Supplementary Materials and Methods

CMR imaging with LGE

CMR in all family members was performed on 1.5 T system (Philips Intera, Best, The Netherlands). Breath-hold cine steady-state free precession images were acquired in multiple short-axis and 3 long-axis planes. Complete LV coverage in short-axis was achieved with contiguous 8-mm-thick slices. LGE imaging protocol was performed 10-15 minutes after intravenous administration of 0.2 mmolkg-1 gadolinium-chelate (Dotarem ®, Guerbet, S.A., Villepinte, France) with breath-held segmented inversion-recovery sequence (inversion time 250 to 350 ms) acquired in the same views as the cine images.

Adapted flow cytometry protocol for analysis of rare LAMP2def cells

Key points in the rare LAMP2def cell analysis protocol were - i) acquisition of large cell numbers (3x106 WBCs) to increase the sensitivity and ii) gating on FSC-A vs. FSC-H parameters to avoid cell aggregates (false LAMP2def events) and also to decrease the background.

In order to obtain concentrated cell suspension, total of 5 ml EDTA drawn peripheral blood was split to three 50 ml conical tubes and erythrocytes were lysed with 48 ml of NH4Cl- containing lysis solution for 15 minutes. After centrifugation, supernatant was discarded and each aliquot was incubated with 3 ml of FACS Lyse followed by FACS Perm according to the manufacturer’s recommendations (BD Biosciences). Then, aliquots were united and incubated with monoclonal antibodies listed in Materials and Methods for 30 minutes. After the final wash, samples were split to two tubes and measured with BD LSRII flow cytometer (total of 3x106 WBCs were evaluated from both tubes). Assay sensitivity values (0.008%. - see Table below) were calculated as 2xSD (standard deviation) above the average values of eight measurements from four LAMP2 mutation negative individuals. As an accurate quantification prerequisite, LAMP2def cells had to form a cluster of more than 100 cells on the scatterplot (Supp. Fig. 2C and Table below).

“Fluorescence minus one” (FMO) and “isotype” (ISO) controls were included to check for autofluorescence/spectral overlap and non-specific antibody binding, respectively.

total granulocyte counts evaluated / frequency of LAMP2def granulocytes within the total granulocyte population / LAMP2def granulocytes - detected cell counts
LAMP2def rare cells -
LAMP2 mutation present
mosaic carrier II.2. (XX) - tube 1 / 1700000 / 0.0580 / 981
mosaic carrier II.2. (XX) - tube 2 / 1200000 / 0.0600 / 720
LAMP2def background - LAMP2 mutation absent
healthy II.1. (XY) - tube 1 / 1740000 / 0.0017 / 29
healthy II.1. (XY) - tube 2 / 1280000 / 0.0017 / 21
healthy I.1. (XY) - tube 1 / 1810000 / 0.0022 / 39
healthy I.1. (XY) - tube 2 / 1610000 / 0.0027 / 43
healthy I.2. (XX) - tube 1 / 1470000 / 0.0055 / 81
healthy I.2. (XX) - tube 2 / 741192 / 0.0074 / 55
healthy - unrelated (XY) - tube 1 / 1590000 / 0.0013 / 21
healthy - unrelated (XY) - tube 2 / 1000000 / 0.0023 / 23
average / 0.0031
average + (2xSD) / 0.0075
sensitivity >0.008

LAMP2 mRNA – PCR cycling conditions

PCR reactions were performed in a total volume of 25 ml containing 1x PCR buffer (Promega, Fitchburg, WI, USA), 0.2 mM of each dNTP, 8% DMSO, 100 nM of each primer, 1U KlenTaq DNA polymerase and 500 ng of template cDNA. The abnormal junction between duplicated exons 5 and 4 was detected using a pair of primers that were specific for exon 5 (forward) and exon 4 (reverse) (PCR ex5-4, Supp. Fig. 3A, B). The amplicon covering the cryptic splice site(s) between exon 9B and 9A was generated using specific primers for exon 8 (forward) and exon 9A (reverse) (PCR ex9B-9A, Supp. Fig. 3A, C).

PCR cycling program for the full-length CDSs of LAMP2B and LAMP2A was as follows: initial denaturation (94°C, 90 seconds) , 34(LAMP2B) or 35(LAMP2A) cycles of denaturation (10 seconds), annealing (10 seconds) at 60°C, extension (72°C, 105 seconds) and final cooling.

PCR program for the abnormal junction between duplicated exons 5 and 4 (PCR ex5-4) was the same except for 20 seconds extension step and 40 cycles in total.

Amplicon covering the cryptic splice site between exon 9B and 9A (PCR ex9B-9A) was generated using cycling conditions identical to the full-length CDS amplification except that the extension time was 24 seconds.

LAMP2 exon copy number (dosage) analysis

qPCR reactions were perfomed in 10 µl triplicates (300 nM forward and reverse primers, 80 nM probe labeled with a reporter dye FAM and a quencher dye (Generi Biotech, Hradec Kralove, Czech Republic or Universal ProbeLibrary, Roche Applied Science), 1.5 ng/µl template gDNA and 1x LightCycler® 480 Probes Master Mix).

qPCR cycling program started with DNA polymerase activation at 95°C for 10 minutes and was followed by 40 cycles of denaturation (95°C, 15 seconds), annealing (60°C, 60 seconds), extension (72°C, 1 second), and final cooling. The PCR specificity was checked by agarose gel electrophoresis. Data quantification was performed using LightCycler® 480 Software (release 1.5.0.).

