The 22q11.2 microdeletion syndrome is also known as DiGeorge syndrome (DGS), velocardiofacial syndrome (VCFS), and conotruncal anomaly face syndrome (CAFS). It has a wide phenotypic spectrum that involves almost all organs, although cardiac deformation is the most common phenotype.1 In addition, some patients show increased rates of cognitive dysfunction and psychiatric disorders.2 Almost all cases result from a common deletion of the chromosome 22q11.2 locus. Recently, more attention has been directed to the early diagnosis of 22q11.2 microdeletion syndrome. Previous researches have demonstrated that imaging data, such as the value of nuchal translucency (NT) in the first and second trimester of pregnancy and the typical heart malformation, can facilitate the prenatal diagnosis of this syndrome.3,4 It should be emphasized that accurate diagnosis is difficult in some cases due to the absence of classic features or family history. Therefore, genetic diagnosis is essential. Together with imaging data, detection of 22q11.2 microdeletion using amniotic fluid cells or placental chorionic villus cells from high-risk fetuses can improve the specificity of prenatal diagnosis.5,6 Currently, researches on 22q11.2 microdeletion syndrome in China are focused primarily on diagnosing the condition by using peripheral blood with only few researches on the prenatal diagnosis of this syndrome. Here we used amniocytes and the fluorescent in situ hybridization (FISH) technique to detect 22q11.2 microdeletion in deformed fetuses and explored the feasibility of using this method in the prenatal diagnosis of the 22q11.2 microdeletion syndrome.
This study was approved by the Ethics Committee of Beijing Anzhen Hospital of the Capital Medical University. Between December 2007 and December 2008, a total of 37 women in our and other hospitals were diagnosed as having fetuses with malformations and voluntarily requested pregnancy termination. Of these cases, 23 showed evidence of a cardiac malformation, including the tetralogy of Fallot, persistent truncus arteriosus, common truncus arteriosus, pulmonary atresia or stenosis, conotruncal defects (CTD), ventricular septal defect, and single ventricle heart, while 14 had non-cardiac malformations, which included conjoined twins, cleft lip/palate, polydactyly, anencephalus, hydrocephalus, spina bifida, and renal agenesis. The average age of the pregnant women was (30.00±2.71) years. The average gestational age at the time of induced labor was (25.27±4.13) weeks. All cases were labor-induced by amniocentesis with injection of rivanol. The stillborn fetuses were sent for autopsy.
During the same period, samples from 20 women who underwent cesarean delivery were used as control. If the sample was contaminated by the blood of matrix, it was abandoned.
The reagents used in this study included Hank's balanced salt solution (BSS), collagenase B (0.001 g/ml in Hank's BSS), KCl hypotonic solution (0.075 mol/L KCl), fixative solution (methanol: acetic acid is 3:1), RNase A (100 µg/ml), pepsin (20 mg/ml), 2×saline sodium citrate (SSC) (pH 7.0), 70% ethanol, 85% ethanol, 0.1 mol/L HCl, and 0.01 mol/L HCl.
The TUPLE1 probe (Beijing Jinpujia BioTech, China) hybridized to chromosome 22q11.21, and the fluorescent signal was red (tetramethylrhodamine). The ARSA probe (Beijing Jinpujia BioTech, China) hybridized to 22q13.31, and the fluorescent signal was green (cell green). The probes served as controls for each other.
Amniotic fluid extraction
When women with malformed fetuses underwent induced labor by intraamniotic injection of rivanol, 20–50 ml of amniotic fluid was extracted. The volume of amniotic fluid was determined according to the gestational age (weeks). For each one-week increase in gestational age, the volume of extracted amniotic fluid also increased by 1 ml.
After extraction, 2–5 ml of amniotic fluid was centrifuged for 10 minutes, resuspended in 5 ml of hypotonic solution, and incubated at 37°C for 30 minutes. Then, 2 ml of fixative solution was added and incubated for 2 minutes. After centrifugation for 10 minutes, the cells were fixed twice with 5 ml of fixative solution. The cells were then spread on glass slides and allowed to stand overnight at room temperature.
