Metaphyseal chondrodysplasia McKusick type in a Chinese fetus,caused by novel compound heterozygosity 64T> A and 79G >T in RMRPgene

We present the first confirmed case by molecular analysis of a metaphyseal chondrodysplasia, McKusick type, in a 22-week fetus. Two novel compound heterozygous mutations, 64T> A and 79G > T, were found in the highly conserved regions of the RMRP gene. Twenty-two heterozygous g.1018 T> C mutations, two homozygous g.1018 T> C mutations, two heterozygous insertion mutations g.799_g.800insC and one heterozygous insertion mutation g.849_g.850insT were found among 100 normal controls. Careful radiological examination of the fetus for skeletal dysplasia allowed definitive diagnosis, proper genetic counselling and future prenatal diagnosis. Copyright © 2006 John Wiley & Sons, Ltd.     

Carbamoyl phosphate synthetase I deficiency: molecular genetic findings and prenatal diagnosis

We report a Japanese boy who died at Day 28 of life because of severe carbamoyl phosphate synthetase I (CPS1) deficiency that was proven by enzyme assay. By analysis of cDNA and genomic DNA, he was shown to be a compound heterozygote with two point mutations of the CPS1 gene, 840G>C leading to an aberrant splicing and 1123C>T (predicting Q375X). The 840G>C was a mutation described in another Japanese family. Since his parents carried each mutation heterozygously, we performed prenatal diagnosis at 16 weeks of his mother's next gestation by multiplex PCR and melting curve analysis in a single capillary containing two-color fluorescent (LC-Red 640 and LC-Red 705) probes on LightCycler. We analyzed genomic DNA extracted from amniotic cells and found that the fetus was homozygous for the wild-type alleles. At term a healthy girl was born without hyperammonemia. Copyright © 2001 John Wiley & Sons, Ltd.     

New contributions to the study of common double mutants in the human <Emphasis Type="Italic">LDL</Emphasis> receptor gene

Variations in the gene encoding the low-density lipoprotein receptor (LDLR) can cause familial hypercholesterolemia (FH), one of the most common inherited metabolic disorders in humans. The functional effects of the p.Gln92Glu and p.Asn564His alterations are predicted as benign, but the c.313 + 1G>C and p.Lys799_Phe801del changes are believed to cause disease. Although p.Gln92Glu and c.313 + 1G>C have been observed only in Spain, p.Asn564His and p.Lys799_Phe801del are widespread in Western Europe. In order to estimate the ages (t generations) of these four variants of the gene, to determine their possible origin and to consider the influence of age and selective pressure on their spread, we analyzed 86 healthy individuals and 126 FH patients in Spain. Most of the FH patients investigated carried two of these four LDLR variants simultaneously, while only one patient carried three of them simultaneously. Haplotype analyses were based on five LDLR SNPs: c.81T>C, c.1413G>A, c.1725C>T, c.1959T>C and c.2232G>A. The results suggest that p.Gln92Glu and c.313 + 1G>C arose at about the same time (99 and 103 generations ago, respectively) in the CACTG haplotype and that p.Asn564His and p.Lys799_Phe801del appeared in the CGCCG haplotype and might be slightly more recent variations (92 and 95 generations ago, respectively). Low selective pressures could explain the maintenance of these variants in spite of their ages. The origin of p.Gln92Glu and c.313 + 1G>C appears to be in Spain whereas p.Asn564His and p.Lys799_Phe801del could have been introduced in Spain by Celtic migrations in the seventh to fifth centuries BC.     

Prenatal exclusion of Leigh syndrome due to T8993C mutation in the mitochondrial DNA

Leigh syndrome (LS) is a mitochondrial encephalopathy that is caused by a mutation either in the mitochondrial DNA (mtDNA) or in the nuclear encoded genes of the mitochondrial proteins. Prenatal diagnosis of defects in the mtDNA is usually problematic because of mtDNA heteroplasmy and tissue specificity. However, the mutations T8993 G/C in the ATP synthase subunit 6 gene of the mtDNA show a more even tissue distribution and do not appear to change significantly over time. There are only few reports of prenatal diagnosis of the T8993G mutation in Leigh disease. Here we describe the first prenatal genetic testing of T8993C in a fetus of a mother whose previous child had died of Leigh syndrome due to the T8993C mutation. Mutant load in the chorionic villus sample (CVS) as well as in amniocytes was undetectable, thus predicting a very high likelihood of an unaffected outcome, indicative of a healthy baby. The diagnosis was confirmed after birth. Gathering data on the prenatal diagnosis of mtDNA mutations is of great importance so that prenatal diagnosis of both T8993G and T8993C mutations can be offered routinely. Copyright © 2002 John Wiley & Sons, Ltd.     

