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1.  Shwachman-Bodian Diamond syndrome is a multi-functional protein implicated in cellular stress responses 
Human Molecular Genetics  2009;18(19):3684-3695.
Shwachman-Diamond syndrome (SDS; OMIM 260400) results from loss-of-function mutations in the Shwachman-Bodian Diamond syndrome (SBDS) gene. It is a multi-system disorder with clinical features of exocrine pancreatic dysfunction, skeletal abnormalities, bone marrow failure and predisposition to leukemic transformation. Although the cellular functions of SBDS are still unclear, its yeast ortholog has been implicated in ribosome biogenesis. Using affinity capture and mass spectrometry, we have developed an SBDS-interactome and report SBDS binding partners with diverse molecular functions, notably components of the large ribosomal subunit and proteins involved in DNA metabolism. Reciprocal co-immunoprecipitation confirmed the interaction of SBDS with the large ribosomal subunit protein RPL4 and with DNA-PK and RPA70, two proteins with critical roles in DNA repair. Function for SBDS in response to cellular stresses was implicated by demonstrating that SBDS-depleted HEK293 cells are hypersensitive to multiple types of DNA damage as well as chemically induced endoplasmic reticulum stress. Furthermore, using multiple routes to impair translation and mimic the effect of SBDS-depletion, we show that SBDS-dependent hypersensitivity of HEK293 cells to UV irradiation can be distinguished from a role of SBDS in translation. These results indicate functions of SBDS beyond ribosome biogenesis and may provide insight into the poorly understood cancer predisposition of SDS patients.
PMCID: PMC2742402  PMID: 19602484
2.  Conformational flexibility and molecular interactions of an archaeal homologue of the Shwachman-Bodian-Diamond syndrome protein 
Defects in the human Shwachman-Bodian-Diamond syndrome (SBDS) protein-coding gene lead to the autosomal recessive disorder characterised by bone marrow dysfunction, exocrine pancreatic insufficiency and skeletal abnormalities. This protein is highly conserved in eukaryotes and archaea but is not found in bacteria. Although genomic and biophysical studies have suggested involvement of this protein in RNA metabolism and in ribosome biogenesis, its interacting partners remain largely unknown.
We determined the crystal structure of the SBDS orthologue from Methanothermobacter thermautotrophicus (mthSBDS). This structure shows that SBDS proteins are highly flexible, with the N-terminal FYSH domain and the C-terminal ferredoxin-like domain capable of undergoing substantial rotational adjustments with respect to the central domain. Affinity chromatography identified several proteins from the large ribosomal subunit as possible interacting partners of mthSBDS. Moreover, SELEX (Systematic Evolution of Ligands by EXponential enrichment) experiments, combined with electrophoretic mobility shift assays (EMSA) suggest that mthSBDS does not interact with RNA molecules in a sequence specific manner.
It is suggested that functional interactions of SBDS proteins with their partners could be facilitated by rotational adjustments of the N-terminal and the C-terminal domains with respect to the central domain. Examination of the SBDS protein structure and domain movements together with its possible interaction with large ribosomal subunit proteins suggest that these proteins could participate in ribosome function.
PMCID: PMC2695463  PMID: 19454024
3.  SBDS Expression and Localization at the Mitotic Spindle in Human Myeloid Progenitors 
PLoS ONE  2009;4(9):e7084.
Shwachman-Diamond Syndrome (SDS) is a hereditary disease caused by mutations in the SBDS gene. SDS is clinically characterized by pancreatic insufficiency, skeletal abnormalities and bone marrow dysfunction. The hematologic abnormalities include neutropenia, neutrophil chemotaxis defects, and an increased risk of developing Acute Myeloid Leukemia (AML). Although several studies have suggested that SBDS as a protein plays a role in ribosome processing/maturation, its impact on human neutrophil development and function remains to be clarified.
Methodology/Principal Findings
We observed that SBDS RNA and protein are expressed in the human myeloid leukemia PLB-985 cell line and in human hematopoietic progenitor cells by quantitative RT-PCR and Western blot analysis. SBDS expression is downregulated during neutrophil differentiation. Additionally, we observed that the differentiation and proliferation capacity of SDS-patient bone marrow hematopoietic progenitor cells in a liquid differentiation system was reduced as compared to control cultures. Immunofluorescence analysis showed that SBDS co-localizes with the mitotic spindle and in vitro binding studies reveal a direct interaction of SBDS with microtubules. In interphase cells a perinuclear enrichment of SBDS protein which co-localized with the microtubule organizing center (MTOC) was observed. Also, we observed that transiently expressed SDS patient-derived SBDS-K62 or SBDS-C84 mutant proteins could co-localize with the MTOC and mitotic spindle.
