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1.  Protein Kinase Cδ Deficiency Causes Mendelian Systemic Lupus Erythematosus With B Cell–Defective Apoptosis and Hyperproliferation 
Arthritis and rheumatism  2013;65(8):2161-2171.
Objective
Systemic lupus erythematosus (SLE) is a prototype autoimmune disease that is assumed to occur via a complex interplay of environmental and genetic factors. Rare causes of monogenic SLE have been described, providing unique insights into fundamental mechanisms of immune tolerance. The aim of this study was to identify the cause of an autosomal-recessive form of SLE.
Methods
We studied 3 siblings with juvenile-onset SLE from 1 consanguineous kindred and used next-generation sequencing to identify mutations in the disease-associated gene. We performed extensive biochemical, immunologic, and functional assays to assess the impact of the identified mutations on B cell biology.
Results
We identified a homozygous missense mutation in PRKCD, encoding protein kinase δ (PKCδ), in all 3 affected siblings. Mutation of PRKCD resulted in reduced expression and activity of the encoded protein PKCδ (involved in the deletion of autoreactive B cells), leading to resistance to B cell receptor– and calcium-dependent apoptosis and increased B cell proliferation. Thus, as for mice deficient in PKCδ, which exhibit an SLE phenotype and B cell expansion, we observed an increased number of immature B cells in the affected family members and a developmental shift toward naive B cells with an immature phenotype.
Conclusion
Our findings indicate that PKCδ is crucial in regulating B cell tolerance and preventing self-reactivity in humans, and that PKCδ deficiency represents a novel genetic defect of apoptosis leading to SLE.
doi:10.1002/art.38008
PMCID: PMC4066615  PMID: 23666743
2.  Dubowitz Syndrome Is a Complex Comprised of Multiple, Genetically Distinct and Phenotypically Overlapping Disorders 
PLoS ONE  2014;9(6):e98686.
Dubowitz syndrome is a rare disorder characterized by multiple congenital anomalies, cognitive delay, growth failure, an immune defect, and an increased risk of blood dyscrasia and malignancy. There is considerable phenotypic variability, suggesting genetic heterogeneity. We clinically characterized and performed exome sequencing and high-density array SNP genotyping on three individuals with Dubowitz syndrome, including a pair of previously-described siblings (Patients 1 and 2, brother and sister) and an unpublished patient (Patient 3). Given the siblings' history of bone marrow abnormalities, we also evaluated telomere length and performed radiosensitivity assays. In the siblings, exome sequencing identified compound heterozygosity for a known rare nonsense substitution in the nuclear ligase gene LIG4 (rs104894419, NM_002312.3:c.2440C>T) that predicts p.Arg814X (MAF:0.0002) and an NM_002312.3:c.613delT variant that predicts a p.Ser205Leufs*29 frameshift. The frameshift mutation has not been reported in 1000 Genomes, ESP, or ClinSeq. These LIG4 mutations were previously reported in the sibling sister; her brother had not been previously tested. Western blotting showed an absence of a ligase IV band in both siblings. In the third patient, array SNP genotyping revealed a de novo ∼3.89 Mb interstitial deletion at chromosome 17q24.2 (chr 17:62,068,463–65,963,102, hg18), which spanned the known Carney complex gene PRKAR1A. In all three patients, a median lymphocyte telomere length of ≤1st centile was observed and radiosensitivity assays showed increased sensitivity to ionizing radiation. Our work suggests that, in addition to dyskeratosis congenita, LIG4 and 17q24.2 syndromes also feature shortened telomeres; to confirm this, telomere length testing should be considered in both disorders. Taken together, our work and other reports on Dubowitz syndrome, as currently recognized, suggest that it is not a unitary entity but instead a collection of phenotypically similar disorders. As a clinical entity, Dubowitz syndrome will need continual re-evaluation and re-definition as its constituent phenotypes are determined.
doi:10.1371/journal.pone.0098686
PMCID: PMC4043752  PMID: 24892279
3.  SHORT COMMUNICATION: Hyperphagia among patients with Bardet-Biedl syndrome 
Pediatric obesity  2013;8(5):e64-e67.
