CDG: Congenital Disorders of Glycosylation; PMM2: Gene coding for the enzyme phosphomannomutase; PMI: Gene coding for phosphomannoseisomerase; FSH:Follicle Stimulant Hormone; LH:Luteinizing Hormone; Tf IEF:Isoelectric Focusing of Transferrin; PMD/MR: psychomotor delay/mental retardation; ALT:alanine aminotransferase; AST:aspartate aminotransferase; PCR: polymerase chain reaction; CK:creatine kinase; PMM: Phophomannomutase; PMI: Phophomannose Isomerase

Congenital Disorders of Glycosylation are pan-ethnic diseases [1,2] caused by the defective glycosylation of glycoproteins and glycolipids. Some 50 CDG have already been recognized [3]. Clinical manifestations range from severe multisystemic disease [4-7] to organspecific involvement [8,9]. The standard method of screening for CDG is plasma Tf IEF. There are two abnormal patterns: CDG type I (defective synthesis and incorporation of the oligosaccharide side chain), and CDG type II (defects in the processing of the oligosaccharide side chain). Some 77-83% of CDG-I patients have deficient phosphomannomutase activity (PMM2-CDG) [4,10]. PMM2-CDG has an estimated frequency of 1:20,000 births [11]. Except for PMI-CDG [12], and some patients with SLC35C1-CDG [13,14], there is no treatment available for CDG.

The general clinical picture of CDG is non-specific: PMD/MR, failure to thrive, seizures, ataxia, hypotonia, cerebellar atrophy, mild facial dysmorphism, strabismus, inverted nipples, hypogonadism, hepatopathy, peripheral neuropathy, coagulopathy, and stroke-like episodes. Due to the non-specificity of the clinical picture, several authors have recommended investigating CDG in patients with at least two affected organs or systems, especially when neurologic disorder (cerebellar hypoplasia, hypotonia, PMD/MR, and seizures), progressive ophtalmopathy or coagulopathy are present [7,8]. Others suggest to screen for CDG in any unexplained clinical disorder [15-18]. Apart from the unspecific Clinical manifestations, these disorders are underdiagnosed for a number of reasons: medical unawareness of the disease, increased mortality in the first years of life, and unavailability of widespread specific laboratory tests. We were unable to find in the literature the frequency of CDG among children with symptoms suggestive of CDG. There is scarce information on Brazilian patients with CDG [19-21].

This study aimed to determine the frequency of the disease in a large cohort of patients using broad inclusion criteria (PMD/MR associated with other symptoms suggestive of CDG), to study its clinical, radiologic, and laboratory characteristics, to determine the frequency of CDG-I and CDG-II, as well as to reveal the mutations in the PMM2 gene among Brazilian patients.

A total of 2619 patients presenting PMD/MR associated with other symptoms suggestive of CDG (ataxia, seizures, stereotypic movements, hypotonia, macro/microcephaly, delay of myelination, cerebellar atrophy, stroke, encephalopathy, iris coloboma, retinitis pigmentosa, strabismus, craniofacial dysmorphism, lipodystrophy, inverted nipples, failure to thrive, hypogonadism, or ichthyosis) were submitted for plasma Tf IEF [22,23]. According to the abnormal Tf IEF pattern, the patients were classified as CDG-I or CDG-II. The PMM2 gene was sequenced in the CDG-I patients, and/or leukocyte PMM and PMI activities determined [10]. According to PMM and PMI activities,CDG-I patients were then subdivided in PMM2-CDG or non-PMM2, non-PMI-CDG-I groups.

Mutation analysis of the PMM2 gene: Genomic DNA was extracted from blood samples anticoagulated with EDTA using standard procedures. The eight protein coding exons and flanking intronic sequences were directly sequenced after PCR [1,24]. Bidirectional sequencing was performed using the Big Dye Terminator Sequencing Kit (Applied Biosystems) according to the manufacturer’s protocol, and analyzed with a 3130xl Genetic Analyzer (Applied Biosystems).

