FWD - prenatal diagnosis from maternal blood
From: acf (evsono@pipeline.com)
Fri Sep 29 08:35:19 2000
I guess you'd actually want to see for yourself - sorry about that but it is Friday :)
Prenatal DNA diagnosis of a single-gene disorder from maternal plasma
Lancet 2000; 356: 1170 Download PDF (144 Kb)
Hiroshi Saito, Akihiko Sekizawa, Taro Morimoto, Makoto Suzuki, Takumi Yanaihara
Achondroplasia is a short-limb disorder caused by a point mutation in a single gene. To diagnose such a disorder prenatally requires the use of invasive procedures such as amniocentesis. However, using PCR and restriction fragment length polymorphism analysis, we were able to detect the mutation in the plasma of a woman carrying a fetus suspected of having achondroplasia. The detection of a fetus-derived mutant gene from maternal plasma may therefore permit non-invasive prenatal diagnosis of single-gene disorders.
Prenatal DNA diagnosis is usually done via invasive procedures such as amniocentesis and chorionic villus sampling. Fetal nucleated erythrocytes in maternal blood have been proposed as potential target cells for non-invasive prenatal diagnosis.1 However, the presence of fetal nucleated erythrocytes in maternal blood is extremely rare, and an efficient method for recovering fetal cells from maternal blood has not yet been established. Since 1997, detection of fetal cell-free DNA in maternal plasma and serum has been done by assessment of Y-chromosomal sequences in pregnant women bearing male fetuses,2 or the rhesus-factor gene in rhesus-negative pregnant women.3 Therefore, maternal plasma may have the potential to provide for non-invasive prenatal diagnosis of single-gene disorders in which the mother does not have genomic alterations in the target sequence.
Although some short-limb disorders are suggested by routine ultrasonography during pregnancy, a definitive diagnosis as to whether or not the disorder is fatal is impossible. Achondroplasia is the most common genetic form of dwarfism and is inherited as an autosomal dominant disorder, although most of the cases are sporadic.4 DNA analysis has revealed that more than 90% of achondroplasia patients have the same mutation--a G-to-A transition at nucleotide 1138 in the gene encoding the fibroblast growth factor receptor 3 (FGFR3). The next most common mutation is a G-to-C transition at the same nucleotide. Both mutations result in the substitution of an arginine for glycine at position 380 in the transmembrane domain of the mature protein.
We cared for a 24-year-old woman whose fetus was suspected to have a short-limb disorder by ultrasonography. At 21 weeks of gestation, fetal growth was within normal limits; however, at 30 weeks of gestation, ultrasonography revealed marked shortening of femur length compared with biparietal diameter and fetal trunk area. The discrepancy in the growth between biparietal diameter and femur length increased as pregnancy progressed. No other abnormality was detected by ultrasonography or magnetic resonance imaging, thus achondroplasia was strongly suggested. We attempted a prenatal DNA diagnosis of achondroplasia from maternal plasma.
Informed consent was obtained, and maternal blood and amniotic fluid were collected at 30 weeks of gestation. DNA was extracted by conventional methods from 2 mL maternal plasma and 3 mL amniotic fluid. Maternal DNA was also obtained from maternal leucocytes to exclude the possibility of maternal inheritance. The segment of FGFR3 containing the nucleotide at which the achondroplasia mutation occurs (nt 1138) was amplified by PCR with specific primers.4 Restriction fragment length polymorphism analysis of PCR products was done with SfcI. The achondroplasia mutation creates an SfcI restriction site such that, upon digestion, two extra bands (of 111 bp and 55 bp) are seen.
PCR amplification and restriction fragment length polymorphism analysis of genomic DNA from maternal plasma and amniotic fluid
Lane 1¾fore SfcI digestion; lane 2¯ter SfcI digestion.
The analysis revealed that DNA from maternal plasma and amnion cells contained the mutant allele (figure). No mutation was found in maternal leucocytes, suggesting that the mutant allele in maternal plasma DNA originated from the fetus. Direct sequencing of amnion-cell DNA confirmed the presence of a G-to-A transition at nt 1138. At 40 weeks of gestation, a girl was born by normal vaginal delivery (birthweight 2834 g, Apgar score 9/10). A radiograph of the infant showed a slight shortness of limbs.
We have successfully carried out a non-invasive prenatal DNA diagnosis of a single-gene disorder, achondroplasia, from maternal plasma. The detection of a fetus-derived mutant gene from maternal plasma could also be used in the prenatal diagnosis of single-gene disorders caused by paternally inherited genes or mutations that are distinguishable from maternally inherited ones.
1 Watanabe A, Sekizawa A, Taguchi A, et al. Prenatal diagnosis of ornitine transcarbamylase deficiency by using a single nucleated erythrocyte from maternal blood. Hum Genet 1998; 102: 611-15 [PubMed].
2 Lo YMD, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CWG. Presence of fetal DNA in maternal plasma and serum. Lancet 1997; 350: 485-87 [PubMed].
3 Lo YMD, Hjelm NM, Fidler C, et al. Prenatal diagnosis of fetal RhD status by molecular analysis of maternal plasma. N Engl J Med 1998; 339: 1734-38 [PubMed].
