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Detection of Turner Syndrome Using High-Throughput Quantitative Genotyping

Section of Perinatal Medicine (H.M., J.R.G.), W. M. Keck Biotechnology Resource Laboratory (K.H.), and Section of Developmental Endocrinology and Biology (S.A.R.), Yale University School of Medicine, New Haven, Connecticut 06520

Address all correspondence and requests for reprints to: Scott A. Rivkees, M.D., Yale Pediatrics, Yale Child Health Research Center, 464 Congress Avenue, New Haven, Connecticut 06520. E-mail: scott.rivkees{at}yale.edu.

	Abstract

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Context: Turner syndrome (TS) is the most common genetic problem affecting women and occurs when an X chromosome is completely deleted, portions of an X chromosome are deleted, or chromosomal mosaicism occurs. Girls with TS may also have occult Y chromosome sequences. Whereas some girls with TS are identified in infancy or early childhood, many girls with TS are not detected until after 10 yr of age, resulting in delayed evaluation and treatment.

Objective: To prevent the delayed recognition and treatment of TS, a quantitative method of genotyping that can be performed as part of newborn screening is needed.

Design: To screen for sex chromosome abnormalities, we assembled a panel of informative single nucleotide polymorphism (SNP) markers that span the X chromosome from the dbSNP database. Pyrosequencing assays suitable for quantitative assessment of signal strength from single nucleotides were designed and used to genotype 46,XX; 46,XY; 45,X; and TS mosaics, examining zygosity and signal strength for individual alleles. Pyrosequencing assays were also designed for the detection of Y chromosome material.

Results: With just four informative SNP markers for the X chromosome, all TS girls with 45,X, partial X chromosome deletions, or mosaicism were identified with 100% sensitivity. In mosaic individuals, Y chromosomal material was detected with 100% sensitivity.

Conclusion: These results suggest that inexpensive high-throughput screening is possible for TS and other sex chromosome disorders using quantitative genotyping approaches.

	Introduction

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TURNER SYNDROME (TS) is the most common genetic condition affecting women (1, 2, 3). The incidence of TS is one in 1500–2000 live female births (1, 2, 3) and occurs when an entire or a portion of an X chromosome is deleted (1, 3). Phenotypic features include primary hypogonadism, renal abnormalities, and structural cardiac problems (1, 3). Girls with TS are short and have an average adult height of 4 ft 6 in. tall (3, 4, 5). Yet with timely initiation of GH therapy, acceptable adult stature can be achieved (5, 6).

Many girls with TS are diagnosed after 10 yr of age (7, 8), and recognition of associated problems may be delayed (9). Final height may be compromised by delayed adjunctive therapy with GH (9). With later recognition, replacement therapy with estrogen and progestin is delayed resulting in late pubertal development (9, 10).

Girls with TS are at risk for gonadal tumor development if Y chromosomal material is present (11, 12). The failure to detect small fragments of Y chromosomal material by standard karyotype analysis (13) precludes recognition of girls with TS with potential tumor risk.

The gold standard for diagnosis of X chromosome aneuploidies remains cytogenetic analysis (karyotype) (13). Whereas cytogenetic analysis by light microscopy has drastically advanced in resolution over the past 50 yr, it remains a labor-intensive and expensive method that is not practical for population screening. Testing of blood spot FSH during early postnatal life has been tested in girls with TS (14). However, perinatal changes in FSH secretion are similar to those in normal girls; thus, FSH measurement will not be effective for neonatal screening of TS (14).

Over the past decade, genotyping techniques have become faster and less expensive. We postulate that a quantitative method of genotyping based on the detection of single nucleotide polymorphisms (SNPs) may prove to be advantageous in identifying girls with TS. SNPs occur about every 100 nucleotide bases (15, 16). There are thousands of SNPs available to interrogate the full length, or any specific segment, of the X and Y chromosomes (15, 16). Pyrosequencing is especially advantageous for detecting SNPs due to a high degree of quantitative accuracy, ease of use, and high throughput capability (15). Thus, we have developed a novel strategy for detecting sex chromosome abnormalities using informative SNP markers spanning the X and Y chromosomes, followed by quantitative assessment of allele signal strength via pyrosequencing.

	Materials and Methods

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DNA samples

DNA samples were obtained from the human genetic cell repository of the National Institute of General Medical Sciences (National Institutes of Health) maintained at the Coriell Institute for Medical Research (Camden, NJ). The karyotype of each sample was determined by the Coriell Institute (see Table 1 for karyotypes).

