Erik Jan Kamsteeg

The tetraspanin CD63 enhances the internalization of the H,K-ATPase β-subunit

The tetraspanin CD63 resides in late endosomes, lysosomes, secretory vesicles, and at the plasma membrane, and it moves among these compartments. We find that CD63 is present also in tubulovesicular elements, the intracellular compartments that contain the H,K-ATPase in unstimulated gastric parietal cells. The H,K-ATPase β-subunit and CD63 colocalize in parietal cells and form a complex that can be coprecipitated. The β-subunit and CD63 also interact when they are coexpressed in COS-7 cells. Furthermore, expression with CD63 induces the redistribution of the β-subunit from the cell surface to CD63+ intracellular compartments. Immunofluorescence and biochemical experiments reveal that this redistribution occurs by enhanced endocytosis of H,K-ATPase β-subunit complexed with CD63. Coexpression of the β-subunit with mutant CD63 polypeptides demonstrates that the enhanced internalization of the β-subunit depends on the capacity of CD63 to interact with adaptor protein complexes 2 and 3. These data indicate that CD63 serves as an adaptor protein that links its interaction partners to the endocytic machinery of the cell and suggest a previously uncharacterized protein-trafficking role for the tetraspanins.

De novo gain-of-function and loss-of-function mutations of SCN8A in patients with intellectual disabilities and epilepsy

Background Mutations of SCN8A encoding the neuronal voltage-gated sodium channel NaV1.6 are associated with early-infantile epileptic encephalopathy type 13 (EIEE13) and intellectual disability. Using clinical exome sequencing, we have detected three novel de novo SCN8A mutations in patients with intellectual disabilities, and variable clinical features including seizures in two patients. To determine the causality of these SCN8A mutations in the disease of those three patients, we aimed to study the (dys)function of the mutant sodium channels. Methods The functional consequences of the three SCN8A mutations were assessed using electrophysiological analyses in transfected cells. Genotype–phenotype correlations of these and other cases were related to the functional analyses. Results The first mutant displayed a 10 mV hyperpolarising shift in voltage dependence of activation (gain of function), the second did not form functional channels (loss of function), while the third mutation was functionally indistinguishable from the wildtype channel. Conclusions Comparison of the clinical features of these patients with those in the literature suggests that gain-of-function mutations are associated with severe EIEE, while heterozygous loss-of-function mutations cause intellectual disability with or without seizures. These data demonstrate that functional analysis of missense mutations detected by clinical exome sequencing, both inherited and de novo, is valuable for clinical interpretation in the age of massive parallel sequencing.

MAL decreases the internalization of the aquaporin-2 water channel.

Body water homeostasis depends critically on the hormonally regulated trafficking of aquaporin-2 (AQP2) water channels in renal collecting duct epithelial cells. Several types of posttranslational modifications are clearly involved in controlling the distribution of AQP2 between intracellular vesicles and the apical plasma membrane. Little is known, however, about the protein interactions that govern the trafficking of AQP2 between these organelles. MAL is a detergent-resistant membrane-associated protein implicated in apical sorting events. We wondered, therefore, whether MAL plays a role in the regulated trafficking of AQP2 between intracellular vesicles and the apical surface. We find that AQP2 and MAL are coexpressed in epithelial cells of the kidney collecting duct. These two proteins interact, both in the native kidney and when expressed by transfection in cultured cells. The S256-phosphorylated form of AQP2 appears to interact more extensively with MAL than does the water channel protein not phosphorylated at this serine. We find that MAL is not involved in detergent-resistant membrane association or apical delivery of AQP2 in LLC-PK1 renal epithelial cells. Instead, MAL increases the S256 phosphorylation and apical surface expression of AQP2. Furthermore, internalization experiments show that MAL induces surface expression of AQP2 by attenuating its internalization. Thus, the involvement of MAL in the cell surface retention of apical membrane proteins could play an important role in regulated absorption and secretion in transporting epithelia.

De Novo Mutations in the Motor Domain of KIF1A Cause Cognitive Impairment, Spastic Paraparesis, Axonal Neuropathy, and Cerebellar Atrophy

KIF1A is a neuron‐specific motor protein that plays important roles in cargo transport along neurites. Recessive mutations in KIF1A were previously described in families with spastic paraparesis or sensory and autonomic neuropathy type‐2. Here, we report 11 heterozygous de novo missense mutations (p.S58L, p.T99M, p.G102D, p.V144F, p.R167C, p.A202P, p.S215R, p.R216P, p.L249Q, p.E253K, and p.R316W) in KIF1A in 14 individuals, including two monozygotic twins. Two mutations (p.T99M and p.E253K) were recurrent, each being found in unrelated cases. All these de novo mutations are located in the motor domain (MD) of KIF1A. Structural modeling revealed that they alter conserved residues that are critical for the structure and function of the MD. Transfection studies suggested that at least five of these mutations affect the transport of the MD along axons. Individuals with de novo mutations in KIF1A display a phenotype characterized by cognitive impairment and variable presence of cerebellar atrophy, spastic paraparesis, optic nerve atrophy, peripheral neuropathy, and epilepsy. Our findings thus indicate that de novo missense mutations in the MD of KIF1A cause a phenotype that overlaps with, while being more severe, than that associated with recessive mutations in the same gene.

