BACKGROUND\nModerately increased plasma concentrations of total homocysteine (tHcy) have been shown to be an important risk factor for vascular diseases. Two common polymorphisms of the methylenetetrahydrofolate reductase (MTHFR) gene, the thermolabile C677T and a more recently reported A1298C polymorphism, may contribute to hyperhomocysteinemia.\n\n\nMETHODS\nUsing PCR and restriction fragment length polymorphism analysis, we studied the prevalence of the C677T and A1298C MTHFR genotypes and the combined effect of these polymorphisms on plasma tHcy concentrations, as measured by HPLC with fluorometric detection, both fasting and post-methionine load (PML), in 1238 individuals.\n\n\nRESULTS\nThe prevalences of the C677T and A1298C genotypes did not differ significantly in 772 individuals with documented coronary artery disease (CAD), 137 individuals with deep-vein thrombosis (DVT), and 329 individuals without documented vascular disease. Individuals homozygous for the 677T allele had significantly increased fasting tHcy, particularly in the presence of low folate, compared with individuals homozygous for the wild-type allele. Neither the 1298AC nor the 1298CC genotype was associated with significantly increased fasting or PML tHcy concentrations irrespective of serum folate. Of the nine combined MTHFR genotypes, six were present in >10% of the population. Of these, the difference in mean fasting tHcy reached statistical significance (P<0.005) only in individuals with the 677TT/1298AA genotype compared with individuals with the wild-type 677CC/1298AA genotype. Differences in mean fasting tHcy did not reach statistical significance in individuals heterozygous for both MTHFR variants. We detected two 677CT/1298CC and three 677TT/1298AC individuals; only one, an 677TT/1298AC individual, had increased tHcy (both fasting and PML). No individuals had the 677TT/1298CC genotype.\n\n\nCONCLUSIONS\nThe prevalences of the C677T and A1298C polymorphisms did not differ among individuals with CAD, DVT, or those without documented vascular disease. In contrast to the C677T polymorphism, the A1298C polymorphism is not associated with increased fasting tHcy. Although the two polymorphisms usually exist in trans configuration, crossover may occur rarely to form recombinant chromosomes.

C677T and A1298C polymorphisms of the methylenetetrahydrofolate reductase gene

Association Between Polymorphism of Methylenetetrahydrofolate Reductase (MTHFR) C677T and Risk of Myocardial Infarction: A Meta-Analysis for 8,140 Cases and 10,522 Controls

BACKGROUND Klinefelter syndrome (KS) has been defined by sex chromosome aneuploidies (classically 47, XXY) in the male patient. The peripubertal timeframe in KS patients has been associated with the initiation of progressive testicular fibrosis, loss of spermatogonial stem cells (SSC), hypogonadism and impaired fertility. Less than half of KS patients are positive for spermatozoa in the ejaculate or testis via semen analysis or testicular sperm extraction, respectively. However, the chance of finding spermatogonia including a sub-population of SSCs in KS testes has not been well defined. Given the recent demonstration of successful cell culture for mouse and human SSCs, it could be feasible to isolate and propagate SSCs and transplant the cells back to the patient or to differentiate them in vitro to haploid cells. OBJECTIVE AND RATIONALE The main objective of this study was to meta-analyse the currently available data from KS patients to identify the prevalence of KS patients with spermatogonia on testicular biopsy across four age groups (year): fetal/infantile (age ≤ 1), prepubertal (age 1 ≤ x ≤ 10), peripubertal/adolescent (age 10   0.05). A similar analysis in the adult group showed the FSH levels in spermatogonia positive and negative patients to be 25.77 ± 2.78 and 36.12 ± 2.90 IU/L, respectively (mean ± SEM level, P < 0.05). All other hormone measurements were not statistically significantly different between groups. WIDER IMPLICATIONS While azoospermia is a common finding in the KS patient population, many patients are positive for spermatogonia. Recent advances in SSC in vitro propagation, transplantation and differentiation open new avenues for these patients for fertility preservation. This would offer a new subset of KS patients a chance of biological paternity. Data surrounding the hormonal profiles of KS patients and their relation to fertility should be interpreted with caution as a paucity of adequately powered data exists. Future work is needed to clarify the utility of FSH, LH, testosterone and inhibin B as biomarkers for successful retrieval of spermatogonia.

