Association of Variants in the SPTLC1 Gene With Juvenile Amyotrophic Lateral Sclerosis

Key Points Question What genetic variants are associated with juvenile amyotrophic lateral sclerosis (ALS)? Findings In this family-based genetic study, exome sequencing was performed in 3 patients diagnosed with juvenile ALS and failure to thrive; this identified de novo variants in SPTLC1 (p.Ala20Ser in 2 patients and p.Ser331Tyr in 1 patient). Variants in SPTLC1 are a known cause of hereditary sensory and autonomic neuropathy, type 1A, and these data extend the phenotype associated with this gene. Meaning De novo variants in the SPTLC1 gene are associated with juvenile ALS, a fatal neurological disorder.


eMethods.
eAppendix.Consortia authors and affiliations eFigure 1. Assays of SPT activity and mitochondrial cellular phenotypes in cell models eFigure 2. SPTLC1 mutations detected in adult-onset ALS patients eFigure 3. Chromatograms of SPTLC1 variants identified in patients eFigure 4. Immunohistochemistry of SPTLC1 in the lumbar spinal cord eFigure 5. Time-series changes of complex sphingolipids in patient 2 on high-dose oral serine supplementation eTable 1. Demographic and clinical features of adult-onset ALS cases screened for SPTLC1 mutations eTable 2. Primer sequences used for Sanger sequencing and mutagenesis of SPTLC1 eTable 3. SPTLC1 mutations identified in adult-onset ALS cases eTable 4. Demographic and clinical features of adult-onset ALS cases carrying SPTLC1 mutations eTable 5. Complex sphingolipid plasma levels in ALS cases and controls eReferences.
This supplementary material has been provided by the authors to give readers additional information about their work.

Detailed description of patient 2
Patient 2 was a right-handed teenage girl with mixed African American and white race/ethnicity who had developed difficulty walking due to lower limb muscle weakness before the age of 10.Over the next few years, she had trouble climbing stairs, and tongue fasciculations were observed.She had generalized weakness and muscle wasting at the time of her referral in her midteens.She could not walk long distances without fatigue, and her weight was more than 7 standard deviations below average for her age.A prior endocrinology workup for her failure to gain weight was unremarkable.Her school performance had also begun to decline in her mid-teens.
Past medical history was otherwise unremarkable, and there was no family history of neurodegenerative disorders (Figure 2B).
Examination at presentation revealed a thin girl with a body mass index below the 1 st percentile based on her age and gender (z-score = -7).There was no evidence of orthostatic hypotension.Cranial nerve examination revealed tongue fasciculations and wasting (Figure 1A).
Limb examination showed an exaggerated lumbar lordosis, generalized weakness involving all muscles, and prominent scapula winging bilaterally (Figure 1B).Her upper limb reflexes were diminished.In the lower limbs, knee jerks were reduced, and ankle jerks were asymmetrically brisk.Pinprick, temperature, vibration, and proprioception sensations were normal.The patient had an abnormal gait and a floridly positive Gower's sign.MRI imaging of the brain and spine were unremarkable, though these imaging studies showed diffusely diminished pelvic and thigh musculature and a notable lack of subcutaneous fat.EMG revealed active and chronic denervation with prominent complex repetitive discharges and giant muscle unit action potentials consistent with a long-standing motor neuropathic or neuronopathic disorder.There was no neurophysiological evidence for a sensory neuropathy.Plasma metanephrine and normetanephrine were normal.A neuropsychological evaluation revealed that her intellectual functioning was within the average range based on her age.However, she demonstrated significant challenges with sustained attention and stamina, aspects of executive functioning, and motor coordination.A swallow study was unremarkable.

