Prenatal diagnosis for severe methylenetetrahydrofolate reductase deficiency by linkage analysis and enzymatic assay

Severe methylenetetrahydrofolate reductase (MTHFR) deficiency is characterized by varying degrees of developmental delay, motor and gait abnormalities, seizures, and thrombosis. Biochemical abnormalities include homocystinuria and hyperhomocysteinemia. Clinical severity correlates with MTHFR activity in cultured fibroblasts; activity can also be assayed in cultured amniocytes and chorionic villus cells (CVC). Forty-four private mutations have been identified, limiting the use of direct mutation detection for prenatal diagnosis. However, intragenic polymorphisms have been identified, making prenatal diagnosis by linkage analysis a possible option, even without knowledge of deleterious mutations. Prenatal diagnosis for severe MTHFR deficiency has been available by biochemical methodologies, but molecular genetic approaches have not yet been reported. We performed prenatal diagnosis for severe MTHFR deficiency in 11 at-risk pregnancies in seven families. A combined approach of linkage analysis and enzymatic assays was used in six pregnancies; linkage analysis alone was performed in one pregnancy. Linkage analysis for the 677C > T or 1298A > C polymorphisms predicted that all seven fetuses were unaffected. For six of these seven fetuses, enzymatic activities were also measured and demonstrated concordant results. Of the 10 pregnancies in which enzymatic assays were performed, activities in cultured amniocytes predicted six unaffected fetuses (1.4-7.1 nmol CHO/mg prot/h (U)) and one affected fetus (0.24 U [control 3.1-9.6 U]). Three pregnancies assessed via CVCs demonstrated two unaffected fetuses (3.6 and 7.7 U) and 1 affected fetus (0 U [control 4.5-7.8 U]). These values were compared to those of the probands (range = 0.02-0.7 U (control 2.4-11.7 U)) in cultured fibroblasts. Our findings suggest that linkage analysis for severe MTHFR deficiency can be a practical approach for prenatal diagnosis.     

Prenatal diagnosis and detection of carriers with DNA probes in Duchenne's muscular dystrophy

We performed genetic analyses for the prenatal diagnosis of Duchenne's muscular dystrophy and detection of the carrier state in five families with seven pregnancies at risk for the disease. As genetic markers for the disorder, we used DNA-sequence polymorphisms detected with 12 different DNA probes derived from the vicinity of the Duchenne's muscular dystrophy locus or from within the gene, on the X chromosome. One male fetus of a proved carrier mother was predicted to be unaffected, and this was confirmed after birth. Another male fetus was predicted to be unaffected (probability, 95 percent or greater), although a crossover event had been identified in a region of the X chromosome thought to be distal to the Duchenne gene. Unfortunately, an elevated serum creatine kinase level after birth indicated that the infant had inherited the Duchenne mutation. Three male fetuses predicted to be affected with 66 percent or 95 percent probabilities were aborted, and the presence of the DNA-marker alleles was confirmed in fetal tissues. In one family, in which the maternal grandparents were unavailable, the initial genetic interpretation had to be revised after a second male fetus was analyzed with intragenic probes. Our experience suggests that despite the large number of intragenic and flanking DNA polymorphisms available, uncertainties often remain in the prenatal diagnosis of Duchenne's muscular dystrophy. Pitfalls are presented by the large size of the region in which Duchenne's mutations can occur. Crossover events in this region, which result in an exchange of DNA between two X chromosomes, can render DNA-marker studies inaccurate. Also, an autosomal recessive mutation can produce the same clinical picture.     

