Downloaded from http://adc.bmj.com/ on January 7, 2015 - Published by group.bmj.com
PostScript
Underdiagnosed Beckwith– Wiedemann syndrome among early onset obese children Childhood obesity is mostly multifactorial, although a proportion of cases have a highly penetrant genetic aetiology. Identifying a genetic cause of childhood obesity can have important implications for patients and relatives. Beckwith–Wiedemann syndrome (BWS) is an overgrowth syndrome with widely variable clinical phenotype that attenuates with age.1 Proposed criteria to warrant its clinical suspicion are the presence of at least three major, or two major plus one minor, features (table 1). Early diagnosis of BWS is relevant because of the increase in risk for malignancies (Wilms tumour and hepatoblastoma). Periodical abdominal ultrasonographic survey and plasma α fetoprotein level determination during infancy and childhood must be done.1 2 Various genetic and epigenetic aberrations affecting the imprinted 11p15.5 locus can cause BWS, by downregulation of maternally expressed genes and/or upregulation of paternally expressed genes (figure 1A).3 The most common defects are the loss of methylation at IC2 (50%), uniparental paternal disomy at 11p15.5 (20%), gain of methylation at IC1 (5%) and point mutations in the maternal allele of CDKN1C. Duplications, inversions or translocations involving 11p15.5 are uncommon.3 Methylation-specific-multiplex ligation-dependent probe amplification (MS-MLPA) can detect methylation defects at 11p15.5 along with copy number variations in one single experiment, resulting in the diagnosis of more than 70% of patients with BWS.4 We have studied 159 children (95 males/64 females) with early onset severe obesity. A custom-made MS-MLPA assay, with HhaI as a methylation-sensitive restriction enzyme, was used to analyse blood cell DNA methylation at the 11p15.5 region, along with other differentially methylated regions also associated with syndromic obesity (15q11.2, 20q12, 14q32 and 7q31). The assay contains 11 probes, including one for the IC1 locus (H19) and one for IC2 (KCNQ1). Hypomethylation at the KCNQ1 locus was identified in two of the 159 patients (figure 1B). The same assay was useful to discard unbalanced genomic rearrangements or uniparental disomy at 11p15.5, considering that methylation at H19 was
Arch Dis Child October 2014 Vol 99 No 10
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Downloaded from http://adc.bmj.com/ on January 7, 2015 - Published by group.bmj.com
PostScript Table 1
Clinical features of patients (both females of European ancestry), with respect to the clinical criteria of BWS
Chronological age at first examination Birth weight Age at the onset of obesity Bone age at first examination (G&P) Ethnic group Sex Pubertal stage BMI Whole body fat (DXA) Height Target height Fasting glucose (mg/dL) Fasting insulin (mU/mL) HOMA index HbA1c Dyslipidaemia Plasma α-fetoprotein (N.V.1-15) Kidney ultrasonography BWS oriented background and exploratory findings
Patient 1
Patient 2
7.84 years −0.43 SDS 4.5 years 7.25 years Caucasian Female Tanner I 26.10 kg/m2 (+3.55 SDS) 18 612 g (42.5%; +3.15 SDS) 130.7 cm (+0.91 SDS) 169.0 cm (+1.35 SDS) 78 16.0 3.08 5.2% No 1.86 ng/mL (age 16.5 years) Normal (age 16.5 years) No
8.33 years +1.94 SDS 1 year 10 years Caucasian Female Tanner I 36.58 kg/m2 (+8.58 SDS) 37 078 g (44.8%; +3.19 SDS) 139.8 cm (+2.27 SDS) 162.1 cm (+0.15 SDS) 89 16.3 3.58 5.6% HDL +8 SDS) while in adolescence. BMI, Body Mass Index; BWS, Beckwith–Wiedemann syndrome; SDS, standard deviation score; HDL, high-density lipoprotein.
