A homozygous FKRP start codon mutation is associated with Walker-Warburg syndrome, the severe end of the clinical spectrum.

Dystroglycanopathies are a heterogeneous group of disorders caused by defects in the glycosylation pathway of alpha-dystroglycan. The clinical spectrum ranges from severe congenital muscular dystrophy with structural brain and eye involvement to a relatively mild adult onset limb-girdle muscular dystrophy without brain abnormalities and normal intelligence. Mutations have been identified in one of six putative or demonstrated glycosyltransferases. Many different FKRP mutations have been identified, which cover the complete clinical spectrum of dystroglycanopathies. In contrast to the other known genes involved in these disorders, genotype-phenotype correlations are not obvious for FKRP mutations. To date, no homozygous or compound heterozygous null mutations have been identified in FKRP, suggesting that null mutations in FKRP could result in embryonic lethality. We report a family with two siblings carrying a homozygous mutation in the start codon of FKRP that is likely to result in a loss of functional FKRP protein. The clinical phenotype of the patients was consistent with Walker-Warburg syndrome, the most severe disorder in the disease spectrum of dystroglycanopathies.

[Sudden blindness: consider Leber's hereditary optic neuropathy]. / Plotselinge blindheid: denk aan hereditaire opticusatrofie van Leber.

In 3 young male patients, aged 10, 19 and 21 years respectively, sequential, severe, painless bilateral visual loss occurred. Ophthalmological examination revealed no other abnormalities and this delayed the diagnosis Leber's hereditary optic neuropathy (LHON). LHON is a mitochondrial genetic disease characterised by bilateral acute or subacute painless loss of central vision. LHON causes blindness, predominantly in young adult males but less frequently in women and children as well. Occasionally, LHON is associated with other neurological and cardiac changes. The first patient recovered his vision within 2 years, but the other 2 remained blind. All 3 patients had a m.11778G > A mutation in the mitochondrial DNA (mtDNA). Over 95% of LHON cases are primarily the result of one of three mitochondrial DNA point mutations. In addition, analysis of patients grouped according to mtDNA mutation has demonstrated differences in both the clinical features of visual failure and in recurrence risks for relatives that are associated with each of the pathogenic mtDNA mutations. Depending on the type of mutation, recovery of vision occurs in 4-58% of the patients. Whilst pathogenic mtDNA mutations are required for the development of LHON, other factors must be responsible for the variable penetrance and male predominance. Familiarity with the clinical spectrum of LHON is necessary for early diagnosis. There is no proven treatment.

A novel mitochondrial ATP8 gene mutation in a patient with apical hypertrophic cardiomyopathy and neuropathy.

PURPOSE: To identify the biochemical and molecular genetic defect in a 16-year-old patient presenting with apical hypertrophic cardiomyopathy and neuropathy suspected for a mitochondrial disorder. METHODS: Measurement of the mitochondrial energy-generating system (MEGS) capacity in muscle and enzyme analysis in muscle and fibroblasts were performed. Relevant parts of the mitochondrial DNA were analysed by sequencing. Transmitochondrial cybrids were obtained by fusion of 143B206 TK(-) rho zero cells with patient-derived enucleated fibroblasts. Immunoblotting techniques were applied to study the complex V assembly. RESULTS: A homoplasmic nonsense mutation m.8529G-->A (p.Trp55X) was found in the mitochondrial ATP8 gene in the patient's fibroblasts and muscle tissue. Reduced complex V activity was measured in the patient's fibroblasts and muscle tissue, and was confirmed in cybrid clones containing patient-derived mitochondrial DNA. Immunoblotting after blue native polyacrylamide gel electrophoresis showed a lack of holocomplex V and increased amounts of mitochondrial ATP synthase subcomplexes. An in-gel activity assay of ATP hydrolysis showed activity of free F(1)-ATPase in the patient's muscle tissue and in the cybrid clones. CONCLUSION: We describe the first pathogenic mutation in the mitochondrial ATP8 gene, resulting in an improper assembly and reduced activity of the complex V holoenzyme.

