Hemizygous Individuals Research Articles

Genotype-phenotype association and biochemical analyses of glucose-6-phosphate dehydrogenase variants: Implications for the hemolytic risk of using 8-aminoquinolines for radical cure.

Background: Plasmodium vivax remains the malaria species posing a major threat to human health worldwide owing to its relapse mechanism. Currently, the only drugs of choice for radical cure are the 8-aminoquinolines (primaquine and tafenoquine), which are capable of killing hypnozoites and thus preventing P. vivax relapse. However, the therapeutic use of primaquine and tafenoquine is restricted because these drugs can cause hemolysis in individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency. This study aimed to assess and understand the hemolytic risk of using 8-aminoquinolines for radical treatment in a malaria endemic area of Thailand. Methods: The prevalence of G6PD deficiency was determined using a quantitative test in 1,125 individuals. Multiplexed high-resolution meltinging (HRM) assays were developed and applied to detect 12 G6PD mutations. Furthermore, biochemical and structural characterization of G6PD variants was carried out to understand the molecular basis of enzyme deficiency. Results: The prevalence of G6PD deficiency was 6.76% (76/1,125), as assessed by a phenotypic test. Multiplexed HRM assays revealed G6PD Mahidol in 15.04% (77/512) of males and 28.38% (174/613) of females, as well as G6PD Aures in one female. G6PD activity above the 30% cut-off was detected in those carrying G6PD Mahidol, even in hemizygous male individuals. Two variants, G6PD Murcia Oristano and G6PD Songklanagarind + Viangchan, were identified for the first time in Thailand. Biochemical characterization revealed that structural instability is the primary cause of enzyme deficiency in G6PD Aures, G6PD Murcia Oristano, G6PD Songklanagarind + Viangchan, and G6PD Chinese 4 + Viangchan, with double G6PD mutations causing more severe enzyme deficiency. Conclusion: In western Thailand, up to 22% of people may be ineligible for radical cure. Routine qualitative tests may be insufficient for G6PD testing, so quantitative tests should be implemented. G6PD genotyping should also be used to confirm G6PD status, especially in female individuals suspected of having G6PD deficiency. People with double G6PD mutations are more likely to have hemolysis than are those with single G6PD mutations because the double mutations significantly reduce the catalytic activity as well as the structural stability of the protein.

PO 8355 HOST GENETIC POLYMORPHISMS AND ASYMPTOMATIC MALARIA IN SOUTHERN GHANA

BackgroundDespite several interventions through malaria control programmes, asymptomatic malaria is a major barrier to control as asymptomatic individuals serve as reservoirs from which others are re-infected. The mechanism by which these individuals remain asymptomatic is not well understood. Much work has been done in relation to human genes and their association to severe, mild and uncomplicated malaria. However, there is limited knowledge regarding host genetic factors and asymptomatic malaria.MethodIn this study, we investigated the association between host genetic polymorphisms of glucose-6-phosphate dehydrogenase gene (G6PD), mannose binding lectin (MBLG54A), tumor necrotic factor alpha (TNF-G308A) and nitric oxide synthase 2 (NOS2-G954C) and the outcome of asymptomatic P. falciparum malaria in 150 healthy individuals in southern Ghana.ResultsWe found a significant association between G6PDd and asymptomatic malaria with a prevalence of 9.6% (p=0.035, by chi-square test). All the individuals who were heterozygous and hemizygote deficient (5.3% and 4.3%) were found to be asymptomatic. Individuals homozygous (GG) for TNF (G308A) were found to be highly asymptomatic (p=0.019, by chi-square test). Regarding MBL (G54A) and NOS (G954C), no significant association was found between these markers and asymptomatic malaria.ConclusionUpon reviewing our data with other data from published work, we conclude that both heterozygous and hemizygous individuals with G6PD A- and homozygous individuals (GG) of TNF (G308A) polymorphisms could be predisposed genetically to asymptomatic malaria.

