Utility and limitations of multiplex ligation-dependent probe amplification technique in the detection of cytogenetic abnormalities in products of conception.

Background and Introduction: Chromosomal abnormality is found in about half of first-trimester abortions. Karyotype is the gold standard to detect chromosomal abnormalities. Multiplex ligation-dependent probe amplification (MLPA) offers advantage over karyotype in terms of lower failure rate, faster turnaround time, and much higher resolution than conventional karyotyping and found to be 98% concordant with conventional karyotype. AIM: We performed this study to look for the utility of MLPA in diagnosing chromosomal abnormalities in first-trimester abortions.
MATERIALS AND METHODS: MLPA using subtelomeric SALSA probe sets (P036 and P070) was used to detect cytogenetic abnormalities in products of conception in missed/spontaneous abortions.
RESULTS: A total of ninety abortus samples were analyzed by MLPA. Successful results were provided in (67) 74.4% of the cases while no conclusion could be drawn in 25.6% (23) of the cases. Fifty-five (82.1%) cases were cytogenetically normal and 17.9% (12) had some abnormality. Aneuploidy was detected in 8 (66.7%) cases, 3 (25%) had double-segment imbalance, and one (8.3%) had partial aneuploidy.
CONCLUSION: We suggest that MLPA is a good substitute to traditional karyotype.

Introduction

Spontaneous abortion is relatively common and occurs in about 10–15% of all clinically recognized pregnancies. Around 50% of first-trimester abortions and one-third of second-trimester abortions are due to cytogenetic abnormalities. The most frequent cytogenetic abnormalities are numerical aberrations (86%), mainly trisomy (75%), polyploidy (13%), and monosomy; followed by structural rearrangements (6%) and other abnormalities such as chromosomal mosaicism or double and triple trisomies (8%).[12] Cytogenetic analysis of products of conception (POC) after spontaneous abortion helps in identifying etiology and providing genetic counseling to the family. The risk of recurrence is very low in case of aneuploidy, polyploidy, and de novo unbalanced chromosomal rearrangements. In case of unbalanced chromosomal rearrangements in which one of the parents is a carrier, the risk of recurrence is around 30%.

Conventional karyotyping of cultured cells is the gold standard technique, but it has some inherent disadvantages such as culture failure (10–40% of cases),[1] maternal contamination (30–40% of cases),[34] requirement of viable tissue, suboptimal quality of metaphase preparation, being labor intensive, longer turnaround time, and requirement of skilled cytogeneticist. Molecular cytogenetic techniques such as fluorescent in situ hybridization (FISH), multiplex ligation-dependent probe amplification (MLPA), quantitative fluorescent polymerase chain reaction (QF-PCR), and array comparative genomic hybridization (aCGH) or cytogenetic microarray are DNA-based tests which offer certain advantages over conventional cytogenetics such as lower failure rate, faster turnaround time, and much higher resolution than conventional karyotyping.[5] FISH and QF-PCR can analyze for few chromosomes in a single reaction while MLPA and aCGH analyze copy number gains/losses in 48 genomic regions and whole genome, respectively, in a single reaction.[67] MLPA is less costly in comparison to aCGH and is shown to be robust in detection of chromosomal numerical aberration and subtelomeric copy number gains/losses in DNA extracted from POC and shown to have 98% success in providing results and >90% concordance with the karyotype.[48] We performed this study to look for utility of MLPA using subtelomeric probe set in detection of cytogenetic abnormalities in spontaneous abortions.

Materials and Methods

This prospective study was carried out in the Department of Medical Genetics, and the POC samples were received from the patients admitted in the Department of Obstetrics and Gynaecology of a tertiary care hospital and was approved by the Institute's Ethics Committee. Samples included were those of spontaneous and missed abortion obtained by dilatation and curettage, excluding those obtained after medical termination of pregnancy. POCs were dissected under microscope to remove any maternal decidua, blood, or mucus. Grossly necrotic or contaminated samples were discarded and not subjected to further analysis. DNA was extracted using Qiagen tissue extraction kit, following manufacturer's protocol. MLPA reaction was done using subtelomeric SALSA probe set P036 or P070 (MRC-Holland). These contain probes which hybridize to specific DNA segments located on subtelomeric regions on both arms of all the chromosomes. DNA extracted from chorionic villous tissue collected in our department to provide prenatal diagnosis was used as control sample in each reaction. MLPA reactions were performed as recommended by the manufacturer's protocol. PCR products were separated by capillary electrophoresis using ABI310 sequencer. Results were analyzed with normalization of peak heights and analysis of peak height ratio using software Coffalyser.net. Peak height ratio >1.35 was interpreted as copy number gain while peak height ratio <0.65 was interpreted as copy number loss. Copy number gains or losses involving one chromosomal arm were interpreted as segmental aneuploidy while copy number gain on both arms of chromosomes was interpreted as trisomy. Deletion of both arms of X chromosomes was interpreted as monosomy X. Copy number gains/losses involving different chromosomes were defined as double-segment imbalance. As the samples were taken from a different hospital, karyotype was not offered as they were unlikely to have any viable tissue.

