Since the identification of cffDNA in maternal plasma in 1997, there have been rapid
developments in exploiting its presence for prenatal diagnosis and screening. The first
proof of principle studies using cfDNA from maternal plasma to detect fetal aneuploidy
were published in 2008 following which there was rapid commercialization. Nowadays,
non-invasive prenatal testing (NIPT) for aneuploidies is widely used across the world as
a screening test for the most frequent fetal trisomies. Unlike non-invasive prenatal
testing, where a positive result requires confirmation following an invasive test,
non-invasive prenatal diagnosis (NIPD) offers the advantage of a definitive diagnosis
without an invasive procedure
- - and its associated miscarriage risk - because confined
placental mosaicism does not occur with NIPD for single-gene disorders (SGD).
NIPD can be offered earlier in pregnancy than invasive testing, from 7 weeks of
gestation. This can reduce parental anxiety and allows more time for decision-making and
planning. Indeed, there are substantial challenges to overcome for SGD-NIPD. i/
Circulating cffDNA, which is released from the placenta from about 4 weeks gestation,
makes up only 5%-20% of total circulating cfDNA in maternal plasma. This percentage
increases with gestation and is influenced by factors such as maternal weight, smoking,
and pregnancy complications such as preeclampsia. Consequently, optimized techniques and
highly sensitive detection approaches are required to detect variants in the fetal DNA.
ii/ fetal fraction must be calculated to confirm that there are sufficient levels of
cffDNA present and to avoid false negative results. iii/ Another issue is the short
fragment length of cffDNA, which makes detection of triplet repeats and large deletions
or duplications challenging.
Besides fetal sex determination and fetal Rhesus D status, principal investigator's team
was the first to propose SGD-NIPD for use in clinical practice in France for autosomal
disorders caused by de novo or paternally inherited mutations, for which variants in the
fetal DNA can easily be distinguished in the high background of maternal cfDNA.
Droplet-digital Polymerase Chain Reaction or Next-Generation Sequencing can be used to
target a single mutation for this analysis.
However, this approach requires mutation-specific developments and is restricted to point
mutations that are absent from maternal DNA. NIPD for X-linked disorders, as well as
autosomal dominant maternally inherited or autosomal recessive disorders for which both
parents are carriers of the same mutation has posed a greater challenge. A quantitative
approach is needed to ascertain fetal inheritance of the maternal mutation. In autosomal
dominant diseases with maternal mutation or autosomal recessive diseases, the ratio
between the total copies of the mutant allele (M) and the wildtype allele (N) in the
maternal plasma contributed by both the maternal and fetal cell-free DNA is expected to
be balanced (M=N) if the genotype of the fetus (M/N) is identical to the mother (M/N).
However, the allelic ratio will be imbalanced if the fetal genotype is different from the
maternal genotype. If the fetus has inherited both parental wildtype alleles (N/N), there
would be additional dosage of the wildtype allele in the maternal plasma contributed from
the fetus, resulting in under-representation in the total copies of the mutant allele
(M
N). The degree of expected allelic imbalance in maternal plasma depends on the DNA
fetal fraction in the maternal plasma.
A quantitative relative mutation dosage (RMD) approach has been developed to detect such
mutant allelic imbalance. This approach has been applied to the non-invasive detection of
recessive disorders such as beta-thalassemia and sickle cell anemia but also for X-linked
disorders like hemophilia . Nevertheless, direct interrogation of the mutation appears to
be difficult- - even impossible - in certain genomic loci due to the presence of
repetitive sequences, homologous pseudogenes, and undefined genomic rearrangement.
Moreover, successful classification of allelic imbalance is statistically dependent on
the available copies of mutant and wildtype alleles in the blood sample, hampered by the
very low absolute concentration of cfDNA. As a result, RMD analysis is still at a
proof-of-concept phase, being evaluated in a limited number of studies, with a limited
number of patients, and has never been implemented in standard care diagnosis to
investigator's knowledge.
