Detection of single nucleotide and copy number variants in the Fabry disease-associated GLA gene using nanopore sequencing

More than 900 variants have been described in the GLA gene. Some intronic variants and copy number variants in GLA can cause Fabry disease but will not be detected by classical Sanger sequence. We aimed to design and validate a method for sequencing the GLA gene using long-read Oxford Nanopore sequencing technology. Twelve Fabry patients were blindly analyzed, both by conventional Sanger sequence and by long-read sequencing of a 13 kb PCR amplicon. We used minimap2 to align the long-read data and Nanopolish and Sniffles to call variants. All the variants detected by Sanger (including a deep intronic variant) were also detected by long-read sequencing. One patient had a deletion that was not detected by Sanger sequencing but was detected by the new technology. Our long-read sequencing-based method was able to detect missense variants and an exonic deletion, with the added advantage of intronic analysis. It can be used as an efficient and cost-effective tool for screening and diagnosing Fabry disease

Diagnostic Yield and Benefits of Whole Exome Sequencing in CAKUT Patients Diagnosed in the First Thousand Days of Life

Infancy; Reverse phenotyping; Whole exome sequencingInfancia; Fenotipado inverso; Secuenciación del exoma completoInfància; Fenotipat invers; Seqüenciació de l'exoma completIntroduction Congenital anomalies of the kidney and urinary tract (CAKUT) are the predominant cause of chronic kidney disease (CKD) and the need for kidney replacement therapy (KRT) in children. Although more than 60 genes are known to cause CAKUT if mutated, genetic etiology is detected, on average, in only 16% of unselected CAKUT cases, making genetic testing unproductive. Methods Whole exome sequencing (WES) was performed in 100 patients with CAKUT diagnosed in the first 1000 days of life with CKD stages 1 to 5D/T. Variants in 58 established CAKUT-associated genes were extracted, classified according to the American College of Medical Genetics and Genomics guidelines, and their translational value was assessed. Results In 25% of these mostly sporadic patients with CAKUT, a rare likely pathogenic or pathogenic variant was identified in 1 or 2 of 15 CAKUT-associated genes, including GATA3, HNF1B, LIFR, PAX2, SALL1, and TBC1D1. Of the 27 variants detected, 52% were loss-of-function and 18.5% de novo variants. The diagnostic yield was significantly higher in patients requiring KRT before 3 years of age (43%, odds ratio 2.95) and in patients with extrarenal features (41%, odds ratio 3.5) compared with patients lacking these criteria. Considering that all affected genes were previously associated with extrarenal complications, including treatable conditions, such as diabetes, hyperuricemia, hypomagnesemia, and hypoparathyroidism, the genetic diagnosis allowed preventive measures and/or early treatment in 25% of patients. Conclusion WES offers significant advantages for the diagnosis and management of patients with CAKUT diagnosed before 3 years of age, especially in patients who require KRT or have extrarenal anomalies.This work was supported by grants from the Else Kröner-Fresenius-Stiftung (2018_Kolleg.12, Clinician Scientist Program TITUS at Hannover Medical School to LW) and the Deutsche Forschungsgemeinschaft (MA 9606/1-1 to HM, and KO 5614/2-1 to AC and RGW)

Anesthetic management for oocyte retrieval: An exploratory analysis comparing outcome in in vitro fertilization cycles with and without pre-implantation genetic diagnosis

Purpose: To date, there has been no comparison of outcomes in women undergoing anesthesia for in vitro fertilization (IVF) oocyte retrieval for the purpose of pre-implantation genetic diagnosis (PGD) because of their or their partner′s genetic disease relative to the outcome in women requiring IVF because of fertility issues. Materials and Methods: A prospective observational study, wherein all demographic and anesthetic management data were collected from IVF and PGD units′ records for a 6-month period. Descriptive analyses and parametric tests were employed. Results: There were 307 cases IVF and 76 cases PGD: most (97.4% and 99.7%, respectively) received general anesthesia with propofol and fentanyl ± dipyrone (90.5% and 93.3%, respectively) with no adverse effects. The only statistically significant difference between IVF and PGD groups that was potentially clinically significant was post-procedure recovery time (23.0 ± 20.4 vs. 29.4 ± 35.8 min, respectively; P < 0.0001), but is explainable as greater caution by Anesthesiologists for higher-risk PGD cases having autosomal dominant diseases that may impact anesthesia management (myotonic dystrophy, neurofibromatosis, Marfan′s); two of these cases also recovered in the general post-anesthesia care unit, as a precaution for early diagnosis and treatment of potential post-procedural complication. Conclusions: Results of this first-ever survey of anesthesia for PGD compared with IVF cases imply that propofol-and-fentanyl-based anesthesia is safe and can be recommended, bearing in mind that with patients who have autosomal dominant diseases impacting anesthetic management it is prudent to be more cautious post-recovery

