Genetics is, in a sense, timeless. DNA sequences stay mostly the same in an individual over a lifetime. Minus the inevitable somatic mutations, the genome of a fertilized ovum is much the same as that of the 80-year-old it might one day become.
A PRECEDENT: DIAGNOSIS FROM A BABY TOOTH
My favorite example of genetic time standing still is the three-year-old who died of Rett syndrome in 1991, but wasn’t diagnosed until 2004. A year earlier, her mother had read a magazine article about the syndrome and recognized her daughter’s symptoms: falling, clumsiness, loss of speech, seizures, and the peculiar repetitive hand-wringing characteristic of the disease. Might her daughter be diagnosed posthumously?
The mom had an idea: instead of disinterring the body, could researchers extract DNA from a stored baby tooth? The Australian Rett Syndrome project connected the astute mother with researchers who indeed probed the daughter’s genome in tooth stem cells, finding the telltale mutation in the MECP2 gene.
Diagnosis after death dispelled guilt – the father had blamed himself when his daughter fell down the stairs, and the mother had blamed a vaccination. DNA testing revealed that the mother hadn’t passed on the mutation – it originated in her daughter. And that meant that their other relatives, including the girl’s siblings, weren’t at risk.
The Rett case is a precedent of sorts for “rescue karyotyping” to explain recurrent pregnancy loss, described in a recent paper in Reproductive Biology and Endocrinology, which I summarized for Medscape. In contrast to recent fetal tests, such as non-invasive prenatal testing (NIPT) using cell-free fetal DNA and sequencing fetal genomes, which provide a look forward, rescue karyotyping looks back.
PREGNANCY LOSS IS COMMON
The birth of a healthy baby is against the biological odds. Of every 100 eggs exposed to sperm, 84 are fertilized, and of these, 69 implant in the uterus. There, 42 survive one week or longer, 37 make it past 6 weeks, and only 31 are born. Of the fertilized ova that cease developing, about half have severe chromosomal abnormalities. The halt comes so early that the event usually goes unnoticed. A late and heavy period.
These odds mean that a miscarriage is a rather common event. Partly for this reason, a couple crushed from their first miscarriage may be comforted, told to try again, and sent home. Devastated.
That may happen with a second miscarriage too. It’s usually at pregnancy loss #3 that a health care provider refers a patient to a genetic counselor, who takes a detailed history and then orders a karyotype – a chromosome check – of the prospective parents. But oftentimes the tests come back with the normal 23 pairs.
The conservative stance in testing after pregnancy loss might be because most chromosomal accidents are just that – an errant chromosome doesn’t part from its homolog and instead follows it along, leading to an egg or sperm with one too many or one too few chromosomes. Because most such “aneuploid” situations are independent events, expensive karyotyping doesn’t make sense, at least not economically. But there are other costs.
“‘We don’t test it. Those are the guidelines.’ That’s what my patients who have had repeat miscarriages tell me. Everyone agrees that you don’t test after the first miscarriage, and most agree not to test even after the second, but to wait for the third,” Zev Williams, M.D., Ph.D., director, Program for Early and Recurrent Pregnancy Loss (PEARL), Montefiore Medical Center/Albert Einstein College of Medicine, recently shared with me. Having had such patients myself as a genetic counselor, seeing them typically after the third loss, I checked. Indeed, the American College of Obstetrics and Gynecology and the American Society for Reproductive Medicine recommend karyotyping only after the third spontaneous abortion, although a woman’s age or other problems may accelerate that timetable.
CHROMOSOMAL ACCIDENT OR REPEAT PROBLEM?
Sometimes a more unusual chromosome glitch occurs that can repeat, such as a translocation in which different chromosomes swap parts. The parents would be carriers, but each can make “unbalanced gametes” – eggs or sperm with hunks of genetic material missing or extra. Each conception then faces the not-so-good odds of a chromosomal imbalance that can be incompatible with life or cause birth defects. Knowing about a translocation can be helpful because it recurs with a known frequency, enabling a couple to use technology such as preimplantation genetic diagnosis to avoid poor outcomes in the future.
