Análisis De Fallas Asociados Con La Calidad

Understanding the Variable Expressivity of ALGS Syndrome

From this work, we have identified correlations between the multisystemic phenotypes in ALGS. There was significant positive correlation between presence of cardiovascular anomalies and two other phenotypes, severe liver disease and presence of butterfly vertebrae. Posterior embryotoxon and renal anomalies were not associated with any of the tested phenotypes. The biological significance of this correlation is unclear, and it may be due to similar functions of JAG1 during development of these three systems. It could be that different organ systems have varying dosage sensitivity to JAG1 haploinsufficiency. The heart, liver, and skeletal systems could be more sensitive to the regulatory effects of JAG1 than the other affected systems. Future work performing functional studies varying the dosage of JAG1 during development can confirm if variable expressivity arises from JAG1 dosage.

In addition, we observed that the type of mutation and transmission characteristics of the JAG1 allele were not associated with the main disease phenotypes of ALGS. Our sib-pair analysis and association test of polymorphisms on JAG1 did not find a significant association between normal JAG1 allele and disease phenotype. We could only conclude the absence of a strong modifying effect emanating from the normal JAG1 allele, as we lack the power to detect small effect sizes.

Gene expression studies we performed exploring the consequences of JAG1 haploinsufficiency revealedsome interesting expression signatures. Genes in the HES and HEY families are known to be important downstream targets of Notch signaling. This study found that HEY2, a co-repressor of transcription, was downregulated in the deleted line of the endoderm, and it was the most significantly altered downstream Notch target and it. Genes regulated by HEY2 may be responsible for variable expressivity due to JAG1 haploinsufficiency. Moreover, pathway analysis revealed an enrichment of upregulated genes that are involved in nucleosome

and chromosome assembly. Additional biological replicates can help confirm the robustness of these gene expression alterations.

In a genome-wide, hypothesis-neutral search for genetic modifiers of liver disease severity, we found a risk allele 14kb upstream of THBS2 on 6q27, rs7382539, from our SNP association study. This region contains H3K27Ac and H3K4Me1 histone marks, suggesting it to be a poised enhancer. Because this genetic association appears to fall in a regulatory region, design and execution of experimental studies disrupting this regulatory region can help predict the biological consequences resulting from this alteration. Functional studies in mice have shown that THBS2 can enhance Jag-Notch binding (Meng et al. 2009), which implicates this gene as a genetic modifier for variable expressivity in ALGS. In the CNV association study, we have identified a deletion in 19q13.2 and duplication in 5p13.3 that may be associated with liver disease severity. Follow-up studies confirming these CNVs and characterization of the breakpoints, especially the duplications, are necessary before undertaking functional studies of CYP2A6, GOLPH3, and PDZD2, the genes affected by these two copy number alterations. Genetic Susceptibility to Biliary Atresia

Our studies to uncover candidate genes and alterations that contribute to the pathogenesis of BA revealed several novel findings and areas for future research. XPNPEP1 and ADD3 flank the 10q25.1 region containing the intergenic SNP signal uncovered from the Chinese GWAS of isolated BA. Fine-mapping of this region in a cohort with European ancestry yielded a stronger association signal in the intronic region of ADD3, and the immunohistochemistry studies suggest that deficiencies of ADD3 may confer susceptibility to BA. However, gene knockdown experiments in model organisms should be conducted in order to confirm the significance of ADD3 in BA.

Whole exome sequencing of 30 isolated BA probands suggest that this disease is genetically heterogeneous. Analysis of candidate genes known to be associated with BA suggest

that inherited, rare variants may contribute to disease etiology. Furthermore, additional gene candidates were identified in the cilia (DNAH3) and planar cell polarity pathways (DCHS1, SMAD9, and VPS13C). These genes have never been studied in the context of BA, and observations in knockdown constructs of these genes in model organisms may help demonstrate their role in this disease.

