COLD-PCR Amplification of Bisulfite-Converted DNA Allows the Enrichment and Sequencing of Rare Un-Methylated Genomic Regions

Aberrant hypo-methylation of DNA is evident in a range of human diseases including cancer and diabetes. Development of sensitive assays capable of detecting traces of un-methylated DNA within methylated samples can be useful in several situations. Here we describe a new approach, fast-COLD-MS-PCR, which amplifies preferentially un-methylated DNA sequences. By employing an appropriate denaturation temperature during PCR of bi-sulfite converted DNA, fast-COLD-MS-PCR enriches un-methylated DNA and enables differential melting analysis or bisulfite sequencing. Using methylation on the MGMT gene promoter as a model, it is shown that serial dilutions of controlled methylation samples lead to the reliable sequencing of un-methylated sequences down to 0.05% un-methylated-to-methylated DNA. Screening of clinical glioma tumor and infant blood samples demonstrated that the degree of enrichment of un-methylated over methylated DNA can be modulated by the choice of denaturation temperature, providing a convenient method for analysis of partially methylated DNA or for revealing and sequencing traces of un-methylated DNA. Fast-COLD-MS-PCR can be useful for the detection of loss of methylation/imprinting in cancer, diabetes or diet-related methylation changes

Hospital Amigo del Ni\uf1o (Baby Friendly): el mejor inicio para la lactancia materna

En 1991, la Organizaci\uf3n Mundial de la Salud y el Fondo para ni\uf1os de las Naciones Unidas lanzaron la Baby-Friendly Hospital Initiative (Iniciativa del Hospital Amigo del Ni\uf1o), la cual es una especie de certificaci\uf3n que puede recibir un hospital o un lugar en el que se atienden partos, al demostrar que cumple con los "Diez pasos para el \ue9xito de la lactancia materna". "Amigo del Ni\uf1o" es m\ue1s que un nombre atractivo o un lema. Con las pol\uedticas de este programa aumentan las tasas de inicio de lactancia materna y disminuyen las enfermedades en lactantes. En este art\uedculo se revisa el desarrollo de la Baby-Friendly Hospital Initiative, se describen los componentes de la iniciativa y se valoran los datos actuales que apoyan su implementaci\uf3n a nivel universal

Maternal birthplace and breastfeeding initiation among term and preterm infants: A statewide assessment for Massachusetts

OBJECTIVES. Among premature infants, formula feeding increases the risk for necrotizing enterocolitis, delayed brainstem maturation, decreased scoring on cognitive and developmental tests, and delayed visual development. With this in mind, many interventions are designed to increase breast milk consumption in preterm infants. Breastfeeding initiation rates among US premature infants are not collected nationally, however, and published data on breastfeeding rates in this population are limited. In addition, national surveys calculate breastfeeding rates among term infants according to maternal race/ethnicity, but maternal birthplace is not recorded. This is likely to be important, because breastfeeding is the cultural norm in the countries of origin for many non-US-born US residents. Massachusetts has a diverse racial/ethnic population, including many non-US-born women. The goals of this study were to compare breastfeeding initiation rates among preterm and term infants in Massachusetts in 2002 and to determine the effect of maternal race/ethnicity and birthplace on breastfeeding initiation rates among term and preterm infants. METHODS. Massachusetts Community Health Information Profile, an online public health database that was created by the Massachusetts Department of Public Health, includes breastfeeding initiation data that are obtained from the electronic birth certificate, which we used to compare breastfeeding rates among preterm and term infants. Birth-linked demographics and data that also were accessed were maternal age, race/ethnicity, birthplace, and health insurance (public or private) as an indicator of socioeconomic status and infant's gestational age. We assessed the association between breastfeeding initiation and maternal birthplace, as well as race/ethnicity and the other potential confounders, using logistic regression. RESULTS. There were 80624 births in Massachusetts in 2002, and 8.2% (6611) of newborns had a gestational age <37 weeks. The state's overall breastfeeding initiation rate was 74.6%. We excluded records of mothers who were younger than 15 years and older than 39 years, nonsingleton births, infants with a gestational age <24 weeks and >42 weeks, and records with missing data. Of the total births in Massachusetts, 67884 (84%) met inclusion criteria for this study. Breastfeeding initiation rates were lowest among preterm infants of the youngest gestational ages. Breastfeeding initiation was 76.8% among term infants born at 37 to 42 weeks, 70.1% among infants born at 32 to 36 weeks, and 62.9% among infants born at 24 to 31 weeks. In univariate analysis, among preterm infants, a lower proportion of US-born black, Asian, and Hispanic mothers initiated breastfeeding than US-born white mothers; non-US-born black and non-US-born Hispanic mothers had the highest breastfeeding initiation rates. Among term infants, US-born black mothers had the lowest initiation rates, and non-US-born black and non-US-born Hispanic mothers had the highest. In multivariate logistic regression, however, after controlling for mother's age, race, birthplace, and insurance, US-born white mothers were least likely to breastfeed either term or preterm infants when compared with any other racial/ethnic group, including US-born black mothers. The likelihood that non-US-born Hispanic mothers would breastfeed was almost 8 times greater than that for US-born white mothers for a preterm infant and almost 10 times greater for a term infant. In multivariate logistic regression analysis stratified by gestational age for both preterm and term infants, older mothers and mothers with private health insurance were most likely to breastfeed. CONCLUSIONS. In Massachusetts, preterm infants were less likely to receive breast milk than term infants, and the likelihood of receiving breast milk was lowest among the youngest preterm infants. In multivariate logistic regression, mothers who were born outside the United States were more likely than US-born mothers to breastfeed either term or preterm infants in all racial and ethnic groups. In an unexpected finding, US-born white mothers were less likely to breastfeed term or preterm infants than US-born black mothers or mothers of any other racial or ethnic group

