Detection of germline rearrangements in patients with α- and ß-thalassemia using high resolution array CGH.

Approximately 80% of α-thalassemia mutations are deletions in the α-globin cluster on chromosome 16 and about 10% of ß-thalassemia mutations are deletions in the ß-globin gene cluster on chromosome 11. Larger deletions involving the ß-globin gene cluster lead to (δß)-, (γδß)-, (εγδß)-thalassemia, or hereditary persistence of fetal hemoglobin (HPFH). Array comparative genomic hybridization (CGH) was applied to screen for deletions in the α- and ß-globin gene clusters not detected by routine gap-PCR. In total, in 13 patients with hypochromia and inclusion bodies (IBs) the α-globin gene cluster was analyzed and in 13 patients with increased fetal hemoglobin levels with or without hypochromia the ß-globin gene cluster was examined. All samples were subsequently investigated by multiplex ligation-dependent probe amplification (MLPA). In 9 out of 13 patients deletions of the α-globin gene cluster were identified; 5 of these deletions remove the entire α-globin cluster and extend to the telomere. Additional sequencing of the remaining 4 patients revealed polyadenylation mutation in 1 of them. 7 deletions were identified in the ß-globin gene cluster in 13 patients. Additional sequencing of the remaining 6 patients revealed mutations in one of the γ-globin gene promoters in 3 of them and a KLF1-mutation in 1 of them. Array CGH is a reliable method to screen for deletions in thalassemia and hemoglobinopathy. The method offers the advantage of a high resolution with the possibility to characterize breakpoints on sequence level.

A new stable alpha chain variant: Hb Basel [alpha14(A12)Trp-->Leu (alpha1)].

We describe a heterozygosity for a new missense mutation on the alpha1-globin gene of an 18-year-old woman of Portuguese ancestry with severe hypochromic anemia and iron deficiency. Hemoglobin (Hb) analysis by high performance liquid chromatography (HPLC) found a prominent peak constituting about 12% of total Hb. Sequencing of the globin genes of the index patient found the mutation alpha14(A12)Trp-->Leu (alpha1), HBA1:c.44G

Phenotypic variability of a distinct deletion in McLeod syndrome.

The X-linked McLeod neuroacanthocytosis syndrome strongly resembles Huntington's disease and has been reported in various countries world-wide. Herein, we report two Chilean brothers with predominant psychiatric features at disease onset including schizophrenia-like psychosis and obsessive compulsive disorder. Molecular genetic analysis revealed a small deletion in the XK gene (938-942delCTCTA), which has been already described in a North American patient of Anglo-Saxon descent and a Japanese family, presenting with seizures, muscle atrophy or chorea yet absence of psychiatric features. These findings argue against a founder effect and indicate a profound phenotypic variability associated with the 938-942delCTCTA deletion. Our report supports the inclusion of McLeod syndrome in the differential diagnosis of Huntington's disease as well as acute psychosis in male subjects.

A novel complex mutation event in the peripherin/RDS gene in a family with retinal pattern dystrophy.

PURPOSE: To report a complex mutation in the peripherin/RDS gene found in a family in whom retinal pattern dystrophy is segregating as an autosomal dominant trait. METHODS: Clinical data were collected from family members of a large Swiss family affected by autosomal dominant retinal pattern dystrophy. Single strand conformation polymorphism (SSCP) analysis of the candidate gene peripherin/RDS and subsequent sequencing of the first exon were performed. RESULTS: Pattern dystrophy of the retina was suspected in 18 family members aged 30 years or older. Assuming a homogeneous phenotype, the candidate locus peripherin/RDS was investigated. SSCP analysis of the first exon of the peripherin/RDS gene showed an aberrant pattern in 18 affected individuals. Direct sequencing of polymerase chain reaction products detected a complex mutation, del265-268GCCA ins AGGGCC, leading to a stop codon at amino acid position 99. CONCLUSION: To our knowledge, we report the first complex mutation in the peripherin/RDS gene as the cause of a mild macular phenotype, supporting the importance of molecular diagnosis in genetic counseling.

The LTR promoter of the rat oncomodulin gene is regulated by cell-line specific accessibility in the LTR U3 region.

