De novo missense variants in exon 9 of SEPHS1 cause a neurodevelopmental condition with developmental delay, poor growth, hypotonia, and dysmorphic features.

Selenophosphate synthetase (SEPHS) plays an essential role in selenium metabolism. Two mammalian SEPHS paralogues, SEPHS1 and SEPHS2, share high sequence identity and structural homology with SEPHS. Here, we report nine individuals from eight families with developmental delay, growth and feeding problems, hypotonia, and dysmorphic features, all with heterozygous missense variants in SEPHS1. Eight of these individuals had a recurrent variant at amino acid position 371 of SEPHS1 (p.Arg371Trp, p.Arg371Gln, and p.Arg371Gly); seven of these variants were known to be de novo. Structural modeling and biochemical assays were used to understand the effect of these variants on SEPHS1 function. We found that a variant at residue Trp352 results in local structural changes of the C-terminal region of SEPHS1 that decrease the overall thermal stability of the enzyme. In contrast, variants of a solvent-exposed residue Arg371 do not impact enzyme stability and folding but could modulate direct protein-protein interactions of SEPSH1 with cellular factors in promoting cell proliferation and development. In neuronal SH-SY5Y cells, we assessed the impact of SEPHS1 variants on cell proliferation and ROS production and investigated the mRNA expression levels of genes encoding stress-related selenoproteins. Our findings provided evidence that the identified SEPHS1 variants enhance cell proliferation by modulating ROS homeostasis. Our study supports the hypothesis that SEPHS1 plays a critical role during human development and provides a basis for further investigation into the molecular mechanisms employed by SEPHS1. Furthermore, our data suggest that variants in SEPHS1 are associated with a neurodevelopmental disorder.

Genotype-phenotype correlations in RHOBTB2-associated neurodevelopmental disorders.

PURPOSE: Missense variants clustering in the BTB domain region of RHOBTB2 cause a developmental and epileptic encephalopathy with early-onset seizures and severe intellectual disability. METHODS: By international collaboration, we assembled individuals with pathogenic RHOBTB2 variants and a variable spectrum of neurodevelopmental disorders. By western blotting, we investigated the consequences of missense variants in vitro. RESULTS: In accordance with previous observations, de novo heterozygous missense variants in the BTB domain region led to a severe developmental and epileptic encephalopathy in 16 individuals. Now, we also identified de novo missense variants in the GTPase domain in 6 individuals with apparently more variable neurodevelopmental phenotypes with or without epilepsy. In contrast to variants in the BTB domain region, variants in the GTPase domain do not impair proteasomal degradation of RHOBTB2 in vitro, indicating different functional consequences. Furthermore, we observed biallelic splice-site and truncating variants in 9 families with variable neurodevelopmental phenotypes, indicating that complete loss of RHOBTB2 is pathogenic as well. CONCLUSION: By identifying genotype-phenotype correlations regarding location and consequences of de novo missense variants in RHOBTB2 and by identifying biallelic truncating variants, we further delineate and expand the molecular and clinical spectrum of RHOBTB2-related phenotypes, including both autosomal dominant and recessive neurodevelopmental disorders.

De Novo Variants in the ATPase Module of MORC2 Cause a Neurodevelopmental Disorder with Growth Retardation and Variable Craniofacial Dysmorphism.

MORC2 encodes an ATPase that plays a role in chromatin remodeling, DNA repair, and transcriptional regulation. Heterozygous variants in MORC2 have been reported in individuals with autosomal-dominant Charcot-Marie-Tooth disease type 2Z and spinal muscular atrophy, and the onset of symptoms ranges from infancy to the second decade of life. Here, we present a cohort of 20 individuals referred for exome sequencing who harbor pathogenic variants in the ATPase module of MORC2. Individuals presented with a similar phenotype consisting of developmental delay, intellectual disability, growth retardation, microcephaly, and variable craniofacial dysmorphism. Weakness, hyporeflexia, and electrophysiologic abnormalities suggestive of neuropathy were frequently observed but were not the predominant feature. Five of 18 individuals for whom brain imaging was available had lesions reminiscent of those observed in Leigh syndrome, and five of six individuals who had dilated eye exams had retinal pigmentary abnormalities. Functional assays revealed that these MORC2 variants result in hyperactivation of epigenetic silencing by the HUSH complex, supporting their pathogenicity. The described set of morphological, growth, developmental, and neurological findings and medical concerns expands the spectrum of genetic disorders resulting from pathogenic variants in MORC2.

