Tabebordbar M, Zhu K, Cheng JKW, Chew WL, Widrick JJ, Yan WX, Maesner C, Wu EY, Xiao R, Ran AF, Cong L, Zhang F, Vandenberghe LH, Church GM, Wagers AJ. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 2015;Abstract

Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we develop and test a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored Dystrophin reading frame in myofibers, cardiomyocytes, and muscle stem cells following local or systemic delivery. AAV-Dmd CRISPR-treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle.

Tang S, Hemberg M, Cansizoglu E, Belin S, Kosik K, Kreiman G, Steen H, Steen J. f-divergence cutoff index to simultaneously identify differential expression in the integrated transcriptome and proteome. Nucleic Acids Res 2016;44(10):e97.Abstract

The ability to integrate 'omics' (i.e. transcriptomics and proteomics) is becoming increasingly important to the understanding of regulatory mechanisms. There are currently no tools available to identify differentially expressed genes (DEGs) across different 'omics' data types or multi-dimensional data including time courses. We present fCI (f-divergence Cut-out Index), a model capable of simultaneously identifying DEGs from continuous and discrete transcriptomic, proteomic and integrated proteogenomic data. We show that fCI can be used across multiple diverse sets of data and can unambiguously find genes that show functional modulation, developmental changes or misregulation. Applying fCI to several proteogenomics datasets, we identified a number of important genes that showed distinctive regulation patterns. The package fCI is available at R Bioconductor and

Telegrafi A, Webb BD, Robbins SM, Speck-Martins CE, FitzPatrick D, Fleming L, Redett R, Dufke A, Houge G, van Harssel JJT, Verloes A, Robles A, Manoli I, Engle EC, Engle EC, Jabs EW, Valle D, Carey J, Hoover-Fong JE, Sobreira NLM. Identification of STAC3 variants in non-Native American families with overlapping features of Carey-Fineman-Ziter syndrome and Moebius syndrome. Am J Med Genet A 2017;173(10):2763-2771.Abstract
Horstick et al. (2013) previously reported a homozygous p.Trp284Ser variant in STAC3 as the cause of Native American myopathy (NAM) in 5 Lumbee Native American families with congenital hypotonia and weakness, cleft palate, short stature, ptosis, kyphoscoliosis, talipes deformities, and susceptibility to malignant hyperthermia (MH). Here we present two non-Native American families, who were found to have STAC3 pathogenic variants. The first proband and her affected older sister are from a consanguineous Qatari family with a suspected clinical diagnosis of Carey-Fineman-Ziter syndrome (CFZS) based on features of hypotonia, myopathic facies with generalized weakness, ptosis, normal extraocular movements, cleft palate, growth delay, and kyphoscoliosis. We identified the homozygous c.851G>C;p.Trp284Ser variant in STAC3 in both sisters. The second proband and his affected sister are from a non-consanguineous, Puerto Rican family who was evaluated for a possible diagnosis of Moebius syndrome (MBS). His features included facial and generalized weakness, minimal limitation of horizontal gaze, cleft palate, and hypotonia, and he has a history of MH. The siblings were identified to be compound heterozygous for STAC3 variants c.851G>C;p.Trp284Ser and c.763_766delCTCT;p.Leu255IlefsX58. Given the phenotypic overlap of individuals with CFZS, MBS, and NAM, we screened STAC3 in 12 individuals diagnosed with CFZS and in 50 individuals diagnosed with MBS or a congenital facial weakness disorder. We did not identify any rare coding variants in STAC3. NAM should be considered in patients presenting with facial and generalized weakness, normal or mildly abnormal extraocular movement, hypotonia, cleft palate, and scoliosis, particularly if there is a history of MH.
