Recessive Nonsense Mutation Research Articles

Neurofilament Light Regulates Axon Caliber, Synaptic Activity, and Organelle Trafficking in Cultured Human Motor Neurons.

Neurofilament light (NFL) is one of the proteins forming multimeric neuron-specific intermediate filaments, neurofilaments, which fill the axonal cytoplasm, establish caliber growth, and provide structural support. Dominant missense mutations and recessive nonsense mutations in the neurofilament light gene (NEFL) are among the causes of Charcot–Marie–Tooth (CMT) neuropathy, which affects the peripheral nerves with the longest axons. We previously demonstrated that a neuropathy-causing homozygous nonsense mutation in NEFL led to the absence of NFL in patient-specific neurons. To understand the disease-causing mechanisms, we investigate here the functional effects of NFL loss in human motor neurons differentiated from induced pluripotent stem cells (iPSC). We used genome editing to generate NEFL knockouts and compared them to patient-specific nonsense mutants and isogenic controls. iPSC lacking NFL differentiated efficiently into motor neurons with normal axon growth and regrowth after mechanical axotomy and contained neurofilaments. Electrophysiological analysis revealed that motor neurons without NFL fired spontaneous and evoked action potentials with similar characteristics as controls. However, we found that, in the absence of NFL, human motor neurons 1) had reduced axonal caliber, 2) the amplitude of miniature excitatory postsynaptic currents (mEPSC) was decreased, 3) neurofilament heavy (NFH) levels were reduced and no compensatory increases in other filament subunits were observed, and 4) the movement of mitochondria and to a lesser extent lysosomes was increased. Our findings elaborate the functional roles of NFL in human motor neurons. NFL is not only a structural protein forming neurofilaments and filling the axonal cytoplasm, but our study supports the role of NFL in the regulation of synaptic transmission and organelle trafficking. To rescue the NFL deficiency in the patient-specific nonsense mutant motor neurons, we used three drugs, amlexanox, ataluren (PTC-124), and gentamicin to induce translational read-through or inhibit nonsense-mediated decay. However, the drugs failed to increase the amount of NFL protein to detectable levels and were toxic to iPSC-derived motor neurons.

A Family Segregating Lethal Primary Coenzyme Q10 Deficiency Due to Two Novel COQ6 Variants.

Primary coenzyme Q10 deficiency-6 (COQ10D6), as a rare autosomal recessive disease caused by COQ6 mutations, is characterized by progressive infantile-onset nephrotic syndrome resulting in end-stage renal failure and sensorineural hearing loss. Here, we report two Chinese siblings with COQ10D6 who primarily presented with severe metabolic acidosis, proteinuria, hypoalbuminemia, growth retardation, and muscle hypotonia and died in early infancy. Using whole-exome sequencing and Sanger sequencing, we identified two rare recessive nonsense mutations in the COQ6 gene segregating with disease in affected family members: c.249C > G (p.Tyr83Ter) and c.1381C > T (p.Gln461Ter), resulting in two truncated protein products. Both mutations are located in a highly conserved area and are predicted to be pathogenic. Indeed, the death of our patients in early infancy indicates the pathogenicity of the p.Tyr83Ter and p.Gln461Ter variants and highlights the significance of the two variants for COQ6 enzyme function, which is necessary for the biosynthesis of coenzyme Q10. In conclusion, we discovered a novel compound heterozygous pathogenic variant of the COQ6 gene as a cause of severe COQ10D6 in the two siblings. Based on the clinical history and genetic characteristics of the patients, our cases expand the genotypic spectrum of COQ10D6 and highlight the heterogeneity and severity of clinical features associated with COQ6 mutations. For patients with clinical manifestations suggestive of COQ10D6, early testing for COQ6 mutations is beneficial for disease diagnosis and therapeutic interventions as well as disease prevention in future generations.

Absence of NEFL in patient-specific neurons in early-onset Charcot-Marie-Tooth neuropathy.

