Diverse Clinical Phenotypes of CASK-Related Disorders and Multiple Functional Domains of CASK Protein.

CASK-related disorders are a form of rare X-linked neurological diseases and most of the patients are females. They are characterized by several symptoms, including microcephaly with pontine and cerebellar hypoplasia (MICPCH), epilepsy, congenital nystagmus, and neurodevelopmental disorders. Whole-genome sequencing has identified various mutations, including nonsense and missense mutations, from patients with CASK-related disorders, revealing correlations between specific mutations and clinical phenotypes. Notably, missense mutations associated with epilepsy and intellectual disability were found throughout the whole region of the CASK protein, while missense mutations related to microcephaly and MICPCH were restricted in certain domains. To investigate the pathophysiology of CASK-related disorders, research groups have employed diverse methods, including the generation of CASK knockout mice and the supplementation of CASK to rescue the phenotypes. These approaches have yielded valuable insights into the identification of functional domains of the CASK protein associated with a specific phenotype. Additionally, recent advancements in the AI-based prediction of protein structure, such as AlphaFold2, and the application of genome-editing techniques to generate CASK mutant mice carrying missense mutations from patients with CASK-related disorders, allow us to understand the pathophysiology of CASK-related disorders in more depth and to develop novel therapeutic methods for the fundamental treatment of CASK-related disorders.

Excessive Laughter-like Vocalizations, Microcephaly, and Translational Outcomes in the Ube3a Deletion Rat Model of Angelman Syndrome.

Angelman syndrome (AS) is a rare genetic neurodevelopmental disorder characterized by intellectual disabilities, motor and balance deficits, impaired communication, and a happy, excitable demeanor with frequent laughter. We sought to elucidate a preclinical outcome measure in male and female rats that addressed communication abnormalities of AS and other neurodevelopmental disorders in which communication is atypical and/or lack of speech is a core feature. We discovered, and herein report for the first time, excessive laughter-like 50 kHz ultrasonic emissions in the Ube3amat-/pat+ rat model of AS, which suggests an excitable, playful demeanor and elevated positive affect, similar to the demeanor of individuals with AS. Also in line with the AS phenotype, Ube3amat-/pat+ rats demonstrated aberrant social interactions with a novel partner, distinctive gait abnormalities, impaired cognition, an underlying LTP deficit, and profound reductions in brain volume. These unique, robust phenotypes provide advantages compared with currently available mouse models and will be highly valuable as outcome measures in the evaluation of therapies for AS.SIGNIFICANCE STATEMENT Angelman syndrome (AS) is a severe neurogenetic disorder for which there is no cure, despite decades of research using mouse models. This study used a recently developed rat model of AS to delineate disease-relevant outcome measures to facilitate therapeutic development. We found the rat to be a strong model of AS, offering several advantages over mouse models by exhibiting numerous AS-relevant phenotypes, including overabundant laughter-like vocalizations, reduced hippocampal LTP, and volumetric anomalies across the brain. These findings are unconfounded by detrimental motor abilities and background strain, issues plaguing mouse models. This rat model represents an important advancement in the field of AS, and the outcome metrics reported herein will be central to the therapeutic pipeline.

Impaired eIF5A function causes a Mendelian disorder that is partially rescued in model systems by spermidine.

The structure of proline prevents it from adopting an optimal position for rapid protein synthesis. Poly-proline-tract (PPT) associated ribosomal stalling is resolved by highly conserved eIF5A, the only protein to contain the amino acid hypusine. We show that de novo heterozygous EIF5A variants cause a disorder characterized by variable combinations of developmental delay, microcephaly, micrognathia and dysmorphism. Yeast growth assays, polysome profiling, total/hypusinated eIF5A levels and PPT-reporters studies reveal that the variants impair eIF5A function, reduce eIF5A-ribosome interactions and impair the synthesis of PPT-containing proteins. Supplementation with 1 mM spermidine partially corrects the yeast growth defects, improves the polysome profiles and restores expression of PPT reporters. In zebrafish, knockdown eif5a partly recapitulates the human phenotype that can be rescued with 1 µM spermidine supplementation. In summary, we uncover the role of eIF5A in human development and disease, demonstrate the mechanistic complexity of EIF5A-related disorder and raise possibilities for its treatment.

