Incidental finding at methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA): how to proceed?

Abstract

Introduction: Since the advent of new generation sequencing, professionals are aware of the possibility of obtaining findings unrelated to the pathology under study. However, this possibility is usually forgotten in the case of studies aimed at a single gene or region. We report a case of a 16-month-old girl with clinical suspicion of Silver-Russell syndrome (SRS). Methods: Following the international SRS consensus, methylation alterations and copy number variations (CNVs) at 11p15 region and maternal uniparental disomy of chromosome 7 were analysed and discarded by MS-MLPA. Results: Unexpectedly, the 11p15 region MS-MLPA showed a decrease in the signal of a copy number reference probe. Deletions affecting a single probe are inconclusive. So, we faced the ethical dilemma of whether it was appropriate to confirm this alteration with independent techniques and to offer a diagnostic possibility that was in no way related to clinical suspicion. Fortunately, in this particular case, the informed consent had not been specific to a particular pathology but to any disorder associated with growth failure. Performed alternative studies allowed the final diagnosis of 22q deletion syndrome. Conclusion: We demonstrate the importance of informing patients about the possibility of obtaining incidental findings in genetic techniques (not only in next generation sequencing) during pre-test genetic counselling consultations. In addition, we highlight the relevance of including in the informed consent the option of knowing these unexpected incidental findings as in some cases, this will help to elucidate the definitive diagnosis and provide the correct follow-up and treatment.

Keywords: 22q11.2 deletion syndrome; MS-MLPA; Silver-Russell syndrome; incidental findings; reference probe.

FIGURE 1

Photography of the front view face of the patient facial signs. Initial clinical suspicion was based on the presence of a high forehead and a triangular-shaped face.

FIGURE 2

Results of the molecular studies (A) The MS-MLPA analysis for 11p15 (ME030-C3 BWS/RSS; lot: C3-0121) with Coffalyser software revealed a decreased signal of a reference probe located at 22q11 (Reference C/M 22–019,079,440-202 nt). For the generation of these results, the software does an intra-normalization, converting absolute fluorescence signal intensities into relative values by normalizing probe signals against the signals of the reference probes in one sample. This is done for each sample. During inter-normalization, it compares each sample to the reference samples. In the present figure, the name of the analysed probes are given on the X-axis. The orange background encompasses the probes located at 11p15; the dark blue the NSD1 gene, involved in Sotos syndrome, a differential diagnosis for BWS/SRS; and the gray one covers the reference probes which are located on different chromosomes and are used for dosage normalization. The 95% confidence interval over the reference samples for each probe is represented by the blue square. The border lines (red for lower border; blue for upper border) are placed −/+ 0.3 from the average probe value of a probe over the reference samples. When a probe is within the borders, it is represented by a black dot; when it is out (either over the blue or below the red lines) it is represented by a red dot. (B) MLPA analysis targeting 22q11 (P250 DiGeorge (lot: B2-0519) performed after revision of the informed consent confirmed the presence of a heterozygous deletion at region 22q11. The blue background encompassed the analyzed probes through the 22q11 region whereas the grey background covers the reference probes. The analysed probes at 22q11 region and intra normalized ratio for CNVs are given on the X-axis and Y-axis, respectively. Probe ratios are indicated by the dots. Black dots indicate the probe ratio is within the 95% confidence interval (CI) of the reference sample population and the red dots indicates the ratio is out of the 95% CI and over the arbitrary borders, lower border: red line (0.7) upper border: blue line (1.3), by default. The boxes represent the 95% CI in reference sample population (by default), the blue boxes compared to test probes and the green ones compared to reference probes. (C) Array CGH analysis of the patient showed a deletion of 2.5 Mb at 22q11.21: arr [GRCh37] 22p11.21 (18894864–21461811)x1, encompassing approximately 40 genes.