A practical workflow for forensic species identification using direct sequencing of real-time PCR products.

BACKGROUND: Forensic scientists are often required to identify species of unknown biological samples. Although methods based on sequencing of DNA barcode regions are the gold standard for species identification in single-source forensic samples, they are cumbersome to implement as routine work in forensic laboratories that perform many tests, including human DNA typing. We have developed a species identification workflow that incorporates direct sequencing with real-time PCR products (real-time PCR-direct sequencing) as the technical trick for easy testing in forensic practice. METHOD AND RESULTS: Following our workflow, DNA samples from vertebrates, such as mammals, amphibians, reptiles, birds, and fish, were subjected to species identification using vertebrate universal primers targeting each of the four DNA barcode regions. In real-time PCR melting curve analysis, humans and animals (nonhuman) could be differentiated by comparing melting temperatures, and subsequent real-time PCR-direct sequencing contributed to simplified sequencing. Searches against public DNA databases using the obtained sequences were compatible with the origin of the samples, indicating that this method might be used to identify animal species at the genus level. Furthermore, this workflow was effective in actual casework, which provided rapid test results according to the needs of the investigating agencies. CONCLUSIONS: The species identification workflow will simply sequence as much as possible and can be integrated into routine forensic practice. The real-time PCR-direct sequencing used in this workflow might be beneficial not only for species identification but also for DNA sequencing by using the Sanger method for a variety of life sciences.

Molecular Status of BRAF Mutation in Epithelial Ovarian Cancer: An Analysis of 57 Cases in the Northeast of Iran.

Background & Objective: Epithelial ovarian cancer (EOC) is the most prevalent type of ovarian cancer. Previous studies have elucidated different pathways for the progression of this malignancy. The mutation in the B-Raf proto-oncogene, serine/threonine kinase (BRAF) gene, a member of the MAPK/ERK signaling pathway, plays a role in the development of EOC. The current study aimed to determine the frequency of the BRAF V600E mutation in ovarian serous and mucinous tumors, including borderline and carcinoma subtypes. Methods: A total of 57 formalin-fixed paraffin-embedded samples, including serous borderline tumors (SBTs), low-grade serous carcinomas (LGSCs), high-grade serous carcinomas (HGSCs), mucinous borderline tumors (MBTs), and mucinous carcinomas, and 57 normal ovarian tissues were collected. The BRAF V600E mutation was analyzed using polymerase chain reaction (PCR) and sequencing. Results: While 40% of the SBT harbor BRAF mutation, we found no BRAF mutation in the invasive serous carcinoma (P=0.017). Also, there was only 1 BRAF mutation in MBT and no mutation in mucinous carcinomas. In addition, we found no mutation in the control group. Conclusion: The BRAF mutation is most frequent in borderline tumors but not in invasive serous carcinomas. It seems that 2 different pathways exist for the development of ovarian epithelial neoplasms: one for borderline tumors and the other for high-grade invasive carcinomas. Our study supports this hypothesis. The BRAF mutation is rare in mucinous neoplasms.

A direct sequencing assay for pharmacogenetic testing of thiopurine-intolerant NUDT15 alleles in an Asian population.

OBJECTIVE: The nucleoside diphosphate linked moiety X (Nudix)-Type motif 15 (NUDT15) enzyme is involved in thiopurine metabolism. Genetic variants in the NUDT15 gene result in decreased NUDT15 activity, which in addition to decreased thiopurine S-methyltransferase (TPMT) activity, contributes to thiopurine toxicity. Current standard approaches of NUDT15 genetic analysis have mainly been targeting several common variants. We aimed to develop a clinical-grade DNA-based assay for genetic analysis of the NUDT15 gene using Sanger di-deoxy sequencing. RESULTS: Sanger sequencing results were fully concordant with the expected NUDT15 genotype in all 17 cell line samples with known NUDT15 variants (accuracy = 100%; 95% CI 80.49 to 100.00%). Precision studies showed 100% intra-run repeatability and 100% inter-run reproducibility, respectively. Genetic analysis of the NUDT15 gene was performed for 80 patients of Asian ethnicity with wildtype TPMT. 76% (N = 61) of the studied individuals had NUDT15 *1/*1 diplotype. 25% (N = 14) of Chinese and 36% (N = 5) of Malays were found to carry at least 1 non-functional NUDT15 allele. Our study confirmed a high frequency of NUDT15 c.415C>T and c.55_56insGAGTCG variants in the Chinese and Malay ethnic groups in Singapore, highlighting the importance of determining NUDT15 genotype prior to thiopurine dosing.

