Targeted Gene Panel Research Articles

Targeted gene panel provides advantages over whole-exome sequencing for diagnosing obesity and diabetes mellitus.

A small fraction of patients diagnosed with obesity or diabetes mellitus has an underlying monogenic cause. Here, we constructed a targeted gene panel consisting of 83 genes reported to be causative for monogenic obesity or diabetes. We performed this panel in 481 patients to detect causative variants and compared these results with whole-exome sequencing (WES) data available for 146 of these patients. The coverage of targeted gene panel sequencing was significantly higher than that of WES. The diagnostic yield in patients sequenced by the panel was 32.9% with subsequent WES leading to three additional diagnoses with two novel genes. In total, 178 variants in 83 genes were detected in 146 patients by targeted sequencing. Three of the 178 variants were missed by WES, although the WES-only approach had a similar diagnostic yield. For the 335 samples only receiving targeted sequencing, the diagnostic yield was 32.2%. In conclusion, taking into account the lower costs, shorter turnaround time, and higher quality of data, targeted sequencing is a more effective screening method for monogenic obesity and diabetes compared to WES. Therefore, this approach could be routinely established and used as a first-tier test in clinical practice for specific patients.

Clinical Utility of Optical Genome Mapping and 523-Gene Next Generation Sequencing Panel for Comprehensive Evaluation of Myeloid Cancers.

The standard-of-care (SOC) for genomic testing of myeloid cancers primarily relies on karyotyping/fluorescent in situ hybridization (FISH) (cytogenetic analysis) and targeted gene panels (usually ≤54 genes) that harbor hotspot pathogenic variants (molecular genetic analysis). Despite this combinatorial approach, ~50% of myeloid cancer genomes remain cytogenetically normal, and the limited sequencing variant profiles obtained from targeted panels are unable to resolve the molecular etiology of many myeloid tumors. In this study, we evaluated the performance and clinical utility of combinatorial use of optical genome mapping (OGM) and a 523-gene next-generation sequencing (NGS) panel for comprehensive genomic profiling of 30 myeloid tumors and compared it to SOC cytogenetic methods (karyotyping and FISH) and a 54-gene NGS panel. OGM and the 523-gene NGS panel had an analytical concordance of 100% with karyotyping, FISH, and the 54-gene panel, respectively. Importantly, the IPSS-R cytogenetic risk group changed from very good/good to very poor in 22% of MDS (2/9) cases based on comprehensive profiling (karyotyping, FISH, and 54-gene panel vs. OGM and 523-gene panel), while additionally identifying six compound heterozygous events of potential clinical relevance in six cases (6/30, 20%). This cost-effective approach of using OGM and a 523-gene NGS panel for comprehensive genomic profiling of myeloid cancers demonstrated increased yield of actionable targets that can potentially result in improved clinical outcomes.

#2684 DIAGNOSTIC YIELD OF A TARGETED GENE PANEL FOR MONOGENIC KIDNEY DISEASES: A EUROPEAN STUDY

Abstract Background and Aims Despite advances in understanding the underlying causes of CKD, 20% of cases remain unexplained (1). A genomic approach has the potential to identify the cause of CKD in a significant portion of pediatric and adult patients, with estimated diagnostic rates of 5-30% (2). However, there is a lack of consensus in the scientific community on the best diagnostic algorithm. The DECIDE project (Diagnostic EffiCacy kIdney Disease European) is a European collaboration that aims to address this issue by evaluating the diagnostic rate of a targeted gene panel in a large cohort of patients. Method DECIDE involved three Italian and two Spanish centers, encompassing both pediatric and adult patients. The study used the Nephropathies Solution Panel (NES, SOPHiA Genetics), which covers 44 kidney-related genes, to test patients with a high suspicion of genetic kidney disease. The clinical presentation was classified into cystic disease, glomerulopathy, CAKUT, tubulopathy, nephrocalcinosis, other, and negative phenotype. The diagnostic yield of the NES panel was calculated. To assess the genotype-phenotype relationship, Kaplan-Meier analyses were performed. Additionally, the diagnostic results obtained from alternative technologies, such as larger panels, WES and hybridization arrays, in cases of NES panel negative results, were also collected. Results As far as now the DECIDE project collected 632 genetic data. To evaluate the diagnostic accuracy of the panel, the number of (likely) pathogenic variants correlated to phenotype was analyzed. The diagnostic yield is shown in Figure 1, with 46% for cystic disease, 41% for glomerulopathy, 33% for tubulopathies, 28% for CAKUT, 14% for nephrocalcinosis, 2% for negative (in the context of segregation analyses or in cascade test) and 8% for other. Table 1 shows that the main genes involved in the diagnosis were PKD1, PKD2, COL4A3, COL4A4, and COL4A5. In patients affected by cystic disease, the mean age of kidney failure was 48 years in the presence of diagnostic variants, 59 years for VUS, and no events were reported in patients with negative tests. The Kaplan-Meier curve confirmed a worse prognosis in patients with diagnostic variants (p-value<0.05). However, no significant genotype-phenotype correlation in terms of time to replacement therapy was observed in glomerulopathic patients. In cases of non-diagnostic results, 21 further investigations were performed. An enriched panel for cystic disease didn't detect any diagnostic variants, while only one patient was diagnosed using WES. The Hybridization Array identified a gene deletion that is under investigation and may be related to the phenotype (Nephronophthisis). Conclusion The preliminary results of the DECIDE project, based on about 600 data, demonstrate the potential of the NES panel in the diagnosis of kidney genomic disease. The panel showed a good diagnostic yield for cystic disease (46%) and glomerulopathy (41%), but it appears less effective in other clinical presentations. The average diagnostic yield of the panel was comparable to published data for gene panel and WES approaches. However, additional tests such as larger gene panels, WES, or CGH-array were found to be of limited usefulness, as they only identified one pathogenic variant. These results suggest that new approaches are necessary to uncover the hidden genetic components of rare renal conditions.

