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The goal of this study was to expand the description of the cognitive development phenotype in boys with Klinefelter syndrome (47,XXY). We tested neuropsychological measures of memory, attention, visual-spatial abilities, visual-motor skills, and language. We examined the influence of age, handedness, genetic aspects (parental origin of the extra X chromosome, CAG(n) repeat length, and pattern of X inactivation), and previous testosterone treatment on cognition. We studied 50 boys with KS (4.1-17.8 years). There was a significant increase in left-handedness (P = 0.002). Specific language, academic, attentional, and motor abilities tended to be impaired. In the language domain, there was relative sparing of vocabulary and meaningful language understanding abilities but impairment of higher level linguistic competence. KS boys demonstrated an array of motor difficulties, especially in strength and running speed. Deficits in the ability to sustain attention without impulsivity were present in the younger boys. Neither genetic factors examined nor previous testosterone treatment accounted for variation in the cognitive phenotype in KS. The cognitive results from this large KS cohort may be related to atypical brain lateralization and have important diagnostic and psychoeducational implications. The difficulty in complex language processing, impaired attention and motor function in boys with KS may be missed. It is critical that boys with KS are provided with appropriate educational support that targets their learning challenges in school in addition to modifications that address their particular learning style. These findings would also be an important component of counseling clinicians and families about this disorder.

Objective To evaluate the growth disorder and phenotype in prepubertal children with Leri-Weill dyschondrosteosis (LWD), a dominantly inherited skeletal dysplasia, and to compare the findings from girls with Turner syndrome (TS). Study design We studied the auxologic and phenotypic characteristics in 34 prepubertal LWD subjects (ages 1 to 10 years; 20 girls, 14 boys) with confirmed short stature homeobox-containing gene ( SHOX ) abnormalities. For comparative purposes, we evaluated similar physical and growth parameters in 76 girls with TS (ages 1 to 19 years) and 24 girls with LWD (ages 1 to 15 years) by using data collected from the postmarketing observational study, GeNeSIS. Results In the clinic sample LWD subjects, height standard deviation score ranged from −5.5 to +0.1 (−2.3 ± 1.3, girls and −1.8 ± 0.6, boys). Wrist changes related to Madelung deformity were present in 18 of 34 (53%) LWD subjects. In comparing the LWD and TS populations in the GeNeSIS sample, Madelung deformity, increased carrying angle, and scoliosis were more prevalent in the LWD population, whereas high arched palate was similarly prevalent in the two populations. Conclusions Short stature is common in both LWD (girls and boys) and TS (girls). Clinical clues to the diagnosis of SHOX haploinsufficiency in childhood include short stature, short limbs, wrist changes, and tibial bowing.

The transgenic mouse line OVE459 carries a transgene-induced insertional mutation resulting in autosomal recessive congenital hydrocephalus. Homozygous transgenic animals experience ventricular dilation with perinatal onset and are noticeably smaller than hemizygous or non-transgenic littermates within a few days after birth. Fluorescence in situ hybridization (FISH) revealed that the transgene inserted in a single locus on mouse Chromosome (chr) 8, region D2-E1. Genetic crosses between hemizygous OVE459 mice and mice heterozygous for the spontaneous mutation hydrocephalus-3 (hy3) produced hydrocephalic offspring with a frequency of 22%, demonstrating that these two mutations are allelic. A genomic library was made by using DNA from homozygous OVE459 mice, and genomic DNA flanking the transgene insertion site was isolated and sequenced. A PCR polymorphism between C57BL/6 DNA and Mus spretus was used to map the location of the transgene insert to 1.06 cM +/- 0.75 proximal to D8Mit152 by using the Jackson Laboratory Backcross DNA Panel Mapping Resource. Furthermore, sequence analysis from a mouse bacterial artificial chromosome (BAC) clone, positive for unique markers on both sides of the transgene insertion site, demonstrated that the genomic DNAs flanking each side of the transgene insertion are physically separated by approximately 51 kb on the wild-type mouse chromosome.

Leri-Weill dyschondrosteosis (LWD) (MIM 127300) is a dominantly inherited skeletal dysplasia characterized phenotypically by Madelung wrist deformity, mesomelia, and short stature. LWD can now be defined genetically by haploinsufficiency of the SHOX (short stature homeobox-containing) gene. We have studied 21 LWD families (43 affected LWD subjects, including 32 females and 11 males, ages 3–56 yr) with confirmed SHOX abnormalities. We investigated the relationship between SHOX mutations, height deficit, and Madelung deformity to determine the contribution of SHOX haploinsufficiency to the LWD and Turner syndrome (TS) phenotypes. Also, we examined the effects of age, gender, and female puberty (estrogen) on the LWD phenotype. SHOX deletions were present in affected individuals from 17 families (81%), and point mutations were detected in 4 families (19%). In the LWD subjects, height deficits ranged from −4.6 to +0.6 sd (mean ± sd = −2.2 ± 1.0). There were no statistically significant effects of age, gender, pubertal status, or parental origin of SHOX mutations on height z-score. The height deficit in LWD is approximately two thirds that of TS. Madelung deformity was present in 74% of LWD children and adults and was more frequent and severe in females than males. The prevalence of the Madelung deformity was higher in the LWD vs. a TS population. The prevalence of increased carrying angle, high arched palate, and scoliosis was similar in the two populations. In conclusion, SHOX deletions or mutations accounted for all of our LWD cases. SHOX haploinsufficiency accounts for most, but not all, of the TS height deficit. The LWD phenotype shows some gender- and age-related differences.

