Bi-allelic variants in neuronal cell adhesion molecule cause a neurodevelopmental disorder characterized by developmental delay, hypotonia, neuropathy/spasticity.

Cell adhesion molecules are membrane-bound proteins predominantly expressed in the central nervous system along principal axonal pathways with key roles in nervous system development, neural cell differentiation and migration, axonal growth and guidance, myelination, and synapse formation. Here, we describe ten affected individuals with bi-allelic variants in the neuronal cell adhesion molecule NRCAM that lead to a neurodevelopmental syndrome of varying severity; the individuals are from eight families. This syndrome is characterized by developmental delay/intellectual disability, hypotonia, peripheral neuropathy, and/or spasticity. Computational analyses of NRCAM variants, many of which cluster in the third fibronectin type III (Fn-III) domain, strongly suggest a deleterious effect on NRCAM structure and function, including possible disruption of its interactions with other proteins. These findings are corroborated by previous in vitro studies of murine Nrcam-deficient cells, revealing abnormal neurite outgrowth, synaptogenesis, and formation of nodes of Ranvier on myelinated axons. Our studies on zebrafish nrcamaΔ mutants lacking the third Fn-III domain revealed that mutant larvae displayed significantly altered swimming behavior compared to wild-type larvae (p < 0.03). Moreover, nrcamaΔ mutants displayed a trend toward increased amounts of α-tubulin fibers in the dorsal telencephalon, demonstrating an alteration in white matter tracts and projections. Taken together, our study provides evidence that NRCAM disruption causes a variable form of a neurodevelopmental disorder and broadens the knowledge on the growing role of the cell adhesion molecule family in the nervous system.

Defective thyroid hormone transport to the brain leads to astroglial alterations.

Allan-Herndon-Dudley syndrome (AHDS) is a rare X-linked disorder that causes severe neurological damage, for which there is no effective treatment. AHDS is due to inactivating mutations in the thyroid hormone transporter MCT8 that impair the entry of thyroid hormones into the brain, resulting in cerebral hypothyroidism. However, the pathophysiology of AHDS is still not fully understood and this is essential to develop therapeutic strategies. Based on evidence suggesting that thyroid hormone deficit leads to alterations in astroglial cells, including gliosis, in this work, we have evaluated astroglial impairments in MCT8 deficiency by means of magnetic resonance imaging, histological, ultrastructural, and immunohistochemical techniques, and by mining available RNA sequencing outputs. Apparent diffusion coefficient (ADC) imaging values obtained from magnetic resonance imaging showed changes indicative of alterations in brain cytoarchitecture in MCT8-deficient patients (n = 11) compared to control subjects (n = 11). Astroglial alterations were confirmed by immunohistochemistry against astroglial markers in autopsy brain samples of an 11-year-old and a 30th gestational week MCT8-deficient subjects in comparison to brain samples from control subjects at similar ages. These findings were validated and further explored in a mouse model of AHDS. Our findings confirm changes in all the astroglial populations of the cerebral cortex in MCT8 deficiency that impact astrocytic metabolic and mitochondrial cellular respiration functions. These impairments arise early in brain development and persist at adult stages, revealing an abnormal distribution, density, morphology of cortical astrocytes, along with altered transcriptome, compatible with an astrogliosis-like phenotype at adult stages. We conclude that astrocytes are potential novel therapeutic targets in AHDS, and we propose ADC imaging as a tool to monitor the progression of neurological impairments and potential effects of treatments in MCT8 deficiency.

A Highly Selective Fluorescent Probe for Monitoring the Thyroid Hormone Transporter Activity in Mammalian Cells.

Monocarboxylate transporter 8 (MCT8) is a trans-membrane transporter, which mediates the cellular delivery of thyroid hormones, L-thyroxine (T4) and 3,5,3'-triiodo-L-thyronine (T3). In humans, the MCT8 protein is encoded by the SLC16A2 gene and mutations in the transporter cause a genetic neurological disorder known as Allan-Herndon-Dudley Syndrome (AHDS). MCT8 deficiency leads to impaired transport of thyroid hormones in the brain. Radiolabelled T4 and T3 or LC/MS-MS methods have been used to monitor the thyroid hormone uptake through MCT8. Herein, we developed a fluorescent based assay to monitor the thyroid hormone uptake through MCT8. A dansyl-based fluorescent probe having L-thyroxine moiety is found to be highly selective towards MCT8 in living cells. The high selectivity of the probe towards MCT8 can be attributed to the halogen bond-mediated recognition by the transporter protein. The presence of a free carboxylic acid group is essential for the specificity of the probe towards MCT8. Additionally, the selectivity of the probe for MCT8 is abolished upon esterification of the carboxylic group. Similarly, MCT8 does not recognize the probe when it contains a free amine group.

