SPTLC1 Mutations Research Articles

SPTLC1 p.Leu38Arg, a novel mutation associated with childhood ALS

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neuromuscular disease. Recently, several gain-of-function mutations in SPTLC1 were associated with juvenile ALS. SPTLC1 encodes for a subunit of the serine-palmitoyltransferase (SPT) - the rate-limiting enzyme in the de novo synthesis of sphingolipids (SL). SPT activity, and thus SL de novo synthesis, is tightly controlled by a homeostatic feedback mechanism mediated by ORMDL proteins. Here we report a novel SPTLC1p.L38R mutation in a young Chinese girl with a signature of juvenile ALS. The patient presented with muscular weakness and atrophy, tongue tremor and fasciculation, breathing problems and positive pyramidal signs. All SPTLC1-ALS mutations including the SPTLC1 p.L38R are located within a single membrane-spanning domain of the protein and impede the interaction with the regulatory ORMDL subunit of SPT. Pertinent to the altered homeostatic control, lipid analysis showed overall increased SL levels in the patient plasma. An increased SPT activity and SL de novo synthesis was confirmed in p.L38R expressing HEK293 cells. Particularily dihydro-sphingolipids (dhSL) were signficantly increased in patient plasma and p.L38R mutant expressing cells. Increased dhSL formation has been previously linked to neurotoxicity and may be involved in the pathomechanism of SPTLC1-ALS mutations.

Deoxy-sphingolipids, oxidative stress, and vitamin C correlate with qualitative and quantitative patterns of small fiber dysfunction and degeneration.

Defined by dysfunction or degeneration of Aδ and C fibers, small fiber neuropathies (SFNs) entail a relevant health burden. In 50% of cases, the underlying cause cannot be identified or treated. In 100 individuals (70% female individuals; mean age: 44.8 years) with an idiopathic, skin biopsy-confirmed SFN, we characterized the symptomatic spectrum and measured markers of oxidative stress (vitamin C, selenium, and glutathione) and inflammation (transforming growth factor beta, tumor necrosis factor alpha), as well as neurotoxic 1-deoxy-sphingolipids. Neuropathic pain was the most abundant symptom (95%) and cause of daily life impairment (72%). Despite the common use of pain killers (64%), the painDETECT questionnaire revealed scores above 13 points in 80% of patients. In the quantitative sensory testing (QST), a dysfunction of Aδ fibers was observed in 70% and of C fibers in 44%, affecting the face, hands, or feet. Despite normal nerve conduction studies, QST revealed Aβ fiber involvement in 46% of patients' test areas. Despite absence of diabetes mellitus or mutations in SPTLC1 or SPTLC2 , plasma 1-deoxy-sphingolipids were significantly higher in the sensory loss patient cluster when compared with those in patients with thermal hyperalgesia ( P < 0.01) or those in the healthy category ( P < 0.1), correlating inversely with the intraepidermal nerve fiber density (1-deoxy-SA: P < 0.05, 1-deoxy-SO: P < 0.01). Patients with arterial hypertension, overweight (body mass index > 25 kg/m 2 ), or hyperlipidemia showed significantly lower L-serine (arterial hypertension: P < 0.01) and higher 1-deoxy-sphingolipid levels (arterial hypertension: P < 0.001, overweight: P < 0.001, hyperlipidemia: P < 0.01). Lower vitamin C levels correlated with functional Aβ involvement ( P < 0.05). Reduced glutathione was lower in patients with Aδ dysfunction ( P < 0.05). Idiopathic SFNs are heterogeneous. As a new pathomechanism, plasma 1-deoxy-sphingolipids might link the metabolic syndrome with small fiber degeneration.

