Flanking Haplotypes Research Articles

Tracing the Distribution of European Lactase Persistence Genotypes Along the Americas.

In adulthood, the ability to digest lactose, the main sugar present in milk of mammals, is a phenotype (lactase persistence) observed in historically herder populations, mainly Northern Europeans, Eastern Africans, and Middle Eastern nomads. As the –13910∗T allele in the MCM6 gene is the most well-characterized allele responsible for the lactase persistence phenotype, the –13910C > T (rs4988235) polymorphism is commonly evaluated in lactase persistence studies. Lactase non-persistent adults may develop symptoms of lactose intolerance when consuming dairy products. In the Americas, there is no evidence of the consumption of these products until the arrival of Europeans. However, several American countries’ dietary guidelines recommend consuming dairy for adequate human nutrition and health promotion. Considering the extensive use of dairy and the complex ancestry of Pan-American admixed populations, we studied the distribution of –13910C > T lactase persistence genotypes and its flanking haplotypes of European origin in 7,428 individuals from several Pan-American admixed populations. We found that the –13910∗T allele frequency in Pan-American admixed populations is directly correlated with allele frequency of the European sources. Moreover, we did not observe any overrepresentation of European haplotypes in the –13910C > T flanking region, suggesting no selective pressure after admixture in the Americas. Finally, considering the dominant effect of the –13910∗T allele, our results indicate that Pan-American admixed populations are likely to have higher frequency of lactose intolerance, suggesting that general dietary guidelines deserve further evaluation across the continent.

Flanking Variation Influences Rates of Stutter in Simple Repeats.

It has been posited that the longest uninterrupted stretch (LUS) of tandem repeats, as defined by the number of exactly matching repeating motif units, is a better predictor of rates of stutter than the parental allele length (PAL). While there are cases where this hypothesis is likely correct, such as the 9.3 allele in the TH01 locus, there can be situations where it may not apply as well. For example, the PAL may capture flanking indel variations while remaining insensitive to polymorphisms in the repeat, and these haplotypic changes may impact the stutter rate. To address this, rates of stutter were contrasted against the LUS as well as the PAL on different flanking haplotypic backgrounds. This study shows that rates of stutter can vary substantially depending on the flanking haplotype, and while there are cases where the LUS is a better predictor of stutter than the PAL, examples to the contrary are apparent in commonly assayed forensic markers. Further, flanking variation that is 7 bp from the repeat region can impact rates of stutter. These findings suggest that non-proximal effects, such as DNA secondary structure, may be impacting the rates of stutter in common forensic short tandem repeat markers.

A method for allocating low-coverage sequencing resources by targeting haplotypes rather than individuals

BackgroundThis paper describes a heuristic method for allocating low-coverage sequencing resources by targeting haplotypes rather than individuals. Low-coverage sequencing assembles high-coverage sequence information for every individual by accumulating data from the genome segments that they share with many other individuals into consensus haplotypes. Deriving the consensus haplotypes accurately is critical for achieving a high phasing and imputation accuracy. In order to enable accurate phasing and imputation of sequence information for the whole population, we allocate the available sequencing resources among individuals with existing phased genomic data by targeting the sequencing coverage of their haplotypes.ResultsOur method, called AlphaSeqOpt, prioritizes haplotypes using a score function that is based on the frequency of the haplotypes in the sequencing set relative to the target coverage. AlphaSeqOpt has two steps: (1) selection of an initial set of individuals by iteratively choosing the individuals that have the maximum score conditional on the current set, and (2) refinement of the set through several rounds of exchanges of individuals. AlphaSeqOpt is very effective for distributing a fixed amount of sequencing resources evenly across haplotypes, which results in a reduction of the proportion of haplotypes that are sequenced below the target coverage. AlphaSeqOpt can provide a greater proportion of haplotypes sequenced at the target coverage by sequencing less individuals, as compared with other methods that use a score function based on haplotype frequencies in the population. A refinement of the initially selected set can provide a larger more diverse set with more unique individuals, which is beneficial in the context of low-coverage sequencing. We extend the method with an approach for filtering rare haplotypes based on their flanking haplotypes, so that only those that are likely to derive from a recombination event are targeted.ConclusionsWe present a method for allocating sequencing resources so that a greater proportion of haplotypes are sequenced at a coverage that is sufficiently high for population-based imputation with low-coverage sequencing. The haplotype score function, the refinement step, and the new approach for filtering rare haplotypes make AlphaSeqOpt more effective for that purpose than previously reported methods for reducing sequencing redundancy.