Development of a high-resolution typing method for SLA-3, swine MHC class I antigen 3.

We developed a high-resolution and comprehensive typing method for swine leukocyte antigen 3 (SLA-3), an MHC class I gene, employing locus-specific genomic PCR followed by subsequent direct sequencing. A total of 292 individuals from nine pure, one cross-breed and six cell lines were successfully typed. A total of 21 SLA-3 alleles were identified, of which four were found to be novel alleles. However, the allelic diversity of SLA-3 was lower than that of previously reported class I genes, SLA-1 and -2. More SLA-3 alleles were observed in the Landrace and Yorkshire breeds than the other breeds. SLA-3*04:01 was identified in seven out of nine breeds and was the most widely distributed allele across all breeds. Therefore, the typing method reported in this study completes our efforts to develop high-resolution typing methods for major SLA molecules, facilitating the combined analysis of major SLA genes from field samples, which is important to understand the relationship between the adaptive immune responses against pathogens and the immunogenetic makeup of an individual.

Subtype Screening of blaIMP Genes Using Bipartite Primers for DNA Sequencing.

Genes conferring carbapenem resistance have spread worldwide among gram-negative bacteria. Subtyping of these genes has epidemiological value due to the global cross-border movement of people. Subtyping of blaIMP genes that frequently detected in Japan appears to be important in public health settings; however, there are few useful tools for this purpose. We developed a subtyping screening tool based on PCR direct sequencing, which targets the internal sequences of almost all blaIMP genes. The tool used bipartite multiplex primers with M13 universal sequences at the 5'-end. According to in silico analysis, among the 78 known IMP-type genes, except for blaIMP-81, 77 detected genes were estimated to be differentiated. In vitro evaluation indicated that sequences of amplicons of IMP-1, IMP-6, IMP-7, and IMP-20 templates were identical to their respective subtypes. Even if the amplicons were small or undetectable through the first PCR, sufficient amplicons for DNA sequencing were obtained through a second PCR using the M13 universal primers. In conclusion, our tool can be possibly used for subtype screening of blaIMP, which is useful for the surveillance of bacteria with blaIMP in clinical and public health settings or environmental fields.

Primary clear cell sarcoma of the femur: a unique case with RT-PCR and direct sequencing confirmation of EWSR1/ATF1 fusion gene.

BACKGROUND: It is very rare for clear cell sarcomas (CCS) to arise in the bone. During diagnosis, it is important to distinguish primary CCS of bone from bone metastasis of melanoma because this difference fundamentally changes the therapeutic options. Recently, characteristic fusion genes of CCS have been detected using reverse transcription polymerase chain reaction (RT-PCR) or direct sequencing which allowed to distinguish CCS from melanoma. However, there was no study applying these analyses with positive results. In this case, we describe the use of fusion gene analysis to diagnose a primary CCS of the bone. CASE PRESENTATION: A 36-year-old male presented with a four-months history of left knee pain. Magnetic resonance imaging showed a lesion in the left femoral medial epicondyle. Histological examination of the biopsy specimen revealed proliferating oval or rounded cells. These cells had clear cytoplasm arranged in fascicles or compact nests with frequent deposits of brown pigment. Furthermore, immunohistochemistry analysis revealed that tumor cells were positive for S-100 protein, HMB-45, Melan-A, and SOX10. It stained negative for CD34 and BRAF v600e. Conclusively, detection of the EWSR1/ATF1 fusion gene using RT-PCR and direct sequencing confirmed that the lesion was a primary CCS of the bone. Wide-margin resection and reconstruction with a tumor endoprosthesis were performed. CONCLUSIONS: Herein, we diagnosed a rare case of primary CCS of the bone by detecting EWSR1/ATF1 fusion gene using RT-PCR and direct sequencing. Since fluorescence-in situ hybridization (FISH) and RT-PCR could show false positive by mainly due to technical problems, it is better to perform direct sequencing to confidently diagnose the tumor as a primary CCS especially at very rare site such as bone.

