Molecular and computational characterization of ABCB11 and ABCG5 variants in Tunisian patients with neonatal/infantile low-GGT intrahepatic cholestasis: Genetic diagnosis and genotype-phenotype correlation assessment.

Many inherited conditions cause hepatocellular cholestasis in infancy, including progressive familial intrahepatic cholestasis (PFIC), a heterogeneous group of diseases with highly overlapping symptoms. In our study, six unrelated Tunisian infants with PFIC suspicion were the subject of a panel-target sequencing followed by an exhaustive bioinformatic and modeling investigations. Results revealed five disease-causative variants including known ones: (the p.Asp482Gly and p.Tyr354 * in the ABCB11 gene and the p.Arg446 * in the ABCC2 gene), a novel p.Ala98Cys variant in the ATP-binding cassette subfamily G member 5 (ABCG5) gene and a first homozygous description of the p.Gln312His in the ABCB11 gene. The p.Gln312His disrupts the interaction pattern of the bile salt export pump as well as the flexibility of the second intracellular loop domain harboring this residue. As for the p.Ala98Cys, it modulates both the interactions within the first nucleotide-binding domain of the bile transporter and its accessibility. Two additional potentially modifier variants in cholestasis-associated genes were retained based on their pathogenicity (p.Gly758Val in the ABCC2 gene) and functionality (p.Asp19His in the ABCG8 gene). Molecular findings allowed a PFIC2 diagnosis in five patients and an unexpected diagnosis of sisterolemia in one case. The absence of genotype/phenotype correlation suggests the implication of environmental and epigenetic factors as well as modifier variants involved directly or indirectly in the bile composition, which could explain the cholestasis phenotypic variability.

Characterisation of the Paenarthrobacter nicotinovorans ATCC 49919 genome and identification of several strains harbouring a highly syntenic nic-genes cluster.

BACKGROUND: Paenarthrobacter nicotinovorans ATCC 49919 uses the pyridine-pathway to degrade nicotine and could provide a renewable source of precursors from nicotine-containing waste as well as a model for studying the molecular evolution of catabolic pathways and their spread by horizontal gene transfer via soil bacterial plasmids. RESULTS: In the present study, the strain was sequenced using the Illumina NovaSeq 6000 and Oxford Nanopore Technology (ONT) MinION platforms. Following hybrid assembly with Unicycler, the complete genome sequence of the strain was obtained and used as reference for whole-genome-based phylogeny analyses. A total of 64 related genomes were analysed; five Arthrobacter strains showed both digital DNA-DNA hybridization and average nucleotide identity values over the species threshold when compared to P. nicotinovorans ATCC 49919. Five plasmids and two contigs belonging to Arthrobacter and Paenarthrobacter strains were shown to be virtually identical with the pAO1 plasmid of Paenarthrobacter nicotinovorans ATCC 49919. Moreover, a highly syntenic nic-genes cluster was identified on five plasmids, one contig and three chromosomes. The nic-genes cluster contains two major locally collinear blocks that appear to form a putative catabolic transposon. Although the origins of the nic-genes cluster and the putative transposon still elude us, we hypothesise here that the ATCC 49919 strain most probably evolved from Paenarthrobacter sp. YJN-D or a very closely related strain by acquiring the pAO1 megaplasmid and the nicotine degradation pathway. CONCLUSIONS: The data presented here offers another snapshot into the evolution of plasmids harboured by Arthrobacter and Paenarthrobacter species and their role in the spread of metabolic traits by horizontal gene transfer among related soil bacteria.

Molecular mechanisms underlying the defects of two novel mutations in the HSD17B3 gene found in the Tunisian population.

