Simplified PGD of common determinants of haemoglobin Bart's hydrops fetalis syndrome using multiplex-microsatellite PCR.

The high incidence of double-gene deletions in α-thalassaemia increases the risk of having pregnancies with homozygous α(0)-thalassaemia, the cause of the lethal haemoglobin (Hb) Bart's hydrops fetalis syndrome. Preimplantation genetic diagnosis (PGD) has played an important role in preventing such cases. However, the current gap-PCR based PGD protocol for deletional α-thalassaemia requires specific primer design for each specific deletion. A universal PGD assay applicable to all common deletional determinants of Hb Bart's hydrops fetalis syndrome has been developed. Microsatellite markers 16PTEL05 and 16PTEL06 within the α-globin gene cluster were co-amplified with a third microsatellite marker outside the affected region in a multiplex-PCR reaction and analysed by capillary electrophoresis. Eight informed couples at risk of having Hb Bart's hydrops fetalis were recruited in this study and all patients underwent standard procedures associated with IVF. A total of 47 embryos were analysed. Three pregnancies were achieved from three couples, with the births of two healthy babies and one ongoing pregnancy. This work has successfully adapted an earlier protocol and developed a simple and reliable single-cell assay applicable to PGD of Hb Bart's hydrops fetalis syndrome regardless of type of deletion. Alpha-thalassaemia is one of the most common inheritable disorders worldwide. It is a blood disorder that, in its lethal form caused by deletion of all four copies of the α-globin gene, results in the demise of the affected fetus, a condition referred to as haemoglobin (Hb) Bart's hydrops fetalis syndrome. Preimplantation genetic diagnosis (PGD) has played an important role in preventing such cases. Current PGD protocols for deletional α-thalassaemia utilize a strategy called gap-PCR, which requires the different assays for different deletion types. We have developed a universal PGD assay applicable to all common deletional determinants of Hb Bart's hydrops fetalis syndrome based on microsatellite marker analysis. Eight informed couples at risk of having Hb Bart's hydrops fetalis were recruited in this study and all patients underwent standard procedures associated with IVF. Forty-five embryos were analysed in total. Three pregnancies were achieved from three couples, with the births of two healthy babies and one pregnancy still ongoing. We have successfully adapted our earlier protocol and developed a simple and reliable single cell assay applicable to PGD of Hb Bart's hydrops fetalis syndrome regardless of the type of deletion.

Phylogenetic and evolutionary relationships and developmental expression patterns of the zebrafish twist gene family.

Four members of the twist gene family (twist1a, 1b, 2, and 3) are found in the zebrafish, and they are thought to have arisen through three rounds of gene duplication, two of which occurred prior to the tetrapod-fish split. Phylogenetic analysis groups most of the vertebrate Twist1 peptides into clade I, except for the Twist1b proteins of the acanthopterygian fish (medaka, pufferfish, stickleback), which clustered within clade III. Paralogies and orthologies among the zebrafish, medaka, and human twist genes were determined using comparative synteny analysis of the chromosomal regions flanking these genes. Comparative nucleotide substitution analyses also revealed a faster rate of nucleotide mutation/substitution in the acanthopterygian twist1b compared to the zebrafish twist1b, thus accounting for their anomalous phylogenetic clustering. We also observed minimal expression overlap among the four twist genes, suggesting that despite their significant peptide similarity, their regulatory controls have diverged considerably, with minimal functional redundancy between them.

Preimplantation genetic diagnosis of chromosome translocations by analysis of polymorphic short tandem repeats.

INTRODUCTION: We aimed to develop and implement a short tandem repeat (STR) polymerase chain reaction alternative to fluorescence in situ hybridisation (FISH) for the preimplantation genetic diagnosis (PGD) of chromosomal translocations. METHODS: Selected informative STRs located on translocated arms of relevant chromosomes were used to discriminate between normal and unbalanced chromosome states in each embryo. RESULTS: PGD cycles were performed on five couples where one spouse carried a balanced translocation. 27 embryos were analysed, of which 12 were normal/balanced, 12 were abnormal/unbalanced and three were indeterminate. Four PGD cycles proceeded to embryo transfer, of which two led to pregnancy. The first pregnancy showed a normal male karyotype, and a healthy baby was delivered at term. A second pregnancy unexpectedly miscarried in the second trimester from unknown causes. CONCLUSION: STR analysis is a simple and suitable alternative to FISH for detecting unbalanced chromosomal states in preimplantation embryos.

Germline transgenesis of zebrafish using the medaka Tol1 transposon system.

