Journal of Molecular Biomarkers & Diagnosis

Joubert syndrome–related disorders (JSRDs) are a group of clinically and genetically pleiotropic conditions that share a midbrain-hindbrain malformation, the pathognomonic molar tooth sign (MTS) visible on brain imaging, with variable involvement of other organs and systems mainly the eyes and the kidneys. Nevertheless, the definition of JSRDs remained problematical due to the extreme phenotypic heterogeneity, often with intrafamilial variability, and the significant clinical overlap among distinct forms. Here we describe two siblings with nephronophthisis (NPHP), the elder is best categorized as JSRD. Nevertheless, his younger sibling lacked the characteristic molar tooth sign; hence best categorized as NPHP- related ciliopathy. Both siblings had NPHP as the common renal phenotype, yet with variable neurological and ocular involvement. Genetic linkage and mutation analysis revealed a novel homozygous, potential loss of function mutation (c.2618dupA, pH is 873Glnfs*14) in the gene NPHP4 in both siblings. This finding extends the phenotypic spectrum associated with NPHP4 mutations, with discernible clinical heterogeneity and intrafamilial variability.

Atherosclerosis has been recognized as an inflammatory disease of the arterial wall. On the other hand, several studies in humans have linked Fatty acid binding protein 4 (FABP4) to coronary artery disease and its risk factors. In the literature, many experimental studies have provided strong evidence for the importance of FABP4 in the pathogenesis of cardiovascular disease. In a recent work, we proposed a potential role of FABP4 by inflammatory proteins in the generation of the atherosclerotic lesions. In conclusion, many results indicate that FABP4 is a key factor connecting vascular and cellular lipid accumulation to inflammation.

Background: Systemic lupus erythematosus (SLE) is an autoimmune disease which may be caused by development of the autoantibodies. On the other hand, SLE is a high-risk group of atherosclerosis, so it is possible that some of autoantibodies in SLE are the result of atherosclerosis-related diseases such as cerebral infarction (CI), cardiovascular disease (CVD) and diabetes mellitus (DM).

Methods: The initial screening of autoantibodies was performed using the protein array method. AlphaLISA was used to analyze the serum antibody levels using synthetic polypeptides as antigens.

Results: After the initial screening using protein array, we identified 67 antigens that were recognized by IgG antibodies in sera of patients with SLE. In the second screening, 170 peptides derived from amino acid sequences of 67 antigens were synthesized and used as antigens for analysis of serum antibody levels by AlphaLISA. The antibody levels for ten peptides were significantly higher in the sera of patients with SLE than in those of healthy donors. Further AlphaLISA analysis of sera of patients with CI, CVD or DM revealed that the serum antibody levels for four peptides derived from SOSTDC1, CTNND1, CLDND1 and CCNG2 were elevated in patients as compared to those of healthy donors.

Conclusions: Serum antibody levels against peptide antigens of SOSTDC1, CTNND1, CLDND1 and CCNG2 are useful markers for diagnosis of the progression of CI, CVD and/or DM.

Background: EGFR tyrosine-kinase inhibitors have shown efficacy in non-small-cell lung cancer (NSCLC) with specific EGFR mutations. The impact of KRAS and BRAF mutations on therapeutic response remains under evaluation. The study aimed validating the use of mutation detection kits in a routine laboratory.

Methods: The mutation status of the EGFR, KRAS and BRAF genes, previously determined by Sanger sequencing, was analyzed with two approaches, pyrosequencing (Therascreen® Pyro® kits) and allele specific amplification (Cobas® mutation tests). A set of 70 DNAs from NSCLC tissue samples was selected and harboured 7 EGFR, 3 KRAS and 4 BRAF mutations.

Results: The Cobas® kit missed one EGFR and all BRAF mutations, and the Therascreen® kit missed one KRAS and 2 BRAF mutations. The Cobas® kit run in a one-step procedure, while the Therascreen® Pyro® kit included several manual steps, a plate’s format change and a final analysis on a separate computer with specific software, allowing access to each experimental result. The Cobas® kit did not give the exact nature in case of mutation. Both kits have thus similar ability to detect mutations.

Conclusions: The Cobas® kit appears suitable for a high-throughput use in a medical laboratory but the synthetic final report presents a limit for full quality of the process. No kit presently integrates flexibility regarding the constant evolution in the set of mutations to be detected.

