Analyses of polymorphism for UGT1*1 exon 1 promoter in neonates with pathologic and prolonged jaundice.

In this study, we investigated whether a TATA box polymorphism in the promoter of the UGT1*1 exon I, the most common detected DNA polymorphism in Gilbert's syndrome, is a contributory factor in unexplained pathologic or prolonged jaundice. 38 neonates who had unexplained pathologic jaundice, 37 neonates who had unexplained prolonged jaundice, and 35 healthy, nonjaundiced neonates were enrolled in the study. Genotypes were assigned as follows: 6/6 (homozygous for a normal allele bearing the sequence [TA](6)TAA), 7/7 (homozygous for an abnormal allele with the sequence [TA](7)TAA), and 6/7 (heterozygous with one of each allele). Of the 110 infants, 10 (9%) had 7/7, 51 (46%) had 6/7, and 49 (45%) had 6/6 genotype; the differences between the three groups were not statistically significant. Also no differences were observed among different genotypes and mean serum total bilirubin concentrations. In conclusion, we showed that TA 7/7 and TA 6/7 genotypes are not rare in our population and that the presence of these polymorphisms alone does not play a significant role in the etiology of unexplained pathologic or prolonged neonatal hyperbilirubinemia.

Technical pitfalls encountered in PCR quantification using microsatellites.

Quantitative PCR has proved useful for different purposes, including the detection of particular genetic changes, such as deletions and duplications in several inherited disorders. Using patients with the known duplication mutation for Charcot-Marie-Tooth disease Type 1A as examples, the importance of selecting informative microsatellite loci and proper electrophoretic conditions so as to eliminate potential sources of error in quantitative PCR studies is discussed.

Charcot-Marie-Tooth disease type 1A: a family study with microsatellites.

Charcot-Marie-Tooth (CMT) disease (also called Hereditary Motor and Sensory Neuropathy) is an inherited peripheral neuropathy with a prevalence rate of 1 in 2,500. Charcot-Marie-Tooth disease type 1A (CMT1A), the most common autosomal dominant form of the disease, is associated with a duplication of a segment of chromosome 17 (17p11.2). In this report we present a three-generation family with CMT1A where simple sequence repeats (di- or tri-nucleotide repeats, also called microsatellites) were used in conjunction with polymerase chain reaction (PCR) to identify the duplication. The presence of three alleles or the presence of two alleles with a dosage ratio of 1:2 for the markers D17S839 and D17S921 indicates the presence of the duplicated segment in affected family members, whereas two alleles with a ratio of 1:1 indicate absence of the duplication. Several markers outside the duplication region which have two alleles with a dosage ratio of 1:1 were used as controls. Seven CMT1A patients in this family carry the CMT1A duplication. One 12-year-old boy who has not exhibited any clinical symptoms does not have the CMT1A duplication. We believe that this is a simple, rapid, and effective method to identify the CMT1A duplication in most patients suspected of having CMT1A.

Preproinsulin I and II mRNAs and insulin electron microscopic immunoreaction are present within the rat fetal nervous system.

Insulin-like substance has been found within the nervous system. In the rat, preproinsulin II mRNA was shown within the brain and preproinsulin I mRNA within the retina. The present study demonstrates the presence of preproinsulin mRNAs within the 15, 17 and 19 day gestational age fetal rat brain, spinal cord and dorsal root ganglia (DRG), employing RNA template-specific polymerase chain reaction (RS-PCR), semi-nested PCR and RNase protection assay. Preproinsulin I mRNA was present in the 17 and 19 day gestational age brain, spinal cord and DRG, and only in the brain of the 15 day gestational age brain. Preproinsulin II mRNA was present in all the gestational ages studied in the brain, spinal cord and DRG. The RS-PCR and the semi-nested PCR demonstrated products that co-migrated with the pancreatic control. The semi-nested products were characterized as preproinsulin I and II by restriction enzyme digestion and sequence. RNase protection assay using specific cRNA for preproinsulin I and II showed a band that co-migrated with pancreatic preproinsulin I and II mRNAs, and confirmed the PCR results. In addition, insulin receptor mRNA was detected by RS-PCR. Ultrastructural studies showed insulin immunoreaction within the endoplasmic reticulum, Golgi apparatus, cytoplasm, axon, dendrites, and in relation to the synapses. Thus, we demonstrated the presence of preproinsulin I and II mRNA, insulin receptor mRNA and insulin immunoreaction within the rat fetal central and peripheral nervous system.

