Resistance to thyroid hormone due to a novel mutation in the thyroid beta receptor (THRß) gene coexisting with autoimmune thyroid disease-A case report.

Resistance to thyroid hormone (RTH) is a syndrome characterized by impaired responsiveness of target tissues to thyroid hormones. The relationship between RTHß and thyroid autoimmunity has been under research. In this study, we demonstrate a case report of a woman with a novel mutation in THRß gene coexisting with autoimmune thyroid disease (AITD). The 36-year-old woman has been treated since childhood for a thyroid disease. Based on high levels of thyroid hormones (THs) and elevated concentrations of thyroperoxidase and thyroglobulin antibodies (TPOAb and TgAb, respectively), she received unnecessary long-term treatment with methimazole and finally underwent subtotal thyroidectomy. After the surgery, her TSH level remained significantly elevated, despite the treatment with 150 + 15 µg of thyroxine and triiodothyronine. A sequence analysis of the THRß gene revealed a novel dinucleotide substitution affecting codon 453, resulting in the replacement of the normal proline with an asparagine (c.1357_1358delinsAA, p.(Pro453Asn)). The mutation has not been described in the literature yet; however, THRß codon 453 represents a mutational hot spot, frequently altered in the TH receptor ß gene. After establishing the diagnosis of RTH, the patient was treated with 300 µg of thyroxine, which showed clinical improvement and normalization of TSH. The coexistence of RTHß and AITD may additionally impede establishment of a proper diagnosis, leading to unnecessary therapy and delayed correct treatment. The presented case encourages a closer cooperation between clinical endocrinologists and geneticists.

Multisite assessment of the impact of cell-free DNA-based screening for rare autosomal aneuploidies on pregnancy management and outcomes.

Cell-free (cf) DNA screening is a noninvasive prenatal screening approach that is typically used to screen for common fetal trisomies, with optional screening for sex chromosomal aneuploidies and fetal sex. Genome-wide cfDNA screening can screen for a wide variety of additional anomalies, including rare autosomal aneuploidies (RAAs) and copy number variants. Here, we describe a multi-cohort, global retrospective study that looked at the clinical outcomes of cases with a high-risk cfDNA screening result for a RAA. Our study cohort included a total of 109 cases from five different sites, with diagnostic outcome information available for 68% (74/109) of patients. Based on confirmatory diagnostic testing, we found a concordance rate of 20.3% for presence of a RAA (15/74) in our study population. Pregnancy outcome was also available for 77% (84/109) of cases in our cohort. Many of the patients experienced adverse pregnancy outcomes, including intrauterine fetal demise, fetal growth restriction, and preterm birth. These adverse outcomes were observed both in patients with fetal or placental confirmation of the presence of a RAA, as well as patients that did not undergo fetal and/or placental diagnostic testing. In addition, we have proposed some suggestions for pregnancy management and counseling considerations for situations where a RAA is noted on a cfDNA screen. In conclusion, our study has shown that genome-wide cfDNA screening for the presence of rare autosomal aneuploidies can be beneficial for both patients and their healthcare practitioners. This can provide a possible explanation for an adverse pregnancy outcome or result in a change in pregnancy management, such as increased monitoring for adverse outcomes.

The prognostic role of BRAF and WNT pathways activation in kinase inhibitors-naïve clinical stage III cutaneous melanoma.

The results of local-regional advanced melanoma (stage III) management are still not satisfactory. Particularly, there is no personalized treatment in stage III melanoma patients due to the lack of useful classical pathological markers for prognostication of indolent or aggressive course of the disease. The aim of this study was to explore melanoma genomic landscape by means of the mutational profiling of 50 genes influencing carcinogenesis pathways in the randomly selected 93 kinase inhibitor-naïve (KI-naïve) stage III patients. The genomic alterations were found in 27 out of 50 tested genes and at least one pathogenic variant was detected in 77 out of 93 cases (82.7%). Survival rate was negatively affected by the presence of the somatic mutations in AKT1, ATM, CDH1 and SMARCB1, while the BRAF+ status in KI-naïve stage III population correlated with the longer median overall survival. Genomic alterations in WNT pathway correlated with extranodal adipocyte tissue involvement (P = 0.027) and higher number of metastatic lymph nodes (P = 0.045). In terms of survival, the Cox model confirmed the worse prognosis in patients with mutation in the WNT pathway [hazard ratio (HR) = 2.9, P = 0.017], and better prognosis in cases with mutations in BRAF pathway (HR = 0.5, P = 0.004). WNT/ß-catenin pathway alteration was associated with more advanced/aggressive disease. From this perspective, the concept of blocking the activity of the WNT pathway in selected cases appears promising and complementary to the BRAF inhibition therapeutic option for the future.

