Heterozygous Loss-of-Function Mutations in DLL4 Cause Adams-Oliver Syndrome

Adams-Oliver syndrome (AOS) is a rare developmental disorder characterized by the presence of aplasia cutis congenita (ACC) of the scalp vertex and terminal limb-reduction defects. Cardiovascular anomalies are also frequently observed. Mutations in five genes have been identified as a cause for AOS prior to this report. Mutations in EOGT and DOCK6 cause autosomal-recessive AOS, whereas mutations in ARHGAP31, RBPJ, and NOTCH1 lead to autosomal-dominant AOS. Because RBPJ, NOTCH1, and EOGT are involved in NOTCH signaling, we hypothesized that mutations in other genes involved in this pathway might also be implicated in AOS pathogenesis. Using a candidate-gene-based approach, we prioritized DLL4, a critical NOTCH ligand, due to its essential role in vascular development in the context of cardiovascular features in AOS-affected individuals. Targeted resequencing of the DLL4 gene with a custom enrichment panel in 89 independent families resulted in the identification of seven mutations. A defect in DLL4 was also detected in two families via whole-exome or genome sequencing. In total, nine heterozygous mutations in DLL4 were identified, including two nonsense and seven missense variants, the latter encompassing four mutations that replace or create cysteine residues, which are most likely critical for maintaining structural integrity of the protein. Affected individuals with DLL4 mutations present with variable clinical expression with no emerging genotype-phenotype correlations. Our findings demonstrate that DLL4 mutations are an additional cause of autosomal-dominant AOS or isolated ACC and provide further evidence for a key role of NOTCH signaling in the etiology of this disorder.     

Mutations in NOTCH1 Cause Adams-Oliver Syndrome

Notch signaling determines and reinforces cell fate in bilaterally symmetric multicellular eukaryotes. Despite the involvement of Notch in many key developmental systems, human mutations in Notch signaling components have mainly been described in disorders with vascular and bone effects. Here, we report five heterozygous NOTCH1 variants in unrelated individuals with Adams-Oliver syndrome (AOS), a rare disease with major features of aplasia cutis of the scalp and terminal transverse limb defects. Using whole-genome sequencing in a cohort of 11 families lacking mutations in the four genes with known roles in AOS pathology (ARHGAP31, RBPJ, DOCK6, and EOGT), we found a heterozygous de novo 85 kb deletion spanning the NOTCH1 5′ region and three coding variants (c.1285T>C [p.Cys429Arg], c.4487G>A [p.Cys1496Tyr], and c.5965G>A [p.Asp1989Asn]), two of which are de novo, in four unrelated probands. In a fifth family, we identified a heterozygous canonical splice-site variant (c.743−1 G>T) in an affected father and daughter. These variants were not present in 5,077 in-house control genomes or in public databases. In keeping with the prominent developmental role described for Notch1 in mouse vasculature, we observed cardiac and multiple vascular defects in four of the five families. We propose that the limb and scalp defects might also be due to a vasculopathy in NOTCH1-related AOS. Our results suggest that mutations in NOTCH1 are the most common cause of AOS and add to a growing list of human diseases that have a vascular and/or bony component and are caused by alterations in the Notch signaling pathway.     

A functional composite cis-element for NF kappa b and RBJ kappa in the rat pregnancy-specific glycoprotein gene.

The rat pregnancy-specific glycoprotein gene rnCGM3 is primarily expressed in the placenta. Previously, three DNase I footprinting sites (FPI, FPII, and FPIII) were identified in the rnCGM3 promoter region, a yeast one-hybrid screen was performed to identify the nuclear factors binding to the FPIII (5'-GCCTGGGAAAAAACTC-3') element, and RBPJ kappa, a downstream effector of the Notch signaling pathway, was identified as one of the FPIII-binding factors. In the present study, the NF kappa B member p65 was identified as another FPIII-binding factor. Electrophoretic mobility shift assays showed that NF kappa B members, including p50 and p65, bound to the FPIII site. The core binding sequence in the FPIII element for p50 and p65 is GGGAAA, which overlaps with that for RBPJ kappa. Competition exists between p50 and RBPJ kappa for binding to the FPIII element. Transient expression analyses revealed that p65 significantly stimulated the expression of a reporter gene directed by the NF kappa B core sequence in the FPIII element. However, RBPJ kappa could block this stimulation. These results suggest that the regulation of rnCGM3 expression involves both NF kappa B and RBPJ kappa, and they are mutually exclusive in the FPIII element.     

