Maria Peñaherrera - Academia.edu (original) (raw)

Papers by Maria Peñaherrera

Research paper thumbnail of Pervasive polymorphic imprinted methylation in the human placenta

Genome Research, 2016

The maternal and paternal copies of the genome are both required for mammalian development, and t... more The maternal and paternal copies of the genome are both required for mammalian development, and this is primarily due to imprinted genes, those that are monoallelically expressed based on parent-of-origin. Typically, this pattern of expression is regulated by differentially methylated regions (DMRs) that are established in the germline and maintained after fertilization. There are a large number of germline DMRs that have not yet been associated with imprinting, and their function in development is unknown. In this study, we developed a genome-wide approach to identify novel imprinted DMRs in the human placenta and investigated the dynamics of these imprinted DMRs during development in somatic and extraembryonic tissues. DNA methylation was evaluated using the Illumina HumanMethylation450 array in 134 human tissue samples, publicly available reduced representation bisulfite sequencing in the human embryo and germ cells, and targeted bisulfite sequencing in term placentas. Forty-thre...

Research paper thumbnail of X-chromosome inactivation (XCI) patterns in placental tissues of a paternally derived bal t(X;20) case

American Journal of Medical Genetics Part A, 2002

Non-random X-chromosome inactivation (XCI) is often seen in female carriers of balanced X-autosom... more Non-random X-chromosome inactivation (XCI) is often seen in female carriers of balanced X-autosome translocations and is generally attributed to a selective growth of cells that inactivate the normal X chromosome. However, little is known concerning when in development the selection acts, and thus whether skewed XCI would also be seen in placental tissues. Furthermore, as males with X-autosome translocations are normally infertile, all translocations studied to date for XCI-skewing have been either maternal or de novo in origin. We now present an analysis of XCI status in cord blood, umbilical cord and four different extraembryonic tissues from a female carrier of a paternally derived balanced (X;20) translocation. Using methylation based assays to determine XCI status, we found preferential inactivation of the non-translocated X in cord blood, umbilical cord and amnion samples of the propositus. Remarkably, random XCI was evident in several placental tissues analyzed (chorion, and chorionic villi trophoblast and mesenchyme). While these findings support the hypothesis of strong selection against cells with an inactive translocated X-chromosome in most embryonic/ fetal tissues, they also suggest weaker selective forces taking place during placental development. Additionally, the finding of normal placental development in the present case, rules out the possibility of a parental bias to XCI in human extraembryonic tissues as a requisite for normal development. The finding of hypomethylation in extraembryonic tissues for two out of three markers used in the study is consistent with previous findings demonstrating low levels of methylation in these tissues.

Research paper thumbnail of Different measures of “genome-wide” DNA methylation exhibit unique properties in placental and somatic tissues

Epigenetics, 2012

performance liquid chromatography (HPLC). Using HPLC, individual nucleotides are counted and a to... more performance liquid chromatography (HPLC). Using HPLC, individual nucleotides are counted and a total measure of 5-methylcytosine (5-mC) content is obtained. 6 5-mC-specific antibody kits have been developed as a less expensive and less labor intensive alternative to HPLC. 7, 8 While both HPLC and antibody kits provide a measure of average global methylation, the distribution of 5-mCs within the genome cannot be determined with these methods. Alternatively, array-based methods sacrifice some genomic coverage for the ability to detect the specific location of 5-mCs. Methylated DNA immunoprecipitation (MeDIP) captures methylated fragments of DNA with a 5-mC-specific antibody and is used in combination with a microarray for comparison against a differentially labeled control. 9 Another array-based method, the Illumina Infinium HumanMethylation27 BeadChip array, allows single-nucleotide resolution of the DNA methylation status of 27578 CpG sites in over 14,000 gene promoters. 10 DNA methylation of two RE families, LINE-1 and Alu, has recently been used to assess genome-wide DNA methylation

Research paper thumbnail of Chromosome-wide DNA methylation analysis predicts human tissue-specific X inactivation

Human genetics, 2011

X-chromosome inactivation (XCI) results in the differential marking of the active and inactive X ... more X-chromosome inactivation (XCI) results in the differential marking of the active and inactive X with epigenetic modifications including DNA methylation. Consistent with the previous studies showing that CpG island-containing promoters of genes subject to XCI are approximately 50% methylated in females and unmethylated in males while genes which escape XCI are unmethylated in both sexes; our chromosome-wide (Methylated DNA ImmunoPrecipitation) and promoter-targeted methylation analyses (Illumina Infinium HumanMethylation27 array) showed the largest methylation difference (D = 0.12, p < 2.2 E-16) between male and female blood at X-linked CpG islands promoters. We used the methylation differences between males and females to predict XCI statuses in blood and found that 81% had the same XCI status as previously determined using expression data. Most genes (83%) showed the same XCI status across tissues (blood, fetal: muscle, kidney and nerual); however, the methylation of a subset o...

