Estimating the relative frequency of leukodystrophies and recommendations for carrier screening in the era of next‐generation sequencing

Leukodystrophies are a heterogeneous group of heritable disorders characterized by abnormal brain white matter signal on magnetic resonance imaging (MRI) and primary involvement of the cellular components of myelin. Previous estimates suggest the incidence of leukodystrophies as a whole to be 1 in 7,000 individuals, however the frequency of specific diagnoses relative to others has not been described. Next generation sequencing approaches offer the opportunity to redefine our understanding of the relative frequency of different leukodystrophies. We assessed the relative frequency of all 30 leukodystrophies (associated with 55 genes) in more than 49,000 exomes. We identified a relatively high frequency of disorders previously thought of as very rare, including Aicardi Goutières Syndrome, TUBB4A‐related leukodystrophy, Peroxisomal biogenesis disorders, POLR3‐related Leukodystrophy, Vanishing White Matter, and Pelizaeus‐Merzbacher Disease. Despite the relative frequency of these conditions, carrier‐screening laboratories regularly test only 20 of the 55 leukodystrophy‐related genes, and do not test at all, or test only one or a few, genes for some of the higher frequency disorders. Relative frequency of leukodystrophies previously considered very rare suggests these disorders may benefit from expanded carrier screening.     

A flow cytometric assay for the detection of antiphospholipid antibodies

The data and the results obtained at the above symposium show that the flow cytometric method closely correlates with the standardized aCL bench ELISA and with the APhL ELISA kit. The aCL/aPS FACS kit is comparable in sensitivity to the APhL ELISA kit and standard aCL bench ELISA, but the aCL/aPS FACS kit is more specific than the standard anticardiolipin assay (particularly for the determination of aPS). In summary, the aCL/aPS FACS kit enables rapid (total run time < or = 1 hr) and simultaneous determination of aCL (aCL) and anti-PS antibodies of the IgG and IgM classes and combines good sensitivity and specificity in a single assay. The method is reproducible (intraassay variations < 5%). Furthermore, it is easy to transform into a partially or fully mechanized process and is well suited for laboratories that test large numbers of samples daily. The assay promises to be useful not only for detecting positive APS sera, but also in evaluating the significance of phospholipid specificity and antibody isotypes in patients with APS.     

Antimicrobial resistance of Salmonella serotypes isolated from slaughter-age pigs and environmental samples

The aim of this study was to determine the antimicrobial resistance patterns of Salmonella strains isolated from slaughter-age pigs and environmental samples collected at modern swine raising facilities in Brazil. Seventeen isolates of six serotypes of Salmonella enterica subsp. enterica were isolated out of 1,026 collected samples: Salmonella Typhimurium (1), Salmonella Agona (5), Salmonella Sandiego (5), Salmonella Rissen (1), Salmonella Senftenberg (4), and Salmonella Javiana (1). Resistance patterns were determined to extended-spectrum penicillin (ampicillin), broad-spectrum cephalosporins (cefotaxime and ceftriaxone), aminoglycosides (streptomycin, neomycin, gentamicin, amikacin, and tobramycin), narrow-spectrum quinolone (nalidixic acid), broad-spectrum quinolone (ciprofloxacin and norfloxacin), tetracycline, trimethoprim, and chloramphenicol. Antimicrobial resistance patterns varied among serotypes, but isolates from a single serotype consistently showed the same resistance profile. All isolates were resistant to tetracycline, streptomycin, and nalidixic acid. One isolate, Salmonella Rissen, was also resistant to cefotaxime and tobramycin. All serotypes were susceptible to ceftriaxone, norfloxacin, ciprofloxacin, ampicillin, gentamicin, and chloramphenicol. The high resistance to tetracycline and streptomycin may be linked to their common use as therapeutic drugs on the tested farms. No relation was seen between nalidixic acid and fluoroquinolone resistance.     

Partitioning of whey proteins, bovine serum albumin and porcine insulin in aqueous two-phase systems

Partitioning of the proteins from cheese whey, bovine serum albumin and porcine insulin were analysed using aqueous two-phase systems (ATPS) prepared with PEG-phosphate, PEG-citrate and PEG-maltodextrin (MD). Proteins were quantified through one of the following methods: FPLC, Bradford and spectrophotometry at 280 nm. Results showed that whey proteins partitioned unevenly on the phases of the systems used, with alpha-lactoalbumin (alpha-La) concentrated in the upper phase and beta-lactoglobulin (beta-Lg) in the lower. Albumin in PEG-MD systems concentrated in the MD-rich lower phase. Porcine insulin showed great affinity with the PEG-rich phase, its partition coefficient was always over 10 and increases with PEG molecular mass.     

