Ignite Creation Date:
2024-05-06 @ 7:15 PM
Last Modification Date:
2024-10-26 @ 3:03 PM
Study NCT ID:
NCT05946915
Status:
RECRUITING
Last Update Posted:
2023-07-14
First Post:
2023-07-07
Brief Title:
miRNA as a Growth Plate Marker After GH
Sponsor:
Faculdade de Ciências Médicas da Santa Casa de São Paulo
Organization:
Faculdade de Ciências Médicas da Santa Casa de São Paulo
Study Overview
Official Title:
The Use of miRNA Panel as a Growth Plate Marker of Short-term Response to GH
Status:
RECRUITING
Status Verified Date:
2023-07
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
The therapeutic use of recombinant human growth hormone usually determines adequate growth response in the majority of approved conditions However it is well recognized an inter-individual responsiveness and the classical biomarker such as GH peak response IGF-I or IGFBP3 levels have poor correlation with clinical outcome We hypothesize that markers directed originated from the growth plate would have the potential to better correlate with growth response during GH treatment Genetic markers as the growth hormone receptor exon 3-deletion polymorphism IGFBP3 polymorphisms were previously tested in an attempt to discriminate the pattern of responsiveness but results were contradictory in the different studies MicroRNAs miRNAs are small non-coding RNAs that regulate gene expression Due to their ability to regulate gene expression miRNAs play an important role both in physiology and pathophysiological conditions and also an important role in the regulation of endochondral ossification and regulation of the hypothalamic-pituitary-IGF axis Several miRNAs are already recognized as involved in the regulation of longitudinal growth and bone development through its action upon WNT-βcatenin Notch PI3KAKT and TGFβ signaling pathways AIM Therefore the aim of this study is to establish a panel of miRNAs correlated to the growth response during GH treatment that can be used of biomarker for early recognition and classification of patients according to GH therapy responsiveness This study will analyze 30 Children and adolescents with GH deficiency associated with Ectopic Posterior Pituitary EPP gland METHODS Clinical biochemical and miRNAs concentration will be measured at four time-points before starting therapy basal and after 1- 3- and 6-months during GH treatment Studied variables include height target height growth velocity and body mass index bone age and pubertal stage Laboratory Assessment at basal condition fasting glucose insulin TSH and free T4 and cortisol and IGF-I at 3 and 6 months Bone age at basal and 6 months of therapy MiRNAs will be measured in peripheral blood sample obtained before starting GH therapy after 1- 3- and 6-months during GH treatment A miRNA panel will be measured by absolute quantitative method digital PCR The identification of a panel of miRNAs that correlates with GH responsiveness offers a huge clinical applicability allowing prompt identification of patients who need differential therapeutic protocols targeted to achieve the best response during GH treatment
Detailed Description:
Introduction The clinical experience with the therapeutic use of recombinant human growth hormone GH has found adequate growth response in the majority of patients treated for each of the approved indications However as the experience accumulates with different treatment regimens it was recognized that individual first-year height responses vary considerably even with individualized treatment regimens 1 Poor short-term response is also translated into an unsatisfactory gain in adult height 2
Some mathematical models were developed in order to predict growth response of an individual patients Prediction models derived from the large Pfizer International Growth Database KIGS database explain approximately 60 of the variability of response to GH therapy in patients with GHD 3 Such models account for the definable variability of responsiveness allowing clinicians to adjust GH dose to individual patient Based on multiple regression analyses these models have identified factors that correlate with growth mainly first year growth velocity such as chronological age GH peak during provocative tests GH dose birth-weight SDS height SDS adjusted for target height SDS 3 Biochemical variables such as the baseline IGF-I and leptin have also been added to the prediction models
Genetic markers were also used in an attempt to discriminate good and bad responders to GH treatment as the exon 3-deleted growth hormone receptor polymorphism IGFBP3 polymorphism and others but their results were contradictory in the different studies
MicroRNAs miRNAs are small non-coding RNAs that regulate gene expression Most genes in the human genome are regulated by one or more miRNAs 4 Due to their ability to regulate gene expression miRNAs appear to play an important role in the pathophysiology and development of physiological processes as well as in human diseases 5 MicroRNAs usually suppress gene expression by binding to the 3UTR domain of mRNA 6 therefore increasing mRNA degradation or preventing translational information and protein synthesis
One of the remarkable characteristics of miRNAs is the plasma circulation inside exosomes determining its resistance to degradation allowing its measurement in the circulating blood performing as a liquid biopsy 7
Scientific evidence in different in-vitro models and in animals suggested that miRNAs have an important role in the regulation of endochondral ossification and in the regulation of the hypothalamic-pituitary-IGF axis 8 In particular miRNA-140 322 and 22 have been described as responsible for growth plate development 9 Catellani et al showed that miR-22-3p miR-30c-5p miR-106a-5p miR-140-5p miR-199a-5p miR-335-5p miR-340- 5p and miR-494-3p are involved in the regulation of longitudinal growth and bone development through its action upon WNT-βcatenin Notch PI3KAKT and TGFβ signaling pathways 10
