June 2010 life.

The 15 breastfed infants were fed every 4 hours, by hospital protocol, starting as early as 2 and as late as 10 hours. The mean age of rst feed was 7.8 (2.6) hours, and infants were not roomed-in. The differences in methods might explain some of the differences in the results. In the Methods section of our article, we state that Only mothers. . . who remained with the infant after birth were included, which means that infants were roomed-in. We did not account for the insensible water loss that might occur during feeding. Casey et al5 corrected the weight on the basis of the insensible water loss calculated by Hendrikson et al.7 They conclude that the insensible water loss was 1.9 g/ kg/h over the rst year of life. They stated that studies were carried out indoors in homes heated in winter. Some used air conditioners in the summer months. Furthermore the authors stated that it should be noted that the humidity in Denver, Colorado, is well below the national average. We considered these conditions to be too different from ours in Brazil. Besides, in our study Infant clothing was standardized before being weighed. Infants with diaper were swaddled until weighed after being breastfed. Swaddling infants might have prevented weight to diminish caused by water loss, because perspiration, urine, and feces would remain in the fabric. We did not obtain any negative weight difference during the study, meaning that the amount of insensible water loss during feeding might not be very signicant in our study. For the regression equation we tried different models. None had signicant associations except for to have a companion. The regression presented in the article was calculated with variables that reached P # .1 in univariate analysis. As stated in our study, Birth weight was not correlated with colostrum intake, either in the univariate or in the regression equation. In their study, Dollberg et al4 stated that In backward stepwise regression analysis where intake on Day 1 was the dependent variable and birth weight (or gestational age), Apgar Score at ve minutes, age at rst feed, 24 hours weight loss and group (formula vs breast feeding) were the independent variables, only the group was signicant. In contrast, Dollberg and Mimouni state in their letter that they found a striking correlation. In any case, the birth weight of the newborns was 3145 309 g in our study and 3258 408 g in the study by Dollberg et al.4 One possibility is that the smaller variation in birth weight of our group did not permit us to show this correlation. Dollberg and Mimouni speculate that more mature infants may have a more mature suck and swallow mechanism. It is not possible to conrm this suggestion with our data. Walter Santoro, Jr. MD gio Martinez, MD Francisco Eulo Rubens Garcia Ricco, MD s Jorge, MD Salim Moyse nicas de Ribeira o Preto Hospital das Cl Departamento de Pediatria o Preto, Sa o Paulo, Brazil Ribeira
10.1016/j.jpeds.2010.02.060

LETTERS TO THE EDITOR

References
1. Saint L, Smith M, Hartmann PE. The yield and nutrient content of colostrum and milk of women from giving birth to 1 month post-partum. Br J Nutr 1984;52:87-95. 2. Hill PD, Aldag JC. Milk volume on day 4 and income predictive of lactation adequacy at 6 weeks of mothers of nonnursing preterm infants. J Perinat Neonatal Nurs 2005;19:273-82. 3. Kent JC, Mitoulas LR, Cregan MD, Ramsay DT, Doherty DA, Hartmann PE. Volume and frequency of breastfeedings and fat content of breast milk throughout the day. Pediatrics 2006;117:e387-95. 4. Dollberg S, Lahav S, Mimouni FB. A comparison of intakes of breast-fed and bottle-fed infants during the rst two days of life. J Am Coll Nutr 2001;20:209-11. 5. Casey CE, Neifert MR, Seacat JM, Neville MC. Nutrient intake by breast-fed infants during the rst ve days after birth. Am J Dis Child 1986;140:933-6. 6. Neville MC, Keller R, Seacat J, Lutes V, Neifert M, Casey C, et al. Studies in human lactation: milk volumes in lactating women during the onset of lactation and full lactation. Am J Clin Nutr 1988;48:1375-86. 7. Hendrikson EC, Seacat JM, Neville MC. Insensible weight loss in children under one year of age. Acta Paediatr Scand 1985;74:678-80.

The overlap between Sotos and BeckwithWiedemann syndromes

To the Editor: We read with great interest the recent report by Kato et al1 describing the noteworthy occurrence of a hepatoblastoma in a child with Sotos syndrome and typical NSD1 microdeletion. This is a remarkable nding, given that only one previous case has been reported.2 Conversely, the occurrence of hepatoblastoma is characteristic of Beckwith-Wiedemann syndrome, another overgrowth condition with cancer predisposition caused by epigenetic deregulation of chromosomal region 11p15. In this case, despite a normal genotype, DNA methylation anomalies led to tissue growth factor overexpression and cyclin deregulation responsible for the phenotype and cancer development.3 The mechanisms causing these methylation anomalies have not yet been fully explained, however. Actually, phenotypical overlap between Sotos and BeckwithWiedemann syndrome can occur. It is notable that molecular overlap also has been demonstrated, with paradoxical 11p15 epimutations in Sotos phenotypes and NSD1 mutations in Beckwith-Wiedemann phenotypes.4 These ndings have led to speculation that the NSD1 protein, a histone methyltransferase involved in epigenetic gene regulation,5 may be involved in establishing or maintaining 11p15 region imprinting.4 Previous studies have reported 11p15 region methylation anomalies in hepatoblastoma tissue6 and NSD1 silencing in other tumors.7 To the best of our knowledge, the cooccurrence of these 2 derangements has not yet been demonstrated or investigated, however. The case reported by Kato et al was investigated by singleucleotide polymorphism array technology, which identied, along with the germline 5q35 microdeletion encompassing NSD1, a somatic gain of chromosome 2 and a uniparental 18q disomy. However, as the authors discuss, that technique does not provide information about DNA epigenetic status. In this respect, we believe that investigating 11p15 region
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epigenetic anomalies in both blood leukocytes and cancer tissues might help unravel the still-unexplained overlap between these two overgrowth syndromes.

