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AEBP1 encodes the aortic carboxypeptidase-like protein (ACLP) that associates with collagens in the extracellular matrix (ECM) and has several roles in development, tissue repair, and fibrosis. ACLP is expressed in bone, the vasculature, and dermal tissues and is involved in fibroblast proliferation and mesenchymal stem cell differentiation into collagen-producing cells. Aebp1−/− mice have abnormal, delayed wound repair correlating with defects in fibroblast proliferation. In this study, we describe four individuals from three unrelated families that presented with a unique constellation of clinical findings including joint laxity, redundant and hyperextensible skin, poor wound healing with abnormal scarring, osteoporosis, and other features reminiscent of Ehlers-Danlos syndrome (EDS). Analysis of skin biopsies revealed decreased dermal collagen with abnormal collagen fibrils that were ragged in appearance. Exome sequencing revealed compound heterozygous variants in AEBP1 (c.1470delC [p.Asn490_Met495delins(40)] and c.1743C>A [p.Cys581∗]) in the first individual, a homozygous variant (c.1320_1326del [p.Arg440Serfs∗3]) in the second individual, and a homozygous splice site variant (c.1630+1G>A) in two siblings from the third family. We show that ACLP enhances collagen polymerization and binds to several fibrillar collagens via its discoidin domain. These studies support the conclusion that bi-allelic pathogenic variants in AEBP1 are the cause of this autosomal-recessive EDS subtype.

PurposeWe delineate the clinical spectrum and describe the histology in arterial tortuosity syndrome (ATS), a rare connective tissue disorder characterized by tortuosity of the large and medium-sized arteries, caused by mutations in SLC2A10.MethodsWe retrospectively characterized 40 novel ATS families (50 patients) and reviewed the 52 previously reported patients. We performed histology and electron microscopy (EM) on skin and vascular biopsies and evaluated TGF-β signaling with immunohistochemistry for pSMAD2 and CTGF.ResultsStenoses, tortuosity, and aneurysm formation are widespread occurrences. Severe but rare vascular complications include early and aggressive aortic root aneurysms, neonatal intracranial bleeding, ischemic stroke, and gastric perforation. Thus far, no reports unequivocally document vascular dissections or ruptures. Of note, diaphragmatic hernia and infant respiratory distress syndrome (IRDS) are frequently observed. Skin and vascular biopsies show fragmented elastic fibers (EF) and increased collagen deposition. EM of skin EF shows a fragmented elastin core and a peripheral mantle of microfibrils of random directionality. Skin and end-stage diseased vascular tissue do not indicate increased TGF-β signaling.ConclusionOur findings warrant attention for IRDS and diaphragmatic hernia, close monitoring of the aortic root early in life, and extensive vascular imaging afterwards. EM on skin biopsies shows disease-specific abnormalities.GENETICS in MEDICINE advance online publication, 11 January 2018; doi:10.1038/gim.2017.253. ispartof: Genetics in Medicine vol:20 issue:10 pages:1236-1245 ispartof: location:United States status: published

Item does not contain fulltext Developmental and epileptic encephalopathies (DEEs) are severe neurodevelopmental disorders often beginning in infancy or early childhood that are characterized by intractable seizures, abundant epileptiform activity on EEG, and developmental impairment or regression. CACNA1E is highly expressed in the central nervous system and encodes the alpha1-subunit of the voltage-gated CaV2.3 channel, which conducts high voltage-activated R-type calcium currents that initiate synaptic transmission. Using next-generation sequencing techniques, we identified de novo CACNA1E variants in 30 individuals with DEE, characterized by refractory infantile-onset seizures, severe hypotonia, and profound developmental impairment, often with congenital contractures, macrocephaly, hyperkinetic movement disorders, and early death. Most of the 14, partially recurring, variants cluster within the cytoplasmic ends of all four S6 segments, which form the presumed CaV2.3 channel activation gate. Functional analysis of several S6 variants revealed consistent gain-of-function effects comprising facilitated voltage-dependent activation and slowed inactivation. Another variant located in the domain II S4-S5 linker results in facilitated activation and increased current density. Five participants achieved seizure freedom on the anti-epileptic drug topiramate, which blocks R-type calcium channels. We establish pathogenic variants in CACNA1E as a cause of DEEs and suggest facilitated R-type calcium currents as a disease mechanism for human epilepsy and developmental disorders.

Overgrowth syndromes are a clinically heterogeneous group of disorders characterized by localized or generalized tissue overgrowth and varying degrees of developmental and intellectual disability. An expanding list of genes associated with overgrowth syndromes include the histone methyltransferase genes EZH2 and NSD1, which cause Weaver and Sotos syndrome, respectively, and the DNA methyltransferase (DNMT3A) gene that results in Tatton-Brown–Rahman syndrome (TBRS). Here, we describe a 5-year-old female with a paternally inherited pathogenic mutation in EZH2 (c.2050C>T, p.Arg684Cys) and a maternally inherited 505-kb duplication of uncertain significance at 2p23.3 (encompassing five genes, including DNMT3A) who presented with intrauterine growth restriction, slow postnatal growth, short stature, hypotonia, developmental delay, and neuroblastoma diagnosed at the age of 8 mo. Her father had tall stature, dysmorphic facial features, and intellectual disability consistent with Weaver syndrome, whereas her mother had short stature, cognitive delays, and chronic nonprogressive leukocytosis. It has been previously shown that EZH2 directly controls DNA methylation through physical association with DNMTs, including DNMT3A, with concomitant H3K27 methylation and CpG promoter methylation leading to repression of EZH2 target genes. Interestingly, NSD1 is involved in H3K36 methylation, a mark associated with transcriptional activation, and exhibits exquisite dosage sensitivity leading to overgrowth when deleted and severe undergrowth when duplicated in vivo. Although there is currently no evidence of dosage effects for DNMT3A, the co-occurrence of a duplication involving this gene and a pathogenic alteration in EZH2 in a patient with severe undergrowth is suggestive of a similar paradigm and further study is warranted.

Objective To understand motivations, educational needs, and concerns of individuals contemplating whole-exome sequencing (WES) and determine what amount of genetic information might be obtained by sequencing a generally healthy cohort so as to more effectively counsel future patients. Patients and Methods From 2012 to 2014, 40 medically educated, generally healthy scientists at Mayo Clinic were invited to have WES conducted on a research basis; 26 agreed to be in a drawing from which 10 participants were selected. The study involved pre- and posttest genetic counseling and completion of 4 surveys related to the experience and outcomes. Whole-exome sequencing was conducted on DNA from blood from each person. Results Most variants (76,305 per person; range, 74,505-77,387) were known benign allelic variants, variants in genes of unknown function, or variants of uncertain significance in genes of known function. The results of suspected pathogenic/pathogenic variants in Mendelian disorders and pharmacogenomic variants were disclosed. The mean number of suspected pathogenic/pathogenic variants was 2.2 per person (range, 1-4). Four pharmacogenomic genes were included for reporting; variants were found in 9 of 10 participants. Conclusion This study provides data that may be useful in establishing reality-based patient expectations, outlines specific points to cover during counseling, and increases confidence in the feasibility of providing adequate preparation and counseling for WES in generally healthy individuals.

Objectives: In an era of precision medicine, our aim was to determine the frequency and theranostic potential of mutations identified in malignant lymph nodes (LNs) sampled by endoscopic ultrasound fine-needle aspiration (EUS FNA) of patients with rectal cancer by targeted next-generation sequencing (NGS). Methods: The NGS Ion AmpliSeq Cancer Hotspot Panel v2 (Life Technologies, Carlsbad, CA) and MiSeq (Illumina, San Diego, CA) sequencers were used to sequence and assess for 2,800 or more possible mutations in 50 established cancer-associated genes. Results: Among 102 patients, 89% had 194 pathogenic alterations identified in 19 genes. The identification of KRAS, NRAS , or BRAF mutations suggests that 42% are likely nonresponders to anti–epidermal growth factor receptor therapy. Among KRAS, NRAS , or BRAF wild-type patients, alterations in eight genes linked to alternative therapies were identified in 44%. Conclusions: Our data demonstrate the successful ability to apply a single multiplex test to allow multigene mutation detection from malignant LN cytology specimen DNA collected by EUS FNA.

