Christina Fuchs

A hierarchical 3D segmentation method and the definition of vertebral body coordinate systems for QCT of the lumbar spine

We have developed a new hierarchical 3D technique to segment the vertebral bodies in order to measure bone mineral density (BMD) with high trueness and precision in volumetric CT datasets. The hierarchical approach starts with a coarse separation of the individual vertebrae, applies a variety of techniques to segment the vertebral bodies with increasing detail and ends with the definition of an anatomic coordinate system for each vertebral body, relative to which up to 41 trabecular and cortical volumes of interest are positioned. In a pre-segmentation step constraints consisting of Boolean combinations of simple geometric shapes are determined that enclose each individual vertebral body. Bound by these constraints viscous deformable models are used to segment the main shape of the vertebral bodies. Volume growing and morphological operations then capture the fine details of the bone-soft tissue interface. In the volumes of interest bone mineral density and content are determined. In addition, in the segmented vertebral bodies geometric parameters such as volume or the length of the main axes of inertia can be measured. Intra- and inter-operator precision errors of the segmentation procedure were analyzed using existing clinical patient datasets. Results for segmented volume, BMD, and coordinate system position were below 2.0%, 0.6%, and 0.7%, respectively. Trueness was analyzed using phantom scans. The bias of the segmented volume was below 4%; for BMD it was below 1.5%. The long-term goal of this work is improved fracture prediction and patient monitoring in the field of osteoporosis. A true 3D segmentation also enables an accurate measurement of geometrical parameters that may augment the clinical value of a pure BMD analysis.

Tracing Myelin Protein Zero (P0) in vivo by construction of P0-GFP fusion proteins

BackgroundMutations in P0, the major protein of the myelin sheath in peripheral nerves, cause the inherited peripheral neuropathies Charcot-Marie-Tooth disease type 1B (CMT1B), Dejerine-Sottas syndrome (DSS) and congenital hypomyelination (CH). We reported earlier a de novo insertional mutation c.662_663GC (Ala221fs) in a DSS patient. The c.662_663GC insertion results in a frame shift mutation Ala221fs altering the C-terminal amino acid sequence. The adhesion-relevant intracellular RSTK domain is replaced by a sequence similar to Na+/K+ ATPase. To further clarify the molecular disease mechanisms in this sporadic patient we constructed wild type P0 and the c.662_663GC mutant expression cassettes by site-specific mutagenesis and transfected the constructs into insect cells (S2, High5). To trace the effects in live cells, green fluorescent protein (GFP) has been added to the carboxyterminus of the wild type and mutated P0 protein.ResultsIn contrast to the membrane-localized wild type P0-GFP the Ala221fs P0-GFP protein was detectable almost only in the cytoplasm of the cells, and a complete loss of adhesion function was observed.ConclusionsThe present study provides evidence that GFP is a versatile tool to trace in vivo effects of P0 and its mutations. Not only a loss of adhesion function as a result of the loss of the RSTK domain, but also altered intracellular trafficking indicated by a loss of membrane insertion are possible consequences of the Ala221fs mutation.

An adhesion test system based on Schneider cells to determine genotype–phenotype correlations for mutated P0 proteins

Myelin protein zero (MPZ, P0) is well known as the adhesion molecule responsible for the compaction of the myelin sheath of peripheral nerves. Mutations are linked to Charcot-Marie-Tooth syndrome type 1B (CMT1B) and the more severe Dejerine-Sottas syndrome (DSS). Three mutations leading to phenotypes of increasing severity (Ser34del/CMT1B, Ser34Cys/DSS, INS663GC/DSS) were expressed in S2 insect cells and resulted in a decreased adhesion capability in correlation with their respective phenotypes.

