Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 583e587
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Taiwanese Journal of Obstetrics & Gynecology journal homepage: www.tjog-online.com
Short Communication
Prenatal diagnosis and molecular cytogenetic characterization of a 1.07-Mb microdeletion at 5q35.2eq35.3 associated with NSD1 haploinsufficiency and Sotos syndrome Chih-Ping Chen a, b, c, d, e, f, *, Chen-Ju Lin a, g, Schu-Rern Chern b, Yu-Peng Liu h, i, Yu-Ling Kuo j, Yen-Ni Chen a, Peih-Shan Wu k, Dai-Dyi Town a, Li-Feng Chen a, Chien-Wen Yang b, Wayseen Wang b, l a
Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei, Taiwan Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan c Department of Biotechnology, Asia University, Taichung, Taiwan d School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan e Institute of Clinical and Community Health Nursing, National Yang-Ming University, Taipei, Taiwan f Department of Obstetrics and Gynecology, School of Medicine, National Yang-Ming University, Taipei, Taiwan g Department of Medicine, Mackay Medical College, New Taipei City, Taiwan h Department of Radiology, Mackay Memorial Hospital Hsinchu Branch, Hsinchu, Taiwan i Mackay Medicine, Nursing and Management College, Taipei, Taiwan j Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan k Gene Biodesign Co. Ltd, Taipei, Taiwan l Department of Bioengineering, Tatung University, Taipei, Taiwan b
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 29 October 2014
Objective: To present prenatal diagnosis and molecular cytogenetic characterization of a de novo 5q35 microdeletion associated with Sotos syndrome. Methods: This was the first pregnancy of a 29-year-old woman. The pregnancy was uneventful until 27 weeks of gestation when left ventriculomegaly was first noted. At 31 weeks of gestation, polyhydramnios, macrocephaly, and ventriculomegaly were prominent on ultrasound, and left pyelectasis and bilateral ventriculomegaly were diagnosed on magnetic resonance imaging. The woman underwent amniocentesis and cordocentesis at 32 weeks of gestation. Conventional cytogenetic analysis was performed using cultured amniocytes and cord blood lymphocytes. Array comparative genomic hybridization (aCGH) was performed on uncultured amniocytes and parental blood. Metaphase fluorescence in situ hybridization (FISH) was performed on cultured lymphocytes. Results: Conventional cytogenetics revealed a karyotype of 46,XX. aCGH on uncultured amniocytes revealed a de novo 1.07-Mb microdeletion at 5q35.2eq35.3 encompassing NSD1. Metaphase FISH analysis on the cord blood lymphocytes confirmed the deletion at 5q35.2. The postnatal phenotype was consistent with Sotos syndrome. Conclusion: Fetuses with Sotos syndrome may present macrocephaly, polyhydramnios, ventriculomegaly, and pyelectasis in the third trimester. aCGH and metaphase FISH are useful for rapid diagnosis of 5q35 microdeletion associated with Sotos syndrome. Copyright © 2014, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC. All rights reserved.
Keywords: 5q35 microdeletion array comparative genomic hybridization NSD1 haploinsufficiency prenatal diagnosis Sotos syndrome
Introduction * Corresponding author. Department of Obstetrics and Gynecology, Mackay Memorial Hospital, 92, Section 2, Chung-Shan North Road, Taipei, Taiwan. E-mail address: [email protected] (C.-P. Chen).
Sotos syndrome (OMIM 117550) is an autosomal dominant disorder that is characterized by cardinal features of macrocephaly with a high broad forehead, an inverted pear-like head, sparse
http://dx.doi.org/10.1016/j.tjog.2014.10.002 1028-4559/Copyright © 2014, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC. All rights reserved.
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Fig. 1. Prenatal ultrasound at 35 weeks of gestation shows (A) ventriculomegaly and (B) left pyelectasis.
frontotemporal hair, molar flushing, down-slanting palpebral fissures, a long face, a pointed chin, learning disability, and overgrowth; major features of advanced bone age, abnormal X-ray findings of the skull, poor feeding, hypotonia, neonatal jaundice, seizures, scoliosis, cardiac and renal abnormalities, joint laxity, and pes planus; and minor features of neoplasm development such as sacrococcygeal teratoma, presacral ganglioma, neuroblastoma, acute lymphoblastic leukemia, small cell lung cancer, Wilms tumor, hepatocellular carcinoma, cardiac/ovarian fibroma, and germ cell tumor [1e5]. The incidence of Sotos syndrome is estimated to be 1:14,000 live births [5]. Prenatal diagnosis of Sotos syndrome associated with a de novo 5q35 microdeletion is very rare. Here, we present molecular cytogenetic characterization in such a case.
horns of lateral ventricles (left side ¼ 1.5 cm; right side ¼ 1.3 cm; Fig. 2). The woman underwent amniocentesis and cordocentesis at 32 weeks of gestation. Conventional cytogenetic analysis was performed using cultured amniocytes and cord blood lymphocytes. Array comparative genomic hybridization (aCGH) was performed using uncultured amniocytes. No cytogenetic abnormality was found by conventional cytogenetics. However, aCGH and metaphase fluorescence in situ hybridization (FISH) detected a 5q35 microdeletion. Sotos syndrome caused by chromosomal microdeletion was diagnosed. At 36 weeks of gestation, a 2800-g female baby was delivered with macrocephaly and characteristic craniofacial appearance of Sotos syndrome.
