Novel Homozygous Variants of SLC13A5 Expand the Functional Heterogeneity of a Homogeneous Syndrome of Early Infantile Epileptic Encephalopathy.

BACKGROUND: Early infantile epileptic encephalopathy 25 (EIEE25) is a distinct type of neonatal epileptic encephalopathy caused by autosomal recessive mutations in the SLC13A5 gene. SLC13A5 encodes a transmembrane sodium/citrate cotransporter required for regulating citrate entry into cells. METHODS: Four families with recessively inherited epileptic encephalopathy were sequenced by clinically accredited laboratories using commercially available epilepsy gene panels. Patients were examined by a neurologist and were clinically diagnosed with infantile epileptic encephalopathy. RESULTS: We present four families with global developmental delay, intellectual disability, and defective tooth development with four novel homozygous mutations in SLC13A5. The neurological examination showed spastic quadriplegia with increased deep tendon reflexes. Brain magnetic resonance imaging showed nonspecific signal abnormality of the bilateral hemispheric white matter. Despite similar clinical features, the conditions were based on different molecular mechanisms acting on SLC13A5 (abnormal splicing, large-scale deletions, and tandem-residue insertion). CONCLUSIONS: Our results extend the landscape of autosomal recessive inherited homozygous mutations in SLC13A5 that cause a distinctive syndrome of severe neonatal epileptic encephalopathy. Our observations confirm the homogeneity of epileptic encephalopathy and dental abnormalities as a distinct clinical marker for EIEE25 despite the heterogeneous functional and mutational background.

An open-source, automated, and cost-effective platform for COVID-19 diagnosis and rapid portable genomic surveillance using nanopore sequencing.

The COVID-19 pandemic, caused by SARS-CoV-2, has emphasized the necessity for scalable diagnostic workflows using locally produced reagents and basic laboratory equipment with minimal dependence on global supply chains. We introduce an open-source automated platform for high-throughput RNA extraction and pathogen diagnosis, which uses reagents almost entirely produced in-house. This platform integrates our methods for self-manufacturing magnetic nanoparticles and qRT-PCR reagents-both of which have received regulatory approval for clinical use-with an in-house, open-source robotic extraction protocol. It also incorporates our "Nanopore Sequencing of Isothermal Rapid Viral Amplification for Near Real-time Analysis" (NIRVANA) technology, designed for tracking SARS-CoV-2 mutations and variants. The platform exhibits high reproducibility and consistency without cross-contamination, and its limit of detection, sensitivity, and specificity are comparable to commercial assays. Automated NIRVANA effectively identifies circulating SARS-CoV-2 variants. Our in-house, cost-effective reagents, automated diagnostic workflows, and portable genomic surveillance strategies provide a scalable and rapid solution for COVID-19 diagnosis and variant tracking, essential for current and future pandemic responses.

Leveraging AI Advances and Online Tools for Structure-Based Variant Analysis.

Understanding how a gene variant affects protein function is important in life science, as it helps explain traits or dysfunctions in organisms. In a clinical setting, this understanding makes it possible to improve and personalize patient care. Bioinformatic tools often only assign a pathogenicity score, rather than providing information about the molecular basis for phenotypes. Experimental testing can furnish this information, but this is slow and costly and requires expertise and equipment not available in a clinical setting. Conversely, mapping a gene variant onto the three-dimensional (3D) protein structure provides a fast molecular assessment free of charge. Before 2021, this type of analysis was severely limited by the availability of experimentally determined 3D protein structures. Advances in artificial intelligence algorithms now allow confident prediction of protein structural features from sequence alone. The aim of the protocols presented here is to enable non-experts to use databases and online tools to investigate the molecular effect of a genetic variant. The Basic Protocol relies only on the online resources AlphaFold, Protein Structure Database, and UniProt. Alternate Protocols document the usage of the Protein Data Bank, SWISS-MODEL, ColabFold, and PyMOL for structure-based variant analysis. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol: 3D Mapping based on UniProt and AlphaFold Alternate Protocol 1: Using experimental models from the PDB Alternate Protocol 2: Using information from homology modeling with SWISS-MODEL Alternate Protocol 3: Predicting 3D structures with ColabFold Alternate Protocol 4: Structure visualization and analysis with PyMOL.

