Deep clinicopathological phenotyping identifies a previously unrecognized pathogenic EMD splice variant

Daniel G Calame 1 2 3, Jawid M Fatih 3, Isabella Herman 1 2 3, Zeynep Coban-Akdemir 3, Haowei Du 3, Tadahiro Mitani 3, Shalini N Jhangiani 4, Dana Marafi 3 5, Richard A Gibbs 3 4, Jennifer E Posey 3, Vidya P Mehta 6, Carrie A Mohila 6, Farida Abid 1 2, Timothy E Lotze 1 2, Davut Pehlivan 1 2 3, Adekunle M Adesina 6, James R Lupski 2 3 4 7

Affiliations

01 October 2021

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doi: 10.1002/acn3.51454

Abstract

Exome sequencing (ES) has revolutionized rare disease management, yet only ~25%-30% of patients receive a molecular diagnosis. A limiting factor is the quality of available phenotypic data. Here, we describe how deep clinicopathological phenotyping yielded a molecular diagnosis for a 19-year-old proband with muscular dystrophy and negative clinical ES. Deep phenotypic analysis identified two critical data points: (1) the absence of emerin protein in muscle biopsy and (2) clinical features consistent with Emery-Dreifuss muscular dystrophy. Sequencing data analysis uncovered an ultra-rare, intronic variant in EMD, the gene encoding emerin. The variant, NM_000117.3: c.188-6A > G, is predicted to impact splicing by in silico tools. This case thus illustrates how better integration of clinicopathologic data into ES analysis can enhance diagnostic yield with implications for clinical practice.

Conflict of interest statement

J.R.L. has stock ownership in 23andMe, is a paid consultant for Regeneron Genetics Center, and is a co‐inventor on multiple United States and European patents related to molecular diagnostics for inherited neuropathies, eye diseases, and bacterial genomic fingerprinting. The Department of Molecular and Human Genetics at Baylor College of Medicine receives revenue from clinical genetic testing conducted at Baylor Genetics (BG) Laboratories; JRL is a member of the Scientific Advisory Board of BG. Other authors have no potential conflicts to report.

Figures

Figure 1 Clinicopathological features of proband with X‐linked Emery‐Dreifuss muscular dystrophy (A) Photograph of proband’s fully extended arms demonstrating limited active range of motion due to elbow contractures (right greater than left, red arrow). (B) Photograph of proband’s fully dorsiflexed foot demonstrating limited active range of motion due to ankle contractures. (C) Hematoxylin and eosin staining of left vastus lateralis muscle biopsy taken at 11 years old. (D) Immunofluorescence staining of lamin A‐C in the proband. Lamin A‐C is a nuclear protein involved in autosomal dominant Emery‐Dreifuss muscular dystrophy 1 (MIM #310300). Lamin A‐C (green) is widely expressed and co‐localizes with the nuclear stain DAPI (blue). (E) Immunofluorescence staining of emerin in the proband. Note the absence of emerin staining (green). Nuclei were counterstained with DAPI (blue). (F) Western blot of muscle biopsy protein lysates from proband and two control patients using antibodies for alpha‐sarcoglycan (αSG) and emerin (EMD). All samples were run in duplicate. Molecular weights are provided in kilodaltons (kDa). A 50 kDa band is seen in all samples corresponding to αSG. A 34 kDa band corresponding to EMD can be seen in the control samples but not in the proband.

Figure 1 Clinicopathological features of proband with X‐linked Emery‐Dreifuss muscular dystrophy (A) Photograph of proband’s fully extended arms demonstrating limited active range of motion due to elbow contractures (right greater than left, red arrow). (B) Photograph of proband’s fully dorsiflexed foot demonstrating limited active range of motion due to ankle contractures. (C) Hematoxylin and eosin staining of left vastus lateralis muscle biopsy taken at 11 years old. (D) Immunofluorescence staining of lamin A‐C in the proband. Lamin A‐C is a nuclear protein involved in autosomal dominant Emery‐Dreifuss muscular dystrophy 1 (MIM #310300). Lamin A‐C (green) is widely expressed and co‐localizes with the nuclear stain DAPI (blue). (E) Immunofluorescence staining of emerin in the proband. Note the absence of emerin staining (green). Nuclei were counterstained with DAPI (blue). (F) Western blot of muscle biopsy protein lysates from proband and two control patients using antibodies for alpha‐sarcoglycan (αSG) and emerin (EMD). All samples were run in duplicate. Molecular weights are provided in kilodaltons (kDa). A 50 kDa band is seen in all samples corresponding to αSG. A 34 kDa band corresponding to EMD can be seen in the control samples but not in the proband.

Figure 2 Research ES identifies a previously unrecognized EMD splice variant. (A) Screenshots of the proband’s BAM file data from Integrated Genomics Viewer (https://software.broadinstitute.org/software/igv/) showing a hemizygous intronic variant in intron 2 of EMD. (B) Heat map showing predicted impact on splicing from SpliceAI and MMSplice of all possible single‐nucleotide variants near c.188‐6A > G. Values in heat map were obtained from CADD v1.6 (https://cadd.gs.washington.edu/snv) and reflect the SpliceAI acceptor gain and MMSplice acceptor submodules, respectively. Darker shades of red indicate a greater predicted splicing impact. c.188‐6A > G and the consensus splice acceptor sites (−1, −2) are highlighted in black. (C) Sanger sequencing of the proband’s parents and unaffected brothers confirms the variant and demonstrates it is only found in the proband and his mother.

Figure 2 Research ES identifies a previously unrecognized EMD splice variant. (A) Screenshots of the proband’s BAM file data from Integrated Genomics Viewer (https://software.broadinstitute.org/software/igv/) showing a hemizygous intronic variant in intron 2 of EMD. (B) Heat map showing predicted impact on splicing from SpliceAI and MMSplice of all possible single‐nucleotide variants near c.188‐6A > G. Values in heat map were obtained from CADD v1.6 (https://cadd.gs.washington.edu/snv) and reflect the SpliceAI acceptor gain and MMSplice acceptor submodules, respectively. Darker shades of red indicate a greater predicted splicing impact. c.188‐6A > G and the consensus splice acceptor sites (−1, −2) are highlighted in black. (C) Sanger sequencing of the proband’s parents and unaffected brothers confirms the variant and demonstrates it is only found in the proband and his mother.

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