Update on the hyper immunoglobulin M syndromes

The Hyper‐immunoglobulin M syndromes (HIGM) are a heterogeneous group of genetic disorders resulting in defects of immunoglobulin class switch recombination (CSR), with or without defects of somatic hypermutation (SHM). They can be classified as defects of signalling through CD40 causing both a humoral immunodeficiency and a susceptibility to opportunistic infections, or intrinsic defects in B cells of the mechanism of CSR resulting in a pure humoral immunodeficiency. A HIGM picture can also be seen as part of generalized defects of DNA repair and in antibody deficiency syndromes, such as common variable immunodeficiency. CD40 signalling defects may require corrective therapy with bone marrow transplantation. Gene therapy, a potential curative approach in the future, currently remains a distant prospect. Those with a defective CSR mechanism generally do well on immunologoblulin replacement therapy. Complications may include autoimmunity, lymphoid hyperplasia and, in some cases, a predisposition to lymphoid malignancy.     

蜱抗凝血肽与RGDS肽重组基因的酵母表达载体构建

目的构建蜱抗凝血肽与RGDS肽重组基因,将其克隆到甲醇酵母,建立分泌表达载体pPIC9K。方法设计、合成蜱抗凝血肽与RGDS肽的双功能分子的重组基因,对人工合成的目的基因片段进行酶切、测序鉴定。运用PCR技术,扩增目的基因,PCR产物经胶回收后克隆至毕赤酵母表达载体pPIC9K,用双酶切和DNA测序鉴定重组质粒。结果PCR、酶切鉴定和DNA测序证明双功能基因重组质粒构建成功。结论该方法能方便、高效地获得所需的多拷贝基因,为下一步在毕赤酵母中表达双功能分子蛋白建立了基础。     

Intramolecular cross-overs generate deleted mitochondrial DNA molecules in Podospora anserina

The unavoidable senescence process that limits the vegetative growth of Podospora anserina is always associated with an accumulation of various classes of circular, tandemly arranged, defective mitochondrial DNA molecules (senDNAs). The monomers of the senDNAs belonging to the so-called β class share a common core, but differ in both their length and termini. To understand the mechanism leading to their formation, we have determined the junction sequence of 36 senDNA β monomers present in various senescent cultures. In most cases, we observe that: (1) short direct repeats precisely bound the senDNA β termini and (2) one copy of the repeats is retained in the senDNA sequence. Moreover, PCR analysis of the mitochondrial DNA of some of the senescent cultures, has allowed us to detect another genome which is exactly lacking the sequence of the senDNA β found in the culture. These results demonstrate that an intramolecular unequal cross-over occurring between short direct repeats can generate deleted mtDNA molecules in P. anserina. In addition, the polymorphism displayed by one pair of repeats allows us to establish that this cross-over may be associated with a short conversion tract spanning a few (about 15) nucleotides. Received: 16 May / 11 November 1996     

The new HLA-Cw*0442 allele possibly generated by a recombination event involving Cw*04010101 and Cw*1801

A new human leukocyte antigen-C (HLA-C) allele Cw*0442 was identified in a Spanish Caucasian patient by sequencing-based typing (SBT). HLA-Cw*0442 differs from Cw*04010101 by three amino acid replacements at positions 9 (S>D), 11 (S>A), and 14 (W>R).     

Quantitation of aberrant interlocus T-cell receptor rearrangements in mouse thymocytes and the effect of the herbicide 2,4-dichlorophenoxyacetic acid

