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Identification of a locus for an autosomal recessive hyaline body myopathy at chromosome 3p22.2–p21.32
Neuromuscular Disorders 14 (2004) 4–9 www.elsevier.com/locate/nmd
Identification of a locus for an autosomal recessive hyaline body myopathy at chromosome 3p22.2 –p21.32 ¨ nengu¨ta,1, Sibel Aylin Ugˇura, Hatice Karasoyb, Nur Yu¨ceyarb, Aslıhan Toluna,* Suna O a
Department of Molecular Biology and Genetics, Bogˇazic¸i University, Bebek 34342 Istanbul, Turkey b Department of Neurology Ege University Medical School Hospital, Izmir, Turkey Received 3 January 2003; received in revised form 16 June 2003; accepted 10 July 2003
Abstract Hyaline body myopathy is a rare congenital disease with distinctive histopathological features. We performed homozygosity mapping in a family with two affected sibs and identified a gene locus with a maximum homozygosity region of 5.35 centi Morgans or 5.59 Megabases at chromosome 3p22.2 – p21.32. The best candidate responsible for the disease is a novel gene that exhibits homology to the myosin heavy chain. q 2003 Elsevier B.V. All rights reserved. Keywords: Hyaline body myopathy; Autosomal recessive; Gene localization; Turkish
1. Introduction Hyaline body myopathy (HBM) is a rare congenital myopathy that manifests with subsarcolemmal hyalinized bodies in type 1 fibers. It was first described in 1971 as familial myopathy with probable lysis of myofibrils in type 1 fibers [1] and later designated ‘myopathy with lysis of type 1 myofibrils’ (MIM 255160). Engel and Baker used the term hyaline bodies because of the appearance in trichromestained sections [2]. HBM has been reported both as sporadic [3 –5] and familial cases, and the familial forms were autosomal dominant [6] or autosomal recessive [1]. The clinical findings were diverse, with either scapuloperoneal weakness [5,6] or limb girdle weakness [1,3,4]. The hyalinized bodies in muscle biopsy samples have been studied extensively [2 – 4,6]. Ultra structurally, they appeared to consist of fine filamentous or granular material and not to have a surrounding membrane. The neighboring myofibrils were normal, with no sign of degeneration. The amorphous bodies contained granules and disorganized filaments and stained intensely with the myofibrillar * Corresponding author. Tel.: þ90-212-358-1540 ext. 1472/90-212-2572095/90-533-433-0377; fax: þ 90-212-265-9778. E-mail address: [email protected] (A. Tolun). 1 Present address: Molecular Genetics Program, Benaroya Research Center, Seattle, WA, USA. 0960-8966/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0960-8966(03)00163-9
ATPase reaction after acidic pre-incubation, but not with stains for oxidative enzymes and polysaccharides. They also reacted with monoclonal antibodies against anti-skeletal heavy myosin. Therefore, they were suggested to be degradation products of myosin [2]. The age of onset in the patients reported varied from early childhood to the fifth decade of life. Intrafamilial variation in the severity of the clinical course has been most obvious in one of the autosomal dominant families, in which the disease onset was in the first decade in one member and in the fifth in another [6]. We report here the results of the genetic analysis as well as the detailed clinical findings in two brothers. We mapped the gene responsible for the disease to 3p23– p21. The best candidate responsible for the disorder is a novel gene with homology to myosin heavy chain.