Research paper thumbnail of Human Placental-Specific Epipolymorphism and its Association with Adverse Pregnancy Outcomes

PLoS ONE, 2009

Interindividual variation in DNA-methylation level is widespread in the human genome, despite its... more Interindividual variation in DNA-methylation level is widespread in the human genome, despite its critical role in regulating gene expression. The nature of this variation, including its tissue-specific nature, and the role it may play in human phenotypic variation and disease is still poorly characterized. The placenta plays a critical role in regulating fetal growth and development in ways that have lifelong effects on health. To identify genes with a high degree of interindividual DNA methylation variation in the human placenta, we surveyed the human genome using the Illumina GoldenGate Methylation Cancer panel targeting 1505 CpG sites of 807 genes. While many sites show a continuous pattern of methylation levels, WNT2, TUSC3 and EPHB4 were identified to have a polymorphic ''on-or-off'' pattern of DNA methylation variation at their promoter region which was confirmed by pyrosequencing. Methylation of these genes can be found in 7%-25% of over 100 placentas tested. The methylation state at the promoter of these genes is concordant with mRNA allelic expression. In three informative cases TUSC3 was observed to be methylated on the maternal allele, and it is thus possible this represents a polymorphically imprinted gene. Furthermore, TUSC3 promoter methylation showed evidence for association with preeclampsia. A biological significance of these methylation allelic polymorphisms (MAPs) to human placental diversity is further implied by their placental specificity and absence in mouse. An extended study of blood suggests that MAPs may also be found in other tissues, implicating their utility for tissue-specific association with complex disorders. The identification of such ''epipolymorphism'' in other tissues and their use in association studies, should improve our understanding of interindividual phenotypic variability and complex disease susceptibility.

Research paper thumbnail of Allele Frequencies of 15 STR Loci in the Population of the City of Quito, Ecuador

Journal of Forensic Sciences, 2008

Whole blood obtained by venipuncture was collected from unrelated individuals residing in Quito C... more Whole blood obtained by venipuncture was collected from unrelated individuals residing in Quito City, Ecuador, South America, using one 1.2-mm micropunch of bloodstains collected on FTA Classic Cards (Whatman FTA Ò 3), and the DNA was extracted following the manufacturer's directions. PCR amplification of the 15 STR loci (D3S1358,

Research paper thumbnail of The dynamics of X-inactivation skewing as women age

Clinical Genetics, 2004

Non-random X-chromosome inactivation (XCI) has been associated with X-linked diseases, neoplastic... more Non-random X-chromosome inactivation (XCI) has been associated with X-linked diseases, neoplastic diseases, recurrent pregnancy loss, and trisomy risk. It also occurs more commonly in older female populations. To understand the etiology of non-random XCI and utilize this assay appropriately in clinical research and practice, the age-related alteration in XCI patterns in normal females needs to be clearly defined. In the present study, we evaluated the XCI status in 350 unselected women aged 0-88 years with unknown history of genetic disorders or abnormal pregnancies. DNA samples were extracted from peripheral blood and analyzed by a methylation-based assay at the androgen receptor locus. A weak but significant positive correlation was observed between age and degree of skewing in XCI over the whole age range (r = 0.23, p &lt; 0.0001), and skewing values become non-normally distributed at older ages. However, the increase in skewed XCI appears to be more pronounced after age 30 than at younger ages. This trend supports the model of increased skewing with age as a consequence of hematopoietic stem cell senescence. An alternative possibility is that there is allele-specific loss of methylation with time that results in the appearance of increased XCI skewing using a methylation-based assay.

Research paper thumbnail of Methylation ofZNF261 as an assay for determining X chromosome inactivation patterns

American Journal of Medical Genetics, 2003

Research paper thumbnail of Patterns of placental development evaluated by X chromosome inactivation profiling provide a basis to evaluate the origin of epigenetic variation

Human Reproduction, 2012

background: Inactivation of the maternally or paternally derived X chromosome (XCI) initially occ... more background: Inactivation of the maternally or paternally derived X chromosome (XCI) initially occurs in a random manner in early development; however as tissues form, a 'patchiness' will occur in terms of which X is inactivated if cells positioned near each other are clonally descended from a common precursor. Determining the relationship between skewed XCI in different tissues and in different samples from the same tissue provides a molecular assessment of the developmental history of a particular tissue that can then be used to understand how genetic and epigenetic variation arises in development. methods: XCI skewing was evaluated in and compared between amnion, chorion, trophoblast and mesenchyme using multiple sampling sites from 14 term placentae. XCI was also evaluated in chorionic villus samples obtained at multiple sites and depths from four additional term placentae. The pattern of variation was then compared with methylation variation associated with the H19/IGF2 imprinting control region (ICR); promoter regions of KISS1, PTPN6, CASP8 and APC; and LINE-1 elements. results: Mean placental level of skewing for amnion and chorion are correlated, consistent with a common developmental origin of at least a component of these membranes from inner cell mass derivatives subsequent to XCI, while trophoblast appears to be derived independently, consistent with its origin from the trophectoderm. Villus samples taken from different depths spanning the fetal to maternal side of the placenta were highly clonally related. Comparing patterns of clonal growth identified through XCI to the distribution of epigenetic variation in other genomic regions suggests that some variation arises early in development (e.g. LINE-1 methylation), whereas other variation arises predominantly after villus tree formation (e.g. methylation at H19/IGF2 ICR). conclusions: The patterns of XCI skewing are consistent with a model whereby each biopsied site of chorionic villi represents one or a few individual villus trees, each of which is clonally derived from only one or a few precursor cells. Sampling of placentae to evaluate changes associated with clinical pathology should be done with consideration of the tree-to-tree differences. A limitation of this study is the small number of placentas used and therefore placental-specific differences in variation could not be assessed.

