Mutation spectrum of hyperphenylalaninemia candidate genes and

the genotype-phenotype correlation in the Chinese population

Supplementary Material

Supplementary Figure

Supplementary Fig. 1. Relative Quantitation results of PTS gene for patient P56, P57 and their parents

RQ: Relative Quantitation; Figure（A）PTS-E2: Exon 2 of PTS gene; Figure(B) PTS-E3: Exon 3 of PTS gene; P56：Patient 56; P56-F：Father of patient 56; P56-M：Mother of patient 56; C1：Control 1; C2：Control 2; C3：Control 3; Figure（C）PTS-E5: Exon 5 of PTS gene; Figure (D) PTS-E6: Exon 6 of PTS gene; P57：Patient 57; P57-F：Father of patient 57; P57-M：Mother of patient 57.

1

Supplementary Tables

Supplementary Table 1. Lists of primer sequences of designed PTS and internal reference ALB gene

Exon of gene Primer sequences（(5’-3’） Primer

length

PTS-E1 F GGAAGATGAGCACGGAAGG 149bp

　 R CCGGTGACGTTCCGGGG 　PTS-E2 F

GGAACAGAGAAGGGGGTTTG

A181bp

　 R TGAAGGGCTGAAATGTCAT 　PTS-E3 F

CTTGTGCTTGGATGTTGATCT

GTT140bp

　 R AAGAGAGCCCAGCAATCTGC 　PTS-E4 F

TATGGAGAGCCTATCACAGTA

AT163bp

　 R GGGGCCACCAAATATTACAGT 　PTS-E5 F

TTTGGAATTTGAGTCGTAAAT

GG168bp

　 RGTTTTACAATCCACATAAGGC

AAG　

PTS-E6 FAACATGAAATTTTATTGTTTGC

A215bp

　 RCTGGGCTTTGTGCAATGCTAA

C　

ALB-Intron 2 FTCCTTGTCATCAGGGTTCAGA

TTC114bp

　 R AGCCATGTGACCAGTGACTAC 　F：Forward primer; R：Reverse primer; E:Exon. cDNA numbering was according to the

reference sequence NM_000317.2.

2

3

Supplementary Table 2. Summary of genetic testing and biochemical investigations in seven PKU patients newly diagnosed by NGS

Samples

Sanger sequencing results Next generation sequencing results Biochemical investigations results at first Visit

geneVariant

allele 1Protein 1 or name Gene

Variant

allele 1

Protein 1or

nameVariant allele 2

Protein 2 or

name

Ge

nd

er

age

(years)

Ph

e（μmol/L）

Phe/Tyr

N

（m

mol/

mol

Cr）

B

（m

mol/

mol

Cr）

B%

[B/

(B+N)

×100]

DHPR

activit

y，nmol/m

in/5 mm

disc

P1 PAH c.194T>C p.I65T PAH c.194T>C p.I65T c.1066-13T>GIVS10-

13T>GF 9 No dataa - 0.2 0.2 44.6 -

P2 PAH c.721C>T p.R241C PAH c.721C>T p.R241C c.913-7A>G IVS8-7A>G M 0.1 339 - 3.1 0.9 22.2 3.7

P3 PAH c.611A>G EX6-96A>G PAH c.611A>G EX6-96A>G c.442-1G>A IVS4-1G>A　 M 3 753 16.7 10.7 2.7 20.1 -

P4 PAH c.1197A>T p.V399V PAH c.1197A>T p.V399V c.913-2A>G IVS8-2A>G M 2.6 825 21.1 1.9 2.6 52.0 1.8

P5 PAH c.728G>A p.R243Q PAH c.728G>A p.R243Q c.1199+19T>CIVS11+19T>

CM 0.1 172 1.8 1.3 1 44.6 2.5

P6 PAH c.1222C>T p.R408W PAH c.1222C>T p.R408W c.1066-14C>GIVS10-

14C>GF 7 512 9.2 0.8 2.2 72.2 2.2

P7 PAH c.722del p.R241Pfs*100 PAH c.722del p.R241Pfs*100 c.721C>T p.R241C M 0.1 279 10.5 2.3 0.5 17.3 3.8

P1-P7: Patient 1-Patient 7; Phe: Phenylalanine; Tyr: Tyrosine; N: Neopterin; B: Biopterin; B%: Biopterin percentage; DHPR: Dihydropteridine reductase; Normal reference: Neopterin: (0.29-

7.49) mmol/mol Cr; Biopterin: (0.35-3.68) mmol/mol Cr; B% : 26.2-75.9; DHPR activity: (1.02-3.35) nmol/min per 5 mmdisc; F: Female; M: Male; aPatient P1 got confirmed diagnosis and

started therapy in the local hospital, phenylalanine concentration before treatment could not get. cDNA numbering was according to the reference sequence NM_000277.1.