Materials and methods
Routine cytogenetic analysis by G-banding techniques at the 550 bands of resolution was performed. Amniotic fluid and umbilical cord blood were collected, and the samples were subjected to cell culture according to the standard cytogenetic protocol.
Clinical description This was the first pregnancy of a 29-year-old woman. Her husband was aged 28 years, and there was no family history of congenital malformations. The pregnancy was uneventful until 27 weeks of gestation when left ventriculomegaly (ventricular diameter ¼ 1.3 cm) was first noted. At 31 weeks of gestation, polyhydramnios, macrocephaly (biparietal diameter ¼ 8.87 cm; 33 weeks) and ventriculomegaly were prominent (Fig. 1). Magnetic resonance imaging at 31 weeks of gestation showed left pyelectasis and mild dilation of the ventricular diameter in bilateral temporal
Conventional cytogenetic analysis
aCGH Whole-genome aCGH on the DNAs extracted from uncultured amniocytes derived from 10 mL of amniotic fluid and parental blood was performed using the NimbleGen ISCA Plus Cytogenetic Array (Roche NimbleGen, Madison, WI, USA), which has 630,000 probes and a median resolution of 15e20 kb across the entire genome according to the manufacturer's instruction. The DNA from uncultured
Fig. 2. Magnetic resonance imaging analysis at 31 weeks of gestation shows (A) mild dilation of bilateral lateral ventricles (arrows) and (B) left pyelectasis and a normal right kidney.
C.-P. Chen et al. / Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 583e587
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amniocytes and lymphocytes was extracted first following the manufacturer's protocol of QIAamp DNA Mini kit (Qiagen, Inc., Valencia, CA, USA). Then, the 0.5 mg of the extracted DNA was labeled in Cy5 dye compared with equivalent amount of normal female genomic DNA (G1521, Promega, Madison, WI, USA) labeled in Cy3 dye to perform the aCGH experiment. The experiment was performed according to the procedures recommended in the user guide for Roche NimbleGen ISCA plus Cytogenetic Array. The data were finally represented using Nexus 6.1 (BioDiscovery, Hawthorne, CA, USA). FISH Metaphase FISH analysis was performed on cultured cord blood lymphocytes using a 5p15.33-specific bacterial artificial chromosome probe RP11-325I22 (1,318,107e1,509,826) [hg 19] (FITC, green spectrum) and a 5q35.2-specific bacterial artificial chromosome probe RP11-627M5 (176,360,255e176,543,816; Texas red, red spectrum) according to the standard FISH protocol. Results G-banded chromosome analysis of cord blood and amniocytes revealed a karyotype of 46,XX. aCGH on the DNA extracted from the uncultured amniocytes detected a de novo 1.07-Mb microdeletion at 5q35.2eq35.3 or arr 5q35.2q35.3 (175,821,192e176,892,141) 1 (Fig. 3). The deleted region contains 31 genes including 19 OMIM genes: CLTB, GPRIN1, SNCB, UNC5A, HK3, UIMC1, ZNF346, FGFR4, NSD1, RAB24, PRELID1, MXD3, LMAN2, RGS14, SLC34A1, PFN3, F12, GRK6, and DBN1. The deletion of NSD1 is associated with Sotos syndrome. aCGH analysis of the parents' blood did not reveal such a microdeletion. Metaphase FISH analysis of the cord blood confirmed the deletion at 5q35.2 (Fig. 4). Discussion The present case had a 1.07-Mb microdeletion at 5q35.2eq35.3 encompassing the NSD1 gene. Sotos syndrome is caused by a
Fig. 4. Metaphase fluorescence in situ hybridization on the cultured cord blood lymphocytes using a 5p15.33-specific probe RP11-325I22 (FITC, green spectrum) and a 5q35.2-specific probe RP11-627M5 (Texas red, red spectrum) shows both red and green signals in the normal chromosome 5 (chr5) and only the green signal in the aberrant chromosome 5 of del(5)(q35.2q35.3).
deletion or mutation in the NSD1 gene (OMIM 606681). The NSD1 gene maps to 5q35.2eq35.3 (176,560,025e176,727,213) [6e8]. Stratton et al [9] reported a patient with deletion of 5q35.3 and the phenotype of macrocephaly and developmental delay. Maroun et al [10] in 1994 first described a female patient with Sotos syndrome and a karyotype of 46,XX,t(5;15) (q35;q22), and suggested that 5q35 as the gene locus associated with Sotos syndrome. Imaizumi et al [11] in 2002 reported a female patient with Sotos syndrome and a karyotype of 46,XX,t(5;8) (q35;q24.1), and suggested that the gene responsible for Sotos syndrome is located at 5q35. Kurotaki et al [7] subsequently in 2002, by positional cloning of the 5q35 breakpoint,
Fig. 3. Array comparative genomic hybridization on the DNA extracted from the uncultured amniocytes shows a 1.07-kb microdeletion at 5q35.2eq35.3 or arr 5q35.2q35.3 (175,821,192e176,892,141) 1. The deleted region contains the NSD1 gene.
