Kyu-Seon Oh

Cancer and neurologic degeneration in xeroderma pigmentosum: long term follow-up characterises the role of DNA repair

Background The frequency of cancer, neurologic degeneration and mortality in xeroderma pigmentosum (XP) patients with defective DNA repair was determined in a four decade natural history study. Methods All 106 XP patients admitted to the National Institutes of Health from 1971 to 2009 were evaluated from clinical records and follow-up. Results In the 65 per cent (n=69) of patients with skin cancer, non-melanoma skin cancer (NMSC) was increased 10 000-fold and melanoma was increased 2000-fold in patients under age 20. The 9 year median age at diagnosis of first non-melanoma skin cancer (NMSC) (n=64) was significantly younger than the 22 year median age at diagnosis of first melanoma (n=38)—a relative age reversal from the general population suggesting different mechanisms of carcinogenesis between NMSC and melanoma. XP patients with pronounced burning on minimal sun exposure (n=65) were less likely to develop skin cancer than those who did not. This may be related to the extreme sun protection they receive from an earlier age, decreasing their total ultraviolet exposure. Progressive neurologic degeneration was present in 24% (n=25) with 16/25 in complementation group XP-D. The most common causes of death were skin cancer (34%, n=10), neurologic degeneration (31%, n=9), and internal cancer (17%, n=5). The median age at death (29 years) in XP patients with neurodegeneration was significantly younger than those XP patients without neurodegeneration (37 years) (p=0.02). Conclusion This 39 year follow-up study of XP patients indicates a major role of DNA repair genes in the aetiology of skin cancer and neurologic degeneration.

Activation of ATM depends on chromatin interactions occurring before induction of DNA damage

Efficient and correct responses to double-stranded breaks (DSB) in chromosomal DNA are crucial for maintaining genomic stability and preventing chromosomal alterations that lead to cancer. The generation of DSB is associated with structural changes in chromatin and the activation of the protein kinase ataxia-telangiectasia mutated (ATM), a key regulator of the signalling network of the cellular response to DSB. The interrelationship between DSB-induced changes in chromatin architecture and the activation of ATM is unclear. Here we show that the nucleosome-binding protein HMGN1 modulates the interaction of ATM with chromatin both before and after DSB formation, thereby optimizing its activation. Loss of HMGN1 or ablation of its ability to bind to chromatin reduces the levels of ionizing radiation (IR)-induced ATM autophosphorylation and the activation of several ATM targets. IR treatments lead to a global increase in the acetylation of Lys 14 of histone H3 (H3K14) in an HMGN1-dependent manner and treatment of cells with histone deacetylase inhibitors bypasses the HMGN1 requirement for efficient ATM activation. Thus, by regulating the levels of histone modifications, HMGN1 affects ATM activation. Our studies identify a new mediator of ATM activation and demonstrate a direct link between the steady-state intranuclear organization of ATM and the kinetics of its activation after DNA damage.

UV-induced histone H2AX phosphorylation and DNA damage related proteins accumulate and persist in nucleotide excision repair-deficient XP-B cells

DNA double strand breaks (DSB) may be caused by ionizing radiation. In contrast, UV exposure forms dipyrimidine photoproducts and is not considered an inducer of DSB. We found that uniform or localized UV treatment induced phosphorylation of the DNA damage related (DDR) proteins H2AX, ATM and NBS1 and co-localization of γ-H2AX with the DDR proteins p-ATM, p-NBS1, Rad51 and FANCD2 that persisted for about 6h in normal human fibroblasts. This post-UV phosphorylation was observed in the absence of nucleotide excision repair (NER), since NER deficient XP-B cells (lacking functional XPB DNA repair helicase) and global genome repair-deficient rodent cells also showed phosphorylation and localization of these DDR proteins. Resolution of the DDR proteins was dependent on NER, since they persisted for 24h in the XP-B cells. In the normal and XP-B cells p53 and p21 was detected at 6h and 24h but Mdm2 was not induced in the XP-B cells. Post-UV induction of Wip1 phosphatase was detected in the normal cells but not in the XP-B cells. DNA DSB were detected with a neutral comet assay at 6h and 24h post-UV in the normal and XP-B cells. These results indicate that UV damage can activate the DDR pathway in the absence of NER. However, a later step in DNA damage processing involving induction of Wip1 and resolution of DDR proteins was not observed in the absence of NER.

