Five years of experience in the Epigenetics and Chromatin Clinic: what have we learned and where do we go from here?

The multidisciplinary Epigenetics and Chromatin Clinic at Johns Hopkins provides comprehensive medical care for individuals with rare disorders that involve disrupted epigenetics. Initially centered on classical imprinting disorders, the focus shifted to the rapidly emerging group of genetic disorders resulting from pathogenic germline variants in epigenetic machinery genes. These are collectively called the Mendelian disorders of the epigenetic machinery (MDEMs), or more broadly, Chromatinopathies. In five years, 741 clinic visits have been completed for 432 individual patients, with 153 having confirmed epigenetic diagnoses. Of these, 115 individuals have one of 26 MDEMs with every single one exhibiting global developmental delay and/or intellectual disability. This supports prior observations that intellectual disability is the most common phenotypic feature of MDEMs. Additional common phenotypes in our clinic include growth abnormalities and neurodevelopmental issues, particularly hypotonia, attention-deficit/hyperactivity disorder (ADHD), and anxiety, with seizures and autism being less common. Overall, our patient population is representative of the broader group of MDEMs and includes mostly autosomal dominant disorders impacting writers more so than erasers, readers, and remodelers of chromatin marks. There is an increased representation of dual function components with a reader and an enzymatic domain. As expected, diagnoses were made mostly by sequencing but were aided in some cases by DNA methylation profiling. Our clinic has helped to facilitate the discovery of two new disorders, and our providers are actively developing and implementing novel therapeutic strategies for MDEMs. These data and our high follow-up rate of over 60% suggest that we are achieving our mission to diagnose, learn from, and provide optimal care for our patients with disrupted epigenetics.

Pathogenic Variants in NUP214 Cause "Plugged" Nuclear Pore Channels and Acute Febrile Encephalopathy.

We report biallelic missense and frameshift pathogenic variants in the gene encoding human nucleoporin NUP214 causing acute febrile encephalopathy. Clinical symptoms include neurodevelopmental regression, seizures, myoclonic jerks, progressive microcephaly, and cerebellar atrophy. NUP214 and NUP88 protein levels were reduced in primary skin fibroblasts derived from affected individuals, while the total number and density of nuclear pore complexes remained normal. Nuclear transport assays exhibited defects in the classical protein import and mRNA export pathways in affected cells. Direct surface imaging of fibroblast nuclei by scanning electron microscopy revealed a large increase in the presence of central particles (known as "plugs") in the nuclear pore channels of affected cells. This observation suggests that large transport cargoes may be delayed in passage through the nuclear pore channel, affecting its selective barrier function. Exposure of fibroblasts from affected individuals to heat shock resulted in a marked delay in their stress response, followed by a surge in apoptotic cell death. This suggests a mechanistic link between decreased cell survival in cell culture and severe fever-induced brain damage in affected individuals. Our study provides evidence by direct imaging at the single nuclear pore level of functional changes linked to a human disease.

Disruption of RFX family transcription factors causes autism, attention-deficit/hyperactivity disorder, intellectual disability, and dysregulated behavior.

PURPOSE: We describe a novel neurobehavioral phenotype of autism spectrum disorder (ASD), intellectual disability, and/or attention-deficit/hyperactivity disorder (ADHD) associated with de novo or inherited deleterious variants in members of the RFX family of genes. RFX genes are evolutionarily conserved transcription factors that act as master regulators of central nervous system development and ciliogenesis. METHODS: We assembled a cohort of 38 individuals (from 33 unrelated families) with de novo variants in RFX3, RFX4, and RFX7. We describe their common clinical phenotypes and present bioinformatic analyses of expression patterns and downstream targets of these genes as they relate to other neurodevelopmental risk genes. RESULTS: These individuals share neurobehavioral features including ASD, intellectual disability, and/or ADHD; other frequent features include hypersensitivity to sensory stimuli and sleep problems. RFX3, RFX4, and RFX7 are strongly expressed in developing and adult human brain, and X-box binding motifs as well as RFX ChIP-seq peaks are enriched in the cis-regulatory regions of known ASD risk genes. CONCLUSION: These results establish a likely role of deleterious variation in RFX3, RFX4, and RFX7 in cases of monogenic intellectual disability, ADHD and ASD, and position these genes as potentially critical transcriptional regulators of neurobiological pathways associated with neurodevelopmental disease pathogenesis.

