Large-scale mitochondrial DNA analysis in Southeast Asia reveals evolutionary effects of cultural isolation in the multi-ethnic population of Myanmar.

BACKGROUND: Myanmar is the largest country in mainland Southeast Asia with a population of 55 million people subdivided into more than 100 ethnic groups. Ruled by changing kingdoms and dynasties and lying on the trade route between India and China, Myanmar was influenced by numerous cultures. Since its independence from British occupation, tensions between the ruling Bamar and ethnic minorities increased. RESULTS: Our aim was to search for genetic footprints of Myanmar's geographic, historic and sociocultural characteristics and to contribute to the picture of human colonization by describing and dating of new mitochondrial DNA (mtDNA) haplogroups. Therefore, we sequenced the mtDNA control region of 327 unrelated donors and the complete mitochondrial genome of 44 selected individuals according to highest quality standards. CONCLUSION: Phylogenetic analyses of the entire mtDNA genomes uncovered eight new haplogroups and three unclassified basal M-lineages. The multi-ethnic population and the complex history of Myanmar were reflected in its mtDNA heterogeneity. Population genetic analyses of Burmese control region sequences combined with population data from neighboring countries revealed that the Myanmar haplogroup distribution showed a typical Southeast Asian pattern, but also Northeast Asian and Indian influences. The population structure of the extraordinarily diverse Bamar differed from that of the Karen people who displayed signs of genetic isolation. Migration analyses indicated a considerable genetic exchange with an overall positive migration balance from Myanmar to neighboring countries. Age estimates of the newly described haplogroups point to the existence of evolutionary windows where climatic and cultural changes gave rise to mitochondrial haplogroup diversification in Asia.

The K-Cl cotransporter KCC3 is mutant in a severe peripheral neuropathy associated with agenesis of the corpus callosum.

Peripheral neuropathy associated with agenesis of the corpus callosum (ACCPN) is a severe sensorimotor neuropathy associated with mental retardation, dysmorphic features and complete or partial agenesis of the corpus callosum. ACCPN is transmitted in an autosomal recessive fashion and is found at a high frequency in the province of Quebec, Canada. ACCPN has been previously mapped to chromosome 15q. The gene SLC12A6 (solute carrier family 12, member 6), which encodes the K+-Cl- transporter KCC3 and maps within the ACCPN candidate region, was screened for mutations in individuals with ACCPN. Four distinct protein-truncating mutations were found: two in the French Canadian population and two in non-French Canadian families. The functional consequence of the predominant French Canadian mutation (2436delG, Thr813fsX813) was examined by heterologous expression of wildtype and mutant KCC3 in Xenopus laevis oocytes; the truncated mutant is appropriately glycosylated and expressed at the cellular membrane, where it is non-functional. Mice generated with a targeted deletion of Slc12a6 have a locomotor deficit, peripheral neuropathy and a sensorimotor gating deficit, similar to the human disease. Our findings identify mutations in SLC12A6 as the genetic lesion underlying ACCPN and suggest a critical role for SLC12A6 in the development and maintenance of the nervous system.

Southeast Asian diversity: first insights into the complex mtDNA structure of Laos.

BACKGROUND: Vast migrations and subsequent assimilation processes have shaped the genetic composition of Southeast Asia, an area of close contact between several major ethnic groups. To better characterize the genetic variation of this region, we analyzed the entire mtDNA control region of 214 unrelated donors from Laos according to highest forensic quality standards. To detail the phylogeny, we inspected selected SNPs from the mtDNA coding region. For a posteriori data quality control, quasi-median network constructions and autosomal STR typing were performed. In order to describe the mtDNA setup of Laos more thoroughly, the data were subjected to population genetic comparisons with 16 East Asian groups. RESULTS: The Laos sample exhibited ample mtDNA diversity, reflecting the huge number of ethnic groups listed. We found several new, so far undescribed mtDNA lineages in this dataset and surrounding populations. The Laos population was characteristic in terms of haplotype composition and genetic structure, however, genetic comparisons with other Southeast Asian populations revealed limited, but significant genetic differentiation. Notable differences in the maternal relationship to the major indigenous Southeast Asian ethnolinguistic groups were detected. CONCLUSIONS: In this study, we portray the great mtDNA variety of Laos for the first time. Our findings will contribute to clarify the migration history of the region. They encourage setting up regional and subpopulation databases, especially for forensic applications. The Laotian sequences will be incorporated into the collaborative EMPOP mtDNA database http://www.empop.org upon publication and will be available as the first mtDNA reference data for this country.