SBDS co-localizes with the mitotic spindle, suggesting a role for SBDS in the cell division process, which corresponds to the decreased proliferation capacity of SDS-patient bone marrow CD34+ hematopoietic progenitor cells in our culture system and also to the neutropenia in SDS patients. A role in chromosome missegregation has not been clarified, since similar spatial and time-dependent localization is observed when patient-derived SBDS mutant proteins are studied. Thus, the increased risk of myeloid malignancy in SDS remains unexplained.
PMCID: PMC2738965  PMID: 19759903
4.  Structural variation and missense mutation in SBDS associated with Shwachman-Diamond syndrome 
BMC Medical Genetics  2014;15:64.
Shwachman–Diamond syndrome (SDS) is an autosomal recessive ribosomopathy caused mainly by compound heterozygous mutations in SBDS. Structural variation (SV) involving the SBDS locus has been rarely reported in association with the disease. We aimed to determine whether an SV contributed to the pathogenesis of a case lacking biallelic SBDS point mutations.
Case presentation
Whole exome sequencing was performed in a patient with SDS lacking biallelic SBDS point mutations. Array comparative genomic hybridization and Southern blotting were used to seek SVs across the SBDS locus. Locus-specific polymerase chain reaction (PCR) encompassing flanking intronic sequence was also performed to investigate mutation within the locus. RNA expression and Western blotting were performed to analyze allele and protein expression. We found the child harbored a single missense mutation in SBDS (c.98A > C; p.K33T), inherited from the mother, and an SV in the SBDS locus, inherited from the father. The missense allele and SV segregated in accordance with Mendelian expectations for autosomal recessive SDS. Complementary DNA and western blotting analysis and locus specific PCR support the contention that the SV perturbed SBDS protein expression in the father and child.
Our findings implicate genomic rearrangements in the pathogenesis of some cases of SDS and support patients lacking biallelic SBDS point mutations be tested for SV within the SBDS locus.
PMCID: PMC4057820  PMID: 24898207
Shwachman-Diamond syndrome; SBDS; Structural variation; Genomic rearrangement; Non-allelic homologous recombination; Low copy repeat; Whole exome sequencing; Copy number variation; Recessive disease
5.  Pluripotent stem cell models of Shwachman-Diamond syndrome reveal a common mechanism for pancreatic and hematopoietic dysfunction 
Cell stem cell  2013;12(6):727-736.
Shwachman-Diamond syndrome (SDS), a rare autosomal recessive disorder characterized by exocrine pancreatic insufficiency and hematopoietic dysfunction, is caused by mutations in the Shwachman-Bodian-Diamond syndrome (SBDS) gene. We created human pluripotent stem cell models of SDS by knock-down of SBDS in human embryonic stem cells (hESCs) and generation of induced pluripotent stem cell (iPSC) lines from two SDS patients. SBDS-deficient hESCs and iPSCs manifest deficits in exocrine pancreatic and hematopoietic differentiation in vitro, enhanced apoptosis and elevated protease levels in culture supernatants, which could be reversed by restoring SBDS protein expression through transgene rescue or by supplementing culture media with protease inhibitors. Protease-mediated auto-digestion provides a mechanistic link between the pancreatic and hematopoietic phenotypes in SDS, highlighting the utility of hESCs and iPSCs in obtaining novel insights into human disease.
PMCID: PMC3755012  PMID: 23602541
6.  The NIP7 protein is required for accurate pre-rRNA processing in human cells 
Nucleic Acids Research  2010;39(2):648-665.
Eukaryotic ribosome biogenesis requires the function of a large number of trans-acting factors which interact transiently with the nascent pre-rRNA and dissociate as the ribosomal subunits proceed to maturation and export to the cytoplasm. Loss-of-function mutations in human trans-acting factors or ribosome components may lead to genetic syndromes. In a previous study, we have shown association between the SBDS (Shwachman–Bodian–Diamond syndrome) and NIP7 proteins and that downregulation of SBDS in HEK293 affects gene expression at the transcriptional and translational levels. In this study, we show that downregulation of NIP7 affects pre-rRNA processing, causing an imbalance of the 40S/60S subunit ratio. We also identified defects at the pre-rRNA processing level with a decrease of the 34S pre-rRNA concentration and an increase of the 26S and 21S pre-rRNA concentrations, indicating that processing at site 2 is particularly slower in NIP7-depleted cells and showing that NIP7 is required for maturation of the 18S rRNA. The NIP7 protein is restricted to the nuclear compartment and co-sediments with complexes with molecular masses in the range of 40S–80S, suggesting an association to nucleolar pre-ribosomal particles. Downregulation of NIP7 affects cell proliferation, consistently with an important role for NIP7 in rRNA biosynthesis in human cells.
