Abnormal Allele Research Articles

Implementing a high-reliability, system-wide program for pre-emptive DPD deficiency testing for patients planned for a systemic fluoropyrimidine.

396 Background: Systemic fluoropyrimidine indications span several solid tumor types including gastrointestinal, breast, and head and neck malignancies. Dihydropyrimidine dehydrogenase (DPD) is the primary enzyme involved in fluoropyrimidine metabolism. Pathogenic polymorphisms of the gene encoding DPD (DPYD), occur at a rate of 4-7% and correlate with DPD enzyme deficiency. Failure to detect patients with DPD deficiency, and adjust the dosage preemptively, may result in severe toxicity, including death. Methods: Between December 2022 and May 2023, Dana-Farber Cancer Institute (DFCI) implemented a program across all sites of care to simplify and standardize preemptive DPYD testing for patient planned for a systemic fluoropyrimidine. The opt-out program includes an automated alert recommending DPYD testing when an oncologist orders a new regimen containing either 5FU or capecitabine, education for providers, pharmacists and nurses, patient education materials, standardized tracking of pending results, closed loop notification of providers of abnormal test results, dose adjustment guidelines, and patient tracking. DPYD allelic variants tested: *2A, *7, *8, *10, *13, rs67376798, rs75017182, rs115232898. Results: DPYD testing has increased from a baseline of 14% to 89%. Abnormal DPYD activity (score <2) has been detected in 5.1% of cases (28 of 553). 23 tests were performed preemptively and 5 reactively (all 5 after > grade 3 toxicity events). 27 cases had a single abnormal allele (activity score 1.5 or 1.0); a single case had 2 abnormal alleles (homozygous for rs75017182). Among 11 of the original 23 preemptively tested patients who continued fluoropyrimidine treatment at our facility, standardized dosage reductions were successfully applied for 10 (90.9%). None have experienced > grade 3 toxicity and subsequent dose escalation was possible for 5 (45.5%) patients. Conclusions: We have implemented a voluntary approach to facilitate routine preemptive DPYD testing for patients planned for a systemic fluoropyrimidine achieving an 89% usage rate. 28 of 553 (5.1%) patients were found to have at least a single allelic variant. Preemptive dose reductions were successfully applied for 90.9% of cases. The results provide proof of principle that pharmacogenomic testing can be applied in routine clinical practice and sets the stage for testing additional genes.

Assessment of major liver enzymes activities among sickle cell subjects in Enugu South East of Nigeria

Objective: Sickle cell anemia is a condition resulting from inheritance of two abnormal allele of genes controlling β-globin formation. This haemoglobinopathy affects multiple organ system in the body. The hepatobiliary system is most commonly affected in sickle cell disease. This study assessed liver enzymes activities in sickle cell subjects in Enugu, Nigeria. Methods: We recruited about hundred subjects age (20-40years) for this study. Fifty sickle cell subjects (Male =22, Female=28) as test subjects and fifty non-sickle cell subjects (Male=24, Female=26) as control. Anthropometric indices were measured and five milliliters of blood sample collected from each participant for analysis of Alanine transaminase (ALT), alkaline phosphatase (ALP), and Aspartate transaminase (AST) using autoanalyser. Data were analysed using statistical package for the social sciences (SPSS) version 25. Results: The sickle cell subjects showed a significant decrease (P<0.05) in mean ± SD of systolic BP, diastolic BP and body mass index compared to control subjects. The liver enzymes analysis showed a statistical increase (P<0.05) in ALP and AST while a non-significant increase (P>0.05) exist in ALT of sickle cell subjects compared to non- sickle cell subjects. There is a positive correlation (P<0.05) in SBP vs. DBP, ALT vs. AST, ALT vs. BMI among the test subjects. Conclusion: This study suggests that there is increase in liver enzymes activities in sickle cell subjects compared to non- sickle cell subjects, which could lead to liver complications and hence, need for regular assessment.

Huntington's Disease in Chile: Epidemiological and Genetic Aspects.

