Increased CpG methylation at the CDH1 locus in inflamed ileal mucosa of patients with Crohn disease.

BACKGROUND: E-cadherin, a major actor of cell adhesion in the intestinal barrier, is encoded by the CDH1 gene associated with susceptibility to Crohn Disease (CD) and colorectal cancer. Since epigenetic mechanisms are suspected to contribute to the multifactorial pathogenesis of CD, we studied CpG methylation at the CDH1 locus. The methylation of the CpG island (CGI) and of the 1st enhancer, two critical regulatory positions, was quantified in surgical specimens of inflamed ileal mucosa and in peripheral blood mononuclear cells (PBMC) of 21 CD patients. Sixteen patients operated on for a non-inflammatory bowel disease, although not normal controls, provided a macroscopically normal ileal mucosa and PBMC for comparison. RESULTS: In ileal mucosa, 19/21 (90%) CD patients vs 8/16 control patients (50%) (p < 0.01) had a methylated CDH1 promoter CGI. In PBMC, CD patients with methylated CGI were 11/21 (52%) vs 7/16 controls (44%), respectively. Methylation in the 1st enhancer of CDH1 was also higher in the CD group for each of the studied CpGs and for their average value (45 ± 17% in CD patients vs 36 ± 17% in controls; p < 0.001). Again, methylation was comparable in PBMC. Methylation of CGI and 1st enhancer were not correlated in mucosa or PBMC. CONCLUSIONS: Methylation of several CpGs at the CDH1 locus was increased in the inflamed ileal mucosa, not in the PBMC, of CD patients, suggesting the association of CDH1 methylation with ileal inflammation. Longitudinal studies will explore if this increased methylation is a risk marker for colorectal cancer.

Long-Term Disease Prevention with a Gene Therapy Targeting Oligodendrocytes in a Mouse Model of Adrenomyeloneuropathy.

Adrenomyeloneuropathy (AMN) is a late-onset axonopathy of spinal cord tracts caused by mutations of the ABCD1 gene that encodes adrenoleukodystrophy protein (ALDP), a peroxisomal transporter of very long-chain fatty acids (VLCFA). Disturbed metabolic interaction between oligodendrocytes (OL) and axons is suspected to play a major role in AMN axonopathy. To develop a vector targeting OL, the human ABCD1 gene driven by a short 0.3 kb part of the human myelin-associated glycoprotein (MAG) promoter was packaged into an adeno-associated viral serotype 9 (rAAV9). An intravenous injection of this vector on postnatal day 10 in Abcd1-/- mice, a model of AMN, allowed a near normal motor performance to persist for 24 months, while age-matched untreated mice developed major defects of balance and motricity. Three weeks postvector, 50-54% of spinal cord white matter OL was expressing human ALDP (hALDP) at the cervical level, and only 6-7% after 24 months. In addition, 29-32% of cervical spinal cord astrocytes at 3 weeks and 16-19% at 24 months also expressed ALDP. C26:0-lysoPC, a sensitive VLCFA marker of AMN, was lower by 41% and 50%, respectively, in the spinal cord and brain of vector-treated compared with untreated mice. In a nonhuman primate, the intrathecal injection of the rAAV9-MAG vector induced abundant ALDP expression at 3 weeks in spinal cord OL (43%, 29%, and 26% at cervical, thoracic, and lumbar levels) and cerebellum OL (35%). In addition, 33-41% of spinal cord astrocytes expressed hALDP, and 27% of cerebellar astrocytes. To our knowledge, OL targeting had not been obtained before in primates with other vectors or promoters. The current results thus provide a robust proof-of-concept not only for the gene therapy of AMN but also for other central nervous system diseases, where the targeting of OL with the rAAV9-MAG vector may be of interest.

Correction of a knock-in mouse model of acrodysostosis with gene therapy using a rAAV9-CAG-human PRKAR1A vector.

