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.

Probabilistic association of differentially expressed genes with cis-regulatory elements.

Differential gene expression in response to perturbations is mediated at least in part by changes in binding of transcription factors (TFs) and other proteins at specific genomic regions. Association of these cis-regulatory elements (CREs) with their target genes is a challenging task that is essential to address many biological and mechanistic questions. Many current approaches rely on chromatin conformation capture techniques or single-cell correlational methods to establish CRE-to-gene associations. These methods can be effective but have limitations, including resolution, gaps in detectable association distances, and cost. As an alternative, we have developed DegCre, a nonparametric method that evaluates correlations between measurements of perturbation-induced differential gene expression and differential regulatory signal at CREs to score possible CRE-to-gene associations. It has several unique features, including the ability to use any type of CRE activity measurement, yield probabilistic scores for CRE-to-gene pairs, and assess CRE-to-gene pairings across a wide range of sequence distances. We apply DegCre to six data sets, each using different perturbations and containing a variety of regulatory signal measurements, including chromatin openness, histone modifications, and TF occupancy. To test their efficacy, we compare DegCre associations to Hi-C loop calls and CRISPR-validated CRE-to-gene associations, establishing good performance by DegCre that is comparable or superior to competing methods. DegCre is a novel approach to the association of CREs to genes from a perturbation-differential perspective, with strengths that are complementary to existing approaches and allow for new insights into gene regulation.

Combined Bioinformatic and Splicing Analysis of Likely Benign Intronic and Synonymous Variants Reveals Evidence for Pathogenicity.

Background: Current clinical variant analysis pipelines focus on coding variants and intronic variants within 10-20 bases of an exon-intron boundary that may affect splicing. The impact of newer splicing prediction algorithms combined with in vitro splicing assays on rare variants currently considered Benign/Likely Benign (B/LB) is unknown. Methods: Exome sequencing data from 576 pediatric cancer patients enrolled in the Texas KidsCanSeq study were filtered for intronic or synonymous variants absent from population databases, predicted to alter splicing via SpliceAI (>0.20), and scored as potentially deleterious by CADD (>10.0). Total cellular RNA was extracted from monocytes and RT-PCR products analyzed. Subsequently, rare synonymous or intronic B/LB variants in a subset of genes submitted to ClinVar were similarly evaluated. Variants predicted to lead to a frameshifted splicing product were functionally assessed using an in vitro splicing reporter assay in HEK-293T cells. Results: KidsCanSeq exome data analysis revealed a rare, heterozygous, intronic variant (NM_177438.3(DICER1):c.574-26A>G) predicted by SpliceAI to result in gain of a secondary splice acceptor site. The proband had a personal and family history of pleuropulmonary blastoma consistent with DICER1 syndrome but negative clinical sequencing reports. Proband RNA analysis revealed alternative DICER1 transcripts including the SpliceAI-predicted transcript.Similar bioinformatic analysis of synonymous or intronic B/LB variants (n=31,715) in ClinVar from 61 Mendelian disease genes yielded 18 variants, none of which could be scored by MaxEntScan. Eight of these variants were assessed (DICER1 n=4, CDH1 n=2, PALB2 n=2) using in vitro splice reporter assay and demonstrated abnormal splice products (mean 66%; range 6% to 100%). Available phenotypic information from submitting laboratories demonstrated DICER1 phenotypes in 2 families (1 variant) and breast cancer phenotypes for PALB2 in 3 families (2 variants). Conclusions: Our results demonstrate the power of newer predictive splicing algorithms to highlight rare variants previously considered B/LB in patients with features of hereditary conditions. Incorporation of SpliceAI annotation of existing variant data combined with either direct RNA analysis or in vitro assays has the potential to identify disease-associated variants in patients without a molecular diagnosis.

Allele biased transcription factor binding across human brain regions gives mechanistic insight into eQTLs.

Transcription Factors (TFs) influence gene expression by facilitating or disrupting the formation of transcription initiation machinery at particular genomic loci. Because genomic localization of TFs is in part driven by TF recognition of DNA sequence, variation in TF binding sites can disrupt TF-DNA associations and affect gene regulation. To identify variants that impact TF binding in human brain tissues, we quantified allele bias for 93 TFs analyzed with ChIP-seq experiments of multiple structural brain regions from two donors. Using graph genomes constructed from phased genomic sequence data, we compared ChIP-seq signal between alleles at heterozygous variants within each tissue sample from each donor. Comparison of results from different brain regions within donors and the same regions between donors provided measures of allele bias reproducibility. We identified thousands of DNA variants that show reproducible bias in ChIP-seq for at least one TF. We found that alleles that are rarer in the general population were more likely than common alleles to exhibit large biases, and more frequently led to reduced TF binding. Combining ChIP-seq with RNA-seq, we identified TF-allele interaction biases with RNA bias in a phased allele linked to 6,709 eQTL variants identified in GTEx data, 3,309 of which were found in neural contexts. Our results provide insights into the effects of both common and rare variation on gene regulation in the brain. These findings can facilitate mechanistic understanding of cis-regulatory variation associated with biological traits, including disease.

