Molecular genetic analysis of 1053 Danish individuals with clinical signs of familial hypercholesterolemia

The lipid disorder familial hypercholesterolemia (FH) predisposes to cardiovascular disease. With a prevalence of approximately one in 500 in the general Caucasian population, FH is one of the most frequent single-gene disorders. As the mutational spectra vary between populations, it is crucial to identify the mutations in a given population in order to implement a molecular genetic screening strategy. A total of 1053 referred individuals with clinical signs of FH were investigated, and mutations were identified in 425 individuals. Fifty-four different mutations were identified, of which 13 are novel. The five most frequent mutations accounted for 56.3% of all disease-causing mutations. The majority of the remaining mutations were of a private nature only encountered in single families. In this study, a reliable molecular genetic screening protocol was established, and the relevance of performing presymptomatic genetic analysis as part of a preventive strategy was documented. We have acquired knowledge of the mutational spectra in the Danish population and thus will be able to trace mutations in their relatives through our index cases.     

Cascade genetic screening for familial hypercholesterolemia

Familial hypercholesterolemia (FH) is caused by a mutation in the low-density lipoprotein receptor gene and is characterized by hypercholesterolemia, xanthomas, and premature coronary heart disease. Heterozygotes typically have values for total serum cholesterol in the range of 7-15 mmol/l and efficient lipid-lowering drug therapy is available. However, only approximately 20% of patients are diagnosed and less than 10% are being adequately treated. The most cost-effective strategy to diagnose patients with FH is to screen close relatives of patients already diagnosed with FH. This is referred to as cascade genetic screening. This review focuses on organization of a cascade genetic screening program for FH as well as cost-efficiency assessments, health benefits, possible adverse effects, and the screening of children. The author concludes that cascade genetic screening for FH leads to health benefits and is cost-effective without causing psychological or social damage. Accordingly, national cascade genetic screening programs for FH should be part of ordinary health care.     

Evaluation of a clinically applicable mutation screening technique for genetic diagnosis of familial hypercholesterolemia and familial defective apolipoprotein B

We have recently developed a simple mutation screening assay based on the denaturing gradient gel electrophoresis (DGGE) technique for detection of mutations in the coding and regulatory regions of the low density lipoprotein receptor (LDLR) gene and the codon 3500 region of the apolipoprotein (apo) B-100 gene leading to familial hypercholesterolemia (FH) and familial defective apo B-100 (FDB), respectively. To evaluate the assay, 14 Danish families suspected of FH were studied. In ten families, the DGGE assay detected seven different point mutations, including mutations undescribed prior to establishing the assay. In addition, in one of these ten families and in one of the remaining four families, Southern blotting detected the FH-DK3 exon 5 deletion. Based on segregation analysis and clinical data, the FH diagnosis was dubious in the remaining three families without DGGE or Southern blotting detectable mutations.
In conclusion, a simple DGGE based mutation screening assay may detect underlying mutations in most FH/FDB families, thus allowing its routine use in genetic counselling of FH-families.     

The molecular genetic basis and diagnosis of familial hypercholesterolemia in Denmark

