SQSTM1 Mutations in Familial and Sporadic Amyotrophic Lateral Sclerosis

ORIGINAL CONTRIBUTION SQSTM1 Mutations in Familial and Sporadic Amyotrophic Lateral Sclerosis

Faisal Fecto, MD; Jianhua Yan, MD, PhD; S. Pavan Vemula; Erdong Liu, MD; Yi Yang, MS; Wenjie Chen, MD; Jian Guo Zheng, MD; Yong Shi, MD, PhD; Nailah Siddique, RN, MSN; Hasan Arrat, MD; Sandra Donkervoort, MS; Senda Ajroud-Driss, MD; Robert L. Sufit, MD; Scott L. Heller, MD; Han-Xiang Deng, MD, PhD; Teepu Siddique, MD

Background: The SQSTM1 gene encodes p62, a major In silico analysis of variants was performed to predict al- pathologic protein involved in neurodegeneration. terations in p62 structure and function.

Objective: To examine whether SQSTM1 mutations con- Results: We identified 10 novel SQSTM1 mutations (9 tribute to familial and sporadic amyotrophic lateral scle- heterozygous missense and 1 deletion) in 15 patients (6 rosis (ALS). with familial ALS and 9 with sporadic ALS). Predictive in silico analysis classified 8 of 9 missense variants as Design: Case-control study. pathogenic.

Setting: Academic research. Conclusions: Using candidate gene identification based on prior biological knowledge and the functional pre- Patients: A cohort of 546 patients with familial diction of rare variants, we identified several novel (n=340) or sporadic (n=206) ALS seen at a major aca- SQSTM1 mutations in patients with ALS. Our findings demic referral center were screened for SQSTM1 muta- provide evidence of a direct genetic role for p62 in ALS tions. pathogenesis and suggest that regulation of protein degradation pathways may represent an important thera- Main Outcome Measures: We evaluated the distri- peutic target in motor neuron degeneration. bution of missense, deletion, silent, and intronic variants in SQSTM1 among our cohort of patients with ALS. Arch Neurol. 2011;68(11):1440-1446

MYOTROPHIC LATERAL SCLE- (also known as sequestosome 1). Ini- rosis (ALS) is a fatal para- tially, p62 was identified as a novel ubiq- lytic disorder caused by uitin-binding protein that acts as a phos- degeneration of motor photyrosine-independent ligand of the neurons in the brain and p56lck Src homology 2 (SH2) do- Author Affiliations: Division of spinal cord. About 90% of ALS is spo- main.10,11 Subsequently, p62 has been Neuromuscular Medicine, Ken A radic (SALS) with unknown cause. Famil- shown to have an important dual role in and Ruth Davee Department of 12 Neurology and Clinical ial ALS (FALS) is genetically heteroge- protein degradation via the proteasome Neurological Sciences neous and represents approximately 5% and as a link between protein aggrega- 13 (Drs Fecto, Yan, Liu, Chen, to 10% of ALS cases. The penetrance of ge- tion and autophagy via its interaction Zheng, Shi, Arrat, Ajroud-Driss, netic mutation–linked FALS may vary sub- with LC3/Atg8.14 Dysfunction in these Sufit, Heller, Deng, and stantially, ranging from classic mende- pathways has been implicated in various T. Siddique and Messrs Vemula lian pattern to apparently sporadic disease. forms of neurodegeneration. For ex- and Yang and Mss N. Siddique Mutations in the copper-zinc superoxide ample, p62 is present in neuronal and glial and Donkervoort) and dismutase gene (SOD1) account for ap- ubiquitin-positive inclusions of Alzhei- Department of Cell and proximately 20% of FALS cases and 1% of mer disease, Pick disease, dementia with Molecular Biology SALS cases and represent the most preva- Lewy bodies, Parkinson disease, and mul- (Dr T. Siddique), Feinberg 1,2 15,16 School of Medicine, and lent known cause of ALS. Several other tiple system atrophy. More recently, Interdepartmental Neuroscience genes, including TARDBP, FUS, OPTN, and p62 was shown to aggregate in patients 3-9 Program (Drs Fecto and VCP, have recently been linked to ALS. with ALS and in the G93A SOD1 mouse T. Siddique), Northwestern The SQSTM1 (OMIM 601530) gene en- model of FALS.17,18 Overexpression of p62 University, Chicago, Illinois. codes the ubiquitin-binding protein p62 with mutant SOD1 in NSC34 cells en-

ARCH NEUROL / VOL 68 (NO. 11), NOV 2011 WWW.ARCHNEUROL.COM 1440

©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 Table 1. SQSTM1 Variants in Patients With Familial Amyotrophic Lateral Sclerosis (FALS) and in Patients With Sporadic Amyotrophic Lateral Sclerosis (SALS)

