Mathilde Varret

TGFB2 mutations cause familial thoracic aortic aneurysms and dissections associated with mild systemic features of Marfan syndrome

A predisposition for thoracic aortic aneurysms leading to acute aortic dissections can be inherited in families in an autosomal dominant manner. Genome-wide linkage analysis of two large unrelated families with thoracic aortic disease followed by whole-exome sequencing of affected relatives identified causative mutations in TGFB2. These mutations—a frameshift mutation in exon 6 and a nonsense mutation in exon 4—segregated with disease with a combined logarithm of odds (LOD) score of 7.7. Sanger sequencing of 276 probands from families with inherited thoracic aortic disease identified 2 additional TGFB2 mutations. TGFB2 encodes transforming growth factor (TGF)-β2, and the mutations are predicted to cause haploinsufficiency for TGFB2; however, aortic tissue from cases paradoxically shows increased TGF-β2 expression and immunostaining. Thus, haploinsufficiency for TGFB2 predisposes to thoracic aortic disease, suggesting that the initial pathway driving disease is decreased cellular TGF-β2 levels leading to a secondary increase in TGF-β2 production in the diseased aorta.

Molecular Spectrum of Autosomal Dominant Hypercholesterolemia in France

Autosomal Dominant Hypercholesterolemia (ADH), characterized by isolated elevation of plasmatic LDL cholesterol and premature cardiovascular complications, is associated with mutations in 3 major genes: LDLR (LDL receptor), APOB (apolipoprotein B) and PCSK9 (proprotein convertase subtilisin‐kexin type 9). Through the French ADH Research Network, we collected molecular data from 1358 French probands from eleven different regions in France. Mutations in the LDLR gene were identified in 1003 subjects representing 391 unique events with 46.0% missense, 14.6% frameshift, 13.6% splice, and 11.3% nonsense mutations, 9.7% major rearrangements, 3.8% small in frame deletions/insertions, and 1.0% UTR mutations. Interestingly, 175 are novel mutational events and represent 45% of the unique events we identified, highlighting a specificity of the LDLR mutation spectrum in France. Furthermore, mutations in the APOB gene were identified in 89 probands and in the PCSK9 gene in 10 probands. Comparison of available clinical and biochemical data showed a gradient of severity for ADH‐causing mutations: FH=PCSK9>FDB>‘Others’ genes. The respective contribution of each known gene to ADH in this French cohort is: LDLR 73.9%, APOB 6.6%, PCSK9 0.7%. Finally, in 19.0% of the probands, no mutation was found, thus underscoring the existence of ADH mutations located in still unknown genes.

Description of a large family with autosomal dominant hypercholesterolemia associated with the APOE p.Leu167del mutation

Apolipoprotein (apo) E mutants are associated with type III hyperlipoproteinemia characterized by high cholesterol and triglycerides levels. Autosomal dominant hypercholesterolemia (ADH), due to the mutations in the LDLR, APOB, or PCSK9 genes, is characterized by an isolated elevation of cholesterol due to the high levels of low‐density lipoproteins (LDLs). We now report an exceptionally large family including 14 members with ADH. Through genome‐wide mapping, analysis of regional/functional candidate genes, and whole exome sequencing, we identified a mutation in the APOE gene, c.500_502delTCC/p.Leu167del, previously reported associated with sea‐blue histiocytosis and familial combined hyperlipidemia. We confirmed the involvement of the APOE p.Leu167del in ADH, with (1) a predicted destabilization of an alpha‐helix in the binding domain, (2) a decreased apo E level in LDLs, and (3) a decreased catabolism of LDLs. Our results show that mutations in the APOE gene can be associated with bona fide ADH.

Prostaglandin transporter mutations cause pachydermoperiostosis with myelofibrosis

Pachydermoperiostosis, or primary hypertrophic osteoarthropathy (PHO), is an inherited multisystem disorder, whose features closely mimic the reactive osteoarthropathy that commonly accompanies neoplastic and inflammatory pathologies. We previously described deficiency of the prostaglandin‐degrading enzyme 15‐hydroxyprostaglandin dehydrogenase (HPGD) as a cause of this condition, implicating elevated circulating prostaglandin E2 (PGE2) as causative of PHO, and perhaps also as the principal mediator of secondary HO. However, PHO is genetically heterogeneous. Here, we use whole‐exome sequencing to identify recessive mutations of the prostaglandin transporter SLCO2A1, in individuals lacking HPGD mutations. We performed exome sequencing of four probands with severe PHO, followed by conventional mutation analysis of SLCO2A1 in nine others. Biallelic SLCO2A1 mutations were identified in 12 of the 13 families. Affected individuals had elevated urinary PGE2, but unlike HPGD‐deficient patients, also excreted considerable quantities of the PGE2 metabolite, PGE‐M. Clinical differences between the two groups were also identified, notably that SLCO2A1‐deficient individuals have a high frequency of severe anemia due to myelofibrosis. These findings reinforce the key role of systemic or local prostaglandin excess as the stimulus to HO. They also suggest that the induction or maintenance of hematopoietic stem cells by prostaglandin may depend upon transporter activity. Hum Mutat 33:1175–1181, 2012.

