Zdravka Poljaković

Novel CCM1, CCM2, and CCM3 mutations in patients with cerebral cavernous malformations: in-frame deletion in CCM2 prevents formation of a CCM1/CCM2/CCM3 protein complex.

Cerebral cavernous malformations (CCM) are prevalent cerebrovascular lesions predisposing to chronic headaches, epilepsy, and hemorrhagic stroke. Using a combination of direct sequencing and MLPA analyses, we identified 15 novel and eight previously published CCM1 (KRIT1), CCM2, and CCM3 (PDCD10) mutations. The mutation detection rate was >90% for familial cases and >60% for isolated cases with multiple malformations. Splice site mutations constituted almost 20% of all CCM mutations identified. One of these proved to be a de novo mutation of the most 3′ acceptor splice site of the CCM1 gene resulting in retention of intron 19. A further mutation affected the 3′ splice site of CCM2 intron 2 leading to cryptic splice site utilization in both CCM2 and its transcript variant lacking exon 2. With the exception of one in‐frame deletion of CCM2 exon 2, which corresponds to the naturally occurring splice variant of CCM2 on the RNA level and is predicted to result in the omission of 58 amino acids (CCM2:p.P11_K68del), all mutations lead to the introduction of premature stop codons. To gain insight into the likely mechanisms underlying the only known CCM2 in‐frame deletion, we analyzed the functional consequences of loss of CCM2 exon 2. The CCM2:p.P11_K68del protein could be expressed in cell culture and complexed with CCM3. However, its ability to interact with CCM1 and to form a CCM1/CCM2/CCM3 complex was lost. These data are in agreement with a loss‐of‐function mechanism for CCM mutations, uncover an N‐terminal CCM2 domain required for CCM1 binding, and demonstrate full‐length CCM2 as the essential core protein in the CCM1/CCM2/CCM3 complex. Hum Mutat 29(5), 709–717, 2008.

Determination of cortical language dominance using functional transcranial Doppler sonography in left-handers.

OBJECTIVE Verbal analytical functions are primarily related to the left hemisphere in right-handers, but there is yet no agreement about cortical language dominance in left-handers. Also, there are some contradictory reports about sex differences in cortical language lateralization. The aim of this study is to investigate cortical language dominance in left-handers and to explore gender influence on cortical language representation. METHODS We performed functional transcranial Doppler sonography (previous validated for determination of cerebral language lateralization) during a word generation task, measuring changes in mean cerebral blood flow velocity (BFVmean) in both middle cerebral arteries (MCA) in 150 healthy subjects (75 left-handers and 75 right-handers). In left-handers we observed significant increase BFVmean in right MCA in 58 (77.3%) subjects. Bilateral increase was observed in 11 (14.7%) subjects and increase in left MCA in 6 (8%) subjects. In right-handed group 93.3% subjects showed left cortical dominance, while 6.7% showed bilateral language representation. RESULTS Current results showed significant (P<0.0001) right hemispheric language dominance in healthy left-handed subjects. CONCLUSIONS Our results showed significant difference in hemispheric dominance for verbal function between righthanders and lefthanders. Also there is statistically insignificant female gender tendency for bilateral hemispheric language representation in both handedness.

Growth hormone and insulin growth factor-I levels in plasma and cerebrospinal fluid of patients with multiple sclerosis

Multiple sclerosis (MS) has several clinically different forms. Whereas the illness progresses slowly in most of the patients, 10% have an aggressively progressive course with fatal outcome without signs of remyelination capability. The process of remyelination depends on numerous interactive factors, including the presence of various growth factors, the most important of which in the adult is insulin growth factor-I (IGF-I). On the other hand, the most powerful postnatal regulator of IGF-I is growth hormone (GH), which also acts as a neuroprotective and an antiapoptotic agent, and has direct influence on myelination. Levels of these growth factors have never been examined in the cerebrospinal fluid (CSF) of patients with MS. The levels of IGF-I and GH were measured in serum and CSF of 46 MS patients and compared with those of 49 patients with no evidence of demyelinating disease. The only positive finding was a deficiency of GH in the CSF of MS patients. The possible implications of those findings in the etiopathogenesis of MS will be discussed.

Economic evaluation of pharmacogenomic-guided warfarin treatment for elderly Croatian atrial fibrillation patients with ischemic stroke.

