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We present the first Turkish family with delta-sarcoglycanopathy (LGMD2F). A novel truncating mutation (E93X) in exon 3 was identified in the gene. The index case showed a severe course and there was no cardiac involvement. LGMD2F seems to be rare in our population.
Non-syndromic cleft lip with or without cleft palate (CL/P, MIM 119530) is among the most common of major birth defects. Homozygosity for a nonsense mutation of PVRL1, W185X, results in an autosomal recessive CL/P syndrome on Margarita Island, CLPED1 (ref. 1). Here we demonstrate highly significant association between heterozygosity for this mutation and sporadic, non-syndromic CL/P in northern Venezuela.
This Commentary discusses new data from the laboratory of Matthias Hentze (Schell et al., in this issue) that begins to dissect the RNA-protein and protein-protein interactions important for the process of nonsense-mediated decay (NMD). Schell and co-workers have developed a novel tool for analysis of such interactions in vivo. Using a proteomics approach, they have identified two different protein-RNA complexes that contain human Upf (up-frameshift) proteins central to the NMD process. Intriguingly, they identified a poly[A]-binding protein associated with these complexes.
Autosomal dominant cataract is a clinically and genetically heterogeneous lens disorder that usually presents as a sight-threatening trait in childhood. Here we have mapped dominant pulverulent cataract to the beta-crystallin gene cluster on chromosome 22q11.2. Suggestive evidence of linkage was detected at markers D22S1167 (LOD score [Z] 2.09 at recombination fraction [theta] 0) and D22S1154 (Z=1.39 at theta=0), which closely flank the genes for betaB1-crystallin (CRYBB1) and betaA4-crystallin (CRYBA4). Sequencing failed to detect any nucleotide changes in CRYBA4; however, a G-->T transversion in exon 6 of CRYBB1 was found to cosegregate with cataract in the family. This single-nucleotide change was predicted to introduce a translation stop codon at glycine 220 (G220X). Expression of recombinant human betaB1-crystallin in bacteria showed that the truncated G220X mutant was significantly less soluble than wild type. This study has identified the first CRYBB1 mutation associated with autosomal dominant cataract in humans.
Epidermodysplasia verruciformis (OMIM 226400) is a rare autosomal recessive genodermatosis associated with a high risk of skin carcinoma that results from an abnormal susceptibility to infection by specific human papillomaviruses (HPVs). We recently mapped a susceptibility locus for epidermodysplasia verruciformis (EV1) to chromosome 17q25. Here we report the identification of nonsense mutations in two adjacent novel genes, EVER1 and EVER2, that are associated with the disease. The gene products EVER1 and EVER2 have features of integral membrane proteins and are localized in the endoplasmic reticulum.
BACKGROUND: Atrichia with papular lesions (APL) is a rare autosomal recessive condition resulting from mutations in the hairless (HR) gene. OBJECTIVE: In the present study, we investigated the molecular basis of APL in a non-consanguineous Korean family. METHODS: Direct automated DNA sequencing of the HR gene and restriction digestion analysis were used to identify and confirm the mutation in our proband. RESULTS: Sequencing of the HR gene revealed two novel nonsense mutations in exons 2 and 4 which were subsequently confirmed via enzymatic restriction. No mutations have previously been detected in this population. CONCLUSION: The growing number of heterozygous mutations in non-consanguineous pedigrees supports the hypothesis that APL is more common than previously expected.
Familial cylindromatosis is a rare dominantly inherited disease characterized by the development of multiple benign tumours of the skin appendages, including cylindromas, trichoepitheliomas and spiradenomas. The gene responsible was positionally cloned recently, and was designated CYLD. We describe a family with cylindromatosis, in which affected individuals have an inherited R758X nonsense mutation of CYLD. Affected members of this family manifest a relatively mild tumour phenotype; the largest tumour was only 30 mm in diameter. Thus far, there is no evident genotype-phenotype relationship in cylindromatosis, although the number of families reported with both phenotypic and genotypic data remains small.
