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Background: Restoration of the long head of the biceps tendon (LHBT) length-tension relationship is critical in preserving muscle strength and efficiency when performing biceps tenodesis. While static anatomic landmarks such as the inferior border of the pectoralis major may be used intraoperatively to achieve this, shoulder position may affect the excursion of the biceps tendon and represents another variable to consider. Purpose/Hypothesis: The purpose of this study was to quantitatively evaluate the normal excursion of LHBT that occurs through a glenohumeral range of motion. We also sought to determine whether elbow position affects LHBT excursion. We hypothesized that LHBT excursion will be affected by glenohumeral flexion and extension, and elbow extension will result in increased excursion at each glenohumeral position compared with a neutral position. Study Design: Controlled laboratory study. Methods: A total of 10 fresh-frozen specimens underwent a standard approach for subpectoral biceps tenodesis. The LHBT was identified and tagged with a radiopaque marker within zone 3 of the bicipital tunnel. A total of 3 K-wires were then drilled into the osseous floor: one at the level of the marker in the LHBT, one at 1 cm proximal, and a third 1 cm distal. All 3 K-wires were then cut flush with the anterior humeral cortex. The specimens were next placed into 8 different positions, and the excursion of the LHBT was measured by referencing the K-wires using static fluoroscopic imaging. The results were analyzed using 1-way analysis of variance testing followed by Tukey honestly significant difference testing for pairwise comparison between each individual position and the reference position. Results: The average total LHBT excursion was 24.4 ± 5.2 mm between the neutral shoulder position and the other shoulder positions tested. The position of the LHBT was significantly different in the reference position compared with each of the other 7 shoulder positions ( P < .001). Additionally, the 2 positions of shoulder extension had different LHBT excursions when compared with each position of shoulder flexion ( P < .0001). For each shoulder position tested, the position of the LHBT was not significantly different in elbow flexion compared with extension. Conclusion: There is approximately 24 mm of LHBT excursion throughout the glenohumeral range of motion, with significantly different amounts of excursion in glenohumeral flexion and extension. Elbow position does not significantly affect LHBT excursion. Positioning the shoulder in extension during biceps tenodesis may overtension the biceps, while positioning the shoulder in flexion may undertension the biceps relative to the neutral position. Further research is needed to identify the optimal shoulder position for biceps tenodesis. Clinical Relevance: Shoulder positioning is an important consideration in establishing a normal length-tension relationship during biceps tenodesis. When compared with flexed shoulder positions, LHBT excursion significantly differs in positions of extension and in a neutral position.

Optimal outcomes following total shoulder arthroplasty TSA and reverse shoulder arthroplasty RSA are dependent on proper implant position. Multiple cadaver studies have demonstrated improved accuracy of implant positioning with use of patient-specific guides/instrumentation compared to traditional methods. At this time, there are 3 commercially available single use patient-specific instrumentation systems and 1 commercially available reusable patient-specific instrumentation system. Currently though, there are no studies comparing the clinical outcomes of patient-specific guides to those of traditional methods of glenoid placement, and limited research has been done comparing the accuracy of each system's 3-dimensional planning software. Future work is necessary to elucidate the ideal indications for the use of patient-specific guides and instrumentation, but it is likely, particularly in the setting of advanced glenoid deformity, that these systems will improve a surgeon's ability to put the implant in the best position possible.

Two important criteria used to select an antibody diluent for robust immunohistochemistry are: (1) optimal signal/noise ratio, and (2) long-term stability when diluted. For immunohistochemistry with Fanconi anemia, complementation group D2 (FANCD2) in formalin-fixed-paraffin-embedded breast cancer tissue, eight experimental and eight commercial diluents were evaluated. Staining intensity was quantitated with Aperio digital image analysis software. An H-Score was calculated for FANCD2 and isotype control staining for each diluent. Signal/noise ratio was defined as H-score of the antibody divided by H-score of the isotype control. The ratios ranged from 0.46 to 135.9 for different diluents. Casein in Tris-based buffer produced the greatest signal/noise ratio. The two best commercial and four best experimental diluents were selected for evaluation of the diluted FANCD2 antibody stability at 4°C, room temperature, and 37°C, respectively. Three diluents were eliminated from further investigation after ...

Background In women at increased risk for breast and ovarian cancer, the identification of a BRCA1/2 mutation has important implications for screening and prevention counseling. Uncertainty regarding the role of BRCA1/2 testing in high-risk women from diverse ancestral backgrounds exists due to variability in prevalence estimates of deleterious (disease-associated) mutations in non-White populations. We examined the prevalence of BRCA1/2 mutations in an ethnically diverse group of women referred for genetic testing.

ACMG previously developed recommendations for standards for interpretation of sequence variations. We now present the updated revised recommendations. Here, we describe six interpretative categories of sequence variations: (1) sequence variation is previously reported and is a recognized cause of the disorder; (2) sequence variation is previously unreported and is of the type which is expected to cause the disorder; (3) sequence variation is previously unreported and is of the type which may or may not be causative of the disorder; (4) sequence variation is previously unreported and is probably not causative of disease; (5) sequence variation is previously reported and is a recognized neutral variant; and (6) sequence variation is previously not known or expected to be causative of disease, but is found to be associated with a clinical presentation. We emphasize the importance of appropriate reporting of sequence variations using standardized terminology and established databases, and of clearly reporting the limitations of sequence-based testing. We discuss follow-up studies that may be used to ascertain the clinical significance of sequence variations, including the use of additional tools (such as predictive software programs) that may be useful in variant classification. As more information becomes available allowing the interpretation of a new sequence variant, it is recommended that the laboratory amend previous reports and provide updated results to the physician. The ACMG strongly recommends that the clinical and technical validation of sequence variation detection be performed in a CLIA-approved laboratory and interpreted by a board-certified clinical molecular geneticist or equivalent.