Summary
Background
The importance of hyperphagia as a cause for energy imbalance in humans with Bardet-Biedl syndrome (BBS) has not been established. We therefore compared hyperphagic symptoms in patients with BBS versus controls.
Methods
We studied 13 patients with BBS and 23 nonsyndromic controls with similar age, sex, and BMI z-score. A 13-item hyperphagia questionnaire was completed by patients’ parents/guardians.
Results
Total hyperphagia questionnaire score was higher in BBS than controls (27.6±9.0 vs. 19.1±7.9, p=0.005). Behavior and drive sub-scales were higher for BBS than controls (12.5±4.1 vs. 7.8±3.2, p=0.001, and 11.2±4.1 vs. 8.3±3.8, p=0.04, respectively); severity was not significantly different between groups (3.8±1.5 vs. 3.0±1.3, p=0.072). After adjustment for demographic variables and BMI-Z score, total and behavior subscale scores remained significantly different between groups, suggesting food-seeking activity, rather than preoccupation with food may be the main hyperphagic feature among patients with BBS.
Conclusion
Appetite dysregulation may contribute to obesity in BBS.
doi:10.1111/j.2047-6310.2013.00182.x
PMCID: PMC3901011  PMID: 23776152
hyperphagia; obesity; Bardet-Biedl syndrome; obesity syndromes; polyphagia; energy intake; questionnaire; child; adolescent
5.  Identification of candidate genes involved in coronary artery calcification by transcriptome sequencing of cell lines 
BMC Genomics  2014;15:198.
Background
Massively-parallel cDNA sequencing (RNA-Seq) is a new technique that holds great promise for cardiovascular genomics. Here, we used RNA-Seq to study the transcriptomes of matched coronary artery disease cases and controls in the ClinSeq® study, using cell lines as tissue surrogates.
Results
Lymphoblastoid cell lines (LCLs) from 16 cases and controls representing phenotypic extremes for coronary calcification were cultured and analyzed using RNA-Seq. All cell lines were then independently re-cultured and along with another set of 16 independent cases and controls, were profiled with Affymetrix microarrays to perform a technical validation of the RNA-Seq results. Statistically significant changes (p < 0.05) were detected in 186 transcripts, many of which are expressed at extremely low levels (5–10 copies/cell), which we confirmed through a separate spike-in control RNA-Seq experiment. Next, by fitting a linear model to exon-level RNA-Seq read counts, we detected signals of alternative splicing in 18 transcripts. Finally, we used the RNA-Seq data to identify differential expression (p < 0.0001) in eight previously unannotated regions that may represent novel transcripts. Overall, differentially expressed genes showed strong enrichment (p = 0.0002) for prior association with cardiovascular disease. At the network level, we found evidence for perturbation in pathways involving both cardiovascular system development and function as well as lipid metabolism.
Conclusions
We present a pilot study for transcriptome involvement in coronary artery calcification and demonstrate how RNA-Seq analyses using LCLs as a tissue surrogate may yield fruitful results in a clinical sequencing project. In addition to canonical gene expression, we present candidate variants from alternative splicing and novel transcript detection, which have been unexplored in the context of this disease.