Connecting Versatile lncRNAs with Heterogeneous Nuclear Ribonucleoprotein K and Pathogenic Disorders.

Heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA-binding protein that regulates multiple biological processes, including paraspeckles formation and cellular signal transduction. Recently, hnRNPK has been shown to interact with SINE-derived nuclear RNA localization (SIRLOIN)-containing RNAs, and orchestrate nuclear enrichment and cellular functions of long noncoding RNAs (lncRNAs). hnRNPK-lncRNAs interaction is potentially implicated in various pathogenic disorders including tumorigenesis, and Kabuki-like, Au-Kline, and Okamoto syndromes.

NMIHBA results from hypomorphic PRUNE1 variants that lack short-chain exopolyphosphatase activity.

Neurodevelopmental disorder with microcephaly, hypotonia and variable brain anomalies (NMIHBA) is an autosomal recessive neurodevelopmental and neurodegenerative disorder characterized by global developmental delay and severe intellectual disability. Microcephaly, progressive cortical atrophy, cerebellar hypoplasia and delayed myelination are neurological hallmarks in affected individuals. NMIHBA is caused by biallelic variants in PRUNE1 encoding prune exopolyphosphatase 1. We provide in-depth clinical description of two affected siblings harboring compound heterozygous variant alleles, c.383G > A (p.Arg128Gln), c.520G > T (p.Gly174*) in PRUNE1. To gain insights into disease biology, we biochemically characterized missense variants within the conserved N-terminal aspartic acid-histidine-histidine (DHH) motif and provide evidence that they result in the destabilization of protein structure and/or loss of exopolyphosphatase activity. Genetic ablation of Prune1 results in midgestational lethality in mice, associated with perturbations to embryonic growth and vascular development. Our findings suggest that NMIHBA results from hypomorphic variant alleles in humans and underscore the potential key role of PRUNE1 exopolyphoshatase activity in neurodevelopment.

Recurrent De Novo NAHR Reciprocal Duplications in the ATAD3 Gene Cluster Cause a Neurogenetic Trait with Perturbed Cholesterol and Mitochondrial Metabolism.

Recent studies have identified both recessive and dominant forms of mitochondrial disease that result from ATAD3A variants. The recessive form includes subjects with biallelic deletions mediated by non-allelic homologous recombination. We report five unrelated neonates with a lethal metabolic disorder characterized by cardiomyopathy, corneal opacities, encephalopathy, hypotonia, and seizures in whom a monoallelic reciprocal duplication at the ATAD3 locus was identified. Analysis of the breakpoint junction fragment indicated that these 67 kb heterozygous duplications were likely mediated by non-allelic homologous recombination at regions of high sequence identity in ATAD3A exon 11 and ATAD3C exon 7. At the recombinant junction, the duplication allele produces a fusion gene derived from ATAD3A and ATAD3C, the protein product of which lacks key functional residues. Analysis of fibroblasts derived from two affected individuals shows that the fusion gene product is expressed and stable. These cells display perturbed cholesterol and mitochondrial DNA organization similar to that observed for individuals with severe ATAD3A deficiency. We hypothesize that the fusion protein acts through a dominant-negative mechanism to cause this fatal mitochondrial disorder. Our data delineate a molecular diagnosis for this disorder, extend the clinical spectrum associated with structural variation at the ATAD3 locus, and identify a third mutational mechanism for ATAD3 gene cluster variants. These results further affirm structural variant mutagenesis mechanisms in sporadic disease traits, emphasize the importance of copy number analysis in molecular genomic diagnosis, and highlight some of the challenges of detecting and interpreting clinically relevant rare gene rearrangements from next-generation sequencing data.

Movement disorders in MCT8 deficiency/Allan-Herndon-Dudley Syndrome.

