SQSTM1 Mutations Research Articles

Zoledronate in the prevention of Paget’s (ZiPP): protocol for a randomised trial of genetic testing and targeted zoledronic acid therapy to prevent SQSTM1-mediated Paget’s disease of bone

IntroductionPaget’s disease of bone (PDB) is characterised by increased and disorganised bone remodelling affecting one or more skeletal sites. Complications include bone pain, deformity, deafness and pathological fractures. Mutations in...

Familial Paget's disease of bone: Long-term follow-up of unaffected relatives with and without Sequestosome 1 mutations.

Familial Paget's disease of bone is inherited as an autosomal-dominant trait and mutations in the sequestosome 1 (SQSTM1) gene have been reported with variable frequency in patients with familial disease. The natural history, however, of the disease in family members with or without SQSTM1 mutations is unknown. To address this question, we investigated members of families with Paget's disease identified and genotyped in 2000 in The Netherlands without clinical, biochemical or radiological signs of Paget's disease. Seventy-five subjects, median age 56 years (range 44-93), with or without SQSTM1 mutations participated in the present study. Medical history was obtained and clinical examination and laboratory investigations were performed in all. When serum biochemical markers of bone turnover were increased, skeletal scintigraphy with SPECT-CT was performed. After a mean period of 15.9 ± 0.32 (SD) years no subject without SQSTM1 mutations (either from positive or negative families) developed Paget's disease. Of 14 carriers of SQSTM1 mutations, Paget's disease of the pelvis was diagnosed in a 74-year old asymptomatic woman. The incidence of new Paget's disease in SQSTM1 positive subjects was 7.1% and no mutation-negative subject developed the disease within 16 years of follow-up. Subjects without SQSTM1 mutations can be reassured whereas mutation carriers should consider screening. Our findings should be confirmed in other populations as currently unknown environmental factors that might be involved in the development of the disease may differ.

ALS-FTLD-linked mutations of SQSTM1/p62 disrupt selective autophagy and NFE2L2/NRF2 anti-oxidative stress pathway

ABSTRACTMacroautophagy (autophagy) is a key catabolic pathway for the maintenance of proteostasis through constant digestion of selective cargoes. The selectivity of autophagy is mediated by autophagy receptors that recognize and recruit cargoes to autophagosomes. SQSTM1/p62 is a prototype autophagy receptor, which is commonly found in protein aggregates associated with major neurodegenerative diseases. While accumulation of SQSTM1 implicates a disturbance of selective autophagy pathway, the pathogenic mechanism that contributes to impaired autophagy degradation remains poorly characterized. Herein we show that amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD)-linked mutations of TBK1 and SQSTM1 disrupt selective autophagy and cause neurotoxicity. Our data demonstrates that proteotoxic stress activates serine/threonine kinase TBK1, which coordinates with autophagy kinase ULK1 to promote concerted phosphorylation of autophagy receptor SQSTM1 at the UBA domain and activation of selective autophagy. In contrast, ALS-FTLD-linked mutations of TBK1 or SQSTM1 reduce SQSTM1 phosphorylation and compromise ubiquitinated cargo binding and clearance. Moreover, disease mutation SQSTM1G427R abolishes phosphorylation of Ser351 and impairs KEAP1-SQSTM1 interaction, thus diminishing NFE2L2/Nrf2-targeted gene expression and increasing TARDBP/TDP-43 associated stress granule formation under oxidative stress. Furthermore, expression of SQSTM1G427R in neurons impairs dendrite morphology and KEAP1-NFE2L2 signaling. Therefore, our results reveal a mechanism whereby pathogenic SQSTM1 mutants inhibit selective autophagy and disrupt NFE2L2 anti-oxidative stress response underlying the neurotoxicity in ALS-FTLD.Abbreviations: ALS: amyotrophic lateral sclerosis; FTLD: frontotemporal lobar degeneration; G3BP1: GTPase-activating protein (SH3 domain) binding protein 1; GSTM1: glutathione S-transferase, mu 1; HMOX/HO-1: Heme oxygenase 1; IP: immunoprecipitation; KEAP1: kelch-like ECH associated protein 1; KI: kinase inactive; KIR: KEAP1 interaction region; KO: knockout; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MBP: maltose binding protein; NBR1: NBR1, autophagy cargo receptor; NFE2L2/Nrf2: nuclear factor, erythroid derived 2, like 2; NQO1: NAD(P)H quinone dehydrogenase 1; SQSTM1/p62: sequestosome 1; SOD1: superoxide dismutase 1, soluble; S.S.: serum starvation; TARDBP/TDP-43: TAR DNA binding protein; TBK1: TANK binding kinase 1; UBA: ubiquitin association; ULK1: unc-51 like autophagy activating kinase 1; WT: wild type.

