Molecular markers reveal that population structure of the human pathogen Candida albicans exhibits both clonality and recombination.

The life history of Candida albicans presents an enigma: this species is thought to be exclusively asexual, yet strains show extensive phenotypic variation. To address the population genetics of C. albicans, we developed a genetic typing method for codominant single-locus markers by screening randomly amplified DNA for single-strand conformation polymorphisms. DNA fragments amplified by arbitrary primers were initially screened for single-strand conformation polymorphisms and later sequenced using locus-specific primers. A total of 12 single base mutations and insertions were detected from six out of eight PCR fragments. Patterns of sequence-level polymorphism observed for individual strains detected considerable heterozygosity at the DNA sequence level, supporting the view that most C. albicans strains are diploid. Population genetic analyses of 52 natural isolates from Duke University Medical Center provide evidence for both clonality and recombination in C. albicans. Evidence for clonality is supported by the presence of several overrepresented genotypes, as well as by deviation of genotypic frequencies from random (Hardy-Weinberg) expectations. However, tests for nonrandom association of alleles across loci reveal less evidence for linkage disequilibrium than expected for strictly clonal populations. Although C. albicans populations are primarily clonal, evidence for recombination suggests that sexual reproduction or some other form of genetic exchange occurs in this species.     

Genetic variations of ��-cardiac actin and cardiac muscle LIM protein in hypertrophic cardiomyopathy in South India

BACKGROUND:

Hypertrophic cardiomyopathy (HCM) is a disease of the heart muscle, with an autosomal dominant mode of inheritance. It is also known as the ‘disease of the sarcomere’, and is a major cause of morbidity and mortality worldwide. Mutations in the sarcomeric genes have been largely implicated in the manifestation of HCM. Modifier genes and environmental factors, along with causative mutation, add to the cumulative effect of the disease.

METHODS:

In the present study, the role of the cardiac actin gene and the cardiac muscle LIM protein as contributors to HCM – through genetic variation – has been elucidated by screening the entire coding region in 100 control and 100 HCM subjects through polymerase chain reaction-based single-strand conformation polymorphism analysis and direct sequencing.

RESULTS:

The authors could not find any novel or reported exonic variations in any of the genes in the studied population; however, intronic variations were revealed in the cardiac actin gene through direct sequencing. A case of compound heterozygosity was observed in a patient with a variation in intron 1, along with a novel heterozygous mutation in exon 7 (S215L) of α-tropomyosin.

CONCLUSIONS:

The particular genes are highly conserved, and account for only 1.5% of HCM cases. They do not seem to play a major role in the genesis of HCM in the present population, thus confirming earlier reports of conserved sequences and ethnicity.     

Routine mutation screening of HNF-1α and GCK genes in MODY diagnosis: How effective are the techniques of DHPLC and direct sequencing used in combination?

Aims/hypothesis. Mutations in the hepatocyte nuclear factor (HNF)-1α and glucokinase (GCK) genes are the major causes of monogenic forms of Type II (non-insulin-dependent) diabetes mellitus (Maturity-Onset Diabetes of the Young subtypes, MODY). We evaluated the effectiveness of fluorescent single-strand conformation polymorphism (F-SSCP), denaturing high-performance liquid chromatography (DHPLC) and sequencing based mutation detection in the molecular diagnosis of MODY. Our goal is to identify a rapid, efficient and cost effective mutation detection method for the molecular diagnosis of MODY and other human genetic disorders. Methods. We evaluated the accuracy of DHPLC in screening for MODY 2 and 3 mutations. In addition, we compared the sensitivity, specificity, cost, handling time and analysis time of fluorescent single-strand conformation polymorphism, denaturing high-performance liquid chromatography and direct sequencing screening methods. Results. Denaturing high-performance liquid chromatography is a recently developed method for mutation detection. It is cost effective, powerful and reliable and quite suitable for 22 out of the 24 fragments required for MODY 2 and 3 testing. However, exons 1 and 7 of the HNF-1α gene are very polymorphic and so direct sequencing is faster as well as more efficient and reliable. Conclusion/interpretation. Our results suggest that combining denaturing high-performance liquid chromatography and direct sequencing is a good approach for the routine detection of HNF-1α and GCK mutations in MODY families. Denaturing high-performance liquid chromatography appears to be a powerful tool in genetic testing and the method could be applied to the molecular diagnosis of other human genetic diseases. [Diabetologia (2001) 44: 775–778] Received: 15 November 2000 and in revised form: 13 February 2001     

Single-strand scissions of chromosomal DNA during commitment to recombination at meiosis.

