Rare Gene Research Articles

Biomarker Testing in Patients With Unresectable Advanced or Recurrent Non–Small Cell Lung Cancer

Biomarker testing for driver mutations is essential for selecting appropriate non-small cell lung cancer (NSCLC) treatment but is insufficient. To investigate the status of biomarker testing and drug therapy for NSCLC in Japan for identifying problems in treatment. The REVEAL cohort study included retrospective data collection and prospective follow-up from 29 institutions across Japan. Of 1500 patients diagnosed with advanced or recurrent NSCLC between January 1 and March 18, 2021, 1479 were eligible. Cases recognized at the wrong clinical stage (n = 12), diagnosed outside the study period (n = 6), not treated according to eligibility criteria before recurrence (n = 2), and with deficient consent acquisition procedure (n = 1) were excluded. The primary end point was the biomarker testing status. Treatment-related factors were examined. Among the 1479 patients included in the analysis, the median age was 72 (range, 30-95) years; 1013 (68.5%) were men; 1161 (78.5%) had an Eastern Cooperative Oncology Group performance status 0 or 1; 1097 (74.2%) were current or past smokers; and 947 (64.0%) had adenocarcinoma. Biomarker status was confirmed in 1273 patients (86.1%). Multigene testing was performed in 705 cases (47.7%); single-gene testing, in 847 (57.3%); and both, in 279 (18.9%). Biomarker testing was performed for EGFR in 1245 cases (84.2%); ALK, in 1165 (78.8%); ROS1, in 1077 (72.8%); BRAF, in 803 (54.3%); and MET, in 805 (54.4%). Positivity rates among 898 adenocarcinoma cases included 305 (34.0%) for EGFR, 29 (3.2%) for ALK, 19 (2.1%) for ROS1, 11 (1.2%) for BRAF, and 14 (1.6%) for MET. Positivity rates among 375 nonadenocarcinoma cases were 14 (3.7%) for EGFR, 6 (1.6%) for ALK, 1 (0.3%) for ROS1, 3 (0.8%) for BRAF, and 8 (2.1%) for MET. Poor physical status, squamous cell carcinoma, and other comorbidities were associated with hampered multigene testing. Targeted therapy was received as first-line treatment by 263 of 278 cases (94.6%) positive for EGFR, 25 of 32 (78.1%) positive for ALK, 15 of 24 (62.5%) positive for ROS1, 9 of 12 (75.0%) positive for BRAF, and 12 of 19 (63.2%) positive for MET. Median overall survival of patients with positive findings for driver gene alteration and who received targeted therapy was 24.3 (95% CI, not reported) months; with positive findings for driver gene alteration and who did not receive targeted therapy, 15.2 (95% CI, 7.7 to not reported) months; and with negative findings for driver gene alteration, 11.0 (95% CI, 10.0-12.5) months. Multigene testing for nonadenocarcinomas and adenocarcinomas accounted for 705 (47.7%) of all NSCLC cases. These findings suggest that multigene testing has not been sufficiently implemented in Japan and should be considered prospectively, even in nonadenocarcinomas, to avoid missing rare driver gene alterations.

Underestimated indirect and persistent effects of decaying macrophytes on aquatic ecosystem through alteration of bacterial community structure and function

The rapid release of nutrients by aquatic macrophyte decomposition directly results in the degradation of water quality and the deterioration of aquatic ecosystems. However, decaying macrophytes have indirect negative effects, mediated by bacteria in aquatic ecosystems, which may have long been underestimated. In this study, 16S rRNA sequencing was used to investigate the structural succession, possible metabolic function shifts, and specific environmental drivers of the bacterial community during the decomposition of four typical aquatic macrophytes over 188 days. Results indicate that nutrients released by macrophyte residues alter the physiochemical properties of overlaying water and have dual concentration effects of low promotion and high inhibition on α- and β-diversity of bacteria, driving their succession in different directions. High nutrient concentrations not only resulted in more substantial changes in the abundance of dominant bacteria at various taxonomic levels, but also inhibited carbon and nitrogen metabolic functions while promoting the phosphorus and sulfur metabolic functions of the bacterial community, which was in contrast to the effects of low nutrient concentrations. These effects persisted for a long time, potentially leading to a decrease in redundant genes and the loss of rare functional genes. This, in turn, indirectly reducing the structural stability of aquatic ecosystems and their resilience to external disturbances. Our findings indicate that the indirect and persistent negative impacts of macrophyte decomposition on aquatic ecosystems should not be ignored and that macrophyte residues should be harvested and properly treated before they undergoing explosive nutrient release.

