Genetic Research Of Ion Channel Gene Mutations And Clinical Study On Primary Hypokalemic Periodic Paralysis

Primary hypokalemic periodic paralysis (HOKPP) is an autosomal dominant genetic muscle disorder characterized by periodic attacks of muscle weakness associated with a decrease in serum potassium. Muscle weakness always involves in limb muscle, and sometimes in respiratory muscle. Severe patient can die of paralysis of respiratory muscle or arhythmia resulted by hypokalemic. According to family history, HOKPP can be divided into two types: familial hypokalemic periodic paralysis (FHOKPP) and sporadical hypokalemic periodic paralysis (SHOKPP).Recent genetic, molecular biologic and electrophysiological combination researches have shown that HOKPP is due to mutations in the skeletal muscle ion channel CACNA1S, SCN4A or KCNE3 gene(which coding involtage-gated calcium al subunit, sodium a subunit or potassium channel 6 subunit respectively). Now people had found that mutations R528H/G and R1239H/G in CACNA1S , R669H, R672H/G/C/S and P1158S in SCN4A , R83H in KCNE3 are correlated with HOKPP. HOKPP linked to CACNA1S mutations was defined as type I , and linked to SCN4A mutations was defined as type II. The literature reported about 69% was type I, about 8.6% was type II, and the rest 22.4% had no identified mutation. HOKPP indicated not only gene heterogeneity , but also phenotype heterogeneity.This work was focused on genetic research of disease-related mutations and clinical study on HOKPP in China. We acquired DNA and clinical data of 18 FHOKPP patients from 12 unrelated families and 71 SHOKPP patients. Using PCR- sequencing, we analyzed the entire coding regions of the CACNA1S, SCN4A and KCNE3 of 12 FHOKPP probands, and CACNA1S exon 11,30 and SCN4A exon 12 of 71 SHOKPP probands.We reviewed clinical data of HOKPP, and identified difference in clinic and genetic characters between patients with SCN4A and CACNA1S mutation , with and without mutation , of FHOKPP and SHOKPP. This research aimed to identify the location, incidence rate of therelevant mutations in Chinese HOKPP, and specify the relativity of genotype and phenotype.lt included three parts.PART ONE: Collecting the genetic resources of HOKPP and construction a screening process for ion channel gene mutations of HOKPPThe objective of this study was to establish a bank for blood DNA samples accompanied with clinical and pedigree data. We collected 18 FHOKPP patients from 12 unrelated families and 71 SHOKPP patients. And then we designed primers for all exons of SCN4A, CACNA1S and KCNE3 gene. At last, the PCR products were sequenced in order to find relevant mutations of HOKPP.PART TWO: Screening the ion channel gene mutations in Chinese patients with HOKPP1.Screening CACNA1S, SCN4A, KCNE3 gene for mutations in Chinese FHOKPPUsing PCR-sequencing, we screened the entire coding region of the CACNA1S, SCN4A and KCNE3 of 12 FHOKPP probands. Two known mutations in three FHOKPP probands were identified,proband of family 6 with R1239H mutation in CACNA1S gene and proband of family 8 and 10 with R672H mutation in SCN4A gene. Moreover, a new mutation of V781I in SCN4A was identified in proband of family 12.CACNA1S R1239H mutation was screened in members of family 6. We found two patients in the family with CACNA1S R1239H mutation, while the other normal relatives of the family without it. Clinic and genetics characters of CACNA1S R1239H mutation were summarized as follows: onset at childhood, aggravation at adolescence and relieve at adult without fixed weakness, effective to treatment with acetazolamide, complete penetrance in male and female.SCN4A R672H mutation was screened in members of family 8 and 10. We found the mutation existed not only in all the patients but also in five normal relatives, including one male(17 years old )and four female(the youngest one was 36 years old). The clinic and genetics characters of patients with SCN4A R672H mutation showed as follows: onset at adolescence and relieve at adult, inefficient to treatment with acetazolamide, incomplete penetrance in male(83%) andnon-penetrance in female.2. Identifying the correlation between SCN4A V781I mutation and HOKPPTo identify correlativity between SCN4A V781I mutation and HOKPP, SCN4A R672H mutation was screened on members of family 12 with PCR sequencing and we found the mutation existed not only in all the patients but also in four normal relatives, including one male and three females. We found an incision enzyme HpyCH4III, which could identify normal and mutation sequence.Using it, we screened the SCN4A V781I mutation in 71 SHOKPP patients and 100 health adults. The entire positive members must be confirmed with PCR-sequencing. The Results showed that 7/71 SHOKPP patients and 7/100 health adults indicated the SCN4A V781I mutation. It could be concluded that SCN4A V781I mutation was an innocence polymorphism. There was little correlativity between SCN4A V781I and HOKPP.3. Screening exon 11,30 of CACNA1S and exon 12 of SCN4A for mutations in Chinese SHOKPPWe didn't find new mutation besides the known mutations when we screened mutation of all FHOKPP probands, Hence, we only screened exon 11,30 of CACNA1S and exon 12 of SCN4A for hot spots of mutation in 71 SHOKPP patients. And one known SCN4A R672C mutation was found in a SHOKPP patient.The other patients were found no known mutation.PART THREE: Clinical characteristic researches on HOKPP 1. The differential diagnosis of HOKPPHOKPP is divided into primary and secondary types. The latter is subdivide into thyrotoxic periodic paralysis (TPP), deficient in potassium intake or excess in potassium excretion(loss of potassium from renal is the most common). We analyzed clinical data of 120 HOKPP patients, including 89 primary and 31 secondary patients.We attempted to identify the useful clues to the diagnosis of HOKPP and set up a fast diagnoses process in this study. We thought that history, serum potassium, ECG, blood gas analysis, urine routine and biochemistry testand thyroid hormone were significant for the fast diagnosis of HOKPP. 2. clinical characteristic researches on primary HOKPPWe analyzed clinical data of primary HOKPP patients and the results showed as follows: ?Male patient was majority, as well as sporadical patient;?The onset usually occurred in the second to the fourth decade of life;(3)We found differences in terms of positive rate, age at onset, response to treatment with acetazolamide, sex penetrance between the SCN4A mutation and CACNA1S mutation. ?Age at onset was more early and serum potassium levels during episode was lower in HOKPP with identified mutation than those without mutation;(5)There were differences in terms of identified mutations rate, age at onset, serum potassium levels during episode, and in diet as an inducement between FHOKPP and SHOKPP patients.