DNA-editing enzymes as potential treatments for heteroplasmic mtDNA diseases.

Mutations in the mitochondrial genome are the cause of many debilitating neuromuscular disorders. Currently, there is no cure or treatment for these diseases, and symptom management is the only relief doctors can provide. Although supplements and vitamins are commonly used in treatment, they provide little benefit to the patient and are only palliative. This is why gene therapy is a promising research topic to potentially treat and, in theory, even cure diseases caused by mutations in the mitochondrial DNA (mtDNA). Mammalian cells contain approximately a thousand copies of mtDNA, which can lead to a phenomenon called heteroplasmy, where both wild-type and mutant mtDNA molecules co-exist within the cell. Disease only manifests once the per cent of mutant mtDNA reaches a high threshold (usually >80%), which causes mitochondrial dysfunction and reduced ATP production. This is a useful feature to take advantage of for gene therapy applications, as not every mutant copy of mtDNA needs to be eliminated, but only enough to shift the heteroplasmic ratio below the disease threshold. Several DNA-editing enzymes have been used to shift heteroplasmy in cell culture and mice. This review provides an overview of these enzymes and discusses roadblocks of applying these to gene therapy in humans.

Manipulation of mtDNA heteroplasmy in all striated muscles of newborn mice by AAV9-mediated delivery of a mitochondria-targeted restriction endonuclease.

Mitochondrial diseases are frequently caused by heteroplasmic mitochondrial DNA (mtDNA) mutations. As these mutations express themselves only at high relative ratios, any approach able to manipulate mtDNA heteroplasmy can potentially be curative. In this study, we developed a system to manipulate mtDNA heteroplasmy in all skeletal muscles from neonate mice. We selected muscle because it is one of the most clinically affected tissues in mitochondrial disorders. A mitochondria-targeted restriction endonuclease (mito-ApaLI) expressed from AAV9 particles was delivered either by intraperitoneal or intravenous injection in neonate mice harboring two mtDNA haplotypes, only one of which was susceptible to ApaLI digestion. A single injection was able to elicit a predictable and marked change in mtDNA heteroplasmy in all striated muscles analyzed, including heart. No health problems or reduction in mtDNA levels were observed in treated mice, suggesting that this approach could have clinical applications for mitochondrial myopathies.

Organ-specific shifts in mtDNA heteroplasmy following systemic delivery of a mitochondria-targeted restriction endonuclease.

Most pathogenic mtDNA mutations are heteroplasmic and there is a clear correlation between high levels of mutated mtDNA in a tissue and pathology. We have found that in vivo double-strand breaks (DSBs) in mtDNA lead to digestion of cleaved mtDNA and replication of residual mtDNA. Therefore, if DSB could be targeted to mutations in mtDNA, mutant genomes could be eliminated and the wild-type mtDNA would repopulate the cells. This can be achieved by using mitochondria-targeted restriction endonucleases as a means to degrade specific mtDNA haplotypes in heteroplasmic cells or tissues. In this work, we investigated the potential of systemic delivery of mitochondria-targeted restriction endonucleases to reduce the proportion of mutant mtDNA in specific tissues. Using the asymptomatic NZB/BALB mtDNA heteroplasmic mouse as a model, we found that a mitochondria-targeted ApaLI (that cleaves BALB mtDNA at a single site and does not cleave NZB mtDNA) increased the proportion of NZB mtDNA in target tissues. This was observed in heart, using a cardiotropic adeno-associated virus type-6 (AAV6) and in liver, using the hepatotropic adenovirus type-5 (Ad5). No mtDNA depletion or loss of cytochrome c oxidase activity was observed in any of these tissues. These results show the potential of systemic delivery of viral vectors to specific organs for the therapeutic application of mitochondria-targeted restriction enzymes in mtDNA disorders.

Modulating mtDNA heteroplasmy by mitochondria-targeted restriction endonucleases in a 'differential multiple cleavage-site' model.