Contraction of fully expanded FMR1 alleles to the normal range: predisposing haplotype or rare events?

Fragile X syndrome (FXS), the most common cause of inherited intellectual disability, is due to the expansion over 200 CGGs and methylation of this polymorphic region, in the 5'-UTR (untranslated region) of FMR1 (Xq27.3). We have identified four FXS mosaic males: M1-(CGG)35/(CGG)>200; M2-(CGG)26/(CGG)>200; M3-(CGG)39/(CGG)>200; and M4-(CGG)18/(CGG)125/(CGG)>200. After genotyping their respective mothers, we suggested that normal alleles of these patients resulted from post-zygotic contractions of full expansions. The detection of these four rare independent cases led us to hypothesize the existence of a large-contraction predisposing haplotype in our population. Next, we questioned whether other normal pure CGGs would have arisen through similar contractions from fully expanded alleles. To address these questions, we identified stable single-nucleotide polymorphism (SNP) lineages and related short tandem repeat (STR) haplotypes (DXS998-DXS548-FRAXAC1-FRAXAC2) of the four mosaics, 123 unrelated FXS patients and 212 controls. An extended flanking haplotype (34-44-38-336) shared by mosaics from lineage A suggested a risk lineage-specific haplotype more prone to large contractions. Other normal pure FMR1 alleles from this SNP background also shared phylogenetically close STR haplotypes, although a single (CGG)exp>(CGG)24 contraction or the loss of AGG interruptions may explain their origin. In both scenarios, multistep FMR1 mutations involving the gain or loss of several CGGs seem to underlie the evolution of the repeat.

Distribution of fragile X mental retardation 1 CGG repeat and flanking haplotypes in a large Chinese population.

Fragile X syndrome is mainly caused by a CGG repeat expansion within the 5′ UTR of the fragile X mental retardation 1 (FMR1) gene. Previous analyses of the FMR1 CGG repeat patterns and flanking haplotypes in Caucasians and African Americans have identified several factors that may influence repeat instability. However, the CGG repeat patterns and distribution for FRAXAC2 have not yet been investigated in mainland Chinese. We surveyed the CGG repeat lengths in 1113 Han Chinese (534 males and 579 females), and the CGG repeat patterns of 534 males were determined by sequence analysis. We also explored the flanking haplotypes (DXS548-FRAXAC1-FRAXAC2) in 566 unaffected and 28 unrelated fragile X Chinese males. The most frequent alleles for DXS548 and FRAXAC1 were identical between our Chinese population and other Asian populations. We identified several low-abundance alleles for DXS548 and FRAXAC1 not found in previous studies in mainland Chinese and Taiwanese cohorts. The most frequent allele was (CGG)29 followed by (CGG)30, and the most frequent patterns were 9 + 9 + 9, 10 + 9 + 9, and 9 + 9 + 6 + 9, similar to those in Singaporeans. We identified only one premutation female carrier with 89 CGG repeats in the 1113 Han Chinese. A few associations between the CGG repeat patterns and flanking haplotypes were determined in this study. In general, the Chinese population had a smaller number of alleles and lower expected heterozygosity for all three STR markers and FRAXA locus when compared with Caucasians and African Americans. We identified a novel haplotype 7-3-5 + that is significantly associated with the full mutation.

