Genotype analysis of the NF1 gene in the French Canadians from the Québec population.

We genotyped 19 NF1 families from the French Canadians of the Québec population with six intragenic polymorphic markers including 2 RFLPs (EcoRI and RsaI) and 4 microsatellites (IVS26-2.3, IVS27AC28.4, IVS27AC33.1, and IVS38GT53.0). Genotype analysis indicated families 7610 and 7473 bear deletions. In Family 7610 the deletion removed the entire NF1 gene except exons 1 to 4b. The breakpoint of the deletion is located between exons 4a and 4b. The deletion 7473 was derived from the maternal chromosome and exons 1 to 5 were deleted. The breakpoint of the deletion is located between exons 7 and 13. Their phenotypes are reported. The allele frequencies of microsatellites IVS27AC28.4 and IVS38GT53.0 are compared to previously reported data from Caucasians, including Spanish and Italians. The difference is statistically significant (P < 0.0036) for marker IVS27AC28.4 between the Québec French Canadian and the Italian population.

Identification and characterization of four novel large deletions in the human neurofibromatosis type 1 (NF1) gene.

We studied 20 unrelated NF1 patients by Southern blots with seven cDNA probes and loss of heterozygosity (LOH) analysis with four intragenic microsatellites (IVS26-2.3, IVS27AC28.4, IVS27AC33.1, and IVS38GT53.0). Four novel large deletions (178, 184, 236, and 237) have been identified and characterized. The breakpoint of deletion 178 was located in between exons 23-2 and 27b and the sequences downstream of the breakpoint were deleted. For deletion 184, the breakpoint was in between exons 27b and 29, and the region upstream of the breakpoint was deleted. With deletion 236, the breakpoint was in between exons 14 and 18 and the region downstream of the breakpoint was deleted. The breakpoint of deletion 237 was in between exons 38 and 45 and the sequences upstream of the breakpoint were deleted. These deletions were distributed randomly across the NF1 gene and no deletion hot spot was found. Our study suggests that the combination of analyses of loss of heterozygosity, southern blotting and southern blot densitometry can be used as a powerful method to detect large deletions, especially when family record is not available or the patient is a sporadic case.

Myocardial tracking, a new method to calculate ejection fraction with gated SPECT: validation with (201)Tl versus planar angiography.

UNLABELLED: Left ventricular ejection fraction (LVEF) and viability are essential variables for the prognosis of myocardial infarction and can be measured simultaneously by (201)Tl gated SPECT; however, most algorithms tend to underestimate LVEF. This study aimed to evaluate a new myocardial tracking algorithm, MyoTrack (MTK), for automatic LVEF calculation. METHODS: A rest/redistribution (20 min/4 h) (201)Tl gated SPECT protocol followed immediately by a (99m)Tc equilibrium radionuclide angiography (ERNA) was performed in 75 patients with history of myocardial infarction. Quality of myocardial uptake was evaluated from count statistics and automatic quantification of defect sizes and severities (CardioMatch). LVEFs were calculated both with Germano's quantitative gated SPECT (QGS) algorithm and with MTK. Briefly, the originality of this algorithm resides in the unique end-diastole segmentation, matching to a template and motion field tracking throughout the cardiac cycle. RESULTS: ERNA LVEF averaged 33% +/- 14%. QGS significantly underestimated this value at 20 min (30% +/- 13%, P < 0.001) and at 4 h (30% +/- 13%, P < 0.0001). By contrast, MTK did not miscalculate LVEF at 20 min (34% +/- 14%, probability value was not significant) though a similar underestimation occurred at 4 h (31% +/- 13%, P < 0.02). Individual differences between early and late gated SPECT values and differences between gated SPECT and ERNA values did not correlate with the extension of perfusion defects, count statistics, or heart rate. CONCLUSION: MTK algorithm accurately calculates LVEF on early/high-count images compared with ERNA [corrected], even in patients with severe perfusion defects, but tends to underestimate LVEF on delayed/low-contrast images, as other algorithms do.

A novel mutation in the neurofibromatosis type 1 (NF1) gene promotes skipping of two exons by preventing exon definition.

