This is a mixed methods evaluation study that used both the quantitative and qualitative research methods to answer complex research questions, and to understand the process and to strengthen the impact evaluation [
This is a diagnostic test accuracy study. The study was conducted at 11 Ministry of Health (MOH) primary care clinics located in the urban and suburban areas at the central administrative region of Malaysia (in the states of Selangor, Kuala Lumpur, and Putrajaya). These areas were chosen due to their commutable proximity to the Universiti Teknologi MARA (UiTM) Lipid Specialist Clinic where patients who are genetically confirmed to have FH are referred for further management and cascade screening of their family members.
To be eligible for selection, the clinics must be equipped with an electronic medical record (EMR) system, have minimum attendance of 500 patients per day, and be led by a family medicine specialist (FMS).
In total, there were 21 MOH primary care clinics (15 clinics in Selangor and 6 clinics in Kuala Lumpur and Putrajaya) that fulfilled the aforesaid selection criteria. The FMS leading these 21 clinics were invited for a briefing to introduce the study. Out of the 21 clinics, 11 clinics were interested in participating and therefore were included in the study (6 are located in Selangor and the other 5 are located in Kuala Lumpur and Putrajaya).
The site selection for this study was completed in February 2020.
Demographic characteristics of the study sites.
| Number | Clinic name | Area | Total number of populations served, n | Description of populations served | Total number of multidisciplinary team members, n |
| 1 | Precinct 18, Putrajaya | Suburban | 91,900 |
Middle to high income Majority are civil servants |
180 |
| 2 | Section 7, Shah Alam | Urban | 511,153 |
Low to middle income Majority are working in the private sector |
103 |
| 3 | Sungai Buloh | Suburban | 466,163 |
Low to middle income Majority are unemployed and self-employed |
183 |
| 4 | Jinjang | Urban | 132,272 |
Low to middle income Majority are self-employed and unemployed |
205 |
| 5 | Pandamaran | Urban | 150,000 |
Low to middle income A mixture of private sector workers and civil servants |
200 |
| 6 | Kelana Jaya | Suburban | 180,000 |
Low to middle income A mixture of private sector workers and civil servants |
171 |
| 7 | Taman Ehsan | Suburban | 78,693 |
Low to middle income A mixture of private sector workers and civil servants |
103 |
| 8 | Selayang Baru | Urban | 212,164 |
Low to middle income A mixture of private sector workers and civil servants |
150 |
| 9 | Tanglin | Urban | 400,000 |
Low to middle income A mixture of private sector workers and civil servants |
170 |
| 10 | Kuala Lumpur | Urban | 105,000 |
Middle income A mixture of private sector workers and civil servants |
217 |
| 11 | Batu Muda | Suburban | 180,000 |
Low to middle income A mixture of private sector workers and civil servants |
158 |
Patients registered at the 11 MOH primary care clinics were recruited according to the inclusion and exclusion criteria. This study included patients aged 18 years or older and who had an LDL-c level of 4.0 mmol/L or more recorded in their EMR. Those who were previously diagnosed with FH by genetic testing, were pregnant, or did not have the mental capacity to provide informed consent were excluded.
This study used the FAMCAT web-based FH Identification Tool, developed by and based at the University of Nottingham, UK. This web-based tool contains 3 index tests, namely, the FAMCAT, SB, and DLCC.
The variables entered into the FAMCAT web-based FH Identification Tool identified patients with suspected FH who were indicated to have genetic testing based on either one of the following definitions:
The FAMCAT algorithm relative risk score >1 (high probability of FH).
Clinical diagnosis of “definite” or “possible” FH by SB criteria.
DLCC score ≥6 (clinical diagnosis of “definite” or “probable” FH by DLCC).
The disease of interest is the HeFH and the reference standard for diagnosis is genetically confirmed HeFH determined by targeted NGS of pathogenic variants (PVs) in 4 FH candidate genes (ie,
Full details of the genetic testing methods are presented in Work stream 2. The laboratory staff analyzing the samples and the specialist interpreting the genetic test results were blinded to the index tests results.
Interface of the FAMCAT web-based FH Identification Tool.
