Antisense oligonucleotide therapy in an individual with KIF1A-associated neurological disorder.

KIF1A-associated neurological disorder (KAND) is a neurodegenerative and often lethal ultrarare disease with a wide phenotypic spectrum associated with largely heterozygous de novo missense variants in KIF1A. Antisense oligonucleotide treatments represent a promising approach for personalized treatments in ultrarare diseases. Here we report the case of one patient with a severe form of KAND characterized by refractory spells of behavioral arrest and carrying a p.Pro305Leu variant in KIF1A, who was treated with intrathecal injections of an allele-specific antisense oligonucleotide specifically designed to degrade the mRNA from the pathogenic allele. The first intrathecal administration was complicated by an epidural cerebrospinal fluid collection, which resolved spontaneously. Otherwise, the antisense oligonucleotide was safe and well tolerated over the 9-month treatment. Most outcome measures, including severity of the spells of behavioral arrest, number of falls and quality of life, improved. There was little change in the 6-min Walk Test distance, but qualitative changes in gait resulting in meaningful reductions in falls and increasing independence were observed. Cognitive performance was stable and did not degenerate over time. Our findings provide preliminary insights on the safety and efficacy of an allele-specific antisense oligonucleotide as a possible treatment for KAND.

Loss of symmetric cell division of apical neural progenitors drives DENND5A-related developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEEs) feature altered brain development, developmental delay and seizures, with seizures exacerbating developmental delay. Here we identify a cohort with biallelic variants in DENND5A, encoding a membrane trafficking protein, and develop animal models with phenotypes like the human syndrome. We demonstrate that DENND5A interacts with Pals1/MUPP1, components of the Crumbs apical polarity complex required for symmetrical division of neural progenitor cells. Human induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division with an inherent propensity to differentiate into neurons. These phenotypes result from misalignment of the mitotic spindle in apical neural progenitors. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles, biasing daughter cells towards a more fate-committed state, ultimately shortening the period of neurogenesis. This study provides a mechanism for DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families.

Evaluation of the Patients with the Diagnosis of Pontocerebellar Hypoplasia: A Multicenter National Study.

Pontocerebellar hypoplasia (PCH) is a heterogeneous group of neurodegenerative disorders characterized by hypoplasia and degeneration of the cerebellum and pons. We aimed to identify the clinical, laboratory, and imaging findings of the patients with diagnosed PCH with confirmed genetic analysis. We collected available clinical data, laboratory, and imaging findings in our retrospective multicenter national study of 64 patients with PCH in Turkey. The genetic analysis included the whole-exome sequencing (WES), targeted next-generation sequencing (NGS), or single gene analysis. Sixty-four patients with PCH were 28 female (43.8%) and 36 (56.3%) male. The patients revealed homozygous mutation in 89.1%, consanguinity in 79.7%, pregnancy at term in 85.2%, microcephaly in 91.3%, psychomotor retardation in 98.4%, abnormal neurological findings in 100%, seizure in 63.8%, normal biochemistry and metabolic investigations in 92.2%, and dysmorphic findings in 51.2%. The missense mutation was found to be the most common variant type in all patients with PCH. It was detected as CLP1 (n = 17) was the most common PCH related gene. The homozygous missense variant c.419G > A (p.Arg140His) was identified in all patients with CLP1. Moreover, all patients showed the same homozygous missense variant c.919G > T (p.A307S) in TSEN54 group (n = 6). In Turkey, CLP1 was identified as the most common causative gene with the identical variant c.419G > A; p.Arg140His. The current study supports that genotype data on PCH leads to phenotypic variability over a wide phenotypic spectrum.

Exome copy number variant detection, analysis, and classification in a large cohort of families with undiagnosed rare genetic disease.

