Anterior Spinal Artery Syndrome Due to Fibrocartilaginous Embolism-Case Report and Treatment Options.

Acute occlusion of the anterior spinal artery and subsequent spinal ischemic infarction leads to anterior spinal artery syndrome characterized by back pain and bilateral flaccid paresis with loss of protopathic sensibility. As a rare cause fibrocartilaginous embolism has been described and is associated with sports or unusual strain.Following gymnastic exercise the day before symptom-onset, a 11 years old girl presented with neck pain, paresis of arms and legs, and impaired deep tendon reflexes. She was unable to lift her arms, grasp, stand, walk and had urinary incontinence. Magnetic resonance imaging revealed a longitudinal T2 hyperintense signal in the anterior spinal cord from C3 to C6 with accompanying bilateral diffusion restriction involving gray matter bilaterally at the level of C4 and C5 and unilaterally at the level of C3/4. The adjacent annulus fibrosus of the intervertebral disc showed a fissure without disc protrusion. Treatment with prednisolone and enoxaparin was started within 12 hours of symptom-onset and continued over 6 days and 8 weeks, respectively. After 2 months, her motor function gradually improved, spinal imaging showed persistent T2 signal hyperintense defects at the level of C4/5. After 5 months, there was only slight impairment affecting elevation and abduction of the right arm.Following physical exercise, the patient suffered from acute anterior spinal cord ischemia with imaging findings in line with a presumed fibrocartilaginous embolism. Unlike most cases, our patient showed almost complete recovery following treatment with prednisolone and enoxaparin. We speculate that the positive outcome is related to rapid treatment initiation.

Specific mosaic KRAS mutations affecting codon 146 cause oculoectodermal syndrome and encephalocraniocutaneous lipomatosis.

Oculoectodermal syndrome (OES) and encephalocraniocutaneous lipomatosis (ECCL) are rare disorders that share many common features, such as epibulbar dermoids, aplasia cutis congenita, pigmentary changes following Blaschko lines, bony tumor-like lesions, and others. About 20 cases with OES and more than 50 patients with ECCL have been reported. Both diseases were proposed to represent mosaic disorders, but only very recently whole-genome sequencing has led to the identification of somatic KRAS mutations, p.Leu19Phe and p.Gly13Asp, in affected tissue from two individuals with OES. Here we report the results of molecular genetic studies in three patients with OES and one with ECCL. In all four cases, Sanger sequencing of the KRAS gene in DNA from lesional tissue detected mutations affecting codon 146 (p.Ala146Val, p.Ala146Thr) at variable levels of mosaicism. Our findings thus corroborate the evidence of OES being a mosaic RASopathy and confirm the common etiology of OES and ECCL. KRAS codon 146 mutations, as well as the previously reported OES-associated alterations, are known oncogenic KRAS mutations with distinct functional consequences. Considering the phenotype and genotype spectrum of mosaic RASopathies, these findings suggest that the wide phenotypic variability does not only depend on the tissue distribution but also on the specific genotype.

[Therapy of inherited diseases of platelet function. Interdisciplinary S2K guideline of the Permanent Paediatric Committee of the Society of Thrombosis and Haemostasis Research (GTH e. V.)]. / Therapie hereditärer Thrombozytopathien. Interdisziplinäre S2K-Leitlinie der Ständigen Kommission Pädiatrie der Gesellschaft für Thrombose- und Hämostaseforschung e. V.

Inherited disorders of platelet function are a heterogeneous group. For optimal prevention and management of bleeding, classification and diagnosis of the underlying defect are highly recommended. An interdisciplinary guideline for a diagnostic approach has been published (AWMF # 086-003 S2K; Hämostaseologie 2014; 34: 201-212). Underlying platelet disorder, platelet count, age and clinical situation modify treatment. Exclusive transfusion of platelet concentrates may be inappropriate as potentially adverse effects can outweigh its benefit. A stepwise and individually adjusted approach for restitution and maintenance of haemostasis is recommended. Administration of antifibrinolytics is generally endorsed, but is of particular use in Quebec disease. Restricted to older children, desmopressin is favourable in storage pool disease and unclassified platelet disorders. Although licensed only for patients with Glanzmann thrombasthenia and alloantibodies, in clinical practice rFVIIa is widely used in inherited platelet disorders with severe bleeding tendency. This guideline aims at presenting the best available advice for the management of patients with inherited platelet function disorders.

