Identification of a DNA methylation episignature for recurrent constellations of embryonic malformations.

The term "recurrent constellations of embryonic malformations" (RCEM) is used to describe a number of multiple malformation associations that affect three or more body structures. The causes of these disorders are currently unknown, and no diagnostic marker has been identified. Consequently, providing a definitive diagnosis in suspected individuals is challenging. In this study, genome-wide DNA methylation analysis was conducted on DNA samples obtained from the peripheral blood of 53 individuals with RCEM characterized by clinical features recognized as VACTERL and/or oculoauriculovertebral spectrum association. We identified a common DNA methylation episignature in 40 out of the 53 individuals. Subsequently, a sensitive and specific binary classifier was developed based on the DNA methylation episignature. This classifier can facilitate the use of RCEM episignature as a diagnostic biomarker in a clinical setting. The study also investigated the functional correlation of RCEM DNA methylation relative to other genetic disorders with known episignatures, highlighting the common genomic regulatory pathways involved in the pathophysiology of RCEM.

De novo variants in FRYL are associated with developmental delay, intellectual disability, and dysmorphic features.

FRY-like transcription coactivator (FRYL) belongs to a Furry protein family that is evolutionarily conserved from yeast to humans. The functions of FRYL in mammals are largely unknown, and variants in FRYL have not previously been associated with a Mendelian disease. Here, we report fourteen individuals with heterozygous variants in FRYL who present with developmental delay, intellectual disability, dysmorphic features, and other congenital anomalies in multiple systems. The variants are confirmed de novo in all individuals except one. Human genetic data suggest that FRYL is intolerant to loss of function (LoF). We find that the fly FRYL ortholog, furry (fry), is expressed in multiple tissues, including the central nervous system where it is present in neurons but not in glia. Homozygous fry LoF mutation is lethal at various developmental stages, and loss of fry in mutant clones causes defects in wings and compound eyes. We next modeled four out of the five missense variants found in affected individuals using fry knockin alleles. One variant behaves as a severe LoF variant, whereas two others behave as partial LoF variants. One variant does not cause any observable defect in flies, and the corresponding human variant is not confirmed to be de novo, suggesting that this is a variant of uncertain significance. In summary, our findings support that fry is required for proper development in flies and that the LoF variants in FRYL cause a dominant disorder with developmental and neurological symptoms due to haploinsufficiency.

The diagnostic yield of genetic and metabolic investigations in syndromic and nonsyndromic patients with autism spectrum disorder, global developmental delay, or intellectual disability from a dedicated neurodevelopmental disorders genetics clinic.

First-tier genetic investigations for patients with neurodevelopmental disorders (NDDs) may include chromosomal microarray, Fragile X testing, and screening for inherited metabolic diseases, but most remain undiagnosed upon completion of testing. Here, we report the diagnostic yields of genetic testing for 537 patients with at least one of autism spectrum disorder, global developmental delay, and/or intellectual disability. Patients were assessed in a single neurodevelopmental genetics clinic, and each underwent a standardized history and physical examination. Each patient was characterized as syndromic or nonsyndromic based on clinical features. Our results demonstrate that multigene sequencing (with an NDD gene panel or exome) had a higher diagnostic yield (8%; 95% confidence interval [CI]: 5%, 13%) than chromosomal microarray and Fragile X testing combined (4%; 95% CI: 3%, 7%). Biochemical screening for inherited metabolic diseases had a diagnostic yield of zero. The diagnostic yield of genetic testing was significantly higher for syndromic patients than for nonsyndromic patients (odds ratio [OR] 3.09; 95% CI: 1.46, 6.83) and higher for female patients than for male (OR 3.21; 95% CI: 1.52, 6.82). These results add to the growing evidence supporting a comprehensive genetic evaluation that includes both copy number analysis and sequencing of known NDD genes for patients with NDDs.

Genetic and metabolic investigations for neurodevelopmental disorders: position statement of the Canadian College of Medical Geneticists (CCMG).

