Börjeson-Forssman-Lehmann Syndrome Research Articles

Loss of PHF6 causes spontaneous seizures, enlarged brain ventricles and altered transcription in the cortex of a mouse model of the Börjeson-Forssman-Lehmann intellectual disability syndrome.

Börjeson-Forssman-Lehmann syndrome (BFLS) is an X-linked intellectual disability and endocrine disorder caused by pathogenic variants of plant homeodomain finger gene 6 (PHF6). An understanding of the role of PHF6 in vivo in the development of the mammalian nervous system is required to advance our knowledge of how PHF6 mutations cause BFLS. Here, we show that PHF6 protein levels are greatly reduced in cells derived from a subset of patients with BFLS. We report the phenotypic, anatomical, cellular and molecular characterization of the brain in males and females in two mouse models of BFLS, namely loss of Phf6 in the germline and nervous system-specific deletion of Phf6. We show that loss of PHF6 resulted in spontaneous seizures occurring via a neural intrinsic mechanism. Histological and morphological analysis revealed a significant enlargement of the lateral ventricles in adult Phf6-deficient mice, while other brain structures and cortical lamination were normal. Phf6 deficient neural precursor cells showed a reduced capacity for self-renewal and increased differentiation into neurons. Phf6 deficient cortical neurons commenced spontaneous neuronal activity prematurely suggesting precocious neuronal maturation. We show that loss of PHF6 in the foetal cortex and isolated cortical neurons predominantly caused upregulation of genes, including Reln, Nr4a2, Slc12a5, Phip and ZIC family transcription factor genes, involved in neural development and function, providing insight into the molecular effects of loss of PHF6 in the developing brain.

PHF6-mediated transcriptional control of NSC via Ephrin receptors is impaired in the intellectual disability syndrome BFLS

The plant homeodomain zinc-finger protein, PHF6, is a transcriptional regulator, and PHF6 germline mutations cause the X-linked intellectual disability (XLID) Börjeson-Forssman-Lehmann syndrome (BFLS). The mechanisms by which PHF6 regulates transcription and how its mutations cause BFLS remain poorly characterized. Here, we show genome-wide binding of PHF6 in the developing cortex in the vicinity of genes involved in central nervous system development and neurogenesis. Characterization of BFLS mice harbouring PHF6 patient mutations reveals an increase in embryonic neural stem cell (eNSC) self-renewal and a reduction of neural progenitors. We identify a panel of Ephrin receptors (EphRs) as direct transcriptional targets of PHF6. Mechanistically, we show that PHF6 regulation of EphR is impaired in BFLS mice and in conditional Phf6 knock-out mice. Knockdown of EphR-A phenocopies the PHF6 loss-of-function defects in altering eNSCs, and its forced expression rescues defects of BFLS mice-derived eNSCs. Our data indicate that PHF6 directly promotes Ephrin receptor expression to control eNSC behaviour in the developing brain, and that this pathway is impaired in BFLS.

Börjeson–Forssman–Lehmann syndrome: delineating the clinical and allelic spectrum in 14 new families

Börjeson-Forssman-Lehmann syndrome (BFLS) is an X-linked intellectual disability syndrome caused by variants in the PHF6 gene. We ascertained 19 individuals from 15 families with likely pathogenic or pathogenic PHF6 variants (11 males and 8 females). One family had previously been reported. Six variants were novel. We analysed the clinical and genetic findings in our series and compared them with reported BFLS patients. Affected males had classic features of BFLS including intellectual disability, distinctive facies, large ears, gynaecomastia, hypogonadism and truncal obesity. Carrier female relatives of affected males were unaffected or had only mild symptoms. The phenotype of affected females with de novo variants overlapped with the males but included linear skin hyperpigmentation and a higher frequency of dental, retinal and cortical brain anomalies. Complications observed in our series included keloid scarring, digital fibromas, absent vaginal orifice, neuropathy, umbilical hernias, and talipes. Our analysis highlighted sex-specific differences in PHF6 variant types and locations. Affected males often have missense variants or small in-frame deletions while affected females tend to have truncating variants or large deletions/duplications. Missense variants were found in a minority of affected females and clustered in the highly constrained PHD2 domain of PHF6. We propose recommendations for the evaluation and management of BFLS patients. These results further delineate and extend the genetic and phenotypic spectrum of BFLS.

Conditional Knock-out of Phf6 Decreases CD44 Expression on Naïve CD4+ T Cells and Skews the Differentiation Toward Regulatory T Cells in Mice

