Epileptic Encephalopathy GABRB Structural Variants Share Common Gating and Trafficking Defects.

Variants in the GABRB gene, which encodes the ß subunit of the GABAA receptor, have been implicated in various epileptic encephalopathies and related neurodevelopmental disorders such as Dravet syndrome and Angelman syndrome. These conditions are often associated with early-onset seizures, developmental regression, and cognitive impairments. The severity and specific features of these encephalopathies can differ based on the nature of the genetic variant and its impact on GABAA receptor function. These variants can lead to dysfunction in GABAA receptor-mediated inhibition, resulting in an imbalance between neuronal excitation and inhibition that contributes to the development of seizures. Here, 13 de novo EE-associated GABRB variants, occurring as missense mutations, were analyzed to determine their impact on protein stability and flexibility, channel function, and receptor biogenesis. Our results showed that all mutations studied significantly impact the protein structure, altering protein stability, flexibility, and function to varying degrees. Variants mapped to the GABA-binding domain, coupling zone, and pore domain significantly impact the protein structure, modifying the ß+/α- interface of the receptor and altering channel activation and receptor trafficking. Our study proposes that the extent of loss or gain of GABAA receptor function can be elucidated by identifying the specific structural domain impacted by mutation and assessing the variability in receptor structural dynamics. This paves the way for future studies to explore and uncover links between the incidence of a variant in the receptor topology and the severity of the related disease.

Novel frameshift variants expand the map of the genetic defects in IRF2BP2.

Background: At present, the knowledge about disease-causing mutations in IRF2BP2 is very limited because only a few patients affected by this condition have been reported. As previous studies have described, the haploinsufficiency of this interferon transcriptional corepressors leads to the development of CVID. Very recently, a more accurate phenotype produced by truncating variants in this gene has been defined, manifesting CVID with gastrointestinal inflammatory symptoms and autoimmune manifestations. Methods: We analyzed 5 index cases with suspected primary immunodeficiency by high throughput sequencing. They were submitted for a genetic test with a panel of genes associated with immune system diseases, including IRF2BP2. The screening of SNVs, indels and CNVs fulfilling the criteria with very low allelic frequency and high protein impact, revealed five novel variants in IRF2BP2. In addition, we isolated both wild-type and mutated allele of the cDNA from one of the families. Results: In this study, we report five novel loss-of-function (LoF) mutations in IRF2BP2 that likely cause primary immunodeficiency, with CVID as more frequent phenotype, variable expression of inflammatory gastrointestinal features, and one patient with predisposition of viral infection. All identified variants were frameshift changes, and one of them was a large deletion located on chromosome 1q42, which includes the whole sequence of IRF2BP2, among other genes. Both de novo and dominant modes of inheritance were observed in the families here presented, as well as incomplete penetrance. Conclusions: We describe novel variants in a delimited low-complex region, which may be considered a hotspot in IRF2BP2. Moreover, this is the first time that a large CNV in IRF2BP2 has been reported to cause CVID. The distinct mechanisms than LoF in IRF2BP2 could cause different phenotype compared with the mainly described. Further investigations are necessary to comprehend the regulatory mechanisms of IRF2BP2, which could be under variable expression of the disease.

The Epithelial Sodium Channel-An Underestimated Drug Target.

Epithelial sodium channels (ENaC) are part of a complex network of interacting biochemical pathways and as such are involved in several disease states. Dependent on site and type of mutation, gain- or loss-of-function generated symptoms occur which span from asymptomatic to life-threatening disorders such as Liddle syndrome, cystic fibrosis or generalized pseudohypoaldosteronism type 1. Variants of ENaC which are implicated in disease assist further understanding of their molecular mechanisms in order to create models for specific pharmacological targeting. Identification and characterization of ENaC modifiers not only furthers our basic understanding of how these regulatory processes interact, but also enables discovery of new therapeutic targets for the disease conditions caused by ENaC dysfunction. Numerous test compounds have revealed encouraging results in vitro and in animal models but less in clinical settings. The EMA- and FDA-designated orphan drug solnatide is currently being tested in phase 2 clinical trials in the setting of acute respiratory distress syndrome, and the NOX1/ NOX4 inhibitor setanaxib is undergoing clinical phase 2 and 3 trials for therapy of primary biliary cholangitis, liver stiffness, and carcinoma. The established ENaC blocker amiloride is mainly used as an add-on drug in the therapy of resistant hypertension and is being studied in ongoing clinical phase 3 and 4 trials for special applications. This review focuses on discussing some recent developments in the search for novel therapeutic agents.

