Asymmetric stem cell division (ASCD) is a fundamental process for generating cellular diversity while maintaining the stem cell pool. This review synthesizes evidence from diverse model systems to establish a paradigm-shifting hypothesis: centrioles are not passive microtubule-organizing centers but active determinants that orchestrate ASCD. We argue that centrioles function as integrative hub-organelles, executing four coordinated roles: as a Compass that fixes the division axis via cortical linkages, a Dispatcher that asymmetrically recruits and segregates fate determinants, a Sensor that transduces niche signals through the primary cilium, and a Chronometer that regulates division timing. The molecular asymmetry between the mother and daughter centriole, established during interphase, is a prerequisite for correct spindle orientation and asymmetric cargo partitioning. Disruption of centriolar integrity, as seen in human "centriolopathies" like primary microcephaly and ciliopathies, leads to randomized divisions and tissue malformation. Conversely, in cancer, centrosome amplification disrupts this intrinsic asymmetry, promoting symmetric, expansive divisions of stem-like cells. This integrative model positions the centriole as the central architect of cell fate, translating extrinsic polarity into intrinsic asymmetry. Understanding this centriole-centric program opens novel avenues in regenerative medicine, by controlling differentiation in vitro, and in oncology, by targeting the self-renewal of cancer stem cells.

Autosomal recessive primary microcephaly (MCPH) is a genetically heterogeneous neurodevelopmental disorder characterized by a markedly reduced head circumference (−3 to −5 standard deviations) at birth, with relatively preserved brain architecture. Affected individuals often present with mild to moderate intellectual disability, and the condition is more prevalent in populations with high rates of consanguinity, such as Pakistan. To date, pathogenic variants in at least 32 genes have been associated with MCPH, with ASPM and WDR62 accounting for the majority of cases (68% and 14%, respectively). In this study, we investigated four consanguineous families with congenital microcephaly and identified three novel variants in CPAP, WDR62, and ASPM. In Family 1, we identified a novel missense variant (c.3947C>A; p. (Thr1316Lys) in CPAP (NM_018451.4) located within the highly conserved TCP domain, which mediates interactions with other MCPH proteins, including STIL and CEP135. Family 2 harbored a previously unreported splice-site variant, c.2867 + 5G>T, in WDR62 (NM_001083961.2). In Families 3 and 4, we identified one novel (c.3188T>G; p. (Leu1063*)) and one previously reported (c.9730C>T; p. (Arg3244*)) pathogenic variant in ASPM (NM_018136.4). Computational analyses and structural modeling indicated that all these variants are likely deleterious, disrupting normal protein function. Our findings expand the mutational spectrum of CPAP and WDR62 and reinforce ASPM as the most frequently mutated gene underlying MCPH in the Pakistani population.

Primary cilia play a pivotal role in cellular signaling and development. Human primary microcephaly is strongly associated with pathogenic variants in primary cilia genes. Here, we examine the role of Ttc21b, a component of the intraflagellar transport-A complex, during mouse forebrain development by using a Ttc21balien null allele. Our findings reveal that significant microcephaly in homozygous mutants is caused by disrupted neural progenitor proliferation and differentiation. Histological and immunohistochemical analyses show an enlarged ventricular zone and reduced cortical plate thickness accompanied by altered mitotic spindle angles, suggesting defects in symmetric versus asymmetric cell divisions. Embryonic expression patterns suggest that perdurant TTC21B protein underlies these phenotypes. Progenitor proliferation kinetics were disrupted along with changes in TBR2-positive intermediate progenitors and TBR1-positive early-developing neurons. Neuronal processes in the cortical plate were significantly shortened. Our findings support a model in which early expression of Ttc21b in neural precursor cells destined for the forebrain is critical to ensure TTC21B protein levels to sustain subsequent neural progenitor proliferation and differentiation. These results advance our understanding of the role primary cilia have in cortical development.

Primary cilia as architects of the neocortex: Roles in brain development, function, and microcephaly.