Patients Patients Patients With SALS Control Exon Change, bp Variant With FALS With SALS or FALS Subjects 1 c.98CϾT A33V 1/340 2/206 3/546 0/724 1 g.3Јϩ7GϾC Intronic 1/340 0/206 1/546 0/724 3 c.457GϾA V153I 0/340 2/206 2/546 0/724 4 g.5Ј − 37CϾT Intronic 1/340 2/206 3/546 0/738 5 c.683CϾT P228L 0/340 1/206 1/546 0/724 5 c.702GϾA V234V 1/340 0/206 1/546 0/724 5 c.714-716delGAA K238del 0/340 1/206 1/546 0/724 6 c.783CϾT H261H 0/340 1/206 1/546 0/738 6 c.952TϾC S318P 1/340 0/206 1/546 0/738 6 c.961CϾT R321C 0/340 1/206 1/546 0/738 7 c.1108TϾC S370P 1/340 0/206 1/546 0/733 8 c.1175CϾT P392L 2/340 1/206 3/546 0/737 8 c.1231GϾA G411S 1/340 0/206 1/546 0/737 8 c.1273GϾA G425R 0/340 1/206 1/546 0/737

Abbreviation: bp, base pair.

hances aggregate formation, and this effect is signifi- supported probable ALS.24 All the ALS cases were negative cantly diminished when the ubiquitin-associated (UBA) for mutations in the SOD1, TARDBP, and FUS genes. By self- domain (UBA) of p62 is deleted.17 Mutant SOD1 can be reported race/ethnicity, 93.9% of the patients with ALS were recognized by p62 in a ubiquitin-independent fashion and white (European American), 2.5% Asian, 1.9% African targeted for autophagy.19 Furthermore, p62 colocalizes American, and 1.3% Latino. The race/ethnicity of 2 patients was unknown. By self-report, 97.6% of controls were white, with TDP-43 in brains of patients having frontotempo- 1.0% Latino, 0.8% Asian, and 0.6% African American. ral lobe degeneration with motor neuron disease.20 It was recently shown that p62 colocalizes with FUS and TDP-43 in ubiquitinated inclusions among motor neurons in spi- SEQUENCING ANALYSIS OF SQSTM1 nal cords from patients with SALS, non-SOD1 FALS, and ALS with dementia.21 In addition, overexpression of p62 Genomic DNA was extracted from transformed lymphoblas- reduces TDP-43 aggregation in an autophagy- and pro- toid cell lines or whole blood using standard protocols (Qia- 22 gen, Valencia, California). Intronic primers covering the teasome-dependent manner. Also, p62 knockout mice coding sequence were designed at least 50 base pairs away develop memory loss after neurodegeneration caused by from the intron and exon boundaries. Primers were designed accumulation of hyperphosphorylated tau and neurofi- using several products (Oligo Analyzer [IDT, Coralville, brillary tangles.23 Although some proteins may partici- Iowa], ExonPrimer [Institute of Human Genetics, Cologne, pate in pathogenic aggregates in a wide variety of neu- Germany], and UCSC Genome Browser [Center for Biomo- rodegenerative disorders, recent investigations indicate lecular Science & Engineering, Santa Cruz, California]). that they cause very specific disease phenotypes when Genomic DNA was amplified according to standard proto- mutant. Choosing candidate genes based on prior bio- cols. Unconsumed deoxyribonucleotide triphosphates and logical knowledge can identify causative genes for neu- primers were digested (ExoSAP-IT; USB, Cleveland, Ohio). rodegenerative disorders, including ALS. If present in ALS, Fluorescent dye–labeled single-strand DNA was amplified using sequencing reagents (GenomeLab DTCS Quick Start SQSTM1 mutations may have an increased aggregation Kit; Beckman Coulter, Fullerton, California), followed by potential. To investigate the role of p62 in ALS, we single-pass bidirectional sequencing (CEQ 8000 Genetic screened a large cohort of patients with ALS for SQSTM1 Analysis System, Beckman Coulter). Forward primer was mutations. used for mutation screening, and all variations were confirmed by reverse sequencing. When a variant was identified, METHODS it was first excluded in the dbSNP Short Genetic Variations (http://www.ncbi.nlm.nih.gov/snp) and 1000 Genomes Proj- ect25 databases, and then more than 724 normal control DNA PARTICIPANTS samples (Table 1) were analyzed to exclude the possibility of a common polymorphism. A cohort of 340 patients with FALS, 206 patients with SALS, and 738 neurologically healthy control subjects was ascer- tained from our neurological diseases registry, in which par- BIOINFORMATICS ticipants are enrolled after informed consent is obtained. Pedigrees and clinical data were collected according to pro- An Internet server (NetPhos 2.0 [http://www.cbs.dtu.dk/services tocols approved by our institutional review board and met /NetPhos/]) was used to predict changes in phosphorylation Health Insurance Portability and Accountability Act stan- sites among variants identified during sequencing.26 Variants dards of confidentiality and disclosure. All the patients were were also analyzed using programs (SIFT27 and PMUT28)topre- diagnosed by board-certified neurologists and met the dict the effect of the mutations on p62. Three-dimensional mod- revised El Escorial World Federation of Neurology criteria eling was performed (Swiss-PdbViewer29) using 1Q02A back- for diagnosis of clinically definite, probable, or laboratory- bone as a template.