We report on a man with neurofibromatosis type 1 (NF1) and Leri-Weill dyschondrosteosis (LWD). His father had NF1. His mother had LWD plus additional findings of Turner syndrome (TS): high arched palate, bicuspid aortic valve, aortic stenosis, and premature ovarian failure. The proband's karyotype was 46,X,dic(X;Y)(p22.3;p11.32). Despite having almost the same genetic constitution as 47,XXY Klinefelter syndrome, he was normally virilized, although slight elevation of serum gonadotropins indicated gonadal dysfunction. His mother's karyotype was mosaic 45,X[17 cells]/46,X,dic(X;Y)(p22.3;p11.32)[3 cells].ish dic(X;Y)(DXZ1 + ,DYZ1 + ). The dic(X;Y) chromosome was also positive for Y markers PABY, SRY, and DYZ5, but negative for SHOX. The dic(X;Y) chromosome was also positive for X markers DXZ1 and a sequence

Bent tail (BN:) is a spontaneous, semi-dominant mutation on the mouse X chromosome that produces tail deformities and, rarely, open neural tube defects. Analysis of 292 normal male and affected male and female progeny from an intraspecific back-cross involving BN: supports a gene order of cen-DXMit89-18.5 +/- 2.3 cM-DXMit166-1.4 +/- 0.7 cM-BN:-1.0 +/- 0.6 cM-DXMit140 -4.8 +/- 1.3 cM-DXBay6-tel. A high frequency of sex chromosomal non-disjunction, unrelated to the BN: mutation, was also identified in the background strain. Refined genetic and physical mapping of the BN: critical region demonstrate that the mutation is associated with a

Mice homozygous for the transgenic insertion in line OVE250 exhibit severe progressive glomerulonephritis. Ultrastructural changes in the glomerular basement membrane (GBM) at 2 weeks of age resemble those in Alport syndrome. The transgenic insertion site was mapped by FISH to mouse chromosome 1 close to Pax3. Genetic and molecular analyses identified a deletion of genomic DNA at the transgene insertion site. Exons 1 through 12 of the collagen IV gene Col4a4, exons 1 and 2 of the adjacent Col4a3 gene, and the intergenic promoter region are deleted. Transcripts of Col4a3 and Col4a4 are undetectable in mutant kidney, and both proteins are missing from the GBM. Persistent cellular proliferation in mutant kidneys suggests that interaction with the extracellular matrix may be important for cell maturation. Evolutionarily conserved sequence elements in the promoter regions of human and mouse Col4a3 and Col4a4 include a 19-bp element that was tandemly duplicated in the human lineage and a CTC box element common to several genes encoding extracellular matrix proteins. This new animal model of Alport syndrome, Col4Delta3-4, lacks both alpha3 and alpha4 chains of collagen IV and exhibits an earlier disease onset than mice lacking alpha3 only.

Carnitine palmitoyltransferase (CPT)-I catalyses the transfer of long-chain fatty acids from CoA to carnitine for translocation across the mitochondrial inner membrane. Expression of the 'liver' isoform of the CPT-I gene (CPT-Ialpha) is subject to developmental, hormonal and tissue-specific regulation. To understand the basis for control of CPT-Ialpha gene expression, we have characterized the proximal promoter of the CPT-Ialpha gene. Here, we report the sequence of 6839 base pairs of the promoter and the localization of the rat CPT-Ialpha gene to region q43 on chromosome 1. Our studies show that the first 200 base pairs of the promoter are sufficient to drive transcription of the CPT-Ialpha gene. Within this region are two sites that bind both Sp1 and Sp3 transcription factors. In addition, nuclear factor Y (NF-Y) binds the proximal promoter. Mutation at the Sp1 or NF-Y sites severely decreases transcription from the CPT-Ialpha promoter. Other protein binding sites were identified within the first 200 base pairs of the promoter by DNase I footprinting, and these elements contribute to CPT-Ialpha gene expression. Our studies demonstrate that CPT-Ialpha is a TATA-less gene which utilizes NF-Y and Sp proteins to drive basal expression.

Two frequent protein variants of glutamate pyruvate transaminase (GPT) (E.C.2.6.1.2) have been used as genetic markers in humans for more than two decades, although chromosomal mapping of the GPT locus in the 1980s produced conflicting results. To resolve this conflict and develop useful DNA markers for this gene, we isolated and characterized cDNA and genomic clones of GPT. We have definitively mapped human GPT to the terminus of 8q using several methods. First, two cosmids shown to contain the GPT sequence were derived from a chromosome 8-specific library. Second, by fluorescencein situhybridization, we mapped the cosmid containing the human GPT gene to chromosome band 8q24.3. Third, we mapped the ratgptcDNA to the syntenic region of rat chromosome 7. Finally, PCR primers specific to human GPT amplify sequences contained within a “half-YAC” from the long arm of chromosome 8, that is, a YAC containing the 8q telomere. The human GPT genomic sequence spans 2.7 kb and consists of 11 exons, ranging in size from 79 to 243 bp. The exonic sequence encodes a protein of 495 amino acids that is nearly identical to the previously reported protein sequence of human GPT-1. The two polymorphic GPT isozymes are the result of a nucleotide substitution in codon 14, coding for a histidine in GPT-1 and an asparagine in GPT-2, which causes a gain or loss of anNlaIII restriction site. In addition, a cosmid containing the GPT sequence also contains a previously unmapped, polymorphic microsatellite sequence, D8S421. The cloned GPT gene and associated polymorphisms will be useful for linkage and physical mapping of disease loci that map to the terminus of 8q, including atypical vitelliform macular dystrophy (VMD1) and epidermolysis bullosa simplex, type Ogna (EBS1). In addition, this will be a useful system for characterizing the telomeric region of 8q. Finally, determination of the molecular basis of the GPT isozyme variants will permit PCR-based detection of this worldwide polymorphism.

