Eiji Nakashima

Novel SBDS mutations caused by gene conversion in Japanese patients with Shwachman-Diamond syndrome.

Shwachman-Diamond syndrome (SDS; OMIM 260400) is an autosomal recessive disorder characterized by exocrine pancreatic insufficiency, bone marrow dysfunction and metaphyseal chondrodysplasia. SDS is caused by mutations in SBDS, an uncharacterized gene. A previous study in SDS patients largely of European ancestry found that most SBDS mutations occurred within a ~240-bp region of exon 2 and resulted from gene conversion due to recombination with a pseudogene, SBDSP. It is unknown, however, whether these findings are applicable to other ethnic groups. To address this question, we examined SBDS mutations in six Japanese families with SDS by direct sequencing. We identified compound heterozygous mutations in four families: two were recurrent (96–97insA, 258+2T>C), and three were novel [292–295delAAAG, (183–184TA>CT +201A>G), (141C>T+183–184TA>CT+201A>G)] mutations. Most of these mutations also appear to result from gene conversion, but the conversion events occurred at various sites between intron 1 and exon 3. Thus, gene conversion mutations in SBDS are common to different ethnic groups, but they are not confined to a limited region of the gene.

Identification of COL2A1 mutations in platyspondylic skeletal dysplasia, Torrance type

Platyspondylic lethal skeletal dysplasias (PLSDs) are a heterogeneous group of chondrodysplasias characterised by severe platyspondyly and limb shortening. The most common form of PLSD is thanatophoric dysplasia (TD), which has been divided into two types, TD1 (MIM 187600) and TD2 (MIM 187610). Three other types of PLSD, or TD variants have been distinguished from TD, the San Diego (PLSD-SD; MIM 270230), Torrance (PLSD-T; MIM 151210), and Luton (PLSD-L; MIM 151210) types.1,2 PLSD-L is now considered to be a mild phenotypic variant of PLSD-T. Mutations in the fibroblast growth factor receptor 3 ( FGFR3 ) gene were identified in TD and PLSD-SD,3 but not in PLSD-T and PLSD-L.3,4 PLSD-T is a common subtype of PLSD.2 The radiological characteristics include wafer-like vertebral bodies, severe hypoplasia of the lower ilia, extremely short long bones with ragged metaphyses, and bowing of the radius. Its chondro-osseous histology is characterised by hypercellularity with slightly large chondrocytes in the resting cartilage and normal columnisation with incorporation of cartilage into bone at the chondro-osseous junction.1,2 These radiological and histological findings of the disorder can be used to discriminate it from other lethal or semilethal skeletal dysplasias including TD. Perinatal death is generally considered to be inevitable in PLSD-T. Recently, however, non-lethal phenotypes of the disorder with better ossified vertebral bodies have been proposed, based on the observations of two affected families. One family included an affected mother who survived to adulthood and her affected daughter who died soon after birth,5,6 and the other family included an affected mother and her son, both of whom are living.4 These observations raise the question of whether PLSD-T represents a single entity with a wide clinical spectrum or a heterogeneous group of disorders with superficial radiological similarities. Here we describe two examples of …

RMRP mutations in Japanese patients with cartilage‐hair hypoplasia

We examined 12 Japanese patients with metaphyseal chondrodysplasia (MCD) for mutations in the ribonuclease mitochondrial RNA processing gene (RMRP), and identified four novel mutations in two patients with typical and atypical cartilage‐hair hypoplasia (CHH), a form of MCD characterized by extra‐skeletal manifestations including hypoplastic hair and defective immunity. A patient with typical CHH had a 17‐bp duplication at +3 and a de novo 182G > A. The other patient with atypical CHH had a 17‐bp insertion at −20 and a 218A > G. Expression analysis revealed that the allele with this insertion mutation in the promoter region silenced the gene. Spectrum analysis of the mutations and polymorphisms in RMRP showed marked difference between the Japanese and other ethnic groups. Such ethnic and phenotypic difference should be taken into account in mutation analysis of the gene.

A type II collagen mutation also results in oto-spondylo-megaepiphyseal dysplasia

Oto-spondylo-megaepiphyseal dysplasia (OSMED) is a skeletal dysplasia characterized by severe sensorineural hearing loss, enlarged epiphyses and early onset of osteoarthritis. COL11A2 has been reported as a causative gene for OSMED. We have identified a novel COL2A1 mutation at a splice-acceptor site within intron 10 (c.709–2A>G) in an OSMED patient. This mutation caused the skipping of exon 11, and of exons 11 and 13. These exon-skipping events are presumed to cause an in-frame deletion of the triple helical region of the COL2A1 product. Thus, our findings highlight the genetic heterogeneity of OSMED and extend the phenotypic spectrum of type II collagenopathy, as well as confirming the overlap between type II and type XI collagenopathies.

