Pathogenic cryptic variants detectable through exome data reanalysis significantly increase the diagnostic yield in Joubert syndrome.

Joubert syndrome (JS) is a genetically heterogeneous neurodevelopmental ciliopathy. Despite exome sequencing (ES), several patients remain undiagnosed. This study aims to increase the diagnostic yield by uncovering cryptic variants through targeted ES reanalysis. We first focused on 26 patients in whom ES only disclosed heterozygous pathogenic coding variants in a JS gene. We reanalyzed raw ES data searching for copy number variants (CNVs) and intronic variants affecting splicing. We validated CNVs through real-time PCR or chromosomal microarray, and splicing variants through RT-PCR or minigenes. Cryptic variants were then searched in additional 44 ES-negative JS individuals. We identified cryptic "second hits" in 14 of 26 children (54%) and biallelic cryptic variants in 3 of 44 (7%), reaching a definite diagnosis in 17 of 70 (overall diagnostic gain 24%). We show that CNVs and intronic splicing variants are a common mutational mechanism in JS; more importantly, we demonstrate that a significant proportion of such variants can be disclosed simply through a focused reanalysis of available ES data, with a significantly increase of the diagnostic yield especially among patients previously found to carry heterozygous coding variants in the KIAA0586, CC2D2A and CPLANE1 genes.

Evaluations of aggressive chasing interactions by 7-month-old infants.

Recent theories of socio-moral development assume that humans evolved a capacity to evaluate others' social actions in different kinds of interactions. Prior infant studies found both reaching and visual preferences for the prosocial over the antisocial agents. However, whether the attribution of either positive or negative valence to agents' actions involved in an aggressive chasing interaction can be inferred by both reaching behaviors and visual attention deployment (i.e., disengagement of visual attention) is still an open question. Here we presented 7-month-old infants (N = 92) with events displaying an aggressive chasing interaction. By using preferential reaching and an attentional task (i.e., overlap paradigm), we assessed whether and how infants evaluate aggressive chasing interactions. The results demonstrated that young infants prefer to reach the victim over the aggressor, but neither agent affects visual attention. Moreover, such reaching preferences emerged only when dynamic cues and emotional face-like features were congruent with agents' social roles. Overall, these findings suggested that infants' evaluations of aggressive interactions are based on infants' sensitivity to some kinematic cues that characterized agents' actions and, especially, to the congruency between such motions and the face-like emotional expressions of the agents.

I like the way you move: how animate motion affects visual attention in early human infancy.

The ability to detect animates (as compared with inanimates) rapidly is advantageous for human survival. Due to its relevance, not only the adult human brain has evolved specific neural mechanisms to discriminate animates, but it has been proposed that selection finely tuned the human visual attention system to prioritize visual cues that signal the presence of living things. Among them, animate motion-i.e., the motion of animate entities -, is one of the most powerful cues that triggers humans' attention. From a developmental point of view, whether such specialization is inborn or acquired through experience is a fascinating research topic. This mini-review aims to summarize and discuss recent behavioral and electrophysiological research that suggests that animate motion has an attentional advantage in the first year of life starting from birth. Specifically, the rationale underlying this paper concerns how attention deployment is affected by animate motion conveyed both by the movement of a single dot and, also, when the single dot is embedded in a complex array, named biological motion. Overall, it will highlight the importance of both inborn predispositions to pay attention preferentially to animate motion, mainly supported by subcortical structures, and the exposure to certain experiences, shortly after birth, to drive the cortical attentional visual system to become the way it is in adults.

Structural Variants at the LMNB1 Locus: Deciphering Pathomechanisms in Autosomal Dominant Adult-Onset Demyelinating Leukodystrophy.

OBJECTIVE: We aimed to elucidate the pathogenic mechanisms underlying autosomal dominant adult-onset demyelinating leukodystrophy (ADLD), and to understand the genotype/phenotype correlation of structural variants (SVs) in the LMNB1 locus. BACKGROUND: Since the discovery of 3D genome architectures and topologically associating domains (TADs), new pathomechanisms have been postulated for SVs, regardless of gene dosage changes. ADLD is a rare genetic disease associated with duplications (classical ADLD) or noncoding deletions (atypical ADLD) in the LMNB1 locus. METHODS: High-throughput chromosome conformation capture, RNA sequencing, histopathological analyses of postmortem brain tissues, and clinical and neuroradiological investigations were performed. RESULTS: We collected data from >20 families worldwide carrying SVs in the LMNB1 locus and reported strong clinical variability, even among patients carrying duplications of the entire LMNB1 gene, ranging from classical and atypical ADLD to asymptomatic carriers. We showed that patients with classic ADLD always carried intra-TAD duplications, resulting in a simple gene dose gain. Atypical ADLD was caused by LMNB1 forebrain-specific misexpression due to inter-TAD deletions or duplications. The inter-TAD duplication, which extends centromerically and crosses the 2 TAD boundaries, did not cause ADLD. Our results provide evidence that astrocytes are key players in ADLD pathology. INTERPRETATION: Our study sheds light on the 3D genome and TAD structural changes associated with SVs in the LMNB1 locus, and shows that a duplication encompassing LMNB1 is not sufficient per se to diagnose ADLD, thereby strongly affecting genetic counseling. Our study supports breaking TADs as an emerging pathogenic mechanism that should be considered when studying brain diseases. ANN NEUROL 2024;96:855-870.

