CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells.

CRISPR-Cas9 is a promising technology for gene therapy. However, the ON-target genotoxicity of CRISPR-Cas9 nuclease due to DNA double-strand breaks has received little attention and is probably underestimated. Here we report that genome editing targeting globin genes induces megabase-scale losses of heterozygosity (LOH) from the globin CRISPR-Cas9 cut-site to the telomere (5.2 Mb). In established lines, CRISPR-Cas9 nuclease induces frequent terminal chromosome 11p truncations and rare copy-neutral LOH. In primary hematopoietic progenitor/stem cells, we detect 1.1% of clones (7/648) with acquired megabase LOH induced by CRISPR-Cas9. In-depth analysis by SNP-array reveals the presence of copy-neutral LOH. This leads to 11p15.5 partial uniparental disomy, comprising two Chr11p15.5 imprinting centers (H19/IGF2:IG-DMR/IC1 and KCNQ1OT1:TSS-DMR/IC2) and impacting H19 and IGF2 expression. While this genotoxicity is a safety concern for CRISPR clinical trials, it is also an opportunity to model copy-neutral-LOH for genetic diseases and cancers.

Identification of a novel alpha1-antitrypsin variant.

Alpha-1-antitrypsin deficiency (A1ATD) is a genetic condition caused by SERPINA1 mutations, which results into decreased protease inhibitor activity in the serum and predisposes to emphysema and/or to liver disease due to accumulation of the abnormal protein in the hepatic cells. In most cases the clinical manifestations of A1ATD are associated with PIZZ (p.Glu366Lys; p.Glu366Lys (p.Glu342Lys; p.Glu342Lys)) or PISZ (p.Glu288Val; p.Glu366Lys (p.Glu264Val; p.Glu342Lys)) genotype, less frequently, deficient or null alleles may be present in compound heterozygous or homozygous A1AT deficient patients. We report the identification of a novel alpha1-antitrypsin variant in a 64-year old woman presenting with dyspnea on exertion. Imaging revealed bilateral bronchiectasis associated with moderate panacinar emphysema. The pulmonary function tests (PFTs) were subnormal but hypoxemia was noticed and A1AT quantitative analysis revealed a severe deficiency. DNA sequencing showed compound heterozygosity for the PIZ variant and a novel missense variant p.Phe232Leu (p.Phe208Leu). No specific treatment was proposed since PFTs were within the normal range at this stage of the disease. Close follow-up of pulmonary and hepatic parameters was recommended.

Congenital erythropoietic porphyria: a single-observer clinical study of 29 cases.

BACKGROUND: Congenital erythropoietic porphyria (CEP) is an autosomal recessive cutaneous porphyria caused by decreased activity of uroporphyrinogen III synthase (UROS). Its predominant characteristics include bullous cutaneous photosensitivity to visible light from early infancy, progressive photomutilation and chronic haemolytic anaemia. Due to its rarity and genetic heterogeneity, clinical phenotypes are unclear and its impact on health-related quality of life (HRQoL) has not been previously assessed. OBJECTIVES: To define comprehensively CEP phenotypes and assess their impact on HRQoL, and to correlate these factors with laboratory parameters. METHODS: A single observer assessed patients with CEP from four European countries. RESULTS: Twenty-seven unrelated patients with CEP, aged between 7.6 and 65 years, participated in the study. The patients came from the U.K. (17), France (4), Switzerland (4) and Germany (2). Additional data were obtained for two deceased patients. Newly characterized features of CEP include acute-onset cutaneous and noncutaneous symptoms immediately following sunlight exposure, and pink erythematous facial papules. There was a lack of consistent genotype-phenotype correlation in CEP. The main poor prognostic factors in CEP are the early age of disease onset and haematological complications. CONCLUSIONS: CEP is a multisystem disease; cutaneous, ocular, oral and skeletal manifestations also contribute to disease severity and impact on HRQoL, in addition to the haematological complications. The rarity of the disease can lead to delayed diagnosis. The lack of consistent genotype-phenotype correlation in CEP suggests a contribution to phenotype from other factors, such as environment, patients' photoprotective behaviour and genes other than UROS. There is currently an unmet need for multidisciplinary management of patients with CEP.

A management algorithm for congenital erythropoietic porphyria derived from a study of 29 cases.

