Exome sequencing as the first-tier test for pediatric respiratory diseases: A single-center study.

The high clinical and genetic heterogeneity makes it difficult to reach a confirmative diagnosis of suspected pediatric respiratory inherited diseases. Many patients with monogenic respiratory disorders could be missed without genetic testing. We performed a single-center study in Beijing Children's Hospital to demonstrate the clinical utility of exome sequencing (ES) as a first-tier test by evaluating the diagnostic yields of ES for inherited diseases with respiratory symptoms. A total of 107 patients were recruited in this study. We identified 51 pathogenic or likely pathogenic variants in 37 patients by ES (with or without copy number variants sequencing). The overall diagnostic yield was 34.6% (37/107). The most frequent disorders in our cohort were primary immunodeficiency disease (PIDs) (18/37, 48.6%) and primary ciliary dyskinesia (PCD) (9/37, 24.3%). We further reviewed the directive outcomes of genetic testing on the 37 positive cases. Our study demonstrated the effectiveness of ES as a first-tier test in China for diagnosing monogenic diseases of the respiratory system. In the era of precision medicine, ES as a first-tier test can rapidly make a molecular diagnosis and direct the intervention of the positive cases in pediatric respiratory medicine.

[Identification of variants in TNNI3 gene in two children with restrictive cardiomyopathy].

OBJECTIVE: To identify the pathogenesis in two patients of restrictive cardiomyopathy (RCM) using high-throughput sequencing. METHODS: Peripheral blood samples from the two patients and their parents were collected and genomic DNAs were extracted to conduct targeted next generation sequencing or whole exome sequencing. Bioinformation analysis was performed to identify the pathogenic variants in genes associated with cardiomyopathy, which were further validated by Sanger sequencing. RESULTS: By high throughput sequencing, we detected a de novo heterozygous variant c.549+1G>T in TNNI3 gene in patient 1. The variant has not been reported previously and was predicted to be pathogenic in line with American College of Medical Genetics and Genomics (ACMG) guidelines (PVS1+PS2+PM2). Another heterozygous variant c.433C>T (p.Arg145Trp) in TNNI3 gene was identified in patient 2 and his father. The variant had been reported as pathogenic variant in Clinvar and HGMD databases; based on ACMG guidelines, the variant was predicted to be likely pathogenic (PS3+PM1+PP3). CONCLUSION: TNNI3 variants may be the causative gene responsible for restrictive cardiomyopathy in the two patients. High throughput sequencing results provide bases for the diagnosis of restrictive cardiomyopathy.

Compound heterozygous LPIN2 pathogenic variants in a patient with Majeed syndrome with recurrent fever and severe neutropenia: case report.

BACKGROUND: Majeed syndrome is a rare, autosomal recessive autoinflammatory disorder first described in 1989. The syndrome starts during infancy with recurrent relapses of osteomyelitis typically associated with fever, congenital dyserythropoietic anemia (CDA), and often neutrophilic dermatosis. Mutations in the LPIN2 gene located on the short arm of chromosome 18 have been identified as being responsible for Majeed syndrome. CASE PRESENTATION: We report an 8-month-old boy, who presented with recurrent fever, mild to moderate anemia, and severe neutropenia. Erythrocyte sedimentation rate and C-reactive protein were elevated. Molecular testing identified a paternal splicing donor site variant c.2327 + 1G > C and a maternal frameshift variant c.1691_1694delGAGA (Arg564Lysfs*3) in LPIN2. CONCLUSIONS: Only a few cases with LPIN2 mutation have been reported, mainly in the Middle East with homozygous variants. Our patient exhibited a mild clinical phenotype and severe neutropenia, different from previous reports.

[Analysis of SMARCA2 gene mutation in a child with Nicolaides-Baraitser syndrome].

OBJECTIVE: To explore the molecular basisfor a child featuring short stature, abnormal facial features and developmental delay. METHODS: Genomic DNA was extracted from peripheral blood samples from the child and his family members. Next-generation sequencing was carried out to screen the whole exomes of the core family. Detected variants were filtered and analyzed according to the standards and guidelines for the interpretation of sequence variants recommended by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. RESULTS: Trio-based sequencing has identified a de novo variant c.3593T>G (p.Val1198Gly) in the SMARCA2 gene in the patient. The variant was located in the Helicase C-terminal domain and was classified as pathogenic based on the guidelines. CONCLUSION: The patient was diagnosed with Nicolaides-Baraitser syndrome caused by SMARCA2 gene mutation.

