Targeted exome sequencing integrated with clinicopathological information reveals novel and rare mutations in atypical, suspected and unknown cases of Alport syndrome or proteinuria

We applied customized targeted next-generation exome sequencing (NGS) to determine if mutations in genes associated with renal malformations, Alport syndrome (AS) or nephrotic syndrome are a potential cause of renal abnormalities in patients with equivocal or atypical presentation. We first sequenced 4,041 exons representing 292 kidney disease genes in a Caucasian woman with a history of congenital vesicoureteral reflux (VUR), recurrent urinary tract infections and hydronephrosis who presented with nephrotic range proteinuria at the age of 45. Her biopsy was remarkable for focal segmental glomerulosclerosis (FSGS), a potential complication of longstanding VUR. She had no family history of renal disease. Her proteinuria improved initially, however, several years later she presented with worsening proteinuria and microhematuria. NGS analysis revealed two deleterious COL4A3 mutations, one novel and the other previously reported in AS, and a novel deleterious SALL2 mutation, a gene linked to renal malformations. Pedigree analysis confirmed that COL4A3 mutations were nonallelic and compound heterozygous. The genomic results in conjunction with subsequent abnormal electron microscopy, Collagen IV minor chain immunohistochemistry and progressive sensorineural hearing loss confirmed AS. We then modified our NGS approach to enable more efficient discovery of variants associated with AS or a subset of FSGS by multiplexing targeted exome sequencing of 19 genes associated with AS or FSGS in 14 patients. Using this approach, we found novel or known COL4A3 or COL4A5 mutations in a subset of patients with clinically diagnosed or suspected AS, APOL1 variants associated with FSGS in African Americans and novel mutations in genes associated with nephrotic syndrome. These studies demonstrate the successful application of targeted capture-based exome sequencing to simultaneously evaluate genetic variations in many genes in patients with complex renal phenotypes and provide insights into etiology of conditions with equivocal clinical and pathologic presentations

Diet modifies pioglitazone’s influence on hepatic PPARγ-regulated mitochondrial gene expression

Pioglitazone (Pio) is a thiazolidinedione (TZD) insulin-sensitizing drug whose effects result predominantly from its modulation of the transcriptional activity of peroxisome proliferator-activated-receptor-gamma (PPARγ). Pio is used to treat human insulin-resistant diabetes and also frequently considered for treatment of nonalcoholic steatohepatitis (NASH). In both settings, Pio’s beneficial effects are believed to result primarily from its actions on adipose PPARγ activity, which improves insulin sensitivity and reduces the delivery of fatty acids to the liver. Nevertheless, a recent clinical trial showed variable efficacy of Pio in human NASH. Hepatocytes also express PPARγ, and such expression increases with insulin resistance and in nonalcoholic fatty liver disease (NAFLD). Furthermore, mice that overexpress hepatocellular PPARγ and Pio-treated mice with extrahepatic PPARγ gene disruption develop features of NAFLD. Thus, Pio’s direct impact on hepatocellular gene expression might also be a determinant of this drug’s ultimate influence on insulin resistance and NAFLD. Previous studies have characterized Pio’s PPARγ-dependent effects on hepatic expression of specific adipogenic, lipogenic, and other metabolic genes. However, such transcriptional regulation has not been comprehensively assessed. The studies reported here address that consideration by genome-wide comparisons of Pio’s hepatic transcriptional effects in wildtype (WT) and liver-specific PPARγ-knockout (KO) mice given either control or high-fat (HFD) diets. The results identify a large set of hepatic genes for which Pio’s liver PPARγ-dependent transcriptional effects are concordant with its effects on RXR-DNA binding in WT mice. These data also show that HFD modifies Pio’s influence on a subset of such transcriptional regulation. Finally, our findings reveal a broader influence of Pio on PPARγ-dependent hepatic expression of nuclear genes encoding mitochondrial proteins than previously recognized. Taken together, these studies provide new insights about the tissue-specific mechanisms by which Pio affects hepatic gene expression and the broad scope of this drug’s influence on such regulation