Somatic serine/threonine kinase 11 (STK11) also known as liver kinase B1 (LKB1) is a tumor suppressor gene and ranks as the third most frequently mutated gene in lung adenocarcinoma. However, current molecular testing guidelines recommend evaluating for epidermal growth factor receptor mutations and ALK fusions to guide therapy in all patients with advanced stage adenocarcinoma, regardless of gender, race, or smoking history. Identifying alternative “driver” mutations and using actionable targeted pharmacotherapy is a key approach to providing effective individualized medical care. The analytical sensitivity and parallel multigene approach of targeted next-generation sequencing is an attractive methodology for use for cytology specimens. The presented lung adenocarcinoma study revealed that STK11 mutations alone and concomitant KRAS/STK11 mutations were identified in 18.2% and 4.5% of solitary adrenal metastases, respectively. Molecular profiling of epidermal growth factor receptor tyrosine kinase inhibitor resistant tumors may help to identify patients who would most benefit from alternative single or dual pathway inhibition potentially leading to a revision in current molecular testing guidelines.

We report a 34-year-old male patient with a novel variant in KMT2D gene, which finally ended a quest for a diagnosis that was clinically suspected in the past, prior the molecular basis of Kabuki Syndrome (KS) was known. The patient showcases the multisystemic features, with involvement of all previously associated with KS body systems, presence of immune deficiency as well as autoimmune disorders, requiring three pancreatic transplants. We also report, for the first time to our knowledge, the presence of epidural lipomatosis and Hodgkin Lymphoma in a patient with KS.

Next-generation sequencing (NGS) is widely used and cost-effective. Depending on the specific methods, NGS can have limitations detecting certain technically challenging variant types even though they are both prevalent in patients and medically important. These types are underrepresented in validation studies, hindering the uniform assessment of test methodologies by laboratory directors and clinicians. Specimens containing such variants can be difficult to obtain; thus, we evaluated a novel solution to this problem in which a diverse set of technically challenging variants was synthesized and introduced into a known genomic background. This specimen was sequenced by 7 laboratories using 10 different NGS workflows. The specimen was compatible with all 10 workflows and presented biochemical and bioinformatic challenges similar to those of patient specimens. Only 10 of 22 challenging variants were correctly identified by all 10 workflows, and only 3 workflows detected all 22. Many, but not all, of the sensitivity limitations were bioinformatic in nature. We conclude that Synthetic controls can provide an efficient and informative mechanism to augment studies with technically challenging variants that are difficult to obtain otherwise. Data from such specimens can facilitate inter-laboratory methodologic comparisons and can help establish standards that improve communication between clinicians and laboratories.

We assessed the performance characteristics of an RNA sequencing (RNA-Seq) assay designed to detect gene fusions in 571 genes to help manage patients with cancer. Polyadenylated RNA was converted to cDNA, which was then used to prepare next-generation sequencing libraries that were sequenced on an Illumina HiSeq 2500 instrument and analyzed with an in-house developed bioinformatic pipeline. The assay identified 38 of 41 gene fusions detected by another method, such as fluorescence in situ hybridization or RT-PCR, for a sensitivity of 93%. No false-positive gene fusions were identified in 15 normal tissue specimens and 10 tumor specimens that were negative for fusions by RNA sequencing or Mate Pair NGS (100% specificity). The assay also identified 22 fusions in 17 tumor specimens that had not been detected by other methods. Eighteen of the 22 fusions had not previously been described. Good intra-assay and interassay reproducibility was observed with complete concordance for the presence or absence of gene fusions in replicates. The analytical sensitivity of the assay was tested by diluting RNA isolated from gene fusion–positive cases with fusion-negative RNA. Gene fusions were generally detectable down to 12.5% dilutions for most fusions and as little as 3% for some fusions. This assay can help identify fusions in patients with cancer; these patients may in turn benefit from both US Food and Drug Administration–approved and investigational targeted therapies.

Maple syrup urine disease (MSUD) is an inborn error of metabolism caused by defects in the branched-chain α-ketoacid dehydrogenase complex, which results in elevations of the branched-chain amino acids (BCAAs) in plasma, α-ketoacids in urine, and production of the pathognomonic disease marker, alloisoleucine. The disorder varies in severity and the clinical spectrum is quite broad with five recognized clinical variants that have no known association with genotype. The classic presentation occurs in the neonatal period with developmental delay, failure to thrive, feeding difficulties, and maple syrup odor in the cerumen and urine, and can lead to irreversible neurological complications, including stereotypical movements, metabolic decompensation, and death if left untreated. Treatment consists of dietary restriction of BCAAs and close metabolic monitoring. Clinical outcomes are generally good in patients where treatment is initiated early. Newborn screening for MSUD is now commonplace in the United States and is included on the Recommended Uniform Screening Panel (RUSP). We review this disorder including its presentation, screening and clinical diagnosis, treatment, and other relevant aspects pertaining to the care of patients.

Bosch–Boonstra–Schaaf optic atrophy syndrome (BBSOAS) is a recently described autosomal dominant disorder caused by mutations in the NR2F1 gene. There are presently 28 cases of BBSOAS described in the literature. Its common features include developmental delay, intellectual disability, hypotonia, optic nerve atrophy, attention deficit disorder, autism spectrum disorder, seizures, hearing defects, spasticity, and thinning of the corpus callosum. Here we report two unrelated probands with novel, de novo, missense variants in NR2F1. The first is a 14-yr-old male patient with hypotonia, intellectual disability, optic nerve hypoplasia, delayed bone age, short stature, and altered neurotransmitter levels on cerebrospinal fluid testing. The second is a 5-yr-old female with severe developmental delay, motor and speech delay, and repetitive motion behavior. Whole-exome sequencing identified a novel missense NR2F1 variant in each case, Cys86Phe in the DNA-binding domain in Case 1, and a Leu372Pro in the ligand-binding domain in Case 2. The presence of clinical findings compatible with BBSOAS along with structural analysis at atomic resolution using homology-based molecular modeling and molecular dynamic simulations, support the pathogenicity of these variants for BBSOAS. Short stature, abnormal CNS neurotransmitters, and macrocephaly have not been previously reported for this syndrome and may represent a phenotypic expansion of BBSOAS. A review of published cases along with new evidence from this report support genotype–phenotype correlations for this disorder.

Pathogenic variants in EBF3 were recently described in three back-to-back publications in association with a novel neurodevelopmental disorder characterized by intellectual disability, speech delay, ataxia, and facial dysmorphisms. In this report, we describe an additional patient carrying a de novo missense variant in EBF3 (c.487C>T, p.(Arg163Trp)) that falls within a conserved residue in the zinc knuckle motif of the DNA binding domain. Without a solved structure of the DNA binding domain, we generated a homology-based atomic model and performed molecular dynamics simulations for EBF3, which predicted decreased DNA affinity for p.(Arg163Trp) compared with wild-type protein and control variants. These data are in agreement with previous experimental studies of EBF1 showing the paralogous residue is essential for DNA binding. The conservation and experimental evidence existing for EBF1 and in silico modeling and dynamics simulations to validate comparable behavior of multiple variants in EBF3 demonstrates strong support for the pathogenicity of p.(Arg163Trp). We show that our patient presents with phenotypes consistent with previously reported patients harboring EBF3 variants and expands the phenotypic spectrum of this newly identified disorder with the additional feature of a bicornuate uterus.

ACTB encodes the β-actin, and pathogenic variations in this gene have typically been associated with Baraitser-Winter cerebrofrontofacial syndrome, a congenital malformation syndrome characterized by short stature, craniofacial anomalies, and cerebral anomalies. Here, we describe the third case with the p.Arg183Trp variant in ACTB causing juvenile-onset dystonia. Our patient has severe, intractable dystonia, developmental delay, and sensorineural hearing loss, besides hyperintensities in the caudate nuclei and putamen on the brain MRI, which is a distinct but overlapping phenotype with the previously reported case of identical twins with the same alteration in ACTB.