textabstractContext: Lynch syndrome is caused primarily by mutations in the mismatch repair genes MLH1 and MSH2. Objectives: To analyze MLH1/MSH2 mutation prevalence in a large cohort of patients undergoing genetic testing and to develop a clinical model to predict the likelihood of finding a mutation in at-risk patients. Design, Setting, and Participants: Personal and family history were obtained for 1914 unrelated probands who submitted blood samples starting in the year 2000 for full gene sequencing of MLH1/MSH2. Genetic analysis was performed using a combination of sequence analysis and Southern blotting. A multivariable model was developed using logistic regression in an initial cohort of 898 individuals and subsequently prospectively validated in 1016 patients. The complex model that we have named PREMM1,2 (Prediction of Mutations in MLH1 and MSH2) was developed into a Webbased tool that incorporates personal and family history of cancer and adenomas. Main Outcome Measure: Deleterious mutations in MLH1/MSH2 genes. Results: Overall, 14.5% of the probands (130/898) carried a pathogenic mutation (MLH1, 6.5%; MSH2, 8.0%) in the development cohort and 15.3% (155/1016) in the validation cohort, with 42 (27%) of the latter being large rearrangements. Strong predictors of mutations included proband characteristics (presence of colorectal cancer, especially ≥2 separate diagnoses, or endometrial cancer) and family history (especially the number of first-degree relatives with colorectal or endometrial cancer). Age at diagnosis was particularly important for colorectal cancer. The multivariable model discriminated well at external validation, with an area under the receiver operating characteristic curve of 0.80 (95% confidence interval, 0.76-0.84). Conclusions: Personal and family history characteristics can accurately predict the outcome of genetic testing in a large population at risk of Lynch syndrome. The PREMM1,2 model provides clinicians with an objective, easy-to-use tool to estimate the likelihood of finding mutations in the MLH1/MSH2 genes and may guide the strategy for molecular evaluation.

This work describes an approach to characterize the clinical significance of genetic variants detected during the genetic testing of BRCA1 in patients from hereditary breast/ovarian cancer families. Results from transgenic mice and extensive clinical testing support the hypothesis that biallelic BRCA1 mutations result in embryonic lethality. Therefore, it is reasonable to conclude that variants of uncertain clinical significance found to reside in trans with known deleterious mutations impart reduced risk for cancer. This approach was applied to a large data set of 55,630 patients who underwent clinical BRCA1 screening by whole gene direct DNA sequencing. Fourteen common single nucleotide polymorphisms (SNPs) were used to assign 10 previously defined common, recurrent, or canonical haplotypes in 99% of these cases. From a total of 1,477 genetic variants detected in these patients, excluding haplotype-tagging SNPs, 877 (59%) could be unambiguously assigned to one or more haplotypes. In 41 instances, variants previously classified as being of uncertain clinical significance, mostly missense variants, were excluded as fully penetrant mutations due to their coincidence in trans with known deleterious mutations. From a total of 1,150 patients that harbored these 41 variants, 956 carried one as the sole variant of uncertain clinical significance reported. This approach could have widespread application to other disease genes where compound heterozygous mutations are incompatible with life or result in obvious phenotypes. This largely computational technique is advantageous because it relies upon existing clinical data and is likely to prove informative for prevalent genetic variants in large data sets.

Polymerase chain reaction (PCR)-based STR DNA typing systems are used extensively in the field of human identification. Under optimal PCR conditions, the amplicon yield from both alleles of an STR locus is expected to be approximately equivalent. However, it is reasonable to expect that rare genomic sequence polymorphisms will co-localize with well-designed primer sets and induce allele imbalance or "dropouts". Two samples were identified in the course of genotyping thousands of individuals with AmpF/STR Profiler Plus that showed strong disparity in amplitude peak height of heterozygous peaks at the loci vWA and FGA. These samples were reamplified at reduced annealing temperature in an attempt to balance the peak heights. Nucleotide sequencing documented polymorphisms at the PCR primer binding sites of the affected alleles. The results indicate that reducing the annealing temperature to improve primer-binding efficiency at the mismatch and employing an alternative multiplex enhanced the data from both samples. Reducing annealing temperatures could provide a simple general solution to improving data quality for samples where polymorphisms are suspected to cause allele imbalance. Finally, we report on additional polymorphisms surrounding the vWA locus in a genetically diverse population.

A powerful method for validating a scientific result is to confirm specific results utilizing independent methodologies and processing pathways. Thus, we have designed, developed and validated an automated allele concordance analysis system (CompareCalls, patent pending) that performs comparisons between two independent DNA analysis platforms to ensure the highest accuracy for allele calls. Application of this system in a quality assurance role has shown the potential to eliminate greater than 90% of the STR analysis required of a DNA data analyst. While this system is broadly applicable for use with any two independent STR analysis programs, either prior to or following human data review, we are presenting its application to data generated with the ABI Prism Genotyper software system versus data generated with the SurelockID system. With the automated allele concordance analysis system, the GeneScan DNA fragment data generated from an ABI 377 gel image are analyzed in two independent pathways. In one analysis pathway, the GeneScan data are imported into Genotyper software where STR labels are assigned to the fragment data based upon the criteria of the Kazam 20% macro. The "Kazam" macro provided with the Genotyper program works by labeling all peaks in a category (or locus) and then filtering (or removing) the labels from peaks, such as those in stutter positions, that meet predefined criteria. In the second pathway, the GeneScan data are imported into the SurelockID analysis platform where STR labels and error messages are assigned to the fragment data based upon hard-coded allele calling criteria and quality parameters. The resulting STR allele calls for each analysis platform are then compared, utilizing the automated allele concordance analysis system. Any differences in the STR allele calls between the two systems are flagged in a discordance report for further review by a qualified DNA data analyst. The automated allele concordance analysis system guides the DNA data analyst to the discordant data generated by either analysis platform. Additionally, the analyst is also directed to data that are of less than pristine quality which may have an increased potential for errors in interpretation by either analysis platform or by a human DNA data analyst. Implementation of an automated allele concordance analysis system will yield high-quality data for CODIS and free the human DNA data analyst to perform other critical duties within the laboratory.

