Ultra High Throughput Sequencing in Human DNA Variation Detection: A Comparative Study on the NDUFA3-PRPF31 Region

BACKGROUND: Ultra high throughput sequencing (UHTS) technologies find an important application in targeted resequencing of candidate genes or of genomic intervals from genetic association studies. Despite the extraordinary power of these new methods, they are still rarely used in routine analysis of human genomic variants, in part because of the absence of specific standard procedures. The aim of this work is to provide human molecular geneticists with a tool to evaluate the best UHTS methodology for efficiently detecting DNA changes, from common SNPs to rare mutations. METHODOLOGY/PRINCIPAL FINDINGS: We tested the three most widespread UHTS platforms (Roche/454 GS FLX Titanium, Illumina/Solexa Genome Analyzer II and Applied Biosystems/SOLiD System 3) on a well-studied region of the human genome containing many polymorphisms and a very rare heterozygous mutation located within an intronic repetitive DNA element. We identify the qualities and the limitations of each platform and describe some peculiarities of UHTS in resequencing projects. CONCLUSIONS/SIGNIFICANCE: When appropriate filtering and mapping procedures are applied UHTS technology can be safely and efficiently used as a tool for targeted human DNA variations detection. Unless particular and platform-dependent characteristics are needed for specific projects, the most relevant parameter to consider in mainstream human genome resequencing procedures is the cost per sequenced base-pair associated to each machine

Two trans-acting eQTLs modulate the penetrance of PRPF31 mutations

Dominant mutations in the gene encoding the ubiquitously-expressed splicing factor PRPF31 cause retinitis pigmentosa, a form of hereditary retinal degeneration, with reduced penetrance. We and others have previously shown that penetrance is tightly correlated with PRPF31 expression, as lymphoblastoid cell lines (LCLs) from affected patients produce less abundant PRPF31 transcripts than LCLs from their unaffected relatives carrying the same mutation. We have investigated the genetic elements determining the variable expression of PRPF31, and therefore possibly influencing the penetrance of its mutations, by quantifying PRPF31 mRNA levels in LCLs derived from 15 CEPH families (200 individuals), representative of the general population. We found that PRPF31 transcript abundance was a highly variable and highly heritable character. Moreover, by linkage analysis we showed that PRPF31 expression was significantly associated with at least one expression quantitative trait locus (eQTL), spanning a 8.2-Mb region on chromosome 14q21-23. We also investigated a previously mapped penetrance factor located near PRPF31 itself in LCLs from individuals belonging to selected families segregating PRPF31 mutations that displayed reduced penetrance. Our results indicate that, despite its constant association with the non-mutant allele, this factor was able to modulate the expression of both PRPF31 alleles. Furthermore, we showed that LCLs from affected patients have less PRPF31 RNA than those of asymptomatic patients, even at the pre-splicing stage. Altogether, these data demonstrate that PRPF31 mRNA expression and consequently the penetrance of PRPF31 mutations is managed by diffusible compounds encoded by at least two modifiers, acting in a co-regulatory system on both PRPF31 alleles during transcriptio

Whole-exome sequencing in a consanguineous Pakistani family identifies a mutational hotspot in the COL7A1 gene, causing recessive dystrophic epidermolysis bullosa

Dystrophic epidermolysis bullosa is a major form of epidermolysis bullosa and may be inherited as an autosomal dominant or recessive trait, with associated mutations in the COL7A1 gene. Here, we describe a consanguineous Pakistani family with four affected individuals suffering from recessive dystrophic epidermolysis bullosa. Exome sequencing of the proband's DNA revealed a homozygous missense variant (c.8038G>A:p.Gly2680Ser) in COL7A1 which cosegregated with disease in the family. The emergence of this particular glycine substitution in patients from diverse ethnic backgrounds such as China, United Kingdom, Poland, Iran, and Pakistan indicates that this variant most likely constitutes a recurrent mutational hotspot in the COL7A1 gene, rather than a germline mutation present at low levels in the general population

Whole exome sequencing and homozygosity mapping reveals genetic defects in consanguineous Iranian families with inherited retinal dystrophies

