Estudo dos vacúolos sugestivos de corpos de inclusão citoplasmática na biópsia muscular : análise clínica, laboratorial, eletroneuromiografica, histoquímica, imunocitoquimica e ultra-estrutural de 16 casos

Orientador: Lineu Cesar WerneckContem 33 fots. coladasDissertação (mestrado) - Universidade Federal do Parana, Setor de Ciencias da Saude, Programa de Pós-Graduação em Medicina InternaResumo: Os vacúolos são formações anormais encontradas nas fibras musculares, podendo ser classificados morfologicamente quanto ao tamanho, número, posição, forma, presença de membranas e se marginados ou não. Os vacúolos marginados, podem ser encontrados em diversas doenças, entre elas a miosite com corpos de inclusão citoplasmática. Esta é caracterizada clinicamente como uma polimiosite crônica tendo na histologia do músculo a presença dos vacúolos marginados, e denominados histologicamente de corpos de inclusão citoplasmáticos, podendo ter filamentos intranucleares e intracitoplasmáticos. Com o objetivo de estudar os vacúolos com aspecto histológico de corpos de inclusão citoplasmática, foram estudados todos os casos entre 1400 biópsias musculares que apresentaram os mesmos, procurando correlacionar com os dados clínicos laboratoriais, afim de verificar a sua especificidade para determinadas doenças. Foram encontrados 16 casos. Dos 16 casos, a idade média foi de 36.0 anos, o início da doença teve a média de 20.5 anos e o tempo de doença de 5.5 anos. Os casos foram classificados conforme a história clínica, hereditariedade, dados laboratoriais, eletrofisiológicos, histoquímicos, imunocitoquímicos e microscopia eletrônica em miosite com corpos de inclusão citoplasmática (4 casos), atrofia muscular espinhal juvenil (6 casos), miopatias distais (3 casos), distrofia de cinturas pélvica e escapular (2 casos) e polineuropatia periférica (1 caso). As enzimas musculares, mais especificamente a creatinoquinase mostrou-se elevada em dez casos. Apenas um caso mostrou moderada redução nas conduções nervosas. A eletromiografia esteve alterada em todos os casos sendo que, em cinco, casos a mesma mostrou sinais de desinervação (ativa e ou crônica), oito foram miopáticos e em dois casos mista (neuromiopática). A biópsia muscular pela histoquímica em cinco casos mostrou histologicamente uma miopatia (ativa e ou crônica); em sete casos, elementos para miopatia e desinervação (misto); em dois casos, desinervação; e em dois casos, miopatia inflamatória. Todos os casos mostravam vacúolos marginados. O estudo imunocitoquímico demonstrou predomínio de linfócitos CD8+ no interstício na maioria dos Casos, e ocasionalmente nas regiões perivasculares e no interior das fibras musculares. As miosites por corpos de inclusão citoplasmática tiveram importante aumento de linfócitos CD8+, em relação a outras doenças. A detecção de imunoglobulinas e complemento foram mais evidentes na miosite com corpos de inclusão citoplasmática, embora não demonstre uma diferença marcante, exceto na polineurite em que não teve nenhuma célula ou deposição de imunoglobulina. A microscopia eletrônica demonstrou a presença de filamentos nucleares e dispersos no citoplasma em cinco casos, um caso demonstrou a presença de filamentos no núcleo e região subsarcolemal, dois casos mostraram filamentos na região subsarcolemal e citoplasma. Em um caso, os filamentos estavam dispersos no citoplasma e núcleo, sendo que em sete casos não foram observados filamentos intracitoplasmáticos ou intranucleares. Foi concluído que: 1) Os filamentos intracitoplasmáticos e intranucleares não são específicos para uma única entidade; 2) A presença de reação inflamatória auxilia na diferenciação das outras doenças com miosite com corpos de inclusão citoplasmática; 3) A creatinaquinase e eletromiografia não são úteis para diferenciar a miosite com corpos de inclusão citoplasmática das outras entidades; 4) Existe um predomínio de linfócitos T no interstício nas miopatias com corpos de inclusão citoplasmática; 5) Foi notado importante aumento de linfócitos CD8+ no interstício, sugerindo relação com o complexo maior de histocompatibilidade 1 (MCH1); 6) Houve inversão da proporção de linfócitos CD4+/CD8+, sugerindo processo mediado pelo MCH1; 7) As imunoglobulinas