An iPSC model of hereditary sensory neuropathy-1 reveals L-serine-responsive deficits in neuronal ganglioside composition and axoglial interactions

SummaryHereditary sensory neuropathy type 1 (HSN1) is caused by mutations in the SPTLC1 or SPTLC2 sub-units of the enzyme serine palmitoyltransferase, resulting in the production of toxic 1-deoxysphingolipid bases (DSBs). We used induced pluripotent stem cells (iPSCs) from patients with HSN1 to determine whether endogenous DSBs are neurotoxic, patho-mechanisms of toxicity and response to therapy. HSN1 iPSC-derived sensory neurons (iPSCdSNs) endogenously produce neurotoxic DSBs. Complex gangliosides, which are essential for membrane micro-domains and signaling, are reduced, and neurotrophin signaling is impaired, resulting in reduced neurite outgrowth. In HSN1 myelinating cocultures, we find a major disruption of nodal complex proteins after 8 weeks, which leads to complete myelin breakdown after 6 months. HSN1 iPSC models have, therefore, revealed that SPTLC1 mutation alters lipid metabolism, impairs the formation of complex gangliosides, and reduces axon and myelin stability. Many of these changes are prevented by l-serine supplementation, supporting its use as a rational therapy.

SPTLC1 Mutations Associated with Early Onset Amyotrophic Lateral Sclerosis Impair ORMDL Regulation

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder characterized by selective degeneration of lower and upper motor neurons leading to progressive muscle weakness, swallowing difficulties, and respiratory insufficiency. The majority of ALS cases occur sporadically, but a significant number (>10%) are hereditary. Although a variety of cellular processes have been implicated in ALS, a specific monogenic metabolic disruption has not been reported. Thus, the recent discovery of four dominantly inherited SPTLC1 mutations that resulting in childhood-onset ALS in seven families is significant. SPTLC1 encodes a subunit of serine palmitoyltransferase (SPT), the committed and rate-limiting enzyme of sphingolipid synthesis. The ALS mutations flank the membrane spanning domain of SPTLC1, which is not required for ER targeting, association with the SPTLC2 and ssSPT subunits, or enzymatic activity. Rather, this domain is critical for binding of the ORMDL proteins that regulate SPT. This suggested that the SPTLC1 mutations likely result in unrestrained SPT activity. Our studies reveal that these highly penetrant SPTLC1 mutations do indeed abrogate negative regulation of SPT by the ORMDL proteins and result in elevated levels of sphingolipids. Using cultured cells, patient fibroblasts, iPSC-induced MNs, and mouse models, we are investigating the importance of ORMDL regulation in the maintenance of sphingolipid homeostasis and potential mechanisms of sphingolipid mediated motor neuron death. Significantly, the ALS SPTLC1 mutations confer disease pathology by a distinctly different mechanism than SPTLC1 and SPTLC2 mutations associated with hereditary sensory and autonomic neuropathy, type 1 (HSAN1). Whereas the ALS mutations result in elevated levels of canonical sphingolipids, the HSAN1 mutations, which compromise amino acid substrate selectivity of SPT, cause accumulation of atypical deoxy-sphingoid bases that are implicated in neuronal cell death.

Serine restriction alters sphingolipid diversity to constrain tumour growth.

SummarySerine, glycine, and other non-essential amino acids are critical for tumor progression, and strategies to limit their availability are emerging as potential cancer therapies1–3. However, the molecular mechanisms driving this response remain unclear, and the impact on lipid metabolism is relatively unexplored. Serine palmitoyltransferase (SPT) catalyzes the de novo biosynthesis of sphingolipids but also produces non-canonical 1-deoxysphingolipids (doxSLs) when using alanine as a substrate4,5. DoxSLs accumulate in the context of SPTLC1 or SPTLC2 mutations6,7 or low serine availability8,9 to drive neuropathy, and deoxysphinganine (doxSA) has been investigated as an anti-cancer agent10. Here we exploit amino acid metabolism and SPT promiscuity to modulate the endogenous synthesis of toxic doxSLs and slow tumor progression. Anchorage-independent growth reprograms a metabolic network involving serine, alanine, and pyruvate resulting in increased susceptibility to endogenous doxSL synthesis. Targeting the mitochondrial pyruvate carrier (MPC) promotes alanine oxidation to mitigate doxSL synthesis and improves spheroid growth, while direct inhibition of doxSL synthesis drives similar phenotypes. Restriction of dietary serine/glycine potently induces accumulation of doxSLs in xenografts while decreasing tumor growth. Pharmacological modulation of SPT rescues xenograft growth on serine/glycine-restricted diets, while reduction of circulating serine by inhibition of phosphoglycerate dehydrogenase (PHGDH) leads to doxSL accumulation and mitigates tumor growth. SPT promiscuity therefore links serine and mitochondrial alanine metabolism to membrane lipid diversity, which sensitizes tumors to metabolic stress.

