The VRONI study is part of DigiMed Bayern, a pilot project in predictive, preventive, personalized and participatory (P4) medicine in Germany, funded by the Bavarian State Ministry of Health and Care. For establishing the VRONI screening program several fundamental factors were considered, including lessons learned from similar trials, logistics and infrastructure, ethical, political and legal requirements, general acceptance and practicability at the doctor’s office, governance, data security and privacy, sustainability, and cost efficiency.(15-19) VRONI was designed as a proof of concept study, providing a population-based screening program for FH in children in Bavaria. The overall conceptual overview of the VRONI study is provided in the Graphic Abstract. Further information can be found on www.myVRONI.de.
Screening and recruitment
Germany offers a pediatric screening program comprising structured preventive medical examinations during childhood and youth – several with mandatory character. Such examinations are conducted by pediatricians in their out-patient offices. However, LDL-C measurements are not part of this routine. To increase gain in efficiency, acceptance and practicability, VRONI was included in already established pediatric examinations.
Upon contract with VRONI, the Bavarian Professional Association of Pediatricians (BVKJ) invited all pediatric doctors to enroll individuals. Necessary laboratory materials and documents were provided from the VRONI main office at the Deutsches Herzzentrum München (DHM). Participating pediatricians offer voluntary enrollment in the context of German preventive examinations (U9, U10, U11, and J1) or as part of any patient visit between 5 and 14 years of age (Figure 1). The child and the parents or legal guardians receive VRONI specific information material and the attending pediatrician also explains the study and answers arising questions. After written informed consent is obtained, a blood sample of the child as well as relevant baseline data and the family history are collected. Filled-in questionnaires and blood samples are sent by mail to the VRONI main office for further evaluation.
Data protection and IT infrastructure
Due to the sensitive nature of medical and genetic data, the primary objective is data security and privacy, particularly regarding identification of personal data. The only document with participant identifying data is the signed consent form, stored separately in a secured cabinet. All other documents and samples only use a unique, randomly generated, 5-character alphanumeric pseudonym. Prefabricated pseudonym barcode labels are used to enable identification of both medical data and blood samples.
The VRONI database was carefully developed in an iterative process based on a modular design and allows collection, processing, and integration of large amounts of disparate data. To maximize process security and efficiency as well as output, it includes the use of various IT technologies including statistical and analytical tools, including machine learning and knowledge management systems.
Research data is collected according to the specifications of the Technology and Methods Platform for networked medical research registered association (TMF) guidelines 2017 for data protection in medical research projects including two-stage pseudonymization. All analytical process steps are thus performed exclusively on pseudonymized data. Furthermore, the IT architecture and platform allow data flow monitoring at various stages and simplify the upkeep of appropriate quality standards. For external access to data, GDPR-compliant (General Data Protection Regulation in its German version) concepts for the exchange of data and samples have been established. The VRONI database and infrastructure is hosted in a secure container environment on university servers of the DHM in strict adherence with all data safety protocols and regulatory requirements. The data protection and security concept were established in concordance with the responsible data protection authorities and local ethics committees and reviewed positively. Beyond compliance necessity, processes and documents were pro-actively and iteratively checked with the Bavarian State Data Protection Officer for advice to secure optimal procedures.
Screening for individuals at risk
EDTA blood samples (200 µl capillary or 1.2 ml venous blood) are sent to the screening center at the DHM by mail. LDL-C measurement is done at the Institute of Laboratory Medicine of the DHM with a quantitative homogeneous enzyme colorimetric method. After centrifugation (2.800 x g, 15 min, 20°C), LDL-C measurement of K3 EDTA plasma (80 µl) is performed by using the LDLC3 test (Roche Diagnostics GmbH, Mannheim, Germany) with the cobas c 501 instrument (Roche Diagnostics GmbH, Mannheim, Germany)
At the same time as the sample is processed, the quality of the measured LDL-C result is assessed by determination of the indices of icterus, hemolysis, and lipemia using the Serum Index Gene 2 test (Roche Diagnostics GmbH, Mannheim, Germany). In case of invalid serum indices, a second sample is required by the VRONI main office at the DHM.
In case of insufficient sample volume 40 µl K3 EDTA plasma is diluted 1:2 with 0.9 % sodium chloride solution (B. Braun Melsungen AG, Melsungen, Germany) before measuring the LDL-C concentration. The suitability of the method was validated previously (data not shown). In this case, the determination of the serum indices is not possible. For samples providing less than 40 µl K3 EDTA plasma a second sample is requested by the VRONI main office.
