Detection of Hypomethylation Syndrome among Patients with

J C E M

O N L I N E

A d v a n c e s

i n

G e n e t i c s — E n d o c r i n e

R e s e a r c h

Detection of Hypomethylation Syndrome among Patients with Epigenetic Alterations at the GNAS Locus Gustavo Perez-Nanclares,* Valeria Romanelli,* Sonia Mayo,* Intza Garin, Celia Zazo, Eduardo Fernandez-Rebollo, Francisco Martínez, Pablo Lapunzina, Guiomar Pe´rez de Nanclares, and the Spanish PHP Group Endocrinology and Diabetes Research Group (G.P.-N.), Hospital de Cruces, E-48903 Barakaldo, Basque Country, Spain; Instituto de Gene´tica Me´dica y Molecular (V.R., P.L.), Instituto de Investigacioń Hospital Universitario La Paz, Universidad Autońoma de Madrid, E-28049 Madrid, Spain; Centro de Investigacioń Biome´dica en Red de Enfermedades Raras U753 (V.R., P.L.), Instituto de Salud Carlos III, E-28029 Madrid, Spain; Unidad de Genetica y Diagnostico Prenatal (S.M., F.M.), Hospital Universitario La Fe, E-46009 Valencia, Spain; Molecular (Epi)Genetics Laboratory (I.G., C.Z., G.P.d.N.), Research Unit, Hospital Txagorritxu, E-01009 Vitoria-Gasteiz, Spain; and Endocrine Unit (E.F.-R.), Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Context: Genomic imprinting is the modification of the genome so that genes from only one (rather than two) of the parental alleles are expressed. The mechanism underlying imprinting is epigenetic, occurring via changes in DNA methylation and histone modifications rather than through alterations in the DNA sequence. To date, nine different imprinting disorders have been clinically and genetically identified and a considerable research effort has been focused on determining the cause of the corresponding methylation defects. Objective: Our objective was to identify multilocus imprinting defects and characterize any mutations in trans-acting genes in patients with pseudohypoparathyroidism (PHP) caused by epigenetic alterations at GNAS locus. Design: We have investigated multilocus imprinting defects in 22 PHP patients with aberrant methylation at the GNAS locus not due to previously described deletions or to paternal uniparental disomy (UPD) of chromosome 20. Results: We found that, in contrast to what has been described in growth disorders, multilocus hypomethylation is an uncommon event in PHP patients. We were also unable to identify any genetic alteration causative of the epigenetic defects in the currently known methylation regulatory genes. Conclusion: Our work suggests that a trans-acting gene regulating the establishment or maintenance of imprinting at GNAS locus, if it exists, should be specific to PHP cases caused by epigenetic defects at GNAS. (J Clin Endocrinol Metab 97: E1060 –E1067, 2012)

G

enome imprinting describes the monoallelic parental-dependent expression of a subset of genes. The majority of them (⬃80%) are physically linked in clusters called differentially methylated regions (DMR) (1).

The most common congenital pathologies associated with imprinted genes are Prader-Willi syndrome (2), Angelman syndrome (2), Beckwith-Wiedemann syndrome (BWS) (3), Silver-Russell syndrome (SRS) (4), transient

ISSN Print 0021-972X ISSN Online 1945-7197 Printed in U.S.A. Copyright © 2012 by The Endocrine Society doi: 10.1210/jc.2012-1081 Received January 11, 2012. Accepted March 12, 2012. First Published Online April 4, 2012

* G.P.-N., V.R., and S.M. contributed equally to this work and should be considered joint first authors. Abbreviations: AD, Autosomal dominant; AHO, Albright’s hereditary osteodystrophy; BWS, Beckwith-Wiedemann syndrome; DMR, differentially methylated regions; LOM, loss of methylation; MLPA, multiplex ligation-dependent probe amplification; MS, methylation-specific; PHP, pseudohypoparathyroidism; QMPSF, quantitative multiplex PCR of short fluorescent fragments; SRS, Silver-Russell syndrome; TNDM, transient neonatal diabetes mellitus; UPD, uniparental disomy.