Clinical diagnostic variables for the FAMCATa, SBb, and DLCCc.
| Index tests | SB criteria | DLCC | FAMCAT |
| Lipid levels |
Total cholesterol >7.5 mmol/L LDL-cd >4.9 mmol/L |
LDL-c ≥4 mmol/L |
The highest LDL-c ever measured The highest total cholesterol ever measured Triglycerides during LDL-c measurement Age during LDL-c measurement Lipid-lowering treatment during LDL-c measurement |
| Physical examination |
Tendon xanthomas |
Tendon xanthomas Corneal arcus <45 years of age |
Nil |
| Personal history |
Nil |
Personal history of premature CADe, cerebrovascular accident, or peripheral vascular disease (<55 years in men, <60 years in women) |
Personal history of premature myocardial infarction (<55 years in men, <60 years in women) Diagnosis of diabetes Diagnosis of chronic kidney disease |
| Family history |
Tendon xanthomas in a first- or second-degree relative Family history of premature myocardial infarction <60 years in a first-degree relative or <50 years in a second-degree relative Family history of elevated total cholesterol >7.5 mmol/L in a first- or second-degree relative Family history of elevated total cholesterol >6.7mmol/L in a child or siblings ≤16 years old |
A first-degree relative with tendon xanthomas or corneal arcus A first-degree relative with premature CAD, cerebrovascular accident, or peripheral vascular disease (<55 years in men, <60 years in women) A first-degree relative with LDL-c >95th percentile Children <18 years with LDL-c >95th percentile |
Family history of premature myocardial infarction (<55 years in men, <60 years in women) Family history of familial hypercholesterolemia |
aFAMCAT: Familial Hypercholesterolemia Case Ascertainment Tool.
bSB: Simon Broome.
cDLCC: Dutch Lipid Clinic Criteria.
dLDL-c: low-density lipoprotein cholesterol.
eCAD: coronary artery disease.
Patient recruitment was conducted by the research assistants (RAs) under the supervision of clinician investigators, who were the FMS. A list of patients with LDL-c levels of 4.0 mmol/L or more was extracted from the EMR. These patients were then stratified into 4 groups (≥7.0 mmol/L, 6.0-6.9 mmol/L, 5.0-5.9 mmol/L, and 4.0-4.9 mmol/L). Patients in these 4 groups were invited to participate via social media messaging service. This comprised a concise invitation message and a flyer containing information about the study. Invitations were sent to all patients in the highest LDL-c groups of 7.0 mmol/L or more and 6.0-6.9 mmol/L. Equal numbers of patients in the other 2 groups (5.0-5.9 mmol/L and 4.0-4.9 mmol/L) were invited to participate. If the patient did not respond to the invitation message sent via the social media messaging service, the RA followed up with a telephone call. Those who agreed to participate were given an appointment to be screened according to the inclusion and exclusion criteria at the primary care clinic.
At the primary care clinic, the patients received the study information sheet and informed consent form. This is available in either Malay or English language according to patients’ preference. It contains important information pertaining to the study and included background; purpose; benefit and risk; information regarding participation, including possibility of genetic testing (if indicated), study procedure, confidentiality status, and contact information. The RA gave the information verbally when the patients required further information or clarification.
Those who verbally consented to participate were screened for eligibility according to the inclusion and exclusion criteria. Screening for the eligibility criteria was conducted by the RA through structured interviewing of the patients. Those who fulfilled the eligibility criteria and agreed to participate were recruited; and written informed consent was obtained by the RA.
Patients were recruited until the sample size was achieved. Sample size calculation for this study is described later in this manuscript.
Data collection at the primary care clinics was conducted by the RA under the supervision of the clinician investigators. Prior to data collection, a training workshop for the RA and the clinician investigators was conducted to ensure standardization of study procedures. The training included physical examinations to identify tendon xanthomata and corneal arcus, and taught the participants how to use the FAMCAT web-based FH Identification Tool. At the primary care clinic, the RA also had an on-site hands-on training on how to interview patients, review the EMR, identify clinical signs of FH, and enter data into the FAMCAT web-based FH Identification Tool. During the first 2 months of the study, the clinician investigator directly observed the RA during the data collection and physical examination procedures. Further, they were trained on how to counsel patients for genetic testing.