Felty's syndrome is an uncommon but severe extra-articular manifestation of rheumatoid arthritis. Felty's syndrome is characterized by the triad of rheumatoid arthritis, neutropenia, and splenomegaly. The lifetime risk of Felty's syndrome for a rheumatoid arthritis patient is less than 1%. We present a case which is a classical presentation of Felty's syndrome with the triad of RA, neutropenia and splenomegaly. We present a case of 51-year old woman on chronic RA treatment who presented with cellulitis. The work up showed splenomegaly and neutropenia which support Felty's syndrome diagnosis. Patients with Rheumatoid arthritis (RA), who developed neutropenia and splenomegaly, should be suspected of developing Felty's syndrome as a complication of Rheumatoid arthritis.

We report on a new case with developmental delay, dysmorphic features, holoprosencephaly that showed deletion in long arm of chromosome 21 (21q22.13-q22.3). The patient is a male 3 months old presented with frontonasal dysplasia, scoliosis, abnormal ears, VSD, hypospadias, undescended testis. MRI has shown holoprosencephaly and agenesis of corpus callosum. Array-comparative genomic hybridization using the Agilent 2×400 oligoarray showed an interstitial deletion in chr21q22.13-q22.3, (start-end: 36,854,967-46,006,008 bp) deletion size is 9 Mb (9,151,042 bp) and includes 75 genes (Data base of genomic variants, hg18). The deletion was found to be maternal in origin. The findings from this report underscore the role of the genes at chromosome 21q22.13-q22.3 in brain development and indicate the usefulness of array-CGH in identification of the deletion size and detection of genes that could be correlated to the patient’s phenotype.

Bronchopulmonary dysplasia (BPD) is a respiratory distress syndrome caused by chronic lung parenchymal injury, occurring primarily in preterm infants. Therefore, research for early biomarkers for BPD is really important. This study was conducted to evaluate levels of Krebs Von den Lungen-6 (KL-6) and adiponectin in cord blood, neutrophil-gelatinase-associated lipocalin 2 (NGAL2) mRNA gene expression in broncho-alveolar lavage and urinary beta 2 microglobulin (β2MG) for early prediction of lung injury or possible involvement of those molecules in BPD pathogenesis and development.

Method: this study was carried out from September 2012 to December 2013 with 58 preterm neonates of gestational age ≤32 weeks. KL-6, adiponectin and urinary β2MG levels by immunoassay, NGAL2 mRNA level by real-time PCR were determined.

Results: cord blood KL-6, urinary β2MG and broncho-alveolar lavage (BAL) fluid NGAL2 mRNA expression levels were significantly increased, while a non-significant decrease in cord blood adiponectin level in BPD preterm relative to preterm without BPD were observed, with the best sensitivity and specificity were for KL-6 and β2MG relative to preterm without BPD.

Conclusion: NGAL2, KL-6, and urinary β2MG may have role in early prediction and development of BPD in preterm neonates that may help in early prevention and treatment for better prognosis and outcome.

Waldenström macroglobulinemia is a lymphoplasmocytic lymphoma involving bone marrow and characterized by the production of a monoclonal IgM gammapathy. Despite conventional chemotherapies it is an incurable disease. The recent discovery of the pro-inflammatory transcription factor MYD88 L265P mutation as a molecular signature of the disease could represent a new therapeutic target. We report here, a case of Waldenström macroglobulinemia associated with a high biological inflammatory syndrome, resistant to conventional therapies and improved by tocilizumab, an anti-interleukin-6 Receptor inhibitor.

Solid organ transplantation is a popular solution for many end stage organ failures. The functional evaluation of these transplanted organs is multi-factorial and involves many aspects involving organ function, overall patient wellbeing and quality of life. Literature reports on vitamin status following transplantation had been collected together in this review article. This review summarizes the current status of research in this area with focus on reported deficiencies in vitamins following solid organ transplantation. The deficiencies in either fat-soluble vitamins like vitamin D, vitamin A and vitamin K as well as water soluble vitamins like vitamin B6, vitamin B12 and thiamine have been summarized. The reported deficiencies are noteworthy and necessitate a critical evaluation and interventions in many transplantation programs

Background: People of East Indian descent account for 40% of the Trinidad and Tobago (TT) population. Most came from Uttar-Pradesh and West Bengal in India where thalassaemia is prevalent. The thalassaemia carrier frequency and exact mutations are unknown in TT. Diagnostic DNA analysis is not routinely available.