SPTLC1 Mutations Associated with Early Onset Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disorder characterized by selective degeneration of lower and upper motor neurons leading to progressive muscle weakness, swallowing difficulties, and respiratory insufficiency that ultimately causes death usually within 2–5 years of diagnosis. The majority of ALS cases occur sporadically, but a significant number (&gt;10%) display Mendelian inheritance. Very recently, four unique de novo or dominantly inherited SPTLC1 mutations associated with early childhood‐onset ALS without clinical sensory involvement have been identified in eight independent families. SPTLC1 encodes a subunit of serine palmitoyltransferase (SPT), the committed and rate‐limiting enzyme of sphingolipid synthesis. The ALS mutations flank the first membrane spanning domain of SPTLC1, which is not required for ER targeting, association with the SPTLC2 and ssSPT subunits, or enzymatic activity. Rather, this domain is critical for binding of the ORMDL proteins that negatively regulate SPT. This suggested that the SPTLC1 mutations might interfere with homeostatic regulation of SPT and that elevated de novo sphingolipid synthesis could underlie the ALS disease pathology. Our studies reveal that these highly penetrant SPTLC1 mutations do indeed abrogate negative regulation of SPT by the ORMDL proteins and result in elevated levels of sphingolipids. Using cultured cells, patient fibroblasts, iPSC‐induced MNs, and mouse models, we are investigating the importance of ORMDL regulation in the maintenance of sphingolipid homeostasis and the mechanisms responsible for sphingolipid mediated motor neuron death. Significantly, the ALS SPTLC1 mutations confer disease pathology by a distinctly different mechanism than the previously characterized SPTLC1 and SPTLC2 mutations associated with hereditary sensory and autonomic neuropathy type 1 (HSAN1). Whereas the ALS mutations result in elevated levels of canonical sphingolipids, the HSAN1 mutations, which compromise amino acid substrate selectivity of SPT, cause accumulation of atypical deoxy‐sphingoid bases that are implicated in neuronal cell death.

Deoxysphingolipids as candidate biomarkers for a novel SPTLC1 mutation associated with HSAN-I.

Hereditary sensory and autonomic neuropathy type 1 (HSAN-I)—an autosomal dominant, mainly sensory neuropathy—typically affects patients in their second and third decades, presenting with ulcerations and lancinating pain attacks.1 Diagnosis is heavily dependent on genetic analysis, focusing on 6 genes: serine palmitoyltransferase ( SPT ) long chain base subunits 1 and 2 ( SPTLC1 and SPTLC2 ), atlastin 1 ( ATL1 ) and 3 ( ATL3 ), DNA methyltransferase 1, and Ras-related protein ( RAB7 ). Most patients carry SPTLC1 mutations (OMIM*605712) that affect sphingolipids biosynthesis by modifying SPT substrate specificity to produce atypical neurotoxic deoxysphingoid bases (DSBs). Along with their accumulation in mutant cells, elevated DSBs were also found in plasma samples, thus becoming a candidate biomarker of disease.2

Mutations in the Sphingolipid Pathway Gene &lt;i&gt;SPTLC1&lt;/i&gt; are a Cause of Amyotrophic Lateral Sclerosis

SPTLC1 encodes a critical subunit of serine palmitoyltransferase, the enzyme catalyzing the first and rate-limiting step in de novo sphingolipid biosynthesis, and mutations in this gene are known to cause hereditary sensory autonomic neuropathy, type 1A. Using exome sequencing, we identified a de novo coding variant in SPTLC1 in an individual diagnosed with juvenile-onset amyotrophic lateral sclerosis (ALS), and confirmed its pathogenicity by showing elevated plasma levels of neurotoxic deoxymethyl-sphinganine. We also found SPTLC1 mutations in 0.34% of 5,607 ALS cases, and immunohistochemically confirmed the expression of SPTLC1 in spinal cord motor neurons, supporting their role in the pathogenesis of this fatal neurodegenerative disease. Toxicity of deoxymethyl-sphinganine was demonstrated in HEK293FT cells, and could be corrected by L-serine supplementation. Our data broaden the phenotype associated with SPTLC1. Furthermore, nutritional supplementation with serine may be beneficial if instituted at an early stage among patients carrying mutations in SPTLC1.

