Mutations in the Chediak-Higashi syndrome gene (CHS1) indicate requirement for the complete 3801 amino acid CHS protein

Chediak-Higashi syndrome (CHS) is a rare, usually fatal, autosomal recessive disorder characterized by severe immunologic defects, reduced pigmentation, progressive neurologic dysfunction and a bleeding diathesis. The hallmark of CHS is giant organelles and giant granules in many different cell types, most likely the result of defective trafficking of specific organellar and granular proteins necessary for the normal genesis, structure or function of these cytoplasmic components. The CHS1 gene has recently been identified and shown to be homologous to the beige locus of the mouse; however, there has been disagreement as to the length of the functional CHS1 mRNA and protein. Here we report homozygous CHS1 gene mutations in two of the original probands we used to map the gene to 1q42-q44. One of these, a frameshift at codon 3197, supports our assertion that the functional CHS protein is a predicted 3801 amino acid polypeptide encoded by a 13.5 kb mRNA.      

In-vitro development of refrozen mouse embryos

To evaluate the effects of sequential, repetitive freezing on their in- vitro development, mouse embryos at the eight- to 16-cell stage were subjected to one of five treatments. They were (i) cultured as unfrozen controls, (ii) frozen once and cultured, (iii) subjected to two consecutive freeze-thaw cycles, (iv) frozen and thawed, and then cultured for 18-30 h before being frozen a second time, and (v) frozen three times in succession without being cultured. To assess their functional survival after freezing and thawing, all embryos were cultured in vitro to the hatched blastocyst stage in Whitten's medium. In one experiment, hatched embryos that developed after one, two or three cycles of freezing and thawing were stained with Hoechst 33342 to determine their mean cell number. More embryos of the culture control group and the once-frozen group developed into hatching blastocysts than those of the refrozen groups. There was no difference in the second post-thaw rate of in-vitro development for embryos refrozen with the culture-refreeze or direct-refreeze procedure. Furthermore, there was no difference among in-vitro development rates for embryos frozen two or three times. However, among those embryos subjected to repeated cycles of freezing and thawing that did not survive, there was a considerable amount of damage to their zonae pellucidae. Furthermore, frozen mouse embryos had fewer cells per embryo at the time of hatching than the unfrozen embryos. Nevertheless, these results demonstrate that mouse embryos can survive even three successive freeze-thaw cycles yet still be capable of in-vitro development.      

Assessment of a radioisotopic assay for vitamin B12 using an intrinsic factor preparation with R proteins blocked by vitamin B12 analogues

A competitive protein binding radioassay kit for serum vitamin B₁₂ has been assessed. Precision, linearity, sensitivity, and specificity have been found to be satisfactory. Falsely-normal assay results in patients with vitamin B₁₂ deficiency have not been observed.     

Insights into the genetics of severe congenital neutropenia

HAX1 deficiency causes autosomal recessive severe congenital neutropenia (Kostmann disease)
Klein et al. (2007)
Nature Genetics 39: 86–92     

Evaluation of a high-throughput repetitive-sequence-based PCR system for DNA fingerprinting of Mycobacterium tuberculosis and Mycobacterium avium complex strains

Repetitive-sequence-based PCR (rep-PCR) is useful for generating DNA fingerprints of diverse bacterial and fungal species. Rep-PCR amplicon fingerprints represent genomic segments lying between repetitive sequences. A commercial system that electrophoretically separates rep-PCR amplicons on microfluidic chips, and provides computer-generated readouts of results has been adapted for use with Mycobacterium species. The ability of this system to type M. tuberculosis and M. avium complex (MAC) isolates was evaluated. M. tuberculosis strains (n = 56) were typed by spoligotyping with rep-PCR as a high-resolution adjunct. Results were compared with those generated by a standard approach of spoligotyping with IS6110-targeted restriction fragment length polymorphism (IS6110-RFLP) as the high-resolution adjunct. The sample included 11 epidemiologically and genotypically linked outbreak isolates and a population-based sample of 45 isolates from recent immigrants to Seattle, Wash., from the African Horn countries of Somalia, Eritrea, and Ethiopia. Twenty isolates exhibited unique spoligotypes and were not analyzed further. Of the 36 outbreak and African Horn isolates with nonunique spoligotypes, 23 fell into four clusters identified by IS6110-RFLP and rep-PCR, with 97% concordance observed between the two methods. Both approaches revealed extensive strain heterogeneity within the African Horn sample, consistent with a predominant pattern of reactivation of latent infections in this immigrant population. Rep-PCR exhibited 89% concordance with IS1245-RFLP typing of 28 M. avium subspecies avium strains. For M. tuberculosis as well as M. avium subspecies avium, the discriminative power of rep-PCR equaled or exceeded that of RFLP. Rep-PCR also generated DNA fingerprints from M. intracellulare (n = 8) and MAC(x) (n = 2) strains. It shows promise as a fast, unified method for high-throughput genotypic fingerprinting of multiple Mycobacterium species.     

Indoor allergens and longitudinal FEV 1 decline in older adults: The Normative Aging Study

We investigated the relationship between home allergen exposure and decline in FEV₁ in 10 asthmatic and 30 randomly selected, age-matched, nonasthmatic participants in the Normative Aging Study. We defined asthma as subject-reported wheezing apart from colds, with either a physician's diagnosis of asthma or a methacholine PD₂₀ FEV₁ of 8.6 μmol or less. We examined the relationship between the annual decline in FEV ₁ and the concentrations of the cockroach (Blattella germanica) allergens Bla g 1 and Bla g 2, the dust mite (Dermatophagoides pteronyssinus and Dermatophagoides farinae) allergens Der p 1 and Der f 1, and the cat (Felis domesticus) allergen Fel d 1 in house dust specimens. Bla g 1 (–79.8 ml/yr, p = 0.0006) and Bla g 2 (–40.81 ml/yr, p = 0.0004) were significant predictors of decline in FEV₁ after adjustment for age, smoking, and baseline FEV₁. These results were unchanged after elimination of the asthmatic subjects from the analysis. We conclude that cockroach allergen levels in homes is a risk factor for accelerated decline in FEV₁ independent of airway responsiveness. (J Allergy Clin Immunol 1998;101:720–5.)     

Feasibility study of repeated fluorescent in-situ hybridization in the same human blastomeres for preimplantation genetic diagnosis

In order to increase the number of chromosomes examined in each blastomere, we have developed a repeated fluorescent in-situ hybridization (FISH) procedure by which six or more chromosomes can be analysed per blastomere of a human embryo. Three consecutive FISH procedures with directly-labelled fluorescent Vysis DNA probes were carried out for examination of chromosomes X, Y, 11, 13, 18 and 21 in the same blastomeres (n = 126) and lymphocytes (n = 164). Based on the initial number of nuclei, the percentages of nuclear loss and presence of signals were 3 and 92% respectively in blastomeres; 6 and 91% respectively in lymphocytes after the first FISH; 7 and 87% respectively in blastomeres and 10 and 86% respectively in lymphocytes, after the second FISH. These percentages were 13 and 78% respectively in blastomeres and 14 and 81% respectively in lymphocytes after the third FISH. The FISH procedure was repeated successfully in a couple for preimplantation genetic diagnosis of chromosomal aneuploidies in biopsied blastomeres of their embryos in our clinic. In conclusion, it is feasible to carry out repeated FISH procedures in the same blastomeres. Six or more chromosomes of a single blastomere may be examined using this procedure.