Family Cascade Screening Research Articles

Novel Mutation in the KCNJ2 Gene Is Associated with a Malignant Arrhythmic Phenotype of Andersen‐Tawil Syndrome

Andersen-Tawil syndrome (ATS) is a rare inherited multisystem disorder associated with mutations in KCNJ2 and low prevalence of life-threatening ventricular arrhythmias. Our aim was to describe the clinical course of ATS in a family, in which the proband survived aborted cardiac arrest (ACA) and genetic screening revealed a previously unknown mutation (c.271_282del12[p.Ala91_Leu94del]) in the KCNJ2 gene. A cascade family screening was performed in a 5-generation family after identification of the KCNJ2 mutation in the proband. Subsequently, 10 of 21 screened individuals appeared to be mutation carriers (median age 38 [range 10-75] years, 3 female). Mutation carriers underwent clinical examination including biochemistry panel, cardiac ultrasound, Holter ECG, and exercise stress test. (1) At baseline, 2 patients had survived ACA, 3 had syncope or presyncopal attacks, and 2 reported palpitations. Exercise-induced nonsustained bidirectional ventricular tachycardia was documented in 4 patients, 2 received implantable cardioverter-defibrillators (ICD) for primary prevention and 2 for secondary prevention. (2) During follow-up, 1 primary prevention and 1 secondary prevention patient received in total 4 adequate ICD shocks. Life-threatening ventricular arrhythmias were documented during childhood in 5 of 10 mutation carriers. (3) All mutation carriers presented with characteristic mild dysmorphic features. Only 1 patient suffered from periodic paralysis. All had normal serum potassium level at repeated assessments and none had any other extracardiac disease manifestation. Our findings suggest that the novel KCNJ2 mutation is associated with a predominantly cardiac phenotype of Andersen-Tawil syndrome with high propensity to life-threatening ventricular arrhythmias presenting from childhood and young adulthood.

Screening methods in the diagnosis and assessment of children and adolescents with familial hypercholesterolemia

Familial hypercholesterolemia is an autosomal dominant disorder. Heterozygous familial hypercholesterolemia (FH) has an estimated incidence of 1 per 300–500 births and is characterized by increased serum total- and low-density lipoprotein-cholesterol levels and an increased risk of coronary heart disease. Early diagnosis and cholesterol-lowering treatment are essential to prevent premature coronary heart disease. Effective screening strategies are therefore of great importance. Screening can be done as selective or general population screening. There is no generally accepted screening program for FH in children and adolescents. In The Netherlands a systematic genetic family cascade screening program has been going on since 1994. In most countries there is no systematic screening for the disease, but clinical and genetic family cascade screening has been applied. Selective screening programs have failed to identify a large number of FH cases. Recommendations for general pediatric population screening have therefore emerged. Controversy exists as to which approach should be adopted. Family cascade screening has been estimated to be the most cost-effective screening method. No general pediatric screening program has been tested on a larger scale, validated or subjected to cost–benefit analyses.

Identification and biochemical analysis of a novel APOB mutation that causes autosomal dominant hypercholesterolemia.

Patients with autosomal dominant hypercholesterolemia (ADH) have a high risk of developing cardiovascular disease that can be effectively treated using statin drugs. Molecular diagnosis and family cascade screening is recommended for early identification of individuals at risk, but up to 40% of families have no mutation detected in known genes. This study combined linkage analysis and exome sequencing to identify a novel variant in exon 3 of APOB (Arg50Trp). Mass spectrometry established that low-density lipoprotein (LDL) containing Arg50Trp APOB accumulates in the circulation of affected individuals, suggesting defective hepatic uptake. Previously reported mutations in APOB causing ADH have been located in exon 26. This is the first report of a mutation outside this region causing this phenotype, therefore, more extensive screening of this large and highly polymorphic gene may be necessary in ADH families. This is now feasible due to the high capacity of recently available sequencing platforms.

