Beyond the exome: What's next in diagnostic testing for Mendelian conditions.

Despite advances in clinical genetic testing, including the introduction of exome sequencing (ES), more than 50% of individuals with a suspected Mendelian condition lack a precise molecular diagnosis. Clinical evaluation is increasingly undertaken by specialists outside of clinical genetics, often occurring in a tiered fashion and typically ending after ES. The current diagnostic rate reflects multiple factors, including technical limitations, incomplete understanding of variant pathogenicity, missing genotype-phenotype associations, complex gene-environment interactions, and reporting differences between clinical labs. Maintaining a clear understanding of the rapidly evolving landscape of diagnostic tests beyond ES, and their limitations, presents a challenge for non-genetics professionals. Newer tests, such as short-read genome or RNA sequencing, can be challenging to order, and emerging technologies, such as optical genome mapping and long-read DNA sequencing, are not available clinically. Furthermore, there is no clear guidance on the next best steps after inconclusive evaluation. Here, we review why a clinical genetic evaluation may be negative, discuss questions to be asked in this setting, and provide a framework for further investigation, including the advantages and disadvantages of new approaches that are nascent in the clinical sphere. We present a guide for the next best steps after inconclusive molecular testing based upon phenotype and prior evaluation, including when to consider referral to research consortia focused on elucidating the underlying cause of rare unsolved genetic disorders.

Diversity of variant alleles encoding Kidd, Duffy, and Kell antigens in individuals with sickle cell disease using whole genome sequencing data from the NHLBI TOPMed Program.

BACKGROUND: Genetic variants in the SLC14A1, ACKR1, and KEL genes, which encode Kidd, Duffy, and Kell red blood cell antigens, respectively, may result in weakened expression of antigens or a null phenotype. These variants are of particular interest to individuals with sickle cell disease (SCD), who frequently undergo chronic transfusion therapy with antigen-matched units. The goal was to describe the diversity and the frequency of variants in SLC14A1, ACKR1, and KEL genes among individuals with SCD using whole genome sequencing (WGS) data. STUDY DESIGN AND METHODS: Two large SCD cohorts were studied: the Recipient Epidemiology and Donor Evaluation Study III (REDS-III) (n = 2634) and the Outcome Modifying Gene in SCD (OMG) (n = 640). Most of the studied individuals were of mixed origin. WGS was performed as part of the National Heart, Lung, and Blood Institute's Trans-Omics for Precision Medicine (TOPMed) program. RESULTS: In SLC14A1, variants included four encoding a weak Jka phenotype and five null alleles (JKnull ). JKA*01N.09 was the most common JKnull . One possible JKnull mutation was novel: c.812G>T. In ACKR1, identified variants included two that predicted Fyx (FY*X) and one corresponding to the c.-67T>C GATA mutation. The c.-67T>C mutation was associated with FY*A (FY*01N.01) in four participants. FY*X was identified in 49 individuals. In KEL, identified variants included three null alleles (KEL*02N.17, KEL*02N.26, and KEL*02N.04) and one allele predicting Kmod phenotype, all in heterozygosity. CONCLUSIONS: We described the diversity and distribution of SLC14A1, ACKR1, and KEL variants in two large SCD cohorts, comprising mostly individuals of mixed ancestry. This information may be useful for planning the transfusion support of patients with SCD.

Whole-Genome Sequencing Identifies Novel Functional Loci Associated with Lung Function in Puerto Rican Youth.

Rationale: Puerto Ricans have the highest childhood asthma prevalence in the United States (23.6%); however, the etiology is uncertain.Objectives: In this study, we sought to uncover the genetic architecture of lung function in Puerto Rican youth with and without asthma who were recruited from the island (n = 836).Methods: We used admixture-mapping and whole-genome sequencing data to discover genomic regions associated with lung function. Functional roles of the prioritized candidate SNPs were examined with chromatin immunoprecipitation sequencing, RNA sequencing, and expression quantitative trait loci data.Measurements and Main Results: We discovered a genomic region at 1q32 that was significantly associated with a 0.12-L decrease in the lung volume of exhaled air (95% confidence interval, -0.17 to -0.07; P = 6.62 × 10-8) with each allele of African ancestry. Within this region, two SNPs were expression quantitative trait loci of TMEM9 in nasal airway epithelial cells and MROH3P in esophagus mucosa. The minor alleles of these SNPs were associated with significantly decreased lung function and decreased TMEM9 gene expression. Another admixture-mapping peak was observed on chromosome 5q35.1, indicating that each Native American ancestry allele was associated with a 0.15-L increase in lung function (95% confidence interval, 0.08-0.21; P = 5.03 × 10-6). The region-based association tests identified four suggestive windows that harbored candidate rare variants associated with lung function.Conclusions: We identified common and rare genetic variants that may play a critical role in lung function among Puerto Rican youth. We independently validated an inflammatory pathway that could potentially be used to develop more targeted treatments and interventions for patients with asthma.

Identification of CFTR variants in Latino patients with cystic fibrosis from the Dominican Republic and Puerto Rico.

BACKGROUND: In cystic fibrosis (CF), the spectrum and frequency of CFTR variants differ by geography and race/ethnicity. CFTR variants in White patients are well-described compared with Latino patients. No studies of CFTR variants have been done in patients with CF in the Dominican Republic or Puerto Rico. METHODS: CFTR was sequenced in 61 Dominican Republican patients and 21 Puerto Rican patients with CF and greater than ​​​​60 mmol/L sweat chloride. The spectrum of CFTR variants was identified and the proportion of patients with 0, 1, or 2 CFTR variants identified was determined. The functional effects of identified CFTR variants were investigated using clinical annotation databases and computational prediction tools. RESULTS: Our study found 10% of Dominican patients had two CFTR variants identified compared with 81% of Puerto Rican patients. No CFTR variants were identified in 69% of Dominican patients and 10% of Puerto Rican patients. In Dominican patients, there were 19 identified CFTR variants, accounting for 25 out of 122 disease alleles (20%). In Puerto Rican patients, there were 16 identified CFTR variants, accounting for 36 out of 42 disease alleles (86%) in Puerto Rican patients. Thirty CFTR variants were identified overall. The most frequent variants for Dominican patients were p.Phe508del and p.Ala559Thr and for Puerto Rican patients were p.Phe508del, p.Arg1066Cys, p.Arg334Trp, and p.I507del. CONCLUSIONS: In this first description of the CFTR variants in patients with CF from the Dominican Republic and Puerto Rico, there was a low detection rate of two CFTR variants after full sequencing with the majority of patients from the Dominican Republic without identified variants.