Pompe disease ascertained through The Lantern Project, 2018-2021: Next-generation sequencing and enzymatic testing to overcome obstacles to diagnosis.

The Lantern Project is an ongoing complimentary diagnostic program for patients in the United States sponsored by Sanofi and implemented by PerkinElmer Genomics. It combines specific enzymatic, biomarker, and genetic testing to facilitate rapid, accurate laboratory diagnosis of Pompe disease and several other lysosomal storage diseases, and a multigene next-generation sequencing panel including Pompe disease, LGMD, and other neuromuscular disorders. This article reports data for Pompe disease collected from October 2018 through December 2021, including acid α-glucosidase (GAA) enzyme assay and GAA sequencing (standard or expedited for positive newborn screening [NBS] to rule out infantile-onset Pompe disease [IOPD]) and the Focused Neuromuscular Panel, which includes GAA. One hundred forty patients (12 received only GAA enzyme testing, 128 had GAA sequencing alone or in addition to enzyme assay) have been confirmed with Pompe disease in this project. Eight of the 140 had a variant of unknown significance, but GAA activity ≤2.10 µmol/L/h, thus were confirmed with Pompe disease. Three diagnosed patients 0-2 years old had cross-reactive immunologic material (CRIM)-negative GAA variants and thus IOPD. One additional infant with presumptive IOPD had a homozygous frameshift c.1846del, likely CRIM-negative; symptoms were not provided. Among the 128 patients with molecular results, the c.-32-13T>G splice variant was homozygous in 11, compound-heterozygous in 98, and absent in 19. Proximal muscle weakness (58 patients) was the most common sign reported at testing; elevated creatine kinase (29 patients) was the most common laboratory result. The most common symptom categories were muscular (73 patients), musculoskeletal (13 patients), and respiratory (23 patients). Clinical information was not available for 42 samples, and 17 infants had only "abnormal NBS" or "low GAA" reported. Cardiac symptoms in 7 included potentially age-related conditions in five c.-32-13T>G-compound-heterozygous adults (myocardial infarction, heart murmur/palpitations, congestive heart failure: 1 each; 2 with atrial fibrillation) and hypertrophic cardiomyopathy in 2 children (1 and 2 years old) with presumptive IOPD. One novel GAA variant was observed in a patient with enzyme activity 0.31 µmol/L/h: c.1853_1854ins49, a frameshift pathogenic variant. The Lantern Project demonstrates the combinatorial utility of enzyme assay, targeted single-gene testing, and a focused neuromuscular next-generation sequencing panel in diagnosing Pompe disease.

A single NGS-based assay covering the entire genomic sequence of the DMD gene facilitates diagnostic and newborn screening confirmatory testing.

Molecular diagnosis for Duchenne and Becker muscular dystrophies (DMD/BMD) involves a two-tiered approach for detection of deletions/duplications using MLPA or array CGH, followed by sequencing of coding and flanking intronic regions to detect sequence variants, which is time-consuming and expensive. We have developed a comprehensive next-generation sequencing (NGS)-based single-step assay to sequence the entire 2.2 Mb of the DMD gene to detect all copy number and sequence variants in both index males and carrier females. Assay validation was 100% concordant with other methodologies. A total of 772 samples have been tested, of which 62% (N = 480) were index cases with a clinical suspicion of DMD. Carrier testing females account for 38% (N = 292). Molecular diagnosis was confirmed in 86% (N = 413) of the index cases. Intragenic deletions and duplications (single-exon or multi-exon) were detected in 60% (N = 247) and 14% (N = 58) of the index cases, respectively. Full-sequence analysis of the entire gene allows for detection of deep intronic pathogenic variants and accurate breakpoint detection of CNVs involving similar exons, which could have an impact on the outcome of clinical trials. This comprehensive assay is highly sensitive for diagnostic testing for DMD and is also suitable for confirmatory testing for newborn screening for DMD.

Focused Exome Sequencing Gives a High Diagnostic Yield in the Indian Subcontinent.

The genetically isolated yet heterogeneous and highly consanguineous Indian population has shown a higher prevalence of rare genetic disorders. However, there is a significant socioeconomic burden for genetic testing to be accessible to the general population. In the current study, we analyzed next-generation sequencing data generated through focused exome sequencing from individuals with different phenotypic manifestations referred for genetic testing to achieve a molecular diagnosis. Pathogenic or likely pathogenic variants are reported in 280 of 833 cases with a diagnostic yield of 33.6%. Homozygous sequence and copy number variants were found as positive diagnostic findings in 131 cases (15.7%) because of the high consanguinity in the Indian population. No relevant findings related to reported phenotype were identified in 6.2% of the cases. Patients referred for testing due to metabolic disorder and neuromuscular disorder had higher diagnostic yields. Carrier testing of asymptomatic individuals with a family history of the disease, through focused exome sequencing, achieved positive diagnosis in 54 of 118 cases tested. Copy number variants were also found in trans with single-nucleotide variants and mitochondrial variants in a few of the cases. The diagnostic yield and the findings from this study signify that a focused exome test is a good lower-cost alternative for whole-exome and whole-genome sequencing and as a first-tier approach to genetic testing.

Combined sequence and copy number analysis improves diagnosis of limb girdle and other myopathies.

OBJECTIVE: Clinical and genetic heterogeneities make diagnosis of limb-girdle muscular dystrophy (LGMD) and other overlapping disorders of muscle weakness complicated and expensive. We aimed to develop a comprehensive next generation sequence-based multi-gene panel ("The Lantern Focused Neuromuscular Panel") to detect both sequence variants and copy number variants in one assay. METHODS: Patients with clinical diagnosis of LGMD or other overlapping muscular dystrophies in the United States were tested by PerkinElmer Genomics in 2018-2021 via "The Lantern Project," a sponsored diagnostic testing program. Sixty-six genes related to LGMD subtypes- and other myopathies were investigated. Main outcomes were diagnostic yield, gene-variant spectrum, and LGMD subtypes' prevalence. RESULTS: Molecular diagnosis was established in 19.6% (1266) of 6473 cases. Major genes contributing to LGMD were identified including CAPN3 (5.4%, 68), DYSF (4.0%, 51), GAA (3.7%, 47), ANO5 (3.6%, 45), and FKRP (2.7%, 34). Genes of other overlapping MD subtypes identified included PABPN1 (10.5%, 133), VCP (2.2%, 28), MYOT (1.2% 15), LDB3 (1.0%, 13), COL6A1 (1.5%, 19), FLNC (1.1%, 14), and DNAJB6 (0.8%, 10). Different sizes of copy number variants including single exon, multi-exon, and whole genes were identified in 7.5% (95) cases in genes including DMD, EMD, CAPN3, ANO5, SGCG, COL6A2, DOK7, and LAMA2. INTERPRETATION: "The Lantern Focused Neuromuscular Panel" enables identification of LGMD subtypes and other myopathies with overlapping clinical features. Prevalence of some MD subtypes was higher than previously reported. Widespread deployment of this comprehensive NGS panel has the potential to ensure early, accurate diagnosis as well as re-define MD epidemiology.