Cindy Melotte

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.

Fusion of NUP214 to ABL1 on amplified episomes in T-cell acute lymphoblastic leukemia.

In T-cell acute lymphoblastic leukemia (T-ALL), transcription factors are known to be deregulated by chromosomal translocations, but mutations in protein tyrosine kinases have only rarely been identified. Here we describe the extrachromosomal (episomal) amplification of ABL1 in 5 of 90 (5.6%) individuals with T-ALL, an aberration that is not detectable by conventional cytogenetics. Molecular analyses delineated the amplicon as a 500-kb region from chromosome band 9q34, containing the oncogenes ABL1 and NUP214 (refs. 5,6). We identified a previously undescribed mechanism for activation of tyrosine kinases in cancer: the formation of episomes resulting in a fusion between NUP214 and ABL1. We detected the NUP214-ABL1 transcript in five individuals with the ABL1 amplification, in 5 of 85 (5.8%) additional individuals with T-ALL and in 3 of 22 T-ALL cell lines. The constitutively phosphorylated tyrosine kinase NUP214-ABL1 is sensitive to the tyrosine kinase inhibitor imatinib. The recurrent cryptic NUP214-ABL1 rearrangement is associated with increased HOX expression and deletion of CDKN2A, consistent with a multistep pathogenesis of T-ALL. NUP214-ABL1 expression defines a new subgroup of individuals with T-ALL who could benefit from treatment with imatinib.

Molecular Karyotyping: Array CGH Quality Criteria for Constitutional Genetic Diagnosis

Array CGH (comparative genomic hybridization) enables the identification of chromosomal copy number changes. The availability of clone sets covering the human genome opens the possibility for the widespread use of array CGH for both research and diagnostic purposes. In this manuscript we report on the parameters that were critical for successful implementation of the technology, assess quality criteria, and discuss the potential benefits and pitfalls of the technology for improved pre- and postnatal constitutional genetic diagnosis. We propose to name the genome-wide array CGH “molecular karyotyping,” in analogy with conventional karyotyping that uses staining methods to visualize chromosomes.

Preimplantation genetic screening for aneuploidy of embryos after in vitro fertilization in women aged at least 35 years: a prospective randomized trial

OBJECTIVE To test the hypothesis that patients with advanced maternal age (AMA) have a higher implantation rate (IR) after embryo transfer of embryos with a normal chromosomal pattern for the chromosomes studied with preimplantation genetic screening (PGS) compared with patients who had an embryo transfer without PGS. DESIGN Prospective randomized controlled trial (RCT). SETTING Academic tertiary setting. PATIENT(S) Patients with AMA (> or =35 years). INTERVENTION(S) In an RCT, the clinical IR per embryo transferred was compared after embryo transfer on day 5 or 6 between the PGS group (analysis of chromosomes 13, 16, 18, 21, 22, X, and Y) and the Control group without PGS. MAIN OUTCOME MEASURE(S) No differences were observed between the PGS group and the Control group for the clinical IR (15.1%; 14.9%; rate ratio 1.01; exact confidence interval [CI], 0.25-5.27), the ongoing IR (at 12 weeks) (9.4%; 14.9%), and the live born rate per embryo transferred (9.4%; 14.9%; rate ratio 0.63; exact CI, 0.08-3.37). Fewer embryos were transferred in the PGS group (1.6 +/- 0.6) than in the Control group (2.0 +/- 0.6). A normal diploid status was observed in 30.3% of the embryos screened by PGS. CONCLUSION(S) In this RCT, the results did not confirm the hypothesis that PGS results in improved reproductive outcome in patients with AMA.

The C20orf133 gene is disrupted in a patient with Kabuki syndrome.

Background: Kabuki syndrome (KS) is a rare, clinically recognisable, congenital mental retardation syndrome. The aetiology of KS remains unknown. Methods: Four carefully selected patients with KS were screened for chromosomal imbalances using array comparative genomic hybridisation at 1 Mb resolution. Results: In one patient, a 250 kb de novo microdeletion at 20p12.1 was detected, deleting exon 5 of C20orf133. The function of this gene is unknown. In situ hybridisation with the mouse orthologue of C20orf133 showed expression mainly in brain, but also in kidney, eye, inner ear, ganglia of the peripheral nervous system and lung. Conclusion: The de novo nature of the deletion, the expression data and the fact that C20orf133 carries a macro domain, suggesting a role for the gene in chromatin biology, make the gene a likely candidate to cause the phenotype in this patient with KS. Both the finding of different of chromosomal rearrangements in patients with KS features and the absence of C20orf133 mutations in 19 additional patients with KS suggest that KS is genetically heterogeneous.