Cell-free DNA BRAF V600E measurements during BRAF inhibitor therapy of metastatic melanoma: long-term analysis.

OBJECTIVE: We assessed the status of the BRAF V600E mutation in cell-free circulating tumor DNA (cfDNA) isolated from the plasma of patients with metastatic melanoma treated with the BRAF inhibitor vemurafenib, collected at different time points during therapy to evaluate the sensitivity and specificity of quantitative polymerase chain reaction and droplet digital polymerase chain reaction (ddPCR) and the correlation between the level of plasma cfDNA p.V600E and the long-term clinical outcome. METHODS: cfDNA in patients with BRAF-mutated melanoma (n = 62) was analyzed at baseline and at 4-8 weeks from the start of vemurafenib therapy. BRAF mutations were assessed using tumor tissue-derived DNA and circulating cfDNA from plasma samples. Quantification of BRAF V600E was performed in cfDNA using ddPCR. RESULTS: cfDNA V600E was detected in the plasma of 48/62 (77%) patients at baseline and in 18/62 (29%) patients after 4-8 weeks of treatment. Patients positive for BRAF mutations in cfDNA at baseline had shorter progression-free survival (PFS) and overall survival (OS) compared with patients with undetectable cfDNA BRAF mutations. Undetectable cfDNA p.V600E at baseline and after 4-8 weeks of therapy was associated with the best prognosis. When treated as a continuous variable, the log-transformed concentration of baseline cfDNA p.V600E was significantly associated with both PFS and OS. This effect was retained in the multivariate OS Cox model adjusted for Eastern Cooperative Oncology Group performance status, the presence of brain metastases, patient age, and previous systemic treatment. CONCLUSIONS: Monitoring of plasma BRAF p.V600E cfDNA concentrations in patients with metastatic melanoma on targeted therapy may have prognostic value. Undetectable cfDNA p.V600E before and during treatment was associated with a favorable prognosis.

Comparison between two widely used laboratory methods in BRAF V600 mutation detection in a large cohort of clinical samples of cutaneous melanoma metastases to the lymph nodes.

AIMS: The study compares detection rates of oncogenic BRAF mutations in a homogenous group of 236 FFPE cutaneous melanoma lymph node metastases, collected in one cancer center. BRAF mutational status was verified by two independent in-house PCR/Sanger sequencing tests, and the Cobas® 4800 BRAF V600 Mutation Test. RESULTS: The best of two sequencing approaches returned results for 230/236 samples. In 140 (60.9%), the mutation in codon 600 of BRAF was found. 91.4% of all mutated cases (128 samples) represented p.V600E. Both Sanger-based tests gave reproducible results although they differed significantly in the percentage of amplifiable samples: 230/236 to 109/143. Cobas generated results in all 236 cases, mutations changing codon V600 were detected in 144 of them (61.0%), including 5 not amplifiable and 5 negative in the standard sequencing. However, 6 cases positive in sequencing turned out to be negative in Cobas. Both tests provided us with the same BRAF V600 mutational status in 219 out of 230 cases with valid results (95.2%). CONCLUSIONS: The total BRAF V600 mutation detection rate didn't differ significantly between the two methodological approaches (60.9% vs. 61.0%). Sequencing was a reproducible method of V600 mutation detection and more powerful to detect mutations other than p.V600E, while Cobas test proved to be less susceptible to the poor DNA quality or investigator's bias. The study underlined an important role of pathologists in quality assurance of molecular diagnostics.

IL-10, IL-12B and IL-17 gene polymorphisms in patients with mixed connective tissue disease.

