Hemanth Tummala

Poly(A)-specific ribonuclease deficiency impacts telomere biology and causes dyskeratosis congenita

Dyskeratosis congenita (DC) and related syndromes are inherited, life-threatening bone marrow (BM) failure disorders, and approximately 40% of cases are currently uncharacterized at the genetic level. Here, using whole exome sequencing (WES), we have identified biallelic mutations in the gene encoding poly(A)-specific ribonuclease (PARN) in 3 families with individuals exhibiting severe DC. PARN is an extensively characterized exonuclease with deadenylation activity that controls mRNA stability in part and therefore regulates expression of a large number of genes. The DC-associated mutations identified affect key domains within the protein, and evaluation of patient cells revealed reduced deadenylation activity. This deadenylation deficiency caused an early DNA damage response in terms of nuclear p53 regulation, cell-cycle arrest, and reduced cell viability upon UV treatment. Individuals with biallelic PARN mutations and PARN-depleted cells exhibited reduced RNA levels for several key genes that are associated with telomere biology, specifically TERC, DKC1, RTEL1, and TERF1. Moreover, PARN-deficient cells also possessed critically short telomeres. Collectively, these results identify a role for PARN in telomere maintenance and demonstrate that it is a disease-causing gene in a subset of patients with severe DC.

DNAJC21 Mutations Link a Cancer-Prone Bone Marrow Failure Syndrome to Corruption in 60S Ribosome Subunit Maturation

A substantial number of individuals with bone marrow failure (BMF) present with one or more extra-hematopoietic abnormality. This suggests a constitutional or inherited basis, and yet many of them do not fit the diagnostic criteria of the known BMF syndromes. Through exome sequencing, we have now identified a subgroup of these individuals, defined by germline biallelic mutations in DNAJC21 (DNAJ homolog subfamily C member 21). They present with global BMF, and one individual developed a hematological cancer (acute myeloid leukemia) in childhood. We show that the encoded protein associates with rRNA and plays a highly conserved role in the maturation of the 60S ribosomal subunit. Lymphoblastoid cells obtained from an affected individual exhibit increased sensitivity to the transcriptional inhibitor actinomycin D and reduced amounts of rRNA. Characterization of mutations revealed impairment in interactions with cofactors (PA2G4, HSPA8, and ZNF622) involved in 60S maturation. DNAJC21 deficiency resulted in cytoplasmic accumulation of the 60S nuclear export factor PA2G4, aberrant ribosome profiles, and increased cell death. Collectively, these findings demonstrate that mutations in DNAJC21 cause a cancer-prone BMF syndrome due to corruption of early nuclear rRNA biogenesis and late cytoplasmic maturation of the 60S subunit.

Triallelic and epigenetic-like inheritance in human disorders of telomerase

Dyskeratosis congenita (DC) and related diseases are a heterogeneous group of disorders characterized by impaired telomere maintenance, known collectively as the telomeropathies. Disease-causing variants have been identified in 10 telomere-related genes including the reverse transcriptase (TERT) and the RNA component (TERC) of the telomerase complex. Variants in TERC and TERT can impede telomere elongation causing stem cells to enter premature replicative senescence and/or apoptosis as telomeres become critically short. This explains the major impact of the disease on highly proliferative tissues such as the bone marrow and skin. However, telomerase variants are not always fully penetrant and in some families disease-causing variants are seen in asymptomatic family members. As a result, determining the pathogenic status of newly identified variants in TERC or TERT can be quite challenging. Over a 3-year period, we have identified 26 telomerase variants (16 of which are novel) in 23 families. Additional investigations (including family segregation and functional studies) enabled these to be categorized into 3 groups: (1) disease-causing (n = 15), (2) uncertain status (n = 6), and (3) bystanders (n = 5). Remarkably, this process has also enabled us to identify families with novel mechanisms of inheriting human telomeropathies. These include triallelic mutations, involving 2 different telomerase genes, and an epigenetic-like inheritance of short telomeres in the absence of a telomerase mutation. This study therefore highlights that telomerase variants have highly variable functional and clinical manifestations and require thorough investigation to assess their pathogenic contribution.