Checking chromosomes of the parents is eventually necessary because most women whose pregnancies were once ending did not have the presence of mind to collect and bring a sample of tissue (“products of conception”) to a doctor to send for testing. But if she had a D&C (dilation-and-curettage) afterwards, a bit of the tragedy may exist on a shelf somewhere, embedded in paraffin. Dr. Williams and his colleagues have gone back to those samples to try to find out why some pregnancies ended.
Hauling out stored samples may seem a low-tech approach in this age of sequencing genomes, but one that can bring peace of mind. For with recurrent pregnancy loss naturally comes guilt.
“Every patient will blame herself. Was it the argument with her husband? Someone smoking nearby? Did she lift something heavy? One woman went on a ski trip and had a miscarriage a few days after and was convinced it was the ski trip. That’s a horrible feeling to have to think that you did something to cause a pregnancy loss,” said Dr. Williams, whose team is questioning 1500 people on the perception and understanding of miscarriage. “It would provide peace of mind to know that it was a trisomy, a triploidy, a tremendous genetic rearrangement and not the stress at work or the fight with the husband. Rescue karyotyping can give a sense of closure to patients who are wracked with guilt,” he explained.
A BRIEF HISTORY OF THE KARYOTYPE
A human karyotype circa mid-1960s would have shown chromosomes of all the same color arranged in groups by size. A child with what was then called mental retardation might have been diagnosed with a “B-group chromosome disorder.”
Karyotyping progressed through ever-more-specific staining, as knowledge of chromosome structure grew, leading to FISH – fluorescence in situ hybridization. FISH uses DNA probes to highlight specific DNA sequences rather than larger-scale structural nuances that affect how dyes bind.
Then came array comparative genomic hybridization (array CGH) and the ability to detect microdeletions and microduplications. This is done during pregnancy and to diagnose children with unexplained developmental delay. But it was the use of array CGH in cancer genetics, on paraffin-embedded tumor samples, that inspired Dr. Williams to retrieve stored tissue from miscarriages past.
“In the cancer field, the push was to do more sensitive testing using higher and higher resolution arrays, to look at small rearrangements. We are looking for higher level anomalies, missing much more, so less stringency is needed. A sample might come back saying ‘insufficient material’ if you want to find a 5 kilobase deletion, but not if it is a question of missing an entire chromosome. That’s easy to answer,” Dr. Williams said. CGH reveals anomalies within that range.
To test the feasibility of rescue karyotyping, the researchers used array CGH on 20 specimens from 17 women who had had recurrent pregnancy loss. Of the four women who’d had fetal chromosomes checked while they were pregnant, three attempts had failed. So rescue karyotyping provided new information on old samples.
Sixteen samples had enough DNA to analyze; the oldest had been stored more than four years. And chromosomal glitches showed up in 8 of the 16: three trisomies (an extra chromosome in all sampled cells), one mosaic trisomy (extra chromosome in some sampled cells), two partial deletions, and two unclassified variants.
As expected, most of the findings indicated a one-time event. But any result is important, Dr. Williams maintains, because of the alleviation of guilt. And the testing seems easy enough to do – once the strategy is validated and standards established, a health care provider would need only find and send tissue blocks to a testing facility.
GENOME SEQUENCING: TMI
Karyotyping is a classic technique, perhaps soon to be supplanted by whole genome sequencing, which Dr. Williams and his group and others are already doing. But is that too much information?
“The problem is interpreting the results. All of us have about 2000 mutations. It’s difficult to tell which ones are completely benign. Some might have some advantage, and others might be the cause of a miscarriage. Whole genome sequencing of a fetus will be a difficult route,” he warns.
But in the meantime, while annotators work furiously to figure out what everything in the genome means, DNA tests on stored products of conception are making past pregnancy losses, for some couples, a little easier to bear.