The treatment effect on peripheral B cell markers in antibody positive myasthenia gravis patients.

B cells play a major role in the pathophysiology of myasthenia gravis (MG) with their ability to produce disease specific, pathogenic antibodies. However, their status during disease development and follow-up stages of the disease in the peripheral blood may need further studies to determine useful markers. In this study, we aimed to detect B cell associated factors concerning immunosuppressive treatment in generalized non-thymomatous MG patients. Although CD19+ B cell distribution did not vary among disease subgroups, expressions of both CD38 and BAFFR were altered on B cells in MG patients under immunosuppressive therapy. Serum levels of BAFF were elevated in untreated MG patients as compared to treated MG patients and healthy controls. B cell activation factors may show profound alterations due to immunosuppression.

Mutation spectrum of 260 dystrophinopathy patients from Turkey and important highlights for genetic counseling.

We genetically evaluated 260 dystrophinopathy patients from Turkey. Karyotyping as an initial test in female patients, followed stepwise by multiplex ligation-dependent probe amplification and by targeted next-generation sequencing of DMD revealed definitive genetic diagnoses in 214 patients (82%), with gross deletions/duplications in 153 (59%), pathogenic sequence variants in 60 (23%), and X-autosome translocation in one. Seven of the gross and 27 of the sequence variants found novel. In silico prediction, co-segregation and transcript assays supported the pathogenic nature of the novel silent (p.Lys534=) and the splice site (c.4345-12C>G) alterations. From a total of 189 singleton cases, 154 (82%) had pathogenic alterations. From 138 of those who had maternal carrier testing, 68 out of 103 (66%) showed gross and 11 out of 35 (31%) showed small pathogenic variants. This suggests that the de novo occurrences in DMD appear approximately 2.1 times more frequently in meiotic unequal crossing-over than in uncorrected replication errors. Our study also disclosed three mothers as obligate gonadal mosaic carriers. Family-based investigation of dystrophinopathy patients is crucial for the ascertainment of novel or rare variants and also for counseling and follow-up care of the families.

Relation of HLA-DRB1 to IgG4 autoantibody and cytokine production in muscle-specific tyrosine kinase myasthenia gravis (MuSK-MG).

A small subset of myasthenia gravis (MG) patients develop autoantibodies against muscle-specific kinase (MuSK), which are predominantly of the immunoglobulin (Ig)G4 isotype. MuSK-MG is strongly associated with HLA-DRB1*14, HLA-DRB1*16 and HLA-DQB1*05. In this study, the possible effects of these HLA associations on MuSK IgG autoantibody or cytokine production were investigated. Samples from 80 MG patients with MuSK antibodies were studied. The disease-associated HLA types were screened in the DNA samples. The IgG1, IgG2, IgG3 and IgG4 titres of the MuSK antibodies and the levels of interleukin (IL)-4, IL-6, IL-17A and IL-10 were measured in the sera. Comparisons were made among the groups with or without HLA-DRB1*14, HLA-DRB1*16 or HLA-DQB1*05. The IgG4 titres of the MuSK antibodies were higher than those of the IgG1, IgG2 and IgG3 isotypes among the whole group of patients. DRB1*14 (+) DRB1*16 (-) patients had higher levels of IgG4 antibodies than those of DRB1*14 (-) DRB1*16 (+) patients. DRB1*14 (+) DRB1*16 (+) patients also had higher levels of IgG4 antibodies than those of DRB1*14 (-) DRB1*16 (+) and DRB1*14 (-) DRB1*16 (-) patients. Higher IL-10 and lower IL-17A levels were measured in DRB1*14 (+) DRB1*16 (-) patients than in DRB1*14 (-) DRB1*16 (-) patients. The higher IgG4 titres of MuSK autoantibodies in patients carrying HLA-DRB1*14 than those in the other patients suggest a role for HLA in the production of the antibodies. The differences in IL-10 and IL-17A support the role of DRB1 in the etiopathogenesis of this autoimmune response.

The effect of interleukin (IL)-21 and CD4+ CD25++ T cells on cytokine production of CD4+ responder T cells in patients with myasthenia gravis.

