A defect in molybdenum cofactor binding causes an attenuated form of sulfite oxidase deficiency.

Isolated sulfite oxidase deficiency (ISOD) is a rare recessive and infantile lethal metabolic disorder, which is caused by functional loss of sulfite oxidase (SO) due to mutations of the SUOX gene. SO is a mitochondrially localized molybdenum cofactor (Moco)- and heme-dependent enzyme, which catalyzes the vital oxidation of toxic sulfite to sulfate. Accumulation of sulfite and sulfite-related metabolites such as S-sulfocysteine (SSC) are drivers of severe neurodegeneration leading to early childhood death in the majority of ISOD patients. Full functionality of SO is dependent on correct insertion of the heme cofactor and Moco, which is controlled by a highly orchestrated maturation process. This maturation involves the translation in the cytosol, import into the intermembrane space (IMS) of mitochondria, cleavage of the mitochondrial targeting sequence, and insertion of both cofactors. Moco insertion has proven as the crucial step in this maturation process, which enables the correct folding of the homodimer and traps SO in the IMS. Here, we report on a novel ISOD patient presented at 17 months of age carrying the homozygous mutation NM_001032386.2 (SUOX):c.1097G > A, which results in the expression of SO variant R366H. Our studies show that histidine substitution of Arg366, which is involved in coordination of the Moco-phosphate, causes a severe reduction in Moco insertion efficacy in vitro and in vivo. Expression of R366H in HEK SUOX-/- cells mimics the phenotype of patient's fibroblasts, representing a loss of SO expression and specific activity. Our studies disclose a general paradigm for a kinetic defect in Moco insertion into SO caused by residues involved in Moco coordination resulting in the case of R366H in an attenuated form of ISOD.

Making sense of missense variants in TTN-related congenital myopathies.

Mutations in the sarcomeric protein titin, encoded by TTN, are emerging as a common cause of myopathies. The diagnosis of a TTN-related myopathy is, however, often not straightforward due to clinico-pathological overlap with other myopathies and the prevalence of TTN variants in control populations. Here, we present a combined clinico-pathological, genetic and biophysical approach to the diagnosis of TTN-related myopathies and the pathogenicity ascertainment of TTN missense variants. We identified 30 patients with a primary TTN-related congenital myopathy (CM) and two truncating variants, or one truncating and one missense TTN variant, or homozygous for one TTN missense variant. We found that TTN-related myopathies show considerable overlap with other myopathies but are strongly suggested by a combination of certain clinico-pathological features. Presentation was typically at birth with the clinical course characterized by variable progression of weakness, contractures, scoliosis and respiratory symptoms but sparing of extraocular muscles. Cardiac involvement depended on the variant position. Our biophysical analyses demonstrated that missense mutations associated with CMs are strongly destabilizing and exert their effect when expressed on a truncating background or in homozygosity. We hypothesise that destabilizing TTN missense mutations phenocopy truncating variants and are a key pathogenic feature of recessive titinopathies that might be amenable to therapeutic intervention.

Clinical and genetic characterization of PYROXD1-related myopathy patients from Turkey.

Congenital myopathies (CMs) are a heterogeneous group of inherited muscle disorders characterized by muscle weakness at birth, while limb-girdle muscular dystrophies (LGMD) have a later onset and slower disease progression. Thus, detailed clinical phenotyping of genetically defined disease entities are required for the full understanding of genotype-phenotype correlations. A recently defined myopathic genetic disease entity is caused by bi-allelic variants in a gene coding for pyridine nucleotide-disulfide oxidoreductase domain 1 (PYROXD1) with unknown substrates. Here, we present three patients from two consanguineous Turkish families with mild LGMD, facial weakness, normal CK levels, and slow progress. Genomic analyses revealed a homozygous known pathogenic missense variant (c.464A>G, p.Asn155Ser) in family 1 with two affected females. In the affected male of family 2, we found this variant in a compound heterozygous state together with a novel frameshift variant (c.329_332delTCTG, p.Leu112Valfs*8), which is the second frameshift variant known so far in PYROXD1. We have been able to define a large homozygous region in family 1 sharing a common haplotype with family 2 in the critical region. Our data suggest that c.464A>G is a Turkish founder mutation. To gain deeper insights, we performed a systematic review of all published PYROXD1-related myopathy cases. Our analysis showed that the c.464A > G variant was found in 87% (20/23) of the patients and that it may cause either a childhood- or adult-onset phenotype, irrespective of its presence in a homozygous or compound heterozygous state. Interestingly, only four patients had elevated CK levels (up to 1000 U/L), and cardiac involvement was found in few compound heterozygous cases.

