Whole Exome Sequencing Analysis Research Articles

A multicenter study investigating the genetic analysis of childhood steroid-resistant nephrotic syndrome: Variants in COL4A5 may not be coincidental.

This study aimed to discuss the pathogenic hereditary factors of children with steroid-resistant nephrotic syndrome (SRNS) in Guangxi, China. We recruited 89 patients with SRNS or infantile NS from five major pediatric nephrology centers in Guangxi, and conducted a retrospective analysis of clinical data. Whole-exome sequencing analysis was also performed on all patients. The risk of progression to chronic kidney disease (CKD) was assessed using the Kaplan-Meier method and Cox proportional hazards model. The study included 69 male and 20 female participants from 86 distinct families, with the median age of disease onset being 48 months (interquartile range: 24-93). Overall, 24.7% had a family history of SRNS, whereas 13.5% exhibited extra-kidney manifestations. We identified disease-causing variants in 24.7% (22/89) of patients across eight screened genes. The most frequently detected variant was found in COL4A5, followed by NPHS2 (5.6%), NPHS1 (2.2%), PAX2 (2.2%), WT1 (1.1%), LMX1B (1.1%), NUP105 (1.1%), and COL4A6 (1.1%). Twelve of the 26 pathogenic variants were determined to be de novo. Based on gene detection results, pathogenic variants were categorized into two groups: identified and unidentified variants. The identified variant group demonstrated a significant association with positive family history, steroid resistant-style, and response to immune therapy (P<0.001). Patients with the identified genetic variant were approximately ten times more likely to develop CKD (P<0.001) than those in the unidentified group at the last follow-up. Kidney biopsy was performed on 66 patients, and minimal change disease was the most prevalent histopathological diagnosis (29 cases; 32.6%). These findings suggest that children diagnosed with SRNS exhibit a diverse range of genetic alterations. We identified the COL4A5 variant as the predominant genetic abnormality and a low frequency of NPHS1 gene involvement in these children. Gene variants may serve as an independent predictor for SRNS progression to CKD.

WES and Transcriptome Analysis Identifies FN1 as a Candidate Gene for Anterior Segment Dysgenesis.

Anterior segment dysgenesis (ASD) disorders are phenotypically diverse and have multiple associated genes. This study reports on a Chinese family of three generations with ASD disorders and identifies several associated genes and pathways of the disorders. The history of illnesses, clinical observations, and blood samples of all family members were collected. Whole exome sequencing (WES) and polymerase chain reaction (PCR) were conducted to detect the genetic variants between patients and control members in the family. Transcriptomic study and quantitative real-time PCR (qRT-PCR) were subsequently performed to validate the differentially expressed genes (DEGs) and investigate the possible mechanisms of ASD caused by the variations. The medical examination and illness history identified four members of the family diagnosed with ASD (III:3, II:3, II:2 and I:2). All four patients suffered various degrees of corneal opacity with abnormally thin cornea. Members II:3, II:2, and I:2 also had cataracts and iris hypoplasia and received an intraocular lens implant before the age of 20. We detected a heterozygous missense variation c.6122G > A (p.R2041Q and rs746145647) in fibronectin1 (FN1) in the four patients in this family that was absent in the other healthy members. The transcriptome and RT-PCR analysis revealed that the FN1 mRNA level was significantly upregulated in the blood samples of patients compared to healthy controls. A total of 909 DEGs were identified, including 607 upregulated genes and 302 downregulated genes. GO and KEGG annotation revealed that many DEGs were involved in biological processes closely related to focal adhesion, extracellular matrix-receptor interaction, TGF-β pathway, and the immune system. This study identified an FN1 mutation associated with ASD patients and probed potential pathways related to it.

C.655C>T Variant of Sepiapterin Reductase Deficiency: Genetic and Bioinformatic Analysis

