Genomic technologies to improve variation identification in undiagnosed diseases.

Human genome variation has increasingly posed challenges and opportunities for patients, medical providers, and an increasing group of stakeholders including advocacy groups, disadvantaged communities, public health experts, and scientists. Here, advances in genomic sequencing and mapping technologies are discussed with particular attention to the increasing ability to detect personal and population genome variation and the potential for accurate integration of variation into health and disease-related care. Genome mapping, one technique used to create genome map scaffolds, has now been combined with long read sequencing. New technologies have led to improved variation detection, including cryptic structural variation and diverse variants with different degrees of disease association. Combined with advances in automated and medical interpretations, variation detection is increasingly being applied in healthcare. These advances promise to make disease diagnostics more rapid, and potentially more accessible, to those with medical needs. Consequentially, the need for medical genetics and genomics experts is increasing. Here, the opportunities and potential challenges for application of genome-scale variation detection in disease are examined. (<300 words).

CXCL14 Promotes a Robust Brain Tumor-Associated Immune Response in Glioma.

PURPOSE: The immunosuppressive tumor microenvironment present in the majority of diffuse glioma limits therapeutic response to immunotherapy. As the determinants of the glioma-associated immune response are relatively poorly understood, the study of glioma with more robust tumor-associated immune responses may be particularly useful to identify novel immunomodulatory factors that can promote T-cell effector function in glioma. EXPERIMENTAL DESIGN: We used multiplex immune-profiling, proteomic profiling, and gene expression analysis to define the tumor-associated immune response in two molecular subtypes of glioma and identify factors that may modulate this response. We then used patient-derived glioma cultures and an immunocompetent murine model for malignant glioma to analyze the ability of tumor-intrinsic factors to promote a CD8+ T-cell response. RESULTS: As compared with isocitrate dehydrogenase (IDH)-mutant astrocytoma, MAPK-activated pleomorphic xanthoastrocytoma (PXA) harbored increased numbers of activated cytotoxic CD8+ T cells and Iba1+ microglia/macrophages, increased MHC class I expression, enrichment of genes associated with antigen presentation and processing, and increased tumor cell secretion of the chemokine CXCL14. CXCL14 promoted activated CD8+ T-cell chemotaxis in vitro, recruited tumor-infiltrating CD8+ T cells in vivo, and prolonged overall survival in a cytotoxic T-cell-dependent manner. The immunomodulatory molecule B7-H3 was also highly expressed in PXA. CONCLUSIONS: We identify the MAPK-activated lower grade astrocytoma PXA as having an immune-rich tumor microenvironment and suggest this tumor may be particularly vulnerable to immunotherapeutic modulation. We also identify CXCL14 as an important determinant of the glioma-associated immune microenvironment, sufficient to promote an antitumor CD8+ T-cell response.

De novo variants in SNAP25 cause an early-onset developmental and epileptic encephalopathy.

PURPOSE: This study aims to provide a comprehensive description of the phenotypic and genotypic spectrum of SNAP25 developmental and epileptic encephalopathy (SNAP25-DEE) by reviewing newly identified and previously reported individuals. METHODS: Individuals harboring heterozygous missense or loss-of-function variants in SNAP25 were assembled through collaboration with international colleagues, matchmaking platforms, and literature review. For each individual, detailed phenotyping, classification, and structural modeling of the identified variant were performed. RESULTS: The cohort comprises 23 individuals with pathogenic or likely pathogenic de novo variants in SNAP25. Intellectual disability and early-onset epilepsy were identified as the core symptoms of SNAP25-DEE, with recurrent findings of movement disorders, cerebral visual impairment, and brain atrophy. Structural modeling for all variants predicted possible functional defects concerning SNAP25 or impaired interaction with other components of the SNARE complex. CONCLUSION: We provide a comprehensive description of SNAP25-DEE with intellectual disability and early-onset epilepsy mostly occurring before the age of two years. These core symptoms and additional recurrent phenotypes show an overlap to genes encoding other components or associated proteins of the SNARE complex such as STX1B, STXBP1, or VAMP2. Thus, these findings advance the concept of a group of neurodevelopmental disorders that may be termed "SNAREopathies."

Automated syndrome diagnosis by three-dimensional facial imaging.

