Evaluation of the procoagulant activity of endogenous phospholipids in the platelet-free plasma of children with sickle cell disease using functional assays

Background

The mechanisms of hypercoagulability in sickle cell disease (SCD) are poorly understood.

Objective

We aimed to explore the procoagulant activity of endogenous phospholipids (ePL) in the platelet-free plasma of SCD children.

Methods

A factor Xa clotting time (XACT), thrombin generation (TG) and a capture-based assay for the detection of procoagulant microparticles (PMP) were used. Forty three SCD children (35 SS, 6 SC and 2 Sβ +) were evaluated at steady-state and compared to 20 controls. Fourteen patients were also evaluated during vaso-occlusive crisis. TG was performed using 10pM tissue factor without addition of exogenous phospholipids. A control condition was also performed using 10pM tissue factor and 4 μM phospholipids. Percentages of the test/control conditions were calculated for the peak height (% peak), endogenous thrombin potential (% ETP) and velocity index (% VI).

Results

XACT times were shorter, PMP levels, peak height and velocity index of thrombin generation were higher in SCD patients than controls. Lag time and ETP were not different between the two groups. % peak, % ETP and % VI were higher in patients than controls. Significant correlations were found between PMP levels and XACT, also between PMP levels and peak height, velocity index, ETP and their respective percentages to the control condition, but not with lag time. Double heterozygous patients showed intermediate values for XACT and TG parameters. No significant difference was observed when comparing patients at steady-state versus vaso-occlusive crisis.

Conclusion

High procoagulant activity of ePL was observed in the platelet-free plasma of SCD children, probably borne by procoagulant microparticles. This may contribute to a high hemostatic potential and predisposition to thrombotic complications in these patients.     

妇科手术中发现的原发性阑尾癌5例分析

 目的  探讨阑尾癌与右侧附件肿瘤的异同点,以期提高阑尾癌术前诊断率。方法  本文就临床诊断附件肿瘤而行妇科手术中确诊为阑尾癌或伴发腹腔假黏液瘤(pseudomyxoma peritonei,PMP)的5例患者进行分析,总结临床症状、影像学检查、胃肠镜检查、血清肿瘤标志物、术中发现等方面的特征。结果  阑尾癌与右侧附件肿瘤在临床表现、影像学、生化指标等方面有很多共同点,但特异性不足。结论  虽然阑尾肿瘤非常罕见,但对于右侧盆腔肿块要充分考虑阑尾来源的可能性,从影像学等各方面入手可提高阑尾癌的术前诊断率。     

Charcot-Marie-Tooth disease type 1A: morphological phenotype of the 17p duplication versus PMP22 point mutations

Charcot-Marie-Tooth disease type 1A (CMT1A) or hereditary motor and sensory neuropathy type Ia (HMSN type Ia) is an autosomal dominant demyelinating polyneuropathy, which may result from duplications as large as 1.5 Mb on chromosome 17p11.2–p12 encompassing the gene for the peripheral myelin protein PMP22, or from point mutations in this gene. In general, it is not possible to distinguish, by clinical and neurophysiological criteria, the cases associated with the duplication mutation from those associated with point mutations of the PMP22 gene, although the latter tend to be more severe. In this study we demonstrated that the two genotypes exhibit different morphological characteristics. In the PMP22 duplicated cases the mean g-ratio (axon diameter versus fibre diameter) is significantly lower than normal, while in cases of PMP22 point mutations nearly all myelinated fibres have an extremely high g-ratio. In cases with point mutations, onion bulbs are abundantly present from an early age, whereas onion bulbs in the duplicated cases develop gradually in the first years of life. Increase in total transverse fascicular area is most pronounced in the point mutation cases. The differences in pathology between these two very different types of mutations involving the same gene likely reflect differences in pathogenesis and may offer clues in understanding the function of PMP22.     

