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.

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.

Frequent allelic deletion at a novel locus on chromosome 5 in human lung cancer.

Frequent allelic deletions in tumor cells are indicative of the inactivation of tumor suppressor genes. Recently, we isolated the single-copy sequence del-27 (I. Wieland, M. Böhm, and S. Bogatz, Proc. Natl. Acad. Sci. USA, 89: 9705-9709, 1992). Here we show that del-27 detects a restriction fragment length polymorphism that allows examination for loss of heterozygosity (LOH) in tumor specimens. LOH at the del-27 locus occurred in 57% (4 of 7) of the informative lung carcinomas independent of the histopathological differentiation grade. LOH for exon 11 of the APC gene occurred in 71% (5 of 7) of the informative cases but was not associated with LOH at the del-27 locus. The del-27 sequence was localized to chromosome 5p13-5q14, proximal to the MCC/APC region, using a somatic cell hybrid panel. Together with our previous finding that del-27 is deleted homozygously in a lung carcinoma cell line, these results suggest that del-27 is linked closely to a novel putative tumor suppressor gene.

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.

Allelic deletion mapping on chromosome 5 in human carcinomas.

We analysed allelic deletions on chromosome 5 in microdissected human non-small cell lung cancers. Thirty-four primary squamous cell carcinomas, 15 primary adenocarcinomas and five regional lymph node metastases were investigated for loss of heterozygosity (LOH) in chromosomal region 5p15-q21. The sites analysed included the APC tumor suppressor gene at 5q21, five polymorphic microsatellite markers and the putative tumor suppressor locus del-27, that was assigned to chromosomal region 5p13-12 by fluorescence in situ hybridization (FISH) analysis. Allelic deletions encompassed larger genomic regions more often in squamous cell carcinomas than in adenocarcinomas. The del-27 amd APC regions were identified as two distinct regions with the highest LOH frequencies within 5p15-q21. In squamous cell carcinomas LOH frequencies were 73% at the del-27 and 70% at the APC locus. In adenocarcinomas LOH at the del-27 and APC loci occurred in 38% of the informative cases. Allelic deletion of the APC gene and at the del-27 locus was also detected in the metastases. The results suggest involvement of at least two tumor suppressor genes on chromosome 5 in lung tumorigenesis.

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.

Differences of E-cadherin expression levels and patterns in primary and metastatic human lung cancer.

Normal lung epithelium and 52 lung carcinomas obtained at surgical resection were examined by immunofluorescence for their expression levels and patterns of the calcium-dependent intercellular adhesion molecule E-cadherin. In dysplastic lung tissue and in well-differentiated squamous cell and adenocarcinomas, expression of E-cadherin was confined to the lateral cell border, similar to the expression level and pattern of normal lung tissue. The E-cadherin level was reduced and expression pattern was spotty or diffuse in moderately and poorly differentiated squamous cell and in small cell carcinomas of the lung. Most metastases resected also had a reduced level and an altered pattern of E-cadherin expression. In contrast, no such correlation was found in adenocarcinomas of the lung. This indicates that different cellular mechanisms are responsible in the progression of squamous cell carcinomas and adenocarcinomas of the lung.

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.

Analysis of tumour-specific alterations in native specimens by PCR.

Native tumours, in contrast to cell lines, are usually heterogeneous, consisting of tumour cells, stroma, infiltrating leukocytes, necrotic cells, and surrounding normal tissue. Therefore, when using non-linear amplification and detection methods such as the PCR, the verification of the DNA from the tumour cells is mandatory to avoid equivocal or false results. Here, current methods to isolate tumour cells from native tumours are reviewed. The methods are: i) a variety of microdissection techniques including microdissection of membrane-mounted native tissue (MOMeNT), ii) Selective ultraviolet radiation fractionation (SURF), iii) antibody-based tumour cell selection and flow cytometric cell or cell nucleus sorting, and iv) in situ PCR. Each of the methods has been used, and overall preference cannot be given to any of them. Accuracy, reproducibility, documentation, cost, and applicability in a routine setting are discussed, from which fields of preferencial use may emerge for the different methods.

Allelic imbalance at the beta-catenin gene (CTNNB1 at 3p22-21.3) in various human tumor types.

beta-catenin is a multifunctional protein: it plays a central role in the cell-cell adhesive junctions, and participates in transduction of the morphogenic Wingless/Wnt-signal. Upon detailed analysis of the human beta-catenin gene, an intragenic polymorphic microsatellite marker could be identified. This marker shows 62% heterozygosity and was used in a study of eleven different tumor types. A high level of beta-catenin allelic imbalance was observed for small cell lung carcinoma, squamous cell lung carcinoma and cervix carcinoma. Other microsatellite markers on 3p24-21 could demonstrate frequent but not invariable codeletion of flanking chromosomal loci. This intragenic polymorphic marker will allow selection of tumor types and tumor samples possibly bearing recessive mutations in the remaining allele of the beta-catenin gene.

Detection of tumor-specific homozygous deletions in human biopsies by polymerase chain reaction.

Cancer is associated with homozygous deletions of specific DNA sequences that are in some instances too small to be detectable by cytogenetic methods or Southern blot analysis. Such small tumor-specific deletions can be detected, however, by the polymerase chain reaction (PCR) provided that tumor cells are meticulously and verifiably isolated from contaminating nontumor cells. Nontumor cells can give positive PCR results and thus obscure the detection of deletions. Using a method that allows accurate and verifiable excision of tumor cells for subsequent PCR analysis, homozygous deletions, one in a putative tumor suppressor gene on chromosome 8 and one in the p53 gene, were detected in two out of 20 human lung carcinomas investigated.

Microsatellite instability and loss of heterozygosity at the hMLH1 locus on chromosome 3p21 occur in a subset of nonsmall cell lung carcinomas.

Microsatellite alterations observed in tumor specimens may reflect genomic instability due to defective mismatch repair genes. To investigate whether this occurs in human nonsmall cell lung carcinomas we have analyzed microsatellite instability at 5 marker loci and loss of heterozygosity (LOH) at the hMLH1 locus on chromosome 3p21 using the polymerase chain reaction. Of a total of 49 nonsmall cell lung carcinomas examined, 43% (13 of 30 informative cases) showed LOH at 3p21 and 29% (14 of 49) exhibited microsatellite instability at one or multiple loci. LOH of the mismatch repair gene hMLH1 at 3p21 occurred in 82% (9 of 11 informative cases) of the tumors with microsatellite instability. This suggests that defects in the mismatch repair gene hMLH1 at 3p21 may be involved in microsatellite instability and tumorigenesis of a subset of nonsmall cell lung carcinomas. However, in those nonsmall cell lung carcinomas without microsatellite instability LOH at 3p21 probably involved another tumor suppressor gene(s) in this chromosomal region.

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.

[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.

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.