Muscle Biopsies Results in Prader-Willi Syndrome (PWS)

This page contains the muscle biopsy reports for two infants subsequently diagnosed with genetically confirmed Prader-Willi syndrome (PWS) and are provided here with the permission of the parents. Both reports reveal impairment in mitochondrial respiratory chain transport activity as well as abnormal muscle morphology (structure) consistent with metabolic myopathy. Parents of those with PWS may wish to print out this page, together with the Stefan study (which found significantly impaired energy metabolism in a PWS mouse model) and the various reports of mitochondrial deficiency in children with PWS (see below), as background materials for their pediatrician and other health care providers, including a metabolic specialist.

The following chart compares the electron transport chain activity from the two biopsies. (For Patient 2, the second percentage in parentheses represents the activity level when adjusted for low citrate synthase activity.)

Muscle Biopsy #1

The following is the report on a right thigh skeletal muscle biopsy performed on a 16-day-old boy who was subsequently diagnosed with genetically confirmed PWS due to maternal uniparental disomy (UPD). The report reveals significant impairment in mitochondrial respiratory chain transport complexes I through IV, as well as abnormal muscle morphology (structure) and glycogen and lipid storage abnormalities.

Surgical Pathology Report

Final diagnosis:

1. Poor fiber type differentiation, with suggestion of fiber type 2 predominance.
2. Slight variation in fiber size.
3. Increased lipid, glycogen and mitochondria appreciable mainly by electron microscopy.
4. No evidence of nemaline, centronuclear, or central core myopathy.
5. No evidence of inflammatory, or a dystrophic myopathy.

Comment: The muscle biopsy in this 16-day-old baby with hypotonia shows findings suggesting a metabolic myopathy. The result of an additional histochemical test (for phosphofructokinase) will follow in an addendum. Clinical correlation is suggested.

Surgical Pathology Report

Clinical History: 16-day-old neonate with hypotonia.

Gross Description: The specimen is received fresh, labeled "muscle biopsy - right thigh" and consists of two portions of tan-red soft tissue measuring 1.0 x 0.7 x 0.3 cm and 0.7 x 0.5 x 0.2 cm. A portion of the tissue is snap frozen for cryostat sections, histochemistry and possible studies. A small portion is submitted in glutaraldehyde for electron microscopy, and the remaining tissue is submitted for routine processing in one cassette.

Microscopic examination:

Supplemental Surgical Pathology Report

Addendum: Additional histochemistry performed to test for phosphofructokinase showed that phosphofructokinase is present in all muscle fibers with no evidence of phosphofructokinase deficiency. The diagnosis is unchanged.

Supplemental Surgical Pathology Report

Addendum:

Mito DNA Mutations
MITDNA Results
mtDNA Point Mutations and Deletions

Results: Point mutations and deletions were not detected.

MITDNA Interpretation

Test results should be interpreted in the context of patient's clinical and family history.

Point mutations and deletions were not detected. We cannot confirm the diagnosis of mitochondrial disorders. We recommend analyses of electron transport chain enzyme complexes (test number ####), which is available from our laboratory. If a more extensive analysis is requested, a clinical summary which includes indications of mitochondrial disorder must be provided. Defects in nuclear genes responsible for mitochondrial disorders should also be considered.

Electron Transport Chain Activities - Muscle

Interpretation

Method

References

Muscle Biopsy #2

The following is the report on a left thigh skeletal muscle biopsy performed on a 2-week-old boy who was subsequently diagnosed with genetically confirmed PWS due to maternal uniparental disomy (UPD). The report reveals impairment in mitochondrial respiratory chain transport activity, as well as abnormal muscle morphology (structure) and lipid storage abnormalities.

Surgical Pathology Report

Final Diagnosis:

Muscle, left thigh, biopsy: mild changes consistent with metabolic myopathy including:

• moderate variation in fiber size and shape
• moderate increase in lipid droplets with Sudan Black [dye]

Comment: The histochemical features of this muscle are consistent with those of metabolic myopathy. Inadvertently, no tissue was submitted for ultrastructural analysis [electron microscopy]. Respiratory chain enzyme studies are pending.