Data were collected using the data collection form and standardized questionnaires, both of which have been translated into the Malay language. Participants were requested to fill in the sociodemographic data (age, ethnicity, education level, marital status, household income) and the Family History Questionnaire [
All of the variables that were collected on paper were double entered by the RA into a Microsoft Excel spreadsheet and the FAMCAT web-based FH Identification Tool to identify patients with suspected FH who were indicated for genetic testing. The criteria for genetic testing have previously been described in the “Study Tool and Variables” section.
Patients with suspected FH who were indicated to have genetic testing were given another set of patient information sheet and informed consent form in either Malay or English language. It contains important information pertaining to genetic testing. Participants were counseled regarding their risk of having FH, the pattern of inheritance, the genetic testing procedure, confirmation of diagnosis after the genetic testing, further management, and the need for cascade screening of family members once they are genetically diagnosed. Written informed consent was obtained from those who agreed to participate. Approximately 4 mL of venous blood sample was collected from each participant into an ethylenediaminetetraacetic acid (EDTA) tube. These samples were kept in a temperature-controlled container and were delivered to the Institute of Pathology, Laboratory and Forensic Medicine (I-PPerForM) Genetic Laboratory within 3 hours.
A previous study, in a UK population, found the detection rate for genetically confirmed FH using the FAMCAT to be 28% [
Comparison of detection rate of genetically confirmed FH between the FAMCAT, SB, and DLCC.
Sensitivity and specificity;
Area under the curve;
Positive and negative predictive values;
Diagnostic odd ratio.
Number of patients who are screened through the EMR and invited to participate.
Number and proportion of patients who disagree to participate or do not respond (out of those invited).
Number and proportion of patients who agree to participate and are given appointment to be seen at the primary care clinic (out of those invited).
Number and proportion of patients who withdraw after being given an appointment (out of those given an appointment).
Number and proportion of patients who are assessed for eligibility according to the inclusion/exclusion criteria at the primary care clinic (out of those given an appointment).
Number and proportion of patients who are excluded due to noneligibility (out of those who are assessed at the clinic).
Number and proportion of patients who are eligible, recruited, and for whom written informed consent was obtained (out of those who are assessed at the clinic).
Number and proportion of patients with complete diagnostic variables data entered into the web-based FH Identification Tool (out of those who are eligible and recruited).
Number and proportion of patients who fulfilled the genetic testing criteria (out of those who are eligible and recruited).
Number and proportion of patients who give consent and have blood sample taken for genetic analysis (out of those who fulfilled the genetic testing criteria).
Number and proportion of patients who are identified to have PV in any of the 4 FH candidate genes (out of those who have blood sample taken for genetic analysis).
Number and proportion of patients who agree to be referred to the lipid specialist for further care (out of those who have PV in any of the 4 FH candidate genes).
Flowchart of the participants screening and recruitment.
The demographics and clinical characteristics of the participants included in the study will be reported using descriptive measures, where frequencies (with percentages) will be used for categorical outcomes and either means (with SDs) or medians (with IQRs) will be used for continuous outcomes. Measures of diagnostic accuracy (detection rate, sensitivity, specificity, positive and negative predictive values, area under the curve, and diagnostic odds ratio) for each of the 3 index tests will be reported with their corresponding 95% CIs, with genetic testing as the reference standard. The diagnostic accuracy of the FAMCAT compared with SB and DLCC will be conducted using paired analyses by constructing a joint classification table and estimating the relative diagnostic odds ratio and relative likelihood ratios, with corresponding 95% CIs. Stratified analyses will be conducted to assess the impact of ethnicity (genotyping profile) and sensitivity analyses will be conducted excluding people with hypothyroidism. Process outcome measures will be reported as frequencies (with percentages).
Targeted NGS of the 4 FH candidate genes (FHCGs), namely,
The EDTA tubes containing venous blood samples were delivered in a temperature-controlled container from the primary care clinics to the I-PPerForM Genetic Laboratory within 3 hours. Once received, the whole blood was stored in a freezer at –20°C before further analysis.