We report on familial occurrence of the Brachmann-de Lange syndrome (BDLS): a mildly affected father and his severely affected son and daughter who have different mothers. Both children are severely affected while the father has a much milder but definite BDLS phenotype. Our report documents the third example of male-to-male transmission and adds to the argument against exclusively maternal transmission in familial cases. In addition, our findings illustrate the occurrence of severe manifestations in cases of familial BDLS.

Pseudoachondroplasia (PSACH) is a well characterized dwarfing condition mapping to chromosome 19p12-13.1. Cartilage oligomeric matrix protein (COMP), a cartilage specific protein, maps to the same location within a contig that spans the PSACH locus. Using single strand conformation polymorphism (SSCP) analysis and nucleotide sequencing we have identified COMP mutations in eight familial and isolated PSACH cases. All mutations involve either a single base-pair change or a three base-pair deletion in exon 17B. Six mutations delete or change a well conserved aspartic acid residue within the calcium-binding type 3 repeats. These results demonstrate that mutations in the COMP gene cause pseudochondroplasia.

A recessive mutation was identified in a family of transgenic mice that resulted in a reversal of left-right polarity (situs inversus) in 100 percent of the homozygous transgenic mice tested. Sequences that flanked the transgenic integration site were cloned and mapped to mouse chromosome 4, between the Tsha and Hxb loci. During early embryonic development, the direction of postimplantation turning, one of the earliest manifestations of left-right asymmetry, was reversed in homozygous transgenic embryos. This insertional mutation identifies a gene that controls embryonic turning and visceral left-right polarity.

We hypothesized that, in esophageal squamous epithelial cells, there are differences among individuals in the signal transduction pathways activated by acid reflux that might underlie the development of Barrett's esophagus. To explore that hypothesis, we immortalized nonneoplastic, esophageal squamous cells from patients with gastroesophageal reflux disease (GERD) with (NES-B3T) and without (NES-G2T) Barrett's esophagus and used those cells to study acid effects on MAPK proteins. During endoscopy in patients with GERD with and without Barrett's esophagus, we took biopsy specimens from the distal squamous esophagus to study MAPK proteins before and after esophageal perfusion with 0.1 N HCl. We used immunoblotting and Western blotting to study MEK1/2 phosphorylation at two activating sites (serines 217/221), MEK1 phosphorylation at an inhibitory site (threonine 286), and MEK1/2 activity. After acid exposure, both cell lines exhibited increased MEK1/2 phosphorylation at the activating sites; the NES-B3T cells had higher levels of MEK1 phosphorylation at the inhibitory site, however, and only the NES-G2T cells showed an acid-induced increase in MEK1/2 activity. Similarly, in the squamous epithelium of patients with GERD with and without Barrett's esophagus, acid perfusion increased MEK1/2 phosphorylation at the activating sites in both patient groups; the Barrett's patients had higher levels of MEK1 phosphorylation at the inhibitory site, however, and only the patients without Barrett's demonstrated an acid-induced increase in ERK1/2 phosphorylation. In esophageal squamous cell lines and biopsies from patients with GERD with and without Barrett's esophagus, we have found differences in MAPK pathways activated by acid exposure. We speculate that these differences might underlie the development of Barrett's metaplasia.

Congenital heart disease (CHD) is the most common type of birth defect, and the etiology of most cases is unknown. CHD often occurs in association with other birth malformations, and only in a minority are disease-causing chromosomal abnormalities identified. We hypothesized that children with CHD and additional birth malformations have cryptic chromosomal abnormalities that might be uncovered using recently developed DNA microarray-based methodologies. We recruited 20 children with diverse forms of CHD and additional birth defects who had no chromosomal abnormality identified by conventional cytogenetic testing. Using whole-genome array comparative genomic hybridization, we screened this population, along with a matched control population with isolated heart defects, for chromosomal copy number variations. We discovered disease-causing cryptic chromosomal abnormalities in five children with CHD and additional birth defects versus none with isolated CHD. The chromosomal abnormalities included three unbalanced translocations, one interstitial duplication, and one interstitial deletion. The genetic abnormalities were predominantly identified in children with CHD and a neurologic abnormality. Our results suggest that a significant percentage of children with CHD and neurologic abnormalities harbor subtle chromosomal abnormalities. We propose that children who meet these two criteria should receive more extensive genetic testing to detect potential cryptic chromosomal abnormalities.

Phenotypically healthy carriers of the balanced 11;22 translocation, the most frequent non-Robertsonian constitutional translocation known in human beings, are at risk of having a progeny with supernumerary derivative (22)t(11;22) syndrome [der(22) syndrome]. We present the cases of 2 male patients with supernumerary der(22) syndrome [47,XY,+der(22)t(11;22)(q23;q11.2)mat], yielding partial trisomy for 22pter-q11 and 11q23-qter. These cases expand the phenotype of the der(22) syndrome, with the first case highlighting the phenotypic overlap of VACTERL and the second adding Hirschsprung's disease and intestinal malrotation to the list of associated anorectal anomalies. Because der(22) syndrome and cat eye syndrome (partial tetrasomy of 22q11) share a similar region of extra dosage on 22q11 and both typically manifest an anorectal phenotype, a dosage-sensitive gene for anorectal anomalies may be present in this locus.

