Determination of the breakpoint and molecular diagnosis of a common alpha-thalassaemia-1 deletion in the Indian population

The previously described South African type alpha-thalassaemia-1 mutation was identified in Indian HbH patients using a polymerase chain reaction (PCR) strategy. A multiplex PCR assay was devised to detect heterozygotes and homozygotes. This alpha-thalassaemia-1 mutation was found to be the commonest determinant causing HbH disease in this population. In one family this mutation was found in combination with a novel splice donor mutation alpha2 IVS I-1 (G-->A). Characterization of the breakpoint junction sequence revealed, in addition to a 23 kb deletion, that there was an addition of approximately 160 bp bridging the breakpoints. Similar to other deletions in the alpha-globin gene cluster, there is an Alu repeat-mediated mechanism for the origin of the deletion.     

A novel beta0-thalassemia mutation at codon 55 (-A) and a rare 17 bp deletion at codons 126-131 in the Indian population

A new mutation at codon 55 (-A) and a rare mutation, a 17 bp deletion at codons 126-131, that gives rise to beta0-thalassemia, were found in the Indian population by means of direct sequencing of two polymerase chain reaction products generated from a 2.3 kb DNA fragment containing the whole beta-globin gene. Each polymerase chain reaction product was sequenced on both strands in a mutation-loading format, showing all nucleotide substitutions or deletions/insertions, including mutations and polymorphisms, in the product. The entire protocol requires four sequencing reactions/gel loadings after two successive polymerase chain reactions, which simplifies the mutation search process and reduces the reading error rate.     

UGT1A1 polymorphism outweighs the modest effect of deletional (-3.7 kb) alpha-thalassemia on cholelithogenesis in sickle cell anemia

Enhanced erythrocyte destruction in sickle cell anemia results in chronic hyperbilirubinemia. Only a subset of patients develop cholelithiasis. UGT1A1 promoter polymorphism is associated both with unconjugated bilirubin level and elevated risk for cholelithiasis in such subset. Here, we investigated the role of alpha-thalassemia, yet another genetic factor that modulates hemolysis, in conferring protection from cholelithiasis. We show that, although alpha-thalassemia is associated with modest reduction in hemolysis and unconjugated bilirubin level, UGT1A1 polymorphism outweighs its effect on cholethiogenesis in sickle cell anemia patients.     

A novel deletion causing (epsilon gamma delta beta) degrees thalassaemia in a Chilean family

We describe a novel deletion causing (epsilongammadeltabeta) degrees thalassaemia segregating in three generations of a Chilean family of Spanish descent. Heterozygotes for the deletion were all affected by neonatal haemolytic anaemia. The deletion of 152,569 bp extends from 77 kb upstream of the epsilon gene to 31 kb downstream of the beta gene, and includes the entire beta-globin gene cluster and two upstream olfactory receptor genes. Comparison of the sequences of the deletion junction with those of the flanking normal DNA suggests that the deletion results from a non-homologous recombination event. The insertion of 16 'orphan' nucleotides in the deletion junction creates a perfect inverted repeat of 12 nucleotides, forming a 12-bp stem with a four-nucleotide loop that could have contributed to the illegitimate recombination. The 3' breakpoint is located within an L1 family repeat that contains a perfect 160-bp palindrome, and is in close proximity to the 3' breakpoints of five other deletions in the beta cluster - Indian (HPFH-3), Italian (HPFH-4) and Vietnamese GgammaAgamma (deltabeta) degrees HPFH, German and Belgian Ggamma (Alphagammadeltabeta) degrees thalassaemia.     

