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Prenatal diagnosis (PD) is available for pregnancies at risk of monogenic disorders. However, PD requires the use of invasive obstetric techniques for fetal-sample collection and therefore, involves a risk of fetal loss. Circulating fetal DNA in the maternal bloodstream is being used to perform non-invasive prenatal diagnosis (NIPD). NIPD is a challenging discipline because of the biological features of the maternal blood sample. Maternal blood is an unequal mixture of small (and fragmented) amounts of fetal DNA within a wide background of maternal DNA. For this reason, initial NIPD studies have been based on the analysis of specific paternally inherited fetal tracts not present in the maternal genome so as to ensure their fetal origin. Following this strategy, different NIPD studies have been carried out, such as fetal-sex assessment for pregnancies at risk of X-linked disorders, RhD determination, and analysis of single-gene disorders with a paternal origin. The study of the paternal mutation can be used for fetal diagnosis of dominant disorders or to more accurately assess the risk of an affected child in case of recessive diseases. Huntington's disease, cystic fibrosis, or achondroplasia are some examples of diseases studied using NIPD. New technologies are opening NIPD to the analysis of maternally inherited fetal tracts. NIPD of trisomy 21 is the latest study derived from the use of next-generation sequencing (NGS).

Resumen Desde que en 1997 se demostrara la presencia de ADN fetal en sangre periferica materna, son numerosos los grupos que se dedican a investigar en este campo para intentar desarrollar e incorporar a la rutina clinica el diagnostico prenatal no invasivo. De la mano de la constatacion de la presencia del ADN fetal en el torrente materno surgio el diagnostico no invasivo del sexo fetal. Este y el estudio del RhD fetal han sido los unicos analisis incorporados a la rutina clinica. Aunque existen grupos investigando en el campo del diagnostico de las enfermedades mendelianas, los esfuerzos se han centrado en el diagnostico de las aneuploidias fetales. En un principio, el alcance de los diagnosticos estuvo limitado por la presencia mayoritaria de ADN materno coexistiendo con el ADN fetal. Sin embargo, el desarrollo reciente de tecnologias mucho mas sensibles, esta permitiendo un avance vertiginoso de este campo.

Resumen Introduccion El presente estudio pretende describir las caracteristicas de los pacientes diagnosticados de deficit grave de alfa-1-antitripsina (AAT) en Espana, calcular la tasa de descenso del FEV1 con y sin tratamiento sustitutivo, e identificar factores asociados a una tasa de descenso acelerada del FEV1. Metodo Estudio retrospectivo de la evolucion de los individuos con deficit de alfa-1-antitripsina (DAAT) incluidos en el registro espanol. La variable principal evaluada en el estudio fue la tasa anual de descenso del FEV1. Resultados Se identificaron 303 pacientes con DAAT grave y fenotipo Pi ZZ. Se dispuso del seguimiento espirometrico de 117 pacientes. Ser fumador activo o ex fumador frente a nunca fumador (odds ratio [OR] = 10,31; intervalo de confianza (IC) del 95% = 1,8-58,8; p = 0,008) y tener un mayor FEV1(%) posbroncodilatador (OR = 1,03; IC del 95% = 1,005-1,06; p = 0,018), se asociaron de manera independiente a una tasa mas acelerada de descenso del FEV1. Se aprecio una tendencia entre tener un indice de masa corporal (IMC) bajo y experimentar una mayor tasa de deterioro del FEV1 (OR = 1,14; IC del 95% = 0,98-1,33; p = 0,085). Conclusiones Ser fumador o ex fumador, tener una funcion pulmonar preservada y un bajo IMC fueron los principales factores de riesgo asociados a una tasa acelerada de descenso del FEV1. Este hallazgo justificaria la necesidad de efectuar un seguimiento estrecho de los pacientes jovenes con un FEV1 mas preservado.

