Classification of non-bacterial osteitis: retrospective study of clinical, immunological and genetic aspects in 89 patients.

OBJECTIVE: To define non-bacterial osteitis (NBO) as a clinical entity possibly associated with autoimmune manifestations. Patients with sterile osteitis were analysed to develop diagnostic criteria. METHODS: A total of 89 patients with non-bacterial inflammatory bone lesions were observed for a median of 49 months. History, diagnostic imaging, laboratory and histological data were obtained. Mutation analysis in the genes PSTPIP1 and PSTPIP2 was performed. RESULTS: Patients had an onset of disease at a median age of 10 yrs [interquartile range (IQR) 7.5-12] and suffered a median period of 21 (IQR 9-52) months with a median of three foci per patient. Twenty percent of all the patients demonstrated associated autoimmune disorders, particularly of the skin and bowel. The majority of bone lesions were located in the vertebrae and metaphyses. Slight-to-moderate elevation of inflammation values were found in all the patients and antinuclear antibodies were elevated in 30%. Non-steroidal anti-inflammatory drugs (NSAIDs) were effective in 85% of the patients. HLA-B27 and Human Leukocyte Antigen-DR (HLA-DR)-classification did not differ from the general population. Autoimmune diseases in 40% of all the families, multiply affected family members, linkage to 18q21 and mouse models strongly indicate a genetic basis for NBO. We observed three different courses of disease regarding the duration of complaints, rate of complications and associated autoimmune manifestations leading to a new classification of NBO. CONCLUSIONS: Clinical analysis of our cohort leads us to define NBO as a distinct disease entity with three clinical presentations: acute NBO, chronic recurrent multifocal osteomyelitis or persistent chronic NBO. Diagnostic criteria were proposed to differentiate NBO from diseases with similar clinical presentation.

A splice site mutation in the methyltransferase gene FTSJ1 in Xp11.23 is associated with non-syndromic mental retardation in a large Belgian family (MRX9).

Mental retardation is the most frequent cause of serious handicap in children and young adults. The underlying causes of this heterogeneous condition are both acquired and genetically based. A recently performed refinement of the linkage interval in a large Belgian family with mild to severe non-syndromic X linked mental retardation, classified as MRX9, revealed a candidate region of 11.3 Mb between markers DXS228 and DXS1204 on the short arm of the X chromosome. In order to identify the underlying disease gene in the MRX9 family, we established a gene catalogue for the candidate region and performed comprehensive mutation analysis by direct sequencing. A human homologue of the bacterial 23S rRNA methyltransferase Fstj, the FTSJ1 gene, is located within this region and displayed a sequence alteration in the conserved acceptor splice site of intron 3 (IVS3-2A>G) in all tested patients and carrier females of this family. In contrast, it was absent in all unaffected male family members tested. The mutation results in skipping of exon 4 and introduces a premature stop codon in exon 5, probably leading to a severely truncated protein. Our finding indicates that a protein, possibly associated with ribosomal stability, can be linked to X linked mental retardation (XLMR).

DNA sequence comparison of human and mouse retinitis pigmentosa GTPase regulator (RPGR) identifies tissue-specific exons and putative regulatory elements.

Retinitis pigmentosa 3 (RP3) is a progressive retinal degeneration due to mutations in the X-linked RPGR gene. Transcription studies in human and mouse tissues have revealed ubiquitously expressed transcripts and also an exceptional high number of tissue-specific alternative splice variants. However, regulation of tissue-specific expression and splicing is unclear, but this is of particular interest as mutations in this ubiquitously expressed gene lead to severe retinal degeneration, while other tissues are unaffected. To elucidate the conservation pattern of RPGR and to identify additional tissue-specific exons and putative regulatory elements we performed comparative genomic sequencing of the human and mouse RPGR gene. Each of the genes spans a region of nearly 59 kb, and all previously identified exons are conserved between the two species. DNA sequence comparison identified 28 conserved sequence elements (CSEs) in introns, upstream of exon 1, within the promotor region, and downstream of the most 3' exon. Some of the intronic CSEs flank tissue-specific exons and therefore may represent important regulatory elements for alternative splicing. Comparative northern blot hybridization of ubiquitous and tissue-specific RPGR probes identified high molecular weight transcripts with similar expression patterns in both human and mouse. These transcripts range from 6 to 15 kb in size and suggest the presence of additional transcribed sequences within RPGR. Our cross-species sequence comparison enables us to define candidate regions that may explain these large transcripts and will therefore contribute to the understanding of RPGR expression and splicing.