An in-depth analysis of the mitochondrial phylogenetic landscape of Cambodia.

Cambodia harbours a variety of human aboriginal populations that have scarcely been studied in terms of genetic diversity of entire mitochondrial genomes. Here we present the matrilineal gene pool of 299 Cambodian refugees from three different ethnic groups (Cham, Khmer, and Khmer Loeu) deriving from 16 Cambodian districts. After establishing a DNA-saving high-throughput strategy for mitochondrial whole-genome Sanger sequencing, a HaploGrep based workflow was used for quality control, haplogroup classification and phylogenetic reconstruction. The application of diverse phylogenetic algorithms revealed an exciting picture of the genetic diversity of Cambodia, especially in relation to populations from Southeast Asia and from the whole world. A total of 224 unique haplotypes were identified, which were mostly classified under haplogroups B5a1, F1a1, or categorized as newly defined basal haplogroups or basal sub-branches of R, N and M clades. The presence of autochthonous maternal lineages could be confirmed as reported in previous studies. The exceptional homogeneity observed between and within the three investigated Cambodian ethnic groups indicates genetic isolation of the whole population. Between ethnicities, genetic barriers were not detected. The mtDNA data presented here increases the phylogenetic resolution in Cambodia significantly, thereby highlighting the need for an update of the current human mtDNA phylogeny.

Annexin A5 interacts with polycystin-1 and interferes with the polycystin-1 stimulated recruitment of E-cadherin into adherens junctions.

Polycystin-1 is the gene product of PKD1, the first gene identified to be causative for the condition of autosomal dominant polycystic kidney disease (ADPKD). Mutations in PKD1 are responsible for the majority of ADPKD cases worldwide. Polycystin-1 is a protein of the transient receptor potential channels superfamily, with 11 transmembrane spans and an extracellular N-terminal region of approximately 3109 amino acid residues, harboring multiple putative ligand binding domains. We demonstrate here that annexin A5 (ANXA5), a Ca(2+) and phospholipid binding protein, interacts with the N-terminal leucine-rich repeats of polycystin-1, in vitro and in a cell culture model. This interaction is direct and specific and involves a conserved sequence of the ANXA5 N-terminal domain. Using Madin-Darby canine kidney cells expressing polycystin-1 in an inducible manner we also show that polycystin-1 colocalizes with E-cadherin at cell-cell contacts and accelerates the recruitment of intracellular E-cadherin to reforming junctions. This polycystin-1 stimulated recruitment is significantly delayed by extracellular annexin A5.

Haemoglobin Noah Mehmet Oeztuerk (alpha(2) delta(2)143 (H21)His-->Tyr: A novel delta-chain variant in the 2,3-DPG binding site.

A new delta-chain variant, delta143 (H21) His-->Tyr or Hb Noah Mehmet Oeztuerk, was discovered during the investigation of the cause of hemolytic anaemia in a 6-month-old infant of Turkish descent. It was detected by Cation exchange high-performance liquid chromatography (CE-HPLC) using PolyCAT A column. P(50) was 20.6+/-0.60 mmHg and 29.3+/-0.40 mmHg for the carrier and the wild-type, respectively. This suggests an increase in oxygen affinity. On routine CE-HPLC Hb A(2) was low (1.2%) and the variant was not detected. An extended family study revealed that the variant was not associated with the anaemia or with any other clinical abnormality.

Spectrum of molecular defects and mutation detection rate in patients with mild and moderate hemophilia A.

