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InternalMed_Harrison. Disorders that are frequently or prominently associated with mutations in a particular gene are shown in boldface. Diseases due to mutations that impair mitochondrial protein synthesis are shown in blue. Diseases due to mutations in protein-coding genes are shown in red. ECM, encephalomyopathy; FBSN, familial bilateral striatal necrosis; LHON, Leber’s hereditary optic neuropathy; LS, Leigh syndrome; MELAS, mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; MERRF, myoclonic epilepsy with ragged red fibers; MILS, maternally inherited Leigh syndrome; NARP, neuropathy, ataxia, and retinitis pigmentosa; PEO, progressive external ophthalmoplegia; PPK, palmoplantar keratoderma; SIDS, sudden infant death syndrome. (Reproduced with permission from S DiMauro, E Schon: Mitochondrial respiratory-chain diseases. N Engl J Med 348:2656, 2003.) (or with the somatic accumulation of mtDNA mutations, see below) can show a mosaic pattern of reduced histochemical staining in


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InternalMed_Harrison. Mitochondrial respiratory-chain diseases. N Engl J Med 348:2656, 2003.) (or with the somatic accumulation of mtDNA mutations, see below) can show a mosaic pattern of reduced histochemical staining in comparison with histochemical staining for the complex II enzyme, succinate dehydrogenase (Fig. 85e-5). Heteroplasmy can also be detected at the genetic level through direct Sanger-type mtDNA genotyping under special conditions, although clinically significant low levels of heteroplasmy can escape detection in genomic samples extracted from whole blood using conventional genotyping and sequencing techniques.


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InternalMed_Harrison. The emerging next-generation sequencing (NGS) techniques and their rapid penetration and recognition as useful clinical diagnostic tools are expected to also dramatically improve the clinical genetic diagnostic evaluation of mitochondrial diseases at the level of both the nuclear genome and mtDNA. In the context of the larger nuclear genome, the ability of NGS techniques to dramatically increase the speed at which DNA can be sequenced at a fraction of the cost of conventional Sanger-type sequencing technology is particularly beneficial. Low sequencing costs and short turnaround time expedite “first-tier” screening of panels of hundreds of previously known or suspected mitochondrial disease genes or screening for the entire exome or genome in an attempt to identify novel genes and mutations affecting different patients or families. In the context of the mtDNA, NGS approaches hold the particular promise for rapid and reliable detection of heteroplasmy in different affected tissues.


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InternalMed_Harrison. affecting different patients or families. In the context of the mtDNA, NGS approaches hold the particular promise for rapid and reliable detection of heteroplasmy in different affected tissues. Although Sanger sequencing allows for complete coverage of the mtDNA, it is limited by the lack of deep coverage and low sensitivity for heteroplasmy detection when it is much less than 50%. In contrast, NGS technology is an excellent tool for rapidly and accurately obtaining a patient’s predominant mtDNA sequence and also lower frequency heteroplasmic variants. This is enabled by deep coverage of the genome through multiple independent sequence reads. Accordingly, recent studies making use of NGS techniques have demonstrated sequence accuracy equivalent to Sanger-type sequencing, but also have uncovered heretofore unappreciated heteroplasmy rates ranging between 10 and 50% and detection of single-nucleotide heteroplasmy down to levels of <10%.


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InternalMed_Harrison. Clinically, the most striking overall characteristic of mitochondrial genetic disease is the phenotypic heterogeneity associated with mtDNA mutations. This extends to intrafamilial phenotypic heterogeneity for the same mtDNA pathogenic mutation and, conversely, to the overlap of phenotypic disease manifestations with distinct mutations. Thus, although fairly consistent and well-defined “classic” syndromes have been attributed to specific mutations, frequently “nonclassic” combinations of disease phenotypes ranging from isolated myopathy to extensive multi-system disease are often encountered, rendering genotype-phenotype correlation challenging. In both classical and nonclassical mtDNA disorders, there is often a clustering of some combination of abnormalities affecting the neurologic system (including optic nerve atrophy, pigment retinopathy, and sensorineural hearing loss), cardiac and skeletal muscle (including extraocular muscles), and endocrine and metabolic systems (including


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InternalMed_Harrison. (including optic nerve atrophy, pigment retinopathy, and sensorineural hearing loss), cardiac and skeletal muscle (including extraocular muscles), and endocrine and metabolic systems (including diabetes mellitus).


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InternalMed_Harrison. Additional organ systems that may be affected include the hematopoietic, renal, hepatic, and gastrointestinal systems, although these are more frequently involved in infants and children. Disease-causing mtDNA coding region mutations can affect either one of the 13 protein encoding genes or one of the 24 protein synthetic genes. Clinical manifestations do not readily distinguish these two categories, although lactic acidosis and muscle pathologic findings tend to be more prominent in the latter. In all cases, either defective ATP production due to disturbances in the ETC or enhanced generation of ROS has been invoked as the mediating biochemical mechanism between mtDNA mutation and disease manifestation.


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InternalMed_Harrison. The clinical presentation of adult patients with mtDNA disease can be divided into three categories: (1) clinical features suggestive of mitochondrial disease (Table 85e-2), but not a well-defined classic syndrome; (2) classic mtDNA syndromes; and (3) clinical presentation confined to one organ system (e.g., isolated sensorineural deafness, cardiomyopathy, or diabetes mellitus). Table 85e-3 provides a summary of eight illustrative classic mtDNA syndromes or disorders that affect adult patients and highlights some of the most interesting features of mtDNA disease in terms of molecular pathogenesis, inheritance, and clinical presentation. The first five of these syndromes result from heritable point mutations in either protein-encoding or protein synthetic mtDNA genes; the other three result from rearrangements or deletions that usually do not involve the germline.


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InternalMed_Harrison. are often homoplasmic for the disease-85e-5 causing mutation. The somewhat later onset in young adulthood and modifying effect of protective background nuclear genomic haplotypes may have enabled homoplasmic pathogenic mutations to have escaped evolutionary censoring.


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InternalMed_Harrison. Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a multisystem disorder with a typical onset between 2 to 10 years of age. Following normal early psychomotor development, the most common initial symptoms are seizures, recurrent headaches, anorexia, and recurrent vomiting. Exercise intolerance or proximal limb weakness can be the initial manifestation, followed by generalized tonic-clonic seizures. Short stature is common. Seizures are often associated with stroke-like episodes of transient FIGURE 85e-5 Cytochrome c oxidase (COX) deficiency in mitochondrial DNA (mtDNA)– hemiparesis or cortical blindness that associated disease. Transverse tissue sections that have been stained for COX and succinate dehy-may produce altered consciousness drogenase (SDH) activities sequentially, with COX-positive cells shown in brown and COX-deficient and may recur. The cumulative residcells shown in blue. A. Skeletal muscle from a patient with a heteroplasmic


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InternalMed_Harrison. (SDH) activities sequentially, with COX-positive cells shown in brown and COX-deficient and may recur. The cumulative residcells shown in blue. A. Skeletal muscle from a patient with a heteroplasmic mitochondrial tRNA ual effects of the stroke-like episodes point mutation. The section shows a typical “mosaic” pattern of COX activity, with many muscle gradually impair motor abilities, vision, fibers harboring levels of mutated mtDNA that are above the crucial threshold to produce a func-and cognition, often by adolescence tional enzyme complex. B. Cardiac tissue (left ventricle) from a patient with a homoplasmic tRNA or young adulthood. Sensorineural mutation that causes hypertrophic cardiomyopathy, which demonstrates an absence of COX in hearing loss adds to the progressive most cells. C. A section of cerebellum from a patient with mtDNA rearrangement that highlights decline of these individuals. A plethora the presence of COX-deficient neurons. D, E. Tissues that show COX deficiency


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InternalMed_Harrison. A section of cerebellum from a patient with mtDNA rearrangement that highlights decline of these individuals. A plethora the presence of COX-deficient neurons. D, E. Tissues that show COX deficiency due to clonal expan-of less common symptoms have been sion of somatic mtDNA mutations within single cells—a phenomenon that is seen in both post-described including myoclonus, ataxia, mitotic cells (D; extraocular muscles) and rapidly dividing cells (E; colonic crypt) in aging humans. episodic coma, optic atrophy, cardio(Reproduced with permission from R Taylor, D Turnbull: Mitochondrial DNA mutations in human disease. myopathy, pigmentary retinopathy,


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Nat Rev Genetics 6:389, 2005.)

InternalMed_Harrison. Nat Rev Genetics 6:389, 2005.)


