The MAP3K7 gene: Further delineation of clinical characteristics and genotype/phenotype correlations.

Mitogen-activated protein 3 kinase 7 (MAP3K7) encodes the ubiquitously expressed transforming growth factor ß-activated kinase 1, which plays a crucial role in many cellular processes. Mutationsin the MAP3K7 gene have been linked to two distinct disorders: frontometaphyseal dysplasia type 2 (FMD2) and cardiospondylocarpofacial syndrome (CSCF). The fact that different mutations can induce two distinct phenotypes suggests a phenotype/genotype correlation, but no side-by-side comparison has been done thus far to confirm this. Here, we significantly expand the cohort and the description of clinical phenotypes for patients with CSCF and FMD2 who carry mutations in MAP3K7. Our findings support that in contrast to FMD2-causing mutations, CSCF-causing mutations in MAP3K7 have a loss-of-function effect. Additionally, patients with pathogenic mutations in MAP3K7 are at risk for (severe) cardiac disease, have symptoms associated with connective tissue disease, and we show overlap in clinical phenotypes of CSCF with Noonan syndrome (NS). Together, we confirm a molecular fingerprint of FMD2- versus CSCF-causing MAP3K7 mutations and conclude that mutations in MAP3K7 should be considered in the differential diagnosis of patients with syndromic congenital cardiac defects and/or cardiomyopathy, syndromic connective tissue disorders, and in the differential diagnosis of NS.

Characterization of a rare mosaic unbalanced translocation of t(3;12) in a patient with neurodevelopmental disorders.

BACKGROUND: Unbalanced translocations may be de novo or inherited from one parent carrying the balanced form and are usually present in all cells. Mosaic unbalanced translocations are extremely rare with a highly variable phenotype depending on the tissue distribution and level of mosaicism. Mosaicism for structural chromosomal abnormalities is clinically challenging for diagnosis and counseling due to the limitation of technical platforms and complex mechanisms, respectively. Here we report a case with a tremendously rare maternally-derived mosaic unbalanced translocation of t(3;12), and we illustrate the unreported complicated mechanism using single nucleotide polymorphism (SNP) array, fluorescence in situ hybridization (FISH), and chromosome analyses. CASE PRESENTATION: An 18-year-old female with a history of microcephaly, pervasive developmental disorder, intellectual disability, sensory integration disorder, gastroparesis, and hypotonia presented to our genetics clinic. She had negative karyotype by parental report but no other genetic testing performed previously. SNP microarray analysis revealed a complex genotype including 8.4 Mb terminal mosaic duplication on chromosome 3 (3p26.3->3p26.1) with the distal 5.7 Mb involving two parental haplotypes and the proximal 2.7 Mb involving three parental haplotypes, and a 6.1 Mb terminal mosaic deletion on chromosome 12 (12p13.33->12p13.31) with no evidence for a second haplotype. Adjacent to the mosaic deletion is an interstitial mosaic copy-neutral region of homozygosity (1.9 Mb, 12p13.31). The mother of this individual was confirmed by chromosome analysis and FISH that she carries a balanced translocation, t(3;12)(p26.1;p13.31). CONCLUSION: Taken together, the proband, when at the stage of a zygote, likely carried the derivative chromosome 12 from this translocation, and a postzygotic mitotic recombination event occurred between the normal paternal chromosome 12 and maternal derivative chromosome 12 to "correct" the partial 3p trisomy and partial deletion of 12p. To the best of our knowledge, it is the first time to report the mechanism utilizing a combined cytogenetic and cytogenomic approach, and we believe it expands our knowledge of mosaic structural chromosomal disorders and provides new insight into clinical management and genetic counseling.

Molecular Diagnostic Outcomes from 700 Cases: What Can We Learn from a Retrospective Analysis of Clinical Exome Sequencing?