Background Turner syndrome (TS) is associated with a neurocognitive phenotype that includes selective nonverbal deficits, e.g., impaired visual-spatial abilities. We previously reported evidence that this phenotype results from haploinsufficiency of one or more genes on distal Xp. This inference was based on genotype/phenotype comparisons of individual girls and women with partial Xp deletions, with the neurocognitive phenotype considered a dichotomous trait. We sought to confirm our findings in a large cohort (n = 47) of adult women with partial deletions of Xp or Xq, enriched for subjects with distal Xp deletions. Methods Subjects were recruited from North American genetics and endocrinology clinics. Phenotype assessment included measures of stature, ovarian function, and detailed neurocognitive testing. The neurocognitive phenotype was measured as a quantitative trait, the Turner Syndrome Cognitive Summary (TSCS) score, derived from discriminant function analysis. Genetic analysis included karyotyping, X inactivation studies, fluorescent in situ hybridization, microsatellite marker genotyping, and array comparative genomic hybridization. Results We report statistical evidence that deletion of Xp22.3, an interval containing 31 annotated genes, is sufficient to cause the neurocognitive phenotype described by the TSCS score. Two other cardinal TS features, ovarian failure and short stature, as well as X chromosome inactivation pattern and subject's age, were unrelated to the TSCS score. Conclusion Detailed mapping suggests that haploinsufficiency of one or more genes in Xp22.3, the distal 8.3 megabases (Mb) of the X chromosome, is responsible for a TS neurocognitive phenotype. This interval includes the 2.6 Mb Xp-Yp pseudoautosomal region (PAR1). Haploinsufficiency of the short stature gene SHOX in PAR1 probably does not cause this TS neurocognitive phenotype. Two genes proximal to PAR1 within the 8.3 Mb critical region, STS and NLGN4X, are attractive candidates for this neurocognitive phenotype.

We present a case of a liveborn male with complete trisomy 1q in mosaic form due to a de novo unbalanced translocation. There are seven previously documented cases of complete trisomy for 1q, which demonstrate that this is a lethal condition. All cases have similar phenotype including weights greater than 50th centile for gestational age, hydrocephalus, microphthalmia, abnormal ears, small mouth or jaw, and abnormal fingers. Single umbilical artery, imperforate anus, and dysplastic kidneys are also seen in more than one patient. Five of the eight translocation cases have identical chromosomal breakpoints involving 1q and Yq. This suggests the possibility of sequence similarities on these two chromosomes as has been documented with several other recurrent chromosomal rearrangements. Further, this case demonstrates the need for postnatal genetics evaluation following prenatal diagnosis. In postnatal testing, the aneuploidy could not be demonstrated in metaphase cells from cultured lymphocytes. More detailed testing prompted by abnormal amniocentesis and neonatal dysmorphology was necessary to confirm the cytogenetic diagnosis. Without the prenatal diagnosis, it is likely that the true cytogenetic aberration would have gone undetected.

Klinefelter syndrome (KS; 47,XXY karyotype and variants) is characterized by tall stature and testicular failure, with marked variation in severity of the phenotype. Previous studies have proposed that genetic factors including mosaicism, parental origin of the supernumerary X-chromosome, skewed X inactivation, and androgen receptor (AR) polyglutamine repeat length may contribute to phenotypic variability in KS.The objective of this study was to investigate the roles of these genetic factors in the variability of the KS phenotype.This was a cross-sectional study.The study was performed at a pediatric endocrinology referral clinic.Thirty-five KS boys and men, aged 0.1-39 yr, were studied.There were no interventions.Auxological measurements, biological indices of testicular function, and clinical assessment of muscle tone were the main outcome measures. Genetic studies included karyotyping to detect mosaicism, genotyping of microsatellite markers to determine parental origin of the supernumerary X-chromosome, and genotyping and methylation studies to measure AR polyglutamine (AR CAGn) repeat length and X inactivation ratio.The only genetic factor that significantly influenced the KS phenotype was the AR CAGn repeat length, which was inversely correlated with penile length, a biological indicator of early androgen action. Mosaicism, imprinting, and skewed X inactivation did not account for the variability of the KS phenotype.Normal genetic variation in the AR coding sequence may be clinically significant in the setting of early testicular failure and subnormal circulating testosterone levels, as occur in KS.

Background/Aims: Klinefelter syndrome (KS) is characterized by the karyotype 47,XXY. In this study, we evaluated the physical and testicular failure phenotypes of infants and young boys with KS. Methods: The evaluation included auxologic measurements, biologic indices of testicular function, and clinical assessment of muscle tone in 22 infants and young boys with KS, ages 1–23 months. Results: Mean length, weight, and head circumference in SDS were generally within the normal range at –0.3 ± 1.0, –0.1 ± 1.4, and 0.0 ± 1.5, respectively. Mean penile length and testicular volume SDS were –0.9 ± 0.8 and –1.1 ± 0.8, indicating significantly reduced penile and testicular size. Mean testosterone levels for the boys ≤6 and >6–23 months were 128 ± 131 (4.4 ± 4.5 nmol/l) and 9.5 ± 7.2 ng/dl (0.3 ± 0.2 nmol/l), respectively. High-arched palate was observed in 6/17 boys and clinodactyly (5th finger) was observed in 15/16 boys. Hypotonia was evaluated clinically and was noted to be present in 12/17 boys. Conclusion: The physical phenotype in infants and young boys with KS (1–23 months old) includes normal auxologic measurements and early evidence of testicular failure. Muscle tone was decreased in most of the boys. Testicular volume and penile length were diminished, indicating early androgen deficiency. The neonatal surge in testosterone was attenuated in our KS population. Thus, infants and young boys with KS have evidence of early testicular failure. The etiology of this failure and the clinical role of early androgen replacement require further study.

