Nephron segment-specific gene expression using AAV vectors.

AAV9 vector provides efficient gene transfer in all segments of the renal nephron, with minimum expression in non-renal cells, when administered retrogradely via the ureter. It is important to restrict the transgene expression to the desired cell type within the kidney, so that the physiological endpoints represent the function of the transgene expressed in that specific cell type within kidney. We hypothesized that segment-specific gene expression within the kidney can be accomplished using the highly efficient AAV9 vectors carrying the promoters of genes that are expressed exclusively in the desired segment of the nephron in combination with administration by retrograde infusion into the kidney via the ureter. We constructed AAV vectors carrying eGFP under the control of: kidney-specific cadherin (KSPC) gene promoter for expression in the entire nephron; N

Molecular cloning and characterisation of the RESA gene, a marker of genetic diversity of Plasmodium falciparum

To identity immunodiagnostic antigen genes, a Plasmodium falciparum (Dd2 clone) expression library was screened using human immune sera. The ring-infected erythrocyte surface antigen (RESA) was isolated: this antigen of the resistant clone presents repeat tandem sequences like the 3D7 clone, albeit in different numbers. RESA has been studied as a marker of genetic diversity, with different sizes being observed in different isolates and clones of Plasmodium falciparum. The native protein was localised in cultures by western-blot and immuno-transmission electron microscopy. The antigenicity of RESA was evaluated by ELISA, using the carboxy-terminal repeat region as antigen. The assay’s sensitivity and specificity were 78.2 and 94% respectively

Clinical aspects and etiologic distribution of the newborn infants with congenital defects registered in the ECEMC

Dismorfología, Citogenética y Clínica: Resultados de estudios sobre los datos del ECEMCAn epidemiological analysis of the main clinical aspects of the infants with congenital defects registered by the ECEMC (Spanish Collaborative Study of Congenital Malformations) between 1980 and 2006, has been performed. Among a total of 2,254,439 newborn surveyed, 35,246 (1.56%), had congenital defects detected during the first 3 days of life. This group of malformed infants was distributed according to their clinical presentation as isolated (74.01%), multiply malformed (13.43%), and syndromes (12.56%). The etiologic distribution of infants with congenital anomalies in the ECEMC showed a 20.42% of genetic cause, 21.03% multifactorial, 1.22% produced by environmental cause, and in the remaining 57.33% the etiology of the defects was unknown. The secular distribution of the 3 main groups of clinical presentation (isolated, multiply malformed and syndromes) was studied and all of them showed a decreasing trend along the years, probably as a consequence of the impact of the interruption of pregnancy of some affected fetuses. The different types of syndromes and their minimal frequency values were also presented separated by type of cause.N

Clinical and genetic aspects of the hamartoneoplastic syndromes that can be diagnosed during the three first days of life.

Dismorfología, Citogenética y Clínica: Resultados de estudios sobre los datos del ECEMCThe hamartoneoplastic syndromes are an heterogeneous group of diseases characterized by their risk to develop malignant tumors among other clinical features that vary from one syndrome to another. Most of these pathologies also share endocrinological abnormalities and sometimes, genetic characteristics, including the autosomal dominant mode of inheritance. Therefore, pediatricians must be aware of their main characteristics in order to prevent as soon as possible further complications and to provide the appropiate genetic counseling to the affected patients and their families. In this chapter, we have classified these pathologies in seven different groups according to the more frequently affected tissue by the development of hamartomas. For each of these groups we reviewed the hamartoneoplastic syndromes that have some manifestations at birth. Finally, some practical guidelines are provided for their clinical, genetic diagnosis, and management.N

Epidemiological surveillance of congenital anomalies in Spain: Analysis of the data from the ECEMC’s Registry in the period 1980-2008