Genotyping HapSTR loci: phase determination from direct sequencing of PCR products

HapSTRs combine information from a microsatellite (or simple tandem repeat, STR) with one or more single nucleotide polymorphisms in the DNA sequence immediately flanking the STR. These loci may offer increased power for the estimation of demographic parameters, but also present some challenges for data collection and analysis. We describe a process for inferring HapSTR alleles, including the flanking haplotypes, STR alleles and their phase relative to each other, directly from DNA sequence electropherograms of PCR products from heterozygous individuals. Our approach eliminates the need for more costly and time-consuming processes, such as cloning or acrylamide gel electrophoresis to separate alleles prior to sequencing.

Non HLA genetic markers association with type-1 diabetes mellitus

The currently available data identified IDDM1 and IDDM2 as 2 susceptibility loci for type 1 diabetes (T1D). The major histocompatibility complex (MHC)/HLA region referred to as IDDM1 contains several 100 genes known to have a great influence on T1D risk. Within IDDM2, a minisatellite variable number of tandem repeats (VNTR) locus in the insulin gene (INS) promoter region is likely to represent the etiologic polymorphism. The aim of the present work was to study the association between genotypes and susceptibility to T1D among Egyptian diabetic children and their family members. Twenty-five nuclear Egyptian families with 27 children having T1D, aged 3–14 years, their non-diabetic 44 sibs, aged 3–15 years and their parents were included in our study. All studied children were subjected to: detailed history and family pedigree. Thorough clinical examination and anthropometric measurements. Laboratory work up of diabetes including random blood sugar (RBS) and HbA 1C. Molecular genetics of INS was studied in four steps; nucleic acid purification, amplification, sequencing and haplotyping using flanking single nucleotide polymorphisms (SNPs) as surrogate markers for minisatellite alleles identification. Analysis of variant repeat distribution among Egyptian families combined with flanking haplotypes revealed that all our diabetic children had class I alleles of INS; 9 had class IC+, 9 had class ID+ and 9 had class ID−, while all non-diabetic family members had class III alleles of INS. Therefore the three class I alleles were considered to be equally predisposing to T1D, while class III alleles are dominantly protective. There was significant positive correlations between body mass index (BMI) and both HbA 1C and AST liver enzyme among diabetic children with class IC+ but not other alleles; indicating that they need close monitoring of their diabetic control and liver functions beside following specific dietary regimens. It can be concluded that all class I alleles (IC+, ID+ and ID−) are equally important susceptibility factors for T1D among Egyptian children, while class III alleles (IIIA and IIIB) are dominantly protective. It is concluded also that our diabetic children with class IC+ are an especially endangered subgroup of diabetics. Genotyping for INS-VNTR alleles is recommended for diabetic children as an important step of diagnostic and follow up regimens and for their non-diabetic family members for family counseling and early identification of potential diabetics. Further studies of INS-VNTR alleles and HLA haplotypes all over Egypt are recommended to define the Egyptian susceptibility loci for T1D and their relations to the clinical and laboratory findings as an important national programs.

Association of TWIST1 gene polymorphisms with bone mineral density in postmenopausal women

A novel polymorphism (+1871A>G) in the 3' flanking region and haplotypes were significantly associated with reduced osteoporosis risk and enhanced bone mineral density (BMD). These results suggest that TWIST1 may be a useful genetic marker for osteoporosis. Our results provide preliminary evidence supporting an association of TWIST1 with osteoporosis in postmenopausal women. TWIST1, a basic helix-loop-helix (bHLH) transcription factor, has been implicated in cell lineage determination and differentiation. To address the genetic variations in the TWIST1 gene associated with osteoporosis, we investigated the potential involvement of three TWIST1 single-nucleotide polymorphisms (SNPs) in osteoporosis in 729 postmenopausal women. BMD was measured using dual-energy X-ray absorptiometry. A novel polymorphism in the 3' flanking region (+1871A>G) was significantly associated with osteoporosis risk (p = 0.007-0.008) and also in multiple comparison (p = 0.02). Consistent with these results, haplotype analysis showed that Block1_ht2 had protective effects in the dominant and additive model (p = 0.006-0.007). Specifically, the +1871A>G polymorphism was overdominantly associated with higher BMD values of the femoral neck (p = 0.039). These results suggest that TWIST1 may be a useful genetic marker for osteoporosis and may have a role on bone metabolism in humans. Our results provide preliminary evidence supporting an association of TWIST1 with osteoporosis in postmenopausal women.

