Study On Clinical, Genomic Features Of Hereditary Neuropathy With Liability To Pressure Palsies

Background:Hereditary neuropathy with liability to pressure palsy (HNPP) is a rare autosomal-dominant inherited disease characterized by recurrent sensory or motor dysfunction, and it is often precipitated by compression or minor trauma. Age at onset of HNPP is frequently between 10 to 20 years old. The dysfunction is painless, usually with acute onset, but progressive numbness and weakness of the limbs often occur. HNPP is electrophysiologically characterized by generalized slowing of nerve conduction velocities and prolonged distal latencies (DLs). The main neuropathological feature of HNPP is sausage-like myelin thickenings (tomacula). In around 85% of the patients, HNPP is caused by a large 1.5-Mb DNA segment deletion of chromosome 17p11.2 that harbors the PMP22 gene. The remaining 15% have a variety of point mutations in PMP22 that may lead to frameshifts or other functional changes in the protein.Objective:(1) To summarize the clinical, electrophysiological, Pathological features on the family of HNPP in China.(2) On the one hand, to explore the causes of HNPP patients in this family. On the other hand, to explore the sensitive and rapid method that be suitable for clinic application to detect HNPP, and improve the understanding and diagnostic accuracy of the disease.(3) To explore the influences of cryopreservation on biological properties of WJ-MSCs and the feasibility of treatment on HNPP using WJ-MSCs.Patients and Methods:(1) A large forty-three-member family with ten members suspected to be affected by HNPP was studied and it’s family tree was made. The proband underwent detailed clinical examinations. Electrophysiological studies were performed with surface stimulation and recording electrodes. MCV, SCV, DL, CMAP and SAP were recorded in the nerves of the upper and lower limbs for the proband and his mother. Sural nerve biopsies were carried out for the proband and his mother, and the specimens were cut into semi-thin sections, stained with toluidine blue for light microscope and were cut into ultrathin sections for electron microscope.(2) Real-time PCR with SYBR Green I dye was used to detect gene deletion on chromosome 17p11.2 in 9 suspected HNPP patients, and molecular cloning and gene sequencing technology were used to detect PMP22 gene point mutation.(3) WJ-MSCs were isolated and amplificated, then they were identificated and frozen for 1 month and 2 months. Meanwhile, to evaluate the influences of cryopreservation on biological properties of WJ-MSCs by using flow cytometry, CFU-F assay and senescence test.Results:(1) This pedigree consisted of forty-three members involving four generations. There were ten suspected patients including five males and five females, and one patient had been dead. Clinically, the proband had limb hyposthenia and atrophy, and his mother showed declined tendon reflexes in the right lower limb. The first generation of suspected patients had had a recurring upper limb hyposthenia when alive. The clinical manifestations of other suspected patients in the kindred were similar to the proband. Their first symptoms appeared in adolescence and they all presented with limb hyposthenia and atrophy after stretching, which spontaneously resolved and relapsed. Electrophysiological study exhibited generalized peripheral nerve damage with reduced nerve conduction velocity predominantly in nerve entrapment sites and delayed distal motor latency, including clinical unaffected nerves. The sural nerve biopsy demostrated some abnormal fibers with focal thickenings of myelin sheath (tomacula), and the axons appear compressed and deformed with the smaller diameter. No onion bulb was found.(2) The deletion of PMP22 gene were found for nine suspected patients by Real-time fluorescent quantitative PCR using SYBR Green I dye, and no PMP22 gene point mutation were explored by gene sequencing.(3) WJ-MSCs were isolated successfully from umbilical cord, and were detected to express positively the surface marker of mesenchymal stem cells by using flow cytometry:namely CD90(+), CD106(+), CD166(+), CD73(+), CD44(+). Moreover, no expression of hematopoietic stem cell surface markers was on WJ-MSCs:namely CD34 (-), CD45 (-), HLA-DR (-). There is no significant difference in cell viability, proliferation, self-renewal ability and senescence between Fresh and cryopreserved WJ-MSCs for 1 month and 2 months.Conclusions:These results indicated that a 1.5-Mb DNA segment deletion on chromosome 17p11.2, harboring the PMP22 gene is the main csuse of HNPP in this pedigree. Real-time PCR with SYBR Green Ⅰ dye is a sensitive, rapid and no-invasive means to detect PMP22 deletions, especially for the asymptomatic cases, which suggests the possibility that this method should be used as a clinical diagnostic tool for HNPP in China. Forthmore, as WJ-MSCs has been isolated successfully and there was no influence on biological characteristics of WJ-MSCs by cryopreservation, it is feasible to provide enough cell sources for the therapy of HNPP using WJ-MSCs.