JAKs and STATs from a Clinical Perspective: Loss-of-Function Mutations, Gain-of-Function Mutations, and Their Multidimensional Consequences.

The JAK/STAT signaling pathway plays a key role in cytokine signaling and is involved in development, immunity, and tumorigenesis for nearly any cell. At first glance, the JAK/STAT signaling pathway appears to be straightforward. However, on closer examination, the factors influencing the JAK/STAT signaling activity, such as cytokine diversity, receptor profile, overlapping JAK and STAT specificity among non-redundant functions of the JAK/STAT complexes, positive regulators (e.g., cooperating transcription factors), and negative regulators (e.g., SOCS, PIAS, PTP), demonstrate the complexity of the pathway's architecture, which can be quickly disturbed by mutations. The JAK/STAT signaling pathway has been, and still is, subject of basic research and offers an enormous potential for the development of new methods of personalized medicine and thus the translation of basic molecular research into clinical practice beyond the use of JAK inhibitors. Gain-of-function and loss-of-function mutations in the three immunologically particularly relevant signal transducers STAT1, STAT3, and STAT6 as well as JAK1 and JAK3 present themselves through individual phenotypic clinical pictures. The established, traditional paradigm of loss-of-function mutations leading to immunodeficiency and gain-of-function mutation leading to autoimmunity breaks down and a more differentiated picture of disease patterns evolve. This review is intended to provide an overview of these specific syndromes from a clinical perspective and to summarize current findings on pathomechanism, symptoms, immunological features, and therapeutic options of STAT1, STAT3, STAT6, JAK1, and JAK3 loss-of-function and gain-of-function diseases.

Relating pathogenic loss-of-function mutations in humans to their evolutionary fitness costs.

Causal loss-of-function (LOF) variants for Mendelian and severe complex diseases are enriched in 'mutation intolerant' genes. We show how such observations can be interpreted in light of a model of mutation-selection balance and use the model to relate the pathogenic consequences of LOF mutations at present to their evolutionary fitness effects. To this end, we first infer posterior distributions for the fitness costs of LOF mutations in 17,318 autosomal and 679 X-linked genes from exome sequences in 56,855 individuals. Estimated fitness costs for the loss of a gene copy are typically above 1%; they tend to be largest for X-linked genes, whether or not they have a Y homolog, followed by autosomal genes and genes in the pseudoautosomal region. We compare inferred fitness effects for all possible de novo LOF mutations to those of de novo mutations identified in individuals diagnosed with one of six severe, complex diseases or developmental disorders. Probands carry an excess of mutations with estimated fitness effects above 10%; as we show by simulation, when sampled in the population, such highly deleterious mutations are typically only a couple of generations old. Moreover, the proportion of highly deleterious mutations carried by probands reflects the typical age of onset of the disease. The study design also has a discernible influence: a greater proportion of highly deleterious mutations is detected in pedigree than case-control studies, and for autism, in simplex than multiplex families and in female versus male probands. Thus, anchoring observations in human genetics to a population genetic model allows us to learn about the fitness effects of mutations identified by different mapping strategies and for different traits.

Different molecular changes underlie the same phenotypic transition: Origins and consequences of independent shifts to homostyly within species.

