Personalized therapy for breast cancer.

Breast cancer is a complex disease characterized by many morphological, clinical and molecular features. For many years, this disease has been classified according to histopathologic criteria, known as the tumor, node and metastasis (TNM) staging system. Clinical criteria that include immunohistochemical markers, such as the estrogen receptor (ER), the progesterone receptor (PR), and the human epidermal growth factor receptor 2 (HER2), provide a classification of breast cancer and dictates the optimal therapeutic approach for treatment. With genomic techniques, such as real-time reverse transcriptase PCR (RT-PCR), microarrays, next-generation sequencing, and whole-exome sequencing, breast cancer diagnostics is going through a significant evolution. Genomic and transcriptomic technologies make the analysis of gene expression signatures and mutation status possible so that tumors may now be classified more accurately with respect to diagnosis and prognosis. The -omic era has also made the possible identification of new biomarkers involved in breast cancer development, survival and invasion that can be gradually incorporated either into clinical testing or clinical trials. Together, clinical and molecular criteria can contribute to a more personalized management of the breast cancer patient. This article will present the progress made in the diagnosis and management of breast cancer using molecular information provided by genomic and transcriptomic technologies.

Neoadjuvant cetuximab, twice-weekly gemcitabine, and intensity-modulated radiotherapy (IMRT) in patients with pancreatic adenocarcinoma.

BACKGROUND: Neoadjuvant therapy has been investigated for localized and locally advanced pancreatic ductal adenocarcinoma (PDAC) but no standard of care exists. Combination cetuximab/gemcitabine/radiotherapy demonstrates encouraging preclinical activity in PDAC. We investigated cetuximab with twice-weekly gemcitabine and intensity-modulated radiotherapy (IMRT) as neoadjuvant therapy in patients with localized or locally advanced PDAC. EXPERIMENTAL DESIGN: Treatment consisted of cetuximab load at 400 mg/m(2) followed by cetuximab 250 mg/m(2) weekly and gemcitabine 50 mg/m(2) twice-weekly given concurrently with IMRT to 54 Gy. Following therapy, patients were considered for resection. RESULTS: Thirty-seven patients were enrolled with 33 assessable for response. Ten patients (30%) manifested partial response and 20 (61%) manifested stable disease by RECIST. Twenty-five patients (76%) underwent resection, including 18/23 previously borderline and 3/6 previously unresectable tumors. Twenty-three (92%) of these had negative surgical margins. Pathology revealed that 24% of resected tumors had grade III/IV tumor kill, including two pathological complete responses (8%). Median survival was 24.3 months in resected patients. Outcome did not vary by epidermal growth factor receptor status. CONCLUSIONS: Neoadjuvant therapy with cetuximab/gemcitabine/IMRT is tolerable and active in PDAC. Margin-negative resection rates are high and some locally advanced tumors can be downstaged to allow for complete resection with encouraging survival. Pathological complete responses can occur. This combination warrants further investigation.

MicroRNAs in graft-versus-host disease: a review of the latest data.

Detectable in biopsies and body fluids, the measurement of a single or panels of microRNAs have been reported to be quite sensitive and specific for the prediction, diagnosis, and prognosis of many diseases. Interest in the use of microRNAs as biomarkers and eventual therapeutic targets has increased exponentially in the last decade. However, in the field of graft-versus-host disease (GVHD), the discovery of their involvement in biological processes and their predictive value is only emerging. With 30-75% of patients developing GVHD following allogeneic hematopoietic cell transplant and the absence of routinely used predictive biomarkers, microRNAs are promising for early detection and follow-up of this condition. We aim to summarize the current knowledge on the involvement of these small RNAs in the pathophysiology of this disease. We also review studies investigating the potential of miRNAs as biomarkers for early detection, follow-up, and prognosis of GVHD.

How close are miRNAs from clinical practice? A perspective on the diagnostic and therapeutic market.

