Study On Detection Of Genes Of Growth Hormone Receptor(GHR) And Its Signaling Pathways In The Children With Short Stature By MLPA

BackgroundShort stature is one of Common symptoms in pediatric endocrine outpatient service. Short stature means that in similar environment the child (or adult) is lower2standard deviations (-2sd) above or below the third percentile than the average height of the normal groups which have the same race, age, and sex. Now the main causes of short stature is divided into two categories:pituitary disease and non-pituitary disease. The non-pituitary disease can be divided into idiopathic short stature, familial short stature, intrauterine growth retardation, pubertal retardation, malnutrition and trace elements disease, non-pituitary endocrine diseases, inherited metabolic diseases and systemic diseases. In the etiology of short stature, the common causes are growth hormone deficiency and idiopathic short stature.Growth hormone (GH) is a single-chain hydrophilic globulin with191amino acids secreted from eosinophils of the anterior pituitary. It has the function of promoting growth and regulating bone metabolism and material metabolism. It is the indispensable ingredient of human growth. Growth hormone deficiency (GHD) refers to the anterior pituitary growth hormone deficiency or growth disorder caused by inadequate secretion. It can be divided to simple growth hormone deficiency and multiple pituitary hormone deficiency.Idiopathic short (idiopathic short stature, ISS) is a disease characterized by short stature which also have no cognitive reasons. ISS is probably a polygenic disease. It is not lack of growth hormone (GH), and the pathological changes include GH insensitivity, normal variant short stature, growth hormone neurosecretory dysfunction, idiopathic growth disorder non GHD, and so on.Growth hormone work mainly through the following ways:play a direct role in conjunction with the GHR and start STAT (STAT1and STAT3, STAT5A and STAT5B) or MAPK or PI3K-AKT signal transduction pathway by combining with GHR. Thus, it can simulate the insulin-like growth factor (IGF) secretion and promote the growth and metabolism regulation.For the diminutive genetic studies most researchers choose direct sequencing method. As genes involved in the etiology are as high as dozens or even hundreds, Choosing the direct sequencing method not only are economically costly but also material resources are costly. For the homozygous deletion of large fragment of the gene, it may not shows abnormal results.MLPA technology is a kind of built on DNA probe hybridization and PCR technology for nucleic acid target sequence under test for qualitative and semi-quantitative analysis of the high throughput technologies. It was invented in2002by the Dutch Schouten, etc. Only need a small amount of DNA, mainly through denaturation, hybridization, connection and augmentation four processes, MLPA technology can detect more than40nucleotide sequence copy number variations in the same reaction tube detection, and carry on quantitative analysis of the test. According to the changes of MLPA’s signal value, it can determine whether a target sequence has a bit mutation or copy number abnormalities.Real-time quantitative PCR (Realtime fluorescent quantitative PCR) is a highly sensitive nucleic acid quantification technology developed on the basis of the PCR technique. The technology monitor of the entire PCR process in real-time through the accumulation of the fluorescent signal.PCR amplification and end product test are all in a closed condition. It has real-time monitoring, pollution-free, rapid, sensitive, accurate, specific characteristics, and vastly overcomes the deficiencies of the original PCR technology. ObjectiveUsing the analysis of the gene kits (SALSA KIT P262MLPA) provided by the Dutch MRC-Holland company, this research detects11GH receptor gene fragments and35GH receptor signaling pathway gene fragments which are in children with idiopathic short (ISS), growth hormone deficiencies and normal children. It can be directly showed the situation of the lack of related genes, repeat and point mutations. Further gene sequencing and pedigree investigation were performed on the patients whose MLPA result was abnormal. To evaluate the clinical significance of mutations, the serum concentration of insulin-like growth factor (IGF)-1were detected using ELISA method (kit provided by the DSL company).MethodResearch object:①96children with short stature who went to the Shenzhen maternal and child health care hospital in March2012-March2013were chosen. These children were aged from4to13years old,52males,44females, and their height was lower than the average height of the same gender and age-2SD. According to the measured height, weight, bone age and serum IGF-1level, growth hormone provocation tests and so on,67cases were diagnosed as growth hormone deficiencies (GHD), and29cases idiopathic short stature(ISS).23children aged4to13years old whose physical development were normal were chosen as normal control group, of which,13males and10females. Compared with control group, sex of the experimental groups had no significant difference.