Background
Prion diseases are a group of lethal neurodegenerative conditions that occur when the normal, cellular form of the prion protein (PrPC) is converted into an abnormal, scrapie, form of the ...protein (PrPSc). Disease may be caused by genetic, infectious, or sporadic etiologies. The genetic form of prion disease comprises~10%–15% of all cases. Prion disease is typically inherited in an autosomal dominant manner. The low incidence of disease makes it highly unlikely that a patient would have two different pathogenic variants. However, we recently identified a case in which the patient did have two pathogenic PRNP variants and presented with an atypical phenotype.
Methods
The patient was evaluated at the Washington Hospital Healthcare System in Fremont, CA. The clinical information for this case report was obtained retrospectively. Variants in the PRNP were identified by polymerase chain reaction (PCR) amplification of exon two of the gene followed by bi‐directional sequence analysis. To determine the phase of the identified variants, a restriction enzyme digestion was utilized, followed by sequence analysis of the products. Cerebral spinal fluid (CSF) was analyzed for surrogate markers of prion disease, 14–3–3 and Tau proteins. CSF real‐time quaking‐induced conversion (RT‐QuIC) assays were also performed.
Results
The patient was a compound heterozygote for the well‐characterized c.628G>A (p.Val210Ile) variant and the rare octapeptide deletion of two repeats c.202_249del48 (p.P68_Q83del). Clinically, the patient presented with an early onset demyelinating peripheral neuropathy, followed by later onset cognitive symptoms.
Conclusion
This presentation is reminiscent of prion protein knockout mice whose predominate symptom, due to complete loss of PrP, was late‐onset peripheral neuropathy. To our knowledge this is the first case reported of a patient with prion disease who had two different pathogenic variants in PRNP.
Genetic prion disease is typically inherited in an autosomal dominant manner and the low incidence of disease makes it highly unlikely that a patient would inherit two different pathogenic mutations, however, we recently identified a case in which the patient did indeed inherit two pathogenic PRNP variants and presented with an atypical phenotype. The well‐characterized c.628G>A (p.V210I) variant and the rare octapeptide deletion of two repeats c.202_249del48 (p.P68_Q83del) was identified in the patient. To our knowledge this is the first case of a patient with prion disease who has two different variants in PRNP.
The Gram-negative bacterium Burkholderia pseudomallei causes melioidosis and is a CDC category B bioterrorism agent. Toll-like receptor (TLR)-2 impairs host defense during pulmonary B.pseudomallei ...infection while TLR4 only has limited impact. We investigated the role of TLRs in B.pseudomallei-lipopolysaccharide (LPS) induced inflammation. Purified B.pseudomallei-LPS activated only TLR2-transfected-HEK-cells during short stimulation but both HEK-TLR2 and HEK-TLR4-cells after 24 h. In human blood, an additive effect of TLR2 on TLR4-mediated signalling induced by B.pseudomallei-LPS was observed. In contrast, murine peritoneal macrophages recognized B.pseudomallei-LPS solely through TLR4. Intranasal inoculation of B.pseudomallei-LPS showed that both TLR4-knockout(-/-) and TLR2x4-/-, but not TLR2-/- mice, displayed diminished cytokine responses and neutrophil influx compared to wild-type controls. These data suggest that B.pseudomallei-LPS signalling occurs solely through murine TLR4, while in human models TLR2 plays an additional role, highlighting important differences between specificity of human and murine models that may have important consequences for B.pseudomallei-LPS sensing by TLRs and subsequent susceptibility to melioidosis.
The management options for the autosomal recessive neurodegenerative disorder spinal muscular atrophy (SMA) are evolving; however, their efficacy may require presymptom diagnosis and continuous ...treatment. To identify presymptomatic SMA patients, we created a DNA-based newborn screening assay to identify the homozygous deletions of the SMN1 (survival of motor neuron 1, telomeric) gene observed in 95%-98% of affected patients.
We developed primers that amplify a 52-bp PCR product from homologous regions in the SMN1 and SMN2 (survival of motor neuron 2, centromeric) genes that flank a divergent site at site c.840. Post-PCR high-resolution melt profiling assessed the amplification product, and we used a unique means of melt calibration to normalize profiles. Samples that we had previously characterized for the numbers of SMN1 and SMN2 copies established genotypes associated with particular profiles. The system was evaluated with approximately 1000 purified DNA samples, 100 self-created dried blood spots, and >1200 dried blood spots from newborn screening tests.
Homozygous deletion of SMN1 exon 7 produced a distinctive melt profile that identified SMA patients. Samples with different numbers of SMN1 and SMN2 copies were resolved by their profiles. All samples with homozygous deletions were unambiguously recognized, and no normal sample was misidentified as a positive.
This assay has characteristics suitable for population-based screening. A reliable screening test will facilitate the identification of an SMA-affected cohort to receive early intervention to maximize the benefit from treatment. A prospective screening trial will allow the efficacy of treatment options to be assessed, which may justify the inclusion of SMA as a target for population screening.