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.

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.

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 around 50%, suggesting some clinically relevant rare variants may be missed by standard analyses. Here we analyze "poison exons" (PEs) which, while often absent from standard gene annotations, are alternative exons whose inclusion results in a premature termination codon. Variants that alter PE inclusion can lead to loss-of-function and may be highly penetrant contributors to disease. Methods: We curated published RNA-seq data from developing mouse cortex to define 1,937 PE regions conserved between humans and mice and potentially relevant to NDDs. We then analyzed variants found by genome sequencing in multiple NDD cohorts. Results: Across 2,999 probands, we found six clinically relevant variants in PE regions that were previously overlooked. Five of these variants are in genes that are part of the sodium voltage-gated channel alpha subunit family ( SCN1A, SCN2A , and SCN8A ), 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 were observed in probands with features consistent with those reported for the associated gene. Conclusion: With only a minimal increase in variant analysis burden (most probands had zero or one candidate PE variants in a known NDD gene, with an average of 0.77 per proband), annotation of PEs can improve diagnostic yield for NDDs and likely other congenital conditions.

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.

Short-Term Administration of HIV Protease Inhibitor Saquinavir Improves Skull Bone Healing with Enhanced Osteoclastogenesis.

BACKGROUND: Using immunomodulatory methods to address the challenging issue of craniofacial bone repair may be a potentially effective approach. The protease inhibitor saquinavir has been shown to inhibit the inflammatory response by targeting the toll-like receptor 4/myeloid differentiation primary response complex. Independently, inhibition of toll-like receptor 4 or myeloid differentiation primary response led to enhanced skull bone repair. Therefore, the authors aimed to investigate the effects of saquinavir on skull bone healing. METHODS: The effects of saquinavir on skull bone healing were assessed by means of gene expression, histology, immunohistochemistry, and tomography in a mouse calvarial defect model. Subsequently, the role of saquinavir in cell viability, migration, and osteogenic and osteoclastogenic differentiation was also evaluated in vitro. RESULTS: One-week saquinavir administration improved skull bone healing based on micro-computed tomographic and histomorphometric analyses. Compared to the vehicle control, 1-week saquinavir treatment (1) enhanced osteoclast infiltration (tartrate-resistant acid phosphatase staining) at day 7, but not at days 14 and 28; (2) induced more CD206 + M2 macrophage infiltration, but not F4/80 + M0 macrophages at days 7, 14, and 28; and (3) elevated osteoclastogenic gene RANKL (quantitative polymerase chain reaction) expression and other osteogenic and cytokine expression. Furthermore, in vitro data showed that saquinavir administration did not influence MC3T3-E1 cell migration or mineralization, whereas higher concentrations of saquinavir inhibited cell viability. Saquinavir treatment also enhanced the osteoclastic differentiation of bone marrow-derived precursors, and partially reversed high-mobility group box 1-driven osteoclastogenesis inhibition and elevated proinflammatory cytokine expression. CONCLUSION: The improved skull bone repair following short-term saquinavir treatment may involve enhanced osteoclastogenesis and modulated inflammatory response following skull injury. CLINICAL RELEVANCE STATEMENT: The authors' work demonstrates improved skull bone healing by short-term application of saquinavir, a drug traditionally used in the treatment of acquired immunodeficiency syndrome. As such, saquinavir may be repurposed for skeletal repair.

Genome sequencing as a first-line diagnostic test for hospitalized infants.

