Person:

Comander, Jason

Background: Gene microarray technology provides the ability to study the regulation of thousands of genes simultaneously, but its potential is limited without an estimate of the statistical significance of the observed changes in gene expression. Due to the large number of genes being tested and the comparatively small number of array replicates (e.g., N = 3), standard statistical methods such as the Student's t-test fail to produce reliable results. Two other statistical approaches commonly used to improve significance estimates are a penalized t-test and a Z-test using intensity-dependent variance estimates.
Results: The performance of these approaches is compared using a dataset of 23 replicates, and a new implementation of the Z-test is introduced that pools together variance estimates of genes with similar minimum intensity. Significance estimates based on 3 replicate arrays are calculated using each statistical technique, and their accuracy is evaluated by comparing them to a reliable estimate based on the remaining 20 replicates. The reproducibility of each test statistic is evaluated by applying it to multiple, independent sets of 3 replicate arrays. Two implementations of a Z-test using intensity-dependent variance produce more reproducible results than two implementations of a penalized t-test. Furthermore, the minimum intensity-based Z-statistic demonstrates higher accuracy and higher or equal precision than all other statistical techniques tested.
Conclusion: An intensity-based variance estimation technique provides one simple, effective approach that can improve p-value estimates for differentially regulated genes derived from replicated microarray datasets. Implementations of the Z-test algorithms are available at http://vessels.bwh.harvard.edu/software/papers/bmcg2004.

Background: Next generation sequencing (NGS) offers a rapid and comprehensive method of screening for mutations associated with retinitis pigmentosa and related disorders. However, certain sequence alterations such as large insertions or deletions may remain undetected using standard NGS pipelines. One such mutation is a recently-identified Alu insertion into the Male Germ Cell-Associated Kinase (MAK) gene, which is missed by standard NGS-based variant callers. Here, we developed an in silico method of searching NGS raw sequence reads to detect this mutation, without the need to recalculate sequence alignments or to screen every sample by PCR. Methods: The Linux program grep was used to search for a 23 bp “probe” sequence containing the known junction sequence of the insert. A corresponding search was performed with the wildtype sequence. The matching reads were counted and further compared to the known sequences of the full wildtype and mutant genomic loci. (See https://github.com/MEEIBioinformaticsCenter/grepsearch.) Results: In a test sample set consisting of eleven previously published homozygous mutants, detection of the MAK-Alu insertion was validated with 100% sensitivity and specificity. As a discovery cohort, raw NGS reads from 1,847 samples (including custom and whole exome selective capture) were searched in ~1 hour on a local computer cluster, yielding an additional five samples with MAK-Alu insertions and solving two previously unsolved pedigrees. Of these, one patient was homozygous for the insertion, one compound heterozygous with a missense change on the other allele (c. 46G>A; p.Gly16Arg), and three were heterozygous carriers. Conclusions: Using the MAK-Alu grep program proved to be a rapid and effective method of finding a known, disease-causing Alu insertion in a large cohort of patients with NGS data. This simple approach avoids wet-lab assays or computationally expensive algorithms, and could also be used for other known disease-causing insertions and deletions.

Purpose Identifying genetic risk factors for developing sarcoidosis-associated uveitis could provide insights into its pathogenesis which is poorly understood. We determine if variants in NOD2 confer an increased risk of developing uveitis in adults with sarcoidosis. Methods: In this genetic case-control study, 51 total subjects were enrolled: 39 patients diagnosed with sarcoid-related uveitis and 12 patients with systemic sarcoidosis without ocular involvement as controls. Sanger sequencing of the eleven exons of the NOD2 gene was performed on DNA obtained from whole blood. Sanger sequencing data were aligned against the NOD2 NCBI-RefSeq reference sequence to identify novel mutations in uveitis patients. For common variants, allele frequencies in cases versus controls were compared using the chi-square test. Results: There were no significant differences in NOD2 common variant allele frequencies between sarcoidosis patients with and without uveitis, and none of the pathogenic NOD2 mutations associated with Blau syndrome were found in this cohort. However, four rare, non-synonymous variants were identified in four patients with ocular sarcoidosis and none of the controls. Variants rs149071116, rs35285618, and 16:g.50745164T > C have never been previously reported to be associated with any disease and may be pathogenic. The fourth variant, rs2066845, is associated with Crohn’s disease and psoriatic arthritis. Conclusions: Despite the phenotypic overlap between sarcoidosis and Blau syndrome, none of the established pathogenic NOD2 variants were present in adults with sarcoidosis. However, four novel, rare, non-synonymous variants were identified in four cases with ocular sarcoidosis. Further investigation is needed to explore the potential clinical significance of these polymorphisms.

