Atypical Phenotype in Two Patients with LAMA2 Mutations

Congenital muscular dystrophy type 1A is caused by mutations in the LAMA2 gene, which encodes the a2-chain of laminin. We report two patients with partial laminin-a2 deficiency and atypical phenotypes, one with almost exclusive central nervous system involvement (cognitive impairment and refractory epilepsy) and the second with marked cardiac dysfunction, rigid spine syndrome and limb-girdle weakness. Patients underwent clinical, histopathological, imaging and genetic studies. Both cases have two heterozygous LAMA2 variants sharing a potentially pathogenic missense mutation c.2461A>C (p.Thr821Pro) located in exon 18. Brain MRI was instrumental for the diagnosis, since muscular examination and motor achievements were normal in the first patient and there was a severe cardiac involvement in the second. The clinical phenotype of the patients is markedly different which could in part be explained by the different combination of mutations types (two missense versus a missense and a truncating mutation).

Sialyl Tn-Expressing Bladder Cancer Cells Induce a Tolerogenic Phenotype in Innate and Adaptive Immune Cells

Despite the wide acceptance that glycans are centrally implicated in immunity, exactly how they contribute to the tilt immune response remains poorly defined. In this study, we sought to evaluate the impact of the malignant phenotype-associated glycan, sialyl-Tn (STn) in the function of the key orchestrators of the immune response, the dendritic cells (DCs). In high grade bladder cancer tissue, the STn antigen is significantly overexpressed and correlated with the increased expression of ST6GALNAC1 sialyltransferase. Bladder cancer tissue presenting elevated expression of ST6GALNAC1 showed a correlation with increased expression of CD1a, a marker for bladder immature DCs and showed concomitant low levels of Th1-inducing cytokines IL-12 and TNF-α. In vitro, human DCs co-incubated with STn+ bladder cancer cells, had an immature phenotype (MHC-IIlow, CD80low and CD86low) and were unresponsive to further maturation stimuli. When contacting with STn+ cancer cells, DCs expressed significantly less IL-12 and TNF-α. Consistent with a tolerogenic DC profile, T cells that were primed by DCs pulsed with antigens derived from STn+ cancer cells were not activated and showed a FoxP3high IFN-γlow phenotype. Blockade of STn antigens and of STn+ glycoprotein, CD44 and MUC1, in STn+ cancer cells was able to lower the induction of tolerance and DCs become more mature. Overall, our data suggest that STn-expressing cancer cells impair DC maturation and endow DCs with a tolerogenic function, limiting their capacity to trigger protective anti-tumour T cell responses. STn antigens and, in particular, STn+ glycoproteins are potential targets for circumventing tumour-induced tolerogenic mechanisms.

Hyper-IgD and Periodic Fever Syndrome: a New MVK Mutation (p.R277G) Associated with a Severe Phenotype

Hyperimmunoglobulinemia D and periodic fever syndrome (HIDS; MIM#260920) is a rare recessively-inherited autoinflammatory condition caused bymutations in the MVK gene, which encodes for mevalonate kinase, an essential enzyme in the isoprenoid pathway. HIDS is clinically characterized by recurrent episodes of fever and inflammation. Herewe report on the case of a 2 year-old Portuguese boy with recurrent episodes of fever, malaise, massive cervical lymphadenopathy and hepatosplenomegaly since the age of 12 months. Rash, arthralgia, abdominal pain and diarrhea were also seen occasionally. During attacks a vigorous acute-phase response was detected, including elevated erythrocyte sedimentation rate, C-reactive protein, serum amyloid A and leukocytosis. Clinical and laboratory improvement was seen between attacks. Despite normal serum IgD level, HIDS was clinically suspected. Mutational MVK analysis revealed the homozygous genotype with the novel p.Arg277Gly (p.R277G) mutation, while the healthy non consanguineous parents were heterozygous. Short nonsteroidal anti-inflammatory drugs and corticosteroid courses were given during attacks with poor benefits, where as anakinra showed positive responses only at high doses. The p.R277Gmutation here described is a novel missense MVK mutation, and it has been detected in this casewith a severe HIDS phenotype. Further studies are needed to evaluate a co-relation genotype, enzyme activity and phenotype, and to define the best therapeutic strategies.

