Necessity of nuclear and mitochondrial genome analysis prior to assisted reproductive techniques/intracytoplasmic sperm injection.

Assisted reproductive technique (ART) has revolutionized the management of severe male factor infertility and in some countries 5% babies are conceived through ART/intra cytoplasmic sperm injection (ICSI). However, the carry-home live birth rate after several ART cycles is low (18-25%) and this is financially, physically and emotionally crippling for the couples. Genetic factors could lead to pre or post-implantation failure and thus explain for low ART success rate. Thus, this study was planned to understand, if infertile men harbour genetic abnormalities which may be iatrogenically transmitted by ART and adversely affect growth potential of embryo. Ninety infertile men underwent semen, cytogenetic, Yq microdeletion and mitochondrial mutation analysis. Of these, 14.4% cases harboured cytogenetic abnormality, and 8.89% Yq microdeletions. A high frequency of mitochondrial mutations was found in 23 men with asthenospermia. It is important to understand that through ART genetic abnormalities are transmitted to offspring, resulting in impaired growth and development potential of embryo and poor take-home live birth rate. Thus, genetic analysis is strongly recommend in all men with idiopathic infertility who opt for ART to counsel couples and provide them with most adapted therapeutics.

Bloom syndrome: is the gene mapped to the point?

Bloom syndrome (BS), an autosomal recessive genetic disorder, is an instructive model to explore variety of questions in "deregulation of normal cell functioning". Most of the BS patients are homozygous for mutant BLM gene and depict high sister chromatid exchanges (SCEs) in almost all the cells. However, a few possess cells with dimorphic SCE phenotype. A majority of patients with dimorphic SCE phenotype have been suggested to be compound heterozygotes, inheriting two different mutations in the BLM locus from each parent. An intragenic somatic recombination event in a precursor stem cell in such patients is envisaged to give rise to a population of cells with functionally wild-type (BLM) gene and normal SCE phenotype. Adopting unique dual approaches of positional mapping through "homozygosity by descent" and "somatic crossover point mapping", a candidate for BLM has been identified and localized to a 250 Kb interval between polymorphic loci, DI5S1108 and D15127. The sequence has been found to encode a 1417 amino acid peptide with homology to the RecQ helicases, a sub-family of DExH box-containing DNA and RNA helicases. The presence of chain terminating mutations in the 'candidate' gene in BS patients has suggested it to be the BLM gene. Apparantly the proposed gene product does not seem to provide answer for a variety of clinical, biochemical and experimental observations made in BS or BS cells till date. Our recent observation of a significant decrease in the activity of pyruvate kinase in three BS B-lymphoblastoid cell lines when compared to a similar cell line established from a normal healthy subject presents with another possible candidate to elucidate the defects in BS. Experiments using okadaic acid, a phosphatase-2a/1 inhibitor, have depicted in our study that many of the clinical features characteristic of BS, not easily explanable by the recently proposed BLM gene, can be explained by the deficiency in the PK alone and/or PP2a/PP1 activity.

Protein phosphatase-1 inhibition induces high SCEs in normal whole blood cultures.

Effect of 1,25,50 and 75 nM of okadaic acid (OA) on human lymphocytes from healthy individuals in culture has been investigated. To our surprise, we observed induction of significantly high sister chromatid exchanges (SCEs) at concentrations known to inhibit both protein phosphatase-1 (PP-1) and protein phosphatase -2A (PP-2A). However, 1 nM okadaic acid, known to inhibit PP-2A alone, did not induce this cellular feature/phenotype. This novel preliminary observation lays foundation for investigating further the role of PP-1 inhibition in governing as yet unknown finer controls in the induction of high SCEs, the mechanism for which has eluded answers till date.

Noise-free chemiluminescent detection of human T cell receptor and interleukin-2 receptor genes after optimization of digoxigenin labeled probe concentration.

Reduction in the background problems to improve the efficiency of nonradioactive labeling and detection procedure has been the focus of attention in the recent past for wider acceptance of the technique in nucleic acid research. We have achieved success in obtaining a relatively background free detection of single copy genes such as, T-cell receptor-delta (TCR-delta) and interleukin-2 receptor (L-2r) in human genome by using optimized concentrations of the digoxigenin labeled probes. Inclusion of such an optimization step for each probe before carrying out the actual hybridization experiment did not require any further modification in hybridization conditions and detection protocols as suggested earlier [Anal Biochem, 210 (1993) 235; Colloq Boehringer Mannheim, 2 (1991) 4.]

Chromatid association in acrocentric chromosomes of abnormal sexual development (ASD) cases.

Concordant/discordant associations at chromatid level were compared and found significant (P less than 0.05) in females with primary amenorrhea. This probably suggested that the acrocentric association pattern in this group of ASD and infertility did not follow a random segregation in subsequent cell divisions and that the concordant acrocentric chromosomes have regularly established physical connections with one another, held together for several cell cycles. It could only be speculated that the association of acrocentric chromosome anomalies in some females with abnormal sex chromosomes are due to this reason. In the event that chromosome association has a bearing on chromosome aberrations, the non-random pattern of acrocentric association probably would increase the choice for translocation and non disjunction in the somatic cells in females with primary amenorrhea during ontogenesis.

Frequency of acrocentric association in abnormal sexual development in males and females.

A significant difference (P less than 0.05) was observed in a chi 2 comparison of DD, GG and DG-DI associations between male hypogonads and females with primary amenorrhea. This difference increased still further (P less than 0.01) when only DD and GG associations were compared between males and females with abnormal sexual development (ASD). Similarly, when normal males and females were compared for DI, TRI, TETRA, DD vs GG and DG vs GG acrocentric chromosome associations, a significant difference (P less than 0.05) was again observed. The sex difference was also apparent in TRI and TETRA acrocentric associations both in abnormal and normal sexual development males and females. These results suggested that probably sex difference (may be hormonal) influences the number and/or type of acrocentric chromosomes involved in association between males and females with ASD and also between normal males and females.