High-resolution profiling of an 11 Mb segment of human chromosome 22 in sporadic schwannoma using array-CGH.

Previous low-resolution schwannoma studies have reported diverse frequencies (30-80%) of 22q deletions, involving the neurofibromatosis-2 tumor suppressor (NF2) gene. We constructed an array spanning 11 million base pairs of 22q encompassing the NF2 gene, with 100% coverage and an average resolution of 58 kb. Moreover, the 220 kb genomic sequence encompassing the NF2 gene was covered by 13 cosmids to further enhance the resolution of analysis. The rationale of this array-CGH study was to map and size 22q deletions around the NF2 gene in sporadic schwannoma using a reliable method with maximal resolution. We studied tumor and constitutional DNA from 47 patients and detected heterozygous deletions in 21 (45%) tumors, which could be classified into three profiles. The predominant profile (12/21) was a continuous deletion of the 11 Mb segment, consistent with monosomy 22. The second profile, comprising five schwannomas, was also in agreement with a continuous 11 Mb heterozygous deletion. However, these displayed a distinctly different level of deletion when compared to the first profile, suggesting a considerable amount of normal tissue in the tumor samples. This is the first report demonstrating the sensitivity of array-CGH to discriminate such samples. The third profile was composed of four cases displaying interstitial deletions of various sizes. Two of these did not encompass the NF2 locus, which further emphasize the importance of other loci in schwannoma development. This is the first high-resolution study performed on a large series of tumors, using an array continuously covering 1/3 of a human chromosome. Our findings warrant further studies of an extended tumor series on a full 22q genomic array, to better define additional, putative 22q-located loci important for schwannoma development. Our array also provides a new diagnostic tool for analysis of NF2 gene deletions in patients affected with neurofibromatosis-2.

[Evaluation of the impact of multiple micronutrient powders on children anemia in three Andean regiones in Peru]. / Evaluación del impacto de los multimicronutrientes en polvo sobre la anemia infantil en tres regiones andinas del Perú

The aim of this research was to determine the impact of the strategy of multi-micronutrient supplementation (MMN) on the childhood anemia in three Andean regions of Peru. A sentinel surveillance system was established in 29 health centers of Andahuaylas, Ayacucho and Huancavelica (Peru) to monitor a cohort of children of 6 to 35 months of age whom been received MMM for a period of 12 months. Data regarding hemoglobin levels were gathered at the beginning and at the end of the research; they included consumption of MMN, and other data from clinical records and from growth and development charts. Between the child who completed the supplementation, the prevalence of anemia decreased from 70.2% to 36.6% (p value <0.01). 55,0% and 69,1% of children with mild and moderate anemia at the beginning of the supplementation got cured. This research shows that supplementation with MMN could be a valuable strategy to fight anemia.

Tissue-specific variation in DNA methylation levels along human chromosome 1.

BACKGROUND: DNA methylation is a major epigenetic modification important for regulating gene expression and suppressing spurious transcription. Most methods to scan the genome in different tissues for differentially methylated sites have focused on the methylation of CpGs in CpG islands, which are concentrations of CpGs often associated with gene promoters. RESULTS: Here, we use a methylation profiling strategy that is predominantly responsive to methylation differences outside of CpG islands. The method compares the yield from two samples of size-selected fragments generated by a methylation-sensitive restriction enzyme. We then profile nine different normal tissues from two human donors relative to spleen using a custom array of genomic clones covering the euchromatic portion of human chromosome 1 and representing 8% of the human genome. We observe gross regional differences in methylation states across chromosome 1 between tissues from the same individual, with the most striking differences detected in the comparison of cerebellum and spleen. Profiles of the same tissue from different donors are strikingly similar, as are the profiles of different lobes of the brain. Comparing our results with published gene expression levels, we find that clones exhibiting extreme ratios reflecting low relative methylation are statistically enriched for genes with high expression ratios, and vice versa, in most pairs of tissues examined. CONCLUSION: The varied patterns of methylation differences detected between tissues by our methylation profiling method reinforce the potential functional significance of regional differences in methylation levels outside of CpG islands.

Evaluación del impacto de los multimicronutrientes en polvo sobre la anemia infantil en tres regiones andinas del Perú / Evaluation of the impact of multiple micronutrient powders on children anemia in three andean regiones in Peru

Con el objetivo de determinar el impacto de la administración con multimicronutrientes (MMN) en polvo sobre la anemia infantil en tres regiones andinas del Perú, se estableció un sistema de vigilancia centinela en 29 establecimientos de Andahuaylas, Ayacucho y Huancavelica, en niños de 6 a 35 meses de edad, a quienes se les indicó MMN por un periodo de 12 meses, entre el 2009 y 2011. Además de los datos sociodemográficos de los menores y las madres, se determinó los niveles de hemoglobina al inicio y al final del estudio. Entre los menores que culminaron la suplementación, la prevalencia de anemia se redujo de 70,2 a 36,6% (p<0,01), y se evidenció que el 55,0% y el 69,1% de niños con anemia leve y moderada al inicio del estudio, la habían superado al término del mismo. Se concluye que la suplementación con MMN en polvo puede ser una estrategia efectiva en la lucha contra la anemia.