PMCID: PMC3025556  PMID: 20798176
7.  Altered Intracellular Localization and Mobility of SBDS Protein upon Mutation in Shwachman-Diamond Syndrome 
PLoS ONE  2011;6(6):e20727.
Shwachman-Diamond Syndrome (SDS) is a rare inherited disease caused by mutations in the SBDS gene. Hematopoietic defects, exocrine pancreas dysfunction and short stature are the most prominent clinical features. To gain understanding of the molecular properties of the ubiquitously expressed SBDS protein, we examined its intracellular localization and mobility by live cell imaging techniques. We observed that SBDS full-length protein was localized in both the nucleus and cytoplasm, whereas patient-related truncated SBDS protein isoforms localize predominantly to the nucleus. Also the nucleo-cytoplasmic trafficking of these patient-related SBDS proteins was disturbed. Further studies with a series of SBDS mutant proteins revealed that three distinct motifs determine the intracellular mobility of SBDS protein. A sumoylation motif in the C-terminal domain, that is lacking in patient SBDS proteins, was found to play a pivotal role in intracellular motility. Our structure-function analyses provide new insight into localization and motility of the SBDS protein, and show that patient-related mutant proteins are altered in their molecular properties, which may contribute to the clinical features observed in SDS patients.
PMCID: PMC3113850  PMID: 21695142
8.  Clinical spectrum and molecular pathophysiology of Shwachman-Diamond syndrome 
Current opinion in hematology  2011;18(1):30-35.
Shwachman Diamond syndrome (SDS) is an inherited bone marrow failure and cancer predisposition syndrome that affects multiple organ systems. Mutations in the SBDS gene are found in the majority of patients, but the molecular function of the SBDS protein product remains unclear. Here, we review recent progress in the clinical and molecular characterization of SDS.
Emerging data support a multifunctional role for the SBDS protein. Current studies indicate that SBDS functions in 60S large ribosomal subunit maturation and in mitotic spindle stabilization. Recent data suggest it may also affect actin polymerization, vacuolar pH regulation and DNA metabolism. SBDS loss results in both hematopoietic cell-intrinsic defects as well as marrow stromal abnormalities.
SDS is a multisystemic disease arising from defects in a protein that participates in several essential cellular processes. Elucidating the molecular function of SBDS will provide important insights into how defects in ribosome biogenesis and mitotic spindle stabilization result in hematopoietic failure, cancer predisposition, and abnormalities.
PMCID: PMC3485416  PMID: 21124213
bone marrow failure; leukemia; neutropenia; ribosome; mitotic spindle
9.  Interaction between Sdo1p and Btn1p in the Saccharomyces cerevisiae model for Batten disease 
Human Molecular Genetics  2009;19(5):931-942.
Juvenile Batten disease is an autosomal recessive pediatric neurodegenerative disorder caused by mutations in the CLN3 gene. The CLN3 protein primarily resides in the lysosomal membrane, but its function is unknown. We demonstrate that CLN3 interacts with SBDS, the protein mutated in Shwachman–Bodian–Diamond syndrome patients. We demonstrate that this protein–protein interaction is conserved between Btn1p and Sdo1p, the respective yeast Saccharomyces cerevisiae orthologs of CLN3 and SBDS. It was previously shown that deletion of BTN1 results in alterations in vacuolar pH and vacuolar (H+)-ATPase (V-ATPase)-dependent H+ transport and ATP hydrolysis. Here, we report that an SDO1 deletion strain has decreased vacuolar pH and V-ATPase-dependent H+ transport and ATP hydrolysis. These alterations result from decreased V-ATPase subunit expression. Overexpression of BTN1 or the presence of ionophore carbonyl cyanide m-chlorophenil hydrazone (CCCP) causes decreased growth in yeast lacking SDO1. In fact, in normal cells, overexpression of BTN1 mirrors the effect of CCCP, with both resulting in increased vacuolar pH due to alterations in the coupling of V-ATPase-dependent H+ transport and ATP hydrolysis. Thus, we propose that Sdo1p and SBDS work to regulate Btn1p and CLN3, respectively. This report highlights a novel mechanism for controlling vacuole/lysosome homeostasis by the ribosome maturation pathway that may contribute to the cellular abnormalities associated with juvenile Batten disease and Shwachman–Bodian–Diamond syndrome.