Aspectos EpidemiológicosThe Spanish Collaborative Study of Congenital Malformations (ECEMC) is a programme for research on congenital anomalies. It was created in 1976 by Prof. Martínez-Frías, as a hospital-based, case-control registry of newborn infants in Spain. Since its foundation, it has surveyed a total population of more than 2.6 million births, and studied more than 39,900 consecutive infants with congenital anomalies. According to the most recent data, the coverage of the registry reaches 21.55% of total births in Spain. The global frequency of infants with congenital defects has significantly decreased along the time, from 2.22% in the base period (1980-1985), to 1.03% in 2008. This significant decrease is mainly attributable to the impact of prenatal diagnosis and further interruption of some affected pregnancies, which is legal in Spain since 1985. Such a decrease has been observed in most of the Spanish Autonomic Regions (see Fig. 1) and participating hospitals, being statistically significant in many of them. The only Autonomic Region in which an increase was detected is Extremadura, where the base frequency was quite low due to a small coverage of births, in a period during which the most complicated pregnancies, and infants with serious birth defects, had to be referred to other regions (a situation that has changed since the basal period). From the periodic analysis of the frequency of a group of 33 defects selected due to their relatively high base frequency or morbidity/ mortality that they bear, only the heart/great vessels defects, and unilateral renal agenesis, have increased along the time, possibly as a result of increasing facilities for their diagnosis. The temporal-spatial analyses mostly detected decreases in the frequency of many of the defects studied in several Spanish Autonomic Regions. However, there were also some increases. Specifically, in the frequency of anophthalmia/microphthalmia in the Región de Murcia, cleft palate in Aragón, oesophageal atresia/stenosis in Galicia, and anal-rectal atresia/stenosis in Canarias. Regarding anophthalmia/ microphthalmia in the Región de Murcia, after excluding one case with a chromosomal abnormality and another one with familial Waardenburg syndrome, the increase lost its statistical significance, and apparently there was not any common denominator among the other cases registered, apart from the area of birth. With respect to the increase of oesophageal atresia/stenosis in Galicia, this was due to the birth of 4 cases in 2008, without any known common characteristic from which a causal relationship could be inferred. This defect will be subject to a special surveillance during the next months. Concerning the increase of cleft palate in Aragón, there was not any sign of a common cause restricted to this geographical area either. The last increase was based on the birth of 3 clinically different cases with anal atresia in Canarias, and no causal agent could be specifically linked to this area. From this report, it is clear the crucial role of the ECEMC system in the epidemiological surveillance of congenital anomalies in Spain, given its long experience since 1976, its huge database, the network of hospitals established, and the close and dynamic collaboration between its participants. This has enabled not only to calculate the birth frequency of congenital defects in Spain on a consecutive series on non-selected newborn infants, in a live and active system, but also to study their evolution along the time, and their geographical distribution, as well as their clinical diagnosis.N

Annual Report of epidemiological surveillance of congenital anomalies in Spain: Data of the period 1980-2010