Tol1 is a DNA-based transposable element first identified from an albino mutant medaka fish. It has been demonstrated to function as an efficient gene transfer vector in mammalian cells. We now demonstrate Tol1 germline transgenesis in zebrafish. A construct containing the green fluorescence protein (GFP) reporter gene inserted between the Tol1 arms was microinjected together with Tol1 transposase mRNA into fertilized eggs. Sustained GFP expression was observed in 88% of 1-month-old fish, suggesting efficient transposon integration into somatic cells. Eleven of 24 adult GFP-positive fish yielded GFP-positive progeny. Sequencing analysis of Tol1 insertion sites in GFP-positive progeny confirmed Tol1 transposition-mediated integrations into zebrafish chromosomes. We also observed functional independence of the Tol1 transposase-substrate system from that of Tol2, another medaka-derived transposon. Coupled with its previously demonstrated maximal cargo capacity of >20 kb, Tol1 could serve as a useful addition to the zebrafish genetic engineering toolbox.

Zebrafish twist1 is expressed in craniofacial, vertebral, and renal precursors.

TWIST1 encodes a transcription factor that contains a highly conserved basic helix-loop-helix DNA-binding domain and a WR motif. We have isolated a full-length complementary DNA of the zebrafish ortholog of TWIST1 and determined its genomic organization. Inter-species comparisons reveal a remarkable degree of conservation at the gene structure, nucleotide, and predicted peptide levels across large evolutionary distances. Using reverse-transcription polymerase chain reaction analysis and in situ hybridization analyses of whole mount and cryosectioned zebrafish embryos, we detected maternal twist1 transcript in the zygote. During somitogenesis, twist1 transcripts were detected in the intermediate mesoderm from the 2-somite to 18-somite stages, followed by expression in the somites from the 5-somite stage to the 24-somite stage. Also, beginning at the two-somite stage, twist1 expression was observed in head mesenchyme and, subsequently, in neural crest-derived pharyngeal arches as the embryo developed. At the 24-hpf stage, twist1 transcripts were also observed in the ventral tail-bud region. These observations are consistent with a role for twist1 in craniofacial, vertebral, and early renal development.

Simplified molecular diagnosis of fragile X syndrome by fluorescent methylation-specific PCR and GeneScan analysis.

BACKGROUND: Fragile X syndrome (FXS), the most common cause of inherited mental impairment, is most commonly related to hyperexpansion and hypermethylation of a polymorphic CGG trinucleotide repeat in the 5' untranslated region of the FMR1 gene. Southern blot analysis is the most commonly used method for molecular diagnosis of FXS. We describe a simplified strategy based on fluorescent methylation-specific PCR (ms-PCR) and GeneScan analysis for molecular diagnosis of fragile X syndrome. METHODS: We used sodium bisulfite treatment to selectively modify genomic DNA from fragile X and normal lymphoblastoid cell lines and from patients. We then performed ms-PCR amplification using fluorescently-labeled primers complementary to modified methylated or unmethylated DNA. Amplification products were resolved by capillary electrophoresis. FMR1 mutational status was determined by a combination of fluorescent peak sizes and patterns on the GeneScan electropherogram. RESULTS: DNA samples from male and female persons with known NL, PM, and FM FMR1 CGG repeats were analyzed. Each FMR1 genotype produced a unique GeneScan electropherogram pattern, thus providing a way to identify the various disease states. The number of CGG repeats in all NL and PM alleles were determined accurately. Analysis by both the new assay and Southern blot of a family segregating with FXS showed complete concordance between both methods. CONCLUSIONS: This simplified molecular diagnostic test, based on fluorescent methylation-specific PCR, may be a suitable alternative or complement to Southern blot analysis for the diagnosis of FXS.

Multiplex minisequencing screen for common Southeast Asian and Indian beta-thalassemia mutations.

BACKGROUND: Beta-thalassemia is endemic to many regions in Southeast Asia and India, and <20 beta-globin gene mutations account for > or =90% of beta-thalassemia alleles in these places. We describe a multiplex minisequencing assay to detect these common mutations. METHODS: Gap-PCR was used to simultaneously amplify the beta-globin gene from genomic DNA and to detect the Delta619bp deletion mutation. Multiplex minisequencing was then performed on the amplified beta-globin fragment to detect an additional 15 common Southeast Asian and Indian beta-thalassemia mutations. Site-specific primers of different lengths were subjected to multiple rounds of annealing and single-nucleotide extension in the presence of thermostable DNA polymerase and the four dideoxynucleotides, each labeled with a different fluorophore. Minisequencing products were separated and detected by capillary electrophoresis, followed by automated genotyping. The optimized assay was subjected to a double-blind validation analysis of 89 beta-thalassemia and wild-type DNA samples of known genotype. RESULTS: Homozygous wild-type or mutant DNA samples produced electropherograms containing only a single colored peak for each mutation site, whereas samples heterozygous for a specific mutation displayed two different-colored peaks for that mutation site. Samples were automatically genotyped based on color and position of primer peaks in the electropherogram. In the double-blind validation analysis, all 89 DNA samples were genotyped correctly (100% assay specificity). CONCLUSIONS: The described semiautomated multiplex minisequencing assay can detect the most common Southeast Asian and Indian beta-thalassemia mutations, is amenable to high-throughput scale up, and may bring population-based screening of beta-thalassemia in endemic regions a step closer to implementation.