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PubMed2001

High-performance genetic analysis using microfabricated capillary array electrophoresis microplates.

This review focuses on some recent advances in realizing microfabricated capillary array electrophoresis (microCAE). In particular, the development of a novel rotary scanning confocal fluorescence detector has facilitated the high-speed collection of sequencing and genotyping data from radially formatted microCAE devices. The concomitant development of a convenient energy-transfer cassette labeling chemistry allows sensitive multicolor labeling of any DNA genotyping or sequencing analyte. High-performance hereditary haemochromatosis and short tandem repeat genotyping assays are demonstrated on these devices along with rapid mitochondrial DNA sequence polymorphism analysis. Progress in supporting technology such as robotic fluid dispensing and batched data analysis is also presented. The ultimate goal is to develop a parallel analysis platform capable of integrated sample preparation and automated electrophoretic analysis with a throughput 10-100 times that of current technology.

Electrophoresis, Capillary↗
PubMed2002

A family of novel DNA sequencing instruments based on single-photon detection.

We have developed a family of high-performance capillary DNA sequencing instruments based on a novel multicolor fluorescent detection technology. This technology is based on two technical innovations: the multilaser excitation of fluorescence of labeled DNA fragments and the "color-blind" single-photon detection of modulated fluorescence. Our machines employ modern digital and broadband techniques that are essential for achieving superior instrument performance. We discuss the design and testing results for several versions of the automated single lane DNA sequencers, as well as our approach to scaling up to multilane instruments.

Color↗
PubMed2002

A powerful, novel, multiplex typing system for six short tandem repeat loci and the allele frequency distributions in two Japanese regional populations.

A new multiplex system for six tetranucleotide short tandem repeat (STR) loci was devised based on multicolor dye technology. Six loci (D20S480, D6S2439, D6S1056, D9S1118, D4S2639, and D17S1290), each with high discriminating power (each unbiased estimates of expected heterozygosity, Exp. Hz, > 0.80 in a preliminary test), were selected from more than 100 tetranucleotide STRs included in a commercially available primer set. These loci were also selected so as not to link with general STRs in commercially released kits including the combined DNA index system (CODIS) 13 core STRs. The primers were newly designed in the present study, in which each amplicon size had a range of less than 200 base pairs (bp), in order to genotype from highly degraded template DNA. Using this system, we genotyped 270 Honshu (mainland)-Japanese and 187 Okinawa-Japanese. From these allele frequencies, we performed three tests, a homozygosity test, a likelihood ratio test and an exact test for Hardy-Weinberg equilibrium (HWE), and no significant deviations (p < 0.05) from HWE were observed. We also compared the allele distributions at six STRs between both populations, and they were significantly different (p < 0.05) at three loci (D6S2439, D9S1118 and D4S2639). Furthermore, the Exp. Hz and the power of discrimination (PD) at all loci in the Honshu-Japanese population were higher than 0.80 and 0.93, respectively. These statistical values for discriminating power in the Honshu-Japanese were almost the same as in the Okinawa-Japanese. This novel, multiplex polymerase chain reaction (PCR) amplification and typing system for six STR loci thus promises to be a convenient and informative new DNA profiling system in the forensic field.

Alleles↗
PubMed2001

CD4+,CD28- T cells in rheumatoid arthritis patients combine features of the innate and adaptive immune systems.