Thomas MG, Maconachie GDE, Constantinescu CS, Chan W-M, Barry B, Hisaund M, Sheth V, Kuht HJ, Dineen RA, Harieaswar S, Engle EC, Gottlob I. Congenital monocular elevation deficiency associated with a novel gene variant. Br J Ophthalmol 2020;104(4):547-550.Abstract
BACKGROUND: The genetic basis of monocular elevation deficiency (MED) is unclear. It has previously been considered to arise due to a supranuclear abnormality. METHODS: Two brothers with MED were referred to Leicester Royal Infirmary, UK from the local opticians. Their father had bilateral ptosis and was unable to elevate both eyes, consistent with the diagnosis of congenital fibrosis of extraocular muscles (CFEOM). Candidate sequencing was performed in all family members. RESULTS: Both affected siblings (aged 7 and 12 years) were unable to elevate the right eye. Their father had bilateral ptosis, left esotropia and bilateral limitation of elevation. Chin up head posture was present in the older sibling and the father. Bell's phenomenon and vertical rotational vestibulo-ocular reflex were absent in the right eye for both children. Mild bilateral facial nerve palsy was present in the older sibling and the father. Both siblings had slight difficulty with tandem gait. MRI revealed hypoplastic oculomotor nerve. Left anterior insular focal cortical dysplasia was seen in the older sibling. Sequencing of revealed a novel heterozygous variant (c.1263G>C, p.E421D) segregating with the phenotype. This residue is in the C-terminal H12 α-helix of β-tubulin and is one of three putative kinesin binding sites. CONCLUSION: We show that familial MED can arise from a variant and could be considered a limited form of CFEOM. Neurological features such as mild facial palsy and cortical malformations can be present in patients with MED. Thus, in individuals with congenital MED, consideration may be made for mutation screening.
Thomas S, Thomas MG, Andrews C, Chan W-M, Proudlock FA, McLean RJ, Pradeep A, Engle EC, Gottlob I. Autosomal-dominant nystagmus, foveal hypoplasia and presenile cataract associated with a novel PAX6 mutation. Eur J Hum Genet 2014;22(3):344-9.Abstract
Autosomal-dominant idiopathic infantile nystagmus has been linked to 6p12 (OMIM 164100), 7p11.2 (OMIM 608345) and 13q31-q33 (OMIM 193003). PAX6 (11p13, OMIM 607108) mutations can also cause autosomal-dominant nystagmus, typically in association with aniridia or iris hypoplasia. We studied a large multigenerational white British family with autosomal-dominant nystagmus, normal irides and presenile cataracts. An SNP-based genome-wide analysis revealed a linkage to a 13.4-MB region on chromosome 11p13 with a maximum lod score of 2.93. A mutation analysis of the entire coding region and splice junctions of the PAX6 gene revealed a novel heterozygous missense mutation (c.227C>G) that segregated with the phenotype and is predicted to result in the amino-acid substitution of proline by arginine at codon 76 p.(P76R). The amino-acid variation p.(P76R) within the paired box domain is likely to destabilise the protein due to steric hindrance as a result of the introduction of a polar and larger amino acid. Eye movement recordings showed a significant intrafamilial variability of horizontal, vertical and torsional nystagmus. High-resolution in vivo imaging of the retina using optical coherence tomography (OCT) revealed features of foveal hypoplasia, including rudimentary foveal pit, incursion of inner retinal layers, short photoreceptor outer segments and optic nerve hypoplasia. Thus, this study presents a family that segregates a PAX6 mutation with nystagmus and foveal hypoplasia in the absence of iris abnormalities. Moreover, it is the first study showing detailed characteristics using eye movement recordings of autosomal-dominant nystagmus in a multigenerational family with a novel PAX6 mutation.