Impairment of the suppressive function of regulatory T (Treg ) cells has been reported in myasthenia gravis (MG). In this study, cytokine-related mechanisms that may lead to the defect of Treg were investigated in patients with anti-acetylcholine receptor antibody-positive MG (AChR + MG). Proliferation and cytokine production of responder T (Tresp ) cells in response to polyclonal activation were measured in a suppression assay. The effect of interleukin (IL)-21 on suppression was evaluated in vitro in co-culture. IL-21 increased the proliferation of Tresp cells in Tresp /Treg co-cultures. Tresp cells from patients with MG secreted significantly lower levels of IL-2. In patients with MG, IL-2 levels did not change with the addition of Treg to cultures, whereas it decreased significantly in controls. In Tresp /Treg co-cultures, IL-4, IL-6 and IL-10 production increased in the presence of Treg in patients. Interferon (IFN)-γ was decreased, whereas IL-17A was increased in both patient and control groups. IL-21 inhibited the secretion of IL-4 in MG and healthy controls (HC), and IL-17A in HC only. The results demonstrated that IL-21 enhances the proliferation of Tresp cells in the presence of Treg . An effect of IL-21 mainly on Tresp cells through IL-2 is implicated.

Titin antibodies in "seronegative" myasthenia gravis--A new role for an old antigen.

Myasthenia gravis (MG) is an autoimmune disease caused by antibodies targeting the neuromuscular junction of skeletal muscles. Triple-seronegative MG (tSN-MG, without detectable AChR, MuSK and LRP4 antibodies), which accounts for ~10% of MG patients, presents a serious gap in MG diagnosis and complicates differential diagnosis of similar disorders. Several AChR antibody positive patients (AChR-MG) also have antibodies against titin, usually detected by ELISA. We have developed a very sensitive radioimmunoprecipitation assay (RIPA) for titin antibodies, by which many previously negative samples were found positive, including several from tSN-MG patients. The validity of the RIPA results was confirmed by western blots. Using this RIPA we screened 667 MG sera from 13 countries; as expected, AChR-MG patients had the highest frequency of titin antibodies (40.9%), while MuSK-MG and LRP4-MG patients were positive in 14.6% and 16.4% respectively. Most importantly, 13.4% (50/372) of the tSN-MG patients were also titin antibody positive. None of the 121 healthy controls or the 90 myopathy patients, and only 3.6% (7/193) of other neurological disease patients were positive. We thus propose that the present titin antibody RIPA is a useful tool for serological MG diagnosis of tSN patients.

MuSK autoantibodies in myasthenia gravis detected by cell based assay--A multinational study.

Seronegative myasthenia gravis (MG) presents a serious gap in MG diagnosis and understanding. We applied a cell based assay (CBA) for the detection of muscle specific kinase (MuSK) antibodies undetectable by radioimmunoassay. We tested 633 triple-seronegative MG patients' sera from 13 countries, detecting 13% as positive. MuSK antibodies were found, at significantly lower frequencies, in 1.9% of healthy controls and 5.1% of other neuroimmune disease patients, including multiple sclerosis and neuromyelitis optica. The clinical data of the newly diagnosed MuSK-MG patients are presented. 27% of ocular seronegative patients were MuSK antibody positive. Moreover, 23% had thymic hyperplasia suggesting that thymic abnormalities are more common than believed.

A comprehensive analysis of the epidemiology and clinical characteristics of anti-LRP4 in myasthenia gravis.

