A mutation in the immunoproteasome subunit PSMB8 causes autoinflammation and lipodystrophy in humans

Proteasomes are multisubunit proteases that play a critical role in maintaining cellular function through the selective degradation of ubiquitinated proteins. When 3 additional β subunits, expression of which is induced by IFN-γ, are substituted for their constitutively expressed counterparts, the structure is converted to an immunoproteasome. However, the underlying roles of immunoproteasomes in human diseases are poorly understood. Using exome analysis, we found a homozygous missense mutation (G197V) in immunoproteasome subunit, β type 8 (PSMB8), which encodes one of the β subunits induced by IFN-γ in patients from 2 consanguineous families. Patients bearing this mutation suffered from autoinflammatory responses that included recurrent fever and nodular erythema together with lipodystrophy. This mutation increased assembly intermediates of immunoproteasomes, resulting in decreased proteasome function and ubiquitin-coupled protein accumulation in the patient's tissues. In the patient's skin and B cells, IL-6 was highly expressed, and there was reduced expression of PSMB8. Downregulation of PSMB8 inhibited the differentiation of murine and human adipocytes in vitro, and injection of siRNA against Psmb8 in mouse skin reduced adipocyte tissue volume. These findings identify PSMB8 as an essential component and regulator not only of inflammation, but also of adipocyte differentiation, and indicate that immunoproteasomes have pleiotropic functions in maintaining the homeostasis of a variety of cell types.     

An EF hand mutation in Stim1 causes premature platelet activation and bleeding in mice

Changes in cytoplasmic Ca2+ levels regulate a variety of fundamental cellular functions in virtually all cells. In nonexcitable cells, a major pathway of Ca2+ entry involves receptor-mediated depletion of intracellular Ca2+ stores followed by the activation of store-operated calcium channels in the plasma membrane. We have established a mouse line expressing an activating EF hand motif mutant of stromal interaction molecule 1 (Stim1), an ER receptor recently identified as the Ca2+ sensor responsible for activation of Ca2+ release-activated (CRAC) channels in T cells, whose function in mammalian physiology is not well understood. Mice expressing mutant Stim1 had macrothrombocytopenia and an associated bleeding disorder. Basal intracellular Ca2+ levels were increased in platelets, which resulted in a preactivation state, a selective unresponsiveness to immunoreceptor tyrosine activation motif-coupled agonists, and increased platelet consumption. In contrast, basal Ca2+ levels, but not receptor-mediated responses, were affected in mutant T cells. These findings identify Stim1 as a central regulator of platelet function and suggest a cell type-specific activation or composition of the CRAC complex.     

A homozygous missense mutation in human KLOTHO causes severe tumoral calcinosis

Familial tumoral calcinosis is characterized by ectopic calcifications and hyperphosphatemia due to inactivating mutations in FGF23 or UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3). Herein we report a homozygous missense mutation (H193R) in the KLOTHO (KL) gene of a 13-year-old girl who presented with severe tumoral calcinosis with dural and carotid artery calcifications. This patient exhibited defects in mineral ion homeostasis with marked hyperphosphatemia and hypercalcemia as well as elevated serum levels of parathyroid hormone and FGF23. Mapping of H193R mutation onto the crystal structure of myrosinase, a plant homolog of KL, revealed that this histidine residue was at the base of the deep catalytic cleft and mutation of this histidine to arginine should destabilize the putative glycosidase domain (KL1) of KL, thereby attenuating production of membrane-bound and secreted KL. Indeed, compared with wild-type KL, expression and secretion of H193R KL were markedly reduced in vitro, resulting in diminished ability of FGF23 to signal via its cognate FGF receptors. Taken together, our findings provide what we believe to be the first evidence that loss-of-function mutations in human KL impair FGF23 bioactivity, underscoring the essential role of KL in FGF23-mediated phosphate and vitamin D homeostasis in humans.     