ACRC codes for a novel nuclear protein with unusual acidic repeat tract and maps to DYT3 (dystonia parkinsonism) critical interval in xq13.1.

We searched for novel genes as candidates of X-linked dystonia parkinsonism (XDP) in the critical interval of Xq13.1 that harbors the disease locus (DYT3). A gene, ACRC (acidic repeat containing), was discovered by a combination of in silico and "wet" experiments. ACRC is composed of at least 12 exons and 11 introns. It is expressed in all tissues tested, including skeletal muscle, liver, kidney, pancreas, heart, lung, and brain. Highest levels of expression are found in skeletal muscle. The ACRC protein is characterized by a previously undescribed acidic repeat tract of 21 units of 8-10 amino acids. The N-terminal portion of the protein is highly acidic (pI=3.2), and the C-terminal region is basic (pI=10.2). There are nuclear localization signals in its C-terminal portion. Extensive mutation analysis of the transcribed region of the gene, including intron-exon boundaries and the 5' and 3' untranslated intervals, did not reveal a mutation in XDP patients. Exclusion of a mutation in the transcribed portion of this and all other known genes within the DYT3 critical interval suggests that XDP is most likely caused by a mutation in a regulatory region of a gene within the critical interval, or by a structural rearrangement.

Complete form of X-linked congenital stationary night blindness: refined mapping and evidence of genetic homogeneity.

A number of distinct, partly non-overlapping genetic loci have been reported for the complete type of X-linked congenital stationary night blindness (CSNB1), suggesting genetic heterogeneity. In order to refine the localization of the CSNB1 gene and to demonstrate genetic homogeneity, linkage analysis was performed in two large CSNB1 families. Clinical features consistent with the diagnosis of CSNB1 were documented in five patients from a German seven-generation kindred by full ophthalmological examination including psychophysical and electroretinographical testing. Haplotype analysis in 30 members of the large German family was performed with 38 polymorphic markers predominantly covering the critical region. Linkage analyses defined a locus for CSNB1 with flanking markers DXS8042 and DXS228, refining the interval to 2.5 cM in Xp11.4. In addition, two-point linkage analysis was carried out using the MLINK computer program. In agreement with meiotic breakpoints, lod scores of 3.0 and greater were obtained for markers located to the proximal site of the former 5 cM CSNB consensus interval. A large Dutch CSNB1 family was re-evaluated with markers from the Xp11.4 region, and supports the CSNB1 minimal interval found in the German family. Together with previous results from three unrelated families from Sweden, Sardinia and Great Britain, our results provide evidence of genetic homogeneity in the disorder. Subsequent mutation analyses in CSNB1 patients revealed no pathogenic sequence alterations in DFFRX and CASK genes, but retain candidates for other diseases mapping to that region.

Physical and transcriptional mapping of the 17p13.3 region that is frequently deleted in human cancer.

Studies of chromosomal losses at 17p13 have suggested the presence of at least two distinct regions for tumor suppressor genes, the TP53 region at 17p13.1 and a more distal region at 17p13.3. Within the latter region, Hypermethylated in Cancer 1 (HIC1) is located, a likely candidate for a tumor suppressor gene that has also been suggested to play a role in the pathogenesis of Miller-Diecker syndrome (MDS). However, single-gene isolation efforts have retrieved additional genes from 17p13.3 that could play a role in tumorigenesis. This indicates that the full potential of this chromosomal region with respect to disease-related genes has not yet been exhausted and that there may exist still unknown genes that contribute to tumorigenesis or to the complex MDS phenotype. To provide a basis for the systematic isolation and evaluation of such genes, we established a physical map over 1.5 Mb of 17p13.3 and assigned 29 transcriptional units within this region.

The DNA sequence of human chromosome 21.

