克隆测序确认HLA新等位基因B*3712

目的：由于技术原理的限制，目前尚不能对所有的HLA等位基因进行严格的区分，特别是以往没有发现的新基因序列只能通过测序的方法解决，然而，当遇到等位基因杂合时，测序给出的结果仍然无法确认新的序列改变发生在等位基因的哪一侧，这时需要用分子生物学方法分离杂合子然后进行测序才能确定新的基因序列。采用基因克隆方法确认HLA新等位基因。 方法：实验于2006-01／05在河南省红十字血液中心HLA实验室，美国海军骨髓库HLA实验室完成。造血干细胞血样由中华骨髓库提供。采用荧光微珠HLA分型方法对中华骨髓库捐献者血样进行HLA分型检测，无法给出确切结果的摸棱两可结果标本用基因克隆（TOPO TA Cloning）、DNA测序的方法确认新的HLA基因序列。 结果：通过克隆分离杂合等位基因，再进行测序确认发现新的序列与B^＊3709相比，出现4个核苷酸改变：1．355nt C〉A，2．363nt C〉G，3．412nt G〉A，4．477ntC〉G，而且均发生在H哺B基因外显子3（exon3）。4处改变引起氨基酸编码改变：①编码95CTC〉ATC，氨基酸改变L〉1（亮氨酸〉异亮氨酸）。②97AGC〉AGG．S〉R（丝氨酸〉精氨酸）。③114GAC〉AACD〉N（天门冬氨酸〉天冬酰胺）。（9135GCC〉GCGA=A无氨基酸改变。 结论：①新的基因序列已经在GenBnak注册，被WHO的HLA因子命名委员会得到正式命名为HLA-B^＊3712基因。②基因克隆是确认HLA新基因的根本方法。     

The use of a recombinant immunoblot assay in the interpretation of anti- hepatitis C virus reactivity among prospectively followed patients, implicated donors, and random donors

Samples from prospectively followed recipients, their respective donors, and a cohort of random donors were used to evaluate the specificity and efficacy of a recombinant immunoblot assay (RIBA) as an adjunct to anti-hepatitis C virus (HCV) testing by enzyme immunoassay (EIA). RIBA reacted (RIBA+) in 100 percent of patients who developed hepatitis associated with anti-HCV seroconversion documented by EIA and in 100 percent of the EIA-positive (EIA+) donors implicated in these cases. In contrast, RIBA reacted in none of 10 recipients who were EIA+ but did not develop hepatitis, in none of 7 EIA+ patients with hepatitis B or cytomegalovirus infection, in 33 percent of EIA+ donors who were not implicated in hepatitis transmission, and in 37 percent of EIA+ random donors. Hence, the vast majority of EIA+ individuals who have ancillary evidence of HCV infection react on RIBA, whereas the majority of EIA+ individuals in low-risk settings do not react (RIBA-negative, or RIBA-). There was a strong association between RIBA reactivity and the presence of a surrogate marker (elevated alanine aminotransferase [ALT] and/or antibody to hepatitis B core antigen); 43 percent of RIBA+ implicated donors had a surrogate marker as compared to none of 14 EIA+, RIBA- donors. Among EIA+ random donors, 77 percent of those with a surrogate marker were RIBA+, as compared with 29 percent of those without a surrogate marker. In addition, in EIA+ donors, RIBA reactivity correlated with the extent of ALT elevation; 86 percent of those with an ALT greater than 135 IU per L were RIBA+ compared with 18 percent of those with an ALT less than 30 IU per L.(ABSTRACT TRUNCATED AT 250 WORDS)     

<Emphasis Type="Italic">For Debate: </Emphasis>Fetal and early postnatal growth restriction lead to diabetes,the metabolic syndrome and renal failure*

We review the progress in testing the thrifty phenotype hypothesis. Many human epidemiological studies both by ourselves and others have confirmed and extended the original observations on which the hypothesis was based. We are not aware of any contradictory findings and we emphasise the strength of the association between birth weight and the subsequent development of the metabolic syndrome. We have worked extensively experimentally to test the hypothesis in a rat model in which pregnant and/or lactating dams are fed a diet moderately restricted in proteins. The range of programming effects that we have discovered in this example of fetal and early postnatal growth restriction is listed and includes changes in hormone receptors, signalling molecules and regulatory enzymes. We have shown the model to develop diabetes, the metabolic syndrome and signs of premature renal failure. We summarise these and other similarities between the phenotype of this model and human Type 2 diabetes and the metabolic syndrome. The number of insults during early development which can lead to a similar outcome is discussed and the suggestion is made that the early life response to stress is limited in its flexibility with outcomes including ageing and decreased longevity. Our preliminary results indicate that some MODY genes could suggest pathways whereby the changes occur and that epigenetic changes during development are involved. We conclude that the way is now clear to discover early human markers of programming by early life growth restriction and to use these to devise strategies for the prevention of Type 2 diabetes.*Based upon the Dorothy Hodgkin Lecture delivered by CNH at the Birmingham Meeting of Diabetes UK, March 2002     

