RESUMO
Resetting of the epigenome in human primordial germ cells (hPGCs) is critical for development. We show that the transcriptional program of hPGCs is distinct from that in mice, with co-expression of somatic specifiers and naive pluripotency genes TFCP2L1 and KLF4. This unique gene regulatory network, established by SOX17 and BLIMP1, drives comprehensive germline DNA demethylation by repressing DNA methylation pathways and activating TET-mediated hydroxymethylation. Base-resolution methylome analysis reveals progressive DNA demethylation to basal levels in week 5-7 in vivo hPGCs. Concurrently, hPGCs undergo chromatin reorganization, X reactivation, and imprint erasure. Despite global hypomethylation, evolutionarily young and potentially hazardous retroelements, like SVA, remain methylated. Remarkably, some loci associated with metabolic and neurological disorders are also resistant to DNA demethylation, revealing potential for transgenerational epigenetic inheritance that may have phenotypic consequences. We provide comprehensive insight on early human germline transcriptional network and epigenetic reprogramming that subsequently impacts human development and disease.
Assuntos
Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Genoma Humano , Células Germinativas/metabolismo , Animais , Metilação de DNA , Embrião de Mamíferos/metabolismo , Feminino , Humanos , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Regiões Promotoras Genéticas , RetroelementosRESUMO
During female germline development, oocytes become a highly specialized cell type and form a maternal cytoplasmic store of crucial factors. Oocyte growth is triggered at the transition from primordial to primary follicle and is accompanied by dynamic changes in gene expression1, but the gene regulatory network that controls oocyte growth remains unknown. Here we identify a set of transcription factors that are sufficient to trigger oocyte growth. By investigation of the changes in gene expression and functional screening using an in vitro mouse oocyte development system, we identified eight transcription factors, each of which was essential for the transition from primordial to primary follicle. Notably, enforced expression of these transcription factors swiftly converted pluripotent stem cells into oocyte-like cells that were competent for fertilization and subsequent cleavage. These transcription-factor-induced oocyte-like cells were formed without specification of primordial germ cells, epigenetic reprogramming or meiosis, and demonstrate that oocyte growth and lineage-specific de novo DNA methylation are separable from the preceding epigenetic reprogramming in primordial germ cells. This study identifies a core set of transcription factors for orchestrating oocyte growth, and provides an alternative source of ooplasm, which is a unique material for reproductive biology and medicine.
Assuntos
Oócitos/metabolismo , Oogênese/genética , Fatores de Transcrição/metabolismo , Animais , Linhagem da Célula , Epigênese Genética , Feminino , Fertilização , Meiose , Metilação , Camundongos , Oócitos/citologia , Folículo Ovariano/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismoRESUMO
Infertility affects around 7% of the male population and can be due to severe spermatogenic failure (SPGF), resulting in no or very few sperm in the ejaculate. We initially identified a homozygous frameshift variant in FKBP6 in a man with extreme oligozoospermia. Subsequently, we screened a total of 2,699 men with SPGF and detected rare bi-allelic loss-of-function variants in FKBP6 in five additional persons. All six individuals had no or extremely few sperm in the ejaculate, which were not suitable for medically assisted reproduction. Evaluation of testicular tissue revealed an arrest at the stage of round spermatids. Lack of FKBP6 expression in the testis was confirmed by RT-qPCR and immunofluorescence staining. In mice, Fkbp6 is essential for spermatogenesis and has been described as being involved in piRNA biogenesis and formation of the synaptonemal complex (SC). We did not detect FKBP6 as part of the SC in normal human spermatocytes, but small RNA sequencing revealed that loss of FKBP6 severely impacted piRNA levels, supporting a role for FKBP6 in piRNA biogenesis in humans. In contrast to findings in piRNA-pathway mouse models, we did not detect an increase in LINE-1 expression in men with pathogenic FKBP6 variants. Based on our findings, FKBP6 reaches a "strong" level of evidence for being associated with male infertility according to the ClinGen criteria, making it directly applicable for clinical diagnostics. This will improve patient care by providing a causal diagnosis and will help to predict chances for successful surgical sperm retrieval.
