Development of pre-implantation genetic testing protocol for monogenic disorders (PGT-M) of Hb H disease.

Hb H disease is the most severe form of α-thalassemia compatible with post-natal life. Compound heterozygous α0-thalassemia- SEA deletion/α+-thalassemia- 3.7kb deletion is the commonest cause of Hb H disease in Thailand. Preimplantation genetics testing for monogenic disorders (PGT-M) is an alternative for couples at risk of the disorder to begin a pregnancy with a healthy baby. This study aims to develop a novel PCR protocol for PGT-M of Hb H disease- SEA/-3.7kb using multiplex fluorescent PCR. A novel set of primers for α+-thalassemia- 3.7kb deletion was developed and tested. The PCR protocol for α0-thalassemia- SEA deletion was combined for Hb H disease- SEA/-3.7kb genotyping. The PCR protocols were applied to genomic DNA extracted from subjects with different thalassemia genotypes and on whole genome amplification (WGA) products from clinical PGT-M cycles of the families at risk of Hb Bart's. The results were compared and discussed. The results showed three PCR products from α+-thalassemia- 3.7kb primer set, and three from α0thalassemiaSEA primer set. The results were consistent with the known thalassemia genotypes. The novel -α3.7 primers protocol was also tested on 37 WGA products from clinical PGT-M cycles giving accurate genotyping results and a satisfying amplification efficiency with the ADO rates of 2.7%, 0%, and 0% for HBA2, HBA1, and internal control fragments, respectively. This novel PCR protocol can precisely distinguish Hb H disease- SEA/-3.7kb from other genotypes. Additionally, this is the first PCR protocol for Hb H disease- SEA/-3.7kb which is optimal for PGT-M.

The successful strategy of comprehensive pre-implantation genetic testing for beta-thalassaemia-haemoglobin E disease and chromosome balance using karyomapping.

Thalassaemia is the commonest monogenic disease and causes a health and economic burden worldwide. Karyomapping can be used for pre-implantation genetic testing of monogenic disorders (PGT-M). This study applied karyomapping in two PGT-M cycles and made a comparison to polymerase chain reaction (PCR). Two families at risk of having beta-thalassaemia-haemoglobin E disease offspring decided to join the project and informed consent was obtained. Karyomapping results of family A (beta-thalassaemia (c.41_42delTCTT)-Hb E (c.26G>A) disease) revealed four normal, two beta-thalassaemia traits, one Hb E trait and six affected. Three embryos exhibited unbalanced chromosomes. One normal male embryo was transferred. Karyomapping results of family B (beta-thalassaemia (c.17A>T)-Hb E (c.26G>A) disease) revealed six Hb E traits and three affected. Three embryos were chromosomally unbalanced. One Hb E trait embryo was transferred. Two successful karyomapping PGT-M were performed, including deletion and single-base mutations. Karyomapping provides accuracy as regards the protocol and copy number variation which is common in pre-implantation embryos. Impact StatementWhat is already known on this subject? Thalassaemia syndrome is the commonest monogenic disease and causes a health and economic burden worldwide. Modern haplotyping using SNP array (aSNP) and karyomapping algorithms can be used for pre-implantation genetic testing of monogenic disorders (PGT-M). However, few clinical karyomapping PGT-M cycles have been done and validated so far.What do the results of this study add? Two successful clinical PGT-M cycles for beta-thalassaemia (c.41_42delTCTT and c.17A>T mutations)-haemoglobin E (c.26G>A) disease were performed using karyomapping. The outcome was two healthy babies. Multiplex fluorescent polymerase chain reaction (PCR) with mini-sequencing was also used for confirmation mutation analysis results. PCR confirmed haplotyping results in all embryos. Six embryos from both PGT-M cycles exhibited unbalanced chromosomes evidenced by aSNP.What are the implications of these findings for clinical practice and/or further research? Karyomapping provides accurate information quickly and the outcomes of the study will save time as regards protocol development, provide a usable universal PGT-M protocol and add additional copy number variation (CNV) information, chromosome number variation being a common issue in pre-implantation embryos.

Pre-implantation genetic testing for Marfan syndrome using mini-sequencing.

