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1.  Using Exome Data to Identify Malignant Hyperthermia Susceptibility Mutations 
Anesthesiology  2013;119(5):1043-1053.
Malignant hyperthermia susceptibility (MHS) is a life-threatening, inherited disorder of muscle calcium metabolism, triggered by anesthetics and depolarizing muscle relaxants. An unselected cohort was screened for MHS mutations using exome sequencing. Our aim was to pilot a strategy for the RYR1 and CACNA1S genes.
Exome sequencing was performed on 870 volunteers not ascertained for MHS. Variants in RYR1 and CACNA1S were annotated using an algorithm that filtered results based on mutation type, frequency, and information in mutation databases. Variants were scored on a six-point pathogenicity scale. Medical histories and pedigrees were reviewed for malignant hyperthermia and related disorders.
We identified 70 RYR1 and 53 CACNA1S variants among 870 exomes. Sixty-three RYR1 and 41 CACNA1S variants passed the quality and frequency metrics but we excluded synonymous variants. In RYR1, we identified 65 missense mutations, one nonsense, two that affected splicing, and one non frameshift indel. In CACNA1S, 48 missense, one frameshift deletion, one splicing and one non frameshift indel were identified. RYR1 variants predicted to be pathogenic for MHS were found in three participants without medical or family histories of MHS. Numerous variants, previously described as pathogenic in mutation databases, were reclassified by us to be of unknown pathogenicity.
Exome sequencing can identify asymptomatic patients at risk for MHS, although the interpretation of exome variants can be challenging. The use of exome sequencing in unselected cohorts is an important tool to understand the prevalence and penetrance of MHS, a critical challenge for the field.
PMCID: PMC4077354  PMID: 24195946
2.  Usefulness of a Darwinian System in a Biotechnological Application: Evolution of Optical Window Fluorescent Protein Variants under Selective Pressure 
PLoS ONE  2014;9(9):e107069.
With rare exceptions, natural evolution is an extremely slow process. One particularly striking exception in the case of protein evolution is in the natural production of antibodies. Developing B cells activate and diversify their immunoglobulin (Ig) genes by recombination, gene conversion (GC) and somatic hypermutation (SHM). Iterative cycles of hypermutation and selection continue until antibodies of high antigen binding specificity emerge (affinity maturation). The avian B cell line DT40, a cell line which is highly amenable to genetic manipulation and exhibits a high rate of targeted integration, utilizes both GC and SHM. Targeting the DT40's diversification machinery onto transgenes of interest inserted into the Ig loci and coupling selective pressure based on the desired outcome mimics evolution. Here we further demonstrate the usefulness of this platform technology by selectively pressuring a large shift in the spectral properties of the fluorescent protein eqFP615 into the highly stable and advanced optical imaging expediting fluorescent protein Amrose. The method is advantageous as it is time and cost effective and no prior knowledge of the outcome protein's structure is necessary. Amrose was evolved to have high excitation at 633 nm and excitation/emission into the far-red, which is optimal for whole-body and deep tissue imaging as we demonstrate in the zebrafish and mouse model.
PMCID: PMC4156574  PMID: 25192257
3.  Postmastectomy/Axillary Node Dissection Chyloma: The Additional Value of SPECT/CT Lymphoscintigraphy 
Journal of Breast Cancer  2014;17(3):291-294.
After mastectomy and axillary node dissection, chylous leakage is rare. However, considerable anatomical variation in the termination of the thoracic duct has been reported. Hence, during breast surgery, injury to the lateral terminating branch is not unlikely and might lead to retrograde chyle leak. Herein, we describe a patient who had a chylous leakage at her wound site after a left simple mastectomy and axillary node dissection and for whom lymphoscintigraphy with Tc-99m albumin nanocolloid was performed. In this case, additional hybrid single-photon emission computed tomography/computed tomography study was done, and has helped with the accurate identification of the chyle leakage site, thus aiding in surgical management.
PMCID: PMC4197361  PMID: 25320629
Breast neoplasms; Chyle; Lymphoscintigraphy; Single-photon emission-computed tomography
4.  Interpreting Secondary Cardiac Disease Variants in an Exome Cohort 
Circulation. Cardiovascular genetics  2013;6(4):10.1161/CIRCGENETICS.113.000039.
