Discordance of insulin-like growth factor-1 results and interpretation on four different platforms.

OBJECTIVE: Inconsistent Insulin-like Growth Factor 1 (IGF-1) measurements among different platforms have been observed. In this study, we compared the IGF-1 assay on four different platforms. METHODS: A total of 110 serum specimens were analyzed in this comparison study. IGF-1 was measured on the three different chemiluminescent automated immunoassay of Siemens Immulite 2000 XPi, DiaSorin Liaison XL, IDS iSYS and LC-MS/MS method. Results were compared with Weighted Deming regression. Bias was evaluated using the Bland-Altman method. RESULTS: Weighted Deming regression analysis showed approximately 36 % negative variation on Immulite, compared to Liaison (Immulite = 0.64 * DiaSorin + 2.95, r2 = 0.95); 8 % negative variation on iSYS, compared to Liaison (iSYS = 0.92 * DiaSorin + 0.51, r2 = 0.97); 17 % negative variation on LC-MS/MS, compared to Liaison (LC-MS/MS = 0.83 * DiaSorin-11.23, r2 = 0.93); 34 % positive variation on LC-MS/MS compared to Immulite (LC-MS/MS = 1.34 * Immulite-21.97, r2 = 0.96); 81 % positive variation on IDS iSYS compared to Immulite (IDS iSYS = 1.81 * Immulite-117.65, r2 = 0.83). The Bland-Altman plot showed a significant negative variation of Immulite versus DiaSorin and positive variation of IDS iSYS versus Immulite. Overall agreement between different platforms was poor, which reflected systematic difference. The variation between platforms increased as IGF-1 values increased. CONCLUSIONS: There are wide variations between different platforms for IGF-1 measurement. The lack of standardization in IGF-1 measurement creates a challenge for clinicians to monitor IGF-1 and treat patients with pituitary disorders, when switching from one platform to another. The potential impact of the variations in IGF-1 measurement between different platforms should be taken into consideration when managing patients.

Detection of SARS-CoV-2 infection in asymptomatic populations using the DiaSorin molecular Simplexa and Roche Cobas EUA assays.

Identification of asymptomatic patients is necessary to control the COVID-19 pandemic and testing is one of the measures to detect this population. We evaluated the clinical correlation of the DiaSorin Molecular Simplexa COVID-19 Direct (DiaSorin Molecular) and Roche Cobas 6800 SARS-CoV-2 (Roche) assays using 253 oropharyngeal (OP) swab specimens collected from asymptomatic patients. Agreement between DiaSorin Molecular and Roche was 97% (95% CI, 0.94 to 0.99), with a κ statistic of 0.90 (95% CI, 0.83 to 0.97) and a PPA of 89% (95% CI, 0.76 to 0.96) and NPA of 99% (95% CI, 0.97 to 0.99). Simple regression analysis of Ct values revealed a regression line of y = 1.065*X - 5.537 with a Pearson's r of 0.8542, indicating a good correlation between both platforms. The DiaSorin Molecular assay demonstrates clinical performance comparable to that of Roche in this population.

Clinical Evaluation and Utilization of Multiple Molecular In Vitro Diagnostic Assays for the Detection of SARS-CoV-2.

OBJECTIVES: To evaluate the clinical performance of 3 molecular assays for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). METHODS: We used 184 nasopharyngeal swab specimens to compare Abbott ID NOW COVID-19 (Abbott ID NOW), DiaSorin Molecular Simplexa COVID-19 Direct (DiaSorin Simplexa), and Roche cobas 6800 SARS-CoV-2 (Roche cobas) assays. In a separate analysis, 3 specimens (nasopharyngeal, oropharyngeal, and nasal) were collected from 182 unique patients presenting to the emergency department with suspicion of coronavirus disease 2019 and were tested utilizing Abbott ID NOW. To further characterize each assay, relative limits of detection were evaluated utilizing positive nasopharyngeal patient samples. RESULTS: The positive percent agreement was 91% (95% confidence interval [CI], 0.76-0.97) for Abbott ID NOW and 100% (95% CI, 0.90-1.00) for DiaSorin Simplexa and Roche cobas. The negative percent agreement was 100% (95% CI, 0.98-1.00) for all 3 assays. All swab types tested with the Abbott assay produced concordant results. Polymerase chain reaction assays had approximately 10 to 100 times lower limits of detection than Abbott ID NOW. CONCLUSIONS: Based on these evaluations, a multiplatform testing approach is proposed, depending on patient population and assay sensitivity, to address testing needs during a public health emergency.

