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.     

A family with homozygous and heterozygous p.Gly337Ser mutations in COL1A2

Osteogenesis imperfecta (OI) is commonly caused by monoallelic mutations in COL1A1 or COL1A2. Biallelic mutations are extremely rare. Only five previous reports have identified seven OI patients with homozygous mutations in COL1A2. OI is a genetically and phenotypically heterogeneous disorder which challenges an establishment of genotype-phenotype correlation. Notably, more than thirty patients with OI possess the heterozygous mutation, p.Gly337Ser, in COL1A2. Their clinical severity ranges from mild OI type I to severe types III and IV. Here, we report a 17-year-old Thai female with recurrent bone fractures, short stature, blue sclerae, triangular face, missing teeth, dentinogenesis imperfecta (DI), skeletal deformities, and scoliosis. She was diagnosed with OI type III. Her parents were second-cousin-once-removed. The father was a professional Thai boxer. Both had normal bone mineral density, no history of bone fractures, and only teeth problems. They were diagnosed with DI without OI. Whole exome sequencing identified that the proband harbored the homozygous mutation, c.1009G > A (p.Gly337Ser), in exon 19 of COL1A2 while her parents were heterozygous for this mutation. This study reports the eighth child with OI and the homozygous mutation in COL1A2; and the first two individuals with the heterozygous p.Gly337Ser mutation in COL1A2 causing an isolated DI without OI.     

Ehlers-Danlos syndrome type VIIA and VIIB result from splice-junction mutations or genomic deletions that involve exon 6 in the COL1A1 and COL1A2 genes of type I collagen

Ehlers-Danlos syndrome (EDS) type VII results from defects in the conversion of type I procollagen to collagen as a consequence of mutations in the substrate that alter the protease cleavage site (EDS type VIIA and VIIB) or in the protease itself (EDS type VIIC). We identified seven additional families in which EDS type VII is either dominantly inherited (one family with EDS type VIIB) or due to new dominant mutations (one family with EDS type VIIA and five families with EDS type VIIB). In six families, the mutations alter the consensus splice junctions, and, in the seventh family, the exon is deleted entirely. The COL1A1 mutation produced the most severe phenotypic effects, whereas those in the COL1A2 gene, regardless of the location or effect, produced congenital hip dislocation and other joint instability that was sometimes very marked. Fractures are seen in some people with EDS type VII, consistent with alterations in mineral deposition on collagen fibrils in bony tissues. These new findings expand the array of mutations known to cause EDS type VII and provide insight into genotype/phenotype relationships in these genes. Am. J. Med. Genet. 72:94–105, 1997. © 1997 Wiley-Liss, Inc.     

COL1A1 and COL1A2 variants in Ehlers-Danlos syndrome phenotypes and COL1-related overlap disorder

Pathogenic variants in COL1A1 and COL1A2 are involved in osteogenesis imperfecta (OI) and, rarely, Ehlers-Danlos syndrome (EDS) subtypes and OI-EDS overlap syndromes (OIEDS1 and OIEDS2, respectively). Here we describe a cohort of 34 individuals with likely pathogenic and pathogenic variants in COL1A1 and COL1A2, 15 of whom have potential OIEDS1 (n = 5) or OIEDS2 (n = 10). A predominant OI phenotype and COL1A1 frameshift variants are present in 4/5 cases with potential OIEDS1. On the other hand, 9/10 potential OIEDS2 cases have a predominant EDS phenotype, including four with an initial diagnosis of hypermobile EDS (hEDS). An additional case with a predominant EDS phenotype had a COL1A1 arginine-to-cysteine variant that was originally misclassified as a variant of uncertain significance despite this type of variant being associated with classical EDS with vascular fragility. Vascular/arterial fragility was observed in 4/15 individuals (including one individual with an original diagnosis of hEDS), which underscores the unique clinical surveillance and management needs in these patients. In comparison to previously described OIEDS1/2, we observed differentiating features that should be considered to refine currently proposed criteria for genetic testing in OIEDS, which will be beneficial for diagnosis and management. Additionally, these results highlight the importance of gene-specific knowledge for informed variant classification and point to a potential genetic resolution (COL1A2) for some cases of clinically diagnosed hEDS.     

