Truncating variants of the sterol recognition region of SHH cause hypertelorism phenotype rather than hypotelorism-holoprosencephaly.

Sonic hedgehog signaling molecule (SHH) is a key molecule in the cilia-mediated signaling pathway and a critical morphogen in embryogenesis. The association between loss-of-function variants of SHH and holoprosencephaly is well established. In mice experiments, reduced or increased signaling of SHH have been shown to be associated with narrowing or excessive expansion of the facial midline, respectively. Herein, we report two unrelated patients with de novo truncating variants of SHH presenting with hypertelorism rather than hypotelorism. The first patient was a 13-year-old girl. Her facial features included hypertelorism, strabismus, telecanthus, malocclusion, frontal bossing, and wide widow's peak. She had borderline developmental delay and agenesis of the corpus callosum. She had a nonsense variant of SHH: Chr7(GRCh38):g.155802987C > T, NM_000193.4:c.1302G > A, p.(Trp434*). The second patient was a 25-year-old girl. Her facial features included hypertelorism and wide widow's peak. She had developmental delay and agenesis of the corpus callosum. She had a frameshift variant of SHH: Chr7(GRCh38):g.155803072_155803074delCGGinsT, NM_000193.4:c.1215_1217delCCGinsA, p.(Asp405Glufs*92). The hypertelorism phenotype contrasts sharply with the prototypical hypotelorism-holoprosencephaly phenotype associated with loss-of-function of SHH. We concluded that a subset of truncating variants of SHH could be associated with hypertelorism rather than hypotelorism.

Biallelic loss of OTUD7A causes severe muscular hypotonia, intellectual disability, and seizures.

The heterozygous deletion of 15q13.3 is a recurrently observed microdeletion syndrome associated with a relatively mild phenotype including learning disability and language impairment. In contrast, the homozygous deletion of 15q13.3 is extremely rare and is associated with a much severer phenotype that includes epileptic encephalopathy, profound intellectual disability, and hypotonia. Which of the genes within the deleted interval is responsible for the more severe features when biallelically deleted is currently unknown. Here, we report a patient with profound hypotonia, severe intellectual disability, and seizures who had biallelic loss-of-function variants in OTUD7A: a 15q13.3 deletion including the OTUD7A locus, and a frameshift OTUD7A variant c.1125del, p.(Glu375Aspfs*11). Unexpectedly, both aberrations occurred de novo. Our experiment using Caenorhabditis elegans showed that worms carrying a corresponding homozygous variant in the homolog OTUB-2 exhibited weakened muscle contraction suggestive of aberrant neuromuscular transmission. We concluded that the biallelic complete loss of OTUD7A in humans represents a presumably new autosomal recessive disorder characterized by profound hypotonia, severe intellectual disability, and seizures.

R3HDM1 haploinsufficiency is associated with mild intellectual disability.

R3HDM1 (R3H domain containing 1) is an uncharacterized RNA-binding protein that is highly expressed in the human cerebral cortex. We report the first case of a 12-year-old Japanese male with haploinsufficiency of R3HDM1. He presented with mild intellectual disability (ID) and developmental delay. He had a pericentric inversion of 46,XY,inv(2)(p16.1q21.3)dn with breakpoints in intron 19 of R3HDM1 (2q21.3) and the intergenic region (2p16.1). The R3HDM1 levels in his lymphoblastoid cells were reduced to approximately half that of the healthy controls. However, the expression of MIR128-1, in intron 18 of R3HDM1, was not affected via the pericentric inversion. Knockdown of R3HDM1 in mouse embryonic hippocampal neurons suppressed dendritic growth and branching. Notably, the Database of Genomic Variants reported the case of a healthy control with a 488-kb deletion that included both R3HDM1 and MIR128-1. miR-128 has been reported to inhibit dendritic growth and branching in mouse brain neurons, which directly opposes the novel functions of R3HDM1. These findings suggest that deleting both R3HDM1 and MIR128-1 alleviates the symptoms of the disease caused by loss-of-function mutations in R3HDM1 only. Thus, haploinsufficiency of R3HDM1 in the patient may be the cause of the mild ID due to the genetic imbalance between R3HDM1 and MIR128-1.

