The evolution of lineage-specific regulatory activities in the human embryonic limb.

The evolution of human anatomical features likely involved changes in gene regulation during development. However, the nature and extent of human-specific developmental regulatory functions remain unknown. We obtained a genome-wide view of cis-regulatory evolution in human embryonic tissues by comparing the histone modification H3K27ac, which provides a quantitative readout of promoter and enhancer activity, during human, rhesus, and mouse limb development. Based on increased H3K27ac, we find that 13% of promoters and 11% of enhancers have gained activity on the human lineage since the human-rhesus divergence. These gains largely arose by modification of ancestral regulatory activities in the limb or potential co-option from other tissues and are likely to have heterogeneous genetic causes. Most enhancers that exhibit gain of activity in humans originated in mammals. Gains at promoters and enhancers in the human limb are associated with increased gene expression, suggesting they include molecular drivers of human morphological evolution.

Coexpression networks implicate human midfetal deep cortical projection neurons in the pathogenesis of autism.

Autism spectrum disorder (ASD) is a complex developmental syndrome of unknown etiology. Recent studies employing exome- and genome-wide sequencing have identified nine high-confidence ASD (hcASD) genes. Working from the hypothesis that ASD-associated mutations in these biologically pleiotropic genes will disrupt intersecting developmental processes to contribute to a common phenotype, we have attempted to identify time periods, brain regions, and cell types in which these genes converge. We have constructed coexpression networks based on the hcASD "seed" genes, leveraging a rich expression data set encompassing multiple human brain regions across human development and into adulthood. By assessing enrichment of an independent set of probable ASD (pASD) genes, derived from the same sequencing studies, we demonstrate a key point of convergence in midfetal layer 5/6 cortical projection neurons. This approach informs when, where, and in what cell types mutations in these specific genes may be productively studied to clarify ASD pathophysiology.

Mitochondrial stress engages E2F1 apoptotic signaling to cause deafness.

Mitochondrial dysfunction causes poorly understood tissue-specific pathology stemming from primary defects in respiration, coupled with altered reactive oxygen species (ROS), metabolic signaling, and apoptosis. The A1555G mtDNA mutation that causes maternally inherited deafness disrupts mitochondrial ribosome function, in part, via increased methylation of the mitochondrial 12S rRNA by the methyltransferase mtTFB1. In patient-derived A1555G cells, we show that 12S rRNA hypermethylation causes ROS-dependent activation of AMP kinase and the proapoptotic nuclear transcription factor E2F1. This retrograde mitochondrial-stress relay is operative in vivo, as transgenic-mtTFB1 mice exhibit enhanced 12S rRNA methylation in multiple tissues, increased E2F1 and apoptosis in the stria vascularis and spiral ganglion neurons of the inner ear, and progressive E2F1-dependent hearing loss. This mouse mitochondrial disease model provides a robust platform for deciphering the complex tissue specificity of human mitochondrial-based disorders, as well as the precise pathogenic mechanism of maternally inherited deafness and its exacerbation by environmental factors.

CRISPR Activation Reverses Haploinsufficiency and Functional Deficits Caused by TTN Truncation Variants.

BACKGROUND: TTN truncation variants (TTNtvs) are the most common genetic lesion identified in individuals with dilated cardiomyopathy, a disease with high morbidity and mortality rates. TTNtvs reduce normal TTN (titin) protein levels, produce truncated proteins, and impair sarcomere content and function. Therapeutics targeting TTNtvs have been elusive because of the immense size of TTN, the rarity of specific TTNtvs, and incomplete knowledge of TTNtv pathogenicity. METHODS: We adapted CRISPR activation using dCas9-VPR to functionally interrogate TTNtv pathogenicity and develop a therapeutic in human cardiomyocytes and 3-dimensional cardiac microtissues engineered from induced pluripotent stem cell models harboring a dilated cardiomyopathy-associated TTNtv. We performed guide RNA screening with custom TTN reporter assays, agarose gel electrophoresis to quantify TTN protein levels and isoforms, and RNA sequencing to identify molecular consequences of TTN activation. Cardiomyocyte epigenetic assays were also used to nominate DNA regulatory elements to enable cardiomyocyte-specific TTN activation. RESULTS: CRISPR activation of TTN using single guide RNAs targeting either the TTN promoter or regulatory elements in spatial proximity to the TTN promoter through 3-dimensional chromatin interactions rescued TTN protein deficits disturbed by TTNtvs. Increasing TTN protein levels normalized sarcomere content and contractile function despite increasing truncated TTN protein. In addition to TTN transcripts, CRISPR activation also increased levels of myofibril assembly-related and sarcomere-related transcripts. CONCLUSIONS: TTN CRISPR activation rescued TTNtv-related functional deficits despite increasing truncated TTN levels, which provides evidence to support haploinsufficiency as a relevant genetic mechanism underlying heterozygous TTNtvs. CRISPR activation could be developed as a therapeutic to treat a large proportion of TTNtvs.