ARCH NEUROL / VOL 68 (NO. 11), NOV 2011 WWW.ARCHNEUROL.COM 1441

1 2 3 4 5 6 7 8

B K238del R321C G411S A33V V153I P228L S318P S370P P392L G425R

SH2 AID ZZ TRAF6 PEST PEST UBA

C A33V V153I P228L K238del S318P R321C S370P P392L G411S G425R Mutation – – – V – – – – – – I – – – – – – L – – – – – – Δ – – – – – – P – – – – – – C – – – – – – P – – – – – – L – – – – – – S – – – – – – R – – –

Homo sapiens EPEAEAE LCSVCEG ASGPSED NFLKNVG ALESEG – – – – RPEE LDPSQ – E EADPRLI DEGGWLT YDIGAAL Pan troglodytes EPEAEAE LCSVCEG ASGPSED NFLKNVG ALESEG – – – – RPEE LDPSQ – E EADPRLI DEGGWLT YDIGAAL Bos taurus EPEAEAE LCSACEG ASGPSED NFLKNVG ALESGG – – – – QHEE LDPSQ – E EADPRLI DEGGWLT YDIGAAL Mus musculus EPEAEAQ LCSVCEG ASAPPED NFLKNVG ALESVG – – – – QPEE LDPSQ – E EADPRLI DEGGWLT YDIGAAL Rattus norvegicus EPEAE – – LCSVCEG ASAPSED NFLKNVG ALESVG – – – – QPEE LDPSQ – E EADPRLI DEGGWLT YDIGAAL Gallus gallus – – – – – – – LCSTCEG TNSQPQD TFLKNVG VIDPVPT GSLQSQE LDVAQ – E EADPRLI DEGGWLT CDIGAAL Danio rerio – – – – – – – LCPTCQS ATASSQ– EYLKNIG – – – – – – – – – – SVSE LNTASGT DADPRLV DEGGWLT YDIGGAL

Figure. SQSTM1 mutations in amyotrophic lateral sclerosis. A, Structure of SQSTM1 indicating coding regions (thick bars) and introns (thin lines). The white boxes indicate the untranslated regions. The location of each mutation identified in amyotrophic lateral sclerosis is shown by diamonds above the exons. B, Primary structure of p62 indicating all the major domains. SH2 indicates Src homology 2–binding domain; AID, acidic interaction domain; ZZ, zinc finger domain; TRAF6, tumor necrosis factor receptor–associated factor 6–binding domain; PEST, proline, glutamic acid, serine, and threonine–rich region; and UBA, ubiquitin-associated domain. The arrowheads indicate the position of each amino acid change identified in our cohort. C, Alignment of p62 sequences from different species. Sequence cluster alignment was performed with a system (HomoloGene; National Center for Biotechnology Information, Bethesda, Maryland) that uses BlastP to compare related sequences. Mutated residues are boxed. Sequences used include NP_003891.1 (Homo sapiens), XP_518154.2 (Pan troglodytes), NP_788814.1 (Bos taurus), NP_035148.1 (Mus musculus), NP_787037.2 (Rattus norvegicus), XP_001233249.1 (Gallus gallus), and NP_998338.1 (Danio rerio).

STATISTICAL ANALYSIS of samples from additional family members. The P392L variant was also present in 1 patient with SALS. The V153I Data were analyzed using available software (PSPP for Win- change was present in 2 patients with SALS. The fre- dows [http://pspp.awardspace.com/]). Case-control genotype quency of these variants in our cohort of patients with ␹2 associations were assessed using analyses, and odds ratios ALS was 2.8%. were calculated. Estimates of departure from Hardy- Weinberg equilibrium were calculated using the ␹2 test. A sta- We also identified 2 silent and 2 intronic variants ex- tistical program (QuickCalcs; GraphPad Software Inc, La Jolla, clusively in our ALS cohort that were not present in con- California) was used to perform the 2-tailed Fisher exact test trols (Table 1). Several other rare and common variants for comparing rare variant frequencies. Clinical data were ana- were identified in cases and controls or were reported in lyzed using the same program, and Kaplan-Meier analysis was the dbSNP Short Genetic Variations database, and their performed using another software package (Epi Info; Centers allele frequencies are given in eTable 1 and eTable 2 (http: for Disease Control and Prevention, Atlanta, Georgia). //www.archneurol.com). We defined rare variants as variations having frequencies of less than 1.0%.30 A few rare RESULTS variants were observed exclusively in controls (eTable 1). We observed a statistically significant difference in SQSTM1 is located on chromosome 5q35, with 8 coding the frequency of all rare variants exclusively present in exons (Figure, A). To identify DNA mutations that pre- patients with ALS vs in controls (22 of 1092 vs 11 of 1448; dispose to ALS, the entire coding region of SQSTM1 was P=.007, 2-tailed Fisher exact test) (Table 3). More- sequenced in a cohort of 546 patients with ALS (340 had over, we also observed a statistically significant differ- FALS and 206 had SALS, representing 1092 chromo- ence in the frequency of only rare missense or deletion somes). Ten novel SQSTM1 mutations (9 heterozygous variants exclusively present in patients with ALS vs in missense and 1 deletion) were identified in 15 patients controls (16 of 1092 vs 9 of 1448; P=.04, 2-tailed Fisher (6 with FALS and 9 with SALS) (Table 1, Table 2, and exact test). eFigure [http://www.archneurol.com]). None of these As shown in panel C of the Figure, p62 is highly con- changes were present in more than 724 controls (repre- served in mammals. All the mutations identified in our senting 1448 chromosomes), the dbSNP Short Genetic ALS cohort were located in conserved regions of p62. Four Variations database, or the 1000 Genomes Project data- of 10 mutations observed in our ALS cohort were fully base (Table 1). There was a personal history of parkin- conserved across 7 species examined. Four mutated resi- sonism in 2 patients. The A33V mutation was present in dues were conserved in 5 species. The V153 residue was 1 patient with FALS and in 2 patients with SALS. The conserved in 4 species. Eight of 9 missense variants were P392L substitution was found in 2 patients with FALS, predicted to have a harmful effect on the structure and but segregation analysis was impossible because of lack function of p62 by at least 1 of 2 protein conformation