4

Supplementary Table 3. Summary of genetic testing and biochemical investigations in 48 patients newly diagnosed by MLPA of PAH gene

Sam

ples

Sanger sequecing results 　MLPA detecting results (copy numbers)b Biochemical investigations results at first Visit

Variant allele 1Protein 1 or

name

Posit

ion

E1-up E2 E3 E4　 E5　 E6　 E7　Gend

er

age

(years)

Ph

e（μm

ol/L）Phe/

Tyr

N

（m

mol/

mol

Cr）

B

（m

mol/

mol

Cr）

B%[B/

(B+N)×1

00]

DHPR

activit

y，nmol/m

in/5 mm

disc

P1 P2 P1 P2 P1 P2 P1 P2 P1 P2 P1 P2 P1 P2

P8 c.611A>G EX6-96A>G E6 1 1 1 2 2 2 2 2 2 2 2 2 2 2 F 0.2 600.0 - - - - -

P9 c.728G>A p.R243Q E7 1 1 1 2 2 2 2 2 2 2 2 2 2 2 M 0.1 1140.0 7.7 1.7 0.8 31.5 3.6

P10 c.1315+6T>A IVS12+6T>A　 I12 1 1 1 2 2 2 2 2 2 2 2 2 2 2 F 0.1 216.0 1.9 0.6 0.1 16.1 3.3

P11 c.442-1G>A IVS4-1G>A　 I4 2 2 2 2 2 2 2 2 2 2 1 1 2 2 F 0.04 1435.9 37.8 0.9 0.1 8.6 3.3

P12 c.1238G>C p.R413P E12 2 2 2 2 2 2 1 1 1 1 2 2 2 2 F 0.08 1097.2 25.0 4.6 1.1 19.3 3.2

P13 c.1199G>C p.R400T E11 2 2 2 2 2 2 2 2 2 2 1 1 2 2 M 13.3 932.0 43 0.1 1.1 90.8 2.7

P14 c.331C>T p.R111* E3 2 2 2 2 2 2 2 2 2 2 1 1 2 2 F 0.4 1800.0 - 0.2 0.2 55.4 -

P15 c.1197A>T p.V399V E11 2 2 2 2 2 2 1 1 1 1 1 1 1 1 F 6.5 277.4 4.3 - - - -

P16 c.1172G>C p.S391T E11 2 2 2 2 2 2 2 2 1 1 2 2 2 2 M 0.2 456.0 4.3 1.5 1.1 42.8 4.6

P17 c.208_210del p.S70del E3 1 1 1 2 2 2 2 2 2 2 2 2 2 2 M 0.4 287.9 3.3 1.0 1.5 58.3 3.8

P18a c.526C>T p.R176* E6 1 1 1 2 2 2 2 2 2 2 2 2 2 2 F 0.75 - - 3.4 1.3 27.2 1.7

P19 c.1197A>T p.V399V E11 1 1 1 2 2 2 2 2 2 2 2 2 2 2 M 0.1 968.6 53.9 10.2 1.3 11.6 2.9

P20 c.907del p.S303Pfs*38 E8 1 1 1 2 2 2 2 2 2 2 2 2 2 2 M 2.1 557.0 - 3.9 4.9 55.7 1.9

P21 c.611A>G EX6-96A>G E6 1 1 1 2 2 2 2 2 2 2 2 2 2 2 F 0.08 1196.0 24.9 6.8 2.0 22.5 4.4

P22 c.611A>G EX6-96A>G E6 1 1 1 2 2 2 2 2 2 2 2 2 2 2 M 0.1 1780.0 25.2 5.8 1.5 20.7 1.9

P23 c.722del p.R241Pfs E7 2 2 2 2 2 2 2 2 2 2 1 1 2 2 M 4.0 1600.0 - - - - -

5

P24 c.611A>G EX6-96A>G E6 1 1 1 2 2 2 2 2 2 2 2 2 2 2 F 0.8 738.0 - 1.4 0.6 31.9 2.0

P25 c.1223G>A p.R408Q E12 1 1 1 2 2 2 2 2 2 2 2 2 2 2 F 0.1 588.0 8.2 4.5 3.6 44.9 2.8

P26 c.721C>T p.R241C E7 2 1 1 1 2 2 2 2 2 2 2 2 2 2 M 0.5 780.0 - 0.9 0.1 11.2 -

P27 c.611A>G EX6-96A>G E6 1 1 1 2 2 2 2 2 2 2 2 2 2 2 M 4.5 1200.0 - 6.5 6.0 47.9 3.7

P28 c.1139C>T p.T380M E11 2 2 2 2 2 2 2 2 2 2 1 1 2 2 M 0.9 474.0 5.8 1.0 1.4 58.8 2.6

P29 c.977G>A p.W326* E10 1 1 1 2 2 2 2 2 2 2 2 2 2 2 M 0.1 845.2 16.0 6.2 2.1 25.3 2.8

P30 c.922C>T p.L308F E9 1 1 1 2 2 2 2 2 2 2 2 2 2 2 F 0.1 467.0 12.4 2.3 1.8 44.7 2.7

P31 c.1057G>T p.E353* E10 1 1 1 2 2 2 2 2 2 2 2 2 2 2 M 0.1 1182.0 11.8 4.8 0.3 6.3 2.5

P32 c.441+2T>A 　IVS4+2T>A I4 2 2 2 2 2 2 2 2 2 2 1 1 2 2 F 8.5 413.0 - 0.7 2.3 75.9 2.3

P33 c.320A>G p.H107R E3 1 1 1 2 2 2 2 2 2 2 2 2 2 2 M 0.1 137.7 2.7 2.5 0.2 7.5 2.5

P34 c.617A>G p.Y206C E6 1 1 1 2 2 2 2 2 2 2 2 2 2 2 F 0.1 914.0 20.1 5.0 1.6 24.7 2.9

P35 c.442-1G>A 　IVS4-1G>A I4 1 1 1 2 2 2 2 2 2 2 2 2 2 2 F 0.1 1756 9.9 0.8 0.1 14.8 2.4

P36 c.1208C>T p.A403V E12 1 1 1 2 2 2 2 2 2 2 2 2 2 2 F 5.0 221.0 4.5 0.7 0.7 49.3 3.3

