Redefining the risks of prenatally ascertained supernumerary marker chromosomes: a collaborative study.

BACKGROUND: A marker chromosome is defined as a structurally abnormal chromosome that cannot be identified by routine cytogenetics. The risk for phenotypic abnormalities associated with a marker chromosome depends on several factors, including inheritance, mode of ascertainment, chromosomal origin, and the morphology, content, and structure of the marker. METHODS: to understand the karyotype-phenotype relationship of prenatally ascertained supernumerary de novo marker chromosomes, we combined data from prenatal cases obtained from 12 laboratories with those from studies in the literature. We were able to obtain cytogenetic and phenotypic data from 108 prenatally ascertained supernumerary de novo marker chromosomes to refine the phenotypic risk associated with these markers. Because of the growing number of cases and because more techniques are available to delineate marker morphology, we have been able to group risk estimates into subcategories, such as by marker type and whether there are ultrasound abnormalities. RESULTS: If a de novo supernumerary marker chromosome is found prenatally, our data suggest there is a 26% risk for phenotypic abnormality when there is no other information defining the marker (such as chromosomal origin or information about the existing phenotype). However, if high resolution ultrasound studies are normal, this risk reduces to 18%. CONCLUSIONS: Our findings strongly support the value of additional genetic studies for more precisely defining the risk in individual cases involving marker chromosomes.

Quantification of aortic regurgitation by real-time 3-dimensional echocardiography in a chronic animal model: computation of aortic regurgitant volume as the difference between left and right ventricular stroke volumes.

BACKGROUND: The accuracy of conventional 2-dimensional echocardiographic and Doppler techniques for the quantification of valvular regurgitation remains controversial. In this study, we examined the ability of real-time 3-dimensional (RT3D) echocardiography to quantify aortic regurgitation by computing aortic regurgitant volume as the difference between 3D echocardiographic-determined left and right ventricular stroke volumes in a chronic animal model. METHODS: Three to 6 months before the study, 6 sheep underwent surgical incision of one aortic valve cusp to create aortic regurgitation. During the subsequent open chest study session, a total of 25 different steady-state hemodynamic conditions were examined. Electromagnetic (EM) flow probes were placed around the main pulmonary artery and ascending aorta and balanced against each other to provide reference right and left ventricular stroke volume (RVSV and LVSV) data. RT3D imaging was performed by epicardial placement of a matrix array transducer on the volumetric ultrasound system, originally developed at the Duke University Center for Emerging Cardiovascular Technology. During each hemodynamic steady state, the left and right ventricles were scanned in rapid succession and digitized image loops stored for subsequent measurement of end-diastolic and end-systolic volumes. Left and right ventricular stroke volumes and aortic regurgitant volumes were then calculated and compared with reference EM-derived values. RESULTS: There was good correlation between RT3D left and right ventricular stroke volumes and reference data (r = 0.83, y = 0.94x + 2.6, SEE = 9.86 mL and r = 0.63, y = 0.8x - 1.0, SEE = 5.37 mL, respectively). The resulting correlation between 3D- and EM-derived aortic regurgitant volumes was at an intermediate level between that for LVSV and that for RVSV (r = 0.80, y = 0.88x + 7.9, SEE = 10.48 mL). RT3D tended to underestimate RVSV (mean difference -4.7 +/- 5.4 mL per beat, compared with -0.03 +/- 9.7 mL per beat for the left ventricle). There was therefore a small overestimation of aortic regurgitant volume (4.7 +/- 10.4 mL per beat). CONCLUSION: Quantification of aortic regurgitation through the computation of ventricular stroke volumes by RT3D is feasible and shows good correlation with reference flow data. This method should also be applicable to the quantification of other valvular lesions or single site intracardiac shunts where a difference between right and left ventricular cavity stroke volumes is produced.

Non-mosaic trisomy 20 presenting at 21 weeks' gestation as a thoraco-abdominal mass.

Non-mosaic trisomy 20 is rare in fetuses surviving beyond the first trimester. We report a case of a fetus with non-mosaic trisomy 20 in amniotic fluid cultures obtained during the prenatal evaluation of an unusual thoraco-abdominal mass which was found at autopsy to be pulmonary sequestration. Gross inspection and autopsy of the fetus revealed multiple anomalies.

