Titin Mutations Research Articles

P.15.8 Hereditary myopathy with early respiratory failure (HMERF) – Rapid increase of identified titinopathy families worldwide

Hereditary myopathy with early respiratory failure (HMERF) was described in 7 families from Sweden in 1990. One titin mutation in the kinase domain of titin gene in two of these families was identified 2005, but the further understanding of the disease remained elusive despite a few reports on families with somewhat similar phenotypes. This has now changed completely: within one year 6 new papers have reported the molecular genetic resolution in 27 different families with a worldwide distribution and occurrence in different ethnic populations: Caucasian, Japanese and Native American. This explosion of detailed clarification of HMERF was possible due to identification of a pathognomonic pattern of muscle involvement on MRI and, above all, due to the use of exome sequencing to clarify the molecular genetic cause. All 27 families have mutations in one single exon 343 of titin gene encoding the fibronectin-like FN3 119 domain (formerly A150) of A-band titin. The most common mutation, also surprisingly identified in different populations, is a C30071R change showing sharing of alleles on a common haplotype in most of the families. One of the new mutations showed semi-recessive inheritance pattern with subclinical myopathy in the heterozygous parents. Typical clinical features were respiratory failure at mid-adulthood in an ambulant patient with very variable degree of muscle weakness. The pathognomonic muscle MRI pattern shows most severe fatty degenerative changes in semitendinosus, obturatorius, gracilis, tibialis posterior and anterior. Cytoplasmic bodies reactive for thin filament and Z-disc proteins combined with rimmed vacuoles constitute the muscle pathology hallmarks. This extraordinary evolution of understanding HMERF disease indicates that the disease is much more common than previously thought and that exon 343 of TTN is the primary target for molecular diagnosis.

P.3.11 Atypical phenotypes in titinopathies explained by second titin mutations and compound heterozygosity

Several patients with previously reported titin gene (<i>TTN</i>) mutations causing tibial muscular dystrophy (TMD) have more complex, severe or unusual phenotypes. This study aimed to clarify the molecular cause of these variant phenotypes in proband patients of six European families. Clinical, histopathological and muscle imaging data of patients and family members was reanalyzed and muscle biopsies of the patients were studied by titin Western blotting and RT-PCR. Western blotting showed a more pronounced C-terminal titin abnormality than expected for heterozygous mutants in all six probands, suggesting the existence of additional <i>TTN</i> mutations. RT-PCR indicated unequal mRNA expression of the two <i>TTN</i> alleles in biopsies of four patients, two of them with an LGMD2J phenotype. <i>TTN</i> was analyzed by Sanger sequencing from genomic DNA of the patients. Three patients proved to be compound heterozygotes with novel <i>TTN</i> frameshift mutations combined with previously reported TMD mutations. One LGMD2J patient was heterozygous for the FINmaj TMD mutation with a likely second yet undiscovered mutation. The unequal expression levels of <i>TTN</i> transcripts in these four probands suggested that the expression of the frameshifted allele was severely reduced, probably through nonsense-mediated decay, explaining the observed more severe phenotypes. One Portuguese patient was homozygote for a previously known Spanish TMD mutation. This TMD mutation seems to cause a more severe TMD rather than LGMD2J when homozygous. One Finnish patient had FINmaj mutation combined with a novel missense mutation in the A-band region of titin causing a new phenotype completely different from previously described titinopathies. Our results further expand the complexity of muscular dystrophies caused by <i>TTN</i> mutations and suggest that the coexistence of second mutations may constitute a more common general mechanism of phenotype variability in muscular dystrophies.