Normal function of the hepatic low-density lipoprotein (LDL) receptor is obligate for normal levels of plasma LDL cholesterol. The LDL receptor regulates the concentration of plasma LDL cholesterol by internalizing apolipoprotein B-100- and apolipoprotein E-containing lipoproteins by receptor-mediated endocytosis. Mutations in the gene encoding the LDL receptor protein give rise to one of the most common classical autosomal dominant inherited disorders in man, familial hypercholesterolemia (FH). The estimated prevalence of heterozygous FH is 0.2% (1:500) in most populations of the world including the Danish. Worldwide, an estimated ten million people are afflicted with FH and in Denmark there are approximately 10,000 subjects with heterozygous FH. Persons with heterozygous FH are characterized by a severely elevated concentration of LDL cholesterol in plasma starting in early childhood, tendon xanthomas and a markedly increased risk of premature coronary heart disease (CHD). Adequate control of plasma LDL cholesterol levels can be achieved in most patients with heterozygous FH, and to a lesser extent in the very rare cases with homozygous FH, using combinations of diet, drug therapy and selective LDL-apheresis. So, it is very important that physicians be aware of this relatively common disorder since there is good evidence that early diagnosis and cholesterol-lowering therapy will delay or even prevent CHD in persons with FH. A large majority of these persons, however, are still not diagnosed or adequately treated. It is believed that the diagnostic abilities molecular biology has to offer will provide the impetus for correcting this situation. The aims of the studies behind the present thesis, therefore, were to obtain important knowledge about current mutation detection technology, prevalence and spectrum of LDL receptor gene mutations in Denmark, methods to evaluate pathogenicity of LDL receptor gene mutations, relationship between FH genotype-phenotype, and clinical versus DNA diagnosis in the Danish FH population. Among different relative laborious and expensive scanning methods for unknown gene mutations we have shown that the polymerase chain reaction (PCR) single-strand conformation polymorphism (SSCP) analysis is a highly efficient and sensitive technique for detection of mutations in the 18 exons including intronic splice-site sequences and the promoter region of the LDL receptor gene, reserving DNA sequencing to the exons revealing variant SSCP patterns. Southern blot analysis or long distance PCR analysis are necessary to identify large gene re-arrangements in the LDL receptor gene in FH patients in whom SSCP analysis did not reveal any smaller sequence alterations. Worldwide, about 700 different mutations in the LDL receptor gene have been reported and in the Danish FH population we have so far identified 60 different mutations localized throughout the LDL receptor gene. In certain populations a small number of mutations predominate due to founder effects. The spectrum of LDL receptor mutations in Danish FH patients is intermediate between such specific founder populations with 5 predominant mutations (W23X, W66G, W556S, 313 + 1G-->A, 1846-1G-->A) accounting for about 40-50% of FH. These frequent mutations can easily and inexpensively be tested for by specific PCR based assays using restriction enzyme cleavage. Future analysis of LDL receptor mutations in heterozygous FH subjects, therefore, should be based on the mutational spectrum present in each relevant specific subset. Most mutations in the LDL receptor gene cause the classical heterozygous form of FH, but a small proportion seem to result in mild or moderate forms of autosomal, dominantly inherited hypercholesterolemia. Differentiation between harmless sequence variations and disease-causing mutations is not always easy without additional work. We have experienced that large re-arrangements, frame-shift and nonsense mutations obviously are pathogenic, but full pathogenicity should not be ascribed to missense mutations and small in-frame deletions, e.g. the N543H and 2393del9 mutations, unless in vitro gene expression in eukaryotic cells have been studied, or to splice-site mutations, e.g. the 1592 + 5G-->A mutation, before mRNA studies in patient cells have been performed. The cumulated LDL cholesterol exposure, mainly determined by the defect LDL receptor, plays a crucial role for the clinical manifestation of FH. The phenotypic expression of homozygous FH appears to be dominated by the consequences of the LDL receptor gene mutations. In heterozygous FH, however, the underlying mutational LDL receptor type determines only to a much lesser extent, if any, the variable phenotypic expression as seen in Danish patients. Extreme low fat dietary habits or major gene interactions may influence the lipid profile and the excess cardiovascular mortality observed in heterozygous FH, whereas minor gene determinants do not seem to play any significant role. The clinical diagnosis of heterozygous FH should be based on an elevated plasma LDL cholesterol concentration above the 95th percentiles for the general population together with either the presence of tendon xanthomas or an autosomal dominant transmission of hypercholesterolemia in the family or a child with hypercholesterolemia. Our studies illustrate clearly that molecular genetics can strengthen an equivocal clinical diagnosis and assist decision-making in diagnosis and tracing family members. If demonstration of a pathogenic mutation in the LDL receptor gene fails, other causes of autosomal dominant inherited hypercholesterolemia should be sought. Familial defective apolipoprotein B (FDB) caused by the R3500Q apolipoprotein B gene mutation may mimic FH but the clinical course, however, is often milder than that seen in patients with LDL receptor gene mutations. A newly discovered third major locus at chromosome 1 may also be of future diagnostic importance although the exact gene remains to be identified. The overall molecular genetic knowledge obtained about FH in Denmark forms the basis for the implementation and use of molecular genetic diagnostics of FH in daily clinical practice.     

