联合采用连锁分析和突变检测进行苯丙酮尿症的产前基因诊断

目的 为经典型苯丙酮尿症(phenylketonuria,PKU)家系提供产前基因诊断.方法 联合采用短串联重复片段(short tandem repeats,STR)多态连锁分析和聚合酶链反应技术扩增苯丙氨酸羟化酶基因突变热区外显子直接测序,对3个经典型PKU先证者及家系成员进行综合分析.结果 家系1的STR连锁分析不能提供遗传信息,家系2、家系3可提供100%信息.突变检测共发现3个先证者均为苯丙氨酸羟化酶基因复合杂合突变,共检测到5种突变:Y166X、R243Q、R413P、EX6-96A＞G和IVS11-1G＞C,其中IVS11-1G＞C为国际首次报道的新突变.综合连锁分析和突变检测结果,确定家系1和家系2胎儿为PKU患者,家系3胎儿正常.结论 联合连锁分析和苯丙氨酸羟化酶基因突变检测,可为PKU家系提供可靠的产前诊断服务.

Abstract：

Objective To explore the prenatal genetic diagnosis for classic phenylketonuria (PKU) families.Methods Probands and their family members from three classic PKU families were analyzed by combining linkage analysis through short tandem repeats (STR) polymorphism and PCR-sequencing for the exons within mutation hot spot of phenylalanine hydroxylase gene.Results Linkage analysis found uninformative for Family 1,while 100 % confirmative information was obtained from Family 2 and 3.Sequencing showed compound heterozygous mutations of phenylalanine hydroxylase gene for all of the three probands.Five mutations were detected,namely Y166X,R243Q,R413P,EX6-96A ＞ G and IVS11-1G＞ C,and IVS11-1G ＞ C was a novel identified muntation.Information from linkage analysis and mutation screening showed clearly that the fetus of Family 1 and 2 were affected,while normal for Family 3.Conclusions For those PKU families,reliable service of prenatal genetic diagnosis could be provided by combining linkage analysis with mutation screening of phenylalanine hydroxylase gene.     

软骨发育不全的产前基因诊断

目的:探讨采用成纤维细胞生长因子受体3(FGFR3)基因测序技术对软骨发育不全(achondroplasia,ACH)进行产前诊断的价值,以为遗传咨询和临床干预提供依据。方法:选择8个ACH家系,其中5个家系中母方为ACH,1个家系父母双方都是ACH,2个家系父母双方正常曾生育过ACH患儿。提取患者及家庭其他成员外周血基因组DNA,应用基因测序对FGFR3基因进行检测;抽取胎儿羊水或脐血,提取基因组DNA后对FGFR3基因进行检测。对超声检查胎儿发现短肢发育异常的4个胎儿进行产前基因诊断。结果:5个家系都是母方c.1138GA杂合型突变,产前基因诊断3个胎儿(其中1个家系是2次妊娠)是c.1138GA杂合型,3个胎儿正常;1个家系父母双方都是c.1138GA杂合型突变,胎儿也是c.1138GA杂合型突变;2个家系父母双方正常而生育的患儿是c.1138GA杂合型突变,再次妊娠的胎儿都正常。4个短肢发育异常胎儿,基因检测均未发现突变。4个c.1138GA杂合型突变胎儿和4个短肢发育异常胎儿均已引产;5个未发现突变的胎儿已分娩,随访均为健康个体。结论:FGFR3基因检测可为ACH患者或生育过ACH的家庭提供遗传咨询和产前基因诊断。     

应用MLPA技术进行脊髓性肌萎缩症高风险胎儿的产前诊断

目的:建立快速、准确的脊肌萎缩症(SMA)家系的产前诊断方法。方法:2个曾生育过SMA患儿(患儿已死亡)的家系,母方再次妊娠。采用多重连接依赖探针扩增(MLPA)技术检测父母双方的生存运动神经元基因(SMN)以及邻近基因拷贝数,明确是否为SMA携带者。经羊膜腔穿刺术抽取胎儿羊水,应用短串联重复序列(STR)位点检测并分析父母及胎儿的遗传关系,排除羊水细胞DNA中孕母DNA的污染,再采用MLPA技术进行胎儿产前诊断。结果:MLPA分析结果显示,2个家系的父母双方SMN1基因7、8外显子均为杂合性缺失,为SMA携带者。STR位点检测分析结果显示,2个家系中的孕母羊水细胞DNA均未受到孕母DNA的污染。家系1的胎儿SMN1基因7、8外显子拷贝数与父母相同,为SMA携带者;家系2的胎儿SMN1基因7、8外显子拷贝数为0,为SMA患者。结论:先证者遗传病因不明确时,可采用MLPA技术对SMA家系进行产前诊断,预防SMA患儿出生。     

