Analytical validation of NeXT Personal®, an ultra-sensitive personalized circulating tumor DNA assay.

We describe the analytical validation of NeXT Personal®, an ultra-sensitive, tumor-informed circulating tumor DNA (ctDNA) assay for detecting residual disease, monitoring therapy response, and detecting recurrence in patients diagnosed with solid tumor cancers. NeXT Personal uses whole genome sequencing of tumor and matched normal samples combined with advanced analytics to accurately identify up to ~1,800 somatic variants specific to the patient's tumor. A personalized panel is created, targeting these variants and then used to sequence cell-free DNA extracted from patient plasma samples for ultra-sensitive detection of ctDNA. The NeXT Personal analytical validation is based on panels designed from tumor and matched normal samples from two cell lines, and from 123 patients across nine cancer types. Analytical measurements demonstrated a detection threshold of 1.67 parts per million (PPM) with a limit of detection at 95% (LOD95) of 3.45 PPM. NeXT Personal showed linearity over a range of 0.8 to 300,000 PPM (Pearson correlation coefficient = 0.9998). Precision varied from a coefficient of variation of 12.8% to 3.6% over a range of 25 to 25,000 PPM. The assay targets 99.9% specificity, with this validation study measuring 100% specificity and in silico methods giving us a confidence interval of 99.92 to 100%. In summary, this study demonstrates NeXT Personal as an ultra-sensitive, highly quantitative and robust ctDNA assay that can be used to detect residual disease, monitor treatment response, and detect recurrence in patients.

Differential modulation of cancer-related genes by mitochondrial DNA haplogroups and the STING DNA sensing system.

Activation of the Simulator of Interferon Genes (STING) system by mitochondrial (mt) DNA can upregulate type 1 interferon genes and enhance immune responses to combat bacterial and viral infections. In cancers, the tumor-derived DNA activates STING leading to upregulation of IFN-beta and induction of antitumor T cells. The entire mtDNA from the cell lines was sequenced using next-generation sequencing (NGS) technology with independent sequencing of both strands in both directions, allowing identification of low-frequency heteroplasmy SNPs. There were 15 heteroplasmy SNPs showing a range from 3.4% to 40.5% occurrence in the K cybrid cell lines. Three H haplogroup cybrids possessed SNP heteroplasmy that ranged from 4.39% to 30.7%. The present study used qRT-PCR to determine if cybrids of H and K haplogroups differentially regulate expression levels of five cancer genes (BRAC1, ALK, PD1, EGFR, and HER2) and seven STING subunits genes (CGAS, TBK1, IRF3, IκBa, NFκB, TRAF2, and TNFRSF19). Some cybrids underwent siRNA knockdown of STING followed by qRT-PCR in order to determine the impact of STING on gene expression. Rho0 (lacking mtDNA) ARPE-19 cells were used to determine if mtDNA is required for the expression of the cancer genes studied. Our results showed that (a) K cybrids have lower expression levels for BRAC1, ALK, PD1, EGFR, IRF3, and TNFRSF19 genes but increased transcription for IκBa and NFκB compared to H cybrids; (b) STING KD decreases expression of EGFR in both H and K cybrids, and (c) PD1 expression is negligible in Rho0 cells. Our findings suggest that the STING DNA sensing pathway may be a previously unrecognized pathway to target modulation of cancer-related genes and the PD1 expression requires the presence of mtDNA.

CONGENITAL POSTERIOR POLAR CHORIORETINAL HYPOPLASIA: Expansion of the Clinical Spectrum, Mutation, and Its Association With PRDM13.