Clinical exome sequencing (CES) aids in the diagnosis of rare genetic disorders. Herein, we report the molecular diagnostic yield and spectrum of genetic alterations contributing to disease in 700 pediatric cases analyzed at the Children's Hospital of Philadelphia. The overall diagnostic yield was 23%, with three cases having more than one molecular diagnosis and 2.6% having secondary/additional findings. A candidate gene finding was reported in another 8.4% of cases. The clinical indications with the highest diagnostic yield were neurodevelopmental disorders (including seizures), whereas immune- and oncology-related indications were negatively associated with molecular diagnosis. The rapid expansion of knowledge regarding the genome's role in human disease necessitates reanalysis of CES samples. To capture these new discoveries, a subset of cases (n = 240) underwent reanalysis, with an increase in diagnostic yield. We describe our experience reporting CES results in a pediatric setting, including reporting of secondary findings, reporting newly discovered genetic conditions, and revisiting negative test results. Finally, we highlight the challenges associated with implementing critical updates to the CES workflow. Although these updates are necessary, they demand an investment of time and resources from the laboratory. In summary, these data demonstrate the clinical utility of exome sequencing and reanalysis, while highlighting the critical considerations for continuous improvement of a CES test in a clinical laboratory.

Genetic Testing in Patients with Neurodevelopmental Disorders: Experience of 511 Patients at Cincinnati Children's Hospital Medical Center.

Our institution developed and continuously improved a Neurodevelopmental Reflex (NDR) algorithm to help physicians with genetic test ordering for neurodevelopmental disorders (NDDs). To assess its performance, we performed a retrospective study of 511 patients tested through NDR from 2018 to 2019. SNP Microarray identified pathogenic/likely pathogenic copy number variations in 27/511 cases (5.28%). Among the 484 patients tested for Fragile X FMR1 CGG repeats, a diagnosis (0.20%) was established for one male mosaic for a full mutation, a premutation, and a one-CGG allele. Within the 101 normocephalic female patients tested for MECP2, two patients were found to carry pathogenic variants (1.98%). This retrospective study suggested the NDR algorithm effectively established diagnoses for patients with NDDs with a yield of 5.87%.

A rare case of postnatal mosaic trisomy 12 with severe congenital heart disease and literature review.

Trisomy 12 is a rare autosomal aneuploidy. All postnatally diagnosed individuals with trisomy 12 have been mosaic for this chromosome abnormality. We herein report an infant girl presented at 2 weeks of age with severe congenital heart defect, tracheobronchomalacia, and dysmorphic features. All of the dysmorphic features of this patient fit into the known phenotype spectrum of mosaic trisomy 12, although this patient uniquely presented with macrocephaly. Tracheo-bronchomalacia has been described once previously but had a significant impact on this patient's clinical course. The patient passed away at 2-month-old due to cardiac and respiratory complications. Chromosomal single nucleotide polymorphism (SNP) microarray analysis on a peripheral blood sample from the patient revealed trisomy 12 in approximately 50% of cells. Concurrent fluorescence in situ hybridization analysis of uncultured blood cells detected a comparable level of trisomy 12 mosaicism. Compared to conventional cytogenetics, SNP microarray examines all nucleated cells without sampling bias, has an increased power to estimate mosaicism level, and can provide a quick assessment of the underlying mechanism. Here we demonstrate the utilization of SNP microarray in the clinical diagnosis of those once considered rare disorders but might have been missed by conventional cytogenetic techniques.

Prenatal diagnosis of Proteus syndrome: Diagnosis of an AKT1 mutation from amniocytes.

Proteus syndrome is a mosaic genetic overgrowth disorder caused by a postzygotic, mosaic activating mutation in AKT1. Rare prenatal presentations include segmental tissue overgrowth, and skeletal and CNS anomalies. We present the first report of prenatally diagnosed and molecularly confirmed Proteus syndrome. Prenatal imaging identified megalencephaly, brain and eye malformations, focal soft tissue enlargement, and ambiguous genitalia. Exome sequencing performed on cultured amniocytes demonstrated an AKT1 pathogenic variant consistent with Proteus syndrome, and postnatal examination confirmed the diagnosis. Postnatal Sanger sequencing could not identify the AKT1 pathogenic variant. This case underscores the importance of prenatal exome sequencing on cultured amniocytes for mosaic overgrowth disorders, as well as provides additional information on the prenatal phenotype of Proteus syndrome, and highlights the impact of prenatal diagnosis on postnatal management.

