Search

Background and Objective Complex regional pain syndrome (CRPS) is a chronic condition characterized by severe regional pain, allodynia, hyperalgesia, and functional impairment. The aim of this systematic review is to investigate whether a familial subtype of CRPS (fCRPS) exists and to determine whether people with fCRPS have specific characteristics. Methods Databases CINAHL, Medline, PsycINFO, and PubMed were searched with no date limitation. Quality of reporting was assessed using the Scottish Intercollegiate Guidelines Network scale and the Joanna Briggs Institute’s checklists. Results Eight studies were included. Family relationships were defined as any immediate (i.e., parents or siblings) or blood relatives. A combination of participants with known or unknown causes for CRPS was recruited. The studies in this review support the potential for the existence of fCRPS, although this included less than 25% of those affected. People with potential fCRPS showed more severe symptoms, more sites involved, a higher percentage of spontaneous onset, and earlier age at onset. An elevated sibling recurrence risk ratio of 5.6 (95% confidence interval [CI], 3.0 to 9.8) was reported for people under 50. None of the studies established a pattern of heritability. Therefore, the most likely explanation for heritability would be a multifactorial model in which cumulative and interactive Gene × Environment effects may be involved. Conclusions This systematic review supports the potential for the existence of fCRPS; however, all identified studies used uncontrolled case reports, case series, and case–control designs that cannot provide evidence of causation. Further studies are required to reveal the heritability and genetic structure of fCRPS.

Runt-related transcription factor 1 translocated to 1 (RUNX1T1; also known as eight-twenty-one [ETO]) encodes a transcription regulator for hematopoietic genes and is well known for its involvement in hematologic malignancies, particularly acute myeloid leukemia (AML). However, its role in congenital disease is less understood. This study provides detailed clinical and molecular information on three cases exhibiting neurodevelopmental and congenital anomalies with germline de novo alterations in RUNX1T1. One case features a de novo nonsense variant in the 5' region of the gene (c.106C>T p.Gln36Ter), while the other two harbor de novo missense variants in the C terminus end (c.1234G>A p.Gly412Arg and c.1561C>T p.His521Tyr). Common features across cases include craniofacial dysmorphism and neurodevelopmental issues, including developmental delay, learning disabilities, attention-deficit hyperactivity disorder, and autism. This study, in conjunction with previously reported germline disruptions of RUNX1T1, provides evidence supporting the role of germline RUNX1T1 variation in human congenital neurodevelopmental disorders., Competing Interests: Declaration of interests E.A.-E., D.A.C., E.A.N., Y.Q., K.M., and E.T. are employees of and may own stock in GeneDx, LLC., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Synonymous variants have been shown to alter the correct splicing of pre-mRNAs and generate disease-causing transcripts. These variants are not an uncommon etiology of genetic disease; however, they are frequently overlooked during genetic testing in the absence of functional and clinical data. Here, we describe the occurrence of a synonymous variant [NM_005422.4 (TECTA):c.327C>T, p.(Gly109=)] in seven individuals with hearing loss from six unrelated families. The variant is not located near exonic/intronic boundaries but is predicted to impact splicing by activating a cryptic splicing donor site in exon 4 of TECTA. In vitro minigene assays show that the variant disrupts the reading frame of the canonical transcript, which is predicted to cause a premature termination codon 48 amino acids downstream of the variant, leading to nonsense-mediated decay. The variant is present in population databases, predominantly in Latinos of African ancestry, but is rare in other ethnic groups. Our findings suggest that this synonymous variant is likely pathogenic for TECTA-associated autosomal recessive hearing loss and seems to have arisen as a founder variant in this specific Latino subpopulation. This study demonstrates that synonymous variants need careful splicing assessment and support from additional testing methodologies to determine their clinical impact., (© 2022 Wiley Periodicals LLC.)

Long-read sequencing (LRS) has been around for more than a decade, but widespread adoption of the technology has been slow due to the perceived high error rates and high sequencing cost. This is changing due to the recent advancements to produce highly accurate sequences and the reducing costs. LRS promises significant improvement over short read sequencing in four major areas: (1) better detection of structural variation (2) better resolution of highly repetitive or nonunique regions (3) accurate long-range haplotype phasing and (4) the detection of base modifications natively from the sequencing data. Several successful applications of LRS have demonstrated its ability to resolve molecular diagnoses where short-read sequencing fails to identify a cause. However, the argument for increased diagnostic yield from LRS remains to be validated. Larger cohort studies may be required to establish the realistic boundaries of LRS's clinical utility and analytical validity, as well as the development of standards for clinical applications. We discuss the limitations of the current standard of care, and contrast with the applications and advantages of two major LRS platforms, PacBio and Oxford Nanopore, for molecular diagnostics of constitutional disorders, and present a critical argument about the potential of LRS in diagnostic settings., (© 2022 Wiley Periodicals LLC.)

KDM5C encodes a demethylase of the histone H3 lysine 4 residue, involved in chromatin regulation and gene expression. Hemizygous KDM5C pathogenic variants cause X-linked intellectual disability of Claes-Jensen type. Because of its mode of inheritance and the low specificity of the clinical phenotype, interpretation of variants can be difficult, hence the need for functional studies and biomarkers specific to this disorder. We present the case of a male patient with intellectual disability, behavioral abnormalities and subtle dysmorphic features, in which genetic investigation identified a hemizygous novel missense KDM5C variant of uncertain significance (VUS), inherited from his asymptomatic mother and present in his paucisymptomatic sister. We assessed the global genomic DNA methylation status from a whole blood sample of the proband. Global DNA methylation profiling specifically identified the recently discovered epi-signature of Claes-Jensen syndrome. This result served as a biomarker which independently highlighted KDM5C as the cause of the disorder in this patient. Because of the X-linked mode of inheritance, variant reclassification had a high impact on genetic counseling in this family. This example highlights the value of global methylome profiling in situations of variants of uncertain significance in genes with a known specific epi-signature., (Copyright © 2022 Elsevier Masson SAS. All rights reserved.)

Li-Fraumeni syndrome (LFS) is one of the most common cancer predisposition syndromes that affects both children and adults. Individuals with LFS are at an increased risk of developing various types of cancer over their lifetime including soft tissue sarcomas, osteosarcomas, breast cancer, leukemia, brain tumors, and adrenocortical carcinoma. Heterozygous germline pathogenic variants in the tumor suppressor gene TP53 are the known causal genetic defect for LFS. Single-nucleotide variants (SNVs) including missense substitutions that occur in the highly conserved DNA binding domain of the protein are the most common alterations, followed by nonsense and splice site variants. Gross copy-number changes in TP53 are rare and account for <1% of all variants. Using next-generation sequencing (NGS) panels, we identified a paternally inherited germline intragenic duplication of TP53 in a child with metastatic osteosarcoma who later developed acute myeloid leukemia (AML). Transcriptome sequencing (RNA-seq) demonstrated the duplication was tandem, encompassing exons 2-6 and 28 nt of the untranslated region (UTR) upstream of the start codon in exon 2. The inclusion of the 28 nt is expected to result in a frameshift with a stop codon 18 codons downstream from the exon 6, leading to a loss-of-function allele. This case highlights the significance of simultaneous identification of both significant copy-number variants as well as SNVs/indels using NGS panels., (© 2022 Xu et al.; Published by Cold Spring Harbor Laboratory Press.)

