Objective To compare the diagnostic effectiveness of methylation-specific PCR ( MS-PCR ) and methylation-specific multiplex ligation-dependent probe amplification ( MS-MLPA ) in clinical suspected Prader-Willi syndrome (PWS).Methods One hundred and two participants (57 males and 45 females) who visited Peking Union Medical College Hospital in the period from October 2005 to February 2014 were included in this retrospective study .Among the 102 participants , 16 were normal controls , 2 PWS cases confirmed by fluores-cence in situ hybridization or high resolutin banding were positive controls , and 84 were suspected PWS cases . Genomic DNA was extracted for MS-PCR and MS-MLPA, based on which diagnoses were made and genetic types distinguished .Chi-squared test was used to compare the sensitivity , specificity , and accuracy of the two meth-ods .Results MS-PCR results showed that all normal controls and positive controls were conform to their pheno -types .Thirty-nine in the 84 suspected patients were shown to be PWS , and the other 45 to be normal .MS-MLPA showed that in the 86 non-normal participants ( positive control and suspected cases ) , 29 had paternal deletions , 9 had maternal uniparental disomy ( mUPD) , 47 were normal;and the assay failed to produce effective result in 1 case due to the overlong DNA storage time .Two participants were diagnosed as PWS cases by MS-PCR but nor-mal by MS-MLPA.The diagnosis of PWS was ruled out by repeat MS-PCR after doubling DNA dosage .Combined with clinical manifestations , 39 participants were definitively diagnosed as PWS , the other 63 were not PWS .The false-positive rate of MS-PCR was 3.17% (2/63).The sensitivity, specificity, and accuracy of MS-PCR were 100%, 96.83%, and 98.03%, respectively; while those indicators of MS-MLPA were 97.43%, 100%, and 99.02%, respectively , showing no significant difference ( all P>0.05 ) .Conclusions Both MS-PCR and MS-MLPA have high sensitivity , specificity , and accuracy , thus are effective for PWS diagnosis .MS-MLPA can dis-tinguish paternal deletion and mUPD .Enough dose of DNA is requisite in MS-PCR to avoid false-positive results . Freshness of DNA samples is essential in MS-MLPA, which also needs more strict performance standards .We suggest a combination of MS-PCR and MS-MLPA in diagnosing PWS to ensure the accuracy of the result .

Objective To assess whether low dose corticosteroid could improve muscle strength and motor function in children with Duchenne muscular dystrophy ( DMD) .Methods In this randomized double-blind con-trolled trial , patients with DMD at the age of 5-10 diagnosed from September 2005 to December 2008 in Peking Union Medical College Hospital were enrolled .The patients were divided into treatment group and control group randomly.The treatment group was given prednisone at a dosage of 0.75 mg/(kg· d) for three months, and the control group was given vitamin C 0.3 mg/d as placebo in the same period .Every patient was followed-up once a month for a total of 4 times, testing muscle strength , motor function ( 9-meter walk test ) , respiratory function [forced vital capacity (FVC)], and serum creatine kinase .Results Thirty-one eligible DMD patients were en-rolled, including 17 in the treatment group and 14 in the control group.The mean muscle strength scores of the con-trol group at the 4 clinic visits were 38.2 ±7.5, 37.0 ±8.0, 36.2 ±7.6, and 36.6 ±7.7, while those of the treat-ment group were 37.7 ±6.1, 39.1 ±6.4, 40.3 ±5.9, and 40.0 ±5.6, respectively .The changes in muscle strength scores at 1, 2, 3 months after treatment compared with the scores at diagnosis were all significantly larger in the treatment group than in the control group (P=0.024, P=0.000, P=0.002).The mean time of 9-meter walk at the 4 visits in the control group were (7.7 ±2.5), (7.6 ±2.8), (8.1 ±2.4), and (7.6 ±2.8) s, and those in the treatment group were (8.5 ±3.4), (7.2 ±3.3), (7.6 ±3.1), and (7.1 ±2.8) s, showing signifi-cantly larger changes of time in the treatment group than in the control group at 2 and 3 months after treatment com-pared with the time at diagnosis ( P=0.013, P=0.003) .The four mean FVCs in the control group were (1.24 ± 0.28), (1.13 ±0.26), (1.21 ±0.31), and (1.16 ±0.26) L, and the mean FVCs in the treatment group were (1.14 ±0.37), (1.40 ±0.35), (1.42 ±0.36), and (1.52 ±0.37) L, showing significantly larger changes in the treatment group than in the control group at 1, 2, and 3 months after treatment compared with the FVCs at diag-nosis (P=0.000, P=0.006, P=0.000).The decrease of creatine kinase in the treatment group was significantly larger after 1-month treatment than that in the control group (P=0.035).There were no serious adverse effects in the treatment group except weight gain .Conclusions Short-term application of low dose prednisone in children with DMD can increase muscle strength , improve motor and respiratory functions .