De novo, deleterious sequence variants that alter the transcriptional activity of the homeoprotein PBX1 are associated with intellectual disability and pleiotropic developmental defects.

We present eight patients with de novo, deleterious sequence variants in the PBX1 gene. PBX1 encodes a three amino acid loop extension (TALE) homeodomain transcription factor that forms multimeric complexes with TALE and HOX proteins to regulate target gene transcription during development. As previously reported, Pbx1 homozygous mutant mice (Pbx1-/-) develop malformations and hypoplasia or aplasia of multiple organs, including the craniofacial skeleton, ear, branchial arches, heart, lungs, diaphragm, gut, kidneys, and gonads. Clinical findings similar to those in Pbx mutant mice were observed in all patients with varying expressivity and severity, including external ear anomalies, abnormal branchial arch derivatives, heart malformations, diaphragmatic hernia, renal hypoplasia and ambiguous genitalia. All patients but one had developmental delays. Previously reported patients with congenital anomalies affecting the kidney and urinary tract exhibited deletions and loss of function variants in PBX1. The sequence variants in our cases included missense substitutions adjacent to the PBX1 homeodomain (p.Arg184Pro, p.Met224Lys, and p.Arg227Pro) or within the homeodomain (p.Arg234Pro, and p.Arg235Gln), whereas p.Ser262Glnfs*2, and p.Arg288* yielded truncated PBX1 proteins. Functional studies on five PBX1 sequence variants revealed perturbation of intrinsic, PBX-dependent transactivation ability and altered nuclear translocation, suggesting abnormal interactions between mutant PBX1 proteins and wild-type TALE or HOX cofactors. It is likely that the mutations directly affect the transcription of PBX1 target genes to impact embryonic development. We conclude that deleterious sequence variants in PBX1 cause intellectual disability and pleiotropic malformations resembling those in Pbx1 mutant mice, arguing for strong conservation of gene function between these two species.

Broadband infrared vibrational nano-spectroscopy using thermal blackbody radiation.

Infrared vibrational nano-spectroscopy based on scattering scanning near-field optical microscopy (s-SNOM) provides intrinsic chemical specificity with nanometer spatial resolution. Here we use incoherent infrared radiation from a 1400 K thermal blackbody emitter for broadband infrared (IR) nano-spectroscopy. With optimized interferometric heterodyne signal amplification we achieve few-monolayer sensitivity in phonon polariton spectroscopy and attomolar molecular vibrational spectroscopy. Near-field localization and nanoscale spatial resolution is demonstrated in imaging flakes of hexagonal boron nitride (hBN) and determination of its phonon polariton dispersion relation. The signal-to-noise ratio calculations and analysis for different samples and illumination sources provide a reference for irradiance requirements and the attainable near-field signal levels in s-SNOM in general. The use of a thermal emitter as an IR source thus opens s-SNOM for routine chemical FTIR nano-spectroscopy.

Perinatal features of the RASopathies: Noonan syndrome, cardiofaciocutaneous syndrome and Costello syndrome.

The RASopathies are a family of developmental disorders caused by heritable defects of the RAS/MAPK signaling pathway. While the postnatal presentation of this group of disorders is well known, the prenatal and neonatal findings are less widely recognized. We report on the perinatal presentation of 10 patients with Noonan syndrome (NS), nine with Cardiofaciocutaneous syndrome (CFCS) and three with Costello syndrome (CS), in conjunction with the results of a comprehensive literature review. The majority of perinatal findings in NS, CS, and CFCS are shared: polyhydramnios; prematurity; lymphatic dysplasia; macrosomia; relative macrocephaly; respiratory distress; hypotonia, as well as cardiac and renal anomalies. In contrast, fetal arrhythmia and neonatal hypoglycemia are relatively specific to CS. NS, CS, and CFCS should all be considered as a possible diagnosis in pregnancies with a normal karyotype and ultrasound findings of a RASopathy. Recognition of the common perinatal findings of these disorders should facilitate both their prenatal and neonatal diagnosis.

