Phenotype And Genotype Association Analysis And The Primary Afferents Function Study For Han Chinese Patients With Congenital Insensitivity To Pain And Anhidrosis

Part oneHeterogeneity of Clinical Features,and Mutation Analysis of NTRK1 in Han Chinese patients with Congenital Insensitivity to Pain with Anhidrosis Background Congenital insensitivity to pain with anhidrosis（CIPA）（OMIM #256800）,also known as hereditary sensory and autonomic neuropathy type IV（HSAN IV）.It is a rare autosomal recessive genetic disorder in which NTRK1 gene loss of function mutation leads to of the encoded Trk A protein,leading to NGF-dependent primary afferent neurons including primary sensory afferent fibers（Aδ and C fibers）and autonomic sympathetic ganglion neurons undergoing apoptosis during development.The main clinical features of this condition are congenital,and they include complete insensitivity to both superficial and deep painful stimuli,absence of sweating,and recurrent episodes of unexplained fever.Moreover,these patients exhibit self-mutilating behaviors such as biting their fingers,lips,and tips of the tongue,multiple repeated bone fractures,and various degrees of mental retardation.Similar cases have been reported around the world,among which Japanese and Israeli scholars have summarized their ethnic characteristics and found specific mutation sites.At present,the research on CIPA in China is mostly based on case reports.It is unclear about the characteristics of the pathogenesis of Chinese CIPA patients and the complex relationship between genotype and clinical phenotype.PurposePatients were collected and pathogenic gene mutations were screened to investigate the incidence of CIPA patients in China,and to analyze the phenotypic and genotypeic characteristics association.In addition,we try to search for the common pathogenic mutation in Han Chinese population.Methods1.We included the CIPA patients through medical institutions,media,and social networks.Personal data and clinical information were investigated by questionnaires,interview and physical examination.Written informed consent was obtained from all participants and parents or legal guardians for participant under the age of 18 years,and that this study was conducted in accordance with the Declaration of Helsinki.2.DNA was extracted from blood samples from these patients and their available familial members.Patients were subjected to genetic analysis by direct Sanger sequencing,targeted next-generation sequencing（NGS）gene panel and whole genome exon sequencing（WES）.Direct sequencing is to detect the 17 exons and 50 intron base sequences of the NTRK1 gene.The NGS gene panel is to screen 57 genetic peripheral neuropathy genes including NTRK1,NGF,PRDM12,TRPV1,SCN9 A,DNMT1 and WNK1.The WES method was a selection that we have not screened any mutation by either of the above methods.The resulting sequences were analyzed using Sequencing Analysis and compared with the current reference sequence for NTRK1 in the NCBI database.3.Bioinformatics analysis for different types of mutations: missense mutations were used to analyze the amino acid conservation of mutants and predicted their effects on Trk A protein function using Clustal Omega and SIFT software,respectively;The splicing mutation on intron was analyzed the effect of mutation on NTRK1 splicing function by means of verification.The insertion and deletion mutations were affected analyzing the frameshift result for the Trk A protein.4.Analyze the genetic characteristics of Chinese patients with CIPA to find out common mutation in Chinese population.Results1.A total of 44 Chinese Han nationality CIPA patients from 38 unrelated families were collected.The family distribution of the patients was mainly concentrated in the central and southern regions of China.The number of patients in Hubei,Henan and Shandong province were the highest;the patients were mainly in prepubertal age,the gender ratio about 3:1 of male with female.And the mortality rate is 9.3% in the present study.2.There are significant differences in the patient’s clinical characteristics,including pain insensitivity,temperature perception,and clinical complications.Interestingly,3 patients developed an evasive response to noxious stimuli after puberty,and some patients reported a feeling of "visceral pain".Quantitative sensory tests found significant differences in patient temperature perception.Most patients were insensitive to cold,warm and thermal stimulus in their limbs,but several patients were able to sense the thermal stimulation.3.There are obvious abnormalities in the bone,immune and neurodevelopment of the patient.The incidence of repeated fractures and dislocations in the study were 47.4% and 13.2%,respectively.The bone mineral density SOS values of a pair of twins were 3356 m/sec and 3270 m/sec,respectively,relative to the normal peers with percentiles 20 and 6.The Gessel pediatric developmental assessment of CIPA in early childhood patients showed that the child was stunted in terms of exercise,medication,language ability and social ability,especially the physiology and language ability.4.This study identified a total of 21 NTRK1 mutation sites in 39 patients,including 12 missense mutations,6 insertional deletion mutations,and 3 splicing mutations.Seven of them were novel mutations,namely c.797G>A,c.1253₁254del TC,c.1729G>C,c.1775T>G,c.1805G>A,c.1806-2A>G and c.429–374_c.717+485del1403.5.Through the whole exon sequencing,it was found that one patient had complete deletion of exon 5 and 6 peaks,and specific mutation sequences were identified by PCR walking.The results showed that large fragment deletion mutations were caused by incorrect recognition of splice sites: c.429-374_c.717+485del1403（p.V144Nfs*8）,a total of 1403 bp base sequence was lost,resulting in amino acid change from proline to asparagine at position 144,terminated by 8 wrong amino acids,forming an abnormal truncated protein.6.More than 52.3% of patients carry the splicing mutation c.851-33T>A,which strongly