| IntroductionCleft lip and/or palate is one of the most common congenital developmental defects among human-beings,and its incidence increases very fast recently.Cleft lip and/or palate can not only cause appearance deformity of children with disease,but also can affect their normal function of sucking,swallowing,pronouncing and face growth et al.Thus,it is very important to diagnose the disease through prenatal diagnosis.At present,ultrasonic inspection is the main methods for prenatal diagnosis, the rate of which about fetal anomaly constantly increases with the development of ultrasonic medicine and the application of new ultrasonic technique.However,it has become a hard point that fetus cleft lip and/or palate,especially cleft palate,is very difficult to show because it is affected by environmental structure sonic shadowing, and hoping that a new ultrasonic technique will appear to improve prenatal diagnosis rate and to provide evidence for guiding pregnant woman's choice about pregnancy outcome.Orthodox 2D ultrasound has had some experience about diagnosis of cleft lip,but the incidence rate of cleft palate is still low.The techniques of three-dimensional surface rendering mode and three orthogonal planar mode have improved the detection rate of cleft palate,otherwise,the recent technique of three-dimensional multi-slice mode for inspecting fetus cleft lip and/or palate, especially cleft palate,has provided more reliable information.However,there are few related reports.Its application value is still among discussion,and there are no reports about these in our country.The pathogenesis of cleft lip and/or palate is still not clear,many results of empirical studies and epidemiologic surveys have made it clear that cleft lip and/or palate is caused by many factors the main of which is genetic factor.At the genetic respect,domestic and abroad scholars have studied the gene locus of cleft lip and/or palate,and have confirmed the relation between part of genes and occurrence of cleft lip and/or palate;however,the conclusion can still not be unified.Because there are differences of gene polymorphism between human races,furthermore,there are differences of incidence rate of congenital cleft lip and/or palate between different human races,so we need different population as samples to explore its pathogenesis and virulence gene from molecular level.For the first time,we study prenatal diagnosis of cleft lip and/or palate and its molecular genetics on the level of fetus.This study is composed of two parts;①After two-dimensional ultrasound examination,we used three orthogonal planar images and new three-dimensional technologies 3DXI and TUI to estimate the value of three-dimensional ultrasound in the diagnosis of fetal cleft lip and palate and explored method that can estimate the integrity of fetal palate effectively and accurately.②mutation detection of related genes and the expression of mRNA;To detect the mutation of MSX1,TGFB3,TGFA and IRF6 and expression of mRNA by PCR-DNA sequencing technology and RT-PCR.So that we can explore the relationship of gene mutation,mRNA expression and happening of human cleft lip and palate and provide clues for seeking pathogenic genes of human cleft lip and/or palate.Materials and methods1.prenatal diagnosis of fetal cleft lip and/or palate by ultrasound;Chose 68 cases of fetuses with cleft lip and/or palate and 20 cases of normal fetuses whose lips can be clearly detected in prenatal examination as study population.Used two-dimensional ultrasound,three orthogonal planar images and new three-dimensional technologies 3DXI and TUI sequentially to observe the face of fetuses and detected the size and depth of cleft lip and palate.And compared the revealing rate of normal and abnormal structures of lip and palate.2.dependable gene study;Applied PCR-DNA sequencing technology to amplify gene MSX1,TGFB3,TGFA and IRF6 and make sequencing and then screen the predisposing genes and detect gene mutation.3.gene expression study;Detected the expression levels of gene MSX1,TGFB3, TGFA and IRF6 in peripheral blood of 46 cases of fetuses with cleft lip and/or palate and 20 normal contrast fetuses.Results1.prenatal ultrasonic diagnosis of cleft lip and/or palate;For 20 cases of normal fetuses,the revealing rate of upper lip is 100%by all the three methods;the revealing rate of upper alveolar ridge is 90%(18/20)by two-dimensional ultrasound,100%(20/20) by both three orthogonal