PMCID: PMC2816617  PMID: 20015955
10.  The Shwachman–Bodian–Diamond syndrome gene mutations cause a neonatal form of spondylometaphysial dysplasia (SMD) resembling SMD Sedaghatian type 
Journal of Medical Genetics  2007;44(4):e73.
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.
PMCID: PMC2598034  PMID: 17400792
11.  Mitotic spindle destabilization and genomic instability in Shwachman-Diamond syndrome 
The Journal of Clinical Investigation  2008;118(4):1511-1518.
Deficiencies in the SBDS gene result in Shwachman-Diamond syndrome (SDS), an inherited bone marrow failure syndrome associated with leukemia predisposition. SBDS encodes a highly conserved protein previously implicated in ribosome biogenesis. Using human primary bone marrow stromal cells (BMSCs), lymphoblasts, and skin fibroblasts, we show that SBDS stabilized the mitotic spindle to prevent genomic instability. SBDS colocalized with the mitotic spindle in control primary BMSCs, lymphoblasts, and skin fibroblasts and bound to purified microtubules. Recombinant SBDS protein stabilized microtubules in vitro. We observed that primary BMSCs and lymphoblasts from SDS patients exhibited an increased incidence of abnormal mitoses. Similarly, depletion of SBDS by siRNA in human skin fibroblasts resulted in increased mitotic abnormalities and aneuploidy that accumulated over time. Treatment of primary BMSCs and lymphoblasts from SDS patients with nocodazole, a microtubule destabilizing agent, led to increased mitotic arrest and apoptosis, consistent with spindle destabilization. Conversely, SDS patient cells were resistant to taxol, a microtubule stabilizing agent. These findings suggest that spindle instability in SDS contributes to bone marrow failure and leukemogenesis.
PMCID: PMC2263145  PMID: 18324336
12.  SBDS Protein Expression Patterns in the Bone Marrow 
Pediatric blood & cancer  2010;55(3):546-549.
Shwachman Diamond Syndrome (SDS) is an inherited bone marrow failure syndrome caused by biallelic SBDS gene mutations. Here we examined SBDS protein levels in human bone marrow. SBDS protein expression was high in neutrophil progenitors, megakaryocytes, plasma cells and osteoblasts. In contrast, SBDS protein levels were low in all hematopoietic cell lineages from patients harboring the common SBDS mutations. We conclude that SBDS protein levels vary widely between specific marrow lineages. Uniformly low SBDS protein expression levels distinguish the majority of SDS patients from controls or other marrow failure syndromes.
PMCID: PMC2913690  PMID: 20658628
Shwachman Diamond Syndrome; SBDS; bone marrow failure; neutropenia; immunohistochemistry
13.  Loss of the Mouse Ortholog of the Shwachman-Diamond Syndrome Gene (Sbds) Results in Early Embryonic Lethality†  
Molecular and Cellular Biology  2006;26(17):6656-6663.
Mutations in SBDS are responsible for Shwachman-Diamond syndrome (SDS), a disorder with clinical features of exocrine pancreatic insufficiency, bone marrow failure, and skeletal abnormalities. SBDS is a highly conserved protein whose function remains largely unknown. We identified and investigated the expression pattern of the murine ortholog. Variation in levels was observed, but Sbds was found to be expressed in all embryonic stages and most adult tissues. Higher expression levels were associated with rapid proliferation. A targeted disruption of Sbds was generated in order to understand the consequences of its loss in an in vivo model. Consistent with recessive disease inheritance for SDS, Sbds+/− mice have normal phenotypes, indistinguishable from those of their wild-type littermates. However, the development of Sbds−/− embryos arrests prior to embryonic day 6.5, with muted epiblast formation leading to early lethality. This finding is consistent with the absence of patients who are homozygous for early truncating mutations. Sbds is an essential gene for early mammalian development, with an expression pattern consistent with a critical role in cell proliferation.
PMCID: PMC1592835  PMID: 16914746
14.  Bone progenitor dysfunction induces myelodysplasia and secondary leukemia 
Nature  2010;464(7290):852-857.
Mesenchymal cell populations contribute to microenvironments regulating stem cells and the growth of malignant cells. Osteolineage cells participate in the hematopoietic stem cell niche. Here, we report that deletion of the miRNA processing endonuclease Dicer1 selectively in mesenchymal osteoprogenitors induces markedly disordered hematopoiesis. Hematopoietic changes affected multiple lineages recapitulating key features of human myelodysplastic syndrome (MDS) including the development of acute myelogenous leukemia. These changes were microenvironment dependent and induced by specific cells in the osteolineage. Dicer1−/− osteoprogenitors expressed reduced levels of Sbds, the gene mutated in the human bone marrow failure and leukemia predisposition Shwachman-Bodian-Diamond Syndrome. Deletion of Sbds in osteoprogenitors largely phenocopied Dicer1 deletion. These data demonstrate that differentiation stage-specific perturbations in osteolineage cells can induce complex hematological disorders and indicate the central role individual cellular elements of ‘estroma’ can play in tissue homeostasis. They reveal that primary changes in the hematopoietic microenvironment can initiate secondary neoplastic disease.
