Mitochondrial augmentation of hematopoietic stem cells in children with single large-scale mitochondrial DNA deletion syndromes.

Patients with single large-scale mitochondrial DNA (mtDNA) deletion syndromes (SLSMDs) usually present with multisystemic disease, either as Pearson syndrome in early childhood or as Kearns-Sayre syndrome later in life. No disease-modifying therapies exist for SLSMDs. We have developed a method to enrich hematopoietic cells with exogenous mitochondria, and we treated six patients with SLSMDs through a compassionate use program. Autologous CD34+ hematopoietic cells were augmented with maternally derived healthy mitochondria, a technology termed mitochondrial augmentation therapy (MAT). All patients had substantial multisystemic disease involvement at baseline, including neurologic, endocrine, or renal impairment. We first assessed safety, finding that the procedure was well tolerated and that all study-related severe adverse events were either leukapheresis-related or related to the baseline disorder. After MAT, heteroplasmy decreased in the peripheral blood in four of the six patients. An increase in mtDNA content of peripheral blood cells was measured in all six patients 6 to 12 months after MAT as compared baseline. We noted some clinical improvement in aerobic function, measured in patients 2 and 3 by sit-to-stand or 6-min walk testing, and an increase in the body weight of five of the six patients suffering from very low body weight before treatment. Quality-of-life measurements as per caregiver assessment and physical examination showed improvement in some parameters. Together, this work lays the ground for clinical trials of MAT for the treatment of patients with mtDNA disorders.

Progressive pulmonary fibrosis in a murine model of Hermansky-Pudlak syndrome.

BACKGROUND: HPS-1 is a genetic type of Hermansky-Pudlak syndrome (HPS) with highly penetrant pulmonary fibrosis (HPSPF), a restrictive lung disease that is similar to idiopathic pulmonary fibrosis (IPF). Hps1ep/ep (pale ear) is a naturally occurring HPS-1 mouse model that exhibits high sensitivity to bleomycin-induced pulmonary fibrosis (PF). Traditional methods of administering bleomycin as an intratracheal (IT) route to induce PF in this model often lead to severe acute lung injury and high mortality rates, complicating studies focusing on pathobiological mechanisms or exploration of therapeutic options for HPSPF. METHODS: To develop a murine model of HPSPF that closely mimics the progression of human pulmonary fibrosis, we investigated the pulmonary effects of systemic delivery of bleomycin in Hps1ep/ep mice using a subcutaneous minipump and compared results to oropharyngeal delivery of bleomycin. RESULTS: Our study revealed that systemic delivery of bleomycin induced limited, acute inflammation that resolved. The distinct inflammatory phase preceded a slow, gradually progressive fibrogenesis that was shown to be both time-dependent and dose-dependent. The fibrosis phase exhibited characteristics that better resembles human disease with focal regions of fibrosis that were predominantly found in peribronchovascular areas and in subpleural regions; central lung areas contained relatively less fibrosis. CONCLUSION: This model provides a preclinical tool that will allow researchers to study the mechanism of pulmonary fibrosis in HPS and provide a platform for the development of therapeutics to treat HPSPF. This method can be applied on studies of IPF or other monogenic disorders that lead to pulmonary fibrosis.

Dietary-Induced Ketogenesis: Adults Are Not Children.

There is increasing interest in the use of a ketogenic diet for various adult disorders; however, the ability of adults to generate ketones is unknown. Our goal was to challenge the hypothesis that there would be no difference between adults and children regarding their ability to enter ketosis. METHODS: Two populations were studied, both treated with identical very low-carbohydrate high-fat diets: a retrospective series of children with epilepsy or/and metabolic disorders (2009-2016) and a prospective clinical trial of adults with glioblastoma. Dietary intake was assessed based upon written food diaries and 24-h dietary recall. Ketogenic ratio was calculated according to [grams of fat consumed]/[grams of carbohydrate and protein consumed]. Ketone levels (ß-hydroxybutyrate) were measured in blood and/or urine. RESULTS: A total of 168 encounters amongst 28 individuals were analyzed. Amongst both children and adults, ketone levels correlated with nutritional ketogenic ratio; however, the absolute ketone levels in adults were approximately one quarter of those seen in children. This difference was highly significant in a multivariate linear regression model, p < 0.0001. CONCLUSIONS: For diets with comparable ketogenic ratios, adults have lower blood ketone levels than children; consequently, high levels of nutritional ketosis are unobtainable in adults.