Establishment of novel neuroendocrine carcinoma patient-derived xenograft models for receptor peptide-targeted therapy

Gastroenteropancreatic neuroendocrine neoplasms (GEP NENs) are rare cancers consisting of neuroendocrine carcinomas (NECs) and neuroendocrine tumors (NETs), which have been increasing in incidence in recent years. Few cell lines and pre-clinical models exist for studying GEP NECs and NETs, limiting the ability to discover novel imaging and treatment modalities. To address this gap, we isolated tumor cells from cryopreserved patient GEP NECs and NETs and injected them into the flanks of immunocompromised mice to establish patient-derived xenograft (PDX) models. Two of six mice developed tumors (NEC913 and NEC1452). Over 80% of NEC913 and NEC1452 tumor cells stained positive for Ki67. NEC913 PDX tumors expressed neuroendocrine markers such as chromogranin A (CgA), synaptophysin (SYP), and somatostatin receptor-2 (SSTR2), whereas NEC1452 PDX tumors did not express SSTR2. Exome sequencing revealed loss o

Rare recessive loss-of-function methionyl-tRNA synthetase mutations presenting as a multi-organ phenotype

BACKGROUND: Methionyl-tRNA synthetase (MARS) catalyzes the ligation of methionine to its cognate transfer RNA and therefore plays an essential role in protein biosynthesis. METHODS: We used exome sequencing, aminoacylation assays, homology modeling, and immuno-isolation of transfected MARS to identify and characterize mutations in the methionyl-tRNA synthetase gene (MARS) in an infant with an unexplained multi-organ phenotype. RESULTS: We identified compound heterozygous mutations (F370L and I523T) in highly conserved regions of MARS. The parents were each heterozygous for one of the mutations. Aminoacylation assays documented that the F370L and I523T MARS mutants had 18 ± 6% and 16 ± 6%, respectively, of wild-type activity. Homology modeling of the human MARS sequence with the structure of E. coli MARS showed that the F370L and I523T mutations are in close proximity to each other, with residue I523 located in the methionine binding pocket. We found that the F370L and I523T mutations did not affect the association of MARS with the multisynthetase complex. CONCLUSION: This infant expands the catalogue of inherited human diseases caused by mutations in aminoacyl-tRNA synthetase genes

Discovery of an unconventional centromere in budding yeast redefines evolution of point centromeres

Centromeres are the chromosomal regions promoting kinetochore assembly for chromosome segregation. In many eukaryotes, the centromere consists of up to mega base pairs of DNA. On such "regional centromeres," kinetochore assembly is mainly defined by epigenetic regulation [1]. By contrast, a clade of budding yeasts (Saccharomycetaceae) has a "point centromere" of 120-200 base pairs of DNA, on which kinetochore assembly is defined by the consensus DNA sequence [2, 3]. During evolution, budding yeasts acquired point centromeres, which replaced ancestral, regional centromeres [4]. All known point centromeres among different yeast species share common consensus DNA elements (CDEs) [5, 6], implying that they evolved only once and stayed essentially unchanged throughout evolution. Here, we identify a yeast centromere that challenges this view: that of the budding yeast Naumovozyma castellii is the first unconventional point centromere with unique CDEs. The N. castellii centromere CDEs are essential for centromere function but have different DNA sequences from CDEs in other point centromeres. Gene order analyses around N. castellii centromeres indicate their unique, and separate, evolutionary origin. Nevertheless, they are still bound by the ortholog of the CBF3 complex, which recognizes CDEs in other point centromeres. The new type of point centromere originated prior to the divergence between N. castellii and its close relative Naumovozyma dairenensis and disseminated to all N. castellii chromosomes through extensive genome rearrangement. Thus, contrary to the conventional view, point centromeres can undergo rapid evolutionary changes. These findings give new insights into the evolution of point centromeres

Development and annotation of perennial Triticeae ESTs and SSR markers.

