A rare LMNA missense mutation causing a severe phenotype of mandibuloacral dysplasia type A: a case report

ABSTRACT Objective: To report the case of a girl presenting a severe phenotype of mandibuloacral dysplasia type A (MADA) characterized by prominent osteolytic changes and ectodermal defects, associated with a rare homozygous LMNA missense mutation (c.1579C>T). Case description: A 6-year-old girl was evaluated during hospitalization exhibiting the following dysmorphic signs: subtotal alopecia, dysmorphic facies with prominent eyes, marked micrognathia and retrognathia, small beaked nose, teeth crowding and thin lips, generalized lipodystrophy, narrow and sloping shoulders, generalized joint stiffness and bone reabsorption in the terminal phalanges. In dermatological examination, atrophic skin, loss of cutaneous elasticity, hyperkeratosis, dermal calcinosis, and hyperpigmented and hypochromic patches were observed. Radiology exams performed showed bilateral absence of the mandibular condyles, clavicle resorption with local amorphous bone mass confluence with the scapulae, shoulder joints with subluxation and severe bone dysplasia, hip dysplasia, osteopenia and subcutaneous calcifications. Comments: MADA is a rare autosomal recessive disease caused by mutations in LMNA gene. It is characterized by craniofacial deformities, skeletal anomalies, skin alterations, lipodystrophy in certain regions of the body and premature ageing. Typical MADA is caused by the p.R527H mutation in the LMNA gene. However, molecular analysis performed from oral epithelial cells obtained from the patient showed the rare mutation c.1579C>T, p. R527C in the exon 9 of LMNA. This is the sixth family identified with this mutation described in the literature.


INTRODUCTION
Mandibuloacral dysplasia (MAD) is a genetic condition belonging to the category of laminopathies, that comprise the progeroid syndromes and lipodystrophies, in which MAD is included, as well as muscular dystrophies, cardiomyopathies and hereditary peripheral neuropathies. 1,2It is a rare autosomal recessive disorder characterized by craniofacial anomalies, skeletal abnormalities including generalized osteoporosis, progressive osteolysis of the distal phalanges and clavicles, growth retardation, joint contractures, pigmentary and atrophic skin changes, lipodystrophy signs and mildly accelerated ageing. 3,4atients with MAD are categorized according to trends of lipodystrophy in types A and B, which are linked to different genetic defects.Mutations in LMNA, which encodes lamin A and C, are associated to type A pattern of lipodystrophy (MADA; OMIM #248370) and mutations on the zinc metalloproteinase ZMPSTE24, encoding a protease involved in post-translational proteolytic processing of prelamin A to form mature lamin A, are linked to type B lipodystrophy (MADB; OMIM #608612). 4,5his study reports the clinical case of a 6-year-old girl presenting a severe phenotype linked to LMNA mutation.

CASE REPORT
The 6-year-old girl is the eldest of three children belonging to a consanguineous couple.The patient in this report was born at term, weighing 2.9 kg and measuring 50 cm, by vaginal delivery, Apgar score of 8/9.In the neonatal period, she evolved with an ischemic stroke in the left middle cerebral artery with hemorrhagic transformation probably associated to polycythemia (hematocrit=69%).However, it was not possible to define by imaging exams whether the alteration had occurred intrauterinely or postnatally.The child was followed up in a hematology service until the age of four, where tests were carried out for hereditary thrombophilia and vasculitis.
Polymerase chain reaction (PCR) analysis performed at 21 months of age evidenced the presence of the factor V Leiden mutation in heterozygosis, an alteration that possibly contributed to the stroke in the neonatal period.There were no mutations in the prothrombin gene, and low levels of factor VIII were reported (39%).Antibody testing for anticardiolipin and lupus anticoagulant, which may be associated with thrombosis, was negative, as well as the investigation for systemic sclerosis, through the determination of anti-fibrillarin, anticentromere, and anti-DNA topoisomerase I antibodies.Tests to identify antinuclear antibodies (ANA), rheumatoid factor, and C-reactive protein were normal.
