Expanding the genetic and phenotypic spectrum of congenital myasthenic syndrome: new homozygous VAMP1 splicing variants in 2 novel individuals.

We report the cases of two Spanish pediatric patients with hypotonia, muscle weakness and feeding difficulties at birth. Whole-exome sequencing (WES) uncovered two new homozygous VAMP1 (Vesicle Associated Membrane Protein 1) splicing variants, NM_014231.5:c.129+5 G > A in the boy patient (P1) and c.341-24_341-16delinsAGAAAA in the girl patient (P2). This gene encodes the vesicle-associated membrane protein 1 (VAMP1) that is a component of a protein complex involved in the fusion of synaptic vesicles with the presynaptic membrane. VAMP1 has a highly variable C-terminus generated by alternative splicing that gives rise to three main isoforms (A, B and D), being VAMP1A the only isoform expressed in the nervous system. In order to assess the pathogenicity of these variants, expression experiments of RNA for VAMP1 were carried out. The c.129+5 G > A and c.341-24_341-16delinsAGAAAA variants induced aberrant splicing events resulting in the deletion of exon 2 (r.5_131del; p.Ser2TrpfsTer7) in the three isoforms in the first case, and the retention of the last 14 nucleotides of the 3' of intron 4 (r.340_341ins341-14_341-1; p.Ile114AsnfsTer77) in the VAMP1A isoform in the second case. Pathogenic VAMP1 variants have been associated with autosomal dominant spastic ataxia 1 (SPAX1) and with autosomal recessive presynaptic congenital myasthenic syndrome (CMS). Our patients share the clinical manifestations of CMS patients with two important differences: they do not show the typical electrophysiological pattern that suggests pathology of pre-synaptic neuromuscular junction, and their muscular biopsies present hypertrophic fibers type 1. In conclusion, our data expand both genetic and phenotypic spectrum associated with VAMP1 variants.

A novel de novo variant in CASK causes a severe neurodevelopmental disorder that masks the phenotype of a novel de novo variant in EEF2.

We report a 9-year-old Spanish boy with severe psychomotor developmental delay, short stature, microcephaly and abnormalities of the brain morphology, including cerebellar atrophy. Whole-exome sequencing (WES) uncovered two novel de novo variants, a hemizygous variant in CASK (Calcium/Calmodulin Dependent Serine Protein Kinase) and a heterozygous variant in EEF2 (Eukaryotic Translation Elongation Factor 2). CASK gene encodes the peripheral plasma membrane protein CASK that is a scaffold protein located at the synapses in the brain. The c.2506-6 A > G CASK variant induced two alternative splicing events that account for the 80% of the total transcripts, which are likely to be degraded by NMD. Pathogenic variants in CASK have been associated with severe neurological disorders such as mental retardation with or without nystagmus also called FG syndrome 4 (FGS4), and intellectual developmental disorder with microcephaly and pontine and cerebellar hypoplasia (MICPCH). Heterozygous variants in EEF2, which encodes the elongation factor 2 (eEF2), have been associated to Spinocerebellar ataxia 26 (SCA26) and more recently to a childhood-onset neurodevelopmental disorder with benign external hydrocephalus. The yeast model system used to investigate the functional consequences of the c.34 A > G EEF2 variant supported its pathogenicity by demonstrating it affects translational fidelity. In conclusion, the phenotype associated with the CASK variant is more severe and masks the milder phenotype of EEF2 variant.

First splicing variant in HECW2 with an autosomal recessive pattern of inheritance and associated with NDHSAL.

We report the clinical and genetic features of a Caucasian girl who presented a severe neurodevelopmental disorder with drug-resistant epilepsy, hypotonia, severe gastro-esophageal reflux and brain magnetic resonance imaging anomalies. WES uncovered a novel variant in homozygosis (g.197092814_197092824delinsC) in HECW2 gene that encodes the E3 ubiquitin-protein ligase HECW2. This protein induces ubiquitination and is implicated in the regulation of several important pathways involved in neurodevelopment and neurogenesis. Furthermore, de novo heterozygous missense variants in this gene have been associated with neurodevelopmental disorder with hypotonia, seizures, and absent language (NDHSAL). The homozygous variant of our patient disrupts the splice donor site of intron 22 and causes the elimination of exon 22 (r.3766_3917+1del) leading to an in-frame deletion of the protein (p.Leu1256_Trp1306del). Functional studies showed a twofold increase of its RNA expression, while the protein expression level was reduced by 60%, suggesting a partial loss-of-function mechanism of pathogenesis. Thus, this is the first patient with NDHSAL caused by an autosomal recessive splicing variant in HECW2.

