Copy Number Variation Analysis Research Articles

O-035 Euploidy’s hidden potential: emerging insights from abnormal fertilized embryos

Abstract Study question Can pregnancies be achieved from abnormally fertilized embryos? Summary answer After conducting parental origin PGTA testing to confirm ploidy status, achieving pregnancy and live births becomes feasible. What is known already A major limitation in IVF is the availability of viable embryos per cycle. Particularly relevant for patients with poor prognosis in response to controlled ovarian stimulation. In these couples, the limited number of oocytes results in a significant reduction of the chance to achieve a live birth. In IVF, zygotes displaying successful extrusion of the 2PB and 2PN are considered “normally fertilized” and cultured further to be used for treatment. In contrast, zygotes showing a single or more than 2PN are deemed abnormally fertilized. However, some can ultimately develop into normal embryos, which may lead to live birth. Study design, size, duration This is a single centre retrospective observational study of 125 blastocysts classified as abnormal through standard fertilization check post-IVF (50 embryos) and ICSI (75 embryos) Embryos were subjected to genetic testing utilizing a validated targeted next-generation sequencing-based PGT-A protocol. The protocol's proven efficacy lies in distinguishing between haploid, diploid and triploid samples, a distinction made possible by the parallel analysis of genotyping data and chromosome copy number variations. Participants/materials, setting, methods The participants in this study were couples who underwent IVF cycles using PGT-A between 2021-2024. Embryos were divided into two groups based on their fertilization method Group A involved blastocysts derived from In vitro fertilization (IVF) = 50. Classified 1PN, micro 3PN, 3PN, and 4PN. Group B blastocysts derived from Intracytoplasmic sperm injection Intracytoplasmic sperm injection (ICSI) =75 Classified 1PN, micro 3PN, 3PN, and 4PN. Main results and the role of chance Group A: Involved embryos resulting from IVF; 54% were diploid, with 15% being euploid from 3PN. From 1PN, 92% were diploid, and 54% were euploid. Micro PN had a 100% diploid rate, of which 33% were euploid. No diploid embryos were reported from 4PN fertilization. Group B: Involved embryos resulting from ICSI; 36% were diploid, and 14% were euploid from 3PN. From 1PN, 53% were diploid, and 40% were euploid. Micro PN had a 100% diploid rate, with 23% being euploid. 4PN had a 50% diploid rate and no euploid embryos. On the other hand, 0.4% (17/4822) of 2PN-classified embryos were reported as haploid, and 1.4% (68/4822) of the 2PN-classified embryos were reported as polyploid. Eighty-nine percent of haploidy and polyploidy in ICSI originated maternally, while 71% of polyploidy in IVF originated paternally and only one embryo was haploid resulted from IVF was maternally originated. Remarkably, three live births were reported: one from 1PN, one from Micro PN, and the third from 3PN. Limitations, reasons for caution The data are observational and were retrospectively analyzed, so unknown confounders could not be assessed. Wider implications of the findings To our knowledge, this is the most extensive cohort study analyzing embryos resulting from abnormal fertilization. It has implications for clinical practice and gives an extra chance for patients, particularly those with a low number of eggs, in response to controlled ovarian stimulation. Trial registration number n/a

Investigating druggable kinases for targeted therapy in retinoblastoma.

Retinoblastoma (RB) is a childhood retinal neoplasm and commonly treated with cytotoxic chemotherapeutic agents. However, these therapeutic approaches often lead to diverse adverse effects. A precise molecular therapy will alleviate these side effects and offer better treatment outcomes. Over the years, kinases have become potential drug targets in cancer therapy. Hence, we aimed to investigate genetic alterations of putative kinase drug targets in RB. Targeted exome sequencing was performed on 35 RB tumors with paired blood samples using a gene panel consisting of 29 FDA-approved kinase genes. Single nucleotide variants were analyzed for pathogenicity using an in-house pipeline and copy number variations (CNVs) were detected by a depth of coverage and CNVPanelizer. The correlation between genetic changes and clinicopathological features was assessed using GraphPad Prism. Three somatic mutations, two in ERBB4 and one in EGFR were identified. Two of these mutations (ERBB4 c.C3836A & EGFR c.A1196T) were not reported earlier. CNV analysis revealed recurrent gains of ALK, MAP2K2, SRC, STK11, and FGFR3 as well as frequent losses of ATM, PI3KCA and ERBB4. Notably, nonresponsive tumors had a higher incidence of amplifications in clinically actionable genes such as ALK. Moreover, ALK gain and ATM loss were strongly correlated with optic nerve head invasion. In conclusion, our study revealed genetic alterations of druggable kinases in RB, providing preliminary insights for the exploration of kinase-targeted therapy in RB.