One-tube, two-stage PCR-directed in vitro mutagenesis using megaprimers

▼Mutations very near to the 5′ or 3′ end (up to ∼ 40 bp) of a target sequence are easy to introduce directly by a onestep PCR protocol using mismatching primers. However, it is more difficult in a one-step PCR protocol to introduce mutations large distances from the ends of the DNA template and to obtain a full-length PCR product avoiding random mutations created during the polymerization reaction (Ref. 1). To circumvent such problems, it is necessary to perform the in vitro mutagenesis in a two-stage PCR reaction using a polymerase such as Pwo polymerase which has proofreading activity instead of a conventional Taq polymerase. Two-stage PCR mutagenesis methods based on at least one useful restriction site in close proximity to the target nucleotide sequence (Ref. 2) or methods which use overlap extension in a second PCR step (Ref. 3, 4) always need a set of four primers. Previously described one-step−threestage or one-tube methods often require special equipment or additional working steps. For example, when using biotinylated primers, classical avidin-coated beads cannot be used as they inactivate the Pwo polymerase and so special encapsulated magnetic beads which are more expensive must be used instead (Ref. 5). Other published protocols specify elaborate stages which could be elimated, such as Geneclean purification of megaprimers (Ref. 6), ddNTPblocked DNA as template in the first PCR step (Ref. 7) to prevent wild-type amplification or the toleration of wildtype amplification because of imbalanced primer relation (Ref. 8) or imbalanced cycling parameters which should be optimized for each mutagenesis (Ref. 7). The treatment with Taq polymerase and dATP to add 3′ protruding adenosine to both ends of the product to be cloned in an TA Cloning Kit vector is also unnecessary (Ref. 7).

Charcot-Marie-Tooth disease type 1A and hereditary neuropathy with liability to pressure palsies: a SacI polymorphism in the proximal CMT1A-REP elements may lead to genetic misdiagnosis.

ABSTRACT A male patient with clinical signs and symptoms of a demyelinating neuropathy was shown to have a duplication of the 1.5-Mb region on chromosome 17p11.2 by means of two-color fluorescence in situ hybridization (FISH). This duplication is typical for the vast majority of Charcot-Marie-Tooth type 1A (CMT1A) cases. Analysis of DNA extracted from peripheral blood used to detect an EcoRI/SacI 3.2-kb junction fragment with probe pLR7.8 confirmed the CMT1A duplication, but also revealed a 7.8-kb fragment usually observed in patients with a hereditary neuropathy with liability to pressure palsies (HNPP). Both fragments observed in one patient canot result from one unequal crossover. In EcoRI/SacI Southern hybridization experiments with probe pLR7.8 DNA of his healthy parents also revealed a 7.8-kB restriction fragment. A subsequent two-color FISH analysis, however, indicated a normal status for interphase nuclei of the parents. Hence we hypothesize that the 7.8-kb fragment observed in our patient and his parents is not the product of unequal crossover during meiosis but due to a polymorphism of the SacI site in a proximal CMT1A-REP element.

High-resolution FISH of stretched chromosome fibers

High resolution fluorescence in situ hybridization (FISH), so-called FIBER-FISH, was first described by Heng et al. (1992) for long stretched DNA-fibers and was later subjected to modifications by other investigators (Fidlerova et al. 1994, Heiskanen et al. 1996). The resolution using cosmids on DNA fibers was restricted by the length of the visible, decondensed DNA strand up to 150 to 450 kb. Using earlier protocols, it was not possible to obtain a coherent DNA strand showing a longer section of cosmids in direct orientation. However, freshly prepared lymphocyte suspension could be used (for lymphocyte preparation see Arnoldus et al., 1990, Verma et al., 1989).This method described here is simple, but very efficient. It allows the presentation of cosmids over a region up to 8 Mb in a linear orientation. Thus, the visualization of the transcriptional orientation of genes is possible within a bigger range. Moreover, the modified treatment allows the use of archival suspensions of cultured lymphocytes and is also available for YACs. The main steps are a special treatment of up to two year old lymphocyte suspensions, the disruption of nuclei by a shortened PBS incubation previous to alkaline treatment and the release of chromosome fibers from interphase nuclei by special handling of the slides. With these modifications, we were able to obtain released chromosome fibers, which were suitable for a high resolution two-color FISH. This method has been specially tested for lymphocyte nuclei (Fuchs et al., 1997).