Multiple risk factors such as exposure to teratogens, advanced parental age, anatomic anomalies of the mother, genetic diseases and chromosomal anomalies are involved in embryo loss. Spontaneous abortions occur in approximately 10–15% of clinically recognised pregnancies. Most women who miscarry are not informed of the cause of their pregnancy loss, thereby causing them anxiety, confusion, low self-esteem and depression [1, 2]. Knowledge of the causes of spontaneous abortion reduces both stress and the feeling of culpability that occur after miscarriage [1]. Cytogenetic advances in the understanding and detection of human chromosomal rearrangements related to certain diseases have allowed for diagnosis of most cases of miscarriage. According to the literature, some 65% of first-trimester spontaneous abortions have been shown to carry chromosomal anomalies [3], although this figure may be below the actual percentage due to culture failure since karyotyping cannot be performed on such cases [4]. Therefore, it is likely that advanced molecular techniques such as multiplex ligation-dependent probe amplification (MLPA) would permit an increase in the percentage of chromosomal anomalies detected and even make it possible to identify other full monosomies which are involved in foetal loss. Loss of a whole chromosome may be lethal possibly because of the haploinsufficiency of essential genes. In fact, Turner syndrome (45X) is the only full monosomy that is compatible with life. Here, we report a full monosomy 21 identified by MLPA and confirmed by array CGH in a molecular study of 310 miscarriages. Full monosomy 21 (FM21) is a very rare chromosomal anomaly and one of the few cases that have appeared in the literature to date is the first full monosomy 21 observed in Spain published by Mori MA et al in 2004 [5].

Purposes We had previously demonstrated surviving sepsis campaign guidelines had not had enough impact at our Emergency Department. Basic Procedures Actions directed to increase the qualification of our staff and residents, to facilitate guidelines divulgation and to improve spatial conditions by creating a High Dependency Unit were implemented as a bundle. The impact of these actions on the achievement of early objectives of the campaign and on mortality was analyzed. Main Findings Following campaign guidelines was more frequent after the implementation of these actions, as shown by less restrictive fluids administration for more severe cases ( P = .001), earlier administration of antibiotics ( P = .001) and lactate determination rate (46% vs. 12%). In-hospital mortality difference did not reach statistical difference. Physicians were able to identify high-risk patients on clinical grounds. Principal Conclusions The bundle of actions has had a moderate beneficial effect on our Emergency Department. High Dependency Units are useful for managing patients not fulfilling criteria for Intensive Care Unit admission.

Background and purpose: The presence of cell-free fetal DNA in maternal plasma could allow performing a non-invasive prenatal diagnosis of Huntington disease (HD). The great advantage of this diagnosis is the absence of risk of fetal loss that it entails. Methods: Maternal plasma from four pregnant women in their first trimester of gestation with a fetus at-risk was studied. In all the four cases, the father was affected. Results: The diagnosis was performed both by a direct study of the mutation and an indirect haplotype study. By the direct analysis, three out of the four fetuses could be correctly diagnosed whilst the indirect analysis was only conclusive in one case. Conclusions: Non-invasive prenatal diagnosis of HD is possible by the analysis of fetal DNA in maternal plasma. Direct analysis of the mutation has shown higher accuracy than the haplotype analysis except for long expansions. Haplotype analysis would need to be improved for the study of Juvenile-onset HD. This diagnostic method would be limited to those couples with an affected male however this situation represents 80–90% of the pregnancies at-risk of HD. Moreover, it could be used as a confirmation test of healthy embryos transferred on pre-implantation genetic studies of HD.

There is limited information about factors associated with mortality of patients with chronic obstructive pulmonary disease (COPD) admitted to hospital because of an acute exacerbation.A retrospective cohort study including all patients admitted to hospital through our emergency department (ED) was conducted. A total of 972 electronic discharge reports were reviewed. Patient baseline features, aspects concerning acute exacerbation, as well as demographic, cardiac ultrasound, and microbiological data were collected.In-hospital mortality rate was 6.4%. Of 315 patients with mild exacerbation according to Anthonisen criteria, only 1 died. In the univariate analysis, moderate to severe acute exacerbation of COPD, age older than 75 years, severe COPD, abnormal blood gas values, onset of complications during hospital stay, radiologic consolidation, a positive result in a microbiological respiratory sample, home oxygenotherapy, admission to the intensive care unit, left ventricular ejection fraction, and department of admission were statistically significant (P.05). The multivariate analysis showed that moderate to severe COPD acute exacerbation (odds ratio [OR] 7.3; 95% confidence interval [CI], 3.6-17.7), age older than 75 years (OR 4.9; 95% CI, 2.3-10.8), severe COPD (OR 4.6; 95% CI, 2.1-10), abnormal blood gas values (OR 4.7; 95% CI, 1.1-19.8), and complication during hospital stay (OR 2.8; 95% CI 1.4-5.4) were independently related to mortality.We found that clinical aspect appears the most relevant of all potential determinants of in-hospital mortality for patients admitted for acute exacerbation of COPD. Thus, the clinical assessment and therapeutic decision taken in this first moment at the ED are the key that predict the prognosis of this patients. These data suggest that the risk of mortality after the admission to hospital of patients with COPD because of an acute exacerbation can be successfully predicted by making a clinical assessment at the ED.