e complemento foram detectadas com maior frequência nas miosites com corpos de inclusão citoplasmática; 8) Os corpos de inclusão citoplasmática ocorrem em diversas entidades, com patogenia e patologia global diferente, sugerindo se tratar de uma reação celular inespecífica, talvez relacionada com tempo de agressão crônica da fibra muscular, tanto nos processos de origem muscular primaria, como de origem neurogênica.Abstract: The vacuoles are abnormal structures of the muscle fibers, who can be morphological classified according the size, number, location, shape, presence of membranes or if they are rimmed. The rimmed vacuoles can be find in several diseases, mainly in the inclusion body myositis. This disease can be presented as chronic polymyositis with rimmed vacuoles in the histology, also called cytoplasmatic inclusion body, with cytoplasmatic or nuclear filaments. With the objectives to study the histopathological aspects of the cytoplasmatic inclusion bodies, we select all the cases who presented rimmed vacuoles among 1400 muscle biopsies, who had the clinical history and laboratory investigation available. We found 16 cases, with mean age of 36.0 years, whose disease started at 20.5 and a mean disease time 5.5 years. The cases where classified regarding the clinical history, hereditary pattern, serum laboratory determinations, electrophysiological tests, histochemical and immunocytochemical analysis and electron microscopic findings in inclusion body myositis (4 cases), juvenile spinal muscular atrophy (6 cases), distal myopathy (3 cases), limb-girdle muscular dystrophy (2 cases) and peripheral neuropathy of unknown etiology (1 case). The serum enzymes, specially the creatinekinases, was increased in ten cases. Only one case had reduced nerve conduction velocity. The electromyography was abnormal in all cases with denervation pattern in five and myopathic pattern in eight and in two cases had a mixed pattern (myopathic and denervation). The muscle biopsy histochemistry had the diagnosis of myopathy (active and chronic) in seven cases, mixed (myopathy and denervation findings) in two cases, denervation in two and inflammatory myopathy in two. All the cases had rimmed vacuoles. The immunocytochemical analysis showed CD8+ lymphocytes in the interstitial in most cases, occasionally ip the perivascular region and rarely inside the muscle fibers. The inclusion body myositis cases had increased of CD8+ lymphocytes comparing with the other diseases. The immunoglobulins and complement deposition were slight more intense in the inclusion body myositis, comparing with the other diseases. The peripheral neuropathy had no cells or immunoglobulins found any time. The electron microscopy detected filaments in the nucleus and diffusely in the cytoplasm in five cases, one case only in the nucleus and sub-sarcolemmal region, two cases with filaments in the sub-sarcolemmal and cytoplasm. One case had filaments where dispersed in the cytoplasm and nucleus. Seven cases had no filaments found in nucleus or cytoplasm. The following conclusion was drawn: 1) The intracytoplasmic or intranuclear filaments is not specific for only one disease. 2) The inflammatory reaction help in the differentiation of the inclusion body myositis from the other diseases studied. 3) The creatinekinase and electromyography where useless in the differentiation the inclusion body myositis from other diseases. 4) A predominance of T lymphocytes was found in the interstitial tissue in the cases of inclusion body myositis. 5) Was noted an important increased of the CD8+ lymphocytes in the interstitial tissue, suggesting a relationship with the major histocompatibilty 1 complex (MCH1). 6) An inversion of the CD4+/CD8+ lymphocyte's proportion was found, suggesting a mediation by the MCH1. 7) The immunoglobulins and complement deposition were found with major frequency in the inclusion body myositis. 8) The cytoplasmatic inclusion bodies can be found in several diseases with different pathogenesis and pathology, suggesting a non specific cellular reaction, maybe related with the time of the chronic aggression to the muscle tissue, who can be similar in the primary muscle lesion and neurogenic etiology

