Difference in allelic expression of the CLCN1 gene and the possible influence on the myotonia congenita phenotype

Mutations in the CLCN1 gene, encoding a muscle-specific chloride channel, can cause either recessive or dominant myotonia congenita (MC). The recessive form, Becker's myotonia, is believed to be caused by two loss-of-function mutations, whereas the dominant form, Thomsen's myotonia, is assumed to be a consequence of a dominant-negative effect. However, a subset of CLCN1 mutations can cause both recessive and dominant MC. We have identified two recessive and two dominant MC families segregating the common R894X mutation. Real-time quantitative RT-PCR did not reveal any obvious association between the total CLCN1 mRNA level in muscle and the mode of inheritance, but the dominant family with the most severe phenotype expressed twice the expected amount of the R894X mRNA allele. Variation in allelic expression has not previously been described for CLCN1, and our finding suggests that allelic variation may be an important modifier of disease progression in myotonia congenita.     

Project Genesis: assessing the efficacy of problem-solving therapy for distressed adult cancer patients

The efficacy of problem-solving therapy (PST) to reduce psychological distress was assessed among a sample of 132 adult cancer patients. A second condition provided PST for both the patient and a significant other. At posttreatment, all participants receiving PST fared significantly better than waiting list control patients. Further, improvements in problem solving were found to correlate significantly with improvements in psychological distress and overall quality of life. No differences in symptom reduction were identified between the 2 treatment protocols. At a 6-month follow-up, however, patients who received PST along with their significant other reported lower levels of psychological distress as compared with members of the PST-alone condition on approximately half of the outcome measures. These effects were further maintained 1-year posttreatment.     

Cellular composition of peripheral lymph and skin of sheep defined by monoclonal antibodies

The surface phenotype of cells in peripheral lymph collected from afferent lymphatics leading to the popliteal lymph node of sheep was determined using a panel of monoclonal antibodies (mAbs). The majority of lymphocytes (83.5%) expressed the sheep pan-T cell antigen and only 13.3% bore surface immunoglobulin molecules. All peripheral T cell subsets occurring in sheep were detected; 50.2% of lymphocytes were positive for mAb SBU-T4 (T helper), 7.3% were positive for mAb ST-8 (T cytotoxic), and 8.4 and 43.0% expressed T subset markers recognized by mAbs 197 and T-80, respectively. Major histocompatibility complex (MHC) class I antigens were detected on 71.1% of lymphocytes and MHC class II antigens on 21.8%. The macrophage/veiled cells found in peripheral lymph did not express lymphocyte subset markers but were positive for MHC class I and II antigens, the sheep homologue of T6 antigen, leukocyte common antigen and mAb 175 (myeloid/erythroid). Macrophage-like cells occurring in the epidermis of skin taken from the lower hindleg gave positive staining reactions to the same mAbs which stained the macrophage/veiled cells in peripheral lymph. These results illustrate differences between the migration of lymphocyte subsets through nonlymphoid as compared to lymphoid tissues and point to a possible developmental or migratory relationship between the macrophage-like cells in skin and those in afferent popliteal lymph.     

Use of multiple displacement amplification to amplify genomic DNA before sequencing of the alpha and beta haemoglobin genes

AIMS: To evaluate the technique of multiple displacement amplification (MDA) for whole genome amplification from small volume blood samples before sequencing in a clinical test to identify haemoglobin gene mutations. METHODS: Phage phi29 DNA polymerase was used to perform MDA, starting with either 1 micro l of blood or 1 ng of previously isolated blood DNA from 23 patients. The amplified products were then evaluated using a clinical test that involves sequencing the haemoglobin genes to detect mutations. The results were compared with the current clinical test method that uses genomic DNA isolated using column based technology. RESULTS: The MDA technique produced large quantities (theoretically approximately 2 mg) of DNA. The amplification procedure was extremely easy and took about four hours (less than one hour of hands on technician time and three hours for amplification). When MDA products were used in the same clinical test protocol as genomic DNA isolated using column technology, there was 100% concordance for detection of a variety of point mutations in the alpha1, alpha2, and beta globin genes. CONCLUSIONS: The MDA technique is useful for overcoming the problem of insufficient genomic DNA in clinical specimens requiring haemoglobin gene sequencing and could be useful for other clinical applications.     

Peptide-Based OspC Enzyme-Linked Immunosorbent Assay for Serodiagnosis of Lyme Borreliosis

Sera from 210 patients with Lyme borreliosis (LB) were studied by an enzyme-linked immunosorbent assay (ELISA) based on a synthetic peptide (pepC10) comprising the C-terminal 10-amino-acid residues of OspC of Borrelia burgdorferi. We found that 36.3 and 45.0% of the serum samples from patients with erythema migrans (EM) and neuroborreliosis (NB), respectively, displayed immunoglobulin M (IgM) anti-pepC10 reactivities, while these samples rarely (≤8%) displayed IgG antibody reactivities. Sera from patients with acrodermatitis chronica atrophicans did not contain anti-pepC10 antibodies. The diagnostic performance of this newly developed peptide ELISA was compared with those of an ELISA based on the full-length recombinant OspC protein (rOspC) and a commercially available ELISA based on the B. burgdorferi flagellum (Fla). The sensitivity of the IgM pepC10 ELISA was slightly lower (P < 0.04) than that of the rOspC ELISA for EM patients (36.3 versus 43.8%), while there was no difference for NB patients (45.0 versus 48.0%). However, the optical density values obtained by the pepC10 ELISA were generally higher than those obtained by the rOspC ELISA, leading to a significantly better quantitative discrimination between seropositive patients with NB and controls (P < 0.008). The specificity of the pepC10 ELISA was similar to those of the rOspC ELISA and the Fla ELISA for relevant controls including patients with syphilis and mononucleosis. Although the overall diagnostic sensitivity of the Fla ELISA was superior, 8.8 and 12.0% of the EM and NB patients, respectively, were antibody positive only by the pepC10 ELISA. Thus, use of a diagnostic test for LB based on the detection of IgM antibodies to pepC10 and Fla has increased sensitivity for the diagnosis of early LB.     