Human PHF6 (plant homeodomain (PHD) finger 6), located in X chromosome, is highly expressed in the central nervous system as well as B- and T-lymphoid cells in human and mice, and involved in multiple physiological pathways through chromatin regulation by interaction with the nucleosome remodeling and deacetylation (NuRD) complex. Germline mutations of PHF6 lead to Börjeson-Forssman-Lehmann syndrome (BFLS), a rare X-linked disorder with distinctive facial features and mental retardation. Meanwhile, somatic PHF6 mutations are present in 11% to 39.5% of adult T-cell acute lymphoblastic leukemia (T-ALL) patients. PHF6 functions as a tumor suppressor gene in T-ALL. Conditional knock-out (cKO) of Phf6 in hematopoietic cells in mice enhances self-renewal potential of hematopoietic stem cells and accelerates the oncogenesis of ICN1 overexpression-induced T-ALL. However, the roles of Phf6 in regulating the development and function of lymphoid cells are not explored yet. In our previous study (YC Hsu et al.Blood adv. 2019), we have shown decreased circulating CD4+ T cells in the Phf6 cKO (Phf6 cKO, Phf6F/F or Phf6F/Y;Vav1-Cre) mice compared with the wild type (WT, Phf6F/F or Phf6F/Y) mice. While the percentages of naïve, effector, and effector memory CD4+ T cells, defined by surface CD44 and CD62L, were comparable between the WT and Phf6 cKO mice, we found significantly lower expression of surface CD44 on naïve CD4+ T cells in the Phf6 cKO mice. To assess the effects of Phf6 on the development of the T cells, we performed bulk RNA sequencing (RNAseq) of thymocytes from 3 pairs of mice. GSEA for these transcriptomes revealed enrichment of the interferon γ, interferon α, and FOXP3 targets. These results support our hypothesis that Phf6 has a role in T cell function and regulation. Consisted with the RNAseq data showing enrichment of the FOXP3 targets in the thymocytes of the Phf6 cKO mice, we discovered that the pooled Treg cells in steady state, containing a majority of natural Treg cells (nTreg cells) and some induced Treg cells (iTreg cells), were significantly increased in the peripheral blood and lymph nodes of the Phf6 cKO mice as revealed by the intracellular Foxp3. (Figure 1a) We further investigated if Phf6 has a role in iTreg cells. To this end, we performed in vitro simulation on splenocytes of the WT and Phf6 cKO mice with CD3 mAB, CD28 mAB, hTGFb1 and mIL2 and found the CD4+Phf6 cKO splenocytes were more prone to differentiate into the CD25+Foxp3+ Treg cells compared with that of the WT mice. (Figure 1b) Collectively, conditional knock-out of Phf6 in mice can skew lymphocyte differentiation toward Treg cells not only in steady state, but also under in vitro regulatory T cell priming condition. To further explore the possible mechanisms, we analyzed single cell RNAseq of splenic T cells of WT and Phf6 cKO mice at steady state and cataloging 7 populations as defined by distinct transcriptomic patterns, including 5 for CD4+ T cells and 2 for CD8+ T cells. (Figure 1c) Compared with the WT, the T cells of Phf6 cKO mice had a larger proportion of Treg cells, which significantly expressed higher Fyb and Eno1. (Figure 1d, e and f) According to the literature, FYB, also known as ADAP (Adhesion and degranulation promoting adaptor protein), is a positive regulator of T-cell development, TCR/CD3-induced T-cell activation, and IL-2 and IFN-g production. (E K Griffiths et al. Science. 2001, Zhang Y et al. Immunology. 2012) Meanwhile, the glycolytic enzyme enolase-1, encoded by Eno1, can stimulate glycolysis, on which the induction and function of induced Treg cells highly depend. (V De Rosa et al. Nat Immunol. 2015) In conclusion, we show that Phf6 is a novel regulator of Treg cells perhaps through ADAP and glycolysis pathways. We will further explore the functions and mechanisms of the Phf6 on T cell regulation. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Molecular and Clinical PHF6 Mutant Myeloid Neoplasia Provides Clues As to Their Pathogenesis and Therapeutic Targeting

