First year of life
The proband is a male first child of first-degree cousin parents from Pakistan (Punjabi ethnic origin). Fetal ultrasound during pregnancy revealed a bilateral clubfoot and NIHF (hydrothorax + ascites). For this reason, amniocentesis for karyotyping was performed and it gave a normal result: 46,XY; maternal-fetal infections and immune-haematological diseases were excluded.
The child was born by caesarean section at 32.5 weeks. His weight was 3613 g (he had generalized oedema), length was 52 cm, and cranial circumference was 36 cm (SDs + 5.6, + 4.0, and + 4.4, respectively, according to Olsen et al. [6]). The Apgar score was 4 at the first minute and the baby was intubated. He had respiratory distress with bilateral hydrothorax that needed right-side drainage for 12 days and mechanical ventilation for 8 days, ascites, and bilateral massive hydrocele. He also exhibited brachycephaly and bilateral clubfoot. No infectious or haematological diseases were seen in the baby. Figure 1 shows the infant at 20 days of life with a mildly coarse face and generalized oedema (Fig. 1a, b).
At 1 month of age, metabolic screening on the urine showed normal free sialic acid (109 mmol/mol creatinine with normal value < 123) while the conjugated and total sialic acid were increased (811 mmol/mol creatinine with normal value < 343 and 920 mmol/mol creatinine with normal value < 454, respectively), and increased GAGs (357 mg/g creatinine with normal value 5.9–60). These results prompted the enzyme analysis on fibroblast culture for MPS I, MPS II, MPS IVA and IVB, MPS VI, sialidosis, and mucolipidosis II, which were all normal.
During the following few months, the clinical evaluation showed worsening of the facial dysmorphism (see Fig. 1c, d), persistence of hydrocele, brachycephaly and bilateral clubfoot, pectus carinatum, gibbus, hepatomegaly, bilateral inguinal hernias, and joint stiffness; a restrictive chest wall deformity was observed with very limited or absent expansion of the cage and only diaphragmatic breathing. At 3 months of age he had axial hypotonia (he did not hold his head up), mild hypertonia in the upper limbs, and moderate hypertonia in the lower limbs. At the same age, heart and kidney functions were normal. Periodic abdominal ultrasound examinations showed progressive disappearance of ascites over 6 months. Brain stem evoked potentials evidenced bilateral severe hypoacusia while the ophthalmological evaluation was normal.
The skeleton x-ray showed dysostosis multiplex. In particular, spine abnormalities (craniocervical junction malformation, wedge-shaped L3 and L4, kyphosis, and scoliosis), oar ribs, hypoplasia of the inferior portion of the iliac bones and flared iliac wings, and squat femurs were seen (Fig. 2); brain magnetic resonance imaging (MRI) showed enlargement of the subarachnoid spaces and ventriculomegaly with spinal canal stenosis at the C1 level (Fig. 3). Growth curves of the patient are presented in (Fig. 4).
All these findings, together with his clinical history, supported the suspicion of MPS, and MPS VII was eventually investigated. The enzymatic assay for β-glucuronidase, performed in cultured fibroblasts, revealed a β-glucuronidase activity of 22 nmol/h/mg (normal value 200–600; residual activity 5.5%) and confirmed the diagnosis of MPS VII.
During the first year of life, the patient had frequent respiratory infections associated with wheezing and desaturation and underwent three surgical interventions: at 5 months for decompression of the spinal cord at the craniocervical junction, and at 10 and 12 months, respectively, for bilateral inguinal hernia and bilateral clubfoot.
At 12 months, a Griffiths test showed mildly delayed psychomotor development (general quotient 70).
Haematopoietic cell transplantation (HCT) and second year of life
At 14 months, the patient underwent successful HCT from an unrelated 5/6 human leukocyte antigen (HLA)-matched cord blood unit (total nucleated cells 7.3 × 107/kg, CD34+ cells 1.83 × 105/kg). The conditioning regimen included Busulfan and cyclophosphamide and graft-versus-host disease (GvHD) prophylaxis, anti-thymocyte globulin (ATG), cyclosporine, and methylprednisolone. Engraftment was achieved on day 31. The post-transplant course was complicated by rotavirus gut infection, Staphylococcus aureus bacteraemia, cytomegalovirus reactivation (the recipient was positive), and acute grade III GvHD, which all resolved. Chimerism had been continuously documented as 100% donor. He made developmental improvements and started walking independently at 20 months of life.
In the second year of life, he developed chronic pulmonary insufficiency with polypnoea and wheezing, needing chronic therapy with inhaled salbutamol and a positive end-expiratory pressure (PEEP) mask. Frequent acute exacerbations with deep desaturations were also observed with fever and/or infections and treated with an increased dosage of beta2 agonist plus anticholinergic bronchodilator, corticosteroids, and O2 therapy. Airway computed tomography (CT) scans at 13 and 20 months of life were similar and showed a restricted rib cage with multiple dystelectatic areas of the lungs and no tracheal abnormalities. From 20 months of age (6 months after transplantation) his maximum O2 saturation outside infection was 93–94% with lower values during sleep; from then on, he started chronic O2 administration at home during the night. At month 9 after HCT he started chronic betamethasone and O2 administration the whole day due to worsening respiratory distress. At 11 months after transplantation, the child had a new acute episode of respiratory distress that required hospitalisation in the Paediatric Intensive Care Unit with intubation and mechanical ventilation. Infection with respiratory syncytial virus was detected and, unfortunately, his respiratory insufficiency did not improve and he deceased at 25 months of age.
Molecular studies
DNA analysis of the proband identified the homozygous genetic variant c.1617C > T, leading to the synonymous mutation p.Ser539=. This nucleotide variation, generating a 5′ splice site (GT) in a non-canonical exonic position, had previously been reported as causing an aberrant partial skipping of the exon 10 (r.1616_1653del38) in a compound heterozygous patient [7]. To confirm the aberrant splicing, reverse transcriptase polymerase chain reaction (RT-PCR) was conducted on GUSB mRNA extracted from the proband’s fibroblast culture. The RT-PCR and sequence analyses showed the expected partial skipping of exon 10 (r.1616_1653del38) already reported by Yamada et al. [7] and, in addition, an abnormal shorter product in which complete skipping of exon 9 also occurred (r.1392_1476del85;r.1616_1653del38). No transcript of normal size was evident. The parents were both carriers of the c.1617C > T mutation.
Genetic counselling and pre-natal diagnosis
Based on the information provided during the genetic counselling, the couple requested pre-natal diagnosis in a subsequent pregnancy. Chorionic villus sampling (CVS) was performed. The molecular analysis revealed that the DNA from the CVS carried the homozygous c.1617C > T variant, as the affected proband. The couple decided to abort. At 18 weeks of gestation, ultrasound examination before abortion showed fetal hydrops, skin oedema, bilateral pleural effusion, and thickness of the placenta. No structural abnormalities of the fetal organs were observed and a normal volume of amniotic fluid was present. The skin and placenta examination by electron microscopy showed the presence of foamy cytoplasmic vacuoles with a weakly electron-dense substrate (Fig. 5), in accordance with the literature [7, 8].