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.

Mitochondrial tRNALeu(UUR) mutation in a patient with steroid-resistant nephrotic syndrome and focal segmental glomerulosclerosis.

BACKGROUND: The heterogeneity of mitochondrial cytopathies is characteristic for this group of disorders, which preferentially affect the muscle and nerve system. The A3243G transition in the tRNA(Leu(UUR)) gene has been associated with slowly progressive forms of focal segmental glomerulosclerosis (FSGS). Here we present a patient who developed a severe nephrotic syndrome during her first pregnancy, which persisted after delivery, and proved resistant to immunosuppressive therapy. A sister of our patient had developed diabetes mellitus. We analysed the DNA for the presence of the mitochondrial DNA (mtDNA) A3243G transition. METHODS: DNA was isolated from peripheral blood leukocytes and urine sediments. Polymerase chain reaction was performed to amplify the mtDNA. Restriction enzyme analysis was used to detect the presence of the A3243G transition. Quantitative analysis of the A3243G mutation was done using the pyrosequencing technique. RESULTS: Quantitative analysis revealed a proportion of mutated mtDNA of 30% in the leukocytes and 68% in the urine sediments of the proband. On further analysis, we also found the transition in the mother, the diabetic sister and the daughter of the proband. CONCLUSION: MtDNA abnormalities can cause a steroid-resistant nephrotic syndrome, histologically characterized by FSGS. Physicians should be especially mindful of mitochondrial abnormalities when hearing loss, diabetes mellitus or neuromuscular disorders are present in the patient or family members.

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.

Array comparative genomic hybridisation analysis of boys with X linked hypopituitarism identifies a 3.9 Mb duplicated critical region at Xq27 containing SOX3.

INTRODUCTION: Array comparative genomic hybridisation (array CGH) is a powerful method that detects alteration of gene copy number with greater resolution and efficiency than traditional methods. However, its ability to detect disease causing duplications in constitutional genomic DNA has not been shown. We developed an array CGH assay for X linked hypopituitarism, which is associated with duplication of Xq26-q27. METHODS: We generated custom BAC/PAC arrays that spanned the 7.3 Mb critical region at Xq26.1-q27.3, and used them to search for duplications in three previously uncharacterised families with X linked hypopituitarism. RESULTS: Validation experiments clearly identified Xq26-q27 duplications that we had previously mapped by fluorescence in situ hybridisation. Array CGH analysis of novel XH families identified three different Xq26-q27 duplications, which together refine the critical region to a 3.9 Mb interval at Xq27.2-q27.3. Expression analysis of six orthologous mouse genes from this region revealed that the transcription factor Sox3 is expressed at 11.5 and 12.5 days after conception in the infundibulum of the developing pituitary and the presumptive hypothalamus. DISCUSSION: Array CGH is a robust and sensitive method for identifying X chromosome duplications. The existence of different, overlapping Xq duplications in five kindreds indicates that X linked hypopituitarism is caused by increased gene dosage. Interestingly, all X linked hypopituitarism duplications contain SOX3. As mutation of this gene in human beings and mice results in hypopituitarism, we hypothesise that increased dosage of Sox3 causes perturbation of pituitary and hypothalamic development and may be the causative mechanism for X linked hypopituitarism.

Exercise intolerance, muscle pain and lactic acidaemia associated with a 7497G>A mutation in the tRNASer(UCN) gene.

A 13-year-old girl with non-familial exercise intolerance, muscle pain and lactic acidaemia underwent a muscle biopsy for suspected mitochondrial disease. Muscle morphology showed 25% ragged-red fibres and 80% COX-negative staining. Enzymatic activities of mitochondrially co-encoded respiratory chain enzymes (complexes I, III, and IV) were decreased in muscle but normal in cultured skin fibroblasts. mtDNA analysis revealed the presence of the 7497G>A mutation in the tRNASer(UCN) gene, homoplasmic in skeletal muscle and 90% in leukocytes. Analysis of the mother's mtDNA showed 10% heteroplasmy in blood. It may be concluded that the 7497G>A mutation is associated with a muscle-only disease presentation for which high levels of mutated mtDNA are required. Exercise intolerance and muscle pain in otherwise normal children warrants further mitochondrial evaluation.

