Diagnóstico prenatal no invasivo: presente y futuro de mano de las nuevas tecnologías / Non-invasive prenatal diagnosis: present and future by means of new technologies

Desde que en 1997 se demostrara la presencia de ADN fetal en sangre periférica materna, son numerosos los grupos que se dedican a investigar en este campo para intentar desarrollar e incorporar a la rutina clínica el diagnóstico prenatal no invasivo. De la mano de la constatación de la presencia del ADN fetal en el torrente materno surgió el diagnóstico no invasivo del sexo fetal. Este y el estudio del RhD fetal han sido los únicos análisis incorporados a la rutina clínica. Aunque existen grupos investigando en el campo del diagnóstico de las enfermedades mendelianas, los esfuerzos se han centrado en el diagnóstico de las aneuploidías fetales. En un principio, el alcance de los diagnósticos estuvo limitado por la presencia mayoritaria de ADN materno coexistiendo con el ADN fetal. Sin embargo, el desarrollo reciente de tecnologías mucho más sensibles, está permitiendo un avance vertiginoso de este campo(AU)

Since the presence of foetal DNA in maternal peripheral blood was demonstrated in 1997, several research groups have developed their activity in this field in order to promote non-invasive prenatal diagnosis into clinical routine. By demonstrating the presence of foetal DNA in the maternal bloodstream, the non-invasive assessment of the sex of the foetus was achieved. This test, together with the foetal RhD determination, has been the only ones incorporated into clinical routine. Although there are research groups working on the diagnosis of Mendelian diseases, efforts have focused on the diagnosis of foetal aneuploidies. At first, the diagnostic scope was limited by the higher presence of maternal DNA co-existing with the foetal DNA. However, recent advances in more sensitive technologies is enabling dramatic progress to be made in this field(AU)

Non-invasive prenatal diagnosis of single-gene disorders from maternal blood.

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).

Noninvasive prenatal diagnosis of monogenic disorders.

INTRODUCTION: 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. AREAS COVERED: 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. EXPERT OPINION: 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.

Improvement in strategies for the non-invasive prenatal diagnosis of Huntington disease.

PURPOSE: We focused on the improvements of prenatal diagnosis by the analysis of DNA from maternal plasma, using Huntington disease as a model of disease. METHODS: We studied plasma from a pregnancy at risk of having a fetus affected with Huntington disease by the use of two direct analysis of the mutation and polymorphic STRs. RESULTS: Direct methods were not informative. Analysis with STRs revealed the presence of the allele that does not co-segregate with the disease, thus the fetus was healthy. CONCLUSIONS: This strategy is very useful to face complex cases when the direct study is not informative not only for Huntington disease but also for many other disorders.

MLPA as a screening method of aneuploidy and unbalanced chromosomal rearrangements in spontaneous miscarriages.

OBJECTIVE: The present study aims to validate multiplex ligation-dependent probe amplification (MLPA) technique with subtelomeric probe mixes as a screening method to detect aneuploidy and unbalanced terminal chromosomal rearrangements in spontaneous abortions (SAs). METHODS: MLPA with P036B and P070 probe mixes was performed on 221 miscarriage DNA samples between the 5th and 24th week of gestation. Cytogenetic culture was attempted on 178 miscarriages. Karyotyped miscarriages served as controls in this blinded study. Results were confirmed by quantitative fluorescent-PCR (QF-PCR). RESULTS: Among the karyotyped miscarriages, MLPA was able to detect all the expected aneuploidies, as well as an unbalanced product from a reciprocal translocation, and revealed cryptic deletions and duplications not visible at the 550-band resolution level. In addition, chromosomal anomalies were found in approximately 37% of cases that failed to grow or could not be cultivated. As expected, ploidy changes were not detected. Copy number variation was found for target sequences of P036B (CYFIP1, MRPL41, CAB45) and P070 (DECR2, TNFRSF18) probe mixes. CONCLUSIONS: We propose the use of MLPA with subtelomeric probe mixes as a reliable, rapid and economical first approach to detect aneuploidy and unbalanced terminal chromosomal rearrangements in SAs.

Double trisomy in spontaneous miscarriages: cytogenetic and molecular approach.

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.

Application of fetal DNA detection in maternal plasma: a prenatal diagnosis unit experience.

Non-invasive prenatal diagnosis tests based on the analysis of fetal DNA in maternal plasma have potential to be a safer alternative to invasive methods. So far, different studies have shown mainly fetal sex, fetal RhD, and quantitative variations of fetal DNA during gestation with fetal chromosomal anomalies or gestations at risk for preeclampsia. The objective of our research was to evaluate the use of fetal DNA in maternal plasma for clinical application. In our study, we have established the methodology needed for the analysis of fetal DNA. Different methods were used, according to the requirements of the assay. We have used quantitative fluorescent polymerase chain reaction (QF-PCR) to perform fetal sex detection with 90% sensitivity. The same technique permitted the detection of fetal DNA from the 10th week of gestation to hours after delivery. We have successfully carried out the diagnosis of two inherited disorders, cystic fibrosis (conventional PCR and restriction analysis) and Huntington disease (QF-PCR). Ninety percent of the cases studied for fetal RhD by real-time PCR were correctly diagnosed. The detection of fetal DNA sequences is a reality and could reduce the risk of invasive techniques for certain fetal disorders in the near future.