Pyrosequencing was used to genotype each genomic DNA sample for SNP markers. Oligonucleotide primer pairs were designed for the PCR amplification of unique DNA flanking each SNP. The reverse primer for all pairs was synthesized with a 5'-T3 tag sequence extension (5'-ATTAACCCTCACTAAAGGGA-3'). In addition to the forward and reverse PCR primers, a universal 5'-biotinylated T3 primer (5'-ATTAACCCTCACTAAAGGGA-3') was added to each PCR. Amplicon sizes ranged from for 88–210 and 168–493 bp for the X chromosome and Y chromosome markers. Biotinylated PCR products were generated in a 20-µl PCR containing 10 ng genomic DNA, 0.4 U Hotstart Taq polymerase (QIAGEN, Valencia CA), 4 pmol of forward PCR primer, 0.4 pmol of reverse PCR primer, 3.6 pmol of biotinylated T3 primer, 2.5 mM MgCl₂, and 200 µM each of dATP, dCTP, dGTP, and dTTP. Thermal cycling was conducted in a 96-well PCR block (Applied Biosystems, Foster City, CA; 15 min at 95 C; 45 cycles of 30 sec at 95 C, 45 sec at 56 C, 60 sec at 72 C; 5 min at 72 C; and a hold at 4 C). Upon completion of PCR, the biotinylated PCR product from the entire reaction was purified by binding to Streptavidin-Sepharose (Amersham, Piscataway, NJ) using a standard protocol (Biotage AB; www.pyrosequencing.com). The resulting single-stranded template was annealed with the extension primer for 2 min at 80 C, cooled to room temperature, and sequenced in a PSQ96MA pyrosequencing instrument (Biotage AB, Uppsala, Sweden). PSQ96MA analysis software (version 2.0.2) automatically scored the quality of each reaction, assigned genotypes, and measured the peak heights of each allele. Genotype and signal strength was exported to standard spreadsheet format. The person who did the pyrosequencing and related analysis was blinded to genotype. After data were tabulated, it was compared with provided genotypes.

Statistics

Mean ± SD values were calculated using GraphPad Prism (San Diego, CA).

	Results

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Development of a panel of SNP markers

To develop a set of SNP markers for TS screening, 22 SNPs with average heterozygosity greater than 25% spanning the X chromosome from Xp22 to Xq28 were selected from the dbSNP database (National Library of Medicine; www.ncbi.nlm.nih.gov/dbSNP). Similarly, a panel of eight SNPs spanning the Y chromosome was selected (Fig. 1).

View larger version (31K): [in this window] [in a new window]   FIG. 1. Location of X and Y chromosome markers.

To assess both qualitative heterozygosity and quantitative signal strength at each SNP, genotyping was performed by pyrosequencing of DNA samples from nine 46,XX females and eight 46,XY males from the Center d’Etude du Polymorphisme Humain pedigree 1331. For each biallelic SNP marker, the frequencies of the three possible genotypes were determined: A/B, A/A, and B/B.

Overall, 17 of the 22 markers were heterozygous (column A/B) in at least one 46,XX subject. Of these 17, one marker was from a known pseudoautosomal region of the X chromosome (marker 1), and one marker behaved like it resides in a pseudoautosomal region, although Blast searching against the reference human genome sequence produced only one significant match (marker 16). Thus, at least 15 SNP markers, widely distributed over the X chromosome, would be informative for interrogating nonmosaic 45,X TS subjects.

To identify TS mosaics, we determined the ratios of A-allele and B-allele signal strength for each marker relative to three possible genotype outcomes: A+B allele equally present (expected: A50%/B50%), A-allele present only (expected: A100%/B0%), and B-allele present only (expected: A0%/B100%).

When A/B alleles were both present (exclusive of markers 1 and 16), the relative ratio of signal strength from each allele was 50.5 ± 2.5% (mean ± SD). When both alleles are present, a ratio of greater than or less than 7.5% from the mean (<43 or >58) represents 3 SD. When only the A-allele was present (A100%/B0%), the mean ratio was 99.8 ± 0.1%. When only the B-allele was present (A0%;B100%), the mean ratio was 100 ± 0. Based on this analysis, we found that we could detect two alleles in 46,XX individuals using markers 2, 4, 5, 8, 9, 10, 11, 13, 14, 17, 18, 19, 20, and 22.

