Current Genomics, Vol. 4, No. 2, 2003
Contents
Gene Mining and Functional Genomics in Human
Osteoarthritis Pp. 109-121
M.G. Attur, M.N. Dave, and A.R. Amin
Clinical and Genetic Aspects of the Joubert Syndrome: a
Disorder Characterised by Cerebellar Vermian Hypoplasia and Accompanying
Brainstem Malformations Pp. 123-129
C.L. Bennett, J. Meuleman, P.F. Chance and I.A. Glass
Review of Common Sequence Alignment Methods: Clues to
Enhance Reliability Pp. 131-146
Christophe Lambert, Jean-Marc Van Campenhout, Xavier
DeBolle and Eric Depiereux
Human Kallikreins: Common Structural Features, Sequence
Analysis and Evolution Pp. 147-165
George M. Yousef and Eleftherios P. Diamandis
Assessment of Gene Transfer Using Imaging Methodology
Pp. 167-184
Annette Altmann, and Uwe Haberkorn
Approaches to Quantification of RNA Targets by PCR Based
Techniques Pp. 185-204
F. Watzinger, and T. Lion
[Back to top]
Gene Mining and Functional Genomics in Human Osteoarthritis
M.G. Attur, M.N. Dave, and A.R. Amin
Human osteoarthritis (OA) is a complex multi-factorial disease
that primarily targets the cartilage. The cartilage is a specialized tissue
where the chondrocytes maintain matrix homeostasis. The paradigm for the post
genomic era of molecular medicine is to identify the function of defective
genes associated with specific diseases. Although the process of identification
and validation of target genes is feasible with the available tools and
technologies, establishing the link between defected gene products and the
disease is still a challenging task. New tools in genomic analysis not only
allow us to profile gene expression in normal and diseased tissues, but also
facilitate the development of unbiased hypothesis at the molecular level.
Several defective heritable genes encoding the matrix components (such as Col2a1
and Col9a1 that code for type II and IX collagen respectively) have been linked
to the development of OA in human and animal models. Several families of genes
expressed differentially in normal and osteoarthritis-affected cartilage have
been identified. These include matrix metalloproteinases (MMPs), matrix
proteins, integrins, transcription factors, cytokines, cytokine receptors,
growth factors and homeostatic genes. A non-conventional functional genomic
approach is required to study the complexity of chondrocyte and matrix
interactions that affect the above mentioned targets.
[Back to top]
Clinical and Genetic Aspects of the Joubert Syndrome: a Disorder
Characterised by Cerebellar Vermian Hypoplasia and Accompanying Brainstem
Malformations
C.L. Bennett, J. Meuleman, P.F. Chance and I.A. Glass
Joubert syndrome (JS) is a rare autosomal recessive
syndrome characterised by agenesis or dysgenesis of the cerebellar vermis with
accompanying brainstem malformations (OMIM: 213300). There is a distinctive
(but not unique) MRI appearance in the posterior fossa of the brain termed the
molar-tooth-sign, and the diagnosis of JS is usually made when this MRI finding
is documented in an infant in combination with hypotonia, episodic hyperpnea or
apnea, abnormal eye movements, with a variety of facial and other dysmorphisms.
Later, developmental delay and cerebellar ataxia develop. In some JS patients a
retinal dystrophy, coloboma and renal abnormalities manifest. It is apparent
that JS is a clinically heterogeneous entity and that both intrafamilial and
interfamilial variation exists. Overlap with other vermal hypoplasias and rare
cerebellar-retinal-renal disorders exists, but whether such disorders are
allelic to forms of JS awaits gene isolation and molecular clarification.
Recently the first JS locus was found on chromosome 9q34 by homozygosity
mapping in a consanguineous family of Omani origin. A second consanguineous
family of similar ethnicity demonstrated haplotype segregation consistent with
linkage to this 9q34 locus. Linkage to the 9q34 region was excluded in two
additional consanguineous families, establishing genetic heterogeneity in the
Joubert syndrome. Haplotype analyses in an additional 26 Joubert pedigrees
including three with consanguinity, showed no evidence of homozygosity within
this interval. These data suggest that the major JS genetic loci are yet to be
discovered. Genes crucial to cerebellar and brainstem development are
functional candidates, particularly homeotic genes specifying the vermal
domain. We have excluded several strong functional candidate genes such as
WNT1, EN1, EN2 and FGF8, by mutation analysis and direct DNA sequencing. To
characterize the underlying molecular mechanism of the JS, it may be necessary
to further define the syndrome. By careful examination, the JS can be
distinguished from the related but genetically distinct syndromes, COACH,
Senior-Loken and Arima.
[Back to top] Review of Common
Sequence Alignment Methods: Clues to Enhance Reliability
Christophe Lambert, Jean-Marc Van Campenhout, Xavier
DeBolle and Eric Depiereux
Today, in various aspects of molecular biology, sequence alignment has become an essential tool to study the structure-function relationships of proteins. With the impressive increase of the number of available sequences, alignments provide a substantial piece of information by way of various computational methods. These approaches have generally become a crucial tool to put forward working hypotheses for time-consuming bench work, as protein engineering and site directed mutagenesis. However alignment methods remain hugely perfectible. All methods are dramatically limited in the twilight zone, taking place around 25% of identity between pairs of sequences. More worrying is the very high rate of false positive results generated by most algorithms, depending of empirical parameters, and hard to validate by statistical criteria.