A Phase I clinical trial of dose-escalated metabolic therapy combined with concomitant radiation therapy in high-grade glioma.

BACKGROUND: Animal brain-tumor models have demonstrated a synergistic interaction between radiation therapy and a ketogenic diet (KD). Metformin has in-vitro anti-cancer activity, through AMPK activation and mTOR inhibition. We hypothesized that the metabolic stress induced by a KD combined with metformin would enhance radiation's efficacy. We sought to assess the tolerability and feasibility of this approach. METHODS: A single-institution phase I clinical trial. Radiotherapy was either 60 or 35 Gy over 6 or 2 weeks, for newly diagnosed and recurrent gliomas, respectively. The dietary intervention consisted of a Modified Atkins Diet (ModAD) supplemented with medium chain triglycerides (MCT). There were three cohorts: Dietary intervention alone, and dietary intervention combined with low-dose or high-dose metformin; all patients received radiotherapy. Factors associated with blood ketone levels were investigated using a mixed-model analysis. RESULTS: A total of 13 patients were accrued, median age 61 years, of whom six had newly diagnosed and seven with recurrent disease. All completed radiation therapy; five patients stopped the metabolic intervention early. Metformin 850 mg three-times daily was poorly tolerated. There were no serious adverse events. Ketone levels were associated with dietary factors (ketogenic ratio, p < 0.001), use of metformin (p = 0. 02) and low insulin levels (p = 0.002). Median progression free survival was ten and four months for newly diagnosed and recurrent disease, respectively. CONCLUSIONS: The intervention was well tolerated. Higher serum ketone levels were associated with both dietary intake and metformin use. The recommended phase II dose is eight weeks of a ModAD combined with 850 mg metformin twice daily.

SCUBE3 loss-of-function causes a recognizable recessive developmental disorder due to defective bone morphogenetic protein signaling.

Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a member of a small family of multifunctional cell surface-anchored glycoproteins functioning as co-receptors for a variety of growth factors. Here we report that bi-allelic inactivating variants in SCUBE3 have pleiotropic consequences on development and cause a previously unrecognized syndromic disorder. Eighteen affected individuals from nine unrelated families showed a consistent phenotype characterized by reduced growth, skeletal features, distinctive craniofacial appearance, and dental anomalies. In vitro functional validation studies demonstrated a variable impact of disease-causing variants on transcript processing, protein secretion and function, and their dysregulating effect on bone morphogenetic protein (BMP) signaling. We show that SCUBE3 acts as a BMP2/BMP4 co-receptor, recruits the BMP receptor complexes into raft microdomains, and positively modulates signaling possibly by augmenting the specific interactions between BMPs and BMP type I receptors. Scube3-/- mice showed craniofacial and dental defects, reduced body size, and defective endochondral bone growth due to impaired BMP-mediated chondrogenesis and osteogenesis, recapitulating the human disorder. Our findings identify a human disease caused by defective function of a member of the SCUBE family, and link SCUBE3 to processes controlling growth, morphogenesis, and bone and teeth development through modulation of BMP signaling.

Bi-allelic Variants in RALGAPA1 Cause Profound Neurodevelopmental Disability, Muscular Hypotonia, Infantile Spasms, and Feeding Abnormalities.

