Deficiency Alleles Research Articles

The ethnogeographic variability of genetic factors underlying G6PD deficiency

Glucose-6-phosphate dehydrogenase (G6PD) deficiency caused by genetic variants in the G6PD gene, constitutes the most common enzymopathy worldwide, affecting approximately 5% of the global population. While carriers are mostly asymptomatic, they are at substantial risk of acute hemolytic anemia upon certain infections or exposure to various medications. As such, information about G6PD activity status in a given patient can constitute an important parameter to guide clinical decision-making. Here, we leveraged whole genome sequencing data from 142,069 unrelated individuals across seven human populations to provide a global comprehensive map of G6PD variability. By integrating established functional classifications with stringent computational predictions using 13 partly orthogonal algorithms for uncharacterized and novel variants, we reveal the large extent of ethnogeographic variability in G6PD deficiency and highlight its population-specific genetic composition. Overall, estimated disease prevalence in males ranged between 12.2% in Africans, 2.7–3.5% across Asia and 2.1% in Middle Easterners to < 0.3% in Europeans, Finnish and Amish. In Africans, the major deficient alleles were A-202A/376 G (minor allele frequency 11.6%) and A-968 C/376 G (0.5%). In contrast, G6PD deficiency in Middle Easterners was primarily due to the Mediterranean allele (1.3%) and the population-specific Cairo variant (0.4%). In South Asia, the most prevalent deficient alleles were Mediterranean (1.7%), Kerala (1.1%), Gond (0.9%) and Orissa (0.2%), whereas in East Asian populations the Canton (1.1%), Kaiping (0.7%) and Viangchan (0.3%) variants were predominant. Combined, our analyses provide a large dataset of G6PD variability across major ethnogeographic groups and can instruct population-specific genotyping strategies to optimize genetically guided therapeutic interventions.

Alpha-1 antitrypsin deficiency hidden in allegedly normal variants

Introduction Rare variants of Alpha-1 antitrypsin (AAT) deficiency (AATD) have been described by the Spanish registry of patients with AATD. The great majority of these rare variants are Mmalton alleles and many recent case series of them have been identified in the Canary Islands. The objective of this study was to analyze the distribution of Mmalton mutations in a Canarian population previously studied for the most common deficient alleles, namely PI*S (S) and PI*Z (Z), with PI*M (M) being the normal variant. Methods A cross-sectional study of 648 patients with allergic asthma was carried out. Mmalton mutation of the SERPINA1 gene was assayed by real-time PCR. Results Of the 648 patients, 3 (0.46%) were carriers of a Mmalton allele. All of them had low levels of AAT (53.9 mg/dL, 90 mg/dL, and 61 mg/dL, respectively) and were asymptomatic, showing normal lung function, radiological images, and levels of hepatic transaminases. Conclusion In conclusion, although the most frequent AATD genotypes are Z and S alleles, it is important to consider other rare variants, particularly when low AAT serum levels are observed. Although individuals with the Mmalton mutation usually have a heterogenous clinical presentation and very low levels of AAT, all the patients in this study were asymptomatic.

An Evaluation of the Diagnostic Accuracy of a Panel of Variants in DPYD and a Single Variant in ENOSF1 for Predicting Common Capecitabine Related Toxicities.