Double-seronegative myasthenia gravis (dSN-MG, without detectable AChR and MuSK antibodies) presents a serious gap in MG diagnosis and understanding. Recently, autoantibodies against the low-density lipoprotein receptor-related protein 4 (LRP4) have been identified in several dSN-MG sera, but with dramatic frequency variation (∼2-50%). We have developed a cell based assay (CBA) based on human LRP4 expressing HEK293 cells, for the reliable and efficient detection of LRP4 antibodies. We have screened about 800 MG patient sera from 10 countries for LRP4 antibodies. The overall frequency of LRP4-MG in the dSN-MG group (635 patients) was 18.7% but with variations among different populations (range 7-32.7%). Interestingly, we also identified double positive sera: 8/107 anti-AChR positive and 10/67 anti-MuSK positive sera also had detectable LRP4 antibodies, predominantly originating from only two of the participating groups. No LRP4 antibodies were identified in sera from 56 healthy controls tested, while 4/110 from patients with other neuroimmune diseases were positive. The clinical data, when available, for the LRP4-MG patients were then studied. At disease onset symptoms were mild (81% had MGFA grade I or II), with some identified thymic changes (32% hyperplasia, none with thymoma). On the other hand, double positive patients (AChR/LRP4-MG and MuSK/LRP4-MG) had more severe symptoms at onset compared with any single positive MG subgroup. Contrary to MuSK-MG, 27% of ocular dSN-MG patients were LRP4 antibody positive. Similarly, contrary to MuSK antibodies, which are predominantly of the IgG4 subtype, LRP4 antibodies were predominantly of the IgG1 and IgG2 subtypes. The prevalence was higher in women than in men (female/male ratio 2.5/1), with an average disease onset at ages 33.4 for females and 41.9 for males. Overall, the response of LRP4-MG patients to treatment was similar to published responses of AChR-MG rather than to MuSK-MG patients.

Sporadic-inclusion body myositis (s-IBM) is not so prevalent in Istanbul/Turkey: a muscle biopsy based survey.

In a muscle biopsy based study, only 9 out of 5450 biopsy samples, received from all parts of greater Istanbul area, had typical clinical and most suggestive light microscopic sporadic-inclusion body myositis (s-IBM) findings. Two other patients with and ten further patients without characteristic light microscopic findings had referring diagnosis of s-IBM. As the general and the age-adjusted populations of Istanbul in 2010 were 13.255.685 and 2.347.300 respectively, the calculated corresponding 'estimated prevalences' of most suggestive s-IBM in the Istanbul area were 0.679 X 10(-6) and 3.834 X 10(-6). Since Istanbul receives heavy migration from all regions of Turkey and ours is the only muscle pathology laboratory in Istanbul, projection of these figures to the Turkish population was considered to be reasonable and an estimate of the prevalence of s-IBM in Turkey was obtained. The calculated 'estimated prevalence' of s-IBM in Turkey is lower than the previously reported rates from other countries. The wide variation in the prevalence rates of s-IBM may reflect different genetic, immunogenetic or environmental factors in different populations.

Oculopharyngodistal myopathy is a distinct entity: clinical and genetic features of 47 patients.

BACKGROUND: Oculopharyngodistal myopathy (OPDM) has been reported as a rare, adult-onset hereditary muscle disease with putative autosomal dominant and autosomal recessive inheritance. Patients with OPDM present with progressive ocular, pharyngeal, and distal limb muscle involvement. The genetic defect causing OPDM has not been elucidated. METHODS: Clinical and genetic findings of 47 patients from 9 unrelated Turkish families diagnosed with OPDM at the Department of Neurology, Istanbul Faculty of Medicine, between 1982 and 2009 were evaluated. RESULTS: The mean age at onset was around 22 years. Both autosomal dominant and autosomal recessive traits were observed, without any clear difference in clinical phenotype or severity. The most common initial symptom was ptosis, followed by oropharyngeal symptoms and distal weakness, which started after the fifth disease year. Intrafamilial variability of disease phenotype and severity was notable in the largest autosomal dominant family. Atypical presentations, such as absence of limb weakness in long-term follow-up in 9, proximal predominant weakness in 4, and asymmetric ptosis in 3 patients, were observed. Swallowing difficulty was due to oropharyngeal dysphagia with myopathic origin. Serum creatine kinase levels were slightly increased and EMG revealed myopathic pattern with occasional myotonic discharges. Myopathologic findings included rimmed and autophagic vacuoles and chronic myopathic changes. Importantly, a considerable proportion of patients developed respiratory muscle weakness while still ambulant. Linkage to the genetic loci for all known muscular dystrophies, and for distal and myofibrillar myopathies, was excluded in the largest autosomal dominant and autosomal recessive OPDM families. CONCLUSIONS: We suggest that OPDM is a clinically and genetically distinct myopathy.

Cortical excitability in Duchenne muscular dystrophy.

