Loss-of-function mutations in the cathepsin C gene result in periodontal disease and palmoplantar keratosis.

Papillon-Lefèvre syndrome, or keratosis palmoplantaris with periodontopathia (PLS, MIM 245000), is an autosomal recessive disorder that is mainly ascertained by dentists because of the severe periodontitis that afflicts patients. Both the deciduous and permanent dentitions are affected, resulting in premature tooth loss. Palmoplantar keratosis, varying from mild psoriasiform scaly skin to overt hyperkeratosis, typically develops within the first three years of life. Keratosis also affects other sites such as elbows and knees. Most PLS patients display both periodontitis and hyperkeratosis. Some patients have only palmoplantar keratosis or periodontitis, and in rare individuals the periodontitis is mild and of late onset. The PLS locus has been mapped to chromosome 11q14-q21 (refs 7, 8, 9). Using homozygosity mapping in eight small consanguineous families, we have narrowed the candidate region to a 1.2-cM interval between D11S4082 and D11S931. The gene (CTSC) encoding the lysosomal protease cathepsin C (or dipeptidyl aminopeptidase I) lies within this interval. We defined the genomic structure of CTSC and found mutations in all eight families. In two of these families we used a functional assay to demonstrate an almost total loss of cathepsin C activity in PLS patients and reduced activity in obligate carriers.

Somatic overgrowth associated with overexpression of insulin-like growth factor II.

Overexpression of the normally imprinted fetal insulin-like growth factor II (IGF2) has been implicated in the pathogenesis of the cancer-predisposing Beckwith-Wiedemann syndrome (BWS). We have detected constitutional relaxation of imprinting of IGF2 in four children with somatic overgrowth who do not show diagnostic features of BWS. Three children showed constitutional abnormalities of H19 methylation. All four children showed nephromegaly and two developed Wilms' tumors. Gene methylation is known to be associated with gene silencing, and three children showed constitutional abnormalities of H19 gene methylation. Disruption of H19 methylation, and concomitant relaxation of IGF2 imprinting, provides another mechanism that can increase IGF2 expression in children with overgrowth. The accumulated data on normal and pathologic IGF2 expression are now sufficient to define an entity, "IGF2 overgrowth disorder," of which BWS may be one extreme manifestation. These findings have broad implications for the characterization of idiopathic overgrowth.

The molecular landscape of ASPM mutations in primary microcephaly.

BACKGROUND: Autosomal recessive primary microcephaly (MCPH) is a model disease to study human neurogenesis. In affected individuals the brain grows at a reduced rate during fetal life resulting in a small but structurally normal brain and mental retardation. The condition is genetically heterogeneous with mutations in ASPM being most commonly reported. METHODS AND RESULTS: We have examined this further by studying three cohorts of microcephalic children to extend both the phenotype and the mutation spectrum. Firstly, in 99 consecutively ascertained consanguineous families with a strict diagnosis of MCPH, 41 (41%) were homozygous at the MCPH5 locus and all but two families had mutations. Thus, 39% of consanguineous MCPH families had homozygous ASPM mutations. Secondly, in 27 non-consanguineous, predominantly Caucasian families with a strict diagnosis of MCPH, 11 (40%) had ASPM mutations. Thirdly, in 45 families with a less restricted phenotype including microcephaly and mental retardation, but regardless of other neurological features, only 3 (7%) had an ASPM mutation. This report contains 27 novel mutations and almost doubles the number of MCPH associated ASPM mutations known to 57. All but one of the mutations lead to the use of a premature termination codon, 23 were nonsense mutations, 28 deletions or insertions, 5 splicing, and 1 was a translocation. Seventeen of the 57 mutations were recurrent. There were no definitive missense mutations found nor was there any mutation/phenotype correlation. ASPM mutations were found in all ethnic groups studied. CONCLUSION: This study confirms that mutations in ASPM are the most common cause of MCPH, that ASPM mutations are restricted to individuals with an MCPH phenotype, and that ASPM testing in primary microcephaly is clinically useful.