CSF ADAM10 levels in AD patients in relationship to ADAM10 gene variants

AbstractBackgroundThe disintegrin metalloproteinase 10 (ADAM10) is the main α‐secretase acting in the non‐amyloidogenic processing of APP. Some ADAM10 gene variants (Q170H, R181G) have been associated with higher susceptibility to develop late‐onset AD and have been shown to promote AD‐type changes in mice models. We tested the hypothesis that some ADAM10 rare variants could increase the risk for AD through conferring an earlier age‐related downregulation of a‐secretase activity.Method ADAM10 gene was sequenced in 103 AD cases (78% with family history) with positive CSF biomarkers, and 96 cognitively preserved nonagenarians. ADAM10 in CSF of the AD cases was analyzed by means of ELISA (total levels) and western blot (segregated by isoforms of 50 kDa, 55 kDa, and 80 kDa) in comparison with controls and non‐AD dementia cases. We examined whether there was an increase of rare gene variants (MAF < 0.01) in the AD group and their potential association with lowers CSF protein levels.ResultRare variants were found in 15,5% of AD cases (in 23% of early‐onset versus 7,8% in late‐onset, p = 0.058) and in 12,5% of nonagenarians, and several of them were exclusive of the group. However, all were intronic variants except the Q170H change found in three AD cases and one nonagenarian. The SNP rs74016945 (3’UTR), with MAF close to 0.01, was found in 6% of the nonagenarians (OR 0.146, Fisher exact test p = 0.057). ELISA ADAM10 levels were not significantly different comparing AD cases with controls, carriers of rare variants versus non‐carriers, and did not differ consistently according to age at onset or APOE genotype. The three ADAM10 species were also similar in AD cases and controls, and did not differ according to genetic variants or age at onset.ConclusionRare ADAM10 exonic variants are uncommon in AD cases and the presence of rare intronic variants (more frequent in early versus late‐onset cases) is not associated with decreased ADAM10 levels in CSF.

A mutation rate model at the basepair resolution identifies the mutagenic effect of polymerase III transcription.

De novo mutations occur at substantially different rates depending on genomic location, sequence context and DNA strand. The success of methods to estimate selection intensity, infer demographic history and map rare disease genes, depends strongly on assumptions about the local mutation rate. Here we present Roulette, a genome-wide mutation rate model at basepair resolution that incorporates known determinants of local mutation rate. Roulette is shown to be more accurate than existing models. We use Roulette to refine the estimates of population growth within Europe by incorporating the full range of human mutation rates. The analysis of significant deviations from the model predictions revealed a tenfold increase in mutation rate in nearly all genes transcribed by polymerase III (Pol III), suggesting a new mutagenic mechanism. We also detected an elevated mutation rate within transcription factor binding sites restricted to sites actively used in testis and residing in promoters.

A Rare and Unusual Case of Ataxia Telangiectasia with Cervical Dystonia from India

We reported a rare and unusual case ATM gene variant responsible of familial Ataxia-Telangiectasia (A-T) with cervical dystonia rather than ataxia as hall mark clinical manifestations in 18-year-old male patient from India. Patient was presented with clinical indications of neck rotatory movements in the last 2 years. Initially intermittent, slowly become constant with more backwards turning and painful movements with severe disability. Examination findings revealed no conjunctival telangiectasia and neurological examination findings suggestive of cervical dystonia with severe retro collis. The patient has a family history of dystonia, his sister started noticing involuntary neck movement in the last 1.5 years with gradual progression. Her physical examination revealed no evidence of conjunctival lesions and neurological examination suggestive of cervical dystonia with lateral collis. MRI findings of patient were normal. Both the patient and her sister are siblings of a consanguineous product. Patient has been referred for whole exome sequencing. The exome data analysis identified a novel homozygous missense variant c.5751A>T, p.Arg1917Ser (chr11:108307973A>T) in ATM gene responsible for familial A-T with cervical dystonia without chorea as hall mark clinical manifestations. In conclusion, to the best of our literature knowledge this is the rare and unusual case from India wherein we are reporting a novel homozygous missense variant c.5751A>T, p.Arg1917Ser (chr11:108307973A>T) in ATM gene responsible for familial A-T with cervical dystonia rather than ataxia as hall mark clinical manifestations affecting both brother and sister in the same family. Thus, early onset of cervical dystonia should be taken into account as a key characteristic of variant A-T, which can manifest without general ataxia and may cause adults with primary dystonia to receive an incorrect diagnosis.

Identification of Hb Lepore, Hb anti-Lepore, and α-globin gene triplications by long-read single-molecule real-time sequencing.

Hemoglobin (Hb) Lepore and Hb anti-Lepore are infrequent fusion gene variants that result from nonhomologous crossovers during meiosis. Conventional molecular testing methods may face challenges in identifying these variants. During Hb analysis using capillary electrophoresis, we encountered 6 cases with unusual Hb variants. Our aim was to identify the alterations in their globin genes. Gap-polymerase chain reaction (PCR), reverse dot-blot assay (RDB), Sanger sequencing, multiplex ligation-dependent probe amplification (MLPA), and long-read single-molecule real-time (SMRT) sequencing were used to confirm the presence of globin gene alterations. The routine thalassemia gene test kit using the gap-PCR and RDB techniques did not detect common gene variations. Direct sequencing failed to identify any known or unknown globin gene alterations. The MLPA analysis, however, revealed the possible presence of α-globin gene triplications as well as 2 types of fusion gene alterations. Further analysis using long-read SMRT sequencing accurately identified 3 rare gene variations: αααanti-3.7, Hb Lepore-Boston-Washington, and Hb anti-Lepore P-India. Conventional methods may overlook rare thalassemias or Hb variants. Long-read SMRT sequencing has the potential to identify breakpoints in fusion genes, demonstrating that it is a promising technique for detecting rare thalassemias.

