N-acetylaspartylglutamate in CNS hypomyelination.

CSF N-acetylaspartylglutamate (NAAG) has been found to be elevated in some hypomyelinating disorders. This study addressed the question whether it could be used as a marker for hypomyelination and as a means to distinguish between hypomyelinating disorders biochemically. We have measured CSF NAAG in a cohort of 28 patients with hypomyelination with known and unknown aetiology. NAAG was found to be elevated in 7 patients, but was normal in the majority, including patients with defined hypomyelinating disorders. CSF NAAG is not a universal marker of hypomyelination, and the mechanism of its elevation remains poorly understood.

Clinical and molecular characteristics of 1qter microdeletion syndrome: delineating a critical region for corpus callosum agenesis/hypogenesis.

BACKGROUND: Patients with a microscopically visible deletion of the distal part of the long arm of chromosome 1 have a recognisable phenotype, including mental retardation, microcephaly, growth retardation, a distinct facial appearance and various midline defects including corpus callosum abnormalities, cardiac, gastro-oesophageal and urogenital defects, as well as various central nervous system anomalies. Patients with a submicroscopic, subtelomeric 1qter deletion have a similar phenotype, suggesting that the main phenotype of these patients is caused by haploinsufficiency of genes in this region. OBJECTIVE: To describe the clinical presentation of 13 new patients with a submicroscopic deletion of 1q43q44, of which nine were interstitial, and to report on the molecular characterisation of the deletion size. RESULTS AND CONCLUSIONS: The clinical presentation of these patients has clear similarities with previously reported cases with a terminal 1q deletion. Corpus callosum abnormalities were present in 10 of our patients. The AKT3 gene has been reported as an important candidate gene causing this abnormality. However, through detailed molecular analysis of the deletion sizes in our patient cohort, we were able to delineate the critical region for corpus callosum abnormalities to a 360 kb genomic segment which contains four possible candidate genes, but excluding the AKT3 gene.

Variable phenotypes associated with 10q23 microdeletions involving the PTEN and BMPR1A genes.

Infantile juvenile polyposis is a rare disease with severe gastrointestinal symptoms and a grave clinical course. Recently, 10q23 microdeletions involving the PTEN and BMPR1A genes were found in four patients with infantile juvenile polyposis. It was hypothesized that a combined and synergistic effect of the deletion of both genes would explain the condition. Subsequently, however, a patient with a larger 10q23 deletion including the same genes but with a mild clinical phenotype was identified. Here, we present four additional patients with 10q23 microdeletions involving the PTEN and BMPR1A genes. The sizes of the deletions were analyzed using single nucleotide polymorphism array analysis. All patients had macrocephaly, dysmorphic features, retardation and congenital abnormalities. One patient developed colorectal cancer. However, only one case had disease onset before 2 years of age and severe symptoms requiring colectomy. No clear correlation was found between ages at onset or severity of gastrointestinal symptoms and the sizes of the deletions. We conclude that patients with 10q23 microdeletions involving the PTEN and BMPR1A genes have variable clinical phenotypes, which cannot be explained merely by the deletion sizes. The phenotypes are not restricted to severe infantile juvenile polyposis but include childhood-onset cases with macrocephaly, retardation, mild gastrointestinal symptoms and possibly early-onset colorectal cancer.

Disruption of the gene Euchromatin Histone Methyl Transferase1 (Eu-HMTase1) is associated with the 9q34 subtelomeric deletion syndrome.

BACKGROUND: A new syndrome has been recognised following thorough analysis of patients with a terminal submicroscopic subtelomeric deletion of chromosome 9q. These have in common severe mental retardation, hypotonia, brachycephaly, flat face with hypertelorism, synophrys, anteverted nares, thickened lower lip, carp mouth with macroglossia, and conotruncal heart defects. The minimum critical region responsible for this 9q subtelomeric deletion syndrome (9q-) is approximately 1.2 Mb and encompasses at least 14 genes. OBJECTIVE: To characterise the breakpoints of a de novo balanced translocation t(X;9)(p11.23;q34.3) in a mentally retarded female patient with clinical features similar to the 9q- syndrome. RESULTS: Sequence analysis of the break points showed that the translocation was fully balanced and only one gene on chromosome 9 was disrupted--Euchromatin Histone Methyl Transferase1 (Eu-HMTase1)--encoding a histone H3 lysine 9 methyltransferase (H3-K9 HMTase). This indicates that haploinsufficiency of Eu-HMTase1 is responsible for the 9q submicroscopic subtelomeric deletion syndrome. This observation was further supported by the spatio-temporal expression of the gene. Using tissue in situ hybridisation studies in mouse embryos and adult brain, Eu-HMTase1 was shown to be expressed in the developing nervous system and in specific peripheral tissues. While expression is selectively downregulated in adult brain, substantial expression is retained in the olfactory bulb, anterior/ventral lateral ventricular wall, and hippocampus and weakly in the piriform cortex. CONCLUSIONS: The expression pattern of this gene suggests a role in the CNS development and function, which is in line with the severe mental retardation and behaviour problems in patients who lack one copy of the gene.