Modeling diploid male dynamics in Hymenoptera: Effects of the number of alleles, dispersal by random walk and simple spatial structuring

Insects in the order Hymenoptera such as wasps, ants and bees exhibit a haplodiploid system of sexual determination in which females result from fertilized eggs and males from unfertilized eggs. Genetic information for sex determination is contained in a single multiallelic locus, so that the heterozygous individuals become females, and the hemizygous or homozygous individuals, haploid or diploid males, respectively. This mechanism is called single locus complementary sex determination (sl-CSD). While females and haploid males are regarded to be fertile, the opposite occurs for diploid males, so the significant production of diploid males (DM) may lead a population to an extinction scenario called the “diploid male vortex”. In the present work, we investigate the dynamics of populations of an organism with sl-CSD under various combinations of three parameters: male flight skills, number of sexual alleles and concentration of prohibited zones in a terrain represented by a square lattice. Sites of this lattice were randomly chosen as prohibited, where individuals could not pass, so the terrain was divided into islands of various sizes where individuals were confined. After a given simulation, the number of DM was measured. The main results that come from our simulations are: (i) the DM number depends more on the number of random steps in the male flight than on the number of alleles; (ii) the DM frequency in the small fragments becomes the same as the entire network when the limit of percolation is reached; (iii) the number of random steps explains the difference between the DM number found in the largest fragment and in the small fragments when the limit of percolation is reached.

Understanding human genetic factors influencing primaquine safety and efficacy to guide primaquine roll-out in a pre-elimination setting in southern Africa

BackgroundPrimaquine (PQ) is recommended as an addition to standard malaria treatments in pre-elimination settings due to its pronounced activity against mature Plasmodium falciparum gametocytes, the parasite stage responsible for onward transmission to mosquitoes. However, PQ may trigger haemolysis in glucose-6-phosphate dehydrogenase (G6PD)-deficient individuals. Additional human genetic factors, including polymorphisms in the human cytochrome P450 2D6 (CYP2D6) complex, may negatively influence the efficacy of PQ. This study assessed the prevalence of G6PD deficiency and two important CYP2D6 variants in representative pre-elimination settings in South Africa, to inform malaria elimination strategies.MethodsVolunteers (n = 248) attending six primary health care facilities in a malaria-endemic region of South Africa were enrolled between October and November 2015. G6PD status was determined phenotypically, using a CareStart™ G6PD rapid diagnostic test (RDT), and genotypically for two common African G6PD variants, namely A+ (A376G) and A− (G202A, A542T, G680T & T968C) by PCR, restriction fragment length polymorphisms (RFLP) and DNA sequencing. CYP2D6*4 and CYP2D6*17 variants were determined with PCR and RFLP.ResultsA prevalence of 13% (33/248) G6PD deficiency was observed in the cohort by G6PD RDT whilst by genotypic assessment, 32% (79/248) were A+ and 3.2% were A−, respectively. Among the male participants, 11% (6/55) were G6PD A− hemizygous; among females 1% (2/193) were G6PD A− homozygous and 16% (32/193) G6PD A− heterozygous. The strength of agreement between phenotyping and genotyping result was fair (Cohens Kappa κ = 0.310). The negative predictive value for the G6PD RDT for detecting hemizygous, homozygous and heterozygous individuals was 0.88 (95% CI 0.85–0.91), compared to the more sensitive genotyping. The CYP2D6*4 allele frequencies for CYP2D6*4 (inferred poor metabolizer phenotype) and CYP2D6*17 (inferred intermediate metabolizer phenotype) were 3.2 and 19.5%, respectively.ConclusionsPhenotypic and genotypic analyses both detected low prevalence of G6PD deficiency and the CYP2D6*4 variants. These findings, combined with increasing data confirming safety of single low-dose PQ in individuals with African variants of G6PD deficiency, supports the deployment of single low-dose PQ as a gametocytocidal drug. PQ would pose minimal risks to the study populations and could be a useful elimination strategy in the study area.

A universal vector concept for a direct genotyping of transgenic organisms and a systematic creation of homozygous lines.

Diploid transgenic organisms are either hemi- or homozygous. Genetic assays are, therefore, required to identify the genotype. Our AGameOfClones vector concept uses two clearly distinguishable transformation markers embedded in interweaved, but incompatible Lox site pairs. Cre-mediated recombination leads to hemizygous individuals that carry only one marker. In the following generation, heterozygous descendants are identified by the presence of both markers and produce homozygous progeny that are selected by the lack of one marker. We prove our concept in Tribolium castaneum by systematically creating multiple functional homozygous transgenic lines suitable for long-term fluorescence live imaging. Our approach saves resources and simplifies transgenic organism handling. Since the concept relies on the universal Cre-Lox system, it is expected to work in all diploid model organisms, for example, insects, zebrafish, rodents and plants. With appropriate adaptions, it can be used in knock-out assays to preselect homozygous individuals and thus minimize the number of wasted animals.

