Zafar Nawaz

Whole exome sequencing unravels disease-causing genes in consanguineous families in Qatar

Whole exome sequencing (WES) has greatly facilitated the identification of causal mutations for diverse human genetic disorders. We applied WES as a molecular diagnostic tool to identify disease‐causing genes in consanguineous families in Qatar. Seventeen consanguineous families with diverse disorders were recruited. Initial mutation screening of known genes related to the clinical diagnoses did not reveal the causative mutations. Using WES approach, we identified the definitive disease‐causing mutations in four families: (i) a novel nonsense homozygous (c.1034C>G) in PHKG2 causing glycogen storage disease type 9C (GSD9C) in a male with initial diagnosis of GSD3; (ii) a novel homozygous 1‐bp deletion (c.915del) in NSUN2 in a male proband with Noonan‐like syndrome; (iii) a homozygous SNV (c.1598C>G) in exon 11 of IDUA causing Hurler syndrome in a female proband with unknown clinical diagnosis; (iv) a de novo known splicing mutation (c.1645+1G>A) in PHEX in a female proband with initial diagnosis of autosomal recessive hypophosphatemic rickets. Applying WES as a diagnostic tool led to the unambiguous identification of disease‐causing mutations in phenotypically complex disorders or correction of the initial clinical diagnosis in ˜25% of our cases.

Loss-of-Function Mutation in APC2 Causes Sotos Syndrome Features

Sotos syndrome, characterized by intellectual disability and characteristic facial features, is caused by haploinsufficiency in the NSD1 gene. We conducted an etiological study on two siblings with Sotos features without mutations in NSD1 and detected a homozygous frameshift mutation in the APC2 gene by whole-exome sequencing, which resulted in the loss of function of cytoskeletal regulation in neurons. Apc2-deficient (Apc2-/-) mice exhibited impaired learning and memory abilities along with an abnormal head shape. Endogenous Apc2 expression was downregulated by the knockdown of Nsd1, indicating that APC2 is a downstream effector of NSD1 in neurons. Nsd1 knockdown in embryonic mouse brains impaired the migration and laminar positioning of cortical neurons, as observed in Apc2-/- mice, and this defect was rescued by the forced expression of Apc2. Thus, APC2 is a crucial target of NSD1, which provides an explanation for the intellectual disability associated with Sotos syndrome.

Nonsense mutation in the WDR73 gene is associated with Galloway-Mowat syndrome

Background Neuroanatomical defects are often present in children with severe developmental delay and intellectual disabilities. Few genetic loci have been associated with disorders of neurodevelopment. Our objective of the present study was to analyse a consanguineous Arab family showing some of the hallmark signs of a rare cerebellar hypoplasia-related neurodevelopmental syndrome as a strategy for discovering a causative genetic mutation. Methods We used whole exome sequencing to identify the causative mutation in two female siblings of a consanguineous Arab family showing some of the hallmark signs of a cerebellar-hypoplasia-related neurodevelopmental disorder. Direct Sanger sequencing was used to validate the candidate mutations that cosegregated with the phenotype. Gene expression and loss of function studies were carried out in the zebrafish model system to examine the role of the candidate gene in neurodevelopment. Results Patients presented with severe global developmental delay, intellectual disability, hypoplasia of the cerebellum and biochemical findings suggestive of nephrotic disease. Whole exome sequencing of the two patients revealed a shared nonsense homozygous variant in WDR73 (p.Q235X (c.703C>T)) resulting in loss of the last 144 amino acids of the protein. The variant segregated according to a recessive mode of inheritance in this family and was absent from public and our inhouse databases. We examined the developmental role of WDR73 using a loss-of-function paradigm in zebrafish. There was a significant brain growth and morphogenesis defect in wdr73 knockdown embryos resulting in a poorly differentiated midbrain and cerebellum. Conclusions The results provide new insight into the functional role of WDR73 in brain development and show that perturbation of its function in an inherited disorder in humans is associated with cerebellar hypoplasia as well as nephrotic disease, consistent with Galloway-Mowat Syndrome.