The use of next-generation sequencing in clinical diagnosis of familial hypercholesterolemia

PurposeFamilial hypercholesterolemia is a common Mendelian disorder associated with early-onset coronary heart disease that can be treated by cholesterol-lowering drugs. The majority of cases in the United Kingdom are currently without a molecular diagnosis, which is partly due to the cost and time associated with standard screening techniques. The main purpose of this study was to test the sensitivity and specificity of two next-generation sequencing protocols for genetic diagnosis of familial hypercholesterolemia.MethodsLibraries were prepared for next-generation sequencing by two target enrichment protocols; one using the SureSelect Target Enrichment System and the other using the PCR-based Access Array platform.ResultsIn the validation cohort, both protocols showed 100% specificity, whereas the sensitivity for short variant detection was 100% for the SureSelect Target Enrichment and 98% for the Access Array protocol. Large deletions/duplications were only detected using the SureSelect Target Enrichment protocol. In the prospective cohort, the mutation detection rate using the Access Array was highest in patients with clinically definite familial hypercholesterolemia (67%), followed by patients with possible familial hypercholesterolemia (26%).ConclusionWe have shown the potential of target enrichment methods combined with next-generation sequencing for molecular diagnosis of familial hypercholesterolemia. Adopting these assays for patients with suspected familial hypercholesterolemia could improve cost-effectiveness and increase the overall number of patients with a molecular diagnosis.Genet Med15 12, 948–957.     

Functional analysis of LDLR promoter and 5' UTR mutations in subjects with clinical diagnosis of familial hypercholesterolemia

Familial hypercholesterolemia (FH) is a dominant disorder due to mutations in the LDLR gene. Several mutations in the LDLR promoter are associated with FH. Screening of 3,705 Spanish FH patients identified 10 variants in the promoter and 5' UTR. Here, we analyse the functionality of six newly identified LDLR variants. Mutations located in the LDLR promoter regulatory elements R2 and R3 (c.-155_-150delACCCCinsTTCTGCAAACTCCTCCC, c.-136C>G, c.-140C>G, and c.-140C>T) resulted in 6 to 15% residual activity in reporter expression experiments and changes in nuclear protein binding affinity compared to wild type. No reduction was observed when cells were transfected with c.-208T, c.-88A, and c.-36G mutant fragments. Our results indicate that mutations localized in R2 and R3 are associated with hypercholesterolemia, whereas mutations outside the LDLR response elements are not a cause of FH. This data emphasizes the importance of functional analysis of variants in the LDLR promoter to determine their association with the FH phenotype.     

Evidence-based diagnosis in clinical practice: a patient with hypercholesterolemia

Evidence-based diagnosis(EBD) is a method for clinical problem solving. The practice of EBD comprises five steps(formulating answerable clinical questions, searching for the best available evidence, critical appraisal of the evidence, application of the evidence to the patients, evaluation of execution of steps 1-4) as well as EBM. I present a case for studying the practice of EBD. The patient is a woman with hypercholesterolemia. It is essential to rule out secondary hypercholesterolemia. A test with high sensitivity is useful to rule out diseases. We call this situation SnNout(a High Sensitivity, a Negative result rules out diagnosis). The level of TSH is SnNout to rule out hypothyroidism with hypercholesterolemia. Clinical score is not SnNout, but may be useful in actual practice. The delay of relaxing phase of tendon reflex is not useful because of low sensitivity. Each clinical findings has a sensitivity and a specificity. It is important for clinicians to consider this fact.     

Homozygous familial hypercholesterolemia in China: Genetic and clinical characteristics from a real-world,multi-center,cohort study

BackgroundThere is a lack of large-scale data on the clinical and genotype characteristics of homozygous familial hypercholesterolemia (HoFH) patients in Asia.ObjectiveTo define the characteristics of phenotypic and genetic HoFH probands from mainland China.MethodsWe collected data from patients with suspected HoFH from ten clinical hospitals across mainland China from 2003 to 2019. Clinical data and DNA testing were obtained in all patients. The Kaplan-Meier method was used to generate survival curves, and the groups were compared with the log-rank test.ResultsA total of 108 unrelated probands with suspected HoFH (mean age 14.9 years) were included. The three most common variants were W483X (c.1448 G>A), A627T (c.1879 G>A), H583Y (c.1747 C>T). The majority (64.8%) were compound heterozygotes (n = 70), 23 (21.3%) were true HoFH patients. True HoFH showed higher LDL-C levels compared to compound HoFH (16.8±3.6 mmol/L vs. 15.0±3.1 mmol/L, P = 0.022). During follow-up, only 21.2% patients exhibited an LDL-C reduction of more than 50%. Kaplan-Meier analysis showed that the true HoFH probands had significantly worse survival rates compared to other genotype probands (13-year survival; 20.3% vs. 76.7%, respectively; P = 0.016). In addition, true HoFH shows that 2.8-fold (P = 0.022) increase any death and 3.0-fold (P = 0.023) increase cardiovascular death risk in relative to other FH.ConclusionsThis report shows that HoFH has devastating consequences, and that patients are often only diagnosed after they have been exposed to severely elevated LDL-C for years. Systematic screening and early intensive treatment are an absolute requirement for these young individuals with HoFH.     