The DNA extraction was conducted by an RA who was blinded to the index tests results (FAMCAT, SB, and DLCC) using standardized method [
The concentration and purity of samples were determined using the SpectraMax QuickDrop Micro-Volume Spectrophotometer (Molecular Devices). TE buffer (1 µL) was then used as the blank and 1 µL sample was pipetted onto the sample port. The reading for the concentration (ng/µL) and purity were recorded. Samples with concentration of 20 ng/µL or more and purity of A260/A280=1.70-2.00 were subjected to electrophoresis that was performed to clarify the intactness of DNA.
The Qubit dsDNA HS (High Sensitivity) Assay Kit (Thermo Fisher Scientific) was used to further assess DNA quality. Samples with concentration of 50 ng/µL or more based on the QuickDrop readings were diluted to 18-30 ng/µL in case the reading was underestimated before proceeding to Qubit quantification.
The Qubit working solution was prepared by diluting the Qubit dsDNA HS Reagent in the Qubit dsDNA HS Buffer with a ratio of 1:200. The Qubit dsDNA HS assay requires 2 standards for calibration. The standards were prepared by mixing 190 μL Qubit working solution and 10 μL of each Qubit standard. The solution was then mixed by vortexing for 2-3 seconds. For sample preparation, 2 μL of the sample was added to 198 μL of the Qubit working solution. The solution was then mixed by vortexing for 2-3 seconds. The tubes were allowed to incubate at room temperature for 2 minutes before being read by the fluorometer. The actual concentration of DNA was obtained by calculating the readings from Qubit and times with the number of dilutions.
One part of sodium acetate solution (3.0 M; pH 5.2; Sigma) and 2 parts of cold absolute EtOH were added to 10 parts of the DNA sample volume. The mixture was vortexed for 10 seconds and kept in a –70°C freezer for 20 minutes. The mixture was then transferred into a column tube and centrifuged for 10 minutes, at 14,000
The NGS [
The samples were diluted to an intermediate concentration of about 20-50 ng/µL using low TE followed by requantification using the Qubit fluorometer. The samples were then further diluted into the desired final concentration of 3.5 ng/µL using low TE.
For each sample, 7 µL of 5× AmpliSeq HiFi Mix and 10.5 µL DNA samples (3.5 ng/µL) were combined in a fresh 1.5-mL microcentrifuge tube to bring the total volume of the master mix to 17.5 µL. About 5 µL of the master mix was transferred into a 96-well PCR plate. Because of the complexity of the panel design, each sample was separated into 3 different wells and separately added with different 5 µL of 2× AmpliSeq Custom DNA Panel Pool (the primer pool). In total, each well contained 5 µL sample master mix and 5 µL primer pool for a total of 10 µL per well. The plate was properly sealed and briefly centrifuged. The plate was placed in a preprogrammed thermal cycler and run with the following settings: preheat lid option, 105°C; reaction volume, 10 µL; 99°C, 2 minutes; 19 cycles of –99°C for 15 minutes and –60°C for 4 minutes; and 10°C for up to 24 hours.
After PCR, the 3 separated products amplified from the same sample were then combined into 1 well, making the total volume per sample as 30 µL. Then, 3 µL of the FuPa reagent was added to each combined sample. The plate was sealed, briefly vortexed at 1600 rpm for 1 minute, followed by centrifugation at 280
Switch solution (6 µL), AmpliSeq CD indexes (3 µL), and finally DNA ligase (3 µL) were added sequentially into each well containing the digested amplicons (33 µL) to a total volume of 45 µL for each sample. The plate was then sealed, vortexed, centrifuged, and placed into the thermal cycler and run with the following settings: preheat lid, 105°C; reaction volume, 45 µL; 22°C for 30 minutes; 68°C for 5 minutes; 72°C for 5 minutes; and 10°C for up to 24 hours.
A total of 45 µL of AMPure XP beads were added to each library. The plate was then sealed, vortexed, centrifuged, and incubated at room temperature for 5 minutes. The plate was then placed on a magnetic stand until the mixture became clear (approximately 2 minutes). While still on the magnetic stand, the plate was unsealed, and the supernatant was carefully pipetted out from each well. The beads were washed by adding 150 µL freshly prepared 70% EtOH to each well, followed by incubation at room temperature until the solution became clear (approximately 30 seconds). Without disturbing the pellets, the supernatant was again discarded from each well. The washing step was repeated once again. The plate was sealed, briefly centrifuged, and placed on the magnetic stand in the same position as before. The plate was unsealed and all residual EtOH was removed from each well.