Genotype-phenotype correlation in seven motor neuron disease families with novel ALS2 mutations.

Autosomal-recessive mutations in the Alsin Rho guanine nucleotide exchange factor (ALS2) gene may cause specific subtypes of childhood-onset progressive neurodegenerative motor neuron diseases (MND). These diseases can manifest with a clinical continuum from infantile ascending hereditary spastic paraplegia (IAHSP) to juvenile-onset forms with or without lower motor neuron involvement, the juvenile primary lateral sclerosis (JPLS) and the juvenile amyotrophic lateral sclerosis (JALS). We report 11 patients from seven unrelated Turkish and Yemeni families with clinical signs of IAHSP or JPLS. We performed haplotype analysis or next-generation panel sequencing followed by Sanger Sequencing to unravel the genetic disease cause. We described their clinical phenotype and analyzed the pathogenicity of the detected variants with bioinformatics tools. We further reviewed all previously reported cases with ALS2-related MND. We identified five novel homozygous pathogenic variants in ALS2 at various positions: c.275_276delAT (p.Tyr92CysfsTer11), c.1044C>G (p.Tyr348Ter), c.1718C>A (p.Ala573Glu), c.3161T>C (p.Leu1054Pro), and c.1471+1G>A (NM_020919.3, NP_065970.2). In our cohort, disease onset was in infancy or early childhood with rapid onset of motor neuron signs. Muscle weakness, spasticity, severe dysarthria, dysphagia, and facial weakness were common features in the first decade of life. Frameshift and nonsense mutations clustered in the N-terminal Alsin domains are most prevalent. We enriched the mutational spectrum of ALS2-related disorders with five novel pathogenic variants. Our study indicates a high detection rate of ALS2 mutations in patients with a clinically well-characterized early onset MND. Intrafamilial and even interfamilial diversity in patients with identical pathogenic variants suggest yet unknown modifiers for phenotypic expression.

The genomic and clinical landscape of fetal akinesia.

PURPOSE: Fetal akinesia has multiple clinical subtypes with over 160 gene associations, but the genetic etiology is not yet completely understood. METHODS: In this study, 51 patients from 47 unrelated families were analyzed using next-generation sequencing (NGS) techniques aiming to decipher the genomic landscape of fetal akinesia (FA). RESULTS: We have identified likely pathogenic gene variants in 37 cases and report 41 novel variants. Additionally, we report putative pathogenic variants in eight cases including nine novel variants. Our work identified 14 novel disease-gene associations for fetal akinesia: ADSSL1, ASAH1, ASPM, ATP2B3, EARS2, FBLN1, PRG4, PRICKLE1, ROR2, SETBP1, SCN5A, SCN8A, and ZEB2. Furthermore, a sibling pair harbored a homozygous copy-number variant in TNNT1, an ultrarare congenital myopathy gene that has been linked to arthrogryposis via Gene Ontology analysis. CONCLUSION: Our analysis indicates that genetic defects leading to primary skeletal muscle diseases might have been underdiagnosed, especially pathogenic variants in RYR1. We discuss three novel putative fetal akinesia genes: GCN1, IQSEC3 and RYR3. Of those, IQSEC3, and RYR3 had been proposed as neuromuscular disease-associated genes recently, and our findings endorse them as FA candidate genes. By combining NGS with deep clinical phenotyping, we achieved a 73% success rate of solved cases.

Correction: The genomic and clinical landscape of fetal akinesia.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Novel mutations in KMT2B offer pathophysiological insights into childhood-onset progressive dystonia.