BACKGROUND AND OBJECTIVES: MCT8 deficiency is a rare genetic leukoencephalopathy caused by a defect of thyroid hormone transport across cell membranes, particularly through blood brain barrier and into neural cells. It is characterized by a complex neurological presentation, signs of peripheral thyrotoxicosis and cerebral hypothyroidism. Movement disorders (MDs) have been frequently mentioned in this condition, but not systematically studied. METHODS: Each patient recruited was video-recorded during a routine outpatient visit according to a predefined protocol. The presence and the type of MDs were evaluated. The type of MD was blindly scored by two child neurologists experts in inherited white matter diseases and in MD. Dystonia was scored according to Burke-Fahn-Marsden Dystonia Rating Scale (BFMDRS). When more than one MD was present, the predominant one was scored. RESULTS: 27 patients were included through a multicenter collaboration. In many cases we saw a combination of different MDs. Hypokinesia was present in 25/27 patients and was the predominant MD in 19. It was often associated with hypomimia and global hypotonia. Dystonia was observed in 25/27 patients, however, in a minority of cases (5) it was deemed the predominant MD. In eleven patients, exaggerated startle reactions and/or other paroxysmal non-epileptic events were observed. CONCLUSION: MDs are frequent clinical features of MCT8 deficiency, possibly related to the important role of thyroid hormones in brain development and functioning of normal dopaminergic circuits of the basal ganglia. Dystonia is common, but usually mild to moderate in severity, while hypokinesia was the predominant MD in the majority of patients.

Homozygous mutation in the Neurofascin gene affecting the glial isoform of Neurofascin causes severe neurodevelopment disorder with hypotonia, amimia and areflexia.

The Neurofascins (NFASCs) are a family of proteins encoded by alternative transcripts of NFASC that cooperate in the assembly of the node of Ranvier in myelinated nerves. Differential expression of NFASC in neurons and glia presents a remarkable example of cell-type specific expression of protein isoforms with a common overall function. In mice there are three NFASC isoforms: Nfasc186 and Nfasc140, located in the axonal membrane at the node of Ranvier, and Nfasc155, a glial component of the paranodal axoglial junction. Nfasc186 and Nfasc155 are the major isoforms at mature nodes and paranodes, respectively. Conditional deletion of the glial isoform Nfasc155 in mice causes severe motor coordination defects and death at 16-17 days after birth. We describe a proband with severe congenital hypotonia, contractures of fingers and toes, and no reaction to touch or pain. Whole exome sequencing revealed a homozygous NFASC variant chr1:204953187-C>T (rs755160624). The variant creates a premature stop codon in 3 out of four NFASC human transcripts and is predicted to specifically eliminate Nfasc155 leaving neuronal Neurofascin intact. The selective absence of Nfasc155 and disruption of the paranodal junction was confirmed by an immunofluorescent study of skin biopsies from the patient versus control. We propose that the disease in our proband is the first reported example of genetic deficiency of glial Neurofascin isoforms in humans and that the severity of the condition reflects the importance of the Nfasc155 in forming paranodal axoglial junctions and in determining the structure and function of the node of Ranvier.

Activated Thiol Sepharose-based proteomic approach to quantify reversible protein oxidation.

Reactive oxygen species (ROS) can act as second messengers in various signaling pathways, and abnormal oxidation contributes to multiple diseases, including cancer. Detecting and quantifying protein oxidation is crucial for a detailed understanding of reduction-oxidation reaction (redox) signaling. We developed an Activated Thiol Sepharose-based proteomic (ATSP) approach to quantify reversible protein oxidation. ATSP can enrich H2O2-sensitive thiol peptides, which are more likely to contain reactive cysteines involved in redox signaling. We applied our approach to analyze hereditary leiomyomatosis and renal cell carcinoma (HLRCC), a type of kidney cancer that harbors fumarate hydratase (FH)-inactivating mutations and has elevated ROS levels. Multiple proteins were oxidized in FH-deficient cells, including many metabolic proteins such as the pyruvate kinase M2 isoform (PKM2). Treatment of HLRCC cells with dimethyl fumarate or PKM2 activators altered PKM2 oxidation levels. Finally, we found that ATSP could detect Src homology region 2 domain-containing phosphatase-2 and PKM2 oxidation in cells stimulated with platelet-derived growth factor. This newly developed redox proteomics workflow can detect reversible oxidation of reactive cysteines and can be employed to analyze multiple physiologic and pathologic conditions.-Xu, Y., Andrade, J., Ueberheide, B., Neel, B. G. Activated Thiol Sepharose-based proteomic approach to quantify reversible protein oxidation.

