Microcephaly Gene Mcph1 Deficiency Induces p19ARF-Dependent Cell Cycle Arrest and Senescence.

MCPH1 has been identified as the causal gene for primary microcephaly type 1, a neurodevelopmental disorder characterized by reduced brain size and delayed growth. As a multifunction protein, MCPH1 has been reported to repress the expression of TERT and interact with transcriptional regulator E2F1. However, it remains unclear whether MCPH1 regulates brain development through its transcriptional regulation function. This study showed that the knockout of Mcph1 in mice leads to delayed growth as early as the embryo stage E11.5. Transcriptome analysis (RNA-seq) revealed that the deletion of Mcph1 resulted in changes in the expression levels of a limited number of genes. Although the expression of some of E2F1 targets, such as Satb2 and Cdkn1c, was affected, the differentially expressed genes (DEGs) were not significantly enriched as E2F1 target genes. Further investigations showed that primary and immortalized Mcph1 knockout mouse embryonic fibroblasts (MEFs) exhibited cell cycle arrest and cellular senescence phenotype. Interestingly, the upregulation of p19ARF was detected in Mcph1 knockout MEFs, and silencing p19Arf restored the cell cycle and growth arrest to wild-type levels. Our findings suggested it is unlikely that MCPH1 regulates neurodevelopment through E2F1-mediated transcriptional regulation, and p19ARF-dependent cell cycle arrest and cellular senescence may contribute to the developmental abnormalities observed in primary microcephaly.

Mcph1, mutated in primary microcephaly, is also crucial for erythropoiesis.

Microcephaly is a common feature in inherited bone marrow failure syndromes, prompting investigations into shared pathways between neurogenesis and hematopoiesis. To understand this association, we studied the role of the microcephaly gene Mcph1 in hematological development. Our research revealed that Mcph1-knockout mice exhibited congenital macrocytic anemia due to impaired terminal erythroid differentiation during fetal development. Anemia's cause is a failure to complete cell division, evident from tetraploid erythroid progenitors with DNA content exceeding 4n. Gene expression profiling demonstrated activation of the p53 pathway in Mcph1-deficient erythroid precursors, leading to overexpression of Cdkn1a/p21, a major mediator of p53-dependent cell cycle arrest. Surprisingly, fetal brain analysis revealed hypertrophied binucleated neuroprogenitors overexpressing p21 in Mcph1-knockout mice, indicating a shared pathophysiological mechanism underlying both erythroid and neurological defects. However, inactivating p53 in Mcph1-/- mice failed to reverse anemia and microcephaly, suggesting that p53 activation in Mcph1-deficient cells resulted from their proliferation defect rather than causing it. These findings shed new light on Mcph1's function in fetal hematopoietic development, emphasizing the impact of disrupted cell division on neurogenesis and erythropoiesis - a common limiting pathway.

Gingival proteomics reveals the role of TGF beta and YAP/TAZ signaling in Raine syndrome fibrosis.

Raine syndrome (RNS) is a rare autosomal recessive osteosclerotic dysplasia. RNS is caused by loss-of-function disease-causative variants of the FAM20C gene that encodes a kinase that phosphorylates most of the secreted proteins found in the body fluids and extracellular matrix. The most common RNS clinical features are generalized osteosclerosis, facial dysmorphism, intracerebral calcifications and respiratory defects. In non-lethal RNS forms, oral traits include a well-studied hypoplastic amelogenesis imperfecta (AI) and a much less characterized gingival phenotype. We used immunomorphological, biochemical, and siRNA approaches to analyze gingival tissues and primary cultures of gingival fibroblasts of two unrelated, previously reported RNS patients. We showed that fibrosis, pathological gingival calcifications and increased expression of various profibrotic and pro-osteogenic proteins such as POSTN, SPARC and VIM were common findings. Proteomic analysis of differentially expressed proteins demonstrated that proteins involved in extracellular matrix (ECM) regulation and related to the TGFß/SMAD signaling pathway were increased. Functional analyses confirmed the upregulation of TGFß/SMAD signaling and subsequently uncovered the involvement of two closely related transcription cofactors important in fibrogenesis, Yes-associated protein (YAP) and transcriptional coactivator with PDZ-binding motif (TAZ). Knocking down of FAM20C confirmed the TGFß-YAP/TAZ interplay indicating that a profibrotic loop enabled gingival fibrosis in RNS patients. In summary, our in vivo and in vitro data provide a detailed description of the RNS gingival phenotype. They show that gingival fibrosis and calcifications are associated with, and most likely caused by excessed ECM production and disorganization. They furthermore uncover the contribution of increased TGFß-YAP/TAZ signaling in the pathogenesis of the gingival fibrosis.