PMCID: PMC3422863  PMID: 20305640
15.  Non-DBA Disorders of Ribosome Function: Shwachman-Diamond Syndrome and 5q- Syndrome 
Seminars in hematology  2011;48(2):136-143.
A number of human disorders, dubbed ribosomopathies, are linked to impaired ribosome biogenesis or function. These include but are not limited to: Diamond Blackfan anemia (DBA), Shwachman Diamond syndrome (SDS) and the 5q- myelodysplastic syndrome. This review focuses on the latter two non-DBA disorders of ribosome function. Both SDS and 5q- syndrome lead to impaired hematopoiesis and a predisposition to leukemia. SDS, due to bi-allelic mutations of the SBDS gene, is a multi-system disorder that also includes bony abnormalities, pancreatic and neurocognitive dysfunction. SBDS associates with the 60S subunit in human cells and has a role in subunit joining and translational activation in yeast models. In contrast, 5q- syndrome is associated with acquired haploinsufficiency of RPS14, a component of the small 40S subunit. RPS14 is critical for 40S assembly in yeast models, and depletion of RPS14 in human CD34+ cells is sufficient to recapitulate the 5q- erythroid defect. Both SDS and the 5q- syndrome represent important models of ribosome function and may inform future treatment strategies for the ribosomopathies.
PMCID: PMC3072806  PMID: 21435510
The Journal of Pediatrics  2005;146(5):675-680.
To determine which features of incomplete or “nonclassic” forms of cystic fibrosis (CF) are associated with deleterious CF transmembrane conductance regulator gene (CFTR) mutations, and to explore other etiologies for features not associated with deleterious CFTR mutations.
Study design
Clinical features were compared between 57 patients with deleterious mutations in each CFTR and 63 with no deleterious mutations. The Shwachman Bodian Diamond syndrome gene (SBDS) was sequenced to search for mutations in patients with no deleterious CFTR mutations and steatorrhea to determine if any had unrecognized Shwachman-Diamond syndrome (SDS).
The presence of a common CF-causing mutation, absence of the vas deferens, and Pseudomona aeruginosa in the sputum correlated with the presence of two deleterious CFTR mutations, whereas sweat chloride concentration, diagnostic criteria for CF, and steatorrhea did not. However, sweat chloride concentration correlated with CFTR mutation status in patients infected with P aeruginosa. One patient had disease-causing mutations in each SBDS.
Presence of a common CF-causing mutation, absence of the vas deferens and/or P aeruginosa infection in a patient with features of nonclassic CF are predictive of deleterious mutations in each CFTR, whereas steatorrhea in the same context is likely to have etiologies other than CF transmembrane conductance regulator (CFTR) dysfunction.
PMCID: PMC3380804  PMID: 15870673
17.  Shwachman-Diamond syndrome: first molecular diagnosis in a Brazilian child 
Herein the first molecular diagnosis of a Brazilian child with Shwachman-Diamond Syndrome is reported. A 6-year-old boy was diagnosed with cystic fibrosis at the age of 15 months due to recurrent respiratory infections, diarrhea and therapeutic response to pancreatic enzymes. Three sweat tests were negative. At the age of 5 years, he began to experience pain in the lower limbs, laxity of joints, lameness and frequent falls. A radiological study revealed metaphyseal chondrodysplasia. A complete blood cell count showed leukopenia (leukocytes: 3.1-3.5 x 103/µL), neutropenia (segmented neutrophils: 15-22%), but normal hemoglobin, hematocrit and platelet count. A molecular study revealed biallelic mutations in the Shwachman-Bodian-Diamond Syndrome gene (183-184TA-CT K62X in exon 2 and a 258+2T-C transition) confirming the diagnosis of Shwachman-Diamond Syndrome. A non-pathologic, silent nucleotide A to G transition at position 201 was also found in heterozygosis in the Shwachman-Bodian-Diamond Syndrome gene. This is the first report to describe a Brazilian child with molecular diagnosis of Shwachman-Diamond Syndrome, a rare autosomal recessive disorder characterized by exocrine pancreatic insufficiency, intermittent or persistent neutropenia and skeletal changes. Other characteristics include immune system, hepatic and cardiac changes and predisposition to leukemia. Recurrent bacterial, viral and fungal infections are common. The possibility of Shwachman-Diamond Syndrome should be kept in mind when investigating children with a diagnosis of cystic fibrosis and normal sweat tests.