COL1A1/COL1A2基因突变分析与成骨不全的产前基因诊断

目的对有成骨不全(Osteogenesis Imperfecta,OI)孕史的患者,进行系统B超及COL1A1/COL1A2基因检测,希望建立OI患儿产前诊断方案,为OI患儿进行产前诊断提供技术保障。方法对于有OI孕史的孕妇,进行系统B超监测;根据胎儿股骨、长骨的超声影像学表现,初诊为成骨不全。抽取羊水,采用直接测序法对羊水DNA的COL1A1和COL1A2基因全编码外显子及启动子区域进行突变位点检测。检出的新突变,对孕妇夫妇及家系其他成员直接测序证实。产前诊断标本均需做母血污染鉴定。结果胎儿超声影像学表现为股骨短小,胫腓骨弯曲成角,颅骨变薄且发现多处骨折,考虑OI。STR法鉴定,羊水无母血污染。DNA序列分析结果显示COL1A1基因鉴定出19个SNP位点,没有鉴定出突变位点;COL1A2基因鉴定出13个SNP位点及第36外显子的第2180位置碱基发生错义突变位点(c.2180G>A,p.Gly727Asp)。孕妇在COL1A2基因的第36外显子亦存在错义突变位点(c.2180G>A,p.Gly727Asp),但其临床特征不一致。其他成员均未检测到Gly727Asp突变。结论有OI孕史的孕妇,采取B超和COL1A1/COL1A2基因诊断技术,可以快速、有效对高危胎儿做出确诊,为预防患病胎儿出生提供技术保障。     

A de novo G+1 → a mutation at the α2(I) exon 16 splice donor site causes skipping of exon 16 in the cDNA of one allele of an OI Type IV proband

We have investigated the procollagen, collagen, α2(I) mRNA, and DNA of a proband with type IV OI. The proband synthesized two α2(I) chains, one with normal electrophoretic migration and one more rapidly migrating. The fast α2(I) chain was relatively retained within the cell and was present in collagens synthesized in the presence of α,α′-dipyridyl. The α2(I) cyanogen bromide peptide CB 4-2 contained both normal and rapidly migrating components. Thermal stability of helices containing the rapidly migrating α2(I) chain was reduced 6°C. Parental fibroblast collagens were normal. RNA/RNA hybrids between proband total RNA and antisense riboprobe complementary to α2(I) nt 236–1390 were digested with RNase A and T1. Digestion products seen exclusively in the proband suggested a structural change in the region coding for exons 16-19. The region which hybridized to the riboprobe was amplified using RNA-PCR and subcloned. Multiple restriction enzyme digestions of the two subcloned alleles suggested a structural change localized to the region coding for exons 16-17. Sequencing revealed a deletion of the 54 bp comprising exon 16 in the cDNA of one allele. The region of the proband's genomic DNA spanning exons 15-17 was amplified by PCR. The subcloned genomic fragments of each allele were distinguished by RNA/DNA hybrid analysis using a riboprobe complementary to normal genomic DNA from this region. Sequencing revealed a G⁺¹ → A mutation at the exon 16 donor site in one allele. The mutation eliminates a Styl site. Digestion of PCR fragments amplified from the proband and parental WBC DNA revealed that only the proband had the undigested mutant fragment. © 1993 Wiley-Liss, Inc.     

Recurrence of lethal osteogenesis imperfecta due to parental mosaicism for a mutation in the COL1A2 gene of type I collagen. The mosaic parent exhibits phenotypic features of a mild form of the disease.

We have determined that a man, ascertained because he fathered a child with lethal osteogenesis imperfecta (OI) with each of two partners, is mosaic in both his germline and somatic tissues for a mutation in the COL1A2 gene which encodes the pro alpha 2(I) chain of type I procollagen. His dermal fibroblasts were previously shown to synthesize a population of cysteine-containing alpha 2(I) chains that were posttranslationally overmodified. DNA sequence analysis of COL1A2 cDNAs demonstrated that the cysteine-containing chain resulted from a point mutation (G to T) in the first position of the codon for the glycine at residue 472 of the triple helical domain. Genomic DNA from the one available affected infant contained the mutant and normal COL1A2 alleles in equal proportion. Examination of DNA from several tissues of the father showed that the mutant allele was present in approximately 40% of his sperm, 80% of his lymphocytes, and nearly 100% of his dermal fibroblasts. Despite the high level of mosaicism detected in somatic tissues, the only phenotypic manifestation of OI in the proband was that he was shorter than his unaffected male relatives and had mild dentinogenesis imperfecta. Thermal stability of type I collagen molecules containing the substitution was decreased, but to a lesser extent than for a nonlethal cysteine for glycine substitution at residue 259 of alpha 2(I), indicating that this measure of molecular stability may be of limited use in explaining the pathogenesis of osteogenesis imperfecta.     

