Bone marrow reinvestigation leading to the diagnosis of VEXAS syndrome.

A 46-year-old male patient presented with inflammatory diseases over more than 3 years. The patient suffered from recurrent pleuritis, polychondritis, orbital phlegmon, fever, and skin lesions. A bone marrow puncture added myelodysplastic syndrome to the patient's history. A focused cytomorphological reinvestigation of the archived bone marrow aspirate smears detected significant vacuolization of erythroid and myeloid precursor cells. Target sequencing revealed the UBA1 (p.Met41Thr) hotspot mutation that established the diagnosis of VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic) syndrome.

Spontaneous reshaping of vertebral fractures in an adolescent with osteogenesis imperfecta.

Background: Vertebral compression fractures (VFs) are a common and severe finding in patients with osteoporosis. In children, VFs have the unique potential to reshape and regain their original configuration. Spontaneous vertebral body reshaping (i.e., medication-unassisted) has been reported in secondary osteoporosis. Here we describe a previously unreported spontaneous vertebral reshaping in an adolescent with osteogenesis imperfecta (OI) with multiple vertebral fractures. Case report: A 17-year-old female was diagnosed with OI type I at 5 years of age caused by a novel frameshift variant in COL1A1 (NM_000088.4: c.540delC; p.Met181TrpfsTer84). Due to parental reservations about medication, she had never received bisphosphonate or any other bone active therapy. A lateral spine X-ray demonstrated transparent bones and no VF. However, previous spine X-rays taken at age of 6 years at an external institution showed VFs in T5-7 (Genant semiquantitative method grade I-II). The two lateral spine x-rays, taken 11 years apart, demonstrate that substantial spontaneous vertebral reshaping occurred without bone active therapy during puberty. Discussion: Vertebral reshaping is explained by the stabilization of bone mineral density (BMD) and the remaining growth capacity the children. We hypothesize that spontaneous reshaping may occur in milder forms of OI, and that puberty may be a key mediator of the phenomenon. In all children with OI and vertebral fractures, we nevertheless recommend bisphosphonate therapy since it improves bone mass, BMD, vertebral shape, physical activity and reduces fracture rates.

Successful kidney transplantation in a patient with pre-existing chronic myeloid leukemia treated with imatinib.

Active malignancy is an absolute contraindication to kidney transplantation. As for chronic myeloid leukemia (CML), a Philadelphia chromosome-positive myeloproliferative neoplasm, the introduction of tyrosine kinase inhibitors has transformed CML from a lethal into a manageable chronic disease with a close-to-normal life expectancy. To date it is unknown whether kidney transplantation can be safely performed in patients with pre-existing CML. We describe the clinical course of a 57-year-old male patient with chronic kidney disease caused by reflux nephropathy. This patient had undergone first kidney transplantation 20 years earlier and had again been on chronic hemodialysis for 6 years when CML was diagnosed. First-line therapy with 400 mg imatinib daily was well tolerated and induced an optimal cytogenetic and molecular response 3 months after initiation. One and a half years after CML diagnosis, a second kidney transplantation from a deceased donor was performed. Immunosuppression included basiliximab, tacrolimus, mycophenolate mofetil, and corticosteroids. Currently, 2 years posttransplant, renal allograft function is stable (serum creatinine 1.09 mg/dL, estimated glomerular filtration rate 75 mL/min per 1.73 m2 ), and CML remains in deep molecular remission with imatinib. Imatinib-treated CML in deep molecular remission could be regarded as inactive malignancy and may therefore not be viewed as an absolute contraindication to kidney transplantation.

Cleft lip/palate and hereditary diffuse gastric cancer: report of a family harboring a CDH1 c.687 + 1G > A germline mutation and review of the literature.

Hereditary diffuse gastric cancer (HDGC) is an autosomal-dominantly inherited cancer syndrome associated with a high risk for diffuse gastric and lobular breast cancer, caused by heterozygous CDH1 germline mutations. Of note, also cleft lip/palate (CLP) has been described in few HDGC families. Here we report on an extensive pedigree presenting with HDGC, CLP and a CDH1 splice site mutation (c.687 + 1G > A) and review the literature for families with CDH1 mutations, HDGC and CLP. Transcript analysis showed that the c.687 + 1G > A mutation leads to loss of the last 42 bp of exon 5 and is consequently predicted to cause loss of 14 amino acids in the first extracellular cadherin repeat (EC) domain. Five mutation carriers developed diffuse gastric cancer and four individuals presented with CLP. Wild type CDH1 expression levels did not differ between CDH1 mutation carriers with CLP compared to those without CLP. Beside this extensive pedigree, we outline another previously unreported HDGC/CLP family with a CDH1 (c.1711 + 1G > C) germline mutation in this study. Review of the literature revealed a significant enrichment of CDH1 mutations within the EC domains in CLP/HDGC families (Fisher's exact test, p = 0.007) in comparison to CDH1 mutations associated with HDGC only. Report of further CLP/HDGC associated mutations is necessary to confirm this observation. This study highlights that CLP represents an important phenotypic feature of CDH1 germline mutation carriers and emphasizes the inclusion of CLP in the HDGC testing criteria. The underlying causes for the appearance of variable phenotypes in CDH1 mutation carriers could include genetic variation, epigenetic changes and environmental factors and should be investigated in future studies.

