Hereditary sensory and autonomic neuropathy type 1 (HSANI) caused by a novel mutation in SPTLC2

Objective: To describe the clinical and neurophysiologic phenotype of a family with hereditary sensory and autonomic neuropathy type 1 (HSANI) due to a novel mutation in SPTLC2 and to characterize the biochemical properties of this mutation. Methods: We screened 107 patients with HSAN who were negative for other genetic causes for mutations in SPTLC2. The biochemical properties of a new mutation were characterized in cell-free and cell-based activity assays. Results: A novel mutation (A182P) was found in 2 subjects of a single family. The phenotype of the 2 subjects was an ulcero-mutilating sensory-predominant neuropathy as described previously for patients with HSANI, but with prominent motor involvement and earlier disease onset in the first decade of life. Affected patients had elevated levels of plasma 1-deoxysphingolipids (1-deoxySLs). Biochemically, the A182P mutation was associated with a reduced canonical activity but an increased alternative activity with alanine, which results in largely increased 1-deoxySL levels, supporting their pathogenicity. Conclusion: This study confirms that mutations in SPTLC2 are associated with increased deoxySL formation causing HSANI.

][4][5][6][7] Mutations in the enzyme serine palmitoyltransferase (SPT) cause HSANI. 2,3SPT is a heteromeric enzyme composed of 3 subunits (SPTLC1-3) located at the outer membrane of the endoplasmic reticulum. 8,9It catalyzes the first and rate-limiting step in de novo sphingolipid synthesis: the condensation of L-serine and palmitoyl-coenzyme A. Mutations in SPTLC1 account for 12% of patients with HSAN. 10 More recently, mutations in SPTLC2 were found to cause HSANI with a similar phenotype. 4SPTLC1 and SPTLC2 mutations generally cause a shift in the substrate specificity of SPT leading to the alternative use of L-alanine and L-glycine over its canonical substrate L-serine. 11,12This forms an atypical category of 1-deoxysphingolipids (1-deoxySLs) lacking the C1 hydroxyl group, which impedes their conversion into complex sphingolipids but also their canonical degradation.Elevated 1-deoxySLs were found in the plasma and lymphoblasts of patients with HSANI 11 and plasma and tissues of transgenic HSANI *Joint first authors.These authors contributed equally to this work.‡Joint senior authors.This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.mice. 13The 1-deoxySLs were shown to be neurotoxic, blocking neurite formation in cultured neurons. 11erein, we report a novel HSANI mutation in SPTLC2 associated with elevated plasma 1-deoxySLs.In vitro and cell culture studies show that this mutant has reduced canonical activity but forms high levels of 1-deoxySLs.
METHODS Standard protocol approvals, registrations, and patient consents.Ethical approval for this study was obtained from the Joint Medical and Ethics Committee at the National Hospital for Neurology and Neurosurgery.Written informed consent was obtained from all patients.
Patients.One hundred seven patients with HSAN were selected from our inherited neuropathy database, including patients seen in the National Hospital for Neurology and Neurosurgery peripheral neuropathy clinics as well as patients whose DNA was referred from other hospitals for diagnostic and research testing.Most patients presented with distal progressive sensory loss, with or without ulcero-mutilating complications or autonomic dysfunction.Because of the overlap between Charcot-Marie-Tooth type 2B and HSANI, we included patients with motor involvement; however, sensory features were predominant.Diagnosis was based on clinical phenotype in addition to neurophysiology.All patients were negative for mutations in SPTLC1 and most were also negative for mutations in RAB7, NGFB, FAM134B, and NTRK1.Three hundred fifty-eight British control chromosomes were screened for control.
Patient assessment.Patients found to have mutations underwent detailed clinical and neurophysiologic assessments including the Charcot-Marie-Tooth Neuropathy Score 2. 14 Nerve conduction studies were performed using standard technique.

