Gladys Zapata

Integrative genomic analysis of the human immune response to influenza vaccination

Identification of the host genetic factors that contribute to variation in vaccine responsiveness may uncover important mechanisms affecting vaccine efficacy. We carried out an integrative, longitudinal study combining genetic, transcriptional, and immunologic data in humans given seasonal influenza vaccine. We identified 20 genes exhibiting a transcriptional response to vaccination, significant genotype effects on gene expression, and correlation between the transcriptional and antibody responses. The results show that variation at the level of genes involved in membrane trafficking and antigen processing significantly influences the human response to influenza vaccination. More broadly, we demonstrate that an integrative study design is an efficient alternative to existing methods for the identification of genes involved in complex traits. DOI: http://dx.doi.org/10.7554/eLife.00299.001

20p12.3 microdeletion predisposes to Wolff-Parkinson-White syndrome with variable neurocognitive deficits

Background: Wolff–Parkinson–White syndrome (WPW) is a bypass re-entrant tachycardia that results from an abnormal connection between the atria and ventricles. Mutations in PRKAG2 have been described in patients with familial WPW syndrome and hypertrophic cardiomyopathy. Based on the role of bone morphogenetic protein (BMP) signalling in the development of annulus fibrosus in mice, it has been proposed that BMP signalling through the type 1a receptor and other downstream components may play a role in pre-excitation. Methods and results: Using the array comparative genomic hybridisation (CGH), we identified five individuals with non-recurrent deletions of 20p12.3. Four of these individuals had WPW syndrome with variable dysmorphisms and neurocognitive delay. With the exception of one maternally inherited deletion, all occurred de novo, and the smallest of these harboured a single gene, BMP2. In two individuals with additional features of Alagille syndrome, deletion of both JAG1 and BMP2 were identified. Deletion of this region has not been described as a copy number variant in the Database of Genomic Variants and has not been identified in 13 321 individuals from other cohort examined by array CGH in our laboratory. Conclusions: Our findings demonstrate a novel genomic disorder characterised by deletion of BMP2 with variable cognitive deficits and dysmorphic features and show that individuals bearing microdeletions in 20p12.3 often present with WPW syndrome.

Association of common variants in ERBB4 with congenital left ventricular outflow tract obstruction defects

BACKGROUND The left ventricular outflow tract (LVOT) defects aortic valve stenosis (AVS), coarctation of the aorta (COA), and hypoplastic left heart syndrome (HLHS) represent an embryologically related group of congenital cardiovascular malformations. They are common and cause substantial morbidity and mortality. Prior evidence suggests a strong genetic component in their causation. METHODS We selected NRG1, ERBB3, and ERBB4 of the epidermal growth factor receptor (EGFR) signaling pathway as candidate genes for investigation of association with LVOT defects based on the importance of this pathway in cardiac development and the phenotypes in knockout mouse models. Single nucleotide polymorphism (SNP) genotyping was performed on 343 affected case-parent trios of European ancestry. RESULTS We identified a specific haplotype in intron 3 of ERBB4 that was positively associated with the combined LVOT defects phenotype (p=0.0005) and in each anatomic defect AVS, COA, and HLHS separately. Mutation screening of individuals with an LVOT defect failed to identify a coding sequence or splice site change in ERBB4. RT-PCR on lymphoblastoid cells from LVOT subjects did not show altered splice variant ratios among those homozygous for the associated haplotype. CONCLUSION These results suggest ERBB4 is associated with LVOT defects. Further replication will be required in separate cohorts to confirm the consistency of the observed association.

Host Transcriptional Response to Influenza and Other Acute Respiratory Viral Infections – A Prospective Cohort Study