Overexpression of the proneural transcription factor ASCL1 in chronic lymphocytic leukemia with a t(12;14)(q23.2;q32.3).

BACKGROUND: Translocations of the IGH locus on 14q32.3 are present in about 8% of patients with chronic lymphocytic leukemia (CLL) and contribute to leukemogenesis by deregulating the expression of the IGH-partner genes. Identification of these genes and investigation of the downstream effects of their deregulation can reveal disease-causing mechanisms. CASE PRESENTATION: We report on the molecular characterization of a novel t(12;14)(q23.2;q32.3) in CLL. As a consequence of the rearrangement ASCL1 was brought into proximity of the IGHJ-Cµ enhancer and was highly overexpressed in the aberrant B-cells of the patient, as shown by qPCR and immunohistochemistry. ASCL1 encodes for a transcription factor acting as a master regulator of neurogenesis, is overexpressed in neuroendocrine tumors and a promising therapeutic target in small cell lung cancer (SCLC). Its overexpression has also been recently reported in acute adult T-cell leukemia/lymphoma.To examine possible downstream effects of the ASCL1 upregulation in CLL, we compared the gene expression of sorted CD5+ cells of the translocation patient to that of CD19+ B-cells from seven healthy donors and detected 176 significantly deregulated genes (Fold Change ≥2, FDR p ≤ 0.01). Deregulation of 55 genes in our gene set was concordant with at least two studies comparing gene expression of normal and CLL B-lymphocytes. INSM1, a well-established ASCL1 target in the nervous system and SCLC, was the gene with the strongest upregulation (Fold Change = 209.4, FDR p = 1.37E-4).INSM1 encodes for a transcriptional repressor with extranuclear functions, implicated in neuroendocrine differentiation and overexpressed in the majority of neuroendocrine tumors. It was previously shown to be induced in CLL cells but not in normal B-cells upon treatment with IL-4 and to be overexpressed in CLL cells with unmutated versus mutated IGHV genes. Its role in CLL is still unexplored. CONCLUSION: We identified ASCL1 as a novel IGH-partner gene in CLL. The neural transcription factor was strongly overexpressed in the patient's CLL cells. Microarray gene expression analysis revealed the strong upregulation of INSM1, a prominent ASCL1 target, which was previously shown to be induced in CLL cells upon IL-4 treatment. We propose further investigation of the expression and potential role of INSM1 in CLL.

Functional characterization, localization, and inhibitor sensitivity of the TPR-FGFR1 fusion in 8p11 myeloproliferative syndrome.

Myeloid and lymphoid neoplasms with fibroblast growth factor receptor 1 (FGFR1) abnormalities, also known as 8p11 myeloproliferative syndrome (EMS), represent rare and aggressive disorders, associated with chromosomal aberrations that lead to the fusion of FGFR1 to different partner genes. We report on a third patient with a fusion of the translocated promoter region (TPR) gene, a component of the nuclear pore complex, to FGFR1 due to a novel ins(1;8)(q25;p11p23). The fact that this fusion is a rare but recurrent event in EMS prompted us to examine the localization and transforming potential of the chimeric protein. TPR-FGFR1 localizes in the cytoplasm, although the nuclear pore localization signal of TPR is retained in the fusion protein. Furthermore, TPR-FGFR1 enables cytokine-independent survival, proliferation, and granulocytic differentiation of the interleukin-3 dependent myeloid progenitor cell line 32Dcl3, reflecting the chronic phase of EMS characterized by myeloid hyperplasia. 32Dcl3 cells transformed with the TPR-FGFR1 fusion and treated with increasing concentrations of the tyrosine kinase inhibitors ponatinib (AP24534) and infigratinib (NVP-BGJ398) displayed reduced survival and proliferation with IC50 values of 49.8 and 7.7 nM, respectively. Ponatinib, a multitargeted tyrosine kinase inhibitor, is already shown to be effective against several FGFR1-fusion kinases. Infigratinib, tested only against FGFR1OP2-FGFR1 to date, is also efficient against TPR-FGFR1. Taking its high specificity for FGFRs into account, infigratinib could be beneficial for EMS patients and should be further investigated for the treatment of myeloproliferative neoplasms with FGFR1 abnormalities.