We report an 8-month-old infant with decreased consciousness after a febrile episode and reduced oral intake. He was profoundly acidotic but his lactate was normal. Serum triglycerides were markedly elevated and HDL cholesterol was very low. The urine organic acid analysis during the acute episode revealed a complex pattern of relative hypoketotic dicarboxylic aciduria, suggestive of a potential fatty acid oxidation disorder. MRI showed extensive brain abnormalities concerning for a primary energy deficiency. Whole exome sequencing revealed heterozygotic HMGCS2 variants. HMGCS2 encodes mitochondrial 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase-2 (HMGCS2), which catalyzes the irreversible and rate-limiting reaction of ketogenesis in the mitochondrial matrix. Autosomal recessive HMG-CoA synthase deficiency (HMGCS2D) is characterized by hypoketotic hypoglycemia, vomiting, lethargy, and hepatomegaly after periods of prolonged fasting or illness. A retrospective analysis of the urine organic acid analysis identified 4-hydrox-6-methyl-2-pyrone, a recently reported putative biomarker of HMGCS2D. There was also a relative elevation of plasma acetylcarnitine as previously reported in one case. Our patient highlights a unique presentation of HMGCS2D caused by novel variants in HMGCS2. This is the first report of HMGCS2D with a significantly elevated triglyceride level and decreased HDL cholesterol level at presentation. Given this, we suggest that HMGCS2D should be considered in the differential diagnosis when hypertriglyceridemia, or low HDL cholesterol levels are seen in a child who presents with acidosis, mild ketosis, and mental status changes after illness or prolonged fasting. Although HMGCS2D is a rare disorder with nonspecific symptoms, with the advent of next-generation sequencing, and the recognition of novel biochemical biomarkers, the incidence of this condition may become better understood.

Loeys-Dietz syndrome (LDS) is a connective tissue disorder characterized by vascular findings of aneurysm and/or dissection of cerebral, thoracic, or abdominal arteries and skeletal findings. We report a case of a novel pathogenic variant in TGFBR2 and phenotype consistent with classic LDS. The proband was a 10-year-old presenting to the genetics clinic with an enlarged aortic root (Z-scores 5-6), pectus excavatum, and congenital contractures of the right 2nd and 3rd digit. Molecular testing of TGFBR2 was sent to a commercial laboratory and demonstrated a novel, likely pathogenic, variant in exon 4, c.1061T>C, p.(L354P). Molecular modeling reveals alteration of local protein structure as a result of this pathogenic variant. This pathogenic variant has not been previously reported in LDS and thus expands the pathogenic variant spectrum of this condition.

BACKGROUND Multigene molecular testing to guide personalized therapy for oncology patients is of increasing clinical relevance. Molecular testing of fine-needle aspiration samples is underused, but when acquired with minimally invasive techniques could become the standard of care to obtain theranostic specimens. The aims of the current study were to identify key cytology specimen selection criteria suitable for next-generation sequencing (NGS) and to determine the prevalence and spectrum of pathogenic alterations in a cohort of patients with AJCC stage IV lung cancer. METHODS A total of 70 adrenal gland cytology specimens with direct smears were screened to identify 56 patients with a single slide containing at least 300 total cells and 20% tumor nuclei. After DNA extraction, the NGS protocols were used to simultaneously detect mutations in >2800 exonic regions in 50 key cancer genes. RESULTS A total of 28 specimens produced acceptable NGS results. Specimens with a combined critical cell mass (>5000 viable cells) and a DNA concentration >5 ng/µL resulted in a 95% chance of successful sequencing. A total of 37 pathogenic alterations were identified in 10 genes and in 25 patients (85%). A pathogenic alteration (≥1) linked to available or developing targeted therapies was revealed in 50% of cases. CONCLUSIONS The data from the current study demonstrate that theranostic NGS can be applied to adrenal gland metastasis using routine cytologic smear specimens. The characteristics of such smears could be evaluated during onsite adequacy assessment by cytopathology professionals. This model greatly augments the opportunity for customized genotype-directed therapy from minimally invasive techniques. Cancer (Cancer Cytopathol) 2014;122:822–832. © 2014 American Cancer Society.

There is increasing recognition that genomic medicine as part of individualized medicine has a defined role in patient care. Rapid advances in technology and decreasing cost combine to bring genomic medicine closer to the clinical practice. There is also growing evidence that genomic-based medicine can advance patient outcomes, tailor therapy and decrease side effects. However the challenges to integrate genomics into the workflow involved in patient care remain vast, stalling assimilation of genomic medicine into mainstream medical practice. In this review we describe the approach taken by one institution to further individualize medicine by offering, executing and interpreting whole exome sequencing on a clinical basis through an enterprise-wide, standalone individualized medicine clinic. We present our experience designing and executing such an individualized medicine clinic, sharing lessons learned and describing early implementation outcomes.

During the past few decades, zebrafish (Danio rerio) have been a workhorse for developmental biology and genetics. Concurrently, zebrafish have proved highly accessible and effective for translational research by providing a vertebrate animal model useful for gene discovery, disease modeling, chemical genetic screening, and other medically relevant studies. Key resources such as an annotated and complete genome sequence, and diverse tools for genetic manipulation continue to spur broad use of zebrafish. Thus, the purpose of this introductory review is to provide a window into the unique characteristics and diverse uses of zebrafish and to highlight in particular the increasing relevance of zebrafish as a translational animal model. This is accomplished by reviewing broad considerations of anatomic and physiological conservation, approaches for disease modeling and creation, general laboratory methods, genetic tools, genome conservation, and diverse opportunities for functional validation. Additional commentary throughout the review also guides the reader to the 4 new reviews found elsewhere in this special issue that showcase the many unique ways the zebrafish is improving understanding of renal regeneration, mitochondrial disease, dyslipidemia, and aging, for example. With many other possible approaches and a rapidly increasing number of medically relevant reports, zebrafish approaches enhance significantly the tools available for translational research and are actively improving the understanding of human disease.

SHBG concentrations correlate inconsistently with metabolic parameters.SHBG assay platforms contribute to nonuniformities according to the literature.The design of the study was a noninterventional quantification of SHBG by two immuno- and two mass spectrometric assays and abdominal visceral fat by computed tomography scan.The study was conducted at the Center for Translational Science Activities.Healthy men (n=120) aged 18-80 years with a body mass index of 20-43 kg/m2 participated I the study.Outcomes of the study included a correlation of log SHBG with age, metabolic surrogates [body mass index, albumin, glucose, insulin, abdominal (total and visceral) fat, homeostasis model assessment insulin resistance index], sex steroids (estrone, 17β-estradiol, T, and dihydrotestosterone by mass spectrometry), and adipocytokines (IL-1β, IL-6, IL-8, IL-10 and IL-12, TNF-α, and adiponectin).By univariate regression, age (P10(-4)), dihydrotestosterone (P10(-4)), T (P≤.00022), and adiponectin (P≤.0084) were positive correlates, and insulin and homeostasis model assessment insulin resistance index were negative correlates (P≤.0060) of SHBG in all four assays. Stepwise multivariate analysis unveiled that age and T together could explain 38.1%-52.5% of the statistical variance in SHBG in all assays (P10(-11)). Multivariate regression without sex steroids unveiled that age (P10(-5)) and insulin (P10(-3)) are jointly associated with SHBG levels in the four assays with overall R2=0.215-0.293 and P10(-6). In one immunological SHBG assay each, abdominal visceral fat and adiponectin were weak multivariates also.Immunological and mass spectrometric SHBG assays yield both consistent and inconsistent correlations with key metabolic variables in healthy men, thereby potentially explaining earlier inconsistencies in the literature.