Huntington's disease (HD) is a neurodegenerative, autosomal dominant disabling condition due to an expansion of the CAG trinucleotide in the HTT gene. Motor, psychiatric, and cognitive disorders characterize it. Chilean reports on HD in the era of molecular diagnosis were wanted. This is a retrospective analysis of a prospective cohort of patients with HD seen at the Center for Movement Disorders (CETRAM) in Chile between 2013 and 2019. Sociodemographic, genotype, and neuropsychiatric features were investigated. One hundred three probands with HD were identified. The majority (63.1%) were born in the metropolitan region, followed by the VIII and V regions with 8.73% and 7.76%, respectively. When pedigrees were analyzed, ninety unrelated families encompassing 1,007 individuals were identified; among relatives, other 35 manifested HD, and 106 died of HD. Besides, five hundred seventy-nine individuals were at genetic risk. The minimum estimated prevalence of HD in Chile in 2019 was 0.72 × 100,000 inhabitants. The mean CAG repeats (CAGR) of 47.2 ± 10.74 for the expanded allele and 17.93 ± 2.05 for the normal allele. The mean age of onset was 41.39 ± 13.47 years. Juvenile cases represented 7.8% of this cohort, and 4.9% had a late onset. There was a negative correlation between the age of onset and the CAGR of the expanded allele (r =-0.84 p < 0.0001). Besides, 79.6% had a family history of HD. This is the first report characterizing genetics, motor, and neuropsychiatric features in patients with HD in Chile. The mean length of CAGR expansion of the abnormal allele was similar to previous reports in North America (i.e., Mexico and Canada) and higher than that reported in the neighboring country of Argentina. According to previous estimations, the minimal prevalence of HD in Chile may be lower than expected.

Real-World Use of Octocog Alfa and Damoctocog Alfa Pegol in Women with Hemophilia a from the ATHNdataset

Background and rationale Women and girls with one or two abnormal alleles of clotting factor genes can experience hemophilia symptoms, but a lack of data exists on the treatment of women with hemophilia. We used the ATHNdataset to identify female patients who received octocog alfa (BAY 81-8973/Kovaltry®) or damoctocog alfa pegol (BAY 94-9027/Jivi®) between January 1, 2010 and April 30, 2022. Baseline demographics, treatment history and medical history of patients aged ≥12 years with hemophilia A for damoctocog alfa pegol and all ages for octocog alfa were collected. The HIPAA-compliant, de-identified database, ATHNdataset, is a national database sponsored by the American Thrombosis and Hemostasis Network. This repository contains patient data, including of those with hemophilia A, gathered from ATHN-affiliated hemophilia treatment centers across the United States. Results: A total of 354 and 205 patients receiving octocog alfa and damoctocog alfa pegol, respectively, were in the ATHNdataset at the time of data cutoff. Of these, 13 patients receiving octocog alfa and four receiving damoctocog alfa pegol were female. Of the patients receiving octocog alfa, there was one pediatric patient (<12 years), two adolescent patients (12-18 years), and ten adult patients (>20 years) of whom seven were older than 30 years. For damoctocog alfa pegol, there were two adolescent patients (12-18 years) and two adults (>50 years). These patients were treated as listed in Table 1. For the octocog alfa patients, out of 11 patients, only one was treated with octocog alfa for <1 year, and six patients were treated for >3 years. Data regarding length of treatment were missing for two patients. Two patients were treated with damoctocog alfa pegol for <1 year, and two for >1 year with one of them for >3 years (Table 2). Of the octocog alfa patients, there was one patient who had four bleeds; all bleeds were traumatic, including three joint bleeds. One patient receiving damoctocog alfa pegol had one traumatic bleed with no joint bleeds. All other patients had zero bleeds. Eight patients on octocog alfa with known data were previously treated with another product. Five of these eight patients were on a previous standard half-life product. Three of the eight had bleeding episodes, including spontaneous and joint bleeds, on their previous product; one patient with five bleeds had two traumatic bleeds and three joint bleeds. None of these patients had bleeds while using octocog alfa. All these patients were classified as having mild hemophilia; there was also one patient with moderate and two with severe hemophilia. Two patients on damoctocog alfa pegol with known data were previously treated with another product, one episodically with desmopressin and the other prophylactically with an extended half-life product. Neither had any bleed data listed in the dataset. Previous product usage of the patient with one traumatic bleed on damoctocog alfa pegol was unknown. Three patients receiving damoctocog alfa pegol had mild hemophilia and one severe. Conclusions: The real-world data obtained from the ATHNdataset confirm that some women and girls in all age groups are being treated with octocog alfa or damoctocog alfa pegol, and indicate the effectiveness of these treatments in this real-world setting. The majority have mild hemophilia A. Approximately 1/3 of the female patients were treated on some form of prophylaxis with 2/3 of these being classified as mild. Only traumatic bleeds, including traumatic joint bleeds, were noted. Three patients treated with different products before using octocog alfa had spontaneous, joint and traumatic bleeds on their previous therapies. More data are needed on the diagnosis and treatment of women with hemophilia A. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Updated Confirmatory Diagnosis for Mucopolysaccharidoses in Taiwanese Infants and the Application of Gene Variants