PURPOSE: SouthSeq is a translational research study that undertook genome sequencing (GS) for infants with symptoms suggestive of a genetic disorder. Recruitment targeted racial/ethnic minorities and rural, medically underserved areas in the Southeastern United States, which are historically underrepresented in genomic medicine research. METHODS: GS and analysis were performed for 367 infants to detect disease-causal variation concurrent with standard of care evaluation and testing. RESULTS: Definitive diagnostic (DD) or likely diagnostic (LD) genetic findings were identified in 30% of infants, and 14% of infants harbored an uncertain result. Only 43% of DD/LD findings were identified via concurrent clinical genetic testing, suggesting that GS testing is better for obtaining early genetic diagnosis. We also identified phenotypes that correlate with the likelihood of receiving a DD/LD finding, such as craniofacial, ophthalmologic, auditory, skin, and hair abnormalities. We did not observe any differences in diagnostic rates between racial/ethnic groups. CONCLUSION: We describe one of the largest-to-date GS cohorts of ill infants, enriched for African American and rural patients. Our results show the utility of GS because it provides early-in-life detection of clinically relevant genetic variations not detected by current clinical genetic testing, particularly for infants exhibiting certain phenotypic features.

Long-read genome sequencing for the molecular diagnosis of neurodevelopmental disorders.

Exome and genome sequencing have proven to be effective tools for the diagnosis of neurodevelopmental disorders (NDDs), but large fractions of NDDs cannot be attributed to currently detectable genetic variation. This is likely, at least in part, a result of the fact that many genetic variants are difficult or impossible to detect through typical short-read sequencing approaches. Here, we describe a genomic analysis using Pacific Biosciences circular consensus sequencing (CCS) reads, which are both long (>10 kb) and accurate (>99% bp accuracy). We used CCS on six proband-parent trios with NDDs that were unexplained despite extensive testing, including genome sequencing with short reads. We identified variants and created de novo assemblies in each trio, with global metrics indicating these datasets are more accurate and comprehensive than those provided by short-read data. In one proband, we identified a likely pathogenic (LP), de novo L1-mediated insertion in CDKL5 that results in duplication of exon 3, leading to a frameshift. In a second proband, we identified multiple large de novo structural variants, including insertion-translocations affecting DGKB and MLLT3, which we show disrupt MLLT3 transcript levels. We consider this extensive structural variation likely pathogenic. The breadth and quality of variant detection, coupled to finding variants of clinical and research interest in two of six probands with unexplained NDDs, support the hypothesis that long-read genome sequencing can substantially improve rare disease genetic discovery rates.

Variants in the degron of AFF3 are associated with intellectual disability, mesomelic dysplasia, horseshoe kidney, and epileptic encephalopathy.

The ALF transcription factor paralogs, AFF1, AFF2, AFF3, and AFF4, are components of the transcriptional super elongation complex that regulates expression of genes involved in neurogenesis and development. We describe an autosomal dominant disorder associated with de novo missense variants in the degron of AFF3, a nine amino acid sequence important for its binding to ubiquitin ligase, or with de novo deletions of this region. The sixteen affected individuals we identified, along with two previously reported individuals, present with a recognizable pattern of anomalies, which we named KINSSHIP syndrome (KI for horseshoe kidney, NS for Nievergelt/Savarirayan type of mesomelic dysplasia, S for seizures, H for hypertrichosis, I for intellectual disability, and P for pulmonary involvement), partially overlapping the AFF4-associated CHOPS syndrome. Whereas homozygous Aff3 knockout mice display skeletal anomalies, kidney defects, brain malformations, and neurological anomalies, knockin animals modeling one of the microdeletions and the most common of the missense variants identified in affected individuals presented with lower mesomelic limb deformities like KINSSHIP-affected individuals and early lethality, respectively. Overexpression of AFF3 in zebrafish resulted in body axis anomalies, providing some support for the pathological effect of increased amount of AFF3. The only partial phenotypic overlap of AFF3- and AFF4-associated syndromes and the previously published transcriptome analyses of ALF transcription factors suggest that these factors are not redundant and each contributes uniquely to proper development.

Genome-wide strand asymmetry in massively parallel reporter activity favors genic strands.

Massively parallel reporter assays (MPRAs) are useful tools to characterize regulatory elements in human genomes. An aspect of MPRAs that is not typically the focus of analysis is their intrinsic ability to differentiate activity levels for a given sequence element when placed in both of its possible orientations relative to the reporter construct. Here, we describe pervasive strand asymmetry of MPRA signals in data sets from multiple reporter configurations in both published and newly reported data. These effects are reproducible across different cell types and in different treatments within a cell type and are observed both within and outside of annotated regulatory elements. From elements in gene bodies, MPRA strand asymmetry favors the sense strand, suggesting that function related to endogenous transcription is driving the phenomenon. Similarly, we find that within Alu mobile element insertions, strand asymmetry favors the transcribed strand of the ancestral retrotransposon. The effect is consistent across the multiplicity of Alu elements in human genomes and is more pronounced in less diverged Alu elements. We find sequence features driving MPRA strand asymmetry and show its prediction from sequence alone. We see some evidence for RNA stabilization and transcriptional activation mechanisms and hypothesize that the effect is driven by natural selection favoring efficient transcription. Our results indicate that strand asymmetry is a pervasive and reproducible feature in MPRA data. More importantly, the fact that MPRA asymmetry favors naturally transcribed strands suggests that it stems from preserved biological functions that have a substantial, global impact on gene and genome evolution.

