Journal of Dermatology 2014; 41: 1047–1052
doi: 10.1111/1346-8138.12657
ORIGINAL ARTICLE
First Japanese case of atypical progeroid syndrome/atypical Werner syndrome with heterozygous LMNA mutation Sei-ichiro MOTEGI,1 Yoko YOKOYAMA,1 Akihiko UCHIYAMA,1 Sachiko OGINO,1 Yuko TAKEUCHI,1 Kazuya YAMADA,1 Tomoyasu HATTORI,1 Hiroaki HASHIZUME,2 Yuichi ISHIKAWA,3 Makoto GOTO,4,5 Osamu ISHIKAWA1 Departments of 1Dermatology, 2Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi, 3 Department of Pathology, Institute of Cancer Research, Japanese Foundation for Cancer Research, 4Division of Orthopedic Surgery and Rheumatology, East Medical Center, Tokyo Women’s Medical University, Tokyo, and 5Division of Anti-aging and Longevity Sciences, Department of Clinical Engineering, Faculty of Medical Engineering, Toin University of Yokohama, Yokohama, Kanagawa, Japan
ABSTRACT Atypical progeroid syndrome (APS), including atypical Werner syndrome (AWS), is a progeroid syndrome involving heterozygous mutations in the LMNA gene encoding the nuclear protein lamin A/C. We report the first Japanese case of APS/AWS with a LMNA mutation (p.D300N). A 53-year-old Japanese man had a history of recurrent severe cardiovascular diseases as well as brain infarction and hemorrhages. Although our APS/AWS patient had overlapping features with Werner syndrome (WS), such as high-pitched voice, scleroderma, lipoatrophy and atherosclerosis, several cardinal features of WS, including short stature, premature graying/alopecia, cataract, bird-like face, flat feet, hyperkeratosis on the soles and diabetes mellitus, were absent. In immunofluorescence staining and electron microscopic analyses of the patient’s cultured fibroblasts, abnormal nuclear morphology, an increase in small aggregation of heterochromatin and a decrease in interchromatin granules in nuclei of fibroblasts were observed, suggesting that abnormal nuclear morphology and chromatin disorganization may be associated with the pathogenesis of APS/AWS.
Key words: atypical progeroid syndrome, atypical Werner syndrome, Hutchinson–Gilford syndrome, lamin A, Werner syndrome.
INTRODUCTION Werner’s syndrome (WS; Online Mendelian Inheritance in Man [OMIM] no. 277700) is an autosomal recessive, adultonset progeroid syndrome caused by a mutation of the RecQ3 helicase gene (WRN) located on chromosome 8p1112.1 WS is clinically characterized by the general appearance of accelerated aging, such as skin atrophy, loss and graying of hair, ocular cataracts, type 2 diabetes mellitus, osteoporosis and atherosclerosis. Approximately 20% of the patients clinically diagnosed with possible WS do not have WRN mutations and are tentatively categorized as “atypical WS” (AWS).2,3 It has recently been reported that patients with AWS carry heterozygous mutations in the LMNA gene, which encodes the nuclear protein lamin A/C.2 These WS-like progeria cases with the LMNA mutation have been reported as “atypical progeroid syndrome” (APS) or AWS.2 We herein report the first Japanese case of APS/AWS with a heterozygous LMNA point mutation. In addition, we examined the
nuclear morphology and chromatin organization in fibroblasts from the patient.
Case report A 53-year-old Japanese man was referred to our department with a 3-year history of sclerodactyly, peripheral coldness and toe ulcers. He had a past history of several cardiovascular diseases: arrhythmia from the age of 38 years, coronary angioplasty and intracoronary stenting for angina at the age of 41 years, valve replacement for combined valvular disease at the age of 46 years and implantation of a pacemaker for thirddegree complete atrioventricular block at the age of 47 years. Furthermore, he suffered from a brain infarction of the left frontal lobe at the age of 51 years and hemorrhages in the thalamus and cerebellum at the age of 52 and 53 years, respectively (Fig. 1e,f). The patient had two siblings, and his younger sister developed similar symptoms (Fig. 1a). She presented a progeroid appearance, underwent aortic valve replacement and suffered from a brain infarction at the age of
Correspondence: Sei-ichiro Motegi, M.D., Ph.D., Department of Dermatology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan. Email: [email protected] Received 16 June 2014; accepted 4 September 2014.
