Prenatal Diagnosis Of Single Gene Disorders Research Articles

Non-invasive prenatal diagnosis of single gene disorders with enhanced relative haplotype dosage analysis for diagnostic implementation.

Non-invasive prenatal diagnosis of single-gene disorders (SGD-NIPD) has been widely accepted, but is mostly limited to the exclusion of either paternal or de novo mutations. Indeed, it is still difficult to infer the inheritance of the maternal allele from cell-free DNA (cfDNA) analysis. Based on the study of maternal haplotype imbalance in cfDNA, relative haplotype dosage (RHDO) was developed to address this challenge. Although RHDO has been shown to be reliable, robust control of statistical error and explicit delineation of critical parameters for assessing the quality of the analysis have not been fully addressed. We present here a universal and adaptable enhanced-RHDO (eRHDO) procedure through an automated bioinformatics pipeline with a didactic visualization of the results, aiming to be applied for any SGD-NIPD in routine care. A training cohort of 43 families carrying CFTR, NF1, DMD, or F8 mutations allowed the characterization and optimal setting of several adjustable data variables, such as minimum sequencing depth, type 1 and type 2 statistical errors, as well as the quality assessment of intermediate steps and final results by block score and concordance score. Validation was successfully performed on a test cohort of 56 pregnancies. Finally, computer simulations were used to estimate the effect of fetal-fraction, sequencing depth and number of informative SNPs on the quality of results. Our workflow proved to be robust, as we obtained conclusive and correctly inferred fetal genotypes in 94.9% of cases, with no false-negative or false-positive results. By standardizing data generation and analysis, we fully describe a turnkey protocol for laboratories wishing to offer eRHDO-based non-invasive prenatal diagnosis for single-gene disorders as an alternative to conventional prenatal diagnosis.

Applying amplification refractory mutation system technique to detecting cell-free fetal DNA for single-gene disorders purpose.

Non-invasive prenatal diagnosis for single-gene disorders (NIPD) is still in development and deserves further study. The advent of next-generation sequencing technology significantly improved the detection of multiple mutations for non-invasive prenatal diagnosis for single-gene disorder purposes. However, bespoke amplicon-based NGS assays are costly. In this study, we developed a new strategy for non-invasive prenatal screening for single-gene disorders based on a capillary electrophoresis (CE) platform using an amplification refractory mutation system (ARMS)-PCR technique. Allele-specific primers for several disease-correlated mutations were designed, and subsequently, sensitivity and specificity assays were conducted. Assays on simulated two-person DNA mixtures showed that three primers targeting the mutant allele could detect minor DNA components in 1:500 mixtures. All primers showed positive results at 0.01ng of the template DNA. Cell-free fetal DNA was extracted from a pregnant woman's peripheral blood for the detection of paternally inherited mutations. Our results showed that one primer successfully amplified the mutant allele of fetal DNA in maternal plasma, which was confirmed by genotyping the genomic DNA extracted from amniotic fluid. This study suggested that the ARMS-PCR technique, a fast and cost-effective method, might be a promising method used to target de novo or paternally inherited pathogenic mutations in maternal plasma.

The current applications of cell-free fetal DNA in prenatal diagnosis of single-gene diseases: A review.

Prenatal diagnosis of hereditary diseases has substantially altered the way medical geneticists are helping families affected by genetic disorders. However, the risk of miscarriage and fear of invasive diagnostic procedures may discourage many couples from seeking prenatal diagnosis. With the discovery of maternal plasma cell-free fetal DNA, prenatal diagnosis has entered a new era of progress. Cell-free DNA is released during normal physiological functions as well as through the cell death programs of apoptosis and necrosis. It can be found in the plasma and other body fluids. Although this method has the advantage of being noninvasive, it is still rather expensive and requires advanced hardware and comprehensive data analysis. Promising implications of noninvasive prenatal diagnosis methods for the diagnosis of common trisomy disorders have paved the way for the development of more complicated assays of single-gene disorders. Relative mutation dosage and relative haplotype dosage are the most widely implemented assays for noninvasive prenatal diagnosis of single-gene disorders. However, each assay has its own advantages and disadvantages. Relative mutation dosage is based on the droplet digital polymerase chain reaction (PCR) technique which includes quantification features of real-time PCR assays. Relative haplotype dosage is based on next-generation sequencing that includes analysis of the maternal and paternal genome followed by sequencing of maternal plasma cell-free DNA. Co-amplification at a lower denaturation temperature PCR is another approach that is based on forming heteroduplexes between alleles to selectively amplify paternal mutations. In this review, we have described the most common noninvasive prenatal diagnosis approaches and compared their applications in genetic disorder diagnosis with different inheritance patterns.