BACKGROUND & METHODS Economic evaluation in genomic medicine is an emerging discipline to assess the cost-effectiveness of genome-guided treatment. Here, we developed a pharmaco-economic model to assess whether pharmacogenomic (PGx)-guided warfarin treatment of elderly ischemic stroke patients with atrial fibrillation in Croatia is cost effective compared with non-PGx therapy. The time horizon of the model was set at 1 year. RESULTS Our primary analysis indicates that 97.07% (95% CI: 94.08-99.34%) of patients belonging to the PGx-guided group have not had any major complications, compared with the control group (89.12%; 95% CI: 84.00-93.87%, p < 0.05). The total cost per patient was estimated at €538.7 (95% CI: €526.3-551.2) for the PGx-guided group versus €219.7 (95% CI: €137.9-304.2) for the control group. In terms of quality-adjusted life-years (QALYs) gained, total QALYs was estimated at 0.954 (95% CI: 0.943-0.964) and 0.944 (95% CI: 0.931-0.956) for the PGx-guided and the control groups, respectively. The true difference in QALYs was estimated at 0.01 (95% CI: 0.005-0.015) in favor of the PGx-guided group. The incremental cost-effectiveness ratio of the PGx-guided versus the control groups was estimated at €31,225/QALY. CONCLUSION Overall, our data indicate that PGx-guided warfarin treatment may represent a cost-effective therapy option for the management of elderly patients with atrial fibrillation who developed ischemic stroke in Croatia.

Long-term angiographic outcome of stent- assisted coiling compared to non-assisted coiling of intracranial saccular aneurysms

Aim To compare angiographic result at long-term follow-up, and rates of progressive occlusion, recurrence, and retreatment of stent-assisted coiled (SAC) and non-assisted coiled (NAC) intracranial saccular aneurysms. Methods Retrospective evaluation of department records identified 260 patients with 283 saccular intracranial aneurysms who had long-term angiographic follow-up (more than 12 months) and were successfully treated with SAC (89 aneurysms) or NAC (194 aneurysms) at the University Hospital Center Zagreb from June 2005 to July 2012. Initial and control angiographic results in both groups were graded using Roy/Raymond scale, converted to descriptive terms, and the differences between them were evaluated for statistical significance. A multivariate analysis was performed to identify factors related to progression of aneurysm occlusion and recurrence at follow-up, and those related to aneurysm retreatment. Results There were more progressively occluded aneurysms in SAC group (38 of 89 aneurysms, 42.7%) than in NAC group (46 of 194, 23.7%) (P = 0.002), but there were no significant differences in the rates of recanalization, regrowth, and stable result. Multivariate logistic regression identified the use of stent as the most important factor associated with progressive occlusion (P = 0.015, odds ratio 2.22, 95% confidence interval 1.17-4.21), and large aneurysm size and posterior circulation location as most predictive of aneurysm recurrence and retreatment. Conclusion The use of stent is associated with delayed occlusion of initially incompletely coiled aneurysms during follow-up, but does not reduce the rate of recurrence and retreatment compared to coiling alone. Long-term angiographic follow-up is needed for both SAC and NAC aneurysms.

Cardiac Papillary Fibroelastoma: Source of Cerebral Embolism Treated With Intravenous Thrombolysis

We present the case of a 41-year-old man with sudden development of left hemiparesis due to infarction of the right middle cerebral artery that was successfully treated with intravenous (IV) thrombolysis with alteplase. Transthoracic echocardiography showed a small mass in the left ventricle. The patient underwent surgical resection, and histological examination of the mass confirmed the diagnosis of papillary fibroelastoma. It remains to be investigated whether heart ultrasound evaluation should be performed before IV thrombolysis in selected patients with stroke, given the apparently increased risk of bleeding. However, IV thrombolysis should not be postponed due to a lengthy investigation, because of its potential for reducing morbidity in patients with stroke.

Clinical application of genotype-guided dosing of warfarin in patients with acute stroke

BACKGROUND Patients with certain types of stroke need urgent anticoagulation and it is extremely important for them to achieve fast and stable anticoagulant effect and receive individualized treatment during the initiation of warfarin therapy. METHODS We conducted a prospective study among 210 acute stroke patients who had an indication for anticoagulation and compared the impact of CYP2C9 and VKORC1 genotype-guided warfarin dosing (PhG) with fixed dosing (NPhG) on anticoagulation control and clinical outcome between groups. RESULTS PhG achieved target INR values earlier, i.e., on average in 4.2 (4.1-4.7, 95% CI) days compared to NPhG (5.2 days [4.7-6.4, 95% CI]) (p = 0.0009), spent a higher percentage of time in the therapeutic INR range (76.3% [74.7-78.5, 95% CI] vs. 67.1% [64.5-69.6, 95% CI] in NPhG), and spent less time overdosed (INR > 3.1) (PhG 0.4 [0.1-0.7, 95% CI], NPhG 1.7 [1.1-2.3, 95% CI] days; p >0.000). PhG reached stable maintenance dose faster (10 [9.9-10.7, 95% CI] vs. 13.9 [13.3-14.7, 95% CI] days in controls; p = 0.0049) and had a better clinical outcome in relation to neurological deficit on admission as compared to NPhG. CONCLUSION We confirmed that warfarin therapy with genotype-guided dosing instead of fixed dosing reduces the time required for stabilization and improves anticoagulant control with better clinical outcome in early stages of warfarin therapy introduction among acute stroke patients, which is essential for clinical practice.

