Dimitra Dafou

Mutations in GATA2 cause primary lymphedema associated with a predisposition to acute myeloid leukemia (Emberger syndrome)

We report an allelic series of eight mutations in GATA2 underlying Emberger syndrome, an autosomal dominant primary lymphedema associated with a predisposition to acute myeloid leukemia. GATA2 is a transcription factor that plays an essential role in gene regulation during vascular development and hematopoietic differentiation. Our findings indicate that haploinsufficiency of GATA2 underlies primary lymphedema and predisposes to acute myeloid leukemia in this syndrome.

Mutations in NOTCH2 cause Hajdu-Cheney syndrome, a disorder of severe and progressive bone loss

We used an exome-sequencing strategy and identified an allelic series of NOTCH2 mutations in Hajdu-Cheney syndrome, an autosomal dominant multisystem disorder characterized by severe and progressive bone loss. The Hajdu-Cheney syndrome mutations are predicted to lead to the premature truncation of NOTCH2 with either disruption or loss of the C-terminal proline-glutamate-serine-threonine-rich proteolytic recognition sequence, the absence of which has previously been shown to increase Notch signaling.

De novo mutations in MLL cause Wiedemann-Steiner syndrome.

Excessive growth of terminal hair around the elbows (hypertrichosis cubiti) has been reported both in isolation and in association with a variable spectrum of associated phenotypic features. We identified a cohort of six individuals with hypertrichosis cubiti associated with short stature, intellectual disability, and a distinctive facial appearance, consistent with a diagnosis of Wiedemann-Steiner syndrome (WSS). Utilizing a whole-exome sequencing approach, we identified de novo mutations in MLL in five of the six individuals. MLL encodes a histone methyltransferase that regulates chromatin-mediated transcription through the catalysis of methylation of histone H3K4. Each of the five mutations is predicted to result in premature termination of the protein product. Furthermore, we demonstrate that transcripts arising from the mutant alleles are subject to nonsense-mediated decay. These findings define the genetic basis of WSS, provide additional evidence for the role of haploinsufficency of histone-modification enzymes in multiple-congenital-anomaly syndromes, and further illustrate the importance of the regulation of histone modification in development.

3D In Vitro Model of a Functional Epidermal Permeability Barrier from Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

Summary Cornification and epidermal barrier defects are associated with a number of clinically diverse skin disorders. However, a suitable in vitro model for studying normal barrier function and barrier defects is still lacking. Here, we demonstrate the generation of human epidermal equivalents (HEEs) from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). HEEs are structurally similar to native epidermis, with a functional permeability barrier. We exposed a pure population of hESC/iPSC-derived keratinocytes, whose transcriptome corresponds to the gene signature of normal primary human keratinocytes (NHKs), to a sequential high-to-low humidity environment in an air/liquid interface culture. The resulting HEEs had all of the cellular strata of the human epidermis, with skin barrier properties similar to those of normal skin. Such HEEs generated from disease-specific iPSCs will be an invaluable tool not only for dissecting molecular mechanisms that lead to epidermal barrier defects but also for drug development and screening.

De Novo Mutations of the Gene Encoding the Histone Acetyltransferase KAT6B Cause Genitopatellar Syndrome

Genitopatellar syndrome (GPS) is a rare disorder in which patellar aplasia or hypoplasia is associated with external genital anomalies and severe intellectual disability. Using an exome-sequencing approach, we identified de novo mutations of KAT6B in five individuals with GPS; a single nonsense variant and three frameshift indels, including a 4 bp deletion observed in two cases. All identified mutations are located within the terminal exon of the gene and are predicted to generate a truncated protein product lacking evolutionarily conserved domains. KAT6B encodes a member of the MYST family of histone acetyltranferases. We demonstrate a reduced level of both histone H3 and H4 acetylation in patient-derived cells suggesting that dysregulation of histone acetylation is a direct functional consequence of GPS alleles. These findings define the genetic basis of GPS and illustrate the complex role of the regulation of histone acetylation during development.