The Expected Impact of the Peace Conduit Project (The Red Sea – Dead Sea Pipeline) on the Dead Sea

The Dead Sea of Israel, Jordan and Syria is a severely disturbed ecosystem, greatly damaged by anthropogenic intervention in its water balance. During the 20 th century, the Dead Sea level dropped by more than 25 meters, and presently (2003) it is at about 416 meters below mean sea level. This negative water balance is mainly due to the diversion of water from the catchment area of the lake by Israel, Jordan and Syria. During the 2002 World Summit on Sustainable Development Israel and Jordan jointly announced their interest in saving the Dead Sea by constructing the ‘Peace Conduit’ that will pipe water from the Red Sea to the Dead Sea. The inflow of seawater (or reject brine after desalinization) into the Dead Sea will have a major impact on its limnology, geochemistry and biology. During the filling stage, relatively diluted surface water will form and the rate of evaporation will therefore increase. Dilution of the surface water will most likely result in microbial blooming whose duration is not known, while the lower water layer is likely to develop reducing conditions, including bacterial sulfate reduction and presence of hydrogen sulfide (H₂S). Mixing between the calcium-rich Dead Sea brine and the sulfate-rich seawater will result in gypsum precipitation (CaSO₄·2H₂O). Once the target level is reached, inflow will be outbalanced by evaporation and salinity of the surface water will increase due to accumulation of sea water-salts. The water column will remix when the density of the surface water will equal that of the lower water column. In spite of its large volume and high salinity relative to that of the inflowing water, over the long run the composition of this unique lake will change. Before a decision is made on the planning and construction of the Conduit, it is essential that the long term evolution and characteristics of the ‘renewed’ Dead Sea be known and anticipated changes examined. Once decided upon, the planning and construction of the Conduit should be conducted so as to minimize possible negative impacts of seawater introduction on the Dead Sea. This can only be achieved through a thorough understanding of the expected changes in the limnological physical/chemical characteristics of the Dead Sea and its unique brine.     

Prenatal diagnosis of free sialic acid storage disorders (SASD)

Free sialic acid storage disorders, Salla disease (SD) and Infantile sialic acid storage disease (ISSD), are lysosomal storage diseases due to impaired function of a sialic acid transporter, sialin, at the lysosomal membrane. Several mutations of the sialin gene, SLC17A5, are known, leading either to the severe neonatal/infantile disease or to the milder, adult-type developmental disorder, Salla disease. Free sialic acid accumulation in lysosomes causes increased tissue concentration and consequently elevated urinary excretion. Prenatal diagnosis of SASD is possible either by determination of free sialic acid concentration or by mutation analysis of the SLC17A5 gene in fetal specimen, in chorionic villus biopsy particularly. Both techniques have been successfully applied in several cases, sialic acid assay more often in ISSD cases but mutation analysis preferentially in SD. Sialic acid assay of amniotic fluid supernatant or cultured amniotic fluid cells may give erroneous results and should not be used for prenatal diagnosis of these disorders. The present comments are mainly based on our experience of prenatal diagnosis of SD in Finnish families. A founder mutation in SLC17A5 gene, 115C-> T, represents 95% of the disease alleles in the Finnish SD patients, which provides a unique possibility to apply mutation analysis. Therefore, molecular studies have successfully been used in 17 families since the identification of the gene and the characterization of the SD mutations. Earlier, eight prenatal studies were performed by measuring the free sialic acid concentration in chorionic villus samples. Copyright © 2006 John Wiley & Sons, Ltd.     

