Differences in peripheral neuropathy in xeroderma pigmentosum complementation groups A and D as evaluated by nerve conduction studies

Background Xeroderma pigmentosum (XP) is a rare autosomal recessive genetic disorder with defective DNA nucleotide excision repair and associated with a high frequency of skin cancer. Approximately 25% of patients develop progressive neurological degeneration. Complementation groups XP-A and XP-D are most frequently associated with neurological disorders. Design/methods This is a retrospective review of patients with XP who were evaluated at NIH from 1986 to 2015 and had nerve conduction studies (NCS). In the complementation groups with peripheral neuropathy, further comparisons of the NCS were made with audiological, brain imaging, neuropsychological assessments that were also performed on most of the patients. Limited neuropathology of XP-A and XP-D patients were examined.. Results The 33 patients had NCS: XP-A (9 patients), XP-C (7 patients), XP-D (10 patients), XP-E (1 patient), XP-V (4 patients), and XP-unknown (2 patients). Peripheral neuropathy based on nerve conduction studies was documented only in two complementation groups: 78% (7/9) of XP-A patients had a sensorimotor neuropathy while 50% (5/10) of XP-D patients had a sensory neuropathy only. Analysis of sural sensory nerve amplitude in both complementation groups XP-A and XP-D correlated with sensorineural hearing loss (SNHL), MRI/CT severity, and Full-scale Intelligence Quotient (IQ). Analysis of fibular motor nerve amplitude in complementation XP-A correlated with SNHL and MRI/CT severity. Limited follow-up studies showed gradual loss of NCS responses compared to an earlier and more rapid progression of the hearing loss. Conclusions Despite similar brain imaging and audiological findings patients, XP-A and XP-D complementation groups differ in the type of neuropathy, sensorimotor versus sensory alone. A few cases suggest that sensorineural hearing loss may precede abnormal NCS in XP and therefore serve as valuable clinical indicators of XP patients that will later develop peripheral neuropathy.

A genetic screen in Drosophila uncovers the multifaceted properties of the NUP98-HOXA9 oncogene

Acute myeloid leukemia (AML) underlies the uncontrolled accumulation of immature myeloid blasts. Several cytogenetic abnormalities have been associated with AML. Among these is the NUP98-HOXA9 (NA9) translocation that fuses the Phe-Gly repeats of nucleoporin NUP98 to the homeodomain of the transcription factor HOXA9. The mechanisms enabling NA9-induced leukemia are poorly understood. Here, we conducted a genetic screen in Drosophila for modifiers of NA9. The screen uncovered 29 complementation groups, including genes with mammalian homologs known to impinge on NA9 activity. Markedly, the modifiers encompassed a diversity of functional categories, suggesting that NA9 perturbs multiple intracellular events. Unexpectedly, we discovered that NA9 promotes cell fate transdetermination and that this phenomenon is greatly influenced by NA9 modifiers involved in epigenetic regulation. Together, our work reveals a network of genes functionally connected to NA9 that not only provides insights into its mechanism of action, but also represents potential therapeutic targets.

Differences in Peripheral Neuropathy in Xeroderma Pigmentosum Complementation Groups A and D

BACKGROUNDXeroderma pigmentosum (XP) is a rare autosomal recessive genetic disorder with defective DNA nucleotide excision repair and associated with a high frequency of skin cancer. Approximately 25% of patients develop progressive neurological degeneration. Complementation groups XP-A and XP-D are most frequently associated with neurological disorders. METHODSThis is a retrospective review of patients with XP who were evaluated at NIH from 1986 to 2015 and had nerve conduction studies (NCS). Audiological, brain imaging, neuropsychological assessments were also performed on most of the patients. Limited neuropathology of XP-A and XP-D patients were compared. RESULTSThe 33 patients had NCS: XP-A (9 patients), XP-C (7 patients), XP-D (10 patients), XP-E (1 patient), XP-V (4 patients), and XP-unknown (2 patients). Peripheral neuropathy was documented only in two complementation groups: 78% (7/9) of XP-A patients had a sensorimotor neuropathy while 50% (5/10) of XP-D patients had a sensory neuropathy only. The severity of the neuropathy correlated with hearing loss. CONCLUSIONSDespite similar brain imaging and audiological findings patients, XP-A and XP-D complementation groups differ in the type of neuropathy, sensorimotor versus sensory alone. A few cases suggest that sensorineural hearing loss and absent deep tendon reflexes may precede abnormal NCS in XP and therefore serve as valuable clinical indicators of XP patients that will later develop peripheral neuropathy. TRIAL REGISTRATION: Patients were evaluated under clinical protocols, NCT00001813 and NCT00046189, approved by the NIH Institutional Review Boards.