Tall stature and progressive overweight in mitochondrial encephalopathy.

We describe two children carrying an inherited T899C mutation in the mitochondrial ATPase 6 gene with mild encephalopathy and normal postnatal growth followed by tall stature and obesity. No familial tall stature, endocrine anomaly or advanced skeletal age were present. Failure to thrive is a characteristic finding in most patients with a mitochondrial disease. Our observations suggest that children with encephalomyopathy, even in the presence of a significant clinical overgrowth, should be screened for a possible defect in oxidative phosphorylation.

Corneal decompensation in a boy with Kearns-Sayre syndrome.

This paper describes the clinical history of a young boy with Kearns-Sayre syndrome. The first presenting symptom of Kearns-Sayre syndrome in this boy was corneal edema with photophobia and tearing.

Identification and characterization of an Xq26-q27 duplication in a family with spina bifida and panhypopituitarism suggests the involvement of two distinct genes.

We investigated a family with a duplication, dup(X)q26-q27, that was present in two brothers, their mother, and their maternal grandmother. The brothers carrying the duplication displayed spina bifida and panhypopituitarism, whereas a third healthy brother inherited the normal X chromosome. Preferential inactivation of the X chromosome containing the duplication was evident in healthy carrier females. We determined the boundaries of the Xq26-q27 duplication. Via interphase FISH analysis we narrowed down each of the two breakpoint regions to approximately 300-kb intervals. The proximal breakpoint is located in Xq26.1 between DXS1114 and HPRT and is contained in YAC yWXD599, while the distal breakpoint is located in Xq27.3 between DXS369 and DXS1200 and contained in YAC yWXD758. The duplication comprises about 13 Mb. Evidence from the literature points to a predisposing gene for spina bifida in Xq27. We hypothesize that the spina bifida in the two brothers may be due to interruption of a critical gene in the Xq27 breakpoint region. Several candidate genes were mapped to the Xq27 critical region but none was shown to be disrupted by the duplication event. Recently, M. Lagerström-Fermér et al. (1997, Am. J. Hum. Genet. 60, 910-916) reported on a family with X-linked recessive panhypopituitarism associated with a duplication in Xq26; however, no details were reported on the extent of the duplication. Our study corroborates their hypothesis that X-linked recessive panhypopituitarism is likely to be caused by a gene encoding a dosage-sensitive protein involved in pituitary development. We place the putative gene between DXS1114 and DXS1200, corresponding to the interval defined by the duplication in the present family.

A deletion encompassing Zic3 in bent tail, a mouse model for X-linked neural tube defects.

Bent tail is a mouse model for human neural tube defects. Bent tail mice are characterized by a shortened, kinked tail. We have observed numerous aberrations in Bent tail embryos including exencephaly, rotation defects and occasionally omphalocele, orofacial schisis and situs abnormalities. Exencephaly was seen in >10% of all embryos and resulted from a closure defect of the hindbrain. Bent tail maps to the proximal part of the X chromosome. By haplotype analysis we have appointed the Bent tail locus to a 1.1 cM interval between markers DXMit159 and DXMit143. Subsequent analysis has revealed the presence of a deletion in all affected animals. The deletion is approximately 1 Mb in size and encompasses the gene for ZIC:3, a zinc finger transcription factor expressed in murine neuroectoderm and dorsal axial mesoderm during neurulation. ZIC:3 is a homolog of the Drosophila segmentation gene odd-paired. Although the Bent tail phenotype probably is the result of the deletion of several genes, combining data on ZIC:3 expression and function of ZIC: genes in the mouse shows that deletion of Zic3 alone is compatible with a major role of this gene in the congenital malformations of the Bent tail mouse. In man, mutations in ZIC3 are associated with situs abnormalities. These patients occasionally also show spina bifida, indicating that genetic variation in human ZIC3 may contribute to other congenital malformations, including neural tube defects.