Partial trisomy 7p defined by analysis of a complex chromosome rearrangement using a BAC clone panel.

PURPOSE: To illustrate the use of bacterial artificial chromosome (BAC) clone panels for molecular cytogenetic analysis of complex chromosome rearrangements (CCRs). METHODS: High resolution cytogenetics followed by fluorescence in situ hybridization (FISH) analysis using chromosome band-specific BAC probes, in addition to commercially available probes. RESULTS: High resolution cytogenetics in conjunction with FISH using commercially available probes proved inadequate to resolve problems in characterizing a balanced CCR in the mother of a patient who had inherited an unbalanced form of the CCR. Accurate interpretation of the CCR and the unbalanced rearrangement in the patient as trisomy 7p12.2-->p21.3 was accomplished only through use of the BAC clone panel. CONCLUSION: Use of BAC clone panels can enhance the power of FISH analysis in defining chromosome rearrangements that cannot be resolved by high resolution chromosome analysis.

Overlaying ultrasonographic images on direct vision.

The objective of this technical advance is to permit in situ visualization of ultrasonographic images so that direct hand-eye coordination can be used during invasive procedures. A method is presented that merges the visual outer surface of a patient with a simultaneous ultrasonographic scan of the patient's interior. The method combines a flat-panel monitor with a half-silvered mirror such that the image on the monitor is reflected precisely at the proper location within the patient. The ultrasonographic image is superimposed in real time on the patient, merging with the operator's hands and any invasive tools in the field of view. Instead of looking away from the patient at an ultrasonographic monitor, the operator sees through skin and underlying tissue as if it were translucent. Two working prototypes have been constructed, demonstrating independence of viewer location and requiring no special apparatus to be worn by the operator. This method could enable needles and scalpels to be manipulated with direct hand-eye coordination under ultrasonographic guidance. Invasive tools would be visible up to where they enter the skin, permitting natural visual extrapolation into the ultrasonographic slice. Biopsy needles would no longer be restricted to lie in the plane of the ultrasonographic scan but could instead intersect it. These advances could lead to increased safety, ease, and reliability in certain invasive procedures.

Real-time three-dimensional ultrasound methods for shape analysis and visualization.

Real-time three-dimensional (RT3D) ultrasound is a relatively new imaging modality that uses a special ultrasound transducer consisting of a matrix array of elements. The array electronically steers an ultrasound beam to interrogate a 3D volume of tissue. The real-time nature of RT3D ultrasound differentiates it from reconstructed 3D ultrasound, in which a conventional ultrasound transducer is moved mechanically through the third dimension. RT3D ultrasound is considerably faster than reconstructed 3D ultrasound, making it suitable for capturing continuous rapid motion such as that of the beating heart. Although RT3D ultrasound has not yet found widespread clinical use, these scanners are presently employed in more than 20 locations worldwide, primarily for cardiac research. The author helped develop the RT3D ultrasound technology as well as specialized analysis and visualization methods for the resulting data. In developing such methods, it has been necessary to consider the physical and mathematical processes by which the ultrasound data are collected. Difficulties arise because of high noise, variation in contrast and intensity between scans, ultrasound's nonrectilinear coordinate system, and the anisotropic nature of the echoes themselves. This article reviews these specific difficulties and provides solutions that are applicable to generalized analysis and visualization of RT3D ultrasound data. Some of the methods presented can also be applied to other imaging modalities with nonrectilinear coordinates.

Human PRRX1 and PRRX2 genes: cloning, expression, genomic localization, and exclusion as disease genes for Nager syndrome.

In this study, we extend our examination of the function of the Prrx1 (a.k.a Mhox, Prx1, K-2, and Pmx1) as well as Prrx2 (a.k.a. S8 and Prx2) genes by characterizing the expression of the human orthologs and their potential for causing specific human malformations. The expression pattern of PRRX2 and its close relative, PRRX1, were analyzed in human tissue by RT-PCR. Although the expression of these human genes is similar to their mouse orthologs, there are notable differences in expression. PRRX2 was detected in the human kidney and lung, whereas in mice and chickens neither of these tissues has been reported to express Prrx2. For PRRX1 the expression pattern was quite similar to other vertebrates, but the ratio of the two isoforms was reversed. To begin the search for the gene-disease connection, both genes were mapped to human chromosomes by FISH. The PRRX1 locus maps to 1q23, whereas the PRRX2 locus maps to 9q34.1. This localization, along with the recently described phenotypes of the gene-targeted Prrx1, Prrx2 and double mutant mice, enabled us to search the human disease databases for similar malformations. This examination suggested that mutations at the PRRX1 and/or PRRX2 loci could result in Nager Acrofacial Dysostosis (NAFD) syndrome. We obtained DNA samples from eight patients with NAFD, as well as two patients with Miller syndrome, and analyzed them for mutations in the PRRX1 and PRRX2 genes. The data excludes mutations in the presumed coding sequences of these genes from causing NAFD.