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proved that haploinsufficiency of NSD1 causes Sotos syndrome. Kılıç et al [12] reported a 6-year-old boy with tall stature, macrocephaly, typical facial appearance, learning disability, megaloencephaly, corpus callosum dysgenesis, and colpocephaly associated with a 5q35 microdeletion spanning the NSD1 locus at 5q35.3. The NSD1 gene encodes nuclear receptor set domain protein 1, which enhances androgen receptor transactivation [13]. About 90% of patients with Sotos syndrome have NSD1 abnormalities including intragenic mutations and microdeletion. However, in about 10% of classic Sotos syndrome cases, NSD1 abnormalities cannot be identified [14,15]. The 5q35 microdeletion causes about 50% of Japanese Sotos syndrome cases, and about 10e15% of nonJapanese Sotos syndrome cases, while intragenic mutations of NSD1 cause about 12% of Japanese Sotos syndrome cases and 27e93% of non-Japanese Sotos syndrome cases [5,14e25]. In a study of 18 unrelated Korean patients with Sotos syndrome, Sohn et al [26] found that eight patients (53%) had 5q35 microdeletions, and 47% had different NSD1 intragenic mutations. Familial Sotos syndrome occurs in < 10% of the cases with Sotos syndrome, and most cases with Sotos syndrome are sporadic [15,22,27]. The present case was associated with prenatal ultrasound findings of fetal overgrowth, macrocephaly, ventriculomegaly, polyhydramnios, and pyelectasis in the third trimester. Prenatal sonographic observations of fetal overgrowth, macrocephaly, polyhydramnios, and renal and central nervous system abnormalities in association with increased nuchal translucency and abnormal maternal serum screen result should include a differential diagnosis of Sotos syndrome [4,28e30]. Chen et al [28] reported prenatal third-trimester ultrasound findings of fetal macrocephaly, ventriculomegaly, corpus callosum hypoplasia, enlarged cistern magna, unilateral hydronephrosis, polyhydramnios, and overgrowth in a pregnancy with familial Sotos syndrome and an abnormal maternal serum screen result for Down syndrome in the second trimester. Thomas and Lemire [29] reported prenatal thirdtrimester ultrasound findings of macrocephaly and polyhydramnios in a pregnancy with familial Sotos syndrome and an abnormal maternal serum screen result for Down syndrome in the second trimester. Schou et al [30] reported increased nuchal translucency and large for date in a fetus with a de novo Sotos syndrome. The central nervous system abnormalities associated with Sotos syndrome include enlargement of lateral ventricles, trigones and occipital horns, corpus callosum hypoplasia, persistence of cavum septum pellucidum, cavum vergae and cavum velum interpositum, enlarged cisterna magna, heterotopias, macrocerebellum, and periventricular leukomalacia [31,32]. Prenatal diagnosis of fetal overgrowth associated with Sotos syndrome should include a differential diagnosis of other overgrowth conditions that may be confused with Sotos syndrome such as Weaver syndrome (EZH2 microdeletions or mutations), BeckwitheWiedemann syndrome (11p15 epigenetic and genomic alterations), SimpsoneGolabieBehmel syndrome (GPC3, GPC4, CXORF5 microdeletions or mutations), BannayaneRileyeRuvalcaba syndrome (PTEN microdeletions or mutations), benign familial macrocephaly (an autosomal dominant disorder), fragile X syndrome [FMR1 microdeletions or mutations by trinucleotide (CGG)n repeat expansion], Gorlin syndrome (PTCH microdeletions or mutations), chromosomal abnormalities of 4p duplications, mosaic 20p trisomy and 22q13.3 deletions, and nonspecific overgrowth [5]. Prenatal diagnosis of Sotos syndrome should include parental genetic testing since there is a 5% incidence of having an affected parent [5]. If a parent is affected with Sotos syndrome, the recurrence risk is about 50%. However, if no parent is affected with Sotos syndrome, the recurrence risk in the following pregnancy is < 1% [2,5]. Germline mosaicism for NSD1 microdeletions or mutations has not been reported [2].