By germline insertion, a long terminal repeat (LTR) of an intracisternal A-particle type IAP retrovirus has overtaken the transcriptional control of the rat oncomodulin (OM) gene, which codes for a high affinity Ca2+-binding protein with modulatory capacity. In order to get insights into regulatory mechanisms of LTR directed OM gene expression we tested promoter activity of this LTR by transient transfection of transformed rat fibroblasts with this sequence placed 5' of the human growth hormone hGH reporter gene. The OM LTR is a strong promoter but does not follow an expression pattern similar to the one of the OM gene. Genomic sequencing showed a good correlation between CpG hypomethylation in the OM LTR and OM transcription among various cell lines and tissues. DNase I mapping of a 18 kb fragment containing the OM gene and 5' flanking sequences revealed cell-line specific hypersensitivity sites located within the U3 region of the LTR element. Several cis-elements in the OM LTR promoter exhibiting cell-line specific occupancy were identified by in vivo DMS-footprinting. Detailed analysis of protein interactions with two such sequence elements in vitro revealed binding of ubiquitously expressed nuclear factors within an AP-1 (activator protein 1) and a intracisternal A-particle upstream enhancer recognition sequence. Protein occupancy to the latter sequence is significantly reduced by CpG methylation. These results indicate that cell-line specificity of OM expression is dictated by factor accessibility to the LTR promoter.

Quantitative monitoring of BCR--ABL transcript--suggestion of a simplified approach considering inaccuracy of measurement and calibration.

According to standard-protocols, real time quantitative RT-PCR (RQ-PCR) for quantification of BCR-ABL fusion transcripts in CML patients is performed with the construction of a standard curve for each run, each sample is analyzed at least in duplicate and 10-40 ml peripheral blood are processed. This approach is appropriate for a research laboratory, but is not suitable for a routine laboratory setting. We show that the calibration curve based on the common 5 dilution standards (between 10 and 10(6) copies) is strongly influenced by the large variability of the measurements below 100 copies of the gene. In other words, including a standard with 10 copies is a source of error, which cannot be reduced through the construction of a standard curve with each run. Adding additional dilutions between 10 and 100 copies to the standard curve, the variance of the obtained curve is much reduced. As a conclusion, it is unnecessary to construct a calibration curve with each run since only negligible inaccuracy of calibration is added to the inaccuracy of measurement. Running of the samples in duplicate seems unnecessary since the inaccuracy of the method can be correctly estimated. Finally, we propose a standardized collection and isolation of total RNA from only 2.5 ml blood using an integrated system, which allows RNA stabilization for up to 5 days and provides snapshots of BCR-ABL fusion transcripts with higher accuracy than with non-stabilized blood samples.

Clinical description and exclusion of candidate genes in a novel autosomal recessively inherited vitreoretinal dystrophy.

OBJECTIVES: To describe the clinical phenotype of a novel autosomal recessively inherited vitreoretinal dystrophy in one generation of a family originating from eastern Switzerland. METHODS: A clinical study including electroretinographic investigations followed by laboratory-based genetic and molecular analysis. Four affected and 3 unaffected members of the family were examined. Ten candidate regions were tested by linkage analysis with highly polymorphic molecular markers or with intragenic restriction fragment length polymorphisms. RESULTS: Of 8 siblings,4 were affected, showing high myopia with pronounced vitreous liquefaction, retinitis pigmentosa-like retinal degeneration, diffuse retinal pigment epithelium atrophy, macular staphylomata, and premature cataract formation. Strikingly abnormal results on electroretinograms, affecting both the rod and the cone systems, revealed an extensive defect of retinal function, unlike those usually found in pathologic myopia. No extraocular manifestations were observed. Three types of nonsyndromic high myopia, Stickler syndrome I, II, and III, Wagner syndrome, Knobloch syndrome, Goldmann-Favre dystrophy, and multiple vitreoretinopathies were excluded by linkage analysis. CONCLUSIONS: The reported phenotype as well as the results of molecular linkage analysis in the siblings described here suggest an autosomal recessively inherited vitreoretinal dystrophy, which, to our knowledge, has not been described until now.

Genomewide homozygosity mapping and molecular analysis of a candidate gene located on 22q13 (fibulin-1) in a previously undescribed vitreoretinal dystrophy.

OBJECTIVES: To localize the gene that causes an autosomal recessively inherited vitreoretinal dystrophy that has not been described, to our knowledge, and to analyze a candidate gene mapped to 22q13 (fibulin-1 [FBLN1]). METHODS: Homozygosity mapping with 500 microsatellite markers spread over the whole genome (mean distance, 7.2 centimorgans [cM]) and mutation analysis of the complete coding region of FBLN1. RESULTS: Homozygosity for all analyzed markers was found in the 4 affected siblings in a region on chromosome 22 encompassing 12 cM from D22S444 (centromeric) to D22S1170 (telomeric). Lod scores were between 0.017 and 2.36 (theta = 0). A mutation analysis of the complete coding region of FBLN1, which encodes interacting extracellular matrix proteins, revealed 4 previously undescribed single nucleotide polymorphisms. CONCLUSIONS: A genomewide homozygosity mapping analysis supported the hypothesis that the gene responsible for a unique vitreoretinal dystrophy is located on chromosome 22q13. No obviously pathogenic mutation was found in the candidate gene, FBLN1.

