Yasutsugu Chinen

Mucopolysaccharidosis Type IVA (Morquio A Disease): Clinical Review and Current Treatment: A Special Review

Mucopolysaccharidosis IVA (MPS IVA), also known as Morquio A, is a rare, autosomal recessive disorder caused by a deficiency of the lysosomal enzyme N-acetylgalatosamine-6-sulfate-sulfatase (GALNS), which catalyzes a step in the catabolism of glycosaminoglycans (GAGs), keratan sulfate (KS) and chondroitin-6-sulfate (C6S). It leads to accumulation of the KS and C6S, mainly in bone and cornea, causing a systemic skeletal chondrodysplasia. MPS IVA has a variable age of onset and variable rate of progression. Common presenting features include elevation of urinary and blood KS, marked short stature, hypoplasia of the odontoid process, pectus carinatum, kyphoscoliosis, genu valgum, laxity of joints and corneal clouding; however there is no central nervous system impairment. Generally, MPS IVA patients with a severe form do not survive beyond the third decade of life whereas those patients with an attenuated form may survive over 70 years. There has been no effective therapy for MPS IVA, and care has been palliative. Enzyme replacement therapy (ERT) and hematopoietic stem cell therapy (HSCT) have emerged as a treatment for mucopolysaccharidoses disorders, including Morquio A disease. This review provides an overview of the clinical manifestations, diagnosis and symptomatic management of patients with MPS IVA and describes potential perspectives of ERT and HSCT. The issue of treating very young patients is also discussed.

Mutations in CD96, a Member of the Immunoglobulin Superfamily, Cause a Form of the C (Opitz Trigonocephaly) Syndrome

The C syndrome is characterized by trigonocephaly and associated anomalies, such as unusual facies, psychomotor retardation, redundant skin, joint and limb abnormalities, and visceral anomalies. In an individual with the C syndrome who harbors a balanced chromosomal translocation, t(3;18)(q13.13;q12.1), we discovered that the TACTILE gene for CD96, a member of the immunoglobulin superfamily, was disrupted at the 3q13.3 breakpoint. In mutation analysis of nine karyotypically normal patients given diagnoses of the C or C-like syndrome, we identified a missense mutation (839C-->T, T280M) in exon 6 of the CD96 gene in one patient with the C-like syndrome. The missense mutation was not found among 420 unaffected Japanese individuals. Cells with mutated CD96 protein (T280M) lost adhesion and growth activities in vitro. These findings indicate that CD96 mutations may cause a form of the C syndrome by interfering with cell adhesion and growth.

