Target Next-generation Research Articles

12488 Exome Sequencing Unravels New Susceptibility Genes For Pheochromocytomas And Paragangliomas

Abstract Disclosure: G.F. Fagundes: None. F.F. Castro: None. L.S. Santana: None. A.F. Afonso: None. A.W. Maciel: None. F.L. Ledesma: None. C.A. Pereira: None. I.C. Soares: None. D.M. Lourenço Junior: None. M.A. Pereira: None. V. Srougi: None. F.Y. Tanno: None. J.L. Chambo: None. M.C. Fragoso: None. A.O. Hoff: None. B.B. Mendonca: None. A. Latronico: None. M.Q. Almeida: None. Background: Pheochromocytomas and paragangliomas (PPGLs) are rare neuroendocrine tumors with a high association with hereditary disease, affecting 40% of the cases. PPGL susceptibility genes are categorized into three clusters based on their pathophysiological and metabolomic mechanisms. Somatic oncogenic variants are identified in 30% of the tumors. Most of the germline and somatic drivers are mutually exclusive in PPGLs. Thus, 30% of the PPGLs remain without known genetic causes. Aim: To investigate new susceptibility genes for PPGLs using whole exome sequencing. Methods: We included 145 index patients with PPGLs (93 female and 52 male) with 91 pheochromocytomas (PHEO) and 60 paragangliomas, of which 43 (29.65%) were metastatic. A target next-generation sequencing panel for susceptibility genes was initially performed. Whole exome sequencing was performed in 56 patients with PPGLs (38 were paired with tumor DNA). Results:SDHB was the most frequently affected gene in 29 of 145 patients (20%). Germline SDHB exon 1 deletion was identified in 14 patients (48.27%). Germline variants were identified as follow: RET 22 (15.17%), VHL 13 (8.96%), SDHA 6 (4.14%), SDHD 5 (3.45%), NF1 5 (3.45%), FH 2 (1.38%), MAX 2 (1.38%), DLST 1 (0.69%), TMEM127 1 (0.69%), SDHC 1 (0.69%) and H3F3A 1 (0.69%) case. Somatic variants were identified in 21 tumors: NF1 6 (4.14%), HRAS 5 (3.45%), VHL 2 (1.38%), FGFR1 2 (1.38%), and RET 1 (0.69%). Six variants (five germline and one somatic) were identified in 3 new candidate genes: 1) Three very rare germline CHEK2 likely pathogenic or pathogenic variants (c.475T>C/ p.Tyr159His; c.362G>A/ p.Cys121Tyr; c.319+2T>A) in one metastatic PHEO and two PGLs (one metastatic). The metastatic PGL did not harbor any somatic oncogenic variant, while exome sequencing of a metastatic lesion from the PHEO had a somatic likely oncogenic HRAS variant; 2) Two very rare germline BRCA2 pathogenic variants (c.3680_3681delTG/ p.Leu1227fs; c.7806-2A>C) in a 33-year-old man with non-metastatic PHEO and in a 25-year-old man with metastatic PGL. All germline variants have not been reported in the Brazilian genomic variant repository. Exome sequencing did not reveal any somatic oncogenic driver in both tumors; 3) The somatic GNAS likely oncogenic variant c.601C>T/ p.Arg201Cys in a pheochromocytoma. The prevalence of CHEK2 and BRCA2 pathogenic or likely pathogenic variants in our cohort were significantly higher compared with those in the gnomAD population database (p < 0.0001 and p = 0.0004, respectively). Co-occurrence of germline and somatic drivers were found in 3 cases: FH and FGFR1, DLST and NF1, and CHEK2 and HRAS. In summary, germline and somatic diagnosis were reached in 63.45% and 15.18% of the PPGLs in our cohort, respectively. Conclusion: Our findings support CHEK2, BRCA2 and GNAS as novel susceptibility genes for PPGLs. Support: Sao Paulo Research Foundation (FAPESP) grant 2019/15873-6. Presentation: 6/2/2024

Targeting next-generation PDE4 inhibitors in search of potential management of rheumatoid arthritis and psoriasis

Immune-mediated inflammatory diseases (IMIDs) comprise a broad spectrum of conditions characterized by systemic inflammation affecting various organs and tissues, for which there is no known cure. The isoform-specific inhibition of phosphodiesterase-4B (PDE4B) over PDE4D constitutes an effective therapeutic strategy for the treatment of IMIDs that minimizes the adverse effects associated with non-selective PDE4 inhibitors. Thus, we report a new class of isoquinolone derivatives as next-generation PDE4 inhibitors for effective management of rheumatoid arthritis (RA) and psoriasis. Among the series, 8 compounds i.e. 1e, 1l, 1m, 1n, 1o, 2m, 2o and 3o showed promising PDE4B inhibition (>80 %) in vitro with IC50 ∼ 1.4–6.2 µM. The compound 1l was identified as an initial hit and was pursued for further studies. According to structure–activity relationship (SAR), an allyl group at C-4 position improved PDE4B inhibition. The correlation between in vitro activity data and binding affinities obtained via molecular docking suggested that the high-affinity binding to PDE4B is a prerequisite for the effective inhibition of PDE4B. Notably, the hit 1l showed selectivity towards PDE4B over PDE4D in vitro. Furthermore, 1l treatment (30 mg/kg) in the adjuvant-induced arthritis (AIA) rat model induced by complete Freund’s adjuvant (CFA) demonstrated anti-arthritic potential via ameliorating paw swelling and body weight, narrowing joint space, reducing excessive immune cells infiltration and pannus formation in addition to reducing mRNA expression of pro-inflammatory cytokines such as TNF-α and IL-6 in synovial tissues of experimental rats. Additionally, 1l reduced the hyper-proliferative state and colony forming potential of IMQ-induced psoriatic keratinocytes. The treatment of these cells with 1l markedly reduced the protein levels of Ki67 and mRNA levels of pro-inflammatory cytokines e.g. IL-17A and TNF-α suggesting its potent anti-psoriatic potential. Furthermore, 1l did not show any significant adverse effects when evaluated in a systematic toxicity (e.g. teratogenicity, hepatotoxicity and cardiotoxicity) studies in zebrafish at the tested concentrations (1–100 µM) and the NOAEL (no-observed-adverse-effect level) was found to be 100 µM. Thus, with promising anti-inflammatory effects both in vitro and in vivo along with PDE4B selectivity with an acceptable safety margin, 1l emerged as a new and promising inhibitor for further studies.