ARCH NEUROL / VOL 68 (NO. 11), NOV 2011 WWW.ARCHNEUROL.COM 1442

©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 Table 2. Characterization of Patients With Familial Amyotrophic Lateral Sclerosis (FALS) and Patients With Sporadic Amyotrophic Lateral Sclerosis (SALS) Having SQSTM1 Mutations

Amino Acid Age at Site of Duration, Pedigree Type Change Race/Ethnicity Sex Onset, y Onset mo 9436 FALS A33V Hispanic Male 47 Limb 15 8588 SALS A33V White Female 69 Limb 94 8253 SALS A33V White Male 62 Bulbar Alive at 85 1216 SALS V153I White Male 55 Limb 25 8655 SALS V153I White Male 65 Limb 65 8913 SALS P228L White Male 33 Limb 51 8187 SALS K238del White Male 57 Limb 20 954 FALS S318P White Female 61 Bulbar 218 8105 SALS R321C White Female 55 Bulbar 5 7165 FALS S370P African American, white Male 43 Limb Alive at 145 1318 FALS P392L NA Male NA NA NA 9064 FALS, parkinsonism P392L White Female 72 Bulbar 29 8257 SALS P392L White Female 54 Limb 81 8516 FALS G411S White Male 45 Limb 168 8796 SALS, parkinsonism G425R White Male 47 Limb 56

Abbreviation: NA, not available.

Table 3. Rare Variant Frequencies in Patients With Amyotrophic Lateral Sclerosis (ALS) and in Control Subjects

All Rare Variants Rare Missense or Deletion Variants

Patients Control Patients Control Variable With ALS Subjects With ALS Subjects Total alleles 1092 1448 1092 1448 All variants 31 26 21 14 P value .10 . . . .06 . . . Exclusive variants 22 11 16 9 P value .007a ... .04a ... Exclusive functional variants ...... 13 6 P value ...... 03a ...

a Exclusive variants refer to rare variants found exclusively in patients with ALS or exclusively in controls. Fisher exact test was used to calculate the significance of rare variant accumulation in patients with ALS, with statistical significance defined as 2-sided PϽ.05. Mutation counts and annotations are given in Table 1 and eTable 1. Functional relevance of all mutations was analyzed in silico (eTable 3).

prediction programs used (eTable 3). The frequency of residues, and phosphorylation in this domain marks pro- functionally relevant rare variants exclusively present in teins for proteolysis.32 Moreover, the S318 residue oc- patients with ALS was significantly higher than that in curs between 2 PEST domains and may remove a cru- controls (13 of 1092 vs 6 of 1448; P=.03, 2-tailed Fisher cial phosphorylation site and make p62 more prone to exact test) (Table 3 and eTable 3). aggregation. In fact, 4 of 10 mutations seen in our ALS The A33V substitution occurs in the SH2-binding do- cohort were predicted to have an effect on p62 phos- main (Figure, B). These domains are generally about 100 phorylation (data not shown). Particularly, the S318P and residues in length and are known to associate with phos- S370P substitutions remove serine residues that are pre- phorylated tyrosine residues. The A33V change may affect dicted to be highly probable phosphorylation sites. The phosphotyrosine ligand binding and specificity, which UBA domain of p62 forms a compact 3-helix bundle (eFig- may lead to altered function of p62 in protein tyrosine ure). The P392L and G411S substitutions are present just kinase pathways. Indeed, several mutations in SH2 do- outside the hydrophobic patch of helix 1 and helix 2, main proteins are associated with human diseases.31 The whereas the G425R change occurs within the hydropho- V153I mutation occurs in the ZZ-type zinc finger do- bic patch of helix 3. These UBA domain mutations may main, which is thought to be involved in protein- affect binding of p62 to ubiquitin or ubiquitinated pro- protein interactions and is present in proteins like dys- teins and may lead to accumulation of the ubiquitin- trophin. The P228L and K238del mutations occur in the positive protein aggregates that are characteristic of ALS. binding site for the tumor necrosis factor receptor– We obtained clinical data on 14 patients having associated factor 6. The S318P and R321C mutations are SQSTM1 mutations for comparison with a cohort of pa- not present in any known domains and may lead to ab- tients having SOD1, TARDBP, and FUS mutations.33 The normal protein folding and aggregation of p62. The S370P mean (SD) age at symptom onset among 14 patients with variant occurs in a PEST domain, which is a region en- SQSTM1 mutations (54.6 [10.9] years) was similar to that riched in proline, glutamic acid, serine, and threonine among 34 patients with TARDBP mutations (54.7 [15.3]