Ung C, Sanchez AV, Shen L, Davoudi S, Ahmadi T, Navarro-Gomez D, Chen CJ, Hancock H, Penman A, Hoadley S, Consugar M, Restrepo C, Shah VA, Arboleda-Velasquez JF, Sobrin L, Gai X, Kim LA. Whole exome sequencing identification of novel candidate genes in patients with proliferative diabetic retinopathy. Vision Res 2017;Abstract
Rare or novel gene variants in patients with proliferative diabetic retinopathy may contribute to disease development. We performed whole exome sequencing (WES) on patients at the phenotypic extremes of diabetic retinal complications: 57 patients diagnosed with proliferative diabetic retinopathy (PDR) as cases and 13 patients with no diabetic retinopathy despite at least 10years of type 2 diabetes as controls. Thirty-one out of the 57 cases and all 13 controls were from the African American Proliferative Diabetic Retinopathy Study (AA). The rest of the cases were of mixed ethnicities (ME). WES identified 721 candidate genes with rare or novel non-synonymous variants found in at least one case with PDR and not present in any controls. After filtering for genes with null alleles in greater than two cases, 28 candidate genes were identified in our ME cases and 16 genes were identified in our AA cases. Our analysis showed rare and novel variants within these genes that could contribute to the development of PDR, including rare non-synonymous variants in FAM132A, SLC5A9, ZNF600, and TMEM217. We also found previously unidentified variants in VEGFB and APOB. We found that VEGFB, VPS13B, PHF21A, NAT1, ZNF600, PKHD1L1 expression was reduced in human retinal endothelial cells (HRECs) cultured under high glucose conditions. In an exome sequence analysis of patients with PDR, we identified variants in genes that could contribute to pathogenesis. Six of these genes were further validated and found to have reduced expression in HRECs under high glucose conditions, suggestive of an important role in the development of PDR.
Unzu C, Planet E, Brandenberg N, Fusil F, Cassano M, Perez-Vargas J, Friedli M, Cosset F-L, Lutolf MP, Wildhaber BE, Trono D. Pharmacological Induction of a Progenitor State for the Efficient Expansion of Primary Human Hepatocytes. Hepatology 2019;69(5):2214-2231.Abstract
The liver is an organ with strong regenerative capacity, yet primary hepatocytes have a low amplification potential in vitro, a major limitation for the cell-based therapy of liver disorders and for ex vivo biological screens. Induced pluripotent stem cells (iPSCs) may help to circumvent this obstacle but often harbor genetic and epigenetic abnormalities, limiting their potential. Here, we describe the pharmacological induction of proliferative human hepatic progenitor cells (HPCs) through a cocktail of growth factors and small molecules mimicking the signaling events involved in liver regeneration. Human HPCs from healthy donors and pediatric patients proliferated vigorously while maintaining their genomic stability and could be redifferentiated in vitro into metabolically competent cells that supported the replication of hepatitis B and delta viruses. Redifferentiation efficiency was boosted by three-dimensional culture. Finally, transcriptome analysis showed that HPCs were more closely related to mature hepatocytes than iPSC-derived hepatocyte-like cells were. Conclusion: HPC induction holds promise for a variety of applications such as ex vivo disease modeling, personalized drug testing or metabolic studies, and development of a bioartificial liver.
Vandenberghe LH. What Is Next for Retinal Gene Therapy?. Cold Spring Harb Perspect Med 2015;Abstract

The field of gene therapy for retinal blinding disorders is experiencing incredible momentum, justified by hopeful results in early stage clinical trials for inherited retinal degenerations. The premise of the use of the gene as a drug has come a long way, and may have found its niche in the treatment of retinal disease. Indeed, with only limited treatment options available for retinal indications, gene therapy has been proven feasible, safe, and effective and may lead to durable effects following a single injection. Here, we aim at putting into context the promise and potential, the technical, clinical, and economic boundaries limiting its application and development, and speculate on a future in which gene therapy is an integral component of ophthalmic clinical care.