within the reference range. In patient 1, a decrease of ∼60% in the methylation level at KCNQ1 locus was detected with respect to controls, while in patient 2 the reduction was ∼33% (figure 1C). In both patients, buccal DNA was also analysed. While a decrease of ∼45% in the methylation level was identified in patient 1, no significant methylation reduction was detected in buccal DNA from patient 2. The different percentage of methylation reduction in different tissues suggests different degrees of mosaicism for the alteration in the two patients. Both patients had been referred due to obesity at around 8 years of age, but neither of them fulfilled the minimum criteria for clinical diagnosis of BWS (table 1). Tumour screening was done at ages 16 and 12 years, respectively. Kidney ultrasound and plasma α-fetoprotein levels were normal (table 1). In summary, patients with BWS can exhibit early onset obesity leading to misdiagnosis and misclassification as ‘common’ obesities preventing appropriate clinical surveillance. Hence, genetic testing for BWS should be considered in children with early onset obesity. MS-MLPA, detecting
966
methylation defects and copy number variations, is a useful and efficient diagnostic tool. Gabriel Á Martos-Moreno,1,2,3 Clara Serra-Juhé,4,5,6 Luis A Pérez-Jurado,4,5,6 Jesús Argente1,2,3 1
Department of Endocrinology, Hospital Infantil Universitario Niño Jesús, Instituto de Investigación La Princesa, Madrid, Spain 2 Department of Pediatrics, Universidad Autónoma de Madrid, Madrid, Spain 3 CIBER Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, Madrid, Spain 4 Genetics Unit, Universitat Pompeu Fabra, Barcelona, Spain 5 Program in Neurosciences, Hospital del Mar Research Institute (IMIM), Barcelona, Spain 6 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Barcelona, Spain Correspondence to Professor Jesús Argente, Department of Pediatrics, Hospital Infantil Universitario Niño Jesús & Universidad Autónoma de Madrid, Avenida Menéndez Pelayo, 65, Madrid 28009, Spain; [email protected] and Professor Luis A PérezJurado; [email protected]. Contributors GAMM, CS-J, contributed equally to the manuscript. GÁM-M wrote the first draft of the manuscript and he was in charge of controlling the patients; CS-J undertook the genetic studies; LAP-J and JA developed the intellectual idea, provided the funds
to undertake the study, analysed the results and read and edited the manuscript. Funding Fondo de Investigación Sanitaria (FIS PI10/ 00747 and PI13/02195 to JA, PI10/02512 and PI13/ 02481 to LAP-J, CIBERobn and CIBERER) and Catalan Department of Economy and Knowledge (2009SGR1274 and ICREA Acadèmia, to LAP-J). Competing interests None. Patient consent Obtained. Ethics approval Ethical Committee University Hospital Niño Jesús. Provenance and peer review Not commissioned; externally peer reviewed.
To cite Martos-Moreno GÁ, Serra-Juhé C, PérezJurado LA, et al. Arch Dis Child 2014;99:965–967. Accepted 11 July 2014 Published Online First 1 August 2014 Arch Dis Child 2014;99:965–967. doi:10.1136/archdischild-2014-307097
REFERENCES 1
Weksberg R, Schuman C, Beckwith B. BeckwithWiedemann syndrome and hemyhyperplasia. In Cassidy SB, Allanson JE, eds. Management of genetic syndromes. 3rd edn. New Jersey: Wiley-Blackwell, 2010:129–48.
Arch Dis Child October 2014 Vol 99 No 10
Downloaded from http://adc.bmj.com/ on January 7, 2015 - Published by group.bmj.com
PostScript
Figure 1 (A) Schematic representation of the imprinted 11p15.5 region. There are two differentially methylated imprinting control regions in the interval, IC1 and IC2, regulating the parent-of-origin expression of two genes, H19 (non-coding RNA expressed from the maternal allele) and IGF2 (coding for insulin-like growth factor 2, expressed from the paternal allele). IC2 is methylated on the maternal allele, leading to non-expression of the non-coding KCNQ1OT1 gene and the activation of CDKN1C (encoding cyclin-dependent kinase inhibitor 1C) and KCNQ1 (encoding the potassium voltage-gated channel, KQT-like subfamily member 1). IGF2 and CDKN1C are growth regulators directly related to the pathogenesis of Beckwith–Wiedemann syndrome (BWS). Paternally expressed IGF2 stimulates growth and cell proliferation, while maternally expressed CDKN1C restrains growth. Hypomethylation at IC2, found in 50% of BWS including these two patients, is shown in the scheme below. This epigenetic alteration leads to the expression of KCNQ1OT1 on the maternal allele and subsequently reduces expression of CDKN1C and KCNQ1; (B) Photographs of patient 1 at age 16; photographs of patient 2 at age 14. (C) MS-MLPA results of both patients. Probes from the 11p15.5 region are shown in grey (H19-M2 and KCNQ1-M5), among other probes used as dosage (C1 and C2) and digestion controls (D1 and D2). In each case, the peak pattern displayed above represents the non-digested products. The patterns below display the methylation status after digestion with a methylation-sensitive restriction enzyme (in blood cell DNA and buccal DNA in patients). No methylation alteration was detected with the H19 probe. However, a reduction in the peak of the KCNQ1 probe was detected in both patients not significant in buccal DNA from patient 2, implying a decrease of methylation status at this locus present in mosaicism. Z-score value was −8.072 (blood cell DNA replica 1), −9.218 (blood cell DNA replica 2) and −5.096 (buccal DNA) for patient 1 and −4.802 (blood cell DNA replica 1) and −5.698 (blood cell DNA replica 2) for patient 2 (reference range: −2.219, 2.733)). 2
Zarate YA, Mena R, Martin LJ, et al. Experience with hemihyperplasia and Beckwith-Wiedemann syndrome surveillance protocol. Am J Med Gen 2009;149A:1691–7.
