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.     

Loss-of-Function Mutations in APPL1 in Familial Diabetes Mellitus

Diabetes mellitus is a highly heterogeneous disorder encompassing several distinct forms with different clinical manifestations including a wide spectrum of age at onset. Despite many advances, the causal genetic defect remains unknown for many subtypes of the disease, including some of those forms with an apparent Mendelian mode of inheritance. Here we report two loss-of-function mutations (c.1655T>A [p.Leu552^∗] and c.280G>A [p.Asp94Asn]) in the gene for the Adaptor Protein, Phosphotyrosine Interaction, PH domain, and leucine zipper containing 1 (APPL1) that were identified by means of whole-exome sequencing in two large families with a high prevalence of diabetes not due to mutations in known genes involved in maturity onset diabetes of the young (MODY). APPL1 binds to AKT2, a key molecule in the insulin signaling pathway, thereby enhancing insulin-induced AKT2 activation and downstream signaling leading to insulin action and secretion. Both mutations cause APPL1 loss of function. The p.Leu552^∗ alteration totally abolishes APPL1 protein expression in HepG2 transfected cells and the p.Asp94Asn alteration causes significant reduction in the enhancement of the insulin-stimulated AKT2 and GSK3β phosphorylation that is observed after wild-type APPL1 transfection. These findings—linking APPL1 mutations to familial forms of diabetes—reaffirm the critical role of APPL1 in glucose homeostasis.     

Loss-of-Function Mutations in the WNT Co-receptor LRP6 Cause Autosomal-Dominant Oligodontia

Tooth agenesis is one of the most common developmental anomalies in man. Oligodontia, a severe form of tooth agenesis, occurs both as an isolated anomaly and as a syndromal feature. We performed exome sequencing on 20 unrelated individuals with apparent non-syndromic oligodontia and failed to detect mutations in genes previously associated with oligodontia. In three of the probands, we detected heterozygous variants in LRP6, and sequencing of additional oligodontia-affected individuals yielded one additional mutation in LRP6. Three mutations (c.1144_1145dupAG [p.Ala383Glyfs^∗8], c.1779dupT [p.Glu594^∗], and c.2224_2225dupTT [p.Leu742Phefs^∗7]) are predicted to truncate the protein, whereas the fourth (c.56C>T [p.Ala19Val]) is a missense variant of a conserved residue located at the cleavage site of the protein’s signal peptide. All four affected individuals harboring a LRP6 mutation had a family history of tooth agenesis. LRP6 encodes a transmembrane cell-surface protein that functions as a co-receptor with members from the Frizzled protein family in the canonical Wnt/β-catenin signaling cascade. In this same pathway, WNT10A was recently identified as a major contributor in the etiology of non-syndromic oligodontia. We show that the LRP6 missense variant (c.56C>T) results in altered glycosylation and improper subcellular localization of the protein, resulting in abrogated activation of the Wnt pathway. Our results identify LRP6 variants as contributing to the etiology of non-syndromic autosomal-dominant oligodontia and suggest that this gene is a candidate for screening in DNA diagnostics.     

Loss-of-Function Mutations in HPSE2 Cause the Autosomal Recessive Urofacial Syndrome

Previously, we localized the defective gene for the urofacial syndrome (UFS) to a region on chromosome 10q24 by homozygosity mapping. We now report evidence that Heparanse 2 (HPSE2) is the culprit gene for the syndrome. Mutations with a loss of function in the Heparanase 2 (HPSE2) gene were identified in all UFS patients originating from Colombia, the United States, and France. HPSE2 encodes a 592 aa protein that contains a domain showing sequence homology to the glycosyl hydrolase motif in the heparanase (HPSE) gene, but its exact biological function has not yet been characterized. Complete loss of HPSE2 function in UFS patients suggests that HPSE2 may be important for the synergic action of muscles implicated in facial expression and urine voiding.     