A cre-lox recombination system for the targeted integration of circular yeast artificial chromosomes into embryonic stem cells.

The ability to produce embryonic stem (ES) cell lines containing different yeast artificial chromosomes (YACs) integrated into the same location in the genome provides a system for comparing the bio-logical effects of YAC transgenes without the confounding influences of integration site and copy number. A targeting system was developed for the directed integration of circular YACs into mouse ES cells. The system combines Cre-lox recombination technology, specifically a positive-selection integration system, with circular YAC lipofection technology to achieve single copy targeted integration of a transgene. Three independent germline competent ES cell lines [lox-containing ES lines (designated LES)] were created that contain a '-neo-lox' cassette integrated at different sites within the ES genome. A plasmid containing YAC vector sequences and a complementary '-neo-lox' cassette was used to circularize two linear YACs containing genomic DNA from human chromosome 21. The circularized YACs were then targeted to the lox sites of the LES cell lines. Polymerase chain reaction and Southern analysis demonstrated that 21% (5 of 24) of lox-recombinants contain a full-length intact YAC. This system will make the study of YAC transgenic mice more reliable and reproducible, allowing the potential for direct comparison of different transgenes expressed from the same site within the genome.

Common trisomy mosaicism diagnosed in amniocytes involving chromosomes 13, 18, 20 and 21: karyotype-phenotype correlations.

Karyotype-phenotype correlations of common trisomy mosaicism prenatally diagnosed via amniocentesis was reviewed in 305 new cases from a collaboration of North American cytogenetic laboratories. Abnormal outcome was noted in 10/25 (40%) cases of 47,+13/46, 17/31 (54%) cases of 47,+18/46, 10/152 (6.5%) cases of 47,+20/46, and in 49/97 (50%) cases of 47,+21/46 mosaicism. Risk of abnormal outcome in pregnancies with less than 50% trisomic cells and greater than 50% trisomic cells were: 26% (4/15) versus 60% (6/10) for 47,+13/46, 52% (11/21) versus 75% (6/8) for 47,+18/46, 4.5% (6/132) versus 20% (4/20) 47,+20/46, and 45% (27/60) versus 59% (22/37) for 47,+21/46. Phenotypically normal liveborns were observed with mean trisomic cell lines of 9.3% for 47,+13/46, 8.6% for 47,+18/46, 27% for 47, +20/46, and 17% for 47,+21/46. Cytogenetic confirmation rates were 46% (6/13 cases) for 47,+13/46 mosaicism, 66% (8/12 cases) for 47, +18/46, 10% (10/97 cases) for 47,+20/46, and 44% (24/54 cases) for 47,+21/46. There were higher confirmation rates in pregnancies with abnormal versus normal outcome: 50% versus 44% for 47,+13/46 mosaicism, 100% versus 33% for 47,+18/46, 66% versus 7% for 47, +20/46, and 55% versus 40% for 47,+21/46. Repeat amniocentesis is not helpful in predicting clinical outcome. It may be considered when there is insufficient number of cells or cultures to establish a diagnosis. Fetal blood sampling may have a role in mosaic trisomy 13, 18, and 21 as the risk for abnormal outcome increases with positive confirmation: 1/5 (20%) normal cases versus 5/8 (62%) abnormal cases. High resolution ultrasound examination(s) is recommended for clinical correlation and to facilitate genetic counselling.

Real-time volumetric echocardiography: the technology and the possibilities.

The heart is a dynamic organ with complexities in its shape. As such, it places special demands on three-dimensional techniques for reconstruction. Real-time volumetric echocardiography, which is based on phased array and parallel processing principles to enhance line density within a scan volume, provides rapid image acquisition. We introduce the principle, potential clinical importance, current limitations, and future of volumetric imaging methods.