The impact of clinical genome sequencing in a global population with suspected rare genetic disease.

There is mounting evidence of the value of clinical genome sequencing (cGS) in individuals with suspected rare genetic disease (RGD), but cGS performance and impact on clinical care in a diverse population drawn from both high-income countries (HICs) and low- and middle-income countries (LMICs) has not been investigated. The iHope program, a philanthropic cGS initiative, established a network of 24 clinical sites in eight countries through which it provided cGS to individuals with signs or symptoms of an RGD and constrained access to molecular testing. A total of 1,004 individuals (median age, 6.5 years; 53.5% male) with diverse ancestral backgrounds (51.8% non-majority European) were assessed from June 2016 to September 2021. The diagnostic yield of cGS was 41.4% (416/1,004), with individuals from LMIC sites 1.7 times more likely to receive a positive test result compared to HIC sites (LMIC 56.5% [195/345] vs. HIC 33.5% [221/659], OR 2.6, 95% CI 1.9-3.4, p < 0.0001). A change in diagnostic evaluation occurred in 76.9% (514/668) of individuals. Change of management, inclusive of specialty referrals, imaging and testing, therapeutic interventions, and palliative care, was reported in 41.4% (285/694) of individuals, which increased to 69.2% (480/694) when genetic counseling and avoidance of additional testing were also included. Individuals from LMIC sites were as likely as their HIC counterparts to experience a change in diagnostic evaluation (OR 6.1, 95% CI 1.1-∞, p = 0.05) and change of management (OR 0.9, 95% CI 0.5-1.3, p = 0.49). Increased access to genomic testing may support diagnostic equity and the reduction of global health care disparities.

DNA methylation analysis in patients with neurodevelopmental disorders improves variant interpretation and reveals complexity.

Analysis of genomic DNA methylation by generating epigenetic signature profiles (episignatures) is increasingly being implemented in genetic diagnosis. Here we report our experience using episignature analysis to resolve both uncomplicated and complex cases of neurodevelopmental disorders (NDDs). We analyzed 97 NDDs divided into (1) a validation cohort of 59 patients with likely pathogenic/pathogenic variants characterized by a known episignature and (2) a test cohort of 38 patients harboring variants of unknown significance or unidentified variants. The expected episignature was obtained in most cases with likely pathogenic/pathogenic variants (53/59 [90%]), a revealing exception being the overlapping profile of two SMARCB1 pathogenic variants with ARID1A/B:c.6200, confirmed by the overlapping clinical features. In the test cohort, five cases showed the expected episignature, including (1) novel pathogenic variants in ARID1B and BRWD3; (2) a deletion in ATRX causing MRXFH1 X-linked mental retardation; and (3) confirmed the clinical diagnosis of Cornelia de Lange (CdL) syndrome in mutation-negative CdL patients. Episignatures analysis of the in BAF complex components revealed novel functional protein interactions and common episignatures affecting homologous residues in highly conserved paralogous proteins (SMARCA2 M856V and SMARCA4 M866V). Finally, we also found sex-dependent episignatures in X-linked disorders. Implementation of episignature profiling is still in its early days, but with increasing utilization comes increasing awareness of the capacity of this methodology to help resolve the complex challenges of genetic diagnoses.

Interface Gain-of-Function Mutations in TLR7 Cause Systemic and Neuro-inflammatory Disease.

