Els Schollen

PTPN11 mutations in LEOPARD syndrome

LEOPARD syndrome is an autosomal dominant disorder with multiple lentigines, congenital cardiac abnormalities, ocular hypertelorism, and retardation of growth. Deafness and genital abnormalities are less frequently found. We report a father and daughter and a third, unrelated patient with LEOPARD syndrome. Recently, missense mutations in the PTPN11 gene located in 12q24 were found to cause Noonan syndrome. All three cases of LEOPARD syndrome reported here have a Y279C mutation in the PTPN11 gene. We hypothesise that some PTPN11 mutations are associated with the typical Noonan syndrome phenotype and that other mutations, such as the Y279C mutation reported here, are associated with both the Noonan syndrome phenotype and with skin pigmentation anomalies, such as multiple lentigines or café au lait spots.

Deficiency of dolichol-phosphate-mannose synthase-1 causes congenital disorder of glycosylation type Ie

Congenital disorders of glycosylation (CDG), formerly known as carbohydrate-deficient glycoprotein syndromes, lead to diseases with variable clinical pictures. We report the delineation of a novel type of CDG identified in 2 children presenting with severe developmental delay, seizures, and dysmorphic features. We detected hypoglycosylation on serum transferrin and cerebrospinal fluid beta-trace protein. Lipid-linked oligosaccharides in the endoplasmic reticulum of patient fibroblasts showed an accumulation of the dolichyl pyrophosphate Man(5)GlcNAc(2) structure, compatible with the reduced dolichol-phosphate-mannose synthase (DolP-Man synthase) activity detected in these patients. Accordingly, 2 mutant alleles of the DolP-Man synthase DPM1 gene, 1 with a 274C>G transversion, the other with a 628delC deletion, were detected in both siblings. Complementation analysis using DPM1-null murine Thy1-deficient cells confirmed the detrimental effect of both mutations on the enzymatic activity. Furthermore, mannose supplementation failed to improve the glycosylation status of DPM1-deficient fibroblast cells, thus precluding a possible therapeutic application of mannose in the patients. Because DPM1 deficiency, like other subtypes of CDG-I, impairs the assembly of N-glycans, this novel glycosylation defect was named CDG-Ie.

Mutations in PMM2 that cause congenital disorders of glycosylation, type Ia (CDG-Ia)

The PMM2 gene, which is defective in CDG‐Ia, was cloned three years ago [Matthijs et al., 1997b]. Several publications list PMM2 mutations [Matthijs et al., 1997b, 1998; Kjaergaard et al., 1998, 1999; Bjursell et al., 1998, 2000; Imtiaz et al., 2000] and a few mutations have appeared in case reports or abstracts [Crosby et al., 1999; Kondo et al., 1999; Krasnewich et al., 1999; Mizugishi et al., 1999; Vuillaumier‐Barrot et al., 1999, 2000b]. However, the number of molecularly characterized cases is steadily increasing and many new mutations may never make it to the literature. Therefore, we decided to collate data from six research and diagnostic laboratories that have committed themselves to a systematic search for PMM2 mutations. In total we list 58 different mutations found in 249 patients from 23 countries. We have also collected demographic data and registered the number of deceased patients. The documentation of the genotype–phenotype correlation is certainly valuable, but is out of the scope of this molecular update. The list of mutations will also be available online (URL: http://www.kuleuven.ac.be/med/cdg) and investigators are invited to submit new data to this PMM2 mutation database. Hum Mutat 16:386–394, 2000.

High Residual Activity of PMM2 in Patients’ Fibroblasts: Possible Pitfall in the Diagnosis of CDG-Ia (Phosphomannomutase Deficiency)

Congenital disorders of glycosylation (CDGs) are a rapidly enlarging group of inherited diseases with abnormal N-glycosylation of glycoconjugates. Most patients have CDG-Ia, which is due to a phosphomannomutase (PMM) deficiency. In this article, we report that a significant portion (9 of 54) of patients with CDG-Ia had a rather high residual PMM activity in fibroblasts included in the normal range (means of the controls +/- 2 SD) and amounting to 35%-70% of the mean control value. The clinical diagnosis of CDG-Ia was made difficult by the fact that most (6 of 9) of these patients belong to a subgroup characterized by a phenotype that is milder than classical CDG-Ia. These patients lack some of the symptoms that are suggestive for the diagnosis, such as inverted nipples and abnormal fat deposition, and, as a mean, had higher residual PMM activities in fibroblasts (2.05+/-0.61 mU/mg protein, n=9; vs. controls 5.34+/-1.74 mU/mg protein, n=22), compared with patients with moderate (1.32+/-0.86 mU/mg protein, n=18) or severe (0.63+/-0.56 mU/mg protein, n=27, P<.001) cases. Yet they all showed mild mental retardation, hypotonia, cerebellar hypoplasia, and strabismus. All of them had an abnormal serum transferrin pattern and a significantly reduced PMM activity in leukocytes. Six of the nine patients with mild presentations were compound heterozygotes for the C241S mutation, which is known to reduce PMM activity by only approximately 2-fold. Our results indicate that intermediate PMM values in fibroblasts may mask the diagnosis of CDG-Ia, which is better accomplished by measurement of PMM activity in leukocytes and mutation search in the PMM2 gene. They also indicate that there is some degree of correlation between the residual activity in fibroblasts and the clinical phenotype.

TMEM165 deficiency causes a congenital disorder of glycosylation.