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InternalMed_Harrison. Leber’s hereditary optic neuropathy (LHON) is a common cause of maternally inherited visual failure. LHON typically presents during young adulthood with subacute painless loss of vision in one eye, with symptoms developing in the other eye 6–12 weeks after the initial onset. In some instances, cerebellar ataxia, peripheral neuropathy, and cardiac conduction defects are observed. In >95% of cases, LHON is due to one of three homoplasmic point mutations of mtDNA that affect genes encoding different subunits of complex I of the mitochondrial ETC; however, not all individuals who inherit a primary LHON mtDNA mutation develop optic neuropathy, and males are four to five times more likely than females to be affected, indicating that additional environmental (e.g., tobacco exposure) or genetic factors are important in the etiology of the disorder. Both the nuclear and mitochondrial genomic backgrounds modify disease penetrance. Indeed, a region of the X chromosome containing a high-risk


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InternalMed_Harrison. factors are important in the etiology of the disorder. Both the nuclear and mitochondrial genomic backgrounds modify disease penetrance. Indeed, a region of the X chromosome containing a high-risk haplotype for LHON was recently identified, supporting the formulation that nuclear genes act as modifiers and affording an explanation for the male prevalence of LHON. This haplotype can be used in predictive genomic testing and prenatal screening for this disease. In contrast to the other classic mtDNA disorders, it is of interest that patients with this syndrome


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InternalMed_Harrison. Neurologic: stroke, epilepsy, migraine headache, peripheral neuropathy, cranial neuropathy (optic atrophy, sensorineural deafness, dysphagia, dysphasia) Skeletal myopathy: ophthalmoplegia, exercise intolerance, myalgia Cardiac: conduction block, cardiomyopathy Respiratory: hypoventilation, aspiration pneumonitis Endocrine: diabetes mellitus, premature ovarian failure, hypothyroidism, hypoparathyroidism


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InternalMed_Harrison. Ophthalmologic: cataracts, pigment retinopathy, neurologic and myopathic (optic atrophy, ophthalmoplegia) ophthalmoplegia, diabetes mellitus, hir sutism, gastrointestinal dysmotility, and nephropathy. The typical age of death ranges from 10 to 35 years, but some individuals live into their sixth decade. Intercurrent infections or intestinal obstructions are often the terminal events. Laboratory investigation commonly demonstrates elevated lactate concentrations at rest with excessive increase after moderate exercise. Brain imaging during stroke-like episodes shows areas of increased T2 signal, typically involving the posterior cerebrum and not conforming to the distribution of major arteries. Electrocardiogram (ECG) may show evidence of cardiomyopathy, preexcitation, or incomplete heart block. Electromyography and nerve conduction studies are consistent with a myopathic process, but axonal and sensory neuropathy may coexist. Muscle biopsy typically shows ragged red fibers with the


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InternalMed_Harrison. block. Electromyography and nerve conduction studies are consistent with a myopathic process, but axonal and sensory neuropathy may coexist. Muscle biopsy typically shows ragged red fibers with the modified Gomori trichrome stain or “ragged blue fibers” resulting from the hyperintense reaction with the histochemical staining for succinate dehydrogenase. The diagnosis of MELAS is based on a combination of clinical findings and molecular genetic testing. Mutations in the mtDNA gene MT-TL1 encoding tRNAleu are causative. The most common mutation, present in approximately 80% of individuals with typical clinical findings, is an A-to-G transition at nucleotide 3243 (m.3243A>G). Mutations can usually be detected in mtDNA from leukocytes in individuals with typical MELAS; however, the occurrence of heteroplasmy can result in varying tissue distribution of mutated mtDNA. In the absence of specific treatment, various manifestations of MELAS are treated according to standard modalities for


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InternalMed_Harrison. of heteroplasmy can result in varying tissue distribution of mutated mtDNA. In the absence of specific treatment, various manifestations of MELAS are treated according to standard modalities for prevention, surveillance, and treatment.


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InternalMed_Harrison. Myoclonic epilepsy with ragged red fibers (MERRF) is a multisystem disorder characterized by myoclonus, seizures, ataxia, and myopathy with ragged red fibers. Hearing loss, exercise intolerance, neuropathy, and short stature are often present. Almost all MERRF patients have mutation in the mtDNA tRNAlys gene, and the m.8344A>G mutation in the mtDNA gene encoding the lysine amino acid tRNA is responsible for 80–90% of MERRF cases. Abbreviations: CSF, cerebrospinal fluid; NARP, neuropathy, ataxia, and retinitis pigmentosa.


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InternalMed_Harrison. Abbreviations: CSF, cerebrospinal fluid; NARP, neuropathy, ataxia, and retinitis pigmentosa. Neuropathy, ataxia, and retinitis pigmentosa (NARP) is characterized by moderate diffuse cerebral and cerebellar atrophy and symmetric lesions of the basal ganglia on magnetic resonance imaging (MRI). A heteroplasmic m.8993T>G mutation in the ATPase 6 subunit gene has been identified as causative. Ragged red fibers are not observed in muscle biopsy. When >95% of mtDNA molecules are mutant, a more severe clinical, neuroradiologic. and neuropathologic picture (Leigh syndrome) emerges. Point mutations in the mtDNA gene encoding the 12S rRNA result in heritable nonsyndromic hearing loss. One such mutation causes heritable ototoxic susceptibility to aminoglycoside antibiotics, which opens a pathway for a simple pharmacogenetic test in the appropriate clinical settings.


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InternalMed_Harrison. Kearns-Sayre syndrome (KSS), sporadic progressive external ophthalmoplegia (PEO), and Pearson syndrome are three disease phenotypes caused by large-scale mtDNA rearrangements including partial deletions or partial duplication. The majority of single large-scale rearrangements of mtDNA are thought to result from clonal amplification of a single sporadic mutational event, occurring in the maternal oocyte or during early embryonic development. Because germline involvement is rare, most cases are sporadic rather than inherited. KSS is characterized by the triad of onset before age 20, chronic progressive external ophthalmoplegia, and pigmentary retinopathy. Cerebellar syndrome, heart block, increased cerebrospinal fluid protein content, diabetes mellitus, and short stature are also part of the syndrome. Single deletions/duplication can also result in milder phenotypes such as PEO, characterized by late-onset progressive external ophthalmoplegia, proximal myopathy, and exercise


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InternalMed_Harrison. part of the syndrome. Single deletions/duplication can also result in milder phenotypes such as PEO, characterized by late-onset progressive external ophthalmoplegia, proximal myopathy, and exercise intolerance. In both KSS and PEO, diabetes mellitus and hearing loss are frequent accompaniments. Pearson syndrome is also characterized by diabetes mellitus from pancreatic insufficiency, together with pancytopenia and lactic acidosis, caused by the large-scale sporadic deletion of several mtDNA genes.


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InternalMed_Harrison. Two important dilemmas in classic mtDNA disease have benefited from recent important research insights. The first relates to the greater involvement of neuronal, muscular, renal, hepatic, and pancreatic manifestations in mtDNA disease in these syndromes. This observation has appropriately been mostly attributed to the high energy utilization of the involved tissues and organ systems and, hence, greater dependency on mitochondrial ETC integrity and health. However, because mutations are stochastic events, mitochondrial mutations should occur in any organ during embryogenesis and development. Recently, additional explanations have been suggested based on studies of the common m.3243A>G transition. The proportion of this mutation in peripheral blood cells was shown to decrease exponentially with age. m.1555A>G mutation in 12S Homoplasmic Maternal rRNA m.7445A>G mutation in 12S Homoplasmic Maternal rRNA Single deletions or duplications Heteroplasmic Mostly sporadic, somatic mutations


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InternalMed_Harrison. m.1555A>G mutation in 12S Homoplasmic Maternal rRNA m.7445A>G mutation in 12S Homoplasmic Maternal rRNA Single deletions or duplications Heteroplasmic Mostly sporadic, somatic mutations Large deletion Heteroplasmic Sporadic, somatic mutations The 5-kb “common deletion” Heteroplasmic Sporadic, somatic mutations A selective process acting at the stem cell level with a strong bias against the mutated form would have its greatest effect to reduce the mutant mtDNA only in highly proliferating cells, such as those derived from the hematopoietic system. Tissues and organs with lower cell turnover, such as those involved with mtDNA mutations, would not benefit from this effect and, thus, would be the most affected.


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InternalMed_Harrison. The other dilemma arises from the observation that only a subset of mtDNA mutations accounts for the majority of the familial mtDNA diseases. The random occurrence of mutations in the mtDNA sequence should yield a more uniform distribution of disease-causing mutations. However, recent studies using the introduction of one severe and one mild point mutation into the female germline of experimental animals demonstrated selective elimination during oogenesis of the severe mutation and selective retention of the milder mutation, with the emergence of mitochondrial disease in offspring after multiple generations. Thus, oogenesis itself can act as an “evolutionary” filter for mtDNA disease.


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InternalMed_Harrison. The clinical presentations of classic syndromes, groupings of disease manifestations in multiple organ systems, or unexplained isolated presentations of one of the disease features of a classic mtDNA syndrome should prompt a systematic clinical investigation as outlined in Fig. 85e-6. Indeed, mitochondrial disease should be considered in the differential diagnosis of any progressive multisystem disorder. Despite the centrality of disruptive oxidative phosphorylation, an elevated blood lactate level is neither specific nor sensitive because there are many causes of blood lactic acidosis, and many patients with mtDNA defects presenting in adulthood have normal blood lactate. An elevated cerebrospinal fluid lactate is a more specific test for mitochondrial disease if there is central nervous system involvement. The serum creatine kinase may be elevated but is often normal, even in the presence of a proximal myopathy. Urinary organic and amino acids may also be abnormal, reflecting


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InternalMed_Harrison. system involvement. The serum creatine kinase may be elevated but is often normal, even in the presence of a proximal myopathy. Urinary organic and amino acids may also be abnormal, reflecting metabolic and kidney proximal tubule dysfunction. Every patient with seizures or cognitive decline should have an electroencephalogram. A brain computed tomography (CT) scan may show calcified basal ganglia or bilateral hypodense regions with cortical atrophy. MRI is indicated in patients with brainstem signs or stroke-like episodes.


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InternalMed_Harrison. For some mitochondrial diseases, it is possible to obtain an accurate diagnosis with a simple molecular genetic screen. For examples, 95% of patients with LHON harbor one of three mtDNA point mutations (m.11778A>G, m.A3460A>G, or m.14484T>C). These patients have very high levels of mutated mtDNA in peripheral blood cells, and Blood: creatine kinase, liver functions, glucose, lactate Urine: organic and amino acids CSF: glucose, protein, lactate Cardiac x-ray, ECG, ECHO EEG, EMG, nerve conduction Brain CT/MRI PCR/RFLP analysis of blood for known mutations Specific point mutation syndrome: e.g., MELAS, MERRF, and LHON


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InternalMed_Harrison. PCR/RFLP analysis of blood for known mutations Specific point mutation syndrome: e.g., MELAS, MERRF, and LHON FIGURE 85e-6 Clinical and laboratory investigation of a suspected mitochondrial DNA (mtDNA) disorder. CSF, cerebrospinal fluid; CT, computed tomography; ECG, electrocardiogram; ECHO, echocardiography; EEG, electroencephalogram; EMG, electromyogram; LHON, Leber’s hereditary optic neuropathy; MELAS, mitochondrial encephalomyopathy, lactic acidosis, and stoke-like episodes; MERFF, myoclonic epilepsy with ragged red fibers; MRI, magnetic resonance imaging; PCR, polymerase chain reaction; RFLP, restriction fragment length polymorphism.