10个甲型血友病家系FⅧ基因突变分析

目的 分析10个家系中甲型血友病(hemophilia A,HA)患者及女性疑似携带者FⅧ基因突变,并指导产前诊断.方法 应用聚合酶链反应(polymerase chin reaction,PCR)、变性高效液相色谱技术(denaturing high performance liquid chromatogramphy,DHPLC)和DNA测序技术对10个家系中8例HA患者、12名女性疑似携带者的FⅧ基因进行突变检测,并应用St14(DXS 52)、13(CA)n、EX18/BclⅠ3个遗传标记位点对HA家系进行连锁分析.产前诊断检测已知突变位点.针对未见报道的新突变,用限制性内切酶进行分析,同时与100名表型正常的无关个体进行比较,排除多态性.结果 (1)在10个家系中发现5例错义突变、3例移码突变、2例无义突变和2个单核苷酸多态性(single nucleotide polymorphism,SNP)位点.错义突变c.878A＞G、c.1015A＞G和c.6870G＞T,移码突变c.1282delA、c3072_3073insT和c.4880_4881insA以及SNP位点c.5000 G＞A均为国际血友病网站和人类突变数据库未记载的突变或多态.在100名正常人中未检测到错义突变c.878A＞G、c.1015A＞G和c.6870G＞T.(2)在12名女性疑似携带者中,基困水平确诊9例为HA携带者,3名为正常人.(3)遗传连锁分析为4个家系提供了X风险染色体的有效信息.(4)产前诊断结果显示2例胎儿正常、1例HA携带者和1例HA患者.结论 发现c.878 A＞G、c.1015A＞G、c.6870G＞T、c.1282delA、c.3072 3073insT及c.4880_4881insA共6种能引起甲型血友病的新突变;PCR、DHPLC和DNA测序技术可有效检测甲型血友病患者基因突变,而联合限制性内切酶分析和遗传连锁分析能快速筛查HA携带者,进行有效的产前诊断.

Abstract：

Objective To identify the F Ⅷ gene mutations of patients and suspected female carriers in 10 Hemophilia A (HA) families, and to guide the prenatal diagnosis. Methods PCR, denaturing high performance liquid chromatogramphy(DHPLC) and DNA sequencing technologies were applied to screen the FⅧ gene of 8 HA patients and 12 suspected female carriers in the 10 families. Linkage analysis was performed by using St 14 (DXS 52), intron 13 (CA)n and EX18/Bcl Ⅰ of the FⅧ gene in the HA families.In prenatal diagnosis, we screened the same mutation found in the patients. PCR-restriction fragment length polymorphism was applied to detect the new missense mutations of F Ⅷ gene in 100 unrelated healthy individuals to exclude the possibility of polymorphism. Results (1) Five missense mutations, 3 frameshift mutations, 2 nonsense mutations and 2 single nucleotide polymorphism(SNP) were identified in 10 the HA families. Among them, c. 878A＞G, c. 1015A＞G, c. 6870G＞T, c. 1282delA, c. 3072_3073insT, c. 4880_4881insA and c. 5000G＞A were novel mutations or polymorphism. No missense mutations c. 878A＞G, c.1015A＞G and c. 6870G＞T, were found in the 100 healthy unrelated controls. (2) Nine suspected female carriers were confirmed at the gene level. (3) X risk chromosome could be determined in 4 HA families by genetic linkage analysis. (4) Among the four fetuses for prenatal diagnosis, 2 were normal, 1 was carrier and the remaining 1 was a patient. Conclusion Six novel mutations, i. e. , c. 878A＞G, c. 1015A＞G, c.6870G＞T, c. 1282delA, c. 3072_3073insT and c. 4880_4881insA, were identified in this study. PCR,DHPLC and DNA sequencing could be used to screen the gene mutations of HA patients, to carry out carrier detection and prenatal diagnosis of HA families efficiently, by combining with restriction endonuclease analysis and genetic linkage analysis.     

Prenatal diagnosis of congenital adrenal hyperplasia (21-hydroxylase deficiency) in Croatia