2. Patients and methods 2.1. Patients The proband (Patient 1) was a 44-year-old male with normal motor and mental development. He had always been thin and not able to run since childhood. He noted difficulty in walking in his late teens. He was discharged early from the military obligation due to his disability. He noticed difficulty
¨ nengu¨t et al. / Neuromuscular Disorders 14 (2004) 4–9 S. O
in lifting objects above his head at age 28 years. The clinical symptoms progressed very slowly in the years that followed. He was examined at the neuromuscular outpatient clinic at age 39 years. Physical examination was unremarkable except for the cachectic appearance, long face, and high-arched palate. Scapular winging and a steppage gait were noticed in the neurological examination. Manual muscle testing revealed weakness of grade 4 (Medical Research Council (MRC) scale) in the neck flexor and shoulder adductors, grade 3 in the shoulder abductors and elbow flexors, and grade 2 in the ankle dorsiflexors. Tendon reflexes were absent except in triceps. Plantar reflexes were flexor. Sensation was normal. Creatine kinase (CK) levels was about two-fold higher than normal. Other laboratory findings were within normal ranges. EMG displayed myopathic features. Electrocardiogram was unremarkable. The neurological examination 2 years later revealed a moderate progression of the weakness in his legs with 0 muscle strength in the ankle dorsiflexors. Echocardiogram showed global systolic dysfunction with an ejection fraction of 30% and left ventricular hypokinesis. Because of the clinical findings for cardiac failure, he was put on medical treatment. He was still ambulant 3 years after the second examination, and no further change was observed in his muscle weakness or cardiologic findings. Patient 2 was the 40-year-old brother of patient 1. He was asymptomatic until the age of 33 years, when he noticed difficulty in raising his arms. His physical examination was unremarkable at age 35 years, but moderate weakness and mild atrophy in shoulder girdle and peroneal muscles were found upon neurological examination. Abnormal manual muscle test results were as follows: grade 2 4 in shoulder abductors and grade 4 in shoulder adductors, elbow flexors, and ankle dorsiflexors. Tendon reflexes and sensation were normal. CK level was three-fold higher than normal. Electromyography (EMG) displayed myopathic changes. His strength had slightly worsened 5 years later, and shoulder abductors showed weakness of grade 3. His ecocardiographic and electrocardiographic examinations were both normal. The parents were in their seventies and had normal strength, so were the two sisters at ages 41 and 36 years. Two siblings of each patient were examined at ages 8 –20 years and found normal. The parents denied consanguinity but reported having origins in neighboring villages. 2.2. Muscle biopsy analysis Muscle biopsy specimens were obtained from the left triceps muscle of patient 1 and the left deltoid muscle of patient 2. Specimens were frozen rapidly in isopentane cooled with liquid nitrogen, and 10 mm cryostat sections were processed for hematoxylin-eosin (H&E), modified Gomori trichrome, Periodic acid-Schiff, Oil-red-O, adenosine triphosphatase (preincubation at pH 4.3 and 9.4), NADH dehydrogenase, succinate dehydrogenase, and Congo red stain.
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2.3. Genome-wide scan Appropriate informed consent was obtained from the subjects for genetic studies. DNA was isolated from peripheral blood samples using standard techniques. The genome-wide scan was performed using the low-density (about 25 centi Morgan (cM) spacing) set Version 8a containing 156 autosomal microsatellite markers (Research Genetics, USA). Marker alleles were resolved on 8% denaturing polyacrylamide gels and visualized by staining with silver nitrate, as described previously [7]. 2.4. Statistical analyses Linkage analysis was performed under the assumption of autosomal recessive inheritance, full penetrance, a disease gene frequency of 1 in 100,000, and equal frequencies of marker alleles. In constructing the pedigree, the grandparents were assumed to be second cousins. We assessed this to be the closest possible relation to be assumed, since the parents were unaware of any consanguinity. Two-point lod scores were calculated using the MLINK program of the FASTLINK 4.1 package [8]. GENEHUNTER version 2.0 beta was used for multipoint parametric linkage analysis and construction of the haplotypes, allowing minimum number of recombination events [9].
3. Results 3.1. Muscle biopsy findings The muscle biopsy findings in the patients were very similar. There was marked variation in fiber size and fiber splittings. Marked type 1 predominance was evident. Approximately 15 – 20% of the fibers had central nuclei. The most striking finding was the presence of subsarcolemmal hyalinized structures. These hyaline structures appeared pale pink with the hematoxylin and eosin stain and pale green with the modified Gomori trichrome stain (Figs. 1A,B). They lacked reactivity for Periodic acid-Schiff stain (PAS) and NADH dehydrogenase and exhibited a dark staining rim at the margins (Figs. 1C,D). Most of the hyaline structures were negative for the ATPase reaction at pH 9.4 and moderately to intensely positive at pH 4.3 (Figs. 1E,F). Subsarcolemmal hyaline bodies were exclusively in type 1 fibers, and they were present in approximately 25 – 30% of the fibers. Staining for amyloid was negative with Congo red. 3.2. Genome-wide scan results The extreme rarity of the trait and the fact that parents originated from neighboring villages prompted us to assume a distant blood relation and apply homozygosity mapping. The genome-wide scan results using the DNA samples of the two patients revealed homozygosity for the same allele
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¨ nengu¨t et al. / Neuromuscular Disorders 14 (2004) 4–9 S. O
Fig. 1. Triceps and deltoid muscle biopsy specimens from patients 1 (A and B) and 2 (C, D, E, and F), respectively. (A) Modified Gomori-trichrome, 20 £ ; (B) H&E, 10 £ ; (C) PAS, 10 £ ; (D) NADH dehydrogenase, 10 £ ; (E) Myofibrillar ATPase at pH 9.4, 10 £ ; and (F) Myofibrillar ATPase at pH 4.3, 10 £ . (A) Trichrome stained section showed variation in fiber size, fiber splitting, central nuclei, some adipose tissue, and pale green subsarcolemmal hyaline bodies. (B) H&E stained sections showed similar findings and pale pink hyaline bodies. Hyaline bodies were devoid of glycogen (C) and oxidative enzyme activity (D). Some fibers showed increased activity at the margin of hyaline bodies. (E) In ATPase reaction after pre-incubation at pH 9.4, type I fibers were predominant and enzyme activity inside hyaline bodies absent. (F) In pre-incubation at pH 4.3, moderate or strong myofibrillar ATPase enzyme activity was seen in hyaline bodies.
