A Highly Sensitive, High-Throughput Assay for the Detection of Turner Syndrome

Turner syndrome (TS) is estimated to occur in about 1 in 1500 to 2000 female live births. It occurs as a result of deletion of an entire X chromosome (45X), of a portion of an X chromosome (46XdelX), or of the X chromosome in a subset of cells (TS mosaicism). With early diagnosis, normal or near-normal adult stature can be achieved with initiation of growth hormone therapy, and late onset of pubertal development can be avoided with estrogen and progestin. Because 1 in 50 to 100 girls with short stature have TS, testing for this condition among short girls (≤5th percentile) is recommended. However, diagnosis is delayed in a high proportion of such girls until ≥10 years of age, and many cases are not identified until 7 years after clear clinical evidence of short stature. Accordingly, the resulting delay of intervention with growth hormone or steroid therapy compromises final adult height and delays onset of puberty. The standard test used to diagnose TS, karyotype testing is labor-intensive and not useful for large-scale population or high-throughput testing. Previous studies have shown that a quantitative, pyrosequencing-based method for TS detection based on the genotyping of single-nucleotide polymorphisms (SNPs) has potential as an alternative to karyotype testing. The test was found to be accurate with high specificity and sensitivity, easy to use, and rapid. However, the DNA used in these studies was obtained from only a modest number of cell lines. The aim of this study was to confirm the clinical usefulness of this test and to establish its specificity and sensitivity among a large population of control subjects and individuals with karyotype-confirmed TS. For genotyping, pyrosequencing was used to quantitate relative allele strength (RAS) from SNPs using 18 informative SNP markers located on the X-chromosome and 1 Y-chromosome marker. Marker RAS cutoff ranges were established for genomic DNA from a cohort of 217 males and 282 females based on homozygous and heterozygous genotypes. Marker value outliers not falling within either homozygous or heterozygous ranges were called out-of-range. Diagnostic criteria for a positive TS score in an individual were defined as complete homozygosity for all 18 markers or the presence of at least 1 out-of-range RAS value. The validity of the test for identifying TS was determined using DNA from 132 females without TS (known 46XX karyotypes) and 74 females with TS (known karyotypes). The sensitivity and specificity of the TS assay in this cohort were 96% and 97%, respectively. Analysis of buccal swab DNA obtained from 19 females without TS and 69 with TS showed that the test identified TS in this group with 97.1% sensitivity and 84.2% specificity. The sensitivity of the assay for detection of individuals with 45X, mosaic, and deletion karyotypes ranged from 86% to 100%. The findings suggest that this high-throughput, pyrosequencing-based assay can detect TS with a high degree of specificity and sensitivity, and has potential as an alternative to karyotype testing.