A duplication at chromosome 11q12.2-11q12.3 is associated with spinocerebellar ataxia type 20.

Spinocerebellar ataxia type 20 (SCA20) has been linked to chromosome 11q12, but the underlying genetic defect has yet to be identified. We applied single-nucleotide polymorphism genotyping to detect structural alterations in the genomic DNA of patients with SCA20. We found a 260 kb duplication within the previously linked SCA20 region, which was confirmed by quantitative polymerase chain reaction and fiber fluorescence in situ hybridization, the latter also showing its direct orientation. The duplication spans 10 known and 2 unknown genes, and is present in all affected individuals in the single reported SCA20 pedigree. While the mechanism whereby this duplication may be pathogenic remains to be established, we speculate that the critical gene within the duplicated segment may be DAGLA, the product of which is normally present at the base of Purkinje cell dendritic spines and contributes to the modulation of parallel fiber-Purkinje cell synapses.

Deletion at ITPR1 underlies ataxia in mice and spinocerebellar ataxia 15 in humans.

We observed a severe autosomal recessive movement disorder in mice used within our laboratory. We pursued a series of experiments to define the genetic lesion underlying this disorder and to identify a cognate disease in humans with mutation at the same locus. Through linkage and sequence analysis we show here that this disorder is caused by a homozygous in-frame 18-bp deletion in Itpr1 (Itpr1(Delta18/Delta18)), encoding inositol 1,4,5-triphosphate receptor 1. A previously reported spontaneous Itpr1 mutation in mice causes a phenotype identical to that observed here. In both models in-frame deletion within Itpr1 leads to a decrease in the normally high level of Itpr1 expression in cerebellar Purkinje cells. Spinocerebellar ataxia 15 (SCA15), a human autosomal dominant disorder, maps to the genomic region containing ITPR1; however, to date no causal mutations had been identified. Because ataxia is a prominent feature in Itpr1 mutant mice, we performed a series of experiments to test the hypothesis that mutation at ITPR1 may be the cause of SCA15. We show here that heterozygous deletion of the 5' part of the ITPR1 gene, encompassing exons 1-10, 1-40, and 1-44 in three studied families, underlies SCA15 in humans.

SNP selection for genes of iron metabolism in a study of genetic modifiers of hemochromatosis.

BACKGROUND: We report our experience of selecting tag SNPs in 35 genes involved in iron metabolism in a cohort study seeking to discover genetic modifiers of hereditary hemochromatosis. METHODS: We combined our own and publicly available resequencing data with HapMap to maximise our coverage to select 384 SNPs in candidate genes suitable for typing on the Illumina platform. RESULTS: Validation/design scores above 0.6 were not strongly correlated with SNP performance as estimated by Gentrain score. We contrasted results from two tag SNP selection algorithms, LDselect and Tagger. Varying r2 from 0.5 to 1.0 produced a near linear correlation with the number of tag SNPs required. We examined the pattern of linkage disequilibrium of three levels of resequencing coverage for the transferrin gene and found HapMap phase 1 tag SNPs capture 45% of the > or = 3% MAF SNPs found in SeattleSNPs where there is nearly complete resequencing. Resequencing can reveal adjacent SNPs (within 60 bp) which may affect assay performance. We report the number of SNPs present within the region of six of our larger candidate genes, for different versions of stock genotyping assays. CONCLUSION: A candidate gene approach should seek to maximise coverage, and this can be improved by adding to HapMap data any available sequencing data. Tag SNP software must be fast and flexible to data changes, since tag SNP selection involves iteration as investigators seek to satisfy the competing demands of coverage within and between populations, and typability on the technology platform chosen.

Whole genome SNP arrays using DNA derived from formalin-fixed, paraffin-embedded ovarian tumor tissue.

Array-based genotyping platforms, such as the Affymetrix mapping array, have been validated as reliable methods for obtaining high-resolution copy number and allele status information when using DNA derived from fresh tissue sources. However, the suitability of such systems for the examination of DNA derived from formalin-fixed, paraffin-embedded (FFPE) tissues has not been tested. Therefore, we analyzed DNA derived from five matching fresh frozen and FFPE ovarian tumors for gene copy number changes and loss of heterozygosity using the Affymetrix GeneChip Human Mapping 10 K Array Xba 131. The data was analyzed using Affymetrix proprietary software, GeneChip DNA Analysis Software, and Chromosome Copy Number Tool. The average SNP call rate (rate of successful genotype identification) of the fresh samples was 89.44% (range 78.72-96.22%, median 92.72%) and was only slightly lower for the matching FFPE samples at 83.48% (range 76.93-93.17%, median 82.60%). The average concordance (rate of agreement between successful genotype calls) between the fresh and matching FFPE samples was 97.06% (range 92.70-99.41%, median 97.52%). Loss of heterozygosity (LOH) profiles of the fresh and FFPE samples were essentially identical across all chromosomes. Copy number data was also comparable, although the quantification of copy number changes appears overstated in the FFPE samples. In conclusion, we have shown that it is possible to achieve high-performance outcomes using FFPE-derived DNA in the Affymetrix 10 K mapping array. This advance will open up vast archival tissue resources to high-resolution genetic analysis and unlock a wealth of biological information.