NHEJ-Mediated Gene Editing, a Versatile Approach to Correct a Variety of Fanconi Anemia Genes in HSCs

Allogeneic hematopoietic stem cell (HSC) transplantation is currently the only curative treatment for the bone marrow failure in Fanconi anemia (FA) patients. However, recent advances in lentiviral-mediated gene therapy have shown that corrected FA HSCs develop an in vivo proliferation advantage, facilitating the engraftment of corrected HSCs in non-conditioned FA patients. Based on these observations, we proposed that gene editing might constitute a promising alternative to correct patients' hematopoietic stem and progenitor cells (HSPCs) in this disorder. Since non-homologous end joining (NHEJ) is the most frequent repair pathway in HSCs, particularly in FA-HSCs, we aimed at exploiting this DNA repair mechanism to remove/compensate specific mutations in different FANC genes by the use of CRISPR/Cas9 system, thus mimicking spontaneous genetic reversions observed in FA mosaic patients. Our results in lymphoblastic cell lines from five different complementation groups (FANCA, FANCB, FANCC, FANCD2 and FANCD1/BRCA2) demonstrated the efficiency of this approach to generate potentially corrective events in all the different complementation groups studied. Importantly, corrected cells showed a marked proliferative advantage after in vitro culture and the analysis by next generation sequencing confirmed the expansion of cells harboring therapeutic events. Functional studies showing the reversion of mitomycin C sensitivity, FANCD2 foci formation and chromosomal instability supported the phenotypic correction of different mutations by NHEJ-mediated gene editing. Moving towards the clinical application of NHEJ-mediated repair we focused on improving the gene editing efficiency in HSCs. To this aim, chemically modified small guide RNAs (MS-sgRNAs) enabled us to increase the editing efficacy 8-fold compared to efficacies obtained with in vitro transcribed sgRNAs, reaching up to 89% indels in healthy donor hematopoietic stem/progenitor cells. Moreover, the CRISPR/Cas9 system demonstrated high editing capacity in the primitive HSCs capable of engrafting immunodeficient NSG mice, confirming the efficacy of NHEJ-editing to correct the phenotype of long-term repopulating HSCs. Finally, studies conducted in mobilized peripheral blood and bone marrow CD34+ cells from FA patients demonstrated the feasibility to correct FA HSCs by NHEJ-mediated gene editing and confirmed the proliferative advantage of NHEJ-mediated corrected cells both in vitro and in vivo. Our results suggest that NHEJ-mediated gene editing should constitute a versatile and simple therapeutic approach to efficiently correct specific mutations in FA and other monogenic disorders of the hematopoietic system. Disclosures Sevilla: Rocket Pharmaceuticals, Inc.: Honoraria, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents; NOVARTIS: Honoraria, Membership on an entity's Board of Directors or advisory committees; Rocket: Membership on an entity's Board of Directors or advisory committees; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sobi: Membership on an entity's Board of Directors or advisory committees; Miltenyi Biotech: Honoraria. Bueren:Rocket Pharmaceuticals, Inc.: Consultancy, Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding. Rio:Rocket Pharmaceuticals, Inc.: Equity Ownership, Patents & Royalties: Inventor on patents on lentiviral vectors filled by CIEMAT, CIBERER and F.J.D and may be entitled to receive financial benefits from the licensing of such patents, Research Funding.