doi:10.1186/1471-2164-15-198
PMCID: PMC4003819  PMID: 24628908
Coronary artery calcification; RNA-Seq; Lymphoblastoid cell lines; Transcriptome profiling
6.  Intentions to receive individual results from whole-genome sequencing among participants in the ClinSeq study 
Genome sequencing has been rapidly integrated into clinical research and is currently marketed to health-care practitioners and consumers alike. The volume of sequencing data generated for a single individual and the wide range of findings from whole-genome sequencing raise critical questions about the return of results and their potential value for end-users. We conducted a mixed-methods study of 311 sequential participants in the NIH ClinSeq study to assess general preferences and specific attitudes toward learning results. We tested how these variables predicted intentions to receive results within four categories of findings ranging from medically actionable to variants of unknown significance. Two hundred and ninety-four participants indicated a preference to learn their genome sequencing results. Most often, participants cited disease prevention as their reason, including intention to change their lifestyle behaviors. Participants held positive attitudes, strongly perceived social norms and strong intentions to learn results, although there were significant mean differences among four categories of findings (P<0.01). Attitudes and social norms for medically actionable and carrier results were most similar and rated the highest. Participants distinguished among the types and quality of information they may receive, despite strong intentions to learn all results presented. These intentions were motivated by confidence in their ability to use the information to prevent future disease and a belief in the value of even uninterpretable information. It behooves investigators to facilitate participants' desire to learn a range of information from genomic sequencing while promoting realistic expectations for its clinical and personal utility.
doi:10.1038/ejhg.2012.179
PMCID: PMC3573208  PMID: 22892536
whole-genome sequencing; individual results; attitudes and intentions
7.  Current and Emerging Technology Approaches in Genomics 
Purpose
To introduce current and emerging approaches that are being utilized in the field of genomics so the reader can conceptually evaluate the literature and appreciate how these approaches are advancing our understanding of health-related issues.
Organizing Construct
Each approach is described and includes information related to how it is advancing research, its potential clinical utility, exemplars of current uses, challenges related to technologies used for these approaches, and when appropriate information related to understanding the evidence base for clinical utilization of each approach is provided. Web-based resources are included for the reader who would like more in-depth information and to provide opportunity to stay up to date with these approaches and their utility.
Conclusions
The chosen approaches– genome sequencing, genome-wide association studies, epigenomics, and gene expression– are extremely valuable approaches for collecting research data to help us better understand the pathophysiology of a variety of health-related conditions, but they are also gaining in utility for clinical assessment and testing purposes.
Clinical Relevance
Our increased understanding of the molecular underpinnings of disease will assist with better development of screening tests, diagnostic tests, tests that allow us to prognosticate, tests that allow for individualized treatments, and tests to facilitate post-treatment surveillance.
doi:10.1111/jnu.12001
PMCID: PMC3773704  PMID: 23294727
Genetics; genomics; next generation sequencing; genome-wide association studies; epigenomics; gene expression; nursing
10.  A Loss-of-Function Variant in the Human Histidyl-tRNA Synthetase (HARS) Gene is Neurotoxic In Vivo 
Human mutation  2012;34(1):191-199.
Aminoacyl-tRNA synthetases (ARSs) are ubiquitously expressed enzymes responsible for ligating amino acids to cognate tRNA molecules. Mutations in four genes encoding an ARS have been implicated in inherited peripheral neuropathy with an axonal pathology, suggesting that all ARS genes are relevant candidates for disease in patients with related phenotypes. Here, we present results from a mutation screen of the histidyl-tRNA synthetase (HARS) gene in a large cohort of patients with peripheral neuropathy. These efforts revealed a rare missense variant (p.Arg137Gln) that resides at a highly conserved amino acid, represents a loss-of-function allele when evaluated in yeast complementation assays, and is toxic to neurons when expressed in a worm model. In addition to the patient with peripheral neuropathy, p.Arg137Gln HARS was detected in three individuals by genome-wide exome sequencing. These findings suggest that HARS is the fifth ARS locus associated with axonal peripheral neuropathy. Implications for identifying ARS alleles in human populations and assessing them for a role in neurodegenerative phenotypes are discussed.
