Application of short tandem repeats (STRs) in the preimplantation genetic diagnosis (PGD) of α-thalassemia.

OBJECTIVES: α-thalassemia is an autosomal recessive monogenic blood disorder, affecting up to 5% of the world's population. The occurrence rate of the disease in Vietnam varies up to up to 51.5%, with high rate of mutation carriers, of couples consisting of two carriers at risk of bearing a child with fetal Hb Bart, which can develop into hydrops fetalis syndrome, threatening the well-being of the mother and the child. Our study aims to facilitate birth of healthy/asymptomatic children of α-thalassemia carrier couples who received reproductive service at our centre during the period of 2019-2022. MATERIALS AND METHODS: 89 couples at risks of having α-thalassemia offsprings requested IVF procedures and PGD at Post Hospital during 2019-2022 were recruited for investigation. Couple and additional family members' peripheral blood samples of couples and additional family members were subjected to haemoglobin electrophoresis, DNA extraction for α-thalassemia gene mutation detection and STRs linkage analysis. Data were observed and analysed on GeneMarker software. RESULTS: 91 cycles of PGD for α-thalassemia were carried out for 89 couples. α-thalassemia large deletion (--SEA/αα) was the most common mutation identified in 88 couples, in which 4 cases also carried ß-thalassemia point mutations. Combining results of PGS and PGD, 278/424 amplified embryos were transferable (HBA-mutation free or carriers of single heterozygous HBA mutation, without chromosomal abnormality). 64/89 couples have been transferred with the embryos (prioritizing mutation free ones over carriers), resulting in the birth of 36 α-thalassemia disease-free children, 17 ongoing pregnancies, and 11 with miscarriages. CONCLUSION: Successful application of microsatellite-based method in PGD facilitated the birth of 36 healthy children and 17 ongoing pregnancies for 53/64 couples with embryo-transferred. All resulted clinical births displayed confirmation results in line with the PGD results, thus demonstrating the feasibility and credibility of the use of STR markers in PGD.

Mutation spectrum of retinoblastoma patients in Vietnam.

BACKGROUND: Retinoblastoma (RB), an intraocular malignancy commonly diagnosed in children, is mostly caused by inactivating mutations of both alleles of the RB1 gene. Early genetic screening for RB1 gene mutations would greatly improve treatment outcomes and patient management. METHODS: In this study, both somatic and germline mutations were detected in blood and tumour samples of 42 RB patients using direct sequencing and multiplex ligation-dependent probe amplification. RESULTS: In total, 34 different mutations were found in 36 patients, including 1 SNP, 4 large deletions, 5 splicing sites, 1 missense, 7 frameshifts and 17 nonsense mutations. There were five novel mutations and one unreported which have not been found in large databases such as Leiden Open Variation Database (LOVD) and ClinVar. CONCLUSION: A higher rate of RB patients carrying heterozygous germline mutation and highly prevalent with pathogenic truncated mutation, hence, early detection of RB is essential for vision salvation and genetic counselling.

Case Report: Novel rare mutation c.6353C > G in the ABCA12 gene causing harlequin ichthyosis identified by whole exome sequencing.

Background: Harlequin ichthyosis (HI) is a severe rare genetic disease that mainly affects the skin. Neonates with this disease are born with thick skin and large diamond-shaped plates covering most of their bodies. Affected neonates lose the ability to control dehydration and regulate temperature and are more susceptible to infections. They also face respiratory failure and feeding problems. These clinical symptoms are factors associated with high mortality rates of neonates with HI. Until now, there are still no effective treatments for HI patients and most patients die in the newborn period. Mutation in the ABCA12 gene, which encodes an adenosine triphosphate-binding cassette (ABC) transporter, has been demonstrated as the major cause of HI. Case presentation: In this study, we report the case who is one infant that was born prematurely at 32 gestational weeks with the whole body covered with thick plate-like scales of skin. The infant was severely infected with mild edema, multiple cracked skins full of the body, yellow discharge, and necrosis of fingers and toes. The infant was suspected to be affected by HI. Whole exome sequencing (WES) was performed as a tool for detecting the novel mutation in one prematurely born Vietnam infant with HI phenotype. And after that, the mutation was confirmed by the Sanger sequencing method in the patient and the members of his family. In this case, one novel mutation c.6353C > G (p.S2118X, Hom) in the ABCA12 gene, was detected in the patient. The mutation has not been reported in any HI patients previously. This mutation was also found in a heterozygous state in the members of the patient's family, including his parents, an older brother, and an older sister who are no symptoms. Conclusions: In this study, we identified a novel mutation in a Vietnamese patient with HI by whole exome sequencing. The results for the patient and the members of his family will be helpful in understanding the etiology of the disease, diagnosing carriers, assisting in genetic counseling, and emphasizing the need for DNA-based prenatal screening for families with a history of the disease.