Biallelic mutations in NALCN: Expanding the genotypic and phenotypic spectra of IHPRF1.

Loss-of function mutations in NALCN on chromosome 13q, a sodium leak channel that maintains baseline neuronal excitability, cause infantile hypotonia with psychomotor retardation and characteristic faces 1 (IHPRF1, OMIM #615419). Here, we document two individuals with early onset hypotonia with poor feeding and intellectual disability who were compatible with a diagnosis of IHPRF1. The two patients had bi-allelic mutations in NALCN through two different genetic mechanisms: Patient 1 had bi-allelic splice site mutations, that is c.1267-2A>G, derived from heterozygous parents, while Patient 2 had a partial maternal uniparental isodisomy that harbored a frameshift mutation, that is c.2022_2023delAT, in chromosome 13 that was detected through a dedicated algorithm for homozygosity data mapping in whole exome sequencing. The delineation of the exact pattern of inheritance provided vital information regarding the risk of recurrence. In animal models with Nalcn mutations, two behavioral phenotypes, that are, postnatal dyspnea and sleep disturbance, have been reported. Our observations of the two patients with postnatal dyspnea and one patient with sleep disturbance support an association between these two behavioral phenotypes and NALCN mutations in humans. The routine use of a detection algorithm for homozygosity data mapping might improve the diagnostic yields of next-generation sequencing.

Pathogenicity evaluation of variants of uncertain significance at exon-intron junction by splicing assay in patients with Mowat-Wilson syndrome.

High-throughput sequencing has identified vast numbers of variants in genetic disorders. However, the significance of variants at the exon-intron junction remains controversial. Even though most cases of Mowat-Wilson syndrome (MOWS) are caused by heterozygous loss-of-function variants in ZEB2, the pathogenicity of variants at exon-intron junction is often indeterminable. We identified four intronic variants in 5/173 patients with clinical suspicion for MOWS, and evaluated their pathogenicity by in vitro analyses. The minigene analysis showed that c.73+2T>G caused most of the transcripts skipping exon 2, while c.916+6T>G led to partial skipping of exon 7. No splicing abnormalities were detected in both c.917-21T>C and c.3067+6A>T. The minigene analysis reproduced the splicing observed in the blood cells of the patient with c.73+2T>G. The degree of the exon skipping was concordant with the severity of MOWS; while the patient with c.73+2T>G was typical MOWS, the patient with c.916+6T>G showed milder phenotype which has been seldom reported. Our results demonstrate that mRNA splicing assays using the minigenes are valuable for determining the clinical significance of intronic variants in patients with not only MOWS but also other genetic diseases with splicing aberrations and may explain atypical or milder cases, such as the current patient.

Atypical Sotos syndrome caused by a novel splice site variant.

Sotos syndrome is usually caused by haploinsufficiency of NSD1; it is characterized by overgrowth, craniofacial features, and learning disabilities. We describe a boy with Sotos syndrome caused by a splicing variant (c.4378+5G>A). The clinical manifestations included severe connective tissue involvement, including joint hypermobility, progressive scoliosis, pectus deformity, and skin hyperextensibility; no overgrowth was observed.

Successful treatment of drug-resistant status epilepticus in an adult patient with Mowat-Wilson syndrome: A case report.

Mowat-Wilson syndrome (MWS) is a rare genetic disorder characterized by intellectual disability, distinctive facial features, epilepsy, and multiple anomalies caused by heterozygous loss-of-function mutations in the zinc finger E-box-binding homeobox-2 gene (ZEB2). Treatment choice is very important as patients with MWS because patients sometimes develop drug-resistant epilepsy. Here, we report the case of a 45-year-old male patient with MWS who developed drug-resistant status epilepticus after a 26-years seizure-free period while taking multiple anti-seizure medications. He showed a characteristic magnetic resonance imaging finding with a focal lesion in his left thalamic pulvinar nucleus, a finding not previously reported in status epilepticus with MWS. We succeeded in controlling seizures in the patient after trying multiple new antiseizure drug combinations. These findings indicate that patients with MWS may develop drug-resistant status epilepticus with age, even after a long-term seizure-free period, which can be managed with anti-seizure medication. Therefore, careful monitoring of seizures is important for the treatment of people with MWS, even in patients who have not experienced seizures for a long time.