OBJECTIVE: To determine whether CD4+,CD28- T cells, which are expanded in patients with rheumatoid arthritis (RA), express receptors that typically regulate the function of natural killer (NK) cells. METHODS: Expression of the NK cell surface molecules CD158, p70, CD94, CD161, and CD8alpha on T cell subsets was determined by multicolor flow cytometric analysis of peripheral blood mononuclear cells from 36 RA patients. Expression of CD161 on tissue-infiltrating CD4 T cells was determined by 2-color immunohistochemistry analysis of synovial tissue samples. RESULTS: Killer cell-inhibitory receptors (KIR) and killer cell-activating receptors (KAR) were exclusively expressed on CD4+,CD28- T cells, with the CD158b molecule being the most frequently detected isoform. A coordinated mechanism inducing KIR/KAR expression was suggested by similarities in the expression of CD158b on CD4 and CD8 T cells. CD4+,CD28- T cells were also positive for CD8-alphaalpha homodimers, another characteristic shared with NK cells. Of the C-type lectin NK cell receptors (NK receptors), CD94 was consistently absent, but CD161 was found on a CD4 T cell population that is significantly expanded in RA patients (P = 0.01). Involvement in disease of NK receptor-expressing CD4 T cells was suggested by the presence of CD4+,CD161+ T cells in follicular microstructures typical of rheumatoid synovitis. CONCLUSION: Patients with RA have an expanded and unusual subset of CD4 T cells that infiltrates the tissue lesions and is characterized by a deficiency of CD28, the expression of CD8-alphaalpha homodimers, and the expression of several types of HLA class I-recognizing NK receptors. CD4 T cells bearing NK receptors can bridge functions of the innate and adaptive immune systems, such as responsiveness to specific antigen, rapid release of interferon-gamma, cytotoxicity, independence from classic costimulatory pathways, and integration of multiple activating and inhibitory signals to control effector functions.

Antigens, Surface↗
PubMed2002

Unique case of mosaicism involving two morphologically similar marker chromosomes of different centric origin in a patient with developmental delay.

A five-year-old Caucasian male presented with developmental delay, minor dysmorphic features, and hyperactivity. Cytogenetic analysis showed the presence of a marker chromosome in the majority of cells analyzed. Fluorescence in situ hybridization (FISH) analyses using several alpha satellite probes, including D13Z1/D21Z1, did not reveal any signal on the marker chromosome. Subsequent multicolor FISH (M-FISH) indicated the marker to be derived from chromosome 13, and FISH with a chromosome 13 paint confirmed this finding. The absence of D13Z1/D21Z1 signal on the marker suggested that it was analphoid in nature. Comparative genomic hybridization (CGH) was utilized to further characterize the region of chromosome 13 from which the marker originated, and unexpectedly revealed a gain of chromosomal material at both the centromeric regions of chromosomes 3 and 13. In view of the CGH results, extensive FISH studies with D3Z1 and D13Z1/D21Z1 were performed and revealed the presence of four cell lines comprising one normal cell line (50.5%), a cell line with a chromosome 3 derived marker (19%), a cell line containing a marker derived from chromosome 13 (20%), and a cell line with both markers (10.5%). As the two markers appeared morphologically similar by GTG banding, all 47,XY metaphases in the initial analysis were thought to comprise only a single marker. This is the first report, to our knowledge, of the presence of a chromosome 3 and a chromosome 13 marker in mosaic condition in a congenital disorder. In light of our experience, we urge caution in interpreting karyotypes with marker chromosomes. Our case illustrates the limitations of fluorescent DNA probes and sampling errors.

Child, Preschool↗
PubMed2001

Noninvasive prenatal diagnosis of chromosomal aneuploidies by isolation and analysis of fetal cells from maternal blood.

The isolation and analysis of nucleated fetal cells (NFCs) from maternal blood may represent a new approach to noninvasive prenatal diagnosis. Although promising, these techniques require highly accurate separation of NFCs from nucleated cells of maternal origin; the two major problems limiting these techniques are the relative rarity of fetal cells in maternal blood and the need to establish their fetal origin. We now report a novel procedure that has allowed accurate separation of NFCs from maternal cells. The technique reported involves direct micromanipulator isolation of histochemically identified hemoglobin F-positive nucleated cells to obtain fetal nucleated red blood cells (FNRBCs) of high yield and purity. Using this technique, followed by cell-by-cell multicolor fluorescence in situ hybridization (FISH) analysis of purified FNRBCs, we were able to detect some of the most common human aneuploidies (including Down syndrome, Klinefelter syndrome, and trisomy 13) in 33 pregnant women referred for amniocentesis. The procedure used, which can be completed in <72 hrs, produced complete concordance with the results of amniocentesis. We also confirm findings of prior studies suggesting that the number of FNRBCs in maternal circulation is remarkably higher in abnormal pregnancies than in normal pregnancies, especially in women carrying a fetus with trisomy 21.

