DBA and DMD are two rare congenital anomalies. Both anomalies occurring in the same child is extremely rare. We report such an unusual case of a nine-year-old boy of north Indian descent, the third of four siblings born to healthy, non-consanguineous parents with a family history of DMD in a maternal first cousin. The boy was presented to our hospital at the age of eight months with progressive pallor and failure to thrive. He was diagnosed with DBA on the basis of peripheral blood showing normocytic, macrocytic anemia and reticulocytopenia with normocellular bone marrow showing erythroblastopenia. Though initially steroid-responsive, the boy later became unresponsive to both steroids and cyclosporine. By the fourth year of life, he was completely transfusion-dependent, and iron chelation was started only in the sixth year of life with intravenous desferrioxamine when his serum ferritin level was found to be 5100 ng/mL.
The patient had normal milestones until the fourth year of life. Thereafter he developed lower-limb weakness and calf muscle pseudohypertrophy. The weakness was progressive in nature, and by the age of five years the boy required support to climb stairs. His CPK level was high (5939 U/L), and his electromyogram was consistent with myopathy. He was diagnosed with DMD. A muscle biopsy was not done as the parents were unwilling to allow their son to undergo the procedure at that time. At this stage, the child was referred to our center for allo-HSCT treatment in view of his transfusion-dependent DBA with hyperferritinemia. The diagnosis was reconfirmed at our center. He also had dysmorphic facial features. However, RPS19 genetic studies could not be done.
The child had an unaffected elder brother who was a human leukocyte antigen (HLA)-identical match (6/6 antigen match) and underwent myeloablative allo-HSCT treatment. The conditioning regimen comprised busulphan (16 mg/kg over four days), cyclophosphamide (200 mg/kg over four days) and equine anti-thymocyte globulin (Atgam; Pfizer(Pharmacia & Upjohn company, a subsidiary of Pharmacia Corporation
Kalamazoo, Michigan 49001, USA) (90 mg/kg over three days). There was a minor blood group mismatch, with the recipient being in the B+ve group and the donor being in the O+ve blood group. Granulocyte colony-stimulating factor primed bone marrow was harvested from the donor while the donor was under general anesthesia. Plasma depletion was done using a Cryofuge 6000i centrifuge(by Heraeus Instruments made in Germany and supplied by Kendro Labarotories (India)) at 3000 rpm, and plasma-depleted marrow was infused with a cell dose of 6 × 108 mononuclear cells (MNCs)/kg body weight. Graft-versus-host disease (GVHD) prophylaxis consisted of standard dose methotrexate and cyclosporine. Neutrophil and platelet engraftment occurred on day+11 and day+16, respectively. RBC engraftment was confirmed with a change of blood group on day+90. The child has been transfusion-free since 2 months post-transplant, and his serum ferritin levels have been reduced to 600 ng/mL with regular phlebotomies. The boy's blood counts were normal 45 months after allo-HSCT treatment: hemoglobin 13.5 g/dL, reticulocyte count 1%, white blood cell count 6.0 × 103/μL with 70% neutrophils and platelet count 1900 × 103/μL.
This patient was evaluated by a neurologist pre-transplant and periodically post-transplant. The patient was wheelchair-bound pre-transplant, and, 45 months post-transplant, there has been no clinical deterioration whatsoever in the boy's motor power. He continues to be wheelchair-bound and is able to sit on his own for more than three hours at a stretch. The CPK level pre-transplant had been in the range of 9000 to 14,000 U/L and showed a declining trend within four weeks of transplantation, reaching a nadir of 300 U/L by six months post-transplantation. The muscle biopsy, which was done twice at 730 and 1250 days post-HSCT, respectively, revealed mixed donor chimerism with 8% to 10% cells of donor origin. Sequential chimerism studies using whole blood established trilineage engraftment with 100% donor chimerism. There were no post-transplant complications in the form of sepsis, hepatic veno-occlusive disease or acute or chronic GVHD.
Chimerism analysis performed on recipient peripheral blood samples on day+30, day+90, day+365, day+730 and day+1250 revealed complete donor chimerism post-transplant. A muscle biopsy was performed twice post-bone marrow transplantation (BMT) at days 730 and 1250 to study histopathology, dystrophin expression and chimerism status. Immunostaining for dystrophin I was reduced, dystrophin II and III were absent and there was up-regulation of utrophin.
Chimerism was performed to monitor donor cell engraftment in the recipient peripheral blood and was also used to detect donor cells in the muscle biopsy from the recipient. Chimerism was carried out using recipient's hair follicle as the pre-transplant sample [11], peripheral whole blood as the post-HSCT sample, and whole blood sample from the donor. Blood was collected in ethylenediaminetetraacetic acid vacutainers. DNA extraction from whole blood was carried out using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Preparation of hair follicles was done with 10 full-length hairs with roots plucked from different areas of the recipient's scalp. The presence of the hair bulb was visually confirmed. Careful washing was done to minimize the risk of blood contamination by rinsing in normal saline. The QIAamp DNA Mini Kit was used for DNA extraction from hair roots and muscle biopsy. The muscle biopsy specimen was preserved in 10.4% formaldehyde for routine histopathological processing and stained with hematoxylin and eosin (Figure 1).
The fragment of muscle biopsy taken for chimerism analysis was washed in saline seven times to remove all traces of RBCs and peripheral blood MNCs (PBMCs). The muscle tissue was homogenized and further rinsed three times in saline, following which a frozen section of the homogenized muscle was taken to check for any contamination with PBMCs (Figure 2). Genomic DNA was extracted from the muscle tissue cryosections serially cut from biopsies using the QIAamp DNA Blood Mini Kit according to the manufacturer's instructions.
Chimerism of all samples was performed using 16 short tandem repeat markers (D8S1179, D21S11, D7S820, CSF1PO, D3S1358, THO1, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, AMEL, D5S818 and FGA) labeled with four types of fluorescent dyes (6FAM, VIC, NED and PET). A multiplex polymerase chain reaction assay was performed in a final reaction volume of 25 μL containing 10.5 μL of reaction mixture, 5.5 μL of primer mix, 0.5 μL of AmpFl STR Identifier kit (Applied Biosystems, Foster City, CA, USA) and 10 μL of DNA at a concentration of 0.2 ng/μL. Cycling parameters were optimized as follows: 95°C for 11 minutes (one hold), 94°C for 60 seconds, 59°C for 60 seconds, 72°C for 60 seconds and 28 cycles, 60°C for 60 minutes (two holds) in a GeneAmp PCR System 9700 (Applied Biosystems). Denaturation was performed for five minutes at 95°C using Hi-Di Formamide and GeneScan 500 LIZ Size Standard (both from Applied Biosystems). The amplicon was resolved by performing capillary electrophoresis using the ABI Prism 3100-Avant Genetic Analyzer System (Applied Biosystems) and analyzed using the Applied Biosystems Gene mapper TM software v 3.5, Foster City CA 94404, USA. To determine the fraction of donor cells in the recipient's peripheral blood and muscle biopsy samples post-transplantation, the informative markers were identified and the percentage of donor cells was estimated. While the post-transplant recipient peripheral blood sample displayed complete donor chimerism, the muscle biopsy sample showed mixed donor chimerism with 8% to 10.4% donor cells on days 730 and 1250, respectively (Figure 3), suggesting the presence of donor-derived cells in the recipient's muscle.