Leber Congenital Amaurosis: Comprehensive Survey of the Genetic Heterogeneity, Refinement of the Clinical Definition, and Genotype-Phenotype Correlations as a Strategy for Molecular Diagnosis

Leber congenital amaurosis (LCA) is the earliest and most severe form of all inherited retinal dystrophies, responsible for congenital blindness. Disease-associated mutations have been hitherto reported in seven genes. These genes are all expressed preferentially in the photoreceptor cells or the retinal pigment epithelium but they are involved in strikingly different physiologic pathways resulting in an unforeseeable physiopathologic variety. This wide genetic and physiologic heterogeneity that could largely increase in the coming years, hinders the molecular diagnosis in LCA patients. The genotyping is, however, required to establish genetically defined subgroups of patients ready for therapy. Here, we report a comprehensive mutational analysis of the all known genes in 179 unrelated LCA patients, including 52 familial and 127 sporadic (27/127 consanguineous) cases. Mutations were identified in 47.5% patients. GUCY2D appeared to account for most LCA cases of our series (21.2%), followed by CRB1 (10%), RPE65 (6.1%), RPGRIP1 (4.5%), AIPL1 (3.4%), TULP1 (1.7%), and CRX (0.6%). The clinical history of all patients with mutations was carefully revisited to search for phenotype variations. Sound genotype-phenotype correlations were found that allowed us to divide patients into two main groups. The first one includes patients whose symptoms fit the traditional definition of LCA, i.e., congenital or very early cone-rod dystrophy, while the second group gathers patients affected with severe yet progressive rod-cone dystrophy. Besides, objective ophthalmologic data allowed us to subdivide each group into two subtypes. Based on these findings, we have drawn decisional flowcharts directing the molecular analysis of LCA genes in a given case. These flowcharts will hopefully lighten the heavy task of genotyping new patients but only if one has access to the most precise clinical history since birth.

Mutations in the MECP2 Gene Are Not a Major Cause of Rett Syndrome-Like or Related Neurodevelopmental Phenotype in Male Patients

Rett syndrome is a genetic neurodevelopmental disorder that affects mainly girls, but mutations in the causative MECP2 gene have also been identified in boys with classic Rett syndrome and Rett syndrome-like phenotypes. We have studied a group of 28 boys with a neurodevelopmental disorder, 13 of which with a Rett syndrome-like phenotype; the patients had diverse clinical presentations that included perturbations of the autistic spectrum, microcephaly, mental retardation, manual stereotypies, and epilepsy. We analyzed the complete coding region of the MECP2 gene, including the detection of large rearrangements, and we did not detect any pathogenic mutations in the MECP2 gene in these patients, in whom the genetic basis of disease remained unidentified. Thus, additional genes should be screened in this group of patients.

Borderline CD30+ Cutaneous Lymphoproliferative Disorder: Report of a Case with Expression of Cytotoxic Markers and Response to Clarithromycin

CD30+ cutaneous lymphoproliferative disorders (CLPDs) are usually characterized by a benign clinical course. The prognostic value of cytotoxic markers in these lymphomas has not been evaluated in large series. We describe a case of borderline CD30+ CLPD with cytotoxic phenotype, presenting in a 22-year-old male patient as an ulcer on the forearm. He reported having had similar ulcers on the buttock and thigh that spontaneously regressed over the course of 1 year. The lesion resolved with a single course of clarithromycin; a subsequent lesion, too, responded to clarithromycin, and no recurrences or systemic involvement have been documented in the 9-month follow-up. A conservative approach in the management of CD30+ CLPD is recommended. We believe that the anti-inflammatory and apoptotic effects of clarithromycin on T cells may have hastened the remission process.

