signaling and inducing cell cycle arrest. Therefore, to demonstrate loss of function due to the c-terminus NF1 p.L2467fs and increased RAS signaling, western blotting for pERK was performed. Significant upregulation of pERK was confirmed in P2RY8-CRLF2+NF1fs in comparison to Ba/F3 control cells (p=0.007) (Figure 1).The MEK inhibitors trametinib and mirdametinib are in clinical trials for NF-1 patients and have shown efficacy in ALL models with RAS mutations. In a 3-day cell death assay, only P2RY8-CRLF2+NF1fs demonstrated sensitivity to trametinib (LD 50 P2RY8-CRLF2 = >6.4 µM, NF1fs = >6.4 µM, P2RY8-CRLF2+NF1fs =1.7µM; p < 0.001) and mirdametinib (LD 50 P2RY8-CRLF2 = >16 µM, NF1fs = >16 µM, P2RY8-CRLF2+NF1fs = 8.3 µM; p < 0.0001) (Figure 2).Here, we have demonstrated a LOF NF1fs mutation using an in-vitro model of ALL. Germline NF1 haploinsufficiency and a second hit NF1 mutation in ALL is limited to one report of monozygotic twins with neurofibromatosis. We propose that NF1 p.L2467fs caused bi-allelic LOF and therefore contributed to relapse in this patient. An understanding of the genomic complexities that lead to relapse may also inform personalized treatment strategies. While this patient subsequently achieved remission with inotuzomab and underwent successful stem cell transplantation, the sensitivity to MEK inhibitors is an exciting development for neurofibromatosis patients with ALL. [Display omitted] DisclosuresYeung: Amgen: Honoraria; BMS: Honoraria, Research Funding; Pfizer: Honoraria; Novartis: Honoraria, Research Funding. White: BMS: Honoraria, Research Funding; Novartis: Research Funding.

DMD – CLINICAL CARE: EP.98 Clinical and molecular characterisation of unusual DMD mutations

The dystrophinopathies encompass a spectrum of clinical phenotypes associated with mutations in the DMD gene. The clinical impact of most mutations can be predicted by the reading frame rule, but uncommon mutations continue to shed light on pathophysiological mechanisms of the disease and inform therapeutic development. We report here clinical, histological, and molecular findings in 5 such patients, including muscle histology, quantitative immunofluorescence (IF) analysis, western blot (WB), and RNA-Seq. Expression of dystrophin of increased molecular weight (536 kDa) was detected in an entirely asymptomatic 16 y.o. boy who was incidentally found to carry an in-frame duplication of exons 10-27. Serum CK, muscle histology, and dystrophin expression by IF were essentially normal. Such complete absence of clinical or laboratory pathology is highly unusual in comparison to other in-frame duplications. An out-of-frame deletion of exon 2 was detected during evaluation for global developmental delay in a 4 y.o. with no observable muscle weakness; consistent with utilization of the downstream (exon 5) internal ribosome entry site (IRES), dystrophin expression was only mildly reduced. In contrast, a boy with exon 3 nonsense mutation (c.103C>T) had a severe phenotype and absent dystrophin expression, indicating lack of IRES-induced translation. An intermediate phenotype was observed in a boy with an exon 3 splice donor site mutation (c.186+1G>A), who showed reduced but not absent dystrophin expression. A final patient had a canonical splice donor site mutation in the last nucleotide of exon 45 (c.6614G>T) that did not result in cryptic splice site activation or exon 45 exclusion, as is typical for splice site mutations. WB showed abundant expression of a 256 kDa dystrophin, undetectable with C-terminal antibodies. RNA-Seq revealed instead the formation of a novel terminal exon, encompassing exon 45 and a portion of intron 45 comprising a pseudo-3’UTR region, a previously unreported molecular mechanism.

Abstract 2057: BRCA1 D11q isoform expression impacts PARP inhibitor responses in high grade serous ovarian carcinoma patient derived xenografts