The aim of this research was to determine the impact of the strategy of multi-micronutrient supplementation (MMN) on the childhood anemia in three Andean regions of Peru. A sentinel surveillance system was established in 29 health centers of Andahuaylas, Ayacucho and Huancavelica (Peru) to monitor a cohort of children of 6 to 35 months of age whom been received MMM for a period of 12 months. Data regarding hemoglobin levels were gathered at the beginning and at the end of the research; they included consumption of MMN, and other data from clinical records and from growth and development charts. Between the child who completed the supplementation, the prevalence of anemia decreased from 70.2% to 36.6% (p value <0.01). 55,0% and 69,1% of children with mild and moderate anemia at the beginning of the supplementation got cured. This research shows that supplementation with MMN could be a valuable strategy to fight anemia.

Analysis of copy number variation in the normal human population within a region containing complex segmental duplications on 22q11 using high-resolution array-CGH.

A previously detected copy number polymorphism (Ep CNP) in patients affected with neuroectodermal tumors led us to investigate its frequency and length in the normal population. For this purpose, a program called Sequence Allocator was developed and applied for the construction of an array that consisted of unique and duplicated fragments, allowing the assessment of copy number variation within regions of segmental duplications. The average resolution of this array was 11 kb and we determined the size of the Ep CNP to be 290 kb. Analysis of normal controls identified 7.7 and 7.1% gains in peripheral blood and lymphoblastoid cell line (LCL) DNA, respectively, while deletions were found only in the LCL group (7.1%). This array platform allows the detection of DNA copy number variation within regions of pronounced genomic complexity, which constitutes an improvement over available technologies.

Chromosome 22 tiling-path array-CGH analysis identifies germ-line- and tumor-specific aberrations in patients with glioblastoma multiforme.

Gliomas are common and frequently malignant tumors of the central nervous system. Recurrent allelic losses of chromosome 22 have been reported in gliomas, indicating tumor-suppressor genes at this location. However, the target genes are still unknown. We applied a high resolution tiling-path chromosome 22 array to a series of 50 glioblastoma samples, with the aim of investigating the underlying abnormalities in both constitutional and tumor-derived DNA. We detected hemizygous deletions in 28% of the tumors (14 of 50), with monosomy 22 (10 of 50) being the predominant pattern. The distribution of overlapping hemizygous deletions delineated two putative tumor-suppressor loci (11.1 and 3.08 Mb in size) across 22q. Most strikingly, we identified two distinct loci affected by regional gains. Both alterations were of germ-line origin and were unique to samples from patients affected with tumors. Analysis of these two amplified regions revealed the presence of two interesting candidate genes: TOP3B and TAFA5. The TOP3B gene encodes a protein that seems to function in the unlinking of parental strands at the final stage of DNA replication and/or in the dissociation of structures in mitotic cells that could lead to recombination. The TAFA5 gene belongs to a novel family of proteins with similarity to chemokines and brain-specific expression. The role of the identified candidate loci should be studied further. Our results demonstrated the power of array-CGH to determine DNA copy number alterations in the context of germ-line- and tumor-specific aberrations.

Localization of a putative low-penetrance ependymoma susceptibility locus to 22q11 using a chromosome 22 tiling-path genomic microarray.

Ependymomas frequently display allelic loss of chromosome 22 in the absence of mutations in the known tumor-suppressor genes on chromosome 22, suggesting the role of an alternative predisposing gene or genes from this chromosome. In an effort to localize these genes, 37 ependymomas derived from 33 patients were analyzed for the presence of copy number changes by use of a high-resolution chromosome 22 genomic microarray. Eighteen ependymomas (49%) displayed an array-CGH profile consistent with monosomy of chromosome 22. However, in 10 of these tumors, the fluorescence ratios for 22q clones scored as deleted were different from those at the single gene copy level. This suggests either analysis of mixed populations of tumor and normal stromal cells or analysis of mixed tumor cell populations with different genetic profiles. Four ependymomas derived from two patients showed overlapping interstitial deletions of 2.2 Mb and approximately 510 kb. Further analyses revealed that these deletions were present in the constitutional DNA of these two patients as well as in some of their unaffected relatives. Detailed microsatellite analysis of these families refined the commonly deleted segment to a region of 320 kb between markers RH13801 and D22S419. Our results provide additional evidence for the involvement of genes on chromosome 22 in the development of ependymoma and suggest the presence of a low-penetrance ependymoma susceptibility locus at 22q11.