Germline heterozygous DDX41 variants in a subset of familial myelodysplasia and acute myeloid leukemia

Germline heterozygous DDX41 variants in a subset of familial myelodysplasia and acute myeloid leukemia

ERCC6L2 Mutations Link a Distinct Bone-Marrow-Failure Syndrome to DNA Repair and Mitochondrial Function

Exome sequencing was performed in three index cases with bone marrow failure and neurological dysfunction and whose parents are first-degree cousins. Homozygous truncating mutations were identified in ERCC6L2 in two of the individuals. Both of these mutations affect the subcellular localization and stability of ERCC6L2. We show here that knockdown of ERCC6L2 in human A549 cells significantly reduced their viability upon exposure to the DNA-damaging agents mitomycin C and Irofulven, but not etoposide and camptothecin, suggesting a role in nucleotide excision repair. ERCC6L2-knockdown cells also displayed H2AX phosphorylation, which significantly increased upon genotoxic stress, suggesting an early DNA-damage response. Intriguingly, ERCC6L2 was seen to translocate to the mitochondria and the nucleus in response to DNA damage, and ERCC6L2 knockdown induced intracellular reactive oxygen species (ROS). Treatment with the ROS scavenger N-acetyl cysteine attenuated the Irofulven-induced cytotoxicity in ERCC6L2-knockdown cells and abolished ERCCGL2 traffic to the mitochondria and nucleus in response to this DNA-damaging agent. Collectively, these observations identify a distinct bone-marrow-failure syndrome due to mutations in ERCC6L2, a gene implicated in DNA repair and mitochondrial function.

Pharmacological inhibition of ATM by KU55933 stimulates ATM transcription

Ataxia-telangiectasia mutated (ATM) kinase is a component of a signalling mechanism that determines the process of decision-making in response to DNA damage and involves the participation of multiple proteins. ATM is activated by DNA double-strand breaks (DSBs) through the Mre11–Rad50–Nbs1 (MRN) DNA repair complex, and orchestrates signalling cascades that initiate the DNA damage response. Cells lacking ATM are hypersensitive to insults, particularly genotoxic stress, induced through radiation or radiomimetic drugs. Here, we investigate the degree of ATM activation during time-dependent treatment with genotoxic agents and the effects of ATM on phospho-induction and localization of its downstream substrates. Additionally, we have demonstrated a new cell-cycle-independent mechanism of ATM gene regulation following ATM kinase inhibition with KU5593. Inhibition of ATM activity causes induction of ATM protein followed by oscillation and this mechanism is governed at the transcriptional level. Furthermore, this autoregulatory induction of ATM is also accompanied by a transient upregulation of p53, pATR and E2F1 levels. Since ATM inhibition is believed to sensitize cancer cells to genotoxic agents, this novel insight into the mechanism of ATM regulation might be useful for designing more precise strategies for modulation of ATM activity in cancer therapy.

Rapid dendritic and axonal responses to neuronal insults.

Glutamate is the major excitatory neurotransmitter in the mammalian central nervous system playing critical roles in basal synaptic transmission and mechanisms of learning and memory. Under normal conditions, glutamate is sequestered within synaptic vesicles (approximately 100 mM) with extracellular glutamate concentrations being limited (<1 microM), via retrieval by plasma-membrane transporters on neuronal and glial cells. In the case of central nervous system trauma, stroke, epilepsy, and in certain neurodegenerative diseases, increased concentrations of extracellular glutamate (by vesicular release, cell lysis and/or decreased glutamate transporter uptake/reversal) stimulate the overactivation of local ionotropic glutamate receptors that trigger neuronal cell death (excitotoxicity). Other natural agonists, such as domoic acid, alcohol and auto-antibodies, have also been reported to induce excitotoxicity.

The D153del Mutation in GNB3 Gene Causes Tissue Specific Signalling Patterns and an Abnormal Renal Morphology in Rge Chickens

Background The GNB3 gene is expressed in cone but not rod photoreceptors of vertebrates, where it acts as the β transducin subunit in the colour visual transduction process. A naturally occurring mutation ‘D153del’ in the GNB3 gene causes the recessively inherited blinding phenotype retinopathy globe enlarged (rge) disease in chickens. GNB3 is however also expressed in most other vertebrate tissues suggesting that the D153del mutation may exert pathological effects that outlie from eye. Principal Findings Recombinant studies in COS-7 cells that were transfected with normal and mutant recombinant GNB3 constructs and subjected to cycloheximide chase showed that the mutant GNB3d protein had a much shorter half life compared to normal GNB3. GNB3 codes for the Gβ3 protein subunit that, together with different Gγ and Gα subunits, activates and regulates phosphorylation cascades in different tissues. As expected, the relative levels of cGMP and cAMP secondary messengers and their activated kinases such as MAPK, AKT and GRK2 were also found to be altered significantly in a tissue specific manner in rge chickens. Histochemical analysis on kidney tissue sections, from rge homozygous affected chickens, showed the chickens had enlargement of the glomerular capsule, causing glomerulomegaly and tubulointerstitial inflammation whereas other tissues (brain, heart, liver, pancreas) were unaffected. Significance These findings confirm that the D153del mutation in GNB3 gene targets GNB3 protein to early degradation. Lack of GNB3 signalling causes reduced phosphorylation activity of ERK2 and AKT leading to severe pathological phenotypes such as blindness and renal abnormalities in rge chickens.