Four novel NIPBL mutations in Japanese patients with Cornelia de Lange syndrome

Cornelia de Lange syndrome (CdLS, OMIM #122470) is a multiple congenital anomaly syndrome characterized by dysmorphic facial features, hirsutism, severe growth and developmental delay, and malformed upper limbs [Ireland et al., 1993; Jackson et al., 1993]. The prevalence is estimated to be 1/10,000 [Opitz, 1985]. Recently, two independent groups proved that CdLS is caused by NIPBL mutations [Krantz et al., 2004; Tonkin et al., 2004]. NIPBL consists of 47 exons and encodes delangin, a 2,804 amino-acid protein, from exon 2 to 47. We analyzed 15 Japanese sporadic patients (CdL 1–15) with typical CdLS features (Table I) and their parents after obtaining written informed consent. All protocols in this study were approved by the Committee for the Ethical Issues on Human Genome and Gene analysis, Nagasaki University. Clinical geneticists diagnosed these patients based on mental and growth retardation, and characteristic facial features. Genomic DNA was extracted using a standard protocol. Fourty-six coding exons (from exon 2 to 47) of NIPBL were amplified by PCR as described previously [Krantz et al., 2004] except for exons 4, 33, 37, and 41, of which primers were originally designed (available on request). Sequence analysis was performed as described previously [Kurotaki et al., 2003]. We identified three novel nonsense mutations and one missense mutation in NIPBL among the 15 Japanese patients examined: 1885C>T (R629X) (CdL 4) and 1921G>T (E641X) (CdL 2) in exon 10, 3346G>T (E1116X) (CdL 15) in exon 12, and 5483G>A (R1828Q) (CdL 10) in exon 29. All the four mutations were not found in any of 97 normal Japanese controls or in the JSNP database (http://snp.ims.u-tokyo.ac.jp/). The altered amino acid (R1828Q) was de novo and located in the evolutionally conserved sequences at least in the human, rat, mouse, and fly homologs, thus the change is likely to be pathological. The C-terminal half 1500 amino acids of delangin is well conserved among homologs of flies, worms, plants, and fungi, and is expected to be biologically important [Tonkin et al., 2004], though it was not found to contain any obvious functional domains by analysis using PROSITE (http://kr. expasy.org/cgi-bin/prosite/PSScan.cgi). Three protein truncation mutations at amino acid positions 629, 641, and 1116 and a missense mutation at amino acid position 1828 could lose or impair the C-terminal half function. The Drosophila homolog of NIPBL, Nipped-B, is involved in activating the Ubx and Cut homeobox genes. Ubx suppresses the limb formation by repressing Dll that requires for the distal limb development, and Cut mutations cause leg and wing abnormalities [Tonkin et al., 2004]. Thus, it is plausible that reduced expression of human NIPBL may lead to limb anomalies in CdLS. Interestingly, limb abnormalities (oligodactyly and ulner deficiency) were observed in three of our four patients with a mutation, but only one of seven patients without any mutation whose clinical information was available did show some limb abnormality (oligodactyly), though Gillis et al. [2004] reported that severity of limb defects was not statistically different between mutation-positive and mutation-negative patients. Additionally, three single nucleotide polymorphisms (SNPs), 1151A>G (N384S) in exon 9, 2021A>G (N674S) in exon 10 and 5874T>C (S1958S) in exon 33, were identified, as they were found among normal controls and the second substitution (2021A>G) was previously reported as a SNP [Gillis et al., 2004]. Allele frequencies of the three SNPs in normal Japanese controls are 3.2% (6/186), 13.0% (25/192), and 64.5% (129/200), respectively. To exclude a submicroscopic deletion around NIPBL and its franking regions, fluorescence in situ hybridization (FISH) analysis was performed in 10 of 15 cases on their metaphase chromosomes using two BAC clones covering the NIPBL gene (Table I), RP11-14I21, and RP11-7M4, selected from the UCSC genome browser, 2003 July version (http://genome.ucsc.edu/ cgi-bin/hgGateway). FISH and subsequent photomicroscopy were performed as described previously [Miyake et al., 2004]. However, none of them showed any deletion. We also investigated core promoter regions in 11 affected individuals not having detectable point mutations in the coding regions. Two core promoter regions were identified, ranging 800 to 500 bp (CPR-A) and 400 to þ 200 bp (CPRB) from the beginning of NIPBL cDNA (NM_015384.3) using four different promoter prediction programs: neural network promoter prediction program (http://www.fruitfly.org/seq_ tools/promoter.html), human core-promoter finder (http:// rulai.cshl.org/tools/genefinder/CPROMOTER/human.htm), promoter 2.0 prediction server (http://www.cbs.dtu.dk/services/ promoter/), bioinformatics & molecular analysis section (http:// bimas.dcrt.nih.gov/molbio/proscan/). No nucleotide changes were detected among the 11 patients in the two core promoter regions except for a part of CPR-B sequence ( 60 þ 60), which was hardly determined due to high GC ratio (75.83%), suggesting that promoter mutations in NIBPL is less likely. In conclusion, we identified four novelNIPBLmutations and three SNPs. It is important to describe a full spectrum of phenotype in more patients with positive mutations and establish comprehensive diagnostic criteria. *Correspondence to: Dr. Naomichi Matsumoto, Department of Human Genetics, Yokohama City University Graduate School of Medicine, Fukuura 3-9, Kanazawa-ku, Yokohama 236-0004, Japan. E-mail: naomat@yokohama-cu.ac.jp

Impact of enzyme replacement therapy and hematopoietic stem cell transplantation in patients with Morquio A syndrome

