Clinical features, lectin staining, and a novel GNE frameshift mutation in HIBM

Abstract

We present a comprehensive report of two siblings of Indian descent with hereditary inclusion body myopathy (HIBM). The clinical features and histological characteristics of the two muscle biopsies show the typical pattern of predominantly distal vacuolar myopathy with quadriceps sparing. This was confirmed by muscle MRI. PNA lectin staining shows a reduced presence of sialic acids at the sarcolemma. Mutation analysis revealed compound heterozygous mutations in the GNE gene (encoding UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase): a missense mutation (c.2086G>A; p.V696M) previously described in HIBM patients of Indian origin, and a novel frameshift mutation (c.1295delA; p.K432RfsX16 ) leading to a premature stopcodon. These findings confirmed the diagnosis HIBM biochemically and genetically.

Introduction

Hereditary inclusion body myopathy (HIMB) or Nonaka distal myopathy is a rare autosomal recessive disorder characterized by distal and proximal weakness with preferential involvement of the anterior tibialis muscle and relative sparing of the quadriceps, facial, respiratory, and cardiac muscles. Disease symptoms generally appear in young adulthood and are gradually progressive leading to wheelchair use overall 12 years after onset of symptoms.[1,2] Muscle biopsy is characterized by the presence of rimmed vacuoles, especially in atrophic fibres. These vacuoles occasionally contain congophilic amyloid material and deposits that are immunoreactive to β-amyloid, ubiquitin, and tau protein. The nucleus may contain tubulofilamentous inclusions.[1] Necrotic and regenerating fibres are rare.[1]

In 2001 mutations in GNEthe gene encoding UDP-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase were identified as cause of autosomal recessive HIBM.[3] Subsequently, distal myopathy with rimmed vacuoles was found to be allelic to HIBM.[2] The most common mutation in HIBM patients was the M712T homozygous missense mutation found in all Middle Eastern families of both Jewish and non-Jewish descent, suggesting a founder effect.[3]

GNE is a bifunctional cytoplasmic enzyme and catalyzes the first two steps in the synthesis of sialic acid from UDP-N-acetylglucosamine. Sialic acids occur on the nonreducing termini of glycans on various sarcolemmal and extracellular matrix glycoproteins, such as the sarcoglycans and dystroglycan. As such, they play an important role in cell-cell and cell-matrix interactions. Reduced GNE activity impairs sialic acid production and thus leads to defective sialylation of a specific subset of proteins [4], mainly affecting O-glycan sialylation.[5,6] This may contribute to disturbed interactions with extracellular matrix proteins and subsequently results in muscle weakness.[7] However, the exact role of hyposialylation in formation of rimmed vacuoles, tubulofilamentous inclusions, and other disease pathology is still unclear.[8,9]

The diagnosis of autosomal recessive HIBM is so far based upon the pattern of predominantly distal muscle weakness with quadriceps sparing, the presence of rimmed vacuoles in muscle biopsy, and detection of GNE mutations. In addition, GNE deficiency can be detected biochemically by aberrant immunohistochemical staining of muscle biopsies with Peanut agglutinin (PNA), a lectin specific for desialylated mucin core 1 O-glycans.[6] We here report the clinical, histological, biochemical, and genetic features of a family of Indian descent with HIBM.

Clinical studies

Patient 1

A 27-year old male patient reported muscle weakness in his lower legs, and reduced ability to walk on toes and heels since two years. As a consequence, he had difficulties running and walking stairs. Muscle force in arms and hands, swallowing, speech, and sensation were normal. At that time, no one in his family had similar complaints. His parents were from Indian origin and not consanguineous. His medical history was unremarkable, and he used no medication. Six years later (age 33), muscle weakness had progressed to include the hip girdle muscles and proximal arm muscles. Walking was possible only for short distances with an orthesis; however, he could ride a bicycle for long distances (50 km). Physical examination at the age of 27 revealed muscle weakness in his upper arms (MRC 4+) and lower legs (MRC 4−), with atrophy of the lower legs. Six year follow-up revealed strong progression of muscle weakness and atrophy in his lower legs (MRC 2)(Figure 1). Iliopsoas and hamstring muscles were also very weak (MRC 1), while the quadriceps muscle had normal strength. Deep tendon reflexes in legs were low, and sensation was normal. Creatine kinase was elevated (1438 U/l) and nerve conduction studies were normal. Electromyography at the age of 27 revealed a myopathic pattern in the anterior tibial, gastrocnemius, biceps brachi, and trapezius muscles, but not in the quadriceps muscle. Needle biopsy of the right quadriceps muscle (age 27) revealed mild increase of fibre diameter variation without the presence of rimmed vacuoles (Figure 3). Muscle MRI (age 33) revealed symmetrical fatty infiltration and atrophy of muscles of hip girdle, lower trunk, upper and lower legs, with still relative sparing of the quadriceps muscle (Figure 2).