for eight markers in the set, namely D2S1400, D3S1766, GATA118002 (chromosome 3), D5S820, D6S1277, D8S1106, D11S1393, and D11S4464. Upon genotyping the patients with markers that flank the above mentioned markers, a shared homozygosity only around marker
D3S1766. Genotype analysis in the family using markers around D3S1766 revealed a candidate gene locus telomeric to the marker. The affected individuals were homozygous for a common haplotype with seven markers at the region, consistent with the disease haplotype being identical by
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Table 1 Haplotypes, genetic positions, and two-point lod scores for 19 markers at 3p Locus
kba
cMb
F
D3S1619 D3S1768 D3S3623 D3S3572 D3S3527 D3S3522 D3S2407 D3S2373 D3S3559 D3S2304 D3S3597 D3S3624 D3S2354 D3S1358 D3S1478 D3S2409 D3S2449 D3S2321 D3S1227
33,963 34,471 37,257 38,842 39,159 40,602 41,208 42,254 42,508 42,634 43,783 44,433 45,369 45,401 46,303 49,272 49,803 52,954 53,864
60.98 61.52 61.52 63.12 63.12 65.26 67.94 – 67.94 67.94 68.47 68.47 – – – 70.61 – 70.61 –
4 1 3 2 2 2 2 1 3 2 3 1 2 3 2 1 2 2 2
a b c d
M 3 2 2 2 3 1 1 2 1 1 2 2 1 1 1 1 1 4 3
1 1 3 1 2 2 2 1 3 2 3 2 2 1 3 1 2 3 4
P1 2 1 1 2 1 1 1 1 2 1 1 3 3 2 1 2 3 1 1
4 1 3 2 2 2 2 1 3 2 3 1 2 3 2 1 2 2 2
P2 1 1 3 1 2 2 2 1 3 2 3 2 2 1 3 1 2 3 4
4 1 3 2 2 2 2 1 3 2 3 1 2 3 2 1 2 2 2
S1 1 1 3 1 2 2 2 1 3 2 3 2 2 1 3 1 2 3 4
4 1 3 2 2 2 2 1 3 2 3 1 2 3 2 1 2 2 2
S2 2 1 1 2 1 1 1 1 2 1 1 3 3 2 1 2 3 1 1
3 2 2 2 3 1 1 2 1 1 2 2 1 1 1 1 1 4 3
2 1 1 2 1 1 1 1 2 1 1 3 3 2 1 2 3 1 1
Zmaxc
uMLEd
0.35 0.94 1.55 0.20 1.55 1.54 1.54 0.94 1.55 1.54 1.55 0.40 1.55 0.40 0.38 1.24 1.55 0.39 0.39
0.11 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.12 0.00 0.12 0.12 0.00 0.00 0.12 0.12
GenBank sequence map. Marshfield genetic map. Maximum two-point lod score. Maximum likelihood estimate for recombination fraction (u).
descent (Table 1). The unaffected siblings were not homozygous for the haplotype. Flanking markers were used to refine the borders of the homozygosity region. The affected individuals were heterozygous for D3S3572 and D3S3624. Thus, D3S3572 defined the telomeric border and D3S3624 the centromeric border of the maximum homozygosity region of the 5.35 cM or 5.59 Megabase gene locus. The markers, their distances, and the haplotypes of the family members are given in Table 1. 3.3. Statistical findings Lod scores were calculated to assess the significance of the results. Two-point lod scores were around 1.55 at a recombination fraction of zero for most of the markers in the homozygosity region (Table 1). The multi-point lod score peaked in the interval D3S3572– D3S3624, with a maximum value of 3.12 (Fig. 2).
