FH ALERT: A feasibility study of a novel approach to identify patients with familial hypercholesterolemia

Background and aims . Diagnosis rates of familial hypercholesterolemia (FH) remain low. We implemented FH ALERT to assess whether alerting physicians for the possibility of FH impacted additional diagnostic activity. Methods. The study was conducted from SYNLAB laboratory Weiden (Bavaria). Beyond common reporting of LDL-C or TC, 1,411 physicians covering approximately a population of 1.5 million people were eligible to receive an alert letter (AL) including information on FH, if laboratory results exceeded thresholds as follows: adults LDL-C ≥190-250 mg/dl (to convert into mmol/l multiply with 0,0259), TC ≥250 to ≤310 mg/dl (probable suspicion); LDL-C >250 mg/dl and TC >310 mg/dl (strong suspicion). Persons below 18 years were alerted for LDL-C ≥140 mg/dl and TC ≥200 mg/dl (strong suspicion). Patients above 60 years were excluded. Our readouts were characteristics of involved physicians, rate of ALs issued, acceptance, and subsequent diagnostic activity. Results were mainly 75% of the ordered tests were 25% We issued 3,512 ALs (~5% of tests). 86% of eligible physicians stayed 32.7% were and 70% were positive upon call-center survey. We registered 101 new visitors of www.fhscore.eu and sent out 93 genetics. Thereof, 26 were returned and 5 patients were positive for FH. Conclusions


Introduction
Elevated low-density lipoprotein cholesterol (LDL-C) is among the most important causes of atherosclerosis 1

. Genetic epidemiological and interventional studies have
shown a continuous relationship between LDL cholesterol and the incidence of atherosclerosis-related vascular events 1 .
Familial hypercholesterolemia (FH) is a genetic disorder of LDL-C metabolism, mainly caused by mutations in the genes encoding the LDL receptor (LDLR), apolipoprotein B100 (APOB) or proprotein convertase subtilisin/kexin type 9 (PCSK9). FH is inherited in an autosomal dominant manner, meaning that 50% of the descendants of a heterozygote parent are affected 2 . FH is the most frequent autosomal dominant genetic disease encountered in adults with a global prevalence estimated at between 1:200-1:300 and more common among populations with founder effects 3 . FH is characterized by pronounced elevations of plasma LDL-C as early as in childhood and premature onset of coronary heart disease 2 . Roughly 2 percent of individuals with an LDL-C above 190 mg/dL (to convert into mmol/l multiply with 0,0259) suffer from FH 4 .
Homozygous FH (HoFH) frequently results in cardiovascular events in the first decade of life, and left untreated those with HoFH may die before the age of 20 years 5 . In heterozygous (HeFH) patients the additional risk of cardiovascular events is 50% in men up to the age of 50 and 30% in women up to the age of 60 6 .
Despite all available data detection remains low. For instance the prevalence rate of HeFH is approximately 1:300, which entails more than 250,000 patients in Germany 6 , yet the current diagnosis rate is estimated to be between 1 to 2% 3 . Unidentified and untreated patients on average lose 15 to 21 years of life 7 . Registry data from the Netherlands have shown that an early screening strategy together with effective and early use of statin therapy to reduce LDL-C can reduce the cardiovascular risk associated with FH similar to that of the general population 8 . Thus, it is crucial that patients are identified early in life. Although, there are several clinical algorithms which help to identify patients with FH 9 10 11 , they are underutilised in clinical practice.
Moreover, these algorithms can be used to increase the pre-test probability of a positive genetic test 12 . The clinical value of genetic diagnostics of FH has been underscored by guidelines and expert consensus in the United States 13,14 , Europe 15,16 , and Germany 17 , yet, utilization of genetic testing remains poor. To facilitate the diagnosis of FH and given that this condition is dependent upon ascertainment of an elevated LDL-C measurement, we designed a pilot study (FH ALERT) to serve as the interface between a clinical laboratory receiving blood samples for lipid measurements and the ambulatory outpatient care, to assess whether a decision support protocol was feasible, acceptable, and improved detection rates compared to historical records.
Here we report the results of this pilot study to evaluate feasibility and acceptance of this approach in a German outpatient care environment.