OBJECTIVES: Mixed connective tissue disease (MCTD) is a rare systemic autoimmune disease with a prevalence of about 10 cases/100,000. It seems that in the pathogenesis of MCTD no individual cytokines/cells, but rather an altered pattern of these markers altogether may contribute to the autoimmune processes and their balance determines disease activity. IL-10, IL-12 and IL-17F as inflammatory cytokines might be an important functional candidate genes for autoimmune diseases including MCTD. METHODS: The study group consisted of 66 patients with MCTD and of 106 (163 for IL-12B) healthy individuals. SNPs in the IL-10 (- 592C/A, - 1082G/A), IL-12B (+ 1188A/C) and IL-17F (His161Arg, Glu126Gly) genes were investigated by PCR-RFLP approach. RESULTS: The frequency of the IL-10-592A and -1082A allele was higher in MCTD patients than in control groups (both p = 0,0000). In addition the -1082G/A IL-10 gene polymorphism was associated with esophageal involvement and with anti-U1-A and -C antibodies. The IL-17 7488A/G variant showed correlation with presence of anti-SmB and anti-dsDNA antibodies, while the IL-17F 7383A/G variant was associated with Sjögren's syndrome and leuco-and thrombocytopenia. Moreover, the IL-12 SNP + 1188A/C showed correlation with sclerodactyly in MCTD patients. CONCLUSION: Present findings indicate that IL-10 gene variants may be considered as genetic risk factors for MCTD susceptibility.

Genetic polymorphisms of Foxp3 in patients with rheumatoid arthritis.

OBJECTIVE: The aim of the study was to identify 2 polymorphic variants in the promoter region of the Foxp3 gene and their possible association with susceptibility to and severity of rheumatoid arthritis (RA). The association between genetic factors and pathogenesis suggests that T cells take part in the induction of RA. The CD4+CD25highFoxp3+ subset of regulatory T cells plays an essential role in preventing autoimmunity and maintaining immune homeostasis. METHODS: Patients with RA (n = 274) and healthy individuals (n = 295) were examined for -3279 C/A and -924 A/G Foxp3 gene polymorphisms by the polymerase chain reaction-restriction fragment-length polymorphism method. Serum Foxp3 levels in patients with RA and controls were measured with ELISA. RESULTS: Foxp3 -3279 A and -924 G alleles were associated with significantly elevated risk of RA in the population tested (p = 0.003 and p = 0.004, respectively) compared to the wild-type alleles. Overall, -3279 C/A and -924 A/G Foxp3 gene polymorphisms were in indistinct linkage disequilibrium with D' = 0.481 and r(2) = 0.225. From 4 possible haplotypes, frequencies of 2 (AG and CA) showed significant differences between both examined groups (respectively, p < 0.001 and p = 0.007). After appropriate adjustment of Bonferroni correction for multiple testing, the genotype-phenotype analysis showed no significant correlation of the Foxp3 -3279 C/A and -924 A/G polymorphisms with the disease activity, joint damage, laboratory variables, and extraarticular manifestation in patients with RA. Serum Foxp3 level was significantly higher in patients than in controls (p < 0.0001). CONCLUSION: Current findings indicated that the Foxp3 genetic polymorphism and the Foxp3 protein level may be associated with susceptibility to RA in the Polish population.

HLA-B27 detection - comparison of genetic sequence-based method and flow cytometry assay.

OBJECTIVES: The presence of human leukocyte antigen B27 (HLA-B27) is strongly associated with ankylosing spondylitis. HLA-B27 testing is routinely applied in the diagnosis of this disease. The aim of the present study was to compare two methods of HLA-B27 detection - a genetic sequence-based method and a flow cytometry assay. MATERIAL AND METHODS: Peripheral blood was obtained from 300 individuals with suspected spondyloarthropathy. Expression of HLA-B27 on the T cell surface was analysed by flow cytometry assay using GS145.2 monoclonal antibody specific for HLA-B27. DNA was isolated from the whole blood. Genes coding for HLA-B27, -B40 and -B47:01 were detected by polymerase chain reaction using the MW02/MW09 primer pair. Then, positive samples were sequenced in order to discriminate allelic variations of the HLA-B27 gene. Results of sequencing were analysed using Chromas LITE 2.1.1 software, BLAST software and the IMGT/HLA database. Ambiguous samples were additionally analysed by polymerase chain reaction using E91 and E136 primers amplifying a 135-bp fragment of the human HLA-B27 gene. RESULTS: Among 300 samples, 76 were HLA-B27-positive on the basis of flow cytometry analysis. Genetic sequence analysis confirmed positivity of 73 from among 76 samples. Two hundred twenty six samples were HLA-B27-negative, whereas the result of one sample analysis was ambiguous. Fifty-three samples were identified as allelic variation 27:05, 19 samples as allelic variation 27:02, and one sample as allelic variation 27:07. CONCLUSIONS: This study shows that the genetic sequence-based method and the flow cytometry assay give consistent results in 99% of cases. The performed genetic analysis proves that the majority of HLA-B27-positive samples belong to the 27:05 allelic variation, which is strongly associated with high risk of ankylosing spondylitis.

