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1.  Exome sequencing: the expert view 
Genome Biology  2011;12(9):128.
To complement our special issue on exome sequencing, Genome Biology asked several leaders in the field for their views on this new approach. Leslie G Biesecker (LGB), Jim C Mullikin (JM) and Kevin V Shianna (KVS) discuss the reasons for the popularity of exome sequencing and its contribution to genomics.
PMCID: PMC3308041  PMID: 21920051
2.  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
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
7.  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.
PMCID: PMC3727274  PMID: 23788249
secondary findings; incidental findings; genome; genomic medicine; personalized medicine; whole-exome; whole-genome; sequencing
8.  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.
PMCID: PMC3790899  PMID: 22344227
9.  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.
PMCID: PMC3461408  PMID: 22729222
11.  Defining Morphology: Hands and Feet 
An international group of clinicians working in the field of Dysmorphology has initiated the (re-) definition of all terms used to describe the external human phenotype. The goal is that through standardization of all terms and consensus regarding their definitions the reliability of description of features in humans will increase, comparisons of findings between patients will become more reliable, and discussions with other workers in the field such as developmental biologists and molecular geneticists will become more accurate. Here we report on the (re-) definition of terms needed to describe the major characteristics of the hands and feet. We provide a limited description of the anatomy of this region, limited background anthropometry, and an illustrated list of definitions.
PMCID: PMC3224990  PMID: 19125433
nomenclature; definitions; feet; hands; limbs; malformations; anomalies; multiple anomalies; anatomy
12.  A splice donor mutation in NAA10 results in the dysregulation of the retinoic acid signaling pathway and causes Lenz microphthalmia syndrome 
Journal of medical genetics  2014;51(3):185-196.
Lenz microphthalmia syndrome (LMS) is a genetically heterogeneous X-linked disorder characterised by microphthalmia/anophthalmia, skeletal abnormalities, genitourinary malformations, and anomalies of the digits, ears, and teeth. Intellectual disability and seizure disorders are seen in about 60% of affected males. To date, no gene has been identified for LMS in the microphthalmia syndrome 1 locus (MCOPS1). In this study, we aim to find the disease-causing gene for this condition.
Methods and results
Using exome sequencing in a family with three affected brothers, we identified a mutation in the intron 7 splice donor site (c.471+2T→A) of the N-acetyltransferase NAA10 gene. NAA10 has been previously shown to be mutated in patients with Ogden syndrome, which is clinically distinct from LMS. Linkage studies for this family mapped the disease locus to Xq27-Xq28, which was consistent with the locus of NAA10. The mutation co-segregated with the phenotype and cDNA analysis showed aberrant transcripts. Patient fibroblasts lacked expression of full length NAA10 protein and displayed cell proliferation defects. Expression array studies showed significant dysregulation of genes associated with genetic forms of anophthalmia such as BMP4, STRA6, and downstream targets of BCOR and the canonical WNT pathway. In particular, STRA6 is a retinol binding protein receptor that mediates cellular uptake of retinol/vitamin A and plays a major role in regulating the retinoic acid signalling pathway. A retinol uptake assay showed that retinol uptake was decreased in patient cells.
We conclude that the NAA10 mutation is the cause of LMS in this family, likely through the dysregulation of the retinoic acid signalling pathway.
PMCID: PMC4278941  PMID: 24431331
13.  The Development of a Clinical Screening Instrument For Tumour Predisposition Syndromes In Childhood Cancer Patients 
Identification of tumour predisposition syndromes in patients who have cancer in childhood is paramount for optimal care. A screening instrument that can help to identify such patients will facilitate physicians caring for children with cancer. The complete screening instrument should consist of a standardized series of pictures and a screening form for manifestations not visible in the pictures. Here we describe the development of such a screening form based on an international two-stage Delphi process and an initial validation of the complete instrument.
Patients and Methods
We identified manifestations that may contribute to the diagnosis of a tumour predisposition syndrome through the Winter-Baraitser Dysmorphology Database and the textbook “Gorlin's Syndromes of the Head and Neck”. In a two-round Delphi process, eight international content-experts scored the contribution of each of these manifestations. We performed a clinical validation of the instrument in a selected cohort of ten paediatric cancer patients from another centre.
In total, 49 manifestations were found to contribute to the diagnosis of a tumour predisposition syndrome and were included in the screening form. The pilot validation study showed that patients suspect for having a tumour predisposition syndrome were recognized. Excellent correlation for indication for referral of a patient between the screening instrument and the reference standard (personal evaluation by an experienced clinical geneticist) was found.).