We report on the prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase in 20 at-risk pregnancies (16 salt-wasting and 4 simple virilizing families). We have diagnosed 3 affected fetuses (2 males and 1 female), 3 healthy homozygotes (2 males and 1 female), and 14 healthy heterozygotes (7 females and 7 males). These data were collected over 4 years. In 16 fetuses, the diagnosis was made with measurements of 17-hydroxyprogesterone (17-OHP) and Δ-4-androstenedione (Δ) in amniotic fluid (AF), human leukocyte antigen (HLA) typing of amniotic cells, as well as karyotypes between the 16th and 18th weeks of gestation. In 4 fetuses, DNA analysis of amniotic cells was also performed. In 3 pregnancies in which affected fetuses were suspected (on the basis of HLA typing and measurements of 17-OHP and Δ concentrations in AF), the fetuses were electively aborted between the 17th to 19th weeks of gestation by parental decision. In all aborted fetuses, diagnosis was confirmed with HLA typing, autopsy findings of hyperplastic adrenal glands, and ambiguous genitalia in female fetuses. Postnatal diagnosis was confirmed in healthy fetuses with HLA typing and serum measurements of 17-OHP concentrations, and in 4 of them with DNA analysis. In 3 of the 4 families, DNA analyses revealed the following mutations: in Family 1, the index case mutation was Intron 2, Exon 3/Exon 6, and the fetus was Normal/Exon 6; in Family 2, the index case mutation was Ex1 Int2 Ex3/Int2, and the fetus was Ex1 Int2 Ex3/Normal; and in Family 3, the index case mutation was Ex8₃₅₆/Ex8₃₅₆, and the fetus was Ex8₃₅₆/Normal. We also report one case of prenatal diagnosis and treatment. Dexamethasone 0.5 mg BID (20 μg/kg/d) was given starting at 6th week of gestation. Prenatal diagnosis suggested, but did not prove, that the female fetus was a heterozygote as the fetus lacked the paternal mutation Ex8₃₁₈. No mutation was found in the mother. The fetus, the mother, and the affected sib shared a haplotype, further suggesting heterozygosity. The unaffected status was confirmed postnatally. Am J. Med. Genet. 72:302–306, 1997. © 1997 Wiley-Liss, Inc.     

90个脊髓性肌萎缩家系的遗传学分析

目的对90个脊髓性肌萎缩(spinal muscular atrophy,SMA)家系进行基因诊断与产前诊断,为SMA的遗传学分析方法提供参考,并初步探讨SMA缺陷基因携带者基因筛査的必要性。方法应用多重连接探针扩增(multiplex ligation dependent probe amplification,MLP A)技术对90个SMA家系进行基因诊断,联合MLPA及等位基因特异性PCR(allele specific PCR,A&PCR)技术对家系进行产前诊断并对产前诊断结果进行分析。结果在90个SMA家系中,84对夫妻无SMA家族史,占比93%;85对夫妻有SMA生育史,占比94%;85个家系夫妻双方及3个家系的孕妇SMN1基因杂合缺失,为SMA缺陷基因携带者;2个家系孕妇SMN1基因纯合缺失,为SMA患者;产前诊断结果显示48名胎儿为SMA缺陷基因携带者,23名胎儿为正常胎儿,19名胎儿为SMA患者,其中无家族史夫妻双方再孕SMA胎儿18例,占总SMA胎儿95%、占总胎儿20%。结论应用MLPA对夫妻双方进行SMA缺陷基因携带者筛查,并联合使用MLPA和AS-PCR对携带者夫妻进行SMA产前诊断十分必要,对预防SMA出生缺陷具有积极意义。     

Prenatal diagnosis of autosomal recessive polycystic kidney disease (ARPKD): Molecular genetics,clinical experience,and fetal morphology

Autosomal recessive polycystic kidney disease (ARPKD) is one of the most common hereditary renal cystic diseases and has a high infant mortality. Prenatal diagnosis using fetal sonography can be unreliable, especially in early pregnancy. The ARPKD locus has been mapped to proximal chromosome 6p allowing haplotype-based prenatal diagnosis in “at-risk” families. From December 1994 to March 1997, we received 258 inquiries regarding prenatal evaluation and we have completed analyses in 212 families. To date, 65 prenatal analyses have been performed in 57 families. In the majority of the requesting families (45/57), the index children are deceased and their DNA was extracted from paraffin-embedded tissue. Eighteen fetuses were homozygous for the disease-associated haplotypes. In 12 of these fetuses, pathoanatomical examination demonstrated typical ARPKD changes consisting of dilated collecting ducts and the characteristic hepatic ductal plate malformation. These changes were detected in two fetuses as early as 13 weeks gestational age. These cases represent the earliest demonstration of ARPKD-associated histopathology reported to date. One high risk fetus was carried to term and turned out to be unaffected. However, the diagnosis of ARPKD remained doubtful in the index patient. Forty-three fetuses were either heterozygous or homozygous for a nondisease-associated haplotype and all infants born were phenotypically unaffected at birth. In four cases, a recombination event occurred between the flanking markers and no genotypic prediction was possible. Three of these pregnancies were terminated and necropsy of the fetuses confirmed ARPKD, while one fetus was carried to term and showed no abnormalities at birth. These results show that haplotype-based prenatal testing is feasible and reliable in pregnancies “at risk” for ARPKD. An absolute prerequisite for these studies is an accurate diagnosis of ARPKD in previously affected sib(s). Am. J. Med. Genet. 76:137–144, 1998. © 1998 Wiley-Liss, Inc.     