Venturini G, Koskiniemi-Kuendig H, Harper S, Berson EL, Rivolta C. Two specific mutations are prevalent causes of recessive retinitis pigmentosa in North American patients of Jewish ancestry. Genet Med 2015;17(4):285-90.Abstract

PURPOSE: Retinitis pigmentosa is a Mendelian disease with a very elevated genetic heterogeneity. Most mutations are responsible for less than 1% of cases, making molecular diagnosis a multigene screening procedure. In this study, we assessed whether direct testing of specific alleles could be a valuable screening approach in cases characterized by prevalent founder mutations. METHODS: We screened 275 North American patients with recessive/isolate retinitis pigmentosa for two mutations: an Alu insertion in the MAK gene and the p.Lys42Glu missense in the DHDDS gene. All patients were unrelated; 35 reported Jewish ancestry and the remainder reported mixed ethnicity. RESULTS: We identified the MAK and DHDDS mutations homozygously in only 2.1% and 0.8%, respectively, of patients of mixed ethnicity, but in 25.7% and 8.6%, respectively, of cases reporting Jewish ancestry. Haplotype analyses revealed that inheritance of the MAK mutation was attributable to a founder effect. CONCLUSION: In contrast to most mutations associated with retinitis pigmentosa-which are, in general, extremely rare-the two alleles investigated here cause disease in approximately one-third of North American patients reporting Jewish ancestry. Therefore, their screening constitutes an alternative procedure to large-scale tests for patients belonging to this ethnic group, especially in time-sensitive situations.Genet Med 17 4, 285-290.

Wan A, Place E, Pierce EA, Comander J. Characterizing variants of unknown significance in rhodopsin: A functional genomics approach. Hum Mutat 2019;40(8):1127-1144.Abstract
Characterizing the pathogenicity of DNA sequence variants of unknown significance (VUS) is a major bottleneck in human genetics, and is increasingly important in determining which patients with inherited retinal diseases could benefit from gene therapy. A library of 210 rhodopsin (RHO) variants from literature and in-house genetic diagnostic testing were created to efficiently detect pathogenic RHO variants that fail to express on the cell surface. This study, while focused on RHO, demonstrates a streamlined, generalizable method for detecting pathogenic VUS. A relatively simple next-generation sequencing-based readout was developed so that a flow cytometry-based assay could be performed simultaneously on all variants in a pooled format, without the need for barcodes or viral transduction. The resulting dataset characterized the surface expression of every RHO library variant with a high degree of reproducibility (r  = 0.92-0.95), recategorizing 37 variants. For example, three retinitis pigmentosa pedigrees were solved by identifying VUS which showed low expression levels (p.G18D, p.G101V, and p.P180T). Results were validated across multiple assays and correlated with clinical disease severity. This study presents a parallelized, higher-throughput cell-based assay for the functional characterization of VUS in RHO, and can be applied more broadly to other inherited retinal disease genes and other disorders.
Wang T, Huang T, Kang JH, Zheng Y, Jensen MK, Wiggs JL, Pasquale LR, Fuchs CS, Campos H, Rimm EB, Willett WC, Hu FB, Qi L. Habitual coffee consumption and genetic predisposition to obesity: gene-diet interaction analyses in three US prospective studies. BMC Med 2017;15(1):97.Abstract
BACKGROUND: Whether habitual coffee consumption interacts with the genetic predisposition to obesity in relation to body mass index (BMI) and obesity is unknown. METHODS: We analyzed the interactions between genetic predisposition and habitual coffee consumption in relation to BMI and obesity risk in 5116 men from the Health Professionals Follow-up Study (HPFS), in 9841 women from the Nurses' Health Study (NHS), and in 5648 women from the Women's Health Initiative (WHI). The genetic risk score was calculated based on 77 BMI-associated loci. Coffee consumption was examined prospectively in relation to BMI. RESULTS: The genetic association with BMI was attenuated among participants with higher consumption of coffee than among those with lower consumption in the HPFS (P interaction  = 0.023) and NHS (P interaction  = 0.039); similar results were replicated in the WHI (P interaction  = 0.044). In the combined data of all cohorts, differences