Molecular insights into an ancient form of Paget’s disease of bone

Paget's disease of bone (PDB) is a chronic skeletal disorder that can affect one or several bones in individuals older than 55 y of age. PDB-like changes have been reported in archaeological remains as old as Roman, although accurate diagnosis and natural history of the disease is lacking. Six skeletons from a collection of 130 excavated at Norton Priory in the North West of England, which dates to medieval times, show atypical and extensive pathological changes resembling contemporary PDB affecting as many as 75% of individual skeletons. Disease prevalence in the remaining collection is high, at least 16% of adults, with age at death estimations as low as 35 y. Despite these atypical features, paleoproteomic analysis identified sequestosome 1 (SQSTM1) or p62, a protein central to the pathological milieu of PDB, as one of the few noncollagenous human sequences preserved in skeletal samples. Targeted proteomic analysis detected >60% of the ancient p62 primary sequence, with Western blotting indicating p62 abnormalities, including in dentition. Direct sequencing of ancient DNA excluded contemporary PDB-associated SQSTM1 mutations. Our observations indicate that the ancient p62 protein is likely modified within its C-terminal ubiquitin-associated domain. Ancient miRNAs were remarkably preserved in an osteosarcoma from a skeleton with extensive disease, with miR-16 expression consistent with that reported in contemporary PDB-associated bone tumors. Our work displays the use of proteomics to inform diagnosis of ancient diseases such as atypical PDB, which has unusual features presumably potentiated by yet-unidentified environmental or genetic factors.

An FTLD-associated SQSTM1 variant impacts Nrf2 and NF-κB signalling and is associated with reduced phosphorylation of p62

Elevated oxidative stress has been implicated in the pathogenesis of amyotrophic lateral sclerosis (ALS) and Frontotemporal Lobar Degeneration (FTLD). In response to oxidative stress, the Nrf2 transcription factor activates protective antioxidant genes. A critical regulator of Nrf2 is the inhibitory protein Keap1, which mediates Nrf2 degradation. In response to cellular stress an interaction between Keap1 and SQSTM1/p62 (p62), a signalling adaptor protein, allows for increased Nrf2 signalling as it escapes degradation. Mutations in SQSTM1 (encoding p62) are linked with ALS-FTLD. Previously, two ALS-FTLD-associated p62 mutant proteins within the Keap1 interacting region (KIR) of p62 were found to be associated with decreased Keap1-p62 binding and Nrf2 activation. Here we report that a non-KIR domain FTLD-associated variant of p62 (p.R110C), affecting a residue close to the N-terminal PB1 oligomerisation domain, also reduces Keap1-p62 binding in cellulo and thereby reduces Nrf2 activity in reporter assays. Further, we observed that expression of p.R110C increased NF-κB activation compared with wild type p62. Altered signalling appeared to be linked with reduced phosphorylation of p62 at Serine residues −349 and −403. Our results confirm that ALS-FTLD mutations affecting multiple domains of p62 result in a reduced stress response, suggesting that altered stress signalling may directly contribute to the pathology of some ALS-FTLD cases.

Beyond dystonia and ataxia: Expanding the phenotype of SQSTM1 mutations

BackgroundHomozygous sequestomosome-1 gene mutations have been recently linked to neurodegeneration with dystonia, ataxia and gaze palsy. Seven affected families were identified thus far. ObjectiveTo describe four new cases with additional phenotypical features. ResultsFour affected patients from two unrelated families were identified. Two compound heterozygous variants of the gene (c.257_259delins35 and c.301+1G > T) were found in one family (cases 1 and 2), and homozygous c.823_824delAG variant was identified in cases 3 and 4. In addition to the previously described syndrome characterized by cerebellar ataxia, dystonia, choreoathetosis, cognitive impairment and gaze palsy, two subjects presented with iridoplegia. Furthermore, we report dysautonomic features such as orthostatic hypotension and sudomotor dysfunction, along with other non-motor symptoms. ConclusionsWe expand the phenotype of dystonia caused by Sequestomosome-1 gene by identifying dysautonomic features along with other non-motor symptoms.