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InternalMed_Harrison. therefore, it is appropriate to send a blood sample for molecular genetic analysis by polymerase chain reaction (PCR) or restriction fragment length polymorphism. The same is true for most MERRF patients who harbor a point mutation in the lysine tRNA gene at position 8344. In contrast, patients with the m.3243A>G MELAS mutation often have low levels of mutated mtDNA in blood. If clinical suspicion is strong enough to warrant peripheral blood testing, then patients with a negative result should be investigated further by performing a skeletal muscle biopsy.


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InternalMed_Harrison. Muscle biopsy histochemical analysis is the cornerstone for investigation of patients with suspected mitochondrial disease. Histochemical analysis may show subsarcolemmal accumulation of mitochondria with the appearance of ragged red fibers. Electron microscopy might show abnormal mitochondria with paracrystalline inclusions. Muscle histochemistry may show cytochrome c oxidase (COX)–deficient fibers, which indicate mitochondrial dysfunction (Fig. 85e-5). Respiratory chain complex assays may also show reduced enzyme function. Either of these two abnormalities confirms the presence of a mitochondrial disease, to be followed by an in-depth molecular genetic analysis.


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InternalMed_Harrison. Recent evidence has provided important insights into the importance of nuclear-mtDNA genomic cross-talk and has provided a descriptive framework for classifying and understanding disorders that emanate from perturbations in this cross-talk. Although not strictly considered as mtDNA genetic disorders, manifestations do overlap those highlighted above (Fig. 85e-7).


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InternalMed_Harrison. The relationship among the degree of heteroplasmy, tissue distribution of the mutant mtDNA, and disease phenotype simplifies inference of a clear causative relationship between heteroplasmic mutation and disease. With the exception of certain mutations (e.g., those causing most cases of LHON), drift to homoplasmy of such mutations would be precluded normally by the severity of impaired oxidative phosphorylation and the consequent reduction in reproductive fitness. Therefore, sequence variants that have reached homoplasmy should be neutral in terms of human evolution and, hence, useful only for tracing human evolution, demography, and migration, as described above. One important exception is in the case of one or more of the homoplasmic population-level variants, which designate the mtDNA haplogroup J, and the interaction with the mtDNA mutations causing LHON. Reduced disease predilection suggests that one or more of the ancient sequence variants designating mtDNA haplogroup J appears


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InternalMed_Harrison. haplogroup J, and the interaction with the mtDNA mutations causing LHON. Reduced disease predilection suggests that one or more of the ancient sequence variants designating mtDNA haplogroup J appears to attenuate predisposition to degenerative disease, in the face of other risk factors. Whether or not additional epistatic interactions between population-level mtDNA haplotypes and common health conditions will be found remains to be determined. If such influences do exist, then they are more likely to be relevant to health conditions in the postreproductive age groups, wherein evolutionary filters would not have had the opportunity to censor deleterious effects and interactions and wherein the effects of oxidative stress may play a role. Although much has been written about the possible associations of population-level common mtDNA variants and human health and disease phenotypes or adaptation to different environmental influences (e.g., climate), a word of caution is in order.


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InternalMed_Harrison. Many studies that purport to show such associations with phenotypes such as longevity, athletic performance, and metabolic and neurodegenerative disease are limited by small sample sizes, possible genotyping inaccuracies, and the possibility of population stratification or ethnic ancestry bias. Because mtDNA haplogroups are so prominently partitioned along phylogeographic lines, it is difficult to rule out the possibility that a haplogroup for which an association has been found is simply a marker for differences in Succinyl-CoA synthase (SUCLA2, SUCLG1)


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InternalMed_Harrison. FIGURE 85e-7 Disorders associated with perturbations in nuclearmitochondrial genomic cross-talk. Clinical features and genes associated with multiple mitochondrial DNA (mtDNA) deletions, mtDNA depletion, and mitochondrial neurogastrointestinal encephalomyopathy syndromes. ANT, adenine nucleotide translocators; adPEO, autosomal dominant progressive external ophthalmoplegia; arPEO, autosomal recessive progressive external ophthalmoplegia; IOSCA, infantile-onset spinocerebellar ataxia; SCAE, spinocerebellar ataxia and epilepsy. (Reproduced with permission from A Spinazzola, M Zeviani: Disorders from perturbations of nuclear-mitochondrial intergenomic cross-talk. J Intern Med 265:174, 2009.) populations with a societal or environmental difference or with different allele frequencies at other genomic loci, which are actually causally related to the heritable trait or disease of interest. The difficulty in generating cellular or animal models to test the functional influence of homoplasmic


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InternalMed_Harrison. genomic loci, which are actually causally related to the heritable trait or disease of interest. The difficulty in generating cellular or animal models to test the functional influence of homoplasmic sequence variants (as a result of mtDNA polyploidy) further compounds the challenge. The most likely formulation is that the risk conferred by different mtDNA haplogroup–defining homoplasmic mutations for common diseases depends on the concomitant nuclear genomic background, together with environmental influences. Progress in minimizing potentially misleading associations in mtDNA heritable trait and disease studies should include ensuring adequate sample size taken from a large sample recruitment base, using carefully matched controls and population structure determination, and performing analysis that takes into account epistatic interactions with other genomic loci and environmental factors.


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InternalMed_Harrison. Studies on aging humans and animals have shown a potentially important correlation of age with the accumulation of heterogeneous mtDNA mutations, especially in those organ systems that undergo the most prominent age-related degenerative tissue phenotype. Sequencing of PCR-amplified single mtDNA molecules has demonstrated an average of two to three point mutations per molecule in elderly subjects when compared with younger ones. Point mutations observed include those responsible for known heritable heteroplasmic mtDNA disorders, such as the m.3344A>G and m.3243A>G mutations responsible for the MERRF and MELAS syndromes, respectively. However, the cumulative burden of these acquired somatic point mutations with age was observed to remain well below the threshold expected for phenotypic expression (<2%). Point mutations at other sites not normally involved in inherited mtDNA disorders have also been shown to accumulate to much higher levels in some tissues of elderly individuals, with


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InternalMed_Harrison. (<2%). Point mutations at other sites not normally involved in inherited mtDNA disorders have also been shown to accumulate to much higher levels in some tissues of elderly individuals, with the description of tissue-specific “hot spots” for mtDNA point mutations. Along the same lines, an age-associated and tissue-specific accumulation of mtDNA deletions has been observed, including deletions involved in known heritable mtDNA disorders, as well as others. The accumulation of functional mtDNA deletions in a given tissue is expected to be associated with mitochondrial dysfunction, as reflected in an age-associated patchy and reduced COX activity on histochemical staining, especially in skeletal and cardiac muscle and brain. A particularly well-studied and potentially important example is the accumulation of mtDNA deletions and COX deficiency observed in neurons of the substantia nigra in Parkinson’s disease patients.


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InternalMed_Harrison. The progressive accumulation of ROS has been proposed as the key factor connecting mtDNA mutations with aging and age-related disease pathogenesis (Fig. 85e-8). As noted above, ROS are a by-product of oxidative phosphorylation and are removed by detoxifying antioxidants into less harmful moieties; however, exaggerated production of ROS or impaired removal results in their accumulation. One of the main targets for ROS-mediated injury is DNA, and mtDNA is particularly vulnerable because of its lack of protective histones and less efficient injury repair systems compared with nuclear DNA. In turn, accumulation of mtDNA mutations results in inefficient oxidative phosphorylation, with the potential for excessive production of ROS, generating a “vicious cycle” of cumulative mtDNA damage. Indeed, measurement of the oxidative stress biomarker 8-hydroxy-2-deoxyguanosine has been used to measure age-dependent increases in mtDNA oxidative damage at a rate exceeding that of nuclear DNA. It should


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InternalMed_Harrison. measurement of the oxidative stress biomarker 8-hydroxy-2-deoxyguanosine has been used to measure age-dependent increases in mtDNA oxidative damage at a rate exceeding that of nuclear DNA. It should be noted that mtDNA mutation can potentially occur in postmitotic cells as well, because mtDNA replication is not synchronized with the cell cycle. Two other proposed links between mtDNA mutation and aging, besides ROS-mediated tissue injury, are the perturbations in efficiency of oxidative phosphorylation with disturbed cellular aerobic function


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InternalMed_Harrison. FIGURE 85e-8 Multiple pathways of mitochondrial DNA (mtDNA) damage and aging. Multiple factors may impinge on the integrity of mitochondria that lead to loss of cell function, apoptosis, and aging. The classic pathway is indicated with blue arrows; the generation of reactive oxygen species (ROS; superoxide anion, hydrogen peroxide, and hydroxyl radicals), as a by-product of mitochondrial oxidative phosphorylation, results in damage to mitochondrial macromolecules, including the mtDNA, with the latter leading to deleterious mutations. When these factors damage the mitochondrial energy-generating apparatus beyond a functional threshold, proteins are released from the mitochondria that activate the caspase pathway, leading to apoptosis, cell death, and aging. (Reproduced with permission from L Loeb et al: The mitochondrial theory of aging and its relationship to reactive oxygen species damage and somatic mtDNA mutations. Proc Natl Acad Sci USA 102:18769, 2005.) and perturbations in


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InternalMed_Harrison. from L Loeb et al: The mitochondrial theory of aging and its relationship to reactive oxygen species damage and somatic mtDNA mutations. Proc Natl Acad Sci USA 102:18769, 2005.) and perturbations in apoptotic pathways, whose execution steps involve mitochondrial activity.