High-resolution array-CGH profiling of germline and tumor-specific copy number alterations on chromosome 22 in patients affected with schwannomas.

Schwannomas may develop sporadically or in association with NF2 and schwannomatosis. The fundamental aberration in schwannomas is the bi-allelic inactivation of the NF2 gene. However, clinical and molecular data suggest that these tumors share a common pathogenetic mechanism related to as yet undefined 22q-loci. Linkage studies in schwannomatosis, a condition related to NF2, have defined a candidate 22q-locus and excluded the NF2 gene as the causative germline mutation. Thus, analysis of aberrations in schwannomas may lead to the identification of putative gene(s) involved in the development of schwannoma/schwannomatosis. We profiled a series of 88 schwannomas and constitutional DNA using a tiling path chromosome 22 array. Array-CGH is a suitable method for high-resolution discrimination between germline and tumor-specific aberrations. Previously reported frequencies of 22q-associated deletions in schwannomas display large discrepancies, ranging from 30% to 80%. We detected heterozygous deletions in 53% of schwannomas and the predominant pattern was monosomy 22. In addition, three tumors displayed terminal deletions and four harbored overlapping interstitial deletions of various sizes encompassing the NF2 gene. When profiling constitutional DNA, we identified eight loci that were affected by copy number variation (CNV). Some of the identified CNVs may not be phenotypically neutral and the possible role of these CNVs in the pathogenesis of schwannomas should be studied further. We observed a correlation between the breakpoint position, present in tumor and/or constitutional DNA and the location of segmental duplications. This association implicates these unstable regions in rearrangements occurring both in meiosis and mitosis.

Chromatin and siRNA pathways cooperate to maintain DNA methylation of small transposable elements in Arabidopsis.

BACKGROUND: DNA methylation occurs at preferred sites in eukaryotes. In Arabidopsis, DNA cytosine methylation is maintained by three subfamilies of methyltransferases with distinct substrate specificities and different modes of action. Targeting of cytosine methylation at selected loci has been found to sometimes involve histone H3 methylation and small interfering (si)RNAs. However, the relationship between different cytosine methylation pathways and their preferred targets is not known. RESULTS: We used a microarray-based profiling method to explore the involvement of Arabidopsis CMT3 and DRM DNA methyltransferases, a histone H3 lysine-9 methyltransferase (KYP) and an Argonaute-related siRNA silencing component (AGO4) in methylating target loci. We found that KYP targets are also CMT3 targets, suggesting that histone methylation maintains CNG methylation genome-wide. CMT3 and KYP targets show similar proximal distributions that correspond to the overall distribution of transposable elements of all types, whereas DRM targets are distributed more distally along the chromosome. We find an inverse relationship between element size and loss of methylation in ago4 and drm mutants. CONCLUSION: We conclude that the targets of both DNA methylation and histone H3K9 methylation pathways are transposable elements genome-wide, irrespective of element type and position. Our findings also suggest that RNA-directed DNA methylation is required to silence isolated elements that may be too small to be maintained in a silent state by a chromatin-based mechanism alone. Thus, parallel pathways would be needed to maintain silencing of transposable elements.

A full-coverage, high-resolution human chromosome 22 genomic microarray for clinical and research applications.

We have constructed the first comprehensive microarray representing a human chromosome for analysis of DNA copy number variation. This chromosome 22 array covers 34.7 Mb, representing 1.1% of the genome, with an average resolution of 75 kb. To demonstrate the utility of the array, we have applied it to profile acral melanoma, dermatofibrosarcoma, DiGeorge syndrome and neurofibromatosis 2. We accurately diagnosed homozygous/heterozygous deletions, amplifications/gains, IGLV/IGLC locus instability, and breakpoints of an imbalanced translocation. We further identified the 14-3-3 eta isoform as a candidate tumor suppressor in glioblastoma. Two significant methodological advances in array construction were also developed and validated. These include a strictly sequence defined, repeat-free, and non-redundant strategy for array preparation. This approach allows an increase in array resolution and analysis of any locus; disregarding common repeats, genomic clone availability and sequence redundancy. In addition, we report that the application of phi29 DNA polymerase is advantageous in microarray preparation. A broad spectrum of issues in medical research and diagnostics can be approached using the array. This well annotated and gene-rich autosome contains numerous uncharacterized disease genes. It is therefore crucial to associate these genes to specific 22q-related conditions and this array will be instrumental towards this goal. Furthermore, comprehensive epigenetic profiling of 22q-located genes and high-resolution analysis of replication timing across the entire chromosome can be studied using our array.