Inhibitor-immunology-study. Evaluation of inhibitor development in haemophilia B.

UNLABELLED: The development of inhibitors in haemophilia B is one of the most important complications of replacement therapy, affecting mortality and morbidity. Inhibitor development is based on complex immunological factors, and to date, only little is known about its underlying mechanisms. Here, we present first results of the haemophilia B group of our Inhibitor-Immunology study. PATIENTS, METHODS: So far we have analysed 15 patients with haemophilia B. Four of them developed a high titre inhibitor; the remaining 11 had no inhibitor. We evaluated 9 SNPs in 8 genes (CD40, CTLA-4 , IL-1ß, IL-10, TLR2 , TLR4, TLR9, TNF-α). We compared the distribution of these alleles between inhibitor and non-inhibitor haemophilia B patients and between haemophilia B patients and a normal male control population. HLA typing was performed in all patients. Results, discussion: There appears to be a trend towards a skewed distribution of TLR 9, IL-10 and CTLA4 alleles in haemophilia B patients. Due to the limited number these differences are, however, not statistically significant. The t-test of all patients with inhibitor versus without inhibitor was significant for HLA-A*03 and DPB1*0401 and borderline for DRB1*0201.

Inhibitor-Immunology-Study. Different HLA-types seem to be involved in the inhibitor development in haemophilia A.

UNLABELLED: The development of inhibitors is one of the most important complications of replacement therapy in haemophilia, affecting mortality and morbidity. Inhibitor development is based on complex immunological factors. Cytokines and their receptors, T-cell receptors, and the Major Histocompatibility Complex may play important roles in the development of inhibitors. Earlier studies showed non significant associations between HLA class and inhibitor development. Later studies found an increased risk of inhibitor development if there was a combination between certain factor VIII mutations and HLA antigens. We performed HLA typing in 50 patients with haemophilia A in an effort to find associations with inhibitor development. RESULTS: 25 patients had developed an inhibitor (11 low titre, 14 high titre), and 25 never had. In logistic regression analysis, HLA-A 34, DRB1 0405, DRB1 1301 seemed to be involved in inhibitor development and HLA-A 30, B 13, B15, B 57, Cw 12, DQB1 0303, DPB1 0201 protection against inhibitor development. In our patients, the HLA-associations with inhibitor development were different from those in previous publications.

Contiguous gene deletions involving EFNB1, OPHN1, PJA1 and EDA in patients with craniofrontonasal syndrome.

Craniofrontonasal syndrome (CFNS [MIM 304110]) is an X-linked malformation syndrome characterized by craniofrontonasal dysplasia and extracranial manifestations in heterozygous females. In the majority of patients CFNS is caused by mutations in the EFNB1 gene (MIM 300035). We identified three girls with classical CFNS and mild developmental delay harboring de novo deletions of the EFNB1 gene. Applying haplotype analysis, Southern blot hybridization and array-comparative genomic hybridization, deletion of EFNB1 was found to be part of contiguous gene deletions in the patients. In one patient the deletion interval includes the genes for oligophrenin-1 (OPHN1 [MIM 300127]) and praja 1 (PJA1 [MIM 300420]). In the second patient the deletion includes OPHN1, PJA1 and the gene for ectodysplasin A (EDA [MIM 300451]). In the third patient EFNB1 gene deletion may include deletion of regulatory regions 5' of OPHN1. Previously, the OPHN1 gene has been shown to be responsible for recessive X-linked mental retardation. Although it is too early to predict the future cognitive performance of the two infant patients with contiguous gene deletions of OPHN1-EFNB1-PJA1, mild learning disabilities have been recognized in the older, third patient. It is important for genetic counseling to be aware that their male offspring may not only be carriers of CFNS but may also be affected by mental retardation and anhidrotic ectodermal dysplasia.