The discovery of miRNAs in the mid-90s has changed the dogma of gene expression regulation. Currently, miRNAs are the main theme of thousands of publications each year and their involvement in human diseases is everyday more deeply understood. With that being known, what are the actual clinical applications of miRNAs and how far are they truly from the patients? To address this question, we reviewed the miRNA diagnostic and therapeutic market. With many companies developing miRNA panels, the activity is high in the diagnostic area. Some products, notably for thyroid cancer (Interpace Diagnostic), are already available to clinician and covered by major insurance companies. In comparison, the therapeutic market, mainly driven by miRNA mimics and antagomiR products, is less advanced. Miravirsen (produced by Roche/Santaris) and RG-101 (produced by Regulus Therapeutics), designed to treat hepatitis C, are considered the flagship products of this class of future drugs. All of the miRNA-based drugs are currently in clinical trials and none have yet reached the pharmaceutical breakthrough. However, acquisition of miRNA-based companies by major pharmas is sending a positive feedback on their potentials. With multiple initiatives on their way, the next years will definitely be determinant for the miRNA market that is still in his infancy.

Partial characterization of nuclear matrix attachment regions from human fibroblast DNA using Alu-polymerase chain reaction.

The proteinaceous nuclear matrix of mammalian cell nuclei has been suggested to be involved in the regulation of chromatin structure, DNA replication, and gene expression. Interaction between cellular DNA and the nuclear matrix is mediated by putative DNA binding sequences, matrix attachment regions (MARs), which may become altered during early events in cellular transformation. Among the cellular changes occurring during the development of neoplasia, all of which may potentially involve the nuclear matrix, are alterations in nuclear structure, loss of control of DNA replication, and significant modifications of cellular gene expression. Therefore, a better understanding of the interaction between DNA and the nuclear matrix is needed. Isolated matrix associated DNA from pulse labeled SV40 transformed human fibroblasts was shown to be enriched in newly replicated DNA, confirming the association of DNA replication with the nuclear matrix as observed by others. Subgenomic fractions of matrix associated DNA enriched in putative MARs sites were prepared from quiescent and logarithmically growing normal human fibroblasts and SV40 transformed human fibroblasts. These fractions of DNA were analyzed by Alu-polymerase chain reaction and agarose gel electrophoresis, revealing complex and unique patterns of DNA products for each cell type investigated. A number of prominent DNA fragments with similar molecular size were found to be present in the amplified DNA products of each DNA source, suggesting that these DNA fragments may represent common DNA sequences which contain MARs sites or which are associated with MARs sites. The application of Alu-polymerase chain reaction to the molecular analysis of nuclear matrix associated DNA may facilitate the isolation and characterization of potentially new human MARs sequences.

Association of putative origins of replication with the nuclear matrix in normal human fibroblasts.

Several metabolic processes, such as DNA organization and replication, transcription, and RNA processing are closely associated with the nuclear matrix. Nuclear matrix attachment regions are nucleotide sequences holding DNA tightly complexed with the nuclear scaffold and are resistant to extractions with detergents and high salt solutions. The role of matrix attachment regions in DNA replication has not been completely clarified, but they have been identified in close association with origins of replication in mammalian cells. We isolated nuclear matrix-associated DNA from normal human fibroblasts synchronized to different phases of the cell cycle and cloned compatible fragments into pUC19. We tested the homology of a fraction of the available clones to DNA replicated at the beginning of the S phase in human fibroblasts. We confirmed that nuclear matrix-associated DNA isolated from cells in G0 and G1 phases of the cell cycle contains sequences that are among the earliest replicated regions in the human genome. This finding supports the hypothesis that matrix attachment regions in human DNA are located in close proximity to origins of DNA replication.

Somatic gene mutation analysis of triple negative breast cancers.

OBJECTIVES: The aims of this study were to analyze triple negative breast cancer (TNBC) using an expanded next generation sequencing (NGS) assay, assess the clinical relevance using a recently described database, and correlate tumor morphology with detected genetic alterations. METHODS: DNA was isolated from twenty primary TNBCs and genes of interest were enriched and sequenced with hybrid capture, followed by variant detection and functional and clinical annotation. The JAX-CTP™ assay detects actionable variants in the form of single nucleotide variations, small insertions and deletions (≤50 bp), and copy number variants in 358 genes in specimens containing a neoplastic cell content of ≥50%. The JAX-CKB is a comprehensive database that curates tumor phenotype, genetic variant and protein effect, therapeutic relevance, and available treatment options. RESULTS: 18/20 (90%) of TNBCs contained at least one somatic mutation detected by the JAX-CTP™. MYC amplification was the most common alteration, present in 75% of tumors. TP53, AURKA, and KDR mutations were each present in 30% (6/20) of cases. Related recruiting clinical trials, extracted from JAX-CKB, included 166 for breast cancer, of which 17 were specific to only the TNBC subtype. All 17 trials were testing at least one therapy that targets a mutation identified in this sample set. The majority (89%) of tumors with basal-like histologic features had MYC amplification. CONCLUSIONS: The expanded gene panel identified a variety of clinically actionable gene alterations in TNBCs. The identification of such variants increases the possibility for new therapeutic interventions and clinical trial eligibility for TNBC patients.