Non-invasive prenatal detection of achondroplasia in size-fractionated cell-free DNA by MALDI-TOF MS assay

Achondroplasia is the most common form of short-limbed dwarfism in humans and is caused by mutations in the FGFR3 gene. Currently, prenatal diagnosis of this disorder relies on invasive procedures. Recent studies have shown that fetal single gene point mutations could be detected in cell-free DNA (cf-DNA) from maternal plasma by either the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) assay with single allele base extension reaction (SABER) approach or the size fractionation of cf-DNA in maternal plasma. Here, we combined the two approaches to non-invasively examine the fetal G1138A mutation in maternal plasma. cf-DNA was extracted from maternal plasma samples obtained from two pregnant women at risk for achondroplasia. The fetal G1138A mutation was determined by the analysis of size-fractionated cf-DNA in maternal plasma using MALDI-TOF MS with SABER approach and homogenous MassEXTEND (hME) assay, respectively. The fetal G1138A mutation was detectable in the two achondroplasia-affected pregnancies by the analysis of cf-DNA in maternal plasma using MALDI-TOF MS. However, the size-fractionation approach led to a more precise detection of the fetal mutation in both analyses. This analysis would be suitable for non-invasive prenatal diagnosis of diseases caused by fetal single gene point mutations. Copyright © 2007 John Wiley & Sons, Ltd.     

Two unrelated fetuses with ITPR1 missense variants and fetal hydrops

We describe two fetuses from unrelated families with likely pathogenic variants in ITPR1 that presented with nonimmune fetal hydrops. Trio exome sequencing revealed a de novo heterozygous likely pathogenic missense variant c.7636G > A (p.Val2531Met) in ITPR1 (NM_001378452.1) in proband 1 and a de novo heterozygous likely pathogenic missense variant c.34G > A [p.Gly12Arg] in proband 2. Variants in ITPR1 have been associated with several genetic conditions, including spinocerebellar ataxia 15, spinocerebellar ataxia 29, and Gillespie syndrome. Our report on two patients details a previously undescribed severe fetal presentation of nonimmune hydrops fetalis associated with missense variants in the ITPR1 gene.     

Prenatal diagnosis of β-thalassemia in Southern Punjab,Pakistan

Pakistan has a large population of more than 150 million people with an overall carrier frequency of approximately 5.6% for β-thalassemia. Punjab is the largest province of the country having more than 50% of the population. The state of β-thalassemia is alarming as consanguinity is very high (>81%) and the literacy rate is low in South Punjab. A thalassemia prevention program is the need of the hour in this part of Pakistan. In this study, we initiated awareness, screening, and characterization of the mutations causing β-thalassemia as well as a genetic counseling program mainly in the districts of Faisalabad and D.G. Khan to establish prenatal diagnosis, a facility previously unavailable in this region for disease prevention. A total of 248 unrelated transfusion-dependent children and the available members of their families were screened to characterize the mutations and identify the carriers. Genetic counseling was provided to these families and prenatal diagnosis offered. In the samples analyzed, 11 β-thalassemia mutations and three hemoglobin variants were detected mainly by using the Monoplex and Multiplex ARMS-PCR. First-trimester prenatal diagnosis was carried out through chorionic villus sampling (CVS) in seven pregnancies at risk. As a result of our campaign, 145 carrier couples planning to have more children gave their consent to have retrospective prenatal diagnosis in every pregnancy in future. A cooperative trend and a positive attitude toward the prevention of β-thalassemia were noticed in the families with affected children and in the general population. Copyright © 2006 John Wiley & Sons, Ltd.     

Rapid molecular prenatal diagnosis of ataxia-telangiectasia by direct mutational analysis

Mutations of the ataxia-telangiectasia-mutated (ATM) gene are responsible for the autosomal recessive disorder ataxia-telangiectasia (A-T). This study reports the first A-T prenatal diagnosis performed in Spain by direct molecular analysis. The pregnant woman had a previous child suffering from A-T due to a deletion in the ATM gene. The ATM coding region was sequenced in the A-T patient and her parents. Then, a specific polymerase chain reaction (PCR) to detect the deletion was performed for prenatal diagnosis. Additionally, polymorphic HLA loci were examined in order to exclude the possible contamination by maternal DNA. In this family of Gypsy origin, we carried out a rapid molecular diagnosis of A-T. Then, a prenatal diagnosis was carried out, identifying the deletion in the fetal DNA. Additionally, we performed a population study in unrelated Spanish Gypsies and in unrelated controls, showing that the deletion described could be a hotspot in the Spanish Gypsy population. The size of the coding region and the genomic structure, together with the absence of hotspots, make the mutation screening of the ATM gene difficult. The ability to identify ATM mutations provides a tool that can be applied in confirmatory diagnosis, genetic counselling, carrier prediction and prenatal diagnosis. Copyright © 2007 John Wiley & Sons, Ltd.     