Massively parallel discovery of human-specific substitutions that alter enhancer activity.

Genetic changes that altered the function of gene regulatory elements have been implicated in the evolution of human traits such as the expansion of the cerebral cortex. However, identifying the particular changes that modified regulatory activity during human evolution remain challenging. Here we used massively parallel enhancer assays in neural stem cells to quantify the functional impact of >32,000 human-specific substitutions in >4,300 human accelerated regions (HARs) and human gain enhancers (HGEs), which include enhancers with novel activities in humans. We found that >30% of active HARs and HGEs exhibited differential activity between human and chimpanzee. We isolated the effects of human-specific substitutions from background genetic variation to identify the effects of genetic changes most relevant to human evolution. We found that substitutions interacted in both additive and nonadditive ways to modify enhancer function. Substitutions within HARs, which are highly constrained compared to HGEs, showed smaller effects on enhancer activity, suggesting that the impact of human-specific substitutions is buffered in enhancers with constrained ancestral functions. Our findings yield insight into how human-specific genetic changes altered enhancer function and provide a rich set of candidates for studies of regulatory evolution in humans.

Rare variant modifier analysis identifies variants in SEC24D associated with orofacial cleft subtypes.

As one of the most common structural birth defects, orofacial clefts (OFCs) have been studied for decades, and recent studies have demonstrated that there are genetic differences between the different phenotypic presentations of OFCs. However, the contribution of rare genetic variation genome-wide to different subtypes of OFCs has been understudied, with most studies focusing on common genetic variation or rare variation within targeted regions of the genome. Therefore, we used whole-genome sequencing data from the Gabriella Miller Kids First Pediatric Research Program to conduct a gene-based burden analysis to test for genetic modifiers of cleft lip (CL) vs cleft lip and palate (CLP). We found that there was a significantly increased burden of rare variants in SEC24D in CL cases compared to CLP cases (p = 6.86 [Formula: see text] 10-7). Of the 15 variants within SEC24D, 53.3% were synonymous, but overlapped a known craniofacial enhancer. We then tested whether these variants could alter predicted transcription factor binding sites (TFBS), and found that the rare alleles destroyed binding sites for 9 transcription factors (TFs), including Pax1 (p = 0.0009), and created binding sites for 23 TFs, including Pax6 (p = 6.12 [Formula: see text] 10-5) and Pax9 (p = 0.0001), which are known to be involved in normal craniofacial development, suggesting a potential mechanism by which these synonymous variants could have a functional impact. Overall, this study indicates that rare genetic variation may contribute to the phenotypic heterogeneity of OFCs and suggests that regulatory variation may also contribute and warrant further investigation in future studies of genetic variants controlling risk to OFC.

An Irf6-Esrp1/2 regulatory axis controls midface morphogenesis in vertebrates.

Irf6 and Esrp1 are important for palate development across vertebrates. In zebrafish, we found that irf6 regulates the expression of esrp1 We detailed overlapping Irf6 and Esrp1/2 expression in mouse orofacial epithelium. In zebrafish, irf6 and esrp1/2 share expression in periderm, frontonasal ectoderm and oral epithelium. Genetic disruption of irf6 and esrp1/2 in zebrafish resulted in cleft of the anterior neurocranium. The esrp1/2 mutant also developed cleft of the mouth opening. Lineage tracing of cranial neural crest cells revealed that the cleft resulted not from migration defect, but from impaired chondrogenesis. Analysis of aberrant cells within the cleft revealed expression of sox10, col1a1 and irf6, and these cells were adjacent to krt4+ and krt5+ cells. Breeding of mouse Irf6; Esrp1; Esrp2 compound mutants suggested genetic interaction, as the triple homozygote and the Irf6; Esrp1 double homozygote were not observed. Further, Irf6 heterozygosity reduced Esrp1/2 cleft severity. These studies highlight the complementary analysis of Irf6 and Esrp1/2 in mouse and zebrafish, and identify a unique aberrant cell population in zebrafish expressing sox10, col1a1 and irf6 Future work characterizing this cell population will yield additional insight into cleft pathogenesis.

