Cargo transport along the cytoplasmic microtubular network is essential for neuronal function, and cytoplasmic dynein-1 is an established molecular motor that is critical for neurogenesis and homeostasis. We performed whole-exome sequencing, homozygosity mapping, and chromosomal microarray studies in five individuals from three independent pedigrees and identified likely-pathogenic variants in DYNC1I2 (Dynein Cytoplasmic 1 Intermediate Chain 2), encoding a component of the cytoplasmic dynein 1 complex. In a consanguineous Pakistani family with three affected individuals presenting with microcephaly, severe intellectual disability, simplification of cerebral gyration, corpus callosum hypoplasia, and dysmorphic facial features, we identified a homozygous splice donor site variant (GenBank: NM_001378.2:c.607+1G>A). We report two additional individuals who have similar neurodevelopmental deficits and craniofacial features and harbor deleterious variants; one individual bears a c.740A>G (p.Tyr247Cys) change in trans with a 374 kb deletion encompassing DYNC1I2, and an unrelated individual harbors the compound-heterozygous variants c.868C>T (p.G1n290*) and c.740A>G (p.Tyr247Cys). Zebrafish larvae subjected to CRISPR-Cas9 gene disruption or transient suppression of dyncli2a displayed significantly altered craniofacial patterning with concomitant reduction in head size. We monitored cell death and cell cycle progression in dync1i2a zebrafish models and observed significantly increased apoptosis, likely due to prolonged mitosis caused by abnormal spindle morphology, and this finding offers initial insights into the cellular basis of microcephaly. Additionally, complementation studies in zebrafish demonstrate that p.Tyr247Cys attenuates gene function, consistent with protein structural analysis. Our genetic and functional data indicate that DYNC1I2 dysfunction probably causes an autosomal-recessive microcephaly syndrome and highlight further the critical roles of the dynein-1 complex in neurodevelopment.

Studying the genetic basis of gene expression and chromatin organization is key to characterizing the effect of genetic variability on the function and structure of the human genome. Here we unravel how genetic variation perturbs gene regulation using a dataset combining activity of regulatory elements, gene expression, and genetic variants across 317 individuals and two cell types. We show that variability in regulatory activity is structured at the intra- and interchromosomal levels within 12,583 cis-regulatory domains and 30 trans-regulatory hubs that highly reflect the local (that is, topologically associating domains) and global (that is, open and closed chromatin compartments) nuclear chromatin organization. These structures delimit cell type-specific regulatory networks that control gene expression and coexpression and mediate the genetic effects of cis- and trans-acting regulatory variants on genes.

FBXL3 (F-Box and Leucine Rich Repeat Protein 3) encodes a protein that contains an F-box and several tandem leucine-rich repeats (LRR) domains. FBXL3 is part of the SCF (Skp1-Cullin-F box protein) ubiquitin ligase complex that binds and leads to phosphorylation-dependent degradation of the central clock protein cryptochromes (CRY1 and CRY2) by the proteasome and its absence causes circadian phenotypes in mice and behavioral problems. No FBXL3-related phenotypes have been described in humans. By a combination of exome sequencing and homozygosity mapping, we analyzed two consanguineous families with intellectual disability and identified homozygous loss-of-function (LoF) variants in FBXL3. In the first family, from Pakistan, an FBXL3 frameshift variant [NM_012158.2:c.885delT:p.(Leu295Phefs*25)] was the onlysegregating variant in five affected individuals in two family loops (LOD score: 3.12). In the second family, from Lebanon, we identified a nonsense variant [NM_012158.2:c.445C>T:p.(Arg149*)]. In a third patient from Italy, a likely deleterious non-synonymous variant [NM_012158.2:c.1072T>C:p.(Cys358Arg)] was identified in homozygosity. Protein 3D modeling predicted that the Cys358Arg change influences the binding with CRY2 by destabilizing the structure of the FBXL3, suggesting that this variant is also likely to be LoF. The eight affected individuals from the three families presented with a similar phenotype that included intellectual disability, developmental delay, short stature and mild facial dysmorphism, mainly large nose with a bulbous tip. The phenotypic similarity and the segregation analysis suggest that FBXL3 biallelic, LoF variants link this gene with syndromic autosomal recessive developmental delay/intellectual disability.