17ß-hydroxysteroid dehydrogenase type 3 (17ß-HSD3) converts Δ4-androstene-3,17-dione (androstenedione) to testosterone. It is expressed almost exclusively in the testes and is essential for appropriate male sexual development. More than 70 mutations in the HSD17B3 gene that cause 17ß-HSD3 deficiency and result in 46,XY Disorders of Sex Development (46,XY DSD) have been reported. This study describes three novel Tunisian cases with mutations in HSD17B3. The first patient is homozygous for the previously reported mutation p.C206X. The inheritance of this mutation seemed to be independent of consanguineous marriage, which can be explained by its high frequency in the Tunisian population. The second patient has a novel splice site mutation in intron 6 at position c.490 -6 T > C. A splicing assay revealed a complete omission of exon 7 in the resulting HSD17B3 mRNA transcript. Skipping of exon 7 in HSD17B3 is predicted to cause a frame shift in exon 8 that affects the catalytic site and results in a truncation in exon 9, leading to an inactive enzyme. The third patient is homozygous for the novel missense mutation p.K202M, representing the first mutation identified in the catalytic tetrad of 17ß-HSD3. Site-directed mutagenesis and enzyme activity measurements revealed a completely abolished 17ß-HSD3 activity of the p.K202M mutant, despite unaffected protein expression, compared to the wild-type enzyme. Furthermore, the present study emphasizes the importance of genetic counselling, detabooization of 46,XY DSD, and a sensitization of the Tunisian population for the risks of consanguineous marriage.

Complete Genome Sequences of Two Closely Related Paenarthrobacter nicotinovorans Strains.

Paenarthrobacter nicotinovorans is a soil bacterium that uses the pyridine pathway to degrade nicotine. The genome of strain ATCC 49919 is composed of a ~4.3-Mbp chromosome and a ~165-kbp plasmid. The second strain, termed here nic-, is a cured derivative lacking the plasmid and not able to degrade nicotine.

Novel cases of Tunisian patients with mutations in the gene encoding 17ß-hydroxysteroid dehydrogenase type 3 and a founder effect.

17ß-Hydroxysteroid dehydrogenase type 3 (17ß-HSD3) is expressed almost exclusively in the testis and converts Δ4-androstene-3,17-dione to testosterone. Mutations in the HSD17B3 gene causing 17ß-HSD3 deficiency are responsible for a rare recessive form of 46, XY Disorders of Sex Development (46, XY DSD). We report novel cases of Tunisian patients with 17ß-HSD3 deficiency due to previously reported mutations, i.e. p.C206X and p.G133R, as well as a case with the novel compound heterozygous mutations p.C206X and p.Q176P. Moreover, the previously reported polymorphism p.G289S was identified in a heterozygous state in combination with a novel non-coding variant c.54G>T, also in a heterozygous state, in a male patient presenting with micropenis and low testosterone levels. The identification of four different mutations in a cohort of eight patients confirms the generally observed genetic heterogeneity of 17ß-HSD3 deficiency. Nevertheless, analysis of DNA from 272 randomly selected healthy controls from the same geographic area (region of Sfax) revealed a high carrier frequency for the p.C206X mutation of approximately 1 in 40. Genotype reconstruction of the affected pedigree members revealed that all p.C206X mutation carriers harbored the same haplotype, indicating inheritance of the mutation from a common ancestor. Thus, the identification of a founder effect and the elevated carrier frequency of the p.C206X mutation emphasize the importance to consider this mutation in the diagnosis and genetic counseling of affected 17ß-HSD3 deficiency pedigrees in Tunisia.

Whole mitochondrial genome screening of a family with maternally inherited diabetes and deafness (MIDD) associated with retinopathy: A putative haplotype associated to MIDD and a novel MT-CO2 m.8241T>G mutation.

Mitochondrial diseases are a clinically heterogeneous group of disorders that arise as a result of dysfunction of the mitochondrial respiratory chain. They can be caused by mutations in both nuclear and mitochondrial DNA. In fact, mitochondrial DNA (mtDNA) defects are known to be associated with a large spectrum of human diseases and patients might present wide range of clinical features with various combinations. Our study reported a Tunisian family with clinical features of maternally inherited diabetes and deafness (MIDD). Accordingly, we performed a whole mitochondrial genome mutational analysis, results revealed a haplotype composed by "A750G, A1438G, G8860A, T12705, T14766C and T16519C", in homoplasmic state, in the mother and transmitted to her daughter and her son. The patient with MIDD2 and retinopathy presented, in addition to this haplotype associated to the MIDD, two de novo variations including a novel one m.8241T>G (p. F219C) in MT-CO2 gene and a known one m.13276G>A (p. M314V) in MT-ND5 gene. The coexistence of these two mutations could explain the retinopathy observed in this patient.

Splicing Defects in the AAAS Gene Leading to both Exon Skipping and Partial Intron Retention in a Tunisian Patient with Allgrove Syndrome.