Assuntos
Azoospermia , Infertilidade Masculina , Animais , Azoospermia/genética , Humanos , Infertilidade Masculina/genética , Elementos Nucleotídeos Longos e Dispersos , Masculino , Camundongos , RNA Interferente Pequeno/metabolismo , Sêmen , Espermatogênese/genética , Proteínas de Ligação a Tacrolimo/genética , Proteínas de Ligação a Tacrolimo/metabolismo , Testículo/patologiaRESUMO
Gametes are highly specialized cells that can give rise to the next generation through their ability to generate a totipotent zygote. In mice, germ cells are first specified in the developing embryo around embryonic day (E) 6.25 as primordial germ cells (PGCs). Following subsequent migration into the developing gonad, PGCs undergo a wave of extensive epigenetic reprogramming around E10.5-E11.5, including genome-wide loss of 5-methylcytosine. The underlying molecular mechanisms of this process have remained unclear, leading to our inability to recapitulate this step of germline development in vitro. Here we show, using an integrative approach, that this complex reprogramming process involves coordinated interplay among promoter sequence characteristics, DNA (de)methylation, the polycomb (PRC1) complex and both DNA demethylation-dependent and -independent functions of TET1 to enable the activation of a critical set of germline reprogramming-responsive genes involved in gamete generation and meiosis. Our results also reveal an unexpected role for TET1 in maintaining but not driving DNA demethylation in gonadal PGCs. Collectively, our work uncovers a fundamental biological role for gonadal germline reprogramming and identifies the epigenetic principles of the PGC-to-gonocyte transition that will help to guide attempts to recapitulate complete gametogenesis in vitro.
Assuntos
Reprogramação Celular/genética , Epigênese Genética , Gametogênese/genética , Células Germinativas/citologia , Células Germinativas/metabolismo , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/metabolismo , Animais , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Feminino , Masculino , Meiose , Camundongos , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismoRESUMO
The ACMG framework for variant interpretation is well-established and widely used. Although formal guidelines have been published on the establishment of the gene-disease relationships as well, these are not nearly as widely acknowledged or utilized, and implementation of these guidelines is lagging. In addition, for many genes so little information is available that the framework cannot be used in sufficient detail. In this manuscript, we highlight the importance of distinguishing between phenotype-first and genotype-first gene-disease relationships. We discuss the approaches currently available to establish gene-disease relationships and suggest a checklist to assist in evaluating gene-disease relationships for genes with very little available information. Several real-life examples from clinical practice are given to illustrate the importance of a thorough thought process on gene-disease relationships. We hope that these considerations and the checklist will provide help for clinicians and clinical scientists faced which variants in genes without robustly ascertained gene-disease relationships.
Assuntos
Doenças Raras , Humanos , Fenótipo , Doenças Raras/diagnóstico , Doenças Raras/genéticaRESUMO
The power of mouse embryonic stem (ES) cells to colonise the developing embryo has revolutionised mammalian developmental genetics and stem cell research. This power is vulnerable, however, to the cell culture environment, deficiencies in which can lead to cellular heterogeneity, adaptive phenotypes, epigenetic aberrations and genetic abnormalities. Here, we provide detailed methodologies for derivation, propagation, genetic modification and primary differentiation of ES cells in 2i or 2i+LIF media without serum or undefined serum substitutes. Implemented diligently, these procedures minimise variability and deviation, thereby improving the efficiency, reproducibility and biological validity of ES cell experimentation.
Assuntos
Diferenciação Celular/genética , Embrião de Mamíferos/citologia , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias Murinas/citologia , Animais , Sistemas CRISPR-Cas , Técnicas de Cultura de Células , Ciclo Celular , Técnicas de Cocultura , Meios de Cultura/química , Humanos , Cariotipagem , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , RNA Interferente Pequeno/genética , Transdução de SinaisRESUMO
Cap analysis of gene expression (CAGE) is a methodology for genome-wide quantitative mapping of mRNA 5' ends to precisely capture transcription start sites at a single nucleotide resolution. In combination with high-throughput sequencing, CAGE has revolutionized our understanding of the rules of transcription initiation, led to discovery of new core promoter sequence features, and discovered transcription initiation at enhancers genome-wide. The biggest limitation of CAGE is that even the most recently improved version (nAnT-iCAGE) still requires large amounts of total cellular RNA (5 µg), preventing its application to scarce biological samples such as those from early embryonic development or rare cell types. Here, we present SLIC-CAGE, a Super-Low Input Carrier-CAGE approach to capture 5' ends of RNA polymerase II transcripts from as little as 5-10 ng of total RNA. This dramatic increase in sensitivity is achieved by specially designed, selectively degradable carrier RNA. We demonstrate the ability of SLIC-CAGE to generate data for genome-wide promoterome with 1000-fold less material than required by existing CAGE methods, by generating a complex, high-quality library from mouse embryonic day 11.5 primordial germ cells.
Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , RNA Mensageiro/genética , Análise de Sequência de RNA/métodos , Sítio de Iniciação de Transcrição , Animais , Biblioteca Gênica , Camundongos , Regiões Promotoras GenéticasRESUMO
Stem cells self-renew and generate specialized progeny through differentiation, but vary in the range of cells and tissues they generate, a property called developmental potency. Pluripotent stem cells produce all cells of an organism, while multipotent or unipotent stem cells regenerate only specific lineages or tissues. Defining stem-cell potency relies upon functional assays and diagnostic transcriptional, epigenetic and metabolic states. Here we describe functional and molecular hallmarks of pluripotent stem cells, propose a checklist for their evaluation, and illustrate how forensic genomics can validate their provenance.
Assuntos
Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Diferenciação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Genômica , HumanosRESUMO
Genomic imprinting directs the allele-specific marking and expression of loci according to their parental origin. Differential DNA methylation at imprinted control regions (ICRs) is established in gametes and, although largely preserved through development, can be experimentally reset by fusing somatic cells with embryonic germ cell (EGC) lines. Here, we show that the Ten-Eleven Translocation proteins Tet1 and Tet2 participate in the efficient erasure of imprints in this model system. The fusion of B cells with EGCs initiates pluripotent reprogramming, in which rapid re-expression of Oct4 is accompanied by an accumulation of 5-hydroxymethylcytosine (5hmC) at several ICRs. Tet2 was required for the efficient reprogramming capacity of EGCs, whereas Tet1 was necessary to induce 5-methylcytosine oxidation specifically at ICRs. These data show that the Tet1 and Tet2 proteins have discrete roles in cell-fusion-mediated pluripotent reprogramming and imprint erasure in somatic cells.
Assuntos
Fusão Celular , Proteínas de Ligação a DNA/fisiologia , Impressão Genômica , Proteínas Proto-Oncogênicas/fisiologia , 5-Metilcitosina/análogos & derivados , Animais , Linfócitos B/citologia , Sequência de Bases , Linhagem Celular , Citosina/análogos & derivados , Citosina/metabolismo , Metilação de DNA , Dioxigenases , Células-Tronco Embrionárias/citologia , Expressão Gênica , Células Germinativas/citologia , Proteínas de Fluorescência Verde/biossíntese , Humanos , Fator de Crescimento Insulin-Like II/genética , Camundongos , Dados de Sequência Molecular , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Polimorfismo de Nucleotídeo Único , Proteínas/genética , Proteínas/metabolismo , RNA Longo não Codificante/genética , Análise de Sequência de DNARESUMO
Mouse pluripotent embryonic stem (ES) cells can exist in distinct yet interchangeable epigenetic states dictated by their culture environment. Previous reports have shown that naïve pluripotent cells grown in the presence of 2i are characterised by global DNA hypomethylation and changes in the abundance and distribution of histone modifications. New research provides insights regarding how this might be achieved.
Assuntos
Metilação de DNA/genética , Células-Tronco Embrionárias , Epigênese Genética , Células-Tronco Pluripotentes , Animais , Diferenciação Celular/genética , Código das Histonas/genética , CamundongosRESUMO
Naive pluripotency refers to the capacity of single cells in regulative embryos to engender all somatic and germline cell types. Only germ cells - conventionally considered to be unipotent - can naturally re-acquire pluripotency, by cycling through fertilisation. Furthermore, primordial germ cells express, and appear to be functionally dependent upon, transcription factors that characterise the pluripotent state. We hypothesise that germ cells require pluripotency factors to control a de-restricted epigenome. Consequently, they harbour latent potential, as manifested in teratocarcinogenesis or direct conversion into pluripotent stem cells in vitro. Thus, we suggest that there exists an unbroken cycle of pluripotency, naive in the early epiblast and latent in the germline, that is sustained by a shared transcription factor network.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/citologia , Óvulo/citologia , Células-Tronco Pluripotentes/citologia , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Diferenciação Celular , Linhagem da Célula , Reprogramação Celular , Metilação de DNA , Epigênese Genética , Gametogênese , Camadas Germinativas/metabolismo , Masculino , Mamíferos , Camundongos , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Óvulo/metabolismo , Células-Tronco Pluripotentes/metabolismo , Espermatozoides/citologia , Espermatozoides/metabolismoRESUMO
Primordial germ cells (PGCs) are the founder cells of the germline. Via gametogenesis and fertilisation this lineage generates a new embryo in the next generation. PGCs are also the cell of origin of multilineage teratocarcinomas. In vitro, mouse PGCs can give rise to embryonic germ (EG) cells - pluripotent stem cells that can contribute to primary chimaeras when introduced into pre-implantation embryos. Thus, PGCs can give rise to pluripotent cells in the course of the developmental cycle, during teratocarcinogenesis and by in vitro culture. However, there is no evidence that PGCs can differentiate directly into somatic cell types. Furthermore, it is generally assumed that PGCs do not contribute to chimaeras following injection into the early mouse embryo. However, these data have never been formally published. Here, we present the primary data from the original PGC-injection experiments performed 40 years ago, alongside results from more recent studies in three separate laboratories. These results have informed and influenced current models of the relationship between pluripotency and the germline cycle. Current technologies allow further experiments to confirm and expand upon these findings and allow definitive conclusions as to the developmental potency of PGCs.