doi:10.1002/humu.22210
PMCID: PMC3535524  PMID: 22930593
Aminoacyl-tRNA Synthetases; Peripheral Neuropathy; HARS; Neurotoxicity
11.  ACMG Recommendations for Reporting of Incidental Findings in Clinical Exome and Genome Sequencing 
In clinical exome and genome sequencing, there is potential for the recognition and reporting of incidental or secondary findings unrelated to the indication for ordering the sequencing but of medical value for patient care. The American College of Medical Genetics and Genomics (ACMG) recently published a policy statement on clinical sequencing, which emphasized the importance of disclosing the possibility of such results in pretest patient discussions, clinical testing, and reporting of results. The ACMG appointed a Working Group on Incidental Findings in Clinical Exome and Genome Sequencing to make recommendations about responsible management of incidental findings when patients undergo exome or genome sequencing. This Working Group conducted a year-long consensus process, including review by outside experts, and produced recommendations that have been approved by the ACMG Board. Specific and detailed recommendations, and the background and rationale for these recommendations, are described herein. We recommend that laboratories performing clinical sequencing seek and report mutations of the specified classes or types in the genes listed here. This evaluation and reporting should be performed for all clinical germline (constitutional) exome and genome sequencing, including the ‘normal’ of tumor-normal subtractive analyses in all subjects, irrespective of age, but excluding fetal samples. We recognize that there are insufficient data on clinical utility to fully support these recommendations and we encourage the creation of an ongoing process for updating these recommendations at least annually as further data are collected.
doi:10.1038/gim.2013.73
PMCID: PMC3727274  PMID: 23788249
secondary findings; incidental findings; genome; genomic medicine; personalized medicine; whole-exome; whole-genome; sequencing
12.  Improving the rigor of mutation reports: Biologic parentage and de novo mutations 
Human mutation  2012;33(11):1501-1502.
The accurate determination and dissemination of the causality or pathogenicity of human DNA sequence variants is a crucial function of genetics journals. Published reports of pathogenic mutations are a common source of information for mutation databases, which are in turn used to make recommendations to patients. One of the strongest pieces of evidence in support of causality or pathogenicity for mutation reports is the occurrence of a de novo mutation. Yet, many publications describing such changes do not demonstrate that the mutation is truly de novo, by performing biologic parentage testing. I argue here that all mutation reports that describe such mutations should include biologic parentage testing, or in the absence of such testing, the mutation should be described as “apparently de novo”. This proposed standard should improve the transparency of the evidence that underlies our literature, and ultimately, improve the databases of mutations in human disease.
doi:10.1002/humu.22131
PMCID: PMC3461126  PMID: 22715147
mutation analysis; mutation databases; de novo mutations; human genetic disease
13.  Effects of informed consent for individual genome sequencing on relevant knowledge 
Clinical genetics  2012;82(5):408-415.
Increasing availability of individual genomic information suggests that patients will need knowledge about genome sequencing to make informed decisions, but prior research is limited. In this study, we examined genome sequencing knowledge before and after informed consent among 311 participants enrolled in the ClinSeq™ sequencing study. An exploratory factor analysis of knowledge items yielded two factors (sequencing limitations knowledge; sequencing benefits knowledge). In multivariable analysis, high pre-consent sequencing limitations knowledge scores were significantly related to education (OR: 8.7, 95% CI: 2.45, 31.10 for postgraduate education and OR: 3.9; 95% CI: 1.05, 14.61 for college degree compared to less than college degree) and race/ethnicity (OR: 2.4, 95% CI: 1.09, 5.38 for non-Hispanic whites compared to other racial/ethnic groups). Mean values increased significantly between pre- and post-consent for the sequencing limitations knowledge subscale (6.9 to 7.7, p<0.0001) and sequencing benefits knowledge subscale (7.0 to 7.5, p<0.0001); increase in knowledge did not differ by sociodemographic characteristics. This study highlights gaps in genome sequencing knowledge, and underscores the need to target educational efforts toward participants with less education or from minority racial/ethnic groups. The informed consent process improved genome sequencing knowledge. Future studies could examine how genome sequencing knowledge influences informed decision making.
doi:10.1111/j.1399-0004.2012.01909.x
PMCID: PMC3469729  PMID: 22694298
genomic knowledge; genome sequencing; informed consent; knowledge
14.  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.