凝血因子Ⅺ基因内含子区受位剪切位点突变导致的遗传性凝血因子Ⅺ缺陷症

目的探讨一个遗传性凝血因子Ⅺ(FⅪ)缺陷症患者及其家系成员中FⅪ基因突变方法用自动凝血仪检测患者及其家系成员凝血酶原时间(PT)、活化部分凝血酶原时间(APTT)，一期法检测血浆F Ⅺ、FⅤ、FⅦ、FⅧ、FⅨ、FⅫ活性；采用ELISA法检测血浆FⅪ：Ag。以外周血中抽提的基因组DNA为模板，PCR扩增FⅪ基因的所有外显子及其侧翼序列，用直接测序的方法查找突变以RT-PCR检测患者FⅪ mRNA的水平，分析突变对FⅪ mRNA剪切造成的影响。结果患者及部分家系成员APTT延长，先证者及其兄血浆FⅪ：C、FⅪ：Ag均低于正常人的10％，其父母血浆FⅪ：C、FⅪ：Ag均低于正常人的．50％。基因测序结果表明FⅪ基因内含子10 3’端4个碱基缺失(IVs J-4delgttg)，先证者及其兄为纯合突变，其父母及侄女、外甥均为杂合型。在患者外周血中未能检测到FFⅪ mRNA。结论该突变导致FⅪ mRNA不能正常剪切，引起FⅪ转录本质和量的改变，不能合成正常的F Ⅺ，是导致该遗传性F Ⅺ缺陷症家系成员发病的分子遗传学基础。     

MCM4 mutation causes adrenal failure, short stature, and natural killer cell deficiency in humans

An interesting variant of familial glucocorticoid deficiency (FGD), an autosomal recessive form of adrenal failure, exists in a genetically isolated Irish population. In addition to hypocortisolemia, affected children show signs of growth failure, increased chromosomal breakage, and NK cell deficiency. Targeted exome sequencing in 8 patients identified a variant (c.71-1insG) in minichromosome maintenance-deficient 4 (MCM4) that was predicted to result in a severely truncated protein (p.Pro24ArgfsX4). Western blotting of patient samples revealed that the major 96-kDa isoform present in unaffected human controls was absent, while the presence of the minor 85-kDa isoform was preserved. Interestingly, histological studies with Mcm4-depleted mice showed grossly abnormal adrenal morphology that was characterized by non-steroidogenic GATA4- and Gli1-positive cells within the steroidogenic cortex, which reduced the number of steroidogenic cells in the zona fasciculata of the adrenal cortex. Since MCM4 is one part of a MCM2-7 complex recently confirmed as the replicative helicase essential for normal DNA replication and genome stability in all eukaryotes, it is possible that our patients may have an increased risk of neoplastic change. In summary, we have identified what we believe to be the first human mutation in MCM4 and have shown that it is associated with adrenal insufficiency, short stature, and NK cell deficiency.     

Pancreatic aspects of cystic fibrosis and other inherited causes of pancreatic dysfunction

Causes of pancreatic dysfunction in childhood can be divided into two general categories: (1) hereditary conditions that directly affect the pancreas and (2) acquired disorders in which loss of pancreatic function is a secondary phenomenon. This article discusses genotypes and phenotypes, clinical features, Shwachman-Diamond syndrome, isolated enzyme deficiencies, and other topics.     

Redundant roles for inflammasome receptors NLRP3 and NLRC4 in host defense against Salmonella