Expanding the genotype-phenotype landscape of PDE10A-associated movement disorders.

BACKGROUND: Phosphodiesterase 10A (PDE10A) controls body movements by regulating cyclic adenosine monophosphate signaling in the basal ganglia. Two classes of PDE10A variants are reported with distinctive genotype-phenotype correlation. The autosomal recessive mutations in the GAF-A and catalytic domains are associated with compromised membrane localization, and manifest with infantile onset chorea, developmental, and cognition delay with normal brain MRI. Conversely, autosomal dominant mutations in the GAF-B domain cause protein aggregates which results in childhood onset chorea in the context of normal cognition and development, with striatal lesions. METHODS: Phenotypic characteristics of affected individuals with PDE10A mutations belonging to a single family were recorded. In addition, Sanger sequencing and in silico analysis were used to identify the mutations. Homozygosity mapping was applied together with whole exome sequencing. RESULTS: Four individuals from a consanguineous family affected with PDE10A mutations were observed for up to 40 years. Although these individuals displayed a clinical phenotype attributed to the recessive GAF-A mutations, they revealed a bi-allelic GAF-B mutation (c.883G > A:p. D295 N; p.Asp295Asn) that was segregated with all affected individuals. In addition to chorea, we observed peculiar foot deformities and pronounced social phobia, with normal brain MRI. In silico structural analysis suggested that the GAF-B mutation blocked allosteric PDE10A activation. The resulting lack of PDE10A activity likely phenocopies GAF-A mutations, and this is achieved through a distinct mechanism. CONCLUSIONS: Collectively, our findings demonstrate the association of recessive and dominant phenotypes of known variants, and further expands the genotype-phenotype landscape of PDE10A-associated movement disorders.

SPTBN5, Encoding the ßV-Spectrin Protein, Leads to a Syndrome of Intellectual Disability, Developmental Delay, and Seizures.

Whole exome sequencing has provided significant opportunities to discover novel candidate genes for intellectual disability and autism spectrum disorders. Variants in the spectrin genes SPTAN1, SPTBN1, SPTBN2, and SPTBN4 have been associated with neurological disorders; however, SPTBN5 gene-variants have not been associated with any human disorder. This is the first report that associates SPTBN5 gene variants (ENSG00000137877: c.266A>C; p.His89Pro, c.9784G>A; p.Glu3262Lys, c.933C>G; p.Tyr311Ter, and c.8809A>T; p.Asn2937Tyr) causing neurodevelopmental phenotypes in four different families. The SPTBN5-associated clinical traits in our patients include intellectual disability (mild to severe), aggressive tendencies, accompanied by variable features such as craniofacial and physical dysmorphisms, autistic behavior, and gastroesophageal reflux. We also provide a review of the existing literature related to other spectrin genes, which highlights clinical features partially overlapping with SPTBN5.

The genome of the zoonotic malaria parasite Plasmodium simium reveals adaptations to host switching.

BACKGROUND: Plasmodium simium, a malaria parasite of non-human primates (NHP), was recently shown to cause zoonotic infections in humans in Brazil. We sequenced the P. simium genome to investigate its evolutionary history and to identify any genetic adaptions that may underlie the ability of this parasite to switch between host species. RESULTS: Phylogenetic analyses based on whole genome sequences of P. simium from humans and NHPs reveals that P. simium is monophyletic within the broader diversity of South American Plasmodium vivax, suggesting P. simium first infected NHPs as a result of a host switch of P. vivax from humans. The P. simium isolates show the closest relationship to Mexican P. vivax isolates. Analysis of erythrocyte invasion genes reveals differences between P. vivax and P. simium, including large deletions in the Duffy-binding protein 1 (DBP1) and reticulocyte-binding protein 2a genes of P. simium. Analysis of P. simium isolated from NHPs and humans revealed a deletion of 38 amino acids in DBP1 present in all human-derived isolates, whereas NHP isolates were multi-allelic. CONCLUSIONS: Analysis of the P. simium genome confirmed a close phylogenetic relationship between P. simium and P. vivax, and suggests a very recent American origin for P. simium. The presence of the DBP1 deletion in all human-derived isolates tested suggests that this deletion, in combination with other genetic changes in P. simium, may facilitate the invasion of human red blood cells and may explain, at least in part, the basis of the recent zoonotic infections.

