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Sequencing Uncovers Rare Variants Linked to Noonan Syndrome, Aids in Diagnosis

NEW YORK (GenomeWeb) – By sequencing a cohort of people diagnosed with Noonan syndrome but without known disease mutations, researchers led by Harvard Medical School's Raju Kucherlapati uncovered rare variants associated with the condition.

As Kucherlapati and his colleagues report in the Proceedings of the National Academy of Sciences this week, they found a number of gain-of-function as well as loss-of-function variants that follow-up functional studies suggested could cause Noonan syndrome. They also, though, found mutations in genes that cause other developmental syndromes, leading to changes in diagnosis for a handful of patients.

"[O]ur data suggest that next-generation sequencing can provide a useful adjunct to RASopathy diagnosis and emphasize that the standard clinical categories for RASopathies might not be adequate to describe all patients," the researchers said in their paper.

Noonan syndrome, which is the most common RASopathy, is marked by short stature, developmental delay, and cardiac defects. While the genetic basis of about three quarters of cases is known, the causes of the remainder have been elusive.

Kucherlapati and his colleagues performed exome or whole-genome sequencing on samples from 27 NS patients, and then narrowed their search for disease-causing variants to mutations affecting the RAS-ERK pathway. All known NS variants, they noted, affect that pathway.

From this, they identified a number of novel variants, including ones in RIT1, RASA2, and NF1. The mutations they found included gain-of-function as well as loss-of-function variants.

For instance, the researchers found that five patients who had four different missense mutations in RIT1, which they confirmed by Sanger sequencing. These mutations were located in regions that are conserved across vertebrates, and three of those four mutations were predicted to affect protein function.

Through functional assays, they reported that the four RIT1 mutations led to increased p-ERK levels and that three of the four mutations were linked to increased EGF-induced ERK activation. RIT1, the researchers reported, does not have a role in normal RAS-ERK signaling, and these mutant forms, rather, act as neomorphs.

Other variants, such as missense mutations in RASA2, led to the loss of function, though those, too, increased RAS-ERK pathway expression. The researchers further reported potential candidate genes in SPRY1 and MAP3K8.

Additionally, some mutations appeared to be working in cahoots. While many of the patients the researchers sequenced had variants in multiple genes linked to the RAS-ERK pathway, in two patients, the researchers suspected that the symptoms exhibited by the patients were due to the influence of two variants.

For instance, one patient had variants in both RIT1 and RASA2, and Kucherlapati and his colleagues suggested that that patient's atypical heart problems stemmed from the combined effects of those mutations.

Some of the researchers' findings prompted re-consideration of diagnoses for a handful of patients.

Two patients were found to have novel mutations in NF1. NF1 encodes neurofibromin, which is a RAS-GTPase-activating protein, and is linked to neurofibromatosis, a related RASopathy.

Based on both the researchers uncovering a nonsense mutation in his NF1 gene and clinical re-examination, one patient's diagnosis was changed to neurofibromatosis-NS. And the authors suggested that a second patient be monitored for NF-1-specific symptoms and complications after finding that she had a likely damaging amino acid substitution in NF1.

Another individual had a mutation in RPS6KA3, which encodes RSK2, and causes Coffin-Lowry syndrome.

"Our results indicate that NGS can aid in the often challenging differential diagnosis of young patients with developmental syndromes," Kucherlapati and his colleagues said.

They noted that as the RAS-ERK pathway has been studied for its role in cancer, there are drugs under development to target it that could potentially also treat RASopathy patients. "[I]dentifying the causative allele in each NS patient is likely to be critical for choosing an effective therapy," they added.