4. Discussion This study describes clinical, morphological, and genetic findings in two brothers with hyaline body myopathy. So far, few individuals have been described to be afflicted with this very rare disorder: six patients from unrelated families [2 – 5], seven patients from an autosomal dominant family [6], and four patients from
two autosomal recessive families including the family we describe here [1]. The clinical phenotypes of our patients consisted of scapuloperoneal weakness and atrophy, very similar to some of the patients reported [5,6], but not limb girdle weakness reported for others [1,3,4]. Intrafamilial variation in the severity of clinical course and the age of onset were observed in our patients, as had been reported for another family [6]. One of our patients (patient 1) additionally had cardiac involvement, a finding not reported previously in any HBM patient. EMG displayed systolic dysfunction at the age of 39 years in his first examination. Such a symptom was not present in his brother at the age of 40 years, consistent with his much milder clinical phenotype in general. Heart biopsy would be necessary to assess whether the heart condition is due to HBM. The hyaline inclusions in our patients were consistent in morphology and staining properties to those in other HBM patients [1 – 6]. These analyzes were sufficient for diagnosis, and EM investigation was not necessary, because there is no other disorder with similar manifestations with respect to hyaline bodies that would necessitate differential diagnosis. Morphological heterogeneity in the inclusion bodies was reported for other families. The hyaline inclusions were centrally located in a few muscle fibers and stained dark green to blue with modified Gomori-trichrome in two members of an autosomal dominant family with scapuloperoneal syndrome but otherwise resembling HBM [10]. Also, Selcen et al. [11] described in two siblings similar
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Fig. 2. The result of 20-point lod score analysis at 3p for HBM. D3S has been omitted from marker designations.
hyaline masses that stained deep red with H&E and dark blue with trichrome and contained intensely congophilic inclusions. The clinical and myopathologic features were also distinct from HBM. An interesting question is whether those families link to the locus identified in this study. We investigated the databases in order to assess the best candidate gene at the locus. An unknown gene (KIAA1042/OIP106; locus ID: 22906) [12] coding for a protein with homology to myosin heavy chain stood out as the best candidate, since the hyaline bodies had been found to react with antibodies against myosin heavy chain [3,4,6]. We have not attempted to screen the patients for mutations in the KIAA1042 gene, because neither the nucleotide sequence nor the exact localization of the region is yet completely determined (GenBank, Build 33). Nevertheless, we believe that the localization of the gene responsible for an autosomal recessive hyaline body myopathy will have benefits for families afflicted with the disorder, as they can be tested for linkage to the novel
locus. Reporting the gene locus would also aid other researchers engaged in a search for the gene(s) responsible for HBM. 5. Electronic database information Online Mendelian Inheritance in Man (OMIM), http:// www.ncbi.nlm.nih.gov/Omim/ (for Myopathy with Lysis of Type I Myofibrils [MIM 255160]). Center for Medical Genetics, http://research. marshfieldclinic.org/genetics/ (for genetic mapping information). National Center for Biotechnology Information, http:// www.ncbi.nlm.nih.gov/ (to access GenBank). Acknowledgements ¨ nengu¨t and Sibel Aylin Ugˇur were fellows of the Suna O Scientific and Technical Research Council of Turkey. This
¨ nengu¨t et al. / Neuromuscular Disorders 14 (2004) 4–9 S. O
work was supported by Turkish State Planning Organization and the Turkish Academy of Sciences.
References [1] Cancilla PA, Kalyanaraman K, Verity MA, Munsat T, Pearson CM. Familial myopathy with probable lysis in type 1 fibres. Neurology 1971;21:579– 85. [2] Engel AG, Banker BQ. Ultrastructural changes in diseased muscle. In: Engel AG, Armstrong CF, editors. Myology, 2nd ed. New York: McGraw-Hill; 1994. p. 889–1017. [3] Barohn RJ, Brumback RA, Mendell JR. Hyaline body myopathy. Neuromuscul Disord 1994;4:257–62. [4] Ceuterick C, Martin JJ, Martens C. Hyaline bodies in skeletal muscle of a patient with a mild chronic non-progressive myopathy. Clin Neuropathol 1993;12:79–83. [5] Sahgal V, Sahgal S. A new congenital myopathy: a morphological, cytochemical and histochemical study. Acta Neuropathol (Berl) 1977; 37:225–30.
