Chia-Cheng Hung

Molecular and clinical analyses of 84 patients with tuberous sclerosis complex

BackgroundTuberous sclerosis complex (TSC) is an autosomal dominant disease characterized by the development of multiple hamartomas in many internal organs. Mutations in either one of 2 genes, TSC1 and TSC2, have been attributed to the development of TSC. More than two-thirds of TSC patients are sporadic cases, and a wide variety of mutations in the coding region of the TSC1 and TSC2 genes have been reported.MethodsMutational analysis of TSC1 and TSC2 genes was performed in 84 Taiwanese TSC families using denaturing high-performance liquid chromatography (DHPLC) and direct sequencing.ResultsMutations were identified in a total of 64 (76 %) cases, including 9 TSC1 mutations (7 sporadic and 2 familial cases) and 55 TSC2 mutations (47 sporadic and 8 familial cases). Thirty-one of the 64 mutations found have not been described previously. The phenotype association is consistent with findings from other large studies, showing that disease resulting from mutations to TSC1 is less severe than disease due to TSC2 mutation.ConclusionThis study provides a representative picture of the distribution of mutations of the TSC1 and TSC2 genes in clinically ascertained TSC cases in the Taiwanese population. Although nearly half of the mutations identified were novel, the kinds and distribution of mutation were not different in this population compared to that seen in larger European and American studies.

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.

Using brachial-ankle pulse wave velocity to associate arterial stiffness with cardiovascular risks

BACKGROUND AND AIMS This study aimed to elucidate the relationship between brachial-ankle pulse wave velocity (baPWV) and conventional cardiovascular risk factors. METHODS AND RESULTS A total of 192 subjects with low to intermediate risk was enrolled in a cardiovascular evaluation program. A multiple regression model was built to find significant cardiovascular biomarkers for predicting baPWV. A logistic regression model was developed to associate baPWV and other biomarkers with the risk of cardiac diastolic dysfunction. A total of 123 men (mean age: 52.6+/-12.0) and 69 women (mean age: 51.7+/-10.4) was included. Age, blood pressure, C-reactive protein, serum homocysteine, heart rate, and blood urea nitrogen were positively predictive of increased pulse wave velocity. In turn, baPWV increased the risk (odds ratio: 1.257 for each m/s, 95% CI: 1.105 approximately 1.430, p<0.001) and high-density lipoprotein decreased the risk for cardiac diastolic dysfunction (0.962 for each mg/dl, 95% CI: 0.925 approximately 1.000, p=0.05). The correlation between baPWV and Framingham 10-year risk was moderate (men: r=0.306, p=0.002; women r=0.548, p<0.001). CONCLUSION The results suggest that baPWV is a composite risk factor for early atherosclerotic change and a predictor for the development of diastolic dysfunction and long-term cardiovascular risk.

Carrier Screening for Spinal Muscular Atrophy (SMA) in 107,611 Pregnant Women during the Period 2005–2009: A Prospective Population-Based Cohort Study

Background Spinal muscular atrophy (SMA) is the most common neuromuscular autosomal recessive disorder. The American College of Medical Genetics has recently recommended routine carrier screening for SMA because of the high carrier frequency (1 in 25–50) as well as the severity of that genetic disease. Large studies are needed to determine the feasibility, benefits, and costs of such a program. Methods and Findings This is a prospective population-based cohort study of 107,611 pregnant women from 25 counties in Taiwan conducted during the period January 2005 to June 2009. A three-stage screening program was used: (1) pregnant women were tested for SMA heterozygosity; (2) if the mother was determined to be heterozygous for SMA (carrier status), the paternal partner was then tested; (3) if both partners were SMA carriers, prenatal diagnostic testing was performed. During the study period, a total of 2,262 SMA carriers with one copy of the SMN1 gene were identified among the 107,611 pregnant women that were screened. The carrier rate was approximately 1 in 48 (2.10%). The negative predictive value of DHPLC coupled with MLPA was 99.87%. The combined method could detect approximately 94% of carriers because most of the cases resulted from a common single deletion event. In addition, 2,038 spouses were determined to be SMA carriers. Among those individuals, 47 couples were determined to be at high risk for having offspring with SMA. Prenatal diagnostic testing was performed in 43 pregnant women (91.49%) and SMA was diagnosed in 12 (27.91%) fetuses. The prevalence of SMA in our population was 1 in 8,968. Conclusion The main benefit of SMA carrier screening is to reduce the burden associated with giving birth to an affected child. In this study, we determined the carrier frequency and genetic risk and provided carrier couples with genetic services, knowledge, and genetic counseling.

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

BackgroundHemophilia 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.MethodsWe 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.ResultsWe 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.ConclusionWe 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.

Newborn genetic screening for hearing impairment: a preliminary study at a tertiary center.