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J Clin Endocrinol Metab, June 2012, 97(6):E1060 –E1067


neonatal diabetes mellitus (TNDM) (5), pseudohypoparathyroidism (PHP) (6), and chromosome 14 uniparental disomy (UPD) (7). It is believed that imprinting alterations may affect more than one locus at a functional level, giving rise to similar phenotypes for different disorders (8). This was supported by some resemblances between BWS and TNDM phenotypes (umbilical hernia, intrauterine growth restriction, macroglossia, tumors, and insulin defects), which drove Arima et al. (8) to look for connections between p57Kip2 (CDKN1C) and ZAC, candidates for BWS and TNDM, respectively. Despite that no direct interaction between both genes was described, they reported that ZAC binds and activates LIT1/KCNQ1OT1, another imprinted gene located in IC2 (imprinting center 2), the causative region for BWS that, besides, simultaneously regulates p57Kip2. Thus, both genes are indirectly related. In their study, they did not find any methylation defect at ZAC in patients affected with BWS, but in patients with TNDM, they found loss of methylation (LOM) at LIT1. Following this hypothesis, Mackay et al. (9) reported two patients with TNDM and one with a methylation defect at the maternal 6q24 region and 11p15 DMR, but with no aberrant methylation patterns in 15q11-13 region (Prader-Willi syndrome/Angelman syndrome related). According to this study, the relationship of both loci did not happen at a functional level, but there was a common mechanism that explained the LOM at both imprinted regions, suggesting that other imprinted regions along the genome could undergo LOM by the same mechanism. In addition, the fact that the TNDM phenotype was more evident than the BWS in their patients drove them to state that there was mosaicism at the methylation level that supported the clinical appearance (9). They concluded that over 50% of individuals with hypomethylation at 6q24 also show mosaic DNA hypomethylation at other imprinted loci through the genome and also a range of additional clinical features (10). Similar studies have been run on different imprinting disorders as BWS (11, 12) and SRS (13, 14), identifying alterations at multiple imprinted genomic regions, leading to the suggestion of the existence of a maternal hypomethylation syndrome (15). The clinical features of patients with this syndrome could be explained by both the deregulation at several loci and the clear tissue mosaicism (blended with alterations from other pathologies) (15). Looking for factors that affect the imprinting establishment during early development, either during the establishment of germline marks or during the maintenance of them after fertilization, new studies are focusing on target genes that may be involved in these processes. Thus, some groups have reported that patients with TNDM carry mu-


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tations in the ZFP57 gene, which is essential in the maintenance of the DNA methylation during the early multicellular stages of the development (10). Other studies have found mutations in the germline at the NLRP2 gene, which is involved in the imprinting establishment of the early stages of the development in BWS patients and hypomethylation at PEG1 (16). Patients affected by PHP due to epigenetic alterations [which should include patients classically diagnosed as PHP type Ib (PHP-Ib; MIM 603233) and also patients with PHP and mild Albright’s hereditary osteodystrophy (AHO) features (17, 18)] show methylation changes at one or several of the four DMR within GNAS complex locus. PHP-Ib can follow an autosomal dominant (AD) trait (AD-PHP-Ib) or it can occur as an apparently sporadic disorder. AD-PHP-Ib is caused most frequently by specific deletions at the syntaxin 16 gene (STX16) referred to as STX16del4-6 or STX16del2-4; however, both deletions are associated, when inherited maternally, with an identical LOM at GNAS exon A/B (also referred to as 1A) alone (19, 20). Other maternally inherited microdeletions that cause AD-PHP-Ib have been identified within GNAS (referred to as delNESP55/delAS3-4 and delAS3-4, respectively), and these lead in affected individuals to a loss of all maternal GNAS methylation imprints (21, 22). Similarly broad GNAS methylation changes are also observed in an apparently sporadic form of PHP-Ib and in patients with mild AHO phenotype and PHP, in which affected patients show clinical and laboratory abnormalities that are indistinguishable from those observed in AD-PHP-Ib (23). None of the previously identified mutations have been revealed in these cases, but some of these patients, all with broad methylation changes, were shown to be affected by paternal uniparental isodisomy (pat20iUPD) involving part of or the whole long arm of chromosome 20, which includes the GNAS locus (24 –26). The aim of the present work was to investigate multilocus imprinting defects in patients with PHP-Ib caused by epigenetic alterations at the GNAS locus.