Molecular cytogenetic analysis and clinical findings in a newborn with prenatally diagnosed rec(7)dup(7q)inv(7)(p22q31.3)pat.

We report prenatal and early postnatal findings in a newborn with a partial trisomy of chromosome 7 (7q31.3-qter), arising from meiotic recombination of a paternal pericentric inversion, inv(7)(p22q31.3). The inversion breakpoints were localized and the regions of duplication and deletion were defined by fluorescence in situ hybridization (FISH) analysis using a series of locus-specific and subtelomeric probes. To our knowledge, only three cases involving a recombinant 7 with duplication of 7q have been reported, two of these being first cousins. The clinical findings in our patient included skeletal abnormalities, facial dysmorphism, dilated cerebral ventricles, microretrognathia and short neck. These findings and some aspects of the neonatal course were consistent with the phenotype previously reported for duplication of distal 7q, without associated monosomy for sequences from another chromosome.

Integration of cytogenetic with recombinational and physical maps of mouse chromosome 16.

To link the cytogenetic map for mouse chromosome 16 (Chr 16) to the more detailed recombinational and physical maps, multiple probes were mapped by fluorescence in situ hybridization (FISH). Sixteen large insert clones (YACs, BACs, and PACs) containing markers that have been assigned to the Chr 16 recombinational map were localized to a cytogenetic band or subband by high-resolution FISH. This linkage of the cytogenetic and recombinational maps provides a useful tool for assigning new probe locations and for defining breakpoints in mice with chromosomal rearrangements. A direct application of these probes is demonstrated by identifying mice trisomic for distal Chr 16 using FISH analysis of interphase nuclei.

X-linked adrenoleukodystrophy: genes, mutations, and phenotypes.

X-linked adrenoleukodystrophy (X-ALD) is a complex and perplexing neurodegenerative disorder. The metabolic abnormality, elevated levels of very long-chain fatty acids in tissues and plasma, and the biochemical defect, reduced peroxisomal very long-chain acyl-CoA synthetase (VLCS) activity, are ubiquitous features of the disease. However, clinical manifestations are highly variable with regard to time of onset, site of initial pathology and rate of progression. In addition, the abnormal gene in X-ALD is not the gene for VLCS. Rather, it encodes a peroxisomal membrane protein with homology to the ATP-binding cassette (ABC) transmembrane transporter superfamily of proteins. The X-ALD protein (ALDP) is closely related to three other peroxisomal membrane ABC proteins. In this report we summarize all known X-ALD mutations and establish the lack of an X-ALD genotype/phenotype correlation. We compare the evolutionary relationships among peroxisomal ABC proteins, demonstrate that ALDP forms homodimers with itself and heterodimers with other peroxisomal ABC proteins and present cDNA complementation studies suggesting that the peroxisomal ABC proteins have overlapping functions. We also establish that there are at least two peroxisomal VLCS activities, one that is ALDP dependent and one that is ALDP independent. Finally, we discuss variable expression of the peroxisomal ABC proteins and ALDP independent VLCS in relation to the variable clinical presentations of X-ALD.

Real-time, three-dimensional echocardiography: feasibility of dynamic right ventricular volume measurement with saline contrast.

BACKGROUND: The asymmetry and complex shape of the right ventricle have made it difficult to determine right ventricular (RV) volume with 2-dimensional echocardiography. Three-dimensional cardiac imaging improves visualization of cardiac anatomy but is also complex and time consuming. A newly developed volumetric scanning system holds promise of obviating past limitations. METHODS: Real-time, transthoracic 3-dimensional echocardiographic images of the right ventricle were obtained with a high-speed volumetric ultrasound system that uses a 16:1 parallel processing schema from a 2.5 MHz matrix phased-array scanner to interrogate an entire pyramidal volume in real time. The instrumentation was used to measure RV volume in 8 excised canine hearts; dynamic real-time 3-dimensional images were also obtained from 14 normal subjects. RESULTS: Three-dimensional images were obtained in vitro and in vivo during intravenous hand-agitated saline injection to determine RV volumes. The RV volumes by real-time 3-dimensional echocardiography are well correlated with those of drained in vitro (y = 1.26x - 9.92, r = 0.97, P <.0001, standard error of the estimate = 3.26 mL). For human subjects, the end-diastolic and end-systolic RV volumes were calculated by tracing serial cross-sectional, inclined C scans; functional data were validated by comparing the scans with conventional 2-dimensional echocardiographic indexes of left ventricular stroke volume. CONCLUSIONS: These data indicate that RV volume measurements of excised heart by real-time 3-dimensional echocardiography are accurate and that beat-to-beat RV quantitative measurement applying this imaging method is possible. The new application of real-time 3-dimensional echocardiography presents the opportunity to develop new descriptors of cardiac performance.