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InternalMed_Harrison. Genetic intervention studies in animal models have sought to clarify the potential causative relationship between acquired somatic mtDNA mutation and the aging phenotype, and the role of ROS in particular. Replication of the mitochondrial genome is mediated by the activity of the nuclear-encoded polymerase gamma gene. A transgenic homozygous mouse knock-in mutation of this gene renders the polymerase enzyme deficient in proofreading and results in a threeto fivefold increase in mtDNA mutation rate. Such mice develop a premature aging phenotype, which includes subcutaneous lipoatrophy, alopecia, kyphonia, and weight loss with premature death. Although the finding of increased mtDNA mutation and mitochondrial dysfunction with age has been solidly established, the causative role and specific contribution of mitochondrial ROS to aging and age-related disease in humans has yet to be proved. Similarly, although many tumors display higher levels of heterogeneous mtDNA mutations, a causal


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InternalMed_Harrison. contribution of mitochondrial ROS to aging and age-related disease in humans has yet to be proved. Similarly, although many tumors display higher levels of heterogeneous mtDNA mutations, a causal relationship to tumorigenesis has not been proved.


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InternalMed_Harrison. Besides the age-dependent acquired accumulation in somatic cells of heterogeneous point mutations and deletions, a quite different effect of nonheritable and acquired mtDNA mutation has been described affecting tissue stem cells. In particular, disease phenotypes attributed to acquired mtDNA mutation have been observed in sporadic and apparently nonfamilial cases involving a single individual or even tissue, usually skeletal muscle. The presentation consists of decreased exercise tolerance and myalgias, sometimes progressing to rhabdomyolysis. As in the case of the sporadic, heteroplasmic, large-scale deletion, classic syndromes of chronic PEO, Pearson syndrome, and KSS, the absence of a maternal inheritance pattern, together with the finding of limited tissue distribution, suggests a molecular pathogenic mechanism emanating from mutations arising de novo in muscle stem cells after germline differentiation (somatic mutations that are not sporadic and occur in tissue-specific stem


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InternalMed_Harrison. molecular pathogenic mechanism emanating from mutations arising de novo in muscle stem cells after germline differentiation (somatic mutations that are not sporadic and occur in tissue-specific stem cells during fetal development or in the postnatal maintenance or postinjury repair stage). Such mutations would be expected to be propagated only within the progeny of that stem cell and affect a particular tissue within a given individual, without evidence of heritability.


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InternalMed_Harrison. No specific curative treatment for mtDNA disorders is currently available; therefore, the management of mitochondrial disease is largely supportive. Management issues may include early diagnosis and treatment of diabetes mellitus, cardiac pacing, ptosis correction, and intraocular lens replacement for cataracts. Less specific interventions in the case of other disorders involve combined treatment strategies including dietary intervention and removal of toxic metabolites. Cofactors and vitamin supplements are widely used in the treatment of diseases of mitochondrial oxidative phosphorylation, although there is little evidence, apart from anecdotal reports, to support their use. This includes administration of artificial electron acceptors, including vitamin K3, vitamin C, and ubiquinone (coenzyme Q10); administration of cofactors (coenzymes) including riboflavin, carnitine, and creatine; and use of oxygen radical scavengers, such as vitamin E, copper, selenium, ubiquinone, and


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InternalMed_Harrison. (coenzyme Q10); administration of cofactors (coenzymes) including riboflavin, carnitine, and creatine; and use of oxygen radical scavengers, such as vitamin E, copper, selenium, ubiquinone, and idebenone. Drugs that could interfere with mitochondrial function, such as the anesthetic agent propofol, barbiturates, and high doses of valproate, should be avoided. Supplementation with the nitric oxide synthase substrate, L-arginine, has been advocated as a vasodilator treatment during stroke-like episodes. The physician should also be familiar with environmental interactions, such as the strong and consistent association between visual loss in LHON and smoking. A clinical penetrance of 93% was found in men who smoked. Asymptomatic carriers of an LHON mtDNA mutation should, therefore, be strongly advised not to smoke and to moderate their alcohol intake. Although not a cure, these interventions might stave off the devastating clinical manifestations of the LHON mutation. Another example is


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InternalMed_Harrison. advised not to smoke and to moderate their alcohol intake. Although not a cure, these interventions might stave off the devastating clinical manifestations of the LHON mutation. Another example is strict avodiance of aminoglycosides in the familial syndrome of ototoxic susceptibility to aminoglycosides in the presence of the mtDNA m.1555A>G mutation of the 12SrRNA encoding gene.


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InternalMed_Harrison. GENETIC COUNSELING, PRENATAL DIAGNOSIS, AND PREIMPLANTATION GENETIC DIAGNOSIS IN MTDNA DISORDERS


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InternalMed_Harrison. The provision of accurate genetic counseling and reproductive options to families with mtDNA mutations is challenging due to the unique genetic features of mtDNA inheritance that distinguish it from Mendelian genetics. mtDNA defects are transmitted by maternal inheritance. mtDNA de novo mutations are often large deletions, affect one family member, and usually represent no significant risk to other members of the family. In contrast, mtDNA point mutations or duplications can be transmitted down the maternal line. Accordingly, the father of an affected individual has no risk of harboring the disease-causing mutation, and a male cannot transmit the mtDNA mutation to his offspring. In contrast, the mother of an affected individual usually harbors the same mutation but might be completely asymptomatic. This wide phenotypic variability is primarily related to the phenomena of heteroplasmy and the mutation load carried by different members of the same family. Consequently, a symptomatic or


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InternalMed_Harrison. This wide phenotypic variability is primarily related to the phenomena of heteroplasmy and the mutation load carried by different members of the same family. Consequently, a symptomatic or asymptomatic female harboring a disease-causing mutation in a heteroplasmic state will transmit to her offspring variable amounts of the mutant mtDNA molecules. The offspring will be symptomatic or asymptomatic primarily according to the mutant load transmitted via the oocyte and, to some extent, subsequent mitotic segregation during development. Interactions with the mtDNA haplotype background or nuclear human genome (as in the case of LHON) serve as an additional important determinant of disease penetrance. Because the severity of the disease phenotype associated with the heteroplasmic mutation load is a function of the stochastic differential segregation and copy number 85e-9 of mutant mtDNA during the oogenesis bottleneck and, subsequently, following tissue and organ development in the


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InternalMed_Harrison. load is a function of the stochastic differential segregation and copy number 85e-9 of mutant mtDNA during the oogenesis bottleneck and, subsequently, following tissue and organ development in the offspring, it is rarely predictable with any degree of accuracy. For this reason, prenatal diagnosis (PND) and PGD techniques that have evolved into integral and well-accepted standards of practice are severely hampered in the case of mtDNA-related diseases.


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InternalMed_Harrison. The value of PND and PGD is limited, partly due to the absence of data on the rules that govern the segregation of wild-type and mutant mtDNA species (heteroplasmy) among tissue in the developing embryo. Three factors are required to ensure the reliability of PND and PGD: (1) a close correlation between the mutant load and the disease severity, (2) a uniform distribution of mutant load among tissues, and (3) no major change in mutant load with time. These criteria are suggested to be fulfilled for the NARP m.8993T>G mutation but do not seem to apply to other mtDNA disorders. In fact, the level of mutant mtDNA in a chorionic villous or amniotic fluid sample may be very different from the level in the fetus, and it would be difficult to deduce whether the mutational load in the prenatal samples provides clinically useful information regarding the postnatal and adult state.


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InternalMed_Harrison. Because the treatment options for patients with mitochondrial disease are rather limited, preventive interventions that eliminate the likelihood of transmission of affected mtDNA into offspring are desirable. The lack of utility of PND and PGD techniques to reliably diagnose and predict mitochondrial disorders at preimplantation-stage products of conception has resulted in the search for alternative preventive approaches for the same problem. One possible approach to “diluting” or even entirely eliminating the mutant mtDNA is applicable only in the earliest embryonic state and in effect represents a form of germline preventive therapy (Fig. 85e-9). This possibility has been explored by using alternative assisted reproduction techniques such as ooplasmic transfer (OT), metaphase chromosome transfer (CT), pronuclear transfer (PNT), and germinal vesicle transfer (GVT) in animal models and, to an extent, in humans. OT is a technique wherein a certain volume (5–15%) of healthy donor oocyte


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InternalMed_Harrison. transfer (CT), pronuclear transfer (PNT), and germinal vesicle transfer (GVT) in animal models and, to an extent, in humans. OT is a technique wherein a certain volume (5–15%) of healthy donor oocyte cytoplasm with normal mitochondria is injected into the patient oocyte containing mutated mitochondria. The reasoning behind OT is to supplement the patient’s oocyte with uncompromised cytoplasmic factors such as mtDNA, mRNA, proteins, and other molecules by injecting cytoplasm from healthy oocytes. In PNT, following fertilization, pronuclei of a patient’s zygote are removed with a cytoplasm (“karyoplast”). The karyoplast is transferred to the perivitelline space of a donated zygote, which has been already enucleated. The karyoplast is then fused with enucleated zygote by electric pulses or inactivated Sendai viruses (HVJ). The reconstructed zygote contains a nucleus from the patient (patient nuclear DNA) and cytoplasm from the donor. Thus, the majority of the patient mtDNA is replaced


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InternalMed_Harrison. inactivated Sendai viruses (HVJ). The reconstructed zygote contains a nucleus from the patient (patient nuclear DNA) and cytoplasm from the donor. Thus, the majority of the patient mtDNA is replaced with mtDNA from the donor oocyte. In CT, meiosis II stage of oocyte maturation provides an opportunity for the reconstruction of oocytes with different nuclear and cytoplasmic components before fertilization takes place. Reconstructed oocytes by metaphase chromosome transfer are then fertilized to produce embryos with desired mtDNA haplotypes. In GVT, replacement of compromised cytoplasm with healthy cytoplasm through germinal vesicle transfer before the start of chromosome segregation is carried out.


