Genome-Wide Screening of Aberrant Methylation Loci for Nonsyndromic Cleft Lip / 中华医学杂志(英文版)

Background@#The pathogenicity of cleft lip (CL) is pretty complicated since it is influenced by the interaction of environment and genetic factors. The purpose of this study was to conduct a genome-wide screening of aberrant methylation loci in partial lesion tissues of patients with nonsyndromic CL (NSCL) and preliminarily validate candidate dysmethylated genes associated with NSCL.@*Methods@#Fifteen healthy and sixteen NSCL fetal lip tissue samples were collected. The Infinium HumanMethylation450 BeadChip was used to screen aberrant methylation loci in three NSCL and three healthy lip tissues. The differential methylation sites and functions of the annotated genes between NSCL and healthy lip tissues were analyzed using minfi package of R software, cluster analysis, Gene Ontology (GO) annotation, and metabolic pathway annotation. Gene expression was assessed in nine differentially methylated genes by real-time polymerase chain reaction (PCR). The transcriptions mRNA levels of three out of nine candidate genes were downregulated remarkably in NSCL lip tissues, and these three genes' abnormal methylation loci were validated by pyrosequencing in 16 NSCL cases and 15 healthy cases.@*Results@#In total, 4879 sites in the genes of NSCL odinopoeia fetuses showed aberrant methylation when compared with normal lip tissue genome. Among these, 3661 sites were hypermethylated and 1218 sites were hypomethylated as compared to methylation levels in healthy specimens. These aberrant methylation sites involved 2849 genes and were widely distributed among the chromosomes. Most differentially methylated sites were located in cytosine-phosphoric acid-guanine islands. Based on GO analysis, aberrantly methylated genes were involved in 11 cellular components, 13 molecular functions, and a variety of biological processes. Notably, the transcription of DAB1, REELIN, and FYN was significantly downregulated in lesion tissues of NSCL fetus (P < 0.05). Pyrosequencing results validated that there were two loci in DAB1 with high methylation status in patient tissues (P < 0.05).@*Conclusions@#We detected numerous aberrantly methylated loci in lesion tissues of NSCL fetus. Aberrant gene expression in the REELIN signaling pathway might be related with NSCL. Decreased transcription of DAB1, a member of REELIN signal pathway, resulted from its abnormal high methylation, which might be one of the factors underlying the occurrence of NSCL.

Comparison of different methods of rat amniotic fluid sample preparation for 2-d electrophoresis / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To optimize the method for preparing samples for amniotic fluid proteomics study.</p><p><b>METHODS</b>Pregnant rats were sacrificed with an overdose of Chloral hydrate at E17. The fetuses and amniotic fluid were harvested. The samples were processed by three different methods including trichloroacetic acid (TCA)-acetone precipitation (protocol 1), TCA-acetone precipitation combined with an Albumin and IgG Removal Kit (protocol 2), and a Centrifugal Filter concentrating combined with an Albumin and IgG Removal Kit (protocol 3). The samples were run through a two-dimensional electrophoresis gel, stained and analyzed with a Image Master 6.0 software. Protein spots were identified with a LCQ Deca XP mass spectrometer.</p><p><b>RESULTS</b>The total numbers of protein spots for samples processed by protocol 1, 2 and 3 were 253 ± 28, 749 ± 32 and 782 ± 27, respectively. And there was a significant difference between protocol 1 has and other two methods. Those with MW > 50 kDa were 57± 14, 45 ± 13 and 41 ± 14, respectively. Protocol 2 differed significantly from protocol 3. Protein number of samples with MW < 50 kDa was 196± 29, 702± 35 and 735 ± 29, respectively. Again, protocol 1 has differed significantly from other two methods.</p><p><b>CONCLUSION</b>By removing albumin and IgG from the serum, low abundance proteins can be enriched with little loss of high abundance proteins. Centrifugal Filter concentrating combined with Albumin and IgG Removal Kit can be effectively applied for amniotic fluid proteomics study.</p>

Scintigraphic assessment of the anorectal function in children with idiopathic constipation / 中华儿科杂志

