Biomechanisms of Comorbidity: Reviewing Integrative Analyses of Multi-omics Datasets and Electronic Health Records.

OBJECTIVES: Disease comorbidity is a pervasive phenomenon impacting patients' health outcomes, disease management, and clinical decisions. This review presents past, current and future research directions leveraging both phenotypic and molecular information to uncover disease similarity underpinning the biology and etiology of disease comorbidity. METHODS: We retrieved ~130 publications and retained 59, ranging from 2006 to 2015, that comprise a minimum number of five diseases and at least one type of biomolecule. We surveyed their methods, disease similarity metrics, and calculation of comorbidities in the electronic health records, if present. RESULTS: Among the surveyed studies, 44% generated or validated disease similarity metrics in context of comorbidity, with 60% being published in the last two years. As inputs, 87% of studies utilized intragenic loci and proteins while 13% employed RNA (mRNA, LncRNA or miRNA). Network modeling was predominantly used (35%) followed by statistics (28%) to impute similarity between these biomolecules and diseases. Studies with large numbers of biomolecules and diseases used network models or naïve overlap of disease-molecule associations, while machine learning, statistics, and information retrieval were utilized in smaller and moderate sized studies. Multiscale computations comprising shared function, network topology, and phenotypes were performed exclusively on proteins. CONCLUSION: This review highlighted the growing methods for identifying the molecular mechanisms underpinning comorbidities that leverage multiscale molecular information and patterns from electronic health records. The survey unveiled that intergenic polymorphisms have been overlooked for similarity imputation compared to their intragenic counterparts, offering new opportunities to bridge the mechanistic and similarity gaps of comorbidity.

A novel mutation in CELSR1 is associated with hereditary lymphedema.

BACKGROUND: Biological evidence reported in the literature supports the role of CELSR1 as being essential for valvular function in murine lymphatics. Yet thus far, there have been no variants in CELSR1 associated with lymphatic dysfunction in humans. CASE PRESENTATION: In this report, a rare early inactivating mutation in CELSR1 is found to be causal for non-syndromic, lower extremity lymphedema in a family across three generations. Near-infrared fluorescence lymphatic imaging shows that instead of being propelled within the lumen of well-defined lymphatic vessels, lymph moved in regions of both legs in an unusual fashion and within sheet-like structures. CONCLUSION: CELSRI may be responsible for primary, non-syndromic lymphedema in humans.

Diversity in the preimmune immunoglobulin repertoire of SHR lines susceptible and resistant to end-organ injury.

We used next-generation sequencing to identify immunoglobulin heavy chain (IGH) genetic variation in two closely related hypertensive rat lines that differ in susceptibility to end-organ disease (SHR-A3 and SHR-B2). The two SHR lines differ extensively at the IGH locus from the rat reference genome sequence and from each other, creating 306 sequence unique IGH genes. Compared with IGH genes mapped in the rat reference genome sequence, 98 are null gene alleles (31 are null in both SHR lines, 45 are null in SHR-A3 only and 23 are null in SHR-B2 only). Of the 306 divergent gene sequences, 126 result in amino acid substitution and, among these, SHR-A3 and SHR-B2 differ from one another at the amino acid level in 96 segments. Twelve pseudogenes in the rat reference genome sequence had changes displacing the stop codon and creating probable functional genes in either or both SHR-A3 and SHR-B2. A further five alleles that encoded functional rat reference genome sequence genes or open reading frames were converted to pseudogenes in either or both SHR-A3 and SHR-B2. These studies reveal that the preimmune immunoglobulin repertoire is highly divergent among SHR lines differing in end-organ injury susceptibility and this may modify immune mechanisms in hypertensive renal injury.

A beta-tubulin leucine cluster involved in microtubule assembly and paclitaxel resistance.

Analysis of beta-tubulin alleles from nine paclitaxel-resistant Chinese hamster ovary cell lines revealed an unexpected cluster of mutations affecting Leu-215, Leu-217, and Leu-228. Six of the mutant alleles encode a His, Arg, or Phe substitution at Leu-215; another mutant allele has an Arg substitution at Leu-217; and the final two mutant alleles have substitutions of His or Phe at Leu-228. Using plasmids that allow tetracycline regulated expression, the L215H, L217R, and L228F mutations were introduced into a hemagglutinin antigen-tagged beta-tubulin cDNA and transfected into wild-type Chinese hamster ovary cells. In all three cases, low to moderate expression of the transfected mutant gene conferred paclitaxel resistance. Higher levels of expression caused disruption of microtubule assembly, cell cycle arrest at mitosis, and failure to proliferate. Consistent with reduced microtubule stability, cells expressing mutant hemagglutinin beta-tubulin had fewer acetylated microtubules than nonexpressing cells in the same population. These data, together with previous studies showing that the paclitaxel-resistant mutant cell lines have less stable microtubules, indicate that the leucine cluster represents an important structural motif for microtubule assembly.

