A common variant association study in ethnic Saudi Arabs reveals novel susceptibility loci for hypertriglyceridemia.

Hypertriglyceridemia (hTG) is a lipid disorder, resulting from an elevation in triglyceride levels, with a strong genetic component. It constitutes a significant risk factor for coronary artery disease (CAD), a leading cause of death worldwide. In this study, we performed a common variant association study for hTG in ethnic Saudi Arabs. We genotyped 5501 individuals in a two-phase experiment using Affymetrix Axiom® Genome-Wide CEU 1 Array (Affymetrix, Santa Cruz, CA) that contains a total of 587,352 single nucleotide polymorphisms (SNPs). The lead variant was the rs1558861 [1.99 (1.73-2.30); p = 7.37 × 10-22 ], residing on chromosome (chr) 11 at the apolipoprotein A-I/A-5 (APOA1/APOA5) locus. The rs780094 [1.34 (1.21-1.49); p = 8.57 × 10-8 ] on chr 2 at the glucokinase regulatory protein (GCKR) locus was similarly significantly associated, while the rs10911205 [1.29 (1.16-1.44); p = 3.52 × 10-6 ] on chr1 at the laminin subunit gamma-1 (LAMC1) locus showed suggestive association with disease. Furthermore, the rs17145738 [0.68 (0.60-0.77); p = 6.69 × 10-9 ] on chr7 at the carbohydrate-responsive element-binding protein-encoding (MLXIPL) gene locus displayed significant protective characteristics, while another variant rs6982502 [0.76 (0.68-0.84); p = 5.31 × 10-7 ] on chr8 showed similar but weaker properties. These findings were replicated in 317 cases vs 1415 controls from the same ethnic Arab population. Our study identified several variants across the human genome that are associated with hTG in ethnic Arabs.

A common variant association study reveals novel susceptibility loci for low HDL-cholesterol levels in ethnic Arabs.

The genetic susceptibility to acquiring low high density lipoprotein-cholesterol (LHDLC) levels is not completely elucidated yet. In this study, we performed a common variant association study for harboring this trait in ethnic Arabs. We employed the Affymetrix high-density Axiom Genome-Wide ASI Array (Asian population) providing a coverage of 598,000 single nucleotide variations (SNPs) to genotype 5495 individuals in a two-phase study involving discovery and validation sets of experiments. The rs1800775 [1.31 (1.22-1.42); p = 3.41E-12] in the CETP gene and rs359027 [1.26 (1.16-1.36); p = 2.55E-08] in the LMCD1 gene were significantly associated with LHDLC levels. Furthermore, rs3104435 [1.26 (1.15-1.38); p = 1.19E-06] at the MATN1 locus, rs9835344 [1.16 (1.08-1.26); p = 8.75E-06] in the CNTN6 gene, rs1559997 [1.3 (1.14-1.47); p = 9.48E-06] in the SDS gene and rs1670273 [1.2 (1.1-1.31); p = 4.81E-06] in the DMN/SYNM gene exhibited suggestive association with the disorder. Seven other variants including rs1147169 in the PLCL1 gene, rs10248618 in the DNAH11, rs476155 in the GLIS3, rs7024300 in the ABCA1, intergenic rs10836699, rs11603691 in P2RX3 and rs750134 in CORO1C gene exhibited borderline protective properties. Validation and joint meta-analysis resulted in rs1800775, rs3104435 and rs359027 retaining their predisposing properties, while rs10836699 and rs11603691 showed protective properties. Our data show several predisposing variants across the genome for LHDLC levels in ethnic Arabs.

Mutation of the matrix metalloproteinase 2 gene (MMP2) causes a multicentric osteolysis and arthritis syndrome.

The inherited osteolyses or 'vanishing bone' syndromes are a group of rare disorders of unknown etiology characterized by destruction and resorption of affected bones. The multicentric osteolyses are notable for interphalangeal joint erosions that mimic severe juvenile rheumatoid arthritis (OMIMs 166300, 259600, 259610 and 277950). We recently described an autosomal recessive form of multicentric osteolysis with carpal and tarsal resorption, crippling arthritic changes, marked osteoporosis, palmar and plantar subcutaneous nodules and distinctive facies in a number of consanguineous Saudi Arabian families. We localized the disease gene to 16q12-21 by using members of these families for a genome-wide search for homozygous-by-descent microsatellite markers. Haplotype analysis narrowed the critical region to a 1.2-cM region that spans the gene encoding MMP-2 (gelatinase A, collagenase type IV; (ref. 3). We detected no MMP2 enzymatic activity in the serum or fibroblasts of affected family members. We identified two family-specific homoallelic MMP2 mutations: R101H and Y244X. The nonsense mutation effects a deletion of the substrate-binding and catalytic sites and the fibronectin type II-like and hemopexin/TIMP2 binding domains. Based on molecular modeling, the missense mutation disrupts hydrogen bond formation within the highly conserved prodomain adjacent to the catalytic zinc ion.