XPC INITIATION CODON MUTATION IN XERODERMA PIGMENTOSUM PATIENTS WITH AND WITHOUT NEUROLOGICAL SYMPTOMS

Two unrelated xeroderma pigmentosum (XP) patients, with and without neurological abnormalities, respectively, had identical defects in the XPC DNA nucleotide excision repair (NER) gene. Patient XP21BE, a 27-year-old woman, had developmental delay and early onset of sensorineural hearing loss. In contrast, patient XP329BE, a 13-year-old boy, had a normal neurological examination. Both patients had marked lentiginous hyperpigmentation and multiple skin cancers at an early age. Their cultured fibroblasts showed similar hypersensitivity to killing by UV and reduced repair of DNA photoproducts. Cells from both patients had a homozygous c.2T>G mutation in the XPC gene which changed the ATG initiation codon to arginine (AGG). Both had low levels of XPC message and no detectable XPC protein on Western blotting. There was no functional XPC activity in both as revealed by the failure of localization of XPC and other NER proteins at the sites of UV-induced DNA damage in a sensitive in vivo immunofluorescence assay. XPC cDNA containing the initiation codon mutation was functionally inactive in a post-UV host cell reactivation (HCR) assay. Microsatellite markers flanking the XPC gene showed only a small region of identity ( approximately 30kBP), indicating that the patients were not closely related. Thus, the initiation codon mutation resulted in DNA repair deficiency in cells from both patients and greatly increased cancer susceptibility. The neurological abnormalities in patient XP21BE may be related to close consanguinity and simultaneous inheritance of other recessive genes or other gene modifying effects rather than the influence of XPC gene itself.

Ocular Manifestations of Trichothiodystrophy

OBJECTIVE Trichothiodystrophy (TTD) is a rare, autosomal recessive disorder characterized by sulfur-deficient brittle hair and multisystem abnormalities. Many TTD patients have a defect in known DNA repair genes. This report systematically evaluates the ocular manifestations of the largest-to-date cohort of TTD patients and xeroderma pigmentosum (XP)/TTD patients. DESIGN Case series. PARTICIPANTS Thirty-two participants, ages 1 to 30 years, referred to the National Eye Institute for examination from 2001 to 2010; 25 had TTD and 7 had XP/TTD. METHODS Complete, age- and developmental stage-appropriate ophthalmic examination. MAIN OUTCOME MEASURES Visual acuity (VA), best-corrected VA, ocular motility, state of the ocular surface and corneal endothelial cell density, corneal diameter, and lens assessment. RESULTS Developmental abnormalities included microcornea (44% TTD), microphthalmia (8% TTD, 14% XP/TTD), nystagmus (40% TTD), and infantile cataracts (56% TTD, 86% XP/TTD). Corrective lenses were required by 65% of the participants, and decreased best-corrected VA was present in 28% of TTD patients and 71% of XP/TTD patients. Degenerative changes included dry eye (32% TTD, 57% XP/TTD) and ocular surface disease identified by ocular surface staining with fluorescein (32% TTD) that usually are exhibited by much older patients in the general population. The 2 oldest TTD patients exhibited clinical signs of retinal/macular degeneration. Four XP/TTD patients presented with corneal neovascularization. CONCLUSIONS These TTD and XP/TTD study participants had a wide variety of ocular findings including refractive error, infantile cataracts, microcornea, nystagmus, and dry eye/ocular surface disease. Although many of these can be ascribed to abnormal development--likely owing to abnormalities in basal transcription of critical genes--patients may also have a degenerative course. FINANCIAL DISCLOSURE(S) Proprietary or commercial disclosures may be found after the references.