A novel Alu-mediated 61-kb deletion of the von Willebrand factor (VWF) gene whose breakpoints co-locate with putative matrix attachment regions

BACKGROUND AND OBJECTIVES: von Willebrand disease (VWD) type 3 is characterized by extremely low levels of von Willebrand factor (VWF) in plasma. To date, only 11 examples of gross deletions have been reported for the VWF gene and the underlying mutational mechanisms remain unclear. A Chinese patient with type 3 VWD was studied to elucidate the underlying mechanism of mutagenesis. DESIGN AND METHODS: PCR was designed to amplify across the putatively deleted region of genomic DNA from the patient and his parents to locate the deletion breakpoints. In silico analysis was then performed to search for repetitive sequence elements, recombination-associated motifs, and scaffold/matrix attachment regions (S/MARs). RESULTS: A novel homozygous gross deletion of the VWF gene, which removes some 61044 bp DNA between introns 5 and 16, was identified in the patient. The deletion junctions were flanked by highly homologous Alu repeats in inverted orientation. These repeats could thus have potentiated the formation of a stem-loop structure thereby bringing the breakpoints into close proximity. A number of recombination-associated motifs were noted in close proximity to both deletion breakpoints. Both the 5' and 3' breakpoints were located in, or near, regions with a high propensity to form S/MARs. INTERPRETATION AND CONCLUSIONS: We report the first example of an Alu-mediated VWF gross gene deletion. Since a number of recombination-associated motifs were also identified in the vicinity of the breakpoints, it may be that multiple sequence elements have acted in concert to give rise to this deletion event.     

Angiotensin-converting enzyme gene insertion/deletion polymorphism and essential hypertension in the Chinese population: a meta-analysis including 21,058 participants

Background: The angiotensin‐converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism has been suggested to influence susceptibility to essential hypertension (EH), but the results of many individual studies are conflicting. Aim and methods: To explore the relationship between the ACE gene I/D polymorphism and EH in the Chinese population, 67 separated studies were analyzed in a meta‐analysis including 21 058 participants. The electronic databases PubMed, Embase, Web of Science, China Biological Medicine Database and China National Knowledge Infrastructure were searched. The pooled odds ratio (ORs) and its corresponding 95% confidence interval were calculated by the random effects model. Results: In this ACE I/D gene polymorphism meta‐analysis, the distribution of the D allele frequency was 0.45 for the EH group and 0.40 for the control group. The summary OR for the distribution frequency of D allele was 1.27 (5% confidence interval 1.17–1.37). The heterogeneity among the 67 studies was also significant (P < 0.00001, I²= 71.4%). There was a significant association between distribution frequency of the D allele and EH risk in Han, Kazakh, Tibetan, Zhuang and unclassified nationalities (P < 0.05). In contrast, in the national minorities, such as Mongolian, Uigur, Yi, Dongxiang, Yugu, Korean and Gamel, the association between distribution frequency of the D allele and EH risk was not significant (P > 0.05). Conclusions: In the whole Chinese population, the D allele was significantly linked with EH susceptibility. However, the relation between the I/D polymorphism and EH is still inconclusive in some national minorities and must await larger scale studies.     

A novel HBV recombinant (genotype I) similar to Vietnam/Laos in a primitive tribe in eastern India

Summary. Genotyping of 20 strains of Hepatitis B virus (HBV) from the Idu Mishmi primitive tribe of northeast India identified multiple genotypes and the presence of a unique cluster grouping with strains from Vietnam and Laos identified as novel recombinants/genotype I. Sequence analysis (similarity and bootscan plots) of three complete HBV genomes from the tribe provided evidence of recombination. Phylogenetic analyses supported recombination between genotypes A, G and C. The Pre‐S gene between nt 2943 and 397 was clearly of genotype A origin, whereas nt 397–1397 represented genotype G and nt 1397–2943 represented genotype C. Percentage divergence from genotypes B, D, E, F, G and H varied from 9.2 ± 0.45% to 13.8 ± 0.53%, whereas genotype A and C differed by 7.9 ± 0.42% and 7.4 ± 0.39% respectively. The identification of similar recombinant viruses in three countries, especially in a primitive tribe with no contact with the outside world suggests that these viruses do not represent recent recombination events, but circulation of closely related viruses highly divergent from known HBV genotypes and should be classified as members of genotype ‘I’.     