The ability to manipulate mitochondrial DNA (mtDNA) heteroplasmy would provide a powerful tool to treat mitochondrial diseases. Recent studies showed that mitochondria-targeted restriction endonucleases can modify mtDNA heteroplasmy in a predictable and efficient manner if it recognizes a single site in the mutant mtDNA. However, the applicability of such model is limited to mutations that create a novel cleavage site, not present in the wild-type mtDNA. We attempted to extend this approach to a 'differential multiple cleavage site' model, where an mtDNA mutation creates an extra restriction site to the ones normally present in the wild-type mtDNA. Taking advantage of a heteroplasmic mouse model harboring two haplotypes of mtDNA (NZB/BALB) and using adenovirus as a gene vector, we delivered a mitochondria-targeted Scal restriction endonuclease to different mouse tissues. Scal recognizes five sites in the NZB mtDNA but only three in BALB mtDNA. Our results showed that changes in mtDNA heteroplasmy were obtained by the expression of mitochondria-targeted ScaI in both liver, after intravenous injection, and in skeletal muscle, after intramuscular injection. Although mtDNA depletion was an undesirable side effect, our data suggest that under a regulated expression system, mtDNA depletion could be minimized and restriction endonucleases recognizing multiple sites could have a potential for therapeutic use.

Mitochondrial DNA depletion: mutations in thymidine kinase gene with myopathy and SMA.

BACKGROUND: The mitochondrial DNA (mtDNA) depletion syndrome (MDS) is an autosomal recessive disorder of early childhood characterized by decreased mtDNA copy number in affected tissues. Recently, MDS has been linked to mutations in two genes involved in deoxyribonucleotide (dNTP) metabolism: thymidine kinase 2 (TK2) and deoxy-guanosine kinase (dGK). Mutations in TK2 have been associated with the myopathic form of MDS, and mutations in dGK with the hepatoencephalopathic form. OBJECTIVES: To further characterize the frequency and clinical spectrum of these mutations, the authors screened 20 patients with myopathic MDS. RESULTS: No patient had dGK gene mutations, but four patients from two families had TK2 mutations. Two siblings were compound heterozygous for a previously reported H90N mutation and a novel T77M mutation. The other siblings harbored a homozygous I22M mutation, and one of them had evidence of lower motor neuron disease. The pathogenicity of these mutations was confirmed by reduced TK2 activity in muscle (28% to 37% of controls). CONCLUSIONS: These results show that the clinical expression of TK2 mutations is not limited to myopathy and that the myopathic form of MDS is genetically heterogeneous.

Barth's syndrome-like disorder: a new phenotype with a maternally inherited A3243G substitution of mitochondrial DNA (MELAS mutation).

An Argentine male child died at 4.5 years of age of a lethal mitochondrial disease associated with a MELAS mutation and a Barth syndrome-like presentation. The child had severe failure to thrive from the early months and for approximately two years thereafter. In addition, the patient had severely delayed gross motor milestones, marked muscle weakness, and dilated cardiomyopathy that progressed to congestive heart failure. He also had persistently elevated urinary levels of 3-methylglutaconic and 2-ethylhydracrylic acids and low blood levels of cholesterol. Detailed histopathologic evaluation of the skeletal muscle biopsy showed high activity of succinate dehydrogenase, a generalized decrease of COX activity, and abundant ragged-red fibers. Electron microscopic studies revealed multiple mitochondrial abnormalities in lymphocytes and monocytes, in the striated muscle, and in the postmortem samples (muscle, heart, liver, and brain). Biochemical analysis showed a pronounced and constant lactic acidosis, and abnormal urinary organic acid excretion (unchanged in the fasting and postprandial states). In addition, in CSF there was a marked increase of lactate and beta-hydroxybutyrate (beta-HOB) and also a high systemic ratio beta-HOB/acetoacetate. Enzymatic assay of the respiratory chain in biopsied muscle showed 10% of complex I activity and 24% of complex IV activity compared with controls. Molecular studies of the mitochondrial genome revealed an A to G mutation at nucleotide pair 3243 in mitochondrial DNA, a well-known pathogenetic mutation (MELAS mutation) in all the patient's tissues and also in the blood specimens of the probands mother and sibs (4 of 5). The diagnosis of MELAS mutation was reinforced by the absence of an identifiable mutation in the X-linked G4.5 gene of the propositus. The present observation gives additional evidence of the variable clinical expression of mtDNA mutations in humans and demonstrates that all clinical variants deserve adequate investigation to establish a primary defect. It also suggests adding Barth-like syndrome to the list of phenotypes with the MELAS mutation.

Muscarinic acetylcholine receptor antibodies as a new marker of dry eye Sjögren syndrome.