Familial translocation t(1;9) associated with macromastia: molecular cloning of the breakpoints.

A familial reciprocal translocation associated with severe macromastia has been characterized by molecular cytogenetic and molecular analysis. Cloning of the translocation breakpoints revealed that no known gene has been disrupted by this translocation. Therefore, a position effect compromising the regulation of a still to be identified gene in the vicinity of the breakpoints can be assumed.

Infrequent genetic alterations of the tumor suppressor gene PTEN/MMAC1 in squamous cell carcinoma of the oral cavity.

BACKGROUND: Fifty tumor specimens from primarily untreated patients were analyzed to elucidate the involvement of the tumor suppressor gene PTEN/MMAC1 in the development of oral squamous cell cancer. METHODS: Eight microsatellite markers, spanning 10 cM of genomic DNA located centromeric, telomeric or intragenic of PTEN/MMAC1 were used for loss of heterozygosity (LOH) and breakpoint analysis. The microsatellite panel within or in close proximity (1 cM) to the 10q23.3 locus showed a LOH rate of 12%. Complete sequence analysis of the genes coding region was performed in all 10 cases that exhibited LOH in one of the eight microsatellite markers within or around the PTEN/MMAC1 gene. Comparative multiplex PCR reactions served to screen for homozygous deletions. RESULTS: There was no association between allelic loss of the gene, overall patient survival and recurrence-free survival. Sequencing did not reveal any mutation in the coding region of PTEN/MMAC1. Differential PCR analysis failed to detect any homozygous deletion. CONCLUSIONS: We conclude that PTEN/MMAC1 gene alterations do not play a key role in tumorigenesis of oral squamous cell cancers.

Mapping of a further locus for X-linked craniofrontonasal syndrome.

Craniofrontonasal syndrome is a rare dysostosis syndrome with an unusual pattern of X-linked inheritance, because males are usually not or less severely affected than females. Previously, a CFNS locus has been localised in Xp22. We report on a haplotype analysis in a German CFNS family, mapping the CFNS locus to the pericentromeric region of the X chromosome. This discrepancy can be explained by locus heterogeneity. Furthermore, random X inactivation could be demonstrated in affected females. The most plausible interpretation for this unusual pattern of X-linked inheritance is metabolic interference. Consequently, we propose that the CFNS gene escapes X inactivation.

Molecular characterization of the DICE1 (DDX26) tumor suppressor gene in lung carcinoma cells.

We have determined the genomic structure of the candidate tumor suppressor gene DICE1 (DDX26). The DICE1 gene colocalizes with microsatellite marker D13S284 telomeric to the RB1 gene in chromosomal region 13q14.3. The DICE1 gene encodes 18 exons that are preceded by a GC-rich promoter region. CpG sites flanking a predicted TATA box were found to be hypermethylated in tumor cells that exhibited decreased DICE1 expression. This suggests tumor-specific transcriptional silencing of the DICE1 gene may occur. Aberrantly spliced products were detected in two of three DICE1 expressing cell lines. The predicted DICE1 amino acid sequence is evolutionarily conserved in mouse, fruit fly (D. melanogaster), and nematode (C. elegans). A DEAD box characteristic of ATP-dependent helicases is the predominant motif found in DICE1 and its mouse and fruit fly homologues. Motifs other than the DEAD box are reminiscent of members of the helicase superfamily II but there is considerable variation from the typical DEAD box helicases. Expression of DICE1 green fluorescent fusion protein showed a preferential localization of DICE1 in the nucleus. This suggests that DICE1 is involved in nuclear processes such as DNA repair, transcription, or RNA splicing.

Loss of heterozygosity analysis on chromosome 5p defines 5p13-12 as the critical region involved in tumor progression of bladder carcinomas.

