Molecular delineation of deletions on 2q37.3 in three cases with an Albright hereditary osteodystrophy-like phenotype.

A minority of the reported cases of terminal 2q37 deletion clinically resemble Albright hereditary osteodystrophy (AHO)/pseudopseudohypoparathyroidism and have only mild-to-moderate mental retardation. Our molecular and cytogenetic fluorescence in situ hybridization (FISH) findings on an additional three patients further reduce the size of the minimal critical region deleted in this syndrome to about 3 Mb. This region includes the G-protein-coupled receptor 35 (GPR35), glypican 1 (GPC1), and serine/threonine protein kinase 25 (STK25) genes on 2q37.3. We have further defined several polymorphic variants within the coding region and flanking regions of GPR35 gene, which could potentially be useful for rapid detection of GPR35 gene deletion. We postulate that the absence of GPR35 may, at least partly, account for the phenotypic resemblance to the AHO. We also believe that the deletion of GPR35 could be responsible for the entity brachydactyly mental retardation syndrome (OMIM #600430), which was coined based on the above minority of patients with terminal 2q37 deletion. We recommend that every patient with AHO phenotype should undergo 2q subtelomeric FISH screen and subsequently a molecular study on the GPR35 gene. GPC1 and/or STK25 haploinsufficiency may also contribute to the AHO-like phenotype.

Inheritance of osteosarcoma and Paget's disease of bone: a familial loss of heterozygosity study.

Pagetoid osteosarcoma is a complication of Paget's disease of bone. Sarcomatous transformation is most often seen in severe, long-standing Paget's disease. Familial clustering of Paget's disease has been described with apparent autosomal dominant inheritance with high penetrance by the sixth decade. Although definitive proof of the specific gene involved remains elusive, some researchers have shown loss of heterozygosity in a region of chromosome 18q in a relatively high percentage of studied patients affected with either Paget's disease alone, in Pagetoid osteosarcoma, and in uncomplicated osteosarcoma. Our patient was diagnosed with Pagetoid osteosarcoma and had a first-degree relative with history of the same. We hypothesized that our patient's tumor samples might contain a similar genetic abnormality. Our analysis of several polymorphic markers from the chromosome 18q21-22 region showed loss of maternally inherited alleles throughout the region. This finding is similar to those described previously and provides further evidence of a susceptibility region relating to this disease. This report describes a father and son, their young ages at diagnosis of Pagetoid sarcoma, the identical sites of disease involvement, and a loss of heterozygosity study illustrating the inheritance of the presumed defective gene.

Cognitive deficits associated with a recently reported familial neurodegenerative disease: familial encephalopathy with neuroserpin inclusion bodies.

BACKGROUND: We recently discovered an autosomal dominant disease causing a progressive dementia. The disease is caused by a point mutation in the gene coding for the serine protease inhibitor (ie, serpin) neuroserpin. The mutation results in an unstable neuroserpin protein that readily aggregates into intraneuronal inclusions that we identify as Collins bodies. The bodies are distributed throughout the cerebral hemispheres but are significantly more numerous in the cortex and the substantia nigra. We have named the disease familial encephalopathy with neuroserpin inclusion bodies (FENIB). OBJECTIVES: To describe the cognitive and neurophysiological changes exhibited by individuals with FENIB and to correlate the phenotypic expression of the disease with the neuropathological findings. DESIGN: Multiple case studies using neuropsychological assessment, electroencephalography (EEG), magnetic resonance imaging (MRI), and single-photon emission computed tomographic (SPECT) studies of family members were performed. Using these measures, we also compared family members in whom the mutation is present with family members in whom the mutation was absent to control for nonspecific familial factors. SUBJECTS: Nine individuals (5 women, aged 31-64 years; 4 men, aged 43-67 years) from 2 generations of family members related to the first reliably identified individual with symptoms of this disease. Symptoms, by self-report and reports of other family members, ranged from asymptomatic to severe dementia. Six of the 9 individuals carried the disease mutation. RESULTS: All subjects with the mutation demonstrated some cognitive changes, with the greatest demonstrated by subjects older than 40 years. The changes included restricted attention, concentration, and response regulation functions, reduced controlled oral fluency (word-list generation), and restricted visuospatial organization. In general, recall memory was not as affected as other cognitive domains. The most severely affected subject demonstrated global dementia with prominent frontal lobe features. Findings on SPECT showed anomalies limited to frontal areas in the less affected subjects and more global, patchy areas of hypoperfusion in the more severely affected subjects. The 3 oldest and most affected subjects demonstrated slowing on EEG findings. The MRI findings were noncontributory except in the 2 most severe cases, which showed global cortical atrophy. CONCLUSIONS: Cognitive changes in mildly to moderately affected subjects were characterized by deficits in frontal and frontal-subcortical area-dependent processes. Continued progressive deterioration of cerebral functions with relative sparing of recall memory suggests a unique dementia associated with this disease.

