[Efficacy and safety of vemurafenib in the treatment of BRAF(V600E)-mutated Erdheim-Chester disease].

Objective: To evaluate the safety and efficacy of vemurafenib in the treatment of BRAF(V600E)-mutated Erdheim-Chester disease (ECD) . Methods: We retrospectively analyzed the clinical data, response rate, adverse events and survival of 12 patients with ECD treated with vemurafenib from March 2015 to October 2020 in Peking Union Medical College Hospital. Results: Of 12 patients [7 males and 5 females, median age = 51.5 (range, 32-66) years old], the median number of organs involved was 6 (range, 4-8) , and the median treatment duration of vemurafenib was 11 (3-60) months. All patients had improvement of clinical symptoms, of which 2 cases were completely relieved, and 10 cases were partially relieved. Seven patients evaluated by (18)F-FDG-positron emission tomography/computed tomography achieved a metabolic response, including 2 patients with a complete metabolic response and 5 patients with a partial metabolic response. The common adverse events in the overall cohort were grade 1 to 2 (Common Terminology Criteria for Adverse Events 5.0) , including skin rash (58.3%) , arthralgia (25.0%) , and digestive tract reactions (16.7%) . The median follow-up time was 13.5 (3-60) months. One patient with central nervous system involvement died due to a cerebrovascular event, and one patient relapsed 10 months after drug withdrawal. The estimated 2-year overall survival rate and 2-year progression free survival rate were 88.89% and 66.67%, respectively. Conclusions: Vemurafenib is safe and effective in the treatment of BRAF(V600E)-mutated ECD.

Genome scanning reveals novel candidate genes for vertebral and teat number in the Beijing Black Pig.

Porcine vertebral and teat numbers are variable and important economic traits in pig production. However, the quantitative trait loci (QTL) and candidate genes for both of these traits in the Beijing Black Pig are not yet known. In the present study, number of vertebrae and number of teats were obtained for 891 individuals of the Beijing Black Pig and genotyped using the Illumina Porcine 50 K BeadChip. Genome scanning was performed to detect associated variants and candidate genes for both traits using a genome-wide association study by tassel software. For vertebral number, 15 significant SNPs were located on SSC7. According to linkage disequilibrium analysis on SSC7, a haplotype block of 221 kb from 97.4 to 97.6 Mb was shown to contain a good candidate gene ABCD4. Interestingly, on SSC12, we recorded a novel QTL containing three significant SNPs and 34 annotated genes from 24.0 to 25.7 Mb for vertebral number. Of the 34 genes, nine Hox family genes (HOXB 1-7, 9, and 13) were found to be good candidate genes. Using the 34 genes, a gene ontology analysis was performed to detect enrichment of anterior/posterior pattern specification. For teat number, a novel chromosome-wide significant QTL was identified on SSC10. In this QTL region, one significant SNP was identified. The nearest gene, NTRK2, was regarded as a candidate gene. The present results expanded the QTL for vertebral and teat numbers and provided useful molecular markers for breeding in the Beijing Black Pig population.

Two fatty acid synthase genes from the integument contribute to cuticular hydrocarbon biosynthesis and cuticle permeability in Locusta migratoria.

Lipids of the insect cuticle have important roles in resistance against the arid environment and invasion of foreign substances. Fatty acid synthase (FAS) is an important enzyme of the insect lipid synthesis pathway. In the present study, we identified three FAS genes from transcriptome data of the migratory locust, Locusta migratoria, based on bioinformatics analyses. Among them, two FAS genes (LmFAS1 and LmFAS3) are highly expressed in the integument of fifth instar nymphs. Suppression of LmFAS1 and LmFAS3 by RNA interference caused lethality during ecdysis or shortly after moulting. The weight of the locusts and the content of lipid droplets were reduced compared with those of the control. The results of gas chromatography-mass spectrometry analysis showed that knockdown of LmFAS3 led to a decrease of both cuticular hydrocarbons and inner hydrocarbons (CHCs and IHCs) contents, especially the content of methyl branched hydrocarbons. By contrast, knockdown of LmFAS1 only resulted in a decrease in the IHC content, but not that of CHCs. By consequence, in LmFAS1- and LmFAS3-suppressed locusts, hydrocarbon deficiency reduced desiccation resistance and enhanced cuticle permeability and sensitivity to insecticides. These results indicate that LmFAS1 and LmFAS3 are essential for hydrocarbon production and cuticle permeability, which play influential roles in waterproofing the insect cuticle.