Background We characterized the pharmacogenomics (PGx) results received by diagnostic odyssey patients as secondary findings during clinical whole exome sequencing (WES) testing as a part of their care in Mayo Clinic's Individualized Medicine Clinic to determine the potential benefits and limitations to this cohort. Methods WES results on 94 patients included a subset of PGx variants in CYP2C19, CYP2C9, and VKORC1 if identified in the patient. Demographic, phenotypic, and medication usage information was abstracted from patient medical data. A pharmacist interpreted the PGx results in the context of the patients’ current medication use and made therapeutic recommendations. Results The majority was young with a median age of 10 years old, had neurological involvement in the disease presentation (71%), and was currently taking medications (90%). Of the 94 PGx-evaluated patients, 91% had at least one variant allele reported and 20% had potential immediate implications on current medication use. Conclusion Due to the disease complexity and medication needs of diagnostic odyssey patients, there may be immediate benefit obtained from early life PGx testing for many and most will likely find benefit in the future. These results require conscientious interpretation and management to be actionable for all prescribing physicians throughout the lifetime of the patient.

Recently, de novo mutations in the gene KCNA2, causing either a dominant-negative loss-of-function or a gain-of-function of the voltage-gated K+ channel Kv1.2, were described to cause a new molecular entity within the epileptic encephalopathies. Here, we report a cohort of 23 patients (eight previously described) with epileptic encephalopathy carrying either novel or known KCNA2 mutations, with the aim to detail the clinical phenotype associated with each of them, to characterize the functional effects of the newly identified mutations, and to assess genotype-phenotype associations. We identified five novel and confirmed six known mutations, three of which recurred in three, five and seven patients, respectively. Ten mutations were missense and one was a truncation mutation; de novo occurrence could be shown in 20 patients. Functional studies using a Xenopus oocyte two-microelectrode voltage clamp system revealed mutations with only loss-of-function effects (mostly dominant-negative current amplitude reduction) in eight patients or only gain-of-function effects (hyperpolarizing shift of voltage-dependent activation, increased amplitude) in nine patients. In six patients, the gain-of-function was diminished by an additional loss-of-function (gain-and loss-of-function) due to a hyperpolarizing shift of voltage-dependent activation combined with either decreased amplitudes or an additional hyperpolarizing shift of the inactivation curve. These electrophysiological findings correlated with distinct phenotypic features. The main differences were (i) predominant focal (loss-of-function) versus generalized (gain-of-function) seizures and corresponding epileptic discharges with prominent sleep activation in most cases with loss-of-function mutations; (ii) more severe epilepsy, developmental problems and ataxia, and atrophy of the cerebellum or even the whole brain in about half of the patients with gain-of-function mutations; and (iii) most severe early-onset phenotypes, occasionally with neonatal onset epilepsy and developmental impairment, as well as generalized and focal seizures and EEG abnormalities for patients with gain- and loss-of-function mutations. Our study thus indicates well represented genotype-phenotype associations between three subgroups of patients with KCNA2 encephalopathy according to the electrophysiological features of the mutations.

Background Cervical dystonias have a variable presentation and underlying etiology, but collectively represent the most common form of focal dystonia. There are a number of known genetic forms of dystonia (DYT1-27); however the heterogeneity of disease presentation does not always make it easy to categorize the disease by phenotype-genotype comparison. Case presentation In this report, we describe a 53-year-old female who presented initially with hand tremor following a total hip arthroplasty. The patient developed a mixed hyperkinetic disorder consisting of chorea, dystonia affecting the upper extremities, dysarthria, and blepharospasm. Whole exome sequencing of the patient revealed a novel heterozygous missense variant (Chr11(GRCh38): g.26525644C > G; NM_031418.2(ANO3): c.702C > G; NP_113606.2. p.C234W) in exon 7 in the ANO3 gene. Conclusions ANO3 encodes anoctamin-3, a Ca+2-dependent phospholipid scramblase expressed in striatal-neurons, that has been implicated in autosomal dominant craniocervical dystonia (Dystonia-24, DYT24, MIM# 615034). To date, only a handful of cases of DYT-24 have been described in the literature. The complex clinical presentation of the patient described includes hyperkinesias, complex motor movements, and vocal tics, which have not been reported in other patients with DYT24. This report highlights the utility of using clinical whole exome sequencing in patients with complex neurological phenotypes that would not normally fit a classical presentation of a defined genetic disease. Electronic supplementary material The online version of this article (doi:10.1186/s12881-016-0354-7) contains supplementary material, which is available to authorized users.

Introduction: Globin gene sequencing has long been impeded by repetitive regions, homology and structural variation. Currently, long-read sequencing technology has been limited by high cost and error rate. These challenges can be mitigated by sample multiplexing of targeted capture sequencing and the use of consensus reads, respectively. This ongoing study analyzes the utility of long range sequencing technology in identifying historically problematic beta globin cluster mutations. Materials and methods: Long range sequencing was performed on complex beta globin gene cluster cases utilizing the Sequel platform (Pacific Biosciences, Menlo Park, California, United States). Sample input was 1 microgram of DNA extracted from whole blood. Circular Consensus Sequence (CCS) FASTQ sequences files were mapped to reference genome (GRCh37/hg19) using NGMLR, followed by annotation of variants using a custom bioinformatics workflow. Comparison to multiplex ligation-dependent probe amplification (MLPA), Sanger sequencing, array CGH, short-range NGS (300 bp), hemoglobin protein, and clinical data was performed. Results: CCS read length varied from 1 to 10 kb, average 3.5. Analysis of the beta globin cluster region showed impressive superiority and comprehensiveness to Sanger, MLPA, array CGH and short range sequencing technology: 1) single nucleotide variants (SNVs) were identified to a sensitivity and specificity of 99.5%; 2) large structural variants (SVs) such as large deletions, duplications, insertions, crossovers and fusions spanning many kilobases were characterized in the heterozygous, homozygous and compound heterozygous state to a precise genomic coordinate (table 1); 3) phasing SNVs identified Hb S haplotypes (Central African region (CAR), Benin, Senegal, Arab-Indian and Cameroon). Conclusion: 1) Long range sequencing holds significant promise in genotyping hemoglobin disorders. This method improves efficiency with potential for a single test offering with increased resolution, particularly in large deletion analysis. Complex cases greatly benefited from improved range and resolution. 2) The decreased requirement of blood volume for extensive testing is of particular interest for the pediatric population. 3) Genotyping of Hb S patients detected by positive newborn screening will help predict need for prophylaxis or therapy by assessing variant zygosity, phasing and haplotypes. In conclusion, we believe that long range sequencing has immense potential to contribute extensively to thalassemia and hemoglobinopathy diagnostics and is far superior to the currently available technologies for beta globin cluster analysis. Disclosures Jen: Celgene: Employment.