Atypical mandibulofacial dysostosis with microcephaly diagnosed through the identification of a novel pathogenic mutation in EFTUD2.

BACKGROUND: Mandibulofacial dysostosis with microcephaly (MFDM, OMIM# 610536) is a rare monogenic disease that is caused by a mutation in the elongation factor Tu GTP binding domain containing 2 gene (EFTUD2, OMIM* 603892). It is characterized by mandibulofacial dysplasia, microcephaly, malformed ears, cleft palate, growth and intellectual disability. MFDM can be easily misdiagnosed due to its phenotypic overlap with other craniofacial dysostosis syndromes. The clinical presentation of MFDM is highly variable among patients. METHODS: A patient with craniofacial anomalies was enrolled and evaluated by a multidisciplinary team. To make a definitive diagnosis, whole-exome sequencing was performed, followed by validation by Sanger sequencing. RESULTS: The patient presented with extensive facial bone dysostosis, upward slanting palpebral fissures, outer and middle ear malformation, a previously unreported orbit anomaly, and spina bifida occulta. A novel, pathogenic insertion mutation (c.215_216insT: p.Tyr73Valfs*4) in EFTUD2 was identified as the likely cause of the disease. CONCLUSIONS: We diagnosed this atypical case of MFDM by the detection of a novel pathogenetic mutation in EFTUD2. We also observed previously unreported features. These findings enrich both the genotypic and phenotypic spectrum of MFDM.

An Ultra-Rare Mixed Phenotype with Combined AP-4 and ERF Mutations: The First Report in a Pediatric Patient and a Literature Review.

The adaptor protein 4 (AP-4) constitutes a conserved hetero-tetrameric complex within the family of adaptor protein (AP) complex, crucial for the signal-mediated trafficking of integral membrane proteins. Mutations affecting all subunits of the AP-4 complex have been linked to autosomal-recessive cerebral palsy and a complex hereditary spastic paraparesis (HSP) phenotype. Our report details the case of a 14-year-old boy born to consanguineous parents, presenting psychomotor delay, severe intellectual disability, microcephaly, and trigonocephaly. Despite a history of febrile seizures, subsequent years were devoid of seizures, with normal EEG. Exome sequencing revealed pathogenic variants in both the AP4B1 and ERF genes. Significantly, the patient exhibited features associated with AP4B1 mutations, including distinctive traits such as cranial malformations. The ERF gene variant, linked to craniosynostosis, likely contributes to the observed trigonocephaly. This case represents the initial documentation of a concurrent mutation in the AP4B1 and ERF genes, underscoring the critical role of exome analysis in unraveling complex phenotypes. Understanding these complex genotypes offers valuable insights into broader syndromic conditions, facilitating comprehensive patient management.

Neuroinflammation, blood-brain barrier dysfunction, hippocampal atrophy and delayed neurodevelopment: Contributions for a rat model of congenital Zika syndrome.