PURPOSE: Deep phenotyping is an emerging trend in precision medicine for genetic disease. The shape of the face is affected in 30-40% of known genetic syndromes. Here, we determine whether syndromes can be diagnosed from 3D images of human faces. METHODS: We analyzed variation in three-dimensional (3D) facial images of 7057 subjects: 3327 with 396 different syndromes, 727 of their relatives, and 3003 unrelated, unaffected subjects. We developed and tested machine learning and parametric approaches to automated syndrome diagnosis using 3D facial images. RESULTS: Unrelated, unaffected subjects were correctly classified with 96% accuracy. Considering both syndromic and unrelated, unaffected subjects together, balanced accuracy was 73% and mean sensitivity 49%. Excluding unrelated, unaffected subjects substantially improved both balanced accuracy (78.1%) and sensitivity (56.9%) of syndrome diagnosis. The best predictors of classification accuracy were phenotypic severity and facial distinctiveness of syndromes. Surprisingly, unaffected relatives of syndromic subjects were frequently classified as syndromic, often to the syndrome of their affected relative. CONCLUSION: Deep phenotyping by quantitative 3D facial imaging has considerable potential to facilitate syndrome diagnosis. Furthermore, 3D facial imaging of "unaffected" relatives may identify unrecognized cases or may reveal novel examples of semidominant inheritance.

Mechanisms of Resistance to EGFR Inhibition Reveal Metabolic Vulnerabilities in Human GBM.

Amplification of the epidermal growth factor receptor gene (EGFR) represents one of the most commonly observed genetic lesions in glioblastoma (GBM); however, therapies targeting this signaling pathway have failed clinically. Here, using human tumors, primary patient-derived xenografts (PDX), and a murine model for GBM, we demonstrate that EGFR inhibition leads to increased invasion of tumor cells. Further, EGFR inhibitor-treated GBM demonstrates altered oxidative stress, with increased lipid peroxidation, and generation of toxic lipid peroxidation products. A tumor cell subpopulation with elevated aldehyde dehydrogenase (ALDH) levels was determined to comprise a significant proportion of the invasive cells observed in EGFR inhibitor-treated GBM. Our analysis of the ALDH1A1 protein in newly diagnosed GBM revealed detectable ALDH1A1 expression in 69% (35/51) of the cases, but in relatively low percentages of tumor cells. Analysis of paired human GBM before and after EGFR inhibitor therapy showed an increase in ALDH1A1 expression in EGFR-amplified tumors (P < 0.05, n = 13 tumor pairs), and in murine GBM ALDH1A1-high clones were more resistant to EGFR inhibition than ALDH1A1-low clones. Our data identify ALDH levels as a biomarker of GBM cells with high invasive potential, altered oxidative stress, and resistance to EGFR inhibition, and reveal a therapeutic target whose inhibition should limit GBM invasion.

Genomic Sequencing Expansion and Incomplete Penetrance.

BACKGROUND: Genetic data have the potential to impact patient care significantly. In primary care and in the ICU, patients are undergoing genetic testing. Genetics is also transforming cancer care and undiagnosed diseases. Optimal personalized medicine relies on the understanding of disease penetrance. In this article, I examine the complexity of penetrance. METHODS: In this article, I assess how variable penetrance can be seen with many diseases, including those of different modes of inheritance, and how genomic testing is being applied effectively for many diseases. In this article, I also identify challenges in the field, including the interpretation of gene variants. RESULTS: Using advancing bioinformatics and detailed phenotypic assessment, we can increase the yield of genomic testing, particularly for highly penetrant conditions. The technologies are useful and applicable to different medical situations. CONCLUSIONS: There are now effective genome diagnostics for many diseases. However, the best personalized application of these data still requires skilled interpretation.

The genetic landscape of anaplastic pleomorphic xanthoastrocytoma.