Nucleotide Excision Repair Proteins Rapidly Accumulate but Fail to Persist in Human XP‐E (DDB2 Mutant) Cells

The xeroderma pigmentosum (XP‐E) DNA damage binding protein (DDB2) is involved in early recognition of global genome DNA damage during DNA nucleotide excision repair (NER). We found that skin fibroblasts from four newly reported XP‐E patients with numerous skin cancers and DDB2 mutations had slow repair of 6‐4 photoproducts (6‐4PP) and markedly reduced repair of cyclobutane pyrimidine dimers (CPD). NER proteins (XPC, XPB, XPG, XPA and XPF) colocalized to CPD and 6‐4PP positive regions immediately (<0.1 h) after localized UV irradiation in cells from the XP‐E patients and normal controls. While these proteins persist in normal cells, surprisingly, within 0.5 h these repair proteins were no longer detectable at the sites of DNA damage in XP‐E cells. Our results indicate that DDB2 is not required for the rapid recruitment of NER proteins to sites of UV photoproducts or for partial repair of 6‐4PP but is essential for normal persistence of these proteins for CPD photoproduct removal.

Effect of mutations in XPD(ERCC2) on pregnancy and prenatal development in mothers of patients with trichothiodystrophy or xeroderma pigmentosum

The XPD(ERCC2) gene encodes a DNA helicase involved in DNA repair and transcription. Patients with mutations in XPD may have different autosomal recessive phenotypes including trichothiodystrophy (TTD) or xeroderma pigmentosum (XP). TTD patients have sulfur-deficient, brittle hair, short stature and developmental delay. In contrast, XP patients have freckle-like pigmentation and a greatly increased risk of sun-induced skin cancers. Mothers of TTD patients have been reported to have a high frequency of pregnancy and neonatal complications. We performed a molecular epidemiological study of 15 mothers of 17 TTD patients and 13 mothers of 17 XP patients, all with XPD mutations. We found that 94% (16/17) of the TTD pregnancies had pre-term delivery, pre-eclampsia, hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome, prematurity or low birth weight. None of the 17 XP pregnancies had these complications (P<0.001). As mutations in XPD may have differential effects on DNA repair and transcription, these observations should provide insights into the role of XPD in human pregnancy and fetal development.