Congenital PHF6 mutations (PHF6MT) cause the X-linked neurodevelopmental disorder, Börjeson-Forssman-Lehmann syndrome (BFLS). In contrast, acquired PHF6MT are frequently detected in hematologic neoplasms including acute myeloid leukemia (AML), myelodysplastic syndrome (MDS), and T-cell acute lymphoblastic leukemia (T-ALL). Indeed, PHF6 is highly expressed in brain/developing CNS as well as in hematopoietic subpopulations suggesting its role in neurogenesis and hematopoiesis consistent with the neurologic BFSL presentation of the germline and leukemias with somatic mutations of this gene. This study focuses on involvement of PHF6 lesions in AML. Here, we hypothesize that there are functional links between lymphoid and myeloid neoplasia (MN) with PHF6-deficiency that can be instructive in terms of the impact of this gene as a tumor suppressor gene (TSG). In total, we analyzed sequencing results of 6886 AML cases, including 616 cases from open sources such as The Beat AML Master dataset (Tyner JW , Tognon CE, Bottomly D, et al. Functional genomic landscape of acute myeloid leukaemia. Nature. 2018;562(7728):526-531). We first investigated the clinical phenotype associated with PHF6MT. We detected a total of 111 (1.6% of cases) PHF6MT. While frameshifts were found across all the coding regions, missense and stop-gains were concentrated in the two PHD-type zinc finger domains and were overlapping between other MN and the previously reported T-ALL cases. PHF6MT were significantly more common in male cases (2.3 vs. 0.8%; p<.001) similar to other X-genes such as UBA1, ZRSR2, and STAG2 but different from ATRX, PIGA, and KDM6A (UTX). When comparing primary AML (pAML) with secondary AML (sAML), sAML had more PHF6MT than pAML (2.9 vs. 1.4%; p<.001). While PHF6MT were not associated with chromosomal abnormalities in male cases, female cases with X chromosome deletion (delX) showed higher frequency of PHF6MT compared to females with diploid X chromosome (6.5 vs. 0.8%; p=.0019). Consequently, when delX were included, a total of 2.3% of female patients had PHF6 hits with 10% of them in a homo/hemizygous configuration. While RUNX1, U2AF1, and ASXL1 mutations coincided with PHF6 hits in males, in female cases, delX and ASXL1 mutations were the most common associated defects (Fig.1). Interestingly, RUNX1 mutations (RUNX1MT) in females were enriched in cases with delX. As a result, PHF6MT were enriched into two distinct subclasses: while PHF6MT were enriched in male cases with sAML and RUNX1MT (8.8%), all female cases with sAML, RUNX1MT, and delX also acquired PHF6MT (Fig.2). A higher frequency of PHF6MT in advanced MN than early diseases (e.g., low-risk MDS), suggestive of a secondary rather than a founder role of PHF6MT prompted us to study the clonal architecture of PHF6MT cases using variant allelic frequencies. RUNX1MT were dominant/co-dominant to PHF6 hits in the clonal evolution of AML in contrast to T-ALL, in which PHF6MT appears to be an earlier event. Similar relationship was found with regard to PHF6 hits and other myeloid gene mutations (e.g., ASXL1, DNMT3A, or SRSF2) suggesting a lineage-restricted TSG function of PHF6 in AML following myeloid commitment (consistent with the physiologic expression of PHF6 in pre-B, pre-T, and erythroid progenitors during hematopoietic ontogeny). PHF6 mutations also affected AML patients’ outcome. Overall, PHF6MT showed worse outcome in male cases, but not in female cases. However, if PHF6MT cases also had RUNX1 mutations, the outcome was extremely low in both sexes. Unsupervised RNAseq analysis of PHF6 mutational status vs. expression pattern revealed that PHF6MT cases showed higher mRNA levels of several lymphoid genes (GCSAM, LY9, or BLNK). This finding was also confirmed by RNAseq of in-house PHF6 knock-out K562 and U-937 clones. Immunohistochemistry unveiled that LY9 was positive in illustrative PHF6MT cases and flow cytometry revealed the presence of lymphoid markers in 2/3 PHF6MT cases. Our study confirms that PHF6MT are more common in male AML. Also we demonstrate the intricate relationships between PHF6MT and RUNX1MT in male sAML patients which extend also to female cases with delX. In AML, PHF6MT are a late event compared to T-ALL, in which they occur early in leukemogenesis. This is also in line with higher frequency of PHF6MT in sAML. Some PHF6MT cases have higher expression of lymphoid genes, which may be candidate for molecular targeted therapy. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

The Role of PHF6 in Hematopoiesis and Hematologic Malignancies.

Epigenetic regulation of gene expression represents an important mechanism in the maintenance of stem cell function. Alterations in epigenetic regulation contribute to the pathogenesis of hematological malignancies. Plant homeodomain finger protein 6 (PHF6) is a member of the plant homeodomain (PHD)-like zinc finger family of proteins that is involved in transcriptional regulation through the modification of the chromatin state. Germline mutation of PHF6 is the causative genetic alteration of the X-linked mental retardation Borjeson-Forssman-Lehmann syndrome (BFLS). Somatic mutations in PHF6 are identified in human leukemia, such as adult T-cell acute lymphoblastic leukemia (T-ALL, ~ 38%), pediatric T-ALL (~ 16%), acute myeloid leukemia (AML, ~ 3%), chronic myeloid leukemia (CML, ~ 2.5%), mixed phenotype acute leukemia (MPAL, ~ 20%), and high-grade B-cell lymphoma (HGBCL, ~ 3%). More recent studies imply an oncogenic effect of PHF6 in B-cell acute lymphoblastic leukemia (B-ALL) and solid tumors. These data demonstrate that PHF6 could act as a double-edged sword, either a tumor suppressor or an oncogene, in a lineage-dependent manner. However, the underlying mechanisms of PHF6 in normal hematopoiesis and leukemogenesis remain largely unknown. In this review, we summarize current knowledge of PHF6, emphasizing the role of PHF6 in hematological malignancies. Epigenetic regulation of PHF6 in B-ALL. PHF6 maintains a chromatin structure that is permissive to B-cell identity genes, but not T-cell-specific genes (left). Loss of PHF6 leads to aberrant expression of B-cell- and T-cell-specific genes resulting from lineage promiscuity and binding of T-cell transcription factors (right).

Further characterization of Borjeson-Forssman-Lehmann syndrome in females due to de novo variants in PHF6.

While inherited hemizygous variants in PHF6 cause X‐linked recessive Borjeson‐Forssman‐Lehmann syndrome (BFLS) in males, de novo heterozygous variants in females are associated with an overlapping but distinct phenotype, including moderate to severe intellectual disability, characteristic facial dysmorphism, dental, finger and toe anomalies, and linear skin pigmentation. By personal communication with colleagues, we assembled 11 additional females with BFLS due to variants in PHF6. We confirm the distinct phenotype to include variable intellectual disability, recognizable facial dysmorphism and other anomalies. We observed skewed X‐inactivation in blood and streaky skin pigmentation compatible with functional mosaicism. Variants occurred de novo in 10 individuals, of whom one was only mildly affected and transmitted it to her more severely affected daughter. The mutational spectrum comprises a two‐exon deletion, five truncating, one splice‐site and three missense variants, the latter all located in the PHD2 domain and predicted to severely destabilize the domain structure. This observation supports the hypothesis of more severe variants in females contributing to gender‐specific phenotypes in addition to or in combination with effects of X‐inactivation and functional mosaicism. Therefore, our findings further delineate the clinical and mutational spectrum of female BFLS and provide further insights into possible genotype–phenotype correlations between females and males.

