Computational approach for deriving cancer progression roadmaps from static sample data.

As with any biological process, cancer development is inherently dynamic. While major efforts continue to catalog the genomic events associated with human cancer, it remains difficult to interpret and extrapolate the accumulating data to provide insights into the dynamic aspects of the disease. Here, we present a computational strategy that enables the construction of a cancer progression model using static tumor sample data. The developed approach overcame many technical limitations of existing methods. Application of the approach to breast cancer data revealed a linear, branching model with two distinct trajectories for malignant progression. The validity of the constructed model was demonstrated in 27 independent breast cancer data sets, and through visualization of the data in the context of disease progression we were able to identify a number of potentially key molecular events in the advance of breast cancer to malignancy.

Differential copy number aberrations in novel candidate genes associated with progression from in situ to invasive ductal carcinoma of the breast.

Only a minority of intraductal carcinomas of the breast give rise to stromally invasive disease. We microdissected 206 paraffin blocks representing 116 different cases of low-grade ductal carcinoma in situ (DCIS). Fifty-five were pure DCIS (PD) cases without progression to invasive carcinoma. Sixty-one cases had a small invasive component. DNA was extracted from microdissected sections and hybridized to high-density bacterial artificial chromosome arrays. Array comparative genomic hybridization analysis of 118 hybridized DNA samples yielded data on 69 samples that were suitable for further statistical analysis. This cohort included 20 pure DCIS cases, 25 mixed DCIS (MD), and 24 mixed invasive carcinoma samples. PD cases had a higher frequency of DNA copy number changes than MD cases, and the latter had similar DNA profiles compared to paired invasive carcinomas. Copy number changes on 13 chromosomal arms occurred at different rates in PD versus MD lesions. Eight of 19 candidate genes residing at those loci were confirmed to have differential copy number changes by quantitative PCR. NCOR2/SMRT and NR4A1 (both on 12q), DYNLRB2 (16q), CELSR1, UPK3A, and ST13 (all on 22q) were more frequently amplified in PD. Moreover, NCOR2, NR4A1, and DYNLRB2 showed more frequent copy number losses in MD. GRAP2 (22q) was more often amplified in MD, whereas TAF1C (16q) was more commonly deleted in PD. A multigene model comprising these candidate genes discriminated between PD and MD lesions with high accuracy. These findings suggest that the propensity to invade the stroma may be encoded in the genome of intraductal carcinomas.

Robust Performance of SARS-CoV-2 Whole-Genome Sequencing from Wastewater with a Nonselective Virus Concentration Method.

The sequencing of human virus genomes from wastewater samples is an efficient method for tracking viral transmission and evolution at the community level. However, this requires the recovery of viral nucleic acids of high quality. We developed a reusable tangential-flow filtration system to concentrate and purify viruses from wastewater for genome sequencing. A pilot study was conducted with 94 wastewater samples from four local sewersheds, from which viral nucleic acids were extracted, and the whole genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was sequenced using the ARTIC V4.0 primers. Our method yielded a high probability (0.9) of recovering complete or near-complete SARS-CoV-2 genomes (>90% coverage at 10× depth) from wastewater when the COVID-19 incidence rate exceeded 33 cases per 100 000 people. The relative abundances of sequenced SARS-CoV-2 variants followed the trends observed from patient-derived samples. We also identified SARS-CoV-2 lineages in wastewater that were underrepresented or not present in the clinical whole-genome sequencing data. The developed tangential-flow filtration system can be easily adopted for the sequencing of other viruses in wastewater, particularly those at low concentrations.

A mouse model of Down syndrome trisomic for all human chromosome 21 syntenic regions.

Down syndrome (DS) is caused by the presence of an extra copy of human chromosome 21 (Hsa21) and is the most common genetic cause for developmental cognitive disability. The regions on Hsa21 are syntenically conserved with three regions located on mouse chromosome 10 (Mmu10), Mmu16 and Mmu17. In this report, we describe a new mouse model for DS that carries duplications spanning the entire Hsa21 syntenic regions on all three mouse chromosomes. This mouse mutant exhibits DS-related neurological defects, including impaired cognitive behaviors, reduced hippocampal long-term potentiation and hydrocephalus. These results suggest that when all the mouse orthologs of the Hsa21 genes are triplicated, an abnormal cognitively relevant phenotype is the final outcome of the elevated expressions of these orthologs as well as all the possible functional interactions among themselves and/or with other mouse genes. Because of its desirable genotype and phenotype, this mutant may have the potential to serve as one of the reference models for further understanding the developmental cognitive disability associated with DS and may also be used for developing novel therapeutic interventions for this clinical manifestation of the disorder.

