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1.  A familial case of alveolar capillary dysplasia with misalignment of pulmonary veins supports paternal imprinting of FOXF1 in human 
Alveolar capillary dysplasia with misalignment of pulmonary veins (ACD/MPV) is a rare developmental lung disorder that is uniformly lethal. Affected infants die within the first few weeks of their life despite aggressive treatment, although a few cases of late manifestation and longer survival have been reported. We have shown previously that mutations and deletions in FOXF1 are a cause of this disorder. Although most of the cases of ACD/MPV are sporadic, there have been infrequent reports of familial cases. We present a family with five out of six children affected with ACD/MPV. DNA analysis identified a missense mutation (c.416G>T; p.Arg139Leu) in the FOXF1 gene that segregated in the three affected siblings tested. The same variant is also present as a de novo mutation in the mother and arose on her paternally derived chromosome 16. The two tested affected siblings share the same chromosome 16 haplotype inherited from their maternal grandfather. Their single healthy sibling has a different chromosome 16 haplotype inherited from the maternal grandmother. The results are consistent with paternal imprinting of FOXF1 in human.
doi:10.1038/ejhg.2012.171
PMCID: PMC3598325  PMID: 22990143
ACD/MPV; FOXF1; imprinting; angiogenesis; lung development
2.  Antibody Correlates and Predictors of Immunity to Naturally Occurring Influenza in Humans and the Importance of Antibody to the Neuraminidase 
The Journal of Infectious Diseases  2013;207(6):974-981.
Background. Serum antibody to the hemagglutinin (HA) of influenza viruses is a correlate and predictor of immunity to influenza in humans; the relative values of other correlates are uncertain.
Methods. Serum and nasal secretions (NS) were collected in fall and spring of 2009–2011 from healthy adults who were monitored for acute respiratory illness (ARI). Serum samples were tested for hemagglutination-inhibition (HAI) antibody increase and secretions for virus if ill; enrollment sera were also tested for neuraminidase-inhibiting (NI) antibody and NS for neutralizing (neut), NI, immunoglobulin A (IgA), and immunoglobulin G (IgG) anti-HA antibody.
Results. Serum anti-HA and anti-neuraminidase (NA) antibody titers to 2009(H1N1) pandemic influenza virus (pH1N1) correlated with titers in NS (including IgA and IgG antibody). Increasing anti-HA and anti-NA titers in serum and NS tests all correlated with reducing infection and infection-associated illness. Multivariate analyses indicated serum HAI and NI each independently predicted immunity to infection and infection-associated illness. Only serum NI independently predicted reduced illness among infected subjects.
Conclusions. Increasing anti-HA and NA antibody in serum and secretions correlated with reducing pH1N1 influenza virus infection and illness in healthy young adults. Both anti-HA and anti-NA antibody are independent predictors of immunity to influenza; ensuring induction of both by vaccination is desirable.
doi:10.1093/infdis/jis935
PMCID: PMC3633450  PMID: 23307936
influenza; hemagglutinin; neuraminidase; antibody; immunity
3.  Zic3 is required in the extra-cardiac perinodal region of the lateral plate mesoderm for left–right patterning and heart development 
Human Molecular Genetics  2012;22(5):879-889.
Mutations in ZIC3 cause human X-linked heterotaxy and isolated cardiovascular malformations. A mouse model with targeted deletion of Zic3 demonstrates an early role for Zic3 in gastrulation, CNS, cardiac and left–right axial development. The observation of multiple malformations in Zic3null mice and the relatively broad expression pattern of Zic3 suggest its important roles in multiple developmental processes. Here, we report that Zic3 is primarily required in epiblast derivatives to affect left–right patterning and its expression in epiblast is necessary for proper transcriptional control of embryonic cardiac development. However, cardiac malformations in Zic3 deficiency occur not because Zic3 is intrinsically required in the heart but rather because it functions early in the establishment of left–right body axis. In addition, we provide evidence supporting a role for Zic3 specifically in the perinodal region of the posterior lateral plate mesoderm for the establishment of laterality. These data delineate the spatial requirement of Zic3 during left–right patterning in the mammalian embryo, and provide basis for further understanding the molecular mechanisms underlying the complex interaction of Zic3 with signaling pathways involved in the early establishment of laterality.
doi:10.1093/hmg/dds494
PMCID: PMC3606008  PMID: 23184148
4.  NPHP4 Variants are Associated with Pleiotropic Heart Malformations 
Circulation research  2012;110(12):1564-1574.