Alu-Sz, Alu-Sg and Alu-Szg PCR amplification

For each 25 µl PCR reaction 100 ng of gDNA was used and all of the resultant PCR products were sequenced.

Pedigree genotype analyses

Elucigene QST*Rplusv2 kit consists of 22 probes located on chromosomes 13, 18, 21, X and Y (see Supp. Tab. 2). Nineteen of these probes identify highly polymorphic short tandem repeat regions of the human gDNA sequence. Multiplex QF-PCR was performed according to the manufacturer’s instructions, 5 ng of DNA was used per reaction. Amplified samples were analyzed by capillary electrophoresis using the same instrument as for XCI measurements.

Supplementary Tables Legends

Supplementary Table 1

Primer sequences and primer combinations used for the individual molecular genetic analyses.

Supplementary Table 2

Results of the polymorphic marker genotyping in all family members. Values correspond to DNA fragment sizes as assessed by the capillary electrophoresis. If possible to establish, parental inheritance was highlighted. Non-informative markers for individual persons were italicized. There was not a single marker value that would, via germline of female II.2 (without being present in her somatic tissues), skip from generation I directly to generation III. Thorough review of the results of this analysis did not imply existence of an additional background genome set (likely chimeric) in either WBCs or buccal swab gDNA of the patients’ mother (II.2). Five markers showed “stutter” as described by the manufacturer of the kit. The fragment size values are presented as values with one decimal place to demonstrate the variability of the results.

Supplementary Figures Legends

Supplementary Fig. 1

(A) Flow cytometry dot plots show the sequence of gating of peripheral blood leukocytes for conventional diagnostic LAMP2 detection. Values represent % fractions. (B) Histograms show LAMP2 presence/absence in the leukocytes’ subsets (columns) of both patients (III.1 and III.2), mosaic carrier (II.2) and healthy unrelated control. LAMP2 protein is depicted in red/blue, irrelevant monoclonal antibody (isotype control) staining is in grey, FMO control is in green. Note that while histograms provide simple overview of the LAMP2 presence in the leukocytes’ subsets, it does not allow assessment of rare LAMP2def cells in the mosaic carrier II.2 (compare to Fig. 1 and Supp. Fig.2).

Supplementary Fig. 2

(A) Flow cytometry contour plots (values represent % fractions) show gating strategy for rare LAMP2def cell detection in the mosaic carrier II.2 (XX) and compare the sensitivity and background of (B) conventional flow cytometry and (C) rare cell adapted detection in granulocytes. Note, that conventional flow cytometry had too low sensitivity and high background to detect LAMP2def granulocytes in the mosaic carrier II.2 (XX).

Supplementary Fig. 3

(A) adapted from Figure 2A. In-scale schematic of the full-length LAMP2B and LAMP2A mRNA profiles in both patients (III.1 and III.2). Asterisks indicate the approximate positions of putative premature (in mutant forms) or regular (in wt forms) STOP codons. (B) Agarose gel electrophoresis for the PCR targeting the abnormal exon 5 to exon 4 duplication junction (PCR ex5-4 in schematic A). Three out of six templates depicted on schematic A give rise to the amplification product. Note the absence of the specific product in the sample of the patients’ mother (II.2) despite the mosaic presence of the mutation in her WBCs. The amount of mutant mRNA species originating from the minute LAMP2def fraction ( Fig. 1C) is likely too small to be detected by the specific PCR (C) Agarose gel electrophoresis for the PCR directed to the 9B|9A LAMP2 mRNA forms resulting from inclusion of the alternative exon 9B (PCR ex9B-9A in the schematic A). The forward primer binding site is in exon 8 and reverse primer at the 3’ end of the coding region of exon 9A. The spectrum of templates of the PCR and the expected sizes of the PCR products are depicted in schematics A (dashed lines). Note the difference in the overall abundance of the 9A and 9B|9A mRNA forms (wt or mutant) between the control, the patients’ mother (II.2) and both patients (III.1 and III.2). The 9B|9A forms (wt or mutant) represent two separate mRNA populations differing in the usage of either of +1311(AAG|GTATAT) or +1271 (AAA|GCAAGT) cryptic splice sites in the LAMP2B mRNA reference sequence (see Materials and Methods). The 40 bp difference becomes clearly apparent in shorter PCR (PCR ex9B-9A) products. The same feature was identified in the corresponding full-length mRNA amplicons when sequenced (not reflected in A schematics). DNA marker sizes are identical for all gels and correspond to Figure 2.

Supplementary Fig. 4

De-novo occurrence of the LAMP2 mutation in the patient’s mother (II.2) was verified by specific PCR detection of the abnormal duplication breakpoint/junction sequence (PCR AluSzg) from her WBC gDNA. Presence of LAMP2 specific Alu-Sz (PCR AluSz) sequence is demonstrated in the rest of generation I. and II. family members.