Multimodal Dimension Reduction and Subtype Classification of Head and Neck Squamous Cell Tumors.

Traditional analysis of genomic data from bulk sequencing experiments seek to group and compare sample cohorts into biologically meaningful groups. To accomplish this task, large scale databases of patient-derived samples, like that of TCGA, have been established, giving the ability to interrogate multiple data modalities per tumor. We have developed a computational strategy employing multimodal integration paired with spectral clustering and modern dimension reduction techniques such as PHATE to provide a more robust method for cancer sub-type classification. Using this integrated approach, we have examined 514 Head and Neck Squamous Carcinoma (HNSC) tumor samples from TCGA across gene-expression, DNA-methylation, and microbiome data modalities. We show that these approaches, primarily developed for single-cell sequencing can be efficiently applied to bulk tumor sequencing data. Our multimodal analysis captures the dynamic heterogeneity, identifies new and refines subtypes of HNSC, and orders tumor samples along well-defined cellular trajectories. Collectively, these results showcase the inherent molecular complexity of tumors and offer insights into carcinogenesis and importance of targeted therapy. Computational techniques as highlighted in our study provide an organic and powerful approach to identify granular patterns in large and noisy datasets that may otherwise be overlooked.

Phase 1 study of arsenic trioxide, high-dose cytarabine, and idarubicin to down-regulate constitutive signal transducer and activator of transcription 3 activity in patients aged <60 years with acute myeloid leukemia.

BACKGROUND: Constitutive activation of signal transducer and activator of transcription-3 (STAT3) was detected in blasts from approximately 50% of patients with acute myeloid leukemia (AML) and was correlated with an adverse outcome. In vitro treatment of AML blasts with arsenic trioxide (ATO) down-regulated STAT3 activity within 6 hours associated with a reduced viability within 48 hours. METHODS: A phase 1 clinical trial to evaluate the biologically effective dose and/or the maximally tolerated dose (MTD) of ATO in vivo in conjunction with high-dose cytarabine (Hidac) and idarubicin (Ida) in patients with AML aged <60 years was conducted. Data were compared with 117 historic AML patients who had received treatment with Hidac/Ida. RESULTS: In total, 61 patients were enrolled onto 11 different dose levels (from 0.01 to 0.65 mg/kg ideal body weight). The MTD was 0.5 mg/kg. Compared with historic controls, patients who received ATO/Hidac/Ida, although they had similar pretreatment characteristics, had better overall survival (P = .039). CONCLUSIONS: ATO priming may have improved the outcome of patients aged <60 years with AML who received Hidac/Ida. The current data suggested that ATO may enhance the effect of chemotherapy. The authors concluded that further studies of this novel combination are warranted.

Genetic analysis of Down syndrome-associated heart defects in mice.

Human trisomy 21, the chromosomal basis of Down syndrome (DS), is the most common genetic cause of heart defects. Regions on human chromosome 21 (Hsa21) are syntenically conserved with three regions located on mouse chromosome 10 (Mmu10), Mmu16 and Mmu17. In this study, we have analyzed the impact of duplications of each syntenic region on cardiovascular development in mice and have found that only the duplication on Mmu16, i.e., Dp(16)1Yey, is associated with heart defects. Furthermore, we generated two novel mouse models carrying a 5.43-Mb duplication and a reciprocal deletion between Tiam1 and Kcnj6 using chromosome engineering, Dp(16Tiam1-Kcnj6)Yey/+ and Df(16Tiam1-Kcnj6)Yey/+, respectively, within the 22.9-Mb syntenic region on Mmu16. We found that Dp(16Tiam1-Kcnj6)Yey/+, but not Dp(16)1Yey/Df(16Tiam1-Kcnj6)Yey, resulted in heart defects, indicating that triplication of the Tiam1-Knj6 region is necessary and sufficient to cause DS-associated heart defects. Our transcriptional analysis of Dp(16Tiam1-Kcnj6)Yey/+ embryos confirmed elevated expression levels for the genes located in the Tiam-Kcnj6 region. Therefore, we established the smallest critical genomic region for DS-associated heart defects to lay the foundation for identifying the causative gene(s) for this phenotype.

The genomic relationship between primary breast carcinomas and their nodal metastases.

