Tafazzin splice variants and mutations in Barth syndrome.

Barth syndrome is caused by mutations in the TAZ (tafazzin) gene on human chromosome Xq28. The human tafazzin gene produces four major mRNA splice variants; two of which have been shown to be functional (TAZ lacking exon 5 and full-length) in complementation studies with yeast and Drosophila. This study characterizes the multiple alternative splice variants of TAZ mRNA and their proportions in blood samples from a cohort of individuals with Barth syndrome (BTHS). Because it has been reported that collection and processing methods can affect the expression of various genes, we tested and chose a stabilizing medium for collecting, shipping and processing of the blood samples of these individuals. In both healthy controls and in BTHS individuals, we found a greater variety of alternatively spliced forms than previously described, with a sizeable proportion of minor splice variants besides the four dominant isoforms. Individuals with certain exonic and intronic splice mutations produce additional mutant mRNAs that could be translated into two or more proteins with different amino acid substitutions in a single individual. A fraction of the minor splice variants is predicted to be non-productive.

A Novel Exonic Splicing Mutation in the TAZ (G4.5) Gene in a Case with Atypical Barth Syndrome.

OBJECTIVE: Barth syndrome is an X-linked recessive disorder characterized by dilated cardiomyopathy, neutropenia, 3-methylglutaconic aciduria, abnormal mitochondria, variably expressed skeletal myopathy, and growth delay. The disorder is caused by mutations in the tafazzin (TAZ/G4.5) gene located on Xq28. We report a novel exonic splicing mutation in the TAZ gene in a patient with atypical Barth syndrome. PATIENT & METHODS: The 4-month-old proband presented with respiratory distress, neutropenia, and dilated cardiomyopathy with reduced ejection fraction of 10%. No 3-methylglutaconic aciduria was detected on repeated urine organic acid analyses. Family history indicated that his maternal uncle died of endocardial fibroelastosis and dilated cardiomyopathy at 26 months. TAZ DNA sequencing, mRNA analysis, and cardiolipin analysis were performed. RESULTS: A novel nucleotide substitution c.553A>G in exon 7 of the TAZ gene was identified in the proband, predicting an amino acid substitution p.Met185Val. However, this mutation created a new splice donor signal within exon 7 causing mis-splicing of the message, producing two messages that only differ in the presence/absence of exon 5; these retain intron 6 and have only 11 bases of exon 7. Cardiolipin analysis confirmed the loss of tafazzin activity. The proband's mother, maternal aunt, and grandmother carry the same mutation. CONCLUSIONS: The identification of a TAZ gene mutation, mRNA analysis, and monolysocardiolipin/cardiolipin ratio determination were important for the diagnosis and genetic counseling in this family with atypical Barth syndrome that was not found to be associated with 3-methylglutaconic aciduria.

Barth syndrome.

First described in 1983, Barth syndrome (BTHS) is widely regarded as a rare X-linked genetic disease characterised by cardiomyopathy (CM), skeletal myopathy, growth delay, neutropenia and increased urinary excretion of 3-methylglutaconic acid (3-MGCA). Fewer than 200 living males are known worldwide, but evidence is accumulating that the disorder is substantially under-diagnosed. Clinical features include variable combinations of the following wide spectrum: dilated cardiomyopathy (DCM), hypertrophic cardiomyopathy (HCM), endocardial fibroelastosis (EFE), left ventricular non-compaction (LVNC), ventricular arrhythmia, sudden cardiac death, prolonged QTc interval, delayed motor milestones, proximal myopathy, lethargy and fatigue, neutropenia (absent to severe; persistent, intermittent or perfectly cyclical), compensatory monocytosis, recurrent bacterial infection, hypoglycaemia, lactic acidosis, growth and pubertal delay, feeding problems, failure to thrive, episodic diarrhoea, characteristic facies, and X-linked family history. Historically regarded as a cardiac disease, BTHS is now considered a multi-system disorder which may be first seen by many different specialists or generalists. Phenotypic breadth and variability present a major challenge to the diagnostician: some children with BTHS have never been neutropenic, whereas others lack increased 3-MGCA and a minority has occult or absent CM. Furthermore, BTHS was first described in 2010 as an unrecognised cause of fetal death. Disabling mutations or deletions of the tafazzin (TAZ) gene, located at Xq28, cause the disorder by reducing remodeling of cardiolipin, a principal phospholipid of the inner mitochondrial membrane. A definitive biochemical test, based on detecting abnormal ratios of different cardiolipin species, was first described in 2008. Key areas of differential diagnosis include metabolic and viral cardiomyopathies, mitochondrial diseases, and many causes of neutropenia and recurrent male miscarriage and stillbirth. Cardiolipin testing and TAZ sequencing now provide relatively rapid diagnostic testing, both prospectively and retrospectively, from a range of fresh or stored tissues, blood or neonatal bloodspots. TAZ sequencing also allows female carrier detection and antenatal screening. Management of BTHS includes medical therapy of CM, cardiac transplantation (in 14% of patients), antibiotic prophylaxis and granulocyte colony-stimulating factor (G-CSF) therapy. Multidisciplinary teams/clinics are essential for minimising hospital attendances and allowing many more individuals with BTHS to live into adulthood.

