Introduction
Methods
Ethics statement
Probands
LSD diagnostic suspicion index
Capture array design, library construction, and NGS
Gene | Lysosomal storage dirorder |
---|---|
SMPD1 | Niemann-Pick Disease, Type A and B |
NPC1 | Niemann-Pick Disease, Type C1 |
NPC2 | Niemann-Pick Disease, Type C2 |
LIPA | Wolman Disease |
GLA | Fabry Disease |
GLB1 | GM1-Gangliosidosis, Type I, II, III |
GM2A | GM2-Gangliosidosis, AB Variant |
HEXA | Tay-Sachs Disease |
HEXB | Sandhoff Disease |
GBA | Gaucher Disease |
GAA | Pompe Disease |
IDUA | MPS I: Hurler/Scheie |
IDS | MPS II: Hunter Syndrome |
SGSH | MPS IIIA: Sanfilippo Type A |
NAGLU | MPS IIIB: Sanfilippo Type B |
HGSNAT | MPS IIIC: Sanfilippo Type C |
GNS | MPS IIID: Sanfilippo Type D |
GALNS | MPS IVA: Morquio A |
GLB1 | MPS IVB: Morquio B |
ARSB | MPS VI: Maroteaux-Lamy Syndrome |
GUSB | MPS VII: Sly Syndrome |
HYAL1 | MPS IX |
ASAH1 | Farber Disease |
ARSA | Metachromatic Leukodystrophy |
GALC | Krabbe Disease |
PSAP | Prosaposin deficiency |
NEU1 | Mucolipidosis I: Sialidosis |
FUCA1 | Fucosidosis |
LAMP2 | Danon Disease: Glycogen Storage Disease IIB |
LAMP3 | Candidate Gene For LSD |
GNPTAB | Mucolipidosis II Alpha/Beta |
GNPTG | Mucolipidosis III Gamma |
MCOLN1 | Mucolipidosis IV: Sialolipidosis |
MAN2B1 | Mannosidosis, Alpha B, Lysosomal |
MANBA | Mannosidosis, Beta A, Lysosomal |
PPT1 | Ceroid Lipofuscinosis, Neuronal, 1 |
TPP1 | Ceroid Lipofuscinosis, Neuronal, 2 |
CLN3 | Ceroid Lipofuscinosis, Neuronal, 3 (Batten Disease) |
CLN5 | Ceroid Lipofuscinosis, Neuronal, 5 |
CLN6 | Ceroid Lipofuscinosis, Neuronal, 6 |
CLN7 | Ceroid Lipofuscinosis, Neuronal, 7 |
CLN8 | Ceroid Lipofuscinosis, Neuronal, 8 |
CLN10 | Ceroid Lipofuscinosis, Neuronal, 10 |
CTSA | Galactosialidosis |
CTNS | Cystinosis |
SLC17A5 | Sialic Acid Storage Disease |
CTSK | Pyknodysostosis |
NAGA | Schindler Disease |
SUMF1 | Multiple Sulfatase Deficiency |
HPS1 | Hermansky-Pudlak Syndrome Type 1 |
AP3B1 | Hermansky-Pudlak Syndrome Type 2 |
HPS3 | Hermansky-Pudlak Syndrome Type 3 |
HPS4 | Hermansky-Pudlak Syndrome Type 4 |
HPS5 | Hermansky-Pudlak Syndrome Type 5 |
HPS6 | Hermansky-Pudlak Syndrome Type 6 |
DTNBP1 | Hermansky-Pudlak Syndrome Type 7 |
BLOC1S3 | Hermansky-Pudlak Syndrome Type 8 |
SOLiD4 platform
HiSeq2000 platform
Data filtering and analysis pipeline
SOLiD4 platform
HiSeq2000 platform
Sanger sequencing
Detection of gross indels
Filtering of annotated sequence data
Assessment of the pathogenicity of variants
Results and discussion
Statistical data from two platforms
