Figure 1:Mutation in Pheo Tissue Confirmed by Targeted Next-Generation Sequencing

Supplement

Figure 1:Mutation in Pheo tissue confirmed by targeted next-generation sequencing.

Targeted next-generation sequencing of DNA extracted from frozen Pheo tissue of patient one detected both G and A at the same locus, implying heterozygous mutation.

Table1: Primers used for PCR and sequencing of VHL gene

Name / Sequence of primers / Amplicon(bp)
hVHL-Exon1-F / AACGGCCTACGGTGCTGGA / 673
hVHL-Exon1-R / AGCAGCGTCACCCTGGATGT
hVHL-Exon2-F / TTAGTAGAGACGAGGTTTCACCACG / 402
hVHL-Exon2-R / AAGATTGGATAACGTGCCTGACAT
hVHL-Exon3-F / CAGCATAACACACTGCCACATACA / 476
hVHL-Exon3-R / TTCTTTTAAGCAATGGTGCCTATTTT

Table2: summary of mutations and phenotypes of VHL disease in Chinese

Type of
mutation / phenotype
Type 1 / Type2
missense / 27 / 18
nonsense / 14 / 1
Micro-deletion / 5 / 0
Micro-insertion / 1 / 2
splice / 3 / 1
Gross deletion / 12 / 0

Table3: Summary of VHL disease type 2 with mutation information in Chinese

Pedigree
No. / type / family history / exon / mutation / consequence / CNS / RA / RCC / PCT / Pheo
1(AH) / 2A / yes / 2 / c.461C>T / p.P154L / + / + / + / + / +
2(AH) / 2A / yes / 1 / c.239G>T / p.S80I / + / / / + / + / +
3(BJ) / 2C / yes / 3 / c.482G>A / p.R161Q / / / / / / / / / +
4(BJ) / 2C / yes / 3 / c.482G>A / p.R161Q / / / / / / / / / +
5(BJ) / 2C / yes / 3 / c.487C>T / p.L163F / / / / / / / / / +
6(BJ) / 2C / yes / 3 / c.499C>T / p.R167W / / / / / / / / / +
7(HK) / 2C / no / 3 / c.482G>A / p.R161Q / / / / / / / / / +
8(HK) / 2B / no / 3 / c.499C>T / p.R167W / + / / / + / / / +
9(HK) / 2C / no / 3 / c.482G>A / p.R161Q / / / / / / / / / +
10(HK) / 2B / yes / 1 / c.239G>T / p.S80I / + / / / + / / / +
11(HZ) / 2A / yes / 1 / c.233A>G / p.N78S / / / / / + / / / +
12(HZ) / 2C / yes / 3 / c.482G>A / p.R161Q / / / / / / / / / +
13(LZ) / 2C / yes / 2 / c.374A>C / p.H125P / / / / / / / / / +
14(LZ) / 2C / no / 2 / c.359G>C / p.R120T / / / / / / / / / +
15(LZ) / 2C / no / 1 / c.410dupT / frameshift / / / / / / / / / +
16(LZ) / 2C / no / 1 / c.411dupT / frameshift / / / / / / / / / +
17(PKU) / 2B / no / 1 / c.256C>T / p.P86S / / / + / + / + / +
18(SH) / 2A / yes / 1 / c.293A>G / p.Y98C / + / / / / / / / +
19(SH) / 2B / yes / 1 / c.446A>G / p.N78S / + / + / + / + / +
20(SH) / 2B / yes / 1 / c.452G>T / p.S80I / + / / / + / + / +
21(SZ) / 2A / yes / intron1 / c.340+5G>C / unknown / + / / / / / / / +
22* / 2B / yes / 1 / c.264G>A / p.W88X / / / / / + / / / +

Abbreviations:

AH:The First Affiliated Hospital of Anhui Medical University, Anhui;

BJ: Peking Union Medical College Hospital, Beijing;

HK: Queen Mary Hospital, Hong Kong;

HZ:The 117th PLA Hospital, Hangzhou;

LZ: Lanzhou General Hospital of PLA, Lanzhou;

PKU: Peking University First Hospital, Beijing;

SH: Renji Hospital, Shanghai;

SZ:The First Affiliated Hospital of Soochow University, Suzhou.

*current pedigree

CNS: hemangioblastoma of CNS; RA: Retinal Angioma; PCT:Pancreatic Cyst or Tumor; RCC Renal Cell Carcinoma; Pheo: pheochromocytoma.