Rapid RHD Zygosity Determination Using Digital PCR.

Paternal zygosity testing is used for determining homo- or hemizygosity of RHD in pregnancies that are at a risk of hemolytic disease of the fetus and newborn. At present, this is achieved by using real-time PCR or the Rhesus box PCR, which can be difficult to interpret and unreliable, particularly for black African populations. DNA samples extracted from 53 blood donors were analyzed using 2 multiplex reactions for RHD-specific targets against a reference (AGO1)2 to determine gene dosage by digital PCR. Results were compared with serological data, and the correct genotype for 2 discordant results was determined by long-range PCR (LR-PCR), next-generation sequencing, and conventional Sanger sequencing. The results showed clear and reliable determination of RHD zygosity using digital PCR and revealed that 4 samples did not match the serologically predicted genotype. Sanger sequencing and long-range PCR followed by next-generation sequencing revealed that the correct genotypes for samples 729M and 351D, which were serologically typed as R1R2 (DCe/DcE), were R2r' (DcE/dCe) for 729M and R1r″ (DCe/dcE), R0ry (Dce/dCE), or RZr (DCE/dce) for 351D, in concordance with the digital PCR data. Digital PCR provides a highly accurate method to rapidly define blood group zygosity and has clinical application in the analysis of Rh phenotyped or genotyped samples. The vast majority of current blood group genotyping platforms are not designed to define zygosity, and thus, this technique may be used to define paternal RH zygosity in pregnancies that are at a risk of hemolytic disease of the fetus and newborn and can distinguish between homo- and hemizygous RHD-positive individuals.

A High-Throughput Standard PCR-Based Genotyping Method for Determining Transgene Zygosity in Segregating Plant Populations.

In crop research programs that implement transgene-based strategies for trait improvement it is necessary to distinguish between transgene homozygous and hemizygous individuals in segregating populations. Direct methods for determining transgene zygosity are technically challenging, expensive, and require specialized equipment. In this report, we describe a standard PCR-based protocol coupled with capillary electrophoresis that can identify transgene homozygous and hemizygous individuals in a segregating population without knowledge of transgene insertion site. PCR primers were designed to amplify conserved T-DNA segments of the 35S promoter, OCS terminator, and NPTII kanamycin resistance gene in the pHellsgate-8 RNAi construct for the Gossypium hirsutum phytochrome A1 gene. Using an optimized multiplexed reaction mixture and an amplification program of only 10 cycles we could discriminate between transgene homozygous and hemizygous cotton control DNA samples based on PCR product peak characteristics gathered by capillary electrophoresis. The protocol was refined by evaluating segregating transgenic progeny from nine BC1S1 populations derived from crosses between the transgenic cotton parent ‘E-1-7-6’ and other cotton cultivars. OCS PCR product peak height and peak area, normalized by amplification of the native cotton gene GhUBC1, revealed clear bimodal distributions of OCS product characteristics for each BC1S1 population indicating the presence of homozygous and hemizygous clusters which was further confirmed via K-means clustering. BC1S1 plants identified as homozygous or hemizygous were self-fertilized to produce BC1S2 progeny. For the homozygous class, 19/20 BC1S2 families confirmed the homozygous BC1S1 prediction while 21/21 BC1S2 families confirmed the hemizygous prediction of the original parent. This relatively simple protocol provides a reliable, rapid, and high-throughput way of evaluating segregating transgenic populations using methods and equipment common to crop molecular breeding labs.

Maternal Haploids Are Preferentially Induced by CENH3-tailswap Transgenic Complementation in Maize.

Doubled haploid plants are invaluable breeding tools but many crop species are recalcitrant to available haploid induction techniques. To test if haploid inducer lines can be engineered into crops, CENH3−∕− and CENH3:RNAi lines were complemented by AcGREEN-tailswap-CENH3 or AcGREEN-CENH3 transgenes. Haploid induction rates were determined following testcrosses to wild-type plants after independently controlling for inducer parent sex and transgene zygosity. CENH3 fusion proteins were localized to centromeres and did not cause vegetative defects or male sterility. CENH3:RNAi lines did not demonstrate consistent knockdown and rarely produced haploids. In contrast, many of the complemented CENH3−∕− lines produced haploids at low frequencies. The rate of gynogenic haploid induction reached a maximum of 3.6% in several hemizygous individuals when backcrossed as males. These results demonstrate that CENH3-tailswap transgenes can be used to engineer in vivo haploid induction systems into maize plants.