Mitochondrial disease diagnosis requires interrogation of both nuclear and mitochondrial (mtDNA) genomes for single-nucleotide variants (SNVs) and copy number alterations, both in the proband and often maternal relatives, together with careful phenotype correlation. We developed a comprehensive mtDNA sequencing test ('MitoGenome') using long-range PCR (LR-PCR) to amplify the full length of the mtDNA genome followed by next generation sequencing (NGS) to accurately detect SNVs and large-scale mtDNA deletions (LSMD), combined with droplet digital PCR (ddPCR) for LSMD heteroplasmy quantification. Overall, MitoGenome tests were performed on 428 samples from 394 patients with suspected or confirmed mitochondrial disease. The positive yield was 11% (43/394), including 34 patients with pathogenic or likely pathogenic SNVs (the most common being m.3243A > G in 8/34 (24%) patients), 8 patients with single LSMD, and 3 patients with multiple LSMD exceeding 10% heteroplasmy levels. Two patients with both LSMD and pathogenic SNV were detected. Overall, this LR-PCR/NGS assay provides a highly accurate and comprehensive diagnostic method for simultaneous mtDNA SNV detection at heteroplasmy levels as low as 1% and LSMD detection at heteroplasmy levels below 10%. Inclusion of maternal samples for variant classification and ddPCR to quantify LSMD heteroplasmy levels further enables accurate pathogenicity assessment and clinical correlation interpretation of mtDNA genome sequence variants and copy number alterations., Competing Interests: Declaration of Competing Interest None., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The NTRK gene family is composed of NTRK1, NTRK2, and NTRK3, which encode three tropomyosin-receptor kinases, belonging to a class of tyrosine kinase receptors. These proteins are known to play roles in cell proliferation, differentiation, apoptosis, and survival. Fusions involving the NTRK genes are long known as drivers in many tumors. Although they occur in less than 5% of all malignancies, their occurrence in a great diversity of tumors has been documented. Several rare tumors including infantile fibrosarcoma, secretory breast carcinoma, and mammary analogue secretory carcinoma are accompanied by NTRK fusions in more than 90% of cases, demonstrating a diagnostic value for the NTRK fusion testing in these tumors. More recently, the development of effective targeted therapies has created a demand for their detection in all malignancies. A variety of approaches are available for testing including immunohistochemistry, fluorescence in situ hybridization (FISH), reverse transcription polymerase chain reaction (RT-PCR), and DNA- and RNA-based next-generation sequencing (NGS). This article reviews the molecular biology and tumorigenesis of NTRK fusions, their prevalence and clinical significance with a focus on available methods for fusion detection. The advantages and limitations of different technologies, the best practice algorithms for NTRK fusion detection, and the future direction of NTRK testing are also discussed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Inc.)

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.

The adaptation of a broad genomic sequencing approach in the clinical setting has been accompanied by considerations regarding the clinical utility, technical performance, and diagnostic yield compared to targeted genetic approaches. We have developed MedExome, an integrated framework for sequencing, variant calling (SNVs, Indels, and CNVs), and clinical assessment of ~4600 medically relevant genes. We compared the technical performance of MedExome with the whole-exome and targeted gene-panel sequencing, assessed the reasons for discordance, and evaluated the added clinical yield of MedExome in a cohort of unresolved subjects suspected of genetic disease. Our analysis showed that despite a higher average read depth in panels (3058 vs. 855), MedExome yielded full coverage of the enriched regions (>20X) and 99% variant concordance rate with panels. The discordance rate was associated with low-complexity regions, high-GC content, and low allele fractions, observed in both platforms. MedExome yielded full sensitivity in detecting clinically actionable variants, and the assessment of 138 patients with suspected genetic conditions resulted in 76 clinical reports (31 full [22.1%], 3 partial, and 42 uncertain/possible molecular diagnoses). MedExome sequencing has comparable performance in variant detection to gene panels. Added diagnostic yield justifies expanded implementation of broad genomic approaches in unresolved patients; however, cost-benefit and health systems impact warrants assessment.

Purpose: Diabetic retinopathy (DR) remains a pressing issue worldwide. Abnormal angiogenesis is a distinct vascular lesion in DR, and research has established that vascular endothelial growth factor A (VEGF-A) is a primary mediator of such changes. However, limitations in current anti-VEGF therapies suggest that our understanding of molecular networks underlying ocular angiogenesis remains far from complete. Based on our long non-coding RNA (lncRNA) array analyses, HOX antisense intergenic RNA (HOTAIR) was identified as one of the top upregulated lncRNAs in high glucose-cultured human retinal endothelial cells (HRECs). Given the well-documented roles of HOTAIR in cancer, no studies have examined the epigenetic implications of HOTAIR in DR, and we investigated such relationships herein., Methods: We used HRECs exposed to various glucose concentrations and epigenetic modulators to examine HOTAIR, angiogenic, and DR-related molecular markers. Oxidative stress, angiogenesis, and mitochondrial dysfunction were assessed. Retinal tissues of diabetic rodents and the vitreous humor and serum of patients with proliferative DR were also investigated., Results: Hyperglycemia significantly augmented HOTAIR expression in HRECs and promoted angiogenesis, oxidative damage, and mitochondrial aberrations. Similarly, vitreous humor and serum from proliferative DR patients and retinas from diabetic animals demonstrated increased HOTAIR expression compared to non-diabetic controls. HOTAIR knockdown protected against glucose-induced increases of angiogenic and diabetes-associated molecules in the retina. Mechanistically, we showed that HOTAIR exerts its capabilities by preventing oxidative stress and modulating epigenetic pathways involving histone methylation, histone acetylation, DNA methylation, and transcription factors., Conclusions: Our findings suggest that HOTAIR is a critical lncRNA in the pathogenesis of DR and may potentially be important for diagnostic and therapeutic targeting.

A growing number of genetic neurodevelopmental disorders are known to be associated with unique genomic DNA methylation patterns, called episignatures, which are detectable in peripheral blood. The intellectual developmental disorder, X-linked, syndromic, Armfield type (MRXSA) is caused by missense variants in FAM50A . Functional studies revealed the pathogenesis to be a spliceosomopathy that is characterized by atypical mRNA processing during development. In this study, we assessed the peripheral blood specimens in a cohort of individuals with MRXSA and detected a unique and highly specific DNA methylation episignature associated with this disorder. We used this episignature to construct a support vector machine model capable of sensitive and specific identification of individuals with pathogenic variants in FAM50A . This study contributes to the expanding number of genetic neurodevelopmental disorders with defined DNA methylation episignatures, provides an additional understanding of the associated molecular mechanisms, and further enhances our ability to diagnose patients with rare disorders.