Objective: To estimate the carrier frequency of thalassaemia among prospective blood donors.

Method: Blood samples were obtained from 125 prospective blood donors of East-Indian origin. CBC was done to screen for microcytosis(MCV≤83 fL). Microcytic samples had ferritin, transferrin saturation, haemoglobin electrophoresis, haemoglobin A2/F quantification and DNA analysis performed performed for thalassaemia.

Results: 72.4% subjects were male and 26.4% female. Microcytosis was found in 14 (11.2%) (9 males, 5 females). Among microcytic subjects, 11(78.6%) (8 males, 3 females) thalassaemia mutations were detected. The range MCV range was wider in β-thalassaemia (63.7-80.7 fL) than α-thalassaemia carriers (78.5-80.1 fL). All subjects with the α-globin gene mutation had the α3.7 deletion which is the commonest α-gene mutation in India. The people with β-thalassaemia mutations had IVS I-5 G/C (common in India) and IVS II-666 T/C.

Conclusion: There were a high percentage of thalassaemia carriers in microcytic individuals, thus showing the importance of testing for this disorder. A larger study is needed to determine the spectrum of α- and β-thalassaemia mutations, to analyze for correlation between the degree of microcytosis and specific genotype and for useful predictors of α- and β-globin gene mutations.

Background: Tumor biopsies obtained from patients are often limited in size and availability, and the ability to perform multiple diagnostic assays depends on the quantity and quality of the tissue. Here we describe and evaluate a method for performing DNA-based mutational analyses after immunohistochemistry analysis has been performed, using a single tissue section.

Method: Immunohistochemistry analysis was performed on 4-5 μm formalin-fixed paraffin-embedded tumor tissue sections and immunohistochemistry-stained sections were stored for subsequent genomic analysis. DNA was isolated from these immunohistochemistry-stained sections and DNA quality was assessed using a multiplexpolymerase chain reaction method as well as real time quantitative polymerase chain reaction of commonly used reference genes. Subsequently, genomic DNA was pre-amplified and mutations in KRAS, BRAF, NRAS and PIK3CA were detected by validated Taqman assays. Comparisons were made with results from unstained formalinfixed paraffin-embedded sections obtained from the same paraffin block.

Results: Our results demonstrate that genomic DNA isolated from immunohistochemistry-stained and unstained formalin-fixed paraffin-embedded tissue sections are comparable in quality and are suitable for down-stream analysis using polymerase chain reaction based assays. We also found that the sensitivity and specificity in detecting hotspot mutations are comparable in both sources of genomic DNA. This study reports 100% concordance in detecting hotspot mutations in KRAS, BRAF, NRAS and PIK3CA using quantitative real-time polymerase chain reaction between stained and unstained formalin-fixed paraffin-embedded sections.

Conclusion: We conclude that by using our novel approach, it is possible to perform immunohistochemistry staining followed by genomic analysis using a single 4-5 μm section of formalin-fixed paraffin-embedded tissue.

Introduction: The management of pediatric high grade osteosarcoma is lacking new approaches to classify closely the patients and adapt thereafter the treatment initially or post-operatively. The objective of this study was to estimate the impact of the tumor molecular response comparatively to initial biopsy and to see if this molecular analysis was correlated to the histological response to neoadjuvant chemotherapy and/or prognosis.

Material and methods: 33 patients were included and allelotyping analyses using 23 micro satellites were performed on biopsy’s and tumor’s DNA versus normal blood DNA. Allelic imbalances were detected in all biopsy samples and the number of persistent AIs or not were quantified on tumors after pre-operative chemotherapy.

Results: We identified 4 subgroups with a significant impact on survival. The first group presents a complete disappearance of the AIs and a complete response independently from the histopathologic measure for tumor necrosis. The second group showed a partial response with persistence of some rearrangements after treatment. The third and the last one were characterized by the same molecular profile even more rearrangements, allowing to considered those subgroups as highly resistant osteosarcomas. This molecular re-stratification was associated with a significant impact on survival and provides evidence that this new approach on tumor resection might be a complementary and useful tool combined with histological response assessment.