Mowat-Wilson syndrome: growth charts.

BACKGROUND: Mowat-Wilson syndrome (MWS; OMIM #235730) is a genetic condition caused by heterozygous mutations or deletions of the ZEB2 gene. It is characterized by moderate-severe intellectual disability, epilepsy, Hirschsprung disease and multiple organ malformations of which congenital heart defects and urogenital anomalies are the most frequent ones. To date, a clear description of the physical development of MWS patients does not exist. The aim of this study is to provide up-to-date growth charts specific for infants and children with MWS. Charts for males and females aged from 0 to 16 years were generated using a total of 2865 measurements from 99 MWS patients of different ancestries. All data were collected through extensive collaborations with the Italian MWS association (AIMW) and the MWS Foundation. The GAMLSS package for the R statistical computing software was used to model the growth charts. Height, weight, body mass index (BMI) and head circumference were compared to those from standard international growth charts for healthy children. RESULTS: In newborns, weight and length were distributed as in the general population, while head circumference was slightly smaller, with an average below the 30th centile. Up to the age of 7 years, weight and height distribution was shifted to slightly lower values than in the general population; after that, the difference increased further, with 50% of the affected children below the 5th centile of the general population. BMI distribution was similar to that of non-affected children until the age of 7 years, at which point values in MWS children increased with a less steep slope, particularly in males. Microcephaly was sometimes present at birth, but in most cases it developed gradually during infancy; many children had a small head circumference, between the 3rd and the 10th centile, rather than being truly microcephalic (at least 2 SD below the mean). Most patients were of slender build. CONCLUSIONS: These charts contribute to the understanding of the natural history of MWS and should assist pediatricians and other caregivers in providing optimal care to MWS individuals who show problems related to physical growth. This is the first study on growth in patients with MWS.

Involvement of T-type Ca2+ channels in the potentiation of synaptic and visual responses during the critical period in rat visual cortex.

Neocortical neuronal circuits are refined by experience during the critical period of early postnatal life. The shift of ocular dominance in the visual cortex following monocular deprivation has been intensively studied to unravel the mechanisms underlying the experience-dependent modification. Synaptic plasticity is considered to be involved in this process. We previously showed in layer 2/3 pyramidal neurons of rat visual cortex that low-frequency stimulation-induced long-term potentiation (LTP) at excitatory synapses, which requires the activation of Ni(2+)-sensitive (R-type or T-type) voltage-gated Ca(2+) channels (VGCCs) for induction, shared a similar age and experience dependence with ocular dominance plasticity. In this study, we examined whether this LTP is involved in ocular dominance plasticity. In visual cortical slices, LTP was blocked by mibefradil, kurtoxin and R-(-)-efonidipine, T-type VGCC blockers, but not by SNX-482, an R-type VGCC blocker, indicating that LTP induction requires T-type VGCC activation. Mibefradil did not affect synaptic transmission even at a dose about 30 times higher than that required for LTP blockade. Therefore, this drug was used to test the effect of T-type VGCC blockade on ocular dominance shift produced by 6 days of monocular deprivation during the critical period using visual evoked potentials (VEPs). Although this monocular deprivation commonly produced both depression of deprived eye responses and potentiation of nondeprived eye responses, only the former change occurred when mibefradil was infused into the visual cortex during monocular deprivation. Mibefradil infusion produced no acute effects on VEPs. These results suggest that T-type VGCC-dependent LTP contributes to the experience-dependent enhancement of visual responses.

Brain-derived neurotrophic factor-mediated retrograde signaling required for the induction of long-term potentiation at inhibitory synapses of visual cortical pyramidal neurons.