Patients with mucopolysaccharidosis IVA (MPS IVA) can present with systemic skeletal dysplasia, leading to a need for multiple orthopedic surgical procedures, and often become wheelchair bound in their teenage years. Studies on patients with MPS IVA treated by enzyme replacement therapy (ERT) showed a sharp reduction on urinary keratan sulfate, but only modest improvement based on a 6-minute walk test and no significant improvement on a 3-minute climb-up test and lung function test compared with the placebo group, at least in the short-term. Surgical remnants from ERT-treated patients did not show reduction of storage materials in chondrocytes. The impact of ERT on bone lesions in patients with MPS IVA remains limited. ERT seems to be enhanced in a mouse model of MPS IVA by a novel form of the enzyme tagged with a bone-targeting moiety. The tagged enzyme remained in the circulation much longer than untagged native enzyme and was delivered to and retained in bone. Three-month-old MPS IVA mice treated with 23 weekly infusions of tagged enzyme showed marked clearance of the storage materials in bone, bone marrow, and heart valves. When treatment was initiated at birth, reduction of storage materials in tissues was even greater. These findings indicate that specific targeting of the enzyme to bone at an early stage may improve efficacy of ERT for MPS IVA. Recombinant N-acetylgalactosamine-6-sulfate sulfatase (GALNS) in Escherichia coli BL21 (DE3) (erGALNS) and in the methylotrophic yeast Pichia pastoris (prGALNS) has been produced as an alternative to the conventional production in Chinese hamster ovary cells. Recombinant GALNS produced in microorganisms may help to reduce the high cost of ERT and the introduction of modifications to enhance targeting. Although only a limited number of patients with MPS IVA have been treated with hematopoietic stem cell transplantation (HSCT), beneficial effects have been reported. A wheelchair-bound patient with a severe form of MPS IVA was treated with HSCT at 15 years of age and followed up for 10 years. Radiographs showed that the figures of major and minor trochanter appeared. Loud snoring and apnea disappeared. In all, 1 year after bone marrow transplantation, bone mineral density at L2–L4 was increased from 0.372 g/cm2 to 0.548 g/cm2 and was maintained at a level of 0.48±0.054 for the following 9 years. Pulmonary vital capacity increased approximately 20% from a baseline of 1.08 L to around 1.31 L over the first 2 years and was maintained thereafter. Activity of daily living was improved similar to the normal control group. After bilateral osteotomies, a patient can walk over 400 m using hip–knee–ankle–foot orthoses. This long-term observation of a patient shows that this treatment can produce clinical improvements although bone deformity remained unchanged. In conclusion, ERT is a therapeutic option for MPS IVA patients, and there are some indications that HSCT may be an alternative to treat this disease. However, as neither seems to be a curative therapy, at least for the skeletal dysplasia in MPS IVA patients, new approaches are investigated to enhance efficacy and reduce costs to benefit MPS IVA patients.

Current and emerging treatments and surgical interventions for Morquio A syndrome: a review.

Patients with mucopolysaccharidosis type IVA (MPS IVA; Morquio A syndrome) have accumulation of the glycosaminoglycans, keratan sulfate, and chondroitin-6-sulfate, in bone and cartilage, causing systemic spondyloepiphyseal dysplasia. Features include lumbar gibbus, pectus carinatum, faring of the rib cage, marked short stature, cervical instability and stenosis, kyphoscoliosis, genu valgum, and laxity of joints. Generally, MPS IVA patients are wheelchair-bound as teenagers and do not survive beyond the second or third decade of life as a result of severe bone dysplasia, causing restrictive lung disease and airway narrowing, increasing potential for pneumonia and apnea; stenosis and instability of the upper cervical region; high risk during anesthesia administration due to narrowed airway as well as thoracoabdominal dysfunction; and surgical complications. Patients often need multiple surgical procedures, including cervical decompression and fusion, hip reconstruction and replacement, and femoral or tibial osteotomy, throughout their lifetime. Current measures to intervene in disease progression are largely palliative, and improved therapies are urgently needed. A clinical trial for enzyme replacement therapy (ERT) and an investigational trial for hematopoietic stem cell transplantation (HSCT) are underway. Whether sufficient enzyme will be delivered effectively to bone, especially cartilage (avascular region) to prevent the devastating skeletal dysplasias remains unclear. This review provides an overview of historical aspects of studies on MPS IVA, including clinical manifestations and pathogenesis of MPS IVA, orthopedic surgical interventions, and anesthetic care. It also describes perspectives on potential ERT, HSCT, and gene therapy.

Long-term therapeutic efficacy of allogenic bone marrow transplantation in a patient with mucopolysaccharidosis IVA.

Mucopolysaccharidosis IVA (MPS IVA) is one of the lysosomal storage diseases. It is caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase. Deficiency of this enzyme leads to accumulation of the specific glycosaminoglycans keratan sulfate and chondroitin-6-sulfate. This accumulation has a direct impact on cartilage and bone development, resulting in systemic skeletal dysplasia. There is no curative therapy for this skeletal dysplasia. This report describes long-term therapeutic efficacy in a 15-year-old boy with a severe form of MPS IVA who received successful allogeneic bone marrow transplantation (BMT) from his HLA-identical carrier sister. The level of the GALNS enzyme in the recipients lymphocytes reached almost half of normal level within two years after BMT. For the successive 9 + years post-BMT, GALNS activity in his lymphocytes maintained the same level as the donors, and the level of urinary uronic acid was reduced. Lumbar bone mineral density increased around 50% one year later post-BMT and was kept consistent. Radiographs showed that the figures of trochanter major and minor appeared, while the epiphyseal dysplasia in the femoral cap was almost unchanged. Loud snoring and apnea disappeared. Vital capacity increased to around 20% for the first two years and was maintained. Activity of daily life (ADL) was improved in work/study efficacy, respiratory status, sleep, joint pain, and frequency of infection. In conclusion, the long-term study of hematopoetic stem cell transplantation has shown clinical improvements in respiratory function, radiograph findings, ADL, and biochemical findings, suggesting that it is a potential therapeutic option for patients with MPS IVA.