DiGeorge/velocardiofacial syndrome: FISH studies of chromosomes 22q11 and 10p14, and clinical reports on the proximal 22q11 deletion

Interphase fluorescence in situ hybridization (FISH) analysis can provide rapid preliminary analysis of chromosome aneuploidy from direct amniocyte and chorionic villus sample (CVS) preparations. Typically, interphase FISH is used in screening for numerical abnormalities of chromosomes X, Y, 13, 18, and 21. More recently, FISH probe sets became available for the subtelomeric region of each chromosome, allowing screening for terminal chromosome rearrangements. The purpose of the current study was to evaluate the use of dual-color interphase FISH analysis with chromosome-specific subtelomere probes for rapid prenatal diagnosis in 14 pregnancies from 12 different translocation carriers. Interphase FISH analysis was performed on direct CVS or amniocyte preparations from 12 reciprocal translocation and two Robertsonian translocation pregnancies with the appropriate chromosome-specific subtelomere probes for each chromosome involved in the translocation. Analysis of the interphase FISH probe signals predicted balanced or normal segregants in each case, thus rapidly excluding a chromosomally unbalanced segregant. Subsequent metaphase analysis showed normal karyotypes in seven fetuses and balanced translocations in the remaining seven. This series illustrates the utility of interphase FISH analysis with chromosome-specific subtelomere probes for rapid prenatal diagnosis in cases of parental reciprocal translocations and Robertsonian translocations.     

Carrier detection of the X-linked primary immunodeficiency diseases using X-chromosome inactivation analysis

Carrier detection of three of the X-linked primary immunodeficiency diseases (X-linked agammaglobulinemia, X-linked severe combined immunodeficiency disease, and the Wiskott-Aldrich syndrome) is possible by analyzing patterns of X-chromosome inactivation in those cells affected by the disorder. Normal women have balanced patterns of X-chromosome inactivation; that is, in a given population of cells, approximately half of their active X chromosomes are of paternal origin and half of their active X chromosomes are of maternal origin. In contrast, female carriers of these X-linked immunodeficiency disorders have an unbalanced pattern of X-chromosome inactivation in those cell lineages that are affected by the disorder; that is, all the active X chromosomes in affected cell lineages are the X chromosomes that carry the normal allele. Two techniques are available for X-chromosome inactivation analysis. One technique depends on methylation differences between the active and inactive X chromosome, and the other technique uses somatic cell hybrids that selectively retain the active X chromosome. In either case, carrier detection can be performed in individuals from families in which only one member of the family has been affected, since neither of these methods depends on linkage analysis.     

血友病甲基因分析技术的改进及其在产前诊断中的应用

目的对血友病甲基因分析技术进行改进并应用于携带者检查和产前诊断。方法长距离聚合酶链反应方法直接检测凝血因子Ⅷ第22内含子倒位,对非倒位家系用FⅧ基因内限制酶切位点XbaⅠ、HindⅢ、二核苷酸重复序列多态性位点STR13和STR22,以及基因外可变数目串联重复序列DXS52(St14)位点进行基因连锁分析。结果52个家系共检出71位携带者。21个家系为第22内含子倒位,28个家系经连锁分析得到明确诊断,3个家系无法诊断,可诊断家系占94.2%。为18个家系做胎儿产前诊断,其中10例诊断为血友病甲胎儿;诊断7例正常男胎和1例携带者女胎,随访1年发育正常。结论应用长距离聚合酶链反应和多位点基因连锁分析技术可以快速有效地进行血友病甲携带者检查和产前诊断。     