Further delineation of the 15q13 microdeletion and duplication syndromes: a clinical spectrum varying from non-pathogenic to a severe outcome.

BACKGROUND: Recurrent 15q13.3 microdeletions were recently identified with identical proximal (BP4) and distal (BP5) breakpoints and associated with mild to moderate mental retardation and epilepsy. METHODS: To assess further the clinical implications of this novel 15q13.3 microdeletion syndrome, 18 new probands with a deletion were molecularly and clinically characterised. In addition, we evaluated the characteristics of a family with a more proximal deletion between BP3 and BP4. Finally, four patients with a duplication in the BP3-BP4-BP5 region were included in this study to ascertain the clinical significance of duplications in this region. RESULTS: The 15q13.3 microdeletion in our series was associated with a highly variable intra- and inter-familial phenotype. At least 11 of the 18 deletions identified were inherited. Moreover, 7 of 10 siblings from four different families also had this deletion: one had a mild developmental delay, four had only learning problems during childhood, but functioned well in daily life as adults, whereas the other two had no learning problems at all. In contrast to previous findings, seizures were not a common feature in our series (only 2 of 17 living probands). Three patients with deletions had cardiac defects and deletion of the KLF13 gene, located in the critical region, may contribute to these abnormalities. The limited data from the single family with the more proximal BP3-BP4 deletion suggest this deletion may have little clinical significance. Patients with duplications of the BP3-BP4-BP5 region did not share a recognisable phenotype, but psychiatric disease was noted in 2 of 4 patients. CONCLUSIONS: Overall, our findings broaden the phenotypic spectrum associated with 15q13.3 deletions and suggest that, in some individuals, deletion of 15q13.3 is not sufficient to cause disease. The existence of microdeletion syndromes, associated with an unpredictable and variable phenotypic outcome, will pose the clinician with diagnostic difficulties and challenge the commonly used paradigm in the diagnostic setting that aberrations inherited from a phenotypically normal parent are usually without clinical consequences.

Leucodysplasia, microcephaly, cerebral malformation (LMC): a novel recessive disorder linked to 2p16.

We report three related and one unrelated child with an apparently novel neurodevelopmental disorder. The clinical course was very similar in all the four patients: congenital microcephaly with severe failure of post-natal brain growth, neonatal onset of intractable seizures associated with lack of developmental progression and death within the first 3 years of life. The appearance on cerebral neuroimaging was almost identical, with simplified gyration associated with a non-thickened cortex, severe hypoplasia of the corpus callosum, a small flattened brain stem, and specific cystic lesions in the white matter around the temporal and occipital horns. To our knowledge these patients represent a previously unreported, autosomal recessive syndrome. Homozygosity mapping in the consanguineous family has identified a candidate region on the chromosome 2p16.

Fanconi anaemia complementation group B presenting as X linked VACTERL with hydrocephalus syndrome.

BACKGROUND: The VACTERL with hydrocephalus (VACTERL-H) phenotype is recognised to be a severe manifestation of autosomal recessive Fanconi anaemia. Several families have been described in which the VACTERL-H phenotype segregates as an X linked syndrome. The mutations which cause X linked VACTERL-H syndrome are not known. OBJECTIVE: To determine if mutations in FANCB, which are known to cause Fanconi anaemia complementation group B, are a cause of X linked VACTERL-H syndrome. METHODS: A three generation pedigree with X linked VACTERL-H syndrome was investigated. X inactivation was tested in carrier females, and fibroblasts from an affected male fetus were analysed for increased sensitivity to diepoxybutane. FANCB coding exons and flanking splice sites were screened for mutations by direct sequencing of polymerase chain reaction (PCR) fragments amplified from genomic DNA. cDNA from affected fetal fibroblasts was analysed by PCR and direct sequencing using specific exonic primers. RESULTS: A FANCB mutation which results in a premature stop codon by causing skipping of exon 7 was identified. Chromosomes from the affected fetus showed increased sensitivity to diepoxybutane, and carrier women were found to have 100% skewed X inactivation in blood. CONCLUSIONS: Mutations in FANCB are a cause of X linked VACTERL-H syndrome. The data presented are of relevance to the genetic counselling of families with isolated male cases of VACTERL-H and Fanconi anaemia.