Preimplantation genetic testing (PGT) for hemophilia A: Experience from one center.

OBJECTIVE: Hemophilia A (HA) is an X-linked recessive bleeding disease caused by a deficiency or dysfunction of blood coagulation factor VIII (FVIII). Available treatment to replenish the missing factor may not reach a good outcome for all patients because of potential complications that include the development of inhibitor antibodies directed against factor VIII. Therefore, the prevention of transmitting pathogenic mutations to the next generation is the best solution for this disease. Preimplantation genetic testing for a monogenic disorder (PGT-M) has become a standard method to prevent the transmission of monogenic heritable disease. The gold standard of the molecular technique used for PGT-M nowadays is the co-amplification of the polymorphic microsatellite linkage markers that use microsatellite DNA technique that overcomes the limitation of other methods. The important issue of this technique is the definition of markers that are specific for each allele on different loci. Each gene locus needs a characteristic design to allow accurate diagnosis that can be applied on PGT-M due to the limited quantity of DNA available. Here we present our study of four specific self-designed linked polymorphic markers applied on screening the embryos before implantation for hemophilia A families in Vietnam. MATERIAL AND METHODS: In this study, we investigated the feasibility of application and diagnostic value of 4 STR loci (FXS1108, DXS9897, F8int22, DXS9901) in the intragenic or neighbouring regions of the F8 gene. 35 hemophilia A families were recruited for STR analysis to define at least two characteristic heterologous markers for each family and 12 cases of pre-implantation genetic testing (PGT-M) for carrier mothers were performed. RESULT: All 4 of these loci (FXS1108, DXS9897, F8int22, DXS9901) were found practical and useful for preimplantation genetic testing (PGT-M). All 12 cases of PGT-M using the method had informative STR results and correct diagnosis was achieved. 9 out of the 12 mothers (75%) were implanted with 1-2 thawed embryos after the biopsy resulting in the birth of 5 healthy babies (55%). CONCLUSION: We conclude that specific 4 STR markers for rapid pre-implantation genetic testing of hemophilia A can be successfully applied with high confidence and accuracy in clinical settings. The results of the study provide solid evidence confirming that the microsatellite DNA technique is a highly reliable method, suitable for hemophilia A families wishing to determine carrier status or having healthy babies.

Microcephaly primary hereditary (MCPH): Report of novel ASPM variants and prenatal diagnosis in a Vietnamese family.

OBJECTIVE: MCPH (microcephaly primary hereditary) is a group of autosomal recessive developmental disorders with microcephaly present at birth and intellectual disability. Since a second trimester ultrasound is not able to detect subtypes with minimal prenatal presentations, only prenatal diagnosis by genetic testing can confirm these cases and allow for effective genetic counseling, especially a family with a previously affected child. CASE REPORT: A 37-year-old women was pregnant for the third time and had two prior children with profound microcephaly and mental retardation. Targeted panel sequencing identified novel compound heterozygous ASPM pathogenic variants: c.1615_1616del (p. Glu539ArgfsTer15); c.∗293T > A (p. Leu98Ter), which confirmed the diagnosis of MCPH5 (#OMIM 608716). Genetic testing was conducted for family members and applied on prenatal diagnosis. CONCLUSION: This is the first cases of MCPH5 to be reported in Vietnam and the genetic result aided in prenatal diagnosis of a high-risk pregnancy. The study highlights the importance of genetic testing in defining definitive diagnosis which allowed for timely prenatal diagnosis and genetic counseling for the family.

