Bi-allelic variants in the ER quality-control mannosidase gene EDEM3 cause a congenital disorder of glycosylation.

EDEM3 encodes a protein that converts Man8GlcNAc2 isomer B to Man7-5GlcNAc2. It is involved in the endoplasmic reticulum-associated degradation pathway, responsible for the recognition of misfolded proteins that will be targeted and translocated to the cytosol and degraded by the proteasome. In this study, through a combination of exome sequencing and gene matching, we have identified seven independent families with 11 individuals with bi-allelic protein-truncating variants and one individual with a compound heterozygous missense variant in EDEM3. The affected individuals present with an inherited congenital disorder of glycosylation (CDG) consisting of neurodevelopmental delay and variable facial dysmorphisms. Experiments in human fibroblast cell lines, human plasma, and mouse plasma and brain tissue demonstrated decreased trimming of Man8GlcNAc2 isomer B to Man7GlcNAc2, consistent with loss of EDEM3 enzymatic activity. In human cells, Man5GlcNAc2 to Man4GlcNAc2 conversion is also diminished with an increase of Glc1Man5GlcNAc2. Furthermore, analysis of the unfolded protein response showed a reduced increase in EIF2AK3 (PERK) expression upon stimulation with tunicamycin as compared to controls, suggesting an impaired unfolded protein response. The aberrant plasma N-glycan profile provides a quick, clinically available test for validating variants of uncertain significance that may be identified by molecular genetic testing. We propose to call this deficiency EDEM3-CDG.

Expanding the genotype-phenotype spectrum in SCN8A-related disorders.

BACKGROUND: SCN8A-related disorders are a group of variable conditions caused by pathogenic variations in SCN8A. Online Mendelian Inheritance in Man (OMIM) terms them as developmental and epileptic encephalopathy 13, benign familial infantile seizures 5 or cognitive impairment with or without cerebellar ataxia. METHODS: In this study, we describe clinical and genetic results on eight individuals from six families with SCN8A pathogenic variants identified via exome sequencing. RESULTS: Clinical findings ranged from normal development with well-controlled epilepsy to significant developmental delay with treatment-resistant epilepsy. Three novel and three reported variants were observed in SCN8A. Electrophysiological analysis in transfected cells revealed a loss-of-function variant in Patient 4. CONCLUSIONS: This work expands the clinical and genotypic spectrum of SCN8A-related disorders and provides electrophysiological results on a novel loss-of-function SCN8A variant.

Dominant negative variants in IKZF2 cause ICHAD syndrome, a new disorder characterised by immunodysregulation, craniofacial anomalies, hearing impairment, athelia and developmental delay.

BACKGROUND: Helios (encoded by IKZF2), a member of the Ikaros family of transcription factors, is a zinc finger protein involved in embryogenesis and immune function. Although predominantly recognised for its role in the development and function of T lymphocytes, particularly the CD4+ regulatory T cells (Tregs), the expression and function of Helios extends beyond the immune system. During embryogenesis, Helios is expressed in a wide range of tissues, making genetic variants that disrupt the function of Helios strong candidates for causing widespread immune-related and developmental abnormalities in humans. METHODS: We performed detailed phenotypic, genomic and functional investigations on two unrelated individuals with a phenotype of immune dysregulation combined with syndromic features including craniofacial differences, sensorineural hearing loss and congenital abnormalities. RESULTS: Genome sequencing revealed de novo heterozygous variants that alter the critical DNA-binding zinc fingers (ZFs) of Helios. Proband 1 had a tandem duplication of ZFs 2 and 3 in the DNA-binding domain of Helios (p.Gly136_Ser191dup) and Proband 2 had a missense variant impacting one of the key residues for specific base recognition and DNA interaction in ZF2 of Helios (p.Gly153Arg). Functional studies confirmed that both these variant proteins are expressed and that they interfere with the ability of the wild-type Helios protein to perform its canonical function-repressing IL2 transcription activity-in a dominant negative manner. CONCLUSION: This study is the first to describe dominant negative IKZF2 variants. These variants cause a novel genetic syndrome characterised by immunodysregulation, craniofacial anomalies, hearing impairment, athelia and developmental delay.