Small studies in human populations have suggested a correlation between the frequency of errors in antigen receptor gene assembly and lymphoid malignancy risk. In particular, agricultural workers exposed to pesticides have both an increased risk for lymphoma and an increased frequency of errors in antigen receptor gene assembly. In order to further investigate the potential of such errors to serve as a mechanistically based biomarker of lymphoid cancer risk, we have developed a sensitive PCR assay for quantifying errors of V(D)J recombination in the thymocytes of mice. This assay measures interlocus rearrangements between two T-cell receptor loci, V-gamma and J-beta, located on chromosomes 13 and 6, respectively. The baseline frequency in four strains of mice was determined at several ages (2-8 weeks of age) and was found to be stable at approximately 1.5 x 10(-5) per thymocyte. Strain AKR, which has a high susceptibility to T-cell lymphomas, did not show an elevated frequency of aberrant V(D)J events. We used this assay to examine the effects of the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) on the frequency of these events. Female B6C3F1 mice, 27 days of age, were exposed to 2,4-D by gavage at doses of 0, 3, 10, 30, and 100 mg/kg/day for 4 successive days and sacrificed on day 5. Thymus DNA was isolated and examined for illegitimate V(D)J recombination-mediated gene rearrangements. In addition, pregnant mice were exposed to 2,4-D and thymocytes from the offspring examined at 2 weeks of age. No significant increase in aberrant V(D)J rearrangements was found, indicating that under these conditions 2,4-D does not appear to effect this important mechanism of carcinogenesis.     

Fas死亡信号激发域cDNA的克隆、表达及体外生物学效应

目的：克隆及表达Fas死亡信号激发域(Fas Activation Domain，FasAD)片段，获得具有生物学活性的FasAD多肽。方法：应用半巢式逆转录-多聚酶链反应(RT-PCR)扩增Fas死亡信号激发域cDNA片段，构建Intein表达型原核表达载体FasAD-pTYB12，应用IMPACT^TM-CN系统表达及进行一步法亲合层析分离、纯化FasAD多肽。结果：DNA序列测定显示克隆的FasAD cDNA碱基排列顺序与Genebank(M67454)所示完全一致。重组表达载体FasAD-pTYB12经IPTG诱导，成功表达可溶性融合蛋白，进一步分离、纯化获得分子量约5000的FasAD多肽，Westem blot显示FasAD多肽能被兔抗人Fas多抗识别。初步的生物学活性鉴定显示，该FasAD多肽抑制rhFasL诱导的细胞凋亡的生物学活性最高可达70％。结论：可利用IMPACT^TM-CN系统制备Fas死亡信号激发域的小分子量多肽，为深入研究Fas结构与功能的关系及与配体的相互作用提供了相关的实验基础，为进一步开发相关的生物免疫调节剂提供了实验依据。     

Extended runs of homozygosity at 17q11.2: an association with type‐2 NF1 deletions?

Large deletions in the NF1 gene region at 17q11.2 are caused by nonallelic homologous recombination (NAHR). The recurrent type‐2 NF1 deletions span 1.2 Mb, with breakpoints in the SUZ12 gene and SUZ12P. Type‐2 NF1 deletions occur preferentially during mitosis and are associated with somatic mosaicism. A panel of 16 type‐2 NF1 deletions was used as a model system in which to investigate whether extended homozygosity across 17q11.2 might be associated with somatic deletion. Using SNP arrays, a 3.2 Mb interval encompassing the NF1 deletion region was found to harbor runs of homozygosity (ROHs) in different human populations. However, ROHs ≥500 kb directly flanking the NF1 deletion region on both sides were not found to occur disproportionately in NF1 patients harboring type‐2 deletions compared to controls. Although low allelic diversity in 17q11.2 is unlikely to be a key factor in promoting NAHR‐mediated somatic type‐2 deletions, a specific ROH of 588 kb (roh1), located some 525 kb proximal to the deletion interval, was found to occur more frequently (P=0.012) in the type‐2 deletion patients compared with controls. We postulate that roh1 may act remotely, via an as yet unknown mechanism, to increase the frequency of somatic recombination between the distally duplicated SUZ12 sequences. Hum Mutat 30:1–10, 2010. © 2010 Wiley‐Liss, Inc.     