Regarding phenotypic changes, the patient at birth had a normal appearance but during the first year of life mild abnormalities appeared, such as hair loss and whitish lesions in the joints of the hands at four months of age, and hyperchromic spots on the body at six months.She evolved with normal developmental parameters, having acquired independent walking at 14 months.At the age of two, she was evaluated in a specialized hospital and, according to the medical report, presented weight, height, and head circumference below the 3 rd percentile, dysmorphic face characterized by thin skin, sharp nose, hypoplastic malar region, prominent cranial veins, irregular skin discoloration, shortage of subcutaneous fat, rigid finger joints, and fine hair with alopecia in the occipital region.
Laboratory tests performed, such as a screening for innate metabolism errors and organic acidemias, transferrin isoelectric focusing, molecular genetic test for mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), karyotype, serum dosage of thyroxine, thyroid stimulating hormone, aspartate aminotransferase and alanine aminotransferase, were all normal.Imaging exams revealed osteolysis of the distal phalanx (fingers and toes), decreased bone density, deformity of the hip joints, hypoplasia of the head of the radius and clavicles, widening of the sagittal sutures.
The patient displayed progressive clinical worsening.At four years of age, after a trauma in both knees, the joints were fixed in a flexible position, and she was unable to walk anymore.At six year of age, the child had the following dysmorphic signs: subtotal alopecia, dysmorphic facies with prominent eyes, marked micrognathia and retrognathia, small beaked nose, teeth crowding and thin lips (Figures 1A, 1B and 1C), generalized lipodystrophy, narrow and sloping shoulders, generalized joint stiffness (Figure 1D), and bone reabsorption in the terminal phalanges (Figures 1E and 1F).
In dermatological examination, atrophic skin, loss of cutaneous elasticity, hyperkeratosis, dermal calcinosis, and hyperpigmented and hypochromic patches were observed (Figures1D to 1G).Diffuse gingivitis with bleeding and presence of significant crowding of the teeth were noted.The marked microretrognathia and glossoptosis, causing significant upper airway obstruction, culminated in the placement of a tracheostomy.The computed tomography (CT) scan of the facial bones allowed the detection of hypoplasia of the mandible branch, with absence of the condyles bilaterally and teeth crowding (Figure 2A).
Further radiographic exams revealed clavicles bilaterally absent with the presence of local amorphous bone mass confluent with the scapulae (fused acromioclavicular joints) (Figure 2B), humerus and shoulder joints with subluxation and major bone dysplasia (Figure 2C), hip dysplasia (Figures 2D and 2E), significant osteopenia, and subcutaneous calcifications (Figures 2E  and 2F).Cardiac CT performed to assess the coronary calcium score did not demonstrate coronary artery disease.
The genomic DNA isolated from oral epithelial cells was amplified by PCR with primers designed to all exons and flanking intronic regions of LMNA and ZMPSTE24 described (http://www.hgmd.cf.ac.uk/ac/all.php,Supplementary Table 1).
After amplification, PCR products were cleaned up and sequenced with a BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, CA, USA).The sequences were analyzed with a 3500 Genetic Analyzer (Applied Biosystems, CA, USA).The genetic analysis detected the mutation c.1579C>T, p. R527C in the exon 9 of LMNA, compatible with the diagnosis of MADA.The analysis of the patient's parents confirmed the autosomal recessive transmission of the c.1579C> T mutation in exon 9 of the LMNA gene (Figure 2G).