planar mode and multi-slice mode;and the revealing rate of hard palate is 5%(1/20)by two-dimensional ultrasound,55%(11/20)by three orthogonal planar mode and 80%(16/20)by multi-slice mode.In the 64 cases of abnormal fetuses, for 14 cases of isolated cleft lip,the diagnostic accuracy is 100%(14/14)by all the three methods.For 52 cases of cleft lip and complete cleft palate,52 cases(100%)were visualized the cleft lip by all the three methods;41cases(82%)were visualized the cleft alveolus by two-dimensional ultrasound and 52 cases(100%)by both three orthogonal planar mode and multi-slice mode;3 cases(5.7%)were visualized the cleft palate by two-dimensional ultrasound,31cases(59.6%)by three orthogonal planar mode and 39 cases(75%)by multi-slice mode.For 2 cases of cleft lip and incomplete cleft palate,all the three methods only revealed the cleft lip but missed the incomplete cleft palate.There was obvious difference between the multi-slice mode and three orthogonal planar mode in the revealing rate of normal palate(p＜0.05);There was obvious difference between the multi-slice mode and two-dimensional ultrasound in the revealing rate of normal palate (p＜0.05);There was obvious difference between three orthogonal planar mode and two-dimensional ultrasound in the revealing rate of normal palate(p＜0.05).There was obvious difference between the multi-slice mode and three orthogonal planar mode in the revealing rate of the cleft palate of cleft lip and complete cleft palate(p＜0.05);There was obvious difference between the multi-slice mode and two-dimensional ultrasound in the revealing rate of the cleft palate of cleft lip and complete cleft palate(p＜0.05);There was obvious difference between three orthogonal planar mode and two-dimensional ultrasound in the revealing rate of the cleft palate of cleft lip and complete cleft palate (p＜0.05).Multi-slice mode can display the fetal lip and palate in a sequential of parallel planes,and we can choose the plane that displays the biggest cleft to measure it.The cleft width of group isolated cleft lip positively correlates with gestational age(r=0.836, P＜0.0005).The width of cleft lip,cleft alveolus and cleft palate of group cleft lip and complete cleft palate all positively correlates with gestational age,with correlation coefficient 0.587,0.514 and 0.648 separately.Four cases of fetal specimen were put in a sink and examined by ultrasound.The results showed that the nostril,cleft of upper lip, upper alveolus all could be displayed clearly,and cleft of hard palate is echoless dark region and communicates with nostril.2.dependable gene study;Applied PCR-DNA sequencing technology to amplify candidate gene fragments of 46 cases of fetuses with cleft lip and/or palate and the results showed MSX1,TGFB3 may be the predisposing genes.Gene sequencing showed gene MSX1 of number 1 sample has one more C at site 1253 and cause frame shift mutation and sequencing of gene TGFB3 had no positive results.3.gene expression study;RT—PCR showed that;the expression of mRNA of gene MSX1 in cleft lip and/or palate patients(1.39±0.36)was obviously lower than normal contrast cases(1.69±0.21),(P=0.02);the expression of mRNA of gene TGFB3 in cleft lip and/or palate patients(0.72±0.28)was obviously lower than normal contrast cases(0.94±0.34),(P=0.045);the expression of mRNA of gene TGFA in cleft lip and/or palate patients(0.45±0.16)was lower than normal contrast cases(0.65±0.58),but has no significant sense(P=0.295);the expression of mRNA of gene IRF6 in cleft lip and/or palate patients(1.39±1.23)was lower than normal contrast cases(1.88±1.29),but has no significant sense(P=0.275).Conclusions1.Three-dimensional multi-slice mode and three orthogonal planar mode can obviously improve the accuracy of prenatal diagnose of cleft lip and palate,especially in diagnose of cleft palate in compare to the two-dimensional ultrasound.2.This study successfully summarized the pictorial characteristics of fetal normal palate and cleft palate by multi-slice mode.Multi-slice mode showed high clinical application value in estimating the integrity of fetal alveolus and palate.3.The width of cleft lip,cleft alveolus and cleft palate of group cleft lip and complete cleft palate all positively correlates with gestational age.4.This study showed mRNA expression of gene MSX1 and TGFB3 was obviously lower in cleft lip and/or palate group than in the normal group.
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