Rapid progress has recently been made in the elucidation of the genetic basis of childhood-onset inherited generalized dystonia (IGD) due to the implementation of genomic sequencing methodologies. We identified four patients with childhood-onset IGD harboring novel disease-causing mutations in lysine-specific histone methyltransferase 2B gene (KMT2B) by whole-exome sequencing. The main focus of this paper is to gain novel pathophysiological insights through understanding the molecular consequences of these mutations. The disease course is mostly progressive, evolving from lower limbs into generalized dystonia, which could be associated with dysarthria, dysphonia, intellectual disability, orofacial dyskinesia, and sometimes distinct dysmorphic facial features. In two patients, motor performances improved after bilateral implantation of deep brain stimulation in the globus pallidus internus (GPi-DBS). Pharmacotherapy with trihexyphenidyl reduced dystonia in two patients. We discovered three novel KMT2B mutations. Our analyses revealed that the mutation in patient 1 (c.7463A > G, p.Y2488C) is localized in the highly conserved FYRC domain of KMT2B. This mutation holds the potential to alter the inter-domain FYR interactions, which could lead to KMT2B instability. The mutations in patients 2 and 3 (c.3596_3697insC, p.M1202Dfs*22; c.4229delA, p.Q1410Rfs*12) lead to predicted unstable transcripts, likely to be subject to degradation by non-sense-mediated decay. Childhood-onset progressive dystonia with orofacial involvement is one of the main clinical manifestations of KMT2B mutations. In all, 26% (18/69) of the reported cases have T2 signal alterations of the globus pallidus internus, mostly at a younger age. Anticholinergic medication and GPi-DBS are promising treatment options and shall be considered early.

Correction to: Novel mutations in KMT2B offer pathophysiological insights on childhood-onset progressive dystonia.

Rapid progress has recently been made in the elucidation of the genetic basis of childhood-onset inherited generalized dystonia (IGD) due to the implementation of genomic sequencing methodologies. We identified four patients with childhood-onset IGD harboring novel disease-causing mutations in lysine-specific histone methyltransferase 2B gene (KMT2B) by whole-exome sequencing. The main focus of this paper is to gain novel pathophysiological insights through understanding the molecular consequences of these mutations.The disease course is mostly progressive, evolving from lower limbs into generalized dystonia, which could be associated with dysarthria, dysphonia, intellectual disability, orofacial dyskinesia, and sometimes distinct dysmorphic facial features. In two patients, motor performances improved after bilateral implantation of deep brain stimulation in the globus pallidus internus (GPi-DBS). Pharmacotherapy with trihexyphenidyl reduced dystonia in two patients.We discovered three novel KMT2B mutations. Our analyses revealed that the mutation in patient 1 (c.7463 A > G, p.Y2488C) is localized in the highly conserved FYRC domain of KMT2B. This mutation holds the potential to alter the inter-domain FYR interactions, which could lead to KMT2B instability. The mutations in patients 2 and 3 (c.3602dupC, p.M1202Dfs*22; c.4229delA, p.Q1410Rfs*12) lead to predicted unstable transcripts, likely to be subject to degradation by non-sense mediated decay.Childhood-onset progressive dystonia with orofacial involvement is one of the main clinical manifestations of KMT2B mutations. In all, 26% (18/69) of the reported cases have T2 signal alterations of the globus pallidus internus, mostly at a younger age. Anticholinergic medication and GPi-DBS are promising treatment options and shall be considered early.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Cleft Palate as Distinguishing Feature in a Patient with GABRB3 Epileptic Encephalopathy.

