Fine Breakpoint Mapping by Genome Sequencing Reveals the First Large X Inversion Disrupting the NHS Gene in a Patient with Syndromic Cataracts

Inversions are structural variants that are generally balanced. However, they could lead to gene disruptions or have positional effects leading to diseases. Mutations in the NHS gene cause Nance-Horan syndrome, an X-linked disorder characterised by congenital cataracts and dental anomalies. Here, we aimed to characterise a balanced pericentric inversion X(p22q27), maternally inherited, in a child with syndromic bilateral cataracts by breakpoint mapping using whole-genome sequencing (WGS). 30× Illumina paired-end WGS was performed in the proband, and breakpoints were confirmed by Sanger sequencing. EdU assays and FISH analysis were used to assess skewed X-inactivation patterns. RNA expression of involved genes in the breakpoint boundaries was evaluated by droplet-digital PCR. We defined the breakpoint position of the inversion at Xp22.13, with a 15 bp deletion, disrupting the unusually large intron 1 of the canonical NHS isoform, and also perturbing topologically-associated domains (TADs). Moreover, a microhomology region of 5 bp was found on both sides. RNA analysis confirmed null and reduced NHS expression in the proband and his unaffected mother, respectively. In conclusion, we report the first chromosomal inversion disrupting NHS, fine-mapped by WGS. Our data expand the clinical spectrum and the pathogenic mechanisms underlying the NHS defects.

The EGFRvIII transcriptome in glioblastoma, a meta-omics analysis

Background EGFR is among the genes most frequently altered in glioblastoma, with exons 2-7 deletions (EGFRvIII) being amongst its most common genomic mutations. There are conflicting reports about its prognostic role and it remains unclear whether and how it differs in signalling compared with wildtype EGFR. Methods To better understand the oncogenic role of EGFRvIII, we leveraged four large datasets into one large glioblastoma transcriptome dataset (n=741) alongside 81 whole-genome samples from two datasets. Results The EGFRvIII/EGFR expression ratios differ strongly between tumours and ranges from 1% to 95%. Interestingly, the slope of relative EGFRvIII expression is near-linear, which argues against a more positive selection pressure than EGFR wildtype. An absence of selection pressure is also suggested by the similar survival between EGFRvIII positive and negative glioblastoma patients. EGFRvIII levels are inversely correlated with pan-EGFR (all wildtype and mutant variants) expression, which indicates that EGFRvIII has a higher potency in downstream pathway activation. EGFRvIII-positive glioblastomas have a lower CDK4 or MDM2 amplification incidence than EGFRvIII-negative (p=0.007), which may point towards crosstalk between these pathways. EGFRvIII-expressing tumours have an upregulation of ‘classical’ subtype genes compared to those with EGFR-amplification only (p=3.873e-6). Genomic breakpoints of the EGFRvIII deletions have a preference towards the 3’ end of the large intron-1. These preferred breakpoints preserve a cryptic exon resulting in a novel EGFRvIII variant and preserve an intronic enhancer. Conclusions These data provide deeper insights into the complex EGFRvIII biology and provide new insights for targeting EGFRvIII mutated tumours.

GCAT dotplots characterize precisely and imprecisely defined topological features of DNA

Two dimensional graphical dotplotting is adopted to identify sequence elements and their variants in lengths of DNA of up to 10 kb. Named GCAT for identification of precisely defined short sequences and their variants, its use complements the precise matching of many computational programs, including BLAST. Short reiterated search sequences are entered in the Y axis of the dotplot program to be matched at their identical and near identical sites in a sequence of interest entered in the X axis. The result is a barcode like representation of the identified sequence elements along the X axis of the dotplot. Alignments of searches and sequence landmarks provide visualization of composition and juxtapositions. The method is described here by example of characterizations of three distinctive sequences available in the annotated Drosophila melanogaster reference genome (www.flybase.org): the Jonah 99C gene region, the transcript of Dipeptidase B and the transposable element roo. Surprising observations emerging from these explorations include in frame STOP codons in the large exonic intron of Dip-B, high A content of the replicative strand of roo as TE example and similarities of its ORF and the large intron of Dip B.

Large Intronic Deletion of the Fragile Site Gene PRKN Dramatically Lowers Its Fragility Without Impacting Gene Expression

Common chromosomal fragile sites (CFSs) are genomic regions prone to form breaks and gaps on metaphase chromosomes during conditions of replication stress. Moreover, CFSs are hotspots for deletions and amplifications in cancer genomes. Fragility at CFSs is caused by transcription of extremely large genes, which contributes to replication problems. These extremely large genes do not encode large proteins, but the extreme sizes of the genes originate from vast introns. Intriguingly, the intron sizes of extremely large genes are conserved between mammals and birds. Here, we have used reverse genetics to address the function and significance of the largest intron in the extremely large gene PRKN, which is highly fragile in our model system. Specifically, we have introduced an 80-kilobase deletion in intron 7 of PRKN. We find that gene expression of PRKN is largely unaffected by this intronic deletion. Strikingly, the intronic deletion, which leads to a 12% reduction of the overall size of the PRKN gene body, results in an almost twofold reduction of the PRKN fragility. Our results stress that while the large intron clearly contributes to the fragility of PRKN, it does not play an important role for PRKN expression. Taken together, our findings further add to the mystery concerning conservation of the seemingly non-functional but troublesome large introns in PRKN.

