A Registered Observational Cohort Study of Facioscapulohumeral Muscular Dystrophy Type 1
Conditions: Facioscapulohumeral Muscular Dystrophy Type 1 (FSHD1)
Sponsor: Ning Wang, MD., PhD.

Official Title: A Registered Observational Cohort Study of Facioscapulohumeral Muscular Dystrophy Type
1
NCT number: NCT04369209
Document date: 2022/2/1

Abstract
Background:
Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is a rare, hereditary, and progressive muscular
dystrophy characterized by weakness and wasting of the facial (facio-) and shoulder-upper arm
(scapulo-humeral-). The clinical severity of varies from nonpenetrant/asymptomatic to disability with
independent ambulation loss or even wheelchair dependence. Expansion of our knowledge on the genetic
model -D4Z4 macrosatellite repeats contraction leading to inappropriate expression of DUX4- has led to
advance in developing (targeted) therapeutic strategies. Thus, clinical trial readiness is now important,
particularly patient registry. However, there is yet any FSHD registry in China, let alone epidemiology of
FSHD), particularly in terms of prevalence and disease progression/disability.
Methods and analysis:
The China FSHD1 Registry (CF1R) is a nationwide, population-based, longitudinal, non-interventional, and a
combination of retrospective and prospective observational cohort clinical study, collecting practice data,
including demographic, disease progression history, clinical and genetic features in genetically-confirmed
FSHD1 patients/families; this study is performed in Fujian Neuromedical Center (FNMC), where is a
diagnosis center for clinical-genetic FSHD1 in China, the only one employing pulsed-field gel electrophoresis
(PFGE)-based Southern blotting. Information regarding clinical suspected FSHD1 patients are obtained from
three sources: FNMC, Genetic and Myopathy Group (branches of the Neurology Society of the Chinese
Medical Association), and “FSHD-China” (an organization supported by FSHD patients). The study will firstly
generate a data analyses concerning on estimated prevalence and disease progression/disability (with
independent ambulation loss or wheelchair dependence); and some other several data analyses, nested as
a sub-study within the CF1R, are expected to be generated via a subsequent protocol amendment.

Conclusion:
The data collected within this rare-disease patient registry (CF1R) will be utilized to synthesise real-world
data on epidemiological information, disease progression/disability, and clinical and (epi)genetic
information which are vital for adequate symptomatic management and clinical trial-readiness of FSHD1 in
China.
Ethics and dissemination
The study was granted ethics committee approval by the First Affiliated Hospital of Fujian Medical
University (MRCTA, ECFAH of FMU [2020]026) in 2020. Findings will be disseminated via presentations and
peer-reviewed publications.
Trial registration: clinicaltrials.gov NCT04369209
Keywords: Facioscapulohumeral muscular dystrophy type 1 (FSHD1), Patient registry, Rare disease,
PFGE-based Southern blotting, Prevalence, Disease progression

1. Introduction
Facioscapulohumeral muscular dystrophy type 1 (FSHD1) is one of rare diseases and is one of the most
common inherited muscular dystrophies with autosomal dominance.[1] The core phenotype of FSHD is
characterized by progressive weakness of the facial (facio-) and shoulder-upper arm (scapulo-humeral-).
Muscle wasting often progresses slowly and typically in a descending pattern to include axial-, distal
anterior leg, and hip girdle muscles.[2, 3] There is a large variability in the severity of symptoms, ranging
from nonpenetrant/asymptomatic to disability with independent ambulation loss or even wheelchair
dependence.[1, 4, 5]
FSHD1 has a unifying genetic model caused by a contraction to 1-10 D4Z4 macrosatellite repeats on a
4qA-specific FSHD1-permissive haplotype,[6-8] which is sensitively identified by pulsed-field gel
electrophoresis (PFGE)-based Southern blotting.[9, 10] FSHD is linked to epigenetic derepression that lead
to chromatin relaxation of the D4Z4 macrosatellite repeats to transcribe full-length DUX4, with transcripts
stabilised by splicing to a poly(A) signal on specific 4qA haplotypes. Inappropriate expression of full-length
DUX4 and activation of its target genes is thought to be the major contributor to FSHD pathology, although
the exact pathophysiological mechanism is still largely unknown.[7, 11] Expansion of our knowledge on the
“DUX4opathy” models[12] has led to advance in developing (targeted) therapeutic strategies, thus, clinical
trial readiness is now important. All of the aspects of patient registries, biomarkers, and clinical outcome
measures are significant for clinical trial readiness in rare diseases such as FSHD, particularly patient
registries,[13] which is one of the most effective methods of collecting all of the required large-scale
patient data in rare diseases. Currently, there are 13 different countries established FSHD registries
collecting data on over 3000 patients (clinical suspected and/or genetically confirmed patients),[13]such as
Dutch[14] , UK[15] , US [16], Italian [17], French[18] , and etc. However, there is yet any reported cohort
internationally of absolute genetically-confirmed FSHD1. In China, there is yet any FSHD registry, hence,
there currently remains a dearth of data reporting on the epidemiology of FSHD (FSHD1), particularly in