Strategies for proprotein convertase subtilisin kexin 9 modulation: a perspective on recent patents

Importance of the field: Proprotein convertase subtilisin kexin 9 (PCSK9) is a new actor discovered in 2003 that is implicated in autosomal dominant hypercholesterolemia, cholesterol homeostasis and coronary heart disease. It has been shown to degrade the low-density lipoprotein (LDL) receptor independently of its catalytic activity. Several pharmacological strategies to reduce PCSK9 are being thoroughly investigated. Areas covered in this review: This article reviews all different strategies that are presently pursued to modulate the functional activity of PCSK9 which is a prime target for controlling LDL-cholesterol. It also provides a briefing of all the patents up to July 2010 from various organizations including pharmaceutical companies and academic institutions that have been submitted and/or approved. What the reader will gain: This review is addressed to researchers from academia and pharmaceutical companies who are engaged in PCSK9 research/cholesterol regulation and in the development of cholesterol lowering drugs. Readers will gain an up-to-date overview of the different strategies that have been investigated to reduce PCSK9 including antisense technology and specific antibodies. Take home message: Clinical trials have been launched using RNA interference approaches to reduce PCSK9 expression or specific antibodies targeting and inhibiting PCSK9 interaction with the LDL receptor. They constitute very promising approaches to reducing cholesterol levels and coronary heart disease.

Exome Sequencing in Suspected Monogenic Dyslipidemias

Background—Exome sequencing is a promising tool for gene mapping in Mendelian disorders. We used this technique in an attempt to identify novel genes underlying monogenic dyslipidemias. Methods and Results—We performed exome sequencing on 213 selected family members from 41 kindreds with suspected Mendelian inheritance of extreme levels of low-density lipoprotein cholesterol (after candidate gene sequencing excluded known genetic causes for high low-density lipoprotein cholesterol families) or high-density lipoprotein cholesterol. We used standard analytic approaches to identify candidate variants and also assigned a polygenic score to each individual to account for their burden of common genetic variants known to influence lipid levels. In 9 families, we identified likely pathogenic variants in known lipid genes (ABCA1, APOB, APOE, LDLR, LIPA, and PCSK9); however, we were unable to identify obvious genetic etiologies in the remaining 32 families, despite follow-up analyses. We identified 3 factors that limited novel gene discovery: (1) imperfect sequencing coverage across the exome hid potentially causal variants; (2) large numbers of shared rare alleles within families obfuscated causal variant identification; and (3) individuals from 15% of families carried a significant burden of common lipid-related alleles, suggesting complex inheritance can masquerade as monogenic disease. Conclusions—We identified the genetic basis of disease in 9 of 41 families; however, none of these represented novel gene discoveries. Our results highlight the promise and limitations of exome sequencing as a discovery technique in suspected monogenic dyslipidemias. Considering the confounders identified may inform the design of future exome sequencing studies.

Novel LRP5 gene mutation in a patient with osteoporosis-pseudoglioma syndrome

Osteoporosis-pseudoglioma syndrome (OPPG) is a rare autosomal recessive disorder characterised by severe juvenile-onset osteoporosis and congenital or early-onset blindness. This serious illness is due to mutations in the low-density lipoprotein receptor-related protein 5 (LRP5) that is a major actor in pathways involved in bone remodelling. Here, we report a novel frameshift mutation identified in a 22 year-old Tunisian boy of a consanguineous family. This patient had low bone mineral density (BMD), experienced multiple fractures during childhood and suffered ocular alterations with blindness. Direct DNA sequencing showed a homozygous 5 base pair insertion in exon 5 of the LRP5 gene. This new mutation is located in the first EGF-like domain and gives rise to a truncated protein of 384 amino acids. The functional significance of this mutation clearly indicates a loss-of-function mutation of the LRP5 gene leading to the observed OPPG phenotype. Rheumatologists must be aware of LRP5 gene that in addition to being a major gene in the mendelian disease that is OPPG syndrome seems to be involved in osteoporosis in the general population through some of its polymorphisms.