Background Although single trisomy is the most common chromosomal abnormality observed within first trimester spontaneous abortions (SA) (>50%), double trisomy (DT) ranges from 0.21 to 2.8% in the literature. Since little is known about mechanisms underlying DT, we report the results of our experience with 517 SA, establishing parental origin and cell stage of non-disjunction when possible in DT cases, and making a revision of those previously reported. Methods Cytogenetic analysis was performed in all aborted specimens. Quantitative fluorescent PCR (QF-PCR) and multiplex ligation-dependent probe amplification (MLPA) were performed in DT cases in order to assess parental origin and stage of error of aneuploidy in addition to its reliability in detecting aneuploidies. Results Karyotyping was successful in 321 miscarriages; the rate of DT was 2.18%. Among the seven DT cases reported, three new combinations were found. Maternal origin was established for all DT SA analysed. Meiotic stage of error was presumed meiosis I (MI) for 48,XX+15+22 and 48,XX+8+21, meiosis II (MII) for 48,XXX+18, and MII and MI respectively for 48,XY+18+22. Molecular results agreed with cytogenetic results. Conclusions Similar maternal age-related mechanisms could be implicated in both single and double trisomy. Molecular techniques could be useful in diagnosing not only single but multiple aneuploidy and determining its origin. This will improve our knowledge about mechanisms underlying human aneuploidy, and enable appropriate genetic counselling.

a b s t r a c t The most common deficiency alleles for alpha-1-antitrypsin deficiency (AATD) are Pi*S and Pi*S, but there are also other deficiency variants. This case report describes the first two cases of AATD detected in Spain resulting from the combination of a null Mattawa allele with a normal PI*M, and a rare Mmalton. Both cases were initially diagnosed as Pi*MM by isoelectric focusing (IEF), but the low serum AAT values led us to suspect the existence of rare deficiency alleles that were undetectable using this technique, and to performing molecular analysis of the gene, which provided the correct diagnosis. Inconsistencies between serum AAT values and the phenotype should make one suspect the existence of one of these rare alleles.

Resumen Los alelos deficitarios mas frecuentes son los Pi*S y Pi*Z, pero existen tambien otras variantes deficientes. En la presente nota clinica se describen los 2 primeros casos detectados en Espana de deficit de alfa-1-antitripsina (DAAT), resultante de la combinacion de un alelo nulo Mattawa con un normal PI*M y con un raro Mmalton. Ambos casos fueron inicialmente diagnosticados como Pi*MM por isoelectroenfoque (IEE), pero los valores sericos bajos de AAT hicieron sospechar la existencia de alelos deficientes infrecuentes indetectables por IEE, por lo que se realizo un analisis molecular del gen que proporciono el diagnostico correcto. Las incoherencias entre los valores sericos de AAT y el fenotipo deben hacer sospechar la existencia de uno de estos alelos infrecuentes.

