Evaluating DNA methylation and gene expression variability in the human term placenta.

UNLABELLED: Obtaining representative samples from a term placenta for gene-expression studies is confounded by both within placental heterogeneity and sampling effects such as sample location and processing time. Epigenetic processes involved in the regulation of gene expression, such as DNA methylation, may show similar variability, but are less well studied. Therefore, we investigated the nature of within and between- placenta variation in gene expression and DNA methylation of genes that were chosen for being differentially expressed or methylated by cell type within the placenta. METHODS: In total, two or more samples from each of 38 normal term placentae were utilized. The expression levels of CDH1, CDH11, ID2, PLAC1 and KISS1 were evaluated by real-time PCR. DNA methylation levels of LINE1 elements and CpGs within the promoter regions of KISS1, PTPN6, CASP8, and APC were similarly quantified by pyrosequencing. RESULTS: Despite considerable sample-to-sample variability within each placenta, the within-placenta correlation for both gene expression and methylation was significant for each studied gene. Most of this variability was not due to sample location. However, between placental differences in gene expression were inflated by the dramatic effect of processing time (0-24 h) on mRNA levels, particularly for PLAC1 and KISS1 (both expressed in the apical syncytiotrophoblast). In contrast, DNA methylation levels remained relatively constant over this same time period. CONCLUSION: Due to extensive site-to-site variability, multiple sampled sites are needed to accurately represent a placenta for molecular studies. Furthermore, mRNA quantitation of some genes may be hampered by its rapid degradation post-delivery (and possibly perinatally) and thus processing time should be considered in such analyses. Within-placenta correlations in expression and methylation from unrelated genes raise the possibility that methylation and expression variation may potentially reflect cell composition differences between samples rather than true differences occurring at the cellular level.

Identification of copy number variants in miscarriages from couples with idiopathic recurrent pregnancy loss.

BACKGROUND: Recurrent pregnancy loss (RPL), defined as two or more miscarriages, affects 3-5% of couples trying to establish a family. Despite extensive evaluation, no factor is identified in ∼40% of cases. In this study, we investigated the possibility that submicroscopic chromosomal changes, not detectable by conventional cytogenetic analysis, exist in miscarriages with normal karyotypes (46,XY or 46,XX) from couples with idiopathic RPL. METHODS: Array comparative genomic hybridization (array-CGH) was used to assess for DNA copy number variants (CNVs) in 26 miscarriages with normal karyotypes. Parental array-CGH analysis was performed to determine if miscarriage CNVs were de novo or inherited. RESULTS: There were 11 unique (previously not described) CNVs, all inherited, identified in 13 miscarriages from 8 couples. The maternal origin of two CNVs was of interest as they involved the imprinted genes TIMP2 and CTNNA3, which are only normally expressed from the maternal copy in the placenta. Two additional cohorts, consisting of 282 women with recurrent miscarriage (RM) and 61 fertile women, were screened for these two CNVs using a Quantitative Multiplex Fluorescent PCR of Short Fragments assay. One woman with RM, but none of the fertile women, carried the CTNNA3-associated CNV. CONCLUSIONS: This preliminary study shows that array-CGH is useful for detecting CNVs in cases of RPL. Further investigations of CNVs, particularly those involving genes that are imprinted in placenta, in women with RPL could be worthwhile.

Estudio colaborativo de abortos espontáneos: empleo de técnicas moleculares en muestras cuyo resultado citogenético no puede ser establecido / Colaborative study of spontaneus abortion: use of molecular techniques in failure culture samples

Introducción. El conocimiento de la causa de abortopermite ofrecer un adecuado consejo genético, además dereducir el estrés psicológico y el sentimiento de culpabilidadgenerado en la mujer tras sufrir un aborto. En la actualidadel estudio de abortos se realiza mediante técnicas citogenéticas,aunque muchas veces no es posible ofrecer un diagnósticodebido al fracaso de cultivo que acontece entre el5-42 % de los casos. En este trabajo se pretende evaluar lasensibilidad de las técnicas moleculares para el estudio dealteraciones cromosómicas en aquellos casos cuyo resultadocitogenético no puede ser establecido.Metodología. Se han estudiado 70 muestras de abortosdel primer y segundo trimestre de gestación mediante citogenéticay genética molecular (Quantitative FluorescentPolymerase chain Reaction [QF-PCR] y Multiplex ligationdependentProbe Amplification [MLPA]).Resultados. No se pudo establecer un resultado citogenéticoen el 37,2 % de las muestras. 44 fueron cariotipadas,obteniéndose 37 cariotipos normales y 7 anómalos. El estudiomolecular permitió establecer una dotación cromosómicaen el 100% de los casos, se encontraron alteraciones en10, de los cuales 3 carecían de resultado citogenético.Conclusiones. Se propone incluir en el protocolo de estudiode abortos el empleo de las técnicas citadas en casode fracaso del cultivo celular. La colaboración entre laboratoriosde citogenética y genética molecular especializados,permitiría ofrecer un resultado a la mayoría de las pacientes,esencial a la hora de poder establecer un adecuado consejogenético(AU)

