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Genetic Variants Associated with Port-Wine Stains.

Frigerio A, Wright K, Wooderchak-Donahue W, Tan OT, Margraf R, Stevenson DA, Grimmer JF, Bayrak-Toydemir P - PLoS ONE (2015)

Bottom Line: Importantly, affected and healthy skin tissue from the same individual were compared.A novel somatic variant GNAQ, c.547C>G, p.Arg183Gly was found in one case with 4% allele frequency.Two novel somatic variants were also found in RASA1, although neither was predicted to be deleterious.

View Article: PubMed Central - PubMed

Affiliation: Carolyn and Peter Lynch Center for Laser and Reconstructive Surgery, Division of Facial Plastic and Reconstructive Surgery, Department of Otology and Laryngology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA, United States of America.

ABSTRACT

Background: Port-wine stains (PWS) are capillary malformations, typically located in the dermis of the head and neck, affecting 0.3% of the population. Current theories suggest that port-wine stains are caused by somatic mutations that disrupt vascular development.

Objectives: Understanding PWS genetic determinants could provide insight into new treatments.

Methods: Our study used a custom next generation sequencing (NGS) panel and digital polymerase chain reaction to investigate genetic variants in 12 individuals with isolated port-wine stains. Importantly, affected and healthy skin tissue from the same individual were compared. A subtractive correction method was developed to eliminate background noise from NGS data. This allowed the detection of a very low level of mosaicism.

Results: A novel somatic variant GNAQ, c.547C>G, p.Arg183Gly was found in one case with 4% allele frequency. The previously reported GNAQ c.548G>A, p.Arg183Gln was confirmed in 9 of 12 cases with an allele frequency ranging from 1.73 to 7.42%. Digital polymerase chain reaction confirmed novel variants detected by next generation sequencing. Two novel somatic variants were also found in RASA1, although neither was predicted to be deleterious.

Conclusions: This is the second largest study on isolated, non-syndromic PWS. Our data suggest that GNAQ is the main genetic determinant in this condition. Moreover, isolated port-wine stains are distinct from capillary malformations seen in RASA1 disorders, which will be helpful in clinical evaluation.

No MeSH data available.


Related in: MedlinePlus

NGS and dPCR results for GNAQ and RASA1 variants.For 2a and 2b, left panels depict the NGS data with the respective somatic GNAQ mutation (arrow). Right panels show corresponding digital PCR results analyzed using the QuantStudio 3D AnalysisSuite software. Relative intensities of FAM were plotted against VIC. The mutant allele (blue) depicts the level of somatic mosaicism in the sample versus the wild type allele (red), both alleles (green), and no amplified alleles (yellow). For 2c and 2d, NGS data with the respective somatic RASA1 mutation (arrow) are shown from cases 5 and 7, respectively.
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pone.0133158.g002: NGS and dPCR results for GNAQ and RASA1 variants.For 2a and 2b, left panels depict the NGS data with the respective somatic GNAQ mutation (arrow). Right panels show corresponding digital PCR results analyzed using the QuantStudio 3D AnalysisSuite software. Relative intensities of FAM were plotted against VIC. The mutant allele (blue) depicts the level of somatic mosaicism in the sample versus the wild type allele (red), both alleles (green), and no amplified alleles (yellow). For 2c and 2d, NGS data with the respective somatic RASA1 mutation (arrow) are shown from cases 5 and 7, respectively.

Mentions: Of 12 PWS skin samples, 9 (75%) harbored the known GNAQ c.548G>A, p.Arg183Gln somatic change (Fig 2), with a mutation frequency of >1%. NGS captured data showed mutant allele frequencies between 1.73–7.42% in affected tissues, 0.00–0.13% in control skins, and 0.06% in healthy control DNA. Two cases had a GNAQ c.548G>A mutation frequency less than 0.2% by NGS indicating that they were negative for this mutation. Digital PCR results for the GNAQ c.548G>A, p.Arg183Gln variant were highly concordant with the NGS panel data, with mutant allele read frequencies ranging between 0.85% ± 0.04 and 7.42% ± 0.04 in affected tissues positive by NGS, 0.00% ± 0.06 and 0.80% ± 1.20 in control skins, and 0.22% ± 0.12 in healthy control DNA (Table 2). Two cases of isolated PWS of the limbs (right wrist from Case #3 and right shin from Case #9, Fig 1B) were negative for somatic GNAQ variants based on the 1% limit of detection for both assays (NGS and digital PCR).