[signatures]

Surgical Pathology Report

Specimen:

Clinical history: hypotonia.

Gross description:

Microscopic examination:

Electron Transport Chain Activities - Muscle

After adjusting for low CS (citrate synthase) activity, no significant abnormalities were observed.

[PWSDots comment: Although a decrease in a single respiratory chain enzyme to 5-10% of normal is often necessary for clinical symptoms to become apparent in single gene disorders, it is difficult to understand the judgment that "no significant abnormalities were observed" in mitochondrial function in this baby with PWS, given his severe hypotonia and that citrate synthase activity was only half of normal, while COX activity was only 28% of normal and still only 57% of normal even after normalization for low citrate synthase activity.]

Background references

• eMedicine - Muscle biopsy

Reports of mitochondrial impairment and abnormal muscle morphology in PWS:

Arch Dis Child. 2003 Apr.
Dual pathology in two hypotonic children with Prader-Willi syndrome and muscle mitochondrial Complex I deficiency.
E. Wassmer, B.H. Robinson, I. Tein.
Dept. of Pediatrics, The Hospital for Sick Children, Toronto, Canada.

Aim & Method: To describe the features of two children with Prader-Willi Syndrome (PWS) and Complex-1 deficiency. Results: Two children presented as neonates with hypotonia, feeding difficulties and growth retardation. Development was subsequently delayed. CPK [creatine kinase], thyroid function, VLCFAs [very long-chain fatty acids], ammonia and organic acids were normal. Serum lactate was normal in Case 1 and mildly elevated in Case 2. ABRs revealed delayed conductions of waves I-III. VEPs, EMG, NCS were normal. Neuroimaging showed mild cerebral volume loss. Muscle biopsy revealed a normal checkerboard pattern of Type I and II fibres and 1 mottled fibre (Gomori-trichrome staining) in Case I. Type II fibres predominated in Case 2. Mitochondrial stains for NADH, LDH [lactate dehydrogenase], SDH [succinate dehydrogenase] and COX [cytochrome C oxidase] were normal. E/M [electron microscopy] revealed normal mitochondria and no lipid or glycogen accumulation. Mitochondrial enzyme analysis demonstrated unmeasurable NADH cytochrome-C-reductase activity [complex I + III] in Case 1 and decreased activity of 11.7 nmol/min/mg mitochondrial protein (controls 94.6± 9.5) in Case 2. Succinate cytochrome-C-reductase [complex II + III] activity was reduced in Case 2 at 47 (controls 102 ± 6.9) but normal in Case 1. COX activities were normal. In skin fibroblasts the lactate/pyruvate ratio was slightly elevated. The Prader-Willi phenotype became evident after 1 year of age. No deletion was detected in region 15q11-q13 with G-banding or FISH, however the methylation pattern was abnormal. Parental samples confirmed maternal uniparental heterodisomy of PWS. Conclusion: The occurence of Complex-1 deficiency in PWS is likely a secondary rather than a primary event, but may contribute to the PWS clinical phenotype in certain cases.

Paediatr Anaesth. 2001 Jul.
Anaesthetic considerations for a child with combined Prader-Willi syndrome and mitochondrial myopathy.
Sharma AD, Erb T, Schulman SR, Sreeram G, Slaughter TF.
Department of Anaesthesiology, Duke University Medical Center, Durham, NC, USA.

We report the anaesthetic management of a child with Prader-Willi syndrome and mitochondrial myopathy for open heart surgery. We used ketamine, fentanyl, rocuronium and caudal morphine together with a propofol infusion with no untoward effects. The implications of both conditions for anaesthesia are discussed.

[According to Butler et al., Management of Prader-Willi Syndrome (2006), the child in this case report was a two-year-old with a combined complex I and IV respiratory chain deficiency diagnosed by muscle biopsy. The open heart surgery was to repair an atrial septal defect.]