The effect of hereditary alpha-1 antitrypsin (AAT) deficiency can manifest clinically in the form of chronic obstructive pulmonary disease (COPD). AAT deficiency (AATD) is defined as a serum concentration lower than 35% of the expected mean value or 50 mg/dl (determined by nephelometry). It is associated in over 95% of cases with Pi*ZZ genotypes, and much less frequently with other genotypes resulting from combinations of Z, S, rare and null alleles. A systematic qualitative review was made of 107 articles, focusing mainly on an active search for AATD in COPD patients and intravenous (iv) treatment with AAT. On the basis of this review, the consultant committee of the Spanish Registry of Patients with AATD recommends that all COPD patients be screened for AATD with the determination of AAT serum concentrations, and when these are low, the evaluation must be completed with phenotyping and, on occasions, genotyping. Patients with severe AATD COPD should receive the pharmacological and non-pharmacological treatment recommended in the COPD guidelines. There is enough evidence from large observational studies and randomized placebo-controlled clinical trials to show that the administration of iv AAT reduces mortality and slows the progression of emphysema, hence its indication in selected cases that meet the inclusion criteria stipulated in international guidelines. The administration of periodic infusions of AAT is the only specific treatment for delaying the progression of emphysema associated with AATD.

Paternal Isodisomy of Chromosome 5 in a Patient With Recessive Multiple Epiphyseal Dysplasia Monica Martinez Garcia, Camilo Velez, Maria Fenollar-Cortes, Ana Bustamante, Isabel Lorda-Sanchez, Leandro Soriano-Guillen, and Maria-Jose Trujillo-Tiebas* Servicio de Genetica del Hospital Fundacion Jimenez Diaz de Madrid, Madrid, Spain Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), Madrid, Spain Seccion de Genetica Clinica, Servicio de Analisis Clinicos en el Hospital Clinico San Carlos de Madrid, Madrid, Spain Servicio de Pediatria del Hospital Fundacion Jimenez Diaz de Madrid, Madrid, Spain

Since the presence of circulating cell-free fetal DNA (ccffDNA) in maternal peripheral blood was demonstrated in 1997, great efforts have been done in order to use this source of fetal material for noninvasive prenatal diagnosis. The advantage that it represents is avoiding the obstetric invasive procedures required for conventional prenatal diagnosis.Efforts are mainly focused on finding the most accurate way to diagnose the most common fetal aneuploidies, paying special attention to trisomy 21. Recent advances in technology offer new diagnostic tools with high degrees of sensitivity thus generating great expectations for this type of diagnosis. However, there are other reasons why pregnant women undergo conventional prenatal diagnosis. Being at risk of transmitting a monogenic disorder is one of them. And although the percentage of those pregnancies may represent a small percentage of the diagnosis performed in the first trimester, these numbers should not be underestimated.Management of pregnancies at risk of an X-linked Mendelian disorder has changed thanks to the noninvasive fetal sex assessment. As for other Mendelian disorders, until recently, their study was limited to those cases paternally inherited. Nevertheless, the new emerging technologies are also opening the scope to maternally inherited disorders.

Introduction The present study intends to describe the characteristics of patients diagnosed with severe alpha-1 antitrypsin deficiency (AATD) in Spain, to observe the rate of decline in forced expiratory volume in 1 s (FEV1) with and without substitutive therapy, and to identify factors associated with a rapid rate of decline in FEV1. Method A retrospective study of the evolution of individuals with AATD was carried out based on data collected from the Spanish registry. The primary response variable was the annual rate of decline in FEV1, calculated using the baseline and last postbronchodilator FEV1 values in an endpoint analysis. Results Three hundred and three patients with severe AATD and Pi ZZ phenotype were identified. Follow-up spirometric data were collected for 117 subjects. Being a smoker or ex-smoker vs never smoker (odds ratio [OR]=10.31; 95% confidence interval (CI)=1.8–58.8; P=.008) and having a higher baseline postbronchodilator FEV1 (% predicted) (OR=1.03; 95% CI=1.005–1.06; P=.018) were independently associated with a more rapid rate of decline in FEV1. There was also a trend towards a relationship between low body mass index (BMI) and a greater rate of deterioration in lung function (OR=1.14; 95% CI=0.98–1.33; P=.085). Conclusion Being a smoker or ex-smoker, greater baseline lung function, and low BMI were the main risk factors associated with an accelerated rate of decline in FEV1. This finding warrants the close observation of younger patients with a better-preserved FEV1.