A PCR assay for detecting clonal rearrangement of the TCR-gamma gene.

BACKGROUND: The T-cell receptor gamma chain (TCR-gamma) gene has 11 functional variable (V) exons that can be organized into four subfamilies and four functional joining (J) exons that can be divided into two subfamilies. METHOD AND RESULTS: Three multiplex PCR reactions amplifying the TCR-gamma gene were developed. Primer combinations for multiplex PCR were chosen so that the V-region subfamily and J-region subfamily involved in a clonal band could be identified. Control primers from the protease inhibitor (PI) gene were also included in each reaction to verify the presence of amplifiable DNA. Fifty-six archived samples that had been tested by Southern blot for clonal rearrangement of the TCR-beta gene were analyzed with the TCR-gamma PCR protocol. Twenty-one of 56 samples were TCR-beta positive by Southern blot and thus expected to be positive with TCR-gamma PCR. Thirty-five of 56 samples were TCR-beta negative by Southern blot. Of these, 14 samples showed clonal rearrangement of the immunoglobulin heavy chain gene. The TCR-gamma PCR protocol showed a diagnostic sensitivity for detecting T-lineage clonality of 90%, with a diagnostic specificity for detecting T-cell lineage of only 74%. CONCLUSION: The PCR protocol described here performed well in comparison with a TCR-beta Southern protocol.

Syndrome of short stature, widow's peak, ptosis, posteriorly angulated ears, and joint problems: exclusion of the Aarskog (FGD1) gene as a candidate gene.

A syndrome encompassing postnatal onset of short stature, widow's peak, ptosis, posteriorly angulated ears, and limitation of forearm supination is reported in a boy and his mother. The boy has not yet experienced dislocation of patella or other joint anomaly except for limitation of supination of the forearms. On the other hand, the mother has a milder limitation of supination only on the left arm and is devoid of ptosis. Their condition is reminiscent of that described in the family reported by Kapur et al. [1989: Am. J. Med. Genet. 33: 357-363.], which showed an X-linked dominant mode of inheritance. DNA study on our family using an intragenic polymorphism of the Aarskog syndrome (FGD1) gene and four other adjacent markers convincingly excludes the possibility that their condition could be caused by a mutation of the FGD1 gene. Our family and the family reported by Kapur et al. may suggest segregation of a novel X-linked dominant condition.

R117H and IVS8-5T cystic fibrosis mutation detection by restriction enzyme digestion.

The number of thymines in a thymine repeat in intron 8 (IVS8) next to the exon 9 of the cystic fibrosis gene (CFTR) affects the efficiency of this site to act as a splice acceptor site and the subsequent inclusion or skipping of exon 9 into the CFTR protein. Another source of genetic variation, responsible for mild cystic fibrosis (CF) and/or congenital bilateral absence of the vas deferens, is the mutation R117H, located in exon 4 of CFTR. Both these genetic variants can be detected using restriction site-generating PCR amplification. The severity of the CF phenotype is partly dependent on the IVS8 background on which R117H occurs; thus, it is important to be able to test clinically for both these variants. A method using restriction digestion of PCR product is presented.

Familial encephalopathy with neuroserpin inclusion bodies.

We report on a new familial neurodegenerative disease with associated dementia that has presented clinically in the fifth decade, in both genders, and in each of several generations of a large family from New York State-a pattern of inheritance consistent with an autosomal dominant mode of transmission. A key pathological finding is the presence of neuronal inclusion bodies distributed throughout the gray matter of the cerebral cortex and in certain subcortical nuclei. These inclusions are distinct from any described previously and henceforth are identified as Collins bodies. The Collins bodies can be isolated by simple biochemical procedures and have a surprisingly simple composition; neuroserpin (a serine protease inhibitor) is their predominant component. An affinity-purified antibody against neuroserpin specifically labels the Collins bodies, confirming their chemical composition. Therefore, we propose a new disease entity-familial encephalopathy with neuroserpin inclusion bodies (FENIB). The conclusion that FENIB is a previously unrecognized neurodegenerative disease is supported by finding Collins bodies in a small kindred from Oregon with familial dementia who are unrelated to the New York family. The autosomal dominant inheritance strongly suggests that FENIB is caused by mutations in the neuroserpin gene, resulting in intracellular accumulation of the mutant protein.