The congenital Zika syndrome (CZS) has been characterized as a set of several brain changes, such as reduced brain volume and subcortical calcifications, in addition to cognitive deficits. Microcephaly is one of the possible complications found in newborns exposed to Zika virus (ZIKV) during pregnancy, although it is an impacting clinical sign. This study aimed to investigate the consequences of a model of congenital ZIKV infection by evaluating the histopathology, blood-brain barrier, and neuroinflammation in pup rats 24 h after birth, and neurodevelopment of the offspring. Pregnant rats were inoculated subcutaneously with ZIKV-BR at the dose 1 × 107 plaque-forming unit (PFU mL-1) of ZIKV isolated in Brazil (ZIKV-BR) on gestational day 18 (G18). A set of pups, 24 h after birth, was euthanized. The brain was collected and later evaluated for the histopathology of brain structures through histological analysis. Additionally, analyses of the blood-brain barrier were conducted using western blotting, and neuroinflammation was assessed using ELISA. Another set of animals was evaluated on postnatal days 3, 6, 9, and 12 for neurodevelopment by observing the developmental milestones. Our results revealed hippocampal atrophy in ZIKV animals, in addition to changes in the blood-brain barrier structure and pro-inflammatory cytokines expression increase. Regarding neurodevelopment, a delay in important reflexes during the neonatal period in ZIKV animals was observed. These findings advance the understanding of the pathophysiology of CZS and contribute to enhancing the rat model of CZS.

A further case of AFG2B-related neurodevelopmental disorder with hearing loss and microcephaly allows further clarification of pathogenicity of the variant c.1313T>C, p.(Leu438Pro).

BACKGROUND: Bi-allelic variants in AFG2B (previously known as SPATA5L1) have recently been associated with a neurodevelopmental disorder with hearing loss and spasticity, as well as isolated hearing loss. We report on a 6 1/2-year-old girl with a history of global developmental delay, subsequent intellectual disability without relevant language acquisition, sensorineural hearing loss, muscular hypotonia and microcephaly. METHODS: We performed trio exome sequencing on the patient and her parents. RESULTS: Trio exome sequencing revealed likely pathogenic compound heterozygous missense variants in AFG2B [c.527G>T, p.(Gly176Val) and c.1313T>C, p.(Leu438Pro)] in the patient. CONCLUSION: Of note, the change c.1313T>C, p.(Leu438Pro) has been observed in a previously published patient as part of a complex disease allele along with a second homozygous missense change, so the exact contribution of the two alterations to this patient's disease had initially remained unclear. Our results support the pathogenic relevance of the c.1313T>C, p.(Leu438Pro) allele while providing detailed insights into the disease manifestation of a further patient.

Novel PNKP mutations associated with reduced DNA single-strand break repair and severe microcephaly, seizures, and developmental delay.

BACKGROUND: Microcephaly with early-onset seizures (MCSZ) is a neurodevelopmental disorder caused by pathogenic variants in the DNA strand break repair protein, polynucleotide kinase 3'-phosphatase (PNKP). METHODS: We have used whole genome sequencing and Sanger sequencing to identify disease-causing variants, followed by a minigene assay, Western blotting, alkaline comet assay, γH2AX, and ADP-ribose immunofluorescence. RESULTS: Here, we describe a patient with compound heterozygous variants in PNKP, including a missense variant in the DNA phosphatase domain (T323M) and a novel splice acceptor site variant within the DNA kinase domain that we show leads to exon skipping. We show that primary fibroblasts derived from the patient exhibit greatly reduced levels of PNKP protein and reduced rates of DNA single-strand break repair, confirming that the mutated PNKP alleles are dysfunctional. CONCLUSION: The data presented show that the detected compound heterozygous variants result in reduced levels of PNKP protein, which affect the repair of both oxidative and TOP1-induced single-strand breaks, and most likely causes MCSZ in this patient.

A novel KNL1 intronic splicing variant likely destabilizes the KMN complex, causing primary microcephaly.

