Lara Al-Olabi

The role and interaction of imprinted genes in human fetal growth.

Identifying the genetic input for fetal growth will help to understand common, serious complications of pregnancy such as fetal growth restriction. Genomic imprinting is an epigenetic process that silences one parental allele, resulting in monoallelic expression. Imprinted genes are important in mammalian fetal growth and development. Evidence has emerged showing that genes that are paternally expressed promote fetal growth, whereas maternally expressed genes suppress growth. We have assessed whether the expression levels of key imprinted genes correlate with fetal growth parameters during pregnancy, either early in gestation, using chorionic villus samples (CVS), or in term placenta. We have found that the expression of paternally expressing insulin-like growth factor 2 (IGF2), its receptor IGF2R, and the IGF2/IGF1R ratio in CVS tissues significantly correlate with crown–rump length and birthweight, whereas term placenta expression shows no correlation. For the maternally expressing pleckstrin homology-like domain family A, member 2 (PHLDA2), there is no correlation early in pregnancy in CVS but a highly significant negative relationship in term placenta. Analysis of the control of imprinted expression of PHLDA2 gave rise to a maternally and compounded grand-maternally controlled genetic effect with a birthweight increase of 93/155 g, respectively, when one copy of the PHLDA2 promoter variant is inherited. Expression of the growth factor receptor-bound protein 10 (GRB10) in term placenta is significantly negatively correlated with head circumference. Analysis of the paternally expressing delta-like 1 homologue (DLK1) shows that the paternal transmission of type 1 diabetes protective G allele of rs941576 single nucleotide polymorphism (SNP) results in significantly reduced birth weight (−132 g). In conclusion, we have found that the expression of key imprinted genes show a strong correlation with fetal growth and that for both genetic and genomics data analyses, it is important not to overlook parent-of-origin effects.

Mosaic Activating Mutations in GNA11 and GNAQ Are Associated with Phakomatosis Pigmentovascularis and Extensive Dermal Melanocytosis

Common birthmarks can be an indicator of underlying genetic disease but are often overlooked. Mongolian blue spots (dermal melanocytosis) are usually localized and transient, but they can be extensive, permanent, and associated with extracutaneous abnormalities. Co-occurrence with vascular birthmarks defines a subtype of phakomatosis pigmentovascularis, a group of syndromes associated with neurovascular, ophthalmological, overgrowth, and malignant complications. Here, we discover that extensive dermal melanocytosis and phakomatosis pigmentovascularis are associated with activating mutations in GNA11 and GNAQ, genes that encode Gα subunits of heterotrimeric G proteins. The mutations were detected at very low levels in affected tissues but were undetectable in the blood, indicating that these conditions are postzygotic mosaic disorders. In vitro expression of mutant GNA11R183C and GNA11Q209L in human cell lines demonstrated activation of the downstream p38 MAPK signaling pathway and the p38, JNK, and ERK pathways, respectively. Transgenic mosaic zebrafish models expressing mutant GNA11R183C under promoter mitfa developed extensive dermal melanocytosis recapitulating the human phenotype. Phakomatosis pigmentovascularis and extensive dermal melanocytosis are therefore diagnoses in the group of mosaic heterotrimeric G-protein disorders, joining McCune-Albright and Sturge-Weber syndromes. These findings will allow accurate clinical and molecular diagnosis of this subset of common birthmarks, thereby identifying infants at risk for serious complications, and provide novel therapeutic opportunities.

Next-Generation Sequencing of Nevus Spilus–Type Congenital Melanocytic Nevus: Exquisite Genotype–Phenotype Correlation in Mosaic RASopathies

