Molecular Genetics of the Usher Syndrome in Lebanon: Identification of 11 Novel Protein Truncating Mutations by Whole Exome Sequencing

Background

Usher syndrome (USH) is a genetically heterogeneous condition with ten disease-causing genes. The spectrum of genes and mutations causing USH in the Lebanese and Middle Eastern populations has not been described. Consequently, diagnostic approaches designed to screen for previously reported mutations were unlikely to identify the mutations in 11 unrelated families, eight of Lebanese and three of Middle Eastern origins. In addition, six of the ten USH genes consist of more than 20 exons, each, which made mutational analysis by Sanger sequencing of PCR-amplified exons from genomic DNA tedious and costly. The study was aimed at the identification of USH causing genes and mutations in 11 unrelated families with USH type I or II.

Methods

Whole exome sequencing followed by expanded familial validation by Sanger sequencing.

Results

We identified disease-causing mutations in all the analyzed patients in four USH genes, MYO7A, USH2A, GPR98 and CDH23. Eleven of the mutations were novel and protein truncating, including a complex rearrangement in GPR98.

Conclusion

Our data highlight the genetic diversity of Usher syndrome in the Lebanese population and the time and cost-effectiveness of whole exome sequencing approach for mutation analysis of genetically heterogeneous conditions caused by large genes.     

Micro-Scale Genomic DNA Copy Number Aberrations as Another Means of Mutagenesis in Breast Cancer

Introduction

In breast cancer, the basal-like subtype has high levels of genomic instability relative to other breast cancer subtypes with many basal-like-specific regions of aberration. There is evidence that this genomic instability extends to smaller scale genomic aberrations, as shown by a previously described micro-deletion event in the PTEN gene in the Basal-like SUM149 breast cancer cell line.

Methods

We sought to identify if small regions of genomic DNA copy number changes exist by using a high density, gene-centric Comparative Genomic Hybridizations (CGH) array on cell lines and primary tumors. A custom tiling array for CGH (244,000 probes, 200 bp tiling resolution) was created to identify small regions of genomic change, which was focused on previously identified basal-like-specific, and general cancer genes. Tumor genomic DNA from 94 patients and 2 breast cancer cell lines was labeled and hybridized to these arrays. Aberrations were called using SWITCHdna and the smallest 25% of SWITCHdna-defined genomic segments were called micro-aberrations (<64 contiguous probes, ∼ 15 kb).

Results

Our data showed that primary tumor breast cancer genomes frequently contained many small-scale copy number gains and losses, termed micro-aberrations, most of which are undetectable using typical-density genome-wide aCGH arrays. The basal-like subtype exhibited the highest incidence of these events. These micro-aberrations sometimes altered expression of the involved gene. We confirmed the presence of the PTEN micro-amplification in SUM149 and by mRNA-seq showed that this resulted in loss of expression of all exons downstream of this event. Micro-aberrations disproportionately affected the 5′ regions of the affected genes, including the promoter region, and high frequency of micro-aberrations was associated with poor survival.

Conclusion

Using a high-probe-density, gene-centric aCGH microarray, we present evidence of small-scale genomic aberrations that can contribute to gene inactivation. These events may contribute to tumor formation through mechanisms not detected using conventional DNA copy number analyses.     

Enrichment of Mutations in Multiple DNA Sequences Using COLD-PCR in Emulsion

Background

Multiplex detection of low-level mutant alleles in the presence of wild-type DNA would be useful for several fields of medicine including cancer, pre-natal diagnosis and infectious diseases. COLD-PCR is a recently developed method that enriches low-level mutations during PCR cycling, thus enhancing downstream detection without the need for special reagents or equipment. The approach relies on the differential denaturation of DNA strands which contain Tm-lowering mutations or mismatches, versus ‘homo-duplex’ wild-type DNA. Enabling multiplex-COLD-PCR that can enrich mutations in several amplicons simultaneously is desirable but technically difficult to accomplish. Here we describe the proof of principle of an emulsion-PCR based approach that demonstrates the feasibility of multiplexed-COLD-PCR within a single tube, using commercially available mutated cell lines. This method works best with short amplicons; therefore, it could potentially be used on highly fragmented samples obtained from biological material or FFPE specimens.