Clinical, radiological, laboratory data were both retrospectively and prospectively obtained from the patients’ medical charts. Comparison of frequencies of clinical and laboratory findings between PMM2- CDG and non-PMM2, non-PMI-CDG-I groups were performed using Fisher’s Exact Test (software SSPS v13.0). The research project was approved by the Research Ethical Committee at the SARAH Network of Rehabilitation Hospitals.

Coagulation factors VIII, IX, XI, antithrombin, protein C, and free protein S were measured with kits from Diagnostica Stago (Asnière, France) using standard procedures. Other routine biochemical tests were performed (hemogram, serum transaminases, glycemia, TSH and T4, renal function tests, blood gases, and inborn errors of metabolism investigation), but only the relevant data were included in this study.

A total of 32 patients (1.2%; 14 males and 18 females) were identified. Based on the patterns found on Tf IEF (type I = 26 patients; type II = 6 patients), molecular studies, and/or enzyme determinations, they were classified into different groups:

CDG-I group (n=26)

Twenty-six patients presented Tf IEF type I pattern (1.0%; 26/2619). PMM2-CDG was confirmed in 13 individuals by demonstration of PMM deficiency and/or PMM2 gene mutations. Eight patients had normal PMM and PMI activities, and were thus classified as non- PMM2, non-PMI-CDG-I. Five individuals represent lost cases and were considered only for the calculation of CDG-I frequency.

PMM2-CDG patients: Clinical findings are summarized in Table 1, and Patients’ faces are shown in Figure 1. Nine patients were females and four were males. Coagulopathy, strabismus and cerebellar atrophy was seen in almost all patients. All other clinical symptoms were consistently found. Patient 4 had isolated protein C value very close above the upper limit, and was considered as not having coagulopathy. Elevated FSH/LH levels were found in 7/8 females and in 0/2 males.

Table 1: Main clinical findings in PMM2-CDG patients.

Figure 1: Dysmorphism of PMM2-CDG patients.

Non-PMM2, non-PMI-CDG-I patients: Clinical findings are summarized in Table 2 and facial features are shown in Figure 2. Four patients were females and four were males. Coagulation disorder was seen in all evaluated patients. PMD/MR and hypotonia was present in 7/8 patients. One patient was considered as not having PMD, because he was evaluated at a very young age (one month of age) and died before the next medical consultation. Abnormal cerebral imaging was detected in 5/7 patients (two boys had normal imaging). We found in this group, not only cerebellar atrophy, but also leukoencephalopathy, cerebral atrophy and stroke. Cryptorchidism/micropenis was found in 3/4 boys. Abnormal serum transaminases levels were seen in 5/7 patients. Three deaths occurred in this group (ages 9 months, 2 y 9 mo, and 3 y 9 mo).

Table 2: Main clinical findings in non-PMM, non-PMI-CDG-I patients.

Figure 2: Facial features with striking variability (from mucopolysaccharidosis-like face in patient B to almost normal face in patient C). Figures A to E (patients 2 to 6; table 2); F (patient 8; table 2).

Comparison between PMM2-CDG and non-PMM2, non-PMICDG- I patients: Most clinical features were equally seen in both groups, as determined by statistical analysis. Statistically significant differences (p<0.05) were limited to ataxia (p=0.032), strabismus (p=0.047), and elevated FSH and LH (p=0.026), which predominated among PMM2- CDG patients, while deaths occurred exclusively among non-PMM2, non-PMI-CDG-I patients (p=0.042). This latter group showed also higher frequency of abnormal results for antithrombin (p=0.035) and protein S (p=0.013), when compared to PMM2-CDG patients (tables 3 and 4). Stroke was found only in one non-PMM2, non-PMI-CDG-I patient.

