Establishment of a Biorepository for Down Syndrome: Experience of the Inter-Institutional Multidisciplinary BioBank - BioBIM.

BACKGROUND: Down syndrome, or Trisomy 21, is the leading genetic cause of cognitive disability in children and is associated with a high risk of several comorbidities, particularly congenital heart defects, early onset Alzheimer's disease, leukaemia, and autoimmune disorders. OBJECTIVE: This study describes the design, methods, and operational procedures employed to establish a biobank dedicated to Down syndrome that can support research projects investigating the effects of various genetic and environmental factors on this complex disease. METHODS: Blood was collected from all recruited subjects, processed, aliquoted and immediately frozen at -80 °C in the Interinstitutional Multidisciplinary BioBank (BioBIM) facilities. A small aliquot of the sample was used to perform blood tests for which analysis would not be feasible at a later date, such as blood cell counts. Each biological sample was coded, assigned a Standard PREanalytical Code, and registered in the oloBIOBANK software connected to a medical card containing all the donor's anamnestic data. All samples were stored under continuous real-time temperature recording using a freezer connected to a T-GUARD alarm system. In addition, a radiofrequency identification tracking system strictly monitored each cryopreservation operation performed throughout the sample lifecycle. RESULTS: Biological samples were collected from 454 individuals with Down syndrome from 2007 to 2023. A total of 2233 biological samples were available for research purposes, including whole blood in different anticoagulants, serum, plasma, and frozen peripheral blood mononuclear cells. The quality of the nucleic acids obtained through specific standard operating procedures demonstrated that these samples were appropriate for clinical and basic research. CONCLUSION: By establishing this biobank, we have gathered a significant number of biological samples and clinical data from individuals with Down syndrome, thereby fostering collaboration between different research groups in an open and transparent manner. Sharing expertise and resources among scientists will ultimately facilitate the transfer of knowledge to clinical practice, leading to the development of more effective therapeutic treatments to improve the outcomes and quality of life of patients with Down syndrome.

Biospecimen Digital Twins: Moving from a "High Quality" to a "Fit-for-Purpose" Concept in the Era of Omics Sciences.

The growing demand for personalized medicine we are currently witnessing has given rise to more in-depth research in the field of biomarker discovery and, thus, in biological banks that hold the ability to process, collect, store, and distribute "high-quality" biological specimens. However, the notion of "specimen quality" is subject to change with technological advancements. In this perspective, we propose that the notion of sample quality should shift from a broad definition of "high-quality" to a "fit-for-purpose" concept more suitable for precision medicine studies. Digital twins are a digital replica of real entities. These are largely adopted in any digitalized domain and are currently finding applications in biomedicine. The adoption of digital twins for biosamples, proposed in this paper, can provide prompt information about the whole lifecycle of the physical twin (i.e., the biosample) and substantially extend the possible matching criteria between the available samples and the researchers' and physicians' requests. This fine-tuning matching could greatly contribute to improving the "fit-for-purpose" quality, not only for studies based on current needs, but also to improve the identification of the best available samples in future situations, determined by the evolution of technologies and biosciences. Assuming and exploiting a data-science view in our biobank perspective, the more (accurate) data there are available, the more information can be extrapolated from them, the more opportunities there are for matching future, currently unknown, needs. This should be a mandatory principle that the 'time machines' called biobanks should follow.

Association of LTA and SOD Gene Polymorphisms with Cerebral White Matter Hyperintensities in Migraine Patients.

White matter hyperintensities (WMHs) in migraine could be related to inflammatory and antioxidant events. The aim of this study is to verify whether migraine patients with WMHs carry a genetic pro-inflammatory/pro-oxidative status. To test this hypothesis, we analyzed lymphotoxin alpha (LTA; rs2071590T and rs2844482G) and superoxide dismutase 1 (SOD1; rs2234694C) and 2 (SOD2; rs4880T) gene polymorphisms (SNPs) in 370 consecutive patients affected by episodic (EM; n = 251) and chronic (CM; n = 119) migraine and in unrelated healthy controls (n = 100). Brain magnetic resonance was available in 183/370 patients. The results obtained show that genotypes and allele frequencies for all tested SNPs did not differ between patients and controls. No association was found between single SNPs or haplotypes and sex, migraine type, cardiovascular risk factors or disorders. Conversely, the LTA rs2071590T (OR = 2.2) and the SOD1 rs2234694C (OR = 4.9) alleles were both associated with WMHs. A four-loci haplotype (TGCT haplotype: rs2071590T/rs2844482G/rs2234694C/rs4880T) was significantly more frequent in migraineurs with WMHs (7 of 38) compared to those without WMHs (4 of 134; OR = 8.7). We may, therefore, conclude by suggesting that that an imbalance between pro-inflammatory/pro-oxidative and antioxidant events in genetically predisposed individuals may influence the development of WMHs.

