Distribution of mutations in the PEX gene in families with X-linked hypophosphataemic rickets (HYP)

Mutations in the PEX gene at Xp22.1 (phosphate-regulating gene with homologies to endopeptidases, on the X-chromosome), are responsible for X-linked hypophosphataemic rickets (HYP). Homology of PEX to the M13 family of Zn2+ metallopeptidases which include neprilysin (NEP) as prototype, has raised important questions regarding PEX function at the molecular level. The aim of this study was to analyse 99 HYP families for PEX gene mutations, and to correlate predicted changes in the protein structure with Zn2+ metallopeptidase gene function. Primers flanking 22 characterised exons were used to amplify DNA by PCR, and SSCP was then used to screen for mutations. Deletions, insertions, nonsense mutations, stop codons and splice mutations occurred in 83% of families screened for in all 22 exons, and 51% of a separate set of families screened in 17 PEX gene exons. Missense mutations in four regions of the gene were informative regarding function, with one mutation in the Zn2+-binding site predicted to alter substrate enzyme interaction and catalysis. Computer analysis of the remaining mutations predicted changes in secondary structure, N-glycosylation, protein phosphorylation and catalytic site molecular structure. The wide range of mutations that align with regions required for protease activity in NEP suggests that PEX also functions as a protease, and may act by processing factor(s) involved in bone mineral metabolism.      

Limiting factors for cytopathological diagnosis of high-grade squamous intraepithelial lesions: a cytohistological correlation between findings in cervical smears and loop electrical excision procedure.

The present study sought possible factors leading to the cytological diagnosis of atypical squamous cells of uncertain significance (ASCUS) in cases of high-grade squamous intraepithelial lesions (HSIL). Based on retrospective histopathological analysis of loop electrical excision procedure (LEEP) products that diagnosed HSIL, two study groups were randomly selected. The first was consisted of cases with two consecutive Papanicolaou (Pap) smears with the diagnosis of ASCUS. The second (control) group was represented by cases diagnosed as HSIL by cytology. From the Pap smears diagnosed as ASCUS, the sampling limitations was different from control group (P < 0.05). The median size of the largest lesion in each case with ASCUS was 2.66 mm (+/- 1.71 mm). In the control group, the median size of the largest lesion was 5.15 mm (+/-2.58 mm) (P < 0.05). The size of the lesion and sample limitations led patients with cervical intraepithelial neoplasms to be diagnosed as ASCUS for two consecutive times, after a 6-mo period.     

Characterization of gait parameters in patients with Charcot-Marie-Tooth disease

The gait of five patients with Charcot-Marie-Tooth(CMT) disease was analyzed using light-emitting diodes and a force plate. The flexion-extension motions of the hips, knees, and ankles, as well as their moments (vector sums of forces acting at the joints) in the flexion-extension and abduction-adduction planes, were quantified. The gait of the CMT patients showed abnormalities consistent with both distal weakness (ankle dorsi- and plantar-flexors) and weakness of the hip abductor muscles. The latter weakness appeared to produce asymmetric hip moments and truncal instability in the mediolateral plane during ambulation. However, the extent to which the gait was abnormal appeared not to be exclusively related to the severity of the sensorimotor conduction deficits in the peripheral nerves. In the four patients for whom nerve conduction velocity studies were available, decrease in the lower-extremity distal conduction velocities and evoked motor amplitude potentials did not correlate with the severity and extent of the gait abnormalities.     

Diagnosis of pulmonary embolism: Ventilation perfusion scintigraphy versus helical computed tomography pulmonary angiography