Autophagy Inhibition Potentiates Ruxolitinib-Induced Apoptosis in JAK2V617F Cells

Introduction: Autophagy is a paradoxical and evolutionarily conserved cellular process that is fundamental to eliminating harmful intracellular components and allowing cells to adapt to nutrient starvation or stress conditions. Ruxolitinib, a selective JAK1/2 inhibitor approved by the FDA for treatment of intermediate and high-risk primary myelofibrosis and polycythemia vera patients, provides clinical benefits, but fail to eliminate the myeloproliferative neoplasm (MPN)-initiating cells. Frequency of mutations in autophagy-related genes has recently been reported in myeloid neoplasms (Visconte et al. Leukemia 2017). Considering that JAK2V617F mimics constitutive growth factors signal and leads to mTOR/PI3K activation, we hypothesized that ruxolitinib induces autophagy as a mechanism of resistance. Aims: To investigate the effects of ruxolitinib treatment on autophagy-related genes and cellular processes, the potential benefit of autophagy inhibitors plus ruxolitinib in JAK2V617F cells, and to verify frequency and clinical impact of autophagy-related genes mutations in MPN patients. Methods: SET2 cells (JAK2V617F-positive) were treated with increasing doses or exposure to ruxolitinib and subjected to protein extraction or autophagy analysis. Ruxotilinib (300 nM) and autophagy inhibitors (3-methyladenine [1 mM], bafilomycin A1 [10 nM] and chloroquine [20 μM]) were used in monotherapy or in combination. The expression of 79 autophagy-related genes was investigated in SET2 cells upon ruxolitinib treatment by RNA-seq (data obtained from Meyer et al. Cancer Cell 2015) and validated by qPCR and Western blot. Autophagy was evaluated by acridine orange staining and LC3BI/II or SQSTM1/p62 consumption, apoptosis by annexin V/PI staining and caspase 3 cleavage, and mitochondrial damage by JC-1 staining. Mutations in 124 autophagy-related genes were investigated by whole genome sequencing in a cohort of 65 MPN patients from a single Institution. ANOVA and Bonferroni post-test and Mann-Whitney test were used as appropriated. Kaplan-Meyer curves, log-rank test, and cox regression analysis were applied for survival analysis. Results: Ruxolitinib treatment promoted an accumulation of acidic vesicular organelles and consumption of LC3BI/II and SQSTM1/p62 proteins, characterizing autophagy induction. Using a 1.3-fold cutoff in both directions, treatment with ruxolitinib modulated the expression of 26 out of 79 autophagy-related genes investigated. BCL2 and BCL2L1 downregulation, BAX upregulation and stable BECN1 expression were validated, which impacted BCL2/BAX and BCL2/BECN1 ratios. In SET2 cells, ruxolitinib treatment reduced important autophagy regulators in a dose-dependent manner, including STAT3Y705, STAT5Y694, mTORS2448, p70S6KT421/S424 and 4EBP1T70 phosphorytation, and BCL2 and BCL-XL expression. All autophagy inhibitors significantly suppressed the ruxolitinib-induced autophagy, being bafilomycin A1 (reduction of 89%) and chroloquine (reduction of 70%) more efficient than 3-methyladenine (reduction of 10%) (all p<0.05). Autophagy pharmacological inhibition significantly increased ruxolitinib-induced apoptosis (all p<0.05) and accumulation of cells containing damaged mitochondria (all p<0.05). Mutations in autophagy-related genes were found in 25% (16 out of 65) of MPN patients and was associated with increased age (p<0.05). TP53 mutations were found in 6 patients and ARSA, TGFB1, APP, PPP2R3A, BECN1, SQSTM1, HGS, TNFSF13B, ATG2A, STAT3, ATG9B, WAC or PRKACB mutations were found in one case each. Mutated patients presented JAK2 (n=14), CALR (n=1) or MPL (n=1) mutations. Median overall survival for autophagy-related genes mutated and wild-type patients were 20 months vs. not reached (p=0.066), respectively. In multivariate analysis using mutations in TP53 only, mutations in other autophagy-related genes, and DIPSS as confounders, only DIPSS independently predicted overall survival (p<0.001). Conclusions: Ruxolitinib induced autophagy in JAK2V617F cells through modulation of mTOR/p70S6K/4EBP1 and STAT/BCL2 axis, which potentially represent a mechanism of resistance to apoptosis. Our results indicate that combination of ruxolitinib with autophagy pharmacological inhibitors, especially chloroquine, may be a promissory strategy to improving apoptosis induction in JAK2V617F cells. DisclosuresNo relevant conflicts of interest to declare.