TLR7 recognizes pathogen-derived single-stranded RNA (ssRNA), a function integral to the innate immune response to viral infection. Notably, TLR7 can also recognize self-derived ssRNA, with gain-of-function mutations in human TLR7 recently identified to cause both early-onset systemic lupus erythematosus (SLE) and neuromyelitis optica. Here, we describe two novel mutations in TLR7, F507S and L528I. While the L528I substitution arose de novo, the F507S mutation was present in three individuals from the same family, including a severely affected male, notably given that the TLR7 gene is situated on the X chromosome and that all other cases so far described have been female. The observation of mutations at residues 507 and 528 of TLR7 indicates the importance of the TLR7 dimerization interface in maintaining immune homeostasis, where we predict that altered homo-dimerization enhances TLR7 signaling. Finally, while mutations in TLR7 can result in SLE-like disease, our data suggest a broader phenotypic spectrum associated with TLR7 gain-of-function, including significant neurological involvement.

Expanding the phenotype of Brunner syndrome from childhood to adulthood: Description of the second pediatric patient and his mother.

Brunner syndrome is a recessive X-linked disorder caused by pathogenic variants in the monoamine oxidase A gene (MAOA). It is characterized by distinctive aggressive behavior, mild intellectual disability, sleep disturbances, and typical biochemical alterations deriving from the impaired monoamine metabolism. We herein describe a 5-year-old boy with developmental delay, autistic features, and myoclonic epilepsy, and his mother, who had mild intellectual disability and recurrent episodes of palpitations, headache, abdominal pain, and abdominal bloating. Whole exome sequencing allowed detection of the maternally-inherited variant c.410A>G, (p.Glu137Gly) in the MAOA gene. The subsequent biochemical studies confirmed the MAOA deficiency both in the child and his mother. Given the serotonergic symptoms associated with high serotonin levels found in the mother, treatment with a serotonin reuptake inhibitor and dietary modifications were carried out, resulting in regression of the biochemical abnormalities and partial reduction of symptoms. Our report expands the phenotypic spectrum of Brunner disease, bringing new perspectives on the behavioral and neurodevelopmental phenotype from childhood to adulthood.

Pathogenic variants in SMARCA1 cause an X-linked neurodevelopmental disorder modulated by NURF complex composition.

Pathogenic variants in ATP-dependent chromatin remodeling proteins are a recurrent cause of neurodevelopmental disorders (NDDs). The NURF complex consists of BPTF and either the SNF2H (SMARCA5) or SNF2L (SMARCA1) ISWI-chromatin remodeling enzyme. Pathogenic variants in BPTF and SMARCA5 were previously implicated in NDDs. Here, we describe 40 individuals from 30 families with de novo or maternally inherited pathogenic variants in SMARCA1. This novel NDD was associated with mild to severe ID/DD, delayed or regressive speech development, and some recurrent facial dysmorphisms. Individuals carrying SMARCA1 loss-of-function variants exhibited a mild genome-wide DNA methylation profile and a high penetrance of macrocephaly. Genetic dissection of the NURF complex using Smarca1, Smarca5, and Bptfsingle and double mouse knockouts revealed the importance of NURF composition and dosage for proper forebrain development. Finally, we propose that genetic alterations affecting different NURF components result in a NDD with a broad clinical spectrum.

Lamin B1 as a key modulator of the developing and aging brain.

Lamin B1 is an essential protein of the nuclear lamina that plays a crucial role in nuclear function and organization. It has been demonstrated that lamin B1 is essential for organogenesis and particularly brain development. The important role of lamin B1 in physiological brain development and aging has only recently been at the epicenter of attention and is yet to be fully elucidated. Regarding the development of brain, glial cells that have long been considered as supporting cells to neurons have overturned this representation and current findings have displayed their active roles in neurogenesis and cerebral development. Although lamin B1 has increased levels during the differentiation of the brain cells, during aging these levels drop leading to senescent phenotypes and inciting neurodegenerative disorders such as Alzheimer's and Parkinson's disease. On the other hand, overexpression of lamin B1 leads to the adult-onset neurodegenerative disease known as Autosomal Dominant Leukodystrophy. This review aims at highlighting the importance of balancing lamin B1 levels in glial cells and neurons from brain development to aging.

Understanding the Ultra-Rare Disease Autosomal Dominant Leukodystrophy: an Updated Review on Morpho-Functional Alterations Found in Experimental Models.