Massively parallel sequencing to identify rare variants is widely practiced in medical research and in the clinic. Genome and exome sequencing can identify the genetic cause of a disease (primary results), but can also identify pathogenic variants underlying diseases that are not being sought (secondary or incidental results). A major controversy has developed surrounding the return of secondary results to research participants. We have piloted a method to analyze exomes to identify participants at-risk for cardiac arrhythmias, cardiomyopathies or sudden death.
Methods and Results
Exome sequencing was performed on 870 participants not selected for arrhythmia, cardiomyopathy, or a family history of sudden death. Exome data from 22 cardiac arrhythmia and 41 cardiomyopathy-associated genes were analyzed using an algorithm that filtered results on genotype quality, frequency, and database information. We identified 1367 variants in the cardiomyopathy genes and 360 variants in the arrhythmia genes. Six participants had pathogenic variants associated with dilated cardiomyopathy (n=1), hypertrophic cardiomyopathy (n=2), left ventricular noncompaction (n=1) or long QT syndrome (n=2). Two of these participants had evidence of cardiomyopathy and one had left ventricular noncompaction on ECHO. Three participants with likely pathogenic variants had prolonged QTc. Family history included unexplained sudden death among relatives.
Approximately 0.5% of participants in this study had pathogenic variants in known cardiomyopathy or arrhythmia genes. This high frequency may be due to self-selection, false positives, or underestimation of the prevalence of these conditions. We conclude that clinically important cardiomyopathy and dysrhythmia secondary variants can be identified in unselected exomes.
PMCID: PMC3887521  PMID: 23861362
arrhythmia (heart rhythm disorders); cardiomyopathy; cardiovascular genomics; genetic heart disease; genetic variation; arrhythmia; genetics; human; genomic medicine
5.  Personalized predictive lung dosimetry by technetium-99m macroaggregated albumin SPECT/CT for yttrium-90 radioembolization 
EJNMMI Research  2014;4:33.
For yttrium-90 (90Y) radioembolization, the common practice of assuming a standard 1,000-g lung mass for predictive dosimetry is fundamentally incongruent with the modern philosophy of personalized medicine. We recently developed a technique of personalized predictive lung dosimetry using technetium-99m (99mTc) macroaggregated albumin (MAA) single photon emission computed tomography with integrated CT (SPECT/CT) of the lung as part of our routine dosimetric protocol for 90Y radioembolization. Its rationales are the technical superiority of SPECT/CT over planar scintigraphy, ease and convenience of lung auto-segmentation CT densitovolumetry, and dosimetric advantage of patient-specific lung parenchyma masses.
This is a retrospective study of our pulmonary clinical outcomes and comparison of lung dosimetric accuracy and precision by 99mTc MAA SPECT/CT versus conventional planar methodology. 90Y resin microspheres (SIR-Spheres) were used for radioembolization. Diagnostic CT densitovolumetry was used as a reference for lung parenchyma mass. Pulmonary outcomes were based on follow-up diagnostic CT chest or X-ray.
Thirty patients were analyzed. The mean lung parenchyma mass of our Southeast Asian cohort was 822 ± 103 g standard deviation (95% confidence interval 785 to 859 g). Patient-specific lung parenchyma mass estimation by CT densitovolumetry on 99mTc MAA SPECT/CT is accurate (bias −21.7 g) and moderately precise (95% limits of agreement −194.6 to +151.2 g). Lung mean radiation absorbed doses calculated by 99mTc MAA SPECT/CT and planar methodology are both accurate (bias <0.5 Gy), but 99mTc MAA SPECT/CT offers better precision over planar methodology (95% limits of agreement −1.76 to +2.40 Gy versus −3.48 to +3.31 Gy, respectively). None developed radiomicrosphere pneumonitis when treated up to a lung mean radiation absorbed dose of 18 Gy at a median follow-up of 4.4 months.
Personalized predictive lung dosimetry by 99mTc MAA SPECT/CT is clinically feasible, safe, and more precise than conventional planar methodology for 90Y radioembolization radiation planning.
PMCID: PMC4086353  PMID: 25024931
Yttrium-90 radioembolization; Selective internal radiation therapy; Partition model; Technetium-99m macroaggregated albumin SPECT/CT; CT lung densitovolumetry; Lung mass
6.  Perinephric Urinoma Due to Locally Advanced Recto-Sigmoid Tumor: Incidental Finding on Bone Scan 
World Journal of Nuclear Medicine  2014;13(2):132-134.
