CFTR-France, a national relational patient database for sharing genetic and phenotypic data associated with rare CFTR variants.

Most of the 2,000 variants identified in the CFTR (cystic fibrosis transmembrane regulator) gene are rare or private. Their interpretation is hampered by the lack of available data and resources, making patient care and genetic counseling challenging. We developed a patient-based database dedicated to the annotations of rare CFTR variants in the context of their cis- and trans-allelic combinations. Based on almost 30 years of experience of CFTR testing, CFTR-France (https://cftr.iurc.montp.inserm.fr/cftr) currently compiles 16,819 variant records from 4,615 individuals with cystic fibrosis (CF) or CFTR-RD (related disorders), fetuses with ultrasound bowel anomalies, newborns awaiting clinical diagnosis, and asymptomatic compound heterozygotes. For each of the 736 different variants reported in the database, patient characteristics and genetic information (other variations in cis or in trans) have been thoroughly checked by a dedicated curator. Combining updated clinical, epidemiological, in silico, or in vitro functional data helps to the interpretation of unclassified and the reassessment of misclassified variants. This comprehensive CFTR database is now an invaluable tool for diagnostic laboratories gathering information on rare variants, especially in the context of genetic counseling, prenatal and preimplantation genetic diagnosis. CFTR-France is thus highly complementary to the international database CFTR2 focused so far on the most common CF-causing alleles.

The improvement of the best practice guidelines for preimplantation genetic diagnosis of cystic fibrosis: toward an international consensus.

Cystic fibrosis (CF) is one of the most common indications for preimplantation genetic diagnosis (PGD) for single gene disorders, giving couples the opportunity to conceive unaffected children without having to consider termination of pregnancy. However, there are no available standardized protocols, so that each center has to develop its own diagnostic strategies and procedures. Furthermore, reproductive decisions are complicated by the diversity of disease-causing variants in the CFTR (cystic fibrosis transmembrane conductance regulator) gene and the complexity of correlations between genotypes and associated phenotypes, so that attitudes and practices toward the risks for future offspring can vary greatly between countries. On behalf of the EuroGentest Network, eighteen experts in PGD and/or molecular diagnosis of CF from seven countries attended a workshop held in Montpellier, France, on 14 December 2011. Building on the best practice guidelines for amplification-based PGD established by ESHRE (European Society of Human Reproduction and Embryology), the goal of this meeting was to formulate specific guidelines for CF-PGD in order to contribute to a better harmonization of practices across Europe. Different topics were covered including variant nomenclature, inclusion criteria, genetic counseling, PGD strategy and reporting of results. The recommendations are summarized here, and updated information on the clinical significance of CFTR variants and associated phenotypes is presented.

Should diffuse bronchiectasis still be considered a CFTR-related disorder?

BACKGROUND: Although several comprehensive studies have evaluated the role of the CFTR gene in idiopathic diffuse bronchiectasis (DB), it remains controversial. METHODS: We analyzed the whole coding region of the CFTR gene, its flanking regions and the promoter in 47 DB patients and 47 controls. Available information about demographic, spirometric, radiological and microbiological data for the DB patients was collected. Unclassified CFTR variants were in vitro functionally assessed. RESULTS: CFTR variants were identified in 24 DB patients and in 27 controls. DB variants were reclassified based on the results of in silico predictive analyses, in vitro functional assays and data from epidemiological and literature databases. Except for the sweat test value, no clear genotype-phenotype correlation was observed. CONCLUSIONS: DB should not be considered a classical autosomal recessive CFTR-RD. Moreover, although further investigations are necessary, we proposed a new class of "Non-Neutral Variants" whose impact on lung disease requires more studies.

Small-scale high-throughput sequencing-based identification of new therapeutic tools in cystic fibrosis.

