Faculty Bibliography

Inversions, deletions and insertions are important mediators of disease and disease susceptibility. We systematically compared the human genome reference sequence with a second genome (represented by fosmid paired-end sequences) to detect intermediate-sized structural variants >8 kb in length. We identified 297 sites of structural variation: 139 insertions, 102 deletions and 56 inversion breakpoints. Using combined literature, sequence and experimental analyses, we validated 112 of the structural variants, including several that are of biomedical relevance. These data provide a fine-scale structural variation map of the human genome and the requisite sequence precision for subsequent genetic studies of human disease.

The human genome contains numerous blocks of highly homologous duplicated sequence. This higher-order architecture provides a substrate for recombination and recurrent chromosomal rearrangement associated with genomic disease. However, an assessment of the role of segmental duplications in normal variation has not yet been made. On the basis of the duplication architecture of the human genome, we defined a set of 130 potential rearrangement hotspots and constructed a targeted bacterial artificial chromosome (BAC) microarray (with 2,194 BACs) to assess copy-number variation in these regions by array comparative genomic hybridization. Using our segmental duplication BAC microarray, we screened a panel of 47 normal individuals, who represented populations from four continents, and we identified 119 regions of copy-number polymorphism (CNP), 73 of which were previously unreported. We observed an equal frequency of duplications and deletions, as well as a 4-fold enrichment of CNPs within hotspot regions, compared with control BACs (P < .000001), which suggests that segmental duplications are a major catalyst of large-scale variation in the human genome. Importantly, segmental duplications themselves were also significantly enriched >4-fold within regions of CNP. Almost without exception, CNPs were not confined to a single population, suggesting that these either are recurrent events, having occurred independently in multiple founders, or were present in early human populations. Our study demonstrates that segmental duplications define hotspots of chromosomal rearrangement, likely acting as mediators of normal variation as well as genomic disease, and it suggests that the consideration of genomic architecture can significantly improve the ascertainment of large-scale rearrangements. Our specialized segmental duplication BAC microarray and associated database of structural polymorphisms will provide an important resource for the future characterization of human genomic disorders.

Genomes of solid tumors are characterized by gains and losses of regions, which may contribute to tumorigenesis by altering gene expression. Often the aberrations are extensive, encompassing whole chromosome arms, which makes identification of candidate genes in these regions difficult. Here, we focused on narrow regions of gene amplification to facilitate identification of genetic pathways important in oral squamous cell carcinoma (SCC) development. We used array comparative genomic hybridization (array CGH) to define minimum common amplified regions and then used expression analysis to identify candidate driver genes in amplicons that spanned <3 Mb. We found genes involved in integrin signaling (TLN1), survival (YAP1, BIRC2), and adhesion and migration (TLN1, LAMA3, MMP7), as well as members of the hedgehog (GLI2) and notch (JAG1, RBPSUH, FJX1) pathways to be amplified and overexpressed. Deregulation of these and other members of the hedgehog and notch pathways (HHIP, SMO, DLL1, NOTCH4) implicates deregulation of developmental and differentiation pathways, cell fate misspecification, in oral SCC development

Males with duplications within the long arm of the X chromosome are rare and most cases are inherited from a maternal heterozygote. We report a male with a de novo Xq duplication and review of the literature. The proband was ascertained prenatally after an abnormal expanded alpha-fetoprotein (AFP) screen and abnormal ultrasound findings. Chromosome analysis on amniocyte and subsequent peripheral blood lymphocyte cultures showed a male karyotype containing additional material on the long arm of the X chromosome. Fluorescence in situ hybridization with an X chromosome whole chromosome paint probe showed that the additional material was derived from the X chromosome, interpreted as a dup(X)(q13.3q24). Further characterization of the duplication by array CGH showed a duplication size between 30-44 Mb as determined by the map position of the flanking clones on the array, and refined the breakpoints of the duplicated region to Xq21.32 --> Xq25. At birth, the proband had multiple craniofacial abnormalities, musculoskeletal anomalies, bilateral cryptorchidism with scrotal hypoplasia, conductive hearing loss, and profound generalized hypotonia despite normal birthweight, length, and head circumference. Although data regarding Xq duplications in males are limited, a clear pattern of characteristic features can be discerned as illustrated in the present case and confirmed in our literature review. Mental, psychomotor and growth retardation, as well as, craniofacial anomalies, muscle hypotonia, hypoplastic genitalia, cryptorchidism, feeding difficulties, and endocrine dysfunction are all significant issues in these individuals.

