Algorithmic prediction of failure modes in healthcare.

BACKGROUND: Preventing medical errors is crucial, especially during crises like the COVID-19 pandemic. Failure Modes and Effects Analysis (FMEA) is the most widely used prospective hazard analysis in healthcare. FMEA relies on brainstorming by multi-disciplinary teams to identify hazards. This approach has two major weaknesses: significant time and human resource investments, and lack of complete and error-free results. OBJECTIVES: To introduce the algorithmic prediction of failure modes in healthcare (APFMH) and to examine whether APFMH is leaner in resource allocation in comparison to the traditional FMEA and whether it ensures the complete identification of hazards. METHODS: The patient identification during imaging process at the emergency department of Sheba Medical Center was analyzed by FMEA and APFMH, independently and separately. We compared between the hazards predicted by APFMH method and the hazards predicted by FMEA method; the total participants' working hours invested in each process and the adverse events, categorized as 'patient identification', before and after the recommendations resulted from the above processes were implemented. RESULTS: APFMH is more effective in identifying hazards (P < 0.0001) and is leaner in resources than the traditional FMEA: the former used 21 h whereas the latter required 63 h. Following the implementation of the recommendations, the adverse events decreased by 44% annually (P = 0.0026). Most adverse events were preventable, had all recommendations been fully implemented. CONCLUSION: In light of our initial and limited-size study, APFMH is more effective in identifying hazards (P < 0.0001) and is leaner in resources than the traditional FMEA. APFMH is suggested as an alternative to FMEA since it is leaner in time and human resources, ensures more complete hazard identification and is especially valuable during crisis time, when new protocols are often adopted, such as in the current days of the COVID-19 pandemic.

Proteomics insights into infantile neuronal ceroid lipofuscinosis (CLN1) point to the involvement of cilia pathology in the disease.

Mutations in the depalmitoylation enzyme, palmitoyl protein thioesterase (PPT1), result in the early onset neurodegenerative disease known as Infantile Neuronal Ceroid Lipofuscinosis. Here, we provide proteomic evidence suggesting that PPT1 deficiency could be considered as a ciliopathy. Analysis of membrane proteins from brain enriched for acylated proteins from neonate Ppt1 knock out and control mice revealed a list of 88 proteins with differential expression levels. Amongst them, we identified Rab3IP, which regulates ciliogenesis in concert with Rab8 and Rab11. Immunostaining analysis revealed that PPT1 is localized in the cilia. Indeed, an unbiased proteomics analysis on isolated cilia revealed 660 proteins, which differed in their abundance levels between wild type and Ppt1 knock out. We demonstrate here that Rab3IP, Rab8 and Rab11 are palmitoylated, and that palmitoylation of Rab11 is required for correct intracellular localization. Cells and brain preparations from Ppt1-/- mice exhibited fewer cells with cilia and abnormally longer cilia, with both acetylated tubulin and Rab3IP wrongly distributed along the length of cilia. Most importantly, the analysis revealed a difference in the distribution and levels of the modified proteins in cilia in the retina of mutant mice versus the wildtype, which may be important in the early neurodegenerative phenotype. Overall, our results suggest a novel link between palmitoylated proteins, cilial organization and the pathophysiology of Neuronal Ceroid Lipofuscinosis.

Notch Activation by Shootin1 Opposing Activities on 2 Ubiquitin Ligases.

The evolutionarily conserved Notch pathway plays an important role in regulation of stem cell renewal and cell fate determination in numerous organs, and as such is a key pathway in normal health and disease processes. Canonical Notch signaling is usually activated by cell contact where transmembrane ligands such as Delta-like and Jagged bind to Notch receptors. Notch activation results in the translocation of the cleaved Notch intracellular domain (NICD) into the nucleus and subsequent activation of transcription. Poly-ubiquitination leading to proteosome degradation of pathway components is one mean of regulating the Notch pathway. Here, we identified that Shootin1 exhibits the surprising propensity of activating the pathway either by interacting with LNX1/2 and promoting poly-ubiquitination of Numb or by complexing with Itch and impairing poly-ubiquitination of NICD. Within the developing brain Shootin1 modulates neuroblasts cell fate by executing 2 opposing activities on ubiquitin ligases, which control Notch signaling on 2 different levels.

Styloid Process Elongation on Cervical Spine Computed Tomography is Associated with the Enthesopathy-Related Diseases of Ankylosing Spondylitis and Diffuse Idiopathic Skeletal Hyperostosis.