Using a protein truncation assay, we have identified a new mutation in the neurofibromatosis type 1 (NF1) gene that causes a severe defect in NF1 pre-mRNA splicing. The mutation, which consists of a G to A transition at position +1 of the 5' splice site of exon 12a, is associated with the loss of both exons 11 and 12a in the NF1 mRNA. Through the use of in vivo and in vitro splicing assays, we show that the mutation inactivates the 5' splice site of exon 12a, and prevents the definition of exon 12a, a process that is normally required to stimulate the weak 3' splice site of exon 12a. Because the 5' splice site mutation weakens the interaction of splicing factors with the 3' splice site of exon 12a, we propose that exon 11/exon 12a splicing is also compromised, leading to the exclusion of both exons 11 and 12a. Our results provide in vivo support for the importance of the exon definition model during NF1 splicing, and suggest that the NF1 region containing exons 11 and 12a plays an important role in the activity of neurofibromin.

Assessment of ejection fraction with Tl-201 gated SPECT in myocardial infarction: Precision in a rest-redistribution study and accuracy versus planar angiography.

BACKGROUND: Viability and left ventricular ejection fraction (LVEF) are essential measures for the assessment of myocardial infarction (MI). These 2 variables may be evaluated simultaneously by means of thallium-201 gated single photon emission computed tomography (SPECT); however, the precision and accuracy of LVEF measurements with this isotope remain controversial, particularly in cases of extended perfusion defects and poor count densities. METHODS AND RESULTS: Fifty patients with a history of MI underwent a 20-minute rest and a 4-hour redistribution Tl-201 gated SPECT viability protocol, immediately followed by a technetium-99m planar equilibrium radionuclide angiography (ERNA). On gated SPECT images, various count statistics were calculated, and perfusion was automatically quantified by means of CardioMatch, which provided both the size and severity of MI defects. Rest and redistribution LVEFs were determined from gated SPECT with Germano's algorithm, whereas LVEFs were calculated from ERNA using the manufacturer's software. Mean LVEF values calculated with rest gated SPECT, redistribution gated SPECT, and planar ERNA were 30% +/- 13%, 30% +/- 13% and 33% +/- 13%, respectively. Significant differences between repeated gated SPECT LVEFs were not shown by means of the paired t test. Correlation coefficients were high between 20-minute and 4-hour scans (r = 0.89) and between gated SPECT and ERNA (r = 0.88 and r = 0.92 at 20 minutes and 4 hours, respectively). Additionally, close agreement between gated SPECT and ERNA was shown by means of the Bland-Altman plot, despite an underestimation of 3 units. Finally, neither the technical conditions (count density, heart rate, lung uptake, etc) nor the perfusion alteration (size, severity, redistribution) appeared to interfere with the precision and accuracy of gated SPECT LVEF measurement. CONCLUSION: Tl-201 gated SPECT is a precise method for assessing LVEF within the same patient at 4-hour intervals, even with a substantial count decay, and it gives accurate results compared with planar ERNA, even in the case of large perfusion defects.

Statistical analysis of normal and abnormal dissymmetry in volumetric medical images.

We present a general method to study the dissymmetry of anatomical structures such as those found in the human brain. Our method relies on the estimate of 3D dissymmetry fields, the use of 3D vector field operators, and T2 statistics to compute significance maps. We also present a fully automated implementation of this method which relies mainly on the intensive use of a 3D non-rigid inter-patient matching tool. Such a tool is applied successively between the images and their symmetric versions with respect to an arbitrary plane, both to realign the images with respect to the mid-plane of the subject and to compute a dense 3D dissymmetry map. Inter-patient matching is also used to fuse the data of a population of subjects. We then describe three main application fields: the study of the normal dissymmetry within a given population, the comparison of the dissymmetry between two populations, and the detection of the significant abnormal dissymmetries of a patient with respect to a reference population. Finally, we present preliminary results illustrating these three applications for the case of the human brain.

Automatic detection of hippocampal atrophy on magnetic resonance images.