Pericentral retinitis pigmentosa (RP) is an atypical form of RP that affects the near-peripheral retina first and tends to spare the far periphery. This study was performed to further define the genetic basis of this phenotype. We identified a cohort of 43 probands with pericentral RP based on a comprehensive analysis of their retinal phenotype. Genetic analyses of DNA samples from these patients were performed using panel-based next-generation sequencing, copy number variations, and whole exome sequencing (WES). Mutations provisionally responsible for disease were found in 19 of the 43 families (44%) analyzed. These include mutations in RHO (five patients), USH2A (four patients), and PDE6B (two patients). Of 28 putatively pathogenic alleles, 15 (54%) have been previously identified in patients with more common forms of typical RP, while the remaining 13 mutations (46%) were novel. Burden testing of WES data successfully identified HGSNAT as a cause of pericentral RP in at least two patients, suggesting it is also a relatively common cause of pericentral RP. While additional sequencing might uncover new genes specifically associated with pericentral RP, the current results suggest that genetically pericentral RP is not a separate clinical entity, but rather is part of the spectrum of mild RP phenotypes.

Purpose To describe in detail cases with an initial diagnosis of Leber congenital amaurosis that were later found to have a hemizygous mutation in the CACNA1F gene. Methods: The patients underwent a detailed ophthalmological evaluation and full-field electroretinography (ERG). Selective targeted capture and whole-exome next-generation sequencing (NGS) were used to find the disease-causing mutations. Results: Patient 1 presented at age 3 months with nystagmus, normal visual attention, and a normal fundus exam. ERG responses were severely decreased. Patient 2 presented with nystagmus, severe hyperopia, esotropia, and visual acuity of 20/360 oculus dexter (OD) and 20/270 oculus sinister (OS) at age 5 months. His fundus exam showed slightly increased pigmentation around the foveae. The scotopic ERG responses were severely decreased and photopic responses mildly decreased. Based on the initial presentation, both patients received the clinical diagnosis of Leber congenital amaurosis (LCA). However, genetic testing showed no mutations in known LCA genes. Instead, broader genetic testing using NGS showed point mutations in the CACNA1F gene, which is reported to be associated with type 2 congenital stationary night blindness (CSNB2). Conclusions: These two cases demonstrate the clinical overlap between LCA and CSNB in infants and young children. Genetic testing is an essential tool in these cases and provides a more accurate diagnosis and prognosis for patients with inherited retinal degenerative disorders.

Base editors are widely used engineered proteins that introduce targeted point mutations into DNA without creating double-stranded DNA breaks. The application of base editors to study and treat genetic diseases is dependent on their in vivo delivery into relevant cell types. Delivery by adeno-associated virus (AAV), a clinically validated delivery method, poses special challenges because the size of base editors exceeds the AAV packaging limit. Here we describe the development and application of in vivo delivery methods for both cytosine base editors (CBEs) and adenine base editors (ABEs). Dual AAVs each provide one half of the editor and trans-splicing inteins reconstitute full base editor activity, circumventing the AAV packaging limit. We optimized each AAV component to greatly improve editing efficiency. The resulting AAVs enable in vivo base editing for the first time in mouse brain, retina, and heart, as well as the most efficient base editing to date in muscle and liver, with therapeutically relevant efficiencies at viral dosages known to be tolerated in humans. A single intravenous injection of split CBE in PHP.eB AAV resulted in editing of up to 59% of unsorted mouse cortical tissue. Intravenous injection of split CBE or ABE in AAV9 mediated up to 38%, 20%, and 9% base editing in unsorted mouse liver, heart, and skeletal muscle, respectively. Subretinal and intracerebroventricular injections of split CBE and ABE in Anc80, PHP.B/PHP.eB, and AAV9 viruses mediated mouse retina and brain editing in up to 38% and 50% of unsorted cells, respectively. We applied this system to directly correct in mouse brain tissue a mutation that causes the neurodegenerative ataxia Niemann-Pick disease type C (NPC), slowing neurodegeneration and increasing lifespan consistent with expectations based on mosaic NPC mice. These findings establish a broadly useful AAV platform for the efficient introduction of targeted point mutations into multiple tissues of therapeutic interest for which in vivo base editing has not been previously reported.