in BMI per increment of 10-risk allele were 1.38 (standard error (SE), 0.28), 1.02 (SE, 0.10), and 0.95 (SE, 0.12) kg/m(2) for coffee consumption of < 1, 1-3 and > 3 cup(s)/day, respectively (P interaction  < 0.001). Such interaction was partly due to slightly higher BMI with higher coffee consumption among participants at lower genetic risk and slightly lower BMI with higher coffee consumption among those at higher genetic risk. Each increment of 10-risk allele was associated with 78% (95% confidence interval (CI), 59-99%), 48% (95% CI, 36-62%), and 43% (95% CI, 28-59%) increased risk for obesity across these subgroups of coffee consumption (P interaction  = 0.008). From another perspective, differences in BMI per increment of 1 cup/day coffee consumption were 0.02 (SE, 0.09), -0.02 (SE, 0.04), and -0.14 (SE, 0.04) kg/m(2) across tertiles of the genetic risk score. CONCLUSIONS: Higher coffee consumption might attenuate the genetic associations with BMI and obesity risk, and individuals with greater genetic predisposition to obesity appeared to have lower BMI associated with higher coffee consumption.
Wang YE, Kakigi C, Barbosa D, Porco T, Chen R, Wang S, Li Y, Singh K, Pasquale LR, Lin SC. Oral Contraceptive Use and Prevalence of Self-Reported Glaucoma or Ocular Hypertension in the United States. Ophthalmology 2016;123(4):729-36.Abstract

PURPOSE: To investigate the association between oral contraceptive (OC) use and glaucoma prevalence in the United States. DESIGN: Cross-sectional study. PARTICIPANTS: A total of 3406 female participants, aged 40 years or older, from the 2005 to 2008 National Health and Nutrition Examination Survey, who reported a presence or absence of glaucoma or ocular hypertension completed both the vision and the reproductive health questionnaires and underwent eye examinations. METHODS: Multivariate regression analysis was used to assess the correlation between OC use and self-reported glaucoma or ocular hypertension (n = 231 cases), controlling for potential confounders, including age, ethnicity, systemic comorbidities such as hypertension and stroke, ocular diseases such as cataract and diabetic retinopathy, and reproductive health factors, including age at menopause, age at menarche, history of hormone replacement therapy, and gynecological surgical history. MAIN OUTCOME MEASURES: The outcome variable was self-reported glaucoma or ocular hypertension. RESULTS: After adjusting for confounders, those with ≥3 years of OC use had greater odds (odds ratio, 1.94; 95% confidence interval, 1.22-3.07) of self-reported glaucoma or ocular hypertension. Other factors associated with higher glaucoma or ocular hypertension prevalence included older age, African American race, and later age at menarche. CONCLUSIONS: Oral contraceptive use may be associated with increased risk of self-reported glaucoma or ocular hypertension.

Wang L, Xiao R, Andres-Mateos E, Vandenberghe LH. Single stranded adeno-associated virus achieves efficient gene transfer to anterior segment in the mouse eye. PLoS One 2017;12(8):e0182473.Abstract
Adeno-associated viruses (AAVs) are used extensively as a gene delivery vehicle for retinal gene therapy, yet its ability to target the anterior segment of the eye, critical to unlocking therapeutic opportunities, is less characterized. Previously, self-complimentary (sc) AAV was shown to be necessary for transduction of the cornea and trabecular meshwork (TM), limiting the size of the gene transfer cassette, likely due to a block in second strand synthesis thought to be required for functional transduction. Here, we evaluated several AAV capsids in a single stranded (ss) genome conformation for their ability to overcome the need for scAAV for targeting corneal endothelium and TM. AAV2, 8, and a recently synthetically developed AAV called Anc80L65 were evaluated in vitro and in vivo by intracameral injection in mice. Results show that although scAAV2 demonstrated superior infectivity in vitro including Human Trabecular meshwork (HTM) immortalized cell lines; Anc80L65 transduced following a single intracameral injection efficiently all components of the mouse anterior segment, including the TM, corneal stroma, and endothelial cells. These results suggest that Anc80L65 is able to overcome the requirement for scAAV genomes to enable TM and corneal targeting, expanding the potential experimental and therapeutic use of AAV gene transfer in the anterior segment of the eye.
