Characterization of an Evolutionarily Conserved Metallophosphoesterase That Is Expressed in the Fetal Brain and Associated with the WAGR Syndrome

Among the human diseases that result from chromosomal aberrations, a de novo deletion in chromosome 11p13 is clinically associated with a syndrome characterized by Wilms' tumor, aniridia, genitourinary anomalies, and mental retardation (WAGR). Not all genes in the deleted region have been characterized biochemically or functionally. We have recently identified the first Class III cyclic nucleotide phosphodiesterase, Rv0805, from Mycobacterium tuberculosis, which biochemically and structurally belongs to the superfamily of metallophosphoesterases. We performed a large scale bioinformatic analysis to identify orthologs of the Rv0805 protein and identified many eukaryotic genes that included the human 239FB gene present in the region deleted in the WAGR syndrome. We report here the first detailed biochemical characterization of the rat 239FB protein and show that it possesses metallophosphodiesterase activity. Extensive mutational analysis identified residues that are involved in metal interaction at the binuclear metal center. Generation of a rat 239FB protein with a mutation corresponding to a single nucleotide polymorphism seen in human 239FB led to complete inactivation of the protein. A close ortholog of 239FB is found in adult tissues, and biochemical characterization of the 239AB protein demonstrated significant hydrolytic activity against 2',3'-cAMP, thus representing the first evidence for a Class III cyclic nucleotide phosphodiesterase in mammals. Highly conserved orthologs of the 239FB protein are found in Caenorhabditis elegans and Drosophila and, coupled with available evidence suggesting that 239FB is a tumor suppressor, indicate the important role this protein must play in diverse cellular events.

Unique Utilization of a Phosphoprotein Phosphatase Fold by a Mammalian Phosphodiesterase Associated with WAGR Syndrome

Metallophosphoesterase-domain-containing protein 2 (MPPED2) is a highly evolutionarily conserved protein with orthologs found from worms to humans. The human MPPED2 gene is found in a region of chromosome 11 that is deleted in patients with WAGR (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) syndrome, and MPPED2 may function as a tumor suppressor. However, the precise cellular roles of MPPED2 are unknown, and its low phosphodiesterase activity suggests that substrate hydrolysis may not be its prime function. We present here the structures of MPPED2 and two mutants, which show that the poor activity of MPPED2 is not only a consequence of the substitution of an active-site histidine residue by glycine but also due to binding of AMP or GMP to the active site. This feature, enhanced by structural elements of the protein, allows MPPED2 to utilize the conserved phosphoprotein-phosphatase-like fold in a unique manner, ensuring that its enzymatic activity can be combined with a possible role as a scaffolding or adaptor protein. (C) 2011 Elsevier Ltd. All rights reserved.

Genetic analysis of an Indian family with members affected with Waardenburg syndrome and Duchenne muscular dystrophy

Purpose: Waardenburg syndrome (WS) is characterized by sensorineural hearing loss and pigmentation defects of the eye, skin, and hair. It is caused by mutations in one of the following genes: PAX3 (paired box 3), MITF (microphthalmia-associated transcription factor), EDNRB (endothelin receptor type B), EDN3 (endothelin 3), SNAI2 (snail homolog 2, Drosophila) and SOX10 (SRY-box containing gene 10). Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder caused by mutations in the DMD gene. The purpose of this study was to identify the genetic causes of WS and DMD in an Indian family with two patients: one affected with WS and DMD, and another one affected with only WS. Methods: Blood samples were collected from individuals for genomic DNA isolation. To determine the linkage of this family to the eight known WS loci, microsatellite markers were selected from the candidate regions and used to genotype the family. Exon-specific intronic primers for EDN3 were used to amplify and sequence DNA samples from affected individuals to detect mutations. A mutation in DMD was identified by multiplex PCR and multiplex ligation-dependent probe amplification method using exon-specific probes. Results: Pedigree analysis suggested segregation of WS as an autosomal recessive trait in the family. Haplotype analysis suggested linkage of the family to the WS4B (EDN3) locus. DNA sequencing identified a novel missense mutation p.T98M in EDN3. A deletion mutation was identified in DMD. Conclusions: This study reports a novel missense mutation in EDN3 and a deletion mutation in DMD in the same Indian family. The present study will be helpful in genetic diagnosis of this family and increases the mutation spectrum of EDN3.

Lacrimal Proline Rich 4 (LPRR4) Protein in the Tear Fluid Is a Potential Biomarker of Dry Eye Syndrome

Dry eye syndrome (DES) is a complex, multifactorial, immune-associated disorder of the tear and ocular surface. DES with a high prevalence world over needs identification of potential biomarkers so as to understand not only the disease mechanism but also to identify drug targets. In this study we looked for differentially expressed proteins in tear samples of DES to arrive at characteristic biomarkers. As part of a prospective case-control study, tear specimen were collected using Schirmer strips from 129 dry eye cases and 73 age matched controls. 2D electrophoresis (2DE) and Differential gel electrophoresis (DIGE) was done to identify differentially expressed proteins. One of the differentially expressed protein in DES is lacrimal proline rich 4 protein (LPRR4). LPRR4 protein expression was quantified by enzyme immune sorbent assay (ELISA). LPRR4 was down regulated significantly in all types of dry eye cases, correlating with the disease severity as measured by clinical investigations. Further characterization of the protein is required to assess its therapeutic potential in DES.

Clinical phenotype and the lack of mutations in the CHRNG, CHRND, and CHRNA1 genes in two Indian families with Escobar syndrome

The objective of this study was to report the clinical phenotype and genetic analysis of two Indian families with Escobar syndrome (ES). The diagnosis of ES in both families was made on the basis of published clinical features. Blood samples were collected from members of both families and used in genomic DNA isolation. The entire coding regions and intron-exon junctions of the ES gene CHRNG (cholinergic receptor, nicotinic, gamma), and two other related genes, CHRND and CHRNA1, were amplified and sequenced to search for mutations in both families. Both families show a typical form of ES. Sequencing of the entire coding regions including the intron-exon junctions of the three genes did not yield any mutations in these families. In conclusion, it is possible that the mutations in these genes are located in the promoter or deep intronic regions that we failed to identify or the ES in these families is caused by mutations in a different gene. The lack of mutations in CHRNG has also been reported in several families, suggesting the possibility of at least one more gene for this syndrome. Clin Dysmorphol 22:54-58 (C) 2013 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.