© 2014 Japanese Dermatological Association
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(a)
(b)
(e)
(f)
(g)
(h)
(i)
(j)
(c)
(d) (k)
Figure 1. (a) Pedigree of the family with atypical progeroid syndrome/atypical Werner syndrome. Squares indicate males, circles indicate females. Filled-in symbols indicate affected individuals. Diagonal lines signify death, and age of death is indicated below. (b–d) Physical examination. (b) Facial appearance. (c,d) Sclerotic and atrophic skin on the hands and feet and toe ulcers. (e–g) Computed tomography of head and abdomen. Hemorrhages in the (e) thalamus and (f) cerebellum. (g) Calcification of the abdominal aorta. (h) A plain radiograph demonstrated calcification of the peripheral arteries, but not of the Achilles tendon. (i,j) Histopathological examination of the forearm skin. Fibrosis of the dermis and degeneration of the fat tissue (left, low magnification, bar = 80 lm; right, high magnification, bar = 20 lm). (k) Von Kossa staining. Calcification of the small dermal blood vessel walls (bar = 20 lm).
47 years. His parents were non-consanguineous, and his father died of a brain infarction at the age of 47 years. Four out of six siblings of the father died of a brain infarction, myocardial infarction or liver failure in middle age, and his grandfather died of a brain infarction at the age of 40 years. A pedigree of the patient’s family indicates that the disease displays an autosomal dominant inheritance pattern (Fig. 1a). Physical examination revealed a low bodyweight (38 kg with a height of 1.64 m), a high-pitched voice, sclerodactyly, atrophic skin on the hands and feet, diffuse pigmentation of skin on face and extremities, and nail-fold bleeding of the fingers and toe ulcers (Fig. 1b–d). However, he had no premature graying hair, alopecia, bird-like face, cataract, abnormal tooth
1048
alignment, nail atrophy, hyperkeratotic lesions on his soles and flat feet. Laboratory findings indicated abnormal liver function due to fatty liver, hyperuricemia and hyperlipidemia. Autoantibodies, including antinuclear antibody, anti-topoisomerase I antibody and anticentromere antibody, were negative. Blood pressure and oral glucose tolerance test results were normal. The patient had exaggerated tendon reflex, osteoporosis, sensorineural deafness and vascular dementia. Computed tomography indicated calcification of the abdominal aorta (Fig. 1g). A plain radiograph demonstrated calcification of the peripheral arteries, but not of the Achilles tendon (Fig. 1h). No malignant neoplasms throughout the whole body were detected by 18F-fluorodeoxyglucose positron emission tomog-
© 2014 Japanese Dermatological Association
Japanese APS/AWS with LMNA mutation
raphy. Histopathological examination of the forearm skin revealed fibrosis of the dermis and degeneration of the subcutaneous fat tissue (Fig. 1i,j). Von Kossa staining revealed the calcification of the small vessel walls in the dermis (Fig. 1k).
METHODS Clinical examination Blood and forearm skin specimens were obtained from the patient, and age- and sex-matched healthy volunteer. This study was approved by the local research ethics committee of Gunma University. The patient provided written informed consent before participation. This study was conducted according to the principles of the Declaration of Helsinki.
Genetic analysis Genomic DNA was isolated from whole blood cells using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). To analyze WRN mutations, genetic analysis was performed based on the mutant allele-specific amplification method as previously described.4 To analyze LMNA mutation, genetic analysis was performed by direct sequencing of the 12 amplified LMNA exons with flanking intronic primers, as previously described.2,5 The polymerase chain reaction products were directly sequenced using an ABI Prism 310 sequence analyzer (Applied Biosystems, Foster City, CA, USA).
Culture for human fibroblasts Primary human dermal fibroblasts were established from dermal explants of skin, and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mmol/L L-glutamine, 10 mmol/L penicillin (100 U/mL), 100 lg/mL streptomycin and 1 mmol/L sodium pyruvate and used before passage 6.