Myocardial cell death and apoptosis in hibernating myocardium.

OBJECTIVES: This study was designed to study apoptosis in hypoperfused hibernating myocardium subtending severe coronary stenosis. BACKGROUND: Apoptosis contributes to myocyte death in acute myocardial infarction. METHODS: A left anterior descending coronary artery stenosis was created in 13 pigs and maintained for 24 h (n = 4), 7 days (n = 5) and 4 weeks (n = 4) to reduce coronary blood flow by a mean of 34% with severe regional myocardial systolic dysfunction, as documented by echocardiography. Apoptosis was detected with an in situ end-labeling method and confirmed by "deoxyribonucleic acid laddering" on agarose-gel electrophoresis. The severity of apoptosis was expressed as the percentage of apoptotic myocyte nuclei and nonapoptotic myocardial nuclei. RESULTS: Myocardial blood flow of the anterior left ventricular wall was reduced from 1.00 +/- 0.18 to 0.66 +/- 0.21 ml/min per g (p < 0.01), with a severe reduction of anterior regional wall thickening from a mean (+/-SD) of 39 +/- 4% to 9 +/- 8% (p < 0.01). There was no myocardial infarction in five pigs and minimal patchy infarction of < or = 6% of the area at risk in eight pigs. Apoptotic myocytes were observed in the hibernating myocardial region in all pigs (4.8 +/- 2.3%). Myocyte apoptosis was patchy in distribution and was found predominantly in the subendocardial myocardium (9.8 +/- 4.6%) and rarely in the subepicardial myocardium (0.32 +/- 0.45%). Apoptosis was found not only around focal fibrosis areas, but also in areas without fibrosis or patchy infarction. Apoptosis was found not only in 24-h hypoperfused myocardium, but also in 4-week hypoperfused myocardium. The severity of myocyte apoptosis correlated significantly with regional coronary blood flow reduction (r = 0.75, p < 0.01). No apoptosis was found in the normal control region. CONCLUSIONS: This study demonstrates that there is ongoing myocyte death through myocyte apoptosis in hypoperfused hibernating myocardium.

Comparative performance evaluation of the HIV-1 LiPA protease and reverse transcriptase resistance assay on clinical isolates.

BACKGROUND: Assays that provide information regarding HIV-1 resistance to antiretroviral drugs are widely used to help manage antiretroviral treatment. The most commonly used HIV genotypic resistance assays are based on DNA sequencing (TRUGENE, ViroSeq, and home-brew) or reverse hybridization (LiPA). OBJECTIVES: This study compares the results from clinical specimens using two assay methods: the LiPA HIV-1 protease (PR) and reverse transcriptase (RT) resistance assay and DNA sequencing. STUDY DESIGN: Operators at each of three sites tested 10-20 randomly selected clinical specimens using LiPA (three strips total with probes for PR codons 30, 46, 48, 50, 54, 82, 84, and 90, and RT codons 41, 69, 70, 74, 75, 103, 106, 151, 181, 184, and 215) and DNA sequencing (TRUGENE) HIV-1 Genotyping Assay or home-brew methodology). Results from the two methods were categorized for each codon as follows: (i) concordant (LiPA and sequencing having the same result for wild-type (WT), mutant, and mixture); (ii) partially concordant (mixture by one method and not by the other); (iii) indeterminate (no result by LiPA); and (iv) discordant (LiPA and sequencing detecting different amino acids). RESULTS: A total of 50 clinical specimens were tested using the LiPA PR strip; 40 of these were also tested using the LiPA RT strip. For PR, 91.3% of the codon results were concordant, 3.0% were partially concordant, 4.5% were indeterminate by LiPA, and 1.3% were discordant. For RT, 88.0% of the codon results were concordant, 5.9% were partially concordant, 5.2% were indeterminate, and 0.9% were discordant. LiPA detected 3.0% (PR) and 6.4% (RT) WT/mutant mixtures, compared to 0.5% (PR) and 3.2% (RT) mixtures by sequencing. CONCLUSIONS: More WT/mutant mixtures were detected using LiPA, possibly indicating increased sensitivity. Relatively high concordance and low discordance rates were observed between LiPA and DNA sequencing. The indeterminate rate for LiPA was moderately high and may limit the clinical utility of this assay.