Research paper thumbnail of Editorial Board

Research paper thumbnail of Quantification of Cell-Free DNA in Normal and Complicated Pregnancies: Overcoming Biological and Technical Issues

PLoS ONE, 2014

The characterization of cell-free DNA (cfDNA) originating from placental trophoblast in maternal ... more The characterization of cell-free DNA (cfDNA) originating from placental trophoblast in maternal plasma provides a powerful tool for non-invasive diagnosis of fetal and obstetrical complications. Due to its placental origin, the specific epigenetic features of this DNA (commonly known as cell-free fetal DNA) can be utilized in creating universal 'fetal' markers in maternal plasma, thus overcoming the limitations of gender-or rhesus-specific ones. The goal of this study was to compare the performance of relevant approaches and assays evaluating the amount of cfDNA in maternal plasma throughout gestation (7.2-39.5 weeks). Two fetal-or placental-specific duplex assays (RPP30/SRY and RASSF1A/b-Actin) were applied using two technologies, real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR). Both methods revealed similar performance parameters within the studied dynamic range. Data obtained using qPCR and ddPCR for these assays were positively correlated (total cfDNA (RPP30): R = 0.57, p = 0.001/placental cfDNA (SRY): R = 0.85, p,0.0001; placental cfDNA (RASSF1A): R = 0.75, p,0.0001). There was a significant correlation in SRY and RASSF1A results measured with qPCR (R = 0.68, p = 0.013) and ddPCR (R = 0.56, p = 0.039). Different approaches also gave comparable results with regard to the correlation of the placental cfDNA concentration with gestational age and pathological outcome. We conclude that ddPCR is a practical approach, adaptable to existing qPCR assays and well suited for analysis of cell-free DNA in plasma. However, it may need further optimization to surpass the performance of qPCR.

Research paper thumbnail of Quantification of DNA methylation at imprinted domains in the placenta

Research paper thumbnail of Origin of amnion and implications for evaluation of the fetal genotype in cases of mosaicism

Prenatal Diagnosis, 2002

Objective To investigate presence of trisomy in amniotic epithelium (uncultured amnion) and mesen... more Objective To investigate presence of trisomy in amniotic epithelium (uncultured amnion) and mesenchyme (cultured amnion) from mosaic cases to understand the origins of these tissues and their relationship to pregnancy outcome.

Research paper thumbnail of Evaluating DNA methylation and gene expression variability in the human term placenta

Placenta, 2010

Obtaining representative samples from a term placenta for gene-expression studies is confounded b... more 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.

Research paper thumbnail of The mechanisms involved in formation of deletions and duplications of 15q11-q13

Journal of Medical Genetics, 1998

Haplotype analysis was undertaken in 20 cases of 15q11-q13 deletion associated with Prader-Willi ... more Haplotype analysis was undertaken in 20 cases of 15q11-q13 deletion associated with Prader-Willi syndrome (PWS) or Angelman syndrome (AS) to determine if these deletions arose through unequal meiotic crossing over between homologous chromosomes. Of these, six cases of PWS and three of AS were informative for markers on both sides of the deletion. For four of six cases of paternal 15q11-q13 deletion (PWS), markers on both sides of the deletion breakpoints were inferred to be of the same grandparental origin, implying an intrachromosomal origin of the deletion. Although the remaining two PWS cases showed evidence of crossing over between markers flanking the deletion, this was not more frequent than expected by chance given the genetic distance between proximal and distal markers. It is therefore possible that all PWS deletions were intrachromosomal in origin with the deletion event occurring after normal meiosis I recombination. Alternatively, both sister chromatid and homologous chromosome unequal exchange during meiosis may contribute to these deletions. In contrast, all three cases of maternal 15ql1-q13 deletion (AS) were associated with crossing over between flanking markers, which suggests significandy more recombination than expected by chance (p=0.002). Therefore, there appears to be more than one mechanism which may lead to PWS/AS deletions or the resolution of recombination intermediates may differ depending on the parental origin of the deletion. Furthermore, 13 of 15 cases of 15qll-ql3 duplication, triplication, or inversion duplication had a distal duplication breakpoint which differed from the common distal deletion breakpoint. The presence of at least four distal breakpoint sites in duplications indicates that the mechanisms of rearrangement may be complex and multiple repeat sequences may be involved. (7Med Genet 1998;35:130-136)