We are presenting a 59 years old man with locally advanced recto-sigmoid colon cancer; complaining of left flank pain and bone scan was done to rule out bone metastases. Findings in bone scan were suggesting left renal obstruction, but further SPECT/CT imaging to localize the uptake revealed a perinephric collection likely due to leak from the left ureter close to tumor site.
PMCID: PMC4150156  PMID: 25191129
Bone scan; single photon emission tomography/computed tomography; urinoma
7.  Non-Target Activity Detection by Post-Radioembolization Yttrium-90 PET/CT: Image Assessment Technique and Case Examples 
High resolution yttrium-90 (90Y) imaging of post-radioembolization microsphere biodistribution may be achieved by conventional positron emission tomography with integrated computed tomography (PET/CT) scanners that have time-of-flight capability. However, reconstructed 90Y PET/CT images have high background noise, making non-target activity detection technically challenging. This educational article describes our image assessment technique for non-target activity detection by 90Y PET/CT, which qualitatively overcomes the problem of background noise. We present selected case examples of non-target activity in untargeted liver, stomach, gallbladder, chest wall, and kidney, supported by angiography and 90Y bremsstrahlung single-photon emission computed tomography with integrated computed tomography (SPECT/CT) or technetium-99m macroaggregated albumin SPECT/CT.
PMCID: PMC3910080  PMID: 24551594
yttrium-90 PET/CT; bremsstrahlung SPECT/CT; non-target activity; yttrium-90 radioembolization; selective internal radiation therapy; diagnostic reporting technique; yttrium-90 resin microspheres; SIR-spheres
8.  Essential Role of the m2R-RGS6-IKACh Pathway in Controlling Intrinsic Heart Rate Variability 
PLoS ONE  2013;8(10):e76973.
Normal heart function requires generation of a regular rhythm by sinoatrial pacemaker cells and the alteration of this spontaneous heart rate by the autonomic input to match physiological demand. However, the molecular mechanisms that ensure consistent periodicity of cardiac contractions and fine tuning of this process by autonomic system are not completely understood.
Here we examined the contribution of the m2R-IKACh intracellular signaling pathway, which mediates the negative chronotropic effect of parasympathetic stimulation, to the regulation of the cardiac pacemaking rhythm. Using isolated heart preparations and single-cell recordings we show that the m2R-IKACh signaling pathway controls the excitability and firing pattern of the sinoatrial cardiomyocytes and determines variability of cardiac rhythm in a manner independent from the autonomic input. Ablation of the major regulator of this pathway, Rgs6, in mice results in irregular cardiac rhythmicity and increases susceptibility to atrial fibrillation. We further identify several human subjects with variants in the RGS6 gene and show that the loss of function in RGS6 correlates with increased heart rate variability. These findings identify the essential role of the m2R-IKACh signaling pathway in the regulation of cardiac sinus rhythm and implicate RGS6 in arrhythmia pathogenesis.
PMCID: PMC3812209  PMID: 24204714
9.  Post-radioembolization yttrium-90 PET/CT - part 1: diagnostic reporting 
EJNMMI Research  2013;3:56.
Yttrium-90 (90Y) positron emission tomography with integrated computed tomography (PET/CT) represents a technological leap from 90Y bremsstrahlung single-photon emission computed tomography with integrated computed tomography (SPECT/CT) by coincidence imaging of low abundance internal pair production. Encouraged by favorable early experiences, we implemented post-radioembolization 90Y PET/CT as an adjunct to 90Y bremsstrahlung SPECT/CT in diagnostic reporting.
This is a retrospective review of all paired 90Y PET/CT and 90Y bremsstrahlung SPECT/CT scans over a 1-year period. We compared image resolution, ability to confirm technical success, detection of non-target activity, and providing conclusive information about 90Y activity within targeted tumor vascular thrombosis. 90Y resin microspheres were used. 90Y PET/CT was performed on a conventional time-of-flight lutetium-yttrium-oxyorthosilicate scanner with minor modifications to acquisition and reconstruction parameters. Specific findings on 90Y PET/CT were corroborated by 90Y bremsstrahlung SPECT/CT, 99mTc macroaggregated albumin SPECT/CT, follow-up diagnostic imaging or review of clinical records.
