Perilipin, the most abundant adipocyte-specific lipid-droplet coat protein, is required for optimal lipid incorporation and release from the droplet. The authors identified mutations in the perilipin ...gene in families with partial lipodystrophy, severe dyslipidemia, and insulin-resistant diabetes.
Although the adverse consequences of an excessive fat mass (obesity) are widely appreciated, adipose tissue also has key metabolic functions, including storage of surplus energy and buffering of the postprandial influx of free fatty acids and their release in the fasting state or during exercise for oxidation by other tissues — all of which are essential for health.
1
Small intracytoplasmic lipid droplets are found in most cell types, whereas white fat cells (adipocytes) store triglycerides within a large, single lipid droplet, which occupies up to 90% of the cell volume. The unilocular droplet of white adipocytes allows optimal energy storage . . .
Context: Berardinelli-Seip congenital lipodystrophy (BSCL) is a rare recessive disease characterized by near absence of adipose tissue, resulting in severe dyslipidemia and insulin resistance. In ...most reported cases, BSCL is due to alterations in either seipin, of unknown function, or 1-acylglycerol-3-phosphate acyltransferase-β (AGPAT2), which catalyzes the formation of phosphatidic acid.
Objective: We sought to determine the genetic origin of the unexplained cases of BSCL. We thus sequenced CAV1, encoding caveolin-1, as a candidate gene involved in insulin signaling and lipid homeostasis. CAV1 is a key structural component of plasma membrane caveolae, and Cav1-deficient mice display progressive loss of adipose tissue and insulin resistance.
Design: We undertook phenotyping studies and molecular screening of CAV1 in four patients with BSCL with no mutation in the genes encoding either seipin or AGPAT2.
Results: A homozygous nonsense mutation (p.Glu38X) was identified in CAV1 in a patient with BSCL born from a consanguineous union. This mutation affects both the α- and β-CAV1 isoforms and ablates CAV1 expression in skin fibroblasts. Detailed magnetic resonance imaging of the proband confirmed near total absence of both sc and visceral adipose tissue, with only vestigial amounts in the dorsal sc regions. In keeping with the lack of adipose tissue, the proband was also severely insulin resistant and dyslipidemic. In addition, the proband had mild hypocalcemia likely due to vitamin D resistance.
Conclusions: These findings identify CAV1 as a new BSCL-related gene and support a critical role for caveolins in human adipocyte function.
Next-generation sequencing (NGS) studies are becoming routinely used for the detection of novel and clinically actionable DNA variants at a pangenomic scale. Such analyses are now used in the ...clinical practice to enable precision medicine. Formalin-fixed paraffin-embedded (FFPE) tissues are still one of the most abundant source of cancer clinical specimen, unfortunately this method of preparation is known to degrade DNA and therefore compromise subsequent analysis. Some studies have reported that variant detection can be performed on FFPE samples sequenced with NGS techniques, but few or none have done an in-depth coverage analysis and compared the influence of different state-of-the-art FFPE DNA extraction kits on the quality of the variant calling. Here, we generated 42 human whole-exome sequencing data sets from fresh-frozen (FF) and FFPE samples. These samples include normal and tumor tissues from two different organs (liver and colon), that we extracted with three different FFPE extraction kits (QIAamp DNA FFPE Tissue kit and GeneRead DNA FFPE kit from Qiagen, Maxwell™ RSC DNA FFPE Kit from Promega). We determined the rate of concordance of called variants between matched FF and FFPE samples on all common variants (representing at least 86% of the total number of variants for SNVs). The concordance rate is very high between all matched FF / FFPE pairs, with equivalent values for the three kits we analyzed. On the other hand, when looking at the difference between the total number of variants in FF and FFPE, we find a significant variation for the three different FFPE DNA extraction kits. Coverage analysis shows that FFPE samples have less good indicators than FF samples, yet the coverage quality remains above accepted thresholds. We detect limited but statistically significant variations in coverage indicator values between the three FFPE extraction kits. Globally, the GeneRead and QIAamp kits have better variant calling and coverage indicators than the Maxwell kit on the samples used in this study, although this kit performs better on some indicators and has advantages in terms of practical usage. Taken together, our results confirm the potential of FFPE samples analysis for clinical genomic studies, but also indicate that the choice of a FFPE DNA extraction kit should be done with careful testing and analysis beforehand in order to maximize the accuracy of the results.