Validation of a targeted gene panel sequencing for the diagnosis of hereditary chronic liver diseases.

Background: The cause of chronic liver diseases (CLD) remains undiagnosed in up to 30% of adult patients. Whole-Exome Sequencing (WES) can improve the diagnostic rate of genetic conditions, but it is not yet widely available, due to the costs and the difficulties in results interpretation. Targeted panel sequencing (TS) represents an alternative more focused diagnostic approach. Aims: To validate a customized TS for hereditary CLD diagnosis. Methods: We designed a customized panel including 82 CLD-associated genes (iron overload, lipid metabolism, cholestatic diseases, storage diseases, specific hereditary CLD and susceptibility to liver diseases). DNA samples from 19 unrelated adult patients with undiagnosed CLD were analyzed by both TS (HaloPlex) and WES (SureSelect Human All Exon kit v5) and the diagnostic performances were compared. Results: The mean depth of coverage of TS-targeted regions was higher with TS than WES (300x vs. 102x; p < 0.0001). Moreover, TS yielded a higher average coverage per gene and lower fraction of exons with low coverage (p < 0.0001). Overall, 374 unique variants were identified across all samples, 98 of which were classified as "Pathogenic" or "Likely Pathogenic" with a high functional impact (HFI). The majority of HFI variants (91%) were detected by both methods; 6 were uniquely identified by TS and 3 by WES. Discrepancies in variant calling were mainly due to variability in read depth and insufficient coverage in the corresponding target regions. All variants were confirmed by Sanger sequencing except two uniquely detected by TS. Detection rate and specificity for variants in TS-targeted regions of TS were 96.9% and 97.9% respectively, whereas those of WES were 95.8% and 100%, respectively. Conclusion: TS was confirmed to be a valid first-tier genetic test, with an average mean depth per gene higher than WES and a comparable detection rate and specificity.

MDB-40. TRANSCRIPTOME-BASED DRUG REPURPOSING IN GROUP 3 MEDULLOBLASTOMA

Abstract Medulloblastoma (MB) is the most common malignant pediatric brain tumor. MB is divided into four molecular subgroups (SHH, WNT, Group 3 [G3], and Group 4 [G4]), each defined by unique genetic and molecular features. G3MB is the most aggressive subgroup, with the lowest five-year survival. Features conferring high risk to G3MB include frequent metastases at diagnosis, MYC amplification, and chromosome i17q. Outcomes for G3MB patients remain dismal, partly due to a lack of G3MB-specific therapies, defining an important gap in MB research. To address this gap, we employed a strategy of comparing the G3MB transcriptomic signature against a repository of chemical perturbagens (LINCS) to identify FDA-approved, blood-brain barrier penetrant drugs capable of reverting the disease signature to a more normal cerebellum-like state. Following preliminary in silico and in vitro screening, we identified four drugs - fluoxetine, sertraline, simvastatin, and nortriptyline - that passed screening criteria and demonstrated cytotoxicity against HDMB03 cells at low micromolar doses. Each compound also inhibited clonogenicity, impaired wound healing, and impeded cell migration and invasion. Further, each compound induced apoptosis as evidenced by annexin V and propidium iodide staining. Western blotting confirmed both dose- and time-dependent increases in the expression of key apoptotic markers. Since nortriptyline possessed the best cytotoxicity profile against HDMB03 cells, we interrogated the transcriptome of HDMB03 cells treated with nortriptyline to identify cancer-related pathways inhibited by drug treatment. Our data revealed strong inhibition of E2F transcription factors, which are critical to cancer cell cycle progression and proliferation. Notably, five of the six E2F transcription factors as well as a panel of E2F target genes (HN1, KIF4A, CDCA3, CDCA8, and SSRP1) are all specifically upregulated in G3MB. This study highlights the promise of drug discovery and repurposing using subgroup-specific transcriptomes, which may deliver effective treatments expeditiously to high-risk patients.

Single-cell multi-gene identification of somatic mutations and gene rearrangements in cancer.

In this proof-of-concept study, we developed a single-cell method that provides genotypes of somatic alterations found in coding regions of messenger RNAs and integrates these transcript-based variants with their matching cell transcriptomes. We used nanopore adaptive sampling on single-cell complementary DNAlibraries to validate coding variants in target gene transcripts, and short-read sequencing to characterize cell types harboring the mutations. CRISPR edits for 16 targets were identified using a cancer cell line,and known variants in the cell line were validated using a 352-gene panel. Variants in primary cancer samples were validated using target gene panels ranging from 161 to 529 genes. A gene rearrangement was also identified in one patient, with the rearrangement occurring in two distinct tumor sites.