PURPOSE: Although 97-99% of CFTR mutations have been identified, great efforts must be made to detect yet-unidentified mutations. METHODS: We developed a small-scale next-generation sequencing approach for reliably and quickly scanning the entire gene, including noncoding regions, to identify new mutations. We applied this approach to 18 samples from patients suffering from cystic fibrosis (CF) in whom only one mutation had hitherto been identified. RESULTS: Using an in-house bioinformatics pipeline, we could rapidly identify a second disease-causing CFTR mutation for 16 of 18 samples. Of them, c.1680-883A>G was found in three unrelated CF patients. Analysis of minigenes and patients' transcripts showed that this mutation results in aberrantly spliced transcripts because of the inclusion of a pseudoexon. It is located only three base pairs from the c.1680-886A>G mutation (1811+1.6kbA>G), the fourth most frequent mutation in southwestern Europe. We next tested the effect of antisense oligonucleotides targeting splice sites on these two mutations on pseudoexon skipping. Oligonucleotide transfection resulted in the restoration of the full-length, in-frame CFTR transcript, demonstrating the effect of antisense oligonucleotide-induced pseudoexon skipping in CF. CONCLUSION: Our data confirm the importance of analyzing noncoding regions to find unidentified mutations, which is essential to designing targeted therapeutic approaches.

A balance between activating and repressive histone modifications regulates cystic fibrosis transmembrane conductance regulator (CFTR) expression in vivo.

The genetic mechanisms that regulate CFTR, the gene responsible for cystic fibrosis, have been widely investigated in cultured cells. However, mechanisms responsible for tissue-specific and time-specific expression are not completely elucidated in vivo. Through the survey of public databases, we found that the promoter of CFTR was associated with bivalent chromatin in human embryonic stem (ES) cells. In this work, we analyzed fetal (at different stages of pregnancy) and adult tissues and showed that, in digestive and lung tissues, which expressed CFTR, H3K4me3 was maintained in the promoter. Histone acetylation was high in the promoter and in two intronic enhancers, especially in fetal tissues. In contrast, in blood cells, which did not express CFTR, the bivalent chromatin was resolved (the promoter was labeled by the silencing mark H3K27me3). Cis-regulatory sequences were associated with lowly acetylated histones. We also provide evidence that the tissue-specific expression of CFTR is not regulated by dynamic changes of DNA methylation in the promoter. Overall, this work shows that a balance between activating and repressive histone modifications in the promoter and intronic enhancers results in the fine regulation of CFTR expression during development, thereby ensuring tissue specificity.

A classification model relative to splicing for variants of unknown clinical significance: application to the CFTR gene.

Molecular diagnosis of cystic fibrosis and cystic fibrosis transmembrane regulator (CFTR)-related disorders led to the worldwide identification of nearly 1,900 sequence variations in the CFTR gene that consist mainly of private point mutations and small insertions/deletions. Establishing their effect on the function of the encoded protein and therefore their involvement in the disease is still challenging and directly impacts genetic counseling. In this context, we built a decision tree following the international guidelines for the classification of variants of unknown clinical significance (VUCS) in the CFTR gene specifically focused on their consequences on splicing. We applied general and specific criteria, including comprehensive review of literature and databases, familial genetics data, and thorough in silico studies. This model was tested on 15 intronic and exonic VUCS identified in our cohort. Six variants were classified as probably nonpathogenic considering their impact on splicing and eight as probably pathogenic, which include two apparent missense mutations. We assessed the validity of our method by performing minigenes studies and confirmed that 93% (14/15) were correctly classified. We provide in this study a high-performance method that can play a full role in interpreting the results of molecular diagnosis in emergency context, when functional studies are not achievable.

CFTR mutation combinations producing frequent complex alleles with different clinical and functional outcomes.

Genotype-phenotype correlations in cystic fibrosis (CF) may be difficult to establish because of phenotype variability, which is associated with certain CF transmembrane conductance regulator (CFTR) gene mutations and the existence of complex alleles. To elucidate the clinical significance of complex alleles involving p.Gly149Arg, p.Asp443Tyr, p.Gly576Ala, and p.Arg668Cys, we performed a collaborative genotype-phenotype correlation study, collected epidemiological data, and investigated structure-function relationships for single and natural complex mutants, p.[Gly576Ala;Arg668Cys], p.[Gly149Arg;Gly576Ala;Arg668Cys], and p.[Asp443Tyr;Gly576Ala;Arg668Cys]. Among 153 patients carrying at least one of these mutations, only three had classical CF and all carried p.Gly149Arg in the triple mutant. Sixty-four had isolated infertility and seven were healthy individuals with a severe mutation in trans, but none had p.Gly149Arg. Functional studies performed on all single and natural complex mutants showed that (1) p.Gly149Arg results in a severe misprocessing defect; (2) p.Asp443Tyr moderately alters CFTR maturation; and (3) p.Gly576Ala, a known splicing mutant, and p.Arg668Cys mildly alter CFTR chloride conductance. Overall, the results consistently show the contribution of p.Gly149Arg to the CF phenotype, and suggest that p.[Arg668Cys], p.[Gly576Ala;Arg668Cys], and p.[Asp443Tyr;Gly576Ala;Arg668Cys] are associated with CFTR-related disorders. The present study emphasizes the importance of comprehensive genotype-phenotype and functional studies in elucidating the impact of mutations on clinical phenotype.

