Peter Papenhausen

UPD detection using homozygosity profiling with a SNP genotyping microarray.

Single nucleotide polymorphism (SNP) based chromosome microarrays provide both a high‐density whole genome analysis of copy number and genotype. In the past 21 months we have analyzed over 13,000 samples primarily referred for developmental delay using the Affymetrix SNP/CN 6.0 version array platform. In addition to copy number, we have focused on the relative distribution of allele homozygosity (HZ) throughout the genome to confirm a strong association of uniparental disomy (UPD) with regions of isoallelism found in most confirmed cases of UPD. We sought to determine whether a long contiguous stretch of HZ (LCSH) greater than a threshold value found only in a single chromosome would correlate with UPD of that chromosome. Nine confirmed UPD cases were retrospectively analyzed with the array in the study, each showing the anticipated LCSH with the smallest 13.5 Mb in length. This length is well above the average longest run of HZ in a set of control patients and was then set as the prospective threshold for reporting possible UPD correlation. Ninety‐two cases qualified at that threshold, 46 of those had molecular UPD testing and 29 were positive. Including retrospective cases, 16 showed complete HZ across the chromosome, consistent with total isoUPD. The average size LCSH in the 19 cases that were not completely HZ was 46.3 Mb with a range of 13.5–127.8 Mb. Three patients showed only segmental UPD. Both the size and location of the LCSH are relevant to correlation with UPD. Further studies will continue to delineate an optimal threshold for LCSH/UPD correlation.

Burkitt lymphoma arising in organ transplant recipients: a clinicopathologic study of five cases.

We report five cases of Burkitt lymphoma arising in organ transplant recipients. There were four men and one woman with a mean age of 35 years. All were solid organ recipients with three renal, one liver, and one double lung transplantation. The time interval between organ transplantation and lymphoma averaged 4.5 years. Patients typically presented with high-stage disease with generalized lymphadenopathy and bone marrow involvement. Histology showed classic Burkitt lymphoma or atypical variant/Burkitt-like morphology. C-MYC rearrangement, including three cases with immunoglobulin heavy chain and two cases with lambda light chain, and Epstein-Barr virus were detected in all the cases. Additional chromosomal abnormalities were present in two of three cases and p53 mutation was found in one of three cases. Aberrant genotype and phenotype were frequently encountered, including minor monoclonal or oligoclonal T-cell populations and undetectable surface immunoglobulin light chain expression. Four patients received antilymphoma regimens, with combination chemotherapy (three patients) and/or Rituximab (three patients), in addition to reduction of immunosuppression. All four patients achieved complete remission. We conclude that posttransplant Burkitt lymphoma represents a characteristic clinicopathologic entity and occurs later after transplantation. Genotypic and phenotypic aberrations are often present. Rituximab may be an effective alternative to conventional combination chemotherapy in the treatment of a posttransplant Burkitt lymphoma.

Prenatal molecular cytogenetic diagnosis of partial tetrasomy 10p due to neocentromere formation in an inversion duplication analphoid marker chromosome

Neocentromeres are fully functional centromeres found on rearranged or marker chromosomes that have separated from endogenous centromeres. Neocentromeres often result in partial tri- or tetrasomy because their formation confers mitotic stability to acentric chromosome fragments that would normally be lost. We describe the prenatal identification and characterization of a de novo supernumerary marker chromosome (SMC) containing a neocentromere in a 20-wk fetus by the combined use of comparative genomic hybridization (CGH) and fluorescence in situ hybridization (FISH). GTG-banding of fetal metaphases revealed a 47,XY,+mar karyotype in 100% of cultured amniocytes; parental karyotypes were both normal. Although sequential tricolor FISH using chromosome-specific painting probes identified a chromosome 10 origin of the marker, a complete panel of chromosome-specific centromeric satellite DNA probes failed to hybridize to any portion of the marker. The presence of a neocentromere on the marker chromosome was confirmed by the absence of hybridization of an all-human-centromere alpha-satellite DNA probe, which hybridizes to all normal centromeres, and the presence of centromere protein (CENP)-C, which is associated specifically with active kinetochores. Based on CGH analysis and FISH with a chromosome 10p subtelomeric probe, the marker was found to be an inversion duplication of the distal portion of chromosome 10p. Thus, the proband’s karyotype was 47,XY,+inv dup(10)(pter→p14∼15::p14∼15→neo→pter), which is the first report of partial tetrasomy 10p resulting from an analphoid marker chromosome with a neocentromere. This study illustrates the use of several molecular strategies in distinguishing centric alphoid markers from neocentric analphoid markers.