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InternalMed_Harrison. These approaches have not yet met with widely reported clinical success, yet there is room for optimism. As noted above, analysis of heteroplasmy and inheritance patterns indicates that even a small increase in copies of nonmutant mtDNA can exceed the threshold required to ameliorate serious clinical disease. All of the approaches described above show promise in achieving this goal and thus reducing the burden of clinical mtDNA disease in the future. Nuclear transfer into Preimplantation donated oocytes: genetic diagnosis a future possibility? Mother’s oocytes fertilized with FIGURE 85e-9 Possible approaches for prevention of mitochondrial DNA (mtDNA) disease. A. No intervention: offspring’s mutant mtDNA load will vary greatly. B. Oocyte donation: currently permitted in some constituencies but limited by the availability of oocyte donors.


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InternalMed_Harrison. C. Preimplantation genetic diagnosis: available for some mtDNA diseases (reliable in determining background nuclear genomic haplotype risk). D. Nuclear transfer: research stage, including initial studies in nonhuman primates. Red represents mutant mtDNA, pink and white represent successively higher proportions of normal mtDNA. Blue represents genetic material from an unrelated donor. (Adapted with permission from J Poulton et al: Preventing transmission of maternally inherited mitochondrial DNA diseases. Br Med J 338:b94, 2009.) and prevention. Key terms used in the discussion of the human micro-86e-1 biome are defined in Table 86e-1. the human Microbiome We are holobionts—collections of human and microbial cells that 86e Jeffrey I. Gordon, Rob Knight function together in an elaborate symbiosis. The aggregate number


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InternalMed_Harrison. The technologies that allowed us to decipher the human genome have revolutionized our ability to delineate the composition and functions of the microbial communities that colonize our bodies and make up our microbiota. Each body habitat, including the skin, nose, mouth, airways, gastrointestinal tract, and vagina, harbors a distinctive community of microbes. Efforts to understand our microbiota and its collection of microbial genes (our microbiome) are changing our views of “self” and deepening our understanding of many normal physiologic, metabolic, and immunologic features and their interpersonal and intrapersonal variations. In addition, this area of research is beginning to provide new insights into diseases not previously known to have microbial “contributors” and is suggesting new strategies for treatment of microbial cells in our microbiota exceeds the number of human cells in our adult bodies by up to 10-fold, and each healthy adult is estimated to harbor 105–106 microbial


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InternalMed_Harrison. strategies for treatment of microbial cells in our microbiota exceeds the number of human cells in our adult bodies by up to 10-fold, and each healthy adult is estimated to harbor 105–106 microbial genes, in contrast to ~20,000 Homo sapiens genes. Members of our microbiota can function as mutualists (i.e., both host and microbe benefit from each other’s presence), as commensals (one partner benefits; the other is seemingly unaffected), and as potential or overt pathogens (one partner benefits; the other is harmed). Many clinicians view pathogens as individual microbial species or strains that can elicit disease in susceptible hosts. An emerging, more ecologic view is that pathogens do not function in isolation; rather, their invasion, emergence, and effects on the host reflect interactions with other members of a microbiota. An even more expansive view is that multiple organisms in a community conspire to


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InternalMed_Harrison. Culture-independent A type of analysis in which the culture of microbes is not required but rather information is extracted directly from environmental analysis samples Diversity (alpha and Alpha diversity measures the effective number of species (kinds of organisms) at the level of individual habitats, sites, or samples. Beta beta) diversity measures differences in the number of kinds of organisms across habitats, sites, or samples. Domains of life The three major branches of life on Earth: the Eukarya (including humans), the Bacteria, and the Archaea Dysbiosis Any deleterious condition arising from a structural and/or functional aberration in one or more of the host organism’s microbial communities Gnotobiotics The rearing of animals under sterile (germ-free) conditions. These animals can subsequently be colonized at various stages of the life cycle with defined collections of microbes.


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InternalMed_Harrison. Gnotobiotics The rearing of animals under sterile (germ-free) conditions. These animals can subsequently be colonized at various stages of the life cycle with defined collections of microbes. Holobiont The biologic entity consisting of a host and all its internal and external symbionts, their gene repertoires, and their functions


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InternalMed_Harrison. Holobiont The biologic entity consisting of a host and all its internal and external symbionts, their gene repertoires, and their functions Human microbiome In ecology, biome refers to a habitat and the organisms in it. In this sense, the human microbiome would be defined as the collection of microorganisms associated with the human body. However, the term microbiome is also used to refer to the collective genomes and genes present in members of a given microbiota (see “Microbiota,” below), and the human metagenome is the sum of the human genome and microbial genes (microbiome). A core human microbiome is defined as everything shared in a given body habitat among all or the vast majority of human microbiomes. A core microbiome may include a common set of genomes and genes encoding various protein families and/or metabolic capabilities. Microbial genes that are variably represented in different humans may contribute to distinctive physiologic/metabolic phenotypes.


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InternalMed_Harrison. Metagenomics An emerging field encompassing culture-independent studies of the structures and functions of microbial communities as well as the interactions of these communities with the habitats they occupy. Metagenomics includes (1) shotgun sequencing of microbial DNA isolated directly from a given environment and (2) high-throughput screening of expression libraries constructed from cloned community DNA to identify specific functions such as antibiotic resistance (functional metagenomics). DNA-level analyses provide the foundation for profiling of mRNAs and proteins produced by a microbiome (metatranscriptomics and metaproteomics) and for identification of a community’s metabolic network (metametabolomics).


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InternalMed_Harrison. Microbial source tracking A collection of methods for assessing the environments of origin for microbes. One method, SourceTracker, uses a Bayesian approach to identify each bacterial taxon’s origins and estimates the proportions of each community made up by bacteria originating from different environments. Microbiota A microbial community—including Bacteria, Archaea, Eukarya, and viruses—that occupies a given habitat Pan-genome The group of genes found in genomes that make up a given microbial phylotype, including both core genes found in all genomes and variably represented genes found in a subset of genomes within the phylotype Phylogenetic analysis Characterization of the evolutionary relationships between organisms and their gene products


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InternalMed_Harrison. Phylogenetic analysis Characterization of the evolutionary relationships between organisms and their gene products Phylogenetic tree A “tree” in which organisms are shown according to their relationships to hypothetical common ancestors. When built from molecular sequences, the branch lengths are proportional to the amount of evolutionary change separating each ancestor–descendant pair. Phylotype A phylogenetic group of microbes, currently defined by a threshold percentage identity shared among their small-subunit rRNA genes (e.g., ≥97% for a species-level phylotype) Principal coordinates An ordination method for visualizing multivariate data based on the similarity/dissimilarity of the measured entities (e.g., visualization analysis of bacterial communities based on their UniFrac distances; see “UniFrac,” below)


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InternalMed_Harrison. Random Forests analysis/ Machine learning is a collection of approaches that allow a computer to learn without being explicitly programmed. Random Forests machine learning is a machine-learning method for classification and regression that uses multiple decision trees during a training step. Rarefaction A procedure in which subsampling is used to assess whether all the diversity present in a given sample or set of samples has been observed at a given sampling depth and to extrapolate how much additional sampling would be needed to observe all the diversity Resilience A community’s ability to return to its initial state after a perturbation Shotgun sequencing A method for sequencing large DNA regions or collections of regions by fragmenting DNA and sequencing the resulting smaller sections


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InternalMed_Harrison. Shotgun sequencing A method for sequencing large DNA regions or collections of regions by fragmenting DNA and sequencing the resulting smaller sections Succession (primary and Succession (in an ecologic context) refers to changes in the structure of a community through time. Primary succession describes the secondary) sequence of colonizations and extinctions that occur in a new habitat. Secondary succession refers to changes in community structure after a disturbance. UniFrac A measure of the phylogenetic dissimilarity between two communities, calculated as the unshared proportion of the phylogenetic tree containing all the organisms present in either community Chapter 86e The Human Microbiome produce pathogenic effects in certain host and environmental contexts (a pathologic community).


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InternalMed_Harrison. Chapter 86e The Human Microbiome produce pathogenic effects in certain host and environmental contexts (a pathologic community). The ability to characterize microbial communities without culturing their component members has spawned the field of metagenomics (Table 86e-1). Metagenomics reflects a confluence of experimental and computational advances in the genome sciences as well as a more ecologic understanding of medical microbiology, according to which the functions of a given microbe and its impact on human biology depend on the context of other microbes in the same community. Traditional microbiology relies on culturing individual microbes, but metagenomics skips this step, instead sequencing DNA isolated directly from a given microbial community. The resulting datasets facilitate follow-up functional studies, such as the profiling of RNA and protein products expressed from the microbiome or the characterization of a microbial community’s metabolic activities.


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InternalMed_Harrison. Metagenomics provides insight into how microbial communities vary in several situations critical to human health. One such situation is how microbial communities are assembled following birth and how they operate over time, including responses of established communities to various perturbations. Another is how microbial communities normally vary between different anatomic sites within an individual and between different groups of people representing different ages, physiologic states, lifestyles, geographies, and gender. Yet another is how microbial communities vary in disease; whether such variations are consistent among individuals grouped according to current criteria for a disease or its subtypes; whether the microbiota or microbiome provides new ways of classifying disease states; and, importantly, whether the structural and functional configurations of microbial communities are a cause or a consequence of disease.


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InternalMed_Harrison. Analysis of our microbiomes also addresses one of the most fundamental questions in genetics: How does environment select our genes and directly influence their function? Each human encounters a unique environment during the course of his or her lifetime. Part of this personally experienced environment is incorporated into the genes and capabilities of our microbial communities. The microbiome therefore expands our conceptualization of “human” genetic potential from a single set of genes “fixed” at birth to a microbiome with additional genes and capabilities acquired via a process influenced by our family and life experiences, including modifiable lifestyle choices such as diet. This view recognizes a previously underappreciated dimension of human evolution that occurs at the level of our microbiomes and inspires us to determine how—and how fast—this microbial evolution effects changes in our human biology. For example, Westernization is associated with loss of bacterial species


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InternalMed_Harrison. our microbiomes and inspires us to determine how—and how fast—this microbial evolution effects changes in our human biology. For example, Westernization is associated with loss of bacterial species diversity (richness) in the microbiota, and this loss may be associated with the suite of Western diseases. The study of our microbiomes also raises important questions about personal identity, how we define the origins of health disparities, and privacy. Further, it offers the possibility of entirely new approaches to disease prevention and treatment, including regenerative medicine, which involves administration of microbial species (probiotics) to individuals harboring communities that have not developed into a mature, fully functional state or that have been perturbed in ways that can be restored by the addition of species that fill unoccupied “jobs” (niches).