We screened the whole tumor genome to identify DNA copy number gains and losses that discriminate between primary breast carcinomas (MP) and their nodal metastases (ML). Six candidate genes were confirmed by quantitative PCR to have differentially distributed copy number changes. Three of the genes (ERRγ, DDX6, and TIAM1) were more commonly amplified in nodal metastases. Principal component analysis revealed that MP-ML pairs varied markedly in their genomic divergence. The latter was larger in PR-negative tumors. Nodal metastases may form early or late in the development of breast carcinomas and PR-negative tumors may metastasize earlier or are genomically less stable.

A de novo 1p34.2 microdeletion identifies the synaptic vesicle gene RIMS3 as a novel candidate for autism.

BACKGROUND: A child with autism and mild microcephaly was found to have a de novo 3.3 Mb microdeletion on chromosome 1p34.2p34.3. The hypothesis is tested that this microdeletion contains one or more genes that underlie the autism phenotype in this child and in other children with autism spectrum disorders. METHODS: To search for submicroscopic chromosomal rearrangements in the child, array comparative genomic hybridisation (aCGH) was performed using a 19 K whole genome human bacterial artificial chromosome (BAC) array and the Illumina 610-Quad BeadChip microarray. Ingenuity pathway analysis (IPA) was used to construct functional biological networks to identify candidate autism genes. To identify putative functional variants in candidate genes, mutation screening was performed using polymerase chain reaction (PCR) based Sanger sequencing in 512 unrelated autism patients and 462 control subjects. RESULTS: A de novo 3.3 Mb deletion containing approximately 43 genes in chromosome 1p34.2p34.3 was identified and subsequently confirmed using fluorescence in situ hybridization (FISH). Literature review and bioinformatics analyses identified Regulating Synaptic Membrane Exocytosis 3 (RIMS3) as the most promising autism candidate gene. Mutation screening of this gene in autism patients identified five inherited coding variants, including one (p.E177A) that segregated with the autism phenotype in a sibship, was predicted to be deleterious, and was absent in 1161 controls. CONCLUSIONS: This case report and mutation screening data suggest that RIMS3 is an autism causative or contributory gene. Functional studies of RIMS3 variants such as p.E177A should provide additional insight into the role of synaptic proteins in the pathophysiology of autism.

A GOG 210 aCGH study of gain at 1q23 in endometrioid endometrial cancer in the context of racial disparity and outcome.

The goal of this study was to identify recurrent regions of genomic gain or loss in endometrial cancer of the endometrioid type in the context of racial disparities in mortality for this disease. Array comparative genomic hybridization (aCGH) analysis was performed on 80 frozen primary tumors from the Gynecologic Oncology Group (GOG)-210 bank using the RPCI 19K BAC arrays. The 80 patients included 20 African American (AA) Stage I, 20 White (W) Stage I, 20 African American (AA) Stage IIIC/IV, and 20 White (W) Stage IIIC/IV. A separate subset of 220 endometrial cancers with outcome data was used for validation. A 1.6-Mbp region of gain at 1q23 was identified by aCGH in all AA patients and high grade W patients, but not W low grade patients. In the validation arm of 220 patients copy number gain at this region was validated using FISH and locus specific BACs. The number of AA patients in the validation arm was too small to confirm the aCGH association with racial disparity. Kaplan-Meier curves for survival showed a significant difference for gain at 1q23 versus no gain (log rank P = 0.0014). When subdivided into various groups of risk by stage and grade the survival curves showed a decreased survival for high grade and/or stage tumors, but not for low grade and/or stage endometrioid tumors. Univariate analyses for gain at 1q23 showed a significant association (P = 0.009) with survival. Multivariate analysis for gain at 1q23 did not show a significant association with survival (P = 0.14).

Recurrent 16p11.2 microdeletions in autism.

Autism is a childhood neurodevelopmental disorder with a strong genetic component, yet the identification of autism susceptibility loci remains elusive. We investigated 180 autism probands and 372 control subjects by array comparative genomic hybridization (aCGH) using a 19K whole-genome tiling path bacterial artificial chromosome microarray to identify submicroscopic chromosomal rearrangements specific to autism. We discovered a recurrent 16p11.2 microdeletion in two probands with autism and none in controls. The deletion spans approximately 500-kb and is flanked by approximately 147-kb segmental duplications (SDs) that are >99% identical, a common characteristic of genomic disorders. We assessed the frequency of this new autism genomic disorder by screening an additional 532 probands and 465 controls by quantitative PCR and identified two more patients but no controls with the microdeletion, indicating a combined frequency of 0.6% (4/712 autism versus 0/837 controls; Fisher exact test P = 0.044). We confirmed all 16p11.2 deletions using fluorescence in situ hybridization, microsatellite analyses and aCGH, and mapped the approximate deletion breakpoints to the edges of the flanking SDs using a custom-designed high-density oligonucleotide microarray. Bioinformatic analysis localized 12 of the 25 genes within the microdeletion to nodes in one interaction network. We performed phenotype analyses and found no striking features that distinguish patients with the 16p11.2 microdeletion as a distinct autism subtype. Our work reports the first frequency, breakpoint, bioinformatic and phenotypic analyses of a de novo 16p11.2 microdeletion that represents one of the most common recurrent genomic disorders associated with autism to date.