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background Blood concentrations of fibrinogen have been associated with coronary heart disease risk in epidemiological studies, but it is uncertain whether this association is causal or reflects ...residual confounding by other risk factors. We investigated the relationship between the single nucleotide polymorphism at position −148 in the beta-fibrinogen gene promoter (β − 148C/T), blood fibrinogen levels, and risk of myocardial infarction (MI) in sufficiently large numbers of coronary disease cases to reliably address this question. Methods Genotyping and measurement of blood fibrinogen concentration were carried out in 4685 cases of confirmed MI and 3460 controls with no history of coronary disease. A meta-analysis of ISIS and 19 other studies of beta-fibrinogen genotypes involving a total of 12 220 coronary disease cases and 18 716 controls was conducted. Results Among the ISIS controls, mean plasma fibrinogen concentrations with the C/C, C/T and T/T genotypes were 3.34 (SE 0.015), 3.48 (0.022), and 3.60 (0.064) g/l, respectively, corresponding to an increase of 0.14 (0.024) g/l per T allele (trend P < 0.0001). In the case–control comparison, 0.14 g/l higher usual plasma fibrinogen concentration was associated with an age-adjusted and sex-adjusted risk ratio for MI of 1.17 95% confidence interval (95% CI) 1.14–1.19; P < 0.0001. But, after further adjustment for smoking, body mass index, and plasma apolipoprotein B/A1 ratio, this risk ratio fell to 1.03 (95% CI 1.00–1.05; P = 0.05). Moreover, fibrinogen genotype was not significantly associated with MI incidence: risk ratio of 1.06 (95% CI 0.96–1.16) per higher-fibrinogen allele in ISIS alone and of 1.00 (95% CI 0.95–1.04) per allele in the meta-analysis. Conclusions Genotypes that produce lifelong differences in fibrinogen concentrations do not materially influence coronary disease incidence. As these genotype-dependent differences in fibrinogen were allocated randomly at conception (Mendelian randomization), this association is not likely to be confounded by other factors. Consequently, these genetic results provide strong evidence that long-term differences in fibrinogen concentrations are not a major determinant of coronary disease risk.
Haploinsufficiency of the transcription factor PAX6 is the main cause of congenital aniridia, a genetic disorder characterized by iris and foveal hypoplasia. 11p13 microdeletions altering PAX6 or its ...downstream regulatory region (DRR) are present in about 25% of patients; however, only a few complex rearrangements have been described to date. Here, we performed nanopore-based whole-genome sequencing to assess the presence of cryptic structural variants (SVs) on the only two unsolved "PAX6-negative" cases from a cohort of 110 patients with congenital aniridia after unsuccessfully short-read sequencing approaches.
Long-read sequencing (LRS) unveiled balanced chromosomal rearrangements affecting the PAX6 locus at 11p13 in these two patients and allowed nucleotide-level breakpoint analysis. First, we identified a cryptic 4.9 Mb de novo inversion disrupting intron 7 of PAX6, further verified by targeted polymerase chain reaction amplification and sequencing and FISH-based cytogenetic analysis. Furthermore, LRS was decisive in correctly mapping a t(6;11) balanced translocation cytogenetically detected in a second proband with congenital aniridia and considered non-causal 15 years ago. LRS resolved that the breakpoint on chromosome 11 was indeed located at 11p13, disrupting the DNase I hypersensitive site 2 enhancer within the DRR of PAX6, 161 Kb from the causal gene. Patient-derived RNA expression analysis demonstrated PAX6 haploinsufficiency, thus supporting that the 11p13 breakpoint led to a positional effect by cleaving crucial enhancers for PAX6 transactivation. LRS analysis was also critical for mapping the exact breakpoint on chromosome 6 to the highly repetitive centromeric region at 6p11.1.