TO THE EDITOR Nevus spilus is a descriptive term used to denote any cutaneous lesion with a cafe-au-lait macular background and superimposed on more pigmented areas. Small single nevus spilus are relatively common, and they have recently been described to be due to somatic activating HRAS mutations (Sarin et al., 2014). Larger superficial lesions with small superimposed junctional nevi in association with phakomatosis pigmentokeratotica have also been found to contain HRAS mutations (Groesser et al., 2013). However, another nevus spilus–type phenotype has also been well described in which large cafe-au-lait macules have superimposed lesions that are indistinguishable both clinically and histopathologically from medium/large congenital melanocytic nevi (CMN), exhibit a wide variety of colors and sizes (Schaffer et al., 2001a, 2001b), and continue to develop postnatally in many cases. This is termed nevus spilus–type CMN. In our experience, there may be delayed appearance of the cafe-au-lait background after birth, but the lesion is still usually predictable on the basis of clustering of many CMN in one anatomical area. Smaller separate lesions in the same individual are often indistinguishable clinically from cafe-au-lait macules, and are so faint that they can be easily missed (Figure 1). Our primary aim in this study was to look for the genetic basis of this defined phenotypic subset of CMN, in the context of the recent discovery that NRAS Q61K and Q61R mutations are the cause of most causes of multiple CMN (Kinsler et al., 2013b). In particular, we were interested in determining the mutation in the background macular portion of the nevus spilus–type CMN, working on the hypothesis that this could be the “first hit” in a two-hit model of nevogenesis. Figure 1 Clinical images of nevus spilus–type CMN in six different patients. The cafe-au-lait macule background is often invisible at birth. Two separate lesions are indicated in one patient. CMN, congenital melanocytic nevi. Written consent was ... These studies were approved by the appropriate Research Ethics Committee, written consent was taken from participants, and the Declaration of Helsinki Principles protocols were followed. A blood sample and skin biopsies of both the cafe-au-lait macule background and a banal superimposed CMN were taken from three children with large nevus spilus–type CMN, from a total of 17 patients from our combined practices (patients 5, 12, and 13 in Supplementary Table S1 online), and DNA was extracted directly by standard methods. Whole-exome sequencing using Nextera library preparation and an ABI Hi-Seq bioanalyser was undertaken on all three samples from two patients, and data were analyzed using an in-house pipeline designed for somatic mosaicism. The principal governing analysis was to look for a mutation present in the cafe-au-lait that was not present in the blood, and a further mutation in the CMN not present in the cafe-au-lait or the blood. In all, 1,991,478 variants were called automatically in the overlying CMN, affecting 20,693 genes. After filtering, the sequencing files of 133 variants in 39 genes were reviewed manually. A single mutation was found in the cafe-au-lait macule and the superimposed CMN, with no further mutation confirmed despite extensive analysis. These mutations were missense activating mutations in NRAS in the skin, absent from the blood, and this pattern of somatic mosaicism was confirmed in the third patient by Sanger sequencing. The mutations, however, are undescribed so far in typical CMN, being 1:115256528 c.183A>C p.Q61H (two patients, Figure 2) and 1:115258745 c.37G>C p.G13R. Both mutations have been described at a somatic level in non-CMN-related malignant melanoma (Forbes et al., 2008). Independently, a fourth patient (patient 17, Supplementary Table S1 online) had targeted exon capture of two skin samples, and analysis also revealed only the same NRAS p.Q61H mutation in both the cafe-au-lait macule and the superimposed CMN. Figure 2 Sequencing results showing NRAS mutation. Next-generation sequencing reads of blood (upper left), cafe-au-lait macule (upper centre), and overlying CMN (upper right) from the same patient showing mutation NRAS c.183A>C p.Q61H. Note the ... In conclusion, nevus spilus–type CMN are a phenotypically and genotypically distinct variant of CMN, and are phenotypically and genotypically distinct from nevus spilus maculosus and papulosus due to HRAS mutations (Groesser et al., 2013; Sarin et al., 2013). This further extends the exquisite mutation specificity of the newly characterized mosaic RASopathies. We found no evidence of a second mutation to explain the superimposed nevi on the macular background. It is, however, possible that there could be a mutation that does not appear pathogenic to us and to the analysis pipelines at the present time. Alternatively, there could be either a translocation that does not disrupt exonic DNA sequence, or a growth-promoting copy number change, although CMN have previously been documented as having few such changes on array comparative genomic hybridization (Bastian et al., 2002). The data at the moment suggest that only one sequence-level mutation occurs, and that these specific NRAS mutations are sufficient to cause cafe-au-lait macular pigmentation, which can lead to macroscopic nevus formation over time. Other experimental evidence supports that nevi can evolve in this way, from a cell or collection of cells in the skin not visible to the naked eye, as nevus cells have been found in normal skin in patients with acquired melanocytic nevi and typical CMN (Dadzie et al., 2008; Kinsler et al., 2013a). It is important to note for clinical management that malignant melanoma has been described in patients with nevus spilus–type CMN (Kinsler et al., 2009), and on current knowledge we would consider nevus spilus–type CMN patients at the same risk of malignancy as other genotypes. Similarly, although interestingly none of the 17 patients described here have neurological abnormalities on magnetic resonance imaging scan (Supplementary Table S1 online), these numbers are too small to draw any conclusions about a possible low risk of neurological phenotype. Management of nevus spilus–type CMN should therefore be the same as for other CMN.

Acute Inhibition of MEK Suppresses Congenital Melanocytic Nevus Syndrome in a Murine Model Driven by Activated NRAS and Wnt Signaling

Congenital melanocytic nevus (CMN) syndrome is the association of pigmented melanocytic nevi with extra-cutaneous features, classically melanotic cells within the central nervous system, most frequently caused by a mutation of NRAS codon 61. This condition is currently untreatable and carries a significant risk of melanoma within the skin, brain, or leptomeninges. We have previously proposed a key role for Wnt signaling in the formation of melanocytic nevi, suggesting that activated Wnt signaling may be synergistic with activated NRAS in the pathogenesis of CMN syndrome. Some familial pre-disposition suggests a germ-line contribution to CMN syndrome, as does variability of neurological phenotypes in individuals with similar cutaneous phenotypes. Accordingly, we performed exome sequencing of germ-line DNA from patients with CMN to reveal rare or undescribed Wnt-signaling alterations. A murine model harboring activated NRASQ61K and Wnt signaling in melanocytes exhibited striking features of CMN syndrome, in particular neurological involvement. In the first model of treatment for this condition, these congenital, and previously assumed permanent, features were profoundly suppressed by acute post-natal treatment with a MEK inhibitor. These data suggest that activated NRAS and aberrant Wnt signaling conspire to drive CMN syndrome. Post-natal MEK inhibition is a potential candidate therapy for patients with this debilitating condition.