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InternalMed_Harrison. This chapter provides a general overview of how human microbial communities are analyzed; reviews ecologic principles that guide our understanding of microbial communities in health and disease; summarizes recent studies that establish correlations and, in some cases, causal relationships between our microbiota/microbiomes and various diseases; and discusses challenges faced in the translation of these findings to new therapeutic interventions.


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InternalMed_Harrison. Life on Earth has been classified into three domains: Bacteria, Archaea, and Eukarya. The habitats of the surface-exposed human body harbor members of each domain plus their viruses. In large part, microbial diversity has not been characterized by culture-based approaches, partly because we do not know how to re-create the metabolic milieu fashioned by these communities in their native habitats and partly because a few organisms tend to outgrow the others. Culture-independent methods readily identify which organisms are present in a microbiota and their relative abundance. The gene widely used to identify microbes and their evolutionary relationships encodes the major RNA component of the small subunit (SSU) of ribosomes. Within each domain of life, the SSU gene is highly conserved, allowing the SSU gene sequences present in different organisms in that domain to be accurately aligned and regions of nucleotide sequence variation to be identified. Pairwise comparisons of SSU ribosomal


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InternalMed_Harrison. the SSU gene sequences present in different organisms in that domain to be accurately aligned and regions of nucleotide sequence variation to be identified. Pairwise comparisons of SSU ribosomal RNA (rRNA) genes from different microbes allow construction of a phylogenetic tree that represents an evolutionary map on which previously unknown organisms can be assigned a position. This approach, known as molecular phylogenetics, permits characterization of each organism on the basis of its evolutionary distance from other organisms. Different phylogenetic types (phylotypes) can be viewed as comprising branches on an evolutionary tree.


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InternalMed_Harrison. Characterization of Bacteria Because members of the Bacteria dominate our microbiota, most studies defining our various body habitat– associated microbial communities have sequenced the bacterial SSU gene that encodes 16S rRNA. This gene has a mosaic structure, with highly conserved domains flanking more variable regions. The most straightforward way to identify bacterial taxonomic groups (taxa) in a given community is to sequence polymerase chain reaction (PCR) products (amplicons) generated from the 16S rRNA genes present in that community. PCR primers directed at the conserved regions of the gene yield PCR amplicons encompassing one or more of that gene’s nine variable regions. PCR primer design is critical: differential annealing with primer pairs designed to amplify different variable regions can lead to overor underrepresentation of specific taxa, and different regions within the 16S rRNA gene can have different patterns of evolution. Therefore, caution must be exercised in


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InternalMed_Harrison. regions can lead to overor underrepresentation of specific taxa, and different regions within the 16S rRNA gene can have different patterns of evolution. Therefore, caution must be exercised in comparisons of the relative abundance of taxa in samples characterized in different studies, as methodologic differences can lead to larger perceived differences in the inferred taxonomy than actually exist.


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InternalMed_Harrison. A key innovation is multiplex sequencing. Amplicons from each microbial-community DNA sample are tagged by incorporation of a unique oligonucleotide barcode into the PCR primer. Amplicons harboring these sample-specific barcodes can then be pooled together so that multiple samples representing multiple communities can be sequenced simultaneously (Fig. 86e-1). One important choice is the tradeoff between the number of samples that can be processed simultaneously and the number of sequences generated per sample. Interpersonal differences in the bacterial components of the microbiota are typically large, as are differences between communities occupying different body habitats in the same individual (see below); thus fewer than 1000 16S rRNA reads are characteristically required to discriminate community type. However, the identification of systematic differences in microbiota composition that correlate with physiologic status or disease state is confounded by the substantial


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InternalMed_Harrison. discriminate community type. However, the identification of systematic differences in microbiota composition that correlate with physiologic status or disease state is confounded by the substantial interpersonal variation that occurs normally.


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InternalMed_Harrison. Sequencing of bacterial 16S rRNA genes creates a challenge for medical microbiology: how to define the taxonomic groups present in a community in a systematic and informative manner, so that one community can be compared with and contrasted to another. Within each domain of life, microbes are classified in a hierarchy beginning with phylum (the broadest group) followed by class, order, family, genus, and species. To determine taxonomy, 16S rRNA sequences are aligned on the basis of their sequence similarity—a process known as picking operational taxonomic units (OTUs). Grouping of 16S rRNA sequences from a given variable region into “bins” that share ≥97% nucleotide sequence identity (97%ID OTUs) is a commonly accepted, albeit arbitrary, way to define a species.


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InternalMed_Harrison. Looking beyond the 16S rRNA gene, we find that different isolates (strains) of a given bacterial species have overlapping but not identical sets of genes in their genomes. The aggregate set of genes identified in all isolates (strains) of a given species-level phylotype represents its pan-genome. Most species are represented by multiple strains, sometimes with markedly different functions (for example, Variable region Community 1 2 3 Align sequences, bin into OTUs, and infer phylogeny by placing OTUs on a master reference phylogenetic tree UniFrac analysis Cluster samples based on UniFrac distances calculated from (iii),


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InternalMed_Harrison. Chapter 86e The Human Microbiome (i) (ii) (iii) DE3 machine learning, feature identification, etc. PC2 PC1 1 2 FIGURE 86e-1 Pipeline for culture-independent studies of a microbiota. (A) DNA is extracted directly from a sampled human body habitat– associated microbial community. The precise location of the community and relevant patient clinical data are collected. Polymerase chain reac-tion (PCR) is used to amplify portions of small-subunit (SSU) rRNA genes (e.g., the genes encoding bacterial 16S rRNA) containing one or more variable regions. Primers with sample-specific, error-correcting barcodes are designed to recognize the more conserved regions of the 16S rRNA gene that flank the targeted variable region(s). (B) Barcoded amplicons from multiple samples (communities 1–3) are pooled and sequenced in batch in a highly parallel next-generation DNA sequencer. (C) The resulting reads are then processed, with barcodes denoting which sample the sequence came from. After barcode sequences


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InternalMed_Harrison. sequenced in batch in a highly parallel next-generation DNA sequencer. (C) The resulting reads are then processed, with barcodes denoting which sample the sequence came from. After barcode sequences are removed in silico, reads are aligned and grouped according to a specified level of shared identity; e.g., sequences that share ≥97% nucleotide sequence identity are regarded as representing a species. Once reads are binned into operational taxonomic units (OTUs) in this fashion, they are placed on a phylogenetic tree of all known bacteria and their phylogeny is inferred.


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InternalMed_Harrison. (D) Communities can be compared to one another by either taxon-based methods, in which phylogeny is not considered and the number of shared taxa are simply scored, or phylogenetic methods, in which community similarity is considered in light of the evolutionary relationships of community members. The UniFrac metric is commonly used for phylogeny-based comparisons. In stylized examples (i), (ii), and (iii), communities with varying degrees of similarity are shown. Each circle represents an OTU colored on the basis of its community of origin and placed on a master phylogenetic tree that includes all lineages from all communities. Branches (horizontal lines) are colored with each community that contains members from that branch. The three examples vary in the amount of branch length shared between the OTUs from each community. In (i), there is no shared branch length, and thus the three communities have a similarity score of 0. In (ii), the communities are identical, and a similarity


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InternalMed_Harrison. between the OTUs from each community. In (i), there is no shared branch length, and thus the three communities have a similarity score of 0. In (ii), the communities are identical, and a similarity score of 1 is assigned. In (iii), there is an intermediate level of similarity: communities represented in red and green share more branch length and thus have a higher similarity score than red vs. blue or green vs. blue. The amount of shared branch length in each pairwise community comparison provides a distance matrix. (E) The results of taxonor phylogeny-based distance matrices can be displayed by principal coordinates analysis (PCoA), which plots each community spatially such that the largest component of variance is captured on the x-axis (PC1) and the second largest component of variance is displayed on the y-axis (PC2). In the example shown, the three communities in example (iii) from panel D are compared. Note that for shotgun sequencing of whole-community DNA (microbiome


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InternalMed_Harrison. variance is displayed on the y-axis (PC2). In the example shown, the three communities in example (iii) from panel D are compared. Note that for shotgun sequencing of whole-community DNA (microbiome analysis), reads are compared with genes that are present in the genomes of sequenced cultured microbes and/or with genes that have been annotated by hierarchical functional classification schemes in various databases, such as the Kyoto Encyclopedia of Genes and Genomes (KEGG). Communities can then be compared on the basis of the distribution of functional groups in their microbiomes—an approach analogous to taxon-based methods for 16S rRNA–based comparisons—and the results plotted with PCoA.


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InternalMed_Harrison. enteropathogenic versus commensal Escherichia coli). Differences in perfused ecosystem exposed to the complex and varying set of sub-genome content among strains of a given species reflect differences stances we ingest, to adapt to changing circumstances rather than in community membership as well as differences in the selective pres-depending on one strain to occupy a given niche important for proper sures these strains experience within and between habitats. Horizontal community functioning. In ecologic studies of different environments, gene transfer among members of a microbiota—mediated by phage, such as grasslands, forests, and reefs, increased diversity within a com-plasmids, and other mechanisms—is a major contributor to this munity increases its capacity to respond to disturbances and to restore strain-level variation. itself (i.e., its resilience); the same is likely true of microbial ecosystems.


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InternalMed_Harrison. Strain-level diversity can be important in any consideration of how When characterizing the mechanisms by which a given species pro-microbial communities differ between individuals and how these com-duces an effect or effects on humans, it is important to consider the munities accommodate perturbations. For example, the great bacte-strain being tested; strain-level diversity has an impact on discovery rial strain-level diversity that exists in the gut is thought to be one of and development efforts aimed at identifying next-generation probiotthe features that allows this microbiota, which occupies a constantly ics that can be used therapeutically to promote health or treat disease.