先天性软骨发育不全的产前快速基因诊断

目的 探讨先天性软骨发育不全(ACH)的产前快速基因诊断方法.方法 选择2007年5～11月南京大学医学院附属鼓楼医院妇产科产前诊断中心收治的3个ACH家系:其中家系1为年龄6个月的ACH患儿;家系2为妊娠18周的孕妇,待采集胎儿羊水细胞进行产前诊断;家系3为孕39周经超声诊断为ACH的胎儿,待采集脐带血进行诊断.同时采用限制性内切酶(Sfc Ⅰ和MspⅠ)酶切、变性高效液相色谱(DHPLC)分析及序列分析3种检测方法,对外周静脉血或脐血中的成纤维细胞生长因子受体3(FGFR3)基因进行突变检测.结果 (1)DHPLC分析:家系1患者的色谱分析结果为异源双链杂合峰,家系2患者和胎儿的结果均为异源双峰,家系3胎儿的结果为异源双峰,正常对照的结果为同源单峰.(2)FGFR3基因10号外显子PCR产物酶切前的聚丙烯酰胺凝胶电泳检测结果:家系1患者、家系2患者及胎儿的PCR产物经Sfc Ⅰ酶切后除有247 bp条带外,还产生162和85 bp两个酶切产物条带,但不能被Msp Ⅰ酶切,且只有247 bp 1条产物条带.家系3胎儿PCR产物不能被Sfc Ⅰ酶切,但经Msp Ⅰ酶切后可出现162和85 bp两个产物条带.正常对照PCR产物均不能被SfcⅠ和Msp Ⅰ酶切.(3)PCR产物测序结果:家系1患者PCR产物经测序发现FGFR3基因1138G→A杂合子突变,家系2患者及胎儿也均为FGFR3基因1138 G→A杂合子突变,家系3胎儿为FGFR3基因1138 G→C杂合子突变,正常对照在FGFR3基因1138位点为G纯合子.家系2胎儿以上各项检测结果均与家系2患者相同,是遗传母亲致病突变基因的结果.结论 DHPLC和限制性内切酶酶切分析均能准确检测致病突变的FGFR3基因,但DHPLC技术操作方便快捷、准确,而且敏感性更高,可在临床上推广用于ACH的产前快速基因诊断.     

三个X-连锁重症联合免疫缺陷病家系的IL2RG基因新突变及产前诊断

目的:对3个X-连锁重症联合免疫缺陷病(SCID)家系进行致病基因突变分析和产前诊断。方法:收集2017～2018年于深圳市妇幼保健院医学遗传中心就诊的3个X-SCID家系中先证者及家庭成员的外周血,提取DNA,应用高通量测序和Sanger测序筛查三个家系IL2RG基因的突变位点,并分析突变位点的致病性,继而对家系中的高危胎儿进行产前诊断。结果:共发现IL2RG基因3个致病突变,均未见报道。3个X-SCID家系分别发现IL2RG基因:c.272dupA(p.Tyr91*),c.245_246insC(p.P82Pfs*15)和c.507delG(p.Q169fs*170)突变。结论:IL2RG基因突变:c.272dupA(p.Tyr91*),c.245_246insC(p.P82Pfs*15)和c.507delG(p.Q169fs*170)分别是3个家系的发病原因。高通量测序结合Sanger测序对X-SCID的基因诊断具有重要的价值。     

先天性代谢病的产前诊断

先天性代谢病除了极少数病种在早期可用饮食控制(例如苯丙酮尿症,PKU)、药物治疗(例如肝豆状核变性)和骨髓移植控制(例如某些免疫缺陷)外,绝大多数迄今尚无有效的治疗方法,而基因治疗目前尚处于实验研究阶段.因此,开展先天性代谢病产前诊断是重要的预防措施.1 基因诊断基因诊断又称DNA 诊断或分析,是在DNA 分子水平上对待测某特定基因进行分析,从而对有关的遗传病作出诊断.常用的产前基因诊断技术有点杂交、限制性内切酶酶谱分析(例如诊断α-地中海贫血)、     