Prophylaxis in Hemophilia A Patients with Inhibitors

The development of antibodies to factor VIII is one of the most serious complications of haemophilia treatment. Approximately 30% of patients with severe haemophilia develop neutralizing inhibitors to replacement FVIII. Although most patients with inhibitors do not bleed more frequently than patients without inhibitors, bleeding is more difficult to control and this patients suffer more severe bleeding and have greater morbidity and mortality. Patients with persistent high-titer inhibitor who are not candidates or fail ITI, pose a great challenge to haemophilia management. The efficacy and safety of prophylaxis with bypassing agents in reducing bleeding tendency, has been described in numerous studies. Patients and methods: We report tree adult severe haemophilia A patients, two with persistent high-titre inhibitors and one who failed ITI, on prophylactic treatment after several significant musculoskeletal and life-threatening haemorrhagic episodes (intrabdominal/intramuscular) and pseudotumor haemorrhage. Treatment regimens consisted of APCC (Feiba®) in doses of 60-70UKg-1, 2-3 times per week, according underlying bleeding phenotype. Breakthrough bleeds were treated with either APCC (Feiba®) or rFVIIa (NovoSeven®). Results and Conclusion: There was reduction in total bleeding episodes in two patients (43% to 80%) and one patient remained stable, while receiving prophylaxis. Absence of severe and life threatening bleeding episodes, as well as inpatient stays, contributing to a better quality of life in those patients, was observed. APCC (Feiba®) was well tolerated and no thrombotic events were observed.

Blastic Plasmacytoid Dendritic Cell Neoplasm

Blastic plasmacytoid dendritic cell neoplasm is a rare and aggressive hematodermic neoplasia with frequent cutaneous involvement and leukemic dissemination. We report the case of a 76-year-old man with a 2 month history of violaceous nodules and a tumor with stony consistency, located on the head, and mandibular, cervical and supraclavicular lymphadenopathies. Multiple thoracic and abdominal adenopathies were identified on computerized tomography. Flow cytometry analysis of the skin, lymph node and bone marrow biopsies demonstrated the presence of plasmocytoid dendritic cell neoplastic precursor cells (CD4+, CD45+, CD56+ and CD123+ phenotype). After initial clinical and laboratorial complete remission with chemotherapy, the patient died due to relapse of the disease associated with the appearance of a cervical mass with medullary compromise.

Cerebrospinal Fluid Synaptic Proteins as Useful Biomarkers in Tyrosine Hydroxylase Deficiency

Tyrosine hydroxylase (TH) deficiency is an inborn error of dopamine biosynthesis and a cause of early parkinsonism. Two clinical phenotypes have been described. Type “B”: early onset severe encephalopathy; type “A”: later onset, less severe and better response to L-dopa. We aimed to study the expression of several key dopaminergic and gabaergic synaptic proteins in the cerebrospinal fluid (CSF) of a series of patients with TH deficiency and their possible relation with the clinical phenotype and response to L-DOPA. Dopamine transporter (DAT), D2-receptor and vesicularmonoamine transporter (VMAT2)weremeasured in the CSF of 10 subjectswith THdeficiency byWestern blot analysis. In 3 patients, data of pre- and post-treatmentwith L-DOPA were available, and in one of them, GABA vesicular transporter was determined. Results were compared to an age-matched control population. The concentration of D2-receptors in CSFwas significantly higher in patients with TH deficiency than in controls. Similarly, DAT and vesicular monoamine transporter type 2 were up-regulated. Studies performed before LDOPA, and on L-DOPA therapy showed a paradoxical response with D2 receptor expression increase as L-Dopa doses and homovanillic concentration gradually raised in a B phenotype patient. The opposite results were found in two patients with A phenotype. However, this is a very small sample, and further studies are needed to conclude robust differences between phenotypes. Synaptic proteins are detectable in the CSF and their quantification can be useful for understanding the pathophysiology of neurotransmitter defects and potentially to adjust and personalize treatments in the future.