Simple Summary5-Fluorouracil (5-FU) is a chemotherapy drug that can cause severe toxicity in some patients. A protein, an action molecule in our cells, called dihydropyrimidine dehydrogenase, or DPD for short, is important in clearing 5-FU from the body. Some people have versions of DPD that do not clear 5-FU very well. This causes active drug to stay in the body too long, causing toxicities such as diarrhoea or low levels of blood cells important for fighting infections. Current guidelines identify four sequence changes in the gene that encodes DPD with high level evidence of an impact on protein activity. Our study aims to calculate the frequency of a set of variants identified in patients with DPD deficiency in patients that were part of a clinical trial called QUASAR 2. We go on to test how well the DPD deficiency variants and a set of common variants previously shown to be associated with 5-FU toxicity can predict a person’s risk of 5-Fluorouracil induced toxicity. Our research is important for working out the best way to identify patients at risk of toxicity so high risk patients can be given lower doses of 5-Fluorouracil or be treated with a different drug all together.Efficacy of 5-Fluorouracil (5-FU)-based chemotherapy is limited by significant toxicity. Tests based upon variants in the Clinical Pharmacogenetics Implementation Consortium (CPIC) guidelines with high level evidence of a link to dihydropyrimidine dehydrogenase (DPD) phenotype and 5-FU toxicity are available to identify patients at high risk of severe adverse events (AEs). We previously reported associations between rs1213215, rs2612091, and NM_000110.3:c.1906-14763G>A (rs12022243) and capecitabine induced toxicity in clinical trial QUASAR 2. We also identified patients with DPD deficiency alleles NM_000110.3: c.1905+1G>A, NM_000110.3: c.2846C>T, NM_000110.3:c.1679T>G and NM_000110.3:c.1651G>A. We have now assessed the frequency of thirteen additional DPYD deficiency variants in 888 patients from the QUASAR 2 clinical trial. We also compared the area under the curve (AUC)—a measure of diagnostic accuracy—of the high-level evidence variants from the CPIC guidelines plus and minus additional DPYD deficiency variants and or common variants associated with 5-FU toxicity. Including additional DPYD deficiency variants retained good diagnostic accuracy for serious adverse events (AEs) and improved sensitivity for predicting grade 4 haematological toxicities (sensitivity 0.75, specificity 0.94) but the improvement in AUC for this toxicity was not significant. Larger datasets will be required to determine the benefit of including additional DPYD deficiency variants not observed here. Genotyping two common alleles statistically significantly improves AUC for prediction of risk of HFS and may be clinically useful (AUC difference 0.177, sensitivity 0.84, specificity 0.31).

Known Mutations at the Cause of Alpha-1 Antitrypsin Deficiency an Updated Overview of SERPINA1 Variation Spectrum.

Alpha-1-Antitrypsin deficiency (AATD), caused by SERPINA1 mutations, is one of the most prevalent Mendelian disorders among individuals of European descend. However, this condition, which is characterized by reduced serum levels of alpha-1-antitrypsin (AAT) and associated with increased risks of pulmonary emphysema and liver disease in both children and adults, remains frequently underdiagnosed. AATD clinical manifestations are often correlated with two pathogenic variants, the Z allele (p.Glu342Lys) and the S allele (p.Glu264Val), which can be combined in severe ZZ or moderate SZ risk genotypes. Yet, screenings of AATD cases and large sequencing efforts carried out in both control and disease populations are disclosing outstanding numbers of rare SERPINA1 variants (>500), including many pathogenic and other likely deleterious mutations. Generally speaking, pathogenic variants can be subdivided into either loss- or gain-of-function according to their pathophysiological effects. In AATD, the loss-of-function is correlated with an uncontrolled activity of elastase by its natural inhibitor, the AAT. This phenomenon can result from the absence of circulating AAT (null alleles), poor AAT secretion from hepatocytes (deficiency alleles) or even from a modified inhibitory activity (dysfunctional alleles). On the other hand, the gain-of-function is connected with the formation of AAT polymers and their switching on of cellular stress and inflammatory responses (deficiency alleles). Less frequently, the gain-of-function is related to a modified protease affinity (dysfunctional alleles). Here, we revisit SERPINA1 mutation spectrum, its origins and population history with a greater emphasis on variants fitting the aforementioned processes of AATD pathogenesis. Those were selected based on their clinical significance and wider geographic distribution. Moreover, we also provide some directions for future studies of AATD clinically heterogeneity and comprehensive diagnosis.

α1-Antitrypsin deficiency and the risk of COVID-19: an urgent call to action

α1-Antitrypsin deficiency and the risk of COVID-19: an urgent call to action

The Clinical Utility of Determining the Allelic Background of Mutations Causing Alpha-1 Antitrypsin Deficiency: The Case with the Null Variant Q0(Mattawa)/Q0(Ourém).

