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1.  Mutations in IFT172 cause isolated retinal degeneration and Bardet–Biedl syndrome 
Human Molecular Genetics  2014;24(1):230-242.
Primary cilia are sensory organelles present on most mammalian cells. The assembly and maintenance of primary cilia are facilitated by intraflagellar transport (IFT), a bidirectional protein trafficking along the cilium. Mutations in genes coding for IFT components have been associated with a group of diseases called ciliopathies. These genetic disorders can affect a variety of organs including the retina. Using whole exome sequencing in three families, we identified mutations in Intraflagellar Transport 172 Homolog [IFT172 (Chlamydomonas)] that underlie an isolated retinal degeneration and Bardet–Biedl syndrome. Extensive functional analyses of the identified mutations in cell culture, rat retina and in zebrafish demonstrated their hypomorphic or null nature. It has recently been reported that mutations in IFT172 cause a severe ciliopathy syndrome involving skeletal, renal, hepatic and retinal abnormalities (Jeune and Mainzer-Saldino syndromes). Here, we report for the first time that mutations in this gene can also lead to an isolated form of retinal degeneration. The functional data for the mutations can partially explain milder phenotypes; however, the involvement of modifying alleles in the IFT172-associated phenotypes cannot be excluded. These findings expand the spectrum of disease associated with mutations in IFT172 and suggest that mutations in genes originally reported to be associated with syndromic ciliopathies should also be considered in subjects with non-syndromic retinal dystrophy.
PMCID: PMC4326328  PMID: 25168386
2.  Age-dependent effects of RPE65 gene therapy for Leber’s congenital amaurosis: a phase 1 dose-escalation trial 
Lancet (London, England)  2009;374(9701):1597-1605.
Gene therapy has the potential to reverse disease or prevent further deterioration of vision in patients with incurable inherited retinal degeneration. We therefore did a phase 1 trial to assess the effect of gene therapy on retinal and visual function in children and adults with Leber’s congenital amaurosis.
We assessed the retinal and visual function in 12 patients (aged 8–44 years) with RPE65-associated Leber’s congenital amaurosis given one subretinal injection of adeno-associated virus (AAV) containing a gene encoding a protein needed for the isomerohydrolase activity of the retinal pigment epithelium (AAV2-hRPE65v2) in the worst eye at low (1·5×1010 vector genomes), medium (4·8×1010 vector genomes), or high dose (1·5×1011 vector genomes) for up to 2 years.
AAV2-hRPE65v2 was well tolerated and all patients showed sustained improvement in subjective and objective measurements of vision (ie, dark adaptometry, pupillometry, electroretinography, nystagmus, and ambulatory behaviour). Patients had at least a 2 log unit increase in pupillary light responses, and an 8-year-old child had nearly the same level of light sensitivity as that in age-matched normal-sighted individuals. The greatest improvement was noted in children, all of whom gained ambulatory vision. The study is registered with, number NCT00516477.
The safety, extent, and stability of improvement in vision in all patients support the use of AAV-mediated gene therapy for treatment of inherited retinal diseases, with early intervention resulting in the best potential gain.
Center for Cellular and Molecular Therapeutics at the Children’s Hospital of Philadelphia, Foundation Fighting Blindness, Telethon, Research to Prevent Blindness, F M Kirby Foundation, Mackall Foundation Trust, Regione Campania Convenzione, European Union, Associazione Italiana Amaurosi Congenita di Leber, Fund for Scientific Research, Fund for Research in Ophthalmology, and National Center for Research Resources.
PMCID: PMC4492302  PMID: 19854499
3.  Suture Forces in Undersized Mitral Annuloplasty: Novel Device and Measurements 
The Annals of thoracic surgery  2014;98(1):305-309.
Demonstrate the first use of a novel technology for quantifying suture forces on annuloplasty rings to better understand the mechanisms of ring dehiscence.
Force transducers were developed, attached to a size 24 Physio™ ring, and implanted in the mitral annulus of an ovine animal. Ring suture forces were measured after implantation and for cardiac cycles reaching peak left ventricular pressures (LVP) of 100, 125, and 150 mmHg.
After implanting the undersized ring to the flaccid annulus, the mean suture force was 2.0±0.6 N. During cyclic contraction, anterior ring suture forces were greater than posterior ring suture forces at peak LVPs of 100 mmHg (4.9±2.0 N vs. 2.1±1.1 N), 125 mmHg (5.4±2.3 N vs. 2.3±1.2 N), and 150 mmHg (5.7±2.4 N vs. 2.4±1.1 N). The largest force was 7.4 N at 150 mmHg.
Preliminary results demonstrate trends in annuloplasty suture forces and their variation with location and LVP. Future studies will significantly contribute to clinical knowledge by elucidating the mechanisms of ring dehiscence while improving annuloplasty ring design and surgical repair techniques.