KMT2B-related disorders: expansion of the phenotypic spectrum and long-term efficacy of deep brain stimulation.

Heterozygous mutations in KMT2B are associated with an early-onset, progressive and often complex dystonia (DYT28). Key characteristics of typical disease include focal motor features at disease presentation, evolving through a caudocranial pattern into generalized dystonia, with prominent oromandibular, laryngeal and cervical involvement. Although KMT2B-related disease is emerging as one of the most common causes of early-onset genetic dystonia, much remains to be understood about the full spectrum of the disease. We describe a cohort of 53 patients with KMT2B mutations, with detailed delineation of their clinical phenotype and molecular genetic features. We report new disease presentations, including atypical patterns of dystonia evolution and a subgroup of patients with a non-dystonic neurodevelopmental phenotype. In addition to the previously reported systemic features, our study has identified co-morbidities, including the risk of status dystonicus, intrauterine growth retardation, and endocrinopathies. Analysis of this study cohort (n = 53) in tandem with published cases (n = 80) revealed that patients with chromosomal deletions and protein truncating variants had a significantly higher burden of systemic disease (with earlier onset of dystonia) than those with missense variants. Eighteen individuals had detailed longitudinal data available after insertion of deep brain stimulation for medically refractory dystonia. Median age at deep brain stimulation was 11.5 years (range: 4.5-37.0 years). Follow-up after deep brain stimulation ranged from 0.25 to 22 years. Significant improvement of motor function and disability (as assessed by the Burke Fahn Marsden's Dystonia Rating Scales, BFMDRS-M and BFMDRS-D) was evident at 6 months, 1 year and last follow-up (motor, P = 0.001, P = 0.004, and P = 0.012; disability, P = 0.009, P = 0.002 and P = 0.012). At 1 year post-deep brain stimulation, >50% of subjects showed BFMDRS-M and BFMDRS-D improvements of >30%. In the long-term deep brain stimulation cohort (deep brain stimulation inserted for >5 years, n = 8), improvement of >30% was maintained in 5/8 and 3/8 subjects for the BFMDRS-M and BFMDRS-D, respectively. The greatest BFMDRS-M improvements were observed for trunk (53.2%) and cervical (50.5%) dystonia, with less clinical impact on laryngeal dystonia. Improvements in gait dystonia decreased from 20.9% at 1 year to 16.2% at last assessment; no patient maintained a fully independent gait. Reduction of BFMDRS-D was maintained for swallowing (52.9%). Five patients developed mild parkinsonism following deep brain stimulation. KMT2B-related disease comprises an expanding continuum from infancy to adulthood, with early evidence of genotype-phenotype correlations. Except for laryngeal dysphonia, deep brain stimulation provides a significant improvement in quality of life and function with sustained clinical benefit depending on symptoms distribution.

Diagnostic utility of telomere length testing in a hospital-based setting.

Telomere length (TL) predicts the onset of cellular senescence in vitro but the diagnostic utility of TL measurement in clinical settings is not fully known. We tested the value of TL measurement by flow cytometry and FISH (flowFISH) in patients with mutations in telomerase and telomere maintenance genes. TL had a discrete and reproducible normal range with definable upper and lower boundaries. While TL above the 50th age-adjusted percentile had a 100% negative predictive value for clinically relevant mutations, the lower threshold in mutation carriers was age-dependent, and adult mutation carriers often overlapped with the lowest decile of controls. The extent of telomere shortening correlated with the age at diagnosis as well as the short telomere syndrome phenotype. Extremely short TL caused bone marrow failure and immunodeficiency in children and young adults, while milder defects manifested as pulmonary fibrosis-emphysema in adults. We prospectively examined whether TL altered treatment decisions for newly diagnosed idiopathic bone marrow failure patients and found abnormally short TL enriched for patients with mutations in some inherited bone marrow failure genes, such as RUNX1, in addition to telomerase and telomere maintenance genes. The result was actionable, altering the choice of treatment regimen and/or hematopoietic stem cell donor in one-fourth of the cases (9 of 38, 24%). We conclude that TL measurement by flowFISH, when used for targeted clinical indications and in limited settings, can influence treatment decisions in ways that improve outcome.