ABSTRACT
Introduction: The aim of this study was to determine the safety and therapeutic potential of L‐carnitine and valproic acid (VPA) in infants with spinal muscular atrophy (SMA). Methods: Our ...investigation was an open‐label phase 2 multicenter trial of L‐carnitine and VPA in infants with SMA type I with retrospective comparison to an untreated, matched cohort. Primary outcomes were: safety and adverse events; secondary outcomes were survival, time to death/>16 hours/day of ventilator support; motor outcomes; and maximum ulnar compound motor action potential amplitude. Results: A total of 245 AEs were observed in 35 of the 37 treated subjects (95%). Respiratory events accounted for 49% of all adverse events, resulting in 14 deaths. Survival was not significantly different between treated and untreated cohorts. Discussion: This trial provides evidence that, in infants with SMA type I, L‐carnitine/VPA is ineffective at altering survival. The substantial proportion of infants reaching end‐points within 6 months of enrollment underscores the urgent need for pre‐symptomatic treatment in SMA type I. Muscle Nerve 57: 193–199, 2018
Mutations in the dystrophin gene result in both Duchenne and Becker muscular dystrophies (DMD and BMD). Approximately two-thirds of the affected patients have large deletions or duplications. Using ...the multiplex polymerase chain reaction and Southern blotting techniques, the detection of these larger mutations is relatively straightforward. Detection of the point mutations in the remaining one-third of the patients has been challenging, mainly due to the large gene size and lack of hotspots or prevalent mutations. However, with the addition of some of the newer molecular screening methods, it is becoming more feasible for clinical laboratories to test for point mutations in the larger genes like dystrophin. Here we review the clinical features, describe the mutation distributions, evaluate current molecular strategies, and illustrate how the genetic findings have impacted the current clinical diagnostics of Duchenne and Becker muscular dystrophies.
Myotonic dystrophy type 1 is an autosomal dominant multisystem condition. Myotonic dystrophy type 1 is the result of an unstable CTG expansion in the 3'-untranslated region of the myotonic dystrophy ...protein kinase gene. The age of onset and the severity of the phenotype are roughly correlated with the size of the CTG expansion. The combination of Southern transfer and polymerase chain reaction provides an accurate means of identifying patients affected by myotonic dystrophy type 1. This document follows the outline format of the general Standards and Guidelines for Clinical Genetics Laboratories. It is designed to be a checklist for genetic testing professionals who are already familiar with the disease and the methods of analysis.
Sphingosine-1-phosphate is a bioactive lipid that is mitogenic for human glioma cell lines by signaling through its G protein-coupled receptors. We investigated the role of sphingosine-1-phosphate ...receptors and the enzymes that form sphingosine-1-phosphate, sphingosine kinase (SphK)-1, and -2 in human astrocytomas. Astrocytomas of various histologic grades expressed three types of sphingosine-1-phosphate receptors, S1P1, S1P2, and S1P3; however, no significant correlation with histologic grade or patient survival was detected. Expression of SphK1, but not SphK2, in human astrocytoma grade 4 (glioblastoma multiforme) tissue correlated with short patient survival. Patients whose tumors had low SphK1 expression survived a median 357 days, whereas those with high levels of SphK1 survived a median 102 days. Decreasing SphK1 expression using RNA interference or pharmacologic inhibition of SphK significantly decreased the rate of proliferation of U-1242 MG and U-87 MG glioblastoma cell lines. Surprisingly, RNA interference to knockdown SphK2 expression inhibited glioblastoma cell proliferation more potently than did SphK1 knockdown. SphK knockdown also prevented cells from exiting G1 phase of the cell cycle and marginally increased apoptosis. Thus, SphK isoforms may be major contributors to growth of glioblastoma cells in vitro and to aggressive behavior of glioblastoma multiforme.
Spinal muscular atrophy (SMA) is an autosomal recessive disorder predominately caused by bi-allelic loss of the SMN1 gene. Increased copies of SMN2, a low functioning nearly identical paralog, are ...associated with a less severe phenotype. SMA was recently recommended for inclusion in newborn screening. Clinical laboratories must accurately measure SMN1 and SMN2 copy number to identify SMA patients and carriers, and to identify individuals likely to benefit from therapeutic interventions. Having publicly available and appropriately characterized reference materials with various combinations of SMN1 and SMN2 copy number variants is critical to assure accurate SMA clinical testing. To address this need, the CDC-based Genetic Testing Reference Materials Coordination Program, in collaboration with members of the genetic testing community and the Coriell Institute for Medical Research, has characterized 15 SMA reference materials derived from publicly available cell lines. DNA samples were distributed to four volunteer testing laboratories for genotyping using three different methods. The characterized samples had zero to four copies of SMN1 and zero to five copies SMN2. The samples also contained clinically important allele combinations (eg, zero copies SMN1, three copies SMN2), and several had markers indicative of an SMA carrier. These and other reference materials characterized by the Genetic Testing Reference Materials Coordination Program are available from the Coriell Institute and are proposed to support the quality of clinical laboratory testing.
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