[Molecular diagnosis and functional study of a pedigree affected with Lubs X-linked mental retardation syndrome].

OBJECTIVE: To explore the genetic basis for a pedigree affected with X-linked mental retardation. METHODS: The proband was subjected to chromosomal karyotyping, FMR1 mutation testing and copy number variation analysis with a single nucleotide polymorphism microarray (SNP array). His family members were subjected to multiplex ligation-dependent probe amplification (MLPA) assaying. Expression of genes within the repeated region were analyzed. RESULTS: The proband had a normal chromosomal karyotype and normal number of CGG repeats within the FMR1 gene. SNP array identified a 370 kb duplication in Xq28 (ChrX: 153 027 633-153 398 515), which encompassed 14 genes including MECP2. The patient was diagnosed as Lubs X-linked mental retardation syndrome (MRXSL). MLPA confirmed the presence of copy number variation, its co-segregation with the disease, in addition with the carrier status of females. Genes from the duplicated region showed higher levels of expression (1.79 to 5.38 folds) within peripheral blood nucleated cells of the proband. CONCLUSION: The patients were diagnosed with MRXSL. The expression of affected genes was up-regulated due to the duplication. Genetic counseling and prenatal diagnosis may be provided based on the results.

De novo exonic deletion of KDM6A in a Chinese girl with Kabuki syndrome: A case report and brief literature review.

Kabuki syndrome (KS) is a rare condition with multiple congenital anomalies and mental retardation. Exonic deletions, disrupting the lysine (K)-specific demethylase 6A (KDM6A) gene have been demonstrated as rare cause of KS. Here, we report a de novo 227-kb deletion in chromosome Xp11.3 of a 7-year-old Chinese girl with KS. Besides the symptoms of KS, the patient also presented with skin allergic manifestations, which were considered to be a new, rare feature of the phenotypic spectrum. The deletion includes the upstream region and exons 1-2 of KDM6A and potentially causes haploinsuffiency of the gene. We also discuss the mutation spectrum of KDM6A and clinical variability of patients with KDM6A deletion through a literature review. © 2016 Wiley Periodicals, Inc.

[Genetic diagnosis and analysis for two cases of ring chromosome 22].

OBJECTIVE: To confirm the genetic diagnosis of two patients with ring chromosome 22 syndrome and investigate the mechanism underlying the formation of r(22) and potential genetic causes for the clinical phenotypes. METHODS: Cytogenetic and molecular analyses using standard G-banding, fluorescence in situ hybridization and single nucleotide polymorphism array (SNP array) were performed. RESULTS: For case 1, the karyotype was 46,XY,r(22)(p11q13). SNP array has identified a 7.0 Mb heterozygous deletion at 22q13.2q13.33. For case 2, the karyotype was 46,XY,r(22)(p11q13)[84]/45,XY,-22[6]; SNP array has detected a heterozygous microdeletion of 1.6 Mb at 22q13.33. CONCLUSION: With combined application of genetic testing, 2 cases of r(22) syndrome were diagnosed, which has improved the understanding of the genotype-phenotype correlation of r(22).

Noninvasive prenatal testing for Wilson disease by use of circulating single-molecule amplification and resequencing technology (cSMART).

BACKGROUND: Noninvasive prenatal testing (NIPT) for monogenic diseases by use of PCR-based strategies requires precise quantification of mutant fetal alleles circulating in the maternal plasma. The study describes the development and validation of a novel assay termed circulating single-molecule amplification and resequencing technology (cSMART) for counting single allelic molecules in plasma. Here we demonstrate the suitability of cSMART for NIPT, with Wilson Disease (WD) as proof of concept. METHODS: We used Sanger and whole-exome sequencing to identify familial ATP7B (ATPase, Cu(++) transporting, ß polypeptide) gene mutations. For cSMART, single molecules were tagged with unique barcodes and circularized, and alleles were targeted and replicated by inverse PCR. The unique single allelic molecules were identified by sequencing and counted, and the percentage of mutant alleles in the original maternal plasma sample was used to determine fetal genotypes. RESULTS: Four families with WD pedigrees consented to the study. Using Sanger and whole-exome sequencing, we mapped the pathogenic ATP7B mutations in each pedigree and confirmed the proband's original diagnosis of WD. After validation of cSMART with defined plasma models mimicking fetal inheritance of paternal, maternal, or both parental mutant alleles, we retrospectively showed in second pregnancies that the fetal genotypes assigned by invasive testing and NIPT were concordant. CONCLUSIONS: We developed a reliable and accurate NIPT assay that correctly diagnosed the fetal genotypes in 4 pregnancies at risk for WD. This novel technology has potential as a universal strategy for NIPT of other monogenic disorders, since it requires only knowledge of the parental pathogenic mutations.

