Mitochondrial Proteostasis Requires Genes Encoded in a Neurodevelopmental Syndrome Locus.

Eukaryotic cells maintain proteostasis through mechanisms that require cytoplasmic and mitochondrial translation. Genetic defects affecting cytoplasmic translation perturb synapse development, neurotransmission, and are causative of neurodevelopmental disorders, such as Fragile X syndrome. In contrast, there is little indication that mitochondrial proteostasis, either in the form of mitochondrial protein translation and/or degradation, is required for synapse development and function. Here we focus on two genes deleted in a recurrent copy number variation causing neurodevelopmental disorders, the 22q11.2 microdeletion syndrome. We demonstrate that SLC25A1 and MRPL40, two genes present in the microdeleted segment and whose products localize to mitochondria, interact and are necessary for mitochondrial ribosomal integrity and proteostasis. Our Drosophila studies show that mitochondrial ribosome function is necessary for synapse neurodevelopment, function, and behavior. We propose that mitochondrial proteostasis perturbations, either by genetic or environmental factors, are a pathogenic mechanism for neurodevelopmental disorders.SIGNIFICANCE STATEMENT The balance between cytoplasmic protein synthesis and degradation, or cytoplasmic proteostasis, is required for normal synapse function and neurodevelopment. Cytoplasmic and mitochondrial ribosomes are necessary for two compartmentalized, yet interdependent, forms of proteostasis. Proteostasis dependent on cytoplasmic ribosomes is a well-established target of genetic defects that cause neurodevelopmental disorders, such as autism. Here we show that the mitochondrial ribosome is a neurodevelopmentally regulated organelle whose function is required for synapse development and function. We propose that defective mitochondrial proteostasis is a mechanism with the potential to contribute to neurodevelopmental disease.

Golgi-Dependent Copper Homeostasis Sustains Synaptic Development and Mitochondrial Content.

Rare genetic diseases preponderantly affect the nervous system causing neurodegeneration to neurodevelopmental disorders. This is the case for both Menkes and Wilson disease, arising from mutations in ATP7A and ATP7B, respectively. The ATP7A and ATP7B proteins localize to the Golgi and regulate copper homeostasis. We demonstrate genetic and biochemical interactions between ATP7 paralogs with the conserved oligomeric Golgi (COG) complex, a Golgi apparatus vesicular tether. Disruption of Drosophila copper homeostasis by ATP7 tissue-specific transgenic expression caused alterations in epidermis, aminergic, sensory, and motor neurons. Prominent among neuronal phenotypes was a decreased mitochondrial content at synapses, a phenotype that paralleled with alterations of synaptic morphology, transmission, and plasticity. These neuronal and synaptic phenotypes caused by transgenic expression of ATP7 were rescued by downregulation of COG complex subunits. We conclude that the integrity of Golgi-dependent copper homeostasis mechanisms, requiring ATP7 and COG, are necessary to maintain mitochondria functional integrity and localization to synapses.SIGNIFICANCE STATEMENT Menkes and Wilson disease affect copper homeostasis and characteristically afflict the nervous system. However, their molecular neuropathology mechanisms remain mostly unexplored. We demonstrate that copper homeostasis in neurons is maintained by two factors that localize to the Golgi apparatus, ATP7 and the conserved oligomeric Golgi (COG) complex. Disruption of these mechanisms affect mitochondrial function and localization to synapses as well as neurotransmission and synaptic plasticity. These findings suggest communication between the Golgi apparatus and mitochondria through homeostatically controlled cellular copper levels and copper-dependent enzymatic activities in both organelles.

Ratiometric two-photon microscopy reveals attomolar copper buffering in normal and Menkes mutant cells.