DISCUSSION
Progeroid laminopathies are a group of rare genetic disorders characterized by the premature appearance of certain signs of physiological aging in a subset of tissues. 6Within the group, Hutchinson-Gilford progeria syndrome (HGPS) is the most common type and this was the diagnosis initially established for the patient.It represents the most widely studied condition in this group of disorders. 7,8Interestingly, while bone, skin, and adipose tissue are severely affected in all forms of LMNAlinked progeria, the cardiovascular system is selectively involved in the HGPS.Atherosclerosis is the most important clinical manifestation, considering that this is the cause of premature death in these patients from myocardial infarction or stroke in early adolescence. 8,9Our patient was submitted to a cardiac CT to assess the coronary calcium score, without evidence of coronary artery disease.
In addition to HGPS, the progeroid laminopathies include MADA, MADB, Nestor-Guillermo progeria syndrome (NGPS) 7 and restrictive dermopathy.While HGPS and MADA are linked to mutations in the LMNA gene, MADB and restrictive dermopathy are associated with a mutation in ZMPSTE24, a gene that encodes a zinc metalloproteinase necessary for the correct processing of prelamin A to mature lamin A. 5,10 NGPS, on the other hand, is due to a mutation in the BANF1 gene, which plays key roles in gene expression, nuclear assembly and chromatin organization. 11The genetic analysis performed in the patient revealed the mutation c.1579C>T (p.R527C) in exon 9 of the LMNA gene, leading to the diagnosis of MADA.
Pronounced acro-osteolysis is a signature symptom of MAD. 10,12Typical MADA is caused by the p.R527H mutation in the LMNA gene, while other LMNA mutations are responsible for different phenotypes. 8Regarding the phenotype, patients with MADA usually have distinguished features compared to MADB, which include more severity of clinical phenotype, early onset, renal disease, calcified skin nodules, premature birth and lack of acanthosis nigricans in the MADB type. 4,10,12Thus, a milder phenotype and slower disease progression is generally expected in MADA.However, the patient described in this case report had an earlier onset of symptoms and more severe phenotype than that usually described for MADA.In agreement with these findings, patients already identified with this mutation have been described in the literature as presenting a similar phenotype and clinical course.Agarwal was the first to describe this mutation, in 2008. 12The clinical manifestations highlighted by him that denote the precocity of onset and severity of the phenotype associated are growth impairment already apparent at one year of age; joint stiffness, skin thinning, and mottled hyperpigmentation developed by 15 months of age; bird face with beak nose, bulbous forehead and very small mouth with limited opening capacity seen at two years of age; progressive clavicular hypoplasia, acro-osteolysis and severe hair loss in the temporal and occipital areas described at three years of age; difficulty swallowing and severe snoring with obstructive apnea; very crowded teeth; lipodystrophy evidenced in the first years of life; severe joint contractures, resulting in abnormal posture and decreased mobility. 12ll these alterations described by Agarwal were also observed at an early age in the patient in this report. 12In addition, radiology exams performed at six years of age showed absence of bilateral mandibular condyles, clavicle resorption with local amorphous bone mass confluence with the scapulae, humeral and shoulder joints with subluxation and severe bone dysplasia, hip dysplasia, osteopenia and subcutaneous calcifications, the latter a feature commonly described in MADB.Another important consideration is that biochemical tests performed revealed metabolic disorder with the presence of hypercholesterolemia and increased low-density lipoprotein cholesterol, probably associated with the generalized lipodystrophy observed in our patient, a pattern of lipodystrophy typically expected for MADB. 4,5,13iang in 2009 also reported the case of two Chinese brothers who presented the homozygous c.1579C>T (R527C) mutation found in our patient, which he describes as having atypical HGPS, not MADA. 14The presence of electrocardiogram (ECG) abnormalities suggesting myocardial ischemia evidenced in the older patient is a commonly expected finding in patients with HGPS.However, the intense osteolysis present at an early age in both siblings is a sign of MAD.In addition, the presence of full cheeks was also a finding contrary to what is expected for typical HGPS.Based on the analysis of the clinical data and the result of the molecular analysis performed by Liang, the evidence points, in our opinion, that the probable diagnosis of the two children was MADA, and not atypical HGPS as described by the authors.