Genetic sequencing. All 12 exons and flanking introns of SPTLC2
were amplified using Roche PCR reagents.Sequence reactions were performed using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and resolved on an ABI 3730xl Sequencer.For primers and primer conditions, see table e-1 on the  Neurology ® Web site at www.neurology.org.Sequence variants were confirmed by repeat sequencing.PolyPhen2 (http://genetics.bwh.harvard.edu/pph/),SIFT (http://blocks.fhcrc.org/sift/SIFT.html),and aGVGD (http:// agvgd.iarc.fr/)were used to predict the effect of the mutation on protein function.
Cloning.The A182P was introduced into the SPTLC2 cDNA by site-directed mutagenesis as described previously 11 (see table e-2 for primers).All constructs were verified by sequencing.
Stable expression of SPTLC2wt and the A182P mutation in HEK293 cells.HEK293 cells (ATCC, Manassas, VA) were stably transfected with the empty vector (control), SPTLC2 wild type (wt), or the A182P mutant as described previously. 11Expression of the constructs was confirmed by reverse transcriptase-PCR and immune blotting (figure e-1).
SPT in vitro activity assay and lipid-base extraction.SPT in vitro activity was measured as described previously. 15In modification to the original protocol, 64 mM L-serine or 320 mM L-alanine (Sigma, St. Louis, MO) was used.

Acid-base extraction and analysis of sphingoid bases.
MetOH 500 mL including 200 pmol internal standard (d7sphinganine and d7-sphingosine; Avanti Polar Lipids, Alabaster, AL) were added to 100 mL of plasma or frozen cell pellets resuspended in 100 mL phosphate-buffered saline.Lipid extraction was performed for 1 hour at 37°C with constant agitation at 1,000 rpm (Thermomixer Comfort; Eppendorf, Hamburg, Germany).Precipitated protein was removed by centrifugation (5 minutes at 16,000g).After transferring the supernatant into a new tube, lipids were extracted and analyzed by liquid chromatography-mass spectrometry as described earlier. 11atistics.One-way analysis of variance was used for statistical analysis.Significance was verified by Bonferroni multiple correction.Unless otherwise stated, p values ,0.001 were considered significant.Statistical analysis was performed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA).

RESULTS
Genetics.Sequencing of SPTLC2 in 107 index patients with HSANI revealed a novel mutation in one family.c.544G.C, A182P, was found in the index case (figure 1, III-2); this mutation was not present in 358 control chromosomes.One other affected member of this family who was tested carried the mutation (figure 1, IV-2).The A182P mutation affects a highly evolutionary conserved amino acid; PolyPhen2 and SIFT both predict that this change would be damaging, whereas aGVGD classifies the variant as class C25, where C65 is most likely to interfere with function and C0 less likely to interfere with function.

