Novel point mutation (W184R) in neonatal type 2 Gaucher disease.

Gaucher disease is the most prevalent inherited sphingolipidosis and results from deficient glucocerebrosidase activity. Three clinical forms of Gaucher disease have been described: type 1, or non-neuronopathic; type 2, or acute neuronopathic; and type 3, or subacute neuronopathic. We have identified a novel mutation in a patient of Russian-British descent who died of type 2 Gaucher disease a few hours after birth. A heterozygous T --> C transition mutation in exon 6, cDNA nucleotide position 667, results in the substitution of tryptophan by arginine at amino acid residue 184 (W184R) of glucocerebrosidase. This mutation creates a new cleavage site for the restriction endonuclease Hinf1. We developed a method that utilizes Hinf1 restriction endonuclease analysis to confirm the presence of the mutation and test family members. The second mutation identified in the other glucocerebrosidase allele of the patient is mutation L444P, a severe mutation frequent in type 2 and 3 Gaucher disease. Since the patient died very shortly after birth, we postulate that the W184R/L444P genotype may result in little or no detectable glucocerebrosidase activity and thus a poor prognosis.

Evaluation of the APOH gene as a positional candidate for prcd in dogs.

PURPOSE: Progressive rod-cone degeneration (prcd) is an autosomal recessive retinal degeneration of dogs characterized by abnormalities in lipid metabolism. It has recently been mapped to the centromeric region of canine chromosome 9, homologous to human 17q, which contains the apolipoprotein H (apoH, protein; APOH, gene) gene involved in lipid metabolism and regulation of triglycerides. The present study was undertaken to evaluate APOH as a positional candidate for prcd. METHODS: Expression of APOH in the retina was examined by reverse transcription-polymerase chain reaction (RT-PCR) and by immunocytochemistry in normal and prcd-affected dogs. The level of apoH in the plasma was determined by western blot analysis. Intragenic polymorphic markers were identified and typed in the prcd pedigree. Canine-rodent hybrid cell lines were analyzed to detect canine APOH. RESULTS: ApoH has been localized to the photoreceptor outer segment layer by immunocytochemistry. Its expression in the retina of normal and prcd-affected dogs was confirmed by RT-PCR. The levels of antihuman apoH cross-reacting material in plasma were similar in all dogs, regardless of disease status. Finally, linkage analysis of the APOH gene with the disease locus in the prcd pedigree detected 3 recombinants among 70 informative offsprings (lod score 15.09 at 0 = 4.3 centimorgan [cM]). CONCLUSIONS: APOH is expressed in the retina and tightly linked to the prcd locus. However, despite its potential role in phenotypes of abnormal lipid metabolism associated with prcd, the gene has been excluded as a primary candidate for prcd by linkage analysis.

Photoreceptor dysplasia (pd) in miniature schnauzer dogs: evaluation of candidate genes by molecular genetic analysis.

Photoreceptor dysplasia (pd) is one of a group of at least six distinct autosomal and one X-linked retinal disorders identified in dogs which are collectively known as progressive retinal atrophy (PRA). It is an early onset retinal disease identified in miniature schnauzer dogs, and pedigree analysis and breeding studies have established autosomal recessive inheritance of the disease. Using a gene-based approach, a number of retina-expressed genes, including some members of the phototransduction pathway, have been causally implicated in retinal diseases of humans and other animals. Here we examined seven such potential candidate genes (opsin, RDS/peripherin, ROM1, rod cGMP-gated cation channel alpha-subunit, and three subunits of transducin) for their causal association with the pd locus by testing segregation of intragenic markers with the disease locus, or, in the absence of informative polymorphisms, sequencing of the coding regions of the genes. Based on these results, we have conclusively excluded four photoreceptor-specific genes as candidates for pd by linkage analysis. For three other photoreceptor-specific genes, we did not find any mutation in the coding sequences of the genes and have excluded them provisionally. Formal exclusion would require investigation of the levels of expression of the candidate genes in pd-affected dogs relative to age-matched controls. At present we are building suitable informative pedigrees for the disease locus with a sufficient number of meiosis to be useful for genomewide screening. This should identify markers linked to the disease locus and eventually permit progress toward the identification of the photoreceptor dysplasia gene and the disease-causing mutation.

Frequency of the codon 807 mutation in the cGMP phosphodiesterase beta-subunit gene in Irish setters and other dog breeds with hereditary retinal degeneration.

