Results
To assay whether the MLPA is able to accurately detect TS cases, we first analyzed a cohort of known mosaic TS patients (“pilot TS group”). Using this group, the ratio able to detect all the mosaicisms and X structural abnormalities was 0.76 at least in one probe (table 1). This ratio permitted to identify also the lowest grade of mosaicism (15%) (mos 45,X[15]/46,XX[85]) among the patients of the pilot TS group. Interestingly, in this population, the MLPA detected Y material presence in two patients (table 1). Applying this 0.76 cut-off in the “TS group”, MLPA did not miss any X aneuploidies or X structural abnormalities represented by X monosomy, mosaicisms 45 X0/46 XX and 45 X0/47 XXX, isochromosome X and deletion of X chromosome. …show more content…
The means of MLPA X probes ratios categorized for karyotype results are shown in table 2. Interestingly, the X monosomy showed mean ratios 1.2 on Xq probes, and the mosaicisms presented high variability of the means of X probes ratios but with means always < 0.76 (table 2). Then, we tested this ratio cut off in the short stature population. Among these patients the MLPA resulted suggestive of X aneuploidies or X structural abnormalities in 10 patients. The karyotype analysis confirmed the diagnosis of Turner syndrome in two patients. The mean of MLPA X probes ratios of the patients resulted true negative to MLPA analysis was about 1 for all the probes (table 3). The MPLA technique did not miss any TS diagnosis and therefore the detection rate appeared to be 100%. The new aneuploidies identified by MLPA were represented by the following karyotypes: 45,X[11]/46,XX[49] and karyotype mos46,X,i(X)(q10)[35]/45,X[15]. Evaluating together TS and short stature group, the ratio of 0.76 in at least one MLPA probe presented a sensibility and specificity of 100% (IC 95%, 0.92-1) and 88.89% (IC 95%, 0.79-0.94), respectively. The MLPA positive predictive value was 88.5% and the negative predictive value …show more content…
However, karyotyping is time consuming and labor-intensive leading to high costs. A standard 30-cell karyotype is recommended to rule out sex chromosome mosaicisms (22). This analysis can identify 10% mosaicism with a confidence level of 95% but a more sensitive level of detection requires analysis of many metaphase cells with further increase of the costs (22). In the last decade, cell-free molecular techniques have become available for rapid aneuploidy detection of the most common chromosome abnormalities (aneuploidies of chromosomes X, Y, 13, 18 and 21), such as QF-PCR or array-CGH. Multiplex Ligation-dependent Probe amplification (MLPA) is a rapid high-throughput technique shown to be robust, cost-effective and flexible. A standard MLPA probe set has been designed to detect aneuploidies of chromosomes X, Y, 13, 18 and 21. We tested it in the routine diagnostics of TS, due to its equivalent accuracy at lower costs (11, 16). Compared to QF-PCR, MLPA has the advantage of detecting over 40 different loci per reaction (23). This is very important for TS, since partial X-chromosome imbalances and mosaicisms make very difficult a cell-free DNA test. MLPA has this power, by increasing the probe number and the quality of results, and it is much cheaper than other techniques such as
To assay whether the MLPA is able to accurately detect TS cases, we first analyzed a cohort of known mosaic TS patients (“pilot TS group”). Using this group, the ratio able to detect all the mosaicisms and X structural abnormalities was 0.76 at least in one probe (table 1). This ratio permitted to identify also the lowest grade of mosaicism (15%) (mos 45,X[15]/46,XX[85]) among the patients of the pilot TS group. Interestingly, in this population, the MLPA detected Y material presence in two patients (table 1). Applying this 0.76 cut-off in the “TS group”, MLPA did not miss any X aneuploidies or X structural abnormalities represented by X monosomy, mosaicisms 45 X0/46 XX and 45 X0/47 XXX, isochromosome X and deletion of X chromosome. …show more content…
The means of MLPA X probes ratios categorized for karyotype results are shown in table 2. Interestingly, the X monosomy showed mean ratios 1.2 on Xq probes, and the mosaicisms presented high variability of the means of X probes ratios but with means always < 0.76 (table 2). Then, we tested this ratio cut off in the short stature population. Among these patients the MLPA resulted suggestive of X aneuploidies or X structural abnormalities in 10 patients. The karyotype analysis confirmed the diagnosis of Turner syndrome in two patients. The mean of MLPA X probes ratios of the patients resulted true negative to MLPA analysis was about 1 for all the probes (table 3). The MPLA technique did not miss any TS diagnosis and therefore the detection rate appeared to be 100%. The new aneuploidies identified by MLPA were represented by the following karyotypes: 45,X[11]/46,XX[49] and karyotype mos46,X,i(X)(q10)[35]/45,X[15]. Evaluating together TS and short stature group, the ratio of 0.76 in at least one MLPA probe presented a sensibility and specificity of 100% (IC 95%, 0.92-1) and 88.89% (IC 95%, 0.79-0.94), respectively. The MLPA positive predictive value was 88.5% and the negative predictive value …show more content…
However, karyotyping is time consuming and labor-intensive leading to high costs. A standard 30-cell karyotype is recommended to rule out sex chromosome mosaicisms (22). This analysis can identify 10% mosaicism with a confidence level of 95% but a more sensitive level of detection requires analysis of many metaphase cells with further increase of the costs (22). In the last decade, cell-free molecular techniques have become available for rapid aneuploidy detection of the most common chromosome abnormalities (aneuploidies of chromosomes X, Y, 13, 18 and 21), such as QF-PCR or array-CGH. Multiplex Ligation-dependent Probe amplification (MLPA) is a rapid high-throughput technique shown to be robust, cost-effective and flexible. A standard MLPA probe set has been designed to detect aneuploidies of chromosomes X, Y, 13, 18 and 21. We tested it in the routine diagnostics of TS, due to its equivalent accuracy at lower costs (11, 16). Compared to QF-PCR, MLPA has the advantage of detecting over 40 different loci per reaction (23). This is very important for TS, since partial X-chromosome imbalances and mosaicisms make very difficult a cell-free DNA test. MLPA has this power, by increasing the probe number and the quality of results, and it is much cheaper than other techniques such as