Further expanding the mutational spectrum and investigation of genotype-phenotype correlation in 3M syndrome.

3M syndrome is characterized by severe pre- and postnatal growth retardation, typical facial features, and normal intelligence. Homozygous or compound heterozygous mutations in either CUL7, OBSL1, or CCDC8 have been identified in the etiology so far. Clinical and molecular features of 24 patients (23 patients and a fetus) from 19 unrelated families with a clinical diagnosis of 3M syndrome were evaluated and genotype-phenotype correlations were investigated with the use of DNA sequencing, chromosomal microarray, and whole exome sequencing accordingly. A genetic etiology could be established in 20 patients (n = 20/24, 83%). Eleven distinct CUL7 or OBSL1 mutations, among which eight was novel, were identified in 18 patients (n = 18/24, 75%). Ten patients had CUL7 (n = 10/18, 56%) while eight had OBSL1 (n = 8/18, 44%) mutations. Birth weight and height standard deviation scores at admission were significantly (p < 0.05) lower in patients with CUL7 mutation compared to that of patients with OBSL1 mutation. Two patients with a similar phenotype had a de novo 20p13p deletion involving BMP2. No genetic etiology could be established in four patients (n = 4/28, 17%). This study yet represents the largest cohort of 3M syndrome patients from a single center in Turkey. Microdeletions involving BMP2 may cause a phenotype similar to 3M syndrome with some distinctive features. Larger cohort of patients are required to establish genotype-phenotype correlations in 3M syndrome.

PIGQ glycosylphosphatidylinositol-anchored protein deficiency: Characterizing the phenotype.

PIGQ (OMIM *605754) encodes phosphatidylinositol glycan biosynthesis class Q (PIGQ) and is required for proper functioning of an N-acetylglucosamine transferase complex in a similar manner to the more established PIGA, PIGC, and PIGH. There are two previous patients reported with homozygous and apparently deleterious PIGQ mutations. Here, we provide the first detailed clinical report of a patient with heterozygous deleterious mutations associated with glycosylphosphatidylinositol-anchored protein (GPI-AP) biosynthesis deficiency. Our patient died at 10 months of age. The rare skeletal findings in this disorder expand the differential diagnosis of long bone radiolucent lesions and sphenoid wing dysplasia. This clinical report describes a new and rare disorder-PIGQ GPI-AP biosynthesis deficiency syndrome.

Functional analysis of monocarboxylate transporter 8 mutations in Japanese Allan-Herndon-Dudley syndrome patients.

Monocarboxylate transporter 8 (MCT8) facilitates T3 uptake into cells. Mutations in MCT8 lead to Allan-Herndon-Dudley syndrome (AHDS), which is characterized by severe psychomotor retardation and abnormal thyroid hormone profile. Nine uncharacterized MCT8 mutations in Japanese patients with severe neurocognitive impairment and elevated serum T3 levels were studied regarding the transport of T3. Human MCT8 (hMCT8) function was studied in wild-type (WT) or mutant hMCT8-transfected human placental choriocarcinoma cells (JEG3) by visualizing the locations of the proteins in the cells, detecting specific proteins, and measuring T3 uptake. We identified 6 missense (p.Arg445Ser, p.Asp498Asn, p.Gly276Arg, p.Gly196Glu, p.Gly401Arg, and p.Gly312Arg), 2 frameshift (p.Arg355Profs*64 and p.Tyr550Serfs*17), and 1 deletion (p.Pro561del) mutation(s) in the hMCT8 gene. All patients exhibited clinical characteristics of AHDS with high free T3, low-normal free T4, and normal-elevated TSH levels. All tested mutants were expressed at the protein level, except p.Arg355Profs*64 and p.Tyr550Serfs*17, which were truncated, and were inactive in T3 uptake, excluding p.Arg445Ser and p.Pro561del mutants, compared with WT-hMCT8. Immunocytochemistry revealed plasma membrane localization of p.Arg445Ser and p.Pro561del mutants similar with WT-hMCT8. The other mutants failed to localize in significant amount(s) in the plasma membrane and instead localized in the cytoplasm. These data indicate that p.Arg445Ser and p.Pro561del mutants preserve residual function, whereas p.Asp498Asn, p.Gly276Arg, p.Gly196Glu, p.Gly401Arg, p.Gly312Arg, p.Arg355Profs*64, and p.Tyr550Serfs*17 mutants lack function. These findings suggest that the mutations in MCT8 cause loss of function by reducing protein expression, impairing trafficking of protein to plasma membrane, and disrupting substrate channel.

