Any given human individual carries multiple genetic variants that disrupt protein-coding genes, through structural variation, as well as nucleotide variants and indels. Predicting the phenotypic consequences of a gene disruption remains a significant challenge. Current approaches employ information from a range of biological networks to predict which human genes are haploinsufficient (meaning two copies are required for normal function) or essential (meaning at least one copy is required for viability). Using recently available study gene sets, we show that these approaches are strongly biased towards providing accurate predictions for well-studied genes. By contrast, we derive a haploinsufficiency score from a combination of unbiased large-scale high-throughput datasets, including gene co-expression and genetic variation in over 6000 human exomes. Our approach provides a haploinsufficiency prediction for over twice as many genes currently unassociated with papers listed in Pubmed as three commonly-used approaches, and outperforms these approaches for predicting haploinsufficiency for less-studied genes. We also show that fine-tuning the predictor on a set of well-studied 'gold standard' haploinsufficient genes does not improve the prediction for less-studied genes. This new score can readily be used to prioritize gene disruptions resulting from any genetic variant, including copy number variants, indels and single-nucleotide variants.

A20, encoded by the TNFAIP3 gene, is a negative regulator of tumor necrosis factor (TNF)-nuclear factor-κB signaling. It was recently demonstrated that A20 haploinsufficiency (HA20), caused by a heterozygous mutation in the TNFAIP3 gene, can present as an early onset autoinflammatory disease resembling Behçet's disease (BD). In addition to autoinflammatory symptoms, HA20 was also reported to be associated with autoimmune diseases and immunodeficiency. Because the phenotypes associated with HA20 are broad, with different severities observed even among individuals in the same family with identical mutations, it has been assumed that the symptoms of HA20 may depend on genetic background and environmental factors. In this review, we summarize the characteristics of patients with HA20 in East Asia and compare these with patients in other regions, mainly the USA and Europe. Patients with HA20 in East Asia developed recurrent fever more frequently than patients in other regions, but were less likely to develop typical BD symptoms such as skin rashes and genital ulcers. In addition, patients with HA20 in East Asia had low rates of complication with autoimmune diseases and low autoantibody detection rates. While anti-TNF-α agents were the primary treatments for severe HA20 in East Asia, anti-interleukin-1 agents and Janus kinase inhibitors were also administered in other regions. Future studies will need to establish methods for analyzing the pathophysiology of HA20 and determining optimal treatment strategies for each patient.

Neuregulin 1 (NRG1) and its receptor ERBB4 are schizophrenia (SZ) risk genes that control the development of both excitatory and inhibitory cortical circuits. Most studies focused on the characterization ErbB4 deficient mice. However, ErbB4 deletion concurrently perturbs the signaling of Nrg1 and Neuregulin 3 (Nrg3), another ligand expressed in the cortex. In addition, NRG1 polymorphisms linked to SZ locate mainly in non-coding regions and they may partially reduce Nrg1 expression. Here, to study the relevance of Nrg1 partial loss-of-function in cortical circuits we characterized a recently developed haploinsufficient mouse model of Nrg1 (Nrg1tm1Lex). These mice display SZ-like behavioral deficits. The cellular and molecular underpinnings of the behavioral deficits in Nrg1tm1Lex mice remain to be established. With multiple approaches including Magnetic Resonance Spectroscopy (MRS), electrophysiology, quantitative imaging and molecular analysis we found that Nrg1 haploinsufficiency impairs the inhibitory cortical circuits. We observed changes in the expression of molecules involved in GABAergic neurotransmission, decreased density of Vglut1 excitatory buttons onto Parvalbumin interneurons and decreased frequency of spontaneous inhibitory postsynaptic currents. Moreover, we found a decreased number of Parvalbumin positive interneurons in the cortex and altered expression of Calretinin. Interestingly, we failed to detect other alterations in excitatory neurons that were previously reported in ErbB4 null mice suggesting that the Nrg1 haploinsufficiency does not entirely phenocopies ErbB4 deletions. Altogether, this study suggests that Nrg1 haploinsufficiency primarily affects the cortical inhibitory circuits in the cortex and provides new insights into the structural and molecular synaptic impairment caused by NRG1 hypofunction in a preclinical model of SZ.