P37 c.442-1G>A IVS4-1G>A　 I4 1 1 1 2 2 2 2 2 2 2 2 2 2 2 F 17.0 784.4 27.3 1.0 0.2 18.1 3.7

P38 c.940C>A p.P314T E9 1 1 1 1 1 1 2 2 2 2 2 2 2 2 F 0.1 184.4 2.5 2.3 2.0 46.2 5.1

P39 c.1223G>A p.R408Q E12 1 1 1 2 2 2 2 2 2 2 2 2 2 2 M 0.1 468.0 9.9 5.0 2.0 28.9 4.2

P40 c.331C>T p.R111* E3 2 2 2 2 2 2 2 2 1 1 2 2 2 2 F 2.3 936.0 21.5 5.4 4.1 43.1 2.3

P41 c.871G>T p.V291L E8 2 2 2 2 2 2 2 2 2 2 1 1 2 2 F 0.2 732.0 18.8 2.3 0.6 21.1 2.8

P42 c.728G>A p.R243Q E7 1 1 1 2 2 2 2 2 2 2 2 2 2 2 F 0.2 610.0 - 1.8 0.7 26.5 2.4

P43 c.728G>A p.R243Q E7 2 2 2 2 2 2 1 1 1 1 2 2 2 2 F 0.2 660.0 - - - - -

P44 c.1068C>A p.Y356* E11 1 1 1 2 2 2 2 2 2 2 2 2 2 2 M 0.1 1109.4 10.1 5.1 1.4 19.5 4.2

P45 c.1223G>A p.R408Q E12 1 1 1 2 2 2 2 2 2 2 2 2 2 2 M 0.1 942.3 - 1.6 0.6 27.7 4.1

P46 c.688G>A p.V230I E6 1 1 1 2 2 2 2 2 2 2 2 2 2 2 M 0.1 126.8 2.5 0.7 0.2 20.1 3.0

P47 c.721C>T p.R241C E7 2 2 2 2 2 2 1 1 1 1 1 1 1 1 M 4.5 851.0 11.5 7.2 1.2 14.1 3.1

P48 c.611A>G EX6-96A>G E6 1 1 1 1 1 2 2 2 2 2 2 2 2 2 F 0.1 1483.3 - 4.9 0.5 9.6 2.9

P49 c.1197A>T p.V399V E11 2 2 2 2 2 2 1 1 1 1 2 2 2 2 M 1.5 1129.3 - - - - -

P50# c.607T>G p.C203G E6 1 1 1 2 2 2 2 2 2 2 2 2 2 2 M 0.2 - - 6.1 3.2 34.5 3.5

P51 c.728G>A p.R243Q E7 2 2 2 2 2 2 2 2 2 2 1 1 2 2 M 0.1 2280.0 - 5.1 1.6 24.0 1.8

P52 c.728G>A p.R243Q E7 2 2 2 2 2 2 1 1 1 1 2 2 2 2 F 0.2 2880.0 - - - - -

6

P53# c.799C>G p.Q267E E7 1 1 1 2 2 2 2 2 2 2 2 2 2 2 F 10.75 - - - - - -

P54# c.728G>A p.R243Q E7 1 1 1 2 2 2 2 2 2 2 2 2 2 2 F 2 - - - - - -

P55 c.618C>A p.Y206* E6 2 2 2 2 2 2 2 2 2 2 1 1 2 2 F 1.6 1375.0 - 0.6 0.4 41.6 -

P8-P55: Patient 8-Patient 55; In gene position of sequencing results, “E” represented exons, and “I” represented introns; Phe: Phenylalanine; Tyr: Tyrosine; N: Neopterin; B: Biopterin; B%:

Biopterin percentage; DHPR: Dihydropteridine reductase; Normal reference: Neopterin: (0.29-7.49) mmol/mol Cr; Biopterin: (0.35-3.68) mmol/mol Cr; B%: 26.2-75.9; DHPR activity: (1.02-

3.35) nmol/min per 5 mmdisc; F: Female; M: Male; a:Patient got confirmed diagnosis and started therapy in the local hospital, phenylalanine concentration before treatment could not get; b:

Copy number of MLPA “1” represent deletion of one copy, “2” represent carrying normal two copies; As detecting results showed all patients carrying normal two copies at Exon 8 to Exon 13

of PAH gene, the results of Exon 8 to Exon 13 were omitted here. cDNA numbering was according to the reference sequence NM_000277.1.

7

8

Supplementary Table 4. Summary of genetic testing and biochemical investigations in five HPA patients carrying with a heterozygosis mutation of PTS gene

Samples

Next generation sequencing results Biochemical investigations results at first Visit

geneVariant allele

1Protein 1

Posi

tion

Gen

der

age

(years

)

Phe(μm

ol/L） Phe/Tyr

N

（mmo

l/

molC

r）

B

（mmo

l/

molC

r）

B%[B/

(B+N)×

100]

DHPR

activit

y，nmol/mi

n/5 mm disc

P56PTS c.259C>T p.P87S E5

M 0.04 1440 -　 4.90 0.04 0.84 2.86PAH c.740G>T p.G247V E7

P57 PTS c.193C>T p.P65S E4 M 0.2 233 2.04 4.07 0.10 2.41 4.00

P58 PTS c.259C>T p.P87S E5 F 0.3 240 2.17 0.17 0.01 2.91 2.13

P59 PTS c.379C>T p.L127F E6 F 0.1 158 - 1.35 0.28 17.05 2.38

P60PTS c.379C>T p.L127F E6

M 0.2 150 - 0.29 0.05 15.18 2.59PAH c.1068C>A p.Y356* E11

P56-P60: Patient 56- Patient 60; In gene position of sequencing results, “E” represented exon; F: Female; M:

Male; Phe: Phenylalanine; Tyr: Tyrosine; N: Neopterin; B: Biopterin; B%: Biopterin percentage; DHPR:

Dihydropteridine reductase. Normal reference: Neopterin: (0.29-7.49) mmol/mol Cr; Biopterin: (0.35-3.68)

mmol/mol Cr; B%: 26.2-75.9; DHPR activity: (1.02-3.35) nmol/min per 5 mmdisc. cDNA numbering of PAH and

PTS according to reference sequence NM_000277.1 and NM_000317.2.

