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Biomedical subjects

Lisa G Shaffer

Publications and source records attributed to Lisa G Shaffer.

At least 19 recordsLinked to original sources
PubMed2006

Detection of low-level mosaicism by array CGH in routine diagnostic specimens.

The advent of microarray-based comparative genomic hybridization (array CGH) promises to revolutionize clinical cytogenetics because of its ability to rapidly screen the genome at an unprecedented resolution. Yet, the ability of array CGH to detect and evaluate low-level mosaicism is not known. Our laboratory has analyzed over 3,600 clinical cases with the SignatureChip which we developed for the detection of microdeletions, microduplications, aneuploidy, unbalanced translocations, and subtelomeric and pericentromeric copy number alterations. Here, we report 18 cases of mosaicism detected by array CGH in a routine diagnostic setting, 14 of which were not known to us at the time of the analysis. These 14 cases represent approximately 8% of all abnormal cases identified in our laboratory. For each case, fluorescence in situ hybridization (FISH) analysis was performed on PHA-stimulated cultures after mosaic chromosome abnormalities were suspected by array CGH. In all cases, FISH confirmed the mosaic chromosome abnormalities which included a variety of marker chromosomes, autosomal trisomies, terminal and interstitial deletions, and derivative chromosomes. Interestingly, confirmatory FISH analyses on direct blood smears indicated that the percentage of abnormal cells in unstimulated cultures was in some cases different than that found in PHA-stimulated cells. We also report the detection of a previously unsuspected case of an isochromosome 12p (associated with Pallister-Killian syndrome) by array CGH using genomic DNA extracted from peripheral blood. These results support a growing body of data that suggests that stimulated peripheral blood cultures likely distort the percentage of abnormal cells and may, for some chromosome abnormalities, make their detection unlikely by conventional analysis. Thus, array CGH, which is based on genomic DNA extracted directly from uncultured peripheral blood, may be more likely to detect low-level mosaicism for unbalanced chromosome abnormalities than traditional cytogenetic techniques.

Chromosome Aberrations↗
PubMed2006

Identification of cryptic imbalance in phenotypically normal and abnormal translocation carriers.

Approximately one in 500 individuals carries a reciprocal translocation. Of the 121 monosomy 1p36 subjects ascertained by our laboratory, three independent cases involved unbalanced translocations of 1p and 9q, all of which were designated t(1;9)(p36.3;q34). These derivative chromosomes were inherited from balanced translocation carrier parents. To understand better the causes and consequences of chromosome breakage and rearrangement in the human genome, we characterized each derivative chromosome at the DNA sequence level and identified the junctions between 1p36 and 9q34. The breakpoint regions were unique in all individuals. Insertions and duplications were identified in two balanced translocation carrier parents and their unbalanced offspring. Sequence analyses revealed that the translocation breakpoints disrupted genes. This study demonstrates that apparently balanced reciprocal translocations in phenotypically normal carriers may have cryptic imbalance at the breakpoints. Because disrupted genes were identified in the phenotypically normal translocation carriers, caution should be exercised when interpreting data on phenotypically abnormal carriers with apparently balanced rearrangements that disrupt putative candidate genes.

Chromosome Breakage↗
PubMed2006

Identification of sequence motifs at the breakpoint junctions in three t(1;9)(p36.3;q34) and delineation of mechanisms involved in generating balanced translocations.

Although approximately 1 in 500 individuals carries a reciprocal translocation, little is known about the mechanisms that result in their formation. We analyzed the sequences surrounding the breakpoints in three unbalanced translocations of 1p and 9q, all of which were designated t(1;9)(p36.3;q34), to investigate the presence of sequence motifs that might mediate nonhomologous end joining (NHEJ). The breakpoint regions were unique in all individuals. Two of three translocations demonstrated insertions and duplications at the junctions, suggesting NHEJ in the formation of the rearrangements. No homology was identified in the breakpoint regions, further supporting NHEJ. We found translin motifs at the breakpoint junctions, suggesting the involvement of translin in the joining of the broken chromosome ends. We propose a model for balanced translocation formation in humans similar to transposition in bacteria, in which staggered nicks are repaired resulting in duplications and insertions at the translocation breakpoints.