PURPOSE: Breast cancer patients treated conservatively with lumpectomy and radiation therapy (LRT) have an estimated lifetime risk of local relapse (ipsilateral breast tumor recurrence [IBTR]) of 10% to 15%. For breast cancer patients carrying BRCA1 or BRCA2 (BRCA1/2) mutations, the outcome of treatment with LRT with respect to IBTR has not been determined. In this study, we estimate the frequency of BRCA1/2 mutations in a study of breast cancer patients with IBTR treated with LRT. PATIENTS AND METHODS: Between 1973 and 1994, there were 52 breast cancer patients treated with LRT who developed an IBTR within the prior irradiated breast and who were willing to participate in the current study. From our database, we also identified 52 control breast cancer patients treated with LRT without IBTR. The control patients were individually matched to the index cases with respect to multiple clinical and pathologic parameters. Lymphocyte DNA specimens from all 52 locally recurrent patients and 15 of the matched control patients under age 40 were used as templates for polymerase chain reaction amplification and dye-primer sequencing of exons 2 to 24 of BRCA1, exons 2 to 27 of BRCA2, and flanking intron sequences. RESULTS: After LRT, eight (15%) of 52 breast cancer patients had IBTR with deleterious BRCA1/2 mutations. By age, there were six (40%) of 15 patients with IBTR under age 40 with BRCA1/2 mutations, one (9.0%) of 11 between ages 40 and 49, and one (3.8%) of 26 older than age 49. In comparison to the six (40%) of 15 of patients under age 40 with IBTR found to have BRCA1/2 mutations, only one (6.6%) of 15 matched control patients without IBTR and had a BRCA1/2 mutation (P = .03). The median time to IBTR for patients with BRCA1/2 mutations was 7.8 years compared with 4.7 years for patients without BRCA1/2 mutations (P = .03). By clinical and histologic criteria, these relapses represented second primary tumors developing in the conservatively treated breast. All patients with BRCA1/2 mutations and IBTR underwent successful surgical salvage mastectomy at the time of IBTR and remain alive without evidence of local or systemic progression of disease. CONCLUSION: In this study, we found an elevated frequency of deleterious BRCA1/2 mutations in breast cancer patients treated with LRT who developed late IBTR. The relatively long time to IBTR, as well as the histologic and clinical criteria, suggests that these recurrent cancers actually represent new primary breast cancers. Early onset breast cancer patients experiencing IBTR have a disproportionately high frequency of deleterious BRCA1/2 mutations. This information may be helpful in guiding management in BRCA1 or BRCA2 patients considering breast-conserving therapy.

The biochemical and genetic characterizations of two variants that occur in BRCA1 intron 8 are presented. The variant IVS8+2T-->C induces an aberrant transcript that deletes exon 8. This exon-skipping deletion disrupts the open reading frame by juxtaposing exon 7 and exon 9 in the aberrant splice product. Theoretically, 50 abnormal residues from reading frame 2 are translated following exon 7 before a stop codon is encountered. The chromosomal contribution to the relevant RNA species was tracked using a silent polymorphism at codon 694 (serine AGC or AGT). Nucleotide sequencing of this polymorphic codon demonstrated that the aberrant transcript was derived solely from the chromosome encoding AGT. The normally spliced productive transcript also displayed loss of heterozygosity and was derived solely from the chromosome encoding AGC at codon 694. Also, a haplotype analysis using a breast cancer patient database showed that the chromosome bearing serine 694-AGT carried IVS8+2T-->C. A second more common variant, IVS8-58delT, was characterized as a polymorphism. Analysis of RNA from patient samples used the same silent polymorphism at codon 694 and showed that the normal message was derived from both chromosomes.

Results and conclusions are presented that characterize BRCA1 IVS16+6TC as a deleterious mutation. BRCA1 transcripts from peripheral blood mononuclear cells of a breast cancer patient with the transition IVS16+6TC show the loss of a heterozygous base within codon 871. Additionally, an aberrant RNA splicing product which incorporates 69 bases of the 5′ end of intron 16 at the junction of exons 16 and 17 is produced solely from the allele with IVS16+6TC. This insertion contains two in-frame stop codons and encodes a protein truncated at residue 1662 (plus 13 residues encoded by the intron). The aberrant transcript is specifically associated with the intronic variant since it was contained within the insertion. Furthermore, sequence analysis of the heterozygous base within codon 871 demonstrates that the two RNA products, productive mRNA and aberrantly spliced RNA, are contributed to exclusively by separate alleles. Finally, the aberrant transcript is produced by the activation of a cryptic splice site which has greater homology with the primate consensus splice sequence than the mutated exon 16 donor site. Am. J. Med. Genet. 85:113–116, 1999. © 1999 Wiley-Liss, Inc.

OBJECTIVE: This study examines the role of fluorescence in situ hybridization on uncultured amniocytes for prenatal diagnosis in a population at high risk for aneuploidies. STUDY DESIGN: All patients undergoing amniocentesis for fetal structural abnormality on ultrasonographic examination (performed from 13 to 39 weeks), abnormal maternal serum aneuploidy screening results, or advanced maternal age with substantial parental anxiety were offered both fluorescence in situ hybridization on uncultured cells and conventional metaphase karyotyping on dividing cells. RESULTS: From 1992 to 1995, 315 patients were studied. Mean time to obtain results was 2.8 days for fluorescence in situ hybridization and 8.3 days for karyotype. Fluorescence in situ hybridization was informative in 254 samples (80.6%), and within this group 21 aneuploidies were correctly identified. Among informative specimens there was 100% sensitivity and specificity, with 100% positive and negative predictive values. Of the 315 samples, 61 (19.4%) were uninformative or unreportable. Of 25 total cases of karyotype-proved aneuploidy, 4 were reported as uninformative by fluorescence in situ hybridization, for a total detection rate of 84%. Overall, amniocenteses performed after 24 weeks were significantly more likely to be uninformative than those performed in the second trimester (45% vs 16%, p = 0.01), peaking at a 56% uninformative rate after 33 weeks. Logistic regression analysis showed an 8% increase in the uninformative rate per week of gestational age (odds ratio 1.08, 95% confidence interval 1.04 to 1.14). CONCLUSIONS: Fluorescence in situ hybridization on uncultured amniocytes is a rapid, clinically useful tool for prenatal diagnosis, with informative specimens being highly accurate. The combination of a structural fetal anomaly and an abnormal fluorescence in situ hybridization result should allow for definitive management decisions. The significant increase in uninformative specimens at later gestational ages limits its usefulness in the third trimester. (Am J Obstet Gynecol 1997;176:769-76.)

