DNA Polymerase Epsilon Deficiency Causes IMAGe Syndrome with Variable Immunodeficiency.

During genome replication, polymerase epsilon (Pol ε) acts as the major leading-strand DNA polymerase. Here we report the identification of biallelic mutations in POLE, encoding the Pol ε catalytic subunit POLE1, in 15 individuals from 12 families. Phenotypically, these individuals had clinical features closely resembling IMAGe syndrome (intrauterine growth restriction [IUGR], metaphyseal dysplasia, adrenal hypoplasia congenita, and genitourinary anomalies in males), a disorder previously associated with gain-of-function mutations in CDKN1C. POLE1-deficient individuals also exhibited distinctive facial features and variable immune dysfunction with evidence of lymphocyte deficiency. All subjects shared the same intronic variant (c.1686+32C>G) as part of a common haplotype, in combination with different loss-of-function variants in trans. The intronic variant alters splicing, and together the biallelic mutations lead to cellular deficiency of Pol ε and delayed S-phase progression. In summary, we establish POLE as a second gene in which mutations cause IMAGe syndrome. These findings add to a growing list of disorders due to mutations in DNA replication genes that manifest growth restriction alongside adrenal dysfunction and/or immunodeficiency, consolidating these as replisome phenotypes and highlighting a need for future studies to understand the tissue-specific development roles of the encoded proteins

Mutations in TOP3A Cause a Bloom Syndrome-like Disorder

Bloom syndrome, caused by biallelic mutations in BLM, is characterized by prenatal-onset growth deficiency, short stature, an erythematous photosensitive malar rash, and increased cancer predisposition. Diagnostically, a hallmark feature is the presence of increased sister chromatid exchanges (SCEs) on cytogenetic testing. Here, we describe biallelic mutations in TOP3A in ten individuals with prenatal-onset growth restriction and microcephaly. TOP3A encodes topoisomerase III alpha (TopIIIα), which binds to BLM as part of the BTRR complex, and promotes dissolution of double Holliday junctions arising during homologous recombination. We also identify a homozygous truncating variant in RMI1, which encodes another component of the BTRR complex, in two individuals with microcephalic dwarfism. The TOP3A mutations substantially reduce cellular levels of TopIIIα, and consequently subjects’ cells demonstrate elevated rates of SCE. Unresolved DNA recombination and/or replication intermediates persist into mitosis, leading to chromosome segregation defects and genome instability that most likely explain the growth restriction seen in these subjects and in Bloom syndrome. Clinical features of mitochondrial dysfunction are evident in several individuals with biallelic TOP3A mutations, consistent with the recently reported additional function of TopIIIα in mitochondrial DNA decatenation. In summary, our findings establish TOP3A mutations as an additional cause of prenatal-onset short stature with increased cytogenetic SCEs and implicate the decatenation activity of the BTRR complex in their pathogenesis

Origins and heterogeneity of adipose tissue: investigating the role of the Wilms’ tumour 1 (Wt1) gene

Largely as a consequence of the ongoing obesity epidemic, research into adipose tissue biology has increased substantially in recent years. Worldwide, the number of people classed as overweight or obese is growing, and this represents a major public health concern. Adipose tissue is broadly divided into two types; white and brown. Whilst white adipose tissue (WAT) functions to store and mobilise triglycerides, brown adipose tissue burns chemical energy to generate heat. WAT is further divided into visceral “bad” fat and subcutaneous “good” fat depots, and it is an increase in the former that is linked to obesity-associated diseases. As well as adipocytes, several other cell types including haematopoietic and endothelial are found within adipose tissue, and comprise the stromal vascular fraction (SVF). Adipocyte precursor cells (APCs) also reside within the SVF and are essential for the maintenance and expansion of adipose tissue. The protein encoded by the Wilms’ tumour 1 (Wt1) gene is predominantly known to function as a transcription factor, but also has a role in post-transcriptional processing. Deletion of Wt1 in adult mice results in a considerable loss of fat tissue. Moreover, recent work has revealed that a proportion of the APCs from all visceral WAT depots express Wt1, therefore revealing heterogeneity within the APC population. Additionally, visceral WAT depots are encapsulated by a WT1 expressing mesothelial layer, which has its origins in the lateral plate mesoderm (LPM), and can give rise to mature adipocytes. Lineage tracing has demonstrated that a significant proportion of the mature adipocytes in all adult visceral WAT depots (but not subcutaneous) are derived from cells that express Wt1 in late gestation. These findings uncovered key ontogenetic differences between visceral and subcutaneous WAT and led us to ask whether Wt1 functions in visceral adipose tissue biology. Preliminary work has shown that adipocytes derived from Wt1 expressing (Wt1+) precursor cells have fewer, larger lipid droplets than those derived from non-Wt1 expressing (Wt1-) precursors. In this thesis, this heterogeneity is explored further using a Wt1GFP/+ knock-in mouse. When Wt1+ and Wt1- APCs are cultured separately, the Wt1+ population differentiate into adipocytes more readily. Moreover, the Wt1+ APCs are more proliferative than the Wt1-. Preliminary results also suggest that the Wt1+ APCs may secrete a factor(s) that causes the Wt1- APCs to exhibit improved adipogenic differentiation, a result that is supported by data from comparative transcriptomic analysis. Finally, the percentage of APCs decreases when mice are fed a high fat diet. Interestingly, this decrease is more pronounced for the Wt1+ population. Therefore, it appears that as well as exhibiting differing behaviours in vitro, the Wt1+ and Wt1- populations respond differently to physiologically relevant conditions in vivo. Whilst the LPM is a major source of visceral WAT, the origin of subcutaneous WAT is currently unknown. Here, the Prx1-Cre and Prx1-CreERT2 mouse lines are used to investigate this. It is shown that the majority of subcutaneous WAT adipocytes and APCs are labelled by Prx1-Cre, however this is not the case for most of the visceral WAT depots. The exception to this is the pericardial (heart fat) depot, in which approximately 70% of the adipocytes and 40% of the APCs are labelled. Moreover, a proportion of the Prx1-Cre labelled pericardial APCs also express Wt1, therefore suggesting additional heterogeneity. Preliminary results show that this heterogeneity may have functional consequences, at least in vitro. Additionally, lineage tracing studies suggest that the somatic LPM may be one source of subcutaneous WAT and pericardial visceral WAT Finally, it is shown that the conditional deletion of Wt1 in the Prx1-Cre lineage results in abnormal diaphragm development. Congenital diaphragmatic hernia (CDH) is severe birth defect, the etiology of which is not well understood. Here, a new model of CDH has been developed, and the cellular and molecular mechanisms responsible for the defect in this model are investigated