Base Sequence↗
PubMed2006

Evidence for involvement of TRE-2 (USP6) oncogene, low-copy repeat and acrocentric heterochromatin in two families with chromosomal translocations.

We report clinical findings and molecular cytogenetic analyses for two patients with translocations [t(14;17)(p12;p12) and t(15;17)(p12;p13.2)], in which the chromosome 17 breakpoints map at a large low-copy repeat (LCR) and a breakage-prone TRE-2 (USP6) oncogene, respectively. In family 1, a 6-year-old girl and her 5-year-old brother were diagnosed with mental retardation, short stature, dysmorphic features, and Charcot-Marie-Tooth disease type 1A (CMT1A). G-banding chromosome analysis showed a der(14)t(14;17)(p12;p12) in both siblings, inherited from their father, a carrier of the balanced translocation. Chromosome microarray and FISH analyses revealed that the PMP22 gene was duplicated. The chromosome 17 breakpoint was mapped within an approximately 383 kb LCR17pA that is known to also be the site of several breakpoints of different chromosome aberrations including the evolutionary translocation t(4;19) in Gorilla gorilla. In family two, a patient with developmental delay, subtle dysmorphic features, ventricular enlargement with decreased periventricular white matter, mild findings of bilateral perisylvian polymicrogyria and a very small anterior commissure, a cryptic duplication including the Miller-Dieker syndrome region was identified by chromosome microarray analysis. The chromosome 17 breakpoint was mapped by FISH at the TRE-2 oncogene. Both partner chromosome breakpoints were mapped on the short arm acrocentric heterochromatin within or distal to the rRNA cluster, distal to the region commonly rearranged in Robertsonian translocations. We propose that TRE-2 together with LCR17pA, located approximately 10 Mb apart, also generated the evolutionary gorilla translocation t(4;19). Our results support previous observations that the USP6 oncogene, LCRs, and repetitive DNA sequences play a significant role in the origin of constitutional chromosome aberrations and primate genome evolution.

Charcot-Marie-Tooth Disease↗
PubMed2006

Microarray detection of a de novo der(X)t(X;11)(q28;p13) in a girl with premature ovarian failure and features of Beckwith-Wiedemann syndrome.

We report an 18-year-old girl with premature ovarian failure (POF), tall stature, and urinary incontinence. Chromosome studies including array comparative genomic hybridization showed that she was the carrier of an unbalanced de novo translocation between the X chromosome and chromosome 11, resulting in partial monosomy Xq and partial trisomy 11p. Microsatellite analysis demonstrated that the patient had paternal duplication of 11p13p15.5, which contributed to some of her features consistent with Beckwith-Wiedemann syndrome (BWS). The combined phenotype of BWS and POF suggests that the translocated portion of 11p remains active.

Adolescent↗
PubMed2006

Characterization of a complex rearrangement with interstitial deletions and inversion on human chromosome 1.

Deletion of the distal band of the short arm of chromosome 1 (monosomy 1p36) is the most common terminal deletion syndrome, occurring in about 1 in 5000 newborns. Of the 121 subjects ascertained for our study to date, 12 (9.9%) have interstitial deletions, three of which are complex rearrangements showing more than one deletion. Herein we report the characterization of a complex rearrangement with two interstitial deletions in the same chromosome 1p36.33-p36.23. We narrowed and analyzed the breakpoints and junctions between the sequence fragments involved in the rearrangement to determine the structure of this deleted chromosome 1. The analyses of the DNA sequence at the junctions showed additional complexity: an inversion and a third de-novo interstitial deletion. We reconstructed this complex rearrangement of 1p36 to understand the mechanism of formation. Analysis of the breakpoint junctions revealed that three of the four breakpoints each interrupted a gene. Alignments of the junctions showed the lack of any sequence similarity between the breakpoints, suggesting the involvement of non-homologous end joining (NHEJ) in the ligation of broken ends following deletion. The identification of translin recognition sites in the breakpoints suggests translin involvement in the repair of broken chromosomes. This report is one of the first to examine constitutional chromosomal rearrangements at the DNA sequence level. The discovery of cryptic events in seemingly simple chromosome rearrangements may provide the basis for proposing mechanisms of formation.

Child, Preschool↗
PubMed2006

Risk estimates for uniparental disomy following prenatal detection of a nonhomologous Robertsonian translocation.

