Efficient detection of alport syndrome COL4a5 mutations with multiplex genomic PCR‐SSCP

We have performed effective mutation screening of COL4A5 with a new method of direct, multiplex genomic amplification that employs a single buffer condition and PCR profile. Application of the method to a consecutive series of 46 United States patients with diverse indications of Alport syndrome resulted in detection of mutations in 31 cases and of five previously unreported polymorphisms. With a correction for the presence of cases that are not likely to be due to changes at the COL4A5 locus, the mutation detection sensitivity is greater than 79%. The test examines 52 segments, including the COL4A6/COL4A5 intergenic promoter region, all 51 of the previously recognized exons and two newly detected exons between exons 41 and 42 that encode an alternatively spliced mRNA segment. New genomic sequence information was generated and used to design primer pairs that span substantial intron sequences on each side of all 53 exons. For SSCP screening, 16 multiplex PCR combinations (15 4‐plex and 1 3‐plex) were used to provide complete, partially redundant coverage of the gene. The selected combinations allow clear resolution of products from each segment using various SSCP gel formulations. One of the 29 different mutations detected initially seemed to be a missense change in exon 32 but was found to cause exon skipping. Another missense variant may mark a novel functional site located in the collagenous domain. © 2001 Wiley‐Liss, Inc.     

High efficiency of mutation detection in type 1 stickler syndrome using a two-stage approach: vitreoretinal assessment coupled with exon sequencing for screening COL2A1

Stickler syndrome is a genetically heterogeneous disorder that affects the ocular, skeletal, and auditory systems. To date three genes, COL2A1, COL11A1, and COL11A2, encoding the heterotypic type II/XI collagen fibrils present in vitreous and cartilage have been shown to have mutations that result in Stickler syndrome. As systemic features in this disorder are variable we have used an ophthalmic examination to differentiate those patients with a membranous vitreous phenotype associated with mutations in COL2A1, from other patients who may have mutations in other genes. Gene amplification and exon sequencing was used to screen 50 families or sporadic cases with this membranous phenotype, for mutations in COL2A1. Mutations were detected in 47 (94%) cases consisting of 166 affected and 78 unaffected individuals. We also demonstrate that the predominantly ocular form of type 1 Stickler syndrome is not confined to mutations in the alternatively spliced exon 2. Using splicing reporter constructs we demonstrate that a mutant GC donor splice site in intron 51 can be spliced normally; this contributed to the predominantly ocular phenotype in the family in which it occurred.     

Missense and nonsense mutations in the alternatively-spliced exon 2 of COL2A1 cause the ocular variant of Stickler syndrome

Stickler syndrome type I (STL1) is a phenotypically heterogeneous disorder characterized by ocular and extraocular features. It is caused by null-allele mutations in the COL2A1 gene that codes for procollagen II. COL2A1 precursor mRNA undergoes alternative splicing, resulting in two isoforms, a long form including exon 2 (type IIA isoform) and a short form excluding exon 2 (type IIB isoform). The short form is predominantly expressed by differentiated chondrocytes in adult cartilage, and the long form in chondroprogenitor cells during early development and in the vitreous of the eye, which is the only adult tissue containing procollagen IIA. Recent evidence indicates that due to the tissue-specific expression of these two isoforms, premature termination codon mutations in exon 2 cause Stickler syndrome with minimal or no extraocular manifestations. We describe here two mutations in exon 2 of COL2A1 in three patients with predominantly ocular Stickler syndrome: Cys64Stop in two patients, and a novel structural mutation, Cys57Tyr, in one patient. RT-PCR of total lymphoblast RNA from one patient with the Cys64Stop mutation revealed that only the normal allele of the IIA form was present, indicating that the mutation resulted either in complete loss of the allele by nonsense-mediated mRNA decay or by skipping of exon 2 via nonsense-mediated altered splicing, resulting in production of the type IIB isoform. The results of COL2A1 minigene expression studies suggest that both Cys64Stop and Cys57Tyr alter positive cis regulatory elements for splicing, resulting in a lower IIA:IIB ratio.     