Genetic backgrounds and diagnosis of familial hypercholesterolemia

Lipid disorders play a critical role in the intricate development of atherosclerosis and its clinical consequences, such as coronary heart disease and stroke. These disorders are responsible for a significant number of deaths in many adult populations worldwide. Familial hypercholesterolemia (FH) is a genetic disorder that causes extremely high levels of LDL cholesterol. The most common mutations occur in genes responsible for low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), or proprotein convertase subtilisin/kexin type 9 (PCSK9). While genetic testing is a dependable method for diagnosing the disease, it may not detect primary mutations in 20%–40% of FH cases.     

Intrafamilial variability in the clinical expression of familial hypercholesterolemia: importance of risk factor determination for genetic counselling

Kotze MJ, Davis HJ, Bissbort S, Langenhoven E, Brusnicky J, Oosthuizen CJJ. Intrafamilial variability in the clinical expression of familial hypercholesterolemia: importance of risk factor determination for genetic counselling.
Clin Genet 1993: 43: 295–299. © Munksgaard, 1993
A specific mutation in the low-density lipoprotein receptor (LDLR) gene causes familial hypercholesterolemia (FH) in about 60% of Afrikaner FH heterozygotes. Molecular diagnosis of this so-called FH Afrikaner-1 mutation was performed in a family with the disease. One individual did not develop coronary heart disease (CHD) by age 84, despite having the FH Afrikaner-1 mutation, while his son who inherited the same gene, developed CHD before age 50 and had to undergo bypass surgery. All the sibs in the third generation inherited the defective LDLR gene allele. This variation in clinical presentation creates a counselling dilemma. It also raises questions about the effect of diet and life style, and the possibility of other genes either contributing to the severity of the disease, or protecting against high lipid levels in plasma. An investigation of the influence of selected factors on the clinical expression of the FH Afrikaner-1 mutation in this family indicated that it was especially the elevated apolipoprotein (a) levels, in addition to low levels of high density lipoprotein cholesterol and raised triglyceride and apolipoprotein B levels, that were associated with a greater risk of developing CHD. These findings are thus in accordance with the view that the severity of CHD in FH patients is not only determined by the nature of the gene defect, but is also influenced by other risk factors.     

建立鼠胚着床前诊断模型的初步研究

目前严重的先天出生缺陷影响与威胁着１３．０７‰的新生儿，其中大部分是遗传病。产前诊断作为预防出生缺陷的手段对优生起着重要作用。但也不能忽视妊娠早期作出诊断，尤其是采用人工授精（ａｒｔｉｆｉｃｉａｌｉｎｓｅｍｉｎａｔｉｏｎ，ＡＩ）辅助妊娠的夫妇。国外在...     

Prenatal diagnosis of homozygous familial hypercholesterolemia: Investigation of a case at risk

Cultivated amnion cells obtained from a pregnancy at risk for the homozygous form of familial hypercholesterolemia were analyzed, as were fibroblasts from normal, heterozygous and homozygous controls. Three different methods were employed in order to compare their diagnostic value: i. Acetate incorporation into the cellular 3beta-OH-sterol fraction; ii. LDL-binding to the cell surface receptor; and iii. Oleate incorporation into the cholesterylester pool of the cells after addition of LDL to lipoprotein-deficient growth medium. The best discrimination between normal, heterozygous and homozygous cells was achieved using the third technique. On the basis of the acetate incorporation analysis, we concluded that the child is not homozygous, but probably completely unaffected. This diagnosis was confirmed by repeated determinations of plasma cholesterol levels during the first 11 months of life. Our investigations further substantiate the specularion that prenatal diagnosis of this disorder is possible.     

Mutational analysis in UK patients with a clinical diagnosis of familial hypercholesterolaemia: relationship with plasma lipid traits, heart disease risk and utility in relative tracing