Arch Dis Child October 2014 Vol 99 No 10
3
Demars J, Gicquel C. Epigenetic and genetic disturbance of the imprinted 11p15 region in Beckwith-Wiedemann and Silver-Russell syndromes. ClinGenet 2012;81:350–61.
4
Priolo M, Sparago A, Mammì C, et al. MS-MLPA is a specific and sensitive technique for detecting all chromosome 11p15.5 imprinting defects of BWS and SRS in a singletube experiment. Eur J Hum Genet 2008;16:565–71.
967
Downloaded from http://adc.bmj.com/ on January 7, 2015 - Published by group.bmj.com
Underdiagnosed Beckwith−Wiedemann syndrome among early onset obese children Gabriel Á Martos-Moreno, Clara Serra-Juhé, Luis A Pérez-Jurado and Jesús Argente Arch Dis Child 2014 99: 965-967 originally published online August 1, 2014
doi: 10.1136/archdischild-2014-307097 Updated information and services can be found at: http://adc.bmj.com/content/99/10/965.2
These include:
References Email alerting service
This article cites 3 articles, 0 of which you can access for free at: http://adc.bmj.com/content/99/10/965.2#BIBL Receive free email alerts when new articles cite this article. Sign up in the box at the top right corner of the online article.
Notes
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PostScript
Underdiagnosed Beckwith– Wiedemann syndrome among early onset obese children Childhood obesity is mostly multifactorial, although a proportion of cases have a highly penetrant genetic aetiology. Identifying a genetic cause of childhood obesity can have important implications for patients and relatives. Beckwith–Wiedemann syndrome (BWS) is an overgrowth syndrome with widely variable clinical phenotype that attenuates with age.1 Proposed criteria to warrant its clinical suspicion are the presence of at least three major, or two major plus one minor, features (table 1). Early diagnosis of BWS is relevant because of the increase in risk for malignancies (Wilms tumour and hepatoblastoma). Periodical abdominal ultrasonographic survey and plasma α fetoprotein level determination during infancy and childhood must be done.1 2 Various genetic and epigenetic aberrations affecting the imprinted 11p15.5 locus can cause BWS, by downregulation of maternally expressed genes and/or upregulation of paternally expressed genes (figure 1A).3 The most common defects are the loss of methylation at IC2 (50%), uniparental paternal disomy at 11p15.5 (20%), gain of methylation at IC1 (5%) and point mutations in the maternal allele of CDKN1C. Duplications, inversions or translocations involving 11p15.5 are uncommon.3 Methylation-specific-multiplex ligation-dependent probe amplification (MS-MLPA) can detect methylation defects at 11p15.5 along with copy number variations in one single experiment, resulting in the diagnosis of more than 70% of patients with BWS.4 We have studied 159 children (95 males/64 females) with early onset severe obesity. A custom-made MS-MLPA assay, with HhaI as a methylation-sensitive restriction enzyme, was used to analyse blood cell DNA methylation at the 11p15.5 region, along with other differentially methylated regions also associated with syndromic obesity (15q11.2, 20q12, 14q32 and 7q31). The assay contains 11 probes, including one for the IC1 locus (H19) and one for IC2 (KCNQ1). Hypomethylation at the KCNQ1 locus was identified in two of the 159 patients (figure 1B). The same assay was useful to discard unbalanced genomic rearrangements or uniparental disomy at 11p15.5, considering that methylation at H19 was
Arch Dis Child October 2014 Vol 99 No 10
965
Downloaded from http://adc.bmj.com/ on January 7, 2015 - Published by group.bmj.com
PostScript Table 1
Clinical features of patients (both females of European ancestry), with respect to the clinical criteria of BWS
