Increased Peripheral Blood Heteroplasmy of the mt.3243A>G Mutation Is Associated with Earlier End-Stage Kidney Disease: A Case Report and Review of the Literature.

The mitochondrial DNA mutation mt.3243A>G is most commonly associated with maternally inherited diabetes and deafness (MIM 52,000), but it has protean phenotypes including renal disease due to focal segmental glomerulosclerosis. We describe monozygotic twins who both harboured this mutation and developed ESRD. Although otherwise genetically identical, the twins differed in their peripheral blood leucocyte levels of circulating mt.3243A>G heteroplasmy: 20 versus 10%, when assessed at 42 years of age. The twin with the higher heteroplasmy load developed end-stage kidney disease 15 years earlier than her sister. A review of the published literature supports a relationship between heteroplasmy level and the age at the development of the end stage of renal failure in patients with mt.3243A>G-related kidney disease.

Bisalbuminaemia due to novel mutation at a critical residue involved in recycling; Albumin Lyon (510His→Arg).

OBJECTIVES: To define the underlying cause of bisalbuminaemia in an individual presenting with spontaneous venous thrombosis. METHOD: Plasma was examined by electrospray time-of-flight mass spectrometry (TOF MS) to assess albumin mutations and to quantify variant expression level. Tryptic peptide mapping and DNA sequencing were used to precisely define the mutation. RESULTS: Whole protein MS indicated a 19Da increase in the mass of 50% of the albumin molecules suggesting a His→Arg substitution. A novel heterozygous 510His→Arg mutation was identified by peptide mass mapping and confirmed by DNA sequencing of exon 12 of the albumin gene. CONCLUSION: The nature and location of the mutation suggest it would have no direct influence on haemostasis through altered warfarin binding or increased fibrinogen attachment and it appears to be incidental to the thrombotic phenotype. However the highly conserved His510 residue is recognised as being of critical importance in albumin recycling through interaction with its savaging neonatal Fc receptor. The normal albumin level of 41.1g/l and the coequal expression of albumin Lyon demonstrate that the conservative 510His→Arg substitution does not interfere with the pH dependant capture and release of albumin by the receptor.

Unique albumin with two silent substitutions (540Thr→Ala and 546Ala→Ser): Insights into how albumin is recycled.

OBJECTIVES: To determine the cause of an albumin abnormality detected by chance on electrospray time-of-flight mass spectrometry (TOF MS) of whole plasma, and to assess its physiological consequences. METHOD: Plasma was examined by TOF MS and tryptic mapping was used to locate mutation sites and determine the relative expression level of the variant and normal albumins. DNA sequencing was used to precisely define mutations. RESULTS: Whole protein electrospray TOF MS indicated a decrease of 14Da in the mass of albumin. Peptide mass mapping and DNA sequencing established the presence of two novel heterozygous point mutations (540Thr→Ala and 546Ala→Ser) whose combined mass changes (-30 and +16Da) indicated both mutations occurred on the same allele. Peptide ratios showed the variant albumin was present at a lower level than normal with an expression ratio of approximately 1:2 (variant:normal). Phylogenetic sequence alignments show Thr540 is highly conserved while Ala546 has wide species variation, suggesting 540Thr→Ala might compromise the protein. CONCLUSION: Both mutations occur close together in domain IIIB, a region involved in albumin scavenging and recycling. In particular, Thr540 is close to His535, a residue directly involved in pH-dependent binding and release of albumin from its recycling neonatal Fc receptor. Compromised receptor binding would explain the low albumin (34g/l) concentration and the diminished variant expression level.

Novel silent albumin variant (191Ala→Thr) detected by TOF MS of whole plasma.

BACKGROUND: Serum protein electrophoresis occasionally reveals multiple albumin bands referred to as bis- or alloalbuminaemia, and whilst the condition can be inherited it may also be acquired. METHODS: We present a new high resolution approach to the investigation of qualitative changes in albumin structure. The on-line reverse phase time-of-flight mass spectrometry procedure (TOF MS) elaborated here requires <0.2 µl of plasma and takes ~10 min to perform. Two plasma samples with classical bisalbuminaemia were used to verify the efficacy of the procedure for detecting genetic variants. When a novel mutation was detected, mass mapping of native unreduced albumin was used to pinpoint its location and inform targeted DNA sequencing of the albumin gene. RESULTS: Normal serum albumin showed its expected major isoform at 66,439 Da and the electrophoretic variants showed co-equal expression of additional components at -14 and +744 Da respectively. Surprisingly, one of the supposed controls showed paired albumin peaks at 66,439 and 66,469 Da and this 30 Da increase in mass was localised to between Arg(114) and Arg(197) and confirmed as being due to a novel 191Ala→Thr (+30 Da) substitution through DNA sequencing. CONCLUSIONS: The electrospray TOF MS approach developed here provides a rapid, sensitive and extremely precise method of revealing minute changes in albumin primary structure.

Association between endothelin type A receptor haplotypes and mortality in coronary heart disease.

AIMS: The endothelin type A receptor, encoded by EDNRA, mediates the effects of endothelin-1 to promote vasoconstriction, vascular cell growth, adhesion, fibrosis and thrombosis. We investigated the association between EDNRA haplotype and cardiovascular outcomes in patients with coronary artery disease. METHODS: Coronary disease patients (n = 1007) were genotyped for the His323His (rs5333) variant and one tag SNP from each of the major EDNRA haplotype blocks (rs6537484, rs1568136, rs5335 and rs10003447). EDNRA haplotype associations with clinical history, natriuretic peptides cardiac function and cardiovascular outcomes were tested over a median 3.8 years. RESULTS: Univariate analysis identified a 'low-risk' EDNRA haplotype associated with later age of Type 2 diabetes onset (p = 0.004) smaller BMI (p = 0.021), and reduced mortality (log rank p = 0.001). Cox proportional hazards analysis including established cardiovascular risk factors revealed an independent association between haplotype and mortality (p < 0.0001). CONCLUSION: These data highlight the potential importance of the endothelin system, and in particular EDNRA in coronary disease.