Publications by authors named "Pierre Lindenbaum"

29 Publications

  • Page 1 of 1

Phenotypic Differences Between Polygenic and Monogenic Hypobetalipoproteinemia.

Arterioscler Thromb Vasc Biol 2021 01 19;41(1):e63-e71. Epub 2020 Nov 19.

Hospices Civils de Lyon, UF Dyslipidémies Service de Biochimie et de Biologie Moléculaire Grand Est, Bron, France (X.V., D.C., O.M., E.D., M.D.F.).

Objective: Primary hypobetalipoproteinemia is characterized by LDL-C (low-density lipoprotein cholesterol) concentrations below the fifth percentile. Primary hypobetalipoproteinemia mostly results from heterozygous mutations in the (apolipoprotein B) and genes, and a polygenic origin is hypothesized in the remaining cases. Hypobetalipoproteinemia patients present an increased risk of nonalcoholic fatty liver disease and steatohepatitis. Here, we compared hepatic alterations between monogenic, polygenic, and primary hypobetalipoproteinemia of unknown cause. Approach and Results: Targeted next-generation sequencing was performed in a cohort of 111 patients with hypobetalipoproteinemia to assess monogenic and polygenic origins using an LDL-C-dedicated polygenic risk score. Forty patients (36%) had monogenic hypobetalipoproteinemia, 38 (34%) had polygenic hypobetalipoproteinemia, and 33 subjects (30%) had hypobetalipoproteinemia from an unknown cause. Patients with monogenic hypobetalipoproteinemia had lower LDL-C and apolipoprotein B plasma levels compared with those with polygenic hypobetalipoproteinemia. Liver function was assessed by hepatic ultrasonography and liver enzymes levels. Fifty-nine percent of patients with primary hypobetalipoproteinemia presented with liver steatosis, whereas 21% had increased alanine aminotransferase suggestive of liver injury. Monogenic hypobetalipoproteinemia was also associated with an increased prevalence of liver steatosis (81% versus 29%, <0.001) and liver injury (47% versus 0%) compared with polygenic hypobetalipoproteinemia.

Conclusions: This study highlights the importance of genetic diagnosis in the clinical care of primary hypobetalipoproteinemia patients. It shows for the first time that a polygenic origin of hypobetalipoproteinemia is associated with a lower risk of liver steatosis and liver injury versus monogenic hypobetalipoproteinemia. Thus, polygenic risk score is a useful tool to establish a more personalized follow-up of primary hypobetalipoproteinemia patients.
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http://dx.doi.org/10.1161/ATVBAHA.120.315491DOI Listing
January 2021

RPL13 Variants Cause Spondyloepimetaphyseal Dysplasia with Severe Short Stature.

Am J Hum Genet 2019 11 17;105(5):1040-1047. Epub 2019 Oct 17.

INSERM U1149/ERL 8252, Inflammation Research Center, 75018 Paris, France; AP-HP, Service d'Hématologie Biologique, Hôpital R. Debré, Université Paris 7 Denis Diderot, Sorbonne Paris Cité, 75019 Paris, France.

Variants in genes encoding ribosomal proteins have thus far been associated with Diamond-Blackfan anemia, a rare inherited bone marrow failure, and isolated congenital asplenia. Here, we report one de novo missense variant and three de novo splice variants in RPL13, which encodes ribosomal protein RPL13 (also called eL13), in four unrelated individuals with a rare bone dysplasia causing severe short stature. The three splice variants (c.477+1G>T, c.477+1G>A, and c.477+2 T>C) result in partial intron retention, which leads to an 18-amino acid insertion. In contrast to observations from Diamond-Blackfan anemia, we detected no evidence of significant pre-rRNA processing disturbance in cells derived from two affected individuals. Consistently, we showed that the insertion-containing protein is stably expressed and incorporated into 60S subunits similar to the wild-type protein. Erythroid proliferation in culture and ribosome profile on sucrose gradient are modified, suggesting a change in translation dynamics. We also provide evidence that RPL13 is present at high levels in chondrocytes and osteoblasts in mouse growth plates. Taken together, we show that the identified RPL13 variants cause a human ribosomopathy defined by a rare skeletal dysplasia, and we highlight the role of this ribosomal protein in bone development.
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http://dx.doi.org/10.1016/j.ajhg.2019.09.024DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6849359PMC
November 2019

Fryns type mesomelic dysplasia of the upper limbs caused by inverted duplications of the HOXD gene cluster.

Eur J Hum Genet 2020 03 7;28(3):324-332. Epub 2019 Oct 7.

Service de Génétique, Hôpital Bretonneau, CHU, Tours, France.

The HoxD cluster is critical for vertebrate limb development. Enhancers located in both the telomeric and centromeric gene deserts flanking the cluster regulate the transcription of HoxD genes. In rare patients, duplications, balanced translocations or inversions misregulating HOXD genes are responsible for mesomelic dysplasia of the upper and lower limbs. By aCGH, whole-genome mate-pair sequencing, long-range PCR and fiber fluorescent in situ hybridization, we studied patients from two families displaying mesomelic dysplasia limited to the upper limbs. We identified microduplications including the HOXD cluster and showed that microduplications were in an inverted orientation and inserted between the HOXD cluster and the telomeric enhancers. Our results highlight the existence of an autosomal dominant condition consisting of isolated ulnar dysplasia caused by microduplications inserted between the HOXD cluster and the telomeric enhancers. The duplications likely disconnect the HOXD9 to HOXD11 genes from their regulatory sequences. This presumptive loss-of-function may have contributed to the phenotype. In both cases, however, these rearrangements brought HOXD13 closer to telomeric enhancers, suggesting that the alterations derive from the dominant-negative effect of this digit-specific protein when ectopically expressed during the early development of forearms, through the disruption of topologically associating domain structure at the HOXD locus.
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http://dx.doi.org/10.1038/s41431-019-0522-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7028936PMC
March 2020

Identification of mobile retrocopies during genetic testing: Consequences for routine diagnosis.