In a fresh 1.5 μL microcentrifuge tube, 1125 µL of 1× Lib Amp Mix was combined with 125 µL of 10× Library Amp Primers for a total of 1250 µL amplification master mix. The master mix was vortexed and centrifuged briefly. The plate was removed from the magnetic stand and 50 µL of the master mix was added to each well and the plate was sealed again, vortexed, centrifuged, and placed into the thermal cycler and run with the following settings: preheat lid, 105°C; reaction volume, 50 µL; 98°C for 2 minutes; 7 cycles of 98°C for 15 seconds and 64°C for 1 minute; and 10°C for up to 24 hours.
About 25 µL of AMPure XP beads were added to each well containing approximately 50 µL library and the plate was sealed again. The library was vortexed, centrifuged briefly, and incubated at room temperature for 5 minutes. The plate was placed on the magnetic stand for another 5 minutes until the liquid is clear and the plate was unsealed. The entire supernatant that contains the desired amplicon library (approximately 75 µL) was transferred to a new plate and 60 µL of AMPure XP beads were added to each well in the new plate. The plate was briefly vortexed, centrifuged, and incubated at room temperature. After 5 minutes, the plate was placed on the magnetic stand for another 5 minutes until the liquid cleared. The plate was unsealed. Without disturbing the beads, the supernatant was discarded from each well. The beads were washed by adding 150 µL freshly prepared 70% EtOH to each well, followed by incubation at room temperature until the solution became clear (approximately 30 seconds). Without disturbing the pellets, the supernatant was discarded from each well. The washing step was repeated once again. All residual EtOH was pipetted out, and the plate was air dried on the magnetic stand for 5 minutes. Low TE (30 µL) was added to each well and mixed well. The plate was sealed again, vortexed, and centrifuged briefly to disperse the beads. It was then placed on the magnetic stand until the liquid cleared and then unsealed. About 27 μL supernatant that contains the amplicon library was transferred to a new plate.
Each library was subjected to Qubit quantification to ensure the concentrations were approximately 1-10 ng/µL.
Each sample was diluted to 0.5-1.0 ng/µL before the bioanalyzer step. Size distribution of the library was checked using the Agilent High Sensitivity DNA Kit (Agilent Technologies). Nearly 15 mL of high-sensitivity DNA dye concentrate was added to a vial containing high-sensitivity DNA gel matrix. The mixture was vortexed and transferred to a spin filter, and subsequently centrifuged to obtain the elution. The gel-dye mix elution was allowed to equilibrate to room temperature for 30 minutes before use. A high-sensitivity DNA chip was put on the chip priming station. About 9 μL of the gel-dye mix was pipetted into the loading well on the chip. A priming plunger was used to push the gel-dye mix to the entire sample wells on the chip. About 5 μL marker was pipetted into each sample well and a ladder well. Nearly 1 μL high-sensitivity DNA ladder was also pipetted in the ladder well. Then, 1 μL sample was also pipetted into each sample well. The chip was vortexed for 1 minute at 2400 rpm, and then run in the Agilent 2100 Bioanalyzer instrument (Agilent Technologies) within 5 minutes after its preparation. Each library size was recorded in a Microsoft Excel sheet and the average library size measured was determined. The molarity of the library was calculated using the following formula [
Samples (about 5 μL) were pipetted into separate microcentrifuge tubes. EB-0.1% Tween 20 mix was pipetted into each sample tube, where the volume varies so the final molarity of each sample was 4 nM. Next, 5 µL of each library was pooled into a single microcentrifuge tube. The library pool was subjected to Qubit to confirm the 4 nM concentration.
The Illumina iSeq 100 is a benchtop high-throughput sequencer, measuring 1 ft3 in size. This sequencer is useful for sequencing small genome, transcriptome, long amplicon, and targeted resequencing applications, generating up to 1.2 Gb data per run.
The cartridge was first thawed overnight in a water bath (20-25°C) in its foil bag. When the cartridge is ready, 4 nM library pool was diluted to 50 pM loading concentration using EB. About 10 nM of PhiX (control library) was diluted to 50 pM using EB. Then, 2 µL of PhiX (50 pM) was added to 98 µL library pool (50 pM), making the final pool volume as 100 µL.
