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Scleroderma Association of BC Research Project

February 2019 Update

SABC funds and co-leads a research study that began recruiting scleroderma patients with and without interstitial lung disease (ILD) in July 2017. Blood samples have also been taken from patients with idiopathic pulmonary fibrosis (IPF) only and both blood and skin samples have been taken from control participants. This research program is creating a firm foundation for intensive research to control lung and skin damage in patients with scleroderma and lung damage in patients with IPF, with the expectation of receiving future support from donations and, hopefully, research funding agencies.

Research Study Progress

Nov 2017 Collection of blood and skin tissue samples completed and stored in The Scleroderma Biobank at St. Paul’s Hospital in Vancouver

Dec 2017 Skin tissue growth completed

Apr 2018 Extraction of micro-RNA (miRNA) from tissue types

May 2018 Frozen tissue samples from three tissue types transferred to the BC Genome Sciences Centre for sequencing

Jul 2018 miRNA sequence data downloaded for one-third of the samples and quality control (QC) checks started by the scientific team members

Nov 2018 Completion of QC assessment for the first two-thirds of the samples

Feb 2019 Completion of QC assessment for the last third of the samples and QC examination for pilot study of fourth tissue.

This proof-of-concept study is to discover miRNA sequences and their differential expression amongst six different types of cells. This study is important for the approximately 15,224 Canadians with scleroderma and the approximately 7,045 Canadians with IPF (as confirmed by CT, biopsy, or bronchoscopy). 

Discovering which miRNA sequences are too low or too high and correcting these imbalances could lead to effective treatment of skin damage in patients with scleroderma and treatment of lung damage in patients with IPF only and in patients with both scleroderma and ILD.

SABC has been behind this project for the last five years, contributing over $240,000 in donations to date. Your donations do make a difference in contributing to research that otherwise would not even be considered for funding for these two orphan diseases.

The research team brings together experts in respirology, rheumatology, bioinformatics, and genetical statistics to uniquely tackle this challenge. The team is led by SABC President Rosanne Queen, SABC past President Bob Buzza, Drs. James Dunne and Kevin Keen, Drs. Raewyn Broady, Robert Holt, Chris Ryerson and Pearce Wilcox round out the scientific research team. Both Rosanne and Bob participate on the leadership team to keep us informed on the progress of this SABC-funded research program and to ensure the interests of patients and their families are at the forefront.

 

Scleroderma Association of B.C. Research Project

Seventh Quarter Report – February 2019

This Project is a hypothesis-generating study for the control of lung damage in patients with idiopathic pulmonary fibrosis (IPF) and both lung and skin damage in systemic scleroderma (SSc) patients either with or without interstitial lung disease (ILD). Enrolment of SSc patients with and without ILD to provide blood and skin tissue samples was completed during the first quarter at the end of August 2017 with samples stored in The Scleroderma Biobank at St. Paul’s Hospital in Vancouver. Enrolment of patients with IPF and normal controls was completed by mid-November 2017. Skin-fibroblast extraction and growth from skin samples (from all but the IPF patients from whom no skin samples were taken due to ethical reasons as their skin is not affected) were completed and stored in the Biobank before the Christmas 2017 break. With some extra efforts by laboratory staff, extraction of miRNA from all five tissue types was completed by mid-April 2018 but miRNA extraction was not sufficient in the fifth tissue for whole genome sequencing (WGS) without additional processing by the laboratory and so this was placed on indefinite hold due to cost. A sixth tissue remains stored in the Biobank because its miRNA can be amplified for miRNA WGS but at additional cost. 

After receiving frozen tissue-samples from the laboratory at St. Paul’s Hospital, on May 16 2018, the British Columbia Genome Sciences Centre (BCGSC) provided miRNA sequencing data on 92 samples on July 26 2018, another 92 samples on August 7 2018, and the last 92 samples on January 2 2019. Quality control assessments on the last 92 samples were completed on February 17 2019, at which point the Project was eight months behind schedule. All 276 samples comprise three tissue-types, with some duplicates, from 11 limited SSc-ILD patients, 12 diffuse SSc-ILD patients, 9 patients with limited SSc only, 5 patients with diffuse SSc only, 19 patients with IPF, and 16 control participants. A limited experiment with 4 samples and 2 different tissues to examine a new procedure for amplifying miRNA for WGS was initiated in December 2018 with promising results in late February 2019, inclusive of a cross-comparison with the expression of 750 miRNA’s as measured on the NanoString platform in October 2018 for 3 different tissue types. The third tissue type examined on the NanoString platform in October was blood serum for the purpose of verifying 3 different methods for preparing serum. The least expensive method for blood serum was deemed the best for NanoString.

