Pavlos Fanis

Haploinsufficiency for the erythroid transcription factor KLF1 causes hereditary persistence of fetal hemoglobin

Hereditary persistence of fetal hemoglobin (HPFH) is characterized by persistent high levels of fetal hemoglobin (HbF) in adults. Several contributory factors, both genetic and environmental, have been identified but others remain elusive. HPFH was found in 10 of 27 members from a Maltese family. We used a genome-wide SNP scan followed by linkage analysis to identify a candidate region on chromosome 19p13.12–13. Sequencing revealed a nonsense mutation in the KLF1 gene, p.K288X, which ablated the DNA-binding domain of this key erythroid transcriptional regulator. Only family members with HPFH were heterozygous carriers of this mutation. Expression profiling on primary erythroid progenitors showed that KLF1 target genes were downregulated in samples from individuals with HPFH. Functional assays suggested that, in addition to its established role in regulating adult globin expression, KLF1 is a key activator of the BCL11A gene, which encodes a suppressor of HbF expression. These observations provide a rationale for the effects of KLF1 haploinsufficiency on HbF levels.

IthaGenes: An Interactive Database for Haemoglobin Variations and Epidemiology

Inherited haemoglobinopathies are the most common monogenic diseases, with millions of carriers and patients worldwide. At present, we know several hundred disease-causing mutations on the globin gene clusters, in addition to numerous clinically important trans-acting disease modifiers encoded elsewhere and a multitude of polymorphisms with relevance for advanced diagnostic approaches. Moreover, new disease-linked variations are discovered every year that are not included in traditional and often functionally limited locus-specific databases. This paper presents IthaGenes, a new interactive database of haemoglobin variations, which stores information about genes and variations affecting haemoglobin disorders. In addition, IthaGenes organises phenotype, relevant publications and external links, while embedding the NCBI Sequence Viewer for graphical representation of each variation. Finally, IthaGenes is integrated with the companion tool IthaMaps for the display of corresponding epidemiological data on distribution maps. IthaGenes is incorporated in the ITHANET community portal and is free and publicly available at http://www.ithanet.eu/db/ithagenes.

Oxidative stress in β-thalassaemia and sickle cell disease

Sickle cell disease and β-thalassaemia are inherited haemoglobinopathies resulting in structural and quantitative changes in the β-globin chain. These changes lead to instability of the generated haemoglobin or to globin chain imbalance, which in turn impact the oxidative environment both intracellularly and extracellularly. The ensuing oxidative stress and the inability of the body to adequately overcome it are, to a large extent, responsible for the pathophysiology of these diseases. This article provides an overview of the main players and control mechanisms involved in the establishment of oxidative stress in these haemoglobinopathies.

Friend of Prmt1, a Novel Chromatin Target of Protein Arginine Methyltransferases

ABSTRACT We describe the isolation and characterization of Friend of Prmt1 (Fop), a novel chromatin target of protein arginine methyltransferases. Human Fop is encoded by C1orf77, a gene of previously unknown function. We show that Fop is tightly associated with chromatin, and that it is modified by both asymmetric and symmetric arginine methylation in vivo. Furthermore, Fop plays an important role in the ligand-dependent activation of estrogen receptor target genes, including TFF1 (pS2). Fop depletion results in an almost complete block of estradiol-induced promoter occupancy by the estrogen receptor. Our data indicate that Fop recruitment to the promoter is an early critical event in the activation of estradiol-dependent transcription.

Five Friends of Methylated Chromatin Target of Protein-Arginine-Methyltransferase[Prmt]-1 (Chtop), a Complex Linking Arginine Methylation to Desumoylation

Chromatin target of Prmt1 (Chtop) is a vertebrate-specific chromatin-bound protein that plays an important role in transcriptional regulation. As its mechanism of action remains unclear, we identified Chtop-interacting proteins using a biotinylation-proteomics approach. Here we describe the identification and initial characterization of Five Friends of Methylated Chtop (5FMC). 5FMC is a nuclear complex that can only be recruited by Chtop when the latter is arginine-methylated by Prmt1. It consists of the co-activator Pelp1, the Sumo-specific protease Senp3, Wdr18, Tex10, and Las1L. Pelp1 functions as the core of 5FMC, as the other components become unstable in the absence of Pelp1. We show that recruitment of 5FMC to Zbp-89, a zinc-finger transcription factor, affects its sumoylation status and transactivation potential. Collectively, our data provide a mechanistic link between arginine methylation and (de)sumoylation in the control of transcriptional activity.