Estudo dos vacuolos sugestivos de corpos de inclusão citoplasmatica na biopsia muscular : analise clinica, laboratorial, eletroneuromiografica, histoquimica, imunocitoquimica e ultra-estrutural de 16 casos

Orientador: Lineu Cesar WerneckContem 33 fots. coladasDissertação (mestrado) - Universidade Federal do Parana, Setor de Ciencias da Saude, Programa de Pós-Graduação em Medicina InternaResumo: Os vacúolos são formações anormais encontradas nas fibras musculares, podendo ser classificados morfologicamente quanto ao tamanho, número, posição, forma, presença de membranas e se marginados ou não. Os vacúolos marginados, podem ser encontrados em diversas doenças, entre elas a miosite com corpos de inclusão citoplasmática. Esta é caracterizada clinicamente como uma polimiosite crônica tendo na histologia do músculo a presença dos vacúolos marginados, e denominados histologicamente de corpos de inclusão citoplasmáticos, podendo ter filamentos intranucleares e intracitoplasmáticos. Com o objetivo de estudar os vacúolos com aspecto histológico de corpos de inclusão citoplasmática, foram estudados todos os casos entre 1400 biópsias musculares que apresentaram os mesmos, procurando correlacionar com os dados clínicos laboratoriais, afim de verificar a sua especificidade para determinadas doenças. Foram encontrados 16 casos. Dos 16 casos, a idade média foi de 36.0 anos, o início da doença teve a média de 20.5 anos e o tempo de doença de 5.5 anos. Os casos foram classificados conforme a história clínica, hereditariedade, dados laboratoriais, eletrofisiológicos, histoquünicos, imunocitoquímicos e microscopia eletrônica em miosite com corpos de inclusão citoplasmática (4 casos), atrofia muscular espinhal juvenil (6 casos), miopatias distais (3 casos), distrofia de cinturas pélvica e escapular (2 casos) e polineuropatia periférica (1 caso). As enzimas musculares, mais especificamente a creatinoquinase mostrou-se elevada em dez casos. Apenas um caso mostrou moderada redução nas conduções nervosas. A eletromiografia esteve alterada em todos os casos sendo que, em cinco, casos a mesma mostrou sinais de desinervação (ativa e ou crônica), oito foram miopáticos e em dois casos mista (neuromiopática). A biópsia muscular pela histoquímica em cinco casos mostrou histologicamente uma miopatia (ativa e ou crônica); em sete casos, elementos para miopatia e desinervação (misto); em dois casos, desinervação; e em dois casos, miopatia inflamatória. Todos os casos mostravam vacúolos marginados. O estudo imunocitoquímico demonstrou predomínio de linfócitos CD8+ no interstício na maioria dos casos, e ocasionalmente nas regiões perivasculares e no interior das fibras musculares. As miosites por corpos de inclusão citoplasmática tiveram importante aumento de linfócitos CD8+, em relação a outras doenças. A detecção de imunoglobulinas e complemento foram mais evidentes na miosite com corpos de inclusão citoplasmática, embora não demonstre uma diferença marcante, exceto na polineurite em que não teve nenhuma célula ou deposição de imunoglobulina. A microscopia eletrônica demonstrou a presença de filamentos nucleares e dispersos no citoplasma em cinco casos, um caso demontrou a presença de filamentos no núcleo e região subsarcolemal, dois casos mostraram filamentos na região subsarcolemal e citoplasma. Em um caso, os filamentos estavam dispersos no citoplasma e núcleo, sendo que em sete casos não foram observados filamentos intracitoplasmáticos q u intranucleares. Foi concluído que: 1) Os filamentos intracitoplasmáticos e intranucleares não são específicos para uma única entidade; 2) A presença de reação inflamatória auxilia na diferenciação das outras doenças com miosite com corpos de inclusão citoplasmática; 3) A creatinaquinase e eletromiografia não são úteis para diferenciar a miosite com corpos de inclusão citoplasmática das outras entidades; 4) Existe um predomínio de linfócitos T no interstício nas miopatias com corpos de inclusão citoplasmática; 5) Foi notado importante aumento de linfócitos CD8+ no interstício, sugerindo relação com o complexo maior de histocompatibilidade 1 (MCH1); 6) Houve inversão da proporção de linfócitos CD4+/CD8+, sugerindo processo mediado pelo MCH1; 7) As imunoglobulinas