Extreme Variability of Phenotype in Patients With an Identical Missense Mutation in the Lamin A/C Gene: Title and subTitle BreakFrom Congenital Onset With Severe Phenotype to Milder Classic Emery-Dreifuss Variant FREE

The LMNA gene encodes the nuclear envelope proteins lamins A and C and is located on chromosome 1q11-q23.¹ Mutations in the LMNA gene are associated with 7 distinct pathologic conditions. These include autosomal dominant (AD) and autosomal recessive forms of Emery-Dreifuss muscular dystrophy (EDMD),^{2 - 3} limb-girdle muscular dystrophy type 1B,⁴ a form of dilated cardiomyopathy with conduction system disease,^{5 - 7} the Dunnigan type of familial partial lipodystrophy,^{8 - 9} a form of autosomal recessive axonal neuropathy (Charcot-Marie-Tooth disease),¹⁰ mandibuloacral dysplasia,¹¹ and, recently, Hutchinson-Gilford progeria syndrome.^{12 - 14}

The way in which these mutations produce different clinical phenotypes remains unclear, and while an association between specific LMNA mutations and definite phenotypes has been suggested in a number of instances, such as specific mutations leading to lipodystrophy, this issue has not been fully resolved. A few studies have reported the coexistence of 2 diseases in the same patient, such as limb-girdle muscular dystrophy 1B and lipodystrophy.^{15 - 16} Other studies have described the same mutation producing different phenotypes with muscle weakness, such as limb-girdle muscular dystrophy type 1B and AD-EDMD, in the same family.^{6 ,17}

We report 5 cases in which the identical missense mutation within the LMNA gene produced phenotypes characterized by muscle involvement of variable severity and additional distinctive features, which expand the spectrum of clinical phenotypes reported in the literature.

Five patients with the same missense LMNA mutation were identified among the clinic patients of the Dubowitz Neuromuscular Centre, Hammersmith Hospital Imperial College, London, England; Institute of Human Genetics and Department of Cardiology, University of Newcastle Upon Tyne, Newcastle, England; and Robert Jones and Agnes Hunt Orthopaedic Hospital, Oswestry, England. Clinical and investigation details were obtained from the records. Patients had been followed up for 1 to 23 years.

Analysis of LMNA was carried out by denaturing high-performance liquid chromatography sequencing techniques for the 12 exons of the gene as described by Bonne et al.² Exon 6 was amplified by polymerase chain reaction with the use of an intronic set of primers in flanking regions (6F 5′-CAAACCCTCCCACCCCCC-3′ and 6R 5′-CCAGTTGCCGGGCCAGAG-3′). The WaveMaker software (Transgenomic, Inc, Omaha, Neb) was used to calculate specific melting curves for each polymerase chain reaction fragment and to determine the optimal temperature for heteroduplex separation. A denaturing high-performance liquid chromatography profile showing both heteroduplexes and homoduplexes (64°C), as compared with unaffected controls showing only homoduplexes, was identified in the index cases for exon 6. Sequencing of the corresponding exon 6 polymerase chain reaction products showed a heterozygous 1072G→A transition in LMNA, resulting in the replacement of the glutamine 358 (GAG) by a lysine (AAG). This abnormal pattern was not found in 150 unrelated control subjects.

Two of the 5 patients had the most common phenotype reported in AD-EDMD.² Another patient also had an AD-EDMD phenotype, but with a relatively early and more severe presentation; this patient has already been described.³ The remaining 2 cases have additional atypical features and will be reported in detail.

A white girl was the second child of nonconsanguineous parents with no family history of neuromuscular disorders. The girl was born at 37 weeks of gestation by emergency low-segment cesarean section.The mother reported the presence of reduced fetal movements in comparison with her first pregnancy. Perinatal and neonatal course were uneventful.

At the age of 5 months the infant lost the ability to roll over (recently acquired at that time). A month later, after a respiratory tract infection, she showed difficulties in lifting her arms and poor head control. These signs persisted, and at 10 months she was found to have elevated serum creatine kinase levels (between 1000 and 2000 U/L) and underwent electromyography, which showed a myopathic pattern; motor and sensory nerve velocities were normal. A muscle biopsy specimen (quadriceps) showed myopathic changes and a mild up-regulation of major histocompatibility complex class I antigens on some fibers, leading to a suspicion of an inflammatory myopathy. Treatment was started with corticosteroids (prednisolone acetate, 1 mg/kg per day) at the age of 12 months. This did not produce any clinical improvement, and corticosteroids were therefore slowly discontinued over 3 months. During this period, there was an increase in weakness and feeding difficulties, which required the introduction of nasogastric tube feeding. At the age of 15 months, the infant had severe feeding difficulties and a marked axial and limb weakness affecting the upper more than the lower limbs. There was some thinning of her biceps and triceps muscles and wasting of the calf muscles in the lower limbs with talipes. At 21 months the patient continued to be nasogastrically fed; although she had acquired better head control in sitting, her upper limb function remained very poor and had possibly deteriorated further. She also had a narrow chest and a predominantly diaphragmatic pattern of breathing.