Comparison of the diagnostic significance of cerebrospinal fluid metagenomic next-generation sequencing copy number variation analysis and cytology in leptomeningeal malignancy

BackgroundDiagnosis and monitoring of leptomeningeal malignancy remain challenging, and are usually based on neurological, radiological, cerebrospinal fluid (CSF) and pathological findings. This study aimed to investigate the diagnostic performance of CSF metagenomic next-generation sequencing (mNGS) and chromosome copy number variations (CNVs) analysis in the detection of leptomeningeal malignancy.MethodsOf the 51 patients included in the study, 34 patients were diagnosed with leptomeningeal malignancies, and 17 patients were diagnosed with central nervous system (CNS) inflammatory diseases. The Sayk’s spontaneous cell sedimentation technique was employed for CSF cytology. And a well-designed approach utilizing the CSF mNGS-CNVs technique was explored for early diagnosis of leptomeningeal malignancy.ResultsIn the tumor group, 28 patients were positive for CSF cytology, and 24 patients were positive for CSF mNGS-CNVs. Sensitivity and specificity of CSF cytology were 82.35% (95% CI: 66.83-92.61%) and 94.12% (95% CI: 69.24-99.69%). In comparison, sensitivity and specificity of CSF mNGS-CNV were 70.59% (95% CI: 52.33-84.29%) and 100% (95% CI: 77.08-100%). There was no significant difference in diagnostic consistency between CSF cytology and mNGS-CNVs (p = 0.18, kappa = 0.650).ConclusionsCSF mNGS-CNVs tend to have higher specificity compared with traditional cytology and can be used as a complementary diagnostic method for patients with leptomeningeal malignancies.

A pedigree analysis of Rotor hyperbilirubinemia combined with hepatitis B virus infection in a SLCO1B1 and SLCO1B3 gene mutations patient

BackgroundRotor syndrome (RS, OMIM#237450) is an extremely rare autosomal digenic recessive disorder characterized by mild non-hemolytic hereditary conjugated hyperbilirubinemia, caused by biallelic variation of SLCO1B1 and SLCO1B3 genes that resulted in OATP1B1/B3 dysfunction in the sinusoidal membrane leading to impaired bilirubin reuptake ability of hepatocytes. MethodsOne RS pedigree was recruited and clinical features were documented. Whole genome second-generation sequencing was used to screen candidate genes and mutations, Sanger sequencing confirmed predicted mutations. ResultsThis study detected a homozygous nonsense variant c.1738C > T (p.R580*) in the coding region of the SLCO1B1 (NM006446) gene in a family with RS and hepatitis B virus infection by Variants analysis and Sanger sequencing, and confirmed by Copy Number Variation (CNV) analysis and Long Range PCR that there was a homozygous insertion of intron 5 of the SLCO1B3 gene into intron 5 of long-interspersed element 1 (LINE1). A few cases of such haplotypes have been reported in East Asian populations. A hepatitis B virus infection with fatty liver disease was indicated by pathology, which revealed mild-moderate lobular inflammation, moderate lobular inflammation, moderate hepatocellular steatosis, and fibrosis stage 1–2 (NAS score: 4 points/S1-2) alterations. Heterozygotes carrying p.R580* and LINE1 insertions were also detected in family members (I1, I2, III2, III3), but they did not develop conjugated hyperbilirubinemia. ConclusionThe mutations may be the molecular genetic foundation for the presence of SLCO1B1 c.1738C > T(p.R580*) and SLCO1B3 (LINE1) in this RS pedigree.