Genomic Landscape of PHD Finger Protein 6 (PHF6) Mutant Myeloid Neoplasia

Genomic Landscape of PHD Finger Protein 6 (PHF6) Mutant Myeloid Neoplasia

Transgenic mice with an R342X mutation in Phf6 display clinical features of Börjeson-Forssman-Lehmann Syndrome.

The PHF6 mutation c.1024C > T; p.R342X, is a recurrent cause of Börjeson-Forssman-Lehmann Syndrome (BFLS), a neurodevelopmental disorder characterized by moderate-severe intellectual disability, truncal obesity, gynecomastia, hypogonadism, long tapering fingers and large ears (MIM#301900). Here, we generated transgenic mice with the identical substitution (R342X mice) using CRISPR technology. We show that the p.R342X mutation causes a reduction in PHF6 protein levels, in both human and mice, from nonsense-mediated decay and nonsense-associated alternative splicing, respectively. Magnetic resonance imaging studies indicated that R342X mice had a reduced brain volume on a mixed genetic background but developed hydrocephaly and a high incidence of postnatal death on a C57BL/6 background. Cortical development proceeded normally, while hippocampus and hypothalamus relative brain volumes were altered. A hypoplastic anterior pituitary was also observed that likely contributes to the small size of the R342X mice. Behavior testing demonstrated deficits in associative learning, spatial memory and an anxiolytic phenotype. Taken together, the R342X mice represent a good preclinical model of BFLS that will allow further dissection of PHF6 function and disease pathogenesis.

Downregulation of the GHRH/GH/IGF1 axis in a mouse model of Börjeson-Forssman-Lehman syndrome.

Börjeson-Forssman-Lehmann syndrome (BFLS) is an intellectual disability and endocrine disorder caused by plant homeodomain finger 6 (PHF6) mutations. Individuals with BFLS present with short stature. We report a mouse model of BFLS, in which deletion of Phf6 causes a proportional reduction in body size compared with control mice. Growth hormone (GH) levels were reduced in the absence of PHF6. Phf6-/Y animals displayed a reduction in the expression of the genes encoding GH-releasing hormone (GHRH) in the brain, GH in the pituitary gland and insulin-like growth factor 1 (IGF1) in the liver. Phf6 deletion specifically in the nervous system caused a proportional growth defect, indicating a neuroendocrine contribution to the phenotype. Loss of suppressor of cytokine signaling 2 (SOCS2), a negative regulator of growth hormone signaling partially rescued body size, supporting a reversible deficiency in GH signaling. These results demonstrate that PHF6 regulates the GHRH/GH/IGF1 axis.

Characterization of a Mouse Model of Börjeson-Forssman-Lehmann Syndrome

SUMMARYMutations of the transcriptional regulator PHF6 cause the X-linked intellectual disability disorder Börjeson-Forssman-Lehmann syndrome (BFLS), but the pathogenesis of BFLS remains poorly understood. Here, we report a mouse model of BFLS, generated using a CRISPR-Cas9 approach, in which cysteine 99 within the PHD domain of PHF6 is replaced with phenylalanine (C99F). Mice harboring the patient-specific C99F mutation display deficits in cognitive functions, emotionality, and social behavior, as well as reduced threshold to seizures. Electrophysiological studies reveal that the intrinsic excitability of entorhinal cortical stellate neurons is increased in PHF6 C99F mice. Transcriptomic analysis of the cerebral cortex in C99F knockin mice and PHF6 knockout mice show that PHF6 promotes the expression of neurogenic genes and represses synaptic genes. PHF6-regulated genes are also overrepresented in gene signatures and modules that are deregulated in neurodevelopmental disorders of cognition. Our findings advance our understanding of the mechanisms underlying BFLS pathogenesis.

PHF6 Somatic Mutations and Their Functional Role in the Pathophysiology of Myelodysplastic Syndromes (MDS) and Acute Myeloid Leukemia (AML)

