Daniel A. Peiffer

Mapping and sequencing of structural variation from eight human genomes

Genetic variation among individual humans occurs on many different scales, ranging from gross alterations in the human karyotype to single nucleotide changes. Here we explore variation on an intermediate scale—particularly insertions, deletions and inversions affecting from a few thousand to a few million base pairs. We employed a clone-based method to interrogate this intermediate structural variation in eight individuals of diverse geographic ancestry. Our analysis provides a comprehensive overview of the normal pattern of structural variation present in these genomes, refining the location of 1,695 structural variants. We find that 50% were seen in more than one individual and that nearly half lay outside regions of the genome previously described as structurally variant. We discover 525 new insertion sequences that are not present in the human reference genome and show that many of these are variable in copy number between individuals. Complete sequencing of 261 structural variants reveals considerable locus complexity and provides insights into the different mutational processes that have shaped the human genome. These data provide the first high-resolution sequence map of human structural variation—a standard for genotyping platforms and a prelude to future individual genome sequencing projects.

Speech delay and autism spectrum behaviors are frequently associated with duplication of the 7q11.23 Williams-Beuren syndrome region

Purpose: Williams-Beuren syndrome is among the most well-characterized microdeletion syndromes, caused by recurrent de novo microdeletions at 7q11.23 mediated by nonallelic homologous recombination between low copy repeats flanking this critical region. However, the clinical phenotype associated with reciprocal microduplication of this genomic region is less well described. We investigated the molecular, clinical, neurodevelopmental, and behavioral features of seven patients with dup(7)(q11.23), including two children who inherited the microduplication from one of their parents, to more fully characterize this emerging microduplication syndrome.Methods: Patients were identified by array-based comparative genomic hybridization. Clinical examinations were performed on seven affected probands, and detailed cognitive and behavioral evaluations were carried out on four of the affected probands.Results: Our findings confirm initial reports of speech delay seen in patients with dup(7)(q11.23) and further delineate and expand the phenotypic spectrum of this condition to include communication, social interactions, and repetitive interests that are often observed in individuals diagnosed with autism spectrum disorders.Conclusions: Array-based comparative genomic hybridization is a powerful means of detecting genomic imbalances and identifying molecular etiologies in the clinic setting, including genomic disorders such as Williams-Beuren syndrome and dup(7)(q11.23). We propose that dup(7)(q11.23) syndrome may be as frequent as Williams-Beuren syndrome and a previously unrecognized cause of language delay and behavioral abnormalities. Indeed, these individuals may first be referred for evaluation of autism, even if they do not ultimately meet diagnostic criteria for an autism spectrum disorder.

Deletion of 7q31.1 supports involvement of FOXP2 in language impairment : Clinical report and review

We report on a young male with moderate mental retardation, dysmorphic features, and language delay who is deleted for 7q31.1‐7q31.31. His full karyotype is 46,XY,der(7)del(7)(q31.1q31.31)ins(10;7)(q24.3;q31.1q31.31)mat. This child had language impairment, including developmental verbal dyspraxia, but did not meet criteria for autism according to standardized ADOS testing. Our patients deletion, which is the smallest reported deletion including FOXP2, adds to the body of evidence that supports the role of FOXP2 in speech and language impairment, but not in autism. A reported association between autism and deletions of WNT2, a gene also deleted in our patient, is likewise not supported by our case. Previously, fine mapping with microsatellites markers within in a large three‐generation family, in which half the members had severe specific language impairment, aided the localization of the SPCH1 locus to 7q31 within markers D7S2459 (107.1 Mb) and D7S643 (120.5 Mb). Additionally, chromosome rearrangement of 7q31 and mutational analyses have supported the growing evidence that FOXP2, a gene within the SPCH1 region, is involved with speech and language development. It is unclear however whether the AUTS1 (autistic spectrum 1) locus, highly linked to 7q31, overlaps with the SPCH1 and FOXP2.