In both cases, the LRS-based identified SVs have been deemed the hidden pathogenic cause of congenital aniridia. Our study underscores the limitations of traditional short-read sequencing in uncovering pathogenic SVs affecting low-complexity regions of the genome and the value of LRS in providing insight into hidden sources of variation in rare genetic diseases.
Polymorphisms in Type II SH2 Domain–Containing Inositol 5-Phosphatase ( INPPL1 , SHIP2) Are Associated With Physiological Abnormalities of the Metabolic Syndrome
Pamela J. Kaisaki 1 ,
Marc Delépine 2 ...,
Peng Y. Woon 1 ,
Liam Sebag-Montefiore 1 ,
Steven P. Wilder 1 ,
Stephan Menzel 1 ,
Nathalie Vionnet 2 ,
Evelyne Marion 3 ,
Jean-Pierre Riveline 4 ,
Guillaume Charpentier 4 ,
Stéphane Schurmans 3 ,
Jonathan C. Levy 5 ,
Mark Lathrop 2 ,
Martin Farrall 6 and
Dominique Gauguier 1
1 The Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford, U.K
2 National Centre for Genotyping, Evry, France
3 Institut de Recherches en Biologie Humaine et Moléculaire, Institut de Biologie et de Médecine Moléculaires, Université Libre
de Bruxelles, Gosselies, Belgium
4 Service d’Endocrinologie-Diabétologie, CH Sud Francilien, Corbeil-Essonnes, France
5 Oxford Centre for Diabetes, Endocrinology and Metabolism, University of Oxford, Oxford, U.K
6 Department of Cardiovascular Medicine, University of Oxford, Oxford, U.K
Address correspondencereprint requests to Pamela J. Kaisaki, The Wellcome Trust Centre for Human Genetics, Roosevelt Drive,
Headington, Oxford OX3 7BN, U.K. E-mail: pamela.kaisaki{at}well.ox.ac.uk
Abstract
Type II SH2 domain–containing inositol 5-phosphatase ( INPPL1, or SHIP2) plays an important role in the control of insulin sensitivity. INPPL1 mutations affecting gene function have been found in rat models of type 2 diabetes and hypertension and in type 2 diabetic
patients. We investigated the influence of nucleotide variation in INPPL1 on components of the metabolic syndrome. Following comprehensive resequencing of the gene, we genotyped 12 informative polymorphisms
in 1,304 individuals from 424 British type 2 diabetes families that were characterized for several metabolic phenotypes. We
have found highly significant associations of single nucleotide polymorphisms (SNPs) and haplotypes of INPPL1 with hypertension as well as with other components of the metabolic syndrome. In a cohort of 905 French type 2 diabetic patients,
we found evidence of association of INPPL1 SNPs with the presence of hypertension. We conclude that INPPL1 variants may impact susceptibility to disease and/or to subphenotypes involved in the metabolic syndrome in some diabetic
patients.
DIF, Diabetes in Families
PDT, pedigree disequilibrium test
SNP, single nucleotide polymorphism
Footnotes
Additional information for this article can be found in an online appendix at http://diabetes.diabetesjournals.org .
Accepted April 5, 2004.
Received November 28, 2003.