Setting
We included all ambulatory care physicians (general practitioners and specialists across all disciplines) referring their laboratory samples to the SYNLAB Medical Care Center (MCC) Weiden GmbH which is located in Bavaria, Germany. We chose MCC Weiden GmbH because it is one of the biggest SYNLAB core laboratories in Germany. All patients aged 60 and below visiting participating physicians who were being evaluated for elevated LDL-C or total cholesterol (TC) measurements were eligible. The study was conducted between March 15, 2018 and June 15, 2018 and approved by the Ethics Committee II of the Medical Faculty Mannheim, Heidelberg University, Theodor-Kutzer-Ufer 1-3, 68167 Mannheim (reference number 2018-849R-MA). As this was a retrospective evaluation of findings generated during regular patient care and no study specific procedures were conducted, the abovementioned ethics committee did not consider it necessary to obtain individual informed consents. All methods were carried out in accordance with relevant guidelines and regulations.

Procedures
Beyond the regular laboratory results, participating physicians received an alert letter (AL) once TC or LDL-C levels exceeded predefined threshold values ( Figure 1). The ALs also included scientific information about FH and the further diagnostic options including genetic testing.
We used six different threshold values to trigger an AL (Table 1). We distinguished among two categories with different wording, namely "suspicion of FH" in adults belonging to the LDL-C threshold category I or TC threshold category I as well as "strong suspicion of FH" in children and in adults belonging to LDL-C threshold II and/or TC threshold II. (Table I). LDL-C was used as the primary criterion to trigger reports, TC was used in cases in which LDL-C was not available. We recommended the Dutch Lipid Clinic Network (DLCN) score to estimate the pre-test probability of a positive genetic test, with a test score of ≥6 indicating for genetic testing.
Approximately two weeks before initiation, we announced FH ALERT by an initiation letter to all physicians regularly referring tests to the SYNLAB MCC Weiden GmbH.
This document described background and aims, design and procedures of the project.
The initiation letter and each subsequent AL informed the participating physician that they had the choice to opt-out from the project at any time.
The software extracting TC or LDL-C from the laboratory information system to produce the ALs was written by CSMed GmbH, Darmstadt, Germany. We established a call center which was specifically instructed and available to participating physicians to provide further information on the diagnoses of FH if requested (inbound calls). The call center was operated by IMS Health GmbH & Co. OHG, Bensheim, Germany. The ALs usually arrived the day following the regular report. They included in depth information on the diagnosis of FH, advice how to use the DLCN score 11 , how to initiate genetic testing, and named lipid specialists in the surroundings of the patients´ residential address. Any medical decisions regarding further diagnostic and therapeutic measures were completely left to the discretion of the responsible physicians ( Figure 1).
Physicians who received at least one AL were contacted pro-actively by the call center and asked to assess the FH Alert project using a standardized questionnaire.
Physicians were also encouraged to express their criticism and to propose improvements of the procedures.

Genetic testing
Current guidelines and expert opinion suggest that the diagnosis of FH is established by genetic testing if accessible [13][14][15][16][17] . To achieve seamless access to genetic testing for FH, we offered physicians the facilities at the SYNLAB Center of Human Genetics in Mannheim, Germany. Interested physicians were provided with kits (including request and consent forms and material for sampling) upon request. We monitored the number of genetic tests requested during the FH ALERT project until 6.5 months after the conclusion of the project on June 15, 2018. This accounted for delays in ordering genetic tests due to waiting for the patients´ next appointments, education of patients or receiving their informed consent.
Patients´ DNA was isolated from a blood sample and then analyzed by next generation sequencing. Following library preparation with TruSight Rapid Capture technology and bridge amplification, the sample was sequenced on an Illumina NextSeq 500 instrument. Coverage of the ROI was >98%, reading depth a minimum of 100-fold. We

Characteristics of the physicians involved
During the project period 1,411 physicians/institutions ordered at least one analysis of any parameter from the SYNLAB MCC Weiden. They were mainly in ambulatory care (general practitioner or medical specialist), but also included occupational health departments of companies or health care institutions. Most letters were issued to  (Table 2).
On average, there were 2.49 ALs per physician, when calculated based on 1,411 (total participating physicians) and 7.6 ALs per physician in relation to physicians who received at least one AL. 462 of participating physicians -predominantly general practitioners and internistswere alerted and received at least one AL (32,74%). 37 medium frequently alerted physicians received 3 to 4 ALs (8.01% of alerted physicians), 48 frequently alerted physicians 5 to 9 ALs (10.39% of alerted physicians) and 121 high frequently alerted physicians were alerted 10 times or more (26.19% of alerted physicians).