The repeated transition from outcrossing to selfing is a key topic in evolutionary biology. However, the molecular basis of such shifts has been rarely examined due to lack of knowledge of the genes controlling these transitions. A classic example of mating system transition is the repeated shift from heterostyly to homostyly. Occurring in 28 angiosperm families, heterostyly is characterized by the reciprocal position of male and female sexual organs in two (or three) distinct, usually self-incompatible floral morphs. Conversely, homostyly is characterized by a single, self-compatible floral morph with reduced separation of male and female organs, facilitating selfing. Here, we investigate the origins of homostyly in Primula vulgaris and its microevolutionary consequences by integrating surveys of the frequency of homostyles in natural populations, DNA sequence analyses of the gene controlling the position of female sexual organs (CYPᵀ), and microsatellite genotyping of both progeny arrays and natural populations characterized by varying frequencies of homostyles. As expected, we found that homostyles displace short-styled individuals, but long-style morphs are maintained at low frequencies within populations. We also demonstrated that homostyles repeatedly evolved from short-styled individuals in association with different types of loss-of-function mutations in CYPᵀ. Additionally, homostyly triggers a shift to selfing, promoting increased inbreeding within and genetic differentiation among populations. Our results elucidate the causes and consequences of repeated transitions to homostyly within species, and the putative mechanisms precluding its fixation in P. vulgaris. This study represents a benchmark for future analyses of losses of heterostyly in other angiosperms.

The rate and role of pseudogenes of the Mycobacterium tuberculosis complex.

Whole-genome sequence analyses have significantly contributed to the understanding of virulence and evolution of the Mycobacterium tuberculosis complex (MTBC), the causative pathogens of tuberculosis. Most MTBC evolutionary studies are focused on single nucleotide polymorphisms and deletions, but rare studies have evaluated gene content, whereas none has comprehensively evaluated pseudogenes. Accordingly, we describe an extensive study focused on quantifying and predicting possible functions of MTBC and Mycobacterium canettii pseudogenes. Using NCBI's PGAP-detected pseudogenes, we analysed 25 837 pseudogenes from 158 MTBC and M. canetii strains and combined transcriptomics and proteomics of M. tuberculosis H37Rv to gain insights about pseudogenes' expression. Our results indicate significant variability concerning rate and conservancy of in silico predicted pseudogenes among different ecotypes and lineages of tuberculous mycobacteria and pseudogenization of important virulence factors and genes of the metabolism and antimicrobial resistance/tolerance. We show that in silico predicted pseudogenes contribute considerably to MTBC genetic diversity at the population level. Moreover, the transcription machinery of M. tuberculosis can fully transcribe most pseudogenes, indicating intact promoters and recent pseudogene evolutionary emergence. Proteomics of M. tuberculosis and close evaluation of mutational lesions driving pseudogenization suggest that few in silico predicted pseudogenes are likely capable of neofunctionalization, nonsense mutation reversal, or phase variation, contradicting the classical definition of pseudogenes. Such findings indicate that genome annotation should be accompanied by proteomics and protein function assays to improve its accuracy. While indels and insertion sequences are the main drivers of the observed mutational lesions in these species, population bottlenecks and genetic drift are likely the evolutionary processes acting on pseudogenes' emergence over time. Our findings unveil a new perspective on MTBC's evolution and genetic diversity.

TP53 loss-of-function causes vulnerability to autophagy inhibition in aggressive prostate cancer.

OBJECTIVES: TP53 loss-of-function is commonly found in aggressive prostate cancer. However, a highly-efficient therapy for this tumor subtype is still lacking. In this study, we investigated the relationship between TP53 mutation status and autophagy in prostate cancer and assessed the efficacy of autophagy inhibitors on TP53-deficient tumors. METHODS: We first evaluated the expression patterns of p53 and autophagy-related proteins, namely LC3B, ULK1 and BECLIN1, as well as their relationship in treatment-naïve and castration-resistant prostate cancer specimens through immunohistochemistry. Subsequently, we generated a Trp53-deleted genetically-engineered mouse model, established prostate tumor organoid lines from the mice and assessed the efficacy of autophagy inhibitors in overcoming Enzalutamide resistance in the tumor organoid model. We also investigated the impact of TP53 re-expression in modulating responses to autophagy inhibitors using LNCaP cell line, which harbored a TP53 missense mutation. Lastly, we attempted to identify potential autophagy-related genes that were crucial for TP53-deficient tumor maintenance. RESULTS: TP53 loss-of-function was associated with increased levels of autophagy-related proteins in aggressive prostate cancers and Trp53-deleted genetically-engineered mouse-derived tumors. Moreover, the generated androgen receptor-independent tumor organoids were highly vulnerable to autophagy inhibition. Upon TP53 re-expression, not only did the surviving LNCaP cells demonstrate resistance, but they also showed growth advantage in response to autophagy inhibition. Lastly, PEX14, an important peroxisomal regulator was differentially upregulated in aggressive tumors with TP53 loss-of-function mutations, thus implying the importance of peroxisome turnover in this tumor subtype. CONCLUSION: Our results support the potential use of autophagy inhibitors in prostate cancers that contain TP53 loss-of-function mutations.