McLeod phenotype associated with a XK missense mutation without hematologic, neuromuscular, or cerebral involvement.

BACKGROUND: The X-linked McLeod neuroacanthocytosis syndrome is a multisystem disorder with hematologic, neuromuscular, and central nervous system (CNS) manifestations. All carriers of the McLeod blood group phenotype examined so far had at least subclinical signs of systemic involvement. STUDY DESIGN AND METHODS: Evaluation of two brothers carrying the McLeod phenotype with neurologic examination, immunohematology, RBC membrane protein Western blotting, analysis of XK DNA sequence and RNA levels, muscle histology including XK/Kell immunohistochemistry, cerebral magnetic resonance imaging (MRI), and quantified positron emission tomography (PET). RESULTS: Immunohematology and Western blotting confirmed presence of the McLeod blood group phenotype. No acanthocytosis or other hematologic anomalies were found. XK gene sequence analysis revealed a missense mutation in exon 3 (E327K). WBC XK RNA levels were not decreased. There were no neuromuscular and CNS signs or symptoms. In addition, no subclinical involvement was discovered on the basis of normal muscle histology with a physiologic pattern of XK and Kell immunohistochemistry, normal cerebral MRI, and quantified PET. CONCLUSION: Known disease-causing XK gene mutations comprised deletions, nonsense, or splice-site mutations predicting absent or truncated XK protein devoid of the Kell-protein binding site. Although the E327K missense mutation was associated with the immunohematologic characteristics of McLeod syndrome, the mutated XK protein seemed to be largely functional. These findings contribute to the understanding of the physiology of XK and Kell proteins, and the pathogenetic mechanisms of acanthocytosis, myopathy, and striatal neurodegeneration in McLeod syndrome.

Spondylo-ocular syndrome: a new entity with crystalline lens malformation, cataract, retinal detachment, osteoporosis, and platyspondyly.

PURPOSE: To define a new clinical entity in a consanguineous family with six children affected by a spondylo-ocular syndrome, including cataract, crystalline lens malformation, retinal detachment, osteoporosis, and platyspondyly. To analyze candidate genes of connective tissue disorders as a possible underlying disorder and to demonstrate especially the ocular phenotype. DESIGN: Observational case series. METHODS: Consanguineous parents, one unaffected sibling and five affected children with clinical features of spondylo-ocular syndrome, were demonstrated. Clinical examination, radiologic, laboratory, and cytogenetic as well as moleculargenetic analyses were performed. The segregation of flanking marker alleles of three collagen genes and the interval for osteoporosis-pseudoglioma syndrome were analyzed. Two microsatellite markers located within Pax6CA/GT region were tested for homozygosity. RESULTS: On laboratory investigation a normal excretion of amino acids, mucopolysaccharides, and oligosaccharides could be found. The karyotype was normal. Complete radiologic examination in one index patient revealed a generalized moderate osteoporosis, platyspondyly with fish bone appearance, and greatly enlarged intervertebral spaces. The candidate genes known to be in Stickler syndrome as well as linkage to the osteoporosis-pseudoglioma syndrome candidate region could be excluded. None of the affected showed homozygosity for the Pax6 microsatellite markers. CONCLUSIONS: We conclude that the phenotype and the clinical features in this family defines a new Mendelian disorder. It remains to be seen what kind of molecule shared by eye and bone is involved.

X-linked retinitis pigmentosa: RPGR mutations in most families with definite X linkage and clustering of mutations in a short sequence stretch of exon ORF15.

PURPOSE: A comprehensive screening was conducted for RP2 and retinitis pigmentosa GTPase regulator (RPGR) gene mutations including RPGR exon ORF15 in 58 index patients. The frequency of RPGR mutations was assessed in families with definite X-linked recessive disease (xlRP), and a strategy for analyzing the highly repetitive mutational hot spot in exon ORF15 is provided. METHODS: Fifty-eight apparently unrelated index-patients were screened for mutations in all coding exons of the RP2 and the RPGR genes, including splice-sites, by single-strand conformation polymorphism (SSCP) analysis, except for RPGR exon ORF15. A strategy for directly sequencing the large repetitive stretch of exon ORF15 from a 1.6-kb PCR-product was developed. According to pedigree size and evidence for X linkage, families were subdivided into three categories. RESULTS: Screening of 58 xlRP families revealed RP2 mutations in 8% and RPGR mutations in 71% of families with definite X-linked inheritance. Mutations clustered within a approximately 500-bp stretch in exon ORF15. In-frame sequence alterations in exon ORF15 ranged from the deletion of 36 bp to the insertion of 75 bp. CONCLUSIONS: Mutations in the RPGR gene are estimated to cause 15% to 20% of all cases of RP, higher than any other single RP locus. This report provides a detailed strategy to analyze the mutational hot spot in RPGR exon ORF15, which cannot be screened by standard procedures. The discrepancy of the proportion of families linked to the RP3 locus and those having RPGR mutations is resolved in a subset of families with definite X linkage.