A second open muscle biopsy specimen of the vastus intermedius at 16 months also showed myopathic changes with occasional basophilic fibers and a few fibers of varying size expressing fetal myosin, suggesting some ongoing muscle damage. Major histocompatibility complex class I antigens were still present on some fibers, and there was no apparent abnormality in immunolabeling of nuclei with antibodies to emerin or lamins A, A/C, B1, or B2. Electron microscopy showed only mild nonspecific changes with well-preserved myofibrils. An electrocardiogram and magnetic resonance images of the brain and spinal cord were all normal.

A 30-year-old white woman was the only child of healthy nonconsanguineous parents. Her mother had had 2 stillbirths, which were attributed to toxemia in pregnancy. There was no family history of any neuromuscular disorder. The mother had died suddenly at the age of 45 years. Postmortem examination did not show any cardiac patterns, so the underlying cause of death was assumed to be a rhythm disturbance.

The patient had been born at 36 weeks' gestation (2.68 kg) after delivery was induced because of toxemia. She was noted to be hypotonic and had feeding difficulties for a few weeks after birth, but motor development was within the normal range (sitting unsupported at 6-7 months, walking independently at 15 months), although she was never able to run. At the age of 7 years she was referred because of generalized hypotonia and a waddling gait. On examination she exhibited severe wasting and weakness, more marked in biceps and triceps in the upper limb and in quadriceps in the lower limb. There were also minimal contractures of the finger flexors and at the right elbow, as well as limited dorsiflexion of both feet. No significant decrease in muscle strength was noted until the age of 16 years, when the patient had difficulty in rising from a sitting to a standing position. The weakness progressed rapidly after that time, and she became wheelchair-bound in her mid-20s.

On examination, the patient was 30 years old and of normal intelligence. She was of short stature (1.47 m in height) and had a marked midface hypoplasia with a broad nasal bridge and some mild weakness of eye closure. There was a striking discrepancy between her rather broad, short "buffalo neck," with increased subcutaneous adipose tissue and fat accumulation also in the facial region, and the extremely thin trunk and extremities (Figure 1). The deltoid and shoulder muscles were relatively well preserved compared with biceps and triceps. There were contractures in the elbows, finger flexors, spine, and Achilles tendons. Muscle power was most markedly reduced in the biceps, triceps, and quadriceps. The patient showed moderate weakness of neck flexion, hip flexion, and hip extension. She also had myalgia along with constant back pain and joint stiffness. At the age of 29 years, the patient had experienced recurrent episodes of palpitations. An electrocardiogram showed recurrent atrial fibrillation and a brady-tachy syndrome treated with amiodarone. A pacemaker was inserted at the age of 30 years, but she continued to experience tachyarrhythymias, and treatment with a β-blocker was added. Also at the age of 29 years, the patient required the institution of noninvasive nighttime ventilation. At examination, she was using ventilation for up to 12 hours overnight.

The patient also gave a history of severe menorrhagia and abnormal hair growth at the age of 17 years. Ultrasound of the pelvis at the age of 19 years disclosed uterine fibroids. Cholesterol level was elevated on 2 occasions up to a maximum of 313 mg/dL (8.1 mmol/L) (reference range, <251 mg/dL [<6.5 mmol/L]) and was within the normal range between these occasions. Levels of triglycerides, low-density lipoprotein cholesterol, apolipoprotein B, and osmolality were at the upper limit of the reference ranges. Levels of creatine kinase, serum glucose, apolipoprotein A1, and calcium were consistently within reference ranges. Nerve conduction velocity in the upper and lower leg were in the reference range.

The other 3 patients all had typical humeroperoneal distribution of muscle weakness. In 1 of the 3 (case 3) there was early onset in the second year of life and a rapidly progressive course with loss of independent ambulation at 5 years. This child also showed progressively reduced respiratory function, and at age 6 years he had forced vital capacity less than 40%. The other 2 patients had onset at school age and a slowly progressive course (Table 1).

Muscle magnetic resonance imaging was performed in cases 3, 4, and 5 (Figure 2) and showed selective involvement of the quadriceps muscles in the thigh in cases 4 and 5 and selective involvement of quadriceps and adductor magnus in case 3. At calf level they all showed more diffuse involvement of the gastrocnemius and soleus muscles, with the medial head of the gastrocnemius relatively spared compared with the lateral one.

The cases described in this article show that the phenotype of patients with laminopathies can be far more severe than has been reported so far and expand the spectrum of clinical phenotype associated with a single LMNA mutation reported in the literature.