DIABETES
Prevalence of Mutations in AGPAT2 Among Human Lipodystrophies
Jocelyne Magré 1 ,
Marc Delépine 2 ,
Lionel Van Maldergem 3 ,
Jean-Jacques Robert 4 ,
J. Antonie Maassen 5 ,
Muriel Meier 1 ,
Vanessa R. ...Panz 6 ,
Chong Ae Kim 7 ,
Nadia Tubiana-Rufi 8 ,
Paul Czernichow 8 ,
Eva Seemanova 9 ,
Charles R. Buchanan 10 ,
Didier Lacombe 11 ,
Corinne Vigouroux 1 ,
Olivier Lascols 1 ,
C. Ronald Kahn 12 ,
Jacqueline Capeau 1 and
Mark Lathrop 2
1 INSERM U.402, Saint-Antoine Faculty of Medicine, University of Pierre and Marie Curie, Paris, France
2 National Center of Genotyping, Evry, France
3 Center of Human Genetics, Institute of Pathology and Genetics, Loverval, Belgium
4 Department of Pediatric Diabetology, Necker Hospital, Paris, France
5 Sylvius Laboratory, University of Leiden Medical Center, Leiden, the Netherlands
6 Department of Medicine, University of the Witwatersrand, Johannesburg, South Africa
7 Department of Pediatrics, da Crianca Institute, University of Sao Paulo, Sao Paulo, Brazil
8 Department of Pediatric Endocrinology, Robert Debré Hospital, Paris, France
9 Department of Clinical Genetics, Charles University, Prague, Czech Republic
10 Department of Child Health, King’s College Hospital, London, U.K
11 Department of Medical Genetics, Pellegrin-Enfants Hospital, Bordeaux, France
12 Joslin Diabetes Center, Harvard University, Boston, Massachusetts
Abstract
Berardinelli-Seip congenital lipodystrophy (BSCL) is a heterogeneous genetic disease characterized by near absence of adipose
tissue and severe insulin resistance. We have previously identified mutations in the seipin gene in a subset of our patients’
cohort. Recently, disease-causing mutations in AGPAT2 have been reported in BSCL patients. In this study, we have performed mutation screening in AGPAT2 and the related AGPAT1 in patients with BSCL or other forms of lipodystrophy who have no detectable mutation in the seipin gene. We found 38 BSCL
patients from 30 families with mutations in AGPAT2 . Three of the known mutations were frequently found in our families. Of the eight new alterations, six are null mutations
and two are missense mutations (Glu172Lys and Ala238Gly). All the patients harboring AGPAT2 mutations presented with typical features of BSCL. We did not find mutations in patients with other forms of lipodystrophies,
including the syndromes of Lawrence, Dunnigan, and Barraquer-Simons, or with type A insulin resistance. In conclusion, mutations
in the seipin gene and AGPAT2 are confined to the BSCL phenotype. Because we found mutations in 92 of the 94 BSCL patients studied, the seipin gene and
AGPAT2 are the two major genes involved in the etiology of BSCL.
Footnotes
Address correspondence and reprint requests to Jocelyne Magré, INSERM U.402, Faculté de Médecine Saint-Antoine, 27 rue Chaligny,
75571 Paris Cedex, France. E-mail: magre{at}st-antoine.inserm.fr .
Received for publication 19 December 2002 and accepted 18 February 2003.
BSCL, Berardinelli-Seip congenital lipodystrophy; FPLD, familial partial lipodystrophy; LPA, lysophosphatidic acid.