Research paper thumbnail of Inactive X chromosome-specific reduction in placental DNA methylation

Human Molecular Genetics, 2009

Genome-wide levels of DNA methylation vary between tissues, and compared with other tissues, the ... more Genome-wide levels of DNA methylation vary between tissues, and compared with other tissues, the placenta has been reported to demonstrate a global decrease in methylation as well as decreased methylation of X-linked promoters. Methylation is one of many features that differentiate the active and inactive X, and it is well established that CpG island promoters on the inactive X are hypermethylated. We now report a detailed analysis of methylation at different regions across the X in male and female placenta and blood. A significant (P < 0.001) placental hypomethylation of LINE1 elements was observed in both males and females. Relative to blood placental promoter hypomethylation was only observed for X-linked, not autosomal promoters, and was significant for females (P < 0.0001) not males (P 5 0.9266). In blood, X-linked CpG island promoters were shown to have moderate female methylation (66% across 70 assays) and low (23%) methylation in males. A similar methylation pattern in blood was observed for 20% of non-island promoters as well as 50% of the intergenic or intragenic CpG islands, the latter is likely due to the presence of unannotated promoters. Both intragenic and intergenic regions showed similarly high methylation levels in male and female blood (68 and 66%) while placental methylation of these regions was lower, particularly in females. Thus placental hypomethylation relative to blood is observed globally at repetitive elements as well as across the X. The decrease in X-linked placental methylation is consistently greater in females than males and implicates an inactive X specific loss of methylation in the placenta.

Research paper thumbnail of Chromosome-wide DNA methylation analysis predicts human tissue-specific X inactivation

Human genetics, 2011

X-chromosome inactivation (XCI) results in the differential marking of the active and inactive X ... more X-chromosome inactivation (XCI) results in the differential marking of the active and inactive X with epigenetic modifications including DNA methylation. Consistent with the previous studies showing that CpG island-containing promoters of genes subject to XCI are approximately 50% methylated in females and unmethylated in males while genes which escape XCI are unmethylated in both sexes; our chromosome-wide (Methylated DNA ImmunoPrecipitation) and promoter-targeted methylation analyses (Illumina Infinium HumanMethylation27 array) showed the largest methylation difference (D = 0.12, p < 2.2 E-16) between male and female blood at X-linked CpG islands promoters. We used the methylation differences between males and females to predict XCI statuses in blood and found that 81% had the same XCI status as previously determined using expression data. Most genes (83%) showed the same XCI status across tissues (blood, fetal: muscle, kidney and nerual); however, the methylation of a subset o...

Research paper thumbnail of DNA methylation profiling of human placentas reveals promoter hypomethylation of multiple genes in early-onset preeclampsia

European Journal of Human Genetics, 2010

Preeclampsia and intrauterine growth restriction (IUGR) are two of the most common adverse pregna... more Preeclampsia and intrauterine growth restriction (IUGR) are two of the most common adverse pregnancy outcomes, but their underlying causes are mostly unknown. Although multiple studies have investigated gene expression changes in these disorders, few studies have examined epigenetic changes. Analysis of the DNA methylation pattern associated with such pregnancies provides an alternative approach to identifying cellular changes involved in these disorders. We analyzed methylation of 1505 CpG sites associated with 807 genes in 26 placentas from early-onset preeclampsia (EOPET), late-onset preeclampsia, IUGR and control subjects using an Illumina GoldenGate Methylation panel. Thirty-four loci were hypomethylated (false discovery rate o10% and methylation difference 410%) in the early-onset preeclamptic placentas while no and only five differentially methylated loci were found in late-onset preeclamptic and IUGR placentas, respectively. Hypomethylation of 4 loci in EOPET was further confirmed by bisulfite pyrosequencing of 26 independent placental samples. The promoter of TIMP3 was confirmed to be significantly hypomethylated in EOPET placentas (P¼0.00001). Our results suggest that gene-specific hypomethylation may be a common phenomenon in EOPET placentas, and that TIMP3 could serve as a potential prenatal diagnostic marker for EOPET.

Research paper thumbnail of Extensive epigenetic reprogramming in human somatic tissues between fetus and adult

Epigenetics & Chromatin, 2011

Background: Development of human tissue is influenced by a combination of intrinsic biological si... more Background: Development of human tissue is influenced by a combination of intrinsic biological signals and extrinsic environmental stimuli, both of which are mediated by epigenetic regulation, including DNA methylation. However, little is currently known of the normal acquisition or loss of epigenetic markers during fetal and postnatal development.