Loss of a 20S proteasome activator in Saccharomyces cerevisiae downregulates genes important for genomic integrity, increases DNA damage, and selectively sensitizes cells to agents with diverse mechanisms of action.

Cytoprotective functions of a 20S proteasome activator were investigated. Saccharomyces cerevisiae Blm10 and human 20S proteasome activator 200 (PA200) are homologs. Comparative genome-wide analyses of untreated diploid cells lacking Blm10 and growing at steady state at defined growth rates revealed downregulation of numerous genes required for accurate chromosome structure, assembly and repair, and upregulation of a specific subset of genes encoding protein-folding chaperones. Blm10 loss or truncation of the Ubp3/Blm3 deubiquitinating enzyme caused massive chromosomal damage and cell death in homozygous diploids after phleomycin treatments, indicating that Blm10 and Ubp3/Blm3 function to stabilize the genome and protect against cell death. Diploids lacking Blm10 also were sensitized to doxorubicin, hydroxyurea, 5-fluorouracil, rapamycin, hydrogen peroxide, methyl methanesulfonate, and calcofluor. Fluorescently tagged Blm10 localized in nuclei, with enhanced fluorescence after DNA replication. After DNA damage that caused a classic G2/M arrest, fluorescence remained diffuse, with evidence of nuclear fragmentation in some cells. Protective functions of Blm10 did not require the carboxyl-terminal region that makes close contact with 20S proteasomes, indicating that protection does not require this contact or the truncated Blm10 can interact with the proteasome apart from this region. Without its carboxyl-terminus, Blm10((-339aa)) localized to nuclei in untreated, nonproliferating (G(0)) cells, but not during G(1) S, G(2), and M. The results indicate Blm10 functions in protective mechanisms that include the machinery that assures proper assembly of chromosomes. These essential guardian functions have implications for ubiquitin-independent targeting in anticancer therapy. Targeting Blm10/PA200 together with one or more of the upregulated chaperones or a conventional treatment could be efficacious.

Microdeletion 9q22.3 syndrome includes metopic craniosynostosis, hydrocephalus, macrosomia, and developmental delay.

Basal cell nevus syndrome (BCNS), also known as Gorlin syndrome (OMIM #109400) is a well-described rare autosomal dominant condition due to haploinsufficiency of PTCH1. With the availability of comparative genomic hybridization arrays, increasing numbers of individuals with microdeletions involving this locus are being identified. We present 10 previously unreported individuals with 9q22.3 deletions that include PTCH1. While 7 of the 10 patients (7 females, 3 males) did not meet strict clinical criteria for BCNS at the time of molecular diagnosis, almost all of the patients were too young to exhibit many of the diagnostic features. A number of the patients exhibited metopic craniosynostosis, severe obstructive hydrocephalus, and macrosomia, which are not typically observed in BCNS. All individuals older than a few months of age also had developmental delays and/or intellectual disability. Only facial features typical of BCNS, except in those with prominent midforeheads secondary to metopic craniosynostosis, were shared among the 10 patients. The deletions in these individuals ranged from 352 kb to 20.5 Mb in size, the largest spanning 9q21.33 through 9q31.2. There was significant overlap of the deleted segments among most of the patients. The smallest common regions shared among the deletions were identified in order to localize putative candidate genes that are potentially responsible for each of the non-BCNS features. These were a 929 kb region for metopic craniosynostosis, a 1.08 Mb region for obstructive hydrocephalus, and a 1.84 Mb region for macrosomia. Additional studies are needed to further characterize the candidate genes within these regions.

Laterally attached kinetochores recruit the checkpoint protein Bub1, but satisfy the spindle checkpoint.

