Leslie A. McNoe

The prevalence of PAX2 mutations in patients with isolated colobomas or colobomas associated with urogenital anomalies.

The PAX2 gene is mutated in patients with ocular colobomas, vesicoureteral reflux (VUR), and kidney anomalies (renal-coloboma syndrome, OMIM 120330). The three abnormalities which make up this syndrome also occur in isolation, but the causal genes are not known. PAX2 encodes a transcription factor of the paired box class of DNA binding proteins, important for the development of the urogenital tract, optic nerve and adjacent retina, inner ear, and CNS. In this paper we have investigated the prevalence of PAX2 mutations in patients with ocular colobomas, microphthalmos, or retinal anomalies, either in isolation or with associated urogenital anomalies. Using PCR-SSCP, most or all exons of PAX2 were examined in blood DNA from 99 patients who have either ocular anomalies alone or a combination of ocular and urogenital conditions. PAX2 mutations were not detected in patients with ocular colobomas, either in isolation or with associated abnormalities, except in one patient with typical renal-coloboma syndrome. We conclude that PAX2 mutations are unlikely to be common in patients with ocular colobomas in isolation or in patients with ocular colobomas and associated anomalies, except for patients with typical renal-coloboma syndrome where PAX2 is known to be the aetiological cause.

Absence of PAX2 gene mutations in patients with primary familial vesicoureteric reflux.

Vesicoureteric reflux (VUR) is a common childhood condition characterised by regurgitation of urine from the bladder to the kidney. It is the commonest cause of end stage renal failure in children and an important cause in adults. Primary VUR is often familial, suggesting that genetic factors play an important role in its aetiology. Recently, VUR was observed as part of a syndrome, involving optic nerve colobomas and renal anomalies, caused by mutations of the PAX2 gene. PAX2 is a member of the paired box family of genes and is expressed in the ureteric bud and differentiating nephrogenic mesenchyme of the developing kidney. PAX2 has been shown to play a critical role in the development of both the kidney and the ureter. The occurrence of VUR in one family with the PAX2 mutation, and the expression pattern of PAX2 in developing ureteric bud, strongly suggested that PAX2 could be the cause of primary familial VUR. Single strand conformational polymorphism (SSCP) analysis of 23 affected subjects in eight families with primary familial VUR showed no alterations in exons 2-5 of the PAX2 gene. In addition, a polymorphic dinucleotide repeat marker located within the PAX2 gene segregated independently of the disease trait in one large family who primarily had VUR or reflux nephropathy. These results suggest that PAX2 is not a major cause of primary familial reflux.

Transposon mutagenesis identifies genes that cooperate with mutant Pten in breast cancer progression

Significance Triple-negative breast cancer (TNBC) is the most aggressive breast cancer subtype. Despite extensive cancer genome-sequencing efforts, there is still an incomplete understanding of the genetic networks driving TNBC. Here, we used Sleeping Beauty transposon mutagenesis to identify genes that cooperate with mutant Pten in the induction of TNBC. We identified 12 candidate TNBC trunk drivers and a larger number of progression genes. Subsequent functional validation studies identified eight human TNBC tumor suppressor genes, including the GATA-like transcriptional repressor TRPS1, which was shown to inhibit lung metastasis by deregulating the expression of multiple serpin and epithelial-to-mesenchymal transition (EMT) pathway genes. Our study provides a better understanding of the genetic forces driving TNBC and discovered genes with clinical importance in TNBC. Triple-negative breast cancer (TNBC) has the worst prognosis of any breast cancer subtype. To better understand the genetic forces driving TNBC, we performed a transposon mutagenesis screen in a phosphatase and tensin homolog (Pten) mutant mice and identified 12 candidate trunk drivers and a much larger number of progression genes. Validation studies identified eight TNBC tumor suppressor genes, including the GATA-like transcriptional repressor TRPS1. Down-regulation of TRPS1 in TNBC cells promoted epithelial-to-mesenchymal transition (EMT) by deregulating multiple EMT pathway genes, in addition to increasing the expression of SERPINE1 and SERPINB2 and the subsequent migration, invasion, and metastasis of tumor cells. Transposon mutagenesis has thus provided a better understanding of the genetic forces driving TNBC and discovered genes with potential clinical importance in TNBC.