Analysis of nuclear morphology Cultured human fibroblasts were seeded in eight-well culture slides (BD Bioscience, San Jose, CA, USA). Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 30 min, and blocked and permeabilized in blocking solution (3% dry milk–PBS supplemented with 5% normal goat serum and 0.1% Triton X-100) for 30 min. After blocking, cells were stained with 40 ,60 -diamidino-2-phenylindole dihydrochloride to visualize nuclei, mounted in ProLong Gold antifade reagent (Invitrogen, San Diego, CA, USA). Quantification of abnormal nuclear structure was performed by counting of abnormally shaped nuclei in 10 random microscopic fields, and was obtained by a blinded observer (K. Y.). For electron microscopy, cultured human fibroblasts grown as monolayers were fixed in 1% glutaraldehyde, subsequently fixed with OsO4, then dehydrated in an ethanol series and embedded in resin.
Statistics P-values were calculated using Student’s t-test (two-sided). Error bars represent standard errors of the mean, and numbers of experiments (n) are as indicated.
© 2014 Japanese Dermatological Association
RESULTS LMNA and WRN mutation analysis We identified a heterozygous LMNA c.898G>A mutation in exon 5, predicted to cause the change of aspartic acid 300 into asparagine at the protein level (p.D300N) (Fig. 2a). No mutations in the WRN gene were found based on the mutant allelespecific amplification method. Because we could not obtain permission from his family, further gene analyses of his family were not performed. Based on clinical and genetic findings, a diagnosis of APS/AWS with LMNA mutation was established.
Analysis of nuclear structure in fibroblasts with LMNA mutation It is known that abnormal nuclear structure is a hallmark of LMNA-based progerias and other laminopathies, such as Hutchinson–Gilford syndrome (HGS; OMIM no. 176670), mandibuloacral dysplasia (MAD; OMIM no. 248370) and familial partial lipodystrophy (FPLD; OMIM no. 151660).6 In addition, it has been reported that the nuclear structure is perturbed in AWS with p.R133L, p.L140R or p.D300G LMNA mutations compared with healthy controls.2,7 We compared the structure of the nuclei in the dermal fibroblasts from the patient and healthy control. The rate of abnormal nuclear morphology in the fibroblasts from the APS/AWS patient was significantly higher than that from the control (41.8% for APS vs 22.2% for control, P = 0.006; Fig. 3a,b). Next, we analyzed the nuclear structure and chromosome organization by electron microscopy. Heterochromatins near the nuclear lamina were lost in HGS cells.8 Ho et al.9 reported that thickening of the nuclear membrane could be observed by electron microscopy in fibroblasts from AWS and HGS patients (heterozygous mutation of LMNA: p.E578V and p.V607_Q656del [also designated as
(a)
(b)
Figure 2. (a) Heterozygous LMNA c.898G>A mutation in exon 5, predicted to cause the change of aspartic acid 300 into asparagine at the protein level (p.D300N). (b) The distribution of mutation of the LMNA gene in atypical progeroid syndrome and atypical Werner syndrome. Red, mutation in our case.
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p.G608G], respectively) compared with that from normal control. We found an invagination of the nuclear envelope (indicated by asterisk in Fig. 3c), an increase in small aggregation
(a)
(b)
of heterochromatin (indicated by arrow in Fig. 3c) and a decrease in interchromatin granules (indicated by arrowhead in Fig. 3c) in the nuclei of fibroblasts from the APS/AWS patient. These results suggest that abnormal nuclear morphology and altered chromatin organization in fibroblasts with LMNA mutation may be associated with the pathogenesis of APS/AWS.
DISCUSSION
(c)
Figure 3. Analysis of nuclear structure and chromatin organization in fibroblasts with LMNA mutation. (a) Representative images of nuclear structure in fibroblasts from atypical progeroid syndrome (APS) patient and control. Arrow highlights abnormal structure of nuclei (bar = 20 lm). (b) Quantitation of abnormal structure of nuclei in fibroblasts from APS patient and control. Values are means standard error of the mean from 10 random confocal microscopic fields in three independent experiments. **P < 0.01. (c) Representative electron microscopic images of nuclear structure in fibroblasts from APS patient and control. Asterisk indicates an invagination of the nuclear envelope. Arrow indicates an increase in small aggregation of heterochromatin. Arrowhead indicates interchromatin granules (upper, bar = 10 lm; middle, bar = 50 nm; lower, bar = 1 lm). DAPI, 40 ,60 -diamidino-2-phenylindole dihydrochloride.