Acrodysostosis is a rare skeletal dysplasia caused by loss-of-function mutations in the regulatory subunit of protein kinase A (PRKAR1A). In a knock-in mouse model (PRKAR1Awt/mut) expressing one copy of the recurrent R368X mutation, we tested the effects of a rAAV9-CAG-human PRKR1A (hPRKAR1A) vector intravenously administered at 4 weeks of age. Caudal vertebrae and tibial diaphyses contained 0.52 ± 0.7 and 0.13 ± 0.3 vector genome per cell (VGC), respectively, at 10 weeks of age and 0.22 ± 0.04 and 0.020 ± 0.04 at 16 weeks while renal cortex contained 0.57 ± 0.14 and 0.26 ± 0.05 VGC. Vector-mediated hPRKAR1A expression was found in growth plate chondrocytes, osteoclasts, osteoblasts, and kidney tubular cells. Chondrocyte architecture was restored in the growth plates. Body length, tail length, and body weight were improved in vector treated PRKAR1Awt/mut mice, not the bone length of their limbs. These results provide one of the few proofs for gene therapy efficacy in a mouse model of chondrodysplasia. In addition, the increased urinary cAMP of PRKAR1Awt/mut mice was corrected almost to normal. In conclusion, gene therapy with hPRKAR1A improved skeletal growth and kidney dysfunction, the hallmarks of acrodysostosis in R368X mutated mice and humans.

Long-Term Follow-Up of Hematopoietic Stem-Cell Gene Therapy for Cerebral Adrenoleukodystrophy.

In 2009, cerebral adrenoleukodystrophy (c-ALD) became the first brain disease to be treated with lentiviral (LV)-based hematopoietic stem cell gene therapy with the ABCD1 gene in four boys (P1-P4) who had demyelinating lesions expected to be lethal in the short term and no bone marrow donor. We report the clinical and magnetic resonance imaging (MRI) follow-up over a mean of 8.8 years posttransplant. In parallel, vector genome copies, expression of transgenic ALD protein (ALDP), and viral integration sites were determined in peripheral blood cells. Prior to transplant, the four patients had a normal or near normal neurocognitive status but gadolinium-enhanced demyelination in various brain regions. Gadolinium diffusion disappeared during the first year posttransplant. P3 kept a near normal status until 8.3 years of follow-up, but P1, P2, and P4 showed major cognitive degradation around 9, 28, and 60 months posttransplant. Neurological status and demyelination stabilized until last evaluation in P2, but deteriorated in both P1 at 10 years and P4 at 3 years posttransplant. The proportion of myeloid and lymphoid cells expressing transgenic ALDP decreased by half within 5 years then stabilized around 5% to 10%. Integration site analysis revealed a durable polyclonal distribution of genetically corrected hematopoietic cells. No adverse effects were observed. The long-term arrest of demyelination at MRI and persistence of transduced hematopoietic progenitors support that LV gene therapy may be a safe and durable treatment of c-ALD. However, the neurological degradation observed in three out of four patients mitigates the benefit of this therapy, calling for an earlier intervention, more potent vectors, and additional therapeutic strategies.

Exploring the risk of hypospadias in children born from mothers living close to a vineyard.

Hypospadias (H) is a common birth defect affecting the male urinary tract. It has been suggested that exposure to endocrine disrupting chemicals might increase the risk of H by altering urethral development. However, whether H risk is increased in places heavily exposed to agricultural pesticides, such as vineyards, remains debated and difficult to ascertain. The objective of the work is to test the possible association of H with residential proximity to vineyards. Residential address at birth of 8,766 H cases born 1980-2011 was taken from 17 specialized surgery centers. The geographical distribution of vineyards was obtained from the European Land Parcel Identification System (LPIS) and the distance of address to the nearest vineyard was computed. A first estimate of the variation of H relative risk with distance to vineyards was obtained using as controls 13,105 cryptorchidism (C) cases operated during the same period in the same centers. A separate estimate was obtained from a case-control study using "virtual controls" (VC) defined as points of the map sampled to match the demographic distribution of births within the recruitment territories of the study centers. Non-exposed patients were defined as those with a residence between 5,000 and 10,000 m from the closest vineyard. The residential distance to vineyard was smaller for H than for C cases (p<10-4). We found 42/8766 H cases (0.48%) and 50/13,105 C cases (0.38%) born to mothers living within 20 m of a vineyard. The odds ratios for H were 2.48 (CI: 1.0 to 5.1) and 2.4 (CI: 1.3 to 4.4), vs C or vs VC, respectively, when pregnant mothers lived 10-20 m from a vineyard. In conclusion, our study supports that children born to mothers living close to a vineyard have a two-fold increased risk of H. For environmental research, the use of VC provides an alternative to classical case control technique.

Laparoscopic Sleeve Gastrectomy for Adolescents Under 18 Years Old with Severe Obesity.