LDL-C results undergo established quality control procedures and subsequently transmitted to the VRONI main office. After LDL-C determination the residual blood clot is resuspended in 100 µl 1 x phosphate-buffered saline (Carl Roth GmbH + Co. KG, Karlsruhe, Germany), transferred to a 300 µl 2D code FluidX Cryo Tube (Brooks, Manchester, United Kingdom), and stored at -80°C until potential genetic analysis. Blood clots from samples with an LDL-C ≥ 130 mg/dl are sent to the Institute of Neurogenomics, Helmholtz Zentrum München (HMGU) for genetic analysis.
In case of clinical suspicion, guidelines recommend to confirm FH genetically by the detection of causative variants.(7) Despite providing an unambiguous diagnosis of FH, the genetic confirmation of FH further has the potential to improve patient management and identify at-risk first-degree relatives through cascade screening.(20)
Cases exceeding the LDL-C threshold of >130 mg/dl (>3.34 mmol/l) undergo genetic investigation at the Helmholtz Zentrum München and the Institute of Human Genetics (IHG) at the Technische Universität München (TUM). Sequencing is performed on a NovaSeq 6000 (Illumina, CA USA) at the HMGU. A targeted NGS-panel (TWIST Bioscience, CA USA), based on DNA from the cellular fraction of the initial capillary blood sample, is utilized for genetic testing. This customized FH-panel contains the exonic regions of 23 genes involved in the lipid metabolism (Supplemental Table 1). In particular, the entire genomic region of the LDLR, APOB and PCSK9 are sequenced, including promoter and intronic regions, excluding repetitive intronic regions, allowing the detection of potentially disease relevant non-coding variants. Sequencing reads are mapped to human genome build GRCh37/hg19. For the analysis and clinical interpretation of sequencing data the genomic database ClinVar (21) and the Exome Variant Annotation Database (EVAdb) at the TUM are utilized. GnomAD is applied for the frequency examination of variants.(22) FH is primarily defined as an elevated LDL-C level (>130 mg/dl or >3.34 mmol/l) in conjunction with pathogenic variants in the LDLR, APOB, PCSK9 and LDLRAP1 genes. Genetic variants are identified either by classification as “likely pathogenic” or “pathogenic” in ClinVar and an allele frequency below 0.1 %, or by a loss-of-function mutation (i.e. stop mutation, frameshift mutation, canonical splice shift mutation or large deletion) according to the American College of Medical Genetics and Genomics 2015 guideline.(23)
Despite the progress made in NGS methodologies, a known causative mutation can only be detected in 60-80% of patients with clinically definite or probable FH.(8) Reasons for this could be variants in novel disease genes, epigenetic mechanisms, secondary cause of disease or most importantly a polygenic background. An increased polygenic risk for FH results from small additive effects of a number of single nucleotide polymorphisms (SNPs) located along the whole genome.
Thus, chip-based genotyping, based on DNA from the cellular fraction of the initial capillary blood sample, is conducted concomitantly to assess the polygenic risk score in all patients with elevated LDL-C levels. A commonly used screening array (GSA, Illumina, CA USA) is used for this purpose.
A scientific genetic report, containing the findings of known pathogenic variants in the LDLR, APOB, PCSK9 and LDLRAP1 (Low-density lipoprotein receptor adapter protein 1) genes is sent to the responsible pediatrician to inform the family about the results. On this occasion, about two to four months after enrollment, a second blood sample (2.7 ml venous blood) is obtained. This allows the replication of initial LDL-C measurements and of genetic findings, resulting in a significant reduction of accidental, undetected sample swapping. Overall, a systematic Failure Mode and Effects Analysis (FMEA) was introduced for minimizing the risk of sample swapping and false results. Along with the second blood sample, a routine follow-up in children with known pathogenic variants is performed by the pediatrician. In children without known pathogenic variants in known FH disease genes, a screening for secondary causes of hypercholesterolemia is carried out.
