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 deg- radation 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 vari- ants 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 con- firmed 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 ©2011 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/29/2021 A 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