Whole Exome Sequencing Analysis Research Articles

FBXO11 variants are associated with intellectual disability and variable clinical manifestation in Chinese affected individuals.

F-box protein 11 (FBXO11) is a member of F-Box protein family, which has recently been proved to be associated with intellectual developmental disorder with dysmorphic facies and behavioral abnormalities (IDDFBA, OMIM: 618089). In this study, 12 intellectual disability individuals from 5 Chinese ID families were collected, and whole exome sequencing (WES), sanger sequencing, and RNA sequencing (RNA-seq) were conducted. Almost all the affected individuals presented with mild to severe intellectual disability (12/12), global developmental delay (10/12), speech and language development delay (8/12) associated with a range of alternate features including increased body weight (7/12), short stature (6/12), seizures (3/12), reduced visual acuity (4/12), hypotonia (1/12), and auditory hallucinations and hallucinations (1/12). Distinguishingly, malformation was not observed in all the affected individuals. WES analysis showed 5 novel FBXO11 variants, which include an inframe deletion variant, a missense variant, two frameshift variants, and a partial deletion of FBXO11 (exon 22-23). RNA-seq indicated that exon 22-23 deletion of FBXO11 results in a new mRNA structure. Conservation and protein structure prediction demonstrated deleterious effect of these variants. The DEGs analysis revealed 148 differentially expressed genes shared among 6 affected individuals, which were mainly associated with genes of muscle and immune system. Our research is the first report of FBXO11-associated IDDFBA in Chinese individuals, which expands the genetic and clinical spectrum of this newly identified NDD/ID syndrome.

The Role of Cilia and the Complex Genetics of Congenital Heart Disease

Congenital heart disease (CHD) can affect up to 1% of live births, and despite abundant evidence of a genetic etiology, the genetic landscape of CHD is still not well understood. A large-scale mouse chemical mutagenesis screen for mutations causing CHD yielded a preponderance of cilia-related genes, pointing to a central role for cilia in CHD pathogenesis. The genes uncovered by the screen included genes that regulate ciliogenesis and cilia-transduced cell signaling as well as many that mediate endocytic trafficking, a cell process critical for both ciliogenesis and cell signaling. The clinical relevance of these findings is supported by whole-exome sequencing analysis of CHD patients that showed enrichment for pathogenic variants in ciliome genes. Surprisingly, among the ciliome CHD genes recovered were many that encoded direct protein–protein interactors. Assembly of the CHD genes into a protein–protein interaction network yielded a tight interactome that suggested this protein–protein interaction may have functional importance and that its disruption could contribute to the pathogenesis of CHD. In light of these and other findings, we propose that an interactome enriched for ciliome genes may provide the genomic context for the complex genetics of CHD and its often-observed incomplete penetrance and variable expressivity.

Osteomesopyknosis associated with a novel ALOX5 variant that impacts the RANKL pathway.

Bone tissue homeostasis relies on the coordinated activity of the bone-forming osteoblasts and bone-resorbing osteoclasts. Osteomesopyknosis is considered a distinctive rare sclerosing skeletal disorder of unelucidated pathophysiology and presumably autosomal dominant transmission. However, the causal genes are unknown. We present a case report encompassing clinical assessments, imaging studies, and whole-exome sequencing analysis, complemented by functional invitro experiments. This new case of osteomesopyknosis was associated with a missense ALOX5 variant predicted to induce protein misfolding and proteasomal degradation. Transfection experiments demonstrated that the variant was associated with reduced protein levels restored by proteasomal inhibition with bortezomib. Likewise, gene expression analysis showed that the mutated gene was associated with a decreased RANKL/OPG ratio, which is a critical driver of osteoclast precursor differentiation. Our data indicate impaired bone resorption as the underlying mechanism of this rare osteosclerosis, implicating ALOX5 pathogenic variants as potential etiological factors.