Identification of a novel duplication CFTRdup2 and functional impact of large rearrangements identified in the CFTR gene.

In European populations, large rearrangements contribute to approximately 2% of CF mutations. Here, we reported a novel duplication, the CFTRdup2, identified in a patient heterozygous for Phe508del and suffering from a mild CF. Using a combination of functional tests, we studied the impact of duplication/deletion on CFTR expression. We showed that the copy number variations of exon 2, in addition to abolishing the rate of the mature CFTR protein, affect the CFTR mRNA levels. These data illustrate the importance to perform functional analysis to better understand the molecular basis responsible for cystic fibrosis. Determining the impact of deletions or duplications is relevant for a more comprehensive diagnosis and prognosis of patients.

Functional analysis of a promoter variant identified in the CFTR gene in cis of a frameshift mutation.

In monogenic diseases, the presence of several sequence variations in the same allele may complicate our understanding of genotype-phenotype relationships. We described new alterations identified in a cystic fibrosis (CF) patient harboring a 48C>G promoter sequence variation associated in cis of a 3532AC>GTA mutation and in trans with the F508del mutation. Functional analyses including in vitro experiments confirmed the deleterious effect of the 3532GTA frameshift mutation through the creation of a premature termination codon. The analyses also revealed that the 48G promoter variant has a negative effect on both transcription and mRNA level, thus demonstrating the importance of analyzing all mutations or sequence variations with potential impact on CF transmembrane conductance regulator processing, even when the two known disease-causing mutations have already been detected. Our results emphasize the need to perform, wherever possible, functional studies that may greatly assist the interpretation of the disease-causing potential of rare mutation-associated sequence variations.

Common CFTR haplotypes and susceptibility to chronic pancreatitis and congenital bilateral absence of the vas deferens.

CFTR mutations enhance susceptibility for idiopathic chronic pancreatitis (ICP) and congenital bilateral absence of the vas deferens (CBAVD); however, it is unknown why CFTR heterozygotes are at increased disease risk. We recently showed that common CFTR variants are associated with aberrantly spliced transcripts. Here, we genotyped for common CFTR variants and tested for associations in two ICP (ICP-A: 126 patients, 319 controls; ICP-B: 666 patients, 1,181 controls) and a CBAVD population (305 patients, 319 controls). Haplotype H10 (TG11-T7-470V) conferred protection (ICP-A: OR 0.19, P<0.0001; ICP-B: OR 0.78, P = 0.06; CBAVD OR 0.08, P<0.001), whereas haplotype H3 (TG10-T7-470M) increased disease risk (ICP-A: OR 8.34, P = 0.003; ICP-B: OR 1.88, P = 0.007; CBAVD: OR 5.67, P = 0.01). The risk of heterozygous CFTR mutations carriers for ICP (OR 2.44, P<0.001) and CBAVD (OR 14.73, P<0.001) was fully abrogated by the H10/H10 genotype. Similarly, ICP risk of heterozygous p.Asn34Ser SPINK1 mutation carriers (OR 10.34, P<0.001) was compensated by H10/H10. Thus, common CFTR haplotypes modulate ICP and CBAVD susceptibility alone and in heterozygous CFTR and p.Asn34Ser mutation carriers. Determination of these haplotypes helps to stratify carriers into high- and low-risk subjects, providing helpful information for genetic counseling.

Variants in CFTR untranslated regions are associated with congenital bilateral absence of the vas deferens.

