A Review of Next-Generation Genetic Testing for the Dermatologist

A Review of Next-Generation Genetic Testingfor the Dermatologist

Eun-Kyung M. Kwon, B.A.,* Donald Basel, M.D.,† Dawn Siegel, M.D.,*,‡ and

Kari L. Martin, M.D.*

Departments of *Dermatology, †Genetics, and ‡Pediatrics, Medical College of Wisconsin, Milwaukee, Wisconsin

Abstract: Dermatologists have been placed in a prime position tomake new genetic discoveries. Tissue is easily obtained from the skin ormucosa for the study of germline and somatic mosaic disorders. This,along with the recent development of next-generation sequencing, makesdermatology an exciting field with essentially endless possibilities fordiscovering genes responsible for disease, better understanding complexmolecular pathways, and eventually developing targeted therapies. Totake advantage of this great opportunity, a basic understanding of theadvances in genetic testing is vital. Herein we give an overview of next-generation sequencing, including some of the applications it may be usedfor. We also review various study designs for genetic discovery, each oftheir benefits and downfalls, and how they may be applied to the study ofdermatologic disease.

Incredible advances in genetic testing haveoccurred over the last few decades. Since completionof the human genome project in April 2003 (1), therehas been an explosion of discoveries uncovering thegenetic basis of disease. Eventually it is likely that itwill be possible to sequence a patient’s genome andidentify not only genetic disease, but also predisposi-tion to complex multifactorial disease, best treatmentoptions for that individual, and prediction as to howthey may respond to specific pharmacogenetic inter-vention. This phenomenon will affect every area ofmedicine, and dermatologists have the opportunity tobe at the forefront of this new era. The skin often actsas a window to genetic disease and also provides greatopportunity for studying mosaicism. Given the prac-

ticality of performing skin biopsies, dermatologistsare able to obtain not only blood and buccal smearsamples, but also actual affected skin tissue foranalysis. Therefore, understanding the large amountof data that can be gleaned from these studies isparamount.

NEXT-GENERATION SEQUENCING

DNA sequencing has come a long way since FredSanger (2) and Maxam and Gilbert (3) first intro-duced it in 1977. New “next-generation” sequencing(NGS) techniques are now readily available to producerapid results, and the costs are quickly dropping (4).Instead of the Sanger method, in which one gene is

Address correspondence to Kari L Martin, M.D., Departmentof Dermatology, MA111. One Hospital Drive, Columbia, MO65212, or e-mail: [email protected].

DOI: 10.1111/pde.12062

© 2012 Wiley Periodicals, Inc. 401

REVIEWS

Pediatric Dermatology Vol. 30 No. 4 401–408, 2013

sequenced at a time by designing primers specific foreach exon, NGS techniques break the DNA intonumerous smaller pieces that are quickly amplified inparallel and then aligned back together to give the finalsequence (Fig. 1). There are various ways of accom-plishingthis (seeResourcesat theendofthisarticle),butall have the same basic methodology. One of thedifficulties with NGS is that structural variantswithin the DNA can be missed because of the alig-nment process, including DNA changes other thansingle nucleotide variants (e.g., block substitutions,insertions or deletions, inversions, and copy numbervariants [CNVs]). Several strategies to reduce this errorhave been described (5). NGS can be used in manydifferent types of studies, as discussed below.

LINKAGE ANALYSIS

Since its initial description in the early 1900s,linkage analysis followed by DNA sequencing hasbeen the traditional method of discovering newgenes responsible for disease. Although slower andmore expensive than today’s standards, great dis-coveries were made using these techniques, includ-ing describing the genes responsible for diseasessuch as cystic fibrosis and sickle cell anemia. In themost basic terms, linkage analysis is the study ofhow DNA loci segregate relative to one anotherand an identifiable phenotype linked to a region ona chromosome (6). The closer they are, the morelikely they are to be carried together throughrecombination processes during cell division. Fam-ilies with a Mendelian disease to be studied are