Ral (Ras-like) GTPases play an important role in the control of cell migration and have been implicated in Ras-mediated tumorigenicity. Recently, variants in RALA were also described as a cause of intellectual disability and developmental delay, indicating the relevance of this pathway to neuropediatric diseases. Here, we report the identification of bi-allelic variants in RALGAPA1 (encoding Ral GTPase activating protein catalytic alpha subunit 1) in four unrelated individuals with profound neurodevelopmental disability, muscular hypotonia, feeding abnormalities, recurrent fever episodes, and infantile spasms . Dysplasia of corpus callosum with focal thinning of the posterior part and characteristic facial features appeared to be unifying findings. RalGAPA1 was absent in the fibroblasts derived from two affected individuals suggesting a loss-of-function effect of the RALGAPA1 variants. Consequently, RalA activity was increased in these cell lines, which is in keeping with the idea that RalGAPA1 deficiency causes a constitutive activation of RalA. Additionally, levels of RalGAPB, a scaffolding subunit of the RalGAP complex, were dramatically reduced, indicating a dysfunctional RalGAP complex. Moreover, RalGAPA1 deficiency clearly increased cell-surface levels of lipid raft components in detached fibroblasts, which might indicate that anchorage-dependence of cell growth signaling is disturbed. Our findings indicate that the dysregulation of the RalA pathway has an important impact on neuronal function and brain development. In light of the partially overlapping phenotype between RALA- and RALGAPA1-associated diseases, it appears likely that dysregulation of the RalA signaling pathway leads to a distinct group of genetic syndromes that we suggest could be named RALopathies.

Whole exome sequencing in childhood-onset lupus frequently detects single gene etiologies.

BACKGROUND: Systemic lupus erythematosus (SLE) comprise a diverse range of clinical manifestations. To date, more than 30 single gene causes of lupus/lupus like syndromes in humans have been identified. In the clinical setting, identifying the underlying molecular diagnosis is challenging due to phenotypic and genetic heterogeneity. METHODS: We employed whole exome sequencing (WES) in patients presenting with childhood-onset lupus with severe and/or atypical presentations to identify cases that are explained by a single-gene (monogenic) cause. RESULTS: From January 2015 to June 2018 15 new cases of childhood-onset SLE were diagnosed in Edmond and Lily Safra Children's Hospital. By WES we identified causative mutations in four subjects in five different genes: C1QC, SLC7A7, MAN2B1, PTEN and STAT1. No molecular diagnoses were established on clinical grounds prior to genetic testing. CONCLUSIONS: We identified a significant fraction of monogenic SLE etiologies using WES and confirm the genetic locus heterogeneity in childhood-onset lupus. These results highlight the importance of establishing a genetic diagnosis for children with severe or atypical lupus by providing accurate and early etiology-based diagnoses and improving subsequent clinical management.

Progressive Pseudorheumatoid Dysplasia resolved by whole exome sequencing: a novel mutation in WISP3 and review of the literature.

BACKGROUND: Progressive pseudorheumatoid dysplasia (PPRD) is a rare autosomal-recessive, non-inflammatory arthropathy, shown to be caused by mutations in the WNT1-inducible signaling pathway protein 3 (WISP3) gene. Although several hundred cases were reported worldwide, the diagnosis remains challenging. Subsequently, the syndrome is often unrecognized and misdiagnosed (for instance, as Juvenile Idiopathic Arthritis), leading to unnecessary procedures and treatments. The objective of the current study was to identify the molecular basis in a family with PPRD and describe their phenotype and course of illness. PATIENTS AND METHODS: We present here a multiply affected consanguineous family of Iraqi-Jewish descent with PPRD. The proband, a 6.5 years old girl, presented with bilateral symmetric bony enlargements of the 1st interphalangeal joints of the hands, without signs of synovitis. Molecular analysis of the family was pursued using Whole Exome Sequencing (WES) and homozygosity mapping. RESULTS: WES analysis brought to the identification of a novel homozygous missense mutation (c.257G > T, p.C86F) in the WISP3 gene. Following this diagnosis, an additional 53 years old affected family member was found to harbor the mutation. Two other individuals in the family were reported to have had similar involvement however both had died of unrelated causes. CONCLUSION: The reported family underscores the importance of recognition of this unique skeletal dysplasia by clinicians, and especially by pediatric rheumatologists and orthopedic surgeons.

Phenotype variability in Hajdu-Cheney syndrome.