P1245 Polymorphic Variants of HSD3B1 Gene Confer Different Outcome in Specific Subgroups of Patients Infected With SARS-CoV-2.

Introduction: Severe respiratory syndrome coronavirus 2 (SARS-CoV-2) uses the androgen receptor (AR), through ACE2 receptor and TMPRSS2, to enter nasal and upper airways epithelial cells. Genetic analyses revealed that HSD3B1 P1245C polymorphic variant increases dihydrotestosterone production and upregulation of TMPRSS2 with respect to P1245A variant, thus possibly influencing SARS-CoV-2 infection. Our aim was to characterize the HSD3B1 polymorphism status and its potential association with clinical outcomes in hospitalized patients with COVID-19 in Southern Switzerland. Materials and Methods: The cohort included 400 patients hospitalized for COVID-19 during the first wave between February and May 2020 in two different hospitals of Canton Ticino. Genomic DNA was extracted from formalin-fixed paraffin-embedded tissue blocks, and HSD3B1 gene polymorphism was evaluated by Sanger sequencing. Statistical associations were verified using different test. Results: HSD3B1 polymorphic variants were not associated with a single classical factor related to worse clinical prognosis in hospitalized patients with SARS-CoV-2. However, in specific subgroups, HSD3B1 variants played a clinical role: intensive care unit admission was more probable in patients with P1245C diabetes compared with P1245A individuals without this comorbidity and death was more associated with hypertensive P1245A>C cases than patients with P1245A diabetes without hypertension. Discussion: This is the first study showing that HSD3B1 gene status may influence the severity of SARS-CoV-2 infection. If confirmed, our results could lead to the introduction of HSD3B1 gene status analysis in patients infected with SARS-CoV-2 to predict clinical outcome.

Triple diagnosis of Wiedemann-Steiner, Waardenburg and DLG3-related intellectual disability association found by WES: A case report.

BACKGROUND: The development of whole-exome sequencing (WES) and whole-genome sequencing (WGS) for clinical purposes now allows the identification of multiple pathogenic variants in patients with a rare disease. This occurs even when a single causative gene was initially suspected. We report the case of an 8-year-old patient with global developmental delays and dysmorphic features, with a possibly pathogenic variant in three distinct genes. METHODS: Trio-based exome sequencing was performed by IntegraGen SA (Evry, France), on an Illumina HiSeq4000 (Illumina, San Diego, CA, USA). Sanger sequencing was performed to confirm the variants that were found. RESULTS: WES showed the presence of three possibly deleterious variants: KMT2A: c.9068delA;p.Gln3023Argfs*3 de novo, PAX3: c.530C>G;p.Ala177Gly de novo and DLG3: c.127delG;p.Asp43Metfs*22 hemizygous inherited from the mother. KMT2A pathogenic variants are involved in Wiedemann-Steiner syndrome, and PAX3 is the gene responsible for Waardenburg syndrome. DLG3 variants have been described in a non-syndromic X-related intellectual disability. CONCLUSIONS: Considering the dysmorphic features and intellectual disability presented by this patient, these three variants were imputed as pathogenic and their association was considered responsible for his phenotype. Dual molecular diagnoses have already been found by WES in several cohorts with an average of diagnostic yield of 7%. This case demonstrates and reminds us of the importance of analyzing exomes rigorously and exhaustively because, in some cases (< 10%), it can explain superimposed traits or blended phenotypes.

Compound heterozygous mutations in ABCG5 or ABCG8 causing Chinese familial Sitosterolemia.