Introduction. The knowledge of miscarriage causepermits offering an appropriate genetic counselling andmoreover, reduces psychological distress and self blamefeelings in women with a miscarriage. Nowadays, chromosomalstudy of abortions is mostly performed usingcytogenetic techniques. In few cases, no diagnosis can beestablished due to the high rate of culture failure (5-42%). Here, we try to evaluate the usefulness of moleculartechniques for the chromosomal study of those casesin which a cytogenetic result can not be established.Methods. A total of 70 miscarriage samples from thefirst and second trimesters of gestation have been studiedby karyotyping and molecular techniques (QF-PCRy MLPA).Results. The karyotype could not be established in37.2% of cases. Karyotype could be obtained in 44 samples,being 37 normal and 7 chromosomally abnormal.Molecular studies permitted to obtain a result in 100% ofthe cases, finding abnormalities in 10 of them, including3 in which the karyotype had not been established.Conclusions. We propose the use of molecular techniquesin those cases in which the culture fails. The collaborationbetween cytogenetic and molecular laboratorieswould permit to establish a result in the majority ofcases, which would represent an improvement for the offeringof an appropriate genetic counselling(AU)

Protocolo recomendado para el estudio cromosómico de abortos espontáneos: abordaje citogenético y molecular / Recommended protocol for the chromosomal study of spontaneous miscarriages: cytogenetic and molecular approach

Las alteraciones cromosómicas son responsables de más del 60 % de las pérdidas embrionarias que acontecen durante el primer trimestre de gestación. El diagnóstico de la causa del aborto reduce significativamente tanto el estrés psicológico a largo plazo como el sentimiento de culpabilidad generado en la mujer tras sufrir un aborto y permite ofrecer un adecuado consejo genético a estas parejas de cara a futuros embarazos. Tradicionalmente, el estudio cromosómico de los abortos espontáneos se ha abordado mediante el cultivo celular de los restos fetales para su posterior cariotipado. Sin embargo, el estudio citogenético de los restos abortivos en particular presenta determinadas limitaciones, como la elevada tasa de fracaso del cultivo celular o el sobrecrecimiento de células de origen materno en el cultivo. El empleo de técnicas moleculares como la QF-PCR y MLPA permite obtener información acerca de la constitución cromosómica del aborto solventando tales limitaciones. En el presente trabajo se propone el protocolo óptimo para el estudio cromosómico de los restos abortivos incluyendo un abordaje citogenético y molecular

Chromosomal abnormalities account for at least 60 % offirst trimester fetal losses. Identification of the cause of fetal loss significantly reduces long-term psychological distress and self-blame feelings in women with a miscarriage and enables offering an improved genetic counselling to these couples in future pregnancies. Routine cytogenetic study of miscarriagesentails high rates of culture failure or misdiagnosis due to maternal cell contamination. On the other hand, molecular techniques such as QF-PCR and MLPA have been proved to be reliable methods to obtain information about chromosomal constitution of miscarriages, solving culture related limitations. In the present manuscript, we propose the optimal protocol for the chromosomal study of spontaneous abortions from a combined cytogenetic and molecular approach

A small and active ring X chromosome in a female with features of Kabuki syndrome.

A ring X chromosome is found in about 6% of patients with Turner syndrome (TS), often with mosaicism for a 45,X cell line. Patients with this karyotype are reported to have a higher incidence of a more severe phenotype including mental retardation. In fact, some studies have shown a correlation between this severity and the presence or absence of an intact and functional X inactivation center (XIST). However, the phenotype of the individuals with r(X) cannot be entirely defined in terms of their X-inactivation patterns. Nevertheless, a small group of these patients have been described to manifest clinical features reminiscent of the Kabuki syndrome. Here we present a female patient with clinical features resembling Kabuki syndrome and a mos 45,X/46,X,r(X) karyotype. Methylation analyses of polymorphic alleles of the androgen receptor gene showed that both alleles were unmethylated suggesting an active ring chromosome. A specific X chromosome array CGH was performed estimating the size of the ring to be 17 Mb, lacking the XIST gene, and including some genes with possible implications in the phenotype of the patient.

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.

Foetal sex determination in maternal blood from the seventh week of gestation and its role in diagnosing haemophilia in the foetuses of female carriers.

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.

New approach for the refinement of the location of the X-chromosome breakpoint in a previously described female patient with choroideremia carrying a X;4 translocation.

Choroideremia (CHM) is an X-linked recessive ophthalmic disease characterized by a progressive degeneration of the choroid and the pigmented epithelium of the retina. We present the genetic characterization of a female patient affected with CHM who has been previously studied cytogenetically and showed a balanced translocation between chromosomes X and 4 [46,X,t(X;4)(q21;p16)]. The breakpoint in the X chromosome lies in the locus of CHM gene and for this reason, we have elucidated whether or not CHM was disrupted in the X chromosome involved in the translocation using different techniques. FISH showed that the 3'UTR and the last exons of the CHM were on the der(X) chromosome, and the 5'UTR and first exons of this gene were on the der(4) chromosome. Expression level analysis revealed that the breakpoint in the der(X) was located between exons 8 and 9 of the CHM gene because the expression level decreased from this point onwards. Based on this result the expression level analysis proved to be a valid method to pinpoint the location of breakpoints when the gene being expressed in peripheral blood is disrupted. Our results confirmed that the CHM gene was indeed disrupted in the X chromosome involved in the translocation. Besides, the nonrandom inactivation of the normal X chromosome observed using a methylation-specific polymerase chain reaction (M-PCR) technique explained the CHM in the female patient.