Carriers of nonhomologous Robertsonian translocations (ROB) are at risk for having offspring with uniparental disomy (UPD). Although risk estimates have been calculated in several independent studies, the estimates have not been optimal because most studies are not of sufficient size and UPD events are rare. However, these collective data have provided the opportunity to derive an overall risk estimate for UPD in the fetus after the prenatal identification of a ROB.

Female↗
PubMed2006

Detecting sex chromosome anomalies and common triploidies in products of conception by array-based comparative genomic hybridization.

OBJECTIVES: In recent years, array-based comparative genomic hybridization (array CGH) has moved to the forefront of molecular cytogenetics with its ability to rapidly characterize chromosome abnormalities at resolutions much higher than routine chromosome banding. However, array CGH, like all CGH procedures, has heretofore been deemed unable to detect ploidy, a major cause of fetal demise and spontaneous miscarriage. METHOD: We recently developed a CGH microarray that is designed for detecting aneuploidy and unbalanced chromosome rearrangements. Here, we introduce the use of a Klinefelter male cell line (47,XXY) as a control for array CGH analyses on products of conception (POCs). RESULTS: This approach facilitates the detection of common trisomies and monosomies of the sex chromosomes by reducing the analysis to the identification of single copy gains or losses. Furthermore, in a blinded study, careful interpretation of the microarray results with particular attention to the sex chromosome ratios between the patient sample and the control allowed for the detection of some common triploidies. CONCLUSION: These results suggest that using a chromosomally abnormal cell line in array CGH analysis can be applied to other CGH platforms and that array CGH, when properly performed and analyzed, is a powerful tool that can detect most chromosomal abnormalities observed in a clinical setting including some polyploidies.

Aneuploidy↗
PubMed2006

Cytogenetic analysis of cardiovascular disease: karyotyping.

Numerical and structural chromosomal rearrangements, such as aneuploidies, deletions, duplications, and other aberrations have been associated with congenital abnormalities, pregnancy loss, and malignancy. Detection of these genetic changes is possible by cytogenetic analysis. The karyotype is determined by analysis of metaphase or prometaphase chromosomes of peripheral blood lymphocytes after banding procedures. This analysis plays an important role in determining patient diagnosis and care. In this chapter, we describe the basic approach of cytogenetic analysis: arresting the cell in metaphase or prometaphase, the obtaining of metaphase chromosome spreads, and staining and chromosome analysis.

Cardiovascular Diseases↗
PubMed2006

Comparative genomic hybridization by microarray for the detection of cytogenetic imbalance.

Chromosomal abnormalities often result in the improper dosage of genes in a particular chromosome or chromosome segment, which may cause specific and complex clinical phenotypes. Comparative genomic hybridization by microarray (array CGH) is a high-throughput and high-resolution method for the detection of microscopic and submicroscopic chromosome abnormalities, some of which may not be detectable by conventional cytogenetic techniques. In addition, with the human genome sequenced and publicly available, array CGH allows for the direct correlation between chromosomal anomalies and genomic sequence. Properly constructed, microarrays have the potential to be a valuable tool for the detection of chromosomal abnormalities in cancer and genetic disease.

Cell Line↗
PubMed2006

Targeted genomic microarray analysis for identification of chromosome abnormalities in 1500 consecutive clinical cases.

OBJECTIVE: To assess the yield of array-based comparative genomic hybridization. STUDY DESIGN: The results of array comparative genomic hybridization were collected on 1500 consecutive clinical cases sent to our laboratory for a variety of developmental problems. Confirmation fluorescence in situ hybridization of metaphase or interphase cells, depending on the aberration, was performed. RESULTS: Of the 1500 cases, 134 (8.9%) showed an abnormality: 36 (2.4%) showed polymorphisms or familial variants, 14 (0.9%) showed alterations of unknown clinical significance, and 84 (5.6%) showed clinically relevant genomic alterations. These included subtelomeric deletions and unbalanced rearrangements, microdeletions and reciprocal duplications, rare abnormalities, and low-level trisomy mosaicism. CONCLUSIONS: A targeted array detects a substantial proportion of abnormalities even in those patients who have already had extensive cytogenetic and/or fluorescence in situ hybridization testing. This study, although not a controlled ascertainment of subjects with specific selection criteria, accurately reflects the reality of clinical cytogenetic practice and provides an estimate of the cytogenetic abnormalities that can be identified with a targeted microarray in a diagnostic laboratory. Microarray analysis likely doubles the current yield of abnormal results detected by conventional cytogenetic analysis.