Diploid cells of the yeast Saccharomyces cerevisiae induced to undergo meiosis accumulate single-strand scissions in both template and newly synthesized DNA during commitment to genetic recombination. No evidence for accumulation of double-strand breaks during meiosis was obtained. When commitment to recombination is at the full meiotic level there are approximately 70 to 200 single-strand scissions per meiotic cell in which approximately 150 recombination events have been reported to occur.     

Mutational analyses in four Japanese families with X-linked liver phosphorylase kinase deficiency type 1

We analysed the gene of the human -subunit of liver phosphorylase kinase (PHKA2) in four Japanese families with X-linked liver phosphorylase kinase deficiency type 1 by RT-PCR followed by PCR–single-strand conformation polymorphism and direct DNA sequencing. In this study, two novel mutations (Y116D and 2675AG) and one mutation previously reported (P1205L) were identified, revealing molecular heterogeneity in Japanese patients. Considering the dissimilarity in phenotype among our patients even with an identical mutation in the PHKA2 gene, it seems that each genetic deficiency in this gene may not be the only factor to determine the clinical heterogeneity in this disease.     

Hepatitis C viral quasispecies

Analysing significant numbers of cDNA clones of the hepatitis C virus (HCV) from single isolates provides unquestionable proof that the viral genome cannot be defined by a single sequence, but rather by a population of variant sequences closely related to one another. This way of organizing the genetic information is referred to as quasispecies. Throughout HCV infection, the number and composition of the variants in the viral population keeps changing owing to environmental influences, resulting in a virus that is constantly redefining itself both genetically and phenotypically. Therefore, the virus has often been investigated in population terms. Many clinical studies have tried to unravel, through the parameters that characterize the HCV quasispecies, prognostic markers of the disease and its response to treatment. Other investigations have focused on discovering how the virus and host interact during chronic infection. The consensus sequence, the rate of fixation of mutations and the complexity of the viral population are useful parameters for describing the viral population behaviour and its interaction with the host. In addition to sequencing, several other methods, based on electrophoretic mobility, have been used to study these parameters, such as temperature gradient-gel electrophoresis, single-strand conformation polymorphism and gel-shift analysis. The viral region examined, the source of clinical specimen, as well as the methodology employed, will be decisive in interpreting the information obtained.     

Characterization of a unique genetic variant in the beta1-adrenoceptor gene and evaluation of its role in idiopathic dilated cardiomyopathy. CARDIGENE Group.

The genetic factors that underlie idiopathic dilated cardiomyopathy (IDCM) have not yet been elucidated. Since beta1-adrenoceptors are downregulated in patients with IDCM, and since beta-blocker therapy is consistently beneficial in this setting, we hypothesized that genetic variation in the beta1-adrenoceptor might affect susceptibility to and/or severity of IDCM. As no intragenic polymorphism was available, a systematic screening of the gene was first performed. The organization and sequence of the human beta1-adrenoceptor gene were established using polymerase chain reaction, single-strand conformation polymorphism analysis and sequencing. The gene comprises 1434 bp and no intron was observed. We found a unique and frequent polymorphism (C1165G) which predicts an Arg389Gly substitution. The association of this polymorphism with IDCM was then analysed using the PCR-restriction fragment length polymorphism method in the CARDIGENE population, a clinically well-characterized population of IDCM. Genotypic distribution was in agreement with Hardy-Weinberg equilibrium. There were no differences in the beta1-adrenoceptor allele frequencies between IDCM (n=426; C/G=0.76/0.24) and age- and sex-matched control subjects (n=395; C/G=0.78/0.22). Within the patient group, no association was observed with the severity of the disease. In conclusion, the genomic organization of beta1-adrenoceptor is described here for the first time. We found a unique and frequent polymorphism in the coding sequence of the gene. No association was observed between IDCM and the genetic variant. Its possible involvement in other cardiac diseases related to the beta1-adrenoceptor remains to be analysed.     