PMCID: PMC3789437  PMID: 24106450
Leukopenia/genetics; Exocrine pancreatic insufficiency/genetics; Cystic fibrosis; Bacterial infections; Humans; Male; Child; Case reports
18.  Role of the loop L4,5 in allosteric regulation in mtHsp70s: in vivo significance of domain communication and its implications in protein translocation 
Molecular Biology of the Cell  2014;25(14):2129-2142.
The SBD loop L4,5 in mtHsp70s functions synergistically with the linker region to maintain the interdomain interface governing protein translocation and mitochondrial biogenesis. Intragenic suppressors of a communication-impaired L4,5 mutant reveal molecular insights into the allosteric regulation of mtHsp70s at the in vivo level.
Mitochondrial Hsp70 (mtHsp70) is essential for a vast repertoire of functions, including protein import, and requires effective interdomain communication for efficient partner-protein interactions. However, the in vivo functional significance of allosteric regulation in eukaryotes is poorly defined. Using integrated biochemical and yeast genetic approaches, we provide compelling evidence that a conserved substrate-binding domain (SBD) loop, L4,5, plays a critical role in allosteric communication governing mtHsp70 chaperone functions across species. In yeast, a temperature-sensitive L4,5 mutation (E467A) disrupts bidirectional domain communication, leading to compromised protein import and mitochondrial function. Loop L4,5 functions synergistically with the linker in modulating the allosteric interface and conformational transitions between SBD and the nucleotide-binding domain (NBD), thus regulating interdomain communication. Second-site intragenic suppressors of E467A isolated within the SBD suppress domain communication defects by conformationally altering the allosteric interface, thereby restoring import and growth phenotypes. Strikingly, the suppressor mutations highlight that restoration of communication from NBD to SBD alone is the minimum essential requirement for effective in vivo function when primed at higher basal ATPase activity, mimicking the J-protein–bound state. Together these findings provide the first mechanistic insights into critical regions within the SBD of mtHsp70s regulating interdomain communication, thus highlighting its importance in protein translocation and mitochondrial biogenesis.
PMCID: PMC4091826  PMID: 24829379
19.  Nom1 Mediates Pancreas Development by Regulating Ribosome Biogenesis in Zebrafish 
PLoS ONE  2014;9(6):e100796.
Ribosome biogenesis is an important biological process for proper cellular function and development. Defects leading to improper ribosome biogenesis can cause diseases such as Diamond-Blackfan anemia and Shwachman-Bodian-Diamond syndrome. Nucleolar proteins are a large family of proteins and are involved in many cellular processes, including the regulation of ribosome biogenesis. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish line carrying mutation in nucleolar protein with MIF4G domain 1 (nom1), which encodes a conserved nulceolar protein with a role in pre-rRNA processing. Zebrafish nom1 mutants exhibit major defects in endoderm development, especially in exocrine pancreas. Further studies revealed that impaired proliferation of ptf1a-expressing pancreatic progenitor cells mainly contributed to the phenotype. RNA-seq and molecular analysis showed that ribosome biogenesis and pre-mRNA splicing were both affected in the mutant embryos. Several defects of ribosome assembly have been shown to have a p53-dependent mechanism. In the nom1 mutant, loss of p53 did not rescue the pancreatic defect, suggesting a p53-independent role. Further studies indicate that protein phosphatase 1 alpha, an interacting protein to Nom1, could partially rescue the pancreatic defect in nom1 morphants if a human nucleolar localization signal sequence was artificially added. This suggests that targeting Pp1α into the nucleolus by Nom1 is important for pancreatic proliferation. Altogether, our studies revealed a new mechanism involving Nom1 in controlling vertebrate exocrine pancreas formation.
PMCID: PMC4072693  PMID: 24967912
20.  A Case of Shwachman-Diamond Syndrome Confirmed with Genetic Analysis in a Korean Child 
Journal of Korean Medical Science  2008;23(1):142-145.
