A Somatic P.G45E GJB2 Mutation Causing Porokeratotic Eccrine Ostial and Dermal Duct Nevus

Research

Case Report/Case Series A Somatic p.G45E GJB2 Mutation Causing Porokeratotic Eccrine Ostial and Dermal Duct Nevus

Jonathan L. Levinsohn, BA; Jennifer M. McNiff, MD; Richard J. Antaya, MD; Keith A. Choate, MD, PhD

Supplemental content at IMPORTANCE Recent data demonstrated somatic mutations in GJB2 that were present in jamadermatology.com affected porokeratotic eccrine ostial and dermal duct nevus (PEODDN) tissue but absent in unaffected skin. Recognizing that PEODDN lesions can also appear in individuals with keratitis-ichthyosis-deafness syndrome and finding somatic mutations in their cohort, the authors concluded that somatic GJB2 mutation may cause PEODDN. By using whole-exome sequencing, we show that somatic GJB2 mutation alone is sufficient to cause PEODDN.

OBSERVATIONS We performed whole-exome sequencing of paired blood and affected tissue samples isolated from a PEODDN lesion of a primary school-aged female patient with bands of hyperkeratotic-affected skin on the upper and lower extremities and trunk, and identified a Author Affiliations: Department of Dermatology, Yale University School single, protein-damaging p.Gly45Glu GJB2 mutation present in tissue samples but not in of Medicine, New Haven, Connecticut blood samples. (Levinsohn, McNiff, Antaya, Choate); Department of Pathology, Yale University School of Medicine, CONCLUSION AND RELEVANCE Our results prove that somatic GJB2 mutation is sufficient to New Haven, Connecticut (Levinsohn, cause PEODDN. Dominantly inherited GJB2 mutations, including the p.Gly45Glu found in our McNiff, Choate); Department of case, have been shown to cause the severe multisystem disorder keratitis-ichthyosis- Genetics, Yale University School of deafness syndrome. GJB2 encodes connexin 26, a gap junction protein, which permits Medicine, New Haven, Connecticut (Levinsohn, Choate); Department of intercellular ion and macromolecule flux. Individuals with somatic mosaicism are at risk for Pediatrics, Yale University School of transmitting systemic disease to their offspring, and all individuals with PEODDN lesions Medicine, New Haven, Connecticut should be counseled regarding the risk of having a child with keratitis-ichthyosis-deafness (Antaya). syndrome. Corresponding Author: Keith A. Choate, MD, PhD, Department of Dermatology, Yale University School JAMA Dermatol. 2015;151(6):638-641. doi:10.1001/jamadermatol.2014.5069 of Medicine, 333 Cedar St, Published online February 18, 2015. New Haven, CT 06519 ([email protected]).

orokeratotic eccrine ostial and dermal duct nevus we aimed to use whole-exome sequencing to determine (PEODDN) is a mosaic disorder presenting clinically whether GJB2 mutation alone is sufficient to cause P as a linear epidermal nevus with spiny hyperkerato- PEODDN. sis, and the results of histologic examination show hyperkeratosis, acanthosis, and porokeratotic plugs emerging from eccrine ducts. Like other epidermal mosaic disorders, Report of a Case PEODDN typically appears in linear patterns following lines of Blaschko,1 which represent migration paths of neuroecto- We identified a primary school–aged female patient with bands dermal precursors during development.2 Our group and of hyperkeratotic-affected skin on the upper and lower ex- others have shown that lesions following these patterns tremities and trunk (Figure 1A and B). These lesions were pres- typically result from somatic mutation affecting keratino- ent at birth and did not appear to change in shape or relative cyte precursors, with the extent of cutaneous lesions deter- size over time. Results of histopathologic examination showed mined by mutation timing.3-6 Notably, PEODDN lesions characteristic hyperkeratosis, acanthosis, and coronoid lamel- have been identified in Blaschkoid patterns in healthy indi- lae (Figure 1C and D), and we made the diagnosis of PEODDN. viduals and in those with keratitis-ichthyosis-deafness The patient’s parent provided written informed consent. Our (KID) syndrome due to autosomal dominant GJB2 mutation, study was approved by the Yale University Human Investiga- which is characterized by keratitis, widespread kerato- tional Committee and complies with the Declaration of Hel- derma, and deafness. It has been hypothesized that sinki principles. PEODDN lesions in individuals with KID syndrome may Suspecting that PEODDN could be caused by somatic ge- result from loss of the wild-type GJB2 allele, although this netic mosaicism, we used paired whole-exome sequencing of hypothesis remains unproven.7 While earlier reports sug- the patient’s blood and affected tissue samples using an a priori gested that PEODDN may be caused by GJB2 mutation,8 approach (eTable in the Supplement) to determine what mu-

