Targeted Gene Sequencing in Children with Crohn's Disease and Their Parents: Implications for Missing Heritability.

Crohn's disease is a complex genetic trait characterized by chronic relapsing intestinal inflammation. Genome wide association studies (GWAS) have identified more than 170 loci associated with the disease, accounting for ∼14% of the disease variance. We hypothesized that rare genetic variation in GWAS positional candidates also contribute to disease pathogenesis. We performed targeted, massively-parallel sequencing of 101 genes in 205 children with Crohn's disease, including 179 parent-child trios and 200 controls, both of European ancestry. We used the gene burden test implemented in VAAST and estimated effect sizes using logistic regression and meta-analyses. We identified three genes with nominally significant p-values: NOD2, RTKN2, and MGAT3 Only NOD2 was significant after correcting for multiple comparisons. We identified eight novel rare variants in NOD2 that are likely disease-associated. Incorporation of rare variation and compound heterozygosity nominally increased the proportion of variance explained from 0.074 to 0.089. We estimated the population attributable risk and total heritability of variation in NOD2 to be 32.9% and 3.4%, respectively, with 3.7% and 0.25% accounted for by rare putatively functional variants. Sequencing probands (as opposed to genotyping) to identify rare variants and incorporating phase by sequencing parents can recover a portion of the missing heritability of Crohn's disease.

Host genetic risk factors for West Nile virus infection and disease progression.

West Nile virus (WNV), a category B pathogen endemic in parts of Africa, Asia and Europe, emerged in North America in 1999, and spread rapidly across the continental U.S. Outcomes of infection with WNV range from asymptomatic to severe neuroinvasive disease manifested as encephalitis, paralysis, and/or death. Neuroinvasive WNV disease occurs in less than one percent of cases, and although host genetic factors are thought to influence risk for symptomatic disease, the identity of these factors remains largely unknown. We tested 360 common haplotype tagging and/or functional SNPs in 86 genes that encode key regulators of immune function in 753 individuals infected with WNV including: 422 symptomatic WNV cases and 331 cases with asymptomatic infections. After applying a Bonferroni correction for multiple tests and controlling for population stratification, SNPs in IRF3 (OR 0.54, p = 0.035) and MX1, (OR 0.19, p = 0.014) were associated with symptomatic WNV infection and a single SNP in OAS1 (OR 9.79, p = 0.003) was associated with increased risk for West Nile encephalitis and paralysis (WNE/P). Together, these results suggest that genetic variation in the interferon response pathway is associated with both risk for symptomatic WNV infection and WNV disease progression.

Mutations in embryonic myosin heavy chain (MYH3) cause Freeman-Sheldon syndrome and Sheldon-Hall syndrome.

The genetic basis of most conditions characterized by congenital contractures is largely unknown. Here we show that mutations in the embryonic myosin heavy chain (MYH3) gene cause Freeman-Sheldon syndrome (FSS), one of the most severe multiple congenital contracture (that is, arthrogryposis) syndromes, and nearly one-third of all cases of Sheldon-Hall syndrome (SHS), the most common distal arthrogryposis. FSS and SHS mutations affect different myosin residues, demonstrating that MYH3 genotype is predictive of phenotype. A structure-function analysis shows that nearly all of the MYH3 mutations are predicted to interfere with myosin's catalytic activity. These results add to the growing body of evidence showing that congenital contractures are a shared outcome of prenatal defects in myofiber force production. Elucidation of the genetic basis of these syndromes redefines congenital contractures as unique defects of the sarcomere and provides insights about what has heretofore been a poorly understood group of disorders.

Clinical characteristics and natural history of Freeman-Sheldon syndrome.

OBJECTIVE: Freeman-Sheldon syndrome (FSS) is a rare, multiple congenital contracture syndrome that is nonetheless relatively well-known, because affected children have a striking appearance: it was historically called "whistling-face syndrome" because of involvement of the facial muscles. FSS is often confused with other congenital contracture syndromes and, as a result, the clinical characteristics and natural history are poorly understood. The objective of this study was to analyze the presentation, natural history, and outcome of a cohort of individuals ascertained using strict diagnostic criteria for FSS. METHODS: Data from questionnaires, medical charts, examination, and photographs were analyzed to describe the physical features, therapeutic interventions, and functional outcomes in 73 individuals referred with the diagnosis of FSS. RESULTS: Only 32 referred cases (approximately 40%) met diagnostic criteria for FSS. In addition to contractures, common features in these cases included severe scoliosis (85%), strabismus (42%), and hearing loss (30%). Most infants required supplementary feedings via a nasogastric (45%) or gastrostomy tube (17%). Children walked by an average age of 19 months, but approximately 80% required ambulation-assist devices. An average of approximately 10 surgeries was performed on each child, and anesthetic and/or surgical complications were reported in 50% of individuals. All individuals were cognitively normal. CONCLUSIONS: The clinical characteristics and natural history of FSS distinguish it from other forms of arthrogryposis, yet FSS is frequently misdiagnosed. Children with FSS require considerable nutritional, surgical, and rehabilitative intervention. Such intensive therapeutic demands differ substantially from most other congenital contracture syndromes. These findings underscore the necessity of making an accurate diagnosis.

Expressivity of Holt-Oram syndrome is not predicted by TBX5 genotype.

Mutations in TBX5, a T-box-containing transcription factor, cause cardiac and limb malformations in individuals with Holt-Oram syndrome (HOS). Mutations that result in haploinsufficiency of TBX5 are purported to cause cardiac and limb defects of similar severity, whereas missense mutations, depending on their location in the T box, are thought to cause either more severe heart or more severe limb abnormalities. These inferences are, however, based on the analysis of a relatively small number of independent cases of HOS. To better understand the relationship between mutations in TBX5 and the variable expressivity of HOS, we screened the coding and noncoding regions of TBX5 and SALL4 for mutations in 55 probands with HOS. Seventeen mutations, including six missense mutations in TBX5 and two mutations in SALL4, were found in 19 kindreds with HOS. Fewer than 50% of individuals with nonsense or frameshift mutations in TBX5 had heart and limb defects of similar severity, and only 2 of 20 individuals had heart or limb malformations of the severity predicted by the location of their mutations in the T box. These results suggest that neither the type of mutation in TBX5 nor the location of a mutation in the T box is predictive of the expressivity of malformations in individuals with HOS.

Mutations in genes encoding fast-twitch contractile proteins cause distal arthrogryposis syndromes.

The distal arthrogryposes (DAs) are a group of disorders characterized by multiple congenital contractures of the limbs. We previously mapped a locus for DA type 2B (DA2B), the most common of the DAs, to chromosome 11. We now report that DA2B is caused by mutations in TNNI2 that are predicted to disrupt the carboxy-terminal domain of an isoform of troponin I (TnI) specific to the troponin-tropomyosin (Tc-Tm) complex of fast-twitch myofibers. Because the DAs are genetically heterogeneous, we sought additional candidate genes by examining modifiers of mutant Drosophila isoforms of TnI. One of these modifiers, Tm2, encodes tropomyosin, another component of the Tc-Tm complex. A human homologue of Tm2, TPM2, encodes beta-tropomyosin and maps to the critical interval of DA type 1 (DA1). We discovered that DA1 is caused by substitution of a highly conserved amino acid residue in beta-tropomyosin. These findings suggest that DAs, in general, may be caused by mutations in genes encoding proteins of the contractile apparatus specific to fast-twitch myofibers. This provides a new opportunity to directly study the etiology and pathogenesis of multiple-congenital-contracture syndromes.