Inhibition of TGFß signaling decreases osteogenic differentiation of fibrodysplasia ossificans progressiva fibroblasts in a novel in vitro model of the disease.

Fibrodysplasia ossificans progressiva is a rare genetic disorder characterized by progressive heterotopic ossification. FOP patients develop soft tissue lumps as a result of inflammation-induced flare-ups which leads to the irreversible replacement of skeletal muscle tissue with bone tissue. Classical FOP patients possess a mutation (c.617G>A; R206H) in the ACVR1-encoding gene which leads to dysregulated BMP signaling. Nonetheless, not all FOP patients with this mutation exhibit equal severity in symptom presentation or disease progression which indicates a strong contribution by environmental factors. Given the pro-inflammatory role of TGFß, we studied the role of TGFß in the progression of osteogenic differentiation in primary dermal fibroblasts from five classical FOP patients based on a novel method of platelet lysate-based osteogenic transdifferentiation. During the course of transdifferentiation the osteogenic properties of the cells were evaluated by the mRNA expression of Sp7/Osterix, Runx2, Alp, OC and the presence of mineralization. During transdifferentiation the expression of osteoblast markers Runx2 (p<0.05) and Alp were higher in patient cells compared to healthy controls. All cell lines exhibited increase in mineralisation. FOP fibroblasts also expressed higher baseline Sp7/Osterix levels (p<0.05) confirming their higher osteogenic potential. The pharmacological inhibition of TGFß signaling during osteogenic transdifferentiation resulted in the attenuation of osteogenic transdifferentiation in all cell lines as shown by the decrease in the expression of Runx2 (p<0.05), Alp and mineralization. We suggest that blocking of TGFß signaling can decrease the osteogenic transdifferentiation of FOP fibroblasts.

Screening for subtelomeric rearrangements in 210 patients with unexplained mental retardation using multiplex ligation dependent probe amplification (MLPA).

BACKGROUND: Subtelomeric rearrangements contribute to idiopathic mental retardation and human malformations, sometimes as distinct mental retardation syndromes. However, for most subtelomeric defects a characteristic clinical phenotype remains to be elucidated. OBJECTIVE: To screen for submicroscopic subtelomeric aberrations using multiplex ligation dependent probe amplification (MLPA). METHODS: 210 individuals with unexplained mental retardation were studied. A new set of subtelomeric probes, the SALSA P036 human telomere test kit, was used. RESULTS: A subtelomeric aberration was identified in 14 patients (6.7%) (10 deletions and four duplications). Five deletions were de novo; four were inherited from phenotypically normal parents, suggesting that these were polymorphisms. For one deletion, DNA samples of the parents were not available. Two de novo submicroscopic duplications were detected (dup 5qter, dup 12pter), while the other duplications (dup 18qter and dup 22qter) were inherited from phenotypically similarly affected parents. All clinically relevant aberrations (de novo or inherited from similarly affected parents) occurred in patients with a clinical score of >or=3 using an established checklist for subtelomeric rearrangements. Testing of patients with a clinical score of >or=3 increased the diagnostic yield twofold to 12.4%. Abnormalities with clinical relevance occurred in 6.3%, 5.1%, and 1.7% of mildly, moderately, and severely retarded patients, respectively, indicating that testing for subtelomeric aberrations among mildly retarded individuals is necessary. CONCLUSIONS: The value of MLPA is confirmed. Subtelomeric screening can be offered to all mentally retarded patients, although clinical preselection increases the percentage of chromosomal aberrations detected. Duplications may be a more common cause of mental retardation than has been appreciated.

[Progressive ataxia and cognitive deficits caused by premutation in the fragile-X-mental retardation gene]. / Progressieve ataxie en cognitieve achteruitgang door een premutatie in het fragiele-X-mentale-retardatiegen.