Small deletions in the type II collagen triple helix produce Kniest dysplasia

Kniest dysplasia is a moderately severe type II collagenopathy, characterized by short trunk and limbs, kyphoscoliosis, midface hypoplasia, severe myopia, and hearing loss. Mutations in the gene that encodes type II collagen (COL2A1), the predominant protein of cartilage, have been identified in a number of individuals with Kniest dysplasia. All but two of these previously described mutations cause in-frame deletions in type II collagen, either by small deletions in the gene or splice site alterations. Furthermore, all but one of these mutations is located between exons 12 and 24 in the COL2A1 gene. We used heteroduplex analysis to identify sequence anomalies in five individuals with Kniest dysplasia. Sequencing of the index patients' genomic DNA identified four new dominant mutations in COL2A1 that result in Kniest dysplasia: a 21-bp deletion in exon 16, an 18-bp deletion in exon 19, and 4-bp deletions in the splice donor sites of introns 14 and 20. A previously described 28-bp deletion at the COL2A1 exon 12–intron 12 junction, deleting the splice donor site, was identified in the fifth case. The latter three mutations are predicted to result in exon skipping in the mRNA encoded from the mutant allele. These data suggest that Kniest dysplasia results from shorter type II collagen monomers, and support the hypothesis that alteration of a specific COL2A1 domain, which may span from exons 12 to 24, leads to the Kniest dysplasia phenotype. Am. J Med. Genet. 85:105–112, 1999. Published 1999 Wiley-Liss, Inc.     

Identification of a new heterozygous point mutation in the COL1A2 gene leading to skipping of exon 9 in a patient with joint laxity,hyperextensibility of skin and blue sclerae

A heterozygous deletion of exon 9 in the COL1A2 - mRNA of a patient with symptoms of both the Ehlers - Danlos - Syndrome and the Osteogenesis Imperfecta is described. In the genomic DNA of the patient, exon 9 is homozygously present. We identified a novel heterozygous point mutation in the splice donor site of intron 9, leading to a G→A substitution in position +5. This mutation leads to heterozygous skipping of exon 9 in the COL1A2 - mRNA of this patient. The deletion results in a shortened (by 18 amino acids) but in frame l2(1) chain, which probably leads to the formation of abberantly processed triple helices. Hum Mutat 12:138, 1998. © 1998 Wiley-Liss, Inc.     

Exon skipping mutations in collagen VI are common and are predictive for severity and inheritance

Mutations in the genes encoding collagen VI (COL6A1, COL6A2, and COL6A3) cause Bethlem myopathy (BM) and Ullrich congenital muscular dystrophy (UCMD), two related conditions of differing severity. BM is a relatively mild dominantly inherited disorder characterized by proximal weakness and distal joint contractures. UCMD was originally regarded as an exclusively autosomal recessive condition causing severe muscle weakness with proximal joint contractures and distal hyperlaxity. We and others have subsequently modified this model when we described UCMD patients with heterozygous in-frame deletions acting in a dominant-negative way. Here we report 10 unrelated patients with a UCMD clinical phenotype and de novo dominant negative heterozygous splice mutations in COL6A1, COL6A2, and COL6A3 and contrast our findings with four UCMD patients with recessively acting splice mutations and two BM patients with heterozygous splice mutations. We find that the location of the skipped exon relative to the molecular structure of the collagen chain strongly correlates with the clinical phenotype. Analysis by immunohistochemical staining of muscle biopsies and dermal fibroblast cultures, as well as immunoprecipitation to study protein biosynthesis and assembly, suggests different mechanisms each for exon skipping mutations underlying dominant UCMD, dominant BM, and recessive UCMD. We provide further evidence that de novo dominant mutations in severe UCMD occur relatively frequently in all three collagen VI chains and offer biochemical insight into genotype-phenotype correlations within the collagen VI-related disorders by showing that severity of the phenotype depends on the ability of mutant chains to be incorporated in the multimeric structure of collagen VI.     

