What causes AIS? Ask the genome!

The most common developmental disorder of the spine is scoliosis, a rotated, lateral deformity in the shape of the spinal column. Scoliosis may be part of the clinical spectrum that is observed in many developmental disorders, but typically presents as an isolated symptom in otherwise healthy adolescent children. Adolescent idiopathic scoliosis (AIS) has defied pathogenic understanding in part due to its genetic complexity, and to the lack of well-defined animal models. The disease is also remarkable in its sexual dimorphism, where girls are at more than five times greater risk of progressive deformity than boys. Breakthroughs have come from recent genome wide association studies (GWAS) and next generation sequencing (NGS) of human AIS cohorts. Post-hoc gene set and pathway-level analyses of genetic datasets have highlighted a role for cartilage biogenesis and the development of the intervertebral disc (IVD) in disease susceptibility. Moreover, next generation sequencing in AIS families, as well as modeling in vertebrate systems, has revealed that rare deficiencies in proteins of the cartilaginous extracellular matrix (ECM) collectively contribute to AIS. Thus, as in a jigsaw puzzle, the pieces coming together from multiple biologic studies suggest that deficiencies in the structural integrity and homeostasis of spinal cartilages are culprits in AIS susceptibility. Here, we update progress in understanding the genetic, biochemical, and cellular determinants of AIS. We also suggest a molecular model in which interaction of the hormonal environment with genetic susceptibility may increase risk of this common disorder of childhood.

Fine-mapping the MHC locus in juvenile idiopathic arthritis (JIA) reveals genetic heterogeneity corresponding to distinct adult inflammatory arthritic diseases.

OBJECTIVES: Juvenile idiopathic arthritis (JIA) is a heterogeneous group of diseases, comprising seven categories. Genetic data could potentially be used to help redefine JIA categories and improve the current classification system. The human leucocyte antigen (HLA) region is strongly associated with JIA. Fine-mapping of the region was performed to look for similarities and differences in HLA associations between the JIA categories and define correspondences with adult inflammatory arthritides. METHODS: Dense genotype data from the HLA region, from the Immunochip array for 5043 JIA cases and 14 390 controls, were used to impute single-nucleotide polymorphisms, HLA classical alleles and amino acids. Bivariate analysis was performed to investigate genetic correlation between the JIA categories. Conditional analysis was used to identify additional effects within the region. Comparison of the findings with those in adult inflammatory arthritic diseases was performed. RESULTS: We identified category-specific associations and have demonstrated for the first time that rheumatoid factor (RF)-negative polyarticular JIA and oligoarticular JIA are genetically similar in their HLA associations. We also observe that each JIA category potentially has an adult counterpart. The RF-positive polyarthritis association at HLA-DRB1 amino acid at position 13 mirrors the association in adult seropositive rheumatoid arthritis (RA). Interestingly, the combined oligoarthritis and RF-negative polyarthritis dataset shares the same association with adult seronegative RA. CONCLUSIONS: The findings suggest the value of using genetic data in helping to classify the categories of this heterogeneous disease. Mapping JIA categories to adult counterparts could enable shared knowledge of disease pathogenesis and aetiology and facilitate transition from paediatric to adult services.

Mutations responsible for Larsen syndrome cluster in the FLNB protein.

BACKGROUND: A gene for Larsen syndrome was recently described, and mutations were reported in five cases. OBJECTIVE: To test whether mutations in this gene, FLNB, could explain the disease in our independent collection of sporadic and dominant Larsen syndrome cases; and to test whether mutations occurred in a non-random pattern. RESULTS: Missense mutations were found in each of five cases. Four of the five were new; one was reported in a sporadic case in the original Larsen syndrome study of five cases. All mutations from the two studies were compiled. Clustered mutations were observed within three filamin B protein domains: the calponin homology 2 domain, repeat 14, and repeat 15. This suggested that as few as five (of the total of 46) coding exons of FLNB could be screened to detect Larsen syndrome mutations. Four of these exons were screened in a sixth (sporadic) case and a previously reported G1691S substitution mutation detected. CONCLUSIONS: Mutations in FLNB may be responsible for all cases of Larsen syndrome. They appear to occur in specific functional domains of the filamin B protein. This should simplify diagnostic screening of the FLNB gene. Analyses in larger patient series are warranted to quantify this. The study confirmed the extreme variability in clinical presentation and the presence of unaffected carriers. A molecular screen would be valuable for diagnosis and genetic counselling.

