Diagnostic utility and reporting recommendations for clinical DNA methylation episignature testing in genetically undiagnosed rare diseases.

PURPOSE: This study aims to assess the diagnostic utility and provide reporting recommendations for clinical DNA methylation episignature testing based on the cohort of patients tested through the EpiSign Clinical Testing Network. METHODS: The EpiSign assay utilized unsupervised clustering techniques and a support vector machine-based classification algorithm to compare each patient's genome-wide DNA methylation profile with the EpiSign Knowledge Database, yielding the result that was reported. An international working group, representing distinct EpiSign Clinical Testing Network health jurisdictions, collaborated to establish recommendations for interpretation and reporting of episignature testing. RESULTS: Among 2399 cases analyzed, 1667 cases underwent a comprehensive screen of validated episignatures, imprinting, and promoter regions, resulting in 18.7% (312/1667) positive reports. The remaining 732 referrals underwent targeted episignature analysis for assessment of sequence or copy-number variants (CNVs) of uncertain significance or for assessment of clinical diagnoses without confirmed molecular findings, and 32.4% (237/732) were positive. Cases with detailed clinical information were highlighted to describe various utility scenarios for episignature testing. CONCLUSION: Clinical DNA methylation testing including episignatures, imprinting, and promoter analysis provided by an integrated network of clinical laboratories enables test standardization and demonstrates significant diagnostic yield and clinical utility beyond DNA sequence analysis in rare diseases.

Clinical findings in individuals with duplication of genes associated with X-linked intellectual disability.

Duplication of all genes associated with X-linked intellectual disability (XLID) have been reported but the majority of the duplications include more than one XLID gene. It is exceptional for whole XLID gene duplications to cause the same phenotype as sequence variants or deletions of the same gene. Duplication of PLP1, the gene associated with Pelizaeus-Merzbacher syndrome, is the most notable duplication of this type. More commonly, duplication of XLID genes results in very different phenotypes than sequence alterations or deletions. Duplication of MECP2 is widely recognized as a duplication of this type, but a number of others exist. The phenotypes associated with gene duplications are often milder than those caused by deletions and sequence variants. Among some duplications that are clinically significant, marked skewing of X-inactivation in female carriers has been observed. This report describes the phenotypic consequences of duplication of 22 individual XLID genes, of which 10 are described for the first time.

Optical Genome Mapping: Integrating Structural Variations for Precise Homologous Recombination Deficiency Score Calculation.

Homologous recombination deficiency (HRD) is characterized by the inability of a cell to repair the double-stranded breaks using the homologous recombination repair (HRR) pathway. The deficiency of the HRR pathway results in defective DNA repair, leading to genomic instability and tumorigenesis. The presence of HRD has been found to make tumors sensitive to ICL-inducing platinum-based therapies and poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) inhibitors (PARPi). However, there are no standardized methods to measure and report HRD phenotypes. Herein, we compare optical genome mapping (OGM), chromosomal microarray (CMA), and a 523-gene NGS panel for HRD score calculations. This retrospective study included the analysis of 196 samples, of which 10 were gliomas, 176 were hematological malignancy samples, and 10 were controls. The 10 gliomas were evaluated with both CMA and OGM, and 30 hematological malignancy samples were evaluated with both the NGS panel and OGM. To verify the scores in a larger cohort, 135 cases were evaluated with the NGS panel and 71 cases with OGM. The HRD scores were calculated using a combination of three HRD signatures that included loss of heterozygosity (LOH), telomeric allelic imbalance (TAI), and large-scale transitions (LST). In the ten glioma cases analyzed with OGM and CMA using the same DNA (to remove any tumor percentage bias), the HRD scores (mean ± SEM) were 13.2 (±4.2) with OGM compared to 3.7 (±1.4) with CMA. In the 30 hematological malignancy cases analyzed with OGM and the 523-gene NGS panel, the HRD scores were 7.6 (±2.2) with OGM compared to 2.6 (±0.8) with the 523-gene NGS panel. OGM detected 70.8% and 66.8% of additional variants that are considered HRD signatures in gliomas and hematological malignancies, respectively. The higher sensitivity of OGM to capture HRD signature variants might enable a more accurate and precise correlation with response to PARPi and platinum-based drugs. This study reveals HRD signatures that are cryptic to current standard of care (SOC) methods used for assessing the HRD phenotype and presents OGM as an attractive alternative with higher resolution and sensitivity to accurately assess the HRD phenotype.