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InternalMed_Harrison. Identification of Archaeal and Eukaryotic Members Surveys based on SSU rRNA gene sequencing have largely focused on Bacteria, yet the census of “who’s there” in human body habitat–associated communities must also include the other two domains of life: Archaea and Eukarya. Differences in the sequences of archaeal and bacterial 16S rRNA genes, first recognized by Carl Woese in 1977, allowed these two domains of life to be distinguished. The representation of Archaea in human microbial communities is less well defined than that of Bacteria, in part due to the difficulty in optimizing the design of PCR primers that specifically target conserved regions of archaeal (versus bacterial) 16S rRNA genes. Identifying archaeal members is important to our understanding of the functional properties of the microbiota. For example, a major challenge faced by microbial communities when breaking down polysaccharides (the most abundant biologic polymers on Earth) is the maintenance of redox balance in


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InternalMed_Harrison. the microbiota. For example, a major challenge faced by microbial communities when breaking down polysaccharides (the most abundant biologic polymers on Earth) is the maintenance of redox balance in the setting of maximal energy production. Many microbial species have branched fermentation pathways that allow them to dispose of reducing equivalents (e.g., by the production of H2, which is energetically efficient). However, there is a caveat: the hydrogen must be removed or it will inhibit reoxidation of pyridine nucleotides. Therefore, hydrogen-consuming (hydrogenotrophic) species are key to maximizing the energy-extracting capacity of primary fermenters.


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InternalMed_Harrison. In the human gut, hydrogenotrophs include a phylogenetically diverse group of bacterial acetogens, a more limited group of sulfate-reducing bacteria that generate hydrogen sulfide, and methane-producing archaeal organisms (methanogens) that can represent up to 10% of the anaerobes present in the feces of some humans. However, the degree of archaeal diversity in the gut microbiota of healthy individuals appears to be low.


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InternalMed_Harrison. Culture-independent surveys of eukaryotic diversity are also confounded by challenges related to the design of PCR primers that target the eukaryotic SSU gene (18S rRNA) as well as the internal transcribed spacer regions of rRNA operons. Metagenomic studies of healthy human adults living in countries with distinct cultural traditions and disparate geographic features and locations have revealed that the degree of eukaryotic diversity is lower than that of bacterial diversity. In the gut, which contains far more microbes than any other body habitat, the representation of fungi is significantly lower in individuals living in Westernized societies than in those living in non-Western societies. The most abundant fungal sequences belong to the phylum-level taxa Ascomycota and Microsporidia. The phyla Ascomycota and Basidiomycota appear to be mutually exclusive, and the presence of Candida in particular correlates with recent consumption of carbohydrates.


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InternalMed_Harrison. Elucidation of Viral Dynamics Viruses are the most abundant biologic entity on Earth. Viral particles outnumber microbial cells by 10:1 in most environments. Humans are no exception in terms of viral colonization; our feces alone contain 108–109 viral particles per gram. Despite this abundance, many eukaryotic viral communities remain incompletely characterized, in part because the identification of viruses within metagenomic sequencing datasets is itself very challenging. Characterizing viral diversity requires different approaches: because no single gene is found in all viruses, no universal phylogenetic “barcode of life” equivalent to the SSU rRNA gene exists. One approach has been to selectively purify virus-like particles from community biospecimens, amplify the small amounts of DNA that are recovered, and randomly fragment the DNA and sequence the fragments (shotgun sequencing). The resulting sequences can be assembled into larger contigs whose function can be computationally


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InternalMed_Harrison. that are recovered, and randomly fragment the DNA and sequence the fragments (shotgun sequencing). The resulting sequences can be assembled into larger contigs whose function can be computationally predicted from homology to known genes, and the information obtained can be used to populate/expand nonredundant viral databases. These annotated nonredundant databases can then be used for more targeted mining of the rapidly expanding number of shotgun sequencing datasets generated from total-community DNA for known or putative DNA viruses.


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InternalMed_Harrison. Given the dominance of bacteria in the gut microbiota, it is not surprising that phages (viruses that infect bacteria) dominate the identifiable components of the gut’s DNA virome. Prophages are a manifestation of a so-called temperate viral–bacterial host dynamic, in which a phage is integrated into its host bacterium’s genome. This temperate dynamic provides a way to constantly refashion the genomes of bacterial species through horizontal gene transfer. Genes encoded by a pro-phage genome may expand the niche and fitness of their bacterial host, for example, by enabling the metabolism of previously inaccessible nutrient sources. Prophage integration can also protect the host strain from superinfection, “immunizing” the strain against infection by closely related phages. A temperate prophage life cycle allows the virus to expand in a 1:1 ratio with its bacterial host. If the integrated virus conveys increased fitness, the prevalence of the bacterial host and its phage will increase


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InternalMed_Harrison. life cycle allows the virus to expand in a 1:1 ratio with its bacterial host. If the integrated virus conveys increased fitness, the prevalence of the bacterial host and its phage will increase in the microbiota. Induction of a lytic cycle, where the prophage replicates and kills the host, may follow. Lytic cycles can cause high bacterial turnover. Lysis debris (e.g., components of capsules) can be used as nutrient sources by surviving bacteria; this change in the energy dynamic in a community is referred to as a phage shunt. A subpopulation of bacteria that undergoes lytic induction may sweep away other sensitive species present in the community, thus increasing the niche space available for survivors (i.e., those bacteria that already have an integrated prophage). Periodic induction of prophages leads to a “constant diversity dynamic” that helps maintain community structure and function.


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InternalMed_Harrison. Interest in viral communities has expanded in recent years, especially given a potentially therapeutic role for phages as an alternative or adjunct to antibiotics. Virome members have evolved elegant survival mechanisms that allow them to evade host defenses, diversify, and establish elaborate and mutually beneficial symbioses with their hosts. A number of recent studies have tried to adapt these mechanisms for therapeutic purposes (e.g., the use of synthetic phages to treat Pseudomonas aeruginosa infections in burn patients or in other settings). Phage therapy is not a new idea: Félix d’Herelle, co-discoverer of phages, recognized their potential medical applications nearly a century ago. However, only recently have our technologic capabilities and our knowledge of the human microbiota made phage therapy realistically attainable within our lifetimes.


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InternalMed_Harrison. At many levels, different people are very much alike: our genomes are >99% identical, and we have similar collections of human cells. However, our microbial communities differ drastically, both between people and between habitats within a single human body. The greatest variation (beta diversity, described below) is between body sites. For example, the difference between the microbial communities residing in a person’s mouth versus the same person’s gut is comparable to the difference in communities residing in soil versus seawater. Even within a body site, the differences among people are not subtle: gut, skin, and oral communities can all differ by 80–90%, even from the broad, bacterial species–level view. The English poet John Donne said that “no man is an island”; however, from a microbial perspective, each of us consists of not just one isolated island but rather a whole archipelago of distinct habitats that exchange microbes with one another and with the outside environment at


InternalMed_Harrison

InternalMed_Harrison. perspective, each of us consists of not just one isolated island but rather a whole archipelago of distinct habitats that exchange microbes with one another and with the outside environment at some as yet undetermined level. Before we can discuss these differences and understand their relevance to human disease, it is important to understand some basic terms and ecologic principles.


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InternalMed_Harrison. Alpha Diversity Alpha diversity is defined as the effective number of species present in a given sample. Communities that are compositionally more diverse (i.e., have more OTUs) or that are phylogenetically more diverse are defined as having greater alpha diversity. Alpha diversity can be measured by plotting the number of different types of SSU rRNA sequences identified at a given phylogenetic level (species, genera, etc.) in a sample as a function of the number of SSU rRNA gene reads collected. The most commonly used metrics of alpha diversity are Sobs (the number of species observed in a given number of sequences), Chao1 (a measure based on the number of species observed only once), the Shannon index (a measure of the number of bits of information gained by revealing the identity of a randomly chosen member of the community), and phylogenetic diversity (a measure of the total branch length of a phylogenetic tree encompassing a sample). Diversity estimators are particularly


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InternalMed_Harrison. of a randomly chosen member of the community), and phylogenetic diversity (a measure of the total branch length of a phylogenetic tree encompassing a sample). Diversity estimators are particularly sensitive to errors introduced during PCR and sequencing.


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InternalMed_Harrison. Beta Diversity Beta diversity refers to the differences between communities and can be defined with phylogenetic or nonphylogenetic distance measurements. UniFrac is a commonly used phylogenetic metric that compares the evolutionary history of different microbial communities, noting the degree to which any two communities share branch length on a tree of microbial life: the more similar communities are to each other, the more branch length they share (Fig. 86e-1). UniFrac-based measurements of distances between communities can be visually represented with principal coordinates analysis or other geometric techniques that project a high-dimensional dataset down onto a small number of dimensions for a more approachable analysis (Fig. 86e-1). Principal coordinates analysis can also be applied to nonphylogenetic methods for comparing communities, such as Euclidean distance, Jensen-Shannon divergence, or Bray-Curtis dissimilarity, which operate independent of evolutionary tree data but can


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InternalMed_Harrison. to nonphylogenetic methods for comparing communities, such as Euclidean distance, Jensen-Shannon divergence, or Bray-Curtis dissimilarity, which operate independent of evolutionary tree data but can make biologic patterns more difficult to identify. The taxonomic data or distance matrices can also be used as input into a range of machine-learning algorithms (such as Random Forests) that employ supervised classification to identify differences between labeled groups of samples. Supervised classification is useful for identifying differences between cases and controls but can obscure important patterns intrinsic to the data, including confounding variables such as different sequencing runs or patient populations.