Dedifferentiation into blastomere-like cancer stem cells via formation of polyploid giant cancer cells.

Our recent perplexing findings that polyploid giant cancer cells (PGCCs) acquired embryonic-like stemness and were capable of tumor initiation raised two important unanswered questions: how do PGCCs acquire such stemness, and to which stage of normal development do PGCCs correspond. Intriguingly, formation of giant cells due to failed mitosis/cytokinesis is common in the blastomere stage of the preimplantation embryo. However, the relationship between PGCCs and giant blastomeres has never been studied. Here, we tracked the fate of single PGCCs following paclitaxel-induced mitotic failure. Morphologically, early spheroids derived from PGCCs were indistinguishable from human embryos at the blastomere, polyploid blastomere, compaction, morula and blastocyst-like stages by light, scanning electron or three-dimensional confocal scanning microscopy. Formation of PGCCs was associated with activation of senescence, while budding of daughter cells was associated with senescence escape. PGCCs showed time- and space-dependent activation of expression of the embryonic stem cell markers OCT4, NANOG, SOX2 and SSEA1 and lacked expression of Xist. PGCCs acquired mesenchymal phenotype and were capable of differentiation into all three germ layers in vitro. The embryonic-like stemness of PGCCs was associated with nuclear accumulation of YAP, a key mediator of the Hippo pathway. Spheroids derived from single PGCCs grew into a wide spectrum of human neoplasms, including germ cell tumors, high-grade and low-grade carcinomas and benign tissues. Daughter cells derived from PGCCs showed attenuated capacity for invasion and increased resistance to paclitaxel. We also observed formation of PGCCs and dedifferentiation in ovarian cancer specimens from patients treated with chemotherapy. Taken together, our findings demonstrate that PGCCs represent somatic equivalents of blastomeres, the most primitive cancer stem cells reported to date. Thus, our studies reveal an evolutionarily conserved archaic embryonic program in somatic cells that can be de-repressed for oncogenesis. Our work offers a new paradigm for cancer origin and disease relapse.

[Clinical value of acoustic radiation force impulse technique to predict esophageal and gastric varices in patients with biliary atresia].

Objective: To investigate the clinical value of acoustic radiation force impulse (ARFI)technique in predicting esophageal and gastric varices in patients with biliary atresia after Kasai portoenterostomy. Methods: A total of 42 patients with biliary atresia after Kasai portoenterostomy were collected from September 2015 to May 2016 in Tianjin First Central Hospital.ARFI technique was used to measure the stiffness of liver and spleen, and 28 healthy children as control.According to the result of CT examination , patients with biliary atresia were divided into two groups , twenty-three patients with esophageal and gastric varices(A group) and nineteen patients without esophageal and gastric varices (B group), Comparing the difference of liver and spleen stiffness between the two groups.The ROC curve analysis was carried out to test the diagnostic power of effective parameter. Results: The ARFI value of liver (2.98±0.80) m/s and spleen (3.00±0.33) m/s of patients with biliary atresia was significantly higher than that of control group((1.10±0.16) m/s, (2.12±0.32) m/s), the differences had statistical significance (both P<0.01). Between group A and group B, the spleen ARFI value of group A(3.16±0.26) m/s was higher than group B(2.83±0.32) m/s, the difference had statistical significance (P<0.01), whereas there was no statistical difference of liver ARFI value between two group((2.93±0.65), (3.02±0.96) m/s)(P>0.05). The cut-off ARFI value of spleen to diagnose esophageal and gastric varices in biliary atresia was 3.02 m/s, and the biggest area under the ROC curve, sensitivity, and specificity were 0.81, 78.6% and 84.5%, respectively. Conclusion: ARFI can be used as a noninvasive method to predict the presence of esophageal and gastric varices in patients with biliary atresia after Kasai portoenterostomy.

Further delineation of a rare recessive encephalomyopathy linked to mutations in GFER thanks to data sharing of whole exome sequencing data.