doi:10.1371/journal.pone.0076973
PMCID: PMC3812209  PMID: 24204714
15.  Opportunities and challenges for the integration of massively parallel genomic sequencing into clinical practice: Lessons from the ClinSeq™ project 
The debate surrounding the return of results from high-throughput genomic interrogation encompasses many important issues including ethics, law, economics, and social policy. As well, the debate is also informed by the molecular, genetic, and clinical foundations of the emerging field of clinical genomics, which is based on this new technology. This manuscript outlines the main biomedical considerations of sequencing technologies and demonstrates some of the early clinical experiences with the technology to enable the debate to stay focused on real-world practicalities. These experiences are based on early data from the ClinSeq project, which has generated exome sequence data on 572 subjects. These data are beginning to be interpreted and returned to the subjects, which provides examples of the potential usefulness and pitfalls of clinical genomics. There are numerous genetic results that can be readily derived from a genome including rare, high penetrance traits and carrier states. However, much work needs to be done to develop the tools and resources for genomic interpretation. The main lesson learned is that a genome sequence may be better considered as a health care resource, rather than a test, one that can be interpreted and used over the lifetime of the patient.
doi:10.1038/gim.2011.78
PMCID: PMC3790899  PMID: 22344227
16.  Exploring Concordance and Discordance for Return of Incidental Findings from Clinical Sequencing 
Purpose
To explore specific conditions and types of genetic variants that specialists in genetics recommend should be returned as incidental findings in clinical sequencing.
Methods
Sixteen specialists in clinical genetics and/or molecular medicine selected variants in 99 common conditions to return to the ordering physician if discovered incidentally through whole genome sequencing. For most conditions, the specialists independently considered 3 molecular scenarios for both adults and minor children: a known pathogenic mutation, a truncating variant presumed pathogenic (where other truncating variants were known to be pathogenic), or a missense variant predicted in silico to be pathogenic.
Results
On average, for adults and children respectively, each specialist selected 83.5 and 79.0 conditions or genes out of 99 in the known pathogenic mutation categories, 57.0 and 53.5 out of 72 in the truncating variant categories, and 33.4 and 29.7 out of 72 in the missense variant categories. Concordance in favor of disclosure within the adult/known pathogenic mutation category was 100% for 21 conditions or genes and 80% or higher for 64 conditions or genes.
Conclusion
Specialists were highly concordant for the return of findings in 64 conditions or genes if discovered incidentally during whole exome or whole genome sequencing.
doi:10.1038/gim.2012.21
PMCID: PMC3763716  PMID: 22422049
whole genome sequencing; incidental findings
17.  A candidate gene for autoimmune myasthenia gravis 
Neurology  2012;79(4):342-347.
Objective:
We sought to identify a causative mutation in a previously reported kindred with parental consanguinity and 5 of 10 siblings with adult-onset autoimmune myasthenia gravis.
Methods:
We performed genome-wide homozygosity mapping, and sequenced all known genes in the one region of extended homozygosity. Quantitative and allele-specific reverse transcriptase PCR (RT-PCR) were performed on a candidate gene to determine the RNA expression level in affected siblings and controls and the relative abundance of the wild-type and mutant alleles in a heterozygote.
Results:
A region of shared homozygosity at chromosome 13q13.3–13q14.11 was found in 4 affected siblings and 1 unaffected sibling. A homozygous single nucleotide variant was found in the 3′-untranslated region of the ecto-NADH oxidase 1 gene (ENOX1). No other variants likely to be pathogenic were found in genes in this region or elsewhere. The ENOX1 sequence variant was not found in 764 controls. Quantitative RT-PCR showed that expression of ENOX1 decreased to about 20% of normal levels in lymphoblastoid cells from individuals homozygous for the variant and to about 50% in 2 unaffected heterozygotes. Allele-specific RT-PCR showed a 55%–60% reduction in the level of the variant transcript in heterozygous cells due to reduced mRNA stability.
