Hereditary sensory and autonomic neuropathy type 1 (HSAN1) is a rare autosomal dominant inherited peripheral neuropathy caused by mutations in the SPTLC1 and SPTLC2 subunits of serine palmitoyltransferase (SPT). The mutations induce a permanent shift in the substrate preference from L-serine to L-alanine, which results in the pathological formation of atypical and neurotoxic 1-deoxy-sphingolipids (1-deoxySL). Here we compared the enzymatic properties of 11 SPTLC1 and six SPTLC2 mutants using a uniform isotope labelling approach. In total, eight SPT mutants (STPLC1p.C133W, p.C133Y, p.S331F, p.S331Y and SPTLC2p.A182P, p.G382V, p.S384F, p.I504F) were associated with increased 1-deoxySL synthesis. Despite earlier reports, canonical activity with l-serine was not reduced in any of the investigated SPT mutants. Three variants (SPTLC1p.S331F/Y and SPTLC2p.I505Y) showed an increased canonical activity and increased formation of C20 sphingoid bases. These three mutations are associated with an exceptionally severe HSAN1 phenotype, and increased C20 sphingosine levels were also confirmed in plasma of patients. A principal component analysis of the analysed sphingoid bases clustered the mutations into three separate entities. Each cluster was related to a distinct clinical outcome (no, mild and severe HSAN1 phenotype). A homology model based on the protein structure of the prokaryotic SPT recapitulated the same grouping on a structural level. Mutations associated with the mild form clustered around the active site, whereas mutations associated with the severe form were located on the surface of the protein. In conclusion, we showed that HSAN1 mutations in SPT have distinct biochemical properties, which allowed for the prediction of the clinical symptoms on the basis of the plasma sphingoid base profile.
Background: Human amniotic membrane has been used clinically in a variety of applications for over the past 100 years and produced a significant amount of data in multiple areas of medicine. Its clinical usage ranges from wound coverage for burn victims to healing of the conjunctiva after pterygium repair. The amniotic membrane natural properties provide an easy to use, safe option for various medical applications. There is need to develop a method for storage of amniotic membrane which can retain the biological properties and as well have long shelf life too.Methods: The experimental technique was standardized for cryopreservation of amniotic membrane. For this, amniotic membrane was obtained from mothers who had delivered through caesarean section with their consent.Results: The standardized protocol for cryopreservation of amniotic membrane was found to be safe and preserved amniotic membrane is expected to have long shelf life. Conclusions: The advantages associated with amniotic membrane such as easily available, inexpensive, non-immunogenic and antimicrobial, anti-inflammatory properties make it a suitable graft to be used in wound healing and diabetic foot ulcers.
Hereditary neuropathies comprise a wide variety of chronic diseases associated to more than 80 genes identified to date. We herein examined 612 index patients with either a Charcot-Marie-Tooth phenotype, hereditary sensory neuropathy, familial amyloid neuropathy, or small fiber neuropathy using a customized multigene panel based on the next generation sequencing technique. In 121 cases (19.8%), we identified at least one putative pathogenic mutation. Of these, 54.4% showed an autosomal dominant, 33.9% an autosomal recessive, and 11.6% an X-linked inheritance. The most frequently affected genes were PMP22 (16.4%), GJB1 (10.7%), MPZ, and SH3TC2 (both 9.9%), and MFN2 (8.3%). We further detected likely or known pathogenic variants in HINT1, HSPB1, NEFL, PRX, IGHMBP2, NDRG1, TTR, EGR2, FIG4, GDAP1, LMNA, LRSAM1, POLG, TRPV4, AARS, BIC2, DHTKD1, FGD4, HK1, INF2, KIF5A, PDK3, REEP1, SBF1, SBF2, SCN9A, and SPTLC2 with a declining frequency. Thirty-four novel variants were considered likely pathogenic not having previously been described in association with any disorder in the literature. In one patient, two homozygous mutations in HK1 were detected in the multigene panel, but not by whole exome sequencing. A novel missense mutation in KIF5A was considered pathogenic because of the highly compatible phenotype. In one patient, the plasma sphingolipid profile could functionally prove the pathogenicity of a mutation in SPTLC2. One pathogenic mutation in MPZ was identified after being previously missed by Sanger sequencing. We conclude that panel based next generation sequencing is a useful, time- and cost-effective approach to assist clinicians in identifying the correct diagnosis and enable causative treatment considerations.
Hereditary sensory neuropathy type 1 (HSN1) is a rare, slowly progressive neuropathy causing profound sensory deficits and often severe motor loss. L-serine supplementation is a possible candidate therapy but the lack of responsive outcome measures is a barrier for undertaking clinical trials in HSN1. We performed a 12-month natural history study to characterise the phenotype of HSN1 and to identify responsive outcome measures.Assessments included Charcot-Marie-Tooth Neuropathy Score version 2 (CMTNSv2), CMTNSv2-Rasch modified, nerve conduction studies, quantitative sensory testing, intraepidermal nerve fibre density (thigh), computerised myometry (lower limbs), plasma 1-deoxysphingolipid levels, calf-level intramuscular fat accumulation by MRI and patient-based questionnaires (Neuropathic Pain Symptom Inventory and 36-Short Form Health Survey version 2 [SF-36v2]).35 patients with HSN1 were recruited. There was marked heterogeneity in the phenotype mainly due to differences between the sexes: males generally more severely affected. The outcome measures that significantly changed over 1 year and correlated with CMTNSv2, SF-36v2-physical component and disease duration were MRI determined calf intramuscular fat accumulation (mean change in overall calf fat fraction 2.36%, 95% CI 1.16 to 3.55, p=0.0004), pressure pain threshold on the hand (mean change 40 kPa, 95% CI 0.7 to 80, p=0.046) and myometric measurements of ankle plantar flexion (median change -0.5 Nm, IQR -9.5 to 0, p=0.0007), ankle inversion (mean change -0.89 Nm, 95% CI -1.66 to -0.12, p=0.03) and eversion (mean change -1.61 Nm, 95% CI -2.72 to -0.51, p=0.006). Intramuscular calf fat fraction was the most responsive outcome measure.MRI determined calf muscle fat fraction shows validity and high responsiveness over 12 months and will be useful in HSN1 clinical trials.
To evaluate the safety and efficacy of l-serine in humans with hereditary sensory autonomic neuropathy type I (HSAN1).In this randomized, placebo-controlled, parallel-group trial with open-label extension, patients aged 18-70 years with symptomatic HSAN1 were randomized to l-serine (400 mg/kg/day) or placebo for 1 year. All participants received l-serine during the second year. The primary outcome measure was the Charcot-Marie-Tooth Neuropathy Score version 2 (CMTNS). Secondary outcomes included plasma sphingolipid levels, epidermal nerve fiber density, electrophysiologic measurements, patient-reported measures, and adverse events.Between August 2013 and April 2014, we enrolled and randomized 18 participants, 16 of whom completed the study. After 1 year, the l-serine group experienced improvement in CMTNS relative to the placebo group (-1.5 units, 95% CI -2.8 to -0.1, p = 0.03), with evidence of continued improvement in the second year of treatment (-0.77, 95% CI -1.67 to 0.13, p = 0.09). Concomitantly, deoxysphinganine levels dropped in l-serine-treated but not placebo-treated participants (59% decrease vs 11% increase; p < 0.001). There were no serious adverse effects related to l-serine.High-dose oral l-serine supplementation appears safe in patients with HSAN1 and is potentially effective at slowing disease progression.NCT01733407.This study provides Class I evidence that high-dose oral l-serine supplementation significantly slows disease progression in patients with HSAN1.
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