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Figure 1. Clinical and Histologic Images of Porokeratotic Eccrine Ostial and Dermal Duct Nevus

A B

C D

A, Spiny pink papules on the upper extremity at presentation. B, Lesions follow Blaschkoid lines. C, Histologic examination of these lesions demonstrates acanthosis and hyperkeratosis (hematoxylin-eosin, original magnification ×10). D, Porokeratotic plugs of eccrine ductal ostia are also found (hematoxylin-eosin, original magnification ×40).

tations could be causing her skin lesions. Whole-exome data were aligned to the hg18 reference genome, and all variations Discussion from the reference were annotated. Recognizing that somatic mutation was likely, we used a Perl script to identify single Porokeratotic eccrine ostial and dermal duct nevus lesions have nucleotide variations present in affected tissue samples but ab- been identified in patients with KID syndrome, raising the pos- sent in blood samples. In so doing, we first filtered for vari- sibility that this disorder could result from type II segmental ants not found in healthy controls (1000 genomes; the Na- mosaicism, in which the patient is heterozygous for a disease- tional Heart, Lung, and Blood Institute exome variant server causing mutation and loss of the wild-type allele gives rise to and 2577 in-house controls) and then identified those that were affected skin.7 While such lesions in those with KID syn- predicted to be protein damaging and specific to tissue. Our drome have not been examined, Easton et al8 provided the first data revealed 1 somatic single nucleotide variation reaching evidence that type I segmental mosaicism resulting from a genome-wide significance: GJB2, c.G134A, p.Gly45Glu (Figure 2 single GJB2 somatic mutation could cause PEODDN. Our study and eAppendix and eFigure 1 in the Supplement). Using proves that PEODDN is caused by GJB2 mutation, finding that ExomeCNV, we assayed for loss of heterozygosity across the a p.Gly45Glu mutation is the only somatic mutation present genome, finding none (eFigure 2 in the Supplement).9 This re- in PEODDN tissue samples without evidence of loss of hetero- sult suggested that somatic GJB2 mutation is sufficient to cause zygosity across the genome. PEODDN. GJB2 encodes connexin 26, a gap junction protein. Con- Using whole-skin biopsy tissue samples for sequencing, we nexins are tetraspan membrane proteins that are translated in found that the tissue-specific p.Gly45Glu GJB2 mutation was the endoplasmic reticulum and undergo homotypic and het- present in only 17.5% of total reads. This finding suggested an erotypic assembly to form hexameric connexons within the admixture of wild-type and mutant DNA, and we used laser Golgi.10 These connexons are transported to the cell mem- capture microdissection to obtain pure affected epidermal tis- brane, where they can either dock with connexons of adja- sue DNA for polymerase chain reaction and sequencing of GJB2. cent cells to form a gap junction or can function as hemichan- This process confirmed that the p.Gly45Glu mutation is pres- nels when unassociated on the cell surface membrane. ent solely within the epidermis, and we found that the The mutation we identified in PEODDN (GJB2, c.G134A, p.Gly45Glu mutation was present in an eqimolar ratio to the p.Gly45Glu) has been found in a severe case of KID syn- wild-type allele (Figure 2). drome, demonstrating complete atrichia, progressive severe

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Figure 2. Whole-Exome Sequencing Reveals a Single Statistically Significant Mutation: GJB2, c.G134A, p.Gly45Glu

Base Protein Tissue, No. of Reads Blood, No. of Reads Position (hg18) Gene Change Effect Ref Nonref Ref Nonref P Value Chr13:19,661,587 GJB2 G>A Gly45Glu 146 31 95 0 5.95 x 10–7

PDN101 Blood PDN101 Tissue WDG WDG/E G T G G G G A G A T T G G G A G A T A

GJB2, c.G134A, p.Gly45Glu

Whole-exome data were run through a Perl script to look for single nucleotide a heterozygous GJB2, c.G134A, p. Gly45Glu tissue-specific mutation. variations that had greater than 2 nonreference (nonref) reads in tissue and Polymerase chain reaction amplicons from laser-microdissected porokeratotic fewer than 6 nonreference reads in blood. These single nucleotide variations eccrine ostial and dermal duct nevus tissue reveal G and A peaks of were ranked by Fisher score, and the top 10 were manually analyzed to detect approximately equimolar amounts. No such mutation was found in polymerase mismapping. The 5 single nucleotide variations with the best Fisher scores are chain reaction amplicons from blood, confirming that this case represents a listed. Genome-wide significance is 1.7 × 10−6 after Bonferroni correction for heterozygous tissue-specific mutation. Ref indicates reference allele. multiple testing of approximately 30 000 genes. Targeted sequencing confirms