P13 – 2298: PCDH19 related epileptic encephalopathy in a 9-year-old girl – Case report

Introduction Mutation in PCDH 19 gene, encoding prothocadherin 19 on chromosome Xq22, results in an epileptic syndrome of seizure onset in infancy, mild to severe intellectual impairment with or without autistic features. This disorder demonstrates an unusual pattern of X-linked inheritance, affecting heterozygous female but sparing hemizygous male individuals. The underlying responsible mechanism is considered to be a “cellular interference”. There is a wide clinical spectrum of seizures, generally starting in infancy or early childhood. A portion of patients manifests a phenotype resembling a Dravet syndrome. The seizures mostly occur in brief clusters even at mild to moderately elevated temperature. In initial course of the disease the seizures become relatively resistant to antiepileptic drugs. However, as the disease progresses, the frequency of seizures and their pharmacoresistance tends to decrease. There may be behavioral difficulties such as autistic, obsessive or aggressive features. Objective is to show the clinical features of PCDH19 related epilepsy in a 9-year-old girl with a focus on early disease characteristics and treatment efficacy. Patient and methods A 9-year-old girl from early childhood suffering and treated of resistant epilepsy that was clinically presented with a series of focal motor seizures accompanied by fear and screaming. Repeated interictal waking and sleeping EEGs, as well as high resolution brain MRI (3T) was normal. Analysis of cerebrospinal fluid excluded inflammatory diseases of the CNS. The rare metabolic diseases with epileptic seizures were excluded by metabolic tests. After the first seizure phenobarbital was recommended, and after recurrence there was a therapy with multiple combinations of various antiepileptic drugs, none of which was effective. Further treatment included a genetic analysis which found mutation PCDH19Xq22.1. The therapy included perampanel with topiramate. Conclusion The authors have the intention to warn to this epileptic encephalopathy that affects only female children and emphasize its importance in the differential diagnosis of uncontrolled epileptic syndromes associated with febrile conditions.

Paternal RHD zygosity determination in Tunisians: evaluation of three molecular tests.

The choice of a molecular test for first intention determination of paternal RHD zygosity, before entering into invasive diagnostics, is important for the management of pregnancies at risk of haemolytic disease of the foetus and newborn related to anti-RhD. RHD zygosity was evaluated in 370 RH:1 Tunisian donors by polymerase chain reaction - sequence-specific polymorphism (PCR-SSP) analysis and polymerase chain reaction - restriction fragment length polymorphism (PCR-RFLP) amplification of hybrid Rhesus box and by real time quantitative polymerase chain reaction (RQ-PCR) specific for RHD exon 5. To evaluate the accuracy of molecular tests in the cases of discordant results, the ten exons of RHD and Rhesus boxes were amplified by PCR and sequenced. Molecular investigations revealed that our 370 donors comprise 193 dizygous and 145 hemizygous individuals and 32 subjects whose zygosity remains unknown. Positive predictive values were higher than 99% for all the methods, reaching 100% for RQ-PCR. Negative predictive values were 83.24%, 87.27% and 98% for PCR-SSP, PCR-RFLP and RQ-PCR respectively. This study also revealed 19 novel Rhesus box polymorphisms and three novel RHD alleles: RHD(Trp185Stop), RHD(Ala176Thr) and RHD(Ile342Ile). RQ-PCR is the most convenient method for first intention determination of paternal RHD zygosity in Tunisians. However, taking into account positive and negative predictive values, PCR-RFLP could be an alternative despite the heterogeneity of Rhesus boxes and the complexity of RHD.

Toll‐like receptor 7 rs179008/Gln11Leu gene variants in chronic hepatitis C virus infection