Background: Phelan-McDermid syndrome is characterized by a range of neurodevelopmental phenotypes with incomplete penetrance and variable expressivity. It is caused by a variable size and breakpoint microdeletions in the distal long arm of chromosome 22, referred to as 22q13.3 deletion syndrome, including the SHANK3 gene. Genetic defects in a growing number of neurodevelopmental genes have been shown to cause genome-wide disruptions in epigenomic profiles referred to as epi-signatures in affected individuals., Results: In this study we assessed genome-wide DNA methylation profiles in a cohort of 22 individuals with Phelan-McDermid syndrome, including 11 individuals with large (2 to 5.8 Mb) 22q13.3 deletions, 10 with small deletions (< 1 Mb) or intragenic variants in SHANK3 and one mosaic case. We describe a novel genome-wide DNA methylation epi-signature in a subset of individuals with Phelan-McDermid syndrome., Conclusion: We identified the critical region including the BRD1 gene as responsible for the Phelan-McDermid syndrome epi-signature. Metabolomic profiles of individuals with the DNA methylation epi-signature showed significantly different metabolomic profiles indicating evidence of two molecularly and phenotypically distinct clinical subtypes of Phelan-McDermid syndrome.

Mendelian neurodevelopmental disorders customarily present with complex and overlapping symptoms, complicating the clinical diagnosis. Individuals with a growing number of the so-called rare disorders exhibit unique, disorder-specific DNA methylation patterns, consequent to the underlying gene defects. Besides providing insights to the pathophysiology and molecular biology of these disorders, we can use these epigenetic patterns as functional biomarkers for the screening and diagnosis of these conditions. This review summarizes our current understanding of DNA methylation episignatures in rare disorders and describes the underlying technology and analytical approaches. We discuss the computational parameters, including statistical and machine learning methods, used for the screening and classification of genetic variants of uncertain clinical significance. Describing the rationale and principles applied to the specific computational models that are used to develop and adapt the DNA methylation episignatures for the diagnosis of rare disorders, we highlight the opportunities and challenges in this emerging branch of diagnostic medicine.

Purpose: Nontruncating variants in SMARCA2, encoding a catalytic subunit of SWI/SNF chromatin remodeling complex, cause Nicolaides-Baraitser syndrome (NCBRS), a condition with intellectual disability and multiple congenital anomalies. Other disorders due to SMARCA2 are unknown., Methods: By next-generation sequencing, we identified candidate variants in SMARCA2 in 20 individuals from 18 families with a syndromic neurodevelopmental disorder not consistent with NCBRS. To stratify variant interpretation, we functionally analyzed SMARCA2 variants in yeasts and performed transcriptomic and genome methylation analyses on blood leukocytes., Results: Of 20 individuals, 14 showed a recognizable phenotype with recurrent features including epicanthal folds, blepharophimosis, and downturned nasal tip along with variable degree of intellectual disability (or blepharophimosis intellectual disability syndrome [BIS]). In contrast to most NCBRS variants, all SMARCA2 variants associated with BIS are localized outside the helicase domains. Yeast phenotype assays differentiated NCBRS from non-NCBRS SMARCA2 variants. Transcriptomic and DNA methylation signatures differentiated NCBRS from BIS and those with nonspecific phenotype. In the remaining six individuals with nonspecific dysmorphic features, clinical and molecular data did not permit variant reclassification., Conclusion: We identified a novel recognizable syndrome named BIS associated with clustered de novo SMARCA2 variants outside the helicase domains, phenotypically and molecularly distinct from NCBRS.

Poly-ADP-ribose-polymerase inhibitor (PARPi) treatment is indicated for advanced-stage ovarian tumors with BRCA1/2 deficiency. The "BRCAness" status is thought to be attributed to a tumor phenotype associated with a specific epigenomic DNA methylation profile. Here, we examined the diagnostic impact of combined BRCA1/2 sequence, copy number, and promoter DNA methylation analysis, and evaluated whether genomic DNA methylation patterns can predict the BRCAness in ovarian tumors. DNA sequencing of 172 human tissue samples of advanced-stage ovarian adenocarcinoma identified 36 samples with a clinically significant tier 1/2 sequence variants (point mutations and in/dels) and 9 samples with a CNV causing a loss of function in BRCA1/2. DNA methylation analysis of the promoter of BRCA1/2 identified promoter hypermethylation of BRCA1 in two mutation-negative samples. Computational modeling of genome-wide methylation markers, measured using Infinium EPIC arrays, resulted in a total accuracy of 0.75, sensitivity: 0.83, specificity: 0.64, positive predictive value: 0.76, negative predictive value: 0.74, and area under the receiver's operating curve (AUC): 0.77, in classifying tumors harboring a BRCA1/2 defect from the rest. These findings indicate that the assessment of CNV and promoter DNA methylation in BRCA1/2 increases the cumulative diagnostic yield by 10%, compared with the 20% yield achieved by sequence variant analysis alone. Genomic DNA methylation data can partially predict BRCAness in ovarian tumors; however, further investigation in expanded BRCA1/2 cohorts is needed, and the effect of other double strand DNA repair gene defects in these tumors warrants further investigations.

The breadth and complexity of genetic testing in patients with suspected Mendelian neurodevelopmental disorders has rapidly expanded in the past two decades. However, in spite of advances in genomic technologies, genetic diagnosis remains elusive in more than half of these patients. Epigenomics, and in particular genomic DNA methylation profiles, are now known to be associated with the underpinning genetic defects in a growing number of Mendelian disorders. These often highly specific and sensitive molecular biomarkers have been used to screen these patient populations, resolve ambiguous clinical cases and interpret genetic variants of unknown clinical significance. Increasing the diagnostic yield beyond genomic sequencing technologies has rapidly propelled epigenomics to clinical utilization, with recent introduction of DNA methylation 'EpiSign' analysis in clinical diagnostic laboratories. This review provides an overview of the principles, applications and limitations of DNA methylation episignature analysis in patients with neurodevelopmental Mendelian disorders, and discusses clinical implications of this emerging diagnostic technology., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