Cloning and characterization of a secreted frizzled-related protein that is expressed by the retinal pigment epithelium.

The Wnt/frizzled cell signaling pathway has been implicated in the determination of polarity in a number of systems, including the Drosophila retina. The vertebrate retina develops from an undifferentiated neuroepithelium into an organized and laminated structure that demonstrates a high degree of polarity at both the tissue and cellular levels. In the process of searching for molecules that are preferentially expressed by the vertebrate retinal pigment epithelium (RPE), we identified secreted frizzled-related protein 5 (SFRP5), a member of the SFRP family that appears to act by modulating Wnt signal transduction. SFRP5 is highly expressed by RPE cells, and is also expressed in the pancreas. Within the retina, the related molecule SFRP2 is expressed specifically by cells of the inner nuclear layer. Thus, photoreceptors are likely to be bathed by two opposing gradients of SFRP molecules. Consistent with SFRP5 's postulated role in modulating Wnt signaling in the retina, it inhibits the ability of Xwnt-8 mRNA to induce axis duplication in Xenopus embryos. The human SFRP5 gene consists of three coding exons and it maps to chromosome 10q24.1; human SFRP2 maps to 4q31.3. Based on the biology and complementary expression patterns of SFRP2 and SFRP5, we suggest that they may be involved in determining the polarity of photoreceptor, and perhaps other, cells in the retina.

Revised genomic organization of FBN1 and significance for regulated gene expression.

FBN1 encodes fibrillin-1, an extracellular matrix protein that is defective in Marfan syndrome. This gene is divided into 65 exons and was previously reported to be approximately 110 kb in length. The existence of 3 exons upstream of the exon containing the putative initiating methionine left open the possibility of alternative fibrillin-1 isoforms that vary at their N-termini. Detailed examination of YACs containing human FBN1 reveal that the gene is 200 kb, almost twice as large as previously thought. Characterization of the porcine FBN1 cDNA and 5' flanking sequence demonstrates extreme conservation between the pig and the human predicted proteins and argues against the possibility of alternative amino-terminal coding sequence. These data further our understanding of the regulatory requirements for gene expression and establish a framework for recombinant expression of fibrillin-1.

Assessment of regional wall motion abnormalities with real-time 3-dimensional echocardiography.

Accurate characterization of regional wall motion abnormalities requires a thorough evaluation of the entire left ventricle (LV). Although 2-dimensional echocardiography is frequently used for this purpose, the inability of tomographic techniques to record the complete endocardial surface is a limitation. Three-dimensional echocardiography, with real-time volumetric imaging, has the potential to overcome this limitation by capturing the entire volume of the LV and displaying it in a cineloop mode. The purpose of this study was to assess the feasibility of using real-time 3-dimensional (RT3D) echocardiography to detect regional wall motion abnormalities in patients with abnormal LV function and to develop a scheme for the systematic evaluation of wall motion by using the 3-dimensional data set. Twenty-six patients with high-quality 2-dimensional echo images and at least 1 regional wall motion abnormality were examined with RT3D echocardiography. For 2-dimensional echocardiography, wall motion was analyzed with a 16-segment model and graded on a 4-point scale from normal (1) to dyskinetic (4), from which a wall motion score index was calculated. Individual segments were then grouped into regions (anterior, inferoposterior, lateral, and apical) and the number of regional wall motion abnormalities was determined. The RT3D echocardiogram was recorded as a volumetric, pyramid-shaped data set that contained the entire LV. Digital images, consisting of a single cardiac cycle cineloop, were analyzed off-line with a computerized display of the apical projection. Two intersecting orthogonal apical projections were simultaneously displayed in cineloop mode, each independently tilted to optimize orientation and endocardial definition. The 2 planes were then slowly rotated about the major axis to visualize the entire LV endocardium. Wall motion was then graded in 6 equally spaced views, separated by 30 degrees, yielding 36 segments per patient. A higher percentage of segments were visualized with 2-dimensional versus RT3D echocardiography (97% vs 83%, respectively, P <.001). With the use of the 2-dimensional echocardiographic results as the standard, RT3D echocardiography detected 55 (96%) of 57 regional wall motion abnormalities. Analysis of the RT3D echocardiograms resulted in 3 false-negative and 5 false-positive findings. The total number of regional wall motion abnormalities was correctly classified by RT3D echocardiography in 19 (73%) of 26 patients. RT3D echocardiography detected 11 of 13 anterior, 19 of 20 inferoposterior, 9 of 9 lateral, and 15 of 15 apical wall motion abnormalities. An excellent correlation was found between the 2 techniques for assessment of the regional wall motion score index (r = 0.89, P <.001). This initial clinical study demonstrates the feasibility and potential advantages of RT3D echocardiography for the assessment of regional LV function. Compared with 2-dimensional echocardiography, this new method permits recording of the entire LV in a single beat, allowing the extent and location of the regional wall motion abnormalities to be determined.