The glass slides were treated sequentially with RNase A, washed with 2×SSC (pH 7.0), incubated in 0.1 mol/L HCl for 7–10 minutes, washed with 2×SSC (pH 7.0) twice, incubated in pepsin solution at 37°C for 7–10 minutes, dehydrated by ethanol, and then fixed, dried and heated to 56°C before hybridization. The probes were mixed (7 µl of hybridization buffer, 1 µl of water, and 2 µl of probes) and protected from light. The glass slides were first denatured in 70% formamide/2×SSC for 5 minutes at (73±1)°C and then dehydrated sequentially at –20°C in 70%, 85%, and 100% ethanol. These slides were then dried and mixed with the probe solution after heating to 45°C–50°C. The hybridization area was covered with a coverslip, sealed with sealing glue, and hybridized at 42°C overnight. On the second day, the cover slips were removed and the slides were incubated in 50% formamide/2×SSC at 46°C for 10 minutes three times. Then, the slides were placed in 2×SSC for 10 minutes, and in 2×SSC/0.1% NP-40 solution for 5 minutes. After being dried, the slides were stained using 15 µl of DAPI for 15–20 minutes with coverslips and protected from light.
The slides were observed under a fluorescence microscope (Olympus BX51, Japan), and the filters used were MBE44720, MBE45600, and MBE41300. The images were analyzed using videotest FISH software (version 2.1, Beijing Jinpujia BioTech). As determined via microscopy, the positive cell hybridization rates were more than 80% in every counted sample, and the cell membranes of the cells were all intact. More than 50 cells were counted in every sample. There were two green dots and two red dots in every normal cell nucleus (Figure 1; no 22q11.2 deletion). If one green dot and one red dot or two green dots and one red dot were seen in one cell, this indicated that there was a 22q11.2 microdeletion in that cell (Figures 2 and 3). The samples having more than 60% of cells showing two green dots and one red dot or one green dot and one red dot were considered to contain a 22q11.2 microdeletion.
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Figure 1. A representative normal cell shows two red and two green dots (FISH).
Figure 2. Representative cells show microdeletion of the TUPLE1 gene (two green and one red dots).
Figure 3. A representative cell shows microdeletion of TUPLE1 gene (one green and one red dot).
Statistical data were expressed as mean ± standard deviation (SD). A Fisher exact test was used as the statistical method to compare the rates of the different groups using SPSS 13.0 (USA). A P value less than 0.05 was considered statistically significant.
The autopsy results of the 37 fetal malformation cases were consistent with those of the imaging data obtained before the induced labor.
Detection of 22q11.2 microdeletion
In the 23 cardiac deformation cases, 10 showed deletion of the TUPLE1 gene, yielding a deletion rate of 43.5%. In the 14 non-cardiac deformation cases, deletion of the TUPLE1 gene was found in eight cases, resulting in a deletion rate of 57.1% (Tables 1 and 2). There were no significant differences between the two groups (P=0.508). In the control group, none showed deletion of the TUPLE1 gene.
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Table 1. Detection results for 22q11.2 microdeletion in different types of fetal cardiac malformation
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Table 2. Detection results for 22q11.2 microdeletion in different types of fetal non-cardiac malformation
The gestational age in the 37 cases varied from 18 to 34 weeks (Table 3). The detection rates of 22q11.2 microdeletion at different gestational ages showed no significant differences (P=0.410).
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Table 3. Detection of 22q11.2 microdeletion in fetal malformations of different gestation ages
The prevalence rate of 22q11.2 microdeletion syndrome is estimated to be 1 in 4000 births.7 The critical genes in the deleted region include Tbx1,8 TUPLE1,9 and Ufd11. The TUPLE1 gene resides in the DiGeorge key region, the core region of the chromosome 22 microdeletion. Haploid deficiency of the TUPLE1 gene product leads to accumulation of proteins and affects the development of the heart and cranial neural crest cells, as well as the apoptosis of immature thymocytes. Therefore, the loss of the TUPLE1 gene causes interrupted aortic arch, cleft palate and other craniofacial defects, and thymic dysplasia. The features of chromosome 22q11.2 microdeletion syndrome vary widely. Congenital heart diseases are the most common symptoms, and there is a wide spectrum of phenotypes, including interrupted aortic arch, tetralogy of Fallot,10 persistent truncus arteriosus, ventricular septal defect, patent ductus arteriosus, right-sided aortic arch, double outlet right ventricle, and absent pulmonary valve or pulmonary atresia. Ventricular outlet and aorta arch malformations in 22q11.2 microdeletion syndrome are relatively common, with incidences of 15% to 20%. It has been reported by foreign researchers that the 22q11.2 microdeletion rate in congenital heart diseases is between 17% and 29%.11,12 Yang et al13 also detected the TUPLE1 gene in heart disease patients using umbilical cord blood and found the loss rate to be 43.75%. Here we found that the 22q11.2 microdeletion rate in congenital heart malformation cases was 43.5%, and that congenital heart malformation was associated with 22q11.2 microdeletion. Our result is consistent with the data of Yang et al,13 but is higher than those of the foreign researchers. One reason for this may be that our hospital accepted many cases that were transferred from other hospitals, and more severe cases were included. In addition, the population we focused on were fetuses with severe heart malformations, which may have led to a relatively high prevalence rate.