Rod-cone dysplasia 1 (rcd1) in Irish setters is caused by a nonsense mutation in the cGMP phosphodiesterase beta-subunit gene (PDE6B). We examined the frequency of the mutant allele in the Irish setter population and determined if the defect is present in dogs of other breeds which are affected with other inherited photoreceptor diseases. Between 1994 and 1997, samples were obtained from 436 clinically normal Irish setters, a red wolf, and dogs from 23 different breeds. The mutation in codon 807 of PDE6B was detected in genomic DNA by heteroduplex analysis, allele-specific PCR, or restriction enzyme digestion. Of the 436 samples from clinically normal setters, 34 contained the mutation in one of the two PDE6B alleles (carrier rate = 7.8%). In contrast, the same mutation was not found in the red wolf or dogs of other breeds affected with PRA or inherited photoreceptor diseases. The high percentage of tested carriers, however, is not representative of the number of carriers in the population since some dogs tested were closely related and did not represent a random sample of the Irish setter breed.

Congenital stationary night blindness in the dog: common mutation in the RPE65 gene indicates founder effect.

PURPOSE: To clone and characterize the canine RPE65 cDNA from normal dog, examine for mutations, and establish if the mutation identified in Swedish briard dogs with retinal dystrophy is present in dogs of the same breed that originated from the United States and other countries, and are affected with congenital stationary night blindness. METHODS: Fifteen briard dogs were studied, of which 10 were affected with csnb, and five were clinically normal. In addition, we tested samples from four Swedish dogs, and samples from a briard affected with progressive retinal atrophy. RPE65 cDNA was cloned a from retinal cDNA library by PCR, and from canine retina by RT-PCR. ERG and morphology were used to characterize csnb. RESULTS: The normal RPE65 cDNA spans 1724 nucleotides (GenBank accession number AF084537), and includes 1602 nucleotides of coding sequence; the deduced amino acid sequence shares 98%, 97%, and 93% identity with homologous human, bovine, and rat sequences, respectively. A homozygous four nucleotide (AAGA) deletion, representing nucleotides 487-490 of wildtype RPE65 sequence, was found only in csnb and retinal dystrophy affected dogs; heterozygous animals had normal and mutant alleles. The mutation produces a frameshift, causing a deduced mistranslation with a premature stop codon. The mutation causes retinal dysfunction and RPE accumulation of lipid vacuoles. CONCLUSIONS: Identification of the same mutation in csnb and retinal dystrophy confirms the molecular identity of the two disorders. A common mutation in dogs derived from different countries suggests a founder effect causing the propagation of a common mutant allele in the population at risk.

The effect of ethnicity on the development of small for gestational age infants associated with hypertension in pregnancy.

The objective of this article is to assess in a hypertensive pregnant population the role of ethnic background on the development of small for gestational age (SGA) infants. A cohort population of 366 pregnant women who developed new hypertension in their pregnancy were interviewed and their ethnic groups defined. We then compared the outcomes of the pregnancies with regard to the development of SGA infants among the various ethnic groups. Preeclamptic women were more likely to deliver a SGA infant than gestational hypertensive women. Women of East Indian descent delivered the highest incidence of SGA infants when they developed preeclampsia (50%) compared to an incidence in the White population of 13.8%. Only the ethnocultural group, mean third-trimester blood pressure and third-trimester hematocrit, significantly correlated with the development of a SGA infant. Chinese and East Indian women who develop preeclampsia are at the highest risk of having a growth-restricted infant.

Canine rod transducin alpha-1: cloning of the cDNA and evaluation of the gene as a candidate for progressive retinal atrophy.

