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.

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.

Adjuvant radiotherapy versus wait-and-see after radical prostatectomy: 10-year follow-up of the ARO 96-02/AUO AP 09/95 trial.

BACKGROUND: Local failure after radical prostatectomy (RP) is common in patients with cancer extending beyond the capsule. Three prospectively randomized trials demonstrated an advantage for adjuvant radiotherapy (ART) compared with a wait-and-see (WS) policy. OBJECTIVE: To determine the efficiency of ART after a 10-yr follow-up in the ARO 96-02 study. DESIGN, SETTING, AND PARTICIPANTS: After RP, 388 patients with pT3 pN0 prostate cancer (PCa) were randomized to WS or three-dimensional conformal ART with 60 Gy. The present analysis focuses on intent-to-treat patients who achieved an undetectable prostate-specific antigen after RP (ITT2 population)--that is, 159 WS plus 148 ART men. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: The primary end point of the study was progression-free survival (PFS) (events: biochemical recurrence, clinical recurrence, or death). Outcomes were compared by log-rank test. Cox regression analysis served to identify variables influencing the course of disease. RESULTS AND LIMITATIONS: The median follow-up was 111 mo for ART and 113 mo for WS. At 10 yr, PFS was 56% for ART and 35% for WS (p<0.0001). In pT3b and R1 patients, the rates for WS even dropped to 28% and 27%, respectively. Of all 307 ITT2 patients, 15 died from PCa, and 28 died for other or unknown reasons. Neither metastasis-free survival nor overall survival was significantly improved by ART. However, the study was underpowered for these end points. The worst late sequelae in the ART cohort were one grade 3 and three grade 2 cases of bladder toxicity and two grade 2 cases of rectum toxicity. No grade 4 events occurred. CONCLUSIONS: Compared with WS, ART reduced the risk of (biochemical) progression with a hazard ratio of 0.51 in pT3 PCa. With only one grade 3 case of late toxicity, ART was safe. PATIENT SUMMARY: Precautionary radiotherapy counteracts relapse after surgery for prostate cancer with specific risk factors.

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.

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.

Phase III postoperative adjuvant radiotherapy after radical prostatectomy compared with radical prostatectomy alone in pT3 prostate cancer with postoperative undetectable prostate-specific antigen: ARO 96-02/AUO AP 09/95.

PURPOSE: Local failure after radical prostatectomy (RP) is common in patients with cancer extending beyond the capsule. Two randomized trials demonstrated an advantage for adjuvant radiotherapy (RT) compared with a wait-and-see policy. We conducted a randomized, controlled clinical trial to compare RP followed by immediate RT with RP alone for patients with pT3 prostate cancer and an undetectable prostate-specific antigen (PSA) level after RP. METHODS: After RP, 192 men were randomly assigned to a wait-and-see policy, and 193 men were assigned to immediate postoperative RT. Eligible patients had pT3 pN0 tumors. Patients who did not achieve an undetectable PSA after RP were excluded from treatment according to random assignment (n = 78; 20%). Of the remaining 307 patients, 34 patients on the RT arm did not receive RT and five patients on the wait-and-see arm received RT. Therefore, 114 patients underwent RT and 154 patients were treated with a wait-and-see policy. The primary end point was biochemical progression-free survival. RESULTS: Biochemical progression-free survival after 5 years in patients with undetectable PSA after RP was significantly improved in the RT group (72%; 95% CI, 65% to 81%; v 54%, 95% CI, 45% to 63%; hazard ratio = 0.53; 95% CI, 0.37 to 0.79; P = .0015). On univariate analysis, Gleason score more than 6 and less than 7, PSA before RP, tumor stage, and positive surgical margins were predictors of outcome. The rate of grade 3 to 4 late adverse effects was 0.3%. CONCLUSION: Adjuvant RT for pT3 prostate cancer with postoperatively undetectable PSA significantly reduces the risk of biochemical progression. Further follow-up is needed to assess the effect on metastases-free and overall survival.

Plant metal chaperones: a novel perspective in dementia therapy.

Dys-homeostasis of copper metabolism and oxidative stress are major hallmarks in the brains of Alzheimer patients. Therefore, metal bioavailability and mechanisms of copper ion homeostasis throughout the body are crucial and potential targets for therapeutic agents. Many of the medications used or suggested, respectively, at present time, may either be toxic, reveal a lack of specificity or have unknown mechanisms of action in vivo. Metal chaperones from medicinal plants are proposed as medications that are relatively free from these disadvantages. Furthermore, these agents are a promising class of molecules for studies aimed at developing innovative and etiological treatments for protein-misfolding diseases, especially Alzheimer's disease.

