Synthesis, biological evaluation, and correlation of cytotoxicity versus redox potential of 1,4-naphthoquinone derivatives.

A series of 1,4-naphthoquinone derivatives of lawsone (1), 6-hydroxy-1,4-naphthoquinone (2), and juglone (3) were synthesized by alkylation, acylation, and sulfonylation reactions. The yields of lawsone derivatives 1a-1k (type A), 6-hydroxy-1,4-naphthoquinone derivatives 2a-2j (type B), and juglone derivatives 3a-3h (type C) were 52-99%, 53-96%, and 28-95%, respectively. All compounds were tested in vitro for the cytotoxicity against human oral epidermoid carcinoma (KB) and cervix epithelioid carcinoma (HeLa) cells and their structure-activity relationship was studied. Compound 3c was found to be most potent in KB cell line (IC50 = 1.39 µM). Some compounds were evaluated for DNA topoisomerase I inhibition. Compounds 2c, 3, 3a, and 3d showed topoisomerase inhibition activity with IC50 values of 8.3-91 µM. Standard redox potentials (E°) of all naphthoquinones in phosphate buffer at pH 7.2 were examined by means of cyclic voltammetry. A definite correlation has been found between the redox potentials and inhibitory effects of type A compounds.

TP53 alterations in relapsed childhood acute lymphoblastic leukemia.

TP53 alterations are frequent relapse-acquired mutations in childhood acute lymphoblastic leukemia (ALL). The present study evaluated the clinical significance of relapsed childhood ALL in Taiwan. Diagnostic and/or relapsed bone marrow or peripheral blood was obtained from 111 children with relapsed ALL who were initially treated by using Taiwan Pediatric Oncology Group (TPOG) ALL protocols from January 1997 to May 2018. Mutations were detected by PCR and sequencing, as well as by multiplex ligation-dependent probe amplification to detect copy number alterations. Copy number and/or sequence alterations of TP53 were detected in 29% (28 of 98) and in 46% (6 of 13) of patients with relapsed B-cell and T-cell ALL, respectively. This incidence was much higher than that in several similar studies conducted in Caucasian populations. Seventy percent of all TP53 alterations were gained at relapse in 67 matched samples by back-tracking matched diagnostic samples. TP53 alterations were associated with lower 5-year event-free survival (EFS) and overall survival (OS) rates (P = .013 and P = .0002, respectively). Multivariate analysis confirmed the prognostic significance of TP53 alterations. Forty-five patients received hematopoietic stem-cell transplantations post-relapse. Patients with TP53 alterations (14/45) had inferior 5-year EFS and OS than patients without TP53 alterations after transplantation (P = .002 and P = .001, respectively). The significance of these TP53 alterations for patients who received transplantations was confirmed by multivariate analysis. In conclusion, TP53 alterations were enriched and useful as prognostic markers in relapsed childhood ALL.

Concurrent Hearing, Genetic, and Cytomegalovirus Screening in Newborns, Taiwan.

OBJECTIVE: To evaluate the feasibility and potential benefits of incorporating genetic and cytomegalovirus (CMV) screenings into the current newborn hearing screening (NHS) programs. STUDY DESIGN: Newborns were recruited prospectively from a tertiary hospital and a maternity clinic between May 2016 and December 2016 and were subjected to hearing screening, CMV screening, and genetic screening for 4 common mutations in deafness genes (p.V37I and c.235delC of GJB2 gene, c.919-2A>G of SLC26A4 gene, and the mitochondrial m.1555A>G). Infants with homozygous nuclear mutations or homoplasmic/heteroplasmic mitochondrial mutation (referred to as "conclusively positive genotypes") and those who tested positive for CMV received diagnostic audiologic evaluations. RESULTS: Of the total 1716 newborns enrolled, we identified 20 (1.2%) newborns with conclusively positive genotypes on genetic screening, comprising 15 newborns (0.9%) with GJB2 p.V37I/p.V37I and 5 newborns (0.3%) with m.1555A>G. Three (0.2%) newborns tested positive on CMV screening. Twelve of the 20 newborns (60%) with conclusively positive genotypes and all 3 newborns who tested positive for CMV (100%) passed NHS at birth. Diagnostic audiologic evaluations conducted at 3 months confirmed hearing impairment in 6 of the 20 infants (30%) with conclusively positive genotypes. CONCLUSIONS: This study confirms the feasibility of performing hearing, genetic, and CMV screenings concurrently in newborns and provides evidence that the incorporation of these screening tests could potentially identify an additional subgroup of infants with impaired hearing that might not be detected by the NHS programs.