Functional PMS2 hybrid alleles containing a pseudogene‐specific missense variant trace back to a single ancient intrachromosomal recombination event

Sequence exchange between PMS2 and its pseudogene PMS2CL, embedded in an inverted duplication on chromosome 7p22, has been reported to be an ongoing process that leads to functional PMS2 hybrid alleles containing PMS2‐ and PMS2CL‐specific sequence variants at the 5′‐and the 3′‐end, respectively. The frequency of PMS2 hybrid alleles, their biological significance, and the mechanisms underlying their formation are largely unknown. Here we show that overall hybrid alleles account for one‐third of 384 PMS2 alleles analyzed in individuals of different ethnic backgrounds. Depending on the population, 14–60% of hybrid alleles carry PMS2CL‐specific sequences in exons 13–15, the remainder only in exon 15. We show that exons 13–15 hybrid alleles, named H1 hybrid alleles, constitute different haplotypes but trace back to a single ancient intrachromosomal recombination event with crossover. Taking advantage of an ancestral sequence variant specific for all H1 alleles we developed a simple gDNA‐based polymerase chain reaction (PCR) assay that can be used to identify H1‐allele carriers with high sensitivity and specificity (100 and 99%, respectively). Because H1 hybrid alleles harbor missense variant p.N775S of so far unknown functional significance, we assessed the H1‐carrier frequency in 164 colorectal cancer patients. So far, we found no indication that the variant plays a major role with regard to cancer susceptibility. Hum Mutat 31:1–8, 2010. © 2010 Wiley‐Liss, Inc.     

Genotoxicity of vesicular fluid and saline extract of Taenia solium metacestodes in somatic cells of Drosophila melanogaster

Neurocysticercosis, the most common parasitic disease of the central nervous system, is caused by cysticerci of the helminth Taenia solium, which is prevalent in developing countries and is reemerging in affluent societies. This helminth is associated with brain tumors and hematological malignancies in humans. In the present study, we analyzed the genotoxicity of vesicular fluid (VF) and a saline extract (SE) of T. solium metacestodes in the Drosophila melanogaster wing somatic mutation and recombination test (SMART). Third-instar larvae derived from standard and high bioactivation crosses were treated for approximately 48 hr with 12.5, 25.0, and 50.0 microg/ml of VF and SE of T. solium metacestodes. Negative (phosphate buffered saline) and positive (10 mM urethane) controls were also included. The results showed that the two test compounds were genotoxic in both crosses of Drosophila. Nevertheless, further research is needed to determine the genotoxic potential of specific compounds present in VF and SE and their role in the development of cancer.     

Changing distribution of norovirus genotypes and genetic analysis of recombinant GIIb among infants and children with diarrhea in Japan

A total of 402 fecal specimens collected during July 2003-June 2004 from infants and children with acute gastroenteritis, encompassing five localities (Maizuru, Tokyo, Sapporo, Saga, and Osaka) of Japan, were tested for the presence of norovirus by RT-PCR. It was found that 58 (14.4%) fecal specimens were positive for norovirus. Norovirus infection was detected throughout the year with the highest prevalence in December. Norovirus GII was the most predominant genogroup (98.3%; 57 of 58). The genotypes detected in this study were GI/4, GII/2, GII/3, GII/4, and GII/6. Of these, NoV GII/3 (known as the Arg320 virus cluster) was the most predominant genotype (43.9%), followed by NoV GII/4 (the Lordsdale virus cluster; 35.1%) and others. Two norovirus strains clustered with a "new variant designated GIIb" and a "new variant of GII/4" were found circulating in Japan for the first time. It was interesting to note that NoV GIIb and NoV GII/3 appeared to be the recombinant strains and the recombination site was demonstrated at the overlap of ORF1 and ORF2. The majority (96%) of the dominant norovirus strains were identified as the recombination of GII/3 capsid and GII/12 polymerase. The recombination in the NoV GIIb capsid gene at the breakpoint located at P1 domain was also identified. Obviously, NoV GIIb isolate in Japan had double recombination. This is the first report demonstrating the existence of different "new variants" co-circulating in Japanese infants and children with acute gastroenteritis.