Comprehensive Computational Analysis of Protein Phenotype Changes Due to Plausible Deleterious Variants of Human SPTLC1 Gene.

Genetic variations found in the coding and non-coding regions of a geneare known to influence the structure as well as the function of proteins. Serine palmitoyltransferase long chain subunit 1 a member of α-oxoamine synthase family is encoded by SPTLC1 gene which is a subunit of enzyme serine palmitoyltransferase (SPT). Mutations in SPTLC1 have been associated with hereditary sensory and autonomic neuropathy type I (HSAN-I). The exact mechanism through which these mutations elicit protein phenotype changes in terms of structure, stability and interaction with other molecules is unknown. Thus, we aimed to perform a comprehensive computational analysis of single nucleotide polymorphisms (SNPs) of SPTLC1 to prioritize a list of potential deleterious SNPs and to investigate the protein phenotype change due to functional polymorphisms. In this study, a diverse set of SPTLC1 SNPs were collected and scrutinized to categorize the potential deleterious variants. Our study concordantly identified 21 non- synonymous SNPs as pathogenic and deleterious that might induce alterations in protein structure, flexibility and stability. Moreover, evaluation of frameshift, 3’ and 5’ UTR variants shows c.*1302T> Gas effective. This comprehensive in silico analysis of systematically characterized list of potential deleterious variants could open avenues as primary filter to substantiate plausible pathogenic structural and functional impact of variants.

Investigation for SPTLC1 mutations in a Taiwanese cohort with hereditary neuropathies

Background: Mutations in the serine palmitoyltransferase long chain base subunit 1 gene (SPLTC1) have been linked to hereditary sensory and autonomic neuropathy type 1 (HSAN1). SPTLC1 mutations alter the canonical activity of serine palmitoyltransferase and consequently induce the production of 1-deoxy-sphingolipid causing neuronal toxicity. Pilot studies for HSAN1 treatment with increasing availability of L-serine has showed promising outcome. To identify SPTLC1 mutations in HSAN1 patients would provide opportunity for the specific management.

Identifying Unique Protein Alterations Caused by SPTLC1 Mutations in a Transfected Neuronal Cell Model

Hereditary sensory neuropathy type I is an autosomal dominant disorder that affects the sensory neurons. Three missense mutations in serine palmitoyltransferase long chain subunit 1 cause hereditary sensory neuropathy type I. The endoplasmic reticulum, where the serine palmitoyltransferase long chain subunit 1 protein resides, and mitochondria are both altered in hereditary sensory neuropathy type I mutant cells. Employing a transfected neuronal cell line (ND15), we have identified and confirmed altered protein expression levels of ubiquinol cytochrome C, Hypoxia Up regulated Protein 1, Chloride Intracellular Channel Protein 1, Ubiqutin-40s Ribosomal Protein S27a, and Coactosin. Additionally, further 14 new proteins that exhibited altered expression within V144D, C133W and C133Y mutants were identified. These data have shown that mutations in SPTLC1 alter the expression of a set of proteins that may help to establish a causal link between the mitochondria and ER and the “dying back” process of dorsal root ganglion neurons that occurs in HSN-I.

Natural history and biomarkers in hereditary sensory neuropathy type 1.