Oncogenic alterations in KIR3DL1 in cutaneous acral CD8+ lymphoproliferative disorder.

Primary cutaneous acral CD8+ T-cell lymphoproliferative disorder (TLPD) is a rare and indolent lymphoma entity. Although TLPD was first identified many years ago, the molecular pathogenesis is still not fully understood. In order to better understand the molecular pathogenesis of cutaneous acral CD8+ TLPD and to identify further discriminatory markers to differentiate this lymphoma subtype from other CD8+ cutaneous lymphomas, we analysed five cases of cutaneous acral CD8+ TLPD for putative molecular alterations. Somatic alterations were assessed using whole-exome and targeted sequencing of paraffin-embedded tissue. Results were evaluated using immunohistochemical staining of respective relevant proteins. CD8+ cutaneous T-cell lymphomas (n = 12) served as control for KIR3DL1 staining. Copy number variation analysis revealed a homozygous deletion of the KIR3DL1 gene in two of the analysed cases. This resulted in loss of KIR3DL1 protein expression, which was observed in all cases of cutaneous acral CD8+ TLPD. In contrast, KIR3DL1 expression was more variable in other CD8+ cutaneous T-cell lymphomas with 50% of analysed cases (n = 12) found to be positive. In addition, one further case of acral CD8+ TLPD harboured a loss-of-function mutation in the PIK3R1 gene, presumably activating the phosphoinositide 3-kinase-AKT pathway. Alterations of the KIR3DL1 gene may be of pathogenetic relevance for acral CD8+ TLPD. Loss of KIR3DL1 protein expression may support the diagnosis of this indolent lymphoma entity; however, this is not a subtype-specific discriminative feature.

The Importance of Copy Number Variant Analysis in Patients with Monogenic Kidney Disease

IntroductionGenetic testing can reveal monogenic causes of kidney diseases, offering diagnostic, therapeutic, and prognostic benefits. While single nucleotide variants (SNVs) and copy number variants (CNVs) can result in kidney disease, CNV analysis is not always included in genetic testing. MethodsWe investigated the diagnostic value of CNV analysis in 2,432 kidney disease patients genetically tested at the University Medical Centre Utrecht between 2014 and May 2022. We combined previous diagnostic testing results, encompassing SNVs and CNVs, with newly acquired results based on retrospective CNV analysis. The reported yield considers both ACMG classification and whether the genotype actually results in disease. ResultsWe report a diagnostic yield of at least 23% for our complete diagnostic cohort. The total diagnostic yield based solely on CNVs was 2.4%. The overall contribution of CNV analysis, defined as the proportion of positive genetic tests requiring CNV analysis, was 10.5% and varied among different disease subcategories, with the highest impact seen in congenital anomalies of the kidney and urinary tract and chronic kidney disease at a young age. We highlight the efficiency of exome-based CNV calling, which reduces the need for additional diagnostic tests. Furthermore, a complex structural variant, likely a COL4A4 founder variant, was identified. Additional findings unrelated to kidney diseases were reported in a small percentage of cases. ConclusionIn summary, this study demonstrates the substantial diagnostic value of CNV analysis, providing insights into its contribution to the diagnostic yield and advocating for its routine inclusion in genetic testing of kidney disease patients.

Identification and analyses of exonic and copy number variants in spastic paraplegia