Limitations of the enrichment method
Filtering of raw data
Assessment of the novel genetic tool for LSD diagnosis
Detection of gross deletions
Limitations of SNP calling software
Diagnosis achieved with the NGS-LSD screening tool
CC | GENE | REF_SEC | NT CHANGE | AA CHANGE | ZIG | MD | DIAGNOSIS | OMIM |
---|---|---|---|---|---|---|---|---|
C1 | ARSA | NM_000487 | c.1046delC | p.P349fs | HO | + | Metachromatic leukodystrophy | 250100 |
C2 | CLN3 | NM_000086 | c.461-280_677 + 382del966 | p.[Gly154Alafs*29, Val155_Gly264del] | HO | + | Ceroid lipofuscinosis, neuronal, 3 | 204200 |
C3 | FUCA1 | NM_000147 | c.464C > T | p.S155F | HT | - | Fucosidosis | 230000 |
FUCA1 | NM_000147 | c.790C > T | p.R264X | HT | - | |||
C4 | GALNS | NM_000512 | c.281G > T | p.R94L | HO | + | Mucopolysaccharidosis type IVA | 253000 |
C5 | GLB1 | NM_000404 | c.1581G > A | p.W527X | HO | + | GM1 gangliosidosis | 230500 |
C6 | GNPTAB | NM_024312 | c.1208 T > C | p.I403T | HT | + | Mucolipidosis III alpha/beta | 252600 |
GNPTAB | NM_024312 | c.1999G > T | p.E667X | HT | + | |||
C7 | GUSB | NM_000181 | c.526C > T | p.L176F | HT | + | Mucopolysaccharidosis VII | 253220 |
GUSB | NM_000181 | c.530C > T | p.T177I | HT | - | |||
C8 | NAGLU | NM_000263 | c.900C > T | p.R234C | HT | + | Sanfilippo B | 252920 |
C9 | NEU1 | NM_000434 | c.700G > A | p.D234N | HT | + | Sialidosis | 256550 |
NEU1 | NM_000434 | c.1021C > T | p.R341X | HT | + | |||
C10 | SGSH | NM_000199 | c.120C > G | p.Y40X | HO | + | Sanfilippo A | 252900 |
C11 | SMPD1 | NM_000543 | c.739G > A | p.G247S | HO | + | Niemann-Pick disease, type A | 257200 |
C12 | TPP1 | NM_000391 | c. 622C > T | p.R208X | HO | + | Ceroid lipofuscinosis, neuronal, 1 | 256730 |
C13 | CTSA | NM_000308 | c.448G > A | p.V150M | HT | - | Galactosialidosis | 256540 |
CTSA | NM_000308 | c.284delC | p.P95Lfs | HT | - | |||
C14 | NPC1 | NM_000271 | c.1552C > T | p.R518W | HT | + | Niemann-Pick disease, type C | 257220 |
C15 | NPC1 | NM_000271 | c.2594C > T | p.S865L | HT | + | Niemann-Pick disease, type C | 257220 |
C16 | SLC17A5 | NM_012434 | c.918 T > G | p.Y306X | HT | + | Sialic acid storage disorder,infantile | 269920 |
SLC17A5 | NM_012434 | c.500 T > C | p.L167P | HT | - | |||
C17 | ARSB | NM_000046 | c.427delG | p.V143Sfs | HO | + | Mucopolysaccharidosis VI | 253200 |
C18 | CLN3 | NM_000086 | c.461-280_677 + 382del966 | p.[Gly154Alafs*29, Val155_Gly264del] | HT | + | Ceroid lipofuscinosis, neuronal, 3 | 204200 |
PC | AO | AD | GD | NGS-LSD | Initial signs | Diagnostic suspicions | Biochemical-histopathology studies* | Other genetic studies* |