Adult-onset MLD: a gene mutation with isolated polyneuropathy.

A 22-year-old man presented with recurrent ulnar mononeuropathies and diffusely slow nerve conduction velocities. Arylsulfatase A (ASA) activity from leukocytes and fibroblasts was reduced, and urinary sulfatides were increased. Sural nerve biopsy revealed a reduction in myelinated fibers and Schwann cell inclusions. Results of studies of CNS integrity, including cranial MRI, evoked potentials, and neuropsychologic tests, were normal. Molecular genetic analyses revealed a novel homozygous missense mutation (Thr286Pro) in the ASA gene.

Correlation of polymorphisms to coagulation and biochemical risk factors for cardiovascular diseases.

Currently, the established risk factors for cardiovascular disease (CVD) are largely environmental in nature. Conflicting studies have suggested that mutations in specific coagulation genes may also provide a genetic basis for CVD risk. We reviewed clinical studies that examined the role of single nucleotide polymorphisms in coagulation and platelet factors, and a biochemical factor to determine if specific genotypes are correlated with patients with a history of arterial thrombotic diseases (acute coronary syndromes or stroke). A meta-analysis was performed on studies for factors II (G20210A variant), V Leiden (G1691A), VII (R353Q), glycoprotein (GP) IIIa receptor (PI(A1/A2)), and methylenetetrahydrofolate reductase (MTHFR, C677T). There was no correlation for factor II or factor V polymorphisms to coronary artery disease (CAD) in 5,607 and 5,431 patients studied, respectively. There was also no correlation for factor II variants and stroke in 3,451 patients studied. For factor V, statistical significance was achieved for the G1691A variant on 3,399 patients with stroke (odds ratio [OR] 1.43, 95% confidence intervals [CI] 1.03 to 1.97). The GP IIIa PI(A1/A2) genotype was associated with increased risk for CAD in 7,920 patients (OR 1.12, 95% CI 1.01 to 1.24), but not for 1,855 patients who had a stroke (OR 0.80, 95% CI 0.62 to 1.04). The combined RQ and RR genotypes of factor VII R353Q were correlated to a reduced risk for CVD in 2,574 patients (OR 0.78, 95% CI 0.65 to 0.93), whereas the QQ genotype had offered more protection (OR 0.53, 95% CI 0.27 to 1.03). The TT homozygous variant of MTHFR was associated with CAD risk in 5,644 patients studied (OR 1.30, 95% CI 1.11 to 1.52) but not for 3,075 patients with stroke. This study shows that for some genes, further studies are unnecessary, whereas for others, no more enrollments are needed. The impact of certain genotypes must be examined in relation to other established risk factors and potentially new therapeutic strategies.

The use of Alu-PCR to distinguish between typical pulmonary carcinoids versus classic midgut carcinoids.

Although histologically 'typical' pulmonary and 'classic' midgut carcinoids are similar, the small intestine tumors are more aggressive than their pulmonary counterpart. We believe that in contrast to pulmonary carcinoids arising from Kulchitsky cells, 'classical' midgut carcinoids develop from crypt stem cells that differentiate into endocrine ('classical') or exocrine-endocrine ('non-classical' adenocarcinoid) tumors. The different progenitor cells may determine different malignant potentials between these types of carcinoids. To identify genetic differences between 'classical' midgut and typical pulmonary carcinoids using an Alu-PCR genomic profiling method, we reviewed 54 cases of carcinoid tumors that were surgically removed at Hartford Hospital from 1996 through 2001. Histologically these cases were selected into three groups: i) foregut or pulmonary carcinoids, ii) 'classic' midgut carcinoids of small intestine and iii) multiple typical pulmonary carcinoids. Genomic-profiling of DNA from these cases was performed using an Alu-PCR method. Metastases were observed in 18/20 'classical' intestinal carcinoid tumors, 3/30 pulmonary carcinoids, and 0/4 multiple pulmonary carcinoids. These results confirm that pulmonary carcinoids behave in a more benign fashion than intestinal carcinoid tumors. Using Alu-PCR to profile tumor cell genomic DNA, we showed that 68% of small intestine carcinoids and 58% of pulmonary carcinoids had allelic banding patterns suggestive of either amplification or deletion of gene sequences. Alu-PCR demonstrated loss or gain of genetic sequences that were unique for each examined group. These findings strongly suggest that pulmonary carcinoids differ from their intestinal counterparts.