Aneuploidy↗
PubMed2003

Autistic disorder and chromosomal mosaicism 46,XY[123]/46,XY,del(20)(pter --> p12.2)[10].

We report on a 3-year-old boy with a moderate to severe mental retardation, autistic behavior patterns, and myoclonic epilepsy of early childhood. The cytogenetic analysis of blood lymphocytes revealed a deletion of chromosome 20pter --> p12.2 occurring as mosaicism in 8% of the analyzed metaphases: 46,XY[123]/46,XY,del(20)(pter --> p12.2)[10]. The deletion was confirmed by the recently developed multicolor banding approach and additionally by region specific fluorescence in situ hybridization (FISH) probes. To the best of our knowledge, this is the first report on a patient with autistic behavior with terminal 20p deletion mosaicism reported up to present.

Abnormalities, Multiple↗
PubMed2004

Molecular cytogenetic analysis of complex chromosomal rearrangements in patients with mental retardation and congenital malformations: delineation of 7q21.11 breakpoints.

Constitutional de novo complex chromosomal rearrangements (CCRs) are a rare finding in patients with mild to severe mental retardation. CCRs pose a challenge to the clinical cytogeneticist: generally CCRs are assumed to be the cause of the observed phenotypic abnormalities, but the complex nature of these chromosomal changes often hamper the accurate delineation of the chromosomal breakpoints and the identification of possible imbalances. In a first step towards a more detailed molecular cytogenetic characterization of CCRs, we studied four de novo CCRs using multicolor fluorescent in situ hybridization (M-FISH), comparative genomic hybridization (CGH), and FISH with region specific probes. These methods allowed a more refined characterization of the breakpoints in three of the four CCRs. The occurrence of 7q breakpoints in three out of these four CCRs and in 30% of reported CCRs suggested preferential involvement of this chromosomal region in the formation of CCRs. Further analysis of these 7q breakpoints revealed a 2 Mb deletion at 7q21.11 in one patient and involvement of the same region in a cryptic insertion in a second patient. This particular region contains at least 5 candidate genes for mental retardation. The other patient had a breakpoint more proximal to this region. The present data together with these from the literature provide evidence that a region within 7q21.11 may be prone to breakage and formation of CCRs.

Child, Preschool↗
PubMed2004

Genotype/phenotype analysis in a patient with pure and complete trisomy 12p.

Reports on patients with pure and complete trisomy 12p are rare. Up to now, 12 cases have been described in the literature. Here, we report on the genotype/phenotype-correlation of a female patient with a pure trisomy 12p. Conventional cytogenetic studies on peripheral blood chromosomes as well as molecular cytogenetic (fluorescence in situ hybridization, FISH) techniques including whole chromosome painting (WCP), comparative genomic hybridization (CGH), multicolor-banding (MCB) detected a female karyotype with an abberant chromosome 12:46,XX,der(12).ish dup(12)(pter --> q24.3::p11.2 --> pter). In addition to the trisomy 12p specific clinical hallmarks, the patient showed some features of Pallister-Killian syndrome (PKS) such as sparse hair, macroglossia, and epilepsy. These findings contribute to the genotype/phenotype correlation in trisomy 12p patients.

Abnormalities, Multiple↗
PubMed2004

A de novo complex karyotype with two independent balanced translocations and a double inversion of chromosome 6 presenting with multiple congenital anomalies.