Copy number variants (CNVs) are significant contributors to the pathogenicity of rare genetic diseases and, with new innovative methods, can now reliably be identified from exome sequencing. Challenges still remain in accurate classification of CNV pathogenicity. CNV calling using GATK-gCNV was performed on exomes from a cohort of 6,633 families (15,759 individuals) with heterogeneous phenotypes and variable prior genetic testing collected at the Broad Institute Center for Mendelian Genomics of the Genomics Research to Elucidate the Genetics of Rare Diseases consortium and analyzed using the seqr platform. The addition of CNV detection to exome analysis identified causal CNVs for 171 families (2.6%). The estimated sizes of CNVs ranged from 293 bp to 80 Mb. The causal CNVs consisted of 140 deletions, 15 duplications, 3 suspected complex structural variants (SVs), 3 insertions, and 10 complex SVs, the latter two groups being identified by orthogonal confirmation methods. To classify CNV variant pathogenicity, we used the 2020 American College of Medical Genetics and Genomics/ClinGen CNV interpretation standards and developed additional criteria to evaluate allelic and functional data as well as variants on the X chromosome to further advance the framework. We interpreted 151 CNVs as likely pathogenic/pathogenic and 20 CNVs as high-interest variants of uncertain significance. Calling CNVs from existing exome data increases the diagnostic yield for individuals undiagnosed after standard testing approaches, providing a higher-resolution alternative to arrays at a fraction of the cost of genome sequencing. Our improvements to the classification approach advances the systematic framework to assess the pathogenicity of CNVs.

Cell-type-resolved mosaicism reveals clonal dynamics of the human forebrain.

Debate remains around the anatomical origins of specific brain cell subtypes and lineage relationships within the human forebrain1-7. Thus, direct observation in the mature human brain is critical for a complete understanding of its structural organization and cellular origins. Here we utilize brain mosaic variation within specific cell types as distinct indicators for clonal dynamics, denoted as cell-type-specific mosaic variant barcode analysis. From four hemispheres and two different human neurotypical donors, we identified 287 and 780 mosaic variants, respectively, that were used to deconvolve clonal dynamics. Clonal spread and allele fractions within the brain reveal that local hippocampal excitatory neurons are more lineage-restricted than resident neocortical excitatory neurons or resident basal ganglia GABAergic inhibitory neurons. Furthermore, simultaneous genome transcriptome analysis at both a cell-type-specific and a single-cell level suggests a dorsal neocortical origin for a subgroup of DLX1+ inhibitory neurons that disperse radially from an origin shared with excitatory neurons. Finally, the distribution of mosaic variants across 17 locations within one parietal lobe reveals that restriction of clonal spread in the anterior-posterior axis precedes restriction in the dorsal-ventral axis for both excitatory and inhibitory neurons. Thus, cell-type-resolved somatic mosaicism can uncover lineage relationships governing the development of the human forebrain.

Neurodevelopmental disorders associated variants in ADAT3 disrupt the activity of the ADAT2/ADAT3 tRNA deaminase complex and impair neuronal migration.

The ADAT2/ADAT3 complex catalyzes the adenosine to inosine modification at the wobble position of eukaryotic tRNAs. Mutations in ADAT3 , the catalytically inactive subunit of the ADAT2/ADAT3 complex, have been identified in patients presenting with severe neurodevelopmental disorders (NDDs). Yet, the physiological function of ADAT2/ADAT3 complex during brain development remains totally unknown. Here we showed that maintaining a proper level of ADAT2/ADAT3 catalytic activity is required for correct radial migration of projection neurons in the developing mouse cortex. In addition, we not only reported 7 new NDD patients carrying biallelic variants in ADAT3 but also deeply characterize the impact of those variants on ADAT2/ADAT3 structure, biochemical properties, enzymatic activity and tRNAs editing and abundance. We demonstrated that all the identified variants alter both the expression and the activity of the complex leading to a significant decrease of I 34 with direct consequence on their steady-state. Using in vivo complementation assays, we correlated the severity of the migration phenotype with the degree of the loss of function caused by the variants. Altogether, our results indicate a critical role of ADAT2/ADAT3 during cortical development and provide cellular and molecular insights into the pathogenicity of ADAT3-related neurodevelopmental disorder.

Loss of symmetric cell division of apical neural progenitors drives DENND5A-related developmental and epileptic encephalopathy.