Mutations in GABAA-receptor subunit genes are associated with a heterogeneous spectrum of epilepsies. Patients with epilepsy caused by mutations in a specific GABAA-receptor (GABRA3) occasionally present with orofacial dysmorphism (e.g., cleft palates). While cleft palates have been described in Gabrb3 knockout mice and in humans with GABRB3 variants without epilepsy, the specific combination of epilepsy and cleft palate in humans with GABRB3 mutations has not yet been reported.We describe a patient with epileptic encephalopathy (EE) who presented with therapy-refractory neonatal-onset myoclonic seizures and severe developmental delay. Electroencephalogram showed burst suppression pattern at neonatal age and hypsarrhythmia at infantile age. Initial magnetic resonance imaging was unremarkable. As he additionally presented with a cleft palate, we were curious whether cleft palate and EE had the same genetic origin. Whole exome sequencing of the index patient revealed a novel pathogenic heterozygous de novo mutation in GABRB3 (c.899T > C; p.I300T). In consistency with Gabrb3 knockout mice data, this is the first report of cleft palate in a patient with GABRB3 associated EE.We suggest to add cleft palate to the phenotypic GABRB3 spectrum and to screen for mutations in GABAA-receptors in patients with EE and orofacial dysmorphism.

Genotype-phenotype correlations in ocular manifestations of Marinesco-Sjögren syndrome: Case report and literature review.

PURPOSE: This study aims to present a family with two children with MSS who presented with different ophthalmic features. We also aim to review MSS patients' ocular manifestations to provide a basis for future clinical trials and improve MSS patients' ophthalmologic care. CASE DESCRIPTION: Both patients presented with global developmental delay, microcephaly, cerebellar ataxia, and myopathy. The older sibling had developed bilateral cataracts at the age of six. Her 2 years younger sister interestingly showed bilateral hyperopic refractive error without cataracts yet. Mendeliome sequencing unraveled a novel homozygous frameshift mutation in the SIL1 gene (SIL1, NM_022464.5, c.1042dupG, p.E348Gfs*4), causing MSS. A systematic literature review revealed that cataracts appear in 96% of MSS cases with a mean onset at 3.2 years. Additional frequent ocular features were strabismus (51.6%) and nystagmus (45.2%). CONCLUSION: SIL1-related MSS is associated with marked clinical variability. Cataracts can develop later than neuromuscular features and cognitive signs. Since cataract is a relatively late finding, patients may refer to ophthalmologists for other reasons such as refractive errors, strabismus, or nystagmus. Molecular genetic testing for SIL1 is essential to facilitate early diagnosis in patients with suspected MSS.

Bi-allelic mutations in PRUNE lead to neurodegeneration with spinal motor neuron involvement and hyperCKaemia.

Okur D, Daimagüler HS, Ersen Danyeli A, Tekgül H, Wang H, Wunderlich G, Çirak S, Yis U. Bi-allelic mutations in PRUNE lead to neurodegeneration with spinal motor neuron involvement and hyperCKaemia. Turk J Pediatr 2019; 61: 931-936. We aimed to systematically investigate the neuromuscular involvement of individuals with PRUNE mutations who may have a major spinal motor neuron involvement as part of the PRUNE-associated neurodegenerative phenotype. The complex neurological phenotypes associated with Prune mutations include microcephaly with brain abnormalities, spasticity, seizures, severe developmental delay and developmental regression. We used whole exome sequencing to identify the mutation and electrophysiological and muscle biopsy studies to evaluate the signs of spinal motor neuron involvement. The affected individuals carry homozygous PRUNE mutation (NM_021222.1, c.316G > A, p.D106N), showing the signs of spinal motor neuron involvement supported by electrophysiological and muscle biopsy findings and also persistent high creatine kinase levels. We confirm that individuals with PRUNE mutations may have a major spinal motor neuron involvement as part of the PRUNE-associated neurodegenerative phenotype. The PRUNE gene should be considered in all the individuals with non-5q spinal muscular atrophy. High creatine kinase values may be a part of PRUNE disease spectrum.

Bi-allelic mutations in uncoordinated mutant number-45 myosin chaperone B are a cause for congenital myopathy.