Limb girdle muscular dystrophy type 2A (LGMD2A) is caused by single or small nucleotide changes widespread along the CAPN3 gene, which encodes the muscle-specific proteolytic enzyme calpain-3. About 356 unique allelic variants of CAPN3 have been identified to date. We performed analysis of the CAPN3 gene in LGMD2A patients at both the mRNA level using reverse transcription-PCR, and at the DNA level using PCR and denaturing high performance liquid chromatography. In four patients, we detected homozygous occurrence of a missense mutation or an in-frame deletion at the mRNA level although the DNA was heterozygous for this mutation in conjunction with a frame-shift mutation. The relationship observed in 12 patients between the quantity of CAPN3 mRNA, determined using real-time PCR, and the genotype leads us to propose that CAPN3 mRNAs which contain frame-shift mutations are degraded by nonsense-mediated mRNA decay. Our results illustrate the importance of DNA analysis for reliable establishment of mutation status, and provide a new insight into the process of mRNA decay in cells of LGMD2A patients.
The present series comprises all families (n = 77) with haemophilia B in Sweden and may be considered to be representative for the purposes of a population-based study of mutational heterogeneity. The 77 families (38 severe, 10 moderate, 29 mild) had 51 different mutations in total. Thirteen families had total, partial or small deletions, two had mutations in the promoter, eight families had splice site mutations, 14 had nonsense and the remaining 41 had missense mutations. Ten of the mutations, all C-->T or G-->A, recurred in 1--6 other families. Using haplotype analysis of seven polymorphisms in the factor IX (FIX) gene, we found that the 77 families carried 65 unique, independent mutations. Of the 48 families with severe or moderate haemophilia, 23 (48%) had a sporadic case of haemophilia compared with 31 families out of 78 (40%) in the whole series. Five of those 23 sporadic cases carried de novo mutations, 11 out of 23 of the mothers were proven carriers and, in the remaining seven families, it was not possible to determine carriership. Eleven of the 48 patients (23%) with severe haemophilia B developed inhibitors and all of them had deletions or nonsense mutations. Thus, 11 out of 37 (30%) patients with severe haemophilia B as a result of deletion/nonsense mutations developed inhibitors compared with 0 out of 11 patients with missense mutations. The ratio of male to female mutation rates was 5.3 and the overall mutation rate was 5.4 x 10(-6) per gamete per generation.
Both viruses and tumors evade cytotoxic T lymphocyte-mediated host immunity by down-regulation of antigen presentation machineries. This can be achieved by either down-regulation of transcription of antigen presentation genes or posttranslational inactivation of proteins involved in antigen presentation. In this study, a major histocompatibility complex (MHC) class I-deficient melanoma cell line, SK-MEL-19, was found deficient in the expression of the transporter associated with antigen processing (TAP)-1 mRNA even after IFN-gamma stimulation, despite its active transcription of the TAP-1 gene. This abnormality was caused by a single-nucleotide deletion at position +1489 of the TAP-1 gene and was corrected by cycloheximide, which inhibits RNA degradation. Using an inducible Tet-Off system, we demonstrated that deletion of the nucleotide resulted in a >2-fold decrease in the half-life of TAP-1 mRNA. However, the decrease of the half-life of TAP-1 mRNA is not mediated by nonsense-mediated mRNA decay because deletions of two additional nucleotides in the region, which corrected the nonsense mutation, did not restore TAP-1 mRNA stability. To our knowledge, this is the first evidence that the degradation of mRNA of an antigen presentation gene is involved in HLA class I down-regulation in malignant cells.
AIMS: To describe mutations in the PAX6 gene in five patients with aniridia from three unrelated families. METHODS: The PAX6 gene was analysed using single stranded conformational polymorphism analysis and direct sequencing. RESULTS: In one family, three individuals from two generations had aniridia, whereas in each of the other families only one member was affected. The first patient had the heterozygous Q221X (1023C --> T) nonsense mutation in exon 8. The same mutation was found in his mother and sister. Another patient had a heterozygous Q297X (1252C --> T) mutation in exon 10. The third patient carried a heterozygous IVS5+2T --> C mutation leading to aberrant splicing of mRNA. CONCLUSIONS: These findings provide further examples of haploinsufficiency of PAX6 in aniridia.