Short Read (Next-Generation) Sequencing

Rapid advances in DNA sequencing technologies have made it increasingly cost-effective to obtain accurate and timely large-scale genomic sequence data on individuals (short read massively parallel or next generation [next-gen]). A next-gen molecular diagnostic approach that has seen rapid deployment in the clinic over the last year is exome sequencing. Whole exome sequencing covers all protein-coding genes in the genome (≈1.1% of genome), and an exome test for a single patient generates ≈6 gigabases (109 bp) of DNA sequence data. A key challenge facing routine use of next-gen data in patient diagnosis and management is data interpretation. What sequence variant findings are relevant to diagnosis (pathogenic mutations)? What sequence variant findings are relevant to clinical care but not necessarily to patient diagnosis (clinically actionable incidental data)? What sequence information should be stored, and where can it be stored? This review provides a tutorial on current approaches to answering these questions. A recent landmark study showed that application of next-gen sequencing to a large cohort of idiopathic dilated cardiomyopathy patients found ≈27% of patients to show mutations of the titin gene, the most complex gene in the genome (363 exons). We use titin in cardiomyopathy as an exemplar for explaining next-gen sequencing approaches and data interpretation. Decreasing sequencing costs and broad dissemination of next-generation (next-gen) equipment and expertise are increasing availability of massively parallel sequencing of patient DNA samples (short read massively parallel or next-gen sequencing).1,2 Most rapidly expanding is exome sequencing, where all protein-coding sequences (exons) are selected from total genomic DNA and selectively sequenced.3 Alternative approaches to next-gen sequencing include targeted sequencing (TS) and whole genome (complete genome) sequencing. Currently, marketed targeted Sanger sequencing panels using traditional individual exon-by-exon sequencing remain expensive and time consuming, and massively parallel next-gen approaches are beginning to supplant …

Hereditary myopathy with early respiratory failure: occurrence in various populations

ObjectiveSeveral families with characteristic features of hereditary myopathy with early respiratory failure (HMERF) have remained without genetic cause. This international study was initiated to clarify epidemiology and the genetic underlying...

Exome sequencing identifies titin mutations causing hereditary myopathy with early respiratory failure (HMERF) in families of diverse ethnic origins

BackgroundHereditary myopathy with early respiratory failure (HMERF) was described in several North European families and recently linked to a titin gene (TTN) mutation. We independently studied HMERF-like diseases with the purpose to identify the cause, refine diagnostic criteria, and estimate the frequency of this disease among myopathy patients of various ethnic origins.MethodsWhole exome sequencing analysis was carried out in a large U.S. family that included seven members suffering from skeletal muscle weakness and respiratory failure. Subsequent mutation screening was performed in further 45 unrelated probands with similar phenotypes. Studies included muscle strength evaluation, nerve conduction studies and concentric needle EMG, respiratory function test, cardiologic examination, and muscle biopsy.ResultsA novel TTN p.Gly30150Asp mutation was identified in the highly conserved A-band of titin that co-segregated with the disease in the U.S. family. Screening of 45 probands initially diagnosed as myofibrillar myopathy (MFM) but excluded based on molecular screening for the known MFM genes led to the identification of a previously reported TTN p.Cys30071Arg mutation in one patient. This same mutation was also identified in a patient with suspected HMERF. The p.Gly30150Asp and p.Cys30071Arg mutations are localized to a side chain of fibronectin type III element A150 of the 10th C-zone super-repeat of titin.ConclusionsMissense mutations in TTN are the cause of HMERF in families of diverse origins. A comparison of phenotypic features of HMERF caused by the three known TTN mutations in various populations allowed to emphasize distinct clinical/pathological features that can serve as the basis for diagnosis. The newly identified p.Gly30150Asp and the p.Cys30071Arg mutation are localized to a side chain of fibronectin type III element A150 of the 10th C-zone super-repeat of titin.

G.P.34 Gene expression profiling in tibial muscular dystrophy reveals new involved molecular pathways

Tibial muscular dystrophy (TMD) is a late onset, autosomal dominant distal myopathy that results from mutations in the two last domains of titin. The cascade of molecular events leading from the causative Titin mutations to the preterm death of muscle cells in TMD is largely unknown. To identify these components we used gene expression profiling from muscle biopsies samples of TMD patients, healthy controls and from phenotypically overlapping Welander distal myopathy (WDM) patients. The profiling results were confirmed through RT-PCR and protein level analysis and we identified an activation of the unfolded protein response (UPR). UPR was then confirmed through elevation of marker genes HSPA5 (BIP) and XBP1 and the presence of ER-stress specific XBP1 splicing events. However, UPR activation appears to be insufficient, leading to build-up of ubiquitinated proteins which in turn cause activation of the autophagic system. Massive accumulation of LC3b positive autophagosomes within the rimmed vacuolated regions of degenerated muscle fibers suggests that this apparently compensatory mechanism is not capable of restoring equilibrium since these fibers degenerate further and disappear in the end stage of the pathology cascade.