Hajdu Cheney syndrome is a rare autosomal dominant skeletal dysplasia, with multi-organ involvement, caused by pathogenic variants in NOTCH2. It is characterized by progressive focal bone destruction, including acro-osteolysis and generalized osteoporosis, craniofacial anomalies, hearing loss, cardiovascular involvement and polycystic kidneys. Distinct radiographic findings, such as a serpentine fibula, may aid in facilitating the diagnosis. Despite several dozens of cases described in the literature, diagnosis often remains elusive, resulting in many cases in a delay in diagnosis reaching adolescence or adulthood. We report herein two unrelated patients of Turkish/Lebanese Jewish and Ashkenazi Jewish descent, each presenting with distinct clinical challenges and subsequently distinct diagnostic odysseys leading to their molecular diagnosis. These illustrative clinical descriptions underscore the wide phenotypic variability of HCS, and further contribute to the current knowledge regarding this rare entity.

Acid-Induced Downregulation of ASS1 Contributes to the Maintenance of Intracellular pH in Cancer.

Downregulation of the urea cycle enzyme argininosuccinate synthase (ASS1) by either promoter methylation or by HIF1α is associated with increased metastasis and poor prognosis in multiple cancers. We have previously shown that in normoxic conditions, ASS1 downregulation facilitates cancer cell proliferation by increasing aspartate availability for pyrimidine synthesis by the enzyme complex CAD. Here we report that in hypoxia, ASS1 expression in cancerous cells is downregulated further by HIF1α-mediated induction of miR-224-5p, making the cells more invasive and dependent on upstream substrates of ASS1 for survival. ASS1 was downregulated under acidic conditions, and ASS1-depleted cancer cells maintained a higher intracellular pH (pHi), depended less on extracellular glutamine, and displayed higher glutathione levels. Depletion of substrates of urea cycle enzymes in ASS1-deficient cancers decreased cancer cell survival. Thus, ASS1 levels in cancer are differentially regulated in various environmental conditions to metabolically benefit cancer progression. Understanding these alterations may help uncover specific context-dependent cancer vulnerabilities that may be targeted for therapeutic purposes. SIGNIFICANCE: Cancer cells in an acidic or hypoxic environment downregulate the expression of the urea cycle enzyme ASS1, which provides them with a redox and pH advantage, resulting in better survival.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/3/518/F1.large.jpg.

Mutations in PPCS, Encoding Phosphopantothenoylcysteine Synthetase, Cause Autosomal-Recessive Dilated Cardiomyopathy.

Coenzyme A (CoA) is an essential metabolic cofactor used by around 4% of cellular enzymes. Its role is to carry and transfer acetyl and acyl groups to other molecules. Cells can synthesize CoA de novo from vitamin B5 (pantothenate) through five consecutive enzymatic steps. Phosphopantothenoylcysteine synthetase (PPCS) catalyzes the second step of the pathway during which phosphopantothenate reacts with ATP and cysteine to form phosphopantothenoylcysteine. Inborn errors of CoA biosynthesis have been implicated in neurodegeneration with brain iron accumulation (NBIA), a group of rare neurological disorders characterized by accumulation of iron in the basal ganglia and progressive neurodegeneration. Exome sequencing in five individuals from two unrelated families presenting with dilated cardiomyopathy revealed biallelic mutations in PPCS, linking CoA synthesis with a cardiac phenotype. Studies in yeast and fruit flies confirmed the pathogenicity of identified mutations. Biochemical analysis revealed a decrease in CoA levels in fibroblasts of all affected individuals. CoA biosynthesis can occur with pantethine as a source independent from PPCS, suggesting pantethine as targeted treatment for the affected individuals still alive.

Long ncRNA Landscape in the Ileum of Treatment-Naive Early-Onset Crohn Disease.

