[Nasopharyngeal carriage of SARS-CoV-2 among health personnel with symptoms suggestive of COVID-19 in a University Hospital in the Paris suburbs]. / Évaluation par RT-PCR du portage nasopharyngé du SARS-Cov-2 chez les personnels de santé symptomatiques suspects de COVID-19 dans un CHU de la banlieue parisienne.

INTRODUCTION: A consultation dedicated to symptomatic health professionals was opened at the beginning of the COVID-19 epidemic in order to meet the specific needs of this population. The objective of this work was to estimate the frequency of SARS-Cov-2 nasopharyngeal carriage in symptomatic healthcare workers suspected of having COVID-19 and to determine the factors associated with this carriage. METHODS: Of the 522 consultants, 308 worked in the Hospital and 214 outside. They had mild forms of COVID-19 and non-specific clinical signs with the exception of agueusia/anosmia, which was significantly more common in those with positive RT-PCR. The rate of RT-PCR positivity was 38% overall, without significant difference according to profession. It was higher among external consultants (47% versus 31%). In the hospital, this rate was significantly lower for symptomatic staff in the care sectors, compared to staff in the technical platforms and laboratories (24%, versus 45%, p = 0.006 and 54%, respectively, p < 0.001), but did not differ between staff in COVID units and other care sectors (30% versus 28%). Among the external consultants, the positivity rates of nursing home and private practices staff (53% and 55% respectively) were more than double that of acute care hospital staff (24%, p < 0.001). CONCLUSIONS: These data confirm the strong impact of COVID-19 on health professionals. The higher positivity rates among symptomatic professionals working outside the hospital compared to those working in hospital may be explained in part by a shortage of protective equipment and by difficulties in accessing virological diagnosis, which were greater outside the hospital when the epidemic began.

A PCSK9 variant and familial combined hyperlipidaemia.

BACKGROUND: Our discovery in 2003 of the first mutations of PCSK9 gene causing autosomal dominant hypercholesterolaemia (ADH) shed light on an unknown factor that strongly influences the level of circulating low density lipoprotein cholesterol (LDL-C). PCSK9 gain of function mutations cause hypercholesterolaemia by a reduction of LDL receptor levels, while PCSK9 loss of function variants are associated with a reduction of LDL-C values and a decreased risk of coronary heart disease. METHODS AND RESULTS: We report an insertion of two leucines (p.L21tri also designated p.L15_L16ins2L) in the leucine stretch of the signal peptide of PCSK9 that is found in two of 25 families with familial combined hyperlipidaemia (FCHL). This mutant is associated with high total cholesterol and LDL-C values in these families and is found also in a patient with familial hypercholesterolaemia and her father. CONCLUSION: PCSK9 variants might contribute to FCHL phenotype and are to be taken into consideration in the study of this complex and multigenic disease with other genes implicated in dyslipidaemia.

Genetic heterogeneity of autosomal dominant hypercholesterolemia.

Autosomal dominant hypercholesterolemia (ADH) is characterized by isolated elevation of plasmatic low-density lipoprotein cholesterol associated with high risk of premature cardiovascular complications. More than 1000 mutations in the LDLR gene and 9 in the APOB gene have been implicated. We have shown further heterogeneity with the discovery of missense mutations in the PCSK9 gene resulting in ADH. Different studies have tried to evaluate the respective contribution of mutations in each gene to the disease, but results were not always in agreement. After a brief overview of mutations reported for each gene, strategies and results of these different studies are reviewed and analyzed. Altogether, numerous reports give evidence for the existence of a greater level of genetic heterogeneity in ADH and the involvement of still unknown genes.

A novel splice site mutation of the LDL receptor gene in a Tunisian hypercholesterolemic family.