The present study compared the accuracy of ventilation perfusion scintigraphy (VQS) and CT pulmonary angiography (CTPA) for the diagnosis of pulmonary embolism. This was a prospective observational study of 112 patients with suspected pulmonary embolism (PE) who could be studied with both investigations within 24 h. Results were compared to final diagnosis at completion of 6-month follow up, using receiver operating characteristic (ROC) analysis. Pulmonary embolism was diagnosed in 27 referred patients (24%). The sensitivity and specificity of VQS and CTPA were similar to that reported from the literature. A normal VQ scan had the highest negative predictive value (100%), while a high-probability VQ scan had the highest positive predictive value (92%). There was no overall difference (area under the ROC curve (AUC)) between VQS (AUC (95% CI) = 0.82 (0.75,0.89)) and CTPA (AUC = 0.88 (0.81,0.94)) for the diagnosis of PE. Among patients with abnormal chest X-rays, CTPA (AUC 0.90 (0.83,0.97)) appeared somewhat better than VQS (AUC 0.78 (0.68,0.88)) but this difference did not reach statistical significance. In this instance, CTPA is at least as accurate as VQS and may provide an opportunity to make alternative diagnoses.     

Assessment of Hageman factor activation in human plasma: quantification of activated Hageman factor-C1 inactivator complexes by an enzyme- linked differential antibody immunosorbent assay

An enzyme-linked immunosorbent assay has been developed for the quantitation of activated Hageman factor-C1 inactivator (HF-C1 INH) complexes. Addition of increasing quantities of either of the major forms of activated Hageman factor (HFa or HFf) to normal plasma or to Hageman factor-deficient plasma leads to a dose-dependent increase in activated HF-C1 INH complexes. As little as 0.5 micrograms/mL of activated HF added to plasma can be detected, corresponding to activation of approximately 2% of plasma HF. The sensitivity of the assay is increased at least tenfold when complexes are formed in HF- deficient plasma, indicating competition between unactivated HF and activated HF-C1 INH complexes for binding to the antibody. Specificity is demonstrated in that addition of activated HF to hereditary angioedema plasma yields less than 1% of the activated HF-C1 INH complex formation obtained with normal plasma. Kaolin activation of HF- deficient plasma yields no detectable complex formation. Kaolin activation of prekallikrein-deficient plasma demonstrates a time- dependent increase in formation of activated HF-C1 INH complex consistent with the ability of HF in this plasma to autoactivate as the time of incubation with the surface is increased. Kaolin treatment of high-molecular weight (HMW) kininogen-deficient plasma yields an even more profound abnormality in the rate of formation of activated HF-C1 INH complexes reflecting the complex role of HMW kininogen in the initiation of contact activation. Although addition of corn inhibitor to plasma prevents activated HF-C1 INH complex formation, it does not inhibit activated HF sufficiently fast to prevent prekallikrein activation.     

A Method to Evaluate Fetal Erythropoiesis from Postnatal Survival of Fetal RBCs

Fetal RBCs are produced during a period of very rapid growth and stimulated erythropoiesis under hypoxic intrauterine conditions. Fetal RBC life span varies with gestational age (GA) and is shorter than that in healthy adults. Due to the special kinetic properties of life span-based survival of human RBCs, a mathematical model-based kinetic analysis of the survival of fetal RBCs shortly after birth provides a unique opportunity to “look backward in time” to evaluate fetal erythropoiesis. This work introduces a novel method that utilizes postnatal in vivo RBC survival data collected within 2 days after birth to study both nonsteady-state (non-SS) in utero RBC production and changing fetal RBC life span over time. The effect of changes in erythropoiesis rate and RBC life span and the effect of multiple postnatal phlebotomies on the RBC survival curves were investigated using model-based simulations. This mathematical model, which considers both changes in the rate of erythropoiesis and RBC life span and which accurately accounts for the confounding effect of multiple phlebotomies, was applied to survival curves for biotin-labeled RBCs from ten anemic very low birth weight preterm infants. The estimated mean fetal RBC production rate scaled by body weight was 1.07 × 10⁷ RBCs/day g, and the mean RBC life span at birth was 52.1 days; these values are consistent with reported values. The in utero RBC life span increased at a rate of 0.51 days per day of gestation. We conclude that the proposed mathematical model and its implementation provide a flexible framework to study in utero non-SS fetal erythropoiesis in newborn infants.KEY WORDS: cord blood RBCs, fetal erythropoiesis, fetal RBC life span, fetal RBC production, red blood cells