Mucopolysaccharidosis (MPS) is a lysosomal storage disease caused by genetic defects that result in deficiency of one specific enzyme activity, consequently impairing the stepwise degradation of glycosaminoglycans (GAGs). Except for MPS II, the other types of MPS have autosomal recessive inheritance in which two copies of an abnormal allele must be present in order for the disease to develop. In this study, we present the status of variant alleles and biochemistry results found in infants suspected of having MPS I, II, IVA, and VI. A total of 324 suspected infants, including 12 for MPS I, 223 for MPS II, 72 for MPS IVA, and 17 for MPS VI, who were referred for MPS confirmation from newborn screening centers in Taiwan, were enrolled. In all of these infants, one specific enzyme activity in dried blood spot filter paper was lower than the cut-off value in the first blood sample, as well asin a second follow-up sample. The confirmatory methods used in this study included Sanger sequencing, next-generation sequencing, leukocyte enzyme fluorometric assay, and GAG-derived disaccharides in urine using tandem mass spectrometry assays. The results showed that five, nine, and six infants had MPS I, II, and IVA, respectively, and all of them were asymptomatic. Thus, a laboratory diagnosis is extremely important to confirm the diagnosis of MPS. The other infants with identified nucleotide variations and reductions in leukocyte enzyme activities were categorized as being highly suspected cases requiring long-term and intensive follow-up examinations. In summary, the final confirmation of MPS depends on the most powerful biomarkers found in urine, i.e., the quantification of GAG-derived disaccharides including dermatan sulfate, heparan sulfate, and keratan sulfate, and analysis of genetic variants can help predict outcomes and guide treatment.

P1026: COMPARATIVE GENOMIC PROFILING OF MYELOPROLIFERATIVE NEOPLASMS PRESENTING WITH AND WITHOUT SPLANCHNIC VEIN THROMBOSIS