The smallest likely pathogenic duplication of a SOX9 enhancer identified to date in a family with 46,XX testicular differences of sex development.

Copy number variants that duplicate distal upstream enhancer elements of the SOX9 gene cause 46,XX testicular differences of sex development (DSD) which is characterized by a 46,XX karyotype in an individual presenting with either ambiguous genitalia or genitalia with varying degrees of virilization, including those resembling typical male genitalia. Reported duplications in this region range in size from 24 to 780 kilobases (kb). Here we report a family with two affected individuals, the proband and his maternal uncle, harboring a 3.7 kb duplication of a SOX9 enhancer identified by clinical genome sequencing. Prior fluorescence in situ hybridization (FISH) for SRY and a multi-gene panel for ambiguous genitalia were non-diagnostic. The unaffected mother also carries this duplication, consistent with previously described incomplete penetrance. To our knowledge, this is the smallest duplication identified to-date, most of which resides in a 5.2 kb region that has been previously shown to possess enhancer activity that promotes the expression of SOX9. The duplication was confirmed by quantitative-PCR and shown to be in tandem by bidirectional Sanger sequencing breakpoint analysis. This finding highlights the importance of non-coding variant interrogation in suspected genetic disorders.

Large Chromosome 2p Duplication-Associated Mechanisms and Clinical Presentations.

Chromosome 2p (chr2p) duplication, also known as trisomy 2p, is a rare chromosome abnormality associated with developmental delay, intellectual disability, behavioral problems, and distinctive facial features. Most of the reported cases involving trisomy 2p include additional copy number variants (CNVs) in other regions of the genome and are usually small in size. Little is known about the clinical outcomes of large duplications of chr2p as the sole cytogenetic abnormality. In this study, 193 samples at the Greenwood Genetic Center (GGC) with CNVs involving chr2p were evaluated, out of which 86 had chr2p duplications. Among them, 8 patients were identified with large chr2p duplications ranging in size from 9.3 Mb to 89 Mb, and no deletions or duplications involving other chromosomes were identified in those patients. These duplications were associated with inverted duplication, tandem duplication, and duplication as the result of translocation, with no additional CNVs identified by microarray analysis. Confirmation by conventional cytogenetics was performed in 7 of the 8 patients, and the translocations were confirmed by fluorescence in situ hybridization. Interestingly, 1 patient was found to have mosaic complete trisomy 2p as the result of an unbalanced de novo (X;2) chromosomal translocation. X-inactivation was skewed toward the derivative X chromosome, yet it did not appear to extend into the chromosome 2 material. Various shared clinical manifestations were observed in the individuals in this study, including developmental delay, hemifacial hypoplasia, cleft palate, and short stature, and they also have distinct features such as hypotonia, cerebellar hypogenesis, and corpus callosum agenesis, which might result from a gene dosage effect of the duplication. In conclusion, single-event large chr2p duplications can result from different mechanisms, including inverted or tandem duplications within chromosome 2, or translocations involving chromosome 2 and other chromosomes. Partial or complete trisomy 2p is commonly associated with developmental delay, and additional clinical features may be related to gene dosage effects.

17q25.3 copy number changes: association with neurodevelopmental disorders and cardiac malformation.

Copy number variants (CNVs) have been identified as common genomic variants that play a significant role in inter-individual variability. Conversely, rare recurrent CNVs have been found to be causal for many disorders with well-established genotype-phenotype relationships. However, the phenotypic implications of rare non-recurrent CNVs remain poorly understood. Herein, we re-investigated 18,542 cases reported from chromosomal microarray at Greenwood Genetic Center from 2010 to 2022 and identified 15 cases with CNVs involving the 17q25.3 region. We report the detailed clinical features of these subjects, and compare with the cases reported in the literature to determine genotype-phenotype correlations for a subset of genes in this region. The CNVs in the 17q25.3 region were found to be rare events, with a prevalence of 0.08% (15/18542) observed in our cohort. The CNVs were dispersed across the entire 17q25.3 region with variable breakpoints and no smallest region of overlap. The subjects presented with a wide range of clinical features, with neurodevelopmental disorders (autism spectrum disorder, intellectual disability, developmental delay) being the most common features (80%), then expressive language disorder (33%), and finally cardiovascular malformations (26%). The association of CNVs involving the critical gene-rich region of 17q25.3 with neurodevelopmental disorders and cardiac malformation, implicates several genes as plausible drivers for these events.