Pleomorphic xanthoastrocytoma (PXA) is an astrocytic neoplasm that is typically well circumscribed and can have a relatively favorable prognosis. Tumor progression to anaplastic PXA (WHO grade III), however, is associated with a more aggressive biologic behavior and worse prognosis. The factors that drive anaplastic progression are largely unknown. We performed comprehensive genomic profiling on a set of 23 PXAs from 19 patients, including 15 with anaplastic PXA. Four patients had tumor tissue from multiple recurrences, including two with anaplastic progression. We find that PXAs are genetically defined by the combination of CDKN2A biallelic inactivation and RAF alterations that were present in all 19 cases, most commonly as CDKN2A homozygous deletion and BRAF p.V600E mutation but also occasionally BRAF or RAF1 fusions or other rearrangements. The third most commonly altered gene in anaplastic PXA was TERT, with 47% (7/15) harboring TERT alterations, either gene amplification (n = 2) or promoter hotspot mutation (n = 5). In tumor pairs analyzed before and after anaplastic progression, two had increased copy number alterations and one had TERT promoter mutation at recurrence. Less commonly altered genes included TP53, BCOR, BCORL1, ARID1A, ATRX, PTEN, and BCL6. All PXA in this cohort were IDH and histone H3 wildtype, and did not contain alterations in EGFR. Genetic profiling performed on six regions from the same tumor identified intratumoral genomic heterogeneity, likely reflecting clonal evolution during tumor progression. Overall, anaplastic PXA is characterized by the combination of CDKN2A biallelic inactivation and oncogenic RAF kinase signaling as well as a relatively small number of additional genetic alterations, with the most common being TERT amplification or promoter mutation. These data define a distinct molecular profile for PXA and suggest additional genetic alterations, including TERT, may be associated with anaplastic progression.

Uveal Ganglioneuroma due to Germline PTEN Mutation (Cowden Syndrome) Presenting as Unilateral Infantile Glaucoma.

PURPOSE: Uveal ganglioneuroma is a rare tumor that usually occurs in association with neurofibromatosis type 1. Here, we present a rare case of a uveal ganglioneuroma leading to a diagnosis of the tumor predisposition condition Cowden syndrome. PROCEDURES: A 5-year-old girl with unilateral refractory glaucoma secondary to diffuse iris and choroidal thickening developed a blind, painful eye. Enucleation was performed, and histopathology revealed infiltration of the entire uveal tract by neoplastic spindle cells containing admixed ganglion cells diagnostic of uveal ganglioneuroma. Targeted next-generation sequencing of 510 cancer-associated genes was performed on tumor tissue and peripheral blood. RESULTS: A germline nonsense mutation in the PTEN gene was found, accompanied by loss of heterozygosity in the tumor. A diagnosis of Cowden syndrome was made, for which the family sought genetic counseling and initiated the recommended cancer screening. CONCLUSIONS: A novel association is found between uveal ganglioneuroma and Cowden syndrome, emphasizing the value of genetic tissue testing in managing patients with rare ocular tumors.

Potential Role of Genomic Sequencing in the Early Diagnosis of Treatable Genetic Conditions.

We present cases of 3 children diagnosed with the same genetic condition, Gitelman syndrome, at different stages using various genetic methods: panel testing, targeted single gene sequencing, and exome sequencing. We discuss the advantages and disadvantages of each method and review the potential of genomic sequencing for early disease detection.

Prioritizing genes for X-linked diseases using population exome data.

Many new disease genes can be identified through high-throughput sequencing. Yet, variant interpretation for the large amounts of genomic data remains a challenge given variation of uncertain significance and genes that lack disease annotation. As clinically significant disease genes may be subject to negative selection, we developed a prediction method that measures paucity of non-synonymous variation in the human population to infer gene-based pathogenicity. Integrating human exome data of over 6000 individuals from the NHLBI Exome Sequencing Project, we tested the utility of the prediction method based on the ratio of non-synonymous to synonymous substitution rates (dN/dS) on X-chromosome genes. A low dN/dS ratio characterized genes associated with childhood disease and outcome. Furthermore, we identify new candidates for diseases with early mortality and demonstrate intragenic localized patterns of variants that suggest pathogenic hotspots. Our results suggest that intrahuman substitution analysis is a valuable tool to help prioritize novel disease genes in sequence interpretation.

Twin Mitochondrial Sequence Analysis.

