MPP1/p55 gene deletion in a hemophilia A patient with ectrodactyly and severe developmental defects
Authors: Daniel I. Fritz, Toshihiko Hanada, Yunzhe Lu, J. Martin Johnston, Athar H. Chishti
Journal: American Journal of Hematology (2018)

Here, we report the first case of MPP1 (p55) gene deletion in a pediatric patient with severe hemophilia A, craniofacial developmental defects, and ectrodactyly of the appendages. Western blotting of erythrocyte membranes from the patient confirmed a complete loss of MPP1 protein. Sequencing revealed a deletion of 128,250 base pairs including most of the MPP1 gene and a substantial portion of the neighboring Factor VIII gene. Our findings showing profound craniofacial and limb ectrodactyly defects in a patient with severe hemophilia A raise the possibility that other hemophilia A patients with developmental abnormalities may be associated with MPP1 deficiency. MPP1 (Membrane Palmitoylated Protein 1) is a founding member of the MPP family of scaffolding proteins termed membrane-associated guanylate kinase homologues (MAGUKs).1 MAGUKs are composed of the conserved functional domains, PDZ (Post synaptic density protein, Drosophila disc large tumor suppressor protein, Zonula occludens-1 protein homology domain), SH3 (SRC Homology 3 domain), and GUK (GUanylate Kinase Domain).2 MPP1 is ubiquitously expressed but enriched in the erythrocytes where it was first discovered and characterized in our laboratory.3 The MPP1 (originally identified as p55) gene is located on the X chromosome and has been implicated in the regulation of membrane-cytoskeletal interactions as well as Ras and MAPK (Mitogen Activated Protein Kinase) signaling pathways.4-8 MPP1 functions as an adaptor protein and is thought to organize and establish cell polarity5, 8 as well as the localization of other proteins5 to the membrane associated cortical actin cytoskeleton. Previously, we generated MPP1 knockout mice showing phenotypes unrelated to erythrocyte stability including defects in neutrophil polarity and Akt signaling.9 With further breeding of the MPP1 KO mouse colony on a C57BL/6 genetic background, we observed developmental abnormalities with increasing frequency. To date, no human case of genetic abnormality in the MPP1 family has been reported. Routine prenatal ultrasound of a 20-year-old G1P0 African-American female in the second trimester revealed severe fetal limb anomalies. This prompted amniocentesis; genomic SNP microarray analysis demonstrated an interstitial deletion of the X chromosome; the most pertinent finding was a large deletion in the Factor VIII gene. On this basis, it was predicted that the fetus would exhibit severe hemophilia A. The patient, delivered by C-section, was noted to have severe craniofacial developmental defects (Figure 1A) and ectrodactyly of the appendages (Figure 1B–D). He also exhibited hypospadias and an undescended right testicle. No obvious malformations of the central nervous system, heart, major vessels, lungs, gastrointestinal tract or kidneys were detected. His clinical course has been characterized by the expected hemorrhagic issues and development of a Factor VIII inhibitor. There has been no clinical evidence of immunodeficiency. The patient has undergone bilateral through-knee amputations. Erythrocyte membranes from the patient were isolated using methods as described previously.10 The membrane protein composition was examined by gel electrophoresis and a complete loss of MPP1 protein expression was observed by Western blotting using a monoclonal antibody against MPP1 (Figure 1E,F, arrowhead). While the patient's erythrocytes clearly lack MPP1, other erythrocyte membrane cytoskeletal-associated proteins including spectrin, ankyrin, band 3, protein 4.1, protein 4.2, and actin appear to be expressed normally (Figure 1E,F). Dematin was used as an internal control to quantify the complete loss of MPP1 protein (Figure 1F). A blood smear indicated no significantly altered morphology of the erythrocytes due to the loss of MPP1 (Figure 1G). Blood parameters of the patient (RBC 4.26 × 106/μL, HGB 9.9 g/dL, HCT (hematocrit) 31%, MCV (mean corpuscular volume) 75.3 fL, MCH 23.4 pg, MCHC 31 g/dL, RDW (red cell distribution width) 19.2%, Reticulocytes 1.67%, WBC 8.65 × 103/μL, PLT (platelet count) 309 × 103/μL, MPV 8.5 fL) appear to be consistent with the normal erythrocyte morphology. The patient does show persistent unexplained normocytic anemia. Bone marrow of the patient is normocellular at 60% cellularity showing maturing trilineage hematopoiesis with an M:E ratio of 1.5:1. Megakaryocytes appeared normal, granulocyte series showed complete maturation, and there is no evidence of significant lymphocytosis or plasmacytosis. Metabolic and lipid markers appeared unremarkable with no hepatic, renal, or electrolyte abnormalities. Aspirate smears are cellular, and the erythroid series shows normoblastic maturation with occasional nuclear irregularities. Iron stains show no detectable storage iron. Future studies using BFU-E (burst-forming unit-erythroid) and CFU-E (colony-forming unit-erythroid) assays would be necessary to rule out a functional role of MPP1 in ineffective erythropoiesis. Prenatal genomic microarray analysis of the patient revealed a large deletion on the X chromosome in the q28 region. This deletion included Factor VIII, a gene that encodes an important clotting protein. MPP1 is a neighboring gene, which was predicted to have been included in the deletion based on the estimated mutation size (Figure 1I). Using genetic material from the patient, we sequenced the genome at Xq28 (Figure 1H), to accurately map the deletion size (Figure 1H–L). We sequentially amplified DNA from Factor VIII and MPP1 utilizing an "exon-hopping" approach, moving "inwards" towards the mutation site to determine an approximate deletion