Newborn genetic screening for hearing impairment: a population-based longitudinal study.

PURPOSE: The feasibility of genetic screening for deafness-causing mutations in newborns has been reported in several studies. The aim of this study was to investigate the long-term results in those who screened positive for deafness mutations; these results are crucial to determine the cost-effectiveness to justify population-wide genetic screening. METHODS: We performed simultaneous hearing screening and genetic screening targeting four common deafness mutations (p.V37I and c.235delC of GJB2, c.919-2A>G of SLC26A4, and the mitochondrial m.1555A>G) in 5173 newborns at a tertiary hospital between 2009 and 2015. Serial audiometric results up to 6 years old were then analyzed in children with conclusive genotypes. RESULTS: Newborn genetic screening identified 82 (1.6%) babies with conclusive genotypes, comprising 62 (1.2%) with GJB2 p.V37I/p.V37I, 16 (0.3%) with GJB2 p.V37I/c.235delC, and 4 (0.1%) with m.1555A>G. Of these, 46 (56.1%) passed hearing screening at birth. Long-term follow-up demonstrated progressive hearing loss in children with the GJB2 p.V37I/p.V37I and p.V37I/c.235delC genotypes; this hearing loss deteriorated by approximately 1 decibel hearing level (dBHL) per year. CONCLUSIONS: We delineated the longitudinal auditory features of the highly prevalent GJB2 p.V37I mutation on a general population basis and confirmed the utility of newborn genetic screening in identifying infants with late-onset or progressive hearing impairment undetectable by newborn hearing screening.Genet Med 19 1, 6-12.

Blastocyst biopsy and vitrification are effective for preimplantation genetic diagnosis of monogenic diseases.

STUDY QUESTION: What is the value of a new strategy for preimplantation genetic diagnosis (PGD) of monogenic diseases: blastocyst biopsy, cryopreservation and thawed embryo transfer? SUMMARY ANSWER: This strategy is highly effective for PGD of monogenic diseases and merits wide use. WHAT IS KNOWN ALREADY: PGD of monogenic diseases is conventionally performed on 6- to 8-cell embryos with fresh transfer. The diagnostic time is restricted and is subjected to amplification failure and allele drop-out (ADO). STUDY DESIGN, SIZE, DURATION: This is a prospective observational cohort study. A total of 33 couples were included from November 2008 to January 2012. PARTICIPANTS/MATERIALS, SETTING, METHODS: A cohort of 33 couples who were carriers of monogenic diseases underwent a total of 40 oocyte pick-up (OPU) cycles, with subsequent blastocyst biopsy, vitrification and thawed embryo transfer. DNA analysis was performed by whole genome amplification using multiple displacement amplification followed by real-time PCR and mini-sequencing. MAIN RESULTS AND THE ROLE OF CHANCE: The diagnostic rate was 90% with 5% amplification failure and 5% ADO. The survival rate of vitrified blastocysts was 94%. Amongst 33 couples, 24 ongoing pregnancies were achieved (60% per OPU cycle) with an implantation rate of 50%. All of the genotyping results of prenatal diagnosis were consistent with those of PGD. There was no severe or late ovarian hyperstimulation syndrome (OHSS) and no hospitalization. LIMITATIONS, REASONS FOR CAUTION: The participants are limited to the carriers of monogenic diseases. WIDER IMPLICATIONS OF THE FINDINGS: This strategy achieves high rates of genotyping success, survival after warming and pregnancy. Cryopreservation of blastocysts after biopsy permits sufficient time for transportation of specimens and molecular diagnosis. In particular, cryopreservation of biopsied embryos without fresh transfer is an important strategy to prevent OHSS and circumvent a suboptimal endometrium in high responders. STUDY FUNDING/COMPETING INTEREST(S): This study is financially supported by the National Science Council of Taiwan (grants NSC 96-2628-B-002-063-MY3, NSC 98-2314-B-002-088-MY3 and 98-FTN13). No competing interests are declared.

