Next-generation sequencing of HLA-G based on long-range polymerase chain reaction.

Clarifying the functional roles of HLA-G and the variation in the HLA-G gene that affects the expression are increasingly important in reproduction, cancer, organ transplantation, and autoimmune diseases. The homology between HLA genes and the genetic variability within each gene complicates the design of HLA gene-specific genotyping assays. We have designed a high-throughput, cost-efficient, robust, and specific assay for sequencing the full HLA-G gene including the 5'-upstream regulatory region, introns, and the 3'-untranslated region, using the next-generation sequencing (NGS) platform Ion Torrent PGM (Thermo Fisher Scientific, Waltham, Massachusetts). Conventional sequencing methods require the design of multiple primer pairs in order to cover the entire HLA-G gene. Designing multiple primer pairs specific for the HLA-G gene that also target all known alleles is difficult. Here, we present a setup that by the use of long-range polymerase chain reaction amplifies the whole HLA-G gene in a single reaction, which only requires a single HLA-G-specific primer pair. Enzymatic DNA shearing is used to break the long-range PCR product into shorter fragments ranging from 75 to 200 bp in length that are sequenced by NGS.

Distribution of HLA-G extended haplotypes and one HLA-E polymorphism in a large-scale study of mother-child dyads with and without severe preeclampsia and eclampsia.

The etiological pathways and pathogenesis of preeclampsia have rendered difficult to disentangle. Accumulating evidence points toward a maladapted maternal immune system, which may involve aberrant placental expression of immunomodulatory human leukocyte antigen (HLA) class Ib molecules during pregnancy. Several studies have shown aberrant or reduced expression of HLA-G in the placenta and in maternal blood in cases of preeclampsia compared with controls. Unlike classical HLA class Ia loci, the nonclassical HLA-G has limited polymorphic variants. Most nucleotide variations are clustered in the 5'-upstream regulatory region (5'URR) and 3'-untranslated regulatory region (3'UTR) of HLA-G and reflect a stringent expressional control. Based on genotyping and full gene sequencing of HLA-G in a large number of cases and controls (n > 900), the present study, which to our knowledge is the largest and most comprehensive performed, investigated the association between the HLA-G 14-bp ins/del (rs66554220) and HLA-E polymorphisms in mother and newborn dyads from pregnancies complicated by severe preeclampsia/eclampsia and from uncomplicated pregnancies. Furthermore, results from extended HLA-G haplotyping in the newborns are presented in order to assess whether a combined contribution of nucleotide variations spanning the 5'URR, coding region, and 3'UTR of HLA-G describes the genetic association with severe preeclampsia more closely. In contrast to earlier findings, the HLA-G 14-bp ins/del polymorphism was not associated with severe preeclampsia. Furthermore, the polymorphism (rs1264457) defining the two nonsynonymous HLA-E alleles, HLA-E*01:01:xx:xx and HLA-E*01:03:xx:xx, were not associated with severe preeclampsia. Finally, no specific HLA-G haplotypes were significantly associated with increased risk of developing severe preeclampsia/eclampsia.

Schizophrenia, neighbourhood, and crime.

OBJECTIVE: To investigate the relationship between the prevalence of schizophrenia and measures of social deprivation in varying areas in the city of Malmö. METHOD: A case-finding study of schizophrenia was combined with a victimization survey. Data was factor-analysed and visualized using geographical information system software. RESULTS: The schizophrenic patients lived predominantly in socially disorganized areas characterized by high levels of disorder, fear of crime and victimization. The local prevalence of schizophrenia covaried significantly with all indices of social disorganization. In addition, a significant but weak association was obtained between prevalence of schizophrenia and fear of specific persons in the neighbourhood. CONCLUSION: In spite of enormous changes in social conditions, psychiatric knowledge and organization of treatment, and political ambitions, schizophrenic patients still aggregate in socially deprived areas. This segregation may result in worsening of the illness as well as increasing the social disorganization in the local domicile.

The Nordic Study on schizophrenic patients living in the community. Subjective needs and perceived help.

In a community sample of 418 persons diagnosed with schizophrenia, subjective needs and perceived help was measured by the Camberwell Assessment of Need (CAN). The mean number of reported needs was 6.2 and the mean number of unmet needs 2.6. The prevalence of needs varied substantially between the need areas from 3.6% ('telephone') to 84.0% ('psychotic symptoms'). The rate of satisfaction estimated as the percentage of persons satisfied with the help provided within an area varied between 20.0% ('telephone') and 80.6% ('food'). The need areas concerning social and interpersonal functioning demonstrated the highest proportion of unmet to total needs. In a majority of need areas the patients received more help from services than from relatives, but in the areas of social relations the informal network provided substantial help. In general the patients reported a need for help from services clearly exceeding the actual amount of help received. In a linear regression model symptom load (BPRS) and impaired functioning (GAF) were significant predictors of the need status, explaining 30% of the variance in total needs and 20% of the variance in unmet needs. It is concluded that the mental health system fails to detect and alleviate needs in several areas of major importance to schizophrenic patients. Enhanced collaboration between the care system and the informal network to systematically map the need profile of the patients seems necessary to minimise the gap between perceived needs and received help.

Predictors of social relations in persons with schizophrenia living in the community: a Nordic multicentre study.

BACKGROUND: Deinstitutionalisation has led to persons with serious mental illness spending most of their time outside psychiatric institutions. Not much is known about their social life. The paper presents the results of structured interviews with non-institutionalised persons with schizophrenia about treatment, care and social network. The network data are analysed from three perspectives: finding predictors of the number and of the quality of social contacts, and establishing the respective variables that characterise persons with high, and those with low, scores on both the quantity and quality dimensions of social integration. METHODS: Random samples of persons with schizophrenia receiving outpatient services in ten psychiatric centres in the four Nordic countries were interviewed. The following instruments were used: Interview Schedule for Social Interaction (ISSI), Camberwell Assessment of Needs, Lancashire Quality of Life Profile, General Assessment of Functioning (GAF) and Brief Psychiatric Rating Scale (BPRS), in addition to a checklist covering the utilisation of different services. The ISSI provided the main data for this paper. A restricted number of possible predictors were used in General Linear Model (GLM) factorial analysis and discriminant analysis. RESULTS: A total of 418 persons took part in the study. The overall participation rate was 55%. Social integration in terms of number of contacts was related to a high GAF score, few BPRS negative and hostility symptoms, having contact with user organisations and living in urban (in contrast to rural) areas. Availability of emotional relations was predicted by female sex, low scores on the BPRS hostility dimension, high GAF score, having contact with one's family more than once a month, and living in urban areas. Work, adequate leisure activities and GAF score discriminated between the best and worst integrated groups. CONCLUSIONS: Living in urban areas, being female, having a high GAF score and low scores on hostility predicted better integration in terms of number of contacts and emotional relations.