Mutations in the histone methyltransferase gene KMT2B cause complex early-onset dystonia

Histone lysine methylation, mediated by mixed-lineage leukemia (MLL) proteins, is now known to be critical in the regulation of gene expression, genomic stability, cell cycle and nuclear architecture. Despite MLL proteins being postulated as essential for normal development, little is known about the specific functions of the different MLL lysine methyltransferases. Here we report heterozygous variants in the gene KMT2B (also known as MLL4) in 27 unrelated individuals with a complex progressive childhood-onset dystonia, often associated with a typical facial appearance and characteristic brain magnetic resonance imaging findings. Over time, the majority of affected individuals developed prominent cervical, cranial and laryngeal dystonia. Marked clinical benefit, including the restoration of independent ambulation in some cases, was observed following deep brain stimulation (DBS). These findings highlight a clinically recognizable and potentially treatable form of genetic dystonia, demonstrating the crucial role of KMT2B in the physiological control of voluntary movement.

Expression of tetraspan protein CD63 activates protein-tyrosine kinase (PTK) and enhances the PTK-induced inhibition of ROMK channels.

In the present study, we tested the role of CD63 in regulating ROMK1 channels by protein-tyrosine kinase (PTK). Immunocytochemical staining shows that CD63 and receptor-linked tyrosine phosphatase α (RPTPα) are expressed in the cortical collecting duct and outer medulla collecting duct. Immunoprecipitation of tissue lysates from renal cortex and outer medulla or 293T cells transfected with CD63 reveals that CD63 was associated with RPTPα both in situ and in transfected cells. Expression of CD63 in 293T cells stimulated the phosphorylation of tyrosine residue 416 of c-Src but decreased the phosphorylation of tyrosine residue 527, indicating that expression of CD63 stimulates the activity of c-Src. Furthermore, c-Src was coimmunoprecipitated with RPTPα and CD63 both in 293T cells transfected with CD63 and in lysates prepared from native rat kidney. Potassium restriction had no effect on the expression of RPTPα, but it increased the association between c-Src and RPTPα in the renal cortex and outer medulla. We also used two-electrode voltage clamp to study the effect of CD63 on ROMK channels in Xenopus oocytes. Expression of CD63 had no significant effect on potassium currents in oocytes injected with ROMK1; however, it significantly enhanced the c-Src-induced inhibition of ROMK channels in oocytes injected with ROMK1+c-Src. The effect of CD63 on the c-Src-induced inhibition was not due to a decreased expression of ROMK1 channels, because blocking PTK with herbimycin A abolished the inhibitory effect of c-Src on ROMK channels in oocytes injected with ROMK1+c-Src+CD63. Furthermore, coexpression of CD63 enhanced tyrosine phosphorylation of ROMK1. We conclude that CD63 plays a role in the regulation of ROMK channels through its association with RPTPα, which in turn interacts with and activates Src family PTK, thus reducing ROMK activity.

Tetraspan proteins: regulators of renal structure and function.

Purpose of reviewMembers of the tetraspan family are widely expressed and poorly understood. An emerging literature suggests that through their interactions with other membrane proteins they play central or regulatory roles in a wide variety of physiological processes. This review will discuss selected tetraspan complexes and highlight their relevance to epithelial cells and the kidney. Recent findingsTetraspans regulate the signaling and trafficking properties of their partner proteins. Tetraspan complexes with integrin molecules, for example, modulate cell adhesion and mobility. Perturbations of tetraspan–integrin assemblies can have dramatic impacts on renal tissue morphogenesis, resulting in a disruption of normal glomerular architecture and selectivity. Tetraspan interactions with renal ion transport proteins appear to affect transporter function by enhancing or inhibiting the endocytic internalization of their transport protein partners. SummaryTetraspans constitute a novel class of proteins whose capacity to alter the cell biological and functional properties of their membrane protein partners is likely to have wide ranging and important physiological ramifications.