BACKGROUND: Enthesopathy may lead to calcification of the stylohyoid ligament and can cause elongation of the styloid process (SP). OBJECTIVES: To evaluate whether SP elongation is associated with two common enthesitis-related diseases: ankylosing spondylitis (AS) and diffuse idiopathic skeletal hyperostosis (DISH). METHODS: Cervical spine computed tomography (CT) examinations of patients with DISH (n=64, Resnick criteria), AS (n=24, New York criteria) and a controls (no radiological signs of DISH or AS, n=54) were retrospectively evaluated. The DISH group was further divided into patients with and without cervical DISH. The length of right and left SP was measured independently by two readers on coronal and sagittal curved reformats. The average right and left styloid length and average length per person were compared among the groups. RESULTS: Demographic characteristics were similar between the DISH and control groups (average age 68.2 ± 15.7, 69.2 ± 12.7 years, male:female ratio 48:16 and 35:19, respectively, P > 0.05), whereas age was significantly lower (average age: 53 ± 15 years, P < 0.0001) in the AS group, which was also composed mainly of men. The AS and DISH groups had significantly longer SP compared to controls (AS 37.9 ± 9.6 mm, DISH 34.4 ± 9 mm, control 30.3 ± 10.1 mm, P < 0.05). There was no correlation between age and SP length. Inter-reader reliability of SP measurements was excellent in all groups (ICC = 0.998, P < 0.0001). CONCLUSIONS: SP elongation is associated with both AS and DISH substantiating the enthesopathy-related pathophysiology of this finding.

Shootin1 acts in concert with KIF20B to promote polarization of migrating neurons.

Shootin1 has been ascribed a role in regulating polarization of primary hippocampal neurons. To better understand the possible role of Shootin1 in the developing brain, we identified a member of the kinesin superfamily, KIF20B, as a novel Shootin1 interacting protein and a potential mediator of Shootin1 interaction with microtubules. KIF20B/Shootin1 binding was mapped to a 57 aa KIF20B sequence, which was used as a dominant-negative fragment. Direct interaction between that peptide (MBD) and Shootin1 was confirmed by surface plasmon resonance-based technology and the affinity was determined in the 10⁻7 m range. The proteins are expressed in the developing brain and formed a complex in vivo based on coimmunoprecipitation experiments and coimmunostaining in primary neurons. In primary hippocampal neurons Kif20b knockdown reduced Shootin1 mobilization to the developing axon, as evidenced by immunostaining and fluorescence recovery after photobleaching analysis, suggesting that Shootin1 is a novel KIF20B cargo. shRNA targeting of Shootin1 reduced PIP3 accumulation in the growth cone, as did Kif20b shRNA. In the developing mouse brain, Kif20b knockdown or expression of the KIF20B minimal binding domain inhibited neuronal migration, and in vivo migration assays suggested that Shootin1/Kif20b acts in the same genetic pathway. Time-lapse imaging of multipolar cells in the subventricular zone revealed that downregulating levels of either Shootin1 or Kif20b hindered the transition from multipolar to bipolar cells. Collectively, our data demonstrate the importance of the Shootin1/KIF20B interaction to the dynamic process of pyramidal neuronal polarization and migration.

Tau's role in the developing brain: implications for intellectual disability.

Microdeletions encompassing the MAPT (Tau) locus resulting in intellectual disability raised the hypothesis that Tau may regulate early functions in the developing brain. Our results indicate that neuronal migration was inhibited in mouse brains following Tau reduction. In addition, the leading edge of radially migrating neurons was aberrant in spite of normal morphology of radial glia. Furthermore, intracellular mitochondrial transport and morphology were affected. In early postnatal brains, a portion of Tau knocked down neurons reached the cortical plate. Nevertheless, they exhibited far less developed dendrites and a striking reduction in connectivity evident by the size of boutons. Our novel results strongly implicate MAPT as a dosage-sensitive gene in this locus involved in intellectual disability. Furthermore, our results are likely to impact our understanding of other diseases involving Tau.

Inhibiting glutamine utilization creates a synthetic lethality for suppression of ATP citrate lyase in KRas-driven cancer cells.

