Gene Panel Research Articles

Nexilin-related cardiomyopathy (NEXN-CMP) - an island's perspective

Abstract Introduction The phenotypic features of Nexilin related cardiomyopathies (NEXN-CMP) are poorly understood and under-reported. A better understanding of the genotype-phenotype association is paramount. Objectives The main objective of this study was to investigate the causative role and phenotypic expression of the NEXN gene in the Maltese Islands. Methodology Probands and relatives referred for a cardiac gene panel were evaluated. Subjects harbouring NEXN variants were identified. Those finally classified as having NEXN-CMP were evaluated for the purpose of this study. Results Out of 444 probands (confirmed or suspected cardiomyopathy), 12 probands were labelled with NEXN-CMP. This equates to a prevalence of 2.7% in the local cardiomyopathy pool, substantially higher to what has been previously reported. Genetic testing identified 19 individuals (probands [n=15] and relatives [n=4]) all harbouring the same novel frameshift variant: NEXN c.1589_1590del p.(Arg530LysfsTer3), classified as likely pathogenic according to ACMG criteria. Three (15.8%) harboured a second pathogenic variant (n=2 MYH7, n=1 TTN). Another 3 were not clinically affected (n=1 FH of SCD, n=2 first degree relatives of NEXN-CMP probands). These individuals were excluded from further analysis. The NEXN-CMP cohort (n=13) consisted of a dominant male (68.8%) population (54±22 years at presentation, 7.7% athletes, 12 families). Most were probands (n=12/13). DCM was the dominant phenotype (n=8, 61.5%) followed by HNDLVC (n=3, 23.1%) and HCM (n=2, 15.4%). One infantile DCM was homozygous for the NEXN variant. Symptoms were the most frequent method of presentation (n=6, 46.2%), followed by a FH of SCD and/or cardiomyopathy (n=3, 23.1%), abnormal echocardiogram (n=2 15.4%), OHCA (n=1, 7.7%) and inutero diagnosis (n=1, 7.7%). A FH of SCD was present in a third (n=4/12, 33.3%). The ECG was abnormal in the majority (n=10/11, 90.9%). Most patients had a LBBB (6/11, 54.5%) followed by inferolateral TWI (n=3, 27.3%), ventricular arrhythmias/high-grade AVB (n=1, 9.1% each). NEXN-CMP appears to be a left dominant cardiomyopathy. LV dilation and reduced LVEF were present in 84.6%. Non-ischaemic scar was present in 3/8 cases (37.5%), 2 had a ring-like pattern. RV morphology and function was normal in all cases. Tachyarrhythmias were frequently observed (n=4/6, 66.7%) during holter monitoring or exercise testing (n=2 NSVTs, n=1 VEs, n=1 SVEs). A fourth (n=3/11, 27.3%) were implanted with an electrophysiological device. No significant predictors for device implantation were identified at univariate analysis. One patient is on the transplant list (infantile DCM). Conclusion The prevalence of NEXN-CMP in Malta appears higher than previously reported (2.7%), possibly because of a founder mutation. DCM and HCM are the dominant presenting phenotypes, frequently associated with symptoms, an abnormal ECG, a FH of SCD and LV dilatation/dysfunction.

Congenital hearing loss - Introduction of the R67 large gene panel in England.

Congenital hearing loss - Introduction of the R67 large gene panel in England.

Identification of Genetic Variants Associated with Hereditary Thoracic Aortic Diseases (HTADs) Using Next Generation Sequencing (NGS) Technology and Genotype-Phenotype Correlations.

Hereditary thoracic aorta diseases (HTADs) are a heterogeneous group of rare disorders whose major manifestation is represented by aneurysm and/or dissection frequently located at the level of the ascending thoracic aorta. The diseases have an insidious evolution and can be encountered as an isolated manifestation or can also be associated with systemic, extra-aortic manifestations (syndromic HTADs). Along with the development of molecular testing technologies, important progress has been made in deciphering the heterogeneous etiology of HTADs. The aim of this study is to identify the genetic variants associated with a group of patients who presented clinical signs suggestive of a syndromic form of HTAD. Genetic testing based on next-generation sequencing (NGS) technology was performed using a gene panel (Illumina TruSight Cardio Sequencing Panel) or whole exome sequencing (WES). In the majority of cases (8/10), de novo mutations in the FBN1 gene were detected and correlated with the Marfan syndrome phenotype. In another case, a known mutation in the TGFBR2 gene associated with Loeys-Dietz syndrome was detected. Two other pathogenic heterozygous variants (one de novo and the other a known mutation) in the SLC2A10 gene (compound heterozygous genotype) were identified in a patient diagnosed with arterial tortuosity syndrome (ATORS). We presented the genotype-phenotype correlations, especially related to the clinical evolution, highlighting the particularities of each patient in a family context. We also emphasized the importance of genetic testing and patient monitoring to avoid acute aortic events.