Actin capping protein CAPZB regulates cell morphology, differentiation, and neural crest migration in craniofacial morphogenesis†.

CAPZB is an actin-capping protein that caps the growing end of F-actin and modulates the cytoskeleton and tethers actin filaments to the Z-line of the sarcomere in muscles. Whole-genome sequencing was performed on a subject with micrognathia, cleft palate and hypotonia that harbored a de novo, balanced chromosomal translocation that disrupts the CAPZB gene. The function of capzb was analyzed in the zebrafish model. capzb(-/-) mutants exhibit both craniofacial and muscle defects that recapitulate the phenotypes observed in the human subject. Loss of capzb affects cell morphology, differentiation and neural crest migration. Differentiation of both myogenic stem cells and neural crest cells requires capzb. During palate morphogenesis, defective cranial neural crest cell migration in capzb(-/-) mutants results in loss of the median cell population, creating a cleft phenotype. capzb is also required for trunk neural crest migration, as evident from melanophores disorganization in capzb(-/-) mutants. In addition, capzb over-expression results in embryonic lethality. Therefore, proper capzb dosage is important during embryogenesis, and regulates both cell behavior and tissue morphogenesis.

From zebrafish to human: A comparative approach to elucidate the role of the thyroid hormone transporter MCT8 during brain development.

Monocarboxylate transporter 8 (MCT8) facilitates transmembrane transport of thyroid hormones (THs) ensuring their action on gene expression during vertebrate neurodevelopment. A loss of MCT8 in humans results in severe psychomotor deficits associated with the Allan-Herndon-Dudley Syndrome (AHDS). However, where and when exactly a lack of MCT8 causes the neurological manifestations remains unclear because of the varying expression pattern of MCT8 between specific brain regions and cells. Here, we elaborate on the animal models that have been generated to elucidate the mechanisms underlying MCT8-deficient brain development. The absence of a clear neurological phenotype in Mct8 knockout mice made it clear that a single species would not suffice. The evolutionary conservation of TH action on neurodevelopment as well as the components regulating TH signalling however offers the opportunity to answer different aspects of MCT8 function in brain development using different vertebrate species. Moreover, the plethora of tools for genome editing available today facilitates gene silencing in these animals as well. Studies in the recently generated mct8-deficient zebrafish and Mct8/Oatp1c1 double knockout mice have put forward the current paradigm of impaired TH uptake at the level of the blood-brain barrier during peri- and postnatal development as being the main pathophysiological mechanism of AHDS. RNAi vector-based, cell-specific induction of MCT8 knockdown in the chicken embryo points to an additional function of MCT8 at the level of the neural progenitors during early brain development. Future studies including also additional in vivo models like Xenopus or in vitro approaches such as induced pluripotent stem cells will continue to help unravelling the exact role of MCT8 in developmental events. In the end, this multispecies approach will lead to a unifying thesis regarding the cellular and molecular mechanisms responsible for the neurological phenotype in AHDS patients.

A homozygous potentially pathogenic variant in the PAXBP1 gene in a large family with global developmental delay and myopathic hypotonia.