Copper is controlled by a sophisticated network of transport and storage proteins within mammalian cells, yet its uptake and efflux occur with rapid kinetics. Present as Cu(I) within the reducing intracellular environment, the nature of this labile copper pool remains elusive. While glutathione is involved in copper homeostasis and has been assumed to buffer intracellular copper, we demonstrate with a ratiometric fluorescent indicator, crisp-17, that cytosolic Cu(I) levels are buffered to the vicinity of 1 aM, where negligible complexation by glutathione is expected. Enabled by our phosphine sulfide-stabilized phosphine (PSP) ligand design strategy, crisp-17 offers a Cu(I) dissociation constant of 8 aM, thus exceeding the binding affinities of previous synthetic Cu(I) probes by four to six orders of magnitude. Two-photon excitation microscopy with crisp-17 revealed rapid, reversible increases in intracellular Cu(I) availability upon addition of the ionophoric complex CuGTSM or the thiol-selective oxidant 2,2'-dithiodipyridine (DTDP). While the latter effect was dramatically enhanced in 3T3 cells grown in the presence of supplemental copper and in cultured Menkes mutant fibroblasts exhibiting impaired copper efflux, basal Cu(I) availability in these cells showed little difference from controls, despite large increases in total copper content. Intracellular copper is thus tightly buffered by endogenous thiol ligands with significantly higher affinity than glutathione. The dual utility of crisp-17 to detect normal intracellular buffered Cu(I) levels as well as to probe the depth of the labile copper pool in conjunction with DTDP provides a promising strategy to characterize perturbations of cellular copper homeostasis.

Trafficking mechanisms of P-type ATPase copper transporters.

Copper is an essential micronutrient required for oxygen-dependent enzymes, yet excess of the metal is a toxicant. The tug-of-war between these copper activities is balanced by chaperones and membrane transporters, which control copper distribution and availability. The P-type ATPase transporters, ATP7A and ATP7B, regulate cytoplasmic copper by pumping copper out of cells or into the endomembrane system. Mutations in ATP7A and ATP7B cause diseases that share neuropsychiatric phenotypes, which are similar to phenotypes observed in mutations affecting cytoplasmic trafficking complexes required for ATP7A/B dynamics. Here, we discuss evidence indicating that phenotypes associated to genetic defects in trafficking complexes, such as retromer and the adaptor complex AP-1, result in part from copper dyshomeostasis due to mislocalized ATP7A and ATP7B.

Systems Analysis of the 22q11.2 Microdeletion Syndrome Converges on a Mitochondrial Interactome Necessary for Synapse Function and Behavior.

Neurodevelopmental disorders offer insight into synaptic mechanisms. To unbiasedly uncover these mechanisms, we studied the 22q11.2 syndrome, a recurrent copy number variant, which is the highest schizophrenia genetic risk factor. We quantified the proteomes of 22q11.2 mutant human fibroblasts from both sexes and mouse brains carrying a 22q11.2-like defect, Df(16)A+/- Molecular ontologies defined mitochondrial compartments and pathways as some of top ranked categories. In particular, we identified perturbations in the SLC25A1-SLC25A4 mitochondrial transporter interactome as associated with the 22q11.2 genetic defect. Expression of SLC25A1-SLC25A4 interactome components was affected in neuronal cells from schizophrenia patients. Furthermore, hemideficiency of the Drosophila SLC25A1 or SLC25A4 orthologues, dSLC25A1-sea and dSLC25A4-sesB, affected synapse morphology, neurotransmission, plasticity, and sleep patterns. Our findings indicate that synapses are sensitive to partial loss of function of mitochondrial solute transporters. We propose that mitoproteomes regulate synapse development and function in normal and pathological conditions in a cell-specific manner.SIGNIFICANCE STATEMENT We address the central question of how to comprehensively define molecular mechanisms of the most prevalent and penetrant microdeletion associated with neurodevelopmental disorders, the 22q11.2 microdeletion syndrome. This complex mutation reduces gene dosage of ∼63 genes in humans. We describe a disruption of the mitoproteome in 22q11.2 patients and brains of a 22q11.2 mouse model. In particular, we identify a network of inner mitochondrial membrane transporters as a hub required for synapse function. Our findings suggest that mitochondrial composition and function modulate the risk of neurodevelopmental disorders, such as schizophrenia.

Rare Disease Mechanisms Identified by Genealogical Proteomics of Copper Homeostasis Mutant Pedigrees.

Rare neurological diseases shed light onto universal neurobiological processes. However, molecular mechanisms connecting genetic defects to their disease phenotypes are elusive. Here, we obtain mechanistic information by comparing proteomes of cells from individuals with rare disorders with proteomes from their disease-free consanguineous relatives. We use triple-SILAC mass spectrometry to quantify proteomes from human pedigrees affected by mutations in ATP7A, which cause Menkes disease, a rare neurodegenerative and neurodevelopmental disorder stemming from systemic copper depletion. We identified 214 proteins whose expression was altered in ATP7A-/y fibroblasts. Bioinformatic analysis of ATP7A-mutant proteomes identified known phenotypes and processes affected in rare genetic diseases causing copper dyshomeostasis, including altered mitochondrial function. We found connections between copper dyshomeostasis and the UCHL1/PARK5 pathway of Parkinson disease, which we validated with mitochondrial respiration and Drosophila genetics assays. We propose that our genealogical "omics" strategy can be broadly applied to identify mechanisms linking a genomic locus to its phenotypes.