9

Supplementary Table 5. Summary of mutation spectrum of PAH gene detected in Chinese Han population

Nucleotide aberration

Protein variant Location Variant type Protein domainAlleles

(n=1616)

Relativefrequency

(%)c.728G>A p.R243Q E7 Missense Catalytic 275 17.0%c.611A>G EX6-96A>G E6 Splice Catalytic 120 7.4%c.721C>T p.R241C E7 Missense Catalytic 117 7.2%

c.1197A>T p.V399V E11 Silence/ Splice Catalytic 87 5.4%c.331C>T p.R111* E3 Nonsense Regulatory 71 4.4%

c.1068C>A p.Y356* E11 Nonsense Catalytic 63 3.9%c.1238G>C p.R413P E12 Missense Tetramerization 62 3.8%c.442-1G>A 　IVS4-1G>A I4 Splice - 59 3.7%Large gene deletiona - - - - 48 3.0%

c.1223G>A p.R408Q E12 Missense Catalytic 39 2.4%c.158G>A p.R53H E2 Missense Regulatory 35 2.2%

c.208_210del S70del E3 Deletion Regulatory 25 1.5%c.498C>G p.Y166* E5 Nonsense Catalytic 20 1.2%c.722del R241Pfs E7 Deletion Catalytic 19 1.2%

c.1174T>A p.F392I E11 Missense Catalytic 18 1.1%c.1301C>A p.A434D E12 Missense Tetramerization 16 1.0%

c.1315+6T>A IVS12+6T>A　 I12 Splice - 15 0.9%c.842+2T>A 　IVS7+2T>A I7 Splice - 14 0.9%c.782G>A p.R261Q E7 Missense Catalytic 13 0.8%c.740G>T p.G247V E7 Missense Catalytic 13 0.8%c.838G>A P.E280K E7 Missense Catalytic 12 0.7%c.770G>T p.G257V E7 Missense Catalytic 11 0.7%c.755G>A p.R252Q E7 Missense Catalytic 11 0.7%c.907del p.S303Pfs*38 E8 Deletion Catalytic 10 0.6%

c.1199G>C p.R400T E11 Missense Catalytic 10 0.6%c.194T>C p.I65T E3 Missense Regulatory 9 0.6%

c.1162G>A p.V388M E11 Missense Catalytic 9 0.6%c.320A>G p.H107R E3 Missense Regulatory 8 0.5%c.1222C>T p.R408W E12 Missense Catalytic 8 0.5%c.482T>C p.F161S E5 Missense Catalytic 8 0.5%c.977G>A p.W326* E10 Nonsense Catalytic 8 0.5%c.1045T>G p.S349A E10 Missense Catalytic 8 0.5%c.526C>T p.R176* E6 Nonsense Catalytic 8 0.5%c.940C>A p.P314T E9 Missense Catalytic 7 0.4%

10

c.856G>A p.E286K E8 Missense Catalytic 7 0.4%c.688G>A p.V230I E6 Missense Catalytic 7 0.4%c.472C>T p.R158W E5 Missense Catalytic 6 0.4%c.739G>C p.G247R E7 Missense Catalytic 6 0.4%c.466G>C p.A156P E5 Missense Catalytic 6 0.4%

c.1199G>A p.R400K E11 Missense Catalytic 6 0.4%c.1252A>C p.T418P E12 Missense Tetramerization 6 0.4%

c.842+1G>A 　IVS7+1G>A I7 Splice - 5 0.3%c.722G>A p.R241H E7 Missense Catalytic 5 0.3%c.1139C>T p.T380M E11 Missense Catalytic 5 0.3%c.975C>G p.Y325* E10 Nonsense Catalytic 5 0.3%c.47_48del p.S16* E1 Deletion Regulatory 5 0.3%c.473G>A p.R158Q E5 Missense Catalytic 5 0.3%c.992T>C p.F331S E10 Missense Catalytic 5 0.3%

c.913-7A>G 　IVS8-7A>G I8 Splice - 5 0.3%c.311C>T p.A104V E3 Missense Regulatory 4 0.2%c.617A>G p.Y206C E6 Missense Catalytic 4 0.2%

c.116_118del p.F39del E2 Deletion Regulatory 4 0.2%c.707-1G>A 　IVS6-1G>A I6 Splice - 4 0.2%

c.727C>T p.R243* E7 Nonsense Catalytic 4 0.2%c.1172G>C p.S391T E11 Missense Catalytic 4 0.2%c.764T>C p.L255S E7 Missense Catalytic 4 0.2%c.833C>T p.T278I E7 Missense Catalytic 4 0.2%

c.1315+4A>G IVS12+4A>G　 I12 Splice - 4 0.2%c.1256A>G p.Q419R E12 Missense Tetramerization 4 0.2%c.1033G>A p.A345T E10 Missense Catalytic 3 0.2%c.842C>T p.P281L E7 Missense Catalytic 3 0.2%