Zellweger syndrome caused by PEX13 deficiency: report of two novel mutations.

Peroxisomal biogenesis disorders represent a group of genetically heterogeneous conditions that have in common failure of proper peroxisomal assembly. Clinically, they are characterized by a spectrum of dysmorphia, neurological, liver, and other organ involvement. To date, mutations in 13 PEX genes encoding peroxins have been identified in patients with peroxisomal biogenesis disorders. Mutations in PEX13, which encodes peroxisomal membrane protein PEX13, are among the least common causes of peroxisomal biogenesis disorders with only three mutations reported so far. Here, we report on two infants whose clinical and biochemical profile was consistent with classical Zellweger syndrome and whose complementation analysis assigned them both to group H of peroxisomal biogenesis disorders. We show that they harbor two novel mutations in PEX13. One patient had a genomic rearrangement resulting in a 147 kb deletion that spans the whole of PEX13, while the other had an out-of-frame deletion of 14 bp. This represents the first report of a PEX13 deletion and suggests that further work is needed to examine the frequency of PEX13 mutations among Arab patients with peroxisomal biogenesis disorders.

The T/G 13915 variant upstream of the lactase gene (LCT) is the founder allele of lactase persistence in an urban Saudi population.

BACKGROUND: The prevalence of lactase persistence is high in Saudi Arabia. OBJECTIVE: To identify a DNA variant for the lactase persistence/non-persistence trait in adult Arabs in Saudi Arabia. METHODS: We sequenced DNA from 432 anonymous neonatal blood donors from five different regions of Saudi Arabia to cover the 400 bp region surrounding the previously identified lactase persistence/non-persistence variant C/T-13910 residing in intron 13 of the MCM6 gene. RESULTS: Two anonymous blood donors carried the C/T-13910 genotype. One variant, T/G -13915, residing 5 bp upstream of the C/T-13910 variant, was present in 332 of 432 (76.9%) of the neonatal samples, compatible with previous prevalence figures of lactase persistence in urban Saudi populations. Determination of disaccharidase activities in 25 intestinal biopsy samples showed a highly significant correlation between lactase activity and the T/G-13915 genotypes (p<0.001; Fisher exact test) as well as between the L:S ratio and the aforementioned genotypes (p<0.001; Fisher exact test). CONCLUSION: The T/G-13915 variant is the founder mutation of lactase persistence in an urban Saudi population. The results obtained here have implications for genetic testing of adult-type hypolactasia and to analysis of human evolution, the origin of cattle domestication and migrations of the populations in the Arabian peninsula.

TSH overcomes Braf(V600E)-induced senescence to promote tumor progression via downregulation of p53 expression in papillary thyroid cancer.

The BRAF(V600E) mutation is found in approximately 40% of papillary thyroid cancers (PTC). Mice with thyroid-specific expression of Braf(V600E) (TPO-Braf(V600E)) develop PTC rapidly with high levels of serum thyroid-stimulating hormone (TSH). It is unclear to what extent the elevated TSH contributes to tumor progression. To investigate the progression of Braf(V600E)-induced PTC (BVE-PTC) under normal TSH, we transplanted BVE-PTC tumors subcutaneously into nude and TPO-Braf(WT) mice. Regression of the transplanted tumors was observed in both nude and TPO-Braf(WT) mice. They were surrounded by heavy lymphocyte infiltration and oncogene-induced senescence (OIS) was demonstrated by strong ß-gal staining and absence of Ki-67 expression. In contrast, BVE-PTC transplants continued to grow when transplanted into TPO-Braf(V600E) mice. The expression of Trp53 was increased in tumor transplants undergoing OIS. Trp53 inactivation reversed OIS and enabled tumor transplants to grow in nude mice with characteristic cell morphology of anaplastic thyroid cancer (ATC). PTC-to-ATC transformation was also observed in primary BVE-PTC tumors. ATC cells derived from Trp53 knockout tumors had increased PI3K/AKT signaling and became resistant to Braf(V600E) inhibitor PLX4720, which could be overcome by combined treatment of PI3K inhibitor LY294002 and PLX4720. In conclusion, BVE-PTC progression could be contained via p53-dependent OIS and TSH is a major disruptor of this balance. Simultaneous targeting of both MAPK and PI3K/AKT pathways offer a better therapeutic outcome against ATC. The current study reinforces the importance of rigorous control of serum TSH in PTC patients.