Biallelic SQSTM1 mutations in early-onset, variably progressive neurodegeneration.

To characterize clinically and molecularly an early-onset, variably progressive neurodegenerative disorder characterized by a cerebellar syndrome with severe ataxia, gaze palsy, dyskinesia, dystonia, and cognitive decline affecting 11 individuals from 3 consanguineous families. We used whole-exome sequencing (WES) (families 1 and 2) and a combined approach based on homozygosity mapping and WES (family 3). We performed in vitro studies to explore the effect of the nontruncating SQSTM1 mutation on protein function and the effect of impaired SQSTM1 function on autophagy. We analyzed the consequences of sqstm1 down-modulation on the structural integrity of the cerebellum in vivo using zebrafish as a model. We identified 3 homozygous inactivating variants, including a splice site substitution (c.301+2T>A) causing aberrant transcript processing and accelerated degradation of a resulting protein lacking exon 2, as well as 2 truncating changes (c.875_876insT and c.934_936delinsTGA). We show that loss of SQSTM1 causes impaired production of ubiquitin-positive protein aggregates in response to misfolded protein stress and decelerated autophagic flux. The consequences of sqstm1 down-modulation on the structural integrity of the cerebellum in zebrafish documented a variable but reproducible phenotype characterized by cerebellum anomalies ranging from depletion of axonal connections to complete atrophy. We provide a detailed clinical characterization of the disorder; the natural history is reported for 2 siblings who have been followed up for >20 years. This study offers an accurate clinical characterization of this recently recognized neurodegenerative disorder caused by biallelic inactivating mutations in SQSTM1 and links this phenotype to defective selective autophagy.

Systemic overexpression of SQSTM1/p62 accelerates disease onset in a SOD1H46R-expressing ALS mouse model

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by a selective loss of upper and lower motor neurons. Recent studies have shown that mutations in SQSTM1 are linked to ALS. SQSTM1 encodes SQSTM1/p62 that regulates not only autophagy via the association with MAP1LC3/LC3 and ubiquitinated proteins but also the KEAP1-NFE2L2/Nrf2 anti-oxidative stress pathway by interacting with KEAP1. Previously, we have demonstrated that loss of SQSTM1 exacerbates disease phenotypes in a SOD1H46R-expressing ALS mouse model. To clarify the effects of SQSTM1 overexpression in this model, we generated SQSTM1 and SOD1H46R double-transgenic (SQSTM1;SOD1H46R) mice. SQSTM1;SOD1H46R mice exhibited earlier disease onset and shorter lifespan than did SOD1H46R mice. Conversely, disease progression after the onset rather slightly but significantly slowed in SQSTM1;SOD1H46R mice. However, there were observable differences neither in the number of Nissl positive neurons nor in the distribution of ubiquitin-positive and/or SQSTM1-positive aggregates between SOD1H46R and SQSTM1;SOD1H46R mice. It was noted that these protein aggregates were mainly observed in neuropil, and partly localized to astrocytes and/or microglia, but not to MAP2-positive neuronal cell bodies and dendrites at the end-stage of disease. Nonetheless, the biochemically-detectable insoluble SQSTM1 and poly-ubiquitinated proteins were significantly and progressively increased in the spinal cord of SQSTM1;SOD1H46R mice compared to SOD1H46R mice. These results suggest that overexpression of SQSTM1 in SOD1H46R mice accelerates disease onset by compromising the protein degradation pathways.

Myopathy With SQSTM1 and TIA1 Variants: Clinical and Pathological Features.

