Discordant phenotype in monozygotic twins with renal coloboma syndrome and a PAX2 mutation
Supplementary material
Paraskevas Iatropoulos1, Erica Daina1, Caterina Mele1, Ramona Maranta1, Giuseppe Remuzzi1,2,
Marina Noris1
Affiliations:
1. Mario Negri Institute for Pharmacological Research, Clinical Research Center for Rare Diseases Aldo e
Cele Daccò, Ranica (BG), Italy
2. Department of Nephrology and Dialysis, Azienda Ospedaliera Ospedali Riuniti di Bergamo, Bergamo
(BG), Italy
Supplementary Figure 1. Panel A shows sequencing electropherograms of twin 1 (i) and a healthy control
person (ii). Twin 1 carries a heterozygous c.155G>A substitution (red arrow) in the PAX2 gene. The lower
electropherogram (ii) shows the same region in a healthy control carrying the homozygous wild-type allele
(red arrow); Panel B shows the sequence alignment of the Pax2 protein and it homologues among various
species around the highly evolutionary conserved Cys52 amino acid (red box). This residue is changed to
Tyr (Y) in both twins. The mutation was predicted to be probably damaging by Polyphen-2 (HumVar score
0.999; sensitivity: 0.09, specificity: 0.99) and damaging by SIFT (score 0.00).
100
Percentage of Mutant A allele
90
80
70
Blood
60
Urine Sediment
50
40
Jugal Cells
30
Hair follicles
20
10
0
Twin 1
Twin 2
Supplementary Figure 2. Semiquantitative analysis of the mutant A allele by qPCR in genomic DNA from
different tissues in the twins. The percentage of the mutant allele was 50.5±1.7, 49.9±2.8, 51.7±2.3, and
51.6±1.7 in peripheral blood, urine sediment, jugal epithelial cells from the interior mouth mucosa and hair
follicles respectively in twin 1, and 49.6±2.3, 50.3±1.7, 51.5±2.2, and 50.3±1.5 respectively in twin 2. No
statistically significant differences were observed among different tissues in each twin (One way ANOVA pvalue of 0.191 and 0.302 for Twin 1 and Twin 2 respectively) or in the same tissue between the two sisters
(t-Test p-value of 0.297, 0.684, 0.871, and 0.167 for peripheral blood, urine sediment, jugal epithelial cells
from the interior mouth mucosa and hair follicles respectively).
Supplementary Table 1. Clinical features of the twin sisters.
Features
Twin 1
Twin 2
+ (right kidney)
-
- Proteinuria
+
+
- End Stage Renal Disease
+
-
- Optic nerve coloboma
-
+ (left eye)
- Visual acuity loss
-
+ (left eye)
- Joint laxity
+
+
- Mild skin hyperextensibility
+
+
Renal findings
- Multiple cystic dysplasia
Opthalmological findings
Additional findings
Supplementary methods
Mutational Analysis
Genomic DNA was extracted from peripheral blood and from the urinary sediment of both patients and their
parents using the NucleonTM BACC2 Genomic DNA extraction kit (GE Healthcare, Little Chalfont, UK).
All twelve exons of the PAX2 gene and their flanking intronic regions were screened for mutations by PCR
amplification followed by purification using ExoSAP-IT® (GE Healthcare, Little Chalfont, UK) and direct
sequencing using a BigDye® terminator kit v.3.1 (Applied Biosystems, Foster City, CA, USA). The
products of the sequencing reaction were purified using the MontageTM Seq96 Sequencing Reaction Cleanup
Kit (Millipore Corporation, Billerica, MA, USA) and then run on an ABIPRISM® 3130xl Genetic Analyzer
(Applied Biosystems, Foster City, CA). Primer sequences and PCR conditions are reported in
Supplementary Table 2.
Primers were designed according to the NCBI PAX2 genomic reference sequence NG_008680.1. Variants
are referred to as isoform a according to the NCBI PAX2 RNA reference sequence NM_003987.3 and
numbering begins with the ATG start codon.
The presence of the c.155G>A variant in the general population was investigated in a control group of 100
healthy volunteers selected from blood donors with no personal or family history of renal disease. Fragments
corresponding to exon 2 were amplified by PCR and subsequently screened by denaturing high performance
liquid chromatography (DHPLC; Transgenomic, Omaha, NE) after determination of the optimal temperature
(66.0°C) for the detection of c.155G>A.
The impact of the non-synonymous variant on protein function was evaluated using PolyPhen-2 software
(http://genetics.bwh.harvard.edu/pph2/) and SIFT software (http://sift.jcvi.org/).
Supplementary Table 2.
