J Pediatr Endocr Met 2014; 27(11-12): 1113–1120
Di Wu, Chunxiu Gong*, Huyong Zheng*, Paul Saenger, Yang Zhao, Yi Gu, Bingyan Cao, Wenjing Li and Chang Su
Clinical characteristics and chromosome 11p15 imprinting analysis of Silver-Russell syndrome – a Chinese experience Abstract Background: Silver-Russell syndrome (SRS) is an imprinting defect disease. This is the first study of Chinese children with SRS caused by chromosome 11p15 imprinting defects. Methods: Twenty-five SRS cases, diagnosed in Beijing Children’s Hospital from 2006 to 2012, were studied retrospectively to detect chromosome 11p15 imprinting defects. Results: Over 80% of the children had (i) small for gestational age and postnatal growth retardation (mean height standard deviation score [HT SDS] was –3.56), (ii) mean body mass index (BMI) SDS was –2.10, and (iii) skeletal malformation. Chromosome 11p15 imprinting defects were examined in 16 of the 25 patients. Six had hypomethylation in chromosome 11p15 imprinting control region 1 (ICR1) of the paternal allele; one had hypomethylation in chromosome 11p15 ICR1 and hypermethylation in imprinting control region 2 (ICR2). Another patient had a duplicated maternal chromosome 11p15 fragment. Six patients had been treated with for 3–24 months. Growth rates ranged from 4 to 10.8 cm/year. Conclusions: This study demonstrated that Chinese children with SRS had more growth retardation than bone retardation, severely low levels of BMI, triangular faces, and limb asymmetry. Chromosome 11p15 imprinting defects
*Corresponding authors: Chunxiu Gong, PhD, Department of Endocrinology, Genetics and Metabolism, Beijing Children’s Hospital, Capital Medical University, 56 Nan Lishi Road, West District, Beijing 100045, China, Phone: +0086-13910136358, Fax: +0086-10-59718682, E-mail: [email protected]; and Huyong Zheng, PhD, Department of Hematology, Beijing Children’s Hospital, Capital Medical University, 56 Nan Lishi Road, West District, Beijing 100045, China, Phone: +0086-13370115036, Fax: +0086-10-59718682, E-mail: [email protected] Di Wu, Yang Zhao, Yi Gu, Bingyan Cao, Wenjing Li and Chang Su: Department of Endocrinology, Genetics, and Metabolism, Beijing Children’s Hospital, Capital Medical University, Beijing 100045, China Paul Saenger: Section of Pediatric Endocrinology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY 10467, USA
contributed to 50% of these cases, and ICR1 hypomethylation was associated with asymmetry. Keywords: chromosome; clinical characteristics; imprinting; methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA); Silver-Russell syndrome. DOI 10.1515/jpem-2013-0490 Received December 18, 2013; accepted May 8, 2014; previously published online June 19, 2014
Introduction Silver– Russell syndrome (SRS) is a growth disorder whose exact cause remains undetermined. Approximately 50% of typical patients have genetic or epigenetic abnormalities, such as DNA imprinting defects (1). Clinically, SRS affects 1/3,000 to 1/100,000 births (2). Diagnosis of SRS mainly depends on clinical symptoms and physical signs in patients (1, 3–5), the accuracy of which often relies on experiences of the physician. Moreover, a number of cases have never had a diagnosis of SRS because the symptoms of skeletal asymmetry gradually improve with age and adult SRS patients have atypical facial features (6). Currently, SRS diagnosis is based on symptoms including a body which is small for gestational age (SGA) at birth, disproportionate head size relative to the small body size, triangular shaped face with a small jaw and a pointed chin, wide and late-closing fontanelle, clinodactyly, body asymmetry, a striking lack of subcutaneous fat, low muscle tone, hypoglycemia, excessive sweating as a baby, gastroesophageal reflux disease, constipation, late closing of the opening between the heart hemispheres, continued poor growth rate, and precocious puberty. SRS treatment includes carefully controlled caloric intake, growth hormone (GH) therapy, or physiotherapy to correct scoliosis (1, 3–6). Although the incidence of SRS is sporadic within families, aggregation and cytogenetic abnormalities of
Brought to you by | University of Florida Authenticated Download Date | 2/17/15 9:52 AM
1114 Wu et al.: Chromosome 11p15 imprinting of SRS children with SRS suggest that genetic alteration is an important part of SRS etiology. Previous studies show that approximately 50% of patients with typical SRS have genetic or epigenetic abnormalities, such as uniparental disomy of maternal chromosome 7, chromosome 11p15 imprinting defects, or submicroscopic structural abnormalities (1, 4, 7, 8). However, to date, there are no relative epidemiological or genetic data available in China, although several previous studies reported a few domestic SRS cases with clinical features (9–12). Therefore, in this study, we investigated 25 Chinese SRS cases from Beijing Children’s Hospital between 2006 and 2012 for clinical characteristics and phenotypic and genotypic association using methylation-specific multiplex ligation dependent probe amplification (MS-MLPA) analysis of methylation and changes in the number of gene copies in chromosome 11p15 of SRS patients (13, 14).