Specific ZNF274 binding interference at SNORD116 activates the maternal transcripts in Prader-Willi syndrome neurons.

Prader-Willi syndrome (PWS) is characterized by neonatal hypotonia, developmental delay and hyperphagia/obesity. This disorder is caused by the absence of paternally expressed gene products from chromosome 15q11-q13. We previously demonstrated that knocking out ZNF274, a Kruppel-associated box-A-domain zinc finger protein capable of recruiting epigenetic machinery to deposit the H3K9me3 repressive histone modification, can activate expression from the normally silent maternal allele of SNORD116 in neurons derived from PWS induced pluripotent stem cells (iPSCs). However, ZNF274 has many other targets in the genome in addition to SNORD116. Depleting ZNF274 will surely affect the expression of other important genes and disrupt other pathways. Here, we used CRISPR/Cas9 to delete ZNF274 binding sites at the SNORD116 locus to determine whether activation of the maternal copy of SNORD116 could be achieved without altering ZNF274 protein levels. We obtained similar activation of gene expression from the normally silenced maternal allele in neurons derived from PWS iPSCs, compared with ZNF274 knockout, demonstrating that ZNF274 is directly involved in the repression of SNORD116. These results suggest that interfering with ZNF274 binding at the maternal SNORD116 locus is a potential therapeutic strategy for PWS.

Epigenomic and Transcriptomic Dynamics During Human Heart Organogenesis.

RATIONALE: There is growing evidence that common variants and rare sequence alterations in regulatory sequences can result in birth defects or predisposition to disease. Congenital heart defects are the most common birth defect and have a clear genetic component, yet only a third of cases can be attributed to structural variation in the genome or a mutation in a gene. The remaining unknown cases could be caused by alterations in regulatory sequences. OBJECTIVE: Identify regulatory sequences and gene expression networks that are active during organogenesis of the human heart. Determine whether these sites and networks are enriched for disease-relevant genes and associated genetic variation. METHODS AND RESULTS: We characterized ChromHMM (chromatin state) and gene expression dynamics during human heart organogenesis. We profiled 7 histone modifications in embryonic hearts from each of 9 distinct Carnegie stages (13-14, 16-21, and 23), annotated chromatin states, and compared these maps to over 100 human tissues and cell types. We also generated RNA-sequencing data, performed differential expression, and constructed weighted gene coexpression networks. We identified 177 412 heart enhancers; 12 395 had not been previously annotated as strong enhancers. We identified 92% of all functionally validated heart-positive enhancers (n=281; 7.5× enrichment; P<2.2×10-16). Integration of these data demonstrated novel heart enhancers are enriched near genes expressed more strongly in cardiac tissue and are enriched for variants associated with ECG measures and atrial fibrillation. Our gene expression network analysis identified gene modules strongly enriched for heart-related functions, regulatory control by heart-specific enhancers, and putative disease genes. CONCLUSIONS: Well-connected hub genes with heart-specific expression targeted by embryonic heart-specific enhancers are likely disease candidates. Our functional annotations will allow for better interpretation of whole genome sequencing data in the large number of patients affected by congenital heart defects.

A bipartite boundary element restricts UBE3A imprinting to mature neurons.

Angelman syndrome (AS) is a severe neurodevelopmental disorder caused by the loss of function from the maternal allele of UBE3A, a gene encoding an E3 ubiquitin ligase. UBE3A is only expressed from the maternally inherited allele in mature human neurons due to tissue-specific genomic imprinting. Imprinted expression of UBE3A is restricted to neurons by expression of UBE3A antisense transcript (UBE3A-ATS) from the paternally inherited allele, which silences the paternal allele of UBE3A in cis However, the mechanism restricting UBE3A-ATS expression and UBE3A imprinting to neurons is not understood. We used CRISPR/Cas9-mediated genome editing to functionally define a bipartite boundary element critical for neuron-specific expression of UBE3A-ATS in humans. Removal of this element led to up-regulation of UBE3A-ATS without repressing paternal UBE3A However, increasing expression of UBE3A-ATS in the absence of the boundary element resulted in full repression of paternal UBE3A, demonstrating that UBE3A imprinting requires both the loss of function from the boundary element as well as the up-regulation of UBE3A-ATS These results suggest that manipulation of the competition between UBE3A-ATS and UBE3A may provide a potential therapeutic approach for AS.

