Biallelic Mutations Research Articles

Identifying novel response markers for spinal muscular atrophy revealed by targeted proteomics following gene therapy.

Spinal muscular atrophy (SMA) is a progressive disease that affects motor neurons, with symptoms usually starting in infancy or early childhood. Recent breakthroughs in treatments targeting SMA have improved both lifespan and quality of life for infants and children with the disease. Given the impact of these treatments, it is essential to develop methods for managing treatment-induced changes in disease characteristics. Zolgensma® is the first effective and approved gene therapy for SMA caused by biallelic mutation in the SMN1 gene. In three children with SMA treated with Zolgensma®, neuronal, glial, inflammation, and vascular markers in the plasma exhibited a quicker response, emphasizing their potential as valuable biomarkers of treatment efficacy in clinical trials. We chose the novel Nucleic acid Linked Immuno-Sandwich Assay, to investigate a predefined panel of neuroinflammatory markers in plasma samples collected from SMA patients at baseline and six months after Zolgensma® treatment. We identified a set of novel targets whose levels differed between pre and post Zolgensma® treatment group and that were responsive to treatment. Even though our results warrant validation in larger SMA cohorts and longer follow-up time, they may pave the way for a panel of responsive proteins solidifying biomarker endpoints in SMA clinical trials.

Biallelic Mutations in ADAM22 Presenting as Ohtahara Syndrome in an Indian Family: Expanding the Electroclinical Phenotype of ADAM22-Related Neurologic Disorder.

Biallelic Mutations in ADAM22 Presenting as Ohtahara Syndrome in an Indian Family: Expanding the Electroclinical Phenotype of ADAM22-Related Neurologic Disorder.

A pilot trial for efficacy confirmation of 6′-sialyllactose supplementation in GNE myopathy: Randomized, placebo-controlled trial

GNE myopathy is a rare genetic muscle disorder characterized by initial ankle dorsiflexor weakness and the presence of rimmed vacuoles in muscle histopathology. Biallelic mutations in the GNE gene are causative, leading to reduced production of sialic acid. In our previous clinical trial, we used 6′-sialyllactose (6SL) as a supplement to increase sialic acid levels and compared the effects of 6SL at doses of 3 g and 6 g. The findings from the trial revealed superior outcomes in muscle strength, attenuation of muscle degeneration, and bioavailability in the 6 g group.This trial was planned to complement the lack of placebo arm from the previous trial and to provide more conclusive evidence for therapeutic value of 6SL in GNE myopathy. Of the 11 participants, five were allocated to the 6SL and six to the placebo group after undergoing 12 weeks of pre-study observation and stratified randomization. At every visit with an interval of 12 weeks for 48 weeks, all participants underwent muscle strength measurement, muscle MRI, biochemical evaluations, 6-min-walk test, and completed a questionnaire.No safety concerns arose during the trial period. Muscle strength, excluding hand grip power, did not show a significant difference between the two groups, which is attributed to the lack of pronounced muscle strength decline in both groups. Hand grip power tended to decrease in both groups, and this decline was statistically significant in the placebo group (p = 0.0004). The fat fraction measured by MRI showed the most significant results in the posterior thigh. The increase in fat fraction, indicating muscle degeneration, was statistically significant between the two groups (p = 0.0004). Although no statistically significant differences were observed between the groups in anterior thigh and both anterior and posterior lower leg, a trend of slowed increase in fat fraction was noted in the 6SL group compared to the placebo group starting from 24 or 36 weeks. Resialylation of cell surface glycoconjugate was demonstrated in 6SL group by measuring lectin bindings on peripheral blood monocytes.The GNEM-FAS, used to assess patient-reported outcomes, did not show statistical significance in the total score or any of the three domains. However, the tendency for scores in the self-care and upper extremity domains to rebound after 24 weeks in the 6SL group suggests the potential for long-term benefits.The effect of 6SL on muscle strength appeared to be minimal compared to our previous clinical trial, likely due to the short duration of the study and the inclusion of relatively early-stage patients. However, the changes in fat fraction measured by muscle MRI and the results of biochemical assays are more promising, suggesting potential benefits with long-term administration in the future.

Modeling sacsin depletion in Danio Rerio offers new insight on retinal defects in ARSACS.