Assuntos
Embrião de Mamíferos/citologia , Células Germinativas/citologia , Animais , Linhagem da Célula , Feminino , Camundongos , GravidezRESUMO
Primordial germ cells (PGCs) and somatic cells originate from postimplantation epiblast cells in mice. As pluripotency is lost upon differentiation of somatic lineages, a naive epigenome and the pluripotency network are re-established during PGC development. Here we demonstrate that Prdm14 contributes not only to PGC specification, but also to naive pluripotency in embryonic stem (ES) cells by repressing the DNA methylation machinery and fibroblast growth factor (FGF) signalling. This indicates a critical role for Prdm14 in programming PGCs and promoting pluripotency in ES cells.
Assuntos
Células-Tronco Embrionárias/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica no Desenvolvimento , Células Germinativas/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição/genética , Animais , Diferenciação Celular , Metilação de DNA , Proteínas de Ligação a DNA , Células-Tronco Embrionárias/citologia , Fatores de Crescimento de Fibroblastos/metabolismo , Perfilação da Expressão Gênica , Células Germinativas/citologia , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Células-Tronco Pluripotentes/citologia , Proteínas de Ligação a RNA , Transdução de Sinais , Fatores de Transcrição/metabolismoRESUMO
Mutations in DNA damage response (DDR) factors are associated with human infertility, which affects up to 15% of the population. The DDR is required during germ cell development and meiosis. One pathway implicated in human fertility is DNA translesion synthesis (TLS), which allows replication impediments to be bypassed. We find that TLS is essential for pre-meiotic germ cell development in the embryo. Loss of the central TLS component, REV1, significantly inhibits the induction of human PGC-like cells (hPGCLCs). This is recapitulated in mice, where deficiencies in TLS initiation (Rev1-/- or PcnaK164R/K164R) or extension (Rev7 -/-) result in a > 150-fold reduction in the number of primordial germ cells (PGCs) and complete sterility. In contrast, the absence of TLS does not impact the growth, function, or homeostasis of somatic tissues. Surprisingly, we find a complete failure in both activation of the germ cell transcriptional program and in DNA demethylation, a critical step in germline epigenetic reprogramming. Our findings show that for normal fertility, DNA repair is required not only for meiotic recombination but for progression through the earliest stages of germ cell development in mammals.
Assuntos
Desmetilação do DNA , Reparo do DNA , DNA Polimerase Dirigida por DNA , Células Germinativas , Animais , Feminino , Humanos , Masculino , Camundongos , Dano ao DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Epigênese Genética , Células Germinativas/metabolismo , Meiose/genética , Camundongos Knockout , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Síntese de DNA TranslesãoRESUMO
Primordial germ cells (PGCs) are the earliest precursors of the gametes. During normal development, PGCs only give rise to oocytes or spermatozoa. However, PGCs can acquire pluripotency in vitro by forming embryonic germ (EG) cells and in vivo during teratocarcinogenesis. Classic embryological experiments directly assessed the potency of PGCs by injection into the pre-implantation embryo. As no contribution to embryos or adult mice was observed, PGCs have been described as unipotent. Here, we demonstrate that PGCs injected into 8-cell embryos can initially survive, divide, and contribute to the developing inner cell mass. Apoptosis-deficient PGCs exhibit improved survival in isolated epiblasts and can form naive pluripotent embryonic stem cell lines. However, contribution to the post-implantation embryo is limited, with no functional incorporation observed. In contrast, PGC-like cells show an extensive contribution to mid-gestation chimeras. We thus propose that PGC formation in vivo establishes a latent form of pluripotency that restricts chimera contribution.
