Homozygosity mapping and whole-exome sequencing to detect SLC45A2 and G6PC3 mutations in a single patient with oculocutaneous albinism and neutropenia.
ABSTRACT: We evaluated a 32-year-old woman whose oculocutaneous albinism (OCA), bleeding diathesis, neutropenia, and history of recurrent infections prompted consideration of the diagnosis of Hermansky-Pudlak syndrome type 2. This was ruled out because of the presence of platelet ?-granules and absence of AP3B1 mutations. As parental consanguinity suggested an autosomal recessive mode of inheritance, we employed homozygosity mapping, followed by whole-exome sequencing, to identify two candidate disease-causing genes, SLC45A2 and G6PC3. Conventional dideoxy sequencing confirmed pathogenic mutations in SLC45A2, associated with OCA type 4 (OCA-4), and G6PC3, associated with neutropenia. The substantial reduction of SLC45A2 protein in the patient's melanocytes caused the mislocalization of tyrosinase from melanosomes to the plasma membrane and also led to the incorporation of tyrosinase into exosomes and secretion into the culture medium, explaining the hypopigmentation in OCA-4. Our patient's G6PC3 mRNA expression level was also reduced, leading to increased apoptosis of her fibroblasts under endoplasmic reticulum stress. To our knowledge, this report describes the first North American patient with OCA-4, the first culture of human OCA-4 melanocytes, and the use of homozygosity mapping, followed by whole-exome sequencing, to identify disease-causing mutations in multiple genes in a single affected individual.
Project description:Severe congenital neutropenia type 4 (SCN4) is an autosomal recessive disorder caused by mutations in the third subunit of the enzyme glucose-6-phosphatase (G6PC3). Its core features are congenital neutropenia and a prominent venous skin pattern, and affected individuals have variable birth defects. Oculocutaneous albinism type 4 (OCA4) is caused by autosomal recessive mutations in SLC45A2.We report a sister and brother from Newfoundland, Canada with complex phenotypes. The sister was previously reported by Cullinane et al., 2011. We performed homozygosity mapping, next generation sequencing and conventional Sanger sequencing to identify mutations that cause the phenotype in this family. We have also summarized clinical data from 49 previously reported SCN4 cases with overlapping phenotypes and interpret the medical histories of these siblings in the context of the literature.The siblings' phenotype is due in part to a homozygous mutation in G6PC3, [c.829C > T, p.Gln277X]. Their ages are 38 and 37 years respectively and they are the oldest SCN4 patients published to date. Both presented with congenital neutropenia and later developed Crohn disease. We suggest that the latter is a previously unrecognized SCN4 manifestation and that not all affected individuals have an intellectual disability. The sister also has a homozygous mutation in SLC45A2, which explains her severe oculocutaneous hypopigmentation. Her brother carried one SLC45A2 mutation and was diagnosed with "partial OCA" in childhood.This family highlights that apparently novel syndromes can in fact be caused by two known autosomal recessive disorders.
Project description:Oculocutaneous albinism (OCA) is a clinically and genetically heterogenic group of pigmentation abnormalities. OCA type IV (OCA4, OMIM 606574) develops due to homozygous or compound heterozygous mutations in the solute carrier family 45, member 2 (SLC45A2) gene. This gene encodes a membrane-associated transport protein, which regulates tyrosinase activity and, thus, melanin content by changing melanosomal pH and disrupting the incorporation of copper into tyrosinase.Here we report two Hungarian siblings affected by an unusual OCA4 phenotype. After genomic DNA was isolated from peripheral blood of the patients, the coding regions of the SLC45A2 gene were sequenced. In silico tools were applied to identify the functional impact of the newly detected mutations.Direct sequencing of the SLC45A2 gene revealed two novel, heterozygous mutations, one missense (c.1226G?>?A, p.Gly409Asp) and one nonsense (c.1459C?>?T, p.Gln437*), which were present in both patients, suggesting the mutations were compound heterozygous. In silico tools suggest that these variations are disease causing mutations.The newly identified mutations may affect the transmembrane domains of the protein, and could impair transport function, resulting in decreases in both melanosomal pH and tyrosinase activity. Our study provides expands on the mutation spectrum of the SLC45A2 gene and the genetic background of OCA4.