Functional Characterization of an Ammonia Transporter RhBG Mutation Identified in Diabetic Kidney Disease Patients

Metabolic acidosis is a major complication of CKD (chronic kidney disease) and DKD (diabetic kidney disease). The kidney responds to metabolic acidosis by eliminating acid, primarily by producing and excreting NH4+ in the urine. Two Rh glycoproteins, RhBG and RhCG, expressed in the collecting duct are directly involved in NH3/NH4+transport. We previously determined that RhBG transports both NH4+ and NH3 across the basolateral membrane of intercalated cells. Impaired NH3/NH4+ production and/or transport may be a factor in increasing acid load and worsening kidney disease. In type 2 diabetes, net endogenous acid production is significantly elevated, and the proportion of acid excreted as NH4+ is decreased even after accounting for diet and renal function. This is significant given that the association between acidosis and faster decline in GFR is now well established. In DKD, impaired NH4+ excretion as a potential risk factor for progression of kidney disease remains poorly understood. Specifically, the genetic risk factors contributing to DKD are unclear. Hypothesis: Defined RhBG variant(s) linked to DKD cause impaired NH3/NH4+ transport that may contribute to DKD development and progression. Methods: We conducted whole-exome sequencing (WES) analysis using data from A therosclerosis R isk i n C ommunities (ARIC) study and the C hronic R enal I nsuffciency C ohort (CRIC) study. We selected DKD patients with the most rapid GFR decline and compared them with: 1) subjects without kidney disease (healthy control); 2) diabetic patients without CKD (DM control); 3) CKD patients without diabetes (CKD control). This approach will identify single nucleotide variants (SNVs) of RhBG associated with DKD. This will also determine if the detected variants contribute to the development of kidney disease per se and whether they contribute to the development of diabetes and/or fast GFR decline. To study function, we expressed RhBG variants (and wild-type RhBG) in Xenopus oocytes by microinjecting oocytes with the respective mRNA. The effect of defined SNV of RhBG on NH3/NH4+ transport was assessed from measurements of changes in intracellular pH (pHi), membrane potential (Vm), whole cell currents (I) or surface pH (pHs) induced by exposing the oocytes to bath solutions containing NH4Cl or methyl ammonium chloride (MA/MA+). Results: We identified an SNV, rs3748569 (minor allele frequency MAF > 0.05), that was significantly associated with DKD. Significance was determined compared to DM control (OR=1.38, p=0.0026) and healthy control (OR=1.26, p=0.01). This variant has also been predicted to be deleterious by 4 bioinformatic tools. Our electrophysiological measurements showed that the resultant amino acid switch, G315R, completely inhibited transport of NH3 and NH4+ (p<0.001) and the transport of MA and MA+ (p<0.0001), compared with RhBG-WT. Conclusions: WES results suggest that this naturally occurring RhBG mutation, G315R, contributes to DKD occurrence. This effect is through the development of kidney disease and less likely due to development of diabetes. It is therefore likely a general CKD risk factor. Inhibition of NH3/NH4+ transport by this mutation is a contributing factor to its deleterious effect. This work provides experimental validation of computational predictions concerning CKD and diabetes. NIH-U54 GM104940, Paul Teschan Research, NIH-R01, Carol Lavin Bernick, Tulane institutional. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Trio-based whole-exome sequencing reveals mutations in early-onset high myopia

PurposeMyopia, especially high myopia (HM), represents a widespread visual impairment with a globally escalating prevalence. This study aimed to elucidate the genetic foundations associated with early-onset HM (eoHM) while delineating...

Biallelic Deletion of PEX26 Exon 4 in a Boy with Phenotypic Features of both Zellweger Syndrome and Infantile Refsum Disease

Introduction: Peroxisome biogenesis disorders (PBDs) encompass a group of diseases marked by clinical and genetic heterogeneity. Phenotypes linked to PBDs include Zellweger syndrome, neonatal adrenoleukodystrophy, infantile Refsum disease (IRD), rhizomelic chondrodysplasia punctata type 1, and Heimler syndrome. PBD phenotypes manifest through hypotonia, developmental delay, facial dysmorphism, seizures, liver dysfunction, sensorineural hearing loss, and retinal dystrophy. Methods: The proband underwent comprehensive clinical evaluation, followed by whole-exome sequencing (WES) coupled with copy number analysis (CNV), aimed at identifying potential disease-causing variants aligning with the observed phenotype. Results: Our findings detail an individual exhibiting developmental delay, hearing loss, visual impairment, hepatomegaly, and splenomegaly, attributed to a biallelic deletion of exon 4 in the PEX26 gene. The WES analysis of the index case did not uncover any pathogenic/likely pathogenic single-nucleotide variations that could account for the observed clinical findings. However, the CNV data derived from WES revealed a homozygous deletion in exon 4 of the PEX26 gene (NM_001127649.3), providing a plausible explanation for the patient’s clinical features. The exon 4 region of PEX26 encodes the transmembrane domain of the protein. The transmembrane domain plays a crucial role in anchoring the protein within lipid bilayers, and its absence can disrupt proper localization and functioning. As a result, this structural alteration may impact the protein’s ability to facilitate essential cellular processes related to peroxisome biogenesis and function. Conclusion: The index patient, which presented with hearing loss, retinal involvement and hepatic dysfunction in adolescence age, has atypical clinical course that can be considered unusual for Zellweger syndrome (ZS) and IRD phenotypes, and its rare genotypic data (in-frame single exon deletion) expands the PBD disease spectrum. This study revealed for the first time that PEX26 protein transmembrane domain loss exhibits an unusual course with clinical findings of IRD and ZS phenotypes. WES studies, incorporating CNV analyses, empower the identification of novel genetic alterations in genes seldom associated with gross deletion/duplication variations, such as those in the PEX26 gene. This not only enhances diagnostic rates in rare diseases but also contributes to broadening the spectrum of causal mutations.