In our data, 22q11.2 microdeletion was found primarily in cases with complex heart malformation, especially aorta malformations. Of the cases with heart disease, 70% (7 out of 10) showed aorta malformation. On the contrary, only one case with a single atrium and a single ventricle malformation was found to have a 22q11.2 microdeletion. This evidence confirms that typical cases of 22q11.2 microdeletion involve malformation of the stem arteries.
22q11.2 microdeletion is one of the primary conditions leading to intrauterine growth retardation and congenital heart malformation.14 Therefore, high-risk fetuses showing growth retardation and malformation should receive screening for 22q11.2 microdeletion. In our study, fetuses with heart malformations have a relatively high prevalence rate of 22q11.2 microdeletion, and thus should be considered a high-risk population. In addition, the following cases should receive screening for 22q11.2 microdeletion due to their increased risk of having the syndrome: the offspring of 22q11.2 microdeletion patients, those having a prevalence rate of 50% and fetuses with congenital heart malformation. In doing so, clinicians can clarify the causes of the fetal malformation as soon as possible and the parents of the fetus can also prepare themselves for the situation emotionally. Additionally, they can choose appropriate hospitals for delivery and be prepared for perinatal handling. When severe fetal malformation is identified, the results of the 22q11.2 microdeletion test can also provide reliable genetic information in order to counsel parents and to provide a basis for them to make decisions about their fetus early on. Meanwhile, some children with 22q11.2 microdeletion syndrome show an atypical malformation phenotype and then develop cognitive dysfunction and psychological problems.2 Therefore, accurate diagnosis is helpful for early intervention in such cases.
Facial malformation is another common phenotype of 22q11.2 microdeletion syndrome. Currently, there is relatively few researches on the relationship between 22q11.2 microdeletion and facial deformities. Qin et al15 found that considering fetal facial deformities as a feature in the clinical diagnosis of the syndrome increased the diagnostic rate of 22q11.2 microdeletion syndrome. Hammond et al16,17 showed that diagnosis sensitivity was 83%, specificity was 92%, and accuracy was more than 95% when they included facial features in the diagnosis of velocardiofacial syndrome, one type of 22q11.2 microdeletion syndrome. As a result, facial deformation is one of the important clinical features. We found that in the 8 out of the 14 cases with non-cardiac malformation, 22q11.2 microdeletion was present. Of the eight cases, four showed cleft lip/palate or cleft lip. Therefore, the prevalence rate of 22q11.2 microdeletion in fetuses with facial deformities is relatively high. Whether all fetuses with evidence of facial deformities should receive 22q11.2 microdeletion screening is a topic that should be further explored.
Thus far, the FISH test of the key gene TUPLE1 is still considered the gold standard for the diagnosis of 22q11.2 microdeletion syndrome. In China, many researches use peripheral blood samples from sick children. There have been relatively few studies with regard to prenatal diagnosis of 22q11.2 microdeletion syndrome. Yang et al13 reported that they successfully detected 22q11.2 microdeletion using umbilical cord blood samples. However, there are drawbacks to this method. Umbilical cord blood is difficult to collect, and the collection procedure requires a technician experienced in the collection method. The detection protocol requires cell culture and takes a longer time. In our study, we used amniotic fluid cells for FISH detection of 22q11.2 microdeletion. Compared with the method of Yang et al, amniotic puncture is easy to perform, and the easily obtained amniotic fluid cells can be used directly in detection. With our method, there is no need for cell culture. The results can be seen in 24 hours. This method not only improves the working efficiency of the operator, but also decreases the psychological burdens of the pregnant women who are eagerly awaiting the results. Abundant amniotic fluid cells can be collected once the fetus reaches a gestational age of 13–14 weeks. In the cases we studied, the earliest gestational age was 18 weeks. The prevalence rate of 22q11.2 microdeletion showed no significant differences among different gestational ages. Therefore, amniotic fluid cells can be used to diagnose 22q11.2 microdeletion syndrome in the middle of pregnancy. Many heart malformations, however, cannot be detected by ultrasonic examination until at least 20 weeks.18 Therefore, using amniotic fluid cells to diagnose 22q11.2 microdeletion syndrome prenatally is especially useful for pregnant women with a clear family history of fetal malformations or with a personal history of deformed fetuses. Taken together, we think that the use amniotic fluid cells and subsequent analysis with FISH has clinical value in the detection of 22q11.2 microdeletion syndrome.
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