Acceptance
Only 200 physicians chose to opt out from the project and 1,211 physicians participated for all 3 months, corresponding to an opt-out rate of 14,2%; 32.74% of all physicians received at least one AL (Table 2).
In the survey conducted by the call center, FH ALERT was evaluated positively by approximately 70% of respondents, 23% did not finally know how to classify and 7% had a negative opinion about FH ALERT. The most frequent criticism arose regarding to the amount of paperwork caused by ALs, followed by a lack of additional time to look through the ALs.
The FH score website was visited 101 times due to the FH ALERT project; 31 visitors recurred at least once, 88 FH score questionnaires were completed whereof 45 had a result of ≥ 3 ( Table 2).

Further diagnostic activity including genetic testing
During the FH ALERT project 93 sampling kits for genetic testing were sent out to 60 physicians and 26 were returned for analysis within the active period and 6.5 months beyond. Of these, 10 samples were inconspicuous, 3 showed variants most likely not related to hypercholesterolemia, 8 samples showed variants or combinations of variants which we classified as polygenic hypercholesterolemia, and 5 samples showed at least one variant that we deemed causal for familial hypercholesterolemia.

Discussion
We successfully established a novel channel of information exchange between the clinical laboratory and requesting physicians. We found that this approach was generally welcome to the majority of the medical community participating. However, we also observed that this poorly translated into further diagnostic stepsas far as this process was visible for us -as they are recommended by international and national expert opinion or guidelines [13][14][15][16] .
Even though TC and LDL-C are amongst the most frequently ordered tests in ambulatory care, the diagnosis rate of FH remains low in Germany, and in many other countries 3 . We started by assuming physicians commonly do not pay attention to extreme TC or LDL-C results included in conventional laboratory reports and hardly consider the diagnosis of FH, and if so, rarely or no consecutive action or further diagnostic steps follow. To enhance awareness for pathologic lipid parameters, we decided to specifically alert physicians once TC or LDL-C exceeded threshold values at which the probability of FH is deemed to be high.

Alert letters generated
Approximately 5% of all the samples tested triggered an alert. This confirms that in the Of the participating physicians, who were predominantly general practitioners and internists, 32.74% received at least one report. This suggests that a small proportion of physicians include cholesterol and/or LDL-C testing into their standard practice of care. The majority of physicians may thus waive for screening for high cholesterol and/or to conduct state of the art risk assessment as it would comply with guidelines 15,16,[21][22][23] . However, at least theoretically there may be physicians caring for patients with well controlled LDL levels or not being at elevated risk according to previous screening. As a consequence of the clustering of ALs to one third of the physicians only it may be worth limiting FH ALERT specifically to general practitioners and internists who regularly order TC and/or LDL-C in the future. In Germany, nephrologists and dialysis centres often provide care for patients with lipid disorders. However, they did not substantially contribute to the total number of ALs, on the one hand because they are small in number, on the other hand because their lipid patients may be well controlled ( Figure 2). Only few ALs originated from paediatricians, indicating that TC and/or LDL cholesterol are infrequently measured in patients < 18 years and that opportunities to identify FH at a young age are vastly missed in Germany. Thus, it appears that the single initiation letter that we issued was not sufficient to provide a broad awareness of the significance of LDL-C. Other reasons to waive cholesterol testing may be that it has been disputed that the cost-effectiveness of systematic risk factor screening in the absence of vascular disease is still a matter of debate 21 or that lipid lowering therapy would not immediately have been considered in a given patient.

Acceptance
Amongst all participating physicians, 200 (14%) decided to opt out. We believe that this discontinuation rate is reasonable since we offered a hitherto completely unknown service. As the most common reason to withdraw physicians quoted that the ALs produced too much additional paperwork and due to this often lead to lacking time in their practice to even read individual ALs. Other withdrawing physicians said that it would need too much time to follow the advice provided with the ALs, disregarding that a clear diagnosis could facilitate treatment decisions and patients´ adherence in the future and might therefore allow time savings alongside. Refinement of the FH ALERT strategy in the future will therefore have to resolve this criticism, for instance by cumulating results within one weekly report. However, of the physicians who finished the pilot study almost more than two thirds assessed the initiative as positive, and only less than 10 % had a negative opinion about the FH ALERT project.