A feasible strategy of preimplantation genetic diagnosis for carriers with chromosomal translocation: Using blastocyst biopsy and array comparative genomic hybridization.

BACKGROUND/PURPOSE: Patients with chromosomal translocation are highly vulnerable to produce unbalanced gametes that result in recurrent miscarriages, affected offspring, or infertility. Preimplantation genetic diagnosis (PGD) with blastomere biopsy and fluorescent in-situ hybridization (FISH) has been used to select normal/balanced embryos for transfer. However, FISH is inherent with some technical difficulties such as cell fixation and signal reading. Here we introduce a strategy of PGD using blastocyst biopsy and array comparative genomic hybridization (aCGH) for reproductive problems of patients with chromosomal translocation. METHODS: Twelve patients diagnosed as having chromosomal translocation who underwent PGD cycles were included in this single-center observational study. Blastocyst biopsy was performed and biopsied blastocysts were cryopreserved individually. Testing was performed with aCGH, and the euploid embryos were transferred in the following thawing cycles. RESULTS: The overall diagnostic efficiency was 90.2% (55/61) and the euploidy rate was 32.7% (18/55). Ten cycles of thawed embryo transfer (ET) were carried out, resulting in three live births and another three ongoing pregnancies with an ongoing pregnancy rate of 60%/transfer cycle. The prenatal diagnosis with chorionic villi sampling confirmed the results of PGD/aCGH in all six pregnant women. No miscarriage happened in our case series. CONCLUSION: Our study demonstrates an effective PGD strategy with promising outcomes. Blastocyst biopsy can retrieve more genetic material and may provide more reliable results, and aCGH offers not only detection of chromosomal translocation but also more comprehensive analysis of 24 chromosomes than traditional FISH. More cases are needed to verify our results and this strategy might be considered in general clinical practice.

IKZF1 deletions predict a poor prognosis in children with B-cell progenitor acute lymphoblastic leukemia: a multicenter analysis in Taiwan.

Despite current risk-directed therapy, approximately 15-20% of pediatric patients with acute lymphoblastic leukemia (ALL) have relapses. Recent genome-wide analyses have identified that an alteration of IKZF1 is associated with very poor outcomes in B-cell progenitor ALL. In this study, we determined the prognostic significance of IKZF1 deletions in patients with childhood ALL. This study analyzed 242 pediatric B-cell progenitor ALL patients in Taiwan. We developed a simple yet sensitive multiplex quantitative PCR coupled with capillary electrophoresis to accurately determine the allele dose of IKZF1, and high resolution melting was used for mutation screening for all coding exons of IKZF1. Twenty-six (10.7%) pediatric B-cell progenitor ALL patients were found to harbor these deletions. Most of the deletions were broader deletions that encompassed exon 3 to exon 6, consistent with previous reports. Genomic sequencing of IKZF1 was carried out in all cases and no point mutations were identified. Patients with IKZF1 deletions had inferior event-free survival (P < 0.001), and overall survival (P = 0.0016). The association between IKZF1 deletions and event-free survival was independent of age, leukocyte count at presentation, and cytogenetic subtype by multivariate Cox analysis (P = 0.003, hazard ratio = 2.45). This study indicates that detection of IKZF1 deletions upon diagnosis of B-cell progenitor ALL may help to identify patients at risk of treatment failure. IKZF1 deletions could be incorporated as a new high-risk prognostic factor in future treatment protocols. To the best of our knowledge, this is the first study to examine the poor prognosis of IKZF1 deletions in an Asian population.

Low penetrance of retinoblastoma for p.V654L mutation of the RB1 gene.