Rationale
Congenital heart malformations are a major cause of morbidity and mortality especially in young children. Failure to establish normal left-right (L-R) asymmetry often results in cardiovascular malformations and other laterality defects of visceral organs.
Objective
To identify genetic mutations causing cardiac laterality defects.
Methods and Results
We performed a genome-wide linkage analysis in patients with cardiac laterality defects from a consanguineous family. The patients had combinations of defects that included dextrocardia, transposition of great arteries, double outlet right ventricle, atrio-ventricular septal defects and caval vein abnormalities. Sequencing of positional candidate genes identified mutations in NPHP4. We performed mutation analysis of NPHP4 in 146 unrelated patients with similar cardiac laterality defects. Forty-one percent of these patients also had laterality defects of the abdominal organs. We identified eight additional missense variants that were absent or very rare in controls. To study the role of nphp4 in establishing L-R asymmetry, we used antisense morpholinos to knockdown nphp4 expression in zebrafish. Depletion of nphp4 disrupted L-R patterning as well as cardiac and gut laterality. Cardiac laterality defects were partially rescued by human NPHP4 mRNA, whereas mutant NPHP4 containing genetic variants found in patients failed to rescue. We show that nphp4 is involved in the formation of motile cilia in Kupffer’s vesicle (KV), which generate asymmetric fluid flow necessary for normal L-R asymmetry.
Conclusions
NPHP4 mutations are associated with cardiac laterality defects and heterotaxy. In zebrafish, nphp4 is essential for the development and function of KV cilia and is required for global L-R patterning.
doi:10.1161/CIRCRESAHA.112.269795
PMCID: PMC3916111  PMID: 22550138
Congenital heart malfortmations; heterotaxy; nphp4; cilia; zebrafish
5.  Rare DNA copy number variants in cardiovascular malformations with extracardiac abnormalities 
Clinically significant cardiovascular malformations (CVMs) occur in 5–8 per 1000 live births. Recurrent copy number variations (CNVs) are among the known causes of syndromic CVMs, accounting for an important fraction of cases. We hypothesized that many additional rare CNVs also cause CVMs and can be detected in patients with CVMs plus extracardiac anomalies (ECAs). Through a genome-wide survey of 203 subjects with CVMs and ECAs, we identified 55 CNVs >50 kb in length that were not present in children without known cardiovascular defects (n=872). Sixteen unique CNVs overlapping these variants were found in an independent CVM plus ECA cohort (n=511), which were not observed in 2011 controls. The study identified 12/16 (75%) novel loci including non-recurrent de novo 16q24.3 loss (4/714) and de novo 2q31.3q32.1 loss encompassing PPP1R1C and PDE1A (2/714). The study also narrowed critical intervals in three well-recognized genomic disorders of CVM, such as the cat-eye syndrome region on 22q11.1, 8p23.1 loss encompassing GATA4 and SOX7 and 17p13.3-p13.2 loss. An analysis of protein-interaction databases shows that the rare inherited and de novo CNVs detected in the combined cohort are enriched for genes encoding proteins that are direct or indirect partners of proteins known to be required for normal cardiac development. Our findings implicate rare variants such as 16q24.3 loss and 2q31.3-q32.1 loss, and delineate regions within previously reported structural variants known to cause CVMs.
doi:10.1038/ejhg.2012.155
PMCID: PMC3548268  PMID: 22929023
rare copy number variations; extracardiac anomalies (ECAs); cardiovascular malformations (CVMs); 16q24.3 microdeletion; protein-interaction network
6.  Assessment of Bone Mineral Status in Children with Marfan syndrome 
Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder with skeletal involvement. It is caused by mutations in fibrillin1 (FBN1) gene resulting in activation of TGF-β, which developmentally regulates bone mass and matrix properties. There is no consensus regarding bone mineralization in children with MFS. Using dual-energy X-ray absorptiometry (DXA) we evaluated bone mineralization in 20 children with MFS unselected for bone problems. Z-scores were calculated based on age, gender, height, and ethnicity matched controls. Mean whole body bone mineral content (BMC) z-score was 0.26 ± 1.42 (p=0.41). Mean bone mineral density (BMD) z-score for whole body was −0.34 ± 1.4 (p=0.29) and lumbar spine was reduced at −0.55 ± 1.34 (p=0.017). On further adjusting for stature, which is usually higher in MFS, mean BMC z-score was reduced at −0.677 ± 1.37 (p=0.04), mean BMD z-score for whole body was −0.82 ± 1.55 (p=0.002) and for lumbar spine was −0.83 ± 1.32 (p=0.001). An increased risk of osteoporosis in MFS is controversial. DXA has limitations in large skeletons because it tends to overestimate BMD and BMC. By adjusting results for height, age, gender, and ethnicity, we found that MFS patients have significantly lower BMC and BMD in whole body and lumbar spine. Evaluation of diet, exercise, vitamin D status, and bone turnover markers will help gain insight into pathogenesis of the reduced bone mass. Further, larger longitudinal studies are required to evaluate the natural history, incidence of fractures and effects of pharmacological therapy.
