A stagewise response to mitochondrial dysfunction in mitochondrial DNA maintenance disorders.

Mitochondrial DNA (mtDNA) deletions which clonally expand in skeletal muscle of patients with mtDNA maintenance disorders, impair mitochondrial oxidative phosphorylation dysfunction. Previously we have shown that these mtDNA deletions arise and accumulate in perinuclear mitochondria causing localised mitochondrial dysfunction before spreading through the muscle fibre. We believe that mito-nuclear signalling is a key contributor in the accumulation and spread of mtDNA deletions, and that knowledge of how muscle fibres respond to mitochondrial dysfunction is key to our understanding of disease mechanisms. To understand the contribution of mito-nuclear signalling to the spread of mitochondrial dysfunction, we use imaging mass cytometry. We characterise the levels of mitochondrial Oxidative Phosphorylation proteins alongside a mitochondrial mass marker, in a cohort of patients with mtDNA maintenance disorders. Our expanded panel included protein markers of key signalling pathways, allowing us to investigate cellular responses to different combinations of oxidative phosphorylation dysfunction and ragged red fibres. We find combined Complex I and IV deficiency to be most common. Interestingly, in fibres deficient for one or more complexes, the remaining complexes are often upregulated beyond the increase of mitochondrial mass typically observed in ragged red fibres. We further find that oxidative phosphorylation deficient fibres exhibit an increase in the abundance of proteins involved in proteostasis, e.g. HSP60 and LONP1, and regulation of mitochondrial metabolism (including oxidative phosphorylation and proteolysis, e.g. PHB1). Our analysis suggests that the cellular response to mitochondrial dysfunction changes depending on the combination of deficient oxidative phosphorylation complexes in each fibre.

Quantitative whole-body muscle MRI in idiopathic inflammatory myopathies including polymyositis with mitochondrial pathology: indications for a disease spectrum.

OBJECTIVE: Inflammatory myopathies (IIM) include dermatomyositis (DM), sporadic inclusion body myositis (sIBM), immune-mediated necrotizing myopathy (IMNM), and overlap myositis (OLM)/antisynthetase syndrome (ASyS). There is also a rare variant termed polymyositis with mitochondrial pathology (PM-Mito), which is considered a sIBM precursor. There is no information regarding muscle MRI for this rare entity. The aim of this study was to compare MRI findings in IIM, including PM-Mito. METHODS: This retrospective analysis included 41 patients (7 PM-Mito, 11 sIBM, 11 PM/ASyS/OLM, 12 IMNM) and 20 healthy controls. Pattern of muscle involvement was assessed by semiquantitative evaluation, while Dixon method was used to quantify muscular fat fraction. RESULTS: The sIBM typical pattern affecting the lower extremities was not found in the majority of PM-Mito-patients. Intramuscular edema in sIBM and PM-Mito was limited to the lower extremities, whereas IMNM and PM/ASyS/OLM showed additional edema in the trunk. Quantitative assessment showed increased fat content in sIBM, with an intramuscular proximo-distal gradient. Similar changes were also found in a few PM-Mito- and PM/ASyS/OLM patients. In sIBM and PM-Mito, mean fat fraction of several muscles correlated with clinical involvement. INTERPRETATION: As MRI findings in patients with PM-Mito relevantly differed from sIBM, the attribution of PM-Mito as sIBM precursor should be critically discussed. Some patients in PM/ASyS/OLM and PM-Mito group showed MR-morphologic features predominantly observed in sIBM, indicative of a spectrum from PM/ASyS/OLM toward sIBM. In some IIM subtypes, MRI may serve as a biomarker of disease severity.

Skeletal muscle fibers produce B-cell stimulatory factors in chronic myositis.

Introduction: We aimed to identify B-cell-mediated immunomechanisms in inclusion body myositis (IBM) and polymyositis (PM) as part of the complex pathophysiology. Materials and methods: Human primary myotube cultures were derived from orthopedic surgery. Diagnostic biopsy specimens from patients with IBM (n=9) and PM (n=9) were analyzed for markers of B cell activation (BAFF and APRIL) and for chemokines that control the recruitment of B cells (CXCL-12 and CXCL-13). Results were compared to biopsy specimens without myopathic changes (n=9) and hereditary muscular dystrophy (n=9). Results: The mRNA expression of BAFF, APRIL, and CXCL-13 was significantly higher in IBM and PM compared to controls. Patients with IBM displayed the highest number of double positive muscle fibers for BAFF and CXCL-12 (48%) compared to PM (25%), muscular dystrophy (3%), and non-myopathic controls (0%). In vitro, exposure of human myotubes to pro-inflammatory cytokines led to a significant upregulation of BAFF and CXCL-12, but APRIL and CXCL-13 remained unchanged. Conclusion: The results substantiate the hypothesis of an involvement of B cell-associated mechanisms in the pathophysiology of IBM and PM. Muscle fibers themselves seem to contribute to the recruitment of B cells and sustain inflammation.