Monozygotic twins discordant for congenital adrenal hyperplasia due to mosaicism.

INTRODUCTION: Genotype-phenotype discordance occurs occasionally in congenital adrenal hyperplasia (CAH). Its causes are largely unknown. We describe a case of monochorionic, diamniotic twins with discordant clinical presentations of CAH, and show evidence for this being due to mosaicism resulting from a postzygotic full gene deletion of CYP21A2 prior to twinning. CASE DESCRIPTION: A 7-day-old 36-week gestation female infant (Twin A) presented to the emergency department with elevated 17-hydroxyprogesterone (17-OHP). Her identical twin (Twin B) had normal 17-OHP on newborn screening. Both twins showed signs of virilization, more pronounced in Twin B. Molecular genetic testing of both twins and their parents showed a WT paternally-inherited CYP21A2 and a maternally-inherited copy containing the c.293-13C>G mutation. Both twins were also found to have a 5'-CYP21A1P/CYP21A2-3' hybrid (product of the common 30-kb deletion), derived from the deletion of the paternally-inherited CYP21A2. Neither mother nor father carried the deletion. CONCLUSIONS: The genetic findings are consistent with mosaicism for two CYP21A2 cell lines in both twins. The first cell line is expected, based on parental results, while the second line is due to a postzygotic full gene deletion of the paternally-inherited WT CYP21A2. The resultant genotype, compound heterozygosity for c.293-13C>G and a CYP21A2 full gene deletion, is consistent with a salt-wasting CAH phenotype. Differential distribution of the second cell line between the twins is most likely the cause for their discrepant phenotypes. We believe this is the first report of somatic CYP21A2 mosaicism, and represents a novel cause for discrepant CAH phenotypes in monozygotic twins.

Vedolizumab quantitation using high-resolution accurate mass-mass spectrometry middle-up protein subunit: method validation.

Background While quantitation methods for small-molecule and tryptic peptide bottom-up mass spectrometry (MS) have been well defined, quantitation methods for top-down or middle-up MS approaches have not been as well defined. Therapeutic monoclonal antibodies (t-mAbs) are a group of proteins that can be used to both demonstrate the advantages of top-down or middle-up detection methods over classic tryptic peptide bottom-up along with the growing need for robust quantitation strategies/software for these top-down or middle-up methods. Bottom-up proteolytic digest methods for the t-mAbs tend to suffer from challenges such as limited peptide selection due to potential interference from the polyclonal immunoglobulin background, complicated workflows, and inadequate sensitivity and specificity without laborious purification steps, and therefore have prompted the search for new detection and quantitation methods. Time-of-flight along with Orbitrap MS have recently evolved from the research and/or pharmaceutical setting into the clinical laboratory. With their superior mass measurement accuracy, resolution and scanning speeds, these are ideal platforms for top-down or middle-up characterization and quantitation. Methods We demonstrate a validated, robust, middle-up protein subunit detection and quantitation method for the IgG1 t-mAb, vedolizumab (VEDO), which takes advantage of the high resolution of the Orbitrap MS detection and quantitation software to increase specificity. Results Validated performance characteristics met pre-defined acceptance criteria with simple workflows and rapid turnaround times: characteristics necessary for implementation into a high-volume clinical MS laboratory. Conclusions While the extraction method can easily be used with other IgG1 t-mAbs, the detection and quantitation method may become an option for measurement of other proteins.

Validation of the Cut-points Recommended for ROMA Using the Roche Elecsys CA125 and HE4 Assays.

Assessing the risk of malignancy in patients with a pelvic mass helps triage women suspected of ovarian cancer to specialized gynecologic oncologists to improve treatment outcomes. To this end, several algorithms have been proposed; most notably, the Risk of Ovarian Malignancy Algorithm (ROMA) based on CA125, HE4, and menopausal status. However, appropriate decision cut-points for the ROMA score depends on the choice of analytical assays used. This study validates the use of the Roche Elecsys CA125 and HE4 assays for ROMA calculation in a cohort of 207 women who presented to Mayo Clinic with a pelvic mass. Results were compared to a definitive histologic diagnosis in each case. Clinical performance of ROMA scores derived using these assays was similar to stated claims and indicates that recommended cut-points are acceptable for clinical use.