PURPOSE: To describe a new ocular phenotype in a single Egyptian family associated with a heterozygous noncoding mutation in the North Carolina macular dystrophy (NCMD/MCDR1) locus, likely affecting the PRDM13 gene. METHODS: A retrospective, clinical chart review of 11 members of a four-generation family. Comprehensive ophthalmic examinations included visual acuity, refraction, fundus imaging, spectral-domain optical coherence tomography, and full-field electroretinography. Molecular genetic analysis of the MCDR1 region was performed using whole genome and targeted sequencing. The main outcome measures were DNA sequence variants, clinical, retinal imaging, and electroretinography findings. RESULTS: The five affected adult family members tested carried a single heterozygous mutation in a noncoding region (Chr6:100,046,783A>C) located 7.8 kb upstream of PRDM13. Visual acuity ranged from 20/200 to 20/400. All members had extensive chorioretinal absence/thinning extending outside of the maculae with extensive posterior bowing of the choroid and sclera centered in the macula giving a large macular coloboma-like appearance. Two additional members had cystoid fluid, and one had macular detachment. Full-field electroretinography revealed reduced cone and rod responses in all affected members. CONCLUSION: The phenotype of this disease falls between the spectrum of progressive bifocal chorioretinal atrophy and NCMD. The findings are most consistent with progressive bifocal chorioretinal atrophy with the exception that there is no bifocal nature to the appearance nor is it progressive. Another view is that the phenotype seems to be an extremely severe form of NCMD. Given that this disease falls in between progressive bifocal chorioretinal atrophy and NCMD, we propose calling it congenital posterior polar chorioretinal hypoplasia.

Altered Retrograde Signaling Patterns in Breast Cancer Cells Cybrids with H and J Mitochondrial DNA Haplogroups.

The aim of this study was to determine the role of retrograde signaling (mitochondria to nucleus) in MCF7 breast cancer cells. Therefore, in the present study, MCF7-H and MCF7-J cybrids were produced using the mitochondria from the same H and J individuals that were already used in our non-diseased retinal pigment epithelium (ARPE19) cybrids. MCF7 cybrids were treated with cisplatin and analyzed for cell viability, mitochondrial membrane potential, ROS, and expression levels of genes associated with the cGAS-STING and cancer-related pathways. Results showed that unlike the ARPE19-H and ARPE19-J cybrids, the untreated MCF7-H and MCF7-J cybrids had similar levels of ATP, lactate, and OCR: ECAR ratios. After cisplatin treatment, MCF7-H and MCF7-J cybrids showed similar (a) decreases in cell viability and ROS levels; (b) upregulation of ABCC1, BRCA1 and CDKN1A/P21; and (c) downregulation of EGFR. Cisplatin-treated ARPE19-H and ARPE19-J cybrids showed increased expression of six cGAS-STING pathway genes, while two were increased for MCF7-J cybrids. In summary, the ARPE19-H and ARPE19-J cybrids behave differentially from each other with or without cisplatin. In contrast, the MCF7-H and MCF7-J cybrids had identical metabolic/bioenergetic profiles and cisplatin responses. Our findings suggest that cancer cell nuclei might have a diminished ability to respond to the modulating signaling of the mtDNA that occurs via the cGAS-STING pathway.

North Carolina Macular Dystrophy: Long-term Follow-up of the Original Family.

PURPOSE: The phenotype of North Carolina macular dystrophy (NCMD) is highly variable and remains poorly appreciated and understood, often causing misdiagnoses in isolated cases. One of the features of NCMD is the general lack of progression despite its original name, "dominant progressive foveal dystrophy," as reported in 1971 by Lefler et al (W.H.L.). The purpose of this study was to report the long-term follow-up of this condition. DESIGN: Systematic, longitudinal, and detailed documentation along with the imaging of the peripheral retina. SUBJECTS: We reexamined 27 of the original family members with NCMD in an office setting 30 to 50 years after they were first reported. METHODS: The evaluation of all the affected subjects included best-corrected visual acuity (BCVA), slit-lamp and dilated-fundus examinations, wide-field fundus and autofluorescent photography, and spectral-domain OCT (SD OCT). Blood was collected for DNA extraction, banking, and sequencing. MAIN OUTCOME MEASURES: Best-corrected visual acuity, slit-lamp and dilated-fundus examinations, wide-field fundus and autofluorescent photography, and SD OCT. RESULTS: The 27 subjects examined were a part of the original family with NCMD that was initially reported in 1971. A point mutation (NC_000006.11:g.100040906G>T) (Hg19) in a noncoding region of a deoxyribonuclease I hypersensitivity binding site was found in all the affected subjects. Nine subjects were the affected children of those originally examined 30 to 50 years ago by Kent W. Small (K.W.S.) and W.H.L., and the remaining 17 subjects (34 eyes) had been examined 30 years previously by K.W.S. Of these 17 subjects (34 eyes), 4 of 34 (11%) eyes showed worsening of vision and evidence of fibrosis due to choroidal neovascular membranes (CNVMs). Fourteen of the 27 (51%) patients showed peripheral retinal drusen, which did not seem to correlate with the severity of the macular disease. CONCLUSIONS: Most patients with NCMD have stable vision and fundus findings throughout their lives. The ones who experienced BCVA decline did so because of the apparent evidence of CNVMs. Patients with grade 2 NCMD seem to be at an increased risk of further or progressive vision loss due to CNVMs. Intravitreal therapy with vascular endothelial growth factor inhibitors may benefit these patients if they are treated in a timely fashion. Peripheral retina drusen of varying degrees of severity were found in slightly more than half of the affected subjects.