Hereditary spastic paraplegias are a diverse group of degenerative disorders that are clinically categorized as isolated; with involvement of lower limb spasticity, or symptomatic, where spastic paraplegia is complicated by further neurological features. We sought to identify the underlying genetic causes of these disorders in the participating patients. Three consanguineous families with multiple affected members were identified by visiting special schools in the Punjab Province. DNA was extracted from blood samples of the participants. Exome sequencing was performed for selected patients from the three families, and the data were filtered to identify rare homozygous variants. ExomeDepth was used for the delineation of the copy number variants. All patients had varying degrees of intellectual disabilities, poor speech development, spasticity, a wide-based gait or an inability to walk and hypertonia. In family RDHR07, a homozygous deletion involving multiple exons and introns of SPG11 (NC000015.9:g.44894055_449028del) was found and correlated with the phenotype of the patients who had spasticity and other complex movement disorders, but not those who exhibited ataxic or indeterminate symptoms as well. In families ANMD03 and RDFA06, a nonsense variant, c.985C > T;(p.Arg329Ter) in DDHD2 and a frameshift insertion‒deletion variant of AP4B1, c.965-967delACTinsC;p.(Tyr322SerfsTer14), were identified which were homozygous in the patients while the obligate carriers in the respective pedigrees were heterozygous. All variants were ultra-rare with none, or very few carriers identified in the public databases. The three loss of function variants are likely to cause nonsense-mediated decay of the respective transcripts. Our research adds to the genetic variability associated with the SPG11 and AP4B1 variants and emphasizes the genetic heterogeneity of hereditary spastic paraplegia.

METB-03. SOLID PEDIATRIC TUMORS IN MICE REVEAL MODEL-SPECIFIC GLOBAL DNA METHYLATION AND CNV PROFILES CORRESPONDING TO HUMAN COUNTERPARTS

Abstract BACKGROUND The analysis and comparison of global DNA methylation patterns has become a gold standard in modern neuropathology. For human central nervous system (CNS) tumors and other cancers, the research community has collected thousands of methylation profiles, leading to the discovery and definition of numerous molecularly distinct tumor types and subtypes. Although reliable preclinical mouse models are essential for the understanding of tumor development and the validation of therapeutic approaches, such DNA methylation analyses have so far not been systematically performed for murine tumors owing to technical limitations. METHODS We employed the Illumina Bead Chip 285k mouse methylation array to profile ~500 samples covering 90 different mouse models representing 33 pediatric solid tumor types, including CNS tumors and sarcomas from 18 laboratories. We compared DNA methylation profiles of these models between each other and to human CNS tumors, performed immune cell deconvolution and conducted an in-depth analysis of copy number variants (CNVs) during tumor evolution. RESULTS Unsupervised clustering showed that mouse models form groups of their presumed tumor types. Using a tailor-made cross-species analysis based on syntenic CpG sites, we found that most tumors also group with their corresponding human counterparts. Immune cell deconvolution revealed distinct tumor microenvironments that showed clear similarities to human tumors. We further identified recurrent, model-specific CNVs that are syntenic to genomic aberrations in respective human tumors, suggesting an essential role of CNVs during tumor initiation or maintenance. CONCLUSION We provide a global resource of well characterized pediatric solid cancer models representing numerous different tumor types. Our comparative analyses reveal novel molecular model characteristics and validate their reliability as human disease models. Our data provide new biological insights into immune composition, tumor cell identities and tumor development by identifying model-specific CNV profiles.

Clinical Relevance of the Systematic Analysis of Copy Number Variants in the Genetic Study of Cardiomyopathies.

Cardiomyopathies (CMs), one of the main causes of sudden death among the young population, are a heterogeneous group of myocardial diseases, usually with a genetic cause. Next-Generation Sequencing (NGS) has expanded the genes studied for CMs; however, the yield is still around 50%. The systematic study of Copy Number Variants (CNVs) could contribute to improving our diagnostic capacity. These alterations have already been described as responsible for cardiomyopathies in some cases; however, their impact has been rarely assessed. We analyzed the clinical significance of CNVs in cardiomyopathies by studying 11,647 affected patients, many more than those considered in previously published studies. We evaluated the yield of the systematic study of CNVs in a production context using NGS and a novel CNV detection software tool v2.0 that has demonstrated great efficacy, maximizing sensitivity and avoiding false positives. We obtained a CNV analysis yield of 0.8% that fluctuated depending on the type of cardiomyopathy studied (0.29% HCM, 1.41% DCM, 1.88% ARVC, 1.8% LVNC, 1.45% RCM), and we present the frequency of occurrence for 18 genes that agglutinate the 95 pathogenic/likely pathogenic CNVs detected. We conclude the importance of including in diagnostic tests a systematic study of these genetic alterations for the different cardiomyopathies.

