Application Of Creatine Kinase Isoenzyme MM In The Screening Of Neonatal Duchenne Muscular Dystrophy

ObjectsDuchenne muscular dystrophy(DMD)is an X-linked hereditary fatal myopathy,with insidious clinical onset and easy to miss diagnosis.The implementation of newborn screening will help early identification and clinical intervention of DMD patients,and ultimately improve the prognosis of patients.Creatine kinase isoenzyme MM(CK-MM)is an important biochemical marker in the early onset of DMD patients.At present,there are few studies on the use of CK-MM for DMD newborn screening,and there is a lack of such studies in Shaanxi.In this study,by detecting the concentration of CK-MM in the residual dried blood spot samples of newborns,preliminary analysis of the CK-MM concentration distribution of random newborns in our center,and analysis of the relevant factors affecting the concentration distribution of CK-MM,to provide a reliable basis for early clinical screening and identification of DMD.MethodsCollected samples of residual dried blood spots from 4572 newborns born in Northwest Women’s and Children’s Hospital from October 1,2019 to May 31,2020,and use time-resolved immunofluorescence to determine the concentration of CK-MM in dried blood spot samples.Calculate the percentile setting cutoff value of 99.9 based on the CK-MM detection value of 2000 samples,and the original blood film is retested on the positive sample,and the children who are still positive in the retest are recalled for clinical evaluation and myocardial enzyme spectrum,genetic testing and regular follow-up.Genetic testing includes the use of multiple ligation-dependent probe amplification(MLPA)technology to detect large deletions/duplications of DMD genes and the use of next generation sequencing(NGS)technology to detect 64 types of hereditary muscles disease-related gene mutations.Spearman correlation analysis was used to analyze the correlation between CK-MM concentration and birth weight,gestational age,blood collection time,and non-parametric test methods were used to analyze the influence of different birth weight,gestational age,blood collection time,delivery method,and gender on CK-MM concentration distribution.Results1.Through the detection of CK-MM concentration in dried blood spot samples of 4572newborns,the median value of CK-MM concentration was 61.60(38.70～103.00)ng/m L.Calculating the percentile setting cutoff value of 99.9 based on the CK-MM detection value of 2000 samples,the cutoff value is 572ng/m L.2.Of the 4572 dried blood spots,5 cases were higher than the cut-off value.After retesting the original blood film,3 cases were positive for screening,1 case of DMD was confirmed by MLPA test,and the other 2 cases were negative for MLPA test.No related pathogenic variants were detected in NGS.3.The results of correlation analysis showed that there was no correlation between CK-MM concentration and birth weight(r_s=0.009,P=0.526);the concentration was negatively correlated with gestational age and blood collection time(r_s=-0.039,P=0.008;r_s=-0.674,P=0.000).The non-parametric Kruskal-Wallis H rank test was used to statistically analyze the CK-MM concentration distribution of the three groups of different birth weight,gestational age,blood collection time,and delivery method.The results show that:there was no statistically significant difference in the distribution of CK-MM concentration values at birth weight and gestational age among the three groups((X~2=5.897,P=0.052;X~2=1.505,P=0.471);the differences in the CK-MM concentration value distribution of the three groups of blood collection time and delivery methods were statistically significant(X~2=1332.744,P=0.000;X~2=850.627,P=0.000).The non-parametric Mann-whitney U test was used to compare different blood collection times,delivery methods,and genders.The results showed that the median CK-MM concentration of newborns whose blood collection time was 3 to 10 days was higher than that of blood collection time for 10 to 20 days and more than 20 days,and the difference was statistically significant(Z=-35.844,P=0.000;Z=-10.203,P=0.000);However,there was no statistically significant difference in CK-MM concentration between 10-20 days and>20 days in blood collection time(Z=-0.241,P=0.810).The concentration of CK-MM detected by cesarean section was higher than normal delivery and forceps-assisted delivery,and the difference was statistically significant(Z=-29.081,P=0.000;Z=-6.214,P=0.000);there was no statistically significant difference in the concentration of CK-MM between normal delivery and forceps-assisted delivery(Z=-1.228,P=0.219).There was a statistically significant difference in the concentration of CK-MM in the screening of newborns of the two sexes(Z=-8.343,P=0.000).Conlusions1.In 4572 random newborns,3 were positive for screening,and 1 child with DMD was diagnosed,the positive rate of screening was 0.07%.The sensitivity of CK-MM in screening neonatal DMD was 100%,the specificity was 99.96%,and the positive predictive value was 33.33%.2.The concentration of CK-MM is affected by gender,delivery method and time of blood collection.The median concentration of CK-MM in male newborns is higher than that of females.The CK-MM concentration in newborns delivered by cesarean section is relatively high.The blood collection time is within 3-10 days,the CK-MM concentration decreases with the prolongation of the blood collection time,and the CK-MM concentration tends to be stable after the blood collection time>10 days.3.The concentration of CK-MM in the confirmed child is>5000ng/m L,and the MLPA result shows a deletion mutation in exon 51 of the DMD gene,indicating that the application of CK-MM can screen neonatal DMD.Combining genetic testing with positive CK-MM screening can reduce the false positive rate.