Developmental and epileptic encephalopathies (DEEs) are a heterogenous group of epilepsies in which altered brain development leads to developmental delay and seizures, with the epileptic activity further negatively impacting neurodevelopment. Identifying the underlying cause of DEEs is essential for progress toward precision therapies. Here we describe a group of individuals with biallelic variants in DENND5A and determine that variant type is correlated with disease severity. We demonstrate that DENND5A interacts with MUPP1 and PALS1, components of the Crumbs apical polarity complex, which is required for both neural progenitor cell identity and the ability of these stem cells to divide symmetrically. Induced pluripotent stem cells lacking DENND5A fail to undergo symmetric cell division during neural induction and have an inherent propensity to differentiate into neurons, and transgenic DENND5A mice, with phenotypes like the human syndrome, have an increased number of neurons in the adult subventricular zone. Disruption of symmetric cell division following loss of DENND5A results from misalignment of the mitotic spindle in apical neural progenitors. A subset of DENND5A is localized to centrosomes, which define the spindle poles during mitosis. Cells lacking DENND5A orient away from the proliferative apical domain surrounding the ventricles, biasing daughter cells towards a more fate-committed state and ultimately shortening the period of neurogenesis. This study provides a mechanism behind DENND5A-related DEE that may be generalizable to other developmental conditions and provides variant-specific clinical information for physicians and families.

The clinical and genetic landscape of developmental and epileptic encephalopathies in Egyptian children.

Developmental and epileptic encephalopathies (DEEs) are a heterogeneous group of epilepsies characterized by early-onset, refractory seizures associated with developmental regression or impairment, with a heterogeneous genetic landscape including genes implicated in various pathways and mechanisms. We retrospectively studied the clinical and genetic data of patients with genetic DEE who presented at two tertiary centers in Egypt over a 10-year period. Exome sequencing was used for genetic testing. We report 74 patients from 63 unrelated Egyptian families, with a high rate of consanguinity (58%). The most common seizure type was generalized tonic-clonic (58%) and multiple seizure types were common (55%). The most common epilepsy syndrome was early infantile DEE (50%). All patients showed variable degrees of developmental impairment. Microcephaly, hypotonia, ophthalmological involvement and neuroimaging abnormalities were common. Eighteen novel variants were identified and the phenotypes of five DEE genes were expanded with novel phenotype-genotype associations. Obtaining a genetic diagnosis had implications on epilepsy management in 17 patients with variants in 12 genes. In this study, we expand the phenotype and genotype spectrum of DEE in a large single ethnic cohort of patients. Reaching a genetic diagnosis guided precision management of epilepsy in a significant proportion of patients.

Unique Capabilities of Genome Sequencing for Rare Disease Diagnosis.

Background: Causal variants underlying rare disorders may remain elusive even after expansive gene panels or exome sequencing (ES). Clinicians and researchers may then turn to genome sequencing (GS), though the added value of this technique and its optimal use remain poorly defined. We therefore investigated the advantages of GS within a phenotypically diverse cohort. Methods: GS was performed for 744 individuals with rare disease who were genetically undiagnosed. Analysis included review of single nucleotide, indel, structural, and mitochondrial variants. Results: We successfully solved 218/744 (29.3%) cases using GS, with most solves involving established disease genes (157/218, 72.0%). Of all solved cases, 148 (67.9%) had previously had non-diagnostic ES. We systematically evaluated the 218 causal variants for features requiring GS to identify and 61/218 (28.0%) met these criteria, representing 8.2% of the entire cohort. These included small structural variants (13), copy neutral inversions and complex rearrangements (8), tandem repeat expansions (6), deep intronic variants (15), and coding variants that may be more easily found using GS related to uniformity of coverage (19). Conclusion: We describe the diagnostic yield of GS in a large and diverse cohort, illustrating several types of pathogenic variation eluding ES or other techniques. Our results reveal a higher diagnostic yield of GS, supporting the utility of a genome-first approach, with consideration of GS as a secondary or tertiary test when higher-resolution structural variant analysis is needed or there is a strong clinical suspicion for a condition and prior targeted genetic testing has been negative.