After reviewing the main methods, this paper draws user’s attention to the fact that algorithm performance evaluations are entirely limited to alignment power (sensibility) evaluation. In reference to a given truth defined from alignment of know structures, the power is defined as the proportion of truth restored in the solution. The power may be overestimated by a lack of independent sets of poorly related sequences and its value depends entirely on the criterion used to define the truth. On the other hand, confidence (selectivity) represents the proportion of the solution that is true. Depending on the method and the parameters used, confidence may be much lower than power, and is usually never evaluated. For non-trivial alignments, when the power is high, confidence is low, which means that correctly aligned positions are embedded in large regions unduly aligned.
One possible solution to these problems is to use
consensus of several multiple alignment methods, which will increase the
confidence of the results. The addition of external information, such as the
prediction of the secondary structure and/or the prediction of solvent
accessibility is also an other way that should increase the performance of
existing multiple alignment methods.
[Back
to top] Human Kallikreins: Common Structural
Features, Sequence Analysis and Evolution
George M. Yousef and Eleftherios P. Diamandis
Kallikreins are a subgroup of serine proteases with
diverse physiological functions. The human kallikrein gene family has now been
fully characterized and includes 15 members tandemly located on chromosome
19q13.4 In this review, we discuss the common structural features of
kallikreins at the DNA, mRNA and protein levels and summarize their tissue
expression and hormonal regulation. Kallikreins are expressed in a wide range
of tissues including the salivary gland, endocrine tissues including testis,
prostate, breast, endometrium, and the central nervous system. Most, if not all
genes are under steroid hormone regulation. The classical kallikreins (KLK1-3)
are thought to represent a distinct evolutionary subgroup of kallikreins. The
occurrence of several splice variants is a very common phenomenon among
kallikreins, and some of the splice variants appear to be tissue-specific and
might be related to certain pathological conditions. We also provide a summary
of predicted and experimentally confirmed promoter elements of kallikrein genes
and describe repeat elements and polymorphisms within this genomic region.
[Back to top]
Assessment of Gene Transfer Using Imaging Methodology
Annette Altmann, and Uwe Haberkorn
Targeting of the therapeutic genes to the malignant or non-malignant tissue may be achieved using recombinant viral vectors for the transfer and tissue-specific or tumor-specific regulatory sequences. However, the efficiency of the recombinant viruses to infect tumor cells, was shown to be rather low. Therefore, repeated injections of the virus might be required to obtain a sufficient therapeutic response and in clinical trials non-invasive procedures have to be employed to assess the transfer and functional activity of the recombinant gene.
In gene therapy based on the transfer and expression of the suicide genes usually genes coding for the non-mammalian enzymes, the Herpes simplex virus thymidine kinase (HSVtk) or the yeast and bacterial cytosine deaminase (CD), have been applied. After infection of the tumor with the recombinant virus, the non-toxic prodrug is applied systemically, which is subsequently converted to a toxic metabolite by the recombinant gene product. Employing a radiolabeled prodrug and scintigraphic procedures to determine the functional activity of the recombinant enzyme in vivo, a therapeutic window of maximal gene expression and consecutive drug administration may be defined. If the gene therapy approach is based on the transduction of receptor genes, the recombinant gene expression in tumor cells will be monitored using radiolabeled ligands. Transfer of transporter genes as the sodium iodide transporter may also lead to the visualization of transduction via accumulation of iodide or pertechnetate. Furthermore, imaging based on transchelation of oxotechnetate to a polypeptide motif from a biocompatible complex with a higher dissociation constant than that of a dicglycilcysteine complex or tyrosinase gene transfer for metal ion scavenging have been described.
In addition, the monitoring of the gene therapy effects is
performed by the evaluation of the morphological changes of the tumor using
magnetic resonance imaging or, more effectively, by the measurement of the
tumor-associated metabolic variations with positron emission tomography (PET)
employing tracers of tumor metabolism and proliferation. The uptake of 18Fluordeoxyglucose
(FDG) has been demonstrated to be a useful parameter for the assessment of the
glucose metabolism and the tumor growth may be elevated by the uptake of (11C)thymidine
or other proliferative markers.
[Back to top] Approaches
to Quantification of RNA Targets by PCR Based Techniques
F. Watzinger, and T. Lion
Reverse transcription polymerase chain reaction (RT-PCR)
techniques are increasingly used for the quantification of gene transcripts.
Compared to other methods permitting relative or absolute quantitation of mRNA,
the RT-PCR approach offers the advantages of superior sensitivity, high
specifity, and great versatility. In this review, we discuss different
approaches to PCR-based quantification of gene expression including
comparative, competitive, and real-time PCR. Different formats of
fluorescence-based real-time PCR and their applications in clinical diagnosis
and research are presented.