A genome-wide linkage scan for cleft lip and cleft palate identifies a novel locus on 8p11-23.

Isolated or nonsyndromic cleft lip and palate (NS CLP) is a complex disorder resulting from multiple genetic and environmental factors. NS CLP has a birth prevalence of 1 per 500 in the Philippines where large families provide an opportunity for gene localization. Genotyping of 392 microsatellite repeat markers at 10 cM intervals over the genome was performed by the Center for Inherited Disease Research (CIDR) on 220 Filipino families with 567 affected and 1,109 unaffected family members genotyped. Among the most statistically significant results from analysis of the genome-wide scan data was a 20 cM region at 8p11-23 in which markers had LODs > or =1.0. This region on 8p11-23 has not been found in any previous genome wide scan nor does it contain any of the candidate genes widely studied in CLP. Fine mapping in 8p11-23 was done in the 220 families plus an additional 51 families, using SNP markers from 10 known genes (FGFR1, NRG1, FZD3, SLC8A1, PPP3CC, EPHX2, BNIP3L, EGR3, PPP2R2A, and NAT1) within the 20 cM region of 8p11-23. Linkage and association analyses of these SNPs yield suggestive results for markers in FGFR1 (recessive multipoint HLOD 1.07) and BAG4 (recessive multipoint HLOD 1.31).

Mutations in PITX2 may contribute to cases of omphalocele and VATER-like syndromes.

Omphalocele is a congenital anomaly with substantial morbidity. Rieger syndrome, an autosomal dominant disorder, is characterized by craniofacial abnormalities and abdominal wall defects. PITX2 mutations are etiologic in >40% of cases of Rieger syndrome. We demonstrate that the birth prevalence of omphalocele is significantly higher in Rieger syndrome than in the general population, with omphaloceles found in 0.03% in the Iowa newborn population and 4.3% of patients with Rieger syndrome. Our objective was to screen coding and conserved non-coding regions of PITX2 for mutations in 209 patients with omphalocele. We identified remarkable evolutionarily conserved regions by comparing the 3'UTR of Pitx2 in 13 vertebrate and 3 invertebrate species. No mutations changing the amino acid sequence were found within the omphalocele population. In one case of omphalocele with VATER-like additional anomalies, a three nucleotide deletion was found in the 3'UTR. This deletion was not seen in 1,186 controls. Also in the 3'UTR, we identified a single nucleotide polymorphism at a highly conserved residue. Our findings suggest additional studies of PITX2 conserved regions will be valuable. We also screened the omphalocele cases for mutations in exon 5 of the gene FLNA. Mutations in FLNA have been shown to cause a broad range of congenital malformations, including otopalatodigital syndrome type 2 in which a missense mutation occurring in exon 5 of FLNA results in omphalocele as part of the phenotype. We did not find any mutations in exon 5 of FLNA in 179 omphalocele cases studied.

Targeted scan of fifteen regions for nonsyndromic cleft lip and palate in Filipino families.

Cleft lip with or without cleft palate (CL/P) is a congenital anomaly with variable birth prevalence based on geographic origins, with the highest rates commonly found in Asian populations. About 70% of cases are nonsyndromic (NS), in which the affected individual has no other abnormalities. NS CL/P is a complex disorder with genetic and environmental effects and no specific genetic loci yet confirmed. Fifteen candidate regions were examined for linkage to NS CL/P. Regions were chosen based on previous suggestive linkage and/or association in human families, or suggestive animal model data. Polymorphic markers in these regions were genotyped for analysis on 36 Filipino families comprised of 126 affected and 218 unaffected individuals. An additional 70 families with 149 affecteds were used for replication of suggestive results. Parametric (LOD score) and nonparametric (SIMIBD) linkage analyses were performed as well as transmission disequilibrium test (TDT) analysis. Five markers yielded suggestive results from the 36 families. The parametric LOD scores for the MSX1-CA and D4S1629 were >1.0 and the SIMIBD P values for D6S1029 and RFC1 are suggestive (<0.06), while the SIMIBD P value of 0.01 for TGFA was significant. Since the Msx1 mouse knockout has cleft palate and MSX1 mutations have been found in rare cases of syndromic CL/P, this locus is especially plausible for linkage. Previous studies have also found linkage of NS CL/P to 4q31 and 6p23. These regions contain several candidate genes, including AP2 at 6p23 and FGF2, BMPR1B, and MADH1 at 4q31. TGFA has both linkage and linkage disequilibrium data supporting it as a candidate gene for NS CL/P. While no region was definitively confirmed for linkage to NS CL/P, the data do support further investigation using larger sample sizes and candidate gene studies at 2p13.2, 4p16.2, 4q31, 6p23, and 16q22-24.