Collagen type IV‐related nephropathies in Portugal: pathogenic COL4A3 and COL4A4 mutations and clinical characterization of 25 families

Pathogenic mutations in genes COL4A3/COL4A4 are responsible for autosomal Alport syndrome (AS) and thin basement membrane nephropathy (TBMN). We used Sanger sequencing to analyze all exons and splice site regions of COL4A3/COL4A4, in 40 unrelated Portuguese probands with clinical suspicion of AS/TBMN. To assess genotype–phenotype correlations, we compared clinically relevant phenotypes/outcomes between homozygous/compound heterozygous and apparently heterozygous patients. Seventeen novel and four reportedly pathogenic COL4A3/COL4A4 mutations were identified in 62.5% (25/40) of the probands. Regardless of the mutated gene, all patients with ARAS manifested chronic renal failure (CRF) and hearing loss, whereas a minority of the apparently heterozygous patients had CRF or extrarenal symptoms. CRF was diagnosed at a significantly younger age in patients with ARAS. In our families, the occurrence of COL4A3/COL4A4 mutations was higher, while the prevalence of XLAS was lower than expected. Overall, a pathogenic COL4A3/COL4A4/COL4A5 mutation was identified in >50% of patients with fewer than three of the standard diagnostic criteria of AS. With such a population background, simultaneous next‐generation sequencing of all three genes may be recommended as the most expedite approach to diagnose collagen IV‐related glomerular basement membrane nephropathies.     

Allelic and non-allelic heterogeneities in pyridoxine dependent seizures revealed by ALDH7A1 mutational analysis

Pyridoxine dependent seizure (PDS) is a disorder of neonates or infants with autosomal recessive inheritance characterized by seizures, which responds to pharmacological dose of pyridoxine. Recently, mutations have been identified in the ALDH7A1 gene in Caucasian families with PDS. To elucidate further the genetic background of PDS, we screened for ALDH7A1 mutations in five PDS families (patients 1-5) that included four Orientals. Diagnosis as having PDS was confirmed by pyridoxine-withdrawal test. Exon sequencing analysis of patients 1-4 revealed eight ALDH7A1 mutations in compound heterozygous forms: five missense mutations, one nonsense mutation, one point mutation at the splicing donor site in intron 1, and a 1937-bp genomic deletion. The deletion included the entire exon 17, which was flanked by two Alu elements in introns 16 and 17. None of the mutations was found in 100 control chromosomes. In patient 5, no mutation was found by the exon sequencing analysis. Furthermore, expression level or nucleotide sequences of ALDH7A1 mRNA in lymphoblasts were normal. Plasma pipecolic acid concentration was not elevated in patient 5. These observations suggest that ALDH7A1 mutation is unlikely to be responsible for patient 5. Abnormal metabolism of GABA/glutamate in brain has long been suggested as the underlying pathophysiology of PDS. CSF glutamate concentration was elevated during the off-pyridoxine period in patient 3, but not in patient 2 or 5. These results suggest allelic and non-allelic heterogeneities of PDS, and that the CSF glutamate elevation does not directly correlate with the presence of ALDH7A1 mutations.     

Identification of a new heterozygous point mutation in the COL1A2 gene leading to skipping of exon 9 in a patient with joint laxity, hyperextensibility of skin and blue sclerae. Mutations in brief no. 166. Online

A heterozygous deletion of exon 9 in the COL1A2-mRNA of a patient with symptoms of both the Ehlers-Danlos-Syndrome and the Osteogensis Imperfecta is described. In the genomic DNA of the patient, exon 9 is homozygously present. We identified a novel heterozygous point mutation in the splice donor site of intron 9, leading to a G-->A substitution in position +5. This mutation leads to heterozygous skipping of exon 9 in the COL1A2-mRNA of this patient. The deletion results in a shortened (by 18 amino acids) but in frame 12(1) chain, which probably leads to the formation of abberantly processed triple helices.     