DNA methylation, a critical epigenetic mechanism, plays an important role in governing gene expressions during biological processes such as aging, which is well known to be accelerated in hyperglycemia (diabetes). In the present study, we investigated the effects of glucose on whole genome DNA methylation in small [human retinal microvascular endothelial cells (HRECs)] and large [human umbilical vein endothelial cells (HUVECs)] vessel endothelial cell (EC) lines exposed to basal or high glucose-containing media for variable lengths of time. Using the Infinium EPIC array, we obtained 773,133 CpG sites (probes) for analysis. Unsupervised clustering of the top 5% probes identified four distinct clusters within EC groups, with significant methylation differences attributed to EC types and the duration of cell culture rather than glucose stimuli alone. When comparing the ECs incubated for 2 days versus 7 days, hierarchical clustering analyses [methylation change >10% and false discovery rate (FDR) <0.05] identified 17,354 and 128 differentially methylated CpGs for HUVECs and HRECs, respectively. Predominant DNA hypermethylation was associated with the length of culture and was enriched for gene enhancer elements and regions surrounding CpG shores and shelves. We identified 88 differentially methylated regions (DMRs) for HUVECs and 8 DMRs for HRECs (all FDR <0.05). Pathway enrichment analyses of DMRs highlighted involvement of regulators of embryonic development (i.e., HOX genes) and cellular differentiation [transforming growth factor-β (TGF-β) family members]. Collectively, our findings suggest that DNA methylation is a complex process that involves tightly coordinated, cell-specific mechanisms. Such changes in methylation overlap genes critical for cellular differentiation and embryonic development.

Genetic syndromes frequently present with overlapping clinical features and inconclusive or ambiguous genetic findings which can confound accurate diagnosis and clinical management. An expanding number of genetic syndromes have been shown to have unique genomic DNA methylation patterns (called "episignatures"). Peripheral blood episignatures can be used for diagnostic testing as well as for the interpretation of ambiguous genetic test results. We present here an approach to episignature mapping in 42 genetic syndromes, which has allowed the identification of 34 robust disease-specific episignatures. We examine emerging patterns of overlap, as well as similarities and hierarchical relationships across these episignatures, to highlight their key features as they are related to genetic heterogeneity, dosage effect, unaffected carrier status, and incomplete penetrance. We demonstrate the necessity of multiclass modeling for accurate genetic variant classification and show how disease classification using a single episignature at a time can sometimes lead to classification errors in closely related episignatures. We demonstrate the utility of this tool in resolving ambiguous clinical cases and identification of previously undiagnosed cases through mass screening of a large cohort of subjects with developmental delays and congenital anomalies. This study more than doubles the number of published syndromes with DNA methylation episignatures and, most significantly, opens new avenues for accurate diagnosis and clinical assessment in individuals affected by these disorders., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: We previously associated HIST1H1E mutations causing Rahman syndrome with a specific genome-wide methylation pattern., Results: Methylome analysis from peripheral blood samples of six affected subjects led us to identify a specific hypomethylated profile. This "episignature" was enriched for genes involved in neuronal system development and function. A computational classifier yielded full sensitivity and specificity in detecting subjects with Rahman syndrome. Applying this model to a cohort of undiagnosed probands allowed us to reach diagnosis in one subject., Conclusions: We demonstrate an epigenetic signature in subjects with Rahman syndrome that can be used to reach molecular diagnosis.

SETD1B is a component of a histone methyltransferase complex that specifically methylates Lys-4 of histone H3 (H3K4) and is responsible for the epigenetic control of chromatin structure and gene expression. De novo microdeletions encompassing this gene as well as de novo missense mutations were previously linked to syndromic intellectual disability (ID). Here, we identify a specific hypermethylation signature associated with loss of function mutations in the SETD1B gene which may be used as an epigenetic marker supporting the diagnosis of syndromic SETD1B-related diseases. We demonstrate the clinical utility of this unique epi-signature by reclassifying previously identified SETD1B VUS (variant of uncertain significance) in two patients.

Nontruncating sequence variants represent a major challenge in variant interpretation and classification. Here, we report a patient with features of Kabuki syndrome who carries two rare heterozygous variants in KMT2D: c.12935C>T, p.(Ser4312Phe) and c.15785-10T>G. The clinical significance of these variants were discordantly interpreted by different diagnostic laboratories. Parental testing showed that the missense variant was inherited from the father with a mild Kabuki phenotype and the intronic variant from the mother with mosaic status. Through genome-wide DNA methylation analysis of peripheral blood, we confirmed that the proband exhibited a previously described episignature of Kabuki syndrome. Parental samples had normal DNA methylation profiles, thus ruling out the involvement of the paternally inherited missense variant. RNA analysis revealed that the intronic change resulted in exon 49 skipping and frameshift, thereby providing a molecular diagnosis of Kabuki syndrome. This study demonstrates the utility of epigenomic and RNA analyses in resolving ambiguous clinical cases., (© 2019 Wiley Periodicals, Inc.)

Objectives: The diagnosis of hematologic malignancies integrates multiple diagnostic and clinical disciplines. Historically, targeted (single-analyte) genetic testing has been used as reflex to initial prescreening by other diagnostic modalities including flow cytometry, anatomic pathology, and clinical cytogenetics. Given the wide range of mutations associated with hematologic malignancies a DNA/RNA-based NGS panel can provide a more effective and economical approach to comprehensive testing of patients as an initial, tier-1 screen., Methods: Using a cohort of 380 patients, we performed clinical validation of a gene panel designed to assess 40 genes (DNA), and 29 fusion driver genes with over 600 gene fusion partners (RNA), including sample exchange data across three clinical laboratories, and correlation with cytogenetic testing results., Results: The clinical validation of this technology demonstrated that its accuracy, sensitivity, and specificity are comparable to the majority of targeted single-gene approaches, while assessment of the initial patient cohort data demonstrated a high diagnostic yield of 50.5%., Conclusions: Implementation of a tier-1 NGS-based protocol for gene panel screening provides a comprehensive alternative to targeted molecular testing in patients with suspected hematologic malignancies, with increased diagnostic yield, scalability, reproducibility, and cost effectiveness, making it ideally suited for implementation in clinical laboratories., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Epigenetic clocks are mathematical models that predict the biological age of an individual using DNA methylation data and have emerged in the last few years as the most accurate biomarkers of the aging process. However, little is known about the molecular mechanisms that control the rate of such clocks. Here, we have examined the human epigenetic clock in patients with a variety of developmental disorders, harboring mutations in proteins of the epigenetic machinery., Results: Using the Horvath epigenetic clock, we perform an unbiased screen for epigenetic age acceleration in the blood of these patients. We demonstrate that loss-of-function mutations in the H3K36 histone methyltransferase NSD1, which cause Sotos syndrome, substantially accelerate epigenetic aging. Furthermore, we show that the normal aging process and Sotos syndrome share methylation changes and the genomic context in which they occur. Finally, we found that the Horvath clock CpG sites are characterized by a higher Shannon methylation entropy when compared with the rest of the genome, which is dramatically decreased in Sotos syndrome patients., Conclusions: These results suggest that the H3K36 methylation machinery is a key component of the epigenetic maintenance system in humans, which controls the rate of epigenetic aging, and this role seems to be conserved in model organisms. Our observations provide novel insights into the mechanisms behind the epigenetic aging clock and we expect will shed light on the different processes that erode the human epigenetic landscape during aging.