Biogeographic range expansion into South America by Coccidioides immitis mirrors New World patterns of human migration

Long-distance population dispersal leaves its characteristic signature in genomes, namely, reduced diversity and increased linkage between genetic markers. This signature enables historical patterns of range expansion to be traced. Herein, we use microsatellite loci from the human pathogen Coccidioides immitis to show that genetic diversity in this fungus is geographically partitioned throughout North America. In contrast, analyses of South American C. immitis show that this population is genetically depauperate and was founded from a single North American population centered in Texas. Variances of allele distributions show that South American C. immitis have undergone rapid population growth, consistent with an epidemic increase in postcolonization population size. Herein, we estimate the introduction into South America to have occurred within the last 9,000-140,000 years. This range increase parallels that of Homo sapiens. Because of known associations between Amerindians and this fungus, we suggest that the colonization of South America by C. immitis represents a relatively recent and rapid codispersal of a host and its pathogen.     

Genomic evidence for a complete sexual cycle in Candida albicans

Candida albicans is a diploid fungus that has become a medically important opportunistic pathogen in immunocompromised individuals. We have sequenced the C. albicans genome to 10.4-fold coverage and performed a comparative genomic analysis between C. albicans and Saccharomyces cerevisiae with the objective of assessing whether Candida possesses a genetic repertoire that could support a complete sexual cycle. Analyzing over 500 genes important for sexual differentiation in S. cerevisiae, we find many homologues of genes that are implicated in the initiation of meiosis, chromosome recombination, and the formation of synaptonemal complexes. However, others are striking in their absence. C. albicans seems to have homologues of all of the elements of a functional pheromone response pathway involved in mating in S. cerevisiae but lacks many homologues of S. cerevisiae genes for meiosis. Other meiotic gene homologues in organisms ranging from filamentous fungi to Drosophila melanogaster and Caenorhabditis elegans were also found in the C. albicans genome, suggesting potential alternative mechanisms of genetic exchange.     

Three novel point mutations causing haemophilia A

Haemophilia A is an X-linked bleeding disorder caused by mutations in the factor VIII gene. In our efforts to elucidate molecular defects in the haemophilia A patients from the Republic of Macedonia, we employed nonradioactive single-strand conformation polymorphism analysis followed by direct sequencing, for identifying point mutations in the factor VIII gene. In the present study we report the detection of three novel missense mutations: Met19 --> Arg; Ala78 --> Pro and Cys2174 --> Gly, all causing haemophilia A.     

Sexual isolation in Drosophila melanogaster: a possible case of incipient speciation.

It is generally believed that Drosophila melanogaster has no closely related species with which it can produce the viable and fertile hybrids that are essential for the genetic analysis of speciation. Following the recent report of molecular differentiation between a Zimbabwe, Africa, population and two United States populations, we provide evidence that strong sexual isolation exists between the D. melanogaster population in Zimbabwe and populations of other continents. In the presence of males of their own kind, females from most isofemale lines of Zimbabwe would not mate with males from elsewhere; the reciprocal mating is also significantly reduced, but to a lesser degree. The genes for sexual behaviors are apparently polymorphic in Zimbabwe and postmating reproductive isolation between this and other populations has not yet evolved. Whole chromosome substitutions indicate significant genetic contributions to male mating success by both major autosomes, whereas the X chromosome effect is too weak to measure. In addition, the relative mating success between hybrid and pure line males supports the interpretation of strong female choice. These observations suggest that we are seeing the early stages of speciation in this group and that it is driven by sexual selection. The genetic and molecular tractability of D. melanogaster offers great promise for the detailed analysis of this apparent case of incipient speciation.     

Spectrum of LDL receptor gene mutations in Denmark: implications for molecular diagnostic strategy in heterozygous familial hypercholesterolemia.

