Mutation spectrum and genotype-phenotype analyses in Cowden disease and Bannayan-Zonana syndrome, two hamartoma syndromes with germline PTEN mutation

The tumour suppressor gene PTEN , which maps to 10q23.3 and encodes a 403 amino acid dual specificity phosphatase (protein tyrosine phosphatase; PTPase), was shown recently to play a broad role in human malignancy. Somatic PTEN deletions and mutations were observed in sporadic breast, brain, prostate and kidney cancer cell lines and in several primary tumours such as endometrial carcinomas, malignant melanoma and thyroid tumours. In addition, PTEN was identified as the susceptibility gene for two hamartoma syndromes: Cowden disease (CD; MIM 158350) and Bannayan-Zonana (BZS) or Ruvalcaba-Riley-Smith syndrome (MIM 153480). Constitutive DNA from 37 CD families and seven BZS families was screened for germline PTEN mutations. PTEN mutations were identified in 30 of 37 (81%) CD families, including missense and nonsense point mutations, deletions, insertions, a deletion/insertion and splice site mutations. These mutations were scattered over the entire length of PTEN , with the exception of the first, fourth and last exons. A 'hot spot' for PTEN mutation in CD was identified in exon 5 that contains the PTPase core motif, with 13 of 30 (43%) CD mutations identified in this exon. Seven of 30 (23%) were within the core motif, the majority (five of seven) of which were missense mutations, possibly pointing to the functional significance of this region. Germline PTEN mutations were identified in four of seven (57%) BZS families studied. Interestingly, none of these mutations was observed in the PTPase core motif. It is also worthy of note that a single nonsense point mutation, R233X, was observed in the germline DNA from two unrelated CD families and one BZS family. Genotype-phenotype studies were not performed on this small group of BZS families. However, genotype-phenotype analysis inthe group of CD families revealed two possible associations worthy of follow-up in independent analyses. The first was an association noted in the group of CD families with breast disease. A correlation was observed between the presence/absence of a PTEN mutation and the type of breast involvement (unaffected versus benign versus malignant). Specifically and more directly, an association was also observed between the presence of a PTEN mutation and malignant breast disease. Secondly, there appeared to be an interdependent association between mutations upstream and within the PTPase core motif, the core motif containing the majority of missense mutations, and the involvement of all major organ systems (central nervous system, thyroid, breast, skin and gastrointestinal tract). However, these observations would need to be confirmed by studying a larger number of CD families.      

Five haplotypes account for fifty-five percent of ATM mutations in Brazilian patients with ataxia telangiectasia: seven new mutations

We have studied the molecular genetics of 27 Brazilian families with ataxia telangiectasia (AT). Five founder effect haplotypes accounted for 55.5% of the families. AT is an autosomal recessive disorder of childhood onset characterized by progressive cerebellar ataxia, ocular apraxia, telangiectasia, immunodeficiency, radiation sensitivity, chromosomal instability, and predisposition to cancer. The ATM gene spans more than 150 kb on chromosome region 11q23.1 and encodes a product of 3056 amino acids. The ATM protein is a member of the phosphatidylinositol 3-kinase (PI-3K) family of proteins and is involved in cell cycle control and DNA repair pathways. DNA was isolated from lymphoblastoid cell lines and haplotyped using four STR markers (D11S1818, NS22, D11S2179, D11S1819) within and flanking the ATM gene; all allele sizes were standardized in advance. In addition to the STR haplotypes, SNP haplotypes were determined using 10 critical polymorphisms. The entire gene was screened sequentially by protein truncation testing (PTT), single strand conformation polymorphism (SSCP), and then denaturing high performance liquid chromatography (dHPLC) to identify the disease-causing mutations. Of the expected 54 mutations, 50 were identified. All mutations but one, led to a truncated or null form of the ATM protein (nonsense, splice site, or frameshift). Five families (18.5%) carried a deletion of 3450nt (from IVS28 to Ex31), making this one of the two most common Brazilian mutations. Mutations were located throughout the entire gene, with no clustering or hotspots. Standardized STR haplotype analysis greatly enhanced the efficiency of mutation screening.     