An automatic method for identifying hippocampal atrophy on magnetic resonance (MR) images obtained from patients with clinical evidence of temporal lobe epilepsy (TLE) is described. The method is based on the analysis of image intensity differences between patients and controls within a volume of interest (VOI) centred on the hippocampus. The core of the method is a fully automatic signal intensity-based inter-subject image registration technique. In particular, a global affine registration to a reference image is performed, followed by a local affine registration within the VOI. A mask produced by manual segmentation of the mean hippocampus for 30 control subjects enabled investigations to be restricted to a specified region of the VOI approximately corresponding to the hippocampus. Normal variations of hippocampal signal intensity were computed from images obtained for the 30 control subjects. The manual method of hippocampal volumetry, currently an important component of the pre-surgical evaluation of patients with clinical evidence of medically intractable TLE, is used to determine the lower 1st percentile limits of normal hippocampal volume. Hippocampi with volumes below this limit are defined as atrophic. We investigated whether the automatic method can correctly distinguish between 15 patients with significant hippocampal atrophy according to absolute volumes and a further 14 controls. ROC curves enabled evaluation of sensitivity and specificity in respect of an intensity threshold. 100% specificity is required when determining suitability of patients for neurosurgery, resulting in levels of 50% and 70% sensitivity in detecting atrophy in the right and left hippocampus, respectively. We propose that the method can be developed as an automatic screening procedure.

Factors affecting and computation of myocardial perfusion reference images.

Many quantitative analysis methods for myocardial perfusion studies require as a central step a comparison with a 'normal' or average density distribution map or reference image. It has been recognized, however, that the normal distribution can be affected by patient attributes, including sex and weight or body habitus, and by acquisition attributes, including the choice of tracer and the position of the patient during imaging. Some authors have proposed separate reference images for the sexes and the tracer. This approach fails if a large number of binary attributes have to be considered, since one would need 2" reference images for each attribute. The problem is compounded when continuous attributes (e.g. age and weight) are included, especially if the approach is to average separate homogeneous groups for each attribute. We propose to create case-specific reference images for the interpretation of myocardial perfusion studies by creating a model based on the influence of each attribute. From a non-homogeneous population of normal cases, or cases presumed to be normal on the basis of the Diamond and Forrester stratification, the effect of patient and study attributes on the density distribution in the stress image and the density differences between rest and stress images were computed. The effects are computed by multi-linear regression, to account for cross-correlation. Significance is assigned on the basis of a partial Fisher test. The data are myocardial perfusion images matched in 3D to a template by an elastic transformation. Even though there was some cross-correlation in the data, we were able to show independent effects of sex, position (prone or supine), age, weight, tracer combination and stress method (exercise, persantine and adenosine). Taken as a whole, the multi-linear regression demonstrated a significant effect in 72% of the pixels within the myocardial volume. In addition, the distribution predicted by the model was equivalent to average images from homogeneous matched groups. In conclusion, our approach makes it possible to produce case-specific reference images without the need for multiple homogeneous large groups to produce averages for each possible patient or study attribute.

Deformation analysis to detect and quantify active lesions in three-dimensional medical image sequences.

Evaluating precisely the temporal variations of lesion volumes is very important for at least three types of practical applications: pharmaceutical trials, decision making for drug treatment or surgery, and patient follow-up. In this paper we present a volumetric analysis technique, combining precise rigid registration of three-dimensional (3-D) (volumetric) medical images, nonrigid deformation computation, and flow-field analysis. Our analysis technique has two outcomes: the detection of evolving lesions and the quantitative measurement of volume variations. The originality of our approach is that no precise segmentation of the lesion is needed but the approximative designation of a region of interest (ROI) which can be automated. We distinguish between tissue transformation (image intensity changes without deformation) and expansion or contraction effects reflecting a change of mass within the tissue. A real lesion is generally the combination of both effects. The method is tested with synthesized volumetric image sequences and applied, in a first attempt to quantify in vivo a mass effect, to the analysis of a real patient case with multiple sclerosis (MS).

Automatic 3-D segmentation of internal structures of the head in MR images using a combination of similarity and free-form transformations: Part I, Methodology and validation on normal subjects.