A

B

Figure 1. Sanger sequencing versus next-generationsequencing. (A) In Sanger sequencing, a single area ofDNA to be sequenced is identified. A primer to target thisarea is created and allowed to bind to the single-stranddenatured sample DNA. It is then added to four separatesolutions of deoxyribonucleotide triphosphates (dNTPs),DNA polymerase, and one of four labeled dideox-ynucleotide triphosphates (ddNTPs). As the DNA strandis replicated, the ddNTPs are taken up and cause chaintermination. This process is repeated numerous times,resulting in replicated DNA strands of varying lengths, allending in the labeled ddNTP. These are then separatedbased on their lengths across an electrophoresis gel, andthe sequence is compiled. (B) There are several methodsof next-generation sequencing, but several steps aresimilar across modalities. The sample DNA is fragm-ented. Templates are then created, typically of theunknown region of DNA to be sequenced along with anadaptor molecule of known sequence. Templates arebound to various structures (e.g., microarray chips,beads) and polymerase and dNTPs are added. Eachnewly transcribed DNA segment is sequenced usingvarious imaging modalities and the sequences arerealigned to decode the original DNA sequence that wastargeted.

402 Pediatric Dermatology Vol. 30 No. 4 July/August 2013

chosen and affected individuals are identified.Marker alleles are chosen along the length of thegenome. Analysis is then performed to determinewhich marker alleles are associated with the phe-notype of interest by determining logarithm of odds(LOD) scores. Traditionally LOD scores greaterthan 3 are considered evidence of linkage, whereasthose less than �2 are considered evidence againstlinkage, although these cutoffs are arbitrary andcan be changed depending on the disease studied.Linkage analysis is best performed for alleles thathave a large effect on the phenotype, although thesetypes of “large effect” alleles are rare (7). Onceareas of interest on specific chromosomes areidentified within one family, confirmation of linkagein other pedigrees is sought. This can be difficult,especially in genetically complex disease becausedifferent loci may be associated with the phenotypein different families. Another challenge with per-forming linkage analyses is that a large pedigreewith many family members displaying the pheno-type or a number of smaller pedigrees are requiredto reach a LOD score of significance. The generesponsible for Kindler syndrome, FERMT1 (alsoknown as KIND1), was discovered using linkageanalysis (8). Although this disease is rare, anisolated Native American tribe was identified offthe coast of Panama with 26 affected members,making this study possible. Linkage analysis con-tinues to be a powerful tool, especially whencombined with other techniques. For example, thecandidate regions identified using linkage analysisare large and have been expensive and timeconsuming to analyze in the past, but by combiningNGS (exome or whole genome) with linkageanalysis, these large candidate areas can easily beanalyzed (7).

GENOME-WIDE ASSOCIATION STUDIES

For common, polygenic diseases such as type 2diabetes, obesity, and Crohn’s disease, genome-wideassociation studies (GWASs) have proven helpful.GWASs scan DNA from a large number ofpatients with a particular disease to look for singlenucleotide polymorphisms (SNPs) (9). SNPs arechanges in a single nucleotide within the DNA. Thecompletion of the Human Genome Project and theInternational HapMap Project made these studiespossible. These are large databases that contain thereference human genome and maps of commongenetic variation in large populations (1,10).GWASs are conducted by collecting DNA from

large cohorts of patients with a certain phenotypeand analyzing it to determine whether there areSNPs present that are more frequent in the affectedpatients than in controls from an ethnically similarpopulation. These associated SNPs discovered inGWASs mark areas of the genome that influencethe risk of developing the studied disease. Furtheranalysis such as DNA sequencing is then conductedto identify potentially causal genes.

Genome-wide association studies are most helpfulin studying common diseases because a large numberof patients need to be analyzed. They are also useful intrying to identify numerous different genetic varia-tions that may all be contributing to the risk ofdeveloping a genetically complex disease. In this samemanner, this may provide numerous genes fortargeted therapies in these often difficult-to-treatconditions. In addition, the entire genome is scannedin these studies, not just the exome, which allowsvariations within introns to be detected as well thatmay be missed in other types of studies such as exomesequencing. A disadvantage of GWASs is that manySNPs are usually discovered (hundreds to thousands),but many have only a minor effect on disease (11,12).In most cases, GWASs are limited not by dataacquisition, but by analysis of those data. In someinstances there may be undetected common geneticvariations. If the genetic variation has a low effect onphenotype, the sample size may be too small toachieve adequate power. Therefore, in cases in whichdisease is due to rare genetic variation, whole-exomeor whole-genome sequencing (WGS) may be morecost effective. Lastly, low-frequency genetic variants(LFGVs) may also be difficult to detect. The 1000Genomes Project, a large database that allows for thedetection of copy number polymorphisms (commonCNVs), other structural variants, and SNPs, wascompleted and published in 2010 and continues to beupdated (13). This will greatly enhance the study ofLFGVs.