The presence of maternal cells in uncultured amniotic fluid may result in error in the interpretation of prenatal tests such as direct DNA analysis and rapid aneuploidy detection by fluorescence in situ hybridization (FISH). Using simultaneous dual colour X and Y FISH, we assessed maternal cell contamination in uncultured amniotic fluids from 500 women carrying male fetuses. The presence of maternal cells was correlated with the amount of blood present in the amniotic fluid as defined by visual examination of the cell pellet after centrifugation. The overall rate of maternal cell contamination in uncultured amniotic fluid as identified using X and Y-specific probes was 21.4 per cent, compared with 0.2 per cent in cultured fluid. Sixteen per cent of slightly bloody and 55 per cent of moderately bloody uncultured fluids had at least 20 per cent maternal cells and were classified as uninformative according to our protocol for rapid aneuploidy detection. Maternal and fetal cells could not be distinguished based on morphological characteristics alone.

The first prenatal diagnosis of a tetraploid fetus via fluorescent in situ hybridization (FISH) technology is reported. FISH analysis of uncultured amniotic fluid cells utilizing probes for chromosomes 13, 21, 18, X, and Y revealed that greater than 65% of hybridized nuclei had four signals. Standard cytogenetic analysis revealed a 92, XXXX karyotype

Ultrasound of a fetus at 17 weeks gestation revealed posterior urethral valve syndrome with anhydramnios. Fluorescence in situ hybridization (FISH) to detect aneuploidies of chromosomes 13, 18, 21, X and Y was performed on transitional cells from the fetal bladder obtained at percutaneous vesicocentesis, followed by conventional cytogenetics. Fetal urine was chosen due to unavailability of amniotic fluid for karyotypic analysis. A nonlethal (disomic) karyotype was suggested by FISH, and thus placement of a vesicoamniotic shunt was performed. The ability to prognosticate in cases of obstructive uropathy is not absolute, and fetal surgery for relief of urinary obstruction is best performed at the earliest possible gestational age. Thus, all available means for rapidly ruling out lethal congenital anomalies should be undertaken in cases of obstructive uropathy prior to any decision regarding fetal surgery.

The critical need for rapid and reliable karyotype analysis can be no greater than in the setting of sonographic fetal anomalies. Fluorescent in situ hybridization (FISH) directly applied to interphase chromosomes can decrease the time required to identify the common aneuploidies. Our retrospective study reviewed 50 consecutive patients with sonographic fetal anomalies who underwent FISH. Within this high risk group, nonmosaic chromosomal aneuploidies were present in 16% of the fetuses (8 of 50), and 2 additional fetuses had cytogenetic abnormalities: 1 case, 46,XY,-12,+der(12)t(12;13)(p13; q14.1), and 1 case a 10% mosaic for trisomy 21. Of the 10 cytogenetically abnormal fetuses, FISH was able to identify correctly all 8 of the nonmosaic aneuploidies within 2 days of receipt of the specimen in the laboratory. Clinical decisions can be made on the basis of concordant FISH and ultrasound abnormalities, shortening the decision-making process for most of the aneuploid cases. However, our experience demonstrates some of the limitations of current FISH protocols and the continued necessity for formal karyotype analysis.

In women at increased risk for breast and ovarian cancer, the identification of a mutation in breast cancer gene 1 (BRCA1) and BRCA2 has important implications for screening and prevention counseling. Uncertainty regarding the role of BRCA1 and BRCA2 testing in high-risk women from diverse ancestral backgrounds exists because of variability in prevalence estimates of deleterious (disease-associated) mutations in non-white populations. In this study, the authors examined the prevalence of BRCA1 and BRCA2 mutations in an ethnically diverse group of women who were referred for genetic testing.In this cross-sectional analysis, the prevalence of BRCA1 and BRCA2 mutations was assessed in a group of non-Ashkenazi Jewish women who underwent genetic testing.From 1996 to 2006, 46,276 women who met study criteria underwent DNA full-sequence analysis of the BRCA1 and BRCA2 genes. Deleterious mutations were identified in 12.5% of women, and recurrent deleterious mutations (prevalence2%) were identified in all ancestral groups. Women of non-European descent were younger (mean age, 45.9 years; standard deviation [SD], 11.6 years) than European women (mean age, 50 years; SD, 11.9 years; P.001). Women of African (15.6%; odds ratio [OR], 1.3 [95% confidence interval (95% CI), 1.1-1.5]) and Latin American (14.8%; OR, 1.2 [95% CI, 1.1-1.4]) ancestries had a significantly higher prevalence of deleterious BRCA1 and BRCA2 mutations compared with women of Western European ancestry (12.1%), primarily because of an increased prevalence of BRCA1 mutations in those 2 groups. Non-European ethnicity was associated strongly with having a variant of uncertain significance; however, reclassification decreased variant reporting (from 12.8%--5.9%), and women of African ancestry experienced the largest decline (58%).Mutation prevalence was found to be high among women who were referred for clinical BRCA1 and BRCA2 testing, and the risk was similar across diverse ethnicities. BRCA1 and BRCA2 testing is integral to cancer risk assessment in all high-risk women.

Two men aged 54 and 73 years, respectively, had oncocytic Schneiderian papilloma (OSP) containing synchronous carcinoma at the time of first biopsy. In both cases, invasive carcinoma involved a small proportion of excised tissue and was in continuity with dysplastic surface epithelium. Our cases document that the epithelial component of OSP can undergo malignant transformation. The focal involvement of OSP with carcinoma underscores the need to examine all excised tissue microscopically.

Many rearrangement mutations in the BRCA1 gene have been identified. It is becoming clear that some of these mutations are prevalent, and therefore their detection is necessary in order for clinical genetic tests to have high sensitivity. Published information on particular rearrangements is frequently limited to a single patient, small groups of patients, or patients of a particular ethnicity. The objectives of this work included characterizing the prevalence of five specific rearrangement mutations in a large North American patient population. A mutation-specific multiplex PCR assay was used for determining the prevalence of five BRCA1 rearrangement mutations that previously had been reported to occur in unrelated patients. The mutation status of these rearrangements, which came from 20,712 patients at high risk for hereditary breast and/or ovarian cancers who had submitted specimens for clinical genetic testing, is presented. The results, obtained from 2,634 mutation carriers, showed a 6-kb duplication of exon 13, identified in 53 patients (2.01%); a 26-kb deletion encompassing exons 14-20, detected in seven patients (0.27%); a 510-bp deletion of exon 22, detected in 5 patients (0.19%); and a 3.4-kb deletion of exon 13, detected in one patient (0.04%). A previously reported 7.1-kb deletion of exons 8-9 was not found. The high frequency of the exon 13 duplication makes it the fourth most prevalent mutation in these patients. These results provide an accurate picture of the prevalence of these mutations in hereditary breast/ovarian cancer patients undergoing genetic testing in North America.