Introduction: Short telomere syndromes (STS) are accelerated aging syndromes affecting hematopoietic, pulmonary, hepatobiliary and/or immunological systems. Clinical assessment of age-appropriate telomere length (TL) is performed using flow cytometry & fluorescence in-situ hybridization (flowFISH). Screening for germline variants in STS-related genes is guided by flowFISH-determined centile categories of TL, with screening recommended for TL 10th centile and integration of clinical phenotype with flowFISH data in predictive algorithms is currently unclear. Methods: FlowFISH testing was done at reference laboratories in Vancouver (Repeat Diagnostics; Canada) & Johns Hopkins University (JHU, USA). Salient clinical features were pre-determined as, personal history of premature hair greying (onset at age < 30 years), idiopathic pulmonary fibrosis (IPF) or IPF/emphysema overlap (in smokers), cryptogenic cirrhosis or NRH, unexplained cytopenias &/or immunodeficiency, & family history of the above (in >1 1st or 2nd degree relatives). Clinical likelihood score (CLS) was assigned as low (1), intermediate (int, 2) or high (>2), based on the number of aforementioned clinical features present prior to flowFISH testing. Genetic testing was performed using either an in-house or commercial bone marrow failure-specific next generation sequencing (NGS) panel or whole exome sequencing (WES), and data for known variants affecting telomerase or telomeric function (TERT, TERC, DKC1, TINF2, NHP2, NOP10, TCAB1, NAF1, & RTEL1) was recorded. Results: One hundred forty-nine patients at our institution underwent TL assessment at Repeat diagnostics (n=38) and JHU (n=111) laboratories, respectively. Median age was 56 (range: 7-79) years; 88 (59%) being males. Significant family history was present in 40 (27%) patients, while premature greying of hair was present in 13 (9%) patients. Organ-specific clinical features included unexplained cytopenias (n=89, 60%) IPF (n=71, 48%), cryptogenic cirrhosis or NRH (n=21, 14%), & unexplained immunodeficiency (n=14, 9%). CLS stratification included low (n=74, 50%), int (n=54, 36%), & high (n=21, 14%), with higher CLS significantly correlating with lower delta TL for L (p=0.0005) but not G (p=0.3). Genetic testing was performed in 51 (35%) patients (NGS-51, WES-1) among which 13 (26%) patients had a telomere-associated variant; 5 (10%) pathogenic (pv, all TERT). CLS alone was unable to predict likelihood of finding a telomere-associated variant (p=0.4). Based on age-appropriate centile categorization of L & G TL (information for both available in 134 patients), patients were stratified into six groups (table 1). TL 1-90th centile in G [C2, n=36, CLS low-19 (53%), 16 (44%), 1 (3%)] of whom 8 (22%) underwent NGS with 1 TERTpv and 2 VUS in TINF2TL >10th centile in L & 1-90th centile in G (n=43, 32%): CLS stratification in this group included 27 (63%) low, 12 (28%) int, 4 (9%) high. NGS testing was done in 13 (30%) patients [CLS low-9(69%), int 2(15%), high 2 (15%)], of whom only 2 (15%) had VUS in TINF2 and TERT gene, but no pathogenic variants (figure 1). Conclusion: Our study demonstrates the importance of using a flowFISH assay based predictive algorithm to screen patients with suspected STS for telomere-related genetic alternations, in comparison to a clinical likelihood score. We also demonstrate a limited role for genetic testing in patients with lymphocyte TL >10th centile, regardless of the clinical likelihood score. Disclosures Patnaik: Stem Line Pharmaceuticals.: Membership on an entity's Board of Directors or advisory committees.

Trichorhinophalangeal syndrome type I (TRPSI) is a rare disorder that causes distinctive ectodermal, facial, and skeletal features affecting the hair (tricho-), nose (rhino-), and fingers and toes (phalangeal) and is inherited in an autosomal dominant pattern. TRPSI is caused by loss of function variants in TRPS1, involved in the regulation of chondrocyte and perichondrium development. Pathogenic variants in TRPS1 include missense mutations and deletions with variable breakpoints, with only a single instance of an intragenic duplication reported to date. Here we report an affected individual presenting with a classic TRPSI phenotype who is heterozygous for a de novo intragenic ∼36.3-kbp duplication affecting exons 2–4 of TRPS1. Molecular analysis revealed the duplication to be in direct tandem orientation affecting the splicing of TRPS1. The aberrant transcripts are predicted to produce a truncated TRPS1 missing the nuclear localization signal and the GATA and IKAROS-like zinc-finger domains resulting in functional TRPS1 haploinsufficiency. Our study identifies a novel intragenic tandem duplication of TRPS1 and highlights the importance of molecular characterization of intragenic duplications.

Mitochondrial disorders are caused by nuclear and mitochondrial pathogenic variants leading to defects in mitochondrial function and cellular respiration. Recently, the nuclear-encoded mitochondrial fusion gene MSTO1 (Misato 1) has been implicated in mitochondrial myopathy and ataxia. Here we report on a 30-yr-old man presenting with a maternally inherited NM_018116.3:c.651C>G, p.F217L missense variant as well as a paternally inherited arr[GRCh37] 1q22(155581773_155706887) × 1 deletion encompassing exons 7–14 of MSTO1. His phenotype included muscle weakness, hypotonia, early motor developmental delay, pectus excavatum, and scoliosis. Testing revealed elevated plasma creatine kinase, and electromyogram results were consistent with longstanding generalized myopathy. These phenotypic features overlap well with previously reported patients harboring biallelic MSTO1 variants. Additionally, our patient presents with dysphagia and restrictive lung disease, not previously reported for MSTO1-associated disorders. The majority of patients with disease-associated variants in MSTO1 present with biallelic variants suggesting autosomal recessive inheritance; however, one family has been reported with a single variant and presumed autosomal dominant inheritance. The pattern of inheritance we observed is consistent with the majority of previous reports suggesting an autosomal recessive disorder. We add to our knowledge of the syndrome caused by variants in MSTO1 and provide additional evidence supporting autosomal recessive inheritance. We also describe phenotypic features not reported in previous cases, although further research is needed to confirm they are associated with defects in MSTO1.

BackgroundRNA sequencing has been proposed as a means of increasing diagnostic rates in studies of undiagnosed rare inherited disease. Recent studies have reported diagnostic improvements in the range of 7.5-35% by profiling splicing, gene expression quantification and allele specific expression. To-date however, no study has systematically assessed the presence of gene-fusion transcripts in cases of germline disease. Fusion transcripts are routinely identified in cancer studies and are increasingly recognized as having diagnostic, prognostic or therapeutic relevance. Isolated reports exist of fusion transcripts being detected in cases of developmental and neurological phenotypes, and thus, systematic application of fusion detection to germline conditions may further increase diagnostic rates. However, current fusion detection methods are unsuited to the investigation of germline disease due to performance biases arising from their development using tumor, cell-line or in-silico data.MethodsWe describe a tailored approach to fusion candidate identification and prioritization in a cohort of 47 undiagnosed, suspected inherited disease patients. We modify an existing fusion transcript detection algorithm by eliminating its cell line-derived filtering steps, and instead, prioritize candidates using a custom workflow that integrates genomic and transcriptomic sequence alignment, biological and technical annotations, customized categorization logic, and phenotypic prioritization.ResultsWe demonstrate that our approach to fusion transcript identification and prioritization detects genuine fusion events excluded by standard analyses and efficiently removes phenotypically unimportant candidates and false positive events, resulting in a reduced candidate list enriched for events with potential phenotypic relevance. We describe the successful genetic resolution of two previously undiagnosed disease cases through the detection of pathogenic fusion transcripts. Furthermore, we report the experimental validation of five additional cases of fusion transcripts with potential phenotypic relevance.ConclusionsThe approach we describe can be implemented to enable the detection of phenotypically relevant fusion transcripts in studies of rare inherited disease. Fusion transcript detection has the potential to increase diagnostic rates in rare inherited disease and should be included in RNA-based analytical pipelines aimed at genetic diagnosis.

Diagnostic exome sequencing yields a single genetic diagnosis in ∼30% of cases, and according to recent studies the prevalence of identifying two genetic conditions in a single individual range between 4.6% and 7%. We present a patient diagnosed with three different rare conditions, each explained by a pathogenic variant in a different gene. A 17-yr-old female was evaluated for a history of motor and speech delay, scoliosis, distinctive craniofacial features, and dry skin in the Department of Clinical Genomics at Mayo Clinic. Her distinctive features included prominent forehead, epicanthus, depressed nasal bridge, narrow mouth, prognathism, malar flattening, and oligodontia. Family history was notable for dry skin in her mother and missing teeth in the paternal grandmother. Previous diagnostic testing was unrevealing including biochemical testing, echocardiogram, abdominal ultrasound, and electroencephalogram. Previous genetic testing included a microarray-based comparative genomic hybridization that was reported normal. Three pathogenic loss-of-function heterozygous variants were identified by exome trio sequencing, each linked to different genetic conditions: SIN3A (Witteveen–Kolk syndrome), FLG (dermatitis), and EDAR (ectodermal dysplasia). Together, these three genetic alterations could explain the patient's overall phenotype. This patient demonstrates the importance of performing a thorough curation of exome data when presented with a complex phenotype. Although phenotypic variability can explain some of these situations, the hypothesis of multiple diseases coexisting in a single patient should never be disregarded completely.

Array comparative genomic hybridization (aCGH) technology is commonly used to estimate genome-wide copy-number variation and to evaluate associations between copy number and disease. Although aCGH technology is well developed and there are numerous algorithms available for estimating copy number, little attention has been paid to the important issue of the statistical experimental design. Herein, we review classical statistical experimental designs and discuss their relevance to aCGH technology as well as their importance for downstream statistical analyses. Furthermore, we provide experimental design guidance for various study objectives.