Personality factors in patients with myasthenia gravis: A prospective study.

INTRODUCTION: In myasthenia gravis (MG), depression and anxiety have frequently been reported as comorbidities. However, little is known about personality characteristics in MG patients. We aimed to characterise personality traits in MG and to correlate them with disease severity and disease course. METHODS: The Big Five Inventory data questionnaire was used to investigate personality traits in 44 MG patients and 45 healthy controls similar in age and gender. In 28 MG patients, a caregiver was also available for patient assessments to limit bias associated with social desirability in patients' responses. Patients were assessed with regard to premorbid personality (before manifestation of MG) and to present condition. In addition, anxiety and depression scales (Hospital Anxiety and Depression Scale and Beck Anxiety Inventory) were applied. RESULTS: Compared to controls, MG patients showed significantly higher levels of neuroticism, whereas openness and extraversion were significantly lower. Agreeableness and conscientiousness did not differ between groups. Neuroticism was influenced by disease severity such as generalization of weakness, presence of thymoma, and bulbar involvement as well as disease duration. Neuroticism correlated with premorbid level of neuroticism but also with depression and anxiety scores. CONCLUSION: A personality profile of increased neuroticism and lower openness and extraversion in MG patients may contribute considerably to the perception of disease severity. It may also be related to frequent comorbidities such as anxiety and depression. Although premorbid levels of neuroticism were increased, this characteristic may also increase considerably during the course of the disease. The data indicate that muscle weakness in MG is accompanied or even complicated by psychological aspects. Therefore, a psychological and behavioral intervention in addition to the specific pharmacological therapy might be of particular value.

Normal Thermostability of p.Ser113Leu and p.Arg631Cys Variants of Mitochondrial Carnitine Palmitoyltransferase II (CPT II) in Human Muscle Homogenate.

Previous fibroblast and recombinant enzyme studies showed a markedly thermolabile p.Ser113Leu variant compared to the wild-type (WT) in muscle carnitine palmitoyltransferase II (CPT II) deficiency. Additionally, it has been shown that cardiolipin (CLP) stimulated or inhibited the p.Ser113Leu recombinant variant depending on the pre-incubation temperatures. In this study, the thermolabilities of mitochondrial enzyme CPT II in muscle homogenates of patients with the p.Ser113Leu (n = 3) and p.Arg631Cys (n = 2) variants were identified to be similar to that of WT. Pre-incubation with CLP on ice stimulated the WT enzyme more than both variants. However, CLP stimulated the variants and WT at 46 °C to about 6-18-fold. The present data indicate that the thermostability of CPT II variant in muscle homogenate is similar to that of WT. This is in contrast to the increased thermolability of enzymes derived from fibroblast and that of recombinant enzymes. Hence, it can be speculated that the disruption of the compartmentation in muscle homogenate mediates a protective effect on the thermolability of the native variant. However, the exact mechanism remains unclear. However, the activating effect of CLP on CPT II in muscle homogenate seems to align with those on recombinant enzymes.

GFPT1-Associated Congenital Myasthenic Syndrome Mimicking a Glycogen Storage Disease - Diagnostic Pitfalls in Myopathology Solved by Next-Generation-Sequencing.