Molecular alterations in clinical stage III cutaneous melanoma: Correlation with clinicopathological features and patient outcome.

The aim of the present study was to evaluate the frequency and type of oncogenic v-raf murine sarcoma viral oncogene homolog B1 (BRAF)/neuroblastoma RAS viral (v-ras) oncogene homolog (NRAS) mutations in cutaneous melanoma with clinically detected nodal metastases (stage IIIB and C) in relation to clinicopathological features and outcome. The clinicopathological data of 250 patients following therapeutic lymphadenectomy (LND) between 1995 and 2010, as well as BRAF/NRAS mutational status in corresponding nodal metastases, were analyzed. The median follow-up time was 53 months. BRAF mutations were detected in 154 (62%) cases (141 p.V600E, nine p.V600K and four others) and mutually exclusive NRAS mutations were detected in 42 (17%) cases. The presence of a BRAF mutation was found to correlate with patients of a younger age. The five-year overall survival (OS) rate was 33 and 43% for LND and primary tumor excision, respectively, and the five-year disease-free survival (DFS) rate for LND was 25%. No correlation was identified between BRAF/NRAS mutational status and RFS or OS (calculated from the date of the LND and primary tumor excision); for BRAF- and NRAS-mutated melanoma, the prognosis was the same for patients with wild-type (WT) melanoma. The important factors which had a negative impact on OS and DFS were as follows: Male gender, >1 metastatic lymph node and extracapsular extension of nodal metastases. The interval between the diagnosis of the initial melanoma to regional nodal metastasis (median, 10 months) was not significantly different between BRAF-mutant and -WT patients. Our largest comprehensive molecular analysis of clinical stage III melanoma revealed that BRAF and NRAS mutational status is not a prognostic marker in stage III melanoma patients with macroscopic nodal involvement, but may have implications for potential adjuvant therapy.

Molecular characterization and patient outcome of melanoma nodal metastases and an unknown primary site.

BACKGROUND: Melanoma of unknown primary site (MUP) is not a completely understood entity with nodal metastases as the most common first clinical manifestation. The aim of this multicentric study was to assess frequency and type of oncogenic BRAF/NRAS/KIT mutations in MUP with clinically detected nodal metastases in relation to clinicopathologic features and outcome. MATERIALS AND METHODS: We analyzed series of 103 MUP patients (period: 1992-2010) after therapeutic lymphadenectomy (LND): 40 axillary, 47 groin, 16 cervical, none treated with BRAF inhibitors. We performed molecular characterization of BRAF/NRAS/KIT mutational status in nodal metastases using direct sequencing of respective coding sequences. Median follow-up time was 53 months. RESULTS: BRAF mutations were detected in 55 cases (53 %) (51 V600E, 93 %; 4 others, 7 %), and mutually exclusive NRAS mutations were found in 14 cases (14 %) (7 p.Q61R, 4 p.Q61K, 2 p.Q61H, 1 p.Q13R). We have not detected any mutations in KIT. The 5-year overall survival (OS) was 34 %; median was 24 months. We have not found significant correlation between mutational status (BRAF/NRAS) and OS; however, for BRAF or NRAS mutated melanomas we observed significantly shorter disease-free survival (DFS) when compared with wild-type melanoma patients (p = .04; 5-year DFS, 18 vs 19 vs 31 %, respectively). The most important factor influencing OS was number of metastatic lymph nodes >1 (p = .03). CONCLUSIONS: Our large study on molecular characterization of MUP with nodal metastases showed that MUPs had molecular features similar to sporadic non-chronic-sun-damaged melanomas. BRAF/NRAS mutational status had negative impact on DFS in this group of patients. These observations might have potential implication for molecular-targeted therapy in MUPs.

What are the current outcomes of advanced gastrointestinal stromal tumors: who are the long-term survivors treated initially with imatinib?

