Spontaneous Akt2 deficiency in a colony of NOD mice exhibiting early diabetes.

Diabetes constitutes a major public health problem, with dramatic consequences for patients. Both genetic and environmental factors were shown to contribute to the different forms of the disease. The monogenic forms, found both in humans and in animal models, specially help to decipher the role of key genes in the physiopathology of the disease. Here, we describe the phenotype of early diabetes in a colony of NOD mice, with spontaneous invalidation of Akt2, that we called HYP. The HYP mice were characterised by a strong and chronic hyperglycaemia, beginning around the age of one month, especially in male mice. The phenotype was not the consequence of the acceleration of the autoimmune response, inherent to the NOD background. Interestingly, in HYP mice, we observed hyperinsulinemia before hyperglycaemia occurred. We did not find any difference in the pancreas' architecture of the NOD and HYP mice (islets' size and staining for insulin and glucagon) but we detected a lower insulin content in the pancreas of HYP mice compared to NOD mice. These results give new insights about the role played by Akt2 in glucose homeostasis and argue for the ß cell failure being the primary event in the course of diabetes.

TAD boundary deletion causes PITX2-related cardiac electrical and structural defects.

While 3D chromatin organization in topologically associating domains (TADs) and loops mediating regulatory element-promoter interactions is crucial for tissue-specific gene regulation, the extent of their involvement in human Mendelian disease is largely unknown. Here, we identify 7 families presenting a new cardiac entity associated with a heterozygous deletion of 2 CTCF binding sites on 4q25, inducing TAD fusion and chromatin conformation remodeling. The CTCF binding sites are located in a gene desert at 1 Mb from the Paired-like homeodomain transcription factor 2 gene (PITX2). By introducing the ortholog of the human deletion in the mouse genome, we recapitulate the patient phenotype and characterize an opposite dysregulation of PITX2 expression in the sinoatrial node (ectopic activation) and ventricle (reduction), respectively. Chromatin conformation assay performed in human induced pluripotent stem cell-derived cardiomyocytes harboring the minimal deletion identified in family#1 reveals a conformation remodeling and fusion of TADs. We conclude that TAD remodeling mediated by deletion of CTCF binding sites causes a new autosomal dominant Mendelian cardiac disorder.

Generation of a patient-specific induced pluripotent stem cell line carrying the DES p.R406W mutation, an isogenic control and a DES p.R406W knock-in line.

Mutations in the DES gene, which encodes the intermediate filament desmin, lead to desminopathy, a rare disease characterized by skeletal muscle weakness and different forms of cardiomyopathies associated with cardiac conduction defects and arrhythmias. We generated human induced pluripotent stem cells (hiPSC) from a patient carrying the DES p.R406W mutation, and employed CRISPR/Cas9 to rectify the mutation in the patient's hiPSC line and introduced the mutation in an hiPSC line from a control individual unrelated to the patient. These hiPSC lines represent useful models for delving into the mechanisms of desminopathy and developing new therapeutic approaches.

Identification of a Gain-of-Function LIPC Variant as a Novel Cause of Familial Combined Hypocholesterolemia.

BACKGROUND: Atherosclerotic cardiovascular disease is the main cause of mortality worldwide and is strongly influenced by circulating low-density lipoprotein (LDL) cholesterol levels. Only a few genes causally related to plasma LDL cholesterol levels have been identified so far, and only 1 gene, ANGPTL3, has been causally related to combined hypocholesterolemia. Here, our aim was to elucidate the genetic origin of an unexplained combined hypocholesterolemia inherited in 4 generations of a French family. METHODS: Using next-generation sequencing, we identified a novel dominant rare variant in the LIPC gene, encoding for hepatic lipase, which cosegregates with the phenotype. We characterized the impact of this LIPC-E97G variant on circulating lipid and lipoprotein levels in family members using nuclear magnetic resonance-based lipoprotein profiling and lipidomics. To uncover the mechanisms underlying the combined hypocholesterolemia, we used protein homology modeling, measured triglyceride lipase and phospholipase activities in cell culture, and studied the phenotype of APOE*3.Leiden.CETP mice after LIPC-E97G overexpression. RESULTS: Family members carrying the LIPC-E97G variant had very low circulating levels of LDL cholesterol and high-density lipoprotein cholesterol, LDL particle numbers, and phospholipids. The lysophospholipids/phospholipids ratio was increased in plasma of LIPC-E97G carriers, suggestive of an increased lipolytic activity on phospholipids. In vitro and in vivo studies confirmed that the LIPC-E97G variant specifically increases the phospholipase activity of hepatic lipase through modification of an evolutionarily conserved motif that determines substrate access to the hepatic lipase catalytic site. Mice overexpressing human LIPC-E97G recapitulated the combined hypocholesterolemic phenotype of the family and demonstrated that the increased phospholipase activity promotes catabolism of triglyceride-rich lipoproteins by different extrahepatic tissues but not the liver. CONCLUSIONS: We identified and characterized a novel rare variant in the LIPC gene in a family who presents with dominant familial combined hypocholesterolemia. This gain-of-function variant makes LIPC the second identified gene, after ANGPTL3, causally involved in familial combined hypocholesterolemia. Our mechanistic data highlight the critical role of hepatic lipase phospholipase activity in LDL cholesterol homeostasis and suggest a new LDL clearance mechanism.

Burden of rare variants in arrhythmogenic cardiomyopathy with right dominant form-associated genes provides new insights for molecular diagnosis and clinical management.

