Hiromi Hatsuse

Human mannose-binding protein is identical to a component of Ra-reactive factor

Human Ra-reactive factor (RaRF) and mannose-binding protein (MBP) were isolated from sera by utilizing their affinity to Ra chemotype Salmonella typhimurium and yeast mannan, respectively. A predominant polypeptide of human RaRF with an Mr of 32 kDa (P32) has the same mobility as human MBP on SDS-PAGE gels. A monoclonal antibody against P32 of human RaRF, 3E7, was found to react not only with P32 but also with human MBP, as assessed by immunoblotting. In addition, 3E7 blocked the complement-activating capacity of human MBP. The NH2-terminal amino acid sequence of P32 of human RaRF was determined and found to coincide with that of human MBP. Based on these results, it is clear that human MBP is identical to a component of RaRF.

Frameshift mutation in the PTCH2 gene 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, encoding a receptor for the secreted protein, sonic hedgehog. Recently, a Chinese family with NBCCS carrying a missense mutation in PTCH2, a close homolog of PTCH1, was reported. However, the pathological significance of missense mutations should be discussed cautiously. Here, we report a 13-year-old girl diagnosed with NBCCS based on multiple keratocystic odontogenic tumors and rib anomalies carrying a frameshift mutation in the PTCH2 gene (c.1172_1173delCT). Considering the deleterious nature of the frameshift mutation, our study further confirmed a causative role for the PTCH2 mutation in NBCCS. The absence of typical phenotypes in this case such as palmar/plantar pits, macrocephaly, falx calcification, hypertelorism and coarse face, together with previously reported cases, suggested that individuals with NBCCS carrying a PTCH2 mutation may have a milder phenotype than those with a PTCH1 mutation.

A mouse with a point mutation in plasma membrane Ca2+-ATPase isoform 2 gene showed the reduced Ca2+ influx in cerebellar neurons

We analyzed mutant mice showing behavioral defects such as severe tremor, up-and-down and side-to-side wriggling of neck without coordination, and found that the gene causing the defects was located between 46 and 60.55 centimorgans (cM) on the mouse chromosome 6. In this region, nucleotide transition of the plasma membrane Ca2+-ATPase isoform 2 (PMCA2) gene was found, which caused a glutamic acid to change into lysine. Since PMCA2 is expressed in the cerebellum and plays an important role to maintain the homeostasis of the intracellular Ca2+ as a Ca2+ pump, the behavioral defect can be ascribed to the impairment of Ca2+ regulation in neurons of the cerebellum. To confirm the defect of Ca2+ homeostasis in the mutant mice, we measured high K+-induced changes in intracellular Ca2+ concentration ([Ca2+]i) in the cerebellar neurons. Contrary to our expectation, the extent of the [Ca2+]i increase in all the regions tested in the cerebellar slice was far smaller than that of the wild type mice, while the resting [Ca2+]i remained almost unaltered. The rate of rise in [Ca2+]i during high K+-induced depolarization was significantly reduced, and the extrusion rate of increased [Ca2+]i was also reduced. These results suggested that voltage-gated Ca2+ channels were down-regulated in the mutant mice in order to regulate [Ca2+]i toward the normal homeostasis. The behavioral defects may be ascribed to the down-regulated Ca2+ homeostasis since dynamic changes in [Ca2+]i are important for various neuronal functions.

Entire PTCH1 deletion is a common event in point mutation-negative cases with nevoid basal cell carcinoma syndrome in Japan.