Prenatal diagnosis of fetal overgrowth, polyhydramnios, macrocephaly, and renal and central nervous system abnormalities should consider a detailed examination of the parents for the phenotypic features of Sotos syndrome, and mutational and microdeletion analysis of NSD1 gene in the fetus. Our case provides an example of the usefulness of aCGH on uncultured amniocytes for rapid molecular cytogenetic diagnosis of Sotos syndrome associated with a 5q35 microdeletion. Conflicts of interest The authors have no conflicts of interest relevant to this article. Acknowledgments This work was supported by research grants NSC-101-2314-B195-011-MY3 and MOST 103-2314-B-195-010 from the Ministry of Science and Technology and MMH-E-103-04 from Mackay Memorial Hospital, Taipei, Taiwan. References [1] Cohen Jr MM. Tumors and nontumors in Sotos syndrome. Am J Med Genet 1999;84:173e5. [2] Baujat G, Cormier-Daire V. Sotos syndrome. Orphanet J Rare Dis 2007;2:36. [3] Tatton-Brown K, Rahman N. Sotos syndrome. Eur J Hum Genet 2007;15: 264e71. [4] Chen CP. Prenatal findings and genetic diagnosis of fetal overgrowth disorders: SimpsoneGolabieBehmel syndrome, Sotos syndrome and BeckwitheWiedemann syndrome. Taiwan J Obstet Gynecol 2012;51:186e91. [5] Tatton-Brown K, Cole TRP, Rahman N. Sotos syndrome. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong CT, et al., editors. GeneReviews [Internet]. Seattle (WA): University of Washington, Seattle; 2004 Dec 17. p. 1993e2014 [updated 2012 Mar 8]. [6] Kurotaki N, Harada N, Yoshiura K, Sugano S, Niikawa N, Matsumoto N. Molecular characterization of NSD1, a human homologue of the mouse Nsd1 gene. Gene 2001;279:197e204. [7] Kurotaki N, Imaizumi K, Harada N, Masuno M, Kondoh T, Nagai T, et al. Haploinsufficiency of NSD1 causes Sotos syndrome. Nat Genet 2002;30:365e6. [8] Niikawa N. Molecular basis of Sotos syndrome. Horm Res 2004;62(Suppl. 3): 60e5. [9] Stratton RF, Tedrowe NA, Tolworthy JA, Patterson RM, Ryan SG, Young RS. Deletion 5q35.3. Am J Med Genet 1994;51:150e2. [10] Maroun C, Schmerler S, Hutcheon RG. Child with Sotos phenotype and a 5:15 translocation. Am J Med Genet 1994;50:291e3. [11] Imaizumi K, Kimura J, Matsuo M, Kurosawa K, Masuno M, Niikawa N, et al. Sotos syndrome associated with a de novo balanced reciprocal translocation t(5;8)(q35;q24.1). Am J Med Genet 2002;107:58e60. lu K. A case of Sotos syndrome with 5q35 micro[12] Kılıç E, Ütine GE, Bodurog deletion and novel clinical findings. Turk J Pediatr 2013;55:207e9. [13] Wang X, Yeh S, Wu G, Hsu CL, Wang L, Chiang T, et al. Identification and characterization of a novel androgen receptor coregulator ARA267-a in prostate cancer cells. J Biol Chem 2001;276:40417e23. [14] Türkmen S, Gillessen-Kaesbach G, Meinecke P, Albrecht B, Neumann LM, Hesse V, et al. Mutations in NSD1 are responsible for Sotos syndrome, but are not a frequent finding in other overgrowth phenotypes. Eur J Hum Genet 2003;11:858e65. [15] Tatton-Brown K, Douglas J, Coleman K, Baujat G, Cole TRP, Das S, et al. Genotype-phenotype associations in Sotos syndrome: an analysis of 266 individuals with NSD1 aberrations. Am J Hum Genet 2005;77:193e204. [16] Douglas J, Hanks S, Temple IK, Davies S, Murray A, Upadhyaya M, et al. NSD1 mutations are the major cause of Sotos syndrome and occur in some cases of Weaver syndrome but are rare in other overgrowth phenotypes. Am J Hum Genet 2003;72:132e43. [17] Rio M, Clech L, Amiel J, Faivre L, Lyonnet S, Le Merrer M, et al. Spectrum of NSD1 mutations in Sotos and Weaver syndromes. J Med Genet 2003;40: 436e40. [18] Cecconi M, Forzano F, Milani D, Cavani S, Baldo C, Selicorni A, et al. Mutation analysis of the NSD1 gene in a group of 59 patients with congenital overgrowth. Am J Med Genet 2005;134A:247e53. [19] Faravelli F. NSD1 mutations in Sotos syndrome. Am J Med Genet C Semin Med Genet 2005;137C:24e31. [20] Melchior L, Schwartz M, Duno M. dHPLC screening of the NSD1 gene identifies nine novel mutationsdsummary of the first 100 Sotos syndrome mutations. Ann Hum Genet 2005;69:222e6. [21] Waggoner DJ, Raca G, Welch K, Dempsey M, Anderes E, Ostrovnaya I, et al. NSD1 analysis for Sotos syndrome: insights and perspectives from the clinical laboratory. Genet Med 2005;7:524e33.
C.-P. Chen et al. / Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 583e587 [22] Kurotaki N, Harada N, Shimokawa O, Miyake N, Kawame H, Uetake K, et al. Fifty microdeletions among 112 cases of Sotos syndrome: low copy repeats possibly mediate the common deletion. Hum Mutat 2003;22:378e87. [23] Miyake N, Kurotaki N, Sugawara H, Shimokawa O, Harada N, Kondoh T, et al. Preferential paternal origin of microdeletions caused by prezygotic chromosome or chromatid rearrangements in Sotos syndrome. Am J Hum Genet 2003;72:1331e7. [24] Tei S, Tsuneishi S, Matsuo M. The first Japanese familial Sotos syndrome with a novel mutation of the NSD1 gene. Kobe J Med Sci 2006;52:1e8. [25] 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;28:1098e107. [26] Sohn YB, Lee CG, Ko JM, Yang JA, Yun JN, Jung EJ, et al. Clinical and genetic spectrum of 18 unrelated Korean patients with Sotos syndrome: frequent 5q35 microdeletion and identification of four novel NSD1 mutations. J Hum Genet 2013;58:73e7.