DMD mutation spectrum analysis in 613 Chinese patients with dystrophinopathy.

Dystrophinopathy is a group of inherited diseases caused by mutations in the DMD gene. Within the dystrophinopathy spectrum, Duchenne and Becker muscular dystrophies are common X-linked recessive disorders that mainly feature striated muscle necrosis. We combined multiplex ligation-dependent probe amplification with Sanger sequencing to detect large deletions/duplications and point mutations in the DMD gene in 613 Chinese patients. A total of 571 (93.1%) patients were diagnosed, including 428 (69.8%) with large deletions/duplications and 143 (23.3%) with point mutations. Deletion/duplication breakpoints gathered mostly in introns 44-55. Reading frame rules could explain 88.6% of deletion mutations. We identified seventy novel point mutations that had not been previously reported. Spectrum expansion and genotype-phenotype analysis of DMD mutations on such a large sample size in Han Chinese population would provide new insights into the pathogenic mechanism underlying dystrophinopathies.

Novel mutation in PARS2 revealed highly variable phenotype of developmental and epileptic encephalopathy-75.

BACKGROUND AND AIMS: Biallelic variants in mitochondrial prolyl-tRNA synthetase 2 (PARS2) are associated with developmental and epileptic encephalopathy-75 (DEE75), which is characterized by global developmental delay, seizures and brain imaging anomalies. To date, fewer than 20 patients with PARS2 mutation have been reported in previous literature, and only ten of them had detailed phenotype information. MATERIALS AND METHODS: In our study, we performed whole exome sequencing for three intellectual disability patients from one family. RESULTS: Two novel missense PARS2 variants, c.467C>G (p. Pro156Arg) and c.1183G>C (p. Asp395His), were identified. All of our patients displayed profound intellectual disability and absent speech, while other features, including seizures, cardiomyopathy, short stature and brain MRI, varied greatly in this family. This is also the first report of ovarian dysfunction in association with PARS2 mutations. CONCLUSIONS: We reported three patients with the longest lifespan in reported cases so far, and our results provided an opportunity to study DEE75 prognosis and symptoms in adulthood. Our results further extended the clinical and genetic spectra of PARS2 gene mutation.

Genetic screening reveals hotspot variants and prevalence rates of Hermansky-Pudlak syndrome in the Chinese population.

BACKGROUND: Hermansky-Pudlak Syndrome (HPS) is a rare autosomal recessive genetic disorder associated with varied clinical manifestations, including oculocutaneous albinism, bleeding tendency, and systemic complications. Early and accurate diagnosis is crucial for medical interventions and genetic counseling. We aimed to characterize the prevalence and spectrum of pathogenic variants of HPS in the Chinese population through genetic screening of newborns. METHODS: Genetic screening for HPS mutations was conducted in 29,622 Chinese newborns from 13 provinces using next-generation sequencing. Pathogenic variants were identified and classified according to ACMG guidelines. Prevalence rates were estimated, and potential hotspot variants were identified. RESULTS: Among screened newborns, 215 carriers with 103 distinct pathogenic variants were identified, including two carriers with additional missense variants. Potential hotspot variants in seven genes were identified, collectively representing over 20 % of carriers in each respective gene. Particularly, the HPS3 c.1838C>G variant was exclusively reported in the Chinese population, suggesting a potential founder effect. The estimated prevalence rate of HPS in China was 2.84/1,000,000. CONCLUSION: Our study provides valuable insights into the genetic landscape of HPS in the Chinese population, aiding in genetic counseling, early diagnosis, and management strategies. These findings contribute to enhancing the understanding and management of HPS in China.