Genotype-phenotype correlations in Down syndrome identified by array CGH in 30 cases of partial trisomy and partial monosomy chromosome 21.

Down syndrome (DS) is one of the most frequent congenital birth defects, and the most common genetic cause of mental retardation. In most cases, DS results from the presence of an extra copy of chromosome 21. DS has a complex phenotype, and a major goal of DS research is to identify genotype-phenotype correlations. Cases of partial trisomy 21 and other HSA21 rearrangements associated with DS features could identify genomic regions associated with specific phenotypes. We have developed a BAC array spanning HSA21q and used array comparative genome hybridization (aCGH) to enable high-resolution mapping of pathogenic partial aneuploidies and unbalanced translocations involving HSA21. We report the identification and mapping of 30 pathogenic chromosomal aberrations of HSA21 consisting of 19 partial trisomies and 11 partial monosomies for different segments of HSA21. The breakpoints have been mapped to within approximately 85 kb. The majority of the breakpoints (26 of 30) for the partial aneuploidies map within a 10-Mb region. Our data argue against a single DS critical region. We identify susceptibility regions for 25 phenotypes for DS and 27 regions for monosomy 21. However, most of these regions are still broad, and more cases are needed to narrow down the phenotypic maps to a reasonable number of candidate genomic elements per phenotype.

An efficient and versatile system for acute and chronic modulation of renal tubular function in transgenic mice.

We describe a transgenic mouse line, Pax8-rtTA, which, under control of the mouse Pax8 promoter, directs high levels of expression of the reverse tetracycline-dependent transactivator (rtTA) to all proximal and distal tubules and the entire collecting duct system of both embryonic and adult kidneys. Using crosses of Pax8-rtTA mice with tetracycline-responsive c-MYC mice, we established a new, inducible model of polycystic kidney disease that can mimic adult onset and that shows progression to renal malignant disease. When targeting the expression of transforming growth factor beta-1 to the kidney, we avoided early lethality by discontinuous treatment and successfully established an inducible model of renal fibrosis. Finally, a conditional knockout of the gene encoding tuberous sclerosis complex-1 was achieved, which resulted in the early outgrowth of giant polycystic kidneys reminiscent of autosomal recessive polycystic kidney disease. These experiments establish Pax8-rtTA mice as a powerful tool for modeling renal diseases in transgenic mice.

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.

Haemoglobin Hokusetsu [beta52 (D3)] Asp-->Gly in German families associated with inclusion body.

Inclusion bodies associated with Hb Hokusetsu have never been published. We investigated the autoxidation of this variant as a cause for the inclusion bodies in three unrelated families. Moreover, haplotype analysis was carried out to unravel the origin of this variant also found in the Japanese population. The presence of inclusion bodies was revealed by incubating the fresh peripheral blood with brilliant cresyl blue. We further characterised this variant using mass spectrometry and DNA analysis. The generation of superoxide radical (ROS) during the autoxidation was assayed by electron spin resonance spectrometry. Inclusion bodies were seen in about 25% of red cells. Hb Hokusetsu turned out to be less thermostable than the control. It showed a tenfold-enhanced ROS formation versus control. The analysis of the beta-globin haplotypes for the three unrelated families showed that Hb Hokosetsu was linked with haplotype I (5' + - - - - + + 3'). This is the first case published in the German population. The inclusion bodies could be due to the instability of the variant. This is supported by the increased autoxidation. The absence of anaemia evokes an elimination of the inclusion bodies by the proteolytic mechanism of the red cells. The association of the variant in three unrelated families with the five polymorphisms of haplotype I indicates a single common mutation event. In the presence of Hb Hokusetsu, HbA 1C standard methods used to assess glycaemic control are mistaken.

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.

Patient survival by Hsp70 membrane phenotype: association with different routes of metastasis.

BACKGROUND: Heat shock proteins (HSPs) play important roles in tumor immunity. The authors prospectively investigated the correlation between the tumor-specific Hsp70 membrane expression as an independent clinicopathological marker and overall survival in tumor entities that differ in their route of metastasis. METHODS: Hsp70 membrane expression was examined by flow cytometry in 58 colon, 19 gastric, 54 lower rectal carcinoma, and 19 squamous cell carcinoma specimens and the corresponding normal tissues at time of first diagnosis. Kaplan-Meier survival curves were analyzed to determine the relation of Hsp70 expression to the patients' prognosis. RESULTS: An Hsp70 membrane-positive phenotype was found in 40% (colon), 37% (gastric), 43% (lower rectal), and 42% (squamous cell) of the analyzed tumor specimens. None of the corresponding normal tissues was found to be Hsp70 membrane-positive. In patients with colon (P = .032) and gastric (P = .045) carcinomas, an Hsp70 membrane expression correlated significantly with an improved overall survival; a negative association was seen in lower rectal (P = .085) and squamous cell carcinoma (P = .048). CONCLUSIONS: The authors hypothesized that differing relations between surface expression of Hsp70 on tumor cells and clinical outcomes may reflect differences in the route of metastases. Colon and gastric carcinomas metastasize into the liver where hepatic natural killer cells may have the capacity to recognize and kill Hsp70 membrane-positive tumor cells and thus account for a better overall survival.

