Clonal Elimination of the Pathogenic Allele as Diagnostic Pitfall in SAMD9L-Associated Neuropathy.

BACKGROUND: Heterozygous gain-of-function variants in SAMD9L are associated with ataxia-pancytopenia syndrome (ATXPC) and monosomy 7 myelodysplasia and leukemia syndrome-1 (M7MLS1). Association with peripheral neuropathy has rarely been described. METHODS: Whole-exome sequencing (WES) from DNA extracted from peripheral blood was performed in a 10-year-old female presenting with demyelinating neuropathy, her similarly affected mother and the unaffected maternal grandparents. In addition to evaluation of single nucleotide variants, thorough work-up of copy number and exome-wide variant allele frequency data was performed. RESULTS: Combined analysis of the mother's and daughter's duo-exome data and analysis of the mother's and her parents' trio-exome data initially failed to detect a disease-associated variant. More detailed analysis revealed a copy number neutral loss of heterozygosity of 7q in the mother and led to reanalysis of the exome data for respective sequence variants. Here, a previously reported likely pathogenic variant in the SAMD9L gene on chromosome 7q (NM_152703.5:c.2956C>T; p.(Arg986Cys)) was identified that was not detected with standard filter settings because of a low percentage in blood cells (13%). The variant also showed up in the daughter at 32%, a proportion well below the expected 50%, which in each case can be explained by clonal selection processes in the blood due to this SAMD9L variant. CONCLUSION: The report highlights the specific pitfalls of molecular genetic analysis of SAMD9L and, furthermore, shows that gain-of-function variants in this gene can lead to a clinical picture associated with the leading symptom of peripheral neuropathy. Due to clonal hematopoietic selection, displacement of the mutant allele occurred, making diagnosis difficult.

Evaluation of prognostic factors after primary chemoradiotherapy of anal cancer: A multicenter study of the German Cancer Consortium-Radiation Oncology Group (DKTK-ROG).

BACKGROUND AND PURPOSE: Prognosis after chemoradiotherapy (CRT) for anal squamous cell carcinoma (ASCC) shows marked differences among patients according to TNM subgroups, however individualized risk assessment tools to better stratify patients for treatment (de-) escalation or intensified follow-up are lacking in ASCC. MATERIALS AND METHODS: Patients' data from eight sites of the German Cancer Consortium - Radiation Oncology Group (DKTK-ROG), comprising a total of 605 patients with ASCC, treated with standard definitive CRT with 5-FU/Mitomycin C or Capecitabine/Mitomycin C between 2004-2018, were used to evaluate prognostic factors based on Cox regression models for disease-free survival (DFS). Evaluated variables included age, gender, Karnofsky performance score (KPS), HIV-status, T-category, lymph node status and laboratory parameters. Multivariate cox models were separately constructed for the whole cohort and the subset of patients with early-stage (cT1-2 N0M0) tumors. RESULTS: After a median follow-up of 46 months, 3-year DFS for patients with early-stage ASCC was 84.9%, and 67.1% for patients with locally-advanced disease (HR 2.4, p < 0.001). T-category (HR vs. T1: T2 2.02; T3 2.11; T4 3.03), N-category (HR versus N0: 1.8 for N1-3), age (HR 1.02 per year), and KPS (HR 0.8 per step) were significant predictors for DFS in multivariate analysis in the entire cohort. The model performed with a C-index of 0.68. In cT1-2N0 patients, T-category (HR 2.14), HIV status (HR 2.57), age (1.026 per year), KPS (HR 0.7 per step) and elevated platelets (HR 1.3 per 100/nl) were associated with worse DFS (C-index of 0.7). CONCLUSION: Classical clinicopathologic parameters like T-category, N-category, age and KPS remain to be significant prognostic factors for DFS in patients treated with contemporary CRT for ASCC. HIV and platelets were significantly associated with worse DFS in patients with early stage ASCC.

Biallelic CSGALNACT1-mutations cause a mild skeletal dysplasia.

Genetic causes of skeletal disorders are manifold and affect, among others, enzymes of bone and connective tissue synthesis pathways. We present a twelve-year-old boy with a mild skeletal dysplasia, hypermobility of joints and axial malalignment of lower limbs and feet. Exome sequencing revealed a biallelic loss of function mutation in CSGALNACT1, which encodes chondroitin sulfate N-acetylgalactosaminyltransferase 1 and plays a major role in the chondroitin sulfate chain biosynthesis and therefore in the synthesis of glycosaminoglycans. Recently, the first case of a pediatric patient with a mild skeletal dysplasia due to a compound heterozygous large intragenic deletion and a damaging missense variant in CSGALNACT1 was reported. We here identify a second case and the first juvenile patient with a homozygous frameshift variant in CSGALNACT1 which corroborates its role in mild and non-progressive skeletal dysplasia with joint laxity.

[Rational use of genetic tests in internal medicine : Possibilities and limitations of next generation sequencing diagnostics]. / Rationaler Einsatz genetischer Tests in der Inneren Medizin : Möglichkeiten und Limitationen der Next-generation-sequencing-Diagnostik.

BACKGROUND: New methods of molecular genetic diagnostics enable a more comprehensive genetic analysis of patients. OBJECTIVES: Rational use and benefits of molecular genetic testing in patients with various internal diseases. METHOD: Evaluation of topic-related literature, discussion of own experiences, as well as consideration of current guidelines. RESULTS: New genetic tests, such as next generation sequencing (NGS), improve the diagnosis of hereditary diseases; however, the use of this technology also leads to additional findings, which must be carefully considered. CONCLUSION: The rational use of genetic tests is a benefit for patients and can significantly influence the prevention and treatment of a disease. The increasing complexity of genetic findings requires interdisciplinary approaches involving human genetics, internal medicine, and other disciplines.

Mutations in CRLF1 cause familial achalasia.

We here report a family from Libya with three siblings suffering from early onset achalasia born to healthy parents. We analyzed roughly 5000 disease-associated genes by a next-generation sequencing (NGS) approach. In the analyzed sibling we identified two heterozygous variants in CRLF1 (cytokine receptor-like factor 1). Mutations in CRLF1 have been associated with autosomal recessive Crisponi or cold-induced sweating syndrome type 1 (CS/CISS1), which among other symptoms also manifests with early onset feeding difficulties. Segregation analysis revealed compound heterozygosity for all affected siblings, while the unaffected mother carried the c.713dupC (p.Pro239Alafs*91) and the unaffected father carried the c.178T>A (p.Cys60Ser) variant. The c.713dupC variant has already been reported in affected CS/CISS1 patients, the pathogenicity of the c.178T>A variant was unclear. As reported previously for pathogenic CRLF1 variants, cytokine receptor-like factor 1 protein secretion from cells transfected with the c.178T>A variant was severely impaired. From these results we conclude that one should consider a CRLF1-related disorder in early onset achalasia even if other CS/CISS1 related symptoms are missing.

[Social support in gender reassignment surgery]. / Soziale Unterstützung bei genitalangleichender Operation.

BACKGROUND: Gender reassignment surgery (GRS) can lead to discrimination. This transition makes great demands on the individual and also affects the social environment. OBJECTIVES: To evaluate the social support of male-to-female (MtF) transgender people. MATERIALS AND METHODS: Group A comprised 254 consecutive MtF transsexuals, who received a penile inversion vaginoplasty between 2004 and 2010. These women were surveyed retrospectively. Group B comprised 144 consecutive MtF transsexuals who presented for preoperative counselling. These patients were asked to answer the survey in advance of the planned GRS. RESULTS: The return rate was 46.9 % (A) and 95.1 % (B). In both groups, approximately two-thirds lived with their parents or children at ease. About 13.4 % (A) and 16.9 % (B) estimated the relationship towards their parents and one- seventh (A) or one-sixth (B) woman rated their relationship towards their children as poor. The acceptance of the parents regarding GRS was 65.6 % (A) and 77.1 % (B). In total 20 % (A) and 9.2 % (B) did not, however, accept GRS in their children. The acceptance of children regarding GRS was 64.9 % (A) and 71.1 % (B) with 10.8 % (A) and 6.7 % (B) who did not approve the decision. DISCUSSION: Social support is an important resource in the context of gender reassignment surgery. Understanding can help to improve the situation for transsexuals and to reduce consecutive healthcare utilisation.

[Sensory and autonomic neuropathies and pain-related channelopathies]. / Sensorisch-autonome Neuropathien und Natriumkanal-assoziierte Schmerzerkrankungen.

Loss of pain perception can result from neurodevelopmental defects, degeneration of nociceptive fibers, or altered excitability of sensory neurons. Hereditary neurodegeneration leading to pain loss is classified as sensory and autonomic neuropathy (HSAN). Mutations in approximately 15 genes have been identified in the group of HSAN disorders. Hallmark of the disease is a liability to injury because of impaired acute pain as a warning system to prevent harm. The clinically overlapping "congenital insensitivity to pain (CIP)" is caused by mutations in voltage-gated sodium channels, which control the excitability of nociceptors. However, mutations in the latter genes can also result in disorders with increased pain susceptibility. This review summarizes the clinical presentation of HSAN and pain-related channelopathies and discusses the underlying disease mechanisms.

A Novel Homozygous WDR72 Mutation in Two Siblings with Amelogenesis Imperfecta and Mild Short Stature.

Amelogenesis imperfecta (AI) is a clinically and genetically heterogeneous group of inherited defects of enamel formation. In isolated AI (no additional segregating features), mutations in at least 7 genes are known so far, causing dominant, recessive or X-linked AI and allowing the identification of the molecular etiology in 40-50% of affected families. We report on 2 siblings (an 11-year-old female and a 7-year-old male) born to consanguineous Turkish parents, with AI and mild, proportionate short stature. Both parents have normal teeth, but mother, maternal grandmother and great-grandfather are/were also of short stature. A spine X-ray performed in the girl excluded brachyolmia. Affymetrix GenomeWide SNP6.0 Array analysis identified no pathogenic copy number changes, but showed sharing of large homozygous regions, including chromosome band 15q21.3 containing the WDR72 gene. WDR72 sequence analysis in both siblings revealed homozygosity for a novel stop mutation in exon 10 (c.997A>T, p.Lys333X) explaining the AI phenotype. Mutations in WDR72 are a very rare cause of autosomal-recessive hypomaturation type of isolated AI. The mutation described in our patients specifies the diagnosis AI IIA3 and represents only the sixth WDR72 mutation reported so far. The WDR72 protein is critical for dental enamel formation, but its exact function is still unknown.

[Acute superficial thrombophlebitis--therapeutic strategies]. / Strategien zur Antikoagulation und Operation bei akuter Thrombophlebitis.

UNLABELLED: Thrombophlebitis (TP) of the superficial venous system is associated to a high percentage with deep venous thrombosis (DVT). References in literature vary between 5 and 65 %, pulmonary embolisms (LE) were described in up to 33 %. PATIENTS: In a retrospective study, 114 patients who had presented themselves with a TP of the superficial venous system between January 1 (st) and December 31 (st) 2004, were analysed in our institution. 50 % (n = 57) exhibited a TP in side branches of the superficial venous system. 19.3 % (n = 22) showed a TP of the great saphenous vein (GSV) of the calf or of the small saphenous vein (SSV) distally, in 28.1 % (n = 32) the GSV or SSV were affected at the thigh or proximally or in total length, 3 patients (2.6 %) exhibited a TP of the arm vein. 11 patients (9.6 %) showed a concomitant DVT. The frequency of DVT depended on the localisation and extension of the TP, and also on additional basic and acute risks for DVT. The incidence of a concomitant DVT was 5.2 % when side branches were affected and amounted to 15.6 % with TP in the area of the GSV or SSV. With varicosis as single risk factor, the frequency of a concomitant DVT was 6 %, varicosis combined with further risks showed a DVT frequency of 15.4 %. RESULTS: All patients were treated with low molecular weight heparin either with prophylactic or therapeutic dosage, depending on localisation, extension and concomitant diseases. 10.5 % of the patients (n = 12) had to undergo urgent surgery with ligation of the sapheno-femoral junction or popliteal junction, if the TP had reached the junction into the deep venous system. By this therapy, we had not to observe any additional DVT. In 9 cases, an extension, respectively a recurrence of the TP could be observed. In each of these cases the dosage of the LMWH had not been adapted to the concomitant risks or had been terminated too early. DISCUSSION: TP of the superficial venous system should be considered and treated as DVT. Consequent anticoagulation is needed, surgery should be performed when the TP reaches the junction into the deep venous system. The duration of the anticoagulation is not quite clear, but is carried out in our institution for three months with therapeutic intention.

[Results of standard varicose vein surgery]. / Ergebnisse nach klassischer Varizenchirurgie.

The principles of modern varicose vein surgery are based on the interruption of the cranial and distal points of venous insufficiency. Especially due to the rise of alternate surgery procedures, we have scrutinized our results of varicose vein surgery. In a retrospective analysis, the results of those patients (pat.) who underwent venous surgery in our institution in 1995 were analysed. In 1995 we performed 1 575 varicose vein operations (n = 1 019 pat., 16.8 % male, 83.2 % female). 63.5 % were on an outpatient basis. During a follow-up period of 4 to 66 months (av. 38 months) the patients were re-examined, 481 (47.2 %) by Duplex sonography, 94 (9.2 %) by clinical examination alone, from 103 (10.1 %) information was obtained through a written inquiry. 341 pat. (33.5 %) had just perioperative follow-ups. We didn't find any signs of varicosis in 301 pat. (33.3 %). Minor side branches could be detected in 515 pat. (56.8 %). In 90 pat. (9.9 %) a clearly visible varicosis could be seen. Based on the results of the Duplex examinations, 86 % of the pat. showed no recurrence after ligation of the sapheno-femoral junction and stripping of the LSV, the results after stripping of the short saphenous vein were similar. Analysis of the inquiry forms concluded that 62.3 % of the pat. were satisfied with surgery and the results. The results of the standard varicose vein surgery are satisfactory regarding recurrence rate and patient satisfaction. Our results are comparable to those published in prospective randomised studies. Alternate procedures, for example the CHIVA method, have still to proof their efficiency, especially in view of long-term results.

Monocyte selectivity and tissue localization suggests a role for breast and kidney-expressed chemokine (BRAK) in macrophage development.

Although numerous chemokines act on monocytes, none of them is specific for these cells. Here, we show that breast and kidney-expressed chemokine (BRAK) is a highly selective monocyte chemoattractant. Migration efficacy and Bordetella pertussis toxin-sensitive Ca(2+) mobilization responses to BRAK were strongly enhanced after treatment of monocytes with the cyclic AMP-elevating agents prostaglandin E(2) and forskolin. BRAK is the first monocyte-selective chemokine, as other types of blood leukocytes or monocyte-derived dendritic cells and macrophages did not respond. Expression in normal skin keratinocytes and dermal fibroblasts as well as lamina propria cells in normal intestinal tissues suggests a homeostatic rather than an inflammatory function for this chemokine. In addition, macrophages were frequently found to colocalize with BRAK-producing fibroblasts. We propose that BRAK is involved in the generation of tissue macrophages by recruiting extravasated precursors to fibroblasts, which are known to secrete essential cytokines for macrophage development.

[Indications and technique for sclerotherapy of varicose veins]. / Indikation und Technik zur Sklerotherapie bei Varikose.

Sclerotherapy is in general a very safe method for the treatment of teleangiectasias and reticular varicose veins done by an experienced therapist. The complication-rate is very low, with the use of Polidocanol the rate of skin-necrosis ranges between 0.001 and 0.2%, the incidence of superficial thrombophlebitis is 0.08%, the incidence of an allergic reaction is reported with 0.2%. The sclerotherapy of large perforating veins and the long or short saphenous veins is controversially discussed. The results of prospective randomized studies show better results with operative treatment in comparison to sclerotherapy. The recurrence-rate of sclerotherapy of the long saphenous vein varies between 22 and 37% after 3 years, between 40 and 70% after 5 years and 94% after 10 years. Before starting the sclerotherapy the extension of the varicose vein disease should be diagnosed correctly. The diagnosis can be obtained with duplex-ultrasound, that gives either functional and morphologic information about the deep and superficial venous system.

Two different epitopes of the signal transducer gp130 sequentially cooperate on IL-6-induced receptor activation.

Cytokines are key mediators for the regulation of hemopoiesis and the coordination of immune responses. They exert their various functions through activation of specific cell surface receptors, thereby initiating intracellular signal transduction cascades which lead to defined cellular responses. As the common signal-transducing receptor subunit of at least seven different cytokines, gp130 is an important member of the family of hemopoietic cytokine receptors which are characterized by the presence of at least one cytokine-binding module. Mutants of gp130 that either lack the Ig-like domain D1 (DeltaD1) or contain a distinct mutation (F191E) within the cytokine-binding module have been shown to be severely impaired with respect to IL-6 induced signal transduction. After cotransfection of COS-7 cells with a combination of both inactive gp130 mutants, signal transduction in response to IL-6 is restored. Whereas cells transfected with DeltaD1 do not bind IL-6/sIL-6R complexes, cells transfected with the F191E mutant bind IL-6/sIL-6R with low affinity. Combination of DeltaD1 and F191E, however, leads to high-affinity ligand binding. These data suggest that two different gp130 epitopes, one on each receptor chain, sequentially cooperate in asymmetrical binding of IL-6/IL-6R in a tetrameric signaling complex. On the basis of our data, a model for the mechanism of IL-6-induced gp130 activation is proposed.

Different epitopes are required for gp130 activation by interleukin-6, oncostatin M and leukemia inhibitory factor.

Gp130 is the common signal transducing receptor subunit of interleukin (IL)-6, IL-11, leukemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor and cardiotrophin-1. IL-6 and IL-11 induce gp130 homodimerization whereas the others lead to the formation of heterodimers with LIFR or OSMR. Binding epitopes for IL-6 and IL-11 are located in the immunoglobulin-like domain and the cytokine binding module (CBM). Here we show that a gp130 mutant lacking domain 1, although unresponsive to IL-6 and IL-11, can still activate signal transducer and activator of transcription (STAT) transcription factors in response to LIF or OSM. Moreover, point mutations in the CBM of gp130 (F191E and V252D) that severely impair signal transduction in response to IL-6 and IL-11 differentially interfere with gp130 activation in response to LIF and OSM. Thus, epitopes involved in gp130 homodimerization are distinct from those leading to the formation of gp130/LIFR or gp130/OSMR heterodimers. These findings may serve as the base for rational design of gp130 antagonists that specifically interfere with bioactivity of distinct IL-6-type cytokines.

Importance of the membrane-proximal extracellular domains for activation of the signal transducer glycoprotein 130.

The transmembrane glycoprotein gp130 is the common signal transducing receptor subunit of the IL-6-type cytokines. The gp130 extracellular part is predicted to consist of six individual domains. Whereas the role of the three membrane-distal domains (D1-D3) in binding of IL-6 and IL-11 is well established, the function of the membrane-proximal domains (D4-D6) is unclear. Mapping of a neutralizing mAb to the membrane-proximal part of gp130 suggests a functional role of D4-D6 in receptor activation. Individual deletion of these three domains differentially interferes with ligand binding of the soluble and membrane-bound receptors. All deletion mutants do not signal in response to IL-6 and IL-11. The deletion mutants Delta4 and, to a lesser extent, Delta6 are still activated by agonistic monoclonal gp130 Abs, whereas the deletion mutant Delta5 does not respond. Because membrane-bound Delta5 binds IL-6/soluble IL-6R as does wild-type gp130, but does not transduce a signal in response to various stimuli, this domain plays a prominent role in coupling of ligand binding and signal transduction. Replacement of the fifth domain of gp130 by the corresponding domain of the homologous G-CSF receptor leads to constitutive activation of the chimera upon overexpression in COS-7 cells. In HepG2 cells this mutant responds to IL-6 comparable to wild-type gp130. Our findings suggest a functional role of the membrane-proximal domains of gp130 in receptor activation. Thus, within the hematopoietic receptor family the mechanism of receptor activation critically depends on the architecture of the receptor ectodomain.

Activation of the signal transducer glycoprotein 130 by both IL-6 and IL-11 requires two distinct binding epitopes.

The coordination and regulation of immune responses are primarily mediated by cytokines that bind to specific cell surface receptors. Glycoprotein 130 (gp130) belongs to the family of class I cytokine receptors and is the common signal-transducing receptor subunit shared by the so-called IL-6 type cytokines (IL-6, IL-11, ciliary neurotrophic factor, leukemia inhibitory factor, oncostatin M, and cardiotrophin-1). The inflammatory cytokines IL-6 and IL-11 induce gp130 homodimerization after binding to their specific alpha receptors, which leads to the activation of the Janus kinase/STAT signal transduction pathway. A molecular model of IL-6/IL-6R/gp130, which is based on the structure of the growth hormone/growth hormone receptor complex, allowed the selection of several amino acids located in the cytokine-binding module of gp130 for mutagenesis. The mutants were analyzed with regard to IL-6- or IL-11-induced STAT activation and ligand binding. It was found that Y190 and F191 are essential for the interaction of gp130 with IL-6 as well as IL-11, suggesting a common mode of recognition of helical cytokines by class I cytokine receptors. Furthermore, the requirement of the gp130 N-terminal Ig-like domain for ligand binding and signal transduction was demonstrated by the use of deletion mutants. Thus, besides the observed analogy to the growth hormone/growth hormone receptor complex, there is a substantial difference in the mechanism of receptor engagement by cytokines that signal via gp130.