BACKGROUND/AIMS: Allgrove syndrome is a rare autosomal recessive disorder characterized by the triad of adrenal insufficiency, achalasia, and alacrima. This syndrome is caused by mutations in the AAAS gene. A major splice site mutation c.1331+1G>A was found previously in North African families affected by Allgrove syndrome. In this study, we analyzed in vivo and in silico the effect of this mutation on the splicing process. METHODS: Using reverse transcriptase-polymerase chain reaction, sequencing and bioinformatics tools, we analyzed all transcripts produced by the AAAS gene containing this splice site mutation. RESULTS: The altered splicing of mRNA produces two aberrant transcripts: one with exon 14 skipping, the other with concurrent exon 14 skipping and retention of 99 bp of intron 14, both outcomes resulting in frameshifts with a new stop codon generation in the untranslated region of the last exon. Using in silico bioinformatics tools, we demonstrated that this mutation abolishes the splice donor site of exon 14 and activates a new intronic cryptic splice site in intron 14. CONCLUSION: This study demonstrated that a single splicing mutation affects the AAAS transcripts and consequently the ALADIN protein structure and function.

A de-novo large deletion of 2.8 kb produced in the ABCD1 gene causing adrenoleukodystrophy disease.

X-linked adrenoleukodystrophy (X-ALD) is a neurodegenerative disorder caused by mutations in the ABCD1 gene, which encodes an ATP-binding cassette transporter protein, ALDP. The disease is characterized by increased concentrations of very long chain fatty acids (VLCFAs) in plasma, adrenal, testicular, and nerve tissues. For this study, our objective was to conduct clinical, molecular, and genetic studies of a Tunisian patient with X-ALD. The diagnosis was based on clinical indications, biochemical analyses, typical brain-scan patterns, and molecular biology; the molecular analyses were based on PCR, long-range PCR, and sequencing. The molecular analysis by long-range PCR and direct sequencing of the ABCD1 gene showed the presence of a de-novo 2794 bp deletion covering the whole of exon 2. Using bioinformatics tools, we demonstrate that the large deletion is located in a region rich with Alu sequences. Furthermore, we suggest that the AluJb sequence could be the cause of the large deletion of intron 1, exon 2, and intron 2, and the creation of a premature stop codon within exon 3. This report is the first report in which we demonstrate the breakpoints and the size of a large deletion in a Tunisian with X-ALD.

Clinical and Genetic Characterization of 26 Tunisian Patients with Allgrove Syndrome.

BACKGROUND AND AIMS: Allgrove syndrome is characterized by achalasia, alacrima, and adrenal insufficiency as well as being associated with progressive neurological signs. This is an autosomal recessive disorder due to mutations in the AAAS gene located on chromosome 12q13. The AAAS gene encodes a protein of 546 amino acids, ALADIN. Mutations in this genwere reported in families from North Africa and Europe. Our objective is to conduct a clinical, molecular and genetic study of 26 Tunisian patients with Allgrove syndrome. METHODS: We report 26 Tunisian patients with between two and four clinical features associated with Allgrove syndrome. Blood samples were collected and isolated DNA derived from subjects was amplified. The entire sequence of the AAAS gene was analyzed by PCR and sequencing. PCR-RFLP method was performed to identify the frequent mutations found. RESULTS: Sequencing of the AAAS gene revealed a major homozygous mutation (c.1331+1G>A) in 25 patients and R286X mutation in one patient. The presence of a major mutation in several unrelated affected individuals suggests the presence of a founder effect in Tunisia and allows for a fast and targeted molecular diagnosis. CONCLUSIONS: We created an easy and rapid molecular enzymatic protocol based on PCR-RFLP using MvaI restriction enzyme that directly targets this major mutation and can be used for prenatal diagnosis and genetic counseling for Tunisian families at risk. To the best of our knowledge, this is the first major series report of Allgrove syndrome in Tunisia.

Molecular Analysis of Libyan Families with Allgrove Syndrome: Geographic Expansion of the Ancestral Mutation c.1331+1G>A in North Africa.

