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Sheffield Diagnostic Genetics Service DNA Analysis in Glycogen storage disease Nick Beauchamp PhD Sheffield Diagnostic Genetics Service, Sheffield Childrens NHS Foundation Trust 2nd February 2011 Sheffield Diagnostic Genetics Service

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Sheffield Diagnostic Genetics Service

DNA Analysis in Glycogen storage disease

Nick Beauchamp PhD

Sheffield Diagnostic Genetics Service, Sheffield Children’s NHS Foundation Trust 2nd February 2011

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Sheffield Diagnostic Genetics Service

Limit dextrin Phosphorylase Debranching Enzyme Glucose G-6-Phosphatase Phosphorylase Kinase Glycogen Glucose 1-P Glucose 6-P Glucose UDPGlucose Glycogen Synthase Branching Enzyme

Glucokinase Phosphoglucose Isomerase Uridine Diphosphoglucose Pyrophosphorylase

Glycogen Synthesis and Breakdown

Alpha-1,4 Alpha-1,6

Type I Type III Type V and VI Type IX Type 0 Type IV

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Sheffield Diagnostic Genetics Service

Limit dextrin Phosphorylase Debranching Enzyme Phosphorylase Kinase Glycogen Glucose 1-P Glucose UDPGlucose Glycogen Synthase Branching Enzyme

Glucokinase Phosphoglucose Isomerase Uridine Diphosphoglucose Pyrophosphorylase

Glycogen Synthesis and Breakdown

Alpha-1,4 Alpha-1,6

Glucose G-6-Phosphatase Glucose 6-P

Type Ia Type Ib G6PC gene SLC37A4 gene

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Sheffield Diagnostic Genetics Service

GSD Type I

  • First described by von Gierke in 1929
  • Approx 1 in 58,000 newborns affected
  • Autosomal recessive
  • Classification:

– Ia: Deficiency of glucose-6-phosphatase enzyme – Ib/Inon-a: Deficiency of glucose-6-phosphate transporter

  • Approx 10% of Type 1 cases are Ib
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Sheffield Diagnostic Genetics Service

GSD Type I

  • Type Ia

– G6PC gene

  • 5 exons, 13 kb on Chr

17q21

– >80 mutations reported – Common changes:

  • p.Arg83Cys - 33%
  • p.Gln347X - 18%
  • Type Ib

– SLC37A4 gene

  • 9 exons, 6 kb on Chr

11q23.3

– >65 mutations reported – Common changes:

  • p.Leu348fs - 28%
  • p.Gly339Cys - 19%
  • Genetic analysis:
  • Avoidance of liver biopsy
  • Confirms diagnosis - type Ia versus Ib
  • Phenotypic heterogeneity
  • No clear genotype/phenotype correlation
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Sheffield Diagnostic Genetics Service

From Foster and Nordlie 2002. Exp Biol Med 227: 601-608.

SLC37A4 gene mutations G6PC gene mutations

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Limit dextrin Phosphorylase Debranching Enzyme Phosphorylase Kinase Glycogen Glucose 1-P Glucose UDPGlucose Glycogen Synthase

Branching Enzyme Glucokinase Phosphoglucose Isomerase Uridine Diphosphoglucose Pyrophosphorylase

Glycogen Synthesis and Breakdown

Alpha-1,4 Alpha-1,6

Glucose G-6-Phosphatase Glucose 6-P

Type V and VI PYGM and PYGL genes

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Sheffield Diagnostic Genetics Service

GSD Type V

  • Also known as McArdle Disease
  • Deficiency of muscle glycogen phosphorylase

– Autosomal recessive – PYGM gene

  • 20 exons, 40kb on Chr 11q12-q13.2
  • 2.5% of GSDs
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Mutations of the PYGM gene

  • Common mutations:

– p.Arg50X - 32% - 81% of alleles – p.Gly205Ser - 0% - 10% of alleles

  • Other mutations

– >85 rare mutations

  • Non-sense mediated mRNA decay
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From Rubio et al 2007. Human Mutation 28: 203-204

Mutations of the PYGM gene

From: Rubio JC et al 2007 Hum Mutat. 28(2): 203-4.

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GSD Type VI

  • Also known as Hers Disease
  • Deficiency of liver glycogen phosphorylase

– Autosomal recessive – PYGL gene

  • 20 exons, 40kb on Chr 14q21-q22
  • Rare
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Sheffield Diagnostic Genetics Service

GSD Type VI - Reported Patients

  • 11 patients published with 17 mutations
  • Majority are missense mutations

– Clustered in exons 16 and 17

p.R399X p.Q13P p.V456M p.R491C p.D634H p.K681T p.S675L p.S675T p.N632I p.E673K c.529-1G>C c.1768+1G>A p.N339S p.N377K c.1620+1G>A p.G233D p.M1?