Background: Splanchnic vein thrombosis (SVT) is the initial manifestation in 5% of myeloproliferative neoplasms (MPN). MPN patients presenting with SVT are usually younger, have less abnormal blood counts and lower JAK2V617F allele burden than other MPN patients. However, it is unknown whether they have a different genomic background. Aims: To describe the clinical and genomic profile of MPN presenting with SVT and to compare it with a matched group of MPN without SVT. Methods: Samples of 190 MPN patients were selected: 64 MPN presenting with SVT (SVT+) and 126 MPN without SVT (SVT-). SVT+ and SVT- were matched according to sex, age, MPN clinical subtype and driver mutation. Next Generation Sequencing was performed using Myeloid Solution by Sophia Genetics, which includes 30 genes recurrently mutated in MPN. Variants were selected using Sophia DDM software and were subsequently classified into 5 categories: 5 (definitely pathogenic), 4 (likely pathogenic), 3 (uncertain), 2 (likely benign), and 1 (benign). Only variants included into 4 and 5 categories were taken into account. Patients were classified according to the genomic classification proposed by Grinfeld and colleagues. Results: One hundred and three patients were women (54.2%) and 87 patients were men (45.8%), without significant differences between SVT+ and SVT- groups. Median age was 44 years (range: 15-85) and 50 years (range: 15-87) in SVT+ and SVT-, respectively (p not significant). Regarding MPN clinical subtype in SVT+ group, 39 (60.9%), 15 (23.4%) and 10 (15.6%) corresponded to Polycythemia Vera (PV), Essential Thrombocythemia (ET) and MPN unclassifiable, respectively. In the SVT- group, 75 (59.5%) and 51 (40.5%) corresponded to PV and ET, respectively. Genomic classification in the SVT+ and SVT- groups is shown in Table 1. The proportion of patients with high molecular risk (TP53 disruption/aneuploidy, chromatin/spliceosome mutation, or homozygous JAK2 mutation) was 14.1% and 26.2% for SVT+ and SVT-, respectively (p=0.06). Median number of pathogenic mutations was 1 (range: 1-4) in both SVT+ and SVT- groups. Pathogenic mutations in TET2, DNMT3A and ASXL1 were present in 10%, 3.7% and 3.7%, respectively, without significant differences between both groups. With a mean follow-up of 10.1 years for cases and 9.7 years for controls, 27 patients died (13 SVT+ and 14 SVT-). Median survival was 24 years and not reached for SVT+ and SVT-, respectively (p=0.1). On multivariate analysis, SVT+ group showed a higher risk of death (HR: 2.4, 95%CI: 1.1-5.5, p=0.03) after correction by age, sex and genomic classification. Transformation to myelofibrosis and acute leukemia was low and similar in both groups: 4.7% vs. 5.6% for myelofibrosis in SVT+ and SVT-, respectively (p not significant); and 0% vs. 2.4% for acute leukemia (p not significant). On multivariate analysis, patients with TP53 disruption/aneuploidy, chromatin/spliceosome mutation, or homozygous JAK2 showed a higher risk of disease progression to myelofibrosis or acute leukemia (HR: 14.5, 95%CI: 3.1-68.7, p=0.001) regardless of the presence of SVT at diagnosis, age and sex. Image:Summary/Conclusion: MPN presenting with SVT and MPN-matched controls showed a low molecular complexity. Genomic classification was helpful in identifying a small proportion of MPN patients presenting with SVT who are at high risk of disease progression. SVT at MPN diagnosis is associated with a higher risk of death independent of sex, age and genomic classification.

HEPES-buffering of bicarbonate-containing culture medium perturbs lysosomal glucocerebrosidase activity.

Glucocerebrosidase (GCase), encoded by the GBA gene, degrades the ubiquitous glycosphingolipid glucosylceramide. Inherited GCase deficiency causes Gaucher disease (GD). In addition, carriers of an abnormal GBA allele are at increased risk for Parkinson's disease. GCase undergoes extensive modification of its four N‐glycans en route to and inside the lysosome that is reflected in changes in molecular weight as detected with sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. Fluorescent activity‐based probes (ABPs) that covalently label GCase in reaction‐based manner in vivo and in vitro allow sensitive visualization of GCase molecules. Using these ABPs, we studied the life cycle of GCase in cultured fibroblasts and macrophage‐like RAW264.7 cells. Specific attention was paid to the impact of 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) supplementation to bicarbonate‐buffered medium. Here, we report how HEPES‐buffered medium markedly influences processing of GCase, its lysosomal degradation, and the total cellular enzyme level. HEPES‐containing medium was also found to reduce maturation of other lysosomal enzymes (α‐glucosidase and β‐glucuronidase) in cells. The presence of HEPES in bicarbonate containing medium increases GCase activity in GD‐patient derived fibroblasts, illustrating how the supplementation of HEPES complicates the use of cultured cells for diagnosing GD.

A family case report of Neurofibromatosis

Neurofibromatosis is a genetic disease inherited in an autosomal, dominant manner. The course of von Recklinghausen’s disease varies from patient to patient. Most cases are mild and uncomplicated. The diagnosis of NF1 is based on the criteria of the National Institutes of Health (NIH). Type 2 neurofibromatosis is inherited, in most cases autosomal dominantly. The abnormal allele can be inherited from either parent, with the risk of passing the gene on to the offspring from the parent, being 50%. This article describes the familial form of neurofibromatosis. In the case in question, all three sisters were found to be mutated in the NF1 gene by genetic testing. In the same family, the clinical manifestation of the disease may be different for sisters and children. Due to compliance with medical recommendations, the disease does not significantly affect the quality of life of the families affected.