Assessment of sensitivity for detecting TS

To test the utility of our X chromosome marker panel in identifying TS, a collection of 25 DNA samples from subjects with TS and other sex chromosome abnormalities was assembled from the National Institute of General Medical Sciences (Table 1) and genotyped by pyrosequencing. First, the ability of the marker panel to detect loss of heterozygosity (LOH) in nonmosaic 45,X TS samples was assessed. Table 1 shows that for the three 45,X samples tested (rows 6–8), there was not a single heterozygote genotype. The odds that no heterozygote genotype could be detected in 46,XX females with 15 consecutive X chromosome markers is 1: (1–0.3)¹⁵ = 1:4.75 x 10^–3 (or about 1 in 200), assuming a heterozygosity value of 0.3 for each marker.

Next, the ability of the marker panel to detect TS mosaics by quantifying the relative signal strength of each SNP allele was examined. When a variety of different TS mosaic DNA samples were interrogated with the X-chromosome SNP panel, it was found that 18 of the 22 markers identified TS mosaicism as defined by a difference of more than 3 SD in relative allele signal strength (Table 1). A combination of a minimum of just four markers (for example 11, 14, 18, and 19) identified 100% of the 13 samples with TS mosaicism (rows 9–21).

Assessment of Y chromosomal material

In addition to testing for X chromosome SNPs, eight Y chromosome markers were tested in 22 46,XY males and seven 46,XX females and in all aneuploid samples. All of the eight Y chromosome SNP markers detected Y sequences in the control males (100% sensitivity); none of the Y chromosome SNP markers detected Y sequences in the control females (100% selectivity). Using just three Y chromosome markers, we detected Y chromosomal sequence in each of the TS subjects known to have Y chromosome material by karyotype (100% sensitivity).

	Discussion

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Many girls with TS are not detected until they are teenagers. The penalty of untimely diagnosis includes delayed treatment and less favorable outcome (1, 10, 17). Our observations show that high-throughput screening for TS using qualitative and quantitative genotyping by pyrosequencing is possible.

Quantitative fluorescent PCR, which is based on the PCR amplification of selected chromosome-specific short tandem nucleotide repeats, has been adapted for the assessment of X chromosome aneuploidies (18). However, this method has limited ability to differentiate quantitative differences in signal strength from different alleles. Thus, the 50% of TS girls with X chromosome mosaicism or partial deletions will not be identified using this approach. In comparison, we show that using pyrosequencing to genotype SNPs provides a comprehensive diagnostic screening strategy that will identify all causes of TS, including mosaicism and partial deletions.

Of the 22 X chromosome markers tested, 14 markers are informative because they identified polymorphisms in nine 46,XX individuals. When two alleles were both present, the ratio of signal strength for both alleles was 50.5 ± 2.5% (mean ± SD). By defining a relative allele difference of more than 3 SD (7.5%) as abnormal, a minimum combination of just four markers (i.e. 11, 14, 18, and 19) identified 100% of girls with TS mosaicism. It was also possible to identify all 45,X individuals because all markers spanning the length of the chromosome showed loss of heterozygosity.

The Y chromosome markers tested were highly effective as well. Using Y chromosome markers, we identified all 46,XY males. We also detected Y chromosomal material in each of the TS individuals known to have Y chromosomal material by karyotype analysis.

We believe that our SNP panels combined with quantitative genotyping has the potential to identify many different kinds of X and Y chromosomal problems, not just TS; these include 47,XXY causing Klinefelter syndrome, translocations, ring chromosome, marker chromosomes, inversions, insertions, isochromes, duplications, dicentric chromosomes, derivative chromosomes, deletions, and complex aneuploidies. Thus, whereas our primary focus is on detecting TS, this testing approach will identify other disorders of sex chromosomes, including 46,XY females and 46,XX males. As such, neonatal screening for TS and other complex conditions involving sex chromosomes and sexual differentiation is now possible using a quantitative genotyping approach.

Future studies are therefore indicated to test the utility of quantitative genotyping in newborn screening, of TS.

	Footnotes

 
First Published Online March 29, 2005

Abbreviations: LOH, Loss of heterozygosity; SNP, single nucleotide polymorphism; TS, Turner syndrome.

Received March 11, 2005.

Accepted March 21, 2005.

	References

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