doi:10.1002/ajmg.a.35540
PMCID: PMC3429634  PMID: 22887731
Marfan syndrome; Bone mineral density; TGF-β
7.  Impact of restricted marital practices on genetic variation in an endogamous Gujarati group 
Recent studies have examined the influence on patterns of human genetic variation of a variety of cultural practices. In India, centuries-old marriage customs have introduced extensive social structuring into the contemporary population, potentially with significant consequences for genetic variation. Social stratification in India is evident as social classes that are defined by endogamous groups known as castes. Within a caste, there exist endogamous groups known as gols (marriage circles), each of which comprises a small number of exogamous gotra (lineages). Thus, while consanguinity is strictly avoided and some randomness in mate selection occurs within the gol, gene flow is limited with populations outside the gol. Gujarati Patels practice this form of “exogamic endogamy.” We have analyzed genetic variation in one such group of Gujarati Patels, the Chha Gaam Patels (CGP), who comprise individuals from six villages. Population structure analysis of 1,200 autosomal loci offers support for the existence of distinctive multilocus genotypes in the CGP with respect to both non-Gujaratis and other Gujaratis, and indicates that CGP individuals are genetically very similar. Analysis of Y-chromosomal and mitochondrial haplotypes provides support for both patrilocal and patrilineal practices within the gol, and a low-level of female gene flow into the gol. Our study illustrates how the practice of gol endogamy has introduced fine-scale genetic structure into the population of India, and contributes more generally to an understanding of the way in which marriage practices affect patterns of genetic variation.
doi:10.1002/ajpa.22101
PMCID: PMC3436606  PMID: 22729696
Endogamy; Gene Flow; HVS1; India; Y-chromosomal
8.  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.
doi:10.1002/bdra.20764
PMCID: PMC3736588  PMID: 21290564
genetics of cardiovascular disease; heart defects; congenital; congenital abnormalities; cardiovascular abnormalities; analysis; genetic association
9.  Small rare recurrent deletions and reciprocal duplications in 2q21.1, including brain-specific ARHGEF4 and GPR148 
Human Molecular Genetics  2012;21(15):3345-3355.
We have identified a rare small (∼450 kb unique sequence) recurrent deletion in a previously linked attention-deficit hyperactivity disorder (ADHD) locus at 2q21.1 in five unrelated families with developmental delay (DD)/intellectual disability (ID), ADHD, epilepsy and other neurobehavioral abnormalities from 17 035 samples referred for clinical chromosomal microarray analysis. Additionally, a DECIPHER (http://decipher.sanger.ac.uk) patient 2311 was found to have the same deletion and presented with aggressive behavior. The deletion was not found in either six control groups consisting of 13 999 healthy individuals or in the DGV database. We have also identified reciprocal duplications in five unrelated families with autism, developmental delay (DD), seizures and ADHD. This genomic region is flanked by large, complex low-copy repeats (LCRs) with directly oriented subunits of ∼109 kb in size that have 97.7% DNA sequence identity. We sequenced the deletion breakpoints within the directly oriented paralogous subunits of the flanking LCR clusters, demonstrating non-allelic homologous recombination as a mechanism of formation. The rearranged segment harbors five genes: GPR148, FAM123C, ARHGEF4, FAM168B and PLEKHB2. Expression of ARHGEF4 (Rho guanine nucleotide exchange factor 4) is restricted to the brain and may regulate the actin cytoskeletal network, cell morphology and migration, and neuronal function. GPR148 encodes a G-protein-coupled receptor protein expressed in the brain and testes. We suggest that small rare recurrent deletion of 2q21.1 is pathogenic for DD/ID, ADHD, epilepsy and other neurobehavioral abnormalities and, because of its small size, low frequency and more severe phenotype might have been missed in other previous genome-wide screening studies using single-nucleotide polymorphism analyses.
doi:10.1093/hmg/dds166
PMCID: PMC3392111  PMID: 22543972
10.  Specific Association of Missense Mutations in CRELD1 with Cardiac Atrioventricular Septal Defects in Heterotaxy Syndrome 
doi:10.1002/ajmg.a.35457
PMCID: PMC3402694  PMID: 22740159
congenital heart defects; atrioventricular septal defect; genetic etiology; heterotaxy syndrome; complex genetic disease; laterality defects
11.  Integrative genomic analysis of the human immune response to influenza vaccination 
eLife  2013;2:e00299.