The evolutionarily conserved RNA exosome complex modulates gene expression during development. Mutations in RNA exosome complex subunits have been implicated in various human brain disorders, suggesting that defects in RNA decay are linked to impaired neural development. In our study, we identified de novo variants of EXOSC10 in microcephalic individuals. The patient's phenotype can be replicated by heterozygous conditional knockout of Exosc10 in the developing mouse forebrain. The heterozygous loss of Exosc10 in the developing mouse cortex leads to increased cell cycle exit, with a premature differentiation of radial glial cells to intermediate progenitors and neurons in the embryonic cortex. This premature neurogenesis at the expense of neural stem cell proliferation coincides with a smaller cortical size. RNA sequencing and RNA immunoprecipitation sequencing revealed upregulation of many Sonic hedgehog (Shh) signalling genes. We further show the direct degradation of Shh pathway transcripts such as Scube1 and Scube3 by Exosc10. In Exosc10 mouse mutants, the reduced cortical size could be largely rescued by reducing Shh activity. We propose that increased Shh activity due to Exosc10 deficiency leads to premature neurogenesis and ultimately to microcephaly. These observations offer new insights into the neurodevelopmental role of Exosc10 and highlight the dosage-dependent regulation of Shh signalling by Exosc10 in cortical development.

The human brain originates from the neural tube that detaches from the ectodermal layer and gradually develops into a mature structure through highly regulated molecular and cellular processes. Here, stem cell technology is combined with 4D bioprinting, a fabrication process that utilizes additive manufacturing, to generate a 4D‐neural tube (4D‐NT). This consists of a scaffold that can self‐fold over time, which is then populated with iPSC‐derived neuroprogenitors, mimicking neural tube cellular architecture. The scaffold's “smart” self‐folding behavior is driven by the differential swelling properties of bilayer films, which create a deformation gradient upon hydration. Cellular analyses reveal a highly efficient induction of neuroprogenitors on 4D‐NTs, demonstrating the ability of this model to mimic the spatial and structural complexity of the developing human neural tube. Furthermore, 4D‐NTs seeded with iPSCs with a mutation in WDR62, associated with autosomal recessive primary microcephaly (MCPH), recapitulate the earlier observations obtained in 2D/3D neural cultures, thereby validating the newly developed 4D‐NT platform and suggesting it represents a tool that can facilitate understanding of human neural development and disease.

DEAH-Box helicase 37 (DHX37) gene, encoding an RNA-helicase, is essential for ribosome biogenesis. Pathogenic variants in the DHX37 gene result in a spectrum of ribosomopathies ranging from neurodevelopmental disorders with possible brain, vertebral, and/or cardiac anomalies (NEDBAVC syndrome, OMIM #618731) as well as disorders of sex development. Here, we describe a young boy with DHX37-related neurodevelopmental disorder with clinical and imaging findings masquerading as cerebral palsy. A 7.5-year-old boy presented with global developmental delay and generalized chorea of 6 months duration. He was born at 37 weeks gestation after an uneventful pregnancy with a birth weight of 2668 g. He had primary microcephaly and intractable epilepsy from infancy. Examination revealed microcephaly, spastic quadriparesis, generalized choreoathetosis and dystonia. MRI of the brain revealed T2-weighted hyperintensity in bilateral corticospinal tracts, posterior limb of the internal capsule (PLIC), corona radiata, external capsule, periventricular and deep white matter, as well as subcortical cysts. Diffusion-weighted images showed high signal in bilateral corticospinal tract and PLIC. As there were red flags pointing away from cerebral palsy such as primary microcephaly, refractory seizures, late-onset movement disorder, and persistent high signal on diffusion-weighted imaging, whole genome sequencing (WGS) was sent. WGS revealed a homozygous variant c.2417G>A (p.Ser806Asn) in the DHX37 gene. He was managed with antiseizure medications and clonazepam. DHX37-related neurodevelopmental disorder should be included in the differential for cerebral palsy mimic as affected children have global developmental delay, primary microcephaly, seizures, and movement disorders and thus may masquerade as sequel of hypoxic ischemic encephalopathy.

Background: Primary microcephaly (MCPH) is a genetically and clinically diverse condition characterized by small head size, structural brain abnormalities, and non-progressive intellectual impairment. To date, variations in 30 genes have been associated with MCPH. Objective: The study aims to identify the genetic variants of MCPH in the Pakistani population, where consanguineous marriages are common, and to explore the functional relationship of MCPH with other neurodevelopmental disorders (NDDs) such as autism spectrum disorder (ASD), intellectual disability (ID), and developmental delay (DD). Methods: Whole-exome sequencing (WES) and Sanger sequencing were applied to identify genetic variants in MCPH patients. The functional relationship between MCPH and other NDD genes was explored using DGH-GO software, employing hierarchical clustering. This cross-sectional study was conducted from September 2023 to October 2024. Results: We identified two novel variants, ERCC2 (c.2255G>A) and ERCC6 (c.1178C>T), and two already reported variants, MCPH1 (c.1254delT) and CENPJ (c.18delC). Machine learning analysis revealed a significant functional overlap between MCPH and other neurodevelopment disorders (NDDs), affected genes. Conclusion: Our study expands the mutational spectrum of MCPH and supports shared genetic etiology between MCPH and other NDDs. These findings provide a deeper understanding of the genetic underpinnings and comorbidities of MCPH, guiding future research toward effective therapeutic strategies.