Recurrent Loss-of-Function Mutations Reveal Costs to OAS1 Antiviral Activity in Primates

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Loss-of-Function Mutations in HOXC13 Cause Pure Hair and Nail Ectodermal Dysplasia

Pure hair and nail ectodermal dysplasia (PHNED) is a congenital condition characterized by hypotrichosis and nail dystrophy. Autosomal-recessive PHNED has previously been mapped to chromosomal region 12q12-q14.1, which contains the type II hair keratin and HOXC clusters. Hoxc13-null mice are known to develop hair and nail defects very similar to those seen in human PHNED. We performed whole-exome sequencing in a consanguineous Chinese family affected by PHNED and identified a homozygous nonsense mutation (c.390C>A [p.Tyr130^∗]) in HOXC13 in all affected individuals. In an additional affected female from a consanguineous Afghan family, we found a 27.6 kb homozygous microdeletion involving the first exon of HOXC13. We examined HOXC13 expression in scalp specimen obtained from the index individual of the Chinese family and detected dramatically reduced mRNA levels in skin tissue and nearly absent protein staining in hair follicles, suggesting a mechanism of nonsense-mediated mRNA decay. We also observed markedly decreased expression of four HOXC13 target genes in the specimen. Taken together, our results demonstrate that loss-of-function mutations in HOXC13 cause autosomal-recessive PHNED and further highlight the importance of HOXC13 in hair and nail development.     

Loss-of-Function Mutations in WDR73 Are Responsible for Microcephaly and Steroid-Resistant Nephrotic Syndrome: Galloway-Mowat Syndrome

Galloway-Mowat syndrome is a rare autosomal-recessive condition characterized by nephrotic syndrome associated with microcephaly and neurological impairment. Through a combination of autozygosity mapping and whole-exome sequencing, we identified WDR73 as a gene in which mutations cause Galloway-Mowat syndrome in two unrelated families. WDR73 encodes a WD40-repeat-containing protein of unknown function. Here, we show that WDR73 was present in the brain and kidney and was located diffusely in the cytoplasm during interphase but relocalized to spindle poles and astral microtubules during mitosis. Fibroblasts from one affected child and WDR73-depleted podocytes displayed abnormal nuclear morphology, low cell viability, and alterations of the microtubule network. These data suggest that WDR73 plays a crucial role in the maintenance of cell architecture and cell survival. Altogether, WDR73 mutations cause Galloway-Mowat syndrome in a particular subset of individuals presenting with late-onset nephrotic syndrome, postnatal microcephaly, severe intellectual disability, and homogenous brain MRI features. WDR73 is another example of a gene involved in a disease affecting both the kidney glomerulus and the CNS.     

Loss-of-Function GAS8 Mutations Cause Primary Ciliary Dyskinesia and Disrupt the Nexin-Dynein Regulatory Complex

Multiciliated epithelial cells protect the upper and lower airways from chronic bacterial infections by moving mucus and debris outward. Congenital disorders of ciliary beating, referred to as primary ciliary dyskinesia (PCD), are characterized by deficient mucociliary clearance and severe, recurrent respiratory infections. Numerous genetic defects, most of which can be detected by transmission electron microscopy (TEM), are so far known to cause different abnormalities of the ciliary axoneme. However, some defects are not regularly discernable by TEM because the ciliary architecture of the axoneme remains preserved. This applies in particular to isolated defects of the nexin links, also known as the nexin-dynein regulatory complex (N-DRC), connecting the peripheral outer microtubular doublets. Immunofluorescence analyses of respiratory cells from PCD-affected individuals detected a N-DRC defect. Genome-wide exome sequence analyses identified recessive loss-of-function mutations in GAS8 encoding DRC4 in three independent PCD-affected families.     

Loss-of-Function Alanyl-tRNA Synthetase Mutations Cause an Autosomal-Recessive Early-Onset Epileptic Encephalopathy with Persistent Myelination Defect

Mutations in genes encoding aminoacyl-tRNA synthetases are known to cause leukodystrophies and genetic leukoencephalopathies—heritable disorders that result in white matter abnormalities in the central nervous system. Here we report three individuals (two siblings and an unrelated individual) with severe infantile epileptic encephalopathy, clubfoot, absent deep tendon reflexes, extrapyramidal symptoms, and persistently deficient myelination on MRI. Analysis by whole exome sequencing identified mutations in the nuclear-encoded alanyl-tRNA synthetase (AARS) in these two unrelated families: the two affected siblings are compound heterozygous for p.Lys81Thr and p.Arg751Gly AARS, and the single affected child is homozygous for p.Arg751Gly AARS. The two identified mutations were found to result in a significant reduction in function. Mutations in AARS were previously associated with an autosomal-dominant inherited form of axonal neuropathy, Charcot-Marie-Tooth disease type 2N (CMT2N). The autosomal-recessive AARS mutations identified in the individuals described here, however, cause a severe infantile epileptic encephalopathy with a central myelin defect and peripheral neuropathy, demonstrating that defects of alanyl-tRNA charging can result in a wide spectrum of disease manifestations.     

