-Thalassemia is very common throughout all tropical and subtropical regions of the world. In Southeast Asia and the Mediterranean regions, compound heterozygotes and homozygotes may have anemia that is mild to severe (hemoglobin [Hb] H disease) or lethal (Hb Bart's hydrops fetalis). We have developed a reliable, single-tube multiplex–polymerase chain reaction (PCR) assay for the 6 most frequently observed determinants of -thalassemia. The assay allows simple, high throughput genetic screening for these common hematological disorders. (Blood. 2000;95:360-362)

Pre-eclampsia (PE) is a leading cause of maternal and fetal mortality and morbidity. Structural or functional alterations of human leukocyte antigen (HLA)-G present at the maternal–fetal interface may predispose women to PE. We tested the HLA-G gene for association with PE in a case–control study of 83 PE and 240 normotensive Malay women. HLA-G was amplified in a single-tube multiplex-PCR reaction and genotyped for 18 single nucleotide polymorphisms (SNPs) by multiplex-minisequencing. Case–control comparisons were performed, and associations with disease were expressed as odds ratios (ORs). Risk for PE was significantly associated with fetal allele G*0106 only in multigravid pregnancies (P = 0.002, OR = 5.0, 95% CI = 1.8–13.8). Among multigravid pregnancies, the frequency of PE babies heterozygous or homozygous for G*0106 was also significantly higher compared with normal control babies (P = 0.002, OR = 5.4, 95% CI = 1.9–15.4). Multivariate analyses with adjustment for factors associated with PE revealed similar results (P = 0.003, OR = 10.1, 95% CI = 2.2–46.8). Additionally, a significantly higher frequency of fetal–maternal G*0106 genotype mismatch was observed in PE compared with normal multigravid pregnancies (P = 0.001, OR = 9.6, 95% CI = 2.4–38.7). Thus, paternal HLA-G G*0106 contribution significantly increases risk for PE in multigravidas who do not carry this allele, potentially mediated by a gradual maternal alloimmune response to repeated exposure to the paternal HLA-G variant.

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.

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. Developmental Dynamics 237:2466–2474, 2008.

BACKGROUND: CGG repeat expansions in the FMR1 (fragile X mental retardation 1) gene are associated with fragile X syndrome, fragile X–associated tremor/ataxia syndrome, and fragile X–associated primary ovarian insufficiency. We evaluated the use of melting curve analysis (MCA) of triplet-primed PCR (TP-PCR) assays as a rapid screening tool for the positive identification of expanded FMR1 alleles in men and women. METHODS: Both 5′- and 3′-weighted direct TP-PCRs (dTP-PCRs) were evaluated on 29 cell line–derived DNA samples and 44 blinded clinical samples. The presence of expansions was identified by the melting curve profiles generated automatically through MCA on the LightCycler 480 Real-Time PCR System. All samples were also analyzed by capillary electrophoresis to confirm the identities of the PCR fragments that gave rise to the observed melt peak profiles. RESULTS: The presence of expanded alleles in samples from both males and females produced melt peak profiles that were distinct from those of individuals with the normal allelic form. In the blinded test, positive and negative calls for the presence of an expanded allele corroborated with previously determined genotype classifications for all samples. CONCLUSIONS: The approach of dTP-PCR plus MCA offers a single-step strategy with high diagnostic sensitivity and specificity for rapid screening detection of FMR1 CGG repeat expansions, regardless of sex. The combined use of 5′- and 3′-weighted dTP-PCR assays minimizes the incidence of false-negative results arising from repeat-flanking deletions.