Assuntos
Células Germinativas , Células-Tronco Pluripotentes , Masculino , Camundongos , Animais , Células Germinativas/metabolismo , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes/metabolismo , Espermatozoides , Camadas Germinativas , Diferenciação CelularRESUMO
Mouse and rat embryonic stem cells can be sustained in defined medium by dual inhibition (2i) of the mitogen-activated protein kinase (Erk1/2) cascade and of glycogen synthase kinase 3. The inhibitors suppress differentiation and enable self-renewal of pluripotent cells that are ex vivo counterparts of naïve epiblast cells in the mature blastocyst. Pluripotent stem cell lines can also be derived from unipotent primordial germ cells via a poorly understood process of epigenetic reprogramming. These are termed embryonic germ (EG) cells to denote their distinct origin. Here we investigate whether EG cell self-renewal and derivation are supported by 2i. We report that mouse EG cells can be established with high efficiency using 2i in combination with the cytokine leukaemia inhibitory factor (LIF). Furthermore, addition of fibroblast growth factor or stem cell factor is unnecessary using 2i-LIF. The derived EG cells contribute extensively to healthy chimaeric mice, including to the germline. Using the same conditions, we describe the first derivations of EG cells from the rat. Rat EG cells express a similar marker profile to rat and mouse ES cells. They have a diploid karyotype, can be clonally expanded and genetically manipulated, and are competent for multilineage colonisation of chimaeras. These findings lend support to the postulate of a conserved molecular ground state in pluripotent rodent cells. Future research will determine the extent to which this is maintained in other mammals and whether, in some species, primordial germ cells might be a more tractable source than epiblast for the capture of naïve pluripotent stem cells.
Assuntos
Células Germinativas/citologia , Células Germinativas/fisiologia , Camadas Germinativas/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Animais , Blastocisto/metabolismo , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Células Cultivadas , Quimera/metabolismo , Cruzamentos Genéticos , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Células Germinativas/metabolismo , Camadas Germinativas/metabolismo , Fator Inibidor de Leucemia/metabolismo , Fator Inibidor de Leucemia/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos , Camundongos Transgênicos , Ratos , Ratos Sprague-DawleyRESUMO
Cultured pluripotent stem cells hold great promise for regenerative medicine. Considerable efforts have been invested into the refinement and definition of improved culture systems that sustain self-renewal and avoid differentiation of pluripotent cells in vitro. Recent studies have, however, found that the choice of culture condition has a significant impact on epigenetic profiles of cultured pluripotent cells. Mouse and human ESCs (embryonic stem cells) show substantial epigenetic differences that are dependent on the culture condition, including global changes to DNA methylation and histone modifications and, in female human ESCs, to the epigenetic process of X chromosome inactivation. Epigenetic perturbations have also been detected during culture of pre-implantation embryos; limited research undertaken in mouse suggests a direct effect of the in vitro environment on epigenetic processes in this system. Widespread epigenetic changes induced by the culture condition in stem cells thus emphasize the necessity for extensive research into both immediate and long-term epigenetic effects of embryo culture during assisted reproductive technologies.
Assuntos
Blastocisto/citologia , Técnicas de Cultura de Células/métodos , Epigênese Genética/fisiologia , Células-Tronco Pluripotentes/citologia , Animais , Blastocisto/metabolismo , Blastocisto/fisiologia , Células Cultivadas , Técnicas de Cultura Embrionária , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Humanos , Camundongos , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologia , Técnicas de Reprodução AssistidaRESUMO
BACKGROUND: Very long-chain fatty acids (VLCFAs) composed of more than 20 carbon atoms are essential in the biosynthesis of cell membranes in the brain, skin, and retina. VLCFAs are elongated beyond 28 carbon atoms by ELOVL4 enzyme. Variants in ELOVL4 are associated with three Mendelian disorders: autosomal dominant (AD) Stargardt-like macular dystrophy type 3, AD spinocerebellar ataxia, and autosomal recessive disorder congenital ichthyosis, spastic quadriplegia and impaired intellectual development (ISQMR). Only seven subjects from five unrelated families with ISQMR have been described, all of which have biallelic single-nucleotide variants. METHODS: We performed clinical exome sequencing on probands from four unrelated families with neuro-ichthyosis. RESULTS: We identified three novel homozygous ELOVL4 variants. Two of the families originated from the same Saudi tribe and had the exact homozygous exonic deletion in ELOVL4, while the third and fourth probands had two different novel homozygous missense variants. Seven out of the eight affected subjects had profound developmental delay, epilepsy, axial hypotonia, peripheral hypertonia, and ichthyosis. Delayed myelination and corpus callosum hypoplasia were seen in two of five subjects with brain magnetic rosonance imaging and cerebral atrophy in three. CONCLUSION: Our study expands the allelic spectrum of ELOVL4-related ISQMR. The detection of the same exonic deletion in two unrelated Saudi family from same tribe suggests a tribal founder mutation.