BACKGROUND: Retinoblastoma is caused by compound heterozygosity or homozygosity of retinoblastoma gene (RB1) mutations. In germline retinoblastoma, mutations in the RB1 gene predispose individuals to increased cancer risks during development. These mutations segregate as autosomal dominant traits with high penetrance (90%). METHODS: We screened 30 family members from one family using high resolution melting assay and DNA direct sequencing for mutations in the RB1 gene. We evaluate the phenotype and penetrance of germline mutations of the RB1 gene in a large Taiwanese family. RESULTS: The molecular analysis and clinical details of this family showed phenotypic variability associated with the p.V654L mutation in exon 19 of the RB1 gene in 11 family members. The phenotype varied from asymptomatic to presence of a unilateral tumor. Only four individuals (2 males and 2 females) developed unilateral retinoblastoma, which resulted in calculated low penetrance of 36% (4/11). The four individuals with retinoblastoma were diagnosed before the age of three years. None of their relatives exhibited variable severity or bilateral retinoblastoma. CONCLUSIONS: The diseased-eye ratio for this family was 0.36, which is lower than current estimates. This suggests that the RB1 p.V654L mutation is a typical mutation associated with low penetrance.

Preimplantation genetic diagnosis of beta-thalassemia using real-time polymerase chain reaction with fluorescence resonance energy transfer hybridization probes.

Preimplantation genetic diagnosis (PGD) is employed increasingly to allow transfer of embryos to the uterus in assisted reproduction procedures. There are three stages of biopsy: polar bodies, one or two blastomeres from the cleavage-stage embryos, and trophectoderm cells ( approximately 5cells) from the blastocyst-stage embryos. Validation of polymerase chain reaction (PCR)-based assays are challenging because only limited genetic material can be obtained for PGD. In the current study, we modified a valid single-cell PCR protocol for PGD using real-time PCR assay with fluorescence resonance energy transfer (FRET) hybridization probes followed by melting curve analysis. We optimized and clinically applied the protocol, permitting molecular genetic analysis to amplify a specific region on the beta-globin (HBB) gene for a couple, carriers of two mutations: c.-78A>G and c.52A>T. Among a total of eight embryos obtained after ovarian stimulation, a single blastomere per embryo at the six- to eight-cell stage was biopsied. This PGD method showed that four embryos were unaffected, two embryos were selected for transfer, and one pregnancy was achieved. Finally, a healthy male baby was delivered at 38weeks' gestation. The results obtained using the new method, FRET hybridization probes, were compared with findings using an existing method, primer extension minisequencing.

Epigenetic profiling of the H19 differentially methylated region and comprehensive whole genome array-based analysis in Silver-Russell syndrome.

Silver-Russell syndrome (SRS) is a clinically and genetically heterogeneous congenital disorder characterized by severe growth retardation. Hypomethylation of the differentially methylated region (DMR) of the H19 gene and uniparental disomy of maternal chromosome 7 is present in ∼45% of the patients with SRS so more than half of these patients have no known genetic etiology. We combined several molecular technologies including multiplex methylation polymerase chain reaction, methylation-sensitive multiple ligation probe-dependent amplification, and methylation-sensitive high-resolution melting to assess the epigenetic status of 34 patients with SRS. Additionally, we applied a whole genome strategy to detect copy number changes and loss of heterozygosity. Thirteen patients (38.2%) had hypomethylation of the DMR of the H19 gene and none had uniparental disomy of maternal chromosome 7. The whole genome arrays identified five patients (14.7%) with microdeletions on chromosomes 1q23q24.3, 7p15.3, 13q31.3, 14q32.31, and 15q26.2qter, respectively. The overall mutation detection rate was 52.9% by the epigenetic study and the whole genome strategy. Although epimutation may be the major cause of SRS and can be identified by multiplex methylation polymerase chain reaction, the whole genome approach also provides information on the etiology of SRS. If no epimutation is identified in the patients with typical SRS, microdeletions should be suspected.

MLPA and DNA index improve the molecular diagnosis of childhood B-cell acute lymphoblastic leukemia.

