Mosaic tissue distribution of the tandem duplication of LAMP2 exons 4 and 5 demonstrates the limits of Danon disease cellular and molecular diagnostics

Abstract

Alu-mediated tandem duplication of exons 4 and 5 (g.15815_22218dup6404) is a novel mutation that has been detected in the LAMP2 gene (Xq24). This exon copy number variation was found in two brothers with the typical phenotype of Danon disease, including characteristic myocardial changes on magnetic resonance imaging. The 6.4 kb duplication was identified in both boys by a combination of exon dosage qPCR analyses and duplication breakpoint/junction mapping. The rearrangement results in a plethora of abnormal LAMP2 splicing variants and also in use of likely cryptic splice sites in the 3' terminus of LAMP2 gene. Although we found minute amounts of normal LAMP2B and LAMP2A mRNAs, no protein was detectable in peripheral blood leukocytes by flow cytometry in both brothers. Uniquely, the fraction of LAMP2-deficient granulocytes (0.06%) assessed by flow cytometry in the patients' asymptomatic mother substantially differed from the random distribution of X-chromosome inactivation in her leukocytes. This discrepancy was later explained by molecular genetic methods as a consequence of mosaic distribution of the mutation in her somatic tissues. Altogether, we report a novel and mosaically distributed exon copy number rearrangement in the LAMP2 gene and comment on obstacles this genetic setup presents to the overall cellular and molecular diagnostic algorithm of Danon disease. Our observations of the mosaicism in the asymptomatic mother suggest that similarly affected females could be a potentially under-diagnosed Danon disease carrier group and that LAMP2 flow cytometry, because of its supreme sensitivity, can be an efficient method for pedigree screening.

LAMP2 flow cytometry in peripheral white blood cells is an established method that facilitates identification of heterozygous Danon disease female patients and mosaic mutation carriers

“…In light of the published data [2][3][4][5], we disagree with the statements by Hashida et al regarding the novelty of the FC LAMP1/LAMP2 assay as well as its usage to identify female DD patients/carriers. Similarly, the utility of WBCs and/or FC in DD screening has been directly implied [2][3][4][5].…”

“…Similarly, the utility of WBCs and/or FC in DD screening has been directly implied [2][3][4][5]. Although briefly referenced in the Introduction, the works by Regelsberger et al [3] and us [4], despite of direct relevance, remain uncited in the rest of the manuscript.…”

“…In May 2013, we published a study [5] in two male DD patients and their clinically unaffected mother all carrying a duplication of LAMP2 exons 4 and 5. To detect the extremely rare LAMP2 deficient WBC populations (0.06% of granulocytes and 0.13% of monocytes), and thus establish the mother's genetic setup as a somatic/ germinal mosaic, we adapted our FC assay to conditions utilized to detect minimal (hemato-oncological) residual disease.…”

“…The methodological setup, including gating strategies and comparison of sensitivity of the two FC protocols (conventional [4] vs. rare deficient cell detection) were presented [5].…”

Danon Disease Due to a Novel LAMP2 Microduplication

“…The majority of reported Danon disease alleles are private, with nonsense and frameshift mutations predominating (Boucek et al 2011). Recently, nonrecurrent microdeletions and microduplications at the LAMP2 locus have been demonstrated in four unrelated cases (Yang et al 2010;Majer et al 2013). Herein, we report the clinical, pathological, and molecular findings in a mother and son with Danon disease due to a small intragenic LAMP2 microduplication, a novel category of mutation in this disorder.…”