Combining genetics and population history in the study of ethnic diversity in the People's Republic of China.

Genomic data have increasingly been used to complement linguistic, archeological, and anthropological evidence in reconstructing the origins and migratory patterns of modern humans. East Asia is a particular hotspot of human migration, especially mainland China, where a large number of human fossils have been unearthed and more than 20% of the world's population now resides. There are 56 officially recognized ethnic populations (minzu) in China. In the present study we investigated the ancestry and genetic diversity of nine populations: the majority Han of Liaoning Province; the Miao, Yao, Kucong, and Tibetan communities of Yunnan Province in southwest China; and four Muslim populations, the Hui, Bonan, Dongxiang, and Sala from central and northern China. We used both biparental and uniparental markers to determine patterns of diversity at autosomal, mitochondrial, and Y-chromosome loci. The study populations displayed several paternal origins but restricted maternal ancestries. From the Y-chromosome data in particular, major demographic changes, such as the Neolithic population expansion and more recent historical events including migration along the Silk Road, could be inferred. Specific aspects of the internal structure and organization of the study populations, including endogamy and consanguinity, were uncovered using autosomal markers. However, we encountered interpretive problems in terms of the definition of the present-day ethnic study populations in China, which appear to reflect past and present political as well as genetic influences.

Localization of a gene for familial recurrent arthritis.

OBJECTIVE: To localize the gene for familial recurrent arthritis via a genome-wide linkage scan in an extended kindred with the disease. METHODS: A 3-generation family in which 9 members were diagnosed with juvenile idiopathic arthritis (JIA) was ascertained. In this family the disease was of very early onset and included episodic inflammation leading to eventual destruction of joints, muscle, and skin. We treated this disorder as a distinct clinical entity that we have named "familial recurrent arthritis." A genome-wide linkage scan with polymorphic microsatellites at 10-15-cM resolution was initiated. RESULTS: The genome-wide scan generated a maximum 2-point logarithm of odds score with D15S211 (Zmax = 3.27 at thetamax = 0.0010). Haplotype reconstruction defined a candidate region of approximately 20 cM flanked proximally by D15S983 and distally by D15S127 on human chromosome 15. CONCLUSION: A gene for familial recurrent arthritis was localized to 15q22-24, as a result of a genome-wide linkage scan in a large, multiply affected kindred. Identification of the altered gene will provide insights into the pathogenesis of autoimmune joint destruction that is reminiscent of JIA.

Localization of susceptibility to familial idiopathic scoliosis.

STUDY DESIGN: Genome-wide linkage surveys in large multiplex families with apparent inherited idiopathic scoliosis. OBJECTIVE: To identify chromosomal loci encoding genes involved in susceptibility to idiopathic scoliosis by positional cloning. SUMMARY OF BACKGROUND DATA: Although the inheritance of idiopathic scoliosis most often exhibits a complex pattern, autosomal dominant inheritance can be identified in some families. Families exhibiting such an inheritance pattern present an opportunity to identify the predisposing gene(s) by positional cloning. METHODS: Probands having clinically relevant idiopathic scoliosis (50 degrees Cobb angle) from large multiplex families were identified. A curve of 15 degrees, made from standing posteroanterior radiographs, was required for a positive diagnosis. A genome-wide search in one large family (seven affected members) was conducted with 385 polymorphic microsatellite markers spaced at an approximate 10-cM resolution. Hot spots identified in this family were subsequently tested in a second large kindred. RESULTS: Maximum evidence of allele-sharing in affected individuals from the first family was detected for three loci on chromosomes 6p, distal 10q, and 18q with nonparametric lod scores of 1.42 (P = 0.020), 1.60 (P = 0.019), and 8.26 (P = 0.002), respectively. Evidence of allele-sharing was also detected in the second family at distal chromosome 10q (nonparametric lod score = 2.02; P = 0.033). CONCLUSIONS: These data indicate a limited number of genetic loci predisposing to idiopathic scoliosis.

Departure from neutrality at the mitochondrial NADH dehydrogenase subunit 2 gene in humans, but not in chimpanzees.