Hum Mutat 2019 11 12;40(11):1993-2000. Epub 2019 Jul 12.

Genetics Department, Hospices Civils de Lyon, Lyon, France.

Human retrocopies, that is messenger RNA transcripts benefitting from the long interspersed element 1 machinery for retrotransposition, may have specific consequences for genomic testing. Next genetration sequencing (NGS) techniques allow the detection of such mobile elements but they may be misinterpreted as genomic duplications or be totally overlooked. We report eight observations of retrocopies detected during diagnostic NGS analyses of targeted gene panels, exome, or genome sequencing. For seven cases, while an exons-only copy number gain was called, read alignment inspection revealed a depth of coverage shift at every exon-intron junction where indels were also systematically called. Moreover, aberrant chimeric read pairs spanned entire introns or were paired with another locus for terminal exons. The 8th retrocopy was present in the reference genome and thus showed a normal NGS profile. We emphasize the existence of retrocopies and strategies to accurately detect them at a glance during genetic testing and discuss pitfalls for genetic testing.
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http://dx.doi.org/10.1002/humu.23845DOI Listing
November 2019

Identification of a new exon and complex splicing alterations in familial erythrocytosis or von Hippel-Lindau disease.

Blood 2018 08 11;132(5):469-483. Epub 2018 Jun 11.

Faculty of Medicine Alexandroupolis, Democritus University of Thrace, Thrace, Greece.

Chuvash polycythemia is an autosomal recessive form of erythrocytosis associated with a homozygous p.Arg200Trp mutation in the von Hippel-Lindau () gene. Since this discovery, additional mutations have been identified in patients with congenital erythrocytosis, in a homozygous or compound-heterozygous state. is a major tumor suppressor gene, mutations in which were first described in patients presenting with VHL disease, which is characterized by the development of highly vascularized tumors. Here, we identify a new cryptic exon (termed E1') deep in intron 1 that is naturally expressed in many tissues. More importantly, we identify mutations in E1' in 7 families with erythrocytosis (1 homozygous case and 6 compound-heterozygous cases with a mutation in E1' in addition to a mutation in coding sequences) and in 1 large family with typical VHL disease but without any alteration in the other exons. In this study, we show that the mutations induced a dysregulation of splicing with excessive retention of E1' and were associated with a downregulation of VHL protein expression. In addition, we demonstrate a pathogenic role for synonymous mutations in exon 2 that altered splicing through E2-skipping in 5 families with erythrocytosis or VHL disease. In all the studied cases, the mutations differentially affected splicing, correlating with phenotype severity. This study demonstrates that cryptic exon retention and exon skipping are new alterations and reveals a novel complex splicing regulation of the gene. These findings open new avenues for diagnosis and research regarding the VHL-related hypoxia-signaling pathway.
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http://dx.doi.org/10.1182/blood-2018-03-838235DOI Listing
August 2018

Rare Coding Variants in ANGPTL6 Are Associated with Familial Forms of Intracranial Aneurysm.

Am J Hum Genet 2018 01;102(1):133-141

INSERM, CNRS, UNIV Nantes, l'institut du thorax, 44007 Nantes, France; CHU Nantes, l'institut du thorax, 44093 Nantes, France. Electronic address:

Intracranial aneurysms (IAs) are acquired cerebrovascular abnormalities characterized by localized dilation and wall thinning in intracranial arteries, possibly leading to subarachnoid hemorrhage and severe outcome in case of rupture. Here, we identified one rare nonsense variant (c.1378A>T) in the last exon of ANGPTL6 (Angiopoietin-Like 6)-which encodes a circulating pro-angiogenic factor mainly secreted from the liver-shared by the four tested affected members of a large pedigree with multiple IA-affected case subjects. We showed a 50% reduction of ANGPTL6 serum concentration in individuals heterozygous for the c.1378A>T allele (p.Lys460Ter) compared to relatives homozygous for the normal allele, probably due to the non-secretion of the truncated protein produced by the c.1378A>T transcripts. Sequencing ANGPTL6 in a series of 94 additional index case subjects with familial IA identified three other rare coding variants in five case subjects. Overall, we detected a significant enrichment (p = 0.023) in rare coding variants within this gene among the 95 index case subjects with familial IA, compared to a reference population of 404 individuals with French ancestry. Among the 6 recruited families, 12 out of 13 (92%) individuals carrying IA also carry such variants in ANGPTL6, versus 15 out of 41 (37%) unaffected ones. We observed a higher rate of individuals with a history of high blood pressure among affected versus healthy individuals carrying ANGPTL6 variants, suggesting that ANGPTL6 could trigger cerebrovascular lesions when combined with other risk factors such as hypertension. Altogether, our results indicate that rare coding variants in ANGPTL6 are causally related to familial forms of IA.
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http://dx.doi.org/10.1016/j.ajhg.2017.12.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5778084PMC
January 2018

bioalcidae, samjs and vcffilterjs: object-oriented formatters and filters for bioinformatics files.

Bioinformatics 2018 04;34(7):1224-1225

Cardiovascular Genetics, L'Institut du Thorax, INSERM, CNRS, UNIV Nantes, Nantes 44007, France.

Motivation: Reformatting and filtering bioinformatics files are common tasks for bioinformaticians. Standard Linux tools and specific programs are usually used to perform such tasks but there is still a gap between using these tools and the programming interface of some existing libraries.

Results: In this study, we developed a set of tools namely bioalcidae, samjs and vcffilterjs that reformat or filter files using a JavaScript engine or a pure java expression and taking advantage of the java API for high-throughput sequencing data (htsjdk).

Availability And Implementation: https://github.com/lindenb/jvarkit.

