Estimation of DNA contamination and its sources in genotyped samples.

Array genotyping is a cost-effective and widely used tool that enables assessment of up to millions of genetic markers in hundreds of thousands of individuals. Genotyping array data are typically highly accurate but sensitive to mixing of DNA samples from multiple individuals before or during genotyping. Contaminated samples can lead to genotyping errors and consequently cause false positive signals or reduce power of association analyses. Here, we propose a new method to identify contaminated samples and the sources of contamination within a genotyping batch. Through analysis of array intensity and genotype data from intentionally mixed samples and 22,366 samples of the Michigan Genomics Initiative, an ongoing biobank-based study, we show that our method can reliably estimate contamination. We also show that identifying sources of contamination can implicate problematic sample processing steps and guide process improvements. Compared to existing methods, our approach can estimate the proportion of contaminating DNA more accurately, eliminate the need for external databases of allele frequencies, and provide contamination estimates that are more robust to the ancestral origin of the contaminating sample.

SARS-CoV-2 receptor networks in diabetic and COVID-19-associated kidney disease.

COVID-19 morbidity and mortality are increased via unknown mechanisms in patients with diabetes and kidney disease. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) for entry into host cells. Because ACE2 is a susceptibility factor for infection, we investigated how diabetic kidney disease and medications alter ACE2 receptor expression in kidneys. Single cell RNA profiling of kidney biopsies from healthy living donors and patients with diabetic kidney disease revealed ACE2 expression primarily in proximal tubular epithelial cells. This cell-specific localization was confirmed by in situ hybridization. ACE2 expression levels were unaltered by exposures to renin-angiotensin-aldosterone system inhibitors in diabetic kidney disease. Bayesian integrative analysis of a large compendium of public -omics datasets identified molecular network modules induced in ACE2-expressing proximal tubular epithelial cells in diabetic kidney disease (searchable at hb.flatironinstitute.org/covid-kidney) that were linked to viral entry, immune activation, endomembrane reorganization, and RNA processing. The diabetic kidney disease ACE2-positive proximal tubular epithelial cell module overlapped with expression patterns seen in SARS-CoV-2-infected cells. Similar cellular programs were seen in ACE2-positive proximal tubular epithelial cells obtained from urine samples of 13 hospitalized patients with COVID-19, suggesting a consistent ACE2-coregulated proximal tubular epithelial cell expression program that may interact with the SARS-CoV-2 infection processes. Thus SARS-CoV-2 receptor networks can seed further research into risk stratification and therapeutic strategies for COVID-19-related kidney damage.

Microcephaly, intellectual impairment, bilateral vesicoureteral reflux, distichiasis, and glomuvenous malformations associated with a 16q24.3 contiguous gene deletion and a Glomulin mutation.

Two hereditary syndromes, lymphedema-distichiasis (LD) syndrome and blepharo-chelio-dontic (BCD) syndrome include the aberrant growth of eyelashes from the meibomian glands, known as distichiasis. LD is an autosomal dominant syndrome primarily characterized by distichiasis and the onset of lymphedema usually during puberty. Mutations in the forkhead transcription factor FOXC2 are the only known cause of LD. BCD syndrome consists of autosomal dominant abnormalities of the eyelid, lip, and teeth, and the etiology remains unknown. In this report, we describe a proband that presented with distichiasis, microcephaly, bilateral grade IV vesicoureteral reflux requiring ureteral re-implantation, mild intellectual impairment and apparent glomuvenous malformations (GVM). Distichiasis was present in three generations of the proband's maternal side of the family. The GVMs were severe in the proband, and maternal family members exhibited lower extremity varicosities of variable degree. A GLMN (glomulin) gene mutation was identified in the proband that accounts for the observed GVMs; no other family member could be tested. TIE2 sequencing revealed no mutations. In the proband, an additional submicroscopic 265 kb contiguous gene deletion was identified in 16q24.3, located 609 kb distal to the FOXC2 locus, which was inherited from the proband's mother. The deletion includes the C16ORF95, FBXO31, MAP1LC3B, and ZCCHC14 loci and 115 kb of a gene desert distal to FOXC2 and FOXL1. Thus, it is likely that the microcephaly, distichiasis, vesicoureteral, and intellectual impairment in this family may be caused by the deletion of one or more of these genes and/or deletion of distant cis-regulatory elements of FOXC2 expression.