Experimental approach to uncover new FH mutations
Although an unambiguous diagnosis of FH is based on the detection of pathogenic variants, a causative genetic alteration can only be detected in 60-80% of patients with clinically elevated LDL-C levels. This suggests that polygenic causes, statistically adding up many weak effects, may be involved in FH disease development, in addition to cases explained by novel FH mutations in either known or unknown FH genes.(24) Furthermore, in a small fraction of FH patients, variants in the non-coding region, causing aberrant splicing and expression of lipid metabolism associated genes, have been detected.(25) A combination of total mRNA sequencing and whole exome sequencing in unsolved cases with repeatedly increased LDL-C levels is applied to identify FH-associated variants in non-coding regions or genes not yet associated with FH. Consequently, in selected cases, PAX tubes are requested for RNA isolation and venous blood samples are used for exome sequencing. Should non-coding variants be identified in cases with no available RNA sample, functional assays such as luciferase assays are employed for validation.
The growing application of next-generation sequencing (NGS) technologies in routine diagnostics facilitates the rapid identification of pathogenic variants. Currently, almost 3,000 LDLR variants have been described (ClinVar), affecting LDLR activity to various degrees. However, the pathogenicity of the great majority of LDLR variants has not been functionally studied.(26) To ensure early treatment and favorable prognosis, early and definitive diagnosis of FH is important. Functional effects of detected variants with uncertain significance (VUS) and functionally uncharacterized variants in known FH disease genes are validated in vitro using relevant cell lines (CHO-ldlA7, HepG2).(27-29)
Long-term treatment and Follow-up
Individuals with confirmed diagnosis of FH will be treated following the guidelines of the European Society of Cardiology (ESC) and European Atherosclerosis Society (EAS).(7, 8) Additionally, VRONI participants with elevated LDL-C levels can be divided into three distinct groups: participants with a known pathogenic genetic variant (Group A); participants without a known pathogenic genetic variant, but with a positive screening for secondary causes of hypercholesterolemia, e.g. obesity, (Group B); and participants with neither a known pathogenic genetic variant, nor a known secondary cause of hypercholesterolemia (Group C) (Figure 2).
For Group A quarterly follow-up visits are planned, either by the attending pediatrician or a pediatric cardiologist. In addition to the routine physical and laboratory examinations, regular ultrasound examinations of the carotid arteries (including measurements of the intima media thickness) as well as echocardiograms are recommended at baseline. Results of these follow-up visits are documented in dedicated questionnaires and sent to the VRONI main office at the DHM. Moreover, affected families are offered a FH-focused training course at a specialized training center in context of the VRONIplus study, educating in a manner suitable for children as well as providing the family with optimized tools to implement and maintain necessary lifestyle changes and pharmacotherapy (Supplemental Material).
Within the framework of a research project, the genetic analysis in VRONI only qualifies as a scientific report and does not equate legally to a valid medical diagnosis in Germany. Consequently, affected families and their pediatricians are advised to confirm the highly suspected FH diagnosis by conventional focused genotyping in accordance with the German health insurance regulations. Further, independent genetic analysis comes with the advantage of further minimizing any mix-up or analysis errors. On the other hand, patients need to consider, that in Germany a registered genetic diagnosis can discriminate affected individuals regarding future insurance contracts or pursuit of a civil service career path. Alongside the follow-up, a reverse cascade screening is strongly recommended and offered to all families in Group A, aiming to detect all affected family members (siblings/children via VRONI and parents/adults in cooperation with CaRe High, a FH Register study in Germany).(30)
All other participants with elevated LDL-C levels and no pathogenic variant associated (Group B and C) undergo a second examination, at the time of notifying the family of the so far negative genetic test result. The goal is to check for secondary causes of hypercholesterolemia and to collect data on phenotypical FH criteria. By means of a second blood sample, the elevated LDL-C levels are validated approximately two to four months after the initial LDL-C measurement, as generally recommended.(4, 31) Moreover, additional laboratory parameters to exclude secondary causes are measured and a dedicated questionnaire about secondary causes and in-depth family history is obtained.
For Group B no additional follow-ups are scheduled in the context of this study. After identification of the secondary cause for elevated LDL-C levels (e.g. obesity, hypothyroidism, nephrotic syndrome, anorexia nervosa, etc.), treatment of the primary cause is organized by the attending pediatrician. In case of obesity, we recommend referring the children to specialized obesity training centers (12 centers in Bavaria) and specific counselling centers to implement and maintain the required lifestyle and nutritional modifications (Figure 3).
In Group C each case will be reviewed individually by a board of experts – encompassing specialists in the fields of cardiology, lipidology, genetics and pediatric cardiology. Recommendations concerning follow-up visits, further diagnostics and therapy will be determined by the board of experts on a case-to-case basis. In selected cases entire exome sequencing or functional analysis will be performed. In either case a lifestyle modification is medically indicated.