Metabolic reprogramming is now considered a hallmark of cancer cells. KRas-driven cancer cells use glutaminolysis to generate the tricarboxylic acid cycle intermediate α-ketoglutarate via a transamination reaction between glutamate and oxaloacetate. We reported previously that exogenously supplied unsaturated fatty acids could be used to synthesize phosphatidic acid-a lipid second messenger that activates both mammalian target of rapamycin (mTOR) complex 1 (mTORC1) and mTOR complex 2 (mTORC2). A key target of mTORC2 is Akt-a kinase that promotes survival and regulates cell metabolism. We report here that mono-unsaturated oleic acid stimulates the phosphorylation of ATP citrate lyase (ACLY) at the Akt phosphorylation site at S455 in an mTORC2 dependent manner. Inhibition of ACLY in KRas-driven cancer cells in the absence of serum resulted in loss of cell viability. We examined the impact of glutamine (Gln) deprivation in combination with inhibition of ACLY on the viability of KRas-driven cancer cells. While Gln deprivation was somewhat toxic to KRas-driven cancer cells by itself, addition of the ACLY inhibitor SB-204990 increased the loss of cell viability. However, the transaminase inhibitor aminooxyacetate was minimally toxic and the combination of SB-204990 and aminooxtacetate led to significant loss of cell viability and strong cleavage of poly-ADP ribose polymerase-indicating apoptotic cell death. This effect was not observed in MCF7 breast cancer cells that do not have a KRas mutation or in BJ-hTERT human fibroblasts which have no oncogenic mutation. These data reveal a synthetic lethality between inhibition of glutamate oxaloacetate transaminase and ACLY inhibition that is specific for KRas-driven cancer cells and the apparent metabolic reprogramming induced by activating mutations to KRas.

Lessons Learned From Five Years of Newborn Screening for Severe Combined Immunodeficiency in Israel.

BACKGROUND: Implementation of newborn screening (NBS) programs for severe combined immunodeficiency (SCID) have advanced the diagnosis and management of affected infants and undoubtedly improved their outcomes. Reporting long-term follow-up of such programs is of great importance. OBJECTIVE: We report a 5-year summary of the NBS program for SCID in Israel. METHODS: Immunologic and genetic assessments, clinical analyses, and outcome data from all infants who screened positive were evaluated and summarized. RESULTS: A total of 937,953 Guthrie cards were screened for SCID. A second Guthrie card was requested on 1,169 occasions (0.12%), which resulted in 142 referrals (0.015%) for further validation tests. Flow cytometry immune-phenotyping, T cell receptor excision circle measurement in peripheral blood, and expression of TCRVß repertoire for the validation of positive cases revealed a specificity and sensitivity of 93.7% and 75.9%, respectively, in detecting true cases of SCID. Altogether, 32 SCID and 110 non-SCID newborns were diagnosed, making the incidence of SCID in Israel as high as 1:29,000 births. The most common genetic defects in this group were associated with mutations in DNA cross-link repair protein 1C and IL-7 receptor α (IL-7Rα) genes. No infant with SCID was missed during the study time. Twenty-two SCID patients underwent hematopoietic stem cell transplantation, which resulted in a 91% survival rate. CONCLUSIONS: Newborn screening for SCID should ultimately be applied globally, specifically to areas with high rates of consanguineous marriages. Accumulating data from follow-up studies on NBS for SCID will improve diagnosis and treatment and enrich our understanding of immune development in health and disease.

Antagonistic effects of doublecortin and MARK2/Par-1 in the developing cerebral cortex.

Abnormal neuronal migration is manifested in brain malformations such as lissencephaly. The impairment in coordinated cell motility likely reflects a faulty mechanism of cell polarization or coupling between polarization and movement. Here we report on the relationship between the polarity kinase MARK2/Par-1 and its substrate, the well-known lissencephaly-associated gene doublecortin (DCX), during cortical radial migration. We have previously shown using in utero electroporation that reduced MARK2 levels resulted in multipolar neurons stalled at the intermediate zone border, similar to the phenotype observed in the case of DCX silencing. However, whereas reduced MARK2 stabilized microtubules, we show here that knock-down of DCX increased microtubule dynamics. This led to the hypothesis that simultaneous reduction may alleviate the phenotype. Coreduction of MARK2 and DCX resulted in a partial restoration of the normal neuronal migration phenotype in vivo. The kinetic behavior of the centrosomes reflected the different molecular mechanisms activated when either protein was reduced. In the case of reducing MARK2 processive motility of the centrosome was hindered, whereas when DCX was reduced, centrosomes moved quickly but bidirectionally. Our results stress the necessity for successful coupling between the polarity pathway and cytoplasmic dynein-dependent activities for proper neuronal migration.