A Novel Nonsense Mutation c.374C>G in CYP21A2 Gene of a Vietnamese Patient with Congenital Adrenal Hyperplasia.

Inactivating mutations of the CYP21A2 gene, encoding for steroid synthesis, have been reported in patients with congenital adrenal hyperplasia (CAH). We report a case of an infant who were diagnosed with CAH and presented with the severe phenotype of CAH with symptoms such as increased testicular volume, elevated of 17-hydroxyprogesteron, testosterone and progesterone. In this study, we established an assay for the detection of unusual genetic in the CYP21A2 gene in the proband and his family. A novel nonsense mutation c.374C > G which caused a substitutions of Serine for a stop codon at codon 125 (p.S125*) within exon 3 was found in the proband. Parental genotype studies confirmed carrier state in the father, but the mother showed a wild allele by PCR and sequencing. This inspired us to find deletions using multiplex ligation-dependent probe amplification (MLPA) technique. The probands were found to have a large deletion in exons 1 and 3, while the mother only had deletion in exon 1. Therefore, mutation c.374C > G (p.S125*) in the proband is considered as a heterozygous deletion. This mutation caused a truncated protein which lead to the salt wasting CAH phenotype of the proband. This novel nonsense mutation expands the CYP21A2 mutation spectrum in CAH disorder. This case also highlights the need of caution when interpreting results of molecular genetic testing during genetic counseling.

Variation of Mitochondrial DNA HV1 AND HV2 of the Vietnamese Population.

BACKGROUND: The sequence polymorphism of mitochondrial DNA (mtDNA) hypervariable segment 1 (HV1) and hypervariable segment 2 (HV2) is studied and applied to genetic diversity and human evolution assessment, forensic genetics, consanguinity determination, and mitochondrial disease diagnosis. METHODS: The study identified the variations of HV1 and HV2 of 517 unrelated Vietnamese individuals in Kinh, Muong, Cham, and Khmer ethnic. We performed sequencing of two hypervariable segments of mitochondrial DNA: HV1 and HV2. RESULTS: Fifty haplogroups were identified in which F1a haplogroup frequency was highest at 15.7%, followed by B5a (10.8%), M (8.9%), and M7b1 (7.7%). The most frequently encountered SNPs in this study were A263G (100%), A73G (99.6%), 315insC (96%), 309insC (56%), C16223T (41%), and T16189C (39%). The genetic diversity was calculated at 99.83%, and the probability of random match of two individuals sharing the same mtDNA haplotype was 0.37%. CONCLUSION: We have assessed the genetic polymorphism of mtDNA HV1 and HV2 of 517 Kinh, Muong, Cham, and Khmer ethnic samples. The result will help in better understanding of Vietnamese's mitochondrial genome diversity and aid in population as well as forensic science.

Assessment of 6 STR loci for prenatal diagnosis of Duchenne Muscular Dystrophy.