Autosomal dominant leukodystrophy (ADLD) is an ultra-rare, slowly progressive, and fatal neurodegenerative disorder associated with the loss of white matter in the central nervous system (CNS). Several years after its first clinical description, ADLD was found to be caused by coding and non-coding variants in the LMNB1 gene that cause its overexpression in at least the brain of patients. LMNB1 encodes for Lamin B1, a protein of the nuclear lamina. Lamin B1 regulates many cellular processes such as DNA replication, chromatin organization, and senescence. However, its functions have not been fully characterized yet. Nevertheless, Lamin B1 together with the other lamins that constitute the nuclear lamina has firstly the key role of maintaining the nuclear structure. Being the nucleus a dynamic system subject to both biochemical and mechanical regulation, it is conceivable that changes to its structural homeostasis might translate into functional alterations. Under this light, this review aims at describing the pieces of evidence that to date have been obtained regarding the effects of LMNB1 overexpression on cellular morphology and functionality. Moreover, we suggest that further investigation on ADLD morpho-functional consequences is essential to better understand this complex disease and, possibly, other neurological disorders affecting CNS myelination.

RNA therapeutics for neurological diseases.

INTRODUCTION: Ribonucleic acid (RNA) therapeutics are a new class of drugs whose importance is highlighted by the growing number of molecules in the clinic. SOURCES OF DATA: We focus on RNA therapeutics for neurogenetic disorders, which are broadly defined as diseases with a genetic background and with at least one clinical sign affecting the nervous system. A systematic search identified 14 RNA drugs approved by FDA and many others in development. AREAS OF AGREEMENT: The field of RNA therapeutics is changing the therapeutic scenario across many disorders. AREAS OF CONTROVERSY: Despite its recent successes, RNA therapeutics encountered several hurdles and some clinical failures. Delivery to the brain represents the biggest challenge. GROWING POINTS: The many advantages of RNA drugs make the development of these technologies a worthwhile investment. AREAS TIMELY FOR DEVELOPING RESEARCH: Clinical failures stress the importance of implementing clinical trial design and optimizing RNA molecules to hold the promise of revolutionizing the treatment of human diseases.

De novo RANBP2 variant in a fetal demise case with cerebral intraparenchymal hemorrhage.

Fetal intracranial hemorrhage (ICH) may result from a wide array of causes, either associated with maternal or fetal risk factors. In the last decade, monogenic causes of susceptibility to fetal ICH have been described, in particular in association with COL4A1 and COL4A2 genes. A peculiar form of ICH is acute necrotizing encephalitis (ANE), which is characterized by a rapid-onset severe encephalopathy following an abnormal inflammatory response to an otherwise banal infection. It usually affects healthy children and it is thought to be multifactorial, with a genetic predisposition. RANBP2 gene has been extensively associated with ANE susceptibility. We hereby present a unique case of a 42-year-old secundigravida with intrauterine fetal demise at 35 weeks of gestation. Trio-based whole-exome sequencing performed on both parents and fetal DNA showed a de novo likely pathogenic variant in the RANBP2 gene on 2q13. At the fetal autopsy, subtentorial hematoma and cerebral intraparenchymal hemorrhage were present. We speculate that this might be a new phenotypic presentation of RANBP2-associated disease. However, more similar fetal cases need to be reported in order to reinforce this hypothesis.

Enhancer hijacking at the ARHGAP36 locus is associated with connective tissue to bone transformation.

Heterotopic ossification is a disorder caused by abnormal mineralization of soft tissues in which signaling pathways such as BMP, TGFß and WNT are known key players in driving ectopic bone formation. Identifying novel genes and pathways related to the mineralization process are important steps for future gene therapy in bone disorders. In this study, we detect an inter-chromosomal insertional duplication in a female proband disrupting a topologically associating domain and causing an ultra-rare progressive form of heterotopic ossification. This structural variant lead to enhancer hijacking and misexpression of ARHGAP36 in fibroblasts, validated here by orthogonal in vitro studies. In addition, ARHGAP36 overexpression inhibits TGFß, and activates hedgehog signaling and genes/proteins related to extracellular matrix production. Our work on the genetic cause of this heterotopic ossification case has revealed that ARHGAP36 plays a role in bone formation and metabolism, outlining first details of this gene contributing to bone-formation and -disease.

The crucial role of titin in fetal development: recurrent miscarriages and bone, heart and muscle anomalies characterise the severe end of titinopathies spectrum.