A Novel Mechanism Involving Four-and-a-half LIM Domain Protein-1 and Extracellular Signal-regulated Kinase-2 Regulates Titin Phosphorylation and Mechanics

Understanding mechanisms underlying titin regulation in cardiac muscle function is of critical importance given recent compelling evidence that highlight titin mutations as major determinants of human cardiomyopathy. We previously identified a cardiac biomechanical stress-regulated complex at the cardiac-specific N2B region of titin that includes four-and-a-half LIM domain protein-1 (Fhl1) and components of the mitogen-activated protein signaling cascade, which impacted muscle compliance in Fhl1 knock-out cardiac muscle. However, direct regulation of these molecular components in mediating titin N2B function remained unresolved. Here we identify Fhl1 as a novel negative regulator of titin N2B levels and phosphorylation-mediated mechanics. We specifically identify titin N2B as a novel substrate of extracellular signal regulated-kinase-2 (Erk2) and demonstrate that Fhl1 directly interferes with Erk2-mediated titin-N2B phosphorylation. We highlight the critical region in titin-N2B that interacts with Fhl1 and residues that are dependent on Erk2-mediated phosphorylation in situ. We also propose a potential mechanism for a known titin-N2B cardiomyopathy-causing mutation that involves this regulatory complex. These studies shed light on a novel mechanism regulating titin-N2B mechano-signaling as well as suggest that dysfunction of these pathways could be important in cardiac disease states affecting muscle compliance.

Mutations in the Sensitive Giant Titin Result in a Broken Heart

### Truncations of Titin Causing Dilated Cardiomyopathy Herman et al N Engl J Med. 2012;366:619–628. The specialized cytoskeleton of striated muscle cells consists of highly ordered structures, the single contractile units termed sarcomeres. Although once believed to be simply a scaffold for the assembly of structural proteins to generate force and motion, it is now widely recognized that the sarcomeric cytoskeleton is dynamic and is intimately involved in numerous cellular signaling processes that respond to a wide range of extracellular cues. Sarcomeres in vertebrates are built of an intricate array of myosin-containing thick filaments, actin-containing thin filaments, and titin filaments (Figure, A), along with a plethora of other structural and regulatory proteins. Titin (also known as connectin) is the largest protein discovered in humans to date.1,2 The human titin gene is enormous; at 363 exons, it encompasses the largest number of exons in any single gene and is predicted to encode up to 38 138 residues, or a 4.2-MDa protein.3 Remarkably, a single molecule of titin spans half the length of a sarcomere with its NH2 terminus embedded in the Z-disc and its COOH terminus anchored at the center of the sarcomere, the M-line (Figure, A). This corresponds to a molecular length of approximately 0.9 to >1.5 μm, depending on sarcomere stretch! Because titin filaments from opposite half-sarcomeres fully overlap in the Z-discs and at the M-line, titin filaments form a continuous system within myofibrils. Figure. Striated muscle sarcomere (Panel A) with locations of titin mutations linked to human dilated cardiomyopathy (Panel B). Mutations are markedly missing from the Z disc and M line; they are highly enriched in the thick filament associated A band region. While some patients presented with identical mutations, numbers reflect unique mutations. Although many unique functions for titin have been described, it is most known for its function as a molecular spring; it limits …

Titin mutation segregates with hereditary myopathy with respiratory failure

Titin mutation segregates with hereditary myopathy with respiratory failure

Titin mutation segregates with hereditary myopathy with early respiratory failure