Heterozygous familial hypercholesterolemia (FH) is one of the most common potentially fatal single-gene diseases leading to premature coronary artery disease, but the majority of heterozygous FH patients have not been diagnosed. FH is due to mutations in the gene coding for the low-density lipoprotein (LDL) receptor, and molecular genetic diagnosis may facilitate identification of more FH subjects. The Danish spectrum of 29 different mutations, five of which account for almost half of heterozygous FH, is intermediate between that of countries such as South Africa, where three mutations cause 95% of heterozygous FH in the Afrikaners, and Germany or England, where there are many more mutations. In clinical practice, a strategy for the genetic diagnosis of heterozygous FH, tailored to the mutational spectrum of patients likely to be seen at the particular hospital/region of the country, will be more efficient than screening of the whole LDL receptor gene by techniques such as single-strand conformation polymorphism (SSCP) analysis in every heterozygous FH candidate. In Aarhus, Denmark, we have chosen to examine all heterozygous FH candidates for the five most common LDL receptor gene mutations (W23X, W66G, W556S, 313 + 1G --> A, 1846 - 1G --> A) and the apoB-3500 mutation by rapid restriction fragment analysis. Negative samples are examined for other mutations by SSCP analysis followed by DNA sequencing of the exon indicated by SSCP to contain a mutation. If no point mutation or small insertion/deletion is detected, Southern blot or Long PCR analysis is performed to look for the presence of large gene rearrangements. In conclusion, our data suggest that an efficient molecular diagnostic strategy depends on the composition of common and rare mutations in a population.     

CYBB Gene Mutation Detection in an Iranian Patient with Chronic Granulomatous Disease

In this study, we report a mutation in CYBB gene in a patient with X-CGD (diagnosed on the base of family history, NDT test, DHR 123 assay). Mutation in CYBB gene was detected using SSCP analysis (single-strand conformation polymorphism) followed by sequencing. During screening for mutations in the CYBB gene we observed 880 CT in exon 8. This mutation resulted in 290 ArgStop. We also observed a change (-270 CA) in the promoter region which needs further investigation.We would like to pursue this study by analyzing more X-CGD patients to find out the CYBB mutation spectrum in Iranian patients.     

卵磷脂胆固醇酰基转移酶基因多态性与动脉粥样硬化性脑梗死的研究

目的探讨卵磷脂胆固醇酰基转移酶(LCAT)基因511C/T多态性在中国汉族人群中的分布及其与动脉粥样硬化性脑梗死(ACI)的关系。方法应用PCR、单链构象多态性技术和DNA测序法检测ACI患者150例(ACI组)和健康体检者122例(对照组)LCAT511C/T多态性。根据基因多态性将ACI组分为2个亚组,511CC组(135例)和511CT组(15例)。结果LCAT第四外显子511位点存在多态现象,此多态位点C/T在ACI组和对照组均符合Hardy-Weinberg平衡定律。ACI组CT基因型频率、T等位基因频率均显著高于对照组(P<0.01)。511CC组HDL-C水平高于511CT组(P<0.05)。结论LCAT第四外显子511C/T多态性可能为中国汉族人群ACI易感因素,T等位基因可能与HDL-C代谢有关。     