Abstract Background: Acquired PARP inhibitor (PARPi) resistance in High Grade Serous Ovarian Carcinoma (HGSOC) commonly occurs via restored homologous recombination DNA repair due to secondary mutations in BRCA1/2. However, overexpression of certain BRCA1 splice isoforms can also contribute to PARPi resistance in HGSOC. This includes the D11q isoform of BRCA1, where deleterious mutations in the 11q region of BRCA1 (exon 10) can be spliced out resulting in a truncated but partially functional BRCA1 protein. Here we describe four HGSOC Patient Derived Xenografts (PDX) with primary BRCA1 11q mutations, where D11q isoform expression has been shown to correlate with platinum and PARPi responses in vivo. Methods: PDX were derived from chemo-naive and pre-treated patients (Table 1). PDX were treated with PARPi rucaparib (300mg/kg) and cisplatin (4mg/kg). D11q isoform expression was assessed using two-step RT-qPCR. DNA sequencing was performed using the BROCA Cancer Risk Panel. Results: PARPi resistant PDX #1032 and #1049 had high D11q expression relative to D11q-high cell line UWB1.289 and the PARPi-responsive models (Table 1). Interestingly, PDX #1049 harboured a second BRCA1 exon 11 donor splice site mutation, shown previously to enhance splicing and increase abundance of the D11q isoform. Indeed, other proximal splice site mutations have been found following relapse in patients with BRCA1 11q mutations post-PARPi. Conclusions: Our work in PDX provides additional functional evidence that BRCA1 D11q expression impacts on PARPi and platinum responses, and is a clinically relevant mechanism of PARPi resistance in a subset of patients with BRCA1 mutations. Whilst high D11q expression can be associated with secondary BRCA1 splice site mutations (PDX #1049), it can also occur in their absence (PDX #1032). Thus, BRCA1 D11q isoform expression should be considered a potential mechanism of PARP-resistance in patients with BRCA1 11q mutations. Table 1.Summary of results.PDX modelPrimary BRCA1 mutationBRCA1 D11q isoform expressionSecondary BRCA1 mutationsOther mutations (BROCA)PDX platinum responsePDX PARPi responsePatient treatments prior to PDX generation#1049Germline BRCA1: c.2475delCVery highBRCA1: c.4096+3A&amp;gt;C (33%) - splice site mutation known to cause increased D11q expressionTP53: c.428_429insCAStable diseaseNon-responsive - progressive disease3 lines of platinum chemotherapy and PARPi#1032Somatic BRCA1: c.1251delTHighNone detectedTP53: c.97-1_97-6del; FANCA: c.3788_3790delResistant - progressive diseaseNon-responsive - progressive disease3 lines of platinum chemotherapy and PARPi#56Germline BRCA1: c.894_895delTGModerateNone detectedNone detectedSensitive - complete responseMixed responsesNo prior treatment#206Germline BRCA1: c.3817C&amp;gt;TLowNone in PDX, one found in post-PARPi patient biopsy: BRCA1:c.3746_4081delTP53:c.451C&amp;gt;T; BLM rearrangementSensitive - complete responseResponsive - complete response1 line of platinum chemotherapy Citation Format: Ksenija Nesic, Cassandra J. Vandenberg, Olga Kondrashova, John J. Krais, Neil Johnson, Elizabeth M. Swisher, Matthew J. Wakefield, Clare L. Scott. BRCA1 D11q isoform expression impacts PARP inhibitor responses in high grade serous ovarian carcinoma patient derived xenografts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2057.

Long-term efficacy of immune checkpoint inhibitors in non-small cell lung cancer patients harboring MET exon 14 skipping mutations.

MET exon 14 skipping mutation, observed in 3-4% of non-small cell lung cancer (NSCLC), is emerging as a targetable alteration. In recent years, immune checkpoint inhibitors (ICI) have been effective in treating several NSCLCs. Our research aimed to investigate the characteristics of patients with NSCLCs harboring MET exon 14 mutations and their response to ICI in Japan. Among the 1954 consecutive NSCLCs diagnosed at Saitama Cancer Center between 2010 and 2019, MET exon 14 skipping mutations were detected in 68 (3.5%) NSCLCs. We evaluated their characteristics such as programmed cell death ligand 1 (PD-L1) expression. Median age of patients with NSCLCs harboring MET exon 14 skipping mutations was 73years. PD-L1 was highly expressed in 17 (70.8%) of the 24 patients examined. Seven patients received ICI monotherapy, and three out of seven had a remarkable treatment response, resulted in objective response rate (ORR) of 42.9% and progression-free survival of 24.7months. Three patients with donor splice-site mutations showed a long-term treatment response, despite the fact that two with acceptor splice-site mutations demonstrated no response and experienced early disease progression with ICI monotherapy. Our results indicated that patients with NSCLCs harboring MET exon 14 mutations presented with a high rate of positive PD-L1 expression. ICI treatment showed a high ORR and long-term efficacy for NSCLCs harboring MET exon 14 mutations. Variants of MET exon 14 splice-site mutations may be associated with ICI response.