PYGL

[c.1964_1969inv6; c.1969+1_+4delGTAC]

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GSD Type VI - Screened Patients

  • All published patients and 17 patients from clinical

service

p.R399X [c.1964_1969inv6; c.1969+1_+4delGTAC] p.Q13P p.V456M p.R491C p.D634H p.K681T p.S675L p.S675T p.N632I p.E673K c.529-1G>C c.1768+1G>A p.N339S p.N377K c.1620+1G>A p.G233D p.S836F p.Y821H c.1518G>A p.G686A p.D307G c.345G>A p.M1? c.1000-1G>A p.Q577X

PYGL

p.W362X c.1768+2dupT

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Sheffield Diagnostic Genetics Service

Pyridoxal Phosphate Active Site

Glycogen + Pi Glycogen + Glucose-1-P

Glycogen Phosphorylase

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Sheffield Diagnostic Genetics Service

Limit dextrin Phosphorylase Debranching Enzyme Phosphorylase Kinase Glycogen Glucose 1-P Glucose UDPGlucose Glycogen Synthase

Branching Enzyme Glucokinase Phosphoglucose Isomerase Uridine Diphosphoglucose Pyrophosphorylase

Glycogen Synthesis and Breakdown

Alpha-1,4 Alpha-1,6

Glucose G-6-Phosphatase Glucose 6-P

Type IX PHKA2 gene PHKG2 gene PHKB gene

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Sheffield Diagnostic Genetics Service

From: Vénien-Bryan et al 2009 Structure 17: 117–127.

Phosphorylase kinase

  • Four copies of each of α, β, γ, δ subunits
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X-linked GSD Type IX

  • Deficiency of liver α subunit (PHKA2 gene)

– (Muscle subunit - PHKA1 gene)

  • Mutations in PHKA2 gene

– 33 exons, 92 kb on Chr Xp22.2-p22.1 – wide range of mutations described

  • X-Linked Glycogenosis type 1 (XLG1)

– Reduced PHK activity in RBC and liver

  • X-Linked Glycogenosis type 2 (XLG2)

– Reduced PHK activity in liver only

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p.H132Y p.R186C p.R186H p.K189E p.K189_T190del p.G193V p.T251del p.R295C p.D299G p.1111_1112insTR p.T1114I p.P498L p.H132P p.R295H Multiphosphorylation domain p.C91Y p.Y116D p.F141del p.R295H p.P399S p.Q818_Y825del8 p.P869R p.R916W p.R1070del p.Y116_T120dup p.M1113I p.E1125K p.P1205L p.G1207W p.G1210E

α Chain

p.I566N p.W43R p.R45W p.C326R p.E893K p.E1117K p.M1170Dup p.R295L p.R45Q p.G292R p.S201Y

XLG1 XLG2

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Case 1

  • Symptoms present at 1 year, diagnosed type VI at 7 years

– Hepatomegaly – Normal fasting – Raised transaminases – Growth retardation – WBC Total GP: 1.4 (NR: 1.0-3.2 µmol Pi/mg alb/h) – WBC Activated GP: 0.3 (NR: 0.5-2.2 µmol Pi/mg alb/h) – RBC PHK: 21.8 (NR: 8.6-45 µmol Pi/min/g Hb)

  • No mutation identified in PYGL gene – Not GSD type VI
  • Analysis of PHKA2: p.Arg182Cys – X-linked GSD Type IX
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Analysis of GSD type VI

  • 17 of 42 (40%) patients initially suspected of having

GSD type VI shown to have GSD type IX

  • 15 (35%) have mutations in PHKA2

– Majority previously described XLG2 mutations

  • Two have PHKB mutations

– c.1207+1G>T and p.Q657X – p.R429X and p.Q516X

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Autosomal GSD Type IX

  • Autosomal recessive
  • Rarer than X-linked form
  • Deficiency of either β or liver γ subunit
  • Mutations in PHKB gene

– 33 exons, 238kb on Chr 16q12-q13 – Majority result in null alleles – Mild symptoms compared to defects in PHKA2 gene

  • or PHKG2 gene

– 10 exons, 9kb on Chr 16 p12-p13 – Missense mutations and null alleles – Severe symptoms compared to defects in PHKA2 gene

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PHKB mutations

  • PHKB missense mutations found in

heterozygous isolation:

– p.Gln657Lys

(Burwinkel B et al 1997 Hum Genet 101: 170-174.)

– p.Ala118Pro

(Burwinkel B et al 2003 Eur J Hum Genet 11 : 516-526.)

– p.Met185Ile

(Beauchamp NJ et al 2007 Mol. Genet. Metab. 92: 88-99.)

