Peter Konings

Chromosome instability is common in human cleavage-stage embryos

Chromosome instability is a hallmark of tumorigenesis. This study establishes that chromosome instability is also common during early human embryogenesis. A new array-based method allowed screening of genome-wide copy number and loss of heterozygosity in single cells. This revealed not only mosaicism for whole-chromosome aneuploidies and uniparental disomies in most cleavage-stage embryos but also frequent segmental deletions, duplications and amplifications that were reciprocal in sister blastomeres, implying the occurrence of breakage-fusion-bridge cycles. This explains the low human fecundity and identifies post-zygotic chromosome instability as a leading cause of constitutional chromosomal disorders.

eXtasy: variant prioritization by genomic data fusion

Massively parallel sequencing greatly facilitates the discovery of novel disease genes causing Mendelian and oligogenic disorders. However, many mutations are present in any individual genome, and identifying which ones are disease causing remains a largely open problem. We introduce eXtasy, an approach to prioritize nonsynonymous single-nucleotide variants (nSNVs) that substantially improves prediction of disease-causing variants in exome sequencing data by integrating variant impact prediction, haploinsufficiency prediction and phenotype-specific gene prioritization.

Improved detection of chromosomal abnormalities in chronic lymphocytic leukemia by conventional cytogenetics using CpG oligonucleotide and interleukin-2 stimulation: A Belgian multicentric study.

We performed a multicentric study to assess the impact of two different culture procedures on the detection of chromosomal abnormalities in 217 consecutive unselected cases with chronic lymphocytic leukemia (CLL) referred for routine analysis either at the time of diagnosis (n = 172) or during disease evolution (n = 45). Parallel cultures of peripheral blood or bone marrow were set up with the addition of either the conventional B‐cell mitogen 12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA) or a combination of CpG oligonucleotide (CpG) and interleukin‐2 (IL‐2). Cytogenetic analyses were performed on both cultures. Clonal abnormalities were identified in 116 cases (53%). In 78 cases (36%), the aberrant clone was detected in both cultures. Among these, the percentages of aberrant metaphases were similar in both conditions in 17 cases, higher in the CpG/IL‐2 culture in 43 cases, and higher in the TPA culture in 18 cases. Clonal aberrations were detected in only one culture, either in CpG/IL‐2 or TPA in 33 (15%) and 5 (2%) cases, respectively. Taken together, abnormal karyotypes were observed in 51% with CpG/IL‐2 and 38% with TPA (P < 0.0001). Application of FISH (n = 201) allowed the detection of abnormalities not visible by conventional cytogenetic analysis in 80 cases: del(13q) (n = 71), del(11q) (n = 5), +12 (n = 2), del(14q) (n = 1), and del(17p) (n = 1). In conclusion, our results confirm that CpG/IL‐2 stimulation increases the detection rate of chromosomal abnormalities in CLL compared with TPA and that further improvement can be obtained by FISH. However, neither conventional cytogenetics nor FISH detected all aberrations, demonstrating the complementary nature of these techniques.

Decomposing socioeconomic health inequalities

This Hints & Kinks paper describes a technique enabling quantification of the contributions of determinants to socioeconomic inequality in health. This technique, differing from an analysis investigating the determinants of average health levels, has received considerable attention from health economists (van Doorslaer and Gerdtham 2003; van Doorslaer and Jones 2003; van Doorslaer et al. 2004; Wagstaff et al. 2003) but only more recently from epidemiologists as well (Harper and Lynch 2007; Lynch 2006; Hosseinpoor et al. 2006). This paper employs the relative concentration index (RCI), described in Konings et al. (2009), to summarize relative inequality across the entire socioeconomic distribution. The RCI of a continuous health outcome y results from a relative concentration curve, which graphs on the x-axis the cumulative percentage of the sample, ranked by an indicator of socioeconomic position such as education or income beginning with the poorest. The y-axis then indicates the cumulative percentage of the health outcome corresponding to each cumulative percentage of the distribution of the socioeconomic indicator. Figure 1 provides an example of a concentration curve, where the health variable is childhood malnutrition in Ghana in 2003. It shows that the level of malnutrition accumulates faster among the poor than among the better-off because the line is above the diagonal. The RCI is defined as twice the area between the concentration curve and the line of equality (the 45 diagonal from the bottom-left corner to the top-right). Details on how to compute the RCI can be found in Konings et al. (2009). If yi is linearly modeled, linking a health variable, y to a set of k health determinants, xk, this can be expressed by: yi 1⁄4 a þ X