Tumor cell enrichment by tissue suspension improves sensitivity to copy number variation in diffuse gastric cancer with low tumor content

The detection of copy number variations (CNVs) and somatic mutations in cancer is important for the selection of specific drugs for patients with cancer. In cancers with sporadic tumor cells, low tumor content prevents the accurate detection of somatic alterations using targeted sequencing. To efficiently identify CNVs, we performed tumor cell enrichment using tissue suspensions of formalin-fixed paraffin-embedded (FFPE) tissue sections with low tumor cell content. Tumor-enriched and residual fractions were separated from FFPE tissue suspensions of intestinal and diffuse-type gastric cancers containing sporadic tumor cells, and targeted sequencing was performed on 225 cancer-related genes. Sequencing of a targeted panel of cancer-related genes using tumor-enriched fractions increased the number of detectable CNVs and the copy number of amplified genes. Furthermore, CNV analysis using the normal cell-enriched residual fraction as a reference for CNV scoring allowed targeted sequencing to detect CNV characteristics of diffuse-type gastric cancer with low tumor content. Our approach improves the CNV detection rate in targeted sequencing with tumor enrichment and the accuracy of CNV detection in archival samples without paired blood.

Exome sequencing in fetuses with congenital diaphragmatic hernia in a nationwide cohort.

To evaluate the diagnostic yield of exome sequencing (ES) in fetuses and neonates with prenatally detected congenital diaphragmatic hernia (CDH) and normal copy number variant (CNV) analysis. We conducted a retrospective cohort study of prenatally diagnosed CDH cases seen between 2019 and 2022. All cases who underwent prenatal or postnatal genetic testing were reviewed. The results from the ES analysis that identified pathogenic or likely pathogenic single nucleotide variants are described. In total, 133 fetuses with CDH were seen, of whom 98 (74%) had an isolated CDH and 35 (26%) had a complex CDH (associated structural anomalies) on prenatal examination. ES was performed in 68 cases, and eight pathogenic or likely pathogenic variants were found, accounting for a 12% diagnostic yield (10% [5/50] in isolated cases and 17% [3/18] in complex CDH). In 12% of fetuses and neonates with CDH and normal CNV analysis results, pathogenic or likely pathogenic variants were identified with ES. These data indicate that there is a substantial diagnostic yield when offering ES in prenatally detected CDH, both in complex and isolated cases.

The role of genomic disorders in chronic kidney failure of undetermined aetiology ≤50 years.

Genomic disorders caused by copy number variations (CNVs) are prevalent in patients with kidney disease; however, their contribution to chronic kidney failure (KF) of undetermined aetiology (uKF) is unclear. We screened patients with uKF aged 50 years or younger to establish the prevalence of causative CNVs. We enrolled patients with an onset of KF ≤50 years from suspected undetermined aetiology for initial review of medical records to exclude patients with clear-cut clinical or histopathological kidney diagnoses or patients with already established genetic kidney diseases. Next, we performed single nucleotide polymorphism (SNP) array-based CNV screening. All the detected CNVs were systematically classified and evaluated as possible causes of the patient's kidney disease. Patients with CNVs not explaining the kidney phenotype were additionally screened for causal variants in 540 genes using whole-genome sequencing. We enrolled 172 patients, of whom 123 underwent SNP-array. Pathogenic CNVs corresponding to known genomic disorders were identified in 12 patients (9.8%). The identified genomic disorders provided a causative kidney diagnosis in three patients, all of whom had reached KF by age 18 years. The remaining nine patients had CNVs with unclear kidney disease causality. Subsequently, whole-genome sequencing provided a causative genetic diagnosis in an additional four patients, including two diagnostic sequence variants unrelated to the detected CNVs. Genomic disorders were prevalent in this cohort with uKF, and causative CNVs were identified in 5 of 123 patients. Further studies combining the analysis of CNVs and sequence variants are needed to clarify the causal role of genomic disorders in kidney disease.