PMCID: PMC4109808  PMID: 24996707
restrictive mitral annuloplasty; dehiscence; functional mitral regurgitation; suture; forces
4.  Targeted Exon Sequencing in Usher Syndrome Type I 
Patients with Usher syndrome type I (USH1) have retinitis pigmentosa, profound congenital hearing loss, and vestibular ataxia. This syndrome is currently thought to be associated with at least six genes, which are encoded by over 180 exons. Here, we present the use of state-of-the-art techniques in the molecular diagnosis of a cohort of 47 USH1 probands.
The cohort was studied with selective exon capture and next-generation sequencing of currently known inherited retinal degeneration genes, comparative genomic hybridization, and Sanger sequencing of new USH1 exons identified by human retinal transcriptome analysis.
With this approach, we were able to genetically solve 14 of the 47 probands by confirming the biallelic inheritance of mutations. We detected two likely pathogenic variants in an additional 19 patients, for whom family members were not available for cosegregation analysis to confirm biallelic inheritance. Ten patients, in addition to primary disease–causing mutations, carried rare likely pathogenic USH1 alleles or variants in other genes associated with deaf-blindness, which may influence disease phenotype. Twenty-one of the identified mutations were novel among the 33 definite or likely solved patients. Here, we also present a clinical description of the studied cohort at their initial visits.
We found a remarkable genetic heterogeneity in the studied USH1 cohort with multiplicity of mutations, of which many were novel. No obvious influence of genotype on phenotype was found, possibly due to small sample sizes of the genotypes under study.
Using targeted exon sequencing of currently known inherited retinal degeneration genes, we analyzed a cohort of 47 patients with Usher syndrome type I. We found a number of new primary disease-causing mutations and found additional variants in Usher-associated genes which may modify the phenotype.
PMCID: PMC4280089  PMID: 25468891
Usher syndrome; retinitis pigmentosa; hearing loss; retina; genetics; next-generation sequencing; selective exon capture; molecular diagnostic
5.  Statistical Modeling of Behavioral Dynamics During Propofol-Induced Loss of Consciousness 
Accurate quantitative analysis of the changes in responses to external stimuli is crucial for characterizing the timing of loss and recovery of consciousness induced by anesthetic drugs. We studied induction and emergence from unconsciousness achieved by administering a computer-controlled infusion of propofol to ten human volunteers. We evaluated loss and recovery of consciousness by having subjects execute every four seconds two interleaved computer delivered behavioral tasks: responding to verbal stimuli (neutral words or the subject's name), or less salient stimuli of auditory clicks.
New Method
We analyzed the data using state-space methods. For each stimulus type the observation model is a two-stage binomial model and the state model is two dimensional random walk in which one cognitive state governs the probability of responding and the second governs the probability of correctly responding given a response. We fit the model to the experimental data using Bayesian Monte Carlo methods.
During induction subjects lost responsiveness to less salient clicks before losing responsiveness to the more salient verbal stimuli. During emergence subjects regained responsiveness to the more salient verbal stimuli before regaining responsiveness to the less salient clicks.
Comparison with Existing Method(s)
The current state-space model is an extension of previous model used to analyze learning and behavioral performance. In this study, the probability of responding on each trial is obtained separately from the probability of behavioral performance.
Our analysis provides a principled quantitative approach for defining loss and recovery of consciousness in experimental studies of general anesthesia.
PMCID: PMC4304648  PMID: 24530701
Bayesian Monte Carlo methods; behavioral data; propofol; state-space models; unconsciousness
6.  Quantitative Evaluation of Annuloplasty on Mitral Valve Chordae Tendineae Forces to Supplement Surgical Planning Model Development 
Computational models of the heart’s mitral valve (MV) exhibit potential for preoperative surgical planning in ischemic mitral regurgitation (IMR). However challenges exist in defining boundary conditions to accurately model the function and response of the chordae tendineae to both IMR and surgical annuloplasty repair. Towards this goal, a ground-truth data set was generated by quantifying the isolated effects of IMR and mitral annuloplasty on leaflet coaptation, regurgitation, and tethering forces of the anterior strut and posterior intermediary chordae tendineae.
MVs were excised from ovine hearts (N=15) and mounted in a pulsatile heart simulator which has been demonstrated to mimic the systolic MV geometry and coaptation of healthy and chronic IMR sheep. Strut and intermediary chordae from both MV leaflets (N=4) were instrumented with force transducers. Tested conditions included a healthy control, IMR, oversized annuloplasty, true-sized annuloplasty, and undersized mitral annuloplasty. A2-P2 leaflet coaptation length, regurgitation, and chordal tethering were quantified and statistically compared across experimental conditions.
IMR was successfully simulated with significant increases in MR, tethering forces for each of the chordae, and decrease in leaflet coaptation (p<.05). Compared to the IMR condition, increasing levels of downsized annuloplasty significantly reduced regurgitation, increased coaptation, reduced posteromedial papillary muscle strut chordal forces, and reduced intermediary chordal forces from the anterolateral papillary muscle (p<.05).
These results provide for the first time a novel comprehensive data set for refining the ability of computational MV models to simulate IMR and varying sizes of complete rigid ring annuloplasty.