The Yin and Yang of Autosomal Recessive Primary Microcephaly Genes: Insights from Neurogenesis and Carcinogenesis.

The stem cells of neurogenesis and carcinogenesis share many properties, including proliferative rate, an extensive replicative potential, the potential to generate different cell types of a given tissue, and an ability to independently migrate to a damaged area. This is also evidenced by the common molecular principles regulating key processes associated with cell division and apoptosis. Autosomal recessive primary microcephaly (MCPH) is a neurogenic mitotic disorder that is characterized by decreased brain size and mental retardation. Until now, a total of 25 genes have been identified that are known to be associated with MCPH. The inactivation (yin) of most MCPH genes leads to neurogenesis defects, while the upregulation (yang) of some MCPH genes is associated with different kinds of carcinogenesis. Here, we try to summarize the roles of MCPH genes in these two diseases and explore the underlying mechanisms, which will help us to explore new, attractive approaches to targeting tumor cells that are resistant to the current therapies.

CDK5RAP2 primary microcephaly is associated with hypothalamic, retinal and cochlear developmental defects.

BACKGROUND: Primary hereditary microcephaly (MCPH) comprises a large group of autosomal recessive disorders mainly affecting cortical development and resulting in a congenital impairment of brain growth. Despite the identification of >25 causal genes so far, it remains a challenge to distinguish between different MCPH forms at the clinical level. METHODS: 7 patients with newly identified mutations in CDK5RAP2 (MCPH3) were investigated by performing prospective, extensive and systematic clinical, MRI, psychomotor, neurosensory and cognitive examinations under similar conditions. RESULTS: All patients displayed neurosensory defects in addition to microcephaly. Small cochlea with incomplete partition type II was found in all cases and was associated with progressive deafness in 4 of them. Furthermore, the CDK5RAP2 protein was specifically identified in the developing cochlea from human fetal tissues. Microphthalmia was also present in all patients along with retinal pigmentation changes and lipofuscin deposits. Finally, hypothalamic anomalies consisting of interhypothalamic adhesions, a congenital midline defect usually associated with holoprosencephaly, was detected in 5 cases. CONCLUSION: This is the first report indicating that CDK5RAP2 not only governs brain size but also plays a role in ocular and cochlear development and is necessary for hypothalamic nuclear separation at the midline. Our data indicate that CDK5RAP2 should be considered as a potential gene associated with deafness and forme fruste of holoprosencephaly. These children should be given neurosensory follow-up to prevent additional comorbidities and allow them reaching their full educational potential. TRIAL REGISTRATION NUMBER: NCT01565005.

Functional analysis of the third identified SLC25A19 mutation causative for the thiamine metabolism dysfunction syndrome 4.

Thiamine metabolism dysfunction syndrome-4 (THMD4) includes episodic encephalopathy, often associated with a febrile illness, causing transient neurologic dysfunction and a slowly progressive axonal polyneuropathy. Until now only two mutations (G125S and S194P) have been reported in the SLC25A19 gene as causative for this disease and a third mutation (G177A) as related to the Amish lethal microcephaly. In this work, we describe the clinical and molecular features of a patient carrying a novel mutation (c.576G>C; Q192H) on SLC25A19 gene. Functional studies on this mutation were performed explaining the pathogenetic role of c.576G>C in affecting the translational efficiency and/or stability of hMTPPT protein instead of the mRNA expression. These findings support the pathogenetic role of Q192H (c.576G>C) mutation on SLC25A19 gene. Moreover, despite in other patients the thiamine supplementation leaded to a substantial improvement of peripheral neuropathy, our patient did not show a clinical improvement.

Homozygous mutation in MFSD2A, encoding a lysolipid transporter for docosahexanoic acid, is associated with microcephaly and hypomyelination.

