© American College of Medical Genetics and Genomics ORIGINAL RESEARCH ARTICLE Genomic diagnostics within a medically underserved population: efficacy and implications Kevin A. Strauss, MD1, Claudia Gonzaga-Jauregui, PhD2, Karlla W. Brigatti, MS1, Katie B. Williams, MD, PhD1, Alejandra K. King, PhD2, Cristopher Van Hout, PhD2, Donna L. Robinson, CRNP1, Millie Young, RNC1, Kavita Praveen, PhD2, Adam D. Heaps, MS1, Mindy Kuebler, MS1, Aris Baras, MD2, Jeffrey G. Reid, PhD2, John D. Overton, PhD2, Frederick E. Dewey, MD2, Robert N. Jinks, PhD3, Ian Finnegan, BA3, Scott J. Mellis, MD, PhD2, Alan R. Shuldiner, MD2 and Erik G. Puffenberger, PhD1 Purpose: We integrated whole-exome sequencing (WES) and Compared to trio analysis, “family” WES (average seven exomes chromosomal microarray analysis (CMA) into a clinical workflow per proband) reduced filtered candidate variants from 22 ± 6to to serve an endogamous, uninsured, agrarian community. 5 ± 3 per proband. Nineteen (51%) alleles were de novo and 17 Methods: Seventy-nine probands (newborn to 49.8 years) who (46%) inherited; the latter added to a population-based diagnostic presented between 1998 and 2015 remained undiagnosed after panel. We found actionable secondary variants in 21 (4.2%) of 502 biochemical and molecular investigations. We generated WES data subjects, all of whom opted to be informed. for probands and family members and vetted variants through Conclusion: CMA and family-based WES streamline and rephenotyping, segregation analyses, and population studies. economize diagnosis of rare genetic disorders, accelerate novel Results: The most common presentation was neurological disease gene discovery, and create new opportunities for community-based (64%). Seven (9%) probands were diagnosed by CMA. Family WES screening and prevention in underserved populations. data were informative for 37 (51%) of the 72 remaining individuals, Genet Med advance online publication 20 July 2017 yielding a specific genetic diagnosis (n = 32) or revealing a novel molecular etiology (n = 5). For five (7%) additional subjects, Key Words: chromosomal microarray; developmental delay; negative WES decreased the likelihood of genetic disease. exome; genomic; intellectual disability INTRODUCTION in state-designated Health Professional Shortage Areas Whole-exome sequencing (WES) and chromosomal micro- (http://www.health.pa.gov/) and the majority are uninsured, array analysis (CMA) have revolutionized investigation of which is the strongest predictor of health disparity in the – rare genetic disorders and intellectual disability,1 6 but United States.14 important diagnostic and service gaps remain. The pretest Uninsured Americans are most commonly served by com- probability of a genetic lesion is high for individuals who munity health centers,15 only 12% of which offer the most basic move through contemporary diagnostic algorithms to arrive forms of genetic testing.16 Such services remain particularly at CMA or WES,7–9 yet many remain undiagnosed at the sparse in rural settings.16,17 Urban areas have meanwhile culmination of the process.2,3,9,10 Moreover, the cost and witnessed the rise of ambitious genomic centers funded by complexity of these methods limit access for people who are academic18 and industry stakeholders19enthused by the promise poor, uninsured, or otherwise medically underserved.11 of precision medicine.20 However, these large-scale genomics The Clinic for Special Children (CSC) is a medical home for initiatives are not necessarily intended to democratize genomic children who derive from endogamous Old Order Amish and testing16 or confront barriers to its broader implementation.11,21 Mennonite (Plain) populations of Pennsylvania and sur- To bridge the gap between technical resources and medical rounding states,12,13 integrating clinical care with an in-house need, CSC and the Regeneron Genetics Center (RGC) forged laboratory for Clinical Laboratory Improvement Amend- a collaboration to make WES freely accessible to uninsured ments–certified targeted testing and research-based CMA members of the Plain community (Supplementary Figure S1 analysis. Approximately 90% of CSC patients are medically online). The arrangement provided benefit to all major underserved, as defined by their geographic, social, and stakeholders: uninsured patients received high-quality geno- economic circumstances (http://www.hrsa.gov/). Many live mic testing at no cost, CSC received genomic data and below the federal poverty threshold (http://www.census.gov/) operating support, and RGC streamlined their investigation of 1Clinic for Special Children, Strasburg, Pennsylvania, USA; 2Regeneron Genetics Center, Regeneron Pharmaceuticals Inc., Tarrytown, New York, USA; 3Department of Biology, Franklin & Marshall College, Lancaster, Pennsylvania, USA. Correspondence: Kevin A. Strauss ([email protected]) Submitted 2 February 2017; accepted 13 March 2017; advance online publication 20 July 2017. doi:10.1038/gim.2017.76 GENETICS in MEDICINE | Volume 20 | Number 1 | January 2018 31 ORIGINAL RESEARCH ARTICLE STRAUSS et al | Clinical genomic diagnostics clinically relevant disease genes and pathways. Through this crossed against an existing panel of more than 200 known partnership, we have been able to optimize the yield of population-specific alleles detected by CSC laboratory using genomic testing, explore its broader social and economic high-resolution melt analysis or Sanger sequencing.12 Vetting value in community practice, and advance precision medicine the process in this way (Figure 1a), we ensured a high pretest while simultaneously redressing health care disparities unique probability of genetic illness while limiting representation of to the genomic era. known recessive “founder alleles” among probands who advanced to WES; institutional knowledge allowed us to MATERIALS AND METHODS enrich for phenotypes caused by de novo, X-linked, We identified 79 probands (36 female, mean age 6.9 ± 9.4 compound heterozygous, and copy-number variants years, range newborn to 49.8 years) who presented to CSC for (CNVs).2,3,7 evaluation between September 1998 and 2015, had clinical Sixty-eight (86%) probands had a 2.6-million marker high- signs of an underlying genetic disorder, and remained without density CMA (CytoScan HD Array, Affymetrix) to detect a diagnosis following focused biochemical and genetic pathogenic CNVs to a resolution of between 25 kb (losses) ± investigations spanning an average of 3.3 3.2 (range 0.1 and 50 kb (gains) using results from Affymetrix Chromosome to 16.9) years. All but three probands descended from Old Analysis Suite software (ChAS 3.1) filtered against CNV data 12,22 Order Amish and Mennonite founder populations. from more than 350 individuals of Amish and Mennonite The Lancaster General Hospital Institutional Review Board descent. We investigated any deletion (regardless of size) that approved the study. Subjects (or their parents) had pretest encompassed at least one exon of an OMIM gene and counseling to explain goals, process, timing, and limitations of impacted at least three separate NspI fragments. CMA and WES before consenting in writing to participate. For probands with an uninformative high-density CMA, we Subjects could choose whether or not to receive medically proceeded to WES in collaboration with RGC. Briefly, 1 μgof actionable secondary findings that fit American College of 23 high-quality genomic DNA was exome-captured using the Medical Genetics and Genomics (ACMG) guidelines, NimbleGen VCRome SeqCap 2.1 reagent. Libraries were including pathogenic variants known to segregate with high sequenced on the Illumina HiSeq 2500 platform using v4 frequency in Plain populations (e.g., APOB c.10580 G > A; chemistry, achieving coverage of > 85% of bases at 20x or p.Arg3527Gln).24 greater. Raw sequence reads were mapped and aligned to the Clinicians phenotyped each proband following a structured GRCh37/hg19 human genome reference assembly using and standardized assessment guided by PhenoTips (https:// BWA/GATK bioinformatics algorithms (https://software. phenotips.org)25 and using Human Phenotype Ontology broadinstitute.org/). Called variants were assessed by standard (HPO) terms. The likelihood of a monogenic disorder was metrics (read depth ≥ 10, genotype quality ≥ 30, allelic based primarily on conventional clinical indices such as balance ≥ 20%), annotated for potential functional effects abnormal brain size or morphology, developmental delay or (e.g., synonymous, missense, frameshift, nonsense), and regression, the presence of craniofacial/skeletal dysmorphisms, subsequently filtered by observed minor allele frequency or characteristic end-organ pathology (e.g., hearing loss, vision ≤ impairment, or epilepsy) in the absence of environmental 1% within public (1000 Genomes, ExAC, and NHLBI antecedents.7 The apparent inheritance of an autosomal ESP6500), RGC internal, and CSC population-specific allele recessive, dominant or X-linked phenotypes supported a genetic frequency databases. etiology in only 14 (17.7%) of 79 cases; 65 remaining probands The annotation process incorporated in silico predictions of presented with a unique clinical phenotype in the context of functional effect (e.g., LRT, Polyphen2, SIFT, CADD, an uninformative family history. MutationTaster) and conservation scores based on multi- Prior to CMA and WES, most probands with develop- species alignment (GERP, PhyloP, PhastCons). Primary ’ mental delay or neurological disease had additional analyte