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The lamins, type V intermediate filament proteins, are components of the nuclear envelope and associated with the surface of the inner nuclear membrane.6 Lamins regulate many functions, such as the size and shape of the nucleus, DNA synthesis and repair, and nuclear migration.6 Mutation of the LMNA gene results in a variety of rare disorders called laminopathies, including HGS, MAD, FPLD and APS/AWS. The comparison of the genetic and clinical features between our case and progeroid syndromes is summarized in Table 1. HGS is a representative progeroid syndrome and most frequently caused by a mutation in exon 11 of the LMNA gene that generates an alternative splicing site, resulting in the formation of an abnormal and truncated lamin A protein called progerin.6 The accumulation of progerin results in an abnormal nuclear shape and abnormal nuclear functions, including altered histone modification patterns, abnormal chromatin remodeling, impaired transport through nuclear pores, reduced DNA repair capacity and increased telomere shortening; these abnormalities may result in a shortened lifespan of cells due to premature senescence.6 The clinical features of HGS manifest as early as the first year of life, and most patients die before 13 years of age. MAD is an autosomal recessive disorder characterized by growth retardation, craniofacial anomalies with mandibular hypoplasia, skeletal abnormalities and cutaneous pigmentation. FPLD is an autosomal dominant disorder with the loss of subcutaneous adipose tissue, and is usually associated with a variety of metabolic disorders including diabetes mellitus and dyslipidemia. There is significant phenotypic overlap in laminopathies. Werner syndrome is also a progeroid syndrome caused by an autosomal recessive mutation in the RECQL DNA helicase gene.1 The clinical features of WS manifest during the third or fourth decade of life, and most patients die during the fourth or fifth decade of life. Both HGS and WS have unique phenotypes with several overlapping clinical features. It has been reported that 26 out of 129 (20%) patients clinically diagnosed with possible WS do not have WRN mutations and have a mutation in the LMNA gene.2 These patients have been diagnosed with APS or AWS.2,10 To date, 24 point mutations of LMNA have been reported to cause APS or AWS (Fig. 2b).2,7,10–13 The analysis of our patient’s LMNA gene revealed a heterozygous point mutation in exon 5, c.898G>A (p.D300N). To the best of our knowledge, only one French case with the same mutation (p.D300N) has been reported.11 In this French patient, they confirmed that the mutation was not present in 200 control chromosomes or the updated LMNA variation database.11 The clinical features of the French case with p.D300N mutation revealed the bird-like facial appearance, scleroderma-like skin
© 2014 Japanese Dermatological Association
Japanese APS/AWS with LMNA mutation
Table 1. Summary of the genetic and clinical features of our patient and progeroid syndromes, including Werner syndrome, atypical progeroid syndrome/atypical Werner syndrome (APS/AWS) and Hutchinson–Gilford syndrome.