Diagnostic Gene Panel Testing in (Non)-Syndromic Patients with Cleft Lip, Alveolus and/or Palate in the Netherlands

Objectives: Clefts of the lip, alveolus and/or palate (CLA/P) are the most common craniofacial congenital malformations in humans. These oral clefts can be divided into non-syndromic (isolated) and syndromic forms. Many cleft-related syndromes are clinically variable and genetically heterogeneous, making it challenging to distinguish syndromic from non-syndromic cases. Recognition of syndromic/genetic causes is important for personalized tailored care, identification of (unrecognized) comorbidities, and accurate genetic counseling. Therefore, next generation sequencing (NGS)-based targeted gene panel testing is increasingly implemented in diagnostics of CLA/P patients. In this retrospective study, we assess the yield of NGS gene panel testing in a cohort of CLA/P cases. Methods: Whole exome sequencing (WES) followed by variant detection and interpretation in an a priori selected set of genes associated with CLA/P phenotypes was performed in 212 unrelated CLA/P patients after genetic counseling between 2015 and 2020. Medical records including family history and results of additional genetic tests were evaluated. Results: In 24 CLA/P cases (11.3%), a pathogenic genetic variant was identified. Twenty out of these 24 had a genetic syndrome requiring specific monitoring and follow-up. Six of these 24 cases (25%) were presumed to be isolated CLA/P cases prior to testing, corresponding to 2.8% of the total cohort. In eight CLA/P cases (3.8%) without a diagnosis after NGS-based gene panel testing, a molecular diagnosis was established by additional genetic analyses (e.g., SNP array, single gene testing, trio WES). Conclusion: This study illustrates NGS-based gene panel testing is a powerful diagnostic tool in the diagnostic workup of CLA/P patients. Also, in apparently isolated cases and non-familial cases, a genetic diagnosis can be identified. Early diagnosis facilitates personalized care for patients and accurate genetic counseling of their families.

Comprehensive ctDNA analysis to identify genome-instability and actionable mutation landscape in gynecologic cancers.

e17503 Background: Gynaecology malignancies are the second most prevalent cancers in women worldwide with rapid progression, high relapse, and mortality. Although HRR pathway inhibitors can greatly benefit clinical outcomes, patients without HRR pathway mutations may remain unbenefited while being profiled for tissue DNA or with a small target gene panel. Compelling data suggest that plasma genotyping shows a wide diversity of cancer mutations along genome instability indicators compared to tumor tissue. Here, we analyzed the mutational landscape of ctDNA and tissue DNA (tDNA) obtained from gynecologic cancer patients. We demonstrate the clinical utility of a comprehensive gene panel to identify multiple actionable mutations and genome instability indicators from ctDNA samples. Methods: We retrospectively analyzed the mutational landscape of 22 ovarian and endometrial cancer patients using target-hybridization enrichment of ctDNA and tDNA. Libraries prepared using a custom-designed comprehensive gene panel OncoIndx, targeting 600 cancer-relevant exons including MSI and HRR pathway genes, were sequenced on the Illumina NGS platform in pair-end mode. Variant calling was performed with an in-house developed bioinformatics pipeline. Results: NGS analysis revealed a total of 127 mutations consisting SNVs (45.6 %), truncations (40.94 %), amplifications (4.72 %), splice variants (4.72 %), and frameshift (3.94 %). Genome-wide mutational analysis showed a slightly higher mean TMB (15.7) for endometrial tumors compared to ovarian samples (11.85). The mean HRD score (45 %) was high for 90 % of patients and corroborated well with the HRR pathway gene mutation. 30 % of patients without HRR gene mutations including BRCA 1/2 had a high average HRD score (43 %) (LOH/TAI/LST) and may qualify for PARP inhibitors (PARPi). Alterations in Ras isoforms (H, K, and N) were the most frequent (36 %) followed by TP53 and PI3K (32 %). At the pathway level, DDR gene-variants were most frequently followed by alterations in proliferative signaling genes. ctDNA showed a wider mutational landscape, including clinically significant mutations, compared to tDNA. Additionally, ctDNA analysis revealed the presence of novel variants of SUFU and KIT, which remain unexplored in the context of cancer stem cells and relapse. Conclusions: Comprehensive mutational landscape showed the presence of genome-wide instability and the presence of mutations in the HRR pathway. Our results suggest that patients with high HRD scores and Ras pathway instability may benefit from Ras pathway inhibitors-PARPi combination. Mutational landscapes obtained from ctDNA were significantly wider and revealed multiple actionable targets compared to tDNA suggesting ctDNA as a viable and informative alternative for genotyping of gynecological cancers.

Clinical and genomic characterization of early-onset pancreatic cancer.