Methods

Following a multiplex pre-amplification of TP53 exons from genomic DNA, emulsions which incorporate the multiplex product, PCR reagents and primers specific for a given TP53 exon are prepared. Emulsions with different TP53 targets are then combined in a single tube and a fast-COLD-PCR program that gradually ramps up the denaturation temperature over several PCR cycles is applied (temperature-tolerant, TT-fast-eCOLD-PCR). The range of denaturation temperatures applied encompasses the critical denaturation temperature (T_c) corresponding to all the amplicons included in the reaction, resulting to a gradual enrichment of mutations within all amplicons encompassed by emulsion.

Results

Validation for TT-fast-eCOLD-PCR is provided for TP53 exons 6–9. Using dilutions of mutated cell-line into wild-type DNA, we demonstrate simultaneous mutation enrichment between 7 to 15-fold in all amplicons examined.

Conclusions

TT-fast-eCOLD-PCR expands the versatility of COLD-PCR and enables high-throughput enrichment of low-level mutant alleles over multiple sequences in a single tube.     

Identifying and Characterizing a Novel Protein Kinase STK35L1 and Deciphering Its Orthologs and Close-Homologs in Vertebrates

Background

The human kinome containing 478 eukaryotic protein kinases has over 100 uncharacterized kinases with unknown substrates and biological functions. The Ser/Thr kinase 35 (STK35, Clik1) is a member of the NKF 4 (New Kinase Family 4 ) in the kinome with unknown substrates and biological functions. Various high throughput studies indicate that STK35 could be involved in various human diseases such as colorectal cancer and malaria.

Methodology/Principal Findings

In this study, we found that the previously published coding sequence of the STK35 gene is incomplete. The newly identified sequence of the STK35 gene codes for a protein of 534 amino acids with a N-terminal elongation of 133 amino acids. It has been designated as STK35L (STK35 long). Since it is the first of further homologous kinases we termed it as STK35L1. The STK35L1 protein (58 kDa on SDS-PAGE), but not STK35 (44 kDa), was found to be expressed in all human cells studied (endothelial cells, HeLa, and HEK cells) and was down-regulated after silencing with specific siRNA. EGFP-STK35L1 was localized in the nucleus and the nucleolus. By combining syntenic and gene structure pattern data and homology searches, two further STK35L1 homologs, STK35L2 (previously known as PDIK1L) and STK35L3, were found. All these protein kinase homologs were conserved throughout the vertebrates. The STK35L3 gene was specifically lost during placental mammalian evolution. Using comparative genomics, we have identified orthologous sets of these three protein kinases genes and their possible ancestor gene in two sea squirt genomes.

Conclusions/Significance

We found the full-length coding sequence of the STK35 gene and termed it as STK35L1. We identified a new third STK35-like gene, STK35L3, in vertebrates and a possible ancestor gene in sea squirt genome. This study will provide a comprehensive platform to explore the role of STK35L kinases in cell functions and human diseases.     

Sexually selected infanticide in a polygynous bat

Background

Adult individuals of many species kill unrelated conspecific infants for several adaptive reasons ranging from predation or resource competition to the prevention of misdirected parental care. Moreover, infanticide can increase the reproductive success of the aggressor by killing the offspring of competitors and thereafter mating with the victimized females. This sexually selected infanticide predominantly occurs in polygynous species, with convincing evidence for primates, carnivores, equids, and rodents. Evidence for bats was predicted but lacking.