Protein glycosylation is a complex process that depends not only on the activities of several enzymes and transporters but also on a subtle balance between vesicular Golgi trafficking, compartmental pH, and ion homeostasis. Through a combination of autozygosity mapping and expression analysis in two siblings with an abnormal serum-transferrin isoelectric focusing test (type 2) and a peculiar skeletal phenotype with epiphyseal, metaphyseal, and diaphyseal dysplasia, we identified TMEM165 (also named TPARL) as a gene involved in congenital disorders of glycosylation (CDG). The affected individuals are homozygous for a deep intronic splice mutation in TMEM165. In our cohort of unsolved CDG-II cases, we found another individual with the same mutation and two unrelated individuals with missense mutations in TMEM165. TMEM165 encodes a putative transmembrane 324 amino acid protein whose cellular functions are unknown. Using a siRNA strategy, we showed that TMEM165 deficiency causes Golgi glycosylation defects in HEK cells.

Rett syndrome in females with CTS hot spot deletions: a disorder profile.

From a series of 107 females with Rett syndrome (RTT), we describe the long‐term history of ten females with a deletion in the C‐terminus of the MECP2 gene. We observed that their disorder profile is clinically recognizable with time and different from other atypical and milder RTT phenotypes. In females with hot spot deletions in the C‐terminus, dystonia is present from childhood and results in a serious spine deformation in spite of preventive measures. Their adaptive behavior is surprisingly better preserved and in contrast with the typical decline in motor functioning. The delineaton of disorder profiles by long‐term clinical observation can teach us about genotype/phenotype relationships and eventually about the effect of epigenetic phenomena on the final phenotype.

Fine mapping of Noonan/cardio-facio cutaneous syndrome in a large family.

Noonan syndrome (NS) is an autosomal dominant condition with facial dysmorphy, congenital cardiac defects and short stature. A gene for NS has previously been linked to a 14 cM region in 12q24. We performed linkage analysis in a four generation Belgian family with NS in some individuals and cardio-facio-cutaneous (CFC) syndrome in others. Clinical data and linkage data in this family indicate that NS and CFC syndrome result from a variable expression of the same genetic defect. We report a maximum lod score of 4.43 at zero recombination for marker D12S84 in 12q24. A crossover in this pedigree narrows the candidate gene region for NS to a 5 cM interval between markers D12S84 and D12S1341.

Effect of mutations found in carbohydrate-deficient glycoprotein syndrome type IA on the activity of phosphomannomutase 2.

Seven mutant forms of human phosphomannomutase 2 were produced in Escherichia coli and purified. These mutants had a V max of 0.2–50% of the wild enzyme and were unstable. The least active protein (R141H) bears a very frequent mutation, which has never been found in the homozygous state whereas the second least active protein (D188G) corresponds to a mutation associated with a particularly severe phenotype. We conclude that total lack of phosphomannomutase 2 is incompatible with life. Another conclusion is that the elevated residual phosphomannomutase activity found in fibroblasts of some patients is contributed by their mutated phosphomannomutase 2.

Kinetic properties and tissular distribution of mammalian phosphomannomutase isozymes.

Human tissues contain two types of phosphomannomutase, PMM1 and PMM2. Mutations in the PMM2 gene are responsible for the most common form of carbohydrate-deficient glycoprotein syndrome [Matthijs, Schollen, Pardon, Veiga-da-Cunha, Jaeken, Cassiman and Van Schaftingen (1997) Nat. Genet. 19, 88-92]. The protein encoded by this gene has now been produced in Escherichia coli and purified to homogeneity, and its properties have been compared with those of recombinant human PMM1. PMM2 converts mannose 1-phosphate into mannose 6-phosphate about 20 times more rapidly than glucose 1-phosphate to glucose 6-phosphate, whereas PMM1 displays identical Vmax values with both substrates. The Ka values for both mannose 1,6-bisphosphate and glucose 1,6-bisphosphate are significantly lower in the case of PMM2 than in the case of PMM1. Like PMM1, PMM2 forms a phosphoenzyme with the chemical characteristics of an acyl-phosphate. PMM1 and PMM2 hydrolyse different hexose bisphosphates (glucose 1,6-bisphosphate, mannose 1,6-bisphosphate, fructose 1,6-bisphosphate) at maximal rates of approximately 3.5 and 0.3% of their PMM activity, respectively. Fructose 1,6-bisphosphate does not activate PMM2 but causes a time-dependent stimulation of PMM1 due to the progressive formation of mannose 1,6-bisphosphate from fructose 1,6-bisphosphate and mannose 1-phosphate. Experiments with specific antibodies, kinetic studies and Northern blots indicated that PMM2 is the only detectable isozyme in most rat tissues except brain and lung, where PMM1 accounts for about 66 and 13% of the total activities, respectively.

Rett syndrome in adolescent and adult females: Clinical and molecular genetic findings

Rett syndrome (RTT) is a neurodevelopmental disorder which is diagnosed clinically. We report on 30 adolescent and adult females with classical or atypical RTT of whom 24 have a MECP2 mutation. In these 24 females, the clinical manifestations, degree of severity, and disorder profiles are discussed as well as the genotype phenotype correlation. After X‐chromosome inactivation (XCI) study in these cases, we found no correlation between skewing and milder phenotype. Three large deletions were found after additional Southern blot analysis in three classical RTT cases. We confirm that early truncating mutations in MECP2 are responsible for a more severe course of the disorder. Three disorder profiles related to the missense mutations R133C and R306C, and to deletions in the C terminal segment are described and are of interest for further clinical study on larger numbers of cases. The R133C genotype has a predominantly autistic presentation while the R306C genotype is associated with a slower disease progression. The phenotype of the “hotspot” deletions in the C terminal segment is predominantly characterized by rapid progressive neurogenic scoliosis. Older women with RTT are underdiagnosed: seven adults were first diagnosed as having RTT between 29 and 60 years of age, and confirmed on finding a MECP2 mutation. Knowledge of the clinical phenotype of RTT at an adult age is important for all involved in the care of these individuals. The involvement of the parent support group is very important in this matter.