ObjectiveWe used patient-specific neuronal cultures to characterize the molecular genetic mechanism of recessive nonsense mutations in neurofilament light (NEFL) underlying early-onset Charcot-Marie-Tooth (CMT) disease.MethodsMotor neurons were differentiated from induced pluripotent stem cells of a patient with early-onset CMT carrying a novel homozygous nonsense mutation in NEFL. Quantitative PCR, protein analytics, immunocytochemistry, electron microscopy, and single-cell transcriptomics were used to investigate patient and control neurons.ResultsWe show that the recessive nonsense mutation causes a nearly total loss of NEFL messenger RNA (mRNA), leading to the complete absence of NEFL protein in patient's cultured neurons. Yet the cultured neurons were able to differentiate and form neuronal networks and neurofilaments. Single-neuron gene expression fingerprinting pinpointed NEFL as the most downregulated gene in the patient neurons and provided data of intermediate filament transcript abundancy and dynamics in cultured neurons. Blocking of nonsense-mediated decay partially rescued the loss of NEFL mRNA.ConclusionsThe strict neuronal specificity of neurofilament has hindered the mechanistic studies of recessive NEFL nonsense mutations. Here, we show that such mutation leads to the absence of NEFL, causing childhood-onset neuropathy through a loss-of-function mechanism. We propose that the neurofilament accumulation, a common feature of many neurodegenerative diseases, mimics the absence of NEFL seen in recessive CMT if aggregation prevents the proper localization of wild-type NEFL in neurons. Our results suggest that the removal of NEFL as a proposed treatment option is harmful in humans.

Prevalence of the AMHR2 mutation in Miniature Schnauzers and genetic investigation of a Belgian Malinois with persistent Müllerian duct syndrome.

Persistent Müllerian duct syndrome (PMDS) is a sex-limited disorder in which males develop portions of the female reproductive tract. Important consequences of PMDS are cryptorchidism and its sequelae of infertility and increased risk of testicular cancer. Anti-Müllerian hormone (AMH) and its receptor (AMHR2) induce the regression of the Müllerian ducts in male embryos. In Miniature Schnauzer dogs, the genetic basis has been identified as an autosomal recessive nonsense mutation in AMHR2, but the allele frequency of the mutation is unknown. Thus, the primary objective of this study was to estimate the prevalence of the AMHR2 mutation in North American Miniature Schnauzers, in order to ascertain the value of genetic testing in this breed. An additional objective was to determine whether mutations in AMH or AMHR2 were responsible for PMDS in a Belgian Malinois; this would aid development of a genetic test for the Belgian Malinois breed. Genomic DNA from 216 Miniature Schnauzers (including one known PMDS case) was genotyped for the AMHR2 mutation, and DNA from a single PMDS-affected Belgian Malinois was sequenced for all coding exons of AMH and AMHR2. The Miniature Schnauzer cohort had an AMHR2 mutation allele frequency of 0.16 and a carrier genotypic frequency of 0.27. The genetic basis for PMDS in the Belgian Malinois was not determined, as no coding or splicing mutations were identified in either AMH or AMHR2. These findings support a benefit to AMHR2 mutation testing Miniature Schnauzers used for breeding or with cryptorchidism.

Fluoxetine prevents dystrophic changes in a zebrafish model of Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a common and relentlessly progressive muscle disease. Some interventions have been identified that modestly slow progression and prolong survival, but more meaningful therapies are lacking. The goal of this study is to identify new therapeutic pathways for DMD using a zebrafish model of the disease. To accomplish this, we performed a non-biased drug screen in sapje, a zebrafish line with a recessive nonsense mutation in dystrophin. We identified 6 positive hits (out of 640 total drugs tested) by their ability to prevent abnormal birefringence in sapje. Follow-up analyses demonstrated that fluoxetine, a selective serotonin reuptake inhibitor (SSRI), provided the most substantial benefit. Morpholino-based experimentation confirmed that modulation of the serotonin pathway alone can prevent the dystrophic phenotype, and transcriptomic analysis revealed changes in calcium homeostasis as a potential mechanism. In all, we demonstrate that monoamine agonists can prevent disease in a vertebrate model of DMD. Given the safe and widespread use of SSRIs in clinical practice, our study identifies an attractive target pathway for therapy development.