PURPOSE: The authors investigated whether circulating autoantibodies against M(3) muscarinic acetylcholine receptors (mAChRs) could be a new marker for diagnosis for primary and secondary Sjögren syndrome (SS) dry eye. METHODS: Enzyme-linked immunosorbent assay (ELISA) using both rat exorbital lacrimal gland acinar cell membranes and synthetic 25-mer peptide as antigens was used to determine autoantibodies against acinar cells and M(3) mAChRs. Also, nitric oxide synthase (NOS) activity was assessed to determine the biological effect of these autoantibodies in relation to the M(3) mAChR. RESULTS: Sera from dry eye primary SS (pSS) or secondary SS (sSS) patients tested by ELISA recognized membrane lacrimal gland acinar cells antigens and the synthetic 25-mer peptide, corresponding to the second extracellular loop of human M(3) mAChRs. Moreover, the IgG fraction and the corresponding affinity-purified anti-M(3) peptide autoantibodies from the same patients were able to activate NOS coupled to lacrimal gland M(3) mAChRs. As controls, IgG and sera from women without dry eye with or without rheumatoid arthritis and from normal control subjects gave negative results on ELISA and biological assay; thus demonstrating the specificity of the reaction. CONCLUSIONS: Autoantibodies against mAChR may be considered among the serum factors implicated in the pathophysiology of the development of pSS dry eyes and could be a new marker to differentiate SS dry eyes from non-SS dry eyes.

Histoenzymology of oxidases and dehydrogenases in peripheral blood lymphocytes and monocytes for the study of mitochondrial oxidative phosphorylation.

Histoenzymological methods usually performed on muscle fibres have been adapted to assess the functioning of oxidative phosphorylation in human circulating blood lymphocytes and monocytes. Oxidases and dehydrogenases were analysed in lymphocyte/monocyte smears. The specificity of each histoenzymological reaction was tested using a specific respiratory chain inhibitor: rotenone for NADH diaphorase, thenoyltrifluoroacetone for succinate dehydrogenase, potassium cyanide for cytochrome c oxidase and oligomycin for ATPase. Complex I activity was detected, but inhibition with rotenone was incomplete. Complexes II, IV and V were almost completely inhibited. These observations indicate that histoenzymology is a valuable method for detecting the activity of these oxidative phosphorylation enzymes in lymphocytes and monocytes. The histoenzymology tests performed on fresh peripheral blood cells resembled those used for muscle biopsies. They could be useful for the diagnosis of respiratory chain disorders in patients.

Sjögren autoantibodies modify neonatal cardiac function via M1 muscarinic acetylcholine receptor activation.

Isolated congenital heart block may be associated with primary Sjögren syndrome. In this work we describe circulating antibodies in the sera of primary Sjögren syndrome patients that are able to interact with neonatal myocardium by activating muscarinic acetylcholine receptors of M1 subtype. We report on the presence of autoantibodies against the second extracellular loop of human M1 muscarinic acetylcholine receptors in primary Sjögren syndrome mothers whose children have congenital heart block using a synthetic peptide in indirect immunofluorescence technique. Autoantibodies from primary Sjögren syndrome patients gave positive image on neonatal atria but not on adult atria slices. The synthetic M1 peptide selectively abrogated indirect immunofluorescence recognition. The primary Sjögren syndrome-immunoglobulin G also displayed an 'agonist like' activity modifying the intracellular events associated with muscarinic acetylcholine receptor activation. The mechanism appears to occur secondarily to stimulation of phosphoinositides turnover via phospholipase C activation. This, in turn, triggers cascade reactions involving calcium/calmodulin and leads to activation of nitric oxide synthase and soluble guanylate cyclase. All of these effects were selectively blunted by pirenzepine and neutralized by M1 synthetic peptide. These biological effects were not obtained using adult instead of neonatal rat atria and neither occurred with the sera of normal healthy women of childbearing age. It could be concluded that antibodies against neonatal M1 muscarinic acetylcholine receptor may be another serum factor to be considered in the pathophysiology of the development of congenital heart block associated with primary Sjögren syndrome mothers.

Autoantibodies against lacrimal gland M3 muscarinic acetylcholine receptors in patients with primary Sjögren's syndrome.