Research paper thumbnail of Genome-wide mapping of imprinted differentially methylated regions by DNA methylation profiling of human placentas from triploidies

Epigenetics & Chromatin, 2011

ABSTRACT:

Research paper thumbnail of Pervasive polymorphic imprinted methylation in the human placenta

Genome Research, 2016

The maternal and paternal copies of the genome are both required for mammalian development, and t... more The maternal and paternal copies of the genome are both required for mammalian development, and this is primarily due to imprinted genes, those that are monoallelically expressed based on parent-of-origin. Typically, this pattern of expression is regulated by differentially methylated regions (DMRs) that are established in the germline and maintained after fertilization. There are a large number of germline DMRs that have not yet been associated with imprinting, and their function in development is unknown. In this study, we developed a genome-wide approach to identify novel imprinted DMRs in the human placenta and investigated the dynamics of these imprinted DMRs during development in somatic and extraembryonic tissues. DNA methylation was evaluated using the Illumina HumanMethylation450 array in 134 human tissue samples, publicly available reduced representation bisulfite sequencing in the human embryo and germ cells, and targeted bisulfite sequencing in term placentas. Forty-thre...

Research paper thumbnail of X-chromosome inactivation (XCI) patterns in placental tissues of a paternally derived bal t(X;20) case

American Journal of Medical Genetics Part A, 2002

Non-random X-chromosome inactivation (XCI) is often seen in female carriers of balanced X-autosom... more Non-random X-chromosome inactivation (XCI) is often seen in female carriers of balanced X-autosome translocations and is generally attributed to a selective growth of cells that inactivate the normal X chromosome. However, little is known concerning when in development the selection acts, and thus whether skewed XCI would also be seen in placental tissues. Furthermore, as males with X-autosome translocations are normally infertile, all translocations studied to date for XCI-skewing have been either maternal or de novo in origin. We now present an analysis of XCI status in cord blood, umbilical cord and four different extraembryonic tissues from a female carrier of a paternally derived balanced (X;20) translocation. Using methylation based assays to determine XCI status, we found preferential inactivation of the non-translocated X in cord blood, umbilical cord and amnion samples of the propositus. Remarkably, random XCI was evident in several placental tissues analyzed (chorion, and chorionic villi trophoblast and mesenchyme). While these findings support the hypothesis of strong selection against cells with an inactive translocated X-chromosome in most embryonic/ fetal tissues, they also suggest weaker selective forces taking place during placental development. Additionally, the finding of normal placental development in the present case, rules out the possibility of a parental bias to XCI in human extraembryonic tissues as a requisite for normal development. The finding of hypomethylation in extraembryonic tissues for two out of three markers used in the study is consistent with previous findings demonstrating low levels of methylation in these tissues.

Research paper thumbnail of Different measures of “genome-wide” DNA methylation exhibit unique properties in placental and somatic tissues

Epigenetics, 2012

performance liquid chromatography (HPLC). Using HPLC, individual nucleotides are counted and a to... more performance liquid chromatography (HPLC). Using HPLC, individual nucleotides are counted and a total measure of 5-methylcytosine (5-mC) content is obtained. 6 5-mC-specific antibody kits have been developed as a less expensive and less labor intensive alternative to HPLC. 7, 8 While both HPLC and antibody kits provide a measure of average global methylation, the distribution of 5-mCs within the genome cannot be determined with these methods. Alternatively, array-based methods sacrifice some genomic coverage for the ability to detect the specific location of 5-mCs. Methylated DNA immunoprecipitation (MeDIP) captures methylated fragments of DNA with a 5-mC-specific antibody and is used in combination with a microarray for comparison against a differentially labeled control. 9 Another array-based method, the Illumina Infinium HumanMethylation27 BeadChip array, allows single-nucleotide resolution of the DNA methylation status of 27578 CpG sites in over 14,000 gene promoters. 10 DNA methylation of two RE families, LINE-1 and Alu, has recently been used to assess genome-wide DNA methylation

Research paper thumbnail of Chromosome-wide DNA methylation analysis predicts human tissue-specific X inactivation

Human genetics, 2011

X-chromosome inactivation (XCI) results in the differential marking of the active and inactive X ... more X-chromosome inactivation (XCI) results in the differential marking of the active and inactive X with epigenetic modifications including DNA methylation. Consistent with the previous studies showing that CpG island-containing promoters of genes subject to XCI are approximately 50% methylated in females and unmethylated in males while genes which escape XCI are unmethylated in both sexes; our chromosome-wide (Methylated DNA ImmunoPrecipitation) and promoter-targeted methylation analyses (Illumina Infinium HumanMethylation27 array) showed the largest methylation difference (D = 0.12, p < 2.2 E-16) between male and female blood at X-linked CpG islands promoters. We used the methylation differences between males and females to predict XCI statuses in blood and found that 81% had the same XCI status as previously determined using expression data. Most genes (83%) showed the same XCI status across tissues (blood, fetal: muscle, kidney and nerual); however, the methylation of a subset o...