Exploring genetic variants in congenital monosaccharide-disaccharide metabolism: Carrier ratiosandphenotypic insights.

Adverse food reactions, often underestimated, encompass congenital monosaccharide-disaccharide metabolism disorders, yielding diverse outcomes such as abdominal pain, diarrhea, bleeding disorders, and even death. This study retrospectively scrutinized genetic variants linked to these disorders in a cohort subjected to whole-exome sequence analysis (WES), determining carrier frequencies and genotype-phenotype correlations. Data from 484 patients, were retrospectively analyzed using a gene panel (ALDOB, FBP1, GALE, GALK1, GALM, GALT, LCT, SLC2A2, SLC5A1, SI) for congenital monosaccharide-disaccharide metabolism disorders. WES was performed on patients using the xGen Exome Research Panel v2 kit, utilizing Next Generation Sequence Analysis (NGS). The study encompassed pathogenic, likely pathogenic, and variant of uncertain significance (VUS) variants. Among 484 patients (244 female, 240 male), 17.35% carried 99 variants (67 distinct) in the analyzed genes. Pathogenic/likely pathogenic allele frequency stood at 0.013, while VUS allele frequency was 0.088. Notably, 44% (37/84) of patients harboring mutations manifested at least one relevant phenotype. Carriage frequencies ranged from 1:25 (SI gene) to 1:968 (GALE gene), with the estimated disease frequency spanning from 1:2500 to 1:3748000. Our study underscores clinical manifestations in heterozygous carriers of recessive genetic disorders, addressing gaps in carrier frequencies and phenotypic effects for congenital monosaccharide-disaccharide metabolism disorders. This knowledge can improve these conditions'diagnosis and management, potentially preventing adverse food reactions and their associated complications.

Current Diagnostic Approaches in the Genetic Diagnosis of Disorders of Sex Development.

Disorders of sex development (DSD) are a clinically and genetically highly heterogeneous group of congenital disorders. The most accurate and rapid diagnosis may be possible with a complementary multidisciplinary diagnostic approach, including comprehensive clinical, hormonal, and genetic investigations. Rapid and accurate diagnosis of DSD requires urgency in terms of gender selection and management of the case. Despite the genetic tests performed in current daily practice, the genetic cause is still not elucidated in a significant proportion of cases. Karyotype analysis can be used as a standard for sex chromosome identification. In addition, quantitative fluorescent polymerase chain reaction (QF-PCR) or fluorescence in situ hybridization (FISH) analysis can be used for faster and more cost-effective detection of the sex chromosome and SRY gene. Multiplex ligation-dependent probe amplification (MLPA), single-gene sequence analysis, next-generation sequence analysis (NGSA), targeted NGSA, whole-exome sequencing (WES), and whole-genome sequencing (WGS) analyses can be performed according to preliminary diagnoses. Microarray analysis (array comparative genomic hybridization (aCGH) and single nucleotide polymorphism array (SNPa)) should be performed in cases with syndromic findings and if no pathology is detected with other tests. In DSD cases, the use of optical genome mapping and techniques, which will probably be in daily practice in near future, may be considered. In conclusion, the clinical and genetic diagnosis of DSD is difficult, and molecular genetic diagnosis is often not available. This has psychosocial and health implications for patients and their families. New genetic techniques, especially those targeting the whole genome, may provide a better understanding of DSD through the identification of little-known genetic causes. This review focuses on conventional genetic and next-generation genetic techniques used in the genetic diagnosis of DSD, as well as possible genetic diagnostic techniques and approaches that may be used in routine practice in near future.