PURPOSE AND SCOPE: The aim of this position statement is to provide recommendations for clinicians regarding the use of genetic and metabolic investigations for patients with neurodevelopmental disorders (NDDs), specifically, patients with global developmental delay (GDD), intellectual disability (ID) and/or autism spectrum disorder (ASD). This document also provides guidance for primary care and non-genetics specialists caring for these patients while awaiting consultation with a clinical geneticist or metabolic specialist. METHODS OF STATEMENT DEVELOPMENT: A multidisciplinary group reviewed existing literature and guidelines on the use of genetic and metabolic investigations for the diagnosis of NDDs and synthesised the evidence to make recommendations relevant to the Canadian context. The statement was circulated for comment to the Canadian College of Medical Geneticists (CCMG) membership-at-large and to the Canadian Pediatric Society (Mental Health and Developmental Disabilities Committee); following incorporation of feedback, it was approved by the CCMG Board of Directors on 1 September 2022. RESULTS AND CONCLUSIONS: Chromosomal microarray is recommended as a first-tier test for patients with GDD, ID or ASD. Fragile X testing should also be done as a first-tier test when there are suggestive clinical features or family history. Metabolic investigations should be done if there are clinical features suggestive of an inherited metabolic disease, while the patient awaits consultation with a metabolic physician. Exome sequencing or a comprehensive gene panel is recommended as a second-tier test for patients with GDD or ID. Genetic testing is not recommended for patients with NDDs in the absence of GDD, ID or ASD, unless accompanied by clinical features suggestive of a syndromic aetiology or inherited metabolic disease.

Deep phenotyping of the neuroimaging and skeletal features in KBG syndrome: a study of 53 patients and review of the literature.

BACKGROUND: KBG syndrome is caused by haploinsufficiency of ANKRD11 and is characterised by macrodontia of upper central incisors, distinctive facial features, short stature, skeletal anomalies, developmental delay, brain malformations and seizures. The central nervous system (CNS) and skeletal features remain poorly defined. METHODS: CNS and/or skeletal imaging were collected from molecularly confirmed individuals with KBG syndrome through an international network. We evaluated the original imaging and compared our results with data in the literature. RESULTS: We identified 53 individuals, 44 with CNS and 40 with skeletal imaging. Common CNS findings included incomplete hippocampal inversion and posterior fossa malformations; these were significantly more common than previously reported (63.4% and 65.9% vs 1.1% and 24.7%, respectively). Additional features included patulous internal auditory canal, never described before in KBG syndrome, and the recurrence of ventriculomegaly, encephalic cysts, empty sella and low-lying conus medullaris. We found no correlation between these structural anomalies and epilepsy or intellectual disability. Prevalent skeletal findings comprised abnormalities of the spine including scoliosis, coccygeal anomalies and cervical ribs. Hand X-rays revealed frequent abnormalities of carpal bone morphology and maturation, including a greater delay in ossification compared with metacarpal/phalanx bones. CONCLUSION: This cohort enabled us to describe the prevalence of very heterogeneous neuroradiological and skeletal anomalies in KBG syndrome. Knowledge of the spectrum of such anomalies will aid diagnostic accuracy, improve patient care and provide a reference for future research on the effects of ANKRD11 variants in skeletal and brain development.

Bridging clinical care and research in Ontario, Canada: Maximizing diagnoses from reanalysis of clinical exome sequencing data.

We examined the utility of clinical and research processes in the reanalysis of publicly-funded clinical exome sequencing data in Ontario, Canada. In partnership with eight sites, we recruited 287 families with suspected rare genetic diseases tested between 2014 and 2020. Data from seven laboratories was reanalyzed with the referring clinicians. Reanalysis of clinically relevant genes identified diagnoses in 4% (13/287); four were missed by clinical testing. Translational research methods, including analysis of novel candidate genes, identified candidates in 21% (61/287). Of these, 24 families have additional evidence through data sharing to support likely diagnoses (8% of cohort). This study indicates few diagnoses are missed by clinical laboratories, the incremental gain from reanalysis of clinically-relevant genes is modest, and the highest yield comes from validation of novel disease-gene associations. Future implementation of translational research methods, including continued reporting of compelling genes of uncertain significance by clinical laboratories, should be considered to maximize diagnoses.

Perturbed hematopoiesis in individuals with germline DNMT3A overgrowth Tatton-Brown-Rahman syndrome.