Alessandro Mussa, MD Biochemical Genetics Laboratory Division of Laboratory Genetics Department of Laboratory Medicine and Pathology Mayo Clinic College of Medicine Department of Pediatrics University of Torino Nicoletta Chiesa, PhD Department of Pediatrics University of Torino Francesco Porta, MD Biochemical Genetics Laboratory Division of Laboratory Genetics Department of Laboratory Medicine and Pathology Mayo Clinic College of Medicine Rochester, Minnesota Department of Pediatrics University of Torino Giuseppina Baldassarre, MD Margherita Cirillo Silengo, MD Giovanni Battista Ferrero, MD, PhD Department of Pediatrics University of Torino Torino, Italy
10.1016/j.jpeds.2010.02.063

To the Editor: We greatly appreciate the interest of Mussa et al in our study and would like to address their concerns. In our genome-wide scanning, copy number abnormalities of 11p were not detected in the tumor sample from our patient; however, as Mussa et al suggest, single-nucleotide polymorphism array technology does not provide information about epigenetic alterations. Because aberrant genomic imprinting of the 11p15 locus plays a pivotal role in the pathogenesis of Beckwith-Wiedemann syndrome,1 we further investigated the methylation status of the H19 gene within this region in both tumor tissue and corresponding normal tissue of our patient, as well as in a normal control, by methylation-specic polymerase chain reaction (PCR).2 In our analysis, the tumor and the peripheral blood samples demonstrated the same DNA methylation pattern, in which PCR products from both methylated and unmethylated alleles were obtained, indicating that the methylation status of the H19 locus in the tumor remained normal. Given that normally, the paternal copy of the H19 gene is methylated and the maternal copy is nonmethylated,3 our ndings indicate that both paternal and maternal 11p15 alleles are preserved in the tumor sample. On the other hand, given that NSD1 encodes a histone methyltransferase,4 loss of NSD1 might lead to epigenetic deregulation of 11p15 genes, including IGF2,5 via altered chromatin status without accompanying DNA methylation. Thus, it would have been intriguing to determine the status of histone methylation of IGF2 in the hepatoblastoma sample of the current case; unfortunately, however, insufcient tumor material precluded further analysis. The molecular mechanisms by which NSD1 deletion is associated with the overgrowth sydrome and tumorigenesis remain to be elucidated.

References
1. Kato M, Takita J, Takahashi K, Mimaki M, Chen Y, Koh K, et al. Hepatoblastoma in a patient with Sotos syndrome. J Pediatr 2009;155:937-9. 2. Saugier-Veber P, Bonnet C, Afenjar A, Drouin-Garraud V, Coubes C, Fehrenbach S, et al. Heterogeneity of NSD1 alterations in 116 patients with Sotos syndrome. Hum Mutat 2007;28:1098-107. 3. Weksberg R, Shuman C, Beckwith JB. Beckwith-Wiedemann syndrome. Eur J Hum Genet 2009;18:8-14. 4. Baujat G, Rio M, Rossignol S, Sanlaville D, Lyonnet S, Le Merrer M, et al. Paradoxical NSD1 mutations in Beckwith-Wiedemann syndrome and 11p15 anomalies in Sotos syndrome. Am J Hum Genet 2004;74:715-20. 5. Rayasam GV, Wendling O, Angrand PO, Mark M, Niederreither K, Song L, et al. NSD1 is essential for early post-implantation development and has a catalytically active SET domain. EMBO J 2003;22:3153-63. 6. Honda S, Arai Y, Haruta M, Sasaki F, Ohira M, Yamaoka H, et al. Loss of imprinting of IGF2 correlates with hypermethylation of the H19 differentially methylated region in hepatoblastoma. Br J Cancer 2008;99:1891-9. 7. Berdasco M, Ropero S, Setien F, Fraga MF, Lapunzina P, Losson R, et al. Epigenetic inactivation of the Sotos overgrowth syndrome gene histone methyltransferase NSD1 in human neuroblastoma and glioma. Proc Natl Acad Sci USA 2009;106:21830-5. 1036

Junko Takita, MD, PhD Department of Cell Therapy and Transplantation Medicine University of Tokyo Tokyo, Japan
10.1016/j.jpeds.2010.02.069

References
1. Reik W, Brown KW, Slatter RE, Sartori P, Elliott M, Maher ER. Allelic methylation of H19 and IGF2 in the Beckwith-Wiedemann syndrome. Hum Mol Genet 1994;3:1297-301. 2. Suzuki M, Kato M, Yuyan C, Takita J, Sanada M, Nannya Y, et al. Wholegenome proling of chromosomal aberrations in hepatoblastoma using high-density single-nucleotide polymorphism genotyping microarrays. Cancer Sci 2008;99:564-70. 3. DeBaun MR, Niemitz EL, McNeil DE, Brandenburg SA, Lee MP, Feinberg AP. Epigenetic alterations of H19 and LIT1 distinguish patients with Beckwith-Wiedemann syndrome with cancer and birth defects. Am J Hum Genet 2002;70:604-11. 4. Rayasam GV, Wendling O, Angrand PO, Mark M, Niederreither K, Song L, et al. NSD1 is essential for early post-implantation development and has a catalytically active SET domain. EMBO J 2003;22:3153-63. 5. Smith AC, Choufani S, Ferreira JC, Weksberg R. Growth regulation, imprinted genes, and chromosome 11p15.5. Pediatr Res 2007;61(5 Pt 2):43R-7.