Detection of HIV-1 antiretroviral resistance from patients with persistently low but detectable viraemia

We modified the Abbott diagnostics HIV-1 Viroseq version 2 assay trade mark in order to detect the presence of HIV-1 drug resistance mutations in patients with viraemia below 1000 copies/ml of plasma. One hundred and forty-four patients with a detectable HIV-1 plasma viral load below 1000 copies/ml were selected and HIV-1 genetic analysis carried out using a modification of the Abbott Diagnostics Viroseq 2.0 assay trade mark. The procedure differs from the standard protocol in that a nested PCR amplification step was introduced. The oligonucleotide primers for the first round of PCR were those supplied in the RT-PCR module of the kit. The nested PCR primers were primers A and H taken from the sequencing module. One hundred and twenty-eight out of 144 (89%) plasma samples with an HIV-1 viral load of less than 1000 copies/ml (ranging from 54 to 992 copies) were successfully sequenced. HIV-1 genotypes were obtained from 68 out of 81 (84%) samples with a viral load of greater than 50 but less than 300 copies/ml and 60/63 (95%) of samples with a viral load of greater than 300 but less than 1000 copies/ml. Serial dilution of a sample with a high viral load did not affect the detection of resistance mutations. Multiple sequencing of samples with low viral load did not result in detection of additional mutations, although, in one sample the K103N mutation was detected in 3/6 replicates while wild-type was detected in 2/6 and a mixture of wild-type/mutant in 1/6. Samples from patients infected with both clade B and non-B clades of HIV-1 could be genotyped at low copy number. Modification of the Abbott Viroseq assay allows reproducible sequencing of the HIV-1 genome from patients with low, but detectable, plasma virus burden.     

Psychological distress among minority and low-income women living with HIV

The growing incidence of HIV infection among low-income and minority women makes it important to investigate how these women adjust to living with HIV and AIDS. Psychological distress associated with HIV infection may compound the adjustment difficulties and increase the barriers to care associated with living in poverty. The authors surveyed 100 women who were receiving HIV care at a public hospital in the southeastern United States on measures of depression, anxiety, life stress, social support, and coping; they also assessed demographic and medical characteristics of the sample. Participants' annual incomes were low (87% < $10,000), and most participants were minorities (84% African American). Their levels of depression, stress, and anxiety symptoms were elevated relative to community norms. Greater anxiety and depression symptoms were associated with women who reported higher stress, using fewer active coping strategies, and perceiving less social support (ps < .001).     

Luteal phase support and severe ovarian hyperstimulation syndrome.

The incidence and statistical associations of the ovarian hyperstimulation syndrome (OHSS) were studied in 304 egg retrievals with gonadotrophin-releasing hormone agonist suppression, gonadotrophin administration and follicular aspiration. In addition to preserving corpus luteum function, the luteal phase administration of human chorionic gonadotrophin (HCG) was associated with a higher incidence of severe OHSS than was supplementation with progesterone alone (12 versus 0%, P less than 0.001). Severe OHSS occurred in 3.7% and 12% of retrievals without and with pregnancy respectively (P less than 0.01). Stepwise logistic regression showed that the occurrence of moderate or severe OHSS was statistically predicted by the log of the serum oestradiol on the day the initial HCG was given (P less than 0.0001), treatment with luteal phase HCG (P less than 0.0003), and fetal number (P less than 0.0079). In the late luteal phase of cycles without luteal HCG, the serum oestradiol concentration was one-tenth and the serum progesterone concentration was one-fifth of the luteal phase value with HCG support (P less than 0.001). Without luteal phase HCG, oestradiol was two-fold higher (P less than 0.001) and progesterone was 1.4-fold higher (P less than 0.005) in pregnant than in non-pregnant women. With luteal phase HCG, oestradiol was 1.4-fold higher in pregnant than in non-pregnant women (P less than 0.05), and progesterone was 1.7-fold higher (P less than 0.001). Oestradiol upper limits of 4400 and 14,700 pmol/l (1200 and 4000 pg/ml) for cycles with and without luteal phase HCG respectively correspond to approximately 5% risk of moderate or severe OHSS with a singleton pregnancy under these conditions.     

Amyloid in prostatic corpora amylacea.

AIM: To determine the presence and nature of amyloid in prostatic corpora amylacea using immunohistological studies. METHODS: Prostatic tissue from 18 transurethral and two open resection specimens was studied. Paraffin wax embedded tissue sections were stained with haematoxylin and eosin and the alkaline Congo red method with and without previous treatment with potassium permanganate. Sections were also stained with antibodies to amyloid A, beta 2 microglobulin, lambda and kappa light chains, prealbumin IgA, G, M, S100 protein, prostatic specific antigen, amyloid P component and CAM 5.2 (control and blocking studies were performed). RESULTS: The prostatic corpora amylacea universally showed the presence of amyloid. In all instances this contained beta 2 microglobulin. CONCLUSION: Prostatic corpora amylacea represents a localised amyloidosis of beta 2 microglobulin origin that is unrelated to chronic renal failure and haemodialysis.