Project description:BACKGROUND:Oculocutaneous albinism (OCA) is a group of heterogeneous autosomal recessive genetic disorder of melanin synthesis results in hypopigmented hair, skin, and eyes. OCA type 1, OCA type 2, and OCA type 4, which are respectively caused by mutations in TYR, OCA2, and SLC45A2 have high morbidity rates in Asia. METHODS:TYR, OCA2, and SLC45A2 mutation analysis was carried out on 18 nonconsanguineous OCA patients and four fetuses were included for prenatal diagnose. Three genes of all individuals were amplified by polymerase chain reaction and examined by Sanger sequencing. The pathogenicity of the detected mutations were analyzed by Mutation Taster, PolyPhen 2, and SIFT software, and the conservation of the substituted amino acids were analyzed by MEGA software. RESULTS:Eleven TYR mutations, three OCA2 mutations, and two SLC45A2 mutations were identified in 14 OCA type 1 patients, two OCA type 2 patients, and two OCA type 4 patients. c.1021A>G, p.R341G in TYR, c.1096_1104del, p.V366*, and c.1079C>T, p.S360F in OCA2 were novel. One of the four fetuses carried compound heterozygous mutation of TYR and became spontaneous abortion, the other three carried no mutations and appeared normal at birth. CONCLUSION:In this study, specific clinical characteristics of OCA patients were described. Three novel pathogenic mutations were identified which will enrich the mutation spectrum of OCA, and the prenatal genetic screening in fetus at risk of OCA can provide vital information for genetic counseling.
Project description:Mutations in more than 15 genes are now known to cause severe congenital neutropenia (SCN); however, the pathologic mechanisms of most genetic defects are not fully defined. Deficiency of G6PC3, a glucose-6-phosphatase, causes a rare multisystem syndrome with SCN first described in 2009. We identified a family with 2 children with homozygous G6PC3 G260R mutations, a loss of enzymatic function, and typical syndrome features with the exception that their bone marrow biopsy pathology revealed abundant neutrophils consistent with myelokathexis. This pathologic finding is a hallmark of another type of SCN, WHIM syndrome, which is caused by gain-of-function mutations in CXCR4, a chemokine receptor and known neutrophil bone marrow retention factor. We found markedly increased CXCR4 expression on neutrophils from both our G6PC3-deficient patients and G6pc3(-/-) mice. In both patients, granulocyte colony-stimulating factor treatment normalized CXCR4 expression and neutrophil counts. In G6pc3(-/-) mice, the specific CXCR4 antagonist AMD3100 rapidly reversed neutropenia. Thus, myelokathexis associated with abnormally high neutrophil CXCR4 expression may contribute to neutropenia in G6PC3 deficiency and responds well to granulocyte colony-stimulating factor.
Project description:The G6PC3 gene encodes the ubiquitously expressed glucose-6-phosphatase enzyme (G-6-Pase β or G-6-Pase 3 or G6PC3). Bi-allelic G6PC3 mutations cause a multi-system autosomal recessive disorder of G6PC3 deficiency (also called severe congenital neutropenia type 4, MIM 612541). To date, at least 57 patients with G6PC3 deficiency have been described in the literature.G6PC3 deficiency is characterized by severe congenital neutropenia, recurrent bacterial infections, intermittent thrombocytopenia in many patients, a prominent superficial venous pattern and a high incidence of congenital cardiac defects and uro-genital anomalies. The phenotypic spectrum of the condition is wide and includes rare manifestations such as maturation arrest of the myeloid lineage, a normocellular bone marrow, myelokathexis, lymphopaenia, thymic hypoplasia, inflammatory bowel disease, primary pulmonary hypertension, endocrine abnormalities, growth retardation, minor facial dysmorphism, skeletal and integument anomalies amongst others. Dursun syndrome is part of this extended spectrum. G6PC3 deficiency can also result in isolated non-syndromic severe neutropenia. G6PC3 mutations in result in reduced enzyme activity, endoplasmic reticulum stress response, increased rates of apoptosis of affected cells and dysfunction of neutrophil activity.In this review we demonstrate that loss of function in missense G6PC3 mutations likely results from decreased enzyme stability. The condition can be diagnosed by sequencing the G6PC3 gene. A number of G6PC3 founder mutations are known in various populations and a possible genotype-phenotype relationship also exists. G6PC3 deficiency should be considered as part of the differential diagnoses in any patient with unexplained congenital neutropenia.Treatment with G-CSF leads to improvement in neutrophil numbers, prevents infections and improves quality of life. Mildly affected patients can be managed with prophylactic antibiotics. Untreated G6PC3 deficiency can be fatal. Echocardiogram, renal and pelvic ultrasound scans should be performed in all cases of suspected or confirmed G6PC3 deficiency. Routine assessment should include biochemical profile, growth profile and monitoring for development of varicose veins or venous ulcers.
