Person: Pollak, Martin
Loading...
Email Address
AA Acceptance Date
Birth Date
Research Projects
Organizational Units
Job Title
Last Name
Pollak
First Name
Martin
Name
Pollak, Martin
18 results
Search Results
Now showing 1 - 10 of 18
Publication Discovery of new glomerular disease-relevant genes by translational profiling of podocytes in vivo(2014) Grgic, Ivica; Hofmeister, Andreas F.; Genovese, Giulio; Bernhardy, Andrea J.; Sun, Hua; Maarouf, Omar H.; Bijol, Vanesa; Pollak, Martin; Humphreys, Benjamin D.Identifying new biomarkers and therapeutic targets for podocytopathies such as focal segmental glomerulosclerosis (FSGS) requires a detailed analysis of transcriptional changes in podocytes over the course of disease. Here we used translating ribosome affinity purification (TRAP) to isolate and profile podocyte-specific mRNA in two different models of FSGS. Expressed eGFP-tagged ribosomal protein L10a in podocytes under the control of the Collagen-1α1 promoter enabled podocyte-specific mRNA isolation in a one-step process over the course of disease. This TRAP protocol robustly enriched known podocyte-specific mRNAs. We crossed col1α1-L10a mice with the actn4−/− and actn4+/K256E models of FSGS and analyzed podocyte transcriptional profiles at 2, 6 and 44 weeks of age. Two upregulated podocyte genes in murine FSGS (CXCL1 and DMPK) were found to be upregulated at the protein level in biopsies from patients with FSGS, validating this approach. There was no dilution of podocyte-specific transcripts during disease. These are the first podocyte-specific RNA expression datasets during aging and in two models of FSGS. This approach identified new podocyte proteins that are upregulated in FSGS and help define novel biomarkers and therapeutic targets for human glomerular disease.Publication Using population admixture to help complete maps of the human genome(2013) Genovese, Giulio; Handsaker, Robert; Li, Heng; Altemose, Nicolas; Lindgren, Amelia M.; Chambert, Kimberly; Pasaniuc, Bogdan; Price, Alkes; Reich, David; Morton, Cynthia; Pollak, Martin; Wilson, James G.; McCarroll, StevenTens of millions of base pairs of euchromatic human genome sequence, including many protein-coding genes, have no known location in the human genome. We describe an approach for localizing the human genome's missing pieces by utilizing the patterns of genome sequence variation created by population admixture. We mapped the locations of 70 scaffolds spanning four million base pairs of the human genome's unplaced euchromatic sequence, including more than a dozen protein-coding genes, and identified eight large novel inter-chromosomal segmental duplications. We find that most of these sequences are hidden in the genome's heterochromatin, particularly its pericentromeric regions. Many cryptic, pericentromeric genes are expressed in RNA and have been maintained intact for millions of years while their expression patterns diverged from those of paralogous genes elsewhere in the genome. We describe how knowledge of the locations of these sequences can inform disease association and genome biology studies.Publication Frequency of Rare Allelic Variation in Candidate Genes among Individuals with Low and High Urinary Calcium Excretion(Public Library of Science, 2013) Toka, Hakan; Genovese, Giulio; Mount, David B.; Pollak, Martin; Curhan, Gary C.Our study investigated the association of rare allelic variants with extremes of 24-hour urinary calcium excretion because higher urinary calcium excretion is a dominant risk factor for calcium-based kidney stone formation. We resequenced 40 candidate genes potentially related to urinary calcium excretion in individuals from the Nurses' Health Studies I & II and the Health Professionals Follow-up Study. A total of 960 participants were selected based on availability of 24-hour urine collection data and level of urinary calcium excretion (low vs. high). We utilized DNA sample pooling, droplet-based target gene enrichment, multiplexing, and high-throughput sequencing. Approximately 64% of samples (n = 615) showed both successful target enrichment and sequencing data with >20-fold deep coverage. A total of 259 novel allelic variants were identified. None of the rare gene variants (allele frequencies <2%) were found with increased frequency in the low vs. high urinary calcium groups; most of these variants were only observed in single individuals. Unadjusted analysis of variants with allele frequencies ≥2% suggested an association of the Claudin14 