Protective Effect of Antioxidants in Nitric Oxide/COX-2 Interaction during Inflammatory Pain: The Role of Nitration.

In clinical practice, inflammatory pain is an important, unresolved health problem, despite the utilization of non-steroidal anti-inflammatory drugs (NSAIDs). In the last decade, different studies have proven that reactive oxygen species (ROS) and reactive nitrogen species (RNS) are involved in the development and maintenance of inflammatory pain and hyperalgesia via the post-translation modification of key proteins, such as manganese superoxide dismutase (MnSOD). It is well-known that inducible cyclooxygenase 2 (COX-2) plays a crucial role at the beginning of the inflammatory response by converting arachidonic acid into proinflammatory prostaglandin PGE2 and then producing other proinflammatory chemokines and cytokines. Here, we investigated the impact of oxidative stress on COX-2 and prostaglandin (PG) pathways in paw exudates, and we studied how this mechanism can be reversed by using antioxidants during hyperalgesia in a well-characterized model of inflammatory pain in rats. Our results reveal that during the inflammatory state, induced by intraplantar administration of carrageenan, the increase of PGE2 levels released in the paw exudates were associated with COX-2 nitration. Moreover, we showed that the inhibition of ROS with Mn (III) tetrakis (4-benzoic acid) porphyrin(MnTBAP) antioxidant prevented COX-2 nitration, restored the PGE2 levels, and blocked the development of thermal hyperalgesia.

Machine learning approach to predict medication overuse in migraine patients.

Machine learning (ML) is largely used to develop automatic predictors in migraine classification but automatic predictors for medication overuse (MO) in migraine are still in their infancy. Thus, to understand the benefits of ML in MO prediction, we explored an automated predictor to estimate MO risk in migraine. To achieve this objective, a study was designed to analyze the performance of a customized ML-based decision support system that combines support vector machines and Random Optimization (RO-MO). We used RO-MO to extract prognostic information from demographic, clinical and biochemical data. Using a dataset of 777 consecutive migraine patients we derived a set of predictors with discriminatory power for MO higher than that observed for baseline SVM. The best four were incorporated into the final RO-MO decision support system and risk evaluation on a five-level stratification was performed. ROC analysis resulted in a c-statistic of 0.83 with a sensitivity and specificity of 0.69 and 0.87, respectively, and an accuracy of 0.87 when MO was predicted by at least three RO-MO models. Logistic regression analysis confirmed that the derived RO-MO system could effectively predict MO with ORs of 5.7 and 21.0 for patients classified as probably (3 predictors positive), or definitely at risk of MO (4 predictors positive), respectively. In conclusion, a combination of ML and RO - taking into consideration clinical/biochemical features, drug exposure and lifestyle - might represent a valuable approach to MO prediction in migraine and holds the potential for improving model precision through weighting the relative importance of attributes.

Circulating Biomarkers in Migraine: New Opportunities for Precision Medicine.

BACKGROUND: Migraine is the most common neurological disorder and the second most disabling human condition, whose pathogenesis is favored by a combination of genetic, epigenetic, and environmental factors. In recent years, several efforts have been made to identify reliable biomarker(s) useful to monitor disease activity and/or ascertain the response to a specific treatment. OBJECTIVE: To review the current evidence on the potential biological markers associated with migraine. METHODS: A structured search of peer-reviewed research literature was performed by searching major publications databases up to December 2017. RESULTS: Several circulating biomarkers have been proposed as diagnostic or therapeutic tools in migraine, mostly related to migraine's inflammatory pathophysiological aspects. Nonetheless, their detection is still a challenge for the scientific community, reflecting, at least in part, disease complexity and clinical diagnostic limitations. At the present time, calcitonin generelated peptide (CGRP) represents probably the most promising candidate as a diagnostic and/or therapeutic biomarker, as its plasma levels are elevated during migraine attack and decrease during successful treatment. Other molecules (including some neuropeptides, cytokines, adipokines, or vascular activation markers) despite promising, do not possess the sufficient prerequisites to be considered as migraine biomarkers. CONCLUSION: The characterization of migraine-specific biomarkers would be fundamental in a perspective of precision medicine, enabling risk assessment and tailored treatments. However, speculating on the clinical validity of migraine biomarkers may be premature and controlled clinical trials are presently needed to investigate both the diagnostic and therapeutic value of these biomarkers in migraine.