border. Then the deletion spanning region was cloned and sequenced using a "primer walking" strategy (details in the Supporting Information).11 Using publicly available genomic databases, we mapped the cloned sequence and determined that 72,289 base pairs of Factor VIII, 25,700 base pairs of MPP1, and 30,261 base pairs of the intergenic space between these genes were deleted; in total, the mutation deleted 128,250 base pairs (Figure 1J–L). Interestingly we detected a conserved 40 base-pair region that mapped to intron 21 of Factor VIII, indicating a small portion from the deleted region of the Factor VIII gene was intact (Figure 1J–L). Therefore, the remaining genetic material exists as a fusion of the MPP1 and Factor VIII genes (Figure 1L). No expression of MPP1 was detected, suggesting that the mutation completely abrogated any protein expression in erythrocytes (Figure 1E,F). The deletion was sufficient to eliminate the capacity of Factor VIII to regulate normal blood clotting mechanisms, causing the patient's severe hemophilia A. We compared the developmental defects seen in the patient (Figure 1A–D) with our established MPP1 null mouse model.9 Sequential backcrosses onto wild type (WT) C57BL/6 mice were performed for at least 10 generations. No discernible limb development abnormality analogous to the patient's condition was observed. However, we did observe frequent abnormalities in the face and eyes in female MPP1 KO mice (Supporting Information Figure S1A–D). Of note, the eye development abnormality frequently occurred unilaterally and observed specifically in females. We also observed frequent hydrocephaly in the MPP1 KO mice (Supporting Information Figure S1A). Generally, the mutant mice suffer from high mortality from unknown causes. No measurable difference in the osmotic fragility of wild type and MPP1 KO erythrocytes was observed (not shown). To probe the role of MPP1 in bone formation, we performed micro-CT analysis (SkyScan 1176) on both live MPP1 KO mice and post-mortem femurs. Cortical and trabecular osseous tissue was analyzed for density and morphology using standard parameters. Trabecular bone pattern factor was determined using methods previously described.12 We detected a significant left/right asymmetry in trabecular bone pattern factor, a value that represents bone architecture integrity and is indicative of trabecular bone strength. The 3D images of cortical bone and trabecular bone of the femur are shown (Supporting Information Figure S1E–H). These findings suggest a developmental role of MPP1 in the trabecular bone architecture and body left/right bone developmental symmetry. Perhaps of note, the patient's skeletal anomalies were similarly asymmetric, with greater deformity on the left side in both upper and lower extremities. Our findings showing profound craniofacial and limb ectrodactyly defects in a patient with severe hemophilia A raise the possibility that other hemophilia A patients with developmental abnormalities may be associated with MPP1 deficiency, thus suggesting a novel role for MPP1 in mammalian morphological development. Here we report the first known case of a loss of function mutation of MPP1 in humans. The mutation resulted in the loss of 128,250 base pairs and encompassed ~96% and ~61% of the MPP1 and Factor VIII genes, respectively. Of note, genes HMGN1P37 and SMIM9 are also located between the MPP1 and Factor VIII loci at Xq28. HMGN1P37 is a non-coding pseudogene with no known function and SMIM9 encodes Small Integral Membrane Protein 9 with no reported function. It is unlikely that the reported phenotypes are due to the loss of these functionally null genes. Until now, morphological developmental defects have never been linked with MPP1 loss of function. However, other homologous proteins in the MAGUK superfamily have been shown to be important for aspects of morphological development in both humans and mice. For example, loss of CASK in humans and mice causes significant defects including microcephaly, facial deformities, and intellectual disabilities.13 Similarly, the loss of discs-large gene in mice leads to craniofacial defects, abnormal eye morphology, cleft palate, and perinatal lethality.14 Considering these findings, MPP1 may emerge as an important target for establishing normal morphological development in mammals. Furthermore, patients who suffer from hemophilia A with morphological defects could also exhibit MPP1/p55 deficiency. These speculations will now be testable in future studies using both human and mouse models of MPP1 deficiency. We thank Prof Gary Sahagian and Dr Min Fang of Tufts Small Animal Imaging/Preclinical Testing Core Facility for the micro-CT analysis of MPP1/p55 null mice. We are particularly grateful to Donna-Marie Mironchuk for her invaluable contributions in the administrative organization and multiple improvements of figures. This study was supported in part by the National Institutes of Health Grants HL051445, HL095050 (AC), and American Heart Association Grant-in-Aid (15GRNT25710346) to AC. Nothing to report All authors read and approved the final version of the manuscript. D.F. performed gene mapping experiments, organized micro-CT scans, genotyped and characterized the MPP1/p55 null mice, and wrote the first draft of the article. T.H. regenerated the MPP1/p55 null mice and performed immunoblotting of MPP1 protein. Y.L. performed erythrocyte osmotic hemolysis assays, helped with the regeneration of MPP1/p55 null mice, and gel electrophoresis experiments. M.J. identified the patient, performed clinical evaluation and initial genetic testing, supplied the patient-derived material, and edited the article. A.C. supervised the project, organized the figures, and wrote multiple drafts of the article as principal investigator. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.