BACKGROUND: Congenital erythropoietic porphyria (CEP) is an autosomal recessive photomutilating porphyria with onset usually in childhood, where haematological complications determine prognosis. Due to its extreme rarity and clinical heterogeneity, management decisions in CEP are often difficult. OBJECTIVES: To develop a management algorithm for patients with CEP based on data from carefully characterized historical cases. METHODS: A single investigator collated data related to treatments and their outcomes in 29 patients with CEP from the U.K., France, Germany and Switzerland. RESULTS: Six children were treated with bone marrow transplantation (BMT); five have remained symptomatically cured up to 11.5 years post-transplantation. Treatments such as oral charcoal, splenectomy and chronic hypertransfusion were either of no benefit or were associated with complications and negative impact on health-related quality of life. Lack of consistent genotype-phenotype correlation meant that this could not be used to predict disease prognosis. The main poor prognostic factors were early age of disease onset and severity of haematological manifestations. CONCLUSIONS: A management algorithm is proposed where every patient, irrespective of disease severity at presentation, should receive a comprehensive, multidisciplinary clinical assessment and should then be reviewed at intervals based on their predicted prognosis, and the rate of onset of complications. A BMT should be considered in those with progressive, symptomatic haemolytic anaemia and/or thrombocytopenia. Uroporphyrinogen III synthase genotypes associated with poor prognosis would additionally justify consideration for a BMT. Rigorous photoprotection of the skin and eyes from visible light is essential in all patients.

Usefulness of a global clinical ichthyosis vulgaris scoring system for predicting common FLG null mutations in an adult caucasian population.

BACKGROUND: Loss of function FLG alleles were first identified as causative of ichthyosis vulgaris (IV) and were subsequently found to be major predisposing factors for atopic dermatitis (AD) and atopic disorders. OBJECTIVES: To identify independent factors associated with the clinical IV phenotype in adult caucasian patients with AD and to assess the performance of a global clinical severity score of IV in predicting common FLG null mutations. PATIENTS AND METHODS: This was a prospective study conducted from January 2007 to June 2008. Adult patients attending the department of dermatology with a diagnosis of AD with or without IV were eligible to participate. For each patient, five clinical signs of IV were scored from 0 to 3 - diffuse xerosis, hyperlinearity of palms, scales on legs, scalp desquamation and keratosis pilaris - and a global IV clinical severity score was derived (0-15). Age of onset of AD, SCORAD (SCORing of Atopic Dermatitis), family and personal history for other signs of atopy, and total immunoglobulin E were recorded. Genotyping was performed for R501X and 2282del4. Univariate and multivariate analysis for factors associated with AD or AD + IV were conducted. RESULTS: In univariate analysis, family history of atopy, global clinical severity scoring and 2282del4 FLG mutation were positively correlated with the AD + IV phenotype. Using multivariate analysis, SCORAD for AD (OR 0·94, P = 0·01) and global clinical severity scoring for AD + IV (OR 2·62, P < 0·0001) were found to be independent factors. CONCLUSIONS: The 2282del4 FLG mutation was confirmed as a good marker of early-onset disease. Moreover, our global clinical severity score yielded a good negative predictive value of common caucasian null FLG mutations.

A prospective study of filaggrin null mutations in keratoconus patients with or without atopic disorders.

BACKGROUND: Atopic dermatitis (AD) is significantly associated with keratoconus (KC). An inherited component for KC has been suggested. Filaggrin (FLG) mutations are a strong genetic risk factor for AD. Since filaggrin is also expressed in the corneal epithelium, we hypothesized a common aetiology for ichthyosis vulgaris (IV), AD and KC. OBJECTIVES: We examined the prevalence of AD and IV in a KC population. We also studied the expression of filaggrin in normal and KC cornea and analysed 2 prevalent loss-of-function FLG alleles (R501X and 2282del4) in a KC population. Finally we examined whether the population with KC and FLG mutations had specific clinical characteristics. RESULTS: Of 89 KC patients, 38 had current or a history of AD and/or IV. Five patients were carriers of at least 1 FLG mutant allele and had a clinical diagnosis of AD and IV with a severer KC. CONCLUSION: The low frequency of FLG mutations is surprising since 42.7% of our KC population had AD associated or not with IV; the expected frequency would have been 12-15%, based on our previous studies. Further studies are required to look at other possible FLG mutations or other candidate genes.

[Congenital erythropoeietic porphyria treated by haematopoietic stem cell allograft]. / Porphyrie érythropoïétique congénitale traitée par allogreffe de cellules souches hématopoïétiques.