Methodology/Principal Findings

Here we report the first case, to our knowledge, of sexually selected infanticide in a bat, the polygynous white-throated round-eared bat, Lophostoma silvicolum. Behavioral studies in a free-living population revealed that an adult male repeatedly attacked and injured the pups of two females belonging to his harem, ultimately causing the death of one pup. The infanticidal male subsequently mated with the mother of the victimized pup and this copulation occurred earlier than any other in his harem.

Conclusions/Significance

Our findings indicate that sexually selected infanticide is more widespread than previously thought, adding bats as a new taxon performing this strategy. Future work on other bats, especially polygynous species in the tropics, has great potential to investigate the selective pressures influencing the evolution of sexually selected infanticide and to study how infanticide impacts reproductive strategies and social structures of different species.     

Pre-Diagnostic Leukocyte Genomic DNA Methylation and the Risk of Colorectal Cancer in Women

Background

Abnormal one-carbon metabolism may lead to general genomic (global) hypomethylation, which may predispose an individual to the development of colorectal neoplasia.

Methods

We evaluated the association between pre-diagnostic leukocyte genomic DNA methylation level and the risk of colorectal cancer in a nested case-control study of 358 colorectal cancer cases and 661 matched controls within the all-female cohort of the Nurses’ Health Study (NHS). Among control subjects, we further examined major plasma components in the one-carbon metabolism pathway in relation to genomic DNA methylation level. Liquid chromatography/tandem mass spectrometry was used to examine leukocyte genomic DNA methylation level. We calculated odds ratios (ORs) and 95% confidence intervals (95% CIs) using logistic regression.

Results

Overall genomic DNA methylation level was not associated with the risk of colorectal cancer (p for trend, 0.45). Compared with women in the lowest quintile of methylation, the multivariate OR of colorectal cancer risk was 1.32 (95% CI, 0.82–2.13) for those in the highest quintile. We did not find significant associations between major plasma components of one-carbon metabolism or risk factors for colorectal cancer and genomic DNA methylation level (all p for trend >0.05). Also, neither one-carbon metabolism-related plasma components nor well-known risk factors for colorectal cancer modified the association between genomic DNA methylation level and the risk of colorectal cancer (all p for interaction >0.05).

Conclusions

We found no evidence that hypomethylation of leukocyte genomic DNA increases risk of colorectal cancer among women. Additional studies are needed to investigate the association between pre-diagnostic genomic DNA methylation level and colorectal cancer risk among diverse populations.     

Comparative genomics of 274 Vibrio cholerae genomes reveals mobile functions structuring three niche dimensions

Background

Vibrio cholerae is a globally dispersed pathogen that has evolved with humans for centuries, but also includes non-pathogenic environmental strains. Here, we identify the genomic variability underlying this remarkable persistence across the three major niche dimensions space, time, and habitat.

Results

Taking an innovative approach of genome-wide association applicable to microbial genomes (GWAS-M), we classify 274 complete V. cholerae genomes by niche, including 39 newly sequenced for this study with the Ion Torrent DNA-sequencing platform. Niche metadata were collected for each strain and analyzed together with comprehensive annotations of genetic and genomic attributes, including point mutations (single-nucleotide polymorphisms, SNPs), protein families, functions and prophages.

Conclusions

Our analysis revealed that genomic variations, in particular mobile functions including phages, prophages, transposable elements, and plasmids underlie the metadata structuring in each of the three niche dimensions. This underscores the role of phages and mobile elements as the most rapidly evolving elements in bacterial genomes, creating local endemicity (space), leading to temporal divergence (time), and allowing the invasion of new habitats. Together, we take a data-driven approach for comparative functional genomics that exploits high-volume genome sequencing and annotation, in conjunction with novel statistical and machine learning analyses to identify connections between genotype and phenotype on a genome-wide scale.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-654) contains supplementary material, which is available to authorized users.     