Marfan syndrome (MFS1) is an autosomal dominant condition causing aortopathy including fatal aortic dissection. This study aimed to perform clinical PGT-M in a family with a history of MFS1 for two generations. A family with two members affected by MFS1 approached the hospital for PGT-M. The couple decided to join the project following extensive counselling and informed consent was obtained. The mutation contributory to MFS1 was identified using whole-exome sequencing (WES). A novel PGT-M protocol using multiplex fluorescent PCR and mini-sequencing was developed and tested. Ten blastocysts were subjected to PGT-M in two clinical PGT cycles. Mini-sequencing revealed four normal and six affected embryos. Microsatellite-based linkage analysis confirmed mutation analysis results in all samples. The embryos diagnosed as normal (non-MFS1) were chosen for transfer. A pregnancy was obtained in the third embryo transfer. Invasive prenatal diagnosis confirmed the normal genotype of the baby. This study demonstrated comprehensive management using the application of clinical-based diagnosis, WES for mutation identification within the MFS1 gene, mini-sequencing for embryo selection and microsatellite-based linkage analysis for backup of PGT-M results and contamination detection to assist couples in having a healthy child when there was a family history of Marfan syndrome.Impact StatementWhat is already known on this subject? Marfan syndrome (MFS1, OMIM#154700) is an autosomal dominant condition causing aortopathy including fatal aortic dissection. Pre-implantation genetic testing (PGT) is an alternative to traditional invasive prenatal diagnosis (PND) giving the couples the chance of starting pregnancy with the confidence that the baby will be unaffected. Most of the previous PGT reports employed microsatellite-based linkage analysis. A few PGT studies used sequencing, mini-sequencing and mutation analysis; however, the details of the techniques were not described.What do the results of this study add? Single-cell PCR protocol using multiplex fluorescent PCR and mini-sequencing was developed and validated. Two clinical PGTs cycles for Marfan syndrome were performed. A healthy baby was resulted. The details of multiplex fluorescent PCR and mini-sequencing protocols are described in this study so that the procedures can be reproduced.What are the implications of these findings for clinical practice and/or further research? Embryo selection can help the family suffering from Marfan syndrome for two generations to start a pregnancy with confidence that their child will be unaffected. This study also shows the use of a mini-sequencing protocol for PGT, which can be a universal protocol for other mutations by changing the PCR primers and mini-sequencing primers.

A study of the association between angiotensinogen (AGT) gene polymorphism (M235T) and preeclampsia in Thai pregnant women.

AGT M235T gene polymorphism may cause increased blood pressure in preeclampsia in pregnancy; however, the evidence remains controversial. This study investigated the association between AGT M235T and preeclampsia in Thai pregnant women. A case-control study was conducted to compare the distributions of AGT M235T genotypes and alleles between 142 normotensive pregnancies as controls and 61 preeclampsia pregnancies as cases in a tertiary-care university hospital in Chiang Mai, Thailand. The results show that the distribution of AGT M235T genotypes (MM, MT and TT) of both groups were not significantly different (preeclampsia: 0.0, 16.4, 83.6%; control: 2.1, 22.5, 75.4%, respectively; p = .30). Additionally, there was no statistical difference in the distribution of AGT M235T alleles (M and T alleles) (preeclampsia: 8.2 and 91.8% versus control: 13.4 and 86.6%, respectively; p = .14). In this study, the distributions of AGT M235T were not different in both groups. Therefore, AGT M235T polymorphism may not play a significant role in preeclampsia pathophysiology in Thai population.Impact statementWhat is already known on this subject? Preeclampsia is one of the major complications during pregnancy; it significantly affects maternal and perinatal morbidity and mortality. Effort has been made to find markers and predictors that are associated with the pathophysiology of preeclampsia. AGT M235T gene polymorphism may cause increased blood pressure in preeclampsia pregnancy; however, evidences are still controversial.What do the results of this study add? We conducted a case-control study to compare the distributions of AGT M235T genotypes and alleles between 142 normotensive pregnancies as controls and 61 preeclampsia pregnancies as cases. The results show that preeclamptic women are more likely to deliver at an earlier gestational age and have a smaller baby in comparison with the normotensive group. In addition, women with preeclampsia had a higher chance of having an operative delivery and caesarean section. However, the distribution of AGT M235T polymorphism of preeclampsia women and the control group were not significantly different.What are the implications of these findings for clinical practice and/or further research? AGT M235T polymorphism may not play a significant role in preeclampsia pathophysiology in Thai population.

Influence of water-soluble channeling agents on the release of diclofenac sodium from Irvingia malayana wax matrix tablets.