Triticeae contains hundreds of species of both annual and perennial types. Although substantial genomic tools are available for annual Triticeae cereals such as wheat and barley, the perennial Triticeae lack sufficient genomic resources for genetic mapping or diversity research. To increase the amount of sequence information available in the perennial Triticeae, three expressed sequence tag (EST) libraries were developed and annotated for Pseudoroegneria spicata, a mixture of both Elymus wawawaiensis and E. lanceolatus, and a Leymus cinereus x L. triticoides interspecific hybrid. The ESTs were combined into unigene sets of 8 780 unigenes for P. spicata, 11 281 unigenes for Leymus, and 7 212 unigenes for Elymus. Unigenes were annotated based on putative orthology to genes from rice, wheat, barley, other Poaceae, Arabidopsis, and the non-redundant database of the NCBI. Simple sequence repeat (SSR) markers were developed, tested for amplification and polymorphism, and aligned to the rice genome. Leymus EST markers homologous to rice chromosome 2 genes were syntenous on Leymus homeologous groups 6a and 6b (previously 1b), demonstrating promise for in silico comparative mapping. All ESTs and SSR markers are available on an EST information management and annotation database (http://titan.biotec.uiuc.edu/triticeae/)

Silent but Not Static: Accelerated Base-Pair Substitution in Silenced Chromatin of Budding Yeasts

Subtelomeric DNA in budding yeasts, like metazoan heterochromatin, is gene poor, repetitive, transiently silenced, and highly dynamic. The rapid evolution of subtelomeric regions is commonly thought to arise from transposon activity and increased recombination between repetitive elements. However, we found evidence of an additional factor in this diversification. We observed a surprising level of nucleotide divergence in transcriptionally silenced regions in inter-species comparisons of Saccharomyces yeasts. Likewise, intra-species analysis of polymorphisms also revealed increased SNP frequencies in both intergenic and synonymous coding positions of silenced DNA. This analysis suggested that silenced DNA in Saccharomyces cerevisiae and closely related species had increased single base-pair substitution that was likely due to the effects of the silencing machinery on DNA replication or repair

Multi-platform assessment of transcriptional profiling technologies utilizing a precise probe mapping methodology

BACKGROUND: The arrival of RNA-seq as a high-throughput method competitive to the established microarray technologies has necessarily driven a need for comparative evaluation. To date, cross-platform comparisons of these technologies have been relatively few in number of platforms analyzed and were typically gene name annotation oriented. Here, we present a more extensive and yet precise assessment to elucidate differences and similarities in performance of numerous aspects including dynamic range, fidelity of raw signal and fold-change with sample titration, and concordance with qRT-PCR (TaqMan). To ensure that these results were not confounded by incompatible comparisons, we introduce the concept of probe mapping directed “transcript pattern”. A transcript pattern identifies probe(set)s across platforms that target a common set of transcripts for a specific gene. Thus, three levels of data were examined: entire data sets, data derived from a subset of 15,442 RefSeq genes common across platforms, and data derived from the transcript pattern defined subset of 7,034 RefSeq genes. RESULTS: In general, there were substantial core similarities between all 6 platforms evaluated; but, to varying degrees, the two RNA-seq protocols outperformed three of the four microarray platforms in most categories. Notably, a fourth microarray platform, Agilent with a modified protocol, was comparable, or marginally superior, to the RNA-seq protocols within these same assessments, especially in regards to fold-change evaluation. Furthermore, these 3 platforms (Agilent and two RNA-seq methods) demonstrated over 80 % fold-change concordance with the gold standard qRT-PCR (TaqMan). CONCLUSIONS: This study suggests that microarrays can perform on nearly equal footing with RNA-seq, in certain key features, specifically when the dynamic range is comparable. Furthermore, the concept of a transcript pattern has been introduced that may minimize potential confounding factors of multi-platform comparison and may be useful for similar evaluations. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12864-015-1913-6) contains supplementary material, which is available to authorized users