Comparative genomic analysis of Acinetobacter baumannii clinical isolates reveals extensive genomic variation and diverse antibiotic resistance determinants

Background

Acinetobacter baumannii is an important nosocomial pathogen that poses a serious health threat to immune-compromised patients. Due to its rapid ability to develop multidrug resistance (MDR), A. baumannii has increasingly become a focus of attention worldwide. To better understand the genetic variation and antibiotic resistance mechanisms of this bacterium at the genomic level, we reported high-quality draft genome sequences of 8 clinical isolates with various sequence types and drug susceptibility profiles.

Results

We sequenced 7 MDR and 1 drug-sensitive clinical A. baumannii isolates and performed comparative genomic analysis of these draft genomes with 16 A. baumannii complete genomes from GenBank. We found a high degree of variation in A. baumannii, including single nucleotide polymorphisms (SNPs) and large DNA fragment variations in the AbaR-like resistance island (RI) regions, the prophage and the type VI secretion system (T6SS). In addition, we found several new AbaR-like RI regions with highly variable structures in our MDR strains. Interestingly, we found a novel genomic island (designated as GI_BJ4) in the drug-sensitive strain BJ4 carrying metal resistance genes instead of antibiotic resistance genes inserted into the position where AbaR-like RIs commonly reside in other A. baumannii strains. Furthermore, we showed that diverse antibiotic resistance determinants are present outside the RIs in A. baumannii, including antibiotic resistance-gene bearing integrons, the bla_OXA-23-containing transposon Tn2009, and chromosomal intrinsic antibiotic resistance genes.

Conclusions

Our comparative genomic analysis revealed that extensive genomic variation exists in the A. baumannii genome. Transposons, genomic islands and point mutations are the main contributors to the plasticity of the A. baumannii genome and play critical roles in facilitating the development of antibiotic resistance in the clinical isolates.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1163) contains supplementary material, which is available to authorized users.     

Molecular Analysis and Phenotypic Study in 14 Chinese Families with Bietti Crystalline Dystrophy

Purpose

To investigate the clinical features and cytochrome P450 family 4 subfamily V polypeptide 2 (CYP4V2) gene mutations in 14 Chinese families with Bietti crystalline dystrophy (BCD).

Methods

Seventeen patients from 14 unrelated Chinese families with BCD were recruited for complete clinical ophthalmic examination and genetic study. The 11 exons of CYP4V2 were amplified from genomic DNA of all patients and their family members by polymerase chain reaction (PCR) and then sequenced. Exons of TIMP3 were also sequenced in BCD patient associated with choroidal neovascularization (CNV). One hundred and seventy unrelated healthy Chinese subjects were screened for mutations in CYP4V2.

Results

All 17 patients with BCD had mutations in CYP4V2; one of these mutations was novel (c.219T>A, p.F73L) and four other mutations had been reported. The p.F73L mutation was a commonly detected mutation in our study (seven out of 34 alleles), either in the homozygous state or in the heterozygous state. Among the patients, considerable phenotypic variability was detected, both within and between families. Screening of TIMP3 did not find any mutation in the BCD patient associated with CNV.

Conclusion

The novel CYP4V2 c.219T>A (p.F73L) mutation may be another recurrent mutation in Chinese patients with BCD. Our study expands the mutation spectrum of CYP4V2 and characterizes novel genotype–phenotype associations in Chinese patients with BCD.     

Potential of genotyping-by-sequencing for genomic selection in livestock populations

Background

Next-generation sequencing techniques, such as genotyping-by-sequencing (GBS), provide alternatives to single nucleotide polymorphism (SNP) arrays. The aim of this work was to evaluate the potential of GBS compared to SNP array genotyping for genomic selection in livestock populations.