BACKGROUND: Alterations in GFER gene have been associated with progressive mitochondrial myopathy, congenital cataracts, hearing loss, developmental delay, lactic acidosis and respiratory chain deficiency in 3 siblings born to consanguineous Moroccan parents by homozygosity mapping and candidate gene approach (OMIM#613076). Next generation sequencing recently confirmed this association by the finding of compound heterozygous variants in 19-year-old girl with a strikingly similar phenotype, but this ultra-rare entity remains however unknown from most of the scientific community. MATERIALS AND METHODS: Whole exome sequencing was performed as part of a "diagnostic odyssey" for suspected mitochondrial condition in 2 patients, presenting congenital cataracts, progressive encephalomyopathy and hypotrophy and detected unreported compound heterozygous variants in GFER. RESULTS: Thanks to an international data sharing, we found 2 additional patients carrying compound heterozygous variants in GFER. Reverse phenotyping confirmed the phenotypical similarities between the 4 patients. Together with the first literature reports, the review of these 8 cases from 4 unrelated families enables us to better describe this apparently homogeneous disorder, with the clinical and biological stigmata of mitochondrial disease. CONCLUSION: This report highlights the clinical utility of whole exome sequencing and reverse phenotyping for the diagnosis of ultra-rare diseases and underlines the importance of a broad data sharing for accurate clinical delineation of previously unrecognized entities.

Linking genomic reorganization to tumor initiation via the giant cell cycle.

To investigate the mechanisms underlying our recent paradoxical finding that mitotically incapacitated and genomically unstable polyploid giant cancer cells (PGCCs) are capable of tumor initiation, we labeled ovarian cancer cells with α-tubulin fused to green fluorescent protein, histone-2B fused to red fluorescent protein and FUCCI (fluorescent ubiquitination cell cycle indicator), and tracked the spatial and time-dependent change in spindle and chromosomal dynamics of PGCCs using live-cell fluorescence time-lapse recording. We found that single-dose (500 nm) treatment with paclitaxel paradoxically initiated endoreplication to form PGCCs after massive cell death. The resulting PGCCs continued self-renewal via endoreplication and further divided by nuclear budding or fragmentation; the small daughter nuclei then acquired cytoplasm, split off from the giant mother cells and acquired competency in mitosis. FUCCI showed that PGCCs divided via truncated endoreplication cell cycle (endocycle or endomitosis). Confocal microscopy showed that PGCCs had pronounced nuclear fragmentation and lacked expression of key mitotic proteins. PGCC-derived daughter cells were capable of long-term proliferation and acquired numerous new genome/chromosome alterations demonstrated by spectral karyotyping. These data prompt us to conceptualize a giant cell cycle composed of four distinct but overlapping phases, initiation, self-renewal, termination and stability. The giant cell cycle may represent a fundamental cellular mechanism to initiate genomic reorganization to generate new tumor-initiating cells in response to chemotherapy-induced stress and contributes to disease relapse.

Radiation-enhancing effect of sodium glycididazole in patients suffering from non-small cell lung cancer with multiple brain metastases: A randomized, placebo-controlled study.

PURPOSE: Median survival of patients with brain metastases from non-small cell lung cancer is poor. This study was to investigate the radiation-enhancing effect of sodium glycididazole combined with whole-brain radiotherapy of multiple brain metastases from non-small cell lung cancer. PATIENTS AND METHODS: Sixty-four patients with multiple brain metastases from non-small cell lung cancer were included: the study group (n=32) received whole-brain radiotherapy combined with sodium glycididazole at a dose of 700mg/m(2) intravenous infusion 30minutes before radiotherapy, three times a week; the control group (n=32) only received whole-brain radiotherapy. The primary end point was central nervous system (CNS) progression-free survival and overall survival. The treatment-related toxicity was also recorded. RESULTS: The CNS disease control rate was better (90.6% vs 65.6%, P=0.016) in the study group than in the control group at 3 month of follow-up. The median CNS progression-free survival time was longer in the study group than in the control group (7.0 months vs 4.0 months, P=0.038). There was no significant difference of the median overall survival time between the study group and the control group (11.0 months vs 9.0 months, P=0.418). On the other hand, the treatment-related toxicity showed no statistically significant difference between these two groups (P>0.05). CONCLUSIONS: The study indicated that sodium glycididazole was an effective, promising radiation-enhancing agent that improved CNS disease control rate, extended the median CNS progression-free survival time and was well tolerated in patients suffering from non-small cell lung cancer with multiple brain metastases.

