Melinda Procter

Genotypes and serum concentrations of human alpha-1-antitrypsin P protein variants in a clinical population

Background: Alpha-1-antitrypsin (AAT) deficiency is a relatively common genetic disorder that can lead to the development of pulmonary disorders. Diagnosis of AAT deficiency is typically performed by isoelectric focusing (IEF) protein phenotyping in concert with determination of AAT serum concentration levels. The “P” phenotypic variant is associated with several known genetic variants that are found at unknown relative frequencies. Aims: To investigate the genetic variation of “P” alleles in patient samples. Methods: A DNA sequencing protocol for the full AAT coding region from serum was developed. Additionally, a retrospective evaluation of AAT concentrations in serum samples containing “P” allele IEF phenotype variants was undertaken. Results: “P” phenotypic variants are observed in ∼1 of every 900 samples received in the reference laboratory. Heterozygous “MP” allele samples exhibited a wide range of serum protein concentrations. Genotyping revealed the presence of the deleterious Plowell variant in six heterozygous MP samples, two heterozygous PZ samples, and one homozygous PP sample. A non-deleterious Pst albans variant was observed in a single MP sample. A novel heterozygous AAT M“P” variant, Psalt lake was identified, that did not exhibit a reduced AAT serum concentration. Conclusions: Genetic heterogeneity is present in clinical “P” phenotype variants identified by IEF, and the deleterious Plowell variant appears to be relatively common. Sequencing of “P” phenotype variants can provide useful clinical information, especially when the “P” phenotype variant is paired with a deficiency phenotype allele.

The Biotinidase Gene Variants Registry: A Paradigm Public Database

The BTD gene codes for production of biotinidase, the enzyme responsible for helping the body reuse and recycle the biotin found in foods. Biotinidase deficiency is an autosomal recessively inherited disorder resulting in the inability to recycle the vitamin biotin and affects approximately 1 in 60,000 newborns. If untreated, the depletion of intracellular biotin leads to impaired activities of the biotin-dependent carboxylases and can result in cutaneous and neurological abnormalities in individuals with the disorder. Mutations in the biotinidase gene (BTD) alter enzymatic function. To date, more than 165 mutations in BTD have been reported. Our group has developed a database that characterizes the known mutations and sequence variants in BTD. (http://arup.utah.edu/database/BTD/BTD_welcome.php). All sequence variants have been verified for their positions within the BTD gene and designated according to standard nomenclature suggested by Human Genome Variation Society (HGVS). In addition, we describe the change in the protein, indicate whether the variant is a known or likely mutation vs. a benign polymorphism, and include the reference that first described the alteration. We also indicate whether the alteration is known to be clinically pathological based on an observation of a known symptomatic individual or predicted to be pathological based on enzymatic activity or putative disruption of the protein structure. We incorporated the published phenotype to help establish genotype-phenotype correlations and facilitate this process for those performing mutation analysis and/or interpreting results. Other features of this database include disease information, relevant links about biotinidase deficiency, reference sequences, ability to query by various criteria, and the process for submitting novel variations. This database is free to the public and will be updated quarterly. This database is a paradigm for formulating databases for other inherited metabolic disorders.

Homozygous deletions of a copy number change detected by array CGH: A new cause for mental retardation?†

We describe two unrelated patients with mental retardation and normal karyotypes found to have relatively large homozygous deletions (>150 kb) of different regions detected by array comparative genomic hybridization (aCGH). Patient 1 showed a 157–214 kb deletion at 8q24.2, containing BAC clone RP11‐17M8. This patient was born to phenotypically normal parents and has microcephaly, distinctive craniofacial features, brachymetacarpia, brachymetatarsia and severe mental retardation. This BAC clone is listed as a copy number variant on the Database of Genomic Variants (http://projects.tcag.ca/variation/). Heterozygosity for the deletion was found in the mother (father is deceased) and uniparental disomy of chromosome 8 was excluded. Patient 2 showed a 812–902 kb deletion at 12q21.1, containing BAC clone RP11‐89P15. This region was not listed in any public database as a known variant. This patient has mild craniofacial dysmorphic features, bifid uvula, peripheral pulmonic stenosis and developmental delay. Heterozygosity for this deletion was confirmed in the phenotypically normal parents and two normal siblings, but surprisingly, homozygosity for the deletion in an apparently normal younger sibling brings into question whether this large homozygous copy number change (CNC) is causal. Homozygous deletions of CNCs have not previously been reported in association with a phenotype or mental retardation. These cases represent homozygosity for presumably benign CNCs, and while causality for the phenotypes cannot be confirmed, similar deletions are bound to be identified more frequently as aCGH is used with increasing regularity. Such homozygous deletions should be viewed as potentially clinically relevant.

Concordance of Butyrylcholinesterase Phenotype With Genotype

Butyrylcholinesterase (BChE) metabolizes the paralytic succinylcholine. Extended paralysis occurs in people with inherited BChE variants that may be identified by measuring BChE activity with and without the inhibitor dibucaine to calculate a dibucaine number (DN). Accurate phenotyping requires phenotype-specific BChE and DN reference intervals. We investigated the concordance between the biochemical BChE phenotype and the BCHE genotype to establish interpretive criteria for biochemical results. DNA was extracted from 45 serum specimens for which BChE activity and DN had been determined. The BCHE gene coding region was amplified and sequenced. Phenotype-genotype concordance and discordance occurred in 16 (36%) and 15 (33%) of specimens, respectively. A phenotype could not be assigned for 14 specimens (31%). An incorrectly assigned phenotype did not change the risk of prolonged paralysis or implied a slightly increased risk when there was none. Accurate BChE phenotyping is difficult using only enzyme activity and DN. The combination of biochemistry and BCHE genotype could improve the assessment of patient risk.