Alpha-1 antitrypsin deficiency (AATD) is caused by genetic variants in the SERPINA1 gene conferring risk of developing emphysema. The clinical expression of AATD-related emphysema mostly occurs in carriers of 2 deficient alleles. By DNA sequencing of SERPINA1, numerous rare variants have been identified. Clarifying whether 2 mutations observed in 1 patient are on the same or distinct alleles has obvious clinical implications. We studied 7 carriers of a rare variant, Leu353Phe_fsTer24, known to lead to undetectable serum levels of AAT. Two of them were also carriers of the S or Z allele. We developed an allele-specific DNA sequencing method to characterize the allelic background of the Leu353Phe_fsTer24 variant. The Leu353Phe_fsTer24 variant was transmitted on the same allele as the M3 variant (E376D) in all patients. This mutation is thus named Q0Ourém on the conventional PI system. We demonstrated that individuals harboring the E264V (S) and E342K (Z) mutations had them on distinct alleles from Q0Ourém and are, thus, compound heterozygotes. The 7 Q0Ourém carriers had AAT levels ranging from 0.18g/l to 0.82g/l. The lowest AAT serum levels were observed in compound heterozygotes (S/Q0Ourém and Z/Q0Ourém) suggesting higher risk of developing emphysema. For the 7 patients, Leu353Phe_fsTer24 is transmitted on the M3 background and they are, thus, carriers of the Q0Ourém allele. Allele-specific DNA sequencing was useful to distinguish 1 or 2 deficient alleles in carriers of 2 mutations. In rare cases, this method is important to understand the clinical significance of genetic variants found in SERPINA1.

Serum Levels of Glutamate-Pyruvate Transaminase, Glutamate-Oxaloacetate Transaminase and Gamma-Glutamyl Transferase in 1494 Patients with Various Genotypes for the Alpha-1 Antitrypsin Gene

Background: Patients with liver disease associated with alpha-1 antitrypsin deficiency (AATD) are homozygous for the Z mutation, leading to chronic liver damage. Objective: To assess the serum levels of glutamate-oxaloacetate transaminase (GOT), glutamate-pyruvate transaminase (GPT), and gamma-glutamyl transpeptidase (GGT) in patients with different genotypes for the alpha-1 antitrypsin (AAT) gene. Methods: Patients (n = 1494) underwent genotyping of the SERPINA1 gene, together with a determination of AAT and GOT and GPT and GGT transaminase levels. Patients with a deficient allele (n = 476) and with a normal genotype were compared. Results: A statistically significant association was found between deficient genotypes and GOT (p < 0.0003), GPT (p < 0.002), and GGT (p < 0.006). Comparing GOT levels in patients with PI*Z deficient variant versus those with normal genotype, an odds ratio (OR) of 2.72 (CI: 1.5–4.87) (p < 0.0005) was obtained. This finding was replicated with the PI*Z allele and the GPT values (OR = 2.31; CI: 1.45–3.67; p < 0.0003). In addition, a statistically significant association was found between liver enzymes and AAT values. Conclusion: The PI*Z allele seemed to be a risk factor for the development of liver damage. AAT deficient genotypes were associated with GOT, GPT, and GGT altered values. Low AAT levels were associated with high GPT and GGT levels.

Pitfalls and challenges associated with phenoconversion in forensic toxcicology

PurposeIn recent years, several publications have demonstrated the interest and the usefulness of pharmacogenetics in forensic toxicology. However, this approach remains namely focused on DNA-based phenotype, which may potentially lead to misinterpretation. Other determinants such as co-medication or physiological parameters may also impact the phenotype. This article aims to highlight the importance of considering such determinants in forensic toxicology, through the original case of a heroin-related fatality. MethodEthanol concentration determination and toxicological screening were performed using gas chromatography with flame ionization detection, liquid chromatography with diode array detection and gas chromatography with mass spectrometry detection. CYP2C19 and CYP2D6 genotypes were determined by Taqman® real-time PCR analyses. ResultsFemoral blood analyses revealed the presence of ethanol, morphine, codeine, venlafaxine (VEN), O-desmethylvenlafaxine (ODV) and N-desmethylvenlafaxine (NDV), paroxetine, and risperidone. 6-acetylmorphine was also identified in urine. VEN, paroxetine and risperidone were quantified at supra-therapeutic or toxic blood concentrations. NDV was not quantified. The metabolic ratio of VEN (ODV to VEN) was exceptionally low (about 0.7). Pharmacogenetics testing showed that the patient was heterozygous for the CYP2C19*2 loss-of-function allele, which predict an intermediate metabolism for CYP2C19. None of the deficient CYP2D6 alleles investigated were identified. Those results suggest an extensive CYP2D6-metabolism phenotype. ConclusionA discrepancy was seen between the results of the genomic evaluation and the observed metabolic ratio of VEN. This tends to exclude a genetic origin and lead us to formulate other hypotheses, such as phenoconversion that may have been induced by drug interaction involving patients’ regular medications. Phenoconversion is as a complex phenomenon that leads to genotype-phenotype mismatch without any genetic abnormality particularly described for cytochromes P450 2D6 and 2C19. Although transient, phenoconversion can have a significant impact on the analysis and interpretation of genotype-focused clinical outcomes correlation and in forensic toxicology conclusions.

