Alpha-1-antitrypsin Deficiency in Fraternal Twins Born with Familial Spontaneous Pneumothorax

Background: Spontaneous pneumothorax is a rare condition that occurs when air accumulates in the pleural space without preceding trauma or obvious underlying lung disease. A positive family history is found in about 11.5% of cases and is referred to as familial spontaneous pneumothorax (FSP). In adults and adolescents, FSP has been mechanistically and genetically linked to Birt-Hogg-Dube syndrome, Marfan syndrome, alpha-1-antitrypsin (AAT) deficiency, homocystinuria, and Ehlers-Danlos syndrome. In neonates, the mechanism of FSP is poorly understood and is likely multifactoral. FSP is uncommon in neonates with four cases reported in the literature. There has not been an etiology established for neonatal FSP. We report a case of fraternal twin boys born with a spontaneous pneumothorax.

Case: The twins’ family history is remarkable for reactive airway disease and a female sibling born with spontaneous pneumothorax. The family had no history of connective tissue disorders, renal cancer, or dermatologic diseases. AAT phenotype analysis revealed identical, uninterpretable patterns in both infants. Additional investigations were performed to definitively identify what appeared to be a novel AAT phenotype.

Methods: Total AAT was measured using an immunoturbidimetric assay. AAT phenotype was determined using isoelectric focusing electrophoresis followed by AAT immunodetection. DNA was extracted from whole blood and all coding exons of the SERPINA1 gene (which codes for the AAT protein) were bidirectionally sequenced.

Results: Total serum AAT concentrations were 117 and 132 mg/dL for twin A and B, respectively (reference interval 100-200mg/dL). Analysis of their AAT genotype revealed that they were both compound heterozygous for rare SERPINA1 alleles. Additional serum and whole blood specimens were collected from the twins and their parents. Total AAT concentrations of the mother and father were 153 and 107 mg/dL, respectively. AAT phenotype analysis of the parents revealed that each expressed one normal and one abnormal AAT variant. Gene sequence analysis revealed that both parents carried one normal allele plus one rare SERPINA1 allele and that each twin had inherited both of the rare parental alleles. Allele S330F was inherited from the mother and A60T;K129E from the father. The A60T (M6passau) mutation has been previously reported to be a benign mutation; A60T has never been reported in cis with K129E. S330F (Smunich) has also been reported as a benign allele. Homozygosity for the rare A60T and S330F has not been reported; thus, the pathogenicity of the variants cannot be confirmed. The total AAT concentration in the second sample collected from twins A and B were 72 and 92 mg/dL, respectively, values compatible with an AAT deficiency. Since AAT is an acute phase reactant, the decrease in AAT concentrations between the two samples (11 days apart) is not unexpected following a traumatic birth. It is also noteworthy that twin A, who consistently had the lower AAT concentration, had a more severe case of FSP, requiring chest tube insertion and needle thoracentesis; twin B’s FSP resolved without additional intervention.

Conclusions: These findings suggest that the combination of A60T;K129E and S330F AAT alleles are likely deleterious and may play a role in neonatal FSP.

Materials and Methods Genotype Results

Conclusions

TABLE 1: Gene sequencing of the SERPINA1 alleles and total AAT concentration for the infant twins and their parents. Reference interval for AAT is 100-200 mg/dL.

Abstract

• Fraternal twin boys with a family history of respiratory problems were born with spontaneous pnemothorax.

• Isoelectric focusing electrophoresis analysis of serum revealed that the neonatal twins harbored an AAT phenotype that we were unable to interpret. Serum and whole blood were obtained from the family to elucidate the AAT results.

• DNA sequencing of the twins’ and their parents revealed that both parents were carriers of a rare allele and that the twins’ had inherited each of the rare alleles. One of these alleles (c.250G>A, c.457A>G) corresponding to the A60T;K129E variant had yet to be described in the literature. This variant was named ISalt Lake.

• The mechanism of FSP in neonates is poorly understood and is likely multifactoral, but our work suggests a role for AAT deficiency in the pathogenesis.

Patient Samples: Original serum samples for the twin infants were sent to the lab for routinephenotype testing. Upon realizing the complexity of the phenotype results, the physician was contacted andthe parents were offered to be tested as controls. The parents and their twin boys submitted additionalwhole blood and serum samples. Consent for testing was obtained from both parents.