The introduction of imatinib to clinical practice revolutionized therapy of advanced gastrointestinal stromal tumors (GIST), but its long-term results have been only just collected. We have attempted to identify factors related to the long-term survival. We have analyzed the data of 430 inoperable/metastatic/recurrent GIST patients treated with imatinib in reference centers, assessed the factors influencing the long-term overall survival (OS), and compared the outcomes in three periods of initiation of imatinib therapy during one decade (2001-2003, 2004-2006, 2007-2010). During analyzed time periods, we have found decrease in median largest tumor size at the start of imatinib therapy: 90.5 mm (2001-2003) versus 74 mm (2004-2006) versus 58 mm (2007-2010) (p = 0.002). Median progression-free survival (PFS) on 1st line imatinib was 37.5 months, without differences in PFS between three groups. Median OS was 5.8 years, 8-year OS rate was 43%, and no difference in OS was demonstrated for patients treated in analyzed time periods. Independent good prognostic factors for longer OS were as follows: surgery of residual disease, initial WHO performance status 0/1, normal baseline albumin level, and the presence of exon 11 KIT mutations. Current median OS in advanced GIST reaches 6 years. The long-term survivors were characterized by smaller maximal tumors at imatinib start, better blood tests results, better performance status, and the surgical removal of residual disease. The latter might reduce the impact of tumor size and equalize the long-term results of therapy during last decade from introduction of imatinib. After introduction of subsequent lines of therapy (as sunitinib), the effect of primary mutational status on the long-term OS is also less visible.

Development of a new, simple and cost-effective diagnostic tool for genetic screening of hereditary colorectal cancer--the DNA microarray assay.

Detection of mutations in families with a hereditary predisposition to colon cancer gives an opportunity to precisely define the high-risk group. 36 patients operated on for colon cancer, with familiar prevalence of this malignancy, were investigated using the DNA microarrays method with the potential detection of 170 mutations in MLH1, MSH2, MSH6, CHEK2, and NOD2 genes. In microarrays analysis of DNA in 9 patients (25% of the investigated group), 6 different mutations were found. The effectiveness of genetic screening using the microarray method is comparable to the effectiveness of other, much more expensive and time-consuming methods.

Structure, expression pattern and biological activity of molecular complex TREM-2/DAP12.

DNAX-activating protein of 12kDa (DAP12) is a member of type I transmembrane adapter proteins containing immunoreceptor tyrosine-based activation motifs (ITAMs). In humans DAP12 gene is located on chromosome 19q13.1. DAP12 forms a molecular complex with triggering receptor expressed on myeloid cells two (TREM-2). TREM-2 ligation leads to the activation of Src family kinases, phosphorylation of tyrosine residues in the ITAM of DAP12, recruitment of the Syk and ZAP70 tyrosine kinases and initiation of an intracellular signaling cascade. Depending on the cell type, DAP12/TREM-2 activation plays an important role in activation and differentiation of osteoclasts, phagocytosis of bacteria, brain and bone homeostasis and inhibition of the toll-like receptor (TLR) signaling in macrophages and dendritic cells. A proper understanding of the function of this complex receptor has been restrained because of the elusive nature of TREM-2 ligands. Here we review the structure, biological functions and signaling pathways of DAP12 and its associated receptor TREM-2.

Downstream and intermediate interactions of synovial sarcoma-associated fusion oncoproteins and their implication for targeted therapy.

Synovial sarcoma (SS), an aggressive type of soft tissue tumor, occurs mostly in adolescents and young adults. The origin and molecular mechanism of the development of SS remain only partially known. Over 90% of SS cases are characterized by the t(X;18)(p11.2;q11.2) translocation, which results mainly in the formation of SS18-SSX1 or SS18-SSX2 fusion genes. In recent years, several reports describing direct and indirect interactions of SS18-SSX1/SSX2 oncoproteins have been published. These reports suggest that the fusion proteins particularly affect the cell growth, cell proliferation, TP53 pathway, and chromatin remodeling mechanisms, contributing to SS oncogenesis. Additional research efforts are required to fully explore the protein-protein interactions of SS18-SSX oncoproteins and the pathways that are regulated by these partnerships for the development of effective targeted therapy.

Clinical utility of the new American Joint Committee on Cancer staging system for gastrointestinal stromal tumors: current overall survival after primary tumor resection.