March 2019 will see the start, in earnest by researchers, of comprehensive statistical analysis of the miRNA WGS data inclusive of seeking to understand the role of miRNA’s in skin damage and calcinosis.

University researchers involved in this project are Drs. James Dunne, Chris Ryerson, and Pearce Wilcox of The University of British Columbia and Dr. Kevin Keen of both The University of British Columbia and The University of Northern British Columbia. Rounding out the Leadership Team are Ms. Rosanne Queen and Mr. Robert Buzza for the Scleroderma Association of B.C.

 

Scleroderma Association of B.C. Research Project

Sixth Quarter Report – November 2018

This Project is a hypothesis-generating study for the control of lung damage in patients with idiopathic pulmonary fibrosis (IPF) and lung and skin damage in systemic scleroderma (SSc) patients with or without interstitial lung disease (ILD). Enrolment of SSc patients with and without ILD to provide blood and skin tissue samples was completed during the first quarter at the end of August 2017 with samples stored in The Scleroderma Biobank at St. Paul’s Hospital in Vancouver. Enrolment of patients with IPF and normal controls was completed by mid-November 2017.  Skin-fibroblast extraction and growth from skin samples (from all but the IPF patients from whom no skin samples were taken due to ethical reasons as their skin is not affected) were completed and stored in the Biobank before the Christmas 2017 break. With some extra efforts by laboratory staff to harvest sufficient miRNA for two of the tissues, extraction of miRNA from all five tissue types was completed by mid-April 2018 but miRNA extraction was not sufficient in the fifth tissue for whole genome sequencing (WGS) without additional processing by the laboratory and so this was placed on indefinite hold due to cost. A sixth tissue remains stored in the Biobank because its miRNA cannot be amplified for miRNA WGS, also for cost reasons, but will be analyzed by NanoString miRNA expression chemistry.

After receiving frozen tissue-samples from the laboratory at St. Paul’s Hospital on May 16 2018, the British Columbia Genome Sciences Centre (BCGSC) provided miRNA sequencing data on 92 samples on July 26 2018 and another 92 samples on August 7 2018. After completing quality control checks for miRNA sequencing for these 184 samples in mid-November and combining a few samples, a further 13 tissue- samples were shipped on November 27 2018 to BCGSC to complete the third set. All 276 samples comprise 3 tissue-types, with some duplicates, from 11 limited SSc-ILD patients, 12 diffuse SSc-ILD patients, 9 patients with limited SSc only, 5 patients with diffuse SSc only, 19 patients with IPF, and 16 control participants. Progress has been affected by vacations understandably taken by laboratory staff during August and November. All will be taking vacations in December. It is expected to receive the miRNA sequences for the last 92 samples and to complete all quality control checks by the end of January 2019.

After training of laboratory staff, two cartridges were processed as a developmental test of the NanoString platform for miRNA expression analysis in October. The first cartridge failed due to a manufacturing defect and a second replacement cartridge plus reagents were provided by NanoString at no extra cost but the samples on the first cartridge were unfortunately lost. The NanoString technology was successfully validated on 3 tissue types in samples from 3 participants, inclusive of verifying 3 different methods for preparing samples of blood serum. Ironically, the least expensive method for blood serum was deemed the best for NanoString.

University researchers involved in this project are Drs. James Dunne, Chris Ryerson, and Pearce Wilcox of The University of British Columbia and Dr. Kevin Keen of both The University of British Columbia and The University of Northern British Columbia. Rounding out the Leadership Team are Ms. Rosanne Queen and Mr. Robert Buzza for the Scleroderma Association of B.C.

 

Scleroderma Association of B.C. Research Project

Fifth Quarter Report – August 2018

This Project is a hypothesis-generating study for the control of lung damage in patients with idiopathic pulmonary fibrosis (IPF) and lung and skin damage in systemic scleroderma (SSc) patients both with and without interstitial lung disease (ILD). Enrolment of SSc patients with and without ILD to provide blood and skin tissue samples closed ahead of schedule during the first quarter by the end of August 2017 with samples stored in The Scleroderma Biobank at St. Paul’s Hospital in Vancouver. Enrolment of patients with idiopathic pulmonary fibrosis (IPF) and normal controls was completed by the middle of November. Skin fibroblast extraction and growth from skin samples (from all but the IPF patients from whom no skin samples were taken due to ethical reasons as their skin is not affected) were completed by the laboratory at St. Paul’s and stored in the Biobank before the Christmas 2017 break. Extra efforts were required by laboratory staff to harvest sufficient miRNA for two of the tissues, which doubled the time required for extraction but not for much-added cost in terms of purchasing reagents and other consumables. Extraction of miRNA from all five tissue types was completed by mid-April 2018 but miRNA extraction was not sufficient in the fifth tissue for whole genome sequencing (WGS) without additional processing by the laboratory and so this was placed on indefinite hold due to cost. A sixth tissue remains stored in the Biobank because its miRNA cannot be amplified for miRNA WGS, also for cost reasons, but will be analyzed by Nanostring miRNA expression chemistry.