MLIP causes recessive myopathy with rhabdomyolysis, myalgia and baseline elevated serum creatine kinase.

Striated muscle needs to maintain cellular homeostasis in adaptation to increases in physiological and metabolic demands. Failure to do so can result in rhabdomyolysis. The identification of novel genetic conditions associated with rhabdomyolysis helps to shed light on hitherto unrecognized homeostatic mechanisms. Here we report seven individuals in six families from different ethnic backgrounds with biallelic variants in MLIP, which encodes the muscular lamin A/C-interacting protein, MLIP. Patients presented with a consistent phenotype characterized by mild muscle weakness, exercise-induced muscle pain, variable susceptibility to episodes of rhabdomyolysis, and persistent basal elevated serum creatine kinase levels. The biallelic truncating variants were predicted to result in disruption of the nuclear localizing signal of MLIP. Additionally, reduced overall RNA expression levels of the predominant MLIP isoform were observed in patients' skeletal muscle. Collectively, our data increase the understanding of the genetic landscape of rhabdomyolysis to now include MLIP as a novel disease gene in humans and solidifies MLIP's role in normal and diseased skeletal muscle homeostasis.

New subtype of PCH1C caused by novel EXOSC8 variants in a 16-year-old Spanish patient.

We report the case of a 16-year-old Spanish boy with cerebellar and spinal muscular atrophy, spasticity, psychomotor retardation, nystagmus, ophthalmoparesis, epilepsy, and mitochondrial respiratory chain (MRC) deficiency. Whole exome sequencing (WES) uncovered three variants (two of them novel) in a compound heterozygous in EXOSC8 gene (NM_181503.3:c.[390+1delG];[628C>T;815G>C]) that encodes the exosome complex component RRP43 protein (EXOSC8). In order to assess the pathogenicity of these variants, expression experiments of RNA and protein for EXOSC8 were carried out. The c.[390+1delG] variant produces the elimination of exon 7 (r.[345_390del]; p.[Ser116LysfsTer27]) and a decrease of the RNA expression in relation to the other allele (p.[Pro210Ser;Ser272Thr]). Furthermore, total mRNA expression is reduced by 30% and the protein level by 65%. EXOSC8 is an essential protein of the exosome core, a ubiquitously expressed complex responsible for RNA processing and degradation. Recessive mutations in EXOSC8 cause pontocerebellar hypoplasia type 1C (PCH1C), and currently, only two homozygous variants in this gene have been described. However, unlike PCH1C-affected individuals with EXOSC8 variants, our patient presents a normal supratentorial cerebral tissue (neither corpus callosum hypoplasia nor hypomyelination) with a less severe phenotype and longer survival. In conclusion, our data expand both genetic and phenotypic spectrum associated with EXOSC8 variants.

Characterization of a complex phenotype (fever-dependent recurrent acute liver failure and osteogenesis imperfecta) due to NBAS and P4HB variants.

We report the clinical, biochemical and genetic findings from a Spanish boy of Caucasian origin who presented with fever-dependent RALF (recurrent acute liver failure) and osteogenesis imperfecta (OI). Whole-exome sequencing (WES) uncovered two compound heterozygous variants in NBAS (c.[1265 T > C];[1549C > T]:p.[(Leu422Pro)];[(Arg517Cys)]), and a heterozygous variant in P4HB (c.[194A > G];[194=]:p.[(Lys65Arg)];[(Lys65=)]) that was transmitted from the clinically unaffected mother who was mosaic carrier of the variant. Variants in NBAS protein have been associated with ILFS2 (infantile liver failure syndrome-2), SOPH syndrome (short stature, optic nerve atrophy, and Pelger-Huët anomaly syndrome), and multisystem diseases. Several patients showed clinical manifestations affecting the skeletal system, such as osteoporosis, pathologic fractures and OI. Experiments in the patient's fibroblasts demonstrated that mutated NBAS protein is overexpressed and thermally unstable, and reduces the expression of MGP, a regulator of bone homeostasis. Variant in PDI (protein encoded by P4HB) has been associated with CLCRP1 (Cole-Carpenter syndrome-1), a type of severe OI. An increase of COL1A2 protein retention was observed in the patient's fibroblasts. In order to study if the variant in P4HB was involved in the alteration in collagen trafficking, overexpression experiments of PDI were carried out. These experiments showed that overexpression of mutated PDI protein produces an increase in COL1A2 retention. In conclusion, these results corroborate that the variants in NBAS are responsible for the liver phenotype, and demonstrate that the variant in P4HB is involved in the bone phenotype, probably in synergy with NBAS variants.