4 Shiang R, Thompson LM, Zhu YZ, et al. Mutations in the transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia. Cell 1994; 78: 335-42 [PubMed]
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-------------------------------------------------------------------------Department of Obstetrics and Gynecology, Showa University School of =
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-------------------------------------------------------------------------Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan (H Saito MD, A Sekizawa MD, T Morimoto MD, M Suzuki MD, T Yanaihara MD)
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-------------------------------------------------------------------------Correspondence to: Dr Hiroshi Saito (e-mail:saito@med.showa-u.ac.jp)
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I guess you'd actually want to see for = yourself - sorry about that but it is Friday :)
Prenatal DNA = diagnosis of a single-gene disorder from maternal plasma
Lancet 2000; 356: 1170 <3d.htm>Download PDF (144 Kb)
Hiroshi Saito, Akihiko Sekizawa, Taro Morimoto, Makoto Suzuki, = Takumi Yanaihara
Achondroplasia is a short-limb disorder caused by a point mutation = in a single gene. To diagnose such a disorder prenatally requires the use of = invasive procedures such as amniocentesis. However, using PCR and restriction = fragment length polymorphism analysis, we were able to detect the mutation in the = plasma of a woman carrying a fetus suspected of having achondroplasia. The = detection of a fetus-derived mutant gene from maternal plasma may therefore permit non-invasive prenatal diagnosis of single-gene disorders.
Prenatal DNA diagnosis is usually done via invasive procedures such = as amniocentesis and chorionic villus sampling. Fetal nucleated = erythrocytes in maternal blood have been proposed as potential target cells for = non-invasive prenatal diagnosis.1 However, the presence of fetal nucleated = erythrocytes in maternal blood is extremely rare, and an efficient = method for recovering fetal cells from maternal blood has not yet been established. = Since 1997, detection of fetal cell-free DNA in maternal plasma and serum has = been done by assessment of Y-chromosomal sequences in pregnant women bearing = male fetuses,2 or the rhesus-factor gene in rhesus-negative = pregnant women.3 Therefore, maternal plasma may have the potential to = provide for non-invasive prenatal diagnosis of single-gene disorders in which = the mother does not have genomic alterations in the target sequence.
Although some short-limb disorders are suggested by routine = ultrasonography during pregnancy, a definitive diagnosis as to whether or not the = disorder is fatal is impossible. Achondroplasia is the most common genetic form of = dwarfism and is inherited as an autosomal dominant disorder, although most of the = cases are sporadic.4 DNA analysis has revealed that more than 90% = of achondroplasia patients have the same mutation--a G-to-A transition at nucleotide 1138 in the gene encoding the fibroblast growth factor = receptor 3 (FGFR3). The next most common mutation is a G-to-C transition at the = same nucleotide. Both mutations result in the substitution of an arginine for = glycine at position 380 in the transmembrane domain of the mature protein.
We cared for a 24-year-old woman whose fetus was suspected to have a short-limb disorder by ultrasonography. At 21 weeks of gestation, fetal = growth was within normal limits; however, at 30 weeks of gestation, = ultrasonography revealed marked shortening of femur length compared with biparietal = diameter and fetal trunk area. The discrepancy in the growth between biparietal = diameter and femur length increased as pregnancy progressed. No other abnormality was = detected by ultrasonography or magnetic resonance imaging, thus = achondroplasia was strongly suggested. We attempted a prenatal DNA diagnosis of = achondroplasia from maternal plasma.
Informed consent was obtained, and maternal blood and amniotic fluid = were collected at 30 weeks of gestation. DNA was extracted by conventional = methods from 2 mL maternal plasma and 3 mL amniotic fluid. Maternal DNA was also = obtained from maternal leucocytes to exclude the possibility of maternal = inheritance. The segment of FGFR3 containing the nucleotide at = which the achondroplasia mutation occurs (nt 1138) was amplified by PCR with = specific primers.4 Restriction fragment length polymorphism analysis = of PCR products was done with SfcI. The achondroplasia mutation creates = an SfcI restriction site such that, upon digestion, two extra bands = (of 111 bp and 55 bp) are seen.
PCR amplification and restriction fragment length = polymorphism analysis of genomic DNA from maternal plasma and = amniotic fluid
Lane 1¾fore SfcI digestion; lane 2¯ter = SfcI digestion.
The analysis revealed that DNA from maternal plasma and amnion cells contained the mutant allele (figure). No mutation was found in maternal leucocytes, suggesting that the mutant allele in maternal plasma DNA = originated from the fetus. Direct sequencing of amnion-cell DNA confirmed the = presence of a G-to-A transition at nt 1138. At 40 weeks of gestation, a girl was born = by normal vaginal delivery (birthweight 2834 g, Apgar score 9/10). A = radiograph of the infant showed a slight shortness of limbs.
We have successfully carried out a non-invasive prenatal DNA = diagnosis of a single-gene disorder, achondroplasia, from maternal plasma. The = detection of a fetus-derived mutant gene from maternal plasma could also be used in the = prenatal diagnosis of single-gene disorders caused by paternally = inherited genes or mutations that are distinguishable from maternally inherited ones.
1 Watanabe A, Sekizawa A, Taguchi A, et al. Prenatal diagnosis of = ornitine transcarbamylase deficiency by using a single nucleated erythrocyte from = maternal blood. Hum Genet 1998; 102: 611-15 [
2 Lo YMD, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CWG. = Presence of fetal DNA in maternal plasma and serum. Lancet 1997; = 350: 485-87 [
3 Lo YMD, Hjelm NM, Fidler C, et al. Prenatal diagnosis of fetal RhD = status by molecular analysis of maternal plasma. N Engl J Med 1998; = 339: 1734-38 [
4 Shiang R, Thompson LM, Zhu YZ, et al. Mutations in the = transmembrane domain of FGFR3 cause the most common genetic form of dwarfism, achondroplasia. = Cell 1994; 78: 335-42 [
----------
Department of Obstetrics and Gynecology, Showa University School = of Medicine, 1-5-8 Hatanodai, Shinagawa-ku, Tokyo 142-8666, Japan (H = Saito MD, A Sekizawa MD, T Morimoto MD, M Suzuki MD, T Yanaihara MD)
----------
Correspondence to: Dr Hiroshi Saito (e-mail:<3d.htm>saito@med.showa-u.ac.jp)<= BR>
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