Conclusion:
These results indicate that this sequence variant in ENOX1 may contribute to the familial autoimmune myasthenia in these patients.
doi:10.1212/WNL.0b013e318260cbd0
PMCID: PMC3400092  PMID: 22744667
18.  Next Generation Sequencing Demands Next Generation Phenotyping 
Human Mutation  2012;33(5):884-886.
Next-Generation Sequencing (NGS) is the most powerful diagnostic tool since the roentgenogram. NGS will facilitate diagnosis on a massive scale –allowing interrogation of all genes in a single assay. It has been suggested that NGS will decrease the need for phenotyping in general, and medical geneticists in particular. We argue that NGS will shift focus and approach of phenotyping. We predict that NGS performed for diagnostic purposes will yield variants in several genes, and consequences of these variants will need to be analyzed and integrated with clinical findings to make a diagnosis. Diagnostic skills of medical specialists will shift from a pre-NGS-test differential diagnostic mode to a post-NGS-test diagnostic assessment mode. In research phenotyping and medical genetic assessments will remain essential as well. NGS can identify primary causative variants in phenotypes inherited in a Mendelian pattern, but biology is much more complex. Phenotypes are caused by the actions of several genes, and epigenetic and environmental influences. Dissecting all influences necessitates ongoing and detailed phenotyping, refinement of clinical diagnostic assignments, and iterative analyses of NGS data. We conclude that there will be a critical need for phenotyping and clinical analysis and that medical geneticists are uniquely positioned to address this need.
doi:10.1002/humu.22048
PMCID: PMC3327792  PMID: 22457028
NGS; whole exome sequencing; whole genome sequencing; phenotype; dysmorphology; Mendelian; monogenic
19.  VarSifter: Visualizing and analyzing exome-scale sequence variation data on a desktop computer 
Bioinformatics  2011;28(4):599-600.
Summary: VarSifter is a graphical software tool for desktop computers that allows investigators of varying computational skills to easily and quickly sort, filter, and sift through sequence variation data. A variety of filters and a custom query framework allow filtering based on any combination of sample and annotation information. By simplifying visualization and analyses of exome-scale sequence variation data, this program will help bring the power and promise of massively-parallel DNA sequencing to a broader group of researchers.
Availability and Implementation: VarSifter is written in Java, and is freely available in source and binary versions, along with a User Guide, at http://research.nhgri.nih.gov/software/VarSifter/.
Contact: mullikin@mail.nih.gov
Supplementary Information: Additional figures and methods available online at the journal's website.
doi:10.1093/bioinformatics/btr711
PMCID: PMC3278764  PMID: 22210868
20.  Mosaic Overgrowth with Fibroadipose Hyperplasia is Caused by Somatic Activating Mutations in PIK3CA 
Nature genetics  2012;44(8):928-933.
The phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway is critical for cellular growth and metabolism. Correspondingly, loss of function of PTEN, a negative regulator of PI3K, or activating mutations in AKT1, AKT2, or AKT3 have been found in distinct disorders featuring overgrowth or hypoglycemia. We performed exome sequencing of DNA from unaffected and affected cells of a patient with an unclassified syndrome of congenital, progressive segmental overgrowth of fibrous and adipose tissue and bone and identified the cancer-associated p.His1047Leu mutation in PIK3CA, which encodes the p110α catalytic subunit of PI3K, only in affected cells. Sequencing of PIK3CA in 10 further patients with overlapping syndromes identified either p.His1047Leu or a second cancer-associated mutation, p.His1047Arg, in 9 cases. Affected dermal fibroblasts showed enhanced basal and EGF-stimulated phosphatidylinositol-3,4,5-trisphosphate (PIP3) generation and concomitant activation of downstream signaling. Our findings characterize a distinct overgrowth syndrome, biochemically demonstrate activation of PI3K signaling and thereby identify a rational therapeutic target.
doi:10.1038/ng.2332
PMCID: PMC3461408  PMID: 22729222
21.  A Recurrent Loss-of-Function Alanyl-tRNA Synthetase (AARS) Mutation in Patients with Charcot-Marie-Tooth Disease Type 2N (CMT2N) 
Human mutation  2011;33(1):244-253.