BACKGROUND: Sitosterolemia (STSL), also known as phytosterolemia, is a rare autosomal recessive hereditary disease caused by mutations in the ABCG5 or ABCG8 genes. The disease is a result of disorders in lipoprotein metabolism, and is characterized by tendinous and tuberous xanthomas, elevated plasma cholesterol and phytosterol levels, and thrombocytopenia and hemolytic anemia in several patients. The manifestations of STSL are diverse and can easily be misdiagnosed. In recent years, cases of this disease in children have been reported in succession. There is therefore a need for clinicians to improve identification of STSL and perform early intervention. METHODS: We evaluated four children with STSL caused by genetic mutations in ABCG5 or ABCG8, as well as their family members, by analyzing their clinical characteristics and performing Trio-whole exome sequencing. The biological consequences of the mutations were analyzed using various bioinformatics software. We also analyzed the consequences of a mutation commonly observed in STSL patients on the structure of the protein involved. RESULTS: We identified five previously unreported pathogenic mutations of different phenotypes of STSL: ABCG5 NM_022436:c.1337G>A; ABCG8 NM_022437:c.965-1G>A, c.323-1G>C, c.1418C>G and c.1534G>A. We also report the structural changes brought about by a mutation common in STSL patients, as well as the possible consequences of these changes. CONCLUSIONS: Our findings further broaden the genotypic and phenotypic profiles of the onset of STSL in the pediatric population and provide information for the diagnosis and treatment of this disease.

Two unrelated pedigrees with achondrogenesis type 1b carrying a Japan-specific pathogenic variant in SLC26A2.

We present two unrelated Japanese pedigrees with achondrogenesis type 1b (ACG1B), characterized by prenatally lethal fetal hydrops and severe micromelia. The affected members in these pedigrees carried a common homozygous missense point mutation in solute carrier family 26 member 2 (SLC26A2), a gene associated with ACG1B (NM_000112:c.1987G>A). This loss-of-function point mutation causes substitution of glycine 663 with arginine in a highly conserved loop domain of SLC26A2. Interestingly, only a few cases of this mutation have been registered in Japanese genomic databases, and there are no reports of this mutation in any major genomic databases outside Japan. Furthermore, we confirmed the presence of a homozygous stretch of approximately 75 kb surrounding the pathogenic variant. Our findings suggest that this missense point mutation in SLC26A2, which is likely the cause of the ACG1B phenotypes in these unrelated fetuses, is distributed exclusively in Japan.

Concurrent immunoglobulin G-lambda type multiple myeloma and mixed cellularity classical Hodgkin lymphoma: A case report.

A 66-year-old man with a swollen right inguinal lymph node (LN) had pain on the lower side of the back. Computed tomography revealed bone disease in the back and swollen right inguinal LNs. Laboratory studies showed anemia and serum immunoglobulin G-lambda (IgG-λ) type monoclonal protein. The bone marrow contained 39.6% plasma cells. He was diagnosed with IgG-λ type multiple myeloma (MM). However, the pathological findings of the right inguinal LN were mixed cellular classical Hodgkin lymphoma (HL). The administration of melphalan, prednisone, and bortezomib (MPB) was started for MM; however, swelling in the right inguinal LN increased. After three cycles of MPB, the administration of doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) was started for HL. However, HL was refractory to ABVD. Pancytopenia subsequently progressed and rapid swelling occurred in his LNs. He died 7 months after diagnosis. Multiple myeloma was diagnosed, based on the typical symptoms, although the pathological findings of the LN indicated a diagnosis of HL. We analyzed the molecular relationship between MM and HL cells using a direct sequencing method. The sequencing results demonstrated that the variable-diversity-joining (VDJ) region of the IgH gene was identified with 94.4% of IGLV3-32*01 in the bone marrow sample at diagnosis. Furthermore, clonotypic IgH sequence was identified in CD30-positive cells from the LN. These results suggested that the clonal HL cells were derived from the same source as the clonal MM cells and demonstrated that MM and HL in this patient may have originated from the same B cell progenitor.

Carriers of the TCF7L2 rs7903146, rs12255372 Risk Alleles in the South Tamil Nadu T2DM Patients Present with Early Incidence and Insulin Dependence.

