multiplex ligation dependent probe amplification mlpa  (Radboud University)

Journal: Advanced Science

Article Title: Large‐Scale Production of Transfusion‐Ready Red Blood Cells From Induced Pluripotent Stem Cells

doi: 10.1002/advs.202504725

Figure Lengend Snippet: In vitro assays confirming functional, definitive erythroid characteristics of the terminally differentiated iRBCs. A) Cytospins combined with benzidine‐giemsa staining on filter‐purified RETs and on nERYs. B) RET‐size measurement by flow cytometry on CD235 + /DRAQ5 ‐ cell population. C) Marker expression pattern of the iRBCs using flow cytometry, n ≥ 58. D) Quantification of Hbe (α2ε2), HbF (α2γ2) and HbA (α2β2) based on HPLC tracks performed on iRBCs, n = 8. E) Oxygen dissociation curve of iRBC lines measured by the Hemox analyzer. F) Deformability assay or RETs and nERY measured by ARCA. G) Blood group phenotype measured on iPSC lines by DNA‐based (MLPA and SNP array)‐ and on iRBCs by protein‐based (flow cytometry)‐ assays, n ≥ 3.

Article Snippet: The multiplex ligation‐dependent probe amplification (MLPA) and SNP array were carried out by the Sanquin Diagnostic Department (Amsterdam, The Netherlands).

Techniques: In Vitro, Functional Assay, Staining, Purification, Flow Cytometry, Marker, Expressing

Journal: International Journal of Molecular Sciences

Article Title: PRPH2 -Related Retinal Dystrophies: Mutational Spectrum in 103 Families from a Spanish Cohort

doi: 10.3390/ijms25052913

Figure Lengend Snippet: Clinical characteristics of patients in our PRPH2 -associated cohort. MD: macular dystrophy; CR/CRD: cone dystrophy/cone–rod dystrophy; RP: retinitis pigmentosa; A: asymptomatic; AAO: age at onset; VAL: visual acuity loss; VFL: visual field loss; NB: night blindness.

Article Snippet: PRPH2 variants were identified using different molecular approaches: (a) commercial genotyping arrays (Asper Biotech, Tartu, Estonia), (b) direct Sanger sequencing of coding regions of PRPH2 , (c) multiplex ligation-dependent probe amplification (MLPA) kits for PRPH2 (MRC-Holland, Amsterdam, the Netherlands), and (d) next-generation sequencing (NGS), including custom IRD gene panels, clinical exome sequencing (CES) and whole-exome sequencing (WES), as previously described [ , , ].

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: PRPH2 -Related Retinal Dystrophies: Mutational Spectrum in 103 Families from a Spanish Cohort

doi: 10.3390/ijms25052913

Figure Lengend Snippet: PRPH2 disease-causing variants identified in FJD-cohort. TMD: transmembrane domain; ACMG: American College of Medical Genetics; P: pathogenic; LP: likely pathogenic; VUS: variants of uncertain significance; Het.: heterozygous state; Homo.: homozygous state.

Article Snippet: PRPH2 variants were identified using different molecular approaches: (a) commercial genotyping arrays (Asper Biotech, Tartu, Estonia), (b) direct Sanger sequencing of coding regions of PRPH2 , (c) multiplex ligation-dependent probe amplification (MLPA) kits for PRPH2 (MRC-Holland, Amsterdam, the Netherlands), and (d) next-generation sequencing (NGS), including custom IRD gene panels, clinical exome sequencing (CES) and whole-exome sequencing (WES), as previously described [ , , ].

Techniques: Variant Assay

Journal: International Journal of Molecular Sciences

Article Title: PRPH2 -Related Retinal Dystrophies: Mutational Spectrum in 103 Families from a Spanish Cohort

doi: 10.3390/ijms25052913

Figure Lengend Snippet: Variant distribution in the PRPH2 Spanish cohort. ( A ) Distribution of alleles by type of variant, considering 103 probands. ( B ) Location of disease-causing coding variants in the protein structure and associated phenotypes. ( C ) Distribution of proband alleles by protein domain location. ( D ) Distribution of non-truncating and truncating alleles found in probands inside and outside the D2-loop domain ( p -value = 0.0003). CNV: copy number variant; C: cytoplasmatic loop; D1: intradiscal loop D1 (D1-loop); D2: intradiscal loop D2 (D2-loop); RP: retinitis pigmentosa.