To test whether patterns of mitochondrial DNA (mtDNA) variation are consistent with a neutral model of molecular evolution, nucleotide sequences were determined for the 1041 bp of the NADH dehydrogenase subunit 2 (ND2) gene in 20 geographically diverse humans and 20 common chimpanzees. Contingency tests of neutrality were performed using four mutational categories for the ND2 molecule: synonymous and nonsynonymous mutations in the transmembrane regions, and synonymous and nonsynonymous mutations in the surface regions. The following three topological mutational categories were also used: intraspecific tips, intraspecific interiors, and interspecific fixed differences. The analyses reveal a significantly greater number of nonsynonymous polymorphisms within human transmembrane regions than expected based on interspecific comparisons, and they are inconsistent with a neutral equilibrium model. This pattern of excess nonsynonymous polymorphism is not seen within chimpanzees. Statistical tests of neutrality, such as TAJIMA's D test, and the D and F tests proposed by FU and LI, indicate an excess of low frequency polymorphisms in the human data, but not in the chimpanzee data. This is consistent with recent directional selection, a population bottleneck or background selection of slightly deleterious mutations in human mtDNA samples. The analyses further support the idea that mitochondrial genome evolution is governed by selective forces that have the potential to affect its use as a "neutral" marker in evolutionary and population genetic studies.

Comparative nuclear and mitochondrial genome diversity in humans and chimpanzees.

Restriction mapping and sequencing have shown that humans have substantially lower levels of mitochondrial genome diversity (d) than chimpanzees. In contrast, humans have substantially higher levels of heterozygosity (H) at protein-coding loci, suggesting a higher level of diversity in the nuclear genome. To investigate the discrepancy further, we sequenced a segment of the mitochondrial genome control region (CR) from 49 chimpanzees. The majority of these were from the Pan troglodytes versus subspecies, which was underrepresented in previous studies. We also estimated the average heterozygosity at 60 short tandem repeat (STR) loci in both species. For a total sample of 115 chimpanzees, d = 0.075 +/0 0.037, compared to 0.020 +/- 0.011 for a sample of 1,554 humans. The heterozygosity of human STR loci is significantly higher than that of chimpanzees. Thus, the higher level of nuclear genome diversity relative to mitochondrial genome diversity in humans is not restricted to protein-coding loci. It seems that humans, not chimpanzees, have an unusual d/H ratio, since the ratio in chimpanzees is similar to that in other catarrhines. This discrepancy in the relative levels of nuclear and mitochondrial genome diversity in the two species cannot be explained by differences in mutation rate. However, it may result from a combination of factors such as a difference in the extent of sex ratio disparity, the greater effect of population subdivision on mitochondrial than on nuclear genome diversity, a difference in the relative levels of male and female migration among subpopulations, diversifying selection acting to increase variation in the nuclear genome, and/or directional selection acting to reduce variation in the mitochondrial genome.

TCOF1 gene encodes a putative nucleolar phosphoprotein that exhibits mutations in Treacher Collins Syndrome throughout its coding region.

Treacher Collins Syndrome (TCS) is the most common of the human mandibulofacial dysostosis disorders. Recently, a partial TCOF1 cDNA was identified and shown to contain mutations in TCS families. Here we present the entire exon/intron genomic structure and the complete coding sequence of TCOF1. TCOF1 encodes a low complexity protein of 1,411 amino acids, whose predicted protein structure reveals repeated motifs that mirror the organization of its exons. These motifs are shared with nucleolar trafficking proteins in other species and are predicted to be highly phosphorylated by casein kinase. Consistent with this, the full-length TCOF1 protein sequence also contains putative nuclear and nucleolar localization signals. Throughout the open reading frame, we detected an additional eight mutations in TCS families and several polymorphisms. We postulate that TCS results from defects in a nucleolar trafficking protein that is critically required during human craniofacial development.

Identification and localization of the gene for EXTL, a third member of the multiple exostoses gene family.

