The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers approximately 99% of the euchromatic genome and is accurate to an error rate of approximately 1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human genome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead.

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Finishing the euchromatic sequence of the human genome

https://www.thelancet.com/pdfs/journals/lancet/PIIS0140-6736(07)60601-1.pdf

Autosomal dominant polycystic kidney disease

A main goal of the Human Genome Project (HGP) is to provide the complete and accurate reference sequence of the euchromatic portions of all human chromosomes (Collins et al. 1998). It has been argued that this endeavor differs from previously sequenced invertebrate models not only in terms of scale but also in terms of repetitive complexity (Green 1997; Eichler 1998). Repetitive complexity leads to misassignment and misassembly of sequence. It has been suggested that segmental duplications may be particularly problematic in this regard because of their inconspicuousness, large size, and high degree of sequence similarity. The inability to identify such duplications, let alone differentiate their true position from paralogous positions, may confound sequence assembly, resulting in merging of distinct loci into the same sequence (Eichler 1998).

Segmental duplications are duplicated blocks of genomic DNA typically ranging in size from 1–200 kb (IHGSC 2001). They often contain sequence features such as high-copy repeats and gene sequences with intron–exon structure. Thus, being composed of apparently normal genomic DNA, segmental duplications cannot be detected a priori; rather, most segmental duplications have to date been discovered based on experimental analyses. Over the past decade a large number of both intra- and interchromosomal segmental duplications have been observed (Wong et al. 1990; Tomlinson et al. 1994; Eichler et al. 1997; Mazzarella and Schlessinger 1997; Regnier et al. 1997; Zimonjic et al. 1997; Eichler 1998; Trask et al. 1998a; Jackson et al. 1999; Ji et al. 1999). These data suggest numerous interchromosomal exchanges during recent hominoid evolution with apparent biases into and between pericentromeric and subtelomeric regions (Eichler et al. 1997, 1999; Monfouilloux et al. 1998; Trask et al. 1998a; Jackson et al. 1999; Horvath et al. 2000a). To date, however, no systematic analysis of the genome has been performed to quantify this bias. Another unanticipated finding has been the important role segmental duplications play in disease (for review, see Ji et al. 2000; Mazzarella and Schlessinger 1998). Aberrant homologous recombination between highly similar paralogs appears to be a major mechanism for many genomic disorders such as velocardiofacial/DiGeorge, Smith-Magenis, and Prader-Willi/Angelman syndromes (Chen et al. 1997; Amos-Landgraf et al. 1999; Christian et al. 1999; Edelmann et al. 1999; Shaikh et al. 2000).

A major step toward developing a final reference sequence has been the completion of the draft-sequencing phase of the HGP and its subsequent assembly. The assembly has occurred in three main steps: (1) Sequenced clones are placed into fingerprint contigs generated from the entire RPCI-11 BAC library; (2) fingerprint contigs are assigned and positioned to chromosomes using all available genetic and STS markers; and (3) the sequence within each contig is assembled by Jim Kent's Gigassembler (IHGMC 2001; IHGSC 2001). This landmark achievement has given us the ability to examine segmental duplications in a genome-wide and systematic manner. We reported an unprecedented amount (3.6%) of sequence was involved in recent segmental duplications with identity between 90%–98%. Additionally we provided examples of pericentromeric and subtelomeric regions that appear to be composed almost entirely of duplicated sequence (IHGSC 2001). However, further characterization of highly duplicated regions has yet to be accomplished.

