p21-activated kinases (PAKs) are serine/threonine protein kinases acting as effectors of CDC42 and RAC, which are members of the RHO family of small GTPases. PAK1’s kinase activity is autoinhibited ...by homodimerization, whereas CDC42 or RAC1 binding causes PAK1 activation by dimer dissociation. Major functions of the PAKs include actin cytoskeleton reorganization, for example regulation of the cellular protruding activity during cell spreading. We report the de novo PAK1 mutations c.392A>G (p.Tyr131Cys) and c.1286A>G (p.Tyr429Cys) in two unrelated subjects with developmental delay, secondary macrocephaly, seizures, and ataxic gait. We identified enhanced phosphorylation of the PAK1 targets JNK and AKT in fibroblasts of one subject and of c-JUN in those of both subjects compared with control subjects. In fibroblasts of the two affected individuals, we observed a trend toward enhanced PAK1 kinase activity. By using co-immunoprecipitation and size-exclusion chromatography, we observed a significantly reduced dimerization for both PAK1 mutants compared with wild-type PAK1. These data demonstrate that the two PAK1 variants function as activating alleles. In a cell spreading assay, subject-derived fibroblasts showed significant enrichment in cells occupied by filopodia. Interestingly, application of the PAK1 inhibitor FRAX486 completely reversed this cellular phenotype. Together, our data reveal that dominantly acting, gain-of-function PAK1 mutations cause a neurodevelopmental phenotype with increased head circumference, possibly by a combined effect of defective homodimerization and enhanced kinase activity of PAK1. This condition, along with the developmental disorders associated with RAC1 and CDC42 missense mutations, highlight the importance of RHO GTPase members and effectors in neuronal development.
Zimmermann-Laband syndrome (ZLS) is a developmental disorder characterized by facial dysmorphism with gingival enlargement, intellectual disability, hypoplasia or aplasia of nails and terminal ...phalanges, and hypertrichosis. We report that heterozygous missense mutations in KCNH1 account for a considerable proportion of ZLS. KCNH1 encodes the voltage-gated K(+) channel Eag1 (Kv10.1). Patch-clamp recordings showed strong negative shifts in voltage-dependent activation for all but one KCNH1 channel mutant (Gly469Arg). Coexpression of Gly469Arg with wild-type KCNH1 resulted in heterotetrameric channels with reduced conductance at positive potentials but pronounced conductance at negative potentials. These data support a gain-of-function effect for all ZLS-associated KCNH1 mutants. We also identified a recurrent de novo missense change in ATP6V1B2, encoding the B2 subunit of the multimeric vacuolar H(+) ATPase, in two individuals with ZLS. Structural analysis predicts a perturbing effect of the mutation on complex assembly. Our findings demonstrate that KCNH1 mutations cause ZLS and document genetic heterogeneity for this disorder.
N-methyl-D-aspartate (NMDA) receptors mediate excitatory neurotransmission in the mammalian brain. Two glycine-binding NR1 subunits and two glutamate-binding NR2 subunits each form highly ...Ca2+-permeable cation channels which are blocked by extracellular Mg2+ in a voltage-dependent manner. Either GRIN2B or GRIN2A, encoding the NMDA receptor subunits NR2B and NR2A, was found to be disrupted by chromosome translocation breakpoints in individuals with mental retardation and/or epilepsy. Sequencing of GRIN2B in 468 individuals with mental retardation revealed four de novo mutations: a frameshift, a missense and two splice-site mutations. In another cohort of 127 individuals with idiopathic epilepsy and/or mental retardation, we discovered a GRIN2A nonsense mutation in a three-generation family. In a girl with early-onset epileptic encephalopathy, we identified the de novo GRIN2A mutation c.1845C>A predicting the amino acid substitution p.N615K. Analysis of NR1-NR2AN615K (NR2A subunit with the p.N615K alteration) receptor currents revealed a loss of the Mg2+ block and a decrease in Ca2+ permeability. Our findings suggest that disturbances in the neuronal electrophysiological balance during development result in variable neurological phenotypes depending on which NR2 subunit of NMDA receptors is affected.
The sodium (Na
):multivitamin transporter (SMVT), encoded by SLC5A6, belongs to the sodium:solute symporter family and is required for the Na
-dependent uptake of biotin (vitamin B7), pantothenic ...acid (vitamin B5), the vitamin-like substance α-lipoic acid, and iodide. Compound heterozygous SLC5A6 variants have been reported in individuals with variable multisystemic disorder, including failure to thrive, developmental delay, seizures, cerebral palsy, brain atrophy, gastrointestinal problems, immunodeficiency, and/or osteopenia. We expand the phenotypic spectrum associated with biallelic SLC5A6 variants affecting function by reporting five individuals from three families with motor neuropathies. We identified the homozygous variant c.1285 A > G p.(Ser429Gly) in three affected siblings and a simplex patient and the maternally inherited c.280 C > T p.(Arg94*) variant and the paternally inherited c.485 A > G p.(Tyr162Cys) variant in the simplex patient of the third family. Both missense variants were predicted to affect function by in silico tools. 3D homology modeling of the human SMVT revealed 13 transmembrane helices (TMs) and Tyr162 and Ser429 to be located at the cytoplasmic facing region of TM4 and within TM11, respectively. The SLC5A6 missense variants p.(Tyr162Cys) and p.(Ser429Gly) did not affect plasma membrane localization of the ectopically expressed multivitamin transporter suggesting reduced but not abolished function, such as lower catalytic activity. Targeted therapeutic intervention yielded clinical improvement in four of the five patients. Early molecular diagnosis by exome sequencing is essential for timely replacement therapy in affected individuals.
