The Y chromosome pool of Jews as part of the genetic landscape of the Middle East.

A sample of 526 Y chromosomes representing six Middle Eastern populations (Ashkenazi, Sephardic, and Kurdish Jews from Israel; Muslim Kurds; Muslim Arabs from Israel and the Palestinian Authority Area; and Bedouin from the Negev) was analyzed for 13 binary polymorphisms and six microsatellite loci. The investigation of the genetic relationship among three Jewish communities revealed that Kurdish and Sephardic Jews were indistinguishable from one another, whereas both differed slightly, yet significantly, from Ashkenazi Jews. The differences among Ashkenazim may be a result of low-level gene flow from European populations and/or genetic drift during isolation. Admixture between Kurdish Jews and their former Muslim host population in Kurdistan appeared to be negligible. In comparison with data available from other relevant populations in the region, Jews were found to be more closely related to groups in the north of the Fertile Crescent (Kurds, Turks, and Armenians) than to their Arab neighbors. The two haplogroups Eu 9 and Eu 10 constitute a major part of the Y chromosome pool in the analyzed sample. Our data suggest that Eu 9 originated in the northern part, and Eu 10 in the southern part of the Fertile Crescent. Genetic dating yielded estimates of the expansion of both haplogroups that cover the Neolithic period in the region. Palestinian Arabs and Bedouin differed from the other Middle Eastern populations studied here, mainly in specific high-frequency Eu 10 haplotypes not found in the non-Arab groups. These chromosomes might have been introduced through migrations from the Arabian Peninsula during the last two millennia. The present study contributes to the elucidation of the complex demographic history that shaped the present-day genetic landscape in the region.

Haplogroup-specific deviation from the stepwise mutation model at the microsatellite loci DYS388 and DYS392.

Deviation from the stepwise mutation model (SMM) at specific human microsatellite loci has implications for population genetic and forensic investigations. In the present study, data on six Y chromosome-specific microsatellites were pooled for 455 paternally unrelated males from six Middle Eastern populations. All chromosomes were assigned to three haplogroups defined by six binary polymorphisms. Two of the microsatellite loci tested, DYS388 and DYS392, displayed marked haplogroup-specific differences in their allele variability. A bimodal distribution of short and long alleles was observed for DYS388 in haplogroup 1 and for DYS392 in haplogroups 1 and 2. Further investigation showed that the short/long alleles segregated almost completely between genealogically distinct haplogroups defined by additional binary markers. Thus, these two loci have a discriminatory power similar to a binary polymorphism. DYS388 was characterised by an extremely low mutation rate in haplogroups 2 and 3, as was DYS392 in haplogroup 3. Sequence analysis of the repeat regions at the two loci revealed no irregularities, indicating that the triplet expansion in these loci is not controlled by sequence variation at the repeat level. A high frequency of long DYS388 alleles has, so far, been found only in populations originating in the Middle East, suggesting that this microsatellite is useful as a region-specific marker.

Diversity of alpha-globin mutations and clinical presentation of alpha-thalassemia in Israel.

Alpha-thalassemia is among the world's most common single gene disorders, caused primarily by gene deletions. In Israel, where alpha(o)-trait thalassemia is uncommon, it is of particular importance because of its phenotypic interactions with beta-thalassemia in hetero- and homozygotes. In a study of 232 individuals referred for molecular evaluation of anemia, 303 chromosomes carried alpha-globin gene abnormalities; 6 gene rearrangements and 11 point mutations were identified. This unexpected heterogeneity is in part due to the many ethnic subgroups represented by these patients. Our findings include nine unique Israeli alleles, 3 of which are described here for the first time. An equal number of point mutations was found in the alpha2-globin gene as compared to alpha1. A threonine deletion in codon 39 of the alpha1-globin gene, found frequently in Arabs, is unique to Israel and probably represents one of several indigenous alleles. Among Arabs, point mutations were more frequent than large deletions. Surprisingly, in Ashkenazi Jews, who resided for many centuries in a nonmalarial environment, a single alpha-globin gene deletion -alpha(3.7) was found in many cases. The clinical presentation of individuals carrying two or more alpha-globin lesions was highly variable. In general, the severity correlated inversely with the number of functional alpha-globin genes. In some cases, impairment of two alpha-globin genes by point mutations led to a thalassemia-intermedia-like picture which could be misdiagnosed as beta-thalassemia. We conclude that alpha-thalassemia is phenotypically and genotypically more heterogeneous than previously recognized. DNA analysis is invaluable as it provides a specific diagnosis and enables reliable genetic counseling.

Molecular analysis of beta-thalassemia in Vietnam.

