Long-read genome sequencing and variant reanalysis increase diagnostic yield in neurodevelopmental disorders.

Variant detection from long-read genome sequencing (lrGS) has proven to be considerably more accurate and comprehensive than variant detection from short-read genome sequencing (srGS). However, the rate at which lrGS can increase molecular diagnostic yield for rare disease is not yet precisely characterized. We performed lrGS using Pacific Biosciences "HiFi" technology on 96 short-read-negative probands with rare disease that were suspected to be genetic. We generated hg38-aligned variants and de novo phased genome assemblies, and subsequently annotated, filtered, and curated variants using clinical standards. New disease-relevant or potentially relevant genetic findings were identified in 16/96 (16.7%) probands, eight of which (8/96, 8.33%) harbored pathogenic or likely pathogenic variants. Newly identified variants were visible in both srGS and lrGS in nine probands (~9.4%) and resulted from changes to interpretation mostly from recent gene-disease association discoveries. Seven cases included variants that were only interpretable in lrGS, including copy-number variants, an inversion, a mobile element insertion, two low-complexity repeat expansions, and a 1 bp deletion. While evidence for each of these variants is, in retrospect, visible in srGS, they were either: not called within srGS data, were represented by calls with incorrect sizes or structures, or failed quality-control and filtration. Thus, while reanalysis of older data clearly increases diagnostic yield, we find that lrGS allows for substantial additional yield (7/96, 7.3%) beyond srGS. We anticipate that as lrGS analysis improves, and as lrGS datasets grow allowing for better variant frequency annotation, the additional lrGS-only rare disease yield will grow over time.

Menke-Hennekam syndrome; delineation of domain-specific subtypes with distinct clinical and DNA methylation profiles.

CREB-binding protein (CBP, encoded by CREBBP) and its paralog E1A-associated protein (p300, encoded by EP300) are involved in histone acetylation and transcriptional regulation. Variants that produce a null allele or disrupt the catalytic domain of either protein cause Rubinstein-Taybi syndrome (RSTS), while pathogenic missense and in-frame indel variants in parts of exons 30 and 31 cause phenotypes recently described as Menke-Hennekam syndrome (MKHK). To distinguish MKHK subtypes and define their characteristics, molecular and extended clinical data on 82 individuals (54 unpublished) with variants affecting CBP (n = 71) or p300 (n = 11) (NP_004371.2 residues 1,705-1,875 and NP_001420.2 residues 1,668-1,833, respectively) were summarized. Additionally, genome-wide DNA methylation profiles were assessed in DNA extracted from whole peripheral blood from 54 individuals. Most variants clustered closely around the zinc-binding residues of two zinc-finger domains (ZZ and TAZ2) and within the first α helix of the fourth intrinsically disordered linker (ID4) of CBP/p300. Domain-specific methylation profiles were discerned for the ZZ domain in CBP/p300 (found in nine out of 10 tested individuals) and TAZ2 domain in CBP (in 14 out of 20), while a domain-specific diagnostic episignature was refined for the ID4 domain in CBP/p300 (in 21 out of 21). Phenotypes including intellectual disability of varying degree and distinct physical features were defined for each of the regions. These findings demonstrate existence of at least three MKHK subtypes, which are domain specific (MKHK-ZZ, MKHK-TAZ2, and MKHK-ID4) rather than gene specific (CREBBP/EP300). DNA methylation episignatures enable stratification of molecular pathophysiologic entities within a gene or across a family of paralogous genes.

Heterozygous loss-of-function variants in DOCK4 cause neurodevelopmental delay and microcephaly.

Neurons form the basic anatomical and functional structure of the nervous system, and defects in neuronal differentiation or formation of neurites are associated with various psychiatric and neurodevelopmental disorders. Dynamic changes in the cytoskeleton are essential for this process, which is, inter alia, controlled by the dedicator of cytokinesis 4 (DOCK4) through the activation of RAC1. Here, we clinically describe 7 individuals (6 males and one female) with variants in DOCK4 and overlapping phenotype of mild to severe global developmental delay. Additional symptoms include coordination or gait abnormalities, microcephaly, nonspecific brain malformations, hypotonia and seizures. Four individuals carry missense variants (three of them detected de novo) and three individuals carry null variants (two of them maternally inherited). Molecular modeling of the heterozygous missense variants suggests that the majority of them affect the globular structure of DOCK4. In vitro functional expression studies in transfected Neuro-2A cells showed that all missense variants impaired neurite outgrowth. Furthermore, Dock4 knockout Neuro-2A cells also exhibited defects in promoting neurite outgrowth. Our results, including clinical, molecular and functional data, suggest that loss-of-function variants in DOCK4 probable cause a variable spectrum of a novel neurodevelopmental disorder with microcephaly.