The identification of intragenic rearrangements is important for a comprehensive understanding of mutations that occur in some clinically important genes. Single nucleotide polymorphism haplotypes obtained from clinical sequence data have been used to identify patients at high risk for rearrangement mutations. Application of this method identified a novel 26-kb deletion of BRCA1 exons 14 through 20 in patients from multiple families with hereditary breast and ovarian cancer. Clinical sequence data from 5911 anonymous patients were screened for genotypes that were inconsistent with known pairs of canonical haplotypes in BRCA1 that could be explained by hemizygous deletions involving exon 16. Long-range polymerase chain reaction demonstrated that two of six samples identified by this search contained a deletion in the expected region encompassing exons 14 through 20. The breakpoint was fully characterized by DNA sequencing and demonstrated that the deletion resulted from Alu-mediated recombination. This mutation was also identified twice in a set of 982 anonymous specimens that had negative clinical test results, but uninformative haplotypes. Three additional occurrences of this mutation were found by testing 10 other patients with the indicative genotype. An assay for this mutation was added to a comprehensive clinical breast/ovarian cancer test and eight more instances were found in 20,649 probands. This multiexon deletion has therefore been detected in 15 different North American families with hereditary breast/ovarian cancer. In conclusion, this primarily computational approach is highly effective and identifies specimens using existing data that are enriched for deletion mutations.

A powerful method for validating a scientific result is to confirm specific results utilizing independent methodologies and processing pathways. Thus, we have designed, developed and validated an automated allele concordance analysis system (CompareCalls, patent pending) that performs comparisons between two independent DNA analysis platforms to ensure the highest accuracy for allele calls. Application of this system in a quality assurance role has shown the potential to eliminate greater than 90% of the STR analysis required of a DNA data analyst. While this system is broadly applicable for use with any two independent STR analysis programs, either prior to or following human data review, we are presenting its application to data generated with the ABI Prism Genotyper software system versus data generated with the SurelockID system. With the automated allele concordance analysis system, the GeneScan DNA fragment data generated from an ABI 377 gel image are analyzed in two independent pathways. In one analysis pathway, the GeneScan data are imported into Genotyper software where STR labels are assigned to the fragment data based upon the criteria of the Kazam 20% macro. The "Kazam" macro provided with the Genotyper program works by labeling all peaks in a category (or locus) and then filtering (or removing) the labels from peaks, such as those in stutter positions, that meet predefined criteria. In the second pathway, the GeneScan data are imported into the SurelockID analysis platform where STR labels and error messages are assigned to the fragment data based upon hard-coded allele calling criteria and quality parameters. The resulting STR allele calls for each analysis platform are then compared, utilizing the automated allele concordance analysis system. Any differences in the STR allele calls between the two systems are flagged in a discordance report for further review by a qualified DNA data analyst. The automated allele concordance analysis system guides the DNA data analyst to the discordant data generated by either analysis platform. Additionally, the analyst is also directed to data that are of less than pristine quality which may have an increased potential for errors in interpretation by either analysis platform or by a human DNA data analyst. Implementation of an automated allele concordance analysis system will yield high-quality data for CODIS and free the human DNA data analyst to perform other critical duties within the laboratory.

Polymerase chain reaction (PCR)-based STR DNA typing systems are used extensively in the field of human identification. Under optimal PCR conditions, the amplicon yield from both alleles of an STR locus is expected to be approximately equivalent. However, it is reasonable to expect that rare genomic sequence polymorphisms will co-localize with well-designed primer sets and induce allele imbalance or "dropouts". Two samples were identified in the course of genotyping thousands of individuals with AmpF/STR Profiler Plus that showed strong disparity in amplitude peak height of heterozygous peaks at the loci vWA and FGA. These samples were reamplified at reduced annealing temperature in an attempt to balance the peak heights. Nucleotide sequencing documented polymorphisms at the PCR primer binding sites of the affected alleles. The results indicate that reducing the annealing temperature to improve primer-binding efficiency at the mismatch and employing an alternative multiplex enhanced the data from both samples. Reducing annealing temperatures could provide a simple general solution to improving data quality for samples where polymorphisms are suspected to cause allele imbalance. Finally, we report on additional polymorphisms surrounding the vWA locus in a genetically diverse population.

Sequence analysis of cDNA from an asymptomatic patient belonging to a high-risk breast cancer family carrying the genetic variant BRCA1 IVS10-2A--C revealed that functional BRCA1 mRNA was derived from only one of the patient's chromosomes. The other chromosome produced an aberrant RNA splicing transcript that deleted exon 11. Analysis of the patient's genomic DNA demonstrated that the chromosome producing the non-functional mRNA carried the genotype BRCA1 IVS10-2A--C. This transversion disrupts a highly conserved base in the consensus splice acceptor motif. These results support the conclusion that BRCA1 IVS10-2A--C is a mutation that confers predisposition to breast and ovarian cancer.

PURPOSE: To assess the characteristics that correlate best with the presence of mutations in BRCA1 and BRCA2 in individuals tested in a clinical setting. PATIENTS AND METHODS: The results of 10,000 consecutive gene sequence analyses performed to identify mutations anywhere in the BRCA1 and BRCA2 genes (7,461 analyses) or for three specific Ashkenazi Jewish founder mutations (2,539 analyses) were correlated with personal and family history of cancer, ancestry, invasive versus noninvasive breast neoplasia, and sex. RESULTS: Mutations were identified in 1,720 (17.2%) of the 10,000 individuals tested, including 968 (20%) of 4,843 women with breast cancer and 281 (34%) of 824 with ovarian cancer, and the prevalence of mutations was correlated with specific features of the personal and family histories of the individuals tested. Mutations were as prevalent in high-risk women of African (25 [19%] of 133) and other non-Ashkenazi ancestries as those of European ancestry (712 [16%] of 4379) and were significantly less prevalent in women diagnosed before 50 years of age with ductal carcinoma in situ than with invasive breast cancer (13% v 24%, P = .0007). Of the 74 mutations identified in individuals of Ashkenazi ancestry through full sequence analysis of both BRCA1 and BRCA2, 16 (21.6%) were nonfounder mutations, including seven in BRCA1 and nine in BRCA2. Twenty-one (28%) of 76 men with breast cancer carried mutations, of which more than one third occurred in BRCA1. CONCLUSION: Specific features of personal and family history can be used to assess the likelihood of identifying a mutation in BRCA1 or BRCA2 in individuals tested in a clinical setting.