BACKGROUND: Titin truncating variants (TTNtvs) have been associated with several forms of myopathies and/or cardiomyopathies. In homozygosity or in compound heterozygosity, they cause a wide spectrum of recessive phenotypes with a congenital or childhood onset. Most recessive phenotypes showing a congenital or childhood onset have been described in subjects carrying biallelic TTNtv in specific exons. Often karyotype or chromosomal microarray analyses are the only tests performed when prenatal anomalies are identified. Thereby, many cases caused by TTN defects might be missed in the diagnostic evaluations. In this study, we aimed to dissect the most severe end of the titinopathies spectrum. METHODS: We performed a retrospective study analysing an international cohort of 93 published and 10 unpublished cases carrying biallelic TTNtv. RESULTS: We identified recurrent clinical features showing a significant correlation with the genotype, including fetal akinesia (up to 62%), arthrogryposis (up to 85%), facial dysmorphisms (up to 73%), joint (up to 17%), bone (up to 22%) and heart anomalies (up to 27%) resembling complex, syndromic phenotypes. CONCLUSION: We suggest TTN to be carefully evaluated in any diagnostic process involving patients with these prenatal signs. This step will be essential to improve diagnostic performance, expand our knowledge and optimise prenatal genetic counselling.

Marked intrafamilial variability of clinical and neuroimaging manifestations in NFIB-related developmental disorder.

NFIB belongs to the nuclear factor I (NFI) family of transcription factors that, by activating or repressing gene expression during embryogenesis, has a relevant role in the development of several organs including the brain. Heterozygous pathogenic variants of NFIB have recently been associated with developmental delay and mild-to-moderate intellectual disability, macrocephaly, nonspecific facial dysmorphisms, and corpus callosum dysgenesis. We identified a heterozygous missense variant in the NFIB gene in a 15-year-old boy with neurodevelopmental disorder and brain malformations, who inherited the variant from his substantially healthy mother presenting only minor physical and neuroanatomical defects.

A genome-wide association study with tissue transcriptomics identifies genetic drivers for classic bladder exstrophy.

Classic bladder exstrophy represents the most severe end of all human congenital anomalies of the kidney and urinary tract and is associated with bladder cancer susceptibility. Previous genetic studies identified one locus to be involved in classic bladder exstrophy, but were limited to a restrict number of cohort. Here we show the largest classic bladder exstrophy genome-wide association analysis to date where we identify eight genome-wide significant loci, seven of which are novel. In these regions reside ten coding and four non-coding genes. Among the coding genes is EFNA1, strongly expressed in mouse embryonic genital tubercle, urethra, and primitive bladder. Re-sequence of EFNA1 in the investigated classic bladder exstrophy cohort of our study displays an enrichment of rare protein altering variants. We show that all coding genes are expressed and/or significantly regulated in both mouse and human embryonic developmental bladder stages. Furthermore, nine of the coding genes residing in the regions of genome-wide significance are differentially expressed in bladder cancers. Our data suggest genetic drivers for classic bladder exstrophy, as well as a possible role for these drivers to relevant bladder cancer susceptibility.

Rare coding variation provides insight into the genetic architecture and phenotypic context of autism.

Some individuals with autism spectrum disorder (ASD) carry functional mutations rarely observed in the general population. We explored the genes disrupted by these variants from joint analysis of protein-truncating variants (PTVs), missense variants and copy number variants (CNVs) in a cohort of 63,237 individuals. We discovered 72 genes associated with ASD at false discovery rate (FDR) ≤ 0.001 (185 at FDR ≤ 0.05). De novo PTVs, damaging missense variants and CNVs represented 57.5%, 21.1% and 8.44% of association evidence, while CNVs conferred greatest relative risk. Meta-analysis with cohorts ascertained for developmental delay (DD) (n = 91,605) yielded 373 genes associated with ASD/DD at FDR ≤ 0.001 (664 at FDR ≤ 0.05), some of which differed in relative frequency of mutation between ASD and DD cohorts. The DD-associated genes were enriched in transcriptomes of progenitor and immature neuronal cells, whereas genes showing stronger evidence in ASD were more enriched in maturing neurons and overlapped with schizophrenia-associated genes, emphasizing that these neuropsychiatric disorders may share common pathways to risk.

Heterozygous NOTCH1 Variants Cause CNS Immune Activation and Microangiopathy.

NOTCH1 belongs to the NOTCH family of proteins that regulate cell fate and inflammatory responses. Somatic and germline NOTCH1 variants have been implicated in cancer, Adams-Oliver syndrome, and cardiovascular defects. We describe 7 unrelated patients grouped by the presence of leukoencephalopathy with calcifications and heterozygous de novo gain-of-function variants in NOTCH1. Immunologic profiling showed upregulated CSF IP-10, a cytokine secreted downstream of NOTCH1 signaling. Autopsy revealed extensive leukoencephalopathy and microangiopathy with vascular calcifications. This evidence implicates that heterozygous gain-of-function variants in NOTCH1 lead to a chronic central nervous system (CNS) inflammatory response resulting in a calcifying microangiopathy with leukoencephalopathy. ANN NEUROL 2022;92:895-901.