Results

During this study, ninety POCs from cases with spontaneous or missed abortion were analyzed for cytogenetic abnormalities using MLPA technique. Out of total ninety samples, no conclusion could be drawn in 23 (25.6%) cases due to inadequate quantity of DNA or unsatisfactory results. In 67 (74.4%) cases with a conclusive result, 55 (82.1%) cases were cytogenetically normal and 12 (17.9%) cases had some abnormality.

The most frequent cytogenetic abnormality was aneuploidy, present in 8 out of the 12 cases (66.7%), followed by double-segment imbalance in 3 (25%) cases and partial aneuploidy in one sample (8.3%). Out of the aneuploid POCs, the most frequent abnormality was trisomy (75%), followed by monosomy X (25%) [Figure 1].

Figure 1

Multiplex ligation-dependent probe amplification result in a sample of products of conception showing Monosomy X

Among the trisomic POCs, trisomy 16 was the most common (33.3%) [Figure 2], followed by one case each of trisomies 7, 15, 18, and 22. Three samples had double-segment imbalance with two samples having gain at 3q29 and loss at 10q26.3, suggesting unbalanced chromosomal rearrangement between chromosome 3 and 10 [Figure 3], whereas one sample had loss at 4p16.3 and gain at 11p15.5, suggesting unbalanced chromosomal rearrangement between chromosome 4 and 11. One of the samples had loss at 19p13.3 suggestive of partial aneuploidy of chromosome 19.

Figure 2

Multiplex ligation-dependent probe amplification result in a sample of products of conception showing Trisomy 16

Figure 3

Multiplex ligation-dependent probe amplification result in a sample of products of conception showing copy number loss at 10q26.3 and copy number gain at 3q29 region suggestive of double-segment imbalance

Discussion

MLPA technique using P036 and P070 probe set was used to analyze the cytogenetic abnormalities in ninety spontaneously aborted fetal tissues. No conclusion could be drawn in 25.6% of the cases due to unsatisfactory results or technical failure which is low as compared to the failure rate of conventional karyotyping (40% of the cases).[1] The cause of failure mainly includes insufficient quantity of DNA (8.8%) and failed MLPA reaction due to poor quality (16.5%). Successful results were provided in 74.4% of the cases which is lower than the previous studies (98%). The causes of failure of MLPA reported in the literature include low mass of DNA, degradation of the sample, and nuclease contamination.[5] The lower results can be due to the fact that samples were transported from outside hospital and many a times after 2–3 days of delivery. Cytogenetic abnormality was present in 17.9% of the cases, the cause presumed to account for about 50% of first-trimester spontaneous abortions. Numerical aberrations were the most common (66.7% cases).

MLPA technique has some inherent limitations. As in this test copy number of target DNA segment is compared with the haploid set of chromosomes, it cannot detect polyploidy. Bruno et al. have performed study on spontaneous abortion samples using FISH in conjunction with MLPA to detect polyploidy which can account for up to 13% of abnormalities in spontaneous abortions.[9] It can also not detect low-level mosaicism, maternal cell contamination, any interstitial segment copy number gain or loss located elsewhere in the genome other than subtelomeric region.[10] aCGH can detect copy number gains or loss located anywhere all over the genome, but using it to detect numerical abnormality in abortus sample will be very expensive.[4] Both MLPA and aCGH cannot detect balanced translocation. However, the presence of double-segment imbalance will point toward the possibility of inherited unbalanced chromosomal rearrangement, and parental karyotype will be necessary to confirm the presence of chromosomal rearrangement. Our study has limitations as karyotype was not offered to all families enrolled in this study as a part of work up. Karyotype would additionally have detected polyploidy or unbalanced chromosomal translocation not involving subtelomeric region which otherwise was missed by MLPA. However, MLPA is a useful substitute to traditional karyotyping due to ease of transportation of sample and higher success rate. Polyploidy has a low risk of recurrence. Diagnosis of balanced translocation is necessary as it has important implications in associated moderate risk of recurrence, small risk of live child with malformations, or intellectual disability and genetic counseling. Recently, cytogenetic analysis on POC has been recommended as first tier of investigation in workup of couples with recurrent pregnancy losses.[11] However, all the couples with two or more spontaneous abortions must have karyotype done from their peripheral blood leukocytes to look for the presence of balanced chromosomal rearrangement.

Financial support and sponsorship

The financial support was provided by the Indian Council for Medical Research.

Conflicts of interest

There are no conflicts of interest.