terms of prevalence and disease progression/disability.
Here, we set up the China FSHD1 Registry (CF1R), which is a nationwide, population-based,
longitudinal, and observational cohort clinical study focused on specific-disease of genetically-confirmed
FSHD1 in all age groups, collecting data retrospectively at study entry and prospectively during follow up.
The data to be collected is intended to help healthcare providers estimate the burden of and make
important medical and financial decisions concerning FSHD in China, through an enhanced understanding
of the prevalence, disease progression/disability, and natural history of genetically-confirmed FSHD1.
2. Objectives
2.1 Primary objectives

To describe the methods of the China FSHD1 Registry to facilitate the development of other rare
disease registries.

To calculate true prevalence of genetically-confirmed FSHD1 in China basing on accurate diagnoses.

To estimate disease progression of FSHD1 (resulting in disability with independent ambulation loss
and/or wheelchair dependence).
2.2 Secondary objectives

To establish a well-characterized baseline cohort for prospective follow-up and recruitment for future
clinical trials.

To collect long-term outcomes of FSHD1.

To collect biological samples for future biomarker studies.
3. Methods
3.1 Registry identification
This rare- and inherited-disease patient registry (CF1R) has been registered with clinicaltrials.gov, under the

identifier NCT04369209.
3.2 Study design
The CF1R study is a nationwide, population-based, longitudinal, non-interventional, and a combination of
retrospective and prospective observational cohort clinical study. The study is performed on
genetically-confirmed FSHD1 patients or families (with at least 1 affected member) accrued through Fujian
Neuromedical Center (FNMC); it is a diagnosis center for clinical-genetic FSHD1 in China, the only one
employing PFGE-based Southern blotting, providing genetic tests for all suspected clinical FSHD1
patients/families.
3.3 Study population
To be eligible for inclusion in the CF1R study, participants must meet the following criteria:

Male or female subjects of all ages at baseline living in China.

Subjects with or without symptoms of FSHD, and genetic confirmation through PFGE-based Southern
blotting: presenting at least one contracted D4Z4 macrosatellite repeats with 4qA-specific
FSHD1-permissive haplotype.[6-8, 19]
Any patient meeting any of the following exclusion criteria at baseline cannot be included in the CF1R:

Subjects or their legally designated representative are inability or unwillingness to provide informed
consent.

Subjects have severe complications.

Subjects cannot cooperate with examination and follow-up.
3.4 Recruitment and screening
Participants will be recruited non-selectively and consecutively starting in January 2001. In order to