MATERNAL-FETAL VITAMIN D RECEPTOR POLYMORPHISMS SIGNIFICANTLY ASSOCIATED WITH PRETERM BIRTH

Aim: Preterm birth (PTB) is a complex trait with strong genetic background, whose etiology is not fully understood. It was recently suggested that pregnancy duration is affected by fetal genetic variation even more than by the maternal genome. Vitamin D receptor (VDR) is involved in embryonic implantation and fertility. We studied the association between both maternal and neonatal vitamin D receptor (VDR) genetic variation and PTB.Materials and Methods: Maternal and fetal (umbilical cord) DNA was isolated from Jewish Israeli idiopathic preterm newborns (24-36 weeks, n=146) and control term newborns (>37 weeks, n=229). Maternal and fetal VDR polymorphisms (FokI, ApaI, BsmI, TaqI) were analyzed by restriction fragment length polymorphism analysis. We have used SPSS analysis in order to determine the correlation between VDR genotypes and phenotypic variation: pregnancy duration, preterm birth and spontaneous miscarriages, adjusted for gravidity, parity and gender of newborn.Results: Women homozygous to VDR ApaI (AA) genotype had significant twofold increase risk for PTB (OR=1.973, [CI] 1.183-3.289, p=0.009) compared to heterozygous women. Male newborns had significant (p<0.05) 1.73 fold increase of PTB. Women with history of previous (≥1) spontaneous miscarriage had a significantly increased risk for PTB if their newborn carried either of the VDR BsmI homozygous (BB or bb) genotypes compared to the heterozygous (Bb) genotype (OR=6.857,[CI] 1.273-36.934, p=0.018 and OR=9.231,[CI] 1.753-48.618, p= 0.008, respectively), or VDR ApaI homozygous (AA or aa) genotype compared to heterozygous (Aa) genotype (OR=4.33, [CI] 1.029-18.257, p= 0.046 and OR=7.2, [CI] 1.34-38.917, p=0.021, respectively).Conclusion: We have shown the association between maternal and fetal VDR genotype variants with PTB

Achievement of Therapeutic Goals with Low-Dose Imiglucerase in Gaucher Disease: A Single-Center Experience

Gaucher disease, a lysosomal storage disorder, is a multisystem disorder with variable and unpredictable onset and severity. Disease-specific enzyme replacement therapy (ERT) has been shown to reverse or ameliorate disease-specific hepatosplenomegaly and anemia and thrombocytopenia. ERT also impacts bone manifestations, including bone crises, bone pain, and appearance of new osteonecrosis, and improves bone mineral density to varying degrees. The objective of this study was to assess achievement of predefined therapeutic goals based on international registry outcomes for Israeli patients with Gaucher disease receiving imiglucerase for four consecutive years on a low-dose regimen followed in a single center. All data were taken from patient files. The therapeutic goals were taken from standards published in the literature for disease-specific clinical parameters. Among 164 patients at baseline, values for spleen and liver volumes, hemoglobin and platelet counts, and Z-scores for lumbar spine and femoral were significantly different from the goal. After four years ERT, there was a significant improvement () in each of the therapeutic goal parameters from baseline. 15.2% of these patients achieved all hematology-visceral goals. In children, there was achievement of linear growth and puberty. This survey highlights the good overall response in symptomatic patients receiving low-dose ERT with imiglucerase in Israel

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background: PGD for fragile X syndrome (FRAX) is inefficient, probably owing to fewer oocytes, poor embryo quality and difficulties in genetic analysis. We investigated IVF -PGD in FRAX mutation carriers compared with controls, looking at the effects of oocyte and embryo number/quality on live birth outcome. methods: We performed IVF -PGD in 27 patients with the FRAX mutation and 33 controls with other genetic diseases. Genetic testing was by multiplex PCR. results: Seventy-nine and 108 IVF -PGD cycles were started in FRAX mutation carriers and controls, respectively. Twenty-two patients had a premutation (CGG repeat number 60 -200) and five had a full mutation (300-2000 CGG repeats). FRAX patients required higher doses of gonadotrophins (6788 + 2379 versus 4360 + 2330, P , 0.001) but had lower peak serum estradiol levels (8166 + 5880 versus 10 211 + 4673, P ¼ 0.03) and fewer oocytes retrieved (9.8 + 6 versus 14 + 8, P ¼ 0.01). The cancelation rate (unsatisfactory ovarian response) was higher in the FRAX group than in the control group (13 versus 1%, P , 0.001). When embryos were transferred, ongoing pregnancy/live birth rates per transfer were similar (29 versus 36%, P ¼ 0.54). conclusions: Ovarian dysfunction in FRAX carriers is more prevalent and profound than previously appreciated, with a high cancelation rate and reduced efficiency of PGD. The main determinant for successful PGD for FRAX is ovarian dysfunction. When embryo transfer is possible, the results are comparable to PGD for other monogenic diseases

DMPK hypermethylation in sperm cells of myotonic dystrophy type 1 patients

International audienceMyotonic dystrophy type 1 (DM1) is an autosomal dominant muscular dystrophy that results from a CTG expansion (50–4000 copies) in the 3′ UTR of the DMPK gene. The disease is classified into four or five somewhat overlapping forms, which incompletely correlate with expansion size in somatic cells of patients. With rare exception, it is affected mothers who transmit the congenital (CDM1) and most severe form of the disease. Why CDM1 is hardly ever transmitted by fathers remains unknown. One model to explain the almost exclusive transmission of CDM1 by affected mothers suggests a selection against hypermethylated large expansions in the germline of male patients. By assessing DNA methylation upstream to the CTG expansion in motile sperm cells of four DM1 patients, together with availability of human embryonic stem cell (hESCs) lines with paternally inherited hypermethylated expansions, we exclude the possibility that DMPK hypermethylation leads to selection against viable sperm cells (as indicated by motility) in DM1 patients