Aneuploidy is a major source of gene dosage imbalance due to copy number alterations (CNA), and viable human trisomies are model disorders of altered gene expression. We study gene and allele-specific expression (ASE) of 9668 single-cell fibroblasts from trisomy 21 (T21) discordant twins and from mosaic T21, T18, T13 and T8. We examine 928 single cells with deep scRNAseq. Expected and observed overexpression of trisomic genes in trisomic vs. diploid bulk RNAseq is not detectable in trisomic vs. diploid single cells. Instead, for trisomic genes with low-to-average expression, their altered gene dosage is mainly due to the higher fraction of trisomic cells simultaneously expressing these genes, in agreement with a stochastic 2-state burst-like model of transcription. These results, confirmed in a further analysis of 8740 single fibroblasts with shallow scRNAseq, suggest that the specific transcriptional profile of each gene contributes to the phenotypic variability of trisomies. We propose an improved model to understand the effects of CNA and, generally, of gene regulation on gene dosage imbalance.

We report two consanguineous families with probands that exhibit intellectual disability, developmental delay, short stature, aphasia, and hypotonia in which homozygous non-synonymous variants were identified in IQSEC1 (GenBank: NM_001134382.3). In a Pakistani family, the IQSEC1 segregating variant is c.1028C>T (p.Thr343Met), while in a Saudi Arabian family the variant is c.962G>A (p.Arg321Gln). IQSEC1-3 encode guanine nucleotide exchange factors for the small GTPase ARF6 and their loss affects a variety of actin-dependent cellular processes, including AMPA receptor trafficking at synapses. The ortholog of IQSECs in the fly is schizo and its loss affects growth cone guidance at the midline in the CNS, also an actin-dependent process. Overexpression of the reference IQSEC1 cDNA in wild-type flies is lethal, but overexpression of the two variant IQSEC1 cDNAs did not affect viability. Loss of schizo caused embryonic lethality that could be rescued to 2nd instar larvae by moderate expression of the human reference cDNA. However, the p.Arg321Gln and p.Thr343Met variants failed to rescue embryonic lethality. These data indicate that the variants behave as loss-of-function mutations. We also show that schizo in photoreceptors is required for phototransduction. Finally, mice with a conditional Iqsec1 deletion in cortical neurons exhibited an increased density of dendritic spines with an immature morphology. The phenotypic similarity of the affecteds and the functional experiments in flies and mice indicate that IQSEC1 variants are the cause of a recessive disease with intellectual disability, developmental delay, and short stature, and that axonal guidance and dendritic projection defects as well as dendritic spine dysgenesis may underlie disease pathogenesis.

The mechanisms underlying cellular and organismal phenotypes due to copy number alterations (CNA) are not fully understood. Aneuploidy is a major source of gene dosage imbalance due to CNA and viable human trisomies are model disorders of altered gene expression. To understand the cellular impact of gene dosage imbalance, we studied gene and allele specific expression (ASE) of 9668 single-cell fibroblasts in trisomies T21, T18, T13 and T8. To limit the bias of interindividual noise, all comparisons between euploid and trisomic single-cells were performed on an isogenic setting for all trisomies studied. Initially we examined 928 single cells with deep RNA-Seq. For T21 we used fibroblasts from one pair of monozygotic twins discordant for T21 and from mosaic T21. For T18, T13 and T8 we analyzed single cells from mosaic individuals. Single-cell analyses revealed inconsistencies concerning the overexpression of some genes observed in differential trisomic vs euploid bulk RNAseq while this imbalance was not detectable in trisomic vs. euploid single cells. Moreover, ASE profiling of all single cells uncovered a substantial monoallelic pattern of expression in the trisomic fraction of the genome. By classifying genes according to the level of mono and bi-allelic transcription, we have observed that, for genes with monoallelic and low-to-average expression, the altered gene dosage is mainly due to the higher fraction of cells simultaneously expressing these genes in the trisomic samples. These results were confirmed in a further experiment of 8740 single fibroblasts from the monozygotic twins discordant for T21 samples. We conclude that gene dosage imbalance is of bidimensional nature: over time (simultaneous expression of all alleles resulting in increased accumulation of RNA of copy altered genes in each single cell) as previously stated, and over space (increased fraction of cells simultaneously expressing copy altered genes). These results strongly suggest that each class of genes contributes to the phenotypic variability of trisomies according to its temporal and spatial behavior and propose an improved model to understand the effects of copy number alterations.