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

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

Sleep disturbances in Phelan-McDermid syndrome: Clinical and metabolic profiling of 56 individuals.

Phelan-McDermid Syndrome (PMS) is caused by deletions at chromosome 22q13.3 or pathogenic/likely pathogenic SHANK3 variants. The clinical presentation is extremely variable and includes global developmental delay/intellectual disability (ID), seizures, neonatal hypotonia, and sleep disturbances, among others. This study investigated the prevalence of sleep disturbances, and the genetic and metabolic features associated with them, in a cohort of 56 individuals with PMS. Sleep data were collected via standardized observer/caregiver questionnaires, while genetic data from array-CGH and sequencing of 9 candidate genes within the 22q13.3 region, and metabolic profiling utilized the Biolog Phenotype Mammalian MicroArray plates. Sleep disturbances were present in 64.3% of individuals with PMS, with the most common problem being waking during the night (39%). Sleep disturbances were more prevalent in individuals with a SHANK3 pathogenic variant (89%) compared to subjects with 22q13.3 deletions of any size (59.6%). Distinct metabolic profiles for individuals with PMS with and without sleep disturbances were also identified. These data are helpful information for recognizing and managing sleep disturbances in individuals with PMS, outlining the main candidate gene for this neurological manifestation, and highlighting potential biomarkers for early identification of at-risk subjects and molecular targets for novel treatment approaches.

Clinical Validation and Diagnostic Utility of Optical Genome Mapping in Prenatal Diagnostic Testing.

The standard-of-care diagnostic prenatal testing includes a combination of cytogenetic methods, such as karyotyping, fluorescence in situ hybridization (FISH), and chromosomal microarray (CMA), using either direct or cultured amniocytes or chorionic villi sampling. However, each technology has its limitations: karyotyping has a low resolution (>5 Mb), FISH is targeted, and CMA does not detect balanced structural variations (SVs). These limitations necessitate the use of multiple tests, either simultaneously or sequentially, to reach a genetic diagnosis. Optical genome mapping (OGM) is an emerging technology that can detect several classes of SVs in a single assay, but it has not been evaluated in the prenatal setting. This validation study analyzed 114 samples that were received in our laboratory for traditional cytogenetic analysis with karyotyping, FISH, and/or CMA. OGM was 100% concordant in identifying the 101 aberrations that included 29 interstitial/terminal deletions, 28 duplications, 26 aneuploidies, 6 absence of heterozygosity regions, 3 triploid genomes, 4 isochromosomes, and 1 translocation; and the method revealed the identity of 3 marker chromosomes and 1 chromosome with additional material not determined by karyotyping. In addition, OGM detected 64 additional clinically reportable SVs in 43 samples. OGM has a standardized laboratory workflow and reporting solution that can be adopted in routine clinical laboratories and demonstrates the potential to replace the current standard-of-care methods for prenatal diagnostic testing.

Functional correlation of genome-wide DNA methylation profiles in genetic neurodevelopmental disorders.

An expanding range of genetic syndromes are characterized by genome-wide disruptions in DNA methylation profiles referred to as episignatures. Episignatures are distinct, highly sensitive, and specific biomarkers that have recently been applied in clinical diagnosis of genetic syndromes. Episignatures are contained within the broader disorder-specific genome-wide DNA methylation changes, which can share significant overlap among different conditions. In this study, we performed functional genomic assessment and comparison of disorder-specific and overlapping genome-wide DNA methylation changes related to 65 genetic syndromes with previously described episignatures. We demonstrate evidence of disorder-specific and recurring genome-wide differentially methylated probes (DMPs) and regions (DMRs). The overall distribution of DMPs and DMRs across the majority of the neurodevelopmental genetic syndromes analyzed showed substantial enrichment in gene promoters and CpG islands, and under-representation of the more variable intergenic regions. Analysis showed significant enrichment of the DMPs and DMRs in gene pathways and processes related to neurodevelopment, including neurogenesis, synaptic signaling and synaptic transmission. This study expands beyond the diagnostic utility of DNA methylation episignatures by demonstrating correlation between the function of the mutated genes and the consequent genomic DNA methylation profiles as a key functional element in the molecular etiology of genetic neurodevelopmental disorders.