Biallelic mutations in the SACS gene, encoding sacsin, cause early-onset autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), a neurodegenerative disease also characterized by unique and poorly understood retinal abnormalities. While two murine models replicate the phenotypic and neuronal features observed in patients, no retinal phenotype has been described so far. In a zebrafish knock-out strain that faithfully mirrors the main aspects of ARSACS, we observed impaired visual function due to photoreceptor degeneration, likely caused by cell cycle defects in progenitor cells. RNA-seq analysis in embryos revealed dysfunction in proteins related to fat-soluble vitamins (e.g., TTPA, RDH5, VKORC) and suggested a key role of neuroinflammation in driving the retinal defects. Our findings indicate that studying retinal pathology in ARSACS could be crucial for understanding the impact of sacsin depletion and may offer insights into halting disease progression.

Calcium level and autophagy defect in GNE mutants of rare neuromuscular disorder.

Rare genetic disorders are low in prevalence and hence there is little or no attention paid to them in the mainstream medical industry. One of the ultra-rare neuromuscular disorders, GNE myopathy is caused due to biallelic mutations in the bifunctional enzyme, GNE (UDP N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase). It catalyses the rate-limiting step in sialic acid biosynthesis. There are no effective treatments for GNE myopathy as the pathomechanism is poorly understood. Pathologically, the disease is characterized by the formation of rimmed vacuoles that contain aggregates of β-amyloid, tau, presenilin etc proteins in muscle biopsy samples. Accumulation of aggregated proteins in the cells may occur due to the failure of the regulated autophagy phenomenon. In the present study, we aim to understand the effect of GNE mutations on autophagy. The cytosolic calcium levels in GNE mutant cells were found to be altered in a GNE mutation-specific manner. The chaperone levels, such as HSP70 and PDI, as well as autophagic markers (LC3II/I ratios) were altered in the GNE mutant cells. Treatment with BAPTA-AM, calcium chelator, significantly restored cytosolic calcium levels in some GNE mutant cells as well as autophagic marker levels and autophagic punctae formation. The effect on the calcium signalling cascade involving CaMKKβ/AMPK/mTOR was studied in the GNE mutant cells. Our study provides insights into the role of calcium in autophagic vacuole formation in the cells with GNE mutations that will have significance towards understanding the pathomechanism of GNE Myopathy and drug target identification for the rare disease.

Novel biallelic nonsense mutation in IGHMBP2 gene linked to neuropathy (CMT2S): A comprehensive clinical, genetic and bioinformatic analysis of a Turkish patient with literature review.

Spinal muscular atrophy with respiratory distress type 1 (SMARD1) and Charcot-Marie-Tooth type 2S (CMT2S) typically present before age 10. Genetic factors account for up to 50% of neuropathies, which often display varied symptoms. Mutations in the IGHMBP2 gene are associated with both CMT2S and SMARD1, resulting in a rare clinical condition marked by axonal neuropathy, spinal muscular atrophy, respiratory distress, and muscle weakness. Detailed family histories and medical data were collected. Segregation analysis was performed using Sanger sequencing and whole exome sequencing. Additionally, a review of molecularly confirmed patients was conducted. Protein tertiary structures expressed in the IGHMBP2 gene were tested for topological and conformational changes using modeling programs and in-silico tools. We identified a novel homozygous nonsense mutation (c.2568_2569del p.Gly857Alafs*27) in a family with a member showing neuropathy. This report details the clinical and genetic findings of the affected individuals, including a Turkish patient with neuropathy, and compares them with literature cases. Understanding the clinical impact of the (c.2568_2569del p.Gly857Alafs*27) mutation will enhance our knowledge of IGHMBP2 gene defects role in neuropathy. This study aims to highlight this severe recessive disease caused by pathogenic IGHMBP2 gene mutations and to examine the mutation spectrum and phenotype differences.

TTC7A missense variants in intestinal disease can be classified by molecular and cellular phenotypes.