Background: Long noncoding RNAs (lncRNA) are key regulators of gene transcription and many show tissue-specific expression. We previously defined a novel inflammatory and metabolic ileal gene signature in treatment-naive pediatric Crohn disease (CD). We now extend our analyses to include potential regulatory lncRNA. Methods: Using RNAseq, we systematically profiled lncRNAs and protein-coding gene expression in 177 ileal biopsies. Co-expression analysis was used to identify functions and tissue-specific expression. RNA in situ hybridization was used to validate expression. Real-time polymerase chain reaction was used to test lncRNA regulation by IL-1ß in Caco-2 enterocytes. Results: We characterize widespread dysregulation of 459 lncRNAs in the ileum of CD patients. Using only the lncRNA in discovery and independent validation cohorts showed patient classification as accurate as the protein-coding genes, linking lncRNA to CD pathogenesis. Co-expression and functional annotation enrichment analyses across several tissues and cell types 1showed that the upregulated LINC01272 is associated with a myeloid pro-inflammatory signature, whereas the downregulated HNF4A-AS1 exhibits association with an epithelial metabolic signature. We confirmed tissue-specific expression in biopsies using in situ hybridization, and validated regulation of prioritized lncRNA upon IL-1ß exposure in differentiated Caco-2 cells. Finally, we identified significant correlations between LINC01272 and HNF4A-AS1 expression and more severe mucosal injury. Conclusions: We systematically define differentially expressed lncRNA in the ileum of newly diagnosed pediatric CD. We show lncRNA utility to correctly classify disease or healthy states and demonstrate their regulation in response to an inflammatory signal. These lncRNAs, after mechanistic exploration, may serve as potential new tissue-specific targets for RNA-based interventions.

A novel inborn error of the coenzyme Q10 biosynthesis pathway: cerebellar ataxia and static encephalomyopathy due to COQ5 C-methyltransferase deficiency.

Primary coenzyme Q10 (CoQ10 ; MIM# 607426) deficiencies are an emerging group of inherited mitochondrial disorders with heterogonous clinical phenotypes. Over a dozen genes are involved in the biosynthesis of CoQ10 , and mutations in several of these are associated with human disease. However, mutations in COQ5 (MIM# 616359), catalyzing the only C-methylation in the CoQ10 synthetic pathway, have not been implicated in human disease. Here, we report three female siblings of Iraqi-Jewish descent, who had varying degrees of cerebellar ataxia, encephalopathy, generalized tonic-clonic seizures, and cognitive disability. Whole-exome and subsequent whole-genome sequencing identified biallelic duplications in the COQ5 gene, leading to reduced levels of CoQ10 in peripheral white blood cells of all affected individuals and reduced CoQ10 levels in the only muscle tissue available from one affected proband. CoQ10 supplementation led to clinical improvement and increased the concentrations of CoQ10 in blood. This is the first report of primary CoQ10 deficiency caused by loss of function of COQ5, with delineation of the clinical, laboratory, histological, and molecular features, and insights regarding targeted treatment with CoQ10 supplementation.

Reversal of cystoid macular edema in gyrate atrophy patients.

PURPOSE: This study reports the presentation of two families with gyrate atrophy (GA). The aim of this study was to characterize the potential effect of therapeutic regimens on macular edema. METHODS: Two unrelated patients with GA were studied for the potential effect of low protein diet (≤ 0.8 g/kg/d), and oral administration of pyridoxine (500 mg/day), on serum ornithine levels, best corrected visual acuity (BCVA), slit-lamp, OCT, and auto-fluorescence findings. Blood samples for DNA, mRNA, and exons of the OAT gene were screened for mutations and splicing effect when relevant. RESULTS: At presentation, both patients manifested typical ophthalmic features of GA including cystoid macular edema (CME). One patient also exhibited optic nerve head hamartoma. Following treatment ornithine levels have lessened, BCVA improved, and central macular thickness (CMT) markedly decreased in all four studied eyes. The molecular pathologic features included a novel splice site mutation (c.900+1G>A). CONCLUSIONS: We have identified a novel mutation and two formerly described mutations in patients with GA. Of them, one patient comprised an unusual phenotype including bilateral astrocytic hamartomas. We have recognized for the first time improvement in CME following treatment with low protein intake and pyridoxine supplement. This finding may have significance in the understanding of treatment options for macular edema regardless of underlying etiology.

Clinical Variability in a Family with an Ectodermal Dysplasia Syndrome and a Nonsense Mutation in the TP63 Gene.

