Gail Norbury

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.

Non‐invasive prenatal determination of fetal sex: translating research into clinical practice

Hill M, Finning K, Martin P, Hogg J, Meaney C, Norbury G, Daniels G, Chitty LS. Non‐invasive prenatal determination of fetal sex: translating research into clinical practice.

What is the clinical utility of DNA testing in patients with familial hypercholesterolaemia

Purpose of review Familial hypercholesterolaemia is a common genetic disorder of lipid metabolism in which patients have a significantly elevated risk of early coronary heart disease, which can be substantially lowered by treatment with the statin class of drugs. In many countries in Europe, tracing of relatives using DNA information, once the family mutation has been identified, is being actively carried out. The present review examines the specificity and clinical utility of DNA testing in patients with familial hypercholesterolaemia. Recent findings Technological progress has improved the detection rate in patients with the strongest clinical suspicion of familial hypercholesterolaemia to more than 70–80%. Patients carrying a mutation have, on average, higher low-density lipoprotein cholesterol levels and greater risk of early coronary heart disease, and studies have reported the utility of DNA information in the identification of affected relatives. More than 1000 different molecular causes of familial hypercholesterolaemia are documented in the University College London database, and although more than 90% of these clearly cause familial hypercholesterolaemia, the remainder require careful interpretation. Summary DNA testing, as an adjunct to the measurement of plasma low-density lipoprotein cholesterol levels, has clinical utility in providing an unequivocal diagnosis in patients and in identifying affected relatives at an early age so that they can be offered lifestyle advice and appropriate lipid-lowering therapies. Researchers and DNA diagnostic laboratories need to interpret novel sequence changes with caution in order to avoid a false positive diagnosis.

Non-invasive prenatal diagnosis of single gene disorders: How close are we?

Analysis of cell free fetal DNA (cffDNA) in maternal plasma provides the opportunity for reliable, timely, safe and cost-effective diagnosis of single gene disorders. The detection of certain fetal loci using cffDNA and conventional molecular analytic approaches is possible from 4 weeks gestation. To date, non-invasive first-trimester analysis for single gene disorders has been limited by assay sensitivity and specificity, due to the background maternal DNA. The anticipated ability to enrich the fetal component of cell free DNA will increase the robustness of tests and permit semi-quantitative analysis, broadening the scope of testing to include recessive disorders such as cystic fibrosis. Testing for large-scale mutations might remain limited by the fragmented nature of cffDNA and, when testing very early in gestation, careful ultrasound examination will be needed to determine the number of gestational sacs, because of the risk of discordant twin pregnancies.

Carrier Frequency of a Nonsense Mutation in the Adenosine Deaminase (ADA) Gene Implies a High Incidence of ADA‐deficient Severe Combined Immunodeficiency (SCID) in Somalia and a Single, Common Haplotype Indicates Common Ancestry

Inherited adenosine deaminase (ADA) deficiency is a rare metabolic disorder that causes immunodeficiency, varying from severe combined immunodeficiency (SCID) in the majority of cases to a less severe form in a small minority of patients. Five patients of Somali origin from four unrelated families, with severe ADA‐SCID, were registered in the Greater London area. Patients and their parents were investigated for the nonsense mutation Q3X (ADA c7C>T), two missense mutations K80R (ADA c239A>G) and R142Q (ADA c425G>A), and a TAAA repeat located at the 3′ end of an Alu element (AluVpA) positioned 1.1 kb upstream of the ADA transcription start site. All patients were homozygous for the haplotype ADA‐7T/ADA‐239G/ADA‐425G/AluVpA7. Among 207 Somali immigrants to Denmark, the frequency of ADA c7C>T and the maximum likelihood estimate of the frequency of the haplotype ADA‐7T/ADA‐239G/ADA‐425G/AluVpA7 were both 0.012 (carrier frequency 2.4%). Based on the analysis of AluVpA alleles, the ADA c7C/T mutation was estimated to be approximately 7,100 years old. Approximately 1 out of 5 – 10000 Somali children will be born with ADA deficiency due to an ADA c7C/T mutation, although within certain clans the frequency may be significantly higher. ADA‐SCID may be a frequent immunodeficiency disorder in Somalia, but will be underdiagnosed due to the prevailing socioeconomic and nutritional deprivation.

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.

Noninvasive prenatal diagnosis of early onset primary dystonia I in maternal plasma

The genetic trait of a fetus may be determined early on in pregnancy using cell‐free fetal DNA extracted from the plasma of pregnant women. The challenges for noninvasive diagnosis include the variable but still low amount of cell‐free fetal DNA in the first trimester (57–761 gE/mL) and the competing high background of maternal DNA in the plasma (∼90%). Prenatal detection of a paternally inherited dystonia 3 bp deletion mutation was undertaken using cell‐free DNA (cfDNA) from the plasma of two at‐risk pregnancies. The predicted fetal genotype was subsequently confirmed in each fetus.

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