Filaggrin loss-of-function mutations are associated with persistence of egg and milk allergy.

BACKGROUND: A genetic defect in the epidermal barrier protein filaggrin (FLG) plays a major role in the etiology of eczema and associated allergic airways diseases. However, it is still controversial to what extend loss-of-function (LOF) mutations in FLG contribute to the development and persistence of food allergies. OBJECTIVES: This study tested association of FLG LOF mutations with allergic reactions to diverse foods and investigated their potential effect on the persistence of early food allergies. METHODS: This study recruited 890 children with challenge-proven food allergy for the German Genetics of Food Allergy Study (GOFA). Longitudinal data were available for 684 children. All children were clinically characterized, including their allergic responses to specific foods, and genotyped for the 4 most common LOF mutations in FLG; R501X, 2282del4, R2447X, and S3247X. Associations between FLG mutations and food allergies were analyzed by logistic regression using the German Multicenter Allergy Study cohort as the control population. RESULTS: FLG mutations were associated with allergies to diverse foods including hen's egg (HE), cow's milk (CM), peanut, hazelnut, fish, soy, cashew, walnut, and sesame with similar risk estimates. Effects remained significant after adjusting for the eczema status. Interestingly, FLG mutations increased the risk of a persistent course of HE and CM allergy. CONCLUSIONS: Using the gold standard for food allergy diagnosis, this study demonstrates that FLG LOF mutations confer a risk of any food allergy independent of eczema. These mutations predispose to the persistence of HE and CM allergy and should be considered in the assessment of tolerance development.

Response to Wyckelsma et al.: Loss of α-actinin-3 during human evolution provides superior cold resilience and muscle heat generation.

The common loss-of-function mutation R577X in the structural muscle protein ACTN3 emerged as a potential target of positive selection from early studies and has been the focus of insightful physiological work suggesting a significant impact on muscle metabolism. Adaptation to cold climates has been proposed as a key adaptive mechanism explaining its global allele frequency patterns. Here, we re-examine this hypothesis analyzing modern (n = 3,626) and ancient (n = 1,651) genomic data by using allele-frequency as well as haplotype homozygosity-based methods. The presented results are more consistent with genetic drift rather than selection in cold climates as the main driver of the ACTN3 R577X frequency distribution in human populations across the world. This Matters Arising paper is in response to Wyckelsma et al. (2021),1 published in The American Journal of Human Genetics. See also the response by Wyckelsma et al. (2022),2 published in this issue.

A Population-Genetic Lens into the Process of Gene Loss Following Whole-Genome Duplication.

Whole-genome duplications (WGDs) have occurred in many eukaryotic lineages. However, the underlying evolutionary forces and molecular mechanisms responsible for the long-term retention of gene duplicates created by WGDs are not well understood. We employ a population-genomic approach to understand the selective forces acting on paralogs and investigate ongoing duplicate-gene loss in multiple species of Paramecium that share an ancient WGD. We show that mutations that abolish protein function are more likely to be segregating in retained WGD paralogs than in single-copy genes, most likely because of ongoing nonfunctionalization post-WGD. This relaxation of purifying selection occurs in only one WGD paralog, accompanied by the gradual fixation of nonsynonymous mutations and reduction in levels of expression, and occurs over a long period of evolutionary time, "marking" one locus for future loss. Concordantly, the fitness effects of new nonsynonymous mutations and frameshift-causing indels are significantly more deleterious in the highly expressed copy compared with their paralogs with lower expression. Our results provide a novel mechanistic model of gene duplicate loss following WGDs, wherein selection acts on the sum of functional activity of both duplicate genes, allowing the two to wander in expression and functional space, until one duplicate locus eventually degenerates enough in functional efficiency or expression that its contribution to total activity is too insignificant to be retained by purifying selection. Retention of duplicates by such mechanisms predicts long times to duplicate-gene loss, which should not be falsely attributed to retention due to gain/change in function.

