Genetic Testing in Familial Hypercholesterolemia: Is It for Everyone?

PURPOSE OF REVIEW: Lipid measurements and genetic testing are the main diagnostic tools for FH screening that are available in many countries. A lipid profile is widely accessible, and genetic testing, although available worldwide, in some countries is only performed in a research context. Still FH is diagnosed late, showing lack of early screening programs worldwide. RECENT FINDINGS: Pediatric screening of FH was recently recognized by the European Commission Public Health Best Practice Portal as one on the best practices in non-communicable disease prevention. The early diagnosis of FH and the lowering of LDL-C values over lifespan can reduce the risk of coronary artery disease and offer health and socioeconomic gains. Current knowledge about FH shows that early detection through appropriate screening needs to become a priority in healthcare systems worldwide. Governmental programs for FH identification should be implemented to unify the diagnosis and increase patient identification.

Structural analysis of APOB variants, p.(Arg3527Gln), p.(Arg1164Thr) and p.(Gln4494del), causing Familial Hypercholesterolaemia provides novel insights into variant pathogenicity.

Familial hypercholesterolaemia (FH) is an inherited autosomal dominant disorder resulting from defects in the low-density lipoprotein receptor (LDLR), in the apolipoprotein B (APOB) or in the proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. In the majority of the cases FH is caused by mutations occurring within LDLR, while only few mutations in APOB and PCSK9 have been proved to cause disease. p.(Arg3527Gln) was the first mutation in APOB being identified and characterized. Recently two novel pathogenic APOB variants have been described: p.(Arg1164Thr) and p.(Gln4494del) showing impaired LDLR binding capacity, and diminished LDL uptake. The objective of this work was to analyse the structure of p.(Arg1164Thr) and p.(Gln4494del) variants to gain insight into their pathogenicity. Secondary structure of the human ApoB100 has been investigated by infrared spectroscopy (IR) and LDL particle size both by dynamic light scattering (DLS) and electron microscopy. The results show differences in secondary structure and/or in particle size of p.(Arg1164Thr) and p.(Gln4494del) variants compared with wild type. We conclude that these changes underlie the defective binding and uptake of p.(Arg1164Thr) and p.(Gln4494del) variants. Our study reveals that structural studies on pathogenic variants of APOB may provide very useful information to understand their role in FH disease.

Totally extraperitoneal (TEP) endoscopic inguinal hernia repair with TAP (transversus abdominis plane) block as a day-case: a prospective cohort study.

BACKGROUND: Totally extraperitoneal (TEP) endoscopic inguinal hernia repair is indicated for recurrent and bilateral inguinal hernias and traditionally is performed under general anesthesia. However, interventions that minimize pain and reduce opioid consumption have certain advantages for patients by avoiding side effects such as nausea and vomiting. The transversus abdominis plane (TAP) block has been used to minimize pain in a diverse range of surgical procedures but its safety on patients undergoing TEP repair has yet to be investigated. AIM: To assess the results of outpatient TEP repair with TAP block without curare. METHODS: Consecutive patients undergoing elective TEP procedure were prospectively enrolled. Patients in two institutes received a similar anesthetic, surgical, and analgesic treatment protocol. RESULTS: Fifty consecutive day-case patients were included in this series. The TEP repair was successful in 49 patients and there was one conversion to transabdominal pre-peritoneal (TAPP) endoscopic inguinal hernia repair. The mean duration of surgery was 20min for unilateral hernia and 40min for bilateral hernia. CONCLUSION: These preliminary results suggest that day-case endoscopic hernia repair (TEP) with TAP block without curare is effective, safe, reproducible and can be proposed in all patients.

In vitro functional characterization of missense mutations in the LDLR gene.

BACKGROUND: Mutations in the LDL receptor gene are the major cause of familial hypercholesterolaemia (FH) but it has been previously shown that the simple finding of a variation in the coding sequence of the LDLR does not confirm that it is the actual cause of FH. The pathogenicity of five missense alterations in the LDLR gene coding sequence found in a previous epidemiologic study was investigated. METHODS: The effects of the different sequence variants on LDLR expression and activity were analysed in vitro stably transfected CHO-ldlA7 cells by immunobloting of cell extracts, by uptake and degradation rates of (125)I-labelled LDL and immunofluorescence microscopy of whole cells. Analysis in silico was also performed. RESULTS: LDLR functional assays showed that variants p.V429L, p.W490R and p.S648P of the LDLR coding sequence severely impaired receptor function, while variant p.P685S had a milder effect and cells carrying p.V859M variant had LDL clearance rates comparable to cells expressing normal LDLR. In silico analysis failed to predict correctly the effect of 4/5 alterations. CONCLUSION: Assessing the pathogenicity of the different variants found in patients with clinical diagnosis of FH is of great importance to distinguish pathogenic mutations from rare silent variants and has clinical implications for determining the associated cardiovascular risk.