1 Targeted next generation sequencing (NGS) of the CFTR locus: Comparison of three technical approaches

Twenty-three years after the discovery of the CFTR gene, more than 1900 anomalies are described in the world, the majority of them being single base-pair substitutions or micro-insertions/deletions. Identification of mutations has important implications for genetic counselling, prenatal diagnosis, cascade screening in families, as well as for understanding the genotype–phenotype relationship. Since a few years, NGS technologies enable us to overcome the classical approaches of whole coding sequence sequencing at single nucleotide resolution. The aim of our study is to compare 3 different strategies with the Ion Torrent technology (Life Technologies, LT): Ampliseq (LT), Haloplex (Agilent) and homemade Long Range PCR (LR). Preliminary results showed that the coverage of the CFTR locus by Ampliseq is limited to 51% and includes 86% of exons with a depth generally consistent between amplicons. Haloplex approach gives more heterogeneous depth and coverage. Finally, LR-PCR showed coverage consistent with the design but differences at depth between each LR amplification. The development of libraries by the two first approaches is rapid (less than 24 h) and technically easy. For LR-PCR, It takes about two days to build a library with long and time-consuming manual steps. Sequencing result for the CFTR gene were validated with wild-type samples and DNA carrying known variants (mutations and polymorphisms) previously identified by DHPLC, HRM and sequencing. In conclusion, the sequencing of the entire CFTR locus by NGS is a tool that can quickly respond to issues such as prenatal diagnostic and the most promising approach seems to be the LR strategy.

2 New insights in CF molecular diagnostics applying the next generation sequencing technologies

Twenty-three years after the discovery of the CFTR gene, more than 1900 anomalies are described in the world, the majority of them being single base-pair substitutions or micro-insertions/deletions. Identification of mutations has important implications for genetic counselling, prenatal diagnosis, cascade screening in families, as well as for understanding the genotype–phenotype relationship. Since a few years, NGS technologies enable us to overcome the classical approaches of whole coding sequence sequencing at single nucleotide resolution. The aim of our study is to compare 3 different strategies with the Ion Torrent technology (Life Technologies, LT): Ampliseq (LT), Haloplex (Agilent) and homemade Long Range PCR (LR). Preliminary results showed that the coverage of the CFTR locus by Ampliseq is limited to 51% and includes 86% of exons with a depth generally consistent between amplicons. Haloplex approach gives more heterogeneous depth and coverage. Finally, LR-PCR showed coverage consistent with the design but differences at depth between each LR amplification. The development of libraries by the two first approaches is rapid (less than 24 h) and technically easy. For LR-PCR, It takes about two days to build a library with long and time-consuming manual steps. Sequencing result for the CFTR gene were validated with wild-type samples and DNA carrying known variants (mutations and polymorphisms) previously identified by DHPLC, HRM and sequencing. In conclusion, the sequencing of the entire CFTR locus by NGS is a tool that can quickly respond to issues such as prenatal diagnostic and the most promising approach seems to be the LR strategy.

Familial hypercholesterolaemia – are we testing everyone we should?

Fewer than 5% of the expected cases of familial hypercholester-olaemia (FH) in Australia have been appropriately identified. FH, which is a potent cause of premature cardiovascular disease, is very responsive to cost-effective treatments. This suggests that relevant diagnostic tests are grossly under-utilised. This review will consider diagnostic options for the detection of FH and the role that these tests could play in an effective preventive strategy. On one hand, the specificity of routine lipid levels such as low-density lipoprotein cholesterol, is poor, particularly in later adulthood. Conversely, positive molecular genetic techniques are diagnostic, but the yield is strongly dependent on pre-test probability. An Australian Model of Care¹ and clinical tools such as the Dutch Lipid Clinic Score² can assist the identification of index cases. This is the critical step in a highly efficient strategy of case detection known as Family Cascade Screening. It will be argued that molecular genetic testing for causative mutations should always be accompanied by a commitment to follow up affected families with Family Cascade Screening. In this sense, genetic diagnosis is as much a management tool as it is a diagnostic test.