Severe obesity and diabetes insipidus in a patient with PCSK1 deficiency.

Non-synonymous mutations affecting both alleles of PCSK1 (proprotein convertase 1/3) are associated with obesity and impaired prohormone processing. We report a proband who was compound heterozygous for a maternally inherited frameshift mutation and a paternally inherited 474kb deletion that encompasses PCSK1, representing a novel genetic mechanism underlying this phenotype. Although pro-vasopressin is not a known physiological substrate of PCSK1, the development of central diabetes insipidus in this proband suggests that PCSK1 deficiency can be associated with impaired osmoregulation.

Formation of novel CENP-A domains on tandem repetitive DNA and across chromosome breakpoints on human chromosome 8q21 neocentromeres

Endogenous human centromeres form on megabase-sized arrays of tandemly repeated alpha satellite DNA. Human neocentromeres form epigenetically at ectopic sites devoid of alpha satellite DNA and permit analysis of centromeric DNA and chromatin organization. In this study, we present molecular cytogenetic and CENP-A chromatin immunoprecipitation (ChIP) on CHIP analyses of two neocentromeres that have formed in chromosome band 8q21 each with a unique DNA and CENP-A chromatin configuration. The first neocentromere was found on a neodicentric chromosome 8 with an inactivated endogenous centromere, where the centromeric activity and CENP-A domain were repositioned to band 8q21 on a large tandemly repeated DNA. This is the first example of a neocentromere forming on repetitive DNA, as all other mapped neocentromeres have formed on single copy DNA. Quantitative fluorescent in situ hybridization (FISH) analysis showed a 60% reduction in the alpha satellite array size at the inactive centromere compared to the active centromere on the normal chromosome 8. This neodicentric chromosome may provide insight into centromere inactivation and the role of tandem DNA in centromere structure. The second neocentromere was found on a neocentric ring chromosome that contained the 8q21 tandemly repeated DNA, although the neocentromere was localized to a different genomic region. Interestingly, this neocentromere is composed of two distinct CENP-A domains in bands 8q21 and 8q24, which are brought into closer proximity on the ring chromosome. This neocentromere suggests that chromosomal rearrangement and DNA breakage may be involved in neocentromere formation. These novel examples provide insight into the formation and structure of human neocentromeres.

CYTOGENETIC, MORPHOLOGIC AND ONCOGENE ANALYSIS OF A CELL LINE DERIVED FROM A HETEROLOGOUS MIXED MULLERIAN TUMOR OF THE OVARY

SummaryA cell line was established from a mixed mullerian tumor of the ovary and designated LN1. Histopathologic analysis of the fresh tumor specimen demonstrated a highly aneuploid heterologous tumor comprised of undifferentiated mesodermal components with carcinomatous cells present as a smaller population. Long-term in vitro culture resulted in the establishment of a cell line that exhibits an epithelial-like morphology and expresses epithelial antigens cytokeratin, epithelial membrane antigen, and carcinoma antigen TAG-72. These cells also express mesenchymal intermediate filaments, vimentin, and desmin. Karyotypic analysis revealed a basic triploid pattern with multiple chromosomal abnormalities, most notably an isochromosome of the short arm of five present in three copies. Analysis of oncogene expression revealed that LN1 cells constitutively express mRNA for c-ras, c-erbB2, and p53. The expression of mRNA for cellular oncogenes correlated with the presence of corresponding oncoproteins, p21H-ras, p21K-ras, and p185erbB2 and mutant p53 protein. In summary, coexpression of epithelial and mesenchymal antigens by LN1 cells lends support to the hypothesis that epithelial and mesenchymal elements comprising mixed mullerian tumors of the ovary are derived from a common stem cell precursor. Furthermore, this cell line represents a functional in vitro model to evaluate the biologic activities of these unusual and highly aggressive ovarian malignancies.