The laboratory at St. Paul’s Hospital transferred 275 frozen tissue samples to the British Columbia Genome Sciences Centre (BCGSC) on May 16 2018, where these were entered in the queue for WGS analysis. On July 26 2018, processed miRNA sequence data from 92 duplicate samples of whole blood comprising 11 patients with both limited SSc and ILD, 19 patients with IPF, and 16 control participants was downloaded from BCGSC and quality control checks were commenced by the Project team. Raw miRNA sequence data for these samples were downloaded from BCGSC on August 7 2018 with quality control checking continuing until mid-August and then suspended due to vacations.

The original design called for miRNA WGS analysis on three tissue types and Nanostring miRNA expression analysis on four tissue types. This experimental design had to be altered because two more tissues were added and miRNA WGS analysis can only be done on three of the four original tissue types. Nanostring analysis requires one-tenth the amount of miRNA. There is ample miRNA for Nanostring analysis for five tissue types. In June, there was to be a developmental run using Nanostring for one tissue type from each of four patients with SSc-ILD, four patients with IPF, and four controls but this and WGS miRNA for the remaining 184 duplicate samples has been delayed until October pending completion of the quality control assessments.

University researchers involved in this project are Drs. James Dunne, Chris Ryerson, and Pearce Wilcox of The University of British Columbia and Dr. Kevin Keen of The University of Northern British Columbia.

 

Scleroderma Association of B.C. Research Project

Fourth Quarter Report – May 2018

This Project is a hypothesis-generating study for the control of lung damage in patients with idiopathic pulmonary fibrosis (IPF) and lung and skin damage in systemic scleroderma (SSc) patients both with and without interstitial lung disease (ILD). Enrolment of SSc patients with and without ILD to provide blood and skin tissue samples closed ahead of schedule during the first quarter by the end of August 2017 with samples stored in The Scleroderma Biobank at St. Paul`s hospital in Vancouver. Enrolment of patients with idiopathic pulmonary fibrosis (IPF) and normal controls was completed by the end of October and the middle of November, respectively, 2017.

Skin fibroblast extraction and growth from skin samples (from all but the IPF patients from whom no skin samples were taken due to ethical reasons as their skin is not affected) were completed by the laboratory at St. Paul’s and stored in the Biobank before the shutdown over the Christmas 2017 break. At the end of February 2018, extraction of micro ribonucleic acid (miRNA) was completed for four tissue types from all participants. Extra efforts were required by laboratory staff to harvest sufficient miRNA for two of the tissues, which doubled the time required for extraction but not for much-added cost in terms of purchasing reagents and other consumables. Extraction of miRNA from the fifth tissue was completed by mid-April but miRNA extraction was not sufficient for whole genome sequencing (WGS) without additional processing by the laboratory and so this was placed on indefinite hold due to cost. A sixth tissue remains stored in the Biobank because its miRNA cannot be amplified for miRNA WGS, also for cost reasons, but will be analyzed by Nanostring miRNA expression chemistry. The laboratory at St. Paul’s Hospital transferred 275 frozen tissue samples to the British Columbia Genome Sciences Centre 16 May 16 2018, where these were entered in the queue for WGS analysis.

The original design called for miRNA WGS analysis on three tissue types and Nanostring miRNA expression analysis on four tissue types. This experimental design had to be altered because two more tissues were added and miRNA WGS analysis can only be done on three of the four original tissue types. Nanostring analysis requires one-tenth the amount of miRNA. There is ample miRNA for Nanostring analysis for five tissue types. In June, there will be a developmental run using Nanostring for one tissue type from each of four patients with SSc-ILD, four patients with IPF, and four controls.

University researchers involved in this project are Drs. James Dunne, Chris Ryerson, and Pearce Wilcox of The University of British Columbia and Dr. Kevin Keen of The University of Northern British Columbia. Rounding out the Leadership Team are Ms. Rosanne Queen and Mr. Robert Buzza for the Scleroderma Association of B.C.

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