The diagnostic options for couples at risk of transmitting a specific inherited disorder to their offspring are preimplantation genetic diagnosis (PGD) or prenatal diagnosis (PND). These two diagnostic procedures share the same purpose, but differ in diagnostic time, type of sampling and laboratory procedures. According to the guidelines of the European Society of Human Reproduction and Embryology (ESHRE), PGD involves the analysis of at least two informative microsatellite markers, namely, short tandem repeats (STR), linked to the gene mutation site, and direct analysis of the mutation, if necessary . Consequently, DNA contamination, allele drop-out and parenthood are tested simultaneously , , , , .
Although PGD has become a well-established diagnostic procedure in at-risk couples, PND is still widely used to detect molecular alterations in the fetus at risk of a monogenic disease , . PND is usually performed between the 11th and 13th week of gestation on chorionic villi samples (CVS) or from the 15th week onwards on amniocytes, and the result is obtained in 7 and 15 days, respectively . The molecular diagnostic strategy consists in a multistep workflow chosen depending on the disease and the type of biological sample (Figure 1A), and it includes: 1) preliminary molecular analysis of the family; 2) molecular analysis of the fetal sample by direct mutation analysis (performed by different methods, i.e. reverse dot-blot or amplification and sequencing of the gene segments at the locus of the parental mutation) when the parental mutations are known; or by linkage analysis (performed by analyzing several STRs) when the parental mutations are unknown ; 3) detection of maternal cell contamination; and 4) parenthood testing .
Next-generation sequencing (NGS) has prompted the development of new molecular strategies that result in a fast and sensitive molecular diagnosis , , . These include the methods used to detect fetal aneuploidies using cell-free fetal DNA (cffDNA) in maternal blood at an early gestational age (about 10 weeks) . At present, the latter method is validated to detect the most frequently observed aneuploidies (50%–70%) in chromosomes 21, 18, 13, X and Y . Notably, non-invasive prenatal testing (NIPT) based on cffDNA is not a diagnostic test but rather a screening procedure. In fact, it determines whether or not the fetus is affected by one of the most common aneuploidies . Furthermore, positive results must be confirmed by a traditional invasive technique .
Very recently, studies have evaluated the application of NIPT using cffDNA in such single gene disorders as β-thalassemia , congenital adrenal hyperplasia  and Duchenne and Becker muscular dystrophies (DMD/BMD) ; however, protocols using cffDNA for the analysis of monogenic diseases are still in an experimental stage , , . In fact, despite the advantages of a shorter analysis time, cost-effectiveness, and the possibility of analyzing several genomic targets in different patients at the same time, some aspects, including data analysis, method repeatability and reliability of results require accurate assessment and further standardization before their routine diagnostic application. Currently, the real diagnostic options for families at-risk of monogenic disease are PGD and PND . Therefore, in the attempt to optimize the laboratory workflow and merge associated procedures (quality controls, paper work, accreditation program, and use of instruments) and to reduce the diagnosis delivery time, we set-up and tested the efficacy of the SEeMORE strategy (Single-tube Electrophoresis analysis-based genotyping to detect MOnogenic diseases Rapidly and Effectively). This strategy is a workflow based on capillary electrophoresis (CE) that enables genetic laboratories to optimize the molecular diagnosis for couples at risk of transmitting a genetic disease from conception until birth.
The SEeMORE strategy consists in a multiplex PCR constituted by linkage analysis, direct analysis, maternal contamination and parenthood testing. It has been optimized on CE because it is the gold standard for the identification of sequence variants at single nucleotide level. Moreover, SEeMORE is neither disease-mutation-dependent nor sample-dependent, which means that laboratories can standardize the diagnosis based on a single procedure.
Here we report the efficacy of this workflow applied to samples previously diagnosed for PND (cystic fibrosis or Duchenne muscular dystrophy), the results of which were unknown to us.
Materials and methods
As examples of diagnosis of monogenic diseases, we analyzed two families affected by an autosomic recessive disease (cystic fibrosis; CF) and two families with an X-linked disease (Duchenne muscular dystrophy; DMD). Villocentesis was performed in all cases. All patients provided informed consent to use anonymously their clinical and laboratory data for scientific purposes. The experiments were performed without knowing the results of traditional PND. In addition, to further assess the method’s reliability, the results were compared to those obtained on the same samples after whole genome amplification (WGA).