Prevalence of genetic polymorphisms of CYP2C9 and VKORC1 - implications for warfarin management and outcome in Croatian patients with acute stroke.

BACKGROUND Data on the prevalence of CYP2C9 and VKORC1 genes and their influence on anticoagulant effect and warfarin dose in stroke patients are scarce. The aim of this study was to determine the occurrence and significance of these gene polymorphisms and to establish pharmacogenetic algorithm to estimate the dose of introduction. Also, the goal was to determine tailored safety and intensity of anticoagulation response depending on the allelic variants and their impact on the clinical outcome in acute stroke patients in Croatia. METHODS A total of 106 consented acute stroke patients were tested for CYP2C9 2, 3 and VKORC1 1173C>T gene polymorphisms. We estimated the dose of introduction and monitored anticoagulant effect obtained by INR values, time to reach stable dose, stable maintenance dose, time spent within the therapeutic/supratherapeutic INR range, occurrence of dosage side effects and clinical outcome depending on genotypes. RESULTS We found that 83% of stroke patients in our study were carriers of multiple allelic variants. The predicted initial dose correlated with the stable warfarin maintenance dose (p=0.0311) and we correctly estimated the dose for 81.5% of 61.3% of study patients who required higher/lower doses than average. Warfarin dosage complications were slightly more frequent among the carriers of CYP2C9 2, 3 compared to the carriers of VKORC1 1173T alleles (68. 9% versus 62.5%), but their occurrence did not affect the final clinical outcome. CONCLUSION Our data indicated rapid and safe anticoagulation achieved by using pharmacogenetically-predicted warfarin dose in high-risk acute stroke patients without increasing the risk of warfarin dosage complications in an elderly population.

Glioblastoma multiforme presenting as Froin’s syndrome: a new face of an old foe

A 23-year-old male presented with acute development of headache and lower back pain. Headache was described as debilitating frontal pressure (VAS 7/10), and was preceded by 2 days of nausea and vomiting. At the time of initial examination he was disoriented, had fever, but showed no signs of focal neurological deficit. As CNS infection was suspected, a spinal tap was performed; cerebrospinal fluid (CSF) coagulated in one of the tubes and analysis showed elevated protein levels (12.05 g/L; normal values \0.37). Brain MRI findings are shown in Fig. 1a, b. The patient was referred to our hospital, at which point he had a mild paresis of the right quadriceps femoris with attenuated right patellar reflex. Another cerebrospinal fluid analysis was performed, this time showing xantochromy and protein level exceeding 17 g/L, with cell count of 53/3 and unremarkable cytology. A fundus examination was performed before the spinal tap and was normal. Spine MRI was performed and is shown in Fig. 1c. Five days after admission, the patient suffered a rapid clinical deterioration, developing quadriplegia, bilateral exophthalmus with ophthalmoplegia, and respiratory failure leading to mechanical ventilation. He was alert with preserved speech understanding. The clinical deterioration was not related to the spinal tap. Following stereotactic biopsy and pathohystological examination, a diagnosis of grade IV glioblastoma was made. After consultation with an oncologist and considering the severity of the disease, chemotherapy with carmustine (BCNU protocol) was started. The patient remained clinically stable until discharge to another hospital. A combination of CSF xanthochromia and hypercoagulation due to high protein level is called Froin’s syndrome, after the French physician who described it for the first time in 1903 [1]. While none of the originally described patients had tumors, today this rare phenomenon is associated, either with tumors causing spinal canal blockage [2], or with inflammation [3]. Intramedullary glioblastoma is a rare entity, representing 1–3 % of all spinal cord tumors, usually presents in a younger age (\30 years) and carries a bleak prognosis [4]. In conclusion, we would like to re-emphasize that Froin’s syndrome can have protean manifestations and a high index of clinical suspicion (especially when imaging of the brain reveals pathology) should be maintained in its diagnosis. J. Ljevak Z. Poljaković I. Adamec M. Habek (&) University Department of Neurology, Referral Center for Demyelinating Diseases of the Central Nervous System, University Hospital Center Zagreb, Kišpatićeva 12, 10000 Zagreb, Croatia e-mail: mhabek@mef.hr

Subacute brainstem angioencephalopathy : Favorable outcome with anticoagulation therapy

We present a patient who developed progressive neurological disease caused by lesions histologically compatible with those observed in subacute brainstem angioencephalopathy. The patient was treated with low-molecular weight heparin, and treatment response was monitored clinically and with MRI. Anticoagulation therapy stopped progression of the neurological deficit and led to improvement of MRI findings. This report further supports the existence of subacute brainstem angioencephalopathy as a characteristic disease entity and gives insight into possible therapeutic approach with anticoagulation treatment.