Non-synonymous Sequence Changes Research Articles

Associations of Multiple NOTCH4 Exonic Variants with Systemic Sclerosis.

Findings from previous genome-wide association studies indicated an association of the NOTCH4 gene with systemic sclerosis (SSc). This is a followup study to fine-map exonic variants of NOTCH4 in SSc. All exons of NOTCH4 were sequenced and analyzed in a total of 1006 patients with SSc and 1004 controls of US white ancestry with the Ion Torrent system. Identified SSc-associated variants were confirmed with Sanger sequencing, and then examined in a Chinese Han cohort consisting of 576 patients with SSc and 574 controls. The NOTCH4 variants were analyzed for association with SSc as a whole and with SSc clinical and autoantibody subtypes with and without the influence of specific HLA-class II alleles that had been previously identified as major genetic factors in SSc. A total of 12 SSc-associated and SSc subtype-associated exonic variants of NOTCH4 were identified in the US cohort. Three of them are nonsynonymous single-nucleotide polymorphisms and 1 is a CTG tandem repeat that encodes for a poly-leucine, all of which are located in the NOTCH4 extracellular domain (NECD). Conditional logistic regression analysis on SSc-associated HLA-class II alleles indicated an independent association of the NOTCH4 variants with SSc autoantibody subtypes. Analysis of the Chinese cohort supported a genetic contribution of NOTCH4 to SSc and its subtypes. Multiple NOTCH4 exonic variants were associated with SSc and/or SSc subtypes. Several of these variants encode nonsynonymous sequence changes occurring in the NECD, which implicates a potentially functional effect of NOTCH4.

Abstract A1-01: Translational genomics of urologic cancer genes

Abstract Next generation sequencing (NGS) of urologic tumor genomes has led to the detection of frequently altered cancer genes. We have characterized bladder (BCa), prostate (PCa), and kidney (RCC) tumor genomes using exome, whole genome, and transcriptome NGS; copy number and SNP arrays, and bisulfite sequencing for methylation to detect genomic alterations associated with disease. We recently examined BCa tumors from 54 U.S. patients and compared these to 99 Chinese patients to identify novel alterations of cancer genes encoding proteins associated with DNA and chromatin, including telomerase (TERT), stromal antigen 2 (STAG2), and BRCA1-associated protein-1 (BAP1). We are the first to show the TERT promoter is altered in BCa by both somatic and germline nucleotide changes in 37/54 (69%) and 30/54 (56%) tumors, respectively; and that TERT somatic alterations do not correlate with other cancer gene mutations examined by exome NGS. Most TERT germline variants were novel (19/20), and three variants were confirmed as both somatic and germline in distinct tumors, including the most frequent variant, c. – 245 T>C (rs2853669). We show somatic promoter alterations are associated with increased TERT expression but, paradoxically, with significantly shorter telomeres in tumors as compared to matched normal tissue. STAG2 was altered by somatic deleterious sequence changes, genomic deletions, and promoter CpG hypermethylation. Mutations are associated with reduced patient survival and tumor cell aneuploidy based on an increased number of chromosomal arm copy number changes. Somatic BAP1 mutations are primarily deleterious sequence changes that occurred preferentially in U.S. Caucasian as compared to Chinese BCa patients. BAP1 mutations are associated with papillary histologic features in a subset of tumors and with mutations in the histone lysine–specific demethylase 6A (KDM6A/UTX) gene. BAP1 mutations contribute to a significant number of tumors with somatic and rare germline variants in BRCA pathway genes, including BRCA1, BRCA2, PALB2, and ATM. We experimentally examined the function of KDM6A since it is the most frequently altered chromatin modifying gene in 24% of tumors. KDM6A loss in human BCa cells enhanced in vitro proliferation, in vivo tumor growth, and cell migration, confirming KDM6A loss drives the BCa phenotype. A comparison of BCa and other urologic tumor genomes allows us to identify several emergent common characteristics. Significant numbers of tumors are altered by rare, disease-associated germline cancer gene variants that likely have substantive effects on cancer risk and which likely influence the subsequent accrual of somatic alterations. These likely contribute to the missing heritability that is observed by cancer epidemiology studies. Many cancer genes are altered by characteristic frequencies of nonsynonymous sequence changes, promoter variants, copy number variation, and hypermethylation, that are routinely observed for a given gene-tissue-patient ethnicity combination. Approximately 4-5 tumor suppressors are altered for each oncogene, and interactions between altered cancer genes affect the resulting biological functions. In BCa, we show KDM6A loss increases cell proliferation which likely results in shorter telomeres even though TERT expression is increased. We identified altered cancer genes such as STAG2 that are associated with discrete clinical aspects of the disease phenotype, namely aneuploidy and reduced patient survival indicating a lethal subtype of disease. Cancer genes on the X chromosome such as STAG2 and KDM6A have a single copy in men and likely contribute to the gender bias observed in cancer. Finally, cancer gene alterations can identify patients likely to respond to a currently approved therapy. BAP1 and BRCA pathway alterations define BCa, PCa, and RCC tumors with a common DNA repair deficiency that might be exploited by targeted therapies such as poly (ADP ribose) polymerase inhibitors. Citation Format: Michael L. Nickerson, Kate M. Im, Sevilay Turan, Guangwu Guo, Lee E. Moore, Dan Theodorescu, Michael Dean. Translational genomics of urologic cancer genes. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-01.

