Mutations in the SLC26A4 gene are responsible for Pendred syndrome and non-syndromic hearing loss (DFNB4). This study analysed non-synonymous SLC26A4 mutations newly identified in East Asians, as well as three common mutations in Caucasians, to characterise their molecular pathogenic mechanisms and to explore the possibility of rescuing their processing defects.
A total of 11 non-synonymous disease associated mutations were generated and their effects on protein processing and on ion transporting activities were examined.
Most of the mutations caused retention of the SLC26A4 gene product (pendrin) in the intracellular region, while wild-type pendrin reached the plasma membrane. Accordingly, these mutations abolished complex glycosylation and Cl–/HCO3– exchange activities of pendrin. However, significant heterogeneity in the processing of mutant pendrin molecules was observed. Each mutant protein exhibited a different cellular localisation, a different degree of N-glycosylation, and a different degree of sensitivity to the treatments that rescue processing defects. For example, H723R-pendrin, the most common mutation in East Asians, was mostly expressed in endoplasmic reticulum (ER), and its defects in protein processing and ion transporting activities were restored considerably by low temperature incubation. On the other hand, L236P-pendrin, the most common mutation in Caucasians, was mainly in the centrosomal region and was temperature insensitive.
These results indicate that the processing of pendrin mutant protein is determined by mutant specific mechanisms, and that a mutant specific method would be required to rescue the conformational defects of each folding mutant.
The ESR1 AGATA haplotype is composed of five markers located within introns 5 and 6 of the human oestrogen receptor 1 (ESR1) gene. This haplotype has been studied in several male urogenital tract anomalies and male infertility. In one of these studies, a deviation from Hardy–Weinberg equilibrium (DHW) was found for the ESR1 AGATA marker rs3020375 in two groups of healthy controls. In the present study, we investigated whether the observed DHW is caused by structural variants present within the ESR1 gene.
229 family units achieving pregnancy through assisted reproductive technologies (n = 129) or by natural means (n = 100), 2465 general Spanish population controls and 162 men with idiopathic infertility.
Segregation analyses of genetic markers in family units and case–control genetic association studies.
We identified a new interstitial deletion of 2244 base pairs within intron 6 of the human ESR1 gene as the cause for the observed DHW. This new variant presents a 10% allelic frequency in the general Spanish population and it is associated with idiopathic male infertility (OR = 1.51; p = 0.03). The percentage of infertile couples in which both members carried the ESR1 deletion (10.08%) was also a higher than expected value of 6% (p = 0.03).
We have characterised a novel structural variation in human ESR1 gene. The available data indicate a deleterious action of the ESR1 deletion in both male and couple fertility. The potential effects of this deletion on other oestrogen-related diseases need to be determined.
Genetic testing for the breast and ovarian cancer susceptibility genes BRCA1 and BRCA2 has important implications for the clinical management of people found to carry a mutation. However, genetic testing is expensive and may be associated with adverse psychosocial effects. To provide a cost-efficient and clinically appropriate genetic counselling service, genetic testing should be targeted at those individuals most likely to carry pathogenic mutations. Several algorithms that predict the likelihood of carrying a BRCA1 or a BRCA2 mutation are currently used in clinical practice to identify such individuals.
We evaluated the performance of the carrier prediction algorithms BOADICEA, BRCAPRO, IBIS, the Manchester scoring system and Myriad tables, using 1934 families seen in cancer genetics clinics in the UK in whom an index patient had been screened for BRCA1 and/or BRCA2 mutations. The models were evaluated for calibration, discrimination and accuracy of the predictions.
Of the five algorithms, only BOADICEA predicted the overall observed number of mutations detected accurately (ie, was well calibrated). BOADICEA also provided the best discrimination, being significantly better (p<0.05) than all models except BRCAPRO (area under the receiver operating characteristic curve statistics: BOADICEA = 0.77, BRCAPRO = 0.76, IBIS = 0.74, Manchester = 0.75, Myriad = 0.72). All models underpredicted the number of BRCA1 and BRCA2 mutations in the low estimated risk category.
Carrier prediction algorithms provide a rational basis for counselling individuals likely to carry BRCA1 or BRCA2 mutations. Their widespread use would improve equity of access and the cost-effectiveness of genetic testing.
Mental retardation can be caused by copy number variations (deletions, insertions, duplications), ranging in size from 1 kb to several megabases. Array based comparative genomic hybridisation (array-CGH) allows detection of an increasing number of genomic alterations.
A series of 46 patients with mental retardation and congenital abnormalities (previously screened for subtelomeric rearrangements) were evaluated for cryptic chromosomal imbalances by array-CGH. This array contains 6465 large-insert BAC/PAC clones, representing sequences uniformly distributed throughout the human genome. The results were confirmed by alternative techniques.
Four pathogenic rearrangements were detected: two of them were novel, a deletion at 2q31.2 and a duplication at 8q12 band; the other two have been previously reported—a duplication of the Williams–Beuren region and a deletion of 3q29. By adding the subtelomeric alterations previously identified, a total rate of 18% of pathogenic rearrangements was found in the series.
Based on our results, ZNF533 is the only gene contained in the overlapping region with other deletions at 2q31.2, and it is most probably the fourth zinc-finger gene implied in mental retardation. On the other hand, we propose that the CHD7 gene, associated with CHARGE syndrome by haploinsufficiency, causes a different phenotype by gain-of-dosage.
Many unclassified variants (UV) of BRCA1 or BRCA2 may have an effect on pre-mRNA splicing. Patient blood samples suitable for RNA extraction are not always available for testing UVs at the RNA level.
Analyses of RNA from patient peripheral blood were performed, using a one-step reverse transcriptase-PCR (RT-PCR) protocol, and were compared with an ex vivo splicing assay based on PCR-amplified patient DNA inserted into a splicing reporter minigene. Using both methods 20 variants found in 17 patients were examined.