Alteration in DNA copy number is one of the many ways in which gene expression and function may be modified. Some variations are found among normal individuals, others occur in the course of normal processes in some species and still others participate in causing various disease states. For example, many defects in human development are due to gains and losses of chromosomes and chromosomal segments that occur before or shortly after fertilization, and DNA dosage-alteration changes occurring in somatic cells are frequent contributors to cancer. Detecting these aberrations and interpreting them in the context of broader knowledge facilitates the identification of crucial genes and pathways involved in biological processes and disease. Over the past several years, array comparative genomic hybridization has proven its value for analyzing DNA copy-number variations. Here, we discuss the state of the art of array comparative genomic hybridization and its applications in cancer, emphasizing general concepts rather than specific results.

CpG islands are present in one-half of all human and mouse genes and typically overlap with promoters or exons. We developed a method for high-resolution analysis of the methylation status of CpG islands genome-wide, using arrays of BAC clones and the methylation-sensitive restriction enzyme NotI. Here we demonstrate the accuracy and specificity of the method. By computationally mapping all NotI sites, methylation events can be defined with single-nucleotide precision throughout the genome. We also demonstrate the unique expandability of the array method using a different methylation-sensitive restriction enzyme, BssHII. We identified and validated new CpG island loci that are methylated in a tissue-specific manner in normal human tissues. The methylation status of the CpG islands is associated with gene expression for several genes, including SHANK3, which encodes a structural protein in neuronal postsynaptic densities. Defects in SHANK3 seem to underlie human 22q13 deletion syndrome. Furthermore, these patterns for SHANK3 are conserved in mice and rats.

Alterations of DNA copy number are believed to be important indicators of tumor progression in human astrocytoma. We used an array of bacterial artificial chromosomes to map relative DNA copy number in 50 primary glioblastoma multiforme tumors at approximately 1.4-Mb resolution. We identified 33 candidate sites for amplification and homozygous deletion in these tumors. We identified three major genetic subgroups within these glioblastoma multiforme tumors: tumors with chromosome 7 gain and chromosome 10 loss, tumors with only chromosome 10 loss in the absence of chromosome 7 gain, and tumors without copy number change in chromosomes 7 or 10. The significance of these genetic groups to therapeutics needs further study.

Structural chromosomal aberrations are common in epithelial tumors. Here, we compared the location of centromeric breaks associated with whole arm translocations in seven adenocarcinoma cell lines and nine squamous cell carcinoma cell lines using SKY, microarray-based comparative genomic hybridization (array CGH) and fluorescence in situ hybridization (FISH). Whole arm translocations were more frequent in squamous cell carcinomas (112 in nine cell lines and nine in one short-term culture) than in adenocarcinomas (13 in seven cases) and most often resulted in copy number alterations. Array CGH analysis demonstrated that in all squamous cell carcinomas and in most adenocarcinomas, the breakpoints of unbalanced whole arm translocations occurred between the two clones on the array flanking the centromeres. However, FISH with centromeric probes revealed that in squamous cell carcinomas, the marker chromosomes with whole arm translocations contained centromeres comprised of material from both participating chromosomes, while in adenocarcinomas centromeric material from only one of the chromosomes was present. These observations suggest that different mechanisms of centromeric instability underlie the formation of chromosomal aberrations in adenocarcinomas and squamous cell carcinomas.

We used arrays of 2069 BACs (1303 nonredundant autosomal clones) to map sequence variation among Mus spretus (SPRET/Ei and SPRET/Glasgow) and Mus musculus (C3H/HeJ, BALB/cJ, 129/J, DBA/2J, NIH, FVB/N, and C57BL/6) strains. We identified 80 clones representing 74 autosomal loci of copy number variation (|log(2)ratio| >/= 0.4). These variant loci distinguish laboratory strains. By FISH mapping, we determined that 63 BACs mapped to a single site on C57BL/6J chromosomes, while 17 clones mapped to multiple chromosomes (n = 16) or multiple sites on one chromosome (n = 1). We also show that small ratio changes (Delta log(2)ratio approximately 0.1) distinguish homozygous and heterozygous regions of the genome in interspecific backcross mice, providing an efficient method for genotyping progeny of backcrosses.