Owing to the risk of fetal loss associated with prenatal diagnostic procedures, the last decade has seen great developments in noninvasive prenatal diagnosis (NIPD). The discovery of circulating cell-free fetal DNA (ccffDNA) in maternal plasma has opened new lines of research in alternative technologies that may facilitate safe diagnosis. Because ccffDNA represents only a small fraction of all DNA present in maternal plasma and it is masked by the background of maternal DNA, the scope of NIPD was, until recently, limited to the study of paternal DNA sequences (i.e., detection of SRY sequences, RhD gene in RhD-negative women and paternally inherited single-gene disorders, such as cystic fibrosis and Huntington’s disease). However, new discoveries and technology are making NIPD a real option for patients and providing for an array of clinical applications, such as molecular studies in high-risk families, general screening and pregnancy management.

PURPOSE. Choroideremia (CHM) is an X-linked ophthalmic disease. The gene associated with CHM (REP-1) encodes a ubiquitously expressed protein that is indispensable for the posttranslational activation of retina-specific Rab protein. Different mutations, including large genomic rearrangements involving the REP-1 gene, are responsible for CHM, but they all cause the protein to be truncated or absent. The authors screened 20 Spanish families with clinical diagnoses of CHM to determine the molecular cause of the disease. METHODS. First, the authors performed haplotype analyses to determine whether the disease is linked to the REP-1 gene. In families in whom the disease segregated with the CHM locus (n = 14), mutational screening of the REP-1 gene was performed. RESULTS. In 13 of the 14 families in which the phenotype segregated with the CHM locus, the authors identified the mutation associated with the disease. Eight different molecular defects that led to truncation and one that led to complete absence of the REP-1 protein were found in nine families and one family, respectively. Furthermore, the authors identified a novel type of mutation in the REP-1 gene in three families. This novel type of mutation did not result in a truncated or absent protein. Rather, these patients lost different parts of the REP-1 mRNA in-frame that in all the cases encode a conserved protein domain implicated in the interaction with Rab proteins. CONCLUSIONS. Based on the different mutations found, the authors propose a four-step protocol for the molecular diagnosis of CHM.

The existence of foetal DNA in maternal blood, discovered in 1997, opened new possibilities for noninvasive prenatal diagnosis. This includes foetal sex assessment by the detection of specific Y chromosome sequences in maternal blood, particularly important when a foetus may be affected by an X-linked disorder such as haemophilia. This study aims to validate this sex assessment method and to test its clinical utility in the diagnosis of 15 potentially affected pregnancies in female carriers of haemophilia. In the validation study, 316 maternal blood samples from 196 pregnant women at gestations ranging from 5 weeks to 12 weeks were analysed. In the clinical study, 15 pregnancies at risk of having a haemophilic foetus were tested. All pregnancies in the validation study were correctly diagnosed. The accuracy and specificity of the methodology from the seventh week of gestation was 100%. The sex of all 15 pregnancies identified as being at risk of bearing a haemophilic foetus was correctly diagnosed. Foetal sex assessment by detecting specific Y chromosome sequences in maternal blood is now routinely used in our hospital because of its high accuracy from the seventh week of gestation. Reliable foetal gender determination from maternal blood of pregnant women carriers of haemophilia in the first trimester of gestation can avoid more conventional, invasive methods of prenatal diagnosis.

Background Since the presence of fetal DNA was discovered in maternal blood, different investigations have focused on non-invasive prenatal diagnosis. The analysis of fetal DNA in maternal plasma may allow the diagnosis of fetuses at risk of cystic fibrosis (CF) without any risk of fetal loss. Here, we present a new strategy for the detection of fetal mutations causing CF in maternal plasma. Methods We have used a mini-sequencing based method, the SNaPshot®, for fetal genotyping of the paternal mutation in maternal blood from three pregnancies at risk of CF. Results The paternal mutation was detected in the analysis of plasma samples from cases 1 and 3 but not in case 2. Results of a posterior conventional molecular analysis of chorionic biopsies were in full agreement with those obtained from analysis of the plasma samples. Conclusions The knowledge about the inheritance of the paternal mutation in a fetus may avoid the conventional prenatal diagnosis in some cases. The SNaPshot® technique has been shown to be a sensitive and accurate method for the detection of fetal mutations in maternal plasma. Its ease handling, rapid and low cost makes it appropriate for a future routine clinical use in non-invasive prenatal diagnosis of cystic fibrosis.