Primary microcephaly (MCPH) is an autosomal recessive disorder characterized by head circumference of at least two standard deviations below the mean. Biallelic variants in the kinetochore gene KNL1 is a known cause of MCPH4. KNL1 is the central component of the KNL1-MIS12-NSL1 (KMN) network, which acts as the signaling hub of the kinetochore and is required for correct chromosomal segregation during mitosis. We identified biallelic KNL1 variants in two siblings from a non-consanguineous family with microcephaly and intellectual disability. The two siblings carry a frameshift variant predicted to prematurely truncate the transcript and undergo nonsense mediated decay, and an intronic single nucleotide variant (SNV) predicted to disrupt splicing. An in vitro splicing assay and qPCR from blood-derived RNA confirmed that the intronic variant skips exon 23, significantly reducing levels of the canonical transcript. Protein modeling confirmed that absence of exon 23, an inframe exon, would disrupt a key interaction within the KMN network and likely destabilize the kinetochore signaling hub, disrupting mitosis. Therefore, this splicing variant is pathogenic and, in trans with a frameshift variant, causes the MCPH phenotype associated with KLN1. This finding furthers the association of splicing variants as a common pathogenic variant class for KNL1.

PHGDH-related microcephalic dwarfism in two fetuses: Expanding the phenotypical spectrum of L-serine biosynthesis defect.

Defects in L-serine biosynthesis are a group of autosomal recessive diseases resulting in a wide phenotypic spectrum ranging from viable to lethal presentations and caused by variants in the three genes encoding the L-serine biosynthesis enzymes, PHGDH, PSAT1, and PSPH. Neu-Laxova syndrome (NLS) is the fetal form of this group, characterized by multiple congenital anomalies including severe intrauterine growth retardation, cutaneous lesions extending from ichthyosis to severe restrictive dermopathy with ectropion and eclabion, edema, microcephaly, central nervous system abnormalities, and flexion contractures. Here we report on two unrelated fetuses with an attenuated phenotype of NLS, that initially evoked Taybi-Linder syndrome. They carry biallelic pathogenic variants in the PHGDH gene. These observations expand the phenotypic continuum of L-serine biosynthesis defects, and illustrate the phenotypic overlap between NLS and microcephalic primordial dwarfism.

Characterization of Vps13b-mutant mice reveals neuroanatomical and behavioral phenotypes with females less affected.

The vacuolar protein sorting-associated protein 13B (VPS13B) is a large and highly conserved protein. Disruption of VPS13B causes the autosomal recessive Cohen syndrome, a rare disorder characterized by microcephaly and intellectual disability among other features, including developmental delay, hypotonia, and friendly-personality. However, the underlying mechanisms by which VPS13B disruption leads to brain dysfunction still remain unexplained. To gain insights into the neuropathogenesis of Cohen syndrome, we systematically characterized brain changes in Vps13b-mutant mice and compared murine findings to 235 previously published and 17 new patients diagnosed with VPS13B-related Cohen syndrome. We showed that Vps13b is differentially expressed across brain regions with the highest expression in the cerebellum, the hippocampus and the cortex with postnatal peak. Half of the Vps13b-/- mice die during the first week of life. The remaining mice have a normal lifespan and display the core phenotypes of the human disease, including microcephaly, growth delay, hypotonia, altered memory, and enhanced sociability. Systematic 2D and 3D brain histo-morphological analyses reveal specific structural changes in the brain starting after birth. The dentate gyrus is the brain region with the most prominent reduction in size, while the motor cortex is specifically thinner in layer VI. The fornix, the fasciculus retroflexus, and the cingulate cortex remain unaffected. Interestingly, these neuroanatomical changes implicate an increase of neuronal death during infantile stages with no progression in adulthood suggesting that VPS13B promotes neuronal survival early in life. Importantly, whilst both sexes were affected, some neuroanatomical and behavioral phenotypes were less pronounced or even absent in females. We evaluate sex differences in Cohen patients and conclude that females are less affected both in mice and patients. Our findings provide new insights about the neurobiology of VPS13B and highlight previously unreported brain phenotypes while defining Cohen syndrome as a likely new entity of non-progressive infantile neurodegeneration.

Zika virus infection histories in brain development.