Novel intragenic duplications and mutations of CASK in patients with mental retardation and microcephaly with pontine and cerebellar hypoplasia (MICPCH)

The CASK gene encoding a member of the membrane-associated guanylate kinase protein family is highly expressed in the mammalian nervous system of both adults and fetuses, playing several roles in neural development and synaptic function. Recently, CASK aberrations caused by both mutations and deletions have been reported to cause severe mental retardation (MR), microcephaly and disproportionate pontine and cerebellar hypoplasia (MICPCH) in females. Here, mutations and copy numbers of CASK were examined in ten females with MR and MICPCH, and the following changes were detected: nonsense mutations in three cases, a 2-bp deletion in one case, mutations at exon–intron junctions in two cases, heterozygous deletions encompassing CASK in two cases and interstitial duplications in two cases. Except for the heterozygous deletions, each change including the intragenic duplications potentially caused an aberrant transcript, resulting in CASK null mutations. The results provide novel mutations and copy number aberrations of CASK, causing MR with MICPCH, and also demonstrate the similarity of the phenotypes of MR with MICPCH regardless of the CASK mutation.

Clinical application of array-based comparative genomic hybridization by two-stage screening for 536 patients with mental retardation and multiple congenital anomalies

Recent advances in the analysis of patients with congenital abnormalities using array-based comparative genome hybridization (aCGH) have uncovered two types of genomic copy-number variants (CNVs); pathogenic CNVs (pCNVs) relevant to congenital disorders and benign CNVs observed also in healthy populations, complicating the screening of disease-associated alterations by aCGH. To apply the aCGH technique to the diagnosis as well as investigation of multiple congenital anomalies and mental retardation (MCA/MR), we constructed a consortium with 23 medical institutes and hospitals in Japan, and recruited 536 patients with clinically uncharacterized MCA/MR, whose karyotypes were normal according to conventional cytogenetics, for two-stage screening using two types of bacterial artificial chromosome-based microarray. The first screening using a targeted array detected pCNV in 54 of 536 cases (10.1%), whereas the second screening of the 349 cases negative in the first screening using a genome-wide high-density array at intervals of approximately 0.7 Mb detected pCNVs in 48 cases (13.8%), including pCNVs relevant to recently established microdeletion or microduplication syndromes, CNVs containing pathogenic genes and recurrent CNVs containing the same region among different patients. The results show the efficient application of aCGH in the clinical setting.

Opitz trigonocephaly C syndrome in a boy with a de novo balanced reciprocal translocation t(3;18)(q13.13;q12.1)

Opitz trigonocephaly C syndrome (OTCS) is a multiple congenital anomaly syndrome characterized by trigonocephaly, mental retardation, a typical facial appearance, redundant skin, joint and limb abnormalities, and visceral anomalies. We describe a patient with the manifestations of OTCS who also had a de novo balanced reciprocal translocation t(3;18)(q13.13q12.1). His phenotype is a mild form with mild developmental delay and no severe visceral anomalies. Our findings suggest the possible existence of a new locus responsible for OTCS either on 3q13.13 or 18q12.1.

Germline Mutation of CBL Is Associated With Moyamoya Disease in a Child With Juvenile Myelomonocytic Leukemia and Noonan Syndrome‐Like Disorder

Germline mutations in CBL have been identified in patients with Noonan syndrome‐like phenotypes, while juvenile myelomonocytic leukemia (JMML) harbors duplication of a germline CBL, resulting in acquired isodisomy. The association between moyamoya disease and Noonan syndrome carrying a PTPN11 mutation has recently been reported. We present a patient with JMML who developed moyamoya disease and neovascular glaucoma. Our patient exhibited a Noonan syndrome‐like phenotype. Genetic analysis revealed acquired isodisomy and a germline heterozygous mutation in CBL. This is a rare case of CBL mutation associated with moyamoya disease. Prolonged RAS pathway signaling may cause disruption of cerebrovascular development. Pediatr Blood Cancer 2015;62:542–544.