INTRODUCTION: Laparoscopic sleeve gastrectomy (LSG) is a widely accepted stand-alone bariatric operation. Data on adolescent patients undergoing LSG are limited. The aim of this study was to demonstrate that LSG is safe and effective for patients strictly under 18 years old with severe obesity. METHODS: Prospectively collected data from consecutive patients undergoing LSG were retrospectively analyzed. Patients with more than 1-year follow-up were included in the analysis for weight loss and comorbidity evaluation. Quality of life (QoL) was evaluated using the Short-Form 36 questionnaire. RESULTS: Eighty-four patients under 18 years old (range: 15-17 years) underwent LSG. Median weight was 128 kg and median body mass index (BMI) 43.7 kg/m2. Median duration of surgery was 68.5 min. One major complication was recorded: a patient developed severe pneumonia that necessitated ventilatory support in intensive care unit and intravenous antibiotic treatment. Mortality was null. Median length of hospital stay was 4 days. Six, 12, and 24 months after LSG, median BMI decreased significantly to 34.3, 29.8, and 28.8 kg/m2, respectively (p < 0.001), with a mean percentage of total body weight loss of 29.1% at 2 years. Obesity-related comorbidities improved at 1 year, while all SF-36 scale scores of QoL assessment improved significantly. CONCLUSION: This study suggests that LSG is safe and effective for patients under 18 years old, resulting in significant weight loss, comorbidity remission, and QoL improvement. Careful patient selection after adequate risk versus benefit evaluation by an expert multidisciplinary team is essential.

Fetal growth is associated with CpG methylation in the P2 promoter of the IGF1 gene.

Background: There are many reasons to think that epigenetics is a key determinant of fetal growth variability across the normal population. Since IGF1 and INS genes are major determinants of intrauterine growth, we examined the methylation of selected CpGs located in the regulatory region of these two genes. Methods: Cord blood was sampled in 159 newborns born to mothers prospectively followed during their pregnancy. A 142-item questionnaire was filled by mothers at inclusion, during the last trimester of the pregnancy and at the delivery. The methylation of selected CpGs located in the promoters of the IGF1 and INS genes was measured in cord blood mononuclear cells collected at birth using bisulfite-PCR-pyrosequencing. Results: Methylation at IGF1 CpG-137 correlated negatively with birth length (r = 0.27, P = 3.5 × 10-4). The same effect size was found after adjustment for maternal age, parity, and smoking: a 10% increase in CpG-137 methylation was associated with a decrease of length by 0.23 SDS. Conclusion: The current results suggest that the methylation of IGF1 CpG-137 contributes to the individual variation of fetal growth by regulating IGF1 expression in fetal tissues.

Genome-Wide Methylation Analysis Identifies Specific Epigenetic Marks In Severely Obese Children.

Obesity is a heterogeneous disease with many different subtypes. Epigenetics could contribute to these differences. The aim of this study was to investigate genome-wide DNA methylation searching for methylation marks associated with obesity in children and adolescents. We studied DNA methylation profiles in whole blood cells from 40 obese children and controls using Illumina Infinium HumanMethylation450 BeadChips. After correction for cell heterogeneity and multiple tests, we found that compared to lean controls, 31 CpGs are differentially methylated in obese patients. A greatest proportion of these CpGs is hypermethylated in obesity and located in CpG shores regions. We next focused on severely obese children and identified 151 differentially methylated CpGs among which 10 with a difference in methylation greater than 10%. The top pathways enriched among the identified CpGs included the "IRS1 target genes" and several pathways in cancer diseases. This study represents the first effort to search for differences in methylation in obesity and severe obesity, which may help understanding these different forms of obesity and their complications.

Deferring surgical treatment of ambiguous genitalia into adolescence in girls with 21-hydroxylase deficiency: a feasibility study.