ARCH NEUROL / VOL 68 (NO. 11), NOV 2011 WWW.ARCHNEUROL.COM 1443

©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 years) but was later than that for 54 patients with FUS than pathogenic mutations. One criterion suggesting that (43.6 [15.8] years, P=.02) or for 164 patients with SOD1 a group of rare variants in a certain gene influences in- mutations (47.7 [13.0] years, P=.05) (2-tailed t test for herited susceptibility is that they are overrepresented in both). We further tested the association between age at disease vs control groups.30 We observed statistically sig- onset and different ALS-linked genes by comparing nificant differences between ALS and controls whether Kaplan-Meier survival curves and then evaluated the ho- we considered all exclusive variants, only missense and mogeneity of the survival curves by using the log-rank deletion variants, or only functionally relevant variants test and Wilcoxon test (data not shown). Using the log- in SQSTM1. We also report several lines of evidence that rank test, we observed no significant differences be- suggest that the variants found in our ALS cohort may tween patients with SQSTM1 mutations vs patients with indeed be pathogenic. First, none of the variants present SOD1, FUS,orTARDBP mutations. However, differ- in our ALS cohort were detected in more than 724 con- ences between patients with SQSTM1 vs FUS mutations trols (representing 1448 chromosomes), the dbSNP Short were significant using the Wilcoxon test (P=.01), which Genetic Variations database, or the 1000 Genomes Proj- is more sensitive than the log-rank test to differences be- ect database. Second, all these variants affect amino ac- tween groups that occur at earlier time points. The mean ids that show a high level of evolutionary conservation. duration of symptoms was longer for 14 patients having Third, in silico analysis predicts that almost all these vari- SQSTM1 mutations (6.3 [5.3] years) compared with 44 ants will have a deleterious effect on the structure and patients having FUS mutations (3.4 [5.7] years), 144 pa- function of p62. tients having SOD1 mutations (4.1 [4.9] years), or 30 pa- Histopathologic studies17,21 have shown that p62 is tients having TARDBP mutations (3.3 [2.3] years). The present in ubiquitinated inclusions of SOD1-positive FALS mean duration of symptoms in patients with SQSTM1 mu- and other forms of ALS, suggesting a common pathogenic tations was almost twice as long as that in patients with mechanism. Our study presents a parallel between p62 and TARDBP mutations (P=.01, 2-tailed t test). The dura- other proteins linked to neurodegeneration, such as TDP- tion of symptoms varied widely. However, 64.3% of pa- 43, FUS, optineurin, ␤-amyloid, ␣-synuclein, and tau. These tients having SQSTM1 mutations survived beyond 4 years, proteins may aggregate in a wide variety of neurological which was remarkably higher compared with 11.4% of disorders, but mutations in their genes cause very specific patients having FUS mutations, 29.9% of patients hav- phenotypes in rare families. Such rare but pathogenic mu- ing SOD1 mutations, and 30.0% of patients having tations provide a novel approach in which the gene and TARDBP mutations. In comparing the site of symptom its product can be investigated in molecular pathways at onset, the proportion of patients with bulbar-onset symp- epigenetic, genetic, and posttranslational levels for rel- toms was similar among patients having SQSTM1 mu- evance to sporadic disease. tations (28.6%) vs patients having FUS mutations (33.3%) Genes linked to 2 distinct clinical syndromes are well or TARDBP mutations (32.1%) but was markedly higher known. For instance, mutations in TRPV4 that were pre- than that among patients having SOD1 mutations (7.6%) viously linked to bony dysplasias were recently linked (P=.05, 2-tailed Fisher exact test). to axonal neuropathies.34 Moreover, mutations in the gene encoding valosin-containing protein (VCP) have been implicated to cause human neurodegeneration in the syn- COMMENT drome of inclusion body myopathy with Paget disease of bone (PDB) and/or frontotemporal lobe degeneration Herein, we describe SQSTM1 mutations in approxi- (IBMPFD).35 Recently, VCP mutations were described by mately 2% to 3% of our large cohort of patients with FALS Johnson et al8 in patients with ALS. Notably, one of the and SALS from unrelated families. Although this fre- mutations (R191Q) described by Johnson and col- quency needs to be confirmed in future independent co- leagues in their ALS cohort had already been described horts, it is similar to what has been reported for other in families with IBMPFD, and 2 other mutations from the genes involved in ALS, namely, FUS, TARDBP, VCP, and same study (R159G and R155H) involved codons that ANG.9 SQSTM1 mutations may confer a toxic gain of func- had been found to be mutated in IBMPFD, highlighting tion through novel protein interactions and subsequent the ability of the same mutation to confer variable clini- deregulation of cell signaling pathways. They may also cal phenotypes. Furthermore, optineurin (mutations of lead to protein misfolding and aggregation. The SQSTM1 which were recently linked to ALS7) was identified as a mutations described in our study may have low pen- genetic risk factor for PDB in a recent genome-wide as- etrance, as most were present in patients with small pedi- sociation study.36 The 3 UBA domain mutations de- gree structure of familial aggregates or sporadic cases and scribed in our ALS cohort have been previously identi- not in large families. Other genes implicated in ALS, such fied in familial and sporadic PDB.37 This is intriguing as the PON genes and ANG, may cause disease by low- because the coexistence of PDB and ALS, although not penetrance mutations, as they have been described in SALS widely recognized, has been previously reported,38 sug- and familial aggregates rather than in large multigenera- gesting a possible common link between these diseases. tional pedigrees. Some low-penetrance mutations in SOD1, It is possible that this coexistence is underreported be- FUS, and TARDBP have been reported in apparent SALS. cause PDB, like ALS, is rarely diagnosed before age 40 Because our approach was based on candidate gene se- years, when symptoms of ALS (being more severe and quencing as opposed to linkage analysis, the possibility lethal) would preclude PDB diagnosis. We found no evi- exists that the identified changes represent rare, possi- dence of a family or personal history of PDB in our co- bly functional, variants conferring increased risk rather hort, and these mutations were absent in our control popu-