The Delphi process performed by international specialists with a function as opinion leaders in their field of expertise has led to a screening form and instrument with which those childhood cancer patients can be identified who may have a tumour predisposition syndrome and thus have an indication to be referred for further genetic analysis.
PMCID: PMC4277698  PMID: 23855994
Paediatric oncology; childhood cancer; screening instrument; tumour predisposition syndromes; Delphi process; questionnaires
14.  Zoom‐in comparative genomic hybridisation arrays for the characterisation of variable breakpoint contiguous gene syndromes 
Journal of Medical Genetics  2007;44(1):e59.
Contiguous gene syndromes cause disorders via haploinsufficiency for adjacent genes. Some contiguous gene syndromes (CGS) have stereotypical breakpoints, but others have variable breakpoints. In CGS that have variable breakpoints, the extent of the deletions may be correlated with severity. The Greig cephalopolysyndactyly contiguous gene syndrome (GCPS‐CGS) is a multiple malformation syndrome caused by haploinsufficiency of GLI3 and adjacent genes. In addition, non‐CGS GCPS can be caused by deletions or duplications in GLI3. Although fluorescence in situ hybridisation (FISH) can identify large deletion mutations in patients with GCPS or GCPS‐CGS, it is not practical for identification of small intragenic deletions or insertions, and it is difficult to accurately characterise the extent of the large deletions using this technique. We have designed a custom comparative genomic hybridisation (CGH) array that allows identification of deletions and duplications at kilobase resolution in the vicinity of GLI3. The array averages one probe every 730 bp for a total of about 14 000 probes over 10 Mb. We have analysed 16 individuals with known or suspected deletions or duplications. In 15 of 16 individuals (14 deletions and 1 duplication), the array confirmed the prior results. In the remaining patient, the normal CGH array result was correct, and the prior assessment was a false positive quantitative polymerase chain reaction result. We conclude that high‐density CGH array analysis is more sensitive than FISH analysis for detecting deletions and provides clinically useful results on the extent of the deletion. We suggest that high‐density CGH array analysis should replace FISH analysis for assessment of deletions and duplications in patients with contiguous gene syndromes caused by variable deletions.
PMCID: PMC2597909  PMID: 17098889
GLI3 ; oligonucleotide array; comparative genomic hybridization
15.  Elements of Morphology: Introduction 
An international group of clinicians working in the field of dysmorphology has initiated the standardization of terms used to describe human morphology. The goals are to standardize these terms and reach consensus regarding their definitions. In this way, we will increase the utility of descriptions of the human phenotype and facilitate reliable comparisons of findings among patients. Discussions with other workers in dysmorphology and related fields, such as developmental biology and molecular genetics, will become more precise. Here we describe the general background of the project and the various issues we have tried to take into account in defining the terms.
PMCID: PMC2774524  PMID: 19127575
nomenclature; definitions; morphology; dysmorphology; birth defects; malformations; minor anomalies; common variants
16.  Databases of genomic variation and phenotypes: existing resources and future needs 
Human Molecular Genetics  2013;22(R1):R27-R31.
Massively parallel sequencing (MPS) has become an important tool for identifying medically significant variants in both research and the clinic. Accurate variation and genotype–phenotype databases are critical in our ability to make sense of the vast amount of information that MPS generates. The purpose of this review is to summarize the state of the art of variation and genotype–phenotype databases, how they can be used, and opportunities to improve these resources. Our working assumption is that the objective of the clinical genomicist is to identify highly penetrant variants that could explain existing disease or predict disease risk for individual patients or research participants. We have detailed how current databases contribute to this goal providing frequency data, literature reviews and predictions of causation for individual variants. For variant annotation, databases vary greatly in their ease of use, the use of standard mutation nomenclature, the comprehensiveness of the variant cataloging and the degree of expert opinion. Ultimately, we need a dynamic and comprehensive reference database of medically important variants that is easily cross referenced to exome and genome sequence data and allows for an accumulation of expert opinion.
PMCID: PMC3782073  PMID: 23962721
17.  Hereditary spastic paraplegia type 43 (SPG43) is caused by mutation in C19orf12 
Human mutation  2013;34(10):10.1002/humu.22378.
We report here the genetic basis for a form of progressive hereditary spastic paraplegia (SPG43) previously described in two Malian sisters. Exome sequencing revealed a homozygous missense variant (c.187G>C; p.Ala63Pro) in C19orf12, a gene recently implicated in neurodegeneration with brain iron accumulation (NBIA). The same mutation was subsequently also found in a Brazilian family with features of NBIA, and we identified another NBIA patient with a three-nucleotide deletion (c.197_199del; p.Gly66del). Haplotype analysis revealed that the p.Ala63Pro mutations have a common origin, but MRI scans showed no brain iron deposition in the Malian SPG43 subjects. Heterologous expression of these SPG43 and NBIA variants resulted in similar alterations in the subcellular distribution of C19orf12. The SPG43 and NBIA variants reported here as well as the most common C19orf12 missense mutation reported in NBIA patients are found within a highly-conserved, extended hydrophobic domain in C19orf12, underscoring the functional importance of this domain.