Gab2-mediated signaling promotes melanoma metastasis.

Metastatic melanoma is a disease with a poor prognosis that currently lacks effective treatments. Critical biological features of metastasis include acquisition of migratory competence, growth factor independence, and invasive potential. In an attempt to identify genes that contribute to melanoma pathogenesis, a genome-wide search using bacterial artificial chromosome array comparative genomic hybridization and single nucleotide polymorphism arrays in a series of 64 metastatic melanoma samples and 20 melanoma cell lines identified increased copy numbers of Gab2 located on 11q14.1. Gab2 is an adaptor protein that potentiates the activation of the Ras-Erk and PI3K-Akt pathways and has recently been implicated in human cancer; however, its role in melanoma has not been explored. In this study, we found that Gab2 was either amplified (approximately 11%) and/or overexpressed (approximately 50%) in melanoma. Gab2 protein expression correlated with clinical melanoma progression, and higher levels of expression were seen in metastatic melanomas compared with primary melanoma and melanocytic nevi. We found that overexpression of Gab2 potentiates, whereas silencing of Gab2 reduces, migration and invasion of melanoma cells. Gab2 mediated the hyperactivation of Akt signaling in the absence of growth factors, whereas inhibition of the PI3K-Akt pathway decreased Gab2-mediated tumor cell migration and invasive potential. Gab2 overexpression resulted in enhanced tumor growth and metastatic potential in vivo. These studies demonstrate a previously undefined role for Gab2 in melanoma tumor progression and metastasis.

A gene expression atlas of the central nervous system based on bacterial artificial chromosomes.

The mammalian central nervous system (CNS) contains a remarkable array of neural cells, each with a complex pattern of connections that together generate perceptions and higher brain functions. Here we describe a large-scale screen to create an atlas of CNS gene expression at the cellular level, and to provide a library of verified bacterial artificial chromosome (BAC) vectors and transgenic mouse lines that offer experimental access to CNS regions, cell classes and pathways. We illustrate the use of this atlas to derive novel insights into gene function in neural cells, and into principal steps of CNS development. The atlas, library of BAC vectors and BAC transgenic mice generated in this screen provide a rich resource that allows a broad array of investigations not previously available to the neuroscience community.

Microcephaly, sensorineural deafness and Currarino triad with duplication-deletion of distal 7q.

Currarino syndrome (CS) is a peculiar form of caudal regression syndrome [also known as autosomal dominant sacral agenesis (OMIM no. 176450)] characterised by (1) partial absence of the sacrum with intact first sacral vertebra, (2) a pre-sacral mass and (3) anorectal anomalies (Currarino triad). We studied a 3-year-old girl with Currarino triad who had additional systemic features and performed array comparative genomic hybridisation to look for chromosomal abnormalities. This girl had the typical spectrum of anomalies of the CS including (a) partial sacral agenesis (hemisacrum with remnants of only sacral S1-S2 vertebrae and a residual S3 vertebral body) associated with complete coccygeal agenesis, (b) pre-intrasacral dermoid, (c) intra-dural lipoma, (d) ectopic anus and (e) tethered cord. She had, in addition, pre- and post-natal growth impairment (<3rd percentile), severe microcephaly (<-3 SD) with normal gyration pattern and lack of cortical thickening associated with a hypoplastic inferior vermis, facial dysmorphism, sensorineural deafness and decreased serum levels of IGF-1. A de novo 10.3-Mb duplication of 7q34-q35 and an 8.8-Mb deletion on 7q36 were identified in this patient. The Homeobox HLXB9 (CS) gene is contained within the deletion accounting for the CS phenotype including microcephaly. The spectrums of associated abnormalities in the IGF-1 deficiency growth retardation with sensorineural deafness and mental retardation syndrome (OMIM no. 608747) are discussed. To the best of our knowledge, this is the first reported case of a patient with distal 7q chromosomal imbalance and features of CS triad (including microcephaly) and the first documented case of a patient with normal gyration pattern microcephaly. The spectrum of associated anomalies in this newly recognised phenotype complex consists of growth failure, typical facial anomalies with additional (previously unreported) nervous system abnormalities (e.g. sensorineural deafness) and somatomedin C deficiency.