Human NDE1 splicing and mammalian brain development.

Exploring genetic and molecular differences between humans and other close species may be the key to explain the uniqueness of our brain and the selective pressures under which it evolves. Recent discoveries unveiled the involvement of Nuclear distribution factor E-homolog 1 (NDE1) in human cerebral cortical neurogenesis and suggested a role in brain evolution; however the evolutionary changes involved have not been investigated. NDE1 has a different gene structure in human and mouse resulting in the production of diverse splicing isoforms. In particular, mouse uses the terminal exon 8 T, while Human uses terminal exon 9, which is absent in rodents. Through chimeric minigenes splicing assay we investigated the unique elements regulating NDE1 terminal exon choice. We found that selection of the terminal exon is regulated in a cell dependent manner and relies on gain/loss of splicing regulatory sequences across the exons. Our results show how evolutionary changes in cis as well as trans acting signals have played a fundamental role in determining NDE1 species specific splicing isoforms supporting the notion that alternative splicing plays a central role in human genome evolution, and possibly human cognitive predominance.

ASPM mutations identified in patients with primary microcephaly and seizures.

BACKGROUND: Human autosomal recessive primary microcephaly (MCPH) is a heterogeneous disorder with at least six genetic loci (MCPH1-6), with MCPH5, caused by ASPM mutation, being the most common. Despite the high prevalence of epilepsy in microcephaly patients, microcephaly with frequent seizures has been excluded from the ascertainment of MCPH. Here, we report a pedigree with multiple affected individuals with microcephaly and seizures. OBJECTIVE: To identify the gene responsible for microcephaly and seizures in this pedigree. METHODS: Clinical assessments of three patients and brain MRIs of two patients were obtained. Genome-wide linkage screen with 10 k SNP microarray, fine mapping with microsatellite markers, and mutational analysis of the genomic DNA were performed on the pedigree. RESULTS: We found that the family was linked to the MCPH5 locus on chromosome 1q31.2-q32.1. We screened ASPM and identified a previously unreported nonsense mutation that introduced a premature stop codon in exon 18 of the ASPM gene. CONCLUSIONS: We thus expand the clinical spectrum of ASPM mutations by showing that they can occur in patients with seizures and that the history of seizures alone should not necessarily preclude the diagnosis of primary microcephaly.

Autosomal recessive primary microcephaly: an analysis of locus heterogeneity and phenotypic variation.

BACKGROUND AND OBJECTIVES: Locus heterogeneity is well established in autosomal recessive primary microcephaly (MCPH) and to date five loci have been mapped. However, the relative contributions of these loci have not been assessed and genotype-phenotype correlations have not been investigated. DESIGN: A study population of 56 consanguineous families resident in or originating from northern Pakistan was ascertained and assessed by the authors. A panel of microsatellite markers spanning each of the MCPH loci was designed, against which the families were genotyped. RESULTS: The head circumference of the 131 affected subjects ranged from 4 to 14 SD below the mean, but there was little intrafamilial variation among affecteds (+/- 1 SD). MCPH5 was the most prevalent, with 24/56 families consistent with linkage; 2/56 families were compatible with linkage to MCPH1, 10/56 to MCPH2, 2/56 to MCPH3, none to MCPH4, and 18/56 did not segregate with any of the loci. CONCLUSIONS: MCPH5 is the most common locus in this population. On clinical grounds alone, the phenotype of families linked to each MCPH locus could not be distinguished. We have also shown that further MCPH loci await discovery with a number of families as yet unlinked.