Shwachman-Diamond syndrome (SDS) is an autosomal recessive genetic disorder, consisting of exocrine pancreatic insufficiency, chronic neutropenia, neutrophil chemotaxis defects, metaphyseal dysostosis, short stature, dental caries, and multiple organ involvements. Although SDS is the second most common hereditary abnormality of exocrine pancreas following cystic fibrosis in the Western countries, it has rarely been reported in Asia. We diagnosed a case of SDS in a 42-month-old girl, and genetic analysis including the relatives of the patient confirmed the diagnosis for the first time in Korea. She had short stature, steatorrhea, dental caries, and recurrent prulent otitis media and pneumonias. Laboratory studies revealed cyclic neutropenia, and serum levels of trypsin, amylase, and lipase were decreased. Simple radiography revealed metaphyseal sclerotic changes at the distal femur. A CT scan demonstrated a fatty infiltration and atrophy of the pancreas. On direct sequencing analysis of Shwachman-Bodian-Diamond Syndrome gene exon 2 region, the patient was homozygous for the c.258+2T>C mutation and heterozygous for the c.183_184TA>CT mutation and c.201A>G single nucleotide polymorphism. Treatment with pancreatic enzyme replacement, multivitamin supplementation, and regular to high fat diet improved her weight gain and steatorrhea.
PMCID: PMC2526486  PMID: 18303216
Shwachman-Diamond Syndrome; Mutation; Korea
21.  Disease-specific induced pluripotent stem (iPS) cells 
Cell  2008;134(5):877-886.
Tissue culture of immortal cell strains from diseased patients is an invaluable resource for medical research, but is largely limited to tumor cell lines or transformed derivatives of native tissues. Here we describe the generation of induced pluripotent stem (iPS) cells from patients with a variety of genetic diseases with either Mendelian or complex inheritance that include: adenosine deaminase deficiency-related severe combined immunodeficiency (ADA-SCID), Shwachman-Bodian-Diamond syndrome (SBDS), Gaucher disease (GD) type III, Duchenne (DMD) and Becker muscular dystrophy (BMD), Parkinson disease (PD), Huntington disease (HD), juvenile-onset, type 1 diabetes mellitus (JDM), Down syndrome (DS)/trisomy 21 and the carrier state of Lesch-Nyhan syndrome. Such patient-specific stem cells offer an unprecedented opportunity to recapitulate both normal and pathologic human tissue formation in vitro, thereby enabling disease investigation and drug development.
PMCID: PMC2633781  PMID: 18691744
22.  A Fluorescent Glycolipid-Binding Peptide Probe Traces Cholesterol Dependent Microdomain-Derived Trafficking Pathways 
PLoS ONE  2008;3(8):e2933.
The uptake and intracellular trafficking of sphingolipids, which self-associate into plasma membrane microdomains, is associated with many pathological conditions, including viral and toxin infection, lipid storage disease, and neurodegenerative disease. However, the means available to label the trafficking pathways of sphingolipids in live cells are extremely limited. In order to address this problem, we have developed an exogenous, non-toxic probe consisting of a 25-amino acid sphingolipid binding domain, the SBD, derived from the amyloid peptide Aβ, and conjugated by a neutral linker with an organic fluorophore. The current work presents the characterization of the sphingolipid binding and live cell trafficking of this novel probe, the SBD peptide. SBD was the name given to a motif originally recognized by Fantini et al [1] in a number of glycolipid-associated proteins, and was proposed to interact with sphingolipids in membrane microdomains.
Methodology/Principal Findings
In accordance with Fantini's model, optimal SBD binding to membranes depends on the presence of sphingolipids and cholesterol. In synthetic membrane binding assays, SBD interacts preferentially with raft-like lipid mixtures containing sphingomyelin, cholesterol, and complex gangliosides in a pH-dependent manner, but is less glycolipid-specific than Cholera toxin B (CtxB). Using quantitative time-course colocalization in live cells, we show that the uptake and intracellular trafficking route of SBD is unlike that of either the non-raft marker Transferrin or the raft markers CtxB and Flotillin2-GFP. However, SBD traverses an endolysosomal route that partially intersects with raft-associated pathways, with a major portion being diverted at a late time point to rab11-positive recycling endosomes. Trafficking of SBD to acidified compartments is strongly disrupted by cholesterol perturbations, consistent with the regulation of sphingolipid trafficking by cholesterol.
The current work presents the characterization and trafficking behavior of a novel sphingolipid-binding fluorescent probe, the SBD peptide. We show that SBD binding to membranes is dependent on the presence of cholesterol, sphingomyelin, and complex glycolipids. In addition, SBD targeting through the endolysosomal pathway in neurons is highly sensitive to cholesterol perturbations, making it a potentially useful tool for the analysis of sphingolipid trafficking in disease models that involve changes in cholesterol metabolism and storage.