Gene Age of onset Inheritance High-pitched voice Scleroderma Lipoatrophy Pigmentation of skin Skin ulcer Atherosclerosis Hyperlipidemia Osteoporosis Premature graying/alopecia Short stature Cataract Bird-like face Nail atrophy Flat feet Hyperkeratosis on soles Calcification of Achilles tendon DM type 2 Infertility Malignant neoplasms Abnormal dentition Hypertension Valvular disease Calcification of vessels Hyperuricemia Hepatic dysfunction Exaggerated tendon reflex Deafness Dementia
Werner syndrome
APS/AWS
Patient
Hutchinson–Gilford syndrome
WRN (RecQ) >20–30 years AR ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + ++ ++ + ++ + + ++ + + + + + + + +
LMNA >2 years AD + + + + + + + + + + + + + +
LMNA 38 years AD + + + + + + + + + + + + + + +
LMNA
doi: 10.1111/1346-8138.12657
ORIGINAL ARTICLE
First Japanese case of atypical progeroid syndrome/atypical Werner syndrome with heterozygous LMNA mutation Sei-ichiro MOTEGI,1 Yoko YOKOYAMA,1 Akihiko UCHIYAMA,1 Sachiko OGINO,1 Yuko TAKEUCHI,1 Kazuya YAMADA,1 Tomoyasu HATTORI,1 Hiroaki HASHIZUME,2 Yuichi ISHIKAWA,3 Makoto GOTO,4,5 Osamu ISHIKAWA1 Departments of 1Dermatology, 2Medicine and Molecular Science, Gunma University Graduate School of Medicine, Maebashi, 3 Department of Pathology, Institute of Cancer Research, Japanese Foundation for Cancer Research, 4Division of Orthopedic Surgery and Rheumatology, East Medical Center, Tokyo Women’s Medical University, Tokyo, and 5Division of Anti-aging and Longevity Sciences, Department of Clinical Engineering, Faculty of Medical Engineering, Toin University of Yokohama, Yokohama, Kanagawa, Japan
ABSTRACT Atypical progeroid syndrome (APS), including atypical Werner syndrome (AWS), is a progeroid syndrome involving heterozygous mutations in the LMNA gene encoding the nuclear protein lamin A/C. We report the first Japanese case of APS/AWS with a LMNA mutation (p.D300N). A 53-year-old Japanese man had a history of recurrent severe cardiovascular diseases as well as brain infarction and hemorrhages. Although our APS/AWS patient had overlapping features with Werner syndrome (WS), such as high-pitched voice, scleroderma, lipoatrophy and atherosclerosis, several cardinal features of WS, including short stature, premature graying/alopecia, cataract, bird-like face, flat feet, hyperkeratosis on the soles and diabetes mellitus, were absent. In immunofluorescence staining and electron microscopic analyses of the patient’s cultured fibroblasts, abnormal nuclear morphology, an increase in small aggregation of heterochromatin and a decrease in interchromatin granules in nuclei of fibroblasts were observed, suggesting that abnormal nuclear morphology and chromatin disorganization may be associated with the pathogenesis of APS/AWS.
Key words: atypical progeroid syndrome, atypical Werner syndrome, Hutchinson–Gilford syndrome, lamin A, Werner syndrome.
INTRODUCTION Werner’s syndrome (WS; Online Mendelian Inheritance in Man [OMIM] no. 277700) is an autosomal recessive, adultonset progeroid syndrome caused by a mutation of the RecQ3 helicase gene (WRN) located on chromosome 8p1112.1 WS is clinically characterized by the general appearance of accelerated aging, such as skin atrophy, loss and graying of hair, ocular cataracts, type 2 diabetes mellitus, osteoporosis and atherosclerosis. Approximately 20% of the patients clinically diagnosed with possible WS do not have WRN mutations and are tentatively categorized as “atypical WS” (AWS).2,3 It has recently been reported that patients with AWS carry heterozygous mutations in the LMNA gene, which encodes the nuclear protein lamin A/C.2 These WS-like progeria cases with the LMNA mutation have been reported as “atypical progeroid syndrome” (APS) or AWS.2 We herein report the first Japanese case of APS/AWS with a heterozygous LMNA point mutation. In addition, we examined the
nuclear morphology and chromatin organization in fibroblasts from the patient.
Case report A 53-year-old Japanese man was referred to our department with a 3-year history of sclerodactyly, peripheral coldness and toe ulcers. He had a past history of several cardiovascular diseases: arrhythmia from the age of 38 years, coronary angioplasty and intracoronary stenting for angina at the age of 41 years, valve replacement for combined valvular disease at the age of 46 years and implantation of a pacemaker for thirddegree complete atrioventricular block at the age of 47 years. Furthermore, he suffered from a brain infarction of the left frontal lobe at the age of 51 years and hemorrhages in the thalamus and cerebellum at the age of 52 and 53 years, respectively (Fig. 1e,f). The patient had two siblings, and his younger sister developed similar symptoms (Fig. 1a). She presented a progeroid appearance, underwent aortic valve replacement and suffered from a brain infarction at the age of
Correspondence: Sei-ichiro Motegi, M.D., Ph.D., Department of Dermatology, Gunma University Graduate School of Medicine, 3-39-22 Showa-machi, Maebashi, Gunma 371-8511, Japan. Email: [email protected] Received 16 June 2014; accepted 4 September 2014.