Kufor-Rakeb syndrome, pallido-pyramidal degeneration with supranuclear upgaze paresis and dementia, maps to 1p36.

Kufor-Rakeb syndrome is an autosomal recessive nigro-striatal-pallidal-pyramidal neurodegeneration. The onset is in the teenage years with clinical features of Parkinson's disease plus spasticity, supranuclear upgaze paresis, and dementia. Brain scans show atrophy of the globus pallidus and pyramids and, later, widespread cerebral atrophy. We report linkage in Kufor-Rakeb syndrome to a 9 cM region of chromosome 1p36 delineated by the markers D1S436 and D1S2843, with a maximum multipoint lod score of 3.6.

Cree encephalitis is allelic with Aicardi-Goutiéres syndrome: implications for the pathogenesis of disorders of interferon alpha metabolism.

Aicardi-Goutiéres syndrome (AGS) is an early onset, progressive encephalopathy characterised by calcification of the basal ganglia, white matter abnormalities, and a chronic cerebrospinal fluid (CSF) lymphocytosis. Cree encephalitis shows phenotypic overlap with AGS although the conditions have been considered distinct because of immunological abnormalities observed in Cree encephalitis. We report that levels of interferon alpha (IFN-alpha), a marker of AGS, are raised in Cree encephalitis. Moreover, linkage analysis indicates that the disorders are allelic and refines the AGS1 locus to a 3.47 cM critical interval. Our data show that a CSF lymphocytosis is not necessary for the diagnosis of AGS and strongly suggest that AGS and pseudo-TORCH syndrome are the same disorder. Recognition of immunological dysfunction as part of the AGS phenotype provides further evidence of a primary pathogenic role for abnormal IFN-alpha production in AGS.

A gene for ataxic cerebral palsy maps to chromosome 9p12-q12.

Cerebral palsy (CP) has an incidence of approximately 1 in 750 births, although this varies between ethnic groups. Genetic forms of the disease account for about 2% of cases in most countries, but contribute a larger proportion in certain sub-types of the condition and in populations with a large proportion of consanguineous marriages. Ataxic cerebral palsy accounts for 5-10% of all forms of CP and it is estimated that approximately 50% of ataxic cerebral palsy is inherited as an autosomal recessive trait. We have identified a complex consanguineous Asian pedigree with four children in two sibships affected with ataxic cerebral palsy and have used homozygosity mapping to map the disorder in this family. A genome-wide search was performed using 343 fluorescently labelled polymorphic markers and linkage to chromosome 9p12-q12 was demonstrated. A maximum Lod score of 3.4 was observed between the markers D9S50 and D9S167 using multipoint analysis, a region of approximately 23cM. We have identified a family that segregates both ataxic CP and ataxic diplegia and have mapped the genetic locus responsible in this family to chromosome 9p12-q12. The identification of gene(s) involved in the aetiology of CP will offer the possibility of prenatal/premarital testing to some families with children affected with the disorder and will greatly increase our understanding of the development of the control of motor function.

The second locus for autosomal recessive primary microcephaly (MCPH2) maps to chromosome 19q13.1-13.2.

Primary microcephaly is a clinical diagnosis made when an individual has a head circumference of greater than 3 standard deviations below the age and sex matched population mean, mental retardation but without other associated malformations and no apparent aetiology. The majority of cases of primary microcephaly exhibit an autosomal recessive mode of inheritance. We now demonstrate the genetic heterogeneity of this condition with the identification of a second primary microcephaly locus (MCPH2) on chromosome 19q13.1-13.2 in two multi-affected consanguineous families. The minimum critical region containing the MCPH2 locus is defined by the polymorphic markers D19S416 and D19S420 spanning a region of approximately 7.6 cM.