SARS-CoV-2 receptor networks in diabetic and COVID-19 associated kidney disease.

COVID-19 morbidity and mortality is increased in patients with diabetes and kidney disease via unknown mechanisms. SARS-CoV-2 uses angiotensin-converting enzyme 2 (ACE2) for entry into host cells. Since ACE2 is a susceptibility factor for infection, we investigated how diabetic kidney disease (DKD) and medications alter ACE2 receptor expression in kidneys. Single cell RNA profiling of healthy living donor (LD) and DKD kidney biopsies revealed ACE2 expression primarily in proximal tubular epithelial cells (PTEC). This cell specific localization was confirmed by in situ hybridization. ACE2 expression levels were unaltered by exposures to renin angiotensin aldosterone system inhibitors in DKD. Bayesian integrative analysis of a large compendium of public -omics datasets identified molecular network modules induced in ACE2-expressing PTEC in DKD (searchable at hb.flatironinstitute.org/covid-kidney) that were linked to viral entry, immune activation, endomembrane reorganization, and RNA processing. The DKD ACE2-positive PTEC module overlapped with expression patterns seen in SARS-CoV-2 infected cells. Similar cellular programs were seen in ACE2-positive PTEC obtained from urine samples of 13 COVID-19 patients who were hospitalized, suggesting a consistent ACE2-coregulated PTEC expression program that may interact with the SARS-CoV-2 infection processes. Thus SARS-CoV-2 receptor networks can seed further research into risk stratification and therapeutic strategies for COVID-19 related kidney damage.

Amplification and overexpression of the dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 (DYRK2) gene in esophageal and lung adenocarcinomas.

Genomic amplification can lead to the activation of cellular proto-oncogenes during tumorigenesis, and is observed in most, if not all, human malignancies, including adenocarcinomas of lung and esophagus. Using a two-dimensional restriction landmark genomic scanning technique, we identified five NotI/HinfI fragments with increased genomic dosage in an adenocarcinoma of the gastroesophageal junction. Four of these amplified fragments were matched within three contigs of chromosome 12 using the bioinformatics tool, Virtual Genome Scan. All three of the contigs map to the 12q13-q14 region, and the regional amplification in the tumor was verified using comparative genomic hybridization analysis. The 12q14 amplicon was characterized using sequence tagged site-amplification mapping with DNA from paired normal-tumor tissues of 75 gastroesophageal and 37 lung adenocarcinomas. The amplicon spans a region of >12 Mb between genes DGKA and BLOV1. The core-amplified domain was determined to be <0.5 Mb between marker WI-12457 and gene IFNG. However, MDM2, a well-documented oncogene of the region, is outside the core-domain. Eleven genes and expressed sequence tags within the amplicon were selected for quantitative reverse transcription-PCR, and DYRK2, a member of the dual-specificity kinase family, was overexpressed in all of the tumors showing gene amplification. Among the sequence tagged site/expressed sequence tag/gene markers tested, DYRK2 demonstrated the highest DNA copy number and the highest level of mRNA overexpression in the tumors. Moreover, DYRK2 mRNA overexpression (>2.5-fold of normal mean) was found in 18.6% of additional 86 lung adenocarcinomas in an assay using oligonucleotide microarrays. DYRK2 mRNA overexpression occurs more frequently than gene amplification in both esophageal and lung adenocarcinomas. This is the first report of amplification and overexpression of DYRK2 in any tumor type.

The hepatocyte nuclear factor 3 alpha gene, HNF3alpha (FOXA1), on chromosome band 14q13 is amplified and overexpressed in esophageal and lung adenocarcinomas.