4018 Background: Pancreatic cancer is a leading cause of cancer-related death worldwide. Early-onset tumours (≤ 50 years) have risen alarmingly in recent years. However, the clinical and genomic particularities of early-onset pancreatic cancer (EOPC) remain poorly defined. We aimed to characterize EOPC and study its implications for treatment and prognosis. Methods: We performed a retrospective analysis of EOPC patients and a control group of patients ≥ 70 years (defined as average-onset pancreatic cancer, AOPC) followed at a tertiary cancer centre from 2010 to 2022. We collected baseline patient characteristics, tumor molecular profiling, germline genetic alterations, survival and treatment outcomes. We used a targeted gene panel to identify somatic genomic events and classified them according to the ESMO scale for clinical actionability of molecular targets (ESCAT). Key molecular findings were validated in an external cohort. We used a propensity score weighting method and multivariate Cox regression analysis to adjust for covariates. Results: We reviewed 824 patients, 336 of whom met all inclusion criteria (EOPC N = 139, AOPC N = 197). EOPC was associated with smoking status (current, 15.9 vs 7.9%, p = 0.03), lower prevalence of diabetes (3.7 vs 39.6%, p &lt; 0.01), better performance status (ECOG 0, 42.9 vs 19.3%, p &lt; 0.01), higher CA19.9 levels (median 574 vs 207.6UI/L, p = 0.07) and higher albumin levels (median 4.2 vs 3.9g/L, p &lt; 0.01). EOPC showed a non-significant higher prevalence of germline alterations (22.4 vs 14.5%, p = 0.18). After adjustment for baseline covariates, we observed no differences in survival (HR 0.94, 95% CI 0.63-1.4). We found a lower prevalence of KRASMUT tumors in EOPC when compared with AOPC (83.1 vs 91.1%, p = 0.12) and validated this finding in an independent cohort of 803 patients (82.9 vs 94.5%, p &lt; 0.01). Notably, EOPC were enriched in potentially actionable alterations when compared with AOPC, both in our cohort (19.1 vs 14.4%) and the validation cohort (14.4 vs 7.9%, p &lt; 0.01). Moreover, 294 patients in our cohort (EOPC N = 130; AOPC N = 164) were diagnosed with or eventually presented metastasis. EOPC more frequently received first-line 5FU-based chemotherapy (44.1 vs 14.7%, p &lt; 0.01) and received a greater number of treatment lines (median 2 vs 1, p &lt; 0.01). EOPC had a longer progression-free survival on first-line chemotherapy when compared with AOPC (HR 0.61, 95% CI 0.43-0.87, p &lt; 0.01) although there were no differences in response rate. Six EOPC patients received matched targeted therapies and 5 patients with AOPC. After adjusting for the number of treatment lines received, EOPC treated with targeted therapies exhibited longer OS compared with EOPC who did not (HR 0.34 95% CI 0.12-0.93, p = 0.04), whereas this trend was not observed in AOPC (HR 0.82 95% CI 0.32-2.11, p = 0.68). Conclusions: EOPC patients harbor unique clinical and molecular features and may particularly benefit from precision-based oncology approaches.

Clinical validity of oncogenic driver genes detected from circulating tumor DNA in the blood of lung cancer patients.

This study aimed to evaluate the concordance of oncogenic driver mutations between tumor tissues and circulating tumor DNA (ctDNA) in patients with lung cancer. In addition, this study attempted to reveal the clinical utility of ctDNA in lung cancer treatment. Recurrent or metastatic non-small cell lung cancer (NSCLC) patients were prospectively enrolled in this study. Tumor tissue and serial blood samples were obtained from newly diagnosed patients (Cohort A) or patients treated with targeted therapy (Cohort B) and targeted gene panel sequencing was conducted to identify tumor mutational profiles. At the time of diagnosis, patients in Cohort A with a high cell-free DNA (cfDNA) concentration had poorer overall survival than those with a low cfDNA concentration. The sensitivity and precision of ctDNA analysis in pre-treatment patients compared with those of tissue sequencing were 58.4% and 61.5%, respectively. Known lung cancer-associated variants of oncogenic driver genes, including EGFR and KRAS, and tumor suppressor genes, including TP53 and APC, were frequently detected in the ctDNA of the patients (76.9%). An association between smoking and TP53 mutation status was observed in both tissues and ctDNA (P=0.005 and 0.037, respectively). In addition, the EGFR T790M resistance mutation was detected solely from the ctDNA of two patients after treatment with an EGFR tyrosine kinase inhibitor. ctDNA may be a reliable prognostic biomarker with an additional role in treating patients with lung cancer. Further analyses are necessary to understand the properties of ctDNA and widen its clinical use.

The mutational landscape of circulating cell free DNA in patients with gastric cancer.

e16055 Background: Gastric cancer (GC) is a malignant tumor with a high lethality rate, with the highest incidence occurring in China. Gastric cancer is phenotypically heterogeneous, therefore the identification of genomic alteration is crucial for guiding clinical treatment. In this study, we aimed to use liquid biopsies to detect somatic mutations of GC patients and describe the mutational landscape of circulating tumor DNA. Methods: The blood samples of 157 Chinese patients with gastric malignancies were collected between 2018 to 2022. Somatic alterations were detected by either whole exome sequencing or high-depth targeted gene panel sequencing of ctDNA, using white blood cells as germline DNA control. Results: In our cohort, 1002 alterations of 323 genes were detected from plasma samples. We used MutSigCV tool to define the significantly mutated genes (SMG) and identified 16 SMG. Overall, the five most common mutated genes were TP53 (52.6%), LRP1B (15.4%), ARID1A(14.7%), SYNE1(12.2%), CDH1(10.9%). Recurrent mutations were identified in TP53 genes including c.524G &gt; A, c.713G &gt; T, which lead to loss of function of P53 protein according to evidence of clinical actionability from OncoKB (http://oncokb.org/). Genetic amplification of ERBB2 was detected in 8 patients (5.1%). 6 out of 95 samples were microsatellite instability (MSI), 3 were MSI-H and 3 were MSI-L.The median blood tumor mutation burden (bTMB) of all samples was 1.97 (range 0–143.51) mutations/Mb, 2 patients (1.27%) to high bTMB (≥ 20 mutations/Mb), 37 patients (23.57%) to intermediate bTMB (5-19 mutations/Mb), 118 patients (75.16%) to low bTMB ( &lt; 5 mutations/Mb). The Co-occurrence and mutual exclusivity of gastric cancer variations was investigated. Mutually exclusive mutation patterns were observed in TP53-KRAS (p &lt; 0.05), TP53-ARID1A (p &lt; 0.05), TP53-CDH1 (p &lt; 0.05) gene pairs. In addition, Gene Ontology (GO) and KEGG analysis were performed to analyze enrichment of somatic mutated genes and understand their biological function. The altered signaling pathway included PI3K−Akt signaling, MAPK signaling, Ras signaling, Rap1 signaling etc. 61 genes with somatic mutations were enriched to PI3K–Akt signaling pathway. Conclusions: In this study, we characterized the somatic mutational landscape of gastric cancer in 157 Chinese patients utilizing ctDNA which might guide clinical treatment.