Three Case Reports of Splenic Artery Steal Syndrome After Liver Transplantation.

Splenic artery steal syndrome (SASS) has gained attention as a complication involving the hepatic artery and can cause ischemia in a grafted liver. This article presents 3 patients with SASS, including their diagnosis and treatment. Contrast-enhanced ultrasonography and angiography are useful in diagnosing SASS, and splenic artery trunk embolization is an effective treatment. The purpose of this article is to reinforce the understanding of the development and progression of SASS.

Molecular cloning and characterization of a proliferating cell nuclear antigen gene by chemically induced male sterility in wheat (Triticum aestivum L.).

Although a number of studies have shown that chemical hybridizing agents (CHAs) affect anther growth and regulate cell-cycle progression, little is known about the molecular and cellular mechanisms involved. Proliferating cell nuclear antigen (PCNA) is an essential factor in DNA replication, and in many other processes in eukaryotic cells. In this study, the open reading frame of TaPCNA, the PCNA in wheat (Triticum aestivum L.), was cloned by reverse transcription polymerase chain reaction (RT-PCR). Sequence analysis revealed that this gene was 792-bp long and encoded a protein with 234 amino acids. Alignment of the TaPCNA-predicted sequence revealed a high degree of identity with PCNAs from other plant species. A subcellular localization assay indicated that TaPCNA was localized in the nucleus. The TaPCNA was cloned into the prokaryotic expression plasmid pET32a, and the recombinant plasmid was transformed into BL21 (DE3). TaPCNA expression was induced by 0.5 mM isopropyl-beta-D-thiogalactopyranoside and verified using sodium dodecyl sulfate polyacrylamide gel electrophoresis and western blot assays, which indicated that the fusion protein was successfully expressed. The gene involved in the G1-to-S transition, Histone H4, was downregulated by 1376- CIMS, which is a chemically induced male sterility line. However, a semi-quantitative RT-PCR revealed that TaPCNA expression was upregulated in 1376-CIMS. Our results suggest that CHAs (SQ-1) induce DNA damage in wheat anthers. DNA damage results in either the delay or arrest of cell-cycle progression, which affects anther development. This study will help to elucidate the mechanisms of SQ-1-induced male sterility.

Relationship between male sterility and ß-1,3-glucanase activity and callose deposition-related gene expression in wheat (Triticum aestivum L.).

In previous studies, we first isolated one different protein ß-1,3-glucanase using two-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry from normal wheat (Triticum aestivum L.) and chemical hybridization agent-induced male sterility (CIMS) wheat. In this experiment, ß-1,3-glucanase activity and the expression of a callose deposition-related gene, UDP-glucose phosphorylase (UGPase), were determinate in normal, CIMS, and genetic male sterility (GS) wheat. ß-1,3-glucanase activity was significantly different between the fertile and sterile lines during callose synthesis and degradation, but there was no difference between CIMS and GS wheat. The UGPase gene of callose deposition was highly expressed in the meiophase and sharply decreased in the tetrad stage. However, the expression of the UGPase gene was significantly different between the fertile and sterile lines. These data indicated that ß-1,3-glucanase activity and the expression of the UGPase gene play important roles in the male sterility of wheat. Consequently, pollen mother cells (PMCs) might degenerate at the early meiosis stage, and differences in UGPase gene expression and ß-1,3-glucanase activity might eventually result in complete pollen collapse. In addition, the critical period of anther abortion might be the meiosis stage to the tetrad stage rather than what we previously thought, the mononuclear period.

Inflammation-related genetic variants predict toxicity following definitive radiotherapy for lung cancer.

Definitive radiotherapy improves locoregional control and survival in inoperable non-small cell lung cancer patients. However, radiation-induced toxicities (pneumonitis/esophagitis) are common dose-limiting inflammatory conditions. We therefore conducted a pathway-based analysis to identify inflammation-related single-nucleotide polymorphisms associated with radiation-induced pneumonitis or esophagitis. A total of 11,930 single-nucleotide polymorphisms were genotyped in 201 stage I-III non-small cell lung cancer patients treated with definitive radiotherapy. Validation was performed in an additional 220 non-small cell lung cancer cases. After validation, 19 single-nucleotide polymorphisms remained significant. A polygenic risk score was generated to summarize the effect from validated single-nucleotide polymorphisms. Significant improvements in discriminative ability were observed when the polygenic risk score was added into the clinical/epidemiological variable-based model. We then used 277 lymphoblastoid cell lines to assess radiation sensitivity and expression quantitative trait loci (eQTL) relationships of the identified single-nucleotide polymorphisms. Three genes (PRKCE, DDX58, and TNFSF7) were associated with radiation sensitivity. We concluded that inflammation-related genetic variants could contribute to the development of radiation-induced toxicities.

