[Genetic testing in a Chinese pedigree with Lowe syndrome].

OBJECTIVE: To identify the pathogenic mutation underlying Lowe syndrome in a Chinese family. METHODS: After obtaining written informed consent of all participating individuals, peripheral blood samples were collected from 11 family members, including one affected male and three obligate female carriers, and an amniotic fluid sample was obtained from a pregnant carrier female in the family. Genomic DNA was extracted using the standard SDS-proteinase K-phenol/chloroform method. Linkage analysis was carried out using microsatellite markers flanking the OCRL gene. All OCRL exons and their flanking intronic sequences were PCR-amplified and sequenced for the proband. Restriction analysis was performed to confirm the pathogenic mutation. Prenatal genetic testing was carried out by combining haplotype analysis, DNA sequencing and restriction analysis. RESULTS: Linkage analysis showed that the affected male and three obligate carrier females shared a same haplotype. Sequence analysis in the proband revealed a nonsense mutation c.2032C→T (p.R678X) in exon 18 of the OCRL gene. Restriction analysis showed that the mutation was present in the proband and three obligate carriers, but not in the other 7 available family members. This nonsense mutation was not found in the amniotic fluid sample. CONCLUSION: A recurrent OCRL nonsense mutation was found to be the pathogenic mutation in the Chinese family and the fetus didn't carry the mutation.

[Study on the correlation of serum folate and red blood cell folate level with birth defects and unexplained recurrent pregnancy loss].

OBJECTIVE: To understand the correlation of lower serum folate, and red blood cell (RBC) folate level with birth defects including unexplained recurrent pregnancy loss, and to evaluate the role of RBC folate level as a suitable marker for folate supplement. METHODS: Two hundred and ninety-nine non-pregnant women at child-bearing age with a birth defect history were selected as birth defect group. The levels of serum and RBC folate, and serum vitamin B(12) were determined. By comparing with the group of non-pregnant women at child-bearing age without any birth defect history (control group), we evaluated the correlation between lower serum folate, RBC folate level and main kinds of birth defects including unexplained recurrent pregnancy loss. And the levels of serum and RBC folate of birth defect group were also determined and compared before and after oral folate intake (5 mg/d) for one month. RESULTS: The serum folate level of birth defect group was not different from the control group (17 - 26 vs 14 nmol/L, P > 0.05). The RBC folate level of birth defect group except the urinary defect was significantly lower compared with the control group (233 - 547 vs 689 nmol/L, P < 0.05). After the oral folate intake (5 mg/d), the serum folate level of unexplained recurrent pregnancy loss group and neural tube defects group were significantly increased than before [(22 +/- 9) vs (27 +/- 12) nmol/L, (19 +/- 10) vs (25 +/- 18) nmol/L; P < 0.05]. The RBC folate level of unexplained recurrent pregnancy loss group and congenital heart defect group were significantly increased than before [(374 +/- 275) vs (567 +/- 397) nmol/L, (322 +/- 205) vs (527 +/- 351) nmol/L, P < 0.05]. CONCLUSION: RBC folate level is more closely correlated than serum folate level with the incidence of main birth defect.

[The power of linkage analysis on PAH gene in prenatal gene diagnosis is improved with three additional short tandem repeat markers].

OBJECTIVE: To increase the success rate of prenatal diagnosis for classical phenylketonuria(PKU). METHODS: Three new short tandem repeat (STR) markers (PAH26, PAH32 and PAH9) within and surrounding phenylalanine hydroxylase(PAH) gene were selected for amplified fragment length polymorphism. The allele frequencies and polymorphism information contests (PIC) were determined in Chinese population. RESULTS: The PIC of these three new STR markers was 0.518 (PAH26), 0.413 (PAH32) and 0.362 (PAH9) respectively. There was linkage disequilibrium between PAH9 marker and PAH-STR marker (TCTA)n in the intron 3 of PAH gene. The linkage phase of the mutant genes and the markers was established using the combination of PAH-STR, PAH26 and PAH32 in 95% families. Prenatal diagnosis was performed successfully with these markers in four cases. CONCLUSION: By selecting or combining the three STR markers, the mutant genes could be distinguished from the normal allele in up to 95% of families with classical PKU.