To better understand the systemic response to naturally acquired acute respiratory viral infections, we prospectively enrolled 1610 healthy adults in 2009 and 2010. Of these, 142 subjects were followed for detailed evaluation of acute viral respiratory illness. We examined peripheral blood gene expression at 7 timepoints: enrollment, 5 illness visits and the end of each year of the study. 133 completed all study visits and yielded technically adequate peripheral blood microarray gene expression data. Seventy-three (55%) had an influenza virus infection, 64 influenza A and 9 influenza B. The remaining subjects had a rhinovirus infection (N = 32), other viral infections (N = 4), or no viral agent identified (N = 24). The results, which were replicated between two seasons, showed a dramatic upregulation of interferon pathway and innate immunity genes. This persisted for 2-4 days. The data show a recovery phase at days 4 and 6 with differentially expressed transcripts implicated in cell proliferation and repair. By day 21 the gene expression pattern was indistinguishable from baseline (enrollment). Influenza virus infection induced a higher magnitude and longer duration of the shared expression signature of illness compared to the other viral infections. Using lineage and activation state-specific transcripts to produce cell composition scores, patterns of B and T lymphocyte depressions accompanied by a major activation of NK cells were detected in the acute phase of illness. The data also demonstrate multiple dynamic gene modules that are reorganized and strengthened following infection. Finally, we examined pre- and post-infection anti-influenza antibody titers defining novel gene expression correlates.

TM4SF20 Ancestral Deletion and Susceptibility to a Pediatric Disorder of Early Language Delay and Cerebral White Matter Hyperintensities

White matter hyperintensities (WMHs) of the brain are important markers of aging and small-vessel disease. WMHs are rare in healthy children and, when observed, often occur with comorbid neuroinflammatory or vasculitic processes. Here, we describe a complex 4 kb deletion in 2q36.3 that segregates with early childhood communication disorders and WMH in 15 unrelated families predominantly from Southeast Asia. The premature brain aging phenotype with punctate and multifocal WMHs was observed in ~70% of young carrier parents who underwent brain MRI. The complex deletion removes the penultimate exon 3 of TM4SF20, a gene encoding a transmembrane protein of unknown function. Minigene analysis showed that the resultant net loss of an exon introduces a premature stop codon, which, in turn, leads to the generation of a stable protein that fails to target to the plasma membrane and accumulates in the cytoplasm. Finally, we report this deletion to be enriched in individuals of Vietnamese Kinh descent, with an allele frequency of about 1%, embedded in an ancestral haplotype. Our data point to a constellation of early language delay and WMH phenotypes, driven by a likely toxic mechanism of TM4SF20 truncation, and highlight the importance of understanding and managing population-specific low-frequency pathogenic alleles.

Recurrent Muscle Weakness with Rhabdomyolysis, Metabolic Crises, and Cardiac Arrhythmia Due to Bi-allelic TANGO2 Mutations

The underlying genetic etiology of rhabdomyolysis remains elusive in a significant fraction of individuals presenting with recurrent metabolic crises and muscle weakness. Using exome sequencing, we identified bi-allelic mutations in TANGO2 encoding transport and Golgi organization 2 homolog (Drosophila) in 12 subjects with episodic rhabdomyolysis, hypoglycemia, hyperammonemia, and susceptibility to life-threatening cardiac tachyarrhythmias. A recurrent homozygous c.460G>A (p.Gly154Arg) mutation was found in four unrelated individuals of Hispanic/Latino origin, and a homozygous ∼34 kb deletion affecting exons 3-9 was observed in two families of European ancestry. One individual of mixed Hispanic/European descent was found to be compound heterozygous for c.460G>A (p.Gly154Arg) and the deletion of exons 3-9. Additionally, a homozygous exons 4-6 deletion was identified in a consanguineous Middle Eastern Arab family. No homozygotes have been reported for these changes in control databases. Fibroblasts derived from a subject with the recurrent c.460G>A (p.Gly154Arg) mutation showed evidence of increased endoplasmic reticulum stress and a reduction in Golgi volume density in comparison to control. Our results show that the c.460G>A (p.Gly154Arg) mutation and the exons 3-9 heterozygous deletion in TANGO2 are recurrent pathogenic alleles present in the Latino/Hispanic and European populations, respectively, causing considerable morbidity in the homozygotes in these populations.

MCTP2 is a dosage-sensitive gene required for cardiac outflow tract development.

Coarctation of the aorta (CoA) and hypoplastic left heart syndrome (HLHS) have been reported in rare individuals with large terminal deletions of chromosome 15q26. However, no single gene important for left ventricular outflow tract (LVOT) development has been identified in this region. Using array-comparative genomic hybridization, we identified two half-siblings with CoA with a 2.2 Mb deletion on 15q26.2, inherited from their mother, who was mosaic for this deletion. This interval contains an evolutionary conserved, protein-coding gene, MCTP2 (multiple C2-domains with two transmembrane regions 2). Using gene-specific array screening in 146 individuals with non-syndromic LVOT obstructive defects, another individual with HLHS and CoA was found to have a de novo 41 kb intragenic duplication within MCTP2, predicted to result in premature truncation, p.F697X. Alteration of Mctp2 gene expression in Xenopus laevis embryos by morpholino knockdown and mRNA overexpression resulted in the failure of proper OT development, confirming the functional importance of this dosage-sensitive gene for cardiogenesis. Our results identify MCTP2 as a novel genetic cause of CoA and related cardiac malformations.