Molecular diagnosis of familial hypercholesterolemia: an important tool for cardiovascular risk stratification.

UNLABELLED: Familial hypercholesterolemia (FH) is associated with an increased risk of premature coronary heart disease. Molecular identification of these patients can reduce the burden of mortality from cardiovascular disorders simply by the correct identification of the disease early in life, followed by counseling and appropriate lifestyle modifications, and therapeutic measures when required. Recent studies show that, in Portugal, this disease is severely under-diagnosed. After more than 10 years of research through the Portuguese FH Study, it is now possible to translate the original research results into clinical application. AIMS: The main aims of the present work were to determine whether clinical characterization is sufficient to identify these individuals at high risk of developing CHD and to evaluate the clinical applicability of molecular diagnosis for FH. METHODS: All patients described in this study were recruited for the Portuguese FH Study. The diagnostic criteria used to select the index patients were adapted from the Simon Broome Heart Research Trust. To analyze the usefulness of the molecular diagnosis, graphs of total and LDL cholesterol values by age were constructed for 622 possible FH patients. The lipid profile of patients genetically identified as having FH, before and under medication, were analyzed to assess whether these patients were receiving appropriate treatment. The data are shown separately for children and adults and for female and male propositi (index cases and hypercholesterolemic relatives), both with and without a detectable mutation in the LDLR gene. RESULTS: The Portuguese FH Study has already genetically identified 404 individuals (171 index patients and 233 relatives) among more than one thousand individuals sent for study. A total of 78 different mutations in the LDLR gene were found in 171 index patients, 2 different mutations were found in the apoB gene of 4 patients and 2 patients had a unique PCSK9 mutation. Statistical analysis revealed that there are significant differences between total cholesterol (p < 0.001) and apoB (p = 0.026) values in the group of children (male and female) with and without a mutation in LDLR. For female children LDL values were also significantly different (p < 0.001) between subgroups but for male children this difference did not reach statistical significance. In adult women there is a statistically significant difference for total cholesterol (p = 0.049), LDL cholesterol (p = 0.031) and apoB (p = 0.003) values in the subgroups with and without a LDLR mutation. In adult males there is a statistical difference for total cholesterol (p = 0.002). LDL cholesterol (p = 0.003) and apoB (p = 0.0023) in subgroups with and without an LDLR mutation. Nevertheless there was considerable dispersion of values and individually it is not possible to distinguish between patients with and without a mutation in the LDLR gene, based only on lipid profile. CONCLUSIONS: By analysis of the clinical data of 696 possible FH patients, the present report shows evidence that clinical characterization is not sufficient to distinguish between patients with genetic or environmental dyslipidemia, and so molecular diagnosis is useful in clinical practice, allowing correct identification of FH patients and their relatives, and the early implementation of therapeutic measures to reduce the elevated cardiovascular risk of these patients. In general, molecular diagnosis of FH is feasible and could be obtained in 1-2 months if the technology is available. In Portugal the test will be offered to the population by our Institute at a cost of about 500 euros, like many other genetic tests or exams such as nuclear magnetic resonance.

Update of the Portuguese Familial Hypercholesterolaemia Study.

The main aim of the Portuguese Familial Hypercholesterolaemia Study is to identify the genetic cause of hypercholesterolaemia in individuals with a clinical diagnosis of Familial Hypercholesterolaemia (FH). A total of 1340 blood samples were collected from 482 index patients and 858 relatives with the collaboration of clinicians from several hospitals all over the country. The genetic diagnosis of FH in this study is based on the analyses of three genes: LDLR, APOB and PCSK9. In the last 10 years, the Portuguese FH Study identified a genetic defect in a total of 171 index patients, corresponding to an overall of 48% of the total received cases with clinical diagnosis of FH. Although the Simon Broome FH register criteria have been adapted to our study, 59 patients that did not fulfil all criteria were included in the study and a mutation causing disease was identified in 8 of these patients. In the LDLR gene were found 80 different mutations in 165 unrelated index patients: 159 heterozygous, 3 compounds heterozygous and 3 true homozygous. The APOB p.Arg3527Gln and the PCSK9 p.Asp374His mutation were not found in any of our patients since our last report, but a novel mutation in the APOB gene, predicted to cause a single amino acid substitution p.Tyr3560Cys, was found in one patient. The cascade screening in relatives of these 171 index patients allowed the identification and genetic characterization of a total of 404 FH patients in Portugal.

