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This study aimed to explore the genotype-phenotype correlations in individuals with Genetic Skeletal Disorders (GSD), evaluate the efficacy of recombinant human Growth Hormone (rhGH) therapy. The retrospective analysis of the medical records of 80 pediatric patients with GSD diagnosed via whole-exome sequencing was conducted. The therapeutic effects of rhGH treatment were analyzed in 30 of these patients who received rhGH therapy. The study included 80 GSD patients, diagnosed at a median age of 4.88 years, with a median height standard deviation score (Ht-SDS) of − 3.58. The most common clinical manifestations included skeletal deformities (87.5%), short stature (81.3%), and distinctive facial features (including triangular face, abnormality of the philtrum, abnormality of the forehead, etc.) (65.0%). A total of 33 pathogenic genes associated with 20 groups of GSD were identified. The most common groups are Type II collagenopathies (related to the COL2A1 gene) (12/80, 15.0%) and the FGFR3 -related chondrodysplasia group (12/80, 15.0%). Those with pathogenic genes linked to Fundamental Cellular Processes had more severe short stature and prenatal phenotypes. Thirty patients received rhGH treatment for a median of 2.25 years (0.33–8.92), showing Ht-SDS increases of 0.66 ± 0.42 and 0.84 ± 0.52, after one and two years, respectively ( p  < 0.001). Eight untreated patients had an average Ht-SDS decrease of − 0.46 ± 0.55. In this cohort, pediatric GSD patients predominantly presented with short stature, skeletal deformities, and distinctive facial features (including triangular face, abnormality of the philtrum, abnormality of the forehead, etc.), indicating a genotype-phenotype correlation. Compared to untreated GSD patients, those receiving rhGH treatment demonstrated varying degrees of height improvement, however, the long-term efficacy of this treatment warrants further investigation.

Background Thalassemia, a hereditary hemolytic blood disorder, is characterized by high global allele prevalence and a gradually expanding variant spectrum. In Guangxi, China, routine genetic screening targeting hotspot variants of thalassemia has been implemented for over a decade, significantly reducing the incidence of severe cases. This study assessed the efficacy of the existing thalassemia screening protocol in Guangxi, China, by applying long-read sequencing (LRS) in family trios/quartets analysis for newborns. Methods This study enrolled 1491 families for the family trios/quartets analysis, among which at least one of the parents had undergone routine genetic screening for thalassemia. A total of 1516 newborns from these families were tested using LRS. The efficacy of routine screening was assessed by comparing detection results across generations. Results α-thalassemia demonstrated higher prevalence than β-thalassemia among newborns, with -- SEA and HBB c.52A > T as the predominant variants. Among the 1516 newborns, 1348 (88.92%) exhibited LRS results that were concordant with the predictions from parental screening. Of these 1348 newborns, 28 had parents who had undergone routine prenatal diagnosis. Discordance between the predictions from parental screening and newborn test results occurred in 168 cases (11.08%), with 154 (10.16%) of these attributable to genetic testing of only one of the parents or incomplete testing of both parents, while the remaining 14 (0.92%) were attributed to limitations of routine methods in detecting complex structural variants and rare point mutations. Conclusions Although routine genetic screening targeting hotspot variants is highly effective, its precision is constrained by strategic and methodological limitations. A comprehensive and accurate LRS-based screening method can serve as a valuable supplement to routine screening, enhancing the precision of thalassemia prevention and management.

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. We used Sanger and whole-exome sequencing to identify familial ATP7B (ATPase, Cu(++) transporting, β 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. 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. 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.

Background Deletion or duplication in the DMD gene is one of the most common causes of Duchenne and Becker muscular dystrophy (DMD/BMD). However, the pathogenicity of complex rearrangements involving DMD , especially segmental duplications with unknown breakpoints, is not well understood. This study aimed to evaluate the structure, pattern, and potential impact of rearrangements involving DMD duplication. Methods Two families with DMD segmental duplications exhibiting phenotypical differences were recruited. Optical genome mapping (OGM) was used to explore the cryptic pattern of the rearrangements. Breakpoints were validated using long-range polymerase chain reaction combined with next-generation sequencing and Sanger sequencing. Results A multi-copy duplication involving exons 64–79 of DMD was identified in Family A without obvious clinical symptoms. Family B exhibited typical DMD neuromuscular manifestations and presented a duplication involving exons 10–13 of DMD . The rearrangement in Family A involved complex in-cis tandem repeats shown by OGM but retained a complete copy (reading frame) of DMD inferred from breakpoint validation. A reversed insertion with a segmental repeat was identified in Family B by OGM, which was predicted to disrupt the normal structure and reading frame of DMD after confirming the breakpoints. Conclusions Validating breakpoint and rearrangement pattern is crucial for the functional annotation and pathogenic classification of genomic structural variations. OGM provides valuable insights into etiological analysis of DMD/BMD and enhances our understanding for cryptic effects of complex rearrangements.