Irvingia malayana wax (IW) is majorly composed of esters of medium chain fatty acids. Its melting point is low and closed to the body temperature. This study aimed at investigating the potential of IW as a matrix-forming agent and evaluate the effect of soluble channeling agents on the release of diclofenac sodium (DS) from IW matrix tablets. The preformulation study by infrared spectroscopy and differential scanning calorimetry showed no incompatibility between IW and DS or soluble channeling agents, namely PEG 4000, PEG 6000 and lactose. IW retarded the release of DS from the matrix tablets more efficiently than carnauba wax due to its greater hydrophobicity and its ability to become partial molten wax at 37° C. Factors affecting the release of DS from IW matrix were drug concentrations, and types and concentrations of channeling agents. The release of DS significantly improved when DS concentration reached approximately 33%. The fast dissolving channeling agent, lactose, could enhance the drug release rate more effectively than PEG 4000 and PEG 6000, respectively. The linear relationship between the DS release rate and the concentration of the chosen channeling agent, PEG 6000, was found (r2=0.9866).

Verification of Thai ethnobotanical medicine "Kamlang Suea Khrong" driven by multiplex PCR and powerful TLC techniques.

Kamlang Suea Khrong (KSK) crude drug, a traditional Thai medicine used for oral tonic and analgesic purposes, is obtained from three origins: the inner stem bark of Betula alnoides (BA) or the stems of Strychnos axillaris (SA) or Ziziphus attopensis (ZA). According to the previous reports, SA contains strychnine-type alkaloids that probably cause poisoning; however, only organoleptic approaches are insufficient to differentiate SA from the other plant materials. To ensure the botanical origin of KSK crude drug, powerful and reliable tools are desperately needed. Therefore, molecular and chemical identification methods, DNA barcoding and thin-layer chromatography (TLC), were investigated. Reference databases, i.e., the ITS region and phytochemical profile of the authentic plant species, were conducted. In case of molecular analysis, multiplex polymerase chain reaction (PCR) based on species-specific primers was applied. Regarding species-specific primers designation, the suitability of three candidate barcode regions (ITS, ITS1, and ITS2) was evaluated by genetic distance using K2P model. ITS2 presented the highest interspecific variability was verified its discrimination power by tree topology. Accordingly, ITS2 was used to create primers that successfully specified plant species of authentic samples. For chemical analysis, TLC with toluene:ethyl acetate:ammonia (1:9:0.025) and hierarchical clustering were operated to identify the authentic crude drugs. The developed multiplex PCR and TLC methods were then applied to identify five commercial KSK crude drugs (CK1-CK5). Both methods correspondingly indicated that CK1-CK2 and CK3-CK5 were originated from BA and ZA, respectively. Molecular and chemical approaches are convenient and effective identification methods that can be performed for the routine quality-control of the KSK crude drugs for consumer reliance. According to chemical analysis, the results indicated BA, SA, and ZA have distinct chemical profiles, leading to differences in pharmacological activities. Consequently, further scientific investigations are required to ensure the quality and safety of Thai ethnobotanical medicine known as KSK.

Clinical Pilot Study of Rectal Suppository Containing Combined Extract of Cissus quadrangularis Linn. and Acmella paniculata (Wall ex. DC.) R. K. Jansen in Acute Hemorrhoids.

BACKGROUND: Cissus quadrangularis Linn. (CQ) is a medicinal plant with good evidence for the treatment of hemorrhoids, listed in the Thai National List of Herbal Products in the oral dosage form. Acmella paniculata (Wall ex. DC.) R. K. Jansen. (AP) is a medicinal plant with a local anesthetic effect. OBJECTIVE: To investigate the potential of rectal suppositories containing CQ and AP extracts to alleviate symptoms of hemorrhoids compared with the commercialized rectal suppository containing hydrocortisone and cinchocaine. MATERIALS AND METHODS: Hemorrhoid outpatients (n = 105) with different severity grades (I, II, or III) from eight hospitals in northern Thailand were included in this study. Hemorrhoid severity was graded by proctoscopy associated with either anal pain or bleeding related to hemorrhoids or both. The patients were randomly allocated to two groups: CQ-AP group (n = 52) or the commercialized rectal suppository group (n = 53). One suppository was rectally administered twice daily in the morning and at bedtime for seven days. Evaluations were performed by physicians on days 1, 4, and 8 of the study. The primary endpoints were bleeding and prolapse size, while the secondary endpoint was anal pain. RESULTS: Baseline demographics, lifestyle, constipation, number of prolapses, grade of hemorrhoid severity, and duration of experiencing hemorrhoids were comparable in both groups of patients. The effects of CQ-AP and the commercialized rectal suppository on bleeding, prolapse size, and anal pain were comparable. The patients in both groups were satisfied with both products at comparable levels and stated a preference for further use in the case of hemorrhoids recurrence. In terms of safety, the patients in the commercialized rectal suppository group experienced a higher incidence of adverse events, including anal pain and bleeding. CONCLUSION: Rectal suppositories containing a combined extract of CQ and AP show potential in alleviating hemorrhoidal symptoms with a good safety profile.