BACKGROUND: Genital surgery in Disorders of Sex Development (DSD) has been an area of debate over the past 20 years. Emerging scientific evidence in the late 1990s defied the then routine practice to surgically align genitalia to the sex of rearing, as early as possible. However, despite multitude of data showing detrimental effects to genital sensation and sexuality, few patients born with ambiguous genitalia have remained unoperated into adolescence. METHODS: We followed up girls with 21 hydroxylase deficiency (21- OHD) in genital morphology during childhood and acceptability among patients and parents of such an approach. RESULTS: Preliminary results from 7 children, aged 1-8 years (median 4.5 years), suggest that it is acceptable among patients and families to defer genital operation in 21-OHD. All patients had a Prader stage III and above. Median clitoral length at birth was 24 mm (20-28 mm) and had diminished to a median of 9 mm (5-15 mm) at their last visit. Height and weight have remained strictly normal in all patients. So far girls and their parents have not expressed significant concerns regarding genital ambiguity. CONCLUSIONS: With this encouraging data at hand, we propose to formally address levels of anxiety, adaptation and quality of life during childhood, with an ultimate goal to assess long term satisfaction and effects on sexuality through deferring genital surgery for adolescence.

Knock-In of the Recurrent R368X Mutation of PRKAR1A that Represses cAMP-Dependent Protein Kinase A Activation: A Model of Type 1 Acrodysostosis.

In humans, activating mutations in the PRKAR1A gene cause acrodysostosis 1 (ACRDYS1). These mutations result in a reduction in PKA activation caused by an impaired ability of cAMP to dissociate mutant PRKAR1A from catalytic PKA subunits. Two striking features of this rare developmental disease are renal resistance to PTH and chondrodysplasia resulting from the constitutive inhibition of PTHR1/Gsa/AC/cAMP/PKA signaling. We developed a knock-in of the recurrent ACRDYS1 R368X PRKAR1A mutation in the mouse. No litters were obtained from [R368X]/[+] females (thus no homozygous [R368X]/[R368X] mice). In [R368X]/[+] mice, Western blot analysis confirmed mutant allele heterozygous expression. Growth retardation, peripheral acrodysostosis (including brachydactyly affecting all digits), and facial dysostosis were shown in [R368X]/[+] mice by weight curves and skeletal measurements (µCT scan) as a function of time. [R368X]/[+] male and female mice were similarly affected. Unexpected, however, whole-mount skeletal preparations revealed a striking delay in mineralization in newborn mutant mice, accompanied by a decrease in the height of terminal hypertrophic chondrocyte layer, an increase in the height of columnar proliferative prehypertrophic chondrocyte layer, and changes in the number and spatial arrangement of proliferating cell nuclear antigen (PCNA)-positive chondrocytes. Plasma PTH and basal urinary cAMP were significantly higher in [R368X]/[+] compared to WT mice. PTH injection increased urinary cAMP similarly in [R368X]/[+] and WT mice. PRKACA expression was regulated in a tissue (kidney not bone and liver) manner. This model, the first describing the germline expression of a PRKAR1A mutation causing dominant repression of cAMP-dependent PKA, reproduced the main features of ACRDYS1 in humans. It should help decipher the specificity of the cAMP/PKA signaling pathway, crucial for numerous stimuli. In addition, our results indicate that PRKAR1A, by tempering intracellular cAMP levels, is a molecular switch at the crossroads of signaling pathways regulating chondrocyte proliferation and differentiation. © 2016 American Society for Bone and Mineral Research.

Growth and descent of the testes in infants with hypogonadotropic hypogonadism receiving subcutaneous gonadotropin infusion.

BACKGROUND: One third of infants with congenital hypogonadotropic hypogonadism (CHH) are said to have micropenis and/or bilateral or unilateral cryptorchidism leading many of them to orchiopexy. Our previous study in two patients suggests that prolonged subcutaneous infusion of large doses of gonadotropins might normalize testicular function and growth. CASE PRESENTATION: To confirm the effects of early and prolonged subcutaneous infusion of large doses of gonadotropins on growth and descent of the testes. Eight boys with CHH, aged 0.25-11 months. Testes were non-palpable in 5 or in high scrotal position in 3. CHH was isolated in 5 infants and part of a syndrome of combined pituitary hormonal deficits in the 3 others. In response to gonadotropin infusion, mean levels of testicular hormones were normalized. Complete testis descent occurred in 6 patients. Partial descent occurred in 2. Testes re-ascended in 1 patient. Testes and penis gained normal dimensions in all cases. CONCLUSION: Subcutaneous gonadotropin infusion seems able to induce testis descent in a large proportion of infants with CHH. If confirmed, this may allow patients to avoid testes surgery but studies in larger series are needed to evaluate the benefits of this treatment versus traditional orchiopexy.

Primordial Influence of Post-operative Compliance on Weight Loss After Adolescent Laparoscopic Adjustable Gastric Banding.

