Autoimmune lymphoproliferative syndrome (ALPS) is a rare genetic disorder featuring chronic lymphadenopathy, splenomegaly, cytopenias, and increased lymphoma risk. Differentiating ALPS from immunodeficiencies with overlapping symptoms is challenging. This study evaluated the performance and the diagnostic yield of a 15-gene NGS panel for ALPS at Cincinnati Children’s Hospital Medical Center. Samples from 802 patients submitted for ALPS NGS panel were studied between May 2014 and January 2023. A total of 62 patients (7.7%) had a definite diagnosis: 52/62 cases (84%) showed 37 unique pathogenic/likely pathogenic germline FAS variants supporting ALPS diagnosis (6.5%, 52/802). The ALPS diagnostic yield increased to 30% in patients who additionally fulfilled abnormal ALPS immunology findings criteria. 17/37 (46%) diagnostic FAS variants were novel variants reported for the first time in ALPS. 10/802 cases (1.2%) showed diagnostic findings in five genes (ADA2, CTLA4, KRAS, MAGT1, NRAS) which are related to autoimmune lymphoproliferative immunodeficiency (ALPID). Family studies enabled the reclassification of variants of unknown significance (VUS) and also the identification of at-risk family members of FAS-positive patients, which helped in the follow-up diagnosis and treatment. Alongside family studies, complete clinical phenotypes and abnormal ALPS immunology and Fas-mediated apoptosis results helped clarify uncertain genetic findings. This study describes the largest cohort of genetic testing for suspected ALPS in North America and highlights the effectiveness of the ALPS NGS panel in distinguishing ALPS from non-ALPS immunodeficiencies. More comprehensive assessment from exome or genome sequencing could be considered for undefined ALPS-U patients or non-ALPS immunodeficiencies after weighing cost, completeness, and timeliness of different genetic testing options.

Exome sequencing (ES) became clinically available in 2011 and promised an agnostic, unbiased next-generation sequencing (NGS) platform for patients with symptoms believed to have a genetic etiology. The diagnostic yield of ES has been estimated to be between 25-40% and may be higher in specific clinical scenarios. Those who remain undiagnosed may have no molecular findings of interest on ES, variants of uncertain significance in genes that are linked to human disease, or variants of uncertain significance in candidate genes that are not definitively tied to human disease. Recent evidence suggests that a post-exome evaluation consisting of clinical re-phenotyping, functional studies of candidate variants in known genes, and variant reevaluation can lead to a diagnosis in 5-15% of additional cases. In this brief research study, we present our experience on post-exome evaluations in a cohort of patients who are believed to have a genetic etiology for their symptoms. We have reached a full or partial diagnosis in approximately 18% (6/33) of cases that have completed evaluations to date. We accomplished this by utilizing NGS-based methods that are available on a clinical basis. A sample of these cases highlights the utility of ES reanalysis with updated phenotyping allowing for the discovery of new genes, re-adjudication of known variants, incorporating updated phenotypic information, utilizing functional testing such as targeted RNA sequencing, and deploying other NGS-based testing methods such as gene panels and genome sequencing to reach a diagnosis.

Biallelic disease-causing variants in the SBDS gene account for over 90 percent of Shwachman-Diamond Syndrome, a congenital autosomal recessive disorder characterized by pancreatic insufficiency, hematological malfunction, and skeletal abnormalities. Molecular genetic tests of SBDS include single-gene Sanger sequencing or next generation sequencing (NGS) panels. The detection of SBDS variants by either method, however, is complicated by the pseudogene, SBDSP1. In fact, common disease variants in SBDS are created by gene conversion events between SBDS and SBDSP1, making it difficult to determine whether a variant affects the functional gene or the pseudogene. We evaluated the performance of SBDS sequencing tests offered at our institution and determined if additional test strategies are needed for accurate variant detection. Sanger sequencing of SBDS identified the 2 known, common disease variants (c.183_184delinsCT and c.258+2T>C), as well as several less common disease variants. NGS testing of SBDS detected the common c.183_184delinsCT as 2 separate variants, such that those 2 variants had to be flagged and manually inspected when they were present. NGS also identified the second common disease variant, c.258+2T>C, and could determine the phase of the 2 common disease variants in individuals carrying them both. It remains possible to miss a rare gene conversion event by both sequencing methods. Extra attention should be given to patients negative by sequencing that are a strong phenotypic match for Shwachman-Diamond Syndrome. A reflex Sanger test can be considered for such patients, as well as analysis of low allele fraction NGS variants that might be constitutively heterozygous. Biallelic disease-causing variants in the SBDS gene account for over 90 percent of Shwachman-Diamond Syndrome, a congenital autosomal recessive disorder characterized by pancreatic insufficiency, hematological malfunction, and skeletal abnormalities. Molecular genetic tests of SBDS include single-gene Sanger sequencing or next generation sequencing (NGS) panels. The detection of SBDS variants by either method, however, is complicated by the pseudogene, SBDSP1. In fact, common disease variants in SBDS are created by gene conversion events between SBDS and SBDSP1, making it difficult to determine whether a variant affects the functional gene or the pseudogene. We evaluated the performance of SBDS sequencing tests offered at our institution and determined if additional test strategies are needed for accurate variant detection. Sanger sequencing of SBDS identified the 2 known, common disease variants (c.183_184delinsCT and c.258+2T>C), as well as several less common disease variants. NGS testing of SBDS detected the common c.183_184delinsCT as 2 separate variants, such that those 2 variants had to be flagged and manually inspected when they were present. NGS also identified the second common disease variant, c.258+2T>C, and could determine the phase of the 2 common disease variants in individuals carrying them both. It remains possible to miss a rare gene conversion event by both sequencing methods. Extra attention should be given to patients negative by sequencing that are a strong phenotypic match for Shwachman-Diamond Syndrome. A reflex Sanger test can be considered for such patients, as well as analysis of low allele fraction NGS variants that might be constitutively heterozygous.

Hereditary hemolytic anemia (HHA) is a group of heterogeneous genetic conditions that are usually caused by pathogenic variants in genes encoding red blood cell (RBC) cytoskeleton proteins, RBC enzymes, and globins. In addition, congenital dyserythropoietic anemias (CDAs) typically present with a phenotype of hemolytic anemia. To utilize the advantages of next generation sequencing (NGS), a 38-gene panel was designed and incorporated in our clinical lab, encompassing 16 genes responsible for RBC membrane disorders, 14 genes responsible for RBC enzymopathies and 8 genes for CDAs.

Our institution developed and continuously improved a Neurodevelopmental Reflex (NDR) algorithm to help physicians with genetic test ordering for neurodevelopmental disorders (NDDs). To assess its performance, we performed a retrospective study of 511 patients tested through NDR from 2018 to 2019. SNP Microarray identified pathogenic/likely pathogenic copy number variations in 27/511 cases (5.28%). Among the 484 patients tested for Fragile X FMR1 CGG repeats, a diagnosis (0.20%) was established for one male mosaic for a full mutation, a premutation, and a one-CGG allele. Within the 101 normocephalic female patients tested for MECP2 , two patients were found to carry pathogenic variants (1.98%). This retrospective study suggested the NDR algorithm effectively established diagnoses for patients with NDDs with a yield of 5.87%.