Fragile X Syndrome (FXS) is an X-linked neurodevelopmental disorder characterised by intellectual disability, developmental delays, anxiety, and social and behavioural challenges. Currently, no effective treatments exist to address the root cause of FXS. Mouse models are the most widely used for studying molecular pathogenesis and conducting preclinical treatment testing. However, therapeutic interventions that show promise in rodent models have yet to succeed in clinical trials. After evaluating the current models, we have developed an ovine model to address this clinical translation gap. We expect this model to more accurately reflect the human condition in brain size, structure, and neurodevelopmental trajectory. We aim to establish this model as a valuable preclinical platform for testing therapies for FXS. To generate the sheep model, we used CRISPR-Cas9 dual-guide editing to knock out the Fragile X Messenger Ribonucleoprotein 1 (FMR1) gene in ovine embryos. Two founder animals were created, one ram (male) and one ewe (female), both of which carried FMR1 gene knockouts. The ewe carries inactivating mutations on both alleles, with the edits in both animals resulting in no detectable Fragile X Messenger Ribonucleoprotein (FMRP) as expected. Both founders have undergone molecular characterisation and basic health checks, with the female founder showing increased joint flexibility, a characteristic of FXS. The ram has been used for breeding, with the successful transmission of the edited allele to his offspring. Importantly, specific lamb cohorts for postnatal treatment testing can be produced efficiently utilising accelerated breeding methods and preimplantation selection.

To characterize the prenatal sonographic features across different trimesters and genomic spectrum of NONO-related X-linked intellectual developmental disorder. We analyzed two fetuses presenting with corpus callosum agenesis and rare cardiac anomalies using genome sequencing and exome sequencing. A systematic literature review was conducted to provide a comprehensive analysis of genotype-phenotype correlations. Two novel cases were reported in this study: one with a de novo 61.7 kb deletion affecting both NONO and ITGB1BP2, and another with a de novo c.1093 C > T(p.Arg365Ter), a recurrent variant reported in the literature. A total of 23 cases with NONO defects and prenatal phenotypes were identified, encompassing 19 distinct variant types. Large deletions and splicing variants accounted for approximately 32% (6/19). Among all cases, 78% (18/23) exhibited typical or concurrent brain abnormalities, such as corpus callosum agenesis, cardiac defects including left ventricular noncompaction (LVNC), or short long bones in the second trimester. Notably, 4% (1/23) of cases were reported with isolated intrauterine growth restriction (IUGR), primarily identified in the third trimester. Genome sequencing facilitates thorough identification of the genetic causes of NONO-related syndrome. The recurrent variant p.Arg365Ter has been reported to cause variable cardiac abnormalities in different patients, suggesting that other genetic or non-genetic factors may contribute to the cardiac manifestations in NONO-related syndromes.

Thyroid hormones (THs), including T4 (3,5,3',5'-tetraiodo-L-thyronine) and T3 (3,5,3'-triiodo-L-thyronine), play critical roles in regulating tissue development and basal metabolism. Monocarboxylate transporter 8 (MCT8) is a key player in TH transport, known for its high specificity and affinity for THs and its direct association with Allan-Herndon-Dudley syndrome (AHDS) caused by pathogenic mutations. In this study, we present the cryo-EM structures of human MCT8 bound to the substrate T3 or the inhibitor silychristin, both in an outward-open conformation at resolutions of 3.0-3.2 Å. MCT8 forms a homodimer with a lipid molecule positioned at the dimerization interface. The carboxyl group of T3 is recognized by Arg371, while its three iodine atoms interact with distinct hydrophobic cavities. Silychristin is also recognized by Arg371, competing with T3 for binding. Complemented by structure-guided biochemical analyses, our research elucidates the mechanisms of substrate recognition and transport, as well as the mode of action of the inhibitor silychristin. These findings may offer insights for developing targeted therapies for TH-related disorders.