ABL oncogene amplification with p16(INK4a) gene deletion in precursor T-cell acute lymphoblastic leukemia/lymphoma: report of the first case

Gene amplification is a relatively rare event in hematologic malignancies. The ABL gene on chromosome band 9q34 is a proto-oncogene and is the well-known translocation partner of the BCR gene on 22q11 giving rise to t(9;22)(q34;q11), which is the hallmark of chronic myeloid leukemia and is the most common chromosomal abnormality in adult acute lymphoblastic leukemia (ALL). Amplification of ABL is an exceedingly rare event, with only less than 5 cases reported in the literature. The p16(INK4a) (or CDKN2A) gene on 9p21 is a tumor suppressor gene, and deletion thereof is recently recognized as one of the most common genetic abnormalities in ALL. The authors herein describe an 8-year-old male patient with precursor T-cell ALL harboring both ABL gene amplification and p16(INK4a) gene deletion. Fluorescence in situ hybridization (FISH) analysis using BCR/ABL probes revealed five or more ABL signals, indicating amplification in 51.5% of interphase nuclei. FISH using p16(INK4a) gene probes showed heterozygous p16(INK4a) deletion in 71.0%. On conventional cytogenetic analysis, however, only 10 metaphases were available, which showed the normal karyotype, 46,XY[10], serving no evidence for the findings on FISH. This is the first report of an ALL case with ABL amplification, and the authors speculate that both ABL proto-oncogene amplification and the p16(INK4a) tumor suppressor gene deletion have been implicated in leukemogenesis in the present case, although whether the ABL amplification truly contributes to the leukemogenesis or merely an epiphenomenon representing underlying genomic instability remains to be determined.     

Compound heterozygosity for a rare small deletion and a common point mutation in the beta‐globin gene: report of two Chinese families

Introduction: Most cases of β‐thalassemia are caused by point mutations in the β‐globin gene. Only a minority of β‐thalassemia mutations are small deletions in the exons of the β‐globin gene. Methods: Here, we report two cases of β‐thalassemia that were caused by compound heterozygosity for a rare small deletion and a common point mutation. Results: Patient A carried a rare 14‐bp deletion (CD89–93) mutation plus the common mutation ?28(A>G). Patient B carried a rare 13‐bp deletion (CD54–58) plus the common mutation IVS‐2‐654(C>T). Conclusion: Patient B is the second report of the CD54–58(?13 bp) deletion. However, our report differs from the previous report in two ways: we performed a family study based on multiple samples; and the carriers and patient showed an elevated level of HbF, which was not observed in the previous case.     

Genetic analysis in FXI deficiency: six novel mutations and the use of a polymerase chain reaction-based test to define a whole gene deletion

The genetic basis of factor XI (FXI) deficiency was investigated in 30 patients from 13 different families of non-Jewish origin. Twelve different mutations were detected (including six novel changes), seven missense mutations and three mutations leading to null alleles. Haplotype analysis suggested a large gene deletion in one family. We confirmed the presence of a recently reported Alu-mediated FXI gene deletion. An unrelated patient with severe deficiency was shown to be compound heterozygous for A412V and this whole gene deletion. We suggest that this recurrent gene deletion should be included in the genetic analysis of FXI deficiency.     

Coexistence of a novel beta-globin gene deletion (codons 81-87) with the codon 30 (G-->C) mutation in an Indian patient with beta0-thalassemia

We identified and characterized a novel beta0-thalassemia mutation due to the deletion of 22 bases from codons 81 through 87, found in a compound heterozygous state with codon 30 (G-->C) in a patient originating from West Bengal State, India. The deletion causes a shift in the reading frame of the coding sequence and creates a stop codon at position 81. Direct and inverted repeat sequences present in the deleted region might be involved in the origin of this mutation. The patient had moderate anemia and did not require blood transfusions (thalassemia intermedia).     

A novel gene STORP (STOmatin-Related Protein) is localized 2 kb upstream of the promyelocytic gene on chromosome 15q22

We generated a 100-kb map of the region 5' of the PML (promyelocytic leukemia) gene on human chromosome 15q22 and identified a new gene provisionally named STORP for stomatin-related protein. The STORP gene is positioned 2 kb upstream of the PML gene in a head-to-head configuration, and contains 7 exons spanning a genomic region of about 11 kb. There is an open reading frame of 398 amino acids, which would encode a protein of 45 kD. Northern blot analysis demonstrated that the STORP gene has a ubiquitous pattern of expression similar to that of the PML gene. Hybridization of STORP cDNA probe to genomic DNA from other species demonstrated that the STORP gene is conserved among mammalian vertebrates and that the physical linkage with PML is conserved in mice. Unlike PML, the STORP gene is not induced by interferon alpha (IFNalpha), and thus can be distinctly regulated from the PML gene. STORP is homologous to the EPB72 gene coding for the erythrocyte band 7 integral membrane protein or stomatin, which is deficient in a certain form of hereditary stomatocytosis. The function of STORP is unknown. Further study will focus on studying its potential role in red cell function and disorders.     