Patients and Methods The Institutional Review Board approved the study. Genetic analyses were performed after informed consent of the patient or parents. We analyzed the methylation status of eight imprinted loci, most of them associated with human imprinting disorders, in 22 PHP-Ib patients with methylation alteration at GNAS locus not due to previously associated deletions (19 –22) nor paternal 20qUPD. Methylation analysis of the GNAS locus had been done by methylation-specific (MS)-multiplex ligation-dependent probe amplification (MLPA), revealing in 16 of 22 PHP-Ib patients complete LOM at the three maternal methylation im-

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Hypomethylation Syndrome in PHP

prints (NESPas, XLas, and A/B) and a gain of methylation at the NESP55 DMR. The rest of the PHP-Ib patients presented partial LOM at XL␣s (25). MS-MLPA dosage results and microsatellite studies at chromosome 20 excluded previously described microdeletions and pat20UPD as the cause of the epigenetic defects. Clinical characteristics are summarized on Table 1. The investigated regions were six maternally methylated loci: ZAC1 at 6q24 implicated in TNDM; IGF2R at 6q26 involved in large offspring syndrome in cattle and sheep (27, 28); PEG1/ MEST at 7q32, which paternal knockout allele causes intrauterine growth restriction and behavior defect in mice (29); LIT1IC2 at 11p15 implicated in BWS; SNRPN at 15q11-13, the locus involved in Angelman and Prader-Willi syndromes; and GRB10 at 7p11.2 involved in SRS. And two paternally methylated loci were investigated: H19-IC1 at 11p15, involved in BWS and SRS, and DLK1/GTL2 at 14q32, implied in the maternal and the paternal UPD14 syndromes (7, 30).

Methylation-specific MLPA Dosage and methylation analyses of SNRPN, LIT1, and H19 were carried out by MS-MLPA using different kits: SALSA ME028 and ME030-B1 (MRC-Holland, Amsterdam, The Netherlands). The protocol was implemented following the manu-


facturer’s recommendations. Analysis of the MS-MLPA PCR products was performed on an ABI3500 genetic analyzer and using the GeneMapper software (Applied Biosystems, Foster City, CA) and analyzed as previously described (31).

Sodium bisulfite conversion and MS-PCR Previously to MS-PCR assay, genomic DNA of controls and patients were treated with bisulfite using the EpiTect bisulfite Kit (QIAGEN, Hilden, Germany) according to the manufacturer’s instructions. MS-PCR was performed for PLAGL1/ZAC, GRB10, IGF2R, and PEG1/MEST loci, as previously described (10).

Quantitative multiplex PCR of short fluorescent fragments (QMPSF) Amplification of a fragment of the GTL2 CpG island was performed together with three DMR of the H19, LIT1, and SNRPN genes, which also present hypermethylation of one of the two alleles (primer sequences and details are available upon request). Undigested and HpaII-digested (methylated) DNA of both patients and controls were used as a template for a FAMlabeled multiplex PCR under semiquantitative conditions. Re-

TABLE 1. Clinical, genetic, and biochemical features of the patient cohort at diagnosis Patient 2 3 8 9 10 11 12 13 16 17 19 20 21 22 1 4 5 6 7 14 15 18