Differential effects of cisplatin on cybrid cells with varying mitochondrial DNA haplogroups.

BACKGROUND: Drug therapy yields different results depending on its recipient population. Cisplatin, a commonly used chemotherapeutic agent, causes different levels of resistance and side effects for different patients, but the mechanism(s) are presently unknown. It has been assumed that this variation is a consequence of differences in nuclear (n) DNA, epigenetics, or some external factor(s). There is accumulating evidence that an individual's mitochondrial (mt) DNA may play a role in their response to medications. Variations within mtDNA can be observed, and an individual's mtDNA can be categorized into haplogroups that are defined by accumulations of single nucleotide polymorphisms (SNPs) representing different ethnic populations. METHODS: The present study was conducted on transmitochondrial cytoplasmic hybrids (cybrids) that possess different maternal-origin haplogroup mtDNA from African (L), Hispanic [A+B], or Asian (D) backgrounds. Cybrids were created by fusing Rho0 ARPE-19 cells (lacking mtDNA) with platelets, which contain numerous mitochondria but no nuclei. These cybrid cells were cultured to passage five, treated with cisplatin, incubated for 48 h, then analyzed for cell metabolic activity (tetrazolium dye (MTT) assay), mitochondrial membrane potential (JC-1 assay), cytotoxicity (lactate dehydrogenase (LDH) assay), and gene expression levels for ALK, BRCA1, EGFR, and ERBB2/HER2. RESULTS: Results indicated that untreated cybrids with varying mtDNA haplogroups had similar relative metabolic activity before cisplatin treatment. When treated with cisplatin, (1) the decline in metabolic activity was greatest in L (27.4%, p < 0.012) < D (24.86%, p = 0.0001) and [A+B] cybrids (24.67%, p = 0.0285) compared to untreated cybrids; (2) mitochondrial membrane potential remained unchanged in all cybrids (3) LDH production varied between cybrids (L >[A+B], p = 0.0270). (4) The expression levels decreased for ALK in L (p < 0.0001) and [A+B] (p = 0.0001) cybrids but not in D cybrids (p = 0.285); and decreased for EGFR in [A+B] cybrids (p = 0.0246) compared to untreated cybrids. CONCLUSION: Our findings suggest that an individual's mtDNA background may be associated with variations in their response to cisplatin treatment, thereby affecting the efficiency and the severity of side effects from the treatment.

Development and Analytical Validation of a DNA Dual-Strand Approach for the US Food and Drug Administration-Approved Next-Generation Sequencing-Based Praxis Extended RAS Panel for Metastatic Colorectal Cancer Samples.