X-chromosome inactivation (XCI) provides a dosage compensation mechanism where, in each female cell, one of the two X chromosomes is randomly silenced. However, some genes on the inactive X chromosome and outside the pseudoautosomal regions escape from XCI and are expressed from both alleles (escapees). We investigated XCI at single-cell resolution combining deep single-cell RNA sequencing with whole-genome sequencing to examine allelic-specific expression in 935 primary fibroblast and 48 lymphoblastoid single cells from five female individuals. In this framework we integrated an original method to identify and exclude doublets of cells. In fibroblast cells, we have identified 55 genes as escapees including five undescribed escapee genes. Moreover, we observed that all genes exhibit a variable propensity to escape XCI in each cell and cell type and that each cell displays a distinct expression profile of the escapee genes. A metric, the Inactivation Score-defined as the mean of the allelic expression profiles of the escapees per cell-enables us to discover a heterogeneous and continuous degree of cellular XCI with extremes represented by "inactive" cells, i.e., cells exclusively expressing the escaping genes from the active X chromosome and "escaping" cells expressing the escapees from both alleles. We found that this effect is associated with cell-cycle phases and, independently, with the XIST expression level, which is higher in the quiescent phase (G0). Single-cell allele-specific expression is a powerful tool to identify novel escapees in different tissues and provide evidence of an unexpected cellular heterogeneity of XCI.

Developmental eye defects often severely reduce vision. Despite extensive efforts, for a substantial fraction of these cases the molecular causes are unknown. Recessive eye disorders are frequent in consanguineous populations and such large families with multiple affected individuals provide an opportunity to identify recessive causative genes. We studied a Pakistani consanguineous family with three affected individuals with congenital vision loss and progressive eye degeneration. The family was analyzed by exome sequencing of one affected individual and genotyping of all family members. We have identified a non-synonymous homozygous variant (NM_001128918.2: c.1708C > G: p.Arg570Gly) in the MARK3 gene as the likely cause of the phenotype. Given that MARK3 is highly conserved in flies (I: 55%; S: 67%) we knocked down the MARK3 homologue, par-1, in the eye during development. This leads to a significant reduction in eye size, a severe loss of photoreceptors and loss of vision based on electroretinogram (ERG) recordings. Expression of the par-1 p.Arg792Gly mutation (equivalent to the MARK3 variant found in patients) in developing fly eyes also induces loss of eye tissue and reduces the ERG signals. The data in flies and human indicate that the MARK3 variant corresponds to a loss of function. We conclude that the identified mutation in MARK3 establishes a new gene-disease link, since it likely causes structural abnormalities during eye development and visual impairment in humans, and that the function of MARK3/par-1 is evolutionarily conserved in eye development.

