XBP1 and MIST1 are not sufficient to induce the regulated secretory pathway in parotid cell lines.

BACKGROUND: Xerostomia causes oral infections, tooth decay, and hindered digestion. Crucial to oral homeostasis is the salivary proteome. How these proteins are trafficked through secretory cells into secretory pathways is unknown. Abundant in the saliva is Parotid Secretory Protein (PSP), secreted through the regulated secretory pathway. HYPOTHESIS: The addition of XBP-1 and Mist1, known for differentiation marker regulation, to ParC10 cells will improve stimulated secretion of PSP. METHODS: Fusion clones were transiently or stably transfected into ParC10 cells. Media from non-stimulated and stimulated cells was harvested and luciferase activity was assayed. RESULTS: Stimulated secretion of cypridina-PSP was not significantly higher than non-stimulated secretion; suggesting cypridina-PSP did not enter the regulated secretory pathway. Stably transfected ParC10 showed similar results. CONCLUSION: Cypridina-PSP, XBP-1, and Mist1 did not show stimulated secretion and thus, no cell differentiation was observed. The ParC10 cells lack a regulated secretory pathway

Mutations in the SPG7 gene cause chronic progressive external ophthalmoplegia through disordered mitochondrial DNA maintenance.

Despite being a canonical presenting feature of mitochondrial disease, the genetic basis of progressive external ophthalmoplegia remains unknown in a large proportion of patients. Here we show that mutations in SPG7 are a novel cause of progressive external ophthalmoplegia associated with multiple mitochondrial DNA deletions. After excluding known causes, whole exome sequencing, targeted Sanger sequencing and multiplex ligation-dependent probe amplification analysis were used to study 68 adult patients with progressive external ophthalmoplegia either with or without multiple mitochondrial DNA deletions in skeletal muscle. Nine patients (eight probands) were found to carry compound heterozygous SPG7 mutations, including three novel mutations: two missense mutations c.2221G>A; p.(Glu741Lys), c.2224G>A; p.(Asp742Asn), a truncating mutation c.861dupT; p.Asn288*, and seven previously reported mutations. We identified a further six patients with single heterozygous mutations in SPG7, including two further novel mutations: c.184-3C>T (predicted to remove a splice site before exon 2) and c.1067C>T; p.(Thr356Met). The clinical phenotype typically developed in mid-adult life with either progressive external ophthalmoplegia/ptosis and spastic ataxia, or a progressive ataxic disorder. Dysphagia and proximal myopathy were common, but urinary symptoms were rare, despite the spasticity. Functional studies included transcript analysis, proteomics, mitochondrial network analysis, single fibre mitochondrial DNA analysis and deep re-sequencing of mitochondrial DNA. SPG7 mutations caused increased mitochondrial biogenesis in patient muscle, and mitochondrial fusion in patient fibroblasts associated with the clonal expansion of mitochondrial DNA mutations. In conclusion, the SPG7 gene should be screened in patients in whom a disorder of mitochondrial DNA maintenance is suspected when spastic ataxia is prominent. The complex neurological phenotype is likely a result of the clonal expansion of secondary mitochondrial DNA mutations modulating the phenotype, driven by compensatory mitochondrial biogenesis

Transmission of Mitochondrial DNA Diseases and Ways to Prevent Them

Recent reports of strong selection of mitochondrial DNA (mtDNA) during transmission in animal models of mtDNA disease, and of nuclear transfer in both animal models and humans, have important scientific implications. These are directly applicable to the genetic management of mtDNA disease. The risk that a mitochondrial disorder will be transmitted is difficult to estimate due to heteroplasmy—the existence of normal and mutant mtDNA in the same individual, tissue, or cell. In addition, the mtDNA bottleneck during oogenesis frequently results in dramatic and unpredictable inter-generational fluctuations in the proportions of mutant and wild-type mtDNA. Pre-implantation genetic diagnosis (PGD) for mtDNA disease enables embryos produced by in vitro fertilization (IVF) to be screened for mtDNA mutations. Embryos determined to be at low risk (i.e., those having low mutant mtDNA load) can be preferentially transferred to the uterus with the aim of initiating unaffected pregnancies. New evidence that some types of deleterious mtDNA mutations are eliminated within a few generations suggests that women undergoing PGD have a reasonable chance of generating embryos with a lower mutant load than their own. While nuclear transfer may become an alternative approach in future, there might be more difficulties, ethical as well as technical. This Review outlines the implications of recent advances for genetic management of these potentially devastating disorders

Clinical Effectiveness of Telemedicine-Based Pediatric Genetics Care

BACKGROUND AND OBJECTIVES: Telemedicine may increase access to medical genetics care. However, in the pediatric setting, how telemedicine may affect the diagnostic rate is unknown, partially because of the perceived importance of the dysmorphology physical examination. We studied the clinical effectiveness of telemedicine for patients with suspected or confirmed genetic conditions. METHODS: We conducted a retrospective cohort study of outpatient encounters before and after the widespread implementation of telemedicine (N = 5854). Visit types, diagnoses, patient demographic characteristics, and laboratory data were acquired from the electronic health record. Patient satisfaction was assessed through survey responses. New molecular diagnosis was the primary end point. RESULTS: Patients seen by telemedicine were more likely to report non-Hispanic White ancestry, prefer to speak English, live in zip codes with higher median incomes, and have commercial insurance (all P \u3c .01). Genetic testing was recommended for more patients evaluated by telemedicine than in person (79.5% vs 70.9%; P \u3c .001). Patients seen in person were more likely to have a sample collected, resulting in similar test completion rates (telemedicine, 51.2%; in person, 55.1%; P = .09). There was no significant difference in molecular diagnosis rate between visit modalities (telemedicine, 13.8%; in person, 12.4%; P = .40). CONCLUSIONS: Telemedicine and traditional in-person evaluation resulted in similar molecular diagnosis rates. However, improved methodologies for remote sample collection may be required. This study reveals the feasibility of telemedicine in a large academic medical genetics practice and is applicable to other pediatric specialties with perceived importance of physical examination

Accurate mitochondrial DNA sequencing using off-target reads provides a single test to identify pathogenic point mutations

PURPOSE: Mitochondrial disorders are a common cause of inherited metabolic disease and can be due to mutations affecting mitochondrial DNA or nuclear DNA. The current diagnostic approach involves the targeted resequencing of mitochondrial DNA and candidate nuclear genes, usually proceeds step by step, and is time consuming and costly. Recent evidence suggests that variations in mitochondrial DNA sequence can be obtained from whole-exome sequence data, raising the possibility of a comprehensive single diagnostic test to detect pathogenic point mutations. METHODS: We compared the mitochondrial DNA sequence derived from off-target exome reads with conventional mitochondrial DNA Sanger sequencing in 46 subjects. RESULTS: Mitochondrial DNA sequences can be reliably obtained using three different whole-exome sequence capture kits. Coverage correlates with the relative amount of mitochondrial DNA in the original genomic DNA sample, heteroplasmy levels can be determined using variant and total read depths, and-providing there is a minimum read depth of 20-fold-rare sequencing errors occur at a rate similar to that observed with conventional Sanger sequencing. CONCLUSION: This offers the prospect of using whole-exome sequence in a diagnostic setting to screen not only all protein coding nuclear genes but also all mitochondrial DNA genes for pathogenic mutations. Off-target mitochondrial DNA reads can also be used to assess quality control and maternal ancestry, inform on ethnic origin, and allow genetic disease association studies not previously anticipated with existing whole-exome data sets