Background/Objectives: Autosomal recessive primary microcephaly is a rare and genetically heterogeneous disorder characterized by congenital non-syndromic microcephaly, with at least 28 causative genes identified to date. Biallelic variants in the CDK5RAP2 gene, an ultra-rare cause of autosomal recessive primary microcephaly, lead to Primary Autosomal Recessive Microcephaly 3 (MCPH3). Methods: We present seven patients from six families diagnosed with MCPH3 in light of clinical and molecular findings using whole-exome sequencing (WES). Furthermore, we investigated the effects of the identified intronic variants on splicing through RNA analysis. Results: Almost all patients had severe microcephaly, mild to moderate intellectual disability, speech delay, and cutaneous pigmentary abnormalities. Four patients presented with postnatal short stature, and two showed weight deficiency. Dysmorphic evaluation revealed that the most prominent features included brachycephaly, hypertelorism, epicanthus, high-arched eyebrows, prominent nasal bridge, and micrognathia. We identified five distinct homozygous CDK5RAP2 variants in our patients, including four novel variants. Segregation analysis verified that the parents were carriers. Two of these variants were intronic (c.3148+5G>C and c.383+4dupA), two were frameshift (c.3168del), and one was a nonsense variant (c.1591C>T). Both intronic variants disrupted splicing, generating a premature stop codon and resulting in a truncated protein. Conclusions: This study broadens the mutational landscape of CDK5RAP2. We also sought to demonstrate the functional consequences of the CDK5RAP2 intronic variants on gene function using RNA analysis. The identification of four novel variants underscores the importance of molecular diagnostics in patients with primary microcephaly and provides valuable data for genetic counseling and future functional studies.

Primary microcephaly, a rare congenital condition characterized by reduced brain size, occurs due to impaired neurogenesis during brain development. Through whole-exome sequencing, we identified compound heterozygous loss-of-function mutations in CENTRIN 3 (CETN3) in a 5-year-old patient with primary microcephaly. As CETN3 has not been previously linked to microcephaly, we investigated its potential function in neurodevelopment in human pluripotent stem cell-derived cerebral organoids. We showed that CETN3-knockout (KO) organoids successfully recapitulated the microcephaly phenotype of reduced size compared to the control organoids. Through transcriptomic, histological, and protein analyses, we found that CETN3 deficiency directly interferes with neuronal differentiation and reduces proliferative capacity in neural stem/progenitor cells by impairing centrosome assembly required in cell cycle progression, consequently activating apoptosis. Furthermore, our data uncovered previously undocumented indirect effects of CETN3 through interaction with RNA splicing machinery involved in brain development. These findings expand the scope of known regulatory mechanisms of CETN3 in brain development and its etiological roles in human brain malformation.

Identification of CDK5RAP2 as a causative gene of focal epilepsy without microcephaly.

BackgroundMicrocephaly, characterized by an abnormally small head size, frequently co-occurs with neurodevelopmental disorders (NDDs). While the genetic basis of NDDs has been widely investigated, the contribution of rare coding variants to microcephaly remains poorly understood.MethodsWe investigated the relationships between head circumference and rare coding variants in 418 individuals with microcephaly, analyzing data from 1050 exomes (312 trios and 106 proband-only samples). Participants were classified into primary microcephaly (PM) and secondary microcephaly (SM) groups, and their clinical and genetic characteristics were systematically assessed. The functional impact of high-priority candidate genes, RTF1 and ASAP2, was further validated using neural progenitor cells (NPCs) and human forebrain organoid models.ResultsExome sequencing revealed 142 causative and 12 candidate genes associated with microcephaly. Pathway analyses indicated that PM genes are linked to early phases of brain development, whereas SM genes are more associated with later stages of neuronal maturation. In addition, the PM group had a significantly higher proportion of autosomal recessive disorders and exhibited more severe microcephaly than the SM group. Notably, females displayed greater microcephaly severity than males, primarily attributable to differences in the origin of the allele and inheritance patterns on the X chromosome. Functional experiments using CRISPR-Cas9 knockout in NPCs and brain organoids demonstrated reduced NPC proliferation, supporting the essential role of RTF1 and ASAP2 in brain development.ConclusionsThis study sheds light on the complex genetic architecture of microcephaly, emphasizing the impact of rare coding variants on brain development and delineating distinct clinical and molecular profiles underlying PM and SM.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13073-025-01513-w.