Development and characterization of a differentiated thyroid cancer cell line resistant to VEGFR-targeted kinase inhibitors.

BACKGROUND: Vascular endothelial growth factor-targeted kinase inhibitors have emerged as highly promising therapies for radioiodine-refractory metastatic differentiated thyroid cancer. Unfortunately, drug resistance uniformly develops, limiting their therapeutic efficacies and thereby constituting a major clinical problem. APPROACH AND METHODS: To study acquired drug resistance and elucidate underlying mechanisms in this setting, BHP2-7 human differentiated thyroid cancer cells were subjected to prolonged continuous in vitro selection with 18 µM pazopanib, a clinically relevant concentration; acquisition of pazopanib resistance was serially assessed, with the resulting resistant cells thereafter subcloned and characterized to assess potential mechanisms of acquired pazopanib resistance. RESULTS: Stable 2- to 4-fold in vitro pazopanib resistance emerged in response to pazopanib selection associated with similar in vitro growth characteristics but with markedly more aggressive in vivo xenograft growth. Selected cells were cross-resistant to sunitinib and to a lesser extent sorafenib but not to MAPK kinase (MEK1/2) inhibition by GSK1120212. Genotyping demonstrated acquisition of a novel activating KRAS codon 13 GGC to GTT (glycine to valine) mutation, consistent with the observed resistance to upstream vascular endothelial growth factor receptor inhibition yet sensitivity to downstream MAPK kinase (MEK1/2) inhibition. CONCLUSIONS: Selection of thyroid cancer cells with clinically utilized therapeutics can lead to acquired drug resistance and altered in vivo xenograft behavior that can recapitulate analogous drug resistance observed in patients. This approach has the potential to lead to insights into acquired treatment-related drug resistance in thyroid cancers that can be subjected to subsequent validation in serially collected patient samples and that has the potential to yield preemptive and responsive approaches to dealing with this important clinical problem.

BIMA V3: an aligner customized for mate pair library sequencing.

Mate pair library sequencing is an effective and economical method for detecting genomic structural variants and chromosomal abnormalities. Unfortunately, the mapping and alignment of mate-pair read pairs to a reference genome is a challenging and time-consuming process for most next-generation sequencing alignment programs. Large insert sizes, introduction of library preparation protocol artifacts (biotin junction reads, paired-end read contamination, chimeras, etc.) and presence of structural variant breakpoints within reads increase mapping and alignment complexity. We describe an algorithm that is up to 20 times faster and 25% more accurate than popular next-generation sequencing alignment programs when processing mate pair sequencing.

A simple method for gene phasing using mate pair sequencing.

BACKGROUND: Recessive genes cause disease when both copies are affected by mutant loci. Resolving the cis/trans relationship of variations has been an important problem both for researchers, and increasingly, clinicians. Of particular concern are patients who have two heterozygous disease-causing mutations and could be diagnosed as affected (one mutation on each allele) or as phenotypically normal (both mutations on the same allele). Several methods are currently used to phase genes, however due to cost, complexity and/or low sensitivity they are not suitable for clinical purposes. METHODS: Long-range amplification was used to select and enrich the target gene (CYP21A2) followed by modified mate-pair sequencing. Fragments that mapped coincidently to two heterozygous sites were identified and used for statistical analysis. RESULTS: Probabilities for cis/trans relationships between heterozygous positions were calculated along with 99% confidence intervals over the entire length of our 10 kb amplicons. The quality of phasing was closely related to the depth of coverage and the number of erroneous reads. Most of the error was found to have been introduced by recombination in the PCR reaction. CONCLUSIONS: We have developed a simple method utilizing massively parallel sequencing that is capable of resolving two alleles containing multiple heterozygous positions. This method stands out among other phasing tools because it provides quantitative results allowing confident haplotype calls.

RNA sequencing identifies multiple fusion transcripts, differentially expressed genes, and reduced expression of immune function genes in BRAF (V600E) mutant vs BRAF wild-type papillary thyroid carcinoma.