Mosaic RAS/MAPK variants cause sporadic vascular malformations which respond to targeted therapy

BACKGROUND. Sporadic vascular malformations (VMs) are complex congenital anomalies of blood vessels that lead to stroke, life-threatening bleeds, disfigurement, overgrowth, and/or pain. Therapeutic options are severely limited, and multidisciplinary management remains challenging, particularly for high-flow arteriovenous malformations (AVM). METHODS. To investigate the pathogenesis of sporadic intracranial and extracranial VMs in 160 children in which known genetic causes had been excluded, we sequenced DNA from affected tissue and optimized analysis for detection of low mutant allele frequency. RESULTS. We discovered multiple mosaic-activating variants in 4 genes of the RAS/MAPK pathway, KRAS, NRAS, BRAF, and MAP2K1, a pathway commonly activated in cancer and responsible for the germline RAS-opathies. These variants were more frequent in high-flow than low-flow VMs. In vitro characterization and 2 transgenic zebrafish AVM models that recapitulated the human phenotype validated the pathogenesis of the mutant alleles. Importantly, treatment of AVM-BRAF mutant zebrafish with the BRAF inhibitor vemurafinib restored blood flow in AVM. CONCLUSION. Our findings uncover a major cause of sporadic VMs of different clinical types and thereby offer the potential of personalized medical treatment by repurposing existing licensed cancer therapies. FUNDING. This work was funded or supported by grants from the AVM Butterfly Charity, the Wellcome Trust (UK), the Medical Research Council (UK), the UK National Institute for Health Research, the L’Oreal-Melanoma Research Alliance, the European Research Council, and the National Human Genome Research Institute (US).

The Baby Bio Bank-A Legacy for Researchers Worldwide into Common Complications of Pregnancy

Two London Universities, University College London and Imperial College London, have established a biobank as a resource for investigating the four main complications of pregnancy (recurrent miscarriage, preterm birth, fetal growth restriction (FGR) and pre-eclampsia), that collectively affect about 20% of families trying to conceive. Samples are being taken from the three key members of the family, mother, father and baby, allowing hereditary factors from both parents to be tracked. Additional samples are being stored to allow parallel analysis of the functional mechanisms of pregnancy including the epigenetic, anatomical, physiological and even metabolic causes for the high loss of fecundity in man. http://www.ucl.ac.uk/babybiobank.

Extending the spectrum of AKT1 mosaicism ‐ not just the Proteus syndrome

DEAR EDITOR, A 5-year-old girl was referred to us for evaluation of a pigmented birthmark, unchanged since birth, on her right cheek. There was no other previous medical history of note and no family history of relevant problems. Cutaneous examination revealed a pigmented keratinocytic epidermal naevus following fine Blaschko lines on the right cheek and neck (Fig. 1), and a solitary, 1-cm caf e-au-lait macule on the lower leg. Subtle hemihypertrophy of the right cheek was noted, including the tragus of the ear, which had appeared to progress over time. In conjunction, she was noted to have rightsided hemihypertrophy of the tongue with intraoral pigmentation of the mucosal surfaces, linear naevoid-looking change on the right side of the tongue and right-sided misalignment of the teeth. Her parents also gave a history of frequent malodorous right ear discharge. The patient was referred to otorhinolaryngology, who diagnosed a severe right-sided conductive hearing loss. The remainder of her physical examination was unremarkable. Magnetic resonance imaging confirmed soft tissue hemihypertrophy of the cheek, with extensive soft tissue overgrowth within the right external auditory canal and middle-ear cleft, and a sclerotic bony spur in the posterior inferior aspect of the external auditory canal (Fig. 1g). Mosaicism for a growth-promoting mutation was suspected. With appropriate consent for genetic investigations a punch skin biopsy was obtained from the epidermal naevus, and DNA extracted directly by standard methods. DNA was also extracted from a venous blood sample. Histology confirmed a keratinocytic epidermal naevus (Fig. 2). Initial sequencing of HRAS and KRAS hotspot mutations, as described in epidermal naevus syndromes, was negative. However, Sanger sequencing for hotspot mutations in AKT1 revealed a heterozygous missense mutation, c.49G>A, p.Glu17Lys, present in the epidermal naevus but not the blood (Fig. 2). Although we only have a single biopsy from this patient, given existing knowledge of this particular mutation, postzygotic mosaicism is highly likely to be the cause of her entire clinical phenotype and not just the epidermal naevus. In mosaic form, this is the characteristic causal mutation in the first cohort of patients with clinically delineated Proteus syndrome, as defined by strict diagnostic criteria. Proteus syndrome is a rare overgrowth syndrome characterized by a normal phenotype or limited features at birth followed by