The genomic landscape of cohesin-associated chromatin interactions.

Cohesin is implicated in establishing tissue-specific DNA loops that target enhancers to promoters, and also localizes to sites bound by the insulator protein CTCF, which blocks enhancer-promoter communication. However, cohesin-associated interactions have not been characterized on a genome-wide scale. Here we performed chromatin interaction analysis with paired-end tag sequencing (ChIA-PET) of the cohesin subunit SMC1A in developing mouse limb. We identified 2264 SMC1A interactions, of which 1491 (65%) involved sites co-occupied by CTCF. SMC1A participates in tissue-specific enhancer-promoter interactions and interactions that demarcate regions of correlated regulatory output. In contrast to previous studies, we also identified interactions between promoters and distal sites that are maintained in multiple tissues but are poised in embryonic stem cells and resolve to tissue-specific activated or repressed chromatin states in the mouse embryo. Our results reveal the diversity of cohesin-associated interactions in the genome and highlight their role in establishing the regulatory architecture of development.

Chromatin state signatures associated with tissue-specific gene expression and enhancer activity in the embryonic limb.

The regulatory elements that direct tissue-specific gene expression in the developing mammalian embryo remain largely unknown. Although chromatin profiling has proven to be a powerful method for mapping regulatory sequences in cultured cells, chromatin states characteristic of active developmental enhancers have not been directly identified in embryonic tissues. Here we use whole-transcriptome analysis coupled with genome-wide profiling of H3K27ac and H3K27me3 to map chromatin states and enhancers in mouse embryonic forelimb and hindlimb. We show that gene-expression differences between forelimb and hindlimb, and between limb and other embryonic cell types, are correlated with tissue-specific H3K27ac signatures at promoters and distal sites. Using H3K27ac profiles, we identified 28,377 putative enhancers, many of which are likely to be limb specific based on strong enrichment near genes highly expressed in the limb and comparisons with tissue-specific EP300 sites and known enhancers. We describe a chromatin state signature associated with active developmental enhancers, defined by high levels of H3K27ac marking, nucleosome displacement, hypersensitivity to sonication, and strong depletion of H3K27me3. We also find that some developmental enhancers exhibit components of this signature, including hypersensitivity, H3K27ac enrichment, and H3K27me3 depletion, at lower levels in tissues in which they are not active. Our results establish histone modification profiling as a tool for developmental enhancer discovery, and suggest that enhancers maintain an open chromatin state in multiple embryonic tissues independent of their activity level.

Mitochondrial ribosomal protein L12 selectively associates with human mitochondrial RNA polymerase to activate transcription.

Basal transcription of human mitochondrial DNA (mtDNA) in vitro requires the single-subunit, bacteriophage-related RNA polymerase, POLRMT, and transcription factor h-mtTFB2. This two-component system is activated differentially at mtDNA promoters by human mitochondrial transcription factor A (h-mtTFA). Mitochondrial ribosomal protein L7/L12 (MRPL12) binds directly to POLRMT, but whether it does so in the context of the ribosome or as a "free" protein in the matrix is unknown. Furthermore, existing evidence that MRPL12 activates mitochondrial transcription derives from overexpression studies in cultured cells and transcription experiments using crude mitochondrial lysates, precluding direct effects of MRPL12 on transcription to be assigned. Here, we report that depletion of MRPL12 from HeLa cells by shRNA results in decreased steady-state levels of mitochondrial transcripts, which are not accounted for by changes in RNA stability. We also show that a significant "free" pool of MRPL12 exists in human mitochondria not associated with ribosomes. "Free" MRPL12 binds selectively to POLRMT in vivo in a complex distinct from those containing h-mtTFB2. Finally, using a fully recombinant mitochondrial transcription system, we demonstrate that MRPL12 stimulates promoter-dependent and promoter-independent transcription directly in vitro. Based on these results, we propose that, when not associated with ribosomes, MRPL12 has a second function in transcription, perhaps acting to facilitate the transition from initiation to elongation. We speculate that this is one mechanism to coordinate mitochondrial ribosome biogenesis and transcription in human mitochondria, where transcription of rRNAs from the mtDNA presumably needs to be adjusted in accordance with the rate of import and assembly of the nucleus-encoded MRPs into ribosomes.