High-frequency stimulation (HFS) induces long-term potentiation (LTP) at inhibitory synapses of layer 5 pyramidal neurons in developing rat visual cortex. This LTP requires postsynaptic Ca2+ rise for induction, while the maintenance mechanism is present at the presynaptic site, suggesting presynaptic LTP expression and the necessity of retrograde signaling. We investigated whether the supposed signal is mediated by brain-derived neurotrophic factor (BDNF), which is expressed in pyramidal neurons but not inhibitory interneurons. LTP did not occur when HFS was applied in the presence of the Trk receptor tyrosine kinase inhibitor K252a in the perfusion medium. HFS produced LTP when bath application of K252a was started after HFS or when K252a was loaded into postsynaptic cells. LTP did not occur in the presence of TrkB-IgG scavenging BDNF or function-blocking anti-BDNF antibody in the medium. In cells loaded with the Ca2+ chelator BAPTA, the addition of BDNF to the medium enabled HFS to induce LTP without affecting baseline synaptic transmission. These results suggest that BDNF released from postsynaptic cells activates presynaptic TrkB, leading to LTP. Because BDNF, expressed activity dependently, regulates the maturation of cortical inhibition, inhibitory LTP may contribute to this developmental process, and hence experience-dependent functional maturation of visual cortex.

Clinical and genetic characterization of a patient with SOX5 haploinsufficiency caused by a de novo balanced reciprocal translocation.

Lamb-Shaffer syndrome (OMIM: 616803) is a neurodevelopmental disorder characterized by developmental delay, mild to moderate intellectual disability, speech delay, and mild characteristic facial appearance caused by SOX5 haploinsufficiency on chromosome 12p12.1. There are clinical variabilities among the patients with genomic alterations, such as intragenic deletions, a point mutation, and a chromosomal translocation of t(11;12)(p13;p12.1), in SOX5. We report herein a 5-year-old Japanese male with a de novo balanced reciprocal translocation t(12;20)(p12.1;p12.3) presenting a mild intellectual disability, speech delay, characteristic facial appearance, and autistic features. We determined the translocation breakpoints of the patient to be in intron 4 of SOX5 and the intergenic region in 20p12.3 via FISH and nucleotide sequence analyses. Thus, the present patient has SOX5 haploinsufficiency affecting 2 long forms of SOX5 and is the second reported case of Lamb-Shaffer syndrome caused by a de novo balanced reciprocal translocation. This report confirmed that haploinsufficiency of the 2 long forms of SOX5 presents common clinical features, including mild intellectual disability and autistic features, which could be useful for the clinical diagnosis of Lamb-Shaffer syndrome.

Distinctive facies, macrocephaly, and developmental delay are signs of a PTEN mutation in childhood.

BACKGROUND: Germline mutations of the PTEN gene are responsible for several PTEN hamartoma tumor syndromes. They are also implicated as a cause of macrocephaly and mild to severe developmental delay, regardless of the presence or absence of hamartomas in childhood. Nevertheless, because of limited information, the clinical features present during childhood in patients with a PTEN mutation are yet to be elucidated. METHODS: PTEN mutations were investigated by multiplex targeted sequencing of genomic DNA from 33 children with increased head circumference (>+2 SD) and developmental delay. The clinical features of all the patients with a PTEN mutation were abstracted by dysmorphologists. RESULTS: We have identified six children with a PTEN mutation. Clinical dissection of these six patients, in addition to patient reports in the literature, revealed distinctive facial features that included frontal bossing, dolichocephaly, horizontal eyebrows, and a depressed nasal bridge. Macrocephaly (+3.2 to +6.0 SD) was noticeable compared to their height (-0.8 to +2.1 SD), and the difference in the SD value of head circumference and height was more than 3 SD in all patients. CONCLUSION: The presence of distinctive facies, extreme macrocephaly with normal to mildly high stature, and developmental delay may be useful for identifying patients with a PTEN mutation in childhood. Early identification of patients with a PTEN mutation would help uncover the natural course of tumor development in this group of individuals who have a possible predisposition to cancer, and be important for the development of an optimal surveillance strategy.