A Síndrome de Smith-Lemli-Opitz: Características Fenotípicas e Genotípicas dos Doentes Portugueses

A síndrome de Smith-Lemli-Opitz (SLOS) é uma síndrome polimalformativa de transmissão autossómica recessiva causada por um défice metabólico da biossíntese do colesterol, que se caracteriza por dismorfias craniofaciais, anomalias congénitas de vários órgãos (salientando-se as do esqueleto e do aparelho urogenital), restrição de crescimento intra-uterino (RCIU), alterações comportamentais e atraso mental. É causada por mutações no gene DHCR7, que codifica para a enzima 7-dehidrocolesterol reductase, responsável pelo último passo da via metabólica da síntese do colesterol. A SLOS caracteriza-se por níveis diminuídos de colesterol e concentrações altas do seu precursor, 7-dehidrocolesterol, no sangue e tecidos. Procedeu-se a uma análise comparativa dos fenótipo e genótipo de quinze casos de SLOS de origem portuguesa, e são tecidas considerações quanto às dificuldades e limitações inerentes ao diagnóstico, e ao facto de esta doença hereditária do metabolismo dever ser considerada no diagnóstico diferencial das situações de (i) hipocolesterolémia, (ii) RCIU e (iii) síndromes polimalformativas,(especialmente quando crianças com atraso de crescimento apresentam simultaneamente sindactilia do segundo e terceiro dedos do pé e microcefalia e/ou narinas antevertidas entre outras anomalias).

Atorvastatin versus Bezafibrate in Mixed Hyperlipidaemia : Randomised Clinical Trial of Efficacy and Safety (the ATOMIX Study)

OBJECTIVE: Combined hyperlipidaemia is a common and highly atherogenic lipid phenotype with multiple lipoprotein abnormalities that are difficult to normalise with single-drug therapy. The ATOMIX multicentre, controlled clinical trial compared the efficacy and safety of atorvastatin and bezafibrate in patients with diet-resistant combined hyperlipidaemia. PATIENTS AND STUDY DESIGN: Following a 6-week placebo run-in period, 138 patients received atorvastatin 10mg or bezafibrate 400mg once daily in a randomised, double-blind, placebo-controlled trial. To meet predefined low-density lipoprotein-cholesterol (LDL-C) target levels, atorvastatin dosages were increased to 20mg or 40mg once daily after 8 and 16 weeks, respectively. RESULTS: After 52 weeks, atorvastatin achieved greater reductions in LDL-C than bezafibrate (percentage decrease 35 vs 5; p < 0.0001), while bezafibrate achieved greater reductions in triglyceride than atorvastatin (percentage decrease 33 vs 21; p < 0.05) and greater increases in high-density lipoprotein-cholesterol (HDL-C) [percentage increase 28 vs 17; p < 0.01 ]. Target LDL-C levels (according to global risk) were attained in 62% of atorvastatin recipients and 6% of bezafibrate recipients, and triglyceride levels <200 mg/dL were achieved in 52% and 60% of patients, respectively. In patients with normal baseline HDL-C, bezafibrate was superior to atorvastatin for raising HDL-C, while in those with baseline HDL-C <35 mg/dL, the two drugs raised HDL-C to a similar extent after adjustment for baseline values. Both drugs were well tolerated. CONCLUSION: The results show that atorvastatin has an overall better efficacy than bezafibrate in concomitantly reaching LDL-C and triglyceride target levels in combined hyperlipidaemia, thus supporting its use as monotherapy in patients with this lipid phenotype.