Celocentesis for early prenatal diagnosis of hemoglobinopathy

Celocentesis is an invasive technique that can provide prenatal diagnosis of single-gene disorders, from as early as 7 weeks' gestation. The objective of this study was to examine the safety of celocentesis. In this prospective study, celocentesis was performed for prenatal diagnosis of hemoglobinopathy in 402 singleton pregnancies in which both parents were carriers of β-thalassemia or sickle cell disease trait. We assessed procedure-related maternal discomfort or pain, success of sampling and obtaining results, pregnancy outcome and postnatal follow-up. First, celocentesis was carried out at a median gestational age of 8.6 (range, 6.9-9.9) weeks and celomic fluid was successfully aspirated in 99.8% of cases. Second, 67% of women had no or only mild discomfort, 18% had moderate discomfort, 12% had mild-to-moderate pain and 3% had severe pain. Third, prenatal diagnosis from analysis of the celomic fluid was successful in 93.8% cases, and in the last 121 cases, it was always successful. Fourth, in all cases of successful sampling and analysis of celomic fluid, the diagnosis was concordant with results obtained from additional prenatal or postnatal testing. Fifth, in addition to diagnosis of hemoglobinopathy, quantitative fluorescence polymerase chain reaction analysis, which was performed to evaluate maternal contamination using several markers for chromosomes X, Y, 21, 18 and 13, led to the accurate diagnosis of chromosomal aneuploidy. Sixth, in all cases of an affected fetus diagnosed by celocentesis in which the parents chose termination of pregnancy, this was carried out < 10 weeks' gestation. Seventh, in 97.1% (298/307) of the continuing pregnancies there was live birth, in seven (2.3%) there was miscarriage and in two (0.7%) there was loss to follow-up. Eighth, fetal abnormalities were diagnosed in three (1%) cases, including unilateral transverse amputation of the forearm, unilateral moderate hydronephrosis and small-bowel duplication. All neonates were examined by a pediatrician and were found to be phenotypically normal, except for the three cases with a prenatally diagnosed defect. Celocentesis can be used for early prenatal diagnosis of genetic abnormalities, and the procedure-related risk of pregnancy complications appears to be low. Copyright © 2020 ISUOG. Published by John Wiley & Sons Ltd.

Bayesian-based noninvasive prenatal diagnosis of single-gene disorders

In the last decade, noninvasive prenatal diagnosis (NIPD) has emerged as an effective procedure for early detection of inherited diseases during pregnancy. This technique is based on using cell-free DNA (cfDNA) and fetal cfDNA (cffDNA) in maternal blood, and hence, has minimal risk for the mother and fetus compared with invasive techniques. NIPD is currently used for identifying chromosomal abnormalities (in some instances) and for single-gene disorders (SGDs) of paternal origin. However, for SGDs of maternal origin, sensitivity poses a challenge that limits the testing to one genetic disorder at a time. Here, we present a Bayesian method for the NIPD of monogenic diseases that is independent of the mode of inheritance and parental origin. Furthermore, we show that accounting for differences in the length distribution of fetal- and maternal-derived cfDNA fragments results in increased accuracy. Our model is the first to predict inherited insertions–deletions (indels). The method described can serve as a general framework for the NIPD of SGDs; this will facilitate easy integration of further improvements. One such improvement that is presented in the current study is a machine learning model that corrects errors based on patterns found in previously processed data. Overall, we show that next-generation sequencing (NGS) can be used for the NIPD of a wide range of monogenic diseases, simultaneously. We believe that our study will lead to the achievement of a comprehensive NIPD for monogenic diseases.