A next-generation sequencing method was developed that can distinguish single-stranded modifications from low-frequency somatic mutations present on both strands of DNA in formalin-fixed paraffin-embedded colorectal cancer samples. We applied this method for analytical validation of the Praxis Extended RAS Panel, a US Food and Drug Administration-approved companion diagnostic for panitumumab, on the Illumina MiSeqDx platform. With the use of the TruSeq amplicon workflow, both strands of DNA from the starting material were interrogated independently. Mutations were reported only if found on both strands; artifacts usually present on only one strand would not be reported. A total of 56 mutations were targeted within the KRAS and NRAS genes. A minimum read depth of 1800× per amplicon is required per sample but averaged >30,000× at maximum multiplexing levels. Analytical validation studies were performed to determine the simultaneous detection of mutations on both strands, reproducibility, assay detection level, precision of the assay across various factors, and the impact of interfering substances. In conclusion, this assay can clearly distinguish single-stranded artifacts from low-frequency mutations. Furthermore, the assay is accurate, precise, and reproducible, can achieve consistent detection of a mutation at 5% mutation frequency, exhibits minimal impact from tested interfering substances, and can simultaneously detect 56 mutations in a single run using 10 samples plus controls.

European mtDNA Variants Are Associated With Differential Responses to Cisplatin, an Anticancer Drug: Implications for Drug Resistance and Side Effects.

Background: Cisplatin, a powerful antitumor agent, causes formation of DNA adducts, and activation of apoptotic pathways. Presently, cisplatin resistance develops in up to 70% of patients but the underlying molecular mechanism(s) are unclear and there are no markers to determine which patients will become resistant. Mitochondria play a significant role not only in energy metabolism but also retrograde signaling (mitochondria to nucleus) that modulates inflammation, complement, and apoptosis pathways. Maternally inherited mitochondrial (mt) DNA can be classified into haplogroups representing different ethnic populations that have diverse susceptibilities to diseases and medications. Methods: Transmitochondrial cybrids, where all cell lines possess identical nuclear genomes but either the H (Southern European) or J (Northern European) mtDNA haplogroups, were treated with cisplatin and analyzed for differential responses related to viability, oxidative stress, and expression levels of genes associated with cancer, cisplatin-induced nephrotoxicity and resistance, apoptosis and signaling pathways. Results: The cisplatin-treated-J cybrids showed greater loss of cell viability along with lower levels of reactive oxygen species and mitochondrial membrane potential compared to cisplatin-treated-H cybrids. After cisplatin treatment, J cybrids showed increased gene expression of BAX, CASP3, and CYP51A, but lower levels of SFRP1 compared to untreated-J cybrids. The cisplatin-treated-H cybrids had elevated expression of CDKN1A/P21, which has a role in cisplatin toxicity, compared to untreated-H cybrids. The cisplatin-treated H had higher transcription levels of ABCC1, DHRS2/HEP27, and EFEMP1 compared to cisplatin-treated-J cybrids. Conclusions: Cybrid cell lines that contain identical nuclei but either H mtDNA mitochondria or J mtDNA mitochondria respond differently to cisplatin treatments suggesting involvement of the retrograde signaling (from mitochondria to nucleus) in the drug-induced cell death. Varying toxicities and transcription levels of the H vs. J cybrids after cisplatin treatment support the hypothesis that mtDNA variants play a role in the expression of genes affecting resistance and side effects of cisplatin.

Clinical validation of the next-generation sequencing-based Extended RAS Panel assay using metastatic colorectal cancer patient samples from the phase 3 PRIME study.