263 PATERNAL ISODISOMY FOR CHROMOSOME 2 AS CAUSE OF BILE SALT EXPORT PUMP (BSEP) DEFICIENCY

Background: Bile salt export pump (BSEP) deficiency, a defect in a canalicular bile acid excretion, falls into the progressive familial intrahepatic cholestasis. It is an autosomal recessive disorder with several mutations being reported in the ABCB11 gene on chromosome 2q24−31. We describe a case of BSEP deficiency with a new mutation, where the patient received both mutated alleles from the father, through the rare phenomenon of uniparental disomy (UPD). Methods: A 4-year-old infant, underwent to orthotopic liver transplantation (OLT) at the age of 9 months because of persistent cholestasis and liver failure. Immunohistochemistry was performed with BSEP antibody. Genomic DNA was extracted from the baby and his parents. Molecular studies of ABCB11 were performed using denaturing high-pressure liquid chromatography (DHPLC) and direct sequencing. Gene copy number determination, segregation of the mutation and reconstruction of the flanking haplotype employed quantitative real time PCR (qPCR), single nucleotide polymorphisms (SNPs) genotyping, and polymorphic microsatellites. Results: Liver histology showed cirrhosis. Immunohistochemistal stain using anti-BSEP antibodies revealed wholly absent marking at canalicular margin of hepatocytes, consistent with the diagnosis of BSEP deficiency. DHPLC and sequencing analysis showed a novel, homozygous c.2620C>T transition in exon 20 of the ABCB11 gene. The mutation replaces arginine for cysteine at residue 833 (p. R833C). This change was absent in a large panel of normal chromosomes, and it was heterozygous in peripheral blood from the father, but not in several tissues obtained from the child’s mother. qPCR analyses did not disclose abnormal gene copy number in both the patient and his mother ruling out a microdeletion of ABCB11. Segregation analysis of seven intragenic SNPs and six flanking microsatellite markers showed data suggestive of UID of the region on chromosome 2q24−31 inherited from the father. Conclusion: In severe forms, infants with BSEP deficiency developed cholestasis and fibrosis, and end-stage liver disease requires OLT. Molecular studies are needed to offer measures as to avoid recurrence of the disease. The present case highlights the importance of full gene analyses to offer a more accurate genetic counselling.

FMR1 alleles in Tasmania: a screening study of the special educational needs population

The distribution of fragile X mental retardation-1 (FMR1) allele categories, classified by the number of CGG repeats, in the population of Tasmania was investigated in 1253 males with special educational needs (SEN). The frequencies of these FMR1 categories were compared with those seen in controls as represented by 578 consecutive male births. The initial screening was based on polymerase chain reaction analysis of dried blood spots. Inconclusive results were verified by Southern analysis of a venous blood sample. The frequencies of common FMR1 alleles in both samples, and of grey zone alleles in the controls, were similar to those in other Caucasian populations. Consistent with earlier reports, we found some (although insignificant) increase of grey zone alleles in SEN subjects compared with controls. The frequencies of predisposing flanking haplotypes among grey zone males FMR1 alleles were similar to those seen in other Caucasian SEN samples. Contrary to expectation, given the normal frequency of grey zone alleles, no premutation (PM) or full mutation (FM) allele was detected in either sample, with only 15 fragile X families diagnosed through routine clinical admissions registered in Tasmania up to 2002. An explanation of this discrepancy could be that the C19th founders of Tasmania carried few PM or FM alleles. The eight to ten generations since white settlement of Tasmania has been insufficient time for susceptible grey zone alleles to evolve into the larger expansions.