Wang T, Huang T, Heianza Y, Sun D, Zheng Y, Ma W, Jensen MK, Kang JH, Wiggs JL, Pasquale LR, Rimm EB, Manson JAE, Hu FB, Willett WC, Qi L. Genetic Susceptibility, Change in Physical Activity, and Long-term Weight Gain. Diabetes 2017;66(10):2704-2712.Abstract
Whether change in physical activity over time modifies the genetic susceptibility to long-term weight gain is unknown. We calculated a BMI-genetic risk score (GRS) based on 77 BMI-associated single nucleotide polymorphisms (SNPs) and a body fat percentage (BF%)-GRS based on 12 BF%-associated SNPs in 9,390 women from the Nurses' Health Study (NHS) and 5,291 men from the Health Professionals Follow-Up Study (HPFS). We analyzed the interactions between each GRS and change in physical activity on BMI/body weight change within five 4-year intervals from 1986 to 2006 using multivariable generalized linear models with repeated-measures analyses. Both the BMI-GRS and the BF%-GRS were associated with long-term increases in BMI/weight, and change in physical activity consistently interacted with the BF%-GRS on BMI change in the NHS (P for interaction = 0.025) and HPFS (P for interaction = 0.001). In the combined cohorts, 4-year BMI change per 10-risk allele increment was -0.02 kg/m(2) among participants with greatest increase in physical activity and 0.24 kg/m(2) among those with greatest decrease in physical activity (P for interaction < 0.001), corresponding to 0.01 kg versus 0.63 kg weight changes every 4 years (P for interaction = 0.001). Similar but marginal interactions were observed for the BMI-GRS (P for interaction = 0.045). Our data indicate that the genetic susceptibility to weight gain may be diminished by increasing physical activity.
Weinberger AD, Gilmore MS. A CRISPR View of Cleavage. Cell 2015;161(5):964-6.Abstract

Seminal studies showed that CRISPR-Cas systems provide adaptive immunity in prokaryotes and promising gene-editing tools from bacteria to humans. Yet, reports diverged on whether some CRISPR systems naturally target DNA or RNA. Here, Samai and colleagues unify the studies, showing that a single type III CRISPR-Cas system cleaves both DNA and RNA targets, independently.

Whitman MC, Miyake N, Nguyen EH, Bell JL, Matos Ruiz PM, Chan W-M, Di Gioia SA, Mukherjee N, Barry BJ, Bosley TM, Khan AO, Engle EC. Decreased ACKR3 (CXCR7) function causes oculomotor synkinesis in mice and humans. Hum Mol Genet 2019;28(18):3113-3125.Abstract
Oculomotor synkinesis is the involuntary movement of the eyes or eyelids with a voluntary attempt at a different movement. The chemokine receptor CXCR4 and its ligand CXCL12 regulate oculomotor nerve development; mice with loss of either molecule have oculomotor synkinesis. In a consanguineous family with congenital ptosis and elevation of the ptotic eyelid with ipsilateral abduction, we identified a co-segregating homozygous missense variant (c.772G>A) in ACKR3, which encodes an atypical chemokine receptor that binds CXCL12 and functions as a scavenger receptor, regulating levels of CXCL12 available for CXCR4 signaling. The mutant protein (p.V258M) is expressed and traffics to the cell surface but has a lower binding affinity for CXCL12. Mice with loss of Ackr3 have variable phenotypes that include misrouting of the oculomotor and abducens nerves. All embryos show oculomotor nerve misrouting, ranging from complete misprojection in the midbrain, to aberrant peripheral branching, to a thin nerve, which aberrantly innervates the lateral rectus (as seen in Duane syndrome). The abducens nerve phenotype ranges from complete absence, to aberrant projections within the orbit, to a normal trajectory. Loss of ACKR3 in the midbrain leads to downregulation of CXCR4 protein, consistent with reports that excess CXCL12 causes ligand-induced degradation of CXCR4. Correspondingly, excess CXCL12 applied to ex vivo oculomotor slices causes axon misrouting, similar to inhibition of CXCR4. Thus, ACKR3, through its regulation of CXCL12 levels, is an important regulator of axon guidance in the oculomotor system; complete loss causes oculomotor synkinesis in mice, while reduced function causes oculomotor synkinesis in humans.