estimate the prevalence, we will try to reach all genetically-confirmed FSHD1 in China through a very
intensive recruitment from three sources distributed across all six spatial zones in China. Genetic and
Myopathy Group (branches of the Neurology Society of the Chinese Medical Association;
https://www.cma.org.cn/) as well as “FSHD-China” (an organization supported by FSHD patients;
http://www.fshd-china.org/), two of the three sources, will provide clinically suspected participants with
FSHD as far as possible to FNMC, the third source, to access genetic test using PFGE-based Southern
blotting; and FNMC will directly recruits participants through outpatients/inpatients to perform genetic test.
For this study, we defined clinically suspected FSHD as: clinical weakness of the facial (facio-) and/or
shoulder-upper arm (scapulo-humeral-) muscles for which the patient sought medical attention with
exclusion of other diagnoses. If the diagnosis is genetically confirmed, the patients can be included in the
study. All available data pertaining to demographics, clinical and (epi)genetic, and disease
progression/disability will be collected within the CF1R Database at FNMC, and baseline data entries are
mandated (Fig. 1).
3.5 Determination of sample size
As this is a combination of retrospective and prospective observational register of patients with
genetically-confirmed FSHD1, there is no specific sample size based on statistical considerations. Since its
initially performing PFGE-based Southern blotting in 2001, FNMC has completely finished 1,744 genetic
tests (total number tests 1,802) for clinical suspected FSHD patients/families, and 997 patients were
genetically confirmed, of whom were eligible participants appropriate to enable robust research and would
be to be conducted on the data contained within the CF1R. The first results concerning prevalence and
disease progression are expected at the end of 2021.
3.6 Patient and public involvement

Patients and the public are not directly involved in the development of the research question or in the
design of the study.
3.7 Ethics
The CF1R is run following the recommendations from the Declaration of Helsinki and has received its
Institutional Review Board approval in January 2020 (Ethics Committee of the First Affiliated Hospital of
Fujian Medical University; approval No.: MRCTA, ECFAH of FMU 20201026). All eligible patients with
genetically-confirmed FSHD1 at FNMC are provided with informed consent. Once a patient or their legal
representative have agreed to participate and signed informed consent, they are enrolled in the CF1R study.
The consent form allows for data and samples collection, future contact, for both communication and
research, to be made and for the additional data to be entered by the data entry officers. Participants are
able to withdraw their consent at any time. All data remain the property of the patient themselves and can
be withdrawn at any time.
4. Biobank management
4.1 Determination of standard operating procedure (SOP)
An additional goal of this study is to obtain a well-documented cohort for long-term follow-up and future
trial acquisition. Patients and their family members (if available) will be asked to participate in the CF1R
study and for biomaterial storage. Hence, standardized written format of policies and procedures, namely,
standard operating procedures (SOPs), must be made to ensure that all biological samples are collected,
processed, and stored uniformly in FNMC Biobank.[20, 21]The SOPs in this CF1R study is developed strictly
meeting the requirements of the Quality Assurance (QA) Program (also termed a Quality Management
System, QMS) and uniquely depending on the characteristics of FSHD1. The original version of SOPs for
CF1R study was drafted firstly by the Principal and Co-investigators; and then it was evaluated by all

members of the Research Committee (or Principal and Co-investigators), logistics company, and the
Biobank of Neurogenctic/neuromuscular Diseases established by FNMC to determine the finally suitable
SOPs.
4.2 Samples collection, processing, and storing
Samples of fresh peripheral blood cells (PBCs), serums, and muscle tissue (if muscle biopsy were performed)
are directly obtained from eligible patients/families who sought medical attention at FNMC of the First
Affiliated Hospital. To avoid degradation, fresh PBCs and serums are collected in vacutainer tube containing
K2EDTA (Changgeng, China) and BD SST tube with silica clot activator, polymer gel, silicone-coated interior
(Becton, Dickinson and Company, China), respectively.[22] 10.0ml PBCs will be drew from participants by
specialized clinician according to SOP, which does not interfere routine diagnosis and treatment, and does
not violate patient’s rights and interests.[23] For the clinical suspected patients/families living in remote
areas who choose to mail PBCs to FNMC for accessing genetic test, we will provide a specific
vacutainer tube, namely, Cell-Free DNA BCT® RUO (Streck, USA), containing anticoagulant K3EDTA and cell
preservative within liquid medium. This guarantees cellular genomic DNA preserving stability for up to 14
days at temperatures 6° C - 37° C,[24] and allows convenient transportation. Except those relatively remote
provinces and municipalities located in west China (e.g., Tibet and Xinjiang), mailing samples (10.0ml PBCs)
will arrive FNMC within 3 days.
To extracted high-quality DNA and gel DNA (high molecular weight DNA), these fresh PBCs (through
direct drawing or mailing) will be either processed immediately or within 2 to 4 hours. The latter samples
(delayed processing) must be preserved at relative low temperature at 4° C (not frozen) according to SOP.
For the 10.0ml PBCs, 6.0ml will be extracted into gel DNA (embedded in low-melting agarose gel; SOP can
be seen in Appendix materials 1); 1.0ml will be performed for routine DNA extraction (for Next Generation
Sequencing or Sanger Sequencing, and Methylation test); and the remaining 3.0ml will be stored frozen at