Whole-exome sequencing reveals a novel variant in two Iranian families with autosomal recessive primary microcephaly.

Primary microcephaly (MCPH) is a rare genetic neurodevelopmental disorder caused by homologous recessive mutations of the MCPH genes. It manifests as a significant reduction in brain volume and intellectual disability at birth. More than 28 genes with several pathogeneses have been identified so far. These genes have a strong effect on DNA damage repair and apoptosis, neuronal proliferation, neuronal differentiation, and neuronal migration. These pathogenesis pathways result in aberrant cell division and cell maturation, as well as an imbalance of the type of neural cells, and eventually a reduction of brain volume. Hence, researching in a multidisciplinary approach promotes research into the different etiologies of MCPH genes and offers a positive outcome for patients. However, investigating the etiology pathways has been given less focus, and limited studies and model systems have been carried out for this complex disease. Research using simple model organisms to study these pathogenic genes is beneficial. Recently, Drosophila melanogaster has been used as a powerful and promising model organism for efficient invivo experiments and for deciphering complex multicellular activities to unravel the function of the MCPH genes. Interestingly, about 80% of the genes that cause genetic diseases in humans have functional counterparts in D. melanogaster . Additionally, genetic similarity, simple genetics, rapid reproduction, high-throughput screening, and ease of generating transgenics make it unique. These features have prompted researchers to widely use it in research, contributing significantly to our understanding of human diseases such as cancer, Alzheimer's disease, Parkinson's disease, MCPH, and muscular dystrophy. In this review, I focus on the various pathways of MCPH genes pathogenesis and the advantage of leveraging the D. melanogaster model to dissect the etiology of MCPH genes. [Correction added on 9 August 2025, after first online publication: In the Abstract section, last sentence, pronoun 'we' has been changed to 'I'.].

The spliceosome is a critical cellular machinery responsible for pre-mRNA splicing that is essential for the proper expression of genes. Mutations in its core components are increasingly linked to neurodevelopmental disorders, such as primary microcephaly. Here, we investigated the role of SNW domain–containing protein 1 (SNW1), a spliceosomal protein, in splicing integrity and neurodevelopment. We identified 9 heterozygous mutations in the SNW1 gene in patients presenting with primary microcephaly. These mutations impaired SNW1’s interactions with core spliceosomal proteins, leading to defective RNA splicing and reduced protein functionality. Using Drosophila melanogaster and human embryonic stem cell–derived cerebral organoids models, we demonstrated that SNW1 depletion resulted in significant reductions in neural stem cell proliferation and increased apoptosis. RNA-Seq revealed disrupted alternative splicing, especially skipping exons, and altered expression of neurodevelopment-associated genes (CENPE, MEF2C, and NRXN2). Our findings provide crucial insights into the molecular mechanisms by which SNW1 dysfunction contributes to neurodevelopmental disorders and underscore the importance of proper spliceosome function in brain development.

WDR62 controls cortical radial migration and callosal projection of neurons in the developing cerebral cortex.

Microcephaly-related global developmental delay (GDD) and intellectual disability (ID) are characterized by a broad spectrum of neurodevelopmental impairments and encompass a multitude of causal factors. METTL5, a critical component involved in 18S rRNA methylation, has garnered considerable attention owing to its pivotal role in the pathogenesis of GDD and ID associated with microcephaly. A comprehensive physical examination and developmental assessment were performed for a 2-year-old girl presenting with symptoms of GDD and primary microcephaly. Whole-exome sequencing (WES) was performed to identify the pathogenic variant, and Sanger sequencing confirmed the mutation. To further investigate the pathogenicity of the mutation, minigene splicing assays, in vivo RT-PCR and bioinformatics analysis were employed. The WES identified a METTL5 homozygous intron mutation (NM_014168.4: c.224+5 G > A) in the proband. Sanger sequencing further validated the mutation in the family. Minigene assays and in vivo RT-PCR assays demonstrated exon 2 skipping, resulting in a 115-bp deletion in the mutated sequence. Bioinformatics analysis confirmed the pathogenicity of the mutation. For the first time, this study reported that a homozygous mutation (c.224+5 G > A) in the METTL5 gene led to microcephaly-related GDD in a Chinese family. Meantime, the report has validated the pathogenicity of intronic mutations and expanded the mutational spectrum of the METTL5 gene. Thus, this study aids our understanding of the role of METTL5 in GDD and provides a theoretical foundation for the prevention of this disease.