Hereditary multiple exostoses (EXT) is an autosomal dominant disorder characterized by multiple bony outgrowths from the juxtaepiphyseal region of long bones. In a small proportion of cases, these exostoses progress to malignant chondrosarcomas. Genetic linkage of this disorder has been described to three independent loci on chromosomes 8q24.1 (EXT1), 11p11-13 (EXT2), and 19p (EXT-3). The EXT1 and EXT2 genes were isolated recently and show extensive sequence homology to each other. These genes are deleted in exostoses-derived tumors, supporting the hypothesis that they encode tumor suppressors. We have identified a third gene that shows striking sequence similarity to both EXT1 and EXT2 at the nucleotide and amino acid sequence levels, and have derived its entire coding sequence. Although the mRNA transcribed from this gene is similar in size to that from EXT1 and EXT2, its pattern of expression is quite different. We have localized this gene by fluorescence in situ hybridization to metaphase chromosomes and by whole genome radiation hybrid mapping to chromosome 1p36.1 between DIS458 and DIS511, region that frequently shows loss of heterozygosity in a variety of tumor types. This gene, EXTL (for EXT-like), is therefore a new member of the EXT gene family and is a potential candidate for several disease phenotypes.

A YAC contig of approximately 3 Mb from human chromosome 5q31-->q33.

The human chromosome 5q31--q33 region contains an interesting cluster of growth factor and receptor genes. In addition, several genetic disease loci have been localized within this region, but have not as yet been isolated as molecular clones. These include those loci involved in autosomal dominant limb-girdle muscular dystrophy, diastrophic dysplasia, Treacher Collins syndrome, and myeloid disorders associated with the 5q- syndrome. A yeast artificial chromosome (YAC) contig of this region would assist in the further localization and isolation of these genes. We have used YACs isolated from the Washington University and Centre d'Etude du Polymorphisme Humain YAC libraries, including YACs from the large insert (mega) YAC library to build a contig greater than 3 Mb in size. An STS content strategy coupled with limited walking from YAC ends was used to isolate 22 overlapping YACs with as much as sixfold coverage. A total of 20 STSs, derived from genes, anonymous sequences, and vector Alu-PCR or inverse PCR products, were used to compile this contig. The order of loci, centromere-GRL-D5S207-D5S70-D5S545-D5S546- D5S547-D5S68-D5S548-D5S210-D5S549- D5S686-ADRB2-D5S559-CSF1R-D5S551-RPS14+ ++-D5S519-SPARC-telomere, was derived from the overlapping clones. This contig and clones derived from it will be useful substrates in selecting candidate cDNAs for the disease loci in this interval.

Molecular analyses of unrelated Charcot-Marie-Tooth (CMT) disease patients suggest a high frequency of the CMTIA duplication.

Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. One form of CMT, CMT type 1A, is characterized by uniformly decreased nerve conduction velocities, usually shows autosomal dominant inheritance, and is associated with a large submicroscopic duplication of the p11.2-p12 region of chromosome 17. A cohort of 75 unrelated patients diagnosed clinically with CMT and evaluated by electrophysiological methods were analyzed molecularly for the presence of the CMT1A DNA duplication. Three methodologies were used to assess the duplication: measurement of dosage differences between RFLP alleles, analysis of polymorphic (GT)n repeats, and detection of a junction fragment by pulsed-field gel electrophoresis. The CMT1A duplication was found in 68% of the 63 unrelated CMT patients with electrophysiological studies consistent with CMT type 1 (CMT1). The CMT1A duplication was detected as a de novo event in two CMT1 families. Twelve CMT patients who did not have decreased nerve conduction velocities consistent with a diagnosis of CMT type 2 (CMT2) were found not to have the CMT1A duplication. The most informative molecular method was the detection of the CMT1A duplication-specific junction fragment. Given the high frequency of the CMT1A duplication in CMT patients and the high frequency of new mutations, we conclude that a molecular test for the CMT1A DNA duplication is very useful in the differential diagnosis of patients with peripheral neuropathies.

Charcot-Marie-Tooth disease type 1A. Association with a spontaneous point mutation in the PMP22 gene.

BACKGROUND: Charcot-Marie-Tooth disease (CMT) is the most common inherited peripheral neuropathy. CMT type 1A is associated with a 1.5-megabase DNA duplication in region p11.2-p12 of chromosome 17 in most patients. An increased dosage of a gene within the duplicated segment appears to cause the disease. The PMP22 gene, which encodes a myelin protein, has been mapped within the duplication and proposed as a candidate gene for CMT type 1A. METHODS: We analyzed DNA samples from a cohort of 32 unrelated patients with CMT type 1 who did not have the 1.5-Mb tandem duplication in 17p11.2-p12 for mutations within the PMP22 coding region. Molecular techniques included the polymerase chain reaction (PCR), heteroduplex analysis to detect point mutations, and direct nucleotide-sequence determination of amplified PCR products. RESULTS: A 10-year-old boy was identified with a point mutation in PMP22, which resulted in the substitution of cysteine for serine in a putative transmembrane domain of PMP22. Analysis of family members revealed that the PMP22 point mutation arose spontaneously and segregated with the CMT type 1 phenotype in an autosomal dominant pattern. The patients with the PMP22 point mutation had clinical and electrophysiologic phenotypes that were similar to those of patients with the 1.5-Mb duplication. CONCLUSIONS: The PMP22 gene has a causative role in CMT type 1. Either a point mutation in PMP22 or a duplication of the region including the PMP22 gene can result in the disease phenotype.