Accurate balance of the polarity kinase MARK2/Par-1 is required for proper cortical neuronal migration.

Radial neuronal migration is key in structuring the layered cortex. Here we studied the role of MARK2/Par-1 in this process. The dual name stands for the MAP/microtubule affinity-regulating kinase 2 (MARK2) and the known polarity kinase 1 (Par-1). Reduced MARK2 levels using in utero electroporation resulted in multipolar neurons stalled at the intermediate zone border. Reintroduction of the wild-type kinase postmitotically improved neuronal migration. Our results indicated that reduction in MARK2 affected centrosomal dynamics in migrating neurons of the cerebral cortex. Increased MARK2 has been shown to destabilize microtubules, and here we show for the first time that reduced MARK2 stabilized microtubules in primary cultured neurons. Kinase-independent activity permitted multipolar-to-bipolar transition but did not restore proper migration. Increased MARK2 levels resulted in a different phenotype, which is loss of neuronal polarity. MARK2 kinase activity reduction hindered migration in the developing brain, which was rescued by increasing kinase activity. Our results stress the necessity of maintaining dynamic microtubules for proper neuronal migration. Furthermore, the exact requirements for MARK2 and its kinase activity vary during the course of neuronal migration. Collectively, our results stress the requirements for the different roles of MARK2 during neuronal migration.

First Year of Israeli Newborn Screening for Severe Combined Immunodeficiency-Clinical Achievements and Insights.

Severe combined immunodeficiency (SCID), the most severe form of T cell immunodeficiency, is detectable through quantification of T cell receptor excision circles (TRECs) in dried blood spots obtained at birth. Herein, we describe the results of the first year of the Israeli SCID newborn screening (NBS) program. This important, life-saving screening test is available at no cost for every newborn in Israel. Eight SCID patients were diagnosed through the NBS program in its first year, revealing an incidence of 1:22,500 births in the Israeli population. Consanguine marriages and Muslim ethnic origin were found to be a risk factor in affected newborns, and a founder effect was detected for both IL7Rα and DCLRE1C deficiency SCID. Lymphocyte subset analysis and TREC quantification in the peripheral blood appear to be sufficient for confirmation of typical and leaky SCID and ruling out false positive (FP) results. Detection of secondary targets (infants with non-SCID lymphopenia) did not significantly affect the management or outcomes of these infants in our cohort. In the general, non-immunodeficient population, TREC rises along with gestational age and birth weight, and is significantly higher in females and the firstborn of twin pairs. Low TREC correlates with both gestational age and birth weight in extremely premature newborns. Additionally, the rate of TREC increase per week consistently accelerates with gestational age. Together, these findings mandate a lower cutoff or a more lenient screening algorithm for extremely premature infants, in order to reduce the high rate of FPs within this group. A significant surge in TREC values was observed between 28 and 30 weeks of gestation, where median TREC copy numbers rise by 50% over 2 weeks. These findings suggest a maturational step in T cell development around week 29 gestation, and imply moderate to late preterms should be screened with the same cutoff as term infants. The SCID NBS program is still in its infancy, but is already bearing fruit in the early detection and improved outcomes of children with SCID in Israel and other countries.

The evolving doublecortin (DCX) superfamily.

BACKGROUND: Doublecortin (DCX) domains serve as protein-interaction platforms. Mutations in members of this protein superfamily are linked to several genetic diseases. Mutations in the human DCX gene result in abnormal neuronal migration, epilepsy, and mental retardation; mutations in RP1 are associated with a form of inherited blindness, and DCDC2 has been associated with dyslectic reading disabilities. RESULTS: The DCX-repeat gene family is composed of eleven paralogs in human and in mouse. Its evolution was followed across vertebrates, invertebrates, and was traced to unicellular organisms, thus enabling following evolutionary additions and losses of genes or domains. The N-terminal and C-terminal DCX domains have undergone sub-specialization and divergence. Developmental in situ hybridization data for nine genes was generated. In addition, a novel co-expression analysis for most human and mouse DCX superfamily-genes was performed using high-throughput expression data extracted from Unigene. We performed an in-depth study of a complete gene superfamily using several complimentary methods. CONCLUSION: This study reveals the existence and conservation of multiple members of the DCX superfamily in different species. Sequence analysis combined with expression analysis is likely to be a useful tool to predict correlations between human disease and mouse models. The sub-specialization of some members due to restricted expression patterns and sequence divergence may explain the successful addition of genes to this family throughout evolution.