In 2001, we described an autosomal dominant myopathy characterized by neuromuscular ventilatory failure in ambulant patients. Here we describe the underlying genetic basis for the disorder, and we define the neuromuscular, respiratory and radiological phenotype in a study of 31 mutation carriers followed for up to 31 years. A combination of genome-wide linkage and whole exome sequencing revealed the likely causal genetic variant in the titin (TTN) gene (g.274375T>C; p.Cys30071Arg) within a shared haplotype of 2.93 Mbp on chromosome 2. This segregated with the phenotype in 21 individuals from the original family, nine subjects in a second family with the same highly selective pattern of muscle involvement on magnetic resonance imaging and a third familial case with a similar phenotype. Comparing the mutation carriers revealed novel features not apparent in our original report. The clinical presentation included predominant distal, proximal or respiratory muscle weakness. The age of onset was highly variable, from early adulthood, and including a mild phenotype in advanced age. Muscle weakness was earlier onset and more severe in the lower extremities in nearly all patients. Seven patients also had axial muscle weakness. Respiratory function studies demonstrated a gradual deterioration over time, reflecting the progressive nature of this condition. Cardiomyopathy was not present in any of our patients despite up to 31 years of follow-up. Magnetic resonance muscle imaging was performed in 21 affected patients and revealed characteristic abnormalities with semitendinosus involvement in 20 of 21 patients studied, including 3 patients who were presymptomatic. Diagnostic muscle histopathology most frequently revealed eosinophilic inclusions (inclusion bodies) and rimmed vacuoles, but was non-specific in a minority of patients. These findings have important clinical implications. This disease should be considered in patients with adult-onset proximal or distal myopathy and early respiratory failure, even in the presence of non-specific muscle pathology. Muscle magnetic resonance imaging findings are characteristic and should be considered as an initial investigation, and if positive should prompt screening for mutations in TTN. With 363 exons, screening TTN presented a major challenge until recently. However, whole exome sequencing provides a reliable cost-effective approach, providing the gene of interest is adequately captured.

Truncating mutations in titin ( TTN ) as a marker of dilated cardiomyopathy

Truncating mutations in titin ( TTN ) as a marker of dilated cardiomyopathy

Single Molecule Studies of a Titin Mutation Linked to Cardiac Disease

Recently, a mutation in the 10th immunoglobulin(Ig)-like domain in titin's elastic I-band was found in a family affected with arrhythmogenic cardiomyopathy (AC). AC is characterized by fibrofatty replacement of myocardium, which can lead to fatal ventricular arrhythmias. Titin is a gigantic sarcomeric protein that is responsible for passive tension development in stretched cardiac muscle. We investigated the effect that the AC-linked point mutation (T16I in Ig10) has on the mechanical and kinetic stability of Ig10 using single molecule force spectroscopy (AFM) and degradation assays. Our AFM data indicate that T16I Ig10 unfolds at a lower force than WT Ig10, which shows that it is mechanically less stable. We also performed force clamp and refolding experiments with AFM and found that the unfolding rate of T16I Ig10 is four times higher than WT Ig10 (.02/s vs. .005/s at zero force), but that this difference is further increased in a force-dependent manner; refolding rates were found to be similar. We also performed degradation assays with the protease tryspin to determine if the T16I mutation affects the highly-stable beta barrel tertiary structure typical of Ig domains. We found that T16I Ig10 has a greater proteolytic susceptibility compared to WT Ig10, and subsequent mass spectrometry analysis determined that the primary cleavage site in T16I Ig10 is the lysine residue that flanks T16I (K17). This strongly suggests that the disease-linked titin mutation disrupts the local structure of Ig10 in the vicinity of T16, which leaves Ig10 vulnerable to cleavage. Peptide bond breakage anywhere along titin's elastic region (which includes Ig10) would likely abolish titin's ability to generate force and lead to severe physiological responses.

EP News: Basic and Translational

Taylor et al (Circulation 2011;124:876, PMID 21810661) evaluated titin (TTN) as a candidate arrhythmogenic right ventricular cardiomyopathy (ARVC) gene. Eight unique TTN variants were detected in 7 families, including a prominent Thr2896Ile mutation that showed complete segregation with the ARVC phenotype in 1 large family. The phenotype of TTN variant carriers was characterized by a history of sudden death, progressive myocardial dysfunction causing death or heart transplantation, frequent conduction disease, and incomplete penetrance. The authors conclude that titin mutations can cause ARVC. Structural impairment of the titin spring is a likely cause of ARVC and constitutes a novel mechanism underlying myocardial remodeling and sudden cardiac death.

Genetic variation in titin in arrhythmogenic right ventricular cardiomyopathy-overlap syndromes.