Figure 1.

Muscle atrophy in lower legs (anterior > distal; asymmetrical right > left in patient 2) of both HIBM patients, with relative sparing of quadriceps femoris muscle.

Figure 3.

Left column: Quadriceps muscle needle biopsy of control patient.

Quadriceps muscle needle biopsy specimen of patient 1, revealing mild myopathic features: increased fibre diameter variance and mild increase of internal nuclei.

Anterior tibial muscle needle biopsy specimen of patient 2, revealing a myopathic pattern, with dystrophic features and rimmed vacuoles (two of which are indicated by an arrow).

Right column: PNA lectin binding on the sarcolemma of muscle sections for patient 1 and patient 2, using FITC-labeled PNA in comparison to a control section (above). Both patients show increased PNA binding along the sarcolemma. Treatment of control section with neuraminidase resulted in similar PNA binding along the sarcolemma.

Figure 2.

Above: Patient 1: Muscle MRI of trunk, upper, and lower leg revealed symmetrical increase of fat of trunk, limb girdle, upper and lower leg muscles. Relative sparing of quadriceps femoris muscle.

Patient 2

A 31-year old woman, the younger sister of patient 1, reported muscle weakness and atrophy in her lower legs and to a lesser extent in her arms since three years. She had difficulties walking stairs predominantly due to foot drop. Swallowing, speech, and sensation were normal. She suffered from psoriasis, for which she used topical agents, but no systemic treatment. Physical examination revealed generalized muscle weakness (MRC 4), with more pronounced weakness in the anterior tibial muscle (MRC 3) than in gastrocnemius muscle (MRC 4+). Muscle atrophy was most pronounced on her right lower leg (Figure 1). Deep tendon reflexes in legs were low, and sensation was normal. Creatine kinase was elevated (705 U/l). Nerve conduction studies were normal. Electromyography revealed a myopathic pattern in the distal leg muscles, which was more pronounced in the anterior tibial than in the gastrocnemius muscle. Muscle MRI revealed asymmetrical fatty infiltration and atrophy of anterior lower leg muscles with normal signal intensity of the quadriceps muscle. Needle biopsy of the right anterior tibial muscle revealed a severe myopathy with signs of dystrophy and presence of rimmed vacuoles (Figure 3).

Serum N- and O-glycosylation by transferrin and apolipoprotein C-III isofocusing

Serum N- and O-glycosylation was tested by transferrin and apolipoprotein C-III (apoCIII) isofocusing as previously described.[10] Whereas the N-glycosylation of serum transferrin was normal, the apoCIII assay showed reduced sialylation in patient 1 and borderline results for patient 2 (Table 1).

Table 1.

Results of apoC-III isofocusing

apoC-III isofocusing* (in %)	Patient 1		Patient 2		Reference
	sample 1	sample 2	sample 1	sample 2	(> 18 yrs)
ApoCIII-0	3.6	6.1	10.7	7.1	2.6–18.9
ApoCIII-1	81.6	79.9	71.3	66.5	42.9–69.2
ApoCIII-2	14.8	13.9	18.0	26.4	23.2–50

^*ApoCIII-0/1/2 indicate the apolipoproteinC-III isoforms with 0, 1 or 2 neuraminic acid residues ApoCIIIisofocusing in two independent serum samples of patient 1 showed reduced sialylation as seen by an increase of monosialylated apoCIII-1 and a decrease of disialylated apoCIII-2. Borderline results were found for patient 2.

PNA Lectin Staining

Deficient muscle protein glycosylation was investigated by staining of muscle biopsies with Peanut agglutinin (PNA), a lectin with specificity for the desialylated core 1 mucin type Oglycan (Galβ1-3GalNAcα1-Ser/Thr)(EY Laboratories Inc. (2007) Lectin and lectin conjugates– Catalog Addendum). Muscle biopsies were obtained from the right quadriceps of patient 1and from the right tibial anterior muscle of patient 2. We used the muscle of a patient with aspecific muscular complaints without histological abnormalities as control tissue. Optimization of FITC-PNA staining was performed by treatment of control biopsies with neuraminidase, which removes terminal neuraminic acid and thereby exposes the PNA binding epitope. Cryostatic sections of the muscle biopsies (10 μm) were fixed in 100% icecoldacetone for ten minutes and pre-incubated with PBS containing 0.1% BSA at room temperature. After washing with PBS, the sections were incubated in the dark at room temperature with 100 μg/ml FITC-labeled PNA (EY Laboratories, Inc. San Mateo, CA, USA) in sodium phosphate buffer for 30 minutes. The sections were then washed in PBS and mounted in Fluortec (Eurodiagnostica, Arnhem, The Netherlands). Fluorescence microscopy was performed on a Leica fluorescence microscope, with emission at 517 nm and absorption measurement at 492 nm. Pictures were recorded using CytoFish Leica cw4000 software. In each series of staining, a control muscle biopsy, treated with or without neuraminidase, was included for comparison. Staining of both quadriceps muscle (patient 1) and right tibialanterior muscle (patient 2) clearly showed positive fluorescence of the sarcolemma, indicating reduced sialylation of mucin core 1 O-glycans on sarcolemmal glycoproteins (Figure 3). Peroxidase-labeled wheat germ agglutinin lectin staining for overall N-glycan sialylation was normal, which reflects that reduced sialylation is limited to O-glycosylation (data not shown).