Age-related presence of spermatogonia in patients with Klinefelter syndrome: a systematic review and meta-analysis

Background: The incidence of aplastic anaemia (AA) is higher in Asia than in the West. The precise incidence of AA in India is not known due to lack of epidemiological study. 20-40% of pancytopenic patients in referral centres are of aplastic anaemia. Patients and Methods: This was an analysis of 1501 patients diagnosed with aplastic anaemia over a period of seven and half years (January 2007– June 2014) attending the Aplastic clinic of department of haematology of All India Institute of Medical Sciences, New Delhi. The details regarding medical history, physical examination, complete blood count, bone marrow aspirate and biopsy, treatment received, were retrieved. Inherited bone marrow failure was screened in patients below 35 years. Treatment response was analysed for various treatment modalities. Results: 1501 patients of AA from 20 different states of India were analysed. The bulk of patients were from Uttar Pradesh (28.7%), Bihar (23.6%), Delhi/NCR (20%) and Haryana (7%). The average number of new aplastic anaemia patients enrolled per year 214 (range: 101-263). The median age at presentation was 25 years (range 2–83), with M;F - 2.3:1. Severity of AA revealed: severe (SAA): 75%, very severe (VSAA): 15%, nonsevere (NSAA):10%. Inherited bone marrow failure syndromes constituted 5% (75 patients) of all aplastic anaemia patients. The most common clinical presentations were pallor (97%), bleeding manifestations (69.6%) and fever (54%). The haematological parameters showed: median level of haemoglobin level: 5.9 gm/dL, WBC: 2700/mm 3 , ANC: 380/mm 3 , platelet: 10000/mm 3 . PNH clone was present in 13.5% of patients. 107 patients (7%) were lost to follow up or expired before any treatment was initiated. Only 69 patients (4.5%) received treatment with HLA-matched sibling stem cell transplantation and another 232 (15.5%) patients received ATG plus cyclosporine as immunosuppressive therapy. Seven hundred thirteen patients (47.5%) received cyclosporine. The overall response to various treatment modalities was: HLA matched sibling haematopoietic stem cell transplant: 75.3%, Anti-thymocyte globulin plus cyclosporine: 58.7%, cyclosporine plus androgen: 45.6%, cyclosporine alone: 32.2%. Conclusion: Management of AA is a real challenge in developing countries. This is one of the largest case series from a single centre from India. It is our endeavour to reduce the detrimental outcome by increasing awareness among patients and referring physicians to reduce the delay between diagnosis and treatment.

Epidemiology, Clinico-Haematological Profile and Management of Aplastic Anaemia: AIIMS Experience.

Background 
The PlA2 polymorphism of glycoprotein IIIa (GPIIIa) has been previously identified as being associated with myocardial infarction (MI), but whether this represents a true association is entirely unclear due to differences in findings from different studies. We performed a meta-analysis to evaluate whether this polymorphism is a risk factor for MI.


Methods 
Electronic databases (MEDLINE and EMBASE) were searched for all articles evaluating genetic polymorphisms of GPIIIa. For studies where acute coronary events were recorded in association with genetic analysis, pooled odds ratios (ORs) were calculated using fixed-effects and random-effects models. The primary outcome measure was MI, and a secondary analysis was also performed for acute coronary syndromes (ACS) more generally.


Findings 
57 studies were eligible for statistical analysis and included 17,911 cases and 24,584 controls. Carriage of the PlA2 allele was significantly associated with MI (n = 40,692; OR 1.077, 95% CI 1.024–1.132; p = 0.004) but with significant publication bias (p = 0.040). The degree of association with MI increased with decreasing age of subjects (≤45 years old: n = 9,547; OR 1.205, 95% CI 1.067–1.360; p = 0.003) and with adjustment of data for conventional cardiovascular risk factors (n = 12,001; OR 1.240, 95% CI 1.117–1.376; p<0.001). There was a low probability of publication bias for these subgroup analyses (all p<0.05).