Chronological age at first examination Birth weight Age at the onset of obesity Bone age at first examination (G&P) Ethnic group Sex Pubertal stage BMI Whole body fat (DXA) Height Target height Fasting glucose (mg/dL) Fasting insulin (mU/mL) HOMA index HbA1c Dyslipidaemia Plasma α-fetoprotein (N.V.1-15) Kidney ultrasonography BWS oriented background and exploratory findings
Patient 1
Patient 2
7.84 years −0.43 SDS 4.5 years 7.25 years Caucasian Female Tanner I 26.10 kg/m2 (+3.55 SDS) 18 612 g (42.5%; +3.15 SDS) 130.7 cm (+0.91 SDS) 169.0 cm (+1.35 SDS) 78 16.0 3.08 5.2% No 1.86 ng/mL (age 16.5 years) Normal (age 16.5 years) No
8.33 years +1.94 SDS 1 year 10 years Caucasian Female Tanner I 36.58 kg/m2 (+8.58 SDS) 37 078 g (44.8%; +3.19 SDS) 139.8 cm (+2.27 SDS) 162.1 cm (+0.15 SDS) 89 16.3 3.58 5.6% HDL +8 SDS) while in adolescence. BMI, Body Mass Index; BWS, Beckwith–Wiedemann syndrome; SDS, standard deviation score; HDL, high-density lipoprotein.
within the reference range. In patient 1, a decrease of ∼60% in the methylation level at KCNQ1 locus was detected with respect to controls, while in patient 2 the reduction was ∼33% (figure 1C). In both patients, buccal DNA was also analysed. While a decrease of ∼45% in the methylation level was identified in patient 1, no significant methylation reduction was detected in buccal DNA from patient 2. The different percentage of methylation reduction in different tissues suggests different degrees of mosaicism for the alteration in the two patients. Both patients had been referred due to obesity at around 8 years of age, but neither of them fulfilled the minimum criteria for clinical diagnosis of BWS (table 1). Tumour screening was done at ages 16 and 12 years, respectively. Kidney ultrasound and plasma α-fetoprotein levels were normal (table 1). In summary, patients with BWS can exhibit early onset obesity leading to misdiagnosis and misclassification as ‘common’ obesities preventing appropriate clinical surveillance. Hence, genetic testing for BWS should be considered in children with early onset obesity. MS-MLPA, detecting
966
methylation defects and copy number variations, is a useful and efficient diagnostic tool. Gabriel Á Martos-Moreno,1,2,3 Clara Serra-Juhé,4,5,6 Luis A Pérez-Jurado,4,5,6 Jesús Argente1,2,3 1
Department of Endocrinology, Hospital Infantil Universitario Niño Jesús, Instituto de Investigación La Princesa, Madrid, Spain 2 Department of Pediatrics, Universidad Autónoma de Madrid, Madrid, Spain 3 CIBER Fisiopatología de la Obesidad y Nutrición, Instituto de Salud Carlos III, Madrid, Spain 4 Genetics Unit, Universitat Pompeu Fabra, Barcelona, Spain 5 Program in Neurosciences, Hospital del Mar Research Institute (IMIM), Barcelona, Spain 6 Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Barcelona, Spain Correspondence to Professor Jesús Argente, Department of Pediatrics, Hospital Infantil Universitario Niño Jesús & Universidad Autónoma de Madrid, Avenida Menéndez Pelayo, 65, Madrid 28009, Spain; [email protected] and Professor Luis A PérezJurado; [email protected]. Contributors GAMM, CS-J, contributed equally to the manuscript. GÁM-M wrote the first draft of the manuscript and he was in charge of controlling the patients; CS-J undertook the genetic studies; LAP-J and JA developed the intellectual idea, provided the funds
to undertake the study, analysed the results and read and edited the manuscript. Funding Fondo de Investigación Sanitaria (FIS PI10/ 00747 and PI13/02195 to JA, PI10/02512 and PI13/ 02481 to LAP-J, CIBERobn and CIBERER) and Catalan Department of Economy and Knowledge (2009SGR1274 and ICREA Acadèmia, to LAP-J). Competing interests None. Patient consent Obtained. Ethics approval Ethical Committee University Hospital Niño Jesús. Provenance and peer review Not commissioned; externally peer reviewed.
