By Abby S. Van Voorhees, MD, August 01, 2013
In this month’s Acta Eruditorum column, Physician Editor Abby S. Van Voorhees, MD, talks with Anne Comi, MD, Douglas Marchuk, PhD, and Jonathan Pevsner, PhD, about their recent New England Journal of Medicine article, “Sturge-Weber Syndrome and Port-Wine Stains Caused by Somatic Mutation in GNAQ.”
Dr. Van Voorhees: Let’s review what has been known about the Sturge-Weber syndrome (SWS). What are the characteristic features? How often is this seen in the population? How often do children who are born with a port-wine (PW) stain have SWS?
Drs. Comi, Marchuk, and Pevsner: The characteristic features of SWS are a facial PW birthmark in the V1 distribution (forehead and/or upper eyelid) associated with abnormal blood vessels in the eye, which frequently cause glaucoma and vision loss, as well as abnormal venous leptomeningeal blood vessels which impair drainage of blood from that area resulting in strokes, seizures, and neurologic and cognitive impairments. About 10 percent of individuals have the characteristic brain involvement but no birthmark. Approximately 1 in 20,000 individuals have SWS. PW birthmarks are much more common, occurring in about 1 in 300 newborns, and may occur anywhere on the head or body. PW birthmarks may be multiple or occur singly and in an isolated fashion, or may be associated with other underlying vascular and tissue malformations, such as in the case of SWS. An infant born with a facial PW birthmark in the V1 distribution has approximately a 20-50 percent chance of brain involvement depending on the size and extent of the birthmark and whether it is unilateral or bilateral. Usually the brain and eye involvement is on the same side as the PW birthmark. The PW birthmark, especially when extensive, is frequently associated with variable degrees of soft and bony hypertrophy of the underlying tissue. [pagebreak]
Dr. Van Voorhees: What has been hypothesized to cause SWS? How about what is thought to cause PW stains — is this thought to be the same pathologic development?
Drs. Comi, Marchuk, and Pevsner: Our overall hypothesis was that SWS would be caused by a somatic mutation in a key gene regulating blood vessel structure and function. By somatic mutation, we mean a mutation that would not be present in the sperm or egg, but which was acquired later in time during fetal development. This mutation would then be present in affected tissue (affected vascular tissue) from the patient, but not in the blood (the typical source of DNA for genetic studies) or in any other unaffected tissue.
The hypothesis that SWS (and other vascular syndromes that are not inherited) might be caused by a somatic mutation has been around for many years. Over 20 years ago, Rudolf Happel suggested that the vascular malformations in SWS, Klippel Trenaunay Weber Syndrome, and others, would be caused by somatic mutations that would be lethal if passed through the germline. This somatic-only occurrence would result in a lack of clear familial inheritance of the trait. This lack of a clear inheritance pattern for nearly all vascular malformations, syndromic or not, fits with this hypothesis. If this hypothesis is true, the identification of the mutated gene would reveal the underlying cause of the vascular malformation and give us the first clue towards a scientifically-based approach to a treatment or cure.
Unfortunately, until very recently, the technology to test Dr. Happel’s hypothesis was not available. The last few years have seen an explosion in new genetic technologies, such that we are now finally in a position to rigorously search for the somatic mutation for vascular lesions. New DNA sequencing technologies have been developed that completely and irrevocably altered the ability to sequence genomes. These new sequencing technologies are being labeled “next-generation” sequencing, and they now sit at the cutting edge of genetic research. We employed these technologies to identify the somatic mutation causing SWS. It is important to point out to SWS patients and families that as these mutations are occurring somatically, they have no bearing on reproductive or other inheritance of the trait in other family members. [pagebreak]
Dr. Van Voorhees: Tell us about your experiment. What did you do, and what did it show?
Drs. Comi, Marchuk, and Pevsner: We performed whole genome sequencing on six samples, using DNA purified from paired affected and unaffected regions of three individuals with SWS.
The presumed affected region consisted of a PW stain region obtained from biopsy, or surgically removed brain tissue. The unaffected region was either a blood sample, unaffected skin, or apparently unaffected brain. We identified a single variant that was predicted to be deleterious, involving a base pair substitution in the GNAQ gene on chromosome 9. Next we confirmed the finding by examining 97 samples from 50 individuals (we used two technologies — deep sequencing and a primer extension assay). Here are the highlights of what we found:
- 9 out of 9 PW stain samples from SWS patients had the mutation.