Genetic diagnosis of familial hypercholesterolaemia: the importance of functional analysis of potential splice-site mutations.

Familial hypercholesterolemia (FH) results from defective low-density lipoprotein receptor (LDLR) activity, mainly due to LDLR gene defects. Of the many different LDLR mutations found in patients with FH, about 6% of single base substitutions are located near or within introns, and are predicted to result in exon skipping, retention of an intron, or activation of cryptic sites during mRNA splicing. This paper reports on the Portuguese FH Study, which found 10 such mutations, 6 of them novel. For the mutations that have not been described before or those whose effect on function have not been analysed, their effect on splicing was investigated, using reverse transcriptase PCR analysis of LDLR mRNA from freshly isolated blood mononuclear cells. Two of these variants (c.313+6 T-->C, c.2389G-->T (p.V776L)) caused exon skipping, and one caused retention of an intron (c.1359-5C-->G), whereas two others (c.2140+5 G-->A and c.1061-8T-->C) had no apparent effect. Any effect of c.1185G-->C (p.V374V) on splicing could not be determined because it was on an allele with a promoter mutation (-42C-->G) that was probably not transcribed. Variants in four patients lost to follow-up could not be tested experimentally, but they almost certainly affect splicing because they disrupt the invariant AG or GT in acceptor (c.818-2A-->G) or donor (c.1060+1G-->A, c.1845+1delG and c.2547+1G-->A) spice sites. These findings emphasise that care must be taken before reporting the presence or absence of a splice-site mutation in the LDLR gene for diagnostic purposes. The study also shows that relatively simple, quick and inexpensive RNA assays can evaluate putative splicing mutations that are not always predictable by available software, thereby reducing genetic misdiagnosis of patients with FH.

Variable phenotypic expression of homozygous familial hypobetalipoproteinaemia due to novel APOB gene mutations.

Homozygous familial hypobetalipoproteinaemia (Ho-FHBL) is a rare co-dominant disorder characterized by extremely low levels of low-density lipoprotein cholesterol (LDL-C) and apolipoprotein B (apoB). Most patients with Ho-FHBL have mutations in APOB gene resulting in truncated apoBs. Some patients are asymptomatic, while others have fatty liver, intestinal fat malabsorption and neurological dysfunctions. We investigated three adult subjects with severe hypobetalipoproteinaemia and a family history of FHBL. Proband FHBL-47 had liver cirrhosis with hepatocarcinoma and a renal carcinoma but no clinical manifestations related to FHBL. He was a compound heterozygote for a 7-bp deletion in exon 21 and a base insertion in exon 26 resulting in truncated apoBs (apoB-22.46/apoB-66.51). Proband FHBL-53, with severe hepatic steatosis and fibrosis, had a nonsense mutation in exon 19 resulting in a truncated apoB (apoB-20.61) and a rare nucleotide substitution in intron 14 (c.2068-4T>A). The latter was also present in her daughter, found to have low plasma LDL-C and apoB. Proband FHBL-82 had chronic diarrhoea and steatorrhoea. She was found to be homozygous for a nonsense mutation in exon 24 resulting in a truncated apoB (apoB-26.65). In adult subjects, the presence of chronic liver disease and chronic diarrhoea, when associated with severe hypobetalipoproteinaemia, may lead to the diagnosis of Ho-FHBL.

Familial hypercholesterolaemia in Portugal.

Familial hypercholesterolaemia (FH) is characterised clinically by an increased level of circulating LDL cholesterol that leads to lipid accumulation in tendons and arteries, premature atherosclerosis and increased risk of coronary heart disease (CHD). Although Portugal should have about 20,000 cases, this disease is severely under-diagnosed in our country, this being the first presentation of Portuguese data on FH. A total of 602 blood samples were collected from 184 index patients and 418 relatives from several centres throughout Portugal. Fifty-three different mutations were found in 83 index patients, 79 heterozygous and 4 with two defective LDLR alleles. Additionally, 4 putative alterations were found in 8 patients but were not considered mutations causing disease, mainly because they did not co-segregate with hypercholesterolaemia in the families. Three unrelated patients were found to be heterozygous for the APOB(3500) mutation and two unrelated patients were found to be heterozygous for a novel mutation in PCSK9, predicted to cause a single amino acid substitution, D374H. Cascade screening increased the number of FH patients identified genetically to 204. The newly identified FH patients are now receiving counselling and treatment based on the genetic diagnosis. The early identification of FH patients can increase their life expectancy and quality of life by preventing the development of premature CHD if patients receive appropriate pharmacological treatment.