A Highlights from MBoC Selection: Systematic identification of pathological lamin A interactors

Laminopathies are a collection of phenotypically diverse diseases that include muscular dystrophies, cardiomyopathies, lipodystrophies, and premature aging syndromes. Laminopathies are caused by >300 distinct mutations in the LMNA gene, which encodes the nuclear intermediate filament proteins lamin A and C, two major architectural elements of the mammalian cell nucleus. The genotype–phenotype relationship and the basis for the pronounced tissue specificity of laminopathies are poorly understood. Here we seek to identify on a global scale lamin A–binding partners whose interaction is affected by disease-relevant LMNA mutations. In a screen of a human genome–wide ORFeome library, we identified and validated 337 lamin A–binding proteins. Testing them against 89 known lamin A disease mutations identified 50 disease-associated interactors. Association of progerin, the lamin A isoform responsible for the premature aging disorder Hutchinson–Gilford progeria syndrome, with its partners was largely mediated by farnesylation. Mapping of the interaction sites on lamin A identified the immunoglobulin G (IgG)–like domain as an interaction hotspot and demonstrated that lamin A variants, which destabilize the Ig-like domain, affect protein–protein interactions more globally than mutations of surface residues. Analysis of a set of LMNA mutations in a single residue, which result in three phenotypically distinct diseases, identified disease-specific interactors. The results represent a systematic map of disease-relevant lamin A interactors and suggest loss of tissue-specific lamin A interactions as a mechanism for the tissue-specific appearance of laminopathic phenotypes.     

Gain-of-Function Mutations in SCN11A Cause Familial Episodic Pain

Many ion channel genes have been associated with human genetic pain disorders. Here we report two large Chinese families with autosomal-dominant episodic pain. We performed a genome-wide linkage scan with microsatellite markers after excluding mutations in three known genes (SCN9A, SCN10A, and TRPA1) that cause similar pain syndrome to our findings, and we mapped the genetic locus to a 7.81 Mb region on chromosome 3p22.3–p21.32. By using whole-exome sequencing followed by conventional Sanger sequencing, we identified two missense mutations in the gene encoding voltage-gated sodium channel Na_v1.9 (SCN11A): c.673C>T (p.Arg225Cys) and c.2423C>G (p.Ala808Gly) (one in each family). Each mutation showed a perfect cosegregation with the pain phenotype in the corresponding family, and neither of them was detected in 1,021 normal individuals. Both missense mutations were predicted to change a highly conserved amino acid residue of the human Na_v1.9 channel. We expressed the two SCN11A mutants in mouse dorsal root ganglion (DRG) neurons and showed that both mutations enhanced the channel’s electrical activities and induced hyperexcitablity of DRG neurons. Taken together, our results suggest that gain-of-function mutations in SCN11A can be causative of an autosomal-dominant episodic pain disorder.     

Mutations in NALCN Cause an Autosomal-Recessive Syndrome with Severe Hypotonia,Speech Impairment,and Cognitive Delay

Sodium leak channel, nonselective (NALCN) is a voltage-independent and cation-nonselective channel that is mainly responsible for the leaky sodium transport across neuronal membranes and controls neuronal excitability. Although NALCN variants have been conflictingly reported to be in linkage disequilibrium with schizophrenia and bipolar disorder, to our knowledge, no mutations have been reported to date for any inherited disorders. Using linkage, SNP-based homozygosity mapping, targeted sequencing, and confirmatory exome sequencing, we identified two mutations, one missense and one nonsense, in NALCN in two unrelated families. The mutations cause an autosomal-recessive syndrome characterized by subtle facial dysmorphism, variable degrees of hypotonia, speech impairment, chronic constipation, and intellectual disability. Furthermore, one of the families pursued preimplantation genetic diagnosis on the basis of the results from this study, and the mother recently delivered healthy twins, a boy and a girl, with no symptoms of hypotonia, which was present in all the affected children at birth. Hence, the two families we describe here represent instances of loss of function in human NALCN.     