To cite Martos-Moreno GÁ, Serra-Juhé C, PérezJurado LA, et al. Arch Dis Child 2014;99:965–967. Accepted 11 July 2014 Published Online First 1 August 2014 Arch Dis Child 2014;99:965–967. doi:10.1136/archdischild-2014-307097
REFERENCES 1
Weksberg R, Schuman C, Beckwith B. BeckwithWiedemann syndrome and hemyhyperplasia. In Cassidy SB, Allanson JE, eds. Management of genetic syndromes. 3rd edn. New Jersey: Wiley-Blackwell, 2010:129–48.
Arch Dis Child October 2014 Vol 99 No 10
Downloaded from http://adc.bmj.com/ on January 7, 2015 - Published by group.bmj.com
PostScript
Figure 1 (A) Schematic representation of the imprinted 11p15.5 region. There are two differentially methylated imprinting control regions in the interval, IC1 and IC2, regulating the parent-of-origin expression of two genes, H19 (non-coding RNA expressed from the maternal allele) and IGF2 (coding for insulin-like growth factor 2, expressed from the paternal allele). IC2 is methylated on the maternal allele, leading to non-expression of the non-coding KCNQ1OT1 gene and the activation of CDKN1C (encoding cyclin-dependent kinase inhibitor 1C) and KCNQ1 (encoding the potassium voltage-gated channel, KQT-like subfamily member 1). IGF2 and CDKN1C are growth regulators directly related to the pathogenesis of Beckwith–Wiedemann syndrome (BWS). Paternally expressed IGF2 stimulates growth and cell proliferation, while maternally expressed CDKN1C restrains growth. Hypomethylation at IC2, found in 50% of BWS including these two patients, is shown in the scheme below. This epigenetic alteration leads to the expression of KCNQ1OT1 on the maternal allele and subsequently reduces expression of CDKN1C and KCNQ1; (B) Photographs of patient 1 at age 16; photographs of patient 2 at age 14. (C) MS-MLPA results of both patients. Probes from the 11p15.5 region are shown in grey (H19-M2 and KCNQ1-M5), among other probes used as dosage (C1 and C2) and digestion controls (D1 and D2). In each case, the peak pattern displayed above represents the non-digested products. The patterns below display the methylation status after digestion with a methylation-sensitive restriction enzyme (in blood cell DNA and buccal DNA in patients). No methylation alteration was detected with the H19 probe. However, a reduction in the peak of the KCNQ1 probe was detected in both patients not significant in buccal DNA from patient 2, implying a decrease of methylation status at this locus present in mosaicism. Z-score value was −8.072 (blood cell DNA replica 1), −9.218 (blood cell DNA replica 2) and −5.096 (buccal DNA) for patient 1 and −4.802 (blood cell DNA replica 1) and −5.698 (blood cell DNA replica 2) for patient 2 (reference range: −2.219, 2.733)). 2
Zarate YA, Mena R, Martin LJ, et al. Experience with hemihyperplasia and Beckwith-Wiedemann syndrome surveillance protocol. Am J Med Gen 2009;149A:1691–7.
Arch Dis Child October 2014 Vol 99 No 10
3
Demars J, Gicquel C. Epigenetic and genetic disturbance of the imprinted 11p15 region in Beckwith-Wiedemann and Silver-Russell syndromes. ClinGenet 2012;81:350–61.
4
Priolo M, Sparago A, Mammì C, et al. MS-MLPA is a specific and sensitive technique for detecting all chromosome 11p15.5 imprinting defects of BWS and SRS in a singletube experiment. Eur J Hum Genet 2008;16:565–71.
967
Downloaded from http://adc.bmj.com/ on January 7, 2015 - Published by group.bmj.com
Underdiagnosed Beckwith−Wiedemann syndrome among early onset obese children Gabriel Á Martos-Moreno, Clara Serra-Juhé, Luis A Pérez-Jurado and Jesús Argente Arch Dis Child 2014 99: 965-967 originally published online August 1, 2014
doi: 10.1136/archdischild-2014-307097 Updated information and services can be found at: http://adc.bmj.com/content/99/10/965.2
These include:
References Email alerting service
This article cites 3 articles, 0 of which you can access for free at: http://adc.bmj.com/content/99/10/965.2#BIBL Receive free email alerts when new articles cite this article. Sign up in the box at the top right corner of the online article.
Notes
To request permissions go to: http://group.bmj.com/group/rights-licensing/permissions To order reprints go to: http://journals.bmj.com/cgi/reprintform To subscribe to BMJ go to: http://group.bmj.com/subscribe/
