Simple, Rapid and Inexpensive Quantitative Fluorescent PCR Method for Detection of Microdeletion and Microduplication Syndromes

<div><p>Because of economic limitations, the cost-effective diagnosis of patients affected with rare microdeletion or microduplication syndromes is a challenge in developing countries. Here we report a sensitive, rapid, and affordable detection method that we have called Microdeletion/Microduplication Quantitative Fluorescent PCR (MQF-PCR). Our procedure is based on the finding of genomic regions with high homology to segments of the critical microdeletion/microduplication region. PCR amplification of both using the same primer pair, establishes competitive kinetics and relative quantification of amplicons, as happens in microsatellite-based Quantitative Fluorescence PCR. We used patients with two common microdeletion syndromes, the Williams-Beuren syndrome (7q11.23 microdeletion) and the 22q11.2 microdeletion syndromes and discovered that MQF-PCR could detect both with 100% sensitivity and 100% specificity. Additionally, we demonstrated that the same principle could be reliably used for detection of microduplication syndromes, by using patients with the Lubs (<i>MECP2</i> duplication) syndrome and the 17q11.2 microduplication involving the <i>NF1</i> gene. We propose that MQF-PCR is a useful procedure for laboratory confirmation of the clinical diagnosis of microdeletion/microduplication syndromes, ideally suited for use in developing countries, but having general applicability as well.</p></div

Schematic overview of MQF-PCR primer selection for Williams-Beuren and Velocardiofacial syndromes critical regions (not drawn to scale).

<p>A) Williams-Beuren critical region at 7q11.23 contains centromeric (white arrow), middle (grey arrow), and telomeric (black arrow) blocks of low copy repeats. The minimal critical region in this study represents the region that is flanked by the inner block of low copy repeats. Sequence similarity search identified a region that can be amplified using the LIMK1-MQF primers (bold) and has significant similarity to homologous region at 18p11.32. B) Multiple sequence alignment of the LIMK1-MQF region with its homologous region on chromosome 18. A deletion of 2 bp (dashed box) differentiates the two fragments amplified using the same primer pair. C) Velocardiofacial critical region at 22q11.2 is delimited by 4 blocks of low copy repeats. The minimal critical region in this study represents the region that is flanked by two blocks of low copy repeats most proximal to the centromere (black boxes). Sequence similarity search identified a region within the critical region that can be amplified using the VCF-MQF primers (bold) with significant similarity to a homologous region at 3p11.1. D) Multiple sequence alignment of the VCF-MQF region with its homologous region on chromosome 3. A deletion of 5 bp (dashed box) differentiates the two fragments amplified using the same primer pair. Microsatellite markers (italicized) and Real-Time PCR primers used in molecular diagnosis of patients are shown in both panels.</p

MQF-PCR results for patients with microdeletion and microduplication syndromes.

<p>MQF-PCR results for patients with microdeletion and microduplication syndromes.</p

Evaluation of MQF-PCR in detection of Williams-Beuren and Velocardiofacial syndromes.

<p>A–D) Representative electropherograms showing the peak areas corresponding to the syndrome-related chromosomes (black) that are reduced by about 50% in comparison with the peaks representing the control chromosomes (white) between controls and affected individuals. Electropherogram depicting change in peak area between chromosome 7 and its control chromosome in normal (A) and individual with WBS syndrome (B). Electropherogram depicting change of the peak area between chromosome 22 and its control chromosome in normal (C) and individual with VCF syndrome (D). E–F) Interactive dot diagrams of ROC curve analysis of Z scores in WBS (E), and PZ scores in VCF syndrome (F). Both diagnostic primers achieved 100% diagnostic sensitivity and 100% diagnostic specificity. The number of cases analyzed and the detection threshold values for both syndromes are given.</p

MQF-PCR primers used in detection of microdeletion and microduplication syndromes.

a<p>Universal M13-40 extension (5′-GTTTTCCCAGTCACGAC-3′) was added to the 5′ end of the primer to allow for cost-efficient fluorescent labelling of amplicons <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061328#pone.0061328-Schuelke1" target="_blank">[31]</a>.</p>b<p>A PIG-tail extension (5′-GTTTCTT-3′) was added to the 5′ end of the primer to promote full adenylation of the 3′ end of the forward strand <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061328#pone.0061328-Brownstein1" target="_blank">[32]</a>.</p

Detection of the Lubs (<i>MECP2</i> duplication) and 17q11.2 microduplication syndromes.