Biochemical and genetic characterizations that support the conclusion that the variant BRCA2 IVS7 + 2T → G represents a deleterious mutation are presented. RNA analysis from a breast cancer patient with BRCA2 IVS7 + 2T → G showed that the productive message was produced from only one chromosome. A haplotype analysis confirmed that the intronic variant resides on the chromosome that does not produce the normal mRNA. Additionally, an RNA splicing product that deletes exon 7 was produced by the chromosome that carries BRCA2 IVS7 + 2T → G. The deletion of exon 7 from the RNA alters the open reading frame by removing residues 249–287 and incorporating 18 abnormal amino acids before terminating with an opal stop codon. The experimental approach presented produces strong evidence of the presence of a deleterious mutation, because the contribution by both chromosomes to each RNA species analyzed was tracked using a coding region polymorphism as a marker. Furthermore, a single nucleotide polymorphism (SNP) haplotype analysis that confirms the location of the intronic variant and an associated family history that shows a high incidence of cancer supported these biochemical data.

Laboratory analysis for genetic susceptibility to cancer is a rapidly evolving discipline. This chapter will provide a brief overview to the types of clinical laboratory tests that are currently being performed for characterization of mutational status in cancer susceptibility genes. The latter portion will focus on comprehensive sequencing of the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 as a representative model of predispositional testing for inherited cancer and to demonstrate the complexity of current diagnostic tests.

One hundred breast and breast-ovarian cancer families identified at the Helsinki University Central Hospital in southern Finland and previously screened for mutations in the BRCA2 gene were now analyzed for mutations in the BRCA1 gene. The coding region and splice boundaries of BRCA1 were analyzed by protein truncation test (PTT) and heteroduplex analysis (HA)/SSCP in all 100 families, and 70 were also screened by direct sequencing. Contrary to expectations based on Finnish population history and strong founder effects in several monogenic diseases in Finland, a wide spectrum of BRCA1 and BRCA2 mutations was found. In the BRCA1 gene, 10 different protein truncating mutations were found each in one family. Six of these are novel Finnish mutations and four have been previously found in other European populations. Six different BRCA2 mutations were found in 11 families. Altogether only 21% of the breast cancer families were accounted for by mutations in these two genes. Linkage to both chromosome 17q21 (BRCA1) and 13q12 (BRCA2) was also excluded in a subset of seven mutation-negative families with four or more cases of breast or ovarian cancer. These data indicate that additional breast and breast-ovarian cancer susceptibility genes are likely to be important in Finland.

OBJECTIVE: Although polymorphonuclear leukocytes are the inflammatory cells most frequently recovered from the amniotic cavity in cases of suspected intrauterine infection, the source of these cells has not been definitively determined. We took advantage of the gender difference between the mother and her male fetus, and we report four cases in which amniotic fluid polymorphonuclear leukocytes were identified as fetal by fluorescence in situ hybridization with probes specific for X and Y chromosomes. Fetal membranes were intact at the time amniotic fluid was obtained in all cases. STUDY DESIGN: Amniotic fluid was obtained from women with male fetuses in premature labor with clinical or laboratory evidence of infection. Cytospin preparations of amniotic fluid samples with polymorphonuclear leukocytes were prepared and sequentially stained with fluorescent reagents. To determine which cells were polymorphonuclear leukocytes, all replicate samples were stained with the fluorescent nuclear stain 4′-6-diamidino-2-phenyl-indole. This allowed definition of the characteristic multilobed polymorphonuclear leukocytes nuclear morphologic features. The sample was then probed with a rhodamine-labeled probe specific for the X chromosome and a fluorescein-labeled probe specific for the Y chromosome to assess whether the polymorphonuclear leukocytes were male or female. RESULTS: Ninety percent to 99% of polymorphonuclear leukocytes identified by normal multiple lobed nuclear morphologic study on 4′-6-diamidino-2-phenyl-indole staining had an X and Y chromosome and were therefore fetal cells. CONCLUSION: These data demonstrate a fetal response during intraamniotic infection. Further investigation of the roles for maternal and fetal polymorphonuclear leukocytes in chorioamnionitis may provide valuable information about the critical interaction of the two immune responses in this setting. (Am J Obstet Gynecol 1997;176:77-81.)

Mutations in the base excision repair gene MYH were recently implicated in recessive inheritance of colorectal adenomas and carcinomas.1 The majority of patient specimens screened for MYH and described in published reports derive from the United Kingdom, where two missense variants—Y165C and G382D—are the most prevalent mutations in the white population.2–5 The carrier frequency for these two mutations is approximately 2% in the British population.1,4 Some additional mutations were detected at lower frequency in these patients. Two other protein truncating mutations—E466X and Y90X—have been found in individuals of Indian and Pakistani descent, respectively.2,3 Finally, a mutation that deletes codon Glu-466 was reported to be prevalent in Italian patients.6 This evidence supports the view that additional mutations will be discovered showing differences in prevalence between ancestries. We determined the mutation spectrum for MYH by direct DNA sequencing in 219 anonymous North American patient specimens found negative for APC mutations during clinical genetic testing for risk …