The aim of this study is to identify the molecular defect of three unrelated individuals with late-onset predominant distal myopathy; to describe the spectrum of phenotype resulting from the contributing role of two variants in genes located on two different chromosomes; and to highlight the underappreciated complex forms of genetic myopathies. Clinical and laboratory data of three unrelated probands with predominantly distal weakness manifesting in the sixth-seventh decade of life, and available affected and unaffected family members were reviewed. Next-generation sequencing panel, whole exome sequencing, and targeted analyses of family members were performed to elucidate the genetic etiology of the myopathy. Genetic analyses detected two contributing variants located on different chromosomes in three unrelated probands: a heterozygous pathogenic mutation in SQSTM1 (c.1175C>T, p.Pro392Leu) and a heterozygous variant in TIA1 (c.1070A>G, p.Asn357Ser). The affected fraternal twin of one proband also carries both variants, while the unaffected family members harbor one or none. Two unrelated probands (family 1, II.3, and family 3, II.1) have a distal myopathy with rimmed vacuoles that manifested with index extensor weakness; the other proband (family 2, I.1) has myofibrillar myopathy manifesting with hypercapnic respiratory insufficiency and distal weakness. The findings indicate that all the affected individuals have a myopathy associated with both variants in SQSTM1 and TIA1, respectively, suggesting that the two variants determine the phenotype and likely functionally interact. We speculate that the TIA1 variant is a modifier of the SQSTM1 mutation. We identify the combination of SQSTM1 and TIA1 variants as a novel genetic defect associated with myofibrillar myopathy and suggest to consider sequencing both genes in the molecular investigation of myopathy with rimmed vacuoles and myofibrillar myopathy although additional studies are needed to investigate the digenic nature of the disease.

TIA1 variant drives myodegeneration in multisystem proteinopathy with SQSTM1 mutations.

Multisystem proteinopathy (MSP) involves disturbances of stress granule (SG) dynamics and autophagic protein degradation that underlie the pathogenesis of a spectrum of degenerative diseases that affect muscle, brain, and bone. Specifically, identical mutations in the autophagic adaptor SQSTM1 can cause varied penetrance of 4 distinct phenotypes: amyotrophic lateral sclerosis (ALS), frontotemporal dementia, Paget's disease of the bone, and distal myopathy. It has been hypothesized that clinical pleiotropy relates to additional genetic determinants, but thus far, evidence has been lacking. Here, we provide evidence that a TIA1 (p.N357S) variant dictates a myodegenerative phenotype when inherited, along with a pathogenic SQSTM1 mutation. Experimentally, the TIA1-N357S variant significantly enhances liquid-liquid-phase separation in vitro and impairs SG dynamics in living cells. Depletion of SQSTM1 or the introduction of a mutant version of SQSTM1 similarly impairs SG dynamics. TIA1-N357S-persistent SGs have increased association with SQSTM1, accumulation of ubiquitin conjugates, and additional aggregated proteins. Synergistic expression of the TIA1-N357S variant and a SQSTM1-A390X mutation in myoblasts leads to impaired SG clearance and myotoxicity relative to control myoblasts. These findings demonstrate a pathogenic connection between SG homeostasis and ubiquitin-mediated autophagic degradation that drives the penetrance of an MSP phenotype.

Identification of a Novel Hemizygous SQSTM1 Nonsense Mutation in Atypical Behavioral Variant Frontotemporal Dementia.

Frontotemporal dementia includes a large spectrum of neurodegenerative disorders. SQSTM1, coding for p62 protein, plays a vital role in the pathogenesis of FTD. Here, we report a case of a female patient with SQSTM1 mutation S224X, who was 59 years old when she initially exhibited memory decline, mild personality changes, and subtle atrophy of frontal/temporal lobes in magnetic resonance imaging (MRI). Genetic testing revealed a nonsense mutation of the SQSTM1 gene (S224X), resulting in premature termination of protein synthesis and a predicted truncated protein 217 amino acids shorter than the normal protein. Moreover, neither intact nor truncated SQSTM1 proteins was detectable in SQSTM1 S224X mutant overexpressing HEK-293T cells. We assayed for SQSTM1 cDNA in samples from the patient's peripheral leucocytes, and did not detect its mutation. The test of quantitative PCR showed significant decreased level of SQSTM1 mRNA from peripheral leucocytes of the patient compared to five dementia controls. Our results identify a novel pathogenic SQSTM1 S224X mutation in an atypical FTD patient accompanied with loss of SQSTM1/p62 protein expression probably due to SQSTM1 gene haploinsufficiency.