The Discovery and Function of Filaggrin.

Keratohyalin granules were discovered in the mid-19th century in cells that terminally differentiate to form the outer, cornified layer of the epidermis. The first indications of the composition of these structures emerged in the 1960s from a histochemical stain for histidine, followed by radioautographic evidence of a high incidence of histidine incorporation into newly synthesized proteins in cells containing the granules. Research during the next three decades revealed the structure and function of a major protein in these granules, which was initially called the 'histidine-rich protein'. Steinert and Dale named the protein 'filaggrin' in 1981 because of its ability to aggregate keratin intermediate filaments. The human gene for the precursor, 'profilaggrin,' was reported in 1991 to encode 10, 11 or 12 nearly identical repeats. Remarkably, the mouse and rat genes encode up to 20 repeats. The lifetime of filaggrin is the time required for keratinocytes in the granular layer to move into the inner cornified layer. During this transition, filaggrin facilitates the collapse of corneocytes into 'building blocks' that become an impermeable surface barrier. The subsequent degradation of filaggrin is as remarkable as its synthesis, and the end-products aid in maintaining moisture in the cornified layer. It was apparent that ichthyosis vulgaris and atopic dermatitis were associated with the absence of this protein. McLean's team in 2006 identified the cause of these diseases by discovering loss-of-function mutations in the profilaggrin gene, which led to dysfunction of the surface barrier. This story illustrates the complexity in maintaining a healthy, functional epidermis.

The rate and role of pseudogenes of the Mycobacterium tuberculosis complex

Whole-genome sequence analyses have significantly contributed to the understanding of virulence and evolution of the Mycobacterium tuberculosis complex (MTBC), the causative pathogens of tuberculosis. Most MTBC evolutionary studies are focused on single nucleotide polymorphisms and deletions, but rare studies have evaluated gene content, whereas none has comprehensively evaluated pseudogenes. Accordingly, we describe an extensive study focused on quantifying and predicting possible functions of MTBC and Mycobacterium canettii pseudogenes. Using NCBI’s PGAP-detected pseudogenes, we analysed 25 837 pseudogenes from 158 MTBC and M. canetii strains and combined transcriptomics and proteomics of M. tuberculosis H37Rv to gain insights about pseudogenes' expression. Our results indicate significant variability concerning rate and conservancy of in silico predicted pseudogenes among different ecotypes and lineages of tuberculous mycobacteria and pseudogenization of important virulence factors and genes of the metabolism and antimicrobial resistance/tolerance. We show that in silico predicted pseudogenes contribute considerably to MTBC genetic diversity at the population level. Moreover, the transcription machinery of M. tuberculosis can fully transcribe most pseudogenes, indicating intact promoters and recent pseudogene evolutionary emergence. Proteomics of M. tuberculosis and close evaluation of mutational lesions driving pseudogenization suggest that few in silico predicted pseudogenes are likely capable of neofunctionalization, nonsense mutation reversal, or phase variation, contradicting the classical definition of pseudogenes. Such findings indicate that genome annotation should be accompanied by proteomics and protein function assays to improve its accuracy. While indels and insertion sequences are the main drivers of the observed mutational lesions in these species, population bottlenecks and genetic drift are likely the evolutionary processes acting on pseudogenes' emergence over time. Our findings unveil a new perspective on MTBC’s evolution and genetic diversity.

Meester-Loeys Syndrome.

Meester-Loeys syndrome is an X-linked form of syndromic thoracic aortic aneurysm, characterized by the involvement of multiple organ systems. More specifically, the cardiovascular, skeletal, craniofacial, cutaneous and neurological systems are affected. Clear clinical overlap with Marfan syndrome and Loeys-Dietz syndrome is observed. Aortic dissections occur typically at young ages and are most often observed in males. Meester-Loeys syndrome is caused by loss-of-function mutations in BGN, encoding the small leucine-rich proteoglycan biglycan. Although functional consequences of these mutations remain largely elusive, increased TGF-ß signaling has been observed. Novel insights will provide opportunities for preventive therapeutic interventions.

