Whole exome sequencing identifies novel pathogenic variants in TGM1 and ALOX12B in patients with hereditary ichthyosis.

BACKGROUND: Hereditary ichthyosis is a clinically and genetically heterogeneous disorder associated with more than 50 genes with TGM1, ALOX12B, and ALOXE3 being the most prevalent. Establishing an accurate diagnosis is important for effective genetic counseling and optimal patient management. OBJECTIVE: We studied the diagnostic value of whole exome sequencing (WES) in a small case series with hereditary ichthyosis. METHODS: During a 1-year period, index cases of 5 unrelated families clinically diagnosed with hereditary ichthyosis went through WES, followed by extensive segregation analysis. Prenatal diagnosis (PND) was conducted where indicated. RESULTS: We identified 4 homozygous variants-2 in TGM1 (c.655A > G and c.797A > G) and 2 in ALOX12B (c.527 + 2 T > G and c.1654G > T)-alongside a heterozygous variant in TGM1 (c.428G > A) in 5 families. The variants were all pathogenic/likely pathogenic according to the ACMG classification and segregation analysis, except for c.797A > G in TGM1 which remained a variant of unknown clinical significance. Four variants were novel. All families were referred either during pregnancy or before reproductive planning; 4 benefited from WES as it identified the mutation in the probands and enabled carrier detection in at-risk relatives; PND was conducted in 2 families. CONCLUSION: Our findings further support WES is a powerful tool for the comprehensive, accurate, and rapid molecular diagnosis of hereditary ichthyosis and can offer opportunities for reproductive planning, carrier screening and prenatal diagnosis to at-risk families.

Molecular characterization of a large cohort of mucopolysaccharidosis patients: Iran Mucopolysaccharidosis RE-diagnosis study (IMPRESsion).

Mucopolysaccharidoses (MPSs) are rare, heterogeneous inborn errors of metabolism (IEM) diagnosed through a combination of clinical, biochemical, and genetic investigations. The aim of this study was molecular characterization of the largest cohort of Iranian MPS patients (302 patients from 289 unrelated families), along with tracking their ethnicity and geographical origins. 185/289 patients were studied using an IEM-targeted NGS panel followed by complementary Sanger sequencing, which led to the diagnosis of 154 MPS patients and 5 non-MPS IEMs (diagnostic yield: 85.9%). Furthermore, 106/289 patients who were referred with positive findings went through reanalysis and confirmatory tests which confirmed MPS diagnosis in 104. Among the total of 258 MPS patients, 225 were homozygous, 90 harbored novel variants, and 9 had copy number variations. MPS IV was the most common type (34.8%) followed by MPS I (22.7%) and MPS VI (22.5%). Geographical origin analysis unveiled a pattern of distribution for frequent variants in ARSB (c.430G>A, c.962T>C [p.Leu321Pro], c.281C>A [p.Ser94*]), GALNS (c.319G>A [p.Ala107Thr], c.860C>T [p.Ser287Leu], c.1042A>G [p.Thr348Ala]), and IDUA (c.1A>C [p.Met1Leu], c.1598C>G [p.Pro533Arg], c.1562_1563insC [p.Gly522Argfs*50]). Our extensive patient cohort reveals the genetic and geographic landscape of MPS in Iran, which provides insight into genetic epidemiology of MPS and can facilitate a more cost-effective, time-efficient diagnostic approach based on the region-specific variants.

Identification of a novel de novo variant in OTX2 in a patient with congenital microphthalmia using targeted next-generation sequencing followed by prenatal diagnosis.

BACKGROUND: Next-generation sequencing has been proven to be a reliable method for the detection of genetic causes in heterogeneous ocular disorders. In this report an NGS-based diagnostic approach was taken to uncover the genetic etiology in a patient with coloboma and microphthalmia, a highly heterogeneous disease with intrafamilial phenotypic variability. MATERIALS AND METHODS: Next generation sequencing using a targeted panel of 316 genes, was carried out in the proband. Prioritized variants were then identified and confirmed using Sanger sequencing. Prenatal diagnosis of the detected variant was then performed in the family. RESULTS: A novel de novo frameshift variant c.157_164delTTCACTCG (p.Phe53fs) in OTX2, leading to a truncated protein, was identified. Prenatal diagnosis identified the same variant in the fetus. CONCLUSIONS: This report demonstrates the importance of genetic counseling and underscores the efficiency and effectiveness of targeted NGS as a means of detecting variants in inherited eye disorders.