Telomere shortening is used for replicative aging in primates and ungulates but not rodents. We examined telomere biology in rabbits to expand the comparative biology of telomere-directed replicative senescence within mammals. The order Lagomorpha consists of two families; Leporidae and Ochotonidae. We examined telomere biology in species representing three leporid genera (European White Rabbit, Black-tailed Jack Rabbit, and Swamp Rabbit) and the monotypic ochotonid genus (North American Pika). Of the leporids one species was a laboratory strain and the others were wild caught. The leporids neither exhibited cellular senescence after sustained periods in culture nor displayed detectable telomerase activity. Continued culture was possible because of their extremely long telomeric arrays. Immunofluorescence showed robust telomere signals at chromosome ends and significant internal chromosomal staining in some instances. Pika was unique in displaying endogenous telomerase activity throughout time in culture. These results show that it is unlikely that lagomorphs use telomere shortening and replicative senescence as a tumor protective mechanism.

The SHOX (short-stature homeobox-containing) gene encodes isoforms of a homeodomain transcription factor important in human limb development. SHOX haploinsufficiency has been implicated in three human growth disorders: Turner syndrome, idiopathic short stature, and Leri-Weill dyschondrosteosis. Langer mesomelic dysplasia is thought to be the homozygous form of dyschondrosteosis. However, complete SHOX deficiency has not been demonstrated for any postnatal patient with the classic Langer phenotype. We studied four adults and one child with Langer mesomelic dysplasia. SHOX abnormalities were detected in all five probands. One was a homozygote or hemizygote and two were compound heterozygotes. The homozygous or hemizygous mutation was in exon 6a, implying that the SHOXa isoform is essential for normal skeletal development. These findings confirm clinical inferences that Langer mesomelic dysplasia is the homozygous form of Leri-Weill dyschondrosteosis and add to our understanding of genotype/phenotype relationships in SHOX deficiency disorders. © 2002 Wiley-Liss, Inc.