Genetic alteration and ctDNA mutation profiling of esophageal cancer in Chinese patients.

e16046 Background: Esophageal cancer is the sixth most common cause of cancer-related mortality. There are two distinct histological types of esophageal cancer: esophageal adenocarcinoma (EAC) and esophageal squamous cell carcinoma (ESCC). In China, ESCC is the major subtype. Higher incidence of EAC is observed in western countries. Liquid biopsy detecting circulating tumor DNA is a non-invasive, real-time and highly sensitive approach that is able to identify and monitor the genetic mutations in tumor. Methods: In this study, we collected 490 Chinese esophageal cancer patients from 2018-2022 and characterized the mutational landscape of esophageal tumor tissue and circulating tumor DNA by whole exome sequencing or deep sequencing of targeted gene panel. The consistency of genetic alteration between tumor and ctDNA was also explored. Results: A total of 176 patients with plasma and 345 patients with cancer tissue were analyzed, among them, 31 patients had both ctDNA and tissue biopsy data. 277 genes with 1257 mutations were identified from plasma samples of 176 patients. The top frequently mutated genes were TP53 (89.1%), KMT2D (24.0%), SYNE1 (14.9%), NOTCH1 (14.3%), EP300 (13.7%), LRP1B (13.7%) and ZNF750 (10.3%). The mutation rates of these genes between ctDNA and tissue biopsy (N = 31 patients) were in concordance by Fisher's exact test (P &gt; 0.05). Most frequent mutations were found in TP53 gene, which account for 89.1% of total plasma samples and 90.4% of tumor tissues. KMT2D gene mutations were detected in 24% of patients which encode histone methyltransferase that methylates the Lys-4 position of histone H3 and regulates transcriptional activation, indicating epigenetic regulation might be involved in the pathogenesis of esophageal cancer. Functional enrichment analysis was performed including Gene Ontology (GO) and KEGG analysis across all the three domains, Biological process, Molecular Function, and Cellular component. The KEGG terms with the highest enrichment score were PI3K−Akt signaling pathway, which is a key driver in carcinogenesis. In addition, we didn’t observe the mutually exclusive mutation or co-occurrence pattern of any gene pairs in our cohort. Conclusions: We demonstrated the utility of liquid biopsy to draw a ctDNA mutation profiling of esophageal cancer in Chinese patients. Genetic mutations identified from ctDNA showed consistency with mutations from tumor tissue.

Clinical Characteristics and Genetic Analyses of Patients with Idiopathic Hypogonadotropic Hypogonadism

Idiopathic hypogonadotropic hypogonadism (IHH) is classified into two groups-Kalman syndrome and normosmic IHH (nIHH). Half of all cases can be explained by mutations in >50 genes. Targeted gene panel testing with nexrt generation sequencing (NGS) is required for patients without typical phenotypic findings. The aim was to determine the genetic etiologies of patients with IHH using NGS, including 54 IHH-associated genes, and to present protein homology modeling and protein stability analyzes of the detected variations. Clinical and demographic data of 16 patients (eight female), aged between 11.6-17.8 years, from different families were assessed. All patients were followed up for a diagnosis of nIHH, had normal cranial imaging, were without anterior pituitary hormone deficiency other than gonadotropins, had no sex chromosome anomaly, had no additional disease, and underwent genetic analysis with NGS between the years 2008-2021. Rare variants were classified according to the variant interpretation framework of the American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology. Changes in protein structure caused by variations were modeled using RoseTTAFold and changes in protein stability resulting from variation were analyzed. Half of the 16 had no detectable variation. Three (18.75%) had a homozygous (pathogenic) variant in the GNRHR gene, one (6.25%) had a compound heterozygous [likely pathogenic-variants of uncertain significance (VUS)] variant in PROK2 and four (25%) each had a heterozygous (VUS) variant in HESX1, FGF8, FLRT3 and DMXL2. Protein models showed that variants interpreted as VUS according to ACMG could account for the clinical IHH. The frequency of variation detection was similar to the literature. Modelling showed that the variant in five different genes, interpreted as VUS according to ACMG, could explain the clinical IHH.

Perinatal-Lethal Non-Immune Fetal Hydrops Attributed to MECOM-associated Bone Marrow Failure.

Pathogenic variants in MECOM, a gene critical to the self-renewal and proliferation of hematopoietic stem cells, are known to cause a rare bone marrow failure syndrome associated with amegakaryocytic thrombocytopenia and bilateral radioulnar synostosis known as RUSAT2. However, the spectrum of disease seen with causal variants in MECOM is broad, ranging from mildly affected adults to fetal loss. We report two cases of infants born preterm who presented at birth with symptoms of bone marrow failure including severe anemia, hydrops, and petechial hemorrhages; radioulnar synostosis was not observed in either patient, and unfortunately, neither infant survived. In both cases, genomic sequencing revealed de novo variants in MECOM considered to be responsible for their severe presentations. These cases add to the growing body of literature that describe MECOM-associated disease, particularly MECOM as a cause of fetal hydrops due to bone marrow failure in utero. Furthermore, they support the use of a broad sequencing approach for perinatal diagnosis, as MECOM is absent from available targeted gene panels for hydrops, and highlight the importance of postmortem genomic investigation.

Molecular diagnostic yield of genome sequencing versus targeted gene panel testing in racially and ethnically diverse pediatric patients

PurposeAdoption of genome sequencing (GS) as a first-line test requires evaluation of its diagnostic yield. We evaluated the GS and targeted gene panel (TGP) testing in diverse pediatric patients (probands) with suspected genetic conditions. MethodsProbands with neurologic, cardiac, or immunologic conditions were offered GS and TGP testing. Diagnostic yield was compared using a fully paired study design. ResultsA total of 645 probands (median age 9 years) underwent genetic testing, and 113 (17.5%) received a molecular diagnosis. Among 642 probands with both GS and TGP testing, GS yielded 106 (16.5%) and TGPs yielded 52 (8.1%) diagnoses (P < .001). Yield was greater for GS vs TGPs in Hispanic/Latino(a) (17.2% vs 9.5%, P < .001) and White/European American (19.8% vs 7.9%, P < .001) but not in Black/African American (11.5% vs 7.7%, P = .22) population groups by self-report. A higher rate of inconclusive results was seen in the Black/African American (63.8%) vs White/European American (47.6%; P = .01) population group. Most causal copy number variants (17 of 19) and mosaic variants (6 of 8) were detected only by GS. ConclusionGS may yield up to twice as many diagnoses in pediatric patients compared with TGP testing but not yet across all population groups.

Assessment of Tumor Mutational Burden and Outcomes in Patients With Diverse Advanced Cancers Treated With Immunotherapy

There are few studies assessing the association of tumor mutational burden (TMB) and clinical outcomes in a large cohort of patients with diverse advanced cancers. To clinically validate a TMB biomarker from a next-generation sequencing targeted gene panel assay. A prespecified cohort study using the deidentified clinicogenomic Tempus database of patients sequenced between 2018 and 2022, which contained retrospective, observational data originating from 300 cancer sites including 199 community sites and 101 academic sites. Patients with advanced solid tumors across 8 cancer types and more than 20 histologies, sequenced with Tempus xT who were treated with immune checkpoint inhibitors (ICIs) in the first-line or second-line setting were included. Data were analyzed from September 2018 to August 2022. Treatment with US Food and Drug Administration (FDA)-approved antiprogrammed cell death-1/programmed cell death-ligand 1 (PD-1/PD-L1) ICI and/or in combination with a cytotoxic T-lymphocyte-associated protein-4 ICI. The primary outcome was the association of tumor mutational burden (TMB) binary category (high [≥10 mut/mb] vs low) with overall survival (OS) in patients treated with ICIs. Secondary outcomes were progression-free survival (PFS), and time to progression (TTP). In the evaluable cohort of 674 patients, the median (IQR) age was 69.4 (28.6-89.8) years, 271 patients (40.2%) were female, and 435 patients (64.5%) were White. The most common advanced cancers were non-small cell lung cancer (330 patients [49.0%]), followed by bladder cancer (148 patients [22.0%]), and head and neck squamous cell carcinoma (96 patients [14.8%]). Median (IQR) follow-up was 7.2 (3.2-14.1) months. High TMB (TMB-H) cancers (206 patients [30.6%]) were significantly associated with longer OS than low TMB (TMB-L) cancers (hazard ratio [HR], 0.72; upper confidence bound [UCB], 0.91; P = .01). In a prospective subset of 403 patients treated with ICIs after TMB testing, TMB-H cancers (135 patients [33.5%]) were significantly associated with longer OS (HR, 0.61; UCB, 0.84; P = .005), PFS (HR, 0.62; UCB, 0.82; P = .003), and TTP (HR, 0.67; UCB, 0.92; P = .02) than TMB-L cancers. An overall survival benefit was seen regardless of the type of ICI used (pembrolizumab, 339 patients; HR, 0.67; UCB, 0.94; P = .03), other ICIs (64 patients; HR, 0.37; UCB, 0.85; P = .03), and after adjusting for PD-L1 and microsatellite stability status (403 patients; HR = 0.67; UCB, 0.92; P = .02). In this cohort study of patients with advanced solid tumors treated with ICIs in diverse clinics, TMB-H cancers were significantly associated with improved clinical outcomes compared with TMB-L cancers.

Evaluating clinical factors for improved utilization of targeted gene panel testing for inborn errors of immunity in a U.S. tertiary care pediatric hospital

RationaleThere has been an increased use of targeted gene panels to evaluate inborn errors of immunity (IEI) due to increased availability and reduced costs. Refinements to clinical indications for targeted gene panels may improve yields for the test. MethodA retrospective chart review on 140 patients aged ≤18 years old with suspected IEI who had clinical targeted gene panel testing (Invitae®) for primary immune deficiency at a tertiary pediatric hospital in the U.S. Relevant clinical information and basic immunology labs, consisting of complete blood count, immunoglobulins, vaccine titers, immune competence, and other relevant lab tests, were evaluated. Additional patients with targeted gene panels sent over the course of 5 years are being reviewed to confirm these initial findings. ResultOf 140 patients (median age 3.9 years [IQR 1.6–9.5], 62% male, 85% Caucasian suspected of IEI and had the targeted gene panel sent, 9% had positive findings that led to a change in diagnosis and/or treatment plan. The most common indications for IEI workups in our cohort were infections (37%), organ-specific inflammation (30%), and periodic fevers (17%), Table 1. Of 52 patients with infections, 3 had pathogenic, or likely pathogenic variants in TNFRSF13B, CD40LG, and XLA genes. All patients with infections and normal clinical-based immunology testing had negative gene panels. Only one patient with organ-specific inflammation had a variant in STAT3 explaining her clinical features. Of 10 patients presenting with systemic autoimmune disease or polyautoimmunity, 5 had positive findings (STAT5B, TNFAIP3, NLRP12, COPA, and NLRC4 genes). Two of 24 patients with periodic fevers had variants in TNFAIP3 and MEFV, one had elevated inflammatory markers and one had positive family history. Of 9 patients who had WES after negative genetic panel, one patient had positive finding that explains his syndromic manifestations (PTEN).Table 1Primary Indication of sending targeted IEI panel and resultsPrimary Indications of sending targeted IEI gene panel N = 140)Number of patients with positive genetic testing that led to changes in diagnosis and/or treatment plans (n(%))Affected genes (each with n = 1)Infections (n = 52, 37%)3(6%)TNFRSF13B CD40LG XLAOrgan-specific inflammation (n = 42, 30%)1(2%)STAT3 LOF• Skin lesion (14)• Inflammatory bowel disease (9)• Cytopenias including ITP, Evan'ssyndrome and HLH (7)• Lymphadenopathy (3)• Others: enteropathy (3), pericarditis(2), arthritis (1), autoimmune hepatitis(1), endocrinologic disease (1), SNHL(1)Periodic fevers (n = 24,17%)2(8%)TNFAIP3 MEFVAbnormal newborn screening or1(9%)ATMabnormal immunology labs (n = 11, 8%)• Abnormal SCID screen (4)• Abnormal immunology labs (7)Systemic autoimmune disease or5 (50%)STAT5Bpolyautoimmunity (n = 10, 7%)TNFAIP3• Systemic autoimmune disease (4)NLRP12• Polyautoimmunity (6)COPANLRC4Family history (n = 1,1%)1 (100%)ATM• Pathogenic variant in ATMgene in identical twinIEI, inborn error of immunity; ITP, immune thrombocytopenia; HLH, hemophagocytic lymphohistiocytosis; SNHL, sensorineural hearing loss; SCID, severe combined immunodeficiency. ConclusionTargeted gene panel testing has the highest yield in patients with polyautoimmunity. Low yields of targeted gene panel were found in organ-specific inflammation, infections with normal clinical testing, and periodic fevers with normal inflammatory markers and a negative family history of IEI. Limitations included potentially missed cases from chart review.

Impact of Variation in Practice in the Prenatal Reporting of Variants of Uncertain Significance by Commercial Laboratories: Need for Greater Adherence to Published Guidelines

ABSTRACT Ultrasound will identify fetal structural anomalies in up to 2% to 3% of pregnancies; these can vary from minor isolated defects to severe multisystem abnormalities. As genetic investigations into the causes can assist with prenatal and perinatal decision-making, various tests exist to assist in this endeavor. Rapid aneuploidy detection and chromosomal microarray analysis are both used for detecting chromosomal aneuploidy and microdeletions or duplications (termed copy number variations) for pregnancies with fetal abnormalities. Approximately 6% of fetuses with identifiable structural abnormalities have pathogenic copy number variations, and 32% have an abnormal karyotype. In the last 8 years, whole exome sequencing (WES) has been used for making a genetic diagnosis in the remaining undiagnosed pregnancies, and a recent meta-analysis determined an exome sequencing diagnostic yield of 31% following nondiagnostic chromosomal microarray analysis or karyotype. Use of exome sequencing is complicated, however, by its association with possibly detecting variants of uncertain significance (VUSs) and pathogenic/likely pathogenic (P/LP) variants in genes possibly noncausal for the prenatal phenotypes. Thus, the value of reporting VUSs is under debate, with various policies worldwide. This study's purpose was to evaluate the clinical impact of WES and targeted panel implementation at the clinical level when fetal structural anomalies suggest a single underlying gene disorder or syndrome. The study focus was the frequency of reporting VUSs, the diagnostic yield, and the contribution of reported VUSs toward a final diagnosis. This retrospective chart review at the Provincial Medical Genetics Program at BC Women's Hospital in Vancouver included patients undergoing WES between January 2016 and December 2020. Amniocentesis was used for obtaining fetal samples, except for 4 cases of sample acquisition at the time of pregnancy termination. A total of 124 patients underwent prenatal WES or panel testing at different commercial laboratories following the identification of fetal structural anomalies by ultrasound. Patients had either WES (n = 90) or panel testing (n = 31), with 3 patients undergoing both, as decided by the ordering clinical geneticist. Genetic diagnoses of variants classified by the laboratory as P or LP were obtained in 20 of 93 patients (21.5%) via prenatal WES and in 9 of 34 patients (26%) who underwent panel testing. The 2 tests' overall diagnostic yield was 23%. Of these, 20 patients (69%) were diagnosed with autosomal dominant disorders. Overall, 32% of patients with panel testing and 42% of patients who had WES had at least 1 VUS, and 23% of WES and 15% of panels reported more than 1 VUS. There was great variation in the reporting of VUSs between laboratories. The study uncovered a lack of strict adherence to guidelines for reporting of prenatal VUSs among various laboratories. One weakness (and strength) of the study is its use of multiple commercial laboratories for the WES, panel testing, and VUS reporting. Although this is beneficial to gain various perspectives (such as the lack of adherence to guidelines), it also diminishes the consistency of the results across the entire study because of varied procedures for diagnoses. In conclusion, this study reports on 124 pregnancies with fetal anomalies at a large tertiary care center experiencing the provision of prenatal WES (or targeted gene panels) with variant interpretation and sequencing outsourced to various laboratories. A result of 23% diagnostic yield for WES and panel testing occurred based on the laboratory-reported LP and P variants. The large number of VUSs reported by laboratories implies significant resource implications due to the time to review evidence and further examine or investigate the fetus and/or parent. This is essential, and the authors emphasize the complex nature of the required posttest genetic counseling. Based on these results, the authors strongly encourage strict adherence to recommendations on VUS reporting in the prenatal setting, with the importance of a multidisciplinary approach bringing laboratory and clinical expertise of prenatal genomics and genetics together.