Identification of Characteristics and Prognostic Impact of FUS-ERG and AML1-MTG16 Fusion Genes in Adult AML Patients

Background FUS-ERG and AML1-MTG16 are two rare fusion genes in acute myeloid leukemia (AML), characterized by similar karyotypic abnormalities, namely t(16;21)(p11;q22) and t(16;21)(q24;q22), respectively. A previous research has demonstrated that FUS-ERG represents a high-risk subtype associated with an extremely poor prognosis, while AML1-MTG16 has favorable outcomes in pediatric cohort. Despite this valuable insight, a comprehensive systematic review of these fusion genes in the adult population remains absent. Method In this study, we conducted an extensive collection of AML patients with FUS-ERG or AML1-MTG16 fusion genes, who were reported in Mitelman Database of Chromosome Aberrations and Gene Fusions in Cancer, PubMed and China national knowledge infrastructure (CNKI) from 1988 to 2023. Patients lacking detailed survival data or those who did not receive treatment were excluded. Additionally, patients diagnosed and treated in our institution between 2018 and 2023 were included if they were identified as carrying FUS-ERG or AML1-MTG16 fusion genes. The reference cohort comprised of 669 adult AML patients (excluding the t(16;21) cases and acute promyelocytic leukemia) admitted to our institution from 2016 to 2021. All patients within the cohort were classified into 3 categories based on the 2022 ELN genetic risk classification, namely favorable (F), intermediate (I) and adverse (A) risk. These patients were then compared with the FUS-ERG and AML1-MTG16 groups to explore their respective clinical features and prognosis. Results A total of 800 patients were included for analysis (Table 1). Patients with FUS-ERG (n=111) exhibited significant distinctions, including a younger onset age (34 years vs. 59 years), lower platelet count (30×10 9/L vs. 51×10 9/L), higher bone marrow blasts proportion (80.0% vs. 60.0%), a higher relapse rate (78.6% vs. 44.7%) and a higher incidence of trisomy 8 (16.4% vs. 9.0%), trisomy 10 (9.1% vs. 0.3%) and complex karyotype (37.8% vs. 11.6%), when compared to the reference cohort (p<0.01). Meanwhile, patients with AML1-MTG16 (n=20) primarily presented as the secondary AML (70.0%), and displayed a higher prevalence of trisomy 8 (40.0%) and complex karyotype (35.0%) than reference cohort (p<0.01). The FUS-ERG patients had a less median overall survival (OS) when compared to the reference cohort (13 vs. 35 months, p<0.001). The median OS in subgroups with ELN 2022 F-risk vs. ELN 2022 I-risk vs. FUS-ERG was not reached (NR) vs. 33.6 months vs. 13.0 months (p<0.001) and the median OS of FUS-ERG group was even inferior to ELN 2022 A-risk group (13.0 vs. 16.8 months, p=0.015). Furthermore, the FUS-ERG patients also displayed a less median disease-free survival (DFS) than the reference cohort (7.5 vs. 20.5 months, p<0.001). The median DFS in subgroups with F-risk vs. I-risk vs. A-risk vs. FUS-ERG was NR vs. 19.8 vs. 13.3 vs. 7.5 months (p<0.001). In contrast, patients with AML1-MTG16 had a median OS of 18.7 months and a median DFS of 16 months, presenting a relatively unfavorable OS trend and indistinctive DFS, when compared to the reference cohort (OS: p=0.083, DFS: p=0.612), F-risk (OS: p<0.001, DFS: p=0.128), I-risk (OS: p=0.162, DFS: p=0.835) and A-risk (OS: p=0.877, DFS: p=0.731). Moreover, the AML1-MTG16 group had a better DFS than the FUS-ERG group (14.0 vs. 7.5 months, p=0.016), but their OS was comparable (16.0 vs. 7.5 months, p=0.219). For adult patients with FUS-ERG (Table 2) multivariate analysis revealed that age >60 years (HR=2.96, 95%CI: 1.14-7.73, p=0.026), WBC >100×10 9/L (HR=2.98, 95%CI: 1.24-7.13, p=0.014) and the presence of monosomy (HR=2.61, 95%CI: 1.21-5.66, p=0.015) were independent risk factors for OS, while monosomy was associated with poor DFS (HR=3.72, 95%CI: 1.69-8.20, p=0.001). However, receiving HSCT could significantly improve both OS (HR=0.22, 95%CI: 0.11-0.42, p<0.001) and DFS (HR=0.31, 95%CI: 0.16-0.57, p<0.001). Pediatric FUS-ERG patients presented similar clinical features, exhibiting a better median OS (19.5 vs.13 months, p=0.014) and a comparable median DFS (9.5 vs. 7.5 months, p=0.496), when compared to the adult population. Conclusion Both FUS-ERG and AML1-MTG16 were identified as high-risk subgroups in adult AML anddiffered from the results in pediatric cohort. Several factors including age, WBC count, HSCT and monosomy influenced the prognosis of adult AML patients with FUS-ERG.

