Frans A. Hol

Mitochondrial disease criteria Diagnostic applications in children

Background: Based on a previous prospective clinical and biochemical study, a consensus mitochondrial disease scoring system was established to facilitate the diagnosis in patients with a suspected mitochondrial disorder. Objective: To evaluate the specificity of the diagnostic system, we applied the mitochondrial disease score in 61 children with a multisystem disease and a suspected oxidative phosphorylation disorder who underwent a muscle biopsy and were consecutively diagnosed with a genetic mutation. Methods: We evaluated data of 44 children diagnosed with a disorder in oxidative phosphorylation, carrying a mutation in the mitochondrial or nuclear DNA. We compared them with 17 children who, based on the clinical and metabolic features, also had a muscle biopsy but were finally diagnosed with a nonmitochondrial multisystem disorder by further genetic analysis. Results: All children with a genetically established diagnosis of a primary oxidative phosphorylation disorder had a mitochondrial disease score above 6 (probable mitochondrial disorder), and 73% of the children had a score above 8 (definite mitochondrial disorder) at evaluation of the muscle biopsy. In the nonmitochondrial multisystem disorder group, the score was significantly lower, and no patients reached a score comparable with a definite respiratory chain disorder. Conclusions: The mitochondrial disease criteria system has a high specificity to distinguish between mitochondrial and other multisystem disorders. The method could also be applied in children with a suspected mitochondrial disorder, prior to performing a muscle biopsy.

Molecular genetic analysis of the gene encoding the trifunctional enzyme MTHFD (methylenetetrahydrofolate-dehydrogenase, methenyltetrahydrofolate-cyclohydrolase, formyltetrahydrofolate synthetase) in patients with neural tube defects

It is now well recognized that periconceptional folic acid or folic acid containing multivitamin supplementation reduces the risk of neural tube defects (NTDs). Recently we were able to show that homozygosity for a thermolabile variant of the enzyme methylenetetrahydrofolate reductase is associated with an increased risk for spina bifida in patients recruited from the Dutch population. However, this genetic risk factor could not account for all folic acid preventable NTDs. In an attempt to identify additional folate related enzymes that contribute to NTD etiology we now studied the methylenetetrahydrofolate dehydrogenase gene on chromosome 14q24 which encodes a single protein with three catalytic properties important in the folate metabolism. The cDNA sequence of 38 familial and 79 sporadic patients was screened for the presence of mutations by single strand conformation polymorphism (SSCP) analysis followed by sequencing. Two amino acid substitutions were identified. The first one (R293H) was detected in a patient with familial spina bifida and not in 300 control individuals. The mutation was inherited from the unaffected maternal grandmother and was also present in two younger brothers of the index patient, one of them displaying spina bifida occulta and the other being unaffected. The second change turned out to be an amino acid polymorphism (R653Q) that was present in both patients and controls with similar frequencies. Our results so far provide no evidence for a major role of the methylenetetrahydrofolate‐dehydrogenase (MTHFD) gene in NTD etiology. However, the identification of a mutation in one family suggests that this gene can act as a risk factor for human NTD.

Susceptibility to spina bifida; an association study of five candidate genes.

Clues regarding candidate genes which influence susceptibility to spina bifida and anencephaly come from the identification of folate‐associated risk factors and from studies of mouse mutants showing neural tube anomalies. On this basis we selected five candidate genes; CBS, MS, MTHFR, T (Brachyury) and BRCA1 for genetic analysis in 31 Dutch and 48 British NTD families. Ten polymorphisms, two for each gene, were used in transmission tests for disequilibrium (TDT). In six instances more than 50 transmissions from heterozygous parents could be examined. Using TDT we find evidence for an association between an allele at the T gene and liability to NTD in the embryo. Data from British and Dutch populations showed the same trend and in combination gave a χ2TDT=4.89, P=0.03 (OR 2.39, CI 95% 1.02–5.61). No association, in either population group, was found for CBS, MS and MTHFR, the enzymes most directly associated with the known risk factors in folate metabolism. The possibility of complex genetic interactions was explored; the data show that a Gly919 MS variant occurs more frequently in combination with the MTHFR thermolabile variant in mothers of NTD offspring (OR 3.94, CI 95% 1.0–16.3).