e complemento foram detectadas com maior freqüência nas miosites com corpos de inclusão citoplasmática; 8) Os corpos de inclusão citoplasmática ocorrem em diversas entidades, com patogenia e patologia global diferente, sugerindo se tratar de uma reação celular inespecífica, talvez relacionada com tempo de agressão crônica da fibra muscular, tanto nos processos de origem muscular primaria, como de origem neurogênica. VIAbstract: The vacuoles are abnormal structures of the muscle fibers, who can be morphological classified according the size, number, location, shape, presence of membranes or if they are rimmed. The rimmed vacuoles can be find in several diseases, mainly in the inclusion body myositis. This disease can be presented as chronic polymyositis with rimmed vacuoles in the histology, also called cytoplasmatic inclusion body, with cytoplasmatic or nuclear filaments. With the objectives to study the histopathological aspects of the cytoplasmatic inclusion bodies, we select all the cases who presented rimmed vacuoles among 1400 muscle biopsies, who had the clinical history and laboratory investigation available. We found 16 cases, with mean age of 36.0 years, whose disease started at 20.5 and a mean disease time 5.5 years. The cases where classified regarding the clinical history, hereditary pattern, serum laboratory determinations, electrophysiological tests, histochemical and immunocytochemical analysis and electron microscopic findings in inclusion body myositis (4 cases), juvenile spinal muscular atrophy (6 cases), distal myopathy (3 cases), limb-girdle muscular dystrophy (2 cases) and peripheral neuropathy of unknown etiology (1 case). The serum enzymes, specially the creatinekinases, was increased in ten cases. Only one case had reduced nerve conduction velocity. The electromyography was abnormal in all cases with denervation pattern in five and myopathic pattern in eight and in two cases had a mixed pattern (myopathic and denervation). The muscle biopsy histochemistry had the diagnosis of myopathy (active and chronic) in seven cases, mixed (myopathy and denervation findings) in two cases, denervation in two and inflammatory myopathy in two. All the cases had rimmed vacuoles. The immunocytocheinical analysis showed CD8+ lymphocytes in the interstitial in most cases, occasionally ip the perivascular region and rarely inside the muscle fibers. The inclusion body myositis cases had increased of CD8+ lymphocytes comparing with the other diseases. The immunoglobulins and complement deposition were slight more intense in the inclusion body myositis, comparing with the other diseases. The peripheral neuropathy had no cells or immunoglobulins found any time. The electron microscopy detected filaments in the nucleus and diffusely in the cytoplasm in five cases, one case only in the nucleus and sub-sarcolemmal region, two cases with filaments in the sub-sarcolemmal and cytoplasm. One case had filaments where dispersed in the cytoplasm and nucleus. Seven cases had no filaments found in nucleus or cytoplasm. The following conclusion was drawn: 1) The intracytoplasmic or intranuclear filaments is not specific for only one disease. 2) The inflammatory reaction help in the differentiation of the inclusion body myositis from the other diseases studied. 3) The creatinekinase and electromyography where useless in the differentiation the inclusion body myositis from other diseases. 4) A predominance of T lymphocytes was found in the interstitial tissue in the cases of inclusion body myositis. 5) Was noted an important increased of the CD8+ lymphocytes in the interstitial tissue, suggesting a relationship with the major histocompatibilty 1 complex (MCH1). 6) An inversion of the CD4+/CD8+ lymphocyte's proportion was found , suggesting a mediation by the MCH1. 7) The immunoglobulins and complement deposition were found with major frequency in the inclusion body myositis. 8) The cytoplasmatic inclusion bodies can be found in several diseases with different pathogenesis and pathology, suggesting a non specific cellular reaction, maybe related with the time of the chronic aggression to the muscle tissue, who can be similar in the primary muscle lesion and neurogenic etiology