PURPOSE: To validate a next-generation sequencing (NGS)-based companion diagnostic using the MiSeqDx® sequencing instrument to simultaneously detect 56 RAS mutations in DNA extracted from formalin-fixed paraffin-embedded metastatic colorectal cancer (mCRC) tumor samples from the PRIME study. The test's ability to identify patients with mCRC likely to benefit from panitumumab treatment was assessed. METHODS: Samples from PRIME, which compared first-line panitumumab + FOLFOX4 with FOLFOX4, were processed according to predefined criteria using a multiplex assay that included input DNA qualification, library preparation, sequencing, and the bioinformatics reporting pipeline. NGS mutational analysis of KRAS and NRAS exons 2, 3, and 4 was performed and compared with Sanger sequencing. RESULTS: In 441 samples, positive percent agreement of the Extended RAS Panel with Sanger sequencing was 98.7% and negative percent agreement was 97.6%. For clinical validation (n = 528), progression-free survival (PFS) and overall survival (OS) were compared between patients with RAS mutations (RAS Positive) and those without (RAS Negative). Panitumumab + FOLFOX4 improved PFS in RAS Negative patients (P = 0.02). Quantitative interaction testing indicated the treatment effect (measured by the hazard ratio of panitumumab + FOLFOX4 versus FOLFOX4) differed for RAS Negative versus RAS Positive for PFS (P = 0.0038) and OS (P = 0.0323). CONCLUSIONS: NGS allows for broad, rapid, highly specific analyses of genomic regions. These results support use of the Extended RAS Panel as a companion diagnostic for selecting patients for panitumumab, and utilization is consistent with recent clinical guidelines regarding mCRC RAS testing. Overall, approximately 13% more patients were detected with the Extended RAS Panel versus KRAS exon 2 alone. CLINICAL TRIAL REGISTRY IDENTIFIER: NCT00364013 (ClinicalTrials.gov).

Diagnostic Mutation Profiling and Validation of Non-Small-Cell Lung Cancer Small Biopsy Samples using a High Throughput Platform.

BACKGROUND: A single platform designed for the synchronous screening of multiple mutations can potentially enable molecular profiling in samples of limited tumor tissue. This approach is ideal for the assessment of advanced non-small-cell lung cancer (NSCLC) diagnostic specimens, which often comprise small biopsies. Therefore, we aimed in this study to validate the mass spectrometry-based Sequenom LungCarta panel and MassARRAY platform using DNA extracted from a single 5 µM formalin-fixed paraffin-embedded tissue section. METHODS: Mutations, including those with an equivocal spectrum, detected in 90 cases of NSCLC (72 lung biopsies, 13 metastatic tissue biopsies, three resections, and two cytology samples) were validated by a combination of standard sequencing techniques, immunohistochemical staining for p53 protein, and next-generation sequencing with the TruSight Tumor panel. RESULTS: Fifty-five mutations were diagnosed in 47 cases (52%) in the following genes: TP53 (22), KRAS (15), EGFR (5), MET (3), PIK3CA (3), STK11 (2), NRF-2 (2), EPHA5 (1), EPHA3 (1), and MAP2K1 (1). Of the 90 samples, one failed testing due to poor quality DNA. An additional 7 TP53 mutations were detected by next-generation sequencing, which facilitated the interpretation of p53 immunohistochemistry but required 5 × 10 µM tumor sections per sample tested. CONCLUSIONS: The LungCarta panel is a sensitive method of screening for multiple alterations (214 mutations across 26 genes) and which optimizes the use of limited amounts of tumor DNA isolated from small specimens.

Human retinal transmitochondrial cybrids with J or H mtDNA haplogroups respond differently to ultraviolet radiation: implications for retinal diseases.

BACKGROUND: It has been recognized that cells do not respond equally to ultraviolet (UV) radiation but it is not clear whether this is due to genetic, biochemical or structural differences of the cells. We have a novel cybrid (cytoplasmic hybrids) model that allows us to analyze the contribution of mitochondrial DNA (mtDNA) to cellular response after exposure to sub-lethal dose of UV. mtDNA can be classified into haplogroups as defined by accumulations of specific single nucleotide polymorphisms (SNPs). Recent studies have shown that J haplogroup is high risk for age-related macular degeneration while the H haplogroup is protective. This study investigates gene expression responses in J cybrids versus H cybrids after exposure to sub-lethal doses of UV-radiation. METHODOLOGY/PRINCIPAL FINDINGS: Cybrids were created by fusing platelets isolated from subjects with either H (n = 3) or J (n = 3) haplogroups with mitochondria-free (Rho0) ARPE-19 cells. The H and J cybrids were cultured for 24 hours, treated with 10 mJ of UV-radiation and cultured for an additional 120 hours. Untreated and treated cybrids were analyzed for growth rates and gene expression profiles. The UV-treated and untreated J cybrids had higher growth rates compared to H cybrids. Before treatment, J cybrids showed lower expression levels for CFH, CD55, IL-33, TGF-A, EFEMP-1, RARA, BCL2L13 and BBC3. At 120 hours after UV-treatment, the J cybrids had decreased CFH, RARA and BBC3 levels but increased CD55, IL-33 and EFEMP-1 compared to UV-treated H cybrids. CONCLUSION/SIGNIFICANCE: In cells with identical nuclei, the cellular response to sub-lethal UV-radiation is mediated in part by the mtDNA haplogroup. This supports the hypothesis that differences in growth rates and expression levels of complement, inflammation and apoptosis genes may result from population-specific, hereditary SNP variations in mtDNA. Therefore, when analyzing UV-induced damage in tissues, the mtDNA haplogroup background may be important to consider.