Microarray analysis of copy number variation in single cells

We present a protocol for reliably detecting DNA copy number aberrations in a single human cell. Multiple displacement-amplified DNAs of a cell are hybridized to a 3,000–bacterial artificial chromosome (BAC) array and to an Affymetrix 250,000 (250K)-SNP array. Subsequent copy number calling is based on the integration of BAC probe-specific copy number probabilities that are estimated by comparing probe intensities with a single-cell whole-genome amplification (WGA) reference model for diploid chromosomes, as well as SNP copy number and loss-of-heterozygosity states estimated by hidden Markov models (HMM). All methods for detecting DNA copy number aberrations in single human cells have difficulty in confidently discriminating WGA artifacts from true genetic variants. Furthermore, some methods lack thorough validation for segmental DNA imbalance detection. Our protocol minimizes false-positive variant calling and enables uniparental isodisomy detection in single cells. Additionally, it provides quality assessment, allowing the exclusion of uninterpretable single-cell WGA samples. The protocol takes 5–7 d.

Single-cell copy number variation detection

Detection of chromosomal aberrations from a single cell by array comparative genomic hybridization (single-cell array CGH), instead of from a population of cells, is an emerging technique. However, such detection is challenging because of the genome artifacts and the DNA amplification process inherent to the single cell approach. Current normalization algorithms result in inaccurate aberration detection for single-cell data. We propose a normalization method based on channel, genome composition and recurrent genome artifact corrections. We demonstrate that the proposed channel clone normalization significantly improves the copy number variation detection in both simulated and real single-cell array CGH data.

Interphase fluorescence in situ hybridization on selected plasma cells is superior in the detection of cytogenetic aberrations in plasma cell dyscrasia.

Interphase fluorescence in situ hybridization (FISH) detects nonrandom cytogenetic abnormalities in plasma cell (PC) dyscrasia according to PC burden. However, when performed on cultured whole bone marrow (BM), it often fails to detect these aberrations. We have compared this interphase FISH technique with FISH after PC purification or identification to detect recurrent aberrations. In this study, 235 BM samples were collected from patients with multiple myeloma (MM) or related PC disorders regardless of disease status. All samples were analyzed in parallel. Clonal abnormalities were detected in 34.9% of cultured samples compared with 71.0% PC selected samples (P < 0.001). Moreover, FISH on PCs allowed to detect more abnormalities per case (P < 0.001) and identified higher percentages of abnormal nuclei (P < 0.001). This study indicates that FISH on PCs is the preferred technique for routine cytogenetic investigation of MM.

Genome-wide copy number variation scan identifies complement component C4 as novel susceptibility gene for Crohn's disease

Background:The genetic component of Crohns disease (CD) is well known, with 140 susceptibility loci identified so far. In addition to single nucleotide polymorphisms typically studied in genome-wide scans, copy number variation is responsible for a large proportion of human genetic variation. Methods:We performed a genome-wide search for copy number variants associated with CD using array comparative genomic hybridization. One of the found regions was validated independently through real-time PCR. Serum levels of the found gene were measured in patients and control subjects. Results:We found copy number differences for the C4S and C4L gene variants of complement component C4 in the central major histocompatibility complex region on chromosome 6p21. Specifically, we saw that CD patients tend to have lower C4L and higher C4S copies than control subjects (P = 5.00 × 10−03 and P = 9.11 × 10−04), which was independent of known associated classical HLA I and II alleles (P = 7.68 × 10−03 and P = 6.29 × 10−03). Although C4 serum levels were not different between patients and control subjects, the relationship between C4 copy number and serum level was different for patients and control subjects with higher copy numbers leading to higher serum concentrations in control subjects, compared with CD patients (P < 0.001). Conclusions:C4 is part of the classical activation pathway of the complement system, which is important for (auto)immunity. Low C4L or high C4S copy number, and corresponding effects on C4 serum level, could lead to an exaggerated response against infections, possibly leading to (auto)immune disease.