Disruption of the ARID1B and ADAMTS6 loci due to a t(5;6)(q12.3;q25.3) in a patient with developmental delay.

Here, we report on a male patient with developmental delay, speech impairment, mild dysmorphic features, and borderline intellectual disability, bearing a de novo balanced t(5;6)(q11;q25.3). By combining FISH and long distance inverse PCR, we identified two genes, ADAMTS6 and ARID1B, which were disrupted at the translocation breakpoints. Due to the opposing transcriptional directions of the two genes, no fusion transcripts could be formed. ADAMTS6 on chromosome 5 encodes a zinc metalloprotease. To date, there has been no information about the substrates and the exact role of this enzyme protein. ARID1B on chromosome 6 is involved in chromatin remodeling and transcriptional activation and is known to play a role in neural development. To our knowledge, this is the fourth translocation involving ARID1B reported in association with intellectual disability. ARID1B haploinsufficiency has already been described in patients with intellectual disabilities with or without corpus callosum abnormalities, Coffin-Siris syndrome and autism (OMIM 614562 and OMIM 614556). A review of patients with ARID1B mutations reveals their broad phenotypic variability. The phenotype of the present patient is of the mildest described to date and further underscores this observation. We conclude that the most prominent and consistent clinical findings in patients with ARID1B haploinsufficiency are developmental delay, speech impairment and intellectual disability and propose that patients with unresolved genetic background and these clinical findings should be considered for ARID1B mutation screening.

Morphological and molecular heterogeneity in colorectal neoplasms with K-RAS mutation. A report of two cases.

Approximately forty percent of colorectal cancers (CRC) are characterized by activating mutations of the K-RAS gene. Determination of K-RAS mutational status as a predictive marker for anti-EGFR therapy is usually based on the assumption of intratumoral homogeneity. We present two cases of CRC in which morphologically distinct tumor components were associated with different activating mutations of K-RAS in one patient and a mutated and a non-mutated portion in the second patient, as demonstrated by laser microdissection and consecutive molecular analyses.

Characterization of a newly identified ETV6-NTRK3 fusion transcript in acute myeloid leukemia.

BACKGROUND: Characterization of novel fusion genes in acute leukemia is important for gaining information about leukemia genesis. We describe the characterization of a new ETV6 fusion gene in acute myeloid leukemia (AML) FAB M0 as a result of an uncommon translocation involving chromosomes 12 and 15. METHODS: The ETV6 locus at 12p13 was shown to be translocated and to constitute the 5' end of the fusion product by ETV6 break apart fluorescence in situ hybridisation (FISH). To identify a fusion partner 3' rapid amplification of cDNA-ends with polymerase chain reaction (RACE PCR) was performed followed by cloning and sequencing. RESULTS: The NTRK3 gene on chromosome 15 was found to constitute the 3' end of the fusion gene and the underlying ETV6-NTRK3 rearrangement was verified by reverse transcriptase PCR. No RNA of the reciprocal NTRK3-ETV6 fusion gene could be detected. CONCLUSION: We have characterized a novel ETV6-NTRK3 fusion transcript which has not been previously described in AML FAB M0 by FISH and RACE PCR. ETV6-NTRK3 rearrangements have been described in secretory breast carcinoma and congenital fibrosarcoma.

[The frequency of wound infections after PEG-placement and utilization of glycogel wound dressing: a randomized controlled trial]. / Häufigkeit von Wundinfektionen bei Verwendung eines Glycogelverbandes nach PEG-Anlage: Ergebnisse einer kontrollierten Studie.