Single nucleotide polymorphism-based genome-wide chromosome copy change, loss of heterozygosity, and aneuploidy in Barrett's esophagus neoplastic progression.

Chromosome copy gain, loss, and loss of heterozygosity (LOH) involving most chromosomes have been reported in many cancers; however, less is known about chromosome instability in premalignant conditions. 17p LOH and DNA content abnormalities have been previously reported to predict progression from Barretts esophagus (BE) to esophageal adenocarcinoma (EA). Here, we evaluated genome-wide chromosomal instability in multiple stages of BE and EA in whole biopsies. Forty-two patients were selected to represent different stages of progression from BE to EA. Whole BE or EA biopsies were minced, and aliquots were processed for flow cytometry and genotyped with a paired constitutive control for each patient using 33,423 single nucleotide polymorphisms (SNP). Copy gains, losses, and LOH increased in frequency and size between early- and late-stage BE (P < 0.001), with SNP abnormalities increasing from <2% to >30% in early and late stages, respectively. A set of statistically significant events was unique to either early or late, or both, stages, including previously reported and novel abnormalities. The total number of SNP alterations was highly correlated with DNA content aneuploidy and was sensitive and specific to identify patients with concurrent EA (empirical receiver operating characteristic area under the curve = 0.91). With the exception of 9p LOH, most copy gains, losses, and LOH detected in early stages of BE were smaller than those detected in later stages, and few chromosomal events were common in all stages of progression. Measures of chromosomal instability can be quantified in whole biopsies using SNP-based genotyping and have potential to be an integrated platform for cancer risk stratification in BE.

Deletion at Fragile Sites is a Common and Early Event in Barrett’s Esophagus

Barretts esophagus (BE) is a premalignant intermediate to esophageal adenocarcinoma, which develops in the context of chronic inflammation and exposure to bile and acid. We asked whether there might be common genomic alterations that could be identified as potential clinical biomarker(s) for BE by whole genome profiling. We detected copy number alterations and/or loss of heterozygosity at 56 fragile sites in 20 patients with premalignant BE. Chromosomal fragile sites are particularly sensitive to DNA breaks and are frequent sites of rearrangement or loss in many human cancers. Seventy-eight percent of all genomic alterations detected by array-CGH were associated with fragile sites. Copy number losses in early BE were observed at particularly high frequency at FRA3B (81%), FRA9A/C (71.4%), FRA5E (52.4%), and FRA 4D (52.4%), and at lower frequencies in other fragile sites, including FRA1K (42.9%), FRAXC (42.9%), FRA 12B (33.3%), and FRA16D (33.3%). Due to the consistency of the region of copy number loss, we were able to verify these results by quantitative PCR, which detected the loss of FRA3B and FRA16D, in 83% and 40% of early molecular stage BE patients, respectively. Loss of heterozygosity in these cases was confirmed through pyrosequencing at FRA3B and FRA16D (75% and 70%, respectively). Deletion and genomic instability at FRA3B and other fragile sites could thus be a biomarker of genetic damage in BE patients and a potential biomarker of cancer risk. Mol Cancer Res; 8(8); 1084–94. ©2010 AACR.

Genomic profiling of 766 cancer-related genes in archived esophageal normal and carcinoma tissues