An exon skipping mutation of a type V collagen gene (COL5A1) in Ehlers-Danlos syndrome.

The Ehlers-Danlos syndrome (EDS) is a heterogeneous group of inherited connective tissue disorders characterised by skin hyperextensibility, joint hypermobility, easy bruising, and cutaneous fragility. Nine discrete clinical subtypes have been classified. We have investigated the molecular defect in a patient with clinical features of Ehlers-Danlos syndromes types I/II and VII. Electron microscopy of skin tissue indicated abnormal collagen fibrillogenesis with longitudinal sections showing a marked disruption of fibril packing giving very irregular outlines to transverse sections. Analysis of the collagens produced by cultured fibroblasts showed that the type V collagen had a population of alpha 1 (V) chains shorter than normal. Peptide mapping suggested a deletion within the triple helical domain. RTPCR amplification of mRNA covering the whole of this domain of COL5A1 showed a deletion of 54 bp. Although six Gly-X-Y triplets were lost, the essential triplet amino acid sequence and C-propeptide structure were maintained allowing mutant protein chains to be incorporated into triple helices. Genomic DNA analysis identified a de novo G+3-->T transversion in a 5' splice site of one COL5A1 allele. This mutation is analogous to mutations causing exon skipping in the major collagen genes, COL1A1, COL1A2, and COL3A1, identified in several cases of osteogenesis imperfecta and EDS type IV. These observations support the hypothesis that type V, although quantitatively a minor collagen, has a critical role in the formation of the fibrillar collagen matrix.     

五个成骨不全家系COL1A1基因的变异分析

目的分析5个成骨不全家系的COL1A1基因致病变异位点,为家系遗传咨询及产前诊断提供依据。方法应用高通量测序方法对5个成骨不全家系的先证者进行225个骨病相关基因进行检测分析,所检岀的可疑变异经PCR扩增后进行Sanger测序,对5个家系的先证者及其家庭成员和100名正常个体进行验证,确定致病性变异后,对1个家系中的高危胎儿进行产前诊断。结果5个家系的先证者分别携带COL1A1基因c.3226G>A(p.Glyl076Ser)杂合错义变异、c.579delT(p.Glyl94Valfs*71)移码变异、c・2911_2912insAG(p.Gly971Glufs*138)插入变异、c.3037G>A(p.Glyl013Arg)杂合错义变异,以及c.642+5G>A杂合剪接变异;家系1胎儿产前诊断结果提示胎儿未携带与先证者相同的变异,与超声检查结果一致。5个家系的父母均未携带相应的变异,均为新发变异。100名正常个体均未检出上述变异。其中COL1A1基因c.3037G>A(p.Glyl013Arg),c.29U_2912insAG(p.Gly971Glufs*138)变异为未报道的新变异。结论COL1A1基因变异是5个成骨不全家系的致病病因。本研究的结果丰富了COL1A1基因的变异谱,为家系遗传咨询和产前诊断提供了依据。     

COL1A2基因生殖腺嵌合变异的分析

目的对1例亲代表型正常但反复妊娠骨骼发育异常胎儿(可疑成骨不全)的家系进行遗传学分析,探讨胎儿的发病原因。方法对胎儿标本及亲代DNA进行全外显子组测序(whole exome sequencing,WES),针对WES检测到的阳性位点进行Sanger测序验证。采集提取丈夫精液与单精子DNA,对致病位点进行PCR扩增并Sanger测序。结果WES检测到胎儿COL1A2基因c.1378G>A(p.G460S)杂合变异,为致病变异,夫妻双方外周血DNA均未检测到该变异。精液DNA检测到该位点野生型与变异型序列,在15个精子中有4个检测到该位点变异。结论为1例骨骼发育异常的胎儿明确了成骨不全的遗传学诊断。亲代的生殖腺嵌合是该家系反复妊娠骨骼发育异常胎儿的原因。在临床遗传咨询过程中,对于表型和和基因型均正常但反复生育或妊娠相同异常表型后代的案例,需要考虑到生殖腺嵌合的可能原因。     

