Simultaneous Screening of the FRAXA and FRAXE Loci for Rapid Detection of FMR1 CGG and/or AFF2 CCG Repeat Expansions by Triplet-Primed PCR.

Moderate to hyper-expansion of trinucleotide repeats at the FRAXA and FRAXE fragile sites, with or without concurrent hypermethylation, has been associated with intellectual disability and other conditions. Unlike molecular diagnosis of FMR1 CGG repeat expansions in FRAXA, current detection of AFF2 CCG repeat expansions in FRAXE relies on low-throughput and otherwise inefficient techniques combining Southern blot analysis and PCR. A novel triplet-primed PCR assay was developed for simultaneous screening for trinucleotide repeat expansions at the FRAXA and FRAXE fragile sites, and was validated using archived clinical samples of known FMR1 and AFF2 genotypes.

Identification of Novel Microsatellite Markers Flanking the and Duplicated Region and Inclusion Into a Single-Tube Tridecaplex Panel for Haplotype-Based Preimplantation Genetic Testing of Spinal Muscular Atrophy.

Preimplantation genetic testing for the monogenic disorder (PGT-M) spinal muscular atrophy (SMA) is significantly improved by supplementation of deletion detection with marker-based linkage analysis. To expand the availability of informative markers for PGT-M of SMA, we identified novel non-duplicated and highly polymorphic microsatellite markers closely flanking the and duplicated region. Six of the novel markers within 0.

Robust Preimplantation Genetic Testing of Huntington Disease by Combined Triplet-Primed PCR Analysis of the HTT CAG Repeat and Multi-Microsatellite Haplotyping.

Huntington disease (HD) is a lethal neurodegenerative disorder caused by expansion of a CAG repeat within the huntingtin (HTT) gene. Disease prevention can be facilitated by preimplantation genetic testing for this monogenic disorder (PGT-M). We developed a strategy for HD PGT-M, involving whole genome amplification (WGA) followed by combined triplet-primed PCR (TP-PCR) for HTT CAG repeat expansion detection and multi-microsatellite marker genotyping for disease haplotype phasing.

FMR1 CGG repeat expansion mutation detection and linked haplotype analysis for reliable and accurate preimplantation genetic diagnosis of fragile X syndrome.

Fragile X mental retardation 1 (FMR1) full-mutation expansion causes fragile X syndrome. Trans-generational fragile X syndrome transmission can be avoided by preimplantation genetic diagnosis (PGD). We describe a robust PGD strategy that can be applied to virtually any couple at risk of transmitting fragile X syndrome.

Improved high sensitivity screen for Huntington disease using a one-step triplet-primed PCR and melting curve assay.

Molecular diagnosis of Huntington disease (HD) is currently performed by fluorescent repeat-flanking or triplet-primed PCR (TP-PCR) with capillary electrophoresis (CE). However, CE requires multiple post-PCR steps and may result in high cost in high-throughput settings. We previously described a cost-effective single-step molecular screening strategy employing the use of melting curve analysis (MCA).

Identification of novel microsatellite markers <1 Mb from the HBB gene and development of a single-tube pentadecaplex PCR panel of highly polymorphic markers for preimplantation genetic diagnosis of beta-thalassemia.

Beta (β)-thalassemia is one of the most common monogenic diseases worldwide. Affected pregnancies can be avoided through preimplantation genetic diagnosis (PGD), which commonly involves customized assays to detect the different combinations of β-globin (HBB) gene mutations present in couples, in conjunction with linkage analysis of flanking microsatellite markers. Currently, the limited number of reported closely linked markers hampers their utility in indirect linkage-based PGD for this disorder.

Single-tube nonaplex microsatellite PCR panel for preimplantation genetic diagnosis of Hb Bart's hydrops fetalis syndrome.

Objective: To develop a single-tube multi-marker assay for improved preimplantation genetic diagnosis (PGD) of deletional and/or non-deletional Hb Bart's hydrops fetalis syndrome, providing haplotype confirmation of deletional status, and maximization of linkage informativity.