Aneuploidy occurs within a significant proportion of childhood B-cell acute lymphoblastic leukemia (B-ALL). Some copy number variations (CNV), associated with novel subtypes of childhood B-ALL, have prognostic significance. A total of 233 childhood B-ALL patients were enrolled into this study. Focal copy number alterations of ERG, IKZF1, PAX5, ETV6, RB1, BTG1, EBF1, CDKN2A/2B, and the Xp22.33/Yp11.31 region were assessed by Multiplex Ligation-dependent Probe Amplification (MLPA). The MLPA telomere kit was used to identify aneuploidy through detection of whole chromosome loss or gain. We carried out these procedures alongside measurement of DNA index in order to identify, aneuploidy status in our cohort. MLPA telomere data and DNA index correlated well with aneuploidy status at higher sensitivity than cytogenetic analysis. Three masked hypodiploid patients, undetected by cytogenetics, and their associated copy number neutral loss of heterozygosity (CN-LOH) were identified by STR and SNP arrays. Rearrangements of TCF3, located to 19p, were frequently associated with 19p deletions. Other genetic alterations including iAMP21, IKZF1 deletions, ERG deletions, PAX5AMP, which have clinical significance or are associated with novel subtypes of ALL, were identified. In conclusion, appropriate application of MLPA aids the identifications of CNV and aneuploidy in childhood B-ALL.

Interaction between PHOX2B and CREBBP mediates synergistic activation: mechanistic implications of PHOX2B mutants.

Congenital central hypoventilation syndrome (CCHS) is a disorder of the autonomic nervous system. The genetic defect is usually caused by heterozygous mutations of the PHOX2B gene such as a 20-alanine tract (+5 to+13 alanines) expansion (approximately 95%) and occasional frameshift or missense mutations. Cytoplasmic aggregates were shown in PHOX2B proteins with longer alanine tract (+9 and longer) expansion and impaired DNA binding was observed in PHOX2B proteins with frameshift, missense, or longer alanine tract (+9 and longer) expansion. Defective transactivation activity was shown in certain PHOX2B mutants. However, PHOX2B proteins with short alanine tract (+5 to+7) expansion in the majority of patients (approximately 75%) did not have cytoplasmic aggregates or DNA binding defects. CREB-binding protein (CREBBP/CBP) is a transcriptional coactivator that interacts with multiple transcription factors to cause synergistic activation. Here we show that CBP interacted with PHOX2B and served as its coactivator to mediate synergistic activation. Wild-type PHOX2B and CBP used specific domains to interact with each other. The domains of CBP that interacted with different PHOX2B mutants were different compared to those interacting with wild-type PHOX2B. Transient cotransfection assays using different PHOX2B mutants and CBP showed the impaired synergistic activation caused by different PHOX2B mutants. An interfering effect was observed in certain PHOX2B mutants. These results demonstrated that aberrant interaction of PHOX2B mutants with CBP and/or an interfering effect of certain PHOX2B mutants may be the critical mechanism to impair synergistic activation, thereby contributing to the phenotypes of CCHS.

Mutation spectrum of the fibrillin-1 (FBN1) gene in Taiwanese patients with Marfan syndrome.

The aim of this study was to establish a national database of mutations in the fibrillin-1 (FBN1) gene that cause Marfan syndrome (MFS) in the Taiwanese population. In this study, we screened 294 patients from 157 families for the presence of FBN1 mutations using polymerase chain reaction/ denaturing high performance liquid chromatography (PCR/DHPLC). We identified 56 mutations in 62 of the 157 (40%) families including 49 single-base substitutions (36 missense mutations, seven nonsense mutations, and six splicing sites), one small insertion, four small deletions, one small indel (insertion and deletion), and one exonic deletion (Exon 36). When family history was taken into consideration, the mutation detection rate rose to 91% (29 of 32). We further investigated the phenotypic data and found that one third (47 of 157) of the families fit the Ghent criteria for MFS. Based on that data, the mutation rate was 98% (46/47). That finding implies that family history and the Ghent criteria play a more important role than clinical manifestations in establishing a clinical diagnosis of Marfan syndrome. Among the 56 mutations found in this study, 40 (71%) have not been registered in the Human Gene Mutation Database (HGMD) or in the Universal Mutation Database (UMD). This is the first study of the mutation spectrum of MFS in a cohort of patients in Taiwan. The database is expected to considerably improve genetic counseling for and medical care of MFS families.

Identification of CpG methylation of the SNRPN gene by methylation-specific multiplex PCR.

