Clinical, biochemical and molecular correlations in MPS II patients
All of the data was available for the MPS II patients except for patients P9-P12 for whom only clinical and biochemical data were known because these patients died before their molecular data could be collected. The delay in diagnosis was explained by a lack of awareness among physicians of the specific MPS II clinical features associated with the adverse socioeconomic conditions of those patients.
Based on the clinical, biochemical, and molecular data, 6 patients (P1-P4 and P6–7) were classified in the MPS II group with a severe disease, and only one patient (P5) presented a mild phenotype.
According to the clinical data, the confirmation of diagnosis in all MPS II patients was done at a mean age of 5 years, unlike what is found in the literature [1]
The urinary GAG concentration ranged from 30.0 to 116 mg of creatinine, according to the age of each patient. The high level of heparan sulfate in the urine was correlated with the severity of the disease as previously described by Tomastsu S et al.,, who demonstrated a significant correlation between the level of heparan sulfate and the severity of this disease [11]
The leukocyte IDS activity in patients (P1-P4; P6-P8) with the severe type of the disease had a mean of 0.13 nmol/h/mg of proteins. Based on the high level of urinary GAGs and the deficiency of IDS activity, a relationship seems to exist between these data and the phenotypic expression of Hunter syndrome, contrasting with what is reported in the literature such as in Filipino patients [12] (Chiong et al., 2017). However, the clinical profiles of the MPS II patients (P1-P7) were in agreement with several studies described in the literature, and the clinical manifestations of the phenotype of Hunter syndrome ranged from moderate to severe Hunter syndrome phenotypes [12].
The most recurrent symptoms observed in this series ranged in degree of severity, including hepatosplenomegaly, coarse facial features including broad noses, macroglossia, psychomotor and mental retardation, multiple dysostoses including joint stiffness, oval vertebrae, respiratory problems including otitis, nasal obstruction, and enlarged tongue and adenoids.
Patient P8 was a girl related to Patient P2 who was hemizygous for the p.R88P mutation. She presented GAG excretion of 125 mg/g/creatinine and leukocyte IDS activity of 1.00%. She died before molecular analysis was conducted, but she probably had the same genetic mutation as patient P2 since she presented the same clinical profile as her cousin P2. MPS II females have been noted to present very rare clinical descriptions, and most of them present the severe form of the disease [13]. Importantly, the identification of MPS II heterozygous females by measurement of IDS activity and urinary GAG levels is unreliable. Therefore, the definitive diagnosis should be determined using genetic analysis [14]
Previous studies [15, 16] showed that the phenotypic expression of this disease in MPS II females is uncommon, and most of the cases described in the literature presented the severe phenotype. MPS II heterozygous females are rarely reported except for the presence of double mutant alleles or a coincidental genetic defect, leading to skewed X-inactivation or hemizygosity in heterozygotes [17]
Patient P12 was diagnosed at the age of three years old when he had an inguinal hernia operation. However, coarse facial features, including macrocrania, macroglossia and small teeth, had been noted at the age of eighteen months. He presented severe hepatosplenomegaly, skeletal disease, and severe mental retardation. The biochemical test showed that the leukocyte IDS activity in this patient was significantly higher than the enzyme activity of other MPS II patients. Patient P12 presented the severe phenotype of the MPS II disease, but he died before the molecular analysis hence the interest of carrier testing.
In this study, cardiovascular involvement, including arrhythmia and congestive heart failure, was identified in all MPS II patients and has been shown to be the cause of morbidity and mortality in most patients, as has been described previously in the literature [18].
Seven different mutations were found in the 12 MPS II patients. These nucleotides variations reflect the genetic heterogeneity leading to the wide spectrum of clinical phenotypes of MPS II in agreement with several other studies [4,12] (Chiong et al., 2017, Hopwood et al., 1993).
Sequence alterations in the IDS gene included five previously reported mutations and two novel mutations. The severe phenotype was found in patients who had the following mutations: c.240+1G>A, p.R88P, Ex1_7del, and p.Q396*. This in agreement with several previous studies (Table 3).
The missense mutation p.G94D was associated with a milder phenotype. This finding agrees with the data reported in Australian patients [4,19]. This mutation occurred within a conserved amino acid of human lysosomal sulfatase, which is essential for the common sulfatase activity [20].
The first novel alteration p.Q204*(c.610C>T) was a nonsense mutation and was identified in a patient who developed a severe form of MPS II. This mutation was due to a cytosine -to- thymine transversion at position 610 of the cDNA resulting in premature glycopolypeptide truncation at the 204th codon in exon 5 of IDS gene. Carrier testing was performed in the mother, who was found conductive.
The second novel frame shift mutation (p.D450Nfs*95) in exon 9 of the IDS gene is caused by a single-base deletion of guanine at genomic DNA position 1565. This mutation in exon 9 changes codon 450 from aspartic acid (GAT) to a chain termination codon (TAG) that leads to the lack of 95 amino acids at the amino terminus of the IDS protein. This novel mutation may lead misfolding of the glycopeptide resulting in a non-functional protein.
Mutations leading to a premature translation codon have frequently been classified as severe mutations; in agreement with this, the novel frame shift (p.D450Nfs*95) was found in a patient (P6) who presented the severe phenotype.
The p.D450Nfs*95 mutation results in exon skipping and introducing premature translation termination codon in exon nine with an abnormal IDS protein and have been classified as severe mutation. The premature stop codon causes a deletion of the last 5 amino acids of the heavy chain which contains the catalytic core (451455) and the entire light chain (456550) of IDS protein (Fig.3). The predicted premature stop codon could affects protein stability. In fact, the light chain of the IDS protein had an important role in the stability of the protein. Furthermore, the four antiparallel strands comprising the light chain are considerably longer than those of other sulfatases, and hence a greater contribution to the shape of the substrate-binding cleft comes directly from the light chain [21]. The expected severity of this mutation was variable and consequence range from local destabilization and misfolding to global unfolding, leading to premature degradation. The K479 residue in the exon 9 was important to the substrate binding [21]. The lack of this residue in our patient (P6) with p.D450Nfs*95 mutation result the nonfonctionnal IDS protein by the absence subtrate binding. Moreover, three too frame shift mutations were described in the exon 9 of IDS gene: p.R443X, p.R443X, p.Y466X and found in the patients who presented severe phenotype [22 ; 23]. However, investigation of mRNA and expression studies will be necessary to prove this conclusively. Correlation between genotype and phenotype was uncertain using genomic DNA. Further investigations such as transcription tests are useful to predict with confidence the disease phenotype.
In this study, there was no relationship between the genotype and phenotype in these MPS II patients except for the significant correlation between the high level of urine GAGs and the severity of the disease. Future studies with a large number of cases of the same age and genotype are needed to confirm this correlation in MPS II patients.