Congenital myopathies (CM) form a genetically heterogeneous group of disorders characterized by perinatal muscle weakness. Here, we report an 11-year old male offspring of consanguineous parents of Lebanese origin. He presented with proximal weakness including Gower's sign, and skeletal muscle biopsy revealed myopathic changes with core-like structures. Whole exome sequencing of this index patient lead to the discovery of a novel genetically defined CM subtype based on bi-allelic mutations in the uncoordinated mutant number-45 myosin chaperone B (UNC45B) NM_173167:c.2261G > A, p.Arg754Gln. The mutation is conserved in evolution and co-segregates within the pedigree with the phenotype, and located in the myosin binding armadillo repeat domain 3 (ARM3), and has a CADD Score of 35. On a multimeric level, UNC45B aggregates to a chain which serves as an assembly line and functions as a "template" defining the geometry, regularity, and periodicity of myosin arranged into muscle thick filaments. Our discovery is in line with the previously described myopathological phenotypes in C. elegans and in vertebrate mutants and knockdown-models. In conclusion, we here report for the first time a patient with an UNC45B mutation causing a novel genetically defined congenital myopathy disease entity.

Clinical outcomes of two patients with a novel pathogenic variant in ASNS: response to asparagine supplementation and review of the literature.

Asparagine synthetase deficiency (ASNSD, OMIM #615574) is a rare autosomal recessive neurometabolic inborn error that leads to severe cognitive impairment. It manifests with microcephaly, intractable seizures, and progressive cerebral atrophy. Currently, there is no established treatment for this condition. In our pediatric cohort, we discovered, by whole-exome sequencing in two siblings from Turkey, a novel homozygous missense mutation in asparagine synthetase at NM_133436.3 (ASNS_v001): c.1108C>T that results in an amino acid exchange p.(Leu370Phe), in the C-terminal domain. After identification of the metabolic defect, treatment with oral asparagine supplementation was attempted in both patients for 24 months. Asparagine supplementation was well tolerated, and no further disease progression was observed during treatment. One of our patients showed mild developmental progress with increased levels of attention and improved nonverbal communication. These results support our hypothesis that asparagine supplementation should be further investigated as a treatment option for ASNSD. We further reviewed all previously reported ASNSD cases with regard for their clinical phenotypes and brain imaging findings to provide an essential knowledge base for rapid diagnosis and future clinical studies.

Long term history of a congenital core-rod myopathy with compound heterozygous mutations in the Nebulin gene.

Mutations in the Nebulin gene (NEB) may cause core-rod myopathy. The large size of the gene so far prevented inclusion of its routine analysis by didesoxy resequencing methodology in the diagnostic regime for muscular dystrophy cases. Here we report a 54-year-old female with a rare histological myopathy presentation of co-occurring cores and rods. The patient reported early childhood onset weakness. Muscle-MRI showed mainly proximal muscle involvement. We identified two compound heterozygous non-sense mutations in NEB (c.19653G > A, p.W6551* exon 127 and c.25441C > T, p.R8481* exon 182) using a comprehensive next generation sequencing (NGS)-based approach named Mendeliome Sequencing. The p.W6551* mutation has not been reported elsewhere. Early diagnosis by NGS shall be chased since even a scoliosis surgery at the age of 18 years had failed to initiate a neurological workup. Rather, cosmetic surgery for facial weakness had been performed recently, albeit with an unsatisfactory outcome.

Childhood onset limb-girdle muscular dystrophies in the Aegean part of Turkey.

The aim of this study is to analyze the epidemiology of the clinical and genetic features of childhood-onset limb-girdle muscular dystrophies (LGMD) in the Aegean part of Turkey. In total fifty-six pediatric cases with LGMD followed in four different pediatric neurology departments in the Aegean region of Turkey were evaluated. Among them, LGMD2C was the most common followed by LGMD2A, LGMD2D, and LGMD2F with equal frequencies. In twenty-eight patients (50%) the diagnosis could be confirmed by genetic analysis, where SGCG proved to be disease-causing in most of the cases. About half of the patients were diagnosed with whole exome or targeted gene sequencing. A positive correlation between muscle biopsy and genetic findings were observed in 11% of the patients. We report one novel frameshifting mutation in TTN. Knowledge on frequencies of childhood-onset limb-girdle muscular dystrophies and related genes in Turkey will lead to a prompt diagnosis of these neuromuscular disorders.