Research paper thumbnail of Human Placental-Specific Epipolymorphism and its Association with Adverse Pregnancy Outcomes

PLoS ONE, 2009

Interindividual variation in DNA-methylation level is widespread in the human genome, despite its... more Interindividual variation in DNA-methylation level is widespread in the human genome, despite its critical role in regulating gene expression. The nature of this variation, including its tissue-specific nature, and the role it may play in human phenotypic variation and disease is still poorly characterized. The placenta plays a critical role in regulating fetal growth and development in ways that have lifelong effects on health. To identify genes with a high degree of interindividual DNA methylation variation in the human placenta, we surveyed the human genome using the Illumina GoldenGate Methylation Cancer panel targeting 1505 CpG sites of 807 genes. While many sites show a continuous pattern of methylation levels, WNT2, TUSC3 and EPHB4 were identified to have a polymorphic ''on-or-off'' pattern of DNA methylation variation at their promoter region which was confirmed by pyrosequencing. Methylation of these genes can be found in 7%-25% of over 100 placentas tested. The methylation state at the promoter of these genes is concordant with mRNA allelic expression. In three informative cases TUSC3 was observed to be methylated on the maternal allele, and it is thus possible this represents a polymorphically imprinted gene. Furthermore, TUSC3 promoter methylation showed evidence for association with preeclampsia. A biological significance of these methylation allelic polymorphisms (MAPs) to human placental diversity is further implied by their placental specificity and absence in mouse. An extended study of blood suggests that MAPs may also be found in other tissues, implicating their utility for tissue-specific association with complex disorders. The identification of such ''epipolymorphism'' in other tissues and their use in association studies, should improve our understanding of interindividual phenotypic variability and complex disease susceptibility.

Research paper thumbnail of Allele Frequencies of 15 STR Loci in the Population of the City of Quito, Ecuador

Journal of Forensic Sciences, 2008

Whole blood obtained by venipuncture was collected from unrelated individuals residing in Quito C... more Whole blood obtained by venipuncture was collected from unrelated individuals residing in Quito City, Ecuador, South America, using one 1.2-mm micropunch of bloodstains collected on FTA Classic Cards (Whatman FTA Ò 3), and the DNA was extracted following the manufacturer's directions. PCR amplification of the 15 STR loci (D3S1358,

Research paper thumbnail of The dynamics of X-inactivation skewing as women age

Clinical Genetics, 2004

Non-random X-chromosome inactivation (XCI) has been associated with X-linked diseases, neoplastic... more Non-random X-chromosome inactivation (XCI) has been associated with X-linked diseases, neoplastic diseases, recurrent pregnancy loss, and trisomy risk. It also occurs more commonly in older female populations. To understand the etiology of non-random XCI and utilize this assay appropriately in clinical research and practice, the age-related alteration in XCI patterns in normal females needs to be clearly defined. In the present study, we evaluated the XCI status in 350 unselected women aged 0-88 years with unknown history of genetic disorders or abnormal pregnancies. DNA samples were extracted from peripheral blood and analyzed by a methylation-based assay at the androgen receptor locus. A weak but significant positive correlation was observed between age and degree of skewing in XCI over the whole age range (r = 0.23, p &lt; 0.0001), and skewing values become non-normally distributed at older ages. However, the increase in skewed XCI appears to be more pronounced after age 30 than at younger ages. This trend supports the model of increased skewing with age as a consequence of hematopoietic stem cell senescence. An alternative possibility is that there is allele-specific loss of methylation with time that results in the appearance of increased XCI skewing using a methylation-based assay.

Research paper thumbnail of Methylation ofZNF261 as an assay for determining X chromosome inactivation patterns

American Journal of Medical Genetics, 2003

Research paper thumbnail of Patterns of placental development evaluated by X chromosome inactivation profiling provide a basis to evaluate the origin of epigenetic variation

Human Reproduction, 2012

background: Inactivation of the maternally or paternally derived X chromosome (XCI) initially occ... more background: Inactivation of the maternally or paternally derived X chromosome (XCI) initially occurs in a random manner in early development; however as tissues form, a 'patchiness' will occur in terms of which X is inactivated if cells positioned near each other are clonally descended from a common precursor. Determining the relationship between skewed XCI in different tissues and in different samples from the same tissue provides a molecular assessment of the developmental history of a particular tissue that can then be used to understand how genetic and epigenetic variation arises in development. methods: XCI skewing was evaluated in and compared between amnion, chorion, trophoblast and mesenchyme using multiple sampling sites from 14 term placentae. XCI was also evaluated in chorionic villus samples obtained at multiple sites and depths from four additional term placentae. The pattern of variation was then compared with methylation variation associated with the H19/IGF2 imprinting control region (ICR); promoter regions of KISS1, PTPN6, CASP8 and APC; and LINE-1 elements. results: Mean placental level of skewing for amnion and chorion are correlated, consistent with a common developmental origin of at least a component of these membranes from inner cell mass derivatives subsequent to XCI, while trophoblast appears to be derived independently, consistent with its origin from the trophectoderm. Villus samples taken from different depths spanning the fetal to maternal side of the placenta were highly clonally related. Comparing patterns of clonal growth identified through XCI to the distribution of epigenetic variation in other genomic regions suggests that some variation arises early in development (e.g. LINE-1 methylation), whereas other variation arises predominantly after villus tree formation (e.g. methylation at H19/IGF2 ICR). conclusions: The patterns of XCI skewing are consistent with a model whereby each biopsied site of chorionic villi represents one or a few individual villus trees, each of which is clonally derived from only one or a few precursor cells. Sampling of placentae to evaluate changes associated with clinical pathology should be done with consideration of the tree-to-tree differences. A limitation of this study is the small number of placentas used and therefore placental-specific differences in variation could not be assessed.