PMCID: PMC2518528  PMID: 18716682
23.  Molecular Diagnosis of Shwachman-Diamond Syndrome Presenting with Pancytopenia at an Early Age: The First Report from Turkey 
A three-month-old boy presented with growth failure, skeletal abnormalities, otitis media and pancytopenia. Exocrine pancreatic insufficiency was confirmed by low levels of fecal elastase. He was diagnosed as Shwachman-Diamond syndrome by clinical and laboratory findings. The diagnosis was confirmed by sequence analysis for SBDS gene on chromosome seven revealing compound heterozygous mutation, which are c.258+2T-C and c.183-184TA-CT. Matched unrelated donor screening for hematopoietic stem cell transplantation was initiated. Unfortunately, he died of respiratory difficulty at 5 months of age. Our case is the youngest patient whose presumptive Shwachman-Diamond syndrome diagnosis was confirmed by molecular analysis.
PMCID: PMC3710552  PMID: 24426364
Immune deficiency; Pancytopenia; Schwachman-Diamond Syndrome
24.  Identification of loss-of-function mutations of SLC35D1 in patients with Schneckenbecken dysplasia, but not with other severe spondylodysplastic dysplasias group diseases 
Journal of medical genetics  2009;46(8):562-568.
Schneckenbecken dysplasia (SBD) is an autosomal recessive lethal skeletal dysplasia that is classified into the severe spondylodysplastic dysplasias (SSDD) group in the international nosology for skeletal dysplasias. The radiological hallmark of SBD is the snaillike configuration of the hypoplastic iliac bone. SLC35D1 (solute carrier-35D1) is a nucleotide-sugar transporter involved in proteoglycan synthesis. Recently, based on human and mouse genetic studies, we showed that loss-of-function mutations of the SLC35D1 gene (SLC35D1) cause SBD.
To explore further the range of SLC35D1 mutations in SBD and elucidate whether SLC35D1 mutations cause other skeletal dysplasias that belong to the SSDD group.
Methods and results
We searched for SLC35D1 mutations in five families with SBD and 15 patients with other SSDD group diseases, including achodrogenesis type 1A, spondylometaphyseal dysplasia Sedaghatian type and fibrochondrogenesis. We identified four novel mutations, c.319C>T (p.R107X), IVS4+3A>G, a 4959-bp deletion causing the removal of exon 7 (p.R178fsX15), and c.193A>C (p. T65P), in three SBD families. Exon trapping assay showed IVS4+3A>G caused skipping of exon 4 and a frameshift (p.L109fsX18). Yeast complementation assay showed the T65P mutant protein lost the transporter activity of nucleotide sugars. Therefore, all these mutations result in loss of function. No SLC35D1 mutations were identified in all patients with other SSDD group diseases.
Our findings suggest that SLC35D1 loss-of-function mutations result consistently in SBD and are exclusive to SBD.
PMCID: PMC4144354  PMID: 19508970
25.  Shwachman-Diamond syndrome: a complex case demonstrating the potential for misdiagnosis as asphyxiating thoracic dystrophy (Jeune syndrome) 
BMC Pediatrics  2012;12:48.
The differential diagnosis of a neonate or fetus presenting with a bell-shaped or long narrow thorax includes a wide range of bony dysplasia syndromes. Where this is accompanied by respiratory distress, asphyxiating thoracic dystrophy (ATD, Jeune syndrome) is an important potential diagnosis. Shwachman-Diamond syndrome (SDS) is widely recognised as a cause of exocrine pancreatic dysfunction, short stature and bone marrow failure. It is not so well appreciated that rib and/or thoracic cage abnormalities occur in 30–50% of patients and that, in severe cases, these abnormalities may lead to thoracic dystrophy and respiratory failure in the newborn. There are, however, at least three previous case reports of children who were initially diagnosed with ATD who were subsequently shown to have SDS.
Case presentation
This report details the case history of a patient misdiagnosed as having ATD as a neonate following the neonatal asphyxial death of her brother. She subsequently developed progressive pancytopenia but was only diagnosed with SDS at 11 years of age after referral for haematopoietic stem cell transplantation for bone marrow failure accompanied by trilineage dysplasia and clonal cytogenetic abnormalities on bone marrow examination. Subsequent testing revealed the presence of fat globules in stools, reduced faecal chymotrypsin, fat-soluble vitamin deficiency, metaphyseal dysplasia on skeletal survey and heterozygous mutations of the SBDS gene.
This report highlights the potential for diagnostic confusion between ATD and SDS. It is important to include SDS in the differential diagnosis of newborns with thoracic dystrophy and to seek expert clinical and radiological assessment of such children.
PMCID: PMC3457858  PMID: 22554078
Shwachman-Diamond syndrome; Asphyxiating thoracic dystrophy; Jeune syndrome; Differential diagnosis; Haematopoietic stem cell transplantation; Isochromosome 7q; Pancreatic insufficiency; Neonatal respiratory distress

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