We have previously observed loss of heterozygosity (LOH) at a single locus (del-27) on human chromosome 5p13-12 to correlate with bladder tumor progression. In this study, we examined 33 bladder tumors for their pattern of allelic loss on chromosome 5p using 7 microsatellite markers. In 14 of 15 bladder tumors with LOH at locus del-27, allelic loss was confined to chromosomal region 5p13-12. This region included the microsatellite marker D5S2025 that showed LOH in 5 of 11 (45%) informative cases with LOH at del-27. This suggests that D5S2025 and del-27 are located within a single critical region of LOH on 5p13-12 harboring a tumor suppressor gene involved in bladder tumor progression. Recurrent LOH at other loci was observed at microsatellite markers located at 5p15. However, these losses appeared to be independent of LOH at 5p13-12 and occurred predominantly in poorly differentiated (G3) and advanced (T3-T4) tumors.

Isolation of DICE1: a gene frequently affected by LOH and downregulated in lung carcinomas.

In the development and progression of sporadic tumors multiple tumor suppressor genes are inactivated that may be distinct from predisposing cancer genes. Previously, a tumor suppressor locus on human chromosome 13q14 that is distinct from the retinoblastoma predisposing gene 1 (RB1) has been identified in lung, head and neck, breast, ovarian and prostate tumors. By an approach that combines genomic difference cloning and positional cloning we isolated the cDNA of a novel gene (DICE1) located at 13q14.12-14.2. The DICE1 gene is highly conserved in evolution and its mRNA is expressed in a wide variety of fetal and adult tissues. The DICE1 cDNA encodes a predicted protein of 887 amino acids corresponding to an 100 kD protein that shows 92.9% identity to the carboxy-terminal half of the mouse EGF repeat transmembrane protein DBI-1. The DBI-1 protein interferes with the mitogenic response to insulin-like growth factor 1 (IGF-I) and is presumably involved in anchorage-dependent growth. When compared to normal lung tissue expression of the DICE1 mRNA was reduced or undetectable in the majority of non-small cell lung carcinomas analysed. The location of the DICE1 gene in the region of allelic loss, its high evolutionary conservation and the downregulation of expression in carcinoma cells suggests that DICE1 is a candidate tumor suppressor gene in non-small cell lung carcinomas and possibly in other sporadic carcinomas.

Genetic alterations of the tumor suppressor gene PTEN/MMAC1 in human brain metastases.

The high mutation rate in advanced brain tumors, recent functional studies, and the high frequency of mutations in prostate metastases all strongly suggest that PTEN/MMAC1 alterations are involved in the formation of metastases. We searched for genetic alterations in the PTEN/MMAC1 gene in 56 consecutive brain metastases from various primary tumors by loss of heterozygosity (LOH), direct sequence analysis, and differential PCR analysis. The highest LOH rates were detected in metastases deriving from lung (67%) and breast (64%) cancers. Three (25%) of the eight detected inactivating mutations (one nonsense mutation, one splice-site mutation, one 11-bp deletion, and five homozygous deletions) were found in metastases originating from 12 different lung carcinomas, suggesting that PTEN/MMAC1 alterations may play a role in the progression of this tumor. With the exception of lung carcinomas, our findings indicate that genetic abnormalities of the PTENM/MMAC1 gene are only involved in a relatively small subset of brain metastases. However, the discrepancy between the high overall LOH rate (50%) and the low frequency of PTEN/MMAC1 mutation detection rate (14%) suggests the presence of one or more additional tumor suppressor genes on chromosome 10q.

A novel member of the NF2/ERM/4.1 superfamily with growth suppressing properties in lung cancer.