Universal newborn hearing screening (UNHS) is of paramount importance for early identification and management of hearing impairment in children. However, infants with slight/mild, progressive, or late-onset hearing impairment might be missed in conventional UNHS. To investigate whether genetic screening for common deafness-associated mutations could assist in identifying these infants, 1017 consecutive newborns in a tertiary hospital were subjected to both newborn hearing screening using a two-step distortion-product otoacoustic emissions (DPOAE) screening and newborn genetic screening (NGS) for deafness. The NGS targeted 4 deafness-associated mutations commonly found in the Taiwanese population, including p.V37I (c.109G>A) and c.235delC of the GJB2 gene, c.919-2A>G of the SLC26A4 gene, and mitochondrial m.1555A>G of the 12S rRNA gene. The results of the NGS were then correlated to the results of the NHS. Of the 1017 newborns, 16 (1.6%) had unilateral DPOAE screening failure, and 22 (2.2%) had bilateral DPOAE screening failure. A total of 199 (19.6%) babies were found to have at least 1 mutated allele on the NGS for deafness, 11 (1.1%) of whom were homozygous for GJB2 p.V37I, 6 (0.6%) compound heterozygous for GJB2 p.V37I and c.235delC, and 1 (0.1%) homoplasmic for m.1555A>G, who may potentially have hearing loss. Among them, 3 babies, 5 babies, and 1 baby, respectively, passed the NHS at birth. Comprehensive audiological assessments in the 9 babies at 3 months identified 1 with slight hearing loss and 2 with mild hearing loss. NGS for common deafness-associated mutations may identify infants with slight/mild or potentially progressive hearing impairment, thus compensating for the inherent limitations of the conventional UNHS.

An update of preimplantation genetic diagnosis in gene diseases, chromosomal translocation, and aneuploidy screening

Preimplantation genetic diagnosis (PGD) is gradually widely used in prevention of gene diseases and chromosomal abnormalities. Much improvement has been achieved in biopsy technique and molecular diagnosis. Blastocyst biopsy can increase diagnostic accuracy and reduce allele dropout. It is cost-effective and currently plays an important role. Whole genome amplification permits subsequent individual detection of multiple gene loci and screening all 23 pairs of chromosomes. For PGD of chromosomal translocation, fluorescence in-situ hybridization (FISH) is traditionally used, but with technical difficulty. Array comparative genomic hybridization (CGH) can detect translocation and 23 pairs of chromosomes that may replace FISH. Single nucleotide polymorphisms array with haplotyping can further distinguish between normal chromosomes and balanced translocation. PGD may shorten time to conceive and reduce miscarriage for patients with chromosomal translocation. PGD has a potential value for mitochondrial diseases. Preimplantation genetic haplotyping has been applied for unknown mutation sites of single gene disease. Preimplantation genetic screening (PGS) using limited FISH probes in the cleavage-stage embryo did not increase live birth rates for patients with advanced maternal age, unexplained recurrent abortions, and repeated implantation failure. Polar body and blastocyst biopsy may circumvent the problem of mosaicism. PGS using blastocyst biopsy and array CGH is encouraging and merit further studies. Cryopreservation of biopsied blastocysts instead of fresh transfer permits sufficient time for transportation and genetic analysis. Cryopreservation of embryos may avoid ovarian hyperstimulation syndrome and possible suboptimal endometrium.

Parallel minisequencing followed by multiplex matrix-assisted laser desorption/ionization mass spectrometry assay for β-thalassemia mutations

Abstractβ-thalassemia is a common monogenic disease caused by mutations in the human β-globin gene (HBB), many of which are differentially represented in human subpopulations stratified by ethnicity. This study describes an efficient and highly accurate method to screen for the eight most-common disease-causing mutations, covering more than 98% of HBB alleles in the Taiwanese population, using parallel minisequencing and multiplex assay by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The MALDI-TOF MS was optimized for sensitivity and resolution by “mass tuning” the PinPoint assay for eight HBB SNPs. Because of the close proximity and clustering of mutations in HBB, primer extension reactions were conducted in parallel. Efficient sequential desalting using POROS and cationic exchange chromatography allowed for an unambiguous multiplex genotyping by MALDI-TOF MS. The embellishing SNP assay allowed for highly accurate identification of the eight most-common β-thalassemia mutations in homozygous normal control, carrier, and eight heterozygous carrier mixtures, as well as the diagnosis of a high-risk family. The results demonstrated a flexible strategy for rapid identification of clustering SNPs in HBB with a high degree of accuracy and specificity. It can be adapted easily for high-throughput diagnosis of various hereditary diseases or to establish family heritage databases for clinical applications.

Successful application of the strategy of blastocyst biopsy, vitrification, whole genome amplification, and thawed embryo transfer for preimplantation genetic diagnosis of neurofibromatosis type 1.

OBJECTIVE Preimplantation genetic diagnosis (PGD) offers an alternative for women to carry an unaffected fetus risk of hereditary diseases. Trophectoderm biopsy may provide more cells for accurate diagnosis. However, the time allowed for transportation of the specimens to the laboratory and performance of molecular diagnosis is limited. We designed a PGD program of trophectoderm biopsy, vitrification of blastocysts, whole genome amplification (WGA), double confirmatory genotypings, and thawed embryo transfer. CASE REPORT We conducted this strategy for a woman of familial neurofibromatosis type I (NF-1). She had a genotype of heterozygous c.6709C>T mutation of NF1 gene. Trophectoderm biopsies were performed on 13 blastocysts. Then, individual blastocyst was vitrified. WGA was performed for the samples, followed by genotypings with both real-time polymerase chain reaction and sequencing. Eight embryos were diagnosed as unaffected, four were affected, and one was inconclusive because of allele drop-out. In the next cycle, two unaffected blastocysts were thawed and transferred, that resulted in a singleton pregnancy. The pregnancy was confirmed as unaffected by means of chorionic villi sampling. CONCLUSION We first demonstrate successful application of blastocyst biopsy, vitrification, WGA, and thawed embryo transfer for PGD of a monogenic disease. Vitrification of blastocysts after biopsy permits sufficient time for shipment of samples and operation of molecular diagnosis.