Biallelic mutations in tetratricopeptide repeat domain 7A (TTC7A) give rise to intestinal and immune disorders. However, our understanding of the genotype-phenotype relationship is limited, because TTC7A variants are mostly compound heterozygous and the disease phenotypes are highly diverse. This study aims to clarify how different TTC7A variants impact the severity of intestinal epithelial disorders. We individually characterized the molecular and cellular consequences of 11 different TTC7A missense mutations in TTC7A knockout Caco-2 cells. We examined variant-specific RNA expression profiles, TTC7A protein abundance, and endoplasmic reticulum (ER) stress by using RNA sequencing and imaging flow cytometry. For six variants we detected no significant alterations on these assays, suggesting that protein function may not be severely compromised. However, for five variants we observed molecular phenotypes, with overlapping gene expression signatures between specific variants. Remarkably, the TTC7AE71K variant displayed a unique expression profile, along with reduced TTC7A RNA and protein expression, which set it apart from all other variants. The findings from this study offer a better understanding of the role of specific TTC7A variants in disease and provide a framework for the classification of the variants based on the severity of impact. We propose a classification system for TTC7A variants that could help diagnosis, guide future treatment decisions and may aid in developing effective molecular therapies for patients that carry specific TTC7A variants.

P59 Clinic and genetic characteristics of patients with generalized pustular psoriasis: a bidirectional cohort study

Abstract Generalized pustular psoriasis (GPP) is an infrequent disease with significant genetic predisposition, the IL36RN gene has been evidenced of pathogenicity, additionally, furthermore, eight genes including CARD14, AP1S3, MPO, SERPINA1, SERPINA3, BTN3A3, TNIP1 and TGFBR2 have been successively identified of involved in the pathogenesis of GPP.1,2 However, a considerable proportion of quintessential GPP patients either exhibit no mutations in the aforementioned genes, or been detected of only single-allele heterozygous variation, or have found two or more pathogenic genes. The prevailing view thought GPP follows an oligogenic inheritance pattern rather than a monogenic one. Currently, genetic testing for GPP employs methodologies such as whole-exome sequencing, next-generation sequencing (NGS) targeted gene panel exome sequencing and Sanger sequencing for specific genes like IL36RN. However, the aforementioned methods do not address the identification of unknown genes, variations in non-coding regions, structural variants (SVs), or copy number variations (CNVs). Whole-genome sequencing (WGS), by comparing individual genomic sequences to a reference genome, provides detailed information on single nucleotide variations (SNVs), insertions and deletions (InDels), SVs and CNVs within the genome. The application of WGS to the research of GPP will undoubtedly contribute to the investigation of its unknown mechanisms. In our study, WGS was carried out on a total of 79 patients diagnosed with pustular psoriasis (PP) up to the present, comprising single GPP (35), psoriasis vulgaris (PsV)-GPP (15), and palmoplantar pustulosis (PPP) or acrodermatitis continua (ACH)-GPP (4), single PPP (10) or ACH (2), clinic characteristics including groove or map-like tongue, nail involvements were also recorded for phenotypic matching. Four variant types (c.115+6T>C, c.334G>A, c.227C>T and c.169G>A) of IL36RN in 43 patients (43/79) were identified, among which c.115 + 6T>C exhibited the highest frequency. Among them, 19 patients had c.115 + 6T>C homozygous mutations, 15 patients had c.115 + 6T>C single heterozygous mutation and 2 patients had c.115 + 6T>C and c.227C>T complex heterozygous mutations. A total of 21/43 patients had IL36RN biallelic mutations, and 22/43 patients had IL36RN monoallelic mutations. In addition, some predicted pathogenic mutations were detected in TNIP1, SERPINA1, MPO, TGFBR2, AP1S3 and CARD14 genes.

Development of a riboflavin-responsive model of riboflavin transporter deficiency in zebrafish.

Riboflavin transporter deficiency (RTD) is a rare and progressive neurodegenerative disease resulting from the disruption of RFVT2- and RFVT3- mediated riboflavin transport caused by biallelic mutations in SLC52A2 and SLC52A3, respectively. The resulting impaired mitochondrial metabolism leads to sensorimotor neurodegeneration and symptoms including muscle weakness, respiratory difficulty, and sensorineural deafness. Although over 70% of patients with RTD improve following high-dose riboflavin supplementation, remaining patients either stabilise or continue to deteriorate. This may be due to the rapid excretion of central nervous system (CNS) riboflavin by organic anion transporter 3 (OAT-3), highlighting the need for alternative or supplemental RTD treatments. Probenecid is a promising therapeutic candidate for RTD due to its known inhibitory effect on OAT-3. Therefore, this study aimed to generate morpholino-mediated knockdowns of human SLC52A3 ortholog slc52a3 in zebrafish larvae for use in therapeutic screening of riboflavin and probenecid. Knockdown of slc52a3 resulted in an RTD-like phenotype indicative of altered neurodevelopment, hearing loss, and reduced mobility. This RTD-like phenotype overlaps with the phenotype of CRISPR/Cas9-mediated knockout of slc52a3 in zebrafish, is maintained following slc52a3 morpholino + p53 morpholino co-injection, and is rescued following slc52a3 morpholino + human SLC52A3 mRNA co-injection, indicating specificity of the knockdown. Riboflavin treatment alone ameliorates locomotor activity and hearing ability in slc52a3 morphants. Riboflavin and probenecid co-treatment provides an additional small benefit to hearing but not to locomotion. Our findings demonstrate that this model recapitulates both the RTD phenotype and the riboflavin-responsiveness of RTD patients, and possible therapeutic benefit conferred by probenecid warrants further investigation.