The major facilitator superfamily domain-containing protein 2A (MFSD2A) is a constituent of the blood-brain barrier and functions to transport lysophosphatidylcholines (LPCs) into the central nervous system. LPCs such as that derived from docosahexanoic acid (DHA) are indispensable to neurogenesis and maintenance of neurons, yet cannot be synthesized within the brain and are dependent on MFSD2A for brain uptake. Recent studies have implicated MFSD2A mutations in lethal and non-lethal microcephaly syndromes, with the severity correlating to the residual activity of the transporter. We describe two siblings with shared parental ancestry, in whom we identified a homozygous missense mutation (c.1205C > A; p.Pro402His) in MFSD2A. Both affected individuals had microcephaly, hypotonia, appendicular spasticity, dystonia, strabismus, and global developmental delay. Neuroimaging revealed paucity of white matter with enlarged lateral ventricles. Plasma lysophosphatidylcholine (LPC) levels were elevated, reflecting reduced brain transport. Cell-based studies of the p.Pro402His mutant protein indicated complete loss of activity of the transporter despite the non-lethal, attenuated phenotype. The aggregate data of MFSD2A-associated genotypes and phenotypes suggest that additional factors, such as nutritional supplementation or modifying genetic factors, may modulate the severity of disease and call for consideration of treatment options for affected individuals.

A partially inactivating mutation in the sodium-dependent lysophosphatidylcholine transporter MFSD2A causes a non-lethal microcephaly syndrome.

The major pathway by which the brain obtains essential omega-3 fatty acids from the circulation is through a sodium-dependent lysophosphatidylcholine (LPC) transporter (MFSD2A), expressed in the endothelium of the blood-brain barrier. Here we show that a homozygous mutation affecting a highly conserved MFSD2A residue (p.Ser339Leu) is associated with a progressive microcephaly syndrome characterized by intellectual disability, spasticity and absent speech. We show that the p.Ser339Leu alteration does not affect protein or cell surface expression but rather significantly reduces, although not completely abolishes, transporter activity. Notably, affected individuals displayed significantly increased plasma concentrations of LPCs containing mono- and polyunsaturated fatty acyl chains, indicative of reduced brain uptake, confirming the specificity of MFSD2A for LPCs having mono- and polyunsaturated fatty acyl chains. Together, these findings indicate an essential role for LPCs in human brain development and function and provide the first description of disease associated with aberrant brain LPC transport in humans.

Inactivating mutations in MFSD2A, required for omega-3 fatty acid transport in brain, cause a lethal microcephaly syndrome.

Docosahexanoic acid (DHA) is the most abundant omega-3 fatty acid in brain, and, although it is considered essential, deficiency has not been linked to disease. Despite the large mass of DHA in phospholipids, the brain does not synthesize it. DHA is imported across the blood-brain barrier (BBB) through the major facilitator superfamily domain-containing 2a (MFSD2A) protein. MFSD2A transports DHA as well as other fatty acids in the form of lysophosphatidylcholine (LPC). We identify two families displaying MFSD2A mutations in conserved residues. Affected individuals exhibited a lethal microcephaly syndrome linked to inadequate uptake of LPC lipids. The MFSD2A mutations impaired transport activity in a cell-based assay. Moreover, when expressed in mfsd2aa-morphant zebrafish, mutants failed to rescue microcephaly, BBB breakdown and lethality. Our results establish a link between transport of DHA and LPCs by MFSD2A and human brain growth and function, presenting the first evidence of monogenic disease related to transport of DHA in humans.

SIP1 expression patterns in brain investigated by generating a SIP1-EGFP reporter knock-in mouse.

A loss of function of SIP1 (Smad interacting protein 1) in the mouse as well as in human of Mowat-Wilson syndrome results in severe and multiple defects in neural tissue development, especially in the brain. However, no detailed expression analysis of SIP1 during brain development has been previously reported. In this study, we describe the generation of an EGFP knock-in reporter mouse for the Sip1 locus and our subsequent analysis of SIP1-EGFP fusion protein expression during brain development. SIP1-EGFP expression was observed in the pyramidal neurons of the hippocampus, the dentate gyrus, and the postmitotic neurons in the cerebral cortex. In layer 5 of the cerebral cortex, SIP1-EGFP expression was complementary to the Ctip2-expressing neurons, most of which are thought to be the cortico-spinal neurons. This suggested that SIP1-EGFP expressing cells might have the specific trajectory targets other than the spinal region. We further observed SIP1-EGFP expression in oligodendrocytes of the corpus callosum and fimbria, Bergmann glial cells of the cerebellum, the olfactory bulb, and in the serotonergic and dopaminergic neurons of the raphe nuclei in the brainstem. These findings may help to clarify the unknown roles of SIP1 in these cells and the pathoetiology of Mowat-Wilson syndrome.