Infantile and childhood-onset cataracts form a heterogeneous group of disorders; among the many genetic causes, numerous pathogenic variants in additional genes associated with autosomal-recessive infantile cataracts remain to be discovered. We identified three consanguineous families affected by bilateral infantile cataracts. Using exome sequencing, we found homozygous loss-of-function variants in DNMBP: nonsense variant c.811C >T (p.Arg271*) in large family F385 (nine affected individuals; LOD score = 5.18 at theta = 0), frameshift deletion c.2947_2948de1 (p.Asp983*) in family F372 (two affected individuals), and frameshift variant c.2852_2855de1 (p.Thr951Metfs*41) in family F3 (one affected individual). The phenotypes of all affected individuals include infantile-onset cataracts. RNAi-mediated knockdown of the Drosophila ortholog still life (sit), enriched in lens-secreting cells, affects the development of these cells as well as the localization of E-cadherin, alters the distribution of septate junctions in adjacent cone cells, and leads to a similar to 50% reduction in electroretinography amplitudes in young flies. DNMBP regulates the shape of tight junctions, which correspond to the septate junctions in invertebrates, as well as the assembly pattern of E-cadherin in human epithelial cells. E-cadherin has an important role in lens vesicle separation and lens epithelial cell survival in humans. We therefore conclude that DNMBP loss-of-function variants cause infantile-onset cataracts in humans.

Genome-wide studies on the genetic basis of gene expression and the structural properties of chromatin have considerably advanced our understanding of the function of the human genome. However, it remains unclear how structure relates to function and, in this work, we aim at bridging both by assembling a dataset that combines the activity of regulatory elements (e.g. enhancers and promoters), expression of genes and genetic variations of 317 individuals and across two cell types. We show that the regulatory activity is structured within 12,583 Cis Regulatory Domains (CRDs) that are cell type specific and highly reflective of the local (i.e. Topologically Associating Domains) and global (i.e. A/B nuclear compartments) nuclear organization of the chromatin. These CRDs essentially delimit the sets of active regulatory elements involved in the transcription of most genes, thereby capturing complex regulatory networks in which the effects of regulatory variants are propagated and combined to finally mediate expression Quantitative Trait Loci. Overall, our analysis reveals the complexity and specificity of cis and trans regulatory networks and their perturbation by genetic variation.

Acute myeloid leukemia (AML) is a particularly aggressive blood cancer that is difficult to treat because of the incomplete eradication of rare blast cells that possess self-renewal and leukemia-initiating properties. To characterize resistant blasts, we analyzed for the first time the transcriptomes of individual CD34+/CD38− blasts by single-cell mRNA sequencing of 359 CD33+/CD34+/CD38−/+ sorted cells from two patients with AML and four unaffected individuals. We demonstrated that the captured blasts possess the transcriptomic hallmarks of self-renewal and leukemia-initiating ability. The effects of somatic mutations on the cancer cells are visible at the transcriptional level, and the cellular signaling pathway activity of the blasts is altered, revealing disease-associated gene networks. We also identified a core set of transcription factors that were co-activated in blasts, which suggests a joint transcription program among blasts. Finally, we revealed that leukemogenesis and putative prognostic gene-expression signatures are present at diagnosis in leukemic CD33+/CD34+/CD38− cells and can be detected using a single-cell RNA sequencing approach.

Genomic imprinting results in parental-specific gene expression. Imprinted genes are involved in the etiology of rare syndromes and have been associated with common diseases such as diabetes and cancer. Standard RNA bulk cell sequencing applied to whole-tissue samples has been used to detect imprinted genes in human and mouse models. However, lowly expressed genes cannot be detected by using RNA bulk approaches. Here, we report an original and robust method that combines single-cell RNA-seq and whole-genome sequencing into an optimized statistical framework to analyze genomic imprinting in specific cell types and in different individuals. Using samples from the probands of 2 family trios and 3 unrelated individuals, 1,084 individual primary fibroblasts were RNA sequenced and more than 700,000 informative heterozygous single-nucleotide variations (SNVs) were genotyped. The allele-specific coverage per gene of each SNV in each single cell was used to fit a beta-binomial distribution to model the likelihood of a gene being expressed from one and the same allele. Genes presenting a significant aggregate allelic ratio (between 0.9 and 1) were retained to identify of the allelic parent of origin. Our approach allowed us to validate the imprinting status of all of the known imprinted genes expressed in fibroblasts and the discovery of nine putative imprinted genes, thereby demonstrating the advantages of single-cell over bulk RNA-seq to identify imprinted genes. The proposed single-cell methodology is a powerful tool for establishing a cell type-specific map of genomic imprinting.