A case of Neonatal generalized atrophic benign epidermolysis bullosa due to variants of COL17A1 gene

To diagnose and explore the genetic etiology of a neonate with Hereditary epidermolysis bullosa. A neonate who was admitted to Suqian Hospital Affiliated to Xuzhou Medical University on July 10, 2021 was selected as the study subject. Peripheral blood samples were collected from the child and his parents for the extraction of genomic DNA. And target gene capture and next-generation sequencing were carried out. Candidate variants were verified by Sanger sequencing and pathogenicity analysis. The child was found to harbor compound heterozygous variants of the COL17A1 gene, namely c.997C>T (p.Q333X) and c.3481dupT (p.Y1161fs*2), which were respectively inherited from his father and mother. Both variants were predicted to be pathogenic. The child was diagnosed with Generalized atrophic benign epidermolysis bullosa due to the compound heterozygous variants of the COL17A1 gene.

Gut microbiota and relevant abundances of<i> Prevotella copri, Lachnospiraceae, Collinsella, Helicobacter cinaedi, Desulfovibrio</i>, and <i>Escherichia coli</i> among cats with <i>Pemphigus foliaceus</i>

The cat gut-inhabitant conjoint microbiota is a peculiar ecosystem in relationship with several bodily functions and immunity. Gut microbiota dysbiosis could participate within autoimmune disease pathogenesis, whereas its niche, whether causative or influencing role regarding systemic immunity in autoimmune diseases, remains fugitive. The purpose of the present study was to identify gut microbiota alterations and probable mechanisms that participated in the development of Pemphigus foliaceus for a better understanding of future effective therapeutical armamentarium based on gut microbiota. In the present article, the authors investigated gut microbiota alterations to those of eight cats diagnosed with Pemphigus foliaceus. Furthermore, the study thoroughly analyzed pathogenic bacteria species as triggers of autoimmunity. The diagnostic algorithm involved two distinct sides: i) on referral with the first criteria was proof of evidence for possible autoimmunity, and ii) Pemphigus foliaceus diagnosis with relevant analytes. A total of eight cats were enrolled, and gut microbiomes were detected by the use of the MIDOG All-in-One Microbial Test targeting Next-Generation DNA Sequencing. By this methodology, we analyzed the dysbiosis network for bacterial kingdoms and determined the relationship between disease activity related to Pemphigus foliaceus and gut microbiota. The data showed increasing abundances of Collinsella, Lachnospiroceae, and Escherichia coli and decreasing Desulfovibrio piger, Prevotella copri, and Helicobacter cianedi among cats with pemphigus foliaceus. For the first time in Turkey, the gut microbiota of cats with pemphigus foliaceus were detected, the results of which could be cautiously taken into consideration for novel and effective therapeutical approaches.

O-305 Next-generation sequencing of a cohort of 100 patients with diminished ovarian reserve reveals an etiology in 27% of cases and may predict the fertility prognosis