BACKGROUND/AIMS: Allgrove syndrome is a rare autosomal recessive disorder characterized by alacrima, achalasia, and adrenal insufficiency. It is caused by mutations of the AAAS gene located on chromosome 12q13 encoding the WD-repeat protein ALADIN. The c.1331+1G>A mutation is one of the most common mutations described in the literature and was identified in Tunisian and Algerian populations. Herein, we describe the clinical and genetic profile of two families from Libya in North Africa associated with Allgrove syndrome. METHODS: Two unrelated families clinically diagnosed with Allgrove syndrome were evaluated for sequence variations in the AAAS gene. Blood samples were collected, and isolated DNA derived from the subjects was amplified. The entire sequence of the AAAS gene was analyzed by PCR-RFLP and direct sequencing. RESULTS: Molecular analysis revealed the major homozygous mutation (c.1331+1G>A) in all patients. The presence of a major mutation in Tunisia, Algeria and, as discovered in this report, in Libya in patients with Allgrove syndrome suggests the existence of an ancestral mutation and a founder effect in North Africa. CONCLUSIONS: The findings allow for a fast genetic counseling in North African families with Allgrove syndrome. To the best of our knowledge, this is the first report of Allgrove syndrome in Libya.

Phenotypic variability in a Tunisian family with X-linked adrenoleukodystrophy caused by the p.Gln316Pro novel mutation.

INTRODUCTION: X-linked adrenoleukodystrophy is a neurodegenerative recessive disorder that affects the brain white matter and associated with adrenal insufficiency. It is characterized by an abnormal function of the peroxisomes, which leads to an accumulation of the Very Long Chain Fatty Acids (VLCFA) in plasma and tissues, especially in the cortex of the adrenal glands and the white matter of the central nervous system. Mutations in the ABCD1 gene affect the function of the encoded protein ALDP, an ATP-binding cassette transporter located in the peroxisomal membrane protein. PATIENTS AND METHODS: The present study reports the clinical, biochemical and molecular investigation in a Tunisian family with two affected males with childhood cerebral adrenoleukodystrophy. RESULTS: The ABCD1 gene sequencing indicated a novel hemizygous missense mutation c.947A>C (p.Gln316Pro) in the exon 2 of the ABCD1 gene in the patients, their mother and their sisters. This missense variation was predicted to be possibly damaging by the PolyPhen and SIFT prediction software. Although presence of the same mutation c.947A>C in both siblings, they present different clinical signs. CONCLUSIONS: Based on the disease's progress, the clinical signs and biochemical aspects between the two siblings, we demonstrate that there is no correlation genotype-phenotype.

Splicing defects in ABCD1 gene leading to both exon skipping and partial intron retention in X-linked adrenoleukodystrophy Tunisian patient.

X-linked adrenoleukodystrophy (X-ALD) affects the nervous system white matter and adrenal cortex secondary to mutations in the ABCD1 gene that encodes a peroxisomal membrane protein: the adrenoleukodystrophy protein. The disease is characterized by high concentrations of very long-chain fatty acids in plasma, adrenal, testicular and nervous tissues. Various types of mutations have been identified in the ABCD1 gene: point mutations, insertions, and deletions. To date, more than 40 point mutations have been reported at the splice junctions of the ABCD1 gene; only few functional studies have been performed to explore these types of mutations. In this study, we have identified de novo splice site mutation c.1780+2T>G in ABCD1 gene in an X-ALD Tunisian patient. Sequencing analysis of cDNA showed a minor transcript lacking exon 7 and a major transcript with a partial intron 7 retention due to activation of a new intronic cryptic splice site. Both outcomes lead to frameshifts with premature stop codon generation in exon 8 and intron 7 respectively. To the best of our knowledge, the current study demonstrates that a single splicing mutation affects the ABCD1 transcripts and the ALDP protein function.

A novel mutation MT-COIII m.9267G>C and MT-COI m.5913G>A mutation in mitochondrial genes in a Tunisian family with maternally inherited diabetes and deafness (MIDD) associated with severe nephropathy.