Carriers of Loss-of-Function Mutations in EXT Display Impaired Pancreatic Beta-Cell Reserve Due to Smaller Pancreas Volume

Exotosin (EXT) proteins are involved in the chain elongation step of heparan sulfate (HS) biosynthesis, which is intricately involved in organ development. Loss of function mutations (LOF) in EXT1 and EXT2 result in hereditary exostoses (HME). Interestingly, HS plays a role in pancreas development and beta-cell function, and genetic variations in EXT2 are associated with an increased risk for type 2 diabetes mellitus. We hypothesized that loss of function of EXT1 or EXT2 in subjects with hereditary multiple exostoses (HME) affects pancreatic insulin secretion capacity and development. We performed an oral glucose tolerance test (OGTT) followed by hyperglycemic clamps to investigate first-phase glucose-stimulated insulin secretion (GSIS) in HME patients and age and gender matched non-affected relatives. Pancreas volume was assessed with magnetic resonance imaging (MRI). OGTT did not reveal significant differences in glucose disposal, but there was a markedly lower GSIS in HME subjects during hyperglycemic clamp (iAUC HME: 0.72 [0.46–1.16] vs. controls 1.53 [0.69–3.36] nmol·l⁻¹·min⁻¹, p<0.05). Maximal insulin response following arginine challenge was also significantly attenuated (iAUC HME: 7.14 [4.22–10.5] vs. controls 10.2 [7.91–12.70] nmol·l⁻¹·min⁻¹ p<0.05), indicative of an impaired beta-cell reserve. MRI revealed a significantly smaller pancreatic volume in HME subjects (HME: 72.0±15.8 vs. controls 96.5±26.0 cm³ p = 0.04). In conclusion, loss of function of EXT proteins may affect beta-cell mass and insulin secretion capacity in humans, and render subjects at a higher risk of developing type 2 diabetes when exposed to environmental risk factors.     

PTHR1 Loss-of-Function Mutations in Familial, Nonsyndromic Primary Failure of Tooth Eruption

Tooth eruption is a complex developmental process requiring coordinated navigation through alveolar bone and oral epithelium. Primary failure of tooth eruption (PFE) is associated with several syndromes primarily affecting skeletal development, but it is also known as a nonsyndromic autosomal-dominant condition. Teeth in the posterior quadrants of the upper and lower jaw are preferentially affected and usually result in an open bite extending from anterior to posterior. In this study, we show that familial, nonsyndromic PFE is caused by heterozygous mutations in the gene encoding the G protein-coupled receptor for parathyroid hormone and parathyroid hormone-like hormone (PTHR1). Three distinct mutations, namely c.1050-3C > G, c.543+1G > A, and c.463G > T, were identified in 15 affected individuals from four multiplex pedigrees. All mutations truncate the mature protein and therefore should lead to a functionless receptor, strongly suggesting that haplo-insufficiency of PTHR1 is the underlying cause of nonsyndromic PFE. Although complete inactivation of PTHR1 is known to underlie the autosomal-recessive Blomstrand osteochondrodysplasia (BOCD), a lethal form of short-limbed dwarfism, our data now imply that dominantly acting PTHR1 mutations that lead to haplo-insufficiency of the receptor result in a nonsyndromic phenotype affecting tooth development with high penetrance and variable expressivity.     

Gain- and Loss-of-Function Mutations in the Breast Cancer Gene GATA3 Result in Differential Drug Sensitivity

Patterns of somatic mutations in cancer genes provide information about their functional role in tumourigenesis, and thus indicate their potential for therapeutic exploitation. Yet, the classical distinction between oncogene and tumour suppressor may not always apply. For instance, TP53 has been simultaneously associated with tumour suppressing and promoting activities. Here, we uncover a similar phenomenon for GATA3, a frequently mutated, yet poorly understood, breast cancer gene. We identify two functional classes of frameshift mutations that are associated with distinct expression profiles in tumours, differential disease-free patient survival and gain- and loss-of-function activities in a cell line model. Furthermore, we find an estrogen receptor-independent synthetic lethal interaction between a GATA3 frameshift mutant with an extended C-terminus and the histone methyltransferases G9A and GLP, indicating perturbed epigenetic regulation. Our findings reveal important insights into mutant GATA3 function and breast cancer, provide the first potential therapeutic strategy and suggest that dual tumour suppressive and oncogenic activities are more widespread than previously appreciated.