BACKGROUND: The objectives of the current study were to assess the reliability of the new revision of the American Joint Committee on Cancer (AJCC) staging system for gastrointestinal stromal tumors (GISTs) based on the National Comprehensive Cancer Network-Armed Forces Institute of Pathology risk classification and to analyze the factors that influence after resection for primary GISTs in 2 AJCC groups: patients with GISTs originating from the stomach and omentum (G-GISTs) and patients with other primary GISTs located mainly in the small bowel (nongastric GISTs [NG-GISTs]). METHODS: The authors prospectively analyzed a group of 640 patients with primary, CD117-positive GISTs who underwent surgery with curative intention (R0/R1 resection), including 340 G-GISTs (55.5%) and 300 NG-GISTs (44.5%). Factors were explored that had an effect on disease-free survival time (DFS), which was calculated from the date of radical operation to the date of recurrence or last follow-up. The median follow-up was 39 months. RESULTS: Compared with NG-GISTs, G-GISTs were characterized by a significantly lower median size (5.3 cm and 8.5 cm, respectively; P < .0001) and lower mitotic activity (median, 3 in 50 high-power fields [HPF] vs 5 in 50 HPF; P < .0001), and they were diagnosed in older patients (median age, 62 years vs 57 years; P = .002). The most commonly detected mutations in G-GIST were those located in KIT exon 11 (60.5%) and platelet-derived growth factor receptor alpha (PDGFRA) exon 18 (19%) versus KIT exons 11 and 9 in NG-GISTs (72% and 17.4%, respectively). The prognosis of patients who had G-GISTs was significantly better compared that of patients who had NG-GISTs, with 5-year DFS rates of 69% (median, 83 months) versus 43% (median, 33 months), respectively (P < .00001). The most significant prognostic factors that correlated with shorter DFS in both G-GISTs and NG-GISTs were primary tumor size >5 cm and >10 cm (P < .0001) and mitotic index >5 in 50 HPF and >10 in 50 HPF (P < .0001). The 5-year DFS rates in G-GISTs according to AJCC stage categories were as follows: 96% for stage IA tumors, 92% for stage IB tumors, 51% for II tumors, 22% for stage IIIA tumors, and 22% for stage IIIB tumors (P < .0001). The 5-year DFS rates in NG-GISTs according to AJCC categories were as follows: 92% for stage I tumors, 66% for stage II tumors, 28% for IIIA tumors, and 16% for IIIB tumors (P < .0001). The high prognostic significance of the AJCC classification also was confirmed for overall survival data, including the impact of therapy with tyrosine kinase inhibitors. CONCLUSIONS: The reliability of AJCC risk classification after resection of primary GIST was confirmed for DFS and overall survival. Patients with primary G-GISTs had a better prognosis than patients with NG-GISTs. In both groups, primary tumor size and mitotic activity were the most important prognostic factors in terms of DFS.

A human postnatal lymphoid progenitor capable of circulating and seeding the thymus.

Identification of a thymus-seeding progenitor originating from human bone marrow (BM) constitutes a key milestone in understanding the mechanisms of T cell development and provides new potential for correcting T cell deficiencies. We report the characterization of a novel lymphoid-restricted subset, which is part of the lineage-negative CD34(+)CD10(+) progenitor population and which is distinct from B cell-committed precursors (in view of the absence of CD24 expression). We demonstrate that these Lin(-)CD34(+)CD10(+)CD24(-) progenitors have a very low myeloid potential but can generate B, T, and natural killer lymphocytes and coexpress recombination activating gene 1, terminal deoxynucleotide transferase, PAX5, interleukin 7 receptor alpha, and CD3epsilon. These progenitors are present in the cord blood and in the BM but can also be found in the blood throughout life. Moreover, they belong to the most immature thymocyte population. Collectively, these findings unravel the existence of a postnatal lymphoid-polarized population that is capable of migrating from the BM to the thymus.

PDZ-binding motifs are unable to ensure correct polarized protein distribution in the absence of additional localization signals.