-80° C for prospective collection. All of the PBCs, serums, muscle tissue, DNA, and gel DNA are proactively
stored in the FNMC Biobank; the first four are stored frozen at -80° C and the last are stored refrigerated at
4℃.
4.3 Quality control and sample management
As a part of QA program, Quality Control (QC) is a system of technical activities measuring attributes and
performance of a process or item, against defined standards.[21] Various forms of QC are applied to ensure
that the both samples and related information meet requirements of investigators.[25, 26] In this study, QC
of the routinely extracted DNA is concentration and purity test, measured by Nanodrop 2000
(Thermo-scientific, USA); while QC of gel DNA will be performed during PFGE-based Southern blotting. In
addition, Research Committee (Principal and Co-investigators) will receive a QC report concerning
nonconforming samples from the FNMC Biobank, and make a decision on sample destruction.
Sample storage in our study is managed by Biobank Information Management Systems (BIMS; Haier,
China), basing on cloud platform. Accurate identification of samples is of critical importance to support
research.[23] BIMS generate an unique code attaching to different samples for whole process. This code
consists of sample type, diagnosis, and item category. Through clicking the link between sample and
Biobank, we can obtain demographic and historical medical information of participants. For samples
accessing and using, “Three Forms”, including EX-Biobank application form, IM-Biobank form of
residual sample after test, and IM-Biobank form of test data, are required. Through this, we can trace
samples trajectory with their unique code. BIMS can also records freezing and thawing time, samples
numbers, and etc. In addition, an automatic temperature monitoring management system (ATMMS; Gsino
Science&Technology, China) is utilized to continually monitor temperature of all equipment in FNMC
Biobank.[21] Alarm system within ATMMS will give an alarm and activate staff “on call” when it detect
abnormal temperature.

4.4 Genetic test (PFGE-based Southern blotting)
Laboratory SOP for PFGE-based Southern blotting are as followed.[27, 28]Five micrograms of high
molecular weight DNA (embedded in gel) were digested with enzymes of EcoRI/HindIII (or EcoRI) and
EcoRI/HindIII/BlnI (or EcoRI/BlnI) for array sizing; for analysis of the allelic variation on 4qter, DNA was
digested with HindIII only (EcoRI and HindIII were purchased from New England Biolabs, and BlnI was
purchased from Takara Bio). DNA was separated by PFGE. After blotting to an Amersham Hybond-N+
Membrane (GE Healthcare), the DNA was hybridised with probes p13E-11 (D4F104S1), 4qA (9B6A), and
4qB. After exposure, the EcoRI/HindIII (or EcoRI) array sizes were measured according to the MidRange PFG
Marker (New England Biolabs) or CHEF DNA Size Standard (Bio-Rad Laboratories). The arrays were assigned
to their respective chromosomes based on their BlnI sensitivity. The D4Z4 repeats number was calculated
from the EcoRI fragment size using the formula: (D4Z4-containing fragment length (in kb, following
EcoRI/HindIII)−4.7)/3.3 or (D4Z4-containing fragment length (in kb, following EcoRI digestion)−6.8)/3.3.[28]
4.5 Epigenetic test (Methylation)
We used the 4qA-allele-specific FasPAS primer (PAS-F 5'-GGATTTATAGGGAGGGGGTATTTTA-3'; PAS-R
5'-CTCCTAACRATCAAAAACATACCTCTATCTA-3') to investigate CpGs hypomethylation in a region distal to the
DRA with sodium bisulfite sequencing (conducted by Genesky Biotechnologies, China).[29] For individuals
carrying 4qA/B arrays, we specifically measured only one 4qA array; for individuals carrying 4qA/A arrays,
we measured the average methylation on both 4qA chromosomes.[28]
5. Data collection
5.1 Data to be collected
When genetically-confirmed FSHD1 patients are included into the CF1R study, great care would be taken to