Diagnostic reporting recommendations were developed from our collective experience across 44 paired scans. Emphasis on the continuity of care improved overall diagnostic accuracy and reporting confidence of the operator. With proper technique, the presence of background noise did not pose a problem for diagnostic reporting. A counter-intuitive but effective technique of detecting non-target activity is proposed, based on the pattern of activity and its relation to underlying anatomy, instead of its visual intensity. In a sub-analysis of 23 patients with a median follow-up of 5.4 months, 90Y PET/CT consistently outperformed 90Y bremsstrahlung SPECT/CT in all aspects of qualitative analysis, including assessment for non-target activity and tumor vascular thrombosis. Parts of viscera closely adjacent to the liver remain challenging for non-target activity detection, compounded by a tendency for mis-registration.
Adherence to proper diagnostic reporting technique and emphasis on continuity of care are vital to the clinical utility of post-radioembolization 90Y PET/CT. 90Y PET/CT is superior to 90Y bremsstrahlung SPECT/CT for the assessment of target and non-target activity.
PMCID: PMC3726297  PMID: 23883566
Yttrium-90 radioembolization; Selective internal radiation therapy; Yttrium-90 PET/CT; Bremsstrahlung SPECT/CT; Diagnostic reporting; Non-target activity
10.  Post-radioembolization yttrium-90 PET/CT - part 2: dose-response and tumor predictive dosimetry for resin microspheres 
EJNMMI Research  2013;3:57.
Coincidence imaging of low-abundance yttrium-90 (90Y) internal pair production by positron emission tomography with integrated computed tomography (PET/CT) achieves high-resolution imaging of post-radioembolization microsphere biodistribution. Part 2 analyzes tumor and non-target tissue dose-response by 90Y PET quantification and evaluates the accuracy of tumor 99mTc macroaggregated albumin (MAA) single-photon emission computed tomography with integrated CT (SPECT/CT) predictive dosimetry.
Retrospective dose quantification of 90Y resin microspheres was performed on the same 23-patient data set in part 1. Phantom studies were performed to assure quantitative accuracy of our time-of-flight lutetium-yttrium-oxyorthosilicate system. Dose-responses were analyzed using 90Y dose-volume histograms (DVHs) by PET voxel dosimetry or mean absorbed doses by Medical Internal Radiation Dose macrodosimetry, correlated to follow-up imaging or clinical findings. Intended tumor mean doses by predictive dosimetry were compared to doses by 90Y PET.
Phantom studies demonstrated near-perfect detector linearity and high tumor quantitative accuracy. For hepatocellular carcinomas, complete responses were generally achieved at D70 > 100 Gy (D70, minimum dose to 70% tumor volume), whereas incomplete responses were generally at D70 < 100 Gy; smaller tumors (<80 cm3) achieved D70 > 100 Gy more easily than larger tumors. There was complete response in a cholangiocarcinoma at D70 90 Gy and partial response in an adrenal gastrointestinal stromal tumor metastasis at D70 53 Gy. In two patients, a mean dose of 18 Gy to the stomach was asymptomatic, 49 Gy caused gastritis, 65 Gy caused ulceration, and 53 Gy caused duodenitis. In one patient, a bilateral kidney mean dose of 9 Gy (V20 8%) did not cause clinically relevant nephrotoxicity. Under near-ideal dosimetric conditions, there was excellent correlation between intended tumor mean doses by predictive dosimetry and those by 90Y PET, with a low median relative error of +3.8% (95% confidence interval, -1.2% to +13.2%).
Tumor and non-target tissue absorbed dose quantification by 90Y PET is accurate and yields radiobiologically meaningful dose-response information to guide adjuvant or mitigative action. Tumor 99mTc MAA SPECT/CT predictive dosimetry is feasible. 90Y DVHs may guide future techniques in predictive dosimetry.
PMCID: PMC3733999  PMID: 23885971
Yttrium-90 radioembolization; Selective internal radiation therapy; PET/CT; Voxel dosimetry; Dose-volume histogram; Predictive dosimetry
11.  Anaplastic supratentorial cortical ependymoma presenting as a butterfly lesion 
Anaplastic cortical ependymomas are rare lesions with few cases reported in the literature.
Case Description:
We present a unique case of an anaplastic cortical ependymoma in a 51-year-old female presenting as a butterfly lesion with involvement of both frontal lobes. The patient underwent gross total resection of her tumor with further adjuvant treatment. We present the findings in our case and review the literature surrounding supratentorial ependymomas and their treatment outcomes.
Rarely, cortical ependymoma can present as a butterfly lesion and should be considered in the differential diagnosis of such lesions in adults.