Deficiency of Adenosine Deaminase 2 (DADA2): Hidden Variants, Reduced Penetrance, and Unusual Inheritance.

PURPOSE: Deficiency of adenosine deaminase 2 (DADA2) is an autosomal recessive disorder that manifests with fever, early-onset vasculitis, strokes, and hematologic dysfunction. This study aimed to identify disease-causing variants by conventional Sanger and whole exome sequencing in two families suspected to have DADA2 and non-confirmatory genotypes. ADA2 enzymatic assay confirmed the clinical diagnosis of DADA2. Molecular diagnosis was important to accurately identify other family members at risk. METHODS: We used a variety of sequencing technologies, ADA2 enzymatic testing, and molecular methods including qRT-PCR and MLPA. RESULTS: Exome sequencing identified heterozygosity for the known pathogenic variant ADA2: c.1358A>G, p.Tyr453Cys in a 14-year-old female with a history of ischemic strokes, livedo, and vasculitis. No second pathogenic variant could be identified. ADA2 enzymatic testing in combination with quantitative RT-PCR suggested a loss-of-function allele. Subsequent genome sequencing identified a canonical splice site variant, c.-47+2T>C, within the 5'UTR of ADA2. Two of her unaffected siblings were found to carry the same two pathogenic variants. A homozygous 800-bp duplication comprising exon 7 of ADA2 was identified in a 5-year-old female with features consistent with Diamond-Blackfan anemia (DBA). The duplication was missed by Sanger sequencing of ADA2, chromosomal microarray, and exome sequencing but was detected by MLPA in combination with long-read PCR sequencing. The exon 7 duplication was also identified in her non-symptomatic father and younger sister. CONCLUSIONS: ADA2 pathogenic variants may not be detected by conventional sequencing and genetic testing and may require the incorporation of additional diagnostic methods. A definitive molecular diagnosis is crucial for all family members to make informed treatment decisions.

Missense variants in the chromatin remodeler CHD1 are associated with neurodevelopmental disability.

BACKGROUND: The list of Mendelian disorders of the epigenetic machinery has expanded rapidly during the last 5 years. A few missense variants in the chromatin remodeler CHD1 have been found in several large-scale sequencing efforts focused on uncovering the genetic aetiology of autism. OBJECTIVES: To explore whether variants in CHD1 are associated with a human phenotype. METHODS: We used GeneMatcher to identify other physicians caring for patients with variants in CHD1. We also explored the epigenetic consequences of one of these variants in cultured fibroblasts. RESULTS: Here we describe six CHD1 heterozygous missense variants in a cohort of patients with autism, speech apraxia, developmental delay and facial dysmorphic features. Importantly, three of these variants occurred de novo. We also report on a subject with a de novo deletion covering a large fraction of the CHD1 gene without any obvious neurological phenotype. Finally, we demonstrate increased levels of the closed chromatin modification H3K27me3 in fibroblasts from a subject carrying a de novo variant in CHD1. CONCLUSIONS: Our results suggest that variants in CHD1 can lead to diverse phenotypic outcomes; however, the neurodevelopmental phenotype appears to be limited to patients with missense variants, which is compatible with a dominant negative mechanism of disease.

Enrolling Genomics Research Participants through a Clinical Setting: the Impact of Existing Clinical Relationships on Informed Consent and Expectations for Return of Research Results.

Genetic counselors working in a clinical setting may find themselves recruiting, enrolling, and returning results for genomic research, and existing clinical relationships with study participants may impact these research interactions. We present a qualitative study using semi-structured interviews of participants enrolled in a genome sequencing/exome sequencing (GS/ES) study at the same institution where they receive clinical care. Interviews were coded for motivations to participate and expectations of this research. The interviews revealed common motivations for participation, including altruism and hope for benefit for themselves, family members, and/or others with their condition. Additionally, themes emerged related to unintentional influence based on trust of the clinical provider that recruited them to the study. Participant trust in the enrolling provider at times appeared to extend to the study team to decide which research results to return and to do so in an appropriate format. Participants also based expectations for research results return on previous clinical genetic testing experiences, which may or may not be realistic depending on study design. It is imperative that genetic counselors enrolling patients into research studies be aware of the potential influence of their clinical relationship on potential subjects, be transparent about their role on the study team, and help set expectations about the study process, including results return.