Multi-tissue cytogenetic analysis for the diagnosis of mosaic Down syndrome: A case report.

Less than one percent of individuals with Down syndrome exhibit mosaicism, a biological phenomenon that describes an individual who has two or more genetically distinct cell lines. The percentage of mosaicism in different tissues can impact the presence of clinical findings and hinder cytogenetic diagnosis. We report a case of mosaicism for trisomy 21 diagnosed after multi-tissue cytogenetic analysis of peripheral blood and buccal mucosa, associated with significant intellectual disability, dysmorphic facial features, congenital heart defects, macropenis, and imperforate anus.

Optical Genome Mapping and Single Nucleotide Polymorphism Microarray: An Integrated Approach for Investigating Products of Conception.

Conventional cytogenetic analysis of products of conception (POC) is of limited utility because of failed cultures, as well as microbial and maternal cell contamination (MCC). Optical genome mapping (OGM) is an emerging technology that has the potential to replace conventional cytogenetic methods. The use of OGM precludes the requirement for culturing (and related microbial contamination). However, a high percentage of MCC impedes a definitive diagnosis, which can be addressed by an additional pre-analytical quality control step that includes histological assessment of H&E stained slides from formalin-fixed paraffin embedded (FFPE) tissue with macro-dissection for chorionic villi to enrich fetal tissue component for single nucleotide polymorphism microarray (SNPM) analysis. To improve the diagnostic yield, an integrated workflow was devised that included MCC characterization of POC tissue, followed by OGM for MCC-negative cases or SNPM with histological assessment for MCC-positive cases. A result was obtained in 93% (29/31) of cases with a diagnostic yield of 45.1% (14/31) with the proposed workflow, compared to 9.6% (3/31) and 6.4% (2/31) with routine workflow, respectively. The integrated workflow with these technologies demonstrates the clinical utility and higher diagnostic yield in evaluating POC specimens.

Novel diagnostic DNA methylation episignatures expand and refine the epigenetic landscapes of Mendelian disorders.

Overlapping clinical phenotypes and an expanding breadth and complexity of genomic associations are a growing challenge in the diagnosis and clinical management of Mendelian disorders. The functional consequences and clinical impacts of genomic variation may involve unique, disorder-specific, genomic DNA methylation episignatures. In this study, we describe 19 novel episignature disorders and compare the findings alongside 38 previously established episignatures for a total of 57 episignatures associated with 65 genetic syndromes. We demonstrate increasing resolution and specificity ranging from protein complex, gene, sub-gene, protein domain, and even single nucleotide-level Mendelian episignatures. We show the power of multiclass modeling to develop highly accurate and disease-specific diagnostic classifiers. This study significantly expands the number and spectrum of disorders with detectable DNA methylation episignatures, improves the clinical diagnostic capabilities through the resolution of unsolved cases and the reclassification of variants of unknown clinical significance, and provides further insight into the molecular etiology of Mendelian conditions.

A SOX3 duplication and lumbosacral spina bifida in three generations.

Chromosomal aneuploidies, microduplications and microdeletions are the most common confirmed genetic causes of spina bifida. Microduplications of Xq27 containing the SOX3 gene have been reported in 11 cases, confirming the existence of an X-chromosomal locus for spina bifida. A three generation kindred reported here with a SOX3 duplication has been identified in one of 17 kindreds with recurrences in the 29 years of the South Carolina Neural Tube Defect Prevention Program. Other recurrences during this time period included siblings with an APAF1 mutation, siblings with a CASP9 mutation, siblings with a microdeletion of 13q, and two sets of siblings with Meckel syndrome who did not have genetic/genomic studies performed.

Identification of a DNA Methylation Episignature in the 22q11.2 Deletion Syndrome.