The molecular basis of the thalassemias has been studied in many of the world's populations. Here we report the results of the first screening for mutations in Vietnam. Twenty-three unrelated patients, of which 17 have Hb E/beta-thalassemia, were diagnosed and beta-globin mutations were detected in all 46 chromosomes. Four previously reported South Asian mutations were found. The most common mutations were the nonsense in codon 17 (A-->T) and the frameshift at codons 41/42 (-TTCT) (30 and 22%, respectively). The rare frameshift mutation at codon 95 (+A) was present in 9% of the 46 chromosomes studied, suggesting that it is indigenous to Vietnam. These results will serve as an initial database for DNA-based prenatal diagnosis of thalassemia in Vietnam.

From a dry bone to a genetic portrait: a case study of sickle cell anemia.

The potential and reliability of DNA analysis for the identification of human remains are demonstrated by the study of a recent bone sample, which represented a documented case of sickle cell anemia. beta-globin gene sequences obtained from the specimen revealed homozygosity for the sickle cell mutation, proving the authenticity of the retrieved residual DNA. Further investigation of mitochondrial and Y chromosome DNA polymorphic markers indicated that this sample came from a male of maternal West African (possibly Yoruban) and paternal Bantu lineages. The medical record, which became available after the DNA analyses had been completed, revealed that it belonged to a Jamaican black male. These findings are consistent with this individual being a descendent of Africans brought to Jamaica during the trans-Atlantic slave trade. This study exemplifies how a "reverse population genetics" approach can be applied to reconstruct a genetic profile from a bone specimen of an unknown individual.

High-resolution Y chromosome haplotypes of Israeli and Palestinian Arabs reveal geographic substructure and substantial overlap with haplotypes of Jews.

High-resolution Y chromosome haplotype analysis was performed in 143 paternally unrelated Israeli and Palestinian Moslem Arabs (I&P Arabs) by screening for 11 binary polymorphisms and six microsatellite loci. Two frequent haplotypes were found among the 83 detected: the modal haplotype of the I&P Arabs (approximately 14%) was spread throughout the region, while its one-step microsatellite neighbor, the modal haplotype of the Galilee sample (approximately 8%), was mainly restricted to the north. Geographic substructuring within the Arabs was observed in the highlands of Samaria and Judea. Y chromosome variation in the I&P Arabs was compared to that of Ashkenazi and Sephardic Jews, and to that of North Welsh individuals. At the haplogroup level, defined by the binary polymorphisms only, the Y chromosome distribution in Arabs and Jews was similar but not identical. At the haplotype level, determined by both binary and microsatellite markers, a more detailed pattern was observed. Single-step microsatellite networks of Arab and Jewish haplotypes revealed a common pool for a large portion of Y chromosomes, suggesting a relatively recent common ancestry. The two modal haplotypes in the I&P Arabs were closely related to the most frequent haplotype of Jews (the Cohen modal haplotype). However, the I&P Arab clade that includes the two Arab modal haplotypes (and makes up 32% of Arab chromosomes) is found at only very low frequency among Jews, reflecting divergence and/or admixture from other populations.

Cost-benefit analysis of a national thalassaemia prevention programme in Israel.

OBJECTIVE: In Israel (population 5.7 million) there are around 200 known living subjects with thalassaemia major, of whom around 80% are from the northern district. This study aims at examining the costs and benefits of a national screening programme to prevent thalassaemia in Israel. MEASUREMENTS AND MAIN RESULTS: The lifetime healthcare costs of caring for a person born with thalassaemia major are $284,154. The costs of the home infusion service (33.1%) actually exceed the costs of the chelating agent itself (22.1%). The remaining 44.8% of costs are due to stay in hospital, operations, outpatient visits, laboratory tests, therapists, etc. Lost earnings and premature mortality costs account for a further $51,843 and $141,944 respectively for each case. A national screening programme would cost $900,197 and prevent around 13.4 homozygotes being born, at a cost of $67,369 for each birth prevented. The benefit-cost ratio of the programme to the health services is 4.22:1, which increases to 6.01:1 when a societal perspective is taken. However, around 13.0 homozygote births are still expected to occur, the majority owing to lack of compliance of patients at various stages in the screening process. The addition of a national health education programme for the higher risk non-Jewish population either nationally or in selected regions will incur extra costs, which may be covered by increased benefits as a result of better compliance with the screening programme. CONCLUSION: Israel should start to provide a nationwide thalassaemia screening programme as the monetary benefits to society (and even to the health services alone) will exceed the screening programmes costs.

Rapid detection of the common Mediterranean alpha-globin deletions/rearrangements using PCR.