Isolated growth hormone (GH) deficiency type 1A associated with a double deletion in the human GH gene cluster

The gene deletions responsible for isolated GH deficiency type 1A were characterized by direct analysis of genomic DNA prepared from the leukocytes of two affected children. The probands had typical symptoms of severe isolated GH deficiency complicated by antibody development and growth arrest after human (h) GH treatment. DNA analysis using the restriction endonucleases Eco RI, Bam HI, and Hind III revealed that the restriction fragment containing the hGH-N gene was absent along with those bearing the human chorionic somatomammotropin (hCS)-A and -B and hGH-V sequences. A total of about 40 kilobases DNA were absent due to two separate deletions flanking the hCS-L gene. The two affected siblings are homozygous for this rearrangement of the hGH/hCS gene cluster, which could have been generated by homologous crossing over between two different chromosomes, one bearing one of the previously described deletions of the hGH-N gene, and one bearing a deletion of DNA containing the hCS-A, hCS-B, and hGH-V sequences. Alternatively, this abnormality could have been generated by a complex intrachromosomal rearrangement. The parents, who are consanguinous, have DNA restriction patterns consistent with heterozygosity for this double deletion. This type of deletional mutation is the first involving multiple deletion of the hGH and hCS gene cluster.     

Primary GH insensitivity '(Laron syndrome) caused by a novel 4 kb deletion encompassing exon 5 of the GH receptor gene: effect of intermittent long-term treatment with recombinant human IGF-I

OBJECTIVE: GH insensitivity syndrome (GHIS; Laron syndrome) is clinically characterized by severe postnatal growth failure and very low serum levels of IGF-I despite increased secretion of GH. This mainly autosomal recessive syndrome is clinically indistinguishable from isolated GH deficiency (IGHD). Fifty-one different mutations in the GH receptor (GHR) gene have been discovered, whereas only three deletions causing the disorder have been reported so far. In this report, we describe a consanguineous family from Sri Lanka with a novel deletion of 4097 bp in length encompassing exon 5. SUBJECTS AND METHODS: Parents of normal phenotype presented their second child (boy) to our clinic at the age of 7 months with severe growth retardation and the clinical features of IGHD (58 cm, -6.1 standard deviation score (SDS); 5.7 kg, -3.4 SDS). Assessment, however, revealed GHIS with absent GH-binding protein. Thereafter, the patient received intermittent recombinant human IGF-I (rhIGF-I; 80 microg/kg twice daily) treatment prepubertally for 5.5 years. Genomic DNA was extracted for genetic analysis and each exon was PCR amplified individually. Further, in order to amplify the GHR gene from exon 4 to 6, Expand Long Template PCR (Roche) was carried out. In addition, RNA isolation and RT-PCR were performed. RESULTS: Separate PCRs of each of the exons of the GHR gene revealed that exon 5 in the patient was missing. Thereafter, "Long PCR" from exons 4 to 6 revealed a 4097 bp deletion encompassing exon 5, in a homozygous state in the patient and in a heterozygous state in both parents. RT-PCR analysis revealed an exact absence of exon 5 resulting in a frameshift, leading to a stop codon in exon 6, which predicts a truncated, non-functional GHR protein. CONCLUSION: Fifty-one different mutations within the GHR gene causing GHIS have been reported so far. In contrast, only three deletions within the GHR gene are known. We describe a patient suffering from GHIS caused by a novel 4 kb deletion of the GHR gene encompassing exon 5 and, additionally, we focus on the effect of intermittent rhIGF-I treatment during prepuberty.