Mitochondrial Sequence Changes in Keratoconus Patients

We investigated whether a group of patients with keratoconus (KTCN) harbor mutations in the mitochondrial genome. We sequenced the full mitochondrial genome in a group of Saudi patients with KTCN (n = 26) and 100 ethnically matched controls who had no KTCN by examination. A total of 10 KTCN patients (38.5%) had potentially pathogenic nonsynonymous mtDNA mutations. Of the nonsynonymous sequence changes detected, 4 (40%) were in Complex I, one was in the tRNA(Glutamine), one was in tRNA(Tryptophan), one was in tRNA(Asparagine), one was in tRNA(Histidine), and two were in the tRNA(Leucine2). One nonsynonymous sequence change was heteroplasmic, whereas all the remaining 9 were homoplasmic. These sequence changes were not detected in controls of similar ethnicity. Four sequence changes were novel (were not reported previously) and 5 were reported previously. Additionally, we detected 54 synonymous (does not result in an amino acid change) sequence changes with no pathologic significance. If our results are confirmed in a larger cohort and multiple ethnicities, then mtDNA mutation may be considered as a genetic risk factor contributing indirectly through the oxidative stress mechanism to the development and/or progression of KTCN.

Divergence and Redundancy in CSLD2 and CSLD3 Function During Arabidopsis Thaliana Root Hair and Female Gametophyte Development

The Arabidopsis cellulose synthase-like D (CSLD) 2 and 3 genes are known to function in root hair development. Here, we show that these genes also play a role in female gametophyte development because csld2 csld3 double mutants were observed to have low seed set that could be traced to defects in female transmission efficiency. Cell biological studies of csld2 csld3 ovules showed synergid cell degeneration during megagametogenesis and reduced pollen tube penetration during fertilization. Although CSLD2 and CSLD3 function redundantly in female gametophyte development, detailed analyses of root hair phenotypes of progeny from genetic crosses between csld2 and csld3, suggest that CSLD3 might play a more prominent role than CSLD2 in root hair development. Phylogenetic and gene duplication studies of CSLD2 and CSLD3 homologs in Arabidopsis lyrata, Populus, Medicago, maize, and Physcomitrella were further performed to investigate the course of evolution for these genes. Our analyses indicate that the ancestor of land plants possibly contained two copies of CSLD genes, one of which developed into the CSLD5 lineage in flowering plants, and the other formed the CSLD1/2/3/4 clade. In addition, CSLD2 and CSLD3 likely originated from a recent genome-wide duplication event explaining their redundancy. Moreover, sliding-window dN/dS analysis showed that most of the coding regions of CSLD2 and CSLD3 have been under strong purifying selection pressure. However, the region that encodes the N-terminus of CSLD3 has been under relatively relaxed selection pressure as indicated by its high dN/dS value, suggesting that CSLD3 might have gained additional functions through more frequent non-synonymous sequence changes at the N-terminus, which could partly explain the more prominent role of CSLD3 during root hair development compared to CSLD2.