Recessive mutations in DOCK6, encoding the guanidine nucleotide exchange factor DOCK6, lead to abnormal actin cytoskeleton organization and Adams-Oliver syndrome

Adams-Oliver syndrome (AOS) is defined by the combination of aplasia cutis congenita (ACC) and terminal transverse limb defects (TTLD). It is usually inherited as an autosomal-dominant trait, but autosomal-recessive inheritance has also been documented. In an individual with autosomal-recessive AOS, we combined autozygome analysis with exome sequencing to identify a homozygous truncating mutation in dedicator of cytokinesis 6 gene (DOCK6) which encodes an atypical guanidine exchange factor (GEF) known to activate two members of the Rho GTPase family: Cdc42 and Rac1. Another homozygous truncating mutation was identified upon targeted sequencing of DOCK6 in an unrelated individual with AOS. Consistent with the established role of Cdc42 and Rac1 in the organization of the actin cytoskeleton, we demonstrate a cellular phenotype typical of a defective actin cytoskeleton in patient cells. These findings, combined with a Dock6 expression profile that is consistent with an AOS phenotype as well as the very recent demonstration of dominant mutations of ARHGAP31 in AOS, establish Cdc42 and Rac1 as key molecules in the pathogenesis of AOS and suggest that other regulators of these Rho GTPase proteins might be good candidates in the quest to define the genetic spectrum of this genetically heterogeneous condition.     

A Novel C-Terminal CIB2 (Calcium and Integrin Binding Protein 2) Mutation Associated with Non-Syndromic Hearing Loss in a Hispanic Family

Hearing loss is a complex disorder caused by both genetic and environmental factors. Previously, mutations in CIB2 have been identified as a common cause of genetic hearing loss in Pakistani and Turkish populations. Here we report a novel (c.556C>T; p.(Arg186Trp)) transition mutation in the CIB2 gene identified through whole exome sequencing (WES) in a Caribbean Hispanic family with non-syndromic hearing loss. CIB2 belongs to the family of calcium-and integrin-binding (CIB) proteins. The carboxy-termini of CIB proteins are associated with calcium binding and intracellular signaling. The p.(Arg186Trp) mutation is localized within predicted type II PDZ binding ligand at the carboxy terminus. Our ex vivo studies revealed that the mutation did not alter the interactions of CIB2 with Whirlin, nor its targeting to the tips of hair cell stereocilia. However, we found that the mutation disrupts inhibition of ATP-induced Ca²⁺ responses by CIB2 in a heterologous expression system. Our findings support p.(Arg186Trp) mutation as a cause for hearing loss in this Hispanic family. In addition, it further highlights the necessity of the calcium binding property of CIB2 for normal hearing.     

Exome Sequencing Identifies Mutations in CCDC114 as a Cause of Primary Ciliary Dyskinesia

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 14 genes, but they collectively account for only ∼60% of all PCD. To identify mutations that cause PCD, we performed exome sequencing on six unrelated probands with ciliary outer dynein arm (ODA) defects. Mutations in CCDC114, an ortholog of the Chlamydomonas reinhardtii motility gene DCC2, were identified in a family with two affected siblings. Sanger sequencing of 67 additional individuals with PCD with ODA defects from 58 families revealed CCDC114 mutations in 4 individuals in 3 families. All 6 individuals with CCDC114 mutations had characteristic oto-sino-pulmonary disease, but none had situs abnormalities. In the remaining 5 individuals with PCD who underwent exome sequencing, we identified mutations in two genes (DNAI2, DNAH5) known to cause PCD, including an Ashkenazi Jewish founder mutation in DNAI2. These results revealed that mutations in CCDC114 are a cause of ciliary dysmotility and PCD and further demonstrate the utility of exome sequencing to identify genetic causes in heterogeneous recessive disorders.     