PMCID: PMC3951125  PMID: 24634699
Mitral Valve; Annuloplasty; Computational Methods; Chordae Tendineae; Heart Valve; Mitral Regurgitation
7.  Mouse genetics and proteomic analyses demonstrate a critical role for complement in a model of DHRD/ML, an inherited macular degeneration 
Human Molecular Genetics  2013;23(1):52-68.
Macular degenerations, inherited and age related, are important causes of vision loss. Human genetic studies have suggested perturbation of the complement system is important in the pathogenesis of age-related macular degeneration. The mechanisms underlying the involvement of the complement system are not understood, although complement and inflammation have been implicated in drusen formation. Drusen are an early clinical hallmark of inherited and age-related forms of macular degeneration. We studied one of the earliest stages of macular degeneration which precedes and leads to the formation of drusen, i.e. the formation of basal deposits. The studies were done using a mouse model of the inherited macular dystrophy Doyne Honeycomb Retinal Dystrophy/Malattia Leventinese (DHRD/ML) which is caused by a p.Arg345Trp mutation in EFEMP1. The hallmark of DHRD/ML is the formation of drusen at an early age, and gene targeted Efemp1R345W/R345W mice develop extensive basal deposits. Proteomic analyses of Bruch's membrane/choroid and Bruch's membrane in the Efemp1R345W/R345W mice indicate that the basal deposits comprise normal extracellular matrix (ECM) components present in abnormal amounts. The proteomic analyses also identified significant changes in proteins with immune-related function, including complement components, in the diseased tissue samples. Genetic ablation of the complement response via generation of Efemp1R345W/R345W:C3−/− double-mutant mice inhibited the formation of basal deposits. The results demonstrate a critical role for the complement system in basal deposit formation, and suggest that complement-mediated recognition of abnormal ECM may participate in basal deposit formation in DHRD/ML and perhaps other macular degenerations.
PMCID: PMC3857944  PMID: 23943789
8.  TMEM14C is required for erythroid mitochondrial heme metabolism 
The Journal of Clinical Investigation  2014;124(10):4294-4304.
The transport and intracellular trafficking of heme biosynthesis intermediates are crucial for hemoglobin production, which is a critical process in developing red cells. Here, we profiled gene expression in terminally differentiating murine fetal liver-derived erythroid cells to identify regulators of heme metabolism. We determined that TMEM14C, an inner mitochondrial membrane protein that is enriched in vertebrate hematopoietic tissues, is essential for erythropoiesis and heme synthesis in vivo and in cultured erythroid cells. In mice, TMEM14C deficiency resulted in porphyrin accumulation in the fetal liver, erythroid maturation arrest, and embryonic lethality due to profound anemia. Protoporphyrin IX synthesis in TMEM14C-deficient erythroid cells was blocked, leading to an accumulation of porphyrin precursors. The heme synthesis defect in TMEM14C-deficient cells was ameliorated with a protoporphyrin IX analog, indicating that TMEM14C primarily functions in the terminal steps of the heme synthesis pathway. Together, our data demonstrate that TMEM14C facilitates the import of protoporphyrinogen IX into the mitochondrial matrix for heme synthesis and subsequent hemoglobin production. Furthermore, the identification of TMEM14C as a protoporphyrinogen IX importer provides a genetic tool for further exploring erythropoiesis and congenital anemias.
PMCID: PMC4191016  PMID: 25157825
9.  AAV2 Gene Therapy Readministration in Three Adults with Congenital Blindness 
Science translational medicine  2012;4(120):120ra15.
Demonstration of safe and stable reversal of blindness after a single unilateral subretinal injection of a recombinant adeno-associated virus (AAV) carrying the RPE65 gene (AAV2-hRPE65v2) prompted us to determine whether it was possible to obtain additional benefit through a second administration of the AAV vector to the contralateral eye. Readministration of vector to the second eye was carried out in three adults with Leber congenital amaurosis due to mutations in the RPE65 gene 1.7 to 3.3 years after they had received their initial subretinal injection of AAV2-hRPE65v2. Results (through 6 months) including evaluations of immune response, retinal and visual function testing, and functional magnetic resonance imaging indicate that readministration is both safe and efficacious after previous exposure to AAV2-hRPE65v2.
PMCID: PMC4169122  PMID: 22323828
10.  Application of Next-Generation Sequencing to Identify Genes and Mutations Causing Autosomal Dominant Retinitis Pigmentosa (adRP) 
The goal of our research is to identify genes and mutations causing auto-somal dominant retinitis pigmentosa (adRP). For this purpose we established a cohort of more than 250 independently ascertained families with adRP in the Houston Laboratory for Molecular Diagnosis of Inherited Eye Diseases. Affected members of each family were screened for disease-causing mutations in genes and gene regions that are commonly associated with adRP. By this approach, we detected mutations in 65 % of the families, leaving 85 families that are likely to harbor mutations outside of the “common” regions or in novel genes. Of these, 32 families were tested by several types of next-generation sequencing (NGS), including (a) targeted polymerase chain reaction (PCR) NGS, (b) whole exome NGS, and (c) targeted retinal-capture NGS. We detected mutations in 11 of these families (31 %) bringing the total detected in the adRP cohort to 70 %. Several large families have also been tested for linkage using Afymetrix single nucleotide polymorphism (SNP) arrays.