A mutation in Drosophila simulans that lengthens the circadian period of locomotor activity.

The length of the Thr-Gly repeat within the period gene of Drosophilids, coevolves with its immediate flanking region to maintain the temperature compensation of the fly circadian clock. In Drosophila simulans, balancing selection appears to maintain a polymorphism in this region, with three repeat lengths carrying 23, 24 or 25 Thr-Gly pairs, each in complete linkage disequilibrium with a distinctive flanking region amino acid moiety. We wondered whether separating a specific length repeat from its associated flanking haplotype might have functional implications for the circadian clock. We fortuitously discovered a population of flies collected in Kenya, in which a chimeric Thr-Gly haplotype was segregating that carried the (Thr-Gly)24 repeat, but the flanking region of a (Thr-Gly)23 allele. One of the five isofemale lines that carried this 'mutant' Thr-Gly sequence showed a dramatically long and temperature-sensitive free-running circadian period. This phenotype was mapped to the X chromosome, close to the D. simulans per gene, but there was also a significant effect of a modifying autosomal locus or loci. It seems remarkable that such a mutant phenotype should be discovered in a screen of chimeric Thr-Gly regions.

Influence of allele lineage on the role of the insulin minisatellite in susceptibility to type 1 diabetes

The insulin minisatellite or variable number of tandem repeats locus (INS VNTR) is the best candidate for the type 1 diabetes mellitus (T1DM) susceptibility locus IDDM2. Small class I alleles associate with predisposition to T1DM, whereas large class III alleles associate with dominant protection. We have analysed variant repeat distribution within the minisatellite and combined this with flanking haplotypes to define five new ancestral allele lineages. Class III alleles divide into two highly diverged lineages, IIIA and IIIB, which correspond perfectly to the previously defined Protective (PH) and Very Protective (VPH) haplotypes, respectively. Class I alleles are divided into three newly defined lineages, IC+, ID+ and ID-, by a combination of variant repeat distributions and flanking haplotypes. All class I alleles are equally predisposing to T1DM except for ID- alleles which are protective when transmitted from ID-/III heterozygous fathers. Similar results have been previously reported for alleles of 42 repeats in length (allele 814) which represent a subset of the ID- lineage. Division of class ID- alleles into those of 42 repeats and those of other sizes suggested that this protective effect was a feature of all ID- alleles, irrespective of size. ID- alleles are only clearly distinguished from all other alleles by an MSPI(-) variant within IGF2 downstream of the minisatellite, suggesting that the apparent role of the minisatellite in susceptibility to T1DM may be modified by neighbouring haplotype and therefore that IDDM2 could have a multi-locus aetiology.

De novo mutations and allelic diversity at minisatellite locus D7S22 investigated by allele-specific four-state MVR-PCR analysis

We have studied the allelic diversity and de novo mutations at the hypervariable minisatellite locus D7S22. A four-state minisatellite variant repeat unit mapping by PCR (MVR-PCR) method was developed for this purpose, and a substitution polymorphism close to the repeat array was used to design allele-specific flanking primers to study individual haplotypes in genomic DNA. A total of 150 alleles from different allele size groups and flanking haplotypes were mapped. On average, MVR-codes extending 65 repeats (2.4 kb) into the repeat array were obtained. The interspersion patterns of variant repeats were highly polymorphic. However, subgroups of alleles close in size and with identical flanking haplotype revealed common MVR-code characteristics indicating a close evolutionary relationship. Unlike the situation in many other hypervariable minisatellites, no polarized variability was revealed at this minisatellite locus. Fifty four small families with D7S22 de novo mutations were analysed by MVR-PCR. The sites where the length change occurred were revealed in 22 cases, while in 32 cases the mutation obviously occurred further into the repeat array. In agreement with a non-polar distribution of the allelic variation, there was no evidence for a hypermutable hot spot for mutation within the repeat array. Comparison of MVR-codes in the mutant and progenitor in gain mutations indicated that at least one, possibly four cases, reflected inter-allelic events. Together with evidence from DNA sequencing of alleles of <2 kb, this indicates that as many as half of the gain mutations might be inter-allelic events in D7S22. Based on these results, different factors which might affect the mutation rate are discussed.