Familial dementia caused by polymerization of mutant neuroserpin.

Aberrant protein processing with tissue deposition is associated with many common neurodegenerative disorders; however, the complex interplay of genetic and environmental factors has made it difficult to decipher the sequence of events linking protein aggregation with clinical disease. Substantial progress has been made toward understanding the pathophysiology of prototypical conformational diseases and protein polymerization in the superfamily of serine proteinase inhibitors (serpins). Here we describe a new disease, familial encephalopathy with neuroserpin inclusion bodies, characterized clinically as an autosomal dominantly inherited dementia, histologically by unique neuronal inclusion bodies and biochemically by polymers of the neuron-specific serpin, neuroserpin. We report the cosegregation of point mutations in the neuroserpin gene (PI12) with the disease in two families. The significance of one mutation, S49P, is evident from its homology to a previously described serpin mutations, whereas that of the other, S52R, is predicted by modelling of the serpin template. Our findings provide a molecular mechanism for a familial dementia and imply that inhibitors of protein polymerization may be effective therapies for this disorder and perhaps for other more common neurodegenerative diseases.

Mapping of a gene (MRXS9) for X-linked mental retardation, microcephaly, and variably short stature to Xq12-q21.31.

Three boys from two families were identified as having a syndrome of X-linked mental retardation (XLMR) with microcephaly and short stature, clinically resembling Renpenning syndrome but with normal size of testicles in affected men. When the effort to map the gene for the above condition was initiated, it was realized that the two families were actually related to each other. Over 50 polymorphic markers of known locations along the X chromosome were scored in this family in a study to map the disease gene. Nine affected and four unaffected males were genotyped to produce a maximum LOD score of 4.42 at zero recombination with markers in proximal Xq. The results indicate that the gene responsible for this disorder is located in the cytogenetic Xq12 to Xq21.31 interval of the X chromosome within a section of chromosome of about 17 cM between the AR and DXS1217 loci over some 25 mb. Since the gene for the X-linked mental retardation from the original Saskatchewan family described by Renpenning [Renpenning et al., 1962: Can Med Assoc J 87:954-956; Fox and Gerrard, 1980: Am J Med Genet 7:491-495] was recently mapped to a different nonoverlapping region [Stevenson et al., 1998: Am J Hum Genet 62:1092-1101] this would appear to be a separate disorder.

Cystic fibrosis mutation frequencies in upstate New York.

Upstate New York patients (100) with cystic fibrosis (i.e., 200 CF chromosomes), 72 from the CF center in Syracuse and 28 from a Buffalo CF center, were analyzed for their CF-causing mutations using restriction enzyme digest, single-strand conformation analysis (SSCA), and Heteroduplex (HA) analysis. Polymerase chain reaction (PCR) amplified products from all 27 CFTR exons using primers that included flanking intron junction sequence were investigated. More than 120 known cystic fibrosis transmembrane conductance regulator (CFTR) disease-causing mutations were screened. Four novel CFTR disease-causing mutations were identified (N287Y in exon 6b, 1259insA in exon 8, R1070P in exon 17b, and CF?20kbdel14b-18). A detection rate of 96% of the combined Syracuse and Buffalo population CF chromosomes was obtained.

A mutation in CFTR produces different phenotypes depending on chromosomal background.

Cystic fibrosis (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene but the association between mutation (genotype) and disease presentation (phenotype) is not straightforward. We have been investigating whether variants in the CFTR gene that alter splicing efficiency of exon 9 can affect the phenotype produced by a mutation. A missense mutation, R117H, which has been observed in three phenotypes, was found to occur on two chromosome backgrounds with intron 8 variants that have profoundly different effects upon splicing efficiency. A close association is shown between chromosome background of the R117H mutation and phenotype. These findings demonstrate that the genetic context in which a mutation occurs can play a significant role in determining the type of illness produced.

Presymptomatic testing for autosomal dominant spinocerebellar ataxia type 1.

Presymptomatic testing was done on four people from a large family in which an autosomal dominant form of spinocerebellar ataxia was segregating. Earlier genetic analysis had shown that in this family the disorder was tightly linked to an informative microsatellite polymorphism on chromosome 6p. Two subjects with prior risks of 50% of developing the disease had final risks after testing of 2%; the other two with prior risks of 25% had final risks of 1%. Chromosome 6p linked spinocerebellar ataxia may now be added to Huntington's disease as a late onset disorder in which genetic linkage may be used to carry out presymptomatic testing.