PMCID: PMC4121110  PMID: 24664689
Retinitis pigmentosa; Next-generation sequencing; Linkage mapping; Mutation prevalence; Retinal gene capture; Whole-exome sequencing
11.  A Transition in Brain State during Propofol-Induced Unconsciousness 
The Journal of Neuroscience  2014;34(3):839-845.
Rhythmic oscillations shape cortical dynamics during active behavior, sleep, and general anesthesia. Cross-frequency phase-amplitude coupling is a prominent feature of cortical oscillations, but its role in organizing conscious and unconscious brain states is poorly understood. Using high-density EEG and intracranial electrocorticography during gradual induction of propofol general anesthesia in humans, we discovered a rapid drug-induced transition between distinct states with opposite phase-amplitude coupling and different cortical source distributions. One state occurs during unconsciousness and may be similar to sleep slow oscillations. A second state occurs at the loss or recovery of consciousness and resembles an enhanced slow cortical potential. These results provide objective electrophysiological landmarks of distinct unconscious brain states, and could be used to help improve EEG-based monitoring for general anesthesia.
PMCID: PMC3891963  PMID: 24431442
α rhythm; anesthesia; cross-frequency coupling; propofol; Slow oscillation; unconsciousness
12.  Three Year Follow-Up after Unilateral Subretinal Delivery of Adeno-Associated Virus in Patients with Leber Congenital Amaurosis Type 2 
Ophthalmology  2013;120(6):1283-1291.
The aim of the current study is to show the clinical data of long-term (3 year) follow-up of five patients affected by Leber Congenital Amaurosis type 2 (LCA2) treated with a single unilateral injection of AAV2-hRPE65v2.
clinical trial
five LCA2 patients with RPE65 gene mutations
After informed consent and confirmation of trial eligibility criteria, the eye with worse visual function was selected for subretinal delivery of Adeno-Associated Virus (AAV2-hRPE65v2). Subjects were evaluated before and after surgery at designated follow-up visits (1, 2, 3, 14, 30, 60, 90, 180, 270, 365 days, 1.5 years and 3 years) by complete ophthalmic examination. Efficacy for each subject was monitored with best corrected visual acuity, kinetic visual field, nystagmus testing and pupillary light reflex.
Main Outcome Measures
best corrected visual acuity, kinetic visual field, nystagmus testing and pupillary light reflex.
The data showed a statistically significant improvement of best corrected visual acuity between baseline and 3 years after treatment in the treated eye (p<0.001). In all patients we observed an enlargement of the areas of visual field, which remained stable till 3 years post injection (average values: baseline 1058 deg2 vs 3 years post treatment: 4630 deg2) and a reduction of the nystagmus frequency compared to baseline at the 3 year time-point. Furthermore, a statistically significant difference was observed in the pupillary constriction of the treated eye (p<0.05) compared to the untreated eye in three patients at 1 and 3-year time-points. No patients suffered serious adverse events related to the vector in the 3 year post-injection period.
The long-term follow-up data (3 years) on the 5-patient Italian cohort involved in the LCA2 gene therapy clinical trial clearly showed a stability of improvement in visual and retinal function that had been achieved a few months after treatment. Longitudinal data analysis showed that the maximum improvement was achieved within six months after treatment, and the visual improvement was stable up to the last observed time-point.
PMCID: PMC3674112  PMID: 23474247
13.  Mitochondrial Disease Genetic Diagnostics: Optimized whole-exome analysis for all MitoCarta nuclear genes and the mitochondrial genome 
Discovery medicine  2012;14(79):389-399.
Discovering causative genetic variants in individual cases of suspected mitochondrial disease requires interrogation of both the mitochondrial (mtDNA) and nuclear genomes. Whole-exome sequencing can support simultaneous dual-genome analysis, although currently available capture kits do not target the mtDNA genome and provide insufficient capture for some nuclear-encoded mitochondrial genes. To optimize interrogation of nuclear and mtDNA genes relevant to mitochondrial biology and disease, a custom SureSelect “Mito-Plus” whole-exome library was formulated by blending RNA “baits” from three separate designs: (A) Agilent Technologies SureSelectXT 50 Mb All Exon PLUS Targeted Enrichment Kit, (B) 16-gene nuclear panel targeting sequences for known MitoCarta proteins not included in the 50 Mb All-Exon design, and (C) sequences targeting the entire mtDNA genome. The final custom formulations consisted of a 1:1 ratio of nuclear baits to which a 1 to 1,000-fold diluted ratio of mtDNA genome baits were blended. Patient sample capture libraries were paired-end sequenced on an Illumina HiSeq 2000 system using v3.0 SBS chemistry. mtDNA genome coverage varied depending on the mtDNA:nuclear blend ratio, where a 1:100 ratio provided optimal dual-genome coverage with 10X coverage for over 97.5% of all targeted nuclear regions and 1,000X coverage for 99.8% of the mtDNA genome. mtDNA mutations were reliably detected to at least an 8% heteroplasmy level, as discriminated both from sequencing errors and potential contamination from nuclear mtDNA transcripts (Numts). The “1:100 Mito-Plus Whole-Exome” Agilent capture kit offers an optimized tool for whole-exome analysis of nuclear and mtDNA genes relevant to the diagnostic evaluation of mitochondrial disease.