BACKGROUND: Accumulating evidence suggests that the benefits seen in adult bariatric surgery can be reproduced in adolescents. In contrast with North America, bariatric surgery in adolescents is still not well accepted in Europe and indications and protocols have still to be formulated. METHODS: This prospective study tested the gastric banding procedure in 49 patients operated in a single French institution since 2008. The mean age at surgery was 16.2 ± 0.9 years with a weight of 118.8 ± 22.3 kg and body mass index of 42.5 ± 5.9 kg/m(2). RESULTS: At 6, 12 and 24 months after surgery, weight was 103.7 ± 20.8 kg, 98.7 ± 21 kg and 93.6 ± 19.3 kg, respectively (p < 0.001), corresponding to excess weight loss (EWL) of 31.6 ± 17.2 %, 41.8 ± 21.4 % and 59.1 ± 24.9 % (p < 0.001), respectively. Multivariate analysis showed that the number of consultations per year was the only variable significantly associated to weight loss. Metabolic disorders were corrected, with a decreased prevalence of insulin resistance from 100 to 17 % and normalisation of homeostasis model assessment-insulin resistance (HOMA-IR) at 24 months (2.09 ± 0.95). Band-related complications were five slippages, one psychological intolerance and two ports repositioning. Six patients (12 %) had the device explanted. The death of a patient was an exceptionally severe adverse event. CONCLUSION: Given frequent follow-up support by a multidisciplinary team, laparoscopic adjustable gastric banding (LAGB) surgery in adolescent results in sustained weight loss. However, even exceptional, potentially serious complications are possible and long-term follow-up is needed to evaluate the risk/benefit ratio at 5 or 10 years after LAGB surgery.

The IGF1 P2 promoter is an epigenetic QTL for circulating IGF1 and human growth.

BACKGROUND: Even if genetics play an important role, individual variation in stature remains unexplained at the molecular level. Indeed, genome-wide association study (GWAS) have revealed hundreds of variants that contribute to the variability of height but could explain only a limited part of it, and no single variant accounts for more than 0.3% of height variance. At the interface of genetics and environment, epigenetics contributes to phenotypic diversity. Quantifying the impact of epigenetic variation on quantitative traits, an emerging challenge in humans, has not been attempted for height. Since insulin-like growth factor 1 (IGF1) controls postnatal growth, we tested whether the CG methylation of the two promoters (P1 and P2) of the IGF1 gene is a potential epigenetic contributor to the individual variation in circulating IGF1 and stature in growing children. RESULTS: Child height was closely correlated with serum IGF1. The methylation of a cluster of six CGs located within the proximal part of the IGF1 P2 promoter showed a strong negative association with serum IGF1 and growth. The highest association was for CG-137 methylation, which contributed 13% to the variance of height and 10% to serum IGF1. CG methylation (studied in children undergoing surgery) was approximately 50% lower in liver and growth plates, indicating that the IGF1 promoters are tissue-differentially methylated regions (t-DMR). CG methylation was inversely correlated with the transcriptional activity of the P2 promoter in mononuclear blood cells and in transfection experiments, suggesting that the observed association of methylation with the studied traits reflects true biological causality. CONCLUSIONS: Our observations introduce epigenetics among the individual determinants of child growth and serum IGF1. The P2 promoter of the IGF1 gene is the first epigenetic quantitative trait locus (QTL(epi)) reported in humans. The CG methylation of the P2 promoter takes place among the multifactorial factors explaining the variation in human stature.

Short-term results of single-port sleeve gastrectomy in adolescents with severe obesity.

BACKGROUND: Dietary and lifestyle modifications, which are commonly proposed to overweight or obese youth, lack efficacy in individuals who are severely obese. Early results with bariatric procedures in obese adolescents suggest that weight loss and safety are comparable or better than seen in adults. One of these procedures, laparoscopic sleeve gastrectomy, is commonly performed using multiple ports. METHODS: We selected single-port sleeve gastrectomy (SPSG) as a minimally invasive surgery to be tested in severely obese adolescents. Prospective clinical and biochemical data were collected from 16 young severely obese patients who underwent SPSG. The setting was a university hospital. RESULTS: The mean age of the cohort was 17.8 years (12 girls, 4 boys). The individuals' average weight was 125.5 kg and their average body mass index was 45.3 kg/m(2). All patients were insulin-resistant and 6 showed hypertriglyceridemia. The median operating time was 66 minutes, and there were no intraoperative complications. No conversion to open surgery was required. No patient required additional trocars and no patient had postoperative complications. The median hospital stay was 3 days. After a one-year follow-up, the average weight decrease was 40.3 kg, resulting in a decrease in excess weight loss by 70.61%. Insulin-resistance decreased in 16/16 patients and hypertriglyceridemia decreased in 5/6 patients. CONCLUSION: SPSG seems safe and effective in the short term in severely obese adolescents.