The interactome of the copper transporter ATP7A belongs to a network of neurodevelopmental and neurodegeneration factors.

Genetic and environmental factors, such as metals, interact to determine neurological traits. We reasoned that interactomes of molecules handling metals in neurons should include novel metal homeostasis pathways. We focused on copper and its transporter ATP7A because ATP7A null mutations cause neurodegeneration. We performed ATP7A immunoaffinity chromatography and identified 541 proteins co-isolating with ATP7A. The ATP7A interactome concentrated gene products implicated in neurodegeneration and neurodevelopmental disorders, including subunits of the Golgi-localized conserved oligomeric Golgi (COG) complex. COG null cells possess altered content and subcellular localization of ATP7A and CTR1 (SLC31A1), the transporter required for copper uptake, as well as decreased total cellular copper, and impaired copper-dependent metabolic responses. Changes in the expression of ATP7A and COG subunits in Drosophila neurons altered synapse development in larvae and copper-induced mortality of adult flies. We conclude that the ATP7A interactome encompasses a novel COG-dependent mechanism to specify neuronal development and survival.

The Proteome of BLOC-1 Genetic Defects Identifies the Arp2/3 Actin Polymerization Complex to Function Downstream of the Schizophrenia Susceptibility Factor Dysbindin at the Synapse.

Proteome modifications downstream of monogenic or polygenic disorders have the potential to uncover novel molecular mechanisms participating in pathogenesis and/or extragenic modification of phenotypic expression. We tested this idea by determining the proteome sensitive to genetic defects in a locus encoding dysbindin, a protein required for synapse biology and implicated in schizophrenia risk. We applied quantitative mass spectrometry to identify proteins expressed in neuronal cells the abundance of which was altered after downregulation of the schizophrenia susceptibility factor dysbindin (Bloc1s8) or two other dysbindin-interacting polypeptides, which assemble into the octameric biogenesis of lysosome-related organelles complex 1 (BLOC-1). We found 491 proteins sensitive to dysbindin and BLOC-1 loss of function. Gene ontology of these 491 proteins singled out the actin cytoskeleton and the actin polymerization factor, the Arp2/3 complex, as top statistical molecular pathways contained within the BLOC-1-sensitive proteome. Subunits of the Arp2/3 complex were downregulated by BLOC-1 loss of function, thus affecting actin dynamics in early endosomes of BLOC-1-deficient cells. Furthermore, we demonstrated that Arp2/3, dysbindin, and subunits of the BLOC-1 complex biochemically and genetically interact, modulating Drosophila melanogaster synapse morphology and homeostatic synaptic plasticity. Our results indicate that ontologically prioritized proteomics identifies novel pathways that modify synaptic phenotypes associated with neurodevelopmental disorder gene defects. SIGNIFICANCE STATEMENT: The mechanisms associated with schizophrenia are mostly unknown despite the increasing number of genetic loci identified that increase disease risk. We present an experimental strategy that impartially and comprehensively interrogates the proteome of neurons to identify effects of genetic mutations in a schizophrenia risk factor, dysbindin. We find that the expression of the actin polymerization complex Arp2/3 is reduced in dysbindin-deficient cells, thus affecting actin-dependent phenotypes in two cellular compartments where dysbindin resides, endosomes and presynapses. Our studies indicate that a central cellular structure affected by schizophrenia susceptibility loci is the actin cytoskeleton, an organelle necessary for synaptic function in the presynaptic and postsynaptic compartment.