Vinblastine induces an interaction between FtsZ and tubulin in mammalian cells.

The Escherichia coli cell division protein FtsZ was expressed in Chinese hamster ovary cells, where it formed a striking array of dots that were independent of the mammalian cytoskeleton. Although FtsZ appears to be a bacterial homolog of tubulin, its expression had no detectable effects on the microtubule network or cell growth. However, treatment of the cells with vinblastine at concentrations that caused microtubule disassembly rapidly induced a network of FtsZ filaments that grew from and connected the dots, suggesting that the dots are an active storage form of FtsZ. Cells producing FtsZ also exhibited vinblastine- and calcium-resistant tubulin polymers that colocalized with the FtsZ network. The FtsZ polymers could be selectively disassembled, indicating that the two proteins were not copolymerized. The vinblastine effects were readily reversible by washing out the drug or by treating the cells with the vinblastine competitor, maytansine. These results demonstrate that FtsZ assembly can occur in the absence of bacterial chaperones or cofactors, that FtsZ and tubulin do not copolymerize, and that tubulin-vinblastine complexes have an enhanced ability to interact with FtsZ.

alpha-Tubulin limits its own synthesis: evidence for a mechanism involving translational repression.

A Chinese hamster alpha-tubulin cDNA was modified to encode an 11-amino acid carboxyl-terminal extension containing the immunodominant epitope from influenza hemagglutinin antigen (to create HA alpha 1-tubulin) and was cloned into a vector for expression in mammalian cells. 12 stable CHO cell lines expressing this HA alpha 1-tubulin were isolated and characterized. HA alpha 1-tubulin incorporated into all classes of microtubules, assembled to the same extent as the endogenous tubulin, and did not perturb the growth of the cells in which it was expressed. However, overexpression of HA alpha 1-tubulin strongly repressed the synthesis of endogenous alpha-tubulin while having little or no effect on the synthesis of beta-tubulin. Treatment of transfected cells with sodium butyrate to induce even greater expression of HA alpha 1-tubulin led to a further decrease in synthesis of endogenous alpha-tubulin that was fully reversible upon removal of the inducer. Decreased synthesis of alpha-tubulin in transfected cells did not result from decreased levels of alpha-tubulin mRNA, as demonstrated by ribonuclease protection assays. On the other hand, colchicine, a drug previously shown to destabilize the tubulin message, caused a clear reduction in both protein synthesis and mRNA levels for transfected HA alpha 1-tubulin and endogenous alpha-tubulin, thus indicating that the decreased alpha-tubulin synthesis observed as a result of HA alpha 1-tubulin overexpression is distinct from the previously described autoregulation of tubulin. The results are consistent with a mechanism in which free alpha-tubulin inhibits the translation of its own message as a way of ensuring stoichiometric synthesis of alpha- and beta-tubulin.

Significant divergence in nucleotide sequences for beta-tubulin from different laboratory strains of Chinese hamster ovary cells.

The nucleotide sequences for isotype 1 beta-tubulin cDNAs cloned from different laboratory strains of Chinese hamster ovary (CHO) cells were compared and found to contain an unexpected number of sequence differences in both translated and untranslated regions of the gene. The results indicate significant changes in the DNA, but not protein, sequence while the cells have been in culture and reveal sequences in the 5' and 3' untranslated regions that have resisted these changes.

Overexpression of an epitope-tagged beta-tubulin in Chinese hamster ovary cells causes an increase in endogenous alpha-tubulin synthesis.

A Chinese hamster beta-tubulin cDNA, engineered to express a 9 amino acid epitope from the influenza hemagglutinin antigen (HA), was transfected into Chinese hamster ovary (CHO) cells. The recombinant protein (HA beta 1-tubulin) appeared to behave normally by the following criteria: immunofluorescence indicated that HA beta 1-tubulin incorporated into all classes of interphase and spindle microtubules as well as microtubule organizing centers. The sensitivity of the cells expressing HA beta 1-tubulin to Colcemid and taxol was unchanged. A 210 kD microtubule associated protein (MAP) remained associated with microtubules that incorporate HA beta 1-tubulin. The synthesis of both endogenous beta-tubulin and HA beta 1-tubulin was repressed by colchicine. The HA beta 1-tubulin incorporated into microtubules to the same extent as the endogenous beta-tubulin, and the overall extent of microtubule assembly in transfected cells was unchanged. Finally, transfected cells had normal growth rates and morphologies. When effects on endogenous tubulin production were measured, it was found that expression of the HA beta 1-tubulin reduced the synthesis of endogenous wild-type beta-tubulin but increased the synthesis of alpha-tubulin. At steady state, a small increase in total tubulin consistent with the increased synthesis of alpha-tubulin was found. The results indicate that expression of excess exogenous beta-tubulin perturbs the synthesis of endogenous alpha-tubulin in a manner that is not easily explained by current models of tubulin regulation. The changes in tubulin synthesis along with degradation of excess tubulin subunits may reflect mechanisms that exist to ensure coordinate levels of alpha- and beta-tubulin for assembly.