Conclusions 
The presence of significant publication bias makes it unclear whether the association between carriage of the PlA2 allele and MI is true for the total population studied. However for younger subjects, the relative absence of conventional cardiovascular risk factors results in a significant association between carriage of the PlA2 allele and MI.

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The PlA1/A2 polymorphism of glycoprotein IIIa as a risk factor for myocardial infarction: a meta-analysis.

Background & objectives: Fluorescence in situ hybridization (FISH) is increasingly being recognized as the most accurate and predictive test for H er0 2/neu gene amplification and response to therapy in breast cancer. In the present study we investigated HER-2/neu gene amplification by FISH in breast carcinoma tissue specimens and compared the results with that of immunohistochemical (IHC) analysis. Methods: A total of 90 breast carcinoma tissue samples were used for immunohistochemical (IHC) and FISH analysis. IHC was performed by using mouse monoclonal antibody to the intracellular domain of HER-2/neu protein. Each slide was scored in a blinded fashion by two pathologists according to the manufacturer's recommended criteria. FISH analysis was performed on paraffin embedded breast tumour tissue sections. The polysomy for centromere 17 (Spec green signal) was read as green signals less than 4 as moderate polysomy, and more than 4 as highly polysomy. Results: Thirty of the 90 patients had negative results by IHC and FISH. Of the 28 patients with the score of 2+ by IHC, 20 were FISH positive for HER-2/neu gene amplification, three were FISH negative and five patients showed equivocal (1.8-2.2) results by FISH. These five cases were retested for IHC and FISH on different paraffin embedded tissue blocks, and all five were found positive for HER-2/neu gene amplification. Twenty five patients with the score of 3+ by IHC were FISH positive for HER-2/neu gene amplification (>2.2). Seven cases with the score of 3+ by IHC were FISH negative for HER-2/neu gene amplification (>2.2), and showed polysomy of chromosome number 17 high polysomy > 4. Interpretation & conclusions: Our results indicated that HER-2/neu status by FISH should be performed in all cases of breast tumour with a 2+ score by IHC. Cases demonstrating a 3+ score by IHC may be subjected to FISH to rule out polysomy of chromosome 17 which could be falsely interpreted as HER-2/neu overexpression by IHC analysis. There is also a need for establishing a clinically validated cut-off value for HER-2/neu FISH amplification against IHC which may be further compared and calibrated.

Evaluation of HER-2/neu status in breast cancer specimens using immunohistochemistry (IHC) & fluorescence in-situ hybridization (FISH) assay

Methylene tetrahydrofolate reductase (MTHFR) plays a significant role in the metabolism of methionine. MTHFR deficiency is an autosomal recessive trait that could be a significant risk factor for a number of defects, for example, vascular events, due to lower dietary folate intake among South Indians. To find the incidence of 677 C>T and 1298 A>C in MTHFR gene single nucleotide polymorphisms (SNPs) among the south Indian population, polymerase chain reaction and restriction fragment length polymorphism were employed among 152 patients with myocardial infarction and 167 controls. The MTHFR 677CT genotype was found among 35 (22.4%) cases and 08 (4.8%) controls, the MTHFR 677CC allele was found among 115 (73.7%) cases and 159 (94.6%) controls. Also, the analysis of the MTHFR 1298A>C SNP identified the MTHFR 1298CC genotype among 16 (10.3%) cases and 01 (0.6%) control, the MTHFR 1298AC genotype was found in 56 (35.9%) cases and 27 (16.2%) controls, and the MTHFR 1298AA genotype was observed in 80 (51.3%) case...