Transcriptional programs in transient embryonic zones of the cerebral cortex defined by high-resolution mRNA sequencing.

Characterizing the genetic programs that specify development and evolution of the cerebral cortex is a central challenge in neuroscience. Stem cells in the transient embryonic ventricular and subventricular zones generate neurons that migrate across the intermediate zone to the overlying cortical plate, where they differentiate and form the neocortex. It is clear that not one but a multitude of molecular pathways are necessary to progress through each cellular milestone, yet the underlying transcriptional programs remain unknown. Here, we apply differential transcriptome analysis on microscopically isolated cell populations, to define five transcriptional programs that represent each transient embryonic zone and the progression between these zones. The five transcriptional programs contain largely uncharacterized genes in addition to transcripts necessary for stem cell maintenance, neurogenesis, migration, and differentiation. Additionally, we found intergenic transcriptionally active regions that possibly encode unique zone-specific transcripts. Finally, we present a high-resolution transcriptome map of transient zones in the embryonic mouse forebrain.

Spatial Multiomics Reveal the Role of Wnt Modulator, Dkk2, in Palatogenesis.

Multiple genetic and environmental etiologies contribute to the pathogenesis of cleft palate, which constitutes the most common among the inherited disorders of the craniofacial complex. Insights into the molecular mechanisms regulating osteogenic differentiation and patterning in the palate during embryogenesis are limited and needed for the development of innovative diagnostics and cures. This study utilized the Pax9-/- mouse model with a consistent phenotype of cleft secondary palate to investigate the role of Pax9 in the process of palatal osteogenesis. While prior research had identified upregulation of Wnt pathway modulators Dkk1 and Dkk2 in Pax9-/- palate mesenchyme, limitations of spatial resolution and technology restricted a more robust analysis. Here, data from single-nucleus transcriptomics and chromatin accessibility assays validated by in situ highly multiplex targeted single-cell spatial profiling technology suggest a distinct relationship between Pax9+ and osteogenic populations. Loss of Pax9 results in spatially restricted osteogenic domains bounded by Dkk2, which normally interfaces with Pax9 in the mesenchyme. These results suggest that Pax9-dependent Wnt signaling modulators influence osteogenic programming during palate formation, potentially contributing to the observed cleft palate phenotype.

Massively parallel disruption of enhancers active in human neural stem cells.

Changes in gene regulation have been linked to the expansion of the human cerebral cortex and to neurodevelopmental disorders, potentially by altering neural progenitor proliferation. However, the effects of genetic variation within regulatory elements on neural progenitors remain obscure. We use sgRNA-Cas9 screens in human neural stem cells (hNSCs) to disrupt 10,674 genes and 26,385 conserved regions in 2,227 enhancers active in the developing human cortex and determine effects on proliferation. Genes with proliferation phenotypes are associated with neurodevelopmental disorders and show biased expression in specific fetal human brain neural progenitor populations. Although enhancer disruptions overall have weaker effects than gene disruptions, we identify enhancer disruptions that severely alter hNSC self-renewal. Disruptions in human accelerated regions, implicated in human brain evolution, also alter proliferation. Integrating proliferation phenotypes with chromatin interactions reveals regulatory relationships between enhancers and their target genes contributing to neurogenesis and potentially to human cortical evolution.

Ube3a unsilencer for the potential treatment of Angelman syndrome.

Deletion of the maternal UBE3A allele causes Angelman syndrome (AS); because paternal UBE3A is epigenetically silenced by a long non-coding antisense (UBE3A-ATS) in neurons, this nearly eliminates UBE3A protein in the brain. Reactivating paternal UBE3A holds promise for treating AS. We previously showed topoisomerase inhibitors can reactivate paternal UBE3A, but their therapeutic challenges prompted our search for small molecule unsilencers with a different mechanism of action. Here, we found that (S)-PHA533533 acts through a novel mechanism to significantly increase paternal Ube3a mRNA and UBE3A protein levels while downregulating Ube3a-ATS in primary neurons derived from AS model mice. Furthermore, peripheral delivery of (S)-PHA533533 in AS model mice induces widespread neuronal UBE3A expression. Finally, we show that (S)-PHA533533 unsilences paternal UBE3A in AS patient-derived neurons, highlighting its translational potential. Our findings provide a lead for developing a small molecule treatment for AS that could be safe, non-invasively delivered, and capable of brain-wide unsilencing of paternal UBE3A.