PMCID: PMC3475880  PMID: 23087823
Adult; Anaplastic; Cortical; Ependymoma; Supratentorial
12.  The Density Factor: The Enigma of Dense Breast 
PMCID: PMC3555393  PMID: 23372436
13.  A Mosaic Activating Mutation in AKT1 Associated with the Proteus Syndrome 
The New England journal of medicine  2011;365(7):611-619.
The Proteus syndrome is characterized by the overgrowth of skin, connective tissue, brain, and other tissues. It has been hypothesized that the syndrome is caused by somatic mosaicism for a mutation that is lethal in the nonmosaic state.
We performed exome sequencing of DNA from biopsy samples obtained from patients with the Proteus syndrome and compared the resultant DNA sequences with those of unaffected tissues obtained from the same patients. We confirmed and extended an observed association, using a custom restriction-enzyme assay to analyze the DNA in 158 samples from 29 patients with the Proteus syndrome. We then assayed activation of the AKT protein in affected tissues, using phosphorylation-specific antibodies on Western blots.
Of 29 patients with the Proteus syndrome, 26 had a somatic activating mutation (c.49G→A, p.Glu17Lys) in the oncogene AKT1, encoding the AKT1 kinase, an enzyme known to mediate processes such as cell proliferation and apoptosis. Tissues and cell lines from patients with the Proteus syndrome harbored admixtures of mutant alleles that ranged from 1% to approximately 50%. Mutant cell lines showed greater AKT phosphorylation than did control cell lines. A pair of single-cell clones that were established from the same starting culture and differed with respect to their mutation status had different levels of AKT phosphorylation.
The Proteus syndrome is caused by a somatic activating mutation in AKT1, proving the hypothesis of somatic mosaicism and implicating activation of the PI3K–AKT pathway in the characteristic clinical findings of overgrowth and tumor susceptibility in this disorder. (Funded by the Intramural Research Program of the National Human Genome Research Institute.)
PMCID: PMC3170413  PMID: 21793738
14.  Schizophrenia susceptibility pathway neuregulin 1–ErbB4 suppresses Src upregulation of NMDA receptors 
Nature Medicine  2011;17(4):470-478.
Hypofunction of the N-methyl D-aspartate subtype of glutamate receptor (NMDAR) is hypothesized to be a mechanism underlying cognitive dysfunction in individuals with schizophrenia. For the schizophrenia-linked genes NRG1 and ERBB4, NMDAR hypofunction is thus considered a key detrimental consequence of the excessive NRG1-ErbB4 signaling found in people with schizophrenia. However, we show here that neuregulin 1β–ErbB4 (NRG1β-ErbB4) signaling does not cause general hypofunction of NMDARs. Rather, we find that, in the hippocampus and prefrontal cortex, NRG1β-ErbB4 signaling suppresses the enhancement of synaptic NMDAR currents by the nonreceptor tyrosine kinase Src. NRG1β-ErbB4 signaling prevented induction of long-term potentiation at hippocampal Schaffer collateral–CA1 synapses and suppressed Src-dependent enhancement of NMDAR responses during theta-burst stimulation. Moreover, NRG1β-ErbB4 signaling prevented theta burst–induced phosphorylation of GluN2B by inhibiting Src kinase activity. We propose that NRG1-ErbB4 signaling participates in cognitive dysfunction in schizophrenia by aberrantly suppressing Src-mediated enhancement of synaptic NMDAR function.
PMCID: PMC3264662  PMID: 21441918 CAMSID: cams1842
15.  “The CUB domain protein Neto1 is an auxiliary protein of native synaptic kainate receptors” 
Ionotropic glutamate receptors of AMPA, NMDA and kainate receptor (KAR) subtypes mediate fast excitatory synaptic transmission in the vertebrate CNS. Auxiliary proteins have been identified for AMPA and NMDA receptor complexes, but little is known about KAR complex proteins. We previously identified the CUB-domain protein, Neto1, as an NMDA receptor-associated polypeptide. Here, we show that Neto1 is also an auxiliary subunit for endogenous synaptic KARs. We found that Neto1 and KARs co-immunoprecipitated from brain lysates, from post-synaptic densities (PSDs) and, in a manner dependent on Neto1 CUB domains, when co-expressed in heterologous cells. In Neto1-null mice, there was an ~50% reduction in the abundance of GluK2-KARs in hippocampal PSDs. Neto1 strongly localized to CA3 stratum lucidum and loss of Neto1 resulted in a selective deficit in KAR-mediated neurotransmission at mossy fiber-CA3 pyramidal cell synapses (MF-CA3): KAR-mediated EPSCs in Neto1-null mice were reduced in amplitude and decayed more rapidly than did those in wild-type mice. In contrast, the loss of Neto2, which also localizes to stratum lucidum and interacts with KARs, had no effect on KAR synaptic abundance or MF-CA3 transmission. Indeed MF-CA3 KAR deficits in Neto1/2 double null mutant mice were indistinguishable from Neto1 single null mice. Thus, our findings establish Neto1 as an auxiliary protein required for synaptic function of KARs. The ability of Neto1 to regulate both NMDARs and KARs reveals a unique dual role in controlling synaptic transmission by serving as an auxiliary protein for these two classes of ionotropic glutamate receptors in a synapse specific fashion.