PURPOSE: Progressive retinal atrophy (PRA) represents a heterogeneous group of retinal dystrophies, distinct forms of which occur in different canine breeds. The present study was undertaken to evaluate the gene for the alpha-1 subunit of the rod specific G-protein transducin (GNAT1), a member of the phototransduction pathway, as a candidate for progressive rod cone degeneration (pred) in poodles, early retinal degeneration (erd) in elkhounds, and rod cone dysplasia 2 (rcd2) in collies. METHODS: Oligonucleotide primers were designed from the consensus region of known cDNA sequences for GNAT1 from other species. Canine GNAT1 cDNA was cloned and sequenced after reverse transcription (RT) and polymerase chain reaction (PCR) of total retinal RNA, and PCR amplification of specific sequences from a canine retinal cDNA library. Large, intron containing fragments of the canine transducin alpha-1 subunit gene were amplified from genomic DNA of individuals in PRA informative pedigrees, using canine-specific primers. PCR products were digested with Nci I, to enable typing of individuals in the PRA affected pedigrees for a previously identified GNAT1 restriction fragment length polymorphism (RFLP). RESULTS: The sequence of canine GNAT1 cDNA is reported (GenBank accession no. U65376). Over the coding region, the canine GNAT1 cDNA sequence presented here shares 92-95% identity with human, bovine and murine sequences. The canine cDNA encodes a polypeptide of 350 amino acids; its theoretical translation is 98-99% identical with the corresponding GNAT1 sequence from each of the other 3 species and it has no unique amino acids. In rcd2 and erd pedigrees informative for both the disease locus and the GNAT1 Nci I RFLP, a minimum of 3 and 2 recombinants were identified, respectively. Similarly, in a prcd pedigree, 3 of 7 progeny informative for both prcd and this RFLP were obligate recombinants. CONCLUSIONS: The canine GNAT1 gene has been excluded as a candidate for prcd, erd and rcd2. Sequence information of canine GNAT1 gene will enable testing this locus as a candidate in other canine hereditary retinal degenerations.

An improved diagnostic test for rod cone dysplasia 1 (rcd1) using allele-specific polymerase chain reaction.

PURPOSE: To develop an improved diagnostic test for rod-cone dysplasia type 1 (rcd1). The rcd1 phenotype is an early onset, autosomal recessive disease caused by a mutation in the canine rod cyclic GMP phosphodiesterase beta-subunit (PDE6B) gene. A G to A transition in codon 807 at nucleotide position 2420 results in a stop codon. This is the only disease causing mutation detected so far in the canine PDE6B gene. METHODS: Allele specific primers were designed in which the 3 end had the nucleotide corresponding to either the wild type or the mutant rcd1 allele. PCR was done using the allele specific primers in combination with a common primer complementary to the opposite strand to distinguish between the wild type and the rcd1 alleles. RESULTS: The wild type and rcd1 alleles were identified successfully in two independent ASPCRs done with two different sets of allele specific primers. Further, both alleles could be amplified in a single tube and distinguished based on the size difference of the PCR products using one allele specific primer of altered length by the addition of a 9 nucleotide long linker. CONCLUSIONS: We have developed an improved diagnostic test for the disease based on ASPCR such that the presence or absence of different size amplified fragments provides direct determination of the genotype. In contrast to previously reported diagnostic tests, this method is more efficient because it eliminates the need for any further manipulation of the PCR product.

Molecular diagnostic tests for ascertainment of genotype at the rod cone dysplasia 1 (rcd1) locus in Irish setters.

Rod-cone dysplasia type 1 (rcd1) is one of several canine photoreceptor degenerations, collectively termed progressive retinal atrophy (PRA), that afflict different breeds of dogs. The rcd1 phenotype is an early onset autosomal recessive disease caused by a nonsense amber mutation, at codon 807, in the canine gene for the beta-subunit of rod cyclic GMP phosphodiesterase (canine PDEB). The mutation involves a G to A transition at nucleotide position 2420, which presumably would cause premature termination of the canine PDEB protein by 49 amino acid residues. In both a small pedigree study of Irish setters from the United Kingdom and in larger canine pedigree studies in the United States, this gene defect has been found to be the only mutation causing rcd1. Here we report development of a diagnostic test which unequivocally distinguishes the three genotypes at the rcd1 locus: rcd1/rcd1 (homozygous mutant, affected); rcd1/+ (heterozygous, carrier); and +/+ (homozygous normal, wildtype).

Cosegregation of codon 807 mutation of the canine rod cGMP phosphodiesterase beta gene and rcd1.