Contact: [email protected]
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http://dx.doi.org/10.1093/bioinformatics/btx734DOI Listing
April 2018

De Novo Mutations in Protein Kinase Genes CAMK2A and CAMK2B Cause Intellectual Disability.

Am J Hum Genet 2017 Nov;101(5):768-788

Nottingham Regional Genetics Service, City Hospital Campus, Nottingham University Hospitals NHS Trust, The Gables, Hucknall Road, Nottingham NG5 1PB, UK.

Calcium/calmodulin-dependent protein kinase II (CAMK2) is one of the first proteins shown to be essential for normal learning and synaptic plasticity in mice, but its requirement for human brain development has not yet been established. Through a multi-center collaborative study based on a whole-exome sequencing approach, we identified 19 exceedingly rare de novo CAMK2A or CAMK2B variants in 24 unrelated individuals with intellectual disability. Variants were assessed for their effect on CAMK2 function and on neuronal migration. For both CAMK2A and CAMK2B, we identified mutations that decreased or increased CAMK2 auto-phosphorylation at Thr286/Thr287. We further found that all mutations affecting auto-phosphorylation also affected neuronal migration, highlighting the importance of tightly regulated CAMK2 auto-phosphorylation in neuronal function and neurodevelopment. Our data establish the importance of CAMK2A and CAMK2B and their auto-phosphorylation in human brain function and expand the phenotypic spectrum of the disorders caused by variants in key players of the glutamatergic signaling pathway.
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http://dx.doi.org/10.1016/j.ajhg.2017.10.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5673671PMC
November 2017

Accurate Identification and Quantification of DNA Species by Next-Generation Sequencing in Adeno-Associated Viral Vectors Produced in Insect Cells.

Hum Gene Ther Methods 2017 06 21;28(3):148-162. Epub 2017 Apr 21.

1 INSERM UMR1089, University of Nantes, Centre Hospitalier Universitaire, Nantes, France.

Recombinant adeno-associated viral (rAAV) vectors have proven excellent tools for the treatment of many genetic diseases and other complex diseases. However, the illegitimate encapsidation of DNA contaminants within viral particles constitutes a major safety concern for rAAV-based therapies. Moreover, the development of rAAV vectors for early-phase clinical trials has revealed the limited accuracy of the analytical tools used to characterize these new and complex drugs. Although most published data concerning residual DNA in rAAV preparations have been generated by quantitative PCR, we have developed a novel single-strand virus sequencing (SSV-Seq) method for quantification of DNA contaminants in AAV vectors produced in mammalian cells by next-generation sequencing (NGS). Here, we describe the adaptation of SSV-Seq for the accurate identification and quantification of DNA species in rAAV stocks produced in insect cells. We found that baculoviral DNA was the most abundant contaminant, representing less than 2.1% of NGS reads regardless of serotype (2, 8, or rh10). Sf9 producer cell DNA was detected at low frequency (≤0.03%) in rAAV lots. Advanced computational analyses revealed that (1) baculoviral sequences close to the inverted terminal repeats preferentially underwent illegitimate encapsidation, and (2) single-nucleotide variants were absent from the rAAV genome. The high-throughput sequencing protocol described here enables effective DNA quality control of rAAV vectors produced in insect cells, and is adapted to conform with regulatory agency safety requirements.
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http://dx.doi.org/10.1089/hgtb.2016.185DOI Listing
June 2017

Dysfunction of the Voltage-Gated K+ Channel β2 Subunit in a Familial Case of Brugada Syndrome.

J Am Heart Assoc 2016 06 10;5(6). Epub 2016 Jun 10.

INSERM, UMR 1087, l'Institut du Thorax, Nantes, France CNRS, UMR 6291, Nantes, France Université de Nantes, Nantes, France CHU Nantes, l'Institut du Thorax, Service de Cardiologie, Nantes, France

Background: The Brugada syndrome is an inherited cardiac arrhythmia associated with high risk of sudden death. Although 20% of patients with Brugada syndrome carry mutations in SCN5A, the molecular mechanisms underlying this condition are still largely unknown.

Methods And Results: We combined whole-exome sequencing and linkage analysis to identify the genetic variant likely causing Brugada syndrome in a pedigree for which SCN5A mutations had been excluded. This approach identified 6 genetic variants cosegregating with the Brugada electrocardiographic pattern within the pedigree. In silico gene prioritization pointed to 1 variant residing in KCNAB2, which encodes the voltage-gated K(+) channel β2-subunit (Kvβ2-R12Q). Kvβ2 is widely expressed in the human heart and has been shown to interact with the fast transient outward K(+) channel subunit Kv4.3, increasing its current density. By targeted sequencing of the KCNAB2 gene in 167 unrelated patients with Brugada syndrome, we found 2 additional rare missense variants (L13F and V114I). We then investigated the physiological effects of the 3 KCNAB2 variants by using cellular electrophysiology and biochemistry. Patch-clamp experiments performed in COS-7 cells expressing both Kv4.3 and Kvβ2 revealed a significant increase in the current density in presence of the R12Q and L13F Kvβ2 mutants. Although biotinylation assays showed no differences in the expression of Kv4.3, the total and submembrane expression of Kvβ2-R12Q were significantly increased in comparison with wild-type Kvβ2.

Conclusions: Altogether, our results indicate that Kvβ2 dysfunction can contribute to the Brugada electrocardiographic pattern.
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http://dx.doi.org/10.1161/JAHA.115.003122DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4937261PMC
June 2016

Targeted resequencing identifies TRPM4 as a major gene predisposing to progressive familial heart block type I.

Int J Cardiol 2016 Mar 11;207:349-58. Epub 2016 Jan 11.