OBJECTIVE: Duchenne Muscular Dystrophy is an X-linked recessive disorder characterized by progressive muscular degeneration, patients often develop cardiac failure in the later stage and death occurs before 20 years of age. For a disease with poor postnatal prognosis such as Duchenne Muscular Dystrophy (DMD), providing the carrier mother with the option of prenatal diagnosis in a subsequent pregnancy is accepted practice in many places where termination of pregnancy is allowed. Though methods of direct sequencing such as Sanger's sequencing has been widely used, Next-Generation Sequencing is been increasingly replacing most of its application. For the DMD gene, being the longest gene in the human genome, methods of direct sequencing is often unpractical and time-consuming, instead, STR analysis for linkage analysis would be a cost-effective option and have been used routinely for prenatal diagnosis of DMD. The diagnostic significance of the STRs is based on several criteria, the most important one being the heterozygosity of the locus, power of discrimination (PD) and power of exclusion (PE). MATERIAL AND METHODS: In this study, we investigated the feasibility of application and diagnostic value of 6 STR loci (DSTR49, DSTR50, DXS1036, DXS1067, DXS890, DXS9907) in the proximity of the DMD gene, 66 healthy individuals were recruited for STR analysis and 5 cases of prenatal diagnosis for carrier mother were performed. RESULT: Allele frequency, heterozygosity, polymorphic information content, the power of discrimination and exclusion and Hardy-Weinberg equilibrium were analyzed and calculated for the 6 STR loci. 5 of these loci (DSTR49, DSTR50, DXS1067, DXS890, DXS9907) were found practical and useful for preimplantation Genetic diagnosis (PGD) and prenatal diagnosis. All 5 cases of prenatal diagnosis using the method had informative STR results and correct diagnosis. CONCLUSION: We concluded that our protocol of STR analysis can be applied for prenatal diagnosis and pre-implantation genetic diagnosis of DMD with high confidence and accuracy, especially in clinical settings where diagnostic resources are more limited.

Acromesomelic dysplasia Maroteaux-type in patients from Vietnam.

Acromesomelic dysplasias are rare skeletal disorders leading to severe short stature and abnormal skeletal morphology. Acromesomelic dysplasia Maroteaux-type is caused by homozygous or compound heterozygous pathogenic variants in NPR2 that encodes for natriuretic peptide receptor B. Here, we reported the first AMDM case in South East Asia and identified a novel pathogenic variant in NPR2 (c. 152T>C, p. (Leu51Pro)). Further analyses reveal the parents and two other family members were heterozygous for the variant. The clinical report highlights the importance of molecular genetic testing in diagnosing rare hereditable disease affecting skeletal abnormalities.

Mosaicism in carrier of Duchenne muscular dystrophy mutation - Implication for prenatal diagnosis.

OBJECTIVE: Duchenne muscular dystrophy (DMD) is a severe disorder caused by mutation in the X-linked dystrophin gene, therefor carrier testing is required for all female family members. However, there are cases mutation analysis cannot detect any mutation due to a phenomenon called mosaicism. The case report describes a case of mosaicism in a DMD carrier and discusses the approach in diagnosis and counseling of familial disorder. CASE REPORT: The proband was diagnosed with DMD at age six. Sequencing of Dystrophin gene identified a 2-nucleotide deletion c.2032_2033delCA, p.Q678DfsX41. Family investigation suggested that the mother was an obligate carrier of Dystrophin mutation. Sequencing of DNA sample from the mother's peripheral blood did not reveal any mutation, there for we take sample from hair follicle for analysis. The result indicated that the mother was a carrier but was masked from initial analysis by mosaicsism. CONCLUSION: We suggested that more care need to be taken in identifying cases when no mutation was detected in probable or obligate carrier and prenatal diagnosis should remain an option.

Three novel mutations in the ATP7B gene of unrelated Vietnamese patients with Wilson disease.

BACKGROUND: Wilson disease (OMIM # 277900) is a autosomal recessive disorder characterized by accumulation of copper in liver and brain. The accumulation of copper resulting in oxidative stress and eventually cell death. The disease has an onset in a childhood and result in a significant neurological impairment or require lifelong treatment. Another serious consequence of the disease is the development of liver damage and acute liver failure leading to liver transplant. The disorder is caused by mutations in the ATP7B gene, encoding a P-type copper transporting ATPase. CASE PRESENTATION: We performed genetic analysis of three unrelated patients from three different Vietnamese families. These patients had clinical features such as numbness of hands and feet, vomiting, insomnia, palsy, liver failure and Kayser-Fleischer (K-F) rings and were diagnosed with Wilson disease in the Human Genetics Department, Vietnam National Children's Hospital. The entire coding region and adjacent splice sites of ATP7B gene were amplified and sequenced by Sanger method. Sequencing data were analyzed and compared with the ATP7B gene sequence published in Ensembl (ENSG00000123191) by using BioEdit software to detect mutations. CONCLUSIONS: In this study, five mutations in the ATP7B gene were found. Among of these, three mutations were novel: c.750_751insG (p.His251Alafs*19) in exon 2, c.2604delC (p.Pro868Profs*5) in exon 11, and c.3077 T > A (p.Phe1026Tyr) in exon 14. Our results of the mutations associated with Wilson disease might facilitate the development of effective treatment plans.