Stable expression of heterologous microtubule-associated proteins (MAPs) in Chinese hamster ovary cells: evidence for differing roles of MAPs in microtubule organization.

To study the effects of microtubule-associated proteins (MAPs) on in vivo microtubule assembly, cDNAs containing the complete coding sequences of a Drosophila 205-kD heat stable MAP, human MAP 4, and human tau were stably transfected into CHO cells. Constitutive expression of the transfected genes was low in most cases and had no obvious effects on the viability of the transfected cell lines. High levels of expression, as judged by Western blots, immunofluorescence, and Northern blots, could be induced by treating cells with sodium butyrate. High levels of MAPs were maintained for at least 24-48 h after removal of the sodium butyrate. Immunofluorescence analysis indicated that all three MAPs bound to cellular microtubules, but only the transfected tau caused a rearrangement of microtubules into bundles. Despite high levels of expression of these exogenous MAPs and the bundling of microtubules in cells expressing tau, transfected cells had normal levels of assembled and unassembled tubulin. With the exception of the tau-induced bundles, microtubules in transfected cells showed the same sensitivity as control cells to microtubule depolymerization by Colcemid. Further, all three MAPs were ineffective in reversing the taxol-dependent phenotype of a CHO mutant cell line. The absence of a quantitative effect of any of these heterologous proteins on the assembly of tubulin suggests that these MAPs may have different roles in vivo from those inferred previously from in vitro experiments.

The presence but not the sequence of the N-terminal peptide in cardiac TnC is important for function.

The most diverged region of the primary amino acid sequence between cardiac (cTnC) and fast skeletal troponin C is the N-terminal ten amino acids. We report here that major changes in the primary sequence of this region in cTnC had a minimal effect on the ability of the mutant proteins to recover maximal activity in TnC-extracted cardiac and fast skeletal muscle myofibrils. However, deletion of the N-terminal nine amino acids resulted in a 60% decrease in maximal Ca(2+)-dependent ATPase activity with only a small change in the pCa50 of activation. Deletion of the N-terminal peptide did not appear to appreciably affect the Ca(2+)-binding properties of cTnC, but it did alter the interaction with hydrophobic fluorescent probes. Thus, the presence but not the sequence, of the N-terminal extension is important for the maximal activity of cTnC. The N-terminal helix may function in a relatively non-specific manner to prevent unfavorable interactions between domains in cTnC or between cTnC and other troponin subunits.

[The molecular diagnosis of hereditary diseases. In memoriam Dr. Eduardo Aguirre Pequeño]. / Diagnóstico molecular de enfermedades hereditarias. En memoria del académico doctor Eduardo Aguirre Pequeño.

Accordingly, we have established in our unit a DNA diagnosis laboratory and have started molecular genetics and epidemiological studies of several inherited diseases. We have started with cystic fibrosis, muscular dystrophy and hemophilia A. We practice the molecular diagnosis with both, Southern transfer and the polymerase chain reaction, using either direct (detection of mutations) or indirect (restriction fragment length polymorphisms) approaches. With the studies we have so far carried out, we have been able to provide genetic counseling and gained valuable information on the type and frequency of mutation associated to these diseases in our region.

Prevalence in two mexican cities of human papillomavirus DNA sequences in cervical cancer.

In Mexico, about 30% of all malignant tumors in women are uterine cervix carcinomas. It is one of their main causes of death. We have previously shown that in Mexico City, 31% (5/16) of the analyzed tumoral samples contained HPV-16 DNA sequences. We have now extended this observation in Mexico City and included the city of Monterrey and found that the prevalence of HPV-16 is similar in both: 26% (6/23) for Monterrey and 29% (4/14) for Mexico City. HPV-18 was detected in only 10% (1/10) and 7% (1/14) of the tumors in these two populations when assayed with an HPV-18 specific probe. In both cities, the majority of the samples analyzed (including samples from the four stages of severity of the disease) contained integrated papillomavirus DNA sequences. Our results suggest that the mexican population contains a rather low proportion of HPV-16 and HPV-18 sequences in uterine-cervix carcinoma.