A gene for pili annulati maps to the telomeric region of chromosome 12q.

Pili annulati (PA) is a rare hair shaft disorder characterized by discrete banding of hairs. We studied two families with PA in which the disorder segregated in an autosomal dominant fashion. All family members were clinically examined and hair samples were examined under the light microscope. In family G, of 19 individuals examined, ten were affected, over three generations. In family B, there were three affected individuals of seven examined over three generations. A genome-wide scan of family G revealed a maximum logarithm of odds (LOD) of linkage score of 3.89 at marker D12S1723 at the telomeric region of chromosome 12q. From one critical recombinant in family G, the locus was narrowed down to a 9.2 cM region between D12S367 and the end of chromosome 12q. In family B linkage at the telomeric region of chromosome 12q also revealed a maximum LOD score of 0.89 at marker D12S1723. A combined LOD score, assuming no locus heterogeneity between the families was 4.78. Frizzled 10, which is located within the region, was sequenced but we were unable to detect a mutation causing PA. This study, for the first time, identifies a genetic locus for PA.

Parametric and non-parametric linkage analysis of several candidate regions for genes for human handedness.

The frequency of left-handedness in the general population is around 11%. Both environmental and genetic models have been proposed to explain the aetiology of human handedness. The majority of genetic models, such as those of Annett, McManus and Klar, propose a single gene determinant with a non-Mendelian inheritance pattern. As left-handedness is correlated with cerebral asymmetry and is a feature of left-right asymmetry, genes involved in the development of left-right asymmetry can be considered as candidate genes. Candidate gene analysis was performed using an informative extended pedigree, and also using nuclear families of right-handed parents with left-handed children. Segregation analysis in the extended pedigree identified allele sharing in the NODAL and DNAHC13 candidate regions on chromosome 10 and 1. Linkage analysis using the models of Klar and McManus, and non-parametric analysis on nuclear families, subsequently excluded all candidate regions tested. This demonstrates the power to identify the genes specifying handedness by the conduct of extended genetic studies on these and similar cohorts.

Parametric and nonparametric genome scan analyses for human handedness.

We have performed a genome scan using 25 nuclear families consisting of right-handed parents with at least two left-handed children. Handedness was assessed as a qualitative trait using a laterality quotient. Laterality quotients indicate the direction of handedness, which is hand preference for performing unimanual tasks. Both parametric and nonparametric linkage analyses were applied. The parametric analysis using the single-locus genetic model of Klar resulted in four different regions with LOD scores higher than 1. The region on chromosome 10q26 gave a suggestive LOD score of 2.02 at a recombination fraction of 0.05. Nonparametric analysis gave an NPL score for this region of 2.16. However, further fine mapping of the region on chromosome 10q26 failed to obtain a higher LOD score. These results suggest that handedness is a human quantitative trait locus and that the proposed non-Mendelian monogenic models are incorrect.

Spinocerebellar ataxia type 20.

Spinocerebellar ataxia type 20 (SCA20) was reported in 2004 in a single Australian Anglo-Celtic pedigree. The phenotype is distinctive, with palatal tremor, and hypermetric saccades, and early dentate (but not pallidal) calcification in the absence of abnormalities of calcium metabolism. Dysarthria, rather than gait ataxia, was the initial symptom in most, and was typically conjoined with dysphonia, clinically resembling adductor spasmodic dysphonia. The onset of these speech abnormalities was abrupt in some cases. MRI scanning showed mild to moderate pancerebellar atrophy with dentate calcification, with olivary pseudohypertrophy in some cases, in the absence of other brainstem or cerebral changes. Nerve conduction studies were normal. Progression appeared to be slow. SCA20 is probably rare, as despite the distinctive phenotype, only this one pedigree has been described. The locus mapped to the pericentromeric region of chromosome 11 with a LOD score of 4.47, and its candidate region overlaps that of SCA5. It seems probable that these two SCAs may be separate genetic entities, on the basis of their divergent clinical features, but formal proof awaits discovery of one or both responsible genes.

Spinocerebellar ataxia type 15.

Spinocerebellar ataxia type 15 (SCA15) was first reported in 2001 on the basis of a single large Anglo-Celtic family from Australia, the locus mapping to chromosomal region 3p24.2-3pter. The characteristic clinical feature was of very slow progression, with two affected individuals remaining ambulant without aids after over 50 years of symptoms. Head and/or upper limb action tremor, and gaze-evoked horizontal nystagmus were seen in several persons. MRI brain scans showed predominant vermal atrophy, sparing the brainstem. In 2004, a Japanese pedigree was reported, which displayed very similar clinical features to the original SCA15 family, and which mapped to an overlapping candidate region. These two families might plausibly reflect a locus homogeneity, but for the present this remains an open question.