Discovery of a novel homozygous SOD1 truncating variant bolsters infantile SOD1 deficiency syndrome.

Superoxide dismutase 1 (SOD1) is an important antioxidant enzyme whose main function is to neutralise superoxide free radicals in the cytoplasm. Heterozygous variants in SOD1 are responsible for a substantial percentage of familial amyotrophic lateral sclerosis (ALS) cases. Recently, several reports have shown that biallelic loss of SOD1 function results in a novel phenotype called infantile SOD1 deficiency syndrome, which is consistent with a recessive pattern of inheritance and can be distinguished from typical (adult-onset) ALS. We documented detailed family histories and clinical data, followed by whole-exome sequencing and family co-segregation analysis through Sanger sequencing. To facilitate comparisons, relevant data from fifteen previously reported patients with SOD1-related neurodevelopmental disorders were included. This study presents a new Turkish family with two affected children exhibiting severe delayed motor development, infancy-onset loss of motor skills, axial hypotonia, tetraspasticity, and impaired cognitive functions. Genetic analysis revealed a novel homozygous frameshift variant in SOD1 (c.248dupG [p.Asp84Argfs*8]), with computational biochemical studies shedding light on the mechanistic aspects of SOD1 dysfunction. Our findings contribute an affirmative report of a fourth biallelic variant resulting in a severe clinical phenotype, reminiscent of those induced by previously identified homozygous loss-of-function SOD1 variants. This research not only advances our understanding of the pathogenesis of this debilitating neurological syndrome but also aligns with ongoing intensive efforts to comprehend and address SOD1-linked ALS.

A multi-omics analysis-based model to predict the prognosis of low-grade gliomas

Lower-grade gliomas (LGGs) exhibit highly variable clinical behaviors, while classic histology characteristics cannot accurately reflect the authentic biological behaviors, clinical outcomes, and prognosis of LGGs. In this study, we carried out analyses of whole exome sequencing, RNA sequencing and DNA methylation in primary vs. recurrent LGG samples, and also combined the multi-omics data to construct a prognostic prediction model. TCGA-LGG dataset was searched for LGG samples. 523 samples were used for whole exome sequencing analysis, 532 for transcriptional analysis, and 529 for DNA methylation analysis. LASSO regression was used to screen genes with significant association with LGG survival from the frequently mutated genes, differentially expressed genes, and differentially methylated genes, whereby a prediction model for prognosis of LGG was further constructed and validated. The most frequently mutated diver genes in LGGs were IDH1 (77%), TP53 (48%), ATRX (37%), etc. Top significantly up-regulated genes were C6orf15, DAO, MEOX2, etc., and top significantly down-regulated genes were DMBX1, GPR50, HMX2, etc. 2077 genes were more and 299 were less methylated in recurrent vs. primary LGG samples. Thirty-nine genes from the above analysis were included to establish a prediction model of survival, which showed that the high-score group had a very significantly shorter survival than the low-score group in both training and testing sets. ROC analysis showed that AUC was 0.817 for the training set and 0.819 for the testing set. This study will be beneficial to accurately predict the survival of LGGs to identify patients with poor prognosis to take specific treatment as early, which will help improve the treatment outcomes and prognosis of LGG.

Broadening the Phenotype and Genotype Spectrum of Glycogen Storage Disease by Unraveling Novel Variants in an Iranian Patient Cohort.

Glycogen storage diseases (GSDs) are a group of rare inherited metabolic disorders characterized by clinical, locus, and allele heterogeneity. This study aims to investigate the phenotype and genotype spectrum of GSDs in a cohort of 14 families from Iran using whole-exome sequencing (WES) and variant analysis. WES was performed on 14 patients clinically suspected of GSDs. Variant analysis was performed to identify genetic variants associated with GSDs. A total of 13 variants were identified, including six novel variants, and seven previously reported pathogenic variants in genes such as AGL, G6PC, GAA, PYGL, PYGM, GBE1, SLC37A4, and PHKA2. Most types of GSDs observed in the cohort were associated with hepatomegaly, which was the most common clinical presentation. This study provides valuable insights into the phenotype and genotype spectrum of GSDs in a cohort of Iranian patients. The identification of novel variants adds to the growing body of knowledge regarding the genetic landscape of GSDs and has implications for genetic counseling and future therapeutic interventions. The diverse nature of GSDs underscores the need for comprehensive genetic testing methods to improve diagnostic accuracy. Continued research in this field will enhance our understanding of GSDs, ultimately leading to improved management and outcomes for individuals affected by these rare metabolic disorders.