PMCID: PMC3819934  PMID: 23857908
SPG43; NBIA; C19orf12; hereditary spastic paraplegia
18.  Characterizing Genetic Variants for Clinical Action 
Genome-wide association studies, DNA sequencing studies, and other genomic studies are finding an increasing number of genetic variants associated with clinical phenotypes that may be useful in developing diagnostic, preventive, and treatment strategies for individual patients. However, few common variants have been integrated into routine clinical practice. The reasons for this are several, but two of the most significant are limited evidence about the clinical implications of the variants and a lack of a comprehensive knowledge base that captures genetic variants, their phenotypic associations, and other pertinent phenotypic information that is openly accessible to clinical groups attempting to interpret sequencing data. As the field of medicine begins to incorporate genome-scale analysis into clinical care, approaches need to be developed for collecting and characterizing data on the clinical implications of variants, developing consensus on their actionability, and making this information available for clinical use. The National Human Genome Research Institute (NHGRI) and the Wellcome Trust thus convened a workshop to consider the processes and resources needed to: 1) identify clinically valid genetic variants; 2) decide whether they are actionable and what the action should be; and 3) provide this information for clinical use. This commentary outlines the key discussion points and recommendations from the workshop.
PMCID: PMC4158437  PMID: 24634402
genomic medicine; clinical actionability; database; electronic health records (EHR); pharmacogenomics; DNA sequencing
19.  Functional analysis of a de novo ACTB mutation in a patient with atypical Baraitser-Winter syndrome 
Human mutation  2013;34(9):1242-1249.
Exome sequence analysis can be instrumental in identifying the genetic etiology behind atypical disease. We report a patient presenting with microcephaly, dysmorphic features, and intellectual disability with a tentative diagnosis of Dubowitz syndrome. Exome analysis was performed on the patient and both parents. A de novo missense variant was identified in ACTB, c.349G>A, p.E117K. Recent work in Baraitser-Winter syndrome has identified ACTB and ACTG1 mutations in a cohort of individuals and we rediagnosed the patient with atypical Baraitser-Winter syndrome. We performed functional characterization of the variant actin and show that it alters cell adhesion and polymer formation supporting its role in disease. We present the clinical findings in the patient, comparison of this patient to other patients with ACTB/ACTG1 mutations, and results from actin functional studies that demonstrate novel functional attributes of this mutant protein.
PMCID: PMC3745514  PMID: 23649928
actin; ACTB; Dubowitz; Baraitser-Winter syndrome
21.  Genomic Inheritances: Disclosing Individual Research Results From Whole-Exome Sequencing to Deceased Participants’ Relatives 
Whole-genome analysis and whole-exome analysis generate many more clinically actionable findings than traditional targeted genetic analysis. These findings may be relevant to research participants themselves as well as for members of their families. Though researchers performing genomic analyses are likely to find medically significant genetic variations for nearly every research participant, what they will find for any given participant is unpredictable. The ubiquity and diversity of these findings complicate questions about disclosing individual genetic test results. We outline an approach for disclosing a select range of genetic results to the relatives of research participants who have died, developed in response to relatives’ requests during a pilot study of large-scale medical genetic sequencing. We also argue that studies that disclose individual research results to participants should, at a minimum, passively disclose individual results to deceased participants’ relatives.
PMCID: PMC4104597  PMID: 22974017
genomics; medical genetics; research; genetic; personal genetic information; bioethical issues; ethics; research
22.  Protein Kinase Cδ Deficiency Causes Mendelian Systemic Lupus Erythematosus With B Cell–Defective Apoptosis and Hyperproliferation 
Arthritis and rheumatism  2013;65(8):2161-2171.
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.
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.
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.
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.
PMCID: PMC4066615  PMID: 23666743
23.  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.
PMCID: PMC4043752  PMID: 24892279
24.  SHORT COMMUNICATION: Hyperphagia among patients with Bardet-Biedl syndrome 
Pediatric obesity  2013;8(5):e64-e67.
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.
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.
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.
Appetite dysregulation may contribute to obesity in BBS.
PMCID: PMC3901011  PMID: 23776152
hyperphagia; obesity; Bardet-Biedl syndrome; obesity syndromes; polyphagia; energy intake; questionnaire; child; adolescent

Results 1-25 (66)