preserve the confidentiality of patient data, with patients being referred to by individual code numbers
(e.g., FSHD_001_II1; 001 stands for family number and II1 stands for algebra). Both retrospective data [past
medical history] and data available at the time of consent [baseline visit] are collected. Prospective data
collected post-baseline at all routine clinical visits through outpatient service or telephone survey/remote
video conferences by the same neurologists should be added periodically (at least annually) to the CF1R
Database. As no investigations or data capture outside of routine clinical practice are mandated, only
information which is available to the treating physician within this framework is recorded within the CF1R.
A summary of the time-points at which specific data is collected is presented in Table 1, alongside
the full list of agreed potential data fields and dictionaries contained within the CF1R (Table 2).
Briefly speaking, clinical assessments were structured according to our previous study. The clinical
phenotypes were assessed by the 2016 Comprehensive Clinical Evaluation Form (CCEF) for FSHD.[30]
Muscle strength was assessed bilaterally by manual muscle testing (MMT) in 14 muscle pairs (deltoid,
triceps, biceps, wrist extensor, wrist flexor, finger extensor, finger flexor, gluteus maximus, gluteus medius,
iliopsoas, hamstring, quadriceps femoris, tibialis anterior and gastrocnemius muscle); and we calculated
the Medical Research Council (MRC) scale sum score by summing all of the average scores of each pair of
muscles.[31] Muscle strength was also evaluated by FSHD clinical score.[32] Both the clinical severity score
(CSS) and the age-corrected clinical severity score (ACSS) were adopted to determine disease severity; the
ACSS is adjusted for the patient’s age at diagnosis: ((CSS×2)/age at diagnosis)×1000.[33, 34] Motor function
was assessed to record the maximum distance walked (in meters) in 6 minutes (6MWT).[35, 36]
Evaluation of disease progression/disability is mainly focusing on independent ambulation, which is
measured by a simple 6-point assessment of modified Rankin Scale (mRS) due to its satisfactory reliability,
only explicit criterion of walking, and idiosyncratic criteria to raters.[37] To date, longitudinal data has been
up to 20 years (2001-2020) available, where the CF1R captured hard endpoints on onset age at first
independent ambulation loss/or even first using of wheelchair. In addition, this CF1R Database can be

extended for future research.
5.2 Data collection tools and data management
The CF1R Database is a stand-alone and diseases-specific system, developed with Microsoft SQL Server
database server. FNMC of the First Affiliated Hospital of Fujian Medical University manages the CF1R
Database through a single online portal (https://168.2.5.202:9110) with an established Research
Committee, including the Principal and Co-investigators, the data entry officers, the data custodian, the
database program manager, and the data inspector. It approves and supervises projects that utilise the data
held by the CF1R Database. The responsibilities include:

Generate an investigation plan and ensure the accuracy, completeness, and legibility of paper case
record form (CRF) (Principal and Co-investigators).

Enter data from paper CRF into eCRF (the data entry officers).

Oversee the confirmation, further investigation, and review of variables outliers, as required (the data
inspector).

Ensure timeliness of the data reported in the eCRF, post eCRF, and lock and export data (the database
program manager).

Data analysis and interpretation (the data custodian).
Patients’ information is firstly collected in a purposely designed paper CRF. The CF1R Database can
check data entered into eCRF automatically using logical checks (e.g., limits set within the database
program). Additional requests for confirming or modifying questioned data may be generated through the
eCRF, and the Principal investigator will be obliged to respond. Gene test report of PFGE-based Southern
blotting is uploaded via a word/pdf secure file transfer process within the Database. Any corrections will
overwrite the previous, initial information, and an audit trail allowing identification of any modifications
will be maintained. A medical coding plan will specify the processes and the dictionaries used for coding. All