Prognostic Significance of Neutrophil-to-lymphocyte Ratio in the Framework of the 8th TNM Edition for Breast Cancer.

BACKGROUND/AIM: To investigate whether neutrophil-to-lymphocyte ratio (NLR) might represent an additional biological criterion able to identify patients with worse prognosis within the 8th edition TNM prognostic staging system for breast cancer (BC). PATIENTS AND METHODS: Pre-treatment NLR was retrospectively analyzed in 475 BC women prospectively followed for a mean time of 3.8 years. The optimal NLR cutoff, identified by ROC analysis, was set at 2. RESULTS: Elevated pre-treatment NLR was associated with worse disease-free survival (DFS) (HR=2.28) and overall survival (OS) (HR=3.39). The prognostic value of NLR was mostly evident in stage I BC (HR for DFS=2.89; HR for OS=1.30), in whom NLR significantly stratified patients who developed distant metastasis (HR= 4.62), but not local recurrence. CONCLUSION: NLR might provide important information in risk stratification, especially in stage I BC patients in whom the presence of a high NLR might raise the question as to whether they should be more aggressively managed.

Clinical significance of glycemic parameters on venous thromboembolism risk prediction in gastrointestinal cancer.

AIM: To investigate the possible predictive role of routinely used glycemic parameters for a first venous thromboembolism (VTE) episode in gastrointestinal (GI) cancer ambulatory patients - with or without clinically diagnosed type 2 diabetes (T2D) or obesity - treated with chemotherapy. METHODS: Pre-treatment fasting blood glucose, insulin, glycated hemoglobin (HbA1c) and homeostasis model of risk assessment (HOMA) were retrospectively evaluated in a cohort study of 342 GI cancer patients. Surgery was performed in 142 (42%) patients with primary cancer, 30 (21%) and 112 (79%) of whom received neoadjuvant and adjuvant therapies, respectively. First-line chemotherapy was administered in 200 (58%) patients with metastatic disease. The study outcome was defined as the occurrence of a first symptomatic or asymptomatic VTE episode during active treatment. RESULTS: Impaired glucose tolerance (IGT) or T2D were diagnosed in 30% of GI cancer patients, while overweight/obesity had an incidence of 41%. VTE occurred in 9.4% of patients (7% of non-diabetic non-obese), especially in those with a high ECOG score (P = 0.025). No significant association was found between VTE incidence and T2D, obesity, different tumor types, metastatic disease, Khorana class of risk, or different anti-cancer drugs, although VTE rates were substantially higher in patients receiving bevacizumab (17% vs 8%, P = 0.044). Conversely, all glucose metabolic indexes were associated with increased VTE risk at ROC analysis. Multivariate Cox proportional analyses confirmed that HOMA index (HR = 4.13, 95%CI: 1.63-10.5) or fasting blood glucose (HR = 3.56, 95%CI: 1.51-8.39) were independent predictors of VTE occurrence during chemotherapy. CONCLUSION: The results here reported demonstrate that evaluating glucose metabolic asset may allow for VTE risk stratification in GI cancer, helping to identify chemotherapy-treated patients who might benefit from thromboprophylaxis. Further multicenter prospective studies involving a larger number of patients are presently needed.

Prognostic value of glycated hemoglobin in colorectal cancer.

