| PART 1:Clinical features of patients with Laron SyndromeObjective:To summarize the clinical data and genetic results of patients with Laron Syndrome and to make comparisons among patients of different ethnic origins.Methods:1.Collect clinical data:Retrospectively review medical records of all the patients diagnosed with Laron Syndrome in Peking Union Medical College Hospital from 2012 to 2017,including serum biological examinations,GH and IGF-1 concentrations.Each patient is required to undertake at least two types of GH stimulation test,and blood sample was taken before administration of medicine and every 3 0 minutes after taking medicine for up to 2 hours(time points for blood collection were:0 min,30min,60min,90min and 120 min)for GH measurement.X-ray of non-dominant hand and wrist was conducted to assess the bone age.2.Take blood sample for GHR gene exons sequencing:Collect peripheral blood sample after informed consent was signed.Genomic DNA was extracted from peripheral blood leukocytes and GHR gene were amplified by PCR.Novel variants were analyzed in silico by online software such as SIFT,Polyphen-2,PROVEAN and Mutation Taster.3.Literature review:By searching for PubMed and several Chinese database with key words of "Laron Syndrome","growth hormone insensitivity syndrome","Laron-type dwarfism","growth hormone resistance syndrome",a literature review was conducted.Articles were included if the original clinical data of each individual were shown in the main text.Results:1.Clinical features of four patients with Laron Syndrome:4 patients with Laron Syndrome were analyzed in the current study.They are all male.The average age of disease onset was 2.5 years old(range from 1.0 to 6.0 ys),and the average age of diagnosis was 7.6 years(range from 3.5 to 14.3 ys).The median height SDS was-4.73(range from-6.71 to-2.80)and median weight SDS was-2.58(range from-2,96 to-1.82),indicating the height defect was much severer than weight defect.Three patients had a normal facial feature,and patient 4 presented with typical facial features of Laron Syndrome(protruding forehead,depressed nasal bridge,mid-facial hypoplasia).Serum IGF-1 levels were extremely low,they were 32 ng/ml,<25 ng/ml,<25 ng/ml and<25 ng/ml,respectively.Peak GH concentration after GH stimulation test were 23.36 ng/ml,31.70 ng/ml and>33.80 ng/ml in patient 1,3 and 4,patients 2 did not perform GH stimulation test.Bone age retardation was 12 months,6 months,28 months and 24 months,and the average retardation time was 17.5 months compared to the chronological age.Patient 1 and patient 4 had received rhGH treatment.Patient 1 received rhGH treatment at a dosage of 0.053 mg/kg/d for 2 months,and IGF-1 level increased from 32 ng/ml to 91 ng/ml,height increased by 3.30 cm.Patient 4 received rhGH treatment at a dosage of 0.057 mg/kg/d for 10 months,IGF-1 level after treatment was<25 ng/ml,and the height increased by 5.75cm.2.GHR gene exons sequencing:A total of five GHR gene variants were discovered.They were c.1483C>A(p.P495T)and c.1630A>C(p.I544L)in patient 1,c.558A>G(P.G186G),c.1319G>T(p.C440F)and c.1735C>A(p.P579T)in patient 2.A GHR gene mutation in patient 4 has previously been reported,c.587A>C(p.Y196S).Each of patient 2,3 and 4 had a novel GHR gene mutation,and the mutations were c.808A>G(p.I270V)(patient 2),c.766C>T(p.Q256X)(patient 3)and c.1707-1710del(p.E570fs)(patient 4),respectively.Functional prediction result of the p.1270V mutation was benign by PolyPhen-2,tolerated by SIFT and a polymorphic site by Mutation Taster.The p.Q256X mutation and the p.E570fsmutation was predicted disease causing by Mutation Taster.3.Characteristics of Chinese patients with Laron Syndrome:A total of 35 Chinese patients were summarized and the sex ratio of male to female was 3.4:1..The average age was 9.1 ±3.4 years old(range from 2.2 to 15.0),and the median height SDS was-4.40(Q1,Q3:-5.90,-2.67).The median GH peak value was 17.01 ng/ml(Q1,Q3:14.56,33.20).IGF-1 SDS and IGFBP-3 SDS was-2.65±0.55(N=23)and 1.82± 1,16(N=17)respectively.13 patients had confirmed GHR gene mutation,and a total of 10 GHR gene mutation sites were found,no significant height SDS difference was observed between different types of mutation.3 patients carried IGFALS gene mutation,and the height defect was relatively mild with a median height SDS of-2.66.4.Clinical features of patiens from different ethnic groups:Comparisons were made among 13 Turkish patients,9 Indian patients,35 European patients and 35 Chinese patients.The age of diagnosis in Chinese patients was significantly later than that of European patients,the median age of diagnosis was 9.0 years old and 5.7 years old(P=0.004).Height defect in Turkish patients was severer than Chinese patients,the height SDS was-7.4 and-4.4 respectively(P =0.001).GH peak value after GH stimulation test in Turkish patients was higher than that of European and Chinese patients(P =0.003,P=0.000).Conclusions:1.Laron Syndrome is a rare genetic disease which could cause severe short stature.We reported 4 patients with Laron Syndrome and three of them had novel GHR gene mutations.Patients presented with a wide spectrum of phenotype,and some of the patients have slight responsiveness to rhGH treatment.With the development of genetic sequencing technology,more and more patients could get a definite genetic diagnosis.2.Among Turks,Indian,European and Chinese,the age of diagnosis of Chinese was the latest,Turks had the most sever height defect and the highest GH peak values after GH stimulation test.PART II: Functional study of GHR gene mutation in cellsObjective: To perform cellular functional study of the novel GHR gene mutations.Methods: The full-length GHR gene wild type was subcloned into pcDNA3.1(+)/Amp expression plasmid.The mutant GHR-Y196 S,GHR-Q256 X and GHR-E570 fs gene was contracted by means of site-directed mutagenesis using the GHR-WT expression plasmid.HEK293 T cell and HepG2 cell were transfected with wild type and mutant overexpression plasmids by Lipofection3000.Western blot(WB)was conducted to estimate the amount of total GHR protein and its size 48 hours after transfection.Immunofluorescence was performed on fixed cells to determine the cellular distribution of GHR.Phosphorylation of STAT5 was examined by means of WB 30 minutes after100 ng/ml rhGH was added into the DMEM medium to evaluate the activation of STAT5.IGF1 gene transcription was calculated by real-time fluorescence quantitative PCR(qRT-PCR)24 hours after 100 ng/ml rhGH was added into the DMEM medium,and the cell culture medium was collected at the same time to measure IGF-1 concentration by enzyme linked immunosorbent assay(ELISA).Results:1.Toal GHR protein expression by WB after transfected with wild type and mutant type overexpression plasmids: WB was performed with anti-HA tag antibody,and a band around 130 kDa could be detected for both GHR-WT and Y196 S mutant,and the relative expression of GHR-WT was significantly higher than Y196S(P<0.05).The Q256 X mutant produced a trancated GHR protein and two bands around 43 kDa were detected.The relative expression of Q256 X was significantly lower than GHR-WT(P<0.05).E570 fs mutation was a frameshift mutation caused by lossing of successive four base pairs.No band was detected at neither 13 OkDa nor 43 kDa.When WB was conducted by anti-GHR antibody,the same result was observed.2.Subcellular localization of GHR protein by immunoflurescence: HepG2 cells were transfected with GHR-WT and three types of mutant overexpression plasmids.Immunofluescence was performed 48 hours after transfection.The GHR-WT protein was present at the plasma membrane,uniformly distributed.Y196 S mutant had a similar distribution as the GHR-WT,while Q256 X and E570 fs mutant had a distinct distribution pattern and they seemed to be trapped ?xmnd the nucleus in a circumferential pattern of fluorescence.3.rhGH-induced STAT5 phosphorylation by WB: HepG2 cells were transfected with GHR-WT and three types of mutant overexpression plasmids.lOOng/ml rhGH was added to cell culture medium for 30min3 and WB was conducted with anti-phosphorylated STAT5 antibody.Phosphorylation of STAT5 in HepG2 cells transfected with Y196 S mutant was decreased by 32.7% compared to cells transfected with GHR-WT,and a decrease of 40.6% and 29.6% was observed for Q256 X and E570 fs mutant(P<0.05).4.rhGH-induced IGF 1 gene transcription by qRT-PCR: HepG2 cells were transfected with GHR-WT and three types of mutant overexpression plasmids.lOOng/ml rhGH was added to cell culture medium for 24 hours,and qRT-PCR was performed.The relative expression of IGF1 mRNA in the E570 fs mutant was 64.1% higher than that of GHR-WT(P<0.05)? no significant difference was observed among other groups.5.Total IGF-1 protein concentration by ELISA: HepG2 cells were transfected with GHR-WT and three types of mutant overexpression plasmids.Cell culture medium was collected 24 hours after rhGH was added,ELISA was used to quantify IGF-1levels.IGF-1 level of cells transfected with GHR-WT was 71.88 pg/ml5 the IGF-1levesl in other cells transfected with Y196 S,Q256X and E570 fs was 94.80 pg/ml595.63 pg/ml and 95.63 pg/ml.The concentration of IGF-1 did not differ significantly among different groups(P=0.238).Conclusions:1.The expression of Y196S mutant GHR was significantly lower than GHR-WT. and the localization of Y196S mutant GHR was similar to GHR-WT.The ability of activating STAT5 was decreased.2.Q256X mutant produced a truncated GHR protein,with a molecular weight of 43kDa,and the expression of Q256X mutant GHR was lower that GHR-WT.Q256 X mutant had a decreased ability to activate STATS.3.The molecular weight of E570fs mutant GHR protein was unknown,and it was trapped in the cytoplasm.It had a decreased ability to activate STAT5. |
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