GFPT1-related congenital myasthenic syndrome (CMS) is characterized by progressive limb girdle weakness, and less prominent involvement of facial, bulbar, or respiratory muscles. While tubular aggregates in muscle biopsy are considered highly indicative in GFPT1-associated CMS, excessive glycogen storage has not been described. Here, we report on three affected siblings with limb-girdle myasthenia due to biallelic pathogenic variants in GFPT1: the previously reported missense variant c.41G > A (p.Arg14Gln) and the novel truncating variant c.1265_1268del (p.Phe422TrpfsTer26). Patients showed progressive proximal atrophic muscular weakness with respiratory involvement, and a lethal disease course in adulthood. In the diagnostic workup at that time, muscle biopsy suggested a glycogen storage disease. Initially, Pompe disease was suspected. However, enzymatic activity of acid alpha-glucosidase was normal, and gene panel analysis including 38 genes associated with limb-girdle weakness (GAA included) remained unevocative. Hence, a non-specified glycogen storage myopathy was diagnosed. A decade later, the diagnosis of GFPT1-related CMS was established by genome sequencing. Myopathological reexamination showed pronounced glycogen accumulations, that were exclusively found in denervated muscle fibers. Only single fibers showed very small tubular aggregates, identified in evaluation of serial sections. This family demonstrates how diagnostic pitfalls can be addressed by an integrative approach including broad genetic analysis and re-evaluation of clinical as well as myopathological findings.

Different K+-release in distal myogenic and neurogenic muscular weakness during non-ischemic exercise.

INTRODUCTION: In myotonic dystrophy, an increased potassium release upon ischemic forearm exercise has been previously described. However, it remains unclear whether this is specific for myotonic dystrophies or just due to distal muscular weakness. METHODS: Non-ischemic forearm test (NIFET) was performed and venous K+ concentration was measured at rest and at three different force levels (20-30%, 50-60%, 70-80%) related to maximal contraction force (MCF) in patients with distal myogenic (n = 7), neurogenic (n = 7) muscular weakness and healthy volunteers (n = 12). The specific K+ release was defined as K+ increase related to workload as force-time-integral during repetitive contraction. RESULTS: Workload was lower at all force levels in both disease groups compared to the control group. With increasing workload, the K+ concentrations increased in all study groups. Analysing individual force levels related to the maximum contraction force (MCF), a higher specific K+ release was measured at low force levels in myopathies (20-30% MCF) in comparison to higher force levels (p = 0.02). At 20-30% MCF, the specific K+ release was significantly higher in myogenic compared to neurogenic muscular weakness (p = 0.005). At 50-60% and 70-80% MCF, the specific K+ values converged and did not significantly differ between the three groups (p = 0.09 and p = 0.37). DISCUSSION: At low force levels, K+ efflux related to workload is higher in patients with myogenic in comparison to neurogenic distal paresis. Our results indicate a different regulation of K+ balance in neurogenic and myogenic muscular weakness possibly due to a different recruitment behaviour of motor units and the firing rate of motor neurons.

[On the Conceptual History of Myotonia Congenita: The Contributions of Julius Thomsen and Adolph Seeligmüller]. / Zur Konzeptgeschichte der Myotonia congenita: Die Beiträge von Julius Thomsen und Adolph Seeligmüller.

Myotonia congenita was first described as an entity in 1876 by Julius Thomsen. Shortly later in the same year it was criticized by Adolph Seeligmüller who extended the clinical findings. Charles Bell, Moritz Benedict and Ernst von Leyden had already partly described the symptoms of the disease before 1876, but did not recognize this as a new entity. A comparison of the publications of Thomsen and Seeligmüller in 1876 and of Seeligmüller's textbook published in 1887, as well as the today's genetically proven disease shows that Seeligmüller correctly criticized two aspects of Thomsen's publication: (i) Thomsen suspected the pathogenesis to be in "one half of the brain's activity, the will" with "seat in the cerebrospinal system" and (ii) he made the assumption of a coordination disorder in the sense of an ataxia [1]. Due to a better understanding of the pathogenesis enabled by Seeligmüller's postulate of a "more difficult mobile muscle substance" [2] without excluding an inborn affection of the lateral cords of the spinal cord, it would have been entirely justified to recognize Seeligmüller's contribution to the conceptual history of Myotonia congenita by including his name in the eponym [3].

Cardiolipin Stabilizes and Increases Catalytic Efficiency of Carnitine Palmitoyltransferase II and Its Variants S113L, P50H, and Y479F.