Institut National de la Santé et de la Recherche Médicale (INSERM) Unité Mixte de Recherche (UMR) 1087, l'institut du thorax, Nantes, France; Centre National de la Recherche Scientifique (CNRS) UMR 6291, l'institut du thorax, Nantes, France; Université de Nantes, l'institut du thorax, Nantes, France; Centre Hospitalier Universitaire (CHU) de Nantes, l'institut du thorax, Service de Cardiologie, Nantes, France. Electronic address:

Background: Progressive cardiac conduction disease (PCCD) is one of the most common cardiac conduction disturbances. It has been causally related to rare mutations in several genes including SCN5A, SCN1B, TRPM4, LMNA and GJA5.

Methods And Results: In this study, by applying targeted next-generation sequencing (NGS) in 95 unrelated patients with PCCD, we have identified 13 rare variants in the TRPM4 gene, two of which are currently absent from public databases. This gene encodes a cardiac calcium-activated cationic channel which precise role and importance in cardiac conduction and disease is still debated. One novel variant, TRPM4-p.I376T, is carried by the proband of a large French 4-generation pedigree. Systematic familial screening showed that a total of 13 family members carry the mutation, including 10 out of the 11 tested affected individuals versus only 1 out of the 21 unaffected ones. Functional and biochemical analyses were performed using HEK293 cells, in whole-cell patch-clamp configuration and Western blotting. TRPM4-p.I376T results in an increased current density concomitant to an augmented TRPM4 channel expression at the cell surface.

Conclusions: This study is the first extensive NGS-based screening of TRPM4 coding variants in patients with PCCD. It reports the third largest pedigree diagnosed with isolated Progressive Familial Heart Block type I and confirms that this subtype of PCCD is caused by mutation-induced gain-of-expression and function of the TRPM4 ion channel.
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http://dx.doi.org/10.1016/j.ijcard.2016.01.052DOI Listing
March 2016

De Novo Truncating Mutations in the Kinetochore-Microtubules Attachment Gene CHAMP1 Cause Syndromic Intellectual Disability.

Hum Mutat 2016 Apr 4;37(4):354-8. Epub 2016 Feb 4.

Inserm, UMR 1087, l'institut du thorax, CHU Nantes, Nantes, France.

A rare syndromic form of intellectual disability with impaired speech was recently found associated with mutations in CHAMP1 (chromosome alignment-maintaining phosphoprotein 1), the protein product of which is directly involved in microtubule-kinetochore attachment. Through whole-exome sequencing in six unrelated nonconsanguineous families having a sporadic case of intellectual disability, we identified six novel de novo truncating mutations in CHAMP1: c.1880C>G p.(Ser627*), c.1489C>T; p.(Arg497*), c.1876_1877delAG; p.(Ser626Leufs*4), c.1043G>A; p.(Trp348*), c.1002G>A; p.(Trp334*), and c.958_959delCC; p.(Pro320*). Our clinical observations confirm the phenotypic homogeneity of the syndrome, which represents therefore a distinct clinical entity. Besides, our functional studies show that CHAMP1 protein variants are delocalized from chromatin and are unable to bind to two of its direct partners, POGZ and HP1. These data suggest a pathogenic mechanism of the CHAMP1-associated intellectual disability syndrome mediated by direct interacting partners of CHAMP1, several of which are involved in chromo/kinetochore-related disorders.
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http://dx.doi.org/10.1002/humu.22952DOI Listing
April 2016

Search for Rare Copy-Number Variants in Congenital Heart Defects Identifies Novel Candidate Genes and a Potential Role for FOXC1 in Patients With Coarctation of the Aorta.

Circ Cardiovasc Genet 2016 Feb 7;9(1):86-94. Epub 2015 Dec 7.

Background: Congenital heart defects are the most frequent malformations among newborns and a frequent cause of morbidity and mortality. Although genetic variation contributes to congenital heart defects, their precise molecular bases remain unknown in the majority of patients.

Methods And Results: We analyzed, by high-resolution array comparative genomic hybridization, 316 children with sporadic, nonsyndromic congenital heart defects, including 76 coarctation of the aorta, 159 transposition of the great arteries, and 81 tetralogy of Fallot, as well as their unaffected parents. We identified by array comparative genomic hybridization, and validated by quantitative real-time polymerase chain reaction, 71 rare de novo (n=8) or inherited (n=63) copy-number variants (CNVs; 50 duplications and 21 deletions) in patients. We identified 113 candidate genes for congenital heart defects within these CNVs, including BTRC, CHRNB3, CSRP2BP, ERBB2, ERMARD, GLIS3, PLN, PTPRJ, RLN3, and TCTE3. No de novo CNVs were identified in patients with transposition of the great arteries in contrast to coarctation of the aorta and tetralogy of Fallot (P=0.002; Fisher exact test). A search for transcription factor binding sites showed that 93% of the rare CNVs identified in patients with coarctation of the aorta contained at least 1 gene with FOXC1-binding sites. This significant enrichment (P<0.0001; permutation test) was not observed for the CNVs identified in patients with transposition of the great arteries and tetralogy of Fallot. We hypothesize that these CNVs may alter the expression of genes regulated by FOXC1. Foxc1 belongs to the forkhead transcription factors family, which plays a critical role in cardiovascular development in mice.

Conclusions: These data suggest that deregulation of FOXC1 or its downstream genes play a major role in the pathogenesis of coarctation of the aorta in humans.
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http://dx.doi.org/10.1161/CIRCGENETICS.115.001213DOI Listing
February 2016

Advanced Characterization of DNA Molecules in rAAV Vector Preparations by Single-stranded Virus Next-generation Sequencing.

Mol Ther Nucleic Acids 2015 Oct 27;4:e260. Epub 2015 Oct 27.

INSERM UMR 1089/Atlantic Gene Therapies, Nantes, France.