SNP rs113831133 with lower urinary calcium excretion (6/520 versus 29/710 haplotypes, P value = 0.003). Our data, together with previous human and animal studies, suggest a possible role for Claudin14 in urinary calcium excretion. Genetic validation studies in larger sample sets will be necessary to confirm our findings for rs113831133. In the tested set of candidate genes, rare allelic variants do not appear to contribute significantly to differences in urinary calcium excretion between individuals.Publication Mutations causing medullary cystic kidney disease type 1 (MCKD1) lie in a large VNTR in MUC1 missed by massively parallel sequencing(2014) Kirby, Andrew; Gnirke, Andreas; Jaffe, David B.; Barešová, Veronika; Pochet, Nathalie; Blumenstiel, Brendan; Ye, Chun; Aird, Daniel; Stevens, Christine; Robinson, James T.; Cabili, Moran N.; Gat-Viks, Irit; Kelliher, Edward; Daza, Riza; DeFelice, Matthew; Hůlková, Helena; Sovová, Jana; Vylet’al, Petr; Antignac, Corinne; Guttman, Mitchell; Handsaker, Robert; Perrin, Danielle; Steelman, Scott; Sigurdsson, Snaevar; Scheinman, Steven J.; Sougnez, Carrie; Cibulskis, Kristian; Parkin, Melissa; Green, Todd; Rossin, Elizabeth; Zody, Michael C.; Xavier, Ramnik; Pollak, Martin; Alper, Seth; Lindblad-Toh, Kerstin; Gabriel, Stacey; Hart, P. Suzanne; Regev, Aviv; Nusbaum, Chad; Kmoch, Stanislav; Bleyer, Anthony J.; Lander, Eric; Daly, MarkWhile genetic lesions responsible for some Mendelian disorders can be rapidly discovered through massively parallel sequencing (MPS) of whole genomes or exomes, not all diseases readily yield to such efforts. We describe the illustrative case of the simple Mendelian disorder medullary cystic kidney disease type 1 (MCKD1), mapped more than a decade ago to a 2-Mb region on chromosome 1. Ultimately, only by cloning, capillary sequencing, and de novo assembly, we found that each of six MCKD1 families harbors an equivalent, but apparently independently arising, mutation in sequence dramatically underrepresented in MPS data: the insertion of a single C in one copy (but a different copy in each family) of the repeat unit comprising the extremely long (~1.5-5 kb), GC-rich (>80%), coding VNTR in the mucin 1 gene. The results provide a cautionary tale about the challenges in identifying genes responsible for Mendelian, let alone more complex, disorders through MPS.Publication Exome sequencing and in vitro studies identified podocalyxin as a candidate gene for focal and segmental glomerulosclerosis(2013) Barua, Moumita; Shieh, Eric; Schlondorff, Johannes; Genovese, Giulio; Kaplan, Bernard S; Pollak, MartinOur understanding of focal and segmental glomerulosclerosis (FSGS) has advanced significantly from the studies of rare, monogenic forms of the disease. These studies have demonstrated the critical roles of multiple aspects of podocyte function in maintaining glomerular function. A substantial body of research has suggested that the integral membrane protein podocalyxin (PODXL) is required for proper function of podocytes, possibly by preserving the patency of the slit diaphragm by negative charge-based repulsion. Exome sequencing of affected cousins from an autosomal dominant pedigree with FSGS identified a co-segregating private variant, PODXL p.L442R, affecting the transmembrane region of the protein. Of the remaining 11 shared gene variants, two segregated with disease but their gene products were not detected in the glomerulus. In comparison to wild type, this disease-segregating PODXL variant facilitated dimerization. By contrast, this change does not alter protein stability, extracellular domain glycosylation, cell surface expression, global subcellular localization, or interaction with its intracellular binding partner ezrin. Thus, a variant form of PODXL remains the most likely candidate causing FSGS in one family with autosomal dominant inheritance, but its full effect on protein function remains unknown. Our work highlights the challenge faced in the clinical interpretation of whole exome data for small pedigrees with autosomal dominant diseases.Publication Genetic testing for nephrotic syndrome and FSGS in the era of next-generation sequencing(2014) Brown, Elizabeth J.; Pollak, Martin; Barua, MoumitaThe haploid human genome is composed of three billion base pairs, about one percent of which consists of exonic regions, the coding sequence for functional proteins, also now known as the “exome”. The development of next-generation sequencing makes it possible from a