OBJECTIVE:Multiple sulfatase deficiency is a rare autosomal recessively inherited lysosomal storage disorder characterized by the accumulation of sulfated lipids and acid mucopolysaccharides. The aim of this study was to explore the clinical manifestations, enzyme activities and SUMF1 gene mutations in two Chinese patients with multiple sulfatase deficiency.METHOD:One boy and one girl from two families were studied. Both patients presented with mental retardation, mild coarse facial features, a neurodegenerative course of disease with loss of sensory and motor function after 2 years of age, ichthyosis and skeletal abnormalities (kyphosis or/and scoliosis). Clinical characteristics indicate multiple sulfatase deficiency.Sulfatases activities in blood leucocytes, plasma or cultured fibroblast of the patients were measured.Genomic DNAs were extracted from peripheral blood leukocytes from the patients and their parents. All SUMF1 gene exons and intron-exon boundaries were amplified by PCR and subjected for direct sequencing.RESULT:In case 1, five sulfatases activities of blood leucocytes and four sulfatases of cultured skin-fibroblasts were analyzed.In case 2, three sulfatases activities of blood leucocytes were tested.Significantly decreased sulfatases activities confirmed the diagnosis of multiple sulfatase deficiency.On SUMF1 gene, c.793_794 insATG (p. P265X)/ c.1045C>T (p.R349W) in case 1 and c.451A>G (p.K151E)/ c.1046G>C (p.R349Q) in case 2 were detected, respectively. Three novel mutations c.793_794insAGT, c.1046G>C and c.451A>G were identified.CONCLUSIONS:Multiple sulfatase deficiency usually results in multi-organ damage, especially neurologic, skeletal and skin.Sulfatases assay and SUMF1 gene analysis are necessary for the diagnosis. Two Chinese cases with multiple sulfatase deficiency were firstly reported. Three novel mutations were found.It should be considered that the mutation profile of SUMF1 gene in Chinese patients is different from other populations.

OBJECTIVE:Wolcott-Rallison syndrome (WRS) is a rare autosomal recessive disorder characterized by the association of permanent neonatal or early-infancy insulin-dependent diabetes, multiple epiphyseal dysplasia and growth retardation, and other variable multisystem clinical manifestations. Here we describe a Chinese boy affected by WRS. Genetic testing of his EIF2AK3 gene was performed in order to elucidate molecular variations and subsequently to provide credible genetic counseling for prenatal diagnosis in his family.METHOD:Based on analysis of a nine-year-old boy's clinical symptoms associated with biochemical examination and imaging, the diagnosis of WRS was therefore made. Genomic DNAs were extracted from peripheral blood leukocytes from the boy and his parents with their informed consent for genetic studies. All EIF2AK3 exons and intron-exon boundaries were amplified by Touch-down polymerase chain reaction (Touch-down PCR) and sequenced.RESULT:Direct sequencing of PCR products revealed the presence of a heterozygous T insertion (c.1408_1409insT) in exon 8 of the EIF2AK3 gene leading to frameshifting and termination, and another heterozygous T to A exchange (c.1596T > A) in exon 9 of the EIF2AK3 gene resulting in nonsense C532X mutation.CONCLUSION:Combining mutation screening of EIF2AK3 gene with clinical manifestations and effective examination may provide a reliable diagnostic method for patients. In this research, two novel mutations identified in the Chinese boy locate in the catalytic domain of the EIF2AK3 gene, disrupting the ability of autophosphorylation, leading to the truncated proteins that are unable to phosphorylate the natural substrate, which are responsible for the phenotype of Wolcott-Rallison syndrome.