MAST1 variant causes mega-corpus-callosum syndrome with cortical malformations but without cerebellar hypoplasia.

We report the case of a Caucasian Spanish origin female who showed severe psychomotor developmental delay, hypotonia, strabismus, epilepsy, short stature, and poor verbal language development. Brain magnetic resonance imaging scans showed thickened corpus callosum, cortical malformations, and dilated and abnormal configuration of the lateral ventricles without hydrocephalus. Whole-exome sequence uncovered a de novo variant in the microtubule associated serine/threonine kinase 1 gene (MAST1; NM_014975.3:c.1565G>A:p.(Gly522Glu)) that encodes for the MAST1. Only 12 patients have been identified worldwide with 10 different variants in this gene: six patients with mega-corpus-callosum syndrome with cerebellar hypoplasia and cortical malformations; two patients with microcephaly and cerebellar hypoplasia; two patients with autism, one patient with diplegia, and one patient with microcephaly and dysmorphism. Our patient shows a new phenotypic subtype defined by mega-corpus-callosum syndrome with cortical malformations without cerebellar hypoplasia. In conclusion, our data expand the phenotypic spectrum associated to MAST1 gene variants.

A novel de novo mutation in the PURA gene associated with a new clinical finding: large brainstem.

We report the case of a Caucasian Spanish boy, who showed profound neonatal hypotonia, feeding difficulties, apnea, severe developmental delay, epilepsy, bilateral convergent strabismus, poor verbal language development and a large brainstem. Whole-exome sequence uncovered a novel de novo mutation in the purine-rich element binding protein A gene (PURA; NM_005859.4:c.72del:p.(-Gly25AlafsTer53)) that encodes the transcriptional activator protein Pur-alpha (PURA). Mutations in this gene have been identified in patients with PURA syndrome, a rare disorder characterized by an early hypotonia, developmental delay, severe intellectual disability with or without epilepsy, and disability in expressive language development. Although, up to 75 cases have been identified worldwide, to the best of our knowledge, this is the first patient described with a brainstem larger than normal. In conclusion, our data expand both geneticand phenotypic spectrum associated with PURA gene mutations.

A novel de novo MTOR gain-of-function variant in a patient with Smith-Kingsmore syndrome and Antiphospholipid syndrome.

We report the clinical, biochemical and genetic findings from a Spanish girl of Caucasian origin who presented with macrocephaly, dysmorphic facial features, developmental delay, hypotonia, combined oxidative phosphorylation (OxPhos) deficiency, epilepsy and anti-phospholipid antibodies (aPL). Whole-exome sequencing (WES) uncovered a heterozygous variant in the MTOR gene (NM_004958.3: c.7235A>T: p.(Asp2412Val)) that encodes for the Serine/threonine-protein kinase mTOR. The substrates phosphorylation experiments demonstrated that this variant exerts its effect by gain-of-function (GOF) and autosomal dominant mechanism. GOF variants in this protein have been associated with Smith-Kingsmore syndrome (SKS), a rare autosomal dominant disorder characterized by intellectual disability, macrocephaly, seizure, developmental delay and dysmorphic facial features. Furthermore, the mTOR pathway has been demonstrated previously to be involved in many types of endothelium injuries including the antiphospholipid syndrome (APS), a systemic autoimmune disease characterized by the production of aPL with recurrent vascular thrombosis. Therefore, our patient is the first one with an mTOR variant and diagnosed with SKS and APS. In conclusion, our data expand both the genetic and phenotypic spectrum associated with MTOR gene variants.

A rare male patient with Fontaine progeroid syndrome caused by p.R217H de novo mutation in SLC25A24.

We report the clinical and genetic findings in a 15-year-old Spanish boy presenting prenatal and postnatal growth retardation, reduced subcutaneous adipose tissue, premature skin wrinkling, sparse hair, short distal phalanges with small nails, umbilical hernia, wide anterior fontanel, and normal cognitive and motor development. Exome sequencing uncovered a heterozygous mutation in SLC25A24 (NM_013386: c.650G>A: p.R217H) that encodes for the calcium-binding mitochondrial carrier protein SCaMC-1. This gain-of-function variant has been previously associated with Fontaine syndrome and Gorlin-Chaudhry-Moss syndrome, two entities that show overlapping features, and have been recently subsumed under the name Fontaine progeroid syndrome (FPS; MIM: 612289) in OMIM. Here, we describe the first male patient with genetically confirmed FPS who survives at least until adolescence.

