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Study Tests Prenatal Sequencing to Diagnose Fetuses with Apparently Balanced Translocations

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An abbreviated paired-end whole-genome sequencing method could be used to identify causal disruptions in cases of balanced translocations identified by karyotyping after abnormal fetal ultrasound more effectively than with microarrays, according to researchers from Harvard Medical School.

The group published a case study in the New England Journal of Medicine last week describing its use of the technique — called large-insert mate-pair, or jumping-library sequencing — to identify disruption of the gene CHD7 in an apparently balanced translocation as the cause of a disorder called CHARGE syndrome, which claimed the life of an infant shortly after birth.

The group is still researching the approach, but Cynthia Morton, the senior author of the study, told Clinical Sequencing News that the team believes strongly that the process could be a useful clinical service.

"At this point, as much as we could handle cases, either from our own lab or from outside hospitals, we would be interested to take them," Morton said.

Currently, some labs offer comparative genomic hybridization microarray analysis after karyotyping indicates an unbalanced translocation, but there are many cases where CGH arrays will not find the causative genetic aberration. "The only way to find these aberrations is sequencing," she said.

"So I think [this approach] will allow us to provide much more precision in the information we give to patients compared to giving them just a risk number."

According to Morton, though the sequencing was not able to inform a timely medical intervention for the fetus in the group's case study, the researchers believe that in other cases the approach could provide important information for parents and physicians who are either making decisions about pregnancy termination or anticipating the birth of a child with an abnormality.

"You could know better what to expect — what you are going to need to do at delivery," such as whether a child will need breathing or cardiac interventions, she said. She acknowledged, however, that "some couples undoubtedly would decide that the information is something they are not comfortable with proceeding with."

According to Morton, the team has already used the technique on several additional subjects, including one, the most recent, from an outside hospital. In the last four cases, there was no abnormality apparent after array-based CGH analysis, but in at least some, the sequencing method was able to identify a potentially causative variant.

In the recently reported case study, the researchers performed their jumping-library sequencing method after array-based CGH analysis failed to identify a clinically significant loss or gain of material at the chromosomal breakpoint in a subject fetus with a confirmed, apparently balanced translocation.

The sequencing approach involves shearing a DNA sample into approximately 2-kb lengths, creating libraries, and sequencing their paired ends to identify chimeric reads, which indicate the presence of a translocation or inversion breakpoint.

"What we are looking for is to find the sequences that cross the inversion or translocation breakpoint and to look carefully right there to see if there was any gene that was broken or dysregulated by that," Morton explained.

"We don't need to deeply sequence the whole genome," she said. "The sequence itself that we are reading is just a little piece at the end of those 2-kb fragments. If the fragment contains the breakpoint, one end would be present on chromosome 6 and the other end would be present on chromosome 8, for example.

The group then amplifies additional DNA using PCR primers designed using the chimeric sequence reads supporting the translocation junction and sequences them to confirm the break.

In their case study, Morton and her colleagues reported that the process yielded 282 million individual reads, or 141 million pairs, using 25-cycle sequencing on a single lane of an Illumina HiSeq 2000. The inserts between aligned pairs covered each base in the genome 68 times, the group wrote, despite a mean coverage of "only two reads spanning each nucleotide."

The group identified only one group of chimeric reads: with one end mapping to chromosome 6 and the other to chromosome 8. This set had 35 read pairs with high mapping quality, according to the group.

The researchers found that the translocation breakpoint disrupted the gene CHD7 in chromosome 8, and the gene LMBRD1 in chromosome 6. According to the group, analysis of copy number variant data suggested that disruptions of a single copy of CHD7 can cause CHARGE syndrome. No relevant disorders appeared to be associated with LMBRD1 variations.

According to the group, the study highlights the fact that "rearrangements appearing to be balanced at karyotypic resolution can be complex at nucleotide resolution."

The team's jumping-library sequencing method — developed by the group initially for more basic research into the function of genes at chromosomal break points in children with congenital malformations — is a temporary measure, Morton said, considering how rapidly the costs of whole-genome sequencing are dropping. Eventually, it will become preferable to do whole-genome sequencing instead of using jumping libraries, she said.

Morton said that though the results in the initial case study did not help the child, the team believes the process can work in a time frame similar to conventional cytogenetic prenatal diagnostic methods, and at a comparable cost.

"This is still a research study, she said, "But we would say it's comparable to the costs that are now in place for prenatal diagnostics such as karyotyping, [which are] in the $1,500 range."

According to Morton, the literature suggests that nine percent of inversions and about six percent of translocations found by karyotyping after an abnormal ultrasound are likely to cause a pathological gene variant or otherwise disrupt a gene to the detriment of the fetus.

"The question for the family is whether they are in that 9 percent or 6 percent," she said. Considering the odds, more often than not, the group would expect their sequencing analysis to offer encouraging news.

For cases where the approach does identify a pathological disruption, the knowledge could help parents either plan for specific treatments, or potentially decide to terminate a pregnancy.

"For sure there are now interventional fetal surgeries that can be done," in addition to post-birth treatments, Morton said.

"And I'm sure that some would also choose to terminate a pregnancy based on a disruption of a gene like the one in this case, which is known to be well-associated with a particular malformation syndrome," she added.

"However," she added. "Parents [currently] make these same decisions based on just ultrasound findings." In fact, in the group's most recent case, the family decided to terminate the pregnancy based on ultrasound findings of a brain abnormality before the researchers had results on whether the fetus's chromosomal abnormality was present in either parent, she said.

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