Introduction: Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is most commonly caused by missense mutations in SPTLC1. In this study we mapped symptom progression and compared the utility of outcomes. Methods: We administered retrospective surveys of symptoms and analyzed results of nerve conduction, autonomic function testing (AFT), and PGP9.5-immunolabeled skin biopsies. Results: The first symptoms were universally sensory and occurred at a median age of 20 years (range 14–54 years). The onset of weakness, ulcers, pain, and balance problems followed sequentially. Skin biopsies revealed universally absent epidermal innervation at the distal leg with relative preservation in the thigh. Neurite density was highly correlated with total Charcot-Marie-Tooth Examination Score (CMTES; r2 = −0.8) and median motor amplitude (r2 = −0.75). Conclusions: These results confirm sensory loss as the initial symptom of HSAN1 and suggest that skin biopsy may be the most promising biomarker for future clinical trials. Muscle Nerve, 2015 Muscle Nerve 51: 489–495, 2015

Early-onset severe hereditary sensory and autonomic neuropathy type 1 with S331F SPTLC1 mutation

Hereditary sensory and autonomic neuropathy type I (HSAN I) is an autosomal dominant disease characterized by prominent sensory impairment, resulting in foot ulcers or amputations and has a juvenile to adult onset. The major underlying causes of HSAN I are mutations in SPTLC1, which encodes the first subunit of serine palmitoyltransferase (SPT). To date, there have been no reports with regard to an HSAN patient of Korean origin. In this report we discussed an HSAN I patient with a missense mutation in SPTLC1 (c.992C>T: p.S331F). The patient had noticed frequent falls, lower leg weakness and hand tremors at age five. The patient also presented with foot ulcers, muscle hypotrophy, cataracts, hoarseness, vocal cord palsy and respiratory difficulties and succumbed to the condition at the age of 28 years. In accordance with previous reports, a mutation in Ser331 in the present patient was associated with early-onset and a severe phenotype. Therefore, Ser331 in SPTLC1 is a crucial amino acid, which characterizes the HSAN I phenotype.

Catecholamine Disorders and Hereditary Sensory and Autonomic Neuropathy

Hereditary disorders affecting catecholamine metabolism or development of sensory and autonomic neurons manifest with a wide range of autonomic disturbance. Genetic catecholamine disorders include deficiencies of tyrosine hydroxylase (TH), tetrahydrobiopterin (BH4), L-amino acid decarboxylase (L-ADCC), dopamine-beta-hydroxylase (DBH), vesicula monoamine transporter (VMAT) 2, and norepinephrine transporter (NET). BH4, L-ADCC and VMAT2 deficiency have similar manifestations; autonomic symptoms include hypersalivation, temperature instability, and pupil abnormalities, associated with various motor (eg, dystonia) and behavioral abnormalities. DBH deficiency manifests neonatally with hypotension, hypothermia, hypoglycemia, vomiting and dehydration; orthostatic hypotension is the major manifestation in childhood and later. NET deficiency produces orthostatic intolerance with postural tachycardia. Hereditary sensory and autonomic neuropathies (HSANs) are a heterogenous group of disorders that present with sensory loss and variable degrees of motor and autonomic involvement. Autosomal dominant HSANs include HSAN-1 (due SPTLC1 mutations), Charcot-Marie-Tooth (CMT) disease 2B (due to Rab7 mutations), and HSAN-1 with dementia and hearing loss (linked to DNTM1 mutations). HSAN-1 has a juvenile-to-adult onset sensory neuropathy with minor autonomic involvement; CMT2B has prominent motor manifestations. Autosomal recesssive forms include HSAN-II (linked to mutations in the WNK1, FAM134B, or KIF1A genes), HSAN-III (linked to IKBKAP mutation), HSAN-IV (linked to NTRK1 mutations), HSAN-V (linked to NGFB mutations), and HSAN with spastic paraplegia (linked to CCT5 mutations). Autosomal recessive HSANs have onset at birth or in early childhood and have prominent autonomic symptoms. In particular, HSAN-III (familial dysautonomia or Riley-Day syndrome) is characterized by severe small fiber sensory loss and life-threatening autonomic dysfunction. Prominent manifestations include absence of cold and pain sensation, temperature instability (hypothermia and excessive perspiration), blood pressure lability (due to impaired baroreflex afferent function), abnormal swallowing, vomiting crises, and recurrent bouts of aspiration pneumonia. HSAN-IV and HSN-V are characterized by congenital insensitivity to pain and reduced or absent sweating.