Complete sequencing shows a role for MSX1 in non-syndromic cleft lip and palate.

MSX1 has been proposed as a gene in which mutations may contribute to non-syndromic forms of cleft lip and/or cleft palate. Support for this comes from human linkage and linkage disequilibrium studies, chromosomal deletions resulting in haploinsufficiency, a large family with a stop codon mutation that includes clefting as a phenotype, and the Msx1 phenotype in a knockout mouse. This report describes a population based scan for mutations encompassing the sense and antisense transcribed sequence of MSX1 (two exons, one intron). We compare the completed genomic sequence of MSX1 to the mouse Msx1 sequence to identify non-coding homology regions, and sequence highly conserved elements. The samples studied were drawn from a panethnic collection including people of European, Asian, and native South American ancestry. The gene was sequenced in 917 people and potentially aetiological mutations were identified in 16. These included missense mutations in conserved amino acids and point mutations in conserved regions not identified in any of 500 controls sequenced. Five different missense mutations in seven unrelated subjects with clefting are described. Evolutionary sequence comparisons of all known Msx1 orthologues placed the amino acid substitutions in context. Four rare mutations were found in non-coding regions that are highly conserved and disrupt probable regulatory regions. In addition, a panel of 18 population specific polymorphic variants were identified that will be useful in future haplotype analyses of MSX1. MSX1 mutations are found in 2% of cases of clefting and should be considered for genetic counselling implications, particularly in those families in which autosomal dominant inheritance patterns or dental anomalies appear to be cosegregating with the clefting phenotype.

Under recognition of late onset ornithine transcarbamylase deficiency.

Late onset ornithine transcarbamylase deficiency (McKusick 311250) is reported in four Finnish patients, two boys and two heterozygous girls. The subtle onset and course of ornithine transcarbamylase deficiency emphasises the need for plasma ammonia and amino acid measurements in clinical situations suggesting a disorder of this nature.

Chromosomal localization of a human mucin gene (MUC8) and cloning of the cDNA corresponding to the carboxy terminus.

A partial cDNA (pAM1) encoding a major airway mucin glycoprotein with novel tandem repetitive sequence has recently been cloned (Shankar, V., M. S. Gilmore, R. C. Elkins, and G. P. Sachdev. 1994. Biochem. J. 300:295-298). In this article, we report additional new sequence derived by 3'-rapid amplification of cDNA ends technique. The sequence corresponds to a stop codon, 3'-untranslated region of 458 bp, a polyadenylation signal, and poly A+ tail, and represents the extreme carboxy terminus of MUC8. A plasmid construct (pAM3) in pBluescript was generated by in-frame ligation of pAM1 to the 479-bp 3'UTR of MUC8. A 5'-end 325-bp fragment of this cDNA subcloned into the protein fusion and expression vector pET28b(+) was used to generate fusion protein under the control of T7 promoter. The purified fusion protein as well as synthetic peptide corresponding to the MUC8 repeat sequence (TSCPRPLQEGTPGS) were used to raise polyclonal antibodies in rabbits. The antiserum to the fusion protein and to the synthetic peptide reacted with the deglycosylated major tracheobronchial mucin. Immunohistochemical studies using the above antibodies localized the MUC8 protein product to submucosal glands in human tracheal epithelium. Furthermore, the gene from which this cDNA is derived, was mapped to chromosome 12 using DNA from a panel of human-mouse somatic cell hybrids. Fluorescence in situ hybridization was used to assign the regional localization to 12q24.3. Since the eight known human mucin genes map to other chromosomes, we have named this gene MUC8, in accordance with mucin gene nomenclature.