Aspectos EpidemiológicosThe Spanish Collaborative Study of Congenital Malformations (ECEMC) annually undertakes the preparation and updating of the report of epidemiological surveillance of congenital anomalies in Spain. ECEMC is a research programme for congenital anomalies, based on an ongoing registry of births in Spain, which is hospital-based and has a case-control design. It has surveyed about 2.8 million births (Table 1), and gathered data on 41,800 consecutive infants with congenital anomalies and a similar number of healthy controls. Present coverage of the registry is 19.8% of total births in Spain (Table 2). The basal frequency of infants with congenital defects in our country is 2.22% (registered in 1980-1985), and it fell up to 1.07% in 2010, mainly as a result of the impact of elective termination of pregnancy after the detection of foetal anomalies (ETOPFA). ETOPFA has been legal in Spain since the end of the year 1985. Such a statistically significant decrease of the global frequency can be observed (Table 3) in many of the participating hospitals and most Spanish Autonomic Regions (see Fig. 1). Some increases in six hospitals were studied in detail. The only Autonomic Region in which an increase was detected is Extremadura, but this finding is probably due to methodological reasons in the first years, and referrals of high-risk pregnancies to other regions in those years, with considerable further changes that allow a better detection and reporting of cases in this region. The corrected global frequency by hospital and Autonomic Region, taking ETOPFA into account, was also analysed. The evolution of the frequency of a selected group of 33 defects with a relatively high base frequency and/or bearing a high morbidity/mortality was studied (Table 4). Most of them diminished along the time, the only increases being observed for heart/great vessels defects and unilateral renal agenesis, possibly as a result of better diagnostic procedures. Down syndrome is the defect for which a more marked decrease was measured (Graphs-1)A group of 18 defects were selected for the temporal-spatial analyses of the frequency, and also many statistically significant decreases were observed in most Spanish Autonomic Regions (Tables 5-10). The only increase was detected for anencephaly in the Balearic Islands, based on two births, and no clue on a local cause was obtained. Geographical heterogeneity could be detected in 2010 for anencephaly, spina bifida, anal/rectal atresia/stenosis, and hypospadias. For anencephaly, heterogeneity was attributable to the previously mentioned relatively high frequency registered in the Balearic Islands. For spina bifida, it was due to a high frequency observed in La Rioja, but based on the birth of just one case. For anal/rectal atresia/stenosis it was due to the high frequency registered in the quite distant regions of the Balearic Islands and La Rioja, and no common factor was identified as a possible cause. In all these cases it is noticeable that in regions where a small number of births is surveyed, the birth of just one case can bring the frequency to unusually high levels, and this can generate some geographical heterogeneity. For hypospadias, it was due to the low frequency observed in 2010 in the Comunidad Valenciana, and the relatively high frequency registered in Andalucia; all cases were balanic and isolated, and the higher frequency was observed in three hospitals in the provinces of Córdoba, Jaén and Malaga. All these findings will be subject to close scrutiny until the next surveillance report. Due to the importance of immigration in Spain in the last years, the ethnic origin of cases and foreign extraction of their parents were also analysed. The percentage of foreign parents has significantly increased with time, and was higher among the cases than among the controls (Graph 4). All ethnic groups had a higher risk for congenital anomalies than the native white group (Graph 6) and, except the oriental group, have increased with time (Graph 5). A reflection is included as a final comment, regarding the need of research on causes of birth defects, as expressed by Olshan et al. [Am J Med Genet A. 2011;155:1794–1797]: ‘For future generations, it is essential that we identify causes so that effective public health and clinical prevention programs can be established’. ECEMC, and other programmes worldwide, collaborate with that aim. For that purpose, ECEMC has a considerable background and experience of more than 35 years, as well as enough flexibility to adapt itself to new challenges, working for the prevention of birth defects.N

Analysis of the main clinical-epidemiological aspects of newborn infants with congenital defects registered through the ECEMC.

Dismorfología, Citogenética y Clínica: Resultados de estudios sobre los datos del ECEMCSome clinical aspects of the total set of infants registered by the Spanish Collaborative Study of Congenital Malformations (ECEMC) during the period 1980-2004, have been analyzed through an epidemiological approach. The ECEMC is defined as a clinical-epidemiological research program on congenital defects, and is built on an ongoing case-control, hospital-based registry of newborn infants in Spain. The ECEMC database for the study period is constituted by a total of 32,883 infants with congenital anomalies detected during the first 3 days of life, that represent 1.61% of the total births surveyed (2,045,751). In order to study the clinical presentation of infants with birth defects, the classification system developed in the ECEMC [Martínez-Frías et al., 2002: Rev Dismor Epidemiol V(1):2-8], which is based in the most modern concepts in Dysmorphology, has been applied. The infants were distributed in the 3 main groups of isolated, multiply malformed, and syndromes, as well as some other subgroups. The analysis of the time distribution of the prevalence of the 3 main groups of clinical presentation confirmed a significant decrease for all of them, that may be considered, at least in great part, as a consequence of the impact of the interruption of some affected pregnancies after the prenatal detection of anomalies. After studying the distribution by clinical presentation of 17 defects that were selected (based on their relative high frequency at birth, or the high morbidity/mortality that they bear, and their monitoring in other countries), a high clinical heterogeneity was evident, since most of them are presented as isolated, or associated to other birth defects. Some, such as gastroschisis, hypospadias, or anencephaly, tend to present isolated, while other, such as anophthalmia/microphthalmia, abdominal wall defects and bilateral renal agenesis, use to be associated to other developmental abnormalities. The etiologic distribution of infants with congenital anomalies has also been studied, and the different syndromes identified in the ECEMC has been detailed, indicating their respective gene map location if known (based on the OMIM database up to July 2005), as well as their minimum birth prevalence in the Spanish population (based on the ECEMC database). The study concludes highlighting the importance of accurate case definition and classification in clinically homogeneous groups in order to optimize, not only the power of epidemiological studies in investigating the causes of birth defects, but the molecular analysis of patients with accurate clinical diagnoses. Thus, probably molecular genetic studies implemented to birth defects epidemiology will also help to reach the final objective of infants being born healthy.N

Localization of serum resistance-associated protein in Trypanosoma brucei rhodesiense and transgenic Trypanosoma brucei brucei.