Genetic and educational assortative mating among US adults

Understanding the social and biological mechanisms that lead to homogamy (similar individuals marrying one another) has been a long-standing issue across many fields of scientific inquiry. Using a nationally representative sample of non-Hispanic white US adults from the Health and Retirement Study and information from 1.7 million single-nucleotide polymorphisms, we compare genetic similarity among married couples to noncoupled pairs in the population. We provide evidence for genetic assortative mating in this population but the strength of this association is substantially smaller than the strength of educational assortative mating in the same sample. Furthermore, genetic similarity explains at most 10% of the assortative mating by education levels. Results are replicated using comparable data from the Framingham Heart Study.Assortative mating occurs when individuals exhibit a preference for those who are either similar, (homogamy) or dissimilar (heterogamy) to themselves. Two expressions—“birds of a feather flock together” and “opposites attract”—are used to explain friendship and spousal pairings but denote opposite assumptions regarding the direction of selection. Critically, no existing research has quantified the degree to which individuals who select into a marriage are genetically similar to one another across the entire genome.Quantifying genome-wide genetic assortative mating (GAM) in the population is important for methodological and substantive reasons. First, statistical models in genetic epidemiology, such as Hardy–Weinberg equilibrium, often assume random mating to forecast population allele frequencies, homozygosity rates, and other parameters of interest across generations (1) and behavior genetics models assume random mating to calculate heritability estimates (2). Second, social scientists have long studied the causes and consequences of assortative mating on a number of phenotypic measures such as height, education, religiosity, and political partisanship (3–5). Although there is research with a focus on the implications of genetic homogamy for phenotypic assortative mating (6), most studies of assortative mating have not considered the possibility that GAM may underlie phenotypic sorting. Social factors clearly limit opportunities to interact with people of different backgrounds (7, 8) but there is no study that simultaneously estimates educational assortative mating (EAM) and GAM in the population. Although much is known about changes in the nature of assortative mating over the past 50 y (5, 8, 9), little is known about the relationship between GAM and EAM.We focus on EAM because it has received the largest amount of attention in the assortative mating literature (4) and, equally important, research has shown that educational attainment reflects genetic influences (10, 11). No existing study has used genome-wide data among spousal pairs to quantify GAM in the population. This observation coupled with the potential bias caused by GAM in traditional heritability estimates (12) makes this line of inquiry both substantively and methodologically important to a large group of biological and social scientists. In this paper we ask three related questions. First, is there any evidence of GAM in the population? Are genetically similar persons more likely to marry than genetically dissimilar persons both inclusive of and net of ethnic intramarriage? Or are spousal genotypes uncorrelated, as is sometimes assumed? Second, how does the magnitude of GAM compare with other phenotypically-based measures of assortative mating in the population—such as education? Third, to what extent is phenotypic assortative mating linked to GAM in the population?     

Multiple G6PD mutations are associated with a clinical and biochemical phenotype similar to that of G6PD Mediterranean

Glucose-6-phosphate dehydrogenase (G6PD) deficiency, one of the most common red cell abnormalities, is characterized by a wide clinical, biochemical, and molecular heterogeneity. In this study we have determined the molecular basis of G6PD deficiency in a sample of 70 male subjects, originating from different parts of Italy, who all shared a clinical and biochemical phenotype identical or very similar to that of G6PD Mediterranean, the most common variant in Italy. In 59 cases (84%) we found the mutation 563 C --> T, previously known to be underlying the G6PD Mediterranean and the two polymorphic variants G6PD Cagliari and G6PD Sassari. From the remaining 11 we amplified the entire coding region of G6PD in 8 different fragments and subjected them to nonradioactive single-strand conformation analysis. Direct sequencing was then performed on abnormal fragments. By this approach we found six cases (8.5%) with 1360 G --> A mutation (G6PD Union) and two cases (2.8%) with 1376 G --> C (G6PD Cosenza). In the remaining three samples we found two other mutations: 1342 A --> G (two cases, 2.8%) and 1052 G --> T (one case, 1.4%). These two molecular defects have never been described before and were designated by us as G6PD S. Antioco and G6PD Partenope, respectively. Haplotype analysis suggested that all the non-Mediterranean mutations occurred independently on a normal G6PD allele. This study shows that the G6PD Union mutation has a high polymorphic frequency in the Italian population and that the genetic heterogeneity of G6PD Mediterranean-like variants is higher at the molecular level than expected from biochemical characterization.     