A 75-year-old man had progressive difficulty with walking, intention tremor, ataxia, and mild cognitive deficits. MRI scan ofthe brain showed symmetrical hyperintensities in the middle cerebellar peduncles. DNA analysis ofthe fragile-X gene revealed an expansion of 150-200 repetitions in the FMR1-gene, compatible with a premutation in the fragile-X gene. Two years later, after progression of the symptoms, the patient was admitted to a nursing home. The clinical picture of intention tremor, parkinsonism and ataxia with white matter lesions and atrophy on MRI occurs in carriers of this premutation and has recently been described as the fragile-X-associated tremor/ataxia syndrome. Recognition of this clinical picture is important for the patient but also for the relatives, since female carriers of the premutation have an increased risk of offspring with the fragile-X syndrome.

Mutation detection in the aspartoacylase gene in 17 patients with Canavan disease: four new mutations in the non-Jewish population.

Canavan disease is a severe progressive autosomal recessive disorder, which is characterised by spongy degeneration of the brain. The disease is caused by mutations in the aspartoacylase gene. Two different mutations were reported on 98% of the alleles of Ashkenazi Jewish patients, in which population the disease is highly prevalent. In non-Jewish patients of European origin, one mutation (914C > A) is found in 50% of the alleles, the other alleles representing all kinds of different mutations. We here describe the results of the mutation analysis in 17 European, non-Jewish patients. Ten different mutations were found, of which four had not been described before (H21P, A57T, R168H, P181T). A deletion of exon4, which until now had only been described once, was revealed in all five alleles of Turkish origin tested, indicating that this is a founder effect in the Turkish population.

Ataxia with vitamin E deficiency: biochemical effects of malcompliance with vitamin E therapy.

To prevent neuronal damage, patients with ataxia with isolated vitamin E deficiency need lifelong supplementation with high doses of vitamin E. Short interruptions of therapy, such as occur in malcompliance, do not lead to clinical symptoms. However, the authors show that even short withdrawals may cause a prolonged decrease of the total radical trapping capacity of plasma; its major contributors, such as urate and sulfhydryl groups, fail to compensate for the missing vitamin E.

Duplication of the proteolipid protein gene is the major cause of Pelizaeus-Merzbacher disease.

BACKGROUND/OBJECTIVE: Pelizaeus-Merzbacher disease (PMD), an X-linked recessive dysmyelination disorder, is caused by mutations in the proteolipid protein (PLP) gene. However, missense mutations were only found in a fraction of PMD patients, even in families that showed linkage with the PLP locus on Xq22. Here we describe the use of an extended protocol that includes screening for both missense mutations and duplications. METHOD: Two groups of patients were analyzed, one group with 10 independent PMD families and one group with 24 sporadic patients suspected of PMD. Missense mutations in the PLP gene were identified by sequencing. PLP gene duplications were detected by quantitative polymerase chain reaction and/or Southern blot analysis. RESULTS: Sequencing of the PLP gene revealed four mutations in group 1 and one mutation in group 2. However, inclusion of duplication analysis in the screening protocol raised the amount of mutations found in group 1 from 40 to 90%, and in group 2 from 4 to 25%. CONCLUSIONS: These results demonstrate that duplications of the PLP gene are the major cause of PMD. Furthermore, it appears that the phenotype resulting from PLP duplications is relatively mild, and that many probands are nontypical PMD patients.

Mutations in noncoding regions of the proteolipid protein gene in Pelizaeus-Merzbacher disease.

BACKGROUND: Pelizaeus-Merzbacher disease (PMD) is an X-linked recessive dysmyelinating disorder of the CNS. Duplications or point mutations in exons of the proteolipid protein (PLP) gene are found in most patients. OBJECTIVE: To describe five patients with PMD who have mutations in noncoding regions of the PLP gene. METHODS: Quantitative multiplex PCR and Southern blot analyses were used to detect duplication of the PLP gene, and DNA sequence analysis, including exon-intron borders, was used to detect mutation of the PLP gene. RESULTS: Duplication of the PLP gene was ruled out, and mutations were identified in noncoding regions of five patients in four families with PMD. In two brothers with a severe form of PMD, a G to T transversion at IVS6+3 was detected. This mutation resulted in skipping of exon 6 in the PLP mRNA of cultured fibroblasts. A patient who developed nystagmus at 16 months and progressive spastic ataxia at 18 months was found to have a 19-base pair (bp) deletion of a G-rich region near the 5' end of intron 3 of the PLP gene. A patient with a T to C transition at IVS3+2 and a patient with an A to G transition at IVS3+4 have the classic form of PMD. These, like the 19-bp deletion, are in intron 3, which is involved in PLP/DM20 alternative splice site selection. CONCLUSIONS: Mutations in introns of the PLP gene, even at positions that are not 100% conserved at splice sites, are an important cause of PMD.