Further diagnostic activity including genetic testing
The 462 physicians who received an alert ordered 93 sampling kits for genetic testing, of which 26 (27.96%) were ultimately returned for genetic testing. We recorded the number of genetic tests requested until 6.5 months after the conclusion of FH ALERT, but evidently the time between ordering kits and actually carrying out the test may be longer so that actual number of tests may yet be slightly higher.
We noted 101 new visitors on the www.fhscore.eu website. Under the assumption that there was an overlap between the 101 new visitors on the website and the 60 physicians who ordered kits for genetic testing, it may be reasonable and conservative to assume that 120 physicians amongst the 462 who had received an AL responded in any of the two ways to the FH ALERT initiative. Thus, the overall response rate may be 25% or more. Since to our knowledge the design of FH ALERT is unprecedented, a comparison to established bench marks is hard to accomplish. In any case, however, the response rates achieved here by far exceed the response rates known from untargeted mailings which are usually 1% or less.
Of the 26 requests for genetic testing 10 were inconspicuous, 3 showed variants most likely not related to hypercholesterolemia, 8 samples showed variants or combinations of variants which we classified as polygenic and 5 (19%) showed at least one variant that we deemed causal for familial hypercholesterolemia. This may be less than obtained in specialized lipid clinics, where we found causal mutations in approximately 80% of patients with a DLCN Score of 6 or more 12 . Further, the 5 true FH cases make up approximately 2% only of the number of patients that would theoretically have FH in our study (see above). Even if one would still account for the cases in which we considered the genetic findings as polygenic hypercholesterolemia, this would increase to no more than 5%. Overall, genetic testing for FH was thus taken up poorly, although the environment for cascade screening, which is recommended as costeffective for identifying FH patients and preventing vascular disease (reviewed in 14  However, it is unlikely that the patients´ attitudes would seriously compromise the use of genetic testing. Rather parents of affected children or patients have been open to genetic testing recognizing its benefit with little evidence for psychological impact [24][25][26][27][28][29] .
Consequently, we consider proactive and comprehensive education of physicians of utmost importance to lead them up to a better understanding of the benefits of genetic testing. This is evidently not only true for genetic lipid disorders but applies to all disease areas in which genetic disease becomes symptomatic in adulthood.

Future directions
3,512 ALs ultimately lead to 26 genetic tests only. Despite this, we consider our pilot study successful, mainly because significant other diagnostic activity (visits to FH score website) developed during the project period. Also the high proportion of physicians rating FH positive has been encouraging and we are convinced that our novel strategy of communication between referrers and the laboratory holds considerable potential for the future.
Based on our experience, conceivable improvements are: i) reducing the amount of paperwork by consolidating individual alert letters to weekly cumulated alert letters; ii) generating increased awareness for TC/LDL-C earlier before starting the initiative by educational events and multiple initiation letters; iii) including medical societies and key opinion leaders in awareness activities; iv)increasing the frequency of calls by the call center to remind physicians to perform genetic testing and send back the genetics kits for analyses; v) yearly repetition of the initiative.

Limitations
Limitations of this project are the exclusion of samples from patients above 60 years of age and the thresholds for ALs at the 95 th percentile of the TC and LDL-C distribution. The latter obviously will miss some FH patients (also including the treated ones), because having an LDL-C of less than 190 mg/dl does not rule out FH 4 . Further, we did not include samples coming from hospitals to which FH patients may present with acute coronary syndromes. The pilot study was restricted to three months only and to a rural area of Germany and may therefore not be representative. It is also a limitation that it was hard to place our findings into the context of similar studies because to the best of our knowledge there is no published report systematically evaluating our approach.

Conclusions
We conclude that bridging the interface between referring physicians and the laboratory by an innovative approach of communication is technically demanding, but feasible. Basically, our initiative was welcome and highly appreciated by the participating physicians. However, even individualized recommendations as they were provided with FH ALERT were not rigorously translated into concrete action. Therapeutics.

Financial support
The initiative FH ALERT was financed equally by SYNLAB Holding Deutschland GmbH and Amgen GmbH.