Automated Clinical Exome Reanalysis Reveals Novel Diagnoses.

Clinical exome sequencing (CES) has a reported diagnostic yield of 20% to 30% for most clinical indications. The ongoing discovery of novel gene-disease and variant-disease associations are expected to increase the diagnostic yield of CES. Performing systematic reanalysis of previously nondiagnostic CES samples represents a significant challenge for clinical laboratories. Here, we present the results of a novel automated reanalysis methodology applied to 300 CES samples initially analyzed between June 2014 and September 2016. Application of our reanalysis methodology reduced reanalysis variant analysis burden by >93% and correctly captured 70 of 70 previously identified diagnostic variants among 60 samples with previously identified diagnoses. Notably, reanalysis of 240 initially nondiagnostic samples using information available on July 1, 2017, revealed 38 novel diagnoses, representing a 15.8% increase in diagnostic yield. Modeling monthly iterative reanalysis of 240 nondiagnostic samples revealed a diagnostic rate of 0.57% of samples per month. Modeling the workload required for monthly iterative reanalysis of nondiagnostic samples revealed a variant analysis burden of approximately 5 variants/month for proband-only and approximately 0.5 variants/month for trio samples. Approximately 45% of samples required evaluation during each monthly interval, and 61.3% of samples were reevaluated across three consecutive reanalyses. In sum, automated reanalysis methods can facilitate efficient reevaluation of nondiagnostic samples using up-to-date literature and can provide significant value to clinical laboratories.

Correction: Novel findings with reassessment of exome data: implications for validation testing and interpretation of genomic data.

In the published version of this article, the degree of author Bo Zhang was incorrectly listed as PhD. The correct degree is BS.

AUDIOME: a tiered exome sequencing-based comprehensive gene panel for the diagnosis of heterogeneous nonsyndromic sensorineural hearing loss.

PURPOSE: Hereditary hearing loss is highly heterogeneous. To keep up with rapidly emerging disease-causing genes, we developed the AUDIOME test for nonsyndromic hearing loss (NSHL) using an exome sequencing (ES) platform and targeted analysis for the curated genes. METHODS: A tiered strategy was implemented for this test. Tier 1 includes combined Sanger and targeted deletion analyses of the two most common NSHL genes and two mitochondrial genes. Nondiagnostic tier 1 cases are subjected to ES and array followed by targeted analysis of the remaining AUDIOME genes. RESULTS: ES resulted in good coverage of the selected genes with 98.24% of targeted bases at >15 ×. A fill-in strategy was developed for the poorly covered regions, which generally fell within GC-rich or highly homologous regions. Prospective testing of 33 patients with NSHL revealed a diagnosis in 11 (33%) and a possible diagnosis in 8 cases (24.2%). Among those, 10 individuals had variants in tier 1 genes. The ES data in the remaining nondiagnostic cases are readily available for further analysis. CONCLUSION: The tiered and ES-based test provides an efficient and cost-effective diagnostic strategy for NSHL, with the potential to reflex to full exome to identify causal changes outside of the AUDIOME test.

Novel findings with reassessment of exome data: implications for validation testing and interpretation of genomic data.

PurposeThe objective of this study was to assess the ability of our laboratory's exome-sequencing test to detect known and novel sequence variants and identify the critical factors influencing the interpretation of a clinical exome test.MethodsWe developed a two-tiered validation strategy: (i) a method-based approach that assessed the ability of our exome test to detect known variants using a reference HapMap sample, and (ii) an interpretation-based approach that assessed our relative ability to identify and interpret disease-causing variants, by analyzing and comparing the results of 19 randomly selected patients previously tested by external laboratories.ResultsWe demonstrate that this approach is reproducible with >99% analytical sensitivity and specificity for single-nucleotide variants and indels <10 bp. Our findings were concordant with the reference laboratories in 84% of cases. A new molecular diagnosis was applied to three cases, including discovery of two novel candidate genes.ConclusionWe provide an assessment of critical areas that influence interpretation of an exome test, including comprehensive phenotype capture, assessment of clinical overlap, availability of parental data, and the addressing of limitations in database updates. These results can be used to inform improvements in phenotype-driven interpretation of medical exomes in clinical and research settings.