Glucose-6-phosphate dehydrogenase deficiency in the Han Chinese population: molecular characterization and genotype\u2013phenotype association throughout an activity distribution

Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common hereditary disorder in China. The existing prevalence and molecular epidemiology of G6PD deficiency in China were geographically limited. In this study, the spectrum of G6PD gene mutations was well characterized in a large and diverse population all over the country; and the correlation of genotype and enzyme activity phenotype was explored for the first time. The results showed that the overall prevalence of G6PD deficiency in China was 2.10% at the national level. The top six common mutations were c.1388 G>A, c.1376 G>T, c.95 A>G, c.392 G>T, c.871 G>A and c.1024 C>T, accounting for more than 90% of G6PD deficient alleles. Compound mutation patterns were frequently observed in females with severe deficiency. The distribution of G6PD activities depended on the type of mutation patterns and genders. Hemizygote, homozygote, and compound heterozygote were predominantly associated with severe G6PD deficiency, whereas heterozygotes with single mutation mainly presented moderate enzyme deficiency. A significant gap between G6PD activities in hemizygous and normal males was observed, and yet, the overall distribution of that in females carrying missense mutations was a continuum from G6PD severely deficient to normal. This is the first report of discussing the association between G6PD genetic variants in the Chinese and enzyme activity phenotypes.

Ethnic differences in alpha-1 antitrypsin deficiency allele frequencies may partially explain national differences in COVID-19 fatality rates.

Infection rates, severity, and fatalities due to COVID‐19, the pandemic mediated by SARS‐CoV‐2, vary greatly between countries. With few exceptions, these are lower in East and Southeast Asian and Sub‐Saharan African countries compared with other regions. Epidemiological differences may reflect differences in border closures, lockdowns, and social distancing measures taken by each county, and by cultural differences, such as common use of face masks in East and Southeast Asian countries. The plasma serine protease inhibitor alpha‐1 antitrypsin was suggested to protect from COVID‐19 by inhibiting TMPRSS2, a cell surface serine protease essential for the SARS‐CoV‐2 cell entry. Here, we present evidence that population differences in alpha‐1 antitrypsin deficiency allele frequencies may partially explain national differences in the COVID‐19 epidemiology. Our study compared reported national estimates for the major alpha‐1 antitrypsin deficiency alleles PiZ and PiS (SERPINA1 rs28929474 and rs17580, respectively) with the Johns Hopkins University Coronavirus Resource Center dataset. We found a significant positive correlation (R = .54, P = 1.98e−6) between the combined frequencies of the alpha‐1 antitrypsin PiZ and PiS deficiency alleles in 67 countries and their reported COVID‐19 mortality rates. Our observations suggest that alpha‐1 antitrypsin deficiency alleles may contribute to national differences in COVID‐19 infection, severity, and mortality rates. Population‐wide screening for carriers of alpha‐1 antitrypsin deficiency alleles should be considered for prioritizing individuals for stricter social distancing measures and for receiving a SARS‐CoV‐2 vaccine once it becomes available.

Abstract 3432: Zinc metallochaperones for mutant p53 reactivation in cancer therapeutics

