GM07441

GM07441 LCL from B-Lymphocyte

Description:

CYSTIC FIBROSIS; CF
CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR; CFTR
5,10-@METHYLENETETRAHYDROFOLATE REDUCTASE; MTHFR
HUMAN GENE MUTATION PANEL - CYSTIC FIBROSIS (VERSION 2)

Affected:

Yes

Sex:

Female

Age:

20 YR (At Sampling)

Overview

Repository

NIGMS Human Genetic Cell Repository

Subcollection

Heritable Diseases

Class

Other Disorders of Known Biochemistry

Biopsy Source

Peripheral vein

Cell Type

B-Lymphocyte

Tissue Type

Blood

Transformant

Epstein-Barr Virus

Sample Source

LCL from B-Lymphocyte

Race

Black/African American

Family Member

Relation to Proband

proband

Confirmation

Biochemical characterization - other

Species

Homo sapiens

Common Name

Human

Remarks

Elevated sweat chloride; pulmonary involvement; 2 similarly affected sibs; donor subject is a compound heterozygote: one allele of the CFTR gene carries a G-to-A substitution at 3120+1 in intron 16 (3120+1G>A ); a second allele of the CFTR gene carries a G-to-T substitution at 621+1 in intron 4 (621+1G>T); both mutations result in mRNA splicing defects; analysis of a DNA variant in a noncoding region of the CFTR gene (polypyrimidine tract in intron 8) showed this donor has alleles 7T/9T; donor subject is also heterozygous for a C>T mutation at nucleotide 677 in exon 4 of the methylenetetrahydrofolate reductase (MTHFR) gene [677C>T] that results in a substitution of a valine for an alanine at codon 222 [Ala222Val (A222V)]

Characterizations

IDENTIFICATION OF SPECIES OF ORIGIN

Species of Origin Confirmed by Nucleoside Phosphorylase, Glucose-6-Phosphate Dehydrogenase, and Lactate Dehydrogenase Isoenzyme Electrophoresis

CYSTIC FIBROSIS GENE: RFLP HAPLOTYPES & MUTATION STUDIES

PROBE: metD metD metH metH XV-2c KM.19 pJ3.11 pJ3.11 JG ENZYME: BanI TaqI MspI TaqI TaqI PstI MspI TaqI PstI RESULTS: 2,2 1,1 2,3 1,2 1,1 2,2 1,2 1,2 2,2 The above results were kindly provided to the Repository by Dr Arthur L. Beaudet, Baylor College of Medicine, Houston, TX. Neither allele shows the F508 deletion mutation (C. Goodpasture, VIVIGEN, Santa Fe, NM, personal communication).

GENE MAPPING & DOSAGE STUDIES - Y CHROMOSOME

PCR analysis of DNA from this cell culture gave a negative result with a primer for Yq11, DYS227.

MUTATION VERIFICATION

The CFTR mutations in this cell line have been verified by 6 laboratories. Methods used for mutation identification include: commercial kits (ABI and Roche); electrophoresis for RFLP and size analysis; sequencing; mutation scanning (heteroduplex analysis, dHPLC, SSCP, DGGE); chip technology; oligonucleotide ligation assay.

Gene

CFTR

Chromosomal Location

7q31.2

Allelic Variant 1

602421.0120; CYSTIC FIBROSIS

Identified Mutation

IVS16,G>A,+1 (3120+1G>A); Macek at al. [Am. J. Hum. Genet. 60: 1122-1127 (1997)] identified a splicing mutation, 3120+1G-A in intron 16 which occurred with a frequency of 12.3% in African Americans. This mutation was also detected in native African cystic fibrosis patients [Carles et al. J. Med. Genet. 33:802-804 (1996)]. Dork et al. [Am. J. Hum. Genet. 63: 656-662 (1998)] concluded that the 3120+1G-A mutation, which is present in African, Arab, and a few Greek families, probably was derived from a common ancestor because the haplotypes are very similar or identical.