As part of a randomised trial [Genetic Risk Assessment for Familial Hypercholesterolaemia (FH) Trial] of the psychological consequences of DNA-based and non-DNA-based diagnosis of FH, 338 probands with a clinical diagnosis of FH (46% with tendon xanthomas) were recruited. In the DNA-based testing arm (245 probands), using single-strand conformation polymorphism of all exons of the low-density lipoprotein receptor (LDLR) gene, 48 different pathogenic mutations were found in 62 probands (25%), while 7 (2.9%) of the patients had the R3500Q mutation in the apolipoprotein B (APOB) gene. Compared to those with no detected mutation, mean untreated cholesterol levels in those with the APOB mutation were similar, while in those with an LDLR mutation levels were significantly higher (None=9.15±1.62 vs LDLR=9.13±1.16 vs APOB=10.26±2.07 mmol/l p<0.001, respectively). Thirty seven percent of the detected mutations were in exon 3/4 of LDLR, and this group had significantly higher untreated cholesterol than those with other LDLR mutations (11.71±2.39 mmol/l vs 9.88±2.44 mmol/l, p=0.03), and more evidence of coronary disease compared to those with other LDLR or APOB mutations (36 vs 13% p=0.04). Of the probands with a detected mutation, 54 first-degree relatives were identified, of whom 27 (50%) had a mutation. Of these, 18 had untreated cholesterol above the 95th percentile for their age and gender, but there was overlap with levels in the non-carrier relatives such that 12% of subjects would have been incorrectly diagnosed on lipid levels alone. In the non-DNA-based testing arm (82 probands) only 19 of the 74 relatives identified had untreated cholesterol above the 95th percentile for their age and gender, which was significantly lower (p<0.0005) than the 50% expected for monogenic inheritance. These data confirm the genetic heterogeneity of LDLR mutations in the UK and the deleterious effect of mutations in exon 3 or 4 of LDLR on receptor function, lipids and severity of coronary heart disease. In patients with a clinical diagnosis of FH but no detectable mutation, there is weaker evidence for a monogenic cause compared with relatives of probands with LDLR mutations. This supports the usefulness of DNA testing to confirm diagnosis of FH for the treatment of hyperlipidaemia and for further cascade screening.     

Common genetic variants do not associate with CAD in familial hypercholesterolemia

In recent years, multiple loci dispersed on the genome have been shown to be associated with coronary artery disease (CAD). We investigated whether these common genetic variants also hold value for CAD prediction in a large cohort of patients with familial hypercholesterolemia (FH). We genotyped a total of 41 single-nucleotide polymorphisms (SNPs) in 1701 FH patients, of whom 482 patients (28.3%) had at least one coronary event during an average follow up of 66 years. The association of each SNP with event-free survival time was calculated with a Cox proportional hazard model. In the cardiovascular disease risk factor adjusted analysis, the most significant SNP was rs1122608:G>T in the SMARCA4 gene near the LDL-receptor (LDLR) gene, with a hazard ratio for CAD risk of 0.74 (95% CI 0.49–0.99; P-value 0.021). However, none of the SNPs reached the Bonferroni threshold. Of all the known CAD loci analyzed, the SMARCA4 locus near the LDLR had the strongest negative association with CAD in this high-risk FH cohort. The effect is contrary to what was expected. None of the other loci showed association with CAD.     

Genetic diagnosis of familial hypercholesterolemia in Han Chinese

Familial hypercholesterolemia (FH) is an inherited autosomal dominant disorder of lipoprotein metabolism resulting in elevated serum levels of low-density lipoprotein cholesterol (LDL-C), which lead to increased risk for premature cardiovascular disease. The recognized cause is mutations of the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), or proprotein convertase subtilisin/kexin type 9 genes. This study reviewed the literature in Han Chinese to investigate the frequency and spectrum of mutations that are recognized by molecular genetics as causes of FH, the clinical characteristics, and mutation detection rates of FH. MEDLINE, EMBASE, BIOSIS, Wanfang, CNKI, and FH websites, were reviewed through December 2014. Sixty-six studies met inclusion criteria. Totally, 143 different LDLR mutations were identified, including 134 point mutations and 9 large rearrangements; functional characteristics of 46 point mutations were studied. The 5 most frequent mutations included APOB 10579C>T, LDLR 986G>A, 1747C>T, 1879G>A, and 268G>A. Most of these mutations were reported in Southeast China, Hong Kong, and Taiwan. DNA detection rates of heterozygous FH were 6.5% to 77.5%, depending on the inclusion criteria and chosen screening method. With the economic growth and Western-like diet patterns being adopted over the past decade in municipalities in mainland China and Taiwan, the mean pretreatment concentration of LDL-C is higher among heterozygous FH patients reported since 2005 than in patients reported before 2005 (231 vs 196 mg/dL, P < .001). This review of DNA data for Han Chinese patients with FH updates the frequency and spectrum of FH scenarios. Large-scale investigations are needed to determine the interactions between mutations and LDL-C level in relation to cardiovascular risk assessment and management.