Rare loss-of-function variants in matrisome genes are enriched in Ebstein’s anomaly

Ebstein's anomaly, a rare congenital heart disease, is distinguished by the failure of embryological delamination of the tricuspid valve leaflets from the underlying primitive right ventricle myocardium. Gaining insight into the genetic basis of Ebstein's anomaly allows a more precise definition of its pathogenesis. In this study, two distinct cohorts from the Chinese Han population were included: a case-control cohort consisting of 82 unrelated cases and 125 controls without cardiac phenotypes and a trio cohort comprising 36 parent-offspring trios. Whole-exome sequencing data from all 315 participants were utilized to identify qualifying variants, encompassing rare (minor allele frequency<0.1% from East Asians in the gnomAD database) functional variants and high-confidence (HC) loss-of-function (LoF) variants. Various statistical models, including burden tests and variance-component models, were employed to identify rare variants, genes, and biological pathways associated with Ebstein's anomaly. Significant associations were noted between Ebstein's anomaly and rare HC LoF variants found in genes related to the matrisome, a collection of extracellular matrix (ECM) components. Specifically, 47 genes with HC LoF variants were exclusively or predominantly identified in cases, while nine genes showed such variants in the probands. Over half of unrelated cases (n= 42) and approximately one-third of probands (n= 12) were found to carry one or two LoF variants in these prioritized genes. These results highlight the role of the matrisome in the pathogenesis of Ebstein's anomaly, contributing to a better understanding of the genetic architecture underlying this condition. Our findings hold the potential to impact the genetic diagnosis and treatment approaches for Ebstein's anomaly.

Genomics of iron refractory iron deficiency anemia phenotype reveals a spectrum of novel pathogenic biallelic and monoallelic TMPRSS6 variants and rare overlapping disorders

The study highlights genomic findings in a series of 13 IRIDA phenotype cases. All had microcytic hypochromic anemia with suboptimal oral iron response to two different oral iron preparations at 4–6 weeks and low-normal ferritin, low transferrin saturation, and inappropriately high hepcidin. Targeted NGS on a 26-gene iron panel revealed pathogenic TMPRSS6 variants in 5/13 (38 %) cases. In addition, 2 (15 %) cases revealed rare SMAD4 and TBXAS1 gene variants that can present with refractory anemia but were consistent with diagnosis of hereditary hemorrhagic telangiectasia and Ghosal hematodiaphyseal dysplasia respectively.

Abstract 15046: Atrial Sarcomeric Dysfunction in Ttntv Mice Indicates a Causal Relationship Between Rare Variation in TTN and Early Onset Atrial Fibrillation in Patients

Background: Atrial fibrillation (AF) is a common cardiac arrhythmia with a substantial genetic component. Rare variation in TTN and other sarcomeric genes have been associated with AF, in particular in early-onset disease. Aim: To describe the burden of rare, functional variants in a Norwegian early-onset AF population and to perform functional characterization in a Ttn truncating variant ( Ttntv ) mouse model. Methods: We performed whole-genome sequencing in 90 individuals with onset of AF before age 50 years and calculated the burden of rare, functional variation for all genes. Variant frequencies and gene burden from gnomAD 3.1.2 NFE were used as controls. The burden was defined as the sum of allele counts for rare (MAF&lt;1%) and damaging variants (high and moderate impact) for each gene, and differences in burden was assessed using Fisher’s exact test. Functional characterization, including echocardiography, electron microscopy and mRNA expression analyses, was performed in atria from haploinsufficient Ttntv mice. Results: We identified an increased burden of rare, functional variation in 57 genes, including the sarcomeric genes TTN , MYH6 and OBSL1 , but also in the cell adhesion gene DCHS2 , in individuals with early-onset AF. Ttntv mice had normal cardiac function assessed by echocardiography, but electron microscopy revealed structural remodeling of the atria, with a patchy pattern of unorganized sarcomeres with focal fibrosis and lipid droplet accumulation. Molecular profiling of atria using RNA sequencing showed differential expression of genes related to cellular stress, sarcomeric structure and fibrosis, including increased levels of Anp , Malat1 and Kcna1 . Dchs2 displayed decreased expression in Ttntv atria. Conclusion: We identified an increased burden of rare, functional variation in genes associated with sarcomeric function in early-onset AF. Atrial myopathy with sarcomeric lesions, focal fibrosis, lipid droplet accumulation and altered gene expression of Anp , Malat1 and Kcna1 was found in atria of mice with Ttntv , suggesting a causal, pathophysiological relationship between TTN variants and AF. Interestingly, DCHS2 displayed both increased burden of rare variation in early-onset AF and decreased expression in Ttntv mouse atria.