Acknowledgements This research was funded by the Swiss National Science Foundation (Grant #176097 to CR). We would like to express gratitude to the patients and all their family members that participated in this study for their valuable cooperation and participation.Peer reviewedPublisher PD

Interactome analysis reveals that FAM161A, deficient in recessive retinitis pigmentosa, is a component of the Golgi-centrosomal network

Defects in FAM161A, a protein of unknown function localized at the cilium of retinal photoreceptor cells, cause retinitis pigmentosa, a form of hereditary blindness. By using different fragments of this protein as baits to screen cDNA libraries of human and bovine retinas, we defined a yeast two-hybrid-based FAM161A interactome, identifying 53 bona fide partners. In addition to statistically significant enrichment in ciliary proteins, as expected, this interactome revealed a substantial bias towards proteins from the Golgi apparatus, the centrosome and the microtubule network. Validation of interaction with key partners by co-immunoprecipitation and proximity ligation assay confirmed that FAM161A is a member of the recently recognized Golgi-centrosomal interactome, a network of proteins interconnecting Golgi maintenance, intracellular transport and centrosome organization. Notable FAM161A interactors included AKAP9, FIP3, GOLGA3, KIFC3, KLC2, PDE4DIP, NIN and TRIP11. Furthermore, analysis of FAM161A localization during the cell cycle revealed that this protein followed the centrosome during all stages of mitosis, likely reflecting a specific compartmentalization related to its role at the ciliary basal body during the G0 phase. Altogether, these findings suggest that FAM161A's activities are probably not limited to ciliary tasks but also extend to more general cellular functions, highlighting possible novel mechanisms for the molecular pathology of retinal diseas

Ultra high throughput sequencing excludes MDH1 as candidate gene for RP28-linked retinitis pigmentosa

PURPOSE: Mutations in IDH3B, an enzyme participating in the Krebs cycle, have recently been found to cause autosomal recessive retinitis pigmentosa (arRP). The MDH1 gene maps within the RP28 arRP linkage interval and encodes cytoplasmic malate dehydrogenase, an enzyme functionally related to IDH3B. As a proof of concept for candidate gene screening to be routinely performed by ultra high throughput sequencing (UHTs), we analyzed MDH1 in a patient from each of the two families described so far to show linkage between arRP and RP28. METHODS: With genomic long-range PCR, we amplified all introns and exons of the MDH1 gene (23.4 kb). PCR products were then sequenced by short-read UHTs with no further processing. Computer-based mapping of the reads and mutation detection were performed by three independent software packages. RESULTS: Despite the intrinsic complexity of human genome sequences, reads were easily mapped and analyzed, and all algorithms used provided the same results. The two patients were homozygous for all DNA variants identified in the region, which confirms previous linkage and homozygosity mapping results, but had different haplotypes, indicating genetic or allelic heterogeneity. None of the DNA changes detected could be associated with the disease. CONCLUSIONS: The MDH1 gene is not the cause of RP28-linked arRP. Our experimental strategy shows that long-range genomic PCR followed by UHTs provides an excellent system to perform a thorough screening of candidate genes for hereditary retinal degeneration

PRPF mutations are associated with generalized defects in spliceosome formation and pre-mRNA splicing in patients with retinitis pigmentosa

Proteins PRPF31, PRPF3 and PRPF8 (RP-PRPFs) are ubiquitously expressed components of the spliceosome, a macromolecular complex that processes nearly all pre-mRNAs. Although these spliceosomal proteins are conserved in eukaryotes and are essential for survival, heterozygous mutations in human RP-PRPF genes lead to retinitis pigmentosa, a hereditary disease restricted to the eye. Using cells from patients with 10 different mutations, we show that all clinically relevant RP-PRPF defects affect the stoichiometry of spliceosomal small nuclear RNAs (snRNAs), the protein composition of tri-small nuclear ribonucleoproteins and the kinetics of spliceosome assembly. These mutations cause inefficient splicing in vitro and affect constitutive splicing ex-vivo by impairing the removal of at least 9% of endogenously expressed introns. Alternative splicing choices are also affected when RP-PRPF defects are present. Furthermore, we show that the steady-state levels of snRNAs and processed pre-mRNAs are highest in the retina, indicating a particularly elevated splicing activity. Our results suggest a role for PRPFs defects in the etiology of PRPF-linked retinitis pigmentosa, which appears to be a truly systemic splicing disease. Although these mutations cause widespread and important splicing defects, they are likely tolerated by the majority of human tissues but are critical for retinal cell surviva