STSs designed to the retina/pineal-expressed EST clusters THC220430 and THC90422 were originally mapped to 17p13.3 (ref. 7) near a retinitis pigmentosa (RP13) candidate region8. Further testing refined the localization to 17p13.1, between SHGC-2251 and SHGC-6095, within the LCA4 candidate region and approximately 2.5 Mb distal to GUCY2D. Fluorescence in situ hybridization (Fig. 1) confirmed the localization. Fig. 1 Fluorescence in situ hybridization (FISH). AIPL1-containing bacterial artificial chromosome (BAC), shown in red, hybridizes to 17p13.1, consistent with placement of AIPL1 in the Stanford G3 radiation hybrid panel. These data refute the original placement ... cDNA sequencing of the two clusters indicated that the ESTs represent transcripts of one gene. THC90422 transcripts bypass the THC220430 polyadenylation signal, resulting in a 3′ UTR longer by 709 bp. The 180-bp 5′ UTR and coding sequence encoded by the six-exon gene are identical in the 1,538-bp and 2,247-bp transcripts (Fig. 2a). Fig. 2 Gene and protein structure of AIPL1. a, AIPL1 consists of six exons, with alternate polyadenylation sites in the 3′ UTR shown by arrows. Cys239Arg denotes the location of the TGC→CGC missense mutation in exon 5 of the RFS128 family. Trp278X ... The protein encoded by AIPL1 was named human aryl hydrocarbon receptor-interacting protein-like 1 (AIPL1) due to its similarity (49% identity, 69% positive) to human aryl hydrocarbon receptor-interacting protein (AIP), a member of the FK506-binding protein (FKBP) family9 (Fig. 2b). The predicted protein consists of 384 amino acids, with a 43,865-dalton molecular mass, and a 5.57 pI. The protein sequence includes three tetratricopeptide repeats (TPR), a 34–amino-acid motif found in proteins with nuclear transport or protein chaperone activity9. Northern-blot hybridization identified mRNA molecules of the predicted sizes in total retinal RNA. The probe also cross-hybridized to 18s rRNA (Fig. 3) in the retina. We detected a weaker signal in skeletal muscle and heart on a poly(A)+ RNA multi-tissue northern blot after very long exposure. It is likely that this signal represents cross-hybridization, as the transcripts differ in size from the retinal mRNAs and are faint. The northern-blot did not indicate AIPL1 expression in brain, but only cerebral tissue was included in the blot. In situ hybridization indicates expression in rat and mouse pineal gland, a high level of expression in adult mouse photoreceptors (Fig. 4) and no expression in cornea (data not shown). Fig. 3 Expression of AIPL1 in human tissues. Northern blots from adult tissues were incubated with an AIPL1 probe. Total retinal RNA blot (top left) and poly(A)+ RNA multi-tissue northern (MTN, top right) are shown. No signal was observed in MTN at 4-, 24- or ... Fig. 4 Retina and pineal expression of Aipl1. a, Digoxygenin in situ hybridization of Aipl1 in adult mouse retina, with expression throughout the outer nuclear layer and photoreceptor inner segments. b, Sense control of (a) with same reaction time. A slight ... Sequencing of the rat Aipl1 cDNA revealed amino acid sequence conservation (87% identity and 96% similarity) between rat and human AIPL1. Rat Aipl1, mouse Aip and human AIP lack a 56–amino-acid carboxy-terminal extension present in AIPL1 (Fig. 2b). This extension includes a ‘hinge’ motif of high flexibility, with multiple O-glycosylation sites, and a casein kinase II (CK2) phosphorylation site, which may be involved in protein complex regulation (as is the CK2 site within the hinge of another FKBP family member, FKBP52; ref. 10). The hinge appears to be conserved in primates, as it is also present in the squirrel monkey (Saimiri sciureus; data not shown). Single-stranded conformational analysis (SSCA) identified three benign nucleotide substitutions within the AIPL1 exon 3 amplimer: G/A at −14, G/A at −10 bp and G/A at codon 100 (Leu100Leu, CTG/CTA). We identified four haplotypes for the combined polymorphisms; the most common, GCG and GAA, have frequencies of 55% and 41%, respectively. Sequencing of AIPL1 from the DNA of one affected individual of the original LCA4 family (Fig. 5a) revealed a homozygous nonsense mutation (Trp278X, TGG→TGA). This allele, if expressed, encodes a protein shorter by 107 amino acids than wild-type AIPL1. The truncated protein includes only 20 of the 34 amino acids of the third TPR motif, a region conserved between human, rat and mouse AIPL1, and AIP. SSCA in other family members confirmed that all affected family members are homozygous for this mutation (Fig. 5a) and that 100 ethnically matched controls did not carry this mutation. Fig. 5 Pedigrees and mutation screen of AIPL1 in families. a, The Trp278X mutation is homozygous in three families: KC, MD and RFS127. SSCA of all living individuals of the KC pedigree demonstrate segregation of the mutant allele. Top electropherogram, an unaffected ... AIPL1 was next analysed in another Pakistani family, MD (Fig. 5a), whose LCA had been mapped to 17p13.1, with GUCY2D excluded by mutational analysis. Sequencing of AIPL1 indicated that affected individuals of this family are homozygous for the Trp278X mutation (Fig. 5a). The MD and KC families differ in haplotype (GCG and GAA, respectively) of the AIPL1 exon 3 polymorphisms, as well as for microsatellite markers tightly linked to AIPL1. These results suggest that the Trp278X mutations causing the LCA in these two families are not derived from a recent, common ancestor. Assay of AIPL1 in 14 families of European descent with LCA that had not been tested previously for linkage to 17p identified apparent disease-causing mutations in three additional families, as follows. Direct sequencing of AIPL1 in the two affected individuals of family RFS121 indicated two mutations, a 2-bp deletion in codon 336 (Ala336Δ2 bp; Fig. 5b) and Trp278X. The deletion results in a frameshift and a termination delayed by 47 codons. The termination signal used in the deletion transcript is upstream of the first AIPL1 polyadenylation signal; therefore, the alternate transcripts from this allele are not predicted to encode alternate proteins. Allele-specific PCR in one affected individual confirmed that the 2-bp deletion and Trp278X are on opposite chromosomes. Therefore, the affected individuals in RFS121 are compound heterozygotes, having received the Trp278X mutation from one parent and the Ala336Δ2 mutation from the other. No unaffected RFS121 family members inherited both mutations. The Ala336Δ2 bp mutation was not observed in 55 unrelated control individuals of European descent. AIPL1 sequencing in two affected individuals from family RFS127 (Fig. 5a) indicated homozygous Trp278X mutations—the same mutation identified in the KC and MD families. Haplo-type analysis of tightly linked microsatellite markers and of the AIPL1 exon 3 polymorphisms suggest that the mutations in the RFS127 and MD families are likely to have descended from a common ancestor; however, there is no indication of Pakistani origin for members of this family. The three affected individuals of family RFS128 (Fig. 5c) are homozygous for a T→C nucleotide substitution predicted to encode a Cys239Arg substitution. This cysteine is conserved in human and rat AIPL1, and in AIP (Fig. 2). This mutation was not identified in over 55 ethnically matched control individuals. Affected members of this family are homozygous for microsatellite markers D17S796 and D17S1881, which are tightly linked, flanking markers of AIPL1. In contrast, affected family members are heterozygous for microsatellite markers D17S960 and D17S1353, which flank GUCY2D. We have identified a new gene that causes LCA4. We detected homozygous AIPL1 mutations in three families in which GUCY2D was excluded as the cause of the disease by linkage or mutation screening: KC, MD and RFS128. AIPL1 is the fourth gene to be associated with LCA. Mutations in AIPL1 may be a common cause of LCA, as an AIPL1 mutation was identified as the apparent cause of the retinal disease in 3 of 14 (21±8%, 90% C.I.) unmapped LCA families. AIPL1 should be assayed in LCA families whose disease locus maps to 17p13 but do not carry disease-causing mutations in GUCY2D, as in 7 of 15 original LCA1 families4. Due to the proximity of AIPL1 and GUCY2D on 17p13, linkage mapping may not distinguish between the genes. Further, is possible that LCA patients who are identical by descent (IBD) at one locus are also IBD at the other. Therefore, both AIPL1 and GUCY2D should be screened for mutations in families whose LCA locus maps to 17p13 or in families with affected individuals who are homozygous for mutations in either gene, unless linkage excludes one of the genes. Of the five families reported here, GUCY2D was excluded by linkage testing or mutation screening in three, the fourth is a compound heterozygote and the fifth is homozygous for a disease-causing mutation confirmed in other families. The similarity of AIPL1 to AIP and the presence of three TPR motifs suggest that it may be involved in retinal protein folding or trafficking. Its role in the pineal gland is also uncertain. The pineal gland contributes to resetting circadian rhythm by diurnal release of melatonin. Additionally, children with destructive pinealomas often display precocious puberty, suggesting a role in long-term periodicity11. Because LCA patients with AIPL1 mutations have grossly abnormal photoreceptors at an early age, the pineal gland also may be affected. Careful clinical characterization of LCA4 patients may reveal pineal-associated abnormalities. Therefore, identifying the exact role of AIPL1 in photoreceptors and the pineal gland will improve our understanding of disease pathology in these patients, and contribute to our understanding of the biology of normal vision and pineal activity.