- 12 out of 13 PW birthmark samples that were not from SWS patients had the mutation, indicating that this mutation underlies both PW birthmarks and SWS.
- 16 of 18 SWS brain samples had the mutation.
- 0 of 6 control brains, as well as 0 of 4 cerebral cavernous malformation (CCM; an unrelated vascular disorder) samples had the mutation.
- Of 669 apparently normal blood or lymphoblastoid cell line samples from the 1000 Genomes Project, 0.7 percent had the mutation; this is consistent with the prevalence of PW birthmark in the general population. [pagebreak]
The finding of a mutation in GNAQ was intriguing. The gene makes a protein, Gq, that has a key role in cell function, including the regulation of blood vessels. When cell surface receptors (such as endothelin receptors) are activated by binding a ligand, Gq is activated, binds and hydrolyzes GTP, and thus initiates an intracellular signaling cascade. The mutation we identified “locks” Gq into a mildly activated state. We demonstrated increased ERK phosphorylation in mammalian cells transfected with the mutant construct.
Dr. Van Voorhees: What other syndromes have similar somatic mosaic mutations? Is there a malignant potential to somatic mutations in some of these syndromes?
Drs. Comi, Marchuk, and Pevsner: Other syndromes occurring by similar somatic mosaic mutations include CLOVES (congenital lipomatous asymmetric overgrowth of the trunk with lymphatic, capillary, venous, and combined-type vascular malformations, epidermal nevi, and skeletal anomalies) and Proteus syndrome. CLOVES syndrome is caused by a somatic mutation in the PIK3CA gene. Proteus syndrome is caused by a somatic mutation in AKT gene. Both of these disorders are somatic overgrowth disorders. PIK3CA and AKT fall along the Ras-Raf-mTOR pathway and both mutations overactivate this pathway. More studies are needed to determine if and how the GNAQ mutation, found to cause SWS and PW birthmarks, impacts this specific pathway. [pagebreak]
There is not known to be an increased risk of cancer in patients with SWS or port-wine birthmarks although this has not been systematically studied yet. Each of the missense mutations observed in participants with CLOVES syndrome has previously been identified in several types of adult-onset cancer. Rather than directly causing cancerous transformation, the PIK3CA
missense mutations increase the tumor’s growth or aggressiveness. When overexpressed, PIK3CA missense mutations identified in participants with CLOVES syndrome have the ability to transform cells. Wilms tumor (MIM 194070) was reported in two CLOVES-affected individuals. It has been hypothesized that the low rate of malignant transformation in individuals with CLOVES syndrome is due to the low level of endogenous PIK3CA expression in most cells. In Proteus syndrome benign tumors occur including lipomas, hemangiomas, vascular malformations, and lymphangiomas. Neoplasms that have been reported include mesothelioma, papillary carcinoma of the thyroid, ovarian serous cystadenoma, meningioma, optic nerve tumor, and endometrial carcinoma. The risk of malignancy is low.
Dr. Van Voorhees: Does the timing of when this mutation occurs during embryonic development affect the presentation or penetrance?Drs. Comi, Marchuk, and Pevsner: We speculate that the timing of the mutation is critical. Perhaps early mutations (e.g. those occurring in the first trimester) might be associated with SWS while relatively later embryonic mutations are associated with the milder phenotype of PW birthmark without SWS. It will be important to determine the cell type(s) that are affected by the GNAQ mutation, a problem we are working on now. It is remarkable that a mutation in the same gene, at the same nucleotide position, is associated with uveal melanoma. Thus the timing and location of the mutation critically affect the clinical presentation.
Dr. Comi is associate professor of neurology and pediatrics at Kennedy Krieger Institute and Johns Hopkins School of Medicine and director of the Hunter Nelson Sturge-Weber Center. Dr. Marchuk is professor and vice-chair in the department of molecular genetics and microbiology at Duke University Medical Center. Dr. Pevsner is associate professor in the department of neurology at Kennedy Krieger Institute and Johns Hopkins School of Medicine. Their article was published online May 8 in the New England Journal of Medicine, 2013; 368:1971-1979. doi:10.1056/NEJMoa1213507.