BACKGROUND: Congenital bilateral absence of the vas deferens (CBAVD), a frequent cause of obstructive azoospermia, is generated by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Despite extensive testing for point mutations and large rearrangements, a small proportion of alleles still remains unidentified in CBAVD patients. METHODS AND RESULTS: Mutation scanning analysis of microsatellite variability in the CFTR gene identified two undescribed 4 bp sequence repeats (TAAA)(6) and (TAAA)(8) in intron 9 in two CBAVD patients heterozygote for either the -33G→A promoter transition or the classical [TG12T5] CBAVD mutation. This study explores the putative impact of this promoter variant by using a combination of web based prediction tools, reporter gene assays, and DNA/proteins interaction analyses. Results of transiently transfected vas deferens cells with either the -33G wild-type or the -33A variant CFTR directed luciferase reporter gene confirmed that the -33A variant, which alters the FOXI1 (Forkhead box I1) binding, significantly decreases the CFTR promoter activity. It was also investigated whether regulatory elements located within the intronic tetrarepeat might influence the CFTR expression. There was evidence that both the (TAAA)(6) and the (TAAA)(8) alleles modulate the CFTR transcription and the binding affinity for FOX transcription factors, involved in the chromatin architecture. CONCLUSIONS: As the vas deferens seems to be one of the tissues most susceptible to a reduction in the normal CFTR transcripts levels, and as two mild mutations are sufficient to induce CBAVD phenotype, these findings raise the possibility that these uncommon variants may be a novel cause of CBAVD.

Measurement of nasal potential difference in young children with an equivocal sweat test following newborn screening for cystic fibrosis.

BACKGROUND: A challenging problem arising from cystic fibrosis (CF) newborn screening is the significant number of infants with hypertrypsinaemia (HIRT) with sweat chloride levels in the intermediate range and only one or no identified CF-causing mutations. OBJECTIVES: To investigate the diagnostic value for CF of assessing CF transmembrane conductance regulator (CFTR) protein function by measuring nasal potential difference in children with HIRT. METHODS: A specially designed protocol was used to assess nasal potential difference (NPD) in 23 young children with HIRT (3 months-4 years) with inconclusive neonatal screening. Results were analysed with a composite score including CFTR-dependent sodium and chloride secretion. Results were correlated with genotype after extensive genetic screening and with clinical phenotype at follow-up 3 years later. RESULTS: NPD was interpretable for 21 children with HIRT: 13 had NPD composite scores in the CF range. All 13 were finally found to carry two CFTR mutations. At follow-up, nine had developed a chronic pulmonary disease consistent with a CF diagnosis. The sweat test could be repeated in nine children, and six had sweat chloride values >or=60 mmol/l. Of the eight children with normal NPD scores, only two had two CFTR mutations, both wide-spectrum mutations. None had developed a CF-like lung disease at follow-up. The sweat test could be reassessed in five of these eight children and all had sweat chloride values <60 mmol/l. CF diagnosis was ruled out in six of these eight children. CONCLUSION: Evaluation of CFTR function in the nasal epithelium of young children with inconclusive results at CF newborn screening is a useful diagnostic tool for CF.

CFTR rearrangements in Spanish cystic fibrosis patients: first new duplication (35kb) characterised in the Mediterranean countries.

Developments in quantitative PCR technologies have greatly improved our ability to detect large genome rearrangements. In particular oligonucleotide-based array comparative genomic hybridisation has become a useful tool for appropriate and rapid detection of breakpoints. In this work, we have analysed 80 samples (42 unknown CF alleles) applying three quantitative technologies (MLPA, qPCR and array-CGH) to detect recurrent as well as novel large rearrangements in the Spanish CF population. Three deletions and one duplication have been identified in five alleles (12%). Interestingly, we provide the comprehensive characterisation of the first duplication in our CF cohort. The new CFTRdupProm-3 mutation spans 35.7 kb involving the 5'-end of the CFTR gene. Additionally, the RNA analysis has revealed a cryptic sequence with a premature termination codon leading to a disrupted protein. This duplication has been identified in five unrelated families from Spain, France and Italy with all patients showing the same associated haplotype, which is further evidence for its likely common Mediterranean origin. Overall, considering this and other previous studies, CFTR rearrangements account for 1.3% of the Spanish CF alleles.

Cystic fibrosis transmembrane conductance regulator mutation spectrum in patients with cystic fibrosis in Tunisia.