Reactivation of super-enhancers by KLF4 in human Head and Neck Squamous Cell Carcinoma.

Head and neck squamous cell carcinoma (HNSCC) is a disease of significant morbidity and mortality and rarely diagnosed in early stages. Despite extensive genetic and genomic characterization, targeted therapeutics and diagnostic markers of HNSCC are lacking due to the inherent heterogeneity and complexity of the disease. Herein, we have generated the global histone mark based epigenomic and transcriptomic cartogram of SCC25, a representative cell type of mesenchymal HNSCC and its normal oral keratinocyte counterpart. Examination of genomic regions marked by differential chromatin states and associated with misregulated gene expression led us to identify SCC25 enriched regulatory sequences and transcription factors (TF) motifs. These findings were further strengthened by ATAC-seq based open chromatin and TF footprint analysis which unearthed Krüppel-like Factor 4 (KLF4) as a potential key regulator of the SCC25 cistrome. We reaffirm the results obtained from in silico and chromatin studies in SCC25 by ChIP-seq of KLF4 and identify ΔNp63 as a co-oncogenic driver of the cancer-specific gene expression milieu. Taken together, our results lead us to propose a model where elevated KLF4 levels sustains the oncogenic state of HNSCC by reactivating repressed chromatin domains at key downstream genes, often by targeting super-enhancers.

Chromosome 11 genomic changes in parathyroid adenoma and hyperplasia: array CGH, FISH, and tissue microarrays.

We have used a combination of gene expression profiling, array comparative genomic hybridization (aCGH), fluorescent in situ hybridization (FISH) and tissue microarrays (TMAs) to investigate chromosome 11 genetic changes in subsets of benign parathyroid tumors. Integration of gene expression profiling and aCGH was done using differential gene locus mapping analysis. We have identified three distinct relatively common chromosome 11 genomic changes in various subsets of parathyroid tumors. The simplest and least common of these genomic changes involves translocation of the CCND1 gene with subsequent strong CCND1 expression. This genetic change is essentially limited to parathyroid adenomas (8%), although expression of CCND1 without translocation is common in uremic hyperparathyroidism. Not surprisingly, deletion of the MEN1 locus at 11q13 or loss of a large portion or an entire chromosome 11 was a common finding. This particular genomic change appears to have a prominent effect on the overall results of gene expression profiling and was present in slightly less than one-half of adenomas. Genomic changes in primary nonfamilial hyperplasia were for the most part restricted to 11q13 deletion or loss of chromosome 11. The third genomic change we identified was 11q23 deletion. This genetic change was relatively independent of other chromosome 11 changes and present in slightly less than one-half of adenomas. 11q23 deletion along with relatively strong CCND1 expression was common in uremic hyperparathyroidism.

Targeting TAZ-Driven Human Breast Cancer by Inhibiting a SKP2-p27 Signaling Axis.

Deregulated expression of the transcriptional coactivator with PDZ-binding motif (WWTR1/TAZ) is a common feature of basal-like breast cancer (BLBC). Yet, how oncogenic TAZ regulates cell-cycle progression and proliferation in breast cancer remains poorly understood, and whether TAZ is required for tumor maintenance has not been established. Here, using an integrative oncogenomic approach, TAZ-dependent cellular programs essential for tumor growth and progression were identified. Significantly, TAZ-driven tumor cells required sustained TAZ expression, given that its withdrawal impaired both genesis and maintenance of solid tumors. Moreover, temporal inhibition of TAZ diminished the metastatic burden in established macroscopic pulmonary metastases. Mechanistic investigation revealed that TAZ controls distinct gene profiles that determine cancer cell fate through cell-cycle networks, including a specific, causal role for S-phase kinase-associated protein 2 (SKP2) in mediating the neoplastic state. Together, this study elucidates the molecular events that underpin the role of TAZ in BLBC and link to SKP2, a convergent communication node for multiple cancer signaling pathways, as a key downstream effector molecule. IMPLICATIONS: Understanding the molecular role of TAZ and its link to SKP2, a signaling convergent point and key regulator in BLBC, represents an important step toward the identification of novel therapeutic targets for TAZ-dependent breast cancer.