Maternal obesity during pregnancy alters daily activity and feeding cycles, and hypothalamic clock gene expression in adult male mouse offspring

An obesogenic diet adversely affects the endogenous mammalian circadian clock, altering daily activity and metabolism, and resulting in obesity. We investigated whether an obese pregnancy can alter the molecular clock in the offspring hypothalamus, resulting in changes to their activity and feeding rhythms. Female mice were fed a control (C, 7% kcal fat) or high fat diet (HF, 45% kcal fat) before mating and throughout pregnancy. Male offspring were fed the C or HF diet postweaning, resulting in four offspring groups: C/C, C/HF, HF/C, and HF/HF. Daily activity and food intake were monitored, and at 15 weeks of age were killed at six time-points over 24 h. The clock genes Clock, Bmal1, Per2, and Cry2 in the suprachiasmatic nucleus (SCN) and appetite genes Npy and Pomc in the arcuate nucleus (ARC) were measured. Daily activity and feeding cycles in the HF/C, C/HF, and HF/HF offspring were altered, with increased feeding bouts and activity during the day and increased food intake but reduced activity at night. Gene expression patterns and levels of Clock, Bmal1, Per2, and Cry2 in the SCN and Npy and Pomc in the ARC were altered in HF diet-exposed offspring. The altered expression of hypothalamic molecular clock components and appetite genes, together with changes in activity and feeding rhythms, could be contributing to offspring obesity

Low-frequency variation in TP53 has large effects on head circumference and intracranial volume.

Cranial growth and development is a complex process which affects the closely related traits of head circumference (HC) and intracranial volume (ICV). The underlying genetic influences shaping these traits during the transition from childhood to adulthood are little understood, but might include both age-specific genetic factors and low-frequency genetic variation. Here, we model the developmental genetic architecture of HC, showing this is genetically stable and correlated with genetic determinants of ICV. Investigating up to 46,000 children and adults of European descent, we identify association with final HC and/or final ICV + HC at 9 novel common and low-frequency loci, illustrating that genetic variation from a wide allele frequency spectrum contributes to cranial growth. The largest effects are reported for low-frequency variants within TP53, with 0.5 cm wider heads in increaser-allele carriers versus non-carriers during mid-childhood, suggesting a previously unrecognized role of TP53 transcripts in human cranial development

Mutations in TOP3A Cause a Bloom Syndrome-like Disorder

Bloom syndrome, caused by biallelic mutations in BLM, is characterized by prenatal-onset growth deficiency, short stature, an erythematous photosensitive malar rash, and increased cancer predisposition. Diagnostically, a hallmark feature is the presence of increased sister chromatid exchanges (SCEs) on cytogenetic testing. Here, we describe biallelic mutations in TOP3A in ten individuals with prenatalonset growth restriction and microcephaly. TOP3A encodes topoisomerase III alpha (TopIIIa), which binds to BLM as part of the BTRR complex, and promotes dissolution of double Holliday junctions arising during homologous recombination. We also identify a homozygous truncating variant in RMI1, which encodes another component of the BTRR complex, in two individuals with microcephalic dwarfism. The TOP3A mutations substantially reduce cellular levels of TopIIIa, and consequently subjects' cells demonstrate elevated rates of SCE. Unresolved DNA recombination and/or replication intermediates persist into mitosis, leading to chromosome segregation defects and genome instability that most likely explain the growth restriction seen in these subjects and in Bloom syndrome. Clinical features of mitochondrial dysfunction are evident in several individuals with biallelic TOP3A mutations, consistent with the recently reported additional function of TopIIIa in mitochondrial DNA decatenation. In summary, our findings establish TOP3A mutations as an additional cause of prenatal-onset short stature with increased cytogenetic SCEs and implicate the decatenation activity of the BTRR complex in their pathogenesis