AIM: To investigate the clinical significance of routinely used glycemic parameters in a cohort of colorectal cancer (CRC) patients. METHODS: Pre-treatment fasting blood glucose, insulin, HbA1c and homeostasis model of risk assessment (HOMA-IR) were retrospectively evaluated in a case-control study of 224 CRC and 112 control subjects matched for sex, obesity and diabetes frequency and blood lipid profile. Furthermore, the prognostic value of routinely used glycemic parameters towards progression-free (PFS) and overall survival (OS) was prospectively evaluated. RESULTS: Fasting blood glucose, insulin, HOMA-IR and HbA1c (all P < 0.0001) levels were higher in non-diabetic CRC patients compared with obesity-matched controls. All parameters were associated with increased CRC risk at ROC analysis, but no relationship with clinical-pathological variables or survival outcomes was observed for glycemia, insulinemia or HOMA-IR. Conversely, advanced CRC stage (P = 0.018) was an independent predictor of increased HbA1c levels, which were also higher in patients who had disease progression compared with those who did not (P = 0.05). Elevated HbA1c levels showed a negative prognostic value both in terms of PFS (HR = 1.24) and OS (HR = 1.36) after adjustment for major confounders, which was further confirmed in a subgroup analysis performed after exclusion of diabetic patients. CONCLUSION: HbA1c might have a negative prognostic value in CRC, thus suggesting that glycemic metabolic markers should be carefully monitored in these patients, independently of overt diabetes.

Ensuring Sample Quality for Biomarker Discovery Studies - Use of ICT Tools to Trace Biosample Life-cycle.

The growing demand of personalized medicine marked the transition from an empirical medicine to a molecular one, aimed at predicting safer and more effective medical treatment for every patient, while minimizing adverse effects. This passage has emphasized the importance of biomarker discovery studies, and has led sample availability to assume a crucial role in biomedical research. Accordingly, a great interest in Biological Bank science has grown concomitantly. In biobanks, biological material and its accompanying data are collected, handled and stored in accordance with standard operating procedures (SOPs) and existing legislation. Sample quality is ensured by adherence to SOPs and sample whole life-cycle can be recorded by innovative tracking systems employing information technology (IT) tools for monitoring storage conditions and characterization of vast amount of data. All the above will ensure proper sample exchangeability among research facilities and will represent the starting point of all future personalized medicine-based clinical trials.

Overexpression of the EMT driver brachyury in breast carcinomas: association with poor prognosis.

BACKGROUND: The epithelial-mesenchymal transition (EMT) has been implicated as an important process in tumor cell invasion, metastasis, and drug resistance. The transcription factor brachyury has recently been described as a driver of EMT of human carcinoma cells. METHODS: Brachyury mRNA and protein expression was analyzed in human breast carcinomas and benign tissues. The role of brachyury in breast tumor prognosis and drug resistance and the ability of brachyury-specific T cells to lyse human breast carcinoma cells were also evaluated. Kaplan-Meier analyses were used to evaluate the association between brachyury expression and survival. All statistical tests were two-sided. RESULTS: The level of brachyury expression in breast cancer cells was positively associated with their ability to invade the extracellular matrix, efficiently form mammospheres in vitro, and resist the cytotoxic effect of docetaxel. A comparison of survival among breast cancer patients treated with tamoxifen in the adjuvant setting who had tumors with high vs low brachyury mRNA expression demonstrated that high expression of brachyury is associated as an independent variable with higher risk of recurrence (hazard ratio [HR] = 7.5; 95% confidence interval [CI] = 2.4 to 23.5; P = 5.14×10(-4)) and distant metastasis (HR = 15.2; 95% CI = 3.5 to 66.3; P = 3.01×10(-4)). We also demonstrated that brachyury-specific T cells can lyse human breast carcinoma cells. CONCLUSIONS: The studies reported here provide the rationale for the use of a vaccine targeting brachyury for the therapy of human breast cancer, either as a monotherapy or in combination therapies.

Association between migraine and ACE gene (insertion/deletion) polymorphism: the BioBIM study.

AIM: In the present case-control study, we investigated the correlation between the common ACE insertion/deletion (I/D) polymorphism and migraine. MATERIALS & METHODS: Genotyping of the ACE I/D variant was performed in 502 Caucasian patients with migraine and 323 age-, sex- and race/ethnicity-matched healthy controls. We investigated associations between ACE genetic variants and sociodemographic and/or clinical features of migraineurs. RESULTS: We found a significant association between ACE insertion/insertion (I/I) polymorphism and lower use of pharmacological prophylaxis in migraine patients with aura and in those with chronic migraine. Moreover, ACE I/I polymorphism was significantly more common in migraine patients with aura who had a negative family history of migraine. CONCLUSION: Our data suggest that although the ACE I/D polymorphism is not a direct risk factor for migraine, the ACE I/I genotype may influence the clinical feature of this disease being associated with reduced use of prophylactic agents in patients with migraine with aura and in those with chronic migraine.

