Michael C. Gorry

Mutations of the UMOD gene are responsible for medullary cystic kidney disease 2 and familial juvenile hyperuricaemic nephropathy

Introduction: Medullary cystic kidney disease 2 (MCKD2) and familial juvenile hyperuricaemic nephropathy (FJHN) are both autosomal dominant renal diseases characterised by juvenile onset of hyperuricaemia, gout, and progressive renal failure. Clinical features of both conditions vary in presence and severity. Often definitive diagnosis is possible only after significant pathology has occurred. Genetic linkage studies have localised genes for both conditions to overlapping regions of chromosome 16p11-p13. These clinical and genetic findings suggest that these conditions may be allelic. Aim: To identify the gene and associated mutation(s) responsible for FJHN and MCKD2. Methods: Two large, multigenerational families segregating FJHN were studied by genetic linkage and haplotype analyses to sublocalise the chromosome 16p FJHN gene locus. To permit refinement of the candidate interval and localisation of candidate genes, an integrated physical and genetic map of the candidate region was developed. DNA sequencing of candidate genes was performed to detect mutations in subjects affected with FJHN (three unrelated families) and MCKD2 (one family). Results: We identified four novel uromodulin (UMOD) gene mutations that segregate with the disease phenotype in three families with FJHN and in one family with MCKD2. Conclusion: These data provide the first direct evidence that MCKD2 and FJHN arise from mutation of the UMOD gene and are allelic disorders. UMOD is a GPI anchored glycoprotein and the most abundant protein in normal urine. We postulate that mutation of UMOD disrupts the tertiary structure of UMOD and is responsible for the clinical changes of interstitial renal disease, polyuria, and hyperuricaemia found in MCKD2 and FJHN.

A Mutation in the SOS1 Gene Causes Hereditary Gingival Fibromatosis Type 1

Hereditary gingival fibromatosis (HGF) is a rare, autosomal dominant form of gingival overgrowth. Affected individuals have a benign, slowly progressive, nonhemorrhagic, fibrous enlargement of the oral masticatory mucosa. Genetic loci for autosomal dominant forms of HGF have been localized to chromosome 2p21-p22 (HGF1) and chromosome 5q13-q22 (HGF2). To identify the gene responsible for HGF1, we extended genetic linkage studies to refine the chromosome 2p21-p22 candidate interval to approximately 2.3 Mb. Development of an integrated physical and genetic map of the interval identified 16 genes. Sequencing of these genes, in affected and unaffected HGF1 family members, identified a mutation in the Son of sevenless-1 (SOS1) gene in affected individuals. In this report, we describe the genomic structure of the SOS1 gene and present evidence that insertion of a cytosine between nucleotides 126,142 and 126,143 in codon 1083 of the SOS1 gene is responsible for HGF1. This insertion mutation, which segregates in a dominant manner over four generations, introduces a frameshift and creates a premature stop codon, abolishing four functionally important proline-rich SH3 binding domains normally present in the carboxyl-terminal region of the SOS1 protein. The resultant protein chimera contains the wild-type SOS1 protein for the N-terminal amino acids 1-1083 fused to a novel 22-amino acid carboxyl terminus. Similar SOS1 deletion constructs are functional in animal models, and a transgenic mouse construct with a comparable SOS1 chimera produces a phenotype with skin hypertrophy. Clarification of the functional role of this SOS1 mutant has implications for understanding other forms of gingival fibromatosis and corrective gingival-tissue management.

Novel ENAM mutation responsible for autosomal recessive amelogenesis imperfecta and localised enamel defects