Birth of healthy children after preimplantation diagnosis of β-thalassemia by whole-genome amplification

Preimplantation genetic diagnosis (PGD) offers couples at risk for transmitting an inherited disorder the possibility to avoid the need to terminate affected pregnancies. PGD for monogenic diseases is most commonly accomplished by blastomere biopsy from cleavage-stage embryos, followed by PCR-based DNA analysis. However, the molecular heterogeneity of many monogenic diseases requires a diagnostic strategy capable of detecting a range of mutations and compound genotypes. With the above considerations, we developed an accurate and reliable strategy for analysis of β-globin gene mutations, applicable for PGD for the wide spectrum of β-thalassemia major mutations in the Chinese population. The strategy involves primer-extension preamplification (PEP), followed by nested PCR and reverse dot blot (RDB) for mutation detection since it facilitates simultaneous analysis of more than one mutation in a single cell. This report describes the application of the strategy in two clinical IVF/PGD cycles at risk for transmitting β-thalassemia major, which resulted in the first thalassemia-free children born after PGD in China. Copyright © 2003 John Wiley & Sons, Ltd.     

Mid–trimester hyperechogenic bowel in a fetus of Japanese origin carrying a new mutation of CFTR gene (L548Q)

We present a case of a fetus with hyperechogenic bowel, in which the L548Q mutation was detected in the mother of Japanese origin and the ΔF508 mutation in the father of Caucasian origin. The fetus proved to be compound heterozygous. Research into cystic fibrosis transmembrane conductance regulator (CFTR) mutations in this case was triggered by the fact that the fetus had a characteristic hyperechogenic bowel image with normal karyotype and no indications of intrauterine infections. Hyperechogenic bowel is highly indicative of a CFTR gene mutation. The incidence of cystic fibrosis (CF) in fetuses with mid-trimester hyperechogenic bowel is 5%, but once the most frequent mutations have been accounted for, rarer mutations must be investigated. Copyright © 2006 John Wiley & Sons, Ltd.     

Identification of a novel β0 - thalassaemia mutation in a greek family and subsequent prenatal diagnosis

We present a case in which a Greek couple was considered not to be at risk of having children with homozygous β-thalassaemia, an assessment based largely on the father's belief that he carried α-thalassaemia. After their first child was diagnosed with homozygous β-thalassaemia, the case was re-assessed and both parents were shown to have the haematological profile of β-thalassaemia trait. Screening for the common Mediterranean mutations demonstrated that the mother carries the IVS-1 nt 110 G→A β⁺ -thalassaemia mutation. Direct nucleotide sequencing of PCR-amplified DNA revealed that the father carries a novel β⁰-thalassaemia mutation, frameshift codons 9/10 (+T). The couple's second pregnancy was terminated after prenatal testing revealed that the fetus had inherited both parental mutations. This case illustrates the need to confirm the carrier status of individuals prior to assessing their genetic risks, and highlights the importance of being able to identify rare or novel β-thalassaemia mutations.     

Anatomical and physiological divergences and compensatory effects in two Leymus chinensis (Poaceae) ecotypes in Northeast China

The purpose of this study was to investigate the anatomical and physiological differences between two Leymus chinensis ecotypes coexisted in semi-humid meadow and semi-arid steppe. The study addressed the hypothesis that, at same habitat, the two ecotypes exhibit remarkable divergences in adaptive strategies under drought and salinity, and the function of these strategies is compensatory. Leaf samples were collected from each type at the two sites in field. Sections of 2 cm × 2 cm were cut from the middle of fully expanded leaves and fixed in FAA. Leaf anatomical traits (e.g. stomatal density, leaf thickness and vessel diameters) were examined, and leaf mass per area (LMA), relative water content (RWC), proline, K⁺ and Na⁺ were measured. Compared with the gray green type (GG), the yellow green type (YG) with relative greater LMA and leaf thickness, lower stomatal density and index exhibited more obvious xerophil-liked anatomical traits, while higher RWC, proline, K⁺ and K⁺/Na⁺ for the GG type suggested that the ability of osmotic adjustment and salt tolerance of the GG type were stronger than the YG type. Stronger xerophytic anatomical traits were the supplementary strategies of the YG type for its low ability of osmotic adjustment and salt tolerance to drought and salinity.Concluding, there exist significant differences in anatomical and physiological strategies between the two ecotypes and the compensatory effects of these strategies enable the two ecotypes coexist at similar habitat.     