Patients with familial hypertrophic cardiomyopathy caused by a Phe110Ile missense mutation in the cardiac troponin T gene have variable cardiac morphologies and a favorable prognosis.

Mutations that cause familial hypertrophic cardiomyopathy have been identified in several genes that encode contractile proteins. Patients with mutations in the cardiac troponin T (cTnT) gene have particularly poor prognosis but only mild hypertrophy. To date, no benign mutation in the cTnT gene has been reported. The clinical characteristics and prognosis of patients with the Phe110Ile mutation in the cTnT gene is unclear because few affected individuals have been identified. Forty-six probands with familial hypertrophic cardiomyopathy were screened for mutations in the cTnT gene. The Phe110Ile missense mutation was found in 6 probands. Individuals in the 6 families were analyzed genetically and clinically. Haplotype analysis was performed with markers encompassing the cTnT gene. Left ventricular hypertrophy was classified as type I, II, III, or IV according to the criteria of Maron et al. The Phe110Ile mutation in the cTnT gene was identified in 16 individuals. Two of the 6 families shared the same flanking haplotype, and 4 were different from each other. Affected individuals exhibited different cardiac morphologies: 4 had type II, 6 had type III, and 3 had type IV hypertrophy with apical involvement. Three individuals with the disease-causing mutation did not fulfill clinical criteria for the disease. The product-limit survival curve analysis demonstrated a favorable prognosis. Multiple independent mutations of residue 340 in the cTnT gene have been described, suggesting that this may be a "hot spot" for such events. The Phe110Ile substitution causes hypertrophic cardiomyopathy with variable cardiac morphologies and a favorable prognosis.

Mechanisms underlying telomere repeat turnover, revealed by hypervariable variant repeat distribution patterns in the human Xp/Yp telomere.

Sequences immediately adjacent to the human Xp/Yp telomere exhibit a high frequency of base substitutional polymorphisms, together with almost complete linkage disequilibrium, to create only a few diverged haplotypes. This sequence divergence has been used to develop a PCR-based system for mapping the distribution of the telomere (TTAGGG) and variant repeats (TGAGGG and TCAGGG) at the proximal end of the telomere repeat array. The distribution of these repeats is extremely variable. Almost all Xp/Yp telomeres are different, indicating a high mutation rate. Some telomere maps associated with the same flanking haplotype show similarities, identifying subsets of telomeres that share a recent common ancestry. Mechanisms underlying the rapid turnover of repeats at the proximal end of the Xp/Yp telomere include intra-allelic processes, such as slippage during replication. Inter-allelic exchanges may occur occasionally, but telomerase activity probably plays only a minor role in the germline turnover of proximally located telomere and variant repeats.

Insert size and flanking haplotype in fragile X and normal populations: possible multiple origins for the fragile X mutation.

A number of recent studies have found non-random association between the fragile X mutation and genotypes for the closest-linked flanking markers, suggesting either a limited number of 'founder' mutations or, alternatively, a predisposing haplotype for the fragile X expansions. Using three microsatellite markers within 150 kb of FRAXA, we have compared haplotypes in a series of fragile X males and in a control population and find a markedly different distribution in the two samples, with apparently greater haplotype diversity in the fragile X sample. In the control sample, various non-random associations of CGG repeat numbers with flanking haplotypes were recorded which provide a clue to the likely origins of the fragile X mutation, suggesting more than one mechanism for the initial expansion event.