Genomewide scan identifies susceptibility locus for dyslexia on Xq27 in an extended Dutch family

Context: Dyslexia is a common disorder with a strong genetic component, but despite significant research effort, the aetiology is still largely unknown. Objective: To identify loci contributing to dyslexia risk. Methods: This was a genomewide linkage analysis in a single large family. Dutch families with at least two first degree relatives suffering from dyslexia participated in the study. Participants were recruited through an advertisement campaign in papers and magazines. The main outcome measure was linkage between genetic markers and dyslexia phenotype. Results: Using parametric linkage analysis, we found strong evidence for a locus influencing dyslexia on Xq27.3 (multipoint lod = 3.68). Recombinations in two family members flanked an 8 cM region, comprising 11 currently confirmed genes. All four males carrying the risk haplotype had very low scores on the reading tests. The presentation in females was more variable, but 8/9 females carrying the risk haplotype were diagnosed dyslexic by our composite score, so we considered the putative risk allele to be dominant with reduced penetrance. Linkage was not found in an additional collection of affected sibling pairs. Conclusions: A locus influencing dyslexia risk is probably located between markers DXS1227 and DXS8091 on the X chromosome, closely situated to a locus indicated by a published genome scan of English sibling pairs. Although the locus may not be a common cause for dyslexia, the relatively small and gene poor region offers hope to identify the responsible gene.

Clinical and biochemical characteristics in patients with a high mutant load of the mitochondrial T8993G/C mutations.

We retrospectively analyzed the clinical, histological, and biochemical data of 11 children, five of which carried the maternally‐inherited mitochondrial T8993C and six carrying the T8993G point mutations in the ATP synthase 6 gene. The percentage of heteroplasmy was 95% or higher in muscle and in blood. All patients had an early clinical presentation with muscle hypotonia, severe extrapyramidal dysfunction and Leigh disease demonstrated by the cranial MRI. A slower clinical progression and more frequent sensory‐neuronal involvement were noted in the patients carrying the T8993C mutation in a high mutation load in muscle and blood. No histological abnormality was found. In 9 out of 11 patients a decreased ATP production was detected, and complex V activity was deficient in all children. The activities of the respiratory enzyme complexes II and IV were normal, whereas an associated combined complex I and III deficiency were present in two patients. No obvious difference was found between the biochemical parameters of the two patient groups harboring different mutations in the same gene. No correlation was found between the degree of complex V enzyme deficiency and the severity of the phenotype. We confirmed an impaired assembly/stability of complex V in our patients. This is the first report of decreased activity and impaired assembly/stability of complex V in patients with T8993C mutations measured in muscle tissue.

Genotype-phenotype correlations in MYCN-related Feingold syndrome.

Feingold syndrome (FS) is the most frequent cause of familial syndromic gastrointestinal atresia and follows autosomal dominant inheritance. FS is caused by germline mutations in or deletions of the MYCN gene. Previously, 12 different heterozygous MYCN mutations and two deletions containing multiple genes including MYCN were described. All these mutations result in haploinsufficiency of both the canonical MYCN protein and the shorter isoform, ΔMYCN. We report 11 novel mutations including seven mutations in exon 2 that result in a premature termination codon (PTC) in the long MYCN transcript. Moreover, we have identified a PTC in exon 1 that only affects the ΔMYCN isoform, without a phenotypic effect. This suggests that mutations in only ΔMYCN do not contribute to the FS. Additionally, we found three novel deletions encompassing MYCN. Together with our previous report we now have a total of four missense mutations in the DNA binding domain, 19 PTCs of which six render the transcript subject to nonsense‐mediated decay (NMD), and five larger deletions in a total of 77 patients. We have reviewed the clinical features of these patients, and found that digital anomalies, e.g., brachymesophalangy and toe syndactyly, are the most consistent features, present in 100% and 97% of the patients, respectively. Small head circumference was present in 89% of the cases. Gastrointestinal atresia remains the most important major congenital anomaly (55%), but cardiac and renal anomalies are also frequent. We suggest that the presence of brachymesophalangy and toe syndactyly in combination with microcephaly is enough to justify MYCN analysis. Hum Mutat 29(9), 1125–1132, 2008.