MA08.08 Association of FDG-PET/CT Findings with Sufficient Amount of Tissue Samples for Gene Panel Testing in TBB/TBNA

MA08.08 Association of FDG-PET/CT Findings with Sufficient Amount of Tissue Samples for Gene Panel Testing in TBB/TBNA

Immune Checkpoint Inhibitor Therapy and Associations with Clonal Hematopoiesis

Cancer cohorts are now known to be associated with increased rates of clonal hematopoiesis (CH). We sort to characterize the hematopoietic compartment of patients with melanoma and non-small cell lung cancer (NSCLC) given our recent population level analysis reporting evolving rates of secondary leukemias. The advent of immune checkpoint blockade (ICB) has dramatically changed our understanding of cancer biology and has altered the standards of care for patients. However, the impact of ICB on hematopoietic myeloid clonal expansion remains to be determined. We studied if exposure to ICB therapy affects hematopoietic clonal architecture and if their evolution contributed to altered hematopoiesis. Blood samples from patients with melanoma and NSCLC (n = 142) demonstrated a high prevalence of CH. Serial samples (or post ICB exposure samples; n = 25) were evaluated in melanoma and NSCLC patients. Error-corrected sequencing of a targeted panel of genes recurrently mutated in CH was performed on peripheral blood genomic DNA. In serial sample analysis, we observed that mutations in DNMT3A and TET2 increased in size with longer ICB exposures in the melanoma cohort. We also noted that patients with larger size DNMT3A mutations with further post ICB clone size expansion had longer durations of ICB exposure. All serial samples in this cohort showed a statistically significant change in VAF from baseline. In the serial sample analysis of NSCLC patients, we observed similar epigenetic expansion, although not statistically significant. Our study generates a hypothesis for two important questions: (a) Can DNMT3A or TET2 CH serve as predictors of a response to ICB therapy and serve as a novel biomarker of response to ICB therapy? (b) As ICB-exposed patients continue to live longer, the myeloid clonal expansion may portend an increased risk for subsequent myeloid malignancy development. Until now, the selective pressure of ICB/T-cell activating therapies on hematopoietic stem cells were less known and we report preliminary evidence of clonal expansion in epigenetic modifier genes (also referred to as inflammatory CH genes).

Expanded phenotypic spectrum in MODY 5 patients with 17q12 deletion syndrome: experience from an Indian tertiary care hospital.

To study the clinical and genotypic spectrum of patients with HNF-1ß deletions (MODY 5) at a tertiary care hospital. This study included four patients from the Department of Endocrinology at Sher-i-Kashmir Institute of Medical Sciences Srinagar with a strong clinical suspicion of MODY 5. Genetic analysis, including a monogenic gene panel comprising 78 genes associated with MODY and other similar forms of monogenic diabetes, was done. Dosage analysis of HNF 1B by Multiplex Ligand-dependent Probe Amplification (MLPA) was performed. The mean age of patients was 22.25 years with a male-to-female ratio of 3:1. Associated phenotypic features included neurodevelopmental disorder in all four patients, insulin resistance in two patients (2/4) and alopecia in three patients (3/4). One patient had clinical and biochemical hyperandrogenism. All patients had renal malformations, and one patient had a Mullerian anomaly. Family history was present in 1 patient. All patients had pancreatic abnormalities, the most common type being dorsal agenesis of the pancreas (3/4), followed by annular pancreas (1/4). All patients had a genetic deletion of the gene HNF1B on chromosome 17 with a deletion interval of (?_37686431)_(37745059_?), (?_37687281)-(37744884_?), comprising exons 1 to 9. It is imperative to maintain a high index of suspicion for MODY 5 in patients presenting with renal anomalies and diabetes, even in the absence of a family history. Early identification allows for screening family members and ensures a comprehensive approach to identifying and managing other abnormalities in these patients.