Table of contents I1 Proceedings of the Fifteenth Annual UT- KBRIN Bioinformatics Summit 2016 Eric C. Rouchka, Julia H. Chariker, Benjamin J. Harrison, Juw Won Park P1 CC-PROMISE: Projection onto the Most Interesting Statistical Evidence (PROMISE) with Canonical Correlation to integrate gene expression and methylation data with multiple pharmacologic and clinical endpoints Xueyuan Cao, Stanley Pounds, Susana Raimondi, James Downing, Raul Ribeiro, Jeffery Rubnitz, Jatinder Lamba P2 Integration of microRNA-mRNA interaction networks with gene expression data to increase experimental power Bernie J Daigle, Jr. P3 Designing and writing software for in silico subtractive hybridization of large eukaryotic genomes Deborah Burgess, Stephanie Gehrlich, John C Carmen P4 Tracking the molecular evolution of Pax gene Nicholas Johnson; Chandrakanth Emani P5 Identifying genetic differences in thermally dimorphic and state specific fungi using in silico genomic comparison Stephanie Gehrlich, Deborah Burgess, John C Carmen P6 Identification of conserved genomic regions and variation therein amongst Cetartiodactyla species using next generation sequencing Kalpani De Silva, Michael P Heaton, Theodore S Kalbfleisch P7 Mining physiological data to identify patients with similar medical events and phenotypes Teeradache Viangteeravat, Rahul Mudunuri, Oluwaseun Ajayi, Fatih Şen, Eunice Y Huang P8 Smart brief for home health monitoring Mohammad Mohebbi, Luaire Florian, Douglas J Jackson, John F Naber P9 Side-effect term matching for computational adverse drug reaction predictions AKM Sabbir, Sally R Ellingson P10 Enrichment vs robustness: A comparison of transcriptomic data clustering metrics Yuping Lu, Charles A Phillips, Michael A Langston P11 Deep neural networks for transcriptome-based cancer classification Rahul K Sevakula, Raghuveer Thirukovalluru, Nishchal K. Verma, Yan Cui P12 Motif discovery using K-means clustering Mohammed Sayed, Juw Won Park P13 Large scale discovery of active enhancers from nascent RNA sequencing Jing Wang, Qi Liu, Yu Shyr P14 Computationally characterizing genomic pipelines and benchmarking results using GATK best practices on the high performance computing cluster at the University of Kentucky Xiaofei Zhang, Sally R Ellingson P15 Development of approaches enabling the identification of abnormal gene expression from RNA-Seq in personalized oncology Naresh Prodduturi, Gavin R Oliver, Diane Grill, Jie Na, Jeanette Eckel-Passow, Eric W Klee P16 Processing RNA-Seq data of plants infected with coffee ringspot virus Michael M Goodin, Mark Farman, Harrison Inocencio, Chanyong Jang, Jerzy W Jaromczyk, Neil Moore, Kelly Sovacool P17 Comparative transcriptomics of three Acinetobacter baumanii clinical isolates with different antibiotic resistance patterns Leon Dent, Mike Izban, Sammed Mandape, Shruti Sakhare, Siddharth Pratap, Dana Marshall P18 Metagenomic assessment of possible microbial contamination in the equine reference genome assembly M Scotty DePriest, James N MacLeod, Theodore S Kalbfleisch P19 Molecular evolution of cancer driver genes Chandrakanth Emani, Hanady Adam, Ethan Blandford, Joel Campbell, Joshua Castlen, Brittany Dixon, Ginger Gilbert, Aaron Hall, Philip Kreisle, Jessica Lasher, Bethany Oakes, Allison Speer, Maximilian Valentine P20 Biorepository Laboratory Information Management System Naga Satya V Rao Nagisetty, Rony Jose, Teeradache Viangteeravat, Robert Rooney, David Hains

STAR syndrome is a rare X-linked dominant disorder characterized by toe Syndactyly, Telecanthus, Anogenital malformations, and Renal malformations, and is caused by loss-of-function variants in FAM58A. Our proband presented with the hallmark features of STAR syndrome, as well as some additional less typical features including tethered cord and hearing loss. The proband's mother and maternal half-sister had similar clinical histories, but had variability in phenotypic severity. Clinical whole exome sequencing revealed a novel pathogenic nonsense variant, c.651G>A (p.Trp217X; NM_152274), in FAM58A in the proband, mother, and maternal half-sister. This pedigree represents the 11-13th patients described with STAR syndrome and the third instance of familial inheritance. To our knowledge, this is the first occurrence of a nonsense variant in FAM58A described in individuals with STAR syndrome and the phenotype in this pedigree suggests that tethered cord and hearing loss are features of STAR syndrome.

Prior studies in with irritable bowel syndrome with diarrhea (IBS-D) patients showed immune activation, secretion, and barrier dysfunction in jejunal or colorectal mucosa. We measured mRNA expression by RT-PCR of 91 genes reflecting tight junction proteins, chemokines, innate immunity, ion channels, transmitters, housekeeping genes, and controls for DNA contamination and PCR efficiency in small intestinal mucosa from 15 IBS-D and 7 controls (biopsies negative for celiac disease). Fold change was calculated using 2(−ΔΔCT) formula. Nominal P values ( P < 0.05) were interpreted with false detection rate (FDR) correction ( q value). Cluster analysis with Lens for Enrichment and Network Studies (LENS) explored connectivity of mechanisms. Upregulated genes (uncorrected P < 0.05) were related to ion transport (INADL, MAGI1, and SONS1), barrier (TJP1, 2, and 3 and CLDN) or immune functions (TLR3, IL15, and MAPKAPK5), or histamine metabolism (HNMT); downregulated genes were related to immune function (IL-1β, TGF-β1, and CCL20) or antigen detection (TLR1 and 8). The following genes were significantly upregulated ( q < 0.05) in IBS-D: INADL, MAGI1, PPP2R5C, MAPKAPK5, TLR3, and IL-15. Among the 14 nominally upregulated genes, there was clustering of barrier and PDZ domains (TJP1, TJP2, TJP3, CLDN4, INADL, and MAGI1) and clustering of downregulated genes (CCL20, TLR1, IL1B, and TLR8). Protein expression of PPP2R5C in nuclear lysates was greater in patients with IBS-D and controls. There was increase in INADL protein (median 9.4 ng/ml) in patients with IBS-D relative to controls (median 5.8 ng/ml, P > 0.05). In conclusion, altered transcriptome (and to lesser extent protein) expression of ion transport, barrier, immune, and mast cell mechanisms in small bowel may reflect different alterations in function and deserves further study in IBS-D.

Current variant discovery approaches often rely on an initial read mapping to the reference sequence. Their effectiveness is limited by the presence of gaps, potential misassemblies, regions of duplicates with a high-sequence similarity and regions of high-sequence divergence in the reference. Also, mapping-based approaches are less sensitive to large INDELs and complex variations and provide little phase information in personal genomes. A few de novo assemblers have been developed to identify variants through direct variant calling from the assembly graph, micro-assembly and whole-genome assembly, but mainly for whole-genome sequencing (WGS) data. We developed SGVar, a de novo assembly workflow for haplotype-based variant discovery from whole-exome sequencing (WES) data. Using simulated human exome data, we compared SGVar with five variation-aware de novo assemblers and with BWA-MEM together with three haplotype- or local de novo assembly-based callers. SGVar outperforms the other assemblers in sensitivity and tolerance of sequencing errors. We recapitulated the findings on whole-genome and exome data from a Utah residents with Northern and Western European ancestry (CEU) trio, showing that SGVar had high sensitivity both in the highly divergent human leukocyte antigen (HLA) region and in non-HLA regions of chromosome 6. In particular, SGVar is robust to sequencing error, k-mer selection, divergence level and coverage depth. Unlike mapping-based approaches, SGVar is capable of resolving long-range phase and identifying large INDELs from WES, more prominently from WGS. We conclude that SGVar represents an ideal platform for WES-based variant discovery in highly divergent regions and across the whole genome.