Aberrant regulation of a poison exon caused by a non-coding variant in a mouse model of Scn1a-associated epileptic encephalopathy.

Dravet syndrome (DS) is a developmental and epileptic encephalopathy that results from mutations in the Nav1.1 sodium channel encoded by SCN1A. Most known DS-causing mutations are in coding regions of SCN1A, but we recently identified several disease-associated SCN1A mutations in intron 20 that are within or near to a cryptic and evolutionarily conserved "poison" exon, 20N, whose inclusion is predicted to lead to transcript degradation. However, it is not clear how these intron 20 variants alter SCN1A expression or DS pathophysiology in an organismal context, nor is it clear how exon 20N is regulated in a tissue-specific and developmental context. We address those questions here by generating an animal model of our index case, NM_006920.4(SCN1A):c.3969+2451G>C, using gene editing to create the orthologous mutation in laboratory mice. Scn1a heterozygous knock-in (+/KI) mice exhibited an ~50% reduction in brain Scn1a mRNA and Nav1.1 protein levels, together with characteristics observed in other DS mouse models, including premature mortality, seizures, and hyperactivity. In brain tissue from adult Scn1a +/+ animals, quantitative RT-PCR assays indicated that ~1% of Scn1a mRNA included exon 20N, while brain tissue from Scn1a +/KI mice exhibited an ~5-fold increase in the extent of exon 20N inclusion. We investigated the extent of exon 20N inclusion in brain during normal fetal development in RNA-seq data and discovered that levels of inclusion were ~70% at E14.5, declining progressively to ~10% postnatally. A similar pattern exists for the homologous sodium channel Nav1.6, encoded by Scn8a. For both genes, there is an inverse relationship between the level of functional transcript and the extent of poison exon inclusion. Taken together, our findings suggest that poison exon usage by Scn1a and Scn8a is a strategy to regulate channel expression during normal brain development, and that mutations recapitulating a fetal-like pattern of splicing cause reduced channel expression and epileptic encephalopathy.

Identifying rare, medically relevant variation via population-based genomic screening in Alabama: opportunities and pitfalls.

PURPOSE: To evaluate the effectiveness and specificity of population-based genomic screening in Alabama. METHODS: The Alabama Genomic Health Initiative (AGHI) has enrolled and evaluated 5369 participants for the presence of pathogenic/likely pathogenic (P/LP) variants using the Illumina Global Screening Array (GSA), with validation of all P/LP variants via Sanger sequencing in a CLIA-certified laboratory before return of results. RESULTS: Among 131 variants identified by the GSA that were evaluated by Sanger sequencing, 67 (51%) were false positives (FP). For 39 of the 67 FP variants, a benign/likely benign variant was present at or near the targeted P/LP variant. Variants detected within African American individuals were significantly enriched for FPs, likely due to a higher rate of nontargeted alternative alleles close to array-targeted P/LP variants. CONCLUSION: In AGHI, we have implemented an array-based process to screen for highly penetrant genetic variants in actionable disease genes. We demonstrate the need for clinical validation of array-identified variants in direct-to-consumer or population testing, especially for diverse populations.

A state-based approach to genomics for rare disease and population screening.

PURPOSE: The Alabama Genomic Health Initiative (AGHI) is a state-funded effort to provide genomic testing. AGHI engages two distinct cohorts across the state of Alabama. One cohort includes children and adults with undiagnosed rare disease; a second includes an unselected adult population. Here we describe findings from the first 176 rare disease and 5369 population cohort AGHI participants. METHODS: AGHI participants enroll in one of two arms of a research protocol that provides access to genomic testing results and biobank participation. Rare disease cohort participants receive genome sequencing to identify primary and secondary findings. Population cohort participants receive genotyping to identify pathogenic and likely pathogenic variants for actionable conditions. RESULTS: Within the rare disease cohort, genome sequencing identified likely pathogenic or pathogenic variation in 20% of affected individuals. Within the population cohort, 1.5% of individuals received a positive genotyping result. The rate of genotyping results corroborated by reported personal or family history varied by gene. CONCLUSIONS: AGHI demonstrates the ability to provide useful health information in two contexts: rare undiagnosed disease and population screening. This utility should motivate continued exploration of ways in which emerging genomic technologies might benefit broad populations.

