Karen A. Johnstone

HNF1B Mutations Associate with Hypomagnesemia and Renal Magnesium Wasting

Mutations in hepatocyte nuclear factor 1B (HNF1B), which is a transcription factor expressed in tissues including renal epithelia, associate with abnormal renal development. While studying renal phenotypes of children with HNF1B mutations, we identified a teenager who presented with tetany and hypomagnesemia. We retrospectively reviewed radiographic and laboratory data for all patients from a single center who had been screened for an HNF1B mutation. We found heterozygous mutations in 21 (23%) of 91 cases of renal malformation. All mutation carriers had abnormal fetal renal ultrasonography. Plasma magnesium levels were available for 66 patients with chronic kidney disease (stages 1 to 3). Striking, 44% (eight of 18) of mutation carriers had hypomagnesemia (<1.58 mg/dl) compared with 2% (one of 48) of those without mutations (P < 0.0001). The median plasma magnesium was significantly lower among mutation carriers than those without mutations (1.68 versus 2.02 mg/dl; P < 0.0001). Because hypermagnesuria and hypocalciuria accompanied the hypomagnesemia, we analyzed genes associated with hypermagnesuria and detected highly conserved HNF1 recognition sites in FXYD2, a gene that can cause autosomal dominant hypomagnesemia and hypocalciuria when mutated. Using a luciferase reporter assay, we demonstrated HNF1B-mediated transactivation of FXYD2. These results extend the phenotype of HNF1B mutations to include hypomagnesemia. HNF1B regulates transcription of FXYD2, which participates in the tubular handling of Mg(2+), thus describing a role for HNF1B not only in nephrogenesis but also in the maintenance of tubular function.

Recessive mutations in the INS gene result in neonatal diabetes through reduced insulin biosynthesis

Heterozygous coding mutations in the INS gene that encodes preproinsulin were recently shown to be an important cause of permanent neonatal diabetes. These dominantly acting mutations prevent normal folding of proinsulin, which leads to beta-cell death through endoplasmic reticulum stress and apoptosis. We now report 10 different recessive INS mutations in 15 probands with neonatal diabetes. Functional studies showed that recessive mutations resulted in diabetes because of decreased insulin biosynthesis through distinct mechanisms, including gene deletion, lack of the translation initiation signal, and altered mRNA stability because of the disruption of a polyadenylation signal. A subset of recessive mutations caused abnormal INS transcription, including the deletion of the C1 and E1 cis regulatory elements, or three different single base-pair substitutions in a CC dinucleotide sequence located between E1 and A1 elements. In keeping with an earlier and more severe beta-cell defect, patients with recessive INS mutations had a lower birth weight (−3.2 SD score vs. −2.0 SD score) and were diagnosed earlier (median 1 week vs. 10 weeks) compared to those with dominant INS mutations. Mutations in the insulin gene can therefore result in neonatal diabetes as a result of two contrasting pathogenic mechanisms. Moreover, the recessively inherited mutations provide a genetic demonstration of the essential role of multiple sequence elements that regulate the biosynthesis of insulin in man.

Behavioural and cognitive abnormalities in an imprinting centre deletion mouse model for Prader–Willi syndrome

The genes in the imprinted cluster on human chromosome 15q11–q13 are known to contribute to psychiatric conditions such as schizophrenia and autism. Major disruptions of this interval leading to a lack of paternal allele expression give rise to Prader–Willi syndrome (PWS), a neurodevelopmental disorder with core symptoms of a failure to thrive in infancy and, on emergence from infancy, learning disabilities and over‐eating. Individuals with PWS also display a number of behavioural problems and an increased incidence of neuropsychiatric abnormalities, which recent work indicates involve aspects of frontal dysfunction. To begin to examine the contribution of genes in this interval to relevant psychological and behavioural phenotypes, we exploited the imprinting centre (IC) deletion mouse model for PWS (PWS‐IC+/−) and the five‐choice serial reaction time task (5‐CSRTT), which is primarily an assay of visuospatial attention and response control that is highly sensitive to frontal manipulations. Locomotor activity, open‐field behaviour and sensorimotor gating were also assessed. PWS‐IC+/− mice displayed reduced locomotor activity, increased acoustic startle responses and decreased prepulse inhibition of startle responses. In the 5‐CSRTT, the PWS‐IC+/− mice showed deficits in discriminative response accuracy, increased correct reaction times and increased omissions. Task manipulations confirmed that these differences were likely to be due to impaired attention. Our data recapitulate several aspects of the PWS clinical condition, including findings consistent with frontal abnormalities, and may indicate novel contributions of the imprinted genes found in 15q11–q13 to behavioural and cognitive function generally.

