Signatures of post-zygotic structural genetic aberrations in the cells of histologically normal breast tissue that can predispose to sporadic breast cancer.

Sporadic breast cancer (SBC) is a common disease without robust means of early risk prediction in the population. We studied 282 females with SBC, focusing on copy number aberrations in cancer-free breast tissue (uninvolved margin, UM) outside the primary tumor (PT). In total, 1162 UMs (1-14 per breast) were studied. Comparative analysis between UM(s), PT(s), and blood/skin from the same patient as a control is the core of the study design. We identified 108 patients with at least one aberrant UM, representing 38.3% of cases. Gains in gene copy number were the principal type of mutations in microscopically normal breast cells, suggesting that oncogenic activation of genes via increased gene copy number is a predominant mechanism for initiation of SBC pathogenesis. The gain of ERBB2, with overexpression of HER2 protein, was the most common aberration in normal cells. Five additional growth factor receptor genes (EGFR, FGFR1, IGF1R, LIFR, and NGFR) also showed recurrent gains, and these were occasionally present in combination with the gain of ERBB2. All the aberrations found in the normal breast cells were previously described in cancer literature, suggesting their causative, driving role in pathogenesis of SBC. We demonstrate that analysis of normal cells from cancer patients leads to identification of signatures that may increase risk of SBC and our results could influence the choice of surgical intervention to remove all predisposing cells. Early detection of copy number gains suggesting a predisposition toward cancer development, long before detectable tumors are formed, is a key to the anticipated shift into a preventive paradigm of personalized medicine for breast cancer.

Single molecule analysis of combinatorial splicing.

Alternative splicing creates diverse mRNA isoforms from single genes and thereby enhances complexity of transcript structure and of gene function. We describe a method called spliceotyping, which translates combinatorial mRNA splicing patterns along transcripts into a library of binary strings of nucleic acid tags that encode the exon composition of individual mRNA molecules. The exon inclusion pattern of each analyzed transcript is thus represented as binary data, and the abundance of different splice variants is registered by counts of individual molecules. The technique is illustrated in a model experiment by analyzing the splicing patterns of the adenovirus early 1A gene and the beta actin reference transcript. The method permits many genes to be analyzed in parallel and it will be valuable for elucidating the complex effects of combinatorial splicing.

Somatic mosaicism for chromosome X and Y aneuploidies in monozygotic twins heterozygous for sickle cell disease mutation.

Somatic genetic variation in health and disease is poorly explored. Monozygotic (MZ) twins are a suitable model for studies of somatic mosaicism since genetic differences in twins derived from the same zygote represent an irrefutable example of somatic variation. We report the analysis of a pair of generally healthy female MZ twins, discordant for somatic mosaicism for aneuploidy of chromosomes X and Y. Both twins are heterozygous carriers of sickle cell disease mutation. Genotyping of blood DNA from both twins using Illumina Human 610 SNP array revealed a copy number imbalance for chromosome X in a proportion of cells in one twin. Fluorescent in situ hybridization (FISH) analysis confirmed monosomy X (45,X) in 7% of proband nucleated blood cells. Unexpectedly, FISH analysis of cells from the other twin revealed 45,X and 46,XY lineages, both present in 1% of cells. The mechanism behind formation of these aneuploidies suggests several aberrant chromosome segregation events in meiosis and mitoses following conception. Our report contributes to the delineation of the frequency of somatic structural genomic variation in normal MZ twins. These results also illustrate the plasticity of the human genome for tolerating large copy number changes in healthy subjects and show the sensitivity of the Illumina platform for detection of aberrations that are present in a minority of the studied cells.

An Immunological Perspective on Neonatal Sepsis.

Despite concerted international efforts, mortality from neonatal infections remains unacceptably high in some areas of the world, particularly for premature infants. Recent developments in flow cytometry and next-generation sequencing technologies have led to major discoveries over the past few years, providing a more integrated understanding of the developing human immune system in the context of its microbial environment. We review these recent findings, focusing on how in human newborns incomplete maturation of the immune system before a full term of gestation impacts on their vulnerability to infection. We also discuss some of the clinical implications of this research in guiding the design of more-accurate age-adapted diagnostic and preventive strategies for neonatal sepsis.

Post-zygotic and inter-individual structural genetic variation in a presumptive enhancer element of the locus between the IL10Rß and IFNAR1 genes.

Although historically considered as junk-DNA, tandemly repeated sequence motifs can affect human phenotype. For example, variable number tandem repeats (VNTR) with embedded enhancers have been shown to regulate gene transcription. The post-zygotic variation is the presence of genetically distinct populations of cells in an individual derived from a single zygote, and this is an understudied aspect of genome biology. We report somatically variable VNTR with sequence properties of an enhancer, located upstream of IFNAR1. Initially, SNP genotyping of 63 monozygotic twin pairs and multiple tissues from 21 breast cancer patients suggested a frequent post-zygotic mosaicism. The VNTR displayed a repeated 32 bp core motif in the center of the repeat, which was flanked by similar variable motifs. A total of 14 alleles were characterized based on combinations of segments, which showed post-zygotic and inter-individual variation, with up to 6 alleles in a single subject. Somatic variation occurred in ∼24% of cases. In this hypervariable region, we found a clustering of transcription factor binding sites with strongest sequence similarity to mouse Foxg1 transcription factor binding motif. This study describes a VNTR with sequence properties of an enhancer that displays post-zygotic and inter-individual genetic variation. This element is within a locus containing four related cytokine receptors: IFNAR2, IL10Rß, IFNAR1 and IFNGR2, and we hypothesize that it might function in transcriptional regulation of several genes in this cluster. Our findings add another level of complexity to the variation among VNTR-based enhancers. Further work may unveil the normal function of this VNTR in transcriptional control and its possible involvement in diseases connected with these receptors, such as autoimmune conditions and cancer.

Posttranscriptional down-regulation of small ribosomal subunit proteins correlates with reduction of 18S rRNA in RPS19 deficiency.

Ribosomal protein S19 (RPS19) is mutated in patients with Diamond-Blackfan anemia (DBA). We hypothesized that decreased levels of RPS19 lead to a coordinated down-regulation of other ribosomal (r-)proteins at the subunit level. We show that small interfering RNA (siRNA) knock-down of RPS19 results in a relative decrease of small subunit (SSU) r-proteins (S20, S21 and S24) when compared to large subunit (LSU) r-proteins (L3, L9, L30 and L38). This correlates with a relative decrease in 18S rRNA with respect to 28S rRNA. The r-protein mRNA levels remain relatively unchanged indicating a post transcriptional regulation of r-proteins at the level of subunit formation.