<p>Representative electropherograms showing changes in the peak area ratios between a control sample (A) and a patient (B) in diagnosis of the Lubs syndrome. The peak area corresponding to the duplicated region on chromosome Xq28 (black) has significantly increased in size compared to its control region on chromosome 5p13.3 (white). Electropherogram depicting change of the peak area between chromosome 17 and its control chromosome in normal (C) and individual with microduplication of 17q11.2 (D) The peak area corresponding to the duplicated region on chromosome 17 (black) has significantly increased in size compared to its control region on chromosome 13q12.11 (white). E–F) Interactive dot diagrams of ROC curve analysis of Z scores in Lubs (E), and 17q11.2 microduplication syndrome (F). Both diagnostic primers achieved 100% diagnostic sensitivity and 100% diagnostic specificity. The number of cases analyzed and the detection threshold values for both syndromes are given.</p

Association of Genetic Variants with Self-Assessed Color Categories in Brazilians

<div><p>The Brazilian population was formed by extensive admixture of three different ancestral roots: Amerindians, Europeans and Africans. Our previous work has shown that at an individual level, ancestry, as estimated using molecular markers, was a poor predictor of color in Brazilians. We now investigate if SNPs known to be associated with human skin pigmentation can be used to predict color in Brazilians. For that, we studied the association of fifteen SNPs, previously known to be linked with skin color, in 243 unrelated Brazilian individuals self-identified as White, Browns or Blacks from Rio de Janeiro and 212 unrelated Brazilian individuals self-identified as White or Blacks from São Paulo. The significance of association of SNP genotypes with self-assessed color was evaluated using partial regression analysis. After controlling for ancestry estimates as covariates, only four SNPs remained significantly associated with skin pigmentation: rs1426654 and rs2555364 within <i>SLC24A5</i>, rs16891982 at <i>SLC45A2</i> and rs1042602 at <i>TYR</i>. These loci are known to be involved in melanin synthesis or transport of melanosomes. We found that neither genotypes of these SNPs, nor their combination with biogeographical ancestry in principal component analysis, could predict self-assessed color in Brazilians at an individual level. However, significant correlations did emerge at group level, demonstrating that even though elements other than skin, eye and hair pigmentation do influence self-assessed color in Brazilians, the sociological act of self-classification is still substantially dependent of genotype at these four SNPs.</p></div

(A) Triangular plots of the genomic proportions of African, European and Amerindian ancestry in three self-reported color groups of 243 Brazilian individuals from Rio de Janeiro samples, self-categorized as White, Brown and Black individuals.

<p>Each point represents a separate individual and the ancestral proportions can be determined by dropping lines parallel to each of the three axes. The graphs were drawn using the <i>Tri-Plot</i> software. (B) Dot plot of the European ancestry proportion for each separate self-assessed color group.</p

Dot-plot of the “Pigmentation Index” (PI) within the three color categories.

<p>The dot-plot was prepared with the MedCalc software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083926#pone.0083926-Schoonjans1" target="_blank">[50]</a>.</p

Numeric regression analysis between self-assessed color categories and SNP genotypes in population samples from Rio de Janeiro and São Paulo.

<p>NR - Numeric full model regression.</p><p>CV - restricted model (partial correlation with ancestry as a covariate). Significant values (<0.05) after Bonferroni correction are shown with an asterisk.</p