Low frequency of melanocortin-4 receptor (MC4R) mutations in a Mediterranean population with early-onset obesity

BACKGROUND: Melanocortin-4 receptor (MC4R) mutations have been reported as the most common single genetic cause of obesity in some populations and it has been suggested that they may be responsible for more than 4% of early-onset obesity. OBJECTIVES: To verify the presence of mutations of the MC4R coding region in children from southern Italy with early-onset obesity. SUBJECTS AND METHODS: Two-hundred and eight unrelated obese children and adolescents were included in the study. The average age at obesity onset was 4.5+/-2.6 y. MC4R coding region was screened using both single-strand conformation polymorphism (SSCP) analysis and denaturing high-performance liquid chromatography (DHPLC). Automatic sequencing of PCR products of all individuals that showed an aberrant SSCP and/or DHPLC pattern was performed. RESULTS: One novel missense mutation and one previously described polymorphism (Vall03Ile) were identified. The missense mutation C142T, resulting in the substitution of proline with serine at codon 48, within the first MC4R transmembrane domain, was detected at the heterozygous state in a 15-y-old obese girl (body mass index (BMI)=35 kg/m(2)) who has been obese since she was 8 y old. The mutation co-segregated with the obesity phenotype for over three generations and was not found in the control population. CONCLUSIONS: Our data show MC4R obesity causing mutations in less than 0.5% of the patients (ie 1 out of 208 patients) and therefore indicate a low prevalence of MC4R variants in the obese population from southern Italy. The specific genetic background of the Mediterranean population could make it difficult for MC4R mutations to produce an essentially polygenic trait such as common obesity, at least during childhood.     

Permanency of response to selection for quantitative characters in finite populations.

To study the permanency of response to selection for a quantitative character in finite populations and the nature of the genetic effects that contribute to this response, we have used the covariance between ancestors and descendents within populations. Effects and variances are defined for an initial equilibrium random mating monoecious population that gives rise to replicate finite populations. After a prescribed history of restricted population size, the replicate populations are expanded, and the covariance between ancestors and descendents is quantified in terms of descent measures and genetic components in the initial population as a means of determining the additive variance within populations. Several dominance components including joint dominance effects of loci contribute to the additive variance, some of which can be negative. There is always a positive contribution of additive by additive variance to the additive variance within populations, which can be large. With the new definitions of components of genetic variance within populations, selection response is formulated in the same manner as for the initial random mating population, but the components have been modified considerably by the restricted population size.     

"Clonal" population structure of the malaria agent Plasmodium falciparum in high-infection regions

The population genetic structure of Plasmodium falciparum, the agent of malignant malaria, has been shown to be predominantly "clonal" (i.e., highly inbred) in regions of low infectivity; in high-infectivity regions, it is often thought to be panmictic, or nearly so, although there is little supporting evidence for this. The matter can be settled by investigating the parasite's genetic makeup in the midgut oocysts of the mosquito vector, where the products of meiosis can directly be observed. The developmental stages of P. falciparum are haploid, except in the oocysts of infected mosquito vectors, where two gametes fuse, diploidy occurs, and meiosis ensues. We have investigated genetic polymorphisms at seven microsatellite loci located on five chromosomes by assaying 613 oocysts in 145 mosquitoes sampled from 11 localities of Kenya, where malignant malaria is perennial and intense. There is considerable allelic variation, 16.3 +/- 2.1 alleles per locus, and considerable inbreeding, approximately 50% on the average. The inbreeding is caused by selfing (approximately 25%) and nonrandom genotype distribution of oocysts among mosquito guts (35%). The observed frequency of heterozygotes is 0.43 +/- 0.03; the expected frequency, assuming random mating, is 0.80 +/- 0.05. Linkage disequilibrium is statistically significant for all 21 pairwise comparisons between loci, even though 19 comparisons are between loci in different chromosomes, which is consistent with strong deviation from panmixia and the consequent reproduction of genomes as clones, without recombination between gene loci. This is of considerable evolutionary significance and of epidemiological consequence, concerning the spread of multilocus drug and vaccine resistance.     

分子遗传技术在血吸虫变异方面的研究进展

分子生物学技术的发展为研究血吸虫遗传变异做出了重要贡献。近年来发展起来的可用于血吸虫遗传变异分 析的分子技术主要包括限制性片段长度多态性 （RFLP）、 随机扩增多态性技术 （RAPD）、 微卫星锚定PCR （SSR?PCR）、 聚合 酶链反应?单链构象多态性 （PCR?SSCP） 等。本文主要就这些分子遗传技术在血吸虫变异方面的研究进展作一综述。