PAX binding protein 1 (PAXBP1) is an adaptor protein linking the transcription factor PAX3 and PAX7 to the histone methylation machinery. PAXBP1 is a nuclear protein and its high expression is known in brain cerebellar hemisphere and cerebellum. Moreover, it is also found in abundance in muscle precursor cells that are involved in myogenesis and skeletal muscles formation. Whole genome SNP genotyping and exome sequencing in a family with distinct syndrome of global developmental delay and hypotonia mapped the disease locus to the chromosome 21q22.11 and identified a homozygous missense variant (c.1612C>T) in the PAXBP1 gene, respectively. This variant is predicted to change the highly conserved strongly basic arginine at position 538 in the PAX7 binding domain of PAXBP1 to a neutral cysteine (p.Arg538Cys) residue. Arg538 is highly conserved and the variant is predicted to be deleterious by variety of in silico tools. Furthermore, protein modeling studies showed that in the mutant protein (Cys538), the shorter cysteine is incapable of forming hydrogen bond with the side chain of nearby Asp517 due to its reduced size and lower polarizability. As a consequence, a slight local perturbation of the loop conformation in the PAX7 binding domain of the PAXBP1 protein was observed. Our findings suggest that the pathogenic variant in PAX binding protein underlies distinct syndrome of global developmental delay and myopathic hypotonia. This clinical report should prompt a search for mutations in PAXBP1 in patients presenting with developmental delay and hypotonia. Moreover, these results imply that establishment of PAXBP1 targets and its spatiotemporal interaction will help in understanding of development of cerebellar and will provide basis for developing therapeutic approaches.

Mutations in SLC25A22: hyperprolinaemia, vacuolated fibroblasts and presentation with developmental delay.

Mutations in SLC25A22 are known to cause neonatal epileptic encephalopathy and migrating partial seizures in infancy. Using whole exome sequencing we identified four novel SLC25A22 mutations in six children from three families. Five patients presented clinical features similar to those in the literature including hypotonia, refractory neonatal-onset seizures and developmental delay. However, the sixth patients presented atypically with isolated developmental delay, developing late-onset (absence) seizures only at 7 years of age. Abnormal metabolite levels have not been documented in the nine patients described previously. One patient in our series was referred to the metabolic clinic because of persistent hyperprolinaemia and another three had raised plasma proline when tested. Analysis of the post-prandial plasma amino acid response in one patient showed abnormally high concentrations of several amino acids. This suggested that, in the fed state, when amino acids are the preferred fuel for the liver, trans-deamination of amino acids requires transportation of glutamate into liver mitochondria by SLC25A22 for deamination by glutamate dehydrogenase; SLC25A22 is an important mitochondrial glutamate transporter in liver as well as in brain. Electron microscopy of patient fibroblasts demonstrated widespread vacuolation containing neutral and phospho-lipids as demonstrated by Oil Red O and Sudan Black tinctorial staining; this might be explained by impaired activity of the proline/pyrroline-5-carboxylate (P5C) shuttle if SLC25A22 transports pyrroline-5-carboxylate/glutamate-γ-semialdehyde as well as glutamate.

Motor hypertonia and lack of locomotor coordination in mutant mice lacking DSCAM.

Down syndrome cell adherence molecule (DSCAM) contributes to the normal establishment and maintenance of neural circuits. Whereas there is abundant literature regarding the role of DSCAM in the neural patterning of the mammalian retina, less is known about motor circuits. Recently, DSCAM mutation has been shown to impair bilateral motor coordination during respiration, thus causing death at birth. DSCAM mutants that survive through adulthood display a lack of locomotor endurance and coordination in the rotarod test, thus suggesting that the DSCAM mutation impairs motor control. We investigated the motor and locomotor functions of DSCAM(2J) mutant mice through a combination of anatomical, kinematic, force, and electromyographic recordings. With respect to wild-type mice, DSCAM(2J) mice displayed a longer swing phase with a limb hyperflexion at the expense of a shorter stance phase during locomotion. Furthermore, electromyographic activity in the flexor and extensor muscles was increased and coactivated over 20% of the step cycle over a wide range of walking speeds. In contrast to wild-type mice, which used lateral walk and trot at walking speed, DSCAM(2J) mice used preferentially less coordinated gaits, such as out-of-phase walk and pace. The neuromuscular junction and the contractile properties of muscles, as well as their muscle spindles, were normal, and no signs of motor rigidity or spasticity were observed during passive limb movements. Our study demonstrates that the DSCAM mutation induces dystonic hypertonia and a disruption of locomotor gaits.

Cohen syndrome is associated with major glycosylation defects.