Background: Sepiapterin reductase deficiency (SRD) is a very rare psychomotor disorder related to enzyme defects in synthesizing tetrahydrobiopterin (BH4) with a spectrum of symptoms. The most common of which are developmental delay and hypotonia. To elucidate the genetic cause of SRD, the patient was analyzed by whole-exome sequencing (WES) followed by mutation analysis. Methods: A complete clinical examination was performed by a pediatric neurologist. Brain imaging and a thorough neuro-metabolic investigation were applied along with biochemical tests including high-performance liquid chromatography (HPLC) to quantify the concentrations of cerebrospinal fluid (CSF) pterins. Genomic DNA was extracted and evaluated through WES. This was followed by bioinformatic analysis of mutated sepiapterin reductase (SPR) protein structure and identification of the functional protein amino acids. Results: Biochemical analysis of biogenic amines of CSF with HPLC showed very low levels of homovanillic acid and 5-hydroxyindolacetic acid in favor of neurotransmitter metabolism disorder. WES analysis displayed a homozygous nonsynonymous variant in exon 3 of the SPR gene (C.655C>T, p.Arg219Ter). The molecular graphic of SPR protein structure with 4HWK PDB code was generated by MOE software in comparison with P.Arg219* mutated protein which is predicted by a Swiss model homology modeling server determining the ligand-binding site residues using COFACTOR software and indicated that the (R219*) mutation destroyed the ligand-binding site from the position of 219. Another important codon including 254 and 256 positions is omitted. Conclusion: Whole exome sequencing and bioinformatic analysis could overcome lengthy, expensive, and emotional diagnostic adventures of challenging neurodevelopmental cases leading to improved management and prevention of irreversible side effects.

A novel pathogenic DICER1 gene variant is associated with hereditary multinodular goiter in an Argentine family as evidenced by clinical, biochemical and molecular genetic analysis.

DICER1 syndrome is an autosomal-dominant disorder that results in malignant or benign tumors. A number of distinct pathogenic germline and somatic variants have been identified as causing multinodular goiter (MNG). The purpose of the present study was to identify and characterize the genetic cause underlying the familial form of MNG through a whole-exome sequencing (WES) analysis in an Argentine family with three affected siblings. Clinical, biochemical and molecular genetics as well as bioinformatics analysis were performed. A novel heterozygous variant in the DICER1 gene was identified in the proband patient by WES. The variant was a single guanine deletion at nucleotide position 2,042 (NM_177438.3:c.2042del) resulting in a frameshift at amino acid 681 with a putative premature stop codon [NP_803187.1:p.Gly681ValfsTer4]. Family segregation analysis showed that his affected sister and his affected brother also were heterozygous for same variant, whereas the father was a healthy heterozygous carrier of the variant and the healthy mother harbor only wild-type alleles in the DICER1 gene. We have also observed that the frameshift variant does not interfere with the pre-mRNA splicing of the exon 13. In addition, two clinically relevant heterozygous variants, not associated with thyroid disease, were also identified in index sibling using the Franklin platform, a frameshift [NP_000234.1:p.Thr55AsnfsTer49] in the MEFV gene (familial mediterranean fever) and a missense [NP_004530.1:p.Ala422Thr] in the NARS1 gene (neurodevelopmental delay and ataxia). In conclusion, in the present study we have identified a novel frameshift variant corresponding to NP_803187.1:p.Gly681ValfsTer4 in the DUF 283 domain of DICER1. The results were in accordance with previous observations confirming the genetic heterogeneity of DICER1 syndrome. Moreover, the identification of this variant in the unaffected father substantiates the hypothesis of incomplete/reduced penetrance.

Use of whole-exome sequencing and pedigree analysis to identify X-linked hypophosphatemia in Saudi Arabian families

Abstract Context X-linked hypophosphatemia (XLH) is the most common form of inherited hypophosphatemic rickets (HR), caused by pathogenic variants in the PHEX gene. Genetic diagnosis of XLH facilitates early treatment optimization, especially for patients suitable for burosumab, a recombinant anti-fibroblast growth factor-23 monoclonal antibody. Objective This study aimed to use whole-exome sequencing (WES) and pedigree analysis to identify patients with XLH. Methods Medical records at a single center in Saudi Arabia were screened between 2014–2024 to identify patients with suggested HR. Of the 800 patients identified, 27 had had suspected XLH. The genetic study comprised 100 patients drawn from these 27 families. Results Clinical manifestations were widespread and variable within families. Severe disease was reported in 55% of children and 25% of adults. At presentation, all children were receiving either conventional therapy (60%) or burosumab (40%); however, 53% of adults were not treated. WES provided a genetic diagnosis in 23 families: alterations in the PHEX gene (20 families), with homozygous ENPP1 and DMP1 variants detected in two and one families, respectively. Pathogenic/likely pathogenic variants were detected in 23 families (diagnostic yield 85%). Ten novel likely pathogenic variants were detected. Pedigree analysis provided information to support disease-specific patient management. Conclusion WES detected a diagnostic molecular abnormality in 85% of families with HR phenotypes; PHEX variants were the most common. Combined use of WES and pedigree analysis highlighted that the under-diagnosis of adult XLH in this population, with most family members being diagnosed after the pedigree analysis.