WENDS-FH Morocco: The Wendish Emigration National Detection Study of FH Morocco in Familial Hypercholesterolaemia: Results of Cascade Family Screening

WENDS-FH Morocco: The Wendish Emigration National Detection Study of FH Morocco in Familial Hypercholesterolaemia: Results of Cascade Family Screening

Community detection of long QT syndrome with a clinical registry: An alternative to ECG screening programs?

Long QT syndrome (LQTS) prevalence is estimated at 4 of 10,000 based on community electrocardiogram (ECG) screening, about which there is disagreement regarding efficacy, accuracy, cost-effectiveness, and practicality. Family studies of autosomal dominant conditions such as LQTS have revealed 8-9 gene-positive family members per proband. To evaluate a cardiac/genetic registry and family screening program as a tool to identify LQTS in the community. Possible LQTS probands were referred to the New Zealand Cardiac Inherited Disease service. The registry was first established in the northern region (population 2.03 million), including central Auckland (population 0.46 million). After clinical evaluation, genetic testing and family cascade screening were initiated. Genotype-positive individuals were classified as definite LQTS, and others were classified as definite or probable LQTS by clinical and ECG criteria. One hundred twelve probands were identified (presentation: 7 sudden death, 82 cardiac event, 16 ECG abnormality, and 7 sudden death of a family member). Following cascade screening, 309 patients with LQTS were identified (248 definite and 61 probable). Two hundred twenty patients had LQTS-causing mutations identified (120 [55%] LQT1, 78 [35%] LQT2, 19 [9%] LQT3, 1 [0.5%] LQT 5, and 2 [1%] LQT7). Thus far, an average of 2.1 definitely or probably affected family members have been identified per proband. The community detection rate is 1.5 of 10,000 for the whole region and 2.2 of 10,000 in Auckland. A high level of community detection of LQTS is possible using a clinical registry. With adequate resourcing, this has the potential to be an effective alternative to community ECG screening.

Should all individuals with a nondiagnostic Brugada-electrocardiogram undergo sodium-channel blocker test?

The diagnostic Brugada-electrocardiogram (Br-ECG) is characterized by "coved-type" ST-segment elevation (type 1) in V(1) to V(2)/V(3). The sodium-channel blocker test is clinically used to unmask diagnostic Br-ECG in patients with nondiagnostic "saddle-back" Br-ECG (type 2 and type 3). To assess the prognostic value of the sodium-channel blockers test in individuals with a nondiagnostic Br-ECG. We studied 153 consecutive patients (128 men; age 41.7 ± 14.0 years) with a type 2/3 Br-ECG who underwent a sodium-channel blocker test with either flecainide (48%) or ajmaline (52%). Nondiagnostic Br-ECGs were identified during the evaluation of cardiac arrest in 5 patients (3%), syncope in 36 (24%), cascade family screening in 48 (31%), and incidental ECG in 64 (42%). A spontaneous type 1 Br-ECG was systematically excluded by serial ECGs (6.1 ± 0.4) and recording of right precordial leads both at standard and second and third intercostal spaces. The sodium-channel blocker test result was positive in 76 (50%) patients. During a follow-up of 59 ± 33 months, 9 (5.9%) patients experienced events such as syncope (n = 4), appropriate interventions of defibrillator (n = 4), or sudden death (n = 1). A positive sodium-channel blocker test was associated with a significantly higher event rate in symptomatic patients (P = .01) but not in asymptomatic individuals (P = .18). No events occurred among asymptomatic individuals with an incidental nondiagnostic Br-ECG. In asymptomatic individuals with a nondiagnostic Br-ECG, the incidence of events is low regardless of the sodium-channel blocker test result while in symptomatic patients a positive sodium-channel blocker test result is associated with an adverse arrhythmic outcome and may contribute to risk stratification.