Hepatitis C virus (HCV) infection affects an estimated 3% of the world's population. The natural outcome of infection and the natural course of disease are highly variable. Sensing of viral single-stranded RNA (ssRNA) by Toll-like receptor 7 (TLR7) is likely involved in early pathogen detection and host response to viral infections. This study analyzed epidemiological and clinical data from 136 patients with HCV infection with regard to rs179008/Gln11Leu, a non-synonymous polymorphism within exon 3 of the X-linked TLR7 gene, the variant allele of which is suggested to code for a functionally impaired protein. Allele-specific transcript quantification (ASTQ) analyses in heterozygous females revealed individual skewed mosaicism in peripheral blood mononuclear cells (PBMCs). Thus, analyses were restricted to homo- and hemizygous individuals. Among the clinical and histological parameters studied, the variant allele T was found to be solely associated with the presence of portal lymphoid aggregates. Whereas hepatic viral load and expression of genes known to be induced in chronic HCV infection were not found to differ in patients with wild-type or variant TLR7 rs179008 genotype, significant lower gene expression of interleukin-29 (IL-29)/lambda(1) interferon (IFN-λ(1)) and both of its receptor subunits was found for T homo- and hemizygous patients. Irrespective of the minor differences in disease phenotype including hepatic viral load, natural, and alpha interferon (IFN-α)-mediated outcome of infection, and disease activity and progression, the significant differences in hepatic IL-29/IFN-λ(1) and IFN-λ receptor gene expression between TLR7 rs179008 T and A allele patients might have implications for responsiveness to future IFN-λ-based approaches.

No Need to Discriminate? Reproductive Diploid Males in a Parasitoid with Complementary Sex Determination

Diploid males in hymenopterans are generally either inviable or sterile, thus imposing a severe genetic load on populations. In species with the widespread single locus complementary sex determination (sl-CSD), sex depends on the genotype at one single locus with multiple alleles. Haploid (hemizygous) individuals are always males. Diploid individuals develop into females when heterozygous and into males when homozygous at the sex determining locus. Our comparison of the mating and reproductive success of haploid and diploid males revealed that diploid males of the braconid parasitoid Cotesia glomerata sire viable and fertile diploid daughters. Females mated to diploid males, however, produced fewer daughters than females mated to haploid males. Nevertheless, females did not discriminate against diploid males as mating partners. Diploid males initiated courtship display sooner than haploid males and were larger in body size. Although in most species so far examined diploid males were recognized as genetic dead ends, we present a second example of a species with sl-CSD and commonly occurring functionally reproductive diploid males. Our study suggests that functionally reproductive diploid males might not be as rare as hitherto assumed. We argue that the frequent occurrence of inbreeding in combination with imperfect behavioural adaptations towards its avoidance promote the evolution of diploid male fertility.

Sex determination and inbreeding depression in an ant with regular sib-mating

Haplodiploidy is one of the most widespread mechanisms of sex determination in animals. In many Hymenoptera, including all hitherto investigated social species, diploid individuals, which are heterozygous at the sex locus, develop as females, whereas haploid, hemizygous individuals develop as males (single-locus complementary sex determination, sl-CSD). Inbreeding leads to homozygosity at the sex locus, resulting in the production of diploid males, which are usually sterile and constitute a considerable fitness cost. Nevertheless, regular inbreeding without diploid male production is known from several solitary wasps, suggesting that in these species sex is not determined by sl-CSD but alternative mechanisms. Here, we examine sex determination in an ant with regular inbreeding, Cardiocondyla obscurior. The almost complete absence of diploid males after 10 generations of brother-sister mating in the laboratory documents for the first time the absence of sl-CSD and CSD with two or a few unlinked sex loci in a species of social Hymenoptera. Queens, which mated with a brother, appeared to decrease the number of males in their brood, as expected from the relatedness relationships under inbreeding. In contrast, some colonies began to show signs of an inbreeding depression after several generations of sib-mating, such as shortened queen life span, higher brood mortality, and a shift to more male-biased sex ratios in some colonies, presumably due to lower insemination capability of sperm.

Immunoquantification of alpha-galactosidase: evaluation for the diagnosis of Fabry disease.

Fabry disease is an X-linked inborn error of glycosphingolipid catabolism resulting from a deficiency of the lysosomal exoglycohydrolase, alpha-galactosidase. Enzyme replacement therapy is currently available for Fabry disease, but early diagnosis before the onset of irreversible pathology will be mandatory for successful treatment. Presymptomatic detection would be possible through the use of a newborn-screening program. We report on the use of sensitive assays for the measurement of alpha-galactosidase protein and activity and for the protein saposin C, which are diagnostic markers for Fabry disease. Two sensitive immunoassays for the measurement of alpha-galactosidase activity and protein were used to determine the concentrations of alpha-galactosidase in dried filter-paper blood spots and plasma samples from control patients and patients with a lysosomal storage disorder (LSD). Fabry hemizygous individuals were clearly identified from control populations by decreases in both alpha-galactosidase activity and protein. Fabry heterozygotes generally fell between the hemizygotes and controls. Including the measurement of saposin C enabled differentiation between Fabry heterozygotes and controls. In blood spots, all Fabry individuals could be distinguished from control blood spots as well as from 16 other LSD patients. The determination of alpha-galactosidase activity or protein in dried filter-paper blood spots could be used for the diagnosis of Fabry patients. With further validation, these assays could be used for the identification of Fabry patients in newborn-screening programs and may also be suitable for screening high-risk populations.