An outbreak of births of microcephalic patients in Brazil motivated multiple studies on this incident. The data left no doubt that infection by Zika virus (ZIKV) was the cause, and that this virus promotes reduction in neuron numbers and neuronal death. Analysis of patients' characteristics revealed additional aspects of the pathology alongside the decrease in neuronal number. Here, we review the data from human, molecular, cell and animal model studies attempting to build the natural history of ZIKV in the embryonic central nervous system (CNS). We discuss how identifying the timing of infection and the pathways through which ZIKV may infect and spread through the CNS can help explain the diversity of phenotypes found in congenital ZIKV syndrome (CZVS). We suggest that intraneuronal viral transport is the primary mechanism of ZIKV spread in the embryonic brain and is responsible for most cases of CZVS. According to this hypothesis, the viral transport through the blood-brain barrier and cerebrospinal fluid is responsible for more severe pathologies in which ZIKV-induced malformations occur along the entire anteroposterior CNS axis.

Potential role of lncRNA in impairing cellular properties of human neural progenitor cells following exposure to Zika virus E protein.

Zika virus (ZIKV) infection during the first trimester of the pregnancy may lead to Congenital zika syndrome in the neonates. The viral infection hampers foetal brain development and causes microcephaly. Human neural progenitor cells (hNPCs) play an important role in brain development, however they are highly susceptible to ZIKV infection. In this study, we elucidated the molecular mechanisms that lead to cellular alterations in hNPCs due to ZIKV E-protein. We investigated proliferation, differentiation, migration and inflammation in hNPCs, which may lead to microcephaly. In our study, we found that ZIKV E-protein causes cell cycle arrest, decrease in proliferation and increase in mitotic length of the dividing hNPCs. We observed CyclinD1 and upstream molecules (p21 and p53) of the pathway are dysregulated, and intracellular calcium at basal level as well as upon ATP stimulation were reduced following over expression of ZIKV E-protein. ZIKV E-protein transfected hNPCs exhibited pre-mature differentiation with pro-neural genes upregulated. Furthermore, ZIKV E-protein disrupted migrational properties of hNPCs and caused elevated levels of inflammatory chemokines and cytokines. To gain insights into molecular mechanisms of these effects on hNPCs, we explored the possible involvement of long non coding RNAs in ZIKV neuropathogenesis. We have shortlisted lncRNAs associated with differentially expressed genes from publicly available transcriptomic data and found some of those lncRNAs are differentially expressed upon E-protein transfection of hNPCs. Gene ontology analysis suggest these lncRNAs play an important role in regulation of viral life cycle, host's defence response and cell proliferation.

Longitudinal evolution of electroencephalogram (EEG): Findings over five years of follow-up in children with Zika-related microcephaly from the Microcephaly Epidemic Research Group Pediatric Cohort (2015-2020).

OBJECTIVE: To assess the longitudinal evolution of EEG findings in children with Zika related-microcephaly (ZRM) and to evaluate the associations of these patterns with the children's clinical and neuroimaging characteristics. METHODS: As part of the follow-up of the Microcephaly Epidemic Research Group Pediatric Cohort (MERG-PC) in Recife, Brazil, we performed serial EEG recordings in a subgroup of children with ZRM to evaluate changes in background rhythms and epileptiform activity (EA). Latent class analysis was used to identify patterns in the evolution of EA over time; clinical and neuroimaging findings were compared across the identified groups. RESULTS: Out of the 72 children with ZRM who were evaluated during 190 EEGs/videoEEGs, all participants presented with abnormal background activity, 37.5% presented with an alpha-theta rhythmic activity, and 25% presented with sleep spindles, which were less commonly observed in children with epilepsy. EA changed over time in 79.2% of children, and three distinct trajectories were identified: (i) multifocal EA over time, (ii) no discharges/focal EA evolving to focal/multifocal EA, and (iii) focal/multifocal EA evolving to epileptic encephalopathy patterns (e.g., hypsarrhythmia or continuous EA in sleep). The multifocal EA over time trajectory was associated with periventricular and thalamus/basal ganglia calcifications, brainstem and corpus callosum atrophy and had less focal epilepsy, whereas the children in the trajectory which evolved to epileptic encephalopathy patterns had more frequently focal epilepsy. SIGNIFICANCE: These findings suggest that, in most children with ZRM, trajectories of changes in EA can be identified and associated with neuroimaging and clinical features.