Screening for hereditary haemochromatosis

Hereditary haemochromatosis (HH) is a common autosomal recessive disorder of iron overload in Caucasian populations. Clinical manifestations usually occur in individuals homozygous for the C282Y mutation in the HFE gene product and who have developed significant iron loading. Current screening methods can detect affected individuals either prior to or early during disease evolution, enabling early introduction of phlebotomy treatment that can normalise life expectancy. Evaluation of possible iron overload, via measurement of serum transferrin saturation and ferritin level, is the most appropriate initial test for those subjects presenting clinically for evaluation. HFE genotyping, when combined with serum biochemical measurements, defines the presence of likely iron overload and the underlying genetic disorder and is the preferred initial screening modality for families of an affected individual. Definitive proof of iron overload requires measurement of hepatic iron concentration or total iron burden via therapeutic phlebotomy; elevated serum ferritin level alone is not adequate. We now recognise that the natural history of HH is not as discrete as previously believed, because genetic and environmental modifiers of disease penetrance are increasingly identified as influencing the clinical expression of HH. In fact, a minority of C282Y homozygotes develop classical 'iron overload disease', although it has recently emerged that the disorder may predispose to breast and colorectal cancer. Uncertainties as to the true clinical impact of the condition at a population level lead to current recommendations of cascade screening of families of affected patients, case-finding in high-risk groups, such as patients with clinical manifestations consistent with the diagnosis, and a high level of clinical awareness in the community to facilitate early diagnosis. Generalised population screening is not presently recommended.

Patients' experiences and views of cascade screening for familial hypercholesterolemia (FH): a qualitative study

Familial DNA cascade screening for familial hypercholesterolemia (FH) has recently been introduced in Scotland. This study investigated index patients' experiences of DNA testing and mediating cascade screening. Thirty-eight patients with a clinical diagnosis of definite or possible FH who had undergone DNA testing in the lipid clinic took part in semi-structured qualitative interviews. All patients were positive about DNA screening being undertaken by familiar and trusted clinicians within the lipid clinic. Most patients had already cascaded close relatives for serum cholesterol testing following their attendance at the lipid clinic. Identified mutation carriers who had attended the genetics clinic (n = 15) for a cascading appointment described finding this consultation helpful because it identified other at-risk family members and provided them with tailored information for their relatives. Participants who expressed a preference said they favoured indirect (patient-mediated) methods of cascading as they considered indirect approaches to be less threatening to family members than direct clinical contact. We conclude that DNA screening and indirect familial cascading is perceived as highly acceptable to index patients with FH. However, while indirect cascading methods may be more acceptable to patients, they do not yield the same numbers as more direct methods. There is, therefore, a need for further systematic research to investigate patients', family members' and staff views of the acceptability of direct versus indirect methods of cascade screening.

Interventional Treatments for Hypertrophic Cardiomyopathy

Hypertrophic cardiomyopathy (HCM) is the most common inherited cardiac disorder. This autosomal dominant condition is defined by left ventricular hypertrophy and associated with functional limitation and premature death. In fact, many individuals are asymptomatic and the annual mortality in most modern series is 1% or less. However, severe symptoms may develop at any age, and the risk of premature death from arrhythmia, stroke, and progressive systolic impairment may complicate asymptomatic disease. The clinical management of patients with HCM therefore encompasses (1) genetic counseling including discussion of indications for genetic testing and cascade family screening, (2) assessment of prognostic risk from ventricular arrhythmia, stroke, and heart failure, and (3) symptom management. This article describes the interventional treatments in the management of severe symptoms associated with left ventricular outflow tract obstruction (LVOTO).

How Effective is Cascade Screening for Familial Hypercholesterolaemia? Preliminary Report from the FHWA Program

Introduction: Familial hypercholesterolaemia (FH) is the most common monogenic cause of premature coronary artery disease. However, the majority of FH cases remain undiagnosed and untreated. Cascade screening of families may be the most cost-effective model for identifying new cases but no experience has hitherto been reported in Australia.

