Alison Taylor

Mutation detection rate and spectrum in familial hypercholesterolaemia patients in the UK pilot cascade project

Taylor A, Wang D, Patel K, Whittall R, Wood G, Farrer M, Neely RDG, Fairgrieve S, Nair D, Barbir M, Jones JL, Egan S, Everdale R, Lolin Y, Hughes E, Cooper JA, Hadfield SG, Norbury G, Humphries SE. Mutation detection rate and spectrum in familial hypercholesterolaemia patients in the UK pilot cascade project.

Multiplex ARMS analysis to detect 13 common mutations in familial hypercholesterolaemia

DNA analysis and mutation identification is useful for the diagnosis of familial hypercholesterolaemia (FH), particularly in the young and in other situations where clinical diagnosis may be difficult, and enables unambiguous identification of at‐risk relatives. Mutation screening of the whole of the three FH‐causing genes is costly and time consuming. We have tested the specificity and sensitivity of a recently developed multiplex amplification refractory mutation system assay of 11 low‐density lipoprotein receptor gene (LDLR) mutations, one APOB (p.R3527Q) and one PCSK9 (p.D374Y) mutation in 400 patients attending 10 UK lipid clinics. The kit detected a mutation in 54 (14%) patients, and a complete screen of the LDLR gene using single‐stranded conformation polymorphism/denaturing high performance liquid chromatography identified 59 different mutations (11 novel) in an additional 87 patients, for an overall detection rate of 35%. The kit correctly identified 38% of all detected mutations by the full screen, with no false‐positive or false‐negative results. In the patients with a clinical diagnosis of definite FH, the overall detection rate was higher (54/110 = 49%), with the kit detecting 52% of the full‐screen mutations. Results can be obtained within a week of sample receipt, and the high detection rate and good specificity make this a useful initial DNA diagnostic test for UK patients.

Multiplex ligation‐dependent probe amplification analysis to screen for deletions and duplications of the LDLR gene in patients with familial hypercholesterolaemia

The most common genetic defect in patients with autosomal dominant hypercholesterolaemia is a mutation of the low‐density lipoprotein receptor (LDLR) gene. An estimate of the frequency of major rearrangements has been limited by the availability of an effective analytical method and testing of large cohorts. We present data from a cohort of 611 patients referred with suspected heterozygous familial hypercholesterolaemia (FH) from five UK lipid clinics, who were initially screened for point mutations in LDLR and the common APOB and PCSK9 mutations. The 377 cases in whom no mutation was found were then screened for large rearrangements by multiplex ligation‐dependent probe amplification (MLPA) analysis. A rearrangement was identified in 19 patients. This represents 7.5% of the total detected mutations of the cohort. Of these, the majority of mutations (12/19) were deletions of more than one exon, two were duplications of more than one exon and five were single exon deletions that need interpreting with care. Five rearrangements (26%) are previously unreported. We conclude that MLPA analysis is a simple and rapid method for detecting large rearrangements and should be included in diagnostic genetic testing for FH.

A functional mutation in the LDLR promoter (-139C > G) in a patient with familial hypercholesterolemia

A novel sequence change in repeat 3 of the promoter of the low-density lipoprotein receptor (LDLR) gene, −139C>G, has been identified in a patient with familial hypercholesterolemia (FH). LDLR -139G has been passed to one offspring who also shows an FH phenotype. Transient transfection studies using luciferase gene reporter assays revealed a considerable reduction (74±1.4% SEM) in reporter gene expression from the −139G variant sequence compared to the wild-type sequence, strongly suggesting that this change is the basis for FH in these patients. Analysis using electrophoretic mobility shift assay demonstrated the loss of Sp1 binding to the variant sequence in vitro, explaining the reduction of transcription.

Mutation screening in patients for familial hypercholesterolaemia (ADH)