Methods

The value of GBS was quantified by simulation analyses in which three parameters were varied: (i) genome-wide sequence read depth (x) per individual from 0.01x to 20x or using SNP array genotyping; (ii) number of genotyped markers from 3000 to 300 000; and (iii) size of training and prediction sets from 500 to 50 000 individuals. The latter was achieved by distributing the total available x of 1000x, 5000x, or 10 000x per genotyped locus among the varying number of individuals. With SNP arrays, genotypes were called from sequence data directly. With GBS, genotypes were called from sequence reads that varied between loci and individuals according to a Poisson distribution with mean equal to x. Simulated data were analyzed with ridge regression and the accuracy and bias of genomic predictions and response to selection were quantified under the different scenarios.

Results

Accuracies of genomic predictions using GBS data or SNP array data were comparable when large numbers of markers were used and x per individual was ~1x or higher. The bias of genomic predictions was very high at a very low x. When the total available x was distributed among the training individuals, the accuracy of prediction was maximized when a large number of individuals was used that had GBS data with low x for a large number of markers. Similarly, response to selection was maximized under the same conditions due to increasing both accuracy and selection intensity.

Conclusions

GBS offers great potential for developing genomic selection in livestock populations because it makes it possible to cover large fractions of the genome and to vary the sequence read depth per individual. Thus, the accuracy of predictions is improved by increasing the size of training populations and the intensity of selection is increased by genotyping a larger number of selection candidates.

Electronic supplementary material

The online version of this article (doi:10.1186/s12711-015-0102-z) contains supplementary material, which is available to authorized users.     

The draft genome of a socially polymorphic halictid bee,Lasioglossum albipes

Background

Taxa that harbor natural phenotypic variation are ideal for ecological genomic approaches aimed at understanding how the interplay between genetic and environmental factors can lead to the evolution of complex traits. Lasioglossum albipes is a polymorphic halictid bee that expresses variation in social behavior among populations, and common-garden experiments have suggested that this variation is likely to have a genetic component.

Results

We present the L. albipes genome assembly to characterize the genetic and ecological factors associated with the evolution of social behavior. The de novo assembly is comparable to other published social insect genomes, with an N50 scaffold length of 602 kb. Gene families unique to L. albipes are associated with integrin-mediated signaling and DNA-binding domains, and several appear to be expanded in this species, including the glutathione-s-transferases and the inositol monophosphatases. L. albipes has an intact DNA methylation system, and in silico analyses suggest that methylation occurs primarily in exons. Comparisons to other insect genomes indicate that genes associated with metabolism and nucleotide binding undergo accelerated evolution in the halictid lineage. Whole-genome resequencing data from one solitary and one social L. albipes female identify six genes that appear to be rapidly diverging between social forms, including a putative odorant receptor and a cuticular protein.

Conclusions

L. albipes represents a novel genetic model system for understanding the evolution of social behavior. It represents the first published genome sequence of a primitively social insect, thereby facilitating comparative genomic studies across the Hymenoptera as a whole.     

Identification and functional characterization of copy number variations in diverse chicken breeds

Background

The detection and functional characterization of genomic structural variations are important for understanding the landscape of genetic variation in the chicken. A recently recognized aspect of genomic structural variation, called copy number variation (CNV), is gaining interest in chicken genomic studies. The aim of the present study was to investigate the pattern and functional characterization of CNVs in five characteristic chicken breeds, which will be important for future studies associating phenotype with chicken genome architecture.

Results

Using a commercial 385 K array-based comparative genomic hybridization (aCGH) genome array, we performed CNV discovery using 10 chicken samples from four local Chinese breeds and the French breed Houdan chicken. The female Anka broiler was used as a reference. A total of 281 copy number variation regions (CNVR) were identified, covering 12.8 Mb of polymorphic sequences or 1.07% of the entire chicken genome. The functional annotation of CNVRs indicated that these regions completely or partially overlapped with 231 genes and 1032 quantitative traits loci, suggesting these CNVs have important functions and might be promising resources for exploring differences among various breeds. In addition, we employed quantitative PCR (qPCR) to further validate several copy number variable genes, such as prolactin receptor, endothelin 3 (EDN3), suppressor of cytokine signaling 2, CD8a molecule, with important functions, and the results suggested that EDN3 might be a molecular marker for the selection of dark skin color in poultry production. Moreover, we also identified a new CNVR (chr24: 3484617–3512275), encoding the sortilin-related receptor gene, with copy number changes in only black-bone chicken.