Table 4: 90 genes responsible for hereditary cancers

ALK / BUB1B / ELAC2 / FANCD2 / HSD3B2 / MSH6 / PMS2 / RNASEL / SUFU
APC / CDC73 / EPCAM / FANCE / KIT / MUTYH / PRF1 / SBDS / TP53
ATM / CDH1 / ERCC2 / FANCF / LMO1 / NBN / PRKAR1A / SDHAF2 / TSC1
AXIN2 / CDK4 / ERCC3 / FANCG / MEN1 / NF1 / PTCH1 / SDHB / TSC2
BARD1 / CDKN2A / ERCC4 / FH / MET / NF2 / PTEN / SDHC / TSHR
BLM / CHEK2 / ERCC5 / FLCN / MLH1 / NTRK1 / RAD50 / SDHD / VHL
BMPR1A / CYLD / EXT1 / GPC3 / MLH3 / PALB2 / RAD51C / SMAD4 / WRN
BRCA1 / DDB2 / EXT2 / HNF1A / MPL / PDGFRA / RB1 / SMARCB1 / WT1
BRCA2 / DICER1 / FANCA / HRAS / MRE11A / PHOX2B / RECQL4 / SRD5A2 / XPA
BRIP1 / EGFR / FANCC / HSD17B3 / MSH2 / PMS1 / RET / STK11 / XPC

