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 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 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.     

Compound Heterozygosity of Low-Frequency Promoter Deletions and Rare Loss-of-Function Mutations in TXNL4A Causes Burn-McKeown Syndrome

Mutations in components of the major spliceosome have been described in disorders with craniofacial anomalies, e.g., Nager syndrome and mandibulofacial dysostosis type Guion-Almeida. The U5 spliceosomal complex of eight highly conserved proteins is critical for pre-mRNA splicing. We identified biallelic mutations in TXNL4A, a member of this complex, in individuals with Burn-McKeown syndrome (BMKS). This rare condition is characterized by bilateral choanal atresia, hearing loss, cleft lip and/or palate, and other craniofacial dysmorphisms. Mutations were found in 9 of 11 affected families. In 8 families, affected individuals carried a rare loss-of-function mutation (nonsense, frameshift, or microdeletion) on one allele and a low-frequency 34 bp deletion (allele frequency 0.76%) in the core promoter region on the other allele. In a single highly consanguineous family, formerly diagnosed as oculo-oto-facial dysplasia, the four affected individuals were homozygous for a 34 bp promoter deletion, which differed from the promoter deletion in the other families. Reporter gene and in vivo assays showed that the promoter deletions led to reduced expression of TXNL4A. Depletion of TXNL4A (Dib1) in yeast demonstrated reduced assembly of the tri-snRNP complex. Our results indicate that BMKS is an autosomal-recessive condition, which is frequently caused by compound heterozygosity of low-frequency promoter deletions in combination with very rare loss-of-function mutations.     

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.     

No Association between Loss-of-Function Mutations in filaggrin and Diabetes,Cardiovascular Disease,and All-Cause Mortality

Background

Common loss-of-function mutations in the filaggrin gene (FLG) are a major predisposing risk factor for atopic disease due to reduced epidermal filaggrin protein levels. We previously observed an association between these mutations and type 2 diabetes and hypothesized that an inherited impairment of skin barrier functions could facilitate low-grade inflammation and hence increase the risk of diabetes and cardiovascular disease. We examined the association between loss-of-function mutations in FLG and diabetes, stroke, ischemic heart disease (IHD), and all-cause mortality in the general population.

Methods

The R501X and 2282del4 loss-of function mutations in FLG were genotyped in four Danish study populations including a total of 13373 adults aged 15-77 years. Two of the studies also genotyped the R2447X mutation. By linkage to Danish national central registers we obtained information for all participants on dates of diagnoses of diabetes, stroke, and IHD, as well as all-cause mortality. Data were analyzed by Cox proportional hazard models and combined by fixed effect meta-analyses.

Results

In meta-analyses combining the results from the four individual studies, carriage of loss-of-function mutations in FLG was not associated with incident diabetes (hazard ratio (HR) (95% confidence intervals (CI)) = 0.95 (0.73, 1.23), stroke (HR (95% CI) = 1.27 (0.97, 1.65), ischemic heart disease (HR (95%CI) = 0.92 (0.71, 1.19), and all-cause mortality (HR (95%CI) = 1.02 (0.83, 1.25)). Similar results were obtained when including prevalent cases in logistic regression models.

Conclusion

Our results suggest that loss-of-function mutations in FLG are not associated with type 2 diabetes, cardiovascular disease, and all-cause mortality. However, larger studies with longer follow-up are needed to exclude any associations.     

Evidence That Loss-of-Function Filaggrin Gene Mutations Evolved in Northern Europeans to Favor Intracutaneous Vitamin D3 Production

Skin pigmentation lightened progressively to a variable extent, as modern humans emigrated out of Africa, but extreme lightening occurred only in northern Europeans. Yet, loss of pigmentation alone cannot suffice to sustain cutaneous vitamin D3 (VD3) formation at the high latitudes of northern Europe. We hypothesized that loss-of-function mutations in the epidermal structural protein, filaggrin (FLG), could have evolved to sustain adequate VD3 status. Loss of FLG results in reduced generation of trans-urocanic acid, the principal endogenous ultraviolet-B (UV-B) filter in lightly-pigmented individuals. Accordingly, we identified a higher prevalence of FLG mutations in northern European populations when compared to more southern European, Asian and African populations that correlates significantly with differences in circulating 25-OH-VD3 levels in these same populations. By allowing additional UV-B penetration and intracutaneous VD3 formation, the latitude-dependent gradient in FLG mutations, likely together with other concurrent mutations in VD3 metabolic pathways, provide a non-pigment-based mechanism that sustains higher levels of circulating VD3 in northern Europeans. At the time that FLG mutations evolved, xerosis due to FLG deficiency was a lesser price to pay for enhanced VD3 production. Yet, the increase in FLG mutations has inadvertently contributed to an epidemic of atopic diseases that has emerged in recent decades.