Brain Dev. 1994 May-Jun.
Muscle histochemistry in the Prader-Willi syndrome.
Sone S.
Department of Ultrastructural Research, National Institute of Neuroscience, National Center of Neurology and Psychiatry, Tokyo, Japan.

In a follow-up study of 259 floppy infants of undetermined cause in my laboratory, 11 patients were later diagnosed as having the Prader-Willi syndrome (PWS). To clarify the pathogenesis of muscle hypotonia in PWS, I examined muscle biopsies by histochemical and morphometric methods and the results were compared with those obtained from floppy infants with only mental retardation but with no other features. The histochemical abnormalities of PWS included (i) fiber size variation of both type 1 and 2 fibers, (ii) type 2 fiber atrophy, (iii) increased numbers of type 2C fibers, and (iv) decreased numbers of type 2B fiber. Although muscle hypotonia in PWS has been thought to be due to central nervous system abnormality, my findings suggest that primary muscle pathology, including muscle fiber immaturity and abnormal muscle fiber type distribution, at least in part, plays a role in muscle hypotonia and weakness.

Brain Dev. 1992 Jan.
A neuropathological study of a case of the Prader-Willi syndrome with an interstitial deletion of the proximal long arm of chromosome 15.
Hayashi M, Itoh M, Kabasawa Y, Hayashi H, Satoh J, Morimatsu Y.
Department of Pediatrics, Tokyo Medical and Dental University, Japan.

An autopsy case, a 6-month-old girl, with an interstitial deletion of the long arm of chromosome 15;del(15)(q11.1q12) was reported. Muscle hypotonia, poor sucking and intermittent ocular deviation were noticed soon after birth. She also exhibited external features peculiar to the Prader-Willi syndrome (PWS). The muscle hypotonia persisted and head control was not achieved. She died at the age of 6 months due to bronchopneumonia. G-banding analysis of prometaphase chromosomes revealed a deletion of chromosome 15. Bronchopneumonia of the lungs and fatty metamorphosis of the liver were found. Neuropathological anomalies recognized were: disturbed undulating structures, resembling cortical micropolygyria and pachygyria, in the dentate nucleus and the inferior olivary nucleus, grumose degeneration of the nerve cells in the dentate nucleus, and heterotopia of middle-sized neurons in the cerebellar white matter. No abnormalities were observed in the hypothalamus-pituitary system. In some autopsy cases of PWS, cerebellar lesions have been reported. These might be related to the muscle hypotonia in PWS.

Brain Dev. 1988.
Atypical Prader-Willi syndrome with severe developmental delay and emaciation.
Miike T, Ogata T, Ohtani Y, Yamaguchi H, Yokoyama Y.
Department of Child Development, Kumamoto University Medical School, Japan.

A young boy showed features of Prader-Willi syndrome including hypotonia, cryptorchidism, a mildly dysmorphic facial appearance, a high-arched palate and an open triangular-shaped mouth, but had additional symptoms such as simian creases and multiple joint ankylosis in early infancy. Deletion of the long arm of chromosome 15(q11.2 to q13) was recognized. A muscle biopsy specimen showed variation in fiber size with hypertrophic type 1 fibers, type 2 fiber smallness, type 2B fiber paucity and an increased number of type 2C fibers. At the age of 4 1/2 years, he still showed severe developmental delay with severe muscle hypotonia, weakness and emaciation.

Arch Neurol. 1984 Jan.
Patterns of muscle fiber-type disproportion in hypotonic infants.
Argov Z, Gardner-Medwin D, Johnson MA, Mastaglia FL.

We studied the histochemical characteristics of muscle in five hypotonic infants. A number of different patterns of disproportion in the sizes and numbers of type 1 and type 2 fibers were found. In three cases, type 1 fibers were smaller than type 2 fibers and type 2b or 2c fibers were largest. In one case, type 2 fibers were smaller than type 1 fibers and were reduced in number, while in a case of Prader-Willi syndrome there was a preponderance of type 2 fibers that were smaller than type 1 fibers. Type 2c fibers were increased in number in all but one case. We postulate that these various patterns of fiber-type disproportion are the result of altered neural influences leading to impaired maturation of type 1 or type 2 motor units.