PMCID: PMC3148853  PMID: 21734292
16.  Correlation of rare coding variants in the gene encoding human glucokinase regulatory protein with phenotypic, cellular, and kinetic outcomes 
Defining the genetic contribution of rare variants to common diseases is a major basic and clinical science challenge that could offer new insights into disease etiology and provide potential for directed gene- and pathway-based prevention and treatment. Common and rare nonsynonymous variants in the GCKR gene are associated with alterations in metabolic traits, most notably serum triglyceride levels. GCKR encodes glucokinase regulatory protein (GKRP), a predominantly nuclear protein that inhibits hepatic glucokinase (GCK) and plays a critical role in glucose homeostasis. The mode of action of rare GCKR variants remains unexplored. We identified 19 nonsynonymous GCKR variants among 800 individuals from the ClinSeq medical sequencing project. Excluding the previously described common missense variant p.Pro446Leu, all variants were rare in the cohort. Accordingly, we functionally characterized all variants to evaluate their potential phenotypic effects. Defects were observed for the majority of the rare variants after assessment of cellular localization, ability to interact with GCK, and kinetic activity of the encoded proteins. Comparing the individuals with functional rare variants to those without such variants showed associations with lipid phenotypes. Our findings suggest that, while nonsynonymous GCKR variants, excluding p.Pro446Leu, are rare in individuals of mixed European descent, the majority do affect protein function. In sum, this study utilizes computational, cell biological, and biochemical methods to present a model for interpreting the clinical significance of rare genetic variants in common disease.
PMCID: PMC3248284  PMID: 22182842
17.  Femtosecond cataract surgery: A review of current literature and the experience from an initial installation 
Saudi Journal of Ophthalmology  2011;26(1):73-78.
Cataract surgery remains the most widely performed intraocular procedure throughout the world. Safety and accuracy of the procedure are paramount and techniques should remain under constant review. Recently, the introduction of the femtosecond laser to assist cataract surgery has provided ophthalmologists with an exciting tool that may further improve outcomes. We review the existing literature and discuss the installation and initial experience of a femtosecond laser into our practice.
PMCID: PMC3729300  PMID: 23960972
Femtosecond laser assisted surgery; Cataract
18.  Identification of a novel LDLR mutation (c.261_262invGA, p.Trp87X): Importance of specifying DNA and protein mutations 
Atherosclerosis  2010;211(2):397-398.
ClinSeq is a large-scale medical sequencing (LSMS) project at the National Institutes of Health (NIH), the goal of which is to pilot the feasibility of using high throughput genome sequencing for clinical research and eventually to improve the delivery of healthcare. In phase one, 1000 participants are being clinically evaluated for cardiovascular phenotypes and DNA is being collected for sequencing of 400 candidate genes to identify genetic variants that may predispose to the early development of atherosclerosis. We report on an individual with familial hypercholesterolemia (OMIM #143890) who has a novel mutation, c.261_262invGA that predicts a premature stop (p.Trp87X) in the LDLR gene. Although the p.Trp87X predicted protein mutation has been reported, c.261_262invGA is distinct from mutations reported in prior families and emphasizes the importance of describing mutations at the DNA level. It is important to describe mutations according to the underlying DNA change as multiple nucleotide changes may underlie a single predicted protein change.
PMCID: PMC2914107  PMID: 20452591
19.  Dosimetric Considerations in Radioimmunotherapy and Systemic Radionuclide Therapies: A Review 
World Journal of Nuclear Medicine  2011;10(2):122-138.