Neurophysiology and Intraepidermal Nerve Fiber Density in Hereditary Sensory and Autonomic Neuropathy Type 1 (HSAN1) (P03.202)

Objective: To quantify the impact of the SPTLC1 mutation upon neurological outcome measures in HSAN1 patients. Background Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is the most common inherited neuropathy to predominantly affect sensory neurons. The disorder is caused by mutations in SPTCL1, which encodes the enzyme serine palmitoyltransferase. Design/Methods: We studied two HSAN1 families with different mutations (n = 8). Five patients carried the C133Y mutation, three the C133W mutation. The median age was 34 years (range 26-59); the median age of onset was 19 (range 15-54). All eight patients underwent neurological examination and were scored using the CMT symptom score (CMTSS). Nerve conduction studies (NCS) of the upper and lower extremities were conducted in all (including 3 motor NCS and 4 sensory NCS). Six patients had skin biopsies at the distal leg and proximal thigh to assess the intraepidermal nerve fiber density (IENFD). Results: The average CMTSS was 12.38 (SD +/-6.74). All patients complained of neuropathic pain apart from two patients who had the highest CMTSS. Sensory symptoms (average score 2.9) were prominent but motor symptoms (average score 1.2) were present as well. No sensory responses were obtainable in 5/8; lower extremity motor responses were absent in 6/8 patients. None of these 6 patients had epidermal nerve fibers detected at the distal leg, while they were present in the thigh in all patients. The correlation coefficient between CMTSS and IENFD was -0.8 and between CMTSS and median motor amplitudes was -0.75. Conclusions: Beyond sensory there is also motor impairment present in HSAN1 patients. In our limited sample size we found an inverse correlation between both CMTSS and IENFD and CMTSS and nerve conduction studies. This study provides important clinical characterization of both neurophysiology and the IENFD in HSAN1. Disclosure: Dr. David has nothing to disclose. Dr. Oaklander has nothing to disclose. Dr. Pan has nothing to disclose. Dr. Novak has nothing to disclose. Dr. Brown has received personal compensation for activities as an advisory board member. Dr. Eichler has nothing to disclose.

Characterization of two mutations in the SPTLC1 subunit of serine palmitoyltransferase associated with hereditary sensory and autonomic neuropathy type I

Hereditary sensory and autonomic neuropathy type I (HSAN-I) is an axonal peripheral neuropathy leading to progressive distal sensory loss and severe ulcerations. Mutations in SPTLC1 and SPTLC2, encoding the two subunits of serine palmitoyltransferase (SPT), the enzyme catalyzing the first and rate-limiting step in the de novo synthesis of sphingolipids, have been reported to cause HSAN-I. Here, we demonstrate that the SPTLC1 mutations p.S331F and p.A352V result in a reduction of SPT activity in vitro and are associated with increased levels of the deoxysphingoid bases 1-deoxy-sphinganine and 1-deoxymethyl-sphinganine in patients' plasma samples. Stably expressing p.S331F-SPTLC1 HEK293T cell lines likewise show accumulation of deoxysphingoid bases, but this accumulation is not observed in HEK293T cells overexpressing p.A352V-SPTLC1. These results confirm that the increased formation of deoxysphingoid bases is a key feature for HSAN-I as it is associated with all pathogenic SPTLC1 and SPTLC2 mutations reported so far, but also warrant for caution in the interpretation of in vitro data.

POG11 A novel mutation in the nerve-specific 5′-UTR of the Cx32 gene causing CMTX1

CMTX1 is the second most common cause of Charcot-Marie-Tooth disease (CMT), usually caused by mutations in the Cx32 gene. Cx32 has two tissue-specific promoters: P1, specific for liver and pancreas,...