Sustained hyperglycemia results in testicular dysfunction and reduced fertility potential in BBWOR diabetic rats.

Rats with short-term diabetes show a greater than 50% reduction of serum testosterone and increased lipid in Leydig cells but normal testicular structure. The purpose of this study was to determine the extent of testicular pathology (morphology index), integrity of the blood-testis barrier, daily sperm production (DSP), number of Leydig cells per testis (LC/T), and total trunk testosterone (TTT) in diabetic rats (BBWORdp) with long-term hyperglycemia (300-350 mg/dl for greater than 180 days) and to evaluate its effects on fertility potential. Results were compared with similarly aged normoglycemic rats (BBWORdr) and normal control Wistar rats. After 6 mo of diabetes, testis weights, DSPs, TTTs, and the morphology index were significantly reduced. The LC/T was not different from BBWORdr rats. The blood-testis barrier appeared intact, although structural abnormalities were noted in Sertoli-Sertoli junction complexes. There was a significant reduction in the number of pregnancies per rat and implantations per pregnancy in matings utilizing the diabetic BBWORdp rat and control Wistar female rats. Results indicate that long-term diabetes with sustained hyperglycemia leads to significant testicular dysfunction associated with decreased fertility potential.

Successful islet/abdominal testis transplantation does not require Leydig cells.

Pancreatic islet allo- and xenografts are not rejected and exhibit long-term beta-cell function if transplanted into the abdominal testis of the diabetic host. Successful transplantation appears dependent on local factors unique to the abdominal testis. Because Leydig cells remain viable in abdominal testes, which also retain high levels of testosterone, the following question was addressed: do Leydig cells and/or their secretory products influence islet transplantability in the successful islet/abdominal testis transplantation model? Streptozotocin-induced diabetic rats (Sprague-Dawley) were injected with 75 mg/kg ethane dimethanesulfonate (EDS) to selectively eliminate Leydig cells prior to or following transplantation with islets isolated from the BBWORdr rat. Subcutaneous silastic tubes packed with estradiol prevented Leydig cell repopulation in the EDS-treated recipient. Grafted diabetic animals, including the EDS-treated rats with serum testosterone at castration levels, became nornoglycemic following islet transplantation and remained so far for up to ten months. Leydig cells were not observed in testes of the EDS- or EDS/estradiol-treated rats, whereas the transplanted islets within these testes appeared structurally normal and highly vascularized. Islets resided within the testicular interstitial compartment and contained alpha-, beta and delta-cells, as identified by electron microscopy. Beta cells were most prominent, contained secretory granules and exhibited a close structural and functional relationship with adjacent intraislet capillaries. We conclude that Leydig cells and Leydig cell secretory products, including testosterone, are not necessary for protecting islets against rejection and they do not play an obligatory role in the success of the islet/abdominal testis transplantation protocol. Leydig cells and Leydig cell secretory products do not promote long-term beta-cell function and are not required for the return to and maintenance of normoglycemia in the grafted diabetic rat.

Clinical and molecular evaluation of acetowhite genital lesions in men.