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InternalMed_Harrison. As noted above, the greatest beta diversity is that among body sites. This fact underscores the need to specify body habitat in microbiota analyses of any type, including microbial surveillance studies examining the flow of normal and pathogenic organisms into and out of different body sites in patients and their health care providers. Several other key points have emerged from beta diversity studies of human-associated microbial communities—notably, that (1) there is a high level of interpersonal variability in every body habitat studied to date, (2) intrapersonal variation in a given body habitat is less pronounced, and (3) family members have more similar communities than unrelated individuals living in separate households. Thus, a person is his/ her own best control, and examination of an individual over time as a function of disease state or treatment intervention is desirable. Similarly, family members serve as logical reference controls, although age is a major covariate that


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InternalMed_Harrison. of an individual over time as a function of disease state or treatment intervention is desirable. Similarly, family members serve as logical reference controls, although age is a major covariate that affects microbiota structure.


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InternalMed_Harrison. Studies of fecal samples obtained from twins over time have shown that the overall degree of phylogenetic similarity of bacterial communities does not differ significantly between monozygotic and dizygotic twin pairs, although monozygotic twin pairs may be more similar in some populations at earlier ages. These results, together with intervention studies in mice and epidemiologic observations in humans, emphasize that early environmental exposures are a very important determinant of adult-gut microbial ecology. In humans, the initial exposures depend on delivery mode: babies sampled within 20 min of birth have relatively undifferentiated microbial communities in the mouth, the skin, and the gut. For vaginally delivered babies, these communities resemble the specific microbial communities found in the mother’s vagina. For babies delivered by cesarean section, the communities resemble skin communities. Although studies of older children and of adults stratified by delivery mode are


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InternalMed_Harrison. found in the mother’s vagina. For babies delivered by cesarean section, the communities resemble skin communities. Although studies of older children and of adults stratified by delivery mode are still rare in the literature, these differences have been shown to persist until at least 4 months of age and perhaps until age 7 years. The infant gut microbiota changes to resemble the adult gut community over the first 3 years of life; comparable studies have not been done in other body habitats to date.


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InternalMed_Harrison. Exposures to environmental microbial reservoirs can continue to influence community structure. For example, unrelated cohabiting adults have more similar microbiotas in all of their body habitats than do non-cohabiting adults, and humans resemble the dogs they live with, at least in terms of skin microbiota. Gender and sexual maturation may also affect the microbiota structure, although efforts to isolate these variables are complicated by many confounding factors; any gender effect must be small compared with the effects of other variables such as diet (except in the case of the female urinary tract, which is influenced 86e-5 by the vaginal microbiota).


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InternalMed_Harrison. The vaginal microbiota illustrates another intriguing aspect of the contributions made by various factors to interpersonal differences in microbial community structure within a given body habitat. Bacterial 16S rRNA–based studies of the midvaginal microbiota in sexually active women have documented significant differences in community configurations between four self-reported ethnic groups: Caucasian, black, Hispanic, and Asian. Unlike most other body habitats that have been surveyed, this ecosystem is dominated by a single genus, Lactobacillus. Four species of this genus together account for more than half of the bacteria in most vaginal communities. Five community categories have been defined: four are dominated by L. iners,


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InternalMed_Harrison. L. crispatus, L. gasseri, and L. jensenii, respectively, and the fifth has proportionally fewer lactobacilli and more anaerobes. The representation of these community categories is distinct within each of the four ethnic groups and correlates with vaginal pH and Nugent score (the latter being a biomarker for bacterial vaginosis). Longitudinal studies of individuals are being conducted to identify factors that determine the assembly of these distinct communities—both within and among ethnic groups—as well as their resistance to or resilience after various physiologic and pathologic disturbances. For example, the menstrual cycle and pregnancy turn out to be surprisingly significant factors (cause larger changes) compared with sexual activity.


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InternalMed_Harrison. Yet another factor affecting beta diversity is spatial location within a habitat. Several surveys show that the skin harbors bacterial communities with predictable, albeit complex, biogeographic features. To determine whether these differences are due to differences in local environmental factors, to the history of a given site’s exposure to microbes, or to a combination of the two, reciprocal microbiota transplantation has been performed. Microbial communities from one region of the skin were depleted by treatment with germicidal agents, and the region (plot) was inoculated with a “foreign” microbiota harvested from different regions of the skin or from different body habitats from the same or another individual. Community assembly at the site of transplantation was then tracked over time. Remarkably, assembly proceeded differently at different sites: forearm plots receiving a tongue microbiota remained more similar to tongue communities than to native forearm communities in terms of


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InternalMed_Harrison. Remarkably, assembly proceeded differently at different sites: forearm plots receiving a tongue microbiota remained more similar to tongue communities than to native forearm communities in terms of their composition and diversity, while forehead plots inoculated with tongue bacteria changed to become more similar to native forehead communities. Thus, in addition to the history of exposure to tongue bacteria, environmental factors operating at the forehead plot likely shape community assembly. Intriguingly, the factors that shape fungal skin communities appear to be entirely different from those that shape bacterial skin communities. The palm and forearm have high bacterial and low fungal diversity, whereas the feet have the opposite diversity pattern. Moreover, fungal communities are generally shaped by location (foot, torso, head), whereas bacterial communities are generally shaped by moisture phenotype (dry, moist, or sebaceous).


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InternalMed_Harrison. Co-Occurrence Analysis Co-occurrence analysis seeks to identify which phylotypes are co-distributed across individuals in a given body habitat and/or between habitats and to determine the factors that explain the observed patterns of co-distribution. Positive correlations tend to reflect shared preferences for certain environmental features, while negative correlations typically reflect divergent preferences or a competitive relationship. Syntrophic (cross-feeding) relationships reflect interdependent interactions based on nutrient-sharing strategies. For example, in food webs, the products of one organism’s metabolism can be used by the other for its own unique metabolic capabilities (e.g., the interactions between fermentative organisms and methanogens).


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InternalMed_Harrison. Enterotype Analysis Enterotype analysis seeks to classify individuals into discrete groups based on the configuration of their microbiotas, essentially drawing boundaries on a map defined by principal coordinates analysis or other ordination techniques. The first enterotype analysis used supervised clustering to define three major types of human-gut microbial configurations across three distinct human studies and provided a view that presupposed the existence of three


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InternalMed_Harrison. Chapter 86e The Human Microbiome clusters. Subsequent work has shown that the range of variability in the gut microbiota of children and of non-Western populations greatly exceeds the variability captured in the populations used to define the original enterotypes; in addition, even in Western populations, the variability follows more of a continuum dominated by a gradient in the abundance of the genera Bacteroides and Prevotella. Another consideration in enterotype analysis is whether location on a map defined by healthy human variation is relevant to predisposition to disease or whether instead rare species with particular functions are more important discriminants.


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InternalMed_Harrison. Functional Redundancy Functional redundancy arises when functions are performed by many bacterial taxa. Thus interpersonal differences in microbial bacterial diversity (i.e., which bacteria are present) are not necessarily accompanied by comparable degrees of difference in functional diversity (i.e., what these bacteria can do). Characterization of a microbiome by shotgun sequencing is important because, unlike SSU rRNA analyses, shotgun sequencing provides a direct readout of the genes (and, via comparative genomics, their functions) in a given community. One fundamental question is the degree to which variations in the species occupying a given body habitat correlate with variations in a community’s functional capabilities. For example, the neutral theory of community assembly developed by macro-ecologists suggests that species are added to the community without respect to function, automatically endowing the community with functional redundancy. If applicable to the microbial


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InternalMed_Harrison. by macro-ecologists suggests that species are added to the community without respect to function, automatically endowing the community with functional redundancy. If applicable to the microbial world, neutral community assembly would predict a high level of variation in the types of microbial lineages that occupy a given body habitat in different individuals, although the broad functions encoded in the microbiomes of these communities could be quite similar.


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InternalMed_Harrison. Shotgun sequencing of the fecal microbiome has revealed that different microbial communities converge on the same functional state: in other words, there is a group of microbial genes represented in the guts of unrelated as well as related individuals. The same principle holds true at other body sites (Fig. 86e-2). The “core” gut microbiome is enriched in functions related to microbial survival (e.g., translation; metabolism of nucleotides, carbohydrates, and amino acids) and in functions that benefit the host (nutrient and energy partitioning from the diet to microbes and host). The latter functions encompass the food webs mentioned above, in which products of one type of microbe become the substrates for other microbes. These webs, which can be incredibly elaborate, change as microbes adjust their patterns of gene expression and metabolism in response to alterations in nutrient availability. Thus the sum of all the activities of the members of a microbial community can be viewed as


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InternalMed_Harrison. their patterns of gene expression and metabolism in response to alterations in nutrient availability. Thus the sum of all the activities of the members of a microbial community can be viewed as an emergent rather than a fixed property.


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InternalMed_Harrison. It is important to note that pairwise comparisons have shown that family members have functionally more similar gut microbiomes than do unrelated individuals. Thus, intrafamilial transmission of a gut microbiome within a given generation and across multiple generations could shape the biologic features of humans belonging to a kinship and modulate/mediate risks for a variety of diseases. Stability Like other ecosystems, human body habitat– associated microbial communities vary over time, and an understanding of this variation is essential for a functional


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InternalMed_Harrison. FIGURE 86e-2 Interpersonal variation in organismal representation in body habitat–associated communities is more extensive than interpersonal variation in gene functional features. Bacterial taxonomy and metabolic function are compared in 107 oral microbiota and microbiome samples (top) and in 139 fecal microbiota and microbiome samples (bottom). Samples represent an arbitrarily chosen subset from 242 healthy young adults living in the United States, with equal numbers of men and women. The same DNA extracts from the same samples were used for both taxonomic and functional classifications; each sample was analyzed by bacterial 16S rRNA amplicon sequencing (mean, 5400 sequences per sample) and by shotgun sequencing of community DNA (mean, 2.9 billion bases per sample). Taxonomic groups vary dramatically in their representation among different samples, with different characteristic bacterial phyla in the oral versus the fecal microbiota; e.g., members of the Actinobacteria and