Insulinlike Growth Factor 1 Gene Variation in Vertebrates

IGF1—a small, single-chain, secreted peptide in mammals—is essential for normal somatic growth and is involved in a variety of other physiological and pathophysiological processes. IGF1 expression appears to be controlled by several different signaling mechanisms in mammals, with GH playing a key role by activating an inducible transcriptional pathway via the Jak2 protein kinase and the Stat5b transcription factor. Here, to understand aspects of Igf1 gene regulation over a substantially longer timeline than is discernible in mammals, Igf1 genes have been examined in 21 different nonmammalian vertebrates representing five different classes and ranging over ∼500 million years of evolutionary history. Parts of vertebrate Igf1 genes resemble components found in mammals. Conserved exons encoding the mature IGF1 protein are detected in all 21 species studied and are separated by a large intron, as seen in mammals; the single promoter contains putative regulatory elements that are similar to those functionally mapped in human IGF1 promoter 1. In contrast, GH-activated Stat5b-binding enhancers found in mammalian IGF1 loci are completely absent, there is no homolog of promoter 2 or exon 2 in any nonmammalian vertebrate, and different types of “extra” exons not present in mammals are found in birds, reptiles, and teleosts. These data collectively define properties of Igf1 genes and IGF1 proteins that were likely present in the earliest vertebrates and support the contention that common structural and regulatory features in Igf1 genes have a long evolutionary history.

An eutherian intronic sequence gave rise to a major satellite DNA in Platyrrhini

Satellite DNAs (satDNAs) are major components of eukaryote genomes. However, because of their quick divergence, the evolutionary origin of a given satDNA family can rarely be determined. Herein we took advantage of available primate sequenced genomes to determine the origin of the CapA satDNA (approx. 1500 bp long monomers), first described in the tufted capuchin monkey Sapajus apella . We show that CapA is an abundant satDNA in Platyrrhini, whereas in the genomes of most eutherian mammals, including humans, this sequence is present only as a single copy located within a large intron of the NOS1AP ( nitric oxide synthase 1 adaptor protein ) gene. Our data suggest that this intronic CapA-like sequence gave rise to the CapA satDNA and we discuss possible mechanisms implicated in this event. This is the first report to our knowledge of a single copy intronic sequence giving origin to a satDNA that reaches up to 100 000 copies in some genomes.

An eutherian intronic sequence gave rise to a major satellite DNA in Platyrrhini

Satellite DNAs (satDNAs) are major components of eukaryote genomes. However, because of their quick divergence, the evolutionary origin of a given satDNA family can rarely be determined. Herein we took advantage of available primate sequenced genomes to determine the origin of the CapA satDNA (~1,500 bp long monomers), first described in Sapajus apella. We show that CapA is an abundant satDNA in Platyrrhini, whereas in the genomes of most eutherian mammals, including humans, this sequence is present only as a single copy located within a large intron of the NOS1AP (nitric oxid synthase 1 adaptor protein) Gene. Our data suggest that this intronic CapA-like sequence gave rise to the CapA satDNA and we discuss possible mechanisms implicated in this event. This is the first report of a single copy intronic sequence giving origin to a satDNA that reaches up to 100,000 copies in some genomes.

Cytochrome b diversity of Hungarian Botrytis cinerea strains

In the mitochondrion of eukaryotes, cytochrome b is a component of respiratory chain complex III. Cytochrome b is encoded by thecytochrome b (CYTB) gene located in the mitochondrial genome. The fungicidal activity of QoIs relies on their ability to inhibit mitochondrial respiration by binding at the so-called Qo site (the outer quinol-oxidation site) of the complex III. Since their introduction, QoIs (like azoxystrobin) have become essential components of plant disease control programs because of their wide-ranging efficacy against many agriculturally important fungal diseases like grey mould on various crops. QoI resistance primarily arises from a target-site-based mechanism involving mutations in the mitochondrial CYTB. As the management of grey mould is often dependent on chemicals, the rational design of control programs requires the information about the diversity of genes connected with resistance in field populations of the pathogen.Monospore B. cinerea field isolates has been collected during 2008-2009 from different hosts in Hungary. PCR fragment length analysisindicated the high frequency presence of type large intron in the isolates while in a few strains G143A substitution could also be detected.These results indicated the heterogeneity of CYTB in the Hungarian B. cinerea populations, which possibly involve the heteroplasmy of thismitochondrial gene, moreover indicates the existence op azoxystrobin resistant populations in Hungary.This work was supported by NKFP-A2-2006/0017 grant. Erzsébet Fekete is a grantee of the János Bolyai Scholarship (BO/00519/09/8).