RNA-seq analysis, targeted long-read sequencing and in silico prediction to unravel pathogenic intronic events and complicated splicing abnormalities in dystrophinopathy.

Dystrophinopathy is caused by alterations in DMD. Approximately 1% of patients remain genetically undiagnosed, because intronic variations are not detected by standard methods. Here, we combined laboratory and in silico analyses to identify disease-causing genomic variants in genetically undiagnosed patients and determine the regulatory mechanisms underlying abnormal DMD transcript generation. DMD transcripts from 20 genetically undiagnosed dystrophinopathy patients in whom no exon variants were identified, despite dystrophin deficiency on muscle biopsy, were analyzed by transcriptome sequencing. Genome sequencing captured intronic variants and their effects were interpreted using in silico tools. Targeted long-read sequencing was applied in cases with suspected structural genomic abnormalities. Abnormal DMD transcripts were detected in 19 of 20 cases; Exonization of intronic sequences in 15 cases, exon skipping in one case, aberrantly spliced and polyadenylated transcripts in two cases and transcription termination in one case. Intronic single nucleotide variants, chromosomal rearrangements and nucleotide repeat expansion were identified in DMD gene as pathogenic causes of transcript alteration. Our combined analysis approach successfully identified pathogenic events. Detection of diseasing-causing mechanisms in DMD transcripts could inform the therapeutic options for patients with dystrophinopathy.

Stability of amino acids, free and acyl-carnitine in stored dried blood spots.

BACKGROUND: Newborn screening of inborn errors of metabolism using tandem mass spectrometry has become a public health strategy in many developed countries. Retrospective analyses using stored dried blood specimens have been limited, mainly due to a lack of biochemical information on the long-term stability of acylcarnitines and amino acids in stored specimens. We studied the characteristic profiles of the stability of amino acid, free carnitine, and acyl carnitines in dried blood specimens stored in a refrigerator after newborn screening. METHODS: Dried blood specimens from 198 healthy newborns, which had been stored in a refrigerator at 5 °C after newborn screening, were prospectively subjected to tandem mass spectrometry analyses after 1, 3, 6 months, 1 and 2 years of storage. We also retrospectively re-analyzed the stored samples from 90 newborns, which had been analyzed and stored at 5 °C for 4 years. RESULTS: We found that proline (Pro) and tyrosine (Tyr) were stable for 2 years, and that alanine (Ala), arginine (Arg), and phenylalanine (Phe) decayed with linear regression. The C0 increased during the time-course of 2 years, whereas most acylcarnitines gradually decayed and some showed a linear correlation. The retrospective analysis of samples stored for 4 years revealed that Ala, Phe, Pro and Tyr were almost stable, leucine (Leu), valine (Val) decayed with linear regression, C0 increased, and C10, C12, C14, C14:1, C16, C18, C18:1 decreased, while maintaining a linear correlation. CONCLUSIONS: These data suggested that some metabolic parameters from refrigerator-stored dried blood specimens were applicable for the detection of inborn errors of metabolism.

Scour ponds from unusually large tsunamis on a beach-ridge plain in eastern Hokkaido, Japan.

Scour ponds from unusually large tsunamis cut across the crest of a beach ridge in Kiritappu marsh, eastern Hokkaido. No fewer than ten of these ponds were imaged by photogrammetry as elongate topographic depressions as large as 5 m by 30 m. Sediments in these ponds are underlain by unconformities that were detected with ground-penetrating radar and observed directly in cores and a slice sample. Sediment deposits in the ponds contain peat and volcanic ash layers, the ages of which suggest that the scouring occurred during tsunamis generated by spatially extensive thrust ruptures along the southern Kuril trench, most recently during the early seventeenth century and its predecessor during the thirteenth-fourteenth century. Some of the ponds appear to have been formed during one tsunami and refreshed during later successors. This evidence of recurrent erosion suggests that the shoreline may retreat as part of earthquake-related cycles of coastal uplift and subsidence.

Alteration in serum neural cell adhesion molecule in patients of schizophrenia.

INTRODUCTION: The neural cell adhesion molecule (N-CAM) plays important roles in neural migration, synaptogenesis and CNS development. Change of N-CAM fragments in CSF of schizophrenic patients was reported previously, and we aimed to detect difference in circulating N-CAM in the serum of schizophrenic patients and healthy controls. METHODS: Samples were from 14 chronic schizophrenic patients including 3 drug naïve patients and 11 healthy controls. After removal of albumin and globulin, N-CAM fragments were measured by Western blot technique with monoclonal antibody. RESULTS: N-CAM immunoreactive bands were detected primarily at 180, 140, 120, 75, 68 and 52 kDa. Samples from patients and controls showed similar patterns of bands, but schizophrenic patients showed increases or decreases at some bands intensity compared to healthy controls. The 68 kDa/73-75 kDa bands intensity ratio was substantially elevated in schizophrenic patients (0.262+/-0.14 in patients, 0.065+/-0.04 in controls) especially, the three drug naïve patients had a higher value of this ratio compared to the medicated patients. One drug naïve patient showed a decrease in this ratio after one month of antipsychotic medication. CONCLUSIONS: The results suggest elevated membrane turnover and/or abnormalities in the regulation of proteolysis of N-CAM in schizophrenia.