ARCH NEUROL / VOL 68 (NO. 11), NOV 2011 WWW.ARCHNEUROL.COM 1444

©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 lation. It has been noted that affected individuals from REFERENCES the same family with PDB have variable and sometimes no expressivity of the disease even with a mutated copy 1. Deng HX, Hentati A, Tainer JA, et al. Amyotrophic lateral sclerosis and structural 39 of SQSTM1. Moreover, reported transgenic mouse mod- defects in Cu,Zn superoxide dismutase. Science. 1993;261(5124):1047- els of PDB do not develop bone disease.40 This suggests 1051. that specific environmental factors or other modifier loci 2. Rosen DR, Siddique T, Patterson D, et al. Mutations in Cu/Zn superoxide dismutase gene are associated with familial amyotrophic lateral sclerosis [pub- in addition to SQSTM1 mutations may be important in lished correction appears in Nature. 1993;364(6435):362]. Nature. 1993;362 determining the specificity of the disease phenotype. (6415):59-62. This study represents the first comprehensive ge- 3. Kwiatkowski TJ Jr, Bosco DA, Leclerc AL, et al. Mutations in the FUS/TLS gene netic screen of SQSTM1 in ALS. Further screening and on chromosome 16 cause familial amyotrophic lateral sclerosis. Science. 2009; functional studies of these variants are needed to con- 323(5918):1205-1208. 4. Vance C, Rogelj B, Hortoba´gyi T, et al. Mutations in FUS, an RNA processing firm their implication in ALS. Our data widen the clini- protein, cause familial amyotrophic lateral sclerosis type 6. Science. 2009; cal spectrum associated with SQSTM1 mutations to in- 323(5918):1208-1211. clude ALS and suggest that patients with ALS should be 5. Kabashi E, Valdmanis PN, Dion P, et al. TARDBP mutations in individuals with monitored for features of PDB and, more broadly, al- sporadic and familial amyotrophic lateral sclerosis. Nat Genet. 2008;40(5): tered bone metabolism. We can hypothesize that SQSTM1 572-574. 6. Sreedharan J, Blair IP, Tripathi VB, et al. TDP-43 mutations in familial and spo- mutations may act directly to cause ALS or through a radic amyotrophic lateral sclerosis. Science. 2008;319(5870):1668-1672. modifier effect involving additional genes or environ- 7. Maruyama H, Morino H, Ito H, et al. Mutations of optineurin in amyotrophic lat- mental factors. The specific effects of these mutations in eral sclerosis. Nature. 2010;465(7295):223-226. SQSTM1 on protein degradation pathways are impor- 8. Johnson JO, Mandrioli J, Benatar M, et al; ITALSGEN Consortium. Exome se- tant to resolve, as this may identify molecular targets for quencing reveals VCP mutations as a cause of familial ALS [published correction appears in Neuron. 2011;69(2):397]. Neuron. 2010;68(5):857-864. developing novel therapeutics in ALS. 9. Ticozzi N, Tiloca C, Morelli C, et al. Genetics of familial amyotrophic lateral sclerosis. Arch Ital Biol. 2011;149(1):65-82. Accepted for Publication: April 13, 2011. 10. Joung I, Strominger JL, Shin J. Molecular cloning of a phosphotyrosine- Correspondence: Teepu Siddique, MD, Division of Neu- independent ligand of the p56lck SH2 domain. Proc Natl Acad SciUSA. 1996; romuscular Medicine, Ken and Ruth Davee Department 93(12):5991-5995. 11. Vadlamudi RK, Joung I, Strominger JL, Shin J. p62, A phosphotyrosine- of Neurology and Clinical Neurological Sciences, Fein- independent ligand of the SH2 domain of p56lck, belongs to a new class of ubiquitin- berg School of Medicine, Northwestern University, Room binding proteins. J Biol Chem. 1996;271(34):20235-20237. 13-715, Tarry Bldg, 303 E Chicago Ave, Chicago, IL 60611 12. Seibenhener ML, Babu JR, Geetha T, Wong HC, Krishna NR, Wooten MW. Se- ([email protected]). questosome 1/p62 is a polyubiquitin chain binding protein involved in ubiquitin proteasome degradation. Mol Cell Biol. 2004;24(18):8055-8068. Author Contributions: Dr T. Siddique had full access to 13. Bjørkøy G, Lamark T, Johansen T. p62/SQSTM1: a missing link between protein all the data in the study and takes responsibility for the aggregates and the autophagy machinery. Autophagy. 