Variable Expressivity of the Beckwith-Wiedemann Syndrome in Four Pedigrees Segregating Loss-of-Function Variants of CDKN1C.

Beckwith-Wiedemann syndrome (BWS) is an imprinting disorder characterized by prenatal and/or postnatal overgrowth, organomegaly, abdominal wall defects and tumor predisposition. CDKN1C is a maternally expressed gene of the 11p15.5 chromosomal region and is regulated by the imprinting control region IC2. It negatively controls cellular proliferation, and its expression or activity are frequently reduced in BWS. In particular, loss of IC2 methylation is associated with CDKN1C silencing in the majority of sporadic BWS cases, and maternally inherited loss-of-function variants of CDKN1C are the most frequent molecular defects of familial BWS. We have identified, using Sanger sequencing, novel CDKN1C variants in three families with recurrent cases of BWS, and a previously reported variant in a woman with recurrent miscarriages with exomphalos. Clinical evaluation of the patients showed variable manifestation of the disease. The frameshift and nonsense variants were consistently associated with exomphalos, while the missense variant caused a less severe phenotype. Pregnancy loss and perinatal lethality were found in the families segregating nonsense mutations. Intrafamilial variability of the clinical BWS features was observed, even between siblings. Our data are indicative of severe BWS phenotypes that, with variable expressivity, may be associated with both frameshift and nonsense variants of CDKN1C.

Gain and loss of function mutations in biological chemical reaction networks: a mathematical model with application to colorectal cancer cells.

This paper studies a system of Ordinary Differential Equations modeling a chemical reaction network and derives from it a simulation tool mimicking Loss of Function and Gain of Function mutations found in cancer cells. More specifically, from a theoretical perspective, our approach focuses on the determination of moiety conservation laws for the system and their relation with the corresponding stoichiometric surfaces. Then we show that Loss of Function mutations can be implemented in the model via modification of the initial conditions in the system, while Gain of Function mutations can be implemented by eliminating specific reactions. Finally, the model is utilized to examine in detail the G1-S phase of a colorectal cancer cell.

Novel mutations in FSIP2 lead to multiple morphological abnormalities of the sperm flagella and poor ICSI prognosis.

Multiple morphological abnormalities of the sperm flagella (MMAF) is defined as deformities that cause sperm motility disorders, further resulting in male infertility. However, the reported genes related to sperm flagellar defects can only explain approximately 60% of human MMAF cases. Here, we report two novel compound heterozygous mutations, c.16246_16247insCCCAAATATCACC (p. T5416fs*7) and c.17323C > T (p.Q5774*), in the fibrous sheath-interacting protein 2 gene (FSIP2; OMIM: 615796) in an infertile patient by whole-exome sequencing (WES). Western blotting and immunofluorescence staining confirmed that the compound heterozygous mutations abrogated FSIP2 protein expression. Notably, our staining revealed that FSIP2 is expressed in the cytoplasm of primary germ cell and flagella of spermatids during the spermiogenesis. Moreover, intracytoplasmic sperm injection (ICSI) was carried out using sperm from this patient; however, pregnancy failed after embryo transfer through one cycle. Our findings may be helpful in establishing a genetic diagnosis for MMAF, as well as provide additional beneficial knowledge for genetic counseling and infertility treatment.

Context Matters: NOTCH Signatures and Pathway in Cancer Progression and Metastasis.

The Notch signaling pathway is a critical player in embryogenesis but also plays various roles in tumorigenesis, with both tumor suppressor and oncogenic activities. Mutations, deletions, amplifications, or over-expression of Notch receptors, ligands, and a growing list of downstream Notch-activated genes have by now been described for most human cancer types. Yet, it often remains unclear what may be the functional impact of these changes for tumor biology, initiation, and progression, for cancer therapy, and for personalized medicine. Emerging data indicate that Notch signaling can also contribute to increased aggressive properties such as invasion, tumor heterogeneity, angiogenesis, or tumor cell dormancy within solid cancer tissues; especially in epithelial cancers, which are in the center of this review. Notch further supports the "stemness" of cancer cells and helps define the stem cell niche for their long-term survival, by integrating the interaction between cancer cells and the cells of the tumor microenvironment (TME). The complexity of Notch crosstalk with other signaling pathways and its roles in cell fate and trans-differentiation processes such as epithelial-to-mesenchymal transition (EMT) point to this pathway as a decisive player that may tip the balance between tumor suppression and promotion, differentiation and invasion. Here we not only review the literature, but also explore genomic databases with a specific focus on Notch signatures, and how they relate to different stages in tumor development. Altered Notch signaling hereby plays a key role for tumor cell survival and coping with a broad spectrum of vital issues, contributing to failed therapies, poor patient outcome, and loss of lives.