Specific thalamic nuclei function as novel 'nociceptive discriminators' in the endogenous control of nociception in rats.

Recently, we hypothesized that supraspinal structures may have important functions in discriminating between noxious mechanically and heat mediated nociception through distinct functions: facilitation and inhibition. In this study, conducted in conscious rats, we explored the role of different thalamic nuclei: the mediodorsal (MD) nucleus, the central medial (CM) nucleus, the submedius (SM) nucleus, the ventralmedial (VM) nucleus and the ventral posterolateral (VPL) nucleus, in the descending control of secondary and contralateral mechanical hyperalgesia and heat hypoalgesia occurring in intramuscularly hypertonic (HT, 5.8%) saline-induced muscle nociception. We found that the MD nuclei participated in the descending facilitation of mechanical hyperalgesia, and that the VM nuclei were specifically involved in the descending inhibition of heat hypoalgesia. Neither descending facilitation nor descending inhibition was affected after electrolytic lesion of the thalamic CM, SM, and VPL nuclei. This descending facilitatory and inhibitory modulation of nociception was strengthened by glutamate, and weakened by GABA, microinjected into the thalamic MD and VM nuclei. It is suggested that (1) thalamic MD nucleus and VM nucleus form two distinct endogenous systems in the control of noxious mechanically and heat evoked responses, and (2) the strengthening of descending inhibition and the weakening of descending facilitation by means of up regulation and down regulation of appropriate receptor expression in the VM and MD nuclei may provide a new strategic policy in treating pathological pain.

Corresponding states principle for the alkaline earth dimers and the van der Waals potential of Ba2.

A simple method based on the corresponding states principle and the Tang-Toennies potential model is used for the determination of the ground state van der Waals potential of barium dimer. The potential energy curve calculated from the theoretical dispersion coefficients and the experimental vibrational frequency is comparable to other quantum chemical calculations. To assess its validity, the same method is used to calculate the potential of the strontium dimer, for which there are many theoretical and experimental determinations. A comparison with experimental measurements suggests that the present method is highly accurate. The potentials of five alkaline earth dimers are compared. The potentials of Mg(2), Ca(2), Sr(2), and Ba(2) are shown to be nearly conformal with each other. The potential of Be(2) has a different shape.

Paroxysmal kinesigenic choreoathetosis: evidence of linkage to the pericentromeric region of chromosome 16 in four Chinese families.

BACKGROUND: Paroxysmal kinesigenic choreoathetosis (PKC) is an autosomal dominant condition characterized by abnormal involuntary movements precipitated by sudden movement. The pericentromeric region of chromosome 16 has been linked to PKC by several reports. This study was to localize and identify PKC gene in four Chinese PKC families. METHODS: Genetic linkage mapping with eight markers spanning chromosome 16p12-q13 was performed in 43 family members. Genome-wide single nucleotide polymorphism (SNP) scans were performed on four individuals in Family 1 in which infantile convulsion (IC) was co-inherited with PKC. RESULTS: Individuals in Family 1 presented with both IC and paroxysmal choreoathetosis (ICCA), and Families 2, 3, and 4 presented only with PKC. Evidence for linkage was found with a maximum two-point LOD score of 4.89 for D16S690 (theta = 0.0) and a maximum multipoint LOD score was 5.34 between D16S3080 and D16S3136. Haplotype analysis showed the disease locus was between D16S3093 and D16S3057. A total of 84 SNPs spanned on 16q12.1-q13 was not segregated with the PKC phenotype, which defined an unlinked region from rs9933187 to rs8044753. Thus, the critical region of the PKC gene is across the pericentromeric region of chromosome 16, and most likely maps to a region of 20.5 Mb (6.2 cM) between D16S3093 and rs9933187 (16p11.2-q12.1). CONCLUSION: The assignment of the locus for PKC to the pericentromeric region of chromosome 16 is confirmed and putatively narrowed in the present study.

