Deletions of Xp22.2 Including PIG-A Locus Lead to Paroxysmal Nocturnal Hemoglobinuria.

Abstract 1158

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematopoietic stem cell disorder characterized by a global deficiency of glycosylphospatidyl inositol-floated proteins (GPI-AP) on the membrane of all cell types derived from the affected stem cell. While the PNH phenotype is non-malignant, outgrowth of GPI-AP (-) clones is mediated by a selective advantage in the context of a permissive condition, such as autoimmune-mediated bone marrow suppression as seen in aplastic anemia (AA). The GPI-AP (-) phenotype results from mutations of the PIG-A gene, located at Xp22.2, which participates in GPI synthesis. Because the gene is X-linked, a single frame-shift or missense inactivating mutation results in loss of enzyme function resulting in GPI-AP deficiency on the cell surface. Due to × chromosome inactivation, the frequency of PNH is similar in men and women.

New cytogenetic technologies such as single nucleotide polymorphism array (SNP-A)-based karyotyping show superb resolution allowing for detection of submicroscopic chromosomal defects not seen on metaphase karyotyping. We applied this technology to identify clonal aberrations in patients with PNH (N=32). The average size of the PNH by percentage of CD55/CD59 double negative granulocytes was 73%.

Among this cohort, we identified 3 patients with a clonal GPI (-) hematopoietic cell population resembling canonical PNH, but who did not harbor an identifiable mutation in PIG-A. Patients had classic symptoms of hemolysis and fairly typical clinical course. By quantitative PCR, these patients also lacked expression of PIG-A. During this molecular screen, a microdeletion of Xp22.2 encompassing the PIG-A locus, was found in GPI (-) sorted myeloid cells in these 3 patients using GPI (+) T cells as the comparator. This finding underscores the acquired somatic nature of this deletion in these patients. The deletions were 555kb, 506kb and 616kb in length. A commonly deleted region (CDR) of 437kb included PIGA as well seven known neighboring genes (ASB9, ASB11, FIGF, PIR, BMX, ACE2, and TMEM27) and predicted LOC100288981. One patient with the Xq22.2 deletion also had copy neutral loss of heterozygosity (CN-LOH) encompassing 9p24.3p21.3; the JAK2 V617F mutation was found in this patient. One of the remaining 29 patients had trisomy 8, 9 and 21, suggesting a concurrent diagnosis of myelodysplastic syndrome.

The PNH phenotype in the male patients (N=2) is easily explained by a hemizygous deletion of the X-linked PIG-A gene. However, in the female patient, the deletion must have occurred on the active (un-lyonized) × chromosome leading to loss of PIG-A transcription and thereby GPI (-) clonal expansion. Nested PCR reactions were performed to amply individual exons of the PIG-A gene and the presence of a wild-type PIG-A gene on the inactive × chromosome was verified by exon amplification, cloning, and sequencing. Our observation indicates that in addition to mutations, the PNH phenotype may arise from an alternate molecular mechanism, namely a segmental chromosomal deletion involving the PIGA locus in addition to inactivating mutations. While three instances of small intragenic deletions, including 2<4kb and one with undefined breakpoints limited to the PIGA gene, have been reported previously, our study describes the first cases of PNH due to a true chromosomal aberration involving Xp22.2 (∼500 kb), spanning a number of genes including PIGA. This clonal deletion of chromosomal material likely leads to the same selective clonal advantage as hypomorphic mutation of the PIG-A gene.