Secondary lymphedema is a debilitating condition, and genetic factors predisposing to its development remain largely unknown. Adrenomedullin (AM) is peptide encoded, together with proadrenomedullin N-terminal peptide (PAMP), by the Adm gene (adrenomedullin gene). AM and its putative receptor calcitonin receptor-like receptor (CLR) are implicated in angiogenesis and lymphangiogenesis during embryogenesis and wound healing, suggesting their possible involvement in secondary lymphedema. To investigate whether AM deficiency predisposes to secondary lymphedema, we used heterozygous adult mice with Adm gene-knockin stop mutation, which selectively abrogated AM, but preserved PAMP, expression (Adm(AM+/Δ) animals). After hind limb skin incision, Adm messenger RNA expression was upregulated in wounded tissue of both Adm(AM+/+) and Adm(AM+/Δ) mice. However, only Adm(AM+/Δ) animals developed limb swelling and histopathological lymphedematous changes, including epidermal thickening, elevated collagen fiber density, and increased microvessel diameter. Secondary lymphedema was prevented when circulating AM levels in Adm(AM+/Δ) mice were restored by systemic peptide delivery. In human skin, CLR was expressed in tissue components affected by lymphedema, including epidermis, lymphatics, and blood vessels. Our study identified a previously unrecognized role for endogenous AM as a key factor in secondary lymphedema pathogenesis and provided experimental in vivo evidence of an underlying germ-line genetic predisposition to developing this disorder.

Craniofacial microsomia (CFM) is the second most common congenital facial anomaly, yet its genetic etiology remains unknown. We perform whole-exome or genome sequencing of 146 kindreds with sporadic (n = 138) or familial (n = 8) CFM, identifying a highly significant burden of loss of function variants in SF3B2 (P = 3.8 × 10-10), a component of the U2 small nuclear ribonucleoprotein complex, in probands. We describe twenty individuals from seven kindreds harboring de novo or transmitted haploinsufficient variants in SF3B2. Probands display mandibular hypoplasia, microtia, facial and preauricular tags, epibulbar dermoids, lateral oral clefts in addition to skeletal and cardiac abnormalities. Targeted morpholino knockdown of SF3B2 in Xenopus results in disruption of cranial neural crest precursor formation and subsequent craniofacial cartilage defects, supporting a link between spliceosome mutations and impaired neural crest development in congenital craniofacial disease. The results establish haploinsufficient variants in SF3B2 as the most prevalent genetic cause of CFM, explaining ~3% of sporadic and ~25% of familial cases.

Notch signaling has been recognized recently as a key regulator of metabolism. Here, we determined the role of Notch1 in adipogenesis in wild-type (WT) and Notch1 hetero-mutant (N1+/-) mice provided with 12-week normal or high-fat diet. Haploinsufficiency of Notch1 was associated with adipose tissue accumulation despite similar food intake. White adipose tissue (WAT) of N1+/- showed accumulation of adipogenic cells (CD34+CD68+ cells), crown-like structures, and upregulation of cell proliferation markers (cyclin D1 and Ki67). Notch1 expression in WAT reached peak levels in 8-week-old WT mice in parallel with fat accumulation, especially under HF/HS-feeding, whereas such increment was blunted in N1+/- mice. Downstream of Notch1 haploinsufficiency, over-expression of adipogenic factors PPARγ and C/EBPα was noted following down-regulation of downstream transcriptional factors of Notch signaling (Hes-1, Pref-1, and Sox9). Both pharmacological Notch signal inhibition and Notch1 knockdown enhanced adipogenesis of 3T3-L1 preadipocytes. N1+/- mice showed impaired glucose and insulin tolerance with downregulation of IRS-1 and GLUT4 in WAT after high-fat diet. Taken together, our results suggest that haploinsufficiency of Notch1 promotes fat accumulation and adipogenesis and provides a mechanistic link between Notch signaling and development of metabolic syndrome.

VPS35, a major component of the retromer complex, is important for endosome-to-Golgi retrieval of membrane proteins. Although implicated in Alzheimer's disease (AD), how VPS35 regulates AD-associated pathology is unknown. In this paper, we show that hemizygous deletion of Vps35 in the Tg2576 mouse model of AD led to earlier-onset AD-like phenotypes, including cognitive memory deficits, defective long-term potentiation, and impaired postsynaptic glutamatergic neurotransmission in young adult age. These deficits correlated well with an increase of β-amyloid peptide (Aβ) level in the mutant hippocampus. We further demonstrate that VPS35 is predominantly expressed in pyramidal neurons of young adult hippocampus and interacts with BACE1, a protease responsible for Aβ production. Loss of VPS35 function in the mouse hippocampus increased BACE1 activity. Suppression of VPS35 expression in culture decreased BACE1 trans-Golgi localization but enriched it in endosomes. These results demonstrate an essential role for VPS35 in suppression of AD neuropathology and in inhibition of BACE1 activation and Aβ production by promoting BACE1 endosome-to-Golgi retrieval.

