Irène Netchine

Mutations in LHX3 result in a new syndrome revealed by combined pituitary hormone deficiency

Combined pituitary hormone deficiency (CPHD) has been linked with rare abnormalities in genes encoding transcription factors necessary for pituitary development. We have isolated LHX3, a gene involved in a new syndrome, using a candidate-gene approach developed on the basis of documented pituitary abnormalities of a recessive lethal mutation in mice generated by targeted disruption of Lhx3 (ref. 2). LHX3, encoding a member of the LIM class of homeodomain proteins, consists of at least six exons located at 9q34. We identified a homozygous LHX3 defect in patients of two unrelated consanguineous families displaying a complete deficit in all but one (adrenocorticotropin) anterior pituitary hormone and a rigid cervical spine leading to limited head rotation. Two of these patients also displayed a severe pituitary hypoplasia, whereas one patient presented secondarily with an enlarged anterior pituitary. These LHX3 mutations consist of a missense mutation (Y116C) in the LIM2 domain at a phylogenetically conserved residue and an intragenic deletion predicting a severely truncated protein lacking the entire homeodomain. These data are consistent with function of LHX3 in the proper development of all anterior pituitary cell types, except corticotropes, and extrapituitary structures.

Syndromic Short Stature in Patients with a Germline Mutation in the LIM Homeobox LHX4

Studies of genetically engineered flies and mice have revealed the role that orthologs of the human LIM homeobox LHX4 have in the control of motor-neuron-identity assignment and in pituitary development. Remarkably, these mouse strains, which bear a targeted modification of Lhx4 in the heterozygous state, are asymptomatic, whereas homozygous animals die shortly after birth. Nevertheless, we have isolated the human LHX4 gene, as well as the corresponding cDNA sequence, to test whether it could be involved in developmental defects of the human pituitary region. LHX4, which encodes a protein 99% identical to its murine counterpart, consists of six coding exons and spans >45 kb of the q25 region of chromosome 1. We report a family with an LHX4 germline splice-site mutation that results in a disease phenotype characterized by short stature and by pituitary and hindbrain (i.e., cerebellar) defects in combination with abnormalities of the sella turcica of the central skull base. This intronic mutation, which segregates in a dominant and fully penetrant manner over three generations, abolishes normal LHX4 splicing and activates two exonic cryptic splice sites, thereby predicting two different proteins deleted in their homeodomain sequence. These findings, which elucidate the molecular basis of a complex Mendelian disorder, reveal the fundamental pleiotropic role played by a single factor that tightly coordinates brain development and skull shaping during head morphogenesis.

Human Prop-1: cloning, mapping, genomic structure: Mutations in familial combined pituitary hormone deficiency1

Prop‐1 is a newly isolated pituitary‐specific paired‐like homeodomain transcription factor whose cDNA sequence is well known in mouse. To study its involvement in human combined pituitary hormone deficiency (CPHD), we have isolated the human cDNA ortholog and determined the exon/intron organization and chromosomal localization of the human gene. A Prop‐1 defect was characterized in three CPHD families. One missense mutation (R73C) involves a residue conserved in 95% of the more than 400 homeodomain proteins so far identified; in vitro splicing assays demonstrated the functional importance of the second defect, whereas the remaining mutation is a frameshift. Given the disease phenotype documented in the patients, these data, which will facilitate molecular investigations in other patients, demonstrate the crucial role of Prop‐1 in the proper development of somatotrophs, lactotrophs, thyreotrophs and gonadotrophs.

Analysis of the IGF2/H19 imprinting control region uncovers new genetic defects, including mutations of OCT-binding sequences, in patients with 11p15 fetal growth disorders

The imprinted expression of the IGF2 and H19 genes is controlled by the imprinting control region 1 (ICR1) located at chromosome 11p15.5. This methylation-sensitive chromatin insulator works by binding the zinc-finger protein CTCF in a parent-specific manner. DNA methylation defects involving the ICR1 H19/IGF2 domain result in two growth disorders with opposite phenotypes: an overgrowth disorder, the Beckwith-Wiedemann syndrome (maternal ICR1 gain of methylation in 10% of BWS cases) and a growth retardation disorder, the Silver-Russell syndrome (paternal ICR1 loss of methylation in 60% of SRS cases). Although a few deletions removing part of ICR1 have been described in some familial BWS cases, little information is available regarding the mechanism of ICR1 DNA methylation defects. We investigated the CTCF gene and the ICR1 domain in 21 BWS patients with ICR1 gain of methylation and 16 SRS patients with ICR1 loss of methylation. We identified four constitutional ICR1 genetic defects in BWS patients, including a familial case. Three of those defects are newly identified imprinting defects consisting of small deletions and a single mutation, which do not involve one of the CTCF binding sites. Moreover, two of those defects affect OCT-binding sequences which are suggested to maintain the unmethylated state of the maternal allele. A single-nucleotide variation was identified in a SRS patient. Our data extends the spectrum of constitutive genetic ICR1 abnormalities and suggests that extensive and accurate analysis of ICR1 is required for appropriate genetic counseling in BWS patients with ICR1 gain of methylation.

