Yoshinaga Takayama

Structures of cDNAs encoding chum salmon pituitary-specific transcription factor, Pit-1/GHF-1

Pit-1/GHF-1, a POU family transcription factor, was originally isolated as a protein that contributes to the pituitary-specific expression of the mammalian growth hormone(GH)-encoding gene. Pit-1/GHF-1 has been recently implicated in the development of three types of hormone-producing cells which secrete GH, prolactin (PRL) and thyroid-stimulating hormone. A presumed pituitary hormone, somatolactin (SL) which belongs to the GH/PRL family, was previously isolated from fish. As the first step for determining the possible involvement of the Pit-1 protein in the pituitary-specific expression of the SL gene and/or in the development of SL-producing cells, chum salmon Pit-1 cDNA clones were isolated. Chum salmon Pit-1 mRNAs were mainly 2 kb and 3 kb in size, and specifically expressed in the pituitaries. They encoded a 365-amino acid (aa) protein, which was 74 aa larger than that found in mammals. The identity of aa sequences between salmon and rat Pit-1 was 69%. These proteins were highly conserved in the region specific for the POU family. Salmon Pit-1 had two additional sequences each consisting of about 30 aa in the N-terminal region. The sequence involved in transcriptional activation is considered to be located in the region including the above sequences, and thus, salmon Pit-1 may possibly have novel functions distinct from those of rat Pit-1.

Localization of the genes for the 100-kDa complement-activating components of Ra-reactive factor (CRARF and Crarf) to human 3q27–q28 and mouse 16B2–B3

Human and mouse genes for the complement-activating component (P100) of Ra-reactive factor, a novel bactericidal factor (CRARF and Crarf), were mapped to R-banded metaphase chromosomes by fluorescence in situ hybridization with human and mouse P100 cDNA 2.7 and 2.0 kb long, respectively. The localization of fluorescent signals showed that CRARF and Crarf mapped to human 3q27-q28 and mouse 16B2-B3, respectively. This evidence is consistent with the previous assumption that the distal portion of the long arm of human chromosome 3 is homologous to the proximal portion of mouse chromosome 16.

Selective haploinsufficiency of longer isoforms of PTCH1 protein can cause nevoid basal cell carcinoma syndrome

Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterized by developmental defects and tumorigenesis. The gene responsible for NBCCS is PTCH1. The PTCH1 gene has five alternatively used first exons resulting in the translation of three isoforms of the PTCH1 protein; that is, PTCHL, PTCHM and PTCHS. However, the biological significance of each isoform is unclear. Here we show an individual with NBCCS carrying a nonsense mutation in PTCH1 exon2, c.387G>A (p.W129X). As the mutation lay upstream of the ATG codon used for PTCHS translation, the mutant allele still expressed RNA isoforms that encode PTCHS. These results clearly demonstrate that a selective haploinsufficiency of longer isoforms of the PTCH1 protein, PTCHL and PTCHM, but not PTCHS is sufficient to cause NBCCS. Although mice selectively deficient in PTCHS isoforms are currently unavailable, this study sheds light on the complex in vivo roles of PTCH1 isoforms.

Nevoid basal cell carcinoma syndrome with cleft lip and palate associated with the novel PTCH gene mutations

Nevoid basal cell carcinoma syndrome (NBCCS) is a rare autosomal dominant disorder characterized by developmental abnormalities and a predisposition to cancers. Two unrelated patients, 21- and 16-year-old males, with cleft lip and palate and multiple jaw cysts, were diagnosed according to clinical criteria. To confirm a diagnosis of NBCCS, we undertook a molecular genetic analysis of the PTCH gene. Their PTCH genes were analyzed by direct sequencing of the PCR product from their DNA, and previously unreported mutations were identified. A heterozygous duplication at the nucleotide position between 3325 and 3328 of the PTCH gene (c.3325_3328dupGGCG) was detected in the 21-year-old patient. It caused a frameshift mutation, resulting in a premature termination of the PTCH protein. A point mutation (G to C) in intron 7 of the PTCH gene (c.1067+1G>C) was detected in the 16-year-old patient. This caused an aberrant splicing of PTCH. It is interesting to note that the non-canonical cryptic splice-donor site was activated, which did not conform to the GT–AG rule.

GENE STRUCTURE OF THE P100 SERINE-PROTEASE COMPONENT OF THE HUMAN RA-REACTIVE FACTOR

The Ra-reactive factor (RaRF) is a complement dependent anti-microbial factor that reacts with numerous microorganisms such as viruses, bacteria, fungi and protozoa. It is a complex of a mannan-binding lectin (MBL) and the serine protease, P100 (MASPI). P100 activates the C4 component of the complement system and its domain organization is similar to C1r and C1s. In this study, determination was made of the structure of the human P100 gene which was found longer than 67 kbp and to be comprised of 16 exons. Its non-protease region consisted of 10 exons, as in the case of C1r and C1s, and the introns were found present in the boundary separating two CUB domains, an EGF-like domain and two CCP domains and each CUB and CCP domain contained extra internal introns. The serine protease region was comprised of 6 exons in contrast to C1r and C1s, either of which consists of a single exon. The exon-intron structure was found to reflect the evolution of these molecules and P100 to have derived earlier in the stage of evolution than C1r or C1s.

Somatic mosaicism containing double mutations in PTCH1 revealed by generation of induced pluripotent stem cells from nevoid basal cell carcinoma syndrome

Background Nevoid basal cell carcinoma syndrome (NBCCS) is an autosomal dominant disorder characterised by developmental defects and tumorigenesis, such as medulloblastomas and basal cell carcinomas, caused by mutations of the patched-1 (PTCH1) gene. In this article, we seek to demonstrate a mosaicism containing double mutations in PTCH1 in an individual with NBCCS. Methods and results A de novo germline mutation of PTCH1 (c.272delG) was detected in a 31-year-old woman with NBCCS. Gene analysis of two out of four induced pluripotent stem cell (iPSC) clones established from the patient unexpectedly revealed an additional mutation, c.274delT. Deep sequencing confirmed a low-prevalence somatic mutation (5.5%–15.6% depending on the tissue) identical to the one found in iPSC clones. Conclusions This is the first case of mosaicism unequivocally demonstrated in NBCCS. Furthermore, the mosaicism is unique in that the patient carries one normal and two mutant alleles. Because these mutations are located in close proximity, reversion error is likely to be involved in this event rather than a spontaneous mutation. In addition, this study indicates that gene analysis of iPSC clones can contribute to the detection of mosaicism containing a minor population carrying a second mutation.