Abstract Study question Identify the genetic cause of a cohort of patients with unexplained Diminished ovarian reserve (DOR) by next-generation sequencing and compare with pregnancy outcome. Summary answer A high-yield positive genetic diagnosis was obtained: 27% of cases. Defects of genes involved in DNA repair/meiosis appeared to have an unfavorable prognosis. What is known already Ten percent of women undergoing Medically Assisted Procreation-MAP have a DOR defined by an AMH level <1.2 and an antral follicle count (AFC) <5. However, most causes of DOR are unknown. There is no known criteria of success in MAP. Primary ovarian insufficiency (POI) corresponds to a complete cessation of ovarian function in 1-4% of women under 40 years. We have very recently shown in a large cohort of POI that a custom-made target next-generation sequencing (NGS) POI panel allowed a genetic diagnosis in 30% of unexplained POI and leads to personalized medicine (Heddar et al., EBioMedicine. 2022 doi: 10.1016/j.ebiom.2022.104246). Study design, size, duration Prospective genetic study of a cohort of 100 patients with undergoing MAP using a custom-made NGS-POI panel comprising 88 validated POI-causing genes. These patients were for 77% European, 20% North African and 3% Asian. The classification of the variants detected were based on the American College of Medical Genetic criteria 2015. Only pathogenic and likely pathogenic variants were used for diagnosis. Candidate gene studies were performed in negative patients. Participants/materials, setting, methods One hundred patients aged 16-35 with DOR, normal karyotype and FMR1 gene, were studied with the NGS POI panel. The segregation of the variants in the available families was performed by Sanger sequencing. Cytogenetic studies of chromosomal fragility was performed if necessary. The outcome of the pregnancy (spontaneous or induced) was recorded. A correlation between the result of the genetic study and the outcome of the pregnancy has been performed. Main results and the role of chance One family included two sisters, one with POI and the other with DOR, highlighting the proximity of the two syndromes. A high positive genetic yield was found: 27% of cases. Genes were involved 1) in follicular growth and gonadal development (SOX8, AR, NOBOX, BMPR1A, BMPR1B, GNAS) (35%) 2) DNA repair (BNC1, ERCC6, BRCA1, ATM) (31%) 3) metabolism and mitochondrial functions (POLG, STAR) (12%), 4) cellular aging (24%), in particular the LMNA gene. In a patient with isolated DOR, we identified for the first time, bi-allelic truncating variants of BRCA1, a major breast/ovarian cancer susceptibility gene. Cytogenetic studies revealed increased chromosomal breaks with radial figures typical of Fanconi Anemia (FA). However there was no sign of FA or cancer in the patient and family. This observation is reminiscent of the family with isolated POI and biallelic mutations of BRCA2 (Heddar et al, J Med Genet 2021; DOI: 10.1136/jmedgenet-2019-106672). Among the 27 patients with an established genetic diagnosis, six were able to obtain a pregnancy. However, no pregnancies were achieved in the DNA repair gene suggesting an unfavorable prognosis for this gene family. On the other hand, patients with mutations of other gene families seem to have a better prognosis of fertility. Limitations, reasons for caution The size of the cohort should be implemented to confirm these results. The issues of pregnancy are not available for the whole cohort. Wider implications of the findings This is the first genetic study of a cohort of DOR and the first implication of BRCA1 in isolated DOR. It shows i) the need for genetic studies of DOR ii) the genetic link between POI and DOR iii) NGS study could give information on the fertility prognosis of DOR. Trial registration number not applicable

Prognostic value of circulating tumor DNA using target next-generation sequencing in extensive-stage small-cell lung cancer

BackgroundChemotherapy remains the mainstay of treatment for small-cell lung cancer (SCLC). Liquid biopsies provide a convenient and non-invasive detection method for monitoring disease progression in patients with SCLC. MethodsWe performed next-generation sequencing of 159 plasma samples from 69 patients with extensive-stage (ES)-SCLC. Circulating tumor (ct)DNA levels were quantified in haploid genome equivalents per mL (hGE/mL). MuTect2 was used to detect single nucleotide variants and short insertions/deletions. The “enrichKEGG” function in the “clusterProfiler” R package was used to enrich the mutated genes that only appeared during disease progression. ResultsIn our cohort, 66 of 69 (95.7%) plasma samples at the time of diagnosis had detectable somatic mutations; TP53 (89%) and RB1(56%) were the most frequent mutations, as well as copy number variations in some common SCLC-related genes such as RB1. Combination ctDNA and tissue testing improved the overall detection rate of actionable mutations from 19.4% to 26.9% compared with that of tissue detection alone. In addition, ctDNA levels changed dynamically during the course of treatment and were significantly associated with decreased progression-free survival. Notably, actionable mutations were detected at the time of diagnosis and during disease progression. ConclusionsOur study revealed a dynamic somatic mutation profile through continuous ctDNA detection and confirmed that ctDNA levels can reflect tumor burden and predict PFS in patients with extensive stage-SCLC. Furthermore, we demonstrated that plasma ctDNA assays can provide real-time information on somatic mutations for potential targeted therapies for SCLC.

Evidence for a Founder Effect of SDHB Exon 1 Deletion in Brazilian Patients With Paraganglioma.

Limited information is available concerning the genetic spectrum of pheochromocytoma and paraganglioma (PPGL) patients in South America. Germline SDHB large deletions are very rare worldwide, but most of the individuals harboring the SDHB exon 1 deletion originated from the Iberian Peninsula. Our aim was to investigate the spectrum of SDHB genetic defects in a large cohort of Brazilian patients with PPGLs. Genetic investigation of 155 index PPGL patients was performed by Sanger DNA sequencing, multiplex ligation-dependent probe amplification, and/or target next-generation sequencing panel. Common ancestrality was investigated by microsatellite genotyping with haplotype reconstruction, and analysis of deletion breakpoint. Among 155 index patients, heterozygous germline SDHB pathogenic or likely pathogenic variants were identified in 22 cases (14.2%). The heterozygous SDHB exon 1 complete deletion was the most frequent genetic defect in SDHB, identified in 8 out of 22 (36%) of patients. Haplotype analysis of 5 SDHB flanking microsatellite markers demonstrated a significant difference in haplotype frequencies in a case-control permutation test (P = 0.03). More precisely, 3 closer/informative microsatellites were shared by 6 out of 8 apparently unrelated cases (75%) (SDHB-GATA29A05-D1S2826-D1S2644 | SDHB-186-130-213), which was observed in only 1 chromosome (1/42) without SDHB exon 1 deletion (X2 = 29.43; P < 0.001). Moreover, all cases with SDHB exon 1 deletion had the same gene breakpoint pattern of a 15 678 bp deletion previously described in the Iberian Peninsula, indicating a common origin. The germline heterozygous SDHB exon 1 deletion was the most frequent genetic defect in the Brazilian PPGL cohort. Our findings demonstrated a founder effect for the SDHB exon 1 deletion in Brazilian patients with paragangliomas.