Gene

MTHFR

Chromosomal Location

1p36.3

Allelic Variant 1

607093.0003; MTHFR THERMOLABILE POLYMORPHISM

Identified Mutation

677C>T; Frosst et al. [Nature Genet. 10: 111-113 (1995)] identified a C-to-T substitution at nucleotide 677 that converted an alanine to a valine residue. The alteration created a HinfI site that was used to screen 114 unselected French-Canadian chromosomes; the allele frequency of the substitution was 0.38. The mutation in the heterozygous or homozygous state correlated with reduced enzyme activity and increased thermolability in lymphocyte extracts; in vitro expression of the mutagenized cDNA containing the mutation confirmed its effect on thermolability of MTHFR. Individuals homozygous for the mutation had significantly elevated plasma homocysteine levels. Thus, the 677C-T mutation may represent an important genetic risk factor in vascular disease.

Gene

CFTR

Chromosomal Location

7q31.2

Allelic Variant 2

mRNA splicing defect; CYSTIC FIBROSIS

Identified Mutation

621+1G>T

Phenotypic Data

Remarks

Publications

Treff NR, Su J, Tao X, Miller KA, Levy B, Scott RT, A novel single-cell DNA fingerprinting method successfully distinguishes sibling human embryos Fertility and sterility94:477-84 2009

PubMed ID: 19394599

Lee HK, Lewis LD, Tsongalis GJ, Schur BC, Jannetto PJ, Wong SH, Yeo KT, Validation of a CYP2D6 genotyping panel on the NanoChip Molecular Biology Workstation Clinical chemistry53:823-8 2007

PubMed ID: 17363417

Bernacki SH, Beck JC, Muralidharan K, Schaefer FV, Shrimpton AE, Richie KL, Levin BC, Pont-Kingdon G, Stenzel TT., Characterization of publicly available lymphoblastoid cell lines for disease-associated mutations in 11 genes. Clin Chem51(11):2156-9 2005

PubMed ID: 16244288

Hadd AG, Laosinchai-Wolf W, Novak CR, Badgett MR, Isgur LA, Goldrick M, Walkerpeach CR, Microsphere bead arrays and sequence validation of 5/7/9T genotypes for multiplex screening of cystic fibrosis polymorphisms The Journal of molecular diagnostics : JMD6:348-55 2004

PubMed ID: 15507674

Matsuzaki H, Dong S, Loi H, Di X, Liu G, Hubbell E, Law J, Berntsen T, Chadha M, Hui H, Yang G, Kennedy GC, Webster TA, Cawley S, Walsh PS, Jones KW, Fodor SP, Mei R, Genotyping over 100,000 SNPs on a pair of oligonucleotide arrays. Nat Methods1(2):109-11 2004

PubMed ID: 15782172

Sebastian S, Spitzer SG, Grosso LE, Amos J, Schaefer FV, Lyon E, Wolff DJ, Hajianpour A, Taylor AK, Millson A, Stenzel TT, Multicenter characterization and validation of the intron-8 poly(T) tract (IVS8-T) status in 25 Coriell cell repository cystic fibrosis reference cell lines for cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation assays. Clin Chem50(1):251-4 2004

PubMed ID: 14709668

Bernacki SH, Farkas DH, Shi W, Chan V, Liu Y, Beck JC, Bailey KS, Pratt VM, Monaghan KG, Matteson KJ, Schaefer FV, Friez M, Shrimpton AE, Stenzel TT, Bioelectronic sensor technology for detection of cystic fibrosis and hereditary hemochromatosis mutations. Arch Pathol Lab Med127(12):1565-72 2003

PubMed ID: 14632577

Spence JE, Rosenbloom CL, O'Brien WE, Seilheimer DK, Cole S, Ferrell RE, Stern RC, Beaudet AL, Linkage of DNA markers to cystic fibrosis in 26 families. Am J Hum Genet39:729-34 1986

PubMed ID: 2879439