We report a 4-year-old female with a de novo complex karyotype with multiple chromosomal rearrangements and a distinctive phenotype. Her medical history is significant for having been a twin born at 35 weeks gestation, breech presentation, with feeding problems and poor growth as an infant, gastroesophageal reflux disease, peripheral pulmonic stenosis, omphalocele, high myopia, and severe mental retardation. She is small for her age with microcephaly, posteriorly sloping forehead, shallow orbits, long palpebral fissures, prominent nose, wide mouth, absent uvula, kyphosis, brachydactyly, bridged palmar crease, and hypertonia. Peripheral blood lymphocytes revealed a karyotype of 46,XX,t(1;12)(p22.3;q21.3),inv(6)(p24q23),t(7;18)(q11.2;q21.2) in all cells. Parental karyotypes and that of her twin were normal. Spectral Karyotyping (SKY) and fluorescence in situ hybridization (FISH) with whole chromosome paints for chromosomes 1, 6, 7, 12, and 18 did not reveal additional rearrangements. Prometaphase G-banding analysis suggested that the "inverted" chromosome 6 might contain a cryptic rearrangement. Although no deletion nor duplication was detected using metaphase comparative genomic hybridization (CGH), multicolor high resolution banding (mBAND) demonstrated a double inversion of chromosome 6, resulting in a final karyotype as above but including der(6)(pter --> p23::q21 --> q22.3::q21 --> p23::q22.3 --> qter).

Abnormalities, Multiple↗
PubMed2006

Delineation of a 2q deletion in a girl with dysmorphic features and epilepsy.

In recent years, the spectrum of available methods for the characterization of chromosomal aberrations has significantly increased. Micro-array technologies now allow the rapid fine mapping of small genomic imbalances. Here we used various technologies to characterize a de novo translocation t(2;15) in a girl with dysmorphic features, severe developmental delay and frequent seizures. Multiplex-FISH (M-FISH) excluded the involvement of other chromosomes than chromosomes 2 and 15. We used an oligonucleotide array containing more than 10.000 SNPs, that is, the GeneChip Mapping 10K 2.0 SNP Affymetrix array, and readily fine-mapped a deletion in chromosomal region 2q24.1 --> 2q31.1. The extent of this deletion was verified with multicolor BAC-clone hybridizations. The deletion has a size of about 13 Mb and is within a gene rich region containing about 76 genes. Interestingly, several of these genes are ion channel genes or genes involved in neuron differentiation, so that the frequently occurring seizures are probably due to loss or haploinsufficiency of one or more of these genes.

Abnormalities, Multiple↗
PubMed2006

Major feeding difficulties in the first reported case of interstitial 20q11.22-q12 microdeletion and molecular cytogenetic characterization.

We report on a 4-year-old female presenting with intrauterine growth retardation, facial dysmorphic features, major feeding difficulties with severe diarrhea and vomiting, mental retardation with abnormal behavior and hypertonia. Feeding difficulties were the most invalidating features with absent oral intake requiring persistent enteral feeding. Standard cytogenetic studies were normal, but high-resolution chromosome analyses revealed a small de novo interstitial deletion of the long arm of chromosome 20, 46,XX,del(20)(q11.21q12). The deletion was confirmed using metaphase comparative genomic hybridization (CGH) and multicolor high resolution banding (mBAND). The deletion breakpoints were characterized using FISH analyses with YACs, PACs, and BACs clones located in the deleted and adjacent regions. A 6.6-Mb deleted region between markers D20S815 (20q11.22) and D20S435 (20q12) could be delineated. None of the nine previously reported cases with interstitial 20q deletion found in the literature involve the same breakpoints. This report further emphasizes the indication of high-resolution chromosome analyses in children with syndromic mental retardation. The description of additional cases would be useful in order to better characterize the phenotype of patients with proximal interstitial 20q deletion.

Abnormalities, Multiple↗
PubMed2006

A report of pure 7p duplication syndrome and review of the literature.

We report on a case of a 9-month-old female infant with a direct duplication of the 7p13-p22.1 chromosome region diagnosed by combining conventional cytogenetic, FISH, and multicolor banding (MCB) studies. Traditional G-banding detected a partial 7p duplication, which was further demonstrated to be entirely of chromosome 7 origin by using a whole chromosome paint for chromosome 7, and derived from 7p13-p22.1 by MCB. The infant presented with characteristic dysmorphic features, psychomotor retardation, and generalized hypotonia. The phenotypic manifestations of partial 7p trisomy with or without other chromosome involvement are briefly reviewed. Our observations in combination with other cases confirm that 7p trisomy due to dir dup(7p) can be regarded as a defined chromosome syndrome.