Immune-complex glomerulonephritis with a membranoproliferative pattern in Frasier syndrome: a case report and review of the literature

BackgroundMutations in the Wilms tumor 1 gene cause a spectrum of podocytopathy ranging from diffuse mesangial sclerosis to focal segmental glomerulosclerosis. In a considerable fraction of patients with Wilms tumor 1 mutations, the distinctive histology of immune-complex-type glomerulonephritis has been reported. However, the clinical relevance and etiologic mechanisms remain unknown.Case presentationA 5-year-old child presented with steroid-resistant nephrotic range proteinuria. Initial renal biopsy revealed predominant diffuse mesangial proliferation with a double-contour and coexisting milder changes of focal segmental glomerulosclerosis. Immunofluorescence and electron microscopy revealed a full-house-pattern deposition of immune complexes in the subendothelial and paramesangial areas. Serial biopsies at 6 and 8 years of age revealed that more remarkable changes of focal segmental glomerulosclerosis had developed on top of the initial proliferative glomerulonephritis. Identification of a de novo Wilms tumor 1 splice donor-site mutation in intron 9 (NM_024426.6:c.1447 + 4C > T) and 46,XY-gonadal dysgenesis led to the diagnosis of Frasier syndrome.ConclusionsOur findings, together with those of others, point to the importance of heterogeneity in clinicopathological phenotypes caused by Wilms tumor 1 mutations and suggest that immune-complex-mediated membranoproliferative glomerulopathy should be considered as a histological variant.

Compound heterozygous RPE65 mutations associated with an early onset autosomal recessive retinitis pigmentosa.

Retinitis pigmentosa (RP) is one of the most common form of inherited retinal dystrophies. Identification of disease-causing mutations is a prerequisite for applying targeted therapeutic approaches. The present study aimed to identify disease-associated mutations in a large Serbian family, in which two brothers have suffered from RP starting in the first decade of their lives. The index patient and 12 additional members of a four-generation family were analyzed. All participants underwent detailed ophthalmic examinations. Genomic DNA was isolated from family members to perform whole exome sequencing (WES) and Sanger sequencing of candidate genes. An early onset RP phenotype was presented in both ocular fundi of the index patient and his brother: arteriolar attenuation, as well as retinal pigmentary changes in peripheral fundus and waxy disc pallor. Both brothers showed foveal thinning. The index patient showed epiretinal membranes in both eyes and a parafoveal cystic lesion in his right eye, whereas the brother of the index patient showed choroid folds and vitreomacular adhesion in his left eye. We identified compound heterozygous mutations in the RPE65 gene (a novel c.1338+1G>A splice donor site mutation in addition to the frame-shifting mutation c.1207_1210dup (p.Glu404Alafs*4)) using an in-house WES pipeline. Evaluation of all previously described RPE65 mutations showed that the sequence variants identified in the present study located to rarely altered exons and likely effect a highly conserved region of the RPE65 protein. Gene augmentation therapies might be a promising treatment option for the patients described.

Impact of RB1 gene mutation type in retinoblastoma patients on clinical presentation and management outcome

Objective/backgroundRetinoblastoma (RB), the most common intraocular malignancy in children, is caused by biallelic inactivation of the human retinoblastoma susceptibility gene (RB1). We are evaluating the impact of the type of RB1 gene mutation on clinical presentation and management outcome. MethodsA retrospective case series of 50 patients with RB. Main outcomes were clinical and pathologic features and types of RB1 gene mutations detected using quantitative multiplex polymerase chain reaction (PCR), allele-specific PCR, next-generation sequencing analysis, and Sanger sequencing. ResultsTwenty (40%) patients had unilateral RB and 30 (60%) had bilateral RB. Overall, 36 (72%) patients had germline disease, 17 (47%) of whom inherited the disease. Of these 17 inherited cases, paternal origin of the RB1 mutation was seen in 15 (88%). The overall eye salvage rate was 74% (n = 49/66; 100% for Groups A + B + C, and 79% for Group D eyes). The most frequent type of mutation was a nonsense mutation generating a stop codon (15/36, 42%). Other mutations that result in a premature stop codon due to deletions or insertions with donor splice site or receptor splice site mutations were detected in 7/36 (19%), 10/36 (28%), and 2/26 (6%) patients, respectively. The remaining two (6%) patients had frameshift mutation. Patients with deletion, acceptor splice site, and frameshift mutations presented with more advanced ICRB (International Classification of Retinoblastoma) stage (75% diagnosed with Group D or E), even though there was no significant difference in eye salvage rate or tumor invasiveness between patients with different types of mutations. ConclusionDespite the heterogeneous nature of RB1 gene mutations, tumor stage remains the most important predictive factor for clinical presentation and outcome. Furthermore, acceptor splice site and frameshift mutations are associated with more advanced tumor stage at diagnosis.