The genetic basis of non-syndromic autosomal recessive forms of amelogenesis imperfecta (AI) is unknown. To evaluate five candidate genes for an aetiological role in AI. In this study 20 consanguineous families with AI were identified in whom probands suggested autosomal recessive transmission. Family members were genotyped for genetic markers spanning five candidate genes: AMBN and ENAM (4q13.3), TUFT1 (1q21), MMP20 (11q22.3–q23), and KLK4 (19q13). Genotype data were evaluated to identify homozygosity in affected individuals. Mutational analysis was by genomic sequencing. Homozygosity linkage studies were consistent for localisation of an AI locus in three families to the chromosome 4q region containing the ENAM gene. ENAM sequence analysis in families identified a 2 bp insertion mutation that introduced a premature stop codon in exon 10. All three probands were homozygous for the same g.13185_13186insAG mutation. These probands presented with a generalised hypoplastic AI phenotype and a class II openbite malocclusion. All heterozygous carriers of the g.13185_13186insAG mutation had localised hypoplastic enamel pitting defects, but none had AI or openbite. The phenotype associated with the g.13185_13186insAG ENAM mutation is dose dependent such that ARAI with openbite malocclusion segregates as a recessive trait, and enamel pitting as a dominant trait.

Evaluation of human leukocyte N-formylpeptide receptor (FPR1) SNPs in aggressive periodontitis patients.

Polymorphonuclear neutrophils (PMNs) are attracted to sites of infection by N-formylpeptide (fMLP) chemoattractants. The high-affinity fMLP receptor (FPR1) of phagocytic cells interacts with bacterial fMLP and mediates chemotaxis, degranulation, and superoxide production. These cellular functions are disrupted in PMN from aggressive periodontitis (AP) patients. Two FPR1 gene single nucleotide polymorphisms (SNPs), c.329T>C and c.378C>G, have been associated with a localized form of AP in African-American patients. To evaluate the generality of these SNPs in AP patients, we sequenced a 363 bp interval of the FPR1 gene in an ethnically diverse group of patients (n=111) and controls (n=115). Neither c.329T>C nor c.378C>G were detected in the 452 alleles sequenced. Six SNPs were identified including two located in the FPR1 second extracellular loop that were significantly associated with the AP phenotype in African-American patients (p.R190W, P=0.0033; and p.N192K, P=0.0018). These two SNPs show three predominant haplotypes, each associated with a different disease risk in African-Americans. These data do not support the hypothesis that the FPR1 SNPs c.329T>C and c.378C>G play an etiologic role in aggressive periodontitis, but do suggest that SNPs in the second extracellular loop may be etiologically important.

Aquaporin expression in developing human teeth and selected orofacial tissues

The aquaporin (AQP) family of membrane channel proteins function as selective pores through which water, glycerol, and other small solutes cross the cell plasma membrane. To date, 11 members of this transporter family, designated AQP0-10, have been cloned and characterized in humans. The AQPs are differentially expressed in temporospatial patterns, where different AQPs demonstrate distinct tissue distributions that may reflect differing cell membrane transport functions. The purpose of this study was to evaluate AQP expression in the developing human teeth by RT-PCR and Western blot analysis. To access the generality of AQP expression, selected other orofacial tissues were studied by RT-PCR. The presence of all eleven human AQPs was screened in each tissue by RT-PCR. Positive amplification products were verified by direct DNA sequencing. AQPs 1, 3, 4, 5, 6, and 10 were identified by RT-PCR in developing teeth, and AQP1, 3, 5, and 6 were confirmed by Western blot analysis. AQP 4 was not detected by Western blot analysis, and we were unable to test for the recently identified AQP10 due to unavailability of antibodies. AQPs detected in other orofacial tissues by RT-PCR included gingiva (AQP3, 7, 10); Meckels cartilage (AQP1, 3, 4, 5, 6); submandibular gland (AQP1, 3, 4, 5, 6, 7); masseter muscle (AQP1, 3, 4, 7, 8, 9,10); and infrahyoid muscle (AQP1, 3, 4,10). These results demonstrate that multiple aquaporins are expressed in developing teeth and in selected orofacial tissues.