Incidence and molecular characteristics of deficient mismatch repair conditions across nine different tumors and identification of germline variants involved in Lynch-like syndrome.

Based on molecular characteristics, deficient DNA mismatch repair (dMMR) solid tumors are largely divided into three categories: somatically MLH1-hypermethylated tumors, Lynch syndrome (LS)-associated tumors, and Lynch-like syndrome (LLS)-associated tumors. The incidence of each of these conditions and the corresponding pathogenic genes related to LLS remain elusive. We identified dMMR tumors in 3609 tumors from 9 different solid organs, including colorectal cancer, gastric cancer, small-bowel cancer, endometrial cancer, ovarian cancer, upper urinary tract cancer, urinary bladder cancer, prostate cancer, and sebaceous tumor, and comprehensively summarized the characterization of dMMR tumors. Characterization of dMMR tumors were performed as loss of at least one of MMR proteins (MLH1, MSH2, MSH6, and PMS2), by immunohistochemistry, followed by MLH1 promotor methylation analysis and genetic testing for MMR genes where appropriate. Somatic variant analysis of MMR genes and whole exome sequencing (WES) were performed in patients with LLS. In total, the incidence of dMMR tumors was 5.9% (24/3609). The incidence of dMMR tumors and the proportion of the three categorized dMMR tumors varied considerably with different tumor types. One to three likely pathogenic/pathogenic somatic MMR gene variants were detected in 15 out of the 16 available LLS tumors. One patient each from 12 patients who gave consent to WES demonstrated non-MMR germline variants affect function (POLQ or BRCA1). Our data regarding the LS to LLS ratio would be useful for genetic counseling in patients who are suspected to have LS, though the genetic backgrounds for the pathogenesis of LLS need further investigation.

Personalizing non-small cell lung cancer treatment through patient-derived xenograft models: preclinical and clinical factors for consideration.

In the pursuit of creating personalized and more effective treatment strategies for lung cancer patients, Patient-Derived Xenografts (PDXs) have been introduced as preclinical platforms that can recapitulate the specific patient's tumor in an in vivo model. We investigated how well PDX models can preserve the tumor's clinical and molecular characteristics across different generations. A Non-Small Cell Lung Cancer (NSCLC) PDX model was established in NSG-SGM3 mice and clinical and preclinical factors were assessed throughout subsequent passages. Our cohort consisted of 40 NSCLC patients, which were used to create 20 patient-specific PDX models in NSG-SGM3 mice. Histopathological staining and Whole Exome Sequencing (WES) analysis were preformed to understand tumor heterogeneity throughout serial passages. The main factors that contributed to the growth of the engrafted PDX in mice were a higher grade or stage of disease, in contrast to the long duration of chemotherapy treatment, which was negatively correlated with PDX propagation. Successful PDX growth was also linked to poorer prognosis and overall survival, while growth pattern variability was affected by the tumor aggressiveness, primarily affecting the first passage. Pathology analysis showed preservation of the histological type and grade; however, WES analysis revealed genomic instability in advanced passages, leading to the inconsistencies in clinically relevant alterations between the PDXs and biopsies. Our study highlights the impact of multiple clinical and preclinical factors on the engraftment success, growth kinetics, and tumor stability of patient-specific NSCLC PDXs, and underscores the importance of considering these factors when guiding and evaluating prolonged personalized treatment studies for NSCLC patients in these models, as well as signaling the imperative for additional investigations to determine the full clinical potential of this technique.