Cloning of humanneuronatin gene and its localization to chromosome-20q11.2–12: the deduced protein is a novel ‘proteolipid’

Human brain development is a continuum governed by differential gene expression. Therefore, we proceeded to identify genes selectively expressed in the developing brain. Using differential display and library screening, a novel rat cDNA,neuronatin, was identified and used to screen a human fetal brain cDNA library. Humanneuronatin cDNA was isolated and sequenced. The cDNA was 1159 by long and corresponded in size to the 1.25 kb message detected on Northern analysis.Neuronatin mRNA was selectively expressed in human brain during fetal development, but became repressed in adulthood. When studied in the rat,neuronatin mRNA first appeared at mid-gestation in association with the onset of neurogenesis, becoming most pronounced later in development when neuroepithelial proliferation and neuroblast commitment are manifest, and declined postnatally coinciding with the completion of neurogenesis. The deduced protein has two distinct domains, a hydrophobic N-terminal and basic C-terminal rich in arginine residues. Both the amino acid sequence and secondary structure of this amphipathic polypeptide exhibited homology to PMPI and phospholamban, members of the ‘proteolipid’ class of proteins which function as regulatory subunits of membrane channels. Theneuronatin gene, 3973 bases long, contains in its 5′-flanking region a neural restrictive silencer element which may govern neuron-specific expression. Based on screening a somatic cell hybrid panel,neuronatin gene was assigned to chromosome-20. And, using deletion constructs of chromosome-20 and fluorescence in situ hybridization,neuronatin was localized to chromosome-20811.2-12. In conclusion,neuronatin is a novel human gene that is developmentally regulated and expressed in the brain. The deduced protein is a proteolipid that may function as a unique regulator of ion channels during brain development. The definitive localization ofneuronatin to human chromosome 20811.2-12 provides the basis to investigate this gene as a candidate in neuro-developmental diseases that may also map to this region.     

Immunohistochemical staining as supportive diagnostic tool for pseudomyxoma peritonei arising from intraductal papillary mucinous neoplasm: A report of two cases and literature review

Background/ObjectivesPseudomyxoma peritonei (PMP) arising from an intraductal papillary mucinous neoplasm of the pancreas (IPMN) is a rare condition. The diagnosis of IPMN as the origin of PMP is mainly inferred from the clinical course and the exclusion of PMP from other organs. The pathological diagnosis has not yet been established. To evaluate the usefulness of immunohistochemical staining for the diagnosis of the primary lesion of PMP as IPMN.MethodsThere are 2 cases of PMP arising from IPMN between March 2010 and December 2019 at National Center for Global Health and Medicine. A PubMed search that reported PMP arising from IPMN identified 16 additional cases. Diagnostic methods and clinicopathological features of 18 cases were compared.ResultsFour cases including our two cases used immunohistochemical staining for the diagnosis of PMP arising from IPMN. The correspondence of the immunohistochemical staining between PMP and IPMN was shown in the three cases including previously reported two cases and one of our two cases to identify the primary lesion of PMP as IPMN. In addition, we revealed that the comparison of the immunostaining pattern of PMP with the representative immunostaining pattern of the candidate primary lesions is helpful for the diagnosis of the primary lesion of PMP.ConclusionsImmunohistochemical staining is helpful to identify the primary lesion of PMP as IPMN.     

The shifting landscape of genetic testing for Charcot–Marie–Tooth disease

Giant axonal neuropathy (GAN) is a rare pediatric neurodegenerative disease. It is best known for the “giant” axons caused by accumulations of intermediate filaments. The disease is progressive, with onset around age 3 years and death by the third decade of life. GAN results from recessive mutations in the GAN gene encoding gigaxonin, and our analysis of all reported mutations shows that they are distributed throughout the protein structure. Precisely how these mutations cause the disease remains to be determined. In addition to changes in peripheral nerves that are similar to those seen in neuropathies such as Charcot–Marie–Tooth type 2, GAN patients exhibit a wide range of central nervous system signs. These features, corroborated by degeneration of central tracts apparent from postmortem pathology, indicate that GAN is also a progressive neurodegenerative disease. To reflect this phenotype more precisely, we therefore propose that the disease should be more appropriately referred to as “giant axonal neurodegeneration.” Muscle Nerve 50 : 467–476, 2014     

A novel PMP22 insertion mutation causing Charcot–Marie–Tooth disease type 3: A case report