A total of 108 male partners of women with cervical condyloma and/or dysplasia underwent evaluation for gross and subclinical condyloma via acetic acid screening with a magnified examination. Biopsies of acetowhite genital skin were obtained for histological and deoxyribonucleic acid hybridization analysis. Of the men 52 (49%) had acetowhite lesions and underwent biopsies, 44 of which were evaluable by histological and deoxyribonucleic acid analyses. Of the lesions 12 had features of condyloma or penile intra-epithelial neoplasia, among which 7 (58%) contained human papillomavirus deoxyribonucleic acid. The remaining 32 lesions revealed minimal histological changes sometimes suggesting condyloma. However, only 5 of the 32 biopsies (16%) contained human papillomavirus deoxyribonucleic acid. A tendency to overdiagnose condyloma based on histological findings is suggested. Criteria by which to identify best human papillomavirus-related morphology are presented. Acetowhite genital epithelia with minor (nonspecific) histological changes correlate poorly with human papillomavirus nucleic acids and in most cases do not represent disease involving common viral types. The application of appropriate histological criteria appears to be particularly relevant to management strategies that avoid overtreatment of minor epithelial abnormalities. It remains unclear whether acetowhite genital epithelia positive for human papillomavirus require treatment given the high tendency for recurrence and lack of demonstrated effect on the natural history of cervical carcinoma.

Comparison of NK-cell (Leu-7+ and Leu-11b+) populations in clinically healthy gingiva, chronic gingivitis and chronic adult periodontitis.

Various investigations have reported the presence of cytotoxic lymphocyte activity in inflammatory periodontal disease. The collective evidence indicates that the inflammatory infiltrates of gingivitis and periodontitis should feature a major component of large granular lymphocytes (NK-cells) possessing cytotoxic potential. Thus, the purpose of this study was to determine and compare, by use of immunohistochemical methods, the numbers of NK-cells in biopsies of clinically healthy gingiva, chronic gingivitis and chronic adult periodontitis and their relationship, if any, to the T- and B-lymphocyte populations. Gingival biopsies were obtained from 8 patients in each of three disease groups selected on the basis of predetermined clinical criteria. Using the avidin-biotin immunoperoxidase technique, four consecutive serial sections from each biopsy specimen were stained with a panel of antihuman monoclonal antibodies for T-lymphocytes (UCHL-1) B-lymphocytes (CD-45R), and NK-cells (Leu-7 and Leu-11b). Analyses of variance yielded a statistically significant main effect for each cell immunophenotype. The Newman-Keuls Sequential Range Test showed statistically significant differences for all but two mean comparisons (p less than 0.01). The comparisons for UCHL-1 and Leu-7 between chronic gingivitis and periodontitis specimens did not demonstrate significance. Although T- and B-lymphocyte populations increased approximately 20 x progressing from healthy to gingivitis to periodontitis specimens, the NK-cell population showed only a 3 x increase which represented 19%, 6.6% and 7% of the total of all positively stained lymphocytes across biopsy groups.

Ureteral duplication with lower pole ectopia to the epididymis.

We report a unique case of a duplex ureter with ureteral ectopia. A 30-year-old man presented with a coliform infection of the epididymis and excretory urography revealed, in addition to a normal-appearing right renal collecting system, a second right ureter that arose from an inferior calix, penetrated the lower pole parenchyma and drained directly into the ipsilateral epididymis. The radiological evaluation and surgical management are discussed, and several embryological explanations for this anomaly are offered.

Value of acetic acid screening for flat genital condylomata in men.

Application of acetic acid solution to the genital skin followed by magnified examination permits the detection of grossly inapparent flat condylomata acuminata. To evaluate the accuracy of this screening method, the male sexual partners of 36 women with genital condylomata were examined by this method and biopsies were obtained when results were positive. Of 47 biopsies of acetowhite (the whitish change that occurs when an epithelial surface is stained with acetic acid) lesions there were 26 cases of histologically confirmed condylomata, 9 of koilocytotic atypia and 12 with false positive results. There were 25 men whose sexual partners had cervical condylomata and cervical dysplasia. In this subgroup, considered to be at higher risk for flat condylomata, the screening method revealed 15 cases of condylomata, 6 of koilocytotic atypia and 4 in which no changes by acetic acid could be found. The extensive involvement of genital skin with flat condylomata in this subgroup raises doubts as to the practicality of treatment. Nevertheless, before treatment is rendered a punch biopsy for confirmation of the screening test is advised.