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
eLife digest
Vaccines increase resistance to disease by priming the immune system to respond to specific viruses or microorganisms. By presenting a weakened (or dead) form of a pathogen, or its toxins or surface proteins, to the immune system, vaccines trigger the production of antibodies against the virus or microorganism. If a vaccinated individual then encounters the pathogen, their immune system should be able to recognize and destroy it. Many vaccines also include a secondary agent, known as an adjuvant, to further stimulate the immune response.
Influenza, an RNA virus commonly referred to as the ‘flu’, is an infectious disease that affects both birds and mammals. Seasonal epidemics occur each year affecting 2–7% of the population. According to the World Health Organization, influenza leads to nearly 5 million hospitalizations each year and causes up to half a million deaths. Vaccination is a primary strategy for the prevention of seasonal influenza, but responses to the vaccine vary markedly, partly because of variation in the genetic makeup or genotype of individuals. However, the details of how genes influence response to vaccination, and indeed susceptibility to influenza, remain unclear.
To investigate the genetic basis of variation in the immune response of healthy adults to the seasonal influenza vaccine, Franco et al. combined information about the genotypes of individuals with measurements of their gene transcription and antibody response to vaccination. They identified 20 genes that contributed to differential immune responses to the vaccine. Almost half of these encode proteins that are not specifically associated with the immune system, but have more general roles in processes such as membrane trafficking and intracellular transport.
Focusing on these genes may enable researchers to spot those individuals who are less likely to respond to a vaccine. It could also open up new avenues of research for vaccine development: rather than designing adjuvants that target known immune mechanisms, researchers should develop adjuvants that target the proteins encoded by these 20 genes.
DOI: http://dx.doi.org/10.7554/eLife.00299.002
doi:10.7554/eLife.00299
PMCID: PMC3713456  PMID: 23878721
Complex-trait genetics; Vaccines; Human genetics; Integrative biology; Systems biology; eQTL; Human
12.  Clan Genomics and the Complex Architecture of Human Disease 
Cell  2011;147(1):32-43.
Human diseases are caused by alleles that encompass the full range of variant types, from single-nucleotide changes to copy-number variants, and these variations span a broad frequency spectrum, from the very rare to the common. The picture emerging from analysis of whole-genome sequences, the 1000 Genomes Project pilot studies, and targeted genomic sequencing derived from very large sample sizes reveals an abundance of rare and private variants. One implication of this realization is that recent mutation may have a greater influence on disease susceptibility or protection than is conferred by variations that arose in distant ancestors.
doi:10.1016/j.cell.2011.09.008
PMCID: PMC3656718  PMID: 21962505
13.  FBN1 Mutations in Patients With Descending Thoracic Aortic Dissections 
Aortic aneurysm and dissection cause significant morbidity and mortality. There are several known single gene disorders that predispose to isolated aortic disease and eventually aneurysm and dissection. FBN1 mutations are associated with multiple clinical phenotypes, including Marfan syndrome (MFS), MASS phenotype, and familial ectopia lentis, but rarely with isolated aortic aneurysm and dissection. In this report, we describe three patients who presented with primary descending thoracic aortic dissection and who were found to have an FBN1 mutation. None of the patients fulfilled clinical criteria for the diagnosis of MFS, and all had few or none of the skeletal features typical of the condition. Two patients had a history of long-term hypertension, and such a history was suspected in the third patient. These observations suggest that some individuals with FBN1 mutations have significant aortic disease involvement of other systems that is typical of FBN1 mutation-related syndromes. Superimposed risk factors, such as hypertension, may weaken the aortic wall and eventually lead to aortic dissection. Given that the cost continues to decrease, we suggest that diagnostic DNA sequencing for FBN1 mutations in patients with thoracic aortic aneurysms and dissection may be a practical clinical step in evaluating such patients and at-risk family members.
doi:10.1002/ajmg.a.32856
PMCID: PMC3593235  PMID: 20082464
FBN1; aortic dissection; hypertension
14.  Prior Infections With Seasonal Influenza A/H1N1 Virus Reduced the Illness Severity and Epidemic Intensity of Pandemic H1N1 Influenza in Healthy Adults 
Preexisting antibody, responses to seasonal and pandemic 2009 vaccine, a low infection-to-illness ratio, and shortened illnesses indicated preexisting immunity to pandemic H1N1 virus that caused the 2009 epidemic to be mild in healthy adults.
