Michael P. Bulman

Mutations in the human delta homologue, DLL3, cause axial skeletal defects in spondylocostal dysostosis.

Spondylocostal dysostosis (SD, MIM 277300) is a group of vertebral malsegmentation syndromes with reduced stature resulting from axial skeletal defects. SD is characterized by multiple hemivertebrae, rib fusions and deletions with a non-progressive kyphoscoliosis. Cases may be sporadic or familial, with both autosomal dominant and autosomal recessive modes of inheritance reported. Autosomal recessive SD maps to a 7.8-cM interval on chromosome 19q13.1–q13.3 (ref. 2) that is homologous with a mouse region containing a gene encoding the Notch ligand delta-like 3 (Dll3). Dll3 is mutated in the X-ray–induced mouse mutant pudgy (pu), causing a variety of vertebrocostal defects similar to SD phenotypes. Here we have cloned and sequenced human DLL3 to evaluate it as a candidate gene for SD and identified mutations in three autosomal recessive SD families. Two of the mutations predict truncations within conserved extracellular domains. The third is a missense mutation in a highly conserved glycine residue of the fifth epidermal growth factor (EGF) repeat, which has revealed an important functional role for this domain. These represent the first mutations in a human Delta homologue, thus highlighting the critical role of the Notch signalling pathway and its components in patterning the mammalian axial skeleton.

Mutations in the Hepatocyte Nuclear Factor–1α Gene Are a Common Cause of Maturity-Onset Diabetes of the Young in the U.K.

Mutations in the hepatocyte nuclear factor–1α (HNF-1α) gene have recently been shown to cause maturityonset diabetes of the young (MODY). We have examined 15 U.K. MODY families for mutations in the coding region of the HNF-1α gene. Eight different mutations, three frameshift (P291fsinsC, P379fsdelCT, and A443fsdelCA) and five missense mutations (P129T, R131W, R159W, P519L, and T620I), were identified in eleven families (73%). The previously reported mutation P291fsinsC was found in four pedigrees. A screen of a further 32 probands with early onset (<40 years of age) NIDDM showed the mutation in two additional families. This common mutation was present on at least three different haplotypes, suggesting that its high frequency is due to recurrent mutation rather than a founder effect. We have demonstrated that mutations in the HNF-1α gene are a common cause of MODY in U.K. families and result in early onset NIDDM with a progressive clinical course. Mutation-based genetic counseling can now be considered for the majority of patients with MODY.

Mutations in the Hepatocyte Nuclear Factor-1β Gene Are Associated with Familial Hypoplastic Glomerulocystic Kidney Disease

Familial glomerulocystic kidney disease (GCKD) is a dominantly inherited condition characterized by glomerular cysts and variable renal size and function; the molecular genetic etiology is unknown. Mutations in the gene encoding hepatocyte nuclear factor (HNF)-1beta have been associated with early-onset diabetes and nondiabetic renal disease-particularly renal cystic disease. We investigated a possible role for the HNF-1beta gene in four unrelated GCKD families and identified mutations in two families: a nonsense mutation in exon 1 (E101X) and a frameshift mutation in exon 2 (P159fsdelT). The family members with HNF-1beta gene mutations had hypoplastic GCKD and early-onset diabetes or impaired glucose tolerance. We conclude that there is genetic heterogeneity in familial GCKD and that the hypoplastic subtype is a part of the clinical spectrum of the renal cysts and diabetes syndrome that is associated with HNF-1beta mutations.

Missense mutations in the insulin promoter factor-1 gene predispose to type 2 diabetes

The transcription factor insulin promoter factor-1 (IPF-1) plays a central role in both the development of the pancreas and the regulation of insulin gene expression in the mature pancreatic β cell. A dominant-negative frameshift mutation in the IPF-l gene was identified in a single family and shown to cause pancreatic agenesis when homozygous and maturity-onset diabetes of the young (MODY) when heterozygous. We studied the role of IPF-1 in Caucasian diabetic and nondiabetic subjects from the United Kingdom. Three novel IPF-1 missense mutations (C18R, D76N, and R197H) were identified in patients with type 2 diabetes. Functional analyses of these mutations demonstrated decreased binding activity to the human insulin gene promoter and reduced activation of the insulin gene in response to hyperglycemia in the human β-cell line Nes2y. These mutations are present in 1% of the population and predisposed the subject to type 2 diabetes with a relative risk of 3.0. They were not highly penetrant MODY mutations, as there were nondiabetic mutation carriers 25‐53 years of age. We conclude that mutations in the IPF-1 gene may predispose to type 2 diabetes and are a rare cause of MODY and pancreatic agenesis, with the phenotype depending upon the severity of the mutation. J. Clin. Invest. 104:R33-R39 (1999).

A Gene for Autosomal Recessive Spondylocostal Dysostosis Maps to 19q13.1-q13.3

In spondylocostal dysostosis (SD), vertebral-segmentation defects are associated with rib anomalies. This results in short-trunk short stature, nonprogressive kyphoscoliosis, and radiological features of multiple hemivertebrae and rib fusions. SD can be familial, and both autosomal dominant and autosomal recessive (AR) inheritance have been reported, but no genes have been identified or localized for nonsyndromic SD in humans. We performed genomewide scanning by homozygosity mapping in a large consanguineous ARSD Arab Israeli family with six definitely affected members. Significant linkage was found to chromosome 19q13, with a LOD score of 6.9. This was confirmed in a second Pakistani family with three affected members, with a LOD score of 2.4. The combined-haplotype data identify a critical region between D19S570 and D19S908, an interval of 8.5 cM on 19q13.1-19q13.3. This is the first study to localize a gene for nonsyndromic SD. ARSD is clinically heterogeneous and is likely to result from mutations in developmental genes or from regulating transcription factors. Identification of these genes will improve the understanding of the molecular processes contributing to both normal and abnormal human vertebral development.