BACKGROUND: Familial Adenomatous Polyposis (FAP) is caused by germline mutations in the APC (Adenomatous Polyposis Coli) gene. The vast majority of APC mutations are point mutations or small insertions/deletions which lead to truncated protein products. Splicing mutations or gross genomic rearrangements are less common inactivating events of the APC gene. METHODS: In the current study genomic DNA or RNA from ten unrelated FAP suspected patients was examined for germline mutations in the APC gene. Family history and phenotype were used in order to select the patients. Methods used for testing were dHPLC (denaturing High Performance Liquid Chromatography), sequencing, MLPA (Multiplex Ligation - dependent Probe Amplification), Karyotyping, FISH (Fluorescence In Situ Hybridization) and RT-PCR (Reverse Transcription - Polymerase Chain Reaction). RESULTS: A 250 Kbp deletion in the APC gene starting from intron 5 and extending beyond exon 15 was identified in one patient. A substitution of the +5 conserved nucleotide at the splice donor site of intron 9 in the APC gene was shown to produce frameshift and inefficient exon skipping in a second patient. Four frameshift mutations (1577insT, 1973delAG, 3180delAAAA, 3212delA) and a nonsense mutation (C1690T) were identified in the rest of the patients. CONCLUSION: Screening for APC mutations in FAP patients should include testing for splicing defects and gross genomic alterations.
KEY POINTS: A novel nonsense variant ( mcy ) in ankyrin repeat and sterile alpha motif domain-containing 6 ( Anks6 ) promotes rapid disease progression in the Han:SPRD- Cy rat carrying a missense variant in Anks6 . mcy-/- rats exhibit prenatal lethality characterized by laterality and cardiovascular abnormalities. These findings indicate that ANKS6 nonfunction in rats leads to prenatal lethality, whereas misfunction leads to polycystic kidney disease development. BACKGROUND: Polycystic kidney disease (PKD) encompasses a group of genetic disorders characterized by the proliferation of fluid-filled renal cysts, leading to progressive renal failure and death. A key feature of PKD is its variable expressivity across patients, even when caused by the same variant, highlighting the importance of genetic background in PKD expression. METHODS: We identified an ostensibly healthy Sprague Dawley rat line with a variant that modifies PKD expressivity in Han:SPRD- Cy rats (caused by a missense variant [p.Arg717Trp] in the ankyrin repeat and sterile alpha motif domain-containing 6 [ Anks6 ] gene), which we named mcy (modifier of Cy ). We used whole-genome sequencing and segregation analysis to identify the mcy variant, quantitative PCR and mRNA sequencing to evaluate its effects on gene expression, western blotting and immunohistochemistry to assess its protein consequences, and ultrasound and histology to examine its impact on rat embryonic development. RESULTS: We identified a nonsense variant in the Anks6 gene as the genetic basis of the mcy phenotype (c.1126G>T [p.Glu376X]). Although mcy+/- rats are ostensibly healthy and do not develop PKD, mcy-/- rats exhibit laterality defects and die prenatally at E16.5 because of apparent perturbations in cardiovascular development. Notably, mcy+/-Cy+/- rats develop PKD much more rapidly than Cy+/- rats, and in a timeframe consistent with Cy-/-rats . Transcripts with the mcy variant allele seem to undergo nonsense-mediated decay, and no ANKS6 protein is detected. However, gene expression patterns in the kidneys did not differ significantly between age-matched mcy+/+ and mcy+/- rats, indicating that ANKS6 insufficiency does not cause PKD. CONCLUSIONS: We identified a novel nonsense variant in Anks6 . The findings indicate that the absence of wild-type ANKS6 accelerates PKD development in the Han:SPRD- Cy rat and that complete ANKS6 deficiency prevents normal embryonic development in rats.
Coffin-Lowry syndrome is a rare X-linked, semi-dominant mental retardation syndrome resulting from mutations of the ribosomal S6 kinase 2 (RSK2) gene. In the present report, a male patient affected with Coffin-Lowry syndrome is shown to have a nonsense mutation of the RSK2 gene. His unaffected mother does not have this mutation in her lymphocytes. In her third pregnancy prenatal diagnosis by mutation analysis has detected gonadal mosaicism. As this is the second report of germinal mosaicism in Coffin-Lowry syndrome, the finding has important implication for genetic counselling.