Construcción de un indicador líder compuesto para el Maule / Construction of a composite leading indicator for Maule

En términos generales, la investigación tiene como objetivo crear un indicador económico para el Maule que permita anticiparse al devenir de su ciclo económico, en consideración de sus principales actividades productivas. En específico, se pretende someter a pruebas estadísticas de significancia y validez a las principales series económicas de la región, de manera tal de seleccionar, por un lado, una serie de referencia de la actividad económica y, por otro, las series componentes del indicador. La metodología utilizada es aquella aplicada por la Nacional Bureau of Economic Research (NBER) en la creación de este tipo de indicadores para los países integrantes de la Organización para la Cooperación y el Desarrollo Económico (OCDE). Como resultado de la investigación se logra seleccionar y validar empíricamente como serie de referencia para el Maule, al Índice de Actividad Económica Regional (INACER), y a las siguientes series componentes del indicador; ocupados, cesantes, buscan trabajo por primera vez, inactivos, edificación aprobada total obras nuevas y total de exportaciones. Con tales series, se construye un indicador predictivo del comportamiento económico para la región, denominado Índice Líder Compuesto para el Maule (ILCM).

Overall, the research aims to create an economic indicator for the Maule that allows anticipate the evolution of the economic cycle, in consideration of its main productive activities. Specifically, it aims to test statistical significance and validity to the main economic series in the region, so as to select the one hand, a number of reference of economic activity and other components series indicator. The methodology used is that applied by the National Bureau of Economic Research (NBER) in the creation of this type of indicators for the member countries of the Organization for Economic Cooperation and Development (OECD). As a result of the research is done select and validate empirically as reference series for the Maule, the Regional Economic Activity Index (INACER), and the following components of the indicator series; employed, unemployed, seeking work for the first time, inactive, all new approved building works Total exports. With such series, a predictive indicator of economic performance for the region, called for the Maule Composite Index (ILCM) Leader is built.

Neuronal copper homeostasis susceptibility by genetic defects in dysbindin, a schizophrenia susceptibility factor.

Environmental factors and susceptible genomes interact to determine the risk of neurodevelopmental disorders. Although few genes and environmental factors have been linked, the intervening cellular and molecular mechanisms connecting a disorder susceptibility gene with environmental factors remain mostly unexplored. Here we focus on the schizophrenia susceptibility gene DTNBP1 and its product dysbindin, a subunit of the BLOC-1 complex, and describe a neuronal pathway modulating copper metabolism via ATP7A. Mutations in ATP7A result in Menkes disease, a disorder of copper metabolism. Dysbindin/BLOC-1 and ATP7A genetically and biochemically interact. Furthermore, disruption of this pathway causes alteration in the transcriptional profile of copper-regulatory and dependent factors in the hippocampus of dysbindin/BLOC-1-null mice. Dysbindin/BLOC-1 loss-of-function alleles do not affect cell and tissue copper content, yet they alter the susceptibility to toxic copper challenges in both mammalian cells and Drosophila. Our results demonstrate that perturbations downstream of the schizophrenia susceptibility gene DTNBP1 confer susceptibility to copper, a metal that in excess is a neurotoxin and whose depletion constitutes a micronutrient deficiency.

The N-ethylmaleimide-sensitive factor and dysbindin interact to modulate synaptic plasticity.

Dysbindin is a schizophrenia susceptibility factor and subunit of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) required for lysosome-related organelle biogenesis, and in neurons, synaptic vesicle assembly, neurotransmission, and plasticity. Protein networks, or interactomes, downstream of dysbindin/BLOC-1 remain partially explored despite their potential to illuminate neurodevelopmental disorder mechanisms. Here, we conducted a proteome-wide search for polypeptides whose cellular content is sensitive to dysbindin/BLOC-1 loss of function. We identified components of the vesicle fusion machinery as factors downregulated in dysbindin/BLOC-1 deficiency in neuroectodermal cells and iPSC-derived human neurons, among them the N-ethylmaleimide-sensitive factor (NSF). Human dysbindin/BLOC-1 coprecipitates with NSF and vice versa, and both proteins colocalized in a Drosophila model synapse. To test the hypothesis that NSF and dysbindin/BLOC-1 participate in a pathway-regulating synaptic function, we examined the role for NSF in dysbindin/BLOC-1-dependent synaptic homeostatic plasticity in Drosophila. As previously described, we found that mutations in dysbindin precluded homeostatic synaptic plasticity elicited by acute blockage of postsynaptic receptors. This dysbindin mutant phenotype is fully rescued by presynaptic expression of either dysbindin or Drosophila NSF. However, neither reduction of NSF alone or in combination with dysbindin haploinsufficiency impaired homeostatic synaptic plasticity. Our results demonstrate that dysbindin/BLOC-1 expression defects result in altered cellular content of proteins of the vesicle fusion apparatus and therefore influence synaptic plasticity.