Abstract The p53 transcription factor functions as one of cancer's most potent tumor suppressors and is the most frequently mutated gene in human cancer. Restoration of wild type structure and function (so-called reactivation) of the high levels of the deficient mutant p53 protein with a small molecule is a highly sought after goal in anti-cancer drug development. We discovered a new class of small molecule zinc chelators named zinc metallochaperones (ZMCs) that reactivate zinc deficient mutant p53 through a novel mechanism by refolding p53 protein and inducing a p53 mediated apoptotic program. We identified the role of cellular zinc homeostasis as the OFF switch in ZMC pharmacodynamics indicating that a brief period of p53 mutant reactivation is sufficient for on-target efficacy. We conducted pre-clinical pharmacokinetic (PK), pharmacodymanic (PD) and efficacy studies in the murine pancreatic cancer (KPC) and BRCA1-deficient breast cancer models and found that ZMC1 and its new formulation of the drug in complex with zinc improved survival and inhibited tumor growth specifically for the zinc deficient allele. Using the BRCA1-deficient breast cancer model, we observed a highly synergistic effect of ZMC1 and the PARP inhibitor olaparib. Olaparib is now approved for the treatment of advanced BRCA1/2 mutant ovarian cancers, and recent clinical data also support its efficacy in BRCA1/2 mutant breast cancers. However acquired resistance to PARP inhibitors is inevitable in most advanced cancers. We investigated the ZMC therapeutic efficacy and found that the cell lines derived from olaparib resistant tumors are still sensitive to ZMC1 treatment. We are currently investigating the mechanism and testing the combination in vivo. In addition, we explored synergistic combinations of ZMC1 and other chemotherapy drugs based on its mechanism of action including ZMC1 with MDM2 antagonists and BCL2 antagonists. Overall, our findings indicate that the mutant p53 reactivational activity of ZMCs is governed by a unique ON/OFF switch mechanism that allows the compounds to be effective with a brief exposure to avoid the off target toxicities. The targeted and synergistic combinatorial treatments represent a significant departure from the traditional paradigm for developing a targeted molecular therapeutic in cancer. Citation Format: Xin Yu, Bing Na, Saif Zaman, Tracy Withers, John Gilleran, Alan J. Blayney, Anthony F. Bencivenga, Adam R. Blanden, Yue Liu, David A. Boothman, Stewart N. Loh, S. David Kimball, Shridar Ganesan, Darren R. Carpizo. Zinc metallochaperones for mutant p53 reactivation in cancer therapeutics [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3432.

Frequency of G6PD Deficiency in General Population at District Bannu, Khyber Pakhtunkhwa, Pakistan

Background: Glucose-6-phosphate dehydrogenase (G6PD) deficiency is an X-linked disease and it is a common enzymaticdisease of RBCs in humans X linked recessive condition are more common in males than females. The said deficiency leads toaffecting &gt;400 million people worldwide Individuals, normally males, with deficient alleles are helpless to neonatal jaundice andintense hemolytic anemia, usually during disease, after treatment with specific medications or subsequent to eating Fava beans..Objective: To measure the frequency of Glucose-6-Phosphate-Dehydrogenase deficiency in general population at district Bannu,Khyber Pakhtunkhwa Pakistan.Material and Methods: This cross-sectional descriptive study was conducted on 500 human subjects, who were referred forG6PD assay, in Samad clinical Laboratory, District Bannu Khyber Pakhtunkhwa, Pakistan, from July 2018 to July 2019. 500ccvenous whole blood was collected in EDTA containing vial, for G6PD Test. (Span Diagnostic S.A.R.L, France). Patients of any Age,Sex &amp; area having fever, hematuria, headache, visible jaundice, family history, malaria and anemia were included in this study,while patients suffering from renal disease, any malignancy &amp; not willing persons were excluded.Results: Out of the total 500 hundred, 370 (74%) were males and females were 130 (26%). Total 64/500 (12.8 %) were G6PDdeficient, with 55 male and 09 were female. Malaria positive with G6PD deficiency were 13/64 (20.31%), with 12 males and onefemale. Statistically significant difference among each group (p= 0.0022) was noted. Mean age of the G6PD deficient persons was(2.8 ± 1.03) years. Anemia was graded as Hemoglobin less than 11.5g/dl was taken as anemia. Severe anemia as Hb &lt; 7 g/dl,moderate anemia as Hb ranged between 7-10 g/dl and mild if Hb ranged between 10-11.5 g/dl.Among 370 males, 86 persons had hemoglobin of less than 11.5 g/dl, 42 had hemoglobin 7-10 g/dl and 7 patients had hemoglobinless than 7 g/dl, only 235 patients had hemoglobin more than 11.5, among 130 females, 33 patients had hemoglobin of less than11.5 g/dl, 17 patients had hemoglobin 7-10 g/dl and 05 patients had hemoglobin less than 7 g/dl, only 75 patients had hemoglobinmore than 11.5 g/dl.Conclusion: This study shows high frequency of G6PD Deficiency in district Bannu Khyber Pakhtunkhwa Pakistan especially veryhigh frequency in males than females.

New cis-Acting Variants in PI*S Background Produce Null Phenotypes Causing Alpha-1 Antitrypsin Deficiency.