Noninvasive Prenatal Diagnosis of Single-Gene Disorders by Use of Droplet Digital PCR.

Prenatal diagnosis in pregnancies at risk of single-gene disorders is currently performed using invasive methods such as chorionic villus sampling and amniocentesis. This is in contrast with screening for common aneuploidies, for which noninvasive methods with a single maternal blood sample have become standard clinical practice. We developed a protocol for noninvasive prenatal diagnosis of inherited single-gene disorders using droplet digital PCR from circulating cell-free DNA (cfDNA) in maternal plasma. First, the amount of cfDNA and fetal fraction is determined using a panel of TaqMan assays targeting high-variability single-nucleotide polymorphisms. Second, the ratio of healthy and diseased alleles in maternal plasma is quantified using TaqMan assays targeting the mutations carried by the parents. Two validation approaches of the mutation assay are presented. We collected blood samples from 9 pregnancies at risk for different single-gene disorders, including common conditions and rare metabolic disorders. We measured cases at risk of hemophilia, ornithine transcarbamylase deficiency, cystic fibrosis, β-thalassemia, mevalonate kinase deficiency, acetylcholine receptor deficiency, and DFNB1 nonsyndromic hearing loss. We correctly differentiated affected and unaffected pregnancies (2 affected, 7 unaffected), confirmed by neonatal testing. We successfully measured an affected pregnancy as early as week 11 and with a fetal fraction as low as 3.7% (0.3). Our method detects single-nucleotide mutations of autosomal recessive diseases as early as the first trimester of pregnancy. This is of importance for metabolic disorders in which early diagnosis can affect management of the disease and reduce complications and anxiety related to invasive testing.

Embryo-fetal erythroid cell selection from celomic fluid allows earlier prenatal diagnosis of hemoglobinopathies.

Celocentesis, which involves aspiration of celomic fluid at 7-9 weeks' gestation, can potentially provide early prenatal diagnosis of single-gene disorders. The main barrier to wide acceptability of this technique is contamination of the sample by maternal cells. This problem can be overcome through selection of embryo-fetal erythroid precursors, which are found in celomatic fluid. Embryo-fetal erythroid precursors were selected by an anti-CD71 MicroBeads method or by direct micromanipulator pickup of the cells selected on the basis of their morphology. In our series of 302 singleton pregnancies at high risk for hemoglobinopathies, Celocentesis provided a sample of celomic fluid in all cases. In 100 (33.1%) samples, maternal contamination was absent or very low (< 5%), and unambiguous results were obtained without the need for any preliminary procedures. In 160 (53%) cases, the contamination was between 5% and 60%, and selection of embryo-fetal erythroid precursors was successfully achieved by anti-CD71 MicroBeads. In 42 (13.9%) cases, the contamination was > 60%, and selection of embryo-fetal cells was achieved by micromanipulation. In all 302 cases, there was concordance between DNA obtained from celomic fluid samples and fetal or newborn DNA. Celocentesis can be a reliable procedure for earlier prenatal diagnosis of fetal monogenic diseases.

Challenges in prenatal and pre-implantation genetic diagnosis studies.