GATA zinc finger domain-containing 2B (GATAD2B) is a subunit of the methyl-CpG-binding protein-1 complex (MECP1), which deacetylates methylated nucleosomes and regresses transcriptional activity. Recently, GATAD2B has been elucidated as a candidate gene in patients with intellectual disability (ID). In this study, we identified two novel heterozygous frameshift mutations of GATAD2B in two unrelated ID cases through next-generation sequencing (NGS). Both of the mutations c.80_81insGATGT and c.552_555delGAAA cause truncated proteins that might be detrimental to neurodevelopment. We performed western blotting and observed a reduction in the target protein compared with normal controls. This is the first report of GATAD2B in Chinese ID patients. Our findings will broaden the spectrum of GATAD2B mutations and facilitate genetic diagnosis and counseling.

Background Thoracic aortic aneurysm/dissection (TAAD) and patent ductus arteriosus (PDA) are serious autosomal-dominant diseases affecting the cardiovascular system. They are mainly caused by variants in the MYH11 gene, which encodes the heavy chain of myosin 11. The aim of this study was to evaluate the genotype–phenotype correlation of MYH11 from a distinctive perspective based on a pair of monozygotic twins. Methods The detailed phenotypic characteristics of the monozygotic twins from the early fetal stage to the infancy stage were traced and compared with each other and with those of previously documented cases. Whole-exome and Sanger sequencing techniques were used to identify and validate the candidate variants, facilitating the analysis of the genotype–phenotype correlation of MYH11 . Results The monozygotic twins were premature and presented with PDA, pulmonary hypoplasia, and pulmonary hypertension. The proband developed heart and brain abnormalities during the fetal stage and died at 18 days after birth, whereas his sibling was discharged after being cured and developed normally post follow-up. A novel variant c.766 A > G p. (Ile256Val) in MYH11 (NM_002474.2) was identified in the monozygotic twins and classified as a likely pathogenic variant according to the American College of Medical Genetics/Association for Molecular Pathology guidelines. Reviewing the reported cases ( n  = 102) showed that the penetrance of MYH11 was 82.35%, and the most common feature was TAAD (41.18%), followed by PDA (22.55%), compound TAAD and PDA (9.80%), and other vascular abnormalities (8.82%). The constituent ratios of null variants among the cases with TAAD (8.60%), PDA (43.8%), or compound TAAD and PDA (28.6%) were significantly different ( P  = 0.01). Further pairwise comparison of the ratios among these groups showed that there were significant differences between the TAAD and PDA groups ( P  = 0.006). Conclusion This study expands the mutational spectrum of MYH11 and provides new insights into the genotype–phenotype correlation of MYH11 based on the monozygotic twins with variable clinical features and outcomes, indicating that cryptic modifiers and complex mechanisms beside the genetic variants may be involved in the condition.