Long QT testing: implications for complex diagnosis in personalized medicine.

compound heterozygous [6,7]. The existence of many rare poly morphisms (and genetic modifiers) in the involved genes further complicates the genetic assessment of LQTS patients [8]. Many disease-causing mutations result in changes in the function of ion channels, for example, gating properties, and require highly specialized electrophysiological ana lysis for their characterization [1]. Such an ana lysis is not readily available and certainly not within a reasonable timeframe. Finally, small family sizes often preclude a certain assessment of disease segregation with the mutation in the pedigree. In reality, most of the mutations described in the literature as ‘disease causing’ have not been confirmed to be so in sensu stricto. For the individual patient, several benefits can be noted if a disease-causing mutation can be found. First, prognostic information can occasionally be gained from knowing the affected gene [9] or the location of the mutation within a specific gene [10]. Second, if the person decides to have children they may be analyzed genetically at birth, or for that matter prenatally, and necessary prophylactic treatment can be instituted prior to occurrence of symptoms, reducing the need for extensive clinical investigations in early childhood. Third, knowledge of the gene involved may also aid in the identification of behavioral risk factors and enable a more precise recommendation of lifestyle changes. Mutations in some genes have been demonstrated to be associated with specific arrhythmia-precipitating factors, for example, physical activity in KCNQ1 mutations and emotional distress in KCNH2 mutations [1]. Thus, the identification of a disease-causing mutation may obviate the need for indiscriminate banning of physical activities. Finally, as the effect of treatment differs between affected genes and mutations in some genes – for example, SCN5A – exhibit complex phenotypes, genotyping may assist in defining optimal treatment or suggest further clinical testing [11]. The latter Long QT syndrome (LQTS) is a cardiac condition characterized by prolonged QT time on the ECG, representing delayed cardiac repolarization and a propensity for ventricular tachycardia. The disease has an estimated prevalence of 1 in 2500 and a clinical presentation that includes sudden death and syncope. Mutations in 12 genes have been demonstrated to cause the disease (LQTS1–12); all the genes code for either cardiac ion channels or proteins interacting with these channels [1]. Presently, LQTS genetic testing, performed by most laboratories, provides comprehensive open reading frame/splice site mutational ana lysis of the five most frequently affected LQTS-causing genes, that is, KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2, via high-throughput DNA sequencing [2,3]. A disease-causing mutation can be identified in 50–70% of symptomatic LQTS cases and cascade screening is routinely performed in affected families [2,3]. This is important as 10% of LQTS gene carriers are asymptomatic and at risk of sudden death [4]; LQTS has been estimated to be responsible for up to 10% of sudden infant death syndrome and 5% of sudden adult death syndrome cases [5]. There is no doubt that the real evidence-based value of LQTS testing at present relates to cascade screening in families of people with symptomatic disease, exemplified by sudden death cases with an identified presumed disease-causing mutation. However, it should be noted that the diagnosis of LQTS in most cases is a clinical one – genetic testing can not replace careful clinical examination as a negative mutation finding does not rule out the condition. The genetics of LQTS is complicated, which is exemplified by the problems in ascertaining the clinical significance of novel mutations. These problems are not merely a consequence of allelic heterogeneity, but also due to the fact that most identified mutations are private, that is, specific for the examined family [1] and approximately 5% of LQTS cases are

Identifying patients with familial hypercholesterolaemia in primary care: an informatics-based approach in one primary care centre

Background:Familial hypercholesterolaemia (FH) is associated with highly raised low-density lipoprotein-cholesterol and causes early-onset cardiovascular disease. Its autosomal dominant inheritance allows family cascade screening to be performed once an index case...

The Clinical Relevance of Compound Heterozygosity for the C282Y and H63D Substitutions in Hemochromatosis