OBJECTIVETo investigate the mutations in protein tyrosine phosphatase, nonreceptor-type 11 (PTPN11) gene in patients with Noonan syndrome (NS).METHODSThree sporadic patients with NS were studied. Genomic DNAs were extracted from peripheral blood leukocytes. All 15 coding exons and their flanking intronic boundaries of the PTPN11 gene were amplified by polymerase chain reaction and followed by direct sequencing. DNAs from parents were sequenced in the corresponding region when the mutation was detected in their affected child. The identified mutation was screened in 100 healthy individuals for exclusion of polymorphism by restriction endonuclease digestion of the PCR products. Protein conservation analysis was performed among 10 species using an online ClustalW tool.RESULTSDirect DNA sequence analysis identified a heterozygous 181G to A change in exon 3 of the PTPN11 gene in one patient, which resulted in the substitution of an aspartic acid residue by an asparagine at codon 61. The mutation was absent in his parents and 100 controls, and is located in a highly conserved amino acid site. No mutation in the coding region of PTPN11 gene was observed in the other two patients.CONCLUSIONThe p.D61N mutation was reported previously in Caucasians and is a de-novo mutation in this patient. Our study further confirmed that the p.D61N is a pathogenic mutation for NS and consistent with the clinical diagnosis. Additional genes may be involved in the other two patients with NS, indicating high genetic heterogeneity of this disease.

Objective To develop a rapid,reliable and convenient method for diagnosing Coffin-Lowry syndrome(CLS).Methods RNA and DNA were extracted from the CLS patients' peripheral blood.The fragments of RPS6KA3 cDNA were amplified by RT-PCR and sequenced.The mutation was verified at the DNA level.The other case that didn't show the nucleotide change was tested by multiplex ligation-dependent probe amplification(MLPA).Results A two nucleotide deletion(r889_890delAG) in RPS6KA3 gene was only detected in the first case.The nucleotide sequence of the first case's mother was normal,suggesting it was a de novo mutation.Conclusion This is the first CLS case diagnosed by molecular method in China,which provided a genetic basis for diagnosis and prenatal diagnosis of CLS.

Objective Diagnose the first case of autosomal dominant Costello syndrome by direct sequencing of HRAS gene.Methods Total genomic DNA was extracted from peripheral blood leukocytes of the patient and her parents.The coding exons 2～5 of the HRAS gene were amplified by polymerase chain reaction(PCR) and sequenced directly.Results A common HRAS missense mutation,p.Gly12Ser,was identified in the patient,but not in the parents.Conclusion Patients with Costello syndrome manifest characteristic facial features,feeding difficulties and developmental delay/mental retardation etc.The diagnosis can be confirmed by mutation analysis of HRAS gene.

Objective To perform mutation analysis in a pseudoachondroplasia (PSACH) family. Methods Mutation screening was carried out by DNA-PCR and direct sequencing the exons 8 to 19 of COMP gene. Results A c.815C > T substitution was identified in exon 8 of COMP gene in the proband and his affected father and brother. Conclusion The disease in the family is caused by mutation of COMP gene.