Charcot-Marie-Tooth type 1A duplication appears to arise from recombination at repeat sequences flanking the 1.5 Mb monomer unit.

We have constructed a 3.1 megabase (Mb) physical map of chromosome 17p11.2-p12, which contains a submicroscopic duplication in patients with Charcot-Marie-Tooth disease type 1A (CMT1A). We find that the CMT1A duplication is a tandem repeat of 1.5 Mb of DNA. A YAC contig encompassing the CMT1A duplication and spanning the endpoints was also developed. Several low copy repeats in 17p11.2-p12 were identified including the large (> 17 kb) CMT1A-REP unit which may be part of a mosaic repeat. CMT1A-REP flanks the 1.5 Mb CMT1A monomer unit on normal chromosome 17 and is present in an additional copy on the CMT1A duplicated chromosome. We propose that the de novo CMT1A duplication arises from unequal crossing over due to misalignment at these CMT1A-REP repeat sequences during meiosis.

Gene dosage is a mechanism for Charcot-Marie-Tooth disease type 1A.

Charcot-Marie-Tooth disease type 1A (CMT1A) is the most common inherited peripheral neuropathy in humans, characterized electrophysiologically by decreased nerve conduction velocities (NCVs). CMT1A is associated with a large submicroscopic DNA duplication in proximal 17p. In this report we demonstrate that a patient with a cytogenetically visible duplication, dup(17)(p11.2p12), has decreased NCV. Molecular analysis demonstrated this patient was duplicated for all the DNA markers duplicated in CMT1A as well as markers both proximal and distal to the CMT1A duplication. These data support the hypothesis that the CMT1A phenotype can result from a gene dosage effect.

Subcutaneous tocolytic infusion therapy for patients at very high risk for preterm birth.

Patients with multiple gestations or recalcitrant preterm labor are at very high risk for preterm birth in spite of adequate tocolysis. Subcutaneous infusion of tocolytic medications on an ambulatory basis has been used in several small series and has effectively prolonged gestation. This retrospective analysis presents data from 992 patients at very high risk for preterm delivery who were prescribed this therapy. The amount of tocolytic medication was individualized by utilizing the patient's volume of distribution and clearance. Pharmacists adjusted the dosage based on uterine activity strips received by nursing personnel. The average basal rate was .073 +/- .020 mg/h. Patients received an average of seven scheduled boluses per day and 1.54 +/- .93 unscheduled boluses per week (.25 +/- .03 mg each). The therapy extended the gestation a mean of 38 +/- 23 days and average gestational age at delivery was 36.3 +/- 2.6 weeks with a mean birthweight of 2759 +/- 681 g. This study, utilizing a large number of patients, confirms earlier reports that for women at very high risk for preterm delivery subcutaneous tocolytic infusion therapy is beneficial. Prospective studies evaluating such treatment on a randomized basis are indicated.

Dramatic size variation of yeast mitochondrial RNAs suggests that RNase P RNAs can be quite small.

The gene coding for the AU-rich RNA required for mitochondrial RNase P activity in Saccharomyces cerevisiae codes for a 490-base RNA while that in Candida glabrata codes for a 227-base RNA. We have detected a 140-nucleotide RNA coded by the mitochondrial DNA from Saccharomycopsis fibuligera by hybridization with an oligonucleotide complementary to a conserved sequence found in mitochondrial and prokaryotic RNase P RNAs. DNA sequence analysis of the mitochondrial DNA from the region coding for this RNA revealed a second conserved sequence block characteristic of RNase P RNA genes and the presence of a downstream tRNA(Pro) gene. Like previously characterized mitochondrial RNase P RNAs, this small RNA is extremely AU-rich. The discovery of this 140-base RNA suggests that naturally occurring RNase P RNAs may be quite small.