Applications of forensic identity testing in the clinical laboratory.

DNA analysis is becoming routine in the clinical laboratory for the diagnosis of human diseases using various tissue sources. Most clinical specimens are followed by tracking forms that include patient demographic data, accession number, and date and time of collection. As part of a thorough quality assurance program, proper documentation of test requisitions and tracking forms is mandatory. Despite these efforts, specimen mislabeling or other mix-ups can, and do, occur. We demonstrate the utility of the PM + DQA1 typing kit and STR analysis using the Visible Genetics automated DNA sequencing system in the proper identification of such clinical specimens as urine, blood, and paraffin-embedded tissues. In each case, sufficient DNA was extracted from these specimen types using a nonorganic extraction protocol for typing purposes. We conclude that DNA typing methods are feasible for distinguishing clinical laboratory specimens of questionable identity and compliment existing quality assurance techniques.

Correlation between morphologic and nonmorphologic prognostic markers of neuroblastoma.

Morphologic (Shimada classification--SC, original and modified histologic grades--OHG and MHG) and nonmorphologic (serum LDH, 1 p del, DNA index, N-myc copy number, telomerase activity, and expression of MRP, MDR1, and TRK) prognostic markers for NB have been reviewed. The functional role of these nonmorphologic markers in the development and progression of this disease include abnormal cell proliferation, resistance to chemotherapeutic agents, and induction of apoptosis. A statistically significant association between high OHG/MHG (grade 3), DNA index of 1 (diploidy), > 1 copy of N-myc per haploid genome and serum LDH of > or = 1500 IU/1 (p < 0.001 for each) has been described. In SC, undifferentiated histology and high MKI are associated with N-myc amplification. However, a lack of correlation between morphology and N-myc amplification has been found in localized NB. Confirmation of these observations must now be obtained on larger numbers of prospectively studied cases. Data on correlation for various prognostic markers could provide guidelines for identification of subsets of NB having strongly significant, readily determinable, reproducible, and relatively inexpensive prognostic markers that could ultimately be used to design an algorithm for risk-specific therapy.

Application of forensic identity testing in a clinical setting. Specimen identification.

Specimen identification is a carefully controlled factor in clinical laboratory testing. However, on occasion, despite surmountable efforts to prevent misidentification, a specimen is either mislabeled or an identifier is lost. Recently, we experienced a case of questionable mix-up of surgical specimens where the surgeon and patient questioned the biopsy site and size of specimen as indicated in the anatomic pathology report. Despite extensive tracking mechanisms, the perception of specimen mix-up warranted further means of identification. We utilized the PM + DQA1 amplification and typing system to confirm that typing results of a questionable biopsy were identical to typing results on a previous biopsy on the same patient and to the patient's blood. We demonstrate that this system is ideal for rapid DNA typing and identification of clinical specimens and that it can be performed on DNA isolated from paraffinembedded tissues.

Coexistence of the methylenetetrahydrofolate reductase single-nucleotide polymorphism (C677T) in patients with the factor V Leiden or prothrombin G20210A polymorphisms.

Venous thromboembolism is a complex disease resulting from the interaction of a multitude of both genetic and environmental factors that can affect the cascade of biochemical reactions involved. Single-nucleotide polymorphisms in the genes that code for coagulation factors V (factor V Leiden) and II (prothrombin G20210A), as well as the methylenetetrahydrofolate reductase (MTHFR C677T) gene, have been implicated in the majority of cases of hereditary thrombophilia. In this study the authors evaluated the coexistence of the MTHFR polymorphism in 96 patients with a clinical suspicion for thrombosis who also have either the factor V Leiden polymorphism or the prothrombin G20210A polymorphism. Results indicate that the frequency of the MTHFR polymorphism was similar between the study and control groups with respect to heterozygosity (36.5% vs. 55.3%) and homozygosity (20.8% vs. 14.9%). These data suggest that the MTHFR polymorphism is not associated preferentially with patients who have had or who are at risk of developing a thrombotic event. In this study, patients with the factor V Leiden polymorphism or the prothrombin G20210A polymorphism were considered to be at risk.

Analysis of the factor V Leiden mutation using the READIT Assay.