PURPOSE: The authors demonstrated that immunoglobulin G, present in the sera of patients with primary Sjögren syndrome (pSS), could recognize and activate muscarinic acetylcholine receptors (mAChRs) of rat exorbital acrimal gland. METHODS: Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, and radioligand binding and biologic assays were used to demonstrate autoantibodies against mAChRs. RESULTS: These autoantibodies recognized by means of SDS-PAGE and immunoblotting assay a band of approximately 70 kDa expressed on lacrimal gland membranes that comigrated with the peak of labeled mAChRs. Moreover, pSS IgG were able to inhibit, in an irreversible manner, the binding of [3H]quinuclidinyl benzilate to mAChRs of rat exorbital lacrimal glands and to simulate the biologic effect of mAChR agonists, because they trigger the activation of phosphoinositide turnover. Atropine and 4-diphenylacetoxy-N-methylpiperidine methiodide blocked the effect and carbachol mimicked it, confirming that the M3 subtype mAChRs mediated pSS IgG action. As control, IgG from sera of women without pSS gave negative results on immunoblotting, binding, and biologic assays, thus demonstrating the specificity of the reaction. CONCLUSIONS: Autoantibodies against mAChRs may be considered among the serum factors implicated in the pathophysiology of the development of pSS dry eyes.

Human primary Sjögren's syndrome autoantibodies as mediators of nitric oxide release coupled to lacrimal gland muscarinic acetylcholine receptors.

IgG obtained from sera of primary Sjögren's syndrome (pSS-IgG) patients and its interaction with M3 muscarinic cholinoceptors of rat exorbital lacrimal glands were studied by indirect immunofluorescence (IFI) and binding assay. Primary Sjögren's syndrome IgG stained epithelial cells with a continuous fluorescence pattern. The IFI imagen was attenuated by incubating the pSS-IgG with a synthetic peptide corresponding to the second extracellular loop of M3 muscarinic cholinoceptor. Primary SS-IgG was also able to bound irreversibly to muscarinic acetylcholine receptors (mAChRs) displacing the specific cholinergic antagonist QNB. Moreover, these antibodies triggered intracellular signals coupled to M3 muscaric cholinoceptors such as nitric oxide synthase (NOS) activation and cGMP production. Both primary Sjögren's syndrome IgG effects mimicked carbachol action and were abrogated by specific muscarinic antagonist 4-DAMP. The nitric oxide pathway through muscarinic cholinoceptors activation by pSS-IgG on rat exorbital lacrimal gland is also described. We proposed that chronic interaction of these autoantibodies on lacrimal gland muscarinic acetylcholine receptors could lead to tissue damage through nitric oxide release after immunological stimulation.

Differential cholinoceptor subtype-dependent activation of signal transduction pathways in neonatal versus adult rat atria.

In this study, we investigated the expression and distribution of muscarinic cholinergic receptors (mAChRs) and the different signaling pathways associated with mAChR activation in atria isolated from adult and neonatal rats. Carbachol stimulation of mAChRs in both neonatal and adult rat atria led to a negative inotropic response with activation of phosphoinositide hydrolysis, an increase in cyclic GMP levels, and a decrease in cyclic AMP production. However, compared with adult atria, neonatal atria showed hypersensitivity in the contractile effect induced by carbachol. Pharmacological analysis with mAChR antagonists indicated that M1 and M2 mAChR subtypes are important mediators of the response to carbachol in neonatal atria. In contrast, in adult atria the effect of the agonist was coupled only to the M2 mAChR subtype. Moreover, an increased number of total mAChRs was labeled in neonatal atrial membranes compared with those of adults. Although a predominant M2 mAChR population is expressed in atria at both stages of development studied, competition binding parameters calculated for carbachol indicated the presence of high-affinity binding sites, with higher affinity in neonates than in adults. These results suggest that the differences observed between neonatal and adult atria in their response to a cholinergic agonist may be related to differential expression of mAChR subtypes and/or changes in functional coupling of mAChR subtypes during development.

Circulating antibodies against neonatal cardiac muscarinic acetylcholine receptor in patients with Sjögren's syndrome.