---|---|---|---|---|---|---|---|---|
P1 | 2Y | - | 12Y | CLN5 | Clumsiness, frequent falls, apraxic gait | CLN | VL; SBIOP + . | PPT1, TTP1 |
P2 | 4Y | - | 7Y | CLN6 | Clumsiness, myoclonic movements | GM2, GCL, MLD, Fabry, Schindler, MANSA, MANBA, MPS, CLN | EA: HEXA, HEXB, GLA, NAGA, MANBA, MAN2B1, MPS, GALC, ARSA, PPT1, TTP1 | MFSD8 |
P3 | 3Y | - | 16Y | CLN3 | Language regression, school inattention, social isolation | CDG, GM1, GM2, GSL, Schindler, GSD II, III, CLN | VL; FC; SPA ; EA: GLB1, HEXB, CTSA, NAGA, GAA, AGL; SBIOP + . | |
P4 | 2Y | - | 23Y | CLN7 | Language delay, clumsiness | CLN | SBIOP +; EA: CLN2. | CLN1 |
P5 | 4Y | - | 16Y | CLN3 | Conduct disorder, attention deficit disorder | CLN | VL +; SBIOP, MBIOP, NBIOP + . | PPT1, TTP1, CLN3, CLN5, CLN8 |
P6 | 3Y | 4Y | 7Y | MLD | Leukodystrophy | Spastic paraparesia | EA: ARSA + | |
P7 | 1Y | - | 9Y | GM1 | Global developmental delay, gait instability | CDG; RCCD; INAD | MBI; NBIOP +; MBS; SPA; EA: HEXA, HEXB | |
P8 | 2Y | 7Y | 4Y | GM2 | Language delay, conduct disorder | Unspecific global developmental delay, Neurodegenerative disease; CLN, GM2 | SBIOP; MBS; EA: HEXA + . | Caryotype; Fragile X; Smith-Magenis |
P9 | 2Y | - | 14Y | GM2 | Clumsiness, frequent falls | Ataxia, Attention deficit disorder | MBI | Caryotype; Fragile X; CGH-60 k array. |
P10 | 3Y | 9Y | 12Y | GM1 | Clumsiness, frequent falls, language delay | RS, MPSIII, GM1 | MBS, U-GAGs; EA: GLB1+ | Caryotype, Fragile-X, MECP2 |
P11 | 5Y | - | 22Y | GM2 | Learning problems, language difficulties | CLN | SBIOP | CLN3, CLN8 |
P12 | 13Y | - | 18Y | NPC2# | Clumsiness, short stature | MTDPS; RCCD; SCA; DRPLA | MBIOP; RCCFS; EA: muscular Coenzima Q10 | mtADN(muscle); MTATP6; tRNALeu(UUR); SCA1,2,3,6,7,12,17; ATN1 |
P13 | 18 M | - | 4Y | ML II,III | Growth delay, macrocephaly | MPS | U-GAGs | SHOX |
P14 | 12 M | 3Y | 3Y | GM2 | Psycomotor regression, language regression | RS, GM2 | EA: HEXA +, HEXB | MECP2 |
P15 | 0 M | 3 M | 1Y | GM1 | Hypotonia, hepatomegaly, cardiomyopathy, bony abnormalities | GSD II | EA: GAA | |
P16 | 9 M | 2Y | 2Y | MPSVI | Bilateral costal deformity, macrocephaly | AA, MPS | U-GAGs, Dermatan sulfate in urine, EA: ARSB | |
P17 | 0 M | - | 2Y | ML II,III | Hypotonia, microcephaly, jaundice | MPS | MBS, U-OLG, U-GAGs | Caryotype; CGH array |
P18 | 2 M | 3Y | 6Y | MPSII | Bilateral inguinal hernia | MPS | U-GAGs; EA: IDS + | |
P19 | 1Y | 4Y | 5Y | MPSI | Clubfoot, arthrogryposis, clawed hands | MPS | U-GAGs; EA: IDUA +, IDS, ARSB | |