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[6] Masuzugawa S, Kuzuhara S, Narita Y, Naito Y, Taniguchi A, Ibi T. Autosomal dominant hyaline body myopathy presenting as scapuloperoneal syndrome: clinical features and muscle pathology. Neurology 1997;48:253–7. ¨ nengu¨t S, Derman O, Kaya A, Tolun A. The novel [7] Kavaslar GN, O genetic disorder microhydranencephaly maps to chromosome 16p13.3–12.1. Am J Hum Genet 2000;66:1705–9. [8] Ott J. Analysis of human genetic linkage. Baltimore: John Hopkins University Press; 1991. [9] Kruglyak L, Daly MJ, Reeve-Daly MP, Lander ES. Parametric and non-parametric linkage analysis: a unified multipoint approach. Am J Hum Genet 1996;58:1357–63. [10] Wilhelmsen KC, Blake DM, Lynch T, et al. Chromosome 12-linked autosomal dominant scapuloperoneal dystrophy. Ann Neurol 1996; 39:507–20. [11] Selcen D, Krueger BR, Engel AG. Familial cardioneuromyopathy with hyaline masses and nemaline rods: a novel phenotype. Ann Neurol 2002;51:224–34. [12] Kikuno R, Nagase T, Ishikawa K, et al. Prediction of the coding sequences of unidentified human genes. XIV. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA Res 1999;30:197 –205.
Identification of a locus for an autosomal recessive hyaline body myopathy at chromosome 3p22.2 –p21.32 ¨ nengu¨ta,1, Sibel Aylin Ugˇura, Hatice Karasoyb, Nur Yu¨ceyarb, Aslıhan Toluna,* Suna O a
Department of Molecular Biology and Genetics, Bogˇazic¸i University, Bebek 34342 Istanbul, Turkey b Department of Neurology Ege University Medical School Hospital, Izmir, Turkey Received 3 January 2003; received in revised form 16 June 2003; accepted 10 July 2003
Abstract Hyaline body myopathy is a rare congenital disease with distinctive histopathological features. We performed homozygosity mapping in a family with two affected sibs and identified a gene locus with a maximum homozygosity region of 5.35 centi Morgans or 5.59 Megabases at chromosome 3p22.2 – p21.32. The best candidate responsible for the disease is a novel gene that exhibits homology to the myosin heavy chain. q 2003 Elsevier B.V. All rights reserved. Keywords: Hyaline body myopathy; Autosomal recessive; Gene localization; Turkish
1. Introduction Hyaline body myopathy (HBM) is a rare congenital myopathy that manifests with subsarcolemmal hyalinized bodies in type 1 fibers. It was first described in 1971 as familial myopathy with probable lysis of myofibrils in type 1 fibers [1] and later designated ‘myopathy with lysis of type 1 myofibrils’ (MIM 255160). Engel and Baker used the term hyaline bodies because of the appearance in trichromestained sections [2]. HBM has been reported both as sporadic [3 –5] and familial cases, and the familial forms were autosomal dominant [6] or autosomal recessive [1]. The clinical findings were diverse, with either scapuloperoneal weakness [5,6] or limb girdle weakness [1,3,4]. The hyalinized bodies in muscle biopsy samples have been studied extensively [2 – 4,6]. Ultra structurally, they appeared to consist of fine filamentous or granular material and not to have a surrounding membrane. The neighboring myofibrils were normal, with no sign of degeneration. The amorphous bodies contained granules and disorganized filaments and stained intensely with the myofibrillar * Corresponding author. Tel.: þ90-212-358-1540 ext. 1472/90-212-2572095/90-533-433-0377; fax: þ 90-212-265-9778. E-mail address: [email protected] (A. Tolun). 1 Present address: Molecular Genetics Program, Benaroya Research Center, Seattle, WA, USA. 0960-8966/$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0960-8966(03)00163-9
ATPase reaction after acidic pre-incubation, but not with stains for oxidative enzymes and polysaccharides. They also reacted with monoclonal antibodies against anti-skeletal heavy myosin. Therefore, they were suggested to be degradation products of myosin [2]. The age of onset in the patients reported varied from early childhood to the fifth decade of life. Intrafamilial variation in the severity of the clinical course has been most obvious in one of the autosomal dominant families, in which the disease onset was in the first decade in one member and in the fifth in another [6]. We report here the results of the genetic analysis as well as the detailed clinical findings in two brothers. We mapped the gene responsible for the disease to 3p23– p21. The best candidate responsible for the disorder is a novel gene with homology to myosin heavy chain.