A rare polymorphism in the low density lipoprotein (LDL) gene that affects mRNA splicing.

Familial hypercholesterolaemia (FH) is usually caused by mutations in the low density lipoprotein (LDL) receptor gene (LDLR) that impair clearance of LDL from the circulation. The increased risk of premature coronary heart disease associated with FH can be reduced by dietary advice and treatment with lipid-lowering drug therapy, but it is important to identify affected individuals at an early stage. Several programmes for genetic diagnosis of FH that rely on identifying nucleotide substitutions in genomic DNA have been initiated, but the validity of these is dependent on distinguishing between a silent nucleotide variant and a mutation that affects LDL-receptor function. Here we describe a single nucleotide substitution in the coding region of exon 9 of LDLR that is an apparently silent polymorphism: CGG (Arg406) to AGG (Arg). Analysis of mRNA from the patient's cells showed that the mutation introduces a new splice site that is used to the exclusion of the natural splice site and causes a deletion of 31 bp from the mRNA, predicted to introduce premature termination four codons after R406. This finding emphasizes the caution needed in genetic diagnosis of FH based on genomic DNA sequence alone.

[The ovarian vein syndrome: eight cases and review of the literature]. / Le syndrome de la veine ovarienne: à propos de huit cas et revue de la littérature.

OBJECTIVE: The ovarian vein syndrome is an uncommon clinical entity occurring secondary to ureteral obstruction caused by dilation of aberrant ovarian veins. After initial scepticism about its reality, many authors currently recognize the ovarian vein syndrome as a separate clinical entity, included among pelvic congestive syndromes. Excretory urography, sonography and retrograde ureteropyelography findings confirm the clinical diagnosis. Our purpose was to demonstrate the efficacy of surgery and the contribution of laparoscopic treatment. METHODS: and material. We rapport eight cases treated from 1980 to 1998. The surgical procedure was laparotomy in seven cases and laparoscopy in one. RESULTS: Ovarian vein resection was successful in all cases without any adverse event. Average hospital stay was about four days. Complete and persistent relief of lumbar pain was achieved. CONCLUSION: Ovarian vein syndrome is an uncommon diagnosis of pelvic pain that should be recognized. Surgery is considered as the appropriated therapy. The laparoscopic approach should be preferred

Determinants of variable response to statin treatment in patients with refractory familial hypercholesterolemia.

Interindividual variability in low density lipoprotein (LDL) cholesterol (LDL-C) response during treatment with statins is well documented but poorly understood. To investigate potential metabolic and genetic determinants of statin responsiveness, 19 patients with refractory heterozygous familial hypercholesterolemia were sequentially treated with placebo, atorvastatin (10 mg/d), bile acid sequestrant, and the 2 combined, each for 4 weeks. Levels of LDL-C, mevalonic acid (MVA), 7-alpha-OH-4-cholesten-3-one, and leukocyte LDL receptor and hydroxymethylglutaryl coenzyme A reductase mRNA were determined after each treatment period. Atorvastatin (10 mg/d) reduced LDL-C by an overall mean of 32.5%. Above-average responders (LDL-C -39.5%) had higher basal MVA levels (34.4+/-6.1 micromol/L) than did below-average responders (LDL-C -23.6%, P<0.02; basal MVA 26.3+/-6.1 micromol/L, P<0.01). Fewer good responders compared with the poor responders had an apolipoprotein E4 allele (3 of 11 versus 6 of 8, respectively; P<0.05). There were no baseline differences between them in 7-alpha-OH-4-cholesten-3-one, hydroxymethylglutaryl coenzyme A reductase mRNA, or LDL receptor mRNA, but the latter increased in the good responders on combination therapy (P<0.05). Severe mutations were not more common in poor than in good responders. We conclude that poor responders to statins have a low basal rate of cholesterol synthesis that may be secondary to a genetically determined increase in cholesterol absorption, possibly mediated by apolipoprotein E4. If so, statin responsiveness could be enhanced by reducing dietary cholesterol intake or inhibiting absorption.

Inheritance of two different alleles of the low-density lipoprotein (LDL)-receptor gene carrying the recurrent Pro664Leu mutation in a patient with homozygous familial hypercholesterolaemia.