BACKGROUND: Familial hypercholesterolemia (FH) is an autosomal dominant inherited disease caused by mutations in either the low-density lipoprotein receptor, the apolipoprotein B or the proprotein convertase subtilisin/kexin type 9 genes. It is characterized by a high concentration of low-density lipoprotein (LDL), which frequently gives rise to premature coronary disease. In this study, we report a novel splice site mutation of the LDL receptor gene in a Tunisian family. METHODS: Seven patients from the family were screened for mutations in the LDLR gene and the apoB gene, using direct sequencing. RT-PCR and study on cultured skin fibroblast were realised to characterize the effect of novel mutation. RESULTS: Direct sequencing of the promoter and 18 exons reveals a G>A substitution in the splice site junction of intron 8 (c.1186+1 G>A). Study on cultured skin fibroblasts showed a residual activity of 10% of the LDL receptor. Reverse transcription, amplification and direct sequencing of RNA from patient's lymphocytes reveal a deletion of the final 51 bp of exon 8 preserving the reading frame. CONCLUSIONS: The study identified a novel splice mutation c.1186+1 G>A in the LDL receptor gene. It causes the utilization of a new cryptic donor splice site 51 bp downstream from the normal site.

[Familial hypercholesterolemia: A largely underestimated cardiovascular risk]. / Hypercholestérolémie familiale : un risque cardiovasculaire largement sous-estimé.

BACKGROUND: Familial hypercholesterolemia is a monogenic autosomal dominant dyslipidemia characterized by a permanent and isolated increase of cholesterol carried by low-density lipoproteins. The prevalence of its heterozygous form is estimated between 1/500 and 1/250, and in the absence of specific treatment, this form is responsible for an increase by a factor of 13 of the risk of premature coronary artery disease compared to patients non-affected by the disease. OBJECTIVES: To perform an inventory of the knowledge of heterozygous familial hypercholesterolemia in France for physicians involved in the management of the disease. METHODS: A survey was conducted (by phone and internet) among a representative sample of 495 physicians (cardiologists, endocrinologists/diabetologists, gynecologists, general practitioners) who, in parallel, completed 579 patient records. RESULTS: Thirty-two percent (95% CI [27.8; 36.2]) of physicians reported the difference between polygenic hypercholesterolemia and familial hypercholesterolemia. The presence of tendinous xanthomas, a key element of diagnosis, was spontaneously mentioned by 44% (95% CI [34; 54.2]) of cardiologists. Six percent (95% CI [2.2; 12.6]) of them gave a correct estimate of the prevalence of familial hypercholesterolemia. The likelihood of transmission of heterozygous familial hypercholesterolemia, when one parent is affected, was known for 59% (95% CI [48.7; 68.7]) of surveyed cardiologists. A cascade screening was performed systematically by 4% (95% CI [1.1; 9.9]) of them. Eighteen percent (95% CI [11; 26.9]) of cardiologists gave an accurate estimation of cardiovascular risk of heterozygous familial hypercholesterolemia. Fifty-seven percent (95% CI [46.7; 66.8]) of cardiologists admitted being misinformed about the heterozygous familial hypercholesterolemia and 83% (95% CI [74.1; 89.7]) expressed a need for information about this disease. CONCLUSION: The lack of knowledge of heterozygous familial hypercholesterolemia and its associated cardiovascular risk is probably the cause of a diagnostic default leading to inappropriate management of this disease.

[After the LDL receptor and apolipoprotein B, autosomal dominant hypercholesterolemia reveals its third protagonist: PCSK9]. / Après le récepteur des LDL et l'apolipoprotéine B, l'hypercholestérolémie familiale révèle son troisième protagoniste: PCSK9.

The genes encoding the low-density lipoproteins receptor and its ligand apolipoprotein B, have been the only two genes classically implicated in autosomal dominant hypercholesterolemia. We have identified in 2003, the third gene implicated in this disease: PCSK9 (Proprotein Convertase Subtilin Kexin 9). Several mutations (p.S127R, p.F216L, p.D374Y...) of this gene have been reported to cause hypercholesterolemia by a gain of function leading to a reduction of LDL receptor levels. Other variations of PCSK9 are conversely associated with hypocholesterolemia particularly the non-sense p.Y142X and p.C679X mutations found in 2% of black Americans and associated with a decrease of LDL levels and coronary heart diseases. PCSK9 substrates and exact role have not been elucidated yet, but it seems that PCSK9 is definitely a major actor in cholesterol homeostasis. PCSK9 inhibitors might constitute new therapeutic targets that would decrease plasma LDL cholesterol levels and be synergistic with statin drugs.

Mutational heterogeneity in low-density lipoprotein receptor gene related to familial hypercholesterolemia in Morocco.