2. Patients and methods 2.1. Patients The proband (Patient 1) was a 44-year-old male with normal motor and mental development. He had always been thin and not able to run since childhood. He noted difficulty in walking in his late teens. He was discharged early from the military obligation due to his disability. He noticed difficulty
¨ nengu¨t et al. / Neuromuscular Disorders 14 (2004) 4–9 S. O
in lifting objects above his head at age 28 years. The clinical symptoms progressed very slowly in the years that followed. He was examined at the neuromuscular outpatient clinic at age 39 years. Physical examination was unremarkable except for the cachectic appearance, long face, and high-arched palate. Scapular winging and a steppage gait were noticed in the neurological examination. Manual muscle testing revealed weakness of grade 4 (Medical Research Council (MRC) scale) in the neck flexor and shoulder adductors, grade 3 in the shoulder abductors and elbow flexors, and grade 2 in the ankle dorsiflexors. Tendon reflexes were absent except in triceps. Plantar reflexes were flexor. Sensation was normal. Creatine kinase (CK) levels was about two-fold higher than normal. Other laboratory findings were within normal ranges. EMG displayed myopathic features. Electrocardiogram was unremarkable. The neurological examination 2 years later revealed a moderate progression of the weakness in his legs with 0 muscle strength in the ankle dorsiflexors. Echocardiogram showed global systolic dysfunction with an ejection fraction of 30% and left ventricular hypokinesis. Because of the clinical findings for cardiac failure, he was put on medical treatment. He was still ambulant 3 years after the second examination, and no further change was observed in his muscle weakness or cardiologic findings. Patient 2 was the 40-year-old brother of patient 1. He was asymptomatic until the age of 33 years, when he noticed difficulty in raising his arms. His physical examination was unremarkable at age 35 years, but moderate weakness and mild atrophy in shoulder girdle and peroneal muscles were found upon neurological examination. Abnormal manual muscle test results were as follows: grade 2 4 in shoulder abductors and grade 4 in shoulder adductors, elbow flexors, and ankle dorsiflexors. Tendon reflexes and sensation were normal. CK level was three-fold higher than normal. Electromyography (EMG) displayed myopathic changes. His strength had slightly worsened 5 years later, and shoulder abductors showed weakness of grade 3. His ecocardiographic and electrocardiographic examinations were both normal. The parents were in their seventies and had normal strength, so were the two sisters at ages 41 and 36 years. Two siblings of each patient were examined at ages 8 –20 years and found normal. The parents denied consanguinity but reported having origins in neighboring villages. 2.2. Muscle biopsy analysis Muscle biopsy specimens were obtained from the left triceps muscle of patient 1 and the left deltoid muscle of patient 2. Specimens were frozen rapidly in isopentane cooled with liquid nitrogen, and 10 mm cryostat sections were processed for hematoxylin-eosin (H&E), modified Gomori trichrome, Periodic acid-Schiff, Oil-red-O, adenosine triphosphatase (preincubation at pH 4.3 and 9.4), NADH dehydrogenase, succinate dehydrogenase, and Congo red stain.
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2.3. Genome-wide scan Appropriate informed consent was obtained from the subjects for genetic studies. DNA was isolated from peripheral blood samples using standard techniques. The genome-wide scan was performed using the low-density (about 25 centi Morgan (cM) spacing) set Version 8a containing 156 autosomal microsatellite markers (Research Genetics, USA). Marker alleles were resolved on 8% denaturing polyacrylamide gels and visualized by staining with silver nitrate, as described previously [7]. 2.4. Statistical analyses Linkage analysis was performed under the assumption of autosomal recessive inheritance, full penetrance, a disease gene frequency of 1 in 100,000, and equal frequencies of marker alleles. In constructing the pedigree, the grandparents were assumed to be second cousins. We assessed this to be the closest possible relation to be assumed, since the parents were unaware of any consanguinity. Two-point lod scores were calculated using the MLINK program of the FASTLINK 4.1 package [8]. GENEHUNTER version 2.0 beta was used for multipoint parametric linkage analysis and construction of the haplotypes, allowing minimum number of recombination events [9].