Identification and Management of Women at High Risk for Hereditary Breast/Ovarian Cancer Syndrome

Abstract:  Despite advances in identifying genetic markers of high risk patients and the availability of genetic testing, it remains challenging to efficiently identify women who are at hereditary risk and to manage their care appropriately. HughesRiskApps, an open-source family history collection, risk assessment, and Clinical Decision Support (CDS) software package, was developed to address the shortcomings in our ability to identify and treat the high risk population. This system is designed for use in primary care clinics, breast centers, and cancer risk clinics to collect family history and risk information and provide the necessary CDS to increase quality of care and efficiency. This paper reports on the first implementation of HughesRiskApps in the community hospital setting. HughesRiskApps was implemented at the Newton-Wellesley Hospital. Between April 1, 2007 and March 31, 2008, 32,966 analyses were performed on 25,763 individuals. Within this population, 915 (3.6%) individuals were found to be eligible for risk assessment and possible genetic testing based on the 10% risk of mutation threshold. During the first year of implementation, physicians and patients have fully accepted the system, and 3.6% of patients assessed have been referred to risk assessment and consideration of genetic testing. These early results indicate that the number of patients identified for risk assessment has increased dramatically and that the care of these patients is more efficient and likely more effective.     

Electronic Health Records and the Management of Women at High Risk of Hereditary Breast and Ovarian Cancer

Abstract:  Currently, management strategies exist that can decrease the morbidity and mortality associated with having a BRCA1 or BRCA2 mutation. Unfortunately, the task of identifying these patients at high risk is a daunting challenge. This problem is intensified because Electronic Health Records (EHRs) today lack the functionality needed to identify these women and to manage those women once they have been identified. Numerous niche software programs have been developed to fill this gap. Unfortunately, these extremely valuable niche programs are prevented from being interoperable with the EHRs, on the premise that each EHR vendor will build their own programs. Effectively, in our efforts to adopt EHRs, we have lost sight of the fact that they can only have a major impact on quality of care if they contain structured data and if they interact with robust Clinical Decision Support (CDS) tools. We are at a cross roads in the development of the health care Information Technology infrastructure. We can choose a path where each EHR vendor develops each CDS module independently. Alternatively, we can choose a path where experts in each field develop external niche software modules that are interoperable with any EHR vendor. We believe that the modular approach to development of niche software programs that are interoperable with current EHRs will markedly increase the speed at which useful and functional EHRs that improve quality of care become a reality. Thus, in order to realize the benefits of CDS, we suggest vendors develop means to become interoperable with external modular niche programs.     

Experimental IUGR and later diabetes

It is widely accepted that an association exists between the intrauterine environment in which a fetus grows and develops and the subsequent development of type 2 diabetes. Any disturbance in maternal ability to provide nutrients and oxygen to the fetus can lead to fetal intrauterine growth restriction (IUGR). Here we will review IUGR in rodent models, in which maternal metabolism has been experimentally manipulated to investigate the molecular basis of the relationship between IUGR and development of type 2 diabetes in later life, and the identification of the molecular derangements in specific metabolically - sensitive organs/tissues.     

G protein-coupled receptor 101 mRNA expression in the mouse brain: altered expression in the posterior hypothalamus and amygdala by energetic challenges

GPCR101 is a recently identified orphan G protein-coupled receptor (GPCR) expressed abundantly in the human and mouse hypothalamus. In the absence of a ligand, a direct approach to determine the function(s) of this receptor is not possible. However, clues to the possible functions of GPCR101 may yield from information on the distribution of the receptor and the effect of in vivo manipulation upon the expression level of the receptor. In situ hybridisation on mouse brain sections revealed GPCR101 expression in a number of nuclei, including the amygdala, lateral parabrachial nucleus and nucleus of the solitary tract, as well as in the arcuate nucleus, posterior hypothalamus and paraventricular nucleus of the hypothalamus. Food-deprivation was found to increase GPCR101 mRNA level in the posterior hypothalamus and amygdala. In obese mice bearing the ob gene mutation, GPCR101 mRNA level decreased in the posterior hypothalamus and remained unaltered in the amygdala. By contrast, in both nuclei, GPCR101 mRNA level did not change significantly in obese ob/ob mice after intraperitoneal injection of leptin or in mice fed with a high fat diet. These data suggest that GPCR101 mRNA expression in the posterior hypothalamus and amygdala is regulated by a factor(s) other than leptin. Dual in situ hybridisation was used to establish the relationship between GPCR101 and neuropeptides expressed in the hypothalamus. In the arcuate nucleus, GPCR101 mRNA was expressed in approximately half of the population of neurones expressing the mRNA for the anorexigenic neuropeptide, pro-opiomelanocortin, which suggests a potential functional relationship.     