© 2014 Japanese Dermatological Association
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(a)
(b)
(e)
(f)
(g)
(h)
(i)
(j)
(c)
(d) (k)
Figure 1. (a) Pedigree of the family with atypical progeroid syndrome/atypical Werner syndrome. Squares indicate males, circles indicate females. Filled-in symbols indicate affected individuals. Diagonal lines signify death, and age of death is indicated below. (b–d) Physical examination. (b) Facial appearance. (c,d) Sclerotic and atrophic skin on the hands and feet and toe ulcers. (e–g) Computed tomography of head and abdomen. Hemorrhages in the (e) thalamus and (f) cerebellum. (g) Calcification of the abdominal aorta. (h) A plain radiograph demonstrated calcification of the peripheral arteries, but not of the Achilles tendon. (i,j) Histopathological examination of the forearm skin. Fibrosis of the dermis and degeneration of the fat tissue (left, low magnification, bar = 80 lm; right, high magnification, bar = 20 lm). (k) Von Kossa staining. Calcification of the small dermal blood vessel walls (bar = 20 lm).
47 years. His parents were non-consanguineous, and his father died of a brain infarction at the age of 47 years. Four out of six siblings of the father died of a brain infarction, myocardial infarction or liver failure in middle age, and his grandfather died of a brain infarction at the age of 40 years. A pedigree of the patient’s family indicates that the disease displays an autosomal dominant inheritance pattern (Fig. 1a). Physical examination revealed a low bodyweight (38 kg with a height of 1.64 m), a high-pitched voice, sclerodactyly, atrophic skin on the hands and feet, diffuse pigmentation of skin on face and extremities, and nail-fold bleeding of the fingers and toe ulcers (Fig. 1b–d). However, he had no premature graying hair, alopecia, bird-like face, cataract, abnormal tooth
1048
alignment, nail atrophy, hyperkeratotic lesions on his soles and flat feet. Laboratory findings indicated abnormal liver function due to fatty liver, hyperuricemia and hyperlipidemia. Autoantibodies, including antinuclear antibody, anti-topoisomerase I antibody and anticentromere antibody, were negative. Blood pressure and oral glucose tolerance test results were normal. The patient had exaggerated tendon reflex, osteoporosis, sensorineural deafness and vascular dementia. Computed tomography indicated calcification of the abdominal aorta (Fig. 1g). A plain radiograph demonstrated calcification of the peripheral arteries, but not of the Achilles tendon (Fig. 1h). No malignant neoplasms throughout the whole body were detected by 18F-fluorodeoxyglucose positron emission tomog-
© 2014 Japanese Dermatological Association
Japanese APS/AWS with LMNA mutation
raphy. Histopathological examination of the forearm skin revealed fibrosis of the dermis and degeneration of the subcutaneous fat tissue (Fig. 1i,j). Von Kossa staining revealed the calcification of the small vessel walls in the dermis (Fig. 1k).
METHODS Clinical examination Blood and forearm skin specimens were obtained from the patient, and age- and sex-matched healthy volunteer. This study was approved by the local research ethics committee of Gunma University. The patient provided written informed consent before participation. This study was conducted according to the principles of the Declaration of Helsinki.
Genetic analysis Genomic DNA was isolated from whole blood cells using the Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA). To analyze WRN mutations, genetic analysis was performed based on the mutant allele-specific amplification method as previously described.4 To analyze LMNA mutation, genetic analysis was performed by direct sequencing of the 12 amplified LMNA exons with flanking intronic primers, as previously described.2,5 The polymerase chain reaction products were directly sequenced using an ABI Prism 310 sequence analyzer (Applied Biosystems, Foster City, CA, USA).
Culture for human fibroblasts Primary human dermal fibroblasts were established from dermal explants of skin, and cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% heat-inactivated fetal bovine serum, 2 mmol/L L-glutamine, 10 mmol/L penicillin (100 U/mL), 100 lg/mL streptomycin and 1 mmol/L sodium pyruvate and used before passage 6.