Dx PHP-Ib PHP-Ib PHP-Ib PHP-Ib PHP-Ib PHP-Ib PHP-Ib PHP-Ib PHP-Ib PHP-Ib PHP-Ib PHP-Ib PHP-Ib PHP-Ib PHP-Ib; mild AHO (Br*, RF) PHP-Ib; mild AHO (Br, RF, Ob) PHP-Ib; mild AHO (Br*, RF) PHP-Ib; mild AHO (Br, RF, Ob) PHP-Ib; mild AHO (Br, Ob) PHP-Ib; mild AHO (Br*, Ow) PHP-Ib; mild AHO (Br, RF, Ow) PHP-Ib; mild AHO (RF, Ow, SS)

Ca (mM); NV ⴝ 2.2–2.6 1.9 NA 1.6 1.6 1.4 1.5 1.6 1.8 2.1 0.9 NA NA NA 2.4 1.6

P (mM)a 1.6 NA 1.8 1.7 2.7 1.9 1.8 1.6 1.6 3.0 NA NA NA 1.3 1.7

Age Dx (yr) 28 29 32 34 13 19 33 23 35 5 NA NA 45 20 19

157

1.7

2.4

19

102

1.5

2.3

31

569

1.6

3.4

8

NA

126

1.8

1.6

34

74.5

25.6

451

1.6

1.9

33

144.5

56.4

27.0

154

1.7

1.6

49

113.3

26.8

976

1.9

2.5

7

PTH (pg/ml); BMI NV ⴝ 40 – 65 NA 456 26.8 NA 24.7 371 25.6 335 24.5 58.5 NA 140.1 23.4 73 23.2 224 35.5 111 NA 771 NA NA NA NA NA 300 NA 471 26.3 500

Sex F F F M F F M F F F F F M M F

Height (cm) NA 147.3 164 176 169 NA 163.3 149.3 149.5 NA NA NA NA NA 156

Weight (kg) NA 58.3 66.5 79.3 70 NA 62.5 51.8 79.3 NA NA NA NA NA 64

F

NA

54.3

NA

F

146

48

22.5

F

142.9

41.5

F

NA

NA

M

170.5

F M

p96

p92

BMI, Body mass index; Br, Brachydactyly, clinically evident; Br*, radiological evidence of brachydactyly; Dx, diagnosis; F, female; M, male; NA, not available; NV, normal value; Ob, obesity defined as BMI above 30 kg/m2 in adults or weight above the 95th percentile in children; Ow, overweight defined as BMI above 25 kg/m2 in adults or weight above the 90th percentile in children; p96, 96th percentile; p92, 92nd percentile; RF, round face; SS, short stature. a

Phosphorus normal values are 1.23–2.1 mM (1–3 yr), 1.19 –1.8 mM (4 –11 yr), 0.9 –1.74 mM (12–15 yr), and 0.77–1.3 mM (⬎15 yr).



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FIG. 1. A, Electropherogram of the MS-PCR results for the PEG1 locus in a control DNA (upper panel) and case 16 (lower panel) showing the complete loss of the methylated allele in the PHP patient; B, coamplification of LIT1, GTL2, H19, and SNRPN DMR by QMPSF. Absence of the GTL2 fragment after HpaII digestion in patient 20’s DNA (upper left panel) indicates a complete maternal LOM. Conversely, dosage analysis in the undigested patient’s sample (upper right panel) compared with control DNA (lower right panel) allows us to discard a deletion of the region as the cause of the observed LOM.

sulting PCR products were analyzed on an ABI 3500 genetic analyzer (Applied Biosystems). To assess the relative rate of methylation and dosage, GTL2 peak areas were normalized against the average of those of DMR and compared with normal controls. Data analysis was performed in Excel (Microsoft Office 2007) (32).

Microsatellites study To characterize chromosome 7 and rule out any deletion or UPD at this locus, polymorphic markers located at 7q32 were typed by fluorescent PCR (primers and conditions are available on request).

Molecular studies of ZFP57, NALP2, and NALP7 The coding exons and intron-exon boundaries of ZFP57, NALP2, and NALP7 genes were amplified, and both strands of amplicons were sequenced as previously described (10, 16).

