CDKN1C mutation affecting the PCNA-binding domain as a cause of familial Russell Silver syndrome.

BACKGROUND: Russell Silver syndrome (RSS) leads to prenatal and postnatal growth retardation. About 55% of RSS patients present a loss-of-methylation of the paternal ICR1 domain on chromosome 11p15. CDKN1C is a cell proliferation inhibitor encoded by an imprinted gene in the 11p15 ICR2 domain. CDKN1C mutations lead to Beckwith Wiedemann syndrome (BWS, overgrowth syndrome) and in IMAGe syndrome which associates growth retardation and adrenal insufficiency. We searched for CDKN1C mutations in a cohort of clinically diagnosed RSS patients with no molecular anomaly. METHOD: The coding sequence and intron-exon boundaries of CDKN1C were analysed in 97 RSS patients. The impact of CDKN1C variants on the cell cycle in vitro were determined by flow cytometry. Stability of CDKN1C was studied by western immunoblotting after inhibition of translation with cycloheximide. RESULTS: We identified the novel c.836G>[G;T] (p.Arg279Leu) mutation in a familial case of intrauterine growth retardation (IUGR) with RSS phenotype and no evidence of IMAGe. All the RSS patients inherited this mutation from their mothers (consistent with monoallelic expression from the maternal allele of the gene). A mutation of this amino acid (p.Arg279Pro) has been reported in cases of IMAGe. Functional analysis showed that Arg279Leu (RSS) did not affect the cell cycle, whereas the Arg279Pro mutation (IMAGe) led to a gain of function. Arg279Leu (RSS) led to an increased stability which could explain an increased activity of CDKN1C. CONCLUSIONS: CDKN1C mutations cause dominant maternally transmitted RSS, completing the molecular mirror with BWS. CDKN1C should be investigated in cases with family history of RSS.

Maintenance of methylation profile in imprinting control regions in human induced pluripotent stem cells.

BACKGROUND: Parental imprinting is an epigenetic mechanism that leads to monoallelic expression of a subset of genes depending on their parental origin. Imprinting disorders (IDs), caused by disturbances of imprinted genes, are a set of rare congenital diseases that mainly affect growth, metabolism and development. To date, there is no accurate model to study the physiopathology of IDs or test therapeutic strategies. Human induced pluripotent stem cells (iPSCs) are a promising cellular approach to model human diseases and complex genetic disorders. However, aberrant hypermethylation of imprinting control regions (ICRs) may appear during the reprogramming process and subsequent culture of iPSCs. Therefore, we tested various conditions of reprogramming and culture of iPSCs and performed an extensive analysis of methylation marks at the ICRs to develop a cellular model that can be used to study IDs. RESULTS: We assessed the methylation levels at seven imprinted loci in iPSCs before differentiation, at various passages of cell culture, and during chondrogenic differentiation. Abnormal methylation levels were found, with hypermethylation at 11p15 H19/IGF2:IG-DMR and 14q32 MEG3/DLK1:IG-DMR, independently of the reprogramming method and cells of origin. Hypermethylation at these two loci led to the loss of parental imprinting (LOI), with biallelic expression of the imprinted genes IGF2 and DLK1, respectively. The epiPS™ culture medium combined with culturing of the cells under hypoxic conditions prevented hypermethylation at H19/IGF2:IG-DMR (ICR1) and MEG3/DLK1:IG-DMR, as well as at other imprinted loci, while preserving the proliferation and pluripotency qualities of these iPSCs. CONCLUSIONS: An extensive and quantitative analysis of methylation levels of ICRs in iPSCs showed hypermethylation of certain ICRs in human iPSCs, especially paternally methylated ICRs, and subsequent LOI of certain imprinted genes. The epiPS™ culture medium and culturing of the cells under hypoxic conditions prevented hypermethylation of ICRs in iPSCs. We demonstrated that the reprogramming and culture in epiPS™ medium allow the generation of control iPSCs lines with a balanced methylation and ID patient iPSCs lines with unbalanced methylation. Human iPSCs are therefore a promising cellular model to study the physiopathology of IDs and test therapies in tissues of interest.

Beckwith-Wiedemann syndrome: growth pattern and tumor risk according to molecular mechanism, and guidelines for tumor surveillance.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is an overgrowth syndrome associated with an increased risk of pediatric tumors. The underlying molecular abnormalities may be genetic (CDKN1C mutations or 11p15 paternal uniparental isodisomy, pUPD) or epigenetic (imprinting center region 1, ICR1, gain of methylation, ICR1 GOM, or ICR2 loss of methylation, ICR2 LOM). AIM: We aimed to describe a cohort of 407 BWS patients with molecular defects of the 11p15 domain followed prospectively after molecular diagnosis. RESULTS: Birth weight and length were significantly higher in patients with ICR1 GOM than in the other groups. ICR2 LOM and CDKN1C mutations were associated with a higher prevalence of exomphalos. Mean adult height (regardless of molecular subtype, n = 35) was 1.8 ± 1.2 SDS, with 18 patients having a final height above +2 SDS. The prevalence of tumors was 8.6% in the whole population; 28.6 and 17.3% of the patients with ICR1 GOM (all Wilms tumors) and 11p15 pUPD, respectively, developed a tumor during infancy. Conversely, the prevalence of tumors in patients with ICR2 LOM and CDKN1C mutations were 3.1 and 8.8%, respectively, with no Wilms tumors. CONCLUSION: Based on these results for a large cohort, we formulated guidelines for the follow-up of these patients according to the molecular subtype of BWS.

[Hypogonadotropic hypogonadism: new aspects in the regulation of hypothalamic-pituitary-gonadal axis]. / Hypogonadisme hypogonadotrope : notions récentes sur la régulation de l'axe hypothalamo-hypophyso-gonadique.

Hypogonadotropic hypogonadism (HH) is defined by the absence of sex steroid synthesis associated with the lack of appropriate gonadotrophin secretion. This leads to a variable degree of impuberism, often diagnosed during childhood or adolescence. Genetics of HH involve many genes. However, molecular defects have been identified in only 30 % of patients. Kallmann syndrome (KS) is defined by the association of HH and anosmia. Six genes are involved in KS (KAL1, FGFR1, FGF8, PROK2, PROKR2 and CHD7). However, genetics of KS is complex, because of the variability of the phenotype for a similar molecular defect. Otherwise, heterozygous anomalies are frequently described. Identification in the same patient of several mutations in some of these genes (digenism) could account for this variability. Autosomal recessive transmission is frequently observed in familial cases of HH without anosmia. Molecular alterations have been identified for several neuropeptides or their corresponding receptors, which are involved in the physiology of the gonadotropic axis : GNRHR, KISS1R/GPR54, neurokinin B (TAC3), TACR3 and GNRH1 (and PROK2, PROKR2 and CHD7). Anomalies of leptin or its receptor are also involved in HH cases. A new negative regulating element has been recently identified in humans : RFRP3, which is ortholog of the avian GnIH (gonadotrophin inhibitory hormone). Recent progress about these neuropeptides leads to a new model of comprehension of the gonadotropic axis physiology, from a linear model to a network model, which regulates the central element of regulation of the gonadotropic axis, represented by the GnRH neurons.