BACKGROUND: Congenital erythropoietic porphyria (CEP) is a genodermatosis associated uroporphyrinogen III synthase deficit that results in porphyrin accumulation in various organs, particularly the skin. It is the most severe form of porphyria associated with haemolytic anaemia and cutaneous phototoxicity. We report a severe case of CEP treated by allogeneic bone marrow transplantation. CASE REPORT: A one-year-old child presented erythrodontia and scarring on exposed areas. The diagnosis of CEP was confirmed by the decline of uroporphyrinogen III synthase activity. Demonstration of p.Cys73Arg mutation confirmed the severity of the disease. Allogeneic bone marrow transplantation resulted in persistent resolution of clinical signs 25 months after grafting. DISCUSSION: Symptomatic treatment is ineffective in this serious disease associated with early mortality. 11 of the 13 patients treated by allogeneic hematopoietic stem cell graft, including our patient, continued to be asymptomatic an average of seven years after transplantation. CONCLUSION: This new case confirms the role of allogeneic hematopoietic stem cell grafting in the treatment of congenital erythropoietic porphyria.

Congenital erythropoietic porphyria: mutation update and correlations between genotype and phenotype.

High quality genotype/phenotype analysis is a difficult issue in rare genetic diseases such as congenital erythropoietic porphyria (CEP) or Günther's disease, a heme biosynthesis defect due to uroporphyrinogen III synthase deficiency. The historical background and the main phenotypic features of the disease are depicted together with an update of published mutants and genotype/phenotype correlations. General rules concerning the prediction of disease severity are drawn as a guide for patient management and therapeutic choices. The phenotypic heterogeneity of the disease is presented in relation with a likely influence of modifying factors, either genetic or acquired.

Catalase overexpression reduces UVB-induced apoptosis in a human xeroderma pigmentosum reconstructed epidermis.

Xeroderma pigmentosum type C (XPC) is a rare autosomal recessive disorder that occurs due to inactivation of the XPC protein, an important DNA damage recognition protein involved in DNA nucleotide excision repair (NER). This defect, which prevents removal of a wide array of direct and indirect DNA lesions, is associated with a decrease in catalase activity. To test the hypothesis of a novel photoprotective approach, we irradiated epidermis reconstructed with XPC human keratinocytes sustainably overexpressing lentivirus-mediated catalase enzyme. Following UVB irradiation, there was a marked decrease in sunburn cell formation, caspase-3 activation and p53 accumulation in human XPC-reconstructed epidermis overexpressing catalase. Moreover, XPC-reconstructed epidermis was more resistant to UVB-induced apoptosis than normal reconstructed epidermis. While not correcting the gene defect, indirect gene therapy using antioxidant enzymes may be of help in limiting photosensitivity in XPC and probably in other monogenic/polygenic photosensitive disorders characterized by ROS accumulation.

Protection of normal human reconstructed epidermis from UV by catalase overexpression.

Reactive oxygen species (ROS) generated by ultraviolet (UV) irradiation are counterbalanced by endogenous antioxidant systems. To test the hypothesis of a novel photoprotective approach, we irradiated epidermis reconstructed with normal human keratinocytes overexpressing sustainably lentivirus-mediated catalase (CAT), copper/zinc superoxide dismutase (CuZnSOD) or manganese superoxide dismutase (MnSOD) enzymes. We found that following UVB irradiation there was a marked decrease in sunburn cell formation, caspase-3 activation and p53 accumulation in human reconstructed epidermis overexpressing CAT. Moreover, UVA-induced hypertrophy and DNA oxidation (8-oxodeoxyguanosine) were decreased by CAT overexpression. These effects were not achieved by overexpression of CuZnSOD or MnSOD. In conclusion, vector-mediated CAT overexpression could be a promising photoprotective tool against deleterious effects of UV irradiation such skin cancer especially in monogenic/polygenic photosensitive disorders characterized by ROS accumulation.

A knock-in mouse model of congenital erythropoietic porphyria.

Congenital erythropoietic porphyria (CEP) is a recessive autosomal disorder characterized by a deficiency in uroporphyrinogen III synthase (UROS), the fourth enzyme of the heme biosynthetic pathway. The severity of the disease, the lack of specific treatment except for allogeneic bone marrow transplantation, and the knowledge of the molecular lesions are strong arguments for gene therapy. An animal model of CEP has been designed to evaluate the feasibility of retroviral gene transfer in hematopoietic stem cells. We have previously demonstrated that the knockout of the Uros gene is lethal in mice (Uros(del) model). This work describes the achievement of a knock-in model, which reproduces a mutation of the UROS gene responsible for a severe UROS deficiency in humans (P248Q missense mutant). Homozygous mice display erythrodontia, moderate photosensitivity, hepatosplenomegaly, and hemolytic anemia. Uroporphyrin (99% type I isomer) accumulates in urine. Total porphyrins are increased in erythrocytes and feces, while Uros enzymatic activity is below 1% of the normal level in the different tissues analyzed. These pathological findings closely mimic the CEP disease in humans and demonstrate that the Uros(mut248) mouse represents a suitable model of the human disease for pathophysiological, pharmaceutical, and therapeutic purposes.