Correction: Novel findings with reassessment of exome data: implications for validation testing and interpretation of genomic data.

In the published version of this article, the name of the 18th author was misspelled as Minjie Lou. The correct name is Minjie Luo. The authors regret the error.

Phenotypic predictors and final diagnoses in patients referred for RASopathy testing by targeted next-generation sequencing.

INTRODUCTION: RASopathies include disorders generally characterized by developmental delay, specific heart defects, short stature, cardiac hypertrophy, and facial dysmorphisms. Next-generation sequencing (NGS)-based panels have widespread acceptance as a diagnostic tool for RASopathies. MATERIALS AND METHODS: The first 126 patients evaluated by clinical examination and the NGS RASopathy panel at the Children's Hospital of Philadelphia were enrolled. We calculated diagnosis rate, correlated reported clinical findings with positive or negative test results, and identified final molecular diagnoses in 28/96 patients who tested negative for RASopathies. RESULTS: Twenty-four patients had pathogenic variants on the RASopathy panel, for a diagnostic yield of 19%. Reported features of pulmonic stenosis and ptosis were significantly correlated with a positive test result; no reported features were significantly correlated with a negative test result. We identified 27 different alternative diagnoses for patients originally suspected of having RASopathies. DISCUSSION: This study provides information that can assist in guiding differential diagnosis and genetic testing for patients suspected of having a RASopathy disorder.Genet Med advance online publication 20 October 2016.

Nutritional control of epigenetic processes in yeast and human cells.

The vitamin folate is required for methionine homeostasis in all organisms. In addition to its role in protein synthesis, methionine is the precursor to S-adenosyl-methionine (SAM), which is used in myriad cellular methylation reactions, including all histone methylation reactions. Here, we demonstrate that folate and methionine deficiency led to reduced methylation of lysine 4 of histone H3 (H3K4) in Saccharomyces cerevisiae. The effect of nutritional deficiency on H3K79 methylation was less pronounced, but was exacerbated in S. cerevisiae carrying a hypomorphic allele of Dot1, the enzyme responsible for H3K79 methylation. This result suggested a hierarchy of epigenetic modifications in terms of their susceptibility to nutritional limitations. Folate deficiency caused changes in gene transcription that mirrored the effect of complete loss of H3K4 methylation. Histone methylation was also found to respond to nutritional deficiency in the fission yeast Schizosaccharomyces pombe and in human cells in culture.

Cellular memory of acquired stress resistance in Saccharomyces cerevisiae.

Cellular memory of past experiences has been observed in several organisms and across a variety of experiences, including bacteria "remembering" prior nutritional status and amoeba "learning" to anticipate future environmental conditions. Here, we show that Saccharomyces cerevisiae maintains a multifaceted memory of prior stress exposure. We previously demonstrated that yeast cells exposed to a mild dose of salt acquire subsequent tolerance to severe doses of H(2)O(2). We set out to characterize the retention of acquired tolerance and in the process uncovered two distinct aspects of cellular memory. First, we found that H(2)O(2) resistance persisted for four to five generations after cells were removed from the prior salt treatment and was transmitted to daughter cells that never directly experienced the pretreatment. Maintenance of this memory did not require nascent protein synthesis after the initial salt pretreatment, but rather required long-lived cytosolic catalase Ctt1p that was synthesized during salt exposure and then distributed to daughter cells during subsequent cell divisions. In addition to and separable from the memory of H(2)O(2) resistance, these cells also displayed a faster gene-expression response to subsequent stress at >1000 genes, representing transcriptional memory. The faster gene-expression response requires the nuclear pore component Nup42p and serves an important function by facilitating faster reacquisition of H(2)O(2) tolerance after a second cycle of salt exposure. Memory of prior stress exposure likely provides a significant advantage to microbial populations living in ever-changing environments.

Multiple means to the same end: the genetic basis of acquired stress resistance in yeast.