BACKGROUND AND AIMS: Metabolic abnormalities in T2DM (Type 2 diabetes mellitus) include classic manifestations such as impaired insulin secretion, synthesis and peripheral insulin resistance. The intronic variants rs7903146 and rs12255372 of the TCF7L2 (transcription factor 7-like 2) gene are strongly associated with risk of incidence of T2DM and impaired ß-cell functions. Studies addressing the early T2DM onset, and early insulin dependence in T2DM patients of south Tamil Nadu are still lacking, and hence the present study focuses in determining the influence of the TCF7L2 polymorphisms in the incidence and disease course in the T2DM patients of south Tamil Nadu. METHODS: Anthropometric measurements and biochemical parameters were carried out in early onset (Group A), early onset insulin dependent T2DM patients (Group B) and non-insulin dependent T2DM patients (Group C). PCR, allele specific PCR (ASP), PCR product sequencing strategies were utilized to determine the genotype and the impact of the TCF7L2 SNPs in the T2DM disease course. RESULTS: Female T2DM patients with the CT/TT rs7903146 genotype (P = 0.005) and the rs12255372 GT/TT genotype (P = 0.036) exhibited a significantly low mean age for T2DM incidence. Correlation/regression analysis in the T2DM patients revealed that rs12255372 (P = 0.042) is associated with early onset in the Group C patients and the rs7903146 (P = 0.018), rs12255372 (P = 0.026) are associated with insulin dependence in the group B patients. CONCLUSION: Screening for the TCF7L2 polymorphisms will prevent T2DM incidence and enable life style changes, appropriate therapeutic strategies that would help combat the accelerated disease course in the T2DM patients.

High-resolution melting analysis for rapid and sensitive MYD88 screening in chronic lymphocytic leukemia.

High resolution melting (HRM) assay is a novel technology for the fast, high-throughput, sensitive, post-PCR analysis of genetic mutations. Myeloid differentiation primary response 88 (MYD88) mutations are frequently reported in chronic lymphocytic leukemia (CLL) and confer a worse prognosis. The objective of the present study was to assess the value of HRM analysis for the rapid screening of MYD88 mutations in patients with CLL. Genomic DNA samples were extracted from the bone marrow of 129 newly diagnosed patients with CLL. A plasmid with an MYD88-L265P mutation was constructed, and the p.L265P substitution, which is the predominant MYD88 mutation in CLL, was detected using HRM analysis and direct sequencing. The plasmid pCMV-MYD88-L265P-Mu was successfully constructed as a positive control, and was verified by direct sequencing. The normalized and shifted melting curves of 6/129 (4.65%) samples were clearly different from those of other patients by HRM analysis. In addition, the 794T>C mutation in MYD88 was identified in 6 (4.65%) patients by direct sequencing. Sensitivity evaluation revealed that the HRM assay had a higher sensitivity (to 1% dilution) than direct sequencing, in addition to being convenient and time-saving. The MYD88 p.L256P mutation has been implicated to be associated with adverse prognosis in CLL. HRM analysis has the potential to be a routine prescreening technique to identify the MYD88 p.L256P mutation and may facilitate the clinical treatment of CLL.

Genetic Analysis of MECP2 Gene in Iranian Patients with Rett Syndrome.

OBJECTIVES: Rett syndrome is an X linked dominant neurodevelopmental disorder which almost exclusively affects females. The syndrome is usually caused by mutations in MECP2 gene, which is a nuclear protein that selectively binds CpG dinucleotides in the genome. MATERIALS & METHODS: To provide further insights into the distribution of mutations in MECP2 gene, we investigated 24 females with clinical characters of Rett syndrome referred to Alzahra University Hospital in Isfahan, Iran during 2015-2017. We sequenced the entire MECP2 coding region and splice sites for detection of point mutations in this gene. Freely available programs including JALVIEW, SIFT, and PolyPhen were used to find out the damaging effects of unknown mutations. RESULTS: Direct sequencing revealed MECP2 mutations in 13 of the 24 patients. We identified in 13 patients, 10 different mutations in MECP2 gene. Three of these mutations have not been reported elsewhere and are most likely pathogenic. CONCLUSION: Defects in MECP2 gene play an important role in pathogenesis of Rett syndrome. Mutations in MECP2 gene can be found in the majority of Iranian RTT patients. We failed to identify mutations in MECP2 gene in 46% of our patients. For these patients, further molecular analysis might be necessary.

Fumaratehydratase-deficient renal cell carcinoma: a clinicopathological and molecular study of 13 cases.