During the last 25 years, a small number of meaningful DNA-based diagnostic tests have been available to aid in the diagnosis and subsequent treatment of heritable disorders. These tests have targeted a limited number of genes and are often ordered in serial testing strategies in which results from one preliminary test dictate the subsequent test orders. This approach can be both time and resource intensive when a patient requires several genes to be sequenced. Recently, there has been much discussion regarding how 'massively parallel' or 'next-generation' DNA sequencing will impact clinical care. While the technology promises to reduce the cost of sequencing an entire human genome to less than US$1000, one must question the diagnostic utility of complete genome sequencing for routine clinical testing, given the many interpretive challenges posed by this approach. At present, it appears next-generation DNA sequencing may provide the greatest benefit to routine clinical testing by enabling comprehensive multigene panel sequencing. This should provide an advantage over traditional Sanger-based sequencing strategies while limiting the total test output to sets to genes with known diagnostic value. This article will discuss the current and near future state of clinical testing approaches and explore what challenges must be addressed in order to extract diagnostic value from whole-exome sequencing and whole-genome sequencing, using hereditary colon cancer as an example.

Advanced prostate cancer has significant long-term morbidity, and there is a growing interest in alternative and complimentary forms of therapy that will improve the outcomes of patients. Azadirachta indica (common name: neem) contains multiple active compounds that have potent anti-inflammatory and anticancer properties. The present study investigates the novel targets of the anticancer activity of ethanol extract of neem leaves (EENL) in vitro and evaluates the in vivo efficacy in the prostate cancer models. Analysis of the components in the EENL by mass spectrometry suggests the presence of 2′,3′-dehydrosalannol, 6-desacetyl nimbinene, and nimolinone. Treatment of C4-2B and PC-3M-luc2 prostate cancer cells with EENL inhibited the cell proliferation. Genome-wide expression profiling, using oligonucleotide microarrays, revealed genes differentially expressed with EENL treatment in prostate cancer cells. Functional analysis unveiled that most of the up-regulated genes were associated with cell death, and drug metabolism, and the down-regulated genes were associated with cell cycle, DNA replication, recombination, and repair functions. Quantitative PCR confirmed significant up-regulation of 40 genes and immunoblotting revealed increase in the protein expression levels of HMOX1, AKR1C2, AKR1C3, and AKR1B10. EENL treatment inhibited the growth of C4-2B and PC-3M-luc2 prostate cancer xenografts in nude mice. The suppression of tumor growth is associated with the formation of hyalinized fibrous tumor tissue and the induction of cell death by apoptosis. These results suggest that EENL-containing natural bioactive compounds could have potent anticancer property and the regulation of multiple cellular pathways could exert pleiotrophic effects in prevention and treatment of prostate cancer. Electronic supplementary material The online version of this article (doi:10.1208/s12248-011-9279-4) contains supplementary material, which is available to authorized users.

Introduction: Zebrafish are emerging as a powerful animal model for studying the molecular and physiological effects of nicotine exposure. The zebrafish have many advantageous physical characteristics, including small size, high fecundity rates, and externally developing transparent embryos. When combined with a battery of molecular-genetic tools and behav- ioral assays, these attributes enable studies to be conducted that are not practical using traditional animal models. Methods: We reviewed the literature on the application of the ze- brafish model as a preclinical model to study the biological effects of nicotine exposure. Results: The identified studies used zebrafish to examine the effects of nicotine exposure on early development, addic- tion, anxiety, and learning. The methods used included green fluorescent protein-labeled proteins to track in vivo nico- tine-altered neuron development, nicotine-conditioned place preference, and locomotive sensitization linked with high-throughput molecular and genetic screens and behav- ioral models of learning and stress response to nicotine. Data are presented on the complete homology of all known hu- man neural nicotinic acetylcholine receptors in zebrafish and on the biological similarity of human and zebrafish dopami- nergic signaling. Conclusions: Tobacco dependence remains a major health problem worldwide. Further understanding of the molecular ef- fects of nicotine exposure and genetic contributions to depen- dence may lead to improvement in patient treatment strategies. While there are limitations to the use of zebrafish as a preclinical model, it should provide a valuable tool to complement existing model systems. The reviewed studies demonstrate the enor- mous opportunity zebrafish have to advance the science of nico - tine and tobacco research.

Background Pathogenic variants in ryanodine receptor 1 (RYR1, MIM# 180901) are the cause of congenital myopathy with fiber‐type disproportion, malignant hyperthermia susceptibility type 1, central core disease of muscle, multiminicore disease and other congenital myopathies. Methods We present a patient with global developmental delay, hypotonia, myopathy, joint hypermobility, and multiple other systemic complaints that were noted early in life. Later she was found to have multiple bone deformities involving her spine, with severe scoliosis that was corrected surgically. She was also diagnosed with ophthalmoplegia, chronic hypercapnic respiratory failure, and hypertension. At 22 years of age she presented to the genetics clinic with a diagnosis of mitochondrial myopathy and underwent whole exome sequencing (WES). Results Whole exome sequencing revealed two novel compound heterozygous variants in RYR1 (c.7060_7062del, p.Val2354del and c.4485_4500del, p.Tyr1495X). Conclusion Review of her clinical, pathologic, and genetic findings pointed to a diagnosis of a congenital myopathy with fiber‐type disproportion.

The emerging scientific field of proteomics encompasses the identification, characterization, and quantification of the protein content or proteome of whole cells, tissues, or body fluids. The potential for proteomic technologies to identify and quantify novel proteins in the plasma that can function as biomarkers of the presence or severity of clinical disease states holds great promise for clinical use. However, there are many challenges in translating plasma proteomics from bench to bedside, and relatively few plasma biomarkers have successfully transitioned from proteomic discovery to routine clinical use. Key barriers to this translation include the need for “orthogonal” biomarkers (i.e., uncorrelated with existing markers), the complexity of the proteome in biological samples, the presence of high abundance proteins such as albumin in biological samples that hinder detection of low abundance proteins, false positive associations that occur with analysis of high dimensional datasets, and the limited understanding of the effects of growth, development, and age on the normal plasma proteome. Strategies to overcome these challenges are discussed.

The secretome is a functionally rich proteome subset, including cellular membrane and extracellular proteins processed through the secretory pathway. In this study, Danio rerio and Homo sapiens RefSeq proteins were analyzed with SignalP, TargetP, Phobius, and pTarget algorithms. About 16.5% of the zebrafish proteome and 17.0% of the human proteome possessed predicted N-terminal signal sequences. Nearly half of these proteins were subsequently classified as soluble, as they lacked predicted transmembrane domains. The soluble proteins were further subclassified, predicting 1345 (3.8%) zebrafish and 1207 (3.2%) human proteins as extracellular. Comparison of the zebrafish and human soluble secretome proteins identified 372 as orthologs, on the basis of reciprocal BLAST best hits. The computational characterization of the zebrafish proteins found many more members of the secretome than annotated in the SwissProt database. Only 180 of the 2078 zebrafish SwissProt protein entries, and 995 of the 19,294 human SwissProt protein entries were annotated with secreted protein locales. A specific investigation of the fibroblast growth factor and matrix metalloproteinase (MMP) protein families confirmed the prediction data and generated annotation of three additional putative MMP zebrafish proteins. This study presents the first known published description of the zebrafish secretome since the completion of the zebrafish genome sequencing project.

Purpose: This paper describes a process for the identification of genes that can report on the aggressiveness of prostate tumors and thereby add to the information provided by current pathologic analysis. Materials and Methods: Expression profiling data from over 100 laser capture microdissection derived samples from nonneoplastic epithelium; Gleason patterns 3, 4, and 5 and node metastasis prostate cancer were used to identify genes at abnormally high levels in only some tumors. These variably overexpressed genes were stratified by their association with aggressive phenotypes and were subsequently filtered to exclude genes with redundant expression patterns. Selected genes were validated in a case-control study in which cases (systemic progression within 5 years) and controls (no systemic progression at 7 years of follow-up) were matched for all clinical and pathologic criteria from time of prostatectomy (n = 175). Both cases and controls, therefore, could have nodal invasion or seminal vesicle involvement at the time of initial treatment. Results: A number of candidate variably overexpressed genes selected for their association with aggressive prostate cancer phenotype were evaluated in the case control study. The most prominent candidates were SSTR1 and genes related to proliferation, including TOP2A. Conclusions: The process described here identified genes that add information not available from current clinical measures and can improve the prognosis of prostate cancer.