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited genetic myocardial disease characterized by fibrofatty replacement of the myocardium and a predisposition to cardiac arrhythmias and sudden death. We evaluated the cardiomyopathy gene titin (TTN) as a candidate ARVC gene because of its proximity to an ARVC locus at position 2q32 and the connection of the titin protein to the transitional junction at intercalated disks. All 312 titin exons known to be expressed in human cardiac titin and the complete 3' untranslated region were sequenced in 38 ARVC families. Eight unique TTN variants were detected in 7 families, including a prominent Thr2896Ile mutation that showed complete segregation with the ARVC phenotype in 1 large family. The Thr2896IIe mutation maps within a highly conserved immunoglobulin-like fold (Ig10 domain) located in the spring region of titin. Native gel electrophoresis, nuclear magnetic resonance, intrinsic fluorescence, and proteolysis assays of wild-type and mutant Ig10 domains revealed that the Thr2896IIe exchange reduces the structural stability and increases the propensity for degradation of the Ig10 domain. The phenotype of TTN variant carriers was characterized by a history of sudden death (5 of 7 families), progressive myocardial dysfunction causing death or heart transplantation (8 of 14 cases), frequent conduction disease (11 of 14), and incomplete penetrance (86%). Our data provide evidence that titin mutations can cause ARVC, a finding that further expands the origin of the disease beyond desmosomal proteins. Structural impairment of the titin spring is a likely cause of ARVC and constitutes a novel mechanism underlying myocardial remodeling and sudden cardiac death.

Interactions with M-band Titin and Calpain 3 Link Myospryn (CMYA5) to Tibial and Limb-girdle Muscular Dystrophies

Mutations in the C terminus of titin, situated at the M-band of the striated muscle sarcomere, cause tibial muscular dystrophy (TMD) and limb-girdle muscular dystrophy (LGMD) type 2J. Mutations in the protease calpain 3 (CAPN3), in turn, lead to LGMD2A, and secondary CAPN3 deficiency in LGMD2J suggests that the pathomechanisms of the diseases are linked. Yeast two-hybrid screens carried out to elucidate the molecular pathways of TMD/LGMD2J and LGMD2A resulted in the identification of myospryn (CMYA5, cardiomyopathy-associated 5) as a binding partner for both M-band titin and CAPN3. Additional yeast two-hybrid and coimmunoprecipitation studies confirmed both interactions. The interaction of myospryn and M-band titin was supported by localization of endogenous and transfected myospryn at the M-band level. Coexpression studies showed that myospryn is a proteolytic substrate for CAPN3 and suggested that myospryn may protect CAPN3 from autolysis. Myospryn is a muscle-specific protein of the tripartite motif superfamily, reported to function in vesicular trafficking and protein kinase A signaling and implicated in the pathogenesis of Duchenne muscular dystrophy. The novel interactions indicate a role for myospryn in the sarcomeric M-band and may be relevant for the molecular pathomechanisms of TMD/LGMD2J and LGMD2A.

Cardiac Titin

Titin constitutes the third myofilament of cardiac muscle, with a single giant polypeptide spanning from Z-disk to the M-band region of the sarcomere1 (Fig. 1). The ∼1.0 MDa region in the I-band is extensible and consists of tandemly arranged immunoglobulin (Ig)-like domains that make up proximal (near Z-disk) and distal (near A-I junction) segments, interspersed by the PEVK sequence (rich in proline, glutamate, valine, and lysine residues) and the N2B element2. Each functions as a distinct spring element3. The C-terminal ∼2 MDa of titin is located in the A-band and is inextensible. It is composed of regular arrays of Ig and fibronectin type 3 (Fn3) modules forming so-called super-repeats2. A-band titin may function as a molecular ruler, regulating assembly of the thick filament2,4,5. Titin's ∼250-kDa COOH-terminal region is an integral part of the M-band and contains a kinase domain6,7. As in the Z-disk, where titin filaments from opposite sarcomeres overlap, titin filaments from opposite half-sarcomeres overlap within the M-band, where they are interconnected by M-band proteins8. Thus, titin filaments with opposite polarity overlap in both Z-disk and M-band, forming a contiguous filament along the myofibril. In this review, we discuss titin's functions in the heart, with an emphasis on its role in diastolic function and the various mechanisms whereby passive stiffness can be tuned. Due to space constraints, it has not been possible to provide inclusive references to all original articles in the field. Figure 1 Schematic of titin in sarcomere. Differential splicing Titin is encoded by a single gene containing 368 exons. Multiple splice pathways in the I-band encoding region (∼230 exons) give rise to isoforms with different spring composition9. The three cardiac isoform classes are shown in Fig. 1. The relatively small ∼3.0 MDa isoform is known as N2B titin (it contains the N2B element)9. A second class also contains the N2A element, and is termed N2BA titin. N2BA titins have a longer PEVK segment and a variable number of additional Ig domains resulting in a ∼3.3-3.5 MDa size9. The third class includes isoforms that predominate in fetal-neonatal life which contain additional spring elements in both tandem Ig and PEVK regions, resulting in a ∼3.6-3.8 MDa size protein10-12. These isoforms gradually disappear during postnatal development. Regulation of the spring composition of titin in fetal-neonatal myocardium allows adjustment of diastolic filling properties during development.