The study presented in this paper tests the hypothesis that the combination of a global similarity transformation and local free-form deformations can be used for the accurate segmentation of internal structures in MR images of the brain. To quantitatively evaluate our approach, the entire brain, the cerebellum, and the head of the caudate have been segmented manually by two raters on one of the volumes (the reference volume) and mapped back onto all the other volumes, using the computed transformations. The contours so obtained have been compared to contours drawn manually around the structures of interest in each individual brain. Manual delineation was performed twice by the same two raters to test inter- and intrarater variability. For the brain and the cerebellum, results indicate that for each rater, contours obtained manually and contours obtained automatically by deforming his own atlas are virtually indistinguishable. Furthermore, contours obtained manually by one rater and contours obtained automatically by deforming this rater's own atlas are more similar than contours obtained manually by two raters. For the caudate, manual intra- and interrater similarity indexes remain slightly better than manual versus automatic indexes, mainly because of the spatial resolution of the images used in this study. Qualitative results also suggest that this method can be used for the segmentation of more complex structures, such as the hippocampus.

A novel and very peculiar HincII polymorphism in the 5' region of the human neurofibromatosis type 1 (NF1) gene.

We report a HincII polymorphism in the 5' end of the neurofibromatosis type 1 gene (NF1) as detected with a probe made of exons 1 to 4a (nucleotides 2 to 401 of the cDNA). This HincII site is most probably in an intron. Evidence presented suggests the probe reveals not one but two similar polymorphisms.

Image matching as a diffusion process: an analogy with Maxwell's demons.

In this paper, we present the concept of diffusing models to perform image-to-image matching. Having two images to match, the main idea is to consider the objects boundaries in one image as semi-permeable membranes and to let the other image, considered as a deformable grid model, diffuse through these interfaces, by the action of effectors situated within the membranes. We illustrate this concept by an analogy with Maxwell's demons. We show that this concept relates to more traditional ones, based on attraction, with an intermediate step being optical flow techniques. We use the concept of diffusing models to derive three different non-rigid matching algorithms, one using all the intensity levels in the static image, one using only contour points, and a last one operating on already segmented images. Finally, we present results with synthesized deformations and real medical images, with applications to heart motion tracking and three-dimensional inter-patients matching.

A scheme for automatically building three-dimensional morphometric anatomical atlases: application to a skull atlas.

We present a general scheme for automatically building a morphometric anatomical atlas. We detail each stage of the method, including the non-rigid registration algorithm, three-dimensional line averaging and statistical processes. We apply the method to obtain a quantitative atlas of skull crest lines. Finally, we use the resulting atlas to study a craniofacial disease; we show how we can obtain qualitative and quantitative results by contrasting a skull affected by a mandible deformation with the atlas.

Automatic analysis of cerebral atrophy.

3D MR data obtained for 10 healthy control subjects have been used to build a brain atlas. The atlas is built in four stages. First, a set of features that are unambiguously definable and anatomically relevant need to be computed for each item in the database. The chosen features are crest lines along which the maximal principal curvature of the surface of the brain is maximal in its associated principal direction. Second, a nonrigid registration algorithm is used to determine the common crest lines among the subjects in the database. These crest lines form the structure of the atlas. Third, a set of crest lines is taken as a reference set and a modal analysis is performed to determine the fundamental deformations that are necessary to bring the individual data in line with the reference set. The deformations are averaged and the set of mean crest lines becomes the atlas. Finally, the standard deviation of the deformations between the atlas and the items in the database defines the normal variation in the relative positions of the crest lines in a healthy population. In a fully automatic procedure, the crest lines on the surface of the brain adjacent to the cerebral ventricles in a patient with primary progressive aphasia were compared to the atlas; confirmation that the brain of this patient demonstrates atrophy was provided by stereological analysis that showed that the volume of the left cerebral hemisphere is 48.8 ml (CE 2.8%) less than the volume of the right cerebral hemisphere in the region of the temporal and frontal lobes. When the amplitude of the deformations necessary to register the crest lines obtained for the patient with the atlas were greater than three standard deviations beyond the variability inherent in the atlas, the deformation was considered significant. Four of the main deformation modes of the longest crest line of the surface of the brain adjacent to the cerebral ventricles were significantly different in the patient with primary progressive aphasia compared to the atlas. The ventricles are preferentially enlarged in the left cerebral hemisphere. Furthermore, they are closer together posteriorly and further apart anteriorly than in the atlas. These observations may be indicative of the atrophy of the temporal and frontal lobes of the left cerebral hemisphere noted in the patient. Ultimately, the approach may provide a useful screening technique for identifying brain diseases involving cerebral atrophy. Serial studies of individual patients may provide insights into the processes controlling or affected by particular disease.