Familial hypercholesterolaemia (FH) is caused by mutations in the low-density lipoprotein (LDL)-receptor gene that result in impaired clearance of plasma LDL and increased risk of coronary heart disease. Numerous different mutations have been found in FH patients worldwide, the majority of which are infrequent in out-bred populations and account for 2% or less of patients with the disorder in large cohorts. Thus, it was surprising to find that two homozygous FH patients referred to a single hospital in the UK were both apparently homozygous for the Pro664Leu mutation. One, an Asian patient, was a true homozygote. The other, of English origin, had inherited two different alleles of the LDL-receptor gene with the same mutation from unrelated parents, as inferred from the haplotype of polymorphic markers. A third, clinically homozygous FH patient, despite being the offspring of first cousins, had inherited one 'Asian' Pro664Leu allele, but an allele with a 1-bp deletion in exon 5 from the other parent. The Pro664Leu mutation in the LDL-receptor gene has now been described in heterozygous patients of very different ethnic origin and is associated with different haplotypes, suggesting that the same base change at a CpG may have recurred as many as six times.

Characterization of a novel cellular defect in patients with phenotypic homozygous familial hypercholesterolemia.

Familial hypercholesterolemia (FH) is characterized by a raised concentration of LDL in plasma that results in a significantly increased risk of premature atherosclerosis. In FH, impaired removal of LDL from the circulation results from inherited mutations in the LDL receptor gene or, more rarely, in the gene for apo B, the ligand for the LDL receptor. We have identified two unrelated clinically homozygous FH patients whose cells exhibit no measurable degradation of LDL in culture. Extensive analysis of DNA and mRNA revealed no defect in the LDL receptor, and alleles of the LDL receptor or apo B genes do not cosegregate with hypercholesterolemia in these families. FACS((R)) analysis of binding and uptake of fluorescent LDL or anti-LDL receptor antibodies showed that LDL receptors are on the cell surface and bind LDL normally, but fail to be internalized, suggesting that some component of endocytosis through clathrin-coated pits is defective. Internalization of the transferrin receptor occurs normally, suggesting that the defective gene product may interact specifically with the LDL receptor internalization signal. Identification of the defective gene will aid genetic diagnosis of other hypercholesterolemic patients and elucidate the mechanism by which LDL receptors are internalized.

[Para-esophageal hiatal hernia with acute gastric volvulus]. / Hernie hiatale para-oesophagienne avec volvulus gastrique aigu.

We report 3 observations of acute gastric volvulus on paraesophageal hiatus hernia requiring emergency laparotomy. The main characteristics of this complication of hiatus hernia are described and the importance of early diagnosis, because of unfavorable prognosis, is stressed.

[Laparoscopic splenorraphy using a resorbable prosthesis in splenic injuries. Apropos of 5 cases]. / Splénorraphie laparoscopique par prothèse résorbable dans les traumatismes spléniques. A propos de cinq cas.

STUDY AIM: The aim of this retrospective study is to report five cases of laparoscopic splenorraphy with an absorbable perisplenic mesh for splenic injury. PATIENTS AND METHOD: From January 1996 to February 1998, three men and two women (mean age: 52 years) were included in this study. The splenic lesions were due to either a fall (n = 3), a traffic accident (n = 1), or pleural paracenthesis in a patient with mediastinitis after valvular replacement. Splenic injury was recognized by ultrasonography. The patients were operated as either emergency cases (n = 2), or within 24 hours (n = 3). The procedure included evacuation of the hemoperitineum, total liberation of the spleen, and splenic hemostasis with a perisplenic mesh which was used in open surgery. The mesh placed behind the spleen, covering its superior and inferior poles, was unrolled forwards and burses progressively tightened. RESULTS: There was no conversion, no mortality, no morbidity. In the four injured patients, the mean duration of surgery was 120 minutes (70-180), without any blood transfusion, and the patients were discharged on d4 or 5. The fifth patient, after valvular replacement, was operated on with anticoagulation. The mean duration of surgery was 270 minutes. Four blood units were necessary. He was discharged at d26. CONCLUSION: This technique combines the advantages of the perisplenic mesh which is efficient and safe, with the advantages of laparoscopic surgery which simplifies the postoperative course. It can only be used in case of isolated splenic injury in patients with stable hemodynamic condition.

[Acute stomach volvulus complicating hiatal hernia revealed by common bile duct calculi]. / Volvulus gastrique aigu sur hernie hiatale révélé par une lithiase cholédocienne.

The authors report about one observation of acute gastric volvulus on paraoesophageal hiatus hernia caused by a choledocholithiasis, requiring gastrectomy and choledochotomy in emergency. On the basis of this case they sum up the main characteristics of this complication of hiatus hernia and insist on a earlier diagnosis before stage of irreversible lesions.