Howard C. Potter

Intermittent severe, symptomatic hyponatraemia due to the nephrogenic syndrome of inappropriate antidiuresis

A 20-year-old fit male soldier presented on two separate occasions 16 months apart with severe, symptomatic hyponatraemia and a clinical and biochemical picture consistent with the syndrome of inappropriate secretion of antidiuretic hormone (SIADH). In the intervening period, repeated plasma sodium values were in the reference range. Intensive investigation failed to reveal a cause for SIADH that was initially considered idiopathic. The description of a family comprising several adults with intermittent or water load induced-hyponatraemia associated with an activating mutation in the arginine vasopressin (AVP) receptor type 2 (AVPR2) raised the question of whether our patient could have a similar ‘nephrogenic syndrome of inappropriate antidiuresis’. Mutational screening of AVPR2 in our patient revealed a single missense mutation (R137C) in the second intracellular loop, which has been associated with constitutive activation of the AVPR2. In conclusion, adults with intermittent, severe hyponatraemia may have a constitutively activating mutation in the AVPR2 with resultant nephrogenic syndrome of inappropriate antidiuresis. Patients with idiopathic SIADH, particularly those with unmeasurable circulating AVP concentrations, should be considered for mutational screening of AVPR2.

Haemoglobin Manukau β67[E11] Val→Gly : transfusion-dependent haemolytic anaemia ameliorated by coexisting alpha thalassaemia

Summary Haemoglobin Manukau (β67 Val→Gly) is a novel haemoglobin variant presenting in two brothers as non‐spherocytic haemolytic anaemia which became transfusion dependent by 6 months of age. The severity of clinical expression seems to be modulated by coexisting alpha thalassaemia: the severely affected children have a normal complement of alpha globin genes with an unusual genotype (‐α3,7/ααα3,7), while their father, who carries the abnormal gene with minimal symptoms, has homozygous α+ thalassaemia (‐α3,7/ ‐α3,7) Another unusual feature of this case is the association of the β67 Val→Gly mutation with modification of β141 Leu to a residue (believed to be hydroxyleucine) that is not detected by standard amino acid analysis. This finding offers an explanation for the previous report of an association of another mutation at this site (Hb Sydney β67 Val→Ala) with Hb Coventry (deletion of β141 Leu).

Catalytic parameters for the hydrolysis of butyrylthiocholine by human serum butyrylcholinesterase variants

Catalysed hydrolysis of butyrylthiocholine (BTCh) by the usual (UU), fluoride-resistant (FS), AK, AJ and atypical (AA) human serum butyrylcholinesterase (EC 3.1.1.8) variants was measured in phosphate buffer pH 7.4 at 25 degrees C. pS-curves for all phenotypes were S-shaped; the activities rose to a plateau with increasing substrate concentration except at 100 mM where there was a small decrease. To obtain the catalytic constants, three equations were applied: Michaelis-Menten equation (Eq. 1), Hill equation (Eq. 2) and an equation which assumes simultaneous binding of the substrate to the catalytic site and to a peripheral site on the enzyme (Eq. 3). Over a range from 0.01 to 50 mM BTCh, the activity versus substrate concentration relationship deviated from Michaelis-Menten kinetics (Eq. 1) while data fitted well with Eqs. 2 and 3. The Michaelis-Menten equation was applied separately to two BTCh concentration ranges: the corresponding Km constants for the UU, FS, AK, AJ and AA phenotypes ranged from 0.1 to 0.2 mM (at 0.01-1.0 mM BTCh) and from 0.3 to 2.0 mM (at 1.0-50 mM BTCh). Hill coefficients (nH) calculated from Eq. 2 were similar for all phenotypes (nH approximately 0.5). The dissociation constants K1 and K2 calculated from Eq. 3 for two sites on the enzyme fell between 0.02 and 0.12 mM (K1) and 0.89 and 4.9 mM (K2) for the five phenotypes. Experimental data support the assumption that the phenotypes studied have two substrate binding sites.

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.