Molecular approach to rapid assessment of p53 tumor suppressor mutations in esophageal tumors from stained histological slides.

The analysis of the tumor suppressor gene, p53, is of fundamental importance in prognosis and staging in many cancers; however, the molecular techniques required to analyze this gene have been expensive, time consuming, and unrelatable to the histological appearance of the samples. This research explored one model of clinically testing for specific mutations in the p53 gene by scraping selected areas of stained histological slides and analyzing for "hot-spot" p53 mutations. Selectively removing samples from the stained histological slide will be of special value in examining suspicious regions in adenomas, potential metastatic regions, and the margins of resected area. A polymerase chain reaction (PCR)-mediated restriction fragment length polymorphism (RFLP) analysis approach in which naturally occurring or primer-mediated mutagenesis-induced restriction enzyme sites were utilized to test seven hot-spot mutations. These assays were able to detect one mutated sequence in 100, and therefore, were sufficiently sensitive to be used with very heterogeneous tumors. Several of the assays could be multiplexed to reduce the number of PCRs necessary to screen for the seven mutational hot spots. Furthermore, an exact determination of the base change could be obtained by direct sequencing of the PCR products. Although this form of analysis may be applicable only to certain types of cancers (e.g., bladder, brain, colon, esophageal, gastric, thyroid, and ovarian tumors), this approach can obtain detailed mutational information from specific regions of a histological slide in a cost-effective and timely manner.

Cystic fibrosis in the southern Midwest United States: molecular characterization of the common mutations.

The identification of different mutations that cause cystic fibrosis in the people of Kansas and Oklahoma has been performed by examining 124 independent cystic fibrosis genes for the 14 most commonly mutated loci. The delta F508 3bp deletion represented 79% of the alleles, and 7% of the remaining alleles were found to harbor the mutations of R553X, G542X, or G551D. None of the remaining 10 common mutations were identified. This pattern of results contrasts with the patterns found in major cities of the United States. The ethnic diversity in these cities is much greater than in the southern Midwest region, and the remaining mutations, therefore, may represent specific ethnic contributions absent in the Midwest population studies. These results directly affect the counseling given to the Midwest patient and impact on any strategies for screening.

Phenotype and TCR gamma delta variable gene repertoire of intestinal intraepithelial lymphocytes in wild mice (Mus musculus domesticus): abundance of V gamma 1 transcripts and extensive delta gene diversity.

In order to study murine intestinal intraepithelial lymphocytes (IEL) independent of factors imparted by conditions of laboratory housing and breeding, and to provide a basis for comparison of IEL studies between inbred and outbred mouse populations, IEL from the domestic house mouse, Mus musculus domesticus, were analyzed by flow cytometric analyses using mAbs to murine lymphocyte markers, and by polymerase chain reaction to study the TCR gamma and delta V gene repertoires. The majority of IEL in wild mice were CD3+, CD8+CD4- T cells. CD4+CD8- also were present in IEL isolates from wild mice, although at low numbers. Among IEL, but not T cells from the spleen or lymph nodes, there was a notable lack of Thy-1 expression, a preponderance of CD8 alpha alpha + T cells, and a relatively high ratio (3:1) of TCR gamma delta + T cells over TCR alpha beta + T cells, suggesting that some IEL in wild mice may develop via an extrathymic pathway similar to that described for laboratory mice. Analyses of the IEL gamma and delta variable genes revealed rearrangements of three of six V region gamma genes (V gamma 1, V gamma 2, and V gamma 5), with an abundance of V gamma 1 transcripts as determined by Northern blot analyses. For the delta gene, rearrangement of five of seven V region elements had occurred (V delta 2, V delta 3, V delta 4, V delta 5, and V delta 6).(ABSTRACT TRUNCATED AT 250 WORDS)

A new high frequency polymorphism in the HER-2/neu oncogene in normal tissue and breast tumors.

The HER-2/neu (erbB-2) oncogene, if amplified and/or overexpressed in breast and ovarian cancers, is associated with a poor prognosis. Employing direct DNA sequencing, we have discovered and sequenced an 80 base pair intron from human placenta which contains an A to G polymorphism. This polymorphism lends itself to restriction fragment length polymorphism analysis of the PCR product spanning this intron. All three genotypes, homozygous A, heterozygous, and homozygous G appear in normal control populations and breast tumors. Also, no difference was seen between the polymorphic form found in five breast cancers and the corresponding normal tissue.