PURPOSE To evaluate the prognostic significance of ipsilateral breast tumor recurrence (IBTR) with respect to the subsequent development of distant metastasis. MATERIALS AND METHODS Between January 1970 and December 1989, 973 patients with invasive breast cancer were treated with conservative surgery and radiation therapy at Yale-New Haven Hospital. The median follow-up time as of December 1993 was 8.6 years. A number of prognostic factors were tested as possible predictors of distant metastases, including whether a patient experienced IBTR. IBTRs were broken down by time to recurrence to determine whether the breast recurrence-free interval had any prognostic relevance with respect to the development of distant metastasis. RESULTS As of December 1993, out of the entire population of 973 patients, 73 patients had developed IBTR and 134 had developed distant metastases. The overall actuarial survival rate at 10 years was .71 +/- .02, with a 10-year actuarial breast recurrence-free rate of .84 +/- .02 and a 10-year distant metastasis-free rate of .77 +/- .02. The overall distant metastasis rate was higher in patients who experienced IBTR compared with patients who had never experienced IBTR. Furthermore, the time to IBTR had a significant effect on distant metastases. Of 32 patients who developed an IBTR within 4 years of original diagnosis, 16 (50%) developed distant metastases. In contrast, of 41 patients who developed later breast relapses (> 4 years from original diagnosis), only seven (17%) developed distant metastases (P < .01). Of 32 patients who developed early breast relapse, the 5-year survival rate following breast relapse was .50 +/- .01, compared with a 5-year post-breast relapse survival rate of .78 +/- .10 among 41 patients with later breast relapses (P < .05). CONCLUSION It appears that early IBTR is a significant predictor for distant metastases. Whether early breast tumor relapse is a marker for or cause of distant metastases remains a controversial and unresolved issue. Implications for adjuvant systemic therapy at the time of breast relapse are discussed.

Four hundred and sixty-five college women were evaluated to determine if specific variables of social and sexual behavior correlated with the presence of human papillomavirus (HPV) DNA in the genital tract, and if these associations differed between women who were HPV DNA positive, HPV DNA positive/clinically (cytologically) negative, or who reported previous HPV-related disease by a history of an abnormal Papanicolaou (Pap) smear or genital warts. HPV positive women had more sexual partners in the recent past, more sexual episodes per month, and used spermacides less commonly than controls. Similarly, self-reporters were more likely to have more lifetime sexual partners and earlier age of onset for sexual activity. Cytologically negative HPV positive women were distinguished only by more sexual episodes per month and sexual partners in the past year (borderline significance). Alcohol use was significantly more frequent in all groups, underscoring this variable as a risk factor for both HPV DNA positivity and related disease in young women. Potential explanations for differences between women with clinically and non-clinically related HPV positivity are discussed, with emphasis on the need for followup studies to determine if an epidemiologically distinct subset of HPV DNA positive but clinically negative women are at risk for subsequent cervical disease.Cancer of the cervix is strongly associated with sexual behavior, and the risk of acquiring both cervical cancer and its precursor lesions increases with the number of sex partners, early age at first intercourse, increasing parity, and cigarette smoking. Low dietary intake of vitamin A and long-term use of oral contraceptives have also been suspected of elevating the risk of cervical intraepithelial neoplasia and cervical cancer. Human papillomavirus (HPV) has been studied intensively in recent years with regard to its role in the genesis of cervical cancer. Cervical infection by the virus occurs sexually and HPV has been isolated from cervical warts, precancers, and invasive carcinomas. Certain HPV types have been associated with cervical neoplasia and extensive experimental data indicate that HPV is an oncogenic virus. The prevalence of clinical HPV infection among young women has increased over the past decade. 465 women enrolled at the University of Virginia were evaluated to determine if certain variables of social and sexual behavior correlated with the presence of HPV DNA in the genital tract, and if the associations differed between women who were HPV DNA positive, HPV DNA positive/clinically negative, or who reported previous HPV-related disease by a history of an abnormal Pap smear or genital warts. The women were of mean age 22.7 years in the range of 17-39 years, 89% White, 91% single, and 65% undergraduate. This study population did not differ significantly from the university population. HPV-positive women had more sex partners in the recent past, more sexual episodes per month, and used spermicides less commonly than controls. Self-reporters were more likely to have more lifetime sex partners and earlier age of onset for sexual activity. Cytologically negative HPV-positive women were distinguished only by more sexual episodes per month and sex partners in the past year. Alcohol use was significantly more frequent in all groups, highlighting that variable as a risk factor for both HPV DNA positivity and related disease in young women. Potential explanations for differences between women with clinically and non-clinically related HPV positivity are discussed, with emphasis upon the need for follow-up studies to determine if an epidemiologically distinct subset of HPV DNA-positive, but clinically negative women are at risk for subsequent cervical disease.

Summary: A fundamental question in Papanicolaou smear screening is the specificity of cytologic criteria for the recognition of genital human papillomavirus (HPV) infection. To address this problem, we conducted a two-phase study of routinely screened women to determine the efficiency with which cytologic findings identified the presence of HPV DNA, focusing on the criteria for identifying smears as “atypical.” In phase 1, 25 of 290 (8.6%) smears were designated atypical, but only 3 (12%) of the samples contained HPV nucleic acids. Four of five (80%) smears designated as diagnostic of HPV/cervical HPV infection were associated with HPV nucleic acids. By applying more stringent criteria for the diagnosis of atypical in phase 2, only 3 of 166 (1.8%) were identified as atypical. Of these, two (67%) contained HPV nucleic acids. The criteria that most efficiently correlated with HPV nucleic acids included prominent nuclear enlargement with either multiple nuclei or nuclear hyperchromatism. On rereview of the 19 HPV-positive and 20 control HPV-negative smears originally diagnosed as cytologically negative, the above criteria identified an additional 3 cytologically atypical/positive smears versus none (0 of 20) in the control group. This study supports the concept that cytologic abnormalities suggesting “subtle” HPV infection may be extremely difficult to distinguish from non-HPV-related changes, and that criteria used to imply “suggestive but not diagnostic for HPV infection” should be continually reevaluated. The potential role of HPV DNA analysis in Papanicolaou smear interpretation is discussed.

9533 Background: Rearrangement mutations may represent 5–10% of clinically significant variants in the BRCA1 tumor suppressor gene. Several of these mutations occur recurrently in unrelated familie...

A patient who exhibited the phenotype of the Pallister mosaic aneuploid syndrome was cytogenetically diagnosed in the newborn period by bone marrow analysis. A 47,XY,i(12p) karyotype was observed in 100% of the metaphases from direct bone marrow preparations, while phytohemagglutinin (PHA)-stimulated bone marrow was 23% isochromosome positive. Initially, 10% of metaphases from a peripheral blood culture were isochromosome positive, but at 2 months of age all metaphases examined were cytogenetically normal. Serial fibroblast cultures were 75%, 100%, and 28% positive, respectively. The isochromosome was also present in all metaphases examined from lung tissue and testes. This karyotypic pattern supports a theory that tissue-limited mosaicism may result from selection due to differing developmental potentials of certain karyotypes in various tissues.