Molecular profiling reveals novel therapeutic targets and clonal evolution in ovarian clear cell carcinoma

BackgroundOvarian clear cell carcinoma (OCCC) has a disproportionately high incidence among women in East Asia. Patients diagnosed with OCCC tend to experience worse clinical outcomes than those with high-grade serous carcinoma (HGSC) at advanced stages. The unfavorable prognosis of OCCC can be partly attributed to its frequent resistance to conventional chemotherapy. Within a precision medicine framework, we sought to provide a comprehensive molecular characterization of OCCC using whole-exome sequencing to uncover potential molecular targets that may inform novel therapeutic strategies.MethodsWe performed whole-exome sequencing analysis on tumor-normal paired samples from 102 OCCC patients. This comprehensive genomic characterization of a substantial cohort of OCCC specimens was coupled with an analysis of clonal progression.ResultsOn analyzing 102 OCCC samples, ARID1A (67%) and PIK3CA (49%) emerged as the most frequently mutated driver genes. We identified tier 1 or 2 clinically actionable molecular targets in 40% of cases. This included DNA mismatch repair deficiency (n = 1), as well as BRCA2 (n = 1), PIK3CA (n = 36), KRASG12C (n = 1), and ATM (n = 4) mutations. Furthermore, 45% of OCCC samples displayed ARID1A biallelic loss. Interestingly, we identified previously unreported mutations in the 5’ untranslated region of the TERT gene that harbored an adverse prognostic significance. Clock-like mutational processes and activated APOBECs were major drivers of somatic point mutations. Mutations arising from DNA mismatch repair deficiency were uncommon. Reconstruction of clonal evolution revealed that early genetic events likely driving tumorigenesis included mutations in the ARID1A, PIK3CA, TERT, KRAS, and TP53 genes.ConclusionsOur study provides a comprehensive characterization of the genomic landscape and clonal evolution in OCCC within a substantial cohort. These findings unveil potentially actionable molecular alterations that could be leveraged to develop targeted therapies.

EPCO-03. METABOLIC INSIGHT INTO GLIOMA HETEROGENEITY: MAPPING WHOLE EXOME SEQUENCING TOIN VIVO IMAGING WITH STEREOTACTIC LOCALIZATION AND DEEP LEARNING

Abstract Intra-tumoral heterogeneity (ITH) complicates the diagnosis and treatment of glioma, partly due to the diverse metabolic profiles driven by genomic alterations. While multiparametric imaging captures spatial and functional variations enabling ITH characterization, it falls short in assessing the metabolic activities underpinning phenotypic differences. This gap stems from the challenge of integrating easily accessible, co-located pathology and genomic data with metabolic insights. This study presents a multi-faceted approach combining stereotactic biopsy with clinical open-craniotomy for sample collection, voxel-wise analysis of MR images, regression-based GAM, and whole-exome sequencing. The aim is to demonstrate the potential of machine learning algorithms to predict variations in cellular and molecular tumor characteristics. This retrospective study enrolled ten treatment-naïve patients with radiologically confirmed glioma who underwent multiparametric MR scans (T1W, T1W-CE, T2W, T2W-FLAIR, DWI) prior to surgery. During craniotomy, at least one stereotactic biopsy was collected from each patient, with the screenshots of sample locations saved for spatial registration to pre-surgical MR data. Whole-exome sequencing was performed on flash-frozen tumor samples, prioritizing five glioma-related genes: IDH1, TP53, EGFR, PIK3CA, and NF1. Regression was implemented with a GAM using a univariate shape function for each predictor. Standard ROC analyses were used to evaluate detection, with AUC calculated for each gene target and MR contrast combination. Mean AUC for five gene targets and 31 MR contrast combinations was 0.75±0.11, with individual AUCs as high as 0.96 for IDH1 and TP53 with T2W-FLAIR and ADC, and 0.99 for EGFR with T2W and ADC. An average AUC of 0.85 across the five mutations was achieved using the combination of T1W, T2W-FLAIR, and ADC. These results suggest the possibility of predicting exome-wide mutation events from non-invasive, in vivo imaging by combining stereotactic localization of glioma samples with a semi-parametric deep learning method. This approach holds potential for refining targeted therapy by better addressing the genomic heterogeneity of glioma tumors.

Inherited Spinocerebellar Ataxia Segregates with Intra-Familial Genetic Heterogeneity in a Consanguineous Pakistani Family: A Report of a Potential Novel Candidate Gene.