suggests that it may be a common pathogenic site in Han patients.ConclusionsThis study details the clinical phenotypes,and mutation characteristics of 41 Chinese CIPA patients,to better understand the disease characteristics in Chinese Han patients with CIPA.Furthermore,heterogeneity of clinical features and molecular pathology of CIPA would provide unique opportunities to explore critical roles of the autonomic sympathetic nervous system,as well as components of the peripheral sensory nervous system that transmit noxious stimuli in humans.Part twoPeripheral sensory nerve function study in patients with congenital insensitivity to pain with anhidrosis BackgroundPrimary afferent fibers of thick myelinated Aβ transmits tactile signals,and myelinated thin fibers Aδ and unmyelinated C fibers participate in the noxious information transmission such as pain.The currently recognized mechanism of CIPA pathogenesis was that NTRK1 gene mutations lead to the NGF-dependent neurons including myelinated A-δ fibers and unmyelinated C fibers,as well as sympathetic postganglionic neuronal developmental abnormalities.Nerve biopsy studies on CIPA patients indicated that almost complete absence of primary afferent neurons with unmyelinated fibers and reduced numbers of primary afferent neurons with small myelinated fibers in individuals with CIPA.They were insensitive to superficial and deep tissue pain stimuli,including visceral sensations,but the sense of touch,vibration,and position are normal.However,our clinical study found that some patients have a tendency to “recover” pain,and there were also significant individual differences in the perception of temperature perception.For this performance,we were unable to determine whether the patient actually perceived the pain signal or acquired behavior for the noxious stimulus,maybe the Aβ fibers alienation to transmit high-intensity tactile information as a pain signal.In addition,a subpopulation of C fibers is believed to mediate pleasure sensory contact.Patients with CIPA have an itchy sensation,but mosquito bites and other irritations do not itch and scratch.Whether they can perceive the sensory（pleasant）touch remains to be determined.PurposeThe aim of this study was to explore the neurological function of primary afferent fibers of Aβ,Aδ,and C fibers of CIPA patients by quantitative sensory testing,electrical stimulation of single nerve fiber microscopy,and axon reflex flare.Methods1.Two CIPA patients aged 10 and 18 years old were included in the study.Nine test parameters were detected,including brush,mechanical sensory threshold（MDT）,mechanical pain threshold（MPT）,pain intensity after repeated stimulation（WUR）,deep muscle tenderness（PPT）,vibration detection threshold（VDT）,and Temperature sensing（CDT,WDT,and HPT）.The testing locations were the dorsum foot,dorsum hand,and the trigeminal innervation area.2.Microneurography（MNG）recording technique was used to identify individual C-fibers.Three neurofibrillary functions of the primary sensory conduction were then detected by three transcutaneous electrical stimulations of rectangular,sinusoidal and half-sinusoidal pulses.3.Drop 1% histamine and itching cowpea into or into the epidermis of the subject,then tell the tester his feelings such as itching,pain or burning sensation every 10 seconds.A laser Doppler imager（Perimed,Sweden）was then used to measure the presence or absence of axonal reflex erythema at the test site and to analyze the size of the axonal reflex flare.4.Under local anesthesia,a skin biopsy ring was used to drill 0.5mm2 skin tissue samples from the patient’s arm and the dorsal of the foot.After PBS washing,we need to fix them in 4 °C 2.5% glutaraldehyde and send them for examination,and observe the nerve ending structure in the skin tissue sample of the patients with CIPA under electron microscope.Results1.Compared with the detect threshold of healthy person,the patients’ tactile sense（brush and MDT）was basically normal,and the MDT threshold of patient 2 was slightly higher.No pain sensation was detected in VDT,MPT,WUR,PPT and HDT,indicating that superficial pain,deep tenderness and thermal pain were complete absent.In both patients,cold and warm sensations were absent in the arms and instep,and only the face could identified the thermal stimulation.2.Two CIPA patients had a NRS score of 0 under rectangular pulse（A-δ）,sinusoidal pulse（CMi）,half-chord pulse（A-δ and CM）and 1 min sinusoidal pulse（CMi）.The patient developed a "hemp" sensation with a high-frequency rectangular pulse stimulus with a strength of 10.The patient has perception under sinusoidal stimulation,but there is no obvious pain.The first patient had a "hemp" of 7 in intensity and a slight burning pain at 0.4 m A.1 min sinusoidal pulse（CMi）stimulation without erythema reaction is a pathological response,indicating the absence of C fiber activation.3.Histamine and itchy cowpea stimulation cannot cause local axonal reflex of the skin,no itching and erythema,indicating that the patient’s histamine-sensitive mechanical insensitive C fiber（CMi-H）and mechanically sensitive C fiber（CM）cannot be activated.4.Scanning electron microscopy revealed that the patient had almost no primary afferent nerve fibers with unmyelinated fibers and small myelin fibers.The myelinated nerve fiber structure was observed in one field of view of the arm skin tissue section.ConclusionsIn this study,we have investigated the function of primary afferent fibers in patients with CIPA through the method of the QST,MNG and axon reflex flare.We found that patients with CIPA have normal perception of non-noxious stimulus which indicated the function of A-beta fiber was normal,and the function of C fiber and A-delta was missing.Although there is no pain sensation,but they could feel the sense of numbness and burning.We consider that the nociception signals may be conducted by residual A-delta fibers,or that the A-beta fibers transmit pain signals in high-intensity nociception similar to the mechanism of allodynia.