Validation and casework application of a Y chromosome specific STR multiplex.

A series of validation experiments was performed for a Y chromosome specific STR multiplex system following the suggestions made by the Technical Working Group DNA Analysis Methods (TWGDAM). The multiplex PCR products were detected on Perkin-Elmer 373 and 377 automated sequencers using two labeling colors. No problems regarding the stability, robustness and sensitivity of the Y STR multiplex were observed. Mixture studies revealed a cut off rate similar to autosomal STRs for mixtures of male DNAs and no interference of any female admixture. The comparison of the Y STR results to the autosomal typing results for 56 nonprobative semen stains and swabs, showed a slightly higher success rate in detecting the semen donor's alleles for the Y STR multiplex. Two examples are shown to illustrate the usefulness of Y STR typing for DNA mixtures. In one case the Y STR results confirmed an isolated exclusion; in the other case, the interpretation of a mixture was clarified since the Y STR results proved the presence of DNA from at least two semen donors. Y STR typing is a valuable addition to the forensic DNA testing panel.

Kidney transplanted children come of age.

BACKGROUND: The aim of renal replacement therapy in children is to restore their potential for normal growth and development in order to reach mature adulthood. Because pediatric kidney transplantation started in the late 1960s, it is now possible to document the progress and outcome of these patients from transplantation in childhood to survival into adulthood. METHODS: In this single-center study, all 150 children born before December 1977 and having received a kidney transplant between 1970 and 1993 were selected for long-term follow-up. The mean age at transplantation was 12.1 years (range 3.2 to 16.7), and the mean follow-up was 13.1 years (range 2.0 to 25.0). In December 1995, 124 grown-up patients with a mean age of 25.4 years (range 18.4 to 40.3) were alive, 89 with a functioning graft. Fifty had the first graft functioning longer than 10 years. The fate of all patients was traced, and those living were analyzed in regard to their somatic and socioeconomic states. RESULTS: The actuarial 25-year survival rate for the patients was 81%, and for the first graft it was 31%. The best graft survival rates were observed after living related donation, preemptive transplantation, and immunosuppression with cyclosporine. The latter benefit, however, vanished after eight years. The mean creatinine clearance declined over the years from 76 to 45 ml/min/1.73 m2, and the incidence of hypertension increased to more than 80% of the patients. Malignancies occurred in 2.6%. Final height was stunted in 44% of noncystinotic patients, whereas all patients with cystinosis were extremely growth retarded. Twenty-seven percent suffered from additional disabilities. A majority of adult patients were rehabilitated in regard to education and socioeconomic status, and 14% were unemployed. CONCLUSIONS: The results indicate that renal transplantation in children leads to a high degree of rehabilitation in adulthood. The life of a kidney transplant, however, is limited, which points out the need for more specific immunosuppression with fewer side-effects in order to reach the goal of lifelong graft function.

Endometrial thickness in tamoxifen-treated patients: correlation with clinical and pathologic findings.

OBJECTIVE: This study was performed to evaluate sonographic measurements of endometrial thickness in postmenopausal breast cancer patients being treated with tamoxifen and to correlate endometrial thickness with pathology, symptoms, and duration of tamoxifen treatment. MATERIALS AND METHODS: Pelvic sonograms and medical records of 91 postmenopausal breast cancer patients being treated with tamoxifen were retrospectively reviewed. Histologic results were available in 46 patients (51%). Endometrial thickness was measured in anteroposterior dimension and was considered normal when less than 8 mm. Endometrial thickness was then correlated with histopathologic findings, symptoms, and duration of tamoxifen treatment. RESULTS: Forty-seven examinations (52%) showed endometrial thickness of less than 8 mm and 44 examinations (48%) showed endometrial thickness of 8 mm or more. Endometrial biopsy was performed in 10 women (21%) in whom the endometrial thickness was less than 8 mm, revealing seven normal endometria, one endometrial polyp, and two insufficient samples. Endometrial biopsy was performed in 36 women (82%) in whom endometrial thickness was 8 mm or more, revealing three cases with more than one diagnosis. In this group, diagnoses included 14 normal endometria, 12 endometrial polyps, four endocervical polyps, three hyperplasias, two endometrial cancers, one papillary syncytial metaplasia, one cystic change, one inflammatory debris, and one insufficient sample. Postmenopausal bleeding prompted 20 studies, 12 of which revealed endometrial thickness of 8 mm or more. We found no difference in endometrial thickness of patients who had bleeding versus those who had no bleeding. Endometrial thickness increased with the duration of tamoxifen treatment. Seventy-three women being treated with tamoxifen for less than 5 years had a median endometrial thickness of 5 mm, and 44% of biopsies yielded abnormal results. Eighteen women receiving tamoxifen 5 years or longer had a median endometrial thickness of 14 mm, and 58% of endometrial biopsies in this group were abnormal. The two endometrial cancers occurred in women who were treated with tamoxifen for 6 years. Correlation between duration of tamoxifen use and endometrial thickness was significant (p < .026). CONCLUSION: The majority of women being treated with tamoxifen were asymptomatic, but 48% of sonograms revealed an endometrial thickness of 8 mm or more. Endometrial polyps, the most common abnormality, were diagnosed in 33% of biopsies performed for endometrial thickness of 8 mm or more. Endometrial thickness showed no correlation with symptoms, but we found a statistically significant correlation between increased endometrial thickness and duration of tamoxifen treatment that was longer than 5 years.