A homozygous double mutation in SMN1: a complicated genetic diagnosis of SMA.

Spinal muscular atrophy (SMA), the most common autosomal recessive cause of infant death, is typically diagnosed by determination of SMN1 copy number. Approximately 3-5% of patients with SMA retain at least one copy of the SMN1 gene carrying pathogenic insertions, deletions, or point mutations. We report a patient with SMA who is homozygous for two mutations carried in cis: an 8 bp duplication (c.48_55dupGGATTCCG; p.Val19fs*24) and a point mutation (c.662C>T; p.Pro221Leu). The consanguineous parents carry the same two mutations within one SMN1 gene copy. We demonstrate that a more accurate diagnosis of the disease is obtained through a novel diagnostic assay and development of a capillary electrophoresis method to determine the copy number of their mutant alleles. This illustrates the complexity of SMN mutations and suggests additional testing (gene sequencing) may be appropriate when based on family lines.

Multiple transmissions of Barth syndrome through an oocyte donor with a de novo TAZ mutation.

OBJECTIVE: To report recurrent transmissions of Barth syndrome through a single oocyte donor carrying a de novo TAZ mutation. DESIGN: Case report. SETTING: Clinical molecular diagnostics laboratory. PATIENT(S): Oocyte donor and individuals conceived with her oocytes. INTERVENTION(S): Molecular testing. MAIN OUTCOME MEASURE(S): Detection of TAZ mutation. RESULT(S): Multiple individuals affected with Barth syndrome conceived from a single oocyte donor who is a carrier of a de novo TAZ mutation. CONCLUSION(S): We report multiple transmissions of Barth syndrome through a single oocyte donor with a de novo TAZ mutation.

Cardiac and clinical phenotype in Barth syndrome.

OBJECTIVE: Barth syndrome, an X-linked disorder that is characterized by cardiomyopathy, neutropenia, skeletal myopathy, and growth delay, is caused by mutations in the taffazin gene at Xq28 that result in cardiolipin deficiency and abnormal mitochondria. The clinical phenotype in Barth syndrome has not been characterized systematically, and the condition may be underrecognized. We sought to evaluate extent of cardioskeletal myopathy, potential for arrhythmia, delays in growth, and biochemical correlates of disease severity in patients with this disorder. METHODS: We conducted an observational, cross-sectional study of the largest cohort of patients with Barth syndrome to date (n = 34; age range: 1.2-22.6 years). Evaluation included echocardiography, electrocardiography (standard and signal-averaged), microvolt T wave alternans analysis, biochemical and hematologic laboratory analyses, and physical therapy evaluation of skeletal myopathy. RESULTS: Family history was positive for confirmed or suspected Barth syndrome in 63%. Ninety percent of patients had a clinical history of cardiomyopathy (mean age at diagnosis of cardiomyopathy: 5.5 months; at genetic confirmation of Barth syndrome: 4.6 years). Echocardiography revealed a mean ejection fraction of 50% +/- 10%, mean fractional shortening of 28% +/- 5%, and mean left ventricular end-diastolic volume z score of 1.9 +/- 1.8. Left ventricular morphology demonstrated increased trabeculations or true noncompaction in 53%. Of 16 patients who were evaluated at > or = 11 years of age, 7 (43%) had documented ventricular arrhythmia. Growth deficiency was present (mean weight percentile: 15%; mean height percentile: 8%). Laboratory analysis revealed low total white blood cell count (absolute count: < 4000 cells per microL) in 25% of those who were not on granulocyte colony-stimulating factor. Hypocholesterolemia was present in 24%, decreased low-density lipoprotein cholesterol in 56%, low prealbumin in 79%, and mildly elevated creatine kinase in 15%. CONCLUSIONS: Our cohort demonstrated clinical variability, but most had cardiomyopathy and diminished growth velocity, with a propensity toward neutropenia and low cholesterol. There was increased incidence of ventricular arrhythmia, predominantly in adolescents and young adults. Barth syndrome should be considered when boys present with cardiomyopathy, especially when associated with increased left ventricular trabeculations, neutropenia, skeletal muscle weakness, or family history indicating an X-linked pattern of inheritance.