Exon
EX1
Primers used in mutational screening
Primer
Length
Tm (°C)
Product Size
63.0
304
63.0
469
63.0
340
68.0
212
63.0
290
63.0
205
63.0
466
63.0
380
63.0
533
68.0
290
68.0
368
F
CGGGCGTTCACTCATCCTC
19
R
GAAAGAACGAAAAGAGGGACCAGA
24
F
TTCTTCTCAAGCTCGGGAACATG
23
R
AGCCACCATCTGAACACTCTCT
22
F
GCAGGAGAGTGGCTCAGCAG
20
R
AGGAGCCAGGAGCTGGAGTC
20
F
GAATTGGCCGGGATAGGAGTGG
22
R
TGGAGCTGCGTTTCCTGCCTT
21
F
GCTTTGGCCTACGATCACAACT
22
R
TGATCTCACAGCAGAGCAGCT
21
F
CTTTCGGGATCTCTCAGTGTTTGT
24
R
CCCTTAGGAACCCACTCTCTGAA
23
F
GCTTCCTCAGCCAGATCTCTGA
22
R
CTGGCTATGCATGTGGTGTTTAGT
24
F
CCACCATCTCTTTCTACCCCATCT
24
R
TGGTTTTAGAAGCCTCGTTCTCTC
24
F
GTGCAGTACCCTGGTGTGAGTAG
23
R
AGGCTCTTCCAAGCAGTGTCAG
22
EX11
F
AGAAGCCACCGGCCGGACTC
20
EX11
R
TGCTGCACTAACAAGCCTGTCCCC
24
EX12
F
GCCCAGCCAAGGTCTCCCAGTC
22
EX12
R
TGAAGGGTTGCGGGGGTCGT
20
EX2
EX3
EX4
EX5
EX6
EX7
EX8
EX9+10
Tm: annealing temperature used during PCR amplification
Twin Zygosity Determination
Molecular zygosity determination was performed by genotyping 11 polymorphic microsatellites (D1S412,
D1S413, D5S818, D7S820, D11S1338, D11S4146, D11S1996, D13S317, D17S798, D17S927, and
D19S425) located on 7 different chromosomes.
For the analysis of markers D1S412, D1S413, D11S1338, D11S4146, D17S798, and D17S927, the probes
of Applied Biosystems were used. For markers D5S818, D7S820, D11S1996, D13S317, and D19S425,
primer sequences of the UniSTS database (http://www.ncbi.nlm.nih.gov/sites/entrez?db=unists) were used
and the forward primers were labelled at the 5’-end by a fluorescent dye (6-Fam or VIC). After PCR
amplification the size of the amplicons was measured by capillary electrophoresis using a 3130xl Genetic
Analyzer and GeneMapper software version 3.7.
Quantification of PAX2 DNA and mRNA in blood and urines
Genomic DNA (gDNA) from peripheral blood and from the urinary sediment was extracted as described
above. To obtain genomic DNA from jugal epithelial cells from the interior mouth mucosa cell sample
collection was performed using a modified version the toothbrush-rinse method described in London et al
(2001). A soft scraper was used instead of the toothbrush. Bottle drinking water was added to the
expectorated saliva to reach a volume of 30 ml and afterwards 6 ml of PBS 5x were added. The samples
were centrifuged at 1300g for 15 min, the supernatant was discarded and the pellet was suspended in 400 µl
of PBS 1x. The DNA was then extracted using PureLink™ Genomic DNA Mini Kit (Invitrogen™)
following manufacturer's protocol. To obtain genomic DNA from hair follicles we collected 12 follicles
from each patient. We added 270 μl of PureLink™ Genomic Digestion Buffer and 30 μl of Proteinase K and
the samples were incubated at 55°C for 3 h with vortexing every 20 min. The samples were then processed
using PureLink™ Genomic DNA Mini Kit (Invitrogen™) following manufacturer's protocol.
RNA was extracted from peripheral blood and urine sediment with Trizol Reagent method (TRIzol®
Reagent Ambion®) and was reverse-transcribed to cDNA with SuperScript® II Reverse Transcriptase
(Invitrogen™).
The real-time quantitative PCR assay was carried out using a Custum TaqMan® SNP Genotyping Assay
(Applied
Biosystem;
Forward
Primer:
CCCAGGATTTTGCTGACACAG;
Reverse
Primer:
AGCTGGCCCACCAGGGT; Probe Allele Mutant: ATGTCATAGGGCCGC; Probe Allele WT:
ATGTCACAGGGCCG).
The real-time amplification mixtures (20 µl) contained gDNA or cDNA template, 2X TaqMan Genotyping
Master Mix (Applied Biosystems), 20X mix with primers and probes and nuclease-free water. The cycling
conditions comprised one step at 50°C for 2 min, the polymerase activation at 95°C for 10 min and 40
cycles at 95°C for 15 sec and 63°C for 60 sec.
The assay included a standard curve of five serial dilution points of twin 2 DNA from peripheral blood in
triplicate (ranging from 200 ng to 0.781 ng), a no-template control, and a calibration curve.
The calibration curve was generated using the amplification product from one twin. The amplicons were
cloned with the Zero Blunt® TOPO® PCR Cloning Kit for Sequencing in DH5α-T1R E. coli cells
(Invitrogen). Plasmid DNA from different colonies was precipitated by isopropanol and then sequenced to
verify the presence of the wild-type or the mutant allele. Plasmids containing the G (wt) and the A alleles
(mutant) were mixed together at different ratios (100:0; 80:20; 60:40; 50:50; 40:60; 20:80; 0:100) to set up
the calibration curve. The results were plotted out and a linear trend line was calculated.
For the study of mosaicism 80 ng of genomic DNA was used. For the expression studies cDNA deriving
from up to 350 ng of RNA per reaction tube was used. All the samples were analyzed in triplicates.
Mutant allele semiquantitative data are expressed as mean ± standard deviation. The two-tailed Student’s ttest was used to compare pairs of subgroups and One way ANOVA test was used when more than two
subgroups were analyzed. All statistical analyses were performed using the MedCalc® software.
References:
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(2001) Collection of buccal cell DNA in seventh-grade children using water and a toothbrush. Cancer
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