Prenatal diagnosis of a severe form of frontonasal dysplasia with severe limb anomalies, hydrocephaly, a hypoplastic corpus callosum, and a ventricular septal defect using 3D ultrasound: a case report and literature review

BackgroundFrontonasal dysplasia (FND) is a rare congenital anomaly resulting from the underdevelopment of the frontonasal process, and it can be syndromic or nonsyndromic. The typical features of FND include a deformed nose and ocular hypertelorism, which are sometimes associated with cleft lip and/or palate. Only approximately 10 cases of prenatally diagnosed nonsyndromic FND have been reported in the past 30 years.Case presentationA 33-year-old woman (G2P1) was referred to our center at 20 gestational weeks for bilateral hydrocephaly. We detected typical features of FND, including severe hypertelorism, median nasal bifidity, a minor cleft lip, and multiple limb anomalies using three-dimensional (3D) ultrasound. A hypoplastic corpus callosum, unilateral microtia, and a ventricular septal defect were also detected. Genetic testing, including karyotype analysis, copy number variation (CNV) analysis, trio-whole exome sequencing (trio-WES), and trio-whole-gene sequencing (trio-WGS), was performed; however, we did not find any de novo gene variants in the fetus as compared to the parents. Postmortem examination confirmed the prenatal diagnosis of FND.ConclusionThe present case expands the wide phenotypic spectrum of prenatal FND patients. 3D ultrasound is a useful tool for detecting facial and limb deformities.

Genetic analysis of two families with abnormal findings upon prenatal diagnosis

To carry out genetic analysis on two families with carriers of small terminal translocations using karyotyping analysis and genomic copy number variation sequencing (CNV-seq). Two couples undergoing prenatal diagnosis at the Tianjin Central Hospital of Obstetrics and Gynecology respectively on April 12, 2020 and December 17, 2021 were selected as the study subjects. With informed consent, amniotic fluid and peripheral blood samples were collected and subjected to conventional karyotyping and CNV-seq analysis for the detection of chromosomal microdeletion/duplications. Both couples had given births to children with chromosomal aberrations previously, and both fetuses were found to have abnormal karyotypes. CNV-seq showed that they had harbored microdeletion/duplications, and their mothers had both carried balanced translocations involving terminal fragments of chromosomes. For fetuses with small chromosomal segmental abnormalities, their parental origin should be traced, and the diagnosis should be confirmed with combined genetic techniques.

A founder deletion in ECM1 of 1163 bp causes lipoid proteinosis in the southeast region of Turkiye.

Lipoid proteinosis (LP) is an inherited disorder characterized by the accumulation of hyaline-like material in the skin, oral cavity, and larynx. The primary symptoms include hoarseness, restricted tongue movements, and various skin lesions. LP is caused by biallelic pathogenic variants in the ECM1 gene. We studied 20 patients from nine different families with LP, 19 of whom are from Şanlıurfa in the southeastern region of Turkiye. Overall, the clinical features of the patient cohort were consistent with those mentioned in the literature, except for one exhibited an atrophoderma vermiculatum-like lesion, which is atypical for LP. The clinical exome sequencing analysis revealed three different homozygous variants in the ECM1 gene (NM_004425). While c.1246C>T p.(Arg416*) on Exon 8 and c.806G>A p.(Cys269Tyr) on Exon 7 were detected in 1 patient each, an intragenic deletion of 1163 base-pairs including Exons 9 and 10 (c.1304 + 33_*300del) was identified in 18 patients from 7 unrelated families. The haplotype analysis of the deletion variant indicated a founder effect in the families from the Şanlıurfa province of Turkiye. Based on all this information, copy number variation analysis is recommended for patients with LP. In addition to this rare observation, this study represents the largest examination of the molecular spectrum of LP patients in Turkiye, alongside the clinical spectrum.