Labelling Alpha-1 antitrypsin deficiency in the medical record – A call to action

Alpha-1 antitrypsin deficiency (AATD) is an autosomal codominant genetic cause of chronic obstructive pulmonary disease (COPD) with over 100 allelic variants described. The normal allele is termed “M”; whereas, the “Z” and “S” alleles are the most common abnormal alleles. The ZZ combination accounts for 95% of cases with severe disease. We described the characteristics of patients given the label of AATD in the medical record. Our analysis found there is significant heterogeneity with regards to labelling subjects with AATD in the medical record, and this was true irrespective of the subjects age, gender, PFT measurements, tobacco pack years, or if the physician was a pulmonologist. In summary, this study highlights the need for continued investigation of the role of other mutations developing disease and options for more specific labelling of subjects with non-severe AATD and severe AATD to provide additional clarity for the patient and healthcare providers.

OBSERVED OUTCOMES OF FMR1 PGT-M ANALYSIS WITH INCORPORATION OF CGG REPEAT EXPANSION CAN LEAD TO EXTRA EMBRYOS SUITABLE FOR TRANSFER

Highlight utility of triplet repeat expansion for the identification of embryos with the abnormal FMR1 allele that may be considered for transfer following preimplantation genetic testing (PGT) FMR1-related disorders, which includes Fragile X syndrome. Trophectoderm biopsies from US-based IVF clinics were sent to CooperGenomics for PGT-M for FMR1-related disorders between January 2018 and February 2021. Samples underwent linkage analysis via Karyomapping (lllumina, USA) and triplet repeat size determination. When requested, screening for aneuploidy (PGT-A) via next generation sequencing (NGS) was completed in conjunction with PGT-M. Patients with FMR1 expansions on both alleles and those with a full expansion (>200 repeats) on one allele were removed from the study population. A total of 1130 blastocysts from 318 IVF cycles underwent triplet repeat size determination and linkage analysis (average 3.55 embryos per cycle). The average age of patient at time of egg retrieval was 34.5 years. 51.4% (581/1130) of embryos were suitable to transfer based on presence of the normal FMR1 allele via linkage analysis. When factoring in PGT-A, 27.7% of the study population were euploid embryos with the normal allele. 48.6% (549/1130) of embryos tested had the abnormal FMR1 allele detected via linkage analysis. Of embryos inheriting the abnormal allele, 30.6% of embryos had experienced an expansion to yield an abnormal allele with >200 repeats (168/549). This expansion places these embryos at increased risk of expressing a FMR1-related disorder. Of these higher risk embryos, 55.4% were male. Importantly, 381 blastocyst samples (69.4%) inheriting the abnormal allele did not experience clinically significant expansion, with repeats remaining <200. 53% (202) of these lower risk embryos further screened as euploid by PGT-A, yielding an additional 17.9% (202/1130) embryos that could be considered for transfer. 49.25% (99/201) were female euploid carriers and 50.74% were male euploid carriers (102/201). Preimplantation testing for monogenic diseases (PGT-M) is classically considered a binary linkage-based test for the determination of the allele present in an at-risk embryo. For triplet repeat conditions like FMR1-related disorders, quantifying the length of the CGG triplet repeat tract inside the FMR1 gene modifies classification of disease risk in the embryo. This results in more embryos available for transfer, which is an important factor to consider in a population where ovarian failure is a common factor associated with carrier status. Knowledge of embryo repeat size is an intangible resource for genetic counseling about which embryos can be considered for transfer. Providing linkage + triplet repeat analysis is a diagnostic advantage for more complete classification of embryo status.

A Distinct Variant of Pseudohypoparathyroidism (PHP) First Characterized Some 41 Years Ago Is Caused by the 3-kbSTX16 Deletion.

