Project description:<p>Combined Immunodeficiency with Multiple Intestinal Atresias (CID-MIA) is a rare genetic disorder with the cause previously unknown. Symptoms of CID-MIA include atresias in the small and/or large intestine, as well as immunodeficiency. In this study, we sequenced the whole exome of 5 unrelated CID-MIA patients as well as their healthy family members, and identified deleterious homozygous or compound heterozygous mutations in the gene <i>TTC7A</i> in all the 5 patients, as well as in 3 additional patients with Sanger sequencing. Our findings strongly indicate <i>TTC7A</i> dysfunction to be the cause for CID-MIA.</p>
Project description:<p>Combined Immunodeficiency with Multiple Intestinal Atresias (CID-MIA) is a rare genetic disorder with the cause previously unknown. Symptoms of CID-MIA include atresias in the small and/or large intestine, as well as immunodeficiency. In this study, we sequenced the whole exome of 5 unrelated CID-MIA patients as well as their healthy family members, and identified deleterious homozygous or compound heterozygous mutations in the gene <i>TTC7A</i> in all the 5 patients, as well as in 3 additional patients with Sanger sequencing. Our findings strongly indicate <i>TTC7A</i> dysfunction to be the cause for CID-MIA.</p>
Project description:Inherited TTC7A loss of function mutations causes intestinal and immune deficiency. TTC7A is expressed in hematopoietic and epithelial cells however its cellular function remains poorly understood. In this work we provided evidence that TTC7A is an intrinsic nucleus factor. In an attempt to link the function of TTC7A in chromatin compaction, histone modifications and general transcriptional regulation we undertook to map the observed interaction of TTC7A to chromatin genome-wide by ChiP-Seq of Flag-tagged WT_TTC7A expressed in B lymphoblastoid cell lines using two anti-Flag antibodies (hereafter IP-Flag1 and IP-Flag2).
Project description:Combined immunodeficiencies are a heterogeneous collection of primary immune disorders that exhibit defects in T cell development or function, along with impaired B cell activity even in light of normal B cell maturation. CARMIL2 (RLTPR) is a protein involved in cytoskeletal organization and cell migration which also plays a role in CD28 co-signaling of T cells. Mutations in this protein have recently been reported to cause a novel primary immunodeficiency disorder with variable phenotypic presentations. Here we deposit genotyping data for seven patients from three unrelated, consanguineous multiplex families that presented with dermatitis, eosophagitis and recurrent skin and chest infections with evidence of combined immunodeficiency. By using this genotyping data to perform autozygome-guided analysis, and coupling it with the results of whole exome sequencing, we uncovered two mutations not previously reported (p.R50T and p.L846Sfs) in CARMIL2.
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