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.

A prospective study validating a clinical scoring system and demonstrating phenotypical-genotypical correlations in Silver-Russell syndrome

Background Multiple clinical scoring systems have been proposed for Silver-Russell syndrome (SRS). Here we aimed to test a clinical scoring system for SRS and to analyse the correlation between (epi)genotype and phenotype. Subjects and methods Sixty-nine patients were examined by two physicians. Clinical scores were generated for all patients, with a new, six-item scoring system: (1) small for gestational age, birth length and/or weight ≤−2SDS, (2) postnatal growth retardation (height ≤−2SDS), (3) relative macrocephaly at birth, (4) body asymmetry, (5) feeding difficulties and/or body mass index (BMI) ≤−2SDS in toddlers; (6) protruding forehead at the age of 1–3 years. Subjects were considered to have likely SRS if they met at least four of these six criteria. Molecular investigations were performed blind to the clinical data. Results The 69 patients were classified into two groups (Likely-SRS (n=60), Unlikely-SRS (n=9)). Forty-six Likely-SRS patients (76.7%) displayed either 11p15 ICR1 hypomethylation (n=35; 58.3%) or maternal UPD of chromosome 7 (mUPD7) (n=11; 18.3%). Eight Unlikely-SRS patients had neither ICR1 hypomethylation nor mUPD7, whereas one patient had mUPD7. The clinical score and molecular results yielded four groups that differed significantly overall and for individual scoring system factors. Further molecular screening led identifying chromosomal abnormalities in Likely-SRS-double-negative and Unlikely-SRS groups. Four Likely-SRS-double negative patients carried a DLK1/GTL2 IG-DMR hypomethylation, a mUPD16; a mUPD20 and a de novo 1q21 microdeletion. Conclusions This new scoring system is very sensitive (98%) for the detection of patients with SRS with demonstrated molecular abnormalities. Given its clinical and molecular heterogeneity, SRS could be considered as a spectrum.

Imprinting disorders: a group of congenital disorders with overlapping patterns of molecular changes affecting imprinted loci

Congenital imprinting disorders (IDs) are characterised by molecular changes affecting imprinted chromosomal regions and genes, i.e. genes that are expressed in a parent-of-origin specific manner. Recent years have seen a great expansion in the range of alterations in regulation, dosage or DNA sequence shown to disturb imprinted gene expression, and the correspondingly broad range of resultant clinical syndromes. At the same time, however, it has become clear that this diversity of IDs has common underlying principles, not only in shared molecular mechanisms, but also in interrelated clinical impacts upon growth, development and metabolism. Thus, detailed and systematic analysis of IDs can not only identify unifying principles of molecular epigenetics in health and disease, but also support personalisation of diagnosis and management for individual patients and families.

New insights into the pathogenesis of Beckwith-Wiedemann and Silver-Russell syndromes: contribution of small copy number variations to 11p15 imprinting defects.

The imprinted 11p15 region is organized in two domains, each of them under the control of its own imprinting control region (ICR1 for the IGF2/H19 domain and ICR2 for the KCNQ1OT1/CDKN1C domain). Disruption of 11p15 imprinting results in two fetal growth disorders with opposite phenotypes: the Beckwith–Wiedemann (BWS) and the Silver–Russell (SRS) syndromes. Various 11p15 genetic and epigenetic defects have been demonstrated in BWS and SRS. Among them, isolated DNA methylation defects account for approximately 60% of patients. To investigate whether cryptic copy number variations (CNVs) involving only part of one of the two imprinted domains account for 11p15 isolated DNA methylation defects, we designed a single nucleotide polymorphism array covering the whole 11p15 imprinted region and genotyped 185 SRS or BWS cases with loss or gain of DNA methylation at either ICR1 or ICR2. We describe herein novel small gain and loss CNVs in six BWS or SRS patients, including maternally inherited cis‐duplications involving only part of one of the two imprinted domains. We also show that ICR2 deletions do not account for BWS with ICR2 loss of methylation and that uniparental isodisomy involving only one of the two imprinted domains is not a mechanism for SRS or BWS. Hum Mutat 32:1171–1182, 2011. ©2011 Wiley‐Liss, Inc.