2006;2(2):138-139. integrity of the data and the accuracy of the data analy- 14. Pankiv S, Clausen TH, Lamark T, et al. p62/SQSTM1 binds directly to Atg8/LC3 sis. Study concept and design: Fecto, Deng, and T. Siddique. to facilitate degradation of ubiquitinated protein aggregates by autophagy. J Biol Acquisition of data: Fecto, Yan, Vemula, Liu, Yang, Chen, Chem. 2007;282(33):24131-24145. 15. Kuusisto E, Salminen A, Alafuzoff I. Ubiquitin-binding protein p62 is present in Zheng, Shi, N. Siddique, Arrat, Donkervoort, Ajroud- neuronal and glial inclusions in human tauopathies and synucleinopathies. Driss, Sufit, Heller, Deng, and T. Siddique. Analysis and Neuroreport. 2001;12(10):2085-2090. interpretation of data: Fecto, Deng, and T. Siddique. Draft- 16. Zatloukal K, Stumptner C, Fuchsbichler A, et al. p62 Is a common component of ing of the manuscript: Fecto and T. Siddique. Critical re- cytoplasmic inclusions in protein aggregation diseases. Am J Pathol. 2002; vision of the manuscript for important intellectual content: 160(1):255-263. 17. Gal J, Stro¨m AL, Kilty R, Zhang F, Zhu H. p62 accumulates and enhances ag- Yan, Vemula, Liu, Yang, Chen, Zheng, Shi, N. Siddique, gregate formation in model systems of familial amyotrophic lateral sclerosis. Arrat, Donkervoort, Ajroud-Driss, Sufit, Heller, and Deng. J Biol Chem. 2007;282(15):11068-11077. Obtained funding: T. Siddique. Study supervision: T. 18. Mizuno Y, Amari M, Takatama M, Aizawa H, Mihara B, Okamoto K. Immunore- Siddique. activities of p62, an ubiquitin-binding protein, in the spinal anterior horn cells of Financial Disclosure: None reported. patients with amyotrophic lateral sclerosis. J Neurol Sci. 2006;249(1):13-18. 19. Gal J, Stro¨m AL, Kwinter DM, et al. Sequestosome 1/p62 links familial ALS mu- Funding/Support: This study was supported by grants tant SOD1 to LC3 via an ubiquitin-independent mechanism. J Neurochem. 2009; NS050641 and T32 AG20506 from the National Insti- 111(4):1062-1073. tutes of Health and by the Les Turner ALS Foundation, 20. Hiji M, Takahashi T, Fukuba H, Yamashita H, Kohriyama T, Matsumoto M. White the Vena E. Schaff ALS Research Fund, the Harold Post matter lesions in the brain with frontotemporal lobar degeneration with motor Research Professorship, the Herbert and Florence C. Wen- neuron disease: TDP-43–immunopositive inclusions co-localize with p62, but not ubiquitin. Acta Neuropathol. 2008;116(2):183-191. ske Foundation, the Ralph and Marian Falk Medical Re- 21. Deng HX, Zhai H, Bigio EH, et al. FUS-immunoreactive inclusions are a common search Trust, the David C. Asselin MD Memorial Fund, feature in sporadic and non-SOD1 familial amyotrophic lateral sclerosis. Ann Neurol. and the Les Turner ALS Foundation/Herbert C. Wenske 2010;67(6):739-748. Foundation Professorship. 22. Brady OA, Meng P, Zheng Y, Mao Y, Hu F. Regulation of TDP-43 aggregation by phosphorylation and p62/SQSTM1. J Neurochem. 2011;116(2):248-259. Previous Presentation: This study was presented at the 23. Babu JR, Lamar Seibenhener M, Peng J, et al. Genetic inactivation of p62 leads 2011 Annual Meeting of the American Academy of Neu- to accumulation of hyperphosphorylated tau and neurodegeneration. rology; April 14, 2011; Honolulu, Hawaii. J Neurochem. 2008;106(1):107-120. Additional Contributions: We thank the patients and 24. Brooks BR; Subcommittee on Motor Neuron Diseases/Amyotrophic Lateral Scle- families who participated in the study. rosis of the World Federation of Neurology Research Group on Neuromuscular Dis- eases and the El Escorial “Clinical Limits of Amyotrophic Lateral Sclerosis” Work- Online-Only Material: The online-only eTables 1 through shop Contributors. El Escorial World Federation of Neurology criteria for the diagnosis 3 and the eFigure are available at http://www.archneurol of amyotrophic lateral sclerosis. J Neurol Sci. 1994;124(suppl):96-107. .com. 25. Durbin RM, Abecasis GR, Altshuler DL, et al; 1000 Genomes Project Consor-