The first GWAS published was in patients withmacular degeneration in 2005 (14). Since that time,more than a thousand studies have been reported (15).Examples of dermatologic diseases studied recentlyusing this method include basal cell carcinoma (BCC),alopecia areata, psoriasis, and atopic dermatitis.Zhang et al used a pathway-based approach for aGWAS of BCCs of the skin in 2001 (16,17). In doingthis, four pathways not previously known to beassociated with BCC risk were discovered. A GWASthat Petukhova et al conducted in patients withalopecia areata revealed 139 SNPs associated withthe disease phenotype (18). Many of these SNPs were

Kwon et al: Review of Genetic Testing 403

found tobe relevant in the innate andacquired immunepathways, as well as newly described ligands up-regulated in the hair follicles themselves.

CNV ANALYSIS

Various comparative studies have demonstrated thatstructural variations rather than single nucleotidepoint mutations contribute to the majority of basepair differences between human genomes (19–22).Structural variants include deletions, duplications,inversions, translocations, and insertions. CNVs spe-cifically are defined as unbalanced structural variantsor segments of DNA that alter the number of basepairs from that of a diploid reference genome (23). ADNA segment size of 1 kb or more in length hastypically been used as the cutoff when screening forCNVs (20,24), but significant improvements in tech-niques have allowed detection of smaller events,incorporating a size of greater than 50 bp into thedefinition (23). Methods used to detect CNVs includearray-based CNV calling (array comparative genomichybridization [CGH] and SNPmicroarrays) and NGS(23). Array CGH involves the hybridization of fluor-escently labeled disease and reference samples toestablished target DNA sequences and the evaluationof the signal hybridization ratio to assess copynumber. SNP microarrays differ from array CGH inthat they apply the fragmented disease DNA sampledirectly to the array and target DNA sequences thatare specific to single nucleotide differences. SNPmicroarrays have the advantage over CGH of beingable to detect a loss of heterozygosity. Finally, NGScompares sequence results to the reference genome toidentify variants (23).

Analyzing the role of CNVs in disease developmentconfers many advantages because CNVs are wide-spread throughout the human genome and affectnumerous genes (20). It is also possible that CNVscontribute to common complex diseases and accountfor the “missing heritability” that cannot be explainedby susceptibility loci discovered using GWASs (25).Improvements in hybridization-based and next-gen-eration sequencing technologies have also made thedetection of CNVs more affordable (23). Conversely,one major disadvantage of CNV analysis is distin-guishing disease CNVs from common polymorphicvariants present in otherwise unaffected populations.This task is particularly challenging given that ahuman reference genome accounting for the completespectrum of structural variations is not available.

Of dermatologic interest, CNV analysis wasrecently applied to investigate whether CNVs within

the filaggrin (FLG) gene contributed to disease risk inatopic dermatitis (26). CNVs within FLG were foundto have a significant dose-dependent effect on atopicdermatitis risk, an effect that was independent ofpreviously identified FLG loss-of-function mutations.CNVs have also been implicated in psoriasis.Deletions in LCE3B and LCE3C, genes involved inskin barrier function, were identified (27) and con-firmed (28) in two studies that revealed a higher allelefrequency of the deletion in patients with psoriasisthan in controls. Before these CNV studies, suscep-tibility loci within the LCE gene cluster were foundusing GWASs (29). GWASs and CNV studies,working in tandem, have been successful in identifyinggenes associated with such dermatologic disorders,once again suggesting that CNV may account for the“missing heritability” that GWAS cannot explain.

EXOME AND WGS

Exome and WGS are useful for studying rare diseasesbecause only a small number of cases or families areneeded. Time and financial constraints had previouslylimited these techniques, but now, using NGS, theyhave become increasingly available and popular. Inboth cases, affected patient’s DNA is sequenced andcompared with ethnically similar controls frominternational databases or their parents to identifyde novo or novel variants. Many variants are usuallydiscovered, so determining which are causative can bechallenging. Often, variants are found outside ofgenes or in genes without a known function, whichmakes evaluation difficult. Recently these techniqueshave been used in studying mosaic disorders. In thesecases, the patient’s affected and unaffected skin aresequenced using exome or WGS and compared witheach other to detect the novel mutations in theaffected tissue.