Whole-exome sequencing in the molecular diagnosis of individuals with congenital anomalies of the kidney and urinary tract and identification of a new causative gene.

PURPOSE: To investigate the utility of whole-exome sequencing (WES) to define a molecular diagnosis for patients clinically diagnosed with congenital anomalies of kidney and urinary tract (CAKUT). METHODS: WES was performed in 62 families with CAKUT. WES data were analyzed for single-nucleotide variants (SNVs) in 35 known CAKUT genes, putatively deleterious sequence changes in new candidate genes, and potentially disease-associated copy-number variants (CNVs). RESULTS: In approximately 5% of families, pathogenic SNVs were identified in PAX2, HNF1B, and EYA1. Observed phenotypes in these families expand the current understanding about the role of these genes in CAKUT. Four pathogenic CNVs were also identified using two CNV detection tools. In addition, we found one deleterious de novo SNV in FOXP1 among the 62 families with CAKUT. The clinical database of the Baylor Miraca Genetics laboratory was queried and seven additional unrelated individuals with novel de novo SNVs in FOXP1 were identified. Six of these eight individuals with FOXP1 SNVs have syndromic urinary tract defects, implicating this gene in urinary tract development. CONCLUSION: We conclude that WES can be used to identify molecular etiology (SNVs, CNVs) in a subset of individuals with CAKUT. WES can also help identify novel CAKUT genes.Genet Med 19 4, 412-420.

KIF5A mutations cause an infantile onset phenotype including severe myoclonus with evidence of mitochondrial dysfunction.

Missense mutations in kinesin family member 5A (KIF5A) cause spastic paraplegia 10. We report on 2 patients with de novo stop-loss frameshift variants in KIF5A resulting in a novel phenotype that includes severe infantile onset myoclonus, hypotonia, optic nerve abnormalities, dysphagia, apnea, and early developmental arrest. We propose that alteration and elongation of the carboxy-terminus of the protein has a dominant-negative effect, causing mitochondrial dysfunction in the setting of an abnormal kinesin "motor." These results highlight the role of expanded testing and whole-exome sequencing in critically ill infants and emphasize the importance of accurate test interpretation. Ann Neurol 2016;80:633-637.

De novo mutations in CSNK2A1 are associated with neurodevelopmental abnormalities and dysmorphic features.

Whole exome sequencing (WES) can be used to efficiently identify de novo genetic variants associated with genetically heterogeneous conditions including intellectual disabilities. We have performed WES for 4102 (1847 female; 2255 male) intellectual disability/developmental delay cases and we report five patients with a neurodevelopmental disorder associated with developmental delay, intellectual disability, behavioral problems, hypotonia, speech problems, microcephaly, pachygyria and dysmorphic features in whom we have identified de novo missense and canonical splice site mutations in CSNK2A1, the gene encoding CK2α, the catalytic subunit of protein kinase CK2, a ubiquitous serine/threonine kinase composed of two regulatory (ß) and two catalytic (α and/or α') subunits. Somatic mutations in CSNK2A1 have been implicated in various cancers; however, this is the first study to describe a human condition associated with germline mutations in any of the CK2 subunits.

Familial TAB2 microdeletion and congenital heart defects including unusual valve dysplasia and tetralogy of fallot.

Haploinsufficiency of TAB2 was recently implicated as a cause for a variety of congenital heart defects. Reported cases have genomic deletions of 2-10 Mbs including TAB2 at 6q24-25 are almost always de novo and show variable cardiac and extra cardiac phenotype. We report on an inherited, 281 kb deletion in a three generation family. This is the smallest reported deletion involving TAB2 that segregates with congenital heart defects. Three affected individuals in this family present with myxomatous cardiac valves in addition to structural heart defects commonly associated with TAB2 deletions. Findings from this family support a key role of TAB2 haploinsufficiency in congenital heart defects and expand the phenotypic spectrum of TAB2-microdeletion syndrome.

The impact of chromosomal microarray on clinical management: a retrospective analysis.