Three-dimensional in vitro cell biology models of ovarian and endometrial cancer

Objectives:  This study aims to establish three‐dimensional (3D) cell culture models of human ovarian and endometrial cancers and to compare biological and morphological characteristics of these models with those of two‐dimensional (2D) models of the same cell lines and the primary tumours.

Mutations in the γ-secretase genes NCSTN, PSENEN, and PSEN1 underlie rare forms of hidradenitis suppurativa (acne inversa).

TO THE EDITOR Hidradenitis suppurativa (HS; OMIM1 42690) is a chronic inflammatory skin disease that presents with nodules, cysts, and abscesses in apocrine gland–bearing sites. It affects 1% of Europeans and is primarily thought to be a disease of follicular occlusion (Alikhan et al., 2009). Associated factors include smoking (B90%), obesity (475%), and infection (Alikhan et al., 2009). HS may segregate as an autosomal dominant trait, and heterozygous mutations in the g-secretase genes NCSTN, PSENEN, and PSEN1 have recently been reported in a small number of kindreds (Wang et al., 2010; Li et al., 2011; Liu et al., 2011; Pink et al., 2011). We previously identified mutations in NCSTN and PSENEN in two of seven multiplex HS pedigrees. In contrast to previous studies, which have predominantly focussed on the identification of mutations in multiplex kindreds, the aim of this study was to determine the prevalence of mutations in each of these genes among a large cohort of subjects sequentially recruited from a tertiary referral HS clinic. We performed mutational analysis of NCSTN, PSENEN, and PSEN1 by direct sequencing of all coding regions and assessment of large-scale deletions and duplications by multiplex ligation phosphorylation assay (MLPA). In all, 48 individuals with HS (diagnostic criteria from Von der Werth et al., 2000) were sequentially consented and recruited from our tertiary referral clinic. The study was conducted in accordance with the Declaration of Helsinki Principles and approved by the East London REC2 (09/H0704/50). Population data are shown in Supplementary Table S1 online. The cohort was of mixed ethnicity, the average body mass index (BMI) was 31.1 (clinically obese), 69% reported a smoking history, and the average Sartorius score (Sartorius et al., 2003) was 49.9. A total of 20 (42%) patients reported a family history of HS, 19 consistent with autosomal dominant inheritance. The DNA was extracted from venous blood or saliva. All coding regions and associated splice sites of NCSTN, PSEN1, and PSENEN were amplified by PCR, using exon flanking intronic primers and Sanger sequenced in all 48 patients. In three subjects we identified heterozygous DNA changes in NCSTN (Supplementary Table S2 online), a missense substitution (NCSTN c.553G4A, p.Asp185Asn), and two single-base substitutions located within 10 bp of donor splice junctions (NCSTN c.996þ 7G4A and NCSTN c.1101þ 10A4G). To our knowledge these variants have not previously been reported and none were observed in dbSNP, 1,000 genomes, or in 400 European controls. All 48 patients were assessed for whole gene/exon deletions and duplications in NCSTN, PSENEN, and PSEN1 using MLPA (Supplementary Methods online; Hills et al., 2010). No genomic deletions or duplications were detected. The heterozygous missense variant in exon 5 of NCSTN (c.553G4A, p.Asp185Asn) was identified in a 45-yearold female (patient HS-01; Supplementary Table S2 online, Figure 1). The missense variant is predicted to lead to the substitution of an evolutionary conserved aspartic acid residue with an asparagine residue. A heterozygous substitution of the conserved seventh base of intron 8 of NCSTN (c.996þ 7G4A) was identified in a 38-year-old female (patient HS-02 Supplementary Table S2 online, Figure 1). Computational splice site analysis (https://splice. uwo.ca) predicts this substitution to have a detrimental effect on splicing. NCSTN mRNA expression levels were significantly lower than wild-type controls, suggesting that the mutant transcript is subject to nonsense-mediated decay (Supplementary Methods and Supplementary Figure S1 online). A third heterozygous variant was detected in the donor splice site of NCSTN exon 9 (c.1101þ 10A4G) in a 24-year-old male (patient HS-03; Supplementary Table S2 online, Figure 1). Computational splice site analysis suggests that this mutation is unlikely to have a significant effect on splicing. Indeed, no aberrant transcripts were detected (via reverse transcriptase–PCR and sequencing of the full-length NCSTN transcript, Supplementary Figures S2 and S3 online) and NCSTN mRNA expression level was within the range of wild-type controls (data not shown). The potential pathogenicity of this variant is therefore unclear and it may represent a rare variant unlinked to HS in this individual. The two individuals with likely pathogenic variants in NCSTN had chronic, severe disease (Sartorius scores of 56 and 160, respectively) comprising painful nodules, cysts, abscesses, sinus tracts, and scarring, involving at least the axillae, groin, and buttocks (see Supplementary Table S2 online). Their