Isolated congenital heart block may be associated with Primary Sjögren's Syndrome. In this work we demonstrated that IgG present in the sera of patients with Primary Sjögren's Syndrome (PSS) could bind and activate muscarinic acetylcholine receptors of rat neonatal atria. These antibodies were able to inhibit in a irreversible manner the binding of 3H-QNB to muscarinic cholinergic receptors of purified rat atria membranes. Moreover, IgG from PSS individuals could modify biological effects mediated by muscarinic cholinoceptors activation, i.e. decrease contractility and cAMP and increase phosphoinositide turnover and cGMP. Atropine blocked all of these effects and carbachol mimicked them; confirming muscarinic cholinergic receptors-mediated PSS IgG action. Neither binding nor biological effect were obtained using adult instead of neonatal rat atria. IgG from sera of normal women were not effective in the studied system. The prevalence of cholinergic antibody was 100% in PSS and was independent of Ro/SS-A and La/SS-B antibodies. It could be concluded that antibody against muscarinic cholinergic receptors may be another serum factor to be considered in the pathophysiology of the development of congenital heart block.

Circulating antibodies against rat parotid gland M3 muscarinic receptors in primary Sjögren's syndrome.

In this study we demonstrate that IgG present in the sera of patients with primary Sjögren's syndrome (PSS) could bind and activate muscarinic acetylcholine receptors (mAChRs) of rat parotid gland. These antibodies were able to inhibit in a non-competitive manner the binding of 3H-quinuclidinyl benzilate (QNB) to mAChRs of purified rat parotid gland membranes. Moreover, IgG from PSS could modify biological effects mediated by mAChR activation; i.e. decrease cAMP, increase phosphoinositide turnover without affecting cGMP. Atropine and 4-DAMP blocked all of these effects, and carbachol mimicked them, confirming the M3 subtype mAChRs mediated PSS IgG action. Neither binding nor biological effect were obtained with IgG from sera of normal women. The prevalence of cholinergic antibody was 100% in PSS, and was independent of Ro/SS-A and La/SS-B antibodies. It could be concluded that antibody against mAChRs may be another serum factor to be considered in the pathophysiology of the development of PSS.

Pharmacological evidence for the existence of different subtypes of muscarinic acetylcholine receptors for phosphoinositide hydrolysis in neonatal versus adult rat atria.

The negative inotropic effect of carbachol, as well as phosphoinositide hydrolysis, was measured in atria from neonatal and adult rats. Carbachol increased phosphoinositide hydrolysis and decreased dF/dt of both neonatal and adult atria; however, the neonatal atria showed hypereactivity to carbachol as compared with adult atria. Inhibition of phospholipase C reduced the supersensitivity to carbachol upon contractility in neonatal atria producing values similar to those of the adult atria, suggesting that muscarinic acetylcholine receptor (mAchR) stimulation is secondary to receptor-mediated hydrolysis of phosphoinositides. Pharmacological analysis with mAchR antagonists tends to support the idea that m1 and m2 subtypes are the most important mediators of the response to carbachol in neonatal atria. In adult atria, the effect of carbachol is coupled only to mAchR m2 subtypes.

Circulating antibodies against neurotransmitter receptor activities in children with congenital heart block and their mothers.

In this work we demonstrated that IgG present in the sera of patients with congenital heart block (CHB) and their mothers could bind and activate beta adrenoceptors and muscarinic cholinergic receptors of neonatal heart. These antibodies were able to inhibit in a noncompetitive manner the binding of [3H]QNB and [3H]CGP to muscarinic cholinergic and beta adrenoceptors of purified neonatal rat myocardial membranes, respectively. Moreover, IgG from children with CHB and their mothers could modify biological effects mediated by these neurotransmitter receptors, i.e., heart contractility and cAMP production. Neither binding nor biological effects were obtained using adult instead of neonatal rat atria. Both reactivities (adrenergic and cholinergic) were independent of Ro/SS-A and La/SS-B antibodies and were absent in the sera of normal women of childbearing age and of normal children. It could be concluded that antibodies against cardiac neurotransmitter receptors may be another serum factor (or factors) to be considered in the pathophysiology of the development of CHB.

Participation of cytoskeleton in the effect of antilaminin IgG on cardiac cholinoceptors.