Recent successful clinical trials with recombinant adeno-associated viral vectors (rAAVs) have led to a renewed interest in gene therapy. However, despite extensive developments to improve vector-manufacturing processes, undesirable DNA contaminants in rAAV preparations remain a major safety concern. Indeed, the presence of DNA fragments containing antibiotic resistance genes, wild-type AAV, and packaging cell genomes has been found in previous studies using quantitative polymerase chain reaction (qPCR) analyses. However, because qPCR only provides a partial view of the DNA molecules in rAAV preparations, we developed a method based on next-generation sequencing (NGS) to extensively characterize single-stranded DNA virus preparations (SSV-Seq). In order to validate SSV-Seq, we analyzed three rAAV vector preparations produced by transient transfection of mammalian cells. Our data were consistent with qPCR results and showed a quasi-random distribution of contaminants originating from the packaging cells genome. Finally, we found single-nucleotide variants (SNVs) along the vector genome but no evidence of large deletions. Altogether, SSV-Seq could provide a characterization of DNA contaminants and a map of the rAAV genome with unprecedented resolution and exhaustiveness. We expect SSV-Seq to pave the way for a new generation of quality controls, guiding process development toward rAAV preparations of higher potency and with improved safety profiles.
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http://dx.doi.org/10.1038/mtna.2015.32DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4881760PMC
October 2015

Short-lived recombinant adeno-associated virus transgene expression in dystrophic muscle is associated with oxidative damage to transgene mRNA.

Mol Ther Methods Clin Dev 2015 8;2:15010. Epub 2015 Apr 8.

INSERM UMR 1089/Atlantic gene therapies , Nantes, France ; University of Nantes , Nantes, France ; Nantes University Hospital , Nantes, France.

Preclinical gene therapy strategies using recombinant adeno-associated virus (AAV) vectors in animal models of Duchenne muscular dystrophy have shown dramatic phenotype improvements, but long-lasting efficacy remains questionable. It is believed that in dystrophic muscles, transgene persistence is hampered, notably by the progressive loss of therapeutic vector genomes resulting from muscle fibers degeneration. Intracellular metabolic perturbations resulting from dystrophin deficiency could also be additional factors impacting on rAAV genomes and transgene mRNA molecular fate. In this study, we showed that rAAV genome loss is not the only cause of reduced transgene mRNA level and we assessed the contribution of transcriptional and post-transcriptional factors. We ruled out the implication of transgene silencing by epigenetic mechanisms and demonstrated that rAAV inhibition occurred mostly at the post-transcriptional level. Since Duchenne muscular dystrophy (DMD) physiopathology involves an elevated oxidative stress, we hypothesized that in dystrophic muscles, transgene mRNA could be damaged by oxidative stress. In the mouse and dog dystrophic models, we found that rAAV-derived mRNA oxidation was increased. Interestingly, when a high expression level of a therapeutic transgene is achieved, oxidation is less pronounced. These findings provide new insights into rAAV transductions in dystrophic muscles, which ultimately may help in the design of more effective clinical trials.
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http://dx.doi.org/10.1038/mtm.2015.10DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4445007PMC
June 2015

Testing the burden of rare variation in arrhythmia-susceptibility genes provides new insights into molecular diagnosis for Brugada syndrome.

Hum Mol Genet 2015 May 3;24(10):2757-63. Epub 2015 Feb 3.

Inserm, UMR 1087, l'institut du thorax, Nantes, France, CNRS, UMR 6291, Nantes, France, Université de Nantes, Nantes, France, CHU Nantes, l'institut du thorax, Service de Cardiologie, Nantes, France,

The Brugada syndrome (BrS) is a rare heritable cardiac arrhythmia disorder associated with ventricular fibrillation and sudden cardiac death. Mutations in the SCN5A gene have been causally related to BrS in 20-30% of cases. Twenty other genes have been described as involved in BrS, but their overall contribution to disease prevalence is still unclear. This study aims to estimate the burden of rare coding variation in arrhythmia-susceptibility genes among a large group of patients with BrS. We have developed a custom kit to capture and sequence the coding regions of 45 previously reported arrhythmia-susceptibility genes and applied this kit to 167 index cases presenting with a Brugada pattern on the electrocardiogram as well as 167 individuals aged over 65-year old and showing no history of cardiac arrhythmia. By applying burden tests, a significant enrichment in rare coding variation (with a minor allele frequency below 0.1%) was observed only for SCN5A, with rare coding variants carried by 20.4% of cases with BrS versus 2.4% of control individuals (P = 1.4 × 10(-7)). No significant enrichment was observed for any other arrhythmia-susceptibility gene, including SCN10A and CACNA1C. These results indicate that, except for SCN5A, rare coding variation in previously reported arrhythmia-susceptibility genes do not contribute significantly to the occurrence of BrS in a population with European ancestry. Extreme caution should thus be taken when interpreting genetic variation in molecular diagnostic setting, since rare coding variants were observed in a similar extent among cases versus controls, for most previously reported BrS-susceptibility genes.
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http://dx.doi.org/10.1093/hmg/ddv036DOI Listing
May 2015

mod_bio: Apache modules for Next-Generation sequencing data.

Bioinformatics 2015 Jan 4;31(1):112-3. Epub 2014 Sep 4.