Determination of Cleaning Limits Considering Toxicological Risk Evaluation to Minimize the Risk of Cross Contamination.

The historical approaches that have been used to establish cleaning validation acceptance limits should be updated to recent approaches to prevent cross contamination. In the present investigation, a cleaning method was validated using high performance liquid chromatography. Method modification critical parameters including spiking, swab sampling from PVC, Stainless Steel, and Polyethylene, extraction technique from swab, solubility, potency, toxicity (LD50), and improvement of limit of detection (LOD) of the method through analytical method validation were studied. In addition, roughness, mechanical and electro-polishing, consideration of dosage form as a quantitative factor, acceptable daily exposure (ADE), and permitted daily exposure (PDE) in the worst-case determination were considered in the study. The method was validated based on USP and ICH guidelines for specificity, limit of detection, limit of quantitation, precision, accuracy, linearity, and range. Linear regression analysis of data for the calibration plot in the range of 7.43, 10.89, 21.78, 43.56, 87.12 µg/mL, and relative standard deviation (R.S.D.) found to be 0.5, 0.4, 0.2, and 0.2, respectively with correlation coefficient of R2 = 0.999997. The LOD and the limit of quantitation (LOQ) were 2.23 and 7.43 µg/mL, respectively. Good recoveries in the range of 73.65-81.20%, and precision with relative standard deviation values lower than 15% have been obtained. The proposed method developed for cleaning validation is specific, precise, and useful for determination of cleaning acceptance limits using health-based limit and Quality Risk Management to develop an appropriate cleaning program for engineering design, safety of patients, and worker protection.

Evaluation of Swab and Rinse Sampling Procedures and Recovery Rate Determination in Cleaning Validation Considering Various Surfaces, Amount and Nature of the Residues and Contaminants.

A cleaning validation for a family of compounds utilizing swab sampling and rinse solution procedures, and high performance liquid chromatography for separation and detection of the analytes was performed.Effective parameters on recovery including sampling method, swab characteristics, solvent, swabbing technique, and material substance of product contact surfaces within the manufacturing equipment for swab and rinse sampling method, quantitative cleaning verification method, and active pharmaceutical ingredient (API) level and nature have been studied.The limit of detection and the limit of quantitation for the HPLC method were determined to be 0.0198 µg/mL, and 0.0495 µg/mL of the analyte, respectively. The linearity on replicate injections of the standard prepared in the range of 0.78, 1.55, 3.1, and 6.2 µg/mL, and relative standard deviation (R.S.D.) found to be 1.2, 1.0, 0.9, and 0.6, respectively with correlation coefficient of R2 = 0.9999. Recovery coverage for each type of surface was acceptable, ranging from 63.88% for swab sampling of stainless steel to 97.85% for rinse sampling of PVC. The acceptance criteria for precision on replicate injections of the analyte prepared in three concentration levels covering the specified range of 50, 100, and 200% was successfully accomplished R.S.D. lower than 15% for recovery results.Thus, choosing the appropriate sampling method, swab type, and surface condition can affect and increase recovery rate determination efficiency.

Prenatal Diagnosis of Tyrosinemia Type 1 Using Next Generation Sequencing.

Prenatal diagnosis using conventional molecular genetic techniques may be encountered with some limitations when the disease causing mutation is unknown. Here, we report on prenatal diagnosis of tyrosinemia in a family with consanguineous marriage and two affected children in whom no disease causing mutation had been identified before pregnancy. Mutation analyses of three genes associated with tyrosinemia including FAH, TAT and HPD were carried out in the fetal DNA sample using Next Generation Sequencing. A heterozygous nonsense mutation (p.Arg237Ter) in FAH gene was detected in the fetus. Further investigations suggested that the fetus was carrier of tyrosinemia type 1. This study demonstrates the successful application of Next Generation Sequencing in prenatal diagnosis, when the time is a limiting factor, more than one (especially large) responsible genes are involved, a "founder" or a "previously detected" mutation is not present and hence the conventional molecular genetic investigations cannot be employed.