Chromosome 21 is the smallest human autosome. An extra copy of chromosome 21 causes Down syndrome, the most frequent genetic cause of significant mental retardation, which affects up to 1 in 700 live births. Several anonymous loci for monogenic disorders and predispositions for common complex disorders have also been mapped to this chromosome, and loss of heterozygosity has been observed in regions associated with solid tumours. Here we report the sequence and gene catalogue of the long arm of chromosome 21. We have sequenced 33,546,361 base pairs (bp) of DNA with very high accuracy, the largest contig being 25,491,867 bp. Only three small clone gaps and seven sequencing gaps remain, comprising about 100 kilobases. Thus, we achieved 99.7% coverage of 21q. We also sequenced 281,116 bp from the short arm. The structural features identified include duplications that are probably involved in chromosomal abnormalities and repeat structures in the telomeric and pericentromeric regions. Analysis of the chromosome revealed 127 known genes, 98 predicted genes and 59 pseudogenes.

Hailey-Hailey disease is caused by mutations in ATP2C1 encoding a novel Ca(2+) pump.

Hailey-Hailey disease (HHD) is an autosomal dominant skin disorder characterized by suprabasal cell separation (acantholysis) of the epidermis. Previous genetic linkage studies localized the gene to a 5 cM interval on human chromosome 3q21. After reducing the disease critical region to <1 cM, we used a positional cloning strategy to identify the gene ATP2C1, which is mutated in HHD. ATP2C1 encodes a new class of P-type Ca(2+)-transport ATPase, which is the homologue for the rat SPLA and the yeast PMR1 medial Golgi Ca(2+)pumps and is related to the sarco(endo)plasmic calcium ATPase (SERCA) and plasma membrane calcium ATPase (PCMA) families of Ca(2+)pumps. The predicted protein has the same apparent transmembrane organization and contains all of the conserved domains present in other P-type ATPases. ATP2C1 produces two alternative splice variants of approximately 4.5 kb encoding predicted proteins of 903 and 923 amino acids. We identified 13 different mutations, including nonsense, frameshift insertion and deletions, splice-site mutations, and non-conservative missense mutations. This study demonstrates that defects in ATP2C1 cause HHD and together with the recent identification of ATP2A2 as the defective gene in Darier's disease, provide further evidence of the critical role of Ca(2+)signaling in maintaining epidermal integrity.

Characterization of a highly complex region in Xq13 and mapping of three isodicentric breakpoints associated with preleukemia.

The chromosomal abnormality represented by an isodicentric X chromosome [idic(X)(q13)] is associated with a subset of acute myeloid leukemia (AML) and preleukemia observed in elderly females. A previous study localized the breakpoints of two acquired isodicentric X chromosomes associated with myelodysplasia to a 450-kb region proximal to the XIST gene. Here we report the construction and extensive characterization of a reliable 1-Mb P1 artificial chromosome and bacterial artificial chromosome contig covering a highly problematic region in Xq13 that includes the previously described isodicentric breakpoint region. In addition to mapping of the brain-specific gene (NAP1L2) and the phosphoglyceryl kinase alpha subunit 1 gene (PHKA1) and generation and mapping of a large number of STSs throughout the contig, we have mapped a putative transcriptional regulatory protein (HDACL1), and 35 ESTs. Sequencing data, Southern blot analysis, and fiber-FISH analysis have permitted characterization of extensive region-specific duplications and triplications in addition to an unusually high concentration of long interspersed repeat elements, both of which could be implicated in isodicentric chromosome formation and other Xq13 chromosome aberrations. FISH analysis of metaphase chromosomes from two previously unpublished AML patients and one preleukemic patient using cosmid clones and selected subclones allowed mapping of the idic(X)(q13) breakpoints to a 100-kb interval, consistent with the involvement of an X-linked gene in the genesis of this form of preleukemia, disruption of which may represent a preliminary step in progression to AML. Assembly and physical mapping of this complex 1-Mb contig establish a foundation for ongoing sequencing and gene identification projects in the region.

Criteria for gene identification and features of genome organization: analysis of 6.5 Mb of DNA sequence from human chromosome 21.

To establish criteria for and the limitations of novel gene identification, to identify novel genes of potential relevance to Down Syndrome and to investigate features of genome organization, 6. 550kb. In total, 41 novel gene models were predicted, and for a subset of these, RT-PCR experiments helped to verify and refine the models, and were used to assess expression in early development and in adult brain regions of potential relevance to Down syndrome. Results suggest generally low and/or restricted patterns of expression, and also reveal examples of complex alternative processing, especially in brain, that may have important implications for regulation of protein function. Analysis of complete gene structures of the known genes identified a number of very large introns, a number of very short intergenic distances, and at least one potentially bi-directional promoter. At least 3/4 of known genes and 1/2 of predicted genes are associated with CpG islands. For novel genes, three cases of overlapping genes are predicted. Results of these analyses illustrate some of the complexities inherent in mammalian genome organization and some of the limitations of current sequence analysis technologies. They also doubled the number of potential genes within the region.