Drug Repurposing Screen for the Rare Disease Ataxia-Telangiectasia

Ataxia Telangiectasia (A-T) is a rare, autosomal recessive genetic disorder characterized by a variety of symptoms, including progressive neurodegeneration, telangiectasia, immunodeficiency, and an increased susceptibility to cancer. It is caused by bi-allelic mutations impacting a gene encoding a serine/threonine kinase ATM (Ataxia Telangiectasia Mutated), which plays a crucial role in DNA repair and maintenance of genomic stability. The disorder primarily affects the nervous system, leading to a range of neurological issues, including cerebellar ataxia. The cause of neurodegeneration due to mutations in ATM is still an area of investigation, and currently there is no known treatment to slow down or stop the progression of the neurological problems.In this collaboration of the A-T Children's Project (ATCP) with Charles River Discovery, we successfully developed a high-throughput assay using induced pluripotent stem cells (iPSC) from A-T donors to measure DNA damage response (DDR). By measuring the changes in levels of activated phosphorylated CHK2 (p-CHK2), which is a downstream signaling event of ATM, we were able to identify compounds that restore this response in the DDR pathway in A-T derived patient cells. Over 6,000 compounds from small molecule drug repurposing libraries were subsequently screened in the assay developed, leading to identification of several promising in vitro hits.Using the assay developed and the identified hits opens avenues to investigate potential therapeutics for A-T.

Anaesthetic management of an infant with MEGD(H)EL syndrome undergoing cochlear implant

BackgroundThe syndrome has these features: 3-methylglutaconic aciduria (MEG), deafness(D), encephalopathy (E), Leigh-like syndrome (L). This disorder is caused by biallelic mutations in serine active site-containing protein 1 (SERAC1) gene. When these patients experience hepatopathy (H) in addition to the above manifestations, the syndrome is referred to as MEGD(H)EL. The pathology of this syndrome shares features with diverse types of inborn errors of metabolism.Case presentationWe discussed the anaesthetic management of an infant 2-year-old suffering from MEGD(H)EL syndrome undergoing cochlear implant. We discuss the pathology, genetics and significant aspects of this sporadic disease which is important for anaesthesiologist.ConclusionsThe usage of dexmedetomidine as the main anaesthetic drug might have the benefit of a non-triggering anaesthetic agent in patients with a mitochondrial disease. Mixture of dexmedetomidine and ketamine provide an effective combination for procedural sedation, predominantly in select populations who are at a high risk of perioperative complications due to underlying co-morbid conditions.

Novel BRAT1 Deep Intronic Variant Affects Splicing Regulatory Elements Causing Cerebellar Hypoplasia Syndrome: Genotypic and Phenotypic Expansion.

Biallelic mutations in BRAT1 result in lethal neonatal rigidity and multifocal seizure syndrome and a milder neurodevelopmental disorder of cerebellar atrophy with or without seizures (NEDCAS, MIM 618056). Combining linkage analysis and whole-genome sequencing (WGS), we identified a novel deep intronic BRAT1 variant, NC_000007.14 (NM_152743.4):c.128-1585 T > G, in 3 siblings of a consanguineous Bedouin family exhibiting NEDCAS. In silico analyses followed by molecular studies demonstrated this variant's impact on splice regulatory elements, forming a cryptic exon, resulting in a deleterious frameshift and aberrant transcript. Previously reported pathogenic BRAT1 splice-site mutations were adjacent to exons, affecting canonical consensus splice sites, and identifiable by whole-exome sequencing. The deep intronic BRAT1 disease-causing variant is thus unique and underscores the potential of intronic splice regulatory elements in BRAT1 disease pathogenesis, demonstrating the utility of WGS in identifying noncoding variants in unresolved cases. The affected individuals (deep into their twenties) are among the longest-surviving patients described to date-delineating the NEDCAS phenotype at these ages. Although sharing homozygosity of the same variant, they show varying penetrance of nystagmus and extreme variability in the extent of ataxia and age of onset of developmental delay. Notably, we summarize all documented BRAT1 splice variants reported to date and their phenotypic associations.