Rentabilidad del trabajo en Chile: análisis de la evolución de los retornos por nivel educativo / Performance of the work in Chile: analysis of the evolution of returns by education level

La presente investigación tiene como propósito realizar un análisis econométrico de la rentabilidad del trabajo calificado en Chile, tanto a nivel general como para sus distintos niveles formativos, con especial referencia en el nivel de educación superior. El análisis de los datos se efectuó considerando la clásica ecuación de ingresos de Mincer, adaptada a través del método spline para determinar las rentabilidades de cada nivel formativo. Se trabaja con datos de panel de corte transversal incluyendo los años 1992, 2000, 2009 y 2011, obtenidos de la Encuesta de Caracterización Socioeconómica Nacional (CASEN). Los resultados destacan que el nivel de educación superior en Chile es el sector más rentable de los distintos niveles educativos, considerando todos los años estudiados.

This research deals with the econometric analysis of the profitability of qualified Chile, both as to its various educational levels generally, with special reference to higher education level work. As a methodology, classical Mincer earnings equation, adapted through the spline method for determining the profitability of each training level is used. Working with panel data from cross section for the years 1992, 2000, 2009 and 2011, obtained from the Encuesta de Caracterización Socioeconómica Nacional (CASEN). As resulting emphasize that higher education in Chile is the most profitable of the different educational levels for all years studied.

Gene dosage in the dysbindin schizophrenia susceptibility network differentially affect synaptic function and plasticity.

Neurodevelopmental disorders arise from single or multiple gene defects. However, the way multiple loci interact to modify phenotypic outcomes remains poorly understood. Here, we studied phenotypes associated with mutations in the schizophrenia susceptibility gene dysbindin (dysb), in isolation or in combination with null alleles in the dysb network component Blos1. In humans, the Blos1 ortholog Bloc1s1 encodes a polypeptide that assembles, with dysbindin, into the octameric BLOC-1 complex. We biochemically confirmed BLOC-1 presence in Drosophila neurons, and measured synaptic output and complex adaptive behavior in response to BLOC-1 perturbation. Homozygous loss-of-function alleles of dysb, Blos1, or compound heterozygotes of these alleles impaired neurotransmitter release, synapse morphology, and homeostatic plasticity at the larval neuromuscular junction, and impaired olfactory habituation. This multiparameter assessment indicated that phenotypes were differentially sensitive to genetic dosages of loss-of-function BLOC-1 alleles. Our findings suggest that modification of a second genetic locus in a defined neurodevelopmental regulatory network does not follow a strict additive genetic inheritance, but rather, precise stoichiometry within the network determines phenotypic outcomes.

Molecular basis of neurodegeneration and neurodevelopmental defects in Menkes disease.

ATP7A mutations impair copper metabolism resulting in three distinct genetic disorders in humans. These diseases are characterized by neurological phenotypes ranging from intellectual disability to neurodegeneration. Severe ATP7A loss-of-function alleles trigger Menkes disease, a copper deficiency condition where systemic and neurodegenerative phenotypes dominate clinical outcomes. The pathogenesis of these manifestations has been attributed to the hypoactivity of a limited number of copper-dependent enzymes, a hypothesis that we refer as the oligoenzymatic pathogenic hypothesis. This hypothesis, which has dominated the field for 25 years, only explains some systemic Menkes phenotypes. However, we argue that this hypothesis does not fully account for the Menkes neurodegeneration or neurodevelopmental phenotypes. Here, we propose revisions of the oligoenzymatic hypothesis that could illuminate the pathogenesis of Menkes neurodegeneration and neurodevelopmental defects through unsuspected overlap with other neurological conditions including Parkinson's, intellectual disability, and schizophrenia.