甲型血友病家系的F8基因检测及产前基因诊断

目的:探讨甲型血友病患者及其家属进行基因诊断与产前诊断的临床价值。方法:应用DHPLC、琼脂糖凝胶电泳及Sanger测序法分别对14个血友病家系进行F8致病基因检测,在明确致病突变基础上,对其中6例疑似致病基因携带者进行产前诊断。结果:14例甲型血友病家系中,检出F8基因内含子22倒位6例,F8基因外显子14缺失1例,F8基因编码区移码突变3例,F8基因编码区错义突变4例。通过羊水穿刺产前基因诊断检出异常胎儿4例。结论:通过基因测序的方法对先证者或高度疑似携带者进行基因诊断,明确突变类型及突变位点后对其进行优生优育指导和产前基因诊断,能有效降低甲型血友病的发病率。     

基于单体型的杜氏肌营养不良无创产前检测研究

目的:探讨利用先证者辅助单体型分析方法(PAHP)对杜氏肌营养不良(DMD)进行无创产前检测(NIPT)的可行性。方法:招募生育过DMD先证者并再次妊娠的家系17例。对孕妇、孕妇丈夫与先证者外周血基因组DNA(g DNA)样本进行目标序列捕获测序,获得孕妇与致病位点连锁的单体型信息。然后在夫妻双方单体型的辅助下,通过对血浆数据中各个信息可供单核苷酸多态性(SNP)位点分析及统计,推断在DMD基因区域胎儿获得的母源单体型是否与先证者一致。携带与先证者相同单体型的男胎为患胎,女胎为携带者,其余为正常胎。将NIPT结果与DMD基因诊断金标准进行比较,验证其准确性。结果:NIPT结果提示17个家系中9例为男性胎儿,8例为女性胎儿;患胎3例,携带者4例,正常胎10例。该结果与胎儿的DMD基因诊断结果一致,无假阳性与假阴性结果。结论:基于先证者辅助单体型分析方法的NIPT取材方便,能避免宫内介入性操作,同时结果准确,应用于DMD的产前检测具有一定的可行性。     

家族性低钾性周期性麻痹家系突变分析及产前诊断研究

目的:分析一较大的汉族家族性低钾性周期性麻痹(HOKPP)家系的致病突变,为其提供产前诊断及遗传咨询。方法:选择与HOKPP疾病相关的基因CACNA1S、SCN4A、KCNE3的部分外显子,采用聚合酶链反应(PCR)方法和序列分析技术对该家系先证者进行突变筛查。针对检测到的变异,检测家系中其他患者,确定是否为致病突变,并以其为依据为胎儿进行产前基因诊断。结果:CACNA1S及KCNE3基因未检测到突变,在先证者检测到SCN4A基因的12号外显子存在错义突变(R672S),对家系中其他患者的序列分析证明均存在该突变,而表型正常者不存在该突变,突变与疾病表型呈共分离。产前诊断证实胎儿未遗传该致病突变。结论:SCN4A基因672位氨基酸的突变是该基因突变的一个热点,可作为该类患者的突变筛查的优选位点。在遗传病基因诊断过程中,根据疾病相关基因的突变特点及热点,选择性进行突变检测,可提高检测效率。     

3例家族性Alport综合征遗传咨询和产前诊断的临床病例分析

目的:利用高通量测序(NGS)技术分析家族性Alport综合征(AS)的致病性突变及遗传方式,为咨询者提供准确的遗传咨询并给予产前诊断。方法:对3例家族性AS先证者进行致病性突变分析及家系验证,对遗传咨询者进行产前诊断。结果:3例先证者均发现AS致病基因COL4A5点突变,突变位点分别位于c2633G→A、c1769A→C、c1352G→A,经家系验证均为致病性突变。对3例咨询者胎儿DNA进行Sanger验证(第一代DNA测序技术),提示1例胎儿携带AS致病基因COL4A5的错义突变,2例未检测出致病性突变。结论:认识AS遗传方式的多样性和遗传特征,强调重视先证者的基因诊断及家系验证,确定遗传方式,建议行遗传咨询并给予生育指导。     

Prenatal diagnosis of phenylketonuria by haplotype analysis

Prenatal diagnosis of classic phenylketonuria (PKU) was performed after chorionic villus sampling by means of linked restriction fragment length polymorphisms (RFLPs) using the cDNA probe ph PAH 247 (Kwok et al. (1985) Biochemistry, 24, 556-561). We report in this paper a PKU family who were only informative for RFLP analysis by a combination of two RFLPs on the basis of haplotype determination of the normal and mutant phenylalanine hydroxylase (PAH) alleles. The DNA analysis detected a PKU fetus homozygous for mutant PAH alleles and the mother opted for termination in the 12th week of gestation.     