DIABETES
SHANK genes code for scaffold proteins located at the post-synaptic density of glutamatergic synapses. In neurons, SHANK2 and SHANK3 have a positive effect on the induction and maturation of ...dendritic spines, whereas SHANK1 induces the enlargement of spine heads. Mutations in SHANK genes have been associated with autism spectrum disorders (ASD), but their prevalence and clinical relevance remain to be determined. Here, we performed a new screen and a meta-analysis of SHANK copy-number and coding-sequence variants in ASD. Copy-number variants were analyzed in 5,657 patients and 19,163 controls, coding-sequence variants were ascertained in 760 to 2,147 patients and 492 to 1,090 controls (depending on the gene), and, individuals carrying de novo or truncating SHANK mutations underwent an extensive clinical investigation. Copy-number variants and truncating mutations in SHANK genes were present in ∼1% of patients with ASD: mutations in SHANK1 were rare (0.04%) and present in males with normal IQ and autism; mutations in SHANK2 were present in 0.17% of patients with ASD and mild intellectual disability; mutations in SHANK3 were present in 0.69% of patients with ASD and up to 2.12% of the cases with moderate to profound intellectual disability. In summary, mutations of the SHANK genes were detected in the whole spectrum of autism with a gradient of severity in cognitive impairment. Given the rare frequency of SHANK1 and SHANK2 deleterious mutations, the clinical relevance of these genes remains to be ascertained. In contrast, the frequency and the penetrance of SHANK3 mutations in individuals with ASD and intellectual disability-more than 1 in 50-warrant its consideration for mutation screening in clinical practice.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background Blood lipid concentrations are causally related to the risk of coronary heart disease (CHD). Various associations between CHD risk and genes that moderately affect plasma lipid levels have ...been described, but previous studies have typically involved too few ‘cases’ to assess these associations reliably. Methods The present study involves 4685 cases of myocardial infarction (MI) and 3460 unrelated controls without diagnosed cardiovascular disease. Six polymorphisms of four ‘lipid-related’ genes were genotyped. Results For the apolipoprotein E ε2/ε3/ε4 polymorphism, the average increase in the plasma ratio of apolipoprotein B to apolipoprotein A1 (apoB/apoA1 ratio) among controls was 0.082 (s.e. 0.007) per stepwise change from ε3/ε2 to ε3/ε3 to ε3/ε4 genotype (trend P < 0.0001). The case-control comparison yielded a risk ratio for MI of 1.16 (95% CI: 1.06, 1.27; P = 0.001) per stepwise change in these genotypes. But, this risk ratio was not as extreme as would have been expected from the corresponding differences in plasma apoB/apoA1 ratio between genotypes. Hence, following adjustment for the measured level of the plasma apoB/apoA1 ratio, the direction of the risk ratio per stepwise change reversed to 0.83 (95% CI: 0.74, 0.92; P < 0.001). Similarly, for the apolipoprotein B Asn4311Ser and Thr71Ile polymorphisms, genotypes associated with more adverse plasma apolipoprotein concentrations were associated with significantly lower risk of MI after adjustment for the apoB/apoA1 ratio. The B2 allele of the cholesteryl ester transfer protein TaqIb polymorphism was associated with a significantly lower plasma apoB/apoA1 ratio, but with no significant difference in the risk of MI. Finally, the lipoprotein lipase Asn291Ser and T4509C (PvuII) polymorphisms did not produce clear effects on either the plasma apoB/apoA1 ratio or the risk of MI. Conclusions It remains unresolved why some of these genetic factors that produce lifelong effects on plasma lipid concentrations have significantly less than the correspondingly expected effects on CHD rates in adult life.
Autism spectrum disorders (ASD) are a heterogeneous group of neurodevelopmental disorders with a complex inheritance pattern. While many rare variants in synaptic proteins have been identified in ...patients with ASD, little is known about their effects at the synapse and their interactions with other genetic variations. Here, following the discovery of two de novo SHANK2 deletions by the Autism Genome Project, we identified a novel 421 kb de novo SHANK2 deletion in a patient with autism. We then sequenced SHANK2 in 455 patients with ASD and 431 controls and integrated these results with those reported by Berkel et al. 2010 (n = 396 patients and n = 659 controls). We observed a significant enrichment of variants affecting conserved amino acids in 29 of 851 (3.4%) patients and in 16 of 1,090 (1.5%) controls (P = 0.004, OR = 2.37, 95% CI = 1.23-4.70). In neuronal cell cultures, the variants identified in patients were associated with a reduced synaptic density at dendrites compared to the variants only detected in controls (P = 0.0013). Interestingly, the three patients with de novo SHANK2 deletions also carried inherited CNVs at 15q11-q13 previously associated with neuropsychiatric disorders. In two cases, the nicotinic receptor CHRNA7 was duplicated and in one case the synaptic translation repressor CYFIP1 was deleted. These results strengthen the role of synaptic gene dysfunction in ASD but also highlight the presence of putative modifier genes, which is in keeping with the "multiple hit model" for ASD. A better knowledge of these genetic interactions will be necessary to understand the complex inheritance pattern of ASD.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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