Growth response in patients during GH treatment is variable depending both on the patients basal conditions and on individual innate sensitivity to therapy 11 Often the measured growth rate does not coincide with the expected one and the degree of correlation between clinical-auxological parameters and dose and GH peak vary enormously both inter and intra-individually during treatment 12
Therefore the aim of this study is to measure the circulating growth plate synthesized miRNAs before and during GH treatment searching for its correlation with clinical and biochemical markers
Methods Children and adolescents n30 patients both genders with GH deficiency GHD followed at the Pediatric Endocrinology Unit of the Irmandade da Santa Casa de Misericórdia de São Paulo Brazil will be screened Written informed consent will be obtained from all participants and their parents as appropriate
Inclusion Criteria GH stimulation test showing GH-peak concentration 5ngml combined to magnetic resonance imaging showing Posterior Pituitary Ectopic gland EPP Patients with combined hormone deficiencies must be compensated during the study period
Exclusion criteria chronic diseases need of prolonged supraphysiologic doses of glucocorticoids bone age 14 years or 16 years girls and boys respectively patients missing adequate follow-up during the study protocol
Clinical follow-up Clinical records at each visit will include age weight height height to target height difference and pubertal stage Clinical evaluation will be taken at five time-points before starting therapy basal and after 1- 3- and 6-months during GH treatment The body mass index BMI will be calculated as weightheight² kgm² Height target height TH and BMI will be expressed as a SD score WHO 2007 Growth velocity SDS will be calculated using Tanner reference 13 Puberty in all subjects will be classified following Marshall and Tanners criteria 14 15 The bone age was will be obtained every 6 months and classified according to Greulich and Pyle 16
Laboratory Assessment Hormone measurements will be measured at 3 and 6 months and will include IGF-I fasting glucose insulin TSH and free T4 and cortisol Gonadotropins and sex steroids will be measured when adequate To evaluate the expression of growth plate synthesized miRNAs a basal blood sample will be obtained before starting GH therapy or after a short-time washout period 15-30 days and after 1- 3- and 6-months during GH treatment
MiRNAs determination quantitative PCR qPCR will be used to measure circulating miRNA levels by employing the droplet digital PCR ddPCR considered to be the gold standard in the application of liquid biopsy because its superior precision and sensitivity being less affected by PCR inhibitors and do not require internalexternal normalization while detecting low concentration of target nucleic acids molecules A whole blood sample will be drawn in vacutainer serum separator tubes and processed within 2h from collection centrifuged at 2000 g for 10 min at 4C Serum will be aliquoted in 15-ml sterile RNase-free tubes and further centrifuged at 2500 g for 10 min at 4C to remove any contaminant cells and debris Serum will be collected in sterile RNase-free tubes and stored at -80C until use Total RNA will be isolated from the sample using 200 ul of serum using the miRNeasy SerumPlasma Advanced Kit ID 217204 - Qiagen
The following miRNAs will be measured miR-22-3p miR-30c-5p miR-106a-5p miR-140-5p miR-199a-5p miR-335-5p miR-340-5p and miR-494-3p For each ddPCR assay 3 μL cDNA sample 10 μL 2 ddPCR supermix for probes Bio-Rad 1 μL 20 TaqMan miRNA probe and 6 μL RNase-free Water will be added in a 20 μL reaction mixture Then the mixture and 70 μL droplet generation oil for probes Bio-Rad will be respectively loaded into the sample wells and oil wells of a disposable droplet generator cartridge Bio-Rad After that droplets will be generated by QX200 droplet generator device Bio-Rad and carefully transferred to a 96-well PCR plate Eppendorf The cycling conditions will be 95 C for 10 min 40 cycles of 95 C for 15 s and 57 C for 1 min and a final step at 98 C for 10 min At the end of the PCR reaction droplets will be read in the QX200 droplet reader and analyzed using the Quantasoft version 174 software Bio-Rad
Benefits Establishing a panel of miRNAs that correlates with GH responsiveness is of huge clinical applicability allowing prompt identification of patients who need differential therapeutic protocols targeted to achieve the best response during GH treatment
Some mathematical models were developed in order to predict growth response of an individual patients Prediction models derived from the large Pfizer International Growth Database KIGS database explain approximately 60 of the variability of response to GH therapy in patients with GHD 3 Such models account for the definable variability of responsiveness allowing clinicians to adjust GH dose to individual patient Based on multiple regression analyses these models have identified factors that correlate with growth mainly first year growth velocity such as chronological age GH peak during provocative tests GH dose birth-weight SDS height SDS adjusted for target height SDS 3 Biochemical variables such as the baseline IGF-I and leptin have also been added to the prediction models
Genetic markers were also used in an attempt to discriminate good and bad responders to GH treatment as the exon 3-deleted growth hormone receptor polymorphism IGFBP3 polymorphism and others but their results were contradictory in the different studies