N-myristoylation is a common form of co-translational protein fatty acylation resulting from the attachment of myristate to a required N-terminal glycine residue. We show that aberrantly acquired ...N-myristoylation of SHOC2, a leucine-rich repeat-containing protein that positively modulates RAS-MAPK signal flow, underlies a clinically distinctive condition of the neuro-cardio-facial-cutaneous disorders family. Twenty-five subjects with a relatively consistent phenotype previously termed Noonan-like syndrome with loose anagen hair (MIM607721) shared the 4A>G missense change in SHOC2 (producing an S2G amino acid substitution) that introduces an N-myristoylation site, resulting in aberrant targeting of SHOC2 to the plasma membrane and impaired translocation to the nucleus upon growth factor stimulation. Expression of SHOC2S2G in vitro enhanced MAPK activation in a cell type-specific fashion. Induction of SHOC2S2G in Caenorhabditis elegans engendered protruding vulva, a neomorphic phenotype previously associated with aberrant signaling. These results document the first example of an acquired N-terminal lipid modification of a protein causing human disease.
P/Q-type channels are the principal presynaptic calcium channels in brain functioning in neurotransmitter release. They are composed of the pore-forming CaV2.1 α1 subunit and the auxiliary α2δ-2 and ...β4 subunits. β4 is encoded by CACNB4, and its multiple splice variants serve isoform-specific functions as channel subunits and transcriptional regulators in the nucleus. In two siblings with intellectual disability, psychomotor retardation, blindness, epilepsy, movement disorder and cerebellar atrophy we identified rare homozygous variants in the genes LTBP1, EMILIN1, CACNB4, MINAR1, DHX38 and MYO15 by whole-exome sequencing. In silico tools, animal model, clinical, and genetic data suggest the p.(Leu126Pro) CACNB4 variant to be likely pathogenic. To investigate the functional consequences of the CACNB4 variant, we introduced the corresponding mutation L125P into rat β4b cDNA. Heterologously expressed wild-type β4b associated with GFP-CaV1.2 and accumulated in presynaptic boutons of cultured hippocampal neurons. In contrast, the β4b-L125P mutant failed to incorporate into calcium channel complexes and to cluster presynaptically. When co-expressed with CaV2.1 in tsA201 cells, β4b and β4b-L125P augmented the calcium current amplitudes, however, β4b-L125P failed to stably complex with α1 subunits. These results indicate that p.Leu125Pro disrupts the stable association of β4b with native calcium channel complexes, whereas membrane incorporation, modulation of current density and activation properties of heterologously expressed channels remained intact. Wildtype β4b was specifically targeted to the nuclei of quiescent excitatory cells. Importantly, the p.Leu125Pro mutation abolished nuclear targeting of β4b in cultured myotubes and hippocampal neurons. While binding of β4b to the known interaction partner PPP2R5D (B56δ) was not affected by the mutation, complex formation between β4b-L125P and the neuronal TRAF2 and NCK interacting kinase (TNIK) seemed to be disturbed. In summary, our data suggest that the homozygous CACNB4 p.(Leu126Pro) variant underlies the severe neurological phenotype in the two siblings, most likely by impairing both channel and non-channel functions of β4b.
Dysfunction of the endolysosomal system is often associated with neurodegenerative disease because postmitotic neurons are particularly reliant on the elimination of intracellular aggregates. ...Adequate function of endosomes and lysosomes requires finely tuned luminal ion homeostasis and transmembrane ion fluxes. Endolysosomal CLC Cl−/H+ exchangers function as electric shunts for proton pumping and in luminal Cl− accumulation. We now report three unrelated children with severe neurodegenerative disease, who carry the same de novo c.1658A>G (p.Tyr553Cys) mutation in CLCN6, encoding the late endosomal Cl−/H+-exchanger ClC-6. Whereas Clcn6−/− mice have only mild neuronal lysosomal storage abnormalities, the affected individuals displayed severe developmental delay with pronounced generalized hypotonia, respiratory insufficiency, and variable neurodegeneration and diffusion restriction in cerebral peduncles, midbrain, and/or brainstem in MRI scans. The p.Tyr553Cys amino acid substitution strongly slowed ClC-6 gating and increased current amplitudes, particularly at the acidic pH of late endosomes. Transfection of ClC-6Tyr553Cys, but not ClC-6WT, generated giant LAMP1-positive vacuoles that were poorly acidified. Their generation strictly required ClC-6 ion transport, as shown by transport-deficient double mutants, and depended on Cl−/H+ exchange, as revealed by combination with the uncoupling p.Glu200Ala substitution. Transfection of either ClC-6Tyr553Cys/Glu200Ala or ClC-6Glu200Ala generated slightly enlarged vesicles, suggesting that p.Glu200Ala, previously associated with infantile spasms and microcephaly, is also pathogenic. Bafilomycin treatment abrogated vacuole generation, indicating that H+-driven Cl− accumulation osmotically drives vesicle enlargement. Our work establishes mutations in CLCN6 associated with neurological diseases, whose spectrum of clinical features depends on the differential impact of the allele on ClC-6 function.