The 22q11.2 deletion syndrome (22q11.2DS) is the most common genomic disorder in humans and is the result of a recurrent 1.5 to 2.5 Mb deletion, encompassing approximately 20-40 genes, respectively. The clinical presentation of the typical deletion includes: Velocardiofacial, Di George, Opitz G/BBB and Conotruncalanomaly face syndromes. Atypical deletions (proximal, distal or nested) are rare and characterized mainly by normal phenotype or mild intellectual disability and variable clinical features. The pathogenetic mechanisms underlying this disorder are not completely understood. Because the 22q11.2 region harbours genes coding for transcriptional factors and chromatin remodelers, in this study, we performed analysis of genome-wide DNA methylation of peripheral blood from 49 patients with 22q11.2DS using the Illumina Infinium Methylation EPIC bead chip arrays. This cohort comprises 43 typical, 2 proximal and 4 distal deletions. We demonstrated the evidence of a unique and highly specific episignature in all typical and proximal 22q11.2DS. The sensitivity and specificity of this signature was further confirmed by comparing it to over 1500 patients with other neurodevelopmental disorders with known episignatures. Mapping the 22q11.2DS DNA methylation episignature provides both novel insights into the molecular pathogenesis of this disorder and an effective tool in the molecular diagnosis of 22q11.2DS.

Copy neutral absence of heterozygosity on chromosome 15 distal long arm: A surrogate marker for Prader-Willi/Angelman syndromes?

BACKGROUND: Copy-neutral absence of heterozygosity (CN-AOH) observed on a single chromosome or part of a chromosome may be indicative of uniparental disomy (UPD) and may require additional testing when such chromosomes or chromosome regions are known to harbor imprinted genes. CASE PRESENTATION: Here we report 2 cases of neonates that presented to clinic with hypotonia, poor oral skills including inability to feed by mouth, weak cry, no response to noxious stimulation and vertical plantar creases (case 1) and hypotonia and respiratory distress (case 2). A preliminary chromosome analysis showed normal karyotypes in both cases while the high-resolution single nucleotide polymorphism (SNP) microarray showed copy neutral absence of heterozygosity involving chromosome 15 distal long arm. In case 1, the CN-AOH involved a 28.7 Mb block from genomic coordinates 73703619_102429049. In case 2, the CN-AOH involved a 15.3 Mb block from genomic coordinates 54729197_70057534. In both cases, methylation-specific PCR did not detect an unmethylated allele for the SNRPN gene suggesting either a deletion of paternal allele or maternal UPD for chromosome 15. Since microarray analysis did not show any copy number alterations on chromosome 15, a microdeletion was ruled out. CONCLUSIONS: Based on our cases, we suggest that CN-AOH on chromosome 15, even if it does not involve the critical region of 15q12q13, should warrant additional studies for diagnosis of Prader-Willi/Angelman syndromes.

Clinical epigenomics: genome-wide DNA methylation analysis for the diagnosis of Mendelian disorders.

PURPOSE: We describe the clinical implementation of genome-wide DNA methylation analysis in rare disorders across the EpiSign diagnostic laboratory network and the assessment of results and clinical impact in the first subjects tested. METHODS: We outline the logistics and data flow between an integrated network of clinical diagnostics laboratories in Europe, the United States, and Canada. We describe the clinical validation of EpiSign using 211 specimens and assess the test performance and diagnostic yield in the first 207 subjects tested involving two patient subgroups: the targeted cohort (subjects with previous ambiguous/inconclusive genetic findings including genetic variants of unknown clinical significance) and the screening cohort (subjects with clinical findings consistent with hereditary neurodevelopmental syndromes and no previous conclusive genetic findings). RESULTS: Among the 207 subjects tested, 57 (27.6%) were positive for a diagnostic episignature including 48/136 (35.3%) in the targeted cohort and 8/71 (11.3%) in the screening cohort, with 4/207 (1.9%) remaining inconclusive after EpiSign analysis. CONCLUSION: This study describes the implementation of diagnostic clinical genomic DNA methylation testing in patients with rare disorders. It provides strong evidence of clinical utility of EpiSign analysis, including the ability to provide conclusive findings in the majority of subjects tested.