Detection of increased gene copy number in DNA from dried blood spot samples allows efficient screening for Klinefelter syndrome

47, XXY Klinefelter syndrome (KS) is the most common sex chromosome disorder affecting one in 660 newborn boys (1). Adolescent and adult patients with KS are characterized by hypergonadotropic hypogonadism, tall stature with eunuchoid body proportions, increased truncal fat and small testes, whereas no specific clinical or physical hallmarks have been identified at birth or during infancy and childhood (2,3). However, the adult phenotype varies greatly, and the symptoms of KS are not exclusive, and, possibly therefore, the syndrome is highly underdiagnosed. In Denmark, <10% of the expected number of boys are diagnosed before puberty, and only approximately 25% of adults with KS are aware of the diagnosis (1). Hence, up to 75% of patients with KS remain undiagnosed. The fate of the undiagnosed majority of men with KS is still subject of much speculation (4). At an International Workshop on KS held in Copenhagen, Denmark, in May 2010, the pros and cons of postnatal screening for KS were discussed (4). There was a general agreement among the participants that to offer the best surveillance and treatment to the KS patients, a large population-based screening study was needed. However, social, legal and ethical issues, and the potential consequences of diagnosis, positive and negative, should be considered thoroughly before such a study can be performed. In the Danish Newborn Screening Biobank (DNSB), dried blood spot cards from the national neonatal screening program have been stored since 1982. Today, the DNSB contains almost 2 million dried blood spot samples, or over 99.9% of all babies born in Denmark since 1982. Samples from this biobank have been shown to represent a reliable resource of DNA for whole-genome amplification and subsequent genome-wide association studies (5). The aim of this study was to test whether detection of KS by a quantitative PCR (qPCR)-based method is possible in DNA extracted from dried blood spot samples. As we report here, the method has proven reliable and can potentially be used for neonatal screening for KS. Dried blood spot cards from the DNSB were identified from 50 patients with diagnosed nonmosaic 47,XXY KS and 97 aged-matched male controls. One 3.2-mm disk (diameter) was punched from the dried blood spot card of each included sample. DNA was extracted using the Extract-N-Amp Blood PCR kit (SigmaAldrich, St. Louis, MO, USA) using the standard protocol. The extracted DNA was hereafter stored at )20 C until use. Subsequently, all samples were re-purified on semiautomatic Maxwell 16 system using LEV Blood and Buccal Swab DNA Purification Kit AS 1290 (Promega, Madison, WI, USA), according to manufacturers’ protocols, except that no Proteinase K or cell lysis treatment was used. In addition, two internal laboratory controls (XY and XX) were used, and DNA was extracted from blood spotted on filter paper, using the Extract-N-Amp Blood PCR kit (Sigma-Aldrich) or QuickGene DNA Tissue Kit (Fuji Photo Film, Tokyo, Japan). No differences in the performance of the DNA samples purified with the two protocols were observed in the quantitative PCR analysis. QPCR with primers for two X-chromosome mapped genes, the AR (Xq) and SHOX (Xp ⁄ Yp), and one autosomal reference gene, GAPDH (12p), was performed on the Abbreviations DNSB, Danish Newborn Screening Biobank; KS, Klinefelter syndrome; LH, Luteinizing hormone; qPCR, Quantitative PCR. Acta Pædiatrica ISSN 0803–5253