Background. A new influenza A/H1N1 (pH1N1) virus emerged in April 2009, proceeded to spread worldwide, and was designated as an influenza pandemic. A/H1N1 viruses had circulated in 1918–1957 and 1977–2009 and were in the annual vaccine during 1977–2009.
Methods. Serum antibody to the pH1N1 and seasonal A/H1N1 viruses was measured in 579 healthy adults at enrollment (fall 2009) and after surveillance for illness (spring 2010). Subjects reporting with moderate to severe acute respiratory illness had illness and virus quantitation for 1 week; evaluations for missed illnesses were conducted over holiday periods and at the spring 2010 visit.
Results. After excluding 66 subjects who received pH1N1 vaccine, 513 remained. Seventy-seven had reported with moderate to severe illnesses; 31 were infected with pH1N1 virus, and 30 with a rhinovirus. Determining etiology from clinical findings was not possible, but fever and prominent myalgias favored influenza and prominent rhinorrhea favored rhinovirus. Tests of fall and spring antibody indicated pH1N1 infection of 23% had occurred, with the rate decreasing with increasing anti-pH1N1 antibody; a similar pattern was seen for influenza-associated illness. A reducing frequency of pH1N1 infections was also seen with increasing antibody to the recent seasonal A/H1N1 virus (A/Brisbane/59/07). Preexisting antibody to pH1N1 virus, responses to a single vaccine dose, a low infection-to-illness ratio, and a short duration of illness and virus shedding among those with influenza indicated presence of considerable preexisting immunity to pH1N1 in the population.
Conclusions. The 2009 A/H1N1 epidemic among healthy adults was relatively mild, most likely because of immunity from prior infections with A/H1N1 viruses.
doi:10.1093/cid/cir809
PMCID: PMC3258274  PMID: 22075792
15.  CCDC39 is required for assembly of inner dynein arms and the dynein regulatory complex and for normal ciliary motility in humans and dogs 
Nature genetics  2010;43(1):72-78.
Primary ciliary dyskinesia (PCD) is an inherited disorder characterized by recurrent infections of the upper and lower respiratory tract, reduced fertility in males and situs inversus in about 50% of affected individuals (Kartagener syndrome). It is caused by motility defects in the respiratory cilia that are responsible for airway clearance, the flagella that propel sperm cells and the nodal monocilia that determine left-right asymmetry1. Recessive mutations that cause PCD have been identified in genes encoding components of the outer dynein arms, radial spokes and cytoplasmic pre-assembly factors of axonemal dyneins, but these mutations account for only about 50% of cases of PCD. We exploited the unique properties of dog populations to positionally clone a new PCD gene, CCDC39. We found that loss-of-function mutations in the human ortholog underlie a substantial fraction of PCD cases with axonemal disorganization and abnormal ciliary beating. Functional analyses indicated that CCDC39 localizes to ciliary axonemes and is essential for assembly of inner dynein arms and the dynein regulatory complex.
doi:10.1038/ng.726
PMCID: PMC3509786  PMID: 21131972
16.  Recurrent Partial Rhombencephalosynapsis and Holoprosencephaly in Siblings with a Mutation of ZIC2 
Rhombencephalosynapsis (RES) is a rare congenital brain malformation typically identified by magnetic resonance imaging and characterized by fusion of the cerebellar hemispheres and dentate nuclei and vermian agenesis or hypogenesis. Although RES is frequently found in conjunction with other brain malformations and/or congenital anomalies, no specific molecular etiology has been discovered to date and no animal models exist. We identified two half sisters with alobar or semi-lobar holoprosencephaly (HPE) and partial RES, suggesting that genes linked to HPE may also contribute to RES. A deletion of seven base pairs in exon one of the ZIC2 gene (c.392_98del7) was identified in each of the two half sisters with HPE and partial RES. To identify genetic causes of RES and to assess whether genes identified in HPE have a role in RES, we tested 11 additional individuals with RES by high resolution chromosome analysis, chromosomal microarray analysis, and sequencing of four HPE genes. No mutations in ZIC2 or in other genes that cause HPE were identified, suggesting that mutation of ZIC2 is a rare cause of, or contributor to, rhombencephalosynapsis associated with HPE. In addition, an individual with a complex rearrangement of chromosome 22q13.3 and RES was identified, suggesting the presence of a dosage-sensitive gene that may contribute to RES in this region.
doi:10.1002/ajmg.a.34029
PMCID: PMC3121908  PMID: 21638761
cerebellum; chromosome microarray; holoprosencephaly; MTHFR; Phelan-McDermid syndrome; SHANK3; 22q13.3 deletion syndrome
17.  Copy number gain at Xp22.31 includes complex duplication rearrangements and recurrent triplications 
Human Molecular Genetics  2011;20(10):1975-1988.