Hereditary spinocerebellar ataxia (SCA) is a group of genetic neurodegenerative disorders caused by a variety of gene variants. At least 44 types of SCAs have been identified to date, and more than 35 genes and hundreds of variants have been reported that are associated with SCAs. We have investigated a Pakistani consanguineous six-generation family with SCA by using whole-exome sequencing analysis. We identified a reported SCA-associated variant, c.C2687G (p.P896R) in CACNA1A, in only a subgroup of the family, while a c.C262T (p.P88S) variant in ARFIP1 serves as a candidate pathogenic variant in the other subgroup as a possible novel cause of SCA. Our study showed that intra-familial heterogeneity may exist in SCA families and presented a candidate new causative gene for SCA.

Evolutionary Characteristics in Primary Aldosteronism Patients.

Primary aldosteronism is predominantly caused by excessive aldosterone production from the adrenal cortex, and the aldosterone-producing structures could take many forms, like adenomas, nodules, micronodules, and so on. Most studies of primary aldosteronism were limited to the hotspot driver genes responsible for autonomous aldosterone production; however, the panoramic genetic architecture and genomic alterations of aldosterone-producing structures and their adjacent hyperplasia glands remain unknown. In this study, whole-exome sequencing and transcriptome sequencing (RNA-seq) analyses were performed using functional nodules and matched hyperplasia tissues, which were microdissected guided by aldosterone synthase immunohistochemistry. Phylogenetic trees were constructed based on the shared and unique mutations, gene mutation spectrums, and clonal characteristics. The rates of mutations represented higher means of functional nodules than hyperplasia samples, and the little mutational overlap was shown between the 2 groups on phylogenetic trees. The mutations of the aldosterone driver gene (KCNJ5 or CACNA1D) were only observed in functional nodules and indicated almost the largest values of cancer cell fraction. Moreover, the functional nodules also harbored some potential variants related to cell proliferation, which were not detected in hyperplasia tissues. Transcriptome analysis suggested that only 25.5% upregulated and 23.3% downregulated genes overlapped between functional nodules and hyperplasia tissues. This study demonstrated a genetic and transcriptome landscape of aldosterone-producing structures and adjacent hyperplasia glands in primary aldosteronism. The results indicated independent clonal origins on functional nodules and hyperplasia tissues, and little mutual evolutionary relationship was found on their phylogenetic trees.

Desmin-related myopathy manifested by various types of arrhythmias: a case report and literature review.

Desmin is a type III intermediate filament protein specifically expressed in muscle cells, which is encoded by the DES gene. Defects in the desmin protein and cytoskeletal instability may interfere with cardiac muscle conduction signals, a fundamental mechanism for arrhythmias in patients with desmin-related myopathy. This current case report presents a female patient in her early 20s who presented with early-onset complete atrioventricular block and complete left bundle branch block over the previous decade. More recently, she had developed ventricular tachycardia, ventricular fibrillation, atrial fibrillation and other arrhythmias. Echocardiography revealed non-compaction of the ventricular myocardium and pulmonary hypertension. Whole-exome sequencing analysis identified a heterozygous missense mutation in the DES gene: c.1216C>T (p.Arg406Trp). She was eventually diagnosed with arrhythmias due to desmin-related myopathy. A literature review of international databases was undertaken to summarise the clinical characteristics of the cardiac involvement associated with this DES gene mutation.

Clinical features and search for genetic determinants of postprandial hypoglycemia.

To test whether postprandial hypoglycaemia is an extreme and repeatable phenotype of glucose metabolism. Secondly, we explored the genetic determinants of this phenotype. We conducted this study using data from Pinggu Metabolic Disease Study database (n = 3,345). We selected subjects after an oral glucose tolerance test (OGTT) (2 h, glucose <3 mmol/L_ and compared clinical features with those of normal glucose tolerance (NGT). We additionally selected 75 subjects as super-healthy control group. Whole-exome sequencing (WES) was performed on postprandial hypoglycaemic and super-healthy controls. We also evaluated several candidate genes believed to be important in pancreatic hypoglycaemia. We found 13 participants (0.39%) had an OGTT 2 h glucose <3 mmol/L. Ten patients were men (76.9%). All 13 participants had insulin > 3 uU/mL when postprandial blood glucose levels were <3 mmol/L. WES analysis identified one gene, paternally expressed 3 (PEG3), which had three rare mutations in four patients (30.8%). Minor allele frequencies (MAF) of rare PEG3 mutations were significantly higher in subjects with postprandial hypoglycaemia than in super-healthy controls. Among all four subjects with PEG3 gene mutations, 71.4% were men, and their body mass index (BMI) was significantly lower than that of the NGT. Postprandial hypoglycaemia is an extreme and reproducible phenotype in the general population. PEG3 mutations may represent a potential genetic aetiology for postprandial hypoglycaemia. Further research with larger and more diverse populations and a broader genetic focus is needed to understand the genetic basis of postprandial hypoglycaemia.