MECP2 Duplication Syndrome, also known as X-linked intellectual developmental disorder Lubs type (MRXSL; MIM: 300260), is a neurodevelopmental disorder caused by copy number gains spanning MECP2. Despite varying genomic rearrangement structures, including duplications and triplications, and a wide range of duplication sizes, no clear correlation exists between DNA rearrangement and clinical features. We had previously demonstrated that up to 38% of MRXSL families are characterized by complex genomic rearrangements (CGRs) of intermediate complexity (2 ≤ copy number variant breakpoints < 5), yet the impact of these genomic structures on regulation of gene expression and phenotypic manifestations have not been investigated. To study the role of the genomic rearrangement structures on an individual's clinical phenotypic variability, we employed a comprehensive genomics, transcriptomics, and deep phenotyping analysis approach on 137 individuals affected by MRXSL. Genomic structural information was correlated with transcriptomic and quantitative phenotypic analysis using Human Phenotype Ontology (HPO) semantic similarity scores. Duplication sizes in the cohort ranging from 64.6 kb to 16.5 Mb were classified into four categories comprising of tandem duplications (48%), terminal duplications (22%), inverted triplications (20%), and other CGRs (10%). Most of the terminal duplication structures consist of translocations (65%) followed by recombinant chromosomes (23%). Notably, 65% of de novo events occurred in the Terminal duplication group in contrast with 17% observed in Tandem duplications. RNA-seq data from lymphoblastoid cell lines indicated that the MECP2 transcript quantity in MECP2 triplications is statistically different from all duplications, but not between other classes of genomic structures. We also observed a significant (p < 0.05) correlation (Pearson R = 0.6, Spearman p = 0.63) between the log-transformed MECP2 RNA levels and MECP2 protein levels, demonstrating that genomic aberrations spanning MECP2 lead to altered MECP2 RNA and MECP2 protein levels. Genotype-phenotype analyses indicated a gradual worsening of phenotypic features, including overall survival, developmental levels, microcephaly, epilepsy, and genitourinary/eye abnormalities in the following order: Tandem duplications, Other complex duplications, Terminal duplications/Translocations, and Triplications encompassing MECP2. In aggregate, this combined analysis uncovers an interplay between MECP2 dosage, genomic rearrangement structure and phenotypic traits. Whereas the level of MECP2 is a key determinant of the phenotype, the DNA rearrangement structure can contribute to clinical severity and disease expression variability. Employing this type of analytical approach will advance our understanding of the impact of genomic rearrangements on genomic disorders and may help guide more targeted therapeutic approaches. FMR1 disorders include fragile X syndrome (FXS), fragile X-associated tremor/ataxia syndrome (FXTAS), and fragile X-associated primary ovarian insufficiency (FXPOI). Fragile X syndrome occurs in individuals with an FMR1 full mutation or other loss-of-function variant and is nearly always characterized in affected males by developmental delay and intellectual disability along with a variety of behavioral issues. Autism spectrum disorder is present in 50%-70% of individuals with FXS. Affected males may have characteristic craniofacial features (which become more obvious with age) and medical problems including hypotonia, gastroesophageal reflux, strabismus, seizures, sleep disorders, joint laxity, pes planus, scoliosis, and recurrent otitis media. Adults may have mitral valve prolapse or aortic root dilatation. The physical and behavioral features seen in males with FXS have been reported in females heterozygous for the FMR1 full mutation, but with lower frequency and milder involvement. FXTAS occurs in individuals who have an FMR1 premutation and is characterized by late-onset, progressive cerebellar ataxia and intention tremor followed by cognitive impairment. Psychiatric disorders are common. Age of onset is typically between 60 and 65 years and is more common among males who are hemizygous for the premutation (40%) than among females who are heterozygous for the premutation (16%-20%). FXPOI, defined as hypergonadotropic hypogonadism before age 40 years, has been observed in 20% of women who carry a premutation allele compared to 1% in the general population. The diagnosis of an FMR1 disorder is established through the use of specialized molecular genetic testing to detect CGG trinucleotide repeat expansion in the 5' UTR of FMR1 with abnormal gene methylation for most alleles with >200 repeats. Typically, a definite diagnosis of FXS requires the presence of a full-mutation repeat size (>200 CGG repeats) while the diagnosis of FXTAS