Progressive patterned scalp hypotrichosis, with wiry hair, onycholysis, and intermittently associated cleft lip and palate: clinical and genetic distinction from Marie Unna.

Marie Unna hereditary hypotrichosis has been described in over a dozen families since 1924. Features include scant or no eyebrows at birth, the development of firm wiry hair in the first few years of life followed by a progressive patterned scalp alopecia in the second or third decade. This is associated with generalized hypotrichosis of the body and the condition is nonsyndromic. We have identified a novel form of autosomal dominant ectodermal dysplasia that resembles Marie Unna hereditary hypotrichosis in a family of 23 members over four generations. Affected individuals have patterned hair loss and associated hair shaft dystrophy similar to that seen in Marie Unna hereditary hypotrichosis. It differs from Marie Unna hereditary hypotrichosis by an absence of signs of affectation at birth, relative sparing of body hair, distal onycholysis, and intermittent cosegregation with autosomal dominant cleft lip and palate. Linkage studies to the known Marie Unna locus at 8p21 near the Hairless gene were performed. Linkage analysis using markers D8S298, D8S560, D8S258, and D8S282 revealed significant exclusion of this locus (Z = -2.0 or lower) at theta = 0.1. This demonstrates that this novel ectodermal dysplasia is both phenotypically and genetically distinct from Marie Unna hereditary hypotrichosis.

Increased gene dosage at Xq26-q27 is associated with X-linked hypopituitarism.

We have identified a novel interstitial duplication at Xq26.1-q27.3 in a previously reported family with X-linked recessive hypopituitarism [1]. Mapping of the duplication was carried out using interphase FISH analysis of over 60 bacterial genomic clones from Xq25-q28. The proximal and distal breakpoints of the duplication are contained within the 432N13 and 91O18 clones, respectively, and are separated by approximately 9 Mb. Comparison with a recently published 13-Mb duplication in another XH family [2] indicated that the duplication break-points in these families were different. Therefore, we conclude that X-linked hypopituitarism is caused by increased dosage of a gene that is critical for pituitary development and that the causative gene is located within the 9-Mb duplicated region that we have defined.

Dominantly inherited ataxia and dysphonia with dentate calcification: spinocerebellar ataxia type 20.

We describe a pedigree of Anglo-Celtic origin with a phenotypically unique form of dominantly inherited spinocerebellar ataxia (SCA) in 14 personally examined affected members. A remarkable observation is dentate nucleus calcification, producing a low signal on MRI sequences. Unusually for an SCA, dysarthria is typically the initial manifestation. Mild pyramidal signs and hypermetric saccades are noted in some. Its distinguishing clinical features, each present in a majority of affected persons, are palatal tremor, and a form of dysphonia resembling spasmodic dysphonia. Repeat expansion detection failed to identify either CAG/CTG or ATTCT/AGAAT repeat expansions segregating with the disease in this family. The testable SCA mutations have been excluded. On linkage analysis, the locus maps to chromosome 11, which rules out all the remaining mapped SCAs except for SCA5. While locus homogeneity with SCA5 is not formally excluded, we consider it rather unlikely on phenotypic grounds, and propose that this condition may represent an addition to the group of neurogenetic disorders subsumed under the rubric SCA. The International Nomenclature Committee has made a provisional assignment of 'SCA20', although firm designation will have to await a definite molecular distinction from SCA5.

Spinocerebellar ataxia type 15 (sca15) maps to 3p24.2-3pter: exclusion of the ITPR1 gene, the human orthologue of an ataxic mouse mutant.

We have studied a large Australian kindred with a dominantly inherited pure cerebellar ataxia, SCA15. The disease is characterised by a very slow rate of progression in some family members, and atrophy predominantly of the superior vermis, and to a lesser extent the cerebellar hemispheres. Repeat expansion detection failed to identify either a CAG/CTG or ATTCT/AGAAT repeat expansions segregating with the disease in this family. A genome-wide scan revealed significant evidence for linkage to the short arm of chromosome 3. The highest two-point LOD score was obtained with D3S3706 (Z = 3.4, theta = 0.0). Haplotype analysis identified recombinants that placed the SCA15 locus within an 11.6-cM region flanked by the markers D3S3630 and D3S1304. The mouse syntenic region contains two ataxic mutants, itpr1-/- and opt, affecting the inositol 1,4,5-triphosphate type 1 receptor, ITPR1 gene. ITPR1 is predominantly expressed in the cerebellar Purkinje cells. Mutation analysis from two representative affected family members excluded the coding region of the ITPR1 gene from being involved in the pathogenesis of SCA15. Thus, the itpr1-/- and opt ITPR1 mouse mutants, which each result in ataxia, are not allelic to the human SCA15 locus.