Table e-1. Summary of clinical, laboratory, pathology, and imaging data in four LGMD patients harboring mutations in ISPD.

Legend: M, male; F, female; y, years; CK, serum creatine kinase levels; FVC, forced vital capacity; ECG, electrocardiogram; Echo, echocardiography; EF, ejection fraction. Patient ID refers to pedigrees shown in figure 1.

Patient (sex)

Onset (age, y) Physical examination (age, y)

Serum CK

%FVC (age, y)

Cardiological examination

Brain MRI

Cognitive examination

Ocular examination

Muscle MRI Muscle biopsy

A-V:5 (M)

Lower limb weakness (childhood)

Scapular winging with wasting of upper girdle muscles. Calf and tongue hypertrophy. Walks for short distances, cannot rise from the floor. Symmetric proximal weakness more prominent in the lower limbs (52)

5-7X 55 (52) ECG: right bundle branch block, left anterior fascicular block. Likely lateral myocardic ischemia. Echo: left ventricular hypetrophy with normal contractile function (EF 50%)

Gliotic ischemic lesions

Normal Normal Pelvis and thigh: severe involvement with relative sparing of iliopsoas, sartorius, gracilis, and biceps femoris short head. Leg: relative sparing of tibialis anterior and posterior, and gastrocnemius lateralis.

Dystrophic. Reduction of α-dystroglycan staining. Normal staining for dystrophin and sarcoglycans.

A-V:6 (F)

Difficulty in climbing stairs (12)

Lost ambulation at age 45. Symmetric proximal weakness more prominent in the lower limbs (50)

5X 35 (50) Normal ECG and Echo

Gliotic ischemic lesions

Normal Normal Pelvis and thigh: severe involvement with relative sparing of sartorius and gracilis Leg: relative sparing of tibialis anterior and posterior.

Dystrophic. Reduction of α-dystroglycan staining. Normal staining for dystrophin and sarcoglycans.

B-II:1 (F)

Quadriceps pain and myoglobinuria after physical exertion (8). Lower limb weakness (12)

Lost ambulation at age 39. Scapular winging, tongue and calf hypetrophy. Symmetric proximal weakness more prominent in the lower limbs (43)

7X 36 (42) Normal ECG and Echo

Not available

Not available

Not available

Fatty-fibrous replacement of all pelvic and lower limb muscles with relative sparing of tibialis posterior and anterior.

Not available

B-II:2 (F)

HyperCKemia (4). Quadriceps cramps and myoglobinuria after exertion, lower limb proximal weakness (9)

Lost ambulation at age 33. Mild scapular winging due to trapezius hypotrophy, tongue and mild calf hypetrophy. Symmetric proximal weakness more prominent in the lower limbs (36)

5X 53 (36) Normal ECG and Echo

Normal Not available

Normal Not available Dystrophic. Reduction of α-dystroglycan staining. Normal staining for dystrophin and sarcoglycans.

Legend to figure e-1. Tongue hypertrophy in patient A-V:5 (up, left) and B-II:2 (up, right). T2-FLAIR brain MRI sequences of patient A-V:5 (down, left) and A-V:6 (down, right) showing absence of white matter changes or structural abnormalities.

ISPD domain 1 47 279 451

LGMD WWS/MEB

nonsense missense

Patients Human Mouse Rat Dog Chicken Zebrafish Xenopus Tetraodon

50 Family A

V L P A A G C G E . . . . . . . . . . . . Y P V V V - S V H F L V L P A G G C G E . . . . . . . . . . . . Y P V V V V S V H F L V L P A G G C G E . . . . . . . . . . . . Y P V V V V L V C F D V L P A G G C G E . . . . . . . . . . . . Y P V V V V L V H C F V L P A G G S G E . . . . . . . . . . . . Y P V V V I S V Y F L V L P A G G S G E . . . . . . . . . . . . H P V V L I S A H L N V L P A G G S G E . . . . . . . . . . . . Y P V V I V W V Q L S V L P A G G C G E . . . . . . . . . . . . Y P L V I V S V H L T V L P A G G T G E . . . . . . . . . . . . C P L V L I W V H L N

Family B 367

Legend to figure e-2. (top) ClustalW2 (www.ebi.ac.uk/Tools/msa/clustalw2/) local sequence alignment of the ISPD gene product among different species. The regions flanking the residues mutated in limb-girdle muscular dystrophy (LGMD) patients (bold red) are illustrated. (bottom) Human ISPD is shown schematically. Red circles indicate nonsense variants and black circles missense mutations reported in association with Walker-Warburg syndrome (WWS) and Muscle-Eye-Brain (MEB) disease (see ref. 3 and 4 in the text). Empty circles indicate new residues mutated in LGMD patients.

G54 A54

Ctrl A-V:5

p.G54A

Polyphen2 (genetics.bwh.harvard.edu/pph/)

MutPred (http://mutpred.mutdb.org/)

score 0.964 0.687

effects Probably damaging Deleterious

Legend to figure e-3. (top) Evaluations in silico of the missense variant p.G54A identified in family A. Data suggest that the mutation is harmful to normal protein function. (bottom) ISPD protein modeling of the novel p.G54A mutation detected in patient A-V:5 was carried out through the HOPE webserver (http://www.nbic.nl/support/brs/project-hope/), which analyzes structural and functional effects of missense mutations retrieving information related to the amino acid sequence and a calculation of the three-dimensional protein structure (using WHAT IF and adopting the available E. coli model of IspD as a template, see ref.3 in the text). The protein is colored grey, the side-chains of the wild-type and mutated residue are represented in green and red, respectively. The mutated residue is located in a domain that is important for the main activity of the protein. The differences between the wild-type (left) and mutant residue (right) can disturb the core structure of this important domain and thereby affect the catalytic activity.