Additive transgene expression and genetic introgression in multiple green-fluorescent protein transgenic crop x weed hybrid generations.

The level of transgene expression in crop x weed hybrids and the degree to which crop-specific genes are integrated into hybrid populations are important factors in assessing the potential ecological and agricultural risks of gene flow associated with genetic engineering. The average transgene zygosity and genetic structure of transgenic hybrid populations change with the progression of generations, and the green fluorescent protein (GFP) transgene is an ideal marker to quantify transgene expression in advancing populations. The homozygous T(1) single-locus insert GFP/ Bacillus thuringiensis (Bt) transgenic canola ( Brassica napus, cv Westar) with two copies of the transgene fluoresced twice as much as hemizygous individuals with only one copy of the transgene. These data indicate that the expression of the GFP gene was additive, and fluorescence could be used to determine zygosity status. Several hybrid generations (BC(1)F(1), BC(2)F(1)) were produced by backcrossing various GFP/Bt transgenic canola ( B. napus, cv Westar) and birdseed rape ( Brassica rapa) hybrid generations onto B. rapa. Intercrossed generations (BC(2)F(2) Bulk) were generated by crossing BC(2)F(1) individuals in the presence of a pollinating insect ( Musca domestica L.). The ploidy of plants in the BC(2)F(2) Bulk hybrid generation was identical to the weedy parental species, B. rapa. AFLP analysis was used to quantify the degree of B. napus introgression into multiple backcross hybrid generations with B. rapa. The F(1) hybrid generations contained 95-97% of the B. napus-specific AFLP markers, and each successive backcross generation demonstrated a reduction of markers resulting in the 15-29% presence in the BC(2)F(2) Bulk population. Average fluorescence of each successive hybrid generation was analyzed, and homozygous canola lines and hybrid populations that contained individuals homozygous for GFP (BC(2)F(2) Bulk) demonstrated significantly higher fluorescence than hemizygous hybrid generations (F(1), BC(1)F(1) and BC(2)F(1)). These data demonstrate that the formation of homozygous individuals within hybrid populations increases the average level of transgene expression as generations progress. This phenomenon must be considered in the development of risk-management strategies.

COMPLEMENTARY SEX DETERMINATION IN HYMENOPTERAN PARASITOIDS AND ITS IMPLICATIONS FOR BIOLOGICAL CONTROL

Abstract In haplodiploid Hymenoptera, unfertilized eggs produce haploid males while fertilized eggs lead to diploid females under most circumstances. Diploid males can also be produced from fertilization under a system of sex determination known as complementary sex determination (CSD). Under single‐locus CSD, sex is determined by multiple alleles at a single sex locus. Individuals heterozygous at the sex locus are female while hemizygous and homozygous individuals develop as haploid and diploid males, respectively. In multiple‐locus CSD, two or more loci, each with two or more alleles, determine sex. Diploid individuals are female if one or more sex loci are heterozygous, while a diploid is male only if homozygous at all sex loci. Diploid males are known to occur in 43 hymenopteran species and single‐locus CSD has been demonstrated in 22 of these species. Diploid males are either developmentally inviable or sterile, so their production constitutes a genetic load. Because diploid male production is more likely under inbreeding, CSD is a form of inbreeding depression. It is crucial to preserve the diversity of sex alleles and reduce the loss of genetic variation in biological control. In the parasitoid species with single‐locus CSD, certain precautionary procedures can prevent negative effects of single‐locus CSD on biological control.

Impact of genetic engineering on the understanding of spermatogenesis.