Tatton-Brown-Rahman syndrome (TBRS) is an overgrowth disorder caused by germline heterozygous mutations in the DNA methyltransferase DNMT3A. DNMT3A is a critical regulator of hematopoietic stem cell (HSC) differentiation and somatic DNMT3A mutations are frequent in hematologic malignancies and clonal hematopoiesis. Yet, the impact of constitutive DNMT3A mutation on hematopoiesis in TBRS is undefined. In order to establish how constitutive mutation of DNMT3A impacts blood development in TBRS we gathered clinical data and analyzed blood parameters in 18 individuals with TBRS. We also determined the distribution of major peripheral blood cell lineages by flow cytometric analyses. Our analyses revealed non-anemic macrocytosis, a relative decrease in lymphocytes and increase in neutrophils in TBRS individuals compared to unaffected controls. We were able to recapitulate these hematologic phenotypes in multiple murine models of TBRS and identified rare hematological and non-hematological malignancies associated with constitutive Dnmt3a mutation. We further show that loss of DNMT3A in TBRS is associated with an altered DNA methylation landscape in hematopoietic cells affecting regions critical to stem cell function and tumorigenesis. Overall, our data identify key hematopoietic effects driven by DNMT3A mutation with clinical implications for individuals with TBRS and DNMT3A-associated clonal hematopoiesis or malignancies.

Further clinical delineation of microcephaly-capillary malformation syndrome.

Microcephaly-Capillary Malformation syndrome (MIC-CAP) is a rare genetic disorder reported in 18 individuals to date. The clinical features typically include microcephaly, multiple cutaneous capillary malformations, seizures, neurologic impairment, and global developmental delay. Currently, there is little published information about the natural history and long-term outcomes for individuals with MIC-CAP. In this report, we provide follow up on two previously published patients and describe four new patients. The included patients highlight increased variability in the clinical spectrum and provide novel information regarding medical complications and recurrent variants.

Neurodevelopmental functioning in probands and non-proband carriers of 22q11.2 microduplication.

Microduplication of the LCR22-A to LCR22-D region on chromosome 22q11.2 is a recurrent copy number variant found in clinical populations undergoing chromosomal microarray, and at lower frequency in controls. Often inherited, there is limited data on intellectual (IQ) and psychological functioning, particularly in those individuals ascertained through a family member rather than because of neurodevelopmental disorders. To investigate the range of cognitive-behavioral phenotypes associated with 22q11.2 duplication, we studied both probands and their non-proband carrier relatives. Twenty-two individuals with 22q11.2 duplication (10 probands, 12 non-proband carriers) were prospectively assessed with a battery of neuropsychological tests, physical examination, and medical record review. Assessment measures with standardized norms included IQ, academic, adaptive, psychiatric, behavioral, and social functioning. IQ and academic skills were within the average range, with a trend toward lower scores in probands versus non-probands. Adaptive skills were within age expectations. Prevalence of attention deficits (probands only) and anxiety (both groups) was high compared with norms. The prevalence of autism spectrum disorder was relatively low (5% of total sample). Assessment of both probands and non-probands with 22q11.2 duplication suggests that the phenotypic spectrum with respect to neurodevelopment overlaps significantly with the general population. IQ and academic abilities are in the average range for most of the individuals with 22q11.2 duplication in our study, regardless of ascertainment as a proband or non-proband relative. Symptoms of attention deficit and anxiety were identified, which require further study. Results of this study further clarify the phenotype of individuals with 22q11.2 duplication, and provides important information for genetic counseling regarding this recurrent copy number variant.

Genetic and metabolic investigations for individuals with neurodevelopmental disorders: A survey of Canadian geneticists' practices.

Neurodevelopmental disorders (NDDs) are genetically heterogeneous. There are many possible etiological investigations for NDDs, and a lack of clear and current guidelines for such testing. Here we characterize the practices of genetic and metabolic physicians in Canada as it pertains to etiological investigation of patients with NDDs, by means of an online questionnaire. The survey response rate was 30% (n = 46). The most commonly ordered first-line tests for patients with non-syndromic NDDs are chromosomal microarray (98%) and Fragile X testing (85%). The most commonly ordered second-line test for non-syndromic NDDs is a multi-gene panel (78%) or exome sequencing (29%). Biochemical screening is ordered as a first line test by 33% of respondents, second line by 31%, and rarely or never by 36% of respondents. Those respondents with metabolics fellowship training were more likely to order biochemical screening than those without. The number of years of clinical experience generally did not affect the types of tests ordered. For patients with NDDs, test-ordering practice among Canadian clinical geneticists is highly variable, in particular with respect to biochemical screening and use of next-generation sequencing technologies. Evidence-based guidelines should be developed to facilitate best practices in Canada.