Functional analysis of two SLC9A6 frameshift variants in lymphoblastoid cells from patients with Christianson syndrome.

BACKGROUND: Christianson syndrome (CS) is caused by mutations in SLC9A6 and is characterized by global developmental delay, epilepsy, hyperkinesis, ataxia, microcephaly, and behavioral disorder. However, the molecular mechanism by which these SLC9A6 mutations cause CS in humans is not entirely understood, and there is no objective method to determine the pathogenicity of single SLC9A6 variants. METHODS: Trio-based whole exome sequencing (WES) was carried out on two individuals with suspicion of CS. qRT-PCR, western blot analysis, filipin staining, lysosomal enzymatic assays, and electron microscopy examination, using EBV-LCLs established from the two patients, were performed. RESULTS: Trio-based WES identified a hemizygous SLC9A6 c.1560dupT, p.T521Yfs*23 variant in proband 1 and a hemizygous SLC9A6 c.608delA, p.H203Lfs*10 variant in proband 2. Both children exhibited typical phenotypes associated with CS. Expression analysis in EBV-LCLs derived from the two patients showed a significant decrease in mRNA levels and no detectable normal NHE6 protein. EBV-LCLs showed a statistically significant increase in unesterified cholesterol in patient 1, but only non-significant increase in patient 2 when stained with filipin. Activities of lysosomal enzymes (ß-hexosaminidase A, ß-hexosaminidase A + B, ß-galactosidase, galactocerebrosidase, arylsulfatase A) of EBV-LCLs did not significantly differ between the two patients and six controls. Importantly, by electron microscopy we detected an accumulation of lamellated membrane structures, deformed mitochondria, and lipid droplets in the patients' EBV-LCLs. CONCLUSIONS: The SLC9A6 p.T521Yfs*23 and p.H203Lfs*10 variants in our patients result in loss of NHE6. Alterations of mitochondria and lipid metabolism may play a role in the pathogenesis of CS. Moreover, the combination of filipin staining with electron microscopy examination of patient lymphoblastoid cells can serve as a useful complementary diagnostic method for CS.

Structural Analysis Implicates CASK-Liprin-α2 Interaction in Cerebellar Granular Cell Death in MICPCH Syndrome.

Microcephaly with pontine and cerebellar hypoplasia (MICPCH) syndrome is a neurodevelopmental disorder caused by the deficiency of the X-chromosomal gene CASK. However, the molecular mechanisms by which CASK deficiency causes cerebellar hypoplasia in this syndrome remain elusive. In this study, we used CASK knockout (KO) mice as models for MICPCH syndrome and investigated the effect of CASK mutants. Female CASK heterozygote KO mice replicate the progressive cerebellar hypoplasia observed in MICPCH syndrome. CASK KO cultured cerebellar granule (CG) cells show progressive cell death that can be rescued by co-infection with lentivirus expressing wild-type CASK. Rescue experiments with CASK deletion mutants identify that the CaMK, PDZ, and SH3, but not L27 and guanylate kinase domains of CASK are required for the survival of CG cells. We identify missense mutations in the CaMK domain of CASK derived from human patients that fail to rescue the cell death of cultured CASK KO CG cells. Machine learning-based structural analysis using AlphaFold 2.2 predicts that these mutations disrupt the structure of the binding interface with Liprin-α2. These results suggest that the interaction with Liprin-α2 via the CaMK domain of CASK may be involved in the pathophysiology of cerebellar hypoplasia in MICPCH syndrome.

YIPF5 (p.W218R) mutation induced primary microcephaly in rabbits.