2017-2023: state of the art of gene therapies in rare diseases in Europe: the dynamics of clinical R&amp;D, new approved treatments and expected therapies in the pipelines

Aim: Gene therapies have been tested over the past three decades, and after a first market authorization in 2017, the field is starting to deliver. The study aims to analyze the current development dynamics of gene therapies for rare diseases using the GENOTRIAL database®, which gathers information on gene therapy clinical trials and studies conducted between 2017 and 2023 in Europe. Methods: The study involved extracting and filtering clinical trial data from the EudraCT database. Trials with the keyword "Gene Therapy" were selected and filtered using the "Rare disease" filter. Manual verification was conducted to ensure that the selected trial only concerned gene therapy treatments authorized in Europe for rare diseases in phases I to III. A total of 300 European country-related clinical investigations representing a total of 93 European-specific clinical studies were included in the GENOTRIAL database. The trials were classified by development phases, temporal status, sponsors and investigating countries, rare diseases with their related therapeutic area, and approval regulatory information of the identified gene therapies. Results: Analysis reveals that rare diseases present a promising area for gene therapy development. On average, eight rare disease gene therapy trials are launched each year in Europe. The main sponsors of European clinical trials of gene therapies for rare diseases are from US, followed by the United Kingdom and France. The United Kingdom conducts the highest number of investigations in Europe, followed by France, Italy, Spain, and Germany. Nutritional and metabolic diseases are the most represented therapeutic area, followed by rare oncology, blood and lymphatic diseases, and ocular diseases. The analysis identifies 73 gene therapy medical products covering 35 diseases at various stages of development, with 12 new therapies approved in recent years for 8 rare diseases, while 15 other gene therapies are at an advanced stage of phase III in their development plan for 11 other rare diseases. Conclusion: Gene therapy has shown significant progress and potential in treating rare genetic diseases. Europe has emerged as a promising region for gene therapy clinical trials in rare diseases. Efforts are now required to catch up with the USA and UK regarding the number of clinical trials sponsored by European groups.

The Use of Targeted RNA- Sequencing Assay in the Diagnostic Evaluation of Acute Myeloid Leukaemia(AML)

Background: Acute Myeloid Leukaemia (AML) is a clinically heterogenous disease characterised by chromosomal and genetic abnormalities which guide risk stratification and management. Hence timely cytogenetic and molecular profiling is essential to initiate appropriate treatment. Approximately 30% of AML cases have fusion genes. We use a combination of fluorescence in situ hybridization (FISH) and the Acute Leukaemia Diagnostic Panel Real-Time PCR assay and targeted RNA-sequencing (Archer Fusion Plex Pan-Heme panel) to rapidly screen for common chromosomal abnormalities and fusion genes. The Fusion Plex Pan-Heme panel is an Anchored Multiplex PCR (AMP™)-based next-generation sequencing (NGS) product to detect and identify fusions, point mutations and expression levels from ribonucleic acid (RNA) input. Both known and novel fusions can be identified. The Acute Leukaemia Diagnostic Panel (TruPCR Acute Leukaemia Diagnostic Panel) is a real time PCR assay that detects 14 fusion genes with 28+ breakpoints commonly associated with AML and ALL, and variants in 5 genes associated with AML. Aims: To evaluate the RNA-sequencing Kit to detect rearrangements, gene fusions on bone marrow specimens from newly diagnosed AML patients; and compare concordance of targeted RNA-sequencing, FISH and qPCR in identifying leukaemia fusion genes. Methods: We retrospectively reviewed FISH, qPCR and targeted RNA-sequencing data for new AML cases diagnosed in the specialist integrated haematological malignancy diagnostic service (SIHMDS) at University College London Hospital over a one year period: 01 January 2022 to 31 December 2022. Results: 87 cases were included. They are classified based on ELN Cytogenetic Risk Group and types of AML as per Table 1. 28 (31%) of 87 patients had a detectable fusion gene by targeted RNA-sequencing (Figure 1). 22/28 were detected by targeted RNA-sequencing, FISH and qPCR. The 6/28 fusion genes detected by targeted RNA-sequencing, but not by FISH /qPCR were: - TFG-ADGRG7 (n=1) - NUP98-NSD1 (n=1) - JAK2-ETV6 (n=1) - LMO1-RIC3(n=1) - SSBP2-CHD1 (n=1)-clinical significance not known - CBFB-MYH11 (n=1) rare breakpoint - detected by FISH, not by PCR The discordant results were due to unavailability of probes/genes in FISH and qPCR panels. Summary/Conclusion: The ability to risk-stratify newly diagnosed AML patients allows earlier initiation of targeted therapies. We found that targeted RNA-sequencing (Archer Fusion Plex) is sensitive and showed 100% concordance in identifying fusions associated with good cytogenetic risk AML. Targeted RNA-sequencing can also identify high risk fusion genes such as NUP98-NSD1 for which FISH fusion probes are not routinely used. These may have been otherwise undetected, but can subsequently be confirmed by RT-PCR and importantly allow the generation of molecular measurable residual disease (MRD) assays to assess treatment response. We conclude that targeted RNA-sequencing can be implemented in a diagnostic laboratory to complement FISH-analysis for the detection of recurrent and rare gene fusions in AML diagnosis.