Visceral left-right asymmetry occurs in all vertebrates, but the inversion of embryo turning (inv) mouse, which resulted following a random transgene insertion, is the only model in which these asymmetries are consistently reversed. We report positional cloning of the gene underlying this recessive phenotype. Although transgene insertion was accompanied by neighbouring deletion and duplication events, our YAC phenotype rescue studies indicate that the mutant phenotype results from the deletion. After extensively characterizing the 47-kb deleted region and flanking sequences from the wild-type mouse genome, we found evidence for only one gene sequence in the deleted region. We determined the full-length 5.5-kb cDNA sequence and identified 16 exons, of which exons 3-11 were eliminated by the deletion, causing a frameshift. The novel gene specifies a 1062-aa product with tandem ankyrin-like repeat sequences. Characterization of complementing and non-complementing YAC transgenic families revealed that correction of the inv mutant phenotype was concordant with integration and intact expression of this novel gene, which we have named inversin (Invs).

Carnitine palmitoyltransferase I (CPT-I) is a key enzyme involved in the regulation of fatty acid oxidation. CPT-IA and CPT-IB are isoforms of carnitine palmitoyltransferase I, of which CPT-IA is expressed in liver, kidney, fibroblasts, and heart and CPT-IB is expressed in skeletal muscle, heart, brown and white adipocytes, and testes. Although the genomic DNA sequence of human CPT-IB is available, the transcription start site and upstream regulatory sequences are not known. For rat CPT-IB, only the cDNA sequence has been published. We have cloned the entire rat CPT-IB gene from a Lambda Fix II rat kidney genomic library. The genomic structure contains 19 exons, with the transcription start site for CPT-IB located in a short first exon, which is a 13-bp extension to the previously published cDNA 5′ sequence. The coding sequence is identical with the rat muscle cDNA. The rat CPT-IB gene contains 18 introns and 19 exons, the latter 18 exons showing 85% homology to the human CPT-IB cDNA. CPT-IB maps to rat chromosome 7 at band q34. A putative promoter region was identified to within 391 bp of the transcription start site. The muscle specificity of the 5′ flanking region was verified by comparison of luciferase expression to that of β-galactosidase in cardiac myocytes and in HepG2 cells.

SummaryDeletions of the distal short arm of chromosome 1 (1p36) represent a common, newly delineated deletion syndrome, characterized by moderate to severe psychomotor retardation, seizures, growth delay, and dysmorphic features. Previous cytogenetic underascertainment of this chromosomal deletion has made it difficult to characterize the clinical and molecular aspects of the syndrome. Recent advances in cytogenetic technology, particularly FISH, have greatly improved the ability to identify 1p36 deletions and have allowed a clearer definition of the clinical phenotype and molecular characteristics of this syndrome. We have identified 14 patients with chromosome 1p36 deletions and have assessed the frequency of each phenotypic feature and clinical manifestation in the 13 patients with pure 1p36 deletions. The physical extent and parental origin of each deletion were determined by use of FISH probes on cytogenetic preparations and by analysis of polymorphic DNA markers in the patients and their available parents. Clinical examinations revealed that the most common features and medical problems in patients with this deletion syndrome include large anterior fontanelle (100%), motor delay/hypotonia (92%), moderate to severe mental retardation (92%), growth delay (85%), pointed chin (80%), eye/vision problems (75%), seizures (72%), flat nasal bridge (65%), clinodactyly and/or short fifth finger(s) (64%), low-set ear(s) (59%), ear asymmetry (57%), hearing deficits (56%), abusive behavior (56%), thickened ear helices (53%), and deep-set eyes (50%). FISH and DNA polymorphism analysis showed that there is no uniform region of deletion but, rather, a spectrum of different deletion sizes with a common minimal region of deletion overlap.

An infant had clinical signs suggestive of Hirschsprung disease as the initial manifestation of leukocyte adhesion deficiency. Chromosome studies showed a deletion of the distal third of the long arm of one chromosome 21, and flow cytometric studies confirmed the defective expression of CD18.

The objective of this project was to assess the efficacy of fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes to identify chromosome number in formalin-fixed, paraffin-embedded placental specimens. Using this approach, 75 per cent of the karyotypes in 20 formalin-fixed placental samples (comprising aneuploids, triploids, and normals) were correctly identified. As this technology improves, the ability to obtain information regarding chromosomal abnormalities in formalin-fixed, paraffin-embedded placental tissue should improve as well. This technology can potentially provide important cytogenetic information even when fresh tissue is not available for standard karyotypic analysis.