Recurrent somatic nonsense PHF6 mutations have been reported in patients with T-acute lymphocytic leukemia, AML and chronic myeloid leukemia in blast crisis. Germ line (GL) PHF6 mutations are responsible for Borjeson−Forssman−Lehmann syndrome (BFLS), a hereditary X-linked disorder characterized by mental retardation and dysmorphic features. PHF6 is a highly conserved 41kDa protein with ubiquitous expression in hematopoietic cells, including CD34+ cells.We screened patients (N=1166) with myeloid neoplasms by targeted multi-amplicon deep NGS targeting all ORFs of PHF6 to determine the prevalence and distribution and molecular context of PHF6 gene alterations. In total, we identified and verified 52 cases with somatic PHF6 mutations, 32 of which were frameshift or nonsense mutations and with a strong male predominance (76%). Mutations were distributed almost equally between 2 DNA binding domains. Previously, PHF6 has been included in other screening panels (Haferlach et al. 2014 and Papaemmanuil 2013) with somatic mutations found in 24/944 and 21/738 MDS cases, respectively. SNP-array karyotyping showed that microdeletions involving the PHF6 locus were present in about 1.2% of myeloid neoplasms, but affected only female patients.The most frequent chromosomal aberration observed in conjunction with PHF6 mutations was trisomy-8 (P=.018). The most commonly associated somatic mutations included RUNX1 (P=.001) and IDH1 (P=.008) but not IDH2 (P>.1). There was no impact on overall survival with respect to PHF6 mutant status in total or within individual risk groups (low risk (RA,RARS) vs. high-risk (RAEB1/2). Concomitant PHF6 and RUNX1 mutations were associated with particularly poor prognosis. RUNX1 mutational status correlated with PHF6 expression levels and PHF6 expression inversely correlated with RUNX1 mRNA levels. Subsequent analysis of clonal architecture using VAF calculations and serial samples for these cases suggested that PHF6 may function as a founder driver gene in 18% of cases. PHF6 variant allelic frequency (VAF) varied between disease subtypes, with the highest clonal burden found in AML patients (P<.01). Within MDS patients we also found lower expression of PHF6 mRNA in CD34+ cells in MDS overall vs. controls (P<.01), as well as lower expression of PHF6 in advanced myeloid neoplasms (P<.05). Lower expression (defined as mean+1SD of controls) was found in 12% and 23% of patients with lower- or higher- risk MDS, respectively.Recent studies have proposed that PHF6 deficiency leads to impaired cell proliferation, cell cycle arrest at G2/M phase and DNA damage. Following shRNA knockdown, hematopoietic cell lines showed only moderately accelerated growth and increased response to growth factors, while EPO-dependent UT7, did not result in growth factor autonomy. To delineate the possible pathophysiological pathway involving PHF6, we compared transcriptional expression profiles of 5 different cell lines with shPHF6 to WT counterparts. We then studied the consequences of PHF6 knockdown on transcriptional profiles. We have found 1020 transcripts differentially expressed (with at least 1.5x change up/down) in the context of shPHF6 knock down. Concordant results among all 5 cell lines resulted in 354 genes that were upregulated and 766 that were down-regulated. Analyses with primary patient data derived from low PHF6 expressors and mutant cases found a concordance of 71 upregulated genes and 80 genes that were downregulated.The most significant functional group of transcripts that was found to be modulated was a family belonging to ribosomal biogenesis pathway (pFDR<1x10-6). Mass spec fingerprinting found protein-protein interaction partners that were found to be dysregulated on a transcriptional level. This finding of protein interaction/transcriptional dependence might suggest feedback mechanisms on a transcriptional level.In conclusion, our results indicate that PHF6 mutations are generally present in more aggressive types of myeloid neoplasms, frequently associated with RUNX1/IDH1 mutations. Our functional in vitro studies along with recently published reports suggest an association of PHF6 deficiency with transcriptional regulation and thereby provide a basis for a phenotype conveyed by ancestral lesions, consistent with its role as a tumor suppressor gene. DisclosuresSekeres:Millenium/Takeda: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees. Makishima:The Yasuda Medical Foundation: Research Funding.