CHD7 mutations causing CHARGE syndrome are predominantly of paternal origin

CHARGE (coloboma, heart defects, atresia of the choanae, retarded growth and development, genital hypoplasia, ear anomalies and deafness) syndrome is a congenital malformation syndrome caused by mutations in the CHD7 gene in approximately 2/3 of cases. In the vast majority of cases, CHARGE syndrome is sporadic. There are only a few reports of parent-to-child transmission and somatic or gonadal mosaicism. To determine the parental origin of CHD7 mutations in sporadic CHARGE syndrome, we screened 30 families for informative exonic or intronic polymorphisms located near the detected CHD7 mutation. An informative polymorphism could be identified in 13 out of 30 families. Linkage analysis was performed between the CHD7 mutation and the polymorphism in the child. In 12 out of 13 families, the mutation affected the paternal allele (92.3%). In our cohort, the mean paternal age at birth was 32.92 years. Comparing the age of fathers of an affected CHARGE patient with the paternal age of the German population in general, we could not observe any paternal age effect. Taken together, we show in this study that de novo CHD7 mutations occur predominantly in the male germ line.     

The application of linkage analysis to genetic counselling in families with Duchenne or Becker muscular dystrophy.

A total of 278 families of probands with Duchenne or Becker muscular dystrophy has been ascertained and offered genetic counselling. Linkage studies have been performed in these families using polymorphic DNA markers identifying loci linked to Duchenne and Becker muscular dystrophy. The clinical features of the probands are discussed: there was marked intrafamilial resemblance in the severity of the disease. We estimate that a complete study of potential carriers in these families would require analysis of samples from approximately 1400 subjects. The results of linkage studies tended to move women's carrier risk estimates (based on CK and pedigree data) towards the extremes of the risk categories, providing a more definitive risk estimate for 81% of the women who were previously in the middle range of carrier risk probabilities. About 70% of the families had only one affected member. Linkage analysis altered carrier risk estimates in 95% of sisters and aunts of index cases, but only affected estimates of the mother's carrier risks in about 11% of isolated cases. Even where linkage studies were not helpful in elucidating carrier risks, information could usually be obtained for use in prenatal diagnosis if required. We have assessed the attitudes to pregnancy and prenatal diagnosis of women at risk of being carriers of Duchenne or Becker muscular dystrophy and report 17 pregnancies in these women.     

Prenatal diagnosis of Fukuyama type congenital muscular dystrophy in eight Japanese families by haplotype analysis using new markers closest to the gene

We conducted prenatal diagnosis by haplotype analysis, using newly developed microsatellite markers, in eight Fukuyama type congenital muscular dystrophy (FCMD) families. In addition to six new families, two previously reported families were re-examined by haplotype analysis including detection of an ancestral founder haplotype (138–183–301) for 3 microsatellite markers closest to the FCMD gene, designated D9S2105–D9S2107–D9S172, the distances of which from the FCMD gene are presumed to be ∼140, ∼20, and ∼280 kb, respectively. Five fetuses from five families were diagnosed as nonaffected, and were subsequently confirmed to be healthy. Three fetuses of the other three families were diagnosed as having a high probability of being affected by FCMD. In the prenatal diagnosis conducted for these eight families, the ancestral founder allele was observed in 13 of 16 (81%) FCMD-bearing chromosomes. Detection of the ancestral haplotype facilitated achieving accurate prenatal diagnosis of FCMD. The brains of all three fetuses prenatally diagnosed as FCMD-affected showed the initial stage of cortical dysplasia, strong evidence of FCMD. Am. J. Med. Genet. 77:310–316, 1998. © 1998 Wiley-Liss, Inc.     

Prenatal prediction of Werdnig-Hoffmann disease using linked polymorphic DNA probes.