<p><b>OBJECTIVE</b>The ability of the rectum to evacuate is valuable information in patients with constipation, or those who find defecation difficult. However, few published studies are available to evaluate this ability in most common examinations of anorectal function. A new, low-radiation, real-time scintigraphic technique has been used by several investigators to outline the 'neo-rectum' following ileoanal pouch anastomosis and the efficiency of rectal evacuation in adult patients with constipation. There was no similar report about the abilities of rectal evacuation in children with constipation. The aim of this study was to investigate the value and reliability of isotope defecography as an objective method for evaluation of anorectal function in patients with chronic idiopathic constipation.</p><p><b>METHODS</b>Twenty five patients (10 were male and 15 were female, range of age was 5 - 15 years, mean age 8.99 years) suffering from chronic idiopathic constipation and 11 normal children were assessed by isotope defecography. The radiopharmaceuticals used were rehydrated potato labelled with 200 MBq (99m)TcO(4)(-). The volume of the potato paste used in each subject was determined by prior balloon proctometrogram studies, which measured the maximum rectal capacity. The patient was seated upright on a commode, and the computer was then set for the dynamic acquisition of 3-second images over 5 min per session. Using a region of interest program on computer, the dynamic radioactivity in anorectum during defecation showed as emptying curves by computer. The half defecation time, the rectal emptying rate and the residual rate could be calculated from the rectal emptying curves. The anorectal angles were measured from the images of anorectum during resting, squeezing and evacuation. All patients underwent X-ray defecography and colonic transit time measurement simultaneously.</p><p><b>RESULTS</b>The rectal emptying curves showed a prompt evacuation in normal children, but it disappeared in most of patients with idiopathic constipation. The abilities of evacuation were damaged severely in patients. In normal group the half defecation time, the rectal emptying rate and the residual rate were 1.97 +/- 0.86 minutes, 78.30% +/- 12.03% and 20.50% +/- 7.67%, respectively, whereas they were 15.16 +/- 3.67 minutes, 44.84% +/- 14.00% and 53.52% +/- 15.02%, respectively, in patients group. There were significant differences between two groups (P < 0.05). According to the results of colonic transit time there were 6 patients with slow transit constipation, 17 patients with outlet dysfunction and 2 patients with normal colonic transit time. The abilities of evacuation were more seriously impaired in patients with outlet dysfunction compared with those patients with slow transit constipation. For the isotope defecography the mean anorectal angles at resting in patient group and normal group were 109 +/- 12 degrees and 101 +/- 17 degrees. There were no significant differences in anorectal angles between patient group and normal group or by both methods of isotope defecography and X-ray defecography. The correlation coefficients for anorectal angles at resting, squeezing and evacuation were 0.87, 0.82 and 0.73, respectively.</p><p><b>CONCLUSION</b>The isotope defecography can be used to evaluate the anorectal function and the ability of defecation dynamically, also it was simple and safe because of the low dose radiation involved.</p>

Determination of total and segmental colonic transit time in constipated children / 中华儿科杂志

<p><b>OBJECTIVE</b>To determine the total and segmental colonic transit time of normal Chinese children and to explore its value in constipation in children.</p><p><b>METHODS</b>The subjects involved in this study were divided into 2 groups. One group was control, which had 33 healthy children (21 males and 12 females) aged 2 - 13 years (mean 5 years). The other was constipation group, which had 25 patients (15 males and 10 females) aged 3 - 14 years (mean 7 years) with constipation according to Benninga's criteria. Written informed consent was obtained from the parents of each subject. In this study the simplified method of radio opaque markers was used to determine the total gastrointestinal transit time and segmental colonic transit time of the normal and constipated children, and in part of these patients X-ray defecography was also used.</p><p><b>RESULTS</b>The total gastrointestinal transit time (TGITT), right colonic transit time (RCTT), left colonic transit time (LCTT) and rectosigmoid colonic transit time (RSTT) of the normal children were 28.7 +/- 7.7 h, 7.5 +/- 3.2 h, 6.5 +/- 3.8 h and 13.4 +/- 5.6 h, respectively. In the constipated children, the TGITT, LCTT and RSTT were significantly longer than those in controls (92.2 +/- 55.5 h vs 28.7 +/- 7.7 h, P < 0.001; 16.9 +/- 12.6 h vs 6.5 +/- 3.8 h, P < 0.01; 61.5 +/- 29.0 h vs 13.4 +/- 5.6 h, P < 0.001), while the RCTT had no significant difference. X-ray defecography demonstrated one rectocele, one perineal descent syndrome and one puborectal muscle syndrome, respectively.</p><p><b>CONCLUSION</b>The TGITT, RCTT, LCTT and RSTT of the normal children were 28.7 +/- 7.7 h, 7.5 +/- 3.2 h, 6.5 +/- 3.8 h and 13.4 +/- 5.6 h, respectively. With the segmental colonic transit time, constipation can be divided into four types: slow-transit constipation, outlet obstruction, mixed type and normal transit constipation. X-ray defecography can demonstrate the anatomical or dynamic abnormalities within the anorectal area, with which constipation can be further divided into different subtypes, and combined use of the gastrointestinal transit time and X-ray defecography is of clinical importance in exploration of etiology of constipation.</p>