Data from patient RNA and from the minigene assay were fully concordant, but the ex vivo splicing assay, which is monoallelic, clarified several ambiguities in the patient RNA data. Two intronic variants induced strong splicing defects: BRCA1 c.4987-5T->A (IVS16-5T->A) induced exon 17 skipping and BRCA2 c.316+5G->C (IVS3+5G->C) induced complete skipping of exon 3. Of the exonic variants, BRCA2 c.7805G->C (p.Arg2602Thr), at the last base of exon 16, induced both exon skipping and activation of a cryptic exonic donor site, and BRCA2 c.8023A->G (p.Ile2675Val) generated a strong donor site within exon 18. These four variants were thus classified as pathogenic, because of the total absence of a normal transcript from the corresponding allele. Variant BRCA2 c.9501+3A->T (IVS25+3A->T) induced incomplete skipping of exon 25, suggesting a mutation with incomplete penetrance, and BRCA2 c.8257_8259del (p.Leu2753del) modified the alternative splicing of exons 17 and 18.
We show that functional analysis using a splicing reporter minigene is sensitive and specific, and should be used for initial screening of potential splicing defects, especially when patient RNA is not readily available.
Music perception and performance are comprehensive human cognitive functions and thus provide an excellent model system for studying human behaviour and brain function. However, the molecules involved in mediating music perception and performance are so far uncharacterised.
To unravel the biological background of music perception, using molecular and statistical genetic approaches.
Methods: 15 Finnish multigenerational families (with a total of 234 family members) were recruited via a nationwide search. The phenotype of all family members was determined using three tests used in defining musical aptitude: a test for auditory structuring ability (Karma Music test; KMT) commonly used in Finland, and the Seashore pitch and time discrimination subtests (SP and ST respectively) used internationally. We calculated heritabilities and performed a genome-wide variance components-based linkage scan using genotype data for 1113 microsatellite markers.
The heritability estimates were 42% for KMT, 57% for SP, 21% for ST and 48% for the combined music test scores. Significant evidence of linkage was obtained on chromosome 4q22 (LOD 3.33) and suggestive evidence of linkage at 8q13-21 (LOD 2.29) with the combined music test scores, using variance component linkage analyses. The major contribution of the 4q22 locus was obtained for the KMT (LOD 2.91). Interestingly, a positive LOD score of 1.69 was shown at 18q, a region previously linked to dyslexia (DYX6) using combined music test scores.
Our results show that there is a genetic contribution to musical aptitude that is likely to be regulated by several predisposing genes or variants.
Dentin phosphoprotein (DPP) is the most abundant non-collagenous protein in dentin, which is highly phosphorylated and plays key roles in dentin biomineralisation. The aetiology of isolated hereditary dentin disorders in most affected families is largely unknown and the association between DPP and dentin disorders has not been well established. This study aims to determine whether there are some involvements for DPP mutations in inherited dentin disorders and to clarify the sequence variation patterns of DPP in normal population.
Genomic DNA was analysed in eight families with hereditary dentin disorders and 110 individuals in the normal population. The full coding sequence of DPP was amplified by polymerase chain reaction (PCR) and screened for mutations and variations by direct sequencing and TOPO TA-cloning sequencing.
Five frameshift mutations in DPP coding region were identified in five of the eight families. The mutations co-segregated with the disease phenotypes in affected families and were not found in 220 control chromosomes. In the normal population, we revealed 14 in-frame indels (insertion/deletion), six non-synonymous single nucleotide polymorphisms (SNPs), and five synonymous SNPs in the DPP coding region. These variants display extensive linkage disequilibrium and constitute a total of 15 haplotypes with three predominant haplotypes in the investigated normal population.
Our data provide the first evidence that DPP mutations can cause hereditary dentin disorders and suggest that in-frame length variations and missense SNPs in DPP have no obvious pathogenetic effects on dentin formation.
More than half of the retinitis pigmentosa (RP) cases are genetically simplex or multiplex. To date, 37 causative genes of RP have been identified; however, the elucidation of gene defects in simplex or multiplex RP patients/families remains problematic. The aim of our study was to identify the genetic causes of RP in patients with unknown or non-Mendelian inheritance.
Since 2003, 52 simplex RP patients, 151 patients from 141 multiplex RP families, and six sporadic patients with retinal degeneration were studied. A total of 108 exons of 30 RP-causing genes that harboured the reported mutations were screened by an efficient denaturing high performance liquid chromatography (dHPLC) based assay. Aberrant fragments were subsequently analysed by automatic sequencing. Twenty-six mutations, including two frameshift mutations, one single amino acid deletion, and 23 missense mutations, were identified in 28 probands (14.07%). Eighteen mutations have not been reported to date. Three pairs of combined mutations in different genes were identified in two sporadic cases and one multiplex family, indicating the possibility of novel digenic patterns. Of the 23 missense mutations, 21 were predicted as deleterious mutations by computational methods using PolyPhen, SIFT, PANTHER, and PMut programs.
We elucidated the mutation spectrum in Japanese RP patients and demonstrated the validity of the mutation detection system using dHPLC sequencing for genetic diagnosis in RP patients independent of familial incidence, which may provide a model strategy for identifying genetic causes in other diseases linked to a wide range of genes.
Ethylmalonic encephalopathy (EE) is a rare autosomal recessive metabolic disorder characterised by progressive encephalopathy, recurrent petechiae, acrocyanosis and chronic diarrhoea, with a fatal outcome in early in life.
14 patients with EE were investigated for mutations in the ETHE1 gene.
Of the 14 patients, 5 were found to carry novel mutations.
This work expands our knowledge of the causative mutations of EE.