Alpha-1 antitrypsin deficiency (AATD) is an inherited condition characterized by reduced levels of serum AAT due to mutations in the SERPINA1 (Serpin family A member 1) gene. The Pi*S (Glu264Val) is one of the most frequent deficient alleles of AATD, showing high incidence in the Iberian Peninsula. Herein, we describe two new alleles carrying an S mutation but producing a null phenotype: QOVigo and QOAachen. The new alleles were identified by sequencing the SERPINA1 gene in three patients who had lower AAT serum levels than expected for the initial genotype. These alleles are the result of combined mutations in cis in a PI*S allele. Sequencing detected the S mutation in cis with Tyr138Cys (S+Tyr138Cys) in two patients, whereas a third one had the S mutation in cis with Pro391Thr variant (S+Pro391Thr). When expressed in a cellular model, these variants caused strong AAT polymerization and very low AAT secretion to almost undetectable levels. The isoelectric focusing method for plasma AAT phenotyping did not show AAT protein encoded by the novel mutant alleles, behaving as null. We called these alleles PI*S-plus because the S variant was phased with another variant conferring more aggressive characteristics to the allele. The current data demonstrate that the clinical variability observed in AATD can be explained by additional genetic variation, such as dual cis-acting variants in the SERPINA1 gene. The possible existence of other unrevealed variants combined in the PI*S alleles should be considered to improve the genetic diagnosis of the patients.

Granularity of SERPINA1 alleles by DNA sequencing in CanCOLD.

DNA sequencing of the SERPINA1 gene to detect α1-antitrypsin (AAT) deficiency (AATD) may provide a better appreciation of the individual and cumulative impact of genetic variants on AAT serum levels and COPD phenotypes.AAT serum level and DNA sequencing of the coding regions of SERPINA1 were performed in 1359 participants of the Canadian Cohort Obstructive Lung Disease (CanCOLD) study. Clinical assessment for COPD included questionnaires, pulmonary function testing and computed tomography (CT) imaging. Phenotypes were tested for association with SERPINA1 genotypes collated into four groups: normal (MM), mild (MS and MI), intermediate (heterozygote MZ, non-S/non-Z/non-I, compound IS, and homozygote SS) and severe (ZZ and SZ) deficiency. Smoking strata and MZ-only analyses were also performed.34 genetic variants were identified including 25 missense mutations. Overall, 8.1% of alleles in this Canadian cohort were deficient and 15.5% of 1359 individuals were carriers of at least one deficient allele. Four AATD subjects were identified and had statistically lower diffusion capacity and greater CT-based emphysema. No COPD phenotypes were associated with mild and intermediate AATD in the overall cohort or stratified by smoking status. MZ heterozygotes had similar CT-based emphysema, but lowered diffusion capacity compared with normal and mild deficiency.In this Canadian population-based cohort, comprehensive genetic testing for AATD reveals a variety of deficient alleles affecting 15.5% of subjects. COPD phenotype was demonstrated in severe deficiency and MZ heterozygotes. This study shows the feasibility of implementing a diagnostic test for AATD using DNA sequencing in a large cohort.

Molecular diagnosis of alpha1-antitrypsin deficiency: A new method based on Luminex technology.

BackgroundAlpha1‐antitrypsin deficiency (AATD) is an under‐diagnosed hereditary disorder characterized by reduced serum levels of alpha1‐antitrypsin (AAT) and increased risk to develop lung and liver diseases at an early age. AAT is encoded by the highly polymorphic SERPINA1 gene. The most common deficiency alleles are S and Z, but more than 150 rare variants lead to low levels of the protein. To identify these pathological allelic variants, sequencing is required. Since traditional sequencing is expensive and time‐consuming, we evaluated the accuracy of A1AT Genotyping Test, a new diagnostic genotyping kit which allows to simultaneously identify and genotype 14 deficiency variants of the SERPINA1 gene based on Luminex technology.MethodsA total of 418 consecutive samples with AATD suspicion and submitted to the Italian Reference laboratory between January and April 2016 were analyzed both by applying the diagnostic algorithm currently in use, and by applying A1AT Genotyping Test.ResultsThe assay gave the following results: 101 samples (24.2%) were positive for at least one of the 14 deficiency variants, 316 (75.6%) were negative for all the variants analyzed. The identified mutations showed a 100% correlation with the results obtained with our diagnostic algorithm. Seventeen samples (4%) resulted negative for the assay but sequencing identified other rare pathological variants in SERPINA1 gene.ConclusionThe A1AT Genotyping Test assay was highly reliable and robust and allowed shorter diagnostic times. In few cases, it has been necessary to sequence the SERPINA1 gene to identify other rare mutations not included in the kit.