Analytical and clinical validity of whole-genome oligonucleotide array comparative genomic hybridization for pediatric patients with mental retardation and developmental delay.

We performed a pilot study to establish the analytical and clinical validity of whole genome oligonucleotide array comparative genomic hybridization (oaCGH) using the 44,000 oligonucleotide array from Agilent Technologies. DNA specimens from 10 patients with different chromosomal abnormalities were used as the test group and sex mismatched normal male or female DNA as references. A series of DNA mixtures containing 50%, 33%, and 25% of a known deletion was generated to evaluate the analytical capacity of oaCGH on detecting mosaic pattern. Receiver operating characteristic (ROC) curves were computed to evaluate sensitivity, specificity, and analytical resolution for detecting deletions, duplications, and mosaic patterns. The oaCGH detected the chromosomally recognized deletions, duplications, and additional genomic aberrations. Fluorescent in situ hybridization (FISH) assays using targeted BAC clone probes confirmed oaCGH findings. Failure in detecting marker chromosomes, a polymorphic inversion, and a Robertsonian translocation was also noted. The oaCGH achieved 99% sensitivity and 99% specificity with a resolution of 300-500 Kb. It also demonstrated 85% sensitivity and 95% specificity in detecting 50% mosaicism; however, increased test-to-test variations and reduced sensitivity were noted as the mosaic percentage decreased. Chromosome and oaCGH analyses on 50 pediatric patients with mental retardation (MR) and developmental delay (DD) delineated the genomic content of chromosomal abnormalities in nine cases, pathogenic genomic disorders in three cases and benign genomic variants in six cases. These results affirmed the analytical and clinical validity of oaCGH and prompted a cytogenomic algorithm to integrate oaCGH, chromosome and FISH analyses for genetic diagnosis.

Estimating the arm-wise false discovery rate in array comparative genomic hybridization experiments.

Array Comparative Genomic Hybridization (aCGH) is an array-based technology which provides simultaneous spot assays of relative genetic abundance (RGA) levels at multiple sites across the genome. These spot assays are spatially correlated with respect to genomic location and, as a result, the univariate tests conducted using data generated from these spot assays are also spatially correlated. In the context of multiple hypothesis testing, this spatial correlation complicates the question of how best to define a 'discovery' and consequently, how best to estimate the false discovery rate (FDR) corresponding to a given rejection region. One can quantify the number of discoveries as the total number of spots for which the spot-based univariate test statistic falls within a given rejection region. Under this spot-based method, separate but correlated discoveries are identified. We show via a simulation study that the method of Benjamini and Hochberg (1995) can provide a reasonable estimate of the spot-wise FDR, but these results require that the simulated spot assays are categorized as true or false discoveries in a particular way. However, laboratory researchers may actually be interested in estimating a 'regional' FDR, rather than a 'local' spot-wise FDR. We describe an example of such circumstances, and present a method for estimating the (chromosome) arm-wise False Discovery Rate. In this framework, one can quantify the number of discoveries as the total number of chromosome arms for which at least one spot-based test statistic falls into a given rejection region. Defining the discoveries in this way, both the biological and testing objectives coincide. We provide results from a series of simulations which involved the analysis of preferentially re-sampled spot assay values from a real aCGH dataset.

Familial 4.3 Mb duplication of 21q22 sheds new light on the Down syndrome critical region.

A 4.3 Mb duplication of chromosome 21 bands q22.13-q22.2 was diagnosed by interphase fluorescent in-situ hybridisation (FISH) in a 31-week gestational age baby with cystic hygroma and hydrops; the duplication was later found in the mother and in her 8-year-old daughter by the same method and confirmed by array comparative genomic hybridisation (aCGH). All had the facial gestalt of Down syndrome (DS). This is the smallest accurately defined duplication of chromosome 21 reported with a DS phenotype. The duplication encompasses the gene DYRK1 but not DSCR1 or DSCAM, all of which have previously been implicated in the causation of DS. Previous karyotype analysis and telomere screening of the mother, and karyotype analysis and metaphase FISH of a chorionic villus sample, had all failed to reveal the duplication. The findings in this family add to the identification and delineation of a "critical region" for the DS phenotype on chromosome 21. Cryptic chromosomal abnormalities can be missed on a routine karyotype for investigation of abnormal prenatal ultrasound findings, lending support to the use of aCGH analysis in this setting.