Radiopharmaceutical therapy, once touted as the “magic bullet” in radiation oncology, is increasingly being used in the treatment of a variety of malignancies; albeit in later disease stages. With ever-increasing public and medical awareness of radiation effects, radiation dosimetry is becoming more important. Dosimetry allows administration of the maximum tolerated radiation dose to the tumor/organ to be treated but limiting radiation to critical organs. Traditional tumor dosimetry involved acquiring pretherapy planar scans and plasma estimates with a diagnostic dose of intended radiopharmaceuticals. New advancements in single photon emission computed tomography and positron emission tomography systems allow semi-quantitative measurements of radiation dosimetry thus allowing treatments tailored to each individual patient.
PMCID: PMC3227338  PMID: 22144871
Radionuclide therapy; radioimmunotherapy; quality of life
20.  Molecular Characterization of Putative Chordoma Cell Lines 
Sarcoma  2010;2010:630129.
Immortal tumor cell lines are an important model system for cancer research, however, misidentification and cross-contamination of cell lines are a common problem. Seven chordoma cell lines are reported in the literature, but none has been characterized in detail. We analyzed gene expression patterns and genomic copy number variations in five putative chordoma cell lines (U-CH1, CCL3, CCL4, GB60, and CM319). We also created a new chordoma cell line, U-CH2, and provided genotypes for cell lines for identity confirmation. Our analyses revealed that CCL3, CCL4, and GB60 are not chordoma cell lines, and that CM319 is a cancer cell line possibly derived from chordoma, but lacking expression of key chordoma biomarkers. U-CH1 and U-CH2 both have gene expression profiles, copy number aberrations, and morphology consistent with chordoma tumors. These cell lines also harbor genetic changes, such as loss of p16, MTAP, or PTEN, that make them potentially useful models for studying mechanisms of chordoma pathogenesis and for evaluating targeted therapies.
PMCID: PMC3022207  PMID: 21253487
21.  Genomic characterization of esophageal squamous cell carcinoma from a high-risk population in China 
Cancer research  2009;69(14):5908-5917.
Genomic instability plays an important role in most human cancers. To characterize genomic instability in esophageal squamous cell carcinoma (ESCC), we examined loss of heterozygosity (LOH), copy number (CN) loss, CN gain, and gene expression using the Affymetrix GeneChip Human Mapping 500K (n=30 cases) and Human U133A (n=17 cases) arrays in ESCC cases from a high-risk region of China. We found that genomic instability measures varied widely among cases and separated them into two groups: a high-frequency instability group (two-thirds of all cases with one or more instability category ≥ 10%) and a low-frequency instability group (one-third of cases with instability < 10%). Genomic instability also varied widely across chromosomal arms, with the highest frequency of LOH on 9p (33% of informative single nucleotide polymorphisms (SNPs)), CN loss on 3p (33%), and CN gain on 3q (48%). Twenty-two LOH regions were identified: four on 9p, seven on 9q, four on 13q, two on 17p, and five on 17q. Three CN loss regions – 3p12.3, 4p15.1, and 9p21.3 – were detected. Twelve CN gain regions were found, including six on 3q, one on 7q, four on 8q, and one on 11q. One of the most gene-rich of these CN gain regions was 11q13.1-13.4, where 26 genes also had RNA expression data available. CN gain was significantly correlated with increased RNA expression in over 80% of these genes. Our findings demonstrate the potential utility of combining CN analysis and gene expression data to identify genes involved in esophageal carcinogenesis.
PMCID: PMC2734334  PMID: 19584285
esophageal cancer; LOH; copy number alteration; 500K SNP array
22.  T (Brachyury) gene duplication confers major susceptibility to familial chordoma 
Nature genetics  2009;41(11):1176-1178.
Using high-resolution array-CGH, we identified unique duplications of a region on 6q27 in four multiplex families with ≥ 3 cases of chordoma, a cancer of presumed notochordal origin. The duplicated region contains only the T gene (Brachyury), which plays an important role in notochord development and is expressed in most sporadic chordomas. Our findings highlight the need to include screening for complex genomic rearrangements in searches for cancer susceptibility genes.
PMCID: PMC2901855  PMID: 19801981
23.  Common Genetic Variants in Candidate Genes and Risk of Familial Lymphoid Malignancies 
British journal of haematology  2009;146(4):418-423.