Marked overlap of four genetic syndromes with dyskeratosis congenita confounds clinical diagnosis

Dyskeratosis congenita is a highly pleotropic genetic disorder. This heterogeneity can lead to difficulties in making an accurate diagnosis and delays in appropriate management. The aim of this study was to determine the underlying genetic basis in patients presenting with features of dyskeratosis congenita and who were negative for mutations in the classical dyskeratosis congenita genes. By whole exome and targeted sequencing, we identified biallelic variants in genes that are not associated with dyskeratosis congenita in 17 individuals from 12 families. Specifically, these were homozygous variants in USB1 (8 families), homozygous missense variants in GRHL2 (2 families) and identical compound heterozygous variants in LIG4 (2 families). All patients had multiple somatic features of dyskeratosis congenita but not the characteristic short telomeres. Our case series shows that biallelic variants in USB1, LIG4 and GRHL2, the genes mutated in poikiloderma with neutropenia, LIG4/Dubowitz syndrome and the recently recognized ectodermal dysplasia/short stature syndrome, respectively, cause features that overlap with dyskeratosis congenita. Strikingly, these genes also overlap in their biological function with the known dyskeratosis congenita genes that are implicated in telomere maintenance and DNA repair pathways. Collectively, these observations demonstrate the marked overlap of dyskeratosis congenita with four other genetic syndromes, confounding accurate diagnosis and subsequent management. This has important implications for establishing a genetic diagnosis when a new patient presents in the clinic. Patients with clinical features of dyskeratosis congenita need to have genetic analysis of USB1, LIG4 and GRHL2 in addition to the classical dyskeratosis congenita genes and telomere length measurements.

Abstract 3103: Differences in the DDR enzymes activation kinetics between normal and cancer cells could be utilized to achieve targeted cellular sensitivity towards genotoxic agents

The DNA damage response (DDR) pathway is a complex signalling network that is activated when eukaryotic cells undergo DNA damage caused by exposure to genotoxic agents. Depending upon the type and scale of DNA damage, the DDR can either cause cell cycle arrest if the DNA lesions are repairable, or trigger apoptosis if the DNA lesions are beyond the repair capacity of the cell. A variety of enzymes take part in this complex signalling network that decides cellular fate after DNA damage, and is comprised of kinases, phosphatases and glycosylases. Here we explored the kinetics of activation of these key enzymes, the rates of their product formation and the interplay between them that impact the decision making process of the cell. We exposed normal (MCF10A, HaCat) and breast cancer cell lines (MCF7, MDA MB231, MDA MB468) to different combinations of radiomimetic drugs, UV radiation and enzyme inhibitors in a time and dose dependent manner and looked at the resulting enzyme activation rates of DDR kinases such as ATM, ATR, Chk1, Chk2, and DDR phosphatases PP2A and WIP1. We studied the effects of such enzymatic activation on their substrates P53, E2F1, BRCA1 and γ-H2AX and examined its outcome on cell survival. Firstly, we have identified key differences in enzyme activation kinetics that impacted the cell fate decision between normal and cancer cell lines. In addition we established treatment regimes based on those differences that bias the cell fate decision towards apoptosis in cancer cells and cell cycle arrest and repair of DNA in their normal counterparts. Thus, we have demonstrated a link between the rate of induction of different enzyme activities in the DDR pathway, the resulting product formation and cellular sensitivity to genotoxic agents. We have successfully used such information to devise a combinatorial drug treatment regime that is selective for cancer cells. Elucidation of the enzyme activation kinetics in a time and dose dependent manner of the proteins governing cell fate decision can be used to achieve targeted cellular selectivity and sensitivity with lesser side effects on normal cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3103. doi:1538-7445.AM2012-3103