Effects of conventional therapeutic interventions on the number and function of regulatory T cells.

Several lines of investigation have revealed the apparent interplay between the immune system of the host and many conventional, "standard-of-care" anticancer therapies, including chemotherapy and small molecule targeted therapeutics. In particular, preclinical and clinical studies have demonstrated the important role of regulatory T cells (Tregs) in inhibiting immune responses elicited by immunotherapeutic regimens such as those based on anticancer vaccines or checkpoint inhibitors. However, how the number and immunosuppressive function of Tregs change in cancer patients undergoing treatment with non-immune anticancer therapies remains to be precisely elucidated. To determine whether immunostimulatory therapies can be employed successfully in combination with conventional anticancer regimens, we have investigated both the number and function of Tregs obtained from the peripheral blood of carcinoma patients before the initiation and during the course of chemotherapeutic and targeted agent regimens. Our studies show that the treatment of breast cancer patients with tamoxifen plus leuprolide, a gonadotropin releasing hormone agonist, has minimal effects on Tregs, while sunitinib appears to exert differential effects on Tregs among patients with metastatic renal carcinoma. However, the administration of docetaxel to patients with metastatic prostate or breast cancer, as well as that of cisplatin plus vinorelbine to non-small cell lung cancer patients, appears to significantly increase the ratio between effector T cells and Tregs and to reduce the immunosuppressive activity of the latter in the majority of patients. These studies provide the rationale for the selective use of active immunotherapy regimens in combination with specific standard-of-care therapies to achieve the most beneficial clinical outcome among carcinoma patients.

A reliable and reproducible technique for DNA fingerprinting in biorepositories: a pilot study from BioBIM.

Standard operating procedures (SOPs) optimization for nucleic acid extraction from stored samples is of crucial importance in a biological repository, considering the large number of collected samples and their future downstream molecular and biological applications. However, the validity of molecular studies using stored specimens depends not only on the integrity of the biological samples, but also on the procedures that ensure the traceability of the same sample, certifying its uniqueness, and ensuring the identification of potential sample contaminations. With this aim, we have developed a rapid, reliable, low-cost, and simple DNA fingerprinting tool for a routine use in quality control of biorepositories samples. The method consists of a double ALU insertion/deletion genotyping panel suitable for uniqueness, identification of sample contaminations, and gender validation. Preliminary data suggest that this easy-to-use DNA fingerprinting protocol could routinely provide assurances of DNA identity and quality in a biorepository setting.

Establishment of a biorepository for migraine research: the experience of Interinstitutional Multidisciplinary BioBank (BioBIM).

The development of Biobanks and recent advances in molecular biology have enhanced the possibility to accelerate translational research studies. The Interinstitutional Multidisciplinary BioBank (BioBIM) is organized in a large healthy donors collection and pathology-based biobanks with the aim to provide a service for development of interdisciplinary studies. A new pathology-based biobank has been organized to specifically collect biospecimen from patients affected by migraine, with the final goal to centralize data, collect blood, plasma, serum, DNA and RNA of patients with this disease. The BioBIM is fully equipped for the automation of sampling/processing, storage and tracking of biospecimens. Standard Operating Procedures have been developed for processing and storage phases as well as archive of clinical data. The availability of biospecimens and clinical data will constitute a resource for various research projects.

SPRECware: software tools for Standard PREanalytical Code (SPREC) labeling - effective exchange and search of stored biospecimens.

Biobanks provide stored material to basic, translational, and epidemiological research and this material should be transferred without institute-dependent intrinsic bias. The ISBER Biospecimen Science Working Group has released a "Standard PREanalytical Code" (SPREC), which is a proposal for a standard coding of the preanalytical options that have been adopted in order to track and make explicit the preanalytical variations in the collection, preparation, and storage of specimens. In this paper we address 2 issues arising in any biobank or biolaboratory aiming at adopting SPREC: (i) reducing the burden required to adopt this standard coding, and (ii) maximize the immediate benefits of this adoption by providing a free, dedicated software tool. We propose SPRECware, a vision encompassing tools and solutions for the best exploitation of SPREC based on information technology (www.sprecware.org). As a first step, we make available SPRECbase, a software tool useful for generating, storing, managing, and exchanging SPREC-related information associated to specimens. Adopting SPREC is useful both for internal purposes (such as finding the samples having some given preanalytical features), and for exchanging the preanalytical information associated to biological samples between Laboratory Information Systems. In case of a common adoption of this coding, it would be easy to find out whether and where, among the participating Biological Resource Centers, the specimens for a given study are available in order to carry out a planned experiment.