Institut National de la Santé et de la Recherche Médicale (INSERM) Unité Mixte de Recherche (UMR) 1087, L'Institut du Thorax, Centre National de la Recherche Scientifique (CNRS) UMR 6291, Centre Hospitalier Universitaire (CHU) de Nantes, L'Institut du Thorax, Service de Cardiologie, 44000 Nantes and Université de Nantes, 44000 Nantes, France Institut National de la Santé et de la Recherche Médicale (INSERM) Unité Mixte de Recherche (UMR) 1087, L'Institut du Thorax, Centre National de la Recherche Scientifique (CNRS) UMR 6291, Centre Hospitalier Universitaire (CHU) de Nantes, L'Institut du Thorax, Service de Cardiologie, 44000 Nantes and Université de Nantes, 44000 Nantes, France Institut National de la Santé et de la Recherche Médicale (INSERM) Unité Mixte de Recherche (UMR) 1087, L'Institut du Thorax, Centre National de la Recherche Scientifique (CNRS) UMR 6291, Centre Hospitalier Universitaire (CHU) de Nantes, L'Institut du Thorax, Service de Cardiologie, 44000 Nantes and Université de Nantes, 44000 Nantes, France Institut National de la Santé et de la Recherche Médicale (INSERM) Unité Mixte de Recherche (UMR) 1087, L'Institut du Thorax, Centre National de la Recherche Scientifique (CNRS) UMR 6291, Centre Hospitalier Universitaire (CHU) de Nantes, L'Institut du Thorax, Service de Cardiologie, 44000 Nantes and Université de Nantes, 44000 Nantes, France.

Summary: We describe mod_bio, a set of modules for the Apache HTTP server that allows the users to access and query fastq, tabix, fasta and bam files through a Web browser. Those data are made available in plain text, HTML, XML, JSON and JSON-P. A javascript-based genome browser using the JSON-P communication technique is provided as an example of cross-domain Web service.

Availability And Implementation: https://github.com/lindenb/mod_bio.
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http://dx.doi.org/10.1093/bioinformatics/btu547DOI Listing
January 2015

NGS library preparation may generate artifactual integration sites of AAV vectors.

Nat Med 2014 Jun;20(6):577-8

1] INSERM, UMR 1089, Nantes, France. [2] University of Nantes, Nantes, France. [3] Nantes University Hospital, Nantes, France.

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http://dx.doi.org/10.1038/nm.3578DOI Listing
June 2014

Mutations in FAM111B cause hereditary fibrosing poikiloderma with tendon contracture, myopathy, and pulmonary fibrosis.

Am J Hum Genet 2013 Dec 21;93(6):1100-7. Epub 2013 Nov 21.

Unité de Génétique Clinique, Service de Génétique Médicale, Centre de Référence Anomalies de Développement et Syndromes Malformatifs de l'Interrégion Grand-Ouest, Centre Hospitalier Universitaire Nantes, 9 Quai Moncousu, 44093 Nantes Cedex 1, France; Institut National de la Santé et de la Recherche Médicale UMR 1089, Atlantic Gene Therapy Institute, University of Nantes, 44007 Nantes, France.

Congenital poikiloderma is characterized by a combination of mottled pigmentation, telangiectasia, and epidermal atrophy in the first few months of life. We have previously described a South African European-descent family affected by a rare autosomal-dominant form of hereditary fibrosing poikiloderma accompanied by tendon contracture, myopathy, and pulmonary fibrosis. Here, we report the identification of causative mutations in FAM111B by whole-exome sequencing. In total, three FAM111B missense mutations were identified in five kindreds of different ethnic backgrounds. The mutation segregated with the disease in one large pedigree, and mutations were de novo in two other pedigrees. All three mutations were absent from public databases and were not observed on Sanger sequencing of 388 ethnically matched control subjects. The three single-nucleotide mutations code for amino acid changes that are clustered within a putative trypsin-like cysteine/serine peptidase domain of FAM111B. These findings provide evidence of the involvement of FAM111B in congenital poikiloderma and multisystem fibrosis.
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http://dx.doi.org/10.1016/j.ajhg.2013.10.013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3853004PMC
December 2013

Common variants at SCN5A-SCN10A and HEY2 are associated with Brugada syndrome, a rare disease with high risk of sudden cardiac death.

Nat Genet 2013 Sep 21;45(9):1044-9. Epub 2013 Jul 21.

Department of Clinical and Experimental Cardiology, Heart Failure Research Center, Academic Medical Center, Amsterdam, The Netherlands.

Brugada syndrome is a rare cardiac arrhythmia disorder, causally related to SCN5A mutations in around 20% of cases. Through a genome-wide association study of 312 individuals with Brugada syndrome and 1,115 controls, we detected 2 significant association signals at the SCN10A locus (rs10428132) and near the HEY2 gene (rs9388451). Independent replication confirmed both signals (meta-analyses: rs10428132, P = 1.0 × 10(-68); rs9388451, P = 5.1 × 10(-17)) and identified one additional signal in SCN5A (at 3p21; rs11708996, P = 1.0 × 10(-14)). The cumulative effect of the three loci on disease susceptibility was unexpectedly large (Ptrend = 6.1 × 10(-81)). The association signals at SCN5A-SCN10A demonstrate that genetic polymorphisms modulating cardiac conduction can also influence susceptibility to cardiac arrhythmia. The implication of association with HEY2, supported by new evidence that Hey2 regulates cardiac electrical activity, shows that Brugada syndrome may originate from altered transcriptional programming during cardiac development. Altogether, our findings indicate that common genetic variation can have a strong impact on the predisposition to rare diseases.
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http://dx.doi.org/10.1038/ng.2712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3869788PMC
September 2013

The 3rd DBCLS BioHackathon: improving life science data integration with Semantic Web technologies.

J Biomed Semantics 2013 Feb 11;4(1). Epub 2013 Feb 11.

Database Center for Life Science, Research Organization of Information and Systems, 2-11-16, Yayoi, Bunkyo-ku, Tokyo, 113-0032, Japan.

Background: BioHackathon 2010 was the third in a series of meetings hosted by the Database Center for Life Sciences (DBCLS) in Tokyo, Japan. The overall goal of the BioHackathon series is to improve the quality and accessibility of life science research data on the Web by bringing together representatives from public databases, analytical tool providers, and cyber-infrastructure researchers to jointly tackle important challenges in the area of in silico biological research.