Double-layered patella in multiple epiphyseal dysplasia is not exclusive to DTDST mutation

Multiple epiphyseal dysplasia (MED) is a common skeletal dysplasia characterized by mild to moderate short stature, early-onset osteoarthritis mainly in the hip and knee joints, and abnormally small or irregular epiphyses of long and short tubular bones. MED is clinically and genetically heterogenous. Six causative genes for MED have been reported—COMP, MATN3, type IX collagen genes (COL9A1,COL9A2,COL9A3), and diastrophic dysplasia sulfate transporter (DTDST) [Briggs and Chapman, 2002]. Mutations in the first five genes cause autosomal dominant MED, whereas those in DTDST result in autosomal recessive MED. Recessive MED is characterized clinically by mild finger deformities including brachydactyly, bilateral clubfoot, and ‘‘double-layered patella.’’ Double-layered patella is a form of partite patella consisting of anterior and posterior components divided by coronal septum [Sheffield, 1998]. It occurs in patients with MED, and so is a reliable sign in the diagnosis of MED [Hodkinson, 1962]. Superti-Furga et al. [1999] firstly described a patient with recessive MED characterized by clubfeet, double-layered patellae, and normal stature, who had homozygous R279W mutations of DTDST. Ballhausen et al. [2003] found a double-layered patella in 7 of the 10 patients with recessive MED and homozygous R279W mutations of DTDST. Their age ranged from 8 to 28 years, an indication that it persists into early adulthood. These authors also mentioned that all patients with double-layered patella referred to them had DTDST mutations (including R279W homozygosity or other genotypes). Double-layered patella is not likely to be a mere abnormal ossification pattern, but is a true anatomic variant, with two ossification centers instead of one. Makitie et al. [2003] reported three patients from two families with recessive MED and homozygous DTDST mutation (C653S). All three patients had a double-layered patella. These studies suggested that double-layered patella is exclusive to recessive MED with DTDST mutation. Here we report a Japanese family with MED carrying a COL9A2 mutation, in which the male proband had doublelayered patellae. The proband was born to non-consanguineous parents. At age 10 years, he started to have occasional pain in the knees and ankles. When he was referred to us at age 12 years, his height was 146 cm ( 1.5 SD); the feet, external ears, and palate were normal. Mild joint laxity was found in the knees. Radiographic examination showed epiphyseal dysplasia in the elbows, hands, knees, and the ankles (Fig. 1a). The spine and hips were normal. The knees showed bilateral double-layered patellae (Fig. 1b). Other members of the family had no joint symptoms, except for a 52-year-old aunt who had episodic knee effusion. Heights of the family members were all above the 2 SD level. We screened COMP and MATN3 and mutation hotspots of COL9A1, COL9A2, and COL9A3 in the proband. Direct sequencing analysis revealed an Ex3-1G>A hemizygous mutation in exon 3 of COL9A2 (Fig. 1c). This mutation has been proved to result in skipping of exon 3 [Holden et al., 1999]. The mutation was examined in the family members by PCRRLFP using a primer set, COL9A2i2i4F (50-taggggacctggacagaaga-30) and COL9A2i2i4R (50-caatgaccctgggtgagagt-30) and BpmI (New England BioLabs, Massachusetts, USA), and was found to segregate with the MED phenotype (data not shown). DTDST mutation was not analyzed. The present finding of double-layered patella in a 12-yearold boy with dominant MED and heterozygous COL9A2 mutation indicates that the sign is not exclusive to recessive MED with homozygous DTDST mutations.

Identification of novel RMRP mutations and specific founder haplotypes in Japanese patients with cartilage-hair hypoplasia

AbstractCartilage-hair hypoplasia (CHH), or metaphyseal dysplasia, McKusick type, is an autosomal recessive disease with diverse clinical manifestations. CHH is caused by mutations in RMRP (ribonuclease mitochondrial RNA processing), the gene encoding the RNA component of the ribonucleoprotein complex RNase MRP. A common founder mutation, 70A>G has been reported in the Finnish and Amish populations. We screened 11 Japanese patients with CHH for RMRP mutations and identified mutations in five probands, including three novel mutations (16-bp dup at +1, 168G>A, and 217C>T). All patients were compound heterozygotes for an insertion or duplication in the promoter or 5′-transcribed regions and a point mutation in the transcribed region. Two recurrent mutations were unique to the Japanese population: a 17-bp duplication at +3 and 218A>G. Haplotype analysis revealed that the two mutations common in Japanese individuals were contained within distinct haplotypes. Through this analysis, we have identified a unique mutation spectrum and founder mutations in the Japanese population.