Novel PTEN mutations in patients with Cowden disease: absence of clear genotype-phenotype correlations.

Cowden disease (CD) is characterised by multiple hamartomas in a variety of tissues. The pathological hallmark is the presence of a number of trichilemmomas. Several neurological symptoms are also part of CD with megalencephaly and Lhermitte-Duclos disease (LDD) as the most important features. Early recognition of CD patients is important because of the increased risk of developing malignancies. Breast cancer is the most frequent malignancy, but also urogenital, digestive tract, and thyroid cancers are found with higher frequencies. CD was localised to chromosome 10q23 and the PTEN gene (also known as MMAC1 or TEP1) was shown to be involved. Germline mutations were identified in both familial and sporadic CD patients. We identified eight PTEN mutations, of which seven were novel, in 13 CD patients. Combined with previous data we have identified 17 independent CD mutations. Gross DNA alterations in CD patients were not detected. Genotype-phenotype relations are discussed. The only correlation suggested to exist is that missense mutations are not detected in LDD patients. However, larger numbers are needed to confirm this. Association of PTEN mutations and the occurrence of malignant breast disease found in an earlier study cannot be confirmed. Clinical features of five CD patients without a PTEN mutation in the coding sequence do not differ from CD patients with a PTEN mutation. Furthermore, it is likely that we have identified the majority of CD patients in the Netherlands. From this we estimate that CD has a prevalence of about 1 in 250,000 in the Dutch population with a low mutation frequency.

Use of monoclonal antibodies to recognise osteoclasts in routinely processed bone biopsy specimens.

In decalcified (5% nitric acid) and undecalcified (glycol-methacrylate or resin embedded) routinely processed bone specimens osteoclasts against resorbing surfaces were identified with monoclonal antibodies directed against leucocyte common antigen (LCA) (PD7/26, 2B11), CD68 (KP1), and gpIIIa (Y2/51) but not against HLA-DR (CR3/43 and Ta11B5). Mononuclear cells on resorbing surfaces and occasional mononuclear cells against or near resting surfaces showed a similar pattern of reactivity. This study shows that immunohistochemistry is a sensitive and useful technique for identifying osteoclasts in routinely processed bone specimens. It also suggests a role for mononuclear cells (possibly pre-osteoclasts) in bone resorption.

Effect of decalcification agents on immunoreactivity of cellular antigens.

The effects of several strong acids, weak acids, a proprietary decalcifier, and edetic acid on the immunohistochemical staining of cryostat and fixed paraffin embedded sections of tissue from a variety of normal and pathological calcified and uncalcified specimens were studied. Even decalcification in strong acids (HCl, HN03, 5% trichloracetic acid, HCl-edetic acid did not diminish the reactivity of many useful antigens (including leucocyte common antigen, intermediate filaments, S100 protein and epithelial membrane antigen). Weaker acids (formic acid, acetic acid) and edetic acid decalcified more slowly and generally showed greater preservation of antigenic reactivity with better morphology and staining quality. Trichloracetic acid was also useful as a quick one step fixation and decalcifying agent for both cryostat and routinely processed sections. Knowledge of the preservation of antigenic reactivity in decalcified tissue will be useful in the diagnosis of tumours of uncertain histogenesis and origin which affect calcified tissues.

Bone changes in polycythaemia vera and myelosclerosis.

To study the problem of new bone formation in myelosclerosis bone biopsies have been performed in cases of polycythaemia vera, which may be regarded as a pre-myelosclerotic condition, and in primary myelosclerosis; morphological studies of bone and reticulin were made and osteoid and bone matrix was quantitatively assessed. Relevant metabolic studies were also carried out, but were predominantly normal. No new bone formation was detected in polycythaemia vera and some bone was osteoporotic; no significant increase in osteoid was found. With the use of polarized light it was found that new bone formation in myelosclerosis resulted from the ossification of wavy argyrophilic fibres in marrow: the presence of a dense network of these wavy fibres was a pre-requisite of new bone formation and ossification of the non-refractile branched reticulin fibres was not observed.