Background: ADNP syndrome is a rare Mendelian disorder characterized by global developmental delay, intellectual disability, and autism. It is caused by truncating mutations in ADNP, which is involved in chromatin regulation. We hypothesized that the disruption of chromatin regulation might result in specific DNA methylation patterns that could be used in the molecular diagnosis of ADNP syndrome., Results: We identified two distinct and partially opposing genomic DNA methylation episignatures in the peripheral blood samples from 22 patients with ADNP syndrome. The "epi-ADNP-1" episignature included ~ 6000 mostly hypomethylated CpGs, and the "epi-ADNP-2" episignature included ~ 1000 predominantly hypermethylated CpGs. The two signatures correlated with the locations of the ADNP mutations. Epi-ADNP-1 mutations occupy the N- and C-terminus, and epi-ADNP-2 mutations are centered on the nuclear localization signal. The episignatures were enriched for genes involved in neuronal system development and function. A classifier trained on these profiles yielded full sensitivity and specificity in detecting patients with either of the two episignatures. Applying this model to seven patients with uncertain clinical diagnosis enabled reclassification of genetic variants of uncertain significance and assigned new diagnosis when the primary clinical suspicion was not correct. When applied to a large cohort of unresolved patients with developmental delay (N = 1150), the model predicted three additional previously undiagnosed patients to have ADNP syndrome. DNA sequencing of these subjects, wherever available, identified pathogenic mutations within the gene domains predicted by the model., Conclusions: We describe the first Mendelian condition with two distinct episignatures caused by mutations in a single gene. These highly sensitive and specific DNA methylation episignatures enable diagnosis, screening, and genetic variant classifications in ADNP syndrome.

Conventional genetic testing of individuals with neurodevelopmental presentations and congenital anomalies (ND/CAs), i.e., the analysis of sequence and copy number variants, leaves a substantial proportion of them unexplained. Some of these cases have been shown to result from DNA methylation defects at a single locus (epi-variants), while others can exhibit syndrome-specific DNA methylation changes across multiple loci (epi-signatures). Here, we investigate the clinical diagnostic utility of genome-wide DNA methylation analysis of peripheral blood in unresolved ND/CAs. We generate a computational model enabling concurrent detection of 14 syndromes using DNA methylation data with full accuracy. We demonstrate the ability of this model in resolving 67 individuals with uncertain clinical diagnoses, some of whom had variants of unknown clinical significance (VUS) in the related genes. We show that the provisional diagnoses can be ruled out in many of the case subjects, some of whom are shown by our model to have other diseases initially not considered. By applying this model to a cohort of 965 ND/CA-affected subjects without a previous diagnostic assumption and a separate assessment of rare epi-variants in this cohort, we identify 15 case subjects with syndromic Mendelian disorders, 12 case subjects with imprinting and trinucleotide repeat expansion disorders, as well as 106 case subjects with rare epi-variants, a portion of which involved genes clinically or functionally linked to the subjects' phenotypes. This study demonstrates that genomic DNA methylation analysis can facilitate the molecular diagnosis of unresolved clinical cases and highlights the potential value of epigenomic testing in the routine clinical assessment of ND/CAs., (Copyright © 2019 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Epigenetic and genetic mechanisms regulate the establishment and maintenance of gene expression in its proper context. Recent genome-wide mapping approaches have identified DNA methylation (DNAm) signatures in patients clinically diagnosed with syndromes manifesting as developmental disabilities with intellectual impairments. Here, we review recent studies in which these DNA methylation signatures have enabled highly sensitive and specific screening of such individuals and have clarified ambiguous cases where subjects present with genetic sequence variants of unknown clinical significance (VUS). We propose that these episignatures be considered as echoes and/or legacies of the initiating mutational events within proteins of the so-called epigenetic machinery. As well, we discuss approaches to directly confirm the functional consequences and the implications of these episignatures to patient management and treatment.

Background: The objective of this study was to examine individual and community factors that influence high-density lipoprotein cholesterol (HDL-C) dyslipidemia in Newfoundland and Labrador (NL), a genetically isolated population in Canada with a high prevalence of HDL-C dyslipidemia., Methods: First, a group of single nucleotide polymorphisms from 10 metabolic trait candidate genes was tested using a multivariate logistic regression model. The significant SNPs were entered into the second phase, where a mixed logistic model incorporated the community disease risk factors together with the individual factors as the fixed part of the model and the geographic region as a random effect., Results: Analysis of 1489 subjects (26.9% HDL-C dyslipidemia) identified rs3758539, a non-coding variant in the 5'UTR of RBP4, to be associated with HDL-C dyslipidemia (odds ratio = 1.45, 95% confidence interval = 1.08-1.97, p = 0.01). The association remained significant, and the effect size did not change after the incorporation of individual and community risk factors from 17 geographic regions (odds ratio: 1.41, 95% confidence interval = 1.03-1.93, p = 0.03) in NL. Besides this variant, sex, BMI, and smoking also showed significant associations with HDL-C dyslipidemia, whereas no role was identified for the community factors., Conclusions: This study demonstrates the use of community-level data in a genetic association testing. It reports a functional variant in the promoter of RBP4, a gene directly involved in lipoprotein metabolism, to be associated with HDL-C dyslipidemia. These findings indicate that individual factors are the main reason for a higher prevalence of HDL-C dyslipidemia in the NL population.

Coffin-Siris and Nicolaides-Baraitser syndromes (CSS and NCBRS) are Mendelian disorders caused by mutations in subunits of the BAF chromatin remodeling complex. We report overlapping peripheral blood DNA methylation epi-signatures in individuals with various subtypes of CSS (ARID1B, SMARCB1, and SMARCA4) and NCBRS (SMARCA2). We demonstrate that the degree of similarity in the epi-signatures of some CSS subtypes and NCBRS can be greater than that within CSS, indicating a link in the functional basis of the two syndromes. We show that chromosome 6q25 microdeletion syndrome, harboring ARID1B deletions, exhibits a similar CSS/NCBRS methylation profile. Specificity of this epi-signature was confirmed across a wide range of neurodevelopmental conditions including other chromatin remodeling and epigenetic machinery disorders. We demonstrate that a machine-learning model trained on this DNA methylation profile can resolve ambiguous clinical cases, reclassify those with variants of unknown significance, and identify previously undiagnosed subjects through targeted population screening.