Chromosome Aberrations↗
PubMed2003

Advances in molecular cytogenetics for the evaluation of mental retardation.

Recent years have witnessed rapid advances in molecular cytogenetics and its impact in studying mental retardation (MR). We review new molecular cytogenetic methods, including interphase fluorescence in situ hyrbridization (FISH), comparative genomic hybridization (CGH), multicolor karyotyping, telomere FISH, primed in situ labeling (PRINS), genotyping, microdissection, and microarray for the evaluation of MR. These new methods are very useful in two major aspects: further characterization of chromosome abnormalities as detected with routine banding analysis, including additions, duplications, deletions, translocations, markers, or complex aberrations; and screening for "hidden" chromosome aberrations in patients with an apparently normal karyotype. These new methods have great diagnostic potential in prenatal, postnatal, and preimplantational settings. Although powerful, at this point, they are primarily research tools in nature. It is essential that these new methods be used in conjunction with standard methods in order to maximize obtainable information for better management of patients with MR.

Chromosome Aberrations↗
PubMed2002

Suppression of autoimmune arthritis in interleukin-1-deficient mice in which T cell activation is impaired due to low levels of CD40 ligand and OX40 expression on T cells.

OBJECTIVE: To elucidate the roles of interleukin-1 (IL-1) in the development of 2 etiologically different rheumatoid arthritis (RA) models: the type II collagen (CII)-induced arthritis (CIA) model and the human T cell leukemia virus type I transgenic (HTLV-I Tg) mouse model. METHODS: For the CIA model, DBA/1J-background IL-1alpha-/-, IL-1beta-/-, IL-1alpha/beta-/-, and wild-type littermate mice were immunized with CII. For the HTLV-I Tg model, BALB/c IL-1beta-/- or IL-1alpha/beta-/- mice were crossed with HTLV-I Tg mice. The effects of IL-1 deficiency were assessed as follows: Development of arthritis was assessed both macroscopically and microscopically. Serum antibody titer was measured by enzyme-linked immunosorbent assay. Proliferative response of lymph node cells was assayed by measurement of (3)H-thymidine incorporation. Expression of T cell surface molecule CD40 ligand (CD40L) and OX40 was determined by multicolor flow cytometric analysis. RESULTS: The development of arthritis was markedly suppressed in IL-1alpha/beta-/- mice in both models, although the effect was less prominent in HTLV-I Tg mice. Deficiency of only IL-1alpha or only IL-1beta was also associated with disease suppression. Antibody production after immunization with CII was normal in IL-1alpha/beta-/- mice, while autoantibody production was suppressed in IL-1alpha/beta-/- HTLV-I Tg mice. In IL-1alpha/beta-/- mice, the T cell proliferative response against CII was greatly reduced in both the CIA and the HTLV-I Tg models, suggesting inefficiency of T cell activation. Furthermore, expression of CD40L and OX40 on T cells was greatly reduced in IL-1alpha/beta-/- mice. CONCLUSION: These observations suggest that T cell activation by IL-1 is important for the development of autoimmunity and arthritis in these mice.

Animals↗
PubMed2005

Tissue trafficking patterns of effector memory CD4+ T cells in rheumatoid arthritis.