African trypanosomes infect a broad range of mammals, but humans and some higher primates are protected by serum trypanosome lytic factors that contain apolipoprotein L1 (ApoL1). In the human-infective subspecies of Trypanosoma brucei, Trypanosoma brucei rhodesiense, a gene product derived from the variant surface glycoprotein gene family member, serum resistance-associated protein (SRA protein), protects against ApoL1-mediated lysis. Protection against trypanosome lytic factor requires the direct interaction between SRA protein and ApoL1 within the endocytic apparatus of the trypanosome, but some uncertainty remains as to the precise mechanism and location of this interaction. In order to provide more insight into the mechanism of SRA-mediated resistance to trypanosome lytic factor, we assessed the localization of SRA in T. b. rhodesiense EATRO3 using a novel monoclonal antibody raised against SRA together with a set of well-characterized endosomal markers. By three-dimensional deconvolved immunofluorescence single-cell analysis, combined with double-labelling immunoelectron microscopy, we found that ≈ 50% of SRA protein localized to the lysosome, with the remaining population being distributed through the endocytic pathway, but apparently absent from the flagellar pocket membrane. These data suggest that the SRA/trypanolytic factor interaction is intracellular, with the concentration within the endosomes potentially crucial for ensuring a high efficiency.MN is funded by grants from the Spanish Ministerio de Ciencia e Innovación, (SAF2012-40029), Junta de Andalucia (CTS-5841) and VI PN de I+D+I 2008–2011, Instituto de Salud Carlos III – Subdirección General de Redes y Centros de Investigación Cooperativa (RICET) RD12/0018/0001 and RD12/0018/0015. J-MB is supported by a Miguel Servet Fellowship (CP09/00300) and funded by ‘Fondo de Investigación Sanitaria’ PI10/01128. JR and MC were funded by a Wellcome Trust Project Grant 093008/Z/10/Z. Work in the Dundee laboratory was funded by the Wellcome Trust (program grant 093008/Z/10/Z) and the Medical Research Council.This is the final version of the article. It first appeared from Wiley via http://dx.doi.org/10.1111/cmi.1245

Clinical-epidemiological analysis of the newborn infants with congenital defects registered by ECEMC: Distribution by etiology and ethnic groups.

Dismorfología y Genética ClínicaIt is presented here the analysis of the main clinical aspects of the infants with congenital defects registered by ECEMC (Spanish Collaborative Study of Congenital Malformations) between 1980 and 2010. Among a total of 2,648,286 newborns surveyed, 39,434 (1.49%) had congenital defects detected during the first 3 days of life. This group of infants with congenital anomalies was distributed according to the clinical presentation of their defects as isolated (73.94%), multiply malformed (13.53%), and syndromes (12.53%). The etiologic distribution of infants with congenital anomalies in the ECEMC showed a 20.47% of genetic cause, 20.28% multifactorial, 1.35% produced by environmental causes, and the etiology of the defects was unknown in the remaining 57.90%. The secular distribution of the 3 main groups of clinical presentation (isolated, multiply malformed and syndromes) was studied and all of them showed a decreasing trend along the years, probably as a consequence of the impact of the interruption of pregnancy of some affected foetuses. The different types of syndromes identified and their minimal frequency values are also presented, separated by type of cause (Tables 4-10). Finally, the proportion of cases with birth defects by ethnic groups, first including (Graph 8) and then excluding (Graph 9) two groups of whites, the autochthones and the immigrant whites group. Due to the small samples in most non-white groups, the differences are not statistically significant, except for a significant higher frequency among Gypsies than in the white groups (both native and foreigner), the black group, and the one of Other (including mix groups).N