Two major mutations are defined within the hemochromatosis gene, HFE. Although the effects of the C282Y substitution have been well characterized, the clinical significance of the C282Y/H63D state remains unclear. This study assessed the phenotypic expression in C282Y/H63D subjects as compared with C282Y homozygotes. Data were obtained from 91 C282Y/H63D probands, 158 C282Y/H63D subjects identified through family screening, and 483 C282Y homozygotes. Subjects underwent clinical evaluation, genotyping, biochemical assessment, and liver biopsy examination where clinically indicated. C282Y/H63D probands had significantly less clinical and biochemical expression than C282Y homozygotes. Biochemical expression was higher in C282Y/H63D probands than in C282Y/H63D subjects identified through family screening (P < .001). Of the C282Y/H63D subjects with serum ferritin levels greater than 1000 mug/L, all had known comorbid factors that could have contributed to the increased ferritin level. Of the 51 C282Y/H63D subjects who underwent liver biopsy examination, significantly increased iron stores were present in 9 subjects and hepatic fibrosis was present in 13. Twelve of the 13 had evidence of hepatic steatosis, excess alcohol consumption, or diabetes. The mobilizable iron level was significantly higher in C282Y homozygous males than in compound heterozygous males (P < .001). Genetic screening of C282Y/H63D first-degree relatives detected 5 C282Y homozygotes. C282Y/H63D subjects referred for assessment had a high prevalence of increased iron indices but did not develop progressive clinical disease without comorbid factors such as steatosis, diabetes, or excess alcohol consumption. When fibrosis was seen, 1 or more comorbid factors almost always were present. Thus, phlebotomy therapy is warranted and cascade screening of relatives should be performed because expressing C282Y homozygotes may be detected.

Recent concepts in iron metabolism and HFE mutations

Abstract Our understanding of the mechanisms by which the body regulates iron absorption and iron homeostasis has improved greatly in recent years with the identification of key molecules such as HFE, ferroportin, transferrin receptor 2, hepcidin and hemojuvelin. Hereditary hemochromatosis, a primary inherited disorder of iron metabolism leading to progressive iron loading of parenchymal cells of the liver and other organs, can result from mutations in any of these genes. However, HFE‐associated hereditary hemochromatosis accounts for over 90% of cases in Caucasian populations. Penetrance is incomplete with variable clinical expression. The majority of cases demonstrate biochemical expression but a lower proportion develops advanced disease. Clinical disease, especially hepatic fibrosis, is related to the level of body iron stores which is reflected primarily in the liver. The available evidence indicates that adequate screening and diagnostic strategies can ensure that early case detection and treatment occur prior to the development of irreversible end‐organ damage. The most cost‐effective methods of early case detection are family (cascade) screening and evaluation of potential cases by primary care physicians with a high index of clinical suspicion.

Follow up after a family based genetic screening programme for familial hypercholesterolaemia: is screening alone enough?

Familial hypercholesterolaemia is an autosomal dominant disorder of lipoprotein metabolism, with an estimated frequency of 1 in 500 in Western countries; it results in excess mortality from coronary artery disease.1...

Complete and rapid scanning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene by denaturing high-performance liquid chromatography (D-HPLC): major implications for genetic counselling.

More than 900 mutations and more than 200 different polymorphisms have now been reported in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Ten years after the cloning of the CFTR gene, the complete scanning of the 27 exons to identify known and novel mutations remains challenging. Rapid accurate identification of mutated alleles is important for prenatal diagnosis, for cascade screening in families at risk of cystic fibrosis (CF) and for understanding the correlation between genotype and phenotype. In this study, we report the successful use of denaturing ion-pair reverse-phase high performance liquid chromatography (D-HPLC) to analyse rapidly the complete coding sequence of the CFTR gene. With 27 pairs of polymerase chain reaction primers, we optimised the temperature conditions required for the analysis of each amplicon and validated thetest conditions on samples from a panel of 1552 CF patients who came from France and other European countries and who had mutations and polymorphisms located in the various melting domains of the gene. D-HPLC identified 415 mutated alleles previously characterised by denaturing gradient gel electrophoresis and DNA sequencing, plus 74 novel mutations reported here. This new technique for screening DNA for sequence variation was extremely accurate (it identified 100% of the CFTR alleles tested so far) and rapid (the complete CFTR gene could be analysed in less than a week). Our approach should reduce the number of untyped CF alleles in populations and thus decrease the residual risk in couples at risk of CF. This technique may be important not only for CF,but also for many other genes with a high frequency of point mutations at a variety of sites.