Patients with small intestinal neuroendocrine tumors (SINETs) frequently present with lymph node and liver metastases at the time of diagnosis, but the molecular changes that lead to the progression of these tumors are largely unknown. Sequencing studies have only identified recurrent point mutations at low frequencies with CDKN1B being the most common harboring heterozygous mutations in less than 10% of all tumors. Although SINETs are genetically stable tumors with a low frequency of point mutations and indels, they often harbor recurrent hemizygous copy number alterations (CNAs) yet the functional implications of these CNA are unclear.

Haploinsufficiency, a dominant phenotype caused by a heterozygous loss-of-function mutation, has been rarely observed. However, high-dimensional single-cell phenotyping of yeast morphological characteristics revealed haploinsufficiency phenotypes for more than half of 1,112 essential genes under optimal growth conditions. Additionally, 40% of the essential genes with no obvious phenotype under optimal growth conditions displayed haploinsufficiency under severe growth conditions. Haploinsufficiency was detected more frequently in essential genes than in nonessential genes. Similar haploinsufficiency phenotypes were observed mostly in mutants with heterozygous deletion of functionally related genes, suggesting that haploinsufficiency phenotypes were caused by functional defects of the genes. A global view of the gene network was presented based on the similarities of the haploinsufficiency phenotypes. Our dataset contains rich information regarding essential gene functions, providing evidence that single-cell phenotyping is a powerful approach, even in the heterozygous condition, for analyzing complex biological systems.

Heterozygous coding mutations in TRIO are associated with neurodevelopmental disorders, including autism, schizophrenia, bipolar disorder, and epilepsy, and impair TRIO's biochemical activities. To model mutant alleles, we ablated one or both Trio alleles from excitatory neurons in the cortex and hippocampus of mice. Trio haploinsufficiency increases anxiety and impairs social preference and motor coordination. Trio loss reduces forebrain size and dendritic arborization but increases dendritic spine densities. Cortical synapses in Trio haploinsufficient mice are small, exhibit pre- and postsynaptic deficits, and cannot undergo long-term potentiation. Similar phenotypes are observed in Trio knockout mice. Overall, Trio haploinsufficiency causes severe disease-relevant deficits in behavior and neuronal structure and function. Interestingly, phosphodiesterase 4A5 (PDE4A5) levels are reduced and protein kinase A (PKA) signaling is increased when TRIO levels are reduced. Elevation of PDE4A5 and drug-based attenuation of PKA signaling rescue Trio haploinsufficiency-related dendritic spine defects, suggesting an avenue for therapeutic intervention for TRIO-related neurodevelopmental disorders.

More than one-half billion people are obese, and despite progress in genetic research, much of the heritability of obesity remains enigmatic. Here, we identify a Trim28-dependent network capable of triggering obesity in a non-Mendelian, "on/off" manner. Trim28(+/D9) mutant mice exhibit a bi-modal body-weight distribution, with isogenic animals randomly emerging as either normal or obese and few intermediates. We find that the obese-"on" state is characterized by reduced expression of an imprinted gene network including Nnat, Peg3, Cdkn1c, and Plagl1 and that independent targeting of these alleles recapitulates the stochastic bi-stable disease phenotype. Adipose tissue transcriptome analyses in children indicate that humans too cluster into distinct sub-populations, stratifying according to Trim28 expression, transcriptome organization, and obesity-associated imprinted gene dysregulation. These data provide evidence of discrete polyphenism in mouse and man and thus carry important implications for complex trait genetics, evolution, and medicine.