Difference in hormone receptor content in breast cancers from Vietnamese and Swedish women.

BACKGROUND: The aim of the present study was to compare both estrogen (ER) and progesterone receptor (PgR) content in operable breast cancers from Vietnamese and Swedish patients. MATERIAL AND METHODS: Primary breast cancer tissues were randomly selected from 249 Vietnamese patients treated in Hanoi, Vietnam between 2002 and 2004 and 1 257 Swedish patients treated in Stockholm, Sweden between 2002 and 2003. Clinical information was available for all patients in the study. The hormone receptor content in tumors from Vietnam was analyzed by immunohistochemistry using an automated slide stainer (Bench MarkXT, Ventana) in combination with anti-ER (SP1 250), and anti-PgR (clone 1E2) rabbit monoclonal antibody. Tumors with ≥ 10% stained nuclei were considered as receptor positive. Tumors from Swedish patients were analyzed with an enzyme immunoassay with a cut-off point of ≥ 0.10 fmol/µg DNA as positive. The hormone receptor frequencies between populations were compared according to clincopathology features. RESULTS: The ER positive rate was higher in premenopausal and lower in postmenopausal Vietnamese patients as compared to Swedish patients with similar menopausal status (71% versus 58%, OR 1.75, p = 0.007; 44% versus 72%, OR 0.32, p < 0.001, respectively). PgR positive tumors were found in 58% and 25% of pre- and postmenopausal Vietnamese patients, respectively. The corresponding figures for Swedish patients were 73% and 66%, respectively. CONCLUSIONS: There were significant differences in the frequency of ER/PgR positivity between Vietnam and Swedish breast cancer patients. These differences were independent on menopausal status and age of patients at diagnosis can not be explained by these factors and they can be contributed to knowledge about both basic biology features and prognoses.

HER2 status in operable breast cancers from Vietnamese women: Analysis by immunohistochemistry (IHC) and automated silver enhanced in situ hybridization (SISH).

BACKGROUND: HER2 amplification has been reported to occur in 19-30% of breast cancers in women from Western countries. Little is known about the HER2 status in breast cancers from Vietnamese women. The aim of this study was to assess the HER2 status in Vietnamese women with operable breast cancer using immunohistochemistry and silver in situ hybridization techniques. MATERIAL AND METHOD: Tissue blocks from 242 Vietnamese women with invasive primary breast carcinoma were stained by immunohistochemistry (IHC) and a dual silver in situ hybridization (SISH) for assessing HER2 status. The analysis followed international recommendations with a semi-quantitative grading of the reaction in four levels, "0", "1+", "2+" and "3+". The HER2 gene amplification was assessed by calculating the ratio of HER2/chromosome 17 in 20 tumor cell nuclei. A ratio of <1.8 was classified as non-amplification and a ratio >2.2 indicated tumors with gene amplification. A ratio between 1.8 and 2.2 was equivocal. RESULTS: Using IHC, 39% of the tumors strongly expressed "3+" the HER2 protein. An intermediate level "2+" of the protein was found in 11% while 50% showed no or low "0/1+" levels of protein expression. Gene amplification was found in 95% and 46% of the tumors with high "3+" and intermediate "2+" levels of protein expression, respectively. Thus, 41% of the patients had tumors with HER2 amplification. The concordance between IHC and SISH was 87%. Gene amplification was more frequent in ER negative than in ER positive tumors, 57% versus 33%, respectively. Additionally, tumors from postmenopausal women were amplified in 55% as compared to 36% in premenopausal women. CONCLUSIONS: HER2 gene amplification was found in 41% of Vietnamese breast cancers; this level is considerably higher than that previously found in a series of Swedish breast cancer women. The HER2-positive tumors were more often found in post-menopausal women than in ER negative tumors.