Characterization of Pathogenic Variants and Clinical Phenotypes in 117 Japanese Fanconi Anemia Patients

Objective: Fanconi anemia (FA) is the most common inherited bone marrow failure syndrome associated with multiple congenital abnormalities and predisposition to malignancies, resulting from mutations in one of the 22 known FA genes (FANCA to W). The proteins encoded by these genes participate in DNA repair pathway (the FA pathway) for endogenous aldehyde damage. Compared to the situation in the US or Europe, the number of Japanese FA patients with genetic diagnosis was relatively limited. In this study, we reveal the genetic subtyping and the characteristics of mutated FA genes in Japanese population and clarify the genotype-phenotype correlations. Results: We studied 117 Japanese FA patients from 103 families (1996 to 2018). The diagnosis of FA was confirmed on the basis of chromosomal breakage tests and clinical features. Molecular diagnosis was obtained in 107 (91.5%) of the 117 patients through direct sequencing of FANCA and FANCG, MLPA analysis for FANCA, targeted exome sequencing (targeted-seq), and whole exome sequencing (WES) analysis (Figure 1). To provide genetic subtyping for the 10 unclassified cases, we tried to apply various technologies. Array CGH revealed large deletions in two FA-B and one FA-T cases. Whole genome sequencing and RNA-sequencing analysis identified splicing site or aberrant splicing mutations among three cases (one FA-B, one FA-C, and one FA-N). Collectively, 113 (97%) of Japanese 117 FA patients were successfully subtyped and a total of 219 mutated alleles were identified. FA-A and FA-G accounted for the disease in 58% and 25% of FA patients, respectively, whereas each of the other complementation groups accounted for less than 5% of FA cases. FANCB was the third most common complementation group (n=4) and only one FA-C case was identified in Japanese FA patients. In the 68 FA-A patients, we identified 130 mutant alleles that included 55 different FANCA variants (17 nucleotide substitutions, 16 small deletions/insertions, 12 large deletions, 1 large duplication and 9 splice site mutation). FANCA c.2546delC was the most prevalent (41/130 alleles; 32%). In the 29 FA-G patients, 57 mutant alleles were identified and seven different FANCG variants were detected. FANCG c.307+1G>C and 1066C>T accounted for most of FANCG mutant alleles (49/57; 88%) in the Japanese FA-G patients. The three hotspot mutations (FANCA c.2546delC, FANCG c.307+1G>C and c.1066C>T) existed at low prevalence (0.04-0.1%) in the whole-genome reference panel of 3554 Japanese individuals (3.5KJPN, Tohoku Megabank). Consistent with the paucity of the FA-C patients as opposed to the previous report (Blood 2000), the FANCC IVS4+4A mutation was absent in the 3.5KJPN database. We were able to examine the hematological outcomes in a subset of our cases (52 FA-A and 23 FA-G). Interestingly, the FA-G patients developed bone marrow failure (BMF) at a significantly younger age than FA-A patients (median age at onset of BMF: 3.1 years vs 5 years). Furthermore, the patients with the FANCA c.2546delC mutation had an increased risk of developing myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), compared to FA-A patients without the mutation. In the rare complementation groups of FA, two FA-B cases with complete loss of FANCB gene and one FA-I patient with N-terminal premature termination codons revealed severe somatic abnormalities, consistent with VACTERL-H association. Two FANCD1 (BRCA2) patients and one FANCN (PALB2) patients did not experience bone marrow failure but developed early-onset malignancies (immature teratoma, T-lymphoblastic lymphoma, adenosquamous lung carcinoma, Wilms tumor). Conclusion: This is the largest series of subtyped Japanese FA patients to date and the results would be useful for future clinical management. To provide molecular diagnosis for FA in Japan, we suggest to start with PCR-direct sequencing of the three common mutations (FANCA c.2546delC, FANCG c.307+1G>C and FANCG c.1066C>T) along with MLPA assay for FANCA. These analyses would enable the identification of about 50% of the mutant alleles. For the rest of the cases, WES or targeted-seq analysis should be useful, however, large deletions and aberrant splicing need to be kept in mind. Disclosures Takaori-Kondo: Pfizer: Honoraria; Novartis: Honoraria; Celgene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Janssen Pharmaceuticals: Honoraria.