OBJECTIVE:Fibrodysplasia ossificans progressiva (FOP) is a rare autosomal dominant inherited disease caused by mutations of ACVR1 gene and can be inherited from either mother or father. FOP is characterized by the presence of malformations of the big toes and of progressive extra-skeletal ossification. Direct sequence analyses of genomic DNA have demonstrated that there is an identical single nucleotide substitution (c617G-->A, R206H) in the glycine-serine (GS) activation domain of ACVR1 gene, responsible for all affected individuals reported so far. We report a Chinese girl with typical FOP characteristics, in whom the same mutation in ACVR1 was identified.METHODS:Clinical diagnosis was based on physical examination, radiological findings, and biochemical tests. For mutation detection, peripheral blood was obtained with informed consent from the patient and the parents. Genomic DNA was extracted from peripheral blood using standard method. Exon 4 of ACVR1 was amplified by polymerase chain reaction (PCR), and the PCR products were subjected to automatic DNA sequencing.RESULTS:The affected girl is 3-year-old and showed typical clinical manifestations of FOP. She had malformations of the halluces at birth and subsequently progressive extra-skeletal ossification developed at the age of 8 - 9 months. Then, she gradually developed stiffness of the knee joint and neck but remained ambulant. Radiographic changes were observable, e.g., the extra-skeletal ossification was found at cervical spine. Her mother has congenital malformations of the halluces, but had no postnatal progressive extra-skeletal ossification. Her father and other family members are normal. With direct sequencing of the PCR products, a G to A substitution at c617 of ACVR1 (R206H) was detected in the patient only but not in her parents. Paternity analysis suggested that it is a de novo mutation.CONCLUSION:This is the first case reported in a Chinese patient with FOP in the mainland of China, which was confirmed by direct sequencing. Although sporadic cases of FOP have been reported in diverse geographic and ethnic group, the mutations of ACVR1 c617 (R206H) are identical up to now. The presence of mutation hot spot facilitates molecular diagnosis in clinical practice. Genetic detection is important for FOP patients to avoid misdiagnosis and further damages, including those from medical intervention.

Objective To establish the diagnosis of juvenile patients with chronic elevation of serum creatine kinase(CK) levels.Methods We made a retrospective evaluation of 25 juvenile patients with hyperCKemia over three months in the genetic clinic of our department between January 2005 and March 2007.Clinical data,neurological examination and laboratory examinations were analysized.All the patients underwent open muscle biopsy.Biochemical and genetic investigation were added in selected cases.Results There were 7 females and 18 males aged between 2 years 9 months and 15 years [mean 7.8y].The disease duration were beween 3 months and 7 years.Serum CK levels were between 285 and 15720 U/L(18~198U/L).We made a diagnosis in 16 patients.The diagnosis included dystrophinopathies in 8 patients,Pompe disease in 2 patients,glycogenosis type III in 1 patient,mitochondrial myopathies in 1 patient; glutaric aciduria type II in 1 patient;spinal muscular atrophy in 1 patients; inflammatory myopathies in 2 patients.Diagnosis were not able to be confirmed in 9 patients;pathological but not conclusive muscle changes were found in 6 patients.Conclusion Neurological and metabolic myopathies disease are the main causes of persistent hyperckemia in children,muscle biopsy as well as biochemical and genetic investigations were essential to the diagnosis.

OBJECTIVE Mucopolysaccharidosis type II (MPS II, Hunter syndrome, OMIM 309900) is an X-linked recessive lysosomal storage disease resulting from a deficiency of iduronte-2-sulphate sulphatase (IDS). The present study aimed to establish an enzyme assay method for IDS activity for carrying out postnatal and prenatal diagnosis of MPS II by means of IDS activity assay on plasma, uncultured chorionic villi (CV) and cultured amniotic fluid cells (AF cell) using a new synthesized substrate. METHODS A fluorigenic substrate (4-methylumbelliferyl-alpha-iduronate-2-sulphate, MU-alpha-Idu-2S) was used for the assay of IDS activity. IDS activity in plasma was determined for diagnosis of the proband. Prenatal diagnosis in 10 pregnancies at risk was carried out according to IDS activity on uncultured CV at 11th week or on cultured AF cell at 18th week of gestation. At the same time, IDS activity was also determined in the maternal plasmas to observe the change of IDS activity in pregnancy. The fetal sex determination was performed by PCR amplification of the ZFX/ZFY genes. RESULT The IDS activity in plasma of normal controls and obligate heterozygotes were 240.2 - 668.2 nmol/(4 hxml) and 88.7 - 547.9 nmol/(4 hxml), respectively, while the enzyme activities in plasmas were in the range of 0.3 - 18.6 nmol/(4 hxml) in affected male. The IDS activities were 37.2 - 54.9 nmol/(4 hxmg protein) and 21.4 - 74.4 nmol/(4 hxmg protein) in CV and cultured AF cells respectively. Out of 50 suspected cases, 46 were diagnosed as having MPS II and 4 were excluded. Prenatal diagnosis was performed on 10 pregnancies at risk. Four of 5 male fetuses [IDS activity were 4.7, 1.8, 7.0 nmol/(4hxmg protein) in CV, 0.6 nmol/(4 hxmg protein) in AF cell] were diagnosed as having MPS II and the other 5 fetuses were normal females [IDS activity were: 48.7, 5.9, 25.2 nmol/(4 hxmg protein) in CV, 55.2, 40.9 nmol/(4 hxmg protein) in AF cell]. Increased IDS activity was observed in plasma of the pregnant women with unaffected fetuses, while the IDS activity decreased in pregnancies with affected fetuses. IDS activity of one female fetus was very low [5.9 nmol/(4 hxmg protein)], but the IDS activity in maternal plasmas increased, this fetus was a normal female. CONCLUSIONS The method using a synthesized fluorigenic 4-methylumbelliferyl-substrate was a sensitive, rapid and convenient assay of IDS activity and was reliable for early prenatal diagnosis. Determination of fetal sex would be helpful in excluding the female fetus with low IDS activity from being considered as an affected male fetus. It would be further helpful if IDS activity in maternal plasma was taken into account.