Endothelial cells (ECs) are primary targets of glucose-induced tissue damage. As a result of hyperglycemia, endothelin-1 (ET-1) is upregulated in organs affected by chronic diabetic complications. The objective of the present study was to identify novel transcriptional mechanisms that influence ET-1 regulation in diabetes. We carried out the investigation in microvascular ECs using multiple approaches. ECs were incubated with 5 mM glucose (NG) or 25 mM glucose (HG) and analyses for DNA methylation, histone methylation, or long non-coding RNA- mediated regulation of ET-1 mRNA were then performed. DNA methylation array analyses demonstrated the presence of hypomethylation in the proximal promoter and 5' UTR/first exon regions of EDN1 following HG culture. Further, globally blocking DNA methylation or histone methylation significantly increased ET-1 mRNA expressions in both NG and HG-treated HRECs. While, knocking down the pathogenetic lncRNAs ANRIL, MALAT1, and ZFAS1 subsequently prevented the glucose-induced upregulation of ET-1 transcripts. Based on our past and present findings, we present a novel paradigm that reveals a complex web of epigenetic mechanisms regulating glucose-induced transcription of ET-1. Improving our understanding of such processes may lead to better targeted therapies.

Background: Dyslipidemia, an increased level of total cholesterol (TC), triglycerides (TG), low-density-lipoprotein cholesterol (LDL-C) and decreased level of high-density-lipoprotein cholesterol (HDL-C), is one of the most important risk factors for cardiovascular disease. We examined the six-year trend of dyslipidemia in Newfoundland and Labrador (NL), a Canadian province with a historically high prevalence of dyslipidemia., Methods: A serial cross-sectional study on all of the laboratory lipid tests available from 2009 to 2014 was performed. Dyslipidemia for every lipid component was defined using the Canadian Guidelines for the Diagnosis and Treatment of Dyslipidemia. The annual dyslipidemia rates for each component of serum lipid was examined. A fixed and random effect model was applied to adjust for confounding variables (sex and age) and random effects (residual variation in dyslipidemia over the years and redundancies caused by individuals being tested multiple times during the study period)., Results: Between 2009 and 2014, a total of 875,208 records (mean age: 56.9 ± 14.1, 47.6% males) containing a lipid profile were identified. The prevalence of HDL-C and LDL-C dyslipidemia significantly decreased during this period (HDL-C: 35.8% in 2009 [95% CI 35.5-36.1], to 29.0% in 2014 [95% CI: 28.8-29.2], P = 0.03, and LDL-C: 35.2% in 2009 [95% CI: 34.9-35.4] to 32.1% in 2014 [95% CI: 31.9-32.3], P = 0.02). A stratification by sex, revealed no significant trend for any lipid element in females; however, in men, the previously observed trends were intensified and a new decreasing trend in dyslipidemia of TC was appeared (TC: 34.1% [95% CI 33.7-34.5] to 32.3% [95%CI: 32.0-32.6], p < 0.02, HDL-C: 33.8% (95%CI: 33.3-34.2) to 24.0% (95% CI: 23.7-24.3)], P < 0.01, LDL-C: 32.9% (95%CI:32.5-33.3) to 28.6 (95%CI: 28.3-28.9), P < 0.001). Adjustment for confounding factors and removing the residual noise by modeling the random effects did not change the significance., Conclusion: This study demonstrates a significant downward trend in the prevalence of LDL-C, HDL-C, and TC dyslipidemia, exclusively in men. These trends could be the result of males being the primary target for cardiovascular risk management.

Despite possessing limited protein-coding potential, long non-coding RNAs (lncRNAs) have been implicated in a myriad of pathologic conditions. Most well documented in cancer, one prominent intergenic lncRNA known as MALAT1 is notorious for its role in impacting epigenetic mechanisms. In this study, we established a novel epigenetic paradigm for MALAT in diabetic retinopathy (DR) by employing siRNA-mediated MALAT1 knockdown in human retinal endothelial cells (HRECs), a Malat1 knockout animal model, vitreous humor from diabetic patients, pharmacological inhibitors for histone and DNA methylation, RNA immunoprecipitation, western blotting, and a unique DNA methylation array to determine glucose-related alterations in MALAT1. Our findings indicated that MALAT1 is capable of impacting the expressions of inflammatory transcripts through its association with components of the PRC2 complex in diabetes. Furthermore, the vitreous humors from diabetic patients revealed increased expressions of MALAT1, TNF-α, and IL-6. Intriguingly, our DNA methylation array demonstrated that transient high glucose exposure in HRECs does not contribute to significant methylation alterations at CpG sites across the MALAT1 gene. However, global inhibition of DNA methyltransferases induced significant increases in MALAT1 and associated inflammatory transcripts in HRECs. Our findings collectively demonstrate the importance of MALAT1 in inflammation and epigenetic regulation in DR.

Introduction: The current methodology involving diagnosis of prostate cancer (PCa) relies on the pathology examination of prostate needle biopsies, a method with high false negative rates partly due to temporospatial, molecular, and morphological heterogeneity of prostate adenocarcinoma. It is postulated that molecular markers have a potential to assign diagnosis to a considerable portion of undetected prostate tumors. This study examines the genome-wide DNA methylation changes in PCa in search of genomic markers for the development of a diagnostic algorithm for PCa screening., Methods: Archival PCa and normal tissues were assessed using genomic DNA methylation arrays. Differentially methylated sites and regions (DMRs) were used for functional assessment, gene-set enrichment and protein interaction analyses, and examination of transcription factor-binding patterns. Raw signal intensity data were used for identification of recurrent copy number variations (CNVs). Non-redundant fully differentiating cytosine-phosphate-guanine sites (CpGs), which did not overlap CNV segments, were used in an L1 regularized logistic regression model (LASSO) to train a classification algorithm. Validation of this algorithm was performed using a large external cohort of benign and tumor prostate arrays., Results: Approximately 6,000 probes and 600 genomic regions showed significant DNA methylation changes, primarily involving hypermethylation. Gene-set enrichment and protein interaction analyses found an overrepresentation of genes related to cell communications, neurogenesis, and proliferation. Motif enrichment analysis demonstrated enrichment of tumor suppressor-binding sites nearby DMRs. Several of these regions were also found to contain copy number amplifications. Using four non-redundant fully differentiating CpGs, we trained a classification model with 100% accuracy in discriminating tumors from benign samples. Validation of this algorithm using an external cohort of 234 tumors and 92 benign samples yielded 96% sensitivity and 98% specificity. The model was found to be highly sensitive to detect metastatic lesions in bone, lymph node, and soft tissue, while being specific enough to differentiate the benign hyperplasia of prostate from tumor., Conclusion: A considerable component of PCa DNA methylation profile represent driver events potentially established/maintained by disruption of tumor suppressor activity. As few as four CpGs from this profile can be used for screening of PCa.