Table 3: Plasma levels of prothrombin time, activated partial thromboplastin time, coagulation factors, and anticlotting factors of PMM2-CDG patients.

Table 4: Plasma levels of coagulation factors, natural anticoagulants, activated partial thromboplastin time, and prothrombin time of non-PMM2, non-PMI-CDG-I patients.

CDG-II group (n=6)

A total of 6 patients with CDG II pattern in Tf IEF (0.2%; 6/2619) were identified in this study. All presented coagulopathy. As shown in Figure 3 and in Table 5, four patients had similar clinical features: cutis laxa, facial dysmorphism, large fontanel, non-lissencephalic cortical dysplasia (Figure 4), and low levels of free protein S. Except for one, all presented also hip luxation and seizures. Low levels of factor XI were seen in three. The other two patients had facial dysmorphism; one presented also failure to thrive, cataract, myopathy and increased level of CK, and factor VIII. The other individual had macrocephahly, obesity, strabismus, myopia and increased levels of factor XI and protein C.

Figure 3: Patients A, B and C share the same features: down-slanting palpebral fissures, cutis laxa, progeroid aspect, and scoliosis. Patient D has only downslanting palpebral fissures. Patient E shows bitemporal narrowing, small nose with short philtrum. Figures A and B (patients 1 and 2; table 4); C (patient 4; table 4), D and E (patients 5 and 6; table 4).

Figure 4: Polymicrogyria/Pachygyriacomplex (CDG II patient 2).

Table 5: Main clinical findings in CDG-II patients.

PMM2 gene

R141H was the most prevalent mutation, present in 10/13 patients. One of these patients had also two other mutations: a novel (p.G79V; c.236G>T) and a common one (p.T226S). Other frequent mutations were p.T237M (n=5) and p.T226S (n=4). Interestingly, the former was found only in patients from Greater Belo Horizonte City area. One patient had another novel mutation (p.R21W; c.61C>T).

There is scarce data in the literature on frequency of CDG in populations. Schollen et al. [11] estimated the frequency of PMM2- CDG as 1:20,000 births, and found no increase of the carrier frequency for the R141H mutation in a cohort of 600 patients with mental retardation. Pérez-Cerdá et al. [25] found 50 CDG patients in a group of 7910 pediatric patients with clinical suspicion of metabolic diseases. This represents a frequency of 0.6%.

We used in the present study PMD/MR, the most prevalent symptom of the disease, as obligatory criterion associated with other symptom suggestive of CDG, in order to make the investigation directed to CDG symptoms. Using at least two symptoms as the inclusion criteria, we intended to increase the frequency of diagnosed cases, and thus, make clinical selection more Effective. This would spare resources.

The frequency of the disease among our patients was relatively high (1.2%; CDG-I 1.0%; CDG-II 0.2%). PMM2-CDG was present in at least 0.5% (13/2619) of the patients, and non-PMM2, non-PMI-CDG-I in at least 0.3% (8/2619).