PMCID: PMC3923327  PMID: 23272691
Exome; Capture; Mitochondria; MitoCarta; heteroplasmy; variants; Agilent; SureSelect; HiSeq; NUMT
14.  Transcriptome analyses of the human retina identify unprecedented transcript diversity and 3.5 Mb of novel transcribed sequence via significant alternative splicing and novel genes 
BMC Genomics  2013;14:486.
The retina is a complex tissue comprised of multiple cell types that is affected by a diverse set of diseases that are important causes of vision loss. Characterizing the transcripts, both annotated and novel, that are expressed in a given tissue has become vital for understanding the mechanisms underlying the pathology of disease.
We sequenced RNA prepared from three normal human retinas and characterized the retinal transcriptome at an unprecedented level due to the increased depth of sampling provided by the RNA-seq approach. We used a non-redundant reference transcriptome from all of the empirically-determined human reference tracks to identify annotated and novel sequences expressed in the retina. We detected 79,915 novel alternative splicing events, including 29,887 novel exons, 21,757 3′ and 5′ alternate splice sites, and 28,271 exon skipping events. We also identified 116 potential novel genes. These data represent a significant addition to the annotated human transcriptome. For example, the novel exons detected increase the number of identified exons by 3%. Using a high-throughput RNA capture approach to validate 14,696 of these novel transcriptome features we found that 99% of the putative novel events can be reproducibly detected. Further, 15-36% of the novel splicing events maintain an open reading frame, suggesting they produce novel protein products.
To our knowledge, this is the first application of RNA capture to perform large-scale validation of novel transcriptome features. In total, these analyses provide extensive detail about a previously uncharacterized level of transcript diversity in the human retina.
PMCID: PMC3924432  PMID: 23865674
RNA-Seq; Transcriptome; Inherited retinal degeneration; Retina; Novel genes; Alternative splicing
15.  Mitochondrial Atpif1 regulates heme synthesis in developing erythroblasts 
Nature  2012;491(7425):608-612.
Defects in the availability of heme substrates or the catalytic activity of the terminal enzyme in heme biosynthesis, ferrochelatase (Fech), impair heme synthesis, and thus cause human congenital anemias1,2. The inter-dependent functions of regulators of mitochondrial homeostasis and enzymes responsible for heme synthesis are largely unknown. To uncover this unmet need, we utilized zebrafish genetic screens and cloned mitochondrial ATPase inhibitory factor 1 (atpif1) from a zebrafish mutant with profound anemia, pinotage (pnt tq209). We now report a direct mechanism establishing that Atpif1 regulates the catalytic efficiency of vertebrate Fech to synthesize heme. The loss of Atpif1 impairs hemoglobin synthesis in zebrafish, mouse, and human hematopoietic models as a consequence of diminished Fech activity, and elevated mitochondrial pH. To understand the relationship among mitochondrial pH, redox potential, [2Fe-2S] clusters, and Fech activity, we used (1) genetic complementation studies of Fech constructs with or without [2Fe-2S] clusters in pnt, and (2) pharmacological agents modulating mitochondrial pH and redox potential. The presence of [2Fe-2S] cluster renders vertebrate Fech vulnerable to Atpif1-regulated mitochondrial pH and redox potential perturbations. Therefore, Atpif1 deficiency reduces the efficiency of vertebrate Fech to synthesize heme, resulting in anemia. The novel mechanism of Atpif1 as a regulator of heme synthesis advances the understanding of mitochondrial heme homeostasis and red blood cell development. A deficiency of Atpif1 may contribute to important human diseases, such as congenital sideroblastic anemias and mitochondriopathies.
PMCID: PMC3504625  PMID: 23135403
16.  Mitochondrial tRNA-serine (AGY) m.C12264T mutation causes severe multisystem disease with cataracts 
Discovery medicine  2012;13(69):143-150.