The investigation of a family with affected, dizygotic twins suggested that a maternally- inherited frameshift variant that resulted in the truncation of IFNK could confer susceptibility to BA. This alteration appears to be three times more prominent in our disease cohort than in the general, healthy population, as it appears in 2 additional probands of our WES cohort. An additional proband was found to have a rare, missense variant in IFNK. Although this gene is not well-studied, we predict that defects in IFNK could impair the immune response to toxins and pathogens. A larger genetic association study may be able to confirm an enrichment of IFNK variants in BA patients.

The second family investigated has a proband that presented with BA and heterotaxy. The father also presented with symptoms at a young age confirmed for heterotaxy, but he did not have BA. From CNV analysis of this family, we uncovered a large deletion on 20p11.21 encompassing only one gene, FOXA2, transmitted from the father to the proband. This critical liver transcription factor has been associated with the Nodal pathway, that is important for the proper development of body symmetry, as well as the development of the liver and biliary system. This deletion is not observed in the healthy control population, and we hypothesize that FOXA2 is a haploinsufficient gene. Furthermore, we suggest that FOXA2 causes heterotaxy and BA in this family. Further studies of this gene in the context of BA may lead to a better understanding of the disease etiology.

APPENDIX

Table A1. Genes Differentially Expressed in Fibroblast, iPSCs, and Endoderm between the deleted (D) and non-deleted (D) lines.

Mean: Mean Expression, FC: Fold Change (Deleted / Non-Deleted), FDR: False Discovery Rate

Gene Symbol Chr Gene Name Mean,

N-D Mean,

D FC p FDR

CAMK1G 1 calcium/calmodulin-dependent protein kinase IG 10.2 6.0 0.05 5.9E-5 0.0%

TRPV2 17 transient receptor potential cation channel, subfamily V, member 2 9.7 5.9 0.07 7.6E-5 0.0%

HOXD10 2 homeobox D10 9.2 5.4 0.07 9.3E-5 0.0%

NCAM2 21 neural cell adhesion molecule 2 8.8 5.5 0.10 1.1E-4 0.0%

VAT1L 16 vesicle amine transport protein 1 homolog (T. californica)-like 9.7 6.6 0.12 1.3E-4 0.0%

PTGS2 1 prostaglandin-endoperoxide synthase 2 11.5 7.8 0.08 1.6E-4 0.0%

NEFM 8 neurofilament, medium polypeptide 10.0 6.4 0.08 1.9E-4 4.5%

CSRP1 1 cysteine and glycine-rich protein 1 9.6 6.7 0.13 5.3E-5 0.0%

NNAT 20 neuronatin 9.5 5.8 0.08 5.8E-5 0.0%

PCDHGA3 5 protocadherin gamma subfamily A, 3 4.6 6.9 4.87 6.3E-5 0.0%

CDYL2 16 chromodomain protein, Y-like 2 7.5 6.3 0.42 8.2E-5 0.0%

C18orf2 18 chromosome 18 open reading frame 2 3.2 4.8 3.00 1.0E-4 0.0%

TMTC1 12 transmembrane and tetratricopeptide repeat containing 1 8.2 9.4 2.34 1.1E-4 0.0%

PKIB 6 protein kinase (cAMP-dependent, catalytic) inhibitor beta 7.3 8.4 2.12 1.3E-4 0.0%