Table 5: Sequencing results of pheochromocytoma DNA of the proband

Mutation site / chromosome / location / Reference base / Variant base / het/homo / RSNo. / frequency / transcript / exon / cDNAvatiation / Amino acid change
ALK:D1529E / chr2 / 29416366 / G / C / het / rs1881421 / 0.8684 / NM_004304 / exon29 / c.4587C>G / p.D1529E
ALK:K1491R / chr2 / 29416481 / T / C / het / rs1881420 / 0.866 / NM_004304 / exon29 / c.4472A>G / p.K1491R
ALK:I1461V / chr2 / 29416572 / T / C / hom / rs1670283 / 0.8947 / NM_004304 / exon29 / c.4381A>G / p.I1461V
APC:V1822D / chr5 / 112176756 / T / A / hom / rs459552 / 0.799 / NM_000038 / exon16 / c.5465T>A / p.V1822D
ATM:N1983S / chr11 / 108183167 / A / G / hom / rs659243 / 0.7177 / NM_000051 / exon40 / c.5948A>G / p.N1983S
AXIN2:P50S / chr17 / 63554591 / G / A / hom / rs2240308 / 0.5622 / NM_004655 / exon2 / c.148C>T / p.P50S
BARD1:V507M / chr2 / 215632255 / C / T / het / rs2070094 / 0.5526 / NM_000465 / exon6 / c.1519G>A / p.V507M
BARD1:R378S / chr2 / 215645464 / C / G / het / rs2229571 / 0.5837 / NM_000465 / exon4 / c.1134G>C / p.R378S
BLM:T298M / chr15 / 91295110 / C / T / het / rs28384991 / 0.1794 / NM_000057 / exon4 / c.893C>T / p.T298M
BLM:V1321I / chr15 / 91354521 / G / A / het / rs7167216 / 0.0431 / NM_000057 / exon21 / c.3961G>A / p.V1321I
BMPR1A:P2T / chr10 / 88635779 / C / A / het / rs11528010 / 0.4593 / NM_004329 / exon3 / c.4C>A / p.P2T
BRCA1:S1613G / chr17 / 41223094 / T / C / het / rs1799966 / 0.6483 / NM_007294 / exon15 / c.4837A>G / p.S1613G
BRCA1:K1183R / chr17 / 41244000 / T / C / het / rs16942 / 0.6172 / NM_007294 / exon10 / c.3548A>G / p.K1183R
BRCA1:E1038G / chr17 / 41244435 / T / C / het / rs16941 / 0.5885 / NM_007294 / exon10 / c.3113A>G / p.E1038G
BRCA1:P871L / chr17 / 41244936 / G / A / het / rs799917 / 0.61 / NM_007294 / exon10 / c.2612C>T / p.P871L
BRCA2:N372H / chr13 / 32906729 / A / C / het / rs144848 / 0.4258 / NM_000059 / exon10 / c.1114A>C / p.N372H
BRCA2:V2466A / chr13 / 32929387 / T / C / hom / rs169547 / 0.8038 / NM_000059 / exon14 / c.7397T>C / p.V2466A
BRIP1:S919P / chr17 / 59763347 / A / G / het / rs4986764 / 0.7847 / NM_032043 / exon19 / c.2755T>C / p.S919P
EGFR:R521K / chr7 / 55229255 / G / A / hom / rs2227983 / 0.701 / NM_201284 / exon13 / c.1562G>A / p.R521K
EPCAM:M115T / chr2 / 47601106 / T / C / hom / rs1126497 / 0.8971 / NM_002354 / exon3 / c.344T>C / p.M115T
FANCA:T1328A / chr16 / 89805914 / T / C / het / rs9282681 / 0.3254 / NM_000135 / exon40 / c.3982A>G / p.T1328A
FANCA:G809D / chr16 / 89836323 / C / T / hom / rs7195066 / 0.9067 / NM_000135 / exon26 / c.2426G>A / p.G809D
FANCA:P643A / chr16 / 89839766 / G / C / het / rs17232910 / 0.299 / NM_000135 / exon22 / c.1927C>G / p.P643A
FANCA:G501S / chr16 / 89849480 / C / T / hom / rs2239359 / 0.7727 / NM_000135 / exon16 / c.1501G>A / p.G501S
FANCA:A412V / chr16 / 89857935 / G / A / het / rs11646374 / 0.3636 / NM_000135 / exon14 / c.1235C>T / p.A412V
FANCA:T266A / chr16 / 89866043 / T / C / hom / rs7190823 / 0.7967 / NM_000135 / exon9 / c.796A>G / p.T266A
FLCN:G303R / chr17 / 17124815 / C / T / het / rs3744124 / 0.4139 / NM_144606 / exon8 / c.907G>A / p.G303R
HNF1A:I27L / chr12 / 121416650 / A / C / het / rs1169288 / 0.6316 / NM_000545 / exon1 / c.79A>C / p.I27L
HNF1A:S487N / chr12 / 121435427 / G / A / het / rs2464196 / 0.7105 / NM_000545 / exon7 / c.1460G>A / p.S487N
HNF1A:S574G / chr12 / 121437382 / A / G / hom / rs1169305 / 0.7727 / NM_000545 / exon9 / c.1720A>G / p.S574G
MEN1:T546A / chr11 / 64572018 / T / C / hom / rs2959656 / 0.8421 / NM_130804 / exon11 / c.1636A>G / p.T546A
MLH3:G1163D / chr14 / 75506696 / C / T / het / rs28757011 / 0.0861 / NM_001040108 / exon5 / c.3488G>A / p.G1163D
MLH3:N826D / chr14 / 75513883 / T / C / hom / rs175081 / 0.8469 / NM_001040108 / exon2 / c.2476A>G / p.N826D
MUTYH:Q324H / chr1 / 45797505 / C / G / hom / rs3219489 / 0.5861 / NM_001048171 / exon12 / c.972G>C / p.Q324H
NBN:E185Q / chr8 / 90990479 / C / G / hom / rs1805794 / 0.5359 / NM_002485 / exon5 / c.553G>C / p.E185Q
PALB2:Q559R / chr16 / 23646191 / T / C / het / rs152451 / 0.2584 / NM_024675 / exon4 / c.1676A>G / p.Q559R
PDGFRA:S478P / chr4 / 55139771 / T / C / het / rs35597368 / 0.3421 / NM_006206 / exon10 / c.1432T>C / p.S478P
PMS2:K541E / chr7 / 6026775 / T / C / hom / rs2228006 / 0.7823 / NM_000535 / exon11 / c.1621A>G / p.K541E
PMS2:T485K / chr7 / 6026942 / G / T / het / rs1805323 / 0.5574 / NM_000535 / exon11 / c.1454C>A / p.T485K
PMS2:R20Q / chr7 / 6045627 / C / T / het / rs10254120 / 0.2081 / NM_000535 / exon2 / c.59G>A / p.R20Q
PTCH1:T1194S / chr9 / 98211572 / T / A / het / rs2236405 / 0.2751 / NM_001083603 / exon22 / c.3580A>T / p.T1194S
RECQL4:R1005Q / chr8 / 145737816 / C / T / het / rs4251691 / 0.5096 / NM_004260 / exon18 / c.3014G>A / p.R1005Q
RECQL4:E267D / chr8 / 145741702 / C / G / het / rs4244612 / 0.5167 / NM_004260 / exon5 / c.801G>C / p.E267D
RECQL4:S92P / chr8 / 145742514 / A / G / hom / rs2721190 / 0.634 / NM_004260 / exon4 / c.274T>C / p.S92P
RET:G691S / chr10 / 43610119 / G / A / het / rs1799939 / 0.3134 / NM_020630 / exon11 / c.2071G>A / p.G691S
RNASEL:D541E / chr1 / 182551337 / A / C / het / rs627928 / 0.7033 / NM_021133 / exon4 / c.1623T>G / p.D541E
TP53:P33R / chr17 / 7579472 / G / C / het / rs1042522 / 0.7847 / NM_001276760 / exon4 / c.98C>G / p.P33R
TSC2:M286V / chr16 / 2108755 / A / G / het / rs1800748 / 0.0191 / NM_000548 / exon10 / c.856A>G / p.M286V
TSHR:P52T / chr14 / 81422178 / C / A / het / rs2234919 / 0.0191 / NM_000369 / exon1 / c.154C>A / p.P52T
TSHR:E727D / chr14 / 81610583 / G / C / hom / rs1991517 / 0.9234 / NM_000369 / exon10 / c.2181G>C / p.E727D
VHL:W88X / chr3 / 10183795 / G / A / het / - / - / NM_198156 / exon1 / c.264G>A / p.W88X
XPC:L16V / chr3 / 14220023 / G / C / het / rs1870134 / 0.3971 / NM_001145769 / exon1 / c.46C>G / p.L16V