Haploinsufficiency leads to neurodegeneration in C9ORF72 ALS/FTD human induced motor neurons.

An intronic GGGGCC repeat expansion in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the pathogenic mechanism of this repeat remains unclear. Using human induced motor neurons (iMNs), we found that repeat-expanded C9ORF72 was haploinsufficient in ALS. We found that C9ORF72 interacted with endosomes and was required for normal vesicle trafficking and lysosomal biogenesis in motor neurons. Repeat expansion reduced C9ORF72 expression, triggering neurodegeneration through two mechanisms: accumulation of glutamate receptors, leading to excitotoxicity, and impaired clearance of neurotoxic dipeptide repeat proteins derived from the repeat expansion. Thus, cooperativity between gain- and loss-of-function mechanisms led to neurodegeneration. Restoring C9ORF72 levels or augmenting its function with constitutively active RAB5 or chemical modulators of RAB5 effectors rescued patient neuron survival and ameliorated neurodegenerative processes in both gain- and loss-of-function C9ORF72 mouse models. Thus, modulating vesicle trafficking was able to rescue neurodegeneration caused by the C9ORF72 repeat expansion. Coupled with rare mutations in ALS2, FIG4, CHMP2B, OPTN and SQSTM1, our results reveal mechanistic convergence on vesicle trafficking in ALS and FTD.

Investigating CCNF mutations in a Taiwanese cohort with amyotrophic lateral sclerosis

Mutations in the cyclin F gene (CCNF) have been recently identified in a small number of patients with amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia, and their role in patients with ALS in Taiwan remains elusive. The aim of this study was to elucidate the frequency and spectrum of CCNF mutations in a Taiwanese ALS cohort of Han Chinese origin. Mutational analyses of the CCNF gene were performed using Sanger sequencing in a cohort of 255 unrelated patients with ALS. Among these patients, the genetic diagnoses of 204 patients remained unclear after mutations in SOD1, C9ORF72, TARDBP, FUS, ATXN2, OPTN, VCP, UBQLN2, SQSTM1, PFN1, HNRNPA1, HNRNPA2B1, MATR3, CHCHD10, TUBA4A, and TKB1 had been investigated. Two novel heterozygous missense mutations in CCNF, p.S222P (c.664T>C) and p.S532R (c.1596C>T), were identified; 1 in each patient with apparently sporadic ALS. In vitro functional study demonstrated that both mutations result in a general and cyclin F-mediated ubiquitin-proteasome pathway dysfunction. The frequency of CCNF mutations in ALS patients in Taiwan is, therefore, approximately 0.8% (2/255). These findings expand the mutational spectrum of CCNF and also emphasize the pathogenic role of CCNF mutations in ALS.

The Changing Presentation of Paget's Disease of Bone in Australia, A High Prevalence Region.

Studies from several countries suggest that the incidence of Paget's disease of bone (PDB) and the severity of newly diagnosed cases are declining. The aim of this study was to examine secular changes in clinical presentation of PDB in Australia, which historically had the highest prevalence outside the United Kingdom. The participants were 293 patients (61% male) diagnosed between 1956 and 2013 with details recorded in the database of the Paget's Disease Research Group of Western Australia. The mean age at diagnosis was 62years (range 28-90); 26% of participants had a family history of PDB and 11% had Sequestosome 1 (SQSTM1) mutations. After adjustment for covariates (SQSTM1 mutation status, family history, country of birth, smoking and dog exposure), there was a significant positive relationship between year of diagnosis and age at diagnosis (P<0.001) and significant negative relationships between year of diagnosis and both pre-treatment total plasma alkaline phosphatase activity (ALP) and number of involved bones (P<0.001 for each). Patients with SQSTM1 mutations had more extensive disease (P<0.001) and higher pre-treatment ALP (P=0.013). In subgroup analyses, relationships between year of diagnosis and each of age at diagnosis, number of involved bones and ALP were similar in patients with sporadic or familial disease, and in patients with and without SQSTM1 mutations. We conclude that the severity of PDB in Western Australia has declined over recent decades. This is likely to reflect altered exposure to one or more environmental agents involved in pathogenesis.