Genome sequencing for detection of pathogenic deep intronic variation: A clinical case report illustrating opportunities and challenges.

Variants in JAM3 have been reported in four families manifesting a severe autosomal recessive disorder characterized by hemorrhagic destruction of the brain, subependymal calcification, and cataracts. We describe a 7-year-old male with a similar presentation found by research-based quad genome sequencing to have two novel splicing variants in trans in JAM3, including one deep intronic variant (NM_032801.4: c.256+1260G>C) not detectable by standard exome sequencing. Targeted sequencing of RNA isolated from transformed lymphoblastoid cell lines confirmed that each of the two variants has a deleterious effect on JAM3 mRNA splicing. The role for genome sequencing as a clinical diagnostic test extends to those patients with phenotypes strongly suggestive of a specific Mendelian disorder, especially when the causal genetic variant(s) are not found by a more targeted approach. Barriers to diagnosis via identification of pathogenic deep intronic variation include lack of laboratory consensus regarding in silico splicing prediction tools and limited access to clinically validated confirmatory RNA experiments.

Tatton-Brown-Rahman syndrome: Six individuals with novel features.

Tatton-Brown Rahman syndrome (TBRS) is an overgrowth-intellectual disability syndrome caused by heterozygous variants in DNMT3A. Seventy-eight individuals have been reported with a consistent phenotype of somatic overgrowth, mild to moderate intellectual disability, and similar dysmorphisms. We present six individuals with TBRS, including the youngest individual thus far reported, first individual to be diagnosed with tumor testing and two individuals with variants at the Arg882 domain, bringing the total number of reported cases to 82. Patients reported herein have additional clinical features not previously reported in TBRS. One patient had congenital diaphragmatic hernia. One patient carrying the recurrent p.Arg882His DNMT3A variant, who was previously reported as having a phenotype due to a truncating variant in the CLTC gene, developed a ganglioneuroblastoma at 18 months and T-cell lymphoblastic lymphoma at 6 years of age. Four patients manifested symptoms suggestive of autonomic dysfunction, including central sleep apnea, postural orthostatic hypotension, and episodic vasomotor instability in the extremities. We discuss the molecular and clinical findings in our patients with TBRS in context of existing literature.

Clinical delineation of GTPBP2-associated neuro-ectodermal syndrome: Report of two new families and review of the literature.

The GTPBP2 gene encodes a guanosine triphosphate (GTP)-binding protein of unknown function. Biallelic loss-of-function variants in the GTPBP2 gene have been previously reported in association with a neuro-ectodermal clinical presentation in six individuals from four unrelated families. Here, we provide detailed descriptions of three additional individuals from two unrelated families in the context of the previous literature. Both families carry nonsense variants in GTPBP2: homozygous p.(Arg470*) and compound heterozygous p.(Arg432*)/p.(Arg131*). Key features of this clinically recognizable condition include prenatal onset microcephaly, tone abnormalities, and movement disorders, epilepsy, dysmorphic features, retinal dysfunction, ectodermal dysplasia, and brain iron accumulation. Our findings suggest that some aspects of the clinical presentation appear to be age-related; brain iron accumulation may appear only after childhood, and the ectodermal findings and peripheral neuropathy are most prominent in older individuals. In addition, we present prenatal and neonatal findings as well as the first Caucasian and black African families with GTPBP2 biallelic variants. The individuals described herein provide valuable additional phenotypic information about this rare, novel, and progressive neuroectodermal condition.

Genotype-phenotype correlations in individuals with pathogenic RERE variants.