Assessment of large copy number variants in patients with apparently isolated congenital left-sided cardiac lesions reveals clinically relevant genomic events

Congenital left‐sided cardiac lesions (LSLs) are a significant contributor to the mortality and morbidity of congenital heart disease (CHD). Structural copy number variants (CNVs) have been implicated in LSL without extra‐cardiac features; however, non‐penetrance and variable expressivity have created uncertainty over the use of CNV analyses in such patients. High‐density SNP microarray genotyping data were used to infer large, likely‐pathogenic, autosomal CNVs in a cohort of 1,139 probands with LSL and their families. CNVs were molecularly confirmed and the medical records of individual carriers reviewed. The gene content of novel CNVs was then compared with public CNV data from CHD patients. Large CNVs (>1 MB) were observed in 33 probands (∼3%). Six of these were de novo and 14 were not observed in the only available parent sample. Associated cardiac phenotypes spanned a broad spectrum without clear predilection. Candidate CNVs were largely non‐recurrent, associated with heterozygous loss of copy number, and overlapped known CHD genomic regions. Novel CNV regions were enriched for cardiac development genes, including seven that have not been previously associated with human CHD. CNV analysis can be a clinically useful and molecularly informative tool in LSLs without obvious extra‐cardiac defects, and may identify a clinically relevant genomic disorder in a small but important proportion of these individuals.

Syndromic congenital myelofibrosis associated with a loss-of-function variant in RBSN

Pilar L. Magoulas,1,2,* Oleg A. Shchelochkov,3,* Matthew N. Bainbridge,4 Shay Ben-Shachar,5 Svetlana Yatsenko,6-8 Lorraine Potocki,1,2 Richard A. Lewis,2,9 Charles Searby,10 Andrea N. Marcogliese,1,11,12 M. Tarek Elghetany,1,11,12 Gladys Zapata,2,13 Paula P. Hernández,2,13 Manasi Gadkari,14 Derek Einhaus,14 Donna M. Muzny,15 Richard A. Gibbs,15 Alison A. Bertuch,1,12 Daryl A. Scott,1,2,16 Silvia Corvera,17 and Luis M. Franco14

Capture-based DNA methylation sequencing facilitates diagnosis and reveals potential pathogenic mechanisms in teratogenic diabetes exposure

Diabetic embryopathy (DE) describes a spectrum of birth defects associated with a teratogenic exposure to maternal diabetes in utero. These defects strongly overlap the phenotypes of known genetic syndromes; however, the pathogenic mechanisms underlying DE remain uncertain and there are no definitive tests that distinguish the diagnosis. Here, we explore the potential of DNA methylation as both a diagnostic biomarker and a means of informing disease pathogenesis in DE. Capture-based bisulfite sequencing was used to compare patterns of DNA methylation at 2,800,516 sites genome-wide in seven DE neonates and 11 healthy neonates, including five with in utero diabetes exposure. DE infants had significantly lower global DNA methylation (ANOVA, Tukey HSD p=0.045) than diabetes-unexposed, healthy controls (UH), with multiple sites showing large (mean methylation difference = 16.6%) and significant (p<0.001) differential methylation between the two groups. We found that a subset of 237 highly differentially methylated loci could accurately distinguish DE infants from both UH and diabetes-exposed healthy infants (sensitivity 80% -100%). Differentially methylated sites were enriched in intergenic (p<3.52×10-15) and intronic (p<0.001) regions found proximal to genes either associated with Mendelian syndromes that overlap the DE phenotype (e.g. TRIO, ANKRD11), or known to influence early organ development (e.g. BRAX1, RASA3). Further, by integrating information on cis-sequence variation, we found that 39.3% of loci with evidence for allele-specific methylation also showed differential methylation between DE and controls. Our study suggests a role for aberrant DNA methylation and cis-sequence variation in the pathogenesis of DE, and highlights the diagnostic potential of DNA methylation for teratogenic birth defects.