Progressive multisystem disease should invoke consideration of potential mitochondrial etiologies. Mitochondrial disease can affect any organ system at any time, particularly involving neurologic, cardiac, muscular, gastroenterologic, and/or ophthalmologic manifestations. We report here a 19-year-old Caucasian man who was followed since birth in multiple pediatric subspecialty clinics for myelomeningocele complications. However, he progressively developed a host of additional problems that were not readily attributable to his neural tube defect but involved developmental, ophthalmologic, cardiac, muscular, endocrine, and intermediary metabolic manifestations. Clinical diagnostic testing limited to analysis for common point mutations and deletions in his blood mitochondrial DNA (mtDNA) was not revealing. Skeletal muscle biopsy revealed abnormal mitochondrial morphology and immunostaining, mitochondrial proliferation, and mildly reduced respiratory chain complex I–III activity. Whole mitochondrial genome sequencing analysis in muscle identified an apparently homoplasmic, novel, 12264C>T transition in the tRNA serine (AGY) gene. The pathogenicity of this mutation was supported by identification of it being present at low heteroplasmy load in his blood (34 percent) as well as in blood from his maternal grandmother (1 percent). Interestingly, the proband developed severe nuclear cataracts that proved to be homoplasmic for the pathogenic mtDNA 12264C>T mutation. This case highlights the value of pursuing whole mitochondrial genome sequencing in symptomatic tissues in the diagnostic evaluation of suspected mitochondrial disease. Furthermore, it is the first report to directly implicate an mtDNA mutation in the pathogenesis of ocular cataracts and clearly illustrates the important contribution of normal metabolic activity to the function of the ocular lens.
PMCID: PMC3618896  PMID: 22369973
17.  NMNAT1 mutations cause Leber congenital amaurosis 
Nature genetics  2012;44(9):1040-1045.
Leber congenital amaurosis (LCA) is an infantile-onset form of inherited retinal degeneration characterized by severe vision loss1, 2. Two-thirds of LCA cases are caused by mutations in 17 known disease genes3 (RetNet Retinal Information Network). Using exome sequencing, we identified a homozygous missense mutation (c.25G>A, p.Val9Met) in NMNAT1 as likely disease-causing in two siblings of a consanguineous Pakistani kindred affected by LCA. This mutation segregated with disease in their kindred, including in three other children with LCA. NMNAT1 resides in the previously identified LCA9 locus and encodes the nuclear isoform of nicotinamide mononucleotide adenylyltransferase, a rate-limiting enzyme in nicotinamide adenine dinucleotide (NAD+) biosynthesis4, 5. Functional studies showed the p.Val9Met mutation decreased NMNAT1 enzyme activity. Sequencing NMNAT1 in 284 unrelated LCA families identified 14 rare mutations in 13 additional affected individuals. These results are the first to link an NMNAT isoform to disease and indicate that NMNAT1 mutations cause LCA.
PMCID: PMC3454532  PMID: 22842227
18.  Knockdown of ttc26 disrupts ciliogenesis of the photoreceptor cells and the pronephros in zebrafish 
Molecular Biology of the Cell  2012;23(16):3069-3078.
The article describes characterization of the cilia protein Ttc26. The data show that Ttc26 is localized in the transition zone of primary cilia and photoreceptor cells. Knockdown of Ttc26 produced defective cilia in murine inner medullary collecting duct 3 cells and ciliogenesis defects in retinal photoreceptor and motile cilia in the pronephros in zebrafish.
In our effort to understand genetic disorders of the photoreceptor cells of the retina, we have focused on intraflagellar transport in photoreceptor sensory cilia. From previous mouse proteomic data we identified a cilia protein Ttc26, orthologue of dyf-13 in Caenorhabditis elegans, as a target. We localized Ttc26 to the transition zone of photoreceptor and to the transition zone of cilia in cultured murine inner medullary collecting duct 3 (mIMCD3) renal cells. Knockdown of Ttc26 in mIMCD3 cells produced shortened and defective primary cilia, as revealed by immunofluorescence and scanning electron microscopy. To study Ttc26 function in sensory cilia in vivo, we utilized a zebrafish vertebrate model system. Morpholino knockdown of ttc26 in zebrafish embryos caused ciliary defects in the pronephric kidney at 27 h postfertilization and distension/dilation of pronephros at 5 d postfertilization (dpf). In the eyes, the outer segments of photoreceptor cells appeared shortened or absent, whereas cellular lamination appeared normal in retinas at 5 dpf. This suggests that loss of ttc26 function prevents normal ciliogenesis and differentiation in the photoreceptor cells, and that ttc26 is required for normal development and differentiation in retina and pronephros. Our studies support the importance of Ttc26 function in ciliogenesis and suggest that screening for TTC26 mutations in human ciliopathies is justified.
PMCID: PMC3418303  PMID: 22718903
19.  Comparative analysis of RNA-Seq alignment algorithms and the RNA-Seq unified mapper (RUM) 
Bioinformatics  2011;27(18):2518-2528.
Motivation: A critical task in high-throughput sequencing is aligning millions of short reads to a reference genome. Alignment is especially complicated for RNA sequencing (RNA-Seq) because of RNA splicing. A number of RNA-Seq algorithms are available, and claim to align reads with high accuracy and efficiency while detecting splice junctions. RNA-Seq data are discrete in nature; therefore, with reasonable gene models and comparative metrics RNA-Seq data can be simulated to sufficient accuracy to enable meaningful benchmarking of alignment algorithms. The exercise to rigorously compare all viable published RNA-Seq algorithms has not been performed previously.