Functional evaluation of 16 SCHAD missense variants: Only amino acid substitutions causing congenital hyperinsulinism of infancy lead to loss-of-function phenotypes in vitro.

Short-chain 3-hydroxyacyl-CoA dehydrogenase (SCHAD), encoded by the HADH gene, is a ubiquitously expressed mitochondrial enzyme involved in fatty acid oxidation. This protein also plays a role in insulin secretion as recessive HADH mutations cause congenital hyperinsulinism of infancy (CHI) via loss of an inhibitory interaction with glutamate dehydrogenase (GDH). Here, we present a functional evaluation of 16 SCHAD missense variants identified either in CHI patients or by high-throughput sequencing projects in various populations. To avoid interactions with endogenously produced SCHAD protein, we assessed protein stability, subcellular localization, and GDH interaction in a SCHAD knockout HEK293 cell line constructed by CRISPR-Cas9 methodology. We also established methods for efficient SCHAD expression and purification in E. coli, and tested enzymatic activity of the variants. Our analyses showed that rare variants of unknown significance identified in populations generally had similar properties as normal SCHAD. However, the CHI-associated variants p.Gly34Arg, p.Ile184Phe, p.Pro258Leu, and p.Gly303Ser were unstable with low protein levels detectable when expressed in HEK293 cells. Moreover, CHI variants p.Lys136Glu, p.His170Arg, and p.Met188Val presented normal protein levels but displayed clearly impaired enzymatic activity in vitro, and their interaction with GDH appeared reduced. Our results suggest that pathogenic missense variants of SCHAD either make the protein target of a post-translational quality control system or can impair the function of SCHAD without influencing its steady-state protein level. We did not find any evidence that rare SCHAD missense variants observed only in the general population and not in CHI patients are functionally affected.

[Progress on loss-of-function hypothesis of presenilin-1 mutations in Alzheimer diseases].

Alzheimer's disease (AD) is an aging-related neurodegenerative disease and is associated with the accumulation of amyloid-ß (Aß) peptides in patient brains. AD can be classified into the familial type and sporadic type. Presenilin-1 (PS1) is the major risk gene for familial AD (fAD) because its mutations comprised over 80%of the total mutations causing fAD. PS1 is the catalytic subunit of the enzyme γ-secretase, which is responsible for the proteolytic cleavage of amyloid precursor protein (APP) to produce Aß. Although novel fAD-causing mutations in PS1 are being reported increasingly, the molecular mechanisms underlying how these mutations induce fAD remain elusive. Since over 90%of the fAD-causing mutations in PS1 leads to a reduction of γ-secretase activity, the PS1 loss-of-function mutation hypothesis has been emerged, which suggests that the loss of PS1 functions may be the root cause of AD. Recently, increasing number of evidence supports this hypothesis. First, PS1 loss-of-function mutations increase the production of long-length Aß by disturbing the cleavage sites of γ-secretase APP, thereby increasing the ratio of Aß42/Aß40; Second, PS1 loss-of-function mutations dysregulate endoplasmic reticulum calcium homeostasis in neurons; Third, PS1 loss-of-function mutations inhibit the autophagy activity of neurons, resulting in the abnormal accumulation of cleaved products from APP; Fourth, PS1 loss-of-function mutations alter the endocytosis and transcytosis processes in neurons, leading to neuratrophy; Fifth, PS1 loss-of-function mutations activate brain immune cells (astrocytes and microglia), which mount a strong neuroinflammation response; Last, PS1 loss-of-function mutations reduce the rates of glycolysis and the production of lactic acid, disrupting the balance of neuronal energy supply. In this article we summary the research progress on the PS1 loss-of-function hypothesis and pose several topics which would guide studies of this field in future.