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€glund P, Kurotaki N, Kyto € la € S, Miyake N, Somer M, Matsumoto N. Familial [27] Ho Sotos syndrome is caused by a novel 1 bp deletion of the NSD1 gene. J Med Genet 2003;40:51e4. [28] Chen CP, Lin SP, Chang TY, Chiu NC, Shih SL, Lin CJ, et al. Perinatal imaging findings of inherited Sotos syndrome. Prenat Diagn 2002;22:887e92. [29] Thomas A, Lemire EG. Sotos syndrome: antenatal presentation. Am J Med Genet 2008;146A:1312e3. [30] Schou KV, Kirchhoff M, Nygaard U, Jørgensen C, Sundberg K. Increased nuchal translucency with normal karyotype: a follow-up study of 100 cases supplemented with CGH and MLPA analyses. Ultrasound Obstet Gynecol 2009;34: 618e22. [31] Schaefer GB, Bodensteiner JB, Buehler BA, Lin A, Cole TRP. Neuroimaging findings in Sotos syndrome. Am J Med Genet 1997;68:462e5. ~o Melo D, Pina-Neto JM, Acosta AX, Daniel J, de Castro V, Santos AC. [32] Gusma Neuroimaging and echocardiographic findings in Sotos syndrome. Am J Med Genet 2000;90:432e4.
Contents lists available at ScienceDirect
Taiwanese Journal of Obstetrics & Gynecology journal homepage: www.tjog-online.com
Short Communication
Prenatal diagnosis and molecular cytogenetic characterization of a 1.07-Mb microdeletion at 5q35.2eq35.3 associated with NSD1 haploinsufficiency and Sotos syndrome Chih-Ping Chen a, b, c, d, e, f, *, Chen-Ju Lin a, g, Schu-Rern Chern b, Yu-Peng Liu h, i, Yu-Ling Kuo j, Yen-Ni Chen a, Peih-Shan Wu k, Dai-Dyi Town a, Li-Feng Chen a, Chien-Wen Yang b, Wayseen Wang b, l a
Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei, Taiwan Department of Medical Research, Mackay Memorial Hospital, Taipei, Taiwan c Department of Biotechnology, Asia University, Taichung, Taiwan d School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan e Institute of Clinical and Community Health Nursing, National Yang-Ming University, Taipei, Taiwan f Department of Obstetrics and Gynecology, School of Medicine, National Yang-Ming University, Taipei, Taiwan g Department of Medicine, Mackay Medical College, New Taipei City, Taiwan h Department of Radiology, Mackay Memorial Hospital Hsinchu Branch, Hsinchu, Taiwan i Mackay Medicine, Nursing and Management College, Taipei, Taiwan j Department of Obstetrics and Gynecology, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan k Gene Biodesign Co. Ltd, Taipei, Taiwan l Department of Bioengineering, Tatung University, Taipei, Taiwan b
a r t i c l e i n f o
a b s t r a c t
Article history: Accepted 29 October 2014
Objective: To present prenatal diagnosis and molecular cytogenetic characterization of a de novo 5q35 microdeletion associated with Sotos syndrome. Methods: This was the first pregnancy of a 29-year-old woman. The pregnancy was uneventful until 27 weeks of gestation when left ventriculomegaly was first noted. At 31 weeks of gestation, polyhydramnios, macrocephaly, and ventriculomegaly were prominent on ultrasound, and left pyelectasis and bilateral ventriculomegaly were diagnosed on magnetic resonance imaging. The woman underwent amniocentesis and cordocentesis at 32 weeks of gestation. Conventional cytogenetic analysis was performed using cultured amniocytes and cord blood lymphocytes. Array comparative genomic hybridization (aCGH) was performed on uncultured amniocytes and parental blood. Metaphase fluorescence in situ hybridization (FISH) was performed on cultured lymphocytes. Results: Conventional cytogenetics revealed a karyotype of 46,XX. aCGH on uncultured amniocytes revealed a de novo 1.07-Mb microdeletion at 5q35.2eq35.3 encompassing NSD1. Metaphase FISH analysis on the cord blood lymphocytes confirmed the deletion at 5q35.2. The postnatal phenotype was consistent with Sotos syndrome. Conclusion: Fetuses with Sotos syndrome may present macrocephaly, polyhydramnios, ventriculomegaly, and pyelectasis in the third trimester. aCGH and metaphase FISH are useful for rapid diagnosis of 5q35 microdeletion associated with Sotos syndrome. Copyright © 2014, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC. All rights reserved.
Keywords: 5q35 microdeletion array comparative genomic hybridization NSD1 haploinsufficiency prenatal diagnosis Sotos syndrome
Introduction * Corresponding author. Department of Obstetrics and Gynecology, Mackay Memorial Hospital, 92, Section 2, Chung-Shan North Road, Taipei, Taiwan. E-mail address: [email protected] (C.-P. Chen).
Sotos syndrome (OMIM 117550) is an autosomal dominant disorder that is characterized by cardinal features of macrocephaly with a high broad forehead, an inverted pear-like head, sparse
http://dx.doi.org/10.1016/j.tjog.2014.10.002 1028-4559/Copyright © 2014, Taiwan Association of Obstetrics & Gynecology. Published by Elsevier Taiwan LLC. All rights reserved.
