Sanger sequencing solved a cryptic case of severe alpha1-antitrypsin deficiency.

AIMS: Alpha(1)-antitrypsin deficiency (AATD) is a clinically under-diagnosed genetic disorder that originates from deleterious mutations in the alpha(1)-antitrypsin (AAT) gene, SERPINA1. Severe deficiency is associated with significant pulmonary and hepatic malfunctions. Conventional clinical diagnosis involves the evaluation of serum AAT level and detection of diseased protein isoforms. In this communication, we describe the investigations of a case of severe AATD in which the AAT levels were well below those expected from the MZ phenotype determined by isoelectric focusing for protease inhibitor type (IEF PI-typing). METHODS: In addition to the traditional diagnostic method that combines the assessment of serum AAT concentration and IEF PI-typing, we investigated the SERPINA1 gene of the proband and participating family members for mutations using Sanger sequencing. RESULTS: We identified a novel mutation (M409T) in the proband, initially missed by the standard diagnostic approach. The novel mutation was present in 4 out of 8 family members who participated in the study. CONCLUSIONS: This report illustrates the diagnostic value of incorporating exon sequencing of the AAT gene into the algorithm for evaluating AATD, particularly when the AAT serum level is significantly lower than expected from IEF PI-typing.

Polyalanine expansion in the ZIC3 gene leading to X-linked heterotaxy with VACTERL association: a new polyalanine disorder?

BACKGROUND: The VACTERL association is a non-random association of congenital defects with an unknown aetiology in the majority of patients. METHODS: A male newborn is reported with features of the VACTERL association, including anal atresia, laryngeal and oesophageal atresia with tracheo-oesophageal fistula, dextroposition of the heart with persistent left superior vena cava, and unilateral multicystic kidney. As the clinical picture of this patient overlaps with that of X-linked heterotaxy caused by ZIC3 mutations, the ZIC3 coding region was sequenced. RESULTS: In a patient with the VACTERL association a 6-nucleotide insertion was found in the GCC repeat of the ZIC3 gene, which is predicted to expand the amino-terminal polyalanine repeat from 10 to 12 polyalanines. The polyalanine expansion is a novel ZIC3 mutation which was not found in 336 chromosomes from 192 ethnically matched controls. The mutation was also not present in the mother, suggesting it occurred de novo in the patient and is therefore a pathogenetic mutation. CONCLUSION: It is hypothesized that this novel and de novo polyalanine expansion in ZIC3 contributes to the VACTERL association in this patient. A newborn male is described with features of the VACTERL association, including anal atresia, laryngeal and oesophageal atresia with tracheo-oesophageal fistula, dextroposition of the heart with persistent left superior vena cava, and unilateral multicystic kidney. As the clinical picture of the VACTERL association overlaps with X-linked heterotaxy caused by ZIC3 mutations, the ZIC3 coding region was sequenced, and a 6-nucleotide insertion was found that is predicted to expand the amino-terminal polyalanine repeat from 10 to 12 polyalanines. This novel mutation was not present in the mother, nor in 336 chromosomes from 192 ethnically matched controls. It is hypothesised that this novel and de novo polyalanine expansion in the ZIC3 gene contributes to the VACTERL association in this patient.

Origin of amnion and implications for evaluation of the fetal genotype in cases of mosaicism.

OBJECTIVE: To investigate presence of trisomy in amniotic epithelium (uncultured amnion) and mesenchyme (cultured amnion) from mosaic cases to understand the origins of these tissues and their relationship to pregnancy outcome. METHODS: Polymerase chain reaction (PCR) of microsatellite loci was used to determine the presence of trisomy (of meiotic origin only) in amnion samples from 33 placentas previously ascertained because of a prenatal diagnosis of trisomy mosaicism that was predominantly confined to the placental tissues. RESULTS: In 16 (48%) of 33 cases, trisomy was confirmed to be present by molecular analysis of uncultured amnion. In contrast, cytogenetic analysis of cultured amnion showed trisomy in only 2 of 20 informative cases. The molecular detection of trisomy in amnion was strongly associated with poor pregnancy outcome (intrauterine growth restriction, fetal anomalies and/or intrauterine/neonatal death) even when analysis was limited to cases negative for the trisomy on amniotic fluid (N = 22, p = 0.0005). CONCLUSIONS: We infer that amniotic mesenchyme (usually diploid) derives from early embryonic mesoderm of the primitive streak and not from the hypoblast as is commonly cited. Trisomy in amniotic epithelium suggests that high numbers of abnormal cells were present in the epiblast, and this correlates with poor outcome even when the subsequently derived fetus and amniotic mesenchyme appear to carry only diploid cells.