Ca2+ and phospholipid-dependent protein kinase (protein kinase C) activity is not necessarily required for secretion by human neutrophils

Ca2+-dependent and phospholipid-dependent protein kinase (PKC) is a receptor for and is activated by phorbol esters. This enzyme is reportedly involved in the mechanism of superoxide anion (O2-) production and the release of intracellular granule contents from human neutrophils. As previously reported by others, we found that greater than 75% of the total cellular PKC activity existed in a soluble form in untreated neutrophils and that this activity was enhanced in a dose- dependent manner by phorbol 12-myristate 13-acetate (PMA) and by phorbol 12,13-dibutyrate (PDBu). Furthermore, mezerein, an analogue of PMA that is thought to be a competitive inhibitor, did not activate PKC, and on the contrary, inhibited PMA-stimulated activity in a dose- dependent manner. Pretreatment of intact neutrophils with PMA or PDBu caused the "translocation" of PKC activity to the insoluble cell fraction; PKC translocation was not detected after mezerein stimulation at any of the tested concentrations. Neither did mezerein cause an increase in intracellular Ca2+, as monitored by Quin 2 fluorescence. Both phorbol esters and mezerein stimulated intact neutrophils to generate O2- and release lysosomal enzymes into the extracellular medium. Finally sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis demonstrated key differences in the patterns of endogenous phosphoproteins of neutrophils stimulated with phorbol as compared with mezerein. We therefore suggest that PKC activation may not be the only pathway required to elicit neutrophil responses.     

Favorably skewed X‐inactivation accounts for neurological sparing in female carriers of Menkes disease

Desai V, Donsante A, Swoboda KJ, Martensen M, Thompson J, Kaler SG. Favorably skewed X‐inactivation accounts for neurological sparing in female carriers of Menkes disease. Classical Menkes disease is an X‐linked recessive neurodegenerative disorder caused by mutations in ATP7A, which is located at Xq13.1‐q21. ATP7A encodes a copper‐transporting P‐type ATPase and plays a critical role in development of the central nervous system. With rare exceptions involving sex chromosome aneuploidy or X‐autosome translocations, female carriers of ATP7A mutations are asymptomatic except for subtle hair and skin abnormalities, although the mechanism for this neurological sparing has not been reported. We studied a three‐generation family in which a severe ATP7A mutation, a 5.5‐kb genomic deletion spanning exons 13 and 14, segregated. The deletion junction fragment was amplified from the proband by long‐range polymerase chain reaction and sequenced to characterize the breakpoints. We screened at‐risk females in the family for this junction fragment and analyzed their X‐inactivation patterns using the human androgen‐receptor (HUMARA) gene methylation assay. We detected the junction fragment in the proband, two obligate heterozygotes, and four of six at‐risk females. Skewed inactivation of the X chromosome harboring the deletion was noted in all female carriers of the deletion (n = 6), whereas random X‐inactivation was observed in all non‐carriers (n = 2). Our results formally document one mechanism for neurological sparing in female carriers of ATP7A mutations. Based on review of X‐inactivation patterns in female carriers of other X‐linked recessive diseases, our findings imply that substantial expression of a mutant ATP7A at the expense of the normal allele could be associated with neurologic symptoms in female carriers of Menkes disease and its allelic variants, occipital horn syndrome, and ATP7A‐related distal motor neuropathy.     

Germline mutation in BRAF codon 600 is compatible with human development: de novo p.V600G mutation identified in a patient with CFC syndrome

Champion KJ, Bunag C, Estep AL, Jones JR, Bolt CH, Rogers RC, Rauen KA, Everman DB. Germline mutation in BRAF codon 600 is compatible with human development: de novo p.V600G mutation identified in a patient with CFC syndrome. BRAF, the protein product of BRAF, is a serine/threonine protein kinase and one of the direct downstream effectors of Ras. Somatic mutations in BRAF occur in numerous human cancers, whereas germline BRAF mutations cause cardio‐facio‐cutaneous (CFC) syndrome. One recurrent somatic mutation, p.V600E, is frequently found in several tumor types, such as melanoma, papillary thyroid carcinoma, colon cancer, and ovarian cancer. However, a germline mutation affecting codon 600 has never been described. Here, we present a patient with CFC syndrome and a de novo germline mutation involving codon 600 of BRAF, thus providing the first evidence that a pathogenic germline mutation involving this critical codon is not only compatible with development but can also cause the CFC phenotype. In vitro functional analysis shows that this mutation, which replaces a valine with a glycine at codon 600 (p.V600G), leads to increased ERK and ELK phosphorylation compared to wild‐type BRAF but is less strongly activating than the cancer‐associated p.V600E mutation.     

To tell or not to tell – what to do about p.C282Y heterozygotes identified by HFE screening

Hereditary hemochromatosis (HH) is a common preventable disorder of iron overload that can result in liver cirrhosis and reduced lifespan. Most HH is due to homozygosity for the HFE p.C282Y substitution. We conducted a study of screening for p.C282Y in high schools where p.C282Y heterozygotes (CY) individuals were informed of their genotype by letter. We studied whether these individuals understood the implications of their genotype, whether this resulted in anxiety or reduced health perception and whether cascade testing was higher in families of CY than wild‐type homozygous (CC) individuals. We found 586 of 5757 (1 in 10) screened individuals were CY. One month after receiving their result, 83% correctly answered that they have one copy of p.C282Y. There was no adverse change in anxiety or health perception from prior to screening to 1 month after receiving results. Significantly more family members of CY individuals than CC individuals were informed about HH and had testing for HH. In conclusion, we found that informing CY individuals of their genotype does not increase anxiety and the implications are generally well understood. This leads to cascade testing in a minority of families. CY individuals should be informed of their genetic status when identified by population screening.     

Neurological symptoms in children with intussusception

Aim:  The classical combination of abdominal pain, vomiting, rectal blood loss and a palpable abdominal mass is only present in a minority of children with intussusception. Neurological signs and symptoms have been described, but are not a well understood phenomenon. We performed a retrospective study to ascertain the frequency and nature of these symptoms and to describe the characteristics of the patients presenting in this atypical way.
Methods:  The records of 58 children presenting with intussusception from 2003 to 2008 were reviewed for abdominal and neurological signs and symptoms, duration of symptoms and effectiveness of treatment.
Results:  In 10 out of 58 patients (17%), one or more neurological symptoms were recorded at presentation, with lethargy being the most frequent, followed by hypotonia and fluctuating consciousness. The patients with neurological abnormalities were significantly younger and presented with a shorter duration of symptoms. Therapy was more invasive, although not statistically significant, in this patient category.
Conclusion:  Intussusception should be considered in the differential diagnosis in young children presenting with lethargy, hypotonia and/or sudden alterations of consciousness even in the absence of the classical symptoms of intussusception.