A frameshift mutation in the gene for PAX3 in a girl with spina bifida and mild signs of Waardenburg syndrome

Neural tube defects (NTD) are among the most prevalent congenital malformations in man. Based on the molecular defect of Splotch, an established mouse model for NTD, and on the clinical association between NTD and Waardenburg syndrome (WS), mutations in the PAX3 gene can be expected to act as factors predisposing to human NTD. To test this hypothesis, 39 patients with familial NTD were screened by SSC analysis for mutations in exons 2 to 6 of the human PAX3 gene. One patient with lumbosacral meningomyelocele was identified with a 5 bp deletion in exon 5 approximately 55 bp upstream of the conserved homeodomain. The deletion causes a frameshift with a stop codon almost immediately after the mutated site. Clinical investigation of the index patient indicated mild signs of WS type I. Varying signs of this syndrome were found to cosegregate with the mutation in the family. Our results support the hypothesis that mutations in the gene for PAX3 can predispose to NTD, but also show that, in general, mutations within or near the conserved domains of the PAX3 protein are only very infrequently involved in familial NTD.

Unmasking of a hemizygous WFS1 gene mutation by a chromosome 4p deletion of 8.3 Mb in a patient with Wolf-Hirschhorn syndrome.

The Wolf–Hirschhorn syndrome (WHS (MIM 194190)), which is characterized by growth delay, mental retardation, epilepsy, facial dysmorphisms, and midline fusion defects, shows extensive phenotypic variability. Several of the proposed mutational and epigenetic mechanisms in this and other chromosomal deletion syndromes fail to explain the observed phenotypic variability. To explain the complex phenotype of a patient with WHS and features reminiscent of Wolfram syndrome (WFS (MIM 222300)), we performed extensive clinical evaluation and classical and molecular cytogenetic (GTG banding, FISH and array-CGH) and WFS1 gene mutation analyses. We detected an 8.3 Mb terminal deletion and an adjacent 2.6 Mb inverted duplication in the short arm of chromosome 4, which encompasses a gene associated with WFS (WFS1). In addition, a nonsense mutation in exon 8 of the WFS1 gene was found on the structurally normal chromosome 4. The combination of the 4p deletion with the WFS1 point mutation explains the complex phenotype presented by our patient. This case further illustrates that unmasking of hemizygous recessive mutations by chromosomal deletions represents an additional explanation for the phenotypic variability observed in chromosomal deletion disorders.

Confirmation of Dyslexia Susceptibility Loci on Chromosomes 1p and 2p, but Not 6p in a Dutch Sib-Pair Collection

In this study, we attempted to confirm genetic linkage to developmental dyslexia and reading‐related quantitative traits of loci that have been shown to be associated with dyslexia in previous studies. In our sample of 108 Dutch nuclear families, the categorical trait showed strongest linkage to 1p36 (NPL‐LOD = 2.1). LOD scores for quantitative traits word‐reading, non‐word reading, and rapid naming peaked near the same location as the categorical trait, as well as on chromosome 2. Non‐word repetition showed little phenotypic correlation with dyslexia or with the other quantitative traits, and this trait showed linkage peaks on 11p and 15q. No evidence for linkage to 6p22‐23 was found for this set of families. Comparison of our results and literature data shows that loci link to different phenotypes in different samples. The mutual connections of these traits and their relation to developmental dyslexia remain elusive.

The mitochondrial 13513G > A mutation is most frequent in Leigh syndrome combined with reduced complex I activity, optic atrophy and/or Wolff-Parkinson-White.

The m.13513G>A transition in the mitochondrial gene encoding the ND5 subunit of respiratory chain complex I, can cause mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS) and has been reported to be a frequent cause of Leigh syndrome (LS). We determined the frequency of the mutation in a cohort of 123 patients with reduced complex I activity in muscle (n=113) or fibroblast (n=10) tissue. We describe a Pyrosequencing™ assay for rapid detection and quantification of the m.13513G>A mutation. Two patients with the mutation were identified; both had LS, optical atrophy and a Wolff–Parkinson–White Syndrome (WPWS)-like cardiac conduction defect. The clinical presentation of the m.13513G>A mutation is discussed. We conclude that the m.13513G>A mutation seems not as frequent as previously suggested and is most likely to be present in patients with Leigh (-like) syndrome combined with a complex I deficiency, optic atrophy and/ or WPWS. In addition, we confirmed that the adjacent m.13514A>G mutation is a rare cause of LS or MELAS since no cases with this transition were found.