Identification and functional characterization of mutations in LPL gene causing severe hypertriglyceridaemia and acute pancreatitis

Hypertriglyceridaemia is a very rare disorder caused by the mutations of LPL gene, with an autosomal recessive mode of inheritance. Here, we identified two unrelated Chinese patients manifested with severe hypertriglyceridaemia and acute pancreatitis. The clinical symptoms of proband 1 are more severe than proband 2. Whole exome sequencing and Sanger sequencing were performed. Functional analysis of the identified mutations has been done. Whole exome sequencing identified two pairs of variants in LPL gene in the proband 1 (c.162C>A and c.1322+1G>A) and proband 2 (c.835C>G and c.1322+1G>A). The substitution (c.162C>A) leads to the formation of a truncated (p.Cys54*) LPL protein. The substitution (c.835C>G) leads to the replacement of leucine to valine (p.Leu279Val). The splice donor site mutation (c.1322+1G>A) leads to the formation of alternative transcripts with the loss of 134 bp in exon 8 of the LPL gene. The proband 1 and his younger son also harbouring a heterozygous variant (c.553G>T; p.Gly185Cys) in APOA5 gene. The relative expression level of the mutated LPL mRNA (c.162C>A, c.835C>G and c.1322+1G>A) showed significant differences compared to wild‐type LPL mRNA, suggesting that all these three mutations affect the transcription of LPL mRNA. These three mutations (c.162C>A, c.835C>G and c.1322+1G>A) showed noticeably decreased LPL activity in cell culture medium but not in cell lysates. Here, we identified three mutations in LPL gene which causes severe hypertriglyceridaemia with acute pancreatitis in Chinese patients. We also described the significance of whole exome sequencing for identifying the candidate gene and disease‐causing mutation in patients with severe hypertriglyceridaemia and acute pancreatitis.

Homozygous mutations in REC114 cause female infertility characterised by multiple pronuclei formation and early embryonic arrest

BackgroundAbnormal pronuclear formation during fertilisation and subsequent early embryonic arrest results in female infertility. In recent years, with the prevalence of assisted reproductive technology, a few genes have been identified...

Recurrent NUS1 canonical splice donor site mutation in two unrelated individuals with epilepsy, myoclonus, ataxia and scoliosis - a case report

BackgroundWe encountered two unrelated individuals suffering from neurological disorders, including epilepsy and scoliosis.Case presentationWhole-exome sequencing identified the same recurrent, de novo, pathogenic variant in NUS1 [NM_138459.4:c.691 + 1C > A] in both individuals. This variant is located in the conserved cis-prenyltransferase domain of the nuclear undecaprenyl pyrophosphate synthase 1 gene (NUS1), which encodes the Nogo-B receptor, an essential catalyst for protein glycosylation. This variant was confirmed to create a new splice donor site, resulting in aberrant RNA splicing resulting in a 91-bp deletion in exon 3 in both individuals. The mutant mRNA was partially degraded by nonsense mediated mRNA decay. To date, only four de novo variants and one homozygous variant have been reported in NUS1, which cause developmental and epileptic encephalopathy, early onset Parkinson’s disease, and a congenital disorder of glycosylation. Seven patients, including our two patients, have presented with epileptic seizures and intellectual disabilities.ConclusionsOur study strongly supports the finding that this recurrent, de novo, variant in NUS1 causes developmental and epileptic encephalopathy with involuntary movement, ataxia and scoliosis.

Combining Engineered U1 snRNA and Antisense Oligonucleotides to Improve the Treatment of a BBS1 Splice Site Mutation

Manipulation of pre-mRNA processing is a promising approach toward overcoming disease-causing mutations and treating human diseases. We show that a combined treatment applying two splice-manipulating technologies improves therapeutic efficacies to correct mutation-induced splice defects. Previously, we identified a family affected by retinitis pigmentosa caused by the homozygous BBS1 splice donor site mutation c.479G > A. The mutation leads to both exon 5 skipping and intron 5 retention. We developed a therapeutic approach applying lentivirus-mediated gene delivery of engineered U1 small nuclear RNA (U1), which resulted in increased levels of correctly spliced BBS1. Herein, we show that the therapeutic effect of the engineered U1 efficiently reverted exon skipping but failed to reduce the intron retention. To complement the engineered U1 treatment, we identified four different antisense oligonucleotides (AONs) that block intron 5 retention in BBS1 transcripts. A treatment using engineered U1 in combination with AONs showed the highest therapeutic efficacy and increased the amount of correctly spliced BBS1 transcripts. We did not detect elevated levels of apoptotic cell death in AON-treated cell lines. In conclusion, engineered U1 or AONs provide efficient therapies with complementary effects and can be combined to increase efficacy of therapeutic approaches to correct splice defects.