Disruption of Urate Transport in Familial Renal Glucosuria and Report on SGLT2 Expression in Normal and Pathological Kidney

Familial renal glucosuria (FRG) is a rare co -dominantly inherited benign phenotype characterized by the presence of glucose in the urine. It is caused by mutations in the SLC5A2 gene that encodes SGLT2, a Na+ -glucose co -transporter. The purpose of our current work was twofold: to characterize the molecular and phenotype findings of an FRG cohort and, in addition, to detail the SGLT2 expression in the adult human kidney. The phenotype of FRG pedigrees was evaluated using direct sequencing for the identification of sequence variations in the SLC5A2 gene. The expression of SGLT2 in the adult human kidney was studied by immunofluorescence on kidney biopsy specimens. In the absence of renal biopsies from FRG individuals, and in order to evaluate the potential disruption of SGLT2 expression in a glucosuric nephropathy, we have selected cases of nucleoside analogues induced proximal tubular toxicity. We identified six novel SLC5A2 mutations in six FRG pedigrees and described the occurrence of hyperuricosuria associated with hypouricaemia in the two probands with the most severe phenotypes. Histopathological studies proved that SGLT2 is localized to the brush -border of the proximal tubular epithelia cell and that this normal pattern was found to be disrupted in cases of nucleoside analogues induced tubulopathy. We present six novel SLC5A2 mutations, further contributing to the allelic heterogeneity in FRG, and identified hyperuricosuria and hypouricaemia as part of the FRG phenotype. SGLT2 is localized to the brush -border of the proximal tubule in the adult human normal kidney, and aberrant expression of the co -transporter may underlie the glucosuria seen with the use of nucleoside analogues.

Rett Syndrome With and Without Detected MECP2 Mutations: an Attempt to Redefine Phenotypes

Background: The diagnosis of Rett syndrome (RTT) is based on a set of clinical criteria, irrespective of mutation status. The aims of this study were (1) to define the clinical differences existing between patients with Rett syndrome with (Group I) and without a MECP2 mutation (Group II), and (2) to characterize the phenotypes associated with the more common MECP2 mutations. Patients and Methods: We analyzed 87 patients fulfilling the clinical criteria for RTT. All were observed and videotaped by the same paediatric neurologist. Seven common mutations were considered separately, and associated clinical features analysed. Results: Comparing Group I and II, we found differences concerning psychomotor development prior to onset, acquisition of propositive manipulation and language, and evolving autistic traits. Based on age at observation, we found differences in eye pointing, microcephaly, growth, number of stereotypies, rigidity, ataxia and ataxic-rigid gait, and severity score. Patients with truncating differed from those with missense mutations regarding acquisition of propositive words and independent gait, before the beginning of the disease, and microcephaly, growth, foot length, dystonia, rigidity and severity score, at the time of observation. Patients with the R168X mutation had a more severe phenotype, whereas those with R133C showed a less severe one. Patients with R294X had a hyperactive behaviour, and those with T158M seemed to be particularly ataxic and rigid. Conclusion: A clear regressive period (with loss of prehension and language, deceleration of growth) and the presence of more than three different stereotypies, rigidity and ataxic-rigid gait seemed to be very helpful in differentiating Group I from Group II.

Evaluation of CSF Neurotransmitters and Folate in 25 Patients with Rett Disorder and Effects of Treatment

Background: Rett disorder (RD) is a progressive neurodevelopmental entity caused by mutations in the MECP2 gene. It has been postulated that there are alterations in the levels of certain neurotransmitters and folate in the pathogenesis of this disease. Here we re-evaluated this hypothesis. Patients and Methods: We evaluated CSF folate, biogenic amines and pterines in 25 RD patients. Treatment with oral folinic acid was started in those cases with low folate. Patients were clinically evaluated and videotaped up to 6 months after therapy. Results: CSF folate was below the reference values in 32% of the patients. Six months after treatment no clinical improvement was observed. Three of the four patients with the R294X mutation had increased levels of a dopamine metabolite associated to a particular phenotype. Three patients had low levels of a serotonin metabolite. Two of them were treated with fluoxetine and one showed clinical improvement. No association was observed between CSF folate and these metabolites, after adjusting for the patients age and neopterin levels. Conclusion: Our results support that folinic acid supplementation has no significant effects on the course of the disease. We report discrete and novel neurotransmitter abnormalities that may contribute to the pathogenesis of RD highlighting the need for further studies on CSF neurotransmitters in clinically and genetically well characterized patients.