Intracellular pathogens and endogenous danger signals in the cytosol engage NOD-like receptors (NLRs), which assemble inflammasome complexes to activate caspase-1 and promote the release of proinflammatory cytokines IL-1β and IL-18. However, the NLRs that respond to microbial pathogens in vivo are poorly defined. We show that the NLRs NLRP3 and NLRC4 both activate caspase-1 in response to Salmonella typhimurium. Responding to distinct bacterial triggers, NLRP3 and NLRC4 recruited ASC and caspase-1 into a single cytoplasmic focus, which served as the site of pro–IL-1β processing. Consistent with an important role for both NLRP3 and NLRC4 in innate immune defense against S. typhimurium, mice lacking both NLRs were markedly more susceptible to infection. These results reveal unexpected redundancy among NLRs in host defense against intracellular pathogens in vivo.IL-1β is a central orchestrator of immunity against various classes of pathogens and a key trigger of inflammatory diseases. It is produced by activated macrophages as a proprotein, which is proteolytically processed to its active form by the cysteine protease caspase-1. Caspase-1 itself is synthesized as a zymogen and must be activated by autocatalytic cleavage. This activation involves recruitment of pro–caspase-1 into multiprotein complexes called inflammasomes, which are comprised of the adapter protein ASC and a NOD-like receptor (NLR), with the latter conferring specificity for microbial and endogenous products (Mariathasan and Monack, 2007).The NLR family of cytosolic receptors responds to a variety of pathogens and endogenous danger signals (Brodsky and Monack, 2009). For example, NLRC4 (also known as Ipaf) is required by cultured primary macrophages to activate caspase-1 after infection with Salmonella spp., Pseudomonas aeruginosa, Listeria monocytogenes, and Legionella pneumophila. NLRC4 is thought to recognize bacterial flagellin that is injected accidentally into the cytoplasm by bacterial virulence-associated secretion systems (Amer et al., 2006; Franchi et al., 2006; Miao et al., 2006; Ren et al., 2006). Recently, it has been reported that NLRC4 also detects the rod subunit of certain type 3 secretion systems (T3SSs; Miao et al., 2010), which explains how NLRC4 can detect nonflagellated bacteria such as Shigella flexneri and some strains of P. aeruginosa (Sutterwala et al., 2007; Suzuki et al., 2007). In contrast to NLRC4, NLRP3 (also known as Nalp3) is activated by diverse molecules originating from viruses (Sendai, influenza, and adenoviral strains; Kanneganti et al., 2006; Muruve et al., 2008), fungi (Candida albicans and Saccharomyces cerevisiae; Gross et al., 2009; Hise et al., 2009), and bacteria (Staphylococcus aureus, Neisseria gonorrhoeae, and L. monocytogenes; Mariathasan et al., 2006; Warren et al., 2008; Duncan et al., 2009). The precise signal that is detected by NLRP3 remains unclear. Common terminal signals appear to involve damage to membranes and/or changes in potassium levels (Brodsky and Monack, 2009). Other NLRs include NLRP1, which is activated by anthrax lethal toxin, and Aim2, which appears to recognize double-stranded DNA in the cytoplasm (Fernandes-Alnemri et al., 2009; Hornung et al., 2009).Although several different inflammasomes have been described, the precise molecular structure and composition of inflammasomes remains largely unknown. In vitro studies with purified inflammasome components suggest that NLRs bind their ligands and oligomerize to form a platform for dimerization and autoproteolytic cleavage of pro–caspase-1 (Faustin et al., 2007). Under certain lysis conditions, inflammasome components can form structures between 500 and 700 kD in size (Martinon et al., 2004; Hsu et al., 2008). Overexpressed ASC also forms large aggregates (Masumoto et al., 1999; Richards et al., 2001; Fernandes-Alnemri et al., 2007), although the physiological significance of these aggregates is unclear.Caspase-1 is important for innate immune defense against many pathogens, but the contributions of specific NLRs to caspase-1 activation during animal infections are less clear. Previous in vivo studies indicated a role for NLRC4 in defense against L. pneumophila and P. aeruginosa but not Salmonella (Amer et al., 2006; Lara-Tejero et al., 2006; Sutterwala et al., 2007). This last finding was surprising, given that NLRC4 was critical for caspase-1 activation by macrophages infected with Salmonella in culture (Mariathasan et al., 2004). Thus far, the only described roles for NLRP3 in vivo during microbial infections are in defense against C. albicans, Plasmodium, and influenza A virus infections (Allen et al., 2009; Dostert et al., 2009; Gross et al., 2009; Hise et al., 2009; Joly et al., 2009; Shio et al., 2009; Thomas et al., 2009), whereas no role has been described in bacterial infections so far.Salmonella enterica serovar typhimurium (Stm) is an intracellular Gram-negative bacterium that causes infections in human and animal hosts. After initial colonization of the gastrointestinal tract, Stm persists in tissue macrophages. Salmonella virulence requires two T3SSs encoded on Salmonella pathogenicity islands: SPI-1 and SPI-2. SPI-1 is necessary to invade epithelial cells in the gut, whereas SPI-2 is necessary for replication in macrophages (Beuzón et al., 2000). Inflammasomes play an important role in innate immune defense against Stm because mice lacking caspase-1, IL-1β, or IL-18 succumb earlier to infection and have higher bacterial loads (Raupach et al., 2006). Stm expressing the SPI-1 T3SS induces caspase-1– and NLRC4–dependent cell death and IL-1β processing in cultured macrophages. Because NLRC4 is not essential for Stm clearance in mice (Lara-Tejero et al., 2006), we investigated whether multiple NLRs respond to Stm infection in the whole animal.     

Arg60 to Leu mutation of the human thromboxane A2 receptor in a dominantly inherited bleeding disorder.