Analysis of nuclear morphology Cultured human fibroblasts were seeded in eight-well culture slides (BD Bioscience, San Jose, CA, USA). Cells were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) at room temperature for 30 min, and blocked and permeabilized in blocking solution (3% dry milk–PBS supplemented with 5% normal goat serum and 0.1% Triton X-100) for 30 min. After blocking, cells were stained with 40 ,60 -diamidino-2-phenylindole dihydrochloride to visualize nuclei, mounted in ProLong Gold antifade reagent (Invitrogen, San Diego, CA, USA). Quantification of abnormal nuclear structure was performed by counting of abnormally shaped nuclei in 10 random microscopic fields, and was obtained by a blinded observer (K. Y.). For electron microscopy, cultured human fibroblasts grown as monolayers were fixed in 1% glutaraldehyde, subsequently fixed with OsO4, then dehydrated in an ethanol series and embedded in resin.
Statistics P-values were calculated using Student’s t-test (two-sided). Error bars represent standard errors of the mean, and numbers of experiments (n) are as indicated.
© 2014 Japanese Dermatological Association
RESULTS LMNA and WRN mutation analysis We identified a heterozygous LMNA c.898G>A mutation in exon 5, predicted to cause the change of aspartic acid 300 into asparagine at the protein level (p.D300N) (Fig. 2a). No mutations in the WRN gene were found based on the mutant allelespecific amplification method. Because we could not obtain permission from his family, further gene analyses of his family were not performed. Based on clinical and genetic findings, a diagnosis of APS/AWS with LMNA mutation was established.
Analysis of nuclear structure in fibroblasts with LMNA mutation It is known that abnormal nuclear structure is a hallmark of LMNA-based progerias and other laminopathies, such as Hutchinson–Gilford syndrome (HGS; OMIM no. 176670), mandibuloacral dysplasia (MAD; OMIM no. 248370) and familial partial lipodystrophy (FPLD; OMIM no. 151660).6 In addition, it has been reported that the nuclear structure is perturbed in AWS with p.R133L, p.L140R or p.D300G LMNA mutations compared with healthy controls.2,7 We compared the structure of the nuclei in the dermal fibroblasts from the patient and healthy control. The rate of abnormal nuclear morphology in the fibroblasts from the APS/AWS patient was significantly higher than that from the control (41.8% for APS vs 22.2% for control, P = 0.006; Fig. 3a,b). Next, we analyzed the nuclear structure and chromosome organization by electron microscopy. Heterochromatins near the nuclear lamina were lost in HGS cells.8 Ho et al.9 reported that thickening of the nuclear membrane could be observed by electron microscopy in fibroblasts from AWS and HGS patients (heterozygous mutation of LMNA: p.E578V and p.V607_Q656del [also designated as
(a)
(b)
Figure 2. (a) Heterozygous LMNA c.898G>A mutation in exon 5, predicted to cause the change of aspartic acid 300 into asparagine at the protein level (p.D300N). (b) The distribution of mutation of the LMNA gene in atypical progeroid syndrome and atypical Werner syndrome. Red, mutation in our case.
1049
S.-i. Motegi et al.
p.G608G], respectively) compared with that from normal control. We found an invagination of the nuclear envelope (indicated by asterisk in Fig. 3c), an increase in small aggregation
(a)
(b)
of heterochromatin (indicated by arrow in Fig. 3c) and a decrease in interchromatin granules (indicated by arrowhead in Fig. 3c) in the nuclei of fibroblasts from the APS/AWS patient. These results suggest that abnormal nuclear morphology and altered chromatin organization in fibroblasts with LMNA mutation may be associated with the pathogenesis of APS/AWS.
DISCUSSION
(c)
Figure 3. Analysis of nuclear structure and chromatin organization in fibroblasts with LMNA mutation. (a) Representative images of nuclear structure in fibroblasts from atypical progeroid syndrome (APS) patient and control. Arrow highlights abnormal structure of nuclei (bar = 20 lm). (b) Quantitation of abnormal structure of nuclei in fibroblasts from APS patient and control. Values are means standard error of the mean from 10 random confocal microscopic fields in three independent experiments. **P < 0.01. (c) Representative electron microscopic images of nuclear structure in fibroblasts from APS patient and control. Asterisk indicates an invagination of the nuclear envelope. Arrow indicates an increase in small aggregation of heterochromatin. Arrowhead indicates interchromatin granules (upper, bar = 10 lm; middle, bar = 50 nm; lower, bar = 1 lm). DAPI, 40 ,60 -diamidino-2-phenylindole dihydrochloride.