Challenging Identification of a Novel PiISF and the Rare PiMmaltonZ α1-Antitrypsin Deficiency Variants in Two Patients

OBJECTIVES α1-Antitrypsin (AAT) deficiency is associated with an increased risk for lung and liver disease. Identification of AAT deficiency as the underlying cause of these diseases is important in correct patient management. METHODS AAT deficiency is commonly diagnosed by demonstrating low concentrations of AAT followed by genotype and/or phenotype testing. However, this algorithm may miss novel AAT phenotypes. RESULTS We report two cases of AAT deficiency in two patients: a case of the novel phenotype PiISF, misclassified as PiII by phenotyping, and a case of the rare phenotype PiMmaltonZ misclassified as PiM2Z. CONCLUSIONS These cases highlight the importance of understanding the limitations of a commonly used diagnostic algorithm, use of further gene sequencing in applicable cases, and the potential for underdiagnosis of AAT deficiency in patients with chronic obstructive pulmonary disease.

Misclassification of an apparent alpha 1-antitrypsin “Z” deficiency variant by melting analysis

BACKGROUND Alpha-1 antitrypsin (AAT) is a protease inhibitor that protects the lungs from degradation by neutrophil elastase. AAT deficiency is associated with both lung and liver diseases. AAT deficiency is diagnosed using a combination of genetic and biochemical tests. Here we demonstrate how polymorphisms in the AAT gene can lead to genotype/phenotype discrepancies in common AAT assays. METHODS Total AAT was measured using an immunoturbidimetric assay. AAT phenotype was determined using isoelectric focusing. Genotypic identification of Z and S AAT alleles was performed by melt curve analysis using a LightCycler. Genotype/phenotype discrepancy was amended using gene sequencing. RESULTS Genotype and phenotype analysis produced conflicting results as a consequence of a polymorphism located in the probe designed to detect the Z allele. Sequencing revealed that the polymorphism had previously been reported as a rare P allele. The probe that caused the discrepancy was designed to match the WT sequence. A new probe was designed to specifically detect the Z allele, eliminating the possibility of future discordance. CONCLUSIONS Laboratories utilizing melt-curve analysis to diagnose patients should be aware of the potential for false positive results caused by polymorphisms located in the binding region of the genotyping probes. Alternatively, the probes should be designed to be specific to the mutation, rather than to the WT sequence.

A Patient with Prolonged Paralysis

A 19-year-old Asian male with no notable medical history presented to the emergency department with a 12-h history of acute abdominal pain. The patients condition was diagnosed as acute appendicitis, and he underwent an emergent laparoscopic appendectomy. A 1-mg dose of vecuronium followed by 120 mg of succinylcholine was administered to induce paralysis and facilitate endotracheal intubation. The progression of the patients muscle relaxation was monitored intraoperatively with a train-of-four twitch monitor and was marked by fewer stimuli making it across the neuromuscular junction. In general surgeries, a neuromuscular block down to 2 twitches is adequate for rapid sequence induction. Normally, a dose of 0.5–2 mg succinylcholine per kilogram body weight completely abolishes the muscle response to nerve stimulation. Within 2 to 2.5 min, the neuromuscular junction starts to show signs of recovery, or twitches. In this case, the patient was administered 1.7 mg/kg succinylcholine. After the appendectomy was completed, however, the patient uncharacteristically remained paralyzed for 1.75 h. He showed no muscle twitches, no spontaneous inspiratory efforts, and no protective airway reflexes. He subsequently required sedation and assisted ventilatory support. ### QUESTIONS TO CONSIDER 1. What are the pharmacodynamic properties of succinylcholine? 2. What is the role of butyrylcholinesterase in the pharmacokinetics of succinylcholine? 3. What conditions can cause delayed recovery from succinylcholine administration? 4. What additional testing should be used to further evaluate this patient? Cholinesterases are enzymes that catalyze the hydrolysis of choline esters. Acetylcholinesterase is distributed in the gray matter of the central nervous system, where it terminates synaptic transmission by specifically hydrolyzing the neurotransmitter acetylcholine (1, 2). Butyrylcholinesterase (BChE),4 also known as pseudocholinesterase, is distributed in the white matter of the central nervous system and in the blood. Although it has no known physiological function, BChE is of pharmacologic and toxicologic importance (1). Unlike acetylcholinesterase, BChE is capable of hydrolyzing exogenous …

Noncontinuously Binding Loop-Out Primers for Avoiding Problematic DNA Sequences in PCR and Sanger Sequencing

We present a method in which noncontinuously binding (loop-out) primers are used to exclude regions of DNA that typically interfere with PCR amplification and/or analysis by Sanger sequencing. Several scenarios were tested using this design principle, including M13-tagged PCR primers, non-M13-tagged PCR primers, and sequencing primers. With this technique, a single oligonucleotide is designed in two segments that flank, but do not include, a short region of problematic DNA sequence. During PCR amplification or sequencing, the problematic region is looped-out from the primer binding site, where it does not interfere with the reaction. Using this method, we successfully excluded regions of up to 46 nucleotides. Loop-out primers were longer than traditional primers (27 to 40 nucleotides) and had higher melting temperatures. This method allows the use of a standardized PCR protocol throughout an assay, keeps the number of PCRs to a minimum, reduces the chance for laboratory error, and, above all, does not interrupt the clinical laboratory workflow.