ABCMdb

PMID: 15371909

Edelmann L, Hashmi G, Song Y, Han Y, Kornreich R, Desnick RJ
Cystic fibrosis carrier screening: validation of a novel method using BeadChip technology.
Genet Med. 2004 Sep-Oct;6(5):431-8., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCC7 p.Arg117His ABCC7 p.Phe508Cys ABCC7 p.Ile506Val ABCC7 p.Ile507Val Key Words: cystic fibrosis, carrier screening, BeadChip technology Cystic fibrosis (CF) results from mutations in the CF transmembrane conductance regulator (CFTR) and is a common autosomal recessive disorder, particularly in individuals of Caucasian and Ashkenazi Jewish (AJ) ancestry.1,2 CF also affects individuals from other ethnic groups, including Hispanics, African Americans, and Asians with carrier frequencies ranging from 1in46to1in90.1 Morethan1000mutationshavebeendescribed in the CFTR gene and although many of them are private mutations, there are a number of mutations that are distributed worldwide and still others that are common to specific ethnic groups.3 In2001,theAmericanCollegesofMedicalGenetics(ACMG)and Obstetrics and Gynecologists (ACOG) established guidelines for prenatal carrier testing for CF that included a panel of 25 panethnic mutations with allele frequencies Ն 0.1% among CF patients inNorthAmerica.1,4 Inaddition,theyrecommendedthatcarriers of R117H be subsequently tested for the 5/7/9T polymorphic alleles in intron 8 and that individuals positive for delF508 and delI507 have reflex testing for interference from the benign variants F508C, I506V, and I507V.1 The ACMG/ACOG recommendations precipitated a dramatic increase in the number of CF tests performed in genetic testing laboratories. Login to comment 35 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Ala455Glu ABCC7 p.Ala455Glu ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Asn1303Lys ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Arg1162* ABCC7 p.Ile148Thr ABCC7 p.Gly85Glu ABCC7 p.Gly85Glu ABCC7 p.Arg560Thr ABCC7 p.Asp1152His Mutation controls included DNA from previously identified positive patient samples (I148T, D1152H, W1282X, R117H, G85E, A455E, delF508, N1303K) and DNA from NIGMS Human Genetic Cell Repositories (Coriell Cell Repositories) (delF508, delI507, G542X, R560T, 3849ϩ10kbCϾT, N1303K, G85E; G551D, R553X, 621ϩ1GϾT, 1717-1GϾA, A455E, R334W, R347P, R1162X, 3659delC; 711ϩ1GϾT, 2789ϩ5GϾA, 3120ϩ1GϾA). Login to comment 40 ABCC7 p.Asp1152His Allele-specific oligonucleotide hybridization Multiplex PCR analysis was performed in two reactions with seven amplimers in Group I and nine amplimers in Group II for the analysis of the ACMG panel of 25 mutations plus D1152H. Login to comment 46 ABCC7 p.Gly551Asp ABCC7 p.Arg117His ABCC7 p.Ala455Glu ABCC7 p.Trp1282* ABCC7 p.Arg553* ABCC7 p.Arg334Trp ABCC7 p.Arg347Pro ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Arg1162* ABCC7 p.Ile148Thr ABCC7 p.Gly85Glu ABCC7 p.Arg560Thr ABCC7 p.Asp1152His Mutant ASOs were end-labeled with ␥-32 P-ATP and pooled into three subgroups (IA-IC) for Group I and four subgroups (IIA-IID) for Group II mutations with the following breakdown of mutations: IA: delF508, delI507, W1282X, R117H; IB: G542X, R560T, 3849ϩ10kbCϾT, N1303K, G85E; IC: G551D, R553X, 621ϩ1GϾT, 1717-1GϾA, I148T; IIA: A455E, R334W, D1152H; IIB: R347P, 1078delT, R1162X, 3659delC; IIC: 711ϩ1GϾT, 1898ϩ1GϾA, 2789ϩ5GϾA, 3120ϩ1GϾA; IID: 2184delA. Login to comment 48 ABCC7 p.Arg117His Reflex testing for the 5T/7T/9T variants of the intron 8 polypyrimidine tract was performed for all R117H-positive samples. Login to comment 49 ABCC7 p.Phe508Cys ABCC7 p.Ile506Thr ABCC7 p.Ile507Thr Reflex testing of delF508 and delI507 positive samples for the F508C, I506T, and I507T variants was not necessary with this methodology. Login to comment 84 ABCC7 p.Arg117His ABCC7 p.Trp1282* ABCC7 p.Gly542* ABCC7 p.Arg560Thr Certain mutations including 711ϩ1GϾA, R117H, G542X, R560T, and W1282X, required a heterozygous allelic ratio with an upper limit set at 2.50. Login to comment 85 ABCC7 p.Gly551Asp ABCC7 p.Arg334Trp G551D and R334W required a heterozygous allelic ratio with an upper limit set at 3.00. Login to comment 87 ABCC7 