Delineating the molecular and phenotypic spectrum of the SETD1B-related syndrome.

PURPOSE: Pathogenic variants in SETD1B have been associated with a syndromic neurodevelopmental disorder including intellectual disability, language delay, and seizures. To date, clinical features have been described for 11 patients with (likely) pathogenic SETD1B sequence variants. This study aims to further delineate the spectrum of the SETD1B-related syndrome based on characterizing an expanded patient cohort. METHODS: We perform an in-depth clinical characterization of a cohort of 36 unpublished individuals with SETD1B sequence variants, describing their molecular and phenotypic spectrum. Selected variants were functionally tested using in vitro and genome-wide methylation assays. RESULTS: Our data present evidence for a loss-of-function mechanism of SETD1B variants, resulting in a core clinical phenotype of global developmental delay, language delay including regression, intellectual disability, autism and other behavioral issues, and variable epilepsy phenotypes. Developmental delay appeared to precede seizure onset, suggesting SETD1B dysfunction impacts physiological neurodevelopment even in the absence of epileptic activity. Males are significantly overrepresented and more severely affected, and we speculate that sex-linked traits could affect susceptibility to penetrance and the clinical spectrum of SETD1B variants. CONCLUSION: Insights from this extensive cohort will facilitate the counseling regarding the molecular and phenotypic landscape of newly diagnosed patients with the SETD1B-related syndrome.

Clinical and Genetic Characterization of Craniosynostosis in Saudi Arabia.

Background: Craniosynostosis (CS) is defined as pre-mature fusion of one or more of the cranial sutures. CS is classified surgically as either simple or complex based on the number of cranial sutures involved. CS can also be classified genetically as isolated CS or syndromic CS if the patient has extracranial deformities. Currently, the link between clinical and genetic patterns of CS in the Saudi population is poorly understood. Methodology: We conducted a retrospective cohort study among 28 CS patients, of which 24 were operated and four were not. Clinical and genetic data were collected between February 2015 and February 2019, from consenting patient's families. The electronic chart data were collected and analyzed including patient demographics, craniofacial features, other anomalies and dysmorphic features, operative data, intra cranial pressure (ICP), parent consanguinity and genetic testing results. Results: The most common deformity in our population was trigonocephaly. The most performed procedure was cranial vault reconstruction with fronto-orbital advancement, followed by posterior vault distraction osteogenesis and suturectomy with barrel staving. Genetics analysis revealed pathogenic mutations in FGFR2 (6 cases), TWIST1 (3 cases), ALPL (2 cases), and TCF12 (2 cases), and FREM1 (2 case). Conclusion: Compared to Western countries, our Saudi cohort displays significant differences in the prevalence of CS features, such as the types of sutures and prevalence of inherited CS. The genomic background allows our phenotype-genotype study to reclassify variants of unknown significance. Worldwide, the sagittal suture is the most commonly affected suture in simple CS, but in the Saudi population, the metopic suture fusion was most commonly seen in our clinic. Further studies are needed to investigate the characteristics of CS in our population in a multicenter setting.

MYH1 is a candidate gene for recurrent rhabdomyolysis in humans.

Rhabdomyolysis is a serious medical condition characterized by muscle injury, and there are recognized genetic causes especially in recurrent forms. The majority of these cases, however, remain unexplained. Here, we describe a patient with recurrent rhabdomyolysis in whom extensive clinical testing failed to identify a likely etiology. Whole-exome sequencing revealed a novel missense variant in MYH1, which encodes a major adult muscle fiber protein. Structural biology analysis revealed that the mutated residue is extremely well conserved and is located in the actin binding cleft. Furthermore, immediately adjacent mutations in that cleft in other myosins are pathogenic in humans. Our results are consistent with the finding that MYH1 is mutated in rhabdomyolysis in horses and suggest that this gene should be investigated in cases with recurrent rhabdomyolysis.