DNAJA4 15 DnaJ (Hsp40) homolog, subfamily A, member 4 7.7 6.4 0.40 1.4E-4 0.0%

TBX20 7 T-box 20 7.4 9.3 3.70 5.9E-5 0.0%

MTNR1A 4 melatonin receptor 1A 7.5 8.9 2.63 7.6E-5 0.0%

C18orf2 18 chromosome 18 open reading frame 2 3.1 5.2 4.29 9.3E-5 0.0%

HIST1H2AI 6 histone cluster 1, H2ai 6.8 8.6 3.50 1.1E-4 0.0%

LOC100506979 6 hypothetical LOC100506979 5.5 6.9 2.58 1.3E-4 0.0%

PDE12 3 phosphodiesterase 12 6.4 7.9 2.93 1.6E-4 0.0%

PLAC8 4 placenta-specific 8 5.3 7.1 3.49 1.8E-4 0.0%

C4orf11 4 chromosome 4 open reading frame 11 5.4 6.7 2.56 2.1E-4 0.0%

MSMB 10 microseminoprotein, beta- 5.3 6.9 3.05 2.3E-4 0.0%

HIST1H2BG 6 histone cluster 1, H2bg 7.6 9.2 2.89 2.4E-4 0.0%

HIST1H3H 6 histone cluster 1, H3h 9.7 10.9 2.40 2.6E-4 0.0%

LGR5 12 leucine-rich repeat containing G protein-coupled receptor 5 9.8 10.9 2.12 3.1E-4 0.0%

PCDHGA3 5 protocadherin gamma subfamily A, 3 4.8 6.8 3.95 3.3E-4 0.0%

DDX58 9 DEAD (Asp-Glu-Ala-Asp) box polypeptide 58 8.4 9.3 1.84 3.6E-4 3.2%

MYCT1 6 myc target 1 7.4 8.5 2.14 4.0E-4 3.2%

PCDHB5 5 protocadherin beta 5 8.8 10.6 3.49 4.3E-4 3.2%

OLR1 12 oxidized low density lipoprotein (lectin-like) receptor 1 6.6 8.5 3.67 4.5E-4 3.2%

PKIB 6 protein kinase (cAMP-dependent, catalytic) inhibitor beta 7.5 8.8 2.54 4.6E-4 3.2%

DLEU2 13 deleted in lymphocytic leukemia 2 (non-protein coding) 7.2 8.4 2.23 4.8E-4 3.2%

VIL1 2 villin 1 7.8 9.4 2.93 5.0E-4 3.2%

ARHGEF35 7 Rho guanine nucleotide exchange factor (GEF) 35 6.8 7.8 1.97 5.2E-4 3.2%

GPR128 3 G protein-coupled receptor 128 5.6 7.1 2.99 5.5E-4 3.2%

SNORA1 11 small nucleolar RNA, H/ACA box 1 4.9 6.2 2.53 6.0E-4 3.2%

EDN1 6 endothelin 1 6.8 8.2 2.59 6.2E-4 3.2%

INPP5F 10 inositol polyphosphate-5-phosphatase F 8.7 9.5 1.76 6.3E-4 3.2%

RPSAP58 19 ribosomal protein SA pseudogene 58 6.8 7.8 1.97 6.5E-4 3.2%

SLC38A5 X solute carrier family 38, member 5 9.4 10.6 2.28 6.7E-4 3.2%

KCNJ13 2 potassium inwardly-rectifying channel, subfamily J, member 13 5.1 6.9 3.64 7.0E-4 3.2%

GINS1 20 GINS complex subunit 1 (Psf1 homolog) 9.4 10.4 2.02 7.3E-4 3.2%

CYP2C8 10 cytochrome P450, family 2, subfamily C, polypeptide 8 4.3 5.1 1.81 7.5E-4 3.2%

HIST1H2BL 6 histone cluster 1, H2bl 7.7 9.3 2.84 8.0E-4 3.2%

ZNF677 19 zinc finger protein 677 5.9 6.9 1.91 8.2E-4 3.2%

TBX22 X T-box 22 5.6 6.9 2.39 8.5E-4 3.2%

LRRTM1 2 leucine rich repeat transmembrane neuronal 1 6.5 7.4 1.91 8.7E-4 3.2%

TXNIP 1 thioredoxin interacting protein 10.6 8.7 0.27 1.4E-4 4.1%

III. Endoderm II. iPSC I. Fibroblast

Figure A1. Screenshots of FileMaker Pro Database Used to Store Phenotype Information for ALGS Patients

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