PHF6 - Somatic Mutations and Their Role in Pathophysiology of MDS and AML

Recently, rare somatic nonsense PHF6 mutations and deletions have been reported in patients with T-ALL, AML and blast crisis CML. Germ line PHF6 mutations have been described in Borjeson−Forssman−Lehmann syndrome (BFLS), a hereditary X-linked disorder characterized by mental retardation and somatic deformities. Patients with BFLS have been also reported to develop leukemia, suggesting PHF6 mutations may predispose to cancer. PHF6 is a highly conserved 41kDa protein showing ubiquitous expression in a variety of tissues, including bone marrow, CD34+ cells and leukocytes. The function and molecular pathogenesis in hematological disorders is unknown. PHF6 has been suggested to be a tumor suppressor gene (TSG) involved in the control of rRNA synthesis. Recent CHIPseq experiments showed that PHF6 binds upstream of the regulatory sequence of RUNX1.In an index case of a young adult female patient with proliferative CMML with dysmorphic features, we have identified remarkable GL mosaicism for PHF6 mutation (p.K44fs), confirmed by deep sequencing of bone marrow, CD3+ cells, spleen and skin tissue. Subsequently, we screened patients with myeloid neoplasms by targeted multi-amplicon sequencing to determine the prevalence and distribution of PHF6 gene alterations. Sequencing results from 1122 cases were analyzed (778 by targeted deep sequencing and 344 by whole exome sequencing). In total, we identified 45 cases with PHF6 mutations, 32 of which were frameshift or nonsense mutations. Previously, PHF6 have been included in screening panels by Haferlach et al., (Leukemia 2014) and Papaemmanuil et al., (Blood 2013) and somatic mutations were found in 24/944 and 21/738 cases of MDS, respectively. The somatic nature of these defects was confirmed by analysis of non-clonal CD3+ lymphocytes, Thus the incidence of PHF6 mutations ranges from 4.3% in current study to 2.8% and 2.5% reported by others and are most frequently observed among patients with secondary AML (33%), suggesting that PHF6 mutations are not uncommon driver events in myeloid neoplasia. Gender distribution showed a strong male predominance (76%), indicating that retention of a single copy of PHF6 may be protective. There was no significant sex difference in the transcriptional expression of PHF6 itself. The most frequent chromosomal aberration observed in conjunction with PHF6 mutations was trisomy-8 (P =.08). The most commonly associated somatic mutations were in RUNX1 (P =.001) and IDH2 (p=.008). Concomitant PHF6 and RUNX1 mutations are associated with a poor prognosis in AML, and occur predominantly in males. There was no association observed between low expressors of PHF6 and RUNX1 mutations or RUNX1 expression levels. Conversely, RUNX1 mutant cases without somatic PHF6 mutations were not observed to have low transcriptional PHF6 levels. Subsequent analysis of clonal architecture using variant allelic frequency calculations and serial sampling suggested that mutated PHF6 may function as a founder driver gene in proportion of cases, while RUNX1 mutations are acquired as secondary events.Recent studies proposed that PHF6 deficiency leads to impaired cell proliferation, cell cycle arrest at the G2/M phase and an increase in DNA damage. To delineate a possible pathophysiological pathway involving PHF6 we compared transcriptional expression profiles of low expressors to those with normal levels of PHF6. The most notably deregulated group of genes were clustered to a functionally related group of ribosomal RNA proteins (p<.00001). To better understand functional properties of PHF6 we conducted PHF6 specific immuno-precipitation followed by mass spectrometric fingerprinting on K562 cells to identify protein partners. We have identified a novel association of PHF6 in RNA degradation/stability and ribosomal proteins, including MOV and PABPC families.In conclusion, our results indicate that PHF6 mutations are generally present in more aggressive types of myeloid neoplasms and arefrequently associated with RUNX1/IDH2 mutations. Our functional in vitro studies, along with recently published reports, suggest an association of PHF6 deficiency with transcriptional regulation and thereby provide a basis for a transcriptional repressor phenotype conveyed by ancestral lesions, consistent with a role for PHF6 as a TSG. DisclosuresLevine:Foundation Medicine: Consultancy; CTI BioPharma: Membership on an entity's Board of Directors or advisory committees; Loxo Oncology: Membership on an entity's Board of Directors or advisory committees. Sekeres:Celgene Corporation: Membership on an entity's Board of Directors or advisory committees.

Impact and Function of Somatic PHF6 Mutations in Myeloid Neoplasms

Borjeson−Forssman−Lehmann syndrome (BFLS), a hereditary X-linked disorder characterized by mental retardation, truncal obesity, gynecomastia, hypogonadism and other dysmorphic features, is known to be caused by germline (GL) mutations of plant homeo domain finger protein 6 (PHF6). PHF6 is a highly conserved 41kDa protein showing ubiquitous expression in a variety of tissues, including bone marrow, CD34+ cells and blood leukocytes. Human PHF6 is located on chrXq26.2. Recently, rare somatic nonsense mutations and deletions have been detected in patients with T-ALL and AML and found in some T-ALL cell lines. Patients with BFLS with PHF6 mutations have been reported to develop leukemia, suggesting PHF6 mutations may predispose cancer. Although the actual function and molecular pathogenesis is unknown, PHF6 has been suggested to be a tumor suppressor gene involved in the control of myeloid development.In an index case of a young adult female patient with proliferative CMML with dysmorphic features, we have identified remarkable GL mosaicism for PHF6 mutation (p.K44fs), confirmed by deep sequencing of marrow, CD3+ cells and skin tissue. Subsequently, we screened patients with myeloid neoplasms by targeted multi-amplicon sequencing to determine the prevalence and distribution of PHF6 gene alterations. Sequencing results from 1072 cases were analyzed (728 by targeted deep sequencing and 344 by whole exome sequencing). In total, we identified 21 cases with PHF6 mutations, 13 of which were frameshift or nonsense mutations. Previously, PHF6 have been included in screening panels by Haferlach et al., (Leukemia 2014) and Papaemmanuil et al., (Blood 2013) and somatic mutations were found in 24/944 and 21/738 cases of MDS, respectively. These results along with ours suggest that PHF6 mutations are common driver events. The somatic nature of these defects was confirmed by analysis of non-clonal CD3+ lymphocytes, thus, PHF6 mutations occur at a frequency of 2.0% and are most frequently observed among patients with secondary AML (33%, P=.0021). Gender distribution showed a strong male predominance (76%), likely due to the location of PHF6 on chrX and indicating that retention of a single copy of PHF6 may be protective. SNP-array karyotyping showed that deletions of Xq, involving the PHF6 locus (Xq26), were present in about 1.2% of myeloid neoplasms and affect only female patients. As a family, plant homeo domain (PHD) finger genes are affected by mutations associated with various cancers. JARID1A, PHF23, NSD1 and NSD3 were described to serve as fusion partners with the NUP98 in a subset of AML cases.The most frequent chromosomal aberration observed in conjunction with PHF6 mutations was trisomy-8 (P=.08). The most commonly associated somatic mutations were in RUNX1 (N=7; P=.001), U2AF1 (N=5), ASXL1 (N=5), IDH1 (N=4), and DNMT3A (N=4). Interestingly, 6/7 cases with concomitant PHF6 and RUNX1 mutations showed a poor prognosis AML. Subsequent analysis of clonal architecture using variant allelic frequency calculations and serial samples for these cases suggested that PHF6 may function as a founder driver gene while RUNX1 mutations are acquired as secondary events.Recent studies proposed that PHF6 deficiency leads to impaired cell proliferation, cell cycle arrest at G2/M phase and an increase of DNA damage. To examine DNA damage and quantify double stranded breaks (DSBs) in primary cells from PHF6-mutants, those with wild-type (WT) PHF6 and normal bone marrow we used a flow cytometric anti-γH2AX assay, following induction of DNA damage with Camptothecin. As judged by greater percentages of anti-γH2AX labeled cells, DSBs were more common in mutant cases consistent with more DNA damage present in PHF6 mutant compared to WT MDS and normal bone marrow cells.In conclusion, our results indicate that PHF6 mutations are generally present in more aggressive types of myeloid neoplasms, frequently associated with RUNX1 mutations. Our functional in vitro studies along with recently published reports suggest an association of PHF6 deficiency with genomic instability and thereby provide a basis for a mutator phenotype conveyed by ancestral lesions, consistent with its role as a tumor suppressor gene. DisclosuresSekeres:Celgene: Membership on an entity’s Board of Directors or advisory committees; Amgen Corp: Membership on an entity’s Board of Directors or advisory committees; Boehringer-Ingelheim Corp: Membership on an entity’s Board of Directors or advisory committees.