Background Disorders of sex development (DSDs) exhibit high genetic and phenotypic heterogeneity, and genotype–phenotype correlations are not fully understood. 5α-Reductase type 2 (5α-RD2) deficiency, a common form of DSD, is caused by SRD5A2 inactivation. This study investigated the role of SRD5A2 haplotypes in DSD, focusing on their corresponding phenotypes, structural changes and impacts on enzyme activity. Methods This study enrolled 216 individuals with DSD who underwent genetic analysis and 2,794 controls. Linkage disequilibrium analysis was performed in individuals with 5α-RD2 deficiency to identify SRD5A2 haplotypes, and haplotype frequencies were analysed across cohorts. The clinical manifestations of individuals with different SRD5A2 haplotypes were characterized. Structural predictions were employed to investigate the impacts of haplotypes on the 5α-RD2 structure and interactions with ligands. Functionally, kinetic assays were conducted to validate the effects of different haplotypes on enzyme activity. Results A SRD5A2 haplotype composed of c.265C > G and c.680G > A (Hap3: G-A) was identified, and the haplotype frequency was 64.71% in individuals with 5α-RD2 deficiency, 2.59% and 1.22% in non-5α-RD2 deficiency DSD cases without or with known DSD-related gene variants, respectively, and 1.57% in in-house controls. Globally, Hap3: G-A was enriched in southern Chinese individuals and showed high population differentiation, indicating a potential founder effect of the haplotype. The majority of homozygotes of Hap3: G-A presented microphallus, and nearly half of them manifested isolated microphallus. Structurally, Hap3: G-A was predicted to result in an increase in the solvent-accessible surface area (10.72 Å 2 ), a redistribution of hydrogen bonds within 5α-RD2, and a loss of key hydrogen bonds with NADPH. Functionally, kinetic assays showed that the catalytic efficiency of the enzyme encoded by Hap3: G-A was between that of Hap1: G-G and that of Hap2: C-A. Conclusions Hap3: G-A, which is prevalent in individuals with 5α-RD2 deficiency, suggests a potential founder effect. Structurally, compared with other haplotypes, Hap3: G-A seems to have a combined effect on the structure and interaction of 5α-RD2, rather than have merely additive effects of its constituent variants. Functionally, kinetic assays suggested a hypomorphic effect of Hap3: G-A. These findings provide valuable insights for understanding genotype–phenotype correlations, genetic counselling, early intervention and clinical management of individuals with 5α-RD2 deficiency or even other DSDs. Highlights On the basis of 216 DSD individuals and 2,794 controls, a novel SRD5A2 haplotype (Hap3: G-A), which is composed of common variants and is especially prevalent in DSD patients with 5α-RD2 deficiency, was identified, indicating the potential founder effect of Hap3: G-A. The majority of 5α-RD2 deficiency individuals with homozygous Hap3: G-A presented microphallus. Three-dimensional structure and model construction predicted that Hap3: G-A resulted in an increase in the solvent-accessible surface area, redistribution of hydrogen bonds within 5α-RD2, and loss of key hydrogen bonds with NADPH. Functionally, kinetic assays showed that the catalytic efficiency of the enzyme encoded by Hap3: G-A was between those of Hap1: G-G and Hap2: C-A, suggesting a hypomorphic effect of this haplotype. Plain Language Summary Individuals with disorders of sex development (DSDs) present variable genotypes and phenotypes, and the genotype–phenotype correlation remains poorly understood. 5α-Reductase type 2 (5α-RD2) deficiency, a common form of DSD, is caused by SRD5A2 inactivation. In this study, genetic testing and analysis were performed in 216 individuals with DSD and 2,794 non-DSD controls. A novel SRD5A2 haplotype (Hap3: G-A) was identified, and the haplotype frequency was 64.71% in individuals with 5α-RD2 deficiency, 2.59% and 1.22% in non-5α-RD2 deficiency DSD cases without or with known DSD-related gene variants, respectively, and 1.57% in in-house controls. Globally, Hap3: G-A was enriched in southern Chinese individuals and showed high population differentiation, indicating a potential founder effect of the haplotype. Analyzing the phenotype spectrum of individuals with 5α-RD2 deficiency, we found that the majority of homozygotes of Hap3: G-A presented microphallus, and nearly half of them manifested isolated microphallus. Structurally, Hap3: G-A was predicted to result in an increase in the solvent-accessible surface area, the redistribution of hydrogen bonds within 5α-RD2, and the loss of key hydrogen bonds with NADPH. Compared with other haplotypes, Hap3: G-A seemed to have a combined effect on the structure and interaction of 5α-RD2, rather than simply additive effects of its constituent variants. Functionally, kinetic assays showed that the catalytic efficiency of the enzyme encoded by Hap3: G-A was between that of Hap1: G-G and that of Hap2: C-A, suggesting a hypomorphic effect of this haplotype. These findings provide valuable insights for understanding genotype–phenotype correlations, genetic counselling, early intervention and clinical management of individuals with 5α-RD2 deficiency or even other DSDs.