Repression of the 5-lipoxygenase gene by c-myb overexpression in differentiated HL-60 cells.

This paper reports on the involvement of c-MYB in the regulation of 5-lipoxygenase gene expression during differentiation of human HL-60 cells. We demonstrate that c-MYB binds the 5-lipoxygenase promoter in undifferentiated cells but not in DMSO-differentiated cells. Also, we show that overexpression of c-myb cDNA in differentiated HL-60 cells represses the 5-lipoxygenase gene expression.

Changes in chromatin conformation regulate the 5-lipoxygenase gene expression during differentiation of HL60 cells.

The HL60 cell line does not express the 5-lipoxygenase gene prior to differentiation with various agents. In this paper we have shown by DNase I sensitivity that the chromatin conformation of the 5-lipoxygenase gene changes following differentiation of HL60 cells with dimethyl sulfoxide (DMSO). Moreover, run-on analysis suggests that transcription of the 5-lipoxygenase gene is enhanced after differentiation. We proposed that the chromatin conformation represses the expression of the 5-lipoxygenase gene in HL60 cell line and that differentiation of these cells with DMSO changes the chromatin conformation, which allows the expression of the 5-lipoxygenase gene. The HeLa cells which, like the HL60 cells, do not express the 5-lipoxygenase, were insensitive to DNase I treatment. These results suggest that chromatin structure might represent a form of regulation for the 5-lipoxygenase gene.

Direct extraction of boundaries from computed tomography scans.

Presents a method, based on the filtered backprojection technique (FBP), to extract directly the boundaries of X-ray images, without previous image reconstruction. The author preprocess the raw data in order to compute directly the reconstructed values of the gradient or of the Laplacian at any location in the plane (defined with real coordinates). The reconstructed value of the gradient and of the Laplacian correspond to the exact mathematical definition of the differentials of the image. For noisy data, the author proposes also to use an extension of existing FBP techniques, adapted to the computation of the gradient and of the Laplacian. Finally, the author shows how to use the corresponding operators to perform the segmentation of a slice, without image reconstruction. Images of the reconstructed gradient, Laplacian, and segmented objects are presented.

Genomic organization and transcriptional units at the myotonic dystrophy locus.

The genomic structure and apparently complete coding sequence of the myotonic dystrophy protein kinase gene have been determined. The gene contains 15 exons distributed over about 13 kb of genomic DNA. It codes for a protein of 624 amino acids with an N-terminal domain highly homologous to cAMP-dependent serine-threonine protein kinases, an intermediate domain with a high alpha-helical content and weak similarity to various filamentous proteins, and a hydrophobic C-terminal segment. Located in close proximity is a second gene, coding for a transcript of about 3 kb, that is homologous to the gene DMR-N9 in the corresponding mouse locus, but has no homologies to other known genes or proteins. Strong expression of the latter gene in brain suggests that it may have a role in the development of mental symptoms in severe cases of the disease.

Selection of mouse cells with amplified metallothionein genes retaining their glucocorticoid inducibility.

Two new mouse cell mutants, resistant to either 80 or 100 mM CdCl2, were isolated to study the regulation of transcription by the glucocorticoid hormones. Their metallothionein mt-1% and mt-2+ genes were amplified coordinately to a maximum of 30 copies per cell. By Southern blot analysis, no gross rearrangement was detectable near the mt+ loci. Contrary to other mutants previously isolated, the metallothionein-specific mRNAs of these mutants are inducible by dexamethasone.

Cloning of the guinea pig 5-lipoxygenase gene and nucleotide sequence of its promoter.

The guinea pig 5-lipoxygenase (5-LO) gene and its promoter were cloned from a guinea pig genomic DNA library. Sequencing analysis of the guinea pig promoter revealed that expression of the 5-LO gene in this rodent is probably governed by cis acting nucleotide sequences quite similar to the human gene. Nucleotide sequences that bind factors like Sp-1, AP-2, NF-kB and c-Ha-ras were identified in the guinea pig 5-LO promoter region.