Detection of α-globin gene deletion and duplication using quantitative multiplex PCR of short fluorescent fragments.

BACKGROUND: The aim of this study was to establish a sensitive method that can detect the presence of not only the common but also the unusual or unknown α-globin gene deletions for screening of α-thalassemia. We used quantitative multiplex PCR of short fluorescent fragments (QMPSF) for the α-globin genes (HBA) to screen α-thalassemia deletions. METHODS: We set up and validated HBA-QMPSF using 50 negative and 100 positive controls of deletional α-thalassemia. To evaluate its ability to detect the presence of the common and unusual or unknown α-globin gene deletions, 579 unrelated samples were simultaneously analyzed using this assay and multiplex Gap polymerase chain reaction (Gap-PCR). The inconsistent results were further confirmed by multiplex ligation-dependent probe amplification (MLPA). RESULTS: HBA-QMPSF was capable of detecting α-globin gene deletions with an acceptable variability as shown by mean values (SD) of allele dosage for the heterozygous deleted control obtained from intra- and inter-experimental replicates [0.63 (0.01) and 0.61 (0.03)]. In 572 out of the 579 unrelated subjects, HBA-QMPSF and multiplex Gap-PCR gave consistent results. In seven cases which were finally proved to be composed of one rare deletion--Thai/-α3.7, one novel deletion--SEA/-α2.8, four αααanti3.7/αα and one αααanti4.2/αα triplications, HBA-QMPSF showed deletion or duplication in the α-globin gene while multiplex Gap-PCR failed to give the correct diagnosis. CONCLUSIONS: HBA-QMPSF is able to detect the presence of the common and unusual or unknown α-thalassemia deletions and duplications. It can be used as an initial screening test for α-thalassemia caused by HBA gene copy number alteration.

[Study on clinical manifestation, genotype and genetic characteristics of two Kennedy disease pedigrees].

OBJECTIVE: To investigate the clinical manifestations, genotypes, and genetic characteristics of two pedigrees with Kennedy disease. METHODS: The clinical data of the patients from two Kennedy disease families were collected. The numbers of trinucleotide CAG repeats in exon 1 of the androgen receptor gene were determined by DNA sequencing and repeat fragment analysis. RESULTS: Family A was composed of 58 individuals in 4 generations. The proband had onset at 39 years old. There were two Kennedy disease patients in family B which included 61 individuals in 5 generations. The two patients had onset at 39 and 41 years old, respectively. All the three patients displayed limbs and bulbar muscular weakness because of the damage of lower motor neurons. They had androgen insensitivity syndrome in common, and showed mild or moderate increase in serum creatine kinase level. The electromyogram showed wild damage in anterior horn of spinal cord. Muscle biopsy displayed neurogenic muscular atrophy. The numbers of the CAG repeat expansion in the androgen receptor gene of the three patients were 49, 48, and 47, respectively. X-linked recessive mode of inheritance was demonstrated by pedigree analysis in the two families. CONCLUSION: Kennedy disease usually occurs in mid-adulthood man. The clinical features are the weakness and wasting of limbs and bulbar muscles. Genetic analysis contributes to diagnosis and identification of carriers, and is beneficial to genetic counseling and prenatal diagnosis.