The cover of the loading reservoir on the thawed cartridge was first teared using a pipette tip. Then, 20 µL final pooled library was pipetted into the bottom of the reservoir. Flow cell was inserted into the cartridge. Then, the cartridge-flow cell assembly was inserted into the iSeq 100 tray. Run was initiated using instrument control software. The total runtime was up to 17.5 hours.
The iSeq100 run monitoring was executed using BaseSpace Sequence Hub (Illumina) cloud-based software, where GRCh37 hg19 human reference assembly [
The variant call format (VCF) files were extracted and downloaded from BaseSpace Sequence Hub. Variants with minor allele frequency of 0.05 or less, based on either the gnomAD or 1000 Genome database, were considered as rare gene mutations.
The VCF files were outsourced (BioEasy Sdn Bhd) for data cleaning and bioinformatics interpretation [
The cleaned and combined VCF files were further analyzed for variant pathogenicity using modified American College of Medical Genetics and Genomics guidelines [
The genetic analysis results for individual patients will be delivered by the clinician investigators. Patients who are genetically confirmed to have FH will be counseled regarding the nature of the genetic mutations, mode of inheritance, the need for pharmacological management, and cascade screening of first- and second-degree relatives. The importance of adherence to lifestyle modification and pharmacotherapy will also be emphasized. Lipid-lowering treatment for each patient will be reviewed and uptitrated to achieve an LDL-c target of <1.8 mmol/L [
This is a nested qualitative study performed in MOH primary care clinics. The study population included patients with suspected FH who have undergone genetic testing. Semistructured interviews and thematic analysis of data are performed.
Patients with suspected FH who have received their genetic analysis results are purposefully sampled to reflect their social, ethnic, and educational diversity, and range of results including those who are genetically confirmed to have FH and those with negative results. Written informed consent is obtained from the participants.
Semistructured in-depth interviews are conducted face-to-face or virtually using the online platform Google Meet (Google LLC/Alphabet Inc.) by an RA, supervised by a Malaysian lead qualitative co-investigator (SAR) who is an expert in qualitative study methods. A topic guide developed for the interview (
Data from the interviews will be transcribed verbatim and translated into English. Data will be organized using NVivo qualitative software (QSR International) [
This is a nested qualitative study conducted in MOH primary care clinics. The study population included PCPs working at the MOH Primary Care Clinics. The “think-aloud” interview method [
PCPs are purposefully sampled to reflect their varied lengths of clinical experience, and range of use of the FAMCAT web-based FH Identification Tool in practice. Written informed consent is obtained from the participants.
Interviews are conducted using the “think-aloud” method [
The “think-aloud” interview method generates direct data on participants’ ongoing thought processes during task performance. The underlying assumption of “think aloud” is based on human cognition where it is postulated that only information capable of being verbalized in situ is being actively processed in the working memory and so is taken as an active representation of task performance [
Video-based observational data are collected to record the participants’ interaction with the interface and functionality of the FAMCAT web-based FH Identification Tool. These data are time-locked for analysis to the verbal protocols. Following the end of the task simulations, participants complete a short questionnaire to capture a subjective measure of their user satisfaction with the interface and functionality of the FAMCAT web-based FH Identification Tool. Recruitment and data generation will continue until saturation of themes and this is expected to be achieved by interviewing approximately 10-15 participants [
Each verbal protocol will be transcribed and coded using a predefined coding scheme following the categories of interest. Similarly, the audio-video–recorded simulation will provide a range of images on all of the tasks including time to completion, types of clinical knowledge, resources accessed, participants’ statements, and the tasks associated therewith. Two qualitative researchers will independently map the statements made by participants and the video observations, identifying both “positive” and “negative” mental heuristics; the former demonstrating potential features that are acceptable to the needs of participants, and the latter to determine whether there are any possible refinements that might be made to the FH Identification Tool.
This study protocol was approved by the respective research ethics committees in Malaysia, namely, the UiTM Research Ethics Committee [(REC/03/2020) (FB/48)] and the Medical Research Ethics Committee of the Ministry of Health Malaysia [NMRR-20-272-52797 (IIR)].