OBJECTIVE: To investigate the probable cortical excitability changes in DMD by electrophysiological means. METHODS: Sixteen cases with DMD, 10 age-matched control children (CC) and 10 healthy adult volunteers (AC) were studied with a transcranial magnetic stimulation (TMS) test battery composed of central conduction time, cortical silent period and paired TMS paradigm. RESULTS: There were no significant differences between DMD and CC groups except for lower amplitude motor responses in DMD cases. These two groups showed a similar pattern of excitability with less short interval intracortical inhibitions and shorter silent period durations as compared to the AC subjects. CONCLUSIONS: The electrophysiological tests performed in our DMD patients did not reveal abnormalities caused particularly by the disorder. SIGNIFICANCE: TMS excitability studies performed in DMD boys may not provide findings other than those related to the developmental age.

Clinical comparison of anti-MuSK- vs anti-AChR-positive and seronegative myasthenia gravis.

We compared 65 anti-acetylcholine receptor (AChR)-negative myasthenia gravis (MG) patients, including 32 anti-muscle-specific tyrosine kinase (MuSK)-positive (49%) and 33 anti-MuSK-negative (seronegative) (51%) patients, with 161 anti-AChR-positive MG patients. The anti-MuSK-positive group had a higher frequency of bulbar involvement and respiratory crises. The seronegative group was in between the anti-MuSK positive and the anti-AChR positive groups, being closer to the latter, with regard to the severity of the disease. At the end of follow-up, the outcome of the anti-MuSK-positive patients was not different from that of the anti-AChR-positive patients, although their maintenance corticosteroid dose was higher. The seronegative patients had better outcome than the other two groups.

Absence of KIF1B mutation in a large Turkish CMT2A family suggests involvement of a second gene.

BACKGROUND: Charcot-Marie-Tooth disease type 2A (CMT2A) was assigned to a 19.3-cM region on chromosome 1p35-36. A missense mutation in the kinesin family member 1B gene (KIF1B) was reported in a single CMT2A family. OBJECTIVE: To report the clinical and genetic data of a Turkish family with CMT2A. METHODS: Linkage to CMT2 loci was investigated in the family. Haplotype analysis of the CMT2A region was completed using additional single-nucleotide polymorphism and short tandem repeat markers. The KIF1B gene was sequenced on genomic DNA and cDNA in two patients. RESULTS: A recombination event narrowed the CMT2A locus to a 9.3-cM region flanked by D1S160 and D1S434. No mutation in KIF1B was found. CONCLUSION: The exclusion of KIF1B gene mutations in this family suggests the involvement of another CMT2A gene in the linked region.

Distribution of extremity muscle weakness in myasthenia gravis: sparing of tibialis anterior muscle.

We evaluated the distribution of muscle weakness in 70 myasthenia gravis (MG) patients, using the MRC scale. Extensors in the upper extremities and proximal flexors in the lower extremities (LE) were found to be weak. Tibialis anterior muscle was rarely affected and only when there was substantial weakness in LE proximal muscles. Attention to this distribution may help in differentiating MG, particularly MG with only limb weakness, from muscular dystrophies.

The range of chronic demyelinating neuropathy of infancy: a clinico-pathological and genetic study of 15 unrelated cases.

The concept of Dejerine-Sottas disease, which corresponds to presumed recessive demyelinating neuropathies with onset in infancy, remains controversial. To learn more on the subject, we performed a clinico-pathological and molecular genetic study in 15 unrelated patients with the Dejerine-Sottas phenotype seen over a 16 year period. There were 12 females and 3 males, born to asymptomatic parents. Study of the PMP22, P0 and Egr2 genes was performed in all cases and 14 underwent a nerve biopsy. First manifestations of neuropathy occurred before 3 years of age in all patients. An inherited disorder was suspected in 10 patients, because of their family history and/or disclosure of a molecular genetic defect in 4 of them. One patient had a recessively transmitted homozygous point mutation (Arg157Trp) of the PMP22 gene. A heterozygous duplication of the 17p11.2-12 segment was detected in one offspring of a consanguineous marriage. One patient carried a "de novo" heterozygous Ser72Leu substitution in the PMP22. A heterozygous double mutation of the P0 gene including a "de novo" Val42 deletion and an Ala221Thr substitution, maternally inherited, were found in an apparently sporadic case. No mutation of the Egr2 gene was identified. A neuropathy with focally folded myelin sheaths (CMT4B) was diagnosed in the nerve biopsy specimens of two patients. In five patients, the clinico-pathological findings along with the absence of an identified mutation suggested the diagnosis of chronic inflammatory demyelinating polyneuropathy of infantile onset. Our findings illustrate the genetic heterogeneity of cases with identified mutations, the scarcity of cases with "demonstrated" recessive transmission and the likelihood of early acquired chronic inflammatory demyelinating polyneuropathy in several patients.