Genomic DNA extracted from the peripheral blood of all family members and from CVSs of the probands were selected from samples collected and stored for “conventional” PND in the biological sample bank of CEINGE-Biotecnologie Avanzate to be analyzed with the proposed strategy. In addition, to simulate single cell analysis in case of blastomeres and trophectoderm in PGD procedures, of a low number of amniocytes or scanty chorionic villi in PND, or of circulating fetal cells in NIPT, single lymphocytes were isolated from peripheral blood, or genomic DNA was diluted at 15 pg/μL, according to ESHRE guidelines .
Isolation of a low number of cells
Lymphocytes were isolated from peripheral blood by FICOLL gradient. Two milliliters of blood were mixed with 2 mL of phosphate buffered saline (PBS). The diluted blood sample (4 mL) was layered on 3 mL of Ficoll. After centrifugation at 400×g for 30 min, the upper plasma layer was drawn off using a clean Pasteur pipette and the lymphocyte layer was transferred to a clean centrifuge tube. Lymphocytes were washed in 6 mL of 0.1% PBS/Poly(vinyl alcohol) solution and centrifuged at 1200×g for 5 min. Single lymphocytes were isolated under a stereomicroscope (Leica MZ7.5). Isolated lymphocytes were washed three times with PBS and transferred into 200 μL tubes containing 2.5 μL of 0.1% PBS/Poly(vinyl alcohol). Tubes containing single cells were frozen at −80 °C prior to WGA.
Whole genome amplification
WGA was performed before the single-tube PCR-based genotyping analysis. It was carried out (in a separate laboratory, with positive pressure, HEPA-filtered air, using dedicated instrumentation and consumables) on single cell isolated as described above. The procedure, based on multiple displacement amplification (MDA) (REPLI-g Single Cell Kit, Qiagen), involves three steps: 1) Alkaline denaturation at 65 °C for 10 min; 2) Neutralization with stop solution; and 3) Amplification at 30 °C for 120 min by Phi 29 polymerase. The reaction was stopped by incubation at 65 °C for 3 min. This modified protocol takes 2.5 h . A positive control, i.e. genomic DNA diluted at 15 pg (Qiagen), and a negative control (amplification mix with no DNA) were included in each experiment. The success of amplification was checked on 0.8% gel electrophoresis.
The panel designed for CF cases consists of three extragenic STRs (D7S2847, D7S486 and D7S23) and two intragenic (D7S677 and IVS17bTA) linked to the CFTR gene. The panel designed for DMD cases consists of five intragenic STRs (DXS1214, DXS1236, DXS997, DXS1238 and DXS1242) linked to the dystrophin genes and sex determination markers, namely, X22, HPRT and AMEL. The features of the primers are detailed in Table 1 , , , . The PCR master mix consists of 2X Qiagen Multiplex Mix, Primer F+R (10 μM) and H2O for a total volume of 15 μL per sample. The PCR master mix was aliquoted to each PCR reaction tube and 1 μL of WGA or 0.5 μL of genomic DNA was added. The PCR protocol was as follows: incubation at 94 °C for 15 min, 55 cycles of 30 s at 94 °C, 90 s at 61 °C, 1 min at 72 °C, and 10 min at 60 °C. When the experiment was set-up, the specific primers of the mutation sites were pooled with linked STR primers in the same multiplex PCR. The PCR products (1 μL) were mixed with 9.5 μL formamide and 0.25 μL of GeneScan LIZ-500 (Applied Biosystems).
The SNaPshot minisequencing-based assay
The PCR temperatures were 96 °C for 1 min, 35 cycles of 15 s at 94 °C, 15 s at 58 °C, 45 s at 65 °C, then 2 min at 65 °C. The PCR products were purified to remove primers and unexhausted dNTPs using the ExoSAP kit (Affymetrix USB): 5 μL of PCR product was incubated with 2 μL of Exo-sapIT for 15 min at 37 °C followed by 15 min at 85 °C to inactivate the enzyme. The single-base extension was obtained using the PCR product diluted 1/50, minisequencing primer forward, and SNaPshot multiplex ready reaction Mix (SNaPshot Kit, Life Technology) in 25 cycles under the following conditions: 10 s at 96 °C, 5 s at 50 °C and 30 s at 60 °C. The SNaPshot Multiplex contained the enzyme, the buffer and the ddNTPs labeled with four fluorescent dyes: ddATP with dR6GTM, ddTTP with dROXTM, ddCTP with dTAMRATM and ddGTP with dR110TM. The minisequencing products (1 μL) were mixed with 9.5 μL formamide and 0.25 μL of GeneScan LIZ-120 (Applied Biosystems, Foster City, CA, USA). The fluorescent PCR products were sized on a 16-capillary sequencer (Applied Biosystems 3500 Genetic Analyzer). Gene Mapper 4.0 software (Applied Biosystems) was used to collect data, track lines and measure fragments.