Molecular and bioenergetic differences between cells with African versus European inherited mitochondrial DNA haplogroups: implications for population susceptibility to diseases.

The geographic origins of populations can be identified by their maternally inherited mitochondrial DNA (mtDNA) haplogroups. This study compared human cybrids (cytoplasmic hybrids), which are cell lines with identical nuclei but mitochondria from different individuals with mtDNA from either the H haplogroup or L haplogroup backgrounds. The most common European haplogroup is H while individuals of maternal African origin are of the L haplogroup. Despite lower mtDNA copy numbers, L cybrids had higher expression levels for nine mtDNA-encoded respiratory complex genes, decreased ATP (adenosine triphosphate) turnover rates and lower levels of reactive oxygen species production, parameters which are consistent with more efficient oxidative phosphorylation. Surprisingly, GeneChip arrays showed that the L and H cybrids had major differences in expression of genes of the canonical complement system (5 genes), dermatan/chondroitin sulfate biosynthesis (5 genes) and CCR3 (chemokine, CC motif, receptor 3) signaling (9 genes). Quantitative nuclear gene expression studies confirmed that L cybrids had (a) lower expression levels of complement pathway and innate immunity genes and (b) increased levels of inflammation-related signaling genes, which are critical in human diseases. Our data support the hypothesis that mtDNA haplogroups representing populations from different geographic origins may play a role in differential susceptibilities to diseases.

Therapeutic potential of the poly(ADP-ribose) polymerase inhibitor rucaparib for the treatment of sporadic human ovarian cancer.

Here, we investigate the potential role of the PARP inhibitor rucaparib (CO-338, formerly known as AG014699 and PF-01367338) for the treatment of sporadic ovarian cancer. We studied the growth inhibitory effects of rucaparib in a panel of 39 ovarian cancer cell lines that were each characterized for mutation and methylation status of BRCA1/2, baseline gene expression signatures, copy number variations of selected genes, PTEN status, and sensitivity to platinum-based chemotherapy. To study interactions with chemotherapy, we used multiple drug effect analyses and assessed apoptosis, DNA fragmentation, and γH2AX formation. Concentration-dependent antiproliferative effects of rucaparib were seen in 26 of 39 (67%) cell lines and were not restricted to cell lines with BRCA1/2 mutations. Low expression of other genes involved in homologous repair (e.g., BCCIP, BRCC3, ATM, RAD51L1), amplification of AURKA or EMSY, and response to platinum-based chemotherapy was associated with sensitivity to rucaparib. Drug interactions with rucaparib were synergistic for topotecan, synergistic, or additive for carboplatin, doxorubicin or paclitaxel, and additive for gemcitabine. Synergy was most pronounced when rucaparib was combined with topotecan, which resulted in enhanced apoptosis, DNA fragmentation, and γH2AX formation. Importantly, rucaparib potentiated chemotherapy independent of its activity as a single agent. PARP inhibition may be a useful therapeutic strategy for a wider range of ovarian cancers bearing deficiencies in the homologous recombination pathway other than just BRCA1/2 mutations. These results support further clinical evaluation of rucaparib either as a single agent or as an adjunct to chemotherapy for the treatment of sporadic ovarian cancer.