Identification of the first nonsenseCDSNmutation with expression of a truncated protein causing peeling skin syndrome type B

Peeling skin disease (PSD), a generalized inflammatory form of peeling skin syndrome, is caused by autosomal recessive nonsense mutations in the corneodesmosin gene (CDSN). To investigate a novel mutation in CDSN. A 50-year-old white woman showed widespread peeling with erythema and elevated serum IgE. DNA sequencing, immunohistochemistry, Western blot and real-time polymerase chain reaction analyses of skin biopsies were performed in order to study the genetics and to characterize the molecular profile of the disease. Histology showed hyperkeratosis and acanthosis of the epidermis, and inflammatory infiltrates in the dermis. DNA sequencing revealed a homozygous mutation leading to a premature termination codon in CDSN: p.Gly142*. Protein analyses showed reduced expression of a 16-kDa corneodesmosin mutant in the upper epidermal layers, whereas the full-length protein was absent. These results are interesting regarding the genotype-phenotype correlations in diseases caused by CDSN mutations. The PSD-causing CDSN mutations identified heretofore result in total corneodesmosin loss, suggesting that PSD is due to full corneodesmosin deficiency. Here, we show for the first time that a mutant corneodesmosin can be stably expressed in some patients with PSD, and that this truncated protein is very probably nonfunctional.

Thomsen or Becker myotonia? A novel autosomal recessive nonsense mutation in the CLCN1 gene associated with a mild phenotype

We describe a large Brazilian consanguineous kindred with 3 clinically affected patients with a Thomsen myotonia phenotype. They carry a novel homozygous nonsense mutation in the CLCN1 gene (K248X). None of the 6 heterozygote carriers show any sign of myotonia on clinical evaluation or electromyography. These findings confirm the autosomal recessive inheritance of the novel mutation in this family, as well as the occurrence of phenotypic variability in the autosomal recessive forms of myotonia.

A novel nonsense mutation in Rhodopsin gene in two Indonesian Families with Autosomal Recessive Retinitis Pigmentosa

Purpose: To report a novel, identical nonsense mutation in the rhodopsin (RHO) gene in two Indonesian families with autosomal recessive retinitis pigmentosa (arRP).Methods: Mutation screening for the RHO gene was performed in 38 unrelated patients with retinitis pigmentosa (RP) by direct sequencing. Clinical features were also characterized, through complete ophthalmologic examination. Family members of RP patients testing positive for the RHO gene were subjected to genetic and clinical examination. To assess the founder effect in the two families, haplotype analysis also was performed.Results: A novel homozygous nonsense mutation was detected in two patients by a G to A transition at nucleotide position 482 in exon 2 of the RHO gene, resulting in substitution of a tryptophan-to-stop at codon 161 (c.482G>A, p.W161X). Examination of family members of these 2 patients showed that the affected members were homozygous and unaffected carriers were heterozygous for the p.W161X mutation. Haplotype analysis revealed that members of the two families carried the same disease-associated variants in markers (IVS1 RHO and D3S2322). No p.W161X mutations were detected in 45 normal Indonesian subjects, nor were any mutations detected in exons 1–5 of the RHO gene in the remaining 36 RP patients.Conclusion: We detected a novel, recessive nonsense mutation (p.W161X) in the RHO gene of two families through mutation screening of RHO in 38 Indonesian RP patients. Haplotype analysis suggested that p.W161X was the founder mutation.

Enhanced transcription of complement and coagulation genes in the absence of adaptive immunity

A recessive nonsense mutation in the zebrafish recombination activating gene 1 (rag1) gene results in defective V(D)J recombination; however, animals homozygous for this mutation (rag1−/−) are reportedly viable and fertile in standard, nonsterile aquarium conditions but display increased mortality after intraperitoneal injection with mycobacteria. Based on their survival in nonsterile environments, we hypothesized that the rag1−/− zebrafish may possess an “enhanced” innate immune response to compensate for the lack of an adaptive immune system. To test this hypothesis, microarray analyses were used to compare the expression profiles of the intestines and hematopoietic kidneys of rag1 deficient zebrafish to the expression profiles of control (heterozygous) siblings. The expression levels of 12 genes were significantly altered in the rag1−/− kidney including the up regulation of a putative interferon stimulated gene, and the down regulation of genes encoding fatty acid binding protein 10, keratin 5 and multiple heat shock proteins. The expression levels of 87 genes were shown to be significantly altered in the rag1−/− intestine; the majority of these differences reflect increased expression of innate immune genes, including those of the coagulation and complement pathways. Subsequent analyses of orthologous coagulation and complement genes in Rag1−/− mice indicate increased transcription of the complement C4 gene in the Rag1−/− intestine.