PRKAR1A and PDE4D mutations cause acrodysostosis but two distinct syndromes with or without GPCR-signaling hormone resistance.

CONTEXT: Acrodysostosis is a rare skeletal dysplasia that is associated with multiple resistance to G protein-coupled receptor (GPCR) signaling hormones in a subset of patients. Acrodysostosis is genetically heterogeneous because it results from heterozygous mutations in PRKAR1A or PDE4D, two key actors in the GPCR-cAMP-protein kinase A pathway. OBJECTIVE: Our objective was to identify the phenotypic features that distinguish the two genotypes causing acrodysostosis. PATIENTS AND METHODS: Sixteen unrelated patients with acrodysostosis underwent a candidate-gene approach and were investigated for phenotypic features. RESULTS: All patients had heterozygous de novo mutations. Fourteen patients carried a PRKAR1A mutation (PRKAR1A patients), five each a novel PRKAR1A mutation (p.Q285R, p.G289E, p.A328V, p.R335L, or p.Q372X), nine the reported PRKAR1A p.R368X mutation; two patients harbored a mutation in PDE4D (PDE4D patients) (one novel mutation, p.A227S; one reported, p.E590A). All PRKAR1A, but none of the PDE4D mutated patients were resistant to PTH and TSH. Two PRKAR1A patients each with a novel mutation presented a specific pattern of brachydactyly. One PDE4D patient presented with acroskyphodysplasia. Additional phenotypic differences included mental retardation in PDE4D patients. In addition, we report the presence of pigmented skin lesions in PRKAR1A and PDE4D patients, a feature not yet described in the acrodysostosis entity. CONCLUSIONS: All PRKAR1A and PDE4D patients present similar bone dysplasia characterizing acrodysostosis. Phenotypic differences, including the presence of resistance to GPCR-cAMP signaling hormones in PRKAR1A but not PDE4D patients, indicate phenotype-genotype correlations and highlight the specific contributions of PRKAR1A and PDE4D in cAMP signaling in different tissues.

XPG and XPF endonucleases trigger chromatin looping and DNA demethylation for accurate expression of activated genes.

Nucleotide excision repair factors, initially characterized as part of DNA repair, have been shown to participate in the transcriptional process in the absence of genotoxic attack. However, their molecular function when recruited at the promoters of activated genes together with the transcription machinery remained obscure. Here we show that the NER factors XPG and XPF are essential for establishing CTCF-dependent chromatin looping between the promoter and terminator of the activated RARß2 gene. Silencing XPG and/or XPF endonucleases, or mutations in their catalytic sites, prevents CTCF recruitment, chromatin loop formation, and optimal transcription of RARß2. We demonstrated that XPG endonuclease promotes DNA breaks and DNA demethylation at promoters allowing the recruitment of CTCF and gene looping, which is further stabilized by XPF. Our results highlight a timely orchestrated activity of the NER factors XPG and XPF in the formation of the active chromatin hub that controls gene expression.

Association of the CpG methylation pattern of the proximal insulin gene promoter with type 1 diabetes.