Research paper thumbnail of Editorial Board

Research paper thumbnail of Quantification of Cell-Free DNA in Normal and Complicated Pregnancies: Overcoming Biological and Technical Issues

PLoS ONE, 2014

The characterization of cell-free DNA (cfDNA) originating from placental trophoblast in maternal ... more The characterization of cell-free DNA (cfDNA) originating from placental trophoblast in maternal plasma provides a powerful tool for non-invasive diagnosis of fetal and obstetrical complications. Due to its placental origin, the specific epigenetic features of this DNA (commonly known as cell-free fetal DNA) can be utilized in creating universal 'fetal' markers in maternal plasma, thus overcoming the limitations of gender-or rhesus-specific ones. The goal of this study was to compare the performance of relevant approaches and assays evaluating the amount of cfDNA in maternal plasma throughout gestation (7.2-39.5 weeks). Two fetal-or placental-specific duplex assays (RPP30/SRY and RASSF1A/b-Actin) were applied using two technologies, real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR). Both methods revealed similar performance parameters within the studied dynamic range. Data obtained using qPCR and ddPCR for these assays were positively correlated (total cfDNA (RPP30): R = 0.57, p = 0.001/placental cfDNA (SRY): R = 0.85, p,0.0001; placental cfDNA (RASSF1A): R = 0.75, p,0.0001). There was a significant correlation in SRY and RASSF1A results measured with qPCR (R = 0.68, p = 0.013) and ddPCR (R = 0.56, p = 0.039). Different approaches also gave comparable results with regard to the correlation of the placental cfDNA concentration with gestational age and pathological outcome. We conclude that ddPCR is a practical approach, adaptable to existing qPCR assays and well suited for analysis of cell-free DNA in plasma. However, it may need further optimization to surpass the performance of qPCR.

Research paper thumbnail of Quantification of DNA methylation at imprinted domains in the placenta

Research paper thumbnail of Origin of amnion and implications for evaluation of the fetal genotype in cases of mosaicism

Prenatal Diagnosis, 2002

Objective To investigate presence of trisomy in amniotic epithelium (uncultured amnion) and mesen... more Objective To investigate presence of trisomy in amniotic epithelium (uncultured amnion) and mesenchyme (cultured amnion) from mosaic cases to understand the origins of these tissues and their relationship to pregnancy outcome.

Research paper thumbnail of Evaluating DNA methylation and gene expression variability in the human term placenta

Placenta, 2010

Obtaining representative samples from a term placenta for gene-expression studies is confounded b... more 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.

Research paper thumbnail of The mechanisms involved in formation of deletions and duplications of 15q11-q13

Journal of Medical Genetics, 1998

Haplotype analysis was undertaken in 20 cases of 15q11-q13 deletion associated with Prader-Willi ... more Haplotype analysis was undertaken in 20 cases of 15q11-q13 deletion associated with Prader-Willi syndrome (PWS) or Angelman syndrome (AS) to determine if these deletions arose through unequal meiotic crossing over between homologous chromosomes. Of these, six cases of PWS and three of AS were informative for markers on both sides of the deletion. For four of six cases of paternal 15q11-q13 deletion (PWS), markers on both sides of the deletion breakpoints were inferred to be of the same grandparental origin, implying an intrachromosomal origin of the deletion. Although the remaining two PWS cases showed evidence of crossing over between markers flanking the deletion, this was not more frequent than expected by chance given the genetic distance between proximal and distal markers. It is therefore possible that all PWS deletions were intrachromosomal in origin with the deletion event occurring after normal meiosis I recombination. Alternatively, both sister chromatid and homologous chromosome unequal exchange during meiosis may contribute to these deletions. In contrast, all three cases of maternal 15ql1-q13 deletion (AS) were associated with crossing over between flanking markers, which suggests significandy more recombination than expected by chance (p=0.002). Therefore, there appears to be more than one mechanism which may lead to PWS/AS deletions or the resolution of recombination intermediates may differ depending on the parental origin of the deletion. Furthermore, 13 of 15 cases of 15qll-ql3 duplication, triplication, or inversion duplication had a distal duplication breakpoint which differed from the common distal deletion breakpoint. The presence of at least four distal breakpoint sites in duplications indicates that the mechanisms of rearrangement may be complex and multiple repeat sequences may be involved. (7Med Genet 1998;35:130-136)