– p.Tyr167Cys

(Unpublished)

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  • History of faintness and

clamminess on fasting

  • PHK activity:

3.9 mmol/min/gHb (10-120)

  • PHKB p.[=]+[Tyr167Cys]
  • 2 year old
  • Recurrent hypoglycaemia

with seizures

  • PHK activity:

0.8 mmol/min/gHb (10-120)

  • PHKB p.[=]+[Tyr167Cys]
  • 6 year old ADHD
  • Sleepy and sweaty if

fasted, better with sugary drinks

  • PHK activity:

0 mmol/min/gHb (10-120)

  • PHKB p.[=]+[Tyr167Cys]

Conclusion: Dominant negative mutations in PHKB result in phosphorylase kinase deficiency.

Case 2

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From: Vénien-Bryan et al 2009 Structure 17: 117–127.

Phosphorylase kinase

  • Four copies of each of α, β, γ, δ subunits
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Female X-Linked GSD type IX

  • Index case is heterozygous for

PHKA2 p.Pro1205Leu

  • Possibilities

– Additional defects in PHKB or PHKG2 genes – Additional PHKA2 gene mutation – Skewed X-inactivation – Monosomy X (Turner syndrome)

PHK activity: 7% of normal PHK activity: 71% of normal PHK activity: Not done

Case 3

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Limit dextrin Phosphorylase Debranching Enzyme Phosphorylase Kinase Glycogen Glucose 1-P Glucose UDPGlucose Glycogen Synthase Branching Enzyme

Glucokinase Phosphoglucose Isomerase Uridine Diphosphoglucose Pyrophosphorylase

Glycogen Synthesis and Breakdown

Alpha-1,4 Alpha-1,6

Glucose G-6-Phosphatase Glucose 6-P

Type III AGL gene

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GSD Type III

  • Also known as Cori or Forbes Disease
  • Deficiency of glycogen debrancher enzyme
  • Autosomal recessive
  • Four subtypes:

– Type IIIa (~85% of patients)

  • Enzyme deficient in both liver and muscle

– Type IIIb (~15% of patients)

  • Enzyme deficient in liver

– Type IIIc

  • Loss of glucosidase activity

– Type IIId

  • Loss of transferase activity
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GSD Type III

  • AGL gene

– 35 exons, 85 kb on Chr 1p21

  • Type IIIa

– Majority (65%) of mutations are nonsense, frameshift or splice site mutations – Common changes

  • p.Arg408X, c.4260-12A>G, p.Arg864X

– Rare changes

  • 118 mutations reported
  • Type IIIb

– Exon 3 nonsense mutations

  • c.16C>T, p.Gln6X,
  • c.18_19delGA, p.Gln6HisfsX20
  • c.22C>T, p.Arg8X
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GSD Type III – AGL mutations

From: JL Goldstein et al 2010. Genet Med 12:424–430

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GSD Type III – AGL mutations

From: JL Goldstein et al 2010. Genet Med 12:424–430

Deletion of exons 29-31

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Cases 4 and 5

  • Patient, aged 5 years
  • Permanent hepatomegaly
  • Fasting <3 hours
  • Permanent CK elevation
  • Cardiomyopathy
  • Fatigue not observed
  • Patient , aged 20 years
  • Hepatomegaly till 16 years
  • Normal feeding
  • Normal CK
  • No Cardiomyopathy
  • Periodic fatigue
  • Leuk Debrancher: 5.7

Normal range: 26.8-105 nmol glu/mg protein/hour

  • RBC Glycogen: 565

Normal range: 5.7-135 mg/g Hb

  • p.Arg408X homozygote
  • Leuk Debrancher: 0.69

Normal range: 26.8-105 nmol glu/mg protein/hour

  • RBC Glycogen: 726

Normal range: 5.7-135 mg/g Hb

  • p.Arg8X and c.4260-12A>G
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Limit dextrin Phosphorylase Debranching Enzyme Phosphorylase Kinase Glycogen Glucose 1-P Glucose UDPGlucose Glycogen Synthase Branching Enzyme

Glucokinase Phosphoglucose Isomerase Uridine Diphosphoglucose Pyrophosphorylase

Glycogen Synthesis and Breakdown

Alpha-1,4 Alpha-1,6

Glucose G-6-Phosphatase Glucose 6-P

Type IV GBE gene

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GSD Type IV

  • Also known as Anderson Disease
  • Deficiency of glycogen branching enzyme
  • Autosomal recessive

– GBE1 gene – 16 exons, 262 kb on Chr 3p14

  • Rare
  • 20 Patients reported

– 37 unique mutations – Some phenotype/genotype correlation

  • Wide range of phenotypes
  • Congenital presentation to polyglucosan body disease
  • Genetic analysis allows prenatal diagnosis
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Limit dextrin Phosphorylase Debranching Enzyme Phosphorylase Kinase Glycogen Glucose 1-P Glucose UDPGlucose Glycogen Synthase