Performance of the eHealth decision support tool, MIPOGG, for recognising children with Li-Fraumeni, DICER1, Constitutional mismatch repair deficiency and Gorlin syndromes.

BACKGROUND: Cancer predisposition syndromes (CPSs) are responsible for at least 10% of cancer diagnoses in children and adolescents, most of which are not clinically recognised prior to cancer diagnosis. A variety of clinical screening guidelines are used in healthcare settings to help clinicians detect patients who have a higher likelihood of having a CPS. The McGill Interactive Pediatric OncoGenetic Guidelines (MIPOGG) is an electronic health decision support tool that uses algorithms to help clinicians determine if a child/adolescent diagnosed with cancer should be referred to genetics for a CPS evaluation. METHODS: This study assessed MIPOGG's performance in identifying Li-Fraumeni, DICER1, Constitutional mismatch repair deficiency and Gorlin (nevoid basal cell carcinoma) syndromes in a retrospective series of 84 children diagnosed with cancer and one of these four CPSs in Canadian hospitals over an 18-year period. RESULTS: MIPOGG detected 82 of 83 (98.8%) evaluable patients with any one of these four genetic conditions and demonstrated an appropriate rationale for suggesting CPS evaluation. When compared with syndrome-specific clinical screening criteria, MIPOGG's ability to correctly identify children with any of the four CPSs was equivalent to, or outperformed, existing clinical criteria respective to each CPS. CONCLUSION: This study adds evidence that MIPOGG is an appropriate tool for CPS screening in clinical practice. MIPOGG's strength is that it starts with a specific cancer diagnosis and incorporates criteria relevant for associated CPSs, making MIPOGG a more universally accessible diagnostic adjunct that does not require in-depth knowledge of each CPS.

Genomic analyses in Cornelia de Lange Syndrome and related diagnoses: Novel candidate genes, genotype-phenotype correlations and common mechanisms.

Cornelia de Lange Syndrome (CdLS) is a rare, dominantly inherited multisystem developmental disorder characterized by highly variable manifestations of growth and developmental delays, upper limb involvement, hypertrichosis, cardiac, gastrointestinal, craniofacial, and other systemic features. Pathogenic variants in genes encoding cohesin complex structural subunits and regulatory proteins (NIPBL, SMC1A, SMC3, HDAC8, and RAD21) are the major pathogenic contributors to CdLS. Heterozygous or hemizygous variants in the genes encoding these five proteins have been found to be contributory to CdLS, with variants in NIPBL accounting for the majority (>60%) of cases, and the only gene identified to date that results in the severe or classic form of CdLS when mutated. Pathogenic variants in cohesin genes other than NIPBL tend to result in a less severe phenotype. Causative variants in additional genes, such as ANKRD11, EP300, AFF4, TAF1, and BRD4, can cause a CdLS-like phenotype. The common role that these genes, and others, play as critical regulators of developmental transcriptional control has led to the conditions they cause being referred to as disorders of transcriptional regulation (or "DTRs"). Here, we report the results of a comprehensive molecular analysis in a cohort of 716 probands with typical and atypical CdLS in order to delineate the genetic contribution of causative variants in cohesin complex genes as well as novel candidate genes, genotype-phenotype correlations, and the utility of genome sequencing in understanding the mutational landscape in this population.

The Clinical Variant Analysis Tool: Analyzing the evidence supporting reported genomic variation in clinical practice.