Project description:The first white Doberman pinscher (WDP) dog was registered by the American Kennel Club in 1976. The novelty of the white coat color resulted in extensive line breeding of this dog and her offspring. The WDP phenotype closely resembles human oculocutaneous albinism (OCA) and clinicians noticed a seemingly high prevalence of pigmented masses on these dogs. This study had three specific aims: (1) produce a detailed description of the ocular phenotype of WDPs, (2) objectively determine if an increased prevalence of ocular and cutaneous melanocytic tumors was present in WDPs, and (3) determine if a genetic mutation in any of the genes known to cause human OCA is causal for the WDP phenotype. WDPs have a consistent ocular phenotype of photophobia, hypopigmented adnexal structures, blue irides with a tan periphery and hypopigmented retinal pigment epithelium and choroid. WDPs have a higher prevalence of cutaneous melanocytic neoplasms compared with control standard color Doberman pinschers (SDPs); cutaneous tumors were noted in 12/20 WDP (<5 years of age: 4/12; >5 years of age: 8/8) and 1/20 SDPs (p<0.00001). Using exclusion analysis, four OCA causative genes were investigated for their association with WDP phenotype; TYR, OCA2, TYRP1 and SLC45A2. SLC45A2 was found to be linked to the phenotype and gene sequencing revealed a 4,081 base pair deletion resulting in loss of the terminus of exon seven of SLC45A2 (chr4?77,062,968-77,067,051). This mutation is highly likely to be the cause of the WDP phenotype and is supported by a lack of detectable SLC45A2 transcript levels by reverse transcriptase PCR. The WDP provides a valuable model for studying OCA4 visual disturbances and melanocytic neoplasms in a large animal model.
Project description:Non-syndromic oculocutaneous albinism (nsOCA) is an inherited disorder of melanin biosynthesis with autosomal recessive mode of inheritance, presenting either hypopigmented or depigmented skin, hair, and eyes. It is genetically heterogeneous with seven loci (OCA1-OCA7) reported to date. In the present study, we have reported three consanguineous families (A, B, C) presenting identical nsOCA phenotypes. Sanger sequencing revealed a novel [NM_000372.5: c.826 T > C, p.(Cys276Arg)] and a recurrent variant [NM_000372.5: c.832C > T, p.(Arg278?)] in tyrosinase (TYR) in families A and B, respectively. Microsatellite marker-based homozygosity mapping linked family C to OCA4. Sequence analysis identified a novel insertion variant (NM_016180.5: c.1331_1332insA) in the SLC45A2. Further, in silico mutagenesis and dynamic simulation approaches revealed that a novel Cys276Arg variant abolished the cysteine bridge and might contribute toward decreased stability of the TYR protein. Our study expands the mutation spectrum of the TYR and SLC45A2 genes and emphasizes that molecular investigations are essential for accurate disease diagnosis.
Project description:Severe Congenital Neutropenia type 4 (SCN4, OMIM 612541) is a rare autosomal recessive disease due to mutations in the G6PC3 gene. The phenotype comprises neutropenia of variable severity and other anomalies including congenital heart defects, prominent superficial veins, uro-genital anomalies, facial dysmorphism, growth and developmental delay and intermittent thrombocytopenia. In some patients, SCN represents the only manifestation of the disease. Variable findings have been reported at bone marrow examination ranging from a maturation arrest at the myelocyte/promyelocyte stage (either in a hypocellular or hypercellular context) to myelokathexis. Here we report two patients harbouring two novel mutations in the G6PC3 gene, including the first Italian patient even described. Both the patients share profound neutropenia with severe infections early in life; in one case non-hematopoietic stigmata of the syndrome, including evident facial dysmorphism and vascular anomalies, appeared gradually over time, prominently in the second decade. Therefore, G6PC3 defects should be considered in any case of congenital, unexplained neutropenia regardless of the clinical phenotype. Both patients are on G-CSF treatment with no evidence of malignant evolution. Even if G6PC3 deficiency seems not to have a propensity towards malignancy, a careful evaluation is warranted.
Project description:BACKGROUND:The main features of severe congenital neutropenia are the onset of severe bacterial infections early in life, a paucity of mature neutrophils, and an increased risk of leukemia. In many patients, the genetic causes of severe congenital neutropenia are unknown. METHODS:We performed genomewide genotyping and linkage analysis on two consanguineous pedigrees with a total of five children affected with severe congenital neutropenia. Candidate genes from the linkage interval were sequenced. Functional assays and reconstitution experiments were carried out. RESULTS:All index patients were susceptible to bacterial infections and had very few mature neutrophils in the bone marrow; structural heart defects, urogenital abnormalities, and venous angiectasia on the trunk and extremities were additional features. Linkage analysis of the two index families yielded a combined multipoint lod score of 5.74 on a linkage interval on chromosome 17q21. Sequencing of G6PC3, the candidate gene encoding glucose-6-phosphatase, catalytic subunit 3, revealed a homozygous missense mutation in exon 6 that abolished the enzymatic activity of glucose-6-phosphatase in all affected children in the two families. The patients' neutrophils and fibroblasts had increased susceptibility to apoptosis. The myeloid cells showed evidence of increased endoplasmic reticulum stress and increased activity of glycogen synthase kinase 3beta (GSK-3beta). We identified seven additional, unrelated patients who had severe congenital neutropenia with syndromic features and distinct biallelic mutations in G6PC3. CONCLUSIONS:Defective function of glucose-6-phosphatase, catalytic subunit 3, underlies a severe congenital neutropenia syndrome associated with cardiac and urogenital malformations.