T cell receptor delta gene repertoire and diversity of intestinal intraepithelial lymphocytes in athymic mice.

T cell receptor (TCR) delta gene rearrangements in intestinal intraepithelial lymphocytes (IEL) were studied in athymic radiation chimeras using polymerase chain reaction (PCR) and sequence analysis of DNAs spanning the variable (V), diversity (D), and junctional (J) genes. In both thymus-bearing and athymic mice, IEL delta gene rearrangements occurred for V delta 3, V delta 4, V delta 5 and V delta 6. V-D-J junctional-site sequence analyses of cloned DNAs from rearranged IEL delta genes in athymic mice revealed a predominance of in-frame rearrangements; junctional diversity consisting of nucleotide removal from V, D and/or J genes; N segment nucleotide insertions; and high overall gene diversity. Evaluation of PCR-amplified cDNAs made from IEL RNA indicated that all four rearranged V delta genes were expressed in IEL from athymic mice. The high diversity observed at the gene level also was present in amino acid sequences encoded by the V-D-J region of IEL delta genes in athymic mice. These data demonstrate that there is extensive diversity of rearranged delta genes in IEL which develop extrathymically, and suggest that the delta chain of IEL TCR-gamma delta+ T cells has the potential for interactions with polymorphic structures.

Rearrangement and junctional-site sequence analyses of T-cell receptor gamma genes in intestinal intraepithelial lymphocytes from murine athymic chimeras.

The molecular organization of rearranged T-cell receptor (TCR) gamma genes intraepithelial lymphocytes (IEL) was studied in athymic radiation chimeras and was compared with the organization of gamma gene rearrangements in IEL from thymus-bearing animals by polymerase chain reaction and by sequence analyses of DNA spanning the junction of the variable (V) and joining (J) genes. In both thymus-bearing mice and athymic chimeras, IEL V-J gamma-gene rearrangements occurred for V gamma 1.2, V gamma 2, and V gamma 5 but not for V gamma 3 or V gamma 4. Sequence analyses of cloned V-J polymerase chain reaction-amplified products indicated that in both thymus-bearing mice and athymic chimeras, rearrangement of V gamma 1.2 and V gamma 5 resulted in in-frame as well as out-of-frame genes, whereas nearly all V gamma 2 rearrangements were out of frame from either type of animal. V-segment nucleotide removal occurred in most V gamma 1.2, V gamma 2, and V gamma 5 rearrangements; J-segment nucleotide removal was common in V gamma 1.2 but not in V gamma 2 or V gamma 5 rearrangements. N-segment nucleotide insertions were present in V gamma 1.2, V gamma 2, and V gamma 5 IEL rearrangements in both thymus-bearing mice and athymic chimeras, resulting in a predominant in-frame sequence for V gamma 5 and a predominant out-of-frame sequence for V gamma 2 genes. These findings demonstrate that (i) TCR gamma-gene rearrangement occurs extrathymically in IEL, (ii) rearrangements of TCR gamma genes involve the same V gene regardless of thymus influence; and (iii) the thymus does not determine the degree to which functional or nonfunctional rearrangements occur in IEL.

Preinjury psychopathology in trauma patients.

One hundred patients admitted to a Level I trauma center were interviewed using the Langner 22-item index of psychiatric symptoms to evaluate pre-existing psychological pathology. The medical chart was also examined for any type of note or evidence of psychiatric consultation efforts by the surgical attending staff. There were 74 males and 26 females in the sample, with a mean age of 33 years. Types of injuries included blunt trauma in 71% and penetrating trauma in 29% of the group, respectively. Eight per cent of these injuries were self inflicted. The mean Injury Severity Score was 17. Alcoholic intoxication was documented in 49%. We found that severe psychopathology as defined by the Langner Index was present in 88% of those admitted with penetrating trauma, in 47% of those admitted with blunt trauma, and in 75% of the intoxicated group. In only 14% of the sample was psychopathology documented in the chart by the attending staff. Preinjury psychopathology in trauma patients was commonly present and seemed to be most highly associated with penetrating trauma and alcohol use.