Distinct phenotype of PHF6 deletions in females

We report on two female patients carrying small overlapping Xq26.2 deletions of 100 kb and 270 kb involving the PHF6 gene. Mutations in PHF6 have been reported in individuals with Borjeson–Forssman–Lehmann syndrome, a condition present almost exclusively in males. Two very recent papers revealed de novo PHF6 defects in seven female patients with intellectual disability and a phenotype resembling Coffin–Siris syndrome (sparse hair, bitemporal narrowing, arched eyebrows, synophrys, high nasal root, bulbous nasal tip, marked clinodactyly with the hypoplastic terminal phalanges of the fifth fingers and cutaneous syndactyly of the toes, Blaschkoid linear skin hyperpigmentation, dental anomalies and occasional major malformations). The clinical presentation of these patients overlaps completely with our first patient, who carries a germline deletion involving PHF6. The second patient has a mosaic deletion and presented with a very mild phenotype of PHF6 loss in females. Our report confirms that PHF6 loss in females results in a recognizable phenotype overlapping with Coffin–Siris syndrome and distinct from Borjeson–Forssman–Lehmann syndrome. We expand the clinical spectrum and provide the first summary of the recommended medical evaluation.

Somatic PHF6 Mutations In Myeloid Malignancies

X chromosome genomics is an important area of hematologic malignancy research because of frequent acquired X-abnormalities, location of important genes on this chromosome, and issues surrounding Lyonization (X-inactivation). For example, we previously described somatic mutations of UTX (KDM6A), a H3K27 demethylase located on chromosome Xp11.3, in aggressive myeloid neoplasms. In a companion abstract to the the results here, we also report loss of function somatic mutations of BRCC3 (Xq28), encoding a subunit of the BRCA1-BRCA2-containing complex.In an index young female case of a proliferative CMML with dysmorphic features, we have identified PHF6 mutation mosaisism (p.K44fs), confirmed by deep sequencing of bone marrow, skin and spleen tissues. Subsequently, we screened our MDS exome project data set, involving 206 patients with MDS and related neoplasms, and have detected and confirmed additional somatic PHF6mutations.Plant homeodomain finger protein 6 (PHF6) is a ubiquitously expressed 41 kDa protein that is conserved and vertebrate-specific. Human PHF6 is located on chrXq26.3. Germline mutations of PHF6 cause Borjeson−Forssman−Lehmann syndrome (BFLS), an X-linked mental retardation disorder characterized by truncal obesity, gynaecomastia, hypogonadism and other dysmorphic features. BFLS patients have been reported to develop leukemias. More recently, rare somatic PHF6mutations were detected in patients with T-ALL, but rarely also in AML.To assess the clinical associations and significance of PHF6 mutations, we analyzed NGS results in a total of 809 patients with MDS, MDS/MPN, MPN and AML. In addition we also investigated for the presence of PHF6 mutation in the TCGA AML data sets (n=199). All mutations in our patients were confirmed by Sanger sequencing and targeted deep NGS. In total, we identified 19/809 cases with PHF6 mutations; they were located throughout the gene including 15 SNVs and 4 indels. In addition TCGA pAML NGS results revealed PHF6 mutations in 6/199 cases, including 4 SNVs and 2 indels. Thus, PHF6 mutation occurs at a frequency of 2.5% in myeloid neoplasm and are most frequently observed in pAML (36%) together with sAML (32%) phenotypes. Gender distribution showed male predominance (84%), likely related to PHF6 locus on chrXq26.3. SNP-array karyotyping showed that deletions of Xq, involving PHF6locus (Xq26) were present in about 2% of myeloid neoplasms.Chromosome 7 abnormalities, including del(7q), were the most frequent lesions seen in conjunction with PHF6 mutations. Most commonly coinciding mutations were in RUNX1 (n=8), TET2 (n=4), ASXL1 (n=3) and U2AF1 (n=3) and unbiased statistical analysis confirmed the significant association between PHF6 and RUNX1 mutations (P=.002). Interestingly, all of 8 cases with concomitant RUNX1 and PHF6 mutations were diagnosed as high-risk diseases; 1 RAEB-2 and 7 AMLs. Deep sequencing analysis of 5 cases with coexisting PHF6 and RUNX1 mutations showed that PHF6 mutated clones were always significantly larger than RUNX1 mutated clones. Such a serial clonal acquisition pattern of ancestral PHF6 and secondary RUNX1 mutations was also observed clearly in an illustrative case with evolution from aplastic anemia (AA) to sAML, in which small clone of PHF6 was detected in AA sample and expanded during MDS stage, followed by secondary driver RUNX1 mutations at the stage. These findings suggest that RUNX1 mutations were acquired as a subclone of the main population with primary driver PHF6mutations.In conclusion, our results indicate that PHF6 mutations, as a recurrent genetic abnormality, were frequently mutated in more aggressive types of myeloid malignancies. Newly identified ancestral nature of PHF6 mutations specifically favor being followed by secondary driver RUNX1 mutations during leukemic evolution. Disclosures:Polprasert:MDS foundation: Research Funding. Maciejewski:Aplastic anemia&MDS International Foundation: Research Funding; NIH: Research Funding. Makishima:Scott Hamilton CARES grant: Research Funding; AA & MDS international foundation: Research Funding.