Conclusions

Here, we report a genome-wide analysis of the CNVs in five chicken breeds using aCGH. The association between EDN3 and melanoblast proliferation was further confirmed using qPCR. These results provide additional information for understanding genomic variation and related phenotypic characteristics.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-934) contains supplementary material, which is available to authorized users.     

Signal transduction protein array analysis links LRRK2 to Ste20 kinases and PKC zeta that modulate neuronal plasticity

Background

Dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are the most common genetic cause of Parkinson''s disease, however, the underlying pathogenic mechanisms are poorly understood. Several in vitro studies have shown that the most frequent mutation, LRRK2(G2019S), increases kinase activity and impairs neuronal survival. LRRK2 has been linked to the mitogen-activated protein kinase kinase kinase family and the receptor-interacting protein kinases based on sequence similarity within the kinase domain and in vitro substrate phosphorylation.

Methodology/Principal Findings

We used an unbiased proteomic approach to identify the kinase signaling pathways wherein LRRK2 may be active. By incubation of protein microarrays containing 260 signal transduction proteins we detected four arrayed Ste20 serine/threonine kinase family members (TAOK3, STK3, STK24, STK25) as novel LRRK2 substrates and LRRK2 interacting proteins, respectively. Moreover, we found that protein kinase C (PKC) zeta binds and phosphorylates LRRK2 both in vitro and in vivo.

Conclusions/Significance

Ste20 kinases and PKC zeta contribute to neuronal Tau phosphorylation, neurite outgrowth and synaptic plasticity under physiological conditions. Our data suggest that these kinases may also be involved in synaptic dysfunction and neurite fragmentation in transgenic mice and in human PD patients carrying toxic gain-of-function LRRK2 mutations.     

Identification of pneumococcal colonization determinants in the stringent response pathway facilitated by genomic diversity

Background

Understanding genetic determinants of a microbial phenotype generally involves creating and comparing isogenic strains differing at the locus of interest, but the naturally existing genomic and phenotypic diversity of microbial populations has rarely been exploited. Here we report use of a diverse collection of 616 carriage isolates of Streptococcus pneumoniae and their genome sequences to help identify a novel determinant of pneumococcal colonization.

Results

A spontaneously arising laboratory variant (SpnYL101) of a capsule-switched TIGR4 strain (TIGR4:19F) showed reduced ability to establish mouse nasal colonization and lower resistance to non-opsonic neutrophil-mediated killing in vitro, a phenotype correlated with in vivo success. Whole genome sequencing revealed 5 single nucleotide polymorphisms (SNPs) affecting 4 genes in SpnYL101 relative to its ancestor. To evaluate the effect of variation in each gene, we performed an in silico screen of 616 previously published genome sequences to identify pairs of closely-related, serotype-matched isolates that differ at the gene of interest, and compared their resistance to neutrophil-killing. This method allowed rapid examination of multiple candidate genes and found phenotypic differences apparently associated with variation in SP_1645, a RelA/ SpoT homolog (RSH) involved in the stringent response. To establish causality, the alleles corresponding to SP_1645 were switched between the TIGR4:19F and SpnYL101. The wild-type SP_1645 conferred higher resistance to neutrophil-killing and competitiveness in mouse colonization. Using a similar strategy, variation in another RSH gene (TIGR4 locus tag SP_1097) was found to alter resistance to neutrophil-killing.

Conclusions

These results indicate that analysis of naturally existing genomic diversity complements traditional genetics approaches to accelerate genotype-phenotype analysis.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1573-6) contains supplementary material, which is available to authorized users.