[Postnatal and prenatal diagnosis of mucopolysaccharidosis type II (Hunter syndrome)].

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.

[Detection of the gene-deleted female carriers of Duchenne/Becker muscular dystrophy using a fluorescent in situ hybridization based method].

OBJECTIVE: To set up a fluorescent in situ hybridization (FISH) based method to detect the gene-deleted female carriers of Duchenne/Becker muscular dystrophy (DMD/BMD). METHODS: Multiplex polymerase chain reaction was used to identify the gene deletion DMD/BMD probands and their female relatives were checked by double-color FISH. RESULTS: Two probands whose exon 46 of dystrophin gene was deleted, one had a positive pedigree and the other was a sporatic patient. In the case of the positive pedigree, four carriers were detected. In the case of the sporatic family, FISH showed that the mother of the proband was a somatic mosaicism. CONCLUSION: Combined with multiplex PCR, double-color FISH is a simple, fast, directly visual and accurate method. It is feasible to identify the carrier status of the female relatives of the gene deletion DMD/BMD probands. The detection of the somatic mosaicism is a prominent feature of FISH.

[Screening by maternal serum markers for Down's syndrome].

OBJECTIVE: To investigate the optimal method of screening for Down's syndrome (DS) with maternal serum mankers. METHODS: Screening by maternal serum markers for Down's syndrome was offered to all 2886 pregnant women in Peking Union Medical Hospital during 1996.11-2001.3. Alpha-fetoprotein (AFP), human chorionic gonadotrophin (free beta-HCG) were used as markers during the first year of pregnancy. Alpha-fetoprotein, free human chorionic gonadotrophin (HCG) and pregnancy-associated plasma protein A (PAPP-A) were used as mid pregnancy and first-trimester markers in next three years. Amniocentesis and (CVS) were done in those defined as risk cases. RESULTS: The detection rate of Down's syndrome by maternal serum markers was 3.8% (11/2886). The proportion of false positive results in group of triple markers (alpha FP, free beta-HCG, PAPP-A) was 5%. CONCLUSIONS: The PAPP-A was a good marker to detect Down's syndrome in early pregnancy and may be used to predict the outcome during mid trimester of pregnancy. The AFP and free beta-HCG can be useful markers to detect Down's syndrome and fetal abnormality. While prenatal diagnostics can be shifted to an early pregnant period.

[Prenatal diagnosis and clinical management of a twin pregnancy consisting of a complete mole and coexisting fetus].

OBJECTIVE: To discuss the differential diagnosis of the hydatidiform mole and a coexisting fetus, to study the prenatal diagnosis and the clinical management of a twin pregnancy consisting of a complete mole and coexisting fetus (CMCF). METHODS: Two cases of CMCF were reported retrospectively. RESULTS: In the first case, the hydatidiform mole and a coexisting fetus was found by B mode ultrasound at the 10th gestational week, the patient asked to terminate the pregnancy. The interphase FISH and karyotype analysis of the normal villi and the mole showed both of them were diploid, thus the CMCF was diagnosed. In the second case, the hydatidiform mole and a coexisting fetus was found by B mode ultrasound at the 21st gestational week. Transabdominal chorionic villi sampling and amniocentesis was performed, interphase FISH and karyotype analysis of the mole and the amniotic fluid showed both of them were diploid, thus the CMCF was diagnosed prenatally. The pregnancy was continued and premature rupture of membrane happened at the 28th gestational week, the cesarean section was performed. The neonate was healthy. The karyotype analysis of the placenta and the neonate was accordant with the prenatal diagnosis. CONCLUSIONS: As long as the hydatidiform mole and a coexisting fetus was found the prenatal diagnosis must be performed in order to differentiate the CMCF and the partial hydatidiform mole (PHM). The transabdominal chorionic villi sampling and the amniocentesis were ideal methods, interphase FISH and karyotype analysis of the mole and the amniotic fluid should be performed. If both of them were diploid, the CMCF could be diagnosed. The clinical management of CMCF should be done individually. If both of them were triploid, the PHM could be diagnosed.