All patients described herein carried the same mutation but different phenotypes, and 2 of the 5 cases had peculiar features that have not been previously described in patients with laminopathy. All the cases were de novo mutations, confirming previous observations that de novo mutations are extremely common in patients with dominant mutations in the LMNA gene.²

Patient 1 had laminopathy associated with congenital onset, with clear signs of muscle involvement such as severe weakness and hypotonia in the first months of life. In patients with AD-EDMD, onset is typically at school age with contractures. An earlier onset, associated with a more severe phenotype, has been reported,^{2 - 3} but even in those cases the onset was after these children had acquired independent ambulation. In our patient, independent sitting posture was never achieved.

Patient 2 had a limb-girdle distribution of weakness and also showed a pattern of abnormal fat accumulation around the face and neck, together with marked lack of subcutaneous fat in the limbs in a manner reminiscent of familial partial lipodystrophy. It has up to now been believed that there was a correlation between the genotype and the phenotype in LMNA gene mutations regarding familial partial lipodystrophy, in which 90% mutations were localized in exon 8 of the LMNA gene. Recently, the association of lipodystrophy in patients with other characteristic presentations of EDMD, limb-girdle muscular dystrophy type 1B, or dilated cardiomyopathy with conduction system disease has been highlighted in patients who demonstrated overlapping phenotypes, ie, laminopathies affecting striated muscles and adipose tissue.^{15 - 16} These patients carried mutations in exons 1 and 9, and so far there is no report of signs of lipodystrophy associated with mutations in exon 6. Patient 2 also showed unique features of short stature and dysmorphisms, indicating skeletal involvement. The phenotype in this case is not the same as mandibuloacral dysplasia, but it does suggest that skeletal involvement may be a broader part of the phenotype than previously thought. So far, only one LMNA mutation, homozygous R527P, was reported in patients with autosomal recessive mandibuloacral dysplasia phenotype.^{11 ,18} It is clear from this and other studies^{6 ,16 - 17 ,19} that there may be more of an overlap of these different features of laminopathy than has previously been reported.

Patient 2 also exhibited early respiratory failure and nocturnal hypoventilation requiring ventilatory support, which has also been previously reported in patients with R453W LMNA mutations.²⁰ It is of interest that another 2 patients (cases 1 and 3) also had signs of respiratory impairment, with patient 1 having a prevalently diaphragmatic breathing pattern and patient 3 a forced vital capacity of 40% at age 6 years.

Muscle biopsy specimens showed myopathic changes in all cases, but patient 1 additionally had other peculiar features that have not previously been reported. Mild up-regulation of major histocompatibility complex I on the sarcolemma of mature fibers was seen in 2 muscle biopsy specimens, leading to the initial diagnosis of an inflammatory myopathy. Up-regulation of major histocompatibility complex class I has not previously been reported in patients with LMNA mutations but is a feature of inflammatory myopathies and has been described in Duchenne and Becker muscular dystrophy^{21 - 22} and in cases with dysferlin deficiency.²³ The reasons for and consequences of this up-regulation for disease are not clear²⁴ and in our case might be related to the age of the patient studied or the severity of the pathological process.

Our cases increase the spectrum of phenotypes associated with this E358K LMNA mutation, which in the past has been found associated with limb-girdle muscular dystrophy type 1D in 1 patient²⁵ and with AD-EDMD in 3 patients.² These findings confirm previous observations that the same mutation in the LMNA gene can be associated with a large spectrum of clinical phenotypes and that overlapping features of the different conditions in which lamin A/C mutations have been described may be seen in individual patients. The way in which the mutations alter the function of the protein to produce the different clinical phenotypes remains unclear,²⁶ although the wide spectrum of conditions caused by mutations in the LMNA gene strongly supports the hypothesis that a multiprotein complex exists at the nuclear envelope whose alterations might be responsible for the different phenotypes described.²⁷ The existence of distinct functional domains in lamin A/C might be essential for the maintenance and integrity of different cell lineages. The interactions of lamin A/C may be diverse in different cell types, and specific mutations may modify some of these interactions, eventually causing tissue- or cell type–specific alterations of the nuclear envelope.

Corresponding author: Eugenio Mercuri, MD, Department of Paediatrics, Hammersmith Hospital, Du Cane Road, London W12 OHN, England (e-mail: e.mercuri@ic.ac.uk).

Accepted for publication November 19, 2003.

This study was supported by grants from the European Union Fifth Framework (contract QLG1-1999-00870 from the MYO-CLUSTER/EUROMEN, Brussels, Belgium). Dr Bonne was also supported by grant RGP0057/2001-M101 from the Human Frontier Science Program, Strasbourg, France. Support from the NSCAG (National Specialist Commissioning Advisory Group) and the Muscular Dystrophy Campaign, London, England, to the Hammersmith and Newcastle muscle centers is also acknowledged.