Whole exome sequencing in patients with recurrent pericarditis

Abstract Background/Introduction About 10% of patients with recurrences may have a family history of pericarditis suggesting a genetic predisposition, that has been confirmed in selected cases with idiopathic recurrent pericarditis with a focused analysis of genes involved in autoinflammatory diseases (TNFRSF1A, MVK, NLRP3 genes, and variants at the Interleukin 1 Gene Locus). Purpose Aim of the present paper is to describe the frequency of detected variants of causative genes by Whole Exome Sequencing (WES) in patients with idiopathic recurrent pericarditis. Methods This is an observational, cohort study including all consecutive adult patients (&amp;gt;18 years) with &amp;gt; 2 episodes of recurrent pericarditis. All patients performed WES. We analyzed the frequency of genetic variants in: 1) known or predicted to be at least likely pathogenic (LP) variants already related to recurrent pericarditis, 2) predicted at least of unknown significance in genes not yet associated with recurrent pericarditis. Moreover, we highlighted incidental findings. Results We studied 108 patients with recurrent pericarditis (median age of 32 years, IQR 18.5; 67.6% females, all Caucasian, idiopathic etiology in 71.1%). The median number of recurrences was 5 (IQR 2). Eleven gene variants related to recurrent pericarditis were detected by WES analysis in 11/108 patients (10.2%): 9 variants were pathogenic/likely pathogenic (P/LP) according the ACMG guidelines and involved NLRP3 and TNFRSF1A genes, 2 VUS associated with familial Mediterranean fever (gene MEFV) (figure 1). We also recorded 3 P/LP variants and 2 VUS related to the inflammatory response (IFIH1, NFKBIA, JAK1, NOD2 and ALPK1 genes); 7 P/LP variants and 3 VUS related to heart’s electrical system (SCN5A, CALM2, TRPM4, KCND2, KCNQ1 genes); 12 P/LP variants and 10 VUS related to heart structural genes (figure 2). Conclusion(s) A substantial proportion of patients with multiple recurrences of pericarditis carries LP or P variants suggesting the importance of genetic predisposition evaluation in this population.

Multiple Pulmonary Sclerosing Pneumocytomas (PSPs): A Comprehensive Analysis of Clinicopathological Characteristics and Whole-exome Sequencing (WES) Results.

Pulmonary sclerosing pneumocytoma (PSP) is a rare neoplasm with indolent clinical behavior and usually presents as a solitary nodule, while only a few cases involving multiple nodules. Recent studies have revealed frequent AKT1 mutations in PSP; however, the molecular genetics of multiple PSPs remain unclear. To better understand the genetic background, eleven patients (4.2%, 11/260) with multiple PSP nodules were identified, and whole-exome sequencing (WES) was performed on 6 patients. Among 5 patients with 2 or 3 PSP nodules, AKT1 alterations were the most common (50%, 7/14), and the predominant alteration was p.E17K (21.4%, 3/14). Novel ARID1A mutations were the second most common driver (14.3%, 2/14), and we first identified these mutations cooccurred with AKT1 p.E17K mutation. Moreover, we observed limited concordance in the mutation spectra and few comutated genes among different lesions from these 5 patients, indicating that PSP with 2 or 3 nodules were independent arising tumors. No AKT1 mutations were identified in 3 PSP samples from a patient with multiple diffuse nodules. However, there were 17 shared genetic alterations among the 3 lesions, but none were typical driver mutations. The findings on multiple diffuse PSP nodules may also have independent origins, but the potential that some of these nodules are metastatic nodules cannot be excluded. In conclusion, this retrospective study is the largest series of multiple PSP cases and provides new insights into the genomic underpinning of PSP. This work has a potential to broaden our understanding of the pathogenesis and development of these lesions and warrants analysis in larger cohorts.