A full-coverage, high-resolution human chromosome 22 genomic microarray for clinical and research applications.

We have constructed the first comprehensive microarray representing a human chromosome for analysis of DNA copy number variation. This chromosome 22 array covers 34.7 Mb, representing 1.1% of the genome, with an average resolution of 75 kb. To demonstrate the utility of the array, we have applied it to profile acral melanoma, dermatofibrosarcoma, DiGeorge syndrome and neurofibromatosis 2. We accurately diagnosed homozygous/heterozygous deletions, amplifications/gains, IGLV/IGLC locus instability, and breakpoints of an imbalanced translocation. We further identified the 14-3-3 eta isoform as a candidate tumor suppressor in glioblastoma. Two significant methodological advances in array construction were also developed and validated. These include a strictly sequence defined, repeat-free, and non-redundant strategy for array preparation. This approach allows an increase in array resolution and analysis of any locus; disregarding common repeats, genomic clone availability and sequence redundancy. In addition, we report that the application of phi29 DNA polymerase is advantageous in microarray preparation. A broad spectrum of issues in medical research and diagnostics can be approached using the array. This well annotated and gene-rich autosome contains numerous uncharacterized disease genes. It is therefore crucial to associate these genes to specific 22q-related conditions and this array will be instrumental towards this goal. Furthermore, comprehensive epigenetic profiling of 22q-located genes and high-resolution analysis of replication timing across the entire chromosome can be studied using our array.

Thirty distinct CACNA1F mutations in 33 families with incomplete type of XLCSNB and Cacna1f expression profiling in mouse retina.

X-linked CSNB patients may exhibit myopia, nystagmus, strabismus and ERG abnormalities of the Schubert-Bornschein type. We recently identified the retina-specific L-type calcium channel alpha1 subunit gene CACNA1F localised to the Xp11.23 region, which is mutated in families showing the incomplete type (CSNB2). Here, we report comprehensive mutation analyses in the 48 CACNA1F exons in 36 families, most of them from Germany. All families were initially diagnosed as having the incomplete type of CSNB, except for two which have been designated as Aland Island eye disease (AIED)-like. Out of 33 families, a total of 30 different mutations were identified, of which 24 appear to be unique for the German population. The mutations, 20 of which are published here for the first time, were found to be equally distributed over the entire gene sequence. No mutation could be found in a classic AIED family previously shown to map to the CSNB2 interval. Cacna1f expression in photoreceptor-negative mice strains indicate that the gene is expressed in the outer nuclear, the inner nuclear, and the ganglion cell layer. Such a distribution points to the central role of calcium regulation in the interaction of retinal cells that mediate signal transmission.

Ancestral founder of mutation W283X in the porphobilinogen deaminase gene among acute intermittent porphyria patients.

Acute intermittent porphyria (AIP) is a low-penetrant autosomal dominant disorder caused by mutations in the porphobilinogen deaminase gene (PBGD). Nearly 60% of all Swiss AIP patients carry a nonsense mutation W283X (G(7916)-->A). In France, the prevalence of W283X is <5%. To determine whether W283X was a founder mutation or originated from multiple de novo events, we studied 25 apparently unrelated W283X families and index patients, 21 of Swiss and 4 of French origins. In the absence of sufficient genealogical data to verify the ancestral background of these W283X families/patients, we identified haplotypes of seven intragenic single nucleotide polymorphisms (SNPs) in the PBGD gene as well as eight microsatellites flanking the PBGD gene covering 9.88 cM in chromosome 11. Molecular cloning and sequencing experiments were required in order to completely resolve the intragenic haplotypes in this study cohort which mainly consisted of single index patients and families with limited members. Thirteen of the 25 W283X families/patients carry a SNP haplotype [C-A-A-A-G-C-W283X-G] and 12 (including four French families) carry a [T-G-G-G-G-C-W283X-G] haplotype. A less conserved microsatellite haplotype was identified among the 25 W283X alleles which allowed us to estimate the age of the mutation. Since W283X is not explained by a methylcytosine mutation, we favor the hypothesis of a single mutational event which took place on the [T-G-G-G-G-C-G] background at approximately 40 generations or 1000 years ago. Around 550 years ago, a recombination event occurred between intron 3 and 10 of the PBGD gene which resulted in the [C-A-A-A-G-C-W283X-G] haplotype only found in a restricted region.