ABSTRACTIn 1980, Farfel and colleagues (NEJM, 1980;303:237–42) provided first evidence for two distinct variants of pseudohypoparathyroidism (PHP) that present with hypocalcemia and impaired parathyroid hormone (PTH)‐stimulated urinary cAMP and phosphate excretion, either in the presence or absence of Albright's hereditary osteodystrophy (AHO). An “abnormal allele” and an “unexpressed allele” were considered as underlying defects, predictions that turned out to be correct for both forms of PHP. Patients affected by the first variant (now referred to as PHP1A) were later shown to be carriers of inactivating mutations involving the maternal GNAS exons encoding Gsα. Patients affected by the second variant (now referred to as PHP1B) were shown in the current study to carry a maternal 3‐kb STX16 deletion, the most frequent cause of autosomal dominant PHP1B, which is associated with loss of methylation at GNAS exon A/B that reduces or abolishes maternal Gsα expression. However, the distinct maternal mutations leading to either PHP1A or PHP1B are disease‐causing only because paternal Gsα expression in the proximal renal tubules is silenced, ie, “unexpressed.” Our findings resolve at the molecular level carefully conducted investigations reported some 41 years ago that had provided first clues for the existence of two distinct PHP variants. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Abnormalities of Hemoglobin and Glucose-6-Phosphate-Dehydrogenase Deficiency in Children with Uncomplicated Malaria and Living in Banfora and Saponé, Two Different Malaria Setting of Burkina Faso

Aims: The aim of this study is to assess the prevalence of hemoglobin abnormalities and G6PD deficiency and their respective influence on anemia occurring in less than five years old children with clinical P. falciparum malaria living in Burkina Faso.&#x0D; Study Design: The study was a cross-sectional survey with descriptive focus conducted from December 2010 to January 2013 in Saponé health district and from May to October 2011 in Banfora health district. Clinical and laboratory data were collected. Blood smears on slides for malaria diagnosis by microscopy, hemoglobin level and filter paper for the detection of human genetic factors were performed.&#x0D; Methodology: A total of 386 subjects from Saponé (131) and Banfora (255) were enrolled. DNA collected from each sample was extracted using chelex-100 method and the human genetic resistance factors background was assessed by RFLP-PCR. Abnormal hemoglobin patients were classified as NonAA while AA was defined the normal hemoglobin.&#x0D; Results: In this study, 70.98% (274/386) were classified normal hemoglobin (AA) while 29.02% (112/386) of subjects were carrying at least one abnormal (NonAA) allele: 24.35%AC, 3.63% AS, 0.78%CC and 0.26%SC. G6PD deficiency was 9.59% (37/386) among which, 4.92% for male and 4.66% in female. However, this gender difference was not statistically significant (p=1.00). 319/367 (86.92%) of the patients were anemic (59.4% with moderate anemia and 20.98% with mild anemia). The prevalence of anemia in G6PD deficient subjects was 83.33% (of which 58.33% were moderate anemia and 22.22% mild anemia). The difference between types of hemoglobin (p=0.64) in the occurrence of anemia (AA 87.64% and Non AA 85.18%) was not statistically significant.&#x0D; Conclusion: This study showed that the prevalence of these genetic factors was relatively low among children with clinical falciparum malaria with high parasite density. In addition, these factors appear to have no effect on anemia.

Germline mosaicism is a pitfall in the diagnosis of “sporadic” X-linked Alport syndrome

Approximately 80% of patients with Alport syndrome have X-linked Alport syndrome (XLAS), which is caused by mutations in the type IV collagen alpha 5 gene (COL4A5). In patients with XLAS, approximately 10-15% of COL4A5 mutations occur as spontaneous events. Here, we describe maternal germline mosaicism in a family of XLAS patients. Since our patient's parents showed normal urinary findings without COL4A5 mutation using genomic DNA isolated from peripheral leukocytes, the patient was initially diagnosed with "sporadic" XLAS. However, genetic analysis of the patient's sister with microscopic hematuria identified the same COL4A5 heterozygous mutation. Therefore, we concluded that our patient and the sister had XLAS caused by maternal germline mosaicism, not "sporadic" XLAS. Our case suggests that "sporadic" XLAS may in some patients be caused by the transmission of an abnormal allele from either parent with germline mosaicism in COL4A5. Germline mosaicism is thought to be rare, but we should consider that even asymptomatic parents of "sporadic" XLAS patients could carry a somatic and/or germline mosaicism. More cautious genetic counseling is advisable for all "sporadic" XLAS patients. Furthermore, urinalysis screening of "sporadic" XLAS patients' siblings is also important to enable an earlier detection of parental germline mosaicism.