Article Snippet: PRPH2 variants were identified using different molecular approaches: (a) commercial genotyping arrays (Asper Biotech, Tartu, Estonia), (b) direct Sanger sequencing of coding regions of PRPH2 , (c) multiplex ligation-dependent probe amplification (MLPA) kits for PRPH2 (MRC-Holland, Amsterdam, the Netherlands), and (d) next-generation sequencing (NGS), including custom IRD gene panels, clinical exome sequencing (CES) and whole-exome sequencing (WES), as previously described [ , , ].

Techniques: Variant Assay

Journal: International Journal of Molecular Sciences

Article Title: PRPH2 -Related Retinal Dystrophies: Mutational Spectrum in 103 Families from a Spanish Cohort

doi: 10.3390/ijms25052913

Figure Lengend Snippet: Pedigrees and segregation of families with atypical genotypes. ( A ) PRPH2-057, carrying homozygously p.Gly208Asp, and ( B ) PRPH2-058 carrying 3 different alleles in 2 genes, PRPH2 and IMPG2 . Arrows indicate the proband in each family. m: pathogenic alleles; wt: wild-type alleles.

Article Snippet: PRPH2 variants were identified using different molecular approaches: (a) commercial genotyping arrays (Asper Biotech, Tartu, Estonia), (b) direct Sanger sequencing of coding regions of PRPH2 , (c) multiplex ligation-dependent probe amplification (MLPA) kits for PRPH2 (MRC-Holland, Amsterdam, the Netherlands), and (d) next-generation sequencing (NGS), including custom IRD gene panels, clinical exome sequencing (CES) and whole-exome sequencing (WES), as previously described [ , , ].

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: PRPH2 -Related Retinal Dystrophies: Mutational Spectrum in 103 Families from a Spanish Cohort

doi: 10.3390/ijms25052913

Figure Lengend Snippet: Retinal imaging of patients carrying PRPH2 . Tomographic, fundoscopic and fundus autofluorescence (FAF) of right eye (RE) and left eye (LE) for 2 patients with disease-causing PRPH2 variants. ( A ) Patient PRPH2-058 is a 56-year-old female, with a diagnosis of early-onset retinitis pigmentosa, carrying the prevalent variant p.Gly208Asp in PRPH2 , and biallelic variants in IMPG2 ((A1–A3): RE; (A4–A6): LE). OCT depicted the absence of extensive outer layers in both eyes affecting the fovea without cystic macular oedema. Fundoscopy revealed pale papilla with vascular attenuation, macula without pigmentary alterations, and absence of bone spicules but scattered pigmentary alterations in the periphery of both eyes. FAF images showed peripapillary atrophy with mottled hypoautofluorescence of the posterior pole, which was more evident in arcades, patches of atrophy in the extreme temporal periphery in RE and some small patches of atrophy in the extreme temporal periphery in LE. ( B ) Patient PRPH2-018-5 is a 61-year-old female, carrying the variant p.Arg142Trp, with a diagnosis of MD, for which clinical imaging shows a different severity in RE (B1–B2) and LE (B3–B4). OCT depicted well-delimited macular atrophy with the disappearance of outer retinal layers in RE and detachment of subfoveal neuroepithelium with thickening of the ellipsoid layer at this level in LE. FAF images showed well-demarcated hypoautofluorescence plaque affecting fovea with surrounding hyperaurofluorescence at its nasal rim and mottled hypo/hyperautofluorescence at its temporal rim in RE and central hypoautofluorescence with areas of reticular hyperautofluorescence within it in LE.

Article Snippet: PRPH2 variants were identified using different molecular approaches: (a) commercial genotyping arrays (Asper Biotech, Tartu, Estonia), (b) direct Sanger sequencing of coding regions of PRPH2 , (c) multiplex ligation-dependent probe amplification (MLPA) kits for PRPH2 (MRC-Holland, Amsterdam, the Netherlands), and (d) next-generation sequencing (NGS), including custom IRD gene panels, clinical exome sequencing (CES) and whole-exome sequencing (WES), as previously described [ , , ].

Techniques: Imaging, Variant Assay