In this article, we show that methylation-specific multiplex PCR (MS-multiplex PCR) is a sensitive and specific single assay for detecting CpG methylation status as well as copy number aberrations. We used MS-multiplex PCR to simultaneously amplify three sequences: the 3' ends of the SNRPN gene (for unmethylated sequences), the KRITI gene (as internal control), and the promoter of the SNRPN gene containing CpG islands (for methylated sequences) after digestion with a methylation-sensitive restriction enzyme (HhaI). We established this duplex assay for the analysis of 38 individuals with Prader-Willi syndrome, 2 individuals with Angelman syndrome, and 28 unaffected individuals. By comparing the copy number of the three regions, the methylation status and the copy number changes can be easily distinguished by MS-multiplex PCR without the need of bisulfite treatment of the DNA. The data showed that MS-multiplex PCR allows for the estimation of the methylation level by comparing the copy number aberrations of unknown samples to the standards with a known methylated status. The in-house-designed MS-multiplex PCR protocol is a relatively simple, cost-effective, and highly reproducible approach as a significant strategy in clinical applications for epigenetics in a routine laboratory.

Identification of fibrillin-1 gene mutations in Marfan syndrome by high-resolution melting analysis.

Marfan syndrome has been associated with approximately 562 mutations in the fibrillin-1 (FBN1) gene. Mutation scanning of the FBN1 gene with DNA direct sequencing is time-consuming and expensive because of its large size. This study analyzed the diagnostic value of high-resolution melting analysis as an alternative method for scanning of the FBN1 gene. A total of 75 polymerase chain reaction (PCR) amplicons (179-301bp, average 256bp) that covered the complete coding regions and splicing sites were evaluated on the 96-well LightCycler system. Melting curves were analyzed as fluorescence derivative plots (-dF/dT vs. temperature). To determine the sensitivity of this method, a total of 82 samples from patients with Marfan syndrome and 50 unaffected individuals were analyzed. All mutations reported in this study had been confirmed previously by direct sequencing analysis. Melting analysis identified 48 heterozygous variants. The variant c.3093 G>T (exon 25) was incorrectly identified by melting curve analysis. The sensitivity of the technique in this sample was 98.78% (81/82). This study demonstrated that high-resolution melting analysis is a reliable gene scanning method with greater speed than DNA sequencing. Our results support the use of this technology as an alternative method for the diagnosis of Marfan syndrome as well as its suitability for high-throughput mutation scanning of other large genes.

Fragile X syndrome carrier screening in pregnant women in Chinese Han population.

Fragile X syndrome (FXS) is the most frequent genetic cause of intellectual disability (ID). It was previously believed that the FXS prevalence was low in Chinese population, and the cost-efficiency of FXS carrier screening was questioned. This retrospective observational study was conducted between September 2014 and May 2017 to determine the prevalence of FXS carriers in a large Chinese cohort of pregnant women. The FMR1 CGG repeat status was determined in 20,188 pregnant Taiwanese women and we identified 26 women with premutation (PM). The PM allele was transmitted to the fetus in 17 pregnancies (56.6%), and six of 17 expanded to full mutation (FM). One asymptomatic woman had a FM allele with 280 CGG repeats. Prenatal genetic diagnosis of her first fetus revealed a male carrying a FMR1 gene deletion of 5' UTR and exon 1. Her second fetus was a female carrying a FM allele as well. This is so far the largest study of the FXS carrier screening in Chinese women. The prevalence of premutation allele for FXS in normal asymptomatic Taiwanese women was found to be as high as 0.13% (1 in 777) in this study. The empirical evidence suggests that reproductive FXS carrier screening in Taiwan might be cost-effective.

Comparison of the mismatch-specific endonuclease method and denaturing high-performance liquid chromatography for the identification of HBB gene mutations.

BACKGROUND: Beta-thalassemia is a common autosomal recessive hereditary disease in the Meditertanean, Asia and African areas. Over 600 mutations have been described in the beta-globin (HBB), of which more than 200 are associated with a beta-thalassemia phenotype. RESULTS: We used two highly-specific mutation screening methods, mismatch-specific endonuclease and denaturing high-performance liquid chromatography, to identify mutations in the HBB gene. The sensitivity and specificity of these two methods were compared. We successfully distinguished mutations in the HBB gene by the mismatch-specific endonuclease method without need for further assay. This technique had 100% sensitivity and specificity for the study sample. CONCLUSION: Compared to the DHPLC approach, the mismatch-specific endonuclease method allows mutational screening of a large number of samples because of its speed, sensitivity and adaptability to semi-automated systems. These findings demonstrate the feasibility of using the mismatch-specific endonuclease method as a tool for mutation screening.