Kinetochore attachment to the ends of dynamic microtubules is a conserved feature of mitotic spindle organization that is thought to be critical for proper chromosome segregation. Although kinetochores have been described to transition from lateral to end-on attachments, the phase of lateral attachment has been difficult to study in yeast due to its transient nature. We have previously described a kinetochore mutant, DAM1-765, which exhibits lateral attachments and misregulation of microtubule length. Here we show that the misregulation of microtubule length in DAM1-765 cells occurs despite localization of microtubule associated proteins Bik1, Stu2, Cin8, and Kip3 to microtubules. DAM1-765 kinetochores recruit the spindle checkpoint protein Bub1, however Bub1 localization to DAM1-765 kinetochores is not sufficient to cause a cell cycle arrest. Interestingly, the DAM1-765 mutation rescues the temperature sensitivity of a biorientation-deficient ipl1-321 mutant, and DAM1-765 chromosome loss rates are similar to wild-type cells. The spindle checkpoint in DAM1-765 cells responds properly to unattached kinetochores created by nocodazole treatment and loss of tension caused by a cohesin mutant. Progression of DAM1-765 cells through mitosis therefore suggests that satisfaction of the checkpoint depends more highly on biorientation of sister kinetochores than on achievement of a specific interaction between kinetochores and microtubule plus ends.

Kip3, the yeast kinesin-8, is required for clustering of kinetochores at metaphase.

In Saccharomyces cerevisiae, chromosome congression clusters kinetochores on either side of the spindle equator at metaphase. Many organisms require one or more kinesin-8 molecular motors to achieve chromosome alignment. The yeast kinesin-8, Kip3, has been well studied in vitro but a role in chromosome congression has not been reported. We investigated Kip3's role in this process using semi-automated, quantitative fluorescence microscopy and time-lapse imaging and found that Kip3 is required for congression. Deletion of KIP3 increases inter-kinetochore distances and increases the variability in the position of sister kinetochores along the spindle axis during metaphase. Kip3 does not regulate spindle length and is not required for kinetochore-microtubule attachment. Instead, Kip3 clusters kinetochores on the metaphase spindle by tightly regulating kinetochore microtubule lengths.

An SMC-domain protein in fission yeast links telomeres to the meiotic centrosome.

Abnormal centrosomal structures similar to those occurring in human cancers are induced in fission yeast by overexpression of the pericentrin homolog Pcp1p. Analysis of abnormal Pcp1p-containing structures with quantitative mass spectrometry and isotope-coded affinity tags identified a coiled-coil, structural maintenance of chromosomes (SMC) domain protein. This protein, termed Ccq1p (coiled-coil protein quantitatively enriched), localizes with Taz1p to telomeres in normal vegetative cells. Fluorescence resonance energy transfer (FRET) measurements indicate that Ccq1p also interacts with centrosomal Pcp1p in mating pheromone-stimulated cells containing centrosomally clustered telomeres. We provide evidence that the Ccq1p-Pcp1p interaction, while essential for meiosis, is deleterious when forced to occur during vegetative growth. Cells lacking one ccq1 allele exhibit a loss-of-function phenotype including abnormally long cell length, chromosome segregation failure, telomeric shortening, and defective telomeric clustering during meiotic prophase. Our data indicate a mechanism underlying meiotic chromosomal bouquet formation and suggest a recruitment model for supernumerary centrosome toxicity.

Tissue-specific translation of murine branched-chain alpha-ketoacid dehydrogenase kinase mRNA is dependent upon an upstream open reading frame in the 5'-untranslated region.

The committed step in the pathway for leucine, isoleucine, and valine catabolism is catalyzed by branched-chain alpha-ketoacid dehydrogenase (BCKD). This multienzyme complex is itself regulated through reversible subunit phosphorylation by a specific kinase (BCKD-kinase). Although BCKD is present in the mitochondria of all mammalian cells, BCKD-kinase has a tissue-specific pattern of expression. Various experimental, nutritional, and hormonal conditions have been used to alter the expression of BCKD-kinase, yet little is known regarding the regulation of basal BCKD-kinase expression under normal conditions including the mechanism of its tissue specificity in any organism. Here we use tissue-derived cultured cells to explore the mechanisms used to control BCKD-kinase expression. Whereas the amount of BCKD-kinase protein is significantly higher in mitochondria from C2C12 myotubes than in BNL Cl.2 liver cells, gene transcription and stability of BCKD-kinase mRNA share similar properties in these two cell types. Our results show that the amount of protein synthesized is regulated at the level of translation of BCKD-kinase mRNA and that an upstream open reading frame in the 5'-untranslated region of this transcript controls its translation. The location and putative 19-residue peptide are conserved in the mouse, rat, chimpanzee, and human genes. Likewise, gene structure of mouse, chimpanzee, and human BCKD-kinase is conserved, whereas the rat gene has lost intron 9.