Moderate phenotypic expression of familial hypercholesterolemia in Tunisia.

BACKGROUND Autosomal Dominant Hypercholesterolemia (ADH) is an autosomal dominant disease caused by mutations in the low density lipoprotein receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. Xanthomas and coronary heart diseases (CHD) at an early age are the major clinical manifestations of the disease. METHODS 16 families with familial hypercholesterolemia from different regions in Tunisia participated in the study. Mutations within the LDLR gene were screened through DNA sequencing. Lipids values were measured by standard enzymatic methods. RESULTS We present here thirty five homozygotes and fifty six heterozygotes. Homozygotes presented extensive xanthomatosis, variable clinical manifestations of CHD, and total cholesterol levels in males and females of 17.26+/-4.18 and 17.64+/-2.59 mmol/L respectively. HDL-cholesterol levels were 0.62+/-0.24 and 1.00+/-0.61 mmol/L for males and females, respectively. None of the heterozygotes had tendon xanthomas (except for one female aged 62), eight had corneal arcus, and nine developed CHD mean between 46 and 88 years old. Total cholesterol levels in males and females ranged from 4.60 to 8.90 and from 4.30 to 10.50 mmol/L, respectively. CONCLUSION Tunisian FH heterozygotes are characterized by a moderate clinical and biological expression of the disease.

Proprotein convertase subtilisin / kexin 9 (PCSK9) inhibitors and the future of dyslipidemia therapy: an updated patent review (2011-2015)

ABSTRACT Introduction: The identification by Abifadel et al. in 2003 of the first mutations of PCSK9 was the major breakthrough in the cholesterol field that led to a new therapeutic target. This discovery paved the way to new lipid lowering drugs reducing LDL-cholesterol levels through the inhibition of PCSK9. Two anti-PCSK9 monoclonal antibodies have received FDA and EMA approvals: Alirocumab and Evolocumab. Areas covered: This article reviews the different strategies that are pursued to modulate the functional activity of PCSK9 for lowering LDL-cholesterol levels. It also provides a brief overview of the patents related to PCSK9 from 2011 until the end of 2015. This review is addressed to researchers from academia and pharmaceutical companies who are engaged in PCSK9 research/cholesterol regulation. Readers will gain an up-to-date overview of the different strategies that have been investigated to reduce PCSK9, focusing on anti-PCSK9 monoclonal antibodies and the related clinical trials. Expert opinion: Anti-PCSK9 antibodies are a new class of lipid lowering drugs with promising results in reducing LDL-cholesterol. Long-term ongoing studies investigating on a large scale the efficacy and safety of the anti-PCSK9 antibodies and their cardiovascular outcomes are eagerly awaited.

Effect of mutations in LDLR and PCSK9 genes on phenotypic variability in Tunisian familial hypercholesterolemia patients

BACKGROUND Autosomal dominant hypercholesterolemia (ADH) is commonly caused by mutations in the low-density lipoprotein (LDL) receptor gene (LDLR), in the apolipoprotein B-100 gene (APOB), or in the proprotein convertase subtilisin kexine 9 gene (PCSK9). ADH subjects carrying a mutation in LDLR present highly variable plasma LDL-cholesterol (LDL-C). This variability might be due to environmental factors or the effect of some modifying genes such as PCSK9 and APOE. AIMS We investigated the molecular basis of thirteen Tunisian ADH families and attempted to determine the impact of PCSK9 and APOE gene variations on LDL-cholesterol levels and on the variable phenotypic expression of the disease. METHODS AND RESULTS Fifty six subjects were screened for mutations in the LDLR gene through direct sequencing. The causative mutation was found to segregate with the disease in each family and a new frameshift mutation, p.Met767CysfsX21, was identified in one family. The distribution of total- and LDL-cholesterol levels, adjusted for age and gender, among homozygous and heterozygous ADH patients varied widely. Within seven families, nine subjects presented low LDL-cholesterol levels despite carrying a mutation in the LDLR gene. To identify the molecular actors underlying this phenotypic variability, the PCSK9 gene was screened using direct sequencing and/or enzymatic restriction analysis, and the apo E genotypes were determined. A new missense variation (p.Pro174Ser) in the PCSK9 gene was identified and characterized as a new putative loss-of-function mutation. CONCLUSION Genetic variations in PCSK9 and APOE genes could explain only part of the variability observed in the phenotypic expression in Tunisian ADH patients carrying mutations in the LDLR gene. Other genetic variants and environmental factors very probably act to fully explain this phenotypic variability.