Genotyping of BCL11A and HBS1L-MYB SNPs associated with fetal haemoglobin levels: a SNaPshot minisequencing approach

BackgroundB-thalassaemia and sickle cell disease (SCD) are two of the most common monogenic diseases that are found in many populations worldwide. In both disorders the clinical severity is highly variable, with the persistence of fetal haemoglobin (HbF) being one of the major ameliorating factors. HbF levels are affected by, amongst other factors, single nucleotide polymorphisms (SNPs) at the BCL11A gene and the HBS1L-MYB intergenic region, which are located outside the β-globin locus. For this reason, we developed two multiplex assays that allow the genotyping of SNPs at these two genomic regions which have been shown to be associated with variable HbF levels in different populations.ResultsTwo multiplex assays based on the SNaPshot minisequencing approach were developed. The two assays can be used to simultaneous genotype twelve SNPs at the BCL11A gene and sixteen SNPs at HBS1L-MYB intergenic region which were shown to modify HbF levels. The different genotypes can be determined based on the position and the fluorescent colour of the peaks in a single electropherogram. DNA sequencing and restriction fragment length polymorphism (PCR-RFLP) assays were used to verify genotyping results obtained by SNaPshot minisequencing.ConclusionsIn summary, we propose two multiplex assays based on the SNaPshot minisequencing approach for the simultaneous identification of SNPs located at the BCL11A gene and HBS1L-MYB intergenic region which have an effect on HbF levels. The assays can be easily applied for accurate, time and cost efficient genotyping of the selected SNPs in various populations.

The DNA binding factor Hmg20b is a repressor of erythroid differentiation

Background In erythroblasts, the CoREST repressor complex is recruited to target promoters by the transcription factor Gfi1b, leading to repression of genes mainly involved in erythroid differentiation. Hmg20b is a subunit of CoREST, but its role in erythropoiesis has not yet been established. Design and Methods To study the role of Hmg20b in erythropoiesis, we performed knockdown experiments in a differentiation-competent mouse fetal liver cell line, and in primary mouse fetal liver cells. The effects on globin gene expression were determined. We used microarrays to investigate global gene expression changes induced by Hmg20b knockdown. Functional analysis was carried out on Hrasls3, an Hmg20b target gene. Results We show that Hmg20b depletion induces spontaneous differentiation. To identify the target genes of Hmg20b, microarray analysis was performed on Hmg20b knockdown cells and controls. In line with its association to the CoREST complex, we found that 85% (527 out of 620) of the deregulated genes are up-regulated when Hmg20b levels are reduced. Among the few down-regulated genes was Gfi1b, a known repressor of erythroid differentiation. Among the consistently up-regulated targets were embryonic β-like globins and the phospholipase HRAS-like suppressor 3 (Hrasls3). We show that Hrasls3 expression is induced during erythroid differentiation and that knockdown of Hrasls3 inhibits terminal differentiation of proerythroblasts. Conclusions We conclude that Hmg20b acts as an inhibitor of erythroid differentiation, through the down-regulation of genes involved in differentiation such as Hrasls3, and activation of repressors of differentiation such as Gfi1b. In addition, Hmg20b suppresses embryonic β-like globins.

The molecular spectrum and distribution of haemoglobinopathies in Cyprus: a 20-year retrospective study

Haemoglobinopathies are the most common monogenic diseases, posing a major public health challenge worldwide. Cyprus has one the highest prevalences of thalassaemia in the world and has been the first country to introduce a successful population-wide prevention programme, based on premarital screening. In this study, we report the most significant and comprehensive update on the status of haemoglobinopathies in Cyprus for at least two decades. First, we identified and analysed all known 592 β-thalassaemia patients and 595 Hb H disease patients in Cyprus. Moreover, we report the molecular spectrum of α-, β- and δ-globin gene mutations in the population and their geographic distribution, using a set of 13824 carriers genotyped from 1995 to 2015, and estimate relative allele frequencies in carriers of β- and δ-globin gene mutations. Notably, several mutations are reported for the first time in the Cypriot population, whereas important differences are observed in the distribution of mutations across different districts of the island.