Molecular cytogenetic detection of 9q34 breakpoints associated with nail patella syndrome.

The nail patella syndrome (NPS1) is an autosomal dominant disorder characterised by dysplasia of the finger nails and skeletal abnormalities. NPS1 has been mapped to 9q34, to a 1 cM interval between D9S315 and the adenylate kinase gene (AK1). We have mapped the breakpoints within the candidate NPS1 region in two unrelated patients with balanced translocations. One patient [46,XY,t(1;9)(q32.1;q34)] was detected during a systematic survey of old cytogenetic files in Denmark and southern Sweden. The other patient [46,XY,t(9;17)(q34.1;q25)] was reported previously. D9S315 and AK1 were used to isolate YACs, from which endclones were used to isolate PACs. Two overlapping PAC clones span the 9q34 breakpoints in both patients, suggesting that NPS1 is caused by haploinsufficiency due to truncation or otherwise inactivation of a gene at or in the vicinity of the breakpoints.

Altered regulation of platelet-derived growth factor receptor-alpha gene-transcription in vitro by spina bifida-associated mutant Pax1 proteins.

Mouse models show that congenital neural tube defects (NTDs) can occur as a result of mutations in the platelet-derived growth factor receptor-alpha gene (PDGFRalpha). Mice heterozygous for the PDGFRalpha-mutation Patch, and at the same time homozygous for the undulated mutation in the Pax1 gene, exhibit a high incidence of lumbar spina bifida occulta, suggesting a functional relation between PDGFRalpha and Pax1. Using the human PDGFRalpha promoter linked to a luciferase reporter, we show in the present paper that Pax1 acts as a transcriptional activator of the PDGFRalpha gene in differentiated Tera-2 human embryonal carcinoma cells. Two mutant Pax1 proteins carrying either the undulated-mutation or the Gln --> His mutation previously identified by us in the PAX1 gene of a patient with spina bifida, were not or less effective, respectively. Surprisingly, Pax1 mutant proteins appear to have opposing transcriptional activities in undifferentiated Tera-2 cells as well as in the U-2 OS osteosarcoma cell line. In these cells, the mutant Pax1 proteins enhance PDGFRalpha-promoter activity whereas the wild-type protein does not. The apparent up-regulation of PDGFRalpha expression in these cells clearly demonstrates a gain-of-function phenomenon associated with mutations in Pax genes. The altered transcriptional activation properties correlate with altered protein-DNA interaction in band-shift assays. Our data provide additional evidence that mutations in Pax1 can act as a risk factor for NTDs and suggest that the PDGFRalpha gene is a direct target of Pax1. In addition, the results support the hypothesis that deregulated PDGFRalpha expression may be causally related to NTDs.

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.

Identification of CXorf1, a novel intronless gene in Xq27.3, expressed in human hippocampus.

We have identified and characterized a novel human gene (Nomenclature Committee of the Genome Database GDB-assigned symbol CXorf1) that maps to the long arm of the X chromosome in Xq27 between loci DXS369 and DXS181, approximately 2.5 Mb centromeric to the FMR1 gene. The CXorf1 gene is conserved in primates, cow, and horse but not in mouse and rat. Northern blot analysis revealed two transcripts, present in the brain and in the G361 melanoma cell line. In situ hybridization experiments performed on sections of human hippocampus showed a clear, uneven localization of the CXorf1 mRNA in specific subfields of this brain area. In particular, CXorf1 was localized in the granular-cell layer of the dentate gyrus and in the CA2-CA3 subfields of Ammon's horn. CXorf1 is one of the first genes from this region to be characterized in detail and, on the basis of its chromosomal location and expression pattern, may have an important function in the brain.