Prenatal diagnosis (PND) of chromosomal anomalies by karyotyping and fluorescence in situ hybridization (FISH) is a routine procedure in many cytogenetic laboratories. However, PND of single gene disorders is available in only a few centres specializing in molecular genetic testing in India. Preimplantation genetic diagnosis (PGD) is a state-of the art technique involving biopsy of a single cell from a cleavage stage embryo obtained after intra-cytoplasmic sperm injection (ICSI). This is an additional step during in-vitro fertilization (IVF). The biopsied cell is then fixed on a slide in a critical step where the cytoplasm is removed to expose the nucleus. Single cells from many embryos of the couple are fixed and then screened for common aneuploidies by FISH using multicolour DNA probes. A maximum of 5 chromosomes can be checked in 1 hybridization. Hence the probes are stripped and slides are rehybridized in 2-4 rounds to study more chromosomes within a limited time frame. Only the chromosomally normal embryos are transferred or cryopreserved for a subsequent transfer. Karyotyping of couples with recurrent early miscarriages helps to identify the cause if the husband or wife is a carrier of a balanced translocation. PGD can help such couples to have a healthy baby earlier than by normal chance. Specific FISH probes have to be ordered especially for reciprocal translocations and a pre-PGD work up is important. This helped to identify an additional cryptic translocation between chromosomes 9 and 12 in a case from a neighbouring country, where the husband had inversion 12. Hence PGD included testing of chromosome 9 in this case. The wife has an ongoing pregnancy in the first attempt. Currently, ours is the only centre in India which offers PGD even for reciprocal/ Robertsonian translocations and microdeletions. We have had success with the birth of healthy babies. PGD for single gene disorders is yet in its infancy in our country. It involves genetic testing from DNA extracted from a single cell. Pre-PGD work up is more demanding, especially in cases where a HLA matched normal savior sibling is desired to cure a child affected with a hematological disorder such as Thalassemia.

Use of fetal nuchal translucency in the first trimester to predict single‐gene disorders

To assess the predictive value of fetal nuchal translucency (NT) measurement in the prenatal diagnosis of single-gene disorders. From January 1996 to December 2006, fetal NT was prospectively measured before chorionic villi sampling in 169 pregnancies at high risk for a single-gene disorder at 11 to 13 weeks of pregnancy. No differences were found between the 63 affected and 116 nonaffected fetuses in pregnancy demographic characteristics, in mean NT measurements, expressed either in millimetres [1.8 (95% CI:1.6-1.9) vs 1.7 (95% CI:1.6-1.8)] or in multiples of the median [1.19 (95%CI: 1.04-1.35) vs 1.14 (95%CI: 1.05-1.23)], or in median NT. The percentage of increased NT above the 95(th) percentile was similar for affected (9.5%) and nonaffected (11.2%) fetuses. Not all single-gene disorders are associated with enlarged NT, therefore NT cannot be regarded as a generic marker for single-gene disorder but only for a limited number of these conditions.

Prenatal diagnosis of single-gene disorders: case studies

In volume 13 issue 3 of this journal, various methods for diagnosing single-gene disorders in the antenatal period were reviewed. The general principles remain the same and have not been discussed again in detail here. Instead, four cases are described that illustrate the points made in this original article, with reference to background information where necessary. Molecular biology is playing an increasingly important role in the prenatal diagnosis of single-gene disorders. In the absence of a family history, or a previously affected sibling, it is usually ultrasound, however, that first raises the possibility of one of these diagnoses in the prenatal period. Clinical investigations, histopathology and molecular testing following birth, or termination of pregnancy, usually combine to provide a precise diagnosis, after which molecular testing in a future pregnancy often becomes possible if the recurrence risks are high.

Prenatal DNA diagnosis of a single-gene disorder from maternal plasma

Achondroplasia is a short-limb disorder caused by a point mutation in a single gene. To diagnose such a disorder prenatally requires the use of invasive procedures such as amniocentesis. However, using PCR and restriction fragment length polymorphism analysis, we were able to detect the mutation in the plasma of a woman carrying a fetus suspected of having achondroplasia. The detection of a fetus-derived mutant gene from maternal plasma may therefore permit non-invasive prenatal diagnosis of single-gene disorders.

The role of cloned genes in the prevention of genetic disease.

The application of recombinant DNA technology to the study of human genetic disease promises to increase the scope for carrier detection and prenatal diagnosis. Here we summarize current experience with prenatal diagnosis of single-gene disorders by DNA analysis and highlight some of the technical and organizational problems that remain to be solved.