c.1200-1G>C IVS11-1G>C　 I11 Splice - 3 0.2%c.1208C>T p.A403V E12 Missense Catalytic 3 0.2%c.971T>A p.I324N E10 Missense Catalytic 3 0.2%c.907T>C p.S303P E8 Missense Catalytic 3 0.2%c.694C>T p.Q232* E6 Nonsense Catalytic 3 0.2%c.505C>T p.R169C E5 Missense Catalytic 3 0.2%c.875C>T p.P292L E8 Missense Catalytic 3 0.2%c.935G>T p.G312V E9 Missense Catalytic 3 0.2%

c.1199+1G>C IVS11+1G>C　 I11 Splice - 3 0.2%c.1084C>A p.P362T E11 Missense Catalytic 3 0.2%c.301G>A p.D101N E3 Missense Regulatory 3 0.2%c.464G>A p.R155H E5 Missense Catalytic 2 0.1%

c.185_188dup p.H64Dfs*4 E3 Duplication Regulatory 2 0.1%c.1154T>C p.L385P E11 Missense Catalytic 2 0.1%c.965C>A p.A322D E9 Missense Catalytic 2 0.1%c.922C>T p.L308F E9 Missense Catalytic 2 0.1%c.505C>A p.R169S E5 Missense Catalytic 2 0.1%

11

c.618C>A p.Y206* E6 Nonsense Catalytic 2 0.1%c.1183G>T p.A395S E11 Missense Catalytic 2 0.1%

c.441+3G>C IVS4+3G>C　 I4 Splice - 2 0.1%c.827T>G p.M276R E7 Missense Catalytic 2 0.1%

c.1123C>G p.Q375E E11 Missense Catalytic 2 0.1%c.478C>T p.Q160* E5 Nonsense Catalytic 2 0.1%c.812A>T p.H271L E7 Missense Catalytic 2 0.1%

c.913-2A>G IVS8-2A>G I8 Splice - 2 0.1%c.724C>T p.L242F E7 Missense Catalytic 2 0.1%c.827T>C p.M276T E7 Missense Catalytic 2 0.1%c.929C>T p.S310F E9 Missense Catalytic 2 0.1%

c.1243G>A p.D415N E12 Missense Tetramerization 2 0.1%c.131_133del p.E44del E2 Deletion Regulatory 2 0.1%

c.1024del p.A342Hfs*58 E10 Deletion Catalytic 2 0.1%c.1125A>C p.Q375H E11 Missense Catalytic 2 0.1%c.1024G>A p.A342T E10 Missense Catalytic 2 0.1%c.800A>G p.Q267R E7 Missense Catalytic 2 0.1%c.470G>T p.R157I E5 Missense Catalytic 1 0.1%c.560G>A p.W187* E6 Nonsense Catalytic 1 0.1%c.257G>C p.R86P E3 Missense Regulatory 1 0.1%

c.441+2T>A 　IVS4+2T>A I4 Splice - 1 0.1%c.228G>C p.E76D E3 Missense Regulatory 1 0.1%c.659A>C p.H220P E6 Missense Catalytic 1 0.1%c.901C>T p.Q301* E8 Nonsense Catalytic 1 0.1%c.609C>A p.C203* E6 Nonsense Catalytic 1 0.1%c.563G>A p.G188D E6 Missense Catalytic 1 0.1%c.871G>T p.V291L E8 Missense Catalytic 1 0.1%c.1057G>T p.E353* E10 Nonsense Catalytic 1 0.1%c.799C>G p.Q267E E7 Missense Catalytic 1 0.1%c.199T>G p.S67A E3 Missense Regulatory 1 0.1%c.204A>T p.R68S E3 Missense Regulatory 1 0.1%c.649T>C p.C217R E6 Missense Catalytic 1 0.1%c.442G>C p.G148R E5 Missense Catalytic 1 0.1%c.673C>G p.P225A E6 Missense Catalytic 1 0.1%c.425T>C p.L142P E4 Missense Regulatory 1 0.1%

c.1002C>A p.C334* E10 Nonsense Catalytic 1 0.1%c.73_74dup p.E26Lfs*13 E2 Duplication Regulatory 1 0.1%

c.584dup p.S196Vfs*4 E6 Duplication Catalytic 1 0.1%c.523C>T p.P175S E6 Missense Catalytic 1 0.1%c.440C>T p.P147L E4 Missense Catalytic 1 0.1%

c.59delinsCC p.Q20Pfs*7 E1Deletion/Insertion

Regulatory 1 0.1%

c.383T>C p.L128P E4 Missense Regulatory 1 0.1%c.212G>A p.R71H E3 Missense Regulatory 1 0.1%

12

c.1115C>G p.T372R E11 Missense Catalytic 1 0.1%c.308G>A p.G130D E3 Missense Regulatory 1 0.1%

c.168+5G>C 　IVS2+5G>C I2 Splice - 1 0.1%c.505C>G p.R169G E5 Missense Catalytic 1 0.1%c.853C>T p.H285Y E8 Missense Catalytic 1 0.1%c.754C>T p.R252W E7 Missense Catalytic 1 0.1%c.506G>A p.R169H E5 Missense Catalytic 1 0.1%

c.706+5G>A 　IVS6+5G>A I6 Splice - 1 0.1%c.763T>G p.L255V E7 Missense Catalytic 1 0.1%c.641A>G p.E214G E6 Missense Catalytic 1 0.1%c.208T>C p.S70P E3 Missense Regulatory 1 0.1%