Renal manifestations of a mutation in the uromodulin (Tamm Horsfall protein) gene

BACKGROUND Uromodulin (Tamm Horsfall glycoprotein) is the most abundant protein found in normal human urine. Its function has yet to be determined. Identifying mutations in the uromodulin gene may be helpful in understanding the function of uromodulin. There has been 1 report of 4 families suffering from mutations in the uromodulin gene, resulting in the autosomal dominant transmission of hypouricosuric hyperuricemia and chronic renal failure. This case report describes another family with similar clinical manifestations. METHODS A family was identified with clinical characteristics of hypouricosuric hyperuricemia and renal failure occurring in a mother and daughter. Clinical characteristics were identified, and laboratory studies were obtained in the proband and the probands daughter. A genetic analysis was performed to evaluate for mutations in the uromodulin gene. RESULTS The proband suffered from hyperuricemia at an early age and progressive renal failure with end-stage renal disease developing at age 49 years. The probands daughter suffered from hyperuricemia, a reduced fractional excretion of uric acid, and mild renal insufficiency. A g.2105G > A mutation in exon 4 of the uromodulin gene resulting in a substitution of tyrosine for cysteine was identified in both the proband and the probands daughter. The clinical characteristics were similar to those of other patients suffering from uromodulin mutations and to those of patients suffering from medullary cystic kidney disease type 2 and familial juvenile hyperuricemic nephropathy. CONCLUSION Uromodulin associated kidney disease results in hyperuricemia and renal failure. The specific uromodulin mutation found in this family is consistent with the hypothesis that mutations disrupt highly conserved cysteine residues in the uromodulin protein. Potential mechanisms for these pathologic changes are discussed. The authors would appreciate referral of other families for screening for mutations.

Fine mapping of the split-hand/split-foot locus (SHFM3) at 10q24: evidence for anticipation and segregation distortion.

Split-hand/split-foot malformation (SHFM, ectrodactyly, or lobster-claw deformity) is a human limb malformation characterized by aberrant development of central digital rays with absence of fingers and toes, a deep median cleft, and fusion of remaining digits. SHFM is clinically heterogeneous, presenting both in an isolated form and in combination with additional abnormalities affecting the tibia and/or other organ systems, including the genitourinary, craniofacial, and ectodermal structures. Three SHFM disease loci have been genetically mapped to chromosomes 7q21 (SHFM1), Xq26 (SHFM2), and 10q24 (SHFM3). We mapped data from a large Turkish family with isolated SHFM to chromosome 10q24 and have narrowed the SHFM3 region from 9 cM to an approximately 2-cM critical interval between genetic markers D10S1147 and D10S1240. In several instances we found evidence for a more severe phenotype in offspring of a mildly affected parent, suggesting anticipation. Finally, data from this family, combined with those from six other pedigrees, mapped to 10q24, demonstrate biased transmission of SHFM3 alleles from affected fathers to offspring. The degree of this segregation distortion is obvious in male offspring and is possibly of the same magnitude for female offspring.

Three epigenetic drugs up-regulate homeobox gene Rhox5 in cancer cells through overlapping and distinct molecular mechanisms.

Epigenetic therapy of cancer using inhibitors of DNA methyltransferases (DNMT) or/and histone deacetylases (HDACs) has shown promising results in preclinical models and is being investigated in clinical trials. Homeodomain proteins play important roles in normal development and carcinogenesis. In this study, we demonstrated for the first time that an epigenetic drug could up-regulate homeobox genes in the reproductive homeobox genes on chromosome X (Rhox) family, including murine Rhox5, Rhox6, and Rhox9 and human RhoxF1 and RhoxF2 in breast, colon, and other types of cancer cells. We examined the molecular mechanisms underlining selective induction of Rhox5 in cancer cells by three epigenetic drugs: 5-aza-2′-deoxycytidine (DAC; decitabine), arsenic trioxide (ATO), and MS-275 [entinostat; N-(2-aminophenyl)-4-[N-(pyridine-3-ylmethoxy-carbonyl)aminomethyl]benzamide]. DAC induced Rhox5 mRNA expression from both distal promoter (Pd) and proximal promoter, whereas MS-275 and ATO induced gene expression from the Pd only. DAC and ATO inhibited both DNMT1 and DNMT3B protein expression, whereas MS-275 significantly reduced DNMT3B protein. In contrast to DAC, neither MS-275 nor ATO induced DNA demethylation on the Pd region. All three drugs led to enhanced acetylation of histones H3 and H4 at the promoter region. The occupancy of the activating histone mark dimethylated lysine 4 of H3 at Pd was enhanced by DAC and MS-275 but not ATO. Because they modulate gene expression with different potencies through shared and distinct epigenetic mechanisms, these epigenetic drugs may possess great potential in different applications for epigenetic therapy of cancer and other diseases.