Results: The theme of BioHackathon 2010 was the 'Semantic Web', and all attendees gathered with the shared goal of producing Semantic Web data from their respective resources, and/or consuming or interacting those data using their tools and interfaces. We discussed on topics including guidelines for designing semantic data and interoperability of resources. We consequently developed tools and clients for analysis and visualization.

Conclusion: We provide a meeting report from BioHackathon 2010, in which we describe the discussions, decisions, and breakthroughs made as we moved towards compliance with Semantic Web technologies - from source provider, through middleware, to the end-consumer.
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http://dx.doi.org/10.1186/2041-1480-4-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3598643PMC
February 2013

Mass spectrometry-based identification of native cardiac Nav1.5 channel α subunit phosphorylation sites.

J Proteome Res 2012 Dec 9;11(12):5994-6007. Epub 2012 Nov 9.

INSERM, UMR1087, CNRS, UMR6291, LUNAM Université, Nantes, France.

Cardiac voltage-gated Na+ (Nav) channels are key determinants of action potential waveforms, refractoriness and propagation, and Nav1.5 is the main Nav pore-forming (α) subunit in the mammalian heart. Although direct phosphorylation of the Nav1.5 protein has been suggested to modulate various aspects of Nav channel physiology and pathophysiology, native Nav1.5 phosphorylation sites have not been identified. In the experiments here, a mass spectrometry (MS)-based proteomic approach was developed to identify native Nav1.5 phosphorylation sites directly. Using an anti-NavPAN antibody, Nav channel complexes were immunoprecipitated from adult mouse cardiac ventricles. The MS analyses revealed that this antibody immunoprecipitates several Nav α subunits in addition to Nav1.5, as well as several previously identified Nav channel associated/regulatory proteins. Label-free comparative and data-driven phosphoproteomic analyses of purified cardiac Nav1.5 protein identified 11 phosphorylation sites, 8 of which are novel. All the phosphorylation sites identified except one in the N-terminus are in the first intracellular linker loop, suggesting critical roles for this region in phosphorylation-dependent cardiac Nav channel regulation. Interestingly, commonly used prediction algorithms did not reliably predict these newly identified in situ phosphorylation sites. Taken together, the results presented provide the first in situ map of basal phosphorylation sites on the mouse cardiac Nav1.5 α subunit.
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http://dx.doi.org/10.1021/pr300702cDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3518584PMC
December 2012

BioStar: an online question & answer resource for the bioinformatics community.

PLoS Comput Biol 2011 Oct 27;7(10):e1002216. Epub 2011 Oct 27.

Nutrition and Genomics Laboratory, Jean Mayer-United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, Massachusetts, United States of America.

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http://dx.doi.org/10.1371/journal.pcbi.1002216DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3203049PMC
October 2011

Knime4Bio: a set of custom nodes for the interpretation of next-generation sequencing data with KNIME.

Bioinformatics 2011 Nov 7;27(22):3200-1. Epub 2011 Oct 7.

Institut du thorax, Inserm UMR 915, Centre Hospitalier Universitaire de Nantes, 44000 Nantes, France.

Summary: Analysing large amounts of data generated by next-generation sequencing (NGS) technologies is difficult for researchers or clinicians without computational skills. They are often compelled to delegate this task to computer biologists working with command line utilities. The availability of easy-to-use tools will become essential with the generalization of NGS in research and diagnosis. It will enable investigators to handle much more of the analysis. Here, we describe Knime4Bio, a set of custom nodes for the KNIME (The Konstanz Information Miner) interactive graphical workbench, for the interpretation of large biological datasets. We demonstrate that this tool can be utilized to quickly retrieve previously published scientific findings.
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http://dx.doi.org/10.1093/bioinformatics/btr554DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3208396PMC
November 2011

Truncating mutations in the last exon of NOTCH2 cause a rare skeletal disorder with osteoporosis.

Nat Genet 2011 Mar 6;43(4):306-8. Epub 2011 Mar 6.

CHU Nantes, Service de Génétique Médicale, Nantes, France.

Hajdu-Cheney syndrome is a rare autosomal dominant skeletal disorder with facial anomalies, osteoporosis and acro-osteolysis. We sequenced the exomes of six unrelated individuals with this syndrome and identified heterozygous nonsense and frameshift mutations in NOTCH2 in five of them. All mutations cluster to the last coding exon of the gene, suggesting that the mutant mRNA products escape nonsense-mediated decay and that the resulting truncated NOTCH2 proteins act in a gain-of-function manner.
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http://dx.doi.org/10.1038/ng.778DOI Listing
March 2011

The Gene Wiki: community intelligence applied to human gene annotation.

Nucleic Acids Res 2010 Jan 15;38(Database issue):D633-9. Epub 2009 Sep 15.

Genomics Institute of the Novartis Research Foundation, San Diego, CA 92121, USA.

Annotating the function of all human genes is a critical, yet formidable, challenge. Current gene annotation efforts focus on centralized curation resources, but it is increasingly clear that this approach does not scale with the rapid growth of the biomedical literature. The Gene Wiki utilizes an alternative and complementary model based on the principle of community intelligence. Directly integrated within the online encyclopedia, Wikipedia, the goal of this effort is to build a gene-specific review article for every gene in the human genome, where each article is collaboratively written, continuously updated and community reviewed. Previously, we described the creation of Gene Wiki 'stubs' for approximately 9000 human genes. Here, we describe ongoing systematic improvements to these articles to increase their utility. Moreover, we retrospectively examine the community usage and improvement of the Gene Wiki, providing evidence of a critical mass of users and editors. Gene Wiki articles are freely accessible within the Wikipedia web site, and additional links and information are available at http://en.wikipedia.org/wiki/Portal:Gene_Wiki.
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http://dx.doi.org/10.1093/nar/gkp760DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2808918PMC
January 2010

Robust physical methods that enrich genomic regions identical by descent for linkage studies: confirmation of a locus for osteogenesis imperfecta.