Mitochondrial involvement in a Bosch-Boonstra-Schaaf optic atrophy syndrome patient with a novel de novo NR2F1 gene mutation.

We report the clinical and biochemical findings from a patient who presented with Bosch-Boonstra-Schaaf optic atrophy syndrome (BBSOAS), an autosomal-dominant disorder characterized by optic atrophy, developmental delay and intellectual disability. In addition, the patient also displays hypotonia, stroke-like episodes, and complex IV deficiency of the mitochondrial respiratory chain. Whole-exome sequencing (WES) uncovered a novel heterozygous mutation in the NR2F1 gene (NM_005654:c.286A>G:p.Lys96Glu) that encodes for the COUP transcription factor 1 protein (COUP-TF1). Loss-of-function mutations in this protein have been associated with BBSOAS, and a luciferase reporter assay showed that this variant, in the zinc-finger DNA-binding domain (DBD) of COUP-TF1 protein, impairs its transcriptional activity. The additional features of this patient are more related with mitochondrial diseases that with BBSOAS, indicating a mitochondrial involvement. Finally, our data expand both the genetic and phenotypic spectrum associated with NR2F1 gene mutations.

Myopathic mtDNA Depletion Syndrome Due to Mutation in TK2 Gene.

Whole-exome sequencing was used to identify the disease gene(s) in a Spanish girl with failure to thrive, muscle weakness, mild facial weakness, elevated creatine kinase, deficiency of mitochondrial complex III and depletion of mtDNA. With whole-exome sequencing data, it was possible to get the whole mtDNA sequencing and discard any pathogenic variant in this genome. The analysis of whole exome uncovered a homozygous pathogenic mutation in thymidine kinase 2 gene ( TK2; NM_004614.4:c.323 C>T, p.T108M). TK2 mutations have been identified mainly in patients with the myopathic form of mtDNA depletion syndromes. This patient presents an atypical TK2-related myopathic form of mtDNA depletion syndromes, because despite having a very low content of mtDNA (<20%), she presents a slower and less severe evolution of the disease. In conclusion, our data confirm the role of TK2 gene in mtDNA depletion syndromes and expanded the phenotypic spectrum.

An innovative strategy to clone positive modifier genes of defects caused by mtDNA mutations: MRPS18C as suppressor gene of m.3946G>A mutation in MT-ND1 gene.

We have developed a new functional complementation approach to clone modifier genes which overexpression is able to suppress the biochemical defects caused by mtDNA mutations (suppressor genes). This strategy consists in transferring human genes into respiratory chain-deficient fibroblasts, followed by a metabolic selection in a highly selective medium. We used a normalized expression cDNA library in an episomal vector (pREP4) to transfect the fibroblasts, and a medium with glutamine and devoid of any carbohydrate source to select metabolically. Growing the patient's fibroblasts in this selective medium, the deficient cells rapidly disappear unless they are rescued by the cDNA of a suppressor gene. The use of an episomal vector allows us to carry out several rounds of transfection/selection (cyclical phenotypic rescue) to enrich the rescue with true clones of suppressor genes. Using fibroblasts from a patient with epileptic encephalopathy with the m.3946G>A (p.E214K) mutation in the MT-ND1 gene, several candidate genes were identified and one of them was characterized functionally. Thus, overexpression of MRPS18C gene (that encode for bS18m protein) suppressed the molecular defects produced by this mtDNA mutation, recovering the complex I activity and reducing the ROS produced by this complex to normal levels. We suggest that modulation of bS18m expression may be an effective therapeutic strategy for the patients with this mutation.

New ATP8A2 gene mutations associated with a novel syndrome: encephalopathy, intellectual disability, severe hypotonia, chorea and optic atrophy.

We report the clinical and biochemical findings from two unrelated patients who presented with a novel syndrome: encephalopathy, intellectual disability, severe hypotonia, chorea and optic atrophy. Whole exome sequencing (WES) uncovered a homozygous mutation in the ATP8A2 gene (NM_016529:c.1287G > T, p.K429N) in one patient and compound heterozygous mutations (c.1630G > C, p.A544P and c.1873C > T, p.R625W) in the other. Only one haploinsufficiency case and a family with a homozygous mutation in ATP8A2 gene (c.1128C > G, p.I376M) have been described so far, with phenotypes that differed slightly from the patients described herein. In conclusion, our data expand both the genetic and phenotypic spectrum associated with ATP8A2 gene mutations.