The association of parental methylenetetrahydrofolate reductase polymorphisms (MTHFR 677C > T and 1298A > C) and fetal loss: a case–control study in South Australia

Objective: To determine the association between parental MTHFR 677C > T (RS1801133) and 1298A > C (RS1801131), and fetal loss (FL).Design: Case–control study.Setting: Department of Obstetrics and Gynecology, Lyell McEwin Hospital (LMH), and the Women’s and Children’s Hospital (WCH) in Adelaide, Australia.Patients: A total of 222 couples with FL and 988 couples with uncomplicated pregnancies.Measurements: The main outcomes were FL and hyperhomocysteinemia (HHcy). All couples were tested for MTHFR 677C > T and 1298A > C. Fasting homocysteine was measured in the women with FL.Results: The main finding was a significant difference between the FL group and controls in couples with ≥4 abnormal alleles compared to <4 [p=.0232, OR 1.9 (95% CI 1.1–3.3)]. None of the couples with FL had zero abnormal alleles (both parents 677CC/1298 AA). However, this was also rare amongst the controls. Maternal carriage of both 677C > T and the 1298A > C polymorphisms was similar between the FL group and controls. The prevalence of paternal 677TT/1298AA and 677CC/1298AC was significantly higher in the FL group compared with controls. HHcy was significantly more common in the FL group compared with controls.Conclusion: The presence of parental MTHFR 677C > T and 1298A > C is associated with FL. The association between maternal MTHFR genotypes with FL is less pronounced than in previously published articles investigating first trimester miscarriages. Maternal HHcy is a significant risk factor for FL.

Fragile X syndrome: diagnosis by molecular characterization of FMR1 gene and clinical correlation

Background One of the most common forms of inherited intellectual disability (ID) is fragile X syndrome (FXS), which is caused by expansion of cytosine–guanine–guanine trinucleotide repeat at the 5′ untranslated region of the fragile X mental retardation gene (FMR1) at Xq27. The cytosine–guanine–guanine repeat expansion leads to hypermethylation and inactive transcription of the gene. The present study aimed to detect expected FMR1 gene alleles by methylation-sensitive PCR and its clinical correlation for rapid screening of FXS among male patients with ID. Patients and methods The study included 50 male patients with ID and clinical features suggestive of FXS, who were compared with 50 healthy age-matched volunteers. All patients were subjected to full history taking, thorough clinical examination using a 15-item checklist [physical (big ears, joint hyperextensibility, Simian crease, wide forehead, macroorchidism, and elongated face) and neurological features (mental retardation, family history of mental retardation, poor eye contact, hand biting, hyperactivity, perservative speech, tactile defensiveness, hand flapping, and short attention span)], and karyotyping using GTG banding. Methylation-sensitive PCR technique after bisulfite treatment of DNA was applied for the detection of expanded alleles of the FMR1 gene. Results Clinical score in patients with abnormal alleles was significantly higher compared with patients with normal alleles. GTG-banding technique showed normal 46XY male karyotype for studied patients. Frequency of normal FMR1 gene alleles was detected in the control group (100%) and 44 (88%) patients. Abnormal alleles were detected in six (12%) patients: three (6%) patients with full mutation (FM) and three (6%) patients with premutation carrier. Conclusion Our study revealed that PCR-positive results of fragile X correlate with the checklist clinical score rather than a single clinical entity.

Complete overview of protein-inactivating sequence variations in 36 sequenced mouse inbred strains.

Mouse inbred strains remain essential in science. We have analyzed the publicly available genome sequences of 36 popular inbred strains and provide lists for each strain of protein-coding genes that acquired sequence variations that cause premature STOP codons, loss of STOP codons and single nucleotide polymorphisms, and short in-frame insertions and deletions. Our data give an overview of predicted defective proteins, including predicted impact scores, of all these strains compared with the reference mouse genome of C57BL/6J. These data can also be retrieved via a searchable website (mousepost.be) and allow a global, better interpretation of genetic background effects and a source of naturally defective alleles in these 36 sequenced classical and high-priority mouse inbred strains.