RNA differential display of scarless wound healing in fetal rabbit indicates downregulation of a CCT chaperonin subunit and upregulation of a glycophorin-like gene transcript.

BACKGROUND/PURPOSE Scars form as wounds heal in adult organisms. In addition to disrupting cosmetic appearance, scar tissue can cause significant morbidity, and even death if it blocks vital organ function. Previous work has established that fetal wounds, especially in early to midgestation, can heal without scarring. Because such inherent physiological mechanisms ultimately are under genetic control, a study was initiated to elucidate the differences in gene expression that produce scarless wound healing in the mammalian fetus but scarring in postnatal wounds. Reverse transcription polymerase chain reaction (RT-PCR) differential display (DD) was used to detect differentially expressed mRNA transcripts in a rabbit model of wound healing. METHODS Adult and 21-day fetal full-thickness rabbit skin specimens from wounded and unwounded sites were harvested 12 hours postwounding. RNA extracted from the tissue was used as a template in DD reactions using anchoring and random primers to generate tissue-specific gene expression fingerprints. The over 2,000 resulting amplimers (gene transcripts) were screened for differential expression among the 4 types of specimens: fetal control (unwounded), fetal wound, adult control, and adult wound. Selected bands distinctly upregulated or downregulated in fetal wound lanes on the DD gels were excised, and the cDNA was extracted, reamplified, cloned into vectors, and sequenced. DD results were confirmed by limiting-dilution RT-PCR using sequence-specific primers. RESULTS Differential display (DD) showed 22 amplimers that were significantly upregulated in all fetal wound samples as compared with little or no expression in fetal control, adult control, or adult wound tissues. Conversely, 5 transcripts were downregulated in the fetal wound specimens but highly expressed in the 3 comparison tissues. Reamplification of selected transcripts by PCR, followed by cloning and DNA sequencing, yielded 7 distinct sequences, each representing a gene expressed differently in fetal wound than in the other 3 tissues. A transcript that was downregulated in fetal wound showed very high sequence homology to part of the human gene for the eta subunit of the hetero-oligomeric particle CCT (the chaperonin containing T-complex polypeptide 1 or TCP-1). An upregulated amplimer showed significant DNA sequence homology to glycophorins A and B. One sequence was identified as 28S rRNA. The remaining 4 candidate sequences showed no significant homology to known genes, but 1 had high homology to expressed sequence tags of unknown function. CONCLUSIONS With careful experimental design and proper controls and verifications, differential display of RNA expression is a potentially powerful method of finding genes that specifically regulate a particular physiological process such as fetal wound healing. No a priori knowledge of what genes might be involved, or why, is necessary. This study indicates that downregulation of a gene that codes for a chaperonin subunit and upregulation of several other genes may be involved in the striking scarless character of wound healing in the mammalian fetus. Results suggest the hypothesis that downregulation of the CCT chaperonin in fetal wound may inhibit the formation of myofibroblasts, a cell type that correlates highly with scarring in postnatal wound healing, by preventing the folding of sufficient alpha-smooth muscle actin to form the stress fibers characteristic of these cells.

Clinico-pathologic findings in medullary cystic kidney disease type 2

Medullary cystic kidney disease type 2 is an uncommon autosomal dominant condition characterized by juvenile onset hyperuricemia, precocious gout and chronic renal failure progressing to end-stage renal disease in the 4th through 7th decades of life. A family suffering from this condition is described. The patient in the index case presented with renal insufficiency as a child. A renal biopsy revealed tubular atrophy, and immunohistochemical staining of the tissue for uromodulin (Tamm Horsfall protein) revealed dense deposits in renal tubular cells. Genetic testing revealed a single nucleotide mutation (c.899G>A) resulting in an exchange of a cysteine residue for tyrosine (C300Y). Medullary cystic kidney disease type 2 (also known as uromodulin-associated kidney disease) likely represents a form of endoplasmic reticulum storage disease, with deposition of the abnormal uromodulin protein in the endoplasmic reticulum, leading to tubular cell atrophy and death.