The effect of rapamycin, NVP-BEZ235, aspirin, and metformin on PI3K/AKT/mTOR signaling pathway of PIK3CA-related overgrowth spectrum (PROS).

The phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR signaling pathway is critical for cellular growth and metabolism. Recently, mosaic or segmental overgrowth, a clinical condition caused by heterozygous somatic activating mutations in PIK3CA, was established as PIK3CA-related overgrowth spectrum (PROS). In this study, we report a Japanese female diagnosed with PROS, who presented with hyperplasia of the lower extremities, macrodactyly, multiple lipomatosis, and sparse hair. Sequencing and mutant allele frequency analysis of PIK3CA from affected tissues revealed that the patient had a heterozygous mosaic mutation (c.3140A>G [p.H1047R]) in PIK3CA and that there were higher mutant allele frequencies from samples with a larger amount of subcutaneous adipose tissue. We established two fibroblast cell lines from the patient, harboring high and low frequencies of the mosaic mutation, in which AKT and S6 showed higher level of phosphorylation compared with three control fibroblasts, indicating that PI3K/AKT/mTOR signaling is activated. We assessed the therapeutic effects of four compounds (rapamycin, NVP-BEZ235, aspirin, and metformin) on PI3K/AKT/mTOR signaling pathway and cell growth. All four compounds suppressed S6 phosphorylation and inhibited cell growth of the patient-derived fibroblast cell lines. However, only metformin mildly inhibited the growth of the control fibroblast cell lines. Since PROS is a congenital disorder, drugs for therapy should take into consideration the natural growth of children. Thus, metformin is a candidate drug for treating PROS in growing children.

Facilitation of low-frequency stimulation-induced long-term potentiation by endogenous noradrenaline and serotonin in developing rat visual cortex.

T-type Ca2+ channel-dependent long-term potentiation (LTP) occurs predominantly during the critical period of ocular dominance plasticity in rat and cat visual cortex. Noradrenaline and serotonin are known to facilitate ocular dominance plasticity. In this study using rat visual cortical slices, we tested whether this LTP is modulated by these neuromodulators in the same way as ocular dominance plasticity. Extracellular field potentials evoked by layer 4 stimulation were recorded from layer 2/3 and LTP was induced by low-frequency (2 Hz) stimulation continued for 15 min. The induction of LTP was suppressed by beta, but not alpha, adrenergic receptor antagonists. LTP induction was also inhibited by selective antagonists for 5-HT(1A) or 5-HT(2) receptors. In slices prepared from rats in which noradrenaline or serotonin was depleted by selective neurotoxins, the magnitude of LTP was significantly smaller compared with control slices. These results indicate that the same types of adrenergic and serotonergic receptors facilitate both LTP and ocular dominance plasticity, supporting our hypothesis that T-type Ca2+ channel-dependent LTP mediates experience-dependent enhancement of visual responses of cortical neurons during the critical period.

A novel Caspr mutation causes the shambling mouse phenotype by disrupting axoglial interactions of myelinated nerves.

The neurological mouse mutation shambling (shm) exhibits ataxia and hindlimb paresis. Positional cloning of shm showed that it encodes contactin-associated protein (Caspr), which is required for formation of the paranodal junction in myelinated nerves. The shm mutation is a TT insertion in the Caspr gene that results in a frame shift and a premature stop codon at the COOH-terminus. The truncated Caspr protein that is generated lacks the transmembrane and cytoplasmic domains. Here, we found that the nodal/paranodal axoplasm of shm mice lack paranodal junctions and contain large mitochondria and abnormal accumulations of cytoplasmic organelles that indicate altered axonal transport. Immunohistochemical analysis of mutant mice showed reduced expression of Caspr, contactin, and neurofascin 155, which are thought to form a protein complex in the paranodal region; protein 4.1B, however, was normally distributed. The mutant mice had aberrant localization of voltage-gated ion channels on the axolemma of nodal/paranodal regions. Electrophysiological analysis demonstrated that the velocity of saltatory conduction was reduced in sciatic nerves and that the visual response was attenuated in the primary visual cortex. These abnormalities likely contribute to the neurological phenotype of the mutant mice.