Clinicopathological characteristics and genomic profiling in patients with transformed lymphoma: a monocentric retrospective study

Background Transformed lymphoma occurs when indolent lymphoma transforms into more aggressive lymphoma usually associated with poor prognosis. Methods In this study, we analysed the immunophenotypes, prognostic factors and outcomes of 35 patients with transformed lymphoma from among 306 marginal zone lymphoma (MZL), 544 follicular lymphoma (FL) and 871 chronic lymphocytic leukaemia/small lymphocytic lymphoma (CLL/SLL) cases. In addition, we performed whole-exome sequencing study of seven transformed MZL (tMZL) cases. Results Our results demonstrate that the median time from indolent lymphoma diagnosis to transformed DLBCL was 35 months (range, 14–53 months). The 5-year overall survival (OS) and progression-free survival (PFS) rates after histological transformation (HT) were 50% and 26%, respectively. Kaplan–Meier survival analysis revealed that asynchronous HT and transformed CLL/SLL (tCLL/SLL) were significant adverse prognostic factors for OS after DLBCL HT. We identified mutations involvement in chromatin remodelling (CREBBP and EP300) and regulators of NF-κB signalling (TNFAIP3, BCL10, MYD88, CD79B and CARD11) were affected in tMZL. Conclusion Whole-exome sequencing and copy-number analysis revealed that tMZL derives from the divergent evolution of an ancestral common progenitor clone (CPC). Collectively, this study provides clinicopathological characteristics of three common types of transformed lymphomas and the genetic profile of tMZL with diagnostic and therapeutic implications.

Melatonin receptor 1A variants as genetic cause of idiopathic osteoporosis.

Idiopathic osteoporosis (IOP) is a rare form of early-onset osteoporosis diagnosed in patients with no known metabolic or hormonal cause of bone loss and unknown pathogenesis. Patients with IOP commonly report both childhood fractures and family history of osteoporosis, raising the possibility of genetic etiologies of IOP. Whole-exome sequencing analyses of different IOP cohorts identified multiple variants in melatonin receptor 1A (MTNR1A) with a potential pathogenic outcome. A rare MTNR1A variant (rs374152717) was found in members of an Ashkenazi Jewish family with IOP, and an MTNR1A variant (rs28383653) was found in a nonrelated female IOP cohort (4%). Both variants occur at a substantially higher frequency in Ashkenazi Jewish individuals than in the general population. We investigated consequences of the heterozygous (rs374152717) variant [MTNR1Ac.184+1G>T (MTNR1Ac.184+1G>T)] on bone physiology. A mouse model of the human rs374152717 variant reproduced the low bone mass (BM) phenotype of young-adult patients with IOP. Low BM occurred because of induction of senescence in mutant osteoblasts followed by compromised differentiation and function. In human cells, introduction of rs374152717 led to translation of a nonfunctional protein and subsequent dysregulation of melatonin signaling. These studies provide evidence that MTNR1A mutations entail a genetic etiology of IOP and establish the rs374152717 variant as a loss-of-function allele that impairs bone turnover by inducing senescence in osteoblasts. The higher prevalence of the MTNR1A variants identified in IOP cohorts versus the general population indicates a greater risk of IOP in those carrying these variants, especially Ashkenazi Jewish individuals bearing the rs374152717 variant.

Targeting DLBCL by mutation-specific disruption of cancer-driving oncogenes.

Diffuse large B cell lymphomas (DLBCL) are highly aggressive tumors. Their genetic complexity and heterogeneity have hampered the development of novel approaches for precision medicine. Our study aimed to develop a personalized therapy for DLBCL by utilizing the CRISPR/Cas system to induce knockouts (KO) of driver genes, thereby causing cancer cell death while minimizing side effects. We focused on OCI-LY3 cells, modeling DLBCL, and compared them with BJAB cells as controls. Analysis of whole exome sequencing revealed significant mutations in genes like PAX5, CD79B, and MYC in OCI-LY3 cells. CRISPR/Cas9-mediated KO of these genes resulted in reduced cancer cell viability. Subsequent single and dual gRNA targeting of PAX5 mutations inhibited proliferation specifically in OCI-LY3 cells. Moreover, dual gRNA targeting of PAX5 and MYC induced chromosomal rearrangements, reducing cell proliferation substantially. However, targeting single intronic mutations did not affect cell viability, highlighting the importance of disrupting protein function. Targeting multiple mutations simultaneously addresses intra-tumoral heterogeneity, and the transient delivery of CRISPR/Cas9 allows for permanent gene disruption. While challenges such as incomplete editing efficiency and delivery limitations exist, further optimization may enhance therapeutic efficacy. Overall, our findings demonstrate the efficacy of CRISPR/Cas9 in targeting oncogenic mutations, opening avenues for precision medicine in DLBCL treatment.