Relationship between driver gene mutations and clinical pathological characteristics in older lung adenocarcinoma.

Lung adenocarcinoma (LUAD) is the most common newly diagnosed malignant tumor in older people. As older patients age, organ function decreases, leading to increased adverse reactions to treatment. The epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase tyrosine (ALK) tyrosine kinase inhibitors (TKIs) therapy are more effective and well-tolerated than chemotherapy, while the rate of genetic testing and subsequent targeted treatment among older patients remains relatively low, the clinical benefit limitation for those patients. This study aims to investigate the mutation characteristics of LUAD diver gene and its relationship with clinicopathological features in older LUAD. A total of 275 patients were diagnosed as LUAD and were over sixty years old. We utilized next-generation sequencing technology to detect and analyze gene mutations in postoperative tissue specimens, including EGFR, KRAS, ALK, ROS1, RET, MET, BRAF, HER2, PIK3CA and NRAS. A total of 90.18% (248/275) of older LUAD patients experienced genetic mutations. The EGFR (192, 69.82%) had the highest mutation rate among ten genes, followed by KRAS (21, 7.64%), MET (21, 7.64%), ERBB2 (15, 5.45%), RET (9, 3.27%), ALK (8, 2.91%), ROS1 (8, 2.91%), PIK3CA (6, 2.18%), BRAF (5, 1.82%) and NRAS (1, 0.36%). We also found thirty patients (15.63%) with EGFR mutations also having other gene mutations. The L858R mutation and exon19 deletion were the predominant EGFR mutations, accounting for 84.90% of EGFR-mutated patients. In addition, fifty-one kinds of EGFR mutations were detected, distributed in the protein tyrosine kinase catalytic domain (43, 84.31%), cysteine enriched domain (4, 7.84%), receptor binding domain (3, 5.88%), and EGFR transmembrane domain (1,1.96%). Ten cases of gene fusion mutation were detected. Two rare partner genes, PKHD1 (P60:R34) and STK39 (R33:S11), were detected by ROS1 gene fusion. RET gene fusion revealed a rare companion gene KCND2 (R11:K2). The EGFR mutations were more prevalent in female, non-smoking patients (p < 0.05), and the KRAS mutations were more common in male and smoking patients (p < 0.01). In addition, the BRAF mutations were more likely to occur in the right lung (p < 0.05). Older LUAD populations exhibit diverse genetic mutations, which may also exist simultaneously. Simultaneous detection of multiple genes by NGS can accelerate and enhance targeted treatment benefits for older LUAD patients, ultimately improving their quality of life.

Dual Role of Dysfunctional Asc-1 Transporter in Distinct Human Pathologies, Human Startle Disease, and Developmental Delay.

Human startle disease is associated with mutations in distinct genes encoding glycine receptors, transporters or interacting proteins at glycinergic synapses in spinal cord and brainstem. However, a significant number of diagnosed patients does not carry a mutation in the common genes GLRA1, GLRB, and SLC6A5 Recently, studies on solute carrier 7 subfamily 10 (SLC7A10; Asc-1, alanine-serine-cysteine transporter) knock-out (KO) mice displaying a startle disease-like phenotype hypothesized that this transporter might represent a novel candidate for human startle disease. Here, we screened 51 patients from our patient cohort negative for the common genes and found three exonic (one missense, two synonymous), seven intronic, and single nucleotide changes in the 5' and 3' untranslated regions (UTRs) in Asc-1. The identified missense mutation Asc-1G307R from a patient with startle disease and developmental delay was investigated in functional studies. At the molecular level, the mutation Asc-1G307R did not interfere with cell-surface expression, but disrupted glycine uptake. Substitution of glycine at position 307 to other amino acids, e.g., to alanine or tryptophan did not affect trafficking or glycine transport. By contrast, G307K disrupted glycine transport similar to the G307R mutation found in the patient. Structurally, the disrupted function in variants carrying positively charged residues can be explained by local structural rearrangements because of the large positively charged side chain. Thus, our data suggest that SLC7A10 may represent a rare but novel gene associated with human startle disease and developmental delay.