Detailed description of patient 3
Patient 3 was an eleven-year-old African American right-handed girl with a history of failure to gain weight and toe-walking since the age of four.She was first evaluated at the age of 10 because of difficulty walking due to her left foot "turning in."In retrospect, her gait had been gradually deteriorating over several years, resulting in occasional falls and difficulty walking upstairs.She was no longer able to participate in her school dance class due to weakness and exercise-induced cramps.Subsequently, her symptoms had spread to her hands.She was unable to undo soda bottle tops, and dressing has been taking longer than usual.The patient had complained of tingling in her feet over the last three years, but the numbness was only noticed on her first neurological evaluation.She had a painless ulcer on her right foot that developed from her ankle-foot orthosis.She has occasional choking episodes, mostly on liquids.She has noticed the pooling of secretions.Her speech has been normal, and there have been no obvious cognitive or behavioral problems.She has worn glasses since age eight.Ophthalmological examination at age eleven revealed bilateral cataracts that did not require surgical intervention.The patient has a history of profuse sweating, but no urinary symptoms or dizziness on standing.
Her past medical history included adenoidectomy, bilateral Achilles tenotomy at the age of 10, and long-standing vitamin D deficiency requiring oral supplementation.She was born one month premature, but she achieved her early developmental milestones appropriately.There was no family history of ALS, and her nineteen-year-old brother was healthy (Figure 2C).Her maternal grandmother, paternal grandfather, and maternal aunt were diagnosed with late-onset dementia.
Examination at the age of eleven revealed a thin girl with a body mass index of 10.9 kg/m2, representing less than the 1 st percentile based on her age and gender (z score = -6.5).Blood pressure was 111/62 mmHg with a heart rate of 107 beats per minute while lying, and 128/62 mmHg with a heart rate of 139 beats per minute on standing.She was alert and articulate, and her conversational speech was normal, though she had difficulty forming K and L sounds.
On cranial nerve examination, her near visual acuity was 20/50 bilaterally without glasses, and fundoscopy confirmed the presence of lens opacities.The tongue was wasted, weak, and slow to move with prominent fasciculations (Figure 1C-D).Jaw jerk was present, and shoulder shrug was weak bilaterally.The rest of the cranial nerves were intact.
Her posture was normal, and there was no evidence of scoliosis.The examination of her limbs showed generalized atrophy and hypotonia.Fasciculations were present in the upper and lower limbs.There was a left greater than right pronator drift with bilateral postural tremor.The patient had bilateral hammertoes, high arches, and a healing ulcer over the right first metatarsal joint.Strength was decreased in a pyramidal pattern that was asymmetric and affecting distal muscles more than proximal.Power in the upper limbs was as follows: bilateral shoulder abductors = 4/5, shoulder adductors = 5/5, right elbow flexor = 5/5, left elbow flexor = 4/5, bilateral elbow extensors = 4/5, wrist dorsiflexor = 4/5, wrist palmar flexion = 5/5, finger flexors = 4/5, right finger extensors = 4/5, left finger extensors = 3/5, bilateral finger adductors = 2/5, finger abductors = 3/5, and thumb abductors = 4/5.Power in the lower limbs was as follows: bilateral hip flexors = 4/5, hip abductors = 4/5, hip adductors = 5/5, knee flexors = 4/5, knee extensors = 5/5, ankle dorsiflexion = 3/5, ankle plantar flexion = 5/5, ankle eversion = 3/5, ankle inversion = 5/5, hallux flexors = 2/5, and hallux extensors = 2/5.Reflexes in the upper limbs were 4+ with spread with a positive Hoffman's sign bilaterally.In the lower limbs, knee jerks were 3+ bilaterally, and ankle jerks were absent.The left Babinski was down going, whereas the right Babinski elicited fanning of her toes.The patient was ataxic on heel-to-shin and finger-to-nose testing with bilateral dysdiadochokinesis, though this appeared to be secondary to muscle weakness.The sensory exam revealed decreased pinprick and temperature sensation in the upper limbs to the level of the wrist, right worse than left, and in the lower limbs to the mid-calf level.Proprioception and vibration sensations were intact.The patient walked abnormally due to generalized weakness and bilateral foot drop.She had difficulty with tandem gait testing, was able to stand on her toes, but she was unable to stand on her heels.Romberg's sign was negative.She had a positive Gower's sign.
Neurophysiological examination at the age of 11 was consistent with sensorimotor axonal neuropathy.Electromyography of the left vastus lateralis and tibialis anterior muscles showed large amplitude, polyphasic potentials with a reduced amplitude pattern.MRI of the brain and spinal cord was unremarkable.Pulmonary function testing demonstrated a mild restrictive ventilatory defect (forced expiratory volume in one second-to-forced vital capacity ratio = 0.9).Creatine phosphokinase, vitamin B12, and folate serum transferrin, iron, copper, metabolic screens were within normal limits.Serine level was within the normal range at 79.3 umol/l.

Patient 4
Patient 4 was a thirty-four-year-old, right-handed, Turkish woman with a history of arm and leg weakness and atrophy since the age of fifteen.Her five siblings, parents, paternal and maternal grandparents, and extended family did not report neurological symptoms.Her mother died at the age of 65, and her father died at the age of 50.