c.23_24delinsT p.N8Ifs*30 E1Deletion/Insertion

Regulatory 1 0.1%

c.223G>C p.D75H E3 Missense Regulatory 1 0.1%c.890G>A p.R297H E8 Missense Catalytic 1 0.1%c.527G>A p.R176Q E6 Missense Catalytic 1 0.1%

c.1066-13G>T IVS10-13G>T I10 Splice - 1 0.1%c.1172G>T p.S391I E11 Missense Catalytic 1 0.1%

c.1316-2A>C IVS12-2A>C　 I12 Splice - 1 0.1%c.1089del p.K363Nfs*37 E11 Deletion Catalytic 1 0.1%

c.1157A>G p.Y386C E11 Missense Catalytic 1 0.1%c.156G>T p.L52F E2 Missense Regulatory 1 0.1%c.1144T>A p.F382I E11 Missense Catalytic 1 0.1%c.1054G>C p.G352R E10 Missense Catalytic 1 0.1%c.932T>G p.L311R E9 Missense Catalytic 1 0.1%c.127G>T p.E43* E2 Nonsense Regulatory 1 0.1%c.868C>T p.H290Y E8 Missense Catalytic 1 0.1%c.182A>G p.N61S E3 Missense Regulatory 1 0.1%

c.1315+5G>C IVS12+5G>C I12 Splice - 1 0.1%c.1216_1220de

lp.I406Sfs*15 E12 Deletion Catalytic 1 0.1%

c.1066-11G>A IVS10-11G>A　 I10 Splice - 1 0.1%c.775G>A p.A259T E7 Missense Catalytic 1 0.1%c.607T>G p.C203G E6 Missense Catalytic 1 0.1%c.793T>C p.C265R E7 Missense Catalytic 1 0.1%

c.541_544del p.E181Kfs*13 E6 Deletion Catalytic 1 0.1%c.470G>A p.R157K E5 Missense Catalytic 1 0.1%c.871G>A p.V291M E8 Missense Catalytic 1 0.1%c.898G>T p.A300S E8 Missense Catalytic 1 0.1%c.716G>A p.G239D E7 Missense Catalytic 1 0.1%c.1043T>C p.L348P E10 Missense Catalytic 1 0.1%

c.1066-14C>G IVS10-14C>G　 I10 Splice - 1 0.1%c.970-14C>G IVS9-14C>G I9 Splice - 1 0.1%c.842+5G>A 　IVS7+5G>A I7 Splice - 1 0.1%

13

c.1250G>A p.Y417C E12 Missense Tetramerization 1 0.1%c.1242C>A Y414* E12 Nonsense Tetramerization 1 0.1%c.781C>T R261* E7 Nonsense Catalytic 1 0.1%

c.169-2A>G IVS2-2A>G I2 Splice - 1 0.1%c.1159T>G p.Y387D E11 Missense Catalytic 1 0.1%c.1289T>C p.L430P E12 Missense Tetramerization 1 0.1%c.1241A>G p.Y414C E12 Missense Tetramerization 1 0.1%c.1147C>T p.Q383* E11 Nonsense Catalytic 1 0.1%c.1090C>T p.L364F E11 Missense Catalytic 1 0.1%

c.509+1G>A IVS5+1G>A　 I5 Splice - 1 0.1%c.671T>C p.I224T E6 Missense Catalytic 1 0.1%

c.1065+1G>T IVS10+1G>T I10 Splice - 1 0.1%c.1262T>C p.I421T E12 Missense Tetramerization 1 0.1%c.1235T>A p.V412D E12 Missense Tetramerization 1 0.1%c.910C>A p.Q304L E8 Missense Catalytic 1 0.1%c.1049C>A p.S350Y E10 Missense Catalytic 1 0.1%c.361T>G p.F121V E4 Missense Regulatory 1 0.1%c.136G>C p.G46R E2 Missense Regulatory 1 0.1%c.532G>A p.E178L E6 Missense Catalytic 1 0.1%

c.158G>A+c.1200-8G>A

p.R53H+IVS11-8G>A

E2-I11Missense/

SpliceRegulatory/- 1 0.1%

c.940C>G p.P314A E9 Missense Catalytic 1 0.1%c.1099del p.L367Wfs*33 E11 Deletion Catalytic 1 0.1%c.3G>C p.M1I E1 Missense Regulatory 1 0.1%

c.1200-12del 　IVS11-12del I11 Splice - 1 0.1%c.490A>G p.I164V E5 Missense Catalytic 1 0.1%c.852C>A p.C284* E8 Nonsense Catalytic 1 0.1%

c.1199+19T>C IVS11+19T>C I11 Splice - 1 0.1%c.845A>G p.D282G E8 Missense Catalytic 1 0.1%c.1012G>T p.D338Y E10 Missense Catalytic 1 0.1%c.650G>A p.C217Y E6 Missense Catalytic 1 0.1%c.158G>A-c.212G>A

p.R53H-p.R71H

E2-E3 Missense Regulatory 1 0.1%

c.1076C>T p.S359L E11 Missense Catalytic 1 0.1%c.134T>C-c.721C>T

p.V45A-p.R241C

E2-E7 MissenseRegulatory/

Catalytic1 0.1%

c.158G>A-c.1250G>A

p.R53H-p.Y417C

E2-E12 MissenseRegulatory/

Tetramerization1 0.1%

No mutation 　 　 　 49 3.0%In gene position of sequencing results, “E” represented exons, and “I” represented introns; “a”:Large Deletion: Large deletion were detected by MLPA; n: number of alleles. cDNA numbering was according to the reference sequence NM_000277.1.