The SEeMORE strategy: the workflow step-by-step (Figure 1B)
Step 1. From sample to DNA. Peripheral blood or a saliva swab is the sample used in commercial kits to extract genomic DNA from all family members. The analysis may be carried out on the proband starting from different types samples. In the case of CVS, the genomic DNA can be extracted with the same methodology used to extract DNA from peripheral blood. In the case of a small amount of amniocytes or scanty CVS, blastomeres, trophectoderm or circulating fetal cells, WGA must be performed.
Step 2. From DNA to the results. Linkage analysis, maternal contamination and parenthood testing are carried out with a multiplex PCR in which one primer of each pair is fluorescently 5′-end labeled with FAM or HEX to enable the analysis of the PCR products by CE. The primers are designed to identify a panel of STRs near the target gene and that occur with a high allelic heterozigosity (including intragenic and extragenic STRs). In addition, for X-linked disorders, a primer for sex determination markers should be included. When the index case is unavailable, the SEeMORE strategy could include the SNaPshot Minisequencing-based assay. This technique is used to analyze specific mutations, it is based on a single-base extension with a labeled ddNTP. The forward and reverse primers for the outer PCR are included in the master mix for analysis with STR, after which an inner PCR is performed using a new inner forward primer and the previously used outer reverse primer. The fluorescent PCR products are sized on a capillary sequencer and a software is used to collect data, track lines, and measure fragments. The results obtained with the SEeMORE strategy are available within 24 h.
We report examples of cases frequently encountered in laboratory practice, namely two couples at risk of transmitting CF and two couples at risk of transmitting DMD.
Case 1: A CF family with two known mutations and index case available
The SEeMORE strategy revealed that the fetus is a CF carrier, excluded DNA contamination and confirmed parenthood. As shown in Figure 2A, the CF son (II-1) inherited the father’s allele (associated with the F508del mutation, in red) and the mother’s allele (associated with the 852del22 mutation, in blue), while the fetus (II-2) inherited the father’s allele (in red) and the mother’s wild-type allele (in green).
The analysis was conducted on a CVS sample and using a panel of intragenic and extragenic STRs closely linked to the CFTR gene by analyzing F508del (c.1521_1523delCTT, pPhe508del) in the same multiplex-PCR (Figure 2B). Figure 2C shows the efficacy of the SEeMORE strategy starting from gDNA or WGA.
Case 2: Both parents are carriers of a known CF mutation
The SEeMORE strategy revealed that the fetus was homozygous for the G542X mutation. Figure 3A shows that the CF fetus (II-1) inherited both the father’s allele (associated with the G542X mutation, in red) and the mother’s allele (associated with the G542X mutation in blue). The analysis was conducted on a CVS sample using a panel of intragenic and extragenic STRs closely linked to the CFTR gene and analyzing the G542X in the same multiplex-PCR (Figure 3B). Despite the known mutation and the unavailability of the index case, the STR analysis was useful to establish the absence of DNA contamination and confirmed parenthood.
Figure 3C shows the efficacy of the SEeMORE strategy, also using SNaPshot Minisequencing starting from gDNA or WGA.
Case 3: DMD family with known mutations and index case available
The SEeMORE strategy revealed that the fetus (II-2) is a DMD female carrier, excluded DNA contamination and confirmed parenthood. Figure 4 shows the STRs related to a DMD-affected son (II-1) and that the fetus (II-2) inherited the mother’s allele (in red) associated with the mutation (c.[(6437+1_6438-1)_(6913+1_6913-1); (7201+1_7202-1)dup]), and female gender. The analysis was conducted on a CVS and with a panel of dystrophin intragenic STRs and sex determination markers in the same multiplex-PCR.
Case 4: DMD family with one known mutation and an uncle as the index case
The analysis was conducted considering that the index case (I-3) was the brother of the pregnant woman (I-2) and a previous analysis showed the c.10798-11G>A mutation in both. As shown in Figure 5A, the SEeMORE strategy revealed that the fetus inherited (II-2) the wild type allele from the mother. Sex determination markers confirmed the male gender of the fetus and confirmed parenthood (Figure 5B).