In another study carried out, patients descended from three unrelated consanguineous families were evaluated. 15,16The same c.1579C>T -p.(Arg527Cys) mutation identified in our study was found in two of the evaluated families.The age at onset of symptoms in the patients was considerably younger than usually described for MADA.These findings are consistent with the results of this research, which is the association of the mutation c.1579C>T with severe MADA disorder.
The same mutation was found by Luo in 2014, in three Chinese sisters. 4He also reports that the phenotype of the patients was more severe than those previously reported in MADA.As a manifestation not previously described for patients with this mutation, there was the possibility of associated muscle damage in two of the sisters, based on the increase in phosphocreatine and an altered electromyography result observed.
Petillo, in 2015, reported the clinical case of four individuals who had a de novo mutation in the LMNA gene.Among these, the genetic study of patient 4 identified the mutation c.1579C>T described as being a de novo missense pathogenic mutation in autosomal dominant inheritance. 17he genetic analysis carried out in our research detected the same mutation c.1579C>T, but with recessive transmission, confirmed by analysis of DNA sample obtained from parents.It is know that any direct correlation between mutations in the LMNA gene and clinical manifestations is hampered by the pleiotropic effect possibly exerted by Lamin A/C gene mutations. 18These pieces of evidence probably explain the very different clinical manifestations in this case when compared to the phenotype observed in our patient, as well as in other cases in the literature reported in this study, and the divergent diagnosis established as Emery-Dreifuss muscular dystrophy.
Of great relevance for the specific diagnosis of genetic syndromes are the recent advances obtained with the use of machine-learning algorithms for image analysis in clinical practice.Through the use of artificial intelligence in the evaluation of images of patients, it is possible to detect similarities of new phenotypes with known disorders and, thus, identify a potential molecular etiology.Using two different image analysis algorithms as a tool, Marbach et al. correctly identified that two individuals from different ethnic backgrounds had highly similar facial dysmorphology and phenotypic proximity to other nuclear envelopathies. 19The hitherto unknown genetic disorder in both was identified as being caused by the same de novo mutation in LEMD2.This disorder exhibits a distinct combination of symptoms with phenotypic overlap with other established segmental progeroid syndromes described in the present study, but with an overall better prognosis when compared to these related syndromes. 19n conclusion, this study led to the identification of an unusual mutation in the LMNA gene, which contributes to the pathogenicity of MADA.This is the sixth family identified with this mutation described in the literature.The clinical manifestations reported so far in patients with the c.1579C>T mutation are consistent with a more severe phenotype, with an earlier age of onset of osteolysis, alopecia, and lipodystrophy.Given the importance and multiple functions of lamina A and lamina C in maintaining the structure and function of the cell nucleus, it would be expected that mutations in the LMNA gene could cause different types of genetic disorders, with different phenotypes.Thus, mutations in different locations of this

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Figure 1 .
Figure 1.Patient pictures (A: frontal and B: lateral view of the patient, with dysmorphic signs: subtotal alopecia, prominent eyes, marked micrognathia and retrognathia, small beaked nose; C: image show thin lips and teeth crowding; D: full body view of the patient, with generalized joint stiffness with abnormal posture and decreased mobility; E, F, G: dermatological changes of the patient, with hypochromic patches, bone reabsorption in the terminal phalanges and dysplastic nails).

Figure 2 .*
Figure 2. Exams result (A: cone-beam computed tomography images: hypoplasia of the mandible branch, with bilateral absence of the mandibular condyles and teeth crowding; B: radiographic findings: clavicle absent, with the presence of local amorphous bone mass confluent with the scapulae; C: humerus and shoulder joints with subluxation and major bone dysplasia; D: high hip dysplasia; E: left hip dysplasia; F: significant osteopenia and subcutaneous calcification; G: sequencing of the patient's genetic material and her parents).