Noncell-autonomous photoreceptor degeneration in a zebrafish model of choroideremia

Choroideremia is an X-linked hereditary retinal degeneration resulting from mutations in the Rab escort protein-1 (REP1). The Rep1 protein facilitates posttranslational modification of Rab proteins, which regulate intracellular trafficking in the retinal pigment epithelium (RPE) and photoreceptors and are likely involved in the removal of outer segment disk membranes by the RPE. A critical question for potential treatment of choroideremia is whether photoreceptor degeneration results from autonomous defects in opsin transport within the photoreceptor or as a nonautonomous and secondary consequence of RPE degeneration. To address this question, we have characterized the retinal pathology in zebrafish rep1 mutants, which carry a recessive nonsense mutation in the REP1 gene. Zebrafish rep1 mutants exhibit degeneration of the RPE and photoreceptors and complete loss of visual function as measured by electroretinograms. In the mutant RPE, photoreceptor outer segment material was not effectively eliminated, and large vacuoles were observed. However, opsin trafficking in photoreceptors occurred normally. Mosaic analysis revealed that photoreceptor degeneration was nonautonomous and required contact with the mutant RPE as mutant photoreceptors were rescued in wild-type hosts and wild-type photoreceptors degenerated in mutant hosts. We conclude that mutations in REP1 disrupt cellular processes in the RPE, which causes photoreceptor death as a secondary consequence. These results suggest that therapies that correct the RPE may successfully rescue photoreceptor loss in choroideremia.

Role of troponin T in disease

Several striated muscle myopathies have been directly linked to mutations in contractile and associated proteins. Troponin T (TnT) is one of the three subunits that form troponin (Tn) which together with tropomyosin is responsible for the regulation of striated muscle contraction. All three subunits of cardiac Tn as well as tropomyosin have been associated with hypertrophic cardiomyopathy (HCM). However, TnT accounts for most of the mutations that cause HCM in these regulatory proteins. To date 30 mutations have been identified in the cardiac TnT (CTnT) gene that results in familial HCM (FHC). The CTnT gene has also been associated with familial dilated cardiomyopathy (DCM). CTnT deficiency is lethal due to impaired cardiac development. A recessive nonsense mutation in the gene encoding slow skeletal TnT has been associated with an unusual, severe form of nemaline myopathy among the Old Order Amish. How each mutation leads to the diverse clinical symptoms associated with FHC, DCM or nemaline myopathy is unclear. However, the use of animal model systems, in particular transgenic mice, has significantly increased our knowledge of normal and myopathic muscle physiology. In this review, we focus on the role of TnT in muscle physiology and disease.

Retinitis pigmentosa and related disorders: Phenotypes of rhodopsin and peripherin/RDS mutations

Retinitis pigmentosa comprises a group of clinically variable and genetically heterogeneous inherited disorders of the retina. It is estimated that approximately 1.5 million people throughout the world are affected by this disease. It is a slowly progressive disorder and causes loss of night vision and peripheral visual field in adolescence. It can be inherited through an autosomal dominant, recessive, or X-linked mode; the autosomal dominant form is considered to be the mildest form. Molecular genetic studies on the autosomal dominant disorder have shown that, in some families, genes encoding the rhodopsin and peripherin/RDS map very close to the disease loci identified previously by the systematic linkage analyses. These results, together with the observation that a recessive nonsense mutation in the Drosophila opsin gene causes photoreceptor degeneration, prompted an extensive search for the alterations in the human rhodopsin and peripherin/RDS genes in families with autosomal dominant retinitis pigmentosa. As a result, several distinct rhodopsin and peripherin/RDS mutations have been found in approximately 30% of all autosomal dominant cases. A wide variety of clinical expression of the disorder even within a family with the same mutation, its late onset, slow progression, and cone degeneration clearly suggest that some other factors or genes in addition to rhodopsin are responsible for the phenotypic expression of the disorder. In this article, an attempt is made to highlight some of these recent developments and to correlate the various mutations and the phenotypes.