Genomic markers of recurrence risk in atypical meningioma following gross total resection.

Meningiomas are the most common primary central nervous system (CNS) tumor in adults and CNS World Health Organization grade 2 (atypical) meningiomas show an intermediate risk of recurrence/progression. Molecular parameters are needed to better inform management following gross total resection (GTR). We performed comprehensive genomic analysis of tumor tissue from 63 patients who underwent radiologically confirmed GTR of a primary grade 2 meningioma, including a CLIA-certified target next-generation sequencing panel (n = 61), chromosomal microarray (n = 63), genome-wide methylation profiling (n = 62), H3K27me3 immunohistochemistry (n = 62), and RNA-sequencing (n = 19). Genomic features were correlated with long-term clinical outcomes (median follow-up: 10 years) using Cox proportional hazards regression modeling and published molecular prognostic signatures were evaluated. The presence of specific copy number variants (CNVs), including -1p, -10q, -7p, and -4p, was the strongest predictor of decreased recurrence-free survival (RFS) within our cohort (P < .05). NF2 mutations were frequent (51%) but did not show a significant association with RFS. DNA methylation-based classification assigned tumors to DKFZ Heidelberg benign (52%) or intermediate (47%) meningioma subclasses and was not associated with RFS. H3K27 trimethylation (H3K27me3) was unequivocally lost in 4 tumors, insufficient for RFS analysis. Application of published integrated histologic/molecular grading systems did not improve prediction of recurrence risk over the presence of -1p or -10q alone. CNVs are strong predictors of RFS in grade 2 meningiomas following GTR. Our study supports incorporation of CNV profiling into clinical evaluation to better guide postoperative patient management, which can be readily implemented using existing, clinically validated technologies.

Clinical, Molecular, and Histopathological Correlates in a Series of 24 Consecutive Cases of Suspected Myeloproliferative Neoplasms with a JAK2V617F Variant Allele Frequency ≤1%

Background: JAK2V617F is detected in >95% of polycythemia vera (PV) and 50-60% of essential thrombocythemia (ET) and primary myelofibrosis (PMF). JAK2V617F variant allele frequency (VAF) is associated with different clinical characteristics and outcome. Highly sensitive molecular assays led to detect JAK2V617F mutation also in healthy subjects, associated with a higher risk of coronary heart disease (N Engl J Med. 2017;377(2):111-121) in the context of CHIP. To date, comprehensive data on suspected MPN patients with a JAK2V617F VAF≤1% are lacking. Aim and methods: The aim was to analyze clinical, molecular, and histopathological correlates in a series of suspected MPN patients (pts) with a JAK2V617F VAF≤1% (from 2017 to 2022) at our Center. JAK2V617F VAF was measured in granulocytes by highly sensitive RTQ-PCR. Conventional karyotype and target next-generation sequencing (NGS) analysis were performed (complete data will be presented at the meeting). Bone marrow (BM) biopsies were reviewed by two independent expert pathologists, R.S. and U.G., the latter included as an author in the International Consensus Classification (Blood 2022; 2022015850). Results: 21 males and 3 females (M/F=7:1), with a median age of 58 y (range, 22-81) were included. Median JAK2V617F VAF was 0.2% (0.1-1). In the suspicion of MPN, complete diagnostic work-up was carried out in pts presenting with erythrocytosis (n=15), thrombocytosis (n=4), history of atypical thrombosis (n=3), splenomegaly (n=1) and leukocytosis (n=1), (Fig.1). In pts with erythrocytosis, median (range) Hb and Hct values were 17 g/dL (15.8-18.5) and 51.5% (50.4-54.7), whereas leukocytes (6.6 x 109/L, 5-11) and platelet (258 x 109/L, 172-440) were within normal range. EPO level (10 mUI/mL; 1.8-12.5) was subnormal in 3 pts. Abnormal karyotype was documented in one case (45,X,-Y[7]/46,XY[13]). Additional mutations were documented in 3 pts: HBB T124N, SH2B3 S186I and cKIT D816V. In pts with thrombocytosis, platelet range was 460-1450 x 109/L; Hb, Hct and leucocytes values were normal. MPL W515K and CALR L367Tfs*46 additional mutations were in one and two pts, respectively. Considering pts with atypical thrombosis, one had a history of arterial events while the others experienced cerebral venous sinus and portal vein thrombosis. Thrombophilia screenings were negative. Karyotype was normal in two pts, whereas one had 45,X,-Y[3]/46,XY[20]. Additional CBL p.D460dup was detected in one case. The subjects investigated for splenomegaly/leukocytosis had normal karyotype, no additional mutations. Morphological features consistent with PV were documented in 4 pts (Fig. 2, A-B). In one of these cases, multifocal dense infiltrates of CD117+/CD25+ mast cells were detected, fulfilling the diagnostic criteria for systemic mastocytosis with associated hematological neoplasm. In the other cases, microscopic examination showed age-adjusted normal or focally increased cellularity, regular or mildly expanded erythroid lineage with no/only minor increase in granulocytic lineage and a higher number of megakaryocytes with PV-like features (i.e. polymorphic forms without atypia and no dense cluster formation) (Fig. 1, C-D). Neither reticulin fibrosis nor excess blasts were detected and these pts had a final diagnosis of MPN unclassifiable (MPN-U), with a PV-like morphology. In pts with thrombocytosis histology was consistent with ET in three cases and overt-PMF in one. Among ET, one pts harbored MPLW515K with grade 1 reticulin fibrosis (Fig. 2, E-F), whereas the overt PMF had CALR type 1 additional mutation. BM biopsies of pts presenting with atypical thrombosis, leukocytosis and mild splenomegaly showed non-specific MPN-like features (including focally increased cellularity, mild expansion of erythroid or granulocytic lineages, higher number of megakaryocytes with a TE-like or a PV-like morphology) and were diagnosed as MPN-U. Conclusions: This study highlights the importance of using highly sensitive assays to detect JAK2V617F, as well as the need to search for CALR and MPL mutations, particularly in pts with thrombocytosis. Moreover, in pts presenting with erythrocytosis and JAK2V617F positivity with VAF≤1%, histopathological diagnostic clues to MPN may be subtle as in the early phase of the disease, emphasizing the need to integrate histopathology with clinical and genetic data in order to address patients to proper clinical management. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Functional analysis of RYR1 variants in patients with confirmed susceptibility to malignant hyperthermia

BackgroundA major bottleneck to the introduction of noninvasive presymptomatic diagnostic tests for the pharmacogenetic disorder malignant hyperthermia is the lack of functional data for associated variants. MethodsWe screened 50 genes having a potential role in skeletal muscle calcium homeostasis using the HaloPlex™ (Agilent Technologies, Santa Clara, CA, USA) target enrichment system and next-generation sequencing. Twenty-one patients with a history of a clinical malignant hyperthermia reaction together with a positive in vitro contracture test were included. Eight variants in RYR1 were subsequently introduced into the cDNA for the human ryanodine receptor gene and tested in cultured human embryonic kidney (HEK293) cells for their effect on calcium release from intracellular stores in response to the ryanodine receptor-1 agonist 4-chloro-m-cresol using fura-2 as calcium indicator. Each variant was subjected to in silico curation using the European Malignant Hyperthermia Group scoring matrix and ClinGen RYR1 variant curation expert panel guidelines. ResultsPotentially causative RYR1 variants were identified in 15 patients. Of these, two families carried two RYR1 variants, five variants had been previously reported as ‘pathogenic’, two variants had been previously reported as ‘likely benign’, and eight were of ‘uncertain significance’. Of these eight variants, four showed hypersensitivity to 4-chloro-m-cresol. Three variants were reclassified as either ‘pathogenic’ or ‘likely pathogenic’. Two were classified as ‘benign’, whilst three remained of ‘uncertain significance’. ConclusionsThree (p.Tyr1711Cys, p.Val2280Ile, and p.Arg4737Gln) additional variants can be added to the list of RYR1 disease-associated variants managed by the European Malignant Hyperthermia Group. These can therefore be used diagnostically in the future. Three variants (p.Glu2348Gly, p.Asn2634Lys, and p.Arg3629Trp) that remained classified as of uncertain significance require further family studies or a different functional test to determine clinical relevance in malignant hyperthermia.

Pan-Cancer analysis of the mutation frequency of genes in TGF-beta pathway.

e15113 Background: The TGF-β signaling pathway governs key cellular processes under physiologic conditions and deregulates in cancer, including a diverse and varied set of gene responses that range from cytostatic and apoptotic tumor-suppressive ones in early-stage tumors to proliferative, invasive, angiogenic, and oncogenic ones in advanced cancer. However, the genomics profiles of TGF-β signaling pathway genes are unclear. In this study, we evaluated the genomic features in Chinese pan-cancer patients and investigated the mutation frequency of genes in the TGF-β pathway. Methods: A total of 5420 patients consisting of 22 cancer types were enrolled in this study. Utilizing target next-generation sequencing (NGS) platform, genomic profiling of 599 cancer-related genes, including genes of TGF-β pathway (SMAD2/3/4, PDGFRB, CDH1, MDM2, CDKN1A, and CDKN2B), was performed on tumor tissue or blood samples. Results: We observed 25.57% (1386/5420) of cases harbored at least one alteration in the TGF-β pathway genes. The mutation frequency ranges 9.09% to 39.77% among the 22 cancer types, including 39.77% (247/621) in gastric cancer, 39.31 (320/814) in bowel cancer, 36.84% (7/19) in endometrial cancer, 11.11% (8/72) in esophageal cancer, 10.46% (7/67) in kidney cancer, and 9.09% (2/22) in the gastrointestinal stromal tumor, respectively. The mutation of tumor-promoting effects genes SMAD2/3/4, PDGFRB, CDH1and MDM2 in the TGF-β pathway were found in all cancer types and mainly occurred in CNS tumor, gastrointestinal stromal tumor, pancreatic cancer, sarcoma, gastric cancer, and bowel cancer. The tumor-suppressing effective genes CDKN1A and CDKN2B primarily mutated in melanoma, endometrial cancer, and kidney cancer. Approximately 21.77% of patients had CNV in the TGF-β pathway of all tumor types. The most frequently mutated TGF-β genes were MDM2 and CDKN2B, mainly altered in sarcoma, melanoma, and CNS Tumor. Conclusions: We found a unique genomic feature of the TGF-β pathway of Chinese pan-cancer patients. Targeting mutant of TGF-β pathway genes remains a potentially effective treatment in the future.

Pan-Cancer analysis of the mutation frequency of genes in DDR pathway in Chinese population.

e15109 Background: The DNA damage repair (DDR) pathway is of vital importance to maintain human genomic stability. And the variation of DDR pathway related genes is closely related to tumorigenesis, tumor progression and therapeutic response. In order to comprehensively study the mutation status of DDR pathway related genes in human cancers, we evaluated the genomic features of Chinese pan-cancer patients and investigated the mutation frequency of genes in DDR pathway. Methods: A total of 5018 patients consisting of more than 22 cancer types were enrolled in this study. Utilizing target next-generation sequencing (NGS) platform, genomic profiling of 599 cancer-related genes was performed on tumor tissue or blood samples. And many DDR pathway related genes are included and detected mutations such as TP53, BRCA2, BRCA1, ATM, BLM, FANCA, POLE and so on. Results: We observed 91.23% (4578/5018) of cases harbored at least one alteration of the DDR pathway genes. The mutation frequency ranges from 71.88% to 100.00% among the 22 cancer types. Among them the DDR gene mutation frequency of lymphoma, esophagus and esophagogastric junction cancer and endometrial cancer are all 100%, and mutation frequency of bowel cancer, hepatobiliary cancer, melanoma, breast cancer, esophageal cancer, gastric cancer and central nervous system cancer are also more than 90% respectively. The highest mutation rate gene is a well-known tumor suppressor gene TP53, which mutated in 2650 patients and the mutation rate is 52.81%. And it primarily mutated in esophageal cancer, with a mutation rate of 81.43%. Besides the mutation rate of TP53 in gastric cancer, bowel cancer, breast cancer, ovarian cancer, pancreatic cancer, bladder cancer and hepatobiliary cancer are all more than 50%. Followed by is BRCA2 gene, which is also a tumor suppressor gene and the mutation rate is 16.04%. Next is ATM, BLM and FANCA, the mutation rate of which is 11.68%, 11.12% and 8.97% respectively. Conclusions: We find a unique genomic feature of DDR pathway in the comprehensively analysis of the mutation profile of the Chinese pan-cancer patients, which may provide potential therapeutic targets in the future.

Analysis of clinical phenotype and genotype of Chinese children with disorders of sex development

Objective: To explore the heterogeneity and correlation of clinical phenotypes and genotypes in children with disorders of sex development (DSD). Methods: A retrospective study of 1 235 patients with clinically proposed DSD in 36 pediatric medical institutions across the country from January 2017 to May 2021. After capturing 277 DSD-related candidate genes, second-generation sequencing was performed to analyzed the heterogeneity and correlation combined with clinical phenotypes. Results: Among 1 235 children with clinically proposed DSD, 980 were males and 255 were females of social gender at the time of initial diagnosis with the age ranged from 1 day of age to 17.92 years. A total of 443 children with pathogenic variants were detected through molecular genetic studies, with a positive detection rate of 35.9%. The most common clinical phenotypes were micropenis (455 cases), hypospadias (321 cases), and cryptorchidism (172 cases) and common mutations detected were in SRD5A2 gene (80 cases), AR gene (53 cases) and CYP21A2 gene (44 cases). Among them, the SRD5A2 mutation is the most common in children with simple micropenis and simple hypospadias, while the AMH mutation is the most common in children with simple cryptorchidism. Conclusions: The SRD5A2 mutation is the most common genetic variant in Chinese children with DSD, and micropenis, cryptorchidism, and hypospadias are the most common clinical phenotypes. Molecular diagnosis can provide clues about the biological basis of DSD, and can also guide clinicians to perform specific clinical examinations. Target sequence capture probes and next-generation sequencing technology can provide effective and economical genetic diagnosis for children with DSD.

Clonal haematopoiesis as a risk factor for therapy-related myeloid neoplasms in patients with chronic lymphocytic leukaemia treated with chemo-(immuno)therapy.

Clonal haematopoiesis of indeterminate potential (CHIP) may predispose for the development of therapy-related myeloid neoplasms (t-MN). Using target next-generation sequencing (t-NGS) panels and digital droplet polymerase chain reactions (ddPCR), we studied the myeloid gene mutation profiles of patients with chronic lymphocytic leukaemia (CLL) who developed a t-MN after treatment with chemo-(immuno)therapy. Using NGS, we detected a total of 30 pathogenic/likely pathogenic (P/LP) variants in 10 of 13 patients with a t-MN (77%, median number of variants for patient: 2, range 0-6). The prevalence of CHIP was then backtracked in paired samples taken at CLL diagnosis in eight of these patients. Six of them carried at least one CHIP-variant at the time of t-MN (median: 2, range: 1-5), and the same variants were present in the CLL sample in five cases. CHIP variants were present in 34 of 285 patients from a population-based CLL cohort, which translates into a significantly higher prevalence of CHIP in patients with a CLL who developed a t-MN, compared to the population-based cohort (5/8, 62.5% vs. 34/285, 12%, p=0.0001). Our data show that CHIP may be considered as a novel parameter affecting treatment algorithms in patients with CLL, and highlight the potential of using chemo-free therapies in CHIP-positive cases.