FAM161A, associated with retinitis pigmentosa, is a component of the cilia-basal body complex and interacts with proteins involved in ciliopathies

Retinitis pigmentosa (RP) is a retinal degenerative disease characterized by the progressive loss of photoreceptors. We have previously demonstrated that RP can be caused by recessive mutations in the human FAM161A gene, encoding a protein with unknown function that contains a conserved region shared only with a distant paralog, FAM161B. In this study, we show that FAM161A localizes at the base of the photoreceptor connecting cilium in human, mouse and rat. Furthermore, it is also present at the ciliary basal body in ciliated mammalian cells, both in native conditions and upon the expression of recombinant tagged proteins. Yeast two-hybrid analysis of binary interactions between FAM161A and an array of ciliary and ciliopathy-associated proteins reveals direct interaction with lebercilin, CEP290, OFD1 and SDCCAG8, all involved in hereditary retinal degeneration. These interactions are mediated by the C-terminal moiety of FAM161A, as demonstrated by pull-down experiments in cultured cell lines and in bovine retinal extracts. As other ciliary proteins, FAM161A can also interact with the microtubules and organize itself into microtubule-dependent intracellular networks. Moreover, small interfering RNA-mediated depletion of FAM161A transcripts in cultured cells causes the reduction in assembled primary cilia. Taken together, these data indicate that FAM161A-associated RP can be considered as a novel retinal ciliopathy and that its molecular pathogenesis may be related to other ciliopathie

Mutational screening of splicing factor genes in cases with autosomal dominant retinitis pigmentosa

Purpose Mutations in genes encoding proteins from the tri-snRNP complex of the spliceosome account for more than 12% of cases of autosomal dominant retinitis pigmentosa (adRP). Although the exact mechanism by which splicing factor defects trigger photoreceptor death is not completely clear, their role in retinitis pigmentosa has been demonstrated by several genetic and functional studies. To test for possible novel associations between splicing factors and adRP, we screened four tri-snRNP splicing factor genes (EFTUD2, PRPF4, NHP2L1, and AAR2) as candidate disease genes. Methods: We screened up to 303 patients with adRP from Europe and North America who did not carry known RP mutations. Exon-PCR and Sanger methods were used to sequence the NHP2L1 and AAR2 genes, while the sequences of EFTUD2 and PRPF4 were obtained by using long-range PCRs spanning coding and non-coding regions followed by next-generation sequencing. Results: We detected novel missense changes in individual patients in the sequence of the genes PRPF4 and EFTUD2, but the role of these changes in relationship to disease could not be verified. In one other patient we identified a novel nucleotide substitution in the 5′ untranslated region (UTR) of NHP2L1, which did not segregate with the disease in the family. Conclusions: The absence of clearly pathogenic mutations in the candidate genes screened in our cohort suggests that EFTUD2, PRPF4, NHP2L1, and AAR2 are either not involved in adRP or are associated with the disease in rare instances, at least as observed in this study in patients of European and North American origin

Sadržaj

Metastasis is a multi-step process in which direct crosstalk between cancer cells and their microenvironment plays a key role. Here, we assessed the effect of paired tumor-associated and normal lung tissue mesenchymal stem cells (MSCs) on the growth and dissemination of primary human lung carcinoma cells isolated from the same patients. We show that the tumor microenvironment modulates MSC gene expression and identify a four-gene MSC signature that is functionally implicated in promoting metastasis. We also demonstrate that tumor-associated MSCs induce the expression of genes associated with an aggressive phenotype in primary lung cancer cells and selectively promote their dissemination rather than local growth. Our observations provide insight into mechanisms by which the stroma promotes lung cancer metastasis