p.Asp1152His RESULTS Validation strategy The BeadChip assay system and eMAP protocol were validated using a panel of 26 CF mutations currently screened for in our laboratory including the ACMG 25 recommended mu- tations plus D1152H, a mutation that is prevalent in the AJ population.15,16 To assess the overall performance and feasibility of this technology for use in the genetic testing laboratory, we blindly assayed 507 patient samples, 12 proficiency samples, and 145 control samples with the CF-26 BeadChip assay system and eMAP protocol, after reporting the testing results obtained by ASOH. Login to comment 105 ABCC7 p.Phe508Cys ABCC7 p.Ile506Val Genomic DNA from an F508C and an I506V carrier were amplified with the Group I multiplex primer mix and analyzed using the BeadChip assay system. Login to comment 106 ABCC7 p.Ile506Met In addition, single-stranded oligonucleotides for the I507V and I506M variants, 40 nucleotides in length, were tested directly on Group I BeadChips. Login to comment 108 ABCC7 p.Phe508Cys ABCC7 p.Ile506Val ABCC7 p.Ile506Met In addition, the PCR products amplified from the genomic DNA of F508C and I506V carriers and the single-stranded oligonucleotides for the I507V and I506M only elongated from the normal probe indicating that these variants did not interfere with allele discrimination. Login to comment 111 ABCC7 p.Arg117His This reflex test is used for screenees who test positive for the R117H mutation or are referred for male factor infertility and is performed by ASOH in our laboratory. Login to comment 130 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp A G551D/R553X compound heterozygote was reported as G551D homozygote, R553X heterozygote. Login to comment 134 ABCC7 p.Arg1162* Result of sample that was heterozygous for Group II mutation, R1162X. Login to comment 135 ABCC7 p.Gly551Asp allele was not able to elongate from the G551 normal oligonucleotide, but the G551D mutant allele was able to elongate from the R553 normal oligonucleotide. Login to comment 137 ABCC7 p.Gly551Asp There is no mismatch between the G551D mutant allele and the R553 normal oligonucleotide because the mutation is outside the region of complementarity. Login to comment 140 ABCC7 p.Gly551Asp However, the reported genotype is unlikely and the sample would have been reflexed to another method for the analysis of the G551D and R553X mutations. Login to comment 160 ABCC7 p.Arg117His ABCC7 p.Arg117His ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Trp1282* ABCC7 p.Asn1303Lys ABCC7 p.Gly542* ABCC7 p.Ile148Thr ABCC7 p.Ile148Thr ABCC7 p.Asp1152His ABCC7 p.Asp1152His ABCC7 p.Asp1152His I II III IV V VI VII VIII Totals Samples tested 87 57 69 72 66 35 72 61 519 Controls testedk 0h 17h 20 29 22 16 20 21 145 PCR Failuresi 4 4 2 1 1 2 1 3 18 (3.5%) Assay Failuresi 2 0 1 0 2 2 1 1 9 (1.7%) Positives 4a 3b 0 3c 4d 2e 2f 1g 19 (3.7%) a W1282X, delF508, D1152H, W1282X b delF508, delF508, D1152H c delF508, R117H, R117H d G542X, delF508, D1152H, N1303K (does not include proficiency samplesj ) e W1282X, delF508 f I148T, 3849ϩ10kbCϾT g I148T h Runs I and II were amplified with the same master mix and used the same control samples. Login to comment 162 ABCC7 p.Gly551Asp ABCC7 p.Gly551Asp ABCC7 p.Arg117His j Proficiency sample ϩs: delF508/G551D, R117H/delF508, R553X, delF508/delF508, 621ϩ1GϾT/delF508, delI507, delF508/3659delC, delF508/G551D k Control samples were not included in calculation of failure rates. Login to comment 163 ABCC7 p.Gly551Asp One control sample (G551D/R553X) failed in this study and is discussed in the text. Login to comment 167 ABCC7 p.Asp1152His Flexibility and limitations The CF-26 panel was developed at Bioarray Solutions, Ltd. specifically for custom use in our laboratory to mimic our current CF panel, which contains the 25 ABMG recommended CF mutations and the D1152H AJ mutation. Login to comment 175 ABCC7 p.Gly551Asp ABCC7 p.Arg553* ABCC7 p.Arg553* The problem encountered with the G551D/R553X control sample was the result of the R553X mutant strand failing to elongate from the G551 normal probe due to reduced annealing efficiency. Login to comment