Defining the phenotype of FHF1 developmental and epileptic encephalopathy.

Fibroblast growth-factor homologous factor (FHF1) gene variants have recently been associated with developmental and epileptic encephalopathy (DEE). FHF1 encodes a cytosolic protein that modulates neuronal sodium channel gating. We aim to refine the electroclinical phenotypic spectrum of patients with pathogenic FHF1 variants. We retrospectively collected clinical, genetic, neurophysiologic, and neuroimaging data of 17 patients with FHF1-DEE. Sixteen patients had recurrent heterozygous FHF1 missense variants: 14 had the recurrent p.Arg114His variant and two had a novel likely pathogenic variant p.Gly112Ser. The p.Arg114His variant is associated with an earlier onset and more severe phenotype. One patient carried a chromosomal microduplication involving FHF1. Twelve patients carried a de novo variant, five (29.5%) inherited from parents with gonadic or somatic mosaicism. Seizure onset was between 1 day and 41 months; in 76.5% it was within 30 days. Tonic seizures were the most frequent seizure type. Twelve patients (70.6%) had drug-resistant epilepsy, 14 (82.3%) intellectual disability, and 11 (64.7%) behavioral disturbances. Brain magnetic resonance imaging (MRI) showed mild cerebral and/or cerebellar atrophy in nine patients (52.9%). Overall, our findings expand and refine the clinical, EEG, and imaging phenotype of patients with FHF1-DEE, which is characterized by early onset epilepsy with tonic seizures, associated with moderate to severe ID and psychiatric features.

A second cohort of CHD3 patients expands the molecular mechanisms known to cause Snijders Blok-Campeau syndrome.

There has been one previous report of a cohort of patients with variants in Chromodomain Helicase DNA-binding 3 (CHD3), now recognized as Snijders Blok-Campeau syndrome. However, with only three previously-reported patients with variants outside the ATPase/helicase domain, it was unclear if variants outside of this domain caused a clinically similar phenotype. We have analyzed 24 new patients with CHD3 variants, including nine outside the ATPase/helicase domain. All patients were detected with unbiased molecular genetic methods. There is not a significant difference in the clinical or facial features of patients with variants in or outside this domain. These additional patients further expand the clinical and molecular data associated with CHD3 variants. Importantly we conclude that there is not a significant difference in the phenotypic features of patients with various molecular disruptions, including whole gene deletions and duplications, and missense variants outside the ATPase/helicase domain. This data will aid both clinical geneticists and molecular geneticists in the diagnosis of this emerging syndrome.

Non-coding and Loss-of-Function Coding Variants in TET2 are Associated with Multiple Neurodegenerative Diseases.

We conducted genome sequencing to search for rare variation contributing to early-onset Alzheimer's disease (EOAD) and frontotemporal dementia (FTD). Discovery analysis was conducted on 435 cases and 671 controls of European ancestry. Burden testing for rare variation associated with disease was conducted using filters based on variant rarity (less than one in 10,000 or private), computational prediction of deleteriousness (CADD) (10 or 15 thresholds), and molecular function (protein loss-of-function [LoF] only, coding alteration only, or coding plus non-coding variants in experimentally predicted regulatory regions). Replication analysis was conducted on 16,434 independent cases and 15,587 independent controls. Rare variants in TET2 were enriched in the discovery combined EOAD and FTD cohort (p = 4.6 × 10-8, genome-wide corrected p = 0.0026). Most of these variants were canonical LoF or non-coding in predicted regulatory regions. This enrichment replicated across several cohorts of Alzheimer's disease (AD) and FTD (replication only p = 0.0029). The combined analysis odds ratio was 2.3 (95% confidence interval [CI] 1.6-3.4) for AD and FTD. The odds ratio for qualifying non-coding variants considered independently from coding variants was 3.7 (95% CI 1.7-9.4). For LoF variants, the combined odds ratio (for AD, FTD, and amyotrophic lateral sclerosis, which shares clinicopathological overlap with FTD) was 3.1 (95% CI 1.9-5.2). TET2 catalyzes DNA demethylation. Given well-defined changes in DNA methylation that occur during aging, rare variation in TET2 may confer risk for neurodegeneration by altering the homeostasis of key aging-related processes. Additionally, our study emphasizes the relevance of non-coding variation in genetic studies of complex disease.