Estudo de 43 pacientes com atrofia muscular espinhal e seu diagnóstico molecular

Apendice e AnexosOrientadora: Rosana Herminia ScolaDissertaçao(mestrado)- Universidade Federal do Paraná. Setor de Ciencias da Saúde. Departamento de Clínica Médica. Curso de Pós-graduaçao em Medicina InternaInclui bibliografiaResumo: A atrofia muscular espinhal e uma doenca degenerativa das celulas do como anterior da medula, com quatro tipos descritos (I, II, IIIa/b, IV). Somente os tipos I, II, Illa e Illb apresentam comprometimento do cromossomo 5ql3, em que dois genes sao os causadores da doenca: o primeiro codifica a proteina de sobrevivencia do neuronio motor (SMN) e o segundo codifica a proteina inibidora da apoptose neuronal (NAIP). Ha delecao dos exons 7 e 8 em mais de 90% dos casos, ambos envolvidos na codificacao do SMN. A delecao do exon 5, envolvido na codificacao do NAIP, ocorre em 45% a 70% dos pacientes com atrofia muscular espinhal tipo I, e em menos de 20% dos casos tipo II e III. Com o proposito de desenvolver e introduzir o diagnostico molecular nas investigacoes de rotina da atrofia muscular espinhal, selecionaram-se 43 pacientes com diagnostico ja estabelecido de atrofia muscular espinhal, baseando-se no quadro clinico, dosagens enzimaticas, resultados de biopsia muscular e eletromiografia. Pesquisou-se a delecao dos exons envolvidos no DNA extraido de biopsias musculares atraves da analise por reacao em cadeia da polimerase. Relacionaram-se os resultados com os respectivos pacientes, por meio de dados de anamnese, exame fisico, achados de biopsia muscular e eletroneuromiografia. Nove pacientes foram classificados como tendo atrofia muscular espinhal tipo I; quatorze pacientes, como atrofia muscular espinhal tipo II, e 20, como atrofia muscular espinhal tipo III (12 Illa e 8 Illb). Nos pacientes com atrofia muscular espinhal tipo I, houve delecao do NAIP em 5 dos 9 casos (55%); naqueles com o tipo II, houve delecao em 3 dos 14 casos (21,4%). Todos os casos considerados como tipo I e II demonstraram delecao simultanea dos exons 7 e 8. No tipo III, o exon 5 esteve deletado em 5 dos 20 casos (25%), mas com delecao do exon 7 em 18 dos 20 pacientes (90%) e do exon 8 em 19 deles (95%). A delecao do NAIP teve relacao estatisticamente significante com os casos de fraqueza muscular da porcao distai dos membros superiores (p < 0,05) e com o achado de nucleos internos na biopsia muscular (p < 0,05). A delecao do exon 8 foi estatisticamente significante na presenca de fibras angulares atroficas (p < 0,05) e de fibras redondas atroficas dispersas (p < 0,05) na biopsia muscular. A biopsia muscular foi condizente com desinervacao em 80% dos casos, e a eletromiografia em 88,8%, enquanto que a delecao concomitante dos exons 7 e 8 esteve presente em 93% dos casos nos tres tipos de atrofia muscular espinhal. Ha estreita relacao entre o diagnostico clinico e o diagnostico molecular, corroborando os indices de delecao descritos na literatura. A analise pela reacao em cadeia da polimerase, portanto, serve como exame diagnostico tao seguro e preciso quanto a biopsia muscular e a eletromiografia. alem de menos invasiva.Abstract: Spinal muscle atrophy results from degeneration of motor neurons in the anterior horn of the spinal cord. There are four clinical types (I, II, IIIa/b, IV), but only three forms (I, II, Ilia and Illb) were found to be linked to the 5ql3 region o f chromosome 5, where 2 genes were postulated: one was designated as the Survival Motor Neuron (SMN) gene, and the other as the Neuronal Apoptosis Inhibitory Protein (NAIP) gene. Deletion of exons 7 and 8 occurs in over 90% of cases, both related to SMN codification. Deletion of exon 5 (related to NAIP gene) occurs in 45% to 70% of type I spinal muscle atrophy patients, and in less than 20% o f type II and III patients. The purpose o f this study is to develop and to establish molecular research in routine investigation of spinal muscle atrophy. Forty-three patients were included in this study, all o f them with previous diagnosis o f spinal muscle atrophy, according to their clinical features, muscular enzymes levels, electromyography and anatomopathologic results. The DNA was isolated from muscle biopsies. Polimerase chain reaction (PCR) amplification of SMN exons 7 and 8, and NAIP exon 5 was carried out and to clinical history, physical exam, electromyography and anatomopathologic results. A population o f 9 type I, fourteen patients type II and 20 patients type III (12 Ilia and 8 Illb) was enrolled into this study. Type I spinal muscle atrophy patients presented deletion of exon 5 (NAIP gene) in 5 cases (55%), and type II patients presented this deletion in 3 cases (21,4%). Type I and type II patients showed concomitant deletion of exons 7 and 8 in 100% o f cases. In type III patients, exon 5 was deleted in 5 cases (25%), with deletion of exon 7 in 18 patients (90%) and deletion o f exon 8 in 19 (95%) of them. Distal impairment of upper limbs was significantly correlated to the cases of NAIP deletion (p < 0,05), as to the presence of internal nucleus in muscle biopsy {p < 0,05). Deletion o f exon 8 was significantly correlated when associated to atrophic angulated fibers (p < 0,05) as well to the presence o f atrophic dispersed rounded fibers (p < 0,05) in muscle biopsy. Muscle biopsy presented with denervation in 80% of cases, and electromyography with the same pattern in 88,8%. Concomitant deletion of exons 7 and 8 occurred in 93% o f cases, in all three types of spinal muscle atrophy. Precise identification of deletion by PCR in spinal muscle atrophy patients provides an important diagnostic achievement, which may be considered as good as the muscle biopsy and electromyography, but less invasive

