Showing papers by "Martha Skinner published in 1999"
TL;DR: A new TTR variant, Ser23-->Asn, was detected and identified using the above method where isoelectric focusing and restriction enzyme analysis failed to identify the nature of the variant.
Abstract: A mass spectrometry approach for the detection and identification of variants of the plasma protein transthyretin (TTR) is presented. The single amino acid substitutions found in TTR are closely associated with familial transthyretin amyloidosis (ATTR), a hereditary degenerative disease. A definitive diagnosis of ATTR relies on the detection and identification of TTR variants. The approach presented here is based on isolation of serum TTR using immunoprecipitation. The detection of the variant is achieved by mass measurement of the intact protein with electrospray ionization mass spectrometry (ESIMS). The liquid chromatography/ESIMS analysis of the tryptic digest of the protein followed by subsequent matrix-assisted laser desorption/ionization (MALDI) time-of-flight mass spectrometry and MALDI postsource decay of the relevant recovered chromatographic fraction containing the variant peptide allows the identification of unknown variants. The method was successfully tested using serum from ATTR patients wit...
102 citations
TL;DR: There are important associations in AL amyloidosis among Ig VL gene utilization, organ‐system tropism and post‐SCT survival, and these findings indicate that there are important associated with immunoglublin light chain variable region (Ig VL) germline gene utilization.
Abstract: AL (primary or immunoglobulin light chain) amyloidosis (AL) differs from myeloma per se in that tissue deposits of amyloid are found, typically in association with small numbers of clonal plasma cells producing lambda light chains, and also in that AL patients typically present with a predominantly dysfunctional organ-system. This constellation of features - fibrillar deposits comprised of light chains, lambda light chain predominance, and organ-system tropism and dysfunction - remains unexplained. Select patients with AL respond to haemopoietic stem cell transplantation (SCT) with clinical improvement and extended survival, particularly those who do not have cardiac involvement. In order to ascertain whether the organ-system tropism of AL was associated with immunoglublin light chain variable region (Ig VL) germline gene utilization, we attempted to clone, sequence and assign germline donors to the clonal Ig VL genes of 62 AL patients, all of whom were treated with SCT. We succeeded in 39 cases, identifying clonal AL genes derived from donors of the lambdaI (n = 10), lambdaII (n = 5), lambdaIII (n = 6), lambdaVI (n = 11) and KI (n = 7) subtypes. The majority of the donors (IGLV6S1, DPL5, DPL2, DPL23 and LFVK431) were genes that appear in the expressed repertoire <5% of the time, suggesting an intrinsic propensity to form amyloid under certain conditions. Patients whose clones derived from the lambdaVI IGLV6S1 donor uniformly presented with dominant renal involvement while those with other Vlambda or unknown donors often had dominant cardiac or other organ involvement, particularly patients whose clones derived from the lambdaI DPL2 donor. In addition, both early (<3 months) and overall post-SCT survival were significantly better in lambdaVI IGLV6S1 patients compared to patients with other Vlambda donors. These findings indicate that there are important associations in AL amyloidosis among Ig VL gene utilization, organ-system tropism and post-SCT survival.
80 citations
TL;DR: No definitive differences were identified between the mobilization regimens and IDM was an active regimen in AL for selected patients, and despite a 19% incidence of grade 4 toxicities, IDM therapy was well tolerated.
Abstract: AL amyloidosis patients ineligible for dose-intensive melphalan (200 mg/m2) were enrolled on a phase II trial to be treated with two cycles of intermediate-dose melphalan (IDM 100 mg/m2) and mobilized blood stem cells (BSC). For mobilization patients were randomized to either GM-CSF 250 μg/m2 for 3 d followed by G-CSF 10 μg/kg for 3 d (GM+G), or G-CSF 10 μg/kg for 6 d (G-alone), with leukaphereses on days 5, 6 and 7. To minimize morbidity, we planned to support each cycle with 3.5 × 106 CD34+ cells/kg and had a collection target of 7 × 106 CD34+ cells/kg. Those who did not achieve the target were treated with one cycle of IDM. 30 patients, a median of 62 years old and 7 months from diagnosis, were enrolled. Both mobilization regimens were generally well tolerated, and similar in terms of CD34+ cells and CFU-GM collected, but only 6/28 patients achieved the collection target (GM+G four, G-alone two). Despite a 19% incidence of grade 4 toxicities, IDM therapy was well tolerated. At a median follow-up of 24 months (19–36) 57% of patients had survived, 17% with durable complete haematological responses and 40% with improved or stable amyloid organ involvement, including 3/9 patients with predominant cardiac amyloid who are alive 2–3 years after treatment. The 100 d mortality was 20%. In conclusion, no definitive differences were identified between the mobilization regimens and IDM was an active regimen in AL for selected patients.
72 citations
TL;DR: The detection and characterization of a new transthyretin (ATTR) variant, Ser23Asn, associated with cardiomyopathy in a Portuguese patient with familial amyloidosis and genetic mutation analysis by DNA sequencing and allele-specific PCR confirmed this finding.
Abstract: The detection and characterization of a new transthyretin (ATTR) variant, Ser2 sn, associated with car-diomyopathy in a Portuguese patient with familial amyloidosis is described Isoelectric focusing (IEF) of serum from the propositus demonstrated heterozygosity for the presence of wild type and variant ATTR. A combination of mass spec-trometric (MS) analyses, including electrospray ionization mass spectrometry (ESI MS), high performance liquid chromatography (HPLC)/ESI MS and matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) performed on the serum-derived TTR were used to identify and locate the amino acid replacement in the variant protein. Genetic mutation analysis by DNA sequencing and allele-specific PCR confirmed this finding.
21 citations
TL;DR: A new TTR variant, Val122Ala, was characterized in an individual who carried the Gly6Ser polymorphism on the opposite allele, and the results were confirmed by DNA analysis.
Abstract: A new TTR variant, Vall22Ala, was characterized in an individual who carried the Gly6Ser polymorphism on the opposite allele. The main clinical feature of this familial transthyretin amyloidosis (ATTR) variant is extensive cardiomyopathy. The detection and characterization of the variant were performed using a combination of isoelectric focusing (IEF), restriction fragment length polymorphism (RFLP), immunoprecipitation, electrospray ionization mass spectrometry (ESIMS), HPLC (high performance liquid chromatography)/ESIMS, and matrix-assisted laser desorption/ionization mass spectrometry (MALDIMS). The results were confirmed by DNA analysis. The propositus has a brother who carries the new variant but not the polymorphism.
18 citations