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C.-P. Chen et al. / Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 583e587
Fig. 1. Prenatal ultrasound at 35 weeks of gestation shows (A) ventriculomegaly and (B) left pyelectasis.
frontotemporal hair, molar flushing, down-slanting palpebral fissures, a long face, a pointed chin, learning disability, and overgrowth; major features of advanced bone age, abnormal X-ray findings of the skull, poor feeding, hypotonia, neonatal jaundice, seizures, scoliosis, cardiac and renal abnormalities, joint laxity, and pes planus; and minor features of neoplasm development such as sacrococcygeal teratoma, presacral ganglioma, neuroblastoma, acute lymphoblastic leukemia, small cell lung cancer, Wilms tumor, hepatocellular carcinoma, cardiac/ovarian fibroma, and germ cell tumor [1e5]. The incidence of Sotos syndrome is estimated to be 1:14,000 live births [5]. Prenatal diagnosis of Sotos syndrome associated with a de novo 5q35 microdeletion is very rare. Here, we present molecular cytogenetic characterization in such a case.
horns of lateral ventricles (left side ¼ 1.5 cm; right side ¼ 1.3 cm; Fig. 2). The woman underwent amniocentesis and cordocentesis at 32 weeks of gestation. Conventional cytogenetic analysis was performed using cultured amniocytes and cord blood lymphocytes. Array comparative genomic hybridization (aCGH) was performed using uncultured amniocytes. No cytogenetic abnormality was found by conventional cytogenetics. However, aCGH and metaphase fluorescence in situ hybridization (FISH) detected a 5q35 microdeletion. Sotos syndrome caused by chromosomal microdeletion was diagnosed. At 36 weeks of gestation, a 2800-g female baby was delivered with macrocephaly and characteristic craniofacial appearance of Sotos syndrome.
Materials and methods
Routine cytogenetic analysis by G-banding techniques at the 550 bands of resolution was performed. Amniotic fluid and umbilical cord blood were collected, and the samples were subjected to cell culture according to the standard cytogenetic protocol.
Clinical description This was the first pregnancy of a 29-year-old woman. Her husband was aged 28 years, and there was no family history of congenital malformations. The pregnancy was uneventful until 27 weeks of gestation when left ventriculomegaly (ventricular diameter ¼ 1.3 cm) was first noted. At 31 weeks of gestation, polyhydramnios, macrocephaly (biparietal diameter ¼ 8.87 cm; 33 weeks) and ventriculomegaly were prominent (Fig. 1). Magnetic resonance imaging at 31 weeks of gestation showed left pyelectasis and mild dilation of the ventricular diameter in bilateral temporal
Conventional cytogenetic analysis
aCGH Whole-genome aCGH on the DNAs extracted from uncultured amniocytes derived from 10 mL of amniotic fluid and parental blood was performed using the NimbleGen ISCA Plus Cytogenetic Array (Roche NimbleGen, Madison, WI, USA), which has 630,000 probes and a median resolution of 15e20 kb across the entire genome according to the manufacturer's instruction. The DNA from uncultured
Fig. 2. Magnetic resonance imaging analysis at 31 weeks of gestation shows (A) mild dilation of bilateral lateral ventricles (arrows) and (B) left pyelectasis and a normal right kidney.
C.-P. Chen et al. / Taiwanese Journal of Obstetrics & Gynecology 53 (2014) 583e587
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amniocytes and lymphocytes was extracted first following the manufacturer's protocol of QIAamp DNA Mini kit (Qiagen, Inc., Valencia, CA, USA). Then, the 0.5 mg of the extracted DNA was labeled in Cy5 dye compared with equivalent amount of normal female genomic DNA (G1521, Promega, Madison, WI, USA) labeled in Cy3 dye to perform the aCGH experiment. The experiment was performed according to the procedures recommended in the user guide for Roche NimbleGen ISCA plus Cytogenetic Array. The data were finally represented using Nexus 6.1 (BioDiscovery, Hawthorne, CA, USA). FISH Metaphase FISH analysis was performed on cultured cord blood lymphocytes using a 5p15.33-specific bacterial artificial chromosome probe RP11-325I22 (1,318,107e1,509,826) [hg 19] (FITC, green spectrum) and a 5q35.2-specific bacterial artificial chromosome probe RP11-627M5 (176,360,255e176,543,816; Texas red, red spectrum) according to the standard FISH protocol. Results G-banded chromosome analysis of cord blood and amniocytes revealed a karyotype of 46,XX. aCGH on the DNA extracted from the uncultured amniocytes detected a de novo 1.07-Mb microdeletion at 5q35.2eq35.3 or arr 5q35.2q35.3 (175,821,192e176,892,141) 1 (Fig. 3). The deleted region contains 31 genes including 19 OMIM genes: CLTB, GPRIN1, SNCB, UNC5A, HK3, UIMC1, ZNF346, FGFR4, NSD1, RAB24, PRELID1, MXD3, LMAN2, RGS14, SLC34A1, PFN3, F12, GRK6, and DBN1. The deletion of NSD1 is associated with Sotos syndrome. aCGH analysis of the parents' blood did not reveal such a microdeletion. Metaphase FISH analysis of the cord blood confirmed the deletion at 5q35.2 (Fig. 4). Discussion The present case had a 1.07-Mb microdeletion at 5q35.2eq35.3 encompassing the NSD1 gene. Sotos syndrome is caused by a
Fig. 4. Metaphase fluorescence in situ hybridization on the cultured cord blood lymphocytes using a 5p15.33-specific probe RP11-325I22 (FITC, green spectrum) and a 5q35.2-specific probe RP11-627M5 (Texas red, red spectrum) shows both red and green signals in the normal chromosome 5 (chr5) and only the green signal in the aberrant chromosome 5 of del(5)(q35.2q35.3).