To the Editor : Nevoid basal cell carcinoma syndrome [NBCCS (OMIM 109400)], also called Gorlin syndrome, is an autosomal dominant neurocutaneous disorder characterized by large body size, developmental and skeletal abnormalities, sensitivity to radiation, and an increased incidence of cancers such as basal cell carcinoma (BCC) and medulloblastoma (1). NBCCS is caused by inactivating mutations in the Patched-1 (PTCH1 ) gene (2, 3). Heterozygous loss of PTCH1 found in certain sporadic and familial cases of BCC indicates that PTCH1 is also a tumor suppressor gene (4, 5). Despite extensive efforts to detect mutations, they are still unidentified in 25–60% of patients (6–8). To date, we have analyzed 38 patients with NBCCS from 32 families. Eight of the families did not harbor any PTCH1 mutations detectable by polymerase chain reaction (PCR)based direct sequencing of the exons. To investigate the possibility of large deletions involving the PTCH1 gene, we employed a high-resolution array-based comparative genomic hybridization technology. Consequently, we identified genomic deletions involving PTCH1 in seven individuals from five of the eight point mutation-negative families (Fig. S1). These patients are listed in Table 1. Some of them have been reported previously by us (9) and one patient reported by others (NS6) (10) is also included in the table, all of which are of Japanese origin. To our knowledge, this table includes all the patients with PTCH1 deletions in which the breakpoints have been identified at the nucleotide level. A schematic representation of each deletion’s size together with the deleted genes is shown in Fig. 1a. Unlike in cases of Sotos syndrome and neurofibromatosis type 1, no recurrent breakpoints were observed in these patients (11, 12). Whereas deletions larger than 2.4 Mb were generated by non-homologous end joining, smaller ones (less than 1.2 Mb) were produced by Alu-mediated nonallelic homologous recombination (Fig. S2). G19 and G36 inherited the deletion from their mothers (G27 and G43, respectively), whereas the deletion in NS6 is of paternal origin. The breakpoint sequences in these cases were completely conserved through generations. Other patients (G35, G10 and G5) did not have a family history of NBCCS and, therefore, the deletions seemed to be de novo. Patients harboring deletions of less than 2.4 Mb did not exhibit any phenotypes atypical for NBCCS despite that up to 22 RefSeq genes (four disease genes) were included in the deleted region. This implies that hemizygous loss of these genes, except for PTCH1, might not have an influence on any observable phenotypes. In contrast, deletions larger than 5.3 Mb led to phenotypes unusual for NBCCS including severe mental and motor retardation, epilepsy, and hypotonia (Table 1). Interestingly, each Alu-mediated deletion was mediated by a distinct path of rearrangement (Fig. 1b). G36/43 had a crossing over point within the Alu elements generating a hybrid Alu element. In G19/27, however, the crossing over occurred near the poly-A tail of the proximal Alu element (9). Therefore, the proximal Alu remained intact while the distal Alu was deleted. In the third case, NS6, crossing over occurred at the 5′ end of the Alu elements and removed both Alu sequences leaving two short direct repeats flanking an Alu element on both sides called target-site duplications (10). To date, we have analyzed 32 NBCCS families and identified entire deletions of PTCH1 in 5 families. This implies that 16% of NBCCS families (five of the eight point mutation-negative families) can be explained by the entire loss of PTCH1. Mutations are not observed in the PTCH1 -coding sequences in considerable numbers of NBCCS cases not only in Japanese but also in other ethnicities and, apart from PTCH1, only one PTCH2 and one SUFU mutation in NBCCS have been

Molecular pathogenesis of keratocystic odontogenic tumors developing in nevoid basal cell carcinoma syndrome.

OBJECTIVE The aim of this study was to investigate the molecular pathogenesis of keratocystic odontogenic tumors (KCOTs) that developed in nevoid basal cell carcinoma syndrome (NBCCS) patients. STUDY DESIGN We analyzed germline and somatic mutations of the PTCH1 and its related genes, SMO and SUFU in 10 KCOTs that developed in 8 unrelated NBCCS patients. Methylation status of the PTCH1 promoter was also investigated by bisulfite sequencing. RESULTS Somatic mutations of PTCH1 were detected in 3 KCOTs. Two out of 3 somatic mutations were either identified as a polymorphism or located on the same allele as the germline mutation. Neither abnormal methylation of the PTCH1 promoter, loss of PTCH1, nor somatic mutation of SMO or SUFU was detected in any of the samples. CONCLUSIONS Our results suggest that the tumorigenesis of most KCOTs associated with NBCCS cannot be explained by the classical 2-hit theory.

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.

TRAV gene usage in pig T-cell receptor alpha cDNA

Pig (Sus scrofa) TRA clones were isolated from cDNA libraries of total RNA from two different sources, the thymus of a 1-month-old LW strain pig and the peripheral blood lymphocytes of a 5-month-old Clawn strain pig. Among 103 complete TRA cDNA clones from both sources, 33 different TRAV genes were identified. By comparing their sequence identities against one another, these pig TRAV genes were grouped into 20 subgroups, including 13 subgroups, each containing only a single member. All of these pig subgroups gave corresponding human and mouse functional counterparts, suggesting their functional commonality. An exception was the Va01 gene segment, which lacked a functional human counterpart. The present report provides groundwork for studies on pig TRA expression.

Immune complex independent activation of complement, Cls secreted from hamster embryo malignant fibroblasts, Nil2C2 in serum free culture medium

Antibody independent activation of complement C1s was examined by immunoblot analysis using an antibody against a synthetic peptide of hamster C1s L chain. Approx. 50% of C1s secreted from hamster embryo malignant fibroblasts Nil2C2 was functionally active in its two-chain form in the serum free culture medium. In contrast, no active C1s was found in a culture medium of hamster embryo fibroblasts (HEF). Active C1s was detectable, however, in the culture medium after HEF became a cell line. The immune complex independent activation of C1s was also observed in rat cell lines but not in secondary rat embryo fibroblasts. C1s in a membrane fraction of Nil2C2 was a proenzyme form and was not activated by incubation of the membrane itself suggesting that C1s was activated after secretion. The activation of C1s was not inhibited by human C1 inhibitor (C1-INH), benzamidine or soy bean trypsin inhibitor (SBTI) but was inhibited by leupeptin, nitrophenyl guanidinobenzoate and DFP. Our results suggest that C1s is activated either by a serine proteinase(s) other than those reported to cleave C1s or by an activator which directly stimulates autoactivation of 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.