Breakage–fusion–bridge cycles leading to inv dup del occur in human cleavage stage embryos

Recently, a high incidence of chromosome instability (CIN) was reported in human cleavage stage embryos. Based on the copy number changes that were observed in the blastomeres it was hypothesized that chromosome breakages and fusions occur frequently in cleavage stage human embryos and instigate subsequent breakage‐fusion‐bridge cycles. In addition, it was hypothesized that the DNA breaks present in spermatozoa could trigger this CIN. To test these hypotheses, we genotyped both parents as well as 93 blastomeres from 24 IVF embryos and developed a novel single nucleotide polymorphism (SNP) array‐based algorithm to determine the parental origin of (aberrant) loci in single cells. Paternal as well as maternal alleles were commonly rearranged in the blastomeres indicating that sperm‐specific DNA breaks do not explain the majority of these structural variants. The parent‐of‐origin analyses together with microarray‐guided FISH analyses demonstrate the presence of inv dup del chromosomes as well as more complex rearrangements. These data provide unequivocal evidence for breakage–fusion–bridge cycles in those embryos and suggest that the human cleavage stage embryo is a major source of chromosomal disorders. Hum Mutat 32:783–793, 2011.

Array painting using microdissected chromosomes to map chromosomal breakpoints

Molecular characterization of breakpoints of chromosomal rearrangements is a successful strategy for the identification of candidate disease genes. Mapping translocation breakpoints and rearranged chromosomal boundaries is labor intensive and/or time consuming. Here, we present a novel and rapid procedure to map such chromosomal breakpoints by hybridizing amplified microdissection derived DNA of aberrant chromosomes to arrays containing genomic clones. We illustrate the potential of the technique by molecularly delineating the breakpoints in five small supernumerary marker chromosomes (sSMC) and mapping the breakpoints of five different chromosomal translocations.

PGD for a complex chromosomal rearrangement by array comparative genomic hybridization

Patients carrying a chromosomal rearrangement (CR) have an increased risk for chromosomally unbalanced conceptions. Preimplantation genetic diagnosis (PGD) may avoid the transfer of embryos carrying unbalanced rearrangements, therefore increasing the chance of pregnancy. Only 7-12 loci can be screened by fluorescence in situ hybridization whereas microarray technology can detect genome-wide imbalances at the single cell level. We performed PGD for a CR carrier with karyotype 46,XY,ins(3;2)(p23;q23q14.2),t(6;14)(p12.2;q13) using array comparative genomic hybridization. Selection of embryos for transfer was only based on copy number status of the chromosomes involved in both rearrangements. In two ICSI-PGD cycles, nine and seven embryos were analysed by array, leaving three and one embryo(s) suitable for transfer, respectively. The sensitivity and specificity of single cell arrays was 100 and 88.8%, respectively. In both cycles a single embryo was transferred, resulting in pregnancy following the second cycle. The embryo giving rise to the pregnancy was normal/balanced for the insertion and translocation but carried a trisomy 8 and nullisomy 9 in one of the two biopsied blastomeres. After 7 weeks of pregnancy the couple miscarried. Genetic analysis following hystero-embryoscopy showed a diploid (90%)/tetraploid (10%) mosaic chorion, while the gestational sac was empty. No chromosome 8 aneuploidy was detected in the chorion, while 8% of the cells carried a monosomy for chromosome 9. In summary, we demonstrate the feasibility and determine the accuracy of single cell array technology to test against transmission of the unbalanced meiotic products that can derive from CRs. Our findings also demonstrate that the genomic constitution of extra-embryonic tissue cannot necessarily be predicted from the copy number status of a single blastomere.

Autosomal-dominant microtia linked to five tandem copies of a copy-number-variable region at chromosome 4p16.

Recently, large-scale benign copy-number variations (CNVs)--encompassing over 12% of the genome and containing genes considered to be dosage tolerant for human development--were uncovered in the human population. Here we present a family with a novel autosomal-dominantly inherited syndrome characterized by microtia, eye coloboma, and imperforation of the nasolacrimal duct. This phenotype is linked to a cytogenetically visible alteration at 4pter consisting of five copies of a copy-number-variable region, encompassing a low-copy repeat (LCR)-rich sequence. We demonstrate that the approximately 750 kb amplicon occurs in exact tandem copies. This is the first example of an amplified CNV associated with a Mendelian disorder, a discovery that implies that genome screens for genetic disorders should include the analysis of so-called benign CNVs and LCRs.

Concurrent Whole-Genome Haplotyping and Copy-Number Profiling of Single Cells

Methods for haplotyping and DNA copy-number typing of single cells are paramount for studying genomic heterogeneity and enabling genetic diagnosis. Before analyzing the DNA of a single cell by microarray or next-generation sequencing, a whole-genome amplification (WGA) process is required, but it substantially distorts the frequency and composition of the cells alleles. As a consequence, haplotyping methods suffer from error-prone discrete SNP genotypes (AA, AB, BB) and DNA copy-number profiling remains difficult because true DNA copy-number aberrations have to be discriminated from WGA artifacts. Here, we developed a single-cell genome analysis method that reconstructs genome-wide haplotype architectures as well as the copy-number and segregational origin of those haplotypes by employing phased parental genotypes and deciphering WGA-distorted SNP B-allele fractions via a process we coin haplarithmisis. We demonstrate that the method can be applied as a generic method for preimplantation genetic diagnosis on single cells biopsied from human embryos, enabling diagnosis of disease alleles genome wide as well as numerical and structural chromosomal anomalies. Moreover, meiotic segregation errors can be distinguished from mitotic ones.