Mutations in the TP63 gene have been associated with a variety of ectodermal dysplasia syndromes, among which the clinically overlapping Ankyloblepharon-Ectodermal defects-Cleft lip/palate (AEC) and the Rapp-Hodgkin syndromes. We report a multiplex nonconsanguineous family of Ashkenazi-Jewish descent, in which the index patient presented with a persistent scalp skin lesion, dystrophic nails and light thin hair. Further evaluation revealed over 10 affected individuals in the kindred, over four generations, exhibiting varying degrees of ectodermal involvement. Analysis of the TP63 gene from four of the patients and from two healthy individuals of the same family was performed. Gene sequencing of the patients revealed a nonsense mutation leading to a premature termination codon (PTC) (p.Gln16X). The same mutation was found in all tested affected individuals in the family, but gave rise to marked phenotypic variability with minor clinical manifestations in some individuals, underscoring the clinical heterogeneity associated with the recently described PTC-causing mutations.

CAOS-Episodic Cerebellar Ataxia, Areflexia, Optic Atrophy, and Sensorineural Hearing Loss: A Third Allelic Disorder of the ATP1A3 Gene.

We describe the molecular basis of a distinctive syndrome characterized by infantile stress-induced episodic weakness, ataxia, and sensorineural hearing loss, with permanent areflexia and optic nerve pallor. Whole exome sequencing identified a deleterious heterozygous c.2452 G>A, p.(E818K) variant in the ATP1A3 gene and structural analysis predicted its protein-destabilizing effect. This variant has not been reported in context with rapid-onset dystonia parkinsonism and alternating hemiplegia of childhood, the 2 main diseases associated with ATP1A3. The clinical presentation in the family described here differs categorically from these diseases in age of onset, clinical course, cerebellar over extrapyramidal movement disorder predominance, and peripheral nervous system involvement. While this paper was in review, a highly resembling phenotype was reported in additional patients carrying the same c.2452 G>A variant. Our findings substantiate this variant as the cause of a unique inherited autosomal dominant neurologic syndrome that constitutes a third allelic disease of the ATP1A3 gene.

Mutant adenosine deaminase 2 in a polyarteritis nodosa vasculopathy.

BACKGROUND: Polyarteritis nodosa is a systemic necrotizing vasculitis with a pathogenesis that is poorly understood. We identified six families with multiple cases of systemic and cutaneous polyarteritis nodosa, consistent with autosomal recessive inheritance. In most cases, onset of the disease occurred during childhood. METHODS: We carried out exome sequencing in persons from multiply affected families of Georgian Jewish or German ancestry. We performed targeted sequencing in additional family members and in unrelated affected persons, 3 of Georgian Jewish ancestry and 14 of Turkish ancestry. Mutations were assessed by testing their effect on enzymatic activity in serum specimens from patients, analysis of protein structure, expression in mammalian cells, and biophysical analysis of purified protein. RESULTS: In all the families, vasculitis was caused by recessive mutations in CECR1, the gene encoding adenosine deaminase 2 (ADA2). All the Georgian Jewish patients were homozygous for a mutation encoding a Gly47Arg substitution, the German patients were compound heterozygous for Arg169Gln and Pro251Leu mutations, and one Turkish patient was compound heterozygous for Gly47Val and Trp264Ser mutations. In the endogamous Georgian Jewish population, the Gly47Arg carrier frequency was 0.102, which is consistent with the high prevalence of disease. The other mutations either were found in only one family member or patient or were extremely rare. ADA2 activity was significantly reduced in serum specimens from patients. Expression in human embryonic kidney 293T cells revealed low amounts of mutant secreted protein. CONCLUSIONS: Recessive loss-of-function mutations of ADA2, a growth factor that is the major extracellular adenosine deaminase, can cause polyarteritis nodosa vasculopathy with highly varied clinical expression. (Funded by the Shaare Zedek Medical Center and others.).

A dominant negative heterozygous G87R mutation in the zinc transporter, ZnT-2 (SLC30A2), results in transient neonatal zinc deficiency.