data coding will be done using internationally recognized and accepted dictionaries.
The web interface is housed on an IE Web Server at the First Affiliated Hospital of Fujian Medical
University site. The CF1R Database is stored on file servers maintained by information department of the
First Affiliated Hospital of Fujian Medical University. This department is responsible for the security of all
hospital network servers, including firewalls, virus checking, network and workstation access passwords,
and backup and disaster recovery. In addition to these security layers, the FNMC further safeguards data
privacy by requiring individual application passwords and restricting access of confidential data to only
those Research Committee members with a direct need. Individual identifiers are limited to a single data
table with limited access by named registry personnel only.
The underlying source documents (paper CRF, informed consent, medical records, and etc.) are
similarly protected. They are locked in file cabinets in secured areas at FNMC. File folders are stored in
sample ID number order (rather than by name) to further protect them from unauthorized access.
5.3 Management and reporting of adverse events/adverse reactions
We will record details of adverse event reports associated with any treatment for FSHD. Adverse events will
be coded with the Medical Dictionary for Regulatory Activities system and described by organ class.
Cumulative adverse event information will be reported in the CF1R’s interim and final analyses.
6. Statistical methods
All patients enrolled in the CF1R study are included in the data analysis set. Analysis as well as generation
of plots and visualizations will be performed using SPSS software (version 25; IBM; USA) and GraphPad
Prism (version 7; GraphPad Software, USA). Missing data will be addressed by deletion or through using of
the last observation carried forward, determined on a case-specific basis. All continuous variables are
described using standard statistical measures (e.g., number of observations, mean, standard deviation,

median, range, and minimum and maximum). All categorical data is summarized in frequency tables.
Patient demographic data and other baseline characteristics are all treated as described above. Since
this study is population-based, one of primary objectives is to estimate prevalence that is calculated using
the number of genetically-confirmed FSHD1 as the numerator, and the denominator is always set to the
2010 population census for China (1,332,810,869 persons; http://www.stats.gov.cn). Detailed prevalence
data stratified by spatial zones, and provinces and municipalities are tabulated. The Kolmogorov-Smirnov
test or Shapiro-Wilk’s test is used to assess the normality of the variables. For normally distributed variables,
group differences will be assessed by independent two-tailed Student’s t-test or analysis of variance. For
variables that are not normally distributed, the Mann-Whitney U or Kruskal-Wall H test will be used, as
appropriate, or the data will be log-transformed for parametric testing. Categorical variables will be
compared for the groups using the Chi-squared test (Fisher’s exact test when the expected value is < 5).
Single variable analysis is implemented to analyze the correlations between exposures, possible
confounding conditions, and outcomes. Cumulative disability curves were used to assess disease
progression with time span as independent ambulation loss since first-ever muscle weakness. A p value <
0.05 was considered statistically significant. Bonferroni correction will be applied to adjust for multiple
comparisons.
7. Discussion
In the area of rare diseases, this China FSHD1 Registry (CF1R) is an invaluable source of collecting
disease-specific, real-world data on epidemiological information, disease progression/disability, natural
history, and clinical and (epi)genetic information which are vital for adequate symptomatic management
and clinical trial-readiness.
To date, 1,744 genetic tests had been completely finished in FNMC (total tests number 1,802), and 997
(57.2%) patients were genetically confirmed with FSHD1. The data of this large number of enrolled patients

have been entered in the CF1R Database in a relatively short time (we have taken great care to preserve
patient and data confidentiality) for prevalence and progression estimation, and that analysis, particularly
in relation to that prediction of disease duration from first-ever muscle weakness to independent
ambulation loss or wheelchair dependent may provide effective measures to delay the onset of disability.
The CF1R allows for collection and analysis of longitudinal data, and next steps are to carry out an in depth
analysis on this and extended data, in particular looking at the biomarkers of progression.
Some factors concerning the content of the CF1R Database have to be addressed. The present CF1R
data are mostly retrospective because patients who were genetically confirmed with FSHD1 in the past and
who are still attending FNMC have also been entered in the Database. The patients entered in the Database
may be biased by regional distance from the clinic genetic center of FNMC or awareness of FSHD1. For
example, CF1R includes more patients from Fujian province (location of FNMC) than other provinces and
municipalities, especially those areas in west China. This bias will be resolved if more genetic test
equipment set up, if more medical professionals in the area of FSHD1 train, and if stronger social awareness
of FSHD1 develop. Despite these shortcomings, these data are important to alert health and political
decision-makers to the possible underestimation of rare diseases, when emphasis is put on mortality,
rather than on the prevalence of chronic diseases and disability rates.
In conclusion, CF1R Database is the first nationwide database for FSHD1 in China. It can serve several
purposes, from patient recruitment for trials to estimation of the burden of FSHD1. The final goal is to
collect uniformly information towards the global FSHD registry.
Declaration of competing interest
All authors report no conflict of interest.
Acknowledgment