De novo variants predicting haploinsufficiency for DIP2C are associated with expressive speech delay.

The disconnected (disco)-interacting protein 2 (DIP2) gene was first identified in D. melanogaster and contains a DNA methyltransferase-associated protein 1 (DMAP1) binding domain, Acyl-CoA synthetase domain and AMP-binding sites. DIP2 regulates axonal bifurcation of the mushroom body neurons in D. melanogaster and is required for axonal regeneration in the neurons of C. elegans. The DIP2 homologues in vertebrates, Disco-interacting protein 2 homolog A (DIP2A), Disco-interacting protein 2 homolog B (DIP2B), and Disco-interacting protein 2 homolog C (DIP2C), are highly conserved and expressed widely in the central nervous system. Although there is evidence that DIP2C plays a role in cognition, reports of pathogenic variants in these genes are rare and their significance is uncertain. We present 23 individuals with heterozygous DIP2C variants, all manifesting developmental delays that primarily affect expressive language and speech articulation. Eight patients had de novo variants predicting loss-of-function in the DIP2C gene, two patients had de novo missense variants, three had paternally inherited loss of function variants and six had maternally inherited loss-of-function variants, while inheritance was unknown for four variants. Four patients had cardiac defects (hypertrophic cardiomyopathy, atrial septal defects, and bicuspid aortic valve). Minor facial anomalies were inconsistent but included a high anterior hairline with a long forehead, broad nasal tip, and ear anomalies. Brainspan analysis showed elevated DIP2C expression in the human neocortex at 10-24 weeks after conception. With the cases presented herein, we provide phenotypic and genotypic data supporting the association between loss-of-function variants in DIP2C with a neurocognitive phenotype.

A qualitative evaluation of patient and parent experiences with an undiagnosed diseases program.

Previous studies have explored patient experiences before being seen or at the beginning of their evaluation by undiagnosed diseases programs. This study provides additional insight into experiences after participation through in-depth, qualitative evaluation, allowing for reflection of current practice and patient/parent needs. Semi-structured interviews were conducted with patients and parents of patients seen at the University of Alabama at Birmingham (UAB)'s unique, clinically focused Undiagnosed Diseases Program (UDP). Analysis of the interviews was guided by a thematic approach. Participants had undergone a diagnostic odyssey before being evaluated by the UDP and remained hopeful for a diagnosis. They appreciated the opportunity to be seen by the UDP. However, perception of experiences differed based on whether evaluation by the UDP led to a diagnosis. Additionally, while participants were pleased with initial communication, they indicated that there were unmet needs regarding follow-up. Patients and parents of patients believe that participation in an undiagnosed diseases program is the best option for diagnosis. The findings of this study provide a general overview of patient experiences and highlight strengths of the UAB UDP while also emphasizing areas to focus the improvement to optimize the benefit to patients and families with undiagnosed and rare diseases, which could be used helpful in the development of similar clinics.

DLG2 intragenic exonic deletions reinforce the link to neurodevelopmental disorders and suggest a potential association with congenital anomalies and dysmorphism.