Primary microcephaly (PMCPH) is a rare autosomal recessive neurodevelopmental disorder with a global prevalence of PMCPH ranging from 0.0013% to 0.15%. Recently, a homozygous missense mutation in YIPF5 (p.W218R) was identified as a causative mutation of severe microcephaly. In this study, we constructed a rabbit PMCPH model harboring YIPF5 (p.W218R) mutation using SpRY-ABEmax mediated base substitution, which precisely recapitulated the typical symptoms of human PMCPH. Compared with wild-type controls, the mutant rabbits exhibited stunted growth, reduced head circumference, altered motor ability, and decreased survival rates. Further investigation based on model rabbit elucidated that altered YIPF5 function in cortical neurons could lead to endoplasmic reticulum stress and neurodevelopmental disorders, interference of the generation of apical progenitors (APs), the first generation of progenitors in the developing cortex. Furthermore, these YIPF5-mutant rabbits support a correlation between unfolded protein responses (UPR) induced by endoplasmic reticulum stress (ERS), and the development of PMCPH, thus providing a new perspective on the role of YIPF5 in human brain development and a theoretical basis for the differential diagnosis and clinical treatment of PMCPH. To our knowledge, this is the first gene-edited rabbit model of PMCPH. The model better mimics the clinical features of human microcephaly than the traditional mouse models. Hence, it provides great potential for understanding the pathogenesis and developing novel diagnostic and therapeutic approaches for PMCPH.

A Novel Variant in VPS13B Underlying Cohen Syndrome.

Pathogenic variants in vacuolar protein sorting 13 homolog B (VPS13B) cause Cohen syndrome (CS), a clinically diverse neurodevelopmental disorder. We used whole exome and Sanger sequencing to identify disease-causing variants in a Pakistani family with intellectual disability, microcephaly, facial dysmorphism, neutropenia, truncal obesity, speech delay, motor delay, and insomnia. We identified a novel homozygous nonsense variant c.8841G > A: p.(W2947∗) in VPS13B (NM_017890.5) which segregated with the disease. Sleep disturbances are commonly seen in neurodevelopmental disorders and can exacerbate medical issues if left untreated. We demonstrate that individuals with Cohen syndrome may also be affected by sleep disturbances. In conclusion, we expand the genetic and phenotypic features of Cohen syndrome in the Pakistani population.

A recurrent de novo variant in NUSAP1 escapes nonsense-mediated decay and leads to microcephaly, epilepsy, and developmental delay.

NUSAP1 encodes a cell cycle-dependent protein with key roles in mitotic progression, spindle formation, and microtubule stability. Both over- and under-expression of NUSAP1 lead to dysregulation of mitosis and impaired cell proliferation. Through exome sequencing and Matchmaker Exchange, we identified two unrelated individuals with the same recurrent, de novo heterozygous variant (NM_016359.5 c.1209C > A; p.(Tyr403Ter)) in NUSAP1. Both individuals had microcephaly, severe developmental delay, brain abnormalities, and seizures. The gene is predicted to be tolerant of heterozygous loss-of-function mutations, and we show that the mutant transcript escapes nonsense mediated decay, suggesting that the mechanism is likely dominant-negative or toxic gain of function. Single-cell RNA-sequencing of an affected individual's post-mortem brain tissue indicated that the NUSAP1 mutant brain contains all main cell lineages, and that the microcephaly could not be attributed to loss of a specific cell type. We hypothesize that pathogenic variants in NUSAP1 lead to microcephaly possibly by an underlying defect in neural progenitor cells.

A PUS7 gene pathogenic variant causing self-injurious behavior, sleep disturbances, and developmental delay: A case report.

PUS7 gene pathogenic variants cause a deficiency in an RNA-independent pseudouridine synthase, which results in a neurodevelopmental phenotype characterized by various degrees of psychomotor delay, acquired microcephaly, aggressive behavior, and intellectual disability. Since 2018, PUS7 deficiency has been described in 15 patients with different pathogenic variants but similar clinical phenotypes. We describe the case of a male infant with a homozygous truncating pathogenic variant in the PUS7 gene (c.329_332delCTGA; p.Thr110Argfs*4) who, in addition to the previously mentioned features, displays self-injurious behavior, sleep disturbances and motor stereotypies.