Methods: We performed in silico mining to identify novel microsatellites within 1 Mb flanking the alpha-globin gene cluster, and optimized a single-tube assay combining detection of α(0) -thalassemia deletions with multi-marker linkage analysis. We performed validation on 100 single cells prior to clinical PGD application.

Fetal polymorphisms at the ABCB1-transporter gene locus are associated with susceptibility to non-syndromic oral cleft malformations.

ATP-binding cassette (ABC) proteins in the placenta regulate fetal exposure to xenobiotics. We hypothesized that functional polymorphisms in ABC genes influence risk for non-syndromic oral clefts (NSOC). Both family-based and case-control studies were undertaken to evaluate the association of nine potentially functional single-nucleotide polymorphisms within four ABC genes with risk of NSOC.

BMP4 was associated with NSCL/P in an Asian population.

Background: The Bone Morphogenetic Protein 4 gene (BMP4) is located in chromosome 14q22-q23 which has shown evidence of linkage for isolated nonsyndromic cleft lip with or without cleft palate (NSCL/P) in a genome wide linkage analysis of human multiplex families. BMP4 has been shown to play crucial roles in lip and palatal development in animal models. Several candidate gene association analyses also supported its potential risk for NSCL/P, however, results across these association studies have been inconsistent.

ROR2 gene is associated with risk of non-syndromic cleft palate in an Asian population.

Background: The receptor tyrosine kinase-like orphan receptor 2 (ROR2) gene has been recently shown to play important roles in palatal development in animal models and resides in the chromosomal region linked to non syndromic cleft lip with or without cleft palate in humans. The aim of this study was to investigate the possible association between ROR2 gene and non-syndromic oral clefts.

Methods: Here we tested 38 eligible single-nucleotide polymorphisms (SNPs) in ROR2 gene in 297 non-syndromic cleft lip with or without cleft palate and in 82 non-syndromic cleft palate case parent trios recruited from Asia and Maryland.

Genome wide study of maternal and parent-of-origin effects on the etiology of orofacial clefts.

We performed a genome wide association analysis of maternally-mediated genetic effects and parent-of-origin (POO) effects on risk of orofacial clefting (OC) using over 2,000 case-parent triads collected through an international cleft consortium. We used log-linear regression models to test individual SNPs. For SNPs with a P-value <10(-5) for maternal genotypic effects, we also applied a haplotype-based method, TRIMM, to extract potential information from clusters of correlated SNPs.

The FGF and FGFR Gene Family and Risk of Cleft Lip With or Without Cleft Palate.

Background : Isolated, nonsyndromic cleft lip with or without cleft palate is a common human congenital malformation with a complex and heterogeneous etiology. Genes coding for fibroblast growth factors and their receptors (FGF/FGFR genes) are excellent candidate genes. Methods : We tested single-nucleotide polymorphic markers in 10 FGF/FGFR genes (including FGFBP1, FGF2, FGF10, FGF18, FGFR1, FGFR2, FGF19, FGF4, FGF3, and FGF9) for genotypic effects, interactions with one another, and with common maternal environmental exposures in 221 Asian and 76 Maryland case-parent trios ascertained through a child with isolated, nonsyndromic cleft lip with or without cleft palate.

Evidence for gene-environment interaction in a genome wide study of nonsyndromic cleft palate.

Nonsyndromic cleft palate (CP) is a common birth defect with a complex and heterogeneous etiology involving both genetic and environmental risk factors. We conducted a genome-wide association study (GWAS) using 550 case-parent trios, ascertained through a CP case collected in an international consortium. Family-based association tests of single nucleotide polymorphisms (SNP) and three common maternal exposures (maternal smoking, alcohol consumption, and multivitamin supplementation) were used in a combined 2 df test for gene (G) and gene-environment (G × E) interaction simultaneously, plus a separate 1 df test for G × E interaction alone.