Whole‐exome sequencing‐based mutational profiling of hepatocellular adenoma malignant transformation to hepatocellular carcinoma

AbstractHepatocellular adenoma (HCA) represents a rare benign hepatic neoplasm with potential for malignant transformation into hepatocellular carcinoma (HCC), yet the underlying mechanism remains elusive. In this study, we investigated the genomic landscape of this process to identify therapeutic strategies for blocking malignant transformation. Using micro‐detection techniques, we obtained specimens of adenoma, cancerous neoplasm and adjacent normal liver from three patients undergoing hepatic resection surgery. Whole‐exome sequencing (WES) was performed, and genomic interactions between HCA and HCC components within the same tumour were evaluated using somatic variant calling, copy number variation (CNV) analysis, clonality evaluation and mutational signature analysis. Our results revealed genomic heterogeneity among patient cases, yet within each sample, HCA and HCC tissues exhibited a similar mutational landscape, suggesting a high degree of homology. Using nonnegative matrix factorization and phylogenetic trees, we identified shared and distinct mutational characteristics and uncovering necessary pathways associated with HCA–HCC malignant transformation. Remarkably, we found that HCA and HCC shared a common monoclonal origin while displaying significant genetic diversity within HCA–HCC tumours, indicating fundamental genetic connections or evolutionary pathways between the two. Moreover, elevated immune therapy‐related markers in these patients suggested heightened sensitivity to immune therapy, providing novel avenues for the treatment of hepatic malignancies. This study sheds light on the genetic mechanisms underlying HCA–HCC progression, offering potential targets for therapeutic intervention and highlighting the promise of immune‐based therapies in managing hepatic malignancies.

Two homozygous adjacent novel missense mutations in DYSF gene caused dysferlinopathy due to splicing abnormalities.

Background: Dysferlinopathy is an autosomal recessive disorder caused by mutations in the DYSF gene. This study reported two homozygous adjacent missense mutations in the DYSF gene, presenting clinically with bilateral lower limb weakness and calf swelling. Two homozygous adjacent missense mutations in the DYSF gene may be associated with the development of dysferlinopathy, but the exact mechanism needs further investigation. Methods: A retrospective analysis of clinical data from a dysferlinopathy-affected family was conducted. Peripheral blood samples were collected from members of this family for whole-exome sequencing (WES) and copy number variation analysis. Sanger sequencing was employed to confirm potential pathogenic variants. The Human Splicing Finder, SpliceAI, and varSEAK database were used to predict the effect of mutations on splicing function. The pathogenic mechanism of aberrant splicing in dysferlinopathy due to two homozygous adjacent missense mutations in the DYSF gene was determined by an in vivo splicing assay and an in vitro minigene assay. Results: The proband was a 42-year-old woman who presented with weakness of the lower limbs for 2 years and edema of the lower leg. Two homozygous DYSF variants, c.5628C>A p. D1876E and c.5633A>T p. Y1878F, were identified in the proband. Bioinformatics databases suggested that the mutation c.5628C>A of DYSF had no significant impact on splicing signals. Human Splicing Finder Version 2.4.1 suggested that the c.5633A>T of DYSF mutation caused alteration of auxiliary sequences and significant alteration of the ESE/ESS motif ratio. VarSEAK and SpliceAI suggested that the c.5633A>T of DYSF mutation had no splicing effect. Both an in vivo splicing assay and an in vitro minigene assay showed two adjacent mutations: c.5628C>A p. D1876E and c.5633A>T p. Y1878F in the DYSF gene leading to an Exon50 jump that resulted in a 32-aa amino acid deletion within the protein. Point mutation c.5628C>A p. D1876E in the DYSF gene affected splicing in vitro, while point mutation c.5633A>T p. Y1878F in the DYSF gene did not affect splicing function. Conclusion: This study confirmed for the first time that two homozygous mutations of DYSF were associated with the occurrence of dysferlinopathy. The c.5628C>A p. D1876E mutation in DYSF affected the splicing function and may be one of the contributing factors to the pathogenicity.