Patients and methods
biochemical tests of the liver and kidney functions; lipids; fasting glucose; fasting insulin, C-peptide (C-P), and G lycated hemoglybin A1C (HbA1C); and serum electrolytes such as potassium, sodium, chloride, bicarbonate, and calcium (Beckman Synchron LX20, Fullerton, CA, USA); arterial blood gas (GEM Premier 3000, Lexington, MA, USA); thyroid functions (Beckman Access2, Fullerton, CA, USA); sex hormones including luteinizing hormone (LH), follicle stimulating hormone (FSH), estrogen, testosterone (Roche Elecsys2010, Basel, Switzerland); adrenal cortical hormones cortisol and adrenocorticotropic hormone (ACTH) (Siemens Immulite2000, Mannheim, Germany); insulin-like growth factor binding protein-3 and insulinlike growth factor 1 (Siemens Immulite2000, Mannheim, Germany). After that, radiological investigations were then performed, which included bone age (DR7500 Kodak, Rochester, NY, USA), magnetic resonance imaging of the sellar region (Philips MR Ailieva intera, Amsterdam, the Netherlands), abdominal ultrasound (GE LOG9, Milwaukee, WI, USA). We also performed genetic tests for chromosomes and chromosome 11p15 imprinting detection. Limited numbers of patients also underwent GH therapy. The efficacy of GH was evaluated by growth velocity (GV). SDS were then calculated as SDS = (test value-mean)/standard deviation. Parents or caretakers of each patient signed an informed consent form to agree to participation of this study.
Study population
MS-MLPA analysis
A total of 25 children with SRS were diagnosed in Beijing Children’s Hospital between 2006 and 2012. The inclusion criteria were based on classical criteria (15, 16), Netchine criteria (17) or Bartholdi clinical Silver-Russell syndrome (SRS) score (18). Classical diagnostic criteria (15, 16) included three of the following: (i) low birth weight (at least 2 standard deviation [SD] below the mean); (ii) short stature at the time of diagnosis; (iii) a characteristic craniofacial appearance; (iv) limb, body, or facial asymmetry; and (v) clinodactyly. Netchine diagnostic criteria (17) were characterized by prenatal growth retardation (birth weight and/or length ≤ –2 standard deviation score [SDS] for gestational age) and at least three of the five following criteria: (i) postnatal growth retardation at 2 years of age or at the nearest measure available, (ii) relative macrocephaly at birth, (iii) prominent forehead during early childhood, (iv) body asymmetry, and (v) feeding difficulties during early childhood. Bartholdi (18) clinical Silver-Russell syndrome (SRS) score was used to identify patients with scores ≥ 8. The scoring criteria are: birth weight ≤ P10, birth length ≤ P10, relative macrocephaly, short stature, height 0.18. To determine numbers of gene copies, the normalized value for each DNA sample was compared with the average corrected value of each control probe generated from the control DNA samples from healthy individuals. No gene variation was defined as a ratio equal to 1.0, deletion was defined as a ratio 1.3.
