title: Molecular diagnostic testing for Klinefelter syndrome and other male sex chromosome aneuploidies.
abstract: BACKGROUND
  Male sex chromosome aneuploidies are underdiagnosed despite concomitant physical and behavioral manifestations.
  
  
  OBJECTIVE
  To develop a non-invasive, rapid and high-throughput molecular diagnostic assay for detection of male sex chromosome aneuploidies, including 47,XXY (Klinefelter), 47,XYY, 48,XXYY and 48,XXXY syndromes.
  
  
  METHODS
  The assay utilizes three XYM and four XA markers to interrogate Y:X and X:autosome ratios, respectively. The seven markers were PCR amplified using genomic DNA isolated from a cohort of 323 males with aneuploid (n = 117) and 46,XY (n = 206) karyotypes. The resulting PCR products were subjected to Pyrosequencing, a quantitative DNA sequencing method.
  
  
  RESULTS
  Receiver operator characteristic (ROC) curves were used to establish thresholds for the discrimination of aneuploid from normal samples. The XYM markers permitted the identification of 47,XXY, 48,XXXY and 47,XYY syndromes with 100% sensitivity and specificity in both purified DNA and buccal swab samples. The 48,XXYY karyotype was delineated by XA marker data from 46,XY; an X allele threshold of 43% also permitted detection of 48,XXYY with 100% sensitivity and specificity. Analysis of X chromosome-specific biallelic SNPs demonstrated that 43 of 45 individuals (96%) with 48,XXYY karyotype had two distinct X chromosomes, while 2 (4%) had a duplicate X, providing evidence that 48,XXYY may result from nondisjunction during early mitotic divisions of a 46,XY embryo.
  
  
  CONCLUSIONS
  Quantitative Pyrosequencing, with high-throughput potential, can detect male sex chromosome aneuploidies with 100% sensitivity.
authors.affiliation: JS Genetics, Inc, 2 Church St, South, B-05, New Haven, CT, 06519, USA. karl.hager@jsgenetics.com.
authors.forename: Karl
authors.initials: K
authors.lastname: Hager
authors.forename: Kori
authors.initials: K
authors.lastname: Jennings
authors.forename: Seiyu
authors.initials: S
authors.lastname: Hosono
authors.forename: Susan
authors.initials: S
authors.lastname: Howell
authors.forename: Jeffrey R
authors.initials: JR
authors.lastname: Gruen
authors.forename: Scott A
authors.initials: SA
authors.lastname: Rivkees
authors.forename: Nicole R
authors.initials: NR
authors.lastname: Tartaglia
authors.forename: Henry M
authors.initials: HM
authors.lastname: Rinder