To the Editor : The UK NICE guidelines (1) recommend DNA testing for patients with a clinical diagnosis of familial hypercholesterolaemia (FH). Cost effective screening strategies are needed to achieve this, given the population prevalence of around 1/500 (2) with approximately 75% undiagnosed cases in UK (3). Furthermore, there are least three genes involved (LDLR, APOB and PCSK9 ) (4) with no specific criteria to differentiate between the underlying defect, and there may be clinical difficulty in distinguishing between FH and familial combined hyperlipidaemia, thereby adding to the potential number of patients referred for testing (5). Here, we describe a comprehensive stepwise screening strategy, based on a study of 110 patients, which can be used as a rapid, cost-effective and efficient method to screen for mutations in patients with FH in the UK population. DNA was extracted from blood using standard methodology (6). The Tepnel Elucigene FH20 ARMs kit was used as an initial screen for 20 relatively common mutations (7, 8). This was followed by direct sequence analysis of the promoter region and 18 exons of the LDLR gene and multiplex ligation-dependent probe amplification (MLPA) to identify larger LDLR rearrangements (9). Table 1 shows the sequence and location of the primers used together with the universal tails that simplify subsequent sequence analysis. Polymerase chain reaction set up, clean up, sequencing and purification was carried out using Beckman robots and standard conditions for all 20 amplicons to maximise efficiency. Data was analysed using Mutation Surveyor software. MLPA was performed using the Salsa P062 LDLR kit from MRCHolland, and data analysed using GeneMarker software (v 1.4 Soft Genetics). The patients were referred from either adult or paediatric lipid clinics in the UK and classified as definite familial hypercholesterolaemia (DFH, n = 19) or possible familial hypercholesterolaemia (PFH, n = 91) using the Simon Broome criteria (10). Overall a mutation was detected in 43 patients, with a detection rate of 63.2% in DFH and 34% in PFH (see Fig. 1). At each stage of the diagnostic algorithm, the proportion of the final number of mutations detected was similar between DFH and PFH subjects (using an X2 test, p = 0.58), and this data is shown (as percentages) in the figure. This suggests that this order of tests is equally valid in subjects with both PFH and DFH. The distribution of detected mutations over the 18 LDLR exons was similar to that reported previously (11, Supporting Information Table S1). The APOB mutation was the most common single mutation, detected in four patients (10% of total) with two other LDLR mutations (p.Val429Met and p.Cys184Tyr) identified in three patients each (7% of total): all of these mutations were in the FH20 Arms kit. Of the 43 different mutations present in this sample, only p.Thr491AsnfsX45 and p.Leu611PhefsX5 were previously unreported. As each cause a frameshift and a truncated protein they are both highly likely to be pathogenic. Two additional sequence changes of unknown significance were identified, in exon 8 c.1066G > C(p.Asp356Asn) and in exon 17c.2479G > A (p.Val827Ile). Using computer prediction algorithms as described (11), p.Asp356Asn was designated “Benign” by POLYPHEN and ‘Tolerated’ by both the SIFT and refined SIFT (11). By contrast, p.Val827Ile was designated as ‘Benign’ by POLYPHEN but ‘Not tolerated’ by SIFT. Neither of these patients had any other sequence change suspected to be of pathogenic significance, and these changes were not detected in any of the other patients, and as a result they do not appear to be common in the UK. In spite of the high sensitivity of the methods used we were unable to detect a mutation in all of the patients investigated, which has been consistently reported in other studies (12–14). In the majority of cases this may be due to misdiagnosis using clinical and blood cholesterol criteria, although, given the genetic heterogeneity of ADH (4), some patients may have a mutation in a

A double heterozygote for familial hypercholesterolaemia and familial defective apolipoprotein B-100

Autosomal dominant hypercholesterolaemia is genetically heterogeneous, but most commonly (∼93%) caused by mutations in low-density lipoprotein receptor (LDLR), where the disease is known as familial hypercholesterolaemia (FH), or apolipoprotein B-100 (APOB) (∼5.5%), where the disease is known as familial defective APOB (FDB), while in ∼2% of patients the mutation is in the proprotein convertase subtilisin/kexin type 9 gene. Homozygous FH having inheritance of two LDLR mutations is a rare but recognized syndrome associated with an extreme hypercholesterolaemia and early-onset coronary artery disease. We present a 15-year-old girl with untreated total cholesterol levels of 8.8 mmol/L who was heterozygous for both the LDLR p.Leu479Pro and APOB p.Arg3527Gln mutation. Cascade testing confirmed the paternal origin of the LDLR mutation and revealed a maternal diagnosis of FDB. This case provides further evidence that the combined effect of an LDLR and an APOB mutation give rise to a phenotype more severe than either mutation alone and is more severe than homozygous FDB, but less severe than homozygous FH. It also highlights the need to consider the presence of additional mutations in families where relatives have varying phenotypes.

Functional analysis of four LDLR 5′UTR and promoter variants in patients with familial hypercholesterolaemia

Familial hypercholesterolaemia (FH) is an autosomal dominant inherited disease characterised by increased low-density lipoprotein cholesterol (LDL-C) levels. The functionality of four novel variants within the LDLR 5′UTR and promoter located at c.-13A>G, c.-101T>C, c.-121T>C and c.-215A>G was investigated using in silico and in vitro assays, and a systemic bioinformatics analysis of all 36 reported promoter variants are presented. Bioinformatic tools predicted that all four variants occurred in sites likely to bind transcription factors and that binding was altered by the variant allele. Luciferase assay was performed for all the variants. Compared with wild type, the c.-101T>C and c.-121T>C variants showed significantly lower mean (±SD) luciferase activity (64±8 and 72±8%, all P<0.001), suggesting that these variants are causal of the FH phenotype. No significant effect on gene expression was seen for the c.-13A>G or c.-215A>G variants (96±15 and 100±12%), suggesting these variants are not FH causing. Similar results were seen for the c.-101T>C and c.-121T>C variants in lipid-depleted serum. However, a significant reduction in luciferase activity was seen in the c.-215A>G variant in lipid-depleted serum. Electrophoretic-mobility shift assays identified allele-specific binding of liver (hepatoma) nuclear proteins to c.-121T>C and suggestive differential binding to c.-101T>C but no binding to c.-215A>G. These data highlight the importance of in vitro testing of reported LDLR promoter variants to establish their role in FH. The functional assays performed suggest that the c.-101T>C and c.-121T>C variants are pathogenic, whereas c.-13A>G variant is benign, and the status of c.-215A>G remains unclear.

Homozygous hypercholesterolaemia and ezetimibe: a case report

A girl of Indian origin presented with unusual nodules on her hands, and total cholesterol was found to be >25 mmol/L. The girl had “mild” P664L mutation and total cholesterol levels fell by 38% when she was on a diet and statin therapy. A further reduction of 26% in total cholesterol and 37% in low‐density lipoprotein (LDL) was achieved by adding ezetimibe to the treatment.