Genomic instability is a feature of the human Xp22.31 region wherein deletions are associated with X-linked ichthyosis, mental retardation and attention deficit hyperactivity disorder. A putative homologous recombination hotspot motif is enriched in low copy repeats that mediate recurrent deletion at this locus. To date, few efforts have focused on copy number gain at Xp22.31. However, clinical testing revealed a high incidence of duplication of Xp22.31 in subjects ascertained and referred with neurobehavioral phenotypes. We systematically studied 61 unrelated subjects with rearrangements revealing gain in copy number, using multiple molecular assays. We detected not only the anticipated recurrent and simple nonrecurrent duplications, but also unexpectedly identified recurrent triplications and other complex rearrangements. Breakpoint analyses enabled us to surmise the mechanisms for many of these rearrangements. The clinical significance of the recurrent duplications and triplications were assessed using different approaches. We cannot find any evidence to support pathogenicity of the Xp22.31 duplication. However, our data suggest that the Xp22.31 duplication may serve as a risk factor for abnormal phenotypes. Our findings highlight the need for more robust Xp22.31 triplication detection in that such further gain may be more penetrant than the duplications. Our findings reveal the distribution of different mechanisms for genomic duplication rearrangements at a given locus, and provide insights into aspects of strand exchange events between paralogous sequences in the human genome.
doi:10.1093/hmg/ddr078
PMCID: PMC3080608  PMID: 21355048
18.  Strategic Approaches to Unraveling Genetic Causes of Cardiovascular Diseases 
Circulation research  2011;108(10):1252-1269.
DNA sequence variants (DSVs) are major components of the “causal field” for virtually all-medical phenotypes, whether single-gene familial disorders or complex traits without a clear familial aggregation. The causal variants in single gene disorders are necessary and sufficient to impart large effects. In contrast, complex traits are due to a much more complicated network of contributory components that in aggregate increase the probability of disease. The conventional approach to identification of the causal variants for single gene disorders is genetic linkage. However, it does not offer sufficient resolution to map the causal genes in small size families or sporadic cases. The approach to genetic studies of complex traits entails candidate gene or Genome Wide Association Studies (GWAS). GWAS provides an unbiased survey of the effects of common genetic variants (common disease - common variant hypothesis). GWAS have led to identification of a large number of alleles for various cardiovascular diseases. However, common alleles account for a relatively small fraction of the total heritability of the traits. Accordingly, the focus has shifted toward identification of rare variants that might impart larger effect sizes (rare variant-common disease hypothesis). This shift is made feasible by recent advances in massively parallel DNA sequencing platforms, which afford the opportunity to identify virtually all common as well as rare alleles in individuals. In this review, we discuss various strategies that are used to delineate the genetic contribution to medically important cardiovascular phenotypes, emphasizing the utility of the new deep sequencing approaches.
doi:10.1161/CIRCRESAHA.110.236067
PMCID: PMC3115927  PMID: 21566222
Genetics; Next-Generation Sequencing; Complex traits; Polymorphism
19.  Inverted genomic segments and complex triplication rearrangements are mediated by inverted repeats in the human genome 
Nature Genetics  2011;43(11):1074-1081.
We identified complex genomic rearrangements consisting of intermixed duplications and triplications of genomic segments at both the MECP2 and PLP1 loci. These complex rearrangements were characterized by a triplicated segment embedded within a duplication in 12 unrelated subjects. Interestingly, only two novel breakpoint junctions were generated during each rearrangement formation. Remarkably, all the complex rearrangement products share the common genomic organization duplication-inverted triplication-duplication (DUP-TRP/INV-DUP) wherein the triplicated segment is inverted and located between directly oriented duplicated genomic segments. We provide evidence that the DUP-TRP/INV-DUP structures are mediated by inverted repeats that can be separated by over 300 kb; a genomic architecture that apparently leads to susceptibility to such complex rearrangements. A similar inverted repeat mediated mechanism may underlie structural variation in many other regions of the human genome. We propose a mechanism that involves both homology driven, via inverted repeats, and microhomologous/nonhomologous events.
doi:10.1038/ng.944
PMCID: PMC3235474  PMID: 21964572
BIR; inversion; MMBIR; MECP2; PLP1; duplication; complex rearrangements
20.  Early Patterns of Gene Expression Correlate With the Humoral Immune Response to Influenza Vaccination in Humans 
The Journal of Infectious Diseases  2011;203(7):921-929.