erythroderma, hyperkeratosis, vegetating plaques, and early lethality.11-13 GJB2 p.Gly45Glu mutations have been shown to Conclusions cause increased hemichannel activity in a dye-uptake assay in HeLa cells and electrophysiologic studies in cultured cells and Like the mutation found in our patient, 1 of the 2 mutations oocytes.14 An inducible mouse model of the p.Gly45Glu GJB2 found by Easton et al8—GJB2 p.Asn14Tyr—has been found in mutation demonstrates a severe skin phenotype similar to that individuals with KID syndrome.15 Since early somatic muta- found in KID syndrome and PEODDN and reproduces the in- tion can affect multiple embryonic lineages, individuals with creased hemichannel activity found in in vitro studies.14 PEODDN are at risk of conceiving children with KID syn- Connexin 26 hemichannels have been shown to increase drome and should be counseled regarding this risk. While the uptake of extracellular calcium. Increased calcium influx greater body surface area involvement is generally corre- can influence cellular proliferation and differentiation as well lated with higher risk, a report of twins with KID syndrome as cell-cell adhesion within the epidermis. The p.Gly45Glu so- born to a mother with limited linear palmoplantar kerato- matic mutation we found to cause PEODDN has been shown derma argues that genetic counseling should be provided to to cause aberrant hemichannel conductance and increased in- all pregnant females and expectant fathers with PEODDN tracellular calcium concentrations.14 lesions.11

ARTICLE INFORMATION Role of the Funder/Sponsor: The funding source 5. Levinsohn JL, Tian LC, Boyden LM, et al. Accepted for Publication: November 16, 2014. had no role in the design and conduct of the study; Whole-exome sequencing reveals somatic collection, management, analysis, and mutations in HRAS and KRAS, which cause nevus Published Online: February 18, 2015. interpretation of the data; preparation, review, or sebaceus. J Invest Dermatol. 2013;133(3):827-830. doi:10.1001/jamadermatol.2014.5069. approval of the manuscript; and decision to submit 6. Groesser L, Herschberger E, Ruetten A, et al. Author Contributions: Dr Choate and Mr the manuscript for publication. Postzygotic HRAS and KRAS mutations cause nevus Levinsohn had full access to all the data in the study sebaceous and Schimmelpenning syndrome. Nat and take responsibility for the integrity of the data REFERENCES Genet. 2012;44(7):783-787. and the accuracy of the data analysis. 1. Blaschko A. Die Nervenverteilung in der Haut in Study concept and design: Levinsohn, Choate. 7. Criscione V, Lachiewicz A, Robinson-Bostom L, ihrer Beziehung zu den Erkrankungen der Haut. Wien, Grenier N, Dill SW. Porokeratotic eccrine duct and Acquisition, analysis, or interpretation of data: All Leipzig: Braumüller; 1901:28-30. authors. hair follicle nevus (PEHFN) associated with Drafting of the manuscript: Levinsohn. 2. Moss C, Larkins S, Stacey M, Blight A, Farndon keratitis-ichthyosis-deafness (KID) syndrome. Critical revision of the manuscript for important PA, Davison EV. Epidermal mosaicism and Pediatr Dermatol. 2010;27(5):514-517. intellectual content: All authors. Blaschko’s lines. J Med Genet. 1993;30(9):752-755. 8. Easton JA, Donnelly S, Kamps MA, et al. Statistical analysis: Levinsohn. 3. Groesser L, Herschberger E, Sagrera A, et al. Porokeratotic eccrine nevus may be caused by Obtained funding: Choate. Phacomatosis pigmentokeratotica is caused by a somatic connexin26 mutations. J Invest Dermatol. Administrative, technical, or material support: postzygotic HRAS mutation in a multipotent 2012;132(9):2184-2191. McNiff, Antaya, Choate. progenitor cell. J Invest Dermatol. 2013;133(8): 9. Sathirapongsasuti JF, Lee H, Horst BA, et al. Conflict of Interest Disclosures: None reported. 1998-2003. Exome sequencing–based copy-number variation Funding/Support: This study was supported by a 4. Lim YH, Ovejero D, Sugarman JS, et al. and loss of heterozygosity detection: ExomeCNV. Doris Duke Charitable Foundation Clinical Scientist Multilineage somatic activating mutations in HRAS Bioinformatics. 2011;27(19):2648-2654. Development Award (Dr Choate) and a Doris Duke and NRAS cause mosaic cutaneous and skeletal 10. Meşe G, Richard G, White TW. Gap junctions: Charitable Foundation Medical Student Research lesions, elevated FGF23 and hypophosphatemia. basic structure and function. J Invest Dermatol. Fellowship (Mr Levinsohn). Hum Mol Genet. 2014;23(2):397-407. 2007;127(11):2516-2524.