Rare Variants Analyses Suggest Novel Cleft Genes in the African Population.

Non-syndromic orofacial clefts (NSOFCs) are common birth defects with a complex etiology. While over 60 common risk loci have been identified, they explain only a small proportion of the heritability for NSOFC. Rare variants have been implicated in the missing heritability. Thus, our study aimed to identify genes enriched with nonsynonymous rare coding variants associated with NSOFCs. Our sample included 814 non-syndromic cleft lip with or without palate (NSCL/P), 205 non-syndromic cleft palate only (NSCPO), and 2150 unrelated control children from Nigeria, Ghana, and Ethiopia. We conducted a gene-based analysis separately for each phenotype using three rare-variants collapsing models: (1) protein-altering (PA), (2) missense variants only (MO); and (3) loss of function variants only (LOFO). Subsequently, we utilized relevant transcriptomics data to evaluate associated gene expression and examined their mutation constraint using the gnomeAD database. In total, 13 genes showed suggestive associations (p = E-04). Among them, eight genes (ABCB1, ALKBH8, CENPF, CSAD, EXPH5, PDZD8, SLC16A9, and TTC28) were consistently expressed in relevant mouse and human craniofacial tissues during the formation of the face, and three genes (ABCB1, TTC28, and PDZD8) showed statistically significant mutation constraint. These findings underscore the role of rare variants in identifying candidate genes for NSOFCs.

A distant global control region is essential for normal expression of anterior HOXA genes during mouse and human craniofacial development.

Craniofacial abnormalities account for approximately one third of birth defects. The regulatory programs that build the face require precisely controlled spatiotemporal gene expression, achieved through tissue-specific enhancers. Clusters of coactivated enhancers and their target genes, known as superenhancers, are important in determining cell identity but have been largely unexplored in development. In this study we identified superenhancer regions unique to human embryonic craniofacial tissue. To demonstrate the importance of such regions in craniofacial development and disease, we focused on an ~600 kb noncoding region located between NPVF and NFE2L3. We identified long range interactions with this region in both human and mouse embryonic craniofacial tissue with the anterior portion of the HOXA gene cluster. Mice lacking this superenhancer exhibit perinatal lethality, and present with highly penetrant skull defects and orofacial clefts phenocopying Hoxa2-/- mice. Moreover, we identified two cases of de novo copy number changes of the superenhancer in humans both with severe craniofacial abnormalities. This evidence suggests we have identified a critical noncoding locus control region that specifically regulates anterior HOXA genes and copy number changes are pathogenic in human patients.

Rare variants analyses suggest novel cleft genes in the African population.

Non-syndromic orofacial clefts (NSOFCs) are common birth defects with a complex etiology. While over 60 common risk loci have been identified, they explain only a small proportion of the heritability for NSOFCs. Rare variants have been implicated in the missing heritability. Thus, our study aimed to identify genes enriched with nonsynonymous rare coding variants associated with NSOFCs. Our sample included 814 non-syndromic cleft lip with or without palate (NSCL/P), 205 non-syndromic cleft palate only (NSCPO), and 2150 unrelated control children from Nigeria, Ghana, and Ethiopia. We conducted a gene-based analysis separately for each phenotype using three rare-variants collapsing models: (1) protein-altering (PA), (2) missense variants only (MO); and (3) loss of function variants only (LOFO). Subsequently, we utilized relevant transcriptomics data to evaluate associated gene expression and examined their mutation constraint using the gnomeAD database. In total, 13 genes showed suggestive associations (p = E-04). Among them, eight genes (ABCB1, ALKBH8, CENPF, CSAD, EXPH5, PDZD8, SLC16A9, and TTC28) were consistently expressed in relevant mouse and human craniofacial tissues during the formation of the face, and three genes (ABCB1, TTC28, and PDZD8) showed statistically significant mutation constraint. These findings underscore the role of rare variants in identifying candidate genes for NSOFCs.