The major difference between exome sequencingand WGS lies in the portions of the genome beingsequenced. Exome sequencing is slightly faster andless expensive than WGS because only exons (theprotein-coding regions) are sequenced. This comprisesapproximately 1% of the total genomic DNA (30). Bydesign, noncoding sections of DNA are not analyzed.Nevertheless, exome sequencing continues to be apowerful tool. Most alleles associated with diseasethat have been discovered involve disruption ofprotein-coding sequences (31). Another challengewith exome sequencing is deciding which DNAsegments are truly part of the exome. With NGSnow widely available, most laboratories have movedfrom the initial conservative group of genes known to


code protein and have begun including more andmore hypothetical protein-coding sequences (31).Therefore the exome is a good place to begin thesearch for small DNA changes that have a largephenotypic effect for rare diseases.

Whole-genome sequencing uses NGS to examinethe entire genomic DNA. This not only includesexons, but also allows for the study of importantsequences in the introns, such as micro-RNA,promoter regions, and ultraconserved elements.These portions of the genome were previouslythought to be mostly “junk,” but may be crucialto understanding the heritability of a disease. Manyof these regions of DNA are involved in theregulation of exon expression or structural stabilityof the DNA. Studies with a narrower focus, suchas exome-only sequencing, typically leave them out.WGS is the most comprehensive study of DNA,but has been limited in the past by cost and lengthof time required to complete sequencing. WithNGS techniques available, WGS has become muchmore efficient and economical and may become soreadily available that it might be applied to largenumbers of patients in much the same way thatGWASs are conducted today (32). This, too, mayhelp discover some of the “missing heritability”that GWASs have not discovered.

As good tools for discovering genetic causes of raredisease, exome sequencing and WGS studies fordermatologic disease have been successful. Mutationsin TRPV3 were recently identified in patients withOlmsted syndrome using whole-exome sequencing(33). Another recent report by Cabral et al used exomesequencing to discover a mutation within CHST8leading to autosomal-recessive peeling skin syndrometype A (34). Exome sequencing also led to the firstreports of somatic mutations in IDH1 and IDH2causing Maffucci syndrome and Ollier syndromesdisease (35,36). A combination of linkage analysis,WGS, and targeted Sanger sequencing was used todescribe deletions in PCLG2 in families with cold-induced urticaria, immunodeficiency, and autoimmu-nity (37).

EPIGENETICS

All of the aforementioned gene discovery techniqueshave relied on the presence of changes in the DNAsequence. In some diseases, the DNA sequenceremains intact, but several modifications can occurthat affect gene expression and ultimately the resul-tant phenotype. These modifications are collectivelyknown as epigenetics and are a rapidly expanding area

of interest for many researchers. Epigenetic changesinclude cytosine modification, histone modification,and non-protein-coding RNA segments.

DNA methylation occurs on cytosine–guaninebase pairs and leads to transcription silencing. Thisoccurs most often in the promoter regions of genes.Studying DNA methylation patterns is different fromDNA sequencing because traditional DNA sequenc-ing techniques destroy the methylation pattern.Previously, methylation studies were limited to selectgenome regions. With the advent of NGS, single-base-pair resolution whole-genome DNA methylationanalysis is now possible (38). Methylation patternshave been emerging for dermatologic disease and haverecently been described for melanoma and psoriasis(39,40).

Another epigenetic process, histone modification,can also lead to altered DNA expression. Immuno-precipitation of DNA bound to specific modifiedhistones can be identified and sequenced. Histonemodifications are beginning to be unveiled in derma-tologic diseases that have been hypothesized to havean environmental component, such as alopecia areata(41). These areas of epigenetics are evolving quickly,but much remains to be learned.