Estudio empírico del trabajo calificado en Chile: productividad y remuneraciones / Empirical study in qualified work in Chile: Productivity and remunerations

EL OBJETIVO del estudio es contrastar las hipótesis más recurrentes en la literatura económica respecto de la productividad y las remuneraciones del trabajo calificado en Chile. Para ello se analiza y evalúa la relación de largo plazo entre las variables productividad y remuneraciones para el trabajo calificado, utilizando series de tiempo que comprenden el período 1990-2009. Los resultados dan cuenta de la estacionariedad de los residuos, lo que implica que las variables no están cointegradas. Por lo que se puede concluir que no existe evidencia empírica, para el caso de Chile, de relaciones de largo plazo entre las variables estudiadas, aunque se observa un aumento significativo de la brecha de ingresos entre los trabajadores cualificados y los que no lo son.

THE OBJECTIVE of the study is to compare the most frequent hypothesis in economic literature on productivity and wages of skilled labor in Chile. This is analyzed and evaluated the long-term relationship between the variables productivity and wages for skilled labor, using time series comprising 1990-2009. The results show stationarity of waste, which means that the variables are not cointegrated. As can be concluded that there is no empirical evidence for the case of Chile, for long-term relationships between the variables studied, although a significant increase in the income gap between skilled workers and those that are not observed.

Relationships between burnout and role ambiguity, role conflict and employee absenteeism among health workers / Burnout y su relación con la ambigüedad de rol, conflicto de rol y absentismo laboral en trabajadores de la salud

El objetivo de esta investigación fue analizar la influencia de algunos factores de riesgo psicosociales en el desarrollo del burnout y analizar la influencia de este fenómeno en el absentismo laboral. La muestra la integraron 142 trabajadores de la salud. El análisis de los datos se efectuó considerando estadística descriptiva y modelos de regresión lineal múltiple. Los resultados confirman la influencia de la ambigüedad y del conflicto de rol sobre el Burnout [F (2.139) = 26.720; p < .001] y no se valida la influencia de éste sobre el absentismo laboral. No obstante, se evidencia una relación significativa y positiva entre la dimensión desgate psíquico y absentismo laboral (beta = 0.197; p< .05). Se concluye que el conflicto de rol es el predictor más intenso del componente emocional del burnout (desgaste psíquico; beta = 0.585; p < .001). Se evidencia que un esfuerzo emocional sostenido pudiese favorecer el absentismo laboral.

The aim of this research was to analyze the influence of some psychosocial risk factors in the development of burnout and to analyze the influence of this phenomenon on employee absenteeism. The study sample included 142 health care workers. The data analysis included descriptive statistics and multiple linear regression models. The results confirmed the influence of role ambiguity and role conflict on burnout [F (2.139) = 26.720; p < .001], but the influence of burnout on employee absenteeism was not confirmed. However, a significant and positive relationship has been shown between burnout and employee absenteeism (beta = 0.197; p < .05). In conclusion, the findings of this study support the claims that role conflict is a more intense predictor of the emotional component of burnout (burnout; beta = 0.585; p < .001). Additionally, there is evidence that prolonged emotional strain could encourage employee absenteeism.

Mutations in the BLOC-1 subunits dysbindin and muted generate divergent and dosage-dependent phenotypes.

Post-mortem analysis has revealed reduced levels of the protein dysbindin in the brains of those suffering from the neurodevelopmental disorder schizophrenia. Consequently, mechanisms controlling the cellular levels of dysbindin and its interacting partners may participate in neurodevelopmental processes impaired in that disorder. To address this question, we studied loss of function mutations in the genes encoding dysbindin and its interacting BLOC-1 subunits. We focused on BLOC-1 mutants affecting synapse composition and function in addition to their established systemic pigmentation, hematological, and lung phenotypes. We tested phenotypic homogeneity and gene dosage effects in the mouse null alleles muted (Bloc1s5(mu/mu)) and dysbindin (Bloc1s8(sdy/sdy)). Transcripts of NMDA receptor subunits and GABAergic interneuron markers, as well as expression of BLOC-1 subunit gene products, were affected differently in the brains of Bloc1s5(mu/mu) and Bloc1s8(sdy/sdy) mice. Unlike Bloc1s8(sdy/sdy), elimination of one or two copies of Bloc1s5 generated indistinguishable pallidin transcript phenotypes. We conclude that monogenic mutations abrogating the expression of a protein complex subunit differentially affect the expression of other complex transcripts and polypeptides as well as their downstream effectors. We propose that the genetic disruption of different subunits of protein complexes and combinations thereof diversifies phenotypic presentation of pathway deficiencies, contributing to the wide phenotypic spectrum and complexity of neurodevelopmental disorders.