Analysis of human neuronal cells carrying ASTN2 deletion associated with psychiatric disorders

Recent genetic studies have found common genomic risk variants among psychiatric disorders, strongly suggesting the overlaps in their molecular and cellular mechanism. Our research group identified the variant in ASTN2 as one of the candidate risk factors across these psychiatric disorders by whole-genome copy number variation analysis. However, the alterations in the human neuronal cells resulting from ASTN2 variants identified in patients remain unknown. To address this, we used patient-derived and genome-edited iPS cells with ASTN2 deletion; cells were further differentiated into neuronal cells. A comprehensive gene expression analysis using genome-edited iPS cells with variants on both alleles revealed that the expression level of ZNF558, a gene specifically expressed in human forebrain neural progenitor cells, was greatly reduced in ASTN2-deleted neuronal cells. Furthermore, the expression of the mitophagy-related gene SPATA18, which is repressed by ZNF558, and mitophagy activity were increased in ASTN2-deleted neuronal cells. These phenotypes were also detected in neuronal cells differentiated from patient-derived iPS cells with heterozygous ASTN2 deletion. Our results suggest that ASTN2 deletion is related to the common pathogenic mechanism of psychiatric disorders by regulating mitophagy via ZNF558.

Fragmentomics of cell-free DNA as a sensitive biomarker for early detection of pancreatic cancer.

4173 Background: Pancreatic ductal adenocarcinoma (PDAC) is one for the most lethal types of cancer. Nearly 80% of the PDAC patients are diagnosed at advanced unresectable stages. Therefore, an accurate early diagnosis assay is urgently needed for reducing PDAC related mortalities. In this study, we aim to utilize cfDNA fragmentomics and machine learning techniques for sensitively detecting PDAC patients. Methods: Total647 cases were prospectively enrolled in this study. Plasma samples were collected from each participant for shallow WGS (~5X). After quality control process, 166 PDAC patients and 167 healthy controls were enrolled as a training cohort. Additional 112 PDAC patients and 111 healthy controls were included as an independent validation cohort. Additional 67 cases with pancreatic benign cystic tumors were also enrolled for model validation including IPMN (Intraductal papillary mucinous neoplasms), MCN (Mucinous cystic neoplasms) and SCN (Serous cystic neoplasms). Four fragmentomics profiles, including copy number variation, fragment size, mutation signatures, and FRAGmentomics-based Methylation Analysis (FRAGMA) were utilized by a machine learning algorithm for developing a predictive model. Results: The predictive model showed an exceptional performance to distinguish PDAC patients from healthy controls, yielding Area Under the Curve (AUC) of 0.993 in the training cohort (5-fold cross validation) and 0.990 in the validation cohort. In the training cohort, our model was capable of detecting PDAC patients at sensitivity of 95.8% and specificity of 95.2% while using 0.49 as cutoff. The validation cohort achieved 99.1% sensitivity at 91.0% specificity while applying the same cutoff. Conclusions: Our model was able to accurately detect PDAC at early stages, by incorporating fragmentomics features through machine learning. It can potentially be used for PDAC early screening, and therefore reducing PDAC related mortalities.

Structural variant calling and clinical interpretation in 6224 unsolved rare disease exomes

Structural variants (SVs), including large deletions, duplications, inversions, translocations, and more complex events have the potential to disrupt gene function resulting in rare disease. Nevertheless, current pipelines and clinical decision support systems for exome sequencing (ES) tend to focus on small alterations such as single nucleotide variants (SNVs) and insertions-deletions shorter than 50 base pairs (indels). Additionally, detection and interpretation of large copy-number variants (CNVs) are frequently performed. However, detection of other types of SVs in ES data is hampered by the difficulty of identifying breakpoints in off-target (intergenic or intronic) regions, which makes robust identification of SVs challenging. In this paper, we demonstrate the utility of SV calling in ES resulting in a diagnostic yield of 0.4% (23 out of 5825 probands) for a large cohort of unsolved patients collected by the Solve-RD consortium. Remarkably, 8 out of 23 pathogenic SV were not found by comprehensive read-depth-based CNV analysis, resulting in a 0.13% increased diagnostic value.