Objective Craniosynostosis is the result of the early fusion of cranial sutures. Syndromic craniosynostosis includes but not limited by Crouzon syndrome and Pfeiffer syndrome. Considerable phenotypic overlap exists among these syndromes and mutations in FGFR2 may cause different syndromes. This study aims to investigate the explanation of the phenotypic variability via clinical and genetic evaluation for eight patients in a large pedigree. Methods For each patient, comprehensive physical examination, cranial plain CT scan with three‐dimensional CT reconstruction (3D‐CT), and eye examinations were conducted. Whole exome sequencing was applied for genetic diagnosis of the proband. Variants were analyzed and interpreted following the ACMG/AMP guidelines. Sanger sequencing was performed to reveal genotypes of all the family members. Results A pathogenic variant in the FGFR2 gene, c.833G > T (p.C278F), was identified and proved to be co‐segregate with the disease. Some symptoms of head, hearing, vision, mouth, teeth expressed differently by affected individuals. Nonetheless, all the eight patients manifested core symptoms of Crouzon syndrome without abnormality in the limbs, which could exclude diagnosis of Pfeiffer syndrome. Conclusion We have established clinical and genetic diagnosis of Crouzon syndrome for eight patients in a five‐generation Chinese family. Variability of clinical features among these familial patients was slighter than that in previously reported sporadic cases. Clinical and genetic evaluations were performed for eight patients in a large Chinese pedigree. All the patients manifested core symptoms of Crouzon syndrome without abnormality in the limbs. A pathogenic variant in the FGFR2 gene, c.833G > T (p.C278F), was identified and proved to be co‐segregate with the disease. Our findings may facilitate diagnosis and genetic counseling of syndromic craniosynostosis.

Background Primary carnitine deficiency (PCD) is an autosomal recessive disorder of carnitine transportation caused by mutations in the SLC22A5 that lead to low serum carnitine levels and decreased intracellular carnitine accumulation. Characteristic clinical findings are hypoketotic hypoglycemia and skeletal and cardiac myopathy. Objective To genetically diagnose 24 unrelated Chinese patients with PCD, including 18 infants and six adults. Methods The entire coding region and the intron–exon boundaries of SLC22A5 were amplified by polymerase chain reaction (PCR). In silico analyses and reverse transcription‐polymerase chain reaction (RT‐PCR) were used to predict variants’ impact on protein structure and function. Results Disease‐causing variants in the SLC22A5 were identified in all 24 subjects, and c.288delG, c.495C>A, c.774_775insTCG, c.824+1G>A, and c.1418G>T were novel. The novel variant c.824+1G>A caused a truncated protein p.Phe276Tyrfs*8. Conclusions We identified 13 variants in the SLC22A5 in 24 PCD patients, and five of these variants are novel mutations. c.824+1G>A was confirmed to alter mRNA splicing by reverse transcription PCR. Furthermore, our findings broaden the mutation spectrum of SLC22A5 and the understanding of the diverse and variable effects of PCD variants. We report 24 cases of PCD confirmed with mutation analysis. Five mutations are novel and c.824+1G>A is a novel splice site mutation. c.51C>G, c.760C>T, and c.1400C>G are the most frequent mutations in China.

ROR2, a member of the ROR family, is essential for skeletal development as a receptor of Wnt5a. The present study aims to investigate the mutational spectrum of in children with short stature and to identify the underlying molecular mechanisms. We retrospectively analyzed clinical phenotype and whole-exome sequencing (WES) data of 426 patients with short stature through mutation screening of . We subsequently examined the changes in protein expression and subcellular location in caused by the mutations. The mRNA expression of downstream signaling molecules of the Wnt5a-ROR2 pathway was also examined. We identified 12 mutations in in 21 patients, including 10 missense, one nonsense, and one frameshift. Among all missense variants, four recurrent missense variants [c.1675G > A(p.Gly559Ser), c.2212C > T(p.Arg738Cys), c.1930G > A(p.Asp644Asn), c.2117G > A(p.Arg706Gln)] were analyzed by experiments . The c.1675G > A mutation significantly altered the expression and the cellular localization of the ROR2 protein. The c.1675G > A mutation also caused a significantly decreased expression of c-Jun. In contrast, other missense variants did not confer any disruptive effect on the biological functions of ROR2. We expanded the mutational spectrum of in patients with short stature. Functional experiments potentially revealed a novel molecular mechanism that the c.1675G > A mutation in might affect the expression of downstream Wnt5a-ROR2 pathway gene by disturbing the subcellular localization and expression of the protein.