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The lamins, type V intermediate filament proteins, are components of the nuclear envelope and associated with the surface of the inner nuclear membrane.6 Lamins regulate many functions, such as the size and shape of the nucleus, DNA synthesis and repair, and nuclear migration.6 Mutation of the LMNA gene results in a variety of rare disorders called laminopathies, including HGS, MAD, FPLD and APS/AWS. The comparison of the genetic and clinical features between our case and progeroid syndromes is summarized in Table 1. HGS is a representative progeroid syndrome and most frequently caused by a mutation in exon 11 of the LMNA gene that generates an alternative splicing site, resulting in the formation of an abnormal and truncated lamin A protein called progerin.6 The accumulation of progerin results in an abnormal nuclear shape and abnormal nuclear functions, including altered histone modification patterns, abnormal chromatin remodeling, impaired transport through nuclear pores, reduced DNA repair capacity and increased telomere shortening; these abnormalities may result in a shortened lifespan of cells due to premature senescence.6 The clinical features of HGS manifest as early as the first year of life, and most patients die before 13 years of age. MAD is an autosomal recessive disorder characterized by growth retardation, craniofacial anomalies with mandibular hypoplasia, skeletal abnormalities and cutaneous pigmentation. FPLD is an autosomal dominant disorder with the loss of subcutaneous adipose tissue, and is usually associated with a variety of metabolic disorders including diabetes mellitus and dyslipidemia. There is significant phenotypic overlap in laminopathies. Werner syndrome is also a progeroid syndrome caused by an autosomal recessive mutation in the RECQL DNA helicase gene.1 The clinical features of WS manifest during the third or fourth decade of life, and most patients die during the fourth or fifth decade of life. Both HGS and WS have unique phenotypes with several overlapping clinical features. It has been reported that 26 out of 129 (20%) patients clinically diagnosed with possible WS do not have WRN mutations and have a mutation in the LMNA gene.2 These patients have been diagnosed with APS or AWS.2,10 To date, 24 point mutations of LMNA have been reported to cause APS or AWS (Fig. 2b).2,7,10–13 The analysis of our patient’s LMNA gene revealed a heterozygous point mutation in exon 5, c.898G>A (p.D300N). To the best of our knowledge, only one French case with the same mutation (p.D300N) has been reported.11 In this French patient, they confirmed that the mutation was not present in 200 control chromosomes or the updated LMNA variation database.11 The clinical features of the French case with p.D300N mutation revealed the bird-like facial appearance, scleroderma-like skin
© 2014 Japanese Dermatological Association
Japanese APS/AWS with LMNA mutation
Table 1. Summary of the genetic and clinical features of our patient and progeroid syndromes, including Werner syndrome, atypical progeroid syndrome/atypical Werner syndrome (APS/AWS) and Hutchinson–Gilford syndrome.
Gene Age of onset Inheritance High-pitched voice Scleroderma Lipoatrophy Pigmentation of skin Skin ulcer Atherosclerosis Hyperlipidemia Osteoporosis Premature graying/alopecia Short stature Cataract Bird-like face Nail atrophy Flat feet Hyperkeratosis on soles Calcification of Achilles tendon DM type 2 Infertility Malignant neoplasms Abnormal dentition Hypertension Valvular disease Calcification of vessels Hyperuricemia Hepatic dysfunction Exaggerated tendon reflex Deafness Dementia
Werner syndrome
APS/AWS
Patient
Hutchinson–Gilford syndrome
WRN (RecQ) >20–30 years AR ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ ++ + ++ ++ + ++ + + ++ + + + + + + + +
LMNA >2 years AD + + + + + + + + + + + + + +
LMNA 38 years AD + + + + + + + + + + + + + + +
LMNA