Recruitment
Patients 1, 2, and 3 were followed by their local neurologists and geneticists.All patients were diagnosed with juvenile ALS by their local physicians.Patients 1 (and her family) was referred to Dr. Traynor at the NIH Clinical Center for evaluation by her pediatric neurologist.Dr. Traynor was contacted by the physicians of patients 2 and 3 through the GeneMatcher program at GeneDx. 1 Patient 2 was followed at her local hospital.Patient 3 (and her family) was referred to Dr. Traynor at the NIH Clinical Center for evaluation by her pediatrician.
Patients 1 and 3 are the only juvenile ALS cases that Dr. Traynor has seen in his ALS clinic since moving to the NIH in 2005.In a separate project, deidentified DNA from three other trios where the proband had juvenile ALS of unknown genetic etiology was sent to Dr. Traynor's laboratory and underwent exome sequencing.The ages of symptom onset in these samples were older than those carrying SPTLC1 mutations (age of onset was 20 years for Coriell repository sample ND14489, 22 years of age for the Italian sample SLA2010-483, and 24 years of age for Italian sample SLA2011-110).De novo SPTLC1 mutations were not identified in these trio samples.Patient 4 was identified by Dr. Başak working at the Koç University, Istanbul, Turkey in a collection of 52 individuals diagnosed with juvenile ALS undergoing whole-exome sequencing.

Immunohistochemistry
The control spinal cord was obtained from the postmortem of a 64-year-old white man, and the postmortem interval was 28 hours.The sporadic ALS spinal cord was obtained from the postmortem of a 66-year old white male, and the postmortem interval was 4.5 hours.The spinal cord carrying the SPTLC1 p.Arg445Gln mutation was obtained from the postmortem of a 78-year-old white man who had been diagnosed with sporadic ALS.The postmortem interval was 3.83 hours, and survival from symptom onset in his upper limbs was 68 months.The autopsy of the SPTLC1 carrier was performed according to the research protocol of the Department of Veterans Affairs Biorepository Brain Bank. 2 The other autopsies were performed at the JHMI Brain Resource Center according to their internal protocol.Formalin-fixed, paraffin-embedded blocks were sectioned to 5 um thickness and stained with primary anti-SPTLC1 antibody (Sigma-Aldrich) at 1:50 dilution overnight at 4°C and counterstained with hematoxylin.

Immunopurification of SPTLC1 protein complex
SPTLC1-FLAG proteins were immuno-purified from HEK293FT cells that were stably expressing SPTLC1.Cells were lysed in lysis buffer (50 mM HEPES, pH8.0, 1 mM EDTA, 0.1% (w/v) sodium monolaurate, phosphatase (Thermo Fisher), and protease inhibitor (Roche AG)).Membrane proteins were further solubilized by sonication for fifteen seconds at 50% power and 50% pulsation for a total of 45 seconds.The lysate was clarified by centrifugation at 21,000g for five minutes at 4°C to remove large cellular debris, followed by immunoprecipitation with EZview Red Anti-Flag M2 Affinity Gel (Sigma-Aldrich) as previously described. 3Purified SPTLC1-FLAG was quantified against a bovine serum albumin protein standard curve (BSA, Thermo Fisher) ran on an SDS-PAGE gel and transferred to a nitrocellulose membrane (Bio-rad).The membrane was stained with Revert Total Protein Stain (LI-COR) and imaged on an Odyssey CLx imaging system.Interaction of SPTLC2 with SPTLC1-FLAG was confirmed by Western blot showing that the purified serine palmitoyltransferase complex was likely functional.

Photometric serine palmitoyltransferase enzymatic assay
Condensation of serine and palmitoyl-CoA by serine palmitoyltransferase enzyme produces 3-ketodihydrosphingosine and releases both carbon dioxide and free coenzyme A (CoA).This photometric assay measures the amount of free CoA released that is reactive to 5,5'-dithiobis-2-nitrobenzoic acid (DTNB, Thermo Fisher), which reflects the enzymatic activity of serine palmitoyltransferase.
This assay was adapted from and shown to be comparable to radioactive assays measuring radiolabeled 3ketodihydrosphingosine. 4 Triplicate wells were assayed per 100ng of purified wild-type or mutant SPTLC1 proteins, and per amino acid tested.The reaction product was measured at 412 nm at the zero-time point and every two minutes for up to one hour.In place of purified SPTLC1-FLAG protein, varying concentrations of CoA were included to build the calibration curve for the estimation of CoA released from the serine palmitoyltransferase enzymatic reaction.Estimated amounts of CoA produced per nanogram of SPTLC1-FLAG protein were plotted over time for each amino acid.