Non-canonical roles of mitotic proteins in cortical neurons.

SummaryBackgroundZika virus (ZIKV) infection during pregnancy is associated with an increased risk of congenital malformations. The prevalence of short and long-term consequences, however, remains uncertain due to heterogeneity across studies. Individual Participant Data Meta-Analysis (IPD-MA) offers an alternative approach to provide more precise and generalisable estimates through data harmonisation across studies, allowing for standardised definitions and exploration of heterogeneity. This project was undertaken to estimate absolute and relative risks of adverse outcomes for individuals with ZIKV infection during pregnancy.MethodsIPD-MA studies and their datasets were identified through a systematic search conducted in 2018 with the following criteria: observational longitudinal or surveillance-based studies investigating ZIKV during pregnancy or at birth, measured fetal, infant, or child outcomes, and included at least 10 participants. Here we used IPD data shared by March 2022 from 18 studies from international health organisations and research networks, comprising 24 unique datasets, in 11 countries. Datasets were harmonised with standardised definitions, using variables related to pregnant individuals, methods used for ZIKV diagnoses, fetal characteristics and outcomes, and pooled for analysis. Frequentist and Bayesian regression methods were applied to estimate outcome prevalence and evaluate the association between maternal ZIKV infection and fetal loss, microcephaly and congenital zika syndrome as primary outcomes.FindingsData including 9568 pregnant individuals and 9608 newborns, were harmonised. The risk of severe primary microcephaly was significantly higher in ZIKV-positive pregnancies (1.5%, CI 0.8%–2.7%) compared to ZIKV-negative ones (0.3%, CI 0.1%–1.0%), with a relative risk of 4.5 (CI 1.5–13.3) in the one-stage meta-analysis. While some risk estimates were consistent between Bayesian and Frequentist methods, estimates for other outcomes varied, underscoring the influence of both the analytical approach and the definition of ZIKV on the associations.InterpretationOur findings align with previously published meta-analyses and indicate an added burden to adverse pregnancy outcomes with higher prevalence compared to pre-epidemic population-based average values. Future research should focus on additional outcomes with clear definitions of maternal infection. Women of reproductive age should be informed about the risks of Zika infection during pregnancy to support reproductive planning.FundingThis project was supported by the 10.13039/100010269Wellcome Trust grant number 206532/Z/17/Z, the 10.13039/100004423WHOHealth Emergencies Programme Global Arbovirus Initiative, and the 10.13039/100004423WHO Department of Sexual and Reproductive Health and Research, including the Human Reproduction Special Programme (HRP).

Autosomal recessive primary microcephaly (MCPH) is a rare, genetically heterogeneous disorder characterized by congenital microcephaly, non-progressive intellectual disability, and absence of neurological abnormalities. Pathogenic variants in CDK5RAP2, linked to MCPH3, represent one of the least common causes of MCPH. Autosomal recessive primary microcephaly (MCPH) is a rare, genetically heterogeneous disorder characterized by reduced head size at birth, variable intellectual disability, and no neurological abnormalities. Among This study aimed to identify and characterize novel genetic and clinical findings in patients with CDK5RAP2 gene variants, contributing to the understanding of this rare disorder. Whole exome sequencing (WES) was conducted in 11 patients from five consanguineous families. Six CDK5RAP2 variants were identified, four of which were novel (c.4421del, c.1968G>C, c.3460C>T, c.625dup). Ten patients harbored homozygous variants, whereas one displayed compound heterozygosity. Segregation analysis confirmed carrier status in parents. Clinical evaluations aligned with typical MCPH3 features, though phenotypic variability was observed. This study expands the CDK5RAP2 variant spectrum and reinforces WES as a critical tool for diagnosing rare MCPH subtypes, guiding carrier screening, and improving genetic counseling. The novel variants highlight the genetic diversity underlying MCPH3, urging broader genomic investigations in undiagnosed cases.