Protective Resistance by Human G6PD Enzyme Deficiency and Hemoglobin Variants Against Malaria and Natural Selection: Further Evidence from Review of New Studies

Main objective of this article is to review and evaluate recent red cell variant studies for protection against malaria and natural selection. Malaria is a parasitic disease highly widespread in tropical and subtropical regions of the world. It is also one of the leading causes of death worldwide and genes involved in malaria resistance are the most important for natural selection in human populations. Multiple red cell variants, which evolved probably to counter the lethal effects of malaria and confer protection against malaria through different mechanisms, show high frequencies in malaria endemic vulnerable populations. Different natural protective/resistance mechanisms including hampering of parasite growth, invasion related immunological responses or rapidly elimination of malaria parasite from the infected erythrocytes of host have briefly been discussed, evaluated, and reviewed. Conclusions drawn have been projected here. High frequency of inherited hemoglobin disorders including thalassemias, and red cell G6PD enzyme deficiency, which seemed to evolve simultaneously in relation to malaria, and high mortality caused by Plasmodium falciparum malaria in different vulnerable populations of tropical and subtropical parts of world, confirm that the natural selection is certainly operating against malaria in one way or another; and human population genetics have distinctly played a significant role in the co-evolution of host and malaria. The inverse relationship between sickle cell trait and G6PD deficiency and vice versa, revealed by allele frequencies distribution shown in our previous studies, is a testimony of disequilibrium, as sickle cell allele being replaced by G6PD deficiency allele in populations of central India. Positive natural selection plays a definite role against malaria for maintaining balance in high frequency endemic populations.

Factor XI Deficiency (FXID) alleles distribution in dairy cow population in Enrekang regency, South Sulawesi

Factor XI Deficiency (FXID) is a genetic defect due to an autosomal recessive allele in dairy cows, thus cause a large economic and productivity loss in livestock. The purpose of this study was to identify the distribution of recessive alleles of FXID in dairy cows in Enrekang regency using the PCR method. A total of 80 DNA samples were isolated from the FH dairy cow blood sample collected from the Enrekang dairy farms (50 samples from Cendana district and 30 samples from Anggeraja District). All DNA samples were amplified by the PCR technique. The identification of the FXID carrier allele was calculated based on the genotype and allele frequencies. Results showed that 1 of 50 samples from Cendana district was FXID heterozygous while none of 30 samples from Anggeraja district showed FXID alleles. This study concluded that there was a normal heterozygous (Ff) FXID carrier in dairy cow population in Enrekang Regency with the allele frequency was 1.25%.

Protein modeling to assess the pathogenicity of rare variants of SERPINA1 in patients suspected of having Alpha 1 Antitrypsin Deficiency

BackgroundAlpha 1 Antitrypsin (AAT) is a key serum proteinase inhibitor encoded by SERPINA1. Sequence variants of the gene can cause Alpha 1 Antitrypsin Deficiency (AATD), a condition associated with lung and liver disease. The majority of AATD cases are caused by the ‘Z’ and ‘S’ variants – single-nucleotide variations (SNVs) that result in amino acid substitutions of E342K and E264V. However, SERPINA1 is highly polymorphic, with numerous potentially clinically relevant variants reported. Novel variants continue to be discovered, and without reports of pathogenicity, it can be difficult for clinicians to determine the best course of treatment.MethodsWe assessed the utility of next-generation sequencing (NGS) and predictive computational analysis to guide the diagnosis of patients suspected of having AATD. Blood samples on serum separator cards were submitted to the DNA1 Advanced Screening Program (Biocerna LLC, Fulton, Maryland, USA) by physicians whose patients were suspected of having AATD. Laboratory analyses included quantification of serum AAT levels, qualitative analysis by isoelectric focusing, and targeted genotyping and NGS of the SERPINA1 gene. Molecular modeling software UCSF Chimera (University College of San Francisco, CA) was used to visualize the positions of amino acid changes as a result of rare/novel SNVs. Predictive software was used to assess the potential pathogenicity of these variants; methods included a support vector machine (SVM) program, PolyPhen-2 (Harvard University, Cambridge, MA), and FoldX (Centre for Genomic Regulation, Barcelona, Spain).ResultsSamples from 23 patients were analyzed; 21 rare/novel sequence variants were identified by NGS, including splice variants (n = 2), base pair deletions (n = 1), stop codon insertions (n = 2), and SNVs (n = 16). Computational modeling of protein structures caused by the novel SNVs showed that 8 were probably deleterious, and two were possibly deleterious. For the majority of probably/possibly deleterious SNVs (I50N, P289S, M385T, M221T, D341V, V210E, P369H, V333M and A142D), the mechanism is probably via disruption of the packed hydrophobic core of AAT. Several deleterious variants occurred in combination with more common deficiency alleles, resulting in very low AAT levels.ConclusionsNGS and computational modeling are useful tools that can facilitate earlier, more precise diagnosis, and consideration for AAT therapy in AATD.