Other abstracts of interest:

Adv Physiol Educ. 2006 Dec.
PGC-1alpha: a key regulator of energy metabolism.
Liang H, Ward WF.
Department of Cellular and Structural Biology, Audie Murphy Veterans Administration Medical Center and University of Texas Health Science Center, San Antonio, Texas, USA.
[ Free full text ]

Peroxisome proliferator-activated receptor-gamma coactivator (PGC)-1alpha is a member of a family of transcription coactivators that plays a central role in the regulation of cellular energy metabolism. It is strongly induced by cold exposure, linking this environmental stimulus to adaptive thermogenesis. PGC-1alpha stimulates mitochondrial biogenesis and promotes the remodeling of muscle tissue to a fiber-type composition that is metabolically more oxidative and less glycolytic in nature, and it participates in the regulation of both carbohydrate and lipid metabolism. It is highly likely that PGC-1alpha is intimately involved in disorders such as obesity, diabetes, and cardiomyopathy. In particular, its regulatory function in lipid metabolism makes it an inviting target for pharmacological intervention in the treatment of obesity and Type 2 diabetes.

From the full text article:

Skeletal muscle fibers are classified into three types: type I, type IIa, and type IIb. Slow-twitch type I and fast-twitch type IIa fibers contain more mitochondria and exhibit relatively higher rates of oxidative metabolism. In contrast, type IIb fibers have fewer mitochondria and are metabolically glycolytic. It is now well established that PGC-1 induces a remodeling of skeletal muscle fiber composition. In general, the ratio of glycolytic type IIb fibers to the more oxidative type I and type IIa fibers decreases. The expression of PGC-1 in skeletal muscle is readily inducible by both short-term exercise and endurance training in rodent models and human subjects (5, 15, 41, 52). Our understanding of the biological role of PGC-1 in skeletal muscle structure and function has been greatly improved through the use of gain of function and loss of function mouse models. In a gain of function transgenic model, PGC-1 is overexpressed in a skeletal muscle-specific manner under the control of the muscle creatine kinase (MCK) promoter (28). PGC-1 overexpression results in the conversion of fast-twitch type IIb muscle fibers to type IIa and slow-twitch type I fibers by 20% and 10%, respectively, in plantaris muscle. In addition, there is an activation of genes involved in mitochondrial oxidative metabolism. The conversion to slow-twitch fibers is also evidenced by the redder muscle color and the expression of contractile proteins characteristic of slow-twitch fibers such as slow troponin I and myoglobin. As would be predicted, based on these alterations, muscles isolated from the MCK-PGC-1 transgenic mouse show increased resistance to electrically stimulated fatigue (28). Consistent with this, Mortenson et al. (39) recently reported that overexpression of PGC-1 in primary rat skeletal muscle cells leads to enhanced levels of mRNA for the slow oxidative-associated myosin heavy chain (MHC) isoform (MHCIb) and decreased mRNA levels for the fast glycolytic-associated MHC isoforms (MHCIIX and MHCIIB) (39). In contrast, PGC-1-deficient mice exhibit decreased mitochondrial number and decreased respiratory capacity in slow-twitch muscle as well as reduced exercise capacity and a reduced fatigue resistance index (26).

Clin Genet. 2005 Oct.
Relationship of primary mitochondrial respiratory chain dysfunction to fiber type abnormalities in skeletal muscle.
Enns GM, Hoppel CL, DeArmond SJ, Schelley S, Bass N, Weisiger K, Horoupian D, Packman S.
Department of Pediatrics, Stanford University, Stanford, CA, USA.