Genetic Variants Associated with Port-Wine Stains.

Frigerio A, Wright K, Wooderchak-Donahue W, Tan OT, Margraf R, Stevenson DA, Grimmer JF, Bayrak-Toydemir P - PLoS ONE (2015)

NGS and dPCR results for GNAQ and RASA1 variants.For 2a and 2b, left panels depict the NGS data with the respective somatic GNAQ mutation (arrow). Right panels show corresponding digital PCR results analyzed using the QuantStudio 3D AnalysisSuite software. Relative intensities of FAM were plotted against VIC. The mutant allele (blue) depicts the level of somatic mosaicism in the sample versus the wild type allele (red), both alleles (green), and no amplified alleles (yellow). For 2c and 2d, NGS data with the respective somatic RASA1 mutation (arrow) are shown from cases 5 and 7, respectively.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4508108&req=5

pone.0133158.g002: NGS and dPCR results for GNAQ and RASA1 variants.For 2a and 2b, left panels depict the NGS data with the respective somatic GNAQ mutation (arrow). Right panels show corresponding digital PCR results analyzed using the QuantStudio 3D AnalysisSuite software. Relative intensities of FAM were plotted against VIC. The mutant allele (blue) depicts the level of somatic mosaicism in the sample versus the wild type allele (red), both alleles (green), and no amplified alleles (yellow). For 2c and 2d, NGS data with the respective somatic RASA1 mutation (arrow) are shown from cases 5 and 7, respectively.
Mentions: Of 12 PWS skin samples, 9 (75%) harbored the known GNAQ c.548G>A, p.Arg183Gln somatic change (Fig 2), with a mutation frequency of >1%. NGS captured data showed mutant allele frequencies between 1.73–7.42% in affected tissues, 0.00–0.13% in control skins, and 0.06% in healthy control DNA. Two cases had a GNAQ c.548G>A mutation frequency less than 0.2% by NGS indicating that they were negative for this mutation. Digital PCR results for the GNAQ c.548G>A, p.Arg183Gln variant were highly concordant with the NGS panel data, with mutant allele read frequencies ranging between 0.85% ± 0.04 and 7.42% ± 0.04 in affected tissues positive by NGS, 0.00% ± 0.06 and 0.80% ± 1.20 in control skins, and 0.22% ± 0.12 in healthy control DNA (Table 2). Two cases of isolated PWS of the limbs (right wrist from Case #3 and right shin from Case #9, Fig 1B) were negative for somatic GNAQ variants based on the 1% limit of detection for both assays (NGS and digital PCR).

Bottom Line: Importantly, affected and healthy skin tissue from the same individual were compared.A novel somatic variant GNAQ, c.547C>G, p.Arg183Gly was found in one case with 4% allele frequency.Two novel somatic variants were also found in RASA1, although neither was predicted to be deleterious.

View Article: PubMed Central - PubMed

Affiliation: Carolyn and Peter Lynch Center for Laser and Reconstructive Surgery, Division of Facial Plastic and Reconstructive Surgery, Department of Otology and Laryngology, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA, United States of America.

ABSTRACT

Background: Port-wine stains (PWS) are capillary malformations, typically located in the dermis of the head and neck, affecting 0.3% of the population. Current theories suggest that port-wine stains are caused by somatic mutations that disrupt vascular development.

Objectives: Understanding PWS genetic determinants could provide insight into new treatments.

Methods: Our study used a custom next generation sequencing (NGS) panel and digital polymerase chain reaction to investigate genetic variants in 12 individuals with isolated port-wine stains. Importantly, affected and healthy skin tissue from the same individual were compared. A subtractive correction method was developed to eliminate background noise from NGS data. This allowed the detection of a very low level of mosaicism.

Results: A novel somatic variant GNAQ, c.547C>G, p.Arg183Gly was found in one case with 4% allele frequency. The previously reported GNAQ c.548G>A, p.Arg183Gln was confirmed in 9 of 12 cases with an allele frequency ranging from 1.73 to 7.42%. Digital polymerase chain reaction confirmed novel variants detected by next generation sequencing. Two novel somatic variants were also found in RASA1, although neither was predicted to be deleterious.

Conclusions: This is the second largest study on isolated, non-syndromic PWS. Our data suggest that GNAQ is the main genetic determinant in this condition. Moreover, isolated port-wine stains are distinct from capillary malformations seen in RASA1 disorders, which will be helpful in clinical evaluation.

No MeSH data available.


Related in: MedlinePlus