Purification of biologically active rubella virus antigens by immunoaffinity chromatography

A general procedure for isolating biologically active rubella virus antigens (VPI, Mr = 61,000; VP2, Mr = 45,000; VP3, Mr = 36,000) by monoclonal antibody affinity chromatography is described. Complexes formed between monoclonal antibodies and rubella virus antigens were found to be stable either at low pH or in Tris buffer containing detergent and high salt, but were efficiently dissociated by 5% diethanolamine, pH 11.5, or 50 mM lithium diiodosalicylate buffer, pH 8.0. Chromatographically purified rubella viral antigens retained their antigenicity as determined by enzyme-linked immunosorbent assays. Biological studies showed that rubella structural proteins VP2 and VP3 had no hemagglutinin function while the mixture of VP1 and VP2 and VP3 directly demonstrated hemagglutination activity. These results indicate that VP1 is at least in part responsible for the hemagglutinin function of rubella virus.     

成人型多囊肾病的基因诊断──PKD_1基因与其两侧四种基因探针的连锁分析

采用位于α３′ＨＶＲ同侧与成人型多囊肾病基因（ＰＫＤ１）更加接近的ｐＧＧＧ１及另一侧的２４－１和２１８ＥＰ６等探针对正常人基因组ＤＮＡ进行限制性片段长度多态性（ＲＥＬＰ）分析，分别检测各等位片段的频率，在此基础上，应用这三个基因探针与３′ＨＶＲ一起对２个成人型多囊肾病家系成员进行单体型分析，在这２个家系中，５个ＡＰＫＤ患者的ＲＦＬＰ单体型被证明与ＰＫＤ１基因相连锁，发现一个重组体，并检测出二个症状前个体。     

RNA interference against enterovirus 71 infection

Enterovirus 71 (EV71) is a highly infectious major causative agent of hand, foot, and mouth disease (HFMD) which could lead to severe neurological complications. There is currently no effective therapy against EV71. In this study, RNA interference (RNAi) is employed as a therapeutic approach for specific viral inhibition. Various regions of the EV71 genome were targeted for inhibition by chemically synthesized siRNAs. Transfection of rhabdomyosarcoma (RD) cells with siRNA targeting the 3'UTR, 2C, 3C, or 3D region significantly alleviated cytopathic effects of EV71. The inhibitory effect was dosage-dependent with a corresponding decrease in viral RNA, viral proteins, and plaque formations by EV71. Viral inhibition of siRNA transfected RD cells was still evident after 48 h. In addition, no significant adverse off-target silencing effects were observed. These results demonstrated the potential and feasibility for the use of siRNA as an antiviral therapy for EV71 infections.     

DNA prime followed by protein boost enhances neutralization and Th1 type immunity against FMDV

Prime-boost strategy has been exhibited its potency to enhance immune responses, which would be important to the success to develop a vaccine against the foot-and-mouth disease virus (FMDV). An eukaryotic expression construct encoding the FMDV capsid VP1 protein with a recombinant VP1 protein or a commercial FMDV vaccine were tested in the prime-boost strategy in mice and cattle trials. The levels of induced specific antibodies, T cell proliferations, and DTH activities were significantly higher in the prime-boost groups than in those vaccinated with DNA, protein or FMDV vaccine alone. More importantly, the levels of neutralizing antibodies in the former groups were significantly higher than others and could last for at least four months in cattle trials. This study suggests that the prime-boost strategy significantly improves the effective immunity and may provide a longer protection against FMDV infection.     

Nocardia osteomyelitis in a pachymeningitis patient: an example of a difficult case to treat with antimicrobial agents

Antimicrobial agents played a miraculous role in the treatment of bacterial infections until resistant bacteria became widespread. Besides antimicrobial-resistant bacteria, many factors can influence the cure of infection. Nocardia infection may be a good example which is difficult to cure with antimicrobial agents alone. A 66-year-old man developed soft tissue infection of the right buttock and thigh. He was given prednisolone and azathioprine for pachymeningitis 3 months prior to admission. Despite surgical and antimicrobial treatment (sulfamethoxazole-trimethoprim), the infection spread to the femur and osteomyelitis developed. The case showed that treatment of bacterial infection is not always as successful as was once thought because recent isolates of bacteria are more often resistant to various antimicrobial agents, intracellular parasites are difficult to eliminate even with the active drug in vitro, and infections in some sites such as bone are refractory to treatment especially when the patient is in a compromised state. In conclusion, for the treatment of infections, clinicians need to rely on laboratory tests more than before and have to consider the influence of various host factors.