Mitochondrial diabetes (MD) is a heterogeneous disorder characterized by a chronic hyperglycemia, maternal transmission and its association with a bilateral hearing impairment. Several studies reported mutations in mitochondrial genes as potentially pathogenic for diabetes, since mitochondrial oxidative phosphorylation plays an important role in glucose-stimulated insulin secretion from beta cells. In the present report, we studied a Tunisian family with mitochondrial diabetes (MD) and deafness associated with nephropathy. The mutational analysis screening revealed the presence of a novel heteroplasmic mutation m.9276G>C in the mitochondrial COIII gene, detected in mtDNA extracted from leukocytes of a mother and her two daughters indicating that this mutation is maternally transmitted and suggest its implication in the observed phenotype. Bioinformatic tools showed that m.9267G>C mutation (p.A21P) is « deleterious ¼ and it can modify the function and the stability of the MT-COIII protein by affecting the assembly of mitochondrial COX subunits and the translocation of protons then reducing the activity of the respective OXPHOS complexes of ATP synthesis. The nonsynonymous mutation (p.A21P) has not been reported before, it is the first mutation described in the COXIII gene which is related to insulin dependent mitochondrial diabetes and deafness and could be specific to the Tunisian population. The m.9267G>C mutation was present with a nonsynonymous inherited mitochondrial homoplasmic variation MT-COI m.5913 G>A (D4N) responsible of high blood pressure, a clinical feature detected in all explored patients.

A novel frameshift mutation in BLM gene associated with high sister chromatid exchanges (SCE) in heterozygous family members.

The Bloom syndrome (BS) is an autosomic recessive disorder comprising a wide range of abnormalities, including stunted growth, immunodeficiency, sun sensitivity and increased frequency of various types of cancer. Bloom syndrome cells display a high level of genetic instability, including a 10-fold increase in the sister chromatid exchanges (SCE) level. Bloom syndrome arises through mutations in both alleles of the BLM gene, which was identified as a member of the RecQ helicase family. In this study, we screened a Tunisian family with three BS patients. Cytogenetic analysis showed several chromosomal aberrations, and an approximately 14-fold elevated SCE frequency in BS cells. A significant increase in SCE frequency was observed in some family members but not reaching the BS patients values, leading to suggest that this could be due to the heterozygous profile. Microsatellite genotyping using four fluorescent dye-labeled microsatellite markers revealed evidence of linkage to BLM locus and the healthy members, sharing higher SCE frequency, showed heterozygous haplotypes as expected. Additionally, the direct BLM gene sequencing identified a novel homozygous frameshift mutation c.3617-3619delAA (p.K1207fsX9) in BS patients and a heterozygous BLM mutation in the family members with higher SCE frequency. Our findings suggest that this latter mutation likely leads to a reduced BLM activity explaining the homologous recombination repair defect and, therefore, the increase in SCE. Based on the present data, the screening of this mutation could contribute to the rapid diagnosis of BS. The genetic confirmation of the mutation in BLM gene provides crucial information for genetic counseling and prenatal diagnosis.

Molecular characterization of X-linked adrenoleukodystrophy in a Tunisian family: identification of a novel missense mutation in the ABCD1 gene.

BACKGROUND: X-linked adrenoleukodystrophy (X-ALD) is a recessive neurodegenerative disorder that affects the brain's white matter and is associated with adrenal insufficiency. It is characterized by an abnormal function of the peroxisomes, which leads to an accumulation of very long-chain fatty acids (VLCFA) in plasma and tissues, especially in the cortex of the adrenal glands and the white matter of the central nervous system, causing demyelinating disease and adrenocortical insufficiency (Addison's disease). X-ALD is caused by a mutation in the ABCD1 gene (ATP-binding cassette, subfamily D, member 1), which encodes the adrenoleukodystrophy protein involved in the transport of fatty acids into the peroxisome for degradation. OBJECTIVE: We report here a disease-related variant in the ABCD1 gene in a 19-year-old Tunisian boy with childhood cerebral adrenoleukodystrophy. METHODS: The diagnosis was based on clinical symptoms, high levels of VLCFA in plasma, typical MRI pattern and molecular analysis. RESULTS: Molecular analysis by direct sequencing of the ABCD1 gene showed the presence of a novel missense mutation c.284C>A (p.Ala95Asp) occurring in the transmembrane domain in the proband, his mother and his sister. CONCLUSION: Using bioinformatic tools we suggest that this novel variant may have deleterious effects on adrenoleukodystrophy protein structure and function.

DNA repair gene polymorphisms at XRCC1 (Arg194Trp, Arg280His, and Arg399Gln) in a healthy Tunisian population: interethnic variation and functional prediction.