P20 | 0 M | 1Y | 1Y | GM1 | Hypotonia, severe psycomotor delay | GM1, GM2, GSL, ML I | MBS; VL; U-GAGs; EA: GBL1 +, HEXA, HEXB, CTSA, NEU1 | |
P21 | 18 M | 3Y | 3Y | GM2 | Unsteady gait, frequent falls | TORCH, Leukodystrophy | EA: | |
P22 | 2Y | - | 6Y | CLN8 | Cognitive regression, epilepsy | Lennox syndrome, CLN | PBIOP + | |
P23 | 3Y | - | 9Y | CLN10 | Psycomotor regression, dystonia, epilepsy | Neuroaxonal dystrophy, sphingolipidosis | MBIO | mtDNA |
P24 | 3Y | 5Y | 23Y | MPSIIIB | Language delay, conduct disorder, learning disabilities, hypoacusia | MPS | Heparan sulfate + | |
P25 | 1Y | - | 6Y | GM2AB | Developmental delay, regression | GM2, GSD II | EA: HEXA, HEXB, GAA (+); HPS GM2 + | GAA |
P26 | 4Y | - | 25Y† | CLN8 | Cognitive regression, epilepsy | CLN | CLN6 |
PC | AO | GDD | IS | GENE | OMIM | REF SEC | NT CHANGE | AA CH | MT | SS | PP | CS | ZIG | MD | BA | DIAGNOS |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
P1 | 2Y | 10Y | Mod | CLN5 | 608102 | NM_006493 | c.335G > C/c.835G > A | p.R112P/p.D279N | 099/0.99 | 0/0.16 | 1/1 | + | HO | + | NA | Finnish variant late infantile CLN |
P2 | 4Y | 4Y | Mod | CLN6 | 606725 | NM_017882 | c.794_796del | p.S265del | - | - | - | HO | + | NA | Early juvenile late infantile CLN | |
P3 | 3Y | 12Y | High | CLN3 | 607042 | NM_000086 | c.461-280_677 + 382del966 | p.[Gly154Alafs*29, Val155_Gly264del] | - | - | - | HO | + | NA | Juvenile CLN | |
P4 | 2Y | 21Y | Mod | MFSD8 | 611124 | NM_152778 | c.881C > A | p. T294K | 0.99 | 0 | 0.99 | + | HO | + | NA | Turkish variant late infantile CLN |
P5 | 4Y | 12Y | High | CLN3 | 607042 | NM_000086 | c.371_372insT | p.Y124fs | - | - | - | HO | - | NA | Juvenile CLN | |
P6 | 3Y | 4.5Y | High | ARSA | 607574 | NM_000487 | c.465 + 1G > A | ---------- | - | - | - | + | HO | + | + | Metachromatic leukodystrophy |
P7 | 1Y | 8Y | Low | GLB1 | 611458 | NM_000404 | c.922 T > C | p.F308L | 0.99 | 0 | 1 | + | HO | - | + | GM1 gangliosidosis |
P8 | 3Y | 1Y | High | HEXA | 606869 | NM_000520 | c.533G > A | p.R178H | 0.99 | 0 | 1 | + | HO | + | + | GM2 gangliosidosis, B1variant |
P9 | 4Y | 10Y | Low | HEXA | 606869 | NM_000520 | c.1496G > A | p.R499H | 0.99 | 0 | 1 | HT | + | + | GM2 gangliosidosis, juvenil (TS) | |
HEXA | NM_000520 | c.1003A > T | p.I335F | 0.99 | 0 | 0.97 | HT | + | ||||||||
P10 | 3Y | 7Y | High | GLB1 | 611458 | NM_000404 | c.602G > A | p.R201H | 0.99 | 0.02 | 1 | HT | + | + | GM1 gangliosidosis | |
GLB1 | NM_000404 | c.1188_1188dupG | p.P397fs | - | - | - | HT | - | ||||||||