Tangier disease is a rare autosomal recessive disorder caused by mutations in the recently identified ATP-binding cassette transporter 1 gene (ABC1). A typical clinical manifestation of Tangier disease is peripheral neuropathy. Former studies differentiated between two manifestations: the more frequent mono- or polyneuropathic form and a syringomyelia-like type. It is unknown whether specific mutations in the ABC1 gene or a particular genetic background are responsible for either of these forms. A family is presented comprising a case with a severe syringomyelia-like phenotype of Tangier disease and absence of cardiovascular disease. Sequencing analysis of the ABC1 gene was performed. A new homozygous C-->T transition in exon 18 was found in the index patient. This mutation results in a stop codon at position 909 (R909X) leading to premature termination of translation. Her clinically asymptomatic daughters, her sister and one of her nieces were heterozygous. Sural nerve biopsies were studied in the index patient at the age of 45 and 54 years; both revealed a severe neuropathy, characterized by a subtotal and finally complete loss of nerve fibres. The entire loss of Schwann cells resulted in an extraordinary form of endoneurial sclerosis. Only rare capillaries, lipid-laden macrophages and fibroblasts had survived in the endoneurium. This case appears to be unique in respect to the underlying novel mutation in the ABC1 gene and its association with complete endoneurial sclerosis of all fascicles in the sural nerve and absence of cardiovascular disease.
We investigated two siblings of a Spanish family presenting with congenital lactic acidosis. They had severe failure to thrive, liver dysfunction, and renal tubulopathy. An isolated biochemical complex III deficiency was detected in liver. A search for mutations in the human bc1 synthesis like (BCS1L) gene was undertaken. Direct sequencing revealed a missense mutation R45C and a nonsense mutation R56X, both located in exon 1 of BCS1L. The missense mutation in combination with a loss of function of the second allele is responsible for the isolated complex III deficiency in this family.
We report on a Japanese family (two brothers and their mother) with ulnar-mammary syndrome (UMS). Clinical features included hypoplasia or aplasia of upper limbs on the ulnar side in the three affected individuals, micropenis with or without cryptorchidism, and hypoplastic nipples in the brothers; and hypoplastic mammary glands and nipples, poor perspiration, and bicornuate uterus in the mother. Endocrine studies performed for the underdeveloped external genitalia when the brothers were 11 6/12 and 7 2/12 years old, respectively, indicated low to low-normal responses of luteinizing hormone (LH) and follicle stimulating hormone (FSH) to gonadotropin releasing hormone stimulation tests (elder brother: LH = < 0.2 --> 2.2 IU/L, FSH = 0.6 --> 2.2 IU/L; younger brother: LH = < 0.2 --> 3.3 IU/L, FSH = 0.7 --> 4.4 IU/L) and normal testosterone responses to human gonadotropin stimulation tests (elder brother: < 0.5 --> 8.8 nmol/L; younger brother: < 0.5 --> 6.3 nmol/L). Testosterone enanthate therapy (25 mg/dose IM twice) was effective in the brothers, with penile length increase being similar between the brothers (approximately 5 mm/dose) and 23 age-matched boys with idiopathic micropenis (mean 4.4 mm/dose, range 2.5-7.5 mm/dose). Sequence analysis of the TBX3 gene showed a novel heterozygous nonsense mutation (A817T, K273X) in exon 4 of the three patients. The results are consistent with the previous finding that UMS is caused by haploinsufficiency of TBX3, and imply that mild gonadotropin deficiency may be the primary cause for underdeveloped external genitalia in males with UMS.
INTRODUCTION: We have described in previous articles a nonsense mutation (4588C>T, R1511X) in exon 22 of the thyroglobulin (TG) gene in a member of a family with a complex history of congenital goiter. In the mutated thyroid gland, full-length thyroglobulin mRNA is almost undetectable. However, a smaller transcript is detected in which the mutated exon 22 is skipped and the reading frame restored. It is conceivable that alternative splicing might be a mechanism involved in the rescue of nonsense mutations. METHODS: To investigate whether the detection of the alternative mRNA is due to an increase in its concentration or its preferential amplification during reverse transcriptase-PCR in the absence of the normal full-length mRNA competitor, we set up an assay in which the competitor mRNA was provided. We also studied the effect of the 4588C>T mutation on exon definition and processing using wild-type and mutated minigenes. RESULTS: The detection of the alternative mRNA lacking exon 22 is not caused by the absence of the full-length competitor. In contrast, our results demonstrate that the alternative transcript preferentially accumulates in the mutated thyroid at a level similar to the full-length transcript in control tissue. Transient expression experiments with wild-type and mutated minigenes indicate that the mutated exon is as efficiently spliced as the wild-type, suggesting that the 4588C>T mutation does not interfere with exon 22 definition and processing. CONCLUSIONS: The alternative splicing of the TG gene described in this article constitutes a new case of nonsense-associated alternative splicing. We have shown that the mutation itself does not interfere with exon definition and processing in vitro. Our results support the hypothesis that the alternative splicing of the mutated exon is driven by the interruption of the reading frame.