Evidence for haploinsufficiency of the human HNF1alpha gene revealed by functional characterization of MODY3-associated mutations.

Abstract Hepatocyte nuclear factor (HNF)1α is a homeodomaincontaining transcription factor participating in the regulation of gene expression in liver, kidney, gut and pancreas of vertebrates. In humans mutations in the HNF1α gene are responsible for one form of maturity onset diabetes of the young (MODY3). To define the molecular mechanism underlying MODY3 we investigated the functional properties of seven MODY3- associated mutations representing the spectrum of different kinds of mutations affecting all functional domains of the protein. The mutations introduced into an expression vector encoding human HNF1α include inframe deletion (ΔN127), nonsense (Q7X, R171X), frameshift (P291fsinsC) and missense (R229Q, P447L, T620I) mutations. Gel retardation and reporter gene assays showed that the functional properties of these mutants differ dramatically, but none of these mutants act in a dominant negative manner. Moreover, the mRNA stability of the mutants ΔN127, R171X, P291fsinsC and T547E548fsdelTG is impaired compared to the wildtype sequence in transfected cells. This decreased RNA stability is independent of the presence of an intron in the expression vector and thus differs from mechanisms known to be involved in nonsensemediated decay (NMD). Our results suggest that haploinsufficiency of HNF1α is responsible for the pathogenesis of MODY3.

A rapid screening method for hepatocyte nuclear factor 1 alpha frameshift mutations; prevalence in maturity-onset diabetes of the young and late-onset non-insulin dependent diabetes

Abstract Non-insulin dependent diabetes (NIDDM) is a polygenic heterogeneous disorder of glucose homeostasis. Maturity-onset diabetes of the young (MODY) is a monogenic subtype of NIDDM characterised by early-onset (< 25 years) and autosomal dominant inheritance. Mutations in the hepatocyte nuclear factor 1 alpha (HNF-1α) gene have recently been shown to cause MODY. The incidence of mutations in this gene in MODY and late-onset NIDDM is not known. We have developed a rapid specific polymerase chain reaction test for HNF-1α mutations; this test involves the use of fluorescently labelled forward primers and modified reverse primers to detect length polymorphisms resulting from frameshift mutations. With this method, we have screened 102 MODY probands, viz. 60 defined according to strict diagnostic criteria (autosomal dominant inheritance and at least one member diagnosed age < 25 years) and 95 late-onset NIDDM probands (diagnosed 35–70 years with ≥ 1 affected relative), for the presence of 9 known HNF-1α frameshift mutations, including 6 that occur at two sites for recurring mutation (residues 291/292 and 379). Mutations were detected in 11 of the strictly defined MODY probands and one mutation was also found in a single subject with early-onset NIDDM but no family history of the disease. The HNF-1α frameshift mutations were not detected in any late-onset NIDDM subjects, suggesting these mutations do not have a major role in the pathogenesis of NIDDM. Our results indicate that the prevalence of the nine frameshift mutations in strictly defined UK MODY is 18%, with the P291fsinsC mutation alone having a frequency of 13%.

Loss of HNF1alpha function in human renal cell carcinoma: frequent mutations in the VHL gene but not the HNF1alpha gene.

Human renal cell carcinoma (RCC) is a common malignant disease of the kidney characterized by dedifferentiation of renal epithelial cells. Our previous experiments showed that most RCCs have a loss of function of the tissue‐specific transcription factor hepatocyte nuclear factor (HNF) 1α. Detailed analyses of the 10 exons encoding HNF1α in 32 human RCCs by single‐strand conformation polymorphism analysis and direct DNA sequencing revealed no tumor‐associated mutation, whereas with the same probes we frequently found mutations in the von Hippel‐Lindau tumor suppressor gene. No mutation leading to loss of HNF1α function was detected by analyzing the integrity of the HNF1α transcripts in the RNA derived from RCCs by the protein truncation test. Investigating human RCC cell lines by western blotting and gel retardation assays showed a dramatic loss in the expression of the tissue‐specific transcription factor HNF1α in eight of 10 cell lines. As the HNF1α‐related transcription factor HNF1β was expressed in all these tumor cell lines, the loss of HNF1α expression was a specific event and was maintained in RCC cell lines. The loss of HNF1α expression in RCC cell lines on the RNA level was confirmed by reverse transcription polymerase chain reaction. We propose that tumor‐associated mutations in the HNF1α gene do not occur in human RCC and that the loss of function is partially due to a transcriptional inactivation of the HNF1α gene. Mol. Carcinog. 24:305–314, 1999.

Proposed mechanism for a novel insertion/deletion frameshift mutation (I414G415ATCG?CCA) in the hepatocyte nuclear factor 1 alpha (HNF-1?) gene which causes maturity-onset diabetes of the young (MODY)

Maturity‐onset diabetes of the young (MODY) is a monogenic subgroup of non‐insulin dependent diabetes (NIDDM) characterized by an early age of diagnosis (usually < 25 years) and an autosomal dominant mode of inheritance. Mutations in the hepatocyte nuclear factor 1 alpha (HNF‐1α) [MODY3] gene represent the most common cause of MODY in the UK and a common cause of MODY in many other populations. Sixty‐three different mutations have been described in a total of 112 families worldwide. This report describes two families, not known to be related, who carry a novel insertion/deletion mutation (I414G415ATCG→CCA) and a 6bp intronic deletion of the HNF‐1α gene in cis. We propose that the insertion/deletion mutation has arisen by formation of a hairpin loop due to the presence of a quasi‐palindromic sequence, followed by insertion of CC and deletion of TCG resulting in the increased stability of the hairpin loop. Hum Mutat 16:273, 2000.