P11 | 5Y | 18Y | High | HEXA | 606869 | NM_000520 | c.155C > A | p.S52X | - | - | - | + | HT | + | + | GM2 gangliosidosis juvenile (TS) |
HEXA | NM_000520 | c.1305C > T | p.Y435Y | - | - | - | HT | - | ||||||||
P12 | 13Y | 6Y | Low | NPC2 | NM_006432 | c.441 + 1G > A | ----------- | - | - | - | HT | - | - | Niemann-Pick disease, type C2? | ||
P13 | 18 M | 3Y | High | GNPTAB | 607840 | NM_024312 | c.2354 T > G | p.L785W | 0.71 | 0.09 | 0.022 | + | HT | - | + | Mucolipidosis II/III |
GNPTAB | NM_024312 | c.1774G > A | p.A592T | 0.98 | 0.01 | 0.955 | HT | - | ||||||||
P14 | 12 M | 4Y | High | HEXA | 606869 | NM_000520 | c.718_719insT | p.K240fs | HT | - | + | GM2 gangliosidosis (TS) | ||||
HEXA | NM_000520 | c.1003A > T | p.I335F | 0.99 | 0.00 | 0.467 | HT | + | ||||||||
P15 | 0 M | 1Y | High | GLB1 | 611458 | NM_000404 | c.671_672delAT | p.H224Qfs | + | HO | - | +* | GM1 gangliosidosis | |||
P16 | 9 M | 1Y | High | ARSB | 611542 | NM_000046 | c.382_384delCTC | p.L128del | HO | - | +* | Mucopolysaccharidosis VI | ||||
P17 | 0 M | 3Y | High | GNPTAB | 607840 | NM_024312 | c.3739_3742delCTTT | p.E1248fs | + | HO | - | + | Mucolipidosis II/III | |||
P18 | 2 M | 6Y | High | IDS | 300823 | NM_000202 | c.425C > T | p.S142F | 0.98 | 0.00 | 0.998 | + | HE | + | +* | Hunter Syndrome |
P19 | 3Y | 5Y | High | IDUA | 252800 | NM_000203 | c.1205G > A | p.W402X | + | HT | + | +* | Hurler Syndrome | |||
IDUA | NM_000203 | c.1874A > G | p.Y625C | 0.98 | 0.00 | 0.99 | HT | - | ||||||||
P20 | 10 M | 2Y | High | GLB1 | 611458 | NM_000404 | c.947A > G | p.Y316C | 0.999 | 0 | 0.79 | HT | + | +* | GM1 gangliosidosis | |
GLB1 | NM_000404 | c.458-401_552 + 1033del1529 | ----------- | HT | + | |||||||||||
P21 | 20 M | 4Y | High | HEXA | 606869 | NM_000520 | c.459 + 5G > A | ----------- | HT | + | +* | GM2 gangliosidosis (TS) | ||||
HEXA | NM_000520 | c.533G > A | p.R178H | 0.99 | 0 | 1 | HT | + | ||||||||
P22 | 2Y | 4Y | High | CLN8 | 607837 | NM_018941 | c.509C > G | p.T170R | 0.999 | 0.00 | 0.999 | + | HO | + | NA | Ceroid lipofuscinosis, neuronal, 8 |
P23 | 3Y | 9Y | High | CTSD | 116840 | NM_001909 | c.470C > T | p.S157L | 0.98 | 0.03 | 0.005 | + | HT | - | NA | Ceroid lipofuscinosis, neuronal, 10 |
CTSD | NM_001909 | c.353-12G > A | ----------- | - | - | - | HT | - | pendε | |||||||
P24 | 3Y | 23Y | High | HGSNAT | 610453 | NM_152419 | c.1250 + 1G > A | ----------- | - | - | - | HT | + | +* | Sanfilippo C | |
HGSNAT | NM_152419 | c.1270G > A | p. G424S | 0.999 | 0.59 | 1 | HT | + | ||||||||