The amount of residual F8 (FVIII:C) determines the clinical severity of hemophilia A. Recently, we showed that the mutation detection rate in severely affected male patients (FVIII:C<1% of normal) is virtually 100% when testing for the common intron 22-/intron 1- inversions and big deletions, followed by genomic sequencing of the F8 gene. Here we report on the spectrum of mutations and their distribution throughout the F8 gene sequence in 135 patients with moderate (n=23) or mild (n=112) hemophilia A. In contrast to the severe form of the disorder, analysis on the genomic level failed to detect the molecular defect in approximately 4% of the moderately and in approximately 12% of the mildly affected patients. A total of 36 of the mutations identified in this study are novel. The vast majority of the detected changes were missense. The newly detected amino acid substitutions were scored for potential distant or local conformational changes and influence on molecular stability for every single F8 domain with available structures, using homology modeling. Two molecular changes in the promoter region of the factor VIII gene (c.-112G>A and -219C>T), affecting the core segment (minimal promoter) were detected in two patients with mild hemophilia A. To our knowledge this is the first report on promoter mutations in the F8 gene.

The ion channel polycystin-2 is required for left-right axis determination in mice.

Generation of laterality depends on a pathway which involves the asymmetrically expressed genes nodal, Ebaf, Leftb, and Pitx2. In mouse, node monocilia are required upstream of the nodal cascade. In chick and frog, gap junctions are essential prior to node/organizer formation. It was hypothesized that differential activity of ion channels gives rise to unidirectional transfer through gap junctions, resulting in asymmetric gene expression. PKD2, which if mutated causes autosomal dominant polycystic kidney disease (ADPKD) in humans, encodes the calcium release channel polycystin-2. We have generated a knockout allele of Pkd2 in mouse. In addition to malformations described previously, homozygous mutant embryos showed right pulmonary isomerism, randomization of embryonic turning, heart looping, and abdominal situs. Leftb and nodal were not expressed in the left lateral plate mesoderm (LPM), and Ebaf was absent from floorplate. Pitx2 was bilaterally expressed in posterior LPM but absent anteriorly. Pkd2 was ubiquitously expressed at headfold and early somite stages, with higher levels in floorplate and notochord. The embryonic midline, however, was present, and normal levels of Foxa2 and shh were expressed, suggesting that polycystin-2 acts downstream or in parallel to shh and upstream of the nodal cascade.

AURKA F31I polymorphism and breast cancer risk in BRCA1 and BRCA2 mutation carriers: a consortium of investigators of modifiers of BRCA1/2 study.

The AURKA oncogene is associated with abnormal chromosome segregation and aneuploidy and predisposition to cancer. Amplification of AURKA has been detected at higher frequency in tumors from BRCA1 and BRCA2 mutation carriers than in sporadic breast tumors, suggesting that overexpression of AURKA and inactivation of BRCA1 and BRCA2 cooperate during tumor development and progression. The F31I polymorphism in AURKA has been associated with breast cancer risk in the homozygous state in prior studies. We evaluated whether the AURKA F31I polymorphism modifies breast cancer risk in BRCA1 and BRCA2 mutation carriers from the Consortium of Investigators of Modifiers of BRCA1/2. Consortium of Investigators of Modifiers of BRCA1/2 was established to provide sufficient statistical power through increased numbers of mutation carriers to identify polymorphisms that act as modifiers of cancer risk and can refine breast cancer risk estimates in BRCA1 and BRCA2 mutation carriers. A total of 4,935 BRCA1 and 2,241 BRCA2 mutation carriers and 11 individuals carrying both BRCA1 and BRCA2 mutations was genotyped for F31I. Overall, homozygosity for the 31I allele was not significantly associated with breast cancer risk in BRCA1 and BRCA2 carriers combined [hazard ratio (HR), 0.91; 95% confidence interval (95% CI), 0.77-1.06]. Similarly, no significant association was seen in BRCA1 (HR, 0.90; 95% CI, 0.75-1.08) or BRCA2 carriers (HR, 0.93; 95% CI, 0.67-1.29) or when assessing the modifying effects of either bilateral prophylactic oophorectomy or menopausal status of BRCA1 and BRCA2 carriers. In summary, the F31I polymorphism in AURKA is not associated with a modified risk of breast cancer in BRCA1 and BRCA2 carriers.

