Physiologic Long Head Biceps Tendon Excursion Throughout Shoulder Range of Motion: A Cadaveric Study.

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.

Patient-Specific Guides/Instrumentation in Shoulder Arthroplasty.

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.

Strategies for H-score normalization of preanalytical technical variables with potential utility to immunohistochemical-based biomarker quantitation in therapeutic response diagnostics.

Digital quantitative immunohistochemical analysis of protein biomarker expression offers a broad dynamic range against which clinical outcomes may be measured. Semi-quantitative expression data represented as an H-score is produced by computer generated average intensity of positive staining given weight by the percentage of cells showing positive staining. While patient H-scores vary for biological reasons, variation may also arise from preanalytic technical issues, such as differences in fixation protocols. In this study, we present data on two candidate calibrator nuclear-localized proteins, SNRPA and SnRNP70, with robust and consistent expression levels across breast cancers. Quantitative expression measurement of these two candidate biomarkers may potentially be used to eliminate the effect of differences in preanalytic processing of specimens by normalizing H-scores derived from test biomarkers of interest. To examine the effects of preanalytical fixation variation on biomarker quantitation and potential utility of candidate calibrators to address such issues, 6 surgically-resected human breast cancer patient specimens were divided into 6 portions and fixed under distinct conditions (fixation following resection in formalin for 2 hr, 8 hr or 48 hr, or held overnight at 4°C in buffered saline prior to formalin fixation for 2 hr, 8 hr, or 48 hr). We find H-score variation between fixation conditions within individual patient's tumors that were stained for XPF, ATM, BRCA1, pMK2 and PARP1. Most interestingly, detectable expression of SNRPA and SnRNP70 is covariant to test biomarkers under distinct fixation conditions and so these hold the potential for serving as calibration standards for general antigen preservation and reactivity.

BRCA1 and BRCA2 mutations in women of different ethnicities undergoing testing for hereditary breast-ovarian cancer.

BACKGROUND: 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. METHODS: 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. RESULTS: 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 (prevalence >2%) 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%). CONCLUSIONS: 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.

ACMG recommendations for standards for interpretation and reporting of sequence variations: Revisions 2007.

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.

Application of embryonic lethal or other obvious phenotypes to characterize the clinical significance of genetic variants found in trans with known deleterious mutations.

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.

Prevalence of five previously reported and recurrent BRCA1 genetic rearrangement mutations in 20,000 patients from hereditary breast/ovarian cancer families.

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.

A multi-exonic BRCA1 deletion identified in multiple families through single nucleotide polymorphism haplotype pair analysis and gene amplification with widely dispersed primer sets.

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.

The application of an automated allele concordance analysis system (CompareCalls) to ensure the accuracy of single-source STR DNA profiles.

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.

Accurate STR allele designations at the FGA and vWA loci despite primer site polymorphisms.

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 biochemical analysis demonstrates that the BRCA1 intronic variant IVS10-2A--> C is a mutation.

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.

Clinical characteristics of individuals with germline mutations in BRCA1 and BRCA2: analysis of 10,000 individuals.

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.