Genetic Identification of Novel Separase regulators in Caenorhabditis elegans

ABSTRACTSeparase is a highly conserved protease required for chromosome segregation. Although observations that separase also regulates membrane trafficking events have been made, it is still not clear how separase achieves this function. Here we present an extensive ENU mutagenesis suppressor screen aimed at identifying suppressors of sep-1(e2406), a temperature sensitive maternal effect embryonic lethal separase mutant. We screened nearly a million haploid genomes, and isolated sixty-eight suppressed lines. We identified fourteen independent intragenic sep-1(e2406) suppressed lines. These intragenic alleles map to seven SEP-1 residues within the N-terminus, compensating for the original mutation within the poorly conserved N-terminal domain. Interestingly, 47 of the suppressed lines have novel mutations throughout the entire coding region of the pph-5 phosphatase, indicating that this is an important regulator of separase. We also found that a mutation near the MEEVD motif of HSP-90, which binds and activates PPH-5, also rescues sep-1(e2406) mutants. Finally, we identified six potentially novel suppressor lines that fall into five complementation groups. These new alleles provide the opportunity to more exhaustively investigate the regulation and function of separase.

Phosphorylated HBO1 at UV irradiated sites is essential for nucleotide excision repair

HBO1, a histone acetyl transferase, is a co-activator of DNA pre-replication complex formation. We recently reported that HBO1 is phosphorylated by ATM and/or ATR and binds to DDB2 after ultraviolet irradiation. Here, we show that phosphorylated HBO1 at cyclobutane pyrimidine dimer (CPD) sites mediates histone acetylation to facilitate recruitment of XPC at the damaged DNA sites. Furthermore, HBO1 facilitates accumulation of SNF2H and ACF1, an ATP-dependent chromatin remodelling complex, to CPD sites. Depletion of HBO1 inhibited repair of CPDs and sensitized cells to ultraviolet irradiation. However, depletion of HBO1 in cells derived from xeroderma pigmentosum patient complementation groups, XPE, XPC and XPA, did not lead to additional sensitivity towards ultraviolet irradiation. Our findings suggest that HBO1 acts in concert with SNF2H–ACF1 to make the chromosome structure more accessible to canonical nucleotide excision repair factors.

Genetic Diversity, Virulence, and Meloidogyne incognita Interactions of Fusarium oxysporum Isolates Causing Cotton Wilt in Georgia

Locally severe outbreaks of Fusarium wilt of cotton (Gossypium spp.) in South Georgia raised concerns about the genotypes of the causal pathogen, Fusarium oxysporum f. sp. vasinfectum. Vegetative complementation tests and DNA sequence analysis were used to determine genetic diversity among 492 F. oxysporum f. sp. vasinfectum isolates obtained from 107 wilted plants collected from seven fields in five counties. Eight vegetative complementation groups (VCG) were found, with VCG 01117B and VCG 01121 occurring in 66% of the infected plants. The newly recognized VCG 01121 was the major VCG in Berrien County, the center of the outbreaks. All eight VCG resulted in significant increases in the percentages of wilted leaves (27 to 53%) and significant reductions in leaf weight (40 to 67%) and shoot weight (33 to 60%) after being stem punctured into Gossypium hirsutum ‘Rowden’. They caused little or no significant reductions in shoot weight and height or increases in foliar symptoms and vascular browning in a soil-infestation assay. Soil infestation with Meloidogyne incognita race 3 (root-knot nematode) alone also failed to cause significant disease. When coinoculated with M. incognita race 3, all VCG caused moderate to severe wilt. Therefore, the VCG identified in this study belong to the vascular-competent pathotype, and should pose similar threats to cotton cultivars in the presence of the root-knot nematode. Use of nematode-resistant cultivars, therefore, is probably the best approach to control the disease in Georgia.