Mutation Analysis

Mutation analysis of the GNE gene on chromosome 9p13.3 revealed two heterozygous mutations in both patients.[7] A previously described missense mutation in exon 12 was identified (c.2086G>A, p.V696M) [11], likely from Indian descent. In addition, a single nucleotide deletion in exon 8 (c.1295delA, p.K432RfsX16) was found, leading to a frameshift and early termination by introduction of a stopcodon.

Discussion

We reported a family of Indian descent in whom hereditary HIBM was diagnosed and compound heterozygous GNE gene mutations were identified, one of which was a novel frameshift mutation. This is the first report of HIBM in the Netherlands. Relative sparing of the quadriceps muscle until late stages of the disease is a characteristic finding in HIBM patients, and HIBM is therefore referred to as Quadriceps Sparing Myopathy. The reason why quadriceps sparing occurs is unknown. We confirmed this pattern, both clinically, histologically, and on MRI in both patients. In fact, rimmed vacuoles could only be found in the anterior tibial muscle in patient 2 and not in the quadriceps muscle biopsy of patient 1. Therefore, muscle biopsy should, as always, preferably be performed in a moderately affected muscle. Nevertheless, PNA lectinstaining clearly showed reduced sialylation in both quadriceps and anterior tibial muscles, indicating that reduced sialylation is present in early stages of disease before the formation of rimmed vacuoles.

Mutation analysis by exon sequencing revealed compound heterozygous mutations in the GNE gene, one of which had previously been described in HIBM patients from Indian descent.[11] The deletion in exon 8 results in a shift of the nucleotide reading frame leading to a premature stopcodon and truncation of the enzyme. The nature of this mutation can be considered pathogenic. Frameshift mutations are rare in HIBM.[2,12]

Absence of systemic or central nervous system features implies that the biochemical consequences of GNE mutations are limited to muscle, whereas abnormal PNA lectin staining suggests restriction to O-glycosylation. In contrast, a complete loss of sialic acids may not be compatible with life [13], or would be expected to result in reduced sialylation of both N- and O-glycans likely resulting in a multisystem disorder. Normal transferrin isofocusing with abnormal apoCIII isofocusing in patient 1 points to reduced mucin type O-glycan sialylation, whereas in previous reports normal profiles have been reported.[7,10] This may partly be explained by the higher sensitivity of the apoCIII protein compared to the transferrin protein towards available levels of CMP-neuraminic acid[14], or alternatively might be related to the stronger disease progression in patient 1.

Since PNA staining indicates reduced sialylation, it could potentially detect other defects in sialic acid biosynthesis leading to a similar clinical phenotype. Nevertheless, in three patients with distal myopathy and rimmed vacuoles without GNE mutations in our centre, normal PNA lectin staining was found (data not shown). As such, FITC-PNA staining may contribute to the early diagnosis of HIBM patients due to GNE mutations.

Early recognition and diagnosis is increasingly important, since experimental treatment strategies for HIBM are being discovered. A recent study found mild benefits in muscle strength experienced by HIBM patients after intravenous immunoglobulin treatment, which may be related to the provision of sialic acid supplied by immunoglobulins.[15] Another option could be the introduction of N-acetylmannosamine [16], the product of the epimerase activity of GNE and a precursor of sialic acid biosynthesis. Even patients with mutations in the kinase domain (the second enzymatic activity of GNE) could likely benefit from such an approach, since HIBM patients with kinase mutations have high residual kinase activities.[17] This may be due to the existence of ancillary kinases such as GlcNAc kinase that can convert ManNAc into ManNAc 6-phosphate for subsequent synthesis of sialicacid.[18]

In short, we have reported the clinical, histological, biochemical, and genetic features of two autosomal recessive HIBM patients of Indian descent. These include a new frameshift mutation in the GNE gene and the biochemical detection of reduced sialic acid by aberrant immunohistochemical staining with PNA, a technique which may contribute to the diagnosis of HIBM.