Mitochondrial DNA variants mediate energy production and expression levels for CFH, C3 and EFEMP1 genes: implications for age-related macular degeneration.

BACKGROUND: Mitochondrial dysfunction is associated with the development and progression of age-related macular degeneration (AMD). Recent studies using populations from the United States and Australia have demonstrated that AMD is associated with mitochondrial (mt) DNA haplogroups (as defined by combinations of mtDNA polymorphisms) that represent Northern European Caucasians. The aim of this study was to use the cytoplasmic hybrid (cybrid) model to investigate the molecular and biological functional consequences that occur when comparing the mtDNA H haplogroup (protective for AMD) versus J haplogroup (high risk for AMD). METHODOLOGY/PRINCIPAL FINDINGS: Cybrids were created by introducing mitochondria from individuals with either H or J haplogroups into a human retinal epithelial cell line (ARPE-19) that was devoid of mitochondrial DNA (Rho0). In cybrid lines, all of the cells carry the same nuclear genes but vary in mtDNA content. The J cybrids had significantly lower levels of ATP and reactive oxygen/nitrogen species production, but increased lactate levels and rates of growth. Q-PCR analyses showed J cybrids had decreased expressions for CFH, C3, and EFEMP1 genes, high risk genes for AMD, and higher expression for MYO7A, a gene associated with retinal degeneration in Usher type IB syndrome. The H and J cybrids also have comparatively altered expression of nuclear genes involved in pathways for cell signaling, inflammation, and metabolism. CONCLUSION/SIGNIFICANCE: Our findings demonstrate that mtDNA haplogroup variants mediate not only energy production and cell growth, but also cell signaling for major molecular pathways. These data support the hypothesis that mtDNA variants play important roles in numerous cellular functions and disease processes, including AMD.

A multiplex assay for detecting genetic variations in CYP2C9, VKORC1, and GGCX involved in warfarin metabolism.

BACKGROUND: Patients differ in responses to warfarin, which is commonly prescribed to treat thromboembolic events. Genetic variations in the cytochrome P450, family 2, subfamily C, polypeptide 9 (CYP2C9), vitamin K epoxide reductase complex, subunit 1 (VKORC1), and gamma-glutamyl carboxylase (GGCX) genes have been shown to contribute to impaired metabolism of warfarin. METHODS: We designed a custom multiplex single-nucleotide polymorphism (SNP) panel to interrogate the CYP2C9 *2, *3, VKORC1 (-1639G-->A), and GGCX (1181T-->G) alleles simultaneously in a single sample by use of single-base extension and capillary electrophoresis after genomic DNA extraction and PCR amplification. RESULTS: Our assay successfully detected various genotypes from known controls and 24 unknown samples. It was found to be 100% concordant with sequencing results. CONCLUSIONS: Our multiplexed SNP panel can be successfully used in genotyping of patient blood samples. Results can be combined with other clinical parameters in an algorithm for warfarin dosing. These data provide a proof-in-principle of multiplexed SNP analysis using rapid single-base extension and capillary electrophoresis, and warrant additional validation using a larger cohort of patient samples.

Candidate gene screening for posterior polymorphous dystrophy.