A codon-based live-cell biomonitoring system for assessing intracellular phenylalanine bioavailability in cyanobacteria

Phenylalanine, as an essential aromatic amino acid, is not only needed for protein and vital molecules such as neurotransmitter and hormone synthesis but also a substrate for the biosynthesis of phenylpropanoids and various bioactive compounds. The metabolism of phenylalanine is dynamic and transitory, which would otherwise inhibit cell growth. Therefore, it is challenging and imperative to monitor intracellular phenylalanine bioavailability in real time, which has great significance for evaluating the effectiveness of introducing pathway-specific genetic modifications to enhance phenylalanine generation. In this study, we proposed a live-cell biomonitoring system to assess phenylalanine bioavailability in real time in cyanobacteria based on codon degeneracy and species-specific usage bias. The biomonitoring system was generated through genetic modification of phenylalanine codons in the chloramphenicol antibiotic resistance gene to wholly preferred and rare codons, in combination with an orthogonal constitutive promoter Trc to express these genes. Cyanobacterial cells equipped with a preferred codon-based gene showed a significant growth advantage over those with rare codons under antibiotic pressure, while the delayed growth caused by rare codon-based genes could be rescued by supplementing phenylalanine in the cultivation medium. Increasing intracellular phenylalanine bioavailability could promote rare codon-based gene containing cell growth to a similar level as wild-type strains harboring preferred codon-based gene, providing a live-cell visualized screening method to relatively define phenylalanine content from either random mutation libraries or pathway-specific engineering cyanobacterial chassis before conducting labor-intensive quantitative measurements.

Impact of Rare and Multiple Concurrent Gene Fusions on Diagnostic DNA Methylation Classifier in Brain Tumors.

DNA methylation signatures need to be interpreted in the context of pathology and discordant results warrant testing for novel and rare gene fusions.

THU335 Hyperinsulinemia Is Associated With An Atherogenic Lipoprotein Profile In Humans With Selective Insulin Resistance And An Atheroprotective Profile In Humans With Non-selective Insulin Resistance

Abstract Disclosure: D. Walzer: None. M. Lightbourne: None. M. Walter: None. Y. Dai: None. R.D. Shamburek: None. R.J. Brown: None. Insulin resistance (IR), a major risk factor for cardiovascular disease (CVD), leads to hyperglycemia and dyslipidemia. In obesity-associated IR, dyslipidemia attributed to increased stimulation of hepatic lipogenesis by insulin, whereas IR in glucoregulatory pathways leads to hyperglycemia. This dichotomy in insulin signaling pathways is termed selective IR. Lipodystrophy syndromes (LD) are rare conditions of severe, selective IR in which, like obesity, hyperinsulinemia with excess insulin receptor signaling is thought to cause high triglycerides (TG) &amp; low HDL. By contrast, in the rare condition of insulin receptor pathogenic variants (INSR) (nonselective IR) lack of insulin signaling through its receptor despite hyperinsulinemia leads to low TG and high HDL. Rodents with hepatic INSR k/o have an atherogenic lipid profile, but the lipoprotein profile in humans with INSR is unknown. Factors influencing atherogenicity of lipoprotein particles, including cholesteryl ester transfer protein (CETP) activity and adiponectin, have not been explored in these human models of severe selective vs. non-selective IR. We characterized atherogenicity of lipoprotein particles and factors influencing these particles in subjects with LD matched 2:1 with subjects with INSR based on age and sex. All subjects had fasting lipoprotein profiles by NMR spectroscopy, apolipoproteins AI (ApoI) and B (ApoB), total adiponectin by ELISA, and CETP activity by fluorometric assay. Subjects with LD (N=24, 75% female; 8 generalized, 16 partial) had a more atherogenic lipoprotein profile vs INSR (N=12, 50% female; 6 homozygous, 6 heterozygous), with 45-fold higher large TRLP, 20-fold higher medium TRLP, 15-fold higher very small TRLP (representing IDL), 3-fold higher small LDLP, 4-fold lower large HDLP, 1.7—fold higher ApoB, and 1.4—fold lower ApoAI (p &amp;lt;0.05 for all). CETP activity was comparable in INSR and LD. Adiponectin was 5.9- fold lower in LD vs INSR (p &amp;lt;0.0001). Among all subjects, adiponectin was positively associated with HDL-C, large HDLP, HDL size, and ApoA-I (p=0.001), and negatively correlated with TRLP number &amp; size, small LDLP, and very small TRLP (p&amp;lt;0.0001). Selective IR in LD is associated with an atherogenic lipoprotein particle profile. By contrast, non-selective IR in INSR is associated with an atheroprotective profile, which differs from rodent models. These divergent lipid phenotypes are not explained by different CETP activity. Although preclinical studies suggest that adiponectin promotes a favorable lipoprotein profile consistent with our observed associations, a causal link in humans is not supported by Mendelian randomization studies or rare ADIPOQ gene deletions. Overall, these findings suggest a key role of selective insulin resistance in the development of an atherogenic lipid profile. Further studies are needed to see if this profile leads to increased CVD risk. Presentation: Thursday, June 15, 2023

Long-Read Sequencing Identified a Large Novel δ/β-Globin Gene Deletion in a Chinese Family