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.

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 common haplotype of the annexin A5 (ANXA5) gene promoter is associated with recurrent pregnancy loss.

We sought to verify whether variation in the promoter of the gene encoding placental anticoagulant protein annexin A5 (ANXA5) represents a risk factor for recurrent pregnancy loss (RPL). Sequence analysis of 70 German RPL patients, all known to carry neither factor V Leiden nor a prothrombin mutation, revealed four consecutive nucleotide substitutions in the ANXA5 promoter, which were transmitted as a joint haplotype (M2). Reporter gene assays revealed that M2 reduces the in vitro activity of the ANXA5 promoter to 37-42% of the normal level. The possible relationship between M2 and RPL was evaluated by comparing RPL patients with two independent control groups recruited from the registry of the Institut für Humangenetik in Münster and the PopGen biobank in Kiel, respectively. Carriers of M2 were found to exhibit a > 2-fold higher RPL risk than non-carriers (odds ratio, 2.42; 95% confidence interval, 1.27-4.58) when using unselected controls (PopGen) and an almost 4-fold higher risk when using the Münster 'super-controls', i.e. women with successful pregnancies and no previous history of pregnancy losses (odds ratio, 3.88; 95% confidence interval, 1.98-7.54). This statistically significant association should facilitate the development of improved prognostic algorithms for RPL, involving a more precise assessment of individual disease risks, and provide a guide to offering adequate therapies where relevant.

A large deletion and novel point mutations in the calpain 3 gene (CAPN3) in Bulgarian LGMD2A patients.

Limb-girdle muscular dystrophy type 2A (LGMD2A) is caused by mutations in the calpain 3 (CAPN3) gene. The clinical diagnoses of these cases in Bulgaria are very complicated, no protein analysis on muscular biopsy is available in our country, and genetic tests are the only possibility to clarify the diagnoses in clinically ambiguous cases. We screened 48 unrelated Bulgarian cases with preliminary diagnoses of different types of muscular dystrophy for mutations in the CAPN3 gene. Altogether, 20 families (42%) were found to carry mutations in the CAPN3 gene. Several misdiagnosed cases were clarified. Three novel and six recurrent mutations were identified. In total, 40% of the patients are homozygous for c.550delA, and 70% carry it at least on one allele. The affected group of women in our sample shows later onset, milder clinical manifestation, slower progression, and later invalidization.

Cardiovascular characterization of Pkd2(+/LacZ) mice, an animal model for the autosomal dominant polycystic kidney disease type 2 (ADPKD2).

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2. Patients with ADPKD have an increased incidence of cardiac valve abnormalities and left ventricular hypertrophy. Systematic analyses of cardiovascular involvement have so far been performed only on genetically unclassified patients or on ADPKD1 patients, but not on genetically defined ADPKD2 patients. Even existing Pkd1 or Pkd2 mouse models were not thoroughly analyzed in this respect. Therefore, the aim of this project was the noninvasive functional cardiovascular characterization of a mouse model for ADPKD2. METHODS: Pkd2(+/LacZ) mice and wildtype controls were classified into 8 groups with respect to gender, age and genotype. In addition, two subgroups of female mice were analyzed for cardiac function before and during advanced pregnancy. Doppler-echocardiographic as well as histological studies were performed. RESULTS: Doppler-echocardiography did not reveal significant cardiovascular changes. Heart rate and left ventricular (LV) length, LV mass, LV enddiastolic and LV endsystolic diameters did not differ significantly among the various groups when comparing wildtype and knockout mice. There were no significant differences except for a tendency towards higher maximal early and late flow velocities over the mitral valve in old wildtype mice. CONCLUSIONS: Non-invasive phenotyping using ultrasound did not reveal significant cardiovascular difference between adult Pkd2(+/LacZ) and WT mice. Due to the lack of an obvious renal phenotype in heterozygous mice, it is likely that in conventional ADPKD knock out mouse models severe cardiac problems appear too late to be identified during the reduced lifespan of the animals.

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.