Heterozygous variants in the arginine-glutamic acid dipeptide repeats gene (RERE) have been shown to cause neurodevelopmental disorder with or without anomalies of the brain, eye, or heart (NEDBEH). Here, we report nine individuals with NEDBEH who carry partial deletions or deleterious sequence variants in RERE. These variants were found to be de novo in all cases in which parental samples were available. An analysis of data from individuals with NEDBEH suggests that point mutations affecting the Atrophin-1 domain of RERE are associated with an increased risk of structural eye defects, congenital heart defects, renal anomalies, and sensorineural hearing loss when compared with loss-of-function variants that are likely to lead to haploinsufficiency. A high percentage of RERE pathogenic variants affect a histidine-rich region in the Atrophin-1 domain. We have also identified a recurrent two-amino-acid duplication in this region that is associated with the development of a CHARGE syndrome-like phenotype. We conclude that mutations affecting RERE result in a spectrum of clinical phenotypes. Genotype-phenotype correlations exist and can be used to guide medical decision making. Consideration should also be given to screening for RERE variants in individuals who fulfill diagnostic criteria for CHARGE syndrome but do not carry pathogenic variants in CHD7.

Improved diagnostic yield compared with targeted gene sequencing panels suggests a role for whole-genome sequencing as a first-tier genetic test.

PurposeGenetic testing is an integral diagnostic component of pediatric medicine. Standard of care is often a time-consuming stepwise approach involving chromosomal microarray analysis and targeted gene sequencing panels, which can be costly and inconclusive. Whole-genome sequencing (WGS) provides a comprehensive testing platform that has the potential to streamline genetic assessments, but there are limited comparative data to guide its clinical use.MethodsWe prospectively recruited 103 patients from pediatric non-genetic subspecialty clinics, each with a clinical phenotype suggestive of an underlying genetic disorder, and compared the diagnostic yield and coverage of WGS with those of conventional genetic testing.ResultsWGS identified diagnostic variants in 41% of individuals, representing a significant increase over conventional testing results (24%; P = 0.01). Genes clinically sequenced in the cohort (n = 1,226) were well covered by WGS, with a median exonic coverage of 40 × ±8 × (mean ±SD). All the molecular diagnoses made by conventional methods were captured by WGS. The 18 new diagnoses made with WGS included structural and non-exonic sequence variants not detectable with whole-exome sequencing, and confirmed recent disease associations with the genes PIGG, RNU4ATAC, TRIO, and UNC13A.ConclusionWGS as a primary clinical test provided a higher diagnostic yield than conventional genetic testing in a clinically heterogeneous cohort.

Assessing the accuracy of the Modified Checklist for Autism in Toddlers: a systematic review and meta-analysis.

AIM: The Modified Checklist for Autism in Toddlers (M-CHAT) could be appropriate for universal screening for autism spectrum disorder (ASD) at 18 months and 24 months. Validation studies, however, reported differences in psychometric properties across sample populations. This meta-analysis summarized its accuracy measures and quantified their change in relation to patient and study characteristics. METHOD: Four electronic databases (MEDLINE, PsycINFO, CINAHL, and Embase) were searched to identify articles published between January 2001 and May 2016. Bayesian regression models pooled study-specific measures. Meta-regressions covariates were age at screening, study design, and proportion of males. RESULTS: On the basis of the 13 studies included, the pooled sensitivity was 0.83 (95% credible interval [CI] 0.75-0.90), specificity was 0.51 (95% CI 0.41-0.61), and positive predictive value was 0.53 (95% CI 0.43-0.63) in high-risk children and 0.06 (95% CI <0.01-0.14) in low-risk children. Sensitivity was higher for screening at 30 months compared with 24 months. INTERPRETATION: Findings indicate that the M-CHAT performs with low to moderate accuracy in identifying ASD among children with developmental concerns, but there was a lack of evidence on its performance in low-risk children or at age 18 months. Clinicians should account for a child's age and presence of developmental concern when interpreting their M-CHAT score. WHAT THIS PAPER ADDS: The Modified Checklist for Autism in Toddlers (M-CHAT) performs with low-to-moderate accuracy in children with developmental concerns. There is limited evidence supporting its use at 18 months or in low-risk children.

Mutations in DCPS and EDC3 in autosomal recessive intellectual disability indicate a crucial role for mRNA decapping in neurodevelopment.