First missense mutation outside of SERAC1 lipase domain affecting intracellular cholesterol trafficking.

We report the clinical and genetic findings in a Spanish boy who presented MEGDEL syndrome, a very rare inborn error of metabolism. Whole-exome sequencing uncovered a new homozygous mutation in the serine active site containing 1 (SERAC1) gene, which is essential for both mitochondrial function and intracellular cholesterol trafficking. Functional studies in patient fibroblasts showed that p.D224G mutation affects the intracellular cholesterol trafficking. Only three missense mutations in this gene have been described before, being p.D224G the first missense mutation outside of the SERAC1 serine-lipase domain. Therefore, we conclude that the defect in cholesterol trafficking is not limited to alterations in this specific part of the protein.

Hepcidin treatment in Hfe-/- mice diminishes plasma iron without affecting erythropoiesis.

BACKGROUND: Iron is essential for mammalian metabolism and its cellular concentration is controlled by regulating its acquisition and storage. Haemochromatosis is a condition involving iron overload that is characterised by increased duodenal iron absorption and a progressive accumulation of iron in vital organs. Hepcidin is the main hormone that regulates iron homoestasis and it is secreted by the liver. MATERIALS AND METHODS: We have studied how extended hepcidin administration affects the iron load status, plasma and tissue iron concentration, erythropoiesis and the expression of proteins involved on iron homeostasis in haemochromatotic (Hfe(-/-)) and wild-type mice. RESULTS: Hepcidin reverted the high plasma iron concentrations in Hfe(-/-) mice to normal values. The high concentration of hepatic iron was not altered in the liver of these Hfe(-/-) mice. Hepcidin administration did not disturb erythropoiesis in either Hfe(-/-) or wild-type mice and likewise, hepcidin did not modify the expression of any protein analysed in the liver, duodenum or spleen of Hfe(-/-) and wild-type mice. These data confirm that hepcidin administration diminishes plasma iron concentrations. CONCLUSION: Treatment with sustained doses of hepcidin diminishes plasma iron concentrations in Hfe(-/-) mice.

Inhibition of p38-MAPK potentiates cisplatin-induced apoptosis via GSH depletion and increases intracellular drug accumulation in growth-arrested kidney tubular epithelial cells.

We were interested in analyzing the regulation by mitogen-activated protein kinases (MAPKs) of cisplatin-provoked toxicity in epithelial renal tubule cell lines, when assayed under culture conditions (cell confluence plus serum deprivation), which mimic the characteristics of a nonproliferating epithelium. Under these restrictive growth conditions, cisplatin induced apoptosis with lower efficacy than in exponentially growing cells, and decreased p38-MAPK phosphorylation in NRK-52E and other (LLC-PK1, MDCK, HK2) cell lines. Moreover, cisplatin-provoked apoptosis was potentiated by cotreatment with p38-MAPK-specific inhibitors (SB203580, SB220025) or transfection with a kinase-negative mutant of MKK6, whereas c-Jun NH2-terminal kinase or extracellular signal-regulated kinase/MAPK and ERK Kinase inhibitors were ineffective. By contrast, when applied to exponentially growing cells, cisplatin stimulated p38-MAPK phosphorylation and apoptosis, was attenuated by kinase inhibitors. Treatment of confluent/serum-deprived cells with cisplatin caused mitochondrial transmembrane potential disruption and activated the mitochondrial apoptotic pathway, as indicated by the decrease in Bcl-X(L) expression, increase in Bax expression and cytochrome c release, and these effects were potentiated by cotreatment with SB203580. Treatment of confluent/serum-deprived cells with cisplatin plus SB203580 decreased the intracellular reduced glutathione (GSH) content, and increased intracellular cisplatin accumulation as well as cisplatin binding to DNA. Cotreatment with the GSH-depleting agent D,L-buthionine-R,S-sulfoximine also potentiated cisplatin-provoked apoptosis. In summary, p38-MAPK inhibition potentiates cisplatin-provoked apoptosis in growth-arrested epithelial renal tubule cells, a result that may be explained at least in part by GSH depletion and drug transport alteration.