Genomic amplification is observed in many, if not all, types of human malignancy and is one of the mechanisms for the activation of dominant-acting oncogenes in tumorigenesis. In the present study, three amplified restriction fragments were identified in an esophageal adenocarcinoma (P16) using the restriction landmark genome scanning two-dimensional gel technique. These fragments were cloned, sequenced, and mapped to chromosome band 14q13. Using the sequence tagged site-amplification mapping approach, we defined the core-amplified domain by screening 75 normal-tumor paired esophageal samples. The frequency of 14q13 amplification is 6.7% in esophageal tumors, and the amplicon spans >6 Mb in 1 tumor but is contained in a region <0.3 Mb in all of the remaining amplified tumors. Quantitative reverse transcription-PCR (RT-PCR) of 8 genes and expressed sequence tags located within the core-amplified domain revealed that the HNF3alpha (FOXA1)(4) gene, a forkhead gene family member, was overexpressed in all of the amplified esophageal tumors. HNF3alpha amplification was confirmed by Southern blot and interphase fluorescence in situ hybridization analyses, and the results of real-time RT-PCR were consistent with that of the regular quantitative RT-PCR. Increased immunohistochemical nuclear staining of the HNF3alpha protein was detected in all of the tumors containing 14q13 amplification. Affymetrix oligonucleotide microarrays of 86 lung adenocarcinomas demonstrated that expression of the HNF3alpha mRNA was elevated (> or =2.5-fold of mean expression in normal lung) in 37% (32 of 86) of the tumors analyzed. Gene amplification of HNF3alpha was detected in 2 of the 5 overexpressed lung tumors examined. This is the first report of HNF3alpha amplification, and overexpression in esophageal and lung adenocarcinomas. Amplification of HNF3alpha in esophageal and lung tumors may suggest a potential oncogenic role for this gene in tumorigenesis.

Foxc2 is expressed in developing lymphatic vessels and other tissues associated with lymphedema-distichiasis syndrome.

The molecular events involved in lymphatic development are poorly understood. Hence, the genes responsible for hereditary lymphedema are of great interest due to the potential for providing insights into the mechanisms of lymphatic development, the diagnosis, prevention and treatment of lymphedema, and lymphangiogenesis during tumor growth. Mutations in the FOXC2 transcription factor cause a major form of hereditary lymphedema, the lymphedema-distichiasis syndrome. We have conducted a study of Foxc2 expression during mouse development using immunohistochemistry, and examined its expression in lymphatics compared to its paralog Foxc1 and to Vegfr-3, Prox1 and other lymphatic and blood vascular proteins. We have found that Foxc2 is expressed in lymphatic primordia, jugular lymph sacs, lymphatic collectors and capillaries, as well as in podocytes, developing eyelids and other tissues associated with abnormalities in lymphedema-distichiasis syndrome.

Mutation of the FOXC2 gene in familial distichiasis.

OBJECTIVE: To examine the FOXC2 gene in a family with hereditary distichiasis. BACKGROUND: Distichiasis, ie, a second row of eyelashes arising from the meibomian glands of the eyelids, can be inherited either alone (Online Mendelian Inheritance in Man [OMIM] no. 126300) or, more commonly, as part of the lymphedema-distichiasis (LD) syndrome (OMIM no. 153400). More than 45 families with mutations in the FOXC2 gene and LD have been described. Both lymphedema and distichiasis are highly penetrant. Distichiasis without lymphedema is not commonly seen. METHODS: We examined three generations of a family (N = nine members) with hereditary distichiasis but without lymphedema or other features of LD syndrome. The FOXC2 gene was polymerase chain reaction--amplified from genomic DNA from all family members and examined for mutations. RESULTS: Clinical examination showed distichiasis of all four lids in two affected family members across two generations. There were no other consistent ophthalmologic abnormalities in the family. A cytosine-to-adenine transversion was identified in DNA from affected study participants at nucleotide position 1076, which would be predicted to cause truncation of the protein at codon 359. This change was not observed in any of the nine unaffected family members participating. CONCLUSIONS: This finding suggests that hereditary distichiasis and LD may not be separate genetic disorders but different phenotypic expressions of the same underlying disorder. Ophthalmologists should be aware that LD may present as distichiasis alone and counsel and refer their patients appropriately.