PURPOSE: Chromosomal microarray has been widely adopted as the first-tier clinical test for individuals with multiple congenital anomalies, developmental delay, intellectual disability, and autism spectrum disorders. Although chromosomal microarray has been extensively shown to provide a higher diagnostic yield than conventional cytogenetic methods, some health insurers refuse to provide coverage for this test, claiming that it is experimental and does not affect patients' clinical management. METHODS: We retrospectively reviewed the electronic medical records of all patients who had abnormal chromosomal microarray findings reported by our laboratory over a 3-year period and quantified the management recommendations made in response to these results. RESULTS: Abnormal chromosomal microarray findings were reported for 12.7% of patients (227/1,780). For patients with clinical follow-up notes available, these results had management implications for 54.5% of patients in the entire abnormal cohort (102/187) and for 42.1% of patients referred for isolated neurodevelopmental disorders (16/38). Recommendations included pharmacological treatment, cancer-related screening or exclusion of screening, contraindications, and referrals for further evaluation. CONCLUSION: These results empirically demonstrate the clinical utility of chromosomal microarray by providing evidence that management was directly affected for the majority of patients in our cohort with abnormal chromosomal microarray findings.

Severe infantile epileptic encephalopathy due to mutations in PLCB1: expansion of the genotypic and phenotypic disease spectrum.

Homozygous deletions of chromosome 20p12.3, disrupting the promoter region and first three coding exons of the phospholipase C ß1 gene (PLCB1), have previously been described in two reports of early infantile epileptic encephalopathy (EIEE). Both children were born to consanguineous parents, one presented with infantile spasms, the other with migrating partial seizures of infancy. We describe an infant presenting with severe intractable epilepsy (without a specific EIEE electroclinical syndrome diagnosis) and neurodevelopmental delay associated with compound heterozygous mutations in PLCB1. A case note review and molecular genetic investigations were performed for a child, approximately 10 months of age, admitted to Johns Hopkins University Hospital for developmental delay and new-onset seizures. The patient presented at 6 months of age with developmental delay, followed by the onset of intractable, focal, and generalized seizures associated with developmental regression from 10 months of age. Presently, at 2 years of age, the child has severe motor and cognitive delays. Diagnostic microarray revealed a heterozygous 476kb deletion of 20p12.3 (encompassing PLCB1), which was also detected in the mother. The genomic breakpoints for the heterozygous deletion were determined. In order to investigate the presence of a second PLCB1 mutation, direct Sanger sequencing of the coding region and flanking intronic regions was undertaken, revealing a novel heterozygous intron 1 splice site variant (c.99+1G>A) in both the index individual and the father. Advances in molecular genetic testing have greatly improved diagnostic rates in EIEE, and this report further confirms the important role of microarray investigation in this group of disorders. PLCB1-EIEE is now reported in a number of different EIEE phenotypes and our report provides further evidence for phenotypic pleiotropy encountered in early infantile epilepsy syndromes.

Three cases of isolated terminal deletion of chromosome 8p without heart defects presenting with a mild phenotype.

Individuals with isolated terminal deletions of 8p have been well described in the literature, however, molecular characterization, particularly by microarray, of the deletion in most instances is lacking. The phenotype of such individuals falls primarily into two categories: those with cardiac defects, and those without. The architecture of 8p has been demonstrated to contain two inversely oriented segmental duplications at 8p23.1, flanking the gene, GATA4. Haploinsufficiency of this gene has been implicated in cardiac defects seen in numerous individuals with terminal 8p deletion. Current microarray technologies allow for the precise elucidation of the size and gene content of the deleted region. We present three individuals with isolated terminal deletion of 8p distal to the segmental duplication telomeric to GATA4. These individuals present with a relatively mild and nonspecific phenotype including mildly dysmorphic features, developmental delay, speech delay, and early behavior issues.

A rasopathy phenotype with severe congenital hypertrophic obstructive cardiomyopathy associated with a PTPN11 mutation and a novel variant in SOS1.