Involvement of Na(+), K(+)-ATPase and endogenous digitalis-like compounds in depressive disorders.

BACKGROUND: Sodium and potassium-activated adenosine triphosphatase (Na(+), K(+)-ATPase) and endogenous digitalis-like compounds (DLC) in the brain have been implicated in the pathogenesis of mood disorders. This hypothesis was examined by the determination of Na(+), K(+)-ATPase/DLC system in parietal cortex of patients with different mood disorders and two animal models of depression. METHODS: Na(+), K(+)-ATPase concentrations in human brain synaptosomal fractions, from patients with mood disorders, schizophrenia, and normal individuals, were determined by (3)H-ouabain binding assay. Alpha isoforms were quantified by Western blotting. Brain DLC were measured using sensitive enzyme linked immunosorbant assay (ELISA). The effects of ouabain and ouabain-antibodies on behavior were determined in two animal models of depression. RESULTS: (3)H-ouabain binding in bipolar patients was significantly lower than in major depressed and schizophrenic patients. Na(+), K(+)-ATPase alpha isoforms in synaptosomal fractions were not different among the groups. DLC levels in the parietal cortex of bipolar patients were significantly higher than in normal individuals and depressed patients. Injection of lipopolysaccharide (intraperitoneally) to rats elicited depression-like symptoms, which were significantly attenuated by pre-injection of ouabain-antibodies. Injection of ouabain and ouabain-antibodies (intracerebroventricular) reduced depression-like symptoms in the forced swimming test in rats. CONCLUSIONS: The results support the possibility that Na(+), K(+)-ATPase and endogenous DLC participate in the pathogenesis of depressive disorders.

Sodium dodecyl sulphate-treated proteins as ligands in ELISA.

Insoluble proteins generally do not adsorb to microtitre wells and, therefore, cannot be used as antigens in enzyme-linked immunosorbent assay (ELISA). However, denaturation and solubilization with 2% sodium dodecyl sulphate (SDS) renders these proteins suitable ligands for ELISA. In quantitative ELISA using polyclonal antibodies as primary antibody, comparable results were obtained with native and SDS-denatured protein ligands. The binding of the antibodies to the SDS-treated ligands was completely inhibited by premixing the primary antibody with the corresponding native antigen. Nonspecific binding of primary and secondary antibodies to SDS-treated ligands was not observed. SDS-treated proteins are able to attach to ELISA microwells, retain their antigenic epitopes and do not engender an elevated background. The concentration of SDS-treated proteins required for coating is the same as that of the native proteins.

Increased LIS1 expression affects human and mouse brain development.

Deletions of the PAFAH1B1 gene (encoding LIS1) in 17p13.3 result in isolated lissencephaly sequence, and extended deletions including the YWHAE gene (encoding 14-3-3epsilon) cause Miller-Dieker syndrome. We identified seven unrelated individuals with submicroscopic duplication in 17p13.3 involving the PAFAH1B1 and/or YWHAE genes, and using a 'reverse genomics' approach, characterized the clinical consequences of these duplications. Increased PAFAH1B1 dosage causes mild brain structural abnormalities, moderate to severe developmental delay and failure to thrive. Duplication of YWHAE and surrounding genes increases the risk for macrosomia, mild developmental delay and pervasive developmental disorder, and results in shared facial dysmorphologies. Transgenic mice conditionally overexpressing LIS1 in the developing brain showed a decrease in brain size, an increase in apoptotic cells and a distorted cellular organization in the ventricular zone, including reduced cellular polarity but preserved cortical cell layer identity. Collectively, our results show that an increase in LIS1 expression in the developing brain results in brain abnormalities in mice and humans.

Common and divergent roles for members of the mouse DCX superfamily.

The doublecortin-like (DCX) domains serve as protein-interaction platforms. DCX tandem domains appear in the product of the X-linked doublecortin (DCX) gene, in retinitis pigmentosa-1 (RP1), as well as in other gene products. Mutations in the human DCX gene are associated with abnormal neuronal migration, epilepsy, and mental retardation; mutations in RP1 are associated with a form of inherited blindness, while DCDC2 has been associated with dyslectic reading disabilities. Motivated by the possible importance of this gene family, a thorough analysis to detect all family members in the mouse was conducted. The DCX-repeat gene superfamily is composed of eleven paralogs, and we cloned the DCX domains from nine different genes. Our study questioned which functions attributed to the DCX domain, are conserved among the different members. Our results suggest that the proteins with the DCX-domain have conserved and unique roles in microtubule regulation and signal transduction. All the tested proteins stimulated microtubule assembly in vitro. Proteins with tandem repeats stabilized the microtubule cytoskeleton in transfected cells, while those with single repeats localized to actin-rich subcellular structures, or the nucleus. All tested proteins interacted with components of the JNK/MAP-kinase pathway, while only a subset interacted with Neurabin 2, and a nonoverlapping group demonstrated actin association. The sub-specialization of some members due to confined intracellular localization, and protein interactions may explain the success of this superfamily.