Guidelines for internal validation of the HLA-DQ alpha DNA typing system.

Validation experiments were performed to evaluate the HLA-DQ alpha DNA typing system for forensic casework. Temperature profiles for two Perkin Elmer TC-1 Thermal Cyclers were measured, and the efficiency of the instruments was tested by amplifying a control sample (DQ alpha 1.2,4) susceptible to allelic drop-out. DNA extraction using chelex was compared to non-organic extraction, and the amplification and hybridization procedures were evaluated at extremes of time and temperature. With the protocol in place, samples exposed to stresses commonly encountered in forensic casework were typed to determine the flexibility of the system. Mixed samples of two different bloods and blood mixed with saliva were typed to determine the threshold at which mixtures could be resolved using the reverse dot blot method. Different storage conditions were evaluated using a set of control bloodstrains, and a set of 12 postmortem blood samples was typed repeatedly over the course of 5 months to determine the effects of natural degradation on the DQ alpha results. Finally, casework stains on a variety of substrates were typed. These experiments demonstrate the flexibility of the HLA-DQ alpha system. Based upon these results, a comprehensive quality assurance program was developed to ensure the integrity of typing results for casework.

DNA-dependent RNA polymerase from bacteriophage T3 transcribes and amplifies an RNA template in vitro.

RNA-based amplification systems that have been recently described are dependent upon the presence of more than one enzyme. In an attempt to minimize the number of polymerases required for efficient amplification, we have studied the template specificity of bacteriophage T3 RNA polymerase. A synthetic bacteriophage T3 promoter was covalently attached to an RNA template. The T3 promoter-RNA complex was found to be selective for its native polymerase, and dependent upon the presence of all four ribonucleoside precursors. The product of the RNA-directed transcription is complementary to the initial template.

Cultured epidermal autografts and allografts: a study of differentiation and allograft survival.

Cultured epidermal sheets were examined before and at various times after grafting on skin ulcer beds. Before grafting, the sheet consisted of four to five layers of keratinocytes with incomplete differentiation. Ten days after grafting, graft recipient sites showed compact hyperkeratosis, a normal-appearing epidermis, and a flat dermoepidermal junction. At 6 months, the stratum corneum had a basket-weave appearance but the dermoepidermal junction remained flat. Monoclonal antibodies to keratins 14 and 10 showed normal basal and suprabasal localization, respectively. Electron microscopy showed a normal basement membrane with anchoring fibrils. LH7:2, a monoclonal antibody that binds to the type VII collagen molecule, stained the dermoepidermal junction in all biopsy specimens. AE-1, an antibody that stains suprabasal cells in hyperproliferative skin, was expressed suprabasally for up to 12 weeks after healing (16 weeks after grafting), but expression was confined to the basal layer at 18 weeks after healing (6 months after grafting). Anti-involucrin staining was found in the deeper layers of the epidermis up to 12 weeks after healing (16 weeks after grafting) but had receded to a normal distribution in upper spinous and granular layers at 18 weeks (6 months after grafting). Overall, the histologic patterns observed in recipient sites during the first 4 months after grafting resembled those observed for 10 to 14 days in newly healed epidermis and in hyperproliferative states such as psoriasis. In four sex-mismatched graft sites, specimens were reacted with a biotinylated probe to the Y chromosome by in situ hybridization. Lack of Y chromosome-positive cells suggested that host keratinocytes had replaced the allografts. Multilocus DNA analysis in one patient confirmed this observation. Our data suggest that an altered state of epithelial maturation persists for several months after culture grafting, with restoration of the normal pattern by 6 months. No differences were detected between autografted and allografted sites.

Site-directed transcription initiation with a mobile promoter.

A method is described for the high-level transcription of any DNA segment using bacteriophage RNA polymerases (RNAPs). A synthetic mobile promoter with a template-complementary 3' extension is ligated to the target sequence of interest. Transcription with an appropriate RNAP results in an amplification of approx. 70-fold. In the presence of heterologous DNA, bacteriophage RNAPs are shown to be specific for their cognate mobile promoters.

Homology maps of the Drosophila alpha-tubulin gene family: one of the four genes is different.

We describe the intron-exon structure of and the homology among the four alpha-tubulin genes of Drosophila melanogaster. Three of the genes share a highly conserved 1.3 kb sequence which corresponds to most of the RNA complementary portion of the genes. The fourth gene is different. Its 5' half has weak homology and its 3' half has moderate homology to the other three genes. The homology maps were first determined by electron microscopy of heteroduplexes between pairs of genes. Higher resolution maps were then obtained by gel analysis of heteroduplexes that had been digested with S1 nuclease.