Preselection of shotgun clones by oligonucleotide fingerprinting: an efficient and high throughput strategy to reduce redundancy in large-scale sequencing projects.

Large-scale genomic sequencing projects generally rely on random sequencing of shotgun clones, followed by different gap closing strategies. To reduce the overall effort and cost of those projects and to accelerate the sequencing throughput, we have developed an efficient, high throughput oligonucleotide fingerprinting protocol to select optimal shotgun clone sets prior to sequencing. Both computer simulations and experimental results, obtained from five PAC-derived shotgun libraries spanning 535 kb of the 17p11.2 region of the human genome, demonstrate that at least a 2-fold reduction in the number of sequence reads required to sequence an individual genomic clone (cosmid, PAC, etc.) can be achieved. Treatment of clone contigs with significant clone overlaps will allow an even greater reduction.

Increased informativeness of RAPD analysis by detection of microsatellite motifs.

The recently developed random-amplified microsatellite polymorphism (RAMPO) technique detects second-level amplification products that are useful as molecular markers. In the first step of the procedure, genomic DNA is amplified with a single arbitrary or microsatellite-complementary primer. PCR products are then electrophoretically separated, photographed, blotted and hybridized to a 32P-labeled microsatellite probe. Autoradiography reveals highly reproducible, polymorphic, probe-dependent fingerprints, which are different from the ethidium bromide staining patterns. In this paper, we report the successful application of various mono-, tri- and tetranucleotide repeat motifs as RAMPO probes. We also compare the efficiency of arbitrary vs. microsatellite primers for the generation of RAMPO patterns. Repeated rehybridization to different probes has expanded the information contained in a single random-amplified polymorphic DNA (RAPD) gel at least fivefold. Pattern complexity varies with the length and sequence of the probe. Application of the technique to a genetic relatedness study in the genus Dioscorea (yam) yielded highly informative markers, mainly at an interspecific level.

Molecular marker based taxonomy and phylogeny of Guinea yam (Dioscorea rotundata - D. cayenensis).

Four different molecular techniques were used to assess relationships among 21 accessions of Guinea yam (Dioscorea rotundata and Dioscorea cayenensis) and 21 accessions belonging to seven putative progenitor species. Random amplified polymorphic DNA (RAPD) and microsatellite-primed PCR (MP-PCR) analysis yielded 246 informative characters that were transformed into a matrix of pairwise distances and analyzed by neighbor joining or split decomposition. Both methods gave congruent results. Well-separated groups were formed that corresponded to their species designation. Dioscorea rotundata and D. cayenensis accessions were clearly separated from each other, supporting the concept that both are distinct species. Two morphological intermediates grouped together with D. rotundata. All investigated species fell into two main clusters, one comprising D. rotundata, D. cayenensis, Dioscorea abyssinica, Dioscorea liebrechtsiana, and Dioscorea praehensilis, the other comprising Dioscorea smilacifolia, Dioscorea minutiflora, Dioscorea burkilliana, and Dioscorea togoensis. The same grouping was also obtained by comparative sequence analysis of chloroplast DNA, which supports earlier studies of nuclear rDNA variation and chloroplast restriction fragment length polymorphisms. We also analyzed the same set of Dioscorea samples with the recently developed random amplified microsatellite polymorphism (RAMPO) technique. A series of diagnostic RAMPO bands was identified that clearly distinguished between D. rotundata and D. cayenensis. Some of these bands could also be traced back to the putative progenitors of both species. The evolutionary origin of Guinea yam is discussed in light of the present results.

Genomic variation and relationships in aerial yam (Dioscorea bulbifera L.) detected by random amplified polymorphic DNA.