Total AAT: Serum AAT was quantified using an immunoturbidimetric assay on the Roche ModularAnalytics P.

Isoelectric Focusing Electrophoresis: IEF was performed using the Hydragel A1AT Isofocusing kit(Sebia, Norcross, GA). Serum samples were applied to a 0.1% agarose (pH 4.2-4.9) IFE gel and electrophoresisperformed for 45 minutes at 20 ⁰C using 700-1000V. AAT bands were visualized using an AAT-antiserumconjugated to peroxidase which produced a pigmented stain after addition of acidic dimethylformamide inhydrogen peroxide. The AAT phenotype is determined by visual inspection and comparison to knownpatterns (Figure 1).

DNA Sequencing: The coding exons 2-5 of the AAT gene were amplified and sequenced bidirectionallyusing Big-dye-terminator chemistry on an ABI PRISM 3100 DNA Sequencer (Applied Biosystems, Foster City,CA). Data analysis was performed using Mutation Surveyor software (SoftGenetics, State College, PA).Sequence data was aligned to the published human chromosome 14 sequence containing the SERPINA1gene (AL132708.3).

Dina N Greene1, Melinda Procter2, Patti Krautscheid2, Rong Mao1,2, Elaine Lyon1,2, and David G Grenache1,2

1Department of Pathology, University of Utah, Salt Lake City, UT 841122ARUP Institute for Clinical and Experimental Pathology, Salt Lake City, UT 84108

Introduction

• AAT is a protease inhibitor that protects the lungs from degradation by neutrophilelastase. It is coded by the SERPINA1 gene, which is located on the long arm ofchromosome 14.

• SERPINA 1 is a polymorphic gene with > 100 alleles reported in the literature. Manyof these polymorphisms result in benign mutations, while others result in an AATdeficiency. AAT deficiencies are inherited in an autosomal codominant fashion and areassociated with both lung and liver diseases. In neonates, AAT deficiency is mostknown for its association with liver disease.

• In this case, AAT phenotype was ordered on twin infants because in adults, FSP is hasbeen associated with AAT deficiency.

• AAT deficiency is diagnosed using an amalgam of biochemical and genetic tests, thegold standard for which is isoelectric focusing electrophoresis. Total AATconcentration is also valuable in assessing AAT deficiency, but should always beinterpreted with caution because AAT is an acute phase reactant.

• FSP is a rare condition that occurs when air accumulates in the pleural space withoutpreceding trauma or obvious underlying lung disease.

• FSP is uncommon in neonates – only 4 cases have been reported in the literature andno etiology has been established.

Phenotype Results

1. Singh SA, Amin H. Familial spontaneous pneumothorax in neonates. Indian J Pediatr 2005; 72:445-4472. Stoller JK, Aboussouan LS. Alpha1-antitrypsin deficiency. Lancet 2005; 365:2225-22363. Greene DG, Elliott-Jelf MC, Straseski JA, et al. Facilitating the Laboratory Diagnosis of Alpha-1-antitrypsin

Deficiency. Manuscript in preparation.4. Faber JP, Poller W, Weidinger S, et al. Identification and DNA sequence analysis of 15 new alpha 1-antitrypsin

variants, including two PI*Q0 alleles and one deficient PI*M allele. Am J Hum Genet 1994; 55:1113-11215. Prins J, van der Meijden BB, Kraaijenhagen RJ, et al. Inherited chronic obstructive pulmonary disease: new

selective-sequencing workup for alpha1-antitrypsin deficiency identifies 2 previously unidentified null alleles. Clin Chem 2008; 54:101-107

6. Daniel R, Teba L. Spontaneous pneumothorax and alpha 1-antitrypsin deficiency. Respir Care 2000; 45:327-329

References

Acknowledgments

FIGURE 1: Examples of different AAT phenotypes using IEF

FIGURE 3: Family study shows that each parent expresses one wild type and one rare AAT variant

FIGURE 2: Twins’ AAT phenotypes are not consistent with any known AAT variants

aFirst draw at 6 days old; bSecond draw at 17 days old

Funding to support this study was provided by ARUP Laboratories and the ARUP Institute for Clinical and Experimental Pathology

FIGURE 4: DNA sequencing of the father identified a novel mutation (c.457A>G) that both twins inherited

Forward reference sequence

Forward sequencing result

Deviation from forward reference

Deviation from reverse reference

Reverse sequencing result

Reverse reference sequence