Novel human PDX1 Asn196Thr variant causes pancreas agenesis and reduces the generation of duodenal enteroendocrine cells

Abstract Introduction/Objective Pancreatic and duodenal homeobox 1 (PDX1) is a well-studied transcription factor required for pancreas organogenesis. PDX1 mutations are associated with the development of diabetes. An 8-month-old female patient with previously uncharacterized compound heterogeneous PDX1 missense mutations (Thr151Met and Asn196Thr) was identified in this study. The patient has permanent neonatal diabetes, pancreas hypoplasia, and a malformed gallbladder. Methods/Case Report PDX1 Thr151Met and Asn196Thr variants in the patient were identified by next-generation sequencing gene panel and confirmed by Sanger sequencing. To examine how the mutations affect PDX1 functions in vitro, we expressed mouse Pdx1 Thr152Met and Asn197Thr, homologous to human PDX1 Thr151Met and Asn196Thr, respectively, in mouse insulinoma MIN6 cells and human embryonic kidney 293T cells and examined protein localization, protein stability, DNA binding, and protein-protein interaction. We also generated embryonic day 18 mouse embryos carrying Pdx1 Asn197Thr missense mutation using CRISPR/Cas9 to examine its roles in the development of the pancreas and other digestive organs in vivo. Results (if a Case Study enter NA) We found that the Pdx1 Asn197Thr variant, but not Thr152Met, altered its nuclear localization and disrupted PDX1-Onecut 1 interaction. Neither variant significantly affected PDX1 protein stability, but both reduced PDX1 binding to the Pdx1 gene promoter. Importantly, we found that the Pdx1 Asn197Thr variant caused pancreas agenesis and a reduction in the generation of enteroendocrine cells in the proximal duodenum in mouse models. Conclusion Our data indicated that the PDX1 Asn196Thr variant impairs its DNA binding and protein-protein interaction, causing pancreas agenesis and a reduction in the generation of duodenal enteroendocrine cells.

Multigene Panel Next-Generation Sequencing Techniques in the Management of Patients with Metastatic Colorectal Carcinoma: The Way Forward for Personalized Treatment? A Single-Center Experience

The efficacy and cost-effectiveness of Multigene Panel Next-Generation Sequencing (NGS) in directing patients towards genomically matched therapies remain uncertain. This study investigated metastatic colorectal cancer (mCRC) patients who underwent NGS analysis on formalin-fixed paraffin-embedded tumor samples. Data from 179 patients were analyzed, revealing no mutations in 39 patients (21.8%), one mutation in 83 patients (46.4%), and two or more mutations in 57 patients (31.8%). KRAS mutations were found in 87 patients (48.6%), including KRAS G12C mutations in 5 patients (2.8%), PIK3CA mutations in 40 patients (22.4%), and BRAF mutations in 26 patients (14.5%). Less common mutations were identified: ERBB2 in five patients (2.8%) and SMO in four patients (2.2%). Additionally, MAP2K1, CTNNB1, and MYC were mutated in three patients (2.4%). Two mutations (1.1%) were observed in ERBB3, RAF1, MTOR, JAK1, and FGFR2. No significant survival differences were observed based on number of mutations. In total, 40% of patients had druggable molecular alterations, but only 1.1% received genomically guided treatment, suggesting limited application in standard practice. Despite this, expanded gene panel testing can identify actionable mutations, aiding personalized treatment strategies in metastatic CRC, although current eligibility for biomarker-guided trials remains limited.

Genetic Biomarkers in Heart Failure: From Gene Panels to Polygenic Risk Scores.