BMC Genet 2009 Mar 30;10:16. Epub 2009 Mar 30.

IntegraGen SA, Evry, France.

Background: The monogenic disease osteogenesis imperfecta (OI) is due to single mutations in either of the collagen genes ColA1 or ColA2, but within the same family a given mutation is accompanied by a wide range of disease severity. Although this phenotypic variability implies the existence of modifier gene variants, genome wide scanning of DNA from OI patients has not been reported. Promising genome wide marker-independent physical methods for identifying disease-related loci have lacked robustness for widespread applicability. Therefore we sought to improve these methods and demonstrate their performance to identify known and novel loci relevant to OI.

Results: We have improved methods for enriching regions of identity-by-descent (IBD) shared between related, afflicted individuals. The extent of enrichment exceeds 10- to 50-fold for some loci. The efficiency of the new process is shown by confirmation of the identification of the Col1A2 locus in osteogenesis imperfecta patients from Amish families. Moreover the analysis revealed additional candidate linkage loci that may harbour modifier genes for OI; a locus on chromosome 1q includes COX-2, a gene implicated in osteogenesis.

Conclusion: Technology for physical enrichment of IBD loci is now robust and applicable for finding genes for monogenic diseases and genes for complex diseases. The data support the further investigation of genetic loci other than collagen gene loci to identify genes affecting the clinical expression of osteogenesis imperfecta. The discrimination of IBD mapping will be enhanced when the IBD enrichment procedure is coupled with deep resequencing.
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http://dx.doi.org/10.1186/1471-2156-10-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2679057PMC
March 2009

Association of autism with polymorphisms in the paired-like homeodomain transcription factor 1 (PITX1) on chromosome 5q31: a candidate gene analysis.

BMC Med Genet 2007 Dec 6;8:74. Epub 2007 Dec 6.

IntegraGen SA, 5 rue Henri Desbruères, Genopole Campus 1, Genavenir 8, 91000 Evry, France.

Background: Autism is a complex, heterogeneous, behaviorally-defined disorder characterized by disruptions of the nervous system and of other systems such as the pituitary-hypothalamic axis. In a previous genome wide screen, we reported linkage of autism with a 1.2 Megabase interval on chromosome 5q31. For the current study, we hypothesized that 3 of the genes in this region could be involved in the development of autism: 1) paired-like homeodomain transcription factor 1 (PITX1), which is a key regulator of hormones within the pituitary-hypothalamic axis, 2) neurogenin 1, a transcription factor involved in neurogenesis, and 3) histone family member Y (H2AFY), which is involved in X-chromosome inactivation in females and could explain the 4:1 male:female gender distortion present in autism.

Methods: A total of 276 families from the Autism Genetic Resource Exchange (AGRE) repository composed of 1086 individuals including 530 affected children were included in the study. Single nucleotide polymorphisms tagging the three candidate genes were genotyped on the initial linkage sample of 116 families. A second step of analysis was performed using tightly linked SNPs covering the PITX1 gene. Association was evaluated using the FBAT software version 1.7.3 for single SNP analysis and the HBAT command from the same package for haplotype analysis respectively.

Results: Association between SNPs and autism was only detected for PITX1. Haplotype analysis within PITX1 showed evidence for overtransmission of the A-C haplotype of markers rs11959298 - rs6596189 (p = 0.0004). Individuals homozygous or heterozygous for the A-C haplotype risk allele were 2.54 and 1.59 fold more likely to be autistic than individuals who were not carrying the allele, respectively.

Conclusion: Strong and consistent association was observed between a 2 SNPs within PITX1 and autism. Our data suggest that PITX1, a key regulator of hormones within the pituitary-hypothalamic axis, may be implicated in the etiology of autism.
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http://dx.doi.org/10.1186/1471-2350-8-74DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2222245PMC
December 2007

RoXaN, a novel cellular protein containing TPR, LD, and zinc finger motifs, forms a ternary complex with eukaryotic initiation factor 4G and rotavirus NSP3.

J Virol 2004 Apr;78(8):3851-62

Virologie Moléculaire et Structurale, Unité Mixte de Recherche, CNRS-INRA, 91198 Gif-sur-Yvette, France.

Rotavirus mRNAs are capped but not polyadenylated, and viral proteins are translated by the cellular translation machinery. This is accomplished through the action of the viral nonstructural protein NSP3, which specifically binds the 3' consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G I. To further our understanding of the role of NSP3 in rotavirus replication, we looked for other cellular proteins capable of interacting with this viral protein. Using the yeast two-hybrid assay, we identified a novel cellular protein-binding partner for rotavirus NSP3. This 110-kDa cellular protein, named RoXaN (rotavirus X protein associated with NSP3), contains a minimum of three regions predicted to be involved in protein-protein or nucleic acid-protein interactions. A tetratricopeptide repeat region, a protein-protein interaction domain most often found in multiprotein complexes, is present in the amino-terminal region. In the carboxy terminus, at least five zinc finger motifs are observed, further suggesting the capacity of RoXaN to bind other proteins or nucleic acids. Between these two regions exists a paxillin leucine-aspartate repeat (LD) motif which is involved in protein-protein interactions. RoXaN is capable of interacting with NSP3 in vivo and during rotavirus infection. Domains of interaction were mapped and correspond to the dimerization domain of NSP3 (amino acids 163 to 237) and the LD domain of RoXaN (amino acids 244 to 341). The interaction between NSP3 and RoXaN does not impair the interaction between NSP3 and eIF4G I, and a ternary complex made of NSP3, RoXaN, and eIF4G I can be detected in rotavirus-infected cells, implicating RoXaN in translation regulation.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC374268PMC
http://dx.doi.org/10.1128/jvi.78.8.3851-3862.2004DOI Listing
April 2004
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