These challenges have inspired an alternative solution with indirect mutational status
inference by relative haplotype dosage analysis (RHDO). SGD-NIPD by indirect mutational
status inference by RHDO has been shown to be successful for -thalassemia and is now in
clinical practice only in the United Kingdom National Health Service for cystic fibrosis,
spinal muscular atrophy and Duchenne muscular dystrophy. Although RHDO has been proven
reliable, quality controls and decisional thresholds are not thoroughly addressed for
clinical implementation.
Principal investigator's team got inspired from the approach developed by Dennis Lo and
enriched the methodological aspect i/ by comprehensively allowing to control the
statistical errors; ii/ by distinctly identifying key parameters that influence the
diagnosis performance of RHDO; iii/ by pinpointing output features illustrating the
overall quality of the test.
All these factors were then merged, resulting in quality scores and decision threshold
definition.
A preliminary fruitful work involving more than 90 at risk families for 5 disorders was
conducted by principal investigator's team.
Altogether, the workflow appears to be:
Specific: 92/92 (100%) fetal genotype correctly identified, among conclusive tests (i.e.
92 concordant + 0 discordant results) Sensitive: 92/98 (94%) conclusive tests, among all
tests (i.e. 92 conclusive concordant results + 6 inconclusive concordant results)
Clinically viable: turn-around-time of 5-6 working days. Universal: applicable to any
mutational profile (point mutation, deletion, duplication, triplet expansion etc.).
Adaptable: can be easily modified for testing of other SGDs. The present project aims to
take advantage of the unique French collaborative network to make SGD-NIPD possible for
theoretically any monogenic disorder and any family. The investigators wish to build on
this preliminary work to broaden the workflow to each disease of interest for a
comprehensive evaluation of the diagnostic performance of SGD-NIPD. Eventually, this
collaborative achievement will allow the redraw of French landscape of prenatal
diagnosis.
One advantage of the approach proposed in this study is that it is targeted. The RHDO
analysis and its result will be specific of DNA locus involved in the family disorder,
and will not affect other regions of the genome. Consecutively, investigators will not be
confronted to ethical and social issues surrounding full exome or genome sequencing in
the prenatal setting, for example the counselling issues that arise through the
identification of variants of uncertain significance or incidental findings.
SGD-NIPD will be proposed by Multidisciplinary Centers for Prenatal Diagnosis to pregnant
women undergoing invasive prenatal diagnosis in a context of family history of
single-gene disorders because of parental pathogenic mutation.s in one of the following
gene: HBB, CFTR, FMR1, SMN1, DMPK, DMD, NF1, HTT, F8, F9, GCK, L1CAM, PKHD1 or ATP7A.
In the case of MODY-GCK diabetes, fetal growth is dependent on fetal genotype. Insulin
treatment should be considered to reduce the risk of macrosomia only in the 50% of
MODY-GCK mothers with unaffected foetus. However, invasive PND is not recommended in
clinical care due to a significant benefit/risk imbalance. For these MODY-GCK cases, the
result of the SGD-NIPD will be compared to the genotype result of the newborn at birth,
already planned as part of routine care. The benefits of NIPD in the pregnancy management
of women with pathogenic GCK variants has been demonstrated by international experts of
monogenic diabetes.
If the baby inherits the maternal GCK variant- - the baby would be expected to have a
normal birthweight as per background population.
Treatment of maternal hyperglycemia is not recommended in those cases as the baby would
not be expected to have a higher risk of being born large-for-gestational-age than the
background population.
However, if the baby does not inherit the maternal GCK variant, it would have a higher
risk of macrosomia / large-for-gestational-age (~800g increase in birth weight).
Monitoring of fetal growth ultrasound (28,32,36 weeks), treatment for maternal
hyperglycemia with insulin, and planning delivery at 37-39 weeks gestation is recommended
to try to limit fetal growth.
Recruitment capacities are assessed by considering the prevalence of rare diseases and
the activities of each co-investigator center as reported annually in the report of the
biomedicine agency.