②Pedigree investigation was conducted in2cases of compatriot sister and1female patient who had ISS and abnormal STAT5B gene results by MLPA, and the family members a total of12people and a total of6people were collected.Measurement index:All subjects’s age, gender, Birth Height were recorded, and height and weight at8o’clock in the morning were measured. Growth hormone stimulation test, thyroid function, liver and kidney function, trace elements, bone age, head MRI examination and chromosome were done.Specimen collection and laboratory testing:①MLPA genetic testing:3ml peripheral blood of all subjects were collected, and disodium citrate anticoagulate was used. Genomic DNA was extracted by conventional phenol chloroform method. DNA denaturation, probe hybridization, ligation, amplification, and product analysis were done by MRC-Holland’s P262kit.②Fluorescence quantitative PCR detection:3ml peripheral blood whose Mlpa results were abnormal and3ml peripheral blood of the healthy control group of normal children were taked, and genomic DNA was extracted by conventional phenol-chloroform method. Then fluorescence quantitative PCR were done. Fluorescence quantitative PCR reagents were ABI SybrGreen PCR Master Mix (2X), and quantitative PCR instruments were the ABI Stepone plus fluorescence quantitative PCR instruments. After the reaction mixture preparation were completed, set PCR cycling conditions and go the PCR cycling. Reference genes, sample gene amplification curve and dissolution curve were maked and Ct values and2-(△△Ct) were Calculated.③Determination of serum IGF-1: Collecting3ml peripheral blood of all subjects, and using enzyme-linked immunosorbent assay (ELISA) method to measure IGF-1concentration after separating of serum. To follow instructions, coefficients of variation intra and inter-assay meet the requirements.Statistical analysis:SPSS17.0software for statistical analysis was used, and Hardy-Weinberg equilibrium test was used to determine a representative sample of the population. R X C table x2test was used to count data, x±s was used to express measurement data, ANOVA was used to analyze the sample mean, and levene variance was used to do homogeneity test. The theoretical frequency<5number was tested by Fisher’s exact probability method. The Dunnett’s test was used for Pairwise comparisons. P<0.05was statistically significant.Results1. The results of short stature children GH receptor gene detected by MLPA technology:The higher signal of GHR gene Ex1～10were found in all the research object, and no signal to reduce or missing was found in the Ex1,2, and4～10.GHR exon3was located in the MLPA map30th gene amplification peaks. The incidence of signal reduction and completely missing were34.3%and1.5%in GHD group,17.2%and3.4%in ISS group and26.1%and8.7%in the control group.Differences of GHR gene Ex3genotype and allele frequencies had no statistically significance among three groups. P>0.05.2. The results of GH receptors signaling pathways in short stature children detected by MLPA technology:All of the subjects for the JAK2(1-7,9,12,14,16,19,21,23,25exons), STAT5b (1-4,7,12,13,15,16,18,19exons) and IGF-1(1,3,4,6exons) of the specific probe signal abnormalities were not found. The signal of intron near to5’end of exon6of STAT5b protein gene was located on the36th gene amplification peaks.The signal intensity of3cases of children in ISS group compared with the normal signal ratio was less than0.6and2cases of compatriot sister and1case of female children. The detection rate was10.34%(a total of three cases).Reducing of signal in the fragment was not found in GHD group and normal control group.3. The results of real-time fluorescent quantitative PCR detection: Amplification of intron near to5’end of exon6of STAT5B protein gene and real-time fluorescent quantitative PCR were done in3children with abnormal MLPA results on exon6of STAT5B gene and normal contros. The results of fluorescence quantitative PCR detection showed that the expression of the patient’s DNA in the region was0.395for the normal control which was lower than the reference value of0.6. It Verified that the gene was heterozygous mutation.4. The results of pedigree investigation for STAT5b Ex6gene deletion:①7members (3males,4females) were found that the genotypes of GHR and Ex6were consistent with the proband in Family Ⅰ which had12members. Height of4cases were lower than the average of the same gender and age-2SD, and2cases’were lower than the average of the same gender and age, and one case had the normal height.②3members (2males,1females) were found that the genotypes of GHR and Ex6were consistent with the proband in Family Ⅱ which had6members. Height of1case was lower than the average of the same gender and age-2SD, and2cases were lower than the average of the same gender and age.Conclusion ①The results of screening of growth hormone receptor gene with MLPA technology showed that the completely deletion in growth hormone receptor gene exon3are found in GHD group, the ISS group and normal control group. Gene deletion had the highest frequency in Growth hormone receptor gene polymorphism. There was no unified conclusion about the significance of gene polymorphism.②The results of screening of growth hormone receptor signaling pathways gene with MLPA technology showed that Gene deletion in intron near to5’end of exon6of STAT5b protein gene is found in Children with idiopathic short stature. Previous studies have found that homozygous mutation of STAT5b gene can cause short stature, and the heterozygous mutation may also cause short stature in children.③Pedigree investigation for STAT5b EX6gene deletion show that10members of the family are found that the genotypes of STAT5b are consistent with the proband.Height of5cases are lower than the average of the same gender and age-2SD, and4cases are lower than the average of the same gender and age. The inheritance mode is autosomal dominant. The specific biological functions of this gene mutation remains to need further research.