CONTEXT: The BRAF V600E mutation (BRAF-MUT) confers an aggressive phenotype in papillary thyroid carcinoma, but unidentified additional genomic abnormalities may be required for full phenotypic expression. OBJECTIVE: RNA sequencing (RNA-Seq) was performed to identify genes differentially expressed between BRAF-MUT and BRAF wild-type (BRAF-WT) tumors and to correlate changes to patient clinical status. DESIGN: BRAF-MUT and BRAF-WT tumors were identified in patients with T1N0 and T2-3N1 tumors evaluated in a referral medical center. Gene expression levels were determined (RNA-Seq) and fusion transcripts were detected. Multiplexed capture/detection and digital counting of mRNA transcripts (nCounter, NanoString Technologies) validated RNA-Seq data for immune system-related genes. PATIENTS: BRAF-MUT patients included nine women, three men; nine were TNM stage I and three were stage III. Three (25%) had tumor infiltrating lymphocytes. BRAF-WT included five women, three men; all were stage I, and five (62.5%) had tumor infiltrating lymphocytes. RESULTS: RNA-Seq identified 560 of 13 085 genes differentially expressed between BRAF-MUT and BRAF-WT tumors. Approximately 10% of these genes were related to MetaCore immune function pathways; 51 were underexpressed in BRAF-MUT tumors, whereas 4 (HLAG, CXCL14, TIMP1, IL1RAP) were overexpressed. The four most differentially overexpressed immune genes in BRAF-WT tumors (IL1B; CCL19; CCL21; CXCR4) correlated with lymphocyte infiltration. nCounter confirmed the RNA-Seq expression level data. Eleven different high-confidence fusion transcripts were detected (four interchromosomal; seven intrachromosomal) in 13 of 20 tumors. All in-frame fusions were validated by RT-PCR. CONCLUSION: BRAF-MUT papillary thyroid cancers have reduced expression of immune/inflammatory response genes compared with BRAF-WT tumors and correlate with lymphocyte infiltration. In contrast, HLA-G and CXCL14 are overexpressed in BRAF-MUT tumors. Sixty-five percent of tumors had between one and three fusion transcripts. Functional studies will be required to determine the potential role of these newly identified genomic abnormalities in contributing to the aggressiveness of BRAF-MUT and BRAF-WT tumors.

Increasing liquid chromatography-tandem mass spectrometry throughput by mass tagging: a sample-multiplexed high-throughput assay for 25-hydroxyvitamin D2 and D3.

BACKGROUND: The limits of chromatographic speed and mechanical frontend capabilities have been reached for many high-volume liquid chromatography-tandem mass spectrometry (LC-MS/MS) tests, curtailing the maximal achievable sample throughput. To overcome these boundaries, we developed and validated a derivatization-based sample-multiplex LC-MS/MS assay for detection of 25-hydroxyvitamins D2 and D3 [25(OH)D2 and 25(OH)D3], which increased sample throughput 5-fold. METHODS: After separate derivatization with 1 of 5 different triazoline-diones (TADs), 5 calibrators, controls, or patient specimens were combined and injected together into an LC-MS/MS. On the basis of mass differences between TADs, the MS/MS quantified analyte and stable isotope internal standards for 25(OH)D2 and 25(OH)D3 for each respective multiplexed sample within the injection. Limits of detection and quantification, spiked recovery, linearity, imprecision, and patient results were determined and compared against our standard LC-MS/MS assay. RESULTS: TAD multiplexing increased throughput on an LC-quadruplexed LC-MS/MS system from 60 samples/h to 300 samples/h. Limits of detection and quantification were 4.9 nmol/L [2 µg/L, 25(OH)D2], 2.2 nmol/L [0.9 µg/L, 25(OH)D3], and 10 nmol/L [4 µg/L, 25(OH)D2], 5 nmol/L [2 µg/L, 25(OH)D3], respectively. The assay was linear to 250 nmol/L (100 µg/L). Interassay CVs across the reportable range were 3.7%-15.2%. Spiked recoveries were 94%-119%. The method comparison with the standard LC-MS/MS method showed slopes of 0.96 and 0.97 (Deming regression) for 25(OH)D2 (n=1733) and 25(OH)D3 (n=7614) (R2=0.96 and 0.97), respectively. CONCLUSIONS: Multiplexing samples by differential mass tagging in LC-MS/MS measurement of 25(OH)D2 and 25(OH)D3 allows for reliable quantification, with throughput increased over standard methods by the multiplexing factor.

Detailed molecular fingerprinting of four new anaplastic thyroid carcinoma cell lines and their use for verification of RhoB as a molecular therapeutic target.