Streptavidin-biotin immunotoxins: a new approach to purging bone marrow.

Immunotoxins have been used both experimentally and clinically to purge bone marrow of tumor cells or T cells before transplantation. We describe the synthesis of a streptavidin-biotin-toxin conjugate using whole ricin. Streptavidin-biotin-ricin (SA-BR) conjugates were synthesized by biotinylation of whole ricin, which was then complexed with streptavidin. Hybrid molecules consisting of a single biotinylated ricin moiety linked to a streptavidin molecule were separated by gel filtration. This SA-BR conjugate was used in an indirect cytotoxicity assay. The assay involved sensitizing of target cells with biotinylated monoclonal antibody (B-MCAB) followed by treatment with dilutions of SA-BR conjugate. The assay demonstrated a specific antibody-directed cytotoxicity. The strength of this SA-BR system is that a single conjugate was able to be used in conjunction with a library of B-MCABs to selectively target phenotypically different cell types. The application of the SA-BR conjugate is thus only restricted by the availability of B-MCABs specific for the desired target cells. The high affinity of avidin for biotin (Kd approximately 10(-15)) and the ability of a single conjugate to target phenotypically different cells through utilization of a library of B-MCABs gives SA-BR conjugates great potential in the selective targeting of individual cell types.

The synthesis of a peanut agglutinin-ricin A chain conjugate: potential as an in vitro purging agent for autologous bone marrow in multiple myeloma.

The lectin peanut agglutinin (PNA) and CD19 monoclonal antibody have been covalently linked to magnetic beads and utilized in an in vitro purging system for autologous bone marrow in multiple myeloma (MM). An alternative to mechanical purging involves the use of immunotoxins to provide specifically targeted cellular toxicity; however, no studies to date have examined the utility of a lectin-ricin A chain (RCA) combination as a purging agent in MM. Initially, we studied the internalization of PNA by target cells (Raji) using flow cytometry. The surface fluorescence intensity of PNA-treated Raji cells was reduced upon incubation at 37 degrees C, and subsequent studies with fixed cells detected the endocytosed PNA. Complete internalization occurred within 120 minutes, indicating the potential of PNA as a purging agent. We manufactured a novel PNA-RCA conjugate and demonstrated its strong and specific binding to PNA reactive cell targets. Subsequent experiments assessed the toxicity of the conjugate to Raji cells and normal bone marrow progenitor cells. 3H-leucine uptake assays showed that PNA-RCA was capable of reducing protein synthesis in Raji cells and that the toxic effects were specific. In addition, at concentrations of conjugate achieving greater than 99% selective cytotoxicity for Raji cells, adequate CFU-GM were preserved in normal marrow. These studies suggest that PNA-RCA may be of value as an in vitro purging agent for MM.

A novel form of autosomal recessive pure hereditary spastic paraplegia maps to chromosome 13q14.

BACKGROUND: Hereditary spastic paraplegia (HSP) is a clinically and genetically heterogeneous disorder characterized by a progressive weakening and spasticity of the lower limbs. HSP is classified according to the presence or absence of accompanying neurologic problems and by the mode of inheritance. Currently, 17 loci have been linked to the various forms of HSP. OBJECTIVE: To determine the chromosomal location of a gene causing pure autosomal recessive spastic paraplegia. METHODS: Genotyping using fluorescently labeled microsatellite markers was performed on three affected individuals and three unaffected individuals from a family displaying pure autosomal recessive HSP (ARHSP) and sensorineural deafness. All family members were then included in the analysis to narrow the genetic interval. Candidate genes were screened for the presence of mutations by heteroduplex analysis. RESULTS: The paraplegic trait linked to a 1.8-Mb region of chromosome 13q14 flanked by the FLJ11712 gene and the microsatellite marker D13S270. The deafness did not link to this region and did not cosegregate with the paraplegic trait. CONCLUSION: The HSP that this family had represents a novel genetic form of pure ARHSP as no other form of HSP (autosomal dominant or recessive) has been linked to chromosome 13.

PER-117: a new human ALL cell line with an immature thymic phenotype.