PURPOSE: Genomic test results, regardless of laboratory variant classification, require clinical practitioners to judge the applicability of a variant for medical decisions. Teaching and standardizing clinical interpretation of genomic variation calls for a methodology or tool. METHODS: To generate such a tool, we distilled the Clinical Genome Resource framework of causality and the American College of Medical Genetics/Association of Molecular Pathology and Quest Diagnostic Laboratory scoring of variant deleteriousness into the Clinical Variant Analysis Tool (CVAT). Applying this to 289 clinical exome reports, we compared the performance of junior practitioners with that of experienced medical geneticists and assessed the utility of reported variants. RESULTS: CVAT enabled performance comparable to that of experienced medical geneticists. In total, 124 of 289 (42.9%) exome reports and 146 of 382 (38.2%) reported variants supported a diagnosis. Overall, 10.5% (1 pathogenic [P] or likely pathogenic [LP] variant and 39 variants of uncertain significance [VUS]) of variants were reported in genes without established disease association; 20.2% (23 P/LP and 54 VUS) were in genes without sufficient phenotypic concordance; 7.3% (15 P/LP and 13 VUS) conflicted with the known molecular disease mechanism; and 24% (91 VUS) had insufficient evidence for deleteriousness. CONCLUSION: Implementation of CVAT standardized clinical interpretation of genomic variation and emphasized the need for collaborative and transparent reporting of genomic variation.

Impact of variation in practice in the prenatal reporting of variants of uncertain significance by commercial laboratories: Need for greater adherence to published guidelines.

OBJECTIVE: To evaluate the impact of implementing commercial whole exome sequencing (WES) and targeted gene panel testing in pregnancies with fetal anomalies. METHODS: A retrospective chart review of 124 patients with sequencing performed by commercial laboratories. RESULTS: The diagnostic yield of WES and panel testing was 21.5% and 26%, respectively, based on likely pathogenic (LP) or pathogenic (P) variants. Forty-two percent of exomes and 32% of panels analysed had one or more variants of uncertain significance (VUS) reported. A multidisciplinary in-depth review of the fetal phenotype, disease phenotype, variant data, and, in some patients, additional prenatal or postnatal investigations increased the diagnostic yield by 5% for exome analysis and 6% for panel analysis. CONCLUSIONS: The diagnostic yield of WES and panel testing combined was 23% based on LP and P variants. Although the reporting of VUS contributed to a 5% increase in diagnostic yield for WES and 6% for panels, the large number of VUS reported by commercial laboratories has significant resource implications. Our results support the need for greater adherence to the recommendations on the prenatal reporting of VUS and the importance of a multidisciplinary approach that brings together clinical and laboratory expertise in prenatal genetics and genomics.

Ulcerated amelanotic melanoma of the ear in an 11 year old with Fitzpatrick VI skin type: A case report.

Melanoma is rare in pediatric patients and even more so in those with darker Fitzpatrick skin types. Although risk factors for conventional melanoma are similar in both adult and pediatric cases, the presentation of melanoma in pediatric patients is often distinct from adults. Here, we describe a case of amelanotic ulcerated nodular melanoma with regional lymph node metastases treated with nivolumab in a patient with Fitzpatrick skin type VI.

Clinical spectrum of individuals with pathogenic NF1 missense variants affecting p.Met1149, p.Arg1276, and p.Lys1423: genotype-phenotype study in neurofibromatosis type 1.