CONTEXT: Anaplastic thyroid carcinoma (ATC) is a highly aggressive carcinoma in need of therapeutic options. One critical component of drug discovery is the availability of well-characterized cell lines for identification of molecular mechanisms related to tumor biology and drug responsiveness. Up to 42% of human thyroid cancer cell lines are redundant or not of correct tissue origin, and a comprehensive analysis is currently nonexistent. Mechanistically, RhoB has been identified as a novel molecular target for ATC therapy. OBJECTIVE: The aim was to develop four ATC cell lines detailing genetic, molecular, and phenotypic characteristics and to test five classes of drugs on the cell lines to determine whether they inhibited cell proliferation in a RhoB-dependent fashion. DESIGN: Four cell lines were derived from ATC tumors. Short tandem DNA repeat and mutational status of the originating tumors and cell lines were performed along with molecular and phenotypic characterizations. Compounds were tested for growth inhibition and ability to up-regulate RhoB. RESULTS: Cell line authenticity was confirmed by DNA short tandem repeat analysis. Each proved unique regarding expression of thyroid markers, oncogene status, amplified and deleted genes, and proliferative growth rates. FTI-277, GGTI-286, lovastatin, romidepsin, and UCN-01 up-regulated RhoB and inhibited cell proliferation in a dose-responsive fashion with only romidepsin and FTI-277 being RhoB dependent. CONCLUSIONS: Molecular descriptions of thyroid lines were matched to the originating tumors, setting a new standard for cell line characterization. Furthermore, suppressed RhoB is implicated as a molecular target for therapy against ATC because five classes of drugs up-regulate RhoB and inhibit growth dose-responsively.

Succinate dehydrogenase gene mutations are strongly associated with paraganglioma of the organ of Zuckerkandl.

Organ of Zuckerkandl paragangliomas (PGLs) are rare neuroendocrine tumors that are derived from chromaffin cells located around the origin of the inferior mesenteric artery extending to the level of the aortic bifurcation. Mutations in the genes encoding succinate dehydrogenase subunits (SDH) B, C, and D (SDHx) have been associated with PGLs, but their contribution to PGLs of the organ of Zuckerkandl PGLs is not known. We aimed to describe the clinical presentation of patients with PGLs of the organ of Zuckerkandl and investigate the prevalence of SDHx mutations and other genetic defects among them. The clinical characteristics of 14 patients with PGL of the organ of Zuckerkandl were analyzed retrospectively; their DNA was tested for SDHx mutations and deletions. Eleven out of 14 (79%) patients with PGLs of the organ of Zuckerkandl were found to have mutations in the SDHB (9) or SDHD (2) genes; one patient was found to have the Carney-Stratakis syndrome (CSS), and his PGL was discovered during surgery for gastrointestinal stromal tumor. Our results show that SDHx mutations are prevalent in pediatric and adult PGLs of the organ of Zuckerkandl. Patients with PGLs of the organ of Zuckerkandl should be screened for SDHx mutations and the CSS; in addition, asymptomatic carriers of an SDHx mutation among the relatives of affected patients may benefit from tumor screening for early PGL detection.

Development and validation of a comprehensive mutation and deletion detection assay for SDHB, SDHC, and SDHD.

BACKGROUND: Lack of sequencing validation and complexity of deletion testing hinder genetic diagnosis of SDH-associated paraganglioma/pheochromocytoma. METHODS: We developed sequencing assays and multiplex ligation-dependent probe amplification (MLPA) deletion detection for SDHB, SDHC and SDHD. Clinical performance was validated on 141 blinded samples, previously tested at NIH. RESULTS: Sequencing and deletion detection were highly reproducible and agreed with previous NIH results in 99.3% and 100%, respectively. CONCLUSIONS: DNA sequencing combined with MLPA allows reliable and simplified genotyping of SDHB, SDHC and SDHD.

Quantification of serum 1-84 parathyroid hormone in patients with hyperparathyroidism by immunocapture in situ digestion liquid chromatography-tandem mass spectrometry.