PURPOSE: Multiple studies suggest an association between DLG2 and neurodevelopmental disorders and indicate the haploinsufficiency of this gene; however, few cases have been thoroughly described. We performed additional studies to confirm this clinical association and DLG2 haploinsufficiency. METHODS: Chromosomal microarray analysis was performed on 11,107 patients at the Cytogenetics Laboratory at the University of Alabama at Birmingham. The Database of Genomic Variants-Gold Standard Variants and the Genome Aggregation Database were selected for the association analysis. Fifty-nine patients from the literature and DECIPHER, all having DLG2 intragenic deletions, were included for comprehensive analysis of the distribution of these deletions. RESULTS: A total of 13 patients with DLG2 intragenic deletions, from 10 families in our cohort, were identified. Nine of 10 probands presented with clinical features of neurodevelopmental disorders. Congenital anomalies and dysmorphism were common in our cohort of patients. Association analysis showed that the frequency of DLG2 deletions in our cohort is significantly higher than those in the Database of Genomic Variants-Gold Standard Variants and the Genome Aggregation Database. Most of DLG2 intragenic deletions identified in 69 unrelated patients from our cohort, the literature, and DECIPHER map to the 5' region of the gene, with a hotspot centered around HPin7, exon 8, and HPin8. CONCLUSION: Our findings reinforce the link between DLG2 intragenic deletions and neurodevelopmental disorders, strongly support the haploinsufficiency of this gene, and indicate that these deletions might also have an association with congenital anomalies and dysmorphism.

Developmental epileptic encephalopathy in DLG4-related synaptopathy.

OBJECTIVE: The postsynaptic density protein of excitatory neurons PSD-95 is encoded by discs large MAGUK scaffold protein 4 (DLG4), de novo pathogenic variants of which lead to DLG4-related synaptopathy. The major clinical features are developmental delay, intellectual disability (ID), hypotonia, sleep disturbances, movement disorders, and epilepsy. Even though epilepsy is present in 50% of the individuals, it has not been investigated in detail. We describe here the phenotypic spectrum of epilepsy and associated comorbidities in patients with DLG4-related synaptopathy. METHODS: We included 35 individuals with a DLG4 variant and epilepsy as part of a multicenter study. The DLG4 variants were detected by the referring laboratories. The degree of ID, hypotonia, developmental delay, and motor disturbances were evaluated by the referring clinician. Data on awake and sleep electroencephalography (EEG) and/or video-polygraphy and brain magnetic resonance imaging were collected. Antiseizure medication response was retrospectively assessed by the referring clinician. RESULTS: A large variety of seizure types was reported, although focal seizures were the most common. Encephalopathy related to status epilepticus during slow-wave sleep (ESES)/developmental epileptic encephalopathy with spike-wave activation during sleep (DEE-SWAS) was diagnosed in >25% of the individuals. All but one individual presented with neurodevelopmental delay. Regression in verbal and/or motor domains was observed in all individuals who suffered from ESES/DEE-SWAS, as well as some who did not. We could not identify a clear genotype-phenotype relationship even between individuals with the same DLG4 variants. SIGNIFICANCE: Our study shows that a subgroup of individuals with DLG4-related synaptopathy have DEE, and approximately one fourth of them have ESES/DEE-SWAS. Our study confirms DEE as part of the DLG4-related phenotypic spectrum. Occurrence of ESES/DEE-SWAS in DLG4-related synaptopathy requires proper investigation with sleep EEG.

Parents' Perspectives on the Utility of Genomic Sequencing in the Neonatal Intensive Care Unit.

BACKGROUND: It is critical to understand the wide-ranging clinical and non-clinical effects of genome sequencing (GS) for parents in the NICU context. We assessed parents' experiences with GS as a first-line diagnostic tool for infants with suspected genetic conditions in the NICU. METHODS: Parents of newborns (N = 62) suspected of having a genetic condition were recruited across five hospitals in the southeast United States as part of the SouthSeq study. Semi-structured interviews (N = 78) were conducted after parents received their child's sequencing result (positive, negative, or variants of unknown significance). Thematic analysis was performed on all interviews. RESULTS: Key themes included that (1) GS in infancy is important for reproductive decision making, preparing for the child's future care, ending the diagnostic odyssey, and sharing results with care providers; (2) the timing of disclosure was acceptable for most parents, although many reported the NICU environment was overwhelming; and (3) parents deny that receiving GS results during infancy exacerbated parent-infant bonding, and reported variable impact on their feelings of guilt. CONCLUSION: Parents reported that GS during the neonatal period was useful because it provided a "backbone" for their child's care. Parents did not consistently endorse negative impacts like interference with parent-infant bonding.

Gain and loss of function variants in EZH1 disrupt neurogenesis and cause dominant and recessive neurodevelopmental disorders.

Genetic variants in chromatin regulators are frequently found in neurodevelopmental disorders, but their effect in disease etiology is rarely determined. Here, we uncover and functionally define pathogenic variants in the chromatin modifier EZH1 as the cause of dominant and recessive neurodevelopmental disorders in 19 individuals. EZH1 encodes one of the two alternative histone H3 lysine 27 methyltransferases of the PRC2 complex. Unlike the other PRC2 subunits, which are involved in cancers and developmental syndromes, the implication of EZH1 in human development and disease is largely unknown. Using cellular and biochemical studies, we demonstrate that recessive variants impair EZH1 expression causing loss of function effects, while dominant variants are missense mutations that affect evolutionarily conserved aminoacids, likely impacting EZH1 structure or function. Accordingly, we found increased methyltransferase activity leading to gain of function of two EZH1 missense variants. Furthermore, we show that EZH1 is necessary and sufficient for differentiation of neural progenitor cells in the developing chick embryo neural tube. Finally, using human pluripotent stem cell-derived neural cultures and forebrain organoids, we demonstrate that EZH1 variants perturb cortical neuron differentiation. Overall, our work reveals a critical role of EZH1 in neurogenesis regulation and provides molecular diagnosis for previously undefined neurodevelopmental disorders.

Poison exon annotations improve the yield of clinically relevant variants in genomic diagnostic testing.

PURPOSE: Neurodevelopmental disorders (NDDs) often result from rare genetic variation, but genomic testing yield for NDDs remains below 50%, suggesting that clinically relevant variants may be missed by standard analyses. Here, we analyze "poison exons" (PEs), which are evolutionarily conserved alternative exons often absent from standard gene annotations. Variants that alter PE inclusion can lead to loss of function and may be highly penetrant contributors to disease. METHODS: We curated published RNA sequencing data from developing mouse cortex to define 1937 conserved PE regions potentially relevant to NDDs, and we analyzed variants found by genome sequencing in multiple NDD cohorts. RESULTS: Across 2999 probands, we found 6 novel clinically relevant variants in PE regions. Five of these variants are in genes that are part of the sodium voltage-gated channel alpha subunit family (SCN1A, SCN2A, and SCN8A), which is associated with epilepsies. One variant is in SNRPB, associated with cerebrocostomandibular syndrome. These variants have moderate to high computational impact assessments, are absent from population variant databases, and in genes with gene-phenotype associations consistent with each probands reported features. CONCLUSION: With a very minimal increase in variant analysis burden (average of 0.77 variants per proband), annotation of PEs can improve diagnostic yield for NDDs and likely other congenital conditions.

Adrenal Insufficiency in Peroxisomal Disorders: A Single Institution Case Series.

INTRODUCTION: There are two major categories of peroxisomal disorders (PDs): peroxisomal biogenesis disorders (PBDs) due to defects in peroxisomal (PEX) genes and deficiency of other peroxisomal enzymes (such as D-bifunctional enzyme deficiency due to HSD17B4). PDs are characterized by abnormal elevations of very-long-chain fatty acids (VLCFA). We aimed to evaluate the clinical phenotype of adrenal insufficiency in patients with PD and to assess any genotype-phenotype correlations with adrenal insufficiency. CASE PRESENTATION: We performed a retrospective electronic medical record review at a single university medical center, of data over 12 years and identified 7 patients with PD. Of the 7 patients identified, 6 patients had a diagnosis of PBD and one had a single peroxisomal enzyme deficiency, HSD17B4. The average age of the patients at diagnosis were 0.61 ± 0.66 years. Four patients (66.7%) had primary adrenal insufficiency: 3, out of the 4, patients had elevated baseline ACTH. Three patients failed to have increased response after the Cortrosyn™ stimulation test. Three patients were on daily hydrocortisone replacement, and 1 patient was on stress-dose hydrocortisone only as needed. Specific genetic variant analysis revealed that all the 3 patients with PBD and adrenal insufficiency who were on steroid supplementation had the compound heterozygous pathogenic variant in exon 13 of PEX1 c.2097dupT (p.Ile700Tyrfs*42) and c.2528G>A (p.Gly843Asp), while the 1 patient with peroxisomal enzyme deficiency and adrenal insufficiency had compound heterozygous pathogenic variants in HSD17B4 c.1369A>T (p.Asn457Tyr) and c.1210 - 1G>A (splice acceptor). Two of these patients with PEX1 variants also required mineralocorticoid supplementation. The 3 PBD patients without adrenal insufficiency did not have a PEX1 variant. DISCUSSION/CONCLUSION: Primary adrenal insufficiency is common in patients with PD. Based on our data, patients with the compound heterozygous PEX1 pathogenic variants of exon 13 (c.2097dupT and c.2528G>A) tend to have adrenal insufficiency. Aldosterone deficiency, though rare, can occur in PD.