Tgfbeta3 regulation of chondrogenesis and osteogenesis in zebrafish is mediated through formation and survival of a subpopulation of the cranial neural crest.

Zebrafish tgfbeta3 is strongly expressed in a subpopulation of the migrating neural crest cells, developing pharyngeal arches and neurocranial cartilages. To study the regulatory role of tgfbeta3 in head skeletal formation, we knocked down tgfbeta3 in zebrafish and found impaired craniofacial chondrogenesis, evident by malformations in selected neurocranial and pharyngeal arch cartilages. Over-expressing tgfbeta3 in embryos resulted in smaller craniofacial cartilages without any gross malformations.

Association between genes on chromosome 4p16 and non-syndromic oral clefts in four populations.

Isolated cleft lip with or without cleft palate and cleft palate are among the most common human birth defects. Several candidate gene studies on MSX1 have shown significant association between markers in MSX1 and risk of oral clefts, and re-sequencing studies have identified multiple mutations in MSX1 in a small minority of cases, which may account for 1-2% of all isolated oral clefts cases. We explored the 2-Mb region around MSX1, using a marker map of 393 single nucleotide polymorphisms (SNPs) in 297 cleft lip, with or without cleft palate, case-parent trios and 84 cleft palate trios from Maryland, Taiwan, Singapore, and Korea.

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.

Evidence that TGFA influences risk to cleft lip with/without cleft palate through unconventional genetic mechanisms.

This study examined the association between markers in transforming growth factor alpha (TGFA) and isolated, non-syndromic cleft lip with/without palate (CL/P) using a case-parent trio design, considering parent-of-origin effects. We also tested for gene-environmental interaction with common maternal exposures, and for gene-gene interaction using markers in TGFA and another recognized causal gene, IRF6. CL/P case-parent trios from four populations (76 from Maryland, 146 from Taiwan, 35 from Singapore, and 40 from Korea) were genotyped for 17 single nucleotide polymorphisms (SNPs) in TGFA.

Maternal transmission effects of the PAX genes among cleft case-parent trios from four populations.

Isolated cleft lip with or without cleft palate (CL/P) is among the most common human birth defects, with a prevalence of 1 in 700 live births. The paired box (PAX) genes have been suggested as candidate genes for CL/P based largely on mouse models; however, few human studies have focused on this gene family. This study tests for association between markers in four PAX genes and CL/P using a case-parent trio design considering parent-of-origin effects.

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.

Excess maternal transmission of markers in TCOF1 among cleft palate case-parent trios from three populations.

Isolated cleft palate is among the most common human birth defects. The TCOF1 gene has been suggested as a candidate gene for cleft palate based on animal models. This study tests for association between markers in TCOF1 and isolated, nonsyndromic cleft palate using a case-parent trio design considering parent-of-origin effects.

Differential parental transmission of markers in RUNX2 among cleft case-parent trios from four populations.

Isolated cleft lip with or without cleft palate (CL/P) is among the most common human birth defects, with a prevalence around 1 in 700 live births. The Runt-related transcription factor 2 (RUNX2) gene has been suggested as a candidate gene for CL/P based largely on mouse models; however, no human studies have focused on RUNX2 as a risk factor for CL/P. This study examines the association between markers in RUNX2 and isolated, nonsyndromic CL/P using a case-parent trio design, while considering parent-of-origin effects.

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.

Genomic, cDNA, and embryonic expression analysis of zebrafish transforming growth factor beta 3 (tgfbeta3).

TGFbeta3, a member of the transforming growth factor beta family, regulates a spectrum of biological processes and is involved in mammalian pulmonary and craniofacial development. Homologs of human TGFbeta3 have been identified in several vertebrate species. We sequenced a cDNA clone of zebrafish tgfbeta3, consisting of a 271-bp 5' untranslated region, a 1,233-bp open reading frame that encodes a predicted 410 amino acid peptide, and a 527-bp 3' untranslated region.