Ancient origin of a Japanese xeroderma pigmentosum founder mutation

Xeroderma pigmentosum (XP (MIM278700)) is a rare autosomal recessive disorder [1–3]. XP patients have sun sensitivity, a 10,000-fold increased risk of skin cancer and defective DNA repair [4]. The frequency of XP in Japan is about 1:22,000 [5;6], which is much more common than in the US and Europe (about 1 per million) [2;6]. There are 8 XP DNA repair genes (XPA to XPG and XP variant). The XPA gene is the predominant XP gene in Japan and is defective in about 55% of Japanese XP patients [3]. These Japanese XP patients have a severe form of XP with progressive neurological degeneration [7]. XPA is located on chromosome 9q22.3 and codes for a 273 amino acid protein that is involved in nucleotide excision repair [8]. More than 90% of the mutant alleles in Japanese XP-A patients have the same G to C base change mutation [7–9]. This founder mutation at the 3′ splice acceptor site of intron 3 (IVS3-1G>C) results in no detectable protein production and markedly reduced DNA repair. Approximately 1% of the Japanese general population are heterozygous carriers of this mutation [5]. In order to estimate the age of the most recent common ancestor of this founder mutation in Japan, we used haplotype analysis. We studied DNA samples from XP-A patients who were homozygous for the founder mutation. Using Sanger sequencing, initially we measured 70 single nucleotide polymorphisms (SNP’s) that were located up to 2.5 MB upstream and 2.5 MB downstream of the mutation on chromosome 9 (Table 1) as indicated on the HapMap http://www.hapmap.org. We then selected SNP’s that were highly polymorphic in the Japanese population as indicated by the Japanese HapMap subjects. Table 1 Haplotype analysis of region surrounding Japanese XPA founder mutation on chromosome 9q22.3 To estimate the age of the most recent common ancestor of the XPA Japanese founder mutation, we used a likelihood-based method [10] that uses multilocus marker data. This method uses multilocus marker data to estimate the age of the most recent common ancestor of the mutation from a small number of patients. This method was originally tested through simulations and shown to be well suited to estimating the age of rare mutations. The basic assumption for the method is that the N affected individuals, all carrying the same mutation at disease locus D, descend from a common ancestor who introduced the mutation ngen generations ago. The likelihood was written as a function of the recombination fraction (θ) between D and each marker, ngen, and the mutation rate and allele frequencies at each marker locus. Twenty-five HapMap-based SNPs flanking D were genotyped for this analysis. Japanese HapMap subjectsa llele frequencies for these SNPs were between 50:50and 71.6:28.4. Since SNPs have a very low mutation rate, the mutation rate was fixed to 0 for this analysis. The closest short tandem repeat markers flanking D had a θ/Mb ratio of approximately 1, thus physical distance (Mb) between each marker and D was converted to θ using this ratio. ngen was estimated from the size of the haplotype shared by the N affected individuals on each side of D and 95% confidence intervals were computed. We received de-identified samples from XP-A patients in 39 Japanese families located throughout Japan except for the northern areas of Tohoku and Hokkaido. At NIH, DNA was extracted from 43 XP-A patients in the 39 families. Forty one XP-A patients in 37 of the families were confirmed to be homozygous for the XPA founder mutation (red column) (Table 1). DNA from 2 XP patients was found to be heterozygous for this splice mutation as well as heterozygous for nearby SNP loci and was not included in the analysis (data not shown). There were 3 affected siblings in one family (family 1) and 2 affected siblings in two other families (families 18 and 28) (Table 1 – first column, blue shading). Six of the families (families 32–37) had a history of consanguinity (4 were first cousins, 1 was second cousins and 1 unspecified) (Table 1 – first column, yellow shading). DNA was tested for 25 SNPs on chromosome 9 in the region of the XPA gene (Table 1 and data not shown). In families 1, 10 and 28 we tested DNA from multiple affected siblings. The siblings in each family had the same haplotypes. We found a small region of SNP homozygosity in all the XP patients extending about 150 kb upstream and 50 kb downstream from the mutation (bright green area). The size of this region is a reflection of the relationship among the patients: a large region indicates a close relationship and a small region indicates a more ancient relationship. Using the method described above, we determined the age of the most recent common ancestor of the XPA founder mutation to be 120 generations (95% CI, 71–205 generations). Assuming a 20-year generation interval this corresponds to 2400 years (95% CI, 1420–4100 years) or 3600 years (95% CI, 2130– 6150 years) based on a generation length of 30 years. The Japanese archipelago was completely separated from the Eurasian continent about 12,000 years ago. Thus this mutation occurred after this separation occurred and spread throughout the isolated Japanese population. XP is recessive and the carriers of this XPA founder mutation do not have overt clinical symptoms. All of the XP-A patients we studied were homozygous for the founder mutation indicating that both parents had the same mutation. However, only 6 of the families were aware of a close relationship between the parents of the affected XP patients. This study indicates that the common ancestor of the other 31 families could have occurred thousands of years ago. Since this mutation is present in about 1 million Japanese carriers [5], genetic counseling for this ancient founder mutation may be considered in the Japanese population.

Dual Roles for Ikaros in Regulation of Macrophage Chromatin State and Inflammatory Gene Expression

Macrophage activation by bacterial LPS leads to induction of a complex inflammatory gene program dependent on numerous transcription factor families. The transcription factor Ikaros has been shown to play a critical role in lymphoid cell development and differentiation; however, its function in myeloid cells and innate immune responses is less appreciated. Using comprehensive genomic analysis of Ikaros-dependent transcription, DNA binding, and chromatin accessibility, we describe unexpected dual repressor and activator functions for Ikaros in the LPS response of murine macrophages. Consistent with the described function of Ikaros as transcriptional repressor, Ikzf1−/− macrophages showed enhanced induction for select responses. In contrast, we observed a dramatic defect in expression of many delayed LPS response genes, and chromatin immunoprecipitation sequencing analyses support a key role for Ikaros in sustained NF-κB chromatin binding. Decreased Ikaros expression in Ikzf1+/− mice and human cells dampens these Ikaros-enhanced inflammatory responses, highlighting the importance of quantitative control of Ikaros protein level for its activator function. In the absence of Ikaros, a constitutively open chromatin state was coincident with dysregulation of LPS-induced chromatin remodeling, gene expression, and cytokine responses. Together, our data suggest a central role for Ikaros in coordinating the complex macrophage transcriptional program in response to pathogen challenge.