Rajagopal Krishnamoorthy

Locus assignment of human a globin mutations by selective amplification and direct sequencing

Summary We describe a simple approach for molecular characterization and locus assignment of structural mutants by direct sequencing of enzymatically amplified DNA selective to α1 and α2 globin gene regions. Nucleotide substitution of two structural variants (Stanleyville II α278Lys and J Mexico α254Glu) were determined and their encoding loci were specified. The amplified segment encompasses sequences upstream of the CAAT box to downstream of the Poly(A) addition signal. Hence all of the α globin structural variants and most of the nondeletion α thalassaemic mutants should be characterizable by this approach.

Rapid analysis of ‐α3.7 thalassaemia and αααanti 3.7 triplication by enzymatic amplification analysis

Summary In this report we describe a PCR‐based method for the diagnosis of the most common form of α thalassaemia, the –α3.7 deletion which occurs throughout all tropical and subtropical regions of the world. The same procedure also identifies the reciprocal recombinant chromosome (αααanti 3.7). Restriction mapping of the PCR products has enabled us to distinguish between the type I (–α3.7I), type II (–α3.7II) and type III (–α3.7III) deletions. This strategy will be very useful in screening programmes of α thalassaemia occurring on its own or in association with β thalassaemia and sickle cell disease.

A novel sickle cell mutation of yet another origin in Africa: the Cameroon type

SummaryThe sickle cell mutation (βs) arose as at least three independent events in Africa and once in Asia, being termed the Senegal, Benin, Bantu and Indian types respectively. An investigation in Cameroon was carried out to determine whether the atypical sickle genes observed in the neighboring countries are the result of recombination or the presence of a sickle cell mutation of a different genetic origin. It was conducted on 40 homozygous SS patients followed at the Blood Transfusion Center in the capital city of Yaoundé. On 80 βs chromosomes, 13 exhibited a novel polymorphic pattern that was observed three times in the homozygous state. This chromosome contains an AγT gene. The restriction fragment length polymorphism haplotype is different from all the other βs chromosomes in both the 5′ and 3′ regions, but has previously been reported in sporadic cases. The (AT)8(T)5 sequence in the — 500 region of the β gene is specific and different from that of the Senegal, Benin, Bantu or Indian βs genes. All the carriers of this specific chromosome belong to the Eton ethnic group and originate from the Sanaga river valley. This observation strongly argues for yet another independent origin of the sickle cell mutation in Africa, here referred to as the “Cameroon type”. The Benin haplotype and a Benin/ Bantu recombinant haplotype have been observed in the other studied populations: Ewondo, Bamiléké, Bassa, Yambassa and Boulou.

Worldwide distribution of NAT2 diversity: Implications for NAT2 evolutionary history

BackgroundThe N-acetyltransferase 2 (NAT2) gene plays a crucial role in the metabolism of many drugs and xenobiotics. As it represents a likely target of population-specific selection pressures, we fully sequenced the NAT2 coding region in 97 Mandenka individuals from Senegal, and compared these sequences to extant data on other African populations. The Mandenka data were further included in a worldwide dataset composed of 41 published population samples (6,727 individuals) from four continental regions that were adequately genotyped for all common NAT2 variants so as to provide further insights into the worldwide haplotype diversity and population structure at NAT2.ResultsThe sequencing analysis of the NAT2 gene in the Mandenka sample revealed twelve polymorphic sites in the coding exon (two of which are newly identified mutations, C345T and C638T), defining 16 haplotypes. High diversity and no molecular signal of departure from neutrality were observed in this West African sample. On the basis of the worldwide genotyping survey dataset, we found a strong genetic structure differentiating East Asians from both Europeans and sub-Saharan Africans. This pattern could result from region- or population-specific selective pressures acting at this locus, as further suggested in the HapMap data by extremely high values of FST for a few SNPs positions in the NAT2 coding exon (T341C, C481T and A803G) in comparison to the empirical distribution of FST values accross the whole 400-kb region of the NAT gene family.ConclusionPatterns of sequence variation at NAT2 are consistent with selective neutrality in all sub-Saharan African populations investigated, whereas the high level of population differentiation between Europeans and East Asians inferred from SNPs could suggest population-specific selective pressures acting at this locus, probably caused by differences in diet or exposure to other environmental signals.