Mosaicism for Dominant Collagen 6 Mutations as a Cause for Intrafamilial Phenotypic Variability

Collagen 6‐related dystrophies and myopathies (COL6‐RD) are a group of disorders that form a wide phenotypic spectrum, ranging from severe Ullrich congenital muscular dystrophy, intermediate phenotypes, to the milder Bethlem myopathy. Both inter‐ and intrafamilial variable expressivity are commonly observed. We present clinical, immunohistochemical, and genetic data on four COL6‐RD families with marked intergenerational phenotypic heterogeneity. This variable expression seemingly masquerades as anticipation is due to parental mosaicism for a dominant mutation, with subsequent full inheritance and penetrance of the mutation in the heterozygous offspring. We also present an additional fifth simplex patient identified as a mosaic carrier. Parental mosaicism was confirmed in the four families through quantitative analysis of the ratio of mutant versus wild‐type allele (COL6A1, COL6A2, and COL6A3) in genomic DNA from various tissues, including blood, dermal fibroblasts, and saliva. Consistent with somatic mosaicism, parental samples had lower ratios of mutant versus wild‐type allele compared with the fully heterozygote offspring. However, there was notable variability of the mutant allele levels between tissues tested, ranging from 16% (saliva) to 43% (fibroblasts) in one mosaic father. This is the first report demonstrating mosaicism as a cause of intrafamilial/intergenerational variability of COL6‐RD, and suggests that sporadic and parental mosaicism may be more common than previously suspected.     

Mutations causing defective splicing in the human hprt gene.

Ten intron mutations and one exon mutation giving rise to defective splicing in the human gene for hypoxanthine phosphoribosyl transferase (hprt) in T-lymphocytes have been characterized. The splicing mutants were detected by PCR amplification of hprt cDNA and direct sequencing. Nine of the mutants showed skipping of whole exons or parts of exons in the cDNA, one mutant had an inclusion of an intron sequence into the cDNA, and one mutant showed both inclusion of an intron sequence and skipping of exons as well as a normal cDNA. Genomic PCR and direct sequencing of the splice sites involved showed one deletion of three base pairs and 10 different single base alterations to be responsible for these splice alterations. One mutation in the last base pair of exon 6 causing skipping of the entire exon 6 was found, whereas an identical mutation in the last base pair of exon 2 caused no aberrant splicing. It was also found that a deletion mutation in the pyrimidine rich stretch of the acceptor site of intron 7 caused skipping of the entire exon 8, whereas a base substitution in the last base of intron 7 caused exclusion of only the first 21 base pairs of exon 8 as a result of the activation of a cryptic acceptor site in exon 8. The results show that many different types of mutations at several different sites can cause splicing errors in the hprt gene and that the sequence differences between the splice sites influence the possible spectrum of mutations in each site.     

Lack of correlation between the type of COL1A1 or COL1A2 mutation and hearing loss in osteogenesis imperfecta patients

Osteogenesis imperfecta (OI) is caused by mutations in COL1A1 and COL1A2 that code for the alpha1 and alpha2 chains of type I collagen. Phenotypes correlate with the mutation types in that COL1A1 null mutations lead to OI type I, and structural mutations in alpha1(I) or alpha2(I) lead to more severe OI types (II-IV). However, correlative analysis between mutation types and OI associated hearing loss has not been previously performed. A total of 54 Finnish OI patients with previously diagnosed hearing loss or age 35 or more years were analyzed here for mutations in COL1A1 or COL1A2. Altogether 49 mutations were identified, of which 41 were novel. The 49 mutations represented the molecular genetic background of 41.1% of the Finnish OI population. A total of 38 mutations were in COL1A1 and 11 were in COL1A2. Of these, 16 were glycine substitutions and 16 were splicing mutations in alpha1(I) or alpha2(I). In addition, 17 null allele mutations were detected in COL1A1. A total of 32 patients (65.3%) with a mutation had hearing loss. That is slightly more than in our previous population study on Finnish adults with OI (57.9%). The association between the mutation types and OI type was statistically evident. Patients with COL1A1 mutations more frequently had blue scleras than those with COL1A2 mutations. In addition, patients with COL1A2 mutations tended to be shorter than those with COL1A1 mutations. However, no correlation was found between the mutated gene or mutation type and hearing pattern. These results suggest that the basis of hearing loss in OI is complex, and it is a result of multifactorial, still unknown genetic effects.