deletion or mutation in the NSD1 gene (OMIM 606681). The NSD1 gene maps to 5q35.2eq35.3 (176,560,025e176,727,213) [6e8]. Stratton et al [9] reported a patient with deletion of 5q35.3 and the phenotype of macrocephaly and developmental delay. Maroun et al [10] in 1994 first described a female patient with Sotos syndrome and a karyotype of 46,XX,t(5;15) (q35;q22), and suggested that 5q35 as the gene locus associated with Sotos syndrome. Imaizumi et al [11] in 2002 reported a female patient with Sotos syndrome and a karyotype of 46,XX,t(5;8) (q35;q24.1), and suggested that the gene responsible for Sotos syndrome is located at 5q35. Kurotaki et al [7] subsequently in 2002, by positional cloning of the 5q35 breakpoint,
Fig. 3. Array comparative genomic hybridization on the DNA extracted from the uncultured amniocytes shows a 1.07-kb microdeletion at 5q35.2eq35.3 or arr 5q35.2q35.3 (175,821,192e176,892,141) 1. The deleted region contains the NSD1 gene.
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proved that haploinsufficiency of NSD1 causes Sotos syndrome. Kılıç et al [12] reported a 6-year-old boy with tall stature, macrocephaly, typical facial appearance, learning disability, megaloencephaly, corpus callosum dysgenesis, and colpocephaly associated with a 5q35 microdeletion spanning the NSD1 locus at 5q35.3. The NSD1 gene encodes nuclear receptor set domain protein 1, which enhances androgen receptor transactivation [13]. About 90% of patients with Sotos syndrome have NSD1 abnormalities including intragenic mutations and microdeletion. However, in about 10% of classic Sotos syndrome cases, NSD1 abnormalities cannot be identified [14,15]. The 5q35 microdeletion causes about 50% of Japanese Sotos syndrome cases, and about 10e15% of nonJapanese Sotos syndrome cases, while intragenic mutations of NSD1 cause about 12% of Japanese Sotos syndrome cases and 27e93% of non-Japanese Sotos syndrome cases [5,14e25]. In a study of 18 unrelated Korean patients with Sotos syndrome, Sohn et al [26] found that eight patients (53%) had 5q35 microdeletions, and 47% had different NSD1 intragenic mutations. Familial Sotos syndrome occurs in < 10% of the cases with Sotos syndrome, and most cases with Sotos syndrome are sporadic [15,22,27]. The present case was associated with prenatal ultrasound findings of fetal overgrowth, macrocephaly, ventriculomegaly, polyhydramnios, and pyelectasis in the third trimester. Prenatal sonographic observations of fetal overgrowth, macrocephaly, polyhydramnios, and renal and central nervous system abnormalities in association with increased nuchal translucency and abnormal maternal serum screen result should include a differential diagnosis of Sotos syndrome [4,28e30]. Chen et al [28] reported prenatal third-trimester ultrasound findings of fetal macrocephaly, ventriculomegaly, corpus callosum hypoplasia, enlarged cistern magna, unilateral hydronephrosis, polyhydramnios, and overgrowth in a pregnancy with familial Sotos syndrome and an abnormal maternal serum screen result for Down syndrome in the second trimester. Thomas and Lemire [29] reported prenatal thirdtrimester ultrasound findings of macrocephaly and polyhydramnios in a pregnancy with familial Sotos syndrome and an abnormal maternal serum screen result for Down syndrome in the second trimester. Schou et al [30] reported increased nuchal translucency and large for date in a fetus with a de novo Sotos syndrome. The central nervous system abnormalities associated with Sotos syndrome include enlargement of lateral ventricles, trigones and occipital horns, corpus callosum hypoplasia, persistence of cavum septum pellucidum, cavum vergae and cavum velum interpositum, enlarged cisterna magna, heterotopias, macrocerebellum, and periventricular leukomalacia [31,32]. Prenatal diagnosis of fetal overgrowth associated with Sotos syndrome should include a differential diagnosis of other overgrowth conditions that may be confused with Sotos syndrome such as Weaver syndrome (EZH2 microdeletions or mutations), BeckwitheWiedemann syndrome (11p15 epigenetic and genomic alterations), SimpsoneGolabieBehmel syndrome (GPC3, GPC4, CXORF5 microdeletions or mutations), BannayaneRileyeRuvalcaba syndrome (PTEN microdeletions or mutations), benign familial macrocephaly (an autosomal dominant disorder), fragile X syndrome [FMR1 microdeletions or mutations by trinucleotide (CGG)n repeat expansion], Gorlin syndrome (PTCH microdeletions or mutations), chromosomal abnormalities of 4p duplications, mosaic 20p trisomy and 22q13.3 deletions, and nonspecific overgrowth [5]. Prenatal diagnosis of Sotos syndrome should include parental genetic testing since there is a 5% incidence of having an affected parent [5]. If a parent is affected with Sotos syndrome, the recurrence risk is about 50%. However, if no parent is affected with Sotos syndrome, the recurrence risk in the following pregnancy is < 1% [2,5]. Germline mosaicism for NSD1 microdeletions or mutations has not been reported [2].