Novel tandem triplication of 1q in a patient with a myelodysplastic syndrome

This is a report of a patient with a myelodysplastic syndrome characterized by symptomatic neutropenia whose bone marrow aspirates have consistently demonstrated an unusual cytogenetic anomaly. The abnormality present in all metaphases consisted of a tandem triplication of a portion of the long arm of chromosome #1, resulting in tetrasomy of a section of this chromosome ( 1q21 -32). Duplications of this portion of chromosome #1 were observed as a nonrandom event in various malignant states. In addition, these precise breakpoints can be increasingly correlated with tandem duplications.

DNA Methylation Profiling of Uniparental Disomy Subjects Provides a Map of Parental Epigenetic Bias in the Human Genome

Genomic imprinting is a mechanism in which gene expression varies depending on parental origin. Imprinting occurs through differential epigenetic marks on the two parental alleles, with most imprinted loci marked by the presence of differentially methylated regions (DMRs). To identify sites of parental epigenetic bias, here we have profiled DNA methylation patterns in a cohort of 57 individuals with uniparental disomy (UPD) for 19 different chromosomes, defining imprinted DMRs as sites where the maternal and paternal methylation levels diverge significantly from the biparental mean. Using this approach we identified 77 DMRs, including nearly all those described in previous studies, in addition to 34 DMRs not previously reported. These include a DMR at TUBGCP5 within the recurrent 15q11.2 microdeletion region, suggesting potential parent-of-origin effects associated with this genomic disorder. We also observed a modest parental bias in DNA methylation levels at every CpG analyzed across ∼1.9 Mb of the 15q11-q13 Prader-Willi/Angelman syndrome region, demonstrating that the influence of imprinting is not limited to individual regulatory elements such as CpG islands, but can extend across entire chromosomal domains. Using RNA-seq data, we detected signatures consistent with imprinted expression associated with nine novel DMRs. Finally, using a population sample of 4,004 blood methylomes, we define patterns of epigenetic variation at DMRs, identifying rare individuals with global gain or loss of methylation across multiple imprinted loci. Our data provide a detailed map of parental epigenetic bias in the human genome, providing insights into potential parent-of-origin effects.

Clinical comparison of overlapping deletions of 19p13.3.

We present three patients with overlapping interstitial deletions of 19p13.3 identified by high resolution SNP microarray analysis. All three had a similar phenotype characterized by intellectual disability or developmental delay, structural heart abnormalities, large head relative to height and weight or macrocephaly, and minor facial anomalies. Deletion sizes ranged from 792 Kb to 1.0 Mb and included a common region arr [hg19] 19p13.3 (3,814,392–4,136,989), containing eight genes: ZFR2, ATCAY, NMRK2, DAPK3, EEF2, PIAS4, ZBTB7A, MAP2K2, and two non‐coding RNAs MIR637 and SNORDU37. The patient phenotypes were compared with three previous single patient reports with similar interstitial 19p13.3 deletions and six additional patients from the DECIPHER and ISCA databases to determine if a common haploinsufficient phenotype for the region can be established.

Three cases of isolated terminal deletion of chromosome 8p without heart defects presenting with a mild phenotype.

Individuals with isolated terminal deletions of 8p have been well described in the literature, however, molecular characterization, particularly by microarray, of the deletion in most instances is lacking. The phenotype of such individuals falls primarily into two categories: those with cardiac defects, and those without. The architecture of 8p has been demonstrated to contain two inversely oriented segmental duplications at 8p23.1, flanking the gene, GATA4. Haploinsufficiency of this gene has been implicated in cardiac defects seen in numerous individuals with terminal 8p deletion. Current microarray technologies allow for the precise elucidation of the size and gene content of the deleted region. We present three individuals with isolated terminal deletion of 8p distal to the segmental duplication telomeric to GATA4. These individuals present with a relatively mild and nonspecific phenotype including mildly dysmorphic features, developmental delay, speech delay, and early behavior issues.