Skeletal muscle bioenergetics in myotonic dystrophy.

Skeletal muscle function of 15 patients with myotonic dystrophy (dystrophia myotonica, DM) was investigated using 31P magnetic resonance spectroscopy to evaluate bioenergetics and intracellular pH at rest and during exercise and recovery. Results from DM patients, normal controls and mitochondrial myopathy patients were compared in order to assess the possible contribution of abnormal mitochondrial metabolism to muscle dysfunction in DM. In resting DM muscle, intracellular pH (pHi) was normal, but there were significant elevations in the concentration ratios of Pi/ATP, phosphomonoesters/ATP and phosphodiesters/ATP. In patients with the most severe exercise intolerance the phosphocreatine/ATP ratio was also reduced. Resting muscle of 11 mitochondrial myopathy patients showed similar changes to those of the most exercise-intolerant DM patients. In exercising DM muscle, energy stores were rapidly depleted as in mitochondrial myopathy. Muscle acidified in all subjects, but in DM the decrease in pHi was less than in normal muscle. Recovery half-times for phosphocreatine, Pi and ADP were normal in DM but slow in mitochondrial myopathy. The initial rate of phosphocreatine repletion after exercise was rapid in DM, consistent with high [ADP], but slow in mitochondrial myopathy in spite of elevated [ADP]. Because recovery is an oxidative process, we conclude that there was no decrease in the oxidative capacity of the muscles in this group of DM patients. In the subjects in whom it could be measured, the rate of recovery of intracellular pH was greater in the 3 DM patients (0.14, 0.15 and 0.16 U/min) than in the 7 normal controls (0.08-0.12 U/min, mean 0.10). The results do not rule out a minor abnormality in glycogenolysis, but they suggest that the failure to acidify normally during exercise is probably due to rapid proton efflux.

Comparison of enzyme mismatch cleavage and chemical cleavage of mismatch on a defined set of heteroduplexes.

Two mutation detection methods based on the cleavage of mismatched heteroduplexes were compared and evaluated. These techniques, chemical cleavage of mismatch (CCM) and enzyme mismatch cleavage (EMC), have the advantages over other available methods of being able to detect and localize mutations in relatively large fragments of DNA (> or = 1 kb). We have constructed clones that enable us to create heteroduplexes of 500 bp, 1 kb, and 1.5 kb and have assessed each of the methods over a range of criteria. Both were able to detect and localize all four types of single-base mismatch and insertion/deletions of 1-5 bp. Whereas EMC was efficient at detection of insertion/deletions in a broad size range of fragments and has the advantage over CCM of using no hazardous chemicals, in our hands it has not been sufficiently robust that we felt confident to consider it for diagnostic use in its present form. CCM using hydroxylamine was efficient over the entire range of fragment sizes tested and using potassium permanganate with tetraethylammonium chloride was efficient up to 1 kb.

Fibrosarcomata and malignant fibrous histiocytomata arising in relation to enchondromata.

Six cases are reported in which a fibrosarcoma or malignant fibrous histiocytoma developed in relation to an enchondroma in a long bone. Four of the tumours were fibrosarcomata, and two were malignant fibrous histiocytomata. Five of the six cases were in the distal femur, which is a common site for old calcified enchondromata or "cartilage rests". The age of the patients was between fifty-six and eighty-six with a mean of seventy. Four were women, Five died less than one year after presentation. The fibrosarcomata and malignant fibrous histiocytomata do not appear to have arisen directly from the tumour cartilage but from the dense fibrous tissue surrounding necrotic areas in the enchondromata by a process analogous to that responsible for the development of fibrosarcomata in bone infarcts and chronic osteomyelitis. The possibility that some "dedifferentiated" chondrosarcomata are forms of collision tumour is discussed.