PURPOSE: To perform candidate gene screening for posterior polymorphous corneal dystrophy (PPCD). The initial 3 genes chosen, ID1, BCL2L1, and VSX1, lie within the region on chromosome 20 to which the PPCD gene has been linked, and mutations in VSX1 have previously been identified in patients with PPCD. METHODS: DNA extraction, PCR amplification, and direct sequencing of the VSX1, BCL2L1, and ID1 genes were performed in 14 affected patients (12 families) as well as in unaffected family members and healthy control subjects. RESULTS: No coding region mutations in the BCL2L1 or ID1 genes were identified in affected patients. In the VSX1 gene, the previously identified Gly160Asp missense change was not present in any of our 12 probands, and the Asp144Glu mutation was identified in 1 affected patient as well as 1 unaffected control individual. Additionally, 2 synonymous substitutions were identified, Ala182Ala (8 affected patients from 8 families) and Gly239Gly (1 affected patient and 1 unaffected patient from the same family). In the ID1 gene, the synonymous substitution Gly216Gly was observed in 2 affected patients (2 families) who also demonstrated a single nucleotide change in both the 5'UTR (2129T>C) and 3'UTR (3267A>G). Another 5'UTR change, 2177T>C, was identified in 1 affected patient and his unaffected parent, both of whom also demonstrated the 2129T>C and 3267A>G changes. CONCLUSIONS: None of the 12 probands with PPCD demonstrated the previously described Gly160Asp mutation within the VSX1 gene. The Asp144Glu missense change, present in an affected patient as well as an unaffected control individual, appears to be a rare polymorphism, not a disease-causing mutation. No coding region changes were identified in the ID1 or BCL2L1 genes. Therefore, although we report a number of novel polymorphisms in the VSX1 and ID1 genes, the failure to identify any sequence variants that sort with the disease phenotype suggests that other genetic factors are involved in PPCD.

Lattice corneal dystrophy associated with the Ala546Asp and Pro551Gln missense changes in the TGFBI gene.

PURPOSE: To report a phenotypic variant of lattice corneal dystrophy associated with two missense changes, Ala546Asp and Pro551Gln, in the transforming growth factor-beta-induced gene (TGFBI). DESIGN: Experimental study. METHODS: Genomic DNA was obtained from the proband as well as affected and unaffected family members. Exons 4, 11, 12, and 14 of the TGFBI gene were amplified and sequenced. Additionally, a corneal button excised from the proband was examined by light and transmission electron microscopy. Haplotype analysis was performed on the proband's family and members of a previously identified pedigree with the same TGFBI gene missense changes. RESULTS: Bilateral, symmetric, radially arranged, branching refractile lines within and surrounding an area of central anterior stromal haze were noted in the proband. Multiple polymorphic, refractile deposits were noted in the mid and posterior stroma in both the proband and her daughter. Light and electron microscopic analyses demonstrated amyloid and excluded the presence of deposits characteristic of granular corneal dystrophy. Screening of TGFBI exon 12 in the proband and her affected daughter revealed two missense changes, Ala546Asp and Pro551Gln (both absent in 250 control chromosomes). Haplotype analysis suggested that the mutations in this family and in a previously identified pedigree reflect a founder effect, rather than an independent occurrence. CONCLUSIONS: We present a phenotypic variant of lattice corneal dystrophy associated with the Ala546Asp and Pro551Gln missense changes in exon 12 of the TGFBI gene. A common ancestor appears to account for the missense mutations observed in this pedigree and in a previously reported family.

Comparative analysis of the FOXL2 gene and characterization of mutations in BPES patients.

Bleparophimosis ptosis epicanthus inversus syndrome (BPES) is a rare disorder characterized by eyelid malformation and in some cases associated with premature ovarian failure. Although the familial form is autosomal dominant, many cases are also sporadic. The mutations causing this disorder were found in a winged/forkhead transcription factor gene named FOXL2. We have sequenced the mouse homolog for the FOXL2 gene and identified the Fugu rubripes (pufferfish) ortholog from the database. By alignment of the three sequences, we found an almost complete conservation of the forkhead domain in the three species. There is 95% and 61% conservation at the protein level between human-mouse and human-pufferfish, respectively. The polyalanine and polyproline tracts within the gene are absent in Fugu rubripes. An overview identifies four breaks in the conservation of the gene within these species. Using a direct sequencing approach, we performed mutation analysis from DNA of nine affected individuals from familial and sporadic cases. The mutations are distributed throughout the coding region of the FOXL2 gene. We identified five novel mutations: g.292delG (E19fsX149); g.530G>A (W98X); g.548A>G (H104R); g.652G>T (E139X); and g.1178_1185del8 (A314fsX530). In addition we also identified two known mutations g.823C>T (Q196X) and g.1092_1108dup17, the latter in individuals from three unrelated pedigrees.