technical and economic standpoint to sequence an individual’s exome but at the cost of generating long lists of gene variants that are not straightforward to interpret. Various public consortiums such as the 1000 Genomes Project and the NHLBI Exome Sequencing Project have sequenced the exomes and a subset of entire genomes of over 2500 control individuals with ongoing efforts to further catalogue genetic variation in humans.1 The use of these public databases facilitates the interpretation of these variant lists produced by exome sequencing and, as a result, novel genetic variants linked to disease are being discovered and reported at a record rate. However, the interpretation of these results and their bearing on diagnosis, prognosis, and treatment is becoming ever more complicated. Here, we discuss the application of genetic testing to individuals with focal and segmental glomerulosclerosis (FSGS), taking a historical perspective on gene identification and its clinical implications along with the growing potential of next-generation sequencing.Publication Innate immunity pathways regulate the nephropathy gene Apolipoprotein L1(2014) Nichols, Brendan; Jog, Prachi; Lee, Jessica; Blackler, Daniel; Wilmot, Michael; D’Agati, Vivette; Markowitz, Glen; Kopp, Jeffrey; Alper, Seth; Pollak, Martin; Friedman, DavidApolipoprotein L1 (APOL1) risk variants greatly elevate the risk of kidney disease in African Americans. Here we report a cohort of patients who developed collapsing focal segmental glomerulosclerosis while receiving therapeutic interferon, all of whom carried the APOL1 high-risk genotype. This finding raised the possibility that interferons and the molecular pattern recognition receptors that stimulate interferon production may contribute to APOL1-associated kidney disease. In cell culture, interferons and toll-like receptor agonists increased APOL1 expression by up to 200-fold, in some cases with the appearance of transcripts not detected under basal conditions. PolyI:C, a double-stranded RNA TLR3 agonist, increased APOL1 expression by upregulating interferons directly or through an interferon-independent, IRF-3 dependent pathway. Using pharmacological inhibitors, shRNA knockdown, and chromatin immunoprecipitation, we found that the interferon-independent TLR3 pathway relied on signaling through TBK1, NF-kB, and Jak kinases, and on binding of IRF1, IRF2, and STAT2 at the APOL1 transcription start site. We also demonstrate that overexpression of the APOL1 risk variants is more injurious to cells than overexpression of the wild-type APOL1 protein. Our study illustrates that anti-viral pathways may be an important inducer of kidney disease in individuals with the APOL1 high-risk genotype and identifies potential targets for prevention or treatment.Publication Copy Number Variation at the APOL1 Locus(Public Library of Science, 2015) Ruchi, Rupam; Genovese, Giulio; Lee, Jessica; Charoonratana, Victoria T.; Bernhardy, Andrea J.; Alper, Seth; Kopp, Jeffrey B.; Thadhani, Ravi; Friedman, David; Pollak, MartinTwo coding variants in the APOL1 gene (G1 and G2) explain most of the high rate of kidney disease in African Americans. APOL1-associated kidney disease risk inheritance follows an autosomal recessive pattern: The relative risk of kidney disease associated with inheritance of two high-risk variants is 7–30 fold, depending on the specific kidney phenotype. We wished to determine if the variability in phenotype might in part reflect structural differences in APOL1 gene. We analyzed sequence coverage from 1000 Genomes Project Phase 3 samples as well as exome sequencing data from African American kidney disease cases for copy number variation. 8 samples sequenced in the 1000 Genomes Project showed increased coverage over a ~100kb region that includes APOL2, APOL1 and part of MYH9, suggesting the presence of APOL1 copy number greater than 2. We reasoned that such duplications should be enriched in apparent G1 heterozygotes with kidney disease. Using a PCR-based assay, we observed the presence of this duplication in additional samples from apparent G0G1 or G0G2 individuals. The frequency of this APOL1 duplication was compared among cases (n = 123) and controls (n = 255) with apparent G0G1 heterozygosity. The presence of APOL1 duplication was observed in 4.06% of cases and 0.78% controls, preliminary evidence that this APOL1 duplication may alter susceptibility to kidney disease (p = 0.03). Taqman-based copy number assays confirmed the presence of 