Structural Characterization of Novel Pseudomonas aeruginosa Type IV Pilins

Pseudomonas aeruginosa type IV pili, composed of PilA subunits, are used for attachment and twitching motility on surfaces. P. aeruginosa strains express one of five phylogenetically distinct PilA proteins, four of which are associated with accessory proteins that are involved either in pilin posttranslational modification or in modulation of pilus retraction dynamics. Full understanding of pilin diversity is crucial for the development of a broadly protective pilus-based vaccine. Here, we report the 1.6-Å X-ray crystal structure of an N-terminally truncated form of the novel PilA from strain Pa110594 (group V), which represents the first non-group II pilin structure solved. Although it maintains the typical T4a pilin fold, with a long N-terminal α-helix and four-stranded antiparallel β-sheet connected to the C-terminus by a disulfide-bonded loop, the presence of an extra helix in the αβ-loop and a disulfide-bonded loop with helical character gives the structure T4b pilin characteristics. Despite the presence of T4b features, the structure of PilA from strain Pa110594 is most similar to the Neisseria gonorrhoeae pilin and is also predicted to assemble into a fiber similar to the GC pilus, based on our comparative pilus modeling. Interactions between surface-exposed areas of the pilin are suggested to contribute to pilus fiber stability. The non-synonymous sequence changes between group III and V pilins are clustered in the same surface-exposed areas, possibly having an effect on accessory protein interactions. However, based on our high-confidence model of group III PilA PA14, compensatory changes allow for maintenance of a similar shape.

Alpha‐synuclein and familial Parkinson's disease

Whole gene duplications and triplications of alpha-synuclein (SNCA) can cause Parkinson's disease (PD), and variation in the promoter region (Rep1) and 3' region of SNCA has been reported to increase disease susceptibility. Within our cohort, one affected individual from each of 92 multiplex PD families showing the greatest evidence of linkage to the region around SNCA was screened for dosage alterations and sequence changes; no dosage or non-synonymous sequence changes were found. In addition, 737 individuals (from 450 multiplex PD families) that met strict diagnostic criteria for PD and did not harbor a known causative mutation, as well as 359 neurologically normal controls, were genotyped for the Rep1 polymorphism and four SNPs in the 3' region of SNCA. The four SNPs were in high LD (r(2) > 0.95) and were analyzed as a haplotype. The effects of the Rep1 genotype and the 3' haplotype were evaluated using regression models employing only one individual per family. Cases had a 3% higher frequency of the Rep1 263 bp allele compared with controls (OR = 1.54; empirical P-value = 0.02). There was an inverse linear relationship between the number of 263 bp alleles and age of onset (empirical P-value = 0.0004). The 3' haplotype was also associated with disease (OR = 1.29; empirical P-value = 0.01), but not age of onset (P = 0.40). These data suggest that dosage and sequence changes are a rare cause of PD, but variation in the promoter and 3' region of SNCA convey an increased risk for PD.

Identification of Novel, Non-Synonymous Sequence Changes in the Tyrosine Kinase Genes of Patients with Acute Myeloid Leukemia.