Background. Annual vaccination is the primary means for preventing influenza. However, great interindividual variability exists in vaccine responses, the cellular events that take place in vivo after vaccination are poorly understood, and appropriate biomarkers for vaccine responsiveness have not been developed.
Methods. We immunized a cohort of healthy male adults with a licensed trivalent influenza vaccine and performed a timed assessment of global gene expression before and after vaccination. We analyzed the relationship between gene expression patterns and the humoral immune response to vaccination.
Results. Marked up regulation of expression of genes involved in interferon signaling, positive IL-6 regulation, and antigen processing and presentation, were detected within 24 hours of immunization. The late vaccine response showed a transcriptional pattern suggestive of increased protein biosynthesis and cellular proliferation. Integrative analyses revealed a 494-gene expression signature—including STAT1, CD74, and E2F2—which strongly correlates with the magnitude of the antibody response. High vaccine responder status correlates with increased early expression of interferon signaling and antigen processing and presentation genes.
Conclusions. The results highlight the role of a systems biology approach in understanding the molecular events that take place in vivo after influenza vaccination and in the development of better predictors of vaccine responsiveness.
doi:10.1093/infdis/jiq156
PMCID: PMC3068032  PMID: 21357945
21.  The use of TeleMedicine in the Treatment of Pediatric Obesity: Feasibility and Acceptability 
Maternal & child nutrition  2011;7(1):71-79.
Objective
To assess the feasibility of conducting empirically supported family based pediatric obesity group treatment via telemedicine.
Methods
Seventeen families were randomly assigned to one of two conditions (physician visit, TeleMedicine). Measures included feasibility, satisfaction, and intervention outcome measures such as BMI percentile, and nutrition and activity behaviors. Measures were completed at baseline, post-treatment, and at one-year follow-up.
Results
Analyses indicate that both feasibility and satisfaction data regarding the TeleMedicine intervention were positive. Intervention outcome indicates no change in BMI percentile or nutrition and activity behaviors for either treatment group.
Conclusions
A behavioral family-based weight loss intervention delivered via TeleMedicine was well received by both parents and providers. Due to the small sample size, null findings regarding intervention outcome should be interpreted with caution. Future research should focus on methods to increase the impact of this intervention on key outcome variables.
doi:10.1111/j.1740-8709.2010.00248.x
PMCID: PMC3071528  PMID: 21108739
TeleMedicine; pediatric obesity; intervention; feasibility
22.  Genome-wide association study identifies a susceptibility locus for thoracic aortic aneurysms and aortic dissections spanning FBN1 at 15q21.1 
Nature genetics  2011;43(10):996-1000.
Although thoracic aortic aneurysms and dissections (TAAD) can be inherited as a single-gene disorder, the genetic predisposition in the majority of affected people is poorly understood. In a multistage genome-wide association study (GWAS), we compared 765 individuals who had sporadic TAAD (STAAD) with 874 controls and identified common SNPs at a 15q21.1 locus that were associated with STAAD, with odds ratios of 1.6–1.8 that achieved genome-wide significance. We followed up 107 SNPs associated with STAAD with P < 1 × 10−5 in the region, in two separate STAAD cohorts. The associated SNPs fall into a large region of linkage disequilibrium encompassing FBN1, which encodes fibrillin-1. FBN1 mutations cause Marfan syndrome, whose major cardiovascular complication is TAAD. This study shows that common genetic variants at 15q21.1 that probably act via FBN1 are associated with STAAD, suggesting a common pathogenesis of aortic disease in Marfan syndrome and STAAD.
doi:10.1038/ng.934
PMCID: PMC3244938  PMID: 21909107
23.  SHROOM3 is a novel candidate for heterotaxy identified by whole exome sequencing 
Genome Biology  2011;12(9):R91.
Background
Heterotaxy-spectrum cardiovascular disorders are challenging for traditional genetic analyses because of clinical and genetic heterogeneity, variable expressivity, and non-penetrance. In this study, high-resolution SNP genotyping and exon-targeted array comparative genomic hybridization platforms were coupled to whole-exome sequencing to identify a novel disease candidate gene.