Recent advances in molecular genetics have revealed the mechanisms underlying a variety of inherited human disorders. Among them, mutations in G protein-coupled receptors have clearly demonstrated two types of abnormalities, namely loss of function and constitutive activation of the receptors. Thromboxane A2 (TXA2) receptor is a member of the family of G protein-coupled receptors and performs an essential role in hemostasis by interacting with TXA2 to induce platelet aggregation. Here we identify a single amino acid substitution (Arg60-->Leu) in the first cytoplasmic loop of the TXA2 receptor in a dominantly inherited bleeding disorder characterized by defective platelet response to TXA2. This mutation was found exclusively in affected members of two unrelated families with the disorder. The mutant receptor expressed in Chinese hamster ovary cells showed decreased agonist-induced second messenger formation despite its normal ligand binding affinities. These results suggest that the Arg60 to Leu mutation is responsible for the disorder. Moreover, dominant inheritance of the disorder suggests the possibility that the mutation produces a dominant negative TXA2 receptor.     

A novel unstable mutation in mitochondrial DNA responsible for maternally inherited diabetes and deafness

OBJECTIVE

The m.3243A>G mutation in mitochondrial DNA (mtDNA) is responsible for maternally inherited diabetes and deafness (MIDD). Other mtDNA mutations are extremely rare.

RESEARCH DESIGN AND METHODS

We studied a patient presenting with diabetes and deafness who does not carry the m.3243A>G mutation.

RESULTS

We identified a deficiency of respiratory chain complex I in the patient’s fibroblasts. mtDNA sequencing revealed a novel mutation that corresponds to an insertion of one or two cytosine residues in the coding region of the MT-ND6 gene (m.14535_14536insC or CC), leading to premature stop codons. This heteroplasmic mutation is unstable in the patient’s somatic tissues.

CONCLUSIONS

We describe for the first time an unstable mutation in a mitochondrial gene coding for a complex I subunit, which is responsible for the MIDD phenotype. This mutation is likely favored by the m.14530T>C polymorphism, which is homoplasmic and leads to the formation of an 8-bp polyC tract responsible for genetic instability.The most common form of maternally inherited diabetes and deafness (MIDD) is associated with the m.3243A>G mutation in mitochondrial DNA (mtDNA), which is located in the tRNA^Leu gene (1). The mutation that affects up to 1% of diabetic patients leads to both impaired glucose-induced insulin secretion (2) and progressive β-cell loss (3). However, in some rare cases characterized by a highly suggestive phenotype but without m.3243A>G mutation, geneticists should look for other diabetes-prone variants (4). Here, we describe a patient presenting an MIDD phenotype who carries a novel unstable mutation in the mitochondrial MT-ND6 gene responsible for a deficiency in the respiratory chain complex I.     

Smad4 loss in mice causes spontaneous head and neck cancer with increased genomic instability and inflammation

Smad4 is a central mediator of TGF-β signaling, and its expression is downregulated or lost at the malignant stage in several cancer types. In this study, we found that Smad4 was frequently downregulated not only in human head and neck squamous cell carcinoma (HNSCC) malignant lesions, but also in grossly normal adjacent buccal mucosa. To gain insight into the importance of this observation, we generated mice in which Smad4 was deleted in head and neck epithelia (referred to herein as HN-Smad4^–/– mice) and found that they developed spontaneous HNSCC. Interestingly, both normal head and neck tissue and HNSCC from HN-Smad4^–/– mice exhibited increased genomic instability, which correlated with downregulated expression and function of genes encoding proteins in the Fanconi anemia/Brca (Fanc/Brca) DNA repair pathway linked to HNSCC susceptibility in humans. Consistent with this, further analysis revealed a correlation between downregulation of Smad4 protein and downregulation of the Brca1 and Rad51 proteins in human HNSCC. In addition to the above changes in tumor epithelia, both normal head and neck tissue and HNSCC from HN-Smad4^–/– mice exhibited severe inflammation, which was associated with increased expression of TGF-β1 and activated Smad3. We present what we believe to be the first single gene–knockout model for HNSCC, in which both HNSCC formation and invasion occurred as a result of Smad4 deletion. Our results reveal an intriguing connection between Smad4 and the Fanc/Brca pathway and highlight the impact of epithelial Smad4 loss on inflammation.     

