Tara Haelle

January 27, 2017

LAS VEGAS — A new type of fetal genomic sequencing has identified the underlying genetic cause of major congenital anomalies that karyotyping and DNA microarray have not been able to explain, new research shows.

Whole-exome sequencing has been used to identify the single-point genetic mutations responsible for 7.7% of major congenital anomalies and the mutations thought to be responsible for 17.9%.

"These are structural anomalies we see frequently and struggle with," said lead investigator Ronald Wapner, MD, professor of obstetrics and gynecology, director of reproductive genetics, and vice-chair of research at the Columbia University Medical Center in New York City.

The exome makes up the 1.5% of the genome that encodes proteins. It is the functional part of the genome that determines the way genes are expressed, and can reveal sex-linked and chromosomal recessive and dominant traits not detectable with microarray.

"Fetal anomalies are among the most frequent problems we see, and they affect approximately 3% of all pregnancies," Dr Wapner said. Although these anomalies are a leading cause of infant death, researchers do not know what causes more than half of them. Currently, karyotyping can identify 25% to 30% of anomalies, and DNA microarray can identify another 5% to 7%.

In this study, "our goal was to determine the incremental value of whole-exome sequencing in sequential, unselected cases of fetal structural anomalies," Dr Wapner explained here at the Society for Maternal-Fetal Medicine 2017 Annual Pregnancy Meeting.

Previous research has shown that whole-exome sequencing identifies 10% to 30% of the pathogenic mutations that cause fetal structural anomalies, but those studies involved very specific populations that were likely to yield a genetic cause for the defects.

Prenatal Testing

In their study, Dr Wapner and his colleagues examined patients who showed evidence of a fetal structural anomaly, including nuchal translucency of at least 3.5 mm. For women already undergoing diagnostic prenatal testing, the investigators conducted whole-exome sequencing, karyotyping, and chromosomal microarray on amniotic fluid or chorionic villus samples; for the other study participants, they used cord blood at birth. The team also sequenced the mother and father so they could distinguish between likely heritable mutations and de novo mutations. If parental blood was not available or if there was a family history of a Mendelian disorder, participants were excluded from the analysis.

Qualifying variants met certain rules on quality and pathogenicity but were not restricted to those in known disease genes. A subset of these, bioinformatic signatures, were plausible but unproven pathogenic variants that were of "increased interest" because "genotype and population genetics demonstrated a high likelihood of disruption in protein formation in humans," Dr Wapner explained.

To be used for diagnosis, a clinical geneticist, the lab, and a clinician had to agree that the genotype fit both the known genetic model of disease for that gene and the observed phenotype.

Of the 168 patients included in the final analysis, 13 (7.7%) received a genetic diagnoses on the basis of whole-exome sequencing: nine were de novo mutations, two were autosomal recessive mutations, one was an inherited mutation, and one was an X-linked recessive mutation. In another 30 patients (17.9%), a bioinformatic signature was identified.

Of the 124 patients with a single anomaly, six (4.8%) had a genetic diagnosis and 18 (14.5%) had a bioinformatic signature. Of the 14 patients with multiple anomalies, seven of 44 total anomalies (15.9%) had a genetic diagnosis and 12 (27.3%) had a bioinformatic signature.

Central Nervous System and Skeletal Defects

The most common anomalies identified with whole-exome sequencing were central nervous system defects, which accounted for 16.1% of genetic diagnoses and 22.6% of bioinformatic signatures, and skeletal defects, which accounted for 22.7% of genetic diagnoses and 22.7% of bioinformatic signatures.

Overall, heart and nuchal defects were the most common anomalies identified — 29.7% and 18.4%, respectively — but genetic diagnoses were identified in only 3.4% of the heart defects and 8.3% of the nuchal defects.

"In the heart defects, we saw fewer malformations than seemed to be caused by a single gene, suggesting that there may be other mechanisms," Dr Wapner told Medscape Medical News. "There is some research going on showing that there are mechanisms other than a single gene — maybe a combination of multiple genes or epigenetics or possibly other mechanisms."

The investigators compared data from their fetus–mother–father trios with those from a control group of 481 trios. A de novo mutation presumed to be associated with disease in the Online Mendelian Inheritance in Man database was more common in the study population than in the control group (12.5% vs 4.2%).

Whole-exome sequencing can help identify phenotypes of de novo mutations that might not fully emerge until later in childhood or adulthood. Therefore, an understanding of the connections between genotypes and phenotypes could uncover the potential effects of mutations or pathologies. Researchers using different registries, including government ones, will have to cooperate to achieve this goal, Dr Wapner explained.

"We really need to work together. All these malformations are rare, and none of us is going to see enough of one, but if we all put our data together, we'll learn," he told Medscape Medical News.

Ethical Considerations

The separation of maternal–fetal records from a child's medical records after birth is an obstacle that will have to be overcome, he pointed out. And the clinical use of whole-exome sequencing will require experienced, knowledgeable researchers.

"Interpretation and analysis of whole-exome sequencing is complex and should be a collaborative effort of the lab, genetic counselors, clinical geneticists, and maternal–fetal medicine specialists experienced in genomic interpretation," he advised.

But Dr Wapner said he foresees a time when whole-exome sequencing is a standard component of prenatal care. In the short term, the technology will be used primarily by specialists so that they can appropriately and accurately counsel families about fetuses with anomalies.

Moving forward, there is "no reason that we won't be able to screen the population for these mutations," he told Medscape Medical News. "It's very possible that, in the future, we'll be able to treat a lot of these things if we diagnose early enough. We're not there yet, but if you've got a single gene that's not working, there are genetic techniques you can use to modify genes if you get to it early enough in pregnancy."

Although whole-exome sequencing is considerably less expensive than whole-genome testing to identify rare, single-point mutations, cost remains a consideration, said Christopher Robinson, MD, from Charleston Maternal and Fetal Medicine in South Carolina, who is associate editor of the American Journal of Perinatology.

It has yet to be determined how this technology will be applied in clinical care, he told Medscape Medical News. Interpretation and genetic counseling its "far too complex" for one clinician to tackle, he emphasized, so multiple specialists will have to work together. "Otherwise, people will fire off a whole-genomic sequence and not know how to interpret it," he explained.

Does this mean Johnny is going to need speech therapy or Johnny is going to need full-time care? If you don't know how to answer that, don't order the test.

This study provides a compelling and "thought-provoking start" to the use of whole-exome sequencing, but many issues need to be addressed before it enters routine clinical use, said Kenneth Chan, MD, a clinical professor of maternal–fetal medicine at the University of California, Irvine.

"We still don't know what some of the findings mean or how practical it is for everyone," Dr Chan told Medscape Medical News. "When you order any test, you want to know what to tell your patient. Does this mean Johnny is going to need speech therapy or Johnny is going to need full-time care? If you don't know how to answer that, don't order the test."

He said he expects to see new findings every 6 months on one gene or another; however, these could be "a red herring or a true pathology." This highlights the need for thorough, conscientious counseling. "Some patients will consider a motor delay nothing whereas others will terminate — but you have to divulge the information," he said.

Like any new technology, there are ethical issues related to this sequencing technique, Dr Wapner acknowledged.

Dr Robinson and Dr Chan have disclosed no relevant financial relationships. Dr Wapner is conducting studies that are funded by Natera, Sequenom, and Illumina, and he has consulted for LabCorp.

Society for Maternal-Fetal Medicine (SMFM) 2017 Annual Pregnancy Meeting. Presented January 26, 2017.


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