Muscle carnitine palmitoyltransferase II (CPT II) deficiency is associated with various mutations in CPT2 gene. In the present study, the impact of the two CPT II variants P50H and Y479F were characterized in terms of stability and activity in vitro in comparison to wildtype (WT) and the well investigated variant S113L. While the initial enzyme activity of all variants showed wild-type-like behavior, the activity half-lives of the variants at different temperatures were severely reduced. This finding was validated by the investigation of thermostability of the enzymes using nano differential scanning fluorimetry (nanoDSF). Further, it was studied whether the protein stabilizing diphosphatidylglycerol cardiolipin (CL) has an effect on the variants. CL indeed had a positive effect on the stability. This effect was strongest for WT and least pronounced for variant P50H. Additionally, CL improved the catalytic efficiency for CPT II WT and the investigated variants by twofold when carnitine was the varied substrate due to a decrease in KM. However, there was no influence detected for the variation of substrate palmitoyl-CoA. The functional consequences of the stabilization by CL in vivo remain open.

Headache and migraine in mitochondrial disease and its impact on life-results from a cross-sectional, questionnaire-based study.

Headache is frequent in patients with mitochondrial disorders. Previous studies point to a higher prevalence of headache in these patients than in the general population. As mitochondrial disorders often present a variety of other symptoms, the question arises how much the presence of headache really influences daily life. We performed a cross-sectional, questionnaire-based study investigation with 61 patients with a genetically proved mitochondrial disease mainly composed of CPEO phenotype. Headache was examined using a standardized questionnaire, and classified according to ICHD-2. Headache-related disability was evaluated by the Headache-Impact-Test-6 (HIT-6). Additionally, depression and anxiety were examined using the Hospital Anxiety and Depression Scale (HADS) and Short-Form-Health Survey (SF-12) was used to investigate the health-related quality of life. Headache was reported by 43/61 (70.5%) of the patients. 35/61 patients (57.4%) described a Tension-type headache (TTH) and 26 patients (42.6%) a migraine. Patients reporting headache had a significantly higher HIT-6 score than those without (mean: 54.47 vs. 38.47, p < 0.001). The HIT-6 score was significantly higher in patients reporting a migraine compared to those with a tension-type headache (mean: 62.13 vs. 46.18, p < 0.001). In the HADS score and in the SF-12 were not significantly influenced by the occurrence of headache. This study confirms the previously reported frequent occurrence of headache in a large cohort of patients with a confirmed mitochondrial disease. Migraine had the greatest impact on daily living, which appeared not to be confounded by depression and anxiety. Thus, we conclude that Migraine may be a substantial contributor for burden of disease in patients with mitochondrial diseases.

Bi-allelic truncating mutations in VWA1 cause neuromyopathy.

The von Willebrand Factor A domain containing 1 protein, encoded by VWA1, is an extracellular matrix protein expressed in muscle and peripheral nerve. It interacts with collagen VI and perlecan, two proteins that are affected in hereditary neuromuscular disorders. Lack of VWA1 is known to compromise peripheral nerves in a Vwa1 knock-out mouse model. Exome sequencing led us to identify bi-allelic loss of function variants in VWA1 as the molecular cause underlying a so far genetically undefined neuromuscular disorder. We detected six different truncating variants in 15 affected individuals from six families of German, Arabic, and Roma descent. Disease manifested in childhood or adulthood with proximal and distal muscle weakness predominantly of the lower limbs. Myopathological and neurophysiological findings were indicative of combined neurogenic and myopathic pathology. Early childhood foot deformity was frequent, but no sensory signs were observed. Our findings establish VWA1 as a new disease gene confidently implicated in this autosomal recessive neuromyopathic condition presenting with child-/adult-onset muscle weakness as a key clinical feature.

Adolph Seeligmüller's contribution to myotonia congenita Thomsen.

In 1876, two articles appeared in Germany in different medical journals under almost the same title "Tonische Krämpfe in willkürlich beweglichen Muskeln in Folge von ererbter psychischer Disposition (Ataxia muscularis?)1" by Julius Thomsen and "Tonische Krämpfe in willkürlich beweglichen Muskeln (Muskelhypertrophie?)2" by Adolph Seeligmüller). The first article was by Julius Thomsen (1815-1896) from Kappeln, the second by Adolph Seeligmüller (1837-1912) from Halle (Saale). Both articles dealt with a disease that has later been referred to as myotonia congenita by Adolf Strümpell (1853-1925) in 1881. Carl Westphal (1833-1890), however, ignored the contribution of Seeligmüller and proposed to name the disease Thomsen'sche Krankheit (Thomsen disease). Despite, the temporal priority of Thomsen, the pathogenesis of the disease was more accurately described by Seeligmüller. He recognized the origin of the myotonia in voluntary muscle whereas Thomsen postulated the myotonia as a result of inherited psychological disposition. Thus, Seeligmüller's contribution to myotonia congenita has to be recognized and honored.