Expression of estrogen receptor subtypes and neuronal nitric oxide synthase in neutrophils from women and men: regulation by estrogen.

OBJECTIVES: (a) To identify the subtype of estrogen receptor (ER) expressed in neutrophils from premenopausal women and in neutrophils from men under different estrogen conditions and (b) to analyze the association between the modifications in the expression of ER subtypes and neuronal nitric oxide synthase (nNOS) expression induced by estrogen. METHODS: Neutrophils were isolated from pre-menopausal women during different stages of the menstrual cycle and from ten men for in vitro estrogen incubations. RESULTS: Neutrophils from premenopausal women expressed both ERalpha and ERbeta subtypes which were increased in the ovulatory phase of the menstrual cycle. Neutrophils derived from men also expressed ERalpha and ERbeta but only ERalpha expression was enhanced by in vitro incubation with 17beta-estradiol (10(-8) mol/l). In vitro incubation of neutrophils from women with 17beta-estradiol enhanced expression of both ER-alpha and ER-beta subtypes. nNOS protein was overexpressed in neutrophils from premenopausal women during the ovulatory phase. 17beta-Estradiol (10(-8) mol/l) also increased nNOS protein expression in neutrophils derived from men. Mithramycin A (10(-6) mol/l) and curcumin (10(-6) mol/l), prevented the upregulation of nNOS and ERalpha in neutrophils derived from men, suggesting the involvement of AP-1 and Sp-1 transcription factors. CONCLUSIONS: Although the in vivo levels of circulating estrogen concentrations seem to be associated with overexpression of both ERalpha and ERbeta in neutrophils from premenopausal women, which was further confirmed by the in vitro experiments with neutrophils from women, in vitro incubation of neutrophils from men with 17beta-estradiol only increased ERalpha protein expression which was associated with enhanced expression of nNOS protein.

Guías de práctica clínica de la Sociedad Española de Cardiología en la gestante con cardiopatía / Guidelines of the Spanish Society of Cardiology for the management of cardiovascular diseases during pregnancy

Durante la gestación normal se producen cambios hormonales, se establece la circulación uteroplacentaria, se incrementa el volumen plasmático, disminuyen las resistencias vasculares periféricas y se producen modificaciones que favorecen la hipercoagulabilidad. Todo ello favorece la aparición de semiología cardiovascular, haciendo que el diagnóstico diferencial con la existencia de cardiopatía subyacente sea a la vez fundamental y complejo. Además, estos cambios fisiológicos aumentan el riesgo de complicaciones maternofetales en las mujeres con cardiopatía. Se considera que la cardiopatía en el embarazo es la primera causa de morbimortalidad materna de causa no obstétrica. Las cardiopatías reumática y congénitas son, hoy día, las más frecuentes en la mujer embarazada, seguidas por la hipertensión arterial, la cardiopatía isquémica y las arritmias. En general, la enfermedad cardíaca tiende a empeorar con el tiempo, por lo que las mujeres con cualquier tipo de cardiopatía que deseen tener niños deberían hacerlo lo antes posible. La mayoría de las pacientes con cardiopatía no tienen problemas para llevar a término un embarazo. Las excepciones a esta regla son las pacientes en grado funcional III-IV con severo compromiso de la función cardíaca, la hipertensión pulmonar de cualquier origen, las cardiopatías congénitas con cianosis y grado funcional IIIIV, el síndrome de Marfan, las lesiones obstructivas izquierdas severas sintomáticas o asintomáticas con datos de disfunción sistólica, las portadoras de válvulas cardíacas artificiales y las mujeres con antecedentes de miocardiopatía asociada al embarazo, en las cuales debe desaconsejarse el embarazo o, en caso de producirse, puede recomendarse su interrupción, asumiendo los problemas éticos que se generan a la embarazada y al médico. Las cardiopatías más graves conllevan una elevada incidencia de aborto espontáneo, y en las cardiopatías congénitas debe valorarse el riesgo asociado de herencia. La intervención médica debe iniciarse precozmente, antes de la concepción, incluyendo la información respecto a las posibles repercusiones sobre la cardiopatía materna y el feto (AU)

La biopsia corial como diagnostico de las anomalias congenitas prenatales. / Choroidal biopsy as diagnosis of congenital prenatal abnormalities

Desde el punto de vista diagnostico prenatal, podemos simplificar diciendo que un feto puede engendrarse con 3 tipos de enfermedades: a) Alteraciones cromosomicas; b) malformaciones o anomalias estructurales; c) metabolopatias. Por medio de la biopsia corial transcervical podemos diagnosticar de manera precoz las anomalias cromosomicas y metabolicas, lo cual redundaria en una disminucion de la mortalidad y morbilidad peri y neonatal