Mutation spectrum of 122 hemophilia A families from Taiwanese population by LD-PCR, DHPLC, multiplex PCR and evaluating the clinical application of HRM.

BACKGROUND: Hemophilia A represents the most common and severe inherited hemorrhagic disorder. It is caused by mutations in the F8 gene, which leads to a deficiency or dysfunctional factor VIII protein, an essential cofactor in the factor X activation complex. METHODS: We used long-distance polymerase chain reaction and denaturing high performance liquid chromatography for mutation scanning of the F8 gene. We designed the competitive multiplex PCR to identify the carrier with exonal deletions. In order to facilitate throughput and minimize the cost of mutation scanning, we also evaluated a new mutation scanning technique, high resolution melting analysis (HRM), as an alternative screening method. RESULTS: We presented the results of detailed screening of 122 Taiwanese families with hemophilia A and reported twenty-nine novel mutations. There was one family identified with whole exons deletion, and the carriers were successfully recognized by multiplex PCR. By HRM, the different melting curve patterns were easily identified in 25 out of 28 cases (89%) and 15 out of 15 (100%) carriers. The sensitivity was 93 % (40/43). The overall mutation detection rate of hemophilia A was 100% in this study. CONCLUSION: We proposed a diagnostic strategy for hemophilia A genetic diagnosis. We consider HRM as a powerful screening tool that would provide us with a more cost-effective protocol for hemophilia A mutation identification.

Genotyping of the G1138A mutation of the FGFR3 gene in patients with achondroplasia using high-resolution melting analysis.

OBJECTIVES: The fibroblast growth factor receptor 3 gene (FGFR3) plays a critical role in cartilage growth-plate differentiation and bony development. It has been shown that 97% of patients with achondroplasia have a G to A transition mutation at position 1138 (c.1138 G>A) of codon 380 of the FGFR3 gene. DESIGN AND METHODS: Exon 8 of the FGFR3 gene was analyzed in 40 patients with achondroplasia, as well as in 50 control individuals for the presence of the c.1138G>A variant using melting curve analysis with a high-resolution melting instrument (HR-1). RESULTS: The high-resolution melting curve analysis successfully genotyped the c.1138G>A mutation in exon 8 of the FGFR3 gene in all 40 patients with achondroplasia without the need of further assays. The technique had a sensitivity and specificity of 100%. CONCLUSION: High-resolution melting analysis is a simple, rapid, and sensitive one tube assay for genotyping the FGFR3 gene. The technique is a low cost high-throughput FGFR3 screening assay.

Rapid prenatal diagnosis of spinal muscular atrophy by denaturing high-performance liquid chromatography system.

OBJECTIVE: Use of Denaturing High-Performance Liquid Chromatography (DHPLC) in prenatal diagnosis of spinal muscular atrophy (SMA). METHODS: Thirty-three members of 7 families participated in carrier test and disease detection of SMA. Prenatal genetic diagnosis was performed if both parents were carriers or any family members had SMA. DNA extracted from blood, chorionic villi and amniotic fluid was amplified and used for DHPLC. RESULTS: Twenty SMA carriers, seven SMA affected cases, and six normal individuals were identified. SMA status was demonstrated by genotyping and total copy number determinations of SMN1 and SMN2. Families 1-3 were classified as group one (SMA affecting previously born child). Group two, comprising families 4 and 5, had lost a child due to an unknown muscular disease. Group three (SMA-affected parent) comprised families 6 and 7; carrier testing was done. DHPLC prenatal genetic diagnosis was made in seven pregnancies, one in each family (affected, n=2; carrier, n=3; normal, n=2). Pregnancy was terminated for the two affected fetuses. The others were delivered uneventfully and SMA free. CONCLUSION: DHPLC prenatal diagnosis of SMA and determination of SMA status in adults is possible, and SMN1 and SMN2 copy numbers can be determined.