Short Communication
Prenatal diagnosis and molecular cytogenetic characterization of a 1.07-Mb
microdeletion at 5q35.2-q35.3 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 and Wayseen Wang b,l
a Department of Obstetrics and Gynecology, Mackay Memorial Hospital, Taipei, Taiwan b 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
* Correspondence to: Chih-Ping Chen, MD
Department of Obstetrics and Gynecology, Mackay Memorial Hospital 92, Section 2, Chung-Shan North Road, Taipei, Taiwan
Tel: +886-2-25433535; Fax: +886-2-25433642, +886-2-25232448 E-mail: [email protected]
Abstract
Objective: To present prenatal diagnosis and molecular cytogenetic characterization of a de novo
5q35 microdeletion associated with Sotos syndrome.
Materials and methods: This was the first pregnancy of a 24-year-old primigravid 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 bloods. 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 5q35.2-q35.3 encompassing NSD1. Metaphase FISH analysis on the cord blood lymphocytes confirmed the deletion at 5q35.2. The postnatal phenotype was consistent with Sotos syndrome.
Conclusions: 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.
Keywords: 5q35 microdeletion; array comparative genomic hybridization; NSD1
Introduction
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 frontotemporal hair, molar flushing, down-slanting palpebral fissures, a long face, a point 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 [1-5]. 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.
Materials and Methods
Clinical description
This was the first pregnancy of a 29-year-old primigravid woman. Her husband was 28 years old, and there was no family history of congenital malformations. The pregnancy was uneventful until 27 weeks of gestation when left ventriculomegaly (ventricular diameter = 1.3 cm) was first noted. At 31 weeks of gestation, polyhydramnios, macrocephaly [biparietal diameter (BPD) = 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 horns of lateral ventricles (left side = 1.5 cm; right side = 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. A 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.
Conventional cytogenetic analysis
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 blood cytogenetic protocol.
aCGH
Whole-genome aCGH on the DNAs extracted from uncultured amniocytes derived from 10 mL of amniotic fluid and parental bloods was performed using NimbleGen ISCA Plus Cytogenetic Array (Roche NimbleGen, Madison, WI, USA). The NimbleGen ISCA Plus Cytogenetic Array has 630,000 probes and a median resolution of 15-20 kb across the entire genome according to the manufacturer's instruction. The DNA from uncultured amniocytes and lymphocytes was extracted first. It was done by following the manufacturer's protocol of QIAamp DNA Mini kit (Qiagen, Inc., Valencia, CA, USA). Then, the 0.5μg of the extracted DNA was labeled in Cy5 dye compared with equivalent amount of normal female gDNA (G1521, Promega) labeled in Cy3 dye to perform the aCGH experiment. The experiment was performed according to the procedures recommended from Roche NimbleGen ISCA plus Cytogenetic Array's user guide. The data were finally represented by
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 (BAC) probe RP11-325I22 (1,318,107-1,509,826) [hg 19] (FITC, green spectrum) and a 5q35.2-specific BAC probe RP11-627M5 (176,360,255-176,543,816) (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 5q35.2-q35.3 or arr 5q35.2q35.3 (175,821,192-176,892,141)1 (Fig. 3). The deleted region contains 31 genes including 19 OMIM genes of 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' bloods 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 5q35.2-q35.3 encompassing the NSD1 gene. Sotos syndrome is caused by a deletion or mutation in the NSD1 gene (OMIM 606681). The NSD1 gene maps to 5q35.2-q35.3 (176,560,025-176,727,213) [6-8]. 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, 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 that 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, NSD1 abnormalities can not been identified [14,15]. The 5q35 microdeletion causes about 50% of Japanese Sotos syndrome cases, and about 10-15% of non-Japanese Sotos syndrome cases, while intragenic mutations of NSD1 cause about 12% of Japanese Sotos syndrome cases and 27%-93% of non-Japanese Sotos syndrome cases [5,14-25]. In a study of 18 unrelated Korean patients with Sotos syndrome, Sohn et al [26] found that 8 patients (53%) has 5q35 microdeletions, and 47% had different NSD1 intragenic mutations. Familial Sotos syndrome occurs in less than 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 (NT) and abnormal maternal serum screen result should include a differential diagnosis of Sotos syndrome [4,28-30]. 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 third-trimester 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 NT 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), Beckwith-Wiedemann syndrome (11p15 epigenetic and genomic alterations), Simpson-Golabi-Behmel syndrome (GPC3, GPC4,
CXORF5 microdeletions or mutations), Bannayan-Riley-Ruvalcaba 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 a 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 follow pregnancy is less that 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.
Acknowledgements
This work was supported by research grants NSC-101-2314-B-195-011-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.
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Figure Legends
Fig. 1. Prenatal ultrasound at 35 weeks of gestation shows (A) ventriculomegaly and (B) left pyelectasis. 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.
Fig. 3. Array comparative genomic hybridization on the DNA extracted from the uncultured amniocytes shows a 1.07-kb microdeletion at 5q35.2-q35.3 or arr 5q35.2q35.3 (175,821,192-176,892,141)1. The deleted region contains the NSD1 gene.
Fig. 4. Metaphase fluorescence in situ hybridization on the cultured cord blood lymphocytes using a 5p15.33-specific probe 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).