7196 Variants in the Neurodevelopmental Gene BRINP2 are Associated with Severe Delayed Puberty

Abstract Disclosure: Y. Al-Sayed: None. R. Oleari: None. A. Cariboni: None. W. He: None. Y. Chan: None. S.R. Howard: None. Gonadotropin-releasing hormone (GnRH) is the master hormone regulating the reproductive axis and its pulsatile secretion is crucial for puberty onset and fertility. Disruption in GnRH neuron development or hypothalamic function can lead to absent or delayed puberty (DP) due to GnRH deficiency, with a phenotypic spectrum from severe delayed puberty to partial or complete Hypogonadotropic Hypogonadism (HH). HH can also be present as a shared trait with other neurodevelopmental disorders (NDDs). The aim of our study was to identify novel genetic etiology of severe DP by identifying variants in associated genes in our patient cohort; and ascertain the functional effects of identified variants of interest. Whole exome sequencing (WES) was performed on DNA samples from 180 probands with DP to identify potentially pathogenic rare coding variants in relevant gene pathways. Integrative analysis was performed on genomic data from human patients combined with transcriptomics analysis of rodent immortalized and primary GnRH neurons to determine novel regulators of GnRH neuronal development and function. Bone morphogenetic protein/retinoic acid inducible neural-specific 2 (BRINP2) was identified as a candidate gene of interest as it was found to be significantly upregulated during GnRH neuronal development in these single cell transcriptomics analyses. Mutations in this gene have been previously associated with NDDs such as autistic spectrum disorder (ASD). BRINP2 is localised to the olfactory bulb, a key site during GnRH neuron migration. Copy number variation in this gene is seen in multiple individuals from the Deciphering Developmental Disorders study with a phenotype including cryptorchidism and intellectual disability (https://www.deciphergenomics.org/gene/BRINP2/patient-overlap/cnvs). WES analysis identified four variants in BRINP2 (p.R726W, p.R649Q, p.I629V and p.D729N) in four unrelated probands with severely DP or partial HH, in combination with ASD or other NDD features. These four variants are all rare or ultra-rare and are predicted to be pathogenic by in silico tools including CADD, REVEL and SIFT. Protein expression of the four mutants was comparable to the reference protein. BRINP2 has been shown to inhibit neuronal cell proliferation by negative regulation of cell cycle transition and Brinp2 knockout mice show hyperactive behaviour representative of human attention-deficit hyperactivity disorder, but pubertal timing has not been assessed. Thus, BRINP2 is a novel candidate for GnRH deficiency with NDD and we have investigated the role of BRINP2 in GnRH biology via wildtype and mutant protein sub-cellular localization, as well as tissue expression in mouse hypothalamic tissue across development. Presentation: 6/2/2024

Next-Generation Integrated Sequencing Identifies Poor Prognostic Factors in Patients with MYD88-Mutated Chronic Lymphocytic Leukemia in Taiwan

Introduction: Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in the Western countries and is very rare in Asia. Methods: Peripheral blood or bone marrow mononuclear cells obtained at initial diagnosis from 215 patients with CLL were analyzed by using next-generation sequencing to investigate the ethnic differences in genetic abnormalities. Results: Whole-genome sequencing and whole-exome sequencing analyses on 30 cases showed that 9 genes, including IGLL5, MYD88, TCHH, DSCAM, AXDND1, BICRA, KMT2D, MYT1L, and RBM43, were more frequently mutated in our Taiwanese cohort compared with those of the Western cohorts. IGLL5, MYD88, and KMT2D genes were further analyzed by targeted sequencing in another 185 CLL patients, unraveling frequencies of 29.3%, 20.9%, and 15.0%, respectively. The most frequent positional mutation of MYD88 was V217F (26/45, 57.8%), followed by L265P (9/45, 20.0%). MYD88 mutations were significantly associated with IGLL5 mutations (p = 0.0004), mutated IGHV (p < 0.0001) and 13q deletion (p = 0.0164). CLL patients with co-occurrence of MYD88 mutations with KMT2D or/and IGLL5 mutations were associated with a significantly inferior survival compared to those with MYD88 mutation alone (not reached vs. 131.8 months, p = 0.007). In multivariate analysis, MYD88 mutation without KMT2D or IGLL5 mutations was an independently favorable predictor. Conclusions: IGLL5, MYD88, and KMT2D mutations were enriched in Taiwanese CLL, and co-occurrence of MYD88 mutations with KMT2D or/and IGLL5 mutations was associated with a poorer prognosis.