Mapping of the second Friedreich's ataxia (FRDA2) locus to chromosome 9p23-p11: evidence for further locus heterogeneity.

Friedreich's ataxia (FRDA), the most-common form of autosomal recessive ataxia, is inherited in most cases by a large expansion of a GAA triplet repeat in the first intron of the frataxin (X25) gene. Genetic heterogeneity in FRDA has been previously reported in typical FRDA families that do not link to the FRDA locus on chromosome 9q13. We report localization of a second FRDA locus (FRDA2) to chromosome 9p23-9p11, and we provide evidence for further genetic heterogeneity of the disease, in a family with the classic FRDA phenotype.

Mutations in Kir2.1 cause the developmental and episodic electrical phenotypes of Andersen's syndrome.

Andersen's syndrome is characterized by periodic paralysis, cardiac arrhythmias, and dysmorphic features. We have mapped an Andersen's locus to chromosome 17q23 near the inward rectifying potassium channel gene KCNJ2. A missense mutation in KCNJ2 (encoding D71V) was identified in the linked family. Eight additional mutations were identified in unrelated patients. Expression of two of these mutations in Xenopus oocytes revealed loss of function and a dominant negative effect in Kir2.1 current as assayed by voltage-clamp. We conclude that mutations in Kir2.1 cause Andersen's syndrome. These findings suggest that Kir2.1 plays an important role in developmental signaling in addition to its previously recognized function in controlling cell excitability in skeletal muscle and heart.

Mutational analysis and genotype/phenotype correlation in Turkish Charcot-Marie-Tooth Type 1 and HNPP patients.

The major Charcot- Marie-Tooth Type 1 (CMT1) locus, CMT1A, and Hereditary neuropathy with liability to pressure palsies (HNPP) cosegregate with a 1.5-Mb duplication and a 1.5-Mb deletion, respectively, in band 17p11.2. Point mutations in peripheral myelin gene 22 (PMP22), myelin protein zero (MPZ), and connexin 32 (Cx32) have been reported in CMT1, and in PMP22 in HNPP patients without deletion. We have screened 54 CMT1 patients, of variable clinical severity, and 25 HNPP patients from Turkey, with no duplication or deletion, for mutations in the PMP22 and Cx32 genes. A novel frameshift mutation affecting the second extracellular domain of PMP22 was found in an HNPP patient, while a point mutation in the second transmembrane domain of the protein was detected in a CMT1 patient. Two point mutations affecting different domains of Cx32 were identified in two CMTX patients. Another patient was found to carry a polymorphism in a non-conserved codon of the Cx32 gene. The clinical phenotypes of the patients correlate well with the effect of the mutation on the protein.

Deletion pattern in the dystrophin gene in Turks and a comparison with Europeans and Indians.

Patterns of dystrophin gene deletions in DMD/BMD patients were compared in four populations: Turks (n = 146 deletions), Europeans (n = 838), North Indians (n = 89), and Indians from all over India (n = 103). Statistical tests revealed that there are differences in the proportions of small deletions. In contrast, the distribution of deletion breakpoints and the frequencies of specific deletions commonly observed in the four populations are not significantly different. The variations strongly suggest that sequence differences exist in the introns, and the differences are in agreement with genetic distances among populations. The similarities suggest that some intronic sequences have been conserved and that those will trigger recurrent deletions, since it is unlikely that gene flow would disperse the deleted chromosomes, which vanish from the gene pool in a few generations.