BACKGROUND: Immunoassays specific for 1-84 parathyroid hormone (PTH) reportedly reflect the bioactivity of PTH; however, PTH immunoassays can be susceptible to interference by cross-reacting PTH fragments. In addition, these assays currently lack standardization. A methodology using immunocapture purification with liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection, along with a stable isotope-labeled internal standard, may help address these issues. METHODS: We isolated 1-84 PTH from 1 mL serum by immunocapture on a 6.5-mm polystyrene bead. The immobilized PTH was digested in situ and analyzed by LC-MS/MS. For quantification, we used the selected reaction monitoring response from the N-terminal tryptic peptide 1-13 PTH ((1)SVSEIQLMHNLGK(13)). RESULTS: The linear range of the assay was 39.1-4560 ng/L, and the limit of detection and limit of quantification were 14.5 ng/L and 39.1 ng/L, respectively. The intraassay CVs ranged from 6% to 11%, and the interassay CVs ranged from 7% to 17%. Interference by PTH fragments 1-44 PTH, 7-84 PTH, 43-68 PTH, 52-84 PTH, 64-84 PTH, and PTH-related protein (PTHrP) was

Mutant BRAF(T1799A) can be detected in the blood of papillary thyroid carcinoma patients and correlates with disease status.

CONTEXT: The BRAF(T1799A) transversion is the most frequent morphotype-specific somatic mutation in papillary thyroid carcinoma (PTC). The ability to detect this mutation in the circulation could aid in diagnosis and follow-up of PTC patients. OBJECTIVE: Our objective was to develop and clinically validate a sensitive and specific assay for the detection of BRAF(T1799A) in blood samples from PTC patients. DESIGN: We developed an allele-specific real-time PCR method for the detection of BRAF(T1799A) in blood samples and studied prospectively blood samples from 193 patients with thyroid cancer (173 PTC, 20 non-PTC) attending for routine follow-up. The results of molecular testing were correlated with disease status and thyroglobulin measurements. BRAF(T1799A) status of the original tumor samples was also confirmed, where available. RESULTS: The assay had a detection sensitivity of fewer than one heterozygote BRAF(T1799A)-carrying cell per 100,000 diploid cells, without detectable cross-reactivity between wild-type BRAF and BRAF(T1799A). Circulating BRAF(T1799A) was detected in 20 of 173 PTC patients and in none of the 20 non-PTC patients. BRAF(T1799A)-positive samples contained between one in 326 and fewer than one in 100,000 copies of BRAF(T1799A). Tissue BRAF status correlated with blood BRAF status, whereas BRAF(T1799A) positivity in blood correlated with the presence of active disease at the time of the blood draw, with eight of the 38 PTC patients with persistent/recurrent disease being positive for circulating BRAF(T1799A) (relative risk vs. circulating BRAF(T1799A)-negative, 2.55; P < 0.04). CONCLUSIONS: BRAF(T1799A) can be detected in the blood of PTC patients with residual or metastatic disease and may provide diagnostic information.

Determinants of intra versus intermolecular self-association within the regulatory domains of Rlk and Itk.

A protein fragment from the Tec family member Rlk (also known as Txk) containing a single proline-rich ligand adjacent to a Src homology 3 (SH3) domain has been investigated by nuclear magnetic resonance (NMR) spectroscopy. Analysis of the concentration dependence of the chemical shifts, NMR linewidths and self-diffusion coefficients reveal that the Rlk fragment dimerizes in solution. Mutation of two critical prolines in the proline-rich ligand abolishes dimerization. Furthermore, analysis of the extrapolated chemical shifts at infinite dilution reveal that intramolecular binding of the proline-rich ligand to the SH3 domain is disfavored. This is in contrast to the corresponding fragment of Itk, for which the proline-rich ligand/SH3 interaction occurs exclusively in an intramolecular fashion and no intermolecular binding is observed. Comparison of the Itk and Rlk sequences reveals that Rlk contains five fewer residues than Itk in the linker region between the proline-rich ligand and the SH3 domain. To assess whether linker length is a molecular determinant of intra- versus intermolecular self-association, we varied the length of the linker in both Rlk and Itk and analyzed the resulting variants by NMR. Intramolecular binding in Itk is reduced by shortening the linker and conversely a longer linker between the proline-rich ligand and the SH3 domain in Rlk enhances intramolecular self-association. Association constants for the binding of peptides corresponding to the proline-rich ligand with their respective SH3 domains were also measured by NMR. The protein/peptide data combined with the association constants for binding of each proline-rich peptide to the corresponding SH3 domain provide an explanation for the opposing modes of self-association within the otherwise closely related Rlk and Itk proteins.