Prenatal detection of an Arg----Ter mutation at codon 111 of the PAH gene using DNA amplification

A CGA----TGA mutation at codon 111 in exon 3 of the phenylalanine hydroxylase (PAH) gene was recently identified in a Chinese phenylketonuria (PKU) patient. This paper reports the prenatal diagnosis of a Chinese fetus at risk for PKU using DNA amplification with PCR and oligonucleotide hybridization. RFLP analysis revealed that the fetus had inherited a PKU gene from his mother, but his paternal PAH gene was uninformative. PCR amplification of 300 bp which included exon 3 plus the flanking intronic sequences of the PAH gene was performed. The amplified DNA was hybridized with a pair of allele-specific oligonucleotide probes. The results indicated that the fetal DNA carried a PAH 111 Arg----Ter mutant gene inherited from his father. Thus, the fetus was predicted to be affected with PKU.     

Undiagnosed maternal phenylketonuria: own clinical experience and literature review

Phenylketonuria (PKU) is an autosomal recessive inborn error of phenylalanine (Phe) metabolism resulting from deficiency of phenylalanine hydroxylase (PAH). Most forms of PKU are caused by mutations in the PAH gene. Untreated PKU is associated with an abnormal phenotype, which includes growth failure, seizures, global developmental delay and severe intellectual impairment. The maternal PKU (MPKU) syndrome is caused by high blood Phe concentrations during pregnancy and presents with serious foetal anomalies, especially microcephaly, congenital heart disease and mental retardation. However, since the introduction of newborn screening programs and with early dietary intervention, children born with PKU can now expect to lead relatively normal lives. We present the case of a 33-year-old woman who had been diagnosed as having PKU only after a pregnancy with MPKU embryopathy, to emphasize that undiagnosed maternal phenylketonuria still exists. On that ground, we reviewed updated literature on the pathogenesis of this syndrome, possibility of prophylaxis and treatment.     

Rapid method for targeted prenatal diagnosis of Duchenne muscular dystrophy in Vietnam

ObjectiveSince there is no effective curative treatment for Duchenne muscular dystrophy (DMD), prevention mostly depends on genetic counseling and prenatal diagnosis. About two-thirds of the affected patients have large deletions or duplications, which can be detected by multiplex ligation-dependent amplification (MLPA). The remaining cases include small mutations, which cannot be easily identified by routine techniques. In such cases, linkage analysis may be a useful tool for prenatal diagnosis. Here we compared results obtained from linkage using short tandem repeats (STRs) with those by MLPA and sequencing analysis.Materials and methodsEight Vietnamese pregnant women at risk of having a baby with DMD and requesting prenatal diagnosis were recruited in this study. MLPA and direct sequencing were applied to screen large rearrangements and point mutations in the dystrophin gene in the DMD probands and the fetal samples. STR linkage was also performed to analyze fetal mutation status.ResultsBy MLPA and sequencing analysis, five DMD patients showed deletions of the dystrophin gene, and no deletions of exons were detected in seven amniotic fluid cell samples; one patient harbored the out-of-frame small deletion of exon 43, which was also found in the fetal sample of this family. STR analysis revealed the transmission of a mutant allele inside each family.ConclusionOur results suggest that the combination of STR and MLPA could be a rapid, reliable, and affordable detection protocol for determination of the carrier's status and prenatal diagnosis of DMD in a developing country such as Vietnam.     

Prenatal diagnosis of classical phenylketonuria by linked restriction fragment length polymorphism analysis

Since the isolation of a recombinant containing a cDNA sequence for human phenylalanine hydroxylase (hPH) (Woo et al., 1983; Speer et al., 1986) prenatal diagnosis by linked restriction fragment length polymorphism (RFLPs) has become possible for families in which phenylketonuria (PKU) occurs (Lidsky et al., 1985a). We describe here the application of a Hind III three-allele RFLP in a single family, which allowed the prenatal diagnosis of an affected fetus.     