Werdnig-Hoffmann disease is a common autosomal recessive neuromuscular disorder that results in paralysis and death. No treatment to prevent this disease or to alter its unremitting course has been found. Recently, linkage analysis with cloned DNA probes has shown that the mutation causing Werdnig-Hoffmann disease is located on chromosome 5q12-q14. We performed genetic analysis for the prenatal diagnosis of Werdnig-Hoffmann disease in seven at risk families. Two fetuses were diagnosed as being affected and the remainder as unaffected, and this was confirmed after birth. This study shows that prenatal diagnosis of Werdnig-Hoffmann disease has become feasible.     

Do parental perceptions and motivations towards genetic testing and prenatal diagnosis for deafness vary in different cultures?

Surveys of attitudes of individuals with deafness and their families towards genetic testing or prenatal diagnosis have mostly been carried out in the West. It is expected that the perceptions and attitudes would vary amongst persons of different cultures and economic background. There is little information on the prevailing attitudes for genetic testing and prenatal diagnosis for deafness in developing countries. Therefore, this study evaluates the motivations of Indian people with inherited hearing loss towards such testing. Twenty‐eight families with history of congenital hearing loss (23 hearing parents with child/family member with deafness, 4 couples with both partners having deafness and 1 parent and child with deafness) participated in a semi‐structured survey investigating their interest, attitudes, and intentions for using genetic and prenatal testing for deafness. Participants opinioned that proper management and care of individuals with deafness were handicapped by limited rehabilitation facilities with significant financial and social burden. Nineteen (68%) opted for genetic testing. Twenty‐six (93%) expressed high interest in prenatal diagnosis, while 19 (73%) would consider termination of an affected fetus. Three hearing couples, in whom the causative mutations were identified, opted for prenatal diagnosis. On testing, all the three fetuses were affected and the hearing parents elected to terminate the pregnancies. This study provides an insight into the contrasting perceptions towards hearing disability in India and its influence on the desirability of genetic testing and prenatal diagnosis. © 2012 Wiley Periodicals, Inc.     

Genetic counseling of isolated carriers of Duchenne muscular dystrophy

It has recently become possible to detect female carriers of Duchenne muscular dystrophy with no affected male relative in the family. These “isolated carriers” represent about 10% of women with high serum creatine phosphokinase (CPK) levels and clinical evidence of a muscle disease. Most isolated carriers ascertained by clinical and/or CPK levels and diagnosed by dystrophin immunostaining of muscle biopsy show symptoms of a muscular dystrophy, and often carry the diagnosis of recessive “limb-girdle muscular dystrophy” prior to dystrophin analysis. It has been difficult to offer genetic counseling and prenatal diagnosis for Duchenne muscular dystrophy in the families of these isolated carriers, largely due to the difficulty in determining which of the dystrophin alleles segregating in the family harbors the mutation in the heterozygote. Here we report genetic counseling of three isolated carriers and their families. In two cases, prenatal diagnosis of at-risk pregnancies was conducted. We determined X inactivation patterns and inheritance of X chromosomes in each family, and used this information to define the at-risk dystrophin gene. In all three families, the mutation was a de novo event, two in the paternal germ-line, and one in the maternal germ-line. In each case we show that sibs of the heterozygous woman are at population risk, while pregnancies of each propositus are at high risk. Our results show that accurate genetic counseling and prenatal diagnosis can be offered to these families. © 1996 Wiley-Liss, Inc.     

Characterization of microsatellite markers flanking FBN1: utility in the diagnostic evaluation for Marfan syndrome

Marfan syndrome (MFS) is an autosomal dominant disorder of connective tissue with marked interfamilial and intrafamilial variation in phenotype. The primary defect in affected patients resides in the gene for fibrillin-1 (FBN1) on 15q21. Linkage analysis has shown no locus heterogeneity in the classic phenotype, although substantial allelic heterogeneity exists. Recently it has been shown that the size of the gene is approximately 200 kb. These and other factors have precluded routine mutation screening for presymptomatic and prenatal diagnosis. Previously we described four intragenic microsatellite polymorphisms that can be used for haplotype segregation analysis. The utility of this approach is limited because the markers do not fully span the gene and show incomplete informativeness, with 16% homozygosity for the most common haplotype. We have now identified and localized highly polymorphic microsatellite markers that fall within 1 Mb of FBN1. Complete haplotype heterozygosity was observed in a population of 50 unrelated control individuals when the flanking markers and existing intragenic polymorphisms were used in combination. We demonstrate the utility of haplotype segregation analysis in the presymptomatic diagnosis and counseling of families showing atypical or equivocal manifestations of MFS. Copyright Wiley-Liss. Inc.     