Med12 gain-of-function mutation causes leiomyomas and genomic instability

Uterine leiomyomas are benign tumors that can cause pain, bleeding, and infertility in some women. Mediator complex subunit 12 (MED12) exon 2 variants are associated with uterine leiomyomas; however, the causality of MED12 variants, their genetic mode of action, and their role in genomic instability have not been established. Here, we generated a mouse model that conditionally expresses a Med12 missense variant (c.131G>A) in the uterus and demonstrated that this alteration alone promotes uterine leiomyoma formation and hyperplasia in both WT mice and animals harboring a uterine mesenchymal cell–specific Med12 deletion. Compared with WT animals, expression of Med12 c.131G>A in conditional Med12–KO mice resulted in earlier onset of leiomyoma lesions that were also greater in size. Moreover, leiomyomatous, Med12 c.131G>A variant–expressing uteri developed chromosomal rearrangements. Together, our results show that the common human leiomyoma–associated MED12 variant can cause leiomyomas in mice via a gain of function that drives genomic instability, which is frequently observed in human leiomyomas.     

Dominantly inherited hyperinsulinism caused by a mutation in the sulfonylurea receptor type 1

ATP-sensitive potassium channels play a major role in linking metabolic signals to the exocytosis of insulin in the pancreatic beta cell. These channels consist of two types of protein subunit: the sulfonylurea receptor SUR1 and the inward rectifying potassium channel Kir6.2. Mutations in the genes encoding these proteins are the most common cause of congenital hyperinsulinism (CHI). Since 1973, we have followed up 38 pediatric CHI patients in Finland. We reported previously that a loss-of-function mutation in SUR1 (V187D) is responsible for CHI of the most severe cases. We have now identified a missense mutation, E1506K, within the second nucleotide binding fold of SUR1, found heterozygous in seven related patients with CHI and in their mothers. All patients have a mild form of CHI that usually can be managed by long-term diazoxide treatment. This clinical finding is in agreement with the results of heterologous coexpression studies of recombinant Kir6.2 and SUR1 carrying the E1506K mutation. Mutant K(ATP) channels were insensitive to metabolic inhibition, but a partial response to diazoxide was retained. Five of the six mothers, two of whom suffered from hypoglycemia in infancy, have developed gestational or permanent diabetes. Linkage and haplotype analysis supported a dominant pattern of inheritance in a large pedigree. In conclusion, we describe the first dominantly inherited SUR1 mutation that causes CHI in early life and predisposes to later insulin deficiency.     

Evaluation of a mitochondrial DNA mutation in maternally inherited and sporadic cases of dupuytren disease

Objective The purpose was to test the hypothesis that Dupuytren disease (DD) is associated with a previously reported mutation in mitochondrial DNA at position 2839. Methods Two hundred sixty-nine cases of DD and an equal number of matched controls were identified in Marshfield Clinic's Personalized Medicine Research Project (PMRP). Clinical data used to describe the cohort were abstracted from the electronic medical records of the population. Genetic analysis of all the cases and controls was done using a custom synthesis TaqMan assay, while genetic analysis of sixteen of the above cases with a familial history of DD was performed by mitochondrial DNA sequencing at position C2839A. Results Cases and controls were evenly distributed with 167 (62%) men and 102 (38%) women. The majority, 264 (98%) of the cases and controls were white non-Hispanic. Of the 269 cases, 16 were found to have a familial history of DD. Two cases had a maternal history, eight a paternal history, five an affected sibling, and one a paternal grandfather. All cases and controls were found to have only the C allele at the site of the reported mitochondrial C2839A polymorphism. Conclusions The previously reported mitochondrial mutation was not present in our small, maternally inherited cohort or in the total population of 538 cases and controls. This finding does not support the reported incidence of this polymorphism in 90% of the affected population with a maternal inheritance, and calls into question the role of the C2839A mitochondrial DNA polymorphism in familial or sporadic cases of DD.     

Mastocytosis in mice expressing human Kit receptor with the activating Asp816Val mutation

Mastocytosis is a rare neoplastic disease characterized by a pathologic accumulation of tissue mast cells (MCs). Mastocytosis is often associated with a somatic point mutation in the Kit protooncogene leading to an Asp/Val substitution at position 816 in the kinase domain of this receptor. The contribution of this mutation to mastocytosis development remains unclear. In addition, the clinical heterogeneity presented by mastocytosis patients carrying the same mutation is unexplained. We report that a disease with striking similarities to human mastocytosis develops spontaneously in transgenic mice expressing the human Asp816Val mutant Kit protooncogene specifically in MCs. This disease is characterized by clinical signs ranging from a localized and indolent MC hyperplasia to an invasive MC tumor. In addition, bone marrow-derived MCs from transgenic animals can be maintained in culture for >24 mo and acquire growth factor independency for proliferation. These results demonstrate a causal link in vivo between the Asp816Val Kit mutation and MC neoplasia and suggest a basis for the clinical heterogeneity of human mastocytosis.