Inducible Cre/loxP recombination in the mouse proximal tubule.

Transgenic technologies in mice became invaluable experimental tools to identify the in vivo function of proteins. However, conventional knockout technology often results in embryonic lethality and because genes are frequently expressed in multiple cell types, the resulting knockout phenotypes can be complex and difficult or impossible to dissect. These issues are particularly important for gene-targeting strategies used to examine renal function. The kidney contains quite a number of different cell types, the function of many of which impacts that of other renal cells. To avoid these limitations conditional knockout strategies have been designed. As one important part of this system we describe the development of a mouse line expressing the tamoxifen-activatable Cre recombinase Cre-ER(T2) specifically in renal proximal tubules. The expression of Cre-ER(T2) is driven by a promoter fragment of the mouse gamma-glutamyl transpeptidase type II gene resulting in the generation of the activatable recombinase in S3 segments of the proximal tubules from which over 80% were positive for Cre activity. In combination with loxP-based conditional mutant mice as a second tool this tamoxifen-inducible Cre-ER(T2) line allows functional analysis of a variety of genes important for renal development and function in a precisely controlled spatiotemporal manner.

A novel TACSTD2 gene mutation in a Turkish family with a gelatinous drop-like corneal dystrophy.

PURPOSE: To identify the molecular defect causing gelatinous drop-like corneal dystrophy in a Turkish family and assign affected and carriership status. METHODS: Visual activity of affected family members was measured using Snellen optotypes. To identify the molecular defect, mutation analysis of the TACSTD2 (M1S1) gene was performed. RESULTS: We report on a new TACSTD2 mutation, c.653delA, in a Turkish family. The identified molecular defect cosegregates with the disease among affected members of the family and is not found in 100 unaffected individuals of various ethnic origin. CONCLUSIONS: A few TACSTD2 gene mutations in the homozygous or compound heterozygous state have been described as causative for this abnormality, mainly in several Japanese families. The newly identified mutation is predicted to generate a shortened protein product, thereby completely altering the COOH-terminal region and deleting the transmembrane domain, required for anchoring at cell membranes and the phosphatidylinosyol2-binding site.

Novel three-break rearrangement and cryptic translocations leading to colocalization of MYC and IGH signals in B-cell acute lymphoblastic leukemia.

Reciprocal translocations involving the MYC locus and immunoglobulin heavy chain (IGH) and light chain (IgK and IgL) loci are characteristic for non-Hodgkin lymphomas, especially Burkitt lymphoma, and have been described in B-cell acute lymphoblastic leukemia (B-ALL). We report on a case of B-ALL of L3 morphology with MYC-IGH translocation. Bone marrow metaphases were characterized using conventional cytogenetics and molecular cytogenetic techniques. G-banding showed a hyperdiploid complex rearranged male karyotype with 51 chromosomes. Additionally to other chromosome changes, a three-break rearrangement involving 6p21, 8q24, and 14q32, as well as cryptic translocations of IGH locus to MYC locus were detected. To our knowledge, this is the first case with colocalizations of MYC and IGH in a three-break rearrangement involving 6p21 and on an additional derivative chromosome as results of cryptic translocations.

Characterization of hemoglobin Würzburg (alpha2beta2 4(A1)Thr-->Asn), a new electrophoretically silent variant, by mass spectrometry and molecular modeling studies.

The first hemoglobin (Hb) variant carrying a mutation at beta4 was identified as beta4(A1)Thr-->Asn or Hb Würzburg and constituted 38% of the total hemoglobin. It showed a slightly elevated oxygen affinity and a slightly decreased cooperativity index (n50 = 2.3 versus n50 = 2.8). The analysis of the electrostatic potential showed an increased negative charge at the site of the mutation with a displacement of beta6(A3)Glu by 1.3A. The replacement of threonine by asparagine seems to stabilize the R conformation. This may explain partially both the high affinity and the reduction in cooperativity.