Whitman MC, Andrews C, Chan W-M, Tischfield MA, Stasheff SF, Brancati F, Ortiz-Gonzalez X, Nuovo S, Garaci F, MacKinnon SE, Hunter DG, Grant EP, Engle EC. Two unique TUBB3 mutations cause both CFEOM3 and malformations of cortical development. Am J Med Genet A 2016;170(2):297-305.Abstract

One set of missense mutations in the neuron specific beta tubulin isotype 3 (TUBB3) has been reported to cause malformations of cortical development (MCD), while a second set has been reported to cause isolated or syndromic Congenital Fibrosis of the Extraocular Muscles type 3 (CFEOM3). Because TUBB3 mutations reported to cause CFEOM had not been associated with cortical malformations, while mutations reported to cause MCD had not been associated with CFEOM or other forms of paralytic strabismus, it was hypothesized that each set of mutations might alter microtubule function differently. Here, however, we report two novel de novo heterozygous TUBB3 amino acid substitutions, G71R and G98S, in four patients with both MCD and syndromic CFEOM3. These patients present with moderately severe CFEOM3, nystagmus, torticollis, and developmental delay, and have intellectual and social disabilities. Neuroimaging reveals defective cortical gyration, as well as hypoplasia or agenesis of the corpus callosum and anterior commissure, malformations of hippocampi, thalami, basal ganglia and cerebella, and brainstem and cranial nerve hypoplasia. These new TUBB3 substitutions meld the two previously distinct TUBB3-associated phenotypes, and implicate similar microtubule dysfunction underlying both. © 2015 Wiley Periodicals, Inc.

Whitman MC, Nguyen EH, Bell JL, Tenney AP, Gelber A, Engle EC. Loss of CXCR4/CXCL12 Signaling Causes Oculomotor Nerve Misrouting and Development of Motor Trigeminal to Oculomotor Synkinesis. Invest Ophthalmol Vis Sci 2018;59(12):5201-5209.Abstract
Purpose: Proper control of eye movements is critical to vision, but relatively little is known about the molecular mechanisms that regulate development and axon guidance in the ocular motor system or cause the abnormal innervation patterns (oculomotor synkinesis) seen in developmental disorders and after oculomotor nerve palsy. We developed an ex vivo slice assay that allows for live imaging and molecular manipulation of the growing oculomotor nerve, which we used to identify axon guidance cues that affect the oculomotor nerve. Methods: Ex vivo slices were generated from E10.5 IslMN-GFP embryos and grown for 24 to 72 hours. To assess for CXCR4 function, the specific inhibitor AMD3100 was added to the culture media. Cxcr4cko/cko:Isl-Cre:ISLMN-GFP and Cxcl12KO/KO:ISLMN-GFP embryos were cleared and imaged on a confocal microscope. Results: When AMD3100 was added to the slice cultures, oculomotor axons grew dorsally (away from the eye) rather than ventrally (toward the eye). Axons that had already exited the midbrain continued toward the eye. Loss of Cxcr4 or Cxcl12 in vivo caused misrouting of the oculomotor nerve dorsally and motor axons from the trigeminal motor nerve, which normally innervate the muscles of mastication, aberrantly innervated extraocular muscles in the orbit. This represents the first mouse model of trigeminal-oculomotor synkinesis. Conclusions: CXCR4/CXCL12 signaling is critical for the initial pathfinding decisions of oculomotor axons and their proper exit from the midbrain. Failure of the oculomotor nerve to innervate its extraocular muscle targets leads to aberrant innervation by other motor neurons, indicating that muscles lacking innervation may secrete cues that attract motor axons.