Statistical analysis The data were analyzed using SPSS 14.0 software (SPSS, Chicago, IL, USA). The clinical characteristics were calculated as occurrence frequency of the symptoms and signs. Correlations between clinical phenotype and genetic variation were analyzed using Pearson correlation, χ2-test, or Student t-test. p-values equal to or 3 years old). Of the 25 patients, the chief complaint was short stature in 22 (88%) patients, external genital abnormalities in two boys (8%), and limb asymmetry in one (4%) patient (Table 1). Five patients had family histories of short stature, and one patient had a family history of genetic clinodactyly of the fifth finger. Furthermore, 11 patients had healthy siblings without SRS-like clinical manifestations. Birth weight records were available for 24 patients, all of which had been small for gestational age. The mean weight standard deviation score (WT SDS) at birth was –2.58. During physical examination, height of all patients was lower than that of the normal healthy children with the same age/ sex to the third percentile line. The mean height standard deviation score (HT SDS) was –3.56, and the mean body mass index (BMI) SDS was –2.10. All patients were of short stature and did not exhibit spontaneous catch-up growth rates after birth. Laboratory tests, including electrolytes, thyroid function, sex hormones, adrenal cortical hormone, ACTH, IGF-1, IGF-BP3, fasting glucose, insulin, C-P, HbA1C, liver/ kidney functions, and lipids profile, were within normal ranges. Pituitary magnetic resonance imaging did not show abnormalities. One patient had advanced bone age of more than a year, while others were normal or retarded. The mean retarded bone age was 1.17 years; retarded bone ages of 0–1 year, 1–2 years, and 2–3 years accounted for 30.4% (7/23), 26.1% (6/23), and 39.1% (9/23), respectively. HT SDS / mean of retarded bone age was –3.56/–1.17 = 3.04 (Tables 1 and 2).
Patients’ follow-up Nine out of 23 children (39.1%) had slightly improved limb asymmetry with increasing age and 48% (12/25) of the children had more obvious skull characteristics before 3 years old. After ascertainment, two children with external genitalia abnormalities were surgically corrected. They were of severely short stature (heights of –7.7 SDS and –2.7 SDS), but neither of them received GH treatments due to their parents’ low expectations for increased height. In contrast, there were six children of short stature who received GH treatments with different doses and courses. Two of
these six children (at a dose of 0.033 mg/kg/d for 1 year treatment) had 1-year growth velocities of 8 cm and 5 cm. Two other children were treated for 8 and 3 months and then stopped treatment due to family financial difficulties. These two children had growth velocities of 8 cm/year and
Di Wu, Chunxiu Gong*, Huyong Zheng*, Paul Saenger, Yang Zhao, Yi Gu, Bingyan Cao, Wenjing Li and Chang Su
Clinical characteristics and chromosome 11p15 imprinting analysis of Silver-Russell syndrome – a Chinese experience Abstract Background: Silver-Russell syndrome (SRS) is an imprinting defect disease. This is the first study of Chinese children with SRS caused by chromosome 11p15 imprinting defects. Methods: Twenty-five SRS cases, diagnosed in Beijing Children’s Hospital from 2006 to 2012, were studied retrospectively to detect chromosome 11p15 imprinting defects. Results: Over 80% of the children had (i) small for gestational age and postnatal growth retardation (mean height standard deviation score [HT SDS] was –3.56), (ii) mean body mass index (BMI) SDS was –2.10, and (iii) skeletal malformation. Chromosome 11p15 imprinting defects were examined in 16 of the 25 patients. Six had hypomethylation in chromosome 11p15 imprinting control region 1 (ICR1) of the paternal allele; one had hypomethylation in chromosome 11p15 ICR1 and hypermethylation in imprinting control region 2 (ICR2). Another patient had a duplicated maternal chromosome 11p15 fragment. Six patients had been treated with for 3–24 months. Growth rates ranged from 4 to 10.8 cm/year. Conclusions: This study demonstrated that Chinese children with SRS had more growth retardation than bone retardation, severely low levels of BMI, triangular faces, and limb asymmetry. Chromosome 11p15 imprinting defects