Spectrum of molecular defects and mutation detection rate in patients with severe hemophilia A.

Hemophilia A is the most frequently occurring X-linked bleeding disorder, affecting one to two out of 10,000 males worldwide. Various types of mutations in the F8 gene are causative for this condition. It is well known that the most common mutation in severely affected patients is the intron 22 inversion, which accounts for about 45% of cases with F8 residual activity of less than 1%. Therefore, the aim of the present study was to determine the spectrum and distribution of mutations in the F8 gene in a large group of patients with severe hemophilia A who previously tested negative for the common intron 22 inversion. Here we report on a mutation analysis of 86 patients collected under the above-mentioned criterion. The pathogenic molecular defect was identified in all patients, and thus our detection rate was virtually 100%. Thirty-four of the identified mutations are described for the first time. The newly detected amino acid substitutions were scored for potential gross or local conformational changes and influence on molecular stability for every single F8 domain with available structures, using homology modeling.

A generalized skeletal hyperostosis in two siblings caused by a novel mutation in the SOST gene.

In this study, a brother and sister of German origin are described with a possible diagnosis of van Buchem disease, a rare autosomal recessive sclerosing bone dysplasia characterized by a generalized hyperostosis of the skeleton mainly affecting the cranial bones. Clinically, patients suffer from cranial nerve entrapment potentially resulting in facial paresis, hearing disturbances, and visual loss. The radiological picture of van Buchem disease closely resembles sclerosteosis, although in the latter patients, syndactyly, tall stature, and raised intracranial pressure are frequently observed, allowing a differential diagnosis with van Buchem disease. Previous molecular studies demonstrated homozygous loss-of-function mutations in the SOST gene in sclerosteosis patients while a chromosomal rearrangement creating a 52-kb deletion downstream of this gene was found in Dutch patients with van Buchem disease. This deletion most likely suppresses SOST expression. Sclerostin, the SOST gene product, has been shown to play a role in bone metabolism. The two siblings reported here were evaluated at the molecular level by carrying out a mutation analysis of the SOST gene. This resulted in the identification of a novel putative disease-causing splice site mutation (IVS1 + 1 G-->C) homozygously present in both siblings.

Prostate-specific antigen persistence after radical prostatectomy as a predictive factor of clinical relapse-free survival and overall survival: 10-year data of the ARO 96-02 trial.

OBJECTIVE: The ARO 96-02 trial primarily compared wait-and-see (WS, arm A) with adjuvant radiation therapy (ART, arm B) in prostate cancer patients who achieved an undetectable prostate-specific antigen (PSA) after radical prostatectomy (RP). Here, we report the outcome with up to 12 years of follow-up of patients who retained a post-RP detectable PSA and received salvage radiation therapy (SRT, arm C). METHODS AND MATERIALS: For the study, 388 patients with pT3-4pN0 prostate cancer with positive or negative surgical margins were recruited. After RP, 307 men achieved an undetectable PSA (arms A + B). In 78 patients the PSA remained above thresholds (median 0.6, range 0.05-5.6 ng/mL). Of the latter, 74 consented to receive 66 Gy to the prostate bed, and SRT was applied at a median of 86 days after RP. Clinical relapse-free survival, metastasis-free survival, and overall survival were determined by the Kaplan-Meier method. RESULTS: Patients with persisting PSA after RP had higher preoperative PSA values, higher tumor stages, higher Gleason scores, and more positive surgical margins than did patients in arms A + B. For the 74 patients, the 10-year clinical relapse-free survival rate was 63%. Forty-three men had hormone therapy; 12 experienced distant metastases; 23 patients died. Compared with men who did achieve an undetectable PSA, the arm-C patients fared significantly worse, with a 10-year metastasis-free survival of 67% versus 83% and overall survival of 68% versus 84%, respectively. In Cox regression analysis, Gleason score ≥8 (hazard ratio [HR] 2.8), pT ≥ 3c (HR 2.4), and extraprostatic extension ≥2 mm (HR 3.6) were unfavorable risk factors of progression. CONCLUSIONS: A persisting PSA after prostatectomy seems to be an important prognosticator of clinical progression for pT3 tumors. It correlates with a higher rate of distant metastases and with worse overall survival. A larger prospective study is required to determine which patient subgroups will benefit most from which treatment option.