This review aims to provide a comprehensive overview of the current understanding of genetic markers associated with heart failure (HF) and its underlying causative diseases, such as cardiomyopathies. It highlights the relevance of genetic biomarkers in diagnosing HF, predicting prognosis, potentially identifying its preclinical stages and identifying targets to enable the implementation of individualized medicine approaches. The prevalence of HF is increasing due to an aging population but with greater access to disease-modifying therapies. Advanced diagnostic tools such as cardiac magnetic resonance, nuclear imaging, and AI-enabled diagnostic testing are now being utilized to further characterize HF patients. Additionally, the importance of genetic testing in HF diagnosis and management is increasingly being recognized. Genetic biomarkers, including single nucleotide polymorphisms (SNPs) and rare genetic variants, are emerging as crucial tools for diagnosing HF substrates, determining prognosis and increasingly directing therapy. These genetic insights are key to optimizing HF management and delivering personalized treatment tailored to individual patients. HF is a complex syndrome affecting millions globally, characterized by high mortality and significant economic burden. Understanding the underlying etiologies of HF is essential for improving management and clinical outcomes. Recent advances highlight the use of multimodal assessments, including AI-enabled diagnostics and genetic testing, to better characterize and manage HF. Genetic biomarkers are particularly promising in identifying preclinical HF stages and providing personalized treatment options. The genetic contribution to HF is heterogeneous, with both monogenic and polygenic bases playing a role. These developments underscore the shift towards personalized medicine in HF management.

Comparative sequencing study of mismatch repair and homology-directed repair genes in endometrial cancer and breast cancer patients from Kazakhstan.

Endometrial cancer has been associated with pathogenic variants in mismatch repair (MMR) genes, especially in the context of the hereditary Lynch Syndrome. More recently, pathogenic variants in genes of homology-directed repair (HDR) have also been suggested to contribute to a subset of endometrial cancers. In the present hospital-based study, we investigated the relative distribution of pathogenic MMR or HDR gene variants in a series of 342 endometrial cancer patients from the Oncology Clinic in Almaty, Kazakhstan. In comparison, we also sequenced 178 breast cancer patients from the same population with the same gene panel. Identified variants were classified according to ClinVar, ESM1b, and AlphaMissense prediction tools. We found 10 endometrial cancer patients (2.9%) carrying pathogenic or likely pathogenic variants in MMR genes (7 MSH6, 1 MSH2, 2 MUTYH), while 14 endometrial cancer patients (4.1%) carried pathogenic variants in HDR genes (4 BRCA2, 3 BRCA1, 3 FANCM, 2 SLX4, 1 BARD1, 1 BRIP1). In the breast cancer series, we found 8 carriers (4.5%) of pathogenic or likely pathogenic variants in MMR genes (2 MSH2, 2 MSH6, 4 MUTYH) while 12 patients (6.7%) harbored pathogenic or likely pathogenic HDR gene variants (5 BRCA1, 3 BRCA2, 1 BRIP1, 1 ERRC4, 1 FANCM, 1 SLX4). One patient who developed breast cancer first and endometrial cancer later carried a novel frameshift variant in MSH6. Our results indicate that MMR and HDR gene variants with predicted pathogenicity occur at substantial frequencies in both breast and endometrial cancer patients from the Kazakh population.

Advancing Precision Oncology: Whole-Exome Sequencing in Endometrial Cancer Liquid-Based Cytology.

Diagnostic strategies for endometrial cancer have been evolving, with cytologic analysis being considered a key method in integrated oncologic diagnostics because of its less invasive nature and adaptability to various assessments. Liquid-based cytology (LBC) has emerged as a promising method for intact DNA preservation; it exhibits improved efficiency in advanced sequencing applications such as next-generation sequencing. However, despite the use of LBC in panel assays, its application in whole-exome sequencing (WES) for comprehensive genomic profiling remains underexplored. To investigate whether molecular classification is possible based on WES using DNA derived from LBC specimens. We combined WES with targeted gene panel analysis to compare genomic findings of LBC and traditional tissue samples obtained from 7 cases of endometrial cancer. We investigated pathogenic mutations, tumor mutational burden, and microsatellite instability, and achieved molecular classification with high accuracy. We found a substantial concordance between LBC and traditional tissue samples in terms of pathogenic mutation detection, with a 95% match in the LBC samples and 94% in the tissue samples. Notably, our results highlight the importance of combining WES with panel-based analysis in identifying the ultramutated status of a case that had been missed during panel analysis. Our findings emphasize the potential of LBC samples in the precise and noninvasive genomic analysis of cases of endometrial cancer and offer a new avenue for developing diagnostic and therapeutic strategies in precision oncology.