A novel MKRN3 nonsense mutation causing familial central precocious puberty

Central or gonadotropin-dependent precocious puberty (CPP) caused by early activation of pulsatile Gonadotropinreleasing hormone (GnRH) secretion is clinically defined by the early maturation of the entire hypothalamic-pituitarygonadal axis and the development of secondary sexual characteristics before the age of 8 years in girls and 9 years in boys [1]. Pubertal timing and growth are influenced by complex interactions of genetic, nutritional, environmental, and socioeconomic factors [2]. Epidemiological studies suggest that around 60–80% of the variation in pubertal onset might be genetically determined [3]. Additionally, family history of CPP has been identified in up to 27.5% of cases with an autosomal mode of inheritance [4]. To date, only a handful of genes, involving both the excitatory and the inhibitory pathways of GnRH secretion, have been reported as causative for CPP and mutations were identified in the kisspeptin system kisspetin 1 (KISS1) and its receptor (KISS1R) [5, 6], and in the makorin RING-finger protein 3 (MKRN3) gene [7]. The initial report by Abreu et al. [7], describing 3 MKRN3 gene frameshift mutations predicted to encode truncated proteins and one missense mutation predicted to disrupt protein function in 2013, was followed by a few more reports of novel loss-of-function mutations resulted to MKRN3 protein deficiency and premature initiation of puberty [8–16]. In the present study, we aimed to identify possible genetic cause in a familial case of CPP in which three out of four girls were affected. The parents were not consanguineous; mother had menarche at normal age (12.5 years), whereas father was considered as an “early developer” although without documentation, but with a final height that is below his midparental height. The proband (Patient II.1, Fig. 1a) was presented with thelarche in 2004 at the age of 6.5 years, the second girl (Patient II.2, Fig. 1a) was presented with thelarche (Tanner staging for breast development: B2) at the age of 8.5 years, whereas the twin, non-monozygotic, younger girls (Patients II.3 and II.4, Fig. 1a) exhibited precocious puberty recently with an interval of 5 months at the ages of 5.1 and 5.6 years respectively. In all affected girls, GnRH stimulation tests were performed by standard techniques and they were all indicative of CPP (Luteinizing hormone (LH) peaked at 18.7, 23.2, and 19.4 μU/ml for the 1st, 3rd, and 4th girl, respectively) and subsequent magnetic resonance imaging (MRI) of the hypothalamic-pituitary region revealed no underlying pathology. The proband started therapy with a long-acting triptorelin and maintained it until the age of 11 years, had menarche at the age of 12.6 years with regular pattern of menses since then. The twin sisters have recently commenced treatment with long-acting triptorelin. Informed consent from the parents and the older sister was obtained in accordance to the national laws. Genomic DNA was extracted from peripheral blood leukocytes samples of the siblings and parents participating * Athanasios Christoforidis christoforidis@doctors.org.uk

A novel MC4R deletion coexisting with FTO and MC1R gene variants, causes severe early onset obesity.

Familial isolated pituitary adenomas (FIPA) constitute 2-3% of pituitary tumours. AIP is the most commonly mutated gene in FIPA. We herein report a novel germline mutation of the AIP gene in a family with FIPA. We present two patients, a father and his 12-year-old daughter, diagnosed clinically and using laboratory measures with acromegaly-gigantism. Both underwent transsphenoidal hypophyseal surgery for macroadenomas. We initially detected a novel heterozygous germline AIP mutation, c.836G>A (p.W279*), in the fathers DNA. We then found the same mutation in his affected daughter. Pituitary adenomas associated with AIP mutations mostly present as FIPA (68%) at an early age (78% occur at <30 years old). They are often growth hormone (GH) - or prolactin - secreting macroadenomas (88%) that have already extended beyond the sella at the time of diagnosis. Acromegalic cases are resistant to somatostatin analogues and multimodal management is frequently essential to control the disease. Our patients had normalized GH/IGF-1 values soon after surgery, although enough time may not have elapsed to reach final cure. While penetrance of the disease can be as low as 10% in FIPA, especially children and young patients with somatotropinoma and prolactinoma should be surveyed for inactivating mutations or deletions in AIP. Determining the causative mutations may be of assistance in early diagnosis, treatment success, and genetic counseling.OBJECTIVE: Heterozygous mutations on the melanocortin-4-receptor gene (MC4R) are the most frequent cause of monogenic obesity. We describe a novel MC4R deletion in a girl with severe early onset obesity, tall stature, pale skin and red hair. CASE REPORT: Clinical and hormonal parameters were evaluated in a girl born full-term by non-consanguineous parents. Her body mass index (BMI) at presentation (3 years) was 30 kg/m2 (z-score: +4.5SDS). By the age of 5.2 years, she exhibited extreme linear growth acceleration and developed hyperinsulinemia. METHODS: Direct sequencing of the MC4R, MC1R and for the known FTO single nucleotide polymorphism (SNP) rs9939609 was performed for the patient and her family. RESULTS: A novel heterozygous MC4R p.Met215del (c.643_645delATG) deletion was identified in the patient, her father and her brother, both of whom exhibited a milder phenotype. 3D structural dynamic simulation studies investigated the conformational changes induced by the p.Met215del. The patient and her mother were also found to be carriers of the obesity risk associated FTO rs9939609 SNP. Finally, the identification of the known p.Arg160Trp MC1R variant in the patient accounts for the red hair and pale skin phenotypic features. CONCLUSION: The p.Met215del causes global conformational and functional changes as it is localized at the alpha-helical transmembrane regions and the membrane spanning regions of the beta-barrel. This novel mutation produces a severe overgrowth phenotype that is apparent as from infancy and is progressive in childhood. The additional negative effect of environmental and unhealthy lifestyle habits as well as a possible co-interaction of FTO rs9939609 SNP may worsen the phenotype.