Inverted papilloma of renal pelvis associated with contralateral ureteral malignancy and bladder recurrence.

We report a case of an inverted papilloma of the renal pelvis diagnosed at the same time as transitional cell carcinoma of the contralateral ureter. The diagnostic studies and surgical management are presented. Recurrence of an inverted papilloma in the bladder 1 year after treatment was confirmed histologically. Recurrence of this lesion and the association with urothelial malignancy suggest the need for close followup of patients with an inverted papilloma.

Comparison of meclofenamate sodium with buffered aspirin and placebo for the relief of postoperative dental pain.

The analgesic effect of meclofenamate sodium at two dose levels (100 mg and 200 mg) was compared with the effects of buffered aspirin (600 mg) and placebo in a double-blind, randomized study of 105 dental outpatients with acute pain following third-molar extraction. Meclofenamate sodium at either dose level was significantly superior to both buffered aspirin and placebo, resulting in significantly greater relief of pain. All four treatments were well tolerated, and side effects were minimal. Meclofenamate sodium is a safe, highly effective analgesic for the relief of acute pain.

Renal preservation utilizing neodymium:YAG laser.

Six patients with malignancies in a solitary kidney were treated with conservative renal parenchymal-sparing surgery utilizing the Neodymium Yttrium Aluminum Garnet (Nd:YAG) laser. Three patients had transitional cell carcinoma in an upper pole calyx of a solitary kidney. The transitional cell carcinoma was treated definitively by the Nd:YAG laser through a nephroscope prior to partial nephrectomy. The 3 patients have been followed up for twenty-eight, eighteen, and six months, respectively. None of the patients has shown evidence of recurrent cancer on follow-up retrograde ureterograms or on urine cytology obtained from the renal pelves. Three patients with renal cell carcinoma in a solitary kidney had the tumor surgically excised utilizing the Nd:YAG laser in conjunction with standard surgical techniques. All the patients were elderly with compromised renal vasculature that prevented bench surgery with autotransplantation or occlusion of the renal artery. At sixteen, fourteen, and three months, respectively, there is no evidence of recurrent cancer on CAT scans obtained on these patients.

Simultaneous flow cytometric deoxyribonucleic acid and acid phosphatase analysis of benign and malignant lesions of the prostate.

Flow cytometry can differentiate benign from malignant lesions of the prostate through deoxyribonucleic acid distribution analysis. A method has been developed that permits simultaneous cytometric determination of deoxyribonucleic acid and acid phosphatase activity in the cell cycle compartments of prostatic biopsy specimens. Histograms of prostatic carcinoma reveal higher acid phosphatase activity and greater deoxyribonucleic acid content in the S and S + G2/M populations than the histograms representing benign lesions. This compartmental difference may have prognostic usefulness.

Mesonephric adenocarcinoma of the bladder.

Mesonephric adenocarcinoma of the bladder may be the malignant counterpart of nephrogenic adenoma. We report the third known case of this invasive, well differentiated, tubular neoplasm resembling nephrogenic adenoma. The invasive potential of nephrogenic neoplasms has altered our approach to the management of these lesions. Nephrogenic adenoma and mesonephric adenocarcinoma appear cytologically similar on a superficial bladder biopsy. The latter is excluded by deeper bladder biopsies. Muscular invasion may indicate a more aggressive behavior and may require radical cystectomy for cure.

Percutaneous ultrasonic lithotripsy: choice of irrigant.

Extravasation of glycine irrigant during percutaneous ultrasonic lithotripsy has caused a transurethral resection syndrome consisting of hypertension, confusion and hyponatremia. With a rabbit model this complication is recreated with the intraperitoneal instillation of 1.5 per cent glycine solution tagged with 14carbon-glycine. Significant quantitative absorption of glycine into blood and other organs is demonstrated. A review of the literature reveals few guidelines as to the choice of irrigant for intrarenal endoscopy. Since this procedure involves no electric current it is suggested that physiological saline rather than glycine be used for ultrasonic stone disintegration.