Marfan syndrome and related disorders are a group of heritable connective tissue disorders and share many clinical features that involve cardiovascular, skeletal, craniofacial, ocular, and cutaneous ...abnormalities. The majority of affected individuals have aortopathies associated with early mortality and morbidity. Implementation of targeted gene panel next-generation sequencing in these individuals is a powerful tool to obtain a genetic diagnosis. Here, we report on clinical and genetic spectrum of 53 families from India with a total of 83 patients who had a clinical diagnosis suggestive of Marfan syndrome or related disorders. We obtained a molecular diagnosis in 45/53 (85%) index patients, in which 36/53 (68%) had rare variants in FBN1 (Marfan syndrome; 63 patients in total), seven (13.3%) in TGFBR1/TGFBR2 (Loeys-Dietz syndrome; nine patients in total) and two patients (3.7%) in SKI (Shprintzen-Goldberg syndrome). 21 of 41 rare variants (51.2%) were novel. We did not detect a disease-associated variant in 8 (15%) index patients, and none of them met the Ghent Marfan diagnostic criteria. We found the homozygous FBN1 variant p.(Arg954His) in a boy with typical features of Marfan syndrome. Our study is the first reporting on the spectrum of variants in FBN1, TGFBR1, TGFBR2, and SKI in Indian individuals.
IFAP syndrome is a rare genetic disorder characterized by ichthyosis follicularis, atrichia, and photophobia. Previous research found that mutations in MBTPS2, encoding site-2-protease (S2P), ...underlie X-linked IFAP syndrome. The present report describes the identification via whole-exome sequencing of three heterozygous mutations in SREBF1 in 11 unrelated, ethnically diverse individuals with autosomal-dominant IFAP syndrome. SREBF1 encodes sterol regulatory element-binding protein 1 (SREBP1), which promotes the transcription of lipogenes involved in the biosynthesis of fatty acids and cholesterols. This process requires cleavage of SREBP1 by site-1-protease (S1P) and S2P and subsequent translocation into the nucleus where it binds to sterol regulatory elements (SRE). The three detected SREBF1 mutations caused substitution or deletion of residues 527, 528, and 530, which are crucial for S1P cleavage. In vitro investigation of SREBP1 variants demonstrated impaired S1P cleavage, which prohibited nuclear translocation of the transcriptionally active form of SREBP1. As a result, SREBP1 variants exhibited significantly lower transcriptional activity compared to the wild-type, as demonstrated via luciferase reporter assay. RNA sequencing of the scalp skin from IFAP-affected individuals revealed a dramatic reduction in transcript levels of low-density lipoprotein receptor (LDLR) and of keratin genes known to be expressed in the outer root sheath of hair follicles. An increased rate of in situ keratinocyte apoptosis, which might contribute to skin hyperkeratosis and hypotrichosis, was also detected in scalp samples from affected individuals. Together with previous research, the present findings suggest that SREBP signaling plays an essential role in epidermal differentiation, skin barrier formation, hair growth, and eye function.
Acute liver failure (ALF) is a life-threatening condition in the absence of preexisting liver disease in children. The main clinical presentation comprises hepatic dysfunction, elevated liver ...biochemical values, and coagulopathy. The etiology of ALF remains unclear in most affected children; however, the recent identification of mutations in the neuroblastoma amplified sequence (NBAS) gene in autosomal recessively inherited ALF has shed light on the cause of a subgroup of fever-triggered pediatric ALF episodes. Previously, biallelic mutations in NBAS have been reported to be associated with a syndrome comprising short stature, optic atrophy, and Pelger-Huët anomaly (SOPH) specifically occurring in the Yakut population. No hepatic phenotype has been observed in individuals with this disorder who all carry the homozygous NBAS founder mutation c.5741G>A p.(Arg1914His). We present the case of a 4-year-old girl with the cardinal features of SOPH syndrome: characteristic facial dysmorphism, postnatal growth retardation, delay of bone age, slender long bones, optic atrophy, and Pelger-Huët anomaly. During the first 2 years of her life, a series of infections with episodes of fever were accompanied by elevated liver enzyme levels, but hyperammonemia, hypoglycemia, coagulopathy, or encephalopathy suggestive of acute and severe liver disease were never observed. Whole exome sequencing in the patient revealed compound heterozygosity of the 2 NBAS variants, p.(Arg1914His) and p.(Glu943*). This case highlights the variability of clinical presentation associated with NBAS deficiency. Absence of severe liver problems in this case and SOPH-affected Yakut subjects suggests that individuals carrying the NBAS missense mutation p.(Arg1914His) are less susceptible to developing ALF.
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