We report 281 individuals carrying a pathogenic recurrent NF1 missense variant at p.Met1149, p.Arg1276, or p.Lys1423, representing three nontruncating NF1 hotspots in the University of Alabama at Birmingham (UAB) cohort, together identified in 1.8% of unrelated NF1 individuals. About 25% (95% confidence interval: 20.5-31.2%) of individuals heterozygous for a pathogenic NF1 p.Met1149, p.Arg1276, or p.Lys1423 missense variant had a Noonan-like phenotype, which is significantly more compared with the "classic" NF1-affected cohorts (all p < .0001). Furthermore, p.Arg1276 and p.Lys1423 pathogenic missense variants were associated with a high prevalence of cardiovascular abnormalities, including pulmonic stenosis (all p < .0001), while p.Arg1276 variants had a high prevalence of symptomatic spinal neurofibromas (p < .0001) compared with "classic" NF1-affected cohorts. However, p.Met1149-positive individuals had a mild phenotype, characterized mainly by pigmentary manifestations without externally visible plexiform neurofibromas, symptomatic spinal neurofibromas or symptomatic optic pathway gliomas. As up to 0.4% of unrelated individuals in the UAB cohort carries a p.Met1149 missense variant, this finding will contribute to more accurate stratification of a significant number of NF1 individuals. Although clinically relevant genotype-phenotype correlations are rare in NF1, each affecting only a small percentage of individuals, together they impact counseling and management of a significant number of the NF1 population.

De Novo Mutations in EBF3 Cause a Neurodevelopmental Syndrome.

Early B cell factor 3 (EBF3) is an atypical transcription factor that is thought to influence the laminar formation of the cerebral cortex. Here, we report that de novo mutations in EBF3 cause a complex neurodevelopmental syndrome. The mutations were identified in two large-scale sequencing projects: the UK Deciphering Developmental Disorders (DDD) study and the Canadian Clinical Assessment of the Utility of Sequencing and Evaluation as a Service (CAUSES) study. The core phenotype includes moderate to severe intellectual disability, and many individuals exhibit cerebellar ataxia, subtle facial dysmorphism, strabismus, and vesicoureteric reflux, suggesting that EBF3 has a widespread developmental role. Pathogenic de novo variants identified in EBF3 include multiple loss-of-function and missense mutations. Structural modeling suggested that the missense mutations affect DNA binding. Functional analysis of mutant proteins with missense substitutions revealed reduced transcriptional activities and abilities to form heterodimers with wild-type EBF3. We conclude that EBF3, a transcription factor previously unknown to be associated with human disease, is important for brain and other organ development and warrants further investigation.

Loss of BRG1 (SMARCA4) Immunoexpression in a Pediatric Non-Central Nervous System Tumor Cohort.

Malignant rhabdoid tumors and atypical teratoid/rhabdoid tumors of the central nervous system are primitive malignancies associated with a poor prognosis. These tumors have previously been characterized by inactivation of the switch/sucrose nonfermenting (SWI/SNF) chromatin remodeling complex protein integrase interactor 1 (INI1), encoded by the SMARCB1 gene. In the last decade, sporadic publications have shown that a different SWI/SNF protein, brahma-related gene 1 (BRG1), encoded by the SMARCA4 gene, is associated with a similar rhabdoid phenotype and possible germline mutation termed rhabdoid tumor predisposition syndrome type 2. We sought to determine the presence of BRG1 expression in pediatric embryonal tumors. Using a local tissue microarray consisting of 28 tumors diagnosed as having an undifferentiated, polyphenotypic, or rhabdoid morphology, expression of BRG1 by immunohistochemistry was performed. Four cases showed loss of INI1, while 3 of the remaining 24 cases demonstrated loss of BRG1. Two cases were diagnosed as soft tissue sarcomas, and 1 case was diagnosed as a small cell carcinoma of the ovary, hypercalcemic type. Survival ranged from less than 6 months after diagnosis to more than 5 years at the time of last follow-up. In conclusion, we demonstrate that BRG1 immunohistochemistry is a useful second-line immunostain for the workup of undifferentiated, polyphenotypic or rhabdoid pediatric tumors that demonstrate retained expression of INI1.

Exome Sequencing and the Management of Neurometabolic Disorders.