Pharmacogenomics-based systematic review of coronary artery disease based on personalized medicine procedure

BackgroundCoronary artery disease (CAD) is the most common reason for mortality and disability-adjusted life years (DALYs) lost globally. This study aimed to suggest a new gene list for the treatment of CAD by a systematic review of bioinformatics analyses of pharmacogenomics impacts of potential genes and variants. MethodsPubMed search was filtered by the title including Coronary Artery Disease during 2020–2023. To find the genes with pharmacogenetic impact on the CAD, additional filtrations were considered according to the variant annotations. Protein-Protein Interactions (PPIs), Gene-miRNA Interactions (GMIs), Protein-Drug Interactions (PDIs), and variant annotation assessments (VAAs) performed by STRING-MODEL (ver. 12), Cytoscape (ver. 3.10), miRTargetLink.2., NetworkAnalyst (ver 0.3.0), and PharmGKB. ResultsResults revealed 5618 publications, 1290 papers were qualified, and finally, 650 papers were included. 4608 protein-coding genes were extracted, among them, 1432 unique genes were distinguished and 530 evidence-based repeated genes remained. 71 genes showed a pharmacogenetics-related variant annotation in at least (entirely 6331 annotations). Variant annotation assessment (VAA) showed 532 potential variants for the final report, and finally, the concluding PGs list represented 175 variants. Based on the function and MAF, 57 nonsynonymous variants of 29 Pharmacogenomics-related genes were associated with CAD. ConclusionConclusively, evaluating circulating miR33a in individuals’ plasma with CAD, and genotyping of rs2230806, rs2230808, rs2487032, rs12003906, rs2472507, rs2515629, and rs4149297 (ABCA1 variants) lead to precisely prescribing of well-known drugs. Also, the findings of this review can be used in both whole-genome sequencing (WGS) and whole-exome sequencing (WES) analysis in the prognosis and diagnosis of CAD.

Exome sequencing and genome-wide association analyses unveils the genetic predisposition in hydroxychloroquine retinopathy.

To unveil the candidate susceptibility genes in chloroquine/hydroxychloroquine (CQ/HCQ) retinopathy using whole exome sequencing (WES) and genome-wide association study (GWAS). Patients with a diagnosis of CQ/HCQ retinopathy based on the comprehensive demographic and ocular examination were included. The peripheral blood was extracted for WES and GWAS analyses. The Chinese Han Southern database from 1000 genomes was used as control group to compare the affected percentage. Multivariate logistic regression analysis adjusted for age, HCQ dose, duration and renal disease were used to analyze the correlation between genetic variants and visual outcome. A poor vision outcome was defined as visual acuity <6/12. An abnormal anatomical outcome was defined as disruption of ellipsoid zone in the fovea. Twenty-nine patients with an average age of 60.9 ± 13.4 years, treatment duration of 12.1 ± 6.2 years, daily dose of 8.5 ± 4.1 mg/kg, and the cumulative dose of 1637.5 ± 772.5 g, were genotyped. Several candidate genes associated with CQ/HCQ retinopathy were found, including RP1L1, RPGR and RPE65, with a difference of affected percentage over 50% in mutation between the case and control groups. New foci in CCDC66: rs56616026 (OR = 63.43, p = 1.63 × 10-8) and rs56616023 (OR = 104.7, p = 5.02 × 10-10) were identified significantly associated with HCQ retinopathy. Multivariate analysis revealed increased genetic variants were significantly associated with poor functional (OR = 1.600, p = 0.004) and structural outcome (OR = 1.318, p = 0.043). Several candidate susceptibility genes including RP1L1, RPGR, RPE65 and CCDC66 were identified to be associated with CQ/HCQ retinopathy. In addition to disease susceptibility, patients with increased genetic variants are more vulnerable to poor visual outcomes.

Central precocious puberty in a boy with X-linked adrenoleukodystrophy caused by a novel ABCD1 mutation

X-linked adrenoleukodystrophy (X-ALD) is a rare genetic disorder caused by pathogenic variants in the ABCD1 gene. The symptoms include primary adrenal insufficiency (PAI), progressive spinal cord disease, inflammatory demyelinating cerebral disease, and primary hypogonadism. It is exceptionally rare that pediatric PAI is accompanied by central precocious puberty (CPP). The purpose of this study was to better understand the diversity of clinical manifestations of X-ALD and to identify the ABCD1 gene mutation in a case of a boy with X-ALD accompanied by CPP. We collected clinical, laboratory and imaging data, and used whole-exome sequencing (WES) analysis to evaluate the pathogenicity of the variant. We also predicted the potential deleterious effects of the novel mutation using Mutation Taster and generated three-dimensional protein structures using Swiss-Model and PyMOL Viewer software. The patient presented with PAI accompanied by CPP. Adrenal gland CT revealed adrenal hypoplasia. Gonadotropin-releasing hormone stimulation tests revealed CPP. WES revealed a novel variant (c.1376dup) in the ABCD1 gene, which resulted in a reading frameshift and a premature termination codon (p.Leu461ProfsTer95). Sanger sequencing confirmed that the variant was inherited from his heterozygous mother. Mutation Taster predicted that the variant could be harmful. The overall three-dimensional structures of the mutant wild-type proteins were visually distinct. Our results shed light on additional aspects of X-ALD. The premature activation of the hypothalamic-pituitary-gonadal axis may possibly be related to the pathogenic ABCD1 gene mutation.