Comprehensive mutational profiling identifies new driver events in cutaneous leiomyosarcoma.

Cutaneous leiomyosarcoma (cLMS) is a rare soft tissue neoplasm, showing smooth muscle differentiation, that arises from the mesenchymal cells of the dermis. To-date, genetic investigation of these tumours has involved studies with small sample sizes and limited analyses that identified recurrent somatic mutations in RB1 and TP53, copy number gain of MYCOD and IGF1R, and copy number loss of PTEN. To better understand the molecular pathogenesis of cLMS, we comprehensively explored the mutational landscape of these rare tumours to identify candidate driver events. In this retrospective, multi-institutional study, we performed whole-exome sequencing and RNA sequencing on 38 cases of cLMS. TP53 and RB1 were identified as significantly mutated, thus, represent validated driver genes of cLMS. COSMIC mutational signatures SBS7a/b and DBS1 were recurrent, thus, ultraviolet light exposure may be an aetiological factor driving cLMS. Analysis of significantly recurrent somatic copy number alterations, which represent candidate driver events, found focal (<10Mb) deletions encompassing TP53 and KDM6B, and amplifications encompassing ZMYM2, MYOCD, MAP2K4 and NCOR1. A larger (24 Mb) recurrent deletion encompassing CYLD was also identified as significant. Significantly recurrent broad copy number alterations, involving at least half of a chromosome arm, included deletions of 6p/q, 10p/q, 11q, 12q, 13q and 16p/q, and amplification of 15q. Notably PTEN is located on 10q, RB1 on 13q and IGFR1 on 15q. Fusion gene analysis identified recurrent CRTC1/3::MAML2 fusions, as well as many novel fusions in individual samples. Our analysis of the largest number of cLMS cases to-date highlights the importance of large cohort sizes and the exploration beyond small targeted gene panels when performing molecular analyses, as it allowed a comprehensive exploration of the mutational landscape of these tumours and identification of novel candidate driver events. It also uniquely afforded the opportunity to compare the molecular phenotype of cLMS with LMS of other tissue types, such as uterine and soft tissue LMS. Given that molecular profiling has resulted in the development of novel targeted treatment approaches for uterine and soft tissue LMS, our study now allows the same opportunities to become available for patients with cLMS.

Whole Exome Sequencing and Panel-Based Analysis in 176 Spanish Children with Neurodevelopmental Disorders: Focus on Autism Spectrum Disorder and/or Intellectual Disability/Global Developmental Delay

Background: Neurodevelopmental disorders (NDDs) represent a significant challenge in pediatric genetics, often requiring advanced diagnostic tools for the accurate identification of genetic variants. Objectives: To determine the diagnostic yield of whole exome sequencing (WES) with targeted gene panels in children with neurodevelopmental disorders (NDDs). Methods: This observational, prospective study included a total of 176 Spanish-speaking pediatric patients with neurodevelopmental disorders (NDDs), encompassing intellectual disability (ID), global developmental delay (GDD), and/or autism spectrum disorder (ASD). Participants were recruited from January 2019 to January 2023 at a University Hospital in Madrid, Spain. Clinical and sociodemographic variables were recorded, along with genetic study results. The age range of the subjects was 9 months to 16 years, and the percentage of males was 72.1%. The diagnostic yield of whole exome sequencing (WES) was calculated both before and after parental testing via Sanger DNA sequencing. Results: The study included 176 children: 67 (38.1%) with ID, 62 (35.2%) with ASD, and 47 (26.7%) with ASD + ID. The diagnostic yield of proband-only exome sequencing was 12.5% (22/176). By group, the diagnostic yield of proband-only exome sequencing was 3.2% in the ASD, 12.7% in the ASD + ID, and 20.8% in the ID group. Variants of uncertain significance (VUS) were found in 39.8% (70/176). After parental testing, some variants were reclassified as “likely pathogenic”, increasing the diagnostic yield by 4.6%, with an overall diagnostic yield of 17.1%. Diagnostic yield was higher in patients with syndromic ID (70.6%% vs. 29.4%; p = 0.036). Conclusions: A sequential approach utilizing WES followed by panel-based analysis, starting with the index case and, when appropriate, including the parents, proves to be a cost-effective strategy. WES is particularly suitable for complex conditions, as it allows for the identification of potentially causative genes beyond those covered by targeted panels, providing a more comprehensive analysis. Including parental testing enhances the diagnostic yield and improves accuracy, especially in cases with variants of uncertain significance (VUS), thereby advancing our understanding of NDDs.

An Analysis of Genetic Polymorphisms in 76 Genes Related to the Development of Ovarian Tumors of Different Aggressiveness.

Borderline ovarian tumors (BOTS) are rare neoplasms of intermediate aggressiveness between cystadenomas and low-grade ovarian cancers (lgOvCa), which they share some molecular resemblances with. In contrast to the most frequent and well-described high-grade ovarian carcinomas (hgOvCa), the molecular background of BOTS and lgOvCa is less thoroughly characterized. Here, we aimed to analyze genetic variants in crucial tumor suppressors and oncogenes in BOTS (with or without the BRAF V600E mutation), lgOvCa, and hgOvCa in two gene panels using next-generation sequencing. Then, we verified the existence of selected polymorphisms by Sanger sequencing. Finally, Western blot analyses were carried out to check the impact of the selected polymorphisms on the expression of the corresponding proteins. Our study contributes to the molecular characterization of ovarian neoplasms, demonstrating divergent polymorphic patterns pointing to distinct signaling pathways engaged in their development. Certain mutations seem to play an important role in BOTS without the BRAF V600E variant (KRAS) and in lgOvCa (KRAS and NRAS), but not in hgOvCa. Additionally, based on multivariable regression analyses, potential biomarkers in BOTS (PARP1) and hgOvCa (FANCI, BRCA2, TSC2, FANCF) were identified. Noteworthy, for some of the analyzed genes, such as FANCI, FANCD2, and FANCI, FANCF, TSC2, the status of BRCA1/2 and TP53, respectively, turned out to be crucial. Our results shed new light on the similarities and differences in the polymorphic patterns between ovarian tumors of diverse aggressiveness. Furthermore, the biomarkers identified herein are of potential use as predictors of the prognosis and/or response to therapy.

Personalized medicine beyond cancer: Impact on other diseases

Personalized medicine beyond cancer: Impact on other diseases With a focus on type 2 diabetes and Parkinson’s disease, Dr Priya Hays explores how personalized medicine approaches are impacting the development of therapies for other chronic conditions beyond cancer. Genetic testing and cancer outcomes have been considerably advanced by precision medicine, or personalized medicine, with gene panel testing conducted routinely in patient care and targeted therapy approaches incorporated into oncology. It may be argued that with these significant advances, diseases such as type 2 diabetes, cardiovascular disease, neurological diseases such as Parkinson’s disease, and Alzheimer’s disease and psychiatric diseases have been unaffected by the gains of personalized medicine. However, this is not entirely accurate as aspects of personalized medicine such as pharmacogenomics, targeted approaches for neurological systems, and single nucleotide polymorphism detection have resulted in safe and effective diagnostics and treatments for cardiovascular disease, diabetes and neurological disorders, and depression and schizophrenia. Outlined here are precision medicine approaches for type 2 diabetes and Parkinson’s disease.

Exploring lipodystrophy gene expression in adipocytes: unveiling insights into the pathogenesis of insulin resistance, type 2 diabetes, and clustering diseases (metabolic syndrome) in Asian Indians.

Studying the molecular mechanisms of lipodystrophy can provide valuable insights into the pathophysiology of insulin resistance (IR), type 2 diabetes (T2D), and other clustering diseases [metabolic syndrome (MetS)] and its underlying adipocentric disease (MetS disease). A high-confidence lipodystrophy gene panel comprising 50 genes was created, and their expressions were measured in the visceral and subcutaneous (both peripheral and abdominal) adipose depots of MetS and non-MetS individuals at a tertiary care medical facility. Most lipodystrophy genes showed significant downregulation in MetS individuals compared to non-MetS individuals in both subcutaneous and visceral depots. In the abdominal compartment, all the genes showed relatively higher expression in visceral depot as compared to their subcutaneous counterpart, and this difference narrowed with increasing severity of MetS. Their expression level shows an inverse correlation with T2D, MetS, and HOMA-IR and with other T2D-related intermediate traits. Results also demonstrated that individualization of MetS patients could be done based on adipose tissue expression of just 12 genes. Adipose tissue expression of lipodystrophy genes shows an association with MetS and its intermediate phenotypic traits. Mutations of these genes are known to cause congenital lipodystrophy syndromes, whereas their altered expression in adipose tissue contributes to the pathogenesis of IR, T2D, and MetS.

Non-Hotspot PIK3CA Variants Have Higher Variant Allele Frequency and are More Common in Syndromic Vascular Malformations.

PIK3CA variants are known to cause vascular malformations. We were interested in studying the phenotypic spectrum, the location within the PIK3CA gene, and the variant allele frequency (VAF) of somatic PI3KCA variants in vascular malformations. Clinical data of consecutive patients with extracranial/extraspinal vascular malformations were collected in the context of the VASCOM cohort (2008-2022, n = 558). Starting October 2020, biopsy samples were tested with the TSO500 gene panel (Illumina). All consenting patients with PIK3CA variants were included in this study. Eighty-nine patients had available genetic results by June 2022. PIK3CA variants (n = 25) were found in 16 simple/combined (nonsyndromic) vascular malformations and in nine vascular malformations associated with other anomalies (syndromic). Four hotspot variants in exons 9 and 20 (c.1624G>A, c.1633G>A, c.3140A>G, c.3140A>T) were identified in 16/25 patients (VAF 0.9%-9.7%). Six non-hotspot variants (c.328_330del, c.323_337del, c.353G>A, c.1258T>C, c.3132T>A, c.3195_3203delinsT) were detected in nine patients (VAF 3.6%-31.7%). Non-hotspot variants were more frequent in syndromic than nonsyndromic vascular malformations (p = 0.0034) and exhibited a higher VAF than hotspot variants (p = 0.0253). Our study contributes to the growing body of knowledge of the genetic background in vascular malformations. Further studies will enrich the ever-growing list of pathogenic PIK3CA variants associated with vascular malformations.

7673 Genetic Analysis and Clinical Characterization of Patients with Congenital Hypopituitarism Due to Gene Defects

Abstract Disclosure: T.M. Sasson: None. I.J. Arnhold: None. B.B. Mendonca: None. L.R. Carvalho: None. Introduction: Congenital hypopituitarism is characterized by the deficiency of one or more hormones secreted by the anterior pituitary gland. The genetics of this pathology is complex, and the molecular diagnosis is established in approximately 13% of cases. Objectives: The aim of the present study is to characterize the phenotype harboring pathogenic variants causing congenital hypopituitarism. Methods: Among 393 subjects with congenital hypopituitarism followed up at a tertiary health center, 57 subjects (14,5%) were selected due to a confirmed molecular diagnosis by exome in 9 subjects or by gene panel or Sanger sequencing in 48 subjects. This was a retrospective study where data from medical records, clinical, laboratory and radiological characteristics were analyzed. Results: The most frequent variants identified by sequencing analyzes were in the following genes: PROP1 (29.8%), GH1 (22.8%), GHRHR (15.7%), followed by GLI2, OTX2, HESX1, POU1F1, TGIF1, GHRS, CDH2, SATB1, SOX3 e LZTR1. Consanguinity was reported in 50% of cases, and there was the presence of familial cases of short stature in 60%. The average age at the start of follow-up was 11.1 years (11 SD), 40% of them were male and 52.3% had physical stigmata typical of GH deficiency. Regarding hormonal deficiencies, 27 (47%) had isolated Growth Hormone Deficiency (DGHI) and the others, multiple deficiencies. MRI study of the pituitary gland showed some alteration in 66%, 54% of whom had adenohypophysis hypoplasia and 31% had an ectopic or non-visualized neurohypophysis. From 29 patients with IGF-1 levels available, 28 were below the reference range for sex and age. The serum GH peak after the stimulus test was on average 1.28 ng/mL (1.4SD), of these, only 4 cases had GH peak between 3.3 and 5.0 ng/mL and 1 patient with low IGF-1 and an ectopic neurohypophysis had GH peak of 6.0 ng/mL in a glucagon test. Regarding growth, the initial height in z-score was -4.8 and the average growth at the end of the first year of treatment was 10.7cm, with the average height after this period of Z-3.35. The average of final delta target height (final height Z-score minus the target height Z-score) of patients who underwent hormonal treatment was -0,53 (good response = delta &amp;lt;-1.5). Conclusion: In subjects with congenital hypopituitarism and an established molecular diagnosis, the most frequently affected genes were PROP1 and GH1, the great majority presented a stimulated GH peak &amp;lt; 3.3 ng/mL (only one patient had GH &amp;gt; 5), low basal serum IGF-1 and good increase in growth speed at the end of the first year of treatment with recovery of final height at the end of GH therapy. Presentation: 6/3/2024

7826 The Identification of an Activating ESR1 Mutation in an Aggressive Prolactinoma Enabled the Use of Personalized Treatment

Abstract Disclosure: T. Paes: None. J.B. Mebarak: None. J. C. Magnotto: None. G. A. Stamatiades: None. Y. Kuang: None. C. Paweletz: None. E.R. Laws: None. R.S. Carroll: None. R. Jeselsohn: None. D. R. Mohan: None. A. Marcondes Lerario: None. W. L. Bi: None. D. A. Reardon: None. D. M. Meredith: None. U.B. Kaiser: None. A. Abreu: None. Introduction: Prolactinomas are benign tumors typically treated with dopamine agonists; few progress on medical therapy, through unclear molecular mechanisms. Although the SF3B1 mutation has been identified in one study as a driver of aggressive prolactinomas, in most cases no genetic mutations have been reported. We aimed to identify genetic alterations associated with prolactinomas using a gene panel. Method and Results: Oncopanel, a massively parallel sequencing panel, was performed to identify genomic variants and copy number variation (CNV) in a cohort of 21 patients with prolactinomas. No SF3B1 pathogenic variants were identified in this cohort. A MEN1 mutation was detected in a macroadenoma resistant to dopamine agonist. In a case of an extremely aggressive prolactinoma diagnosed in a 49-year-old woman, a somatic ESR1 [encoding estrogen receptor alpha (ERα)] mutation (ESR1Y537S) was identified in tissue from the patient’s fourth surgery. Using droplet digital PCR (ddPCR), ESR1Y537S was also detected in circulating cell-free DNA. We aimed to investigate whether ESR1Y537S may have driven initial tumor formation, or whether it was acquired as the tumor progressed. To this end, Sanger sequencing and ddPCR analyses of DNA from the first two resected tumors were performed and failed to identify ESR1Y537S. In the CNV analysis, the ESR1-mutated prolactinoma had the highest number of CNVs, compared to the rest of the cohort. Despite efforts to control tumor growth with multiple therapeutic modalities, the mutated prolactinoma had progressively enlarged post-menopausally, with invasion of surrounding structures. ESR1Y537S is a hotspot mutation in metastatic breast cancer. In breast cancer cells, ESR1Y537S activates the ERα independent of ligand binding, resulting in high cell proliferation and conferring resistance to classic hormonal treatment in ER-positive breast cancer. Elacestrant, a second-line ER degrader, increases overall free-survival in patients with resistant breast cancer and ESR1Y537S. Given the lack of response to multimodality therapy, off-label elacestrant was initiated after the third cycle of radiotherapy was completed in the patient with the aggressive prolactinoma. The combination of radiotherapy with this personalized treatment targeting ESR1 activity (elacestrant) was able to control tumor growth and significantly reduce prolactin levels. Conclusion:ESR1 regulates lactotroph differentiation and proliferation, possibly contributing to prolactinoma tumorigenesis. Our data support a role for ESR1Y537S in this prolactinoma’s unusually aggressive behavior in the postmenopausal setting. Molecular profiling revealing the mutation (ESR1Y537S) allowed the patient to receive a targeted therapy which, together with radiotherapy, resulted in a decrease in prolactin levels and a remarkable improvement in disease control. Presentation: 6/2/2024

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&amp;gt;C/ p.Tyr159His; c.362G&amp;gt;A/ p.Cys121Tyr; c.319+2T&amp;gt;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&amp;gt;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&amp;gt;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 &amp;lt; 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