AIMS: Hereditary leiomyomatosis and renal cell carcinoma (HLRCC) is a newly recognised entity in the WHO 2016 classification defined as the germline mutation of FH gene. Fumaratehydratase-deficient renal cell carcinoma (FH-deficient RCC) is recommended for tumours with FH deficiency but lacking of genetic evidences of FH germline mutation. In this study, we described the clinicopathological and molecular changes of 13 FH-deficient RCCs. METHODS AND RESULTS: Histology features, clinicopathological data, radiology performance and outcomes were collected for each patient. Next-generation sequencing and DNA sequencing of FH gene were performed to examine FH mutations. The patient group included five females and eight males. Different morphological patterns of papillary, nested, adenoid, foam adenoid, cribriform, tubular, tubulocystic, cystic and loose oedema stroma were observed. Except typical big nuclei with or without eosinophilic nucleoli and perinucleolar halos, raisin-like, hobnail-like and even low-grade nuclei were also observed in these tumours. Eleven cases with high-grade nuclei showed disease progression or death, but no disease progression was detected in two cases with low-grade nuclei and eosinophilic cytoplasm. FH expression was absent in tumour cells except for case 11. Next-generation sequencing and DNA sequencing verified seven FH germline mutations and four somatic mutations out of 13 cases. CONCLUSIONS: FH-deficient RCC is a rare renal tumour and has a wide morphological spectrum. Most of the tumours had high-grade nuclei and were aggressive. However, we observed a morphological subtype of FH-deficient RCC with low-grade nuclei and eosinophilic cytoplasm, which might mainly occur in young women and show a relatively good prognosis.

Developing a New qPCR-Based System for Screening Mutation.

An accurate genotyping analysis is one of the critical prerequisites for lung cancer targeted therapy. Here, a quantitative polymerase chain reaction (qPCR)-based mutation detection system, mutation-selected amplification-specific system PCR (MASS-PCR), is developed. The specific primers and probes used in MASS-PCR exactly match with the mutant sequence that only allows mutant gene to emit the fluorescence peak. To determine the sensitivity of MASS-PCR, 717 lung cancer specimens, 61 formalin-fixed paraffin-embedded (FFPE) tissues, and 656 fresh reaction tissues are collected and undergo mutation detection of lung cancer driver genes (EGFR, KRAS, BRAF, HER2, MET, ALK, and ROS1). These samples are divided into two groups. Mutations in Group I, which has 631 fresh reaction tissues, are analyzed by MASS-PCR and the amplification refractory mutation system PCR (ARMS-PCR). While group II samples, 25 fresh reaction tissues and 61 FFPE tissues, are screened through MASS-PCR and next-generation sequencing (NGS). All results are verified by direct sequencing. MASS-PCR shows high consistency with ARMS-PCR (kappa value > 0.733) and NGS (kappa value = 0.79) (P < 0.001). For the samples with inconsistent MASS-PCR and ARMS-PCR results, DS results more likely support the MASS-PCR results. These data suggest that MASS-PCR is a convenient, accurate, and economical method for the detection of lung cancer driver gene mutations in clinical practice.

A Multiplex-PCR Method for Diagnosis of AY-Group Phytoplasmas.

Polymerase chain reaction (PCR) methods using phytoplasma-specific primers are widely used to detect phytoplasmas from infected plants and insects. Here, I describe a method of multiplex-PCR to amplify nine gene fragments in PCR reactions from AY-group phytoplasmas. Strain-identification was possible after electrophoresis and direct sequencing was also possible after PCR. The combinations of primers can be easily modified, so this method could be applied to other phytoplasma strains.

IGF1R Gene Alterations in Children Born Small for Gestitional Age (SGA).