When breastfeeding is beset by stormy seas

An exclusively breastfed newborn may normally lose less than 7% of birth weight during the first few days of life. A weight loss of over 7% requires careful assessment of mother and child. The following mnemonic (SAILBOAT) is a handy tool that offers a differential diagnosis for recalling the possibilities that may cause above-average weight loss

Detection of un-methylated DNA within different abundances of methylated DNA background by conventional or <i>fast</i>-COLD-MS-PCR.

<p><b>Panel A.</b> Post-PCR melting profile of the 255 bp bisulfite-converted-specific amplicon after conventional PCR or COLD-PCR. Serial dilutions of un-methylated (U) to methylated (M) genomic DNA are depicted (top half). Higher abundances of un-methylated DNA can be discriminated from methylated DNA by the melt peak, whereas lower abundances are only detectable if <i>fast</i>-COLD-MS-PCR replaces conventional PCR. <b>Panel B.</b> Sanger sequencing results of the 0.05% un-methylated (U): methylated (M) DNA sample as amplified by conventional and <i>fast</i>-COLD-MS-PCR are shown (bottom half). Chromatograms are aligned and compared using SeqDoc, and the CpG methylation positions are revealed in the middle panel.</p

Melting profiles of bisulfite-converted DNA from clinical samples following conventional PCR.

<p>Post-PCR melting profiles of the 255 bp bisulfite-converted <i>MGMT</i> gene amplicon after conventional PCR. Examples of fully un-methylated DNA samples isolated from infant blood (<b>Panel A</b>) and glioma samples (<b>Panel B</b>) are depicted. 100% methylated (M) and 100% un-methylated (U) DNA controls are used as reference standards, demonstrating a ∼5°C melting temperature difference among the two. <b>Panel C.</b> A glioma sample with mixed methylation/unmethylation pattern is shown.</p

Melting profiles of bisulfite-converted DNA from clinical samples following <i>fast</i>-COLD-MS-PCR.

<p>The effect of lowering the denaturation temperature in PCR is depicted. <b>Panel A.</b> Glioma sample no. 3 was subjected to different critical denaturation temperature-T_c during <i>fast</i>-COLD-MS-PCR. The modulation of the preferential amplification of the un-methylated DNA fraction is shown. <b>Panel B. </b><i>fast</i>-COLD-MS-PCR performed at a T_c of 84°C demonstrates that the amplification of the methylated DNA fraction is completely inhibited.</p