Deleterious, protein-altering variants in the transcriptional coregulator ZMYM3 in 27 individuals with a neurodevelopmental delay phenotype.

Neurodevelopmental disorders (NDDs) result from highly penetrant variation in hundreds of different genes, some of which have not yet been identified. Using the MatchMaker Exchange, we assembled a cohort of 27 individuals with rare, protein-altering variation in the transcriptional coregulator ZMYM3, located on the X chromosome. Most (n = 24) individuals were males, 17 of which have a maternally inherited variant; six individuals (4 male, 2 female) harbor de novo variants. Overlapping features included developmental delay, intellectual disability, behavioral abnormalities, and a specific facial gestalt in a subset of males. Variants in almost all individuals (n = 26) are missense, including six that recurrently affect two residues. Four unrelated probands were identified with inherited variation affecting Arg441, a site at which variation has been previously seen in NDD-affected siblings, and two individuals have de novo variation resulting in p.Arg1294Cys (c.3880C>T). All variants affect evolutionarily conserved sites, and most are predicted to damage protein structure or function. ZMYM3 is relatively intolerant to variation in the general population, is widely expressed across human tissues, and encodes a component of the KDM1A-RCOR1 chromatin-modifying complex. ChIP-seq experiments on one variant, p.Arg1274Trp, indicate dramatically reduced genomic occupancy, supporting a hypomorphic effect. While we are unable to perform statistical evaluations to definitively support a causative role for variation in ZMYM3, the totality of the evidence, including 27 affected individuals, recurrent variation at two codons, overlapping phenotypic features, protein-modeling data, evolutionary constraint, and experimentally confirmed functional effects strongly support ZMYM3 as an NDD-associated gene.

Perspectives on the future of dysmorphology.

The field of clinical genetics and genomics continues to evolve. In the past few decades, milestones like the initial sequencing of the human genome, dramatic changes in sequencing technologies, and the introduction of artificial intelligence, have upended the field and offered fascinating new insights. Though difficult to predict the precise paths the field will follow, rapid change may continue to be inevitable. Within genetics, the practice of dysmorphology, as defined by pioneering geneticist David W. Smith in the 1960s as "the study of, or general subject of abnormal development of tissue form" has also been affected by technological advances as well as more general trends in biomedicine. To address possibilities, potential, and perils regarding the future of dysmorphology, a group of clinical geneticists, representing different career stages, areas of focus, and geographic regions, have contributed to this piece by providing insights about how the practice of dysmorphology will develop over the next several decades.

A cost-utility analysis of BRCA1 and BRCA2 testing in high-risk breast cancer patients and family members in Thailand: a cost-effective policy in resource-limited settings.