Hereditary Sensory Neuropathy Type 1 Is Caused by the Accumulation of Two Neurotoxic Sphingolipids

HSAN1 is an inherited neuropathy found to be associated with several missense mutations in the SPTLC1 subunit of serine palmitoyltransferase (SPT). SPT catalyzes the condensation of serine and palmitoyl-CoA, the initial step in the de novo synthesis of sphingolipids. Here we show that the HSAN1 mutations induce a shift in the substrate specificity of SPT, which leads to the formation of the two atypical deoxy-sphingoid bases (DSBs) 1-deoxy-sphinganine and 1-deoxymethyl-sphinganine. Both metabolites lack the C(1) hydroxyl group of sphinganine and can therefore neither be converted to complex sphingolipids nor degraded. Consequently, they accumulate in the cell, as demonstrated in HEK293 cells overexpressing mutant SPTLC1 and lymphoblasts of HSAN1 patients. Elevated DSB levels were also found in the plasma of HSAN1 patients and confirmed in three groups of HSAN1 patients with different SPTLC1 mutations. The DSBs show pronounced neurotoxic effects on neurite formation in cultured sensory neurons. The neurotoxicity co-occurs with a disturbed neurofilament structure in neurites when cultured in the presence of DSBs. Based on these observations, we conclude that HSAN1 is caused by a gain of function mutation, which results in the formation of two atypical and neurotoxic sphingolipid metabolites.

P59 A novel mutation in the nerve-specific 5 UTR of the Cx32 gene causing CMTX1

CMTX1 is the second most common cause of Charcot-Marie-Tooth disease (CMT), usually caused by mutations in the Cx32 gene. Cx32 has two tissue-specific promoters: P1, specific for liver and pancreas, and P2 which is nerve specific. Over 300 mutations have been described in Cx32 , spread throughout the coding region. However, 10% of CMTX1 families do not have mutations in the coding region. To date, 5 noncoding region mutations have been reported. We describe two families with X-linked inheritance and a phenotype consistent with CMTX1 who did not have mutations in the Cx32 coding region. Family 1 were negative for mutations in PMP22, MPZ, SPTLC1 and GDAP1 Family 2 were negative for mutations in MPZ and rearrangements at chromosome 17p11.2. The noncoding region of Cx32 was sequenced and an upstream exon-splicing variant found at c.-373G>A which segregated with the disease in both families. This variant is located at the last base of the nerve-specific 5′UTR exon and thus may disrupt splicing of the nerve-specific transcript. Online consensus splice-site programs predict a reduced score for the mutant sequence vs the normal sequence. Two other mutations have previously been described within the 5′UTR region, which created a potential donor splice site and were shown to prevent translation of mutant mRNA. It is likely that other mutations within the Cx32 noncoding regions account for the CMTX1 families who do not have coding region mutations.

Genes for hereditary sensory and autonomic neuropathies: a genotype-phenotype correlation

Hereditary sensory and autonomic neuropathies (HSAN) are clinically and genetically heterogeneous disorders characterized by axonal atrophy and degeneration, exclusively or predominantly affecting the sensory and autonomic neurons. So far, disease-associated mutations have been identified in seven genes: two genes for autosomal dominant (SPTLC1 and RAB7) and five genes for autosomal recessive forms of HSAN (WNK1/HSN2, NTRK1, NGFB, CCT5 and IKBKAP). We performed a systematic mutation screening of the coding sequences of six of these genes on a cohort of 100 familial and isolated patients diagnosed with HSAN. In addition, we screened the functional candidate gene NGFR (p75/NTR) encoding the nerve growth factor receptor. We identified disease-causing mutations in SPTLC1, RAB7, WNK1/HSN2 and NTRK1 in 19 patients, of which three mutations have not previously been reported. The phenotypes associated with mutations in NTRK1 and WNK1/HSN2 typically consisted of congenital insensitivity to pain and anhidrosis, and early-onset ulcero-mutilating sensory neuropathy, respectively. RAB7 mutations were only found in patients with a Charcot-Marie-Tooth type 2B (CMT2B) phenotype, an axonal sensory-motor neuropathy with pronounced ulcero-mutilations. In SPTLC1, we detected a novel mutation (S331F) corresponding to a previously unknown severe and early-onset HSAN phenotype. No mutations were found in NGFB, CCT5 and NGFR. Overall disease-associated mutations were found in 19% of the studied patient group, suggesting that additional genes are associated with HSAN. Our genotype–phenotype correlation study broadens the spectrum of HSAN and provides additional insights for molecular and clinical diagnosis.