Prenatal diagnosis for the cystic fibrosis mutation 1717-1, G-->A using arms.

A family carrying two cystic fibrosis mutations, delta F508 and 1717-1, G-->A, requested prenatal diagnosis. In order to eliminate the need for labelling of allele-specific oligonucleotides and to simplify the analysis, 1717-1, G-->A was detected using an ARMS (amplification refractory mutation system) method (Newton et al., 1989). Fetal DNA was obtained by chorionic villus sampling (CVS) and the ARMS technique was used to exclude the 1717-1, G-->A mutation. The fetus was found to be heterozygous for the delta F508 mutation. ARMS is a simple, quick, non-radioactive method suitable for detecting DNA mutations in various clinical situations.

Molecular screening of partners of cystic fibrosis heterozygotes.

We have screened 100 partners of known or suspected CF heterozygotes for ten CF mutations which account for 88% of the CF mutations seen by us on CF chromosomes. We have identified six CF heterozygotes amongst the 100 low-risk people screened. As two of the six people at high-risk of being CF carriers were subsequently shown not to be CF carriers this gave rise to four couples with a one in four risk of CF in a pregnancy and so far to two PND's. The risk of CF in the remaining 94 couples was reduced to less than one in 800. We have concentrated on the screening of partners for the commoner CF mutations rather than haplotyping them for the CF linked markers XV-2c/TaqI and KM19/PstI which are in linkage disequilibrium with CF. For individuals shown not to carry these ten mutations, a five fold difference in risk of being a CF carrier remains between those who have the XV-2c/KM19 genotypes associated with the highest risk(BB), as compared to those with the lowest risk(CC). Nevertheless we feel that effort is better expended in mutation screening rather than haplotyping.

Linkage disequilibrium and recombination make a telomeric site for the Huntington's disease gene unlikely.

In a Scottish family in which Huntington's disease (HD) was segregating, recombination was observed between the D4S115/S111 and D4S43/S95 loci, with the HD gene associated with the more proximal D4S43/S95 locus. Analysis of linkage disequilibrium in Scottish families showed significant non-random association between the HD gene and alleles at the D4S95 and D4S98 loci. This adds to previous evidence that the HD locus is not sited at the telomere of chromosome 4.

Isolation of probes specific to human chromosomal region 6p21 from immunoselected irradiation-fusion gene transfer hybrids.

A hybrid cell line (R21/B1) containing a truncated human chromosome 6 (6pter-6q21) and a human Y chromosome on a hamster background was irradiated and fused to A23 (TK-) or W3GH (HPRT-) hamster cells. Clones containing expressed HLA class I genes (4/40) were selected using monoclonal antibodies. These clones were recloned and analyzed with a panel of probes from the HLA region. One hybrid (4G6) contained the entire HLA complex. Two other hybrids (4J4 and 4H2) contained only the HLA class I region, while the fourth hybrid (5P9) contained HLA class I and III genes in addition to other genes located in the 6p21 chromosomal region. In situ hybridization showed that the hybrid cells contained more than one fragment of human DNA. Alu and LINE PCR products were derived from these cells and compared to each other as well as to products from two somatic cell hybrids having the 6p21 region in common. The PCR fragments were then screened on conventional Southern blots of the somatic cell hybrids to select a panel of novel probes encompassing the 6p21 region. In addition, the origin of the human DNA fragments in hybrid 4J4 was determined by regional mapping of PCR products.

The incidence of different cystic fibrosis mutations in the Scottish population: effects on prenatal diagnosis and genetic counselling.

We present an analysis of the frequency of 16 different cystic fibrosis (CF) mutant alleles in the Scottish population. Each allele was detected in DNA amplified by the polymerase chain reaction (PCR) either directly on polyacrylamide gels, on agarose gels after restriction enzyme digestion, or by using allele specific oligonucleotides. Among 506 CF chromosomes, of predominantly Scottish origin, the frequencies of the different mutations were delta F508 0.71, G551D 0.05, G542X 0.04, R117H 0.01, 1717-1G----A 0.01, A455E + delta I507 + R553X + R560T + W1282X + 621 + 1G----T combined 0.03, unpublished 0.01, and unknown 0.13. No examples of D110H, R347P, S549N, S549I, or 2566ins AT mutations were found. The relevance of this type of analysis for both prenatal diagnosis and heterozygote screening is discussed.