P25 | 1Y | 6Y | High | GM2A | 613109 | NM_000405 | c.333delC | p. C112Vfs | - | - | - | HO | - | +# | GM2 gangliosidosis, AB variant | |
P26 | 4Y | >20Y | High | CLN8 | 607837 | NM_018941 | c.792C > G | p. N264K | 0.97 | 0 | 0.99 | + | HO | - | NA | Ceroid lipofuscinosis, neuronal, 8 |
PC | IS | Gene | Ref sec | Coding variants* | dbSNP ID | MAF | Zig | MD | BA | |
---|---|---|---|---|---|---|---|---|---|---|
P27 | High | MANBA | NM_005908 | c.1922G > A | p.R641H | -- | HT | + | NegΨ | |
P28 | High | HYAL1 | NM_033159 | c.676C > T | p.R226C | -- | HT | - | NCI | |
HEXB | NM_000521 | c.383 T > G | p.L128R | -- | HT | - | NegΨ | |||
P29 | High | SMPD1 | NM_000543 | c.1460C > T | p.A487V | -- | HT | + | NegΨ | |
P30 | Low | NEU1 | NM_000434 | c.1070G > A | p.R357Q | -- | HT | - | NCI | |
P31 | Mod | MFSD8 | NM_152778 | c.50C > G | p.T17R | -- | HT | - | NCI | |
P32 | Low | NAGA | NM_000262 | c.697G > A | p.V233M | -- | HT | - | NCI | |
NPC1 | NM_000271 | c.665A > G | p.N222S | rs1805081 | 0.001 | HT | + | NCI | ||
P33 | Low | SMPD1 | NM_000543 | c.1550A > T | p.E517V | -- | HT | + | NCI | |
P34 | High | SGSH | NM_000199 | c.308A > G | p.K103R | -- | HT | - | NCI | |
CLN6 | NM_017882 | c.755G > C | p.R252P | -- | HT | - | NCI | |||
P35 | Low | TPP1 | NM_000391 | c.1117C > G | p.Q373E | -- | HT | - | NCI | |
GAA | NM_000152 | c.1367G > T | p.R456M | -- | HT | - | NCI | |||
P36 | High | CLN5 | NM_006493 | c.606G > A | p.M202I | -- | HT | - | NCI | |
P37 | High | SMPD1 | NM_000543 | c.8G > A | p.R3H | -- | HT | - | NCI | |
P38 | Low | IDUA | NM_000203 | c.251G > C | p.G84A | -- | HT | - | NCI | |
P39 | Low | IDS | NM_000202 | c.754G > A | p.D252N | -- | HO | + | Neg* | |
P40 | Low | CLN3 | NM_000086 | c.995C > T | p.A332V | -- | HT | - | NA | |
P41 | High | CLN5 | NM_006493 | c.726C > A | p.N242K | -- | HT | - | NA | |
P42 | High | IDUA | NM_000203 | c.650G > A | p.R217Q | -- | HT | - | NCI | |
NPC1 | NM_000271 | c.2257G > A | p.V753M | -- | HT | - | NCI | |||
P43 | Mod | ASAH1 | NM_177924 | c.2 T > C | p.M1T | -- | HT | - | NCI | |
P44 | Low | SMPD1 | NM_000543 | c.1460C > T | p.A487V | -- | HT | + | NCI | |
P45 | Low | CLN3 | NM_000086 | c.995C > T | p.A332V | -- | HT | - | NA | |
P46 | Low | CLN5 | NM_006493 | c.606G > A | p.M202I | -- | HT | - | NA | |
GALNS | NM_000512 | c.1127G > A | p.R376Q | -- | HT | + | NCI | |||
P47 | Low | GNPTG | NM_032520 | c.857C > T | p.T286M | -- | HT | + | NCI | |
NPC1 | NM_000271 | c.3535A > G | p.M1179V | rs61731969 | 0.002 | HT | - | NCI | ||
P48 | Low | MAN2B1 | NM_000528 | c.844C > T | p.P282S | rs45576136 | 0.003 | HT | - | NCI |