The expected impact of the “Peace Conduit” project (the Red Sea - Dead Sea pipeline) on the Dead Sea

The Dead Sea is a severely disturbed ecosystem, greatly damaged by anthropogenic intervention in its water balance. During the 20th century, the Dead Sea level dropped by more than 25 meters, and presently (2003) it is at about 416 meters below mean sea level. This negative water balance is mainly due to the diversion of water from the catchment area of the lake by Israel, Jordan and Syria. During the 2002 World Summit on Sustainable Development Israel and Jordan jointly announced their interest in saving the Dead Sea by constructing the “Peace Conduit” that will pipe water from the Red Sea to the Dead Sea. The inflow of seawater (or reject brine after desalinization) into the Dead Sea will have a major impact on its limnology, geochemistry and biology. During the filling stage, relatively diluted surface water will form and the rate of evaporation will therefore increase. Dilution of the surface water will most likely result in microbial blooming whose duration is not known, while the lower water layer is likely to develop reducing conditions, including bacterial sulfate reduction and presence of hydrogen sulfide (H₂S). Mixing between the calcium-rich Dead Sea brine and the sulfate-rich seawater will result in gypsum precipitation (CaSO₄⋅ 2H₂O). Once the target level is reached, inflow will be outbalanced by evaporation and salinity of the surface water will increase due to accumulation of seawater-salts. The water column will re-mix when the density of the surface water will equal that of the lower water column. In spite of its large volume and high salinity relative to that of the inflowing water, over the long run the composition of this unique lake will change. Before a decision is made on the planning and construction of the Conduit, it is essential that the long term evolution and characteristics of the “renewed” Dead Sea be known and anticipated changes examined. Once decided upon, the planning and construction of the Conduit should be conducted so as to minimize possible negative impacts of seawater introduction on the Dead Sea. This can only be achieved through a thorough understanding of the expected changes in the limnological physical/chemical characteristics of the Dead Sea and its unique brine.     

Denaturing high-performance liquid chromatography (DHPLC)-based prenatal diagnosis for tuberous sclerosis

Tuberous sclerosis (TSC) is a frequent autosomal-dominant condition (affecting 1 in 6000 individuals) caused by various mutations in either the hamartin (TSC1) or the tuberin gene (TSC2). This allelic and non-allelic heterogeneity makes genetic counseling and prenatal diagnosis difficult, especially as a significant proportion of TSC cases are due to de novo mutations. For this reason the identification of the disease causing mutation is mandatory for accurate counseling, yet current mutation detection methods such as single-strand conformation polymorphism (SSCP) or denaturing gradient gel electrophoresis (DGGE) are labor intensive with limited detection efficiency. Denaturing high-performance liquid chromatography (DHPLC) is a high-throughput, semi-automated mutation detection system with a reported mutation detection rate close to 100% for PCR fragments of up to 800 bp. We used a recently described DHPLC assay allowing the efficient detection of mutations in TSC1 to analyze the DNA extracted from a chorion villus sample in order to perform a prenatal diagnosis for TSC. The fetus was found not to have inherited the deleterious mutation and the DHPLC diagnosis was confirmed by haplotype analysis. This represents the first DHPLC-based prenatal diagnosis of a genetic disease. Copyright © 2001 John Wiley & Sons, Ltd.     

Usefulness of automated chromatography for rapid fetal blood analysis for second trimester prenatal diagnosis of β-thalassemia

Prenatal diagnosis of β-thalassemia is now ideally done in the first trimester of pregnancy by chorionic villus tissue DNA analysis. Nevertheless, fetal blood analysis in the second trimester is required either when the mutation in both parents cannot be characterised or when the couple comes late for investigations. We evaluated the usefulness of analysis of fetal blood on the Biorad Variant Hemoglobin Testing System using the β-thalassemia short programme in comparison with the conventional globin biosynthesis in 58 pregnancies. The β/α biosynthesis ratios in 13 homozygous fetuses ranged from 0 to 0.03 and the adult hemoglobin (HbA) levels by automated chromatography varied from 0% to 0.4%. The normal or heterozygous fetuses had β/α ratios of >0.04 and HbA levels ranging from 2.1% to 10.6%. In 17 fetuses we also correlated the β gene mutations with the predicted genotypes using automated high-performance liquid chromatography (HPLC). Follow-up of 18 unaffected fetuses using the Variant System at birth showed a significant increase in HbA levels. Copyright © 2002 John Wiley & Sons, Ltd.