Effect of Recanalization on Cerebral Edema, Long-Term Outcome, and Quality of Life in Patients with Large Hemispheric Infarctions.

OBJECTIVES: Space-occupying cerebral edema is the main cause of mortality and poor functional outcome in patients with large cerebral artery occlusion (LVO). We aimed to determine whether recanalization of LVO would augment cerebral edema volume and the impact on functional outcome and quality of life (QoL). MATERIALS AND METHODS: Prospectively, 43 patients with large middle cerebral artery territory infarction or NIHSS ≥ 12 on admission were enrolled. The degree of recanalization (partial and complete versus no recanalization) was assessed by computed tomography (CT)-angiography or Duplex ultrasound more than 24 h after symptom onset. Cerebral edema volume was measured on follow up CTs by computer-based planimetry. Mortality, functional outcome (by modified Ranking Scale (mRS) and Barthel Index (BI)) were assessed at discharge and 12 months, and QoL (by SF-36 and EQ-5D-3L) at 12 months. RESULTS: Mean cerebral edema volume was 333±141 ml without recanalization (n=13, group 1) and 276±140 ml with partial or complete recanalization (n=30, group 2, p= 0.23). There were no significant differences in mortality at discharge (38% versus 23%), at 12 months (58% versus 48%), in functional outcome at discharge (mRS 0-3: 0% both; mRS 4-5: 62% versus 77%) and at 12 months (mRS 0-3: 0% versus 11%; mRS 4-5: 42% versus 41%). The BI improved significantly from discharge to 12 months only in group 2 (p=0.001). Mean physical component score in SF-36 was 25.6±6.4, psychological component score was 41.9±14.1. In the EQ-5D-3L, most patients reported problems with activities of daily living, reduced mobility, and selfcare. CONCLUSIONS: Recanalization of a large cerebral artery occlusion in the anterior circulation territories is not associated with amplification of post-ischemic cerebral edema but may be correlated with better long-term functional outcome. QoL was low and mainly dependent on physical disability. The association between recanalization, collateral status and development of cerebral edema after LVO and the effect on functional outcome and quality of life should be explored in a larger patient population.

Palmitate but Not Oleate Exerts a Negative Effect on Oxygen Utilization in Myoblasts of Patients with the m.3243A>G Mutation: A Pilot Study.

It is known that exposure to excess saturated fatty acids, especially palmitate, can trigger cellular stress responses interpreted as lipotoxicity. The effect of excessive free fatty acids on oxidative phosphorylation capacity in myoblasts of patients with the m.3243A>G mutation was evaluated with the mitochondrial (Mito) stress test using a Seahorse XF96 analyzer. ß-oxidation, measured with the Seahorse XF96 analyzer, was similar in patients and controls, and reduced in both patients and controls at 40 °C compared to 37 °C. Mito stress test in the absence of fatty acids showed lower values in patients compared to controls. The mitochondrial activity and ATP production rates were significantly reduced in presence of palmitate, but not of oleate in patients, showing a negative effect of excessive palmitate on mitochondrial function in patients. Diabetes mellitus is a frequent symptom in patients with m.3243A>G mutation. It can be speculated that the negative effect of palmitate on mitochondrial function might be related to diacylglycerols (DAG) and ceramides (CER) mediated insulin resistance. This might contribute to the elevated risk for diabetes mellitus in m.3243A>G patients.

Cellular Stress in the Pathogenesis of Muscular Disorders-From Cause to Consequence.

Cellular stress has been considered a relevant pathogenetic factor in a variety of human diseases. Due to its primary functions by means of contractility, metabolism, and protein synthesis, the muscle cell is faced with continuous changes of cellular homeostasis that require rapid and coordinated adaptive mechanisms. Hence, a prone susceptibility to cellular stress in muscle is immanent. However, studies focusing on the cellular stress response in muscular disorders are limited. While in recent years there have been emerging indications regarding a relevant role of cellular stress in the pathophysiology of several muscular disorders, the underlying mechanisms are to a great extent incompletely understood. This review aimed to summarize the available evidence regarding a deregulation of the cellular stress response in individual muscle diseases. Potential mechanisms, as well as involved pathways are critically discussed, and respective disease models are addressed. Furthermore, relevant therapeutic approaches that aim to abrogate defects of cellular stress response in muscular disorders are outlined.