A monoclonal human B-lymphoblastoid cell line (UTMB-460) arose spontaneously from the bone marrow of a normal healthy woman who was seropositive for an EB-virus infection. Chromosomally, the UTMB-460 cells are near tetraploid, with a specific translocation (8;9) (p11.2; p24), and have surface IgMk. The UTMB-460 cells are resistant to killing in vitro by spontaneous and rIFNα2 and rIL-2 stimulated NK cells from the patient and other normal subjects, but are killed by lymphokine activated killer cells. The index patient has not developed leukemia/lymphoma during the follow-up interval of 22 months. The growth of UTMB-460 cells is supported by undefined growth factors in FCS and by BCGF in the absence of FCS. rIL-2 stimulates DNA synthesis by UTMB-460 cells. The UTMB-460 cells were adherent to the normal MSC in the primary culture and show specific heterotypic adherence to normal MSC when compared to skin fibroblasts. In addition, 6/6 normal marrow stromal cells and 4/6 normal skin fibroblasts induced growth of colonies from UTMB-460 cells. These data suggest that MSC interacted with the transformed cells (UTMB-460) in vitro and played a critical role in the establishment of the UTMB-460 cell line.

Translocation (4;11)(q21;q23) is characteristic of a distinct acute leukemic syndrome. We report an 8-wk-old male patient with the clinical features ascribed to t(4;11), but with an unusual chromosome rearrangement consisting of an insertion of the 11q23 band into the q21 region of chromosome #4.

Recently, i(8q) was suggested to be a nonrandom chromosomal abnormality characteristic of adenocarcinoma of the lung. To further investigate this observation, a chromosomal analysis of five cases of pleural effusions representing metastatic adenocarcinoma from different primary sites (two lung, two breast, and one stomach) was undertaken. The i(8q) occurred in three of the tumors, one from each of the three different primary sites. In addition, abnormalities of the short arm of chromosome 3 and extra copies of chromosome 7, both of which have been associated with adenocarcinoma of the lung, were simultaneously present in the same three tumors. Our findings demonstrate that i(8q) is not specific for adenocarcinoma of the lung and that it may have a role in the pathogenesis of adenocarcinomas from multiple organs. The simultaneous presence of i(8q), abnormalities of 3p, and extra copies of chromosome 7 may indicate a relationship among these abnormalities in multistep carcinogenesis or the development of metastatic potential.

the dermal microcirculation, which are influenced by the autonomic sympathetic nervous system, s,9 Skin hyperthermia caused by a direct noxious injury or a local reflex caused by an inflammatory reaction occurring deeper in the extremity can be detected easily and imaged by thermography)'9 This localized thermographic abnormality is distinct from the pattern seen in patients with RSD, which consists of difl'use thermal alterations in the extremities in a "glove and stocking" pattern, s, 9 Relative coldness is the most common manifestation. However, for unknown reasons, an affected hand or foot may be hyperthermic) ,9 These changes have been related to sympathetic vasoconstriction and to compensatory and rebound vasodilation of skin capillaries, respectively. 6,~,9 The RSD was characterized by hypothermia in the majority of our patients, although hyperthermia was also seen. Both hypothermic and hyperthermic reactions are abnormal and may represent different stages of the disease, because there should be no significant temperature differential between normal extremities.6.8-/0 Thermography is a noninvasive technique, without known biologic hazards, that readily confirms the diagnosis of RSD by graphically documenting the subjective complaint of pain. It therefore facilitates appropriate therapy, preventing permanent disability. We have shown that results parallel therapeutic response and may be useful in evaluating and monitoring responses to various therapies. R E F E R E N C E S 1. Bernstein BH, Singsen BH, Kent JT, et al. Reflex neurovascular dystrophy in childhood. J PEDIATR 1978;93:211. 2. Fermaglich DR. Reflex sympathetic dystrophy in children. Pediatrics 1977;60:881. 3. Jaeobs JC. Pediatric rheumatology for the practitioner. New York: Springer-Verlag, 1982:165-7. 4. Kozin F, McCarty D J, Sims J~ et al. The reflex sympathetic dystrophy syndrome. I. Clinical and histologic studies: evidence for bilaterality, response to eorticosteroids and articular involvement. Am J Med 1976;60:321. 5. Kozin FF, Ryan LM, Carerra GF, et al. The reflex sympathetic dystrophy syndrome (RSDS). III. Scintigraphic studies: further evidence for the therapeutic efficacy of systemic corticosteroids, and proposed diagnostic criteria. Am J Med 1981;70:23. 6. Hendler N, Uematsu S, Long D. Thermographic validation of physical complaints in psychogenic pain patients. Psychosomatics 1982;23:283. 7. Laxer RM, Malleson RM, Morrison RT. Technetium 99m-methylene diphosphate bone scans in children with reflex neurovascular dystrophy. J PED~ATR 1985;106:437. 8. Pochaczevsky R, Wexler CE, Meyers PH, et al. Liquid crystal thermography of the spine and extremities: its value in the diagnosis of spinal root syndromes. J Neurosurg 1982; 56:386. 9. Pochaczevsky R. Thermography in skeletal and soft tissue trauma. In: Taveras JM, Ferrucei F, Norman A, eds. Radiology: diagnostic imaging and intervention, vol 5. Philadelphia: JB Lippincott, 1987:1-7. 10. Feldman F, Niekoloff EL. Normal thermographic standards for the cervical spine and upper extremities. Skeletal Radiol 1984;12:235.