*Corresponding authors: Chunxiu Gong, PhD, Department of Endocrinology, Genetics and Metabolism, Beijing Children’s Hospital, Capital Medical University, 56 Nan Lishi Road, West District, Beijing 100045, China, Phone: +0086-13910136358, Fax: +0086-10-59718682, E-mail: [email protected]; and Huyong Zheng, PhD, Department of Hematology, Beijing Children’s Hospital, Capital Medical University, 56 Nan Lishi Road, West District, Beijing 100045, China, Phone: +0086-13370115036, Fax: +0086-10-59718682, E-mail: [email protected] Di Wu, Yang Zhao, Yi Gu, Bingyan Cao, Wenjing Li and Chang Su: Department of Endocrinology, Genetics, and Metabolism, Beijing Children’s Hospital, Capital Medical University, Beijing 100045, China Paul Saenger: Section of Pediatric Endocrinology, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, NY 10467, USA
contributed to 50% of these cases, and ICR1 hypomethylation was associated with asymmetry. Keywords: chromosome; clinical characteristics; imprinting; methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA); Silver-Russell syndrome. DOI 10.1515/jpem-2013-0490 Received December 18, 2013; accepted May 8, 2014; previously published online June 19, 2014
Introduction Silver– Russell syndrome (SRS) is a growth disorder whose exact cause remains undetermined. Approximately 50% of typical patients have genetic or epigenetic abnormalities, such as DNA imprinting defects (1). Clinically, SRS affects 1/3,000 to 1/100,000 births (2). Diagnosis of SRS mainly depends on clinical symptoms and physical signs in patients (1, 3–5), the accuracy of which often relies on experiences of the physician. Moreover, a number of cases have never had a diagnosis of SRS because the symptoms of skeletal asymmetry gradually improve with age and adult SRS patients have atypical facial features (6). Currently, SRS diagnosis is based on symptoms including a body which is small for gestational age (SGA) at birth, disproportionate head size relative to the small body size, triangular shaped face with a small jaw and a pointed chin, wide and late-closing fontanelle, clinodactyly, body asymmetry, a striking lack of subcutaneous fat, low muscle tone, hypoglycemia, excessive sweating as a baby, gastroesophageal reflux disease, constipation, late closing of the opening between the heart hemispheres, continued poor growth rate, and precocious puberty. SRS treatment includes carefully controlled caloric intake, growth hormone (GH) therapy, or physiotherapy to correct scoliosis (1, 3–6). Although the incidence of SRS is sporadic within families, aggregation and cytogenetic abnormalities of
Brought to you by | University of Florida Authenticated Download Date | 2/17/15 9:52 AM
1114 Wu et al.: Chromosome 11p15 imprinting of SRS children with SRS suggest that genetic alteration is an important part of SRS etiology. Previous studies show that approximately 50% of patients with typical SRS have genetic or epigenetic abnormalities, such as uniparental disomy of maternal chromosome 7, chromosome 11p15 imprinting defects, or submicroscopic structural abnormalities (1, 4, 7, 8). However, to date, there are no relative epidemiological or genetic data available in China, although several previous studies reported a few domestic SRS cases with clinical features (9–12). Therefore, in this study, we investigated 25 Chinese SRS cases from Beijing Children’s Hospital between 2006 and 2012 for clinical characteristics and phenotypic and genotypic association using methylation-specific multiplex ligation dependent probe amplification (MS-MLPA) analysis of methylation and changes in the number of gene copies in chromosome 11p15 of SRS patients (13, 14).
Patients and methods
biochemical tests of the liver and kidney functions; lipids; fasting glucose; fasting insulin, C-peptide (C-P), and G lycated hemoglybin A1C (HbA1C); and serum electrolytes such as potassium, sodium, chloride, bicarbonate, and calcium (Beckman Synchron LX20, Fullerton, CA, USA); arterial blood gas (GEM Premier 3000, Lexington, MA, USA); thyroid functions (Beckman Access2, Fullerton, CA, USA); sex hormones including luteinizing hormone (LH), follicle stimulating hormone (FSH), estrogen, testosterone (Roche Elecsys2010, Basel, Switzerland); adrenal cortical hormones cortisol and adrenocorticotropic hormone (ACTH) (Siemens Immulite2000, Mannheim, Germany); insulin-like growth factor binding protein-3 and insulinlike growth factor 1 (Siemens Immulite2000, Mannheim, Germany). After that, radiological investigations were then performed, which included bone age (DR7500 Kodak, Rochester, NY, USA), magnetic resonance imaging of the sellar region (Philips MR Ailieva intera, Amsterdam, the Netherlands), abdominal ultrasound (GE LOG9, Milwaukee, WI, USA). We also performed genetic tests for chromosomes and chromosome 11p15 imprinting detection. Limited numbers of patients also underwent GH therapy. The efficacy of GH was evaluated by growth velocity (GV). SDS were then calculated as SDS = (test value-mean)/standard deviation. Parents or caretakers of each patient signed an informed consent form to agree to participation of this study.