Western blotting
Western blot was performed as previously described. 3We used the following primary antibodies at the indicated dilutions: mouse anti-Flag (1:5,000, Sigma-Aldrich), rabbit anti-SPTLC1 (1:1,000, Sigma-Aldrich), and rabbit anti-SPTLC2 (1:1000, LifeSpan BioSciences Inc.).eFigure 5. Time-series changes of complex sphingolipids in patient 2 on high-dose oral serine supplementation Glucosylceramides, lactosylceramides, ceramides, and sphingomyelins levels were measured in patient 2 before and throughout serine supplementation.The purple lines represent the linear regression lines over time, with the 95% confidence interval demarcated by dotted lines.Pearson R = regression slope, R 2 = slope correlation and p = significance of correlation.Green arrows show the serine doses, and the duration of each dosage is given in brackets.The x-axis shows the age in years, the left y-axis shows the z-score weights, and the right y-axis is the normalized peak area ratio from lipid mass spectra on a log 10 scale.

A B eFigure 1 .
Assays of SPT activity and mitochondrial cellular phenotypes in cell models(A) Western blot of purified SPTLC1-FLAG proteins showing that the purified complex contains SPTLC2.(B) Western blot showing comparable SPTLC1 expression across all lines of HEK293 cells stably overexpressing wild-type (WT) or mutant proteins.Exogenous SPTLC1 expression was qualitatively estimated to be ~10x higher than endogenous levels.(C) Representative image from CX7 LZR high-content analysis instrument of MitoTracker staining in wild-type and mutant SPTLC1 cells with and without treatment with 400 μM L-serine for 48 hours.Treatment with 400 μM L-serine rescued mitochondrial abnormalities in cells overexpressing mutant SPTLC1.Scale bar = 25μm.C © 2021 Johnson JO et al.JAMA Neurology.

eFigure 2 .
SPTLC1 mutations detected in adult-onset ALS patientsThe top panel is a graphical representation showing the domains of SPTLC1 based on ENST00000262554.2.Mutations detected in ALS cases are indicated in red, and mutations previously described to cause HSAN1 are shown in green.The p.Ala305Thr variant was found in three ALS patients, and the other variants were found in single cases of adult-onset ALS.The middle panel shows the conservation of amino acid residue across species generated using the Clustal Omega online tool.

eFigure 4 .
Immunohistochemistry of SPTLC1 in the lumbar spinal cordThe control spinal cord (top panel) exhibits SPLTC1 immunoreactivity in the lamina IX motor neurons of the anterior horns.The spinal cord from a subject with sporadic ALS (middle panel) shows weaker immunoreactivity in the same region, consistent with a decrease in the number and size of surviving motor neurons.The spinal cord from an ALS case carrying the p.Arg445Gln mutation in SPTLC1 (bottom panel) shows a similar diminished staining pattern as the sporadic ALS sample.Scale bars in magnified inset represent 100 m.
Demographic and clinical features of adult-onset ALS cases screened for SPTLC1 mutations position is in build 19; amino acid change is based on the canonical transcript NP_006406, except for p.Tyr509X which is based on NP_001268232; No. cases, number of ALS cases carrying the variant; only variants with frequency less than 3ꞏ3x10 -5 are shown; gnomAD, frequency shown is the maximum in either the Finnish or the non-Finnish European populations (non-neurological samples, version 2ꞏ1); 'damaging' means the variant is designated as such by at least four out of five prediction algorithms consisting of FATHMM, M-CAP, MetaLR, MetaSVM and VEST3; 'conserved' means the position is considered as such by the GERP++, phastCons100way_vertebrate, phyloP100way_vertebrate, and SiPhy_29way algorithms; dbscSNV adaptive boosting score greater than 0ꞏ6 indicates a deleterious splice mutation; NA, not available.Complex sphingolipid plasma levels in ALS cases and controlsValues represent normalized peak area ratios.In patient 2, the measurements were made before serine supplementation and 10.5 months after commencing serine.