Objective. Increasingly rare thalassemia has been identified with the advanced use of long-read sequencing based on long-read technology. Here, we aim to present a novel δ/β-globin gene deletion identified by long-read sequencing technology. Methods. Enrolled in this study was a family from the Quanzhou region of Southeast China. Routine blood analysis and hemoglobin (Hb) capillary electrophoresis were used for hematological screening. Genetic testing for common α- and β-thalassemia was carried out using the reverse dot blot hybridization technique. Long-read sequencing was performed to detect rare globin gene variants. Specific gap-polymerase chain reaction (gap-PCR) and/or Sanger sequencing were further used to verify the detected variants. Results. None of the common α- and β-thalassemia mutations or deletions were observed in the family. However, decreased levels of MCV, MCH, and abnormal Hb bands were observed in the family members, who were suspected as rare thalassemia carriers. Further, long-read sequencing demonstrated a large novel 7.414 kb deletion NG_000007.3:g.63511_70924del partially cover HBB and HBD globin genes causing delta-beta fusion gene in the proband. Parental verification indicated that the deletion was inherited from the proband’s father, while none of the globin gene variants were observed in the proband’s mother. In addition, the novel δ/β-globin gene deletion was further verified by gap-PCR and Sanger sequencing. Conclusion. In this study, we first present a large novel δ/β-globin gene deletion in a Chinese family using long-read sequencing, which may cause δβ-thalassemia. This study further enhances that long-read sequencing would be applied as a sharp tool for detecting rare and novel globin gene variants.

Expert consensus on the diagnosis and treatment of RET gene fusion non-small cell lung cancer in China.

The rearranged during transfection (RET) gene is one of the receptor tyrosine kinases and cell-surface molecules responsible for transmitting signals that regulate cell growth and differentiation. In non-small cell lung cancer (NSCLC), RET fusion is a rare driver gene alteration associated with a poor prognosis. Fortunately, two selective RET inhibitors (sRETi), namely pralsetinib and selpercatinib, have been approved for treating RET fusion NSCLC due to their remarkable efficacy and safety profiles. These inhibitors have shown the ability to overcome resistance to multikinase inhibitors (MKIs). Furthermore, ongoing clinical trials are investigating several second-generation sRETis that are specifically designed to target solvent front mutations, which pose a challenge for first-generation sRETis. The effective screening of patients is the first crucial step in the clinical application of RET-targeted therapy. Currently, four methods are widely used for detecting gene rearrangements: next-generation sequencing (NGS), reverse transcription-polymerase chain reaction (RT-PCR), fluorescence in situ hybridization (FISH), and immunohistochemistry (IHC). Each of these methods has its advantages and limitations. To streamline the clinical workflow and improve diagnostic and treatment strategies for RET fusion NSCLC, our expert group has reached a consensus. Our objective is to maximize the clinical benefit for patients and promote standardized approaches to RET fusion screening and therapy.

Analysis of copy number variation in AZF region of Y chromosome in patients with spermatogenic failure

To explore the characteristics of copy number variation (CNV) within the Y chromosome azoospermia factor (AZF) region in patients with spermatogenesis disorders in the Shenzhen area. A total of 123 patients with spermatogenesis disorders who had visited Shenzhen People's Hospital from January 2016 to October 2022 (including 73 patients with azoospermia and 50 patients with oligozoospermia) and 100 normal semen males were selected as the study subjects. The AZF region was detected with multiplex ligation-dependent probe amplification (MLPA), and the correlation between the CNV in the AZF region and spermatogenesis disorders was analyzed using the chi-square test or Fisher's exact test. 19 CNV were detected among 53 patients from the 223 samples, including 20 cases (27.40%, 20/73) from the azoospermia group, 19 cases (38%, 19/50) from the oligozoospermia group, and 14 cases (14%, 14/100) from the normal control group. In the azoospermia, oligozoospermia, and normal control groups, the detection rates for CNV related to the AZFa region (including AZFab and AZFabc) were 5.48% (4/73), 2.00% (1/50), and 0 (0/100), respectively. The detection rates for the AZFb region (including the AZFbc region) were 6.85% (5/73), 0 (0/50), and 0 (0/100), respectively. The detection rates for gr/gr deletions in the AZFc region were 2.74% (2/73), 6.00% (3/50), and 9.00% (9/100), respectively, and those for b2/b4 deletions in the AZFc region were 2.74% (2/73), 10.00% (5/50), and 0 (0/100), respectively. The detection rates for complex rearrangements in the AZFc region were 6.85% (5/73), 18.00% (9/50), and 3.00% (3/100), respectively. Statistical analysis showed no significant difference in the detection rate of gr/gr deletions between the three groups (Fisher's Exact Test value = 2.712, P = 0.249); the differences in the detection rate of b2/b4 deletions between the three groups were statistically significant (Fisher's Exact Test value = 9.489, P = 0.002); the differences in the detection rate of complex rearrangements in the AZFc region between the three groups were statistically significant (Fisher's Exact Test value = 9.493, P = 0.006). In this study, a rare AZFa region ARSLP1 gene deletion (involving SY86 deletion) was detected in a patient with oligozoospermia. CNV in the AZFa and AZFb regions have a severe impact on spermatogenesis, but partial deletion in the AZFa region (ARSLP1 gene deletion) has a minor impact on spermatogenesis. The b2/b4 deletion and complex rearrangement in the AZFc region may be risk factors for male infertility. The gr/gr deletion may not serve as a risk factor for male infertility in the Shenzhen area.