Comprehensive multi-omics analysis of G6PC3 deficiency-related congenital neutropenia with inflammatory bowel disease.

Autosomal recessive mutations in G6PC3 cause isolated and syndromic congenital neutropenia which includes congenital heart disease and atypical inflammatory bowel disease (IBD). In a highly consanguineous pedigree with novel mutations in G6PC3 and MPL, we performed comprehensive multi-omics analyses. Structural analysis of variant G6PC3 and MPL proteins suggests a damaging effect. A distinct molecular cytokine profile (cytokinome) in the affected proband with IBD was detected. Liquid chromatography-mass spectrometry-based proteomics analysis of the G6PC3-deficient plasma samples identified 460 distinct proteins including 75 upregulated and 73 downregulated proteins. Specifically, the transcription factor GATA4 and LST1 were downregulated while platelet factor 4 (PF4) was upregulated. GATA4 and PF4 have been linked to congenital heart disease and IBD respectively, while LST1 may have perturbed a variety of essential cell functions as it is required for normal cell-cell communication. Together, these studies provide potentially novel insights into the pathogenesis of syndromic congenital G6PC3 deficiency.

Interleukin-26 activates macrophages and facilitates killing of Mycobacterium tuberculosis.

Tuberculosis-causing Mycobacterium tuberculosis (Mtb) is transmitted via airborne droplets followed by a primary infection of macrophages and dendritic cells. During the activation of host defence mechanisms also neutrophils and T helper 1 (TH1) and TH17 cells are recruited to the site of infection. The TH17 cell-derived interleukin (IL)-17 in turn induces the cathelicidin LL37 which shows direct antimycobacterial effects. Here, we investigated the role of IL-26, a TH1- and TH17-associated cytokine that exhibits antimicrobial activity. We found that both IL-26 mRNA and protein are strongly increased in tuberculous lymph nodes. Furthermore, IL-26 is able to directly kill Mtb and decrease the infection rate in macrophages. Binding of IL-26 to lipoarabinomannan might be one important mechanism in extracellular killing of Mtb. Macrophages and dendritic cells respond to IL-26 with secretion of tumor necrosis factor (TNF)-α and chemokines such as CCL20, CXCL2 and CXCL8. In dendritic cells but not in macrophages cytokine induction by IL-26 is partly mediated via Toll like receptor (TLR) 2. Taken together, IL-26 strengthens the defense against Mtb in two ways: firstly, directly due to its antimycobacterial properties and secondly indirectly by activating innate immune mechanisms.

Novel Missense Variant in Heterozygous State in the BRPF1 Gene Leading to Intellectual Developmental Disorder With Dysmorphic Facies and Ptosis.

Intellectual developmental disorder with dysmorphic facies and ptosis is an autosomal dominant condition characterized by delayed psychomotor development, intellectual disability, delayed speech, and dysmorphic facial features, mostly ptosis. Heterozygous mutations in bromodomain and plant homeodomain (PHD) finger containing one (BRPF1) gene have been reported. In this study, whole exome sequencing (WES) was performed as a molecular diagnostic test. Bioinformatics of WES data and candidate gene prioritization identified a novel variant in heterozygous state in the exon 3 of BRPF1 gene (ENST383829: c.1054G > C and p.Val352Leu). Autosomal dominant inheritance in the family affected individuals and exclusion of non-pathogenicity in the ethnically matched healthy controls (n = 100) were performed by Sanger sequencing. To the best of our knowledge, this is the first evidence of BRPF1 variant in a Saudi family. Whole exome sequencing analysis has been proven as a valuable tool in the molecular diagnostics. Our findings further expand the role of WES in efficient disease diagnosis in Arab families and explained that the mutation in BRPF1 gene plays an important role for the development of IDDFP syndrome.

A Novel Biallelic STING1 Gene Variant Causing SAVI in Two Siblings.