3. Results 3.1. Muscle biopsy findings The muscle biopsy findings in the patients were very similar. There was marked variation in fiber size and fiber splittings. Marked type 1 predominance was evident. Approximately 15 – 20% of the fibers had central nuclei. The most striking finding was the presence of subsarcolemmal hyalinized structures. These hyaline structures appeared pale pink with the hematoxylin and eosin stain and pale green with the modified Gomori trichrome stain (Figs. 1A,B). They lacked reactivity for Periodic acid-Schiff stain (PAS) and NADH dehydrogenase and exhibited a dark staining rim at the margins (Figs. 1C,D). Most of the hyaline structures were negative for the ATPase reaction at pH 9.4 and moderately to intensely positive at pH 4.3 (Figs. 1E,F). Subsarcolemmal hyaline bodies were exclusively in type 1 fibers, and they were present in approximately 25 – 30% of the fibers. Staining for amyloid was negative with Congo red. 3.2. Genome-wide scan results The extreme rarity of the trait and the fact that parents originated from neighboring villages prompted us to assume a distant blood relation and apply homozygosity mapping. The genome-wide scan results using the DNA samples of the two patients revealed homozygosity for the same allele
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¨ nengu¨t et al. / Neuromuscular Disorders 14 (2004) 4–9 S. O
Fig. 1. Triceps and deltoid muscle biopsy specimens from patients 1 (A and B) and 2 (C, D, E, and F), respectively. (A) Modified Gomori-trichrome, 20 £ ; (B) H&E, 10 £ ; (C) PAS, 10 £ ; (D) NADH dehydrogenase, 10 £ ; (E) Myofibrillar ATPase at pH 9.4, 10 £ ; and (F) Myofibrillar ATPase at pH 4.3, 10 £ . (A) Trichrome stained section showed variation in fiber size, fiber splitting, central nuclei, some adipose tissue, and pale green subsarcolemmal hyaline bodies. (B) H&E stained sections showed similar findings and pale pink hyaline bodies. Hyaline bodies were devoid of glycogen (C) and oxidative enzyme activity (D). Some fibers showed increased activity at the margin of hyaline bodies. (E) In ATPase reaction after pre-incubation at pH 9.4, type I fibers were predominant and enzyme activity inside hyaline bodies absent. (F) In pre-incubation at pH 4.3, moderate or strong myofibrillar ATPase enzyme activity was seen in hyaline bodies.
for eight markers in the set, namely D2S1400, D3S1766, GATA118002 (chromosome 3), D5S820, D6S1277, D8S1106, D11S1393, and D11S4464. Upon genotyping the patients with markers that flank the above mentioned markers, a shared homozygosity only around marker
D3S1766. Genotype analysis in the family using markers around D3S1766 revealed a candidate gene locus telomeric to the marker. The affected individuals were homozygous for a common haplotype with seven markers at the region, consistent with the disease haplotype being identical by
¨ nengu¨t et al. / Neuromuscular Disorders 14 (2004) 4–9 S. O
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Table 1 Haplotypes, genetic positions, and two-point lod scores for 19 markers at 3p Locus
kba
cMb
F
D3S1619 D3S1768 D3S3623 D3S3572 D3S3527 D3S3522 D3S2407 D3S2373 D3S3559 D3S2304 D3S3597 D3S3624 D3S2354 D3S1358 D3S1478 D3S2409 D3S2449 D3S2321 D3S1227
33,963 34,471 37,257 38,842 39,159 40,602 41,208 42,254 42,508 42,634 43,783 44,433 45,369 45,401 46,303 49,272 49,803 52,954 53,864
60.98 61.52 61.52 63.12 63.12 65.26 67.94 – 67.94 67.94 68.47 68.47 – – – 70.61 – 70.61 –
4 1 3 2 2 2 2 1 3 2 3 1 2 3 2 1 2 2 2
a b c d
M 3 2 2 2 3 1 1 2 1 1 2 2 1 1 1 1 1 4 3
1 1 3 1 2 2 2 1 3 2 3 2 2 1 3 1 2 3 4
P1 2 1 1 2 1 1 1 1 2 1 1 3 3 2 1 2 3 1 1
4 1 3 2 2 2 2 1 3 2 3 1 2 3 2 1 2 2 2
P2 1 1 3 1 2 2 2 1 3 2 3 2 2 1 3 1 2 3 4
4 1 3 2 2 2 2 1 3 2 3 1 2 3 2 1 2 2 2
S1 1 1 3 1 2 2 2 1 3 2 3 2 2 1 3 1 2 3 4
4 1 3 2 2 2 2 1 3 2 3 1 2 3 2 1 2 2 2
S2 2 1 1 2 1 1 1 1 2 1 1 3 3 2 1 2 3 1 1
3 2 2 2 3 1 1 2 1 1 2 2 1 1 1 1 1 4 3
2 1 1 2 1 1 1 1 2 1 1 3 3 2 1 2 3 1 1
Zmaxc
uMLEd
0.35 0.94 1.55 0.20 1.55 1.54 1.54 0.94 1.55 1.54 1.55 0.40 1.55 0.40 0.38 1.24 1.55 0.39 0.39
0.11 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.12 0.00 0.12 0.12 0.00 0.00 0.12 0.12
GenBank sequence map. Marshfield genetic map. Maximum two-point lod score. Maximum likelihood estimate for recombination fraction (u).