Brachyury, a driver of the epithelial-mesenchymal transition, is overexpressed in human lung tumors: an opportunity for novel interventions against lung cancer.

PURPOSE: The epithelial-mesenchymal transition (EMT) is emerging as a critical factor for the progression and metastasis of carcinomas, as well as drug resistance. The T-box transcription factor Brachyury has been recently characterized as a driver of EMT in human carcinoma cells. The purpose of this study was to characterize Brachyury as a potential target for lung cancer therapy. EXPERIMENTAL DESIGN: The expression of Brachyury was evaluated by PCR and by immunohistochemistry in human lung tumors and adult normal tissues. Brachyury gene copy number and promoter methylation status were analyzed in tumor tissues with various levels of Brachyury expression. Lung carcinoma cells' susceptibility to T-cell lysis and EGF receptor (EGFR) kinase inhibition were also evaluated relative to the levels of Brachyury. RESULTS: Our results showed Brachyury protein expression in 41% of primary lung carcinomas, including 48% of adenocarcinomas and 25% of squamous cell carcinomas. With the exception of normal testis and some thyroid tissues, the majority of normal tissues evaluated in this study were negative for the expression of Brachyury protein. Brachyury-specific T cells could lyse Brachyury-positive tumors and the level of Brachyury corresponded to resistance of tumor cells to EGFR kinase inhibition. CONCLUSION: We hypothesize that the elimination of Brachyury-positive tumor cells may be able to prevent and/or diminish tumor dissemination and the establishment of metastases. The ability of Brachyury-specific T-cell lines to lyse Brachyury-positive tumor cells, in vitro, supports the development of Brachyury-based immunotherapeutic approaches for the treatment of lung cancer.

Impact of preanalytical handling and timing for peripheral blood mononuclear cells isolation and RNA studies: the experience of the Interinstitutional Multidisciplinary BioBank (BioBIM).

Multicenter studies and biobanking projects require blood transportation from the participating center to a central collection or diagnostic laboratory. The impact of time delays between venous blood collection and peripheral blood mononuclear cells (PBMC) isolation prior to RNA extraction may affect the quality and quantity of isolated nucleic acids for genomic applications. Thus, standard operating procedure (SOP) optimization for the treatment of biological samples before RNA extraction is crucial in a biological repository. In order to define SOPs for whole blood preservation prior to RNA extraction, we sought to determine whether different blood storage times (0, 3, 6, 10, 24, and 30 hours) prior to PBMCs isolation and storage at -80°C, could affect the quality and quantity of extracted RNA. After spectrophotometric quantification, the quality and integrity of RNA were assessed by agarose gel electrophoresis, RNA integrity number and real time-PCR (RT-PCR).
Across the different time points we did not observe significant differences within the first 24 hours of blood storage at room temperature, while a significant loss in RNA yield and integrity was detected between 24 and 30 hours. We conclude that time delays before PBMCs isolation prior to RNA extraction may have a significant impact on downstream molecular biological applications.

An AT-rich region in the APC gene may cause misinterpretation of familial adenomatous polyposis molecular screening.

Familial adenomatous polyposis (FAP) is an autosomal-dominant condition mainly due to a mutation of the adenomatous polyposis coli (APC) gene. The present study reports evidence of a technical issue occurring during the mutational analysis of APC exon 4. Genetic conventional direct sequence analysis of a repetitive AT-rich region in the splice acceptor site of APC intron 3 could be misinterpreted as a pathogenetic frameshift result. However, this potential bias may be bypassed adopting a method for random mutagenesis of DNA based on the use of a triphosphate nucleoside analogues mixture. Using this method as a second-level analysis, we also demonstrated the nonpathogenic nature of the variant in the poly A trait in APC exon 4 region (c.423-4delA) that do not result in aberrant splicing of APC exons 3-4; conversely, we did not find a previously reported T deletion/insertion polymorphism.