A new face of Borjeson–Forssman–Lehmann syndrome? De novo mutations in PHF6 in seven females with a distinct phenotype

BackgroundBorjeson–Forssman–Lehmann syndrome (BFLS) is an X-linked recessive intellectual disability (ID) disorder caused by mutations in the PHF6 gene and characterised by variable cognitive impairment, a distinct facial gestalt, obesity, and...

The X-Linked Intellectual Disability Protein PHF6 Associates with the PAF1 Complex and Regulates Neuronal Migration in the Mammalian Brain

Intellectual disability is a prevalent disorder that remains incurable. Mutations of the X-linked proteinPHF6 cause the intellectual disability disorder Börjeson-Forssman-Lehmann syndrome (BFLS). However, the biological role of PHF6 relevant to BFLS pathogenesis has remained unknown. We report that knockdown of PHF6 profoundly impairs neuronal migration in the mouse cerebral cortex invivo, leading to the formation of white matter heterotopias displaying neuronal hyperexcitability. We find that PHF6 physically associates with the PAF1 transcription elongation complex, and inhibition of PAF1 phenocopies the PHF6 knockdown-induced migration phenotype invivo. We also identify Neuroglycan C/Chondroitin sulfate proteoglycan 5 (NGC/CSPG5), a potential schizophrenia susceptibility gene, as a critical downstream target of PHF6 in the control of neuronal migration. These findings define PHF6, PAF1, and NGC/CSPG5 as components of a cell-intrinsic transcriptional pathway that orchestrates neuronal migration in the brain, with important implications for the pathogenesis of developmental disorders of cognition.

1094 BöRjeson - Forssman - Lehmann Syndrome: A Rare But Important Cause of Obesity

Background & Aims: Borjeson - Forssman - Lehmann Syndrome (BFLS) is a rare X-linked condition characterized by obesity and developmental delay. Only nineteen unrelated cases of BFLS, with confirmed PHF6 mutations, have been reported since its first description in 1962. The phenotype is better characterized in males with moderate to severe developmental delay, microcephaly and hypogonadism. The phenotype of affected females ranges from normal through to developmental delay with obesity and hypothyroidism. Our aim is to report three cases of BFLS, describe the associated endocrine dysfunction and further delineate the female phenotype. Method: Endocrine and genetic results were reviewed of three patients (2 female and 1 male) with a clinical diagnosis of BFLS. Results: Confirmation of the diagnosis was established in one patient and is pending in the other two. All three have developmental delay. Patient 1 is an obese female (BMI 41 kg/m2) with genetically proven BFLS and pubertal delay, autoimmune hypothyroidism and hypercholesterolaemia. Patient 2 is an obese female (BMI 39kg/m2) with autoimmune hypothyroidism, hyperandrogenism and hypercholesterolaemia. Patient 3 is an obese male (BMI 30 kg/m2) with typical dysmorphic features and pubertal delay. Conclusion: BFLS is an important condition to consider when reviewing patients with obesity and developmental delay. We suspect it is more prevalent than reported in the literature and may be missed particularly in females where the phenotype is poorly characterized. Endocrine abnormalities were seen in all of our patients. Because of the X-linked pattern of inheritance, diagnosis and genetic counselling are important.

T‐cell acute lymphoblastic leukemia in association with Börjeson–Forssman–Lehmann syndrome due to a mutation in PHF6

Börjeson-Forssman-Lehmann syndrome (BFLS) is a rare X-linked mental retardation syndrome that is caused by germline mutations in PHF6. We describe a 9-year-old male with BFLS, who developed T-cell acute lymphoblastic leukemia (T-ALL). The PHF6 gene is located on the X chromosome and encodes a protein with two PHD-type zinc finger domains and four nuclear localization sequences. Previously, overexpression of Phf6 was observed in murine T-cell lymphomas. Our observation indicates that BFLS may represent a cancer predisposition syndrome and that mutations of PHF6 contribute to T-ALL.