Targeted gene sequencing and hearing follow-up in 7501 newborns reveals an improved strategy for newborn hearing screening.

Hearing loss is a common congenital condition. Concurrent newborn hearing and limited genetic screening has been implemented in China for the last decade. However, the role of gene sequencing screening has not been evaluated. In this study, we enrolled 7501 newborns (52.7% male, 47.3% female) in our Newborn Screening with Targeted Sequencing (NESTS) program, and 90 common deafness genes were sequenced for them. Hearing status assessments were conducted via telephone from February 2021 to August 2022, for children aged 3 to 48 months. Of the universal newborn hearing screening, 126 (1.7%) newborns did not pass. Targeted sequencing identified 150 genetically positive newborns (2.0%), with 25 exhibiting dual-positive results in both screening. Following diagnostic audiometry revealed 18 hearing loss newborns and half of them had abnormal results in both screening. The positive predictive value for universal newborn hearing screening alone was merely 14.3% (18/126). However, when combined with targeted sequencing, this rate increased to 36.0% (9/25). Furthermore, limited genetic screening identified 316 carriers of hot-spot variants, but none exhibited biallelic variants. All 15 hot-spot carriers who failed physical screening demonstrated normal hearing during follow-up. In this cohort study of 7501 Newborns, Combining targeted sequencing with universal newborn hearing screening demonstrated technical feasibility and clinical utility of identifying individuals with hearing loss, especially when coupled with genetic counseling and closed-loop management. It is suggested to use this integrated method as an improved strategy instead of the current limited genetic screening program in some regions of China.

DVsc: An Automated Framework for Efficiently Detecting Viral Infection from Single-cell Transcriptomics Data.

Single-cell RNA sequencing (scRNA-seq) has emerged as a valuable tool for studying cellular heterogeneity in various fields, particularly in virological research. By studying the viral and cellular transcriptomes, the dynamics of viral infection can be investigated at a single-cell resolution. However, limited studies have been conducted to investigate whether RNA transcripts from clinical samples contain substantial amounts of viral RNAs, and a specific computational framework for efficiently detecting viral reads based on scRNA-seq data has not been developed. Hence, we introduce DVsc, an open-source framework for precise quantitative analysis of viral infection from single-cell transcriptomics data. When applied to approximately 200 diverse clinical samples that were infected by more than 10 different viruses, DVsc demonstrated high accuracy in systematically detecting viral infection across a wide array of cell types. This innovative bioinformatics pipeline could be crucial for addressing the potential effects of surreptitiously invading viruses on certain illnesses, as well as for designing novel medicines to target viruses in specific host cell subsets and evaluating the efficacy of treatment. DVsc supports the FASTQ format as an input and is compatible with multiple single-cell sequencing platforms. Moreover, it could also be applied to sequences from bulk RNA sequencing data. DVsc is available at http://62.234.32.33:5000/DVsc.

Phenylalanyl-tRNA synthetase deficiency caused by biallelic variants in FARSA gene and literature review.

BACKGROUND: Aminoacyl-tRNA synthetases (ARSs) are indispensable enzymes for protein biosynthesis in cells. The phenylalanyl-tRNA synthetase (FARS1) located in cytoplasm which consists of two FARS alpha subunits (FARSA) and two FARS beta subunits (FARSB). Autosomal recessive inheritance of pathogenic variants of FARSA or FARSB can result in defective FARS1 which are characterized by interstitial lung disease, liver disease, brain abnormalities, facial dysmorphism and growth restriction. METHODS: Exome sequencing was used to detect the candidate variants. The in silico prediction and expressional level analysis were performed to evaluate the pathogenicity of the variations. Additionally, we presented the patient's detailed clinical information and compared the clinical feature with other previously reported patients with FARSA-deficiency. RESULTS: We identified compound heterozygous rare missense variants (c.1172 T > C/ p.Leu391Pro and c.1211G > A/ p.Arg404His) in FARSA gene in a Chinese male patient. The protein structure prediction and the analysis of levels of FARSA and FARSB subunits indicated both variants pathogenic. Clinical feature review indicated inflammatory symptoms in young infants may be an additional key feature. Thyroid dysfunction should be considered as a phenotype with variable penetrance. CONCLUSIONS: Our results expanded the current phenotypic and genetic spectrum of FARSA-deficiency.