3 APOL1 copies in individuals positive for this specific duplication by PCR assay, but also identified a small number of individuals with additional APOL1 copies of presumably different structure. These observations motivate further studies to better assess the contribution of APOL1 copy number on kidney disease risk and on APOL1 function. Investigators and clinicians genotyping APOL1 should also consider whether the particular genotyping platform used is subject to technical errors when more than two copies of APOL1 are present.Publication Functional Validation of an Alpha-Actinin-4 Mutation as a Potential Cause of an Aggressive Presentation of Adolescent Focal Segmental Glomerulosclerosis: Implications for Genetic Testing(Public Library of Science, 2016) Feng, Di; Steinke, Julia M.; Krishnan, Ramaswamy; Birrane, Gabriel; Pollak, MartinGenetic testing in the clinic and research lab is becoming more routinely used to identify rare genetic variants. However, attributing these rare variants as the cause of disease in an individual patient remains challenging. Here, we report a patient who presented with nephrotic syndrome and focal segmental glomerulosclerosis (FSGS) with collapsing features at age 14. Despite treatment, her kidney disease progressed to end-stage within a year of diagnosis. Through genetic testing, an Y265H variant with unknown clinical significance in alpha-actinin-4 gene (ACTN4) was identified. This variant has not been seen previously in FSGS patients nor is it present in genetic databases. Her clinical presentation is different from previous descriptions of ACTN4 mediated FSGS, which is characterized by sub-nephrotic proteinuria and slow progression to end stage kidney disease. We performed in vitro and cellular assays to characterize this novel ACTN4 variant before attributing causation. We found that ACTN4 with either Y265H or K255E (a known disease-causing mutation) increased the actin bundling activity of ACTN4 in vitro, was associated with the formation of intracellular aggregates, and increased podocyte contractile force. Despite the absence of a familial pattern of inheritance, these similar biological changes caused by the Y265H and K255E amino acid substitutions suggest that this new variant is potentially the cause of FSGS in this patient. Our studies highlight that functional validation in complement with genetic testing may be required to confirm the etiology of rare disease, especially in the setting of unusual clinical presentations.Publication Human Kidney Disease-causing INF2 Mutations Perturb Rho/Dia Signaling in the Glomerulus(Elsevier, 2014) Sun, Hua; Al-Romaih, Khaldoun I.; MacRae, Calum; Pollak, MartinMutations in Inverted Formin 2 (INF2), a diaphanous formin family protein that regulates actin cytoskeleton dynamics, cause focal segmental glomerulosclerosis (FSGS) and Charcot–Marie–Tooth Disease (CMT) in humans. In addition to directly remodeling actin filaments in vitro, we have shown that INF2 regulates intracellular actin dynamics and actin dependent cellular behavior by opposing Rhoa/Dia signaling. As a step towards a better understanding of the human kidney disease, we wanted to explore the relevance of these findings to the in vivo situation. We used dose dependent knockdown of INF2 to first define an in vivo model and establish an overt glomerular phenotype in zebrafish. This simple assay was validated by rescue with wild type INF2 confirming the specificity of the findings. The edema, podocyte dysfunction, and an altered glomerular filtration barrier observed in the zebrafish pronephros correlate with mistrafficking of glomerular slit diaphragm proteins, defective slit-diaphragm signaling, and disinhibited diaphanous formin (mDia) activity. In contrast to wild-type human INF2, INF2 mutants associated with kidney disease fail to rescue the zINF2 morphant phenotype. Of particular interest, this INF2 knockdown phenotype is also rescued by loss of either RhoA or Dia2. This simple assay allows the demonstration that INF2 functions, at least in part, to modulate Dia-mediated Rho signaling, and that disease causing mutations specifically impair this regulatory function. These data support a model in which disease-associated diaphanous inhibitory domain (DID) mutants in INF2 interfere with its binding to and inhibition of Dia, leading to uncontrolled Rho/Dia signaling and perturbed actin dynamics. Methods to fine tune Rho signaling in the glomerulus may lead to new approaches to therapy in humans.