Current Aspects of Genetic Diagnosis of 21-Hydroxylase Insufficiency

Objective of the Review: To discuss the current peculiarities of 21-hydroxylase insufficiency diagnostics. Key Points. Congenital adrenal hyperplasia (CAH) is a group of autosomal-recessive pathologies, associated with a defective enzyme or transport protein participating in cortisol biosynthesis. Currently, there is information on CAH genetics including information on 21-hydroxylase insufficiency. Neonatal screening, antenatal and postnatal methods to diagnose 21-hydroxylase deficit have been developed. Timely diagnosis and correct therapy have been found to facilitate normal physical and mental development of patients. Molecular genetic testing for CAH associated with 21-hydroxylase deficit is widely used both in Russia and globally and is of importance for differential diagnosis, identification of pathogenic CYP21A2 gene carriers and adequate genetic counselling. The best strategy to diagnose 21-hydroxylase insufficiency is 2-stage CYP21A2 gene analysis. The test should include both gene sequence analysis and identification of point replacements, minor deletions and duplication (e.g., dideoxynucleotide chain-termination method or Next Generation Sequencing), and identification of extended deletions and duplication (multiplex ligation dependent probe amplification, real-time polymerase chain reaction). Such a comprehensive approach can help finding a majority of types of possible changes. Conclusion. Correct genetic testing methods ensure detection of pathogen variants in CYP21A2 gene; evaluation of the possible rate of clinical manifestations of the disease, both during antenatal testing and if an unknown clinical CAH form is encountered; prescribing an adequate therapy; and ensuring genetic counselling in order to develop a management strategy, including when planning for pregnancy in a woman with confirmed CAH. Keywords: congenital adrenal hyperplasia, CYP21A2 gene, genetic counselling, 21-hydroxylase

First Report of the Occurrence of Wheat dwarf virus in Wheat in China

In May of 2004 and 2005, several diseased wheat (Triticum aestivum L.) plants showing extreme dwarfing, various types of yellowing, and reduced or no heading were found in the breeding fields of the Institute of Crop Science, Shanxi Academy of Agricultural Sciences, Taiyuan, Shanxi Province, China. On the basis of these symptoms, infection with Wheat dwarf virus (WDV) was suspected. Total DNA was extracted from diseased plants with the DNeasy Plant Mini kit (Qiagen, Hilden, Germany). Primers were designed based on the WDV-Enkoping1 genome sequence (NC_003326) (1), including: 245f, 5′-CGACTACGCCTGGCGAACATTTG-3′ (residues 245–267); 806r, 5′-TCTGGCATTGCCTGTTTCGG-3′ (complementary to residues 787–806); 1381f, 5′-CAGTGACATCTTCGCCGGAG-3′ (residues 1381–1400); and, 1886r, 5′-ACTCCGTAAGCCTCGAATCC-3′ (complementary to residues 1867–1886). With primer pairs 245f/806r, 1381f/1886r, 245f/1886r, and 1381f/806r, PCR products of 560, 506, 1642, and 2,275 bp were expected, respectively. After amplification, fragments of the expected sizes were seen on 1% (w/v) agarose gels. The fragments were purified by using a DNA gel extraction kit (TaKaRa, Dalian, China) and cloned into the pGEM-T vector (Promega, Madison, WI). The plasmids were transformed into E. coli strain DH5α and plasmid DNA was isolated from overnight cultures by alkaline lysis. Insert sequences were determined using the dideoxynucleotide chain termination method with an automated sequencer (ABI BigDye 3.1, Applied Biosystems, Foster City, CA). At least three independently isolated clones were analyzed for each PCR product. The compiled 2,750 nt sequence (GenBank Accession No. DQ868525) was 98.1, 98.5, 97.8, and 97.9% identical to WDV-Enkoping1 (NC_003326), WDV-SE (X02869), WDV-B (AM040732), and WDV-F (AM040733), respectively. Therefore, the virus isolate (WDV-TY) was identified as WDV (genus Mastrevirus, family Geminividae). Wheat samples collected from different provinces from 2004-2006 were also infected with WDV as indicated by PCR using the same primer pairs. For Shijiazhuang (Hebei Province), Yangling (Shanaxi Province), Kunming (Yunnan Province), Yuncheng (Shanxi Province), Tianshui (Gansu Province), Gangu (Gansu Province), and Zhenzhou (Henan Province), 13 of 14, 6 of 6, 5 of 5, 4 of 4, 2 of 3, 1 of 2, and 1 of 1 samples were positive, respectively, indicating a broad distribution of WDV in China. To our knowledge, this is the first report of WDV in wheat in China. Reference: (1) A. Kvarnheden et al. Arch Virol. 147:205, 2002.

Application of RT-PCR for Indexing Avocado Sunblotch Viroid

A method for the routine detection of avocado sunblotch viroid (ASBVd) in nucleic acid extracts of infected avocado tissues by reverse transcription-polymerase chain reaction (RT-PCR) was developed using ASBVd-specific primers. Amplified cDNA products were analyzed by electrophoresis on nondenaturing 6% polyacrylamide slab gels. The size of the major RT-PCR product from ASBVd-infected tissue was estimated to be 250 bp. This product was absent from amplified extracts of uninfected tissue. The amplification product from ASBVd was sequenced by the dideoxynucleotide chain termination method, and the sequence was over 97% identical to the published sequence. The RT-PCR assay is sensitive enough to allow viroid detection without requiring large amounts of tissue, highly purified ASBVd, or molecular hybridization.