BACKGROUND: Percutaneous endoscopic gastrostomy (PEG) is the method of choice for long- term artificial enteral feeding. Standardized wound management such as daily dressing changes and local disinfection of the exit site helps to keep complication rates low. New bacteriostatic glycogel wound dressing has not yet been tested. We compared glycogel dressing to the usual method of wound aftercare with regard to wound infections. METHODS: The standard wound management was compared to glycogel dressing. 100 consecutive patients were investigated in a prospective randomized trial from Aug. 2004 to Jan. 2006 regarding wound infections. We also compared indications for PEG placement, complications other than wound infection, and mortality. The exit site was examined and scored daily using a specific wound scoring system. After 30 days, the patients were followed up by phone calls to determine if any infection had occurred after discharge. RESULTS: During our study, 98 out of 100 patients had a successful PEG procedure performed. Out of these 98 patients, 48 patients received standard wound dressing care and 50 patients used glycogel dressing. The indications for PEG placement were not significantly different between the two groups. A total of 88% of patients (n = 42) with standard wound care had no relevant infection (50%, n = 24 with score 0 or 1; 38%, n = 18 had score 2), 10% (n = 5) presented with serious local infection (score 3) and one patient (2%) had severe infection necessitating PEG removal (score 4). In the group using glycogel dressing, 88% of the patients (n = 44) did not show any relevant sign of infection (54%, n = 27 with score 0 or 1; 34%, n = 17 had score 2), 8% (n = 4) had serious local infection (score 3), 2% (n = 1) had severe infection (score 4) and 2% (n = 1) were lost to follow up. CONCLUSION: Regarding wound infection rates after PEG placement, glycogel wound dressing was found to be as effective as standard wound dressing. Thus, omitting daily changes of regular wound dressings by using glycogel dressing instead may be advantageous for patients and generally help to decrease overall cost.

Comparison of molecular assays for detection and typing of human papillomavirus.

OBJECTIVE: The objective of the study was to compare the performance of 3 different extraction instruments in conjunction with 4 different amplification and detection kits for detection and typing of human papillomavirus (HPV) deoxyribonucleic acid (DNA). STUDY DESIGN: A total of 42 cervical swabs were investigated. HPV DNA was extracted on the 3 different instruments. Each of the extracts was then amplified, and HPV DNA amplification products were detected with 4 different kits. RESULTS: In 31 samples, HPV DNA was detected by both the Amplicor HPV test and the LINEAR ARRAY HPV genotyping test in conjunction with DNA extraction on the easyMAG instrument. In another 6 samples, only low-risk types were detected with the linear array HPV genotyping test. After extraction on the easyMAG instrument, 32 samples tested positive when the PapilloCheck with the HotStarTaq DNA polymerase was used. CONCLUSION: Together with extraction on the easyMAG instrument, the Amplicor HPV test, the linear array HPV genotyping test, and the new PapilloCheck with the HotStarTaq DNA polymerase provide comparable results allowing reliable and safe HPV diagnostics in the routine laboratory. Use of alternative assays may lead to an increase of invalid and divergent HPV typing results.

Long-term outcome following placement of percutaneous endoscopic gastrostomy in younger and older patients.

OBJECTIVE: Percutaneous endoscopic gastrostomy (PEG) is the method of choice in maintaining enteral nutrition in patients with swallowing and nutritional disorders of different etiology. The aim of this study was to assess the long-term outcome of patients following placement of a PEG. MATERIAL AND METHODS: All patients who received a PEG between October 1999 and September 2000 were included in this prospective study. Long-term function, replacement or removal of the PEG, complications and survival of the patients were analyzed in group A (younger than 75 years) and group B (75 years or older). RESULTS: The indications for PEG placement in group A (54 patients, mean age 54.5 years) were neurological (66.7%) and malignant (31.5%) disorders, whereas in group B (40 patients, mean age 81 years) the indications were predominantly neurological diseases (87.5%). The majority of patients (91 of 94 patients; 96.8%) could be followed long term or until death. In group A, 46 patients (85.2%) had uncomplicated long-term function of their PEG and interventions were necessary in only 8 patients. Removal of the PEG was possible during the course in 17 patients (31.5%). In group B, uncomplicated long-term function was observed in 34 patients (85%) and interventions were required in only 6 patients. Removal of the PEG was not possible in group B. Survival rates for 1-, 2- and 5 years in group A were 73.9%, 61.8% and 43.9%, respectively, and in group B 41.4%, 31.9% and 15.9%, respectively; the difference was statistically significant (p=0.002). CONCLUSIONS: Excellent long-term function of PEG was seen in this study of 94 consecutive patients, and interventions were necessary only in a minority of patients. The prognosis for older patients was worse; however, the 2-year survival rate of 32% justified the PEG insertion.

[Indications and complications of percutaneous endoscopic gastrostomy]. / Indikationen und Komplikationen der perkutanen endoskopischen Gastrostomie.

BACKGROUND: Percutaneous endoscopic gastrostomy (PEG) plays an important role in maintaining enteral nutrition in patients with swallowing disorders of different etiologies. The aim of our study was to record indications and complications of PEG-placement in a one-year period. METHODS: All patients were investigated prospectively regarding indications, wound infections, other complications and mortality between 1999-10-01 and 2000-09-30. The exit site was examined daily, after 30 days a follow-up by telephone was carried out. RESULTS: The PEG-procedure was performed in 93 patients, one patient received a percutaneous endoscopic jejunostomy. The mean age of the patients was 65.4 years (range 7 months--92 years). The most frequent indications were neurological diseases (n = 61, 65%). 21 patients had a PEG-placement because of malignancies (22%), 9 patients following brain injury (10%) and 3 patients (3%) due to other benign swallowing disorders. 63 patients (67%) had no complications, 28 patients (30%) had wound infections, and in two patients hemorrhage was observed (small hematoma requiring no further intervention). One patient had laparotomy because of suspected perforation--however, laparotomy was negative. In 7 patients (7%) wound infections (n = 28) were mild and needed only local or no therapy. In 18 patients (19%) we found a relevant infection that required systemic antibiotic therapy. 2 patients had serious local infections that caused further interventions. One patient died from sepsis caused by wound infection. Patients receiving antibiotic therapy at the time of PEG-placement suffered from wound infections in 25%. Patients with malignant diseases more often had wound infections. 8 patients died after 7 days and 19 patients after 30 days (8% and 19%, respectively) from their underlying disease. CONCLUSIONS: PEG is regarded as a small intervention with low morbidity and mortality. However, our analysis of daily practice shows a remarkable rate of complications. The high mortality in our study reflects the seriousness of the comorbidities. Antibiotic therapy failed to prevent wound infection in 25% of our patients.

The molecular basis for the autoregulation of calponin by isoform-specific C-terminal tail sequences.

The three genetic isoforms of calponin (CaP), h1, h2 and acidic, are distinguished mostly by their individual C-terminal tail sequences. Deletion of these sequences beyond the last homologous residue Cys273 increases actin filament association for all three isoforms, indicating a negative regulatory role for the unique tail regions. We have tested this hypothesis by constructing a series of deletion and substitution mutants for all three CaP isoforms. Here we demonstrate that the C-terminal sequences regulate actin association by altering the function of the second actin-binding site, ABS2, in CaP comprised of the three 29-residue calponin repeats. Removal of the inhibitory tail resulted in an increased binding and bundling activity, and caused a prominent re-localization of h2 CaP from the peripheral actin network to the central actin stress fibers in transfected A7r5 smooth muscle cells. Domain-swap experiments demonstrated that the tail sequence of h2 CaP can downregulate cytoskeletal association efficiently in all three CaP isoforms, whereas the tail of the smooth-muscle-specific h1 CaP variant had little effect. Site-directed mutagenesis further revealed that the negatively charged residues within the tail region are essential for this regulatory function. Finally we demonstrate that the tail sequences regulate the second actin-binding site (ABS2) and not the strong actin-binding ABS1 region in CaP.

Functional plasticity of CH domains.

With the refinement of algorithms for the identification of distinct motifs from sequence databases, especially those using secondary structure predictions, new protein modules have been determined in recent years. Calponin homology (CH) domains were identified in a variety of proteins ranging from actin cross-linking to signaling and have been proposed to function either as autonomous actin binding motifs or serve a regulatory function. Despite the overall structural conservation of the unique CH domain fold, the individual modules display a quite striking functional variability. Analysis of the actopaxin/parvin protein family suggests the existence of novel (type 4 and type 5) CH domain families which require special attention, as they appear to be a good example for how CH domains may function as scaffolds for other functional motifs of different properties.

Modulation of substrate adhesion dynamics via microtubule targeting requires kinesin-1.

Recent studies have shown that the targeting of substrate adhesions by microtubules promotes adhesion site disassembly (Kaverina, I., O. Krylyshkina, and J.V. Small. 1999. J. Cell Biol. 146:1033-1043). It was accordingly suggested that microtubules serve to convey a signal to adhesion sites to modulate their turnover. Because microtubule motors would be the most likely candidates for effecting signal transmission, we have investigated the consequence of blocking microtubule motor activity on adhesion site dynamics. Using a function-blocking antibody as well as dynamitin overexpression, we found that a block in dynein-cargo interaction induced no change in adhesion site dynamics in Xenopus fibroblasts. In comparison, a block of kinesin-1 activity, either via microinjection of the SUK-4 antibody or of a kinesin-1 heavy chain construct mutated in the motor domain, induced a dramatic increase in the size and reduction in number of substrate adhesions, mimicking the effect observed after microtubule disruption by nocodazole. Blockage of kinesin activity had no influence on either the ability of microtubules to target substrate adhesions or on microtubule polymerisation dynamics. We conclude that conventional kinesin is not required for the guidance of microtubules into substrate adhesions, but is required for the focal delivery of a component(s) that retards their growth or promotes their disassembly.