Clinical and Genetic Analysis of Multiple Idiopathic Cervical Root Resorption.

To explore the genetic background and clinical phenotypes of multiple idiopathic cervical root resorption (MICRR) in a Chinese family. The proband and his three family members were clinically examined and had radiographs taken with a radiovisiography (RVG) system and CBCT to define the diagnosis of MICRR. Genomic DNA (gDNA) was extracted from peripheral blood samples of the patient, his father, mother and younger sister for whole exome sequencing (WES). The pathogenicity of rare variants with minor allele frequency (MAF) less than 0.005 were analysed following possible inheritance patterns, predicted results from 12 software programs, the American College of Medical Genetics (ACMG) 2015 criteria, and information from ClinVar, OMIM and HGMD databases as well as gene function. The proband presented the typical MICRR phenotypes such as thin cervical pulp wall and apple core-like lesions in radiographs. Following the recessive inheritance pattern, WES analysis identified SHROOM2, SYTL5, MAGED1 and FLNA with a higher chance of causing MICRR. Four genes with compound heterozygous variants and another 27 genes with de novo variants either in autosomal-dominant or autosomal-recessive pattern were also found to have the potential pathogenicity. A total of 35 novel potential pathogenic genes were found to be associated with MICRR from a Chinese family through WES. The new genetic background of MICRR may be helpful for clinical and molecular diagnosis.

Deciphering the Impact of Rare Missense Variants in EGFR-TKI-Resistant Non-Small-Cell Lung Cancer through Whole Exome Sequencing: A Computational Approach.

Targeted therapy revolutionizes the treatment of non-small-cell lung cancer (NSCLC), harboring molecular change. Epidermal growth factor receptor(EGFR) mutations play a crucial role in the development of NSCLC, serving as a pivotal factor in its pathogenesis. We elucidated the mechanisms of resistance and potential therapeutic strategies in NSCLC resistant to the EGFR-tyrosine kinase inhibitor (EGFR-TKI). This is achieved by identifying rare missense variants through whole exome sequencing (WES). The goal is to enhance our understanding, identify biomarkers, and lay the groundwork for targeted interventions, thereby offering hope for an improved NSCLC treatment landscape. We conducted WES analysis on 16 NSCLC samples with EGFR-TKI-resistant NSCLC obtained from SRA-NCBI (PRJEB50602) to reveal genomic profiles within the EGFR-TKI. Our findings showed that 48% of the variants were missense, and after filtering with the Ensembl variant effect predictor, 53 rare missense variants in 23 genes were identified as highly deleterious. Further examination using pathogenic tools like PredictSNP revealed 12 deleterious rare missense variants in 7 genes: ZNF717, PSPH, ESRRA, SEMA3G, PTPN7, CAVIN4, and MYBBP1A. Molecular dynamics simulation (MDS) suggested that the L385P variant alters the structural flexibility of ESRRA, potentially leading to unfolding of ERRα proteins. This could impact their function and alter ERRα expression. These insights from MDS enhance our understanding of the structural and dynamic consequences of the L385P ESRRA variant and provide valuable implications for subsequent therapeutic considerations and targeted interventions.

Abstract 3354: Capturing tumor heterogeneity to personalize therapy in young patients with hormone receptor positive breast cancer