Combined effects of niacin and chromium treatment on vascular endothelial dysfunction in hyperlipidemic rats.

Combined treatment with niacin and chromium has been found to have a protective effect against oxidative damage to different tissues of hyperlipidemic rats. But its effects on vascular endothelial dysfunction are less clear. This study was performed to investigate the effect of combined treatment with niacin and chromium on vascular endothelial dysfunction, with the aim of gaining insight to the mechanisms by detecting the expression levels of ox-LDL and LOX-1. Twenty-four male, 4-week-old Wistar rats were randomly divided into three groups: control group (CG; n = 8), high-fat group (HF; n = 8), and drug control group (DG; n = 8). In CG group, rats were fed with pellet chow. In HF group, rats were fed with high-fat diet for 12 weeks. In DG group, rats were fed with the same high-fat diet and treated with CrCl(3) x 6 H(2)O (250 microg/kg days) and niacin (100 mg/kg days) by gavage technique for 12 weeks. At the end of the 12th week, samples from aorta and blood were collected. In HF group, the serum levels of total cholesterol (TC), low-density lipoprotein (LDL), oxidized low-density lipoprotein (ox-LDL) and endothelin (ET) were higher, whereas the levels of high-density lipoprotein (HDL), serum NO were lower than those in CG group. The levels of serum TC, LDL, ox-LDL and ET decreased and HDL, NO levels increased in DG group when compared with HF group. The levels of LOX-1, ICAM-1 were also observed in abdominal artery. In HF group, the protein and mRNA expression of LOX-1, ICAM-1 were elevated comparing with CG group. In DG group, the protein and mRNA expression of LOX-1, ICAM-1 were decreased obviously, but still differed significantly from those in CG group. ox-LDL was related positively to TC, LDL, ET, ICAM-1 and LOX-1, but related negatively to NO and HDL. These findings indicated that combined treatment with niacin and chromium has potential therapeutic protection of endothelial function by down-regulating ox-LDL/LOX-1 signaling pathway.

M129V polymorphism in the prion protein gene is not associated with mesial temporal lobe epilepsy in a Han Chinese population.

BACKGROUND AND PURPOSE: Prion protein (PrP) predominantly localized at synapses can modulate neuronal excitability. The prion protein gene (PRNP) has been considered one of the candidate genes that play a role in seizure susceptibility. A recent study demonstrated that the 129V allele in the PRNP gene was associated with susceptibility to temporal lobe epilepsy (TLE) in female patients in an Italian population. We screened variations in the open-reading frame (ORF) of the PRNP gene and also replicated the association of the M129V polymorphism with TLE in a Han Chinese population. METHODS: The M129V polymorphism was genotyped in 320 MTLE patients and 558 non-epilepsy controls. All subjects were Han Chinese. RESULTS: No novel polymorphism in the ORF of the PRNP gene was detected. Differences in the genotype distributions and allele frequencies of this polymorphism between cases and controls were insignificant (P = 0.24). Further analysis with stratification of the results by gender or age and analysis of clinical features in relation to M129V genotypes also yielded negative findings. CONCLUSIONS: The present study provides evidence that the M129V polymorphism in the PRNP gene is not associated with MTLE in a Han Chinese population.

Multiple missense mutations in the diazepam binding inhibitor (DBI) gene identified in schizophrenia but lack of disease association.

The diazepam binding inhibitor (DBI), alternatively known as the acyl-CoA binding protein (ACBP), is involved in multiple biological actions. The polypeptide binds to the peripheral, or mitochondrial, benzodiazepine receptor and facilitates transport of cholesterol to the inner membrane to stimulate steroid synthesis. Through this action, DBI indirectly modulates gamma-aminobutyric acid (GABA)-mediated inhibitory neurotransmission. DBI can be postulated as a candidate gene for psychiatric phenotypes including anxiety, mood, and psychotic disorders. In an examination of the DBI gene among 112 individuals with schizophrenia, our laboratory has identified 18 novel single nucleotide polymorphisms (SNPs), including three missense changes in conserved amino acids, a coding region microdeletion, and multiple SNPs in the putative promoter region. Case-control association analyses were performed for the missense changes, but none was found to be significantly associated with disease.