Molecular genetic analysis of consanguineous families with primary microcephaly identified pathogenic variants in the ASPM gene

Autosomal recessive primary microcephaly is a rare genetic disorder that is characterized by reduced head circumference and a varying degree of intellectual disability. Genetic studies on consanguineous families with primary microcephaly have identified 15 (MCPH) causative genes that include MCPH1, WDR62, CDK5RAP2, CASC5, ASPM, CENPJ, STIL, CEP135, CEP152, ZNF335, PHC1, CDK6, CENPE, SASS6MFSD2AANKLE2 and CIT (Khan et al.2014; Yamamoto et al.2014; Alakbarzade et al.2015; Morris-Rosendahl and Kaindl 2015; Basit et al.2016). Physiologically, most of these MCPH proteins are involved in cell cycle and its regulation. In the present clinical genetic study, we have present two consanguineous Pakistani families segregating primary microcephaly and intellectual disability. These families were ascertained from the Saraiki ethnic part of Khyber-Pakhtunkhwa province in Pakistan. Whole exome sequencing in one family revealed a novel 1-bp deletion NM_018136.4: c.10013delA (p.Asp3338Valfs*2), while the other family showed a previously reported nonsense mutation NM_018136.4: c.9730C > T (rs199422195 (p.Arg3244*)) in ASPM gene. The novel frame-shift mutation (p.Asp3338Valfs*2) in ASPM presumably truncates the protein synthesis that results in loss of armadillo-type fold domain.

Abstract A52: Investigation of p53 restoration therapy in a mouse model of pineoblastoma

Background: Pineoblastoma is a malignant childhood tumor with poor outcomes. We have previously characterized a mouse model of pineoblastoma induced by transgenic expression of Cyclin D1 in a p53 null background. We showed that Cyclin D1 expression in the setting of intact p53 results in pineal hyperplasia, but no onset of invasive tumors, reminiscent of pineal enlargement seen in a subset of children with RB haploinsufficiency. In the setting of p53 loss, pineoblastoma occurs with 100% penetrance. We now investigated the role of p53 restoration therapy in prevention and treatment of pineoblastoma using this mouse model. Results: We found that restoration of p53 in mouse pre-malignant proliferating pineal tumors results in tumor prevention, shown by effective cell cycle exit and induction of cellular senescence, a tumor suppressor mechanism. Conversely, p53 reactivation in established malignant pineal tumors had no effect cell proliferation, unless paired with DNA damaging therapy. The response to p53 restoration did not correlate with levels of p19Arf expression. Interestingly, in premalignant lesions, p53 restoration was associated with secondary amplification of MAPK signaling and induction of p19Arf expression. Subsequent inactivation of p53 after senescence resulted in diffuse re-entry into the cell cycle and rapid tumor progression. Evaluation of a panel of human supratentorial primitive neuroectodermal tumors showed low activity of the p53 pathway, while only one of 6 tumors had p53 deletion or mutation. Conclusion: Our data shows that, in a mouse model of Cyclin D1-driven pineoblastoma, restoration of p53 has different effects in premalignant versus invasive pineal tumors, where it may need to be paired with DNA damaging agents for effective tumor suppression. Furthermore, p53 activation may need to be continually sustained in such lesions undergoing senescence. Finally, p53 restoration approaches may be worth exploring in sPNET, where the p53 gene seems to be intact but the pathway inactive in the majority of examined tumors. Citation Format: Mohammad Harajly, Hassan Zalzali, Zafar Nawaz, Raya Saab. Investigation of p53 restoration therapy in a mouse model of pineoblastoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A52.