BACKGROUND: Small for gestational age (SGA)-born children are a heterogeneous group with few genetic causes reported. Genetic alterations in the IGF1 receptor (IGF1R) are found in some SGA children. AIM: To investigate whether alterations in IGF1R gene are present in SGA born children. PATIENTS AND METHODS: We analysed 64 children born SGA who stayed short (mean -3.25 ± 0.9 SDS) within the first 4 years of age, and 36 SGA children who caught up growth (0.20 ± 1.1 SDS). PCR products of all coding IGF1R exons were screened by dHPLC followed by direct sequencing of conspicuous fragments to identify small nucleotide variants. The presence of IGF1R gene copy number alterations was determined by Multiplex Ligation-dependent Probe Amplification (MLPA). RESULTS: The cohort of short SGA born children revealed a heterozygous, synonymous variant c.3453C > T in one patient and a novel heterozygous 3 bp in-frame deletion (c.3234_3236delCAT) resulting in one amino acid deletion (p.Ile1078del) in another patient. The first patient had normal serum levels of IGF1. The second patient had unusually low IGF1 serum concentrations (-1.57 SD), which contrasts previously published data where IGF1 levels rarely are found below the age-adjusted mean. CONCLUSIONS: IGF1R gene alterations were present in 2 of 64 short SGA children. The patients did not have any dysmorphic features or developmental delay. It is remarkable that one of them had significantly decreased serum concentrations of IGF1. Growth response to GH treatment in one of the patients was favourable, while the second one discontinued the treatment, but with catch-up growth.

Hepatitis B virus precore G1896A mutation in chronic liver disease patients with HBeAg negative serology from North India.

Hepatitis B with precore stop codon mutation is related with severe liver damage in HBeAg negative patients. It is of utmost importance to screen the G1896A precore mutation. The study was designed to assess the impact of G1986A mutations in patients with different clinical spectra of the liver disease by PCR-LCR. 210 HBV positive patients with HBeAg negative serology of different kind of liver diseases (AVH = 72, FH = 21, CH = 79, Cirrhosis = 20 and HCC = 18) were screened. Patients were screened for the presence or absence of precore G1896A mutation by PCR-LCR. Direct nucleotide sequencing was done to confirm the results of LCR. Precore mutant in HCC was 94.4% (17/18), 85.7% (18/21) in FH, 60% (12/20) in liver cirrhosis, 48.1% (38/79) in chronic hepatitis and 27.7% (20/72) in AVH cases. The serum ALT level was statistically significant between HBeAg negative WT and G1896A mutants in chronic hepatitis cases. ALT level and HBV DNA level was slightly raised in the pre core mutant but and was not significant. Genotype D had a higher prevalence (79.5%) as compared to genotype A (20.5%). The mutations detected by PCR-LCR were in 100% concordance with direct sequencing. The exceptionally high prevalence of G1896A in FH and HCC demonstrates that the precore mutants are strongly associated with the progression of liver diseases in patients with HBeAg negative serology. The findings are also suggestive of screening HBV precore G1896A mutation particularly in HBeAg negative cases. The precore G1896A mutation increases proportionately in severe form of liver diseases. LCR can be a suitable tool for screening of G1896A mutations.

Methodological comparison of the allele refractory mutation system and direct sequencing for detecting EGFR mutations in NSCLC, and the association of EGFR mutations with patient characteristics.

Gefitinib is effective for patients with non-small cell lung cancer (NSCLC) with a mutation in the epidermal growth factor receptor (EGFR) gene, which makes the detection of EGFR mutations a critical step prior to determining a treatment schedule. Therefore, the present study determined the EGFR mutation status in patients with NSCLC using an allele refractory mutation system (ARMS) and analyzed the detection ratio for different specimen types. A total of 1,596 NSCLS samples were collected and EGFR gene mutations were detected on exons 18-21 using ARMS and direct sequencing. The concordance of two methods reached 89.21%, with a total mutation rate of 45.55% (727/1,596), in which the mutation rate in lung adenocarcinoma samples was markedly increased compared with squamous cell carcinoma (51.77 vs. 8.68%). In patients with lung adenocarcinoma, EGFR mutations were more frequent in female patients than male patients (65.53 vs. 39.80%, P<0.01); there was no observable difference depending on age. Similar results were obtained for squamous cell carcinoma. In the present study, certain rare mutations were also identified; these may be subjects for further study. The impact of different sample types on the consistency between the methods was determined to be insignificant. ARMS is a more applicable approach for large-scale clinical detection than direct sequencing, and we hypothesize that ARMS may replace direct sequencing if the drawbacks of ARMS, including its narrow detection range, can be amended.