BACKGROUND: Whole-exome sequencing has transformed gene discovery and diagnosis in rare diseases. Translation into disease-modifying treatments is challenging, particularly for intellectual developmental disorder. However, the exception is inborn errors of metabolism, since many of these disorders are responsive to therapy that targets pathophysiological features at the molecular or cellular level. METHODS: To uncover the genetic basis of potentially treatable inborn errors of metabolism, we combined deep clinical phenotyping (the comprehensive characterization of the discrete components of a patient's clinical and biochemical phenotype) with whole-exome sequencing analysis through a semiautomated bioinformatics pipeline in consecutively enrolled patients with intellectual developmental disorder and unexplained metabolic phenotypes. RESULTS: We performed whole-exome sequencing on samples obtained from 47 probands. Of these patients, 6 were excluded, including 1 who withdrew from the study. The remaining 41 probands had been born to predominantly nonconsanguineous parents of European descent. In 37 probands, we identified variants in 2 genes newly implicated in disease, 9 candidate genes, 22 known genes with newly identified phenotypes, and 9 genes with expected phenotypes; in most of the genes, the variants were classified as either pathogenic or probably pathogenic. Complex phenotypes of patients in five families were explained by coexisting monogenic conditions. We obtained a diagnosis in 28 of 41 probands (68%) who were evaluated. A test of a targeted intervention was performed in 18 patients (44%). CONCLUSIONS: Deep phenotyping and whole-exome sequencing in 41 probands with intellectual developmental disorder and unexplained metabolic abnormalities led to a diagnosis in 68%, the identification of 11 candidate genes newly implicated in neurometabolic disease, and a change in treatment beyond genetic counseling in 44%. (Funded by BC Children's Hospital Foundation and others.).

RAPIDOMICS: rapid genome-wide sequencing in a neonatal intensive care unit-successes and challenges.

Genetic disorders are one of the leading causes of infant mortality and are frequent in neonatal intensive care units (NICUs). Rapid genome-wide sequencing (GWS; whole genome or exome sequencing (ES)), due to its diagnostic capabilities and immediate impacts on medical management, is becoming an appealing testing option in the NICU setting. RAPIDOMICS was a trio-based rapid ES pilot study of 25 babies with suspected genetic disorders in the BC Women's Hospital NICU. ES and bioinformatic analysis were performed after careful patient ascertainment. Trio analysis was performed using an in-house pipeline reporting variants in known disease-causing genes. Variants interpreted by the research team as definitely or possibly causal of the infant's phenotype were Sanger validated in a clinical laboratory. The average time to preliminary diagnosis was 7.2 days. Sanger validation was pursued in 15 patients for 13 autosomal dominant and 2 autosomal recessive disorders, with an overall diagnostic rate (partial or complete) of 60%.Conclusion: In total, 72% of patients enrolled had a genomic diagnosis achieved through ES, multi-gene panel testing or chromosomal microarray analysis. Among these, there was an 83% rate of significant and immediate impact on medical decision-making directly related to new knowledge of the diagnosis. Health service implementation challenges and successes are discussed. What is Known: • Rapid genome-wide sequencing in the neonatal intensive care setting has a greater diagnostic hit rate and impact on medical management than conventional genetic testing. However, the impact of consultation with genetics and patient ascertainment requires further investigation. What is New: • This study demonstrates the importance of genetic consultation and careful patient selection prior to pursuing exome sequencing (ES). • In total, 15/25 (60%) patients achieved a diagnosis through ES and 18/25 (72%) through ES, multi-gene panel testing or chromosomal microarray analysis with 83% of those having immediate effects on medical management.

Comprehensive whole genome sequence analyses yields novel genetic and structural insights for Intellectual Disability.