Glucose-6-phosphate dehydrogenase deficiency genotypes and allele frequencies in the Kavango and Zambezi regions of northern Namibia

Namibia has made significant gains in the fight against malaria, with a target of elimination by 2023. We examined the genotype and allele frequencies of glucose-6-phosphate dehydrogenase (G6PD) deficiency to inform decisions on primaquine use, as we recently detected clusters of Plasmodium ovale curtisi in Kavango. A multistaged cross-sectional sampling method was used to enrol 212 children 2-9 y of age from schools and clinics in the Okavango and Zambezi regions of northern Namibia. Genotypes for the 202 G→A and 376 A→G mutations were assigned by polymerase chain reaction restriction fragment length polymorphism. Of the 212 subjects enrolled, genotypes were available for 210, made up of 61 males and 149 females. G6PD-deficient males (hemizygotes) and females (homozygotes) constituted 3.27% (2/61) and 0.0% (0/149), respectively. Female heterozygotes (AA- and BA-) constituted 10.07% (15/149), while G6PD wild-type males (with A or B haplotype) and females (with AA, BB or AB haplotypes) consisted of 96.72% (59/61) and 89.93% (134/149), respectively. The A-, A and B allele frequencies were 0.0474, 0.3036 and 0.6490, respectively. Hardy-Weinberg equilibrium tests for female genotype frequencies did not show deviation (p=0.29). The frequency of G6PD deficiency alleles in males in the Kavango and Zambezi regions of northern Namibia constitute 3.27%, a first report to inform policy on primaquine role out.

Clinical Effect of Alpha-1 Antitrypsin Deficiency in Antineutrophil Cytoplasmic Antibody-associated Vasculitis: Results from a French Retrospective Monocentric Cohort.

Deficiency in alpha-1 antitrypsin (AAT) is a possible pathogenic cofactor in antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV). However, the clinical effect of AAT deficiency remains poorly established in this setting. This study aimed to describe the clinical phenotypes and outcomes of AAV according to AAT phenotypes. This study was conducted retrospectively at Caen University Hospital and included all consecutive granulomatosis with polyangiitis (GPA) and microscopic polyangiitis (MPA) patients with positive proteinase 3-ANCA or myeloperoxidase-ANCA, from January 2000 or September 2011, respectively, to June 2016. AAT dosage (nephelometry) and phenotyping (isoelectric focusing in agarose gel) were performed. Among the 142 patients with AAV, including 88 GPA and 54 MPA, 102 (72%) had the MM phenotype, 5 (4%) had a nonpolymerogenic M-variant phenotype, 18 (13%) had the deficient allele MZ, 12 (8%) had MS, 2 (1%) had ZZ, 2 (1%) had SZ, and 1 (1%) had SS. M, Z, and S allele frequencies were 84%, 8%, and 6%, respectively. No association was observed between AAT deficiency and ANCA subtype or AAV phenotype, except for intraalveolar hemorrhage (IAH), which was more frequent in patients harboring at least 1 of the deficient Z or S alleles than in those without any deficient alleles (p < 0.01). Global, renal, or relapse-free survival rates were similar for all subgroups. This study shows that AAT deficiency confers, independently of ANCA subtype, a higher risk of IAH. Prospective studies are required to refine these data and to assess the need for replacement therapy in AAT-deficient patients with AAV.