Identification of novel homozygous nonsense SLC10A7 variant causing short stature, amelogenesis imperfecta, and skeletal dysplasia with scoliosis and surgical management of spine.

BACKGROUND: Short stature, amelogenesis imperfecta, and skeletal dysplasia with scoliosis is a rare, autosomal recessive, skeletal disorder first described in 2018. This syndrome starts with pre- and postnatal developmental delay, and gradually presents with variable facial dysmorphisms, a short stature, amelogenesis imperfecta, and progressive skeletal dysplasia affecting the limbs, joints, hands, feet, and spine. CASE PRESENTATION: We identified a homozygous novel nonsense mutation in exon 1 of SLC10A7 (NM_001300842.2: c.100G > T / p.Gly34*) segregating with the typical disease phenotype in a Han Chinese family. We reviewed the 12-year surgical treatment history with seven interventions on spine. CONCLUSION: To date, only 12 cases of the SLC10A7 mutation have been reported, mainly from consanguineous families. Our patient showed a relatively severe and broad clinical phenotype compared with previously reported cases. In this patient, annual check-ups and timely surgeries led to a good outcome.

NeuroCNVscore: a tissue-specific framework to prioritise the pathogenicity of CNVs in neurodevelopmental disorders.

BACKGROUND: Neurodevelopmental disorders (NDDs) are associated with altered development of the brain especially in childhood. Copy number variants (CNVs) play a crucial role in the genetic aetiology of NDDs by disturbing gene expression directly at linear sequence or remotely at three-dimensional genome level in a tissue-specific manner. Despite the substantial increase in NDD studies employing whole-genome sequencing, there is no specific tool for prioritising the pathogenicity of CNVs in the context of NDDs. METHODS: Using an XGBoost classifier, we integrated 189 features that represent genomic sequences, gene information and functional/genomic segments for evaluating genome-wide CNVs in a neuro/brain-specific manner, to develop a new tool, neuroCNVscore. We used Human Phenotype Ontology to construct an independent NDD-related set. RESULTS: Our neuroCNVscore framework (https://github.com/lxsbch/neuroCNVscore) achieved high predictive performance (precision recall=0.82; area under curve=0.85) and outperformed an existing reference method SVScore. Notably, the predicted pathogenic CNVs showed enrichment in known genes associated with autism. CONCLUSIONS: NeuroCNVscore prioritises functional, deleterious and pathogenic CNVs in NDDs at whole genome-wide level, which is important for genetic studies and clinical genomic screening of NDDs as well as for providing novel biological insights into NDDs.

Behavioural deficits of autism spectrum disorder and associations with different gene clusters: a study with the whole-genome transmission disequilibrium test.

BACKGROUND: Autism spectrum disorder (ASD) is a diverse neurodevelopmental disease primarily distinguished by limited and stereotyped activities as well as impaired social interaction. Due to the high heritability of ASD, research on the disorder has emphasised on identifying the underlying genetic and epigenetic aetiology. Many ASD loci have been identified by genome-wide association studies (GWASs). However, GWASs are more susceptible to bias due to population stratification. Moreover, GWASs barely reflect the genetic aetiology of subtypes of behavioural deficits. METHODS: We applied whole-genome transmission disequilibrium test (TDT) to reveal the gene sets that are significantly associated with the four behavioural subtypes of restricted repetitive behaviours in 334 ASD trios. We further mapped the clustered genes to pathways and enriched the SFARI genes in these pathways. RESULTS: Four unique gene clusters (181 genes in total) that are related to four different behavioural subtypes in ASD were identified. 23 SFARI genes were enriched in these four clusters. Through pathway analysis, nine non-SFARI genes (CNDP1, ETNK1, ITPKB, KCNQ5, PDE4D, PDGFRA, PPARGC1A, ULK2, SYNJ2) were found to be linked to the SFARI genes, which may contribute to the development of ASD. Furthermore, we found that the mTOR pathway enriched with the CNDP1, PDE4D, ULK2 genes is associated with neurodevelopment. CONCLUSIONS: Whole-genome TDT test is a unique tool in clustering genes related to ASD subtypes of behavioural deficits. Several new candidate genes for ASD are revealed by pathway analysis of the clustered genes. These findings are useful for understanding the underlying mechanism of ASD.