Prenatal diagnosis of fetal overgrowth, polyhydramnios, macrocephaly, and renal and central nervous system abnormalities should consider a detailed examination of the parents for the phenotypic features of Sotos syndrome, and mutational and microdeletion analysis of NSD1 gene in the fetus. Our case provides an example of the usefulness of aCGH on uncultured amniocytes for rapid molecular cytogenetic diagnosis of Sotos syndrome associated with a 5q35 microdeletion. Conflicts of interest The authors have no conflicts of interest relevant to this article. Acknowledgments This work was supported by research grants NSC-101-2314-B195-011-MY3 and MOST 103-2314-B-195-010 from the Ministry of Science and Technology and MMH-E-103-04 from Mackay Memorial Hospital, Taipei, Taiwan. References [1] Cohen Jr MM. Tumors and nontumors in Sotos syndrome. Am J Med Genet 1999;84:173e5. [2] Baujat G, Cormier-Daire V. Sotos syndrome. Orphanet J Rare Dis 2007;2:36. [3] Tatton-Brown K, Rahman N. Sotos syndrome. Eur J Hum Genet 2007;15: 264e71. [4] Chen CP. Prenatal findings and genetic diagnosis of fetal overgrowth disorders: SimpsoneGolabieBehmel syndrome, Sotos syndrome and BeckwitheWiedemann syndrome. Taiwan J Obstet Gynecol 2012;51:186e91. [5] Tatton-Brown K, Cole TRP, Rahman N. Sotos syndrome. In: Pagon RA, Adam MP, Ardinger HH, Bird TD, Dolan CR, Fong CT, et al., editors. GeneReviews [Internet]. Seattle (WA): University of Washington, Seattle; 2004 Dec 17. p. 1993e2014 [updated 2012 Mar 8]. [6] Kurotaki N, Harada N, Yoshiura K, Sugano S, Niikawa N, Matsumoto N. Molecular characterization of NSD1, a human homologue of the mouse Nsd1 gene. Gene 2001;279:197e204. [7] Kurotaki N, Imaizumi K, Harada N, Masuno M, Kondoh T, Nagai T, et al. Haploinsufficiency of NSD1 causes Sotos syndrome. Nat Genet 2002;30:365e6. [8] Niikawa N. Molecular basis of Sotos syndrome. Horm Res 2004;62(Suppl. 3): 60e5. [9] Stratton RF, Tedrowe NA, Tolworthy JA, Patterson RM, Ryan SG, Young RS. Deletion 5q35.3. Am J Med Genet 1994;51:150e2. [10] Maroun C, Schmerler S, Hutcheon RG. Child with Sotos phenotype and a 5:15 translocation. Am J Med Genet 1994;50:291e3. [11] Imaizumi K, Kimura J, Matsuo M, Kurosawa K, Masuno M, Niikawa N, et al. Sotos syndrome associated with a de novo balanced reciprocal translocation t(5;8)(q35;q24.1). Am J Med Genet 2002;107:58e60. lu K. A case of Sotos syndrome with 5q35 micro[12] Kılıç E, Ütine GE, Bodurog deletion and novel clinical findings. Turk J Pediatr 2013;55:207e9. [13] Wang X, Yeh S, Wu G, Hsu CL, Wang L, Chiang T, et al. Identification and characterization of a novel androgen receptor coregulator ARA267-a in prostate cancer cells. J Biol Chem 2001;276:40417e23. [14] Türkmen S, Gillessen-Kaesbach G, Meinecke P, Albrecht B, Neumann LM, Hesse V, et al. Mutations in NSD1 are responsible for Sotos syndrome, but are not a frequent finding in other overgrowth phenotypes. Eur J Hum Genet 2003;11:858e65. [15] Tatton-Brown K, Douglas J, Coleman K, Baujat G, Cole TRP, Das S, et al. Genotype-phenotype associations in Sotos syndrome: an analysis of 266 individuals with NSD1 aberrations. Am J Hum Genet 2005;77:193e204. [16] Douglas J, Hanks S, Temple IK, Davies S, Murray A, Upadhyaya M, et al. NSD1 mutations are the major cause of Sotos syndrome and occur in some cases of Weaver syndrome but are rare in other overgrowth phenotypes. Am J Hum Genet 2003;72:132e43. [17] Rio M, Clech L, Amiel J, Faivre L, Lyonnet S, Le Merrer M, et al. Spectrum of NSD1 mutations in Sotos and Weaver syndromes. J Med Genet 2003;40: 436e40. [18] Cecconi M, Forzano F, Milani D, Cavani S, Baldo C, Selicorni A, et al. Mutation analysis of the NSD1 gene in a group of 59 patients with congenital overgrowth. Am J Med Genet 2005;134A:247e53. [19] Faravelli F. NSD1 mutations in Sotos syndrome. Am J Med Genet C Semin Med Genet 2005;137C:24e31. [20] Melchior L, Schwartz M, Duno M. dHPLC screening of the NSD1 gene identifies nine novel mutationsdsummary of the first 100 Sotos syndrome mutations. Ann Hum Genet 2005;69:222e6. [21] Waggoner DJ, Raca G, Welch K, Dempsey M, Anderes E, Ostrovnaya I, et al. NSD1 analysis for Sotos syndrome: insights and perspectives from the clinical laboratory. Genet Med 2005;7:524e33.
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