Epigenetics, genomic imprinting and assisted reproductive technology

Epigenetic mechanisms play a key role in regulating gene expression. One hallmark of these modifications is DNA methylation at cytosine residues of CpG dinucleotides in gene promoters, transposons and imprinting control regions. Genomic imprinting refers to an epigenetic marking of genes that results in monoallelic expression depending on their parental origin. There are two critical time periods in epigenetic reprogramming: gametogenesis and early preimplantation development. Major reprogramming takes place in primordial germ cells in which parental imprints are erased and totipotency is restored [1]. Imprint marks are then and re-established during spermatogenesis or oogenesis, depending on sex [1-3]. Upon fertilization, genome-wide demethylation occurs followed by a wave of de novo methylation, both of which are resisted by imprinted loci [4]. Epigenetic patterns are usually faithfully maintained during development. However, this maintenance sometimes fails, resulting in the disturbance of epigenetic patterns and human disorders. For example, two fetal growth disorders, the Beckwith-Wiedemann (BWS) and the Silver-Russell (SRS) syndromes with opposite phenotypes, are caused by abnormal DNA methylation at the 11p15 imprinted locus [5-7]: respectively loss of methylation at the Imprinting Region Center (ICR2) or gain of methylation at ICR1 in BWS and loss of methylation at ICR1 in SRS. Early embryogenesis is a critical time for epigenetic regulation, and this process is sensitive to environmental factors. The use of assisted reproductive technology (ART) has been shown to induce epigenetic alterations and to affect fetal growth and development [8-11]. In humans, several imprinting disorders, including BWS, occur at significantly higher frequencies in children conceived with the use of ART than in children conceived spontaneously [12,13]. The cause of these epigenetic imprinting disorders (following ART, unfertility causes, hormonal hyperstimulation, in vitro fertilization-IVF, Intracytoplasmic sperm injection-ICSI, micro-manipulation of gametes, exposure to culture medium, in vitro ovocyte maturation, time of transfer) remains unclear. However, recent data have shown that in patients with BWS or SRS, including those born following the use of ART, the DNA methylation defect involves imprinted loci other than 11p15 [14,15] (11p15 region: CTCF binding sites at ICR1, H19 and IGF2 DMRs, KCNQ1OT1 [ICR2], SNRPN [chromosome 15 q11-13], PEG/MEST1 [chromosome 7q31], IGF type2 receptor and ZAC1 [chromosome 6q26 et 6q24 respectively], DLK1/GTL2-IG-DMR [chromosome 14q32] and GNAS locus [chromosome 20q13.3]). This suggests that unfaithful maintenance of DNA methylation marks following fertilization involves the dysregulation of a trans-acting regulatory factor that could be altered by ART.

Simultaneous Hyper‐ and Hypomethylation at Imprinted Loci in a Subset of Patients with GNAS Epimutations Underlies a Complex and Different Mechanism of Multilocus Methylation Defect in Pseudohypoparathyroidism Type 1b

Most patients with pseudohypoparathyroidism type 1b (PHP‐1b) display a loss of imprinting (LOI) encompassing the GNAS locus resulting in PTH resistance. In other imprinting disorders, such as Russell–Silver or Beckwith–Wiedemann syndrome, we and others have shown that the LOI is not restricted to one imprinted locus but may affect other imprinted loci for some patients. Therefore, we hypothesized that patients with PHP‐1b might present multilocus imprinting defects. We investigated, in 63 patients with PHP‐1b, the methylation pattern of eight imprinted loci: GNAS, ZAC1, PEG1/MEST, ICR1, and ICR2 on chromosome 11p15, SNRPN, DLK1/GTL2 IG‐DMR, and L3MBTL1. We found multilocus imprinting defects in four PHP‐1b patients carrying broad LOI at the GNAS locus (1) simultaneous hypermethylation at L3MBTL1 differentially methylated region 3 (DMR3), and hypomethylation at PEG1/MEST DMR (n = 1), (2) hypermethylation at the L3MBTL1 (DMR3) (n = 1) and at the DLK1/GTL2 IG‐DMR (n = 1), and (3) hypomethylation at the L3MBTL1 DMR3 (n = 1). We suggest that mechanisms underlying multilocus imprinting defects in PHP‐1b differ from those of other imprinting disorders having only multilocus loss of methylation. Furthermore, our results favor the hypothesis of “epidominance”, that is, the phenotype is controlled by the most severely affected imprinted locus.