Results We could exclude methylation defects of the imprinted regions in 11p15, 15q11, 6q24-q27, and 7p12 in the an-

alyzed patients with PHP due to epigenetic defects at the GNAS locus. However, two patients did present complete LOM: one patient (case 16) at PEG1 (Fig. 1A) and the other one (case 20) at GTL2 (Fig. 1B) (Table 2). To exclude any deletion or UPD at these loci, we performed a microsatellite analysis close to PEG1 (7q32) and a QMPSF at the GTL2 locus. These results excluded deletion and UPD at these regions as the cause of the observed LOM (Figs. 1B and 2). By direct sequencing of the coding region and exonintron boundaries of ZFP57, we identified the variant p.Arg187Cys (c.559C⬎T, rs61730330) in homozygosis in one patient (case 7) and heterozygosis in two unrelated ones (cases 8 and 12). Analysis of 161 healthy people revealed that it was present in heterozygosis in eight individuals, so this variant was excluded as pathogenic. No genetic variation except for reported single-nucleotide polymorphisms was found at NALP2 and NALP7 genes.

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TABLE 2. Results of epigenetic analysis of imprinted loci Patient 1 4 6 7 8 9 10 11 12 13 14 16 18 20 21 22 2 3 5 15 17 19

NESP55 GOM GOM GOM GOM GOM GOM GOM GOM GOM GOM GOM GOM GOM GOM GOM GOM GOM GOM GOM GOM GOM GOM

NESPAS LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM

XL␣s LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM Partial LOM Partial LOM Partial LOM Partial LOM Partial LOM Partial LOM

A/B LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM LOM

PLAGL1/ ZAC N N N N N N N N N N N N N N N N N N N N N N

LIT1-IC2 ND N N N N N N N N N N N N N N N N N ND N N N

H19-IC1 N N N N N N N N N N N N N N N N N N N N N N

SNRPN ND N N N N N N N N N N N N N N N N N ND N N N

IGF2R ND N N N N N N N N N N N N N N N N N N N N N

GRB10 ND N ND N N N N N N N N N N N N N N N ND N N N

PEG1/ MEST ND N N N N N N N N N N LOM N N N N N N N N N N

DLK1/ GTL2 ND N N N N N N N N N N N N LOM N N N ND ND N N N

GOM, Gain of methylation; N, normal; ND, not done.

Discussion Patients with PHP-Ib present with PTH and, occasionally, mild TSH resistance, but resistance to other hormones is usually absent (33–35). They usually lack evidence of AHO and exhibit normal Gs␣ activity in erythrocytes and fibroblasts (33), although recent reports point to overlapping clinical and biochemical characteristics (17, 18, 36). Nevertheless, genomic DNA from PHP-Ib patients show a LOM at exon A/B, which is sometimes combined with epigenetic defects at other GNAS DMR (19, 24, 37). Maternally inherited deletions within STX16, NESP55, or exons 3 and 4 of NESPas have been identified in some of these patients (19 –22), leading to reduced Gs␣ expression in the proximal tubule. However, some patients with overall methylation defect at the GNAS locus do not present any underlying genetic cause. So, the question of whether alternative mechanisms could explain the methylation changes remains open. Isolated LOM at specific and exclusive DMR during development may be due to a random event or even other in cis variants remaining to be found; however, an alternative possible explanation would be the existence of a mutation at a trans-acting gene that directly or indirectly regulates methylation, as has been proposed for other imprinted genes (10, 16). For example, it has been reported that ZFP57, a multizinc-finger gene, is implicated in the etiology of multiple hypomethylation in TNDM (10). This protein, which is enriched in undifferentiated embryonic stem cells (38), may be required for the maintenance of DNA methylation at specific