BACKGROUND: A variety of methods exist for the detection of single-nucleotide polymorphisms (SNPs) present in amplified segments of genomic DNA. We show the application of a novel SNP scoring tool for analysis of the factor V Leiden mutation. METHODS AND RESULTS: We have developed a novel method for analyzing SNPs. The luciferase-based technique, known as the READIT Technology (Promega Corp, Madison, WI), was used to analyze 510 residual human samples sent for factor V Leiden testing from three independent testing laboratories. A blinded retrospective analysis of the factor V Leiden mutation was used to determine the accuracy and throughput capabilities of the technology. One hundred percent concordance was observed between the READIT Assay and genotype assignments made in the testing laboratories. In addition, greater than 6 SDs of separation were observed between the means of wild-type and heterozygote sample populations. Repetitive sample measurements with representative wild-type, heterozygote, and mutant samples showed that greater than 9 SDs separated the means of heterozygote and homozygote sample populations. Confidence intervals based on the means of wild-type, heterozygote, and mutant sample populations were determined. CONCLUSION: Perfect concordance using the READIT Assay showed its effectiveness as a SNP scoring tool. The design of the factor V READIT Assay was straightforward, requiring the design of two unmodified oligonucleotides that differ at the 3' penultimate position to form perfect hybrids with the wild-type or Leiden form of the factor V sequence. The use of previously published amplification primers and conditions minimized the time needed to optimize and validate the assay. The READIT Calculator supplied with the assay allowed automated genotype assignments and statistical analysis from the READIT Assay data. Confidence-interval analysis validated the ability to distinguish between wild-type, heterozygote, and mutant samples using the READIT Assay.

Single nucleotide polymorphisms in the methylenetetrahydrofolate reductase gene are common in US Caucasian and Hispanic American populations.

To investigate the prevalence of the C677T and A1298C single nucleotide polymorphisms (SNPs) in the methylenetetrahydrofolate reductase (MTHFR) gene in Caucasian and Hispanic populations, EDTA-anticoagulated whole blood specimens were collected from a total of 100 random patients, 50 Caucasians and 50 Hispanics of Puerto Rican dissent. The prevalence of the two MTHFR SNPs was determined by polymerase chain reaction (PCR) mediated restriction fragment length polymorphism analysis. In the Caucasian population, homozygosity for the MTHFR A1298C SNP was detected in 4% (2/50) of the individuals tested, while 42% (21/50) were heterozygous for this SNP. Among Hispanics, 4% (2/50) were homozygous and 38% (19/50) heterozygous for the A1298C SNP. Homozygosity for the C677T MTHFR SNP was detected in 16% (8/50) and 10% (5/50) of Caucasians and Hispanics, respectively. In this study, the frequency of the C677T heterozygotes was very high at 56% (28/50) and 52% (26/50) Caucasians and Hispanics, respectively. C677T and A1298C are common SNPs in the MTHFR gene. The high prevalence of these SNPs in both Caucasian and Hispanic populations demonstrates the possibility of compounding effects of these SNPs in the pathogenesis of human diseases. While subgroups of patients may exhibit some clinical phenotype linked to these SNPs, our analysis demonstrates the need for careful interpretation of SNP data in the context of population screening.

Homocysteine screening of a female Hispanic population.

A total of 821 women of Hispanic descent aged 21-65 years, were screened for total and high density lipoprotein (HDL) cholesterol through outpatient clinics and public screening. Of this group, 78 were invited back for further testing because they had a total cholesterol/HDL cholesterol ratio exceeding 4.5 indicative of high risk for cardiovascular disease. Written consent and a fasting blood sample was obtained from these women, and tested for serum homocysteine. The concentrations for 77 of the 78 women (mean 8. 40+/-2.24, range 4.21-13.99 micromol/l) were within the pre-established normal range for women. One subject had an exceptionally high homocysteine concentration of 137 micromol/l. This subject subsequently developed a stroke and has been institutionalized since that time. Blood from the subject and immediate family members were tested for the 5'-10'-methylenetetrahydrofolate reductase (MTHFR) polymorphism. The subject and her children were both hyperhomocysteinemic and heterozygous for the mutation. One of the children also had a low vitamin B12 concentration in blood. Although the high homocysteine and cardiovascular risk in these subjects were likely due to a dietary deficiency of the vitamins, the MTHFR mutation may have also been a contributing factor. With the availability of rapid assays, screening blood for homocysteine in subjects deemed at high risk for cardiovascular disease may be justified.