The C-terminal PDZ-binding motifs are required for polarized apical/basolateral localization of many membrane proteins. To determine the specificity of the PDZ-binding motifs in establishing cellular distribution, we utilized a 111-amino acid region from the C-terminus of cystic fibrosis transmembrane conductance regulator (CFTR) that is able to direct apical localization of fused reporter proteins. Substitution of the C-terminal PDZ-binding motif of CFTR with corresponding motifs necessary for basolateral localization of other membrane proteins did not lead to the redistribution of the fusion protein to the basolateral membrane. Instead, some fusion proteins remained localized to the apical membrane, whereas others showed no specific distribution. The specificity of the PDZ-based interactions was substantially increased when specific amino acids located upstream of the classical PDZ-binding motifs were included. However, even the presence of a longer C-terminal motif from a basolateral protein could not ensure basolateral distribution of the fusion protein. Our results indicate that the C-terminal PDZ-binding motifs are not the primary signals for polarized protein distribution, although they are required for targeting and/or stabilization of protein at the given location.

[Genetic polymorphism and outcome in acute lymphoblastic leukaemia of childhood]. / Polimorfizm genetyczny a skutecznosc leczenia ostrych bialaczek limfoblastycznych u dzieci.

Current treatment strategies of leukaemia use risk factors existing at the time of diagnosis to establish risk-adapted therapy. This approach currently results in overall 95% rate of complete remission in paediatric acute lymphoblastic leukemia (ALL). However, as many as 30% of patients suffer from relapse and majority of children with ALL is profoundly affected by toxicity of anticancer drugs. In our study the probability of event free survival in children presenting with normal blast karyotype and treated according to BFM 90 protocol was 75%. This suggests the existence of factors independent from leukaemia genetic background, which influences the outcome of patients with ALL. This review is focused on genetic and functional polymorphism of enzymes that have or may have the influence on metabolism of drugs included in treatment protocol for ALL in children. Basic enzymes of anticancer drug metabolic pathways are described and frequency of their polymorphisms in Caucasian population is discussed.

The search for a genetic defect in Polish patients with chronic granulomatous disease.

INTRODUCTION: Chronic granulomatous disease (CGD)is a rare inherited disorder in which phagocytic cells are unable to generate superoxide anions. Patients with CGD are predisposed to recurrent bacterial and fungal infections because the superoxide-generating NADPH oxidase activity is needed for efficient killing of microbes. Among the at least 5 subunits cre-ating a functional NADPH oxidase, a molecular defect located in any of the gp91phox, p22phox, p47phox, or p67phox subunits may cause CGD. MATERIAL/METHODS: In this study,8 patients were diagnosed with CGD on the basis of clinical findings and absence of nitroblue tetrazolium reduction in phagocytes. Southern blot analysis, GeneScan, and direct sequencing were performed to define particular DNA mutations. RESULTS: Among 6 X-linked CGD (X-CGD)patients, 4 different mutations were identified in the X-linked CYBB gene (encoding gp91phox)by direct sequencing. A novel missense mutation, located in the NADPH-binding region of gp91phox,was found in 2 brothers. One frameshift 1578delA, one splicing 252G->A mutation, and one partial gene deletion were also identified. The molecular defect in the NCF1 gene (encoding p47phox)was established in 2 patients. One was DeltaGT/DeltaGT homozygote, the other carried, besides this GT deletion on one allele, a unique Phe118stop mutation on the other. CONCLUSIONS: In general, the X-CGD patients within the group followed a more severe clinical course than the patients with an NCF1 defect. However, the lack of a straightforward genotype phenotype correlation indicates that the clinical severity of CGD depends also on other antimicrobial host-defense systems.

Genetic and biochemical background of chronic granulomatous disease.

Chronic granulomatous disease (CGD) is a rare inherited immunodeficiency syndrome caused by a profound defect in the oxygen metabolic burst machinery. Activity of NADPH oxidase is absent or profoundly diminished, as at least one of its components (gp91(phox), p22(phox), p47(phox) and p67(phox)) is lacking or non-functional. This review explains the molecular basis of NADPH oxidase dysfunction by the effects of mutations in genes coding for particular oxidase components. Among the four types of CGD, the most common is X-linked CGD (approximately 65%), with defects in the CYBB gene encoding gp91(phox). A wide spectrum of mutations has been described in the CYBB gene with no predominant genotype. The second most common subtype of CGD caused by NCF1 mutation accounts for 30% of CGD patients and is inherited in an autosomal recessive manner, with predominance of a homozygotous deltaGT deletion in the genotype. The other two CGD subtypes having an autosomal recessive pattern together account for no more than 10% of CGD cases. A strategy for the molecular diagnostics in CGD patients is proposed and principles of genetic counseling are discussed here.