Familial aggregation, linkage, and case-control studies support the role of germline genes in the etiology of lymphoid malignancies. To further examine the role of genetic variation underlying susceptibility, we analyzed 1536 SNPs in 152 genes involved in apoptosis, DNA repair, immune response, and oxidative stress pathways among a unique sample of 165 unrelated familial cases including patients with chronic lymphocytic leukemia (CLL), Waldenström's (WM), and Hodgkin lymphoma (HL), and 107 spouse controls. We confirmed previous studies showing a polymorphism in the IL10 promoter (rs1800890/-3575T>A) to be associated with non-Hodgkin lymphoma since we found this allele to be associated with both CLL and WM. We also confirmed the role of IL6 variation to be associated with HL. Polymorphisms in the TRAIL gene were associated with both CLL and WM. Future replication and functional studies are needed to clarify the role of these genetic variants. Finally, our data further support the close association of WM and CLL.
PMCID: PMC2890251  PMID: 19573080
Candidate genes; association; familial; lymphoid malignancies; chronic lymphocytic leukemia; Waldenström's macroglobulinemia; Hodgkin lymphoma
24.  Putative tumour-suppressor gene DAB2 is frequently down regulated by promoter hypermethylation in nasopharyngeal carcinoma 
BMC Cancer  2010;10:253.
Human Disabled-2 (DAB2), is a multi-function signalling molecule that it is frequently down-regulated in human cancers. We aimed to investigate the possible tumour suppressor effect of DAB2 in nasopharyngeal carcinoma (NPC).
We studied the expression of DAB2 in NPC cell lines, xenografts and primary tumour samples. The status of promoter methylation was assessed by methylation specific PCR and bisulfite sequencing. The functional role of DAB2 in NPC was investigated by re-introducing DAB2 expression into NPC cell line C666-1.
Decrease or absent of DAB2 transcript was observed in NPC cell lines and xenografts. Loss of DAB2 protein expression was seen in 72% (33/46) of primary NPC as demonstrated by immunohistochemistry. Aberrant DAB2 promoter methylation was detected in 65.2% (30/46) of primary NPC samples by methylation specific PCR. Treatment of the DAB2 negative NPC cell line C666-1 with 5-aza-2'-deoxycytidine resulted in restoration of DAB2 expression in a dose-dependent manner. Overexpression of DAB2 in NPC cell line C666-1 resulted in reduced growth rate and 35% reduction in anchorage-dependent colony formation, and inhibition of serum-induced c-Fos expression compared to vector-transfected controls. Over expression of DAB2 resulted in alterations of multiple pathways as demonstrated by expression profiling and functional network analysis, which confirmed the role of DAB2 as an adaptor molecule involved in multiple receptor-mediated signalling pathways.
We report the frequent down regulation of DAB2 in NPC and the promoter hypermethylation contributes to the loss of expression of DAB2. This is the first study demonstrating frequent DAB2 promoter hypermethylation in human cancer. Our functional studies support the putative tumour suppressor effect of DAB2 in NPC cells.
PMCID: PMC2891638  PMID: 20525238
25.  Association of a de novo 16q copy number variant with a phenotype that overlaps with Lenz microphthalmia and Townes-Brocks syndromes 
BMC Medical Genetics  2009;10:137.
Anophthalmia and microphthalmia are etiologically and clinically heterogeneous. Lenz microphthalmia is a syndromic form that is typically inherited in an X-linked pattern, though the causative gene mutation is unknown. Townes-Brocks syndrome manifests thumb anomalies, imperforate anus, and ear anomalies. We present a 13-year-old boy with a syndromic microphthalmia phenotype and a clinical diagnosis of Lenz microphthalmia syndrome.
Case Presentation
The patient was subjected to clinical and molecular evaluation, including array CGH analysis. The clinical features included left clinical anophthalmia, right microphthalmia, anteriorly placed anus with fistula, chordee, ventriculoseptal defect, patent ductus arteriosus, posteriorly rotated ears, hypotonia, growth retardation with delayed bone age, and mental retardation. The patient was found to have an approximately 5.6 Mb deletion of 16q11.2q12.1 by microarray based-comparative genomic hybridization, which includes the SALL1 gene, which causes Townes-Brocks syndrome.
Deletions of 16q11.2q12.2 have been reported in several individuals, although those prior reports did not note microphthalmia or anophthalmia. This region includes SALL1, which causes Townes-Brocks syndrome. In retrospect, this child has a number of features that can be explained by the SALL1 deletion, although it is not clear if the microphthalmia is a rare feature of Townes-Brocks syndrome or caused by other mechanisms. These data suggest that rare copy number changes may be a cause of syndromic microphthalmia allowing a personalized genomic medicine approach to the care of patients with these aberrations.
PMCID: PMC2806267  PMID: 20003547

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