Branching Enzyme Glucokinase Phosphoglucose Isomerase Uridine Diphosphoglucose Pyrophosphorylase

Glycogen Synthesis and Breakdown

Alpha-1,4 Alpha-1,6

Glucose G-6-Phosphatase Glucose 6-P

Type 0 GYS2 gene

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GSD Type 0

  • Deficiency of glycogen synthase
  • Clinical symptoms

– Ketotic hypoglycaemia – Post-prandial hyperglycaemia and hyperlactataemia – Low activity in liver biopsy

  • Autosomal recessive

– GYS2 gene

  • 16 exons, 69kb on Chr 12p12.2
  • 18 mutations described
  • Rare - 3 of 59 (6%) patients with suspected GSD type 0 have mutations
  • Molecular analysis avoids biopsy
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Limit dextrin Phosphorylase Debranching Enzyme Glucose G-6-Phosphatase Phosphorylase Kinase Glycogen Glucose 1-P Glucose 6-P Glucose UDPGlucose Glycogen Synthase Branching Enzyme

Glucokinase Phosphoglucose Isomerase Uridine Diphosphoglucose Pyrophosphorylase

Glycogen Synthesis and Breakdown

Alpha-1,4 Alpha-1,6

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Debranching Enzyme Glucose G-6-Phosphatase Glucose 1-P Glucose 6-P Glucose

Glucokinase Phosphoglucose Isomerase Uridine Diphosphoglucose Pyrophosphorylase

Alpha-1,4 Alpha-1,6

Glycogen Synthesis and Breakdown

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Debranching Enzyme Glucose G-6-Phosphatase Glucose 1-P Glucose 6-P Glucose

Glucokinase Phosphoglucose Isomerase Uridine Diphosphoglucose Pyrophosphorylase

Alpha-1,4 Alpha-1,6

Glycogen Synthesis and Breakdown

Fructose 6-P Fructose-1,6-bisphosphate Phosphofructokinase

Glucose Phosphate Isomerase

Fructose-1,6-bisphosphatase Pyruvate

Type VII FBP1 deficiency

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GSD Type VII

  • Also known as Tarui disease
  • First identified in 1965
  • Deficiency of muscle phosphofructokinase

– Three isoforms muscle, liver and platelet types

  • Autosomal recessive inheritance

– PFKM gene

  • Located on 12q13
  • 24 exons covering 30kb (differential splicing of 5’ region)
  • Rare - 15 mutations reported -

– No genotype/phenotype correlation described

  • Genetic analysis

– ? avoid muscle biopsy and allows prenatal diagnosis

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GSD Type II

  • Also known as Pompe Disease,

acid α-glucosidase deficiency or acid maltase deficiency

  • Autosomal recessive
  • Lysomal storage disease - accumulation of glycogen in

all tissues

  • 1 in 40,000 live births
  • GAA gene on Chr 17q25.2-q25.3

– Common mutations:

  • c.-45T>G - Mild phenotype
  • c.525del - severe phenotype

– >150 listed on mutation database at: www.pompecenter.nl

  • Part of Newborn Screening in USA
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Type Ia Ib III IV VI IXauto IXx Totals Total Samples 59 45 26 81 32 42 38 99 422 Confirmed Diagnosis 3 (6%) 27 (60%) 15 (58%) 56 (69%) 7 (22%) 12 (29%) 14 (37%) 61 (62%) 195 (46%) Investigated Further 5 10 5 10 1 23 9 29 92 Diagnosis Changed (0%) 4 (9%) 4 (15%) 3 (4%) 1 (5%) 18 (43%) 2 (5%) 12 (12%) 44 (10%) 1 1 Ia 2 2 Ib 4 4 III 1 1 VI 1 2 2 5 IX X-linked 2 2 15 19 IX Autosomal 1

PHKG2

2

PHKB

6

PHKG2

3

PHKB

7

PHKG2

5

PHKB

Analysis of liver type GSD Patients in Sheffield

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Type II V VII IXx Muscle Total Grand Total Total Samples 37 67 9 4 117 539 Confirmed Diagnosis 25 (68%) 28 (42%) 1 (11%) (0%) 54 (46%) 249 (46%) Investigated Further 1 3 1 3 8 102 Diagnosis Changed (0%) (0%) (0%) (0%) (0%) 44 (8%)

Analysis of muscle type GSD Patients in Sheffield

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Conclusions

  • Genetics analysis can:

– Provide a definitive diagnosis – Avoids liver or muscle biopsy – Allow carrier testing and prenatal diagnosis – Change diagnosis and inheritance patterns – In some cases indicate prognosis