BACKGROUND: Intellectual Disability (ID) is among the most common global disorders, yet etiology is unknown in ~30% of patients despite clinical assessment. Whole genome sequencing (WGS) is able to interrogate the entire genome, providing potential to diagnose idiopathic patients. METHODS: We conducted WGS on eight children with idiopathic ID and brain structural defects, and their normal parents; carrying out an extensive data analyses, using standard and discovery approaches. RESULTS: We verified de novo pathogenic single nucleotide variants (SNV) in ARID1B c.1595delG and PHF6 c.820C > T, potentially causative de novo two base indels in SQSTM1 c.115_116delinsTA and UPF1 c.1576_1577delinsA, and de novo SNVs in CACNB3 c.1289G > A, and SPRY4 c.508 T > A, of uncertain significance. We report results from a large secondary control study of 2081 exomes probing the pathogenicity of the above genes. We analyzed structural variation by four different algorithms including de novo genome assembly. We confirmed a likely contributory 165 kb de novo heterozygous 1q43 microdeletion missed by clinical microarray. The de novo assembly resulted in unmasking hidden genome instability that was missed by standard re-alignment based algorithms. We also interrogated regulatory sequence variation for known and hypothesized ID genes and present useful strategies for WGS data analyses for non-coding variation. CONCLUSION: This study provides an extensive analysis of WGS in the context of ID, providing genetic and structural insights into ID and yielding diagnoses.

A girl with developmental delay, ataxia, cranial nerve palsies, severe respiratory problems in infancy-Expanding NDST1 syndrome.

NDST1 encodes an enzyme involved in the first steps in the synthesis of heparan sulfate chains, proteoglycans that are regulators found on the cell surface and in the extracellular matrix. Eight individuals homozygous for one of four family-specific missense mutations in the sulfotransferase domain of the enzyme have been described. They have intellectual disability. Some additionally had hypotonia, ataxia. seizures, and/or short stature, but none had history of respiratory problems. No humans with homozygous null mutations are known. ndst1b (orthologous to NDST1) morpholino knockdown in zebrafish (Danio rerio) causes delayed development, craniofacial cartilage abnormalities, shortened body and pectoral fin length. Ndst1 homozygous null mice have craniofacial abnormalities and die within the first 10 h of life of respiratory failure. We report a girl upon whom deep phenotyping, extensive genetic and biochemical investigations, and exome sequencing were performed. She had cranial nerves dysfunction, gastroesophageal reflux, history of a seizure, ataxia, developmental delays, head sparing failure to thrive, and minor malformations including distinctive facial features and a bifid uvula. Compound heterozygous mutations in NDST1 were identified, in the heparan sulfate N deacetylatase domain of one allele and the sulfotransferase domain of the other allele. This report expands the phenotypic spectrum of Ndst1 deficiency in humans. © 2017 Wiley Periodicals, Inc.

Case Series: A Kindred With Eruptive Vellus Hair Cysts and Systemic Features.

Eruptive vellus hair cysts (EVHCs) often occur on the trunk and limbs. Facial involvement is uncommon. Autosomal dominant inheritance has been described, but associated extracutaneous anomalies have not. We describe a 4-patient kindred presenting with multiple facial EVHCs and an association of preauricular pits, lipomas, joint hypermobility, and cardiac defects. Histopathologic examination confirmed the diagnosis of EVHCs in 3 affected individuals. We propose that facial EVHCs may indicate the presence of an inherited autosomal dominant disorder with extracutaneous manifestations. Extracutaneous manifestations noted in the kindred have been sporadically described in association with steatocystoma multiplex (SM), a condition occasionally noted in the presence of EVHCs, further supporting an association between these disorders.

Successful umbilical cord blood hematopoietic stem cell transplantation in pediatric patients with MDS/AML associated with underlying GATA2 mutations: two case reports and review of literature.

Germline GATA2 mutations have been associated with a vast array of clinical manifestations, as well as hematological deficiencies and a propensity to AML or MDS. We present two cases of pediatric AML/MDS with underlying GATA2 mutations who underwent a successful umbilical cord hematopoietic stem cell transplantation using two different conditioning regimens. These cases illustrate the importance of recognizing the clinical features associated with GATA2 mutations and performing the appropriate molecular testing. Diagnosis of heritable gene mutations associated with familial AML/MDS has significant clinical implication for the patients and affected families.