Abstract Background: Breast cancer (BC) in young women (Y) is a distinct entity in terms of prognosis. Although triple negative and HER2 positive subtypes are enriched in Y patients, the hormone receptor (HR) positive subtype (luminal, LBC) remains the most common. A thorough understanding of LBC biology in Y women has not been reached yet, also hampered by lack of appropriate models that faithfully recapitulate these tumors in molecular and preclinical studies. Methods: To investigate molecular determinants of progression and therapeutic resistance, we collected a large panel of fresh specimens (N=226) from Y (&amp;lt; 40 years) and nY (&amp;gt;55 years) LBC patients, characterized by targeted sequencing and used to generate Patient-Derived Organoids (PDO) and Xenografts (PDX). A selected panel of Y and nY LBC patients (N=96), sharing similar clinical-pathological features, was used for whole exome sequencing (WES) and transcriptomic analyses. Results: We generated stable LBC PDO (N=32, 13 from Y patients), with a success rate of 50% and stable LBC PDX (N=15, 11 from Y patients), with a success rate 17%. Our targeted panel of NGS, covering most mutated genes in LBC at high coverage (1000x), revealed a significantly higher number of Y patients displaying mutations in PIK3CA and in PIK3CA+TP53. However, these data were not observable in WES analyses (200x), likely because many of those mutations displayed a VAF &amp;lt; 5% (20% of PIK3CA and 30% of TP53). The transcriptomic analysis showed an enrichment in signatures of aggressiveness in the Y LBC subgroup. The EGF family member AREG (Amphiregulin) was the most enriched gene in tumors from pre- versus post-menopausal patients. Following validation in a larger panel of LBC, both at RNA and protein level, we tested the impact of AREG in LBC cell lines. AREG expression was timely induced by growth factor and hormonal stimulation. Furthermore, stimulation using amphiregulin induced the activation of EGFR and its downstream pathway, creating a positive feedback loop. Relevant to the Y LBC phenotype, a consistent and time dependent induction of AREG transcription was induced following estrogen and progesterone stimulation, indicating that, in LBC, AREG might act as a transcriptional target of ESR1. Finally, high levels of amphiregulin reduced response to tamoxifen and CDK4/6 inhibitors in LBC cells and PDOs. Conclusions and Perspectives: Our study provides valuable molecular and biological insights into LBC in Y women. We identified distinct genetic and transcriptomic profiles, including PIK3CA and TP53 mutations and AREG overexpression, which may be exploited to plan more targeted and efficient treatments. The establishment of a living biobank of PDO and PDX from LBC patients of Y age will enable preclinical trials, facilitating discovery of biomarkers and therapeutic strategies tailored to the unique biology of Y LBC. Citation Format: Ilenia Segatto, Maria Chiara Mattevi, Alessandra Dall'Acqua, Lorena Musco, Federica Ruggiero, Gabriele Di Giustino, Andrea Favero, Gian Luca Rampioni Vinciguerra, Nicole Crestan, Luca Pellizzari, Riccardo Spizzo, Riccardo Bomben, Filippo Vit, Andrea Vecchione, Lorenzo Gerratana, Fabio Puglisi, Samuele Massarut, Tiziana Perin, Gustavo Baldassarre, Barbara Belletti. Capturing tumor heterogeneity to personalize therapy in young patients with hormone receptor positive breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3354.

Diverse genetic causes of amenorrhea in an ethnically homogeneous cohort and an evolving approach to diagnosis

Research questionPremature ovarian insufficiency (POI) is characterised by amenorrhea associated with elevated follicle stimulating hormone (FSH) under the age of 40 years and affects 1–3.7% women. Genetic factors explain 20–30% of POI cases, but most causes remain unknown despite genomic advancements. DesignWe used whole exome sequencing (WES) in four Iranian families, validated variants via Sanger sequencing, and conducted the Acyl-cLIP assay to measure HHAT enzyme activity. ResultsDespite ethnic homogeneity, WES revealed diverse genetic causes, including a novel homozygous nonsense variant in SYCP2L, impacting synaptonemal complex (SC) assembly, in the first family. Interestingly, the second family had two independent causes for amenorrhea – the mother had POI due to a novel homozygous loss-of-function variant in FANCM (required for chromosomal stability) and her daughter had primary amenorrhea due to a novel homozygous GNRHR (required for gonadotropic signalling) frameshift variant. WES analysis also provided cytogenetic insights. WES revealed one individual was in fact 46, XY and had a novel homozygous missense variant of uncertain significance in HHAT, potentially responsible for complete sex reversal although functional assays did not support impaired HHAT activity. In the remaining individual, WES indicated likely mosaic Turners with the majority of X chromosome variants having an allelic balance of ∼85% or ∼15%. Microarray validated the individual had 90% 45,XO. ConclusionsThis study demonstrates the diverse causes of amenorrhea in a small, isolated ethnic cohort highlighting how a genetic cause in one individual may not clarify familial cases. We propose that, in time, genomic sequencing may become a single universal test required for the diagnosis of infertility conditions such as POI.