Variants in SLC18A3, vesicular acetylcholine transporter, cause congenital myasthenic syndrome

Objective: To describe the clinical and genetic characteristics of presynaptic congenital myasthenic syndrome secondary to biallelic variants in SLC18A3. Methods: Individuals from 2 families were identified with biallelic variants in SLC18A3, the gene encoding the vesicular acetylcholine transporter (VAChT), through whole-exome sequencing. Results: The patients demonstrated features seen in presynaptic congenital myasthenic syndrome, including ptosis, ophthalmoplegia, fatigable weakness, apneic crises, and deterioration of symptoms in cold water for patient 1. Both patients demonstrated moderate clinical improvement on pyridostigmine. Patient 1 had a broader phenotype, including learning difficulties and left ventricular dysfunction. Electrophysiologic studies were typical for a presynaptic defect. Both patients showed profound electrodecrement on low-frequency repetitive stimulation followed by a prolonged period of postactivation exhaustion. In patient 1, this was unmasked only after isometric contraction, a recognized feature of presynaptic disease, emphasizing the importance of activation procedures. Conclusions: VAChT is responsible for uptake of acetylcholine into presynaptic vesicles. The clinical and electrographic characteristics of the patients described are consistent with previously reported mouse models of VAChT deficiency. These findings make it very likely that defects in VAChT due to variants in SLC18A3 are a cause of congenital myasthenic syndrome in humans.

Mosaicism for dominant collagen 6 mutations as a cause for intrafamilial phenotypic variability.

Collagen 6‐related dystrophies and myopathies (COL6‐RD) are a group of disorders that form a wide phenotypic spectrum, ranging from severe Ullrich congenital muscular dystrophy, intermediate phenotypes, to the milder Bethlem myopathy. Both inter‐ and intrafamilial variable expressivity are commonly observed. We present clinical, immunohistochemical, and genetic data on four COL6‐RD families with marked intergenerational phenotypic heterogeneity. This variable expression seemingly masquerades as anticipation is due to parental mosaicism for a dominant mutation, with subsequent full inheritance and penetrance of the mutation in the heterozygous offspring. We also present an additional fifth simplex patient identified as a mosaic carrier. Parental mosaicism was confirmed in the four families through quantitative analysis of the ratio of mutant versus wild‐type allele (COL6A1, COL6A2, and COL6A3) in genomic DNA from various tissues, including blood, dermal fibroblasts, and saliva. Consistent with somatic mosaicism, parental samples had lower ratios of mutant versus wild‐type allele compared with the fully heterozygote offspring. However, there was notable variability of the mutant allele levels between tissues tested, ranging from 16% (saliva) to 43% (fibroblasts) in one mosaic father. This is the first report demonstrating mosaicism as a cause of intrafamilial/intergenerational variability of COL6‐RD, and suggests that sporadic and parental mosaicism may be more common than previously suspected.

Affected female carriers of MTM1 mutations display a wide spectrum of clinical and pathological involvement: delineating diagnostic clues

X-linked myotubular myopathy (XLMTM), a severe congenital myopathy, is caused by mutations in the MTM1 gene located on the X chromosome. A majority of affected males die in the early postnatal period, whereas female carriers are believed to be usually asymptomatic. Nevertheless, several affected females have been reported. To assess the phenotypic and pathological spectra of carrier females and to delineate diagnostic clues, we characterized 17 new unrelated affected females and performed a detailed comparison with previously reported cases at the clinical, muscle imaging, histological, ultrastructural and molecular levels. Taken together, the analysis of this large cohort of 43 cases highlights a wide spectrum of clinical severity ranging from severe neonatal and generalized weakness, similar to XLMTM male, to milder adult forms. Several females show a decline in respiratory function. Asymmetric weakness is a noteworthy frequent specific feature potentially correlated to an increased prevalence of highly skewed X inactivation. Asymmetry of growth was also noted. Other diagnostic clues include facial weakness, ptosis and ophthalmoplegia, skeletal and joint abnormalities, and histopathological signs that are hallmarks of centronuclear myopathy such as centralized nuclei and necklace fibers. The histopathological findings also demonstrate a general disorganization of muscle structure in addition to these specific hallmarks. Thus, MTM1 mutations in carrier females define a specific myopathy, which may be independent of the presence of an XLMTM male in the family. As several of the reported affected females carry large heterozygous MTM1 deletions not detectable by Sanger sequencing, and as milder phenotypes present as adult-onset limb-girdle myopathy, the prevalence of this myopathy is likely to be greatly underestimated. This report should aid diagnosis and thus the clinical management and genetic counseling of MTM1 carrier females. Furthermore, the clinical and pathological history of this cohort may be useful for therapeutic projects in males with XLMTM, as it illustrates the spectrum of possible evolution of the disease in patients surviving long term.