Clinical details. The family had AD inheritance (figure 1). Clinical features are documented in table 1.
Both patients had onset in the first decade, presenting with reduced sensation in the feet, with later development of motor weakness.Sensory loss occurred in a glove and stocking distribution; pinprick perception was affected to a greater extent than vibration perception, similar to patients with SPTLC1 mutations. 16atient III-2 had painful tingling in the hands whereas IV-2 had no history of pain.Sensory complications including ulcers and accidental burns occurred in both patients, and severe wasting and weakness were present (figure e-2).There were no symptoms of hearing loss or autonomic dysfunction.
Neurophysiology.Table 2 describes the neurophysiology of the patients with HSANI.Although this was predominantly an axonal neuropathy, motor conduction velocities in the upper limbs were slow (,38 m/s), even in patient III-2 who had reasonable upper limb motor amplitudes, a finding that we have previously documented in our HSANI families with SPTLC1 mutations. 16asma sphingoid and deoxysphingoid base levels.Total sphinganine (SA) or sphingosine (SO) levels showed no difference between SPTLC2-A182P patients and healthy controls, whereas the 1-deoxySL levels were elevated in the plasma of the two A182P carriers.Higher 1-deoxySL plasma levels were found in the more severely affected patient, IV-2 (figure 2).Effect of the SPTLC2-A182P mutation on SPT activity.
4]17 The A182P mutant showed a 65% reduced in vitro activity with L-serine and a 3.5-fold increased activity with L-alanine (figure 3, A  and B).These results were confirmed by a metabolic labeling assay using isotope-labeled (d 3 , 15 N) L-serine (1 mM) and (d 4 ) L-alanine (5 mM), which leads to the formation of isotope-labeled (M13)SA and (M13)de-oxySA because one deuterium is lost during the condensation reaction. 18The assay was performed in the presence or absence of FB 1 , an inhibitor for ceramide synthase.The inhibition of ceramide synthase leads to the accumulation of SA over time, which is a measure for cellular SPT activity.In the presence of FB 1 , the SPTLC2wt cells showed a 2-to 3-fold higher accumulation of (M13)SA and (M13)deoxySA compared with controls (figure 3, C and D).In the A182P cells, the formation of (M13)SA was reduced 2-to 3-fold (figure 3C).(M13)deoxySA formation was 15-fold higher in A182P than in SPTLC2wt cells (figure 3D).In the absence of FB 1 , the sphingoid bases are mostly metabolized into ceramides and complex sphingolipids, which results in a predominant release of (M13)SO after acid hydrolysis (figure 3C).In contrast, deoxySLs were predominantly present as (M13)deox-ySA (figure 3D).Interestingly, in SPTLC2wt cells, the total sum of isotope-labeled sphingoid bases was 50% higher in FB 1 -treated than in nontreated cells.In the absence of FB 1 , the activity with serine was the same in A182P and SPTLC2wt cells (figure 3C).No isotopelabeled 1-deoxySLs were found in control or SPTLC2wt cells when FB 1 was absent.However, labeled 1-deoxySLs were formed in A182P cells, although total levels were about 8-fold lower compared with FB 1 -treated A182P cells (figure 3D).][21] No differences in the spatial distribution were seen between SPTLC2 and the A182P mutant.Immunohistochemistry showed in both cases a colocalization with the endoplasmic reticulum marker calnexin (figure e-3).
DISCUSSION Mutations in SPTLC1 are a wellestablished cause of HSANI. 2,3,16An initial study of 12 families with HSANI did not find any patients with mutations in SPTLC2. 22However, because of the known association of SPT with HSANI, SPTLC2 remained a functional candidate and a later study established that mutations in SPTLC2 were also associated with HSANI. 4 Our report provides genetic and functional data demonstrating that a novel A182P mutation found in one family is pathogenic, and gives detailed clinical and neurophysiologic descriptions of the phenotype.The age at onset in this family was in the first decade, younger than is typically seen in patients with HSANI.To date, 3 mutations in SPTLC2 have been described (V359M, G382V, and I504F). 4One of these was associated with early onset in the first decade and other atypical features such as motor conduction velocities in the demyelinating range, suggesting a wide phenotypic spectrum of HSANI.
Elevated blood 1-deoxySL levels were confirmed in both A182P carriers; higher levels were found in the more severely affected daughter than the mother.In vitro, the A182P mutant showed significantly reduced activity with L-serine and increased activity with L-alanine.This was confirmed by 2 metabolic labeling assays.
The combined genetic, clinical, and functional data confirm the association of this novel SPTLC2 mutation with HSANI.It further supports the concept that a pathologic 1-deoxySL formation underlies the pathogenesis of HSANI, indicating that HSANI, in contrast to most other inherited neuropathies, is a metabolic disorder.The 1-deoxySLs are hereby relevant HSANI biomarkers to validate the functional consequences of genetic SPTLC1 and SPTLC2 variants.The Academy's Neurology Career Center is working to bring experienced members together with members who seek guidance on their career path.AAN Mentor Connect needs volunteer Mentors who are willing to share their expertise, insights, and experiences with Mentees.This flexible program, available only to AAN members, matches prospective Mentors and Mentees, and enables you to develop a plan with the Mentee that has a mutually agreeable schedule and expectations.
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From
the MRC Centre for Neuromuscular Diseases (S.M.M., M.L., H.H., M.M.R.) and Department of Molecular Neuroscience (S.M.M., M.L., Y.-T.L., H.H., M.M.R.), UCL Institute of Neurology, Queen Square, London, UK; Department of Neurology (S.M.M.), Adelaide & Meath Hospitals Incorporating the National Children's Hospital, Tallaght, Dublin, Ireland; Institute for Clinical Chemistry (D.E., Y.W., T.H.) and Institute of Physiology and Zurich Center for Integrative Human Physiology (D.E., T.H.), University Hospital Zurich, Switzerland; Department of Neurology (Y.-T.L.), Neurological Institute, Taipei Veterans General Hospital; National Yang-Ming University School of Medicine (Y.-T.L.), Taipei, Taiwan; Neurogenetics Unit (J.P.), National Hospital for Neurology and Neurosurgery, Queen Square; Department of Clinical Neurophysiology (J.B.),The National Hospital for Neurology and Neurosurgery and Department of Molecular Neuroscience, UCL Institute of Neurology, London; Department of Clinical Neurophysiology (J.B.), Norfolk and Norwich University Hospital, Norwich; and Department of Clinical Neurosciences (J.W.), Queen Elizabeth Hospital, Birmingham, UK.Go to Neurology.orgfor full disclosures.Funding information and disclosures deemed relevant by the authors, if any, are provided at the end of the article.

Figure 3 SPT
Figure 3 SPT activity in vitro (A, B) and in HEK293 cells (C, D)