The HMG box transcription factor Sox9 plays a critical role in progenitor cell expansion during pancreas organogenesis and is required for proper endocrine cell development in the embryo. Based on in vitro studies it has been suggested that Sox9 controls expression of a network of important developmental regulators, including Tcf2/MODY5, Hnf6, and Foxa2, in pancreatic progenitor cells. Here, we sought to: 1) determine whether Sox9 regulates this transcriptional network in vivo and 2) investigate whether reduced Sox9 gene dosage leads to impaired glucose homeostasis in adult mice. Employing two genetic models of temporally-controlled Sox9 inactivation in pancreatic progenitor cells, we demonstrate that contrary to in vitro findings, Sox9 is not required for Tcf2, Hnf6, or Foxa2 expression in vivo. Moreover, our analysis revealed a novel role for Sox9 in maintaining the expression of Pdx1/MODY4, which is an important transcriptional regulator of beta-cell development. We further show that reduced beta-cell mass in Sox9-haploinsufficient mice leads to glucose intolerance during adulthood. Sox9-haploinsufficient mice displayed 50% reduced beta-cell mass at birth, which recovered partially via a compensatory increase in beta-cell proliferation early postnatally. Endocrine islets from mice with reduced Sox9 gene dosage exhibited normal glucose stimulated insulin secretion. Our findings show Sox9 plays an important role in endocrine development by maintaining Ngn3 and Pdx1 expression. Glucose intolerance in Sox9-haploinsufficient mice suggests that mutations in Sox9 could play a role in diabetes in humans.

Autoimmunity can occur when a checkpoint of self-tolerance fails. The study of familial autoimmune diseases can reveal pathophysiological mechanisms involved in more common autoimmune diseases. Here, by whole-exome/genome sequencing we identify heterozygous, autosomal-dominant, germline loss-of-function mutations in the SOCS1 gene in ten patients from five unrelated families with early onset autoimmune manifestations. The intracellular protein SOCS1 is known to downregulate cytokine signaling by inhibiting the JAK-STAT pathway. Accordingly, patient-derived lymphocytes exhibit increased STAT activation in vitro in response to interferon-γ, IL-2 and IL-4 that is reverted by the JAK1/JAK2 inhibitor ruxolitinib. This effect is associated with a series of in vitro and in vivo immune abnormalities consistent with lymphocyte hyperactivity. Hence, SOCS1 haploinsufficiency causes a dominantly inherited predisposition to early onset autoimmune diseases related to cytokine hypersensitivity of immune cells.

Successful embryogenesis relies on the coordinated interaction between genes and tissues. The transcription factors Pax9 and Msx1 genetically interact during mouse craniofacial morphogenesis, and mice deficient for either gene display abnormal tooth and palate development. Pax9 is expressed specifically in the pharyngeal endoderm at mid-embryogenesis, and mice deficient for Pax9 on a C57Bl/6 genetic background also have cardiovascular defects affecting the outflow tract and aortic arch arteries giving double-outlet right ventricle, absent common carotid arteries and interruption of the aortic arch.

A novel microdeletion of 14q13.1q13.3 was identified in a patient with developmental delay and intractable epilepsy. The 2.2-Mb deletion included 15 genes, of which TULIP1 (approved gene symbol: RALGAPA1)was the only gene highly expressed in the brain. Western blotting revealed reduced amount of TULIP1 in cell lysates derived from immortalized lymphocytes of the patient, suggesting the association between TULIP1 haploinsufficiency and the patient's phenotype, then 140 patients were screened for TULIP1 mutations and four missense mutations were identified. Although all four missense mutations were common with parents, reduced TULIP1 was observed in the cell lysates with a P297T mutation identified in a conserved region among species. A full-length homolog of human TULIP1 was identified in zebrafish with 72% identity to human. Tulip1 was highly expressed in zebrafish brain, and knockdown of which resulted in brain developmental delay. Therefore, we suggest that TULIP1 is a candidate gene for developmental delay.

Haploinsufficiency, wherein a single functional copy of a gene is insufficient to maintain normal function, is a major cause of dominant disease. Human disease studies have identified several hundred haploinsufficient (HI) genes. We have compiled a map of 1,079 haplosufficient (HS) genes by systematic identification of genes unambiguously and repeatedly compromised by copy number variation among 8,458 apparently healthy individuals and contrasted the genomic, evolutionary, functional, and network properties between these HS genes and known HI genes. We found that HI genes are typically longer and have more conserved coding sequences and promoters than HS genes. HI genes exhibit higher levels of expression during early development and greater tissue specificity. Moreover, within a probabilistic human functional interaction network HI genes have more interaction partners and greater network proximity to other known HI genes. We built a predictive model on the basis of these differences and annotated 12,443 genes with their predicted probability of being haploinsufficient. We validated these predictions of haploinsufficiency by demonstrating that genes with a high predicted probability of exhibiting haploinsufficiency are enriched among genes implicated in human dominant diseases and among genes causing abnormal phenotypes in heterozygous knockout mice. We have transformed these gene-based haploinsufficiency predictions into haploinsufficiency scores for genic deletions, which we demonstrate to better discriminate between pathogenic and benign deletions than consideration of the deletion size or numbers of genes deleted. These robust predictions of haploinsufficiency support clinical interpretation of novel loss-of-function variants and prioritization of variants and genes for follow-up studies.