A new cell line, PER-117, was established from bone marrow cells of an eighteen months old boy with an acute lymphoblastic leukaemia (ALL). The leukaemic origin of cell line PER-117 is indicated by its cytochemical, immunological and cytogenetic similarity to the patient's fresh leukaemic cells. PER-117 carries a marker chromosome which was identified as a translocation between chromosomes 1 and 11. The surface marker analysis revealed that the phenotype of PER-117 is RFB-1+, RFT-1+ (CD5), 3A1+ (CD7), OKT 9+, OKT 10+ and HLA-DR-. Thus, this cell line appears to represent a prothymocyte or stage I thymocyte and preliminary data suggest that it can be induced in vitro to further differentiate.

Sanjad-Sakati and autosomal recessive Kenny-Caffey syndromes are allelic: evidence for an ancestral founder mutation and locus refinement.

The Sanjad-Sakati syndrome (SSS; MIM241410), an autosomal recessive trait characterized by congenital hypoparathyroidism, growth and mental retardation, seizures, and a characteristic physiognomy, was recently linked to chromosome area 1q42-q43. SSS resembles the autosomal recessive form of Kenny-Caffey syndrome (KCS; MIM244460), with similar manifestations but lacking osteosclerosis. Since KCS was recently linked to the region 1q42-q43, the possibility that this disorder is allelic with SSS was considered. Eight Sanjad-Sakati families from Saudi Arabia were genotyped with polymorphic short tandem repeat markers from the SSS/KCS critical region. A maximum multipoint LOD score of 14.32 was obtained at marker D1S2649, confirming linkage of SSS to the same region as autosomal recessive KCS. Haplotype analysis refined the critical region to 2.6 cM and identified a rare haplotype present in all the SSS disease alleles, indicative of a common founder. In addition to the assignment of the Saudi SSS and Kuwaiti KCS syndromes to overlapping genetic intervals, comparison of the haplotypes unexpectedly demonstrated that the diseases shared an identical haplotype. This finding, combined with the clinical similarity between the two syndromes, suggests that the two conditions are not only allelic but are also caused by the same ancestral mutation.

Prompt haemopoietic reconstitution following hyperthermia purged autologous marrow and peripheral blood stem cell transplantation in acute myeloid leukaemia.

We report a case of acute myeloid leukaemia in a 35-year-old woman where marrow and chemotherapy-mobilized peripheral blood stem cells (PBSC) were obtained in first complete remission and heat treated at 42 degrees C for 1 h to minimize the likelihood of leukaemic relapse. Transplantation of marrow and PBSC was undertaken following busulphan/cyclophosphamide conditioning 9 months after diagnosis in CR1. Hyperthermic purging did not impair the rate of engraftment and the patient was independent of blood product support by day 21. Studies on this patient's committed normal granulocyte-macrophage progenitor cells (CFU-GM) on samples from marrow and peripheral blood following treatment at 42 degrees C for 1 h, showed minimal and acceptable loss of activity comparable with the loss seen in other marrow progenitor activity treated in a similar fashion in our laboratory. We conclude that hyperthermia-purged PBSC can be used to hasten recovery in autologous haemopoietic progenitor transplantation without compromising rate of engraftment. This is part of an ongoing pilot study to evaluate the role of hyperthermic purging to reduce the risk of leukaemic relapse.

Hyperthermia potentiates the activity of immunotoxin conjugates against common acute lymphoblastic leukaemia cells in vitro.

We studied the in-vitro cytotoxic effect of hyperthermia at 42 degrees C, both alone and in combination with ricin-linked immunotoxins, reactive with the common acute lymphoblastic leukaemia cell lines Reh and KM-3. Assessment of cytotoxicity was by incorporation of 3H-leucine and limiting dilutions analysis. The effect of immunotoxins alone and in combination with hyperthermia on normal human marrow progenitor cells was assessed by conventional colony forming units-granulocyte macrophage (CFU-GM) assay. We found that incubation of either of the cell lines with a mixture of the two immunotoxins, RPH-7-ricin and PHM-6-ricin, at 42 degrees C for one hour (h) potentiated the cytotoxic activity of the immunotoxins at 37 degrees C. At a concentration of 10(-8) mol/L, a 2.2-log kill was seen with KM-3 leukaemic cells at 37 degrees C and a 3.3-log kill at 42 degrees C, an increase of approximately 10 fold in cytotoxic activity. Survival of CFU-GM following treatment at 42 degrees C for one h with a similar concentration of immunotoxins was 26.2% (+/- 13.7%) (equivalent to 0.6 log kill) and 76.0% (+/- 1.83%) (0.1 log kill) when normal marrow was incubated with immunotoxins at 37 degrees C. This suggests relative sparing of normal marrow cells compared with the leukaemic cells tested as indicated by the 2.1-log kill difference (approximately 100 fold) between normal and leukaemic cells at 37 degrees C and the 2.7-log kill (approximately 500-fold) difference at 42 degrees C. We conclude that hyperthermia may have a role in addition to immunotoxins in the purging of marrow ex vivo to remove leukaemic cells.