Cohen syndrome (CS) is a rare autosomal recessive disorder with multisytemic clinical features due to mutations in the VPS13B gene, which has recently been described encoding a mandatory membrane protein involved in Golgi integrity. As the Golgi complex is the place where glycosylation of newly synthesized proteins occurs, we hypothesized that VPS13B deficiency, responsible of Golgi apparatus disturbance, could lead to glycosylation defects and/or mysfunction of this organelle, and thus be a cause of the main clinical manifestations of CS. The glycosylation status of CS serum proteins showed a very unusual pattern of glycosylation characterized by a significant accumulation of agalactosylated fucosylated structures as well as asialylated fucosylated structures demonstrating a major defect of glycan maturation in CS. However, CS transferrin and α1-AT profiles, two liver-derived proteins, were normal. We also showed that intercellular cell adhesion molecule 1 and LAMP-2, two highly glycosylated cellular proteins, presented an altered migration profile on SDS-PAGE in peripheral blood mononuclear cells from CS patients. RNA interference against VPS13B confirmed these glycosylation defects. Experiments with Brefeldin A demonstrated that intracellular retrograde cell trafficking was normal in CS fibroblasts. Furthermore, early endosomes were almost absent in these cells and lysosomes were abnormally enlarged, suggesting a crucial role of VPS13B in endosomal-lysosomal trafficking. Our work provides evidence that CS is associated to a tissue-specific major defect of glycosylation and endosomal-lysosomal trafficking defect, suggesting that this could be a new key element to decipher the mechanisms of CS physiopathology.

Hypomyelinating leukodystrophies - a molecular insight into the white matter pathology.

Hypomyelinating leukodystrophies (HLDs) are a group of neurodevelopmental disorders that affect proper formation of the myelin sheath in the central nervous system. They are characterized by developmental delay, hypotonia, spasticity, and variable intellectual disability. In the past various classification systems for HLDs have been used, based on imaging findings, clinical manifestation, and organelle-specific disorders. Here we present a molecular insight into HLDs based on a defect in specific gene engaged in myelination. We discuss recent findings on pathogenesis, clinical presentation, and imaging related to these disorders. We focus on HLDs that are in use in differential diagnostics of Pelizaeus-Merzbacher disease (PMD), with a special emphasis on Allan-Herndon-Dudley syndrome (AHDS), an X-linked condition with delayed myelination due to thyroid transport disturbances. On the background of previously published patients we describe a proband initially considered as presenting with a severe PMD, whose diagnosis of AHDS due to a novel nonsense SLC16A2 mutation unraveled two previously undiagnosed generations of affected males who died in infancy from unexplained reasons. Since AHDS is found to be a relatively frequent cause of X-linked intellectual disability, we emphasize the need for determining the whole thyroid profile especially in hypotonic males with a delay of psychomotor development.

Hypomyelination and developmental delay associated with VPS11 mutation in Ashkenazi-Jewish patients.

BACKGROUND: The genetic heterogeneity of developmental delay and cognitive impairment is vast. The endocytic network is essential for neural development and synaptic plasticity by regulating the sorting of numerous transmembrane proteins. Disruption of the pathway can lead to neuronal pathology. Endosomal biogenesis relies on two Rab proteins, Rab5 and Rab7, which bind to two hexameric tethering complexes, the endosomal class C core vacuole/endosome tethering complex (CORVET) and the late endosomal/lysosomal homotypic fusion and protein sorting complex (HOPS). Both complexes consist of four core proteins and differ by their specific Rab-binding proteins. OBJECTIVES: To identify the molecular basis of a neurological disease, which consists of global developmental stagnation at 3-8 months, increasing appendicular spasticity, truncal hypotonia and acquired microcephaly, with variable seizure disorder, accompanied by thin corpus callosum, paucity of white matter and delayed myelination in eight patients from four unrelated Ashkenazi-Jewish (AJ) families. METHODS: Exome analysis, homozygosity mapping and Mup1-GFP transport assay in mutant yeast. RESULTS: Homozygosity for a missense mutation, p.Cys846Gly, in one of the endosomal biogenesis core proteins, VPS11, was identified in all the patients. This was shown to be a founder mutation with a carrier frequency of 0.6% in the AJ population. The homologous yeast mutant had moderate impairment of fusion of the late endosome to the vacuole in Mup1-GFP transport assay. CONCLUSIONS: We speculate that in neuronal cells, impairment of fusion of the late endosome to the vacuole would attenuate the degradation of plasma membrane receptors, thereby underlying the progressive neuronal phenotype in our patients. The VPS11 p.Cys846Gly mutation should be added to the AJ carrier screening panel.