In this article, we present our methodology for the analysis of such duplications and an in-depth analysis of segmental duplications in the current working draft assembly (January 2001, oo23 assembly), paying particular attention to the quality of assignment and assembly for the duplication-rich clones and regions. Because of the estimated error rates of sequence and the potential for misassembly in the draft assembly, we consider two categories of duplications: segments with >98% nucleotide identity, and segments with 90%–98% identity. For the first time, we quantify the genome-wide enrichment of duplicated sequence in both pericentromeric and subtelomeric regions. In addition, we examine more specifically the impact of these segments on the current assembly. We find duplicated sequences are enriched in sequence contigs that have not been mapped within the current assembly. We also find that clones containing duplications are often assigned to a chromosome inconsistent with FISH and only ∼50% of the chromosomes with FISH signals from these clones have a corresponding sequence similarity by BLAST analysis. This underrepresentation may be attributable to many factors: misassignment, merging, or reduced coverage in these paralogous regions. Taken together, the clustering of duplications combined with the difficulty in positioning and assembling them, suggests that large tracts of segmental duplications, particularly those located at pericentromeres, will be refractory to currently employed assembly methods. Specialized methods will be necessary to correctly integrate these regions into the reference human genome sequence. We propose that the determination of whether an observed overlap is allelic or paralogous will facilitate the final assembly of the human genome, helping to eliminate many gaps both within paralogous as well as unique sequence regions.

Segmental Duplications: Organization and Impact Within the Current Human Genome Project Assembly

We report the identification of a recurrent, 520-kb 16p12.1 microdeletion associated with childhood developmental delay. The microdeletion was detected in 20 of 11,873 cases compared with 2 of 8,540 controls (P = 0.0009, OR = 7.2) and replicated in a second series of 22 of 9,254 cases compared with 6 of 6,299 controls (P = 0.028, OR = 2.5). Most deletions were inherited, with carrier parents likely to manifest neuropsychiatric phenotypes compared to non-carrier parents (P = 0.037, OR = 6). Probands were more likely to carry an additional large copy-number variant when compared to matched controls (10 of 42 cases, P = 5.7 x 10(-5), OR = 6.6). The clinical features of individuals with two mutations were distinct from and/or more severe than those of individuals carrying only the co-occurring mutation. Our data support a two-hit model in which the 16p12.1 microdeletion both predisposes to neuropsychiatric phenotypes as a single event and exacerbates neurodevelopmental phenotypes in association with other large deletions or duplications. Analysis of other microdeletions with variable expressivity indicates that this two-hit model might be more generally applicable to neuropsychiatric disease.

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A recurrent 16p12.1 microdeletion supports a two-hit model for severe developmental delay

The sulfonation of endogenous molecules is a pervasive biological phenomenon that is not always easily understood, and although it is increasingly recognized as a function of fundamental importance, there remain areas in which significant cognizance is still lacking or at most minimal. This is particularly true in the field of endocrinology, in which the sulfoconjugation of hormones is a widespread occurrence that is only partially, if at all, appreciated. In the realm of steroid/sterol sulfoconjugation, the discovery of a novel gene that utilizes an alternative exon 1 to encode for two sulfotransferase isoforms, one of which sulfonates cholesterol and the other pregnenolone, has been an important advance. This is significant because cholesterol sulfate plays a crucial role in physiological systems such as keratinocyte differentiation and development of the skin barrier, and pregnenolone sulfate is now acknowledged as an important neurosteroid. The sulfonation of thyroglobulin and thyroid hormones has been extensively investigated and, although this transformation is better understood, there remain areas of incomplete comprehension. The sulfonation of catecholamines is a prevalent modification that has been extensively studied but, unfortunately, remains poorly understood. The sulfonation of pituitary glycoprotein hormones, especially LH and TSH, does not affect binding to their cognate receptors; however, sulfonation does play an important role in their plasma clearance, which indirectly has a significant effect on biological activity. On the other hand, the sulfonation of distinct neuroendocrine peptides does have a profound influence on receptor binding and, thus, a direct effect on biological activity. The sulfonation of specific extracellular structures plays an essential role in the binding and signaling of a large family of extracellular growth factors. In summary, sulfonation is a ubiquitous posttranslational modification of hormones and extracellular components that can lead to dramatic structural changes in affected molecules, the biological significance of which is now beginning to be appreciated.

Steve Mitchell

Papers

Genome duplications and other features in 12 Mb of DNA sequence from human chromosome 16p and 16q.

Human 3'-phosphoadenosine 5'-phosphosulfate synthetase 1 (PAPSS1) and PAPSS2: gene cloning, characterization and chromosomal localization.