Supplemental Digital Content 1. Methods
Screening for Point Mutations and Micro-Insertions/Micro-Deletions in the CASR Gene
All six coding exons (i.e., exons 2-7) of the CASR gene were analyzed by direct sequencing
for point mutations and micro-insertions/micro-deletions, with the primer sequences and
amplicon sizes being supplied below:
Exon
Amplicon size (bp)
Primer Sequence (5 to 3')
2
Forward: GTTGCAGTGGTCATCACAAG
570
Reverse: TATTTTGCGTTTGGTGCAG
3
Forward: CGATGATTCAAACCCAGCT
448
Reverse: CTGCTTCTTCTGATCCTGC
4
Forward: AGAAAGCCACCTCCACAACA
1030
Reverse: GCAGCCCAACTCTGCTTTAT
5
Forward: TGGGGCTTGTACTCATTCTT
290
Reverse: CTGGTTTTCTGATGGACAGC
6
Forward: GCCCAACGTCTGTCACACT
324
Reverse: TCCATGGGCTTCACTGAC
7
Forward: ACACATTTTAGTCTGTGCC
1588
Reverse: CTCCCTAGCCCAGTCTT
PCR pertaining to this analysis was performed in a 15 µL reaction mixture using the
AmpliTaq® DNA polymerase (Applied Biosystems, Foster City, CA) with 10 ng genomic
DNA according to the manufacturer’s protocol. The PCR programme comprised an initial
step of denaturation at 94°C for 2min, followed by 40 cycles of denaturation at 94°C for 30
sec, annealing at 60°C for 30 sec and extension at 72°C for 30 sec or 90 sec (according to the
amplicon size), and a final extension at 72°C for 10 min. Amplicons were purified by
ExoSAP-IT (GE Healthcare, Orsay, France) and sequenced using the ABI PRISM Big-Dye
Terminator v3.1 Cycle Sequencing kit (Applied Biosystems).
Screening for Gross Genomic Rearrangements or Copy Number Variation in the CASR
Gene
Gross genomic rearrangements or copy number mutations in the CASR gene were screened
for by means of quantitative fluorescent multiplex-PCR. Seven primer pairs were designed to
span its seven exons with a control primer pair targeting the MGAM gene located on
chromosome 7:
Exon
Primer sequence (5 to 3')
1
Forward: TGCAGACCTGCCATGCCAATT
Reverse: TGTGCGGCCCATATACTTGGA
Forward: ACTCAAGGACCACCCACAT
Reverse: AATGCCATGGTTCTGCCGT
Forward: ATTTCCGTGGGTTTCGCTGGT
Reverse: AGGCCTTAGAAACGGTGTTGC
Forward: TGGGCACAATTGCAGCTGAT
Reverse: ACGATGACTTTGGCCGTGGAAT
Forward: AGATGGCTCCATCGTGTT
Reverse: AGAACCCACTCCACAGGAT
Forward: TCACCTTTGTGCTGTCTGTCCT
Reverse: ACACACTCAAAGCAGCAGGT
Forward: TCCTGCATTGCCAAGGAGAT
2
3
4
5
6
7
Primer
concentration (µM)
0.4
Amplicon
size (bp)
154
0.4
209
0.2
132
0.4
178
0.4
104
0.4
115
0.6
142
MGAM
exon 7
Reverse: TGACAATGGGTGTGTTGCGGA
Forward: TTTGCTGACCAGTTCTTGCAGCTC
Reverse: GGGCCCATTTGAAAGCTTACTCCA
0.1
164
All eight targeted regions were amplified in a single PCR reaction with 100 ng genomic DNA
in a 10 µL reaction mixture using the Qiagen Multiplex PCR Kit (Qiagen, Courtaboeuf,
France). After an initial step of denaturation at 95°C for 15 min, the PCR program comprised
25 cycles of denaturation at 95°C for 30 sec, annealing at 58°C for 60 sec and extension at
72°C for 90 sec, with a final extension at 72°C for 30 min. Amplified DNA fragments were
separated on an ABI Prism 310 sequencer (Applied Biosystems, Foster City, CA, USA) and
data were analysed with Genemapper v3.4 (Applied Biosystems). Peak heights of the seven
CASR amplicons for each patient sample were first normalized against the peak height of the
control MGAM amplicon before being superimposed upon those of a control sample. The
presence of a deletion was indicated by the two-fold reduction of the corresponding peak.
Statistical Analysis
Variant frequencies in cases and controls were compared using the χ2 test wherever
appropriate. In case of significant differences (P < 0.05), the strength of the association was
assessed by calculating the odds-ratio (OR) with 95% confidence interval (CI).