Rationale:Charcot–Marie–Tooth disease (CMT) is a group of hereditary neuropathies with clinical features of muscle atrophy, sensory loss, and foot deformities. CMT is related to a number of genes, such as peripheral myelin protein 22 gene (PMP22). Missense mutations, small deletion mutations, and duplications of PMP22 are common in CMT patients, but few insertion mutation cases of PMP22 have been reported.Patient concerns:A 26-year-old male patient with the complaint of general weakness, peroneal atrophy, and deformities in the extremities visited our hospital. The patient was born with bilateral thumbs and feet dystonia. Additionally, delayed feet arch development and delayed walking was observed when he was a child.Diagnosis:Using whole-exome sequencing and electrophysiological test, we identified a novel insertion mutation of PMP22 ({"type":"entrez-nucleotide","attrs":{"text":"NM_153322","term_id":"1674986609"}}NM_153322, c.54_55insGTGCTG, p.(L19delinsVLL)) in a 26-year-old male patient with peroneal atrophy and nerve conduction was not elicited in electromyography (EMG) study. The Protein Variation Effect Analyzer (PROVEAN) program analysis predicted that the variant is likely to be “deleterious.” SWISS-MODEL program predicted that alpha helix in original location was disrupted by inserted 6 bases, which may account for the occurrence of CMT3.Interventions:The patient received symptomatic and supportive treatments, and routine rehabilitation exercises during hospitalization.Outcomes:The condition of the patient was improved, but the disease could not be cured. At 1- and 3-months follow-up, manifestations of the patient were unchanged, and he could take care of himself.Lessons:Our findings link a novel PMP22 mutation with a clinical diagnosis of CMT3. The link between gene variation and CMT phenotype may help to reveal the structure and function of PMP22 protein and the pathogenesis of CMT. This study adds further support to the heterogeneity of PMP22 related CMT and provides solid functional evidence for the pathogenicity of the p.(L19delinsVLL) PMP22 variant. Moreover, with the development of high-throughput sequencing technology, the combination of next-generation sequencing (NGS) and conventional Sanger sequencing is becoming one of the comprehensive, inexpensive, and convenient tools for genetic diagnosis of CMT.     

Glycans of myelin proteins

Human P0 is the main myelin glycoprotein of the peripheral nervous system. It can bind six different glycans, all linked to Asn⁹³, the unique glycosylation site. Other myelin glycoproteins, also with a single glycosylation site (PMP22 at Asn³⁶, MOG at Asn³¹), bind only one glycan. The MAG has 10 glycosylation sites; the glycoprotein OMgp has 11 glycosylation sites. Aside from P0, no comprehensive data are available on other myelin glycoproteins. Here we review and analyze all published data on the physicochemical structure of the glycans linked to P0, PMP22, MOG, and MAG. Most data concern bovine P0, whose glycan moieties have an MW ranging from 1,294.56 Da (GP3) to 2,279.94 Da (GP5). The pI of glycosylated P0 protein varies from pH 9.32 to 9.46. The most charged glycan is MS2 containing three sulfate groups and one glucuronic acid; whereas the least charged one is the BA2 residue. All glycans contain one fucose and one galactose. The most mannose rich are the glycans MS2 and GP4, each of them has four mannoses; OPPE1 contains five N‐acetylglucosamines and one sulfated glucuronic acid; GP4 contains one sialic acid. Furthermore, human P0 variants causing both gain and loss of glycosylation have been described and cause peripheral neuropathies with variable clinical severity. In particular, the substitution T⁹⁵→M is a very common in Europe and is associated with a late‐onset axonal neuropathy. Although peripheral myelin is made up largely of glycoproteins, mutations altering glycosylation have been described only in P0. This attractive avenue of research requires further study. © 2014 Wiley Periodicals, Inc.     

Comparative study of cephalometric measurements using 3 imaging modalities

Background

The authors conducted a study to compare 2-dimensional (2D) lateral cephalometric radiography (LCR), 2D cone-beam computer tomographic (CBCT)–generated cephalogram and 3-dimensional (3D) CBCT for assessing cephalometric measurements.