Transcription is required to establish maternal imprinting at the Prader-Willi syndrome and Angelman syndrome locus.

The Prader-Willi syndrome (PWS [MIM 17620]) and Angelman syndrome (AS [MIM 105830]) locus is controlled by a bipartite imprinting center (IC) consisting of the PWS-IC and the AS-IC. The most widely accepted model of IC function proposes that the PWS-IC activates gene expression from the paternal allele, while the AS-IC acts to epigenetically inactivate the PWS-IC on the maternal allele, thus silencing the paternally expressed genes. Gene order and imprinting patterns at the PWS/AS locus are well conserved from human to mouse; however, a murine AS-IC has yet to be identified. We investigated a potential regulatory role for transcription from the Snrpn alternative upstream exons in silencing the maternal allele using a murine transgene containing Snrpn and three upstream exons. This transgene displayed appropriate imprinted expression and epigenetic marks, demonstrating the presence of a functional AS-IC. Transcription of the upstream exons from the endogenous locus correlates with imprint establishment in oocytes, and this upstream exon expression pattern was conserved on the transgene. A transgene bearing targeted deletions of each of the three upstream exons exhibited loss of imprinting upon maternal transmission. These results support a model in which transcription from the Snrpn upstream exons directs the maternal imprint at the PWS-IC.

Cre-loxP chromosome engineering of a targeted deletion in the mouse corresponding to the 3p21.3 region of homozygous loss in human tumours.

Chromosomal deletions are a common feature of epithelial tumours and when further defined by homozygous deletions, are often the location of tumour suppressor genes. Deletions within the short arm of chromosome 3 occur very frequently in human carcinomas: a minimal region of loss at 3p21.3 (the Luca) region has been defined by overlapping homozygous deletions in lung and breast cancer cell lines. Using a rapid strategy for Cre-loxP chromosome engineering, a deletion of approximately 370 kb was created in the mouse germline corresponding to the deleted region at 3p21.3. The deletion when homozygous is embryonic lethal. Heterozygotes develop normally despite being haplo-insufficient for twelve genes including the candidate tumour suppressor gene Rassf1. Because damage to 3p21.3 often occurs very early in the sequence of genetic changes that lead to malignancy, particularly in lung and breast cancer, further genetic damage to these mice will provide the opportunity to model multi-step tumorigenesis of these tumours.

A new deletion refines the boundaries of the murine Prader-Willi syndrome imprinting center

The human chromosomal 15q11-15q13 region is subject to both maternal and paternal genomic imprinting. Absence of paternal gene expression from this region results in Prader-Willi syndrome (PWS), while absence of maternal gene expression leads to Angelman syndrome. Transcription of paternally expressed genes in the region depends upon an imprinting center termed the PWS-IC. Imprinting defects in PWS can be caused by microdeletions and the smallest commonly deleted region indicates that the PWS-IC lies within a region of 4.3 kb. The function and location of the PWS-IC is evolutionarily conserved, but delineation of the PWS-IC in mouse has proven difficult. The first targeted mutation of the PWS-IC, a deletion of 35 kb spanning Snrpn exon 1, exhibited a complete PWS-IC deletion phenotype. Pups inheriting this mutation paternally showed a complete loss of paternal gene expression and died neonatally. A reported deletion of 4.8 kb showed only a reduction in paternal gene expression and incomplete penetrance of neonatal lethality, suggesting that some PWS-IC function had been retained. Here, we report that a 6 kb deletion spanning Snrpn exon 1 exhibits a complete PWS-IC deletion phenotype. Pups inheriting this mutation paternally lack detectable expression of all PWS genes and paternal silencing of Ube3a, exhibit maternal DNA methylation imprints at Ndn and Mkrn3 and suffer failure to thrive leading to a fully penetrant neonatal lethality.