We tested the “SEeMORE strategy” in the attempt to optimize workflows, to reduce equipment, timing and costs, and so to enable genetic laboratories to provide a better diagnostic service for family at risk of genetic disease from conception until birth. This strategy is based on the analysis of an STR panel specific for the disease-causing gene regardless of the mutation in the family. The STRs included in the panel are intragenic and extragenic, have a high degree of heterozygosity, and produce a clearly interpretable peak pattern. Analysis of at least two loci closely linked to the disease-causing gene reduces the risk of misdiagnosis due to allele drop out (from about 5% to a minimum of 0.1%) in case of WGA , . More than two STR markers make the test more robust. The advantage of SEeMORE is that the workflow is independent: (1) of the diagnostic procedures (PND or PGD), (2) of the starting samples (embryo cells, CVS, amniocytes, circulating pure fetal cells) and (3) of the type of mutation present in the family (Figure 6).
Considering CF, in which the direct analysis is carried out when the mutation is known (94%), and the indirect analysis when the mutation has not been identified (7%), the gene-linked STR panel leads to a diagnosis even when the pathogenic mutation is not identified in the analysis of the whole coding region of the gene of interest . Furthermore, it is important to emphasize that DNA contamination and parenthood can be identified even when the STRs are not informative (i.e. parental consanguinity) or the index case is not available, as shown in Figure 4. In this case, direct analysis was performed by the SNaPshot Minisequencing-based assay.
To test the effectiveness of the SEeMORE strategy, we applied to samples previously diagnosed for PND in four families at risk of transmission of CF or DMD. These diseases are well-known examples of diseases (autosomal recessive and X-linked) that can be diagnosed before birth and are the most frequent indication for PGD after hemoglobin disorders . The results obtained with the SEeMORE strategy concurred with those of traditional PND, but with a significant improvement of turnaround time and with less use of techniques and handwork.
The advantages of the SEeMORE strategy for diagnostic laboratories are an easy to use diagnostic workflow, rapid results and sustainable costs. In detail, it foresees the use of customized oligonucleotides for CE. The latter method is the gold standard for identification of STR (three base pairs) and is used for paternity and forensic testing in which a no error-prone procedure is essential. However, our strategy revealed point mutations (only one bp change). In case of deletion(s) or insertion(s) of only two or one nucleotide, the indirect analysis will identify the allele carrying the genetic alteration.
Based on these considerations, the SEeMORE strategy, which is neither mutation- nor sample-dependent, allows workflow optimization, provides the results within 24 h and consequently shortens the diagnosis delivery time also to laboratories that do not have NGS.
The most recent challenge is to develop a method for NIPT in order to reduce physical discomfort and the associated 1%–2% risk of procedure-induced miscarriage . The major problem in NIPT is that the abundance of maternal DNA in plasma impairs the detection of subchromosomal defects. This problem can be overcome by isolating circulating pure fetal cells from maternal blood and performing genetic analysis using the WGA product . Haplotype based on single nucleotide polymorphism analysis is also an effective non-invasive prenatal diagnosis strategy with which to detect the normal allele , , . However, non-invasive prenatal diagnosis for monogenic diseases as well as the isolation of circulating pure fetal cells remains a challenge.
Previous studies demonstrate that low numbers of amniocytes or chorionic villi (without cell culture), and fetal cells can be amplified by WGA and used to genotype genes of interest , , , , . Consequently, the SEeMORE strategy, which includes WGA, can be applied also to such samples, that include circulating pure fetal cells once a method to isolate them will be validated for diagnostic purposes from the appropriate regulatory bodies. Notably, the allele drop out artifact due to WGA is very low in case of several cells recovered or pooling WGA product from single cells (1/10,000) thereby reducing the risk of misdiagnosis .
In conclusion, SEeMORE is an optimal strategy for the molecular diagnostic laboratory because it optimizes the laboratory workflow and merges the associated procedure. Finally, SEeMORE is particularly useful for patients and families as it reduces the diagnosis delivery time. The strategy is neither disease- nor sample-dependent and effective for the molecular diagnosis of monogenic diseases. It can be used for prenatal or preimplantation diagnosis, and paves the way for the NIPT on fetal cells.
The authors thank Jean Ann Gilder (Scientific Communication srl., Naples, Italy) for editing the text, and Vittorio Lucignano, CEINGE-Biotecnologie Avanzate, for technical assistance.
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About the article
Published Online: 2017-08-08
Published in Print: 2017-11-27
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and have approved submission.
Research Funding: This work was supported by POR CAMPANIA FSE 2007–2013 Project DIAINTECH, Italy (to F.S.); Società Italiana di Biochimica Clinica e Biologia Molecolare Clinica (grant 08/14) (to R.T.); and Consorzio Interuniversitario Biotecnologie (grant 11/15) (to F.C.).
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.