Erratum to: The first Italian family with tibial muscular dystrophy caused by a novel titin mutation

Erratum to: J NeurolDOI 10.1007/s00415-009-5372-3Under the heading Introduction in line 12, the correctsentence should read: Homozygous mutations moreN-terminal of the TMD/LGMD2J mutations and in theC-terminus of TTN cause lethal cardiac and skeletalmyopathy [8].Under the heading Case description in line 11, thecorrectsentenceshould read:Electromyogram(EMG)showedmyopathic and neurogenic changes only in the muscles ofthe anterolateral compartment of both legs, without signs inthe quadriceps and gastrocnemii.

The first Italian family with tibial muscular dystrophy caused by a novel titin mutation

Tibial muscular dystrophy (TMD) or Udd myopathy is an autosomal dominant distal myopathy with late onset, at first described in the Finnish population. We report here the first Italian cases of TTN mutated titinopathy. The proband, a 60 year-old female, had the first muscular signs at the age of 59 years, with difficulty in walking and right foot drop. Muscle imaging showed selective fatty degenerative change in the anterior compartment of leg muscles. Her 67 year-old brother, started to show muscle weakness, pain at lower limbs and hypertrophy of calf muscles at the age of 66 years. Their mother began to show foot drop and impaired walking from the age of 60 years. Other relatives are reported to be affected in a similar way. Because the phenotype appeared compatible with TMD, we analyzed the TTN gene in the DNA of the proband and we identified a heterozygous mutation 293326A>C. This mutation is also present in the brother and in the other affected individuals of the same family. The mutation predicts a His33378Pro change located next to the previously known Belgian TMD mutation. The mutation was not found in 100 Italian control DNA samples. Then, since the introduction of a proline in the last domain of titin was previously known to cause TMD in French families, we can conclude that this missense mutation is the obvious pathogenetic mutation in the affected patients. No other disease causing mutations in the TTN gene have so far been reported in the Italian population.

Stress-induced dilated cardiomyopathy in a knock-in mouse model mimicking human titin-based disease

Mutations in a variety of myofibrillar genes cause dilated cardiomyopathy (DCM) in humans, usually with dominant inheritance and incomplete penetrance. Here, we sought to clarify the functional effects of the previously identified DCM-causing TTN 2-bp insertion mutation (c.43628insAT) and generated a titin knock-in mouse model mimicking the c.43628insAT allele. Mutant embryos homozygous for the Ttn knock-in mutation developed defects in sarcomere formation and consequently died before E9.5. Heterozygous mice were viable and demonstrated normal cardiac morphology, function and muscle mechanics. mRNA and protein expression studies on heterozygous hearts demonstrated elevated wild-type titin mRNA under resting conditions, suggesting that up-regulation of the wild-type titin allele compensates for the unstable mutated titin under these conditions. When chronically exposed to angiotensin II or isoproterenol, heterozygous mice developed marked left ventricular dilatation (p<0.05) with impaired fractional shortening (p<0.001) and diffuse myocardial fibrosis (11.95±2.8% vs. 3.7±1.1%). Thus, this model mimics typical features of human dilated cardiomyopathy and may further our understanding of how titin mutations perturb cardiac function and remodel the heart.

Truncating mutations in C-terminal titin may cause more severe tibial muscular dystrophy (TMD)

Mutations in C-terminal titin cause autosomal dominant tibial muscular dystrophy (TMD) as reported previously. Samples from 25 new families and 25 sporadic new distal myopathy cases were screened for titin mutations. Three novel mutations were discovered in two families from Spain and two families from France. Two mutations, g.292998delT and g.293376delA lead to frameshift and premature stop codons in the second last and the last titin gene ( TTN) exons, Mex5 and Mex6, respectively. The third was a nonsense mutation g.293379C>T (p.Q33396X) in Mex6. Patients with the upstream Mex5 mutation showed a more severe phenotype with earlier onset implying a genotype–phenotype correlation.