14

Supplementary Table 6. Summary of mutation spectrum of PTS gene detected in Chinese Han population

Nucleotide aberration

Protein variant Location Variant typeAlleles

(n=408)Relative

frequency (%)

c.259C>T p.P87S E5 Missense 156 38.2%c.84-291A>G IVS1-291A>G I1 Splice 45 11.0%

c.155A>G p.N52S E2 Missense 35 8.6%c.286G>A p.D96N E5 Missense 35 8.6%c.166G>A p.V56M E3 Missense 21 5.1%

c.186+1G>A IVS3+1G>A I3 Splice 15 3.7%c.317C>T p.T106M E6 Missense 15 3.7%c.379C>T p.L127F E6 Missense 13 3.2%c.272A>G p.K91R E5 Missense 11 2.7%c.73C>G p.R25G E1 Missense 7 1.7%c.73C>T p.R25* E1 Nonsense 6 1.5%

c.331G>A p.A111T E6 Missense 5 1.2%c.200C>T p.T67M E4 Missense 4 1.0%c.293C>A p.P98Q E5 Missense 3 0.7%c.34G>C p.A12P E1 Missense 2 0.5%

c.116_119del p.F40Gfs*17 E2 Deletion 2 0.5%c.421G>T p.V141F E6 Missense 2 0.5%c.181G>A p.G61R E3 Missense 1 0.2%c.62G>A p.S21N E1 Missense 1 0.2%

c.84-6insTTTTIVS1-

6insTTTTI1 Splice 1 0.2%

c.164-37C>T IVS2-37C>T I2 Splice 1 0.2%c.374G>A p.G125E E6 Missense 1 0.2%c.143G>A p.G48D E2 Missense 1 0.2%

c.244-2A>T IVS4-2A>T I4 Splice 1 0.2%c.3G>A p.M1I E1 Missense 1 0.2%

c.193C>T p.P65S E4 Missense 1 0.2%c.1A>G p.M1V E1 Missense 2 0.5%c.35C>A p.A12E E1 Missense 1 0.2%c.212T>C p.M71T E4 Missense 1 0.2%c.65C>G p.A22G E1 Missense 1 0.2%c.280G>C p.D94H E5 Missense 1 0.2%c.260C>T p.P87L E5 Missense 1 0.2%c.322G>T p.E108* E6 Nonsense 1 0.2%

15

c.313A>T p.S105C E5 Missense 1 0.2%c.244-2A>G IVS4-2A>G I4 Splice 1 0.2%c.84-252G>T IVS1-252G>T I1 Splice 1 0.2%

c.289_290insCTT p.V97delinsAL E5 Insertion 1 0.2%c.342C>G p.I114M E6 Missense 1 0.2%c.393A>C p.K131N E6 Missense 1 0.2%c.402A>C p.E134D E6 Missense 1 0.2%c.432dup p.E145Rfs*18 E6 Duplication 1 0.2%c.200C>T p.T67M E4 Missense 1 0.2%

Deletion of exon 2 and 3

E2 and E3 Deletion 1 0.2%

Deletion of exon 5 and 6

E5 and E6 Deletion 1 0.2%

No mutation 3 0.7%In gene position of sequencing results, “E” represented exons, and “I” represented introns; n : number of alleles. cDNA numbering was according to the reference sequence NM_000317.2.

16

Supplementary Table 7. Prediction of the effects of 25 novel variants of PAH gene on protein functions