Relationship between Toxoplasma gondii infection and bipolar disorder in a French sample

BACKGROUND Prenatal exposure to viruses or parasites with tropism for the central nervous system is one of the risk factors for psychotic disorders. However, the relationship between past exposure to Toxoplasma gondii (T. gondii) and incidence of bipolar disorders (BD) is poorly documented across populations. METHODS We explored the potential association between T. gondii exposure and BD in France, a country of high prevalence of Toxoplasmosis, comparing the prevalence of serological markers (IgG/IgM class antibodies) for T. gondii infection in 110 BD patients and 106 healthy controls all living in France. In a subgroup of 42 patients and 42 controls we also evaluated the levels of interleukin 6 (IL-6) transcripts, an adjunct marker of inflammation. RESULTS We found that the sero-positive group for IgG antibodies to T. gondii had a 2.7 fold odds of having BD as compared to the sero-negative group (OR=2.17 CI 95%=1.09-4.36, p=0.028). Despite the fact that BD patients had significantly higher levels of IL-6 than the non-patient controls, no notable association between T. gondii status and IL-6 transcript levels was found. We did not find any clinical or demographic correlates of Toxoplasma exposure in the study population. LIMITATIONS Our results are to be interpreted with caution because of our small sample size. RESULTS We confirm the association between seropositive status to T. gondii and bipolar disorders reported in other populations and extend it to French patients. Our data strengthen the importance of early detection of T. gondii infected patients in order to propose specific and adequate treatments.

Genotyping of the polymorphic N-acetyltransferase (NAT2*) gene locus in two native African populations.

The hepatic N-acetyltransferase enzyme encoded by the NAT2* gene locus is responsible for the human polymorphic acetylation of numerous arylamine or hydrazine-containing drugs and xenobiotics including AIDS-related therapeutic agents such as isoniazid and sulphonamides. The genetic basis underlying the human acetylation polymorphism has been extensively studied in several populations but native African populations were poorly documented. In the present study, 117 unrelated black Africans, namely Dogons from Mali and Gabonese, were investigated for NAT2* allelic variability and genotype distribution. Thirteen NAT2* alleles were unambiguously identified by combined use of allele-specific reamplifications and restriction endonuclease digestions. Our results confirm the African origin of G191->A substitution in the NAT2* coding region which was previously associated with slow acetylation in African-Americans. The finding of high allelic diversity in the studied populations is consistent with the hypothesis of a single African origin for NAT2*-associated polymorphism. Finally, no excess of the slow acetylator phenotype is predicted in these populations, implying no need for fitting NAT2* polymorphism-sensitive therapies to black Africans, compared to Caucasians.

Protein titration curves by combined isoelectric focusing-electrophoresis with hemoglobin mutants as models

Abstract By performing electrphoresis perpendicular to a stationary pH gradient generated by focused carrier ampholytes in a gel slab, a pictorial representation of a protein titration curve is obtained. By running a protein and its genetic mutants in parallel, the shape of the relative titration curve reveals which charged amino acid has been substituted. Within a given family of proteins, under a constant set of experimental conditions, the relative mobility at any given pH can be correlated with the number of protons lost or acquired by the protein.