Cell-surface sialoglycoconjugate structures in wild-type and mutant Crithidia fasciculata

The cell-surface expression of sialoglycoconjugate structures in wild-type Crithidia fasciculata and its TFR^R1 drug-resistant mutant was analyzed with the aid of an influenza C virus strain, lectin, enzymatic treatment, and flow cytofluorimetry analysis probed with fluorescein isothiocyanate-labeled (FITC) lectins. 9-O-Acetyl-N-acetyl neuraminic acid (Neu5,9Ac₂) structures mediate influenza C virus cell-binding. The SAα2,3Gal and SAα2,6Gal sequences are specifically recognized by Maackia amurensis (MAA) and Sambucus nigra (SNA) lectins, respectively. On the basis of these param- eters the TFR^R1 mutant strain of C. fasciculata was found to contain exposed sialoglycoconjugates bearing Neu5,9Ac₂ surface structures. After the removal of sialic acid residues by neuraminidase activity the marked increases in PNA (peanut agglutinin)-mediated agglutinating activity showed that those acidic units on C. fasciculata cells were glycosidically linked to d-galactose. The bond involves SAα2,6Gal and SAα2,3Gal linkages as suggested by the use of FITC-SNA and FITC-MAA lectins, respectively. Both SAα2,3Gal and SAα2,6Gal sequences were preferentially expressed by the TFR^R1 mutant. The SAα2,6 linkage markedly predominated. In the TFR^R1 mutant, but not in wild-type cells, two distinct populations of cells were distinguished by reactivity with FITC-SNA, one of which was enriched with surface SAα2,6Gal sequences. These diverse findings suggest that sialoglycoconjugate structures present on the flagellate surface may be associated with mutation and the cell growth cycle in C. fasciculata. Received: 17 September 1998 / Accepted: 22 October 1998     

A murine plasmacytoma with a variant (15;16)(D2/3;B1) translocation that involves the c-myc and lambda light chain gene-carrying chromosomes

A murine plasmacytoma (MPC) with a reciprocal translocation between chromosomes 15 and 16 with breakpoints in 15D2/3 and 16B1 is reported. The breakpoint on chromosome 15 is identical to the breakpoint in the MPC-associated typical (12;15) and kappa variant (6;15) translocation. Therefore it probably involves the c-myc gene as well. Unlike the Burkitt lymphoma (BL) system, a lambda/myc variant translocation has not been described in the MPC system. Chromosome 16 is known to carry the lambda gene. Therefore, the 15;16 translocation probably represents the "missing" lambda/myc variant in MPC, suggesting that the lambda gene is localized at 16B1.     

Indoor mite allergens in patients with respiratory allergy living in Porto, Portugal

We investigated the levels of mite allergens (Der p 1, Der f 1, Der 2, and Lep d 1) in dust samples from the homes of 59 patients with asthma, 36 sensitized to house-dust mites (HDM) and 23 to grass pollen (controls), living in Porto, northern Portugal. The relationship between exposure and sensitization to HDM and the influence of housing conditions on mite-allergen levels were also evaluated. Der p 1 (median 9.2 μg/g) and Der 2 (4.6 μg/g) were the main allergens, while Der f 1 and Lep d 1 levels were always <1 μg/g dust and undetectable in 11% and 47% of samples, respectively. All HDM-sensitized asthmatics were exposed to Der p 1 levels >2 μg/g and their homes contained significantly higher levels of Der p 1 (median 12.5 vs 6.4 μg/g; P=0.008) and Der 2 (6.2 vs 3.0 μg/g; P=0.004) when compared to the control group. A significant correlation was observed between the exposure to Der p 1 and the wheal area at skin testing with the Dermatophagoides pteronyssinus (Dp) extract (P=0.01) as well as with serum specific IgE levels to Dp (P=0.03). Patients with higher levels of serum specific IgE (≥17.5 HRU/ml) were also more frequently exposed to Der p 1 levels ≥10 (μg/g (P=0.002). Old homes, presence of carpets, and signs of dampness were conditions associated with significantly higher levels of mite allergens. In conclusion, we found high levels of Der p 1 and Der 2 particularly in the homes of HDM-sensitized patients and we confirm the relationship between exposure and sensitization to HDM, assessed by both in vivo and in vitro methods. In addition to a favorable outdoor climate, we found in our region housing conditions propitious to mite growth, suggesting that specific geographic characteristics must also be taken into account for the correct planning of mite-avoidance measures.     

Stromal cell-derived factor-1 production by spleen cells is affected by nitric oxide in protective immunity against blood-stage Plasmodium chabaudi CR in C57BL/6j mice

Malaria, a major endemic tropical disease, is caused by the infection of blood cells by Plasmodium protozoa. Most patients control their parasitemia by a not fully understood spleen-dependent mechanism. SDF-1alpha is a chemokine produced by stromal cells such as reticular spleen cells. Nitric oxide (NO) has several immune functions, including killing of intracellular pathogens and its function in malaria is debated. We have previously shown that SDF-1alpha production peaks during the ascending parasitemia in Plasmodium chabaudi infection and its supplementation in lethal models could reduce the parasitemia. In the present study, we analyzed SDF-1 production by spleen cells as related to NO metabolism in the P. chabaudi rodent malaria model using IFN-gamma; TNFR and iNOS-knockout mice or iNOS-blocked, L-NAME- or aminoguanidine-treated mice. Parasitemia and production of SDF-1alpha and SDF-1beta were determined by RT-PCR. In vitro NO production by spleen adherent cells was also tested. The data showed that parasitemia was less intense in both iNOS(-/-) or NO-inhibited mice than in controls, with increased and long-lasting production of SDF-1alpha mRNA. In the absence of cytokines involved in the final regulation of NO production by effector cells, as is the case for TNFR(-/-) and GKO mice, the infection progressed in an uncontrolled manner regardless of SDF-1alpha production, suggesting that these cytokines must be involved in the control of parasitemia after the SDF-1alpha dependent process. The SDF-1beta isoform was constitutive in all experiments, with elevated levels only clearly seen in TNFR(-/-) mice. We conclude that SDF-1 is involved in the promotion of parasitemia control in malaria, and excessive NO could affect its production.