Sperm chromosome analysis to assess potential germ cell mosaicism

Human sperm chromosome complements were examined to assess the possibility that the conceptions of two children with the same chromosomal defect, del(13)(q22q32), from chromosomally normal parents were the result of a paternal germ cell mosaicism. Analysis of 216 complements, both by quinacrine banding and by measuring the relative length of chromosome 13, showed no unusual subpopulation of 13s; this decreased the likelihood of a paternal origin of the deletion. Sperm chromosomal analysis is a useful adjunct to available techniques in clinical genetics. When counseling cases involving either structural or numerical de novo chromosome abnormality, it is of importance to discuss the possibility of germ cell line mosaicism as well as to offer prenatal diagnosis for subsequent pregnancies.     

Mutational spectrum and DNA-based prenatal diagnosis in carnitine-acylcarnitine translocase deficiency

Carnitine-acylcarnitine translocase (CAC) deficiency is a rare autosomal recessive disorder of long-chain fatty acid oxidation with a severe outcome. We report mutation analysis in a cohort of 12 patients. Twelve mutations were identified of which 9 have not been reported so far (G28C, D32N, R178Q, P230R, D231H, 179delG, 802delG, 69-70insTGTGC, and 609-1g>a). Altogether, including our results, 22 mutations of the CAC gene have been published to date in 23 patients demonstrating the allelic heterogeneity of CAC deficiency. DNA-based prenatal diagnosis was performed for the first time in pregnancies at risk for CAC deficiency. Two fetuses were affected and one pregnancy was terminated by family decision. Two other fetuses had normal genotype and five others were heterozygotes. All the offspring of these seven pregnancies are alive and apparently healthy.     

Determination of the parent of origin in nine cases of prenatally detected chromosome aberrations found after intracytoplasmic sperm injection

Prenatal cytogenetic analysis of 71 fetuses conceived by intracytoplasmic sperm injection (ICSI) resulted in the detection of nine (12.7%) chromosome aberrations including two cases of 47,XXY, four cases involving a 45,X cell line and three autosomal trisomies. Molecular analysis of the parental origin of the deleted or supernumerary chromosome was performed by using polymorphic microsatellite markers. Six cases involving a sex chromosome abnormality were found to be of paternal origin while the two trisomic cases that could be analysed were of maternal origin. Two cases involved the same infertile couple who had two consecutive ICSI pregnancies terminated because of a chromosome abnormality. The replaced embryos in both cases originated from a single batch of ICSI fertilized oocytes of which part was used to initiate the first pregnancy and part was cryopreserved and used to initiate the second pregnancy.      

应用变性高效液相色谱技术进行肝豆状核变性的基因突变筛查及产前诊断

目的 探讨变性高效液相色谱(denature high performance liquid chromatography,DHPLC)技术在肝豆状核变性(Wilson's disease,WD)的突变筛查及产前诊断中的临床应用.方法 以6个WD家系中的患者及其父母的DNA为模板,采用PCR技术扩增ATP7B基因的21个外显子及5'非翻译区,PCR产物经DHPLC技术进行突变筛查,对峰型有改变者进行测序验证.在确定了先证者突变类型的基础上,采用相同方法对其中4个家系(1个双胎和3个单胎)进行产前诊断.结果 6例患者中检测出5种已知的致病突变及8种多态类型.患者的父母均为相应突变类型的携带者.产前诊断结果显示,两例妊娠为异常胎儿,其中1例双胎为Arg778Leu/IVS4-1G>C双重杂合子,1例单胎为Ser975Tyr/Pro992Leu双重杂合子,这两对妊娠夫妇选择了终止妊娠.另两例妊娠中,1例为Ser975Tyr杂合子,1例完全正常,他们选择了继续妊娠,出生了表型正常儿.结论 DHPLC在Wilson病的突变检测和产前诊断中有良好的应用前景.