When applying genome-wide sequencing technologies to disease investigation, it is increasingly important to resolve sequence variation in regions of the genome that may have homologous sequences. The human mitochondrial genome challenges interpretation given the potential for heteroplasmy, somatic variation, and homologous nuclear mitochondrial sequences (numts). Identical twins share the same mitochondrial DNA (mtDNA) from early life, but whether the mitochondrial sequence remains similar is unclear. We compared an adult monozygotic twin pair using high throughput-sequencing and evaluated variants with primer extension and mitochondrial pre-enrichment. Thirty-seven variants were shared between the twin individuals, and the variants were verified on the original genomic DNA. These studies support highly identical genetic sequence in this case. Certain low-level variant calls were of high quality and homology to the mitochondrial DNA, and they were further evaluated. When we assessed calls in pre-enriched mitochondrial DNA templates, we found that these may represent numts, which can be differentiated from mtDNA variation. We conclude that twin identity extends to mitochondrial DNA, and it is critical to differentiate between numts and mtDNA in genome sequencing, particularly since significant heteroplasmy could influence genome interpretation. Further studies on mtDNA and numts will aid in understanding how variation occurs and persists.

Disorders of left ventricular trabeculation/compaction or right ventricular wall formation.

Cardiomyopathies are remarkably variable in form. Although hearts may be dilated or hypertrophic, the spectrum of cardiomyopathies includes left ventricular noncompaction/hypertrabeculation and right ventricular wall disorders. These conditions have been increasingly recognized in patients given advances in clinical diagnostics. Here we present information on cardiac pathophysiology, from ventricular wall formation and trabeculae in model organisms to pediatric and adult disease. Many genes to affect the ventricular phenotype, and this has implications for deciphering developmental and disease pathways and for applying testing for clinical care.

Implications of genetic testing in noncompaction/hypertrabeculation.

Noncompaction/hypertrabeculation is increasingly being recognized in children and adults, yet we understand little about the causes of disease. Genes associated with noncompaction/hypertrabeculation have been identified, but how can these assist in clinical management? Genomic technologies have also expanded tremendously, making testing more comprehensive, but they also present new questions given the tremendous diversity of phenotypes and variability of genomes. Here we present genetic evaluation strategies and assess clinical testing options for noncompaction/hypertrabeculation. We assess genes/gene panels offered by clinical laboratories and the potential for high-throughput sequencing to fuel further discovery. We discuss challenges in cardiovascular genetics, such as interpretation of genomic variants, prediction and disease penetrance.

Monoamniotic monochorionic twins discordant for noncompaction cardiomyopathy.

Occasionally "identical twins" are phenotypically different, raising the question of zygosity and the issue of genetic versus environmental influences during development. We recently noted monochorionic-monoamniotic twins, one of which had an isolated cardiac abnormality, noncompaction cardiomyopathy, a condition characterized by cardiac ventricular hypertrabeculation. We examined the prenatal course and subsequent pathologic correlation since ventricular morphogenesis may depend on early muscular contraction and blood flow. The monochorionic-monoamniotic female twin pair was initially identified since one fetus presented with increased nuchal translucency. Complete heart block was later identified in the fetus with nuchal translucency who did not survive after delivery. In contrast, the unaffected twin had normal cardiac studies both prenatally and postnatally. Pathologic analysis of the affected twin demonstrated noncompaction of the left ventricle with dysplasia of the aortic and pulmonary valves. Dissection of the cardiac conduction system disclosed atrioventricular bundle fibrosis. Maternal lupus studies, amniocentesis with karyotype, and studies for 22q11.2 were normal. To test for zygosity, we performed multiple STR marker analysis and found that all markers were shared even using nonblood tissues from the affected twin. These studies demonstrate that monozygotic twins that are monochorionic monoamniotic can be discordant for cardiac noncompaction. The results suggest further investigation into the potential roles of pathologic fibrosis, contractility, and blood flow in cardiac ventricle development.

Copy number variation analysis in 98 individuals with PHACE syndrome.