Seven novel and four recurrent point mutations in the factor VIII (F8C) gene.

Haemophilia A is a X-linked bleeding disorder, caused by deficiency in the activity of coagulation factor VIII due to mutations in the corresponding gene. The most common defect in patients is an inversion of the factor VIII gene that accounts for nearly 45% of individuals with severe hemophilia A. Point mutations and small deletions/insertions are responsible for the majority of cases with moderate to mild clinical course and for half of the severe hemophilia A occurrences. The majority of these mutations are "private", because of the high mutation rate for this particular gene. We report on eleven pathological changes in the factor VIII sequence detected in male patients with haemophilia A or in female obligate carriers. Seven of these mutations are novel [E204N, E265X, M320T, F436C, S535C, N2129M and R2307P] and four have been previously identified [V162M, R527W, R1966X, and R2159C]. Genotype-phenotype correlations and computer prediction analysis on the effect of missense mutations on the secondary structure of the factor VIII protein are performed and the relationships evaluated.

Prevalence of small rearrangements in the factor VIII gene F8C among patients with severe hemophilia A.

Hemophilia A is a common X-linked bleeding disorder caused by various types of mutations in the factor VIII gene F8C. The most common intron 22-inversion is responsible for about 40% of the severe hemophilia A cases while large deletions, point mutations and small (less than 100 bp) deletions or insertions are responsible for the disease in the rest of patients. We report on nine novel (6 deletions, two indels and one partial duplication) and five recurrent small rearrangements identified in 15 German patients with severe hemophilia A, negative for the intron 22-inversion. c.2208-2214delTTATTAC/c.2207-2215insCTCTT and c.4665-4678del/c.4664-4678insAAGGAA identified in the present study are the first small indels described in the factor VIII gene. Our analyses suggest that the prevalence of this type of mutations (predominantly located in exon 14) among patients with severe phenotype and negative for the common intron 22-inversion, is about 30%. The correlation between these molecular defects and formation of factor VIII inhibitors as well as the parental origin of the de novo mutations are evaluated. Finally we show that denaturing HPLC (DHPLC) and classic heteroduplex analysis (HA) are able to detect these sequence alterations on 100% and could be preferred as a screening approach when analysing for mutations in factor VIII in severely affected patients.

Expression of two protein isoforms of PAX7 is controlled by competing cleavage-polyadenylation and splicing.

The PAX7 gene encodes an evolutionary conserved transcription factor that is involved in the determination of the myogenic cell lineage during the development of vertebrates. In the postnatal period, the function of PAX7 is ultimately required for the specification of muscle satellite cells. The fact that PAX7 is expressed in fast proliferating embryonal myoblasts and in quiescent satellite cells of adults raised the question whether different PAX7 protein isoforms may have distinct roles in these myogenic precursors. Previously, we identified a human PAX7 mRNA encoding a C-terminus which did not show any sequence similarity to the PAX7 proteins of other organisms. So far, there was no further information available concerning the biological nature and significance of this form of PAX7. Here, we show that expression of PAX7 can be regulated by differential transcriptional termination either in exon 9 or in exon 8. Thereby, differential mRNA cleavage-polyadenylation and splicing of PAX7 may result in production of two alternative protein forms that contain or exclude the evolutionary conserved carboxy-terminal domain, respectively. The existence of both protein isoforms in vivo was confirmed by Western blot analysis. These data imply that the alternative C-termini of PAX7 may convey different functions to the corresponding protein isoforms.