[Hemoglobin H disease with a rare α-thalassemia gene mutation (--SEA/α*92A>Gα): pedigree analysis and genetic diagnosis].

OBJECTIVE: To identify a rare α-thalassemia gene mutation in a family from south China and perform a pedigree analysis and genetic diagnosis of hemoglobin H (HbH) disease caused by this mutation. METHODS: Peripheral blood samples were collected from the family members for analysis of the hematological phenotype and routine test of thalassemia genes. DNA sequencing was carried out for samples that showed genotype and phenotype inconsistency. RESULTS: A rare α-thalassemia *92A>G gene mutation was detected within this family. The proband and his sister were confirmed to have non-deletional HbH disease with α--SEA/α*92A>Gα genotype. The proband's brother was confirmed to have an α-thalassemia trait with the genotype of -α3.7/α*92A>Gα. The proband's father was identified as an α-thalassemia silent carrier with the genotype of αα/α*92A>Gα. CONCLUSION: A rare α-thalassemia *92A>G gene mutation was identified for first time in south China. The description of the basic phenotypic characteristics of α-thalassemia trait and silent carrier caused by this mutation enriches the α-thalassemia gene mutation spectrum in Chinese population and helps in population screening, clinical molecular diagnosis and genetic counseling.

Rapid detection of Down's syndrome using quantitative real-time PCR (qPCR) targeting segmental duplications on chromosomes 21 and 11.

OBJECTIVE: Development of a qPCR test for the detection of trisomy 21 using segmental duplications. METHODS: Segmental duplications in the TTC3 gene on chromosome 21 and the KDM2A gene on chromosome 11 were selected as molecular markers for the diagnostic qPCR assay. A set of consensus primers selected from the conserved regions of these segmental duplications were used to amplify internal diverse sequences that were detected and quantified with different probes labeled with distinct fluorescence. The copy numbers of these two fragments were determined based on the ΔCq values of qPCR. The results of qPCR for prenatal and neonatal screening of Down's syndrome were compared with the conventional karyotype analysis by testing 82 normal individuals and 50 subjects with Down's syndrome. RESULTS: The ΔCq values of segmental duplications on chr21 and 11 ranged between 0.33 and 0.75 in normal individuals, and between 0.91 and 1.18 in subjects with Down's syndrome. The ΔCq values of these two segmental duplications clearly discriminated Down's syndrome from normal individuals (P<0.001). Furthermore, the qPCR results were consistent with karyotype analysis. CONCLUSION: Our qPCR can be used for rapid prenatal and neonatal screening of Down's syndrome.

Rapid diagnosis of aneuploidy using segmental duplication quantitative fluorescent PCR.

The aim of this study was use a simple and rapid procedure, called segmental duplication quantitative fluorescent polymerase chain reaction (SD-QF-PCR), for the prenatal diagnosis of fetal chromosomal aneuploidies. This method is based on the co-amplification of segmental duplications located on two different chromosomes using a single pair of fluorescent primers. The PCR products of different sizes were subsequently analyzed through capillary electrophoresis, and the aneuploidies were determined based on the relative dosage between the two chromosomes. Each primer set, containing five pairs of primers, was designed to simultaneously detect aneuploidies located on chromosomes 21, 18, 13, X and Y in a single reaction. We applied these two primer sets to DNA samples isolated from individuals with trisomy 21 (n = 36); trisomy 18 (n = 6); trisomy 13 (n = 4); 45, X (n = 5); 47, XXX (n = 3); 48, XXYY (n = 2); and unaffected controls (n = 40). We evaluated the performance of this method using the karyotyping results. A correct and unambiguous diagnosis with 100% sensitivity and 100% specificity, was achieved for clinical samples examined. Thus, the present study demonstrates that SD-QF-PCR is a robust, rapid and sensitive method for the diagnosis of common aneuploidies, and these analyses can be performed in less than 4 hours for a single sample, providing a competitive alternative for routine use.

Rapid diagnosis of aneuploidy in chromosomes 13, 18, 21, X and Y by quantitative fluorescence-PCR combined with short tandem repeat and fluorescence-labeled homologous gene quantitative­PCR using 4-color fluorescently labeled universal primers.

The present study aimed to develop a rapid diagnostic test of aneuploidy in chromosomes 13, 18, 21, X and Y through a program combining short tandem repeat (STR) typing with fluorescence-labeled homologous gene quantitative­polymerase chain reaction (fHGQ-PCR), which avoids misjudgment risks by using one method alone. Furthermore, fluorescently labeled universal primers not only ensure the accuracy of the results but also reduces the cost of fluorescent labels. The verification of DNA extracted from samples confirmed by karyotype analysis with quantitative fluorescence (QF)-PCR shows that the results obtained using the QF-PCR program are consistent with the results of karyotype analysis in rapidly diagnosing the aneuploidy of chromosomes 13, 18, 21, X and Y.

Detection of three common α-thalassemia in non-deletion types and six common thalassemia in deletion types by QF-PCR.

OBJECTIVE: Thalassemia is one of the most common monogenic hereditary diseases in tropical and subtropical regions. An effective way to avoid the birth of severe thalassemia patients is to strengthen the thalassemia screening of couples before wives are pregnant. Thalassemia gene carriers can be diagnosed by molecular biology in order to conduct effective guidance for fertility. DESIGNS AND METHODS: For --(SEA) and --(THAI) of α-thalassemia and HPFH-SEA and DBT of ß-thalassemia, we design the fGap-PCR primer; for α(CS)α, α(QS)α and α(WS)α, we design the fAS-PCR primer; for -α(3.7)and -α(4.2), we design the QF-PCR primer; and lastly, we use universal primers and multiple-tailed primers to make a single-tube QF-PCR system. RESULTS: When the QF-PCR system is used to diagnose 123 screening samples of thalassemia genotyping, the typing result is consistent with conventional diagnosis of Gap-PCR and PCR-RDB. CONCLUSIONS: Compared with conventional Gap-PCR and PCR-RDB, this QF-PCR system is easy to operate, has high precision, and can diagnose genotypes in a large scale. Its automatic operation is more suitable for the large-scale screening of the thalassemia gene.

[Construction and identification of the chromosome specific DNA library.].

The objective of the present study was to construct a human chromosome 21 specific DNA library for further use in research of genetic disease. Human chromosome 21 microdissected from the peripheral blood cells were subjected to repeatedly incubation in gradient temperature bath to release DNA. The library of chromosome 21 was constructed using the DNA fragment of 100-500 bp and 500-2 000 bp recovered from the products of DOP-PCR. Florescence in situ hy-bridization (FISH) and dot blotting analyses were carried out to assess the chromosome 21 specificity of the DNA library. The results indicated that DNA of chromosome 21 was released easily after repeatedly incubation in gradient temperature bath. Recovery of DNA fragments from DOP-PCR in different size ranges improved the efficiency of cloning of large fragments. Both FISH and dot blotting analyses revealed that the DNA library constructed in this study was chromosome 21-specific. This DNA library facilitates identification and investigation of the chromosome 21 related abnormality.