The insulin (INS) region is the second most important locus associated with Type 1 Diabetes (T1D). The study of the DNA methylation pattern of the 7 CpGs proximal to the TSS in the INS gene promoter revealed that T1D patients have a lower level of methylation of CpG -19, -135 and -234 (p = 2.10(-16)) and a higher methylation of CpG -180 than controls, while methylation was comparable for CpG -69, -102, -206. The magnitude of the hypomethylation relative to a control population was 8-15% of the corresponding levels in controls and was correlated in CpGs -19 and -135 (r = 0.77) and CpG -135 and -234 (r = 0.65). 70/485 (14%) of T1D patients had a simultaneous decrease in methylation of CpG -19, -135, -234 versus none in 317 controls. CpG methylation did not correlate with glycated hemoglobin or with T1D duration. The methylation of CpG -69, -102, -180, -206, but not CpG -19, -135, -234 was strongly influenced by the cis-genotype at rs689, a SNP known to show a strong association with T1D. We hypothesize that part of this genetic association could in fact be mediated at the statistical and functional level by the underlying changes in neighboring CpG methylation. Our observation of a CpG-specific, locus-specific methylation pattern, although it can provide an epigenetic biomarker of a multifactorial disease, does not indicate whether the reported epigenetic pattern preexists or follows the establishment of T1D. To explore the effect of chronic hyperglycemia on CpG methylation, we studied non obese patients with type 2 diabetes (T2D) who were found to have decreased CpG-19 methylation versus age-matched controls, similar to T1D (p = 2.10(-6)) but increased CpG-234 methylation (p = 5.10(-8)), the opposite of T1D. The causality and natural history of the different epigenetic changes associated with T1D or T2D remain to be determined.

Lentiviral hematopoietic cell gene therapy for X-linked adrenoleukodystrophy.

X-linked adrenoleukodystrophy (X-ALD) is a severe genetic demyelinating disease caused by a deficiency in ALD protein, an adenosine triphosphate-binding cassette transporter encoded by the ABCD1 gene. When performed at an early stage of the disease, allogeneic hematopoietic stem cell transplantation (HCT) can arrest the progression of cerebral demyelinating lesions. To overcome the limitations of allogeneic HCT, hematopoietic stem cell (HSC) gene therapy strategy aiming to perform autologous transplantation of lentivirally corrected cells was developed. We demonstrated the preclinical feasibility of HSC gene therapy for ALD based on the correction of CD34+ cells from X-ALD patients using an HIV1-derived lentiviral vector. These results prompted us to initiate an HSC gene therapy trial in two X-ALD patients who had developed progressive cerebral demyelination, were candidates for allogeneic HCT, but had no HLA-matched donors or cord blood. Autologous CD34+ cells were purified from the peripheral blood after G-CSF stimulation, genetically corrected ex vivo with a lentiviral vector encoding wild-type ABCD1 cDNA, and then reinfused into the patients after they had received full myeloablative conditioning. Over 3 years of follow-up, the hematopoiesis remained polyclonal in the two patients treated with 7-14% of granulocytes, monocytes, and T and B lymphocytes expressing the lentivirally encoded ALD protein. There was no evidence of clonal dominance or skewing based on the retrieval of lentiviral insertion repertoire in different hematopoietic lineages by deep sequencing. Cerebral demyelination was arrested 14 and 16months, respectively, in the two treated patients, without further progression up to the last follow-up, a clinical outcome that is comparable to that observed after allogeneic HCT. Longer follow-up of these two treated patients and HSC gene therapy performed in additional ALD patients are however needed to evaluate the safety and efficacy of lentiviral HSC gene therapy in cerebral forms of X-ALD.

A pilot study of discontinuous, insulin-like growth factor 1-dosing growth hormone treatment in young children with FGFR3 N540K-mutated hypochondroplasia.

OBJECTIVE: To assess the growth promoting effect of a recombinant growth hormone (rGH) treatment protocol adjusted on insulin-like growth factor 1 (IGF-1) dosing in children affected by the most severe forms of FGFR3 N540K-mutated hypochondroplasia. STUDY DESIGN: Midterm results of an open-label, single-center, nonrandomized, 2003-2020 pilot trial to final stature, including 6 children (mean age, 2.6 ± 0.7 years; mean height SDS, -3.0 ± 0.5) with the N540K mutation of FGFR3 gene who received an rGH dosage titrated to an IGF-1 level close to 1.5 SDS of the normal range. rGH therapy was interrupted 1 day per week, 1 month per year, and 6 months every 2 years. RESULTS: The mean height SDS increased by 1.9 during the 6.1 ± 0.9-year study period, reaching -0.8 to -1.3 at age 8.7 ± 1 years. The mean±SDS baseline IGF-1 value was -1.6 ± 0.5 before rGH treatment and 1.4±0.3 during the last year of observation. The average cumulative rGH dose was 0.075 ± 0.018 mg/kg/day (range, 0.059-0.100 mg/kg/day). Trunk/leg disproportion was improved. CONCLUSION: IGF-1-dosing rGH treatment durably improves growth and reduces body disproportion in children with severe forms of hypochondroplasia.