Supplementary data to:
Epidemiological, Clinical and Biochemical Characterization of the p.(Ala359Asp)
SMPD1 Variant Causing Niemann-Pick Disease Type B
Mariana Acuña,1,2 Pablo Martínez,1,2 Carol Moraga,2,3 Xingxuan He,4 Mauricio Moraga,5 Bessie
Hunter,6 Peter Nuernberg,7 Rodrigo A. Gutiérrez,2,3 Mauricio González,2,8 Edward H. Schuchman,4
José Luis Santos,9 Juan Francisco Miquel,1,2 Paulina Mabe,10 Silvana Zanlungo,1,2.
1
Departamento de Gastroenterología, Facultad de Medicina, Pontificia Universidad Católica de
Chile, Santiago, 8330024, Chile. 2FONDAP Center for Genome Regulation (CGR). 3Departamento de
Genética Molecular y Microbiología, Facultad de Ciencias Biológicas, Pontificia Universidad Católica
de Chile, Santiago, 8331150, Chile. 4Department of Genetics & Genomic Sciences, Icahn School of
Medicine at Mount Sinai, New York, NY, 10029, USA. 5Programa de Genética Humana, Instituto de
Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, 8380453, Chile.
6
Hospital Luis Calvo Mackenna, Santiago, 7500539, Chile. 7Cologne Center for Genomics (CCG),
University of Cologne, Köln, D-50931, Germany. 8INTA, Universidad de Chile, Santiago, 7830490,
Chile. 9Departamento de Nutrición, Pontificia Universidad Católica de Chile, Santiago, 8330024,
Chile. 10Hospital Exequiel González Cortés, Santiago, 8910095, Chile.
Table of Contents
Supplementary Materials and Methods....................................................................................2-4
Taqman allelic Discrimination Assay...………………………………………………...2
Clinical Characteristics of NPDB in Homozygous p.(Ala359Asp) Patients……..............2
Common Ancestor Haplotype Determination and Population Origin…………................2
DNA Genomic Sequencing...………………………………...………………………...3
Cell Transfection and ASM Activity Assay ...…………………………………………..3
Immunoblotting...………………...............................………………………………...3
Supplementary Tables................................................................................................................4
1
Supplementary Materials and methods
Taqman Allelic Discrimination Assay
The assay mix was developed using the Applied Biosystems bioinformatics assay design process
and included forward (GAGCCCTGGCTGCCT; 15 nucleotides in length) and reverse primers
(GTTTCCACGGACGATAAGTACCT; 23 nucleotides in length). Also included two TaqMan MGB probes
designed to detect the normal allele (CGCAGGGCTTCG; linked to VIC in the 5' end), and the mutant
allele (CGCAGGTCTTCG; linked to FAM in the 5' end).
Clinical Characteristics of NPDB in Homozygous p.(Ala359Asp) Patients
The laboratory analysis included blood count, lipid profile, serum alanine aminotransferase,
aspartate aminotransferase, total and direct bilirubin, prothrombin time and abdominal
ultrasonography.
Common Ancestor Haplotype Determination and Population Origin
The SNPs genotypes analyzed were determined by PCR-RFLP or sequencing methods. The primers
used are described in Supplementary Table I. Of the 14 SNPs analyzed, 8 were covering the SMPD1
gene and flanking regions (~10 Kb). The other 6 SNPs were located 75, 68 and 41 Kb upstream and
72, 200 and 404 Kb downstream of the gene.
In addition, 3 Short Tandem Repeats (STRs) were analyzed: D11S1323 (STR1) (PCR products 200207bp), D11S4531 (STR2) (PCR products 160-188bp) and D11S1331 (STR3) (PCR products 191205bp). STR1 and STR2 are located 135 and 99 Kb upstream of the SMPD1 gene, respectively, while
STR3 is located 800 Kb downstream of the SMPD1 gene.
DNA Genomic Sequencing
The sequenced 14.5 Kb DNA fragment included 4.5 Kb encoding the entire SMPD1 genomic region
and an additional 5.0 Kb upstream and downstream of the gene.
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Cell Transfection
Cells were transfected with 2 μg of wild-type or p.(Ala359Asp) mutant plasmid using the XtremeGEN transfection reagent (Roche, Indianapolis, IN, USA). Cells transfected with an empty
pcDNA3.1(–) vector were used as a negative control. Forty-eight hours after transfection, cell
pellets were washed twice with phosphate buffered saline (PBS) and resuspended in CellLytic
reagent (Sigma) for ASM activity and immunoblotting.
Immunoblotting
Cell extracts (10 μg) were resolved by SDS-PAGE and transferred onto nitrocellulose membranes.
Membranes were blocked by overnight incubation at 4°C in 5% (w/v) milk powder dissolved in Tris
buffered saline (TBS) and 0.1 % (w/v) Tween 20 (TBST). They were then rinsed in TBST and
incubated at room temperature for 2 h in the presence of a polyclonal anti-ASM antibody (1:2000
dilution) donated by Dr. Edward Schuchman. The membrane was washed again in TBST, and
incubated at room temperature for 1 h in the presence of a HRP conjugated anti-rabbit IgG
antibody (1:5000 dilution).
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Supplementary Table
Supplementary Table 1: Gene-specific primer sequences for SNP analysis
SNP
Forward primer
Reverse primer
rs11040859
GCCACATGTGCTGGATGACA
TGACCCCTAGAAACCCAAAGG
rs2947030
CCCCACTCATCAAAGCTGGT
CAGCTTCATTCCAGGCCTCA
rs7128818
TGCCAGCAACTTCTGTCTGA
CGGCCAGGGTCCATTTTTAC
rs7940008
CTGGTCAACAACAGCGAAAA
GGTTAAGGACATGGCTGGAA
rs2723659
AGACATGTTTTGCTTGACACATCCA
AGGTACTTTTCTGAGGACTCTGCCT
rs1542705
AAGGGGCTCATCTTCCTTAGA
CCTTGGTCCCTGCAAATACT
rs7951904
CCTCACTGACCTGCACTGGGA
CACCTGGCCGGGATGCGG
rs67992843*
GGGCACTGGGACATTTTCT
CATGGTGAAACCCCATCTCT
rs113960387*
GGGCACTGGGACATTTTCT
CATGGTGAAACCCCATCTCT
rs72896270
CCACCACACCCAGCTAATTT
TGGGAAAAACTGGAGCACTT
rs74053351
CAGTTCTTTGGCCACACTCA
GGCAAGGAGCAAGGGTAACT
rs11601088
GGCCTTAATCCTGGTGAGTG
CGAGCCCTGTAGAGAAGCTG
rs1050239
GGCCTTAATCCTGGTGAGTG
CGAGCCCTGTAGAGAAGCTG
rs1800606
CTCTAGACCCCTCCCTGACC
AGTAGGGTGGTGTGGTGGTG
rs2682092
CCACTTTGCCAGCCTACATT
ATTGGGTCTTGGGAGAGGAC
rs2723663
GCTGCAGCTCTCGTTTTCTT
TTTCCAAGGCTACACAGTAAGT
rs11040930
TCACTGCTGATTGCTCTGGG
AGGATGGGCAGGTTGTTAGC
rs497681
TAGGGGTCATGTCCCAAGGT
TCCACAGCACCAGACAAGAC
Thermal cycling consisted of 30 cycles at 94ºC for 30 s, 58ºC for 30 s, and 72ºC for 30 s. The melting
curves were performed according to the manufacturer’s procedure. *Both SNPs were determined
by sequencing of the same PCR reaction, which generated a fragment of approximately 640 bp that
also contained the A359D mutation.
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