Researchers from Baylor College of Medicine and Texas Children's Hospital have published a study in which Complete Genomics' whole-genome sequencing and long-fragment read analysis revealed a mutation in MAGEL2 as the likely cause of a child's Prader-Willi-like symptoms.
Further retrospective analysis of Baylor's clinical whole-exome database identified another three subjects with similar phenotypes and probable pathogenic mutations in MAGEL2, the group reported in a letter in Nature Genetics online Sept. 29.
Brock Peters, Complete Genomics' director of research, told Clinical Sequencing News this week that the discovery underscores the importance of being able to distinguish between maternal and paternal variants, something he said requires the use of LFR, or a similar approach that is able to resolve haplotype.
Because MAGEL2 is silenced on the maternal side, and only expressed in paternally inherited alleles, only point mutations on the paternal copy of the MAGEL2 gene should influence the disorder.
"Sequencing the parents and the child, we found this one-base deletion in the MAGEL2 gene," Peters said. "It was de novo, so then the question became, since it's an imprinted gene and not expressed from the mother's chromosome, can we show that this mutation is coming from the father and therefore potentially the cause of this child's disorder."
"LFR, or something like it, is the only way you can do it short of designing a special assay," he explained.
Complete Genomics previously has said that it would launch the long-fragment read technology commercially for the clinical market in early 2013 (CSN 3/14/2012), but Peters said this week that LFR "in its current form" will not be part of the company's clinical sequencing service. However, the approach in some other form is "almost certainly going to be part of [Complete's strategy] once we start providing clinical genomes," he said.
In the study, the researchers describe a single initial case sequenced by Complete Genomics as part of a larger cohort of medical mystery cases.
In this individual, the company's sequencing service identified a heterozygous de novo point mutation in MAGEL2, which is one of the protein-coding genes in the Prader-Willi domain, and LFR analysis determined that the mutation was indeed on the paternal allele.
Christian Schaaf, a professor of molecular and human genetics at Baylor and first author of the study told CSN that he was consulted to assess the subject's phenotype in light of the discovery, to determine whether the mutation might be a likely cause of his symptoms, which were not completely typical of PWS but shared many features of the disorder.
"Till now the gold standard for Prader-Willi has been methylation testing, which is abnormal in more than 90 percent of PW cases. But this patient was negative," Schaaf said.
"As he grew up, it became clear that he was not a typical case, but he continued to have a lot of the features [of the disease.] He had other testing when chromosome microarrays later became standard of care for all individuals with intellectual disability and autism — both of which he has — but that was also negative."
"This mutation, because of the physiological context of Prader-Willi, stood out as a very strong candidate in this individual, and then it was shown that it was a de novo mutation and Complete Genomics was also able to show [based on their long-fragment read method] that the mutation is on the paternal copy," Schaaf explained.
Based on this discovery, Schaaf said, the researchers were then able to go back to a retrospective dataset from Baylor's clinical whole-exome sequencing service; reviewing more than 1,200 exome sequencing reports to identify another three subjects with nonsense or frameshift indel mutations in MAGEL2 who also had symptoms of PWS.
All three, like the first subject, also had normal methylation testing for PWS and normal chromosome microarray analysis results.
In analyzing these additional three patients, Schaaf said he and his colleagues developed a specific assay relying on a methylation-specific restriction enzyme to determine the maternal or paternal lineage, and so did not need to use Completed Genomics' long-fragment analysis.
"As more patients became available, we actually came up with another design which is [easier], and much less expensive, and could be done in a high-throughput manner," Schaaf said. "But this was only possible after the initial mutation was identified in the first subject," he explained.
"The [three additional subjects] had undergone exome sequencing, but because MAGEL2 was not a known disease gene yet, these cases had been signed out as unsolved," he said.
"It's a neat example of how even if you have parental whole-genome information, without doing something like we did in this study you still can't pin down whether this particular mutation came from mom or dad," Complete Genomics' Peters added.
According to Schaaf, one of the challenges of sequencing MAGEL2 is that the gene is relatively GC-rich. "In some segments the content is higher than 75 percent," he said. "And when we went back and looked at some of the exome data and the exome capture [when we identified the three additional cases] MAGEL2 was not even captured very well."
"I think that probably is one of the reasons it's been challenging to identify and will continue to be challenging. You really need a very accurate sequencing to capture this and identify mutations in this gene."
LFR, beyond being able to identify the maternal or paternal chromosomal location of a mutation, is also something that allows the company to increase the accuracy of its genome sequencing method overall, Peters said.
"With de novo mutations you typically pick up about 50 each generation. More and more we are finding that these sorts of mutations are really important in various diseases, so being able to accurately identify these is really important," he said.
"We didn’t go into it in this paper, but LFR also helps us improve our error rates," Peters said. "You need a process that is incredibly accurate since there are only about 50 of these, and if your whole sequencing process gives thousands of errors, you can't distinguish those errors from de novo mutations. So, in the future, LFR or a similar technology is going to be critical for being able to identify these things."
Peters said the company is excited to be involved in any study where it can highlight the benefits of the "additional layer of data" LFR provides on top of high quality whole-genome sequencing.
"We do have other collaborations [like this]," he said. "But we haven’t published anything else yet."
"This is just one example, but if you have somebody who, for instance, has two mutations in a gene separated by a couple of kilobases … LFR is able to sort that out, to see if you got both bad copies from one or both parents. That's another example of the kinds of analysis we think we can do with this," he said.
According to Schaaf, the group's discovery has real significance for the management of children with symptoms of PWS, specifically those who, like the subjects in the recent study, also show signs of autism and intellectual disability.
"This is certainly something to be looked at in those cases of individuals with Prader-Willi syndrome with normal methylation tests," Schaaf said. "Even more, since all these individuals have autism, this is a gene that should be considered in autism cases where some of these [mild Prader-Willi] features are also present."
In their Nature Genetics letter, the authors wrote that on the basis of the study results, they recommend considering MAGEL2 or whole-exome sequencing in cases of complex autism, "especially in individuals with a history of neonatal hypotonia, feeding difficulties, or hypogonadism."