The wide genetic landscape of clinical frontotemporal dementia: systematic combined sequencing of 121 consecutive subjects

PurposeTo define the genetic spectrum and relative gene frequencies underlying clinical frontotemporal dementia (FTD).MethodsWe investigated the frequencies and mutations in neurodegenerative disease genes in 121 consecutive FTD subjects using an unbiased, combined sequencing approach, complemented by cerebrospinal fluid Aβ1-42 and serum progranulin measurements. Subjects were screened for C9orf72 repeat expansions, GRN and MAPT mutations, and, if negative, mutations in other neurodegenerative disease genes, by whole-exome sequencing (WES) (n = 108), including WES-based copy-number variant (CNV) analysis.ResultsPathogenic and likely pathogenic mutations were identified in 19% of the subjects, including mutations in C9orf72 (n = 8), GRN (n = 7, one 11-exon macro-deletion) and, more rarely, CHCHD10, TARDBP, SQSTM1 and UBQLN2 (each n = 1), but not in MAPT or TBK1. WES also unraveled pathogenic mutations in genes not commonly linked to FTD, including mutations in Alzheimer (PSEN1, PSEN2), lysosomal (CTSF, 7-exon macro-deletion) and cholesterol homeostasis pathways (CYP27A1).ConclusionOur unbiased approach reveals a wide genetic spectrum underlying clinical FTD, including 11% of seemingly sporadic FTD. It unravels several mutations and CNVs in genes and pathways hitherto not linked to FTD. This suggests that clinical FTD might be the converging downstream result of a delicate susceptibility of frontotemporal brain networks to insults in various pathways.

SQSTM1 MUTATIONS IN FRONTOTEMPORAL DEMENTIA ARE ASSOCIATED WITH ASYMMETRICAL FOCAL TEMPORAL LOBE ATROPHY

FTD is a common cause of young onset dementia and in around a third of cases is genetic. Recently mutations in SQSTM1 have been shown to be a rare cause of familial FTD. Little is known about the clinical or neuroanatomical phenotype at present. In this study, we investigated the pattern of grey matter atrophy in a group of patients with SQSTM1-associated FTD. SQSTM1 variants known to be a cause of familial FTD, or strongly increase risk, were found in four patients in the UCL FTD DNA cohort (n=440): two P392L, 1 E155K and 1 E396 frameshift mutation. The patient with the E396fs mutation was also found to have a C9orf72 expansion. The clinical diagnoses in the four patients were behavioural variant FTD, FTD with motor neurone disease, corticobasal syndrome and primary progressive aphasia. The mean (standard deviation) age at onset was 59.0 (5.9). All patients had undergone a T1-weighted magnetic resonance imaging scan acquired on a 3T Siemens Trio scanner: age at scan 65.8 (6.4). Their imaging was compared with 24 age- and gender-matched healthy controls (age: 66.5 (5.2)). We undertook a voxel-based morphometry (VBM) analysis comparing the SQSTM1 mutation carriers and the controls, correcting for age, gender and total intracranial volume. Statistical parametric maps were thresholded at p<0.05 after family-wise error correction and rendered on a study-specific average group T1-weighted MRI template image in MNI space. Grey matter atrophy was asymmetrical with three patients having right-sided dominant atrophy and one left-sided. For the analysis we flipped the scan in the midsagittal plane of the patient with left-sided atrophy so that the hemisphere in which there was predominant atrophy was the same in all four cases. The VBM analysis showed evidence of focal involvement of the antero-inferior-medial temporal lobe, particularly affecting the temporal pole, amygdala, hippocampus, entorhinal cortex, parahippocampal gyrus, and fusiform gyrus. There was an anterior-posterior gradient of atrophy as well as inferior-superior and medial-lateral gradients. In summary, patients with SQSTM1 mutations can have a varied clinical phenotype but appear to be particularly associated with focal temporal lobe atrophy that can predominantly affect the right or left hemisphere.

Adult Paget's disease of bone: a review.