Results: We developed an RNA-Seq simulator that models the main impediments to RNA alignment, including alternative splicing, insertions, deletions, substitutions, sequencing errors and intron signal. We used this simulator to measure the accuracy and robustness of available algorithms at the base and junction levels. Additionally, we used reverse transcription–polymerase chain reaction (RT–PCR) and Sanger sequencing to validate the ability of the algorithms to detect novel transcript features such as novel exons and alternative splicing in RNA-Seq data from mouse retina. A pipeline based on BLAT was developed to explore the performance of established tools for this problem, and to compare it to the recently developed methods. This pipeline, the RNA-Seq Unified Mapper (RUM), performs comparably to the best current aligners and provides an advantageous combination of accuracy, speed and usability.
Availability: The RUM pipeline is distributed via the Amazon Cloud and for computing clusters using the Sun Grid Engine (
Supplementary Information:The RNA-Seq sequence reads described in the article are deposited at GEO, accession GSE26248.
PMCID: PMC3167048  PMID: 21775302
20.  Expression of Wild-Type Rp1 Protein in Rp1 Knock-in Mice Rescues the Retinal Degeneration Phenotype 
PLoS ONE  2012;7(8):e43251.
Mutations in the retinitis pigmentosa 1 (RP1) gene are a common cause of autosomal dominant retinitis pigmentosa (adRP), and have also been found to cause autosomal recessive RP (arRP) in a few families. The 33 dominant mutations and 6 recessive RP1 mutations identified to date are all nonsense or frameshift mutations, and almost exclusively (38 out of 39) are located in the 4th and final exon of RP1. To better understand the underlying disease mechanisms of and help develop therapeutic strategies for RP1 disease, we performed a series of human genetic and animal studies using gene targeted and transgenic mice. Here we report that a frameshift mutation in the 3rd exon of RP1 (c.686delC; p.P229QfsX35) found in a patient with recessive RP1 disease causes RP in the homozygous state, whereas the heterozygous carriers are unaffected, confirming that haploinsufficiency is not the causative mechanism for RP1 disease. We then generated Rp1 knock-in mice with a nonsense Q662X mutation in exon 4, as well as Rp1 transgenic mice carrying a wild-type BAC Rp1 transgene. The Rp1-Q662X allele produces a truncated Rp1 protein, and homozygous Rp1-Q662X mice experience a progressive photoreceptor degeneration characterized disorganization of photoreceptor outer segments. This phenotype could be prevented by expression of a normal amount of Rp1 protein from the BAC transgene without removal of the mutant Rp1-Q662X protein. Over-expression of Rp1 protein in additional BAC Rp1 transgenic lines resulted in retinal degeneration. These findings suggest that the truncated Rp1-Q662X protein does not exert a toxic gain-of-function effect. These results also imply that in principle gene augmentation therapy could be beneficial for both recessive and dominant RP1 patients, but the levels of RP1 protein delivered for therapy will have to be carefully controlled.
PMCID: PMC3424119  PMID: 22927954
21.  Bayesian Analysis of Trinomial Data in Behavioral Experiments and Its Application to Human Studies of General Anesthesia 
Conference Proceedings  2011;2011:4705-4708.
Accurate quantification of loss of response to external stimuli is essential for understanding the mechanisms of loss of consciousness under general anesthesia. We present a new approach for quantifying three possible outcomes that are encountered in behavioral experiments during general anesthesia: correct responses, incorrect responses and no response. We use a state-space model with two state variables representing a probability of response and a conditional probability of correct response. We show applications of this approach to an example of responses to auditory stimuli at varying levels of propofol anesthesia ranging from light sedation to deep anesthesia in human subjects. The posterior probability densities of model parameters and the response probability are computed within a Bayesian framework using Markov Chain Monte Carlo methods.
PMCID: PMC3282046  PMID: 22255388
22.  Robust Time-Varying Multivariate Coherence Estimation: Application to Electroencephalogram Recordings During General Anesthesia 
Conference Proceedings  2011;2011:4725-4728.
Coherence analysis characterizes frequency-dependent covariance between signals, and is useful for multivariate oscillatory data often encountered in neuroscience. The global coherence provides a summary of coherent behavior in high-dimensional multivariate data by quantifying the concentration of variance in the first mode of an eigenvalue decomposition of the cross-spectral matrix. Practical application of this useful method is sensitive to noise, and can confound coherent activity in disparate neural populations or spatial locations that have a similar frequency structure. In this paper we describe two methodological enhancements to the global coherence procedure that increase robustness of the technique to noise, and that allow characterization of how power within specific coherent modes change through time.
PMCID: PMC3293406  PMID: 22255393
23.  Dynamic Assessment of Baroreflex Control of Heart Rate during Induction of Propofol Anesthesia Using a Point Process Method 
Annals of biomedical engineering  2010;39(1):260-276.