STING-associated vasculopathy of infantile-onset (SAVI) is one of the newly identified types of interferonopathies. SAVI is caused by heterozygous gain-of-function mutations in the STING1. We herein report for the first time a homozygous variant in the STING1 gene in two siblings that resulted in constitutive activation of STING gene and the SAVI phenotype. Exome sequencing revealed a novel homozygous NM_198282.3: c.841C>T; p.(Arg281Trp) variant in exon 7 of the STING1 gene. The variant segregated in the family to be homozygous in all affected and either heterozygous or wild type in all healthy. Computational structural analysis of the mutants revealed changes in the STING protein structure/function. Elevated serum beta-interferon levels were observed in the patients compared to the control family members. Treatment with Janus kinase inhibitor (JAK-I) Ruxolitinib suppressed the inflammatory process, decreased beta-interferon levels, and stopped the progression of the disease.

Truncating biallelic variant in DNAJA1, encoding the co-chaperone Hsp40, is associated with intellectual disability and seizures.

Intellectual disability poses a huge burden on the health care system, and it is one of the most common referral reasons to the genetic and child neurology clinic. Intellectual disability (ID) is genetically heterogeneous, and it is associated with several other neurological conditions. Exome sequencing is a robust genetic tool and has revolutionized the process of molecular diagnosis and novel gene discovery. Besides its diagnostic clinical value, novel gene discovery is prime in reverse genetics, when human mutations help to understand the function of a gene and may aid in better understanding of the human brain and nervous system. Using WES, we identified a biallelic truncating variant in DNAJA1 gene (c.511C>T p.(Gln171*) in a multiplex Saudi consanguineous family. The main phenotype shared between the siblings was intellectual disability and seizure disorder.

Loss of tubulin deglutamylase CCP1 causes infantile-onset neurodegeneration.

A set of glutamylases and deglutamylases controls levels of tubulin polyglutamylation, a prominent post-translational modification of neuronal microtubules. Defective tubulin polyglutamylation was first linked to neurodegeneration in the Purkinje cell degeneration (pcd) mouse, which lacks deglutamylase CCP1, displays massive cerebellar atrophy, and accumulates abnormally glutamylated tubulin in degenerating neurons. We found biallelic rare and damaging variants in the gene encoding CCP1 in 13 individuals with infantile-onset neurodegeneration and confirmed the absence of functional CCP1 along with dysregulated tubulin polyglutamylation. The human disease mainly affected the cerebellum, spinal motor neurons, and peripheral nerves. We also demonstrate previously unrecognized peripheral nerve and spinal motor neuron degeneration in pcd mice, which thus recapitulated key features of the human disease. Our findings link human neurodegeneration to tubulin polyglutamylation, entailing this post-translational modification as a potential target for drug development for neurodegenerative disorders.

Biallelic UFM1 and UFC1 mutations expand the essential role of ufmylation in brain development.

The post-translational modification of proteins through the addition of UFM1, also known as ufmylation, plays a critical developmental role as revealed by studies in animal models. The recent finding that biallelic mutations in UBA5 (the E1-like enzyme for ufmylation) cause severe early-onset encephalopathy with progressive microcephaly implicates ufmylation in human brain development. More recently, a homozygous UFM1 variant was proposed as a candidate aetiology of severe early-onset encephalopathy with progressive microcephaly. Here, we establish a locus for severe early-onset encephalopathy with progressive microcephaly based on two families, and map the phenotype to a novel homozygous UFM1 mutation. This mutation has a significantly diminished capacity to form thioester intermediates with UBA5 and with UFC1 (the E2-like enzyme for ufmylation), with resulting impaired ufmylation of cellular proteins. Remarkably, in four additional families where eight children have severe early-onset encephalopathy with progressive microcephaly, we identified two biallelic UFC1 mutations, which impair UFM1-UFC1 intermediate formation with resulting widespread reduction of cellular ufmylation, a pattern similar to that observed with UFM1 mutation. The striking resemblance between UFM1- and UFC1-related clinical phenotype and biochemical derangements strongly argues for an essential role for ufmylation in human brain development. The hypomorphic nature of UFM1 and UFC1 mutations and the conspicuous depletion of biallelic null mutations in the components of this pathway in human genome databases suggest that it is necessary for embryonic survival, which is consistent with the embryonic lethal nature of knockout models for the orthologous genes.