Dendritic cell functions in vivo: A user's guide to current and next- generation mutant mouse models.

DCs do not just excel in antigen presentation. They orchestrate information transfer from innate to adaptive immunity by sensing and integrating a variety of danger signals, and translating them to naïve T cells, to mount specifically tailored immune responses. This is accomplished by distinct DC types specialized in different functions and because each DC is functionally plastic, assuming different activation states depending on the input signals received. Mouse models hold the key to untangle this complexity and determine which DC types and activation states contribute to which functions. Here, we aim to provide comprehensive information for selecting the most appropriate mutant mouse strains to address specific research questions on DCs, considering three in vivo experimental approaches: (i) interrogating the roles of DC types through their depletion; (ii) determining the underlying mechanisms by specific genetic manipulations; (iii) deciphering the spatiotemporal dynamics of DC responses. We summarize the advantages, caveats, suggested use, and perspectives for a variety of mutant mouse strains, discussing in more detail the most widely used or accurate models. Finally, we discuss innovative strategies to improve targeting specificity and next-generation mutant mouse models, and briefly address how humanized mouse models can accelerate translation into the clinic.

Prevalence and Spectrum of Predisposition Genes With Germline Mutations Among Chinese Patients With Bowel Cancer.

Background: Bowel cancer is the third-most common cancer and the second leading cause of cancer-related death worldwide. Bowel cancer has a substantial hereditary component; however, additional hereditary risk factors involved in bowel cancer pathogenesis have not been systematically defined. Materials and Methods: A total of 573 patients with bowel cancer were enrolled in the present study, of whom 93.72% had colorectal cancer (CRC). Germline mutations were integrated with somatic mutation information via utilizing target next-generation sequencing. Results: Pathogenic/Likely Pathogenic (P/LP) germline alterations were identified in 47 (8.2%) patients with bowel cancer and the ratio of the number of these patients with family history was significantly higher in the P/LP group than that noted in the non-pathogenic (Non-P) group. Certain rare germline alterations were noted, such as those noted in the following genes: FANCD2, CDH1, and FLCN. A total of 32 patients (68.1%) had germline alterations in the DNA-damage repair (DDR) genes and homologous recombination (HR) accounted for the highest proportion of this subgroup. By comparing 573 patients with bowel cancer with reference controls (China_MAPs database), significant associations (p < 0.01) were observed between the incidence of bowel cancer and the presence of mutations in APC, ATM, MLH1, FANCD2, MSH3, MSH6, PMS1, and RAD51D. Somatic gene differential analysis revealed a marked difference in 18 genes and a significant difference was also noted in tumor mutation burden (TMB) between germline mutation carriers and non-germline mutation subjects (p < 0.001). In addition, TMB in DDR mutation groups indicated a dramatic difference compared with the non-DDR mutation group (p < 0.01). However, no statistically significant differences in TMB were noted among detailed DDR pathways for patients with bowel cancer, irrespective of the presence of germline mutations. Moreover, a significantly higher level (p < 0.0001) of mutation count was observed in the DDR group from The Cancer Genome Atlas (TCGA) database and the DDR and non-DDR alteration groups displayed various immune profiles. Conclusion: Chinese patients with bowel cancer exhibited a distinct spectrum of germline variants, with distinct molecular characteristics such as TMB and DDR. Furthermore, the information on somatic mutations obtained from TCGA database indicated that a deeper understanding of the interactions among DDR and immune cells would be useful to further investigate the role of DDR in bowel cancer.

Genomic Profiling of Chinese Cervical Cancer Patients Reveals Prevalence of DNA Damage Repair Gene Alterations and Related Hypoxia Feature.

BackgroundCervical cancer is responsible for 10–15% of cancer-related deaths in women worldwide. In China, it is the most common cancer in the female genital tract. However, the genomic profiles of Chinese cervical cancer patients remain unclear.Materials and MethodsA total of 129 cervical cancer patients were enrolled in this study (113 squamous, 12 adenocarcinoma, 2 adenosquamous, and 2 neuroendocrine carcinoma). To classify the clinical features and molecular characteristics of cervical cancer, the genomic alterations of 618 selected genes were analyzed in the samples of these patients, utilizing target next-generation sequencing (NGS) technology. Furthermore, the findings from the Chinese cohort were then compared with the data of Western patients downloaded from The Cancer Genome Atlas (TCGA) database, in terms of gene expression files, mutation data, and clinical information.ResultsAll studied patients had valid somatic gene alterations, and the most frequently altered genes were PIK3C, TP53, FBXW7, ARID1A, ERBB2, and PTEN. Comparison of genomic profiling showed significantly different prevalence of genes, including TP53, KMT2C, and RET, between the Chinese and the TCGA cohorts. Moreover, 57 patients (44.19%) with 83 actionable alterations were identified in our cohort, especially in PI3K and DNA damage repair (DDR) pathways. After an in-depth analysis of cervical cancer data from the TCGA cohort, DDR alteration was found to be associated with extremely higher tumor mutation burden (TMB) (median mutation count: 149.5 vs 66, p <0.0001), and advanced stages (p <0.05). Additionally, DDR alteration, regardless of its function, was positively correlated with hypoxia feature and score. Moreover, patients with a high hypoxia score were positively correlated with a high abundance of mast cell resting, but lower abundance of CD8+ T cells and activated mast cell. Finally, CDHR5 was identified as the hub gene to be involved in the DDR–hypoxia network, which was negatively correlated with both the DDR alteration and hypoxia score.ConclusionsOverall, a unique genomic profiling of Chinese patients with cervical cancer was uncovered. Besides, the prevalent actionable variants, especially in PI3K and DDR pathways, would help promote the clinical management. Moreover, DDR alteration exerted the significant influence on the tumor microenvironment in cervical cancer, which could guide the clinical decisions for the treatment. CDHR5 was the first identified hub gene to be negatively correlated with DDR or hypoxia in cervical cancer, which had potential effects on the treatment of immune checkpoint inhibitors (ICIs).