Deciphering the molecular basis of arsenic/interferon-alpha induced eradication of leukemia initiating cells in adult T cell leukemia

Adult T-cell leukemia (ATL) is a severe, chemotherapy-resistant malignancy caused by chronic HTLV-I infection and associated with dismal long-term prognosis. The HTLV-I oncoprotein Tax initiates ATL in transgenic mice. We have previously shown that arsenic trioxide and interferon-α (IFN) combination, known to trigger proteasome mediated Tax proteolysis and apoptosis in HTLV-I transformed or fresh ATL cells, cures Tax-driven ATL in mice. Unexpectedly, this combination therapy of primary donor mice abrogated leukemia initiating cells (LIC) engraftment into untreated secondary recipients, whereas the primary tumor bulk still grew in the primary hosts, only to ultimately abate later on. This loss of initial transplantability required proteasomal function. Furthermore, these preclinical results led to very promising improvement in ATL patients when arsenic was added to zidovudine and IFN. To decipher the molecular basis of LIC eradication in the murine ATL Tax transgenic model, primary ATL donor mice were treated with arsenic/IFN for 3 days starting on day 18 post-leukemic cells inoculation and sacrificed at day 21. Spleen derived ATL cells were intraperitoneally injected into secondary recipient SCID mice that were left untreated and were sacrificed on a weekly basis. Whereas, secondary mice derived from untreated primary succumbed from ATL between 3 and 4 weeks after transplantation, we observed an unprecedented biphasic growth in secondary mice transplanted from arsenic/interferon treated primary mice. Indeed, the tumor increased progressively until week 5 with organ infiltration and leukocytosis, followed by a spontaneous and dramatic tumor regression for 2 weeks before a second phase of tumor growth that culminates in death from ATL at around 9-10 weeks. ATL cells derived from the initial phase of growth (week 4) were healthy, TUNEL negative and showed a normal capacity in inducing ATL in tertiary hosts. Strikingly, ATL cells derived from the second phase of growth (weeks 7 to 9) showed a progressive increase in p53 expression and TUNEL positivity reaching more than 50% at week 9, and lost their capacity to induce ATL in tertiary hosts. Finally, gene expression profile revealed an arsenic/interferon signature that was similar at Week 4 and Week 8 as well as a specific signature at Week 8 concomitant with the loss of LIC activity. These results represent a unique and novel model of studying treatment-induced LIC eradication.

Abstract 1227: Differential efficacy of p53 restoration in induction and maintenance of senescence in premalignant and malignant cells

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Restoration of p53 in tumors has been suggested as a possible therapeutic approach, based on preclinical in vivo and in vitro evidence of possible efficacy. However, the relationship between the timing of p53 restoration and its efficacy is still unclear. We now show that restoration of p53 in murine pre-malignant proliferating pineal tumors results in effective cell cycle exit and induction of cellular senescence, while p53 reactivation in established malignant pineal tumors does not impact cell proliferation, unless paired with DNA damaging therapies. This differential effect was not related to levels of p19Arf expression, as its expression was more pronounced in malignant tumors, nor to MAPK pathway activity. Interestingly, in premalignant lesions induced to senesce by p53 restoration, inactivation of p53 after senescence resulted in re-entry into the cell cycle, and rapid tumor progression. These findings were also validated in cell culture models of Cyclin D1-induced and RasV12-induced senescence in murine pineal cells and mouse embryo fibroblasts, respectively. Evaluation of a panel of human supratentorial primitive neuroectodermal tumors (sPNET), of which pineoblastoma is a subtype, showed low activity of the p53 pathway, while only one of 6 tumors had p53 deletion or mutation. Together, this data shows that restoration of the p53 pathway has different effects in premalignant versus invasive pineal tumors, where it may need to be paired with DNA damaging agents, to engage it in tumor suppression. Furthermore, p53 activation may need to be continually sustained for effective tumor suppression in such lesions undergoing senescence as a tumor suppressor mechanism. Finally, p53 restoration approaches may be worth exploring in sPNET, where the p53 gene seems to be intact but the pathway inactive in the majority of examined tumors. Citation Format: Mohammad Harajly, Hassan Zalzali, Sandra Ghayad, Farrah Ghamloush, Mark Jabbour, Hussein Basma, Ayman Tawil, Zafar Nawaz, Raya H. Saab. Differential efficacy of p53 restoration in induction and maintenance of senescence in premalignant and malignant cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1227. doi:10.1158/1538-7445.AM2015-1227