PURPOSE: To determine if a previously reported nonsense mutation (G to A transition at nucleotide position 2420) in the canine rod cyclic GMP (cGMP) phosphodiesterase beta (PDEB) subunit gene cosegregates with the rod-cone dysplasia 1 disease allele (rcd1) in the rcd1-dog reference colony; to establish the prevalence of this mutation among rcd1-affected Irish setters in the United States; and to screen for this mutation in other forms of canine hereditary progressive retinal atrophy (PRA). METHODS: Exon 21 of canine PDEB, previously reported to contain a nonsense mutation in rcd1-affected dogs, was amplified by polymerase chain reaction from genomic DNA isolated from peripheral blood samples. The mutation was detected in amplified DNA by restriction enzyme digestion and double-stranded conformational polymorphism. Linkage between rcd1 and the PDEB mutation was tested using the computer program LIPED: RESULTS: Three different rcd1-informative canine pedigrees were tested for the PDEB nonsense mutation. The first was a multigenerational pedigree representing the rcd1 reference colony. The other two pedigrees represented purebred Irish setter breeding lines in which rcd1 was known to be segregating. In all three pedigrees, the same point mutation was present and segregated with no discordance with the rcd1 allele. Linkage analysis established a maximum logarithm of odds (LOD) score of 12.05 at a linkage distance (theta) of 0.0. In a representative sampling of Irish setters in the United States diagnosed clinically as affected with typical rcd1 phenotype, all dogs were demonstrated to have the same (codon 807) PDEB mutation. Three of four Irish setters affected with atypical, relatively slower disease also had this mutation, but one dog did not. This point mutation in the canine PDEB gene was absent in other forms of canine hereditary retinal degeneration. CONCLUSIONS: In three informative pedigrees, the codon 807 mutation in canine PDEB cosegregates with the rcd1 disease allele with zero discordance. A linkage distance (theta) of zero, with an LOD score of 12.05, indicates identity of this mutation and rcd1. This appears to be the only mutation causing rcd1 in the United States. In all other forms of canine hereditary retinal degeneration tested (cd, erd, prcd, rcd2, X-linked PRA, and in one Iris degeneration tested (cd, erd, prcd, rcd2, X-linked PRA, and in one Irish setter with late onset PRA), this PDEB point mutation was absent.

Delayed delivery of second twin: report of four cases and review of the literature.

In four cases of delayed delivery of a twin pregnancy with survival of the second twin, the interval ranged at 41-143 days. Review of the literature and our cases supports the following approach: high ligation of the umbilical cord with an absorbable suture, cervical suture in the presence of cervical dilatation, and serial monitoring of fetal growth and maternal coagulation indices. Disseminated intravascular coagulation has not been reported in such cases.

Necrotizing funisitis.

Necrotizing funisitis is an umbilical cord lesion characterized by perivascular bands of necrotic Wharton jelly containing inflammatory cells in various stages of degeneration. Sixty cases were reviewed histologically. Clinical information was available in 45. Forty-five age-matched infants with acute (nonspecific) funisitis only were used as controls. Infants with necrotizing funisitis had more stillbirths, birth weights below the tenth percentile (small for gestational age [SGA]), infectious complications, and necrotizing enterocolitis. No consistent infectious agents or predisposing maternal factors were found. Cord neovascularization correlated with SGA infants. Necrotizing funisitis occurred in 0.1% of deliveries greater than 20 weeks' gestation. The perivascular bands, likened to the pattern of an Ouchterlony diffusion plate, suggest the presence of a diffusible toxin in the amniotic fluid. The stillbirths and SGA infants may represent the toxin's effect on the fetus. The lack of perivascular necrotic bands around vessels on the placental surface suggests neutralization or more effective clearing of the agent in this region, for reasons as yet undetermined. The factors underlying the cord lesion may contribute to superimposed acute nonspecific vasculitis and chorioamnionitis.

Optimal positioning of endotracheal tubes for ventilation of preterm infants.

Accurate knowledge of upper-airway dimensions is required to prevent malpositioning of endotracheal tubes in preterm infants. We measured vocal cord-carina, oral-carina, and nasal-carina distances in situ at autopsy of two groups of infants (less than 1000 and greater than or equal to 1000 g). In all 24 infants, crown-heel length, crown-rump length, and occipitofrontal circumference were better than weight in predicting upper-airway dimensions. Flexion of the neck decreased and extension increased both nasal-carina and oral-carina distances. Lateral rotation produced no significant changes. The postmortem data were not different from nasal-carina distances measured radiologically in 40 living, nasally intubated and ventilated infants, confirming the clinical validity of our findings. Regression equations were derived to predict optimal endotracheal tube lengths based on the external measurements of crown-rump length and crown-heel length.

Chorionic villus sampling: analysis of fetal losses to delivery, placental pathology, and cervical microbiology.

A population of 1639 patients were seen for chorionic villus sampling (CVS). Embryonic death was identified at ultrasound in 5.3 per cent of patients. The number of patients undergoing CVS was 1551, with 1416 transcervical procedures and 135 transabdominal procedures. The most common indication for CVS was advanced maternal age. Spontaneous pregnancy losses identified by increased risk of pregnancy loss with increasing aspiration attempts. The total fetal loss for this population was 5.4 per cent with the pregnancy loss estimated due to procedure being 1.2 per cent. Analysis of placentae from patients having CVS and amniocentesis showed no differences. Microbiological assessment prior to CVS was similar to previous publications.