Alzheimer disease was transmitted in a pattern consistent with an autosomal dominant trait in three families. This brings to 50 the number of such families reported. In one of our families, one patient had histologically confirmed Alzheimer disease, whereas her sister had proved spongiform encephalopathy. Other data suggest a link between familial Alzheimer disease and transmissible dementia. Alzheimer disease is associated with abnormal neurofibrillary structures, Down syndrome, and abnormal numbers of chromosomes in lymphocytes (aneuploidy). These observations are consistent with a disorder in the physiology of tubular-filamentous structures involving different cell types.

Certain human papillomavirus (HPV) types (such as type 16) have been linked to high-grade precancers and invasive carcinomas of the cervix. However, the accuracy with which morphologic characteristics will predict the presence and type of HPV infection is controversial. Three pathologists independently classified 102 consecutive cervical biopsies with the use of specific criteria and correlated their findings with the presence of HPV 11, 16, and 18 RNA sequences by in situ hybridization. Based on the presence and distribution of nuclear atypia, abnormal mitotic figures, and koilocytosis, biopsies were classified into borderline condyloma, condyloma, borderline cervical intraepithelial neoplasia (CIN) with koilocytotic atypia (CINK), and CIN. Two or more observers agreed on the diagnosis in 96% of cases. HPV 16-related sequences alone were detected in 0% of borderline condylomata, 17% of flat condylomata, 43% of borderline CINK, 67% of CINK, and 77% of CIN lesions. Other HPVs, including those producing signals with more than one probe, were present in 0, 50, 14, 9, and 0% of these lesions, respectively. The authors data suggest that consistent identification of HPV-related cervical disease requires the presence of specific cytologic changes. In the authors' series, when HPV-related disease is present, CIN is the most common lesion and most (71%) contain HPV 16-related nucleic acids. Thus, a high proportion (88%) of histologic abnormalities associated with HPV-16 could be distinguished as CIN by morphologic characteristics alone, and this distinction could be made by most observers.

The purpose of this study was to determine if cytogenetic changes are present in Alzheimer disease, one of the presenile dementias. The chromosomes of three groups of people were studied: 1) sporadic cases of Alzheimer disease (eight cases), 2) familial cases of Alzheimer disease with affected individuals in at least two generations of their families (five cases), and 3) currently unaffected siblings of the affected individuals in these families (nine cases). One hundred cells per individual were examined using GTG banding to allow chromosome identification. A statistically significant increase in aneuploidy was found in five of eight patients in group 1 (P less than 0.05) and in each of five patients in group 2 (P less than 0.001) when compared with the rate of aneuploidy in age- and sex-matched controls. In addition, two individuals in group 3 exhibited a significant increase in aneuploidy over the control group, raising the possibility that finding increased aneuploidy may allow one to anticipate the clinical expression of the disease state.

The authors describe two women with locally infiltrative neoplasms of the nipple that exhibited sweat duct differentiation. The findings are compared with those previously recorded for syringomatous adenoma of the nipple. The patients presented with firm, subareolar nodules. Nests, cords, and ducts composed of cytologically uniform squamous cells were dispersed throughout the dermis and also involved subcutaneous tissue and adjacent breast parenchyma. Small keratocysts formed a minor component of the lesions. Additional observations in the present study included connections between tumor and epidermis; distinctive fibrous stroma; sparse mitotic figures; perineural invasion; carcinoembryomic antigen in luminal content and periluminal cytoplasm; tumor cells that contained S-100 protein; and Langerhans' cells within neoplastic cellular aggregates. Neither of the lesions had recurred after follow-up of 9 and 12 months. The foregoing neoplasms of the nipple appear to be part of a family of microscopically similar tumors that have a predilection for the face, breast and possibly the axilla and salivary tissue. Although capable of local recurrence, syringomatous tumors of the nipple and related lesions at other sites have not been reported to metastasize.

Octext. —A mutation in theBRCA1gene may confer substantial risk for breast and/or ovarian cancer. However, knowledge regarding all possible mutations and the relationship between risk factors and mutations is incomplete. Objectives. —To identifyBRCA1mutations and to determine factors that best predict presence of a deleteriousBRCA1mutation in patients with breast and/or ovarian cancer. Design. —A complete sequence analysis of theBRCA1coding sequence and flanking intronic regions was performed in 798 women in a collaborative effort involving institutions from the United States, Italy, Germany, Finland, and Switzerland. Participanta. —Institutions selected 798 persons representing families (1 person for each family) thought to be at elevated a priori risk ofBRCA1mutation due to potential risk factors, such as multiple cases of breast cancer, early age of breast cancer diagnosis, and cases of ovarian cancer. No participant was from a family in which genetic markers showed linkage to theBRCA1locus. Major Outcome Measures. —Sequence variants detected in this sample are presented along with analyses designed to determine predictive characteristics of those testing positive forBRCA1mutations. Results. —In 102 women (12.8%), clearly deleterious mutations were detected. Fifty new genetic alterations were found including 24 deleterious mutations, 24 variants of unknown significance, and 2 rare polymorphisms. In a subset of 71 Ashkenazi Jewish women, only 2 distinct deleterious mutations were found: 185delAG in 17 cases and 5382insC in 7 cases. A bias in prior reports for mutations in exon 11 was revealed. Characteristics of a patient's specific diagnosis (unilateral or bilateral breast cancer, with or without ovarian cancer), early age at diagnosis, Ashkenazi Jewish ethnicity, and family history of cancer were positively associated with the probability of her carrying a deleteriousBRCA1mutation. Conclusions. —Using logistic regression analysis, we provide a method for evaluating the probability of a woman's carrying a deleteriousBRCA1mutation for a wide range of cases, which can be an important tool for clinicians as they incorporate genetic susceptibility testing into their medical practice.

Inverted tandem duplications in man are a rare form of structural rearrangements involving a single chromosome.1 We describe a patient with partial trisomy of chromosome 14 due to an inverted tandem duplication of the long arm. CASE REPORT The proband was a term product of a 24-year-old mother's second pregnancy. The first terminated in a miscarriage at six weeks. After the birth of the proband, the mother had two miscarriages at six weeks' gestation followed by two term pregnancies resulting in the delivery of normal female infants. The proband was a male infant whose birth weight was 5 lb 0 oz.