The genetic polymorphisms in DNA repair genes might affect the repair activities of the enzymes, predisposing individuals to cancer risk. Due to these genetic variants, interethnic differences in DNA repair capacity were observed in various populations. Hence, our study aimed to determine the prevalence of three nonsynonymous single-nucleotide polymorphisms (SNPs) in an X-ray repair cross-complementation group 1 gene (XRCC1) (Arg194Trp, Arg280His, and Arg399Gln) in a healthy Tunisian population (TUN) and to compare that with HapMap ( www.hapmap.org ) populations. Also, we predicted their eventual functional effect based on the protein conservation analysis by Sorting Intolerant From Tolerant (SIFT; http://sift.jcvi.org/www/SIFT_dbSNP.html ) software. The genotypes of 154 healthy individuals were determined by the polymerase chain reaction-restriction fragment length polymorphism. Tunisians showed a relative relatedness with Caucasians (European ancestry) for Arg194Trp and Arg399Gln that may be explained by the strategic geographic location of Tunisia in the Mediterranean, allowing exchanges with European countries. However, a characteristic pattern was observed in Arg280His polymorphism, which could be explained by the high inbreeding rate in TUN. The analysis of protein conservation showed that the three amino acid substitutions were predicted as damaged. The results presented here provide the first report on XRCC1 polymorphisms about Tunisians and may establish baseline database for our future clinical and genetic studies.

An interethnic variability and a functional prediction of DNA repair gene polymorphisms: the example of XRCC3 (p.Thr241>Met) and XPD (p.Lys751>Gln) in a healthy Tunisian population.

Genetic polymorphisms in DNA repair genes might influence the repair activities of the enzymes predisposing individuals to cancer risk. Owing to the presence of these genetic variants, interethnic differences in DNA repair capacity have been observed in various populations. The present study was undertaken to determine the allele and genotype frequencies of two common non-synonymous SNPs, XRCC3 p.Thr241>Met (C > T, rs861539) and XPD p.Lys751>Gln (T > G, rs13181) in a healthy Tunisian population and to compare them with HapMap ( http://www.hapmap.org/ ) populations. Also, we predicted their eventual functional effect based on bioinformatics tools. The genotypes of 154 healthy and unrelated individuals were determined by PCR-RFLP procedure. Our findings showed a close relatedness with Caucasians from European ancestry which might be explained by the strategic geographic location of Tunisia in the Mediterranean, thus allowing exchanges with Europeans countries. The in silico predictions showed that p.Thr241>Met substitution in XRCC3 protein was predicted as possibly damaging, indicating that it is likely to have functional consequences as well. To the best of our knowledge, this is the first study in this regard in Tunisia. So, these data could provide baseline database and help us to explore the relationship of XRCC3 and XPD polymorphisms with both cancer risk and DNA repair variability in our population.

Polymorphisms of glutathione S-transferases M1, T1, P1 and A1 genes in the Tunisian population: an intra and interethnic comparative approach.

Genetic polymorphisms in glutathione S-transferases (GSTs) genes might influence the detoxification activities of the enzymes predisposing individuals to cancer risk. Owing to the presence of these genetic variants, inter-individual and ethnic differences in GSTs detoxification capacity have been observed in various populations. Therefore, the present study was performed to determine the prevalence GSTM1 0/0, GSTT1 0/0, GSTP1 Ile(105)Val, and GSTA1 A/B polymorphisms in 154 healthy individuals from South Tunisia, and to compare them with those observed in North and Centre Tunisian populations and other ethnic groups. GSTM1 and GSTT1 polymorphisms were analyzed by a Multiplex-PCR approach, whereas GSTP1 and GSTA1 polymorphisms were examined by PCR-RFLP. The frequencies of GSTM10/0 and GSTT1 0/0 genotypes were 53.9% and 27.9%, respectively. The genotype distribution of GSTP1 was 47.4% (Ile/Ile), 40.9% (Ile/Val), and 11.7% (Val/Val). For GSTA1, the genotype distribution was 24.7% (A/A), 53.9% (A/B), and 21.4% (B/B). The combined genotypes distribution of GSTM1, GSTT1, GSTP1 and GSTA1 polymorphisms showed that thirty one of the 36 possible genotypes were present in our population; eight of them have a frequency greater than 5%. To the best of our knowledge, this is the first report of GSTs polymorphisms in South Tunisian population. Our findings demonstrate the impact of ethnicity and reveal a characteristic pattern for Tunisian population. The molecular studies in these enzymes provide basis for further epidemiological investigations in the population where these functional polymorphisms alter therapeutic response and act as susceptibility markers for various clinical conditions.