Study population
MS-MLPA analysis
A total of 25 children with SRS were diagnosed in Beijing Children’s Hospital between 2006 and 2012. The inclusion criteria were based on classical criteria (15, 16), Netchine criteria (17) or Bartholdi clinical Silver-Russell syndrome (SRS) score (18). Classical diagnostic criteria (15, 16) included three of the following: (i) low birth weight (at least 2 standard deviation [SD] below the mean); (ii) short stature at the time of diagnosis; (iii) a characteristic craniofacial appearance; (iv) limb, body, or facial asymmetry; and (v) clinodactyly. Netchine diagnostic criteria (17) were characterized by prenatal growth retardation (birth weight and/or length ≤ –2 standard deviation score [SDS] for gestational age) and at least three of the five following criteria: (i) postnatal growth retardation at 2 years of age or at the nearest measure available, (ii) relative macrocephaly at birth, (iii) prominent forehead during early childhood, (iv) body asymmetry, and (v) feeding difficulties during early childhood. Bartholdi (18) clinical Silver-Russell syndrome (SRS) score was used to identify patients with scores ≥ 8. The scoring criteria are: birth weight ≤ P10, birth length ≤ P10, relative macrocephaly, short stature, height 0.18. To determine numbers of gene copies, the normalized value for each DNA sample was compared with the average corrected value of each control probe generated from the control DNA samples from healthy individuals. No gene variation was defined as a ratio equal to 1.0, deletion was defined as a ratio 1.3.
Statistical analysis The data were analyzed using SPSS 14.0 software (SPSS, Chicago, IL, USA). The clinical characteristics were calculated as occurrence frequency of the symptoms and signs. Correlations between clinical phenotype and genetic variation were analyzed using Pearson correlation, χ2-test, or Student t-test. p-values equal to or 3 years old). Of the 25 patients, the chief complaint was short stature in 22 (88%) patients, external genital abnormalities in two boys (8%), and limb asymmetry in one (4%) patient (Table 1). Five patients had family histories of short stature, and one patient had a family history of genetic clinodactyly of the fifth finger. Furthermore, 11 patients had healthy siblings without SRS-like clinical manifestations. Birth weight records were available for 24 patients, all of which had been small for gestational age. The mean weight standard deviation score (WT SDS) at birth was –2.58. During physical examination, height of all patients was lower than that of the normal healthy children with the same age/ sex to the third percentile line. The mean height standard deviation score (HT SDS) was –3.56, and the mean body mass index (BMI) SDS was –2.10. All patients were of short stature and did not exhibit spontaneous catch-up growth rates after birth. Laboratory tests, including electrolytes, thyroid function, sex hormones, adrenal cortical hormone, ACTH, IGF-1, IGF-BP3, fasting glucose, insulin, C-P, HbA1C, liver/ kidney functions, and lipids profile, were within normal ranges. Pituitary magnetic resonance imaging did not show abnormalities. One patient had advanced bone age of more than a year, while others were normal or retarded. The mean retarded bone age was 1.17 years; retarded bone ages of 0–1 year, 1–2 years, and 2–3 years accounted for 30.4% (7/23), 26.1% (6/23), and 39.1% (9/23), respectively. HT SDS / mean of retarded bone age was –3.56/–1.17 = 3.04 (Tables 1 and 2).
Patients’ follow-up Nine out of 23 children (39.1%) had slightly improved limb asymmetry with increasing age and 48% (12/25) of the children had more obvious skull characteristics before 3 years old. After ascertainment, two children with external genitalia abnormalities were surgically corrected. They were of severely short stature (heights of –7.7 SDS and –2.7 SDS), but neither of them received GH treatments due to their parents’ low expectations for increased height. In contrast, there were six children of short stature who received GH treatments with different doses and courses. Two of
these six children (at a dose of 0.033 mg/kg/d for 1 year treatment) had 1-year growth velocities of 8 cm and 5 cm. Two other children were treated for 8 and 3 months and then stopped treatment due to family financial difficulties. These two children had growth velocities of 8 cm/year and