ARNT2 mutation causes hypopituitarism, post-natal microcephaly, visual and renal anomalies.

We describe a previously unreported syndrome characterized by secondary (post-natal) microcephaly with fronto-temporal lobe hypoplasia, multiple pituitary hormone deficiency, seizures, severe visual impairment and abnormalities of the kidneys and urinary tract in a highly consanguineous family with six affected children. Homozygosity mapping and exome sequencing revealed a novel homozygous frameshift mutation in the basic helix-loop-helix transcription factor gene ARNT2 (c.1373_1374dupTC) in affected individuals. This mutation results in absence of detectable levels of ARNT2 transcript and protein from patient fibroblasts compared with controls, consistent with nonsense-mediated decay of the mutant transcript and loss of ARNT2 function. We also show expression of ARNT2 within the central nervous system, including the hypothalamus, as well as the renal tract during human embryonic development. The progressive neurological abnormalities, congenital hypopituitarism and post-retinal visual pathway dysfunction in affected individuals demonstrates for the first time the essential role of ARNT2 in the development of the hypothalamo-pituitary axis, post-natal brain growth, and visual and renal function in humans.

A variable degree of intrauterine and postnatal growth retardation in a family with a missense mutation in the insulin-like growth factor I receptor.

CONTEXT: The type 1 IGF-I receptor (IGF1R) mediates the biological functions of IGF-I. Binding of IGF-I to the IGF1R results in autophosphorylation of the intracellular beta-subunit and activation of intracellular signaling. OBJECTIVE: The objective of this study was to evaluate the functional characteristics of a novel IGF1R mutation and describe the phenotypic features of two patients with this mutation. DESIGN: The study was performed in a university hospital. PATIENTS: We describe a 35-yr-old female with mild intrauterine growth failure, progressive postnatal growth retardation, severe failure to thrive, and microcephaly. Her daughter was born with severe intrauterine growth retardation and also showed postnatal failure to thrive and microcephaly. RESULTS: We found a heterozygous G3148-->A nucleotide substitution in the IGF1R gene, changing a negatively charged glutamic acid at position 1050 into a positively charged lysine residue (E1050K). E1050 is a conserved residue in the intracellular kinase domain. Dermal fibroblasts of the mother showed normal binding of iodinated IGF-I, but autophosphorylation and activation of downstream signaling cascades upon challenging with IGF-I was markedly reduced. Consequently, the maximal [(3)H]thymidine incorporation upon challenge with a dose range of IGF-I was reduced compared with a panel of control cells (3.65 +/- 1.79-fold vs. 6.75 +/- 4.7-fold stimulation; P < 0.01). These data suggest that the mutation results in the inactivation of one copy of the IGF1R gene. CONCLUSIONS: These two patients support the key role for IGF-I in intrauterine and postnatal growth. The different phenotypes of these and earlier described patients may be associated with variability in IGF-I signaling. The degree of intrauterine growth retardation may be partially determined by the presence or absence of maternal IGF-I resistance.

Congenital microcephaly and seizures due to 3-phosphoglycerate dehydrogenase deficiency: outcome of treatment with amino acids.

Congenital microcephaly, intractable seizures and severe psychomotor retardation characterize 3-phosphoglycerate dehydrogenase (3-PGDH) deficiency, a disorder of L-serine biosynthesis. The enzyme defect results in low concentrations of serine and to a variable degree of glycine in plasma and cerebrospinal fluid. Short-term beneficial effects have been reported of oral treatment with the deficient amino acids. In this paper, we report the first follow-up data of amino acid therapy in five patients treated for 3-7.5 years. Different treatment regimes were used, but a favourable response to amino acids was observed in all patients. A major reduction in seizure frequency occurred in all patients; two patients became free of seizures. Amino acids were well tolerated and no adverse effects were documented. A progress of psychomotor development was only observed in one patient, diagnosed early and treated with a high dosage of L-serine. A favourable outcome of 3-PGDH deficiency depends on early diagnosis and treatment.