A NOVEL MISSENSE MUTATION PATTERN OF THE GCH1 GENE IN DOPA-RESPONSIVE DYSTONIA

Dopa-responsive dystonia (DRD) is an inherited metabolic disorder now classified as DYT5 with two different biochemical defects: autosomal dominant GTP cyclohydrolase 1 (GCH1) deficiency or autosomal recessive tyrosine hydroxylase deficiency. We report the case of a 10-years-old girl with progressive generalized dystonia and gait disorder who presented dramatic response to levodopa. The phenylalanine to tyrosine ratio was significantly higher after phenylalanine loading test. This condition had two different heterozygous mutations in the GCH1 gene: the previously reported P23L mutation and a new Q182E mutation. The characteristics of the DRD and the molecular genetic findings are discussed

Acute effects of physiotherapeutic respiratory maneuvers in critically ill patients with craniocerebral trauma

OBJECTIVE: To evaluate the effects of physiotherapeutic respiratory maneuvers on cerebral and cardiovascular hemodynamics and blood gas variables. METHOD: A descriptive, longitudinal, prospective, nonrandomized clinical trial that included 20 critical patients with severe craniocerebral trauma who were receiving mechanical ventilation and who were admitted to the intensive care unit. Each patient was subjected to the physiotherapeutic maneuvers of vibrocompression and increased manual expiratory flow (5 minutes on each hemithorax), along with subsequent airway suctioning with prior instillation of saline solution, hyperinflation and hyperoxygenation. Variables related to cardiovascular and cerebral hemodynamics and blood gas variables were recorded after each vibrocompression, increased manual expiratory flow and airway suctioning maneuver and 10 minutes after the end of airway suctioning. RESULTS: The hemodynamic and blood gas variables were maintained during vibrocompression and increased manual expiratory flow maneuvers; however, there were increases in mean arterial pressure, intracranial pressure, heart rate, pulmonary arterial pressure and pulmonary capillary pressure during airway suctioning. All of the values returned to baseline 10 minutes after the end of airway suctioning. CONCLUSION: Respiratory physiotherapy can be safely performed on patients with severe craniocerebral trauma. Additional caution must be taken when performing airway suctioning because this technique alters cerebral and cardiovascular hemodynamics, even in sedated and paralyzed patients

Identification and Functional Characterization of a Novel Mutation in theNKX2-1Gene: Comparison with the Data in the Literature

Background: NKX2-1 mutations have been described in several patients with primary congenital hypothyroidism, respiratory distress, and benign hereditary chorea, which are classical manifestations of the brain-thyroid-lung syndrome (BTLS). Methods: The NKX2-1 gene was sequenced in the members of a Brazilian family with clinical features of BTLS, and a novel monoallelic mutation was identified in the affected patients. We introduced the mutation in an expression vector for the functional characterization by transfection experiments using both thyroidal and lung-specific promoters. Results: The mutation is a deletion of a cytosine at position 834 (ref. sequence NM-003317) (c.493delC) that causes a frameshift with formation of an abnormal protein from amino acid 165 and a premature stop at position 196. The last amino acid of the nuclear localization signal, the whole homeodomain, and the carboxy-terminus of NKX2-1 are all missing in the mutant protein, which has a premature stop codon at position 196 (p.Arg165Glyfs*32). The p.Arg165Glyfs*32 mutant does not bind DNA, and it is unable to transactivate the thyroglobulin (Tg) and the surfactant protein-C (SP-C) promoters. Interestingly, a dose-dependent dominant negative effect of the p.Arg165Glyfs*32 was demonstrated only on the Tg promoter, but not on the SP-C promoter. This effect was also noticed when the mutation was tested in presence of PAX8 or cofactors that synergize with NKX2-1 (P300 and TAZ). The functional effect was also compared with the data present in the literature and demonstrated that, so far, it is very difficult to establish a specific correlation among NKX2-1 mutations, their functional consequence, and the clinical phenotype of affected patients, thus suggesting that the detailed mechanisms of transcriptional regulation still remain unclear. Conclusions: We describe a novel NKX2-1 mutation and demonstrate that haploinsufficiency may not be the only explanation for BTLS. Our results indicate that NKX2-1 activity is also finely regulated in a tissue-specific manner, and additional studies are required to better understand the complexities of genotype-phenotype correlations in the NKX2-1 deficiency syndrome

Clinical and molecular genetic findings in COLQ-mutant congenital myasthenic syndromes

n/