ARCH NEUROL / VOL 68 (NO. 11), NOV 2011 WWW.ARCHNEUROL.COM 1445

©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 tium. A map of human genome variation from population-scale sequencing. Nature. 34. Deng HX, Klein CJ, Yan J, et al. Scapuloperoneal spinal muscular atrophy and 2010;467(7319):1061-1073. CMT2C are allelic disorders caused by alterations in TRPV4. Nat Genet. 2010; 26. Blom N, Gammeltoft S, Brunak S. Sequence and structure–based prediction of 42(2):165-169. eukaryotic protein phosphorylation sites. J Mol Biol. 1999;294(5):1351-1362. 35. Weihl CC, Pestronk A, Kimonis VE. Valosin-containing protein disease: inclu- 27. Ng PC, Henikoff S. SIFT: predicting amino acid changes that affect protein function. sion body myopathy with Paget’s disease of the bone and fronto-temporal dementia. Nucleic Acids Res. 2003;31(13):3812-3814. Neuromuscul Disord. 2009;19(5):308-315. 28. Ferrer-Costa C, Gelpı´ JL, Zamakola L, Parraga I, de la Cruz X, Orozco M. PMUT: 36. Albagha OM, Visconti MR, Alonso N, et al. Genome-wide association study iden- a web-based tool for the annotation of pathological mutations on proteins. tifies variants at CSF1, OPTN and TNFRSF11A as genetic risk factors for Paget’s Bioinformatics. 2005;21(14):3176-3178. disease of bone. Nat Genet. 2010;42(6):520-524. 29. Guex N, Peitsch MC. SWISS-MODEL and the Swiss-PdbViewer: an environment 37. Michou L, Collet C, Laplanche JL, Orcel P, Corne´lis F. Genetics of Paget’s dis- for comparative protein modeling. Electrophoresis. 1997;18(15):2714-2723. ease of bone. Joint Bone Spine. 2006;73(3):243-248. 30. Bodmer W, Bonilla C. Common and rare variants in multifactorial susceptibility 38. Varelas PN, Bertorini TE, Kapaki E, Papageorgiou CT. Paget’s disease of bone to common diseases. Nat Genet. 2008;40(6):695-701. 31. Lappalainen I, Thusberg J, Shen B, Vihinen M. Genome-wide analysis of patho- and motor neuron disease. Muscle Nerve. 1997;20(5):630. genic SH2 domain mutations. Proteins. 2008;72(2):779-792. 39. Leach RJ, Singer FR, Ench Y, Wisdom JH, Pina DS, Johnson-Pais TL. Clinical 32. Rogers S, Wells R, Rechsteiner M. Amino acid sequences common to rapidly and cellular phenotypes associated with sequestosome 1 (SQSTM1) mutations. degraded proteins: the PEST hypothesis. Science. 1986;234(4774):364-368. J Bone Miner Res. 2006;21(suppl 2):45-50. 33. Yan J, Deng HX, Siddique N, et al. Frameshift and novel mutations in FUS in fa- 40. Kurihara N, Hiruma Y, Zhou H, et al. Mutation of the sequestosome 1 (p62) gene milial amyotrophic lateral sclerosis and ALS/dementia. Neurology. 2010;75 increases osteoclastogenesis but does not induce Paget disease. J Clin Invest. (9):807-814. 2007;117(1):133-142.

Correction

Incorrect Positioning. In the Observation titled “Novel POLG Splice Site Mutation and Optic Atro- phy” by Milone et al, published in the June issue of the Archives (2011;68[6]:806-811), on page 807 Figure 1A and B should have been turned 90° coun- terclockwise for proper positioning.

ARCH NEUROL / VOL 68 (NO. 11), NOV 2011 WWW.ARCHNEUROL.COM 1446