Association between Parkinson's Disease and the HLA-DRB1 Locus

Two genome‐wide association studies (GWASs) recently highlighted the HLA‐DRA and HLA‐DRB5 genes as associated with Parkinson disease (PD). However, because HLA‐DRA displays a low level of polymorphisms and HLA‐DRB5 is only present in approximately 20% of the population, these findings are difficult to interpret. Our aims were: (1) to replicate and investigate in greater detail the association between PD and the HLA‐DR region; (2) to identify PD‐associated HLA alleles; and (3) to perform a meta‐analysis of our top finding. As part of 2 French population‐based case–control studies of PD including highly ethnically homogeneous participants, we investigated the association between PD and 51 Single‐nucleotide polymorphisms (SNPs) in the HLA‐DR region. HLA‐DRB1 alleles were imputed using the HLA*IMP software. HLA typing was performed in a subsample of the participants. We performed a meta‐analysis of our top finding based on 4 GWAS data sets. Among 499 cases and 1123 controls, after correction for multiple testing, we found an association with rs660895 (OR/minor allele, 0.70; 95% CI, 0.57–0.87) within the HLA‐DRB1 gene, which encodes the most polymorphic HLA‐DR chain (DRβ). A meta‐analysis (7996 cases, 36455 controls) confirmed this association (OR, 0.86; 95% CI, 0.82–0.91; P < .0001). SNP‐based imputation of HLA alleles showed an inverse association between PD and the HLA‐DRB1*04 allele. We replicated an association between PD and the HLA‐DR region and provided further insight into the loci and alleles involved. The highly polymorphic HLA‐DRB1 locus contains rs660895, which represents a more legitimate candidate than previous ones. Our finding is in agreement with the hypothesis of an immune component in PD pathophysiology.

Effect of α-thalassemia on sickle-cell anemia linked to the Arab-Indian haplotype in India

Two population groups from Western India with a high prevalence of the βs gene, one tribal (Valsad) and the other nontribal (Nagpur), were studied. The βs gene frequency in both populations was similar (0.22 vs. 0.23), but not the clinical expression of sickle‐cell anemia (SS): the sickle homozygotes in the tribal group appeared to have a mild clinical course, whereas the majority in the nontribal group exhibited a more severe clinical phenotype. Both tribal and nontribal SS patients had a similarly high mean hemoglobin (Hb)F expression (18.5% vs. 15.5%) and a high number of F cells (72.3% vs. 66.6%). DNA analysis of the β‐globin gene cluster region revealed that in these two populations, this portion of DNA was identical with and corresponded to the typical Arab‐Indian haplotype. Nevertheless, in heterozygotes, the mean βs expression was lower (27.9%) in the tribal as compared to the nontribal group (35.5%). The major epistatic factor distinguishing the milder presentation in tribals vs. a more severe manifestation in nontribals was the very high frequency (0.97) of the α‐thalassemia gene in the former as compared to the latter (0.24). We conclude that the phenotypic expression of sickle‐cell anemia, linked to the Arab‐India haplotype and expressing similar levels of HbF and F cells, is not uniformly mild in India and that α‐thalassemia is a powerful and additional epistatic factor in the Indian subcontinent. Am. J. Hematol. 55:104‐109, 1997.

Human desmin gene: cDNA sequence, regional localization and exclusion of the locus in a familial desmin-related myopathy

Abstract Desmin is a muscle-specific intermediate filament that is encoded by a gene assigned to human chromosome 2q35. Desmin-related myopathies are inherited disorders characterized by an intrasarcoplasmic accumulation of desmin. Recently, the knockout of the desmin gene was shown to generate a myopathic syndrome in transgenic mice, suggesting that functional abnormality of desmin may generate similar clinical symptoms in mouse and human. To determine the potential role of the desmin gene in a well-defined desmin-related myopathy (autosomal dominant form of Fardeau), human desmin cDNAs obtained from affected and unaffected individuals were cloned, sequenced and compared. No obvious mutation was detected. A BssHII restriction fragment length polymorphism (RFLP) was identified in exon 6 of the desmin gene. This RFLP was associated with a previously identified EcoRV RFLP in exon 4 to generate a tetra-allelic system, which was tested for linkage to the desmin-related myopathy in three families. The human desmin gene was localized within an 11-cM interval on chromosome 2q using a panel of radiation hybrids. This 11-cM region was clearly excluded by linkage analysis in the three desmin-related myopathy families using a set of highly polymorphic microsatellite markers. These results suggest that the desmin gene is not primarily involved in this disease.