Genetic diagnostic yields of 354 Chinese ASD children with rare mutations by a pipeline of genomic tests.

Purpose: To establish an effective genomic diagnosis pipeline for children with autism spectrum disorder (ASD) for its genetic etiology and intervention. Methods: A cohort of 354 autism spectrum disorder patients were obtained from Beijing Children's Hospital, Capital Medical University. Peripheral blood samples of the patients were collected for whole genome sequencing (WGS) and RNA sequencing (RNAseq). Sequencing data analyses were performed for mining the single nucleotide variation (SNV), copy number variation (CNV) and structural variation (SV). Sanger sequencing and quantitative PCR were used to verify the positive results. Results: Among 354 patients, 9 cases with pathogenic/likely pathogenic copy number variation and 10 cases with pathogenic/likely pathogenic single nucleotide variations were detected, with a total positive rate of 5.3%. Among these 9 copy number variation cases, 5 were de novo and 4 were inherited. Among the 10 de novo single nucleotide variations, 7 were previously unreported. The pathological de novo mutations account for 4.2% in our cohort. Conclusion: Rare mutations of copy number variations and single nucleotide variations account for a relatively small proportion of autism spectrum disorder children, which can be easily detected by a genomic testing pipeline of combined whole genome sequencing and RNA sequencing. This is important for early etiological diagnosis and precise management of autism spectrum disorder with rare mutations.

Molecular identification of T-box transcription factor 6 and prognostic assessment in patients with congenital scoliosis: A single-center study.

Background: Congenital scoliosis (CS) is characterized by vertebral malformations. The precise etiology of CS is not fully defined. A compound inheritance of TBX6 was identified in 10% of patients with CS in Han Chinese and formed a distinguishable subtype named TBX6-associated congenital scoliosis (TACS). Methods: To investigate the variants and risk haplotype of TBX6, we recruited 121 patients with CS at Beijing Children's Hospital. We collected the clinical characteristics and surgical treatment options and followed their postoperative prognoses. Results: Eight patients (6.6%) were molecularly diagnosed with TACS and carried the previously defined pathogenic TBX6 compound heterozygous variants. All the eight patients with TACS had the typical TACS clinical feature of hemivertebrae in the lower part of the spine. These patients received posterior hemivertebra resection combined with segmental fusion. Follow-ups revealed satisfactory correction without postoperative complications. Conclusion: We observed a 6.6% prevalence of TACS in our CS cohort. Follow-ups further highlighted that surgical treatment of hemivertebra resection combined with segmental fusion performed well with prognosis for patients with TACS. This could provide valuable information for CS individuals with compound heterozygosity in TBX6.

Genotypes and clinical intervention of patients with neurofibromatosis type 1 associated dystrophic scoliosis.

Objective: To analyze the genotypic characteristics of patients with neurofibromatosis type 1 (NF1) associated dystrophic scoliosis and to summarize the outcomes of the surgical treatment of these patients. Methods: Exome sequencing (ES) combined with multiplex ligation-dependent probe amplification (MLPA) was used for genotypic identification. All patients underwent surgical treatments for spinal deformities, and the outcomes of the surgery was summarized by analyzing the clinical and imaging parameters before and after the surgery. Results: Fourteen patients (six males and eight females) were clinically diagnosed as NF1 associated dystrophic scoliosis with common symptoms including café-au-lait spots, paravertebral tumors, and dystrophic scoliosis. NF1 mutations were detected in 12 (85.7%) patients, including four nonsense mutations, three splicing mutations, three frameshift mutations, and two exon deletions. The first surgical procedure included growing-rod surgery in 10 patients and posterior spinal fusion in four patients. The follow-up duration was 2.3 years (1.0-10.3 years), and the Cobb angle of the main curve improved from 61.5° (30°-125°) pre-operatively to 14.5° (0°-42°) at the last follow-up, with an average correction rate of 74.0% (44-100%). Instrumentation-related complications occurred in four patients during the follow-up period. Conclusions: In patients with dystrophic scoliosis who met the clinical diagnostic criteria for NF1, the mutation detection rate of ES combined with MLPA was 85.7%. There was no mutation hotspot in NF1 gene, molecular diagnosis could offer information about genetic counseling, prenatal diagnosis and eugenics. Surgical treatment according to patient's age and severity could effectively correct the spinal deformities.