descent (Table 1). The unaffected siblings were not homozygous for the haplotype. Flanking markers were used to refine the borders of the homozygosity region. The affected individuals were heterozygous for D3S3572 and D3S3624. Thus, D3S3572 defined the telomeric border and D3S3624 the centromeric border of the maximum homozygosity region of the 5.35 cM or 5.59 Megabase gene locus. The markers, their distances, and the haplotypes of the family members are given in Table 1. 3.3. Statistical findings Lod scores were calculated to assess the significance of the results. Two-point lod scores were around 1.55 at a recombination fraction of zero for most of the markers in the homozygosity region (Table 1). The multi-point lod score peaked in the interval D3S3572– D3S3624, with a maximum value of 3.12 (Fig. 2).
4. Discussion This study describes clinical, morphological, and genetic findings in two brothers with hyaline body myopathy. So far, few individuals have been described to be afflicted with this very rare disorder: six patients from unrelated families [2 – 5], seven patients from an autosomal dominant family [6], and four patients from
two autosomal recessive families including the family we describe here [1]. The clinical phenotypes of our patients consisted of scapuloperoneal weakness and atrophy, very similar to some of the patients reported [5,6], but not limb girdle weakness reported for others [1,3,4]. Intrafamilial variation in the severity of clinical course and the age of onset were observed in our patients, as had been reported for another family [6]. One of our patients (patient 1) additionally had cardiac involvement, a finding not reported previously in any HBM patient. EMG displayed systolic dysfunction at the age of 39 years in his first examination. Such a symptom was not present in his brother at the age of 40 years, consistent with his much milder clinical phenotype in general. Heart biopsy would be necessary to assess whether the heart condition is due to HBM. The hyaline inclusions in our patients were consistent in morphology and staining properties to those in other HBM patients [1 – 6]. These analyzes were sufficient for diagnosis, and EM investigation was not necessary, because there is no other disorder with similar manifestations with respect to hyaline bodies that would necessitate differential diagnosis. Morphological heterogeneity in the inclusion bodies was reported for other families. The hyaline inclusions were centrally located in a few muscle fibers and stained dark green to blue with modified Gomori-trichrome in two members of an autosomal dominant family with scapuloperoneal syndrome but otherwise resembling HBM [10]. Also, Selcen et al. [11] described in two siblings similar
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¨ nengu¨t et al. / Neuromuscular Disorders 14 (2004) 4–9 S. O
Fig. 2. The result of 20-point lod score analysis at 3p for HBM. D3S has been omitted from marker designations.
hyaline masses that stained deep red with H&E and dark blue with trichrome and contained intensely congophilic inclusions. The clinical and myopathologic features were also distinct from HBM. An interesting question is whether those families link to the locus identified in this study. We investigated the databases in order to assess the best candidate gene at the locus. An unknown gene (KIAA1042/OIP106; locus ID: 22906) [12] coding for a protein with homology to myosin heavy chain stood out as the best candidate, since the hyaline bodies had been found to react with antibodies against myosin heavy chain [3,4,6]. We have not attempted to screen the patients for mutations in the KIAA1042 gene, because neither the nucleotide sequence nor the exact localization of the region is yet completely determined (GenBank, Build 33). Nevertheless, we believe that the localization of the gene responsible for an autosomal recessive hyaline body myopathy will have benefits for families afflicted with the disorder, as they can be tested for linkage to the novel
locus. Reporting the gene locus would also aid other researchers engaged in a search for the gene(s) responsible for HBM. 5. Electronic database information Online Mendelian Inheritance in Man (OMIM), http:// www.ncbi.nlm.nih.gov/Omim/ (for Myopathy with Lysis of Type I Myofibrils [MIM 255160]). Center for Medical Genetics, http://research. marshfieldclinic.org/genetics/ (for genetic mapping information). National Center for Biotechnology Information, http:// www.ncbi.nlm.nih.gov/ (to access GenBank). Acknowledgements ¨ nengu¨t and Sibel Aylin Ugˇur were fellows of the Suna O Scientific and Technical Research Council of Turkey. This
¨ nengu¨t et al. / Neuromuscular Disorders 14 (2004) 4–9 S. O
work was supported by Turkish State Planning Organization and the Turkish Academy of Sciences.
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