目的了解窒息新生儿在听觉刺激诱发脑神经活动时的脑氧合代谢和脑血流量的改变.方法 1998～2003年北京中日友好医院儿科选择窒息新生儿34例为窒息组,健康新生儿40名为对照组.使用近红外光谱仪,观察听觉刺激试验诱发的脑氧合血红蛋白[Hb O2]、还原血红蛋白[Hb H]和总血红蛋白[Hb tot]浓度的变化,并比较两组脑氧合代谢和脑血流量的改变.根据[Hb O2]、[Hb H]和[Hb tot]不同的变化,将氧合代谢曲线分为A([Hb O2]、[Hb H]和[Hb tot]均增加); B([Hb O2]和[Hb tot]增加, [Hb H]降低);C([Hb O2]和[Hb tot]降低, [Hb H]增加)3种曲线类型.结果窒息组中25例(25/34、73.5%)显示C型变化,对照组中28例(28/40、70.0%)显示A型变化,两组中A、C两型例数比较差异显著(P<0.05).两组[Hb O2]和[Hb tot]数值变化幅度比较差异显著(P<0.05).结论窒息新生儿听觉刺激诱发相应皮层的神经活动时,显示局部脑血流量下降、氧合代谢降低,重度窒息儿更明显.

一、新生儿窒息在中国 每年约2000万新生儿出生的中国,鼓励一对夫妻只生一个孩子,随着国民经济迅速发展,生活水平不断提高,每一个新生儿的健康都受到家庭及社会的高度关注[1].1991年中国婴儿死亡率为50.2‰,1998年降至33.3‰,地区性差别很大,贫困地区最高可达56‰,一些条件较好的城市低至7.5‰[2,3].

患儿女,16.5岁.因反复发作、严重肌肉痛性痉挛约10年为主诉就诊.患儿出生至6岁生长发育正常,尔后生长发育迟缓,9岁后发育停滞.头发脱落、不长.全身肌肉(包括面颊部、颈部、腹部、大腿、小腿部)经常痉挛,发作时全身大汗、疼痛难忍,神志清醒.牙齿疼痛、自行断裂而只剩残根.常年腹泻,食用水果后为甚.左膝内翻畸形.至今第二性征未发育,无月经.患儿父母及家族中无类同患者.外院曾予以抗感染,助消化,营养、钙剂、多种维生素包括VitD治疗,均未见疗效.近期在我院多科就诊,未能明确诊断而转至我科.

目的总结儿童肝豆状核变性(WD)的诊治经验,探讨监测疗效的敏感指标.方法分析77例儿童WD临床资料.结果77例中以肝脏症状、神经/精神症状和其他症状起病者分别为59、13、6例.以肝病起病59例中坚持治疗者可达到或维持临床无症状.暴发性肝炎型5/6例死亡或自动出院.以神经/精神症状起病者4/5例症状明显好转.监测治疗前尿铜平均728.4μg/24 h,青霉胺、硫酸锌及青霉胺+硫酸锌治疗后尿铜分别为792.0、286.1、388.5μg/24 h.结论儿童WD中以肝病为首发症状者最多见.青霉胺仍是治疗WD的经典药物,治疗期间定期重点随诊肝功能、血常规及尿铜最为重要.