A novel VEGFR3 mutation causes Milroy disease.

Milroy disease, also known as primary congenital lymphedema, is a hereditary form of lymphedema with autosomal dominant inheritance. Individuals with Milroy disease are typically characterized by congenital onset of lymphedema of the lower limbs due to hypoplasia of the lymphatic vessels. The genetic basis of most cases of Milroy disease has not been established, although mutations in the vascular endothelial growth factor receptor VEGFR3 (FLT-4) are responsible for some cases with 17 mutations described to date. In this report, we describe a novel VEGFR3 mutation in exon 22 in a four-generation family in which congenital lymphedema segregates in an autosomal dominant manner. In addition to lymphedema, affected family members had other clinical manifestations associated with Milroy disease including hydrocele, ski jump toenails, large caliber veins, and subcutaneous thickening. We screened VEGFR3 for mutations which revealed a novel 3059A>T transversion in exon 22 resulting in Q1020L missense mutation in the second tyrosine kinase domain of VEGFR3. This mutant allele segregated with lymphedema among affected individuals with incomplete penetrance. This is the first report of an exon 22 mutation in Milroy disease.

Molecular characterization and mapping of ATOH7, a human atonal homolog with a predicted role in retinal ganglion cell development.

The human ATOH7 gene encodes a basic helix-loop-helix (bHLH) transcription factor that is highly similar to Drosophila Atonal within the conserved bHLH domain. The ATOH7 coding region is contained within a single exon. We mapped ATOH7 to Chromosome (Chr) 10q21.3-22.1, a region syntenic to the segment of mouse Chr 10 where Atoh7 (formerly Math5) is located. The evolutionary relationship between ATOH7 and other atonal homologs was investigated using parsimony analysis. A direct comparison of ATH5/7 and ATH1 protein subgroups to Atonal also revealed a nonrandom distribution of amino acid changes across the bHLH domain, which may be related to their separate visual and proprioceptive sensory functions. Among bHLH genes, ATOH7 is most closely related to Atoh7. This sequence conservation extends significantly beyond the coding region. We define blocks of strong homology in flanking human and mouse genomic DNA, which are likely to include cis regulatory elements. Because targeted deletion of Atoh7 causes optic nerve agenesis in mice, we propose ATOH7 as a candidate for human optic nerve aplasia and related clinical syndromes.

FOXC2 haploinsufficient mice are a model for human autosomal dominant lymphedema-distichiasis syndrome.

Lymphedema-distichiasis (LD) (OMIM 153400) is a rare autosomal-dominant condition characterized by pubertal onset of lower limb lymphedema and an aberrant second row of eyelashes arising from the meibomian glands. In some patients cardiac, skeletal and other defects coexist. We previously identified inactivating, nonsense and frameshift mutations in the forkhead transcription factor FOXC2 in affected members of LD families. To further delineate the relationship of FOXC2 deficiency to the clinical (and lymphangiodysplastic) phenotype in this syndrome, we performed dynamic lymphatic imaging and immunohistochemical examination of lymphatic tissues in mice heterozygous (+/-) for a targeted disruption of Foxc2. Adult heterozygote mice characteristically exhibited a generalized lymphatic vessel and lymph node hyper plasia and rarely exhibited hindlimb swelling. Retrograde lymph flow through apparently incompetent interlymphangion valves into the mesenteric nodes, intestinal wall and liver was also observed. In addition, Foxc2 +/- mice uniformly displayed distichiasis. We conclude that Foxc2 haploinsufficient mice mimic closely the distinctive lymphatic and ocular phenotype of LD patients. Furthermore, the craniofacial, cardiovascular and skeletal abnormalities sometimes associated with LD have previously been shown to be fully penetrant in homozygous Foxc2 null mice. This Foxc2 mutant mouse thus provides an ideal model for exploring molecular mechanisms and physiologic events in mesenchymal differentiation associated with lymphatic growth and development and the clinical abnormalities seen in human LD syndrome.