Age-related vision loss has been associated with degeneration of the retina and decline in Müller glia cell activity. Sox2 is a critical transcription factor for the development and maintenance of the mammalian retina. Here we determined the role of Sox2 in retinal aging. We observed a decline in the number of Sox2-positive Müller, amacrine and ganglion cells with age. We also explored the impact of Sox2 haploinsufficiency (Sox2GFP ) on the activity of Müller glia cells and vision loss with age. Reduction of Sox2-positive cells promoted impaired Müller glia cell function at advanced age of Sox2GFP . These findings correlated with a significant decline in electroretinographic response in Sox2 haploinsufficient mice. Together, these results indicate that Sox2 is required for the maintenance of the transmission of visual information from cones and rods, and suggest that decline in Sox2 expression is responsible for retinal cell aging and age-related vision loss.

The link between autoimmune diseases and primary immunodeficiency syndromes has been increasingly appreciated. Immunologic evaluation of a young man with autoimmune enterocolopathy and unexplained infections revealed evidence of immunodeficiency, including IgG subclass deficiency, impaired Ag-induced lymphocyte proliferation, reduced cytokine production by CD8(+) T lymphocytes, and decreased numbers of NK cells. Genetic evaluation identified haploinsufficiency of NFAT5, a transcription factor regulating immune cell function and cellular adaptation to hyperosmotic stress, as a possible cause of this syndrome. Inhibition or deletion of NFAT5 in normal human and murine cells recapitulated several of the immune deficits identified in the patient. These results provide evidence of a primary immunodeficiency disorder associated with organ-specific autoimmunity linked to NFAT5 deficiency.

Congenital mirror movements (CMM) are characterized by involuntary movements of one side of the body that mirror intentional movements on the opposite side. CMM reflect dysfunctions and structural abnormalities of the motor network and are mainly inherited in an autosomal-dominant fashion. Recently, heterozygous mutations in DCC, the gene encoding the receptor for netrin 1 and involved in the guidance of developing axons toward the midline, have been identified but CMM are genetically heterogeneous. By combining genome-wide linkage analysis and exome sequencing, we identified heterozygous mutations introducing premature termination codons in RAD51 in two families with CMM. RAD51 mRNA was significantly downregulated in individuals with CMM resulting from the degradation of the mutated mRNA by nonsense-mediated decay. RAD51 was specifically present in the developing mouse cortex and, more particularly, in a subpopulation of corticospinal axons at the pyramidal decussation. The identification of mutations in RAD51, known for its key role in the repair of DNA double-strand breaks through homologous recombination, in individuals with CMM reveals a totally unexpected role of RAD51 in neurodevelopment. These findings open a new field of investigation for researchers attempting to unravel the molecular pathways underlying bimanual motor control in humans.

In human, myosin VI (MYO6) haploinsufficiency causes postlingual progressive hearing loss. Because the usefulness of mouse models remains unclear, we produced novel Myo6 null (-/-) mutant mice and analyzed the hearing phenotypes of Myo6+/- (+/-) heterozygous mutants. We first recorded and compared the auditory brainstem responses and distortion product otoacoustic emissions in control Myo6+/+ (+/+) wild-type and +/- mice. These hearing phenotypes of +/- mice were mild; however, we confirmed that +/- mice developed progressive hearing loss. In particular, the hearing loss of female +/- mice progressed faster than that of male +/- mice. The stereocilia bundles of +/- mice exhibited progressive taper loss in cochlear inner hair cells (IHCs) and outer hair cells (OHCs). The loss of OHCs in +/- heterozygotes occurred at an earlier age than in +/+ mice. In particular, the OHCs at the basal area of the cochlea were decreased in +/- mice. IHC ribbon synapses from the area at the base of the cochlea were significantly reduced in +/- mice. Thus, our study indicated that MYO6 haploinsufficiency affected the detection of sounds in mice, and we suggest that +/- mice with Myo6 null alleles are useful animal models for gene therapy and drug treatment in patients with progressive hearing loss due to MYO6 haploinsufficiency.