Results
SNP genotyping identified absence-of-heterozygosity regions in the heterotaxy proband on chromosomes 1, 4, 7, 13, 15, 18, consistent with parental consanguinity. Subsequently, whole-exome sequencing of the proband identified 26,065 coding variants, including 18 non-synonymous homozygous changes not present in dbSNP132 or 1000 Genomes. Of these 18, only 4 - one each in CXCL2, SHROOM3, CTSO, RXFP1 - were mapped to the absence-of-heterozygosity regions, each of which was flanked by more than 50 homozygous SNPs, confirming recessive segregation of mutant alleles. Sanger sequencing confirmed the SHROOM3 homozygous missense mutation and it was predicted as pathogenic by four bioinformatic tools. SHROOM3 has been identified as a central regulator of morphogenetic cell shape changes necessary for organogenesis and can physically bind ROCK2, a rho kinase protein required for left-right patterning. Screening 96 sporadic heterotaxy patients identified four additional patients with rare variants in SHROOM3.
Conclusions
Using whole exome sequencing, we identify a recessive missense mutation in SHROOM3 associated with heterotaxy syndrome and identify rare variants in subsequent screening of a heterotaxy cohort, suggesting SHROOM3 as a novel target for the control of left-right patterning. This study reveals the value of SNP genotyping coupled with high-throughput sequencing for identification of high yield candidates for rare disorders with genetic and phenotypic heterogeneity.
doi:10.1186/gb-2011-12-9-r91
PMCID: PMC3308054  PMID: 21936905
24.  Recurrent reciprocal 16p11.2 rearrangements associated with global developmental delay, behavioural problems, dysmorphism, epilepsy, and abnormal head size 
Journal of medical genetics  2009;47(5):332-341.
Background
Deletion and the reciprocal duplication in 16p11.2 were recently associated with autism and developmental delay.
Method
We indentified 27 deletions and 18 duplications of 16p11.2 were identified in 0.6% of all samples submitted for clinical array-CGH (comparative genomic hybridisation) analysis. Detailed molecular and phenotypic characterisations were performed on 17 deletion subjects and ten subjects with the duplication.
Results
The most common clinical manifestations in 17 deletion and 10 duplication subjects were speech/language delay and cognitive impairment. Other phenotypes in the deletion patients included motor delay (50%), seizures (~40%), behavioural problems (~40%), congenital anomalies (~30%), and autism (~20%). The phenotypes among duplication patients included motor delay (6/10), behavioural problems (especially attention deficit hyperactivity disorder (ADHD)) (6/10), congenital anomalies (5/10), and seizures (3/10). Patients with the 16p11.2 deletion had statistically significant macrocephaly (p<0.0017) and 6 of the 10 patients with the duplication had microcephaly. One subject with the deletion was asymptomatic and another with the duplication had a normal cognitive and behavioural phenotype. Genomic analyses revealed additional complexity to the 16p11.2 region with mechanistic implications. The chromosomal rearrangement was de novo in all but 2 of the 10 deletion cases in which parental studies were available. Additionally, 2 de novo cases were apparently mosaic for the deletion in the analysed blood sample. Three de novo and 2 inherited cases were observed in the 5 of 10 duplication patients where data were available.
Conclusions
Recurrent reciprocal 16p11.2 deletion and duplication are characterised by a spectrum of primarily neurocognitive phenotypes that are subject to incomplete penetrance and variable expressivity. The autism and macrocephaly observed with deletion and ADHD and microcephaly seen in duplication patients support a diametric model of autism spectrum and psychotic spectrum behavioural phenotypes in genomic sister disorders.
doi:10.1136/jmg.2009.073015
PMCID: PMC3158566  PMID: 19914906
25.  Argentine Population Genetic Structure: Large Variance in Amerindian Contribution 
Argentine population genetic structure was examined using a set of 78 ancestry informative markers (AIMs) to assess the contributions of European, Amerindian, and African ancestry in 94 individuals members of this population. Using the Bayesian clustering algorithm STRUCTURE, the mean European contribution was 78%, the Amerindian contribution was 19.4%, and the African contribution was 2.5%. Similar results were found using weighted least mean square method: European, 80.2%; Amerindian, 18.1%; and African, 1.7%. Consistent with previous studies the current results showed very few individuals (four of 94) with greater than 10% African admixture. Notably, when individual admixture was examined, the Amerindian and European admixture showed a very large variance and individual Amerindian contribution ranged from 1.5 to 84.5% in the 94 individual Argentine subjects. These results indicate that admixture must be considered when clinical epidemiology or case control genetic analyses are studied in this population. Moreover, the current study provides a set of informative SNPs that can be used to ascertain or control for this potentially hidden stratification. In addition, the large variance in admixture proportions in individual Argentine subjects shown by this study suggests that this population is appropriate for future admixture mapping studies.
doi:10.1002/ajpa.20534
PMCID: PMC3142769  PMID: 17177183
ancestry informative markers; admixture; population stratification

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