In this paper, we present a point process method to assess dynamic baroreflex sensitivity by estimating the baroreflex gain as focal component of a simplified closed-loop model of the cardiovascular system. Specifically, an inverse Gaussian probability distribution is used to model the heartbeat interval, whereas the instantaneous mean is identified by linear and bilinear bivariate regressions on both the previous R-R intervals (RR) and blood pressure (BP) beat-to-beat measures. The instantaneous baroreflex gain is estimated as the feedback branch of the loop with a point-process filter, while the RR→BP feedforward transfer function representing heart contractility and vasculature effects is simultaneously estimated by a recursive least-squares (RLS) filter. These two closed-loop gains provide a direct assessment of baroreflex control of heart rate. In addition, the dynamic coherence, cross-bispectrum, and their power ratio can also be estimated. All statistical indices provide a valuable quantitative assessment of the interaction between heartbeat dynamics and hemodynamics. To illustrate the application, we have applied the proposed point process model to experimental recordings from eleven healthy subjects in order to monitor cardiovascular regulation under propofol anesthesia. We present quantitative results during transient periods, as well as statistical analyses on steady state epochs before and after propofol administration. Our findings validate the ability of the algorithm to provide a reliable and fast-tracking assessment of baroreflex sensitivity (BRS), and show a clear overall reduction in baroreflex gain from the baseline period to the start of propofol anesthesia, confirming that instantaneous evaluation of arterial baroreflex control of heart rate may yield important implications in clinical practice, particularly during anesthesia and in postoperative care.
PMCID: PMC3010293  PMID: 20945159
Baroreflex control; Baroreflex sensitivity; Heart rate variability; Hemodynamics; Point processes; Adaptive filters; Volterra series; Closed-loop feedback control; Cardiovascular system
24.  TTC21B contributes both causal and modifying alleles across the ciliopathy spectrum 
Nature genetics  2011;43(3):189-196.
Ciliary dysfunction leads to a broad range of overlapping phenotypes, termed collectively as ciliopathies. This grouping is underscored by genetic overlap, where causal genes can also contribute modifying alleles to clinically distinct disorders. Here we show that mutations in TTC21B/IFT139, encoding a retrograde intraflagellar transport (IFT) protein, cause both isolated nephronophthisis (NPHP) and syndromic Jeune Asphyxiating Thoracic Dystrophy (JATD). Moreover, although systematic medical resequencing of a large, clinically diverse ciliopathy cohort and matched controls showed a similar frequency of rare changes, in vivo and in vitro evaluations unmasked a significant enrichment of pathogenic alleles in cases, suggesting that TTC21B contributes pathogenic alleles to ∼5% of ciliopathy patients. Our data illustrate how genetic lesions can be both causally associated with diverse ciliopathies, as well as interact in trans with other disease-causing genes, and highlight how saturated resequencing followed by functional analysis of all variants informs the genetic architecture of disorders.
PMCID: PMC3071301  PMID: 21258341
25.  Generation of Cre Transgenic Mice with Postnatal RPE-Specific Ocular Expression 
The authors characterize a new transgenic mouse line, BEST1-cre, that provides RPE-specific ocular cre expression. These mice begin expressing cre at postnatal day 10 and maintain expression into adulthood without causing retinal dysfunction. Cre expression is present in up to 90% of RPE nuclei. Therefore, these mice provide a useful tool for studying the postnatal function of genes within the RPE.
To generate and characterize a constitutively active, RPE-specific, cre-expressing transgenic mouse line. This line can be used to create RPE-specific knockouts by crossing with mice harboring loxP-flanked (floxed) genes.
A transgene construct was assembled with the BEST1 promoter driving cre expression. Transgenic mice were generated on a C57BL/6 background. Cre expression was assessed by immunofluorescence and Western blot analysis. Cre enzymatic activity was tested by crossing to three lines with floxed DNA regions and detecting deletion of the intervening sequences or through histochemical detection of lacZ activity. Potential cre-mediated toxicity was assessed by retinal histology up to 24 months of age and by electroretinography.
The BEST1-cre line with expression in the highest percentage of RPE cells displayed a patchy mosaic expression pattern, with 50% to 90% of RPE cells expressing cre. In mice outcrossed to a mixed B6/129 background, expression was consistently found in 90% of RPE cells. Within the eye, only the RPE cells were immunoreactive with an anti-cre antibody. Maximum cre expression quantified by Western blot analysis occurred at P28. Crosses with three lines containing floxed sequences revealed RPE-specific cre activity in the eye and extraocular expression limited to the testes. Histology and electroretinography showed no cre-mediated RPE toxicity.
This BEST1-cre transgenic line enables generation of RPE-specific knockout mice. The mosaic expression pattern provides an internal control; the non–cre-expressing RPE cells continue to express the floxed genes. These mice should facilitate study of the multifunctional RPE and the generation of mouse models of human retinal disease.
PMCID: PMC3101664  PMID: 21212186

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