A unique T-cell monoclonal antibody with potential uses in autologous bone marrow transplantation.

A monoclonal antibody reactive with an antigen expressed by a T-cell subset has been produced. The antibody designated RPH-1 reacts with a subset of normal T-cells and thymocytes. The antibody was strongly reactive with blast cells from 3 patients with T-acute lymphoblastic leukemia (T-ALL) and also reacted with cells of 2 patients with non-T-ALL. The antibody did not react with leukemic cells from patients with acute myeloid leukemia (AML) or B-chronic lymphocytic leukemia (B-CLL)). RPH-1 reacted with 2 T-cell lines, no B-cell lines and no myeloid cell lines. Absence of toxicity to granulocyte macrophage colony forming units (CFU-GM) by RPH-1 in the presence of complement was demonstrated, and in view of strong reactivity with certain leukemic cells, potential application of RPH-1 as an immunological means of selectively removing tumour cells is indicated.

Monoclonal antibody-ricin conjugate cytotoxic to cells expressing the common acute lymphoblastic leukemia antigen (CALLA).

The monoclonal antibody PHM-6, which is specific for the common acute lymphoblastic leukemia antigen (CALLA), was conjugated to the plant toxin ricin. Binding of the PHM-6-ricin conjugate to cells via the ricin molecule was blocked by the presence of 100 mM lactose. The IC50 (concentration resulting in 50% inhibition) of the PHM-6-ricin conjugate for the CALLA-positive KM-3 cell line was 280-fold greater than for bone marrow stem cells, indicating the potential of this conjugate for immunological purging of autologous remission marrow.

Angiotensin-converting enzyme polymorphism and the risk of coronary heart disease in the Saudi male population.

OBJECTIVE: To determine the relevance of angiotensin I-converting enzyme (ACE) gene polymorphism for coronary artery disease (CAD) in the Saudi population. METHODS AND RESULTS: DNA of 84 male Saudi patients with established CAD, 36 male controls who underwent angiography, and 327 healthy Saudi male blood donors was amplified by polymerase chain reaction, using oligonucleotide primers flanking the insertion (I)/deletion (D) sites in the polymorphic region of intron 16 of the ACE gene. Polymerase chain reaction amplification resulted in 490-bp (II), 190-bp (DD), or 490- and 190-bp (ID) fragments. The genotype II distribution was 16.7% in the control group, 7.3% in the blood donor group, and 7.2% in the patients with CAD, and the distribution for DD was 58.3%, 47.1%, and 41.0%, respectively. Notably, 61.9% (P <.0001) of CAD patients presented with angina on admission, and 52.4% had diabetes mellitus. CONCLUSIONS: The results show no increased risk of CAD in association with either the II or DD genotypes in the Saudi population. However, further investigation of genotype II as a predictor for atherosclerosis rather than increased risk of coronary heart disease may be indicated.

Relevance of apolipoprotein E polymorphism for coronary artery disease in the Saudi population.

BACKGROUND: The apolipoprotein E alleles epsilon2 and epsilon4 have been reported as independent risk factors for coronary artery disease (CAD) and as predictors for the development of atherosclerosis. METHODS AND RESULTS: We determined by polymerase chain reaction the distribution of apolipoprotein E polymorphism in 320 Saudi blood donors (BD), 96 CAD patients, and 40 control subjects who had undergone angiography. Compared to controls, only epsilon4 was elevated in CAD patients. More than 61% (P <.0001) of the patients had angina, and 52.1% (P <.05) were diabetic; both of these factors were strongly associated with the presence of allele epsilon2. The epsilon2 allele was also associated with hypertension, elevated serum triglycerides, and total cholesterol. On the other hand, the allele epsilon4 appeared to be associated with increased risk of CAD and was also associated with hypertension, 3-vessel disease, and restenosis. CONCLUSIONS: Accordingly, epsilon4 may be associated with increased risk of CAD, whereas epsilon2 appears to be a predictor of several risk factors for atherosclerosis.