To date, about 100 genes have been found, by genetic engineering, to be implicated in spermatogenesis. Primordial germ cells, spermatogonia, spermatocytes I and elongating spermatids are particularly sensitive. Transgenic and knockout mice permit an approach to be made to the question of genetic factors involved in DNA damage repair, thermal injury, sperm chromatin compaction and sex-specific recombination. Knockout mice reveal unexpected functional redundancies of testis-specific genes. This review considers how functional divergences can exist among homologous genes from different species, and to what extent the phenotypes of knockout mice can be similar to those from spontaneous mutations. Additional anomalies in reproductive function have frequently been found in these mice, as were found factors leading to tumour susceptibility and/or various diseases. Finally, knockout mice remind us that, in nearly all cases, hemizygous individuals retain a fertility and a wild-type sperm phenotype, although half of the spermatozoa share a genetic defect. The findings strongly emphasize the importance of understanding epidemiology in male infertility, to identify hereditary forms of impaired spermatogenesis, and to create DNA and pathological germ cell banks.

Single-Locus Complementary Sex Determination in the Ichneumonid Venturia Canescens (Gravenhorst) (Hymenoptera)

Some Hymenoptera have single locus Complementary Sex Determination (sl-CSD); heterozygous individuals are female (diploid fertilised eggs) and hemizygous individuals are male (haploid unfertilised eggs). Through inbreeding homozygous diploids can arise which develop into males that are often sterile and sometimes inviable. The phylogenetic distribution of sl-CSD is unclear. In this study, we used inbred crosses to demonstrate that the parasitoid wasp Venturia canescens (Gravenhorst) has sl-CSD. Diploid males were detected through heterozygosity at a marker locus (Virus Like Particle protein). They were fully viable at 20°, 25° and 30°C as the sex ratios of inbred crosses did not deviate from controls after adjustment for diploid male frequency. These results further confirm the existence of sl-CSD in the family Ichneumonidae.

Phospholipase D activity is altered in X-linked adrenoleukodystrophy heterozygous carriers, but not in hemizygous patients

Abnormalities in levels of choline and its metabolites have been reported in the lesions of brains of X-linked adrenoleukodystrophy (X-ALD) patients. We have examined the turnover of the major choline-containing phospholipid, phosphatidylcholine (PtdCho), in fibroblasts from hemizygous X-ALD, heterozygous X-ALD, Zellweger syndrome (ZW), and male and female control individuals to assess possible alterations in PtdCho metabolism mediated by activation of protein kinase C (PKC). Hydrolysis of PtdCho by phospholipase D (PLD) and resynthesis of PtdCho from labeled choline were stimulated 2- to 4-fold by PKC activation with the phorbol ester, 4β-12- O-tetradecanoylphorbol-13-acetate (β-TPA), in all cells except those from heterozygous X-ALD individuals. No differences in quantity or intracellular distribution of PKC activity, PKC isoforms by Western blot analysis, or of the PKC substrate, myristoylated alanine-rich C kinase substrate (MARCKS), were apparent in any of the cells. Thus, altered PtdCho metabolism was not directly linked to either of these inherited defects that result in abnormal peroxisomal functions. Further, altered responsiveness of PLD in X-ALD heterozygotes was independent of changes in PKC and MARCKS.

Different missense mutations in histidine-108 of lysosomal acid lipase cause cholesteryl ester storage disease in unrelated compound heterozygous and hemizygous individuals.

Cholesteryl ester storage disease (CESD) and Wolman disease (WD) are both autosomal recessive disorders associated with reduced activity of lysosomal acid lipase (LAL), that leads to the tissue accumulation of cholesteryl esters in endosomes and lysosomes. WD is caused by genetic defects of LAL that leave no residual enzymatic activity, while in CESD patients a residual LAL activity can be identified. We have analyzed the LAL cDNA in three CESD patients from two nonrelated families and identified the mutations responsible for the disease. The associated genetic defects characterized revealed compound heterozygosity for a splice defect leading to skipping of exon 8, due to a G-->A transition at position -1 of the exon 8 splice donor site, and a point mutation leading to a Hisl08Pro change (CAT-->CCT) in two patients (siblings) with mild CESD phenotype. A further CESD patient was hemizygous for a His108-->Arg missense mutation (CAT-->CGT) in combination with a partial deletion of the LAL gene and was affected more severely. Expression of the LAL enzymes with the His108-->Pro and His108-->Arg mutation in insect cells revealed residual enzymatic activities of 4.6% versus 2.7%, respectively, compared with controls. Therefore, His108 seems to play a crucial role in folding or catalytic activity of the lysosomal acid lipase. This is the first description of two different, naturally occurring mutations involving the same amino acid residue in the lysosomal acid lipase in unrelated CESD patients. Moreover, our results demonstrate that the variable manifestation of CESD can be explained by mutation-dependent, variable inactivation of the LAL enzyme.