Variation in the size and relative proportion of type 1 and type 2 muscle fibers can occur in a number of conditions, including structural myopathies, neuropathies, and various syndromes. In most cases, the pathogenesis of such fiber type changes is unknown and the etiology is heterogeneous. Skeletal muscle mitochondrial respiratory chain analysis was performed in 10 children aged 3 weeks to 5 years with abnormalities in muscle fiber type, size, and proportion. Five children were classified as having definite, four as probable, and one as possible mitochondrial disease. Type 1 fiber predominance was the most common histological finding (six of 10). On light microscopy, four cases had subtle concomitants of a mitochondriopathy, including mildly increased glycogen, lipid, and/or succinate dehydrogenase staining, and one case had more prominent evidence of underlying mitochondrial disease with marked subsarcolemmal staining. Most cases (nine of 10) had abnormal mitochondrial morphology on electron microscopy. All were found to have mitochondrial electron transport chain (ETC) abnormalities and met diagnostic criteria for mitochondrial disease. We did not ascertain any patients who had isolated fiber type abnormalities and normal respiratory chain analysis during the period of study. We conclude that mitochondrial ETC disorders may represent an etiology of at least a subset of muscle fiber type abnormalities. To establish an etiologic diagnosis and to determine the frequency of such changes in mitochondrial disease, we suggest analysis of ETC function in individuals with fiber type changes in skeletal muscle, even in the absence of light histological features suggestive of mitochondrial disorders.

Can J Neurol Sci. 2005 May.
Pathology of mitochondrial encephalomyopathies.
Sarnat HB, Marin-Garcia J.
University of Calgary Faculty of Medicine, Alberta, Canada.

Muscle biopsy provides the best tissue to confirm a mitochondrial cytopathy. Histochemical features often correlate with specific syndromes and facilitate the selection of biochemical and genetic studies. Ragged-red fibres nearly always indicate a combination defect of respiratory complexes I and IV. Increased punctate lipid within myofibers is a regular feature of Kearns-Sayre and PEO, but not of MELAS and MERRF. Total deficiency of succinate dehydrogenase indicates a severe defect in Complex II; total absence of cytochrome-c-oxidase activity in all myofibres correlates with a severe deficiency of Complex IV or of coenzyme-Q10. The selective loss of cytochrome-c-oxidase activity in scattered myofibers, particularly if accompanied by strong succinate dehydrogenase staining in these same fibres, is good evidence of mitochondrial cytopathy and often of a significant mtDNA mutation, though not specific for Complex IV disorders. Glycogen may be excessive in ragged-red zones. Ultrastructure provides morphological evidence of mitochondrial cytopathy, in axons and endothelial cells as well as myocytes. Abnormal axonal mitochondria may contribute to neurogenic atrophy of muscle, a secondary chronic feature. Quantitative determinations of respiratory chain enzyme complexes, with citrate synthase as an internal control, confirm the histochemical impressions or may be the only evidence of mitochondrial disease. Biological and technical artifacts may yield falsely low enzymatic activities. Genetic studies screen common point mutations in mtDNA. The brain exhibits characteristic histopathological alterations in mitochondrial diseases. Skin biopsy is useful for mitochondrial ultrastructure in smooth erector pili muscles and axons; skin fibroblasts may be grown in culture. Mitochondrial alterations occur in many nonmitochondrial diseases and also may be induced by drugs and toxins.

Am J Clin Pathol. 2001 Sep.
Diagnostic yield muscle biopsy in patients with clinical evidence of mitochondrial cytopathy.
Rollins S, Prayson RA, McMahon JT, Cohen BH.
Ohio State University College of Medicine and Public Health, Cleveland, USA.