Site-specific recombination of asymmetric lox sites mediated by a heterotetrameric Cre recombinase complex.

Previous reports have demonstrated that new Cre recombinase specificities can be developed for symmetrically designed lox mutants through directed evolution. The development of Cre variants that allow the recombination of true asymmetric lox mutant sites has not yet been addressed, however. In the present study, we demonstrate that a mixture of two different site-specific Cre recombinase molecules (wt Cre and a mutant Cre) catalyzes efficient recombination between two asymmetric lox sites in vitro, presumably via formation of a functionally active heterotetrameric complex. The results may broaden the application of site-specific recombination in basic and applied research, including the custom-design of recombinases for natural, asymmetric, and lox-related target sequences present in the genome. Future applications may potentially include genomic manipulations, for example, site-specific integrations, deletions or substitutions within precise regions of the genomes of mammalians and other organisms.

DCX's phosphorylation by not just another kinase (JNK).

The mammalian cortex is generally subdivided into six organized layers, which are formed during development in an organized fashion. This organized cortical layering is disrupted in case of mutations in the doublecortin (DCX) gene. DCX is a Microtubule Associated Protein (MAP). However, besides stabilization of microtubules, it may be involved in additional functions. The participation of this molecule in signal transduction is beginning to emerge via discovery of interacting molecules and its regulation by phosphorylation using several different kinases. We raise the hypothesis, that the combinatorial phosphorylation of DCX by different kinases and at different sites may be a molecular regulatory switch in the transition of a migrating neuron through multiple phases of migration. Our recent research has suggested the involvement of DCX in the JNK (Jun-N-terminal Kinase) pathway. The JNK pathway is linked to the reelin pathway, known to regulate cortical layering. Positioning of DCX in this signaling pathway opens up additional possibilities of understanding how migrating neurons are controlled.

DCX, a new mediator of the JNK pathway.

Mutations in the X-linked gene DCX result in lissencephaly in males, and abnormal neuronal positioning in females, suggesting a role for this gene product during neuronal migration. In spite of several known protein interactions, the involvement of DCX in a signaling pathway is still elusive. Here we demonstrate that DCX is a substrate of JNK and interacts with both c-Jun N-terminal kinase (JNK) and JNK interacting protein (JIP). The localization of this signaling module in the developing brain suggests its functionality in migrating neurons. The localization of DCX at neurite tips is determined by its interaction with JIP and by the interaction of the latter with kinesin. DCX is phosphorylated by JNK in growth cones. DCX mutated in sites phosphorylated by JNK affected neurite outgrowth, and the velocity and relative pause time of migrating neurons. We hypothesize that during neuronal migration, there is a need to regulate molecular motors that are working in the cell in opposite directions: kinesin (a plus-end directed molecular motor) versus dynein (a minus-end directed molecular motor).

LIS1 missense mutations: variable phenotypes result from unpredictable alterations in biochemical and cellular properties.

Mutations in one allele of the human LIS1 gene cause a severe brain malformation, lissencephaly. Although most LIS1 mutations involve deletions, several point mutations with a single amino acid alteration were described. Patients carrying these mutations reveal variable phenotypic manifestations. We have analyzed the functional importance of these point mutations by examining protein stability, folding, intracellular localization, and protein-protein interactions. Our data suggest that the mutated proteins were affected at different levels, and no single assay could be used to predict the lissencephaly phenotype. Most interesting are those mutant proteins that retain partial folding and interactions. In the case of LIS1 mutated in F31S, the cellular phenotype may be modified by overexpression of specific interacting proteins. Overexpression of the PAF-AH alpha1 subunit dissolved aggregates induced by this mutant protein and increased its half-life. Overexpression of NudE or NudEL localized this mutant protein to spindle poles and kinetochores but had no effect on protein stability. Our results implicate that there are probably different biochemical and cellular mechanisms obstructed in each patient yielding the varied lissencephaly phenotypes.