COVID-19-Related Encephalitis in a Child with PIK3CD Defect

IntroductionCOVID-19 related encephalitis has been reported in pediatric patients; however, there are no reports in patients with inborn errors of immunity (IEI). Activated PI3K Delta Syndrome (APDS) is a disease of immune dysregulation with immunodeficiency, autoimmunity, and abnormal lymphoproliferation resulting from autosomal dominant gain-offunction variants in PIK3CD or PIK3R1 genes. We investigate a family with APDS, one mother and three children, one of whom developed COVID-19 related encephalitis. MethodsPatients were consented to an IRB-approved protocol at our institution. Medical records and detailed immunophenotyping were reviewed. Family members were sequenced for IEI with a targeted gene panel. ResultsThe index case is a 10-year-old female with a known pathogenic variant in PIK3CD (c.3061 G > A, p.Glu1021Lys), who contracted SARS-COV-2 despite one COVID-19 vaccination in the series. Her disease course included COVID-related encephalitis with cerebellitis and compression of the pons, resulting in lasting truncal ataxia and cerebellar mutism. At that time, the patient was not on immunoglobulin replacement therapy (IgRT), but was receiving Sirolimus. Besides the index case, 3 family members (2 brothers, 1 mother) also share the same PIK3CD variant with variable clinical and immunological phenotypes. All children exhibited high transitional B-cells, consistent with developmental block to follicular B cell stage. Increased non-class switched IgM+ memory B cells and skewing towards CD21lo B cell subset, which is considered autoreactive-like, was observed in all patients. Of note, the patient had low plasmablasts, but normal immunoglobulins. Of her family members, only one was receiving both sirolimus and IgRT. ConclusionsWe describe a rare case of COVID-19-related encephalitis in a patient with inborn error of immunity while not on IgRT. This may indicate infection susceptibility because of a lack of sufficient immunity to SARS-CoV-2, unlike the rest of her family with the same PIK3CD variant.

Multiple Intestinal Atresia with Combined Immunodeficiency, a New Gene Arises

We present here a single case report of a 2-day-old female born to a 29-year-old G4P3→4 Swartzentruber Amish mother at 35 weeks gestation that presented with abdominal distension and failure to pass meconium.Family history was positive for a cousin born with multiple intestinal atresia (MIA) and combined immunodeficiency (CID) who died in his first month of life. A primary immune deficiency targeted gene panel was done, covering TTC7A, in addition to 206 other genes, with no known mutations identified.An exploratory laparotomy was performed on DOL4 with MIA observed, as well as malrotation without volvulus. Repair of pyloric atresia and multiple small bowel resections and anastomosis were performed. She was kept NPO with TPN and NG suctioning of gastric contents for bowel rest.Immunologic profiling revealed low CD3+ cells (578/µL), very low CD8+ T cells (23/µL), low CD4+ T cells (485/µL), normal NK and B cell counts, low levels of serum IgM (< 10 mg/dL) with normal levels of IgG (likely maternal) and IgA. Mitogen stimulation testing was normal. Sanger sequencing showed a homozygous c.4867 T>G (p.Tyr1623Asp) mutation in PIK4A confirming the diagnosis of Gastrointestinal Defects and Immunodeficiency Syndrome 2 (GIDID2). Despite surgery, the patient continued to have large volume NG output without return of bowel function. Other treatment options, including long term TPN, potential small bowel and liver transplants, and HSCT were discussed, but the family elected for hospice given the poor prognosis. She passed away on DOL 28. PI4KIIIα, encoded by PI4KA, catalyzes the conversion of phosphatidyl inositol to phosphatidyl inositol-4-phosphate while using TTC7A as a scaffolding protein. This process is critical for development of enterocyte polarity and actomyosin contractility in multiple cell types. PI4KA mutations have only recently been implicated in causing MIA with CID and can be missed in many genetic screens. GIDID2 is a devastating disease in which intestinal surgery provides only an immediate mechanical fix, without improving mortality. Further research on the molecular mechanisms that govern GIDID2 is critically needed to identify new treatment modalities. Furthermore, awareness of PI4KA mutations in newborns with MIA with CID is critical, particularly in Amish populations.