The ability to identify classically secreted proteins is an important component of targeted therapeutic studies and the discovery of circulating biomarkers. Here, we review some of the most recent programs available for the in silico prediction of secretory proteins, the performance of which is benchmarked with an independent set of annotated human proteins. The description of these programs and the results of this benchmarking provide insights into the most recently developed prediction programs, which will enable investigators to make more informed decisions about which program best addresses their research needs.

To describe the experience and outcome of performing whole-exome sequencing (WES) for resolution of patients on a diagnostic odyssey in the first 18 months of an individualized medicine clinic (IMC).The IMC offered WES to physicians of Mayo Clinic practice for patients with suspected genetic disease. DNA specimens of the proband and relatives were submitted to WES laboratories. We developed the Genomic Odyssey Board with multidisciplinary expertise to determine the appropriateness for IMC services, review WES reports, and make the final decision about whether the exome findings explain the disease. This study took place from September 30, 2012, to March 30, 2014.In the first 18 consecutive months, the IMC received 82 consultation requests for patients on a diagnostic odyssey. The Genomic Odyssey Board deferred 7 cases and approved 75 cases to proceed with WES. Seventy-one patients met with an IMC genomic counselor. Fifty-one patients submitted specimens for WES testing, and the results have been received for all. There were 15 cases in which a diagnosis was made on the basis of WES findings; thus, the positive diagnostic yield of this practice was 29%. The mean cost per patient for this service was approximately $8000. Medicaid supported 27% of the patients, and 38% of patients received complete or partial insurance coverage.The significant diagnostic yield, moderate cost, and notable health marketplace acceptance for WES compared with conventional genetic testing make the former method a rational diagnostic approach for patients on a diagnostic odyssey.

Current clinical laboratory practice guidelines for next-generation sequencing (NGS) do not provide definitive guidance on confirming NGS variants. Sanger confirmation of NGS results can be inefficient, redundant, and expensive. We evaluated the accuracy of NGS-detected single-nucleotide variants (SNVs) and insertion/deletion variants (indels) and the necessity of NGS variant confirmation using four NGS target-capture gene panels covering 117 genes, 568 Kbp, and 77 patient DNA samples. Unique NGS-detected variants (1080 SNVs and 124 indels) underwent Sanger confirmation and/or were compared to data from the 1000 Genomes Project (1000G). Recurrent variants in unrelated samples resulted in 919 comparisons between NGS and Sanger, with 100% concordance. In a second comparison, 762 unique NGS results (736 SNVs, 26 indels) from seven 1000G samples were found to have 97.1% concordance with 1000G phase 1 data. Sanger sequencing and 1000G phase 3 data confirmed the accuracy of the NGS results for all 1000G phase 1 discrepancies. In all samples, the depth of coverage exceeded 100× in >99.7% of bases in the target regions. In conclusion, confirmatory analysis by Sanger sequencing of SNVs detected via capture-based NGS testing that meets appropriate quality thresholds is unnecessarily redundant. In contrast, Sanger sequencing for indels may be required for defining the correct genomic location, and Sanger may be used for quality-assurance purposes.

BACKGROUND Next generation sequencing (NGS)-based assays continue to redefine the field of genetic testing. Owing to the complexity of the data, bioinformatics has become a necessary component in any laboratory implementing a clinical NGS test. CONTENT The computational components of an NGS-based work flow can be conceptualized as primary, secondary, and tertiary analytics. Each of these components addresses a necessary step in the transformation of raw data into clinically actionable knowledge. Understanding the basic concepts of these analysis steps is important in assessing and addressing the informatics needs of a molecular diagnostics laboratory. Equally critical is a familiarity with the regulatory requirements addressing the bioinformatics analyses. These and other topics are covered in this review article. SUMMARY Bioinformatics has become an important component in clinical laboratories generating, analyzing, maintaining, and interpreting data from molecular genetics testing. Given the rapid adoption of NGS-based clinical testing, service providers must develop informatics work flows that adhere to the rigor of clinical laboratory standards, yet are flexible to changes as the chemistry and software for analyzing sequencing data mature.

AMOD is a web-based program that aids in the functional evaluation of nucleotide sequences through sequence characterization and antisense morpholino oligonucleotide (target site) selection. Submitted sequences are analyzed by translation initiation site prediction algorithms and sequence-to-sequence comparisons; results are used to characterize sequence features required for morpholino design. Within a defined subsequence, base composition and homodimerization values are computed for all putative morpholino oligonucleotides. Using these properties, morpholino candidates are selected and compared with genomic and transcriptome databases with the goal to identify target-specific enriched morpholinos. AMOD has been used at the University of Minnesota to design approximately 200 morpholinos for a functional genomics screen in zebrafish. The AMOD web server and a tutorial are freely available to both academic and commercial users at http://www.secretomes.umn.edu/AMOD/.

Bosch–Boonstra–Schaaf optic atrophy syndrome (BBSOAS) is a recently described autosomal dominant disorder caused by mutations in the NR2F1 gene. There are presently 28 cases of BBSOAS described in the literature. Its common features include developmental delay, intellectual disability, hypotonia, optic nerve atrophy, attention deficit disorder, autism spectrum disorder, seizures, hearing defects, spasticity, and thinning of the corpus callosum. Here we report two unrelated probands with novel, de novo, missense variants in NR2F1. The first is a 14-yr-old male patient with hypotonia, intellectual disability, optic nerve hypoplasia, delayed bone age, short stature, and altered neurotransmitter levels on cerebrospinal fluid testing. The second is a 5-yr-old female with severe developmental delay, motor and speech delay, and repetitive motion behavior. Whole-exome sequencing identified a novel missense NR2F1 variant in each case, Cys86Phe in the DNA-binding domain in Case 1, and a Leu372Pro in the ligand-binding domain in Case 2. The presence of clinical findings compatible with BBSOAS along with structural analysis at atomic resolution using homology-based molecular modeling and molecular dynamic simulations, support the pathogenicity of these variants for BBSOAS. Short stature, abnormal CNS neurotransmitters, and macrocephaly have not been previously reported for this syndrome and may represent a phenotypic expansion of BBSOAS. A review of published cases along with new evidence from this report support genotype–phenotype correlations for this disorder.

TGF-β-related heritable connective tissue disorders are characterized by a similar pattern of cardiovascular defects, including aortic root dilatation, mitral valve prolapse, vascular aneurysms, and vascular dissections and exhibit incomplete penetrance and variable expressivity. Because of the phenotypic overlap of these disorders, panel-based genetic testing is frequently used to confirm the clinical findings. Unfortunately in many cases, variants of uncertain significance (VUSs) obscure the genetic diagnosis until more information becomes available. Here, we describe and characterize the functional impact of a novel VUS in the TGFBR2 kinase domain (c.1255G>T; p.Val419Leu), in a patient with the clinical diagnosis of Marfan syndrome spectrum. We assessed the structural and functional consequence of this VUS using molecular modeling, molecular dynamic simulations, and in vitro cell-based assays. A high-quality homology-based model of TGFBR2 was generated and computational mutagenesis followed by refinement and molecular dynamics simulations were used to assess structural and dynamic changes. Relative to wild type, the V419L induced conformational and dynamic changes that may affect ATP binding, increasing the likelihood of adopting an inactive state, and, we hypothesize, alter canonical signaling. Experimentally, we tested this by measuring the canonical TGF-β signaling pathway activation at two points; V419L significantly delayed SMAD2 phosphorylation by western blot and significantly decreased TGF-β-induced gene transcription by reporter assays consistent with known pathogenic variants in this gene. Thus, our results establish that the V419L variant leads to aberrant TGF-β signaling and confirm the diagnosis of Loeys–Dietz syndrome in this patient.