Zinc is an essential mineral, and infants are particularly vulnerable to zinc deficiency as they require large amounts of zinc for their normal growth and development. We have recently described the first loss-of-function mutation (H54R) in the zinc transporter ZnT-2 (SLC30A2) in mothers with infants harboring transient neonatal zinc deficiency (TNZD). Here we identified and characterized a novel heterozygous G87R ZnT-2 mutation in two unrelated Ashkenazi Jewish mothers with infants displaying TNZD. Transient transfection of G87R ZnT-2 resulted in endoplasmic reticulum-Golgi retention, whereas the WT transporter properly localized to intracellular secretory vesicles in HC11 and MCF-7 cells. Consequently, G87R ZnT-2 showed decreased stability compared with WT ZnT-2 as revealed by Western blot analysis. Three-dimensional homology modeling based on the crystal structure of YiiP, a close zinc transporter homologue from Escherichia coli, revealed that the basic arginine residue of the mutant G87R points toward the membrane lipid core, suggesting misfolding and possible loss-of-function. Indeed, functional assays including vesicular zinc accumulation, zinc secretion, and cytoplasmic zinc pool assessment revealed markedly impaired zinc transport in G87R ZnT-2 transfectants. Moreover, co-transfection experiments with both mutant and WT transporters revealed a dominant negative effect of G87R ZnT-2 over the WT ZnT-2; this was associated with mislocalization, decreased stability, and loss of zinc transport activity of the WT ZnT-2 due to homodimerization observed upon immunoprecipitation experiments. These findings establish that inactivating ZnT-2 mutations are an underlying basis of TNZD and provide the first evidence for the dominant inheritance of heterozygous ZnT-2 mutations via negative dominance due to homodimer formation.

Cellular and clinical report of new Griscelli syndrome type III cases.

The RAB27A/Melanophilin/Myosin-5a tripartite protein complex is required for capturing mature melanosomes in the peripheral actin network of melanocytes for subsequent transfer to keratinocytes. Mutations in any one member of this tripartite complex cause three forms of Griscelli syndrome (GS), each with distinct clinical features but with a similar cellular phenotype. To date, only one case of GS type III (GSIII), caused by mutations in the Melanophilin (MLPH) gene, has been reported. Here, we report seven new cases of GSIII in three distinct Arab pedigrees. All affected individuals carried a homozygous missense mutation (c.102C>T; p.R35W), located in the conserved Slp homology domain of MLPH, and had hypomelanosis of the skin and hair. We report the first cellular studies on GSIII melanocytes, which demonstrated that MLPH(R35W) causes perinuclear aggregation of melanosomes in melanocytes, typical for GS. Additionally, co-immunoprecipitation assays showed that MLPH(R35W) lost its interaction with RAB27A, indicating pathogenicity of the R35W mutation.

Gray platelet syndrome: natural history of a large patient cohort and locus assignment to chromosome 3p.

Gray platelet syndrome (GPS) is an inherited bleeding disorder characterized by macrothrombocytopenia and absence of platelet α-granules resulting in typical gray platelets on peripheral smears. GPS is associated with a bleeding tendency, myelofibrosis, and splenomegaly. Reports on GPS are limited to case presentations. The causative gene and underlying pathophysiology are largely unknown. We present the results of molecular genetic analysis of 116 individuals including 25 GPS patients from 14 independent families as well as novel clinical data on the natural history of the disease. The mode of inheritance was autosomal recessive (AR) in 11 and indeterminate in 3 families. Using genome-wide linkage analysis, we mapped the AR-GPS gene to a 9.4-Mb interval on 3p21.1-3p22.1, containing 197 protein-coding genes. Sequencing of 1423 (69%) of the 2075 exons in the interval did not identify the GPS gene. Long-term follow-up data demonstrated the progressive nature of the thrombocytopenia and myelofibrosis of GPS resulting in fatal hemorrhages in some patients. We identified high serum vitamin B(12) as a consistent, novel finding in GPS. Chromosome 3p21.1-3p22.1 has not been previously linked to a platelet disorder; identification of the GPS gene will likely lead to the discovery of novel components of platelet organelle biogenesis. This study is registered at www.clinicaltrials.gov as NCT00069680 and NCT00369421.