parkin基因的一个新的点突变

目的：研究parkin基因外显子2-10点突变与散发性早发帕金森病发病的关系。方法：应用聚合酶链反应（polymerase chain reaction ，PCR）、琼脂糖电泳、单链构象多态性（single strand conformation polymorphism，SSCP）、DNA测序及限制性核酸内切酶酶切方法，检测了60例散发性早发帕金森病患者以及120名正常人外周血白细胞DNA的parkin基因外显子2-10点突变。结果：发现1例患者的parkin基因外显子2存在纯合突变（G237→C），限制性内切酶酶切证实，其它外显子未见突变，120名正常对照也未见突变。结论：parkin基因外显子存在点突变，可能与部分散发性早发帕金森病发病有关。     

Stickler-like syndrome due to a dominant negative mutation in the COL2A1 gene

The type II collagenopathies include a wide spectrum of phenotypes ranging from mild spondylo epiphyseal dysplasia (SED) to severe achondrogenesis/ hypochondrogenesis. Several attempts have been made at providing phenotype-genotype correlations in this group of disorders. In this report we discuss a South African family in which four members have a phenotype resembling Stickler syndrome type 1. Ocular problems and conductive deafness predominate, while skeletal changes resemble those of a mild form of multiple epiphyseal dysplasia (MED). In distinction to the classical form of Stickler syndrome, the affected persons have stubby digits. DNA analysis of the exons of the COL2A1 gene documented a C-T transversion in exon 39, resulting in an Arg704Cys substitution in the triple helical domain of the type II collagen peptide; this nontermination mutation may be indicative of further heterogeneity in the Stickler group of disorders or of a new syndrome amongst the type II collagenopathies. Am. J. Med. Genet. 80:6–11, 1998. © 1998 Wiley-Liss, Inc.     

Functional analysis of a new splice site mutation, c.605-3C>A, in the cystinuria gene SLC7A9 leading to exon skipping

Cystinuria is a hereditary disorder of cystine and dibasic amino acid transport across the luminal membrane of renal tubules and intestine, resulting in recurrent nephrolithiasis. While mutations in the SLC3A1 gene cause type I cystinuria, patients with non-type I cystinuria carry mutations in the SLC7A9 gene. Up to now, more than 80 mutations in SLC3A1 and 50 in SLC7A9 have been reported in the literature. While deletions, duplications, and truncating mutations can often unambiguously classified to be pathogenic, the functional relevance of base pair substitutions is often difficult to predict. To determine the functional relevance of a new splice site mutation in intron 5 of SLC7A9, c.605-3C>A, we transfected COS7 cells with expression constructs containing the wild-type and mutant allele, respectively. cDNAs derived from the resulting SLC7A9 mRNAs were sequenced. By this approach we could demonstrate that the mutant allele c.605-3A causes exon skipping and therefore represents a splice site mutation. To the best of our knowledge, this is the first splice site mutation in a cystinuria gene with a proven functional consequence.     

A misquoted mutation in exon16 of the BRCA2 gene

A pathogenic mutation in the BRCA2 gene, nt7602del16, has been misquoted as a mutation, possibly due to the incorrect inclusion of the last 16 nucleotides of exon15 of the BRCA2 gene as part of the intron15–exon16 BRCA2 gene sequence in publicly available databases. This was concluded following mutational screening by sequencing and enzymatic mapping of the BRCA2 gene exon15–exon16 region in DNA from peripheral blood samples from a total of 74 breast cancer and non-breast cancer patients as well as healthy individuals and the cell line MCF7. Careful interpretation of genetic variants and direct feedback to the corresponding sequence databases prevent systemic errors by integrating updated data into broadly referenced sources.     

Erratum: A novel splice site mutation (3157+1G>T) in the dystrophin gene causing total exon skipping and DMD phenotype

Erratum: An error was printed in the original version of this article in the Comments section, paragraph 2, relating to the size of exon 22 and the RT‐PCR product size described as resulting from the mutation 3157+1G>T. The paragraph should read: “We report a case of a 5 year old DMD patient with a novel splice site mutation affecting the GT dinucleotide splice donor of exon 22. The RT‐PCR analysis with primer sets spanning dystrophin exons 17‐25 amplified no normal size fragment (1251 bp), but a product shorter by 146 bp (the length of exon 22). Direct sequencing of the faster migrating fragment revealed total skipping of exon 22.”