or FXPOI is associated with a premutation-sized repeat (55-200 CGG repeats). It should be noted that typical multigene panels and comprehensive genomic testing (exome or genome sequencing) are useful only when no CGG repeat expansion is detected but FXS is still suspected. Treatment of manifestations: Fragile X syndrome: supportive and symptom-based therapy for children and adults typically consisting of a dual approach of psychopharmacologic treatment of symptoms as needed in conjunction with therapeutic services, such as behavioral intervention, speech and language therapy, occupational therapy, and individualized educational support; routine treatment of medical problems. FXTAS: symptomatic and supportive and should be tailored to the individual. FXPOI: Gynecologic or reproductive endocrinologic evaluation can provide appropriate treatment and counseling for reproductive considerations and hormone replacement. Agents/circumstances to avoid: FXTAS: typical and atypical antipsychotics with significant anti-dopaminergic effects and metoclopramide, which can exacerbate parkinsonism; anticholinergic agents, which can exacerbate cognitive complaints; excessive alcohol, which can enhance cerebellar dysfunction and postural instability; agents with known cerebellar toxicity or side effects. FXPOI: tobacco use as this decreases ovarian reserve and the age of onset of FXPOI. FMR1 disorders are inherited in an X-linked manner. All mothers of individuals with an FMR1 full mutation (expansion >200 CGG trinucleotide repeats and abnormal methylation) are heterozygous for an FMR1 pathogenic variant. Mothers and their female relatives who are heterozygous for a premutation are at increased risk for FXTAS, FXPOI, and fragile X-associated neuropsychiatric disorders (FXAND); those with a full mutation may have findings of fragile X syndrome. All are at increased risk of having offspring with fragile X syndrome, FXTAS, FXPOI, or FXAND. Males with premutations are at increased risk for FXTAS. Males with FXTAS will transmit their FMR1 premutation expansion to all of their daughters, who will be heterozygous for a premutation and at increased risk for FXTAS, FXPOI, and FXAND. Males with FXTAS do not transmit their FMR1 premutation to sons. Prenatal testing for a pregnancy at increased risk and preimplantation genetic testing are possible once an expanded (or altered) FMR1 allele has been identified in a family member.

Global developmental delay or intellectual disability usually accompanies various genetic disorders as a part of the syndrome, which may include seizures, autism spectrum disorder and multiple congenital abnormalities. Next-generation sequencing (NGS) techniques have improved the identification of pathogenic variants and genes related to developmental delay. This study aimed to evaluate the yield of whole exome sequencing (WES) and neurodevelopmental disorder gene panel sequencing in a pediatric cohort from Ukraine. Additionally, the study computationally predicted the effect of variants of uncertain significance (VUS) based on recently published genetic data from the country's healthy population. The study retrospectively analyzed WES or gene panel sequencing findings of 417 children with global developmental delay, intellectual disability, and/or other symptoms. Variants of uncertain significance were annotated using CADD-Phred and SIFT prediction scores, and their frequency in the healthy population of Ukraine was estimated. A definitive molecular diagnosis was established in 66 (15.8%) of the individuals. WES diagnosed 22 out of 37 cases (59.4%), while the neurodevelopmental gene panel identified 44 definitive diagnoses among the 380 tested patients (12.1%). Non-diagnostic findings (VUS and carrier) were reported in 350 (83.2%) individuals. The most frequently diagnosed conditions were developmental and epileptic encephalopathies associated with severe epilepsy and GDD/ID (associated genes ARX, CDKL5, STXBP1, KCNQ2, SCN2A, KCNT1, KCNA2). Additionally, we annotated 221 VUS classified as potentially damaging, AD or X-linked, potentially increasing the diagnostic yield by 30%, but 18 of these variants were present in the healthy population of Ukraine. This is the first comprehensive study on genetic causes of GDD/ID conducted in Ukraine. This study provides the first comprehensive investigation of the genetic causes of GDD/ID in Ukraine. It presents a substantial dataset of diagnosed genetic conditions associated with GDD/ID. The results support the utilization of NGS gene panels and WES as first-line diagnostic tools for GDD/ID cases, particularly in resource-limited settings. A comprehensive approach to resolving VUS, including computational effect prediction, population frequency analysis, and phenotype assessment, can aid in further reclassification of deleterious VUS and guide further testing in families.