There is a general agreement between our findings and cohorts with neurologic forms of PMM2-CDG [4,26,27]. The frequency of PMM2-CDG (13/21; 62%) in our sample was similar to that reported in the literature (77-83% of CDG-I) [4,10]. PMD/MR, cerebellar atrophy, strabismus, coagulopathy, hypotonia, hyporeflexia, facial dysmorphism, short stature, and hypergonadotropic hypogonadism (in females) were present in at least 69% of the PMM2-CDG patients. The presence of ataxia, strabismus and hypergonadotropic hypogonadism (in females), although not exclusively seen in PMM2-CDG, should favor this diagnosis. We identified 11 mutations in the PMM2 gene. The allele p.R141H is present in 47-81% of PMM2-CDG families in Europe [4,26,28]. In our study this allele was found in 8/11 families (73%). Two previously unreported mutations in the PMM2 gene were found. The p.R21W mutation was considered to be pathogenic. In the PMM1 gene Arg28 is the sole core domain contact, which interacts with the C-4 hydroxyl group of the substrate. This amino acid is located in the position 21 in the PMM2 gene [29]. Its substitution for trytptofan will prevent binding to the substrate. Patient 7, who has this novel mutation and the known p.T226S, shows enzyme activity close to zero. The p.G79V mutation was associated with two known pathogenic mutations (p.R141H and p.T226S). We submitted this mutation to algorithms (SNPs3D and nsSNPAnalyzer) that predict pathogenicity [30,31]. Both algorithms predicted p.G79V to be pathogenic. As shown in Table 1, this additional mutation seems to afffect significantly the protein function, as compared with other patients (patients 3 and 4). The mutation p.T237M was found exclusively in patients from Belo Horizonte city area and is likely to represent a founder effect. Brazilian population’s ancestry is composed largely by Portuguese and Italian immigrants. The mutation p.T237M has been identified among Italians [28]. Interestingly, all these patients bear Portuguese surnames. Hence, it could represent either a mutation in Portuguese genes or reveal an unknown Italian ancestry. The mutation p.D223E found in one family in the present study had only been identified among Danish [28]. The other mutations found in our study reveal the Iberic ancestry of Brazilian population. We could not find a genotype-phenotype correlation.

The most frequent clinical findings in our non-PMM2, non-PMICDG- I patients, such as PMD/MR, hypotonia, liver involvement, cerebellar atrophy, and coagulopathy, are similar to those previously reported [32]. In our series, they did not show distinctive symptoms from PMM2-CDG, except for the higher frequency of death, probably because they represent more severe subtypes. Laboratory data suggestive of hypergonadotropic hypogonadism were not found among these patients.

Clotting factors and anticlotting factors are glycoproteins. As many as 80% of the patients present coagulation abnormalities, but it seems that cerebral infarction is rare [33,34]. Coagulopathy, defined by abnormal levels of anticlotting and clotting factors, was present in 16 of the 17 CDG-I patients (Tables 1 and 2), but cerebral infarction was seen in only one non-PMM2, non-PMI-CDG-I patient. Mechanisms underlying clotting disorders in CDG are complex and not fully understood. In our study, non-PMM2, non-PMI-CDG-I patients showed, in general, more severe deficiency of clotting and anticlotting factors. There was no correlation between the coagulopathy and the degree of neurologic involvement, except for the patient 2, who had cerebral infarction. It is important to notice that levels of abnormal glycosylated glycoproteins fluctuate with age, are likely to worsen with fever or other stresses [35], and represent solely a static photography at the moment of the testing. So, direct inference from glycoprotein levels might be misleading.

All CDG-II patients presented coagulopathy. Mental retardation, cutis laxa, facial dysmorphysm (down-slanting palpebral fissures, and posteriorly rotated ears), and cerebral malformation were found in four patients with CDG-II, and probably represent a single entity. These features are consistent with the diagnosis of ATP6V0A2-CDG [36-38]. Myopathy is an uncommon feature in CDG-II and was reported in a patient with B4GALT1-CDG (formerly CDG IId) [39]. Similarly to this reported patient, CDG-II patient number 5 showed electroneuromyographic data suggestive of myopathy, elevated plasma CK, and asialo profile of Tf IEF (increased mono- to trisialotransferrin). We speculate that this is likely to be the diagnosis of this patient.

We thank the patients and their families. We are indebted with Dr. Eva Morava, Dirk Lefeber, and Ron Wever for the productive discussions on our patient’s diagnosis. We thank also Dr. Hudson Freeze for helping us with the technique of PMM and PMI assays, Savana Camilla de Lima Santos for helping with PMM2 gene sequencing, Guilherme Dottto Brand for the structural analyses of the mutations, and Luiz Guilherme Nadal Nunes for the statistical analysis. Finally, we are grateful to Dr. Jaak Jaeken for helping with interpretation of the transferrin IEF results.