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11. Jonard L, Feldmann D, Parsy C, et al. A familial 13. van Steensel MA, van Geel M, Nahuys M, Smitt 15. Arita K, Akiyama M, Aizawa T, et al. case of keratitis-ichthyosis-deafness (KID) JH, Steijlen PM. A novel connexin 26 mutation in a A novel N14Y mutation in connexin26 in syndrome with the GJB2 mutation G45E. Eur J Med patient diagnosed with keratitis-ichthyosis-deafness keratitis-ichthyosis-deafness syndrome: analyses Genet. 2008;51(1):35-43. syndrome. J Invest Dermatol. 2002;118(4):724-727. of altered gap junctional communication and 12. Janecke AR, Hennies HC, Günther B, et al. GJB2 14. Mese G, Sellitto C, Li L, et al. The Cx26-G45E molecular structure of N terminus of mutated mutations in keratitis-ichthyosis-deafness syndrome mutation displays increased hemichannel connexin26. Am J Pathol. 2006;169(2):416-423. including its fatal form. Am J Med Genet A. 2005;133A activity in a mouse model of the lethal form of (2):128-131. keratitis-ichthyosis-deafness syndrome. Mol Biol Cell. 2011;22(24):4776-4786.

NOTABLE NOTES

True Colors

Leonard J. Hoenig, MD

“True colors are beautiful like a rainbow” Pink From the song True Colors by Billy Steinberg and Tom Kelly Fortunately, pink disease, also called acrodynia, is now rare. During the first half of the 20th century, many young children developed this Dermatology may be medicine’s most colorful specialty. Just about ev- potentially fatal condition caused by a hypersensitivity to mercury (calo- ery color has a dermatology-related eponym, including the patriotic col- mel) present in teething powder. A striking clinical feature was the bright ors of red, (erythroderma), white (leukoderma), and blue (blue nevus, pink color of the hands and feet. blue man syndrome [argyria], and cyanosis [Latin for “blue disease”]). Here are some interesting facts behind dermatology’s colorful Green eponyms. Chlorosis (from the Greek “chloros” meaning “pale green”) referred to agreenskintintonceseeninirondeficiencyanemia.Itwascommonlycalled Yellow “greensickness.”JeanVarandal,aFrenchprofessorofmedicine,introduced There are many beautiful yellow birds, one of which was believed, the term in 1615. during ancient times, to cure jaundice (Old French for “yellowness”). The Roman naturalist, Pliny the Elder, relates this myth as follows: “There is Bronze a bird, known as the ‘icterus,’ from its peculiar color: if the patient looks By contrast, iron overload, as occurs in hereditary hemochromato- at it, he will be cured of jaundice, they say, and the bird will die.”1 Icterus sis, has been called bronze diabetes. The brownish skin discoloration is is Latin for jaundice, which is derived from the ancient Greek “ikteros.” due to hypermelanosis caused by iron excess. The yellow nail syndrome was first described by Peter Derrick Samman (1914-1992), a leading experts on nail pathology.2 This condition fea- tures a triad of lymphedema, pulmonary disease, and yellow nails. Purple If purple is your favorite color, you can enjoy 2 medical eponyms: (1) Salmon Color porphyria (from the Greek “porphyros” meaning “purple”) and (2) pur- pura, a Latin word meaning “purple dye.”In antiquity,purple dye was de- The noted British physician, Sir Jonathan Hutchinson (1828-1913), rived from secretions produced by sea snails in the family muricidae. described a salmon-colored neovascularized area (patch) of the cornea Yes, dermatology may be medicine’s most colorful specialty. We seen in syphilitic interstitial keratitis. He proposed the famed triad of should be proud of its true colors, which are beautiful like a rainbow. keratitis, notched teeth, and deafness for the diagnosis of congenital syphilis. Author Affiliation: Private practice. Corresponding Author: Leonard J. Hoenig, MD, 601 N Flamingo Rd, Ste 201, Pembroke Pines, FL 33028 ([email protected]). Black Additional Information: This article was written in honor of Dr Walter H. C. Black hairy tongue was important in medical history. In 1937, Con- Burgdorf, one of today’s great teachers of medicine, in recognition of his many rad Elvehjem showed that niacin cured pellagra in dogs, which mani- contributions to the field of dermatology. fested as black tongue. Melanoma (Latin for “tumor containing black 1. Pliny the Elder. Remedies for jaundice. In: Bostock J, Riley HT, eds. The [dark] pigment”) was first described as a disease entity by Rene Laen- Natural History. London, England: Taylor and Francis; 1855. Book XXX, Chapter 28. nec (1781-1826), the French physician who invented the stethoscope. He 2. Baran RL, Dawber R. Peter Derrick Samman. In: Loser C, Plewig G, Burgdorf used the term “les melanoses.” WHC, eds. Pantheon of Dermatology. Berlin, Germany: Springer; 2013:977-979.

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