OBJECTIVE: Clonal populations of CD4+,CD28- T cells accumulating in rheumatoid arthritis functionally resemble end-differentiated, nondividing, short-lived effector memory cells that reside in peripheral tissues. We undertook this study to examine the tissue niche for CD4+,CD28- T cells and the signals regulating their survival and tissue homing patterns. METHODS: Chemokine receptor expression on CD4+,CD28- T cell clones and peripheral blood lymphocytes was assessed by multicolor cytometry. In vitro chemotaxis and transendothelial migration were examined in a Transwell system. In vivo tissue-homing patterns were established by adoptively transferring fluorescence-labeled T cell clones into SCID mice engrafted either with rheumatoid synovium or with human lymph nodes. RESULTS: CD4+,CD28- T cell clones adoptively transferred into human tissue-SCID mouse chimeras infiltrated rheumatoid synovium but preferentially homed to lymph nodes. Such T cells coexpressed the chemokine receptors CCR7, CCR5, and CXCR4 and migrated in response to both inflammatory chemokines (CCL5) and homing chemokines (CXCL12). T cell receptor crosslinking abrogated chemotactic responsiveness. In contrast, interleukin-12 stimulation induced the up-regulation of CCR5 and a shift in the in vivo homing pattern away from the lymph nodes toward the inflamed synovium. CONCLUSION: CD4+,CD28- T cells resemble both short-lived effector memory cells and long-lived central memory cells, and they find a niche both in inflamed synovium and in lymph nodes. Nonspecific cytokine stimulation, not antigen recognition, triggers the transition from the lymph node to the synovium. By maintaining CCR7 expression, these end-differentiated T cells can home to lymphoid organs, enhance their survival, support clonal expansion, and perpetuate autoreactivity.

Adoptive Transfer↗
PubMed2006

Immunoregulatory effects of allogeneic mixed chimerism induced by nonmyeloablative bone marrow transplantation on chronic inflammatory arthritis and autoimmunity in interleukin-1 receptor antagonist-deficient mice.

OBJECTIVE: To investigate the immunoregulatory effects of allogeneic mixed chimerism induced by T cell-depleted, nonmyeloablative bone marrow transplantation (BMT) on chronic inflammatory arthritis and autoimmunity in mice deficient in interleukin-1 receptor antagonist (IL-1Ra). METHODS: IL-1Ra(-/-) mice (H-2K(d)) were treated with antibody to asialoganglioside G(M1) (anti-natural killer cell), total body irradiation (500 cGy), and T cell-depleted, nonmyeloablative BMT derived from C57BL/6 mice (H-2K(b)). Engraftment and chimerism were evaluated in peripheral blood, lymph nodes, and spleen by multicolor flow cytometry. The severity of arthritis was evaluated by clinical scoring and histopathologic assessment. Levels of IgG1 and IgG2a subtypes of anti-type II collagen (anti-CII) antibodies were measured in serum samples. After T cells were stimulated with CII, ovalbumin, and phytohemagglutinin, T cell proliferative responses and levels of cytokine production (interferon-gamma [IFNgamma], tumor necrosis factor alpha [TNFalpha], interleukin-10 [IL-10], and IL-17) were assayed in culture supernatants. RESULTS: All IL-1Ra(-/-) mice receiving BMT showed marked improvement in arthritis within 3 weeks, as well as successful induction of mixed chimerism. These mice showed higher levels of IgG1, and lower levels of IgG2a anti-CII antibodies and weaker T cell proliferative responses than did mice in the control groups (either no treatment or conditioning alone without bone marrow rescue). In mixed chimeras, the levels of IFNgamma, TNFalpha, and IL-17 produced from CII-stimulated T cells were significantly suppressed and IL-10 production was significantly higher as compared with controls. CONCLUSION: The introduction of allogeneic mixed chimerism showed a strong immunoregulatory potential to correct established chronic inflammatory arthritis and autoimmunity originating from a dysregulated proinflammatory cytokine network.

Animals↗
PubMed1985

Color matching throughout the work week: an industrial application of the swallowtail-difference equation.

This article considers a multilevel system problem involving color perception of individual human beings, man-machine interactions, and changing social factors. Previous research on the same system found critical differences in color matching performance over a 24-hour period, modeled by a cusp catastrophe. The hypothesis now tested is whether critical differences in group performance occur in a regular weekly cycle. Subjects were 13 color matchers and 30 printers who worked rotating shifts. Data were drawn from production records for 27 pairs of multicolor jobs performed over three four-week periods. The swallowtail hypothesis was upheld for three criteria: color matching time (R2 = .55), printing press time (R2 = .55), and printing paper conserved or wasted (R2 = .83). Job length (difficulty), time period (denoting organizational dynamics taking place), and whether the job pair occurred in the Monday-Wednesday, or Thursday-Saturday part of the week (semicycle) served as control variables.

Color Perception↗