A mitochondrial tRNA(Val) gene mutation (G1642A) in a patient with mitochondrial myopathy, lactic acidosis, and stroke-like episodes.

We studied a patient with the diagnosis of mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) for mitochondrial DNA mutations in muscle. Established MELAS mutations were excluded. Mitochondrial DNA was further analyzed for mutations in the 22 tRNA genes by single-strand conformation polymorphism (SSCP) analysis; a tRNA(Val) mutation (G1642A) was found. The structure of the altered tRNA, the heteroplasmy, and the absence of the mutation in the mother and in 100 control subjects suggests that the tRNA(Val) mutation is associated with the MELAS syndrome.

A novel homozygous 5 bp deletion in FKBP10 causes clinically Bruck syndrome in an Indonesian patient.

We report an Indonesian patient with bone fragility and congenital joint contractures. The initial diagnosis was Osteogenesis Imperfecta type III (OI type III) based on clinical and radiological findings. Because of (i) absence of COL1A1/2 mutations, (ii) a consanguineous pedigree with a similarly affected sibling and (iii) the existence of congenital joint contractures with absence of recessive variants in PLOD2, mutation analysis was performed of the FKBP10 gene, recently associated with Bruck syndrome and/or recessive OI. A novel homozygous deletion in FKBP10 was discovered. Our report of the first Indonesian patient with clinically Bruck syndrome, confirms the role of causative recessive FKBP10 mutations in this syndrome.

Pure subtelomeric microduplications as a cause of mental retardation.

Submicroscopic subtelomeric aberrations are a common cause of mental retardation (MR). New molecular techniques allow the identification of subtelomeric microduplications, but their frequency and significance are largely unknown. We determined the frequency of subtelomeric, pure microduplications in a cohort of 624 patients with MR and/or multiple congenital anomalies using multiplex ligation dependent probe amplification (MLPA) and delineated the identified microduplications using array based comparative genomic hybridization (array CGH). In 11 patients, MLPA revealed a subtelomeric duplication without a concurrent deletion. Additional fluorescence in situ hybridization studies and parental analyses showed that three had occurred de novo: one duplication 5q34qter (12.7 Mb), one duplication 9q34.13qter (7.2 Mb) and one duplication 9p24.2pter (4.1 Mb). Five microduplications (9p, 11q, 12q, 15q and 16p) appeared to be inherited from an unaffected parent, while in three cases (9p, 12p and 17p) the parents were not available for testing. Based on our findings and data from the literature, the three de novo duplications were the only ones likely to be disease-causing, leading to a frequency of pathogenic subtelomeric, pure microduplications of 0.5%. Our study shows that subtelomeric microduplications are an infrequent cause of MR and that additional clinical and family studies are required to assess their clinical significance.

A unique second donor splice site in the intron 5 sequence of the HLA-A*11 alleles results in a class I transcript encoding a molecule with an elongated cytoplasmic domain.

Full-length cDNA sequencing of the A*1103 allele revealed an insertion of 18 bp between exon 5 and 6. We hypothesized that this could be the result of alternative splicing. Sequencing of intron-5 of A*1101, *1102 and *1103 alleles demonstrated that this 18-bp insertion consisted of the 5'-end of intron 5, concluded by a second in-frame donor splice site. Alignment of the 5'-end intron 5 sequence of A*1101-3 with that of A*0101, *0201 and 0301 revealed a unique polymorphism at position 17 of the intron (A to G) that created this second donor splice site. To exclude the possibility of an Epstein-Barr virus (EBV)-induced event, reverse transcriptase-polymerase chain reaction (RT-PCR) analysis was performed on both peripheral blood mononuclear cells (PBMC) and EBV-transformed b-LcL's of several A*11-positive individuals, using primers spanning exons 5 and 6. Without exception, both cell types revealed two products for A*11. Densitometric analysis using EBV-transformed b-LcL's and PBMC indicated a ratio of approximately 4:1 in favor of the alternative splice product. Notably, except for the A*11's none of the other A-locus alleles yields this alternative splice product. Translation of this product will result in a protein that has an additional 6 amino acids in its cytoplasmic domain. This introduces a negative charge just behind the basic anchor residues of the cytoplasmic segment and results in the loss of the single potential protein kinase C phosphorylation site.