27 research outputs found

    Additional file 5: of Analytical parameters and validation of homopolymer detection in a pyrosequencing-based next generation sequencing system

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    Table S2. Self-designed primers used for assessing HP regions in the CFTR gene. Flopping bases on known SNPs are in brackets. “Tag sequences” also included in the beginning of the primers, separated by a space. (DOCX 58 kb

    Additional file 2: of Analytical parameters and validation of homopolymer detection in a pyrosequencing-based next generation sequencing system

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    Table S1. Mutagenesis, amplification and sequencing primers in the plasmid system. Amplification/sequencing primers contain a starting “Tag sequence,” which was separated by a space within the primer sequence. (DOCX 35 kb

    Molecular findings, family with JAM1 deficiency (index patient ID F1RO453).

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    (A) Pedigree; arrow, index patient. (B) Sanger-sequencing electropherogram of F11R exon 2 amplified from the index patient´s gDNA. Arrow, homozygous F11R c.65-2A>T splice-site variant. (C) Agarose-gel electropherogram of PCR products encompassing exons 1–10 amplified from nasopharyngeal swabs F11R cDNA. (D) Sanger-sequencing electropherogram of the 983-bp fragment amplified from nasopharyngeal-swab cDNA of proband. Arrow, skipped exon 2. (E) Sanger-sequencing electropherogram of the 1052-bp fragment amplified from control nasopharyngeal-swab cDNA (wild-type sequence). Arrows, breakpoints. (F) Graphical representation of F11R exons 2, 3, and 4 coverage by RNAseq of nasopharyngeal-swab mRNA in IGV Viewer.</p

    List of referring centres.

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    Background and aimGene defects contribute to the aetiology of intrahepatic cholestasis. We aimed to explore the outcome of whole-exome sequencing (WES) in a cohort of 51 patients with this diagnosis.Patients and methodsBoth paediatric (n = 33) and adult (n = 18) patients with cholestatic liver disease of unknown aetiology were eligible. WES was used for reassessment of 34 patients (23 children) without diagnostic genotypes in ABCB11, ATP8B1, ABCB4 or JAG1 demonstrable by previous Sanger sequencing, and for primary assessment of additional 17 patients (10 children). Nasopharyngeal swab mRNA was analysed to address variant pathogenicity in two families.ResultsWES revealed biallelic variation in 3 ciliopathy genes (PKHD1, TMEM67 and IFT172) in 4 clinically unrelated index subjects (3 children and 1 adult), heterozygosity for a known variant in PPOX in one adult index subject, and homozygosity for an unreported splice-site variation in F11R in one child. Whereas phenotypes of the index patients with mutated PKHD1, TMEM67, and PPOX corresponded with those elsewhere reported, how F11R variation underlies liver disease remains unclear. Two unrelated patients harboured different novel biallelic variants in IFT172, a gene implicated in short-rib thoracic dysplasia 10 and Bardet-Biedl syndrome 20. One patient, a homozygote for IFT172 rs780205001 c.167A>C p.(Lys56Thr) born to first cousins, had liver disease, interpreted on biopsy aged 4y as glycogen storage disease, followed by adult-onset nephronophthisis at 25y. The other, a compound heterozygote for novel frameshift variant IFT172 NM_015662.3 c.2070del p.(Met690Ilefs*11) and 2 syntenic missense variants IFT172 rs776310391 c.157T>A p.(Phe53Ile) and rs746462745 c.164C>G p.(Thr55Ser), had a severe 8mo cholestatic episode in early infancy, with persisting hyperbilirubinemia and fibrosis on imaging studies at 17y. No patient had skeletal malformations.ConclusionOur findings suggest association of IFT172 variants with non-syndromic cholestatic liver disease.</div

    Antibodies.

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    Background and aimGene defects contribute to the aetiology of intrahepatic cholestasis. We aimed to explore the outcome of whole-exome sequencing (WES) in a cohort of 51 patients with this diagnosis.Patients and methodsBoth paediatric (n = 33) and adult (n = 18) patients with cholestatic liver disease of unknown aetiology were eligible. WES was used for reassessment of 34 patients (23 children) without diagnostic genotypes in ABCB11, ATP8B1, ABCB4 or JAG1 demonstrable by previous Sanger sequencing, and for primary assessment of additional 17 patients (10 children). Nasopharyngeal swab mRNA was analysed to address variant pathogenicity in two families.ResultsWES revealed biallelic variation in 3 ciliopathy genes (PKHD1, TMEM67 and IFT172) in 4 clinically unrelated index subjects (3 children and 1 adult), heterozygosity for a known variant in PPOX in one adult index subject, and homozygosity for an unreported splice-site variation in F11R in one child. Whereas phenotypes of the index patients with mutated PKHD1, TMEM67, and PPOX corresponded with those elsewhere reported, how F11R variation underlies liver disease remains unclear. Two unrelated patients harboured different novel biallelic variants in IFT172, a gene implicated in short-rib thoracic dysplasia 10 and Bardet-Biedl syndrome 20. One patient, a homozygote for IFT172 rs780205001 c.167A>C p.(Lys56Thr) born to first cousins, had liver disease, interpreted on biopsy aged 4y as glycogen storage disease, followed by adult-onset nephronophthisis at 25y. The other, a compound heterozygote for novel frameshift variant IFT172 NM_015662.3 c.2070del p.(Met690Ilefs*11) and 2 syntenic missense variants IFT172 rs776310391 c.157T>A p.(Phe53Ile) and rs746462745 c.164C>G p.(Thr55Ser), had a severe 8mo cholestatic episode in early infancy, with persisting hyperbilirubinemia and fibrosis on imaging studies at 17y. No patient had skeletal malformations.ConclusionOur findings suggest association of IFT172 variants with non-syndromic cholestatic liver disease.</div
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