1. We have previously demonstrated a molecular relationship between laminin and cardiac cholinoceptors. 2. We have now explored the participation of cytoskeletal proteins in the interaction between an antilaminin IgG with cardiac cholinoceptors. 3. Antilaminin IgG, whilst it specifically reacts with laminin molecules was able to induce cardiac cholinoceptor activation; acting like an agonist, decreasing cyclic AMP concentrations, reducing heart contractility and increasing phosphoinositide turnover. 4. Antilaminin IgG also interfered with the binding of a radiolabelled muscarinic antagonist, [3H]-quinuclidinyl benzilate. Colchicine and cytochalasin B, drugs that are able to prevent microfilament and microtubule polimerization, impaired the binding of antilaminin IgG to muscarinic cholinoceptors. 5. Cytochalasin B but not colchicine modified the muscarinic cholinoceptor effects mediated by regulatory G proteins (cyclic AMP and contractility) induced by antilaminin IgG. 6. It was demonstrated, by immunofluorescence, that none of these disrupting drugs altered the specific recognition of the antibody by its antigen. 7. These data indirectly suggest the participation of the cytoskeleton in the laminin and cholinergic receptor association.

Antibodies against beta adrenoceptors in mothers of children with congenital heart block.

To determine whether antibodies against beta adrenergic activity interact with neonatal cardiac cell receptors and alter their physiological behaviour. An "in vitro" experimental model measuring the frequency of contraction, the production of cAMP and binding assay on neonatal and adult rat atria was employed. Sera and IgG fraction from mothers of infants with congenital heart block (CHB) interact with neonatal rat atria increasing the frequency of contraction and cAMP production. These effects were abolished by propranolol, pointing to a beta adrenergic participation. IgG also competed with 3H-CGP to beta adrenergic receptors on neonatal cardiac membranes. Neither the contractile nor the cAMP effects or binding assay were obtained using adult instead of neonatal rat atria. Reactivity against cardiac neurotransmitter receptors may be another serum factor(s) to be considered in the pathophysiology of the development of CHB.

Antibodies against beta adrenoceptors in mothers of children with congenital heart block

En este trabajo se empleó un modelo experimental "in vitro" en el cual se midió la frecuencia de las contracciones, la producción de AMPc y la la unión de ligandos específicos al receptor ß adrenérgicos en las aurículas de ratas neonatales y adultas; a fin de, determinar si anticuerpos con actividad ß adrenérgica interactuaban con receptores ß adrenérgicos cardíacos y alteraban su compartimiento fisiológico. Los sueros y la fracción IgG de madres de infantes con bloqueo cardíaco neonatal congénito incrementaron la frecuencia de las contracciones y la producciín de AMPc en el miocardio auricular neonato. Este efecto fue inhibido por propranolol, puntalizando una participación ß adrenérgica. La IgG también compitió con el radiologando específico por los receptores ß adrenérgicos (3H-CGP) en las membranas purificadas de miocardio neonatal. Ni los efectos biológicos ni los ensayos de unión específica fueron modificados cuando se usó miocardio de rata adulta. La reactividad contra adrenoreceptores cardíacos por parte de anticuerpos provenientes de madres de niños con bloqueo neonatal congénito, podría ser otro factor sérico que debería considerarse en la patofisiología del desarrollo del bloqueo neonatal congénito

Antibodies against beta adrenoceptors in mothers of children with congenital heart block

En este trabajo se empleó un modelo experimental "in vitro" en el cual se midió la frecuencia de las contracciones, la producción de AMPc y la la unión de ligandos específicos al receptor ß adrenérgicos en las aurículas de ratas neonatales y adultas; a fin de, determinar si anticuerpos con actividad ß adrenérgica interactuaban con receptores ß adrenérgicos cardíacos y alteraban su compartimiento fisiológico. Los sueros y la fracción IgG de madres de infantes con bloqueo cardíaco neonatal congénito incrementaron la frecuencia de las contracciones y la producciín de AMPc en el miocardio auricular neonato. Este efecto fue inhibido por propranolol, puntalizando una participación ß adrenérgica. La IgG también compitió con el radiologando específico por los receptores ß adrenérgicos (3H-CGP) en las membranas purificadas de miocardio neonatal. Ni los efectos biológicos ni los ensayos de unión específica fueron modificados cuando se usó miocardio de rata adulta. La reactividad contra adrenoreceptores cardíacos por parte de anticuerpos provenientes de madres de niños con bloqueo neonatal congénito, podría ser otro factor sérico que debería considerarse en la patofisiología del desarrollo del bloqueo neonatal congénito (AU)