CLONING AND MAPPING OF MURINE NFE2L1

The murine homologue of the human NFE2L1 basic leucine-zipper gene was isolated from an early embryo library. The deduced amino acid sequence shows 97% identity between the two proteins. Significant sequence similarity is also seen with the p45 subunit of NF-E2 and with the Drosophila CNC protein. Murine Nfe2l1 maps to chromosome 11DE with similar sequence at 7D1-7F1 and 2E4-2G.

A targeted deletion upstream of Snrpn does not result in an imprinting defect

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) result from the disturbance of imprinted gene expression within human chromosome 15q11–q13. Some cases of PWS and AS are caused by microdeletions near the SNRPN gene that disrupt a regulatory element termed the imprinting center (IC). The IC has two functional components; an element at the promoter of SNRPN involved in PWS (PWS-IC) and an element 35 kilobases (kb) upstream of SNRPN involved in AS (AS-IC). To further understand the function of the IC, we sought to create a mouse model for AS-IC mutations. We have generated two deletions at a location analogous to that of the human AS-IC. Neither deletion produced an imprinting defect as indicated by DNA methylation and gene expression analyses. These results indicate that no elements critical for AS-IC function in mouse reside within the 12.8-kb deleted region and suggest that the specific location of the AS-IC is not conserved between human and mouse.

Regulatory Elements Associated with Paternally-Expressed Genes in the Imprinted Murine Angelman/Prader-Willi Syndrome Domain

The Angelman/Prader-Willi syndrome (AS/PWS) domain contains at least 8 imprinted genes regulated by a bipartite imprinting center (IC) associated with the SNRPN gene. One component of the IC, the PWS-IC, governs the paternal epigenotype and expression of paternal genes. The mechanisms by which imprinting and expression of paternal genes within the AS/PWS domain – such as MKRN3 and NDN – are regulated by the PWS-IC are unclear. The syntenic region in the mouse is organized and imprinted similarly to the human domain with the murine PWS-IC defined by a 6 kb interval within the Snrpn locus that includes the promoter. To identify regulatory elements that may mediate PWS-IC function, we mapped the location and allele-specificity of DNase I hypersensitive (DH) sites within the PWS-IC in brain cells, then identified transcription factor binding sites within a subset of these DH sites. Six major paternal-specific DH sites were detected in the Snrpn gene, five of which map within the 6 kb PWS-IC. We postulate these five DH sites represent functional components of the murine PWS-IC. Analysis of transcription factor binding within multiple DH sites detected nuclear respiratory factors (NRFs) and YY1 specifically on the paternal allele. NRFs and YY1 were also detected in the paternal promoter region of the murine Mrkn3 and Ndn genes. These results suggest that NRFs and YY1 may facilitate PWS-IC function and coordinately regulate expression of paternal genes. The presence of NRFs also suggests a link between transcriptional regulation within the AS/PWS domain and regulation of respiration. 3C analyses indicated Mkrn3 lies in close proximity to the PWS-IC on the paternal chromosome, evidence that the PWS-IC functions by allele-specific interaction with its distal target genes. This could occur by allele-specific co-localization of the PWS-IC and its target genes to transcription factories containing NRFs and YY1.

Temporal and developmental requirements for the Prader–Willi imprinting center

Imprinted gene expression associated with Prader–Willi syndrome (PWS) and Angelman syndrome (AS) is controlled by two imprinting centers (ICs), the PWS-IC and the AS-IC. The PWS-IC operates in cis to activate transcription of genes that are expressed exclusively from the paternal allele. We have created a conditional allele of the PWS-IC to investigate its developmental activity. Deletion of the paternal PWS-IC in the embryo before implantation abolishes expression of the paternal-only genes in the neonatal brain. Surprisingly, deletion of the PWS-IC in early brain progenitors does not affect the subsequent imprinted status of PWS/AS genes in the newborn brain. These results indicate that the PWS-IC functions to protect the paternal epigenotype at the epiblast stage of development but is dispensable thereafter.