BACKGROUND: Familial hypercholesterolemia (FH) is an autosomal dominant disorder caused by mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB) and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. Until now, molecular data concerning FH in Morocco is still limited. To gain more information in this field and to assess the contribution of these three genes in the cause of FH determinism, we analyzed six unrelated Moroccan probands and twenty-five of their family's members. METHODS: After LDLR and APOB genotype analysis, we screened the LDLR gene for mutations using southern blot and PCR-sequencing analysis. We also screened the APOB gene for the two common mutations R3500Q and R3531C by PCR-mediated site-directed mutagenesis. The PCSK9 gene was analyzed by direct sequencing. RESULTS: We identified three novel mutations (C25X, IVS3+5G>T, D558A) and two mutations previously described (D151N, A480E) in the LDLR gene. The R3500Q and R3531C mutations are absent in our probands and for 1 proband, the implication of LDLR, APOB and PCSK9 genes was excluded, supporting the implication of a fourth gene in the determination of FH. CONCLUSION: These data are in agreement with our previous study that suggests a heterogeneous mutational spectrum of FH in Morocco.

R3531C mutation in the apolipoprotein B gene is not sufficient to cause hypercholesterolemia.

Familial hypercholesterolemia and familial ligand-defective apolipoprotein B-100 (FDB) are dominantly inherited disorders leading to impaired low-density lipoprotein receptor (LDLR) and apolipoprotein B-100 (APOB) interaction, plasma LDL elevation, and hypercholesterolemia. We previously identified the first French FDB-R3531C proband, a woman with very high total cholesterol, in a group of type IIa hypercholesterolemic families. We report here the investigation of her family at large that revealed the total absence of cosegregation with hypercholesterolemia. Six of the 10 subjects heterozygous for the R3531C mutation had plasma cholesterol lower than the 97.5th percentile for their age and gender, and mean cholesterol levels were not significantly different between affected and unaffected persons. Furthermore, 2 family members with similar high LDL-cholesterol levels were not carriers of the R3531C substitution, suggesting the implication of another mutation. Segregation analysis of the LDLR gene revealed statistically significant genetic linkage with hypercholesterolemia, and analysis of the proband LDLR gene led to the identification of the 664 proline to leucine defective mutation and its detection in all 6 hypercholesterolemic-related members of this family. Therefore, our results show that the family presents with familial hypercholesterolemia and give evidence that the R3531C substitution in the APOB gene is not an allelic variant leading to FDB. Furthermore, thorough analysis of our data suggests that the APOB-R3531C mutation enhances the hypercholesterolemic effect of the LDLR-P664L defect, suggesting that it is a susceptibility mutation.

Autosomal dominant type IIa hypercholesterolemia: evaluation of the respective contributions of LDLR and APOB gene defects as well as a third major group of defects.

Autosomal dominant type IIa hypercholesterolaemia (ADH) is characterised by an elevation of total plasma cholesterol associated with increased LDL particles. Numerous different molecular defects have been identified in the LDL receptor (LDLR) and few specific mutations in the apolipoprotein B (APOB) gene resulting in familial hypercholesterolaemia and familial defective apoB-100 respectively. To estimate the respective contribution of LDLR, APOB and other gene defects in this disease, we studied 33 well characterised French families diagnosed over at least three generations with ADH through the candidate gene approach. An estimation of the proportions performed with the HOMOG3R program showed that an LDLR gene defect was involved in approximately 50% of the families (P = 0.001). On the other hand, the estimated contribution of an APOB gene defect was only 15%. This low estimation of ADH due to an APOB gene defect is further strengthened by the existence of only two probands carrying the APOB (R3500Q) mutation in the sample. More importantly and surprisingly, 35% of the families in the sample were estimated to be linked to neither LDLR nor APOB genes. These data were confirmed by the exclusion of both genes through direct haplotyping in three families. Our results demonstrate that the relative contributions of LDLR and APOB gene defects to the disease are very different. Furthermore, our results also show that genetic heterogeneity is, generally, underestimated in ADH, and that at least three major groups of defects are involved. At this point, the contribution of the recently mapped FH3 gene to ADH cannot be assessed nor its importance in the group of 'non LDLR/non APOB' families.

Limited mutational heterogeneity in the LDLR gene in familial hypercholesterolemia in Tunisia.