The Shwachman–Bodian–Diamond syndrome gene mutations cause a neonatal form of spondylometaphysial dysplasia (SMD) resembling SMD Sedaghatian type

The Shwachman–Bodian–Diamond syndrome (SBDS) gene is a causative gene for Shwachman–Diamond syndrome, an autosomal recessive disorder with exocrine pancreatic insufficiency, bone marrow dysfunction and skeletal dysplasia. We report here on two patients with skeletal manifestations at the severest end of the phenotypic spectrum of SBDS mutations. An 11-year-old Japanese girl presented with neonatal respiratory failure necessitating lifelong ventilation support, severe short stature and severe developmental delay. She developed neutropenia in infancy, and decreased serum amylase was noted in childhood. A British boy was a stillbirth with pulmonary hypoplasia and hepatic fibrosis found on autopsy. Both cases had neonatal skeletal manifestations that included platyspondyly, lacy iliac crests and severe metaphysial dysplasia, and thus did not fall in the range of the known Shwachman–Diamond syndrome skeletal phenotype but resembled spondylometaphysial dysplasia (SMD) Sedaghatian type. The girl harboured a recurrent mutation (183TA→CT) and a novel missense mutation (79T→C), whereas the boy carried two recurrent mutations (183TA→CT and 258+2T→C). We also examined SBDS in one typical case with SMD Sedaghantian type and eight additional cases with neonatal SMD, but failed to discover SBDS mutations. Our experience expands the phenotypic spectrum of SBDS mutations, which, at its severest end, results in severe neonatal SMD.

Comprehensive screening of multiple epiphyseal dysplasia mutations in Japanese population

Multiple epiphyseal dysplasia (MED) is among the most genetically heterogeneous skeletal dysplasias. Six genes involved in MED, COMP, MATN3, COL9A1, COL9A2, COL9A3, and DTDST have been identified; however, the presence of additional disease genes has been reported, and the detection rate for mutations in known genes accounts for no more than 50% of patients with MED in Western populations. Here, we screened the six known disease genes in 35 consecutive Japanese MED patients. We analyzed the entire coding region of each gene, along with flanking intron–exon junctions, by direct sequencing. A total of 19 mutations were identified in COMP, MATN3, COL9A2, COL9A3, and DTDST. The detection rate for known mutations was higher in this study than in previous reports, and we identified a substantially different spectrum of mutations. Mutations in MATN3 were more prevalent among these Japanese patients, whereas no DTDST mutations were detected. Most of the mutations were localized within specific regions of each gene: COMP mutations were found in the calmodulin‐like repeat domains; MATN3 mutations in the von Willebrand factor type A domain; and type IX collagen gene mutations occurred in the third collagenous domains. Based on the integration of clinical and genetic information, we propose an algorithm for detecting mutations in Japanese MED patients. Our study further supports the existence of additional MED gene(s).

Novel COL9A3 mutation in a family with multiple epiphyseal dysplasia.

Multiple epiphyseal dysplasia (MED) is a common skeletal dysplasia characterized by mild to moderate short stature, early‐onset of osteoarthritis (OA) mainly in the hip and knee joints, and abnormally small and/or irregular epiphyses. MED is clinically and genetically heterogeneous. Six causative genes of MED have been reported, including type IX collagen genes (COL9A1, COL9A2, COL9A3). All the type IX collagen mutations previously reported cause exon skipping that loses the COL3 domain. Here we have identified a novel COL9A3 mutation co‐segregating in a three‐generation family with MED. The mutation (IVS3 + 5G > A) was speculated to lose the COL3 domain by skipping of exon 3, which was confirmed by in vitro analysis. The patients were of normal height and had minimal complaints with phenotypes being more severe in male patients. The radiographic phenotypes of the patients were relatively milder than those of previously reported cases, and were indistinguishable to common, idiopathic OA.

A compound heterozygote harboring novel and recurrent DTDST mutations with intermediate phenotype between atelosteogenesis type II and diastrophic dysplasia.

Diastrophic dysplasia sulfate transporter (DTDST) is a sulfate transporter required for the synthesis of sulfated proteoglycans in the cartilage. Over 30 mutations have been described in the DTDST gene, which result in a continuous clinical spectrum of recessively inherited chondrodysplasias, including, in order of increasing severity, a recessive form of multiple epiphyseal dysplasia (rMED), diastrophic dysplasia (DTD), atelosteogenesis type II (AO‐II) and achondrogenesis 1B (ACG‐1B). Correlation between disease severity and residual sulfate transport activity has been reported. Here we report a patient with DTDST mutations, whose manifestations fell in a range between AO‐II and DTD. The patient was a compound heterozygote for the recurrent c.835C>T (p.R279W) and novel c.1987G>A (p.G663R) mutations. Immunocytochemical analysis in HEK293 cells showed that the p.G663R mutation was localized within the cytoplasm, and not to the cell membrane, suggesting p.G663R is a loss‐of‐function mutation. Our case supports the previously described correlation between the severity of the phenotype and the putative level of residual transport function.