AIM: To determine the frequency and types of mutations causing cystic fibrosis (CF) in Tunisia. METHODS: We analyzed the complete coding region and flanking intronic sequences of the cystic fibrosis transmembrane conductance regulator (CFTR) gene in 68 unrelated patients suffering from the classical form of the disease. RESULTS: Twelve different CFTR mutations accounted for 90% (123/136) of CF alleles, including F508del (47.06%), E1104X (16.18%), N1303K (6.62%), 711 + 1T > G (5.88%), W1282X (4.41%), G542X (3.67%), R1158X (1.47%), 4016insT (0.74%), and R785X (0.74%). Three novel mutations were detected in this study: I1203V (1.47%), 1811 + 5A > G (0.74%), and 4268 + 2T > G (1.47%). Fifty patients (74%) were homozygous, among which 28 (41.17%) for F508del and 10 (14.7%) for E1104X. CONCLUSIONS: Ninety-seven percent of patients were found with at least one CFTR mutation. This study contributes to a better knowledge on CF-causing mutations in different regions in Tunisia and demonstrates that a complete scanning of CFTR sequences is necessary to implement efficient programs for CF genetic screening and counseling in this part of North Africa.

Mutations in the amiloride-sensitive epithelial sodium channel in patients with cystic fibrosis-like disease.

We investigated whether mutations in the genes that code for the different subunits of the amiloride-sensitive epithelial sodium channel (ENaC) might result in cystic fibrosis (CF)-like disease. In a small fraction of the patients, the disease could be potentially explained by an ENaC mutation by a Mendelian mechanism, such as p.V114I and p.F61L in SCNN1A. More importantly, a more than three-fold significant increase in incidence of several rare ENaC polymorphisms was found in the patient group (30% vs. 9% in controls), indicating an involvement of ENaC in some patients by a polygenetic mechanism. Specifically, a significantly higher number of patients carried c.-55+5G>C or p.W493R in SCNN1A in the heterozygous state, with odds ratios (ORs) of 13.5 and 2.7, respectively.The p.W493R-SCNN1A polymorphism was even found to result in a four-fold more active ENaC channel when heterologously expressed in Xenopus laevis oocytes. About 1 in 975 individuals in the general population will be heterozygous for the hyperactive p.W493R-SCNN1A mutation and a cystic fibrosis transmembrane conductance regulator (CFTR) gene that results in very low amounts (0-10%) functional CFTR. These ENaC/CFTR genotypes may play a hitherto unrecognized role in lung diseases.

Best practice guidelines for molecular genetic diagnosis of cystic fibrosis and CFTR-related disorders--updated European recommendations.

The increasing number of laboratories offering molecular genetic analysis of the CFTR gene and the growing use of commercial kits strengthen the need for an update of previous best practice guidelines (published in 2000). The importance of organizing regional or national laboratory networks, to provide both primary and comprehensive CFTR mutation screening, is stressed. Current guidelines focus on strategies for dealing with increasingly complex situations of CFTR testing. Diagnostic flow charts now include testing in CFTR-related disorders and in fetal bowel anomalies. Emphasis is also placed on the need to consider ethnic or geographic origins of patients and individuals, on basic principles of risk calculation and on the importance of providing accurate laboratory reports. Finally, classification of CFTR mutations is reviewed, with regard to their relevance to pathogenicity and to genetic counselling.

WGA allows the molecular characterization of a novel large CFTR rearrangement in a black South African cystic fibrosis patient.

By performing extensive scanning of whole coding and flanking sequences of the cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene, we had previously identified the CF-causing mutations in black South African patients of different ethnic groups suspected with the disease. Of ten samples analyzed, there were six remaining that had either one (n = 2) or two (n = 4) unidentified CFTR alleles that have now been tested for large rearrangements using a semiquantitative fluorescent PCR assay. A novel deletion encompassing CFTR exon 2 was detected in one patient who was heterozygous for the mutation 3120+1G>A. The Caucasian deletion involving the same exon [c.54-5811_c.164+2186del8108ins182] was ruled out. The DNA had been stored for more than 12 years and only minute quantities remained. We thus used a whole-genome amplification method based on multiple displacement amplification to generate sufficient amounts of DNA to characterize the intronic breakpoints and identify the deletion at the genomic level. Mapping and sequencing the breakpoint junctions revealed a novel large deletion [c.54-1161_c.164+1603del2875]. We have designed a simple test to specifically detect the presence or absence of this large rearrangement. This study reports the first large CFTR rearrangement in a black South African CF patient, further defining the molecular spectrum of CF that will be useful for improving genetic testing and counseling in this region.