Umbilical cord ulceration and intestinal atresia: a new association?

In three fetuses, congenital intestinal atresia was associated with linear ulcerations of the umbilical cord. In two cases, hemorrhage was seen from the cord ulcer. Both fetuses required emergency cesarean section for fetal distress and were born anemic. The third fetus was mildly hydropic, attributed to hemorrhage, and was stillborn. The mechanism of the association could not be determined. These cases suggest a risk of prenatal umbilical cord hemorrhage in infants with intestinal atresia.

Syndrome of mental retardation, facial anomalies, hypopituitarism, and distal arthrogryposis in sibs.

A brother and a sister presented with a malformation syndrome consisting of facial anomalies, distal arthrogryposis with camptodactyly of fingers and "hammer toes," severe mental retardation, and hypopituitarism. The girl is now 6 1/2 years old and exhibits severe mental retardation. She has abnormal secretion of growth hormone and responded to growth hormone therapy. Her brother was born with the same facial manifestations, distal contractures, and hypopituitarism. He died unexpectedly at age 3 months of no apparent cause. The occurrence of the syndrome in 2 sibs of different sex suggests autosomal recessive inheritance.

Apparent postnatal onset of some manifestations of the Wiedemann-Beckwith syndrome.

We report on 2 patients who were apparently normal at birth but later developed characteristics of Wiedemann-Beckwith syndrome (WBS). Both had hypoglycemia neonatally and gradually developed coarse facial changes, umbilical hernia, and macroglossia. Renal sonography done after the macroglossia developed showed large kidneys in both. The placentas were carefully examined in both cases but findings described as typical of WBS were only found in one. The clinical evolution of these infants suggests that some WBS manifestations may have their onset postnatally in some cases. We postulate that the cellular hyperplasia and hypertrophy characteristic of WBS may be caused by persistent rests of embryonal cells that secrete paracrine and/or endocrine growth factors.

Pathology of intragestational intervention in twin-to-twin transfusion syndrome.

Selective intervention in multiple pregnancy is being used to enhance the chances of survival of at least one conceptus when the risks for the combined conceptuses and mother are considered too great. These procedures have been applied to induced polyembryonic conceptions (selective continuance) and discordant dichorionic twins (selective birth). We report attempts at selective intervention in three monochorionic twin gestations affected by twin-to-twin transfusion syndrome. In all three cases, both fetuses seemed doomed and the mother was in significant distress. The selected survivor in the first case is doing well; both twins were stillborn in the second case; in the third case, the selected survivor died as a neonate but the other twin survived and is doing well. We suggest possible explanations for the clinical outcome of each case based on detailed pathologic examination of the delivered placentas and autopsy examination of the nonsurviving twins. The shared chorionic circulation is the source of both the clinical disorder and the potential complications of any attempt to alleviate the disorder. This situation is unique to monochorionic twins, and we discuss the implications of this for intrauterine therapy of twin-to-twin transfusion syndrome.

Biochemical abnormalities in rhizomelic chondrodysplasia punctata.

Biochemical studies with emphasis on peroxisomal functions were conducted in six patients with well-documented rhizomelic chondrodysplasia punctata (RCDP) and compared with findings in patients with Zellweger syndrome and neonatal adrenoleukodystrophy (ALD). Patients with RCDP had three characteristic biochemical abnormalities: (1) profound defect in plasmalogen (ether lipid) synthesis, which is significantly greater than the analogous defect in Zellweger syndrome or neonatal ALD; (2) reduction of phytanic acid oxidation activity to 1% to 5% of control, similar to that observed in Refsum disease, Zellweger syndrome, and neonatal ALD; (3) presence of the unprocessed form of peroxisomal 3-oxoacyl-coenzyme A thiolase in the postmortem liver of two patients. Other peroxisomal functions were normal, including levels of very long chain fatty acids, pipecolic acid, and bile acid intermediates, and immunoblot studies of peroxisomal acyl-CoA oxidase and bifunctional enzyme in postmortem liver. Unlike what is observed in Zellweger syndrome and neonatal ALD, catalase activity in cultured skin fibroblasts was sedimentable, indicating that peroxisome structure is not grossly deficient in RCDP. The biochemical abnormalities in RCDP were consistent and set it apart from all the other known peroxisomal disorders.