In nature, stressful environments often occur in combination or close succession, and thus the ability to prepare for impending stress likely provides a significant fitness advantage. Organisms exposed to a mild dose of stress can become tolerant to what would otherwise be a lethal dose of subsequent stress; however, the mechanism of this acquired stress tolerance is poorly understood. To explore this, we exposed the yeast gene-deletion libraries, which interrogate all essential and non-essential genes, to successive stress treatments and identified genes necessary for acquiring subsequent stress resistance. Cells were exposed to one of three different mild stress pretreatments (salt, DTT, or heat shock) and then challenged with a severe dose of hydrogen peroxide (H(2)O(2)). Surprisingly, there was little overlap in the genes required for acquisition of H(2)O(2) tolerance after different mild-stress pretreatments, revealing distinct mechanisms of surviving H(2)O(2) in each case. Integrative network analysis of these results with respect to protein-protein interactions, synthetic-genetic interactions, and functional annotations identified many processes not previously linked to H(2)O(2) tolerance. We tested and present several models that explain the lack of overlap in genes required for H(2)O(2) tolerance after each of the three pretreatments. Together, this work shows that acquired tolerance to the same severe stress occurs by different mechanisms depending on prior cellular experiences, underscoring the context-dependent nature of stress tolerance.

Impact of nonsense-mediated mRNA decay on the global expression profile of budding yeast.

Nonsense-mediated mRNA decay (NMD) is a eukaryotic mechanism of RNA surveillance that selectively eliminates aberrant transcripts coding for potentially deleterious proteins. NMD also functions in the normal repertoire of gene expression. In Saccharomyces cerevisiae, hundreds of endogenous RNA Polymerase II transcripts achieve steady-state levels that depend on NMD. For some, the decay rate is directly influenced by NMD (direct targets). For others, abundance is NMD-sensitive but without any effect on the decay rate (indirect targets). To distinguish between direct and indirect targets, total RNA from wild-type (Nmd(+)) and mutant (Nmd(-)) strains was probed with high-density arrays across a 1-h time window following transcription inhibition. Statistical models were developed to describe the kinetics of RNA decay. 45% +/- 5% of RNAs targeted by NMD were predicted to be direct targets with altered decay rates in Nmd(-) strains. Parallel experiments using conventional methods were conducted to empirically test predictions from the global experiment. The results show that the global assay reliably distinguished direct versus indirect targets. Different types of targets were investigated, including transcripts containing adjacent, disabled open reading frames, upstream open reading frames, and those prone to out-of-frame initiation of translation. Known targeting mechanisms fail to account for all of the direct targets of NMD, suggesting that additional targeting mechanisms remain to be elucidated. 30% of the protein-coding targets of NMD fell into two broadly defined functional themes: those affecting chromosome structure and behavior and those affecting cell surface dynamics. Overall, the results provide a preview for how expression profiles in multi-cellular eukaryotes might be impacted by NMD. Furthermore, the methods for analyzing decay rates on a global scale offer a blueprint for new ways to study mRNA decay pathways in any organism where cultured cell lines are available.

Ebs1p, a negative regulator of gene expression controlled by the Upf proteins in the yeast Saccharomyces cerevisiae.

Mutations in EBS1 were identified in Saccharomyces cerevisiae that cosuppress missense, frameshift, and nonsense mutations. Evidence from studies of loss of function and overexpression of EBS1 suggests that Ebs1p affects gene expression by inhibiting translation and that a loss of EBS1 function causes suppression by increasing the rate of translation. Changes in EBS1 expression levels alter the expression of wild-type genes, but, in general, no changes in mRNA abundance were associated with a loss of function or overexpression of EBS1. Translation of a lacZ reporter was increased in strains carrying an ebs1-Delta mutant gene, whereas translation was decreased when EBS1 was overexpressed. The cap binding protein eIF-4E copurifies with Ebs1p in the absence of RNA, suggesting that the two proteins interact in vivo. Although physical and genetic interactions were detected between Ebs1p and Dcp1p, copurification was RNase sensitive, and changes in the expression of Ebs1p had little to no effect on decapping of the MFA2 transcript. The combined results suggest that Ebs1p inhibits translation, most likely through effects on eIF-4E rather than on decapping. Finally, EBS1 transcript levels are under the control of nonsense-mediated mRNA decay (NMD), providing the first example of an NMD-sensitive transcript whose protein product influences a step in gene expression required for NMD.