Charcot-Marie-Tooth (CMT) disease comprises a heterogeneous group of peripheral neuropathies characterized by muscle weakness and wasting, and impaired sensation in the extremities. Four genes encoding an aminoacyl-tRNA synthetase (ARS) have been implicated in CMT disease. ARSs are ubiquitously expressed, essential enzymes that ligate amino acids to cognate tRNA molecules. Recently, a p.Arg329His variant in the alanyl-tRNA synthetase (AARS) gene was found to segregate with dominant axonal CMT type 2N (CMT2N) in two French families; however, the functional consequence of this mutation has not been determined. To investigate the role of AARS in CMT, we performed a mutation screen of the AARS gene in patients with peripheral neuropathy. Our results showed that p.Arg329His AARS also segregated with CMT disease in a large Australian family. Aminoacylation and yeast viability assays showed that p.Arg329His AARS severely reduces enzyme activity. Genotyping analysis indicated that this mutation arose on three distinct haplotypes, and the results of bisulfite sequencing suggested that methylation-mediated deamination of a CpG dinucleotide gives rise to the recurrent p.Arg329His AARS mutation. Together, our data suggest that impaired tRNA charging plays a role in the molecular pathology of CMT2N, and that patients with CMT should be directly tested for the p.Arg329His AARS mutation.
doi:10.1002/humu.21635
PMCID: PMC3240693  PMID: 22009580
AARS; Charcot-Marie-Tooth disease; CMT2N; Peripheral Neuropathy; Axonopathy
22.  Motivators for participation in a whole-genome sequencing study: implications for translational genomics research 
European Journal of Human Genetics  2011;19(12):1213-1217.
The promise of personalized medicine depends on the ability to integrate genetic sequencing information into disease risk assessment for individuals. As genomic sequencing technology enters the realm of clinical care, its scale necessitates answers to key social and behavioral research questions about the complexities of understanding, communicating, and ultimately using sequence information to improve health. Our study captured the motivations and expectations of research participants who consented to participate in a research protocol, ClinSeq, which offers to return a subset of the data generated through high-throughput sequencing. We present findings from an exploratory study of 322 participants, most of whom identified themselves as white, non-Hispanic, and coming from higher socio-economic groups. Participants aged 45–65 years answered open-ended questions about the reasons they consented to ClinSeq and about what they anticipated would come of genomic sequencing. Two main reasons for participating were as follows: a conviction to altruism in promoting research, and a desire to learn more about genetic factors that contribute to one's own health risk. Overall, participants expected genomic research to help improve understanding of disease causes and treatments. Our findings offer a first glimpse into the motivations and expectations of individuals seeking their own genomic information, and provide initial insights into the value these early adopters of technology place on information generated by high-throughput sequencing studies.
doi:10.1038/ejhg.2011.123
PMCID: PMC3230362  PMID: 21731059
whole-genome sequencing; personalized medicine; early adopters
23.  Approaches to informed consent for hypothesis-testing and hypothesis-generating clinical genomics research 
BMC Medical Genomics  2012;5:45.
Background
Massively-parallel sequencing (MPS) technologies create challenges for informed consent of research participants given the enormous scale of the data and the wide range of potential results.
Discussion
We propose that the consent process in these studies be based on whether they use MPS to test a hypothesis or to generate hypotheses. To demonstrate the differences in these approaches to informed consent, we describe the consent processes for two MPS studies. The purpose of our hypothesis-testing study is to elucidate the etiology of rare phenotypes using MPS. The purpose of our hypothesis-generating study is to test the feasibility of using MPS to generate clinical hypotheses, and to approach the return of results as an experimental manipulation. Issues to consider in both designs include: volume and nature of the potential results, primary versus secondary results, return of individual results, duty to warn, length of interaction, target population, and privacy and confidentiality.