The RAS-MAPK pathway is critical for human growth and development. Abnormalities at different steps of this signaling cascade result in neuro-cardio-facial-cutaneous syndromes, or the RASopathies, a group of disorders with overlapping yet distinct phenotypes. RASopathy patients have variable degrees of intellectual disability, poor growth, relative macrocephaly, ectodermal abnormalities, dysmorphic features, and increased risk for certain malignancies. Congenital heart disease, particularly hypertrophic cardiomyopathy (HCM) and pulmonic stenosis, are prominent features in these disorders. Significant locus heterogeneity exists for many of the RASopathies. Traditionally, these diseases were thought to be inherited in an autosomal dominant manner. However, recently patients with defects in two components of this pathway and overlapping features of various forms of Noonan syndrome and neurofibromatosis 1 and have been reported. Here we present a patient with severe, progressive neonatal HCM, elevated urinary catecholamine metabolites, and dysmorphic features in whom we identified a known LEOPARD syndrome-associated PTPN11 mutation (c.1403 C > T; p.T468M) and a novel, potentially pathogenic missense SOS1 variant (c.1018 C > T; p.P340S) replacing a rigid nonpolar imino acid with a polar amino acid at a highly conserved position. We describe detailed clinical manifestations, cardiac histopathology, and the molecular genetic findings. Oligogenic models of inheritance with potential synergistic effects should be considered in the RASopathies.

Exploring Genes and Phenotypes Within Chromosomal Regions Using OMIM's GeneScout.

Structural variation in genomes, such as copy number variants (CNVs), is under scrutiny for its contribution to phenotypic expression and evolution. Regions of homozygosity (ROH) are ripe for phenotype-gene discovery. Determining the genes and related phenotypes within genomic regions is key to studying potential functional and phenotypic consequences. Because individuals have multiple CNVs and ROHs in their genome, identifying genomic regions that are phenotypically significant is challenging. GeneScout is a web-based tool that can be used to search genomic regions to display and filter the genes and their associated phenotypes within regions of interest. Phenotypes and their associated gene(s) can then be filtered to show only the genes with phenotypes that have a particular inheritance pattern and/or specific clinical feature(s). Phenotypes can then be selected to compare the clinical synopses side-by-side in Online Mendelian Inheritance in Man (OMIM® ). Additionally, two coordinate sets can be compared to determine either the regions of overlap or the unique regions (subtraction). The resulting coordinate ranges are displayed on the results page, and the results table displays only the genes and phenotypes present within the coordinate ranges. The interactive table includes gene-specific links to external resources such as ClinVar, ClinGen validity, ClinGen dosage, and gnomAD, and a diamond symbol next to the gene name indicates a gene that spans the start or end of a coordinate range. Searches and comparisons may be performed for coordinates in assemblies GRCh37 (hg19) and GRCh38 (hg38). The results page offers the option to liftover coordinates entered in GRCh37 to GRCh38 and updates the results table to display the gene content based on assembly GRCh38. The search coordinates and results table can be downloaded in a tab-delimited or Excel file. © 2022 Wiley Periodicals LLC. Basic Protocol: Searching GeneScout.

The Role of Genetic Testing in Pulmonary Fibrosis: A Perspective From the Pulmonary Fibrosis Foundation Genetic Testing Work Group.

Patients with familial pulmonary fibrosis represent a subset of patients with pulmonary fibrosis in whom inherited gene variation predisposes them to disease development. In the appropriate setting, genetic testing allows for personalized assessment of disease, recognition of clinically relevant extrapulmonary manifestations, and assessing susceptibility in unaffected relatives. However currently, the use of genetic testing is inconsistent, partly because of the lack of guidance regarding high-yield scenarios in which the results of genetic testing can inform clinical decision-making. To address this, the Pulmonary Fibrosis Foundation commissioned a genetic testing work group comprising pulmonologists, geneticists, and genetic counselors from the United States to provide guidance on genetic testing in patients with pulmonary fibrosis. This CHEST special feature presents a concise review of these proceedings and reviews pulmonary fibrosis susceptibility, clinically available genetic testing methods, and clinical scenarios in which genetic testing should be considered.

Haploinsufficiency of KMT2D is sufficient to cause Kabuki syndrome and is compatible with life.

We present the first patient described with haploinsufficency of KMT2D leading to Kabuki syndrome. Deletion of KMT2D has been thought to be lethal, but here we describe a patient with KMT2D deletion and classical Kabuki syndrome phenotype.