MOSAICISM

As dermatologists, we observe mosaicism perhapsmore than any other specialist. Blaschko first notedand diagrammed these patterns in the skin that arenow known to be migration paths of cells duringembryonic development. Many dermatologic diseaseswith Blaschko linear patterns are the result of somaticmosaicism or lyonization. Somatic mosaicism occurswhen there is a mutation in one group of cells (e.g.,fibroblasts, vascular progenitor cells, keratinocytes)early in development. As those cells migrate, theyconstitute a population of cells different from thosearound them in genetic make-up and often in clinicalappearance as well (42). Perhaps the most common ofthese is pigmentary mosaicism. Other examplesinclude epidermal nevi, lichen striatus, and Proteussyndrome. The somatic mosaic mutation responsiblefor Proteus syndrome was recently discovered (43).Exome sequencing was performed from tissuesaffected by the disease and normal-appearing tissue,and activating mutations of AKT1 were found only inclinically affected tissues.

In females, sporadic inactivation of one of the Xchromosomes in each cell leads to another form ofmosaicism: lyonization. This can be easily observed inthe skin of certain X-linked diseases such as


incontinentia pigmenti and focal dermal hypoplasia.These disorders are lethal in males (except in somecases of XXY males and hypomorphic or mosaicmutations), but females survive through lyonization.Early in development, each cell in a female silencesone of the X chromosomes. Those groups of cellsmigrate along Blaschko’s lines, and in a female infant,it is possible to see which areas of skin have cellsexpressing the X chromosome with the wild-type alleleand which have the mutation (42).

When studying mosaic disorders, determining thecell line affected had traditionally been the firstquestion posed. Classically, fibroblast cultures weregrown from skin biopsies, and DNA sequencing wasrun on these cell lines. If the mosaic disorder primarilyaffected the keratinocytes or vasculature, then thecausative mutation would have been missed. Now, insome instances, all of the tissue obtained in a skinbiopsy is processed for DNA extraction. As long asthe DNA of the cell of interest comprises at least acertain percentage of the total DNA (depending onthe filters applied during analysis), the genetic varia-tion from unaffected skin or hematopoietic cells canbe detected. One study using exome sequencingfollowed by deep resequencing (sequencing eachnucleotide numerous times) has shown that NGStechniques are sensitive enough to detect a somaticmosaic mutation with an allele frequency as low as18% (44). Mosaic disorders offer an excellent oppor-tunity for genetic research; a built-in control lies in theunaffected tissue of the patient. The only differencebetween the affected and unaffected tissues should bethe genetic variation leading to the phenotype inquestion.

CLINICAL RESEARCH APPLICATION

Often the first step in determining the genetic causeof disease is to solidify the phenotype in question asmuch as possible. Having a group of patients withvariations in phenotype may result in a cohort withslight genetic differences as well, which can makediscovery of the underlying genetic cause challengingor even impossible. Deciding which method to usewhen trying to discover the underlying genetic causeof disease depends on several factors (Table 1). Forcommon genetically complex diseases such as pso-riasis or atopic dermatitis, GWASs or CNV studiesare usually best suited. These methods are able toidentify numerous genetic variations that may becontributing to one phenotype. For rare diseases,searching for genetic variation with large effects is

more easily done using exome or WGS. If a pedigreewith numerous affected family members is available,linkage analysis can be a good first step to identifyloci associated with the phenotype before perform-ing exome or WGS.

CONCLUSION

Many ethical questions will arise as we come closer tothe era of WGS for every patient we encounter. Whatdo we do with the incidental information found whenwe are looking for the answer to a particular clinicalquestion? Does disclosure of that information dependon whether it would change prevention or treatmentstrategies for the patient (45)? How do we obtaininformed consent while obtaining blood or tissuesamples when the information gleaned from it maycontinue to change, especially if stored in a biobank(46)?

Genetic analysis of the patients we see every daywill soon be commonplace. New techniques and waysto use NGS will be abundant as well. Just asimportant will be the software and database systemsto analyze the incomprehensible amount of data thatwill be gained from the increased use of NGS. It isimportant for dermatologists to understand the con-cepts of NGS and the ways it can be applied. Whenpresented with a clinical question, it is important tounderstand the breadth of genetic testing availableand what would be best suited to answer the questionat hand.

RESOURCES

Glossary: http://www.genome.gov/glossary/index.cfm.Animations of Sanger sequencing and various

types of NGS: http://www.wellcome.ac.uk/News/2009/Features/WTX056032.htm.

ACKNOWLEDGMENTS

We would like to thank Eric Larson for the graphicdesign of the figures.

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A Review of Next-Generation Genetic Testing for the Dermatologist

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