Adult PD of bone is the second commonest metabolic bone condition after osteoporosis. The condition is characterized by increased bone cell activity, with bone-resorbing osteoclasts often larger and containing more nuclei than normal, and osteoblasts producing increased amounts of disorganized bone. This leads to expanded bone of poor quality possessing both sclerotic and lytic areas. PD of bone has a strong genetic element, with a family history being noted in 10-20% of cases. A number of genetic defects have been found to be associated with the condition. The most common disease-associated variants identified affect the SQSTM1 gene, providing insights into disease aetiology, with the clinical value of knowledge of SQSTM1 mutation status currently under active investigation. The diagnosis may be suggested by an isolated raised total ALP without other identifiable causes. This can be confirmed on plain X-rays and the extent determined by isotope bone scan. The mainstays of treatment are the bisphosphonates, especially i.v. zoledronate, which results in long-term suppression of bone turnover. ALP is the usual means of monitoring the condition, although more specific bone turnover markers can be helpful, especially in coincident liver disease. Patients should be followed up to monitor for biochemical relapse or development of complications, which may require medical or surgical intervention.

ALS-FTLD associated mutations of SQSTM1 impact on Keap1-Nrf2 signalling.

The transcription factor Nrf2 and its repressor protein Keap1 play key roles in the regulation of antioxidant stress responses and both Keap1-Nrf2 signalling and oxidative stress have been implicated in the pathogenesis of the ALS-FTLD spectrum of neurodegenerative disorders. The Keap1-binding partner and autophagy receptor SQSTM1/p62 has also recently been linked genetically to ALS-FTLD, with some missense mutations identified in patients mapping within or close to its Keap1-interacting region (KIR, residues 347–352). Here we report the effects on protein function of four different disease associated mutations of SQSTM1/p62 which affect the KIR region. Only mutations mapping precisely to the KIR (P348L and G351A) were associated with a loss of Keap1 binding in co-immunoprecipitations comparable to wild-type SQSTM1/p62. These selective effects on Keap1 recognition were entirely rational based on protein structural models. Consistent with impaired Keap1 binding, the P348L and G351A KIR mutants showed reduced ability to activate Nrf2 signalling compared to wild-type SQSTM1/p62 in antioxidant response element (ARE)-luciferase reporter assays. The results suggest that SQSTM1 mutations within the KIR of SQSTM1/p62 contribute to aetiology of some cases of ALS-FTLD through a mechanism involving aberrant expression or regulation of oxidative response genes.

Neuroimaging Correlates of Frontotemporal Dementia Associated with SQSTM1 Mutations.

Frontotemporal lobar degeneration (FTLD) is a progressive dementia characterized by focal atrophy of frontal and/or temporal lobes caused by mutations in the gene coding for sequestosome 1 (SQSTM1), among other genes. Rare SQSTM1 gene mutations have been associated with Paget's disease of bone, amyotrophic lateral sclerosis, and, more recently, frontotemporal lobar degeneration (FTLD). The aim of the study was to determine whether a characteristic pattern of grey and white matter loss is associated with SQSTM1 dysfunction. We performed a voxel-based morphometry (VBM) study in FTD subjects carrying SQSTM1 pathogenic variants (FTD/SQSTM1 mutation carriers; n = 10), compared with FTD subjects not carrying SQSTM1 mutations (Sporadic FTD; n = 20) and healthy controls with no SQSTM1 mutations (HC/SQSTM1 noncarriers; n = 20). The groups were matched according to current age, disease duration, and gender. After comparing FTD/SQSTM1 carriers with Sporadic FTD, a predominantly right cortical atrophy pattern was localized in the inferior frontal, medial orbitofrontal, precentral gyri, and the anterior insula. White matter atrophy was found in both medial and inferior frontal gyri, pallidum, and putamen. FTD/SQSTM1 carriers compared with HC/SQSTM1 noncarriers showed atrophy at frontal, temporal, and parietal lobes of both hemispheres whereas the MRI pattern found in Sporadic FTD compared with controls was frontal and left temporal lobe atrophy, extending toward parietal and occipital lobes of both hemispheres. These results suggest that fronto-orbito-insular regions including corticospinal projections as described in ALS are probably more susceptible to the damaging effect of SQSTM1 mutations delineatinga specific gene-linked atrophy pattern.