A novel putative tumor suppressor gene and member of the NF2/ERM/ 4.1 superfamily was isolated using Differential Display PCR (DDPCR) on primary lung tumors. When reintroduced into nonexpressing non-small cell lung carcinoma cell lines, this gene, named DAL-1 (for Differentially expressed in Adenocarcinoma of the Lung), was shown to suppress growth. In addition, significantly reduced expression (>50%) of DAL-1 was measured in 39 primary non-small cell lung carcinoma tumors as compared with patient-matched normal lung tissue. Immunocytochemical staining with a polyclonal anti-DAL-1 antibody localized the protein to the plasma membrane, particularly at cell-cell contact points, a pattern reminiscent of other members of the protein 4.1 superfamily including ezrin and NF2. The data suggest DAL-1 is a novel membrane-associated protein with potential to play an important role in the origin and progression of lung cancer.

Microbeam MOMeNT: non-contact laser microdissection of membrane-mounted native tissue.

The analysis of tissue-specific genetic alterations depends on the selective procurement of homogeneous cell populations. Microbeam microdissection of membrane-mounted native tissue (MOMeNT) permits the rapid, selective, and low-contamination procurement of tumor or other cells from histological sections by non-thermic non-contact laser microdissection. Tissue sections are mounted on a specifically designed ultrathin transparent supporter membrane. Tissue together with the membrane are then dissected with an ultraviolet (337-nm) pulsed laser microbeam coupled into a robot-stage microscope. The ultraviolet laser causes dissection by cold photolysis due to the high photon density of the microbeam rather than by local heating. The track of the laser microbeam can be preselected freely on a video screen, and the size and form of the dissectates can thus be adapted to the histological features of the section with a delineation accuracy in the micron range. Polymerase chain reaction amplification of DNA from the dissectates is not impaired, and tumor-specific loss of heterozygosity of the APC gene as well as homozygous deletion of the MTS1 gene are demonstrated in bladder carcinomas. Taken together, microbeam MOMeNT is a novel technique that utilizes membrane-based microdissection by an ultraviolet laser microbeam, thus providing a flexible, easy-to-use high-performance tool for the molecular pathologist.

Deletion analysis at the DEL-27, APC and MTS1 loci in bladder cancer: LOH at the DEL-27 locus on 5p13-12 is a prognostic marker of tumor progression.

Inactivation of relevant tumor-suppressor genes by allelic or homozygous deletion is a characteristic event in tumor cells. Here, the prognostic value of allelic deletions on 5p13-12 at the putative del-27 tumor-suppressor locus and in the APC tumor-suppressor gene on 5q21, as well as homozygous deletions of the MTS1 (p16INK4, CDKN 2) tumor-suppressor gene on 9p21 was assessed in 87 bladder cancers using microdissection and PCR-based assays. Tumor-specific LOH was detected in 10 of 38 (26%, del-27), and 15 of 30 (50%, APC) informative specimens. Homozygous deletion of the MTS1 gene was detected in 33% of 84 tumors investigated. These deletion frequencies implicate the 3 tumor-suppressor regions in the genesis of transitional-cell carcinoma. In contrast to deletions of the APC or MTS1 genes, LOH at the del-27 locus correlated with tumor progression. This suggests that loss of the putative tumor-suppressor gene DEL-27 is involved in an aggressive behavior of the tumor cells and appears to be a prognostic marker for the clinical outcome of patients with transitional-cell carcinoma.

Detection of loss of heterozygosity in the APC tumor suppressor gene in nonpapillary renal cell carcinoma by microdissection and polymerase chain reaction.

The role of the APC (adenomatous polyposis coli) tumor suppressor gene in the genesis of nonpapillary renal cell carcinoma is addressed. The frequency of allelic deletion in the APC gene was analyzed using microdissection of the tumor specimens and a PCR (polymerase chain reaction)-based assay for the detection of intragenic loss of heterozygosity (LOH). Twelve of 29 carcinomas investigated were informative (41%). In five of these (42%) LOH was detected in the APC gene, LOH did not correlate with tumor grade or stage. This high frequency of intragenic LOH suggests an implication of the APC gene or a closely linked gene in the genesis of a subset of nonpapillary renal cell carcinoma. The use of a microdissection technique allows the reliable detection of tumor-specific LOH when using a PCR-based assay.