Nucleotide

aberration

Protein

variant

Variant

typeLocation Conservation SIFT prediction

PolyPhen

prediction

Mutation

taster

prediction

ACMG/AMP

variants

classificationa

Accession

number in

PAHvdb

c.257G>C p.R86P Missense E3 Moderate Tolerated BenignPolymorp

hism

Uncertain

significancePAH1019

c.199T>G p.S67A Missense E3 ModerateAffect protein

function

Probably

damaging

Disease

causing

Uncertain

significancePAH1020

c.425T>C p.L142P Missense E4 ModerateAffect protein

function

Probably

damaging

Disease

causing

Uncertain

significancePAH1021

c.383T>C p.L128P Missense E4Moderate Affect protein

function

Probably

damaging

Disease

causing

Uncertain

significancePAH1022

c.641A>G p.E214G Missense E6High

ToleratedPossible

damaging

Disease

causing

Uncertain

significancePAH1023

c.1183G>T p.A395S Missense E11High Affect protein

function

Probably

damaging

Disease

causing

Likely

pathogenicPAH1024

c.156G>T p.L52F Missense E2Moderate Affect protein

function

Probably

damaging

Disease

causing

Uncertain

significancePAH1025

c.1144T>A p.F382I Missense E11High Affect protein

functionBenign

Disease

causing

Uncertain

significancePAH1026

c.932T>G p.L311R Missense E9 HighAffect protein

function

Probably

damaging

Disease

causing

Likely

pathogenicPAH1027

c.182A>G p.N61S Missense E3 Moderate Tolerated BenignDisease

causing

Uncertain

significancePAH1028

c.607T>G p.C203G Missense E6High Affect protein

function

Probably

damaging

Disease

causing

Likely

pathogenicPAH1030

c.1125A>C p.Q375H Missense E11High Affect protein

function

Probably

damaging

Disease

causing

Likely

pathogenicPAH1031

c.1090C>T p.L364F Missense E11 Moderate Tolerated BenignPolymorp

hism

Uncertain

significancePAH1032

c.1235T>A p.V412D Missense E12High Affect protein

function

Probably

damaging

Disease

causing

Uncertain

significancePAH1033

c.940C>G p.P314A Missense E9High

ToleratedPossible

damaging

Disease

causing

Uncertain

significancePAH1034

c.706+5G>A IVS6+5G>A Splice I6 - - - - Uncertain PAH1035

17

significance

c.1199+19T>

C

IVS11+19T

>CSplice I11 - - - -

Uncertain

significancePAH1036

c.1200-12del IVS11-12del Splice I11 - - - -Uncertain

significancePAH1037

c.1057G>T p.E353* Nonsense E10 High - - - Pathogenic PAH1038

c.609C>A p.C203* Nonsense E6 High - - - Pathogenic PAH1039

c.852C>A p.C284* Nonsense E8 High - - - Pathogenic PAH1040

c.73_74dup p.E26Lfs*13Duplicatio

nE2 Moderate - - - Pathogenic PAH1041

c.59delinsCC p.Q20Pfs*7Deletion/

InsertionE1 Moderate - - - Pathogenic PAH1042

c.23_24delin

sTp.N8Ifs*30

Deletion/

InsertionE1 Low - - - Pathogenic PAH1043

c.541_544delp.E181Kfs*

13Deletion E6 Moderate - - - Pathogenic PAH1044

In gene position of sequencing results, “E” represented exons, and “I” represented introns. cDNA numbering was

according to the reference sequence NM_000277.1. “a”: Classification of each six novel variant was following the

variant interpretation guideline by the American College of Medical Genetics and Genomics/the Association for

Molecular Pathology [23].

18

Supplementary Table 8. Prediction of the effects of 17 novel variants of PTS gene on protein functions

Nucleotide

aberrationProtein variant Variant type

Locatio

n

Conservatio

n

SIFT

prediction

PolyPhen

prediction

Mutation

taster

prediction

ACMG/AMP

variants

classificatio

na

Accession

number in

PNDdb

c.34G>C p.A12P Missense E1 High Tolerated

Possibly

damagin

g

Polymorphis

m

Uncertain

significance

PNDDB25

9

c.421G>T p.V141F Missense E6 High Tolerated BenignDisease

causing

Uncertain

significance

PNDDB26

0

c.181G>A p.G61R Missense E3 High

Affect

protein

function

Probably

damagin

g

Disease

causing

Uncertain

significance

PNDDB26

1

c.62G>A p.S21N Missense E1 High

Affect

protein

function

Probably

damagin

g

Disease

causing

Uncertain

significance

PNDDB26

2

c.374G>A p.G125E Missense E6 High Tolerated BenignDisease

causing

Uncertain

significance

PNDDB26

3

c.143G>A p.G48D Missense E2 High

Affect

protein

function

Probably

damagin

g

Disease

causing

Uncertain

significance

PNDDB26

4

c.193C>T p.P65S Missense E4 High Tolerated BenignDisease

causing

Uncertain

significance

PNDDB26

5

c.1A>G p.M1V Missense E1 High

Affect

protein

function

BenignDisease

causing

Uncertain

significance

PNDDB26

6

c.35C>A p.A12E Missense E1 High

Affect

protein

function

Probably

damagin

g

Polymorphis

m

Uncertain

significance

PNDDB26

7

c.280G>C p.D94H Missense E5 High

Affect

protein

function

Probably

damagin

g

Disease

causing

Likely

pathogenic

PNDDB26

8

c.313A>T p.S105C Missense E5 High Affect

protein

Probably

damagin

Disease

causing

Uncertain

significance

PNDDB26

9

19

function g

c.289_290insCT

Tp.V97delinsAL Insertion E5

High- - -

Uncertain

significance

PNDDB27

0

c.322G>T p.E108* Nonsense E6High

- - - PathogenicPNDDB27

1

c.432dup p.E145Rfs*18 Duplication E6High

- - - PathogenicPNDDB27

2

c.84-6insTTTTIVS1-

6insTTTTSplice I1 - - - -

Uncertain

significance

PNDDB27

3

c.84-252G>T IVS1-252G>TSplice

I1 - - - -Uncertain

significance

PNDDB27

4

c.164-37C>T IVS2-37C>TSplice

I2 - - - -Uncertain

significance

PNDDB27

5

In gene position of sequencing results, “E” represented exons, and “I” represented introns. cDNA numbering was

according to the reference sequence NM_000317.2. “a”: Classification of each six novel variant was following the

variant interpretation guideline by the American College of Medical Genetics and Genomics/the Association for

Molecular Pathology [23].

20

Supplementary Table 9. Prediction of the effects of novel variants of GCH1 and QDPR gene on protein functions

GeneNucleotide

aberration

Protein

variantVariant type Location Conservation

SIFT

prediction

PolyPhen

prediction

Mutation

taster

prediction

ACMG/AMP

variants

classificationa

Accession

number in

PNDdb

GCH1 c.166G>A p.E56K Missense E1 Moderate Tolerated BenignDisease

causing

Uncertain

significancePNDDB276

QDPR c.143C>T p.A48V Missense E2 High

Affect

protein

function

Possibly

damaging

Disease

causing

Uncertain

significancePNDDB282

c.81dup p.Q28Afs*14 Duplication E1 Moderate - - - Pathogenic PNDDB278

c.328C>T p.Q110* Nonsense E4 High - - - Pathogenic PNDDB279

c.229G>T p.E77* Nonsense E3 Low - - - Pathogenic PNDDB280

In gene position of sequencing results, “E” represented exons. cDNA numbering of GCH1 and QDPR according to

reference sequence NM_000161.2 and NM_000320.2. “a”: Classification of each six novel variant was following

the variant interpretation guideline by the American College of Medical Genetics and Genomics/the Association

for Molecular Pathology [23].

21

22