Background: Screening for germline pathogenic BRCA1 or BRCA2 variants (gBRCA) in high-risk breast cancer patients is known to be cost-effective in high-income countries. Nationwide adoption of genetics testing in high-risk breast cancer population remains poor. Our study aimed to assess gBRCA health economics data in the middle-income country setting of Thailand. Methods: Decision tree and Markov model were utilized to assess cost-utility between the testing vs. no-testing groups from a societal and lifetime perspective and lifetime. We interviewed 264 patients with breast/ovarian cancer and their family members to assess relevant costs and quality of life using EQ-5D-5L. One-way sensitivity, probabilistic sensitivity (Monte Carlo simulation), and budget impact analyses were done to estimate the outcome under Thailand's Universal Health Coverage scheme. Results: The predicted lifetime cost and Quality-adjusted Life Years (QALY) for those with breast cancer were $13,788 and 10.22 in the testing group and $13,702 and 10.07 in the no-testing group. The incremental cost-effectiveness ratio for gBRCA testing in high-risk breast cancer patients was $573/QALY. The lifetime cost for the family members of those with gBRCA was $14,035 (QALY 9.99), while the no-testing family members group was $14,077 (QALY 9.98). Performing gBRCA testing in family members was cost-saving. Conclusion: Cost-utility analysis demonstrated a cost-effective result of gBRCA testing in high-risk breast cancer patients and cost-saving in familial cascade testing. The result was endorsed in the national health benefits package in 2022. Other middle-income countries may observe the cost-effective/cost-saving aspects in common genetic diseases under their national health schemes.

Return of non-ACMG recommended incidental genetic findings to pediatric patients: considerations and opportunities from experiences in genomic sequencing.

BACKGROUND: The uptake of exome/genome sequencing has introduced unexpected testing results (incidental findings) that have become a major challenge for both testing laboratories and providers. While the American College of Medical Genetics and Genomics has outlined guidelines for laboratory management of clinically actionable secondary findings, debate remains as to whether incidental findings should be returned to patients, especially those representing pediatric populations. METHODS: The Sequencing Analysis and Diagnostic Yield working group in the Clinical Sequencing Evidence-Generating Research Consortium has collected a cohort of pediatric patients found to harbor a genomic sequencing-identified non-ACMG-recommended incidental finding. The incidental variants were not thought to be associated with the indication for testing and were disclosed to patients and families. RESULTS: In total, 23 "non-ACMG-recommended incidental findings were identified in 21 pediatric patients included in the study. These findings span four different research studies/laboratories and demonstrate differences in incidental finding return rate across study sites. We summarize specific cases to highlight core considerations that surround identification and return of incidental findings (uncertainty of disease onset, disease severity, age of onset, clinical actionability, and personal utility), and suggest that interpretation of incidental findings in pediatric patients can be difficult given evolving phenotypes. Furthermore, return of incidental findings can benefit patients and providers, but do present challenges. CONCLUSIONS: While there may be considerable benefit to return of incidental genetic findings, these findings can be burdensome to providers and present risk to patients. It is important that laboratories conducting genomic testing establish internal guidelines in anticipation of detection. Moreover, cross-laboratory guidelines may aid in reducing the potential for policy heterogeneity across laboratories as it relates to incidental finding detection and return. However, future discussion is required to determine whether cohesive guidelines or policy statements are warranted.

Applying the Clinician-reported Genetic testing Utility InDEx (C-GUIDE) to genome sequencing: further evidence of validity.

Genome sequencing (GS) outperforms other rare disease diagnostics, but standardized approaches to assessing its clinical utility are limited. This study assessed the validity of the Clinician-reported Genetic testing Utility InDEx (C-GUIDE), a novel tool for assessing the utility of genetic testing from a clinician's perspective, for GS. C-GUIDE ratings were completed for patients who received GS results. For each patient, total C-GUIDE and single item global scores were calculated. Construct validity was assessed using linear regression to determine the association between C-GUIDE total and global item scores and measure the effects of potential explanatory variables. Ratings were completed for 67 pediatric and 36 adult patients. GS indications were neurological for 70.9% and results were diagnostic for 28.2%. When the C-GUIDE assessed primary (PV), secondary (SV), and pharmacogenomic (PGx) variants, on average, a one unit increase in the global item score was associated with an increase of 7.3 in the C-GUIDE score (p < 0.05). Diagnostic results were associated with an increase in C-GUIDE score of 5.0 compared to non-diagnostic results (p < 0.05) and an increase of one SV was associated with an increase of 2.5 (p < 0.05). For children, decreased age of one year was associated with an increase in C-GUIDE score of 0.3 (p < 0.05). Findings provide evidence that C-GUIDE measures the construct of clinical utility in pediatric and adult rare disease populations and is sensitive to changes in utility related to variant type. Quantifying the clinical utility of GS using C-GUIDE can inform efforts to optimize its use in patient care.