Purpose Leber congenital amaurosis (LCA) is one of the most severe inherited retinal dystrophies with the earliest age of onset. Mutations in the Crumbs homologue 1 (CRB1; OMIM 600105) gene explain 10%–24% of cases with LCA depending on the population. The aim of the present work was to study a fetal mutation associated to LCA in maternal plasma by a new methodology in the noninvasive prenatal diagnosis field: the denaturing High Performance Liquid Chromatography (dHPLC). Methods This study presents the case of a compound heterozygous fetus for two mutations in CRB1 (1q3.1-q32.2). dHPLC and automated DNA sequencing were used to detect the paternally inherited fetal mutation in a maternal plasma sample collected at the 12th week of gestation. To test the detection limit of dHPLC, we made serial dilutions of paternal DNA in control DNA. Results We were able to detect the presence of the paternally inherited fetal CRB1 mutation in maternal plasma by dHPLC. Moreover, by comparing chromatograms of serial dilutions to the plasma sample, we could ascertain that the percentage of fetal DNA in maternal plasma was at least 2%. However, the detection of the fetal mutation was not possible by automated DNA sequencing. Conclusions dHPLC seems to be sensitive enough to detect small amounts of fetal DNA in maternal plasma samples. It could be a useful tool for the noninvasive prenatal detection of paternally inherited point mutations associated with retinopathies.

The discovery of circulating fetal DNA in maternal blood has been an encouraging step forward in the prenatal diagnostic field. It has opened up the possibility of development of a noninvasive method for the genetic analysis of the fetus. Many techniques have been applied to the study of this fetal DNA, but automated sequencing has been seldom used. The intention of this study was to use the automated sequencing technique for the detection of a paternally inherited fetal mutation in maternal plasma. Maternal plasma samples from a pregnant woman, whose husband had a mutation (Q134X) in the RP2 gene, which is located in the X-chromosome, were collected at two different gestational ages (10th and 19th week of gestation) in order to determine whether the paternally inherited fetal mutation could be detected by automated sequencing. Restriction analysis was also performed to confirm the results. The fetal mutation was clearly detected in the maternal plasma by the use of automated sequencing. The automated sequencing enables the possibility of analyzing fetal sequences, at a nucleotide level, in order to detect mutations or polymorphisms which are distinguishable from maternal sequences.

Aims Different epidemiological studies have demonstrated that some ethanol containing beverages intake could be associated with a reduction of cardiovascular mortality, effect attributed in part to its antioxidant properties. Nuclear factor-kappa B (NF-kappaB) is a redox sensitive transcription factor implicated in the pathogenesis of atherosclerosis. We have examined the effect of four different ethanol containing beverages on the activation of NF-kappaB in peripheral blood mononuclear cells (PBMC) and circulating concentrations of monocyte chemoattractant protein-1 (MCP-1) in healthy volunteers receiving a fat-enriched diet. Methods and results Sixteen volunteers received 16 g/m2 of ethanol in form of red wine, spirits (vodka, rum, and brandy) or no ethanol intake along with a fat-enriched diet during 5 days and all of them took all alcohols at different periods. NF-kappaB activation (electrophoretic mobility shift assay) and circulating MCP-1 levels (ELISA) were examined in blood samples taken before and after 5 days of ethanol intake. Subjects receiving a fat-enriched diet had increased NF-kappaB activation in PBMC at day 5. Furthermore, MCP-1 levels were increased in plasma at day 5. Red wine intake and some ethanol beverages containing polyphenols (brandy and rum) prevented NF-kappaB activation and decreased MCP-1 release. Conclusion Consumption of moderate amounts of alcoholic drinks containing polyphenols decreases NF-kappaB activation in PBMCs and MCP-1 plasma levels during a fat-enriched diet. Our results provide additional evidence of the anti-inflammatory effects of some ethanol containing beverages, further supporting the idea that its moderate consumption may help to reduce overall cardiovascular mortality.