随着常频机械通气(CMV)策略的改进,高频通气(HFV)、肺表面活性物质、体外膜肺(ECMO)以及吸入一氧化氮(NO)的应用,新生儿呼吸道疾病死亡率显著降低,愈来愈多的危重新生儿、早产儿、极低体重儿存活.

OBJECTIVE:The study aimed to investigate the clinical characteristics of enteral feeding in very low birthweight infants (VLBWI), to determine the risk factors associated with feeding intolerance, and to analysis the beneficial factors in order to improve gut motility and maturation.METHODS:The study was carried out in 38 VLBWI, birthweight (1,314 +/- 180) g, in the NICU of authors' department. They were divided into feeding tolerance and intolerance groups, and earlier enteral feeding and later groups. Comparison was made between two groups about the associate factors.RESULTS:The incidence of feeding intolerance was 55 per cent. There was a significant difference in two groups about the clinical factors (gestational age, birth weight, the age of the first feeding, time of full enteral feeding and the hospitalized days). The significant risk factors associated with feeding intolerance were the smaller gestational age, umbilical catheterization, theophylline therapy, and delay of the time when the first feed was commenced.CONCLUSION:If vital signs are stable, trophic feeding in VLBWI should be commenced as soon as possible during the first 6 days of life. Early trophic feeding, slowly increase the feeding volume, carefully fast, and moving bowel were suggested and will improve feeding tolerance and gastrointestinal maturation.

早产带来的高围产儿死亡及体格、智能发育障碍是儿童医疗保健的难题[1,2].早产导致的高额花费与其预后对比,是多年来医学伦理学争论的重点.为救治危重早产儿特有的多系统、多器官的合并症,新生儿学界在基础、临床和康复等领域做了大量工作,使预后明显改善.最近在青岛召开的以"早产儿"为主要议题的第六届全国暨第三届海峡两岸新生儿学术会议对进一步提高早产儿存活率及生存质量起到了促进作用.

Objective To establish regional critical neonatal transport network,and to reduce mortality rate, decrease disability and improve prognosis of newborn infants.Methods Having established regional critical neonatal transport network with some hospitals of dongcheng district in Beijing.There were 251 critical newborn infants by transport from November 1999 to May 2003,including 183 premature infants(72.9 %),19 babies with 26-30 weeks of gestational age,28 babies with birth weight 1500 g, most babies with respiratory diseases. Recent 3 years,167 out-call of doctors,102 by transport is incubator and emergency ambulance,65 resuscitated in referring hospitals.Results The number of neonatal transport is increasing year after year,230 recurred(91.6 %),17 abandoned and 4 died in total 251 infants.Conclusions Establishing regional neonatal transport network in Ⅲ level is very important for reducing neonatal mortality rate. An active transport pattern with giving first-aid treatment and well-trained staff is necessary to rescue critical newborn infants in initial stage of life and improve the quality of treatment.It is an assurance for successful transportation to transfer premature infants as soon as early and pay attention to the details of stabilization before transport.It is recommended that the mother is transferred to the Ⅲ level center before delivery of a high-risk infant for reducing maternal and neonatal mortality rate.An appeal is being made for establishing the regional critical neonatal transport network in Ⅲ level and that is necessary to reducing maternal and neonatal mortality rate, improving prognosis of critical newborn infants, and enhancing quality of population.

评价高频振荡通气(HFOV)治疗新生儿呼吸衰竭的疗效,11例患儿中早产儿9例,合并双侧气胸1例,合并肺出血1例,合并新生儿持续肺动脉高压(PPHN)3例.结果显示:治疗半小时,PaCO2由74.38±23.85 mmHg降至41.85±20.53 mmHg(P＜0.05),肺泡氧分压/动脉血氧分压比(a/AP02)由0.23±0.58上升至0.36±0.15(P＜0.05),12小时为0.37±0.11(P＜0.01),氧合指数(OI)和FiO2稳步降低.10例患儿对HFOV有效,1例无效;8例治愈,无1例发生气漏,6例存活早产儿无慢性肺部疾患(CLD)发生.以上提示HFOV对新生儿呼吸衰竭,特别是合并气漏者十分有效,对部分PPHN也有疗效.