The application of SNP array analysis, homozygosity mapping, and whole exome sequencing to a patient with OCA and neutropenia allowed us to diagnose two different disorders in a single, unique patient. This was possible in part because the patient was the product of a consanguineous mating. Furthermore, the identified mutations were present in genes previously associated with similar phenotypes (Boztug et al., 2009
; Rundshagen et al., 2004
), allowing for greater confidence that these mutations are causing the phenotypes seen in our patient.
One of the homozygous variants identified in our patient involved SLC45A2, associated with OCA-4. Reduced mRNA expression, consistent with nonsense-mediated decay, combined with absence of SLC45A2 protein detected by western blotting and immunofluorescence in cultured melanocytes, strongly suggested that the SLC45A2 mutation, c.986delC, is pathogenic. This case represents the first North American patient shown to have OCA-4, and to our knowledge, her melanocytes are the only such cultures obtained from an individual with OCA-4.
Relatively little is known about the function of SLC45A2 in human melanocytes, although mutations in the gene were identified as the cause of OCA-4 nearly a decade ago (Newton et al., 2001
). We showed that human SLC45A2-deficient melanocytes exhibited a normal distribution of PMEL17-tagged melanosomes, similar to what was observed in the Underwhite (uw
) mouse model of OCA-4 (Costin et al., 2003
). We also demonstrated that, in our patient’s cells, tyrosinase was mislocalized to the plasma membrane, suggesting that SLC45A2 functions in the correct trafficking of melanocyte-specific proteins to melanosomes, probably at the level of TGN sorting. Moreover, the presence of tyrosinase, in the growth medium of the patient’s cells, and specifically in exosomes (), suggests that tyrosinase is also prematurely secreted out of the cell without passing through melanosomes. These findings explain the hypopigmentation phenotype of OCA-4, and are consistent with observations in melanocytes from the uw
OCA-4 mouse model (Costin et al., 2003
). However, our results should be interpreted with caution, since the patient’s cells have a mutation in another disease-causing gene, with unknown effects on melanocytes.
The second mutation in our patient involves G6PC3. Three genes are known to encode enzymes that have glucose-6-phosphatase activity, i.e., G6PC1
. All three proteins have the same enzymatic activity, i.e., removal of the phosphate group from glucose-6-phosphate to produce glucose. G6PC1 is expressed in the liver, kidney and small intestine and catalyzes the essential hydrolysis of glucose-6-phosphate in the gluconeogenic and glycogenolytic pathways (Lei et al., 1993
). G6PC2 is expressed only in pancreatic islet cells where it is thought to function in glucose-dependent insulin secretion by controlling free glucose levels (Martin et al., 2001
; Petrolonis et al., 2004
). In contrast, G6PC3 is ubiquitously expressed, as evidenced by a wide range of affected tissues in patients with G6PC3
mutations. For example, such patients have atrial septal defects and low platelet counts (Boztug et al., 2009
), features that were also observed in our patient.
mutation identified in our patient, c.829C>T, was previously described in a French patient in the heterozygous state (Boztug et al., 2009
). However, since it is a null allele, this mutation was not functionally investigated. Furthermore, expression at the protein level could not be assessed because there is no commercially available antibody against the G6PC3. We did demonstrate a reduction in G6PC3
mRNA expression in our patient’s cells (), consistent with nonsense-mediated decay. This finding strongly implies decreased protein expression as well. Functional G6PC3 is required to maintain neutrophil viability; absence of the enzyme results in reduced neutrophil number and increased spontaneous apoptosis due to ER stress (Boztug et al., 2009
; Cheung et al., 2007
). We demonstrated a higher rate of spontaneous apoptosis in our patient’s fibroblasts compared to control cells. If the same phenomenon were to occur in neutrophils, the decreased G6PC3 protein expression could explain the neutropenia. It could also account for the relative paucity of mature neutrophils in the blood and bone marrow, since the older cells may be more likely to undergo apoptosis than survive.
Our patient’s inflammatory bowel disease could not readily be attributed to either SLC45A2 or G6PC3 mutations, but may be related to another mutated gene due to consanguinity.
Syndromic, multisystemic pigmentary disorders are excellent examples of diseases whose identification can be difficult, especially when consanguinity introduces the possible involvement of multiple genes. One approach in this situation is to perform homozygosity mapping based upon SNP array results, followed by whole exome sequencing.