We employed the BeadArray™ technology to perform a genetic analysis in 33 formalin‐fixed, paraffin‐embedded (FFPE) human esophageal carcinomas, mostly squamous‐cell‐carcinoma (ESCC), and their adjacent normal tissues. A total of 1,432 single nucleotide polymorphisms (SNPs) derived from 766 cancer‐related genes were genotyped with partially degraded genomic DNAs isolated from these samples. This directly targeted genomic profiling identified not only previously reported somatic gene amplifications (e.g., CCND1) and deletions (e.g., CDKN2A and CDKN2B) but also novel genomic aberrations. Among these novel targets, the most frequently deleted genomic regions were chromosome 3p (including tumor suppressor genes FANCD2 and CTNNB1) and chromosome 5 (including tumor suppressor gene APC). The most frequently amplified genomic region was chromosome 3q (containing DVL3, MLF1, ABCC5, BCL6, AGTR1 and known oncogenes TNK2, TNFSF10, FGF12). The chromosome 3p deletion and 3q amplification occurred coincidently in nearly all of the affected cases, suggesting a molecular mechanism for the generation of somatic chromosomal aberrations. We also detected significant differences in germline allele frequency between the esophageal cohort of our study and normal control samples from the International HapMap Project for 10 genes (CSF1, KIAA1804, IL2, PMS2, IRF7, FLT3, NTRK2, MAP3K9, ERBB2 and PRKAR1A), suggesting that they might play roles in esophageal cancer susceptibility and/or development. Taken together, our results demonstrated the utility of the BeadArray technology for high‐throughput genetic analysis in FFPE tumor tissues and provided a detailed genetic profiling of cancer‐related genes in human esophageal cancer.

Delineation of the Proximal 3q Microdeletion Syndrome

Interstitial deletions of the proximal long arm of chromosome 3 are very rare and a defined clinical phenotype is not established yet. We report on the clinical, cytogenetic and molecular findings of a 20‐month‐old Hispanic male with a 2.5 Mb de novo deletion on q13.11q13.12. Up to now, this is the smallest deletion reported among patients with the proximal 3q microdeletion syndrome. The patient has distinct facial features including brachycephaly, broad and prominent forehead, flat nasal bridge, prominent ears, anteverted nose, tetralogy of Fallot, bilateral cryptorchidism, and peripheral skeletal abnormalities. To further delineate the proximal 3q deletion syndrome, the phenotype of our patient was compared with 10 other patients previously described. We found that ALCAM and CBLB are the only genes deleted in our patient and based on previously published data, we propose that the CBLB gene is responsible for the craniofacial phenotype in patients with deletions of proximal 3q region.

Mosaic tetrasomy 12p with triplication of 12p detected by array-based comparative genomic hybridization of peripheral blood DNA†

A patient whose dysmorphism at birth was not diagnostic for Pallister–Killian syndrome (PKS) was found to have mosaic tetrasomy 12p by an array‐based comparative genomic hybridization of peripheral blood DNA. He was determined to be mosaic for 46,XY,trp(12)(p11.2 → p13) in cultured skin fibroblasts. His appearance was typical for PKS at 4 months of age.

Design of Tag SNP Whole Genome Genotyping Arrays

Whole genome association studies have recently been enabled by combining tag SNP information derived from the International HapMap project with novel whole genome genotyping array technologies. In particular, Infinium whole genome genotyping (WGG) technology now has the power to genotype over 1 million SNPs on a single array. Additionally, this assay provides access to virtually any SNP in the genome enabling selection of optimized SNP content . In this chapter, we provide an overview of the tag SNP-based selection strategy for Infinium whole-genome genotyping BeadChips, including the Human 1 M BeadChip. These advances in both SNP content and technology have enabled both large-scale whole-genome disease association (WGAS) and copy number variation (CNV) studies with the ultimate goal of identifying common genetic variants, disease-associated loci, proteins, and biomarkers.

Integrated Molecular Analyses of Biological Samples on a Bead-Based Microarray Platform

Molecular analyses of biological samples have traditionally been pursued in parallel, with those researchers studying genetic diversity having few technical approaches in common with those studying gene expression. Increasingly, scientists recognize the importance of integrating analytical technologies to further research, particularly into emerging fields such as epigenetics and the genetics of gene expression. In this chapter, we describe a suite of applications that take advantage of the Illumina® bead-based microarrays, all of which are read out on a single analytical instrument. The integration of whole genome genotyping, high throughput focused genotyping, whole transcriptome expression profiling, focused expression profiling of fresh or preserved tissues, allele-specific expression profiling and DNA methylation assays on the BeadArray™ Reader allows researchers to expand their perspectives, from whole genomes to single bases, from genetics to expression and on to epigenetics.