Simultaneous CNV-seq and WES: An effective strategy for molecular diagnosis of unexplained fetal structural anomalies

BackgroundFetal structural anomalies are detected by ultrasound in approximately 3 % of pregnancies. Numerous genetic diagnostic strategies have been widely applied to identify the genetic causes of prenatal abnormalities. We aimed to assess the value of simultaneous copy number variation sequencing (CNV-seq) and whole exome sequencing (WES) in diagnosing fetuses with structural anomalies. MethodsFetuses with structural anomalies detected by ultrasound were included for eligibility. After genetic counseling, WES and CNV-seq were performed on DNA samples of fetuses and their parents. All detected variants were evaluated for pathogenicity according to ACMG criteria, with the final diagnosis was determined based on ultrasound results and relevant family history. ResultsThe diagnostic rate of 174 fetuses with prenatal ultrasound abnormalities was 26.44 %, higher than that achieved through either CNV or WES analysis alone. Furthermore, the highest diagnostic rate was observed in fetuses with multiple system anomalies, accounting for 50 % of the total diagnostic yield, followed by skeletal system anomalies at 45.45 %. Three cases with multiple system abnormalities were found to have a dual diagnosis of pathogenic CNVs and SNV variants, representing 1.72 % of the total cohort. 38 pregnant women in their third trimester of pregnancy (27 weeks+) participated in this study, and 23.68 % received a confirmed genetic diagnosis. Finally, 31 women (67.39 %) voluntarily terminated their pregnancy following the testing and extensive genetic counseling. ConclusionsOur study demonstrated that the simultaneous CNV-seq and WES analyses are beneficial for the molecular diagnosis of underlying unexplained structural anomalies in fetuses. This strategy is more efficient in elucidating prenatal abnormalities with compound problems, such as dual diagnoses. Furthermore, the simultaneous strategy has a shorter turnaround time and is particularly suitable for families with structural anomalies found in the third trimester of pregnancy.

A heterozygous SPRY4 variant identified in female infertility characterized by reduced oocyte potential and early embryonic arrest.

Can novel genetic factors contributing to early embryonic arrest in infertile patients be identified, along with the underlying mechanisms of the pathogenic variant? We identified a heterozygous variant in the SPRY4 (sprouty RTK signaling antagonist 4) in infertile patients and conducted in vitro and in vivo studies to investigate the effects of the variant/deletion, highlighting its critical role in female reproductive health. SPRY4 acts as a negative regulator of receptor tyrosine kinases (RTKs) and functions as a tumor suppressor. Its abnormal expression can lead to recurrent miscarriage by affecting trophoblast function. In mice, Spry4 knockout (KO) leads to craniofacial anomalies and growth defects. A human study links the SPRY4 variant to a male patient with isolated hypogonadotropic hypogonadism (IHH), hypothetically impacting gonadotropin-releasing hormone (GnRH) neurons, and causing reproductive dysfunctions. SPRY4 is thus potentially integral in regulating endocrine homeostasis and reproductive function. To date, no study has reported SPRY4 variants associated with female fertility, and a causal relationship has not been established with functional evidence. Whole-exome sequencing (WES) was performed in 392 infertile women who suffered from primary infertility of unknown reason, and the heterozygous SPRY4 variant were identified in one independent family. The infertile patients presenting were recruited from July 2017 to November 2023. Women diagnosed with primary infertility were recruited from the Reproduction Center of Zhongshan Hospital, Fudan University. Genomic DNA was extracted from peripheral blood for WES analysis. The SPRY4 variant were identified through WES, in silico analysis, and variant screening. All variants were confirmed by Sanger sequencing. The effects of the variants were investigated in human embryonic kidney (HEK) 293T (HEK293T) cells via western blotting, and in mouse oocytes and embryos through complementary RNA (cRNA) injection, RNA sequencing, fluorescence, absorbance, and RT-qPCR assays. Gene function was further examined in Spry4 KO mice via histology, western blotting, ELISA, and RT-qPCR assays. We identified a missense heterozygous pathogenic variant in SPRY4 (GRCh38, GenBank: NM_030964.5, c.157C>T p.(Arg53Trp), rs200531302) that reduces SPRY4 protein levels in HEK293T cells and disrupts the redox system and mitochondrial function in mouse oocyte, and perturbs developmental potential in mouse embryos. These phenotypes could be partially reversed by the exogenous addition of Nrf1 cRNA. Additionally, Spry4-/- mice exhibit ovarian oxidative stress and decreased ovarian function. Due to the limited WES data and population, we identified only one family with a SPRY4 mutation. The deeper mechanism and therapeutic strategy should be further investigated through mutant mice and recovery experiment. Our study has identified a pathogenic variant in SPRY4 associated with early embryonic arrest in humans. These findings enhance our understanding of the role of SPRY4 in early embryonic development and present a new genetic marker for female infertility. This work was supported by the National Natural Science Foundation of China (82071643 and 82171655) and Natural Science Foundation of Shanghai (22ZR1456200). None of the authors have any competing interests. N/A.