Assessment of 6 STR loci for prenatal diagnosis of Duchenne Muscular Dystrophy

ObjectiveDuchenne Muscular Dystrophy is an X-linked recessive disorder characterized by progressive muscular degeneration, patients often develop cardiac failure in the later stage and death occurs before 20 years of age. For a disease with poor postnatal prognosis such as Duchenne Muscular Dystrophy (DMD), providing the carrier mother with the option of prenatal diagnosis in a subsequent pregnancy is accepted practice in many places where termination of pregnancy is allowed. Though methods of direct sequencing such as Sanger's sequencing has been widely used, Next-Generation Sequencing is been increasingly replacing most of its application. For the DMD gene, being the longest gene in the human genome, methods of direct sequencing is often unpractical and time-consuming, instead, STR analysis for linkage analysis would be a cost-effective option and have been used routinely for prenatal diagnosis of DMD. The diagnostic significance of the STRs is based on several criteria, the most important one being the heterozygosity of the locus, power of discrimination (PD) and power of exclusion (PE).Material and methodsIn this study, we investigated the feasibility of application and diagnostic value of 6 STR loci (DSTR49, DSTR50, DXS1036, DXS1067, DXS890, DXS9907) in the proximity of the DMD gene, 66 healthy individuals were recruited for STR analysis and 5 cases of prenatal diagnosis for carrier mother were performed.ResultAllele frequency, heterozygosity, polymorphic information content, the power of discrimination and exclusion and Hardy–Weinberg equilibrium were analyzed and calculated for the 6 STR loci. 5 of these loci (DSTR49, DSTR50, DXS1067, DXS890, DXS9907) were found practical and useful for preimplantation Genetic diagnosis (PGD) and prenatal diagnosis. All 5 cases of prenatal diagnosis using the method had informative STR results and correct diagnosis.ConclusionWe concluded that our protocol of STR analysis can be applied for prenatal diagnosis and pre-implantation genetic diagnosis of DMD with high confidence and accuracy, especially in clinical settings where diagnostic resources are more limited.     

Preimplantation testing for phenylketonuria

OBJECTIVE: To use preimplantation genetic diagnosis to achieve a phenylketonuria-free pregnancy in a couple at 50% risk for producing an affected child. DESIGN: DNA analysis of the first and second polar bodies (PB1 and PB2) obtained from oocytes of a heterozygous mother in IVF-ET, with the goal of identifying and transferring back to the patient the embryos resulting from mutation-free oocytes. SETTING: IVF program of Reproductive Genetics Institute, Chicago, Illinois. PATIENT(S): A mother carrying the R408W mutation and a father with compound heterozygosity for R408 and Y414C mutations in phenylalanine hydroxylase (PAH) gene. INTERVENTION(S): Removal and testing for maternal mutation in PB1 and PB2 from each oocyte after standard IVF. MAIN OUTCOME MEASURE(S): DNA analysis of PB1 and PB2 indicating whether corresponding oocytes were mutation-free, for the purposes of transferring only unaffected embryos resulting from these oocytes. RESULT(S): Of 11 zygotes with both PB1 and PB2, 6 were predicted to be free of phenylketonuria. Of these, 4 were transferred, resulting in an unaffected twin pregnancy and birth of two healthy children. CONCLUSION(S): Preimplantation genetic diagnosis of phenylketonuria resulted in the birth of phenylketonuria-free children. Preimplantation genetic diagnosis by PB analysis in couples with a compound heterozygous male partner is clinically useful.     

Hyperphenylalaninemia in a premature infant with heterozygosity for phenylketonuria

Hyperphenylalaninemia in preterm neonates with heterozygosity for phenylketonuria has previously not been described. We report on a very low birth weight infant, born at a gestational age of 27+5 weeks with a birth weight of 1080 g. Due to a positive family history prenatal diagnosis for phenylketonuria was performed, revealing heterozygosity for classic phenylketonuria. Yet the girl showed hyperphenylalaninemia with a maximum serum phenylalanine concentration of 515 micromol/l on the eighth day of life. Phenylalanine-restrictive parenteral and enteral nutrition was kept from the eighth until the 41st day of life. At term serum phenylalanine concentrations had normalized. We hypothesize that heterozygosity for phenylketonuria may be a risk factor for hyperphenylalaninemia in preterm born infants. Prematurity and the resulting immaturity of liver function with the genetically determined reduced activity of phenylalanine hydroxylase might have caused hyperphenylalaninemia in this girl.     

A new case of maternal phenylketonuria treated with sapropterin dihydrochloride (6R-BH4)

Purpose: A woman with phenylketonuria (PKU) was diagnosed through neonatal screening, her PAH mutation was p.V388M/p.I65T, for which she received treatment with phenylalanine restriction, and was administered oral sapropterin dihydrochloride (6R-BH₄) from the age of thirty. The purpose of this article is to describe the treatment with BH₄ during her pregnancy and to evaluate a plan for its use.

Methods: The patient had an unplanned pregnancy at 34 years of age, for which she received a phenylalanine-free supplement enriched with essential fatty acids, vitamins and trace elements.

Results: The dose of 6R-BH₄ was reduced from 500?mg/day to 100?mg/day until its suspension in the 28th week of gestation, and was well tolerated. Blood phenylalanine control was easily accomplished during this pregnancy, and no nutritional deficiency was seen.

Conclusion: The pregnancy had a normal outcome, and so we consider that adaptation of the dose of 6R-BH₄ to the prenatal periods aided a greater efficiency and a lower risk in the treatment of maternal PKU.