Research paper thumbnail of Inactive X chromosome-specific reduction in placental DNA methylation

Human Molecular Genetics, 2009

Genome-wide levels of DNA methylation vary between tissues, and compared with other tissues, the ... more Genome-wide levels of DNA methylation vary between tissues, and compared with other tissues, the placenta has been reported to demonstrate a global decrease in methylation as well as decreased methylation of X-linked promoters. Methylation is one of many features that differentiate the active and inactive X, and it is well established that CpG island promoters on the inactive X are hypermethylated. We now report a detailed analysis of methylation at different regions across the X in male and female placenta and blood. A significant (P < 0.001) placental hypomethylation of LINE1 elements was observed in both males and females. Relative to blood placental promoter hypomethylation was only observed for X-linked, not autosomal promoters, and was significant for females (P < 0.0001) not males (P 5 0.9266). In blood, X-linked CpG island promoters were shown to have moderate female methylation (66% across 70 assays) and low (23%) methylation in males. A similar methylation pattern in blood was observed for 20% of non-island promoters as well as 50% of the intergenic or intragenic CpG islands, the latter is likely due to the presence of unannotated promoters. Both intragenic and intergenic regions showed similarly high methylation levels in male and female blood (68 and 66%) while placental methylation of these regions was lower, particularly in females. Thus placental hypomethylation relative to blood is observed globally at repetitive elements as well as across the X. The decrease in X-linked placental methylation is consistently greater in females than males and implicates an inactive X specific loss of methylation in the placenta.

Research paper thumbnail of Chromosome-wide DNA methylation analysis predicts human tissue-specific X inactivation

Human genetics, 2011

X-chromosome inactivation (XCI) results in the differential marking of the active and inactive X ... more X-chromosome inactivation (XCI) results in the differential marking of the active and inactive X with epigenetic modifications including DNA methylation. Consistent with the previous studies showing that CpG island-containing promoters of genes subject to XCI are approximately 50% methylated in females and unmethylated in males while genes which escape XCI are unmethylated in both sexes; our chromosome-wide (Methylated DNA ImmunoPrecipitation) and promoter-targeted methylation analyses (Illumina Infinium HumanMethylation27 array) showed the largest methylation difference (D = 0.12, p < 2.2 E-16) between male and female blood at X-linked CpG islands promoters. We used the methylation differences between males and females to predict XCI statuses in blood and found that 81% had the same XCI status as previously determined using expression data. Most genes (83%) showed the same XCI status across tissues (blood, fetal: muscle, kidney and nerual); however, the methylation of a subset o...

Research paper thumbnail of DNA methylation profiling of human placentas reveals promoter hypomethylation of multiple genes in early-onset preeclampsia

European Journal of Human Genetics, 2010

Preeclampsia and intrauterine growth restriction (IUGR) are two of the most common adverse pregna... more Preeclampsia and intrauterine growth restriction (IUGR) are two of the most common adverse pregnancy outcomes, but their underlying causes are mostly unknown. Although multiple studies have investigated gene expression changes in these disorders, few studies have examined epigenetic changes. Analysis of the DNA methylation pattern associated with such pregnancies provides an alternative approach to identifying cellular changes involved in these disorders. We analyzed methylation of 1505 CpG sites associated with 807 genes in 26 placentas from early-onset preeclampsia (EOPET), late-onset preeclampsia, IUGR and control subjects using an Illumina GoldenGate Methylation panel. Thirty-four loci were hypomethylated (false discovery rate o10% and methylation difference 410%) in the early-onset preeclamptic placentas while no and only five differentially methylated loci were found in late-onset preeclamptic and IUGR placentas, respectively. Hypomethylation of 4 loci in EOPET was further confirmed by bisulfite pyrosequencing of 26 independent placental samples. The promoter of TIMP3 was confirmed to be significantly hypomethylated in EOPET placentas (P¼0.00001). Our results suggest that gene-specific hypomethylation may be a common phenomenon in EOPET placentas, and that TIMP3 could serve as a potential prenatal diagnostic marker for EOPET.

Research paper thumbnail of Extensive epigenetic reprogramming in human somatic tissues between fetus and adult

Epigenetics & Chromatin, 2011

Background: Development of human tissue is influenced by a combination of intrinsic biological si... more Background: Development of human tissue is influenced by a combination of intrinsic biological signals and extrinsic environmental stimuli, both of which are mediated by epigenetic regulation, including DNA methylation. However, little is currently known of the normal acquisition or loss of epigenetic markers during fetal and postnatal development.

Research paper thumbnail of Genome-wide mapping of imprinted differentially methylated regions by DNA methylation profiling of human placentas from triploidies

Epigenetics & Chromatin, 2011

ABSTRACT: