RESUMEN

Clonality assessment by the detection of immunoglobulin (IG) gene rearrangements is an important method to determine whether two concurrent or subsequent lymphoid malignancies in one patient are clonally related. Here, we report the detailed clonality analysis in a patient with a diagnosis of B-cell acute lymphoblastic leukemia (B-ALL) followed by a histiocytic sarcoma (HS), in which we were able to study clonal evolution by applying next generation sequencing (NGS) to identify IG rearrangements and gene mutations. Using the sequence information of the NGS-based IG clonality analysis, multiple related subclones could be distinguished in the PAX5 P80R-mutated B-ALL. Notably, only one of these subclones evolved into HS after acquiring a RAF1 mutation. This case demonstrates that NGS-based IG clonality assessment and mutation analysis provide clear added value for clonal comparison and thereby improves clinicobiological understanding.

Asunto(s)

Linfoma de Burkitt , Sarcoma Histiocítico , Leucemia-Linfoma Linfoblástico de Células Precursoras , Humanos , Sarcoma Histiocítico/genética , Sarcoma Histiocítico/patología , Inmunoglobulinas/genética , Reordenamiento Génico , Linfoma de Burkitt/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Factor de Transcripción PAX5/genética

RESUMEN

OBJECTIVES: In 2010, a new subtype of salivary gland cancer (SGC), (mammary analogue) secretory carcinoma (SC), was defined, characterized by the ETV6-NTRK3 fusion gene. As clinical behavior and outcome data of this histological subtype tumor are still sparse, we aimed to describe the clinicopathological course and outcome of a series of translocation positive SC patients. PATIENT AND METHODS: We re-evaluated the pathological diagnosis of a subset of SGCs, diagnosed in 4 of 8 Dutch head and neck centers. Subsequently, tumors with a morphological resemblance to SC were tested for the ETV6-NTRK3 fusion gene using RT-PCR. Furthermore, patients prospectively diagnosed with SC were included. The clinical characteristics and outcomes were retrieved from the patient files. RESULTS: Thirty-one patients with ETV6-NTRK3 fusion gene positive SC were included. The median age was 49 years, 17 patients (55%) were male. Eighteen tumors (58%) arose in the parotid gland. One patient presented with lymph node metastasis. All patients underwent tumor resection and 4 patients had a neck dissection. Four patients had re-resection and 15 patients (48%) received postoperative radiotherapy. One patient developed a local recurrence, no regional recurrences or distant metastases were observed. After a median follow-up of 49 months the 5- and 10-year overall survival were 95%, the 5- and 10-year disease free survival were 89%. CONCLUSION: The clinical course of SC is favorable with a low rate of locoregional recurrence and excellent survival. Given the low incidence of nodal metastases, elective neck treatment, i.e. surgery and/or radiotherapy, does not seem to be indicated.

Asunto(s)

Proteínas de Fusión Oncogénica/genética , Neoplasias de las Glándulas Salivales/patología , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Recurrencia Local de Neoplasia , Neoplasias de las Glándulas Salivales/genética , Análisis de Supervivencia , Adulto Joven

Asunto(s)

Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Neoplasias/tratamiento farmacológico , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor , Estudios de Casos y Controles , Terapia Combinada , Femenino , Estudios de Seguimiento , Trastornos Histiocíticos Malignos , Humanos , Técnicas para Inmunoenzimas , Inmunofenotipificación , Metástasis Linfática , Linfoma de Células B Grandes Difuso , Masculino , Persona de Mediana Edad , Clasificación del Tumor , Invasividad Neoplásica , Recurrencia Local de Neoplasia/tratamiento farmacológico , Recurrencia Local de Neoplasia/patología , Estadificación de Neoplasias , Neoplasias/patología , Leucemia-Linfoma Linfoblástico de Células Precursoras , Pronóstico , Estudios Prospectivos , Tasa de Supervivencia

RESUMEN

PCR-based immunoglobulin (Ig)/T-cell receptor (TCR) clonality testing in suspected lymphoproliferations has largely been standardized and has consequently become technically feasible in a routine diagnostic setting. Standardization of the pre-analytical and post-analytical phases is now essential to prevent misinterpretation and incorrect conclusions derived from clonality data. As clonality testing is not a quantitative assay, but rather concerns recognition of molecular patterns, guidelines for reliable interpretation and reporting are mandatory. Here, the EuroClonality (BIOMED-2) consortium summarizes important pre- and post-analytical aspects of clonality testing, provides guidelines for interpretation of clonality testing results, and presents a uniform way to report the results of the Ig/TCR assays. Starting from an immunobiological concept, two levels to report Ig/TCR profiles are discerned: the technical description of individual (multiplex) PCR reactions and the overall molecular conclusion for B and T cells. Collectively, the EuroClonality (BIOMED-2) guidelines and consensus reporting system should help to improve the general performance level of clonality assessment and interpretation, which will directly impact on routine clinical management (standardized best-practice) in patients with suspected lymphoproliferations.

Asunto(s)

Inmunoglobulinas/genética , Trastornos Linfoproliferativos/diagnóstico , Receptores de Antígenos de Linfocitos T/genética , ADN/análisis , Reordenamiento Génico , Guías como Asunto , Humanos , Trastornos Linfoproliferativos/genética , Reacción en Cadena de la Polimerasa Multiplex

RESUMEN

B-cell clonality detection in whole tissue is considered indicative of B-cell non-Hodgkin lymphoma (NHL). We tested frozen tissue of 24 classical Hodgkin lymphomas (cHL) with a varying tumor cell load with the multiplex polymerase chain reaction (PCR) primer sets for IGH and IGK gene rearrangement (BIOMED-2). A clonal population was found in 13 cases with the IGH FR1 and/or FR2/FR3 PCRs. Using the IGK-VJ and IGK-DE PCRs, an additional six cases had a dominant clonal cell population, resulting in a detection rate of 79% in frozen tissue. Of 12 cases, also the formalin-fixed and paraffin-embedded (FFPE) tissue was tested. Surprisingly, in eight of the 12 FFPE cases with acceptable DNA quality (allowing PCR amplification of >200 nt fragments), the IGK multiplex PCRs performed better in detecting clonality (six out of eight clonal IGK rearrangements) than the IGH PCRs (four out of nine clonal rearrangements), despite a rather large amplicon size. There was no evidence of B-cell lymphoma during follow-up of 1 to 6 years and no correlation was found between the presence of a clonal result and Epstein-Barr virus in the tumor cells. Our results indicate that the present routine PCR methods are sensitive enough to detect small numbers of malignant cells in cHL. Therefore, the presence of a clonal B-cell population does not differentiate between cHL and NHL.

RESUMEN

This review deals with podocyte proteins that play a significant role in the structure and function of the glomerular filter. Genetic linkage studies has identified several genes involved in the development of nephrotic syndrome and contributed to the understanding of the pathophysiology of glomerular proteinuria and/or focal segmental glomerulosclerosis. Here, we describe already well-characterized genetic diseases due to mutations in nephrin, podocin, CD2AP, alpha-actinin-4, WT1, and laminin beta2 chain, as well as more recently identified genetic abnormalities in TRPC6, phospholipase C epsilon, and the proteins encoded by the mitochondrial genome. In addition, the role of the proteins which have shown to be important for the structure and functions by gene knockout studies in mice, are also discussed. Furthermore, some rare syndromes with glomerular involvement, in which molecular defects have been recently identified, are briefly described. In summary, this review updates the current knowledge of genetic causes of congenital and childhood nephrotic syndrome and provides new insights into mechanisms of glomerular dysfunction.

Asunto(s)

Marcadores Genéticos/genética , Glomeruloesclerosis Focal y Segmentaria/genética , Glomérulos Renales/fisiopatología , Podocitos , Actinina/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas del Citoesqueleto/genética , Genoma , Glomeruloesclerosis Focal y Segmentaria/fisiopatología , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Laminina/genética , Proteínas de la Membrana/genética , Mutación , Fosfoinositido Fosfolipasa C/genética , Proteinuria/genética , Canales Catiónicos TRPC/genética , Canal Catiónico TRPC6 , Proteínas WT1/genética

RESUMEN

OBJECTIVE: Patients with a gastrointestinal stromal tumour (GIST) suffering from non-islet cell tumour-induced hypoglycaemia (NICTH), being associated with increased plasma levels of pro-insulin-like growth factor (IGF)-IIE[68-88], have been reported occasionally. We studied the clinical relevance of pro-IGF-IIE[68-88] and other IGF-related proteins in GIST patients. PATIENTS AND METHODS: Twenty-four patients were included. Plasma samples were collected before 1 week and median 5 months after start of treatment with imatinib, and levels of IGF-I, total IGF-II, pro-IGF-IIE[68-88], insulin-like growth factor-binding protein (IGFBP)-2, -3 and -6 were determined. GIST specimens from 17 patients and tumour cyst fluid from two patients were analysed for IGF-II and IGFBP-2. RESULTS: Before treatment and/or during follow-up, 3 of 24 (13%) patients showed increased plasma levels of pro-IGF-IIE[68-88]. All three developed NICTH. Overall, patients with metastatic disease, elevated serum lactate dehydrogenase activity or total tumour size >12 cm had the highest pro-IGF-IIE[68-88] levels. Most patients had increased plasma IGFBP-2 levels and these levels were significantly higher in patients with progressive disease. (Pro-)IGF-II was expressed in 82% of GISTs and IGFBP-2 only in one case. CONCLUSION: We identified pro-IGF-IIE[68-88] as a marker that may be used in the surveillance of GIST.

Asunto(s)

Tumores del Estroma Gastrointestinal/complicaciones , Hipoglucemia/etiología , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Somatomedinas/análisis , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Líquido Quístico/química , Líquido Quístico/metabolismo , Femenino , Tumores del Estroma Gastrointestinal/sangre , Tumores del Estroma Gastrointestinal/patología , Humanos , Hipoglucemia/sangre , Hipoglucemia/epidemiología , Incidencia , Masculino , Persona de Mediana Edad , Adulto Joven

Asunto(s)

Dermatitis Exfoliativa/patología , Síndromes de Inmunodeficiencia/patología , Inmunodeficiencia Combinada Grave/patología , Biopsia , Dermatitis Exfoliativa/genética , Diagnóstico Diferencial , Humanos , Síndromes de Inmunodeficiencia/genética , Lactante , Masculino , Inmunodeficiencia Combinada Grave/genética , Piel/patología

RESUMEN

Focal segmental glomerulosclerosis (FSGS) is a histologic diagnosis in several kidney diseases characterized by proteinuria and a severe decrease in kidney function. Mutations in several genes were found in patients with primary FSGS, one of which is a CD2-associated protein CD2AP (originally referred to as CMS). This gene encodes an adaptor protein that plays a role in endocytosis, cell motility, and cell survival. Mice deficient in Cd2ap (the mouse homolog) die due to kidney failure, while heterozygous mice develop lesions similar to those of FSGS patients. In the kidney, CD2AP regulates the actin cytoskeleton. The only previously described patient with CD2AP mutation had a severely truncated protein. In this study, we describe a patient with a novel mutation resulting in a premature stop codon yielding a protein truncated by only 4%. This shortened CD2AP protein displays a significantly decreased F-actin binding efficiency in vitro with no expression of the mutated allele in the patient's lymphocytes. Heterozygous expression of the CD2AP mutation in both parents did not lead to any kidney pathology, as both have normal glomerular filtration rates and no proteinuria.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas del Citoesqueleto/genética , Glomeruloesclerosis Focal y Segmentaria/genética , Glomeruloesclerosis Focal y Segmentaria/patología , Homocigoto , Mutación , Actinas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Biopsia , Cadáver , Preescolar , Codón de Terminación/genética , Consanguinidad , Tasa de Filtración Glomerular , Glomeruloesclerosis Focal y Segmentaria/cirugía , Humanos , Glomérulos Renales/metabolismo , Glomérulos Renales/fisiología , Glomérulos Renales/ultraestructura , Trasplante de Riñón , Masculino , Datos de Secuencia Molecular , Técnicas de Amplificación de Ácido Nucleico , Unión Proteica , Estructura Terciaria de Proteína , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Resultado del Tratamiento

RESUMEN

Polymerase chain reaction (PCR) assessment of clonal T-cell receptor (TCR) and immunoglobulin (Ig) gene rearrangements is an important diagnostic tool in mature T-cell neoplasms. However, lack of standardized primers and PCR protocols has hampered comparability of data in previous clonality studies. To obtain reference values for Ig/TCR rearrangement patterns, 19 European laboratories investigated 188 T-cell malignancies belonging to five World Health Organization-defined entities. The TCR/Ig spectrum of each sample was analyzed in duplicate in two different laboratories using the standardized BIOMED-2 PCR multiplex tubes accompanied by international pathology panel review. TCR clonality was detected in 99% (143/145) of all definite cases of T-cell prolymphocytic leukemia, T-cell large granular lymphocytic leukemia, peripheral T-cell lymphoma (unspecified) and angioimmunoblastic T-cell lymphoma (AILT), whereas nine of 43 anaplastic large cell lymphomas did not show clonal TCR rearrangements. Combined use of TCRB and TCRG genes revealed two or more clonal signals in 95% of all TCR clonal cases. Ig clonality was mostly restricted to AILT. Our study indicates that the BIOMED-2 multiplex PCR tubes provide a powerful strategy for clonality assessment in T-cell malignancies assisting the firm diagnosis of T-cell neoplasms. The detected TCR gene rearrangements can also be used as PCR targets for monitoring of minimal residual disease.

Asunto(s)

Genes de Inmunoglobulinas , Leucemia de Células T/genética , Linfoma de Células T/genética , Reacción en Cadena de la Polimerasa/métodos , Receptores de Antígenos de Linfocitos T/genética , Amplificación de Genes , Reordenamiento Génico , Genotipo , Humanos , Inmunohistoquímica , Leucemia Prolinfocítica/genética , Leucemia Prolinfocítica/inmunología , Leucemia Prolinfocítica/patología , Leucemia de Células T/inmunología , Leucemia de Células T/patología , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/inmunología , Linfoma de Células B Grandes Difuso/patología , Linfoma de Células T/inmunología , Linfoma de Células T/patología , Linfocitos T/inmunología

RESUMEN

Polymerase chain reaction (PCR) assessment of clonal immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements is an important diagnostic tool in mature B-cell neoplasms. However, lack of standardized PCR protocols resulting in a high level of false negativity has hampered comparability of data in previous clonality studies. In order to address these problems, 22 European laboratories investigated the Ig/TCR rearrangement patterns as well as t(14;18) and t(11;14) translocations of 369 B-cell malignancies belonging to five WHO-defined entities using the standardized BIOMED-2 multiplex PCR tubes accompanied by international pathology panel review. B-cell clonality was detected by combined use of the IGH and IGK multiplex PCR assays in all 260 definitive cases of B-cell chronic lymphocytic leukemia (n=56), mantle cell lymphoma (n=54), marginal zone lymphoma (n=41) and follicular lymphoma (n=109). Two of 109 cases of diffuse large B-cell lymphoma showed no detectable clonal marker. The use of these techniques to assign cell lineage should be treated with caution as additional clonal TCR gene rearrangements were frequently detected in all disease categories. Our study indicates that the BIOMED-2 multiplex PCR assays provide a powerful strategy for clonality assessment in B-cell malignancies resulting in high Ig clonality detection rates particularly when IGH and IGK strategies are combined.

Asunto(s)

Genes de Inmunoglobulinas , Leucemia de Células B/genética , Linfoma de Células B/genética , Reacción en Cadena de la Polimerasa/métodos , Cromosomas Humanos Par 11 , Cromosomas Humanos Par 14 , Cromosomas Humanos Par 18 , Reordenamiento Génico , Genotipo , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Leucemia de Células B/diagnóstico , Leucemia de Células B/inmunología , Linfoma de Células B/diagnóstico , Linfoma de Células B/inmunología , Receptores de Antígenos de Linfocitos T/genética , Translocación Genética

RESUMEN

The diagnosis of malignant lymphoma is a recognized difficult area in histopathology. Therefore, detection of clonality in a suspected lymphoproliferation is a valuable diagnostic criterion. We have developed primer sets for the detection of rearrangements in the B- and T-cell receptor genes as reliable tools for clonality assessment in lymphoproliferations suspected for lymphoma. In this issue of Leukemia, the participants of the BIOMED-2 Concerted Action CT98-3936 report on the validation of the newly developed clonality assays in various disease entities. Clonality was detected in 99% of all B-cell malignancies and in 94% of all T-cell malignancies, whereas the great majority of reactive lesions showed polyclonality. The combined BIOMED-2 results are summarized in a guideline, which can now be implemented in routine lymphoma diagnostics. The use of this standardized approach in patients with a suspect lymphoproliferation will result in improved diagnosis of malignant lymphoma.

Asunto(s)

Linfoma/genética , Linfoma/patología , Reacción en Cadena de la Polimerasa/métodos , Reacciones Falso Negativas , Reordenamiento Génico , Humanos , Linfoma de Células B/genética , Linfoma de Células B/patología , Linfoma de Células T/genética , Linfoma de Células T/patología , Receptores de Antígenos de Linfocitos T/genética , Reproducibilidad de los Resultados

RESUMEN

The BIOMED-2 Concerted Action BMH4-CT98-3936 on 'Polymerase chain reaction (PCR)-based clonality studies for early diagnosis of lymphoproliferative disorders' developed standardized PCR protocols for detection of immunoglobulin (Ig) and T-cell receptor (TCR) rearrangements, including TCR beta (TCRB). As no comparable TCRB PCR method pre-existed and only a limited number of samples was tested within the BIOMED-2 study, we initiated this study for further validation of the newly developed TCRB PCR approach by comparing PCR data with previously generated Southern blot (SB) data in a series of 66 immature (ALL) and 36 mature T-cell malignancies. In 91% of cases, concordant PCR and SB results were found. Discrepancies consisted of either failure to detect SB-detected TCRB rearrangements by PCR (6.5%) or detection of an additional non-SB defined rearrangement (2.5%). In 99% of cases (99/100), at least one clonal TCRB rearrangement was detected by PCR in the SB-positive cases. A predominance of complete Vbeta-Jbeta rearrangements was seen in TCRalphabeta(+) T-cell malignancies and CD3-negative T-ALL (100 and 90%, respectively), whereas in TCRgammadelta(+) T-ALL, more incomplete Dbeta-Jbeta TCRB rearrangements were detected (73%). Our results underline the reliability of this new TCRB PCR method and its strategic applicability in clonality diagnostics of lymphoproliferative disorders and MRD studies.

Asunto(s)

Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T , Genes Codificadores de la Cadena beta de los Receptores de Linfocito T/genética , Leucemia-Linfoma de Células T del Adulto/genética , Reacción en Cadena de la Polimerasa/métodos , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Southern Blotting , ADN de Neoplasias/análisis , Humanos , Receptores de Antígenos de Linfocitos T gamma-delta/genética

RESUMEN

INTRODUCTION: Refractory coeliac disease (RCD) is a rare syndrome with a poor prognosis, defined by malabsorption due to gluten-related enteropathy after initial or subsequent failure of a strict gluten-free diet and after exclusion of any disorder mimicking coeliac disease. PATIENTS AND METHODS: Nineteen patients were included and treated. Based on intraepithelial T-lymphocyte(IEL) phenotyping, patients were recorded as having RCD type I with normal IELs, or RCD type II with phenotypically immature IELs defined by a lack of characteristic T-cell markers. Treatment consisted of azathioprine combined with prednisone for 1 year, which was tapered and, if possible, stopped. RESULTS: Clinical improvement was seen in nearly all patients in both groups. Eight of 10 RCD type I patients responded histologically, and complete normalization of villi was seen in four patients. In RCD type II, 6/8 patients developed enteropathy-associated T-cell lymphoma (EATL) and 7/8 patients died. CONCLUSIONS: For the first time we report a promising therapeutic treatment option for RCD type I. In RCD type II, azathioprine and prednisone therapy (APT) is not effective, therefore we suggest that other (chemo)therapeutic agents are considered. Not all RCD type II patients presented with a monoclonal TCRgamma-gene rearrangement and immunohistological changes as is currently reported in the literature. Therefore, immunophenotyping seems mandatory in the work-up of RCD.

Asunto(s)

Antiinflamatorios/uso terapéutico , Azatioprina/uso terapéutico , Enfermedad Celíaca/tratamiento farmacológico , Inmunosupresores/uso terapéutico , Prednisona/uso terapéutico , Adulto , Anciano , Anciano de 80 o más Años , Quimioterapia Combinada , Femenino , Genes Codificadores de los Receptores de Linfocitos T , Humanos , Masculino , Persona de Mediana Edad , Linfocitos T

RESUMEN

Myotonic dystrophy (DM) is the most prevalent inherited neuromuscular disease in adults. The genetic defect is a CTG triplet repeat expansion in the 3'-untranslated region of the myotonic dystrophy protein kinase ( DMPK ) gene, consisting of 15 exons. Using a transgenic DMPK-overexpressor mouse model, we demonstrate here that the endogenous mouse DMPK gene and the human DMPK transgene produce six major alternatively spliced mRNAs which have almost identical cell type-dependent distribution frequencies and expression patterns. Use of a cryptic 5' splice site in exon 8, which results in absence or presence of 15 nucleotides specifying a VSGGG peptide motif, and/or use of a cryptic 3' splice site in exon 14, which leads to a frameshift in the mRNA reading frame, occur as independent stochastic events in all tissues examined. In contrast, the excision of exons 13/14 that causes a frameshift and creates a C-terminally truncated protein is clearly cell type dependent and occurs predominantly in smooth muscle. We generated all six full-length mouse cDNAs that result from combinations of these three major splicing events and show that their transfection into cells in culture leads to production of four different approximately 74 kDa full-length (heart-, skeletal muscle- or brain-specific) and two C-terminally truncated approximately 68 kDa (smooth muscle-specific) isoforms. Information on DMPK mRNA and protein isoform expression patterns will be useful for recognizing differential effects of (CTG)(n)expansion in DM manifestation.

Asunto(s)

Empalme Alternativo , Distrofia Miotónica/genética , Proteínas Serina-Treonina Quinasas/genética , Animales , Células COS , ADN Complementario/metabolismo , Exones , Humanos , Isoenzimas/genética , Isoenzimas/metabolismo , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Proteína Quinasa de Distrofia Miotónica , Procesamiento Proteico-Postraduccional , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transgenes

RESUMEN

Myotonic dystrophy (DM), the most prevalent muscular disorder in adults, is caused by (CTG)n-repeat expansion in a gene encoding a protein kinase (DM protein kinase; DMPK) and involves changes in cytoarchitecture and ion homeostasis. To obtain clues to the normal biological role of DMPK in cellular ion homeostasis, we have compared the resting [Ca2+]i, the amplitude and shape of depolarization-induced Ca2+ transients, and the content of ATP-driven ion pumps in cultured skeletal muscle cells of wild-type and DMPK[-/-] knockout mice. In vitro-differentiated DMPK[-/-] myotubes exhibit a higher resting [Ca2+]i than do wild-type myotubes because of an altered open probability of voltage-dependent l-type Ca2+ and Na+ channels. The mutant myotubes exhibit smaller and slower Ca2+ responses upon triggering by acetylcholine or high external K+. In addition, we observed that these Ca2+ transients partially result from an influx of extracellular Ca2+ through the l-type Ca2+ channel. Neither the content nor the activity of Na+/K+ ATPase and sarcoplasmic reticulum Ca2+-ATPase are affected by DMPK absence. In conclusion, our data suggest that DMPK is involved in modulating the initial events of excitation-contraction coupling in skeletal muscle.

Asunto(s)

Calcio/metabolismo , Músculo Esquelético/metabolismo , Distrofia Miotónica/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Acetilcolina/farmacología , Animales , Canales de Calcio/efectos de los fármacos , Canales de Calcio/fisiología , ATPasas Transportadoras de Calcio/metabolismo , Células Cultivadas , Homeostasis , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Músculo Esquelético/efectos de los fármacos , Proteína Quinasa de Distrofia Miotónica , Nifedipino/farmacología , Cloruro de Potasio/farmacología , Proteínas Serina-Treonina Quinasas/deficiencia , Rianodina/farmacología , Retículo Sarcoplasmático/enzimología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Tetrodotoxina/farmacología

RESUMEN

Myotonic dystrophy (DM) is commonly associated with CTG repeat expansions within the gene for DM-protein kinase (DMPK). The effect of altered expression levels of DMPK, which is ubiquitously expressed in all muscle cell lineages during development, was examined by disrupting the endogenous Dmpk gene and overexpressing a normal human DMPK transgene in mice. Nullizygous (-/-) mice showed only inconsistent and minor size changes in head and neck muscle fibres at older age, animals with the highest DMPK transgene expression showed hypertrophic cardiomyopathy and enhanced neonatal mortality. However, both models lack other frequent DM symptoms including the fibre-type dependent atrophy, myotonia, cataract and male-infertility. These results strengthen the contention that simple loss- or gain-of-expression of DMPK is not the only crucial requirement for development of the disease.

Asunto(s)

Cardiomegalia/patología , Distrofia Miotónica/enzimología , Proteínas Serina-Treonina Quinasas/biosíntesis , Animales , Secuencia de Bases , Cardiomegalia/genética , Regulación del Desarrollo de la Expresión Génica , Homocigoto , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Fibras Musculares Esqueléticas/patología , Mutación , Distrofia Miotónica/genética , Distrofia Miotónica/patología , Proteína Quinasa de Distrofia Miotónica , Proteínas Serina-Treonina Quinasas/deficiencia , Proteínas Serina-Treonina Quinasas/genética , ARN Mensajero/análisis , Distribución Tisular

RESUMEN

Thirteen recombinant alpha A-crystallin mutants were constructed that differed in the type of amino acid residue directly preceding the sole amine donor lysine for transglutaminases in this protein. The capacity of these mutants to be cross-linked to amine acceptor substrates by tissue transglutaminase and factor XIII was assessed. Two different biotinylated glutamine-containing oligopeptides were used as amine acceptor probes. It appears that the type of residue preceding the amine donor lysine has a considerable influence on the substrate potential of alpha A-crystallin for transglutaminases. This influence shows qualitatively similar trends for tissue transglutaminase and factor XIII and is irrespective of the amine acceptor probe. In general, glycine or aspartic acid before the amine donor lysine has the strongest adverse effects on substrate reactivity, and proline, histidine, and tryptophan are less favorable. Valine, arginine, and phenylalanine, and to a more variable or somewhat lesser extent also serine, alanine, leucine, tyrosine, and asparagine, have an enhancing effect. This pattern of preference is largely in agreement with that observed for the limited number of characterized amine donor lysines in protein substrates for transglutaminases. It can be concluded that tissue transglutaminase and factor XIII have a rather broad yet clearly differentiated tolerance with respect to the residue preceding the amine donor lysine substrate in native proteins.

Asunto(s)

Cristalinas/metabolismo , Factor XII/metabolismo , Transglutaminasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Clonación Molecular , Cristalinas/química , ADN Complementario , Escherichia coli , Lisina , Datos de Secuencia Molecular , Mutagénesis , Oligopéptidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Mapeo Restrictivo , Especificidad por Sustrato

RESUMEN

alpha-Crystallin is a high-molecular-mass protein that for many decades was thought to be one of the rare real organ-specific proteins. This protein exists as an aggregate of about 800 kDa, but its composition is simple. Only two closely related subunits termed alpha A- and alpha B-crystallin, with molecular masses of approximately 20 kDa, form the building blocks of the aggregate. The idea of organ-specificity had to be abandoned when it was discovered that alpha-crystallin occurs in a great variety of nonlenticular tissues, notably heart, kidney, striated muscle and several tumors. Moreover alpha B-crystallin is a major component of ubiquinated inclusion bodies in human degenerative diseases. An earlier excitement arose when it was found that alpha B-crystallin, due to its very similar structural and functional properties, belongs to the heat-shock protein family. Eventually the chaperone nature of alpha-crystallin could be demonstrated unequivocally. All these unexpected findings make alpha-crystallin a subject of great interest far beyond the lens research field. A survey of structural data about alpha-crystallin is presented here. Since alpha-crystallin has resisted crystallization, only theoretical models of its three-dimensional structure are available. Due to its long life in the eye lens, alpha-crystallin is one of the best studied proteins with respect to post-translational modifications, including age-induced alterations. Because of its similarities with the small heat-shock proteins, the findings about alpha-crystallin are illuminative for the latter proteins as well. This review deals with: structural aspects, post-translational modifications (including deamidation, racemization, phosphorylation, acetylation, glycation, age-dependent truncation), the occurrence outside of the eye lens, the heat-shock relation and the chaperone activity of alpha-crystallin.

Asunto(s)

Cristalinas/química , Cristalinas/metabolismo , Proteínas de Choque Térmico/química , Cristalino/fisiología , Acetilación , Secuencia de Aminoácidos , Animales , Bovinos , Cristalinas/biosíntesis , Proteínas de Choque Térmico/metabolismo , Humanos , Cristalino/patología , Datos de Secuencia Molecular , Fosforilación , Procesamiento Proteico-Postraduccional , Homología de Secuencia de Aminoácido , Vertebrados

RESUMEN

The amine-donor substrate specificity of tissue-type transglutaminase has been studied in a series of recombinant alpha A-crystallin mutants. These mutant proteins have been provided with a potential substrate lysine residue, flanked by different amino acid residues, in the C-terminal extended arm of alpha A-crystallin. A biotinylated amine-acceptor hexapeptide was used as a probe for labelling the amine-donor sites. Wild-type bovine alpha A-crystallin does not function as an amine-donor substrate for tissue-type transglutaminase. Yet, upon introduction of a lysine residue at the C-terminal or penultimate position, all mutant alpha A-crystallins act as amine-donor substrates, although to different extents. This shows that accessibility is the primary requirement for a lysine residue to function as an amine-donor substrate for transglutaminase and that the enzyme has a broad tolerance towards the neighbouring residues. However, the nature of the flanking amino acid residues does clearly affect the reactivity of the substrate lysine residue. Notably, we found that a proline or glycine residue in front of the substrate lysine has a strong adverse effect on the substrate reactivity as compared to a preceding leucine, serine, alanine or arginine residue.

Asunto(s)

Cristalinas/metabolismo , Transglutaminasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Bovinos , Cristalinas/química , Lisina/química , Datos de Secuencia Molecular , Relación Estructura-Actividad , Especificidad por Sustrato