Allele-specific and multiplex PCR based tools for cost-effective and comprehensive genetic testing in Congenital Adrenal Hyperplasia

Congenital Adrenal Hyperplasia (CAH) is an autosomal recessive disorder due to enzyme defects in adrenal steroidogenesis. Several genes code for these enzymes, out of which mutations in the CYP21A2 gene resulting in 21 hydroxylase deficiency, contribute to the most common form of CAH. However, pseudogene imposed challenges complicate genotyping CYP21A2 gene, and there is also a lack of comprehensive molecular investigations in other genetic forms of CAH in India. Here, we describe a cost-effective, highly specific, and sensitive Allele Specific PCR (ASPCR) assay designed and optimized in-house to screen eight common pathogenic mutations in the CYP21A2 gene. We have also established and utilized a multiplex PCR assay for target enrichment and Next-generation sequencing (NGS) of CYP11B1, CYP17A1, POR, and CYP19A1 genes. Following preliminary amplification of the functional gene CYP21A2, ASPCR based genotyping of eight common mutations - P30L, I2G, 8BPdel, I172N, E6CLUS (I235N, V236E, M238K) V281L, Q318X, and R356W was carried out. These results were further validated using Sanger and Next-generation sequencing. Once optimized to be specific and sensitive, the advantage of ASPCR in CYP21A2 genotyping extends to provide genetic screening for both adult and paediatric subjects and carrier testing at a low cost and less time. Furthermore, multiplex PCR coupled NGS has shown to be cost-effective and robust for parallel multigene sequencing in CAH.

A Custom Target Next-Generation Sequencing 70-Gene Panel and Replication Study to Identify Genetic Markers of Diabetic Kidney Disease.

Diabetic kidney disease (DKD) has been pointed out as a prominent cause of chronic and end-stage renal disease (ESRD). There is a genetic predisposition to DKD, although clinically relevant loci are yet to be identified. We utilized a custom target next-generation sequencing 70-gene panel to screen a discovery cohort of 150 controls, DKD and DKD-ESRD patients. Relevant SNPs for the susceptibility and clinical evolution of DKD were replicated in an independent validation cohort of 824 controls and patients. A network analysis aiming to assess the impact of variability along specific pathways was also conducted. Forty-eight SNPs displayed significantly different frequencies in the study groups. Of these, 28 with p-values lower than 0.01 were selected for replication. MYH9 rs710181 was inversely associated with the risk of DKD (OR = 0.52 (0.28–0.97), p = 0.033), whilst SOWAHB rs13140552 and CNDP1 rs4891564 were not carried by cases or controls, respectively (p = 0.044 and 0.023). In addition, the RGMA rs1969589 CC genotype was significantly correlated with lower albumin-to-creatinine ratios in the DKD patients (711.8 ± 113.0 vs. 1375.9 ± 474.1 mg/g for TC/TT; mean difference = 823.5 (84.46–1563.0); p = 0.030). No biological pathway stood out as more significantly affected by genetic variability. Our findings reveal new variants that could be useful as biomarkers of DKD onset and/or evolution.

Target-enriched sequencing enables accurate identification of bloodstream infections in whole blood

Bloodstream infections are within the top ten causes of death globally, with a mortality rate of up to 70%. Gold standard blood culture testing is time-consuming, resulting in delayed, but accurate, treatment. Molecular methods, such as RT-qPCR, have limited targets in one run. We present a new Ampliseq detection system (ADS) combining target amplification and next-generation sequencing for accurate identification of bacteria, fungi, and antimicrobial resistance determinants directly from blood samples. In this study, we included removal of human genomic DNA during nucleic acid extraction, optimized the target sequence set and drug resistance genes, performed antimicrobial resistance profiling of clinical isolates, and evaluated mock specimens and clinical samples by ADS. ADS successfully identified pathogens at the species-level in 36 h, from nucleic acid extraction to results. Besides pathogen identification, ADS can also present drug resistance profiles. ADS enabled detection of all bacteria and accurate identification of 47 pathogens. In 20 spiked samples and 8 clinical specimens, ADS detected at least 92.81% of reads mapped to pathogens. ADS also showed consistency with the three culture-negative samples, and correctly identified pathogens in four of five culture-positive clinical blood specimens. This Ampliseq-based technology promises broad coverage and accurate pathogen identification, helping clinicians to accurately diagnose and treat bloodstream infections.