Case report: 11-ketotestosterone may potentiate advanced bone age as seen in some cases of Wiedemann-Steiner Syndrome.

Context: Wiedemann-Steiner Syndrome (WSS) is a genetic disorder associated with an array of clinical phenotypes, including advanced bone age and short stature. 11-ketotestosterone (11KT) is a member of the group known as 11-oxygenated C19 androgens that are implicated in premature adrenarche. Case description: Case 1: The patient is a 3 year and 11-month-old female diagnosed with WSS due to deletion of KMT2A detected on CGH microarray. At two years and 11 months, imaging revealed an advanced bone age. We obtained an 11KT level on this patient. 11KT in case 1 was elevated at 26.3 ng/dL, while the normal reference range is 7.3-10.9 ng/dL and the reference interval for premature adrenarche is 12.3-22.9 ng/dL, The repeat 11KT at follow up (chronological age 4 years and 6 months) was still elevated at 33.8 ng/dL Case 2: A second child with WSS and a 5kb intragenic KMT2A deletion was evaluated at 11 months of age; his 11KT was 4.5 ng/dL. Conclusions: The elevated 11KT may indicate maturational changes related to increasing adrenal gland androgenic activation and may explain the advanced bone age seen in some patients with WSS. To our knowledge, this is the first case report that describes 11KT as a bioactive androgen potentially causing bone age advancement in WSS. Lack of elevation of 11KT in the second child who is an infant suggests increasing androgenic precursors and metabolites related to premature adrenarche may need to be longitudinally followed.

DNA Methylation Signature for JARID2-Neurodevelopmental Syndrome.

JARID2 (Jumonji, AT Rich Interactive Domain 2) pathogenic variants cause a neurodevelopmental syndrome, that is characterized by developmental delay, cognitive impairment, hypotonia, autistic features, behavior abnormalities and dysmorphic facial features. JARID2 encodes a transcriptional repressor protein that regulates the activity of various histone methyltransferase complexes. However, the molecular etiology is not fully understood, and JARID2-neurodevelopmental syndrome may vary in its typical clinical phenotype. In addition, the detection of variants of uncertain significance (VUSs) often results in a delay of final diagnosis which could hamper the appropriate care. In this study we aim to detect a specific and sensitive DNA methylation signature for JARID2-neurodevelopmental syndrome. Peripheral blood DNA methylation profiles from 56 control subjects, 8 patients with (likely) pathogenic JARID2 variants and 3 patients with JARID2 VUSs were analyzed. DNA methylation analysis indicated a clear and robust separation between patients with (likely) pathogenic variants and controls. A binary model capable of classifying patients with the JARID2-neurodevelopmental syndrome was constructed on the basis of the identified episignature. Patients carrying VUSs clustered with the control group. We identified a distinct DNA methylation signature associated with JARID2-neurodevelopmental syndrome, establishing its utility as a biomarker for this syndrome and expanding the EpiSign diagnostic test.

Endocannabinoid dysfunction in neurological disease: neuro-ocular DAGLA-related syndrome.

The endocannabinoid system is a highly conserved and ubiquitous signalling pathway with broad-ranging effects. Despite critical pathway functions, gene variants have not previously been conclusively linked to human disease. We identified nine children from eight families with heterozygous, de novo truncating variants in the last exon of DAGLA with a neuro-ocular phenotype characterized by developmental delay, ataxia and complex oculomotor abnormality. All children displayed paroxysms of nystagmus or eye deviation accompanied by compensatory head posture and worsened incoordination most frequently after waking. RNA sequencing showed clear expression of the truncated transcript and no differences were found between mutant and wild-type DAGLA activity. Immunofluorescence staining of patient-derived fibroblasts and HEK cells expressing the mutant protein showed distinct perinuclear aggregation not detected in control samples. This report establishes truncating variants in the last DAGLA exon as the cause of a unique paediatric syndrome. Because enzymatic activity was preserved, the observed mislocalization of the truncated protein may account for the observed phenotype. Potential mechanisms include DAGLA haploinsufficiency at the plasma membrane or dominant negative effect. To our knowledge, this is the first report directly linking an endocannabinoid system component with human genetic disease and sets the stage for potential future therapeutic avenues.