Hypothalamic thyroid hormone catabolism acts as a gatekeeper for the seasonal control of body weight and reproduction

Seasonal adaptations in physiology exhibited by many animals involve an interface between biological timing and specific neuroendocrine systems, but the molecular basis of this interface is unknown. In this study of Siberian hamsters, we show that the availability of thyroid hormone within the hypothalamus is a key determinant of seasonal transitions. The expression of the gene encoding type III deiodinase (Dio3) and Dio3 activity in vivo (catabolism of T(4) and T(3)) is dynamically and temporally regulated by photoperiod, consistent with the loss of hypothalamic T(3) concentrations under short photoperiods. Chronic replacement of T(3) in the hypothalamus of male hamsters exposed to short photoperiods, thus bypassing synthetic or catabolic deiodinase enzymes located in cells of the ependyma of the third ventricle, prevented the onset of short-day physiology: hamsters maintained a long-day body weight phenotype and failed to undergo testicular and epididymal regression. However, pelage moult to a winter coat was not affected. Type II deiodinase gene expression was not regulated by photoperiod in these hamsters. Collectively, these data point to a pivotal role for hypothalamic DIO3 and T(3) catabolism in seasonal cycles of body weight and reproduction in mammals.     

Mechanisms of disease: the developmental origins of disease and the role of the epigenotype

There is accumulating evidence that many chronic diseases such as type 2 diabetes and coronary heart disease might originate during early life. This evidence gives rise to the developmental origins of disease hypothesis, and is supported by epidemiological data in humans and experimental animal models. A perturbed environment in early life is thought to elicit a range of physiological and cellular adaptive responses in key organ systems. These adaptive changes result in permanent alterations and might lead to pathology in later life. Aging organs and cells seem therefore to retain a 'memory' of their fetal history and adaptive responses. The mechanisms underlying the developmental origins of disease remain poorly defined. Epigenetic tagging of genes, such as DNA methylation and histone modification, controls the function of the genome at different levels and maintains cellular memory after many cellular divisions; importantly, tagging can be modulated by the environment and is involved in onset of diseases such as cancer. Here we review the evidence for the developmental origins of disease and discuss the role of the epigenotype as a contributing mechanism. Environmentally induced changes in the epigenotype might be key primary events in the developmental origins of disease, with important clinical implications.     

Sinus pericranii: diagnostic and therapeutic considerations in 15 patients

INTRODUCTION: Sinus pericranii (SP) is a rare, usually asymptomatic condition characterized by a large communication between the intra- and the extracranial venous drainage pathways in which blood may circulate bidirectionally through dilated veins of the skull. We describe our diagnostic and therapeutic experience with SP, with a special focus on the vascular analysis of digital subtraction angiography (DSA). METHODS: DSA images of 15 patients were evaluated with regard to the delay in opacification of the scalp vessels, the absence or distortion of the superficial cortical veins in the vicinity of the SP, the drainage patterns of the superior sagittal sinus, and the degree of maturation of the venous outlets of the brain. SP were classified either as "dominant", if the main stream of contrast flow used the SP to drain the brain bypassing usual venous outlets, or as "accessory", if only a small part of the venous outflow occurred through the extradiploic vessels. RESULTS: All patients presented with a nonpulsatile, soft-tissue mass. The lesion was on the midline in 14 of 15 patients, frontal in 12 patients, and parietal in 2 patients. In 13 patients, associated intracranial venous anomalies were present, eight of which were developmental venous anomalies. Seven patients had a dominant SP, and eight an accessory SP. CONCLUSION: SP can be considered the cutaneous sign of an underlying venous anomaly. If treatment is contemplated, analysis of the drainage pattern of the SP has to be performed. Treatment should be avoided in dominant SP or if its accessory role constitutes the only collateral pathway of an underlying venous anomaly.