DMR in early embryogenesis as the 6q24 region. On the other hand, NALP2 and NALP7 encode members of the NLRP family of CATERPILLER proteins that have not been extensively studied but seem to have a role in the establishment of imprinting marks during the oogenesis and/or in their maintenance in the early embryo (39) and have been involved in some imprinting disorders (16). It is possible that a defect in a similar regulatory protein is involved in methylation establishment and maintenance in PHP. Works by other groups have shown that some imprinting disorders, such as TNDM, BWS, and SRS, can include methylation abnormalities at multiple different genomic regions in the context of what is called hypomethylation syndrome (9, 11–14). Our results suggest that PHP-Ib not due to proximal deletions can occasionally also show LOM at other loci. However, the maternal hypomethylation syndrome is not frequent among patients diagnosed as PHP, suggesting that multiple imprinting defects are not infrequent in growth disorders, but they are in metabolic disorders (PHP). However, given that PHP-Ib is a rare endocrine disease, only a relatively small number of patients have been analyzed, and it remains possible that there is generalized hypomethylation in some patients with PHP-Ib. In addition, it is possible that extending the analysis to other maternally methylated DMR beyond those studied here will increase the proportion of PHP-Ib patients with maternal hypomethylation syndrome. Regarding the influence of the other methylation alterations on the phenotype of the patients with maternal hy-



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(10, 40, 41). Because no clinical data are available for patient 20, nothing can be discussed about the role of GTL2 on her phenotype. However, regarding patient 16, PEG1/MEST is believed to be involved in fetal growth, although its precise function is unknown. In fact, disruption of the PEG1/MEST gene causes embryonic growth retardation when paternally transmitted (29). So, a loss of maternal methylation marks at this locus may therefore result in the biallelic expression of PEG1/MEST and fetal overgrowth. Indeed, patient 16 presents the highest body mass index of the series, maybe influenced by the LOM at MEST, as has been suggested for other cases with multiple hypomethylation defects (9, 10, 15). On the other hand, the existence of a multilocus hypomethylation syndrome suggests that the establishment and/or the maintenance of methylation at imprinted regions is/are regulated by specific trans-acting elements. So far, however, no such regulating element has been identified for the GNAS locus. Indeed, no evidence of the existence of this trans-acting agent has been reported for this specific locus, and in the present study, no patient presented mutations at the three analyzed genes (ZFP57, NALP2, and NALP7). Although the imprinted defects in several diseases are not exclusive to the corresponding DMR, the regulating elements apparently act through a more specific disease-dependent pathway. In fact, to our knowledge, no cases of BWS caused by mutations at ZFP57 (42) or TNDM cases caused by mutations at NLRP2 have been reported. This suggests that the GNAS-controlling element, if it exists, should be searched for in PHP patients.

Acknowledgments We thank all family members for their participation and their physicians for sending us the samples. Members of the Spanish PHP Group include the following institutions: Complejo Hospitalario de Caćeres, Caćeres (J. Arroyo); Complejo Hospitalario Dr. Negrin, Las Palmas de Gran Canaria (F. Saéz and A. Sańchez); Complejo Hospitalario Materno Insular, Las Palmas de Gran Canaria (A. Domínguez and A. Santana); Complejo Hospitalario Universitario de Albacete, Albacete (R. Ruiz); Complexo Hospitalario Universitario Santiago de Com-

FIG. 2. A, Scheme showing the locations of the analyzed microsatellites relative to PEG1; B, electropherograms of the microsatellite analysis of four markers close to PEG1 (D7S2544, D7S2519, D7S2531, and D7S649) performed in case 16 and her parents, in which UPD or deletion was excluded as the cause for the observed LOM.

pomethylation syndrome, it has recently been proposed that the principal phenotype is under the effect of the symptoms of other diseases, depending on the loci and tissues affected, supporting the mosaicism-based hypothesis

