Genetic Alterations in Gorlin Syndrome

Gorlin syndrome (GS) is an autosomal dominant disorder that predisposes affected individuals to developmental defects and tumorigenesis, and caused mainly by heterozygous germline PTCH1 mutations. Despite exhaustive analysis, PTCH1 mutations are often unidentifiable in some patients; the failure to detect mutations is presumably because of mutations occurred in other causative genes or outside of analyzed regions of PTCH1, or copy number alterations (CNAs). In this study, we subjected a cohort of GS-affected individuals from six unrelated families to next-generation sequencing (NGS) analysis for the combined screening of causative alterations in Hedgehog signaling pathway-related genes. Specific single nucleotide variations (SNVs) of PTCH1 causing inferred amino acid changes were identified in four families (seven affected individuals), whereas CNAs within or around PTCH1 were found in two families in whom possible causative SNVs were not detected. Through a targeted resequencing of all coding exons, as well as simultaneous evaluation of copy number status using the alignment map files obtained via NGS, we found that GS phenotypes could be explained by PTCH1 mutations or deletions in all affected patients. Because it is advisable to evaluate CNAs of candidate causative genes in point mutation-negative cases, NGS methodology appears to be useful for improving molecular diagnosis through the simultaneous detection of both SNVs and CNAs in the targeted genes/regions

Theoretical Consideration on Influences of Cavity or Pillar Shape on Band Structures of Silicon-Based Photonic Crystals

This paper describes the physical manifestations of the various influences of cavity (or pillar) shape and the filling factor of dielectric material on band structures in two-dimensional photonic crystals. The influences of circular or rectangular cross-sections of cavity (or pillar) arrays on photonic band structures are considered theoretically, and significant aspects of square and triangular lattices are compared. It is shown that both the averaged dielectric constant of the photonic crystal and the distribution profile of photon energy play important roles in defining optical properties. For the triangular lattice, especially, it is shown that cavity array with a rectangular cross-section breaks the band structure symmetry. So, we go on to discuss this from the perspective of band structure, and consider the optical properties of a lattice with a circular cross-section cavity

Converting cytochrome c into a DyP-like metalloenzyme

Dye-decolorizing peroxidase (DyP), which can degrade anthraquinone dyes using H2O2, is an attractive prospect for potential biotechnological applications for environmental purification. We previously designed an artificial DyP with an optimal pH for reactive blue 19 (RB19) degradation shifting from pH 4.5 to 6.5. We then attempted to degrade RB19 using Escherichia coli expressing this mutant, but RB19 was degraded equally compared with bacteria expressing wild-type (WT) DyP because most DyP was expressed in a heme-free form. In this study, we attempted to design an artificial peroxidase based on cytochrome c (cyt c), whose heme is covalently bound to the protein. We found that cyt c can degrade RB19, but its ability at pH 7.0 was ~60% of that of DyP from Vibrio cholerae at pH 4.5. To enhance this activity we constructed several mutants using three approaches. Initially, to improve reactivity with H2O2, Met80 was replaced with a noncoordinating residue, Ala or Val, but catalytic efficiency (kcat/Km) was increased by only ~1.5-fold. To enhance the substrate binding affinity we introduced an additional Trp by replacing Pro76 (P76W). The catalytic efficiency of this mutant was ~3-fold greater than that of WT cyt c. Finally, to form a hydrogen bond to axial histidine Gly29 was replaced with Asp (G29D). This mutant exhibited an ~80-fold greater dye-decolorizing activity. Escherichia coli expressing the G29D mutant was unable to degrade RB19 in solution due to degradation of heme itself, but this study provides new insights into the design of artificial DyPs

Radical transfer but not heme distal residues is essential for pH dependence of dye-decolorizing activity of peroxidase from Vibrio cholerae

Dye-decolorizing peroxidase (DyP) is a heme-containing enzyme that catalyzes the degradation of anthraquinone dyes. A main feature of DyP is the acidic optimal pH for dye-decolorizing activity. In this study, we constructed several mutant DyP enzymes from Vibrio cholerae (VcDyP), with a view to identifying the decisive factor of the low pH preference of DyP. Initially, distal Asp144, a conserved residue, was replaced with His, which led to significant loss of dye-decolorizing activity. Introduction of His into a position slightly distant from heme resulted in restoration of activity but no shift in optimal pH, indicating that distal residues do not contribute to the pH dependence of catalytic activity. His178, an essential residue for dye decolorization, is located near heme and forms hydrogen bonds with Asp138 and Thr278. While Trp and Tyr mutants of His178 were inactive, the Phe mutant displayed ~35% activity of wild-type VcDyP, indicating that this position is a potential radical transfer route from heme to the active site on the protein surface. The Thr278Val mutant displayed similar enzymatic properties as WT VcDyP, whereas the Asp138Val mutant displayed significantly increased activity at pH 6.5. On the basis of these findings, we propose that neither distal amino acid residues, including Asp144, nor hydrogen bonds between His178 and Thr278 are responsible while the hydrogen bond between His178 and Asp138 plays a key role in the pH dependence of activity

Simultaneous Detection of Both Single Nucleotide Variations and Copy Number Alterations by Next-Generation Sequencing in Gorlin Syndrome.

Gorlin syndrome (GS) is an autosomal dominant disorder that predisposes affected individuals to developmental defects and tumorigenesis, and caused mainly by heterozygous germline PTCH1 mutations. Despite exhaustive analysis, PTCH1 mutations are often unidentifiable in some patients; the failure to detect mutations is presumably because of mutations occurred in other causative genes or outside of analyzed regions of PTCH1, or copy number alterations (CNAs). In this study, we subjected a cohort of GS-affected individuals from six unrelated families to next-generation sequencing (NGS) analysis for the combined screening of causative alterations in Hedgehog signaling pathway-related genes. Specific single nucleotide variations (SNVs) of PTCH1 causing inferred amino acid changes were identified in four families (seven affected individuals), whereas CNAs within or around PTCH1 were found in two families in whom possible causative SNVs were not detected. Through a targeted resequencing of all coding exons, as well as simultaneous evaluation of copy number status using the alignment map files obtained via NGS, we found that GS phenotypes could be explained by PTCH1 mutations or deletions in all affected patients. Because it is advisable to evaluate CNAs of candidate causative genes in point mutation-negative cases, NGS methodology appears to be useful for improving molecular diagnosis through the simultaneous detection of both SNVs and CNAs in the targeted genes/regions