There are two known mRNA degradation pathways, 3' to 5' and 5' to 3'. We identified likely pathogenic variants in two genes involved in these two pathways in individuals with intellectual disability. In a large family with multiple branches, we identified biallelic variants in DCPS in three affected individuals; a splice site variant (c.636+1G>A) that results in an in-frame insertion of 45 nucleotides and a missense variant (c.947C>T; p.Thr316Met). DCPS decaps the cap structure generated by 3' to 5' exonucleolytic degradation of mRNA. In vitro decapping assays showed an ablation of decapping function for both variants in DCPS. In another family, we identified a homozygous mutation (c.161T>C; p.Phe54Ser) in EDC3 in two affected children. EDC3 stimulates DCP2, which decaps mRNAs at the beginning of the 5' to 3' degradation pathway. In vitro decapping assays showed that altered EDC3 is unable to enhance DCP2 decapping at low concentrations and even inhibits DCP2 decapping at high concentration. We show that individuals with biallelic mutations in these genes of seemingly central functions are viable and that these possibly lead to impairment of neurological functions linking mRNA decapping to normal cognition. Our results further affirm an emerging theme linking aberrant mRNA metabolism to neurological defects.

Mutations in the histamine N-methyltransferase gene, HNMT, are associated with nonsyndromic autosomal recessive intellectual disability.

Histamine (HA) acts as a neurotransmitter in the brain, which participates in the regulation of many biological processes including inflammation, gastric acid secretion and neuromodulation. The enzyme histamine N-methyltransferase (HNMT) inactivates HA by transferring a methyl group from S-adenosyl-l-methionine to HA, and is the only well-known pathway for termination of neurotransmission actions of HA in mammalian central nervous system. We performed autozygosity mapping followed by targeted exome sequencing and identified two homozygous HNMT alterations, p.Gly60Asp and p.Leu208Pro, in patients affected with nonsyndromic autosomal recessive intellectual disability from two unrelated consanguineous families of Turkish and Kurdish ancestry, respectively. We verified the complete absence of a functional HNMT in patients using in vitro toxicology assay. Using mutant and wild-type DNA constructs as well as in silico protein modeling, we confirmed that p.Gly60Asp disrupts the enzymatic activity of the protein, and that p.Leu208Pro results in reduced protein stability, resulting in decreased HA inactivation. Our results highlight the importance of inclusion of HNMT for genetic testing of individuals presenting with intellectual disability.

Noncompaction cardiomyopathy in an infant with Walker-Warburg syndrome.

Walker-Warburg syndrome (WWS) is a rare autosomal recessive, congenital muscular dystrophy that is associated with brain and eye anomalies. Several genes encoding proteins involved in α-dystroglycan glycosylation have been implicated in the aetiology of WWS. We describe a patient with nonclassical features of WWS presenting with heart failure related to noncompaction cardiomyopathy resulting in death at 4 months of age. Muscle biopsy revealed absent α-dystroglycan on immunostaining and genetic testing confirmed the diagnosis with two previously described POMT2 mutations. This is the first reported case of WWS syndrome associated with noncompaction cardiomyopathy.

Implication of LRRC4C and DPP6 in neurodevelopmental disorders.

We performed whole-genome sequencing on an individual from a family with variable psychiatric phenotypes that had a sensory processing disorder, apraxia, and autism. The proband harbored a maternally inherited balanced translocation (46,XY,t(11;14)(p12;p12)mat) that disrupted LRRC4C, a member of the highly specialized netrin G family of axon guidance molecules. The proband also inherited a paternally derived chromosomal inversion that disrupted DPP6, a potassium channel interacting protein. Copy Number (CN) analysis in 14,077 cases with neurodevelopmental disorders and 8,960 control subjects revealed that 60% of cases with exonic deletions in LRRC4C had a second clinically recognizable syndrome associated with variable clinical phenotypes, including 16p11.2, 1q44, and 2q33.1 CN syndromes, suggesting LRRC4C deletion variants may be modifiers of neurodevelopmental disorders. In vitro, functional assessments modeling patient deletions in LRRC4C suggest a negative regulatory role of these exons found in the untranslated region of LRRC4C, which has a single, terminal coding exon. These data suggest that the proband's autism may be due to the inheritance of disruptions in both DPP6 and LRRC4C, and may highlight the importance of the netrin G family and potassium channel interacting molecules in neurodevelopmental disorders. © 2016 Wiley Periodicals, Inc.