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postela, A Corunã (L. Castro); Hospital Central de Asturias, Asturias (C. Rivas); Hospital Clínico San Carlos, Madrid (O. Pe´rez); Hospital de Barbanza, A Corunã (S. Molinos), Hospital Universitario de Cruces, Bizkaia (L. Castanõ, S. Gaztambide, M. D. Moure, A. Rodríguez, and A. Vela); Hospital de Donostia, Gipuzkoa (R. Saez and G. Unanue); Hospital de Navarra, Navarra (E. Meneńdez and E. Anda); Hospital de Nens, Barcelona (C. Pavia); Hospital Universitario de Alava-Txagorritxu, A´lava (I. Diez-Lopez); Hospital del Mar, Barcelona (M. Bonet); Hospital Do Meixoeiro, Vigo (M. J. Morales); Hospital General de Alicante, Alicante (M. Zapico); Hospital General de Ciudad Real, Ciudad Real (M. Aguirre); Hospital Infantil Universitario del Ninõ Jesu´s, Madrid (M. T. Munõz, O. Rubio-Cabezas, and J. Argente); Hospital Infantil Vall D⬘Hebron, Barcelona (L. Audi and D. Yeste); Hospital Materno Infantil Carlos Haya, Ma´laga (F. Soriguer); Hospital Príncipe de Viana, Navarra (M. García, R. M. Rodríguez, and M. J. Gon˜i); Hospital Puerta del Mar; Ca´diz (D. Armenta and D. Gonzalez-Duarte); Hospital Ramoń y Cajal, Madrid (R. Barrio); Hospital San Jorge, Huesca (A. Ca´mara); Hospital Sant Joan de Deú, Barcelona (L. Martorell, L. Sua´rez, R. Cardona, and E. Gean); Hospital Severo Ochoa, Madrid (B. García-Cuartero); Hospital Teresa de Herrera, A Corunã (M. S. Pereira and B. Rodríguez); Hospital Universitario 12 de Octubre, Madrid (S. Azriel and J. Sanchez del Pozo); Hospital Universitario de Guadalajara, Guadalajara (J. M. Jimeńez and L. Sentchordi); Hospital Universitario de Valme, Sevilla (R. Espino-Aguilar); Hospital Universitario La Fe, Valencia (M. Beneyto); Hospital Universitario La Paz, Madrid (C. A´lvarez and B. Lecumberri); Hospital Universitario Marques de Valdecilla, Cantabria (C. Luzuriaga); Hospital Universitario Miguel Servet, Zaragoza (M. T. Calvo and J. I. Labarta); Hospital Universitario Príncipe de Asturias, Madrid (P. Saavedra); Hospital Universitario Reina Sofía, Co´rdoba (R. Can˜ete Estrada); Hospital Universitario San Cecilio, Granada (R. Ordunã); Hospital Universitario Virgen de la Arriaxaca, Murcia (E. Guillen-Navarro, C. Guillen, and F. Gon˜i); Hospital Universitario Virgen del Rocío, Sevilla (J. Del Valle); Hospital Virgen de la Salud, Toledo (I. Luque, A. Meneńdez, and A. Vicente); and Hospital Virgen del Camino, Navarra (S. Berrade and M. Oyarzabal). Address all correspondence and requests for reprints to: Guiomar Perez de Nanclares, Ph.D., Molecular (Epi)Genetics Lab Research Unit, Hospital Txagorritxu, E-01009, VitoriaGasteiz, Alava, Spain. E-mail: [email protected]. G.P.d.N. is cofunded by the I3SNS Program of the Spanish Ministry of Health (CP03/0064; SIVI 1395/09). G.P.-N. and I.G. are supported by FIS programs I3SNS-ECA07/036 and I3SNSCA10/01056, respectively. S.M. is supported by Fundacioń para la Investigacioń del Hospital la Fe/Fundacioń Bancaja fellowship. This work was partially supported by Grants GV2008/ 111035 from the Basque Department of Health, BIO08/ER/001, Fundacion Eugenio Rodriguez Pascual, and grants from the Instituto de Salud Carlos III to G.P.D.N. (PI10/0148) and to V.R. and P.L. (PI08/1360). Disclosure Summary: There is no conflict of interest.


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