The Effect of Resveratrol on Mitochondrial Function in Myoblasts of Patients with the Common m.3243A>G Mutation.

Mitochondrial function is essential for ATP-supply, especially in response to different cellular stressors. Increased mitochondrial biogenesis resulting from caloric restriction (CR) has been reported. Resveratrol (RSV) is believed to mimic the physiological effects of CR mainly via a sirtuin (SIRT) 1-dependent pathway. The effect of RSV on the physiological function of mitochondrial respiratory complexes was evaluated using a Seahorse XF96. Myoblasts of five patients harboring the m.3243A>G mutation and five controls were analyzed. The relative expression of several genes involved in mitochondrial biogenesis was evaluated for a better understanding of the coherent mechanisms. Additionally, media-dependent effects of nutritional compounds and hormonal restrictions (R) on myoblasts from patients and controls in the presence or absence of RSV were investigated. Culturing of myoblasts under these conditions led to an upregulation of almost all the investigated genes compared to normal nutrition. Under normal conditions, there was no positive effect of RSV on mitochondrial respiration in patients and controls. However, under restricted conditions, the respiratory factors measured by Seahorse were improved in the presence of RSV. Further studies are necessary to clarify the involved mechanisms and elucidate the controversial effects of resveratrol on SIRT1 and SIRT3 expression.

Whole-body muscle MRI of patients with MATR3-associated distal myopathy reveals a distinct pattern of muscular involvement and highlights the value of whole-body examination.

OBJECTIVE: MATR3-associated distal myopathy is a rare distal myopathy predominantly affecting lower legs as well as wrist- and finger extensors. Whilst most distal myopathies are clinically and genetically well characterized, diagnosis often remains challenging. Pattern-based magnetic resonance imaging (MRI) approaches offer valuable additional information. However, a consistent pattern of muscular affection is missing for most distal myopathies. Thus, the aim of the present study was to establish a disease-specific pattern of muscular involvement in MATR3-associated distal myopathy using whole-body MRI. METHODS: 15 patients (25-79 years of age, 7 female) with MATR3-associated distal myopathy were subjected to whole-body MRI. The grade of fatty involution for individual muscles was determined using Fischer-Grading. Results were compared to established MRI-patterns of other distal myopathies. RESULTS: There was a predominant affection of the distal lower extremities. Lower legs showed a severe fatty infiltration, prominently affecting gastrocnemius and soleus muscle. In thighs, a preferential involvement of semimembranous and biceps femoris muscle was observed. Severe affection of gluteus minimus muscle as well as axial musculature, mainly affecting the thoracic segments, was seen. A sufficient discrimination to other forms of distal myopathy based solely on MRI-findings of the lower extremities was not possible. However, the inclusion of additional body parts seemed to yield specificity. INTERPRETATION: Muscle MRI of patients with MATR3-associated distal myopathy revealed a distinct pattern of muscular involvement. The usage of whole-body muscle MRI provided valuable additional findings as compared to regular MRI of the lower extremities to improve distinction from other disease entities.

Exon skipping in Duchenne Muscle dystrophy due to a silent p.Ser443= mutation in the DMD gene.

Duchenne Muscle dystrophy (DMD) is a X-linked inherited disease predominantly caused by severe mutations in DMD gene leading to absence of dystrophin protein. Here we report a 14-year-old Mongolian boy suffering from proximal muscle weakness, pseudohypertrophic deltoid and gastrocnemius muscles since early childhood. Lactate dehydrogenase (LDH) and creatine kinase (CK) levels were elevated. Mutation analysis including MLPA and sequencing of the DMD gene revealed a hemizygous silent variant, c.1329C>T (p.Ser443=) in exon 11. This silent mutation, listed in the SNP database (rs1060502631), was described as a variant of unknown significance (VUS) in ClinVar database. cDNA analysis demonstrated partial skipping of exon 11 due to this mutation. Although silent mutations are usually considered non-pathogenic, our case emphasizes that silent mutations can be potentially pathogenic. Hence, if silent variants are not annotated in database or not known to be benign, they should be analysed further at cDNA level.