An Update of Phenotypic-Genotypic IMNEPD Cases and a Bioinformatics Analysis of the New PTRH2 Gene Variants.

Biallelic mutations in the PTRH2 gene are associated with a rare genetic disease known as infantile-onset multisystem neurologic, endocrine, and pancreatic disease (IMNEPD). In this study, we describe a new case carrying a previously identified mutation, provide an updated analysis of the relative frequencies of the clinical features across all published cases (including the three latest studies), and perform a bioinformatics analysis of the newly identified PTRH2 protein variants from a structural perspective. Clinical examination of the patients was carried out, and genetic testing was performed using a genome sequencing strategy. A bioinformatics analysis was carried out for the newly reported mutations using PYMOL that was utilized to view the structure and analyze the mutations. Additionally, the ThermoMPNN webserver was employed to check the effect of point mutations on the overall stability of the protein. Our findings indicate that motor delay, neuropathy, intellectual disability, distal weakness, hearing impairment, and ataxia are the most common symptoms, while the other clinical features fall into two frequency categories: moderately common ones and the least common ones. The bioinformatics analysis revealed that the Q85 residue is highly conserved, suggesting that mutations at this position could disrupt key signaling pathways or cellular functions. Indeed, the Q85R mutation was shown to significantly impair the stability and functionality of the protein. The clinical presentation of IMNEPD remains highly variable in terms of both severity and progression. Mutations at the Q85 residue have been identified in nearly 50% of reported cases, highlighting this position as a potential mutational hotspot in the PTRH2 protein.

Human glyoxylate metabolism revisited: New insights pointing to multi-organ involvement with implications for siRNA-based therapies in primary hyperoxaluria.

Glyoxylate is a toxic metabolite because of its rapid conversion into oxalate, as catalyzed by the ubiquitous enzyme lactate dehydrogenase. This requires the presence of efficient glyoxylate detoxification systems in multiple subcellular compartments, as glyoxylate is produced in peroxisomes, mitochondria, and the cytosol. Alanine glyoxylate aminotransferase (AGT) and glyoxylate reductase/hydroxypyruvate reductase (GRHPR) are the key enzymes involved in glyoxylate detoxification. Bi-allelic mutations in the genes coding for these enzymes cause primary hyperoxaluria type 1 (PH1) and 2 (PH2), respectively. Glyoxylate is derived from various sources, including 4-hydroxyproline, which is degraded in mitochondria, generating pyruvate and glyoxylate, as catalyzed by the mitochondrial enzyme 4-hydroxy-2-oxoglutarate aldolase (HOGA); however, counterintuitively, a defect in HOGA1 is the molecular basis of primary hyperoxaluria type 3 (PH3). Irrespective of its underlying cause, hyperoxaluria in humans leads to nephrocalcinosis, recurrent urolithiasis, and kidney damage, which may culminate in kidney failure requiring combined liver-kidney transplantation in severely affected patients. In the past few years, therapeutic options, especially for primary hyperoxaluria type 1 (PH1), have greatly been improved thanks to the introduction of two RNAi-based therapies that inhibit either the production of glycolate oxidase (lumasiran) or lactate dehydrogenase (nedosiran). While lumasiran only targets PH1 patients, nedosiran was specifically developed to target all three subtypes of PH. Inspired by the findings reported in the literature that nedosiran effectively reduced urinary oxalate excretion in PH1 patients but not in PH2 or PH3 patients, we have now revisited glyoxylate metabolism in humans and performed a thorough literature study which revealed that glyoxylate/oxalate metabolism is not confined to the liver but instead involves multiple different organs. This new view on glyoxylate/oxalate metabolism in humans may well explain the disappointing results of nedosiran in PH2 and PH3, and provides new clues for the future generation of new therapeutic strategies for PH2 and PH3.

