Kristin M Noonan

Comprehensive assessment of array-based platforms and calling algorithms for detection of copy number variants.

We have systematically compared copy number variant (CNV) detection on eleven microarrays to evaluate data quality and CNV calling, reproducibility, concordance across array platforms and laboratory sites, breakpoint accuracy and analysis tool variability. Different analytic tools applied to the same raw data typically yield CNV calls with <50% concordance. Moreover, reproducibility in replicate experiments is <70% for most platforms. Nevertheless, these findings should not preclude detection of large CNVs for clinical diagnostic purposes because large CNVs with poor reproducibility are found primarily in complex genomic regions and would typically be removed by standard clinical data curation. The striking differences between CNV calls from different platforms and analytic tools highlight the importance of careful assessment of experimental design in discovery and association studies and of strict data curation and filtering in diagnostics. The CNV resource presented here allows independent data evaluation and provides a means to benchmark new algorithms.

Mutations in LRP2 , which encodes the multiligand receptor megalin, cause Donnai-Barrow and facio-oculo-acoustico-renal syndromes

Donnai-Barrow syndrome is associated with agenesis of the corpus callosum, congenital diaphragmatic hernia, facial dysmorphology, ocular anomalies, sensorineural hearing loss and developmental delay. By studying multiplex families, we mapped this disorder to chromosome 2q23.3–31.1 and identified LRP2 mutations in six families with Donnai-Barrow syndrome and one family with facio-oculo-acoustico-renal syndrome. LRP2 encodes megalin, a multiligand uptake receptor that regulates levels of diverse circulating compounds. This work implicates a pathway with potential pharmacological therapeutic targets.

Protective and Therapeutic Capacity of Human Single-Chain Fv-Fc Fusion Proteins against West Nile Virus

ABSTRACT West Nile virus has spread rapidly across the United States, and there is currently no approved human vaccine or therapy to prevent or treat disease. Passive immunization with antibodies against the envelope protein represents a promising means to provide short-term prophylaxis and treatment for West Nile virus infection. In this study, we identified a panel of 11 unique human single-chain variable region antibody fragments (scFvs) that bind the envelope protein of West Nile virus. Selected scFvs were converted to Fc fusion proteins (scFv-Fcs) and were tested in mice for their ability to prevent lethal West Nile virus infection. Five of these scFv-Fcs, 11, 15, 71, 85, and 95, protected 100% of mice from death when given prior to infection with virus. Two of them, 11 and 15, protected 80% of mice when given at days 1 and 4 after infection. In addition, four of the scFv-Fcs cross-neutralized dengue virus, serotype 2. Binding assays using yeast surface display demonstrated that all of our scFvs bind to sites within domains I and II of West Nile virus envelope protein. These recombinant human scFvs are potential candidates for immunoprophylaxis and therapy of flavivirus infections.

A review of Donnai‐Barrow and facio‐oculo‐acoustico‐renal (DB/FOAR) syndrome: Clinical features and differential diagnosis

Mutations in the gene LRP2 have recently been identified as the cause of Donnai-Barrow and Facio-oculo-acoustico-renal (DB/FOAR) syndrome. More than two dozen cases, the first reported more than 30 years ago by Holmes, have been published. Summarizing available information, we highlight the cardinal features of the disorder found in >or=90% of published cases. These features include: agenesis of the corpus callosum, developmental delay, enlarged anterior fontanelle, high myopia, hypertelorism, proteinuria, and sensorineural hearing loss. Congenital diaphragmatic hernia and omphalocele are reported in only half of the patients. There is no evidence for genotype-phenotype correlation, though the sample size is too small to preclude this with certainty. Although several conditions to consider in the differential diagnosis are highlighted, the diagnosis of DB/FOAR syndrome should not be difficult to establish as its constellation of findings is strikingly characteristic.

Donnai–Barrow syndrome (DBS/FOAR) in a child with a homozygous LRP2 mutation due to complete chromosome 2 paternal isodisomy†‡

Donnai–Barrow syndrome [Faciooculoacousticorenal (FOAR) syndrome; DBS/FOAR] is a rare autosomal recessive disorder resulting from mutations in the LRP2 gene located on chromosome 2q31.1. We report a unique DBS/FOAR patient homozygous for a 4‐bp LRP2 deletion secondary to paternal uniparental isodisomy for chromosome 2. The propositus inherited the mutation from his heterozygous carrier father, whereas the mother carried only wild‐type LRP2 alleles. This is the first case of DBS/FOAR resulting from uniparental disomy (UPD) and the fourth published case of any paternal UPD 2 ascertained through unmasking of an autosomal recessive disorder. The absence of clinical symptoms above and beyond the classical phenotype in this and the other disorders suggests that paternal chromosome 2 is unlikely to contain imprinted genes notably affecting either growth or development. This report highlights the importance of parental genotyping in order to give accurate genetic counseling for autosomal recessive disorders.

Characterization of the chromosome 1q41q42.12 region, and the candidate gene DISP1, in patients with CDH.