CYP2C19 genetic variation and individualized clopidogrel prescription in a cardiology clinic

ABSTRACTBackground: Clopidogrel (Plavix) is an antiplatelet medication that is routinely used in patients with cardiovascular disease. Cytochrome P2C19 enzymes play a major role in its metabolism, which determines its varied therapeutic level and its effectiveness.Objectives: To customize clopidogrel therapy and evaluate its efficacy by using CYP2C19 genotypic and phenotypic information to improve clinical outcomes in patients.Methods: A total of 465 patients with underlying cardiovascular disease were selected from our out-patient cardiology clinic. DNA sequences of CYP2C19 were analyzed in 465 patients.Results: Of 465 patients, 183 were wild-type homozygous (*1/*1) and 18.8% gain-of function and 19.8% loss-of-function alleles in our patient population The following changes were made: 1) Switching to prasugrel in patients whose genotype noted them to be “Slow metabolizers. This medication adjustment improved clinical outcomes in this patient group.2) Discontinuing or lowering clopidogrel doses in patients whose genotypes noted them to be “Fast or ultra-fast metabolizes” to decrease bleeding risk. For those who were not on clopidogrel but carried abnormal allele(s), “clopidogrel caution” was documented. These individuals were followed up for 3 years and there has not been any cardiac clinical symptoms, cardiac death or excessive bleeding reported.Conclusions: Given the varied effectiveness of clopidogrel due to its metabolism by CYP2C19 enzyme, and the relatively high frequency of both gain-of-function (18.8%) and loss-of-function (19.8%) alleles in our patient population, we believe that genotyping CYP2C19 is clinically important in order to improve patient outcomes and minimize patient risk.

Congenital protein C deficiency with renal vein thrombosis and central retinal venous occlusion in a term neonate due to novel mutation in the PROC gene

Severe protein C (PC) deficiency is a rare disorder caused by homozygous or compound heterozygous genetic anomalies. PC deficiency is inherited as autosomal dominant, and most patients are heterozygous for a genetic defect. Homozygous or compound heterozygous for a mutation or other genetic defect affecting PC is typically due to inheritance of an abnormal allele from both parents. In the neonatal period, homozygous PC deficiency is usually associated with the development of severe, and often fatal, purpura fulminans and disseminated intravascular coagulation and if untreated is incompatible with life. Here, we report a case of congenital PC deficiency due to novel homozygous mutation in the protein coding gene (PROC) in a term neonate which presented with severe purpura fulminans, perinatal right renal vein thrombosis (RVT), bilateral adrenal hemorrhage, and early neonatal central retinal venous occlusion with retinal detachment. Perinatal RVT and early neonatal central retinal vein occlusion have not been reported previously in the literature.

The allele frequency of CYP2C9 and VKORC1 in the Southern Khorasan population.

The genetic factors are determinants in required dosage changes of warfarin among which are polymorphisms of CYP2C9 and VKORC1 genes. The present study aimed to determine the allele and genotype frequency of CYP2C9 and VKORC1 genes in Birjand population. This study was conducted on 120 individuals who referred to Imam Reza and Vali-Asr hospitals for PT/INR test. After extracting the genomic DNA, the considered sequences were amplified by PCR, and restriction fragment length polymorphism analysis was done by AvaII and KpnI enzymes to determine allele polymorphisms. Moreover, related sequences of VKORC1, after amplification, were sequenced for determining the genotype. Allelic and genotypic frequencies as well as Hardy-Weinberg equilibrium, observed heterozygosity, expected heterozygosity, and polymorphism information content were calculated by PowerMarker V 3.25 software. Amongst 120 individuals in this study with the mean age of 58.12 ± 12.7 years, 80.8%, 9.1%, and 10% exhibited the alleles of 1, 2, and 3 CYP2C9 gene, respectively. The genotype frequencies of 1/1, 1/2, 2/2, 3/1, 3/2, and 3/3 of this gene were found to be 64.1, 15.8, 0, 17.5, 2.5, and 0 %, respectively. In -1639 G>A region, VKORC1 had normal homozygote genotype (GG) and in 1173 C>T region, heterozygote (CT) with the frequency of 48.7% and 45.9% had the most prevalence. Compared with other populations, there is a considerable difference between the allele frequency of CYP2C9 and VKORC1 genetic variance. Since 35.8% of the selected populations carry an abnormal allele causing sensitivity to warfarin, the specialists at medical centers must be informed about the genotypes of patients before prescribing warfarin.

An Audit on Management of G6PD Deficiency Acute Hemolytic Crisis

An Audit on Management of G6PD Deficiency Acute Hemolytic Crisis