The most frequent molecular lesions causing alpha-thalassemia are deletions of one or more alpha-globin genes. Detection of these deletions generally requires genomic Southern analysis, which is cumbersome and time consuming. We have designed new sets of primers for PCR identification of the common Mediterranean alpha-globin gene rearrangements, including the -alpha3.7 deletion and the alphaalphaalpha(anti3.7) triplication, the -alpha4.2 deletion, and the --Med allele. We have established reaction conditions that provide easily interpretable, unambiguous diagnoses. Some of the PCR reactions are multiplex, simultaneously identifying several genotypes, thus reducing the time and cost of screening and prenatal testing. The use of these methods should facilitate carrier screening and identification of couples at risk for alpha-thalassemia.

A novel mechanism generating short deletion/insertions following slippage is suggested by a mutation in the human alpha2-globin gene.

A novel mechanism generating short deletion/insertions is described based on a mutation in the human alpha2-globin gene. A deletion of 9 bp (codons 39-41) is replaced by an eight nucleotide insertion, duplicating the adjacent downstream sequence. We propose that the mutation arose by slipped strand mispairing (SSM), creating a single-stranded loop, followed by DNA elongation, strand breathing and the formation of a mismatch bubble. An extensive literature search has revealed six additional deletion/insertion mutations in humans in which the inserted nucleotides come from the same DNA strand. Our model explains all six mutations, suggesting that rearrangement of a mismatch loop or bubble during DNA replication may be not uncommon.

Genetic analysis of beta-thalassemia intermedia in Israel: diversity of mechanisms and unpredictability of phenotype.

Molecular analysis was performed on 95 Israeli patients with thalassemia intermedia, representing 60 families of Arab (Moslem and Christian), Jewish, Druze, and Samaritan origin. There was a wide range of phenotypic severity, with baseline hemoglobin levels ranging from 5.5 to 10.7. Eighteen thalassemia mutations were found (29 genotypes), which were subdivided into groups, according to the severity of mutations. A consistently mild phenotype (10 families) was caused by compound heterozygosity for a silent mutation, such as -101 C-T or by coexistence of triplicated alpha-globin genes with thalassemia trait. In 39 thalassemia intermedia families, the genotype which was found was one which led to severe thalassemia intermedia, or, in other families, was associated with thalassemia major. Elevated hemoglobin F ameliorated the disease in some patients with a severe genotype. We did not find a beneficial effect of concurrent alpha-thalassemia in any of the families studied. In 11 families, only one beta-thalassemia allele was identified. One was a dominant thalassemia intermedia allele. Three additional families with heterozygous beta-thalassemia had excess alpha-globin genes (5 or 6 total). In 7 of these heterozygotes, no explanation was found for the thalassemia intermedia phenotype. Our results suggest a substantial influence of as yet unknown genetic modifiers. These findings have important implications for prenatal diagnosis and for the genetic counseling of families with thalassemia intermedia.

Second transplantation using allogeneic peripheral blood stem cells in a beta-thalassaemia major patient featuring stable mixed chimaerism.

Allogeneic bone marrow transplantation (BMT) for beta-thalassaemia major carries the risks of disease recurrence due to residual thalassaemic stem cells or true immune-mediated rejection. We report a thalassaemic patient who displayed stable mixed chimaerism with only 5% donor-derived cells for about 5 years after BMT. Displacement of host cells was accomplished by ambulatory non-myeloablative conditioning and allogeneic G-CSF mobilized peripheral blood stem cell transplantation from the same donor, resulting in full reconstitution. Patients featuring stable mixed chimaerism after BMT may benefit from allogeneic cell therapy with immunocompetent lymphocytes and stem cells, whilst avoiding supralethal conditioning.

Familial pheochromocytoma associated with a novel mutation in the von Hippel-Lindau gene.

We report a three generation, 25 member kindred with familial pheochromocytoma. Seven subjects of generations I and II had pheochromocytoma, in five of the seven, the tumors were bilateral, and in two of the seven, the tumors were both adrenal and extraadrenal. One patient also had a carotid body chemodectoma, and one patient had a malignant adrenal tumor and abdominal paraganglioma. In the patient with the chemodectoma, a cerebellar hemangioblastoma became manifest 25 yr after his initial diagnosis with pheochromocytoma, leading only then to a clinical diagnosis of von Hippel-Lindau disease (VHL). A mutational analysis of the VHL gene revealed a novel nucleotide 709 G-->T transversion present in all affected subjects and in four presymptomatic children. In familial pheochromocytoma the diagnosis of VHL should be considered, even when the formal criteria for diagnosis of the syndrome are lacking.

Sex identification of archaeological human remains based on amplification of the X and Y amelogenin alleles.