Background: Claes-Jensen syndrome is an X-linked inherited intellectual disability caused by mutations in the KDM5C gene. Kdm5c is a histone lysine demethylase involved in histone modifications and chromatin remodeling. Males with hemizygous mutations in KDM5C present with intellectual disability and facial dysmorphism, while most heterozygous female carriers are asymptomatic. We hypothesized that loss of Kdm5c function may influence other components of the epigenomic machinery including DNA methylation in affected patients., Results: Genome-wide DNA methylation analysis of 7 male patients affected with Claes-Jensen syndrome and 56 age- and sex-matched controls identified a specific DNA methylation defect (epi-signature) in the peripheral blood of these patients, including 1769 individual CpGs and 9 genomic regions. Six healthy female carriers showed less pronounced but distinctive changes in the same regions enabling their differentiation from both patients and controls. Highly specific computational model using the most significant methylation changes demonstrated 100% accuracy in differentiating patients, carriers, and controls in the training cohort, which was confirmed on a separate cohort of patients and carriers. The 100% specificity of this unique epi-signature was further confirmed on additional 500 unaffected controls and 600 patients with intellectual disability and developmental delay, including other patient cohorts with previously described epi-signatures., Conclusion: Peripheral blood epi-signature in Claes-Jensen syndrome can be used for molecular diagnosis and carrier identification and assist with interpretation of genetic variants of unknown clinical significance in the KDM5C gene., Competing Interests: This study has been approved by the Western University Research Ethics Boards (REB ID 106302) and the Hamilton Integrated Research Ethics Board (REB ID 13-653-T). All of the samples and records were de-identified and anonymized prior to the study. Consent was obtained from all individuals for publication. No individual-level data is reported.The authors declare that they have no competing interests.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Pediatric developmental syndromes present with systemic, complex, and often overlapping clinical features that are not infrequently a consequence of Mendelian inheritance of mutations in genes involved in DNA methylation, establishment of histone modifications, and chromatin remodeling (the "epigenetic machinery"). The mechanistic cross-talk between histone modification and DNA methylation suggests that these syndromes might be expected to display specific DNA methylation signatures that are a reflection of those primary errors associated with chromatin dysregulation. Given the interrelated functions of these chromatin regulatory proteins, we sought to identify DNA methylation epi-signatures that could provide syndrome-specific biomarkers to complement standard clinical diagnostics. In the present study, we examined peripheral blood samples from a large cohort of individuals encompassing 14 Mendelian disorders displaying mutations in the genes encoding proteins of the epigenetic machinery. We demonstrated that specific but partially overlapping DNA methylation signatures are associated with many of these conditions. The degree of overlap among these epi-signatures is minimal, further suggesting that, consistent with the initial event, the downstream changes are unique to every syndrome. In addition, by combining these epi-signatures, we have demonstrated that a machine learning tool can be built to concurrently screen for multiple syndromes with high sensitivity and specificity, and we highlight the utility of this tool in solving ambiguous case subjects presenting with variants of unknown significance, along with its ability to generate accurate predictions for subjects presenting with the overlapping clinical and molecular features associated with the disruption of the epigenetic machinery., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Next-generation sequencing (NGS) technology has rapidly replaced Sanger sequencing in the assessment of sequence variations in clinical genetics laboratories. One major limitation of current NGS approaches is the ability to detect copy number variations (CNVs) approximately >50 bp. Because these represent a major mutational burden in many genetic disorders, parallel CNV assessment using alternate supplemental methods, along with the NGS analysis, is normally required, resulting in increased labor, costs, and turnaround times. The objective of this study was to clinically validate a novel CNV detection algorithm using targeted clinical NGS gene panel data. We have applied this approach in a retrospective cohort of 391 samples and a prospective cohort of 2375 samples and found a 100% sensitivity (95% CI, 89%-100%) for 37 unique events and a high degree of specificity to detect CNVs across nine distinct targeted NGS gene panels. This NGS CNV pipeline enables stand-alone first-tier assessment for CNV and sequence variants in a clinical laboratory setting, dispensing with the need for parallel CNV analysis using classic techniques, such as microarray, long-range PCR, or multiplex ligation-dependent probe amplification. This NGS CNV pipeline can also be applied to the assessment of complex genomic regions, including pseudogenic DNA sequences, such as the PMS2CL gene, and to mitochondrial genome heteroplasmy detection., (Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Genomic imprinting involves a DNA methylation-dependent and parent-of-origin-specific regulation of gene expression. Clinical assays for imprinting disorders are genomic locus, disorder, and molecular defect specific. We aimed to clinically validate a genome-wide approach for simultaneous testing of common imprinting disorders in a single assay. Using genome-wide DNA methylation arrays, epigenetic profiles from peripheral blood of patients with Angelman, Prader-Willi, Beckwith-Wiedemann, or Silver-Russell syndromes were compared to a reference cohort of 361 unaffected individuals. The analysis was of developmental delay and intellectual disabilities. This approach has allowed 100% sensitivity and specificity in detecting imprinting defects in all 28 patients and enabled identification of defects beyond the classically tested imprinted loci. Analysis of the cohort of patients with developmental delay and intellectual disabilities identified two patients with Prader-Willi syndrome, one with Beckwith-Wiedemann syndrome, and several other patients with DNA methylation defects in novel putative imprinting loci. These findings demonstrate clinical validation of a sensitive and specific genome-wide DNA methylation array-based approach for molecular testing of imprinting disorders to allow simultaneous assessment of genome-wide epigenetic defects in a single analytical procedure, enabling replacement of multiple locus-specific molecular tests while allowing discovery of novel clinical epigenomic associations and differential diagnosis of other epigenomic disorders., (Copyright © 2017 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Genetic service for couples plays an increasingly important role in diagnosis and risk management. This study investigated the status of consanguinity and the medical genetic history (effectiveness and coverage of medical genetic services) in couples residing in a city in southern Iran. We questioned couples who were referred to Behbahan Marital Counseling Center, Behbahan, Iran, during the period from January to November 2014, to obtain information on consanguinity, disease history, and previous referral to a medical genetics center. For the collected data was obtained descriptive statistics with STATA 11.0 software. A total of 500 couples were questioned. Mean age was 24.8 ± 5.2 years. Almost one quarter (23.4%) of the couples were consanguineous. Consanguinity was almost twice as common in rural areas as in urban areas (33.9 vs . 19.2%, p = 0.001). Only a few couples (~3.0%) had ever been referred for genetic counseling. The main reason for previous genetic counseling was consanguinity (85.7%). The majority of the participants (96.3%) had never been tested for any genetic conditions. Our findings suggest that only a small proportion of couples in Khuzestan Province, Iran (Behbahan City) were receiving adequate genetics care. This may reflect the limited accessibility of such services, and inadequate awareness and education among the care providers.