Random amplified polymorphic DNA (RAPD) markers were used to assess intraspecific variability and relationships in aerial yam (Dioscorea bulbifera L.). A total of 23 accessions from different geographic locations in Africa, Asia, and Polynesia were analyzed by 10 arbitrarily chosen GC-rich decamer primers. Using cesium chloride purified genomic template DNA, highly reproducible polymorphic fingerprints were generated by all 10 primers, resulting in a total of 375 informative characters. Only eight bands were monomorphic among all investigated accessions. A binary character matrix was generated by scoring for presence/absence of a band at a particular position, transformed into a matrix of pairwise distances using either the Jaccard or a simple matching coefficient, and analyzed by neighbour joining, UPGMA (unweighted pair group method with arithmetic averaging) cluster analysis, or split decomposition. All methods of data evaluation resulted in similar groupings that reflected the geographical origin of the samples. The African accessions formed a distinct isolated group, whereas Asian and Polynesian accessions proved to be more heterogeneous. With two exceptions (var. suavior and var. sativa), the RAPD data supported previous varietal classification based on morphological characters. Stepwise reduction of the number of evaluated characters did not affect branching patterns of the trees above a minimum threshold of 150. Key words : Dioscorea bulbifera, random amplified polymorphic DNA (RAPD), genetic variation, genetic relatedness.

Multilocus DNA fingerprinting and genetic relatedness in plants: a case study with banana and tomato.

The technique of DNA fingerprinting is frequently used for studies of genetic diversity and relatedness in a wide range of organisms. In humans and animals, multilocus fingerprints are mainly applied to paternity and identity test cases, behavioral ecology, and the analysis of population structures. In plants and fungi, the frequent occurrence of "low-variability" fingerprint patterns additionally allows to use multilocus fingerprinting for studying taxonomical problems at an intraspecific level. In the present article, we (1) present an overview of such approaches in a series of plant species, (2) summarize our attempts to estimate genetic relationships within two cultivated plant species, banana and tomato, by band sharing data derived from oligonucleotide fingerprints, and (3) discuss the limitations and potentials of multilocus fingerprinting for the determination of genetic relatedness.

A comparison of femoral neck fixation with the reconstruction nail versus cancellous screws in anatomic specimens.

Femoral neck fixation techniques were applied to five matched pairs of autopsy specimens to evaluate the fixation of the Russell-Taylor femoral nail in ipsilateral neck and shaft fractures of the femur. Reconstruction nail fixation of the femoral neck was compared with that of three parallel screws. The intact and postfixation femora were subjected to an applied bending moment in 0 degrees, 30 degrees, and 90 degrees of simulated hip flexion. The bending stiffness was determined from the load deformation data for each intact femur and then after the appropriate fixation. The fatigue response of the fixation, presence of osteopenia, degree of fracture reduction, and device alignment showed that the stiffness ratio (fixed to normal) of the nail was greater in most specimens. There was no statistical difference in retained stiffness after cyclic loading between the nail and cancellous screw fixations. The ultimate strength of the nail was 2.5 times the strength of the screw fixation of the femoral neck. Thus, the nail provided biomechanically sound fixation of the femoral neck.

Plant DNA fingerprinting with radioactive and digoxigenated oligonucleotide probes complementary to simple repetitive DNA sequences.

The existence of hypervariable DNA sequences in nuclear genomes, and the use of appropriate "fingerprinting" probes to detect them, has gained widespread scientific interest, and also led to multiple applications in diverse areas. Two years ago, the new technique of "DNA fingerprinting" was also introduced into the analysis and characterization of plant genomes, initially by using human or M13 minisatellites as probes. In the present article, we demonstrate the applicability for plant DNA fingerprinting of oligonucleotide probes specific for simple repetitive DNA sequences. We show that various levels of intra- and interspecific polymorphisms can be detected; the information to be gained depends on the optimal combination of probe and species. Variety-specific patterns were obtained in several cases. Some probes revealed variability between individuals. Somatic variability was not observed. Different DNA isolation and purification procedures were tested in order to introduce a fast and easy-to-perform isolation method suitable for a large variety of plant species. Nonradioactive fingerprinting was performed using digoxigenated oligonucleotides as probes. Banding patterns obtained with radioactive and digoxigenin-based labeling techniques proved to be of similar quality.

Technique for treatment of a bent Russell-Taylor femoral nail.

A case is presented in which a 14-mm Russell-Taylor intramedullary nail, placed to secure a right femoral shaft fracture, was deformed 22 months postoperatively by subsequent trauma. The nail was straightened in situ, removed, and replaced with a larger nail. The femur subsequently healed without complication.