Sudden Cardiac Death Due to Deficiency of the Mitochondrial Inorganic Pyrophosphatase PPA2

We have used whole-exome sequencing in ten individuals from four unrelated pedigrees to identify biallelic missense mutations in the nuclear-encoded mitochondrial inorganic pyrophosphatase (PPA2) that are associated with mitochondrial disease. These individuals show a range of severity, indicating that PPA2 mutations may cause a spectrum of mitochondrial disease phenotypes. Severe symptoms include seizures, lactic acidosis, cardiac arrhythmia, and death within days of birth. In the index family, presentation was milder and manifested as cardiac fibrosis and an exquisite sensitivity to alcohol, leading to sudden arrhythmic cardiac death in the second decade of life. Comparison of normal and mutant PPA2-containing mitochondria from fibroblasts showed that the activity of inorganic pyrophosphatase was significantly reduced in affected individuals. Recombinant PPA2 enzymes modeling hypomorphic missense mutations had decreased activity that correlated with disease severity. These findings confirm the pathogenicity of PPA2 mutations and suggest that PPA2 is a cardiomyopathy-associated protein, which has a greater physiological importance in mitochondrial function than previously recognized.

Familial dysalbuminaemic hyperthyroxinaemia: a rapid and novel mass spectrometry approach to diagnosis.

Background Familial dysalbuminaemic hyperthyroxinaemia is an important cause of discordant thyroid function test results (due to an inherited albumin variant); however, the diagnosis can be challenging. A 51-year-old man had persistently elevated free thyroxine (T4), with discordant normal thyroid-stimulating hormone and normal free triiodothyronine. He was clinically euthyroid and had a daughter with similar thyroid function test results. We aimed to apply a whole protein mass spectrometry method to investigate this case of suspected familial dysalbuminaemic hyperthyroxinaemia. Methods Intact serum albumin was assessed directly using electrospray time-of-flight mass spectrometry. Results were confirmed using tryptic peptide m/z mapping and targeted DNA sequencing (exons 3 and 7 of the albumin gene). We also used this sequencing to screen 14 archived DNA samples that were negative for thyroid hormone receptor mutations (in suspected thyroid hormone resistance). Results Mass spectrometry analysis demonstrated heterozygosity for an albumin variant with a 19 Da decrease in mass, indicative of an Arg→His substitution. The familial dysalbuminaemic hyperthyroxinaemia variant was confirmed with peptide mapping (showing the precise location of the substitution, 218Arg→His) and DNA sequencing (showing guanine to adenine transition at codon 218 of exon 7). The same familial dysalbuminaemic hyperthyroxinaemia variant was identified in one additional screened sample. Conclusions Time-of-flight mass spectrometry is a novel procedure for diagnosing familial dysalbuminaemic hyperthyroxinaemia. The test is rapid (<10 min), can be performed on <2 μL of serum and requires minimal sample preparation.

Three cases of congenital adrenal hypoplasia with novel mutations in the (NROB1) DAX-1 gene

Abnormalities in the DAX-1 gene (dosage-sensitive sex reversal–adrenal hypoplasia gene on the X chromosome) are a well-recognized cause of congenital adrenal hypoplasia. DAX-1 is expressed in the adrenal cortex, gonads, hypothalamus and anterior pituitary, which gives rise to the clinical features of this deletion. Presentations are varied but salt-wasting and/or hypoglycaemia are the most common in an infant, with late onset of hypogonadotrophic hypogonadism. Over 80 different mutations in this gene have been identified. We present three unrelated cases with variable clinical presentations, all with novel mutations in the DAX-1 gene.

Elevated Second Trimester Amniotic Fluid Myoglobin from a Fetus with Duchenne Muscular Dystrophy

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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.

Familial dysalbuminaemic hyperthyroxinaemia: a novel mass spectrometry approach to diagnosis

Background and aims: Familial dysalbuminaemic hyperthyroxinaemia (FDH) is an important cause of discordant thyroid function tests (TFT). Free thyroxine (T4) levels are falsely elevated on some assays and current diagnostic tests for FDH are laborious. We aimed to investigate a novel, mass spectrometry approach to diagnosis of FDH in a 51-year-old man with persistently elevated free T4 levels, discordant normal TSH and normal free triiodothyronine. He was clinically euthyroid and had a daughter with similar TFT profile. Methods: Serum albumin was assessed using electrospray time-of-flight (TOF) liquid chromatography/mass spectrometry (LC/MS). Targeted sequencing of exons 3 and 7 of the albumin gene was used to confirm mutations and to screen 15 archived DNA samples that were negative for thyroid hormone receptor mutations. Results: LC/MS demonstrated heterozygosity for an albumin variant with a 19 Da decrease in mass, indicative of an Arg→His substitution. This was confirmed by DNA sequencing which showed a guanine to adenine transition at codon 218 of exon 7, a mutation known to increase T4 binding. Discussion: TOF LC/MS is a novel procedure for diagnosing FDH. The test is rapid (taking < 5 min), requires minimal sample preparation and can be performed on < 2 &mgr;L of serum or plasma.