BackgroundSilver-Russell syndrome (SRS) is an imprinting disorder characterised by prenatal and postnatal growth restriction, but its clinical features are non-specific and its differential diagnosis is broad. Known molecular causes of SRS include imprinting disturbance, single nucleotide variant (SNV), CNV or UPD affecting several genes; however, up to 40% of individuals with a clinical diagnosis of SRS currently receive no positive molecular diagnosis.MethodsTo determine whether whole-genome sequencing (WGS) could uncover pathogenic variants missed by current molecular testing, we analysed data of 72 participants recruited to the 100,000 Genomes Project within the clinical category of SRS.ResultsIn 20 participants (27% of the cohort) we identified genetic variants plausibly accounting for SRS. Coding SNVs were identified in genes including CDKN1C, IGF2, IGF1R and ORC1. Maternal-effect variants were found in mothers of five participants, including two participants with imprinting disturbance and one with multilocus imprinting disorder. Two regions of homozygosity were suggestive of UPD involving imprinted regions implicated in SRS and Temple syndrome, and three plausibly pathogenic CNVs were found, including a paternal deletion of PLAGL1. In 48 participants with no plausible pathogenic variant, unbiased analysis of SNVs detected a potential association with STX4.ConclusionWGS analysis can detect UPD, CNV and SNV and is potentially a valuable addition to diagnosis of SRS and related growth-restricting disorders.

The alternative lengthening of telomeres (ALT) facilitates telomere lengthening by a DNA strand invasion and copying mechanism. The nuclear receptors (NRs), NR2F2 and NR2C2, can bind to (TCAGGG)n variant repeats within telomeres and it has been proposed that this facilitates telomere interactions in ALT+ cells. Here we show that the frequency of cells with detectable NR2F2 and NR2C2 nuclear foci varies considerably between ALT+ cell lines and does not correlate with the level of protein expression. In addition, four of five ALT+ cell lines lack (TCAGGG)n repeats in some telomeres, indicating that direct NR binding does not play a role in ALT at these telomeres. NR2F2-depletion altered the abundance of C-circles and APBs but the direction of the response was inconsistent between three ALT+ cell lines. Moreover, transcriptome analysis following NR2F2-depletion in the ALT+ cell lines revealed different very responses. For example, NR2F2-depletion down-regulated many genes in U2OS cells, consistent with the cell cycle arrest and changes to ALT markers, but these features were not shared by the other two ALT+ cell lines. Among 86 ALT-associated genes, only MND1 showed consistent down-regulation across three NR2F2-depleted ALT+ cell lines. Altogether our data suggest that NR2F2 does not play a direct role in ALT and we speculate about an alternative role for this NR in a DNA damage response at telomeres.

Lung squamous cell carcinoma (LUSC) accounts for a significant proportion of cancer deaths worldwide, and is preceded by the appearance of progressively disorganised pre-invasive lesions in the airway epithelium. Yet the biological mechanisms underlying progression of pre-invasive lesions into invasive LUSC are not fully understood. LRIG1 is downregulated in pre-invasive airway lesions and invasive LUSC tumours and this correlates with decreased lung cancer patient survival.Using an Lrig1 knock-in reporter mouse and human airway epithelial cells collected at bronchoscopy, we show that during homeostasis LRIG1 is heterogeneously expressed in the airway epithelium. In basal airway epithelial cells, the suspected cell of origin of LUSC, LRIG1 identifies a subpopulation of progenitor cells with higher in vitro proliferative and self-renewal potential in both the mouse and human. Using the N-nitroso-tris-chloroethylurea (NTCU)-induced murine model of LUSC, we find that Lrig1 loss-of-function leads to abnormally high cell proliferation during the earliest stages of pre-invasive disease and to the formation of significantly larger invasive tumours, suggesting accelerated disease progression.Together, our findings identify LRIG1 as a marker of basal airway progenitor cells with high proliferative potential and as a regulator of pre-invasive lung cancer progression. This work highlights the clinical relevance of LRIG1 and the potential of the NTCU-induced LUSC model for functional assessment of candidate tumour suppressors and oncogenes.

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