The objective of this study was to define the optimal algorithm to identify patients with dyslipidemia using electronic medical records (EMRs). EMRs of patients attending primary care clinics in St. John's, Newfoundland and Labrador (NL), Canada during 2009-2010, were studied to determine the best algorithm for identification of dyslipidemia. Six algorithms containing three components, dyslipidemia ICD coding, lipid lowering medication use, and abnormal laboratory lipid levels, were tested against a gold standard, defined as the existence of any of the three criteria. Linear discriminate analysis, and bootstrapping were performed following sensitivity/specificity testing and receiver's operating curve analysis. Two validating datasets, NL records of 2011-2014, and Canada-wide records of 2010-2012, were used to replicate the results. Relative to the gold standard, combining laboratory data together with lipid lowering medication consumption yielded the highest sensitivity (99.6%), NPV (98.1%), Kappa agreement (0.98), and area under the curve (AUC, 0.998). The linear discriminant analysis for this combination resulted in an error rate of 0.15 and an Eigenvalue of 1.99, and the bootstrapping led to AUC: 0.998, 95% confidence interval: 0.997-0.999, Kappa: 0.99. This algorithm in the first validating dataset yielded a sensitivity of 97%, Negative Predictive Value (NPV) = 83%, Kappa = 0.88, and AUC = 0.98. These figures for the second validating data set were 98%, 93%, 0.95, and 0.99, respectively. Combining laboratory data with lipid lowering medication consumption within the EMR is the best algorithm for detecting dyslipidemia. These results can generate standardized information systems for dyslipidemia and other chronic disease investigations using EMRs.

Objective: To assess the validity of the International Classification of Disease (ICD) codes for identifying patients with dyslipidemia in electronic medical record (EMR) data., Methods: The EMRs of patients receiving primary care in St. John's, Newfoundland and Labrador (NL), Canada, were retrieved from the Canadian Primary Care Sentinel Surveillance Network database. International Classification of Disease codes were first compared with laboratory lipid data as an independent criterion standard, and next with a "comprehensive criterion standard," defined as any existence of abnormal lipid test, lipid-lowering medication record, or dyslipidemia ICD codes. The ability of ICD coding alone or combined with other components was evaluated against the two criterion standards using receiver operating characteristic (ROC) analysis, sensitivity, specificity, negative predictive value (NPV) and Kappa agreement. (No specificity was reported for the comparison of ICD codes against the comprehensive criterion standard as this naturally leads to 100% specificity.)., Results: The ICD codes led to a poor outcome when compared with the serum lipid levels (sensitivity, 27%; specificity, 76%; PPV, 71%; NPV, 33%; Kappa, 0.02; area under the receiver operating characteristic curve (AUC), 0.51) or with the comprehensive criterion standard (sensitivity, 32%; NPV, 25%; Kappa, 0.15; AUC, 66%). International Classification of Disease codes combined with lipid-lowering medication data also resulted in low sensitivity (51.2%), NPV (32%), Kappa (0.28), and AUC (75%). The addition of laboratory lipid levels to ICD coding marginally improved the algorithm (sensitivity, 94%; NPV, 79%; Kappa, 0.85; AUC, 97%)., Conclusions: The use of ICD coding, either alone or in combination with laboratory data or lipid-lowering medication records, was not an accurate indicator in identifying dyslipidemia., Competing Interests: DECLARATION OF CONFLICTING INTERESTS: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Kabuki syndrome (KS) is caused by mutations in KMT2D, which is a histone methyltransferase involved in methylation of H3K4, a histone marker associated with DNA methylation. Analysis of >450,000 CpGs in 24 KS patients with pathogenic mutations in KMT2D and 216 controls, identified 24 genomic regions, along with 1,504 CpG sites with significant DNA methylation changes including a number of Hox genes and the MYO1F gene. Using the most differentiating and significant probes and regions we developed a "methylation variant pathogenicity (MVP) score," which enables 100% sensitive and specific identification of individuals with KS, which was confirmed using multiple public and internal patient DNA methylation databases. We also demonstrated the ability of the MVP score to accurately reclassify variants of unknown significance in subjects with apparent clinical features of KS, enabling its potential use in molecular diagnostics. These findings provide novel insights into the molecular etiology of KS and illustrate that DNA methylation patterns can be interpreted as 'epigenetic echoes' in certain clinical disorders.

Objective: To identify novel biomarker(s) for knee osteoarthritis (OA) using a metabolomics approach., Method: We utilized a two-stage case-control study design. Plasma samples were collected from knee OA patients and healthy controls after 8-h fasting and metabolically profiled using a targeted metabolomics assay kit. Linear regression was used to identify novel metabolic markers for OA. Receiver operating characteristic (ROC) analysis was used to examine diagnostic values. Gene expression analysis was performed on human cartilage to explore the potential mechanism for the novel OA marker(s)., Results: Sixty-four knee OA patients and 45 controls were included in the discovery stage and 72 knee OA patients and 76 age and sex matched controls were included in the validation stage. We identified and confirmed six metabolites that were significantly associated with knee OA, of which arginine was the most significant metabolite (P < 3.5 × 10(-13)) with knee OA patients having on average 69 μM lower than that in controls. ROC analysis showed that arginine had the greatest diagnostic value with area under the curve (AUC) of 0.984. The optimal cutoff of arginine concentration was 57 μM with 98.3% sensitivity and 89% specificity. The depletion of arginine in OA patients was most likely due to the over activity of arginine to ornithine pathway, leading to imbalance between cartilage repair and degradation., Conclusion: Arginine is significantly depleted in refractory knee OA patients. Further studies within a longitudinal setting are required to examine whether arginine can predict early OA changes., (Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: To compare SMAD3 gene expression between human osteoarthritic and healthy cartilage and to examine whether expression is regulated by the promoter DNA methylation of the gene., Methods: Human cartilage samples were collected from patients undergoing total hip/knee joint replacement surgery due to primary osteoarthritis (OA), and from patients with hip fractures as controls. DNA/RNA was extracted from the cartilage tissues. Real-time quantitative PCR was performed to measure gene expression, and Sequenom EpiTyper was used to assay DNA methylation. Mann-Whitney test was used to compare the methylation and expression levels between OA cases and controls. Spearman rank correlation coefficient was calculated to examine the association between the methylation and gene expression., Results: A total of 58 patients with OA (36 women, 22 men; mean age 64 ± 9 yrs) and 55 controls (43 women, 12 men; mean age 79 ± 10 yrs) were studied. SMAD3 expression was on average 83% higher in OA cartilage than in controls (p = 0.0005). No difference was observed for DNA methylation levels in the SMAD3 promoter region between OA cases and controls. No correlation was found between SMAD3 expression and promoter DNA methylation., Conclusion: Our study demonstrates that SMAD3 is significantly overexpressed in OA. This overexpression cannot be explained by DNA methylation in the promoter region. The results suggest that the transforming growth factor-β/SMAD3 pathway may be overactivated in OA cartilage and has potential in developing targeted therapies for OA.