Summary
The categorization of MPS studies as hypothesis-testing versus hypothesis-generating can help to clarify the issue of so-called incidental or secondary results for the consent process, and aid the communication of the research goals to study participants.
doi:10.1186/1755-8794-5-45
PMCID: PMC3495642  PMID: 23046515
Whole genome sequencing; Whole exome sequencing; Informed consent
24.  Assessment and management of the orthopedic and other complications of Proteus syndrome 
Purpose
A multidisciplinary workshop was convened at the National Institutes of Health (NIH) to discuss the management of the orthopedic and other complications of Proteus syndrome (PS), a progressive, disproportionate overgrowth disorder. While PS poses many complex challenges, the focus of the workshop was the management of the asymmetric and disorganized skeletal overgrowth that characterizes this multisystem disorder.
Methods
Workshop participants developed recommendations for clinical research and patient management and surveillance to maximize the benefits and reduce the risks of surgical and other interventions.
Results
Recommendations for clinical care and management included assessments of skeletal overgrowth and its progression with modalities such as X-ray, magnetic resonance imaging (MRI), dual-energy X-ray absorptiometry, and computerized tomography (CT) imaging. The recommendations also cover the assessment of non-orthopedic complications of PS that significantly impact surgical risk, such as pulmonary embolism and lung bullae. Surgical considerations in PS include assessment of the contribution of contractures to deformities and prophylactic soft-tissue release, aggressive and early use of epiphysiodesis and epiphysiostasis, amputation, and spinal bracing.
Conclusion
Decisions on the timing of orthopedic procedures in children with PS are challenging because they entail balancing the risks of intervention in this high-risk and complex population against the increasing morbidity that patients experience with progressive bony overgrowth. If surgery is delayed too long, the condition may become inoperable. We hope that these recommendations will help clinicians gather appropriate data and assist their patients in making timely treatment decisions.
doi:10.1007/s11832-011-0350-6
PMCID: PMC3179535  PMID: 23024722
Proteus syndrome; Overgrowth; Scoliosis; Limb-length inequality
25.  Long Term Survival in TARP Syndrome and Confirmation of RBM10 as the Disease Causing Gene 
TARP syndrome, comprising Talipes equinovarus, Atrial septal defect, Robin sequence (micrognathia, glossoptosis, and cleft palate), and Persistence of the left superior vena cava, is an X-linked condition with pre- or postnatal lethality in affected males. Based on linkage studies and massively parallel sequencing of X-chromosome exons in two families, the disease causing gene was identified as RBM10. We identified a maternally inherited frameshift mutation in an unrelated patient, confirming RBM10 as the disease gene. This is the first reported individual with TARP syndrome who survived past early infancy, thus expanding the phenotypic spectrum of this disorder. In addition to the characteristic cleft palate, atrial septal defect and persistent superior vena cava, he had low-set and posteriorly angulated ears, upslanting palpebral fissures, cryptorchidism and structural brain abnormalities including partial agenesis of the corpus callosum, dysplastic enlarged caudate, and cerebellar hypoplasia with megacisterna magna. Preterm delivery, suspected pulmonary hypoplasia and pulmonary hypertension resulted in chronic lung disease. At the age of 3 7/12 years, he remained ventilator-dependent at night, and he was fed exclusively through a gastro-jejunal tube. Sensorineural hearing loss required a hearing aid. Optic atrophy and cortical visual impairment were noted. He was unable to sit independently, was non-communicative and he had severe intellectual disability. Atrial flutter required recurrent ablation of intra-atrial re-entry pathways. The mother's heterozygosity for the RBM10 mutation underscored the importance of accurate diagnosis and counseling for TARP syndrome.
doi:10.1002/ajmg.a.34190
PMCID: PMC3183328  PMID: 21910224
ASD; cryptorchidism; persistent left superior vena cava; Pierre-Robin sequence; RBM10; talipes equinovarus; X-linked

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