Wiggs JL, Langgurth AM, Allen KF. Carrier frequency of CYP1B1 mutations in the United States (an American Ophthalmological Society thesis). Trans Am Ophthalmol Soc 2014;112:94-102.Abstract
PURPOSE: CYP1B1 mutations cause autosomal recessive congenital glaucoma. Disease risk assessment for families with CYP1B1 mutations requires knowledge of the population mutation carrier frequency. The purpose of this study is to determine the CYP1B1 mutation carrier frequency in clinically normal individuals residing in the United States. Because CYP1B1 mutations can exhibit variable expressivity, we hypothesize that the mutation carrier frequency is higher than expected. METHODS: Two hundred fifty individuals without glaucoma or a family history of glaucoma were enrolled. CYP1B1 mutations were identified by DNA sequencing, and pathogenicity was estimated by PolyPhen-2 or a previous report of disease causality. RESULTS: Based on the disease frequency (1 in 10,000) and prevalence of CYP1B1-related congenital glaucoma (15% to 20%), the frequency of CYP1B1-related congenital glaucoma in the United States is approximately 1 in 50,000. Assuming Hardy-Weinberg equilibrium, the expected CYP1B1 mutation carrier frequency would be 1 in 112, or 0.89%. Among the 250 study participants, 11 (4.4%) are carriers of a single pathogenic mutation, representing a carrier frequency of 1 in 22, which is 5.1 times the expected frequency. A higher-than-expected carrier frequency (1 in 33, 3.0%) was also observed in 4300 white individuals sequenced by the National Heart Lung and Blood Institute Exome Sequencing Project. CONCLUSIONS: Our results show that the CYP1B1 mutation carrier frequency in the US population is between 1 in 22 and 1 in 33, which is 5.1 to 3.4 times the expected frequency. These results suggest that more individuals than expected are carriers of a deleterious CYP1B1 mutation, and that the prevalence of CYP1B1-related disease may be higher than expected.
Wiggs JL, Pasquale LR. Genetics of Glaucoma. Hum Mol Genet 2017;Abstract
Genetic and genomic studies, including genome-wide association studies (GWAS) have accelerated the discovery of genes contributing to glaucoma, the leading cause of irreversible blindness world-wide. Glaucoma can occur at all ages, with Mendelian inheritance typical for rare early onset disease (before age 40) and complex inheritance evident in common adult-onset forms of disease. Recent studies have suggested possible therapeutic targets for some patients with early-onset glaucoma based on the molecular and cellular events caused by MYOC, OPTN and TBK1 mutations. Diagnostic genetic tests using early-onset glaucoma genes are also proving useful for pre-symptomatic disease detection and genetic counseling. Recent GWAS completed for three types of common adult-onset glaucoma have identified novel loci for POAG (primary-open-angle glaucoma) (ABCA1, AFAP1, GMDS, PMM2, TGFBR3, FNDC3B, ARHGEF12, GAS7, FOXC1, ATXN2, TXNRD2); PACG (primary angle-closure glaucoma (EPDR1, CHAT, GLIS3, FERMT2, DPM2-FAM102); and exfoliation syndrome (XFS) glaucoma (CACNA1A). In total sixteen genomic regions have been associated with POAG (including the normal tension glaucoma (NTG) subgroup), 8 with PACG and 2 with XFS. These studies are defining important biological pathways and processes that contribute to disease pathogenesis.