Child↗
PubMed2006

Application of array-based comparative genomic hybridization to clinical diagnostics.

Microarray-based comparative genomic hybridization (array CGH) is a revolutionary platform that was recently adopted in the clinical laboratory. This technology was first developed as a research tool for the investigation of genomic alterations in cancer. It allows for a high-resolution evaluation of DNA copy number alterations associated with chromosome abnormalities. Array CGH is based on the use of differentially labeled test and reference genomic DNA samples that are simultaneously hybridized to DNA targets arrayed on a glass slide or other solid platform. In this review, we examine the technology and its transformation from a research tool into a maturing diagnostic instrument. We also evaluate the various approaches that have shaped the current platforms that are used for clinical applications. Finally, we discuss the advantages and shortcomings of "whole-genome" arrays and compare their diagnostic use to "targeted" arrays. Depending on their design, microarrays provide distinct advantages over conventional cytogenetic analysis because they have the potential to detect the majority of microscopic and submicroscopic chromosomal abnormalities. This new platform is poised to revolutionize modern cytogenetic diagnostics and to provide clinicians with a powerful tool to use in their increasingly sophisticated diagnostic capabilities.

Chromosome Aberrations↗
PubMed2005

Allele-specific methylation of a functional CTCF binding site upstream of MEG3 in the human imprinted domain of 14q32.

The gene MEG3 is located in the imprinted human chromosomal region on 14q32. Imprinting of a structurally homologous region IGF2/H19 on 11p15 is mediated through cytosine methylation-controlled binding of the protein CTCF to target sites upstream of H19. We identified five new CTCF binding sites around the promoter of MEG3. Using an electrophoretic mobility shift assay, we showed that these sites bind CTCF in vitro. Using one of these sites, chromatin immunoprecipitation (ChIP) analysis confirmed CTCF binding in-vivo, and differential allele-specific methylation was demonstrated in seven individuals with either maternal or paternal uniparental disomy 14 (UPD14). The site was unmethylated on the maternally inherited chromosomes 14 and methylated on the paternally inherited chromosomes 14, suggesting parent-specific methylation of sequences upstream of MEG3. We speculate that this CTCF-binding region may provide a mechanism for the transcriptional regulation of MEG3 and DLK1.

Alleles↗
PubMed2005

Use of targeted array-based CGH for the clinical diagnosis of chromosomal imbalance: is less more?

Chromosome analysis is an important component to the diagnosis of congenital anomalies, developmental delay, and mental retardation. Routine chromosome analysis identifies aneuploidy and structural rearrangements greater than 5 Mb but cannot identify abnormalities of the telomeric regions or microdeletions reliably. Molecular cytogenetic techniques were developed to overcome these limitations. High-resolution comparative genomic hybridization (CGH)-based microarrays (array CGH) were developed to increase the resolution of chromosomal studies and to provide a comprehensive assay by using large-insert clones as the target for analysis. We constructed a microarray for the clinical diagnosis of medically significant and relatively common chromosomal alterations. Nine hundred six bacterial artificial chromosome (BAC) clones were chosen, the chromosomal locations of which were confirmed by fluorescence in situ hybridization (FISH). FISH-testing showed that 7% of the clones were mismapped based on map locations obtained from two publicly available databases (58 mapped to the wrong chromosome and three mapped to a different locus on the same chromosome), 16% cross-hybridized to other chromosomes, and 12% did not hybridize or showed poor hybridization signals under uniform FISH conditions. Thus, from a total of 906 BAC clones that were evaluated, only 589 (65%) were deemed adequate for arraying on this clinical device. The performance of this array was tested in a set of blinded experiments on a cohort of phenotypically normal individuals and on individuals with known chromosome abnormalities. The array identified deletion/duplication polymorphisms not seen by FISH in the phenotypically normal individuals and detected single copy dosage differences in all of the cases with known chromosomal abnormalities. All abnormalities detected by the array were confirmed by FISH with BACs from the appropriate loci. Our data demonstrate that the rigorous assessment of BACs and their use in array CGH is especially important when the microarray is used for clinical diagnosis. In addition, this study illustrates that when constructed carefully with proper attention to the quality of the BACs that are arrayed, array CGH is an effective and efficient tool for delineating chromosomal aberrations and an important adjunct to FISH and conventional cytogenetics.