Analyzing tumor heterogeneity and driver genes in single myeloid leukemia cells with SBCapSeq

A central challenge in oncology is how to kill tumors containing heterogeneous cell populations defined by different combinations of mutated genes. Identifying these mutated genes and understanding how they cooperate requires single-cell analysis, but current single-cell analytic methods, such as PCR-based strategies or whole-exome sequencing, are biased, lack sequencing depth or are cost prohibitive. Transposon-based mutagenesis allows the identification of early cancer drivers, but current sequencing methods have limitations that prevent single-cell analysis. We report a liquid-phase, capture-based sequencing and bioinformatics pipeline, Sleeping Beauty (SB) capture hybridization sequencing (SBCapSeq), that facilitates sequencing of transposon insertion sites from single tumor cells in a SB mouse model of myeloid leukemia (ML). SBCapSeq analysis of just 26 cells from one tumor revealed the tumors major clonal subpopulations, enabled detection of clonal insertion events not detected by other sequencing methods and led to the identification of dominant subclones, each containing a unique pair of interacting gene drivers along with three to six cooperating cancer genes with SB-driven expression changes.

Aortic dissection, patent ductus arteriosus, iris hypoplasia and brachytelephalangy in a male adolescent

We describe a 14-year-old male with dissection of the descending aorta, bilateral iris hypoplasia, striae distensae and brachytelephalangy, the latter being most marked in the thumbs. Inguinal herniae and a patent ductus arteriosus were surgically repaired in infancy. The pattern of abnormalities may constitute a previously undescribed syndrome. The proband died suddenly at the age of 17 years.

Molecular and clinical studies of three cases of female pseudohermaphroditism with caudal dysplasia suggest multiple etiologies

Female pseudohermaphroditism with caudal dysplasia is a clinical entity in which normal‐appearing male genitalia may occur in the apparent absence of testosterone or the sex‐determining gene (SRY). We have extended observations of two previously reported cases, and report a third case, which strongly suggests multiple etiologies. The first case was one of identical twins. The other identical twin did not show female pseudohermaphroditism with caudal dysplasia, but both patients had the rare birth defect of neonatal cataracts. We have explored skewed X‐inactivation as a possible difference between the two twins, with a negative result. The second case had a deletion at 10q25.3→ter. This is near the location of PAX2, and we searched for mutations in PAX2 in both this and the first case, with negative results. Neither patient had a scrotal raphe, suggesting that a failure of division of the cloacal membrane was an important step in their development of female pseudohermaphroditism. The final case is newly described and differed from the above two in the presence of a scrotal raphe and an elevated testosterone level. Although no source for the testosterone was found, this case suggests that the etiology in this patient was different and that the presence of a scrotal raphe can be used to distinguish between at least two etiologies.

Familial fibronectin glomerulopathy: analysis of chromosome 1q32 and uteroglobin gene loci in a large New Zealand family

SUMMARY: Recently, a newly recognized familial glomerulopathy with predominant fibronectin deposits has been reported. This is the first report of a family with this condition in Australasia and spans two generations over a 30‐year period, with the histologically confirmed glomerulopathy present in the father and five out of eight siblings. The clinical presentations have ranged from asymptomatic proteinuria, pregnancy‐associated proteinuria and the nephrotic syndrome to hypertension and proteinuria with progressive renal failure. The time‐course from presentation to renal failure was over a 20 years. Histology demonstrated global and diffuse thickening of capillary loops, but no cellular proliferation. Immunofluorescence demonstrated granular positivity for IgM in the capillary loops only. Electron microscopy demonstrated massive electron‐dense subendothelial granular deposits with occasional small fibrils and unremarkable epithelial cell foot processes. Immunohistochemical staining was strongly positive for fibronectin and negative for type I or type IV collagen and transforming growth factor β in all biopsies. Genetic studies of familial fibronectin glomerulopathy have recently highlighted two genetic loci. Firstly, a large five‐generation pedigree has been described with linkage of fibronectin glomerulopathy to chromosome 1q32. Secondly, fibronectin glomerulopathy has been reported in uteroglobin gene knockout mice. In our studies, DNA sequence analysis of the uteroglobin gene showed that it was normal in all family members, and a DNA polymorphism in the uteroglobin gene did not co‐segregate with the disease. In addition, DNA microsatellite markers at the 1q32 locus did not co‐segregate with the disease in our family. We presume that the underlying abnormality involves as yet undefined glomerular extracellular matrix regulation and is inherited as an autosomal dominant condition. These data favour genetic heterogeneity for the aetiology of fibronectin glomerulopathy.