Cytogenetic and molecular cytogenetic studies demonstrate association between congenital diaphragmatic hernia (CDH) and chromosome 1q41q42 deletions. In this study, we screened a large CDH cohort (N = 179) for microdeletions in this interval by the multiplex ligation‐dependent probe amplification (MLPA) technique, and also sequenced two candidate genes located therein, dispatched 1 (DISP1) and homo sapiens H2.0‐like homeobox (HLX). MLPA analysis verified deletions of this region in two cases, an unreported patient with a 46,XY,del(1)(q41q42.13) karyotype and a previously reported patient with a Fryns syndrome phenotype [Kantarci et al., 2006 ]. HLX sequencing showed a novel but maternally inherited single nucleotide variant (c.27C>G) in a patient with isolated CDH, while DISP1 sequencing revealed a mosaic de novo heterozygous substitution (c.4412C>G; p.Ala1471Gly) in a male with a left‐sided Bochdalek hernia plus multiple other anomalies. Pyrosequencing demonstrated the mutant allele was present in 43%, 12%, and 4.5% of the patients lymphoblastoid, peripheral blood lymphocytes, and saliva cells, respectively. We examined Disp1 expression at day E11.5 of mouse diaphragm formation and confirmed its presence in the pleuroperitoneal fold, as well as the nearby lung which also expresses Sonic hedgehog (Shh). Our report describes the first de novo DISP1 point mutation in a patient with complex CDH. Combining this finding with Disp1 embryonic mouse diaphragm and lung tissue expression, as well as previously reported human chromosome 1q41q42 aberrations in patients with CDH, suggests that DISP1 may warrant further consideration as a CDH candidate gene.

Nucleocapsid-RNA interactions are essential to structural stability but not to assembly of retroviruses.

ABSTRACT The process of RNA incorporation into nascent virions is thought to be critical for efficient retroviral particle assembly and production. Here we show that human immunodeficiency virus type 1 mutant particles (which are highly unstable and break down soon after release from the cell) lacking nucleocapsid (NC) core protein-mediated RNA incorporation are produced efficiently and can be recovered at the normal density when viral protease function is abolished. These results demonstrate that RNA binding by Gag is not necessary for retroviral particle assembly. Rather, the RNA interaction with NC is critical for retroviral particle structural stability subsequent to release from the membrane and protease-mediated Gag cleavage. Thus, the NC-RNA interaction, and not simply the presence of RNA, provides the virus with a structural function that is critical for stable retroviral particle architecture.

Congenital diaphragmatic hernia interval on chromosome 8p23.1 characterized by genetics and protein interaction networks

Chromosome 8p23.1 is a common hotspot associated with major congenital malformations, including congenital diaphragmatic hernia (CDH) and cardiac defects. We present findings from high‐resolution arrays in patients who carry a loss (n = 18) or a gain (n = 1) of sub‐band 8p23.1. We confirm a region involved in both diaphragmatic and heart malformations. Results from a novel CNVConnect algorithm, prioritizing protein–protein interactions between products of genes in the 8p23.1 hotspot and products of previously known CDH causing genes, implicated GATA4, NEIL2, and SOX7 in diaphragmatic defects. Sequence analysis of these genes in 226 chromosomally normal CDH patients, as well as in a small number of deletion 8p23.1 patients, showed rare unreported variants in the coding region; these may be contributing to the diaphragmatic phenotype. We also demonstrated that two of these three genes were expressed in the E11.5–12.5 primordial mouse diaphragm, the developmental stage at which CDH is thought to occur. This combination of bioinformatics and expression studies can be applied to other chromosomal hotspots, as well as private microdeletions or microduplications, to identify causative genes and their interaction networks.

Genetic tools and algorithms for gene discovery in major congenital anomalies

Major congenital malformations are a leading cause of infant mortality in this country, as improved obstetrical and neonatal care have successfully addressed many other previously lethal disorders (Centers for Disease Control and Prevention [CDC], 2008). Patients with congenital anomalies of the brain, heart, lung, and skeleton as well as gastrointestinal, craniofacial, and other organs fill as many as one third of all beds in childrens hospitals in the developed world (McCandless et al., 2004), with little known about the underlying mechanisms. Of the 4 million births per year in the United States, 3% (120,000) have major congenital abnormalities (CDC, 2008). Survivors require extensive and repeated hospitalizations, often with complex reconstructive surgery, to achieve a good quality of life. For others, only prolonged supportive care can be offered. The economic, social, and emotional burden is enormous (CDC, 2007). It is clearly an imperative to discover new ways to diagnose and treat these patients. Even one discovery that ameliorates a single condition can have a substantial effect if it lessens or even averts the phenotype—for example, the preventive effect of folic acid on reducing the prevalence of neural tube defects (de Wals et al., 2007; Wald, 2004; Oakley et al., 2001; Czeizel et al., 1992). Recent progress made in discovering genomic alterations requires expensive genetic and genomic tools, novel statistical and integrative analyses, a wide variety of molecular functional assays to analyze gene variations and to conceive potential therapeutics, and a well-organized infrastructure in which to conduct broad studies and to archive patient and sample collections. We have applied new and evolving genetic tools for gene discovery in the study of congenital diaphragmatic hernia (CDH) that we think are applicable to other congenital anomalies. Although a genetic contribution to congenital anomalies has long been recognized, traditional linkage cannot be applied to these early lethal disorders such as CDH. However, increased recurrence risk in siblings (Piacentini et al., 2005), the 20-fold occurrence in consanguineous unions of having multiple affected children (Stoltenberg et al., 1999), the association of malformations with chromosomal abnormalities (Pober et al., 2005), and a recent discovery of patients with de novo mutations in crucial developmental genes (Ackerman et al., 2005; Kantarci et al., 2006) offer correlative proof that a genetic component is responsible. Standard cytogenetic analyses have been augmented by array comparative genomic hybridization (aCGH) to discover de novo or inherited structural variants such as microdeletions and microduplications. High-density single nucleotide polymorphism (SNP) analyses (genotyping) are used both for homozygosity mapping and to assess the contribution of SNP differences or similarities in individual cohorts being analyzed concurrently with our colleagues studying heart, brain, and neurosensory hearing disorders, each having collected significant cohorts of patients for each major anomaly (Fig. 1). We will integrate these findings using novel computational and functional analyses. It is clear that such approaches will be necessary to make significant changes in the care of these patients, but that the technologies discussed are rapidly evolving. Figure 1 Systems approach to gene discovery.