Medial-node models to identify and measure objects in real-time 3-D echocardiography.

A method is proposed for the automatic, rapid, and stable identification and measurement of objects in three-dimensional (3-D) images. It is based on local shape properties derived statistically from populations of medial primitives sought throughout the image space. These shape properties are measured at medial locations within the object and include scale, orientation, endness, and medial dimensionality. Medial dimensionality is a local shape property differentiating sphere-like, cylinder-like, and slab-like structures, with intermediate dimensionality also possible. Endness is a property found at the cap of a cylinder or the edge of a slab. In terms of an application, the cardiac left ventricle (LV) during systole is modeled as a large dark cylinder with an apical cap, terminated at the other end by a thin bright slab-like mitral valve (MV). Such a model, containing medial shape properties at just a few locations, along with the relative distances and orientations between these locations, is intuitive and robust and permits automated detection of the LV axis in vivo, using real-time 3-D (RT3D) echocardiography. The statistical nature of these shape properties allows their extraction, even in the presence of noise, and permits statistical geometric measurements without exact delineation of boundaries, as demonstrated in determining the volume of balloons in RT3D scans. The inherent high speed of the method is appropriate for real-time clinical use.

Isolation and characterization of the human gene encoding Ito: further diversity by alternative mRNA splicing.

The transient outward K+ current (Ito) in the heart is responsible for the initial phase of repolarization and for setting the plateau voltage of the ventricular action potential. Recently, Kv4.3 has emerged as the leading candidate alpha-subunit gene that underlies Ito in larger mammals such as dogs and humans. We have cloned the human Kv4.3 homolog and describe a carboxyl-terminal splice variant that inserts 19 amino acids with a consensus protein kinase C (PKC) phosphorylation site into the protein after the last membrane-spanning segment. The coding region of Kv4.3 is comprised of at least five exons and is located on chromosome 1p13.3. In the basal state the basic biophysical properties of both of the splice variants are identical.

Region-specific FISH probes used to identify and characterize an interstitial paracentric inv(21)(q22.1q22.3).

Region-specific probes developed for the diagnosis of specific syndrome, can be adapted to elucidate the exact nature of certain chromosomal structural anomalies. We describe the use of FISH probes in characterizing a prenatally diagnosed chromosome rearrangement. An abnormal chromosome 21 was detected during amniocentesis for maternal age indication, and a similar appearing chromosome 21 was found in the mother. The exact nature of the rearrangement was not immediately evident from G-banded karyotypes. FISH was performed using a whole chromosome painting probe, as well as the region-specific probes D21S65 (21q21-22.1), D21S55 (21q22.3) and D21S1219/D21S1220 (21q22.3-qter) (Oncor). Results showed an interstitial paracentric inversion, with breakpoints in bands 21q22.1 and 21q22.3, which was identical in the mother and the fetus: 46,XX,?inv(21)(q).ish inv(21)(q22.1q22.3)(wcp+.D21S65 mv, D21S55 mv, D21S1219/D21S1220 st). In this case, FISH using region-specific probes was helpful in characterizing the inversion and aided in the genetic counselling of risk assessment for the family.