MicroRNAs miRNAs are small non-coding RNAs that regulate gene expression Most genes in the human genome are regulated by one or more miRNAs 4 Due to their ability to regulate gene expression miRNAs appear to play an important role in the pathophysiology and development of physiological processes as well as in human diseases 5 MicroRNAs usually suppress gene expression by binding to the 3UTR domain of mRNA 6 therefore increasing mRNA degradation or preventing translational information and protein synthesis
One of the remarkable characteristics of miRNAs is the plasma circulation inside exosomes determining its resistance to degradation allowing its measurement in the circulating blood performing as a liquid biopsy 7
Scientific evidence in different in-vitro models and in animals suggested that miRNAs have an important role in the regulation of endochondral ossification and in the regulation of the hypothalamic-pituitary-IGF axis 8 In particular miRNA-140 322 and 22 have been described as responsible for growth plate development 9 Catellani et al showed that miR-22-3p miR-30c-5p miR-106a-5p miR-140-5p miR-199a-5p miR-335-5p miR-340- 5p and miR-494-3p are involved in the regulation of longitudinal growth and bone development through its action upon WNT-βcatenin Notch PI3KAKT and TGFβ signaling pathways 10
Growth response in patients during GH treatment is variable depending both on the patients basal conditions and on individual innate sensitivity to therapy 11 Often the measured growth rate does not coincide with the expected one and the degree of correlation between clinical-auxological parameters and dose and GH peak vary enormously both inter and intra-individually during treatment 12
Therefore the aim of this study is to measure the circulating growth plate synthesized miRNAs before and during GH treatment searching for its correlation with clinical and biochemical markers
Methods Children and adolescents n30 patients both genders with GH deficiency GHD followed at the Pediatric Endocrinology Unit of the Irmandade da Santa Casa de Misericórdia de São Paulo Brazil will be screened Written informed consent will be obtained from all participants and their parents as appropriate
Inclusion Criteria GH stimulation test showing GH-peak concentration 5ngml combined to magnetic resonance imaging showing Posterior Pituitary Ectopic gland EPP Patients with combined hormone deficiencies must be compensated during the study period
Exclusion criteria chronic diseases need of prolonged supraphysiologic doses of glucocorticoids bone age 14 years or 16 years girls and boys respectively patients missing adequate follow-up during the study protocol
Clinical follow-up Clinical records at each visit will include age weight height height to target height difference and pubertal stage Clinical evaluation will be taken at five time-points before starting therapy basal and after 1- 3- and 6-months during GH treatment The body mass index BMI will be calculated as weightheight² kgm² Height target height TH and BMI will be expressed as a SD score WHO 2007 Growth velocity SDS will be calculated using Tanner reference 13 Puberty in all subjects will be classified following Marshall and Tanners criteria 14 15 The bone age was will be obtained every 6 months and classified according to Greulich and Pyle 16
Laboratory Assessment Hormone measurements will be measured at 3 and 6 months and will include IGF-I fasting glucose insulin TSH and free T4 and cortisol Gonadotropins and sex steroids will be measured when adequate To evaluate the expression of growth plate synthesized miRNAs a basal blood sample will be obtained before starting GH therapy or after a short-time washout period 15-30 days and after 1- 3- and 6-months during GH treatment
MiRNAs determination quantitative PCR qPCR will be used to measure circulating miRNA levels by employing the droplet digital PCR ddPCR considered to be the gold standard in the application of liquid biopsy because its superior precision and sensitivity being less affected by PCR inhibitors and do not require internalexternal normalization while detecting low concentration of target nucleic acids molecules A whole blood sample will be drawn in vacutainer serum separator tubes and processed within 2h from collection centrifuged at 2000 g for 10 min at 4C Serum will be aliquoted in 15-ml sterile RNase-free tubes and further centrifuged at 2500 g for 10 min at 4C to remove any contaminant cells and debris Serum will be collected in sterile RNase-free tubes and stored at -80C until use Total RNA will be isolated from the sample using 200 ul of serum using the miRNeasy SerumPlasma Advanced Kit ID 217204 - Qiagen
The following miRNAs will be measured miR-22-3p miR-30c-5p miR-106a-5p miR-140-5p miR-199a-5p miR-335-5p miR-340-5p and miR-494-3p For each ddPCR assay 3 μL cDNA sample 10 μL 2 ddPCR supermix for probes Bio-Rad 1 μL 20 TaqMan miRNA probe and 6 μL RNase-free Water will be added in a 20 μL reaction mixture Then the mixture and 70 μL droplet generation oil for probes Bio-Rad will be respectively loaded into the sample wells and oil wells of a disposable droplet generator cartridge Bio-Rad After that droplets will be generated by QX200 droplet generator device Bio-Rad and carefully transferred to a 96-well PCR plate Eppendorf The cycling conditions will be 95 C for 10 min 40 cycles of 95 C for 15 s and 57 C for 1 min and a final step at 98 C for 10 min At the end of the PCR reaction droplets will be read in the QX200 droplet reader and analyzed using the Quantasoft version 174 software Bio-Rad
Benefits Establishing a panel of miRNAs that correlates with GH responsiveness is of huge clinical applicability allowing prompt identification of patients who need differential therapeutic protocols targeted to achieve the best response during GH treatment
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
False
Is a FDA Regulated Device?:
False
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None