PHACE syndrome is the association of large segmental facial hemangiomas and congenital anomalies, such as posterior fossa malformations, cerebral arterial anomalies, coarctation of the aorta, eye anomalies, and sternal defects. To date, the reported cases of PHACE syndrome have been sporadic, suggesting that PHACE may have a complex pathogenesis. We report here genomic copy number variation (CNV) analysis of 98 individuals with PHACE syndrome as a first step in deciphering a potential genetic basis of PHACE syndrome. A total of 3,772 CNVs (2,507 duplications and 1,265 deletions) were detected in 98 individuals with PHACE syndrome. CNVs were then eliminated if they failed to meet established criteria for quality, spanned centromeres, or did not contain genes. CNVs were defined as "rare" if not documented in the database of genomic variants. Ten rare CNVs were discovered (size range: 134-406 kb), located at 1q32.1, 1q43, 3q26.32-3q26.33, 3p11.1, 7q33, 10q24.32, 12q24.13, 17q11.2, 18p11.31, and Xq28. There were no rare CNV events that occurred in more than one subject. Therefore, further study is needed to determine the significance of these CNVs in the pathogenesis of PHACE syndrome.

Candidate locus analysis for PHACE syndrome.

PHACE syndrome (OMIM #606519) is a neurocutaneous syndrome of unknown etiology and pathogenesis. We report on an individual with PHACE syndrome with a complete deletion of SLC35B4 on 7q33. In order to further analyze this region, SLC35B4 was sequenced for 33 individuals with PHACE syndrome and one parental set. Common polymorphisms with a possible haplotype but no disease causing mutation were identified. Sixteen of 33 samples of the PHACE syndrome patients were also analyzed for copy number variations using high-resolution oligo-comparative genomic hybridization (CGH) microarray. A second individual in this cohort had a 26.5 kb deletion approximately 80 kb upstream of SLC35B4 with partial deletion of the AKR1B1 on 7q33. The deletions observed on 7q33 are not likely the singular cause of PHACE syndrome; however, it is possible that this region provides a genetic susceptibility to phenotypic expression with other confounding genetic or environmental factors.

Consanguinity and the risk of congenital heart disease.

Consanguineous unions have been associated with an increased susceptibility to various forms of inherited disease. Although consanguinity is known to contribute to recessive diseases, the potential role of consanguinity in certain common birth defects is less clear, particularly since the disease pathophysiology may involve genetic and environmental/epigenetic factors. In this study, we ask whether consanguinity affects one of the most common birth defects, congenital heart disease, and identify areas for further research into these birth defects, since consanguinity may now impact health on a near-global basis. A systematic review of consanguinity in congenital heart disease was performed, focusing on non-syndromic disease, with the methodologies and results from studies of different ethnic populations compared. The risks for congenital heart disease have been assessed and summarized collectively and by individual lesion. The majority of studies support the view that consanguinity increases the prevalence of congenital heart disease, however, the study designs differed dramatically. Only a few (n = 3) population-based studies that controlled for potential sociodemographic confounding were identified, and data on individual cardiac lesions were limited by case numbers. Overall the results suggest that the risk for congenital heart disease is increased in consanguineous unions in the studied populations, principally at first-cousin level and closer, a factor that should be considered in empiric risk estimates in genetic counseling. However, for more precise risk estimates a better understanding of the underlying disease factors is needed.

Elevated miR-499 levels blunt the cardiac stress response.

BACKGROUND: The heart responds to myriad stresses by well-described transcriptional responses that involve long-term changes in gene expression as well as more immediate, transient adaptations. MicroRNAs quantitatively regulate mRNAs and thus may affect the cardiac transcriptional output and cardiac function. Here we investigate miR-499, a microRNA embedded within a ventricular-specific myosin heavy chain gene, which is expressed in heart and skeletal muscle. METHODOLOGY/PRINCIPAL FINDINGS: We assessed miR-499 expression in human tissue to confirm its potential relevance to human cardiac gene regulation. Using a transgenic mouse model, we found that elevated miR-499 levels caused cellular hypertrophy and cardiac dysfunction in a dose-dependent manner. Global gene expression profiling revealed altered levels of the immediate early stress response genes (Egr1, Egr2 and Fos), ß-myosin heavy chain (Myh7), and skeletal muscle actin (Acta1). We verified the effect of miR-499 on the immediate early response genes by miR-499 gain- and loss-of-function in vitro. Consistent with a role for miR-499 in blunting the response to cardiac stress, asymptomatic miR-499-expressing mice had an impaired response to pressure overload and accentuated cardiac dysfunction. CONCLUSIONS: Elevated miR-499 levels affect cardiac gene expression and predispose to cardiac stress-induced dysfunction. miR-499 may titrate the cardiac response to stress in part by regulating the immediate early gene response.