Activating mutations in receptor tyrosine kinase (RTK) genes (including FLT3 and KIT) occur in more than 30% of newly diagnosed patients with acute myeloid leukemia (AML); we and others have speculated that mutations in other TK genes may be present in the remaining 70%. We therefore examined the expression of all annotated RTK and cytoplasmic tyrosine kinase (CTK) genes to prioritize these genes for sequencing. We performed high-throughput re-sequencing of the kinase domains of 24 TK genes (9 RTK and 15 CTK) using amplified genomic DNA from the bone marrow (tumor) and matched skin biopsy samples (“germline”) from 94 patients with de novo AML, and validated positive findings in an additional 94 AML tumor samples (14.4 million base pairs of double-stranded coverage). In addition to previously reported somatic mutations in FLT3, KIT, and JAK2 (which occurred at expected frequencies), we found novel somatic mutations in four patients in JAK1, NTRK1 and DDR1. Unexpectedly, we also identified novel non-synonymous germline sequence changes in 14 genes, including TYK2. We examined frequencies of known polymorphisms in our patients versus controls. We determined that the previously reported JAK3P132T allele is a germline variant that occurs in 19% of normal African Americans. Even when controlling for race, the TYK2G363S allele was found significantly less frequently in AML samples (12/376 alleles, 3.2%) compared to 147 normal controls (27/294 alleles, 9.2%, p=0.0013). Notably, there was loss of heterozygosity (LOH) at TYK2 in 2 patients. Additional population based studies and biologic validation will be required to define the significance of these sequence changes for AML pathogenesis. Lastly, we compared the expression of RTK and CTK genes in AML samples (n=92) to highly enriched normal human CD34+, promyelocyte, or polymorphonuclear neutrophil populations (n=5 each). We found several RTKs (FLT3, KIT, LTK) and CTKs (FYN, LCK, ITK, HCK and FGR) were tightly regulated in normal hematopoietic development but were dysregulated in many AML samples. Taken together, our data suggest that RTK or CTK mutations are not required for AML development but may be disease modifying events. Our data also suggest that germline variants and dysregulated expression of RTK and CTK genes may play significant roles AML pathogenesis.

Age-dependent prevalence of mutations at the GLC1A locus in primary open-angle glaucoma

PURPOSE: To screen a population with primary open-angle glaucoma for mutations in the gene that encodes the trabecular meshwork inducible glucocorticoid response protein (TIGR), also known as myocilin (MYOC). METHODS: Ophthalmologic information was collected for study subjects with primary open-angle glaucoma and their relatives. Mutation screening of 74 primary open-angle glaucoma probands was conducted by sequencing TIGR/MYOC coding sequence and splice sites. RESULTS: In 23 families we detected 13 nonsynonymous sequence changes, nine of which appear to be mutations likely to cause or contribute to primary open-angle glaucoma. Two mutations, Arg272Gly and Ile499Ser, and one nonsynonymous sequence variant, Asn57Asp, are novel. We found mutations in nine of 25 juvenile glaucoma probands (36%) and two of 49 adult-onset glaucoma probands (4%). Age classification of families rather than individual probands revealed mutations in three of nine families with strictly juvenile primary open-angle glaucoma (33%), and no mutations in 39 families with strictly adult-onset primary open-angle glaucoma (0%). In families with mixed-onset primary open-angle glaucoma containing both juvenile primary open-angle glaucoma and adult-onset primary open-angle glaucoma cases, we found mutations in eight of 26 families (31%). CONCLUSIONS: Our data suggest that Gly252Arg, Arg272Gly, Glu323Lys, Gln368STOP, Pro370Leu, Thr377Met, Val426Phe, Ile477Asn, and Ile499Ser are likely to play roles that cause or contribute to the etiology of autosomal dominant primary open-angle glaucoma. Our finding of more TIGR/MYOC mutations in families with mixed-onset primary open-angle glaucoma than in the families with strictly adult-onset primary open-angle glaucoma implies that the presence of relatives with juvenile primary open-angle glaucoma in a family could be used as a basis for identifying a subset of the population with adult-onset primary open-angle glaucoma with higher prevalence of TIGR/MYOC mutations. To address this issue, and to refine estimations of mutation prevalence in these age-defined subpopulations, prospective study of a larger population ascertained entirely through adult-onset primary open-angle glaucoma probands will be needed.