Paternal bias in parental origin of HRAS mutations in Costello syndrome.

Costello syndrome (CS) is a rare congenital condition caused by heterozygous de novo missense mutations affecting the codon for glycine 12 or 13 of the HRAS gene. We have identified 39 CS patients harboring the p.Gly12Ser mutation (NM_005343.2:c.34 G > A), two patients with c.35G > C mutations resulting in p.Gly12Ala substitutions, and one patient carrying the p.Gly13Cys substitution (c.37G > A). We analyzed the region flanking the mutated sites in 42 probands and 59 parents, and used four polymorphic markers to trace the parental origin of the germline mutations: one highly polymorphic hexanucleotide (GGGCCT) repeat region, defining three alleles with different numbers of repeat units (two, three, or four), and three SNPs. One of the SNPs, rs12628 (c.81T > C), was found in strong linkage disequilibrium with the hexanucleotide repeat region. Out of a total of 24 probands with polymorphic markers, 16 informative families were tested and the paternal origin of the germline mutation was found in 14 CS probands; a distribution that is neither consistent with an equal likelihood of mutations arising in either parent (P = 0.0018), nor with exclusive paternal origin.

HRAS mutation analysis in Costello syndrome: genotype and phenotype correlation.

Costello syndrome is a rare condition comprising mental retardation, distinctive facial appearance, cardiovascular abnormalities (typically pulmonic stenosis, hypertrophic cardiomyopathy, and/or atrial tachycardia), tumor predisposition, and skin and musculoskeletal abnormalities. Recently mutations in HRAS were identified in 12 Japanese and Italian patients with clinical information available on 7 of the Japanese patients. To expand the molecular delineation of Costello syndrome, we performed mutation analysis in 34 North American and 6 European (total 40) patients with Costello syndrome, and detected missense mutations in HRAS in 33 (82.5%) patients. All mutations affected either codon 12 or 13 of the protein product, with G12S occurring in 30 (90.9%) patients of the mutation-positive cases. In two patients, we found a mutation resulting in an alanine substitution in position 12 (G12A), and in one patient, we detected a novel mutation (G13C). Five different HRAS mutations have now been reported in Costello syndrome, however genotype-phenotype correlation remains incomplete.

Barth syndrome: TAZ gene mutations, mRNAs, and evolution.

Barth syndrome (MIM 302060) is an X-linked condition that includes dilated cardiomyopathy, neutropenia, failure to thrive, abnormal mitochondria, and 3-methylglutaconic aciduria. The mutated gene, TAZ, first described in 1996, appeared to produce a large set of alternatively spliced mRNAs with initiations of transcription upstream of exons 1 and 3. Since then, disease-causing mutations have been found in all exons including, most recently, a missense mutation in the controversial exon 5. Because of the initially described second initiation of transcription in intron 2, with in-frame initiation of translation in exon 3, we hypothesized that subjects with mutations in exons 1 and 2 would produce more normal "short product" that might attenuate their phenotype. Moreover, it was of interest to determine which splice variants were potentially functional as exon 5 is not present in yeast and rodents, and the variant lacking this exon is the most abundant. Using RT-PCR, we characterized TAZ mRNAs in cultured lymphocytes from nine subjects with Barth syndrome and two healthy controls. The TAZ genes and mRNAs of primates were also included. We found the following: (1) there is only one site for initiation of transcription, and the normal alternatively spliced assortment is limited to full-length, delta5, delta7, delta5delta7; (2) there are two alternative splice sites within introns 1 and 2 that could potentially produce an in-frame product; (3) exon 5 evolved into "exonhood" in the primate lineage after the split between Old World monkeys and hominoid primates; and (4) our results suggest that only two functional protein variants exist in lymphocytes: delta5 and full-length. Although exon 5 does not appear to be required for TAZ function in yeast and monkeys, its evolution to a highly conserved spliced exon in hominoid primates and the recent finding of an exon 5 mutation in a patient with Barth syndrome suggest that the full-length variant is important to TAZ function.