We retrospectively reviewed 118 muscle biopsy specimens from 113 patients with clinical and/or biochemical evidence of mitochondrial cytopathy. Light microscopic evaluation revealed histologic abnormalities in 65 specimens. The most common histologic findings included angular atrophic esterase-positive muscle fibers, type II muscle atrophy, regenerating muscle fibers, and scattered cytochrome-oxidase deficient fibers. Ragged red fibers were noted in 3 specimens on a Gomori trichrome stain. Electron microscopic evaluation was performed in 113 muscle specimens, and in 34, no abnormalities were identified. Increased numbers of mitochondria, particularly in the subsarcolemmal region, were identified in 54 specimens. Increased mitochondrial size was seen in 8 specimens and paracrystalline mitochondrial inclusions in 3. Other ultrastructural findings included focally increased glycogen deposition, focal Z-band streaming, and focally increased lipid accumulation. For 39 cases, concomitant skin biopsy specimens were available; abnormalities were identified by electron microscopy in 12. The majority of biopsy specimens demonstrated some light or electron microscopic abnormality. Specific histologic findings suggestive of mitochondrial abnormalities (partial cytochrome oxidase deficiency, ragged red fibers) were noted in a minority of cases. Ultrastructural evidence of mitochondrial abnormalities was noted in the majority of cases.

Arch Pathol Lab Med. 2000 Dec.
Succinate dehydrogenase deficiency.
Vladutiu GD, Heffner RR.
Department of Pediatrics, School of Medicine and Biomedical Sciences, State University of New York at Buffalo, NY, USA.

Background: Partial succinate dehydrogenase deficiency (15% to 50% of normal reference enzyme activity) in skeletal muscle causes mitochondrial myopathy with various symptoms, for example, brain involvement, cardiomyopathy, and/or exercise intolerance. The deficiency may be isolated or may coexist with other respiratory-chain enzyme defects. The histopathologic assessment of succinate dehydrogenase activity in muscle biopsies of patients with suspected mitochondrial myopathies has focused on the finding of increased staining, usually in ragged-red fibers, rather than on reduced staining. Objectives: To determine the prevalence of muscle succinate dehydrogenase deficiency among patients with respiratory-chain defects and to determine whether the reduced activity is present histochemically and is comparable to the quantitative reduction found in muscle homogenates. Patients and methods: One hundred eight muscle biopsies were evaluated from patients with suspected mitochondrial myopathies by qualitative histochemical analysis and quantitative biochemical analyses of respiratory-chain enzymes using standard methodologies. Results: Fifty-two patients had defects in respiratory-chain complexes; of these patients, 12 (23%) had partial deficiencies in succinate dehydrogenase activity either alone or together with reductions in other enzymes. The reduced activity was detectable histochemically in muscle biopsies with residual enzyme activity of up to 34% of the normal reference activity, while 2 biopsies with higher residual activity (49% and 68% of normal) could not be distinguished from normal biopsies. Conclusions: Of the patients with respiratory-chain enzyme defects, 23% had partial deficiencies of succinate dehydrogenase activity in muscle biopsies. This reduction could be detected histochemically in biopsies in most cases. The marked prevalence of succinate dehydrogenase deficiency among patients with respiratory-chain defects and its detection initially by histochemical analysis are important findings.

Br J Ophthalmol. 1996 Jan.
Compensatory elevation of complex II activity in Leber's hereditary optic neuropathy.
Yen MY, Lee HC, Liu JH, Wei YH.
Department of Ophthalmology, Taipei Veterans General Hospital, Taiwan.

AIMS: To evaluate the mitochondrial respiratory enzyme activities in blood cells of Leber's hereditary optic neuropathy (LHON) with 11778 point mutation of mitochondrial DNA. METHODS: Assays for the activities of NADH-cytochrome c reductase (complex I+complex III), succinate-cytochrome c reductase (complex II+complex III), and cytochrome c oxidase (complex IV) on blood cell mitochondria of seven LHON patients and 15 normal controls. RESULTS: There was no statistically significant difference in NADH-cytochrome c reductase and cytochrome c oxidase activities between LHON patients and controls, but activities of succinate-cytochrome c reductase in LHON patients was significantly elevated compared with normal controls. CONCLUSION: The observations that the activity of NADH-cytochrome c reductase is normal but that of succinate-cytochrome c reductase is increased in LHON patients with 11778 point mutation of mitochondrial DNA indicate an elevation of complex II activity, which may be due to a nuclear compensatory effect for defects of the respiratory function of mitochondria.