Clinical Phenotypic Characterization of the SLC26A4 Mutation in Pendred Syndrome/Nonsyndromic Enlarged Vestibular Aqueduct.

To summarize the Solute Carrier Family 26 Member 4 (SLC26A4) mutations and clinical phenotypic characteristics of patients with Pendred syndrome/nonsyndromic enlarged vestibular aqueduct (PS/NSEVA). A retrospective cohort study for the Chinese population was performed to analyze the hearing test results of 406 patients with PS/NSEVA who had a SLC26A4 mutation and the relationship between inner ear imaging and audiology. There was a significant difference in the mean hearing threshold in patients with biallelic mutations (M2), monoallelic mutations (M1), and nonallelic mutations (M0) and between patients with isolated vestibular aqueduct enlargement (IEVA) and patients with IEVA combined with Mondini malformation. There was no significant difference between patients with different gene mutation types or different sexes, or between the width of the vestibular aqueduct (VA) and the mean hearing threshold. The degree of hearing loss was linearly correlated with age. We propose that the presence and absence of SLC26A4 mutation, whether combined with Mondini malformation and patient age, are essential factors affecting the degree of hearing loss in the Chinese population. However, the number and type of mutations, degree of VA expansion, and sex of the patients did not affect the clinical audiological phenotype. 3 Laryngoscope, 2024.

Bmpr1aa modulates the severity of the skeletal phenotype in an fkbp10-deficient Bruck syndrome zebrafish model.

Rare monogenic disorders often exhibit significant phenotypic variability among individuals sharing identical genetic mutations. Bruck syndrome (BS), a prime example, is characterized by bone fragility and congenital contractures, although with a pronounced variability among family members. BS arises from recessive biallelic mutations in FKBP10 or PLOD2. FKBP65, the protein encoded by FKBP10, collaborates with the LH2 enzyme (PLOD2) in type I collagen telopeptide lysine hydroxylation, crucial for collagen cross-linking. To identify potential modifier genes and to investigate the mechanistic role of FKBP10 in BS pathogenesis, we established an fkbp10a knockout zebrafish model. Mass-spectrometry analysis in fkbp10a-/- mutants revealed a generally decreased type I collagen lysyl hydroxylation, paralleled by a wide skeletal variability similar to human patients. Ultrastructural examination of the skeleton in severely affected mutants showed enlarged type I collagen fibrils and disturbed elastin layers. Whole-exome sequencing of 7 mildly and 7 severely affected mutant zebrafish siblings, followed by SNP-based linkage analysis, indicated a linked region on chromosome 13, which segregates with phenotypic severity. Transcriptome analysis identified six differentially expressed genes (DEGs) between mildly and severely affected mutants. The convergence of genes within the linked region and DEGs highlighted bmpr1aa as a potential modifier gene, as its reduced expression correlates with increased skeletal severity. In summary, our study provides deeper insights into the role of FKBP10 in BS pathogenesis. Additionally, we identified a pivotal gene that influences phenotypic severity in a zebrafish model of BS. These findings hold promise for novel treatments in the field of bone diseases.

Prognostic significance of mutation type and chromosome fragility in Fanconi anemia.

Fanconi anemia (FA) is a rare genetic disease characterized by high phenotypic and genotypic heterogeneity, and extreme chromosome fragility. To better understand the natural history of FA, identify genetic risk and prognostic factors, and develop novel therapeutic strategies, the Spanish Registry of Patients with FA collects data on clinical features, chromosome fragility, genetic subtypes, and DNA sequencing with informed consent of participating individuals. In this article, we describe the clinical evolution of 227 patients followed up for up to 30 years, for whom our data indicate a cumulative cancer incidence of 86% by age 50. We found that patients with lower chromosome fragility had a milder malformation spectrum and better outcomes in terms of later-onset hematologic impairment, less severe bone marrow failure, and lower cancer risk. We also found that outcomes were better for patients with mutations leading to mutant FANCA protein expression (genetic hypomorphism) than for patients lacking this protein. Likewise, prognosis was consistently better for patients with biallelic mutations in FANCD2 (mainly hypomorphic mutations) than for patients with biallelic mutations in FANCA and FANCG, with the lack of the mutant protein in patients with biallelic mutations in FANCG contributing to their poorer outcomes. Our results regarding the clinical impact of chromosome fragility and genetic hypomorphism suggest that mutant FA proteins retain residual activity. This finding should encourage the development of novel therapeutic strategies aimed at partially or fully enhancing mutant FA function, thereby preventing or delaying bone marrow failure and cancer in patients with FA. Clinical Trial Registration number: NCT06490510.