Sequence Variation of the Methylene Tetrahydrofolate Reductase Gene (677C>T and 1298 A>C) and Traditional Risk Factors in a South Indian Population

Karyotyping was done in 100 children suspected of having chromosomal abnormalities of genetically uncertain syndromes, multiple congenital anomalies, short stature, dysmorphic features, unclassified mental retardation, and Down syndrome. A total of 56 patients had an abnormal karyotype: ring chromosome of 13 was seen in 1 patient (1.78%), and trisomy 21 was seen in 29 patients (51.78%) who were diagnosed as Down syndrome patients. Among them, 9 were male patients (31.03%) (47,XY+21) and 18 were female patients (47,XX+21) (62.06%); 2 patients showed 47,XY+21/46,XY (mosaicism) (6.89%). Chromosomal rearrangements involving chromosome numbers 13, 14, and 21 were seen in three patients. Among them, one patient had t(13;21) [45,XX,t(13;21)] and two patients had 45,XY,t(14;21). Trisomy 22 was seen in three patients (5.3%), marker chromosome was seen in two patients (3.57%), 46,XY,16qh variant was seen in one patient (1.78%), 46,XX,der(2) was seen in one patient (1.78%), 46,XX,14ps+ was seen in two patients (3.57...

Increasing role of cytogenetics in pediatric practice.

The disease man agement of a plastic anemia patien ts is, to an extent, based on the etiology i.e., constitutional or acquired. Chromosomal breakage study using Mitomycin-C is widely used for this differential diagnosis in India. The present study was undertaken to find out the frequency of constitutional aplastic anemia. This prospective study was carried out at Immunology and Molecular biology Lab of Apollo hospital during July, 2007 to June, 2009. Clinico-hematologically classified 300 aplastic anemic patients that have been catered to the hospital for their differential dia gnosis of aplastic anemia and their res pective age and sex matched healthy controls were processed f or chromosomal breakage study. Patient's habitat, clini cal symptoms, differential blood count and history of dr ug exposure and post vir al devel opment of t he disease were documented. The survival rate was documented after 2 years of diagnosis. Relative risk was estimated by odds ratio (OR) with 95% confidence interval (CI) in m atched cas es an d con trols. A significant inc rease of chromosomal breakages seen in 9.40% patients. 8 of 83 (9.64%) patients of > 21 yrs of age and 19 of 204 (9.31%) of ≤21 years of age showed increased breaks. The sex ratio was 3.2:1. Moderate, severe and very- severe levels of diseas e w ere seen in 27.6, 69.8 and 2.6% of the patients respectively. The su rvival data documented for 100 patients suggest 60% mortality. 9.40% had evidence of constitutional aplastic anemia in contrast to previously published data from India where the proportion ranges from 11-42%. The skewed sex ratio in our study probably reflects the gender bias in our society. No significant difference (p 21 yrs age grou ps (28 .9%). Patien ts with constitutional disease could not be differentiated on the basis severity. The high mortality rate raise a need to analyze these patients on a molecular platform to dig out the genetic factors involved, if any.

Chromosomal Breakage Study in Aplastic Anemia Patients in India

The mixed lineage leukemia (MLL) gene at chromosome band 11q23 is commonly involved in reciprocal translocations that is detected in acute leukemia. The MLL gene, commonly known as mixed lineage leukemia or myeloid lymphoid leukemia, has been independently identified and cloned from the 11q23 breakpoint of acute leukemia. We describe a patient with acute lymphoblastic leukemia whose cells had shown reciprocal translocation between short arm (p21) of chromosome 2 and long arm (q23) of chromosome number 11 [t(2;11) (p21;q23)] by cytogenetic analysis. Fluorescence in situ hybridization analysis (FISH) was also performed for reconfirmation with a probe for MLL which showed split signals, hybridizing to both the derivative 2 and 11 chromosomes. Our study confirmed FISH as the most suitable assay for detecting MLL rearrangements because of its sensitivity and speed. It recommended that FISH should be used as complementary to conventional cytogenetic analysis. In conclusion, evaluation of the t(2;11)(p21;q23) was done by molecular clarification and flow cytometry.

Seetha Dayakar

Papers

Evaluation of HER-2/neu status in breast cancer specimens using immunohistochemistry (IHC) & fluorescence in-situ hybridization (FISH) assay

Sequence Variation of the Methylene Tetrahydrofolate Reductase Gene (677C>T and 1298 A>C) and Traditional Risk Factors in a South Indian Population

Increasing role of cytogenetics in pediatric practice.

Chromosomal Breakage Study in Aplastic Anemia Patients in India

Molecular diagnosis of lymphoblastic leukemia