Arrhythmogenic cardiomyopathy with right dominant form (ACR) is a rare heritable cardiac cardiomyopathy disorder associated with sudden cardiac death. Pathogenic variants (PVs) in desmosomal genes have been causally related to ACR in 40% of cases. Other genes encoding nondesmosomal proteins have been described in ACR, but their contribution in this pathology is still debated. A panel of 71 genes associated with inherited cardiopathies was screened in an ACR population of 172 probands and 856 individuals from the general population. PVs and uncertain significance variants (VUS) have been identified in 36% and 18.6% of patients, respectively. Among the cardiopathy-associated genes, burden tests show a significant enrichment in PV and VUS only for desmosomal genes PKP2 (plakophilin-2), DSP (desmoplakin), DSC2 (desmocollin-2), and DSG2 (desmoglein-2). Importantly, VUS may account for 15% of ACR cases and should then be considered for molecular diagnosis. Among the other genes, no evidence of enrichment was detected, suggesting an extreme caution in the interpretation of these genetic variations without associated functional or segregation data. Genotype-phenotype correlation points to (1) a more severe and earlier onset of the disease in PV and VUS carriers, underlying the importance to carry out presymptomatic diagnosis in relatives and (2) to a more prevalent left ventricular dysfunction in DSP variant carriers.

Rare pathogenic variants in WNK3 cause X-linked intellectual disability.

PURPOSE: WNK3 kinase (PRKWNK3) has been implicated in the development and function of the brain via its regulation of the cation-chloride cotransporters, but the role of WNK3 in human development is unknown. METHOD: We ascertained exome or genome sequences of individuals with rare familial or sporadic forms of intellectual disability (ID). RESULTS: We identified a total of 6 different maternally-inherited, hemizygous, 3 loss-of-function or 3 pathogenic missense variants (p.Pro204Arg, p.Leu300Ser, p.Glu607Val) in WNK3 in 14 male individuals from 6 unrelated families. Affected individuals had ID with variable presence of epilepsy and structural brain defects. WNK3 variants cosegregated with the disease in 3 different families with multiple affected individuals. This included 1 large family previously diagnosed with X-linked Prieto syndrome. WNK3 pathogenic missense variants localize to the catalytic domain and impede the inhibitory phosphorylation of the neuronal-specific chloride cotransporter KCC2 at threonine 1007, a site critically regulated during the development of synaptic inhibition. CONCLUSION: Pathogenic WNK3 variants cause a rare form of human X-linked ID with variable epilepsy and structural brain abnormalities and implicate impaired phospho-regulation of KCC2 as a pathogenic mechanism.

APOB CRISPR-Cas9 Engineering in Hypobetalipoproteinemia: A Promising Tool for Functional Studies of Novel Variants.

Hypobetalipoproteinemia is characterized by LDL-cholesterol and apolipoprotein B (apoB) plasma levels below the fifth percentile for age and sex. Familial hypobetalipoproteinemia (FHBL) is mostly caused by premature termination codons in the APOB gene, a condition associated with fatty liver and steatohepatitis. Nevertheless, many families with a FHBL phenotype carry APOB missense variants of uncertain significance (VUS). We here aimed to develop a proof-of-principle experiment to assess the pathogenicity of VUS using the genome editing of human liver cells. We identified a novel heterozygous APOB-VUS (p.Leu351Arg), in a FHBL family. We generated APOB knock-out (KO) and APOB-p.Leu351Arg knock-in Huh7 cells using CRISPR-Cas9 technology and studied the APOB expression, synthesis and secretion by digital droplet PCR and ELISA quantification. The APOB expression was decreased by 70% in the heterozygous APOB-KO cells and almost abolished in the homozygous-KO cells, with a consistent decrease in apoB production and secretion. The APOB-p.Leu351Arg homozygous cells presented with a 40% decreased APOB expression and undetectable apoB levels in cellular extracts and supernatant. Thus, the p.Leu351Arg affected the apoB secretion, which led us to classify this new variant as likely pathogenic and to set up a hepatic follow-up in this family. Therefore, the functional assessment of APOB-missense variants, using gene-editing technologies, will lead to improvements in the molecular diagnosis of FHBL and the personalized follow-up of these patients.

Phenotypic Differences Between Polygenic and Monogenic Hypobetalipoproteinemia.

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 APOB (apolipoprotein B) and PCSK9 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%, P<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.

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

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.

RPL13 Variants Cause Spondyloepimetaphyseal Dysplasia with Severe Short Stature.

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.

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

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.

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

Chuvash polycythemia is an autosomal recessive form of erythrocytosis associated with a homozygous p.Arg200Trp mutation in the von Hippel-Lindau (VHL) gene. Since this discovery, additional VHL mutations have been identified in patients with congenital erythrocytosis, in a homozygous or compound-heterozygous state. VHL 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 VHL 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 VHL coding sequences) and in 1 large family with typical VHL disease but without any alteration in the other VHL exons. In this study, we show that the mutations induced a dysregulation of VHL 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 VHL 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 VHL alterations and reveals a novel complex splicing regulation of the VHL gene. These findings open new avenues for diagnosis and research regarding the VHL-related hypoxia-signaling pathway.

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

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.

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

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: pierre.lindenbaum@univ-nantes.fr.

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

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.

Dysfunction of the Voltage-Gated K+ Channel ß2 Subunit in a Familial Case of Brugada Syndrome.

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 ß2-subunit (Kvß2-R12Q). Kvß2 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 Kvß2 revealed a significant increase in the current density in presence of the R12Q and L13F Kvß2 mutants. Although biotinylation assays showed no differences in the expression of Kv4.3, the total and submembrane expression of Kvß2-R12Q were significantly increased in comparison with wild-type Kvß2. CONCLUSIONS: Altogether, our results indicate that Kvß2 dysfunction can contribute to the Brugada electrocardiographic pattern.

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

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.

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

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.

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.

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.

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

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.