Comprehensive and rapid genotyping of mutations and haplotypes in congenital bilateral absence of the vas deferens and other cystic fibrosis transmembrane conductance regulator-related disorders.

Available commercial kits only screen for the most common cystic fibrosis transmembrane conductance regulator (CFTR) mutations causing classic cystic fibrosis and for the Tn variant in IVS8. However, full scanning of CFTR is needed for the diagnosis of patients with cystic fibrosis or CFTR-related disorders (including congenital bilateral absence of the vas deferens) bearing rare mutations. Standard strategies for detecting point mutations rely on extensive scanning of the gene by denaturing gradient gel electrophoresis or denaturing high performance liquid chromatography, which are time-consuming. Moreover, the haplotyping of IVS8-(TG)m and Tn tracts is still challenging despite several recent improvements. We have optimized both the detection of mutations and the haplotyping of IVS8 polyvariants in developing two methods: i) a rapid and robust direct sequence analysis of all exons/flanking introns of the CFTR gene based on single condition touchdown amplification/sequencing in 96-well plates, and ii) a fluorescent assay that allows haplotyping of IVS8-(TG)mTn even without family linkage study. Combined with search for rare large rearrangements, this strategy detected 87.9% of CFTR defects in congenital bilateral absence of the vas deferens patients, a proportion considerably higher than those usually reported. These highly efficient tests, scanning each sample in a few days, greatly improve the genotyping of patients with CFTR-related symptoms and may be particularly important in emergency situations such as fetus with hyperechogenic bowel suggestive of cystic fibrosis.

Large genomic rearrangements in the CFTR gene contribute to CBAVD.

BACKGROUND: By performing extensive scanning of whole coding and flanking sequences of the CFTR (Cystic Fibrosis Transmembrane Conductance Regulator) gene, we had previously identified point mutations in 167 out of 182 (91.7%) males with isolated congenital bilateral absence of the vas deferens (CBAVD). Conventional PCR-based methods of mutation analysis do not detect gross DNA lesions. In this study, we looked for large rearrangements within the whole CFTR locus in the 32 CBAVD patients with only one or no mutation. METHODS: We developed a semi-quantitative fluorescent PCR assay (SQF-PCR), which relies on the comparison of the fluorescent profiles of multiplex PCR fragments obtained from different DNA samples. We confirmed the gross alterations by junction fragment amplification and identified their breakpoints by direct sequencing. RESULTS: We detected two large genomic heterozygous deletions, one encompassing exon 2 (c.54-5811_c.164+2186del8108ins182) [or CFTRdele2], the other removing exons 22 to 24 (c.3964-3890_c.4443+3143del9454ins5) [or CFTRdele 22_24], in two males carrying a typical CBAVD mutation on the other parental CFTR allele. We present the first bioinformatic tool for exon phasing of the CFTR gene, which can help to rename the exons and the nomenclature of small mutations according to international recommendations and to predict the consequence of large rearrangements on the open reading frame. CONCLUSION: Identification of large rearrangements further expands the CFTR mutational spectrum in CBAVD and should now be systematically investigated. We have designed a simple test to specifically detect the presence or absence of the two rearrangements identified in this study.

Haplotype block structure study of the CFTR gene. Most variants are associated with the M470 allele in several European populations.

An average of about 1700 CFTR (cystic fibrosis transmembrane conductance regulator) alleles from normal individuals from different European populations were extensively screened for DNA sequence variation. A total of 80 variants were observed: 61 coding SNSs (results already published), 13 noncoding SNSs, three STRs, two short deletions, and one nucleotide insertion. Eight DNA variants were classified as non-CF causing due to their high frequency of occurrence. Through this survey the CFTR has become the most exhaustively studied gene for its coding sequence variability and, though to a lesser extent, for its noncoding sequence variability as well. Interestingly, most variation was associated with the M470 allele, while the V470 allele showed an 'extended haplotype homozygosity' (EHH). These findings make us suggest a role for selection acting either on the M470V itself or through an hitchhiking mechanism involving a second site. The possible ancient origin of the V allele in an 'out of Africa' time frame is discussed.