Amido-3-hydroxypyridin-4-ones as iron(III) ligands.

The synthesis and physicochemical properties of a range of 2- and 6-amido-3-hydroxypyridin-4-ones are described. All the amido-substituted 3-hydroxypyridin-4-ones have lower pK(a) values than 1,2-dimethyl-3-hydroxypyridin-4-one (deferiprone). This is due to the inductive effect of the amido group. Furthermore, the pK(a) values of the 3-hydroxy group in 1-nonsubstituted pyridinones are dramatically lower than those of the corresponding 1-alkyl analogues, indicating that a strong hydrogen bond exists between the 2-amido function and the 3-oxygen anion, which stabilises the anion. As a result of the decreased competition with protons, the pFe(3+) values of this group of molecules are higher than that of deferiprone. The distribution coefficients of these molecules are also increased despite the lack of a hydrophobic 1-alkyl substituent and this is ascribed to the intramolecular hydrogen bond. X-ray diffraction studies confirm the existence of the intramolecular hydrogen bond.

A successful strategy for Preimplantation Genetic Diagnosis of beta-thalassemia and simultaneous detection of Down's syndrome using multiplex fluorescent PCR.

OBJECTIVES: Preimplantation Genetic Diagnosis (PGD) is an alternative to prenatal diagnosis providing couples the chance to start a pregnancy with an unaffected fetus. The objective of the present study was to develop and apply quick, sensitive and accurate single cell PCR protocols for PGD of beta-thalassemia and Down's syndrome detection. MATERIAL AND METHOD: Two couples carrying beta-thalassemia codon41-42 mutation underwent routine IVF procedures. Embryo biopsy was performed on Day-3 post-fertilisation and single cell multiplex fluorescent PCR was employed for mutation analysis, contamination detection and diagnosis of trisomy 21 cases. RESULTS: Seventeen embryos were tested in two clinical PGD cycles. This resulted in the first birth following PGD for a single gene disorder in Thailand and South East Asia, confirmed by prenatal testing. Two embryos were shown to be affected by Down's syndrome. CONCLUSION: Successful strategy for PGD of beta-thalassemia and Down's syndrome detection using multiplex fluorescent PCR was introduced.

Bioactive deoxypreussomerins and dimeric naphthoquinones from Diospyros ehretioides fruits: deoxypreussomerins may not be plant metabolites but may be from fungal epiphytes or endophytes.

Deoxypreussomerin derivatives, palmarumycins JC1 (1) and JC2 (2), and two dimeric naphthoquinones, isodiospyrin (3) and its new derivative isodiospyrol A (4), were isolated from dried fruits of Diospyros ehretioides. Structures of the isolated compounds were elucidated by spectroscopic analyses. Palmarumycins were not found in the extract of freshly collected fruits; however, they were present in dried fruit extract. The absence of palmarumycins in fresh fruits of D. ehretioides, together with the chemotaxonomic point of view, we proposed that palmarumycins JC1 (1) and JC2 (2) are more likely to be fungal metabolites, i.e., endophytes or epiphytes. The isolation of palmarumycins 1 and 2 from dried D. ehretioides fruits could be reproducible; both plant samples collected in the years 2002 and 2004 provided the same result, and, therefore, symbiont fungal strains should be specific to the plant host, D. ehretioides, and they can grow on the fruits during drying the sample. Palmarumycin JC1 (1) did not exhibit antimalarial, antifungal, antimycobacterial, and cytotoxic activities. Palmarumycin JC2 (2) exhibited antimalarial (IC50 4.5 microg/ml), antifungal (IC50 12.5 microg/ml), antimycobacterial (MIC 6.25 microg/ml), and cytotoxic (IC50 11.0 microg/ml for NCI-H187 cell line) activities. In our bioassay systems, isodiospyrin (3) did not exhibit antimycobacterial, antifungal, antimalarial, and cytotoxic activities. Isodiospyrol A (4) exhibited antimalarial (IC50 2.7 microg/ml) and antimycobacterial (MIC 50 microg/ml) activities, but was inactive towards Candida albicans. Compound 4 also exhibited cytotoxicity against BC cells (IC50 12.3 microg/ml), but not towards KB and Vero cell lines.