Sex identification of archaeological human remains is essential for the exploration of gender differences in past populations. Traditional morphometric analyses fail to identify the gender of incomplete skeletal remains and that of immature individuals. In the present work, we have established a sensitive and reliable method, based on amplification of the single-copy amelogenin-encoding gene (AMG). The Y allele carries a small deletion in the first intron, facilitating the design of distinct X- and Y-specific polymerase chain reactions. Amplification with three primers, two of which are allele-specific, allows unambiguous identification of both X and Y chromosome signals in a single reaction, providing an internal control. For added confidence, the reaction may be performed in separate tubes for each allele. Using this method, the sex was determined from the skeletal remains of 18 individuals, including young children, out of 22 examined from periods ranging from 200 to around 8000 years ago. The state of skeletal preservation ranged from poor to good. Cortical and cranial bones, as well as teeth, were found to provide sufficiently preserved DNA. The success of retrieval of amplifiable DNA was not related either to the period or to the burial site. On the other hand, the method of DNA purification was critical. In our hands, direct DNA purification by Chelex from minute samples of bone/tooth powder gave the best results. This study demonstrates the applicability of the method for gender determination in skeletal remains from different periods.

Analysis of beta-globin mutations shows stable mixed chimerism in patients with thalassemia after bone marrow transplantation.

Beta-thalassemia major (TM) is caused by any of approximately 150 mutations within the beta-globin gene. To establish the degree of chimerism after bone marrow transplantation (BMT), we have performed molecular analysis of beta-globin mutations in 14 patients with TM over a period of 10 years. All patients underwent T cell-depleted allogeneic BMT from HLA-identical related donors, using either in vitro T-cell depletion with CAMPATH 1M and complement or in vivo depletion using CAMPATH 1G in the bone marrow collection bag. To date, at different time periods after BMT, seven patients have some degree of chimerism; six of these patients, all blood transfusion-independent, have donor cells in the range of 70% to 95%, with stable mixed chimerism (MC). The seventh patient has less than 10% donor cells with, surprisingly, only minimal transfusion requirements. The detection of beta-globin gene point mutation, as used here, is a highly specific and sensitive marker for engraftment and MC in patients with thalassemia. In light of its specificity, the method is applicable in all cases of TM, as it is independent of sex and other non-globin-related DNA markers. The high incidence of MC found in our patients may be a consequence of the pre-BMT T-cell depletion. Because MC was associated with transfusion independence, complete eradication of residual host cells for effective treatment of TM and possibly other genetic diseases may prove not to be essential.

Sequence analysis reveals a beta-thalassaemia mutation in the DNA of skeletal remains from the archaeological site of Akhziv, Israel.

beta-Thalassaemia is manifested by severe anaemia and extensive bone pathology. Similar pathology may also result from other forms of anaemia. To clarify the precise cause, we performed DNA analyses on archaeological remains of a child with severe bone pathology. We found homozygosity for frameshift in codon 8 of beta-globin, causing a beta-null phenotype. Paradoxically, the child died when eight years old, whereas such patients are transfusion dependent from early infancy. An infrequent polymorphic marker in the child's DNA, and information from present-day patients, indicated that amelioration of the clinical condition was due to elevated fetal haemoglobin production. Thus this analysis provided not only precise diagnosis of a genetic disease but also allowed clarification of the molecular mechanism underlying the clinical presentation.

Hemoglobin switching in humans is accompanied by changes in the ratio of the transcription factors, GATA-1 and SP1.

BACKGROUND: Understanding the mechanism of developmental regulation of hemoglobin switching has scientific as well as clinical relevance because of the influence of fetal hemoglobin (HbF) production in adulthood on the clinical manifestation of thalassemia and sickle cell anemia. We have previously found that the normal developmental patterns of globin gene expression are recapitulated in an experimental system of primary cultures that support differentiation of erythroid progenitors. We further found that high activities of the transcriptional activators, GATA-1 and SP1, are associated with normal adult erythroid differentiation. MATERIALS AND METHODS: In the present work, we have studied, the activities of GATA-1 and SP1 during differentiation of cultured erythroid progenitors derived from cord blood and from fetal livers, as well as from beta zero-thalassemia patients. RESULTS: The results showed high GATA-1 binding activity and very low SP1 activity in the fetal liver cultures. This pattern was in contrast to cultures derived from normal adult peripheral blood, in which both GATA-1 and SP1 activities were high. Cord blood cultures showed an additive combination of "adult" and "fetal" patterns. The progenitors derived from a beta zero-thalassemia patient with high HbF production showed "fetal" pattern. On the other hand, in cultures of 2 beta zero-thalassemia patients without high HbF, "adult" pattern was observed. CONCLUSIONS: In the present work, we show that human fetal and adult erythroid progenitors are distinct in their transcription factors, and that the commitment to fetal or adult program occurs at a very early differentiation stage. Our studies also demonstrate that under anemic stress, recruitment of fetal progenitors may occur in adulthood.