We sincerely thank all participants for their contributions to this study and Professor Silvere Van der
Maarel of Leiden University Medical Center for kindly providing the p13E-11, 4qA/4qB and B31 plasmids.

Figures
Fig. 1 Flow chart of inclusion

Tables
Note: Table 2 was located in the last of the manuscript.

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Appendix materials
Appendix materials 1: SOP of gel DNA (high molecular weight DNA) extraction
1.
6ml fresh or refrigerated (within 6 hours, at 4° C) peripheral blood cells (PBCs) was put into a 25ml
centrifuge tube.
2.
Gently invert centrifuge tube upside down 10 times for mixxing.
3.
To lyse red blood cells (RBCs), the PBCs was added 18ml (triple volume of PBCs) commercial 10% Red
Cell Lysis Buffer (TIANGEN) and incubated on ice for 15 minutes.
4.
Centrifuge the mixxed liquid at 3800rpm at 4° C for 10 min, and gently discard supernatant (RBCs).
5.
Sediments (white blood cells, WBCs) was resuspend with 24ml (quadruple volume of PBCs) 10% RBC
pyrolysis buffer (155mM NH4Cl, 10mM KHCO3, 1mM EDTA; pH 7.4), and was incubated on ice for 5
minutes.
6.
Repeat step 4.
7.
Resuspend sediments with 10ml 0.01M washing buffer (0.25M EDTA and 0.75M NaCl; pH 7.4).
8.
Centrifuge the mixture at 3000rpm at 4° C for 10 minutes and gently discard the supernatant.
9.
Resuspend sediments with 10ml 0.01M PBS (phosphate buffered saline) (0.14M NaCl, 30mM KCl,
10mM Na2HPO4 and 2mM KH2PO4; pH 7.4).
10. Repeat step 8.
11. Resuspend sediments with 1ml 0.01M PBS, and transfer the mixture into a 2.0ml centrifuge tube.
12. Centrifuge the mixture (in the 2.0ml centrifuge tube) at 2000rpm at 4° C for 10 minutes and gently
discard the supernatant.
13. Melt 1.4% agarose (InCert agarose, FMC) on water bath at 60° C.
14. Resuspend final sediments with 600ul 0.01M PBS, then add 600ul of melted 1.4% agaros (10ml PBCs ≈
(14-28)×106 cells ≈100μg DNA).
15. Transfer the above mixture to a plastic mold to obtain gel DNA (0.1ml mixxted liquid ≈a piece of gel.
16. Cooling and solidification of gel DNA at 4° C at least for 30 minutes.
17. Add 1ml ESP buffer (0.5M EDTA and 0.1M sarcosine [SIGMA]; pH 9.0) to a new 2.0ml EP tube.
18. Transfer solid gel DNA to the mixxted EP tube, and add proteinase K (SIGMA Aldrich ) to a final
concentration of 2 mg/ml, incubate on water bath at 51° C for 48-72 hours (4 pieces of gel ≈1 ml ESP ≈
140 μl proteinase K).
19. Wah solid gel DNA with 10% TE buffer (10mM Tris-HCl, 0.1mM EDTA; pH 7.4) at 4° C three times, 30
minutes each time.
20. Digest residual proteinase K with adding 10ul PMSF (Beyotime) to TE buffer and incubating on water

bath at 51° C twice times, 30minutes each time.
21. Wah solid gel DNA with 10 TE buffer.
22. Store solide gel DNA in 0.5M EDTA buffer (pH 8.0) at 4° C.

Continued