Novel Mutations and Deletions of the KIT (Steel Factor Receptor) Gene in Human Piebaldism

Piebaldism is an autosomal dominant genetic disorder of pigmentation characterized by white patches of skin and hair. Melanocytes are lacking in these hypopigmented regions, the result of mutations of the KIT gene, which encodes the cell surface receptor for steel factor (SLF). We describe the analysis of 26 unrelated patients with piebaldism-like hypopigmentation—17 typical patients, 5 with atypical clinical features or family histories, and 4 with other disorders that involve white spotting. We identified novel pathologic mutations or deletions of the KIT gene in 10 (59%) of the typical patients, and in 2 (40%) of the atypical patients. Overall, we have identified pathologic KIT gene mutations in 21 (75%) of 28 unrelated patients with typical piebaldism we have studied. Of the patients without apparent KIT mutations, none have apparent abnormalities of the gene encoding SLF itself (MGF), and genetic linkage analyses in two of these families are suggestive of linkage of the piebald phenotype to KIT. Thus, most patients with typical piebaldism appear to have abnormalities of the KIT gene.     

An N-ethyl-N-nitrosourea-induced mutation in N-acetyltransferase 1 in mice

Genetic variation in human N-acetyltransferases (NAT) has been implicated in susceptibility to aromatic amine and hydrazine carcinogens and therapeutic drugs. There are mouse models for variability of human NAT1; however mice with genetic differences in Nat1 (corresponding to human NAT2), have not been available. N-Ethyl-N-nitrosourea (ENU) mutagenesis was used to create genetic variation in Nat1. Among a number of mutations identified, a base-pair change substituting threonine for isoleucine at position 95 was recovered and studied. Molecular models suggested that this substitution would alter substrate binding. Analysis of hepatic Nat1 activity with the selective substrate isoniazid showed that there was a significant reduction in enzymatic activity in the homozygous mutants compared to the parental strain.     

Hereditary leukonychia, or porcelain nails, resulting from mutations in PLCD1

Hereditary leukonychia (porcelain nails or white nails) is a rare nail disorder with an unknown genetic basis. To identify variants in a gene underlying this phenotype, we identified four families of Pakistani origin showing features of hereditary leukonychia. All 20 nails of each affected individual were chalky and white in appearance, consistent with total leukonychia, with no other cutaneous, appendageal, or systemic findings. By using Affymetrix 10K chip, we established linkage to chromosome 3p21.3-p22 with a LOD score (Z) of 5.1. We identified pathogenic mutations in PLCD1 in all four families, which encodes phosphoinositide-specific phospholipase C delta 1 subunit, a key enzyme in phosphoinositide metabolism. We then identified localization of PLCD1 in the nail matrix. It was recently shown that PLCD1 is a component of the human nail plate by proteomic analysis and is localized in the matrix of human nails. Furthermore, mutations detected in PLCD1 resulted in reduced enzymatic activity in vitro. Our data show that mutations in PLCD1 underlie hereditary leukonychia, revealing a gene involved in molecular control of nail growth.     

Mutations in BICD2 Cause Dominant Congenital Spinal Muscular Atrophy and Hereditary Spastic Paraplegia

Dominant congenital spinal muscular atrophy (DCSMA) is a disorder of developing anterior horn cells and shows lower-limb predominance and clinical overlap with hereditary spastic paraplegia (HSP), a lower-limb-predominant disorder of corticospinal motor neurons. We have identified four mutations in bicaudal D homolog 2 (Drosophila) (BICD2) in six kindreds affected by DCSMA, DCSMA with upper motor neuron features, or HSP. BICD2 encodes BICD2, a key adaptor protein that interacts with the dynein-dynactin motor complex, which facilitates trafficking of cellular cargos that are critical to motor neuron development and maintenance. We demonstrate that mutations resulting in amino acid substitutions in two binding regions of BICD2 increase its binding affinity for the cytoplasmic dynein-dynactin complex, which might result in the perturbation of BICD2-dynein-dynactin-mediated trafficking, and impair neurite outgrowth. These findings provide insight into the mechanism underlying both the static and the slowly progressive clinical features and the motor neuron pathology that characterize BICD2-associated diseases, and underscore the importance of the dynein-dynactin transport pathway in the development and survival of both lower and upper motor neurons.