TNF gene cluster deletion abolishes lipopolysaccharide-mediated sensitization of the neonatal brain to hypoxic ischemic insult

In the current study, we explored the role of TNF cluster cytokines on the lipopolysaccharide (LPS)-mediated, synergistic increase in brain injury after hypoxic ischemic insult in postnatal day 7 mice. Pretreatment with moderate doses of LPS (0.3 μg/g) resulted in particularly pronounced synergistic injury within 12 h. Systemic application of LPS alone resulted in a strong upregulation of inflammation-associated cytokines TNFα, LTβ, interleukin (IL) 1β, IL6, chemokines, such as CXCL1, and adhesion molecules E-Selectin, P-Selectin and intercellular adhesion molecule-1 (ICAM1), as well as a trend toward increased LTα levels in day 7 mouse forebrain. In addition, it was also associated with strong activation of brain blood vessel endothelia and local microglial cells. Here, deletion of the entire TNF gene cluster, removing TNFα, LTβ and LTα completely abolished endotoxin-mediated increase in the volume of cerebral infarct. Interestingly, the same deletion also prevented endothelial and microglial activation following application of LPS alone, suggesting the involvement of these cell types in bringing about the LPS-mediated sensitization to neonatal brain injury.

Common alleles in candidate susceptibility genes associated with risk and development of epithelial ovarian cancer

Common germline genetic variation in the population is associated with susceptibility to epithelial ovarian cancer. Microcell‐mediated chromosome transfer and expression microarray analysis identified nine genes associated with functional suppression of tumorogenicity in ovarian cancer cell lines; AIFM2, AKTIP, AXIN2, CASP5, FILIP1L, RBBP8, RGC32, RUVBL1 and STAG3. Sixty‐three tagging single nucleotide polymorphisms (tSNPs) in these genes were genotyped in 1,799 invasive ovarian cancer cases and 3,045 controls to look for associations with disease risk. Two SNPs in RUVBL1, rs13063604 and rs7650365, were associated with increased risk of serous ovarian cancer [HetOR = 1.42 (1.15–1.74) and the HomOR = 1.63 (1.10–1.42), p‐trend = 0.0002] and [HetOR = 0.97 (0.80–1.17), HomOR = 0.74 (0.58–0.93), p‐trend = 0.009], respectively. We genotyped rs13063604 and rs7650365 in an additional 4,590 cases and 6,031 controls from ten sites from the United States, Europe and Australia; however, neither SNP was significant in Stage 2. We also evaluated the potential role of tSNPs in these nine genes in ovarian cancer development by testing for allele‐specific loss of heterozygosity (LOH) in 286 primary ovarian tumours. We found frequent LOH for tSNPs in AXIN2, AKTIP and RGC32 (64, 46 and 34%, respectively) and one SNP, rs1637001, in STAG3 showed significant allele‐specific LOH with loss of the common allele in 94% of informative tumours (p = 0.015). Array comparative genomic hybridisation indicated that this nonrandom allelic imbalance was due to amplification of the rare allele. In conclusion, we show evidence for the involvement of a common allele of STAG3 in the development of epithelial ovarian cancer.

In vitro three-dimensional modelling of human ovarian surface epithelial cells.

Objectives:  Ninety percent of malignant ovarian cancers are epithelial and thought to arise from the ovarian surface epithelium (OSE). We hypothesized that biological characteristics of primary OSE cells would more closely resemble OSE in vivo if established as three‐dimensional (3D) cultures.