Cell Line, Transformed↗
PubMed2005

Microduplication and triplication of 22q11.2: a highly variable syndrome.

22q11.2 microduplications of a 3-Mb region surrounded by low-copy repeats should be, theoretically, as frequent as the deletions of this region; however, few microduplications have been reported. We show that the phenotype of these patients with microduplications is extremely diverse, ranging from normal to behavioral abnormalities to multiple defects, only some of which are reminiscent of the 22q11.2 deletion syndrome. This diversity will make ascertainment difficult and will necessitate a rapid-screening method. We demonstrate the utility of four different screening methods. Although all the screening techniques give unique information, the efficiency of real-time polymerase chain reaction allowed the discovery of two 22q11.2 microduplications in a series of 275 females who tested negative for fragile X syndrome, thus widening the phenotypic diversity. Ascertainment of the fragile X-negative cohort was twice that of the cohort screened for the 22q11.2 deletion. We also report the first patient with a 22q11.2 triplication and show that this patient's mother carries a 22q11.2 microduplication. We strongly recommend that other family members of patients with 22q11.2 microduplications also be tested, since we found several phenotypically normal parents who were carriers of the chromosomal abnormality.

Abnormalities, Multiple↗
PubMed2005

Interstitial deletion 11(p11.12p11.2) and analphoid marker formation results in inherited Potocki-Shaffer syndrome.

We report a family with inherited Potocki-Shaffer syndrome. The phenotypically normal mother has an interstitial deletion of 11(p11.12p11.2) with neocentric marker chromosome formation. The marker chromosome contains the deleted material on 11p11.2 and is likely a ring. The patient inherited a maternal deleted chromosome 11 but not the marker chromosome, thus resulting in an unbalanced karyotype along with the phenotype of Potocki-Shaffer syndrome. The deleted region in our case-11p11.12p11.2-is a newly reported site of constitutional neocentromere formation. This is also the first report describing deletion of 11p11.12-p11.2 and neocentromere formation resulting in inherited Potocki-Shaffer syndrome.

Abnormalities, Multiple↗
PubMed2005

Acute lymphoblastic leukemia in a patient with Greig cephalopolysyndactyly and interstitial deletion of chromosome 7 del(7)(p11.2 p14) involving the GLI3 and ZNFN1A1 genes.

Greig cephalopolysyndactyly (GCPS; OMIM 175700) is an autosomal dominant condition caused by mutations of the gene GLI3, located on 7p13. To date, several cases of deletions and/or translocations involving this locus have been reported in patients with GCPS. GLI3 is a transcription factor from the GLI-Kruppel gene family that has been implicated in three distinct entities: GCPS, Pallister-Hall syndrome, and postaxial polydactyly type A. The zinc finger protein, subfamily 1, member 1 gene (ZNFN1A1; OMIM 603023), on 7p12, codes for a lymphoid-restricted zinc finger transcription factor, ZNFN1A1, also called IKAROS, that regulates lymphocyte differentiation and has been associated with the development of childhood leukemia. We present the case of a 9-year-old Latin-American boy who was referred for stem cell transplantation because of recurrent acute lymphoblastic leukemia (ALL). On evaluation, he was found to have dysmorphic features consistent with GCPS, including a prominent forehead, down-slanting palpebral fissures, 1-2-3 toe syndactyly, broad thumbs and first toes, and mild developmental delay. He had developed ALL at 5 years of age. Chromosome analysis of bone marrow and fibroblastic cells showed an interstitial deletion of chromosome arm 7p, del(7)(p11.2p14), in 74% and 44% of the cells, respectively. We performed FISH analysis with a BAC clone containing the ZNFN1A1 gene and demonstrated that it is contained in the deleted segment. To our knowledge, this is the first report of a patient with GCPS and leukemia. We hypothesize that constitutional deletion of the ZNFN1A1 gene in this patient may have resulted in an increased risk of lymphoid malignancy.

Bone Marrow Cells↗