Identification of biallelic intronic EPM2A mutations in a Lafora disease kindred.

Lafora disease (LD) is a severe autosomal recessive disease, which usually presents as seizure and myoclonus, followed by behavioral changes, dysarthria, intellectual decline, and finally progressed to dementia and a vegetative state. The main cause of LD is the loss-of-function mutations in EPM2A and NHLRC1 that encode laforin and malin, respectively. Targeted genetic testing is the gold standard to confirm the diagnosis of LD. To describe the pathogenic role of biallelic EPM2A intronic mutations carried by patients in a family diagnosed as LD. Here, we present clinical findings in a patient presenting with epileptic seizures and Lafora bodies in muscle biopsy. Long-read DNA and RNA sequencing were performed to identify the causative mutation. Western blot and qPCR confirmed the pathogenic role of biallelic EPM2A intronic mutations. Genetic testing identified two intronic mutations in EPM2A which caused aberrant mRNA splicing. c.301+1 G > A in EPM2A caused aberrant splicing at donor site and resulted in intron retention in transcript NM_005670.4, while c.476+14860 C > A caused aberrant splicing in transcript NM_001368129.2 and NM_001368132.1. Our findings expand the spectrum of variants in LD disease, additionally providing evidence linking non-coding regulatory regions mutations to LD disease.

Efficient multi-allelic genome editing via CRISPR-Cas9 ribonucleoprotein-based delivery to Brassica napus mesophyll protoplasts.

Canola (Brassica napus L.) is a valuable oilseed crop worldwide. However, trait improvement by breeding has been limited by its low genetic diversity and polyploid genetics. Whilst offering many potential benefits, the application of transgenic technology is challenged by the stringent and expensive regulatory processes associated with the commercialisation of genetically modified organisms, coupled with a prevailing low public acceptance of such modifications. DNA-free genome editing using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 ribonucleoproteins (RNPs) offers a promising way to achieve trait improvements without the limitations of transgenic methods. Here, we present a method for DNA-free genome editing via the direct delivery of RNPs to canola mesophyll protoplasts. This method allows high-throughput in vivo testing of the efficacy of gRNA design as part of the transformation process to facilitate the selection of optimal designs prior to the generation of edited events. Of the 525 shoots regenerated via tissue culture from RNP-transfected protoplasts and screened for the presence of mutations in the targeted gene, 62% had one or more mutated target alleles, and 50% had biallelic mutations at both targeted loci. This high editing efficiency compares favourably with similar CRISPR-Cas9 approaches used in other crop plants.

The Microbiome Modifies Manifestations of Hemophagocytic Lymphohistiocytosis in Perforin-Deficient Mice.

Primary hemophagocytic lymphohistiocytosis (HLH) is a life-threatening hyperinflammatory syndrome caused by inborn errors of cytotoxicity. Patients with biallelic PRF1 null mutations (encoding perforin) usually develop excessive immune cell activation, hypercytokinemia, and life-threateningimmunopathology in the first 6 months of life, often without an apparent infectious trigger. In contrast, perforin-deficient (PKO) mice only develop HLH after systemic infection with lymphocytic choriomeningitis virus (LCMV). We hypothesized that restricted microbe-immune cell interactions due to specific pathogen-free (SPF) housing might explain the need for this specific viral trigger in PKO mice. To investigate the influence of a "wild" microbiome in PKO mice, we fostered PKO newborns with Wildling microbiota ('PKO-Wildlings') and monitored them for signs of HLH. PKO-Wildlings survived long-term without spontaneous disease. Also, systemic infection with vaccinia virus did not reach the threshold of immune activation required to trigger HLH in PKO-Wildlings. Interestingly, after infection with LCMV, PKO-Wildlings developed an altered HLH pattern. This included lower IFN-γ serum levels along with improved IFN-γ-driven anemia, but more elevated levels of IL-17 and increased liver inflammation compared with PKO-SPF mice. Thus, wild microbiota alone is not sufficient to trigger HLH in PKO mice, but host-microbe interactions shape inflammatory cytokine patterns, thereby influencing manifestations of HLH immunopathology.