Hematopoietic stem cell gene therapy of murine protoporphyria by methylguanine-DNA-methyltransferase-mediated in vivo drug selection.

Erythropoietic protoporphyria (EPP) is an inherited defect of the ferrochelatase (FECH) gene characterized by the accumulation of toxic protoporphyrin in the liver and bone marrow resulting in severe skin photosensitivity. We previously described successful gene therapy of an animal model of the disease with erythroid-specific lentiviral vectors in the absence of preselection of corrected cells. However, the high-level of gene transfer obtained in mice is not translatable to large animal models and humans if there is no selective advantage for genetically modified hematopoietic stem cells (HSCs) in vivo. We used bicistronic SIN-lentiviral vectors coexpressing EGFP or FECH and the G156A-mutated O6-methylguanine-DNA-methyltransferase (MGMT) gene, which allowed efficient in vivo selection of transduced HSCs after O6-benzylguanine and BCNU treatment. We demonstrate for the first time that the correction and in vivo expansion of deficient transduced HSC population can be obtained by this dual gene therapy, resulting in a progressive increase of normal RBCs in EPP mice and a complete correction of skin photosensitivity. Finally, we developed a novel bipromoter SIN-lentiviral vector with a constitutive expression of MGMT gene to allow the selection of HSCs and with an erythroid-specific expression of the FECH therapeutic gene.

Lentivirus-mediated gene transfer of uroporphyrinogen III synthase fully corrects the porphyric phenotype in human cells.

Congenital erythropoietic porphyria (CEP) is an inherited disease due to a deficiency in the uroporphyrinogen III synthase, the fourth enzyme of the heme biosynthesis pathway. It is characterized by accumulation of uroporphyrin I in the bone marrow, peripheral blood and other organs. The prognosis of CEP is poor, with death often occurring early in adult life. For severe transfusion-dependent cases, when allogeneic cell transplantation cannot be performed, the autografting of genetically modified primitive/stem cells may be the only alternative. In vitro gene transfer experiments have documented the feasibility of gene therapy via hematopoietic cells to treat this disease. In the present study lentiviral transduction of porphyric cell lines and primary CD34(+) cells with the therapeutic human uroporphyrinogen III synthase (UROS) cDNA resulted in both enzymatic and metabolic correction, as demonstrated by the increase in UROS activity and the suppression of porphyrin accumulation in transduced cells. Very high gene transfer efficiency (up to 90%) was achieved in both cell lines and CD34(+) cells without any selection. Expression of the transgene remained stable over long-term liquid culture. Furthermore, gene expression was maintained during in vitro erythroid differentiation of CD34(+) cells. Therefore the use of lentiviral vectors is promising for the future treatment of CEP patients by gene therapy.

Gene therapy of a mouse model of protoporphyria with a self-inactivating erythroid-specific lentiviral vector without preselection.

Successful treatment of blood disorders by gene therapy has several complications, one of which is the frequent lack of selective advantage of genetically corrected cells. Erythropoietic protoporphyria (EPP), caused by a ferrochelatase deficiency, is a good model of hematological genetic disorders with a lack of spontaneous in vivo selection. This disease is characterized by accumulation of protoporphyrin in red blood cells, bone marrow, and other organs, resulting in severe skin photosensitivity. Here we develop a self-inactivating lentiviral vector containing human ferrochelatase cDNA driven by the human ankyrin-1/beta-globin HS-40 chimeric erythroid promoter/enhancer. We collected bone marrow cells from EPP male donor mice for lentiviral transduction and injected them into lethally irradiated female EPP recipient mice. We observed a high transduction efficiency of hematopoietic stem cells resulting in effective gene therapy of primary and secondary recipient EPP mice without any selectable system. Skin photosensitivity was corrected for all secondary engrafted mice and was associated with specific ferrochelatase expression in the erythroid lineage. An erythroid-specific expression was sufficient to reverse most of the clinical and biological manifestations of the disease. This improvement in the efficiency of gene transfer with lentiviruses may contribute to the development of successful clinical protocols for erythropoietic diseases.