Familial hypercholesterolemia (FH) 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. In previous studies, we have identified novel mutations in Tunisian FH families. In this study, we have extended our investigation to additional families. Five unrelated probands were screened for mutations in the LDLR and APOB genes, using direct sequencing and enzymatic restriction. We identified two novel LDLR mutations: a missense mutation in exon 7: p.Gly343Cys (c.1027G>T), and a nonsense mutation in exon 17: p.Lys816X (c.2446A>T). Using the PolyPhen and SIFT prediction computer programs the p.Gly343Cys is predicted to have a deleterious effect on LDL receptor activity. The missense mutation we found in exon 3, p.Cys89Trp (c.267C>G), has previously been identified in patients from United Kingdom and Spain, and is reported here for the first time in the Tunisian population. Finally, the framshift mutation in exon 10, p.Ser493ArgfsX44, is reported here for the fourth and fifth time in Tunisian families. The latter is the most frequent FH-causing mutation in Tunisia. These LDLR gene mutations enrich the spectrum of mutations causing FH in the Tunisian population. The framshift mutation, p.Ser493ArgfsX44, seems to be a founder mutation in this population.

Software and database for the analysis of mutations in the human LDL receptor gene.

The low-density lipoprotein receptor (LDLr) plays a pivotal role in cholesterol homeostasis. Mutations in the LDLr gene (LDLR), which is located on chromosome 19, cause familial hypercholesterolemia (FH), an autosomal dominant disorder characterized by severe hypercholesterolemia associated with premature coronary atherosclerosis. To date almost 300 mutations have been identified in the LDLR gene. To facilitate the mutational analysis of the LDLR gene, and promote the analysis of the relationship between genotype and phenotype, a software package along with a computerized database (currently listing 210 entries) have been created.

Myotonic dystrophy: absence of CTG enlarged transcript in congenital forms, and low expression of the normal allele.

Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease. The mutation has been identified as an unstable trinucleotide CTG repeat in a sequence encoding a putative cAMP-dependent protein kinase. The CTG repeat varies in length between affected siblings, and generally increases through generations in parallel with increasing severity of the disease. Congenital myotonic dystrophy, which represents the most severe phenotype, is exclusively maternally inherited. In this report, we show, by Northern blot analysis, that no mutated enlarged transcript is detectable in a 20-week-old DM fetus and in two congenitally affected infants. Furthermore, in skeletal and cardiac muscle of the DM fetus, we observed by RNA analysis, including Northern blot and RT-PCR, an unexpectedly low expression of the paternal wild type allele. Varying degrees of expression of the mutant and/or the normal allele might therefore account for the characteristic features of the congenital form and the extreme variability of the disease.

Myotonic dystrophy: size- and sex-dependent dynamics of CTG meiotic instability, and somatic mosaicism.

Myotonic dystrophy (DM) is a progressive neuromuscular disorder which results from elongations of an unstable (CTG)n repeat, located in the 3' untranslated region of the DM gene. A correlation has been demonstrated between the increase in the repeat number of this sequence and the severity of the disease. However, the clinical status of patients cannot be unambiguously ascertained solely on the basis of the number of CTG repeats. Moreover, the exclusive maternal inheritance of the congenital form remains unexplained. Our observation of differently sized repeats in various DM tissues from the same individual may explain why the size of the mutation observed in lymphocytes does not necessarily correlate with the severity and nature of symptoms. Through a molecular and genetic study of 142 families including 418 DM patients, we have investigated the dynamics of the CTG repeat meiotic instability. A positive correlation between the size of the repeat and the intergenerational enlargement was observed similarly through male and female meioses for < or = 0.5-kb CTG sequences. Beyond 0.5 kb, the intergenerational variation was more important through female meioses, whereas a tendency to compression was observed almost exclusively in male meioses, for > or = 1.5-kb fragments. This implies a size- and sex-dependent meiotic instability. Moreover, segregation analysis supports the hypothesis of a maternal as well as a familial predisposition for the occurrence of the congenital form. Finally, this analysis reveals a significant excess of transmitting grandfathers partially accounted for by increased fertility in affected males.

Familial ligand-defective apolipoprotein B-100: simultaneous detection of the ARG3500-->GLN and ARG3531-->CYS mutations in a French population.

Familial ligand-defective apolipoprotein B-100 (FDB) is an autosomal dominant disorder leading to plasma LDL cholesterol elevation and coronary artery disease (CAD). Two specific mutations in the APOB gene--R3500Q and R3531C--induce FDB. We report an original method to detect both mutations simultaneously, based upon PCR-mediated, site-directed mutagenesis and double restriction of a unique PCR product. With this method we have investigated the prevalence of these mutations in 1,040 French patients. The R3500Q mutation was found in five probands. Genotypes were determined for 10 APOB polymorphic markers and were consistent with the common European ancestral haplotype previously reported. The only exception was one FDB proband who did not harbor the 48 repeat allele of the 3'HVR. Additionally, the first two R3531C mutations were identified in French probands. Genotypes were consistent with a previously reported haplotype, suggesting that this is another mutation of European ancestry.

French myotonic dystrophy families show expansion of a CTG repeat in complete linkage disequilibrium with an intragenic 1 kb insertion.

The molecular basis of myotonic dystrophy (DM) has been characterised. All DM mutations characterised to date appear as an unstable elongation of a fragment containing a tandem repeat of a CTG motif, which can be visualised in both EcoRI and BamHI digests. It has been shown that the fragment is polymorphic in the normal population. Another 1 kb insertion/deletion polymorphism located near the unstable CTG repeat region has been identified. The 1 kb insertion allele is present in all DM patients. These different polymorphic systems can be distinguished using cDNA25 and BamHI, because this enzyme cuts between the site of the 1 kb insertion and the CTG repeat. We thus haplotyped DM patients from 72 French families and clearly showed that all chromosomes (100%) with the DM mutation carried the 1 kb insertion as well. In addition to this association, we detected significant linkage disequilibrium between the DM locus and D19S63 for which allelic frequencies were different from other European populations. Our results in the French DM population are thus in agreement with the hypothesis that the CTG expansion occurred on one or a few ancestral chromosomes carrying the large 1 kb insertion allele.

A third major locus for autosomal dominant hypercholesterolemia maps to 1p34.1-p32.

Autosomal dominant hypercholesterolemia (ADH), one of the most frequent hereditary disorders, is characterized by an isolated elevation of LDL particles that leads to premature mortality from cardiovascular complications. It is generally assumed that mutations in the LDLR and APOB genes account for ADH. We identified one large French pedigree (HC2) and 12 additional white families with ADH in which we excluded linkage to the LDLR and APOB, implicating a new locus we named "FH3." A LOD score of 3.13 at a recombination fraction of 0 was obtained at markers D1S2892 and D1S2722. We localized the FH3 locus to a 9-cM interval at 1p34.1-p32. We tested four regional markers in another set of 12 ADH families. Positive LOD scores were obtained in three pedigrees, whereas linkage was excluded in the others. Heterogeneity tests indicated linkage to FH3 in approximately 27% of these non-LDLR/non-APOB ADH families and implied a fourth locus. Radiation hybrid mapping located four candidate genes at 1p34.1-p32, outside the critical region, showing no identity with FH3. Our results show that ADH is genetically more heterogeneous than conventionally accepted.

LDLR Database (second edition): new additions to the database and the software, and results of the first molecular analysis.

Mutations in the LDL receptor gene (LDLR) cause familial hypercholesterolemia (FH), a common autosomal dominant disorder. The LDLR database is a computerized tool that has been developed to provide tools to analyse the numerous mutations that have been identified in the LDLR gene. The second version of the LDLR database contains 140 new entries and the software has been modified to accommodate four new routines. The analysis of the updated data (350 mutations) gives the following informations: